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Microbiological and biochemical characterization


of seera: a traditional fermented food of
Himachal Pradesh
Article January 2012

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INTERNATIONAL JOURNAL OF FOOD AND


FERMENTATION TECHNOLOGY
VOL. 2, NO. 1, JUNE 2012

Editor-in-Chief
Prof. VK Joshi
Professor and Head,
Fermentation Technology Lab, Department of Food Science and Technology
Dr YS Parmar University of Horticulture and Forestry
Nauni, Solan, Himachal Pradesh, India
E Mail:vkjoshipht@rediffmail.com

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EDITORIAL BOARD
Editor-In-chief
Prof. V.K. Joshi
Professor and Head,
Fermentation Technology Lab,
Department of Food Science and Technology,
Dr YS Parmar University of Horticulture and
Forestry, Nauni, Solan, Himachal Pradesh, India
vkjoshipht@rediffmail.com
editorijfft@gmail.com

Executive Editorial Board


Dr (Mrs) Sunita Garg,
Editor, Natural Product Radiance,
NISCAIR, New Delhi, India
sunitaq@niscair.in
Dr ( Mrs) Devina Vaidya
Department of Food Science and Technology
Dr. Y.S. Parmar University of Horticulture and
Forestry, Nauni, Solan, Himachal Pradesh, India
devinavaidya@yahoo.com
Dr NS Thakur
Department of Food Science and Technology
Dr. Y.S. Parmar University of Horticulture and
Forestry, Nauni, Solan, Himachal Pradesh, India
nsthakur1964@gmail.com
Dr JK Gupta
Department of Entomology and Apiculture
Dr YS Parmar University of Horticulture and Forestry
Nauni-Solan , Himachal Pradesh, India
jkg393@gmail.com
Dr B.L.Attri
Central Institute of temperate Horticulture,
Regional Station ,Mukteshwar,-Kunam,
Nainital, Uttarakhand
Attriblnrcwa@rediffmail.com,
Dr Wamik Azmi
Department of Biotechnology,
Himachal Pradesh University,
Summer Hill, Shimla, Himachal Pradesh, India
wamikazmi@rediffmail.com

Dr Neerja S. Rana
Department of Basic Sciences
Dr. Y.S. Parmar University of Horticulture and
Forestry, Nauni-Solan, Himachal Pradesh, India
drneerjauhf24@yahoo.co.in
Dr Shashi Bhushan
Division of Biotechnology,
Institute of Himalayan Bio-resource Technology (CSIR),
Palampur,DisttKangra, Himachal Pradesh, India
sbhushan@ihbt.res.in,
Dr Satish Kumar Sharma
G B Pant University of Agriculture & Technology, Hill campus,
Ranichauri, Distt Tehri-Garhwal,
Uttarakhand, India
drsatish10@yahoo.com
Dr Om Prakash Chauhan
Scientist
Fruits and Vegetables Technology Division
Defence Food Research Laboratory
Siddarthanagar, Mysore, India
opchauhan@gmail.com
Dr PS Panesar
Associate Professor
Biotechnology Research Laboratory
Department of Food Engineering and Technology
SL Institute of Engineering and Technology,
Longowal, Punjab,India
pspanesarrr@yahoo.com

EDITORIAL ADVISORY BOARD


Dr Sumit Arora ( Dairy Science)
Dairy Chemistry Division, National Dairy Research Institute
Karnal,India
sumitak123@yahoo.com
Prof Tek Chand Bhalla
Department of Biotechnology,
Himachal Pradesh University,
Shimla, Himachal Pradesh, India
bhallatc@rediffmail.com
Dr M C Pandey, ( Meat and Meat products)
Deptt of Freeze Drying and Animal Product Technology,
Defense Food Research lab, Mysore, India
dfrlmysore@sancharnet.in
Dr V M Pratape, (Grain Science and Technology),
Deptt of Grain Science and Technology,
CFTRI Mysore, India
gst@cftri.res.in
Dr Pura Naik J (Plantation crops)
Division of Plantation, Spices and Flavour Tech.,
CFTRI Mysore, India
puranaik@yahoo.com
Professor Pradeep Khanna
Coordinator
College of Basic Science, PAU Ludhiana, Punjab, India
pkhanna_pau@rediffmail.com
Dr Y S Dhaliwal ( Food and Nutrition)
Head, Department of Food Science and Nutrition, College of Home
Science, CSK HPKV, Palampur , Himachal Pradesh, India
Ysdhaliwal44@yahoo.co.in
Dr R.S. Singh ( Food fermentation and enzyme technology)
Department of Biotechnology,
Punjabi University, Patiala, Punjab, India.
rssingh11@lycos.com
Dr SS Kanwar
Department of Microbiology, CSK HPKV Palampur, Distt Kangra
Himachal Pradesh, India
sskanwar1956@gmail.com
Dr. RC Ray
Principal Scientist (Microbiology)
Regional Centre of Central Tuber Crops Research Institute
Dumuduma Housing Board, Bhubaneswar, Orissa, India
Dr. Rintu Banerjee ( Microbial Technology)
Microbial Biotechnology and Downstream Processing
Laboratory
Agricultural & Food Engineering Department
Indian Institute of Technology, Kharagpur ,West Bengal, India
rin_ tuin@yahoo.com

Dr Eveline Bartowsky (Wine Microbiology)


The Australian Wine Research Institute
P.O. Box 197, Glen Osmond, SA, 5064, Australia.
Eveline.Bartowsky@awri.com.au
Dr L. Rebordinos ( Food Microbiology)
Laboratorio de Microbiologa y Gentica.
Facultad de Ciencias del Mar y Ambientales.
Universidad de Cdiz. Polgono del ro San
Pedro. 11510 Puerto Real, Cdiz, Spain.
laureana.rebordinos@uca.es
Dr Aline Lonvaud (Wine and Brandy)
Faculty of Enology, University Victor Segalen
Bordeaux 2, France.
aline.lonvaud@oenologie.u-bordeaux2.fr
Dr Creina S. Stockley ( Wine and Health)
The Australian Wine Research Institute
Australia.
Creina.Stockley@awri.com.au
Dr M. Remedios Marn
Universidad Publica de Navarra Nafarroako
Unibertsitste Publikoa Area de Tecnologia de
Alimentos Universidad Publica de Navarra
Campus Arrosadia S/N 31006 Pamplona, (Navarra), Spain.
remedios.marin@unavarra.es
Dr Philippe Jeandet
Laboratory of Enology and Applied Chemistry,
Unit de Recherche sur la Vigne et le Vin de
Champagne, Research Unit N2069 University of
Reims, Faculty of Science, France.
philippe.jeandet@univ-reims.fr
Dr Luca Cocolin
Dipartimento di Scienze degli Alimenti,
Universit degli studi di Udine,
Facolt di Agraria, via Marangoni
Udine, Italy.
lucasimone.cocolin@unito.it
Dr Gins NAVARRO
Departamento de Qumica Agrcola,
Geologa y Edafologa, Facultad de Qumica.
Universidad de Murcia. Campus Universitario de
Espinardo, Murcia. Spain.
gnavarro@um.es

Contents
International Journal of Food and Fermentation Technology
Vol. 2 No. 1, June 2012

Review paper
A Panorama of lactic acid bacterial fermentation of vegetables
V.K. Joshi and Somesh Sharma

1-12

Research Paper
Application of thermostable -amylase in Streptomyces erumpens liquefaction of cassava bagasse for production of lactic acid 13-18
Ramesh C. Ray and Shaktimay Kar
Changes in phytochemicals during fermentation of wine grapes
A.K. Sharma, S.V. Navale, S.N. Aute, G. S. Karibasappa, D. P. Oulkar and P. G. Adsule

19-25

Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach
Balvir Kumar, P. P. Balgir and B. Kaur

27-36

Effect of growth conditions on extracellular -amylase production by Bacillus thuringiensis


V.R. Tembhurkar, M.K. Bannatwala, S.S. Udare and M.G. Kalyankar

37-41

Design of cascade membrane filtration process for clarification of whey proteins and membrane fouling
Pranav Kaushik Pidatala and Senthil R. Kumar

43-48

Microbiological and biochemical characterization of Seera: A traditional fermented food of Himachal Pradesh
Savitri, N. Thakur, D. Kumar and T.C. Bhalla

49-56

Preparation and evaluation of Mahua (Bassia latifolia) Vermouth


Preeti Yadav, Neelima Garg and Deepa Dwivedi

57-61

-amylase production from Endomyces fibuliger an indigenous yeast isolate of Western Himalayas
Keshani Bhushan, Anamika Jain, O.P. Sharma, B. Singh and S.S. Kanwar

63-69

Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7
Baljinder Kaur and Rajinder Kaur

71-79

Effect of nitrogen source on the fermentation behaviour of musts and quality of wine from two Cvs. of pineapple
B.L. Attri

81-86

Research Note
Effect of Solid state fermentation and yeast species on composition of apple pomace: Application of PCA
V.K. Joshi and D.K. Sandhu

87-91

Processing potential of newly introduced Amla cultivars grown in lower Himalayan Region of Himachal Pradesh
Manisha Kaushal and Shashi K. Sharma
Evaluation of the stability of plum anthocyanin powder in RTS based model solution
M. Preema Devi, V.K. Joshi and Y. Indrani Devi
Book Review

93-97

99-101

103

From the Desk of Editor- in-Chief


It is with great pleasure and a sense of fulfilment to state that we have successfully brought out two issues of
inaugural volume of our journal International journal of Food and Fermentation Technology during 2011 to
cater to the needs of food scientists and R and D workers, and the academicians in the fields. The inaugural
issue of the journal was released by the honourable Vice-chancellor of Dr YS Parmar university of Horticulture
and Forestry, Nauni, Solan (India) Dr K.R.Dhiman on 26th June 2012 in a function held in the university to mark
the beginning of the ICAR sponsored training on Advance in Fruit and Vegetable Processing and Preservation
with me as a Course Director.
I am equalty happly to inform you that the journal has received an overwhelming response from the scientists
and academician from the field of food and fermentation technology.The journal during the past one year has
published papers on various aspects of food technology including food fermentation technology. Form this
issues, we could be achieved only with the help of all the members of editorial board, the contributors and the
publisher. I hope you would have relished the style, general get-up, cover page, coverage of the journal; scientific
and technical contents, with an attractive of look A4 Size format. At the same time, we would welcome suggestions
from the readers, contributors and peers of the field. Now, I am placing before you the first issue of Vol. 2
Number 1of the journal. I would like to express my deep gratitude to all those who have extended their help,
guidance and co-operation in bringing out this issue.
I siege this opportunity to request all the members of editorial board to exert more so that the journal gets
high recognition among the peers and we are able to get the NAAS ratings to the journal. I am sure through your
efforts we would get more number of papers of good quality. They should themselves contribute papers of high
quality to encourage others to do so. From this issue, we are introducing conceptual editorial for which I invite
the members of editorial board to contribute liberally. Certainly, there would not be any page charges from them
for this contribution. The scientists abroad can certainly contribute to improve the quality of this journal being in
infancy. On behalf of the editorial board and the publisher, I assure the readers and the contributors that we
would strive hard to ensure high level of scientific integrity, transparency, impartiality and accuracy in selection
of articles and finally, production of the journal. I earnestly call upon all the scientists of food and fermentation
technology to contribute their valuable research articles to the journal as a vehicle of transmission of their
scientific findings. The contributors of review or mini-review can consult the editor- in- chief, member of the
editorial board for the topic of review to contribute. At the same time, I must express very clearly that just
because of some page charges we will not accept and publish substandard articles, but only peer reviewed and
accepted papers would be published. I understand very well that the final scientific quality of any journal would
be determined by the type of papers submitted to the journal so, the cooperation of all would be a pre-requisite in
this regard and would be welcomed from the core of my heart.

V.K. Joshi

Conceptual Editorial

Modern Post-Harvest Technology


A Panacea for Developing Countries
The Green Revolution and subsequent efforts made through the application of science and technology for increasing food
production in India and world have brought self-reliance in food. With advent of advanced agriculture practices, the production of
various crops have increased linearly. This phenomenon has been recorded not only from the developed countries but also in the
developing nations of the world. At present, the world production of wheat is 653654525 MT, maize 840308214 MT, rice and
paddy 696324394 MT, Buffalo milk, whole, fresh milk 92473371 MT, Cow milk, whole, fresh 600838992 MT, Indigenous cattle
meat 63782689 MT, Indigenous pig meat 109100198 MT, Indigenous chicken meat 85860953 MT, Indigenous sheep meat
8689557 MT, fruits 609213509 MT, vegetables 965650533 MT. Out of these, milk, meat, fruits and vegetables highly perishable
crops. Food being a living commodity respire and therefore, is liable to be spoiled. The major causes of spoilage of food include
spoilage by microorganisms, enzymes of microorganisms or the food. The non-enzymatic or purely chemical reactions, environmental
conditions. After the crop is harvested, these factors lead to a considerable postharvest spoilage. In the developed countries, the
quantam of spoilages is bare minimum but in the developing countries, like India. The postharvest losses are staggering high.
These have been estimated to be as high as 25-30% leading to a loss of Rs. 52,000 crores annually. It may be astonishing but is
a fact that the United Kingdom produces the amount of the fruit crop equal to what India wastes. The major difference lies in lack
of proper infrastructure in the developing countries in contrast to the developed nations.
Postharvest handling is the stage of crop production immediately following harvest. It largely determines the final quality and
it includes cooling, cleaning, sorting and packing. After harvest, foods (e.g. fruits, vegetables, milk, meat, fish) are liable to
accelerated physiological, chemical, and microbial processes that invariably lead to deterioration and loss of wholesomeness. It is
then, necessary to institute some measure of processing such as reduction in moisture content, denaturation of endogenous

Figure 1: World production of various agricultural commodities


Source: FAO, Stat: 2010

Figure 2: Production of various agricultural commodities in India


Source: FAO, Stat: 2010

enzymes and microorganisms, or packaging in order to curtail the loss of perishables. Utilizing improved postharvest practices
thus often results in reduced food losses, improved overall quality and food safety, and higher profits for growers and marketers.
The most important goals of post-harvesting handling are to keep the product cool, to avoid moisture loss and slow down
undesirable chemical changes, and avoiding physical damage such as bruising, to delay spoilage. The way the grains are stored
after harvest becomes the major cause of contamination with fungi some of which are producer of toxin like aflatoxin. The fruit
like apple after contamination has a toxin called patulin, if not stored properly. Sanitation is also an important factor, to reduce the
possibility of pathogens that could be carried by fresh produce, for example, as residue from contaminated washing water.
Consumption of such food results in the diseases of gastro-intestinal track which sometimes prove to be fatal.
Postharvest loss reduction technology encompasses the usage of optimum harvest factors, reduction of losses in handling,
packaging, transportation and storage with modern infrastructure machinery, processing into a wide variety of products, home
scale preservation with low cost technology. Use of thermal processing, low temperature storage drying chemical and biological
reactions coupled with other preservation techniques are applied to enhance the storability of the perishables.
There are different constraints for postharvest management like: large number of small and marginal farmers with primitive
system of cultivation, poor infrastructure in terms of handling, transport, storage, processing and marketing, lack of adequate quality
systems and procedure like grading and sorting, hot and humid climates, cost of installation of post-harvest treatment facilities is
expensive, lack of awareness training for the rural farmers and inconsistency in supply due to seasonality of the produce.
Adoption of latest techniques could make available a large quantity of food by avoiding losses and provide quality food and
nutrition, more raw materials for processing. Postharvest technology has the capability to meet food requirement of growing
population. By adopting improper and inefficient methods of storage we are losing a substantial portion of production. If better
methods of processing and storage are adopted, the losses could be reduced to a large extent. The developing countries need
proper infrastructure for this and a strong will to adopt modern postharvest technology to save the wastage. Thus, it could
certainty prove to be a panacea for the problems faced by the developing countries.

V K Joshi

Intl. J. of Food. Ferment. Technol. 2(1): 1-12, June, 2012

Review paper

A Panorama of lactic acid bacterial


fermentation of vegetables
V.K. Joshi1* and Somesh Sharma2

Department of Food Science and Technology, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal
Pradesh,India
2
Department of Biotechnology, School of Bioengineering and Food Technology, Shoolini University, Bajhol, Solan, Himachal
Pradesh,India
*Email: vkjoshipht@rediffmail.com
Paper no: 31

Received: 14 February,2011

Received in revised form: 19 April, 2012

Accepted: 17 May, 2012

Abstract
Vegetables occupy a prominent position in our diet and form the most nourishing diet.Preservation of
vegetables is carried out to prevent their postharvest losses of functional quality whilst providing safe
and stable products. In general, preservation process consists of a combination of mild heat stress and
low concentration of chemical preservatives to control the food spoilage and the growth of pathogenic
spore- forming bacteria. But, consumer demand today is for natural and minimally processed foods, with
a fresh like appearance and taste, easy-to-eat and with high level of safety.It is also dependent on a wide
range of technologies to ensure that food is maintained at an acceptable level of quality from the time of
manufacture till the time of consumption. As a result, research and development of new products is
leading to the reduction or even replacement of heat treatments and encouraging the use of traditional
preservatives as these are capable of assuring the sensory and nutritional properties of the product
without reducing food safety. Non-thermal preservation methods are thus gaining interest as alternative
treatments in the food industry due to their capability of assuring the quality and safety of foods.
Among them, use of natural antimicrobials from plants (essential oil) and microorganisms (LAB and
Bacteriocins) have gained wide range of popularity as bio-preservatives. Traditionally, microorganisms
have been used in fermentation from ages but now modern large scale production of foods exploits use
of defined strains to ensure consistency and quality in the final product. The biopreservation offer
potential application in food preservation and helps in reducing the addition of chemical preservatives as
well as the intensity of heat treatment, resulting in foods which are more naturally preserved and richer in
sensory and nutritional qualities. Beside this, biopreservation also provides health benefits such as
reduction in cardiovascular diseases, improving gastro intestinal microorganisms and immune system,
helps in curing diarrhea and lowering cholesterol in the consumers of lactic acid fermented foods. The
preservation is accomplished through the production of antimicrobial substances such as lactic acid,
diacetyl, hydrogen peroxide, ethanol reutrin and bacteriocin. The bacteria from the genera Lactobacillus,
Leuconostoc, Pediococcus and Streptococcus are the main species involved in the lactic acid fermentation.
Thus,the lactic acid fermentation of vegetables is an important method of bio-preservation and providing
healthful foods to the consumers.

2012 New Delhi Publishers. All rights reserved


Keywords: Vegetables,Lactic acid bacteria, LAB fermentation, Biopreservation, Bacteriocin

Joshi and Sharma

Vegetables form the most nourishing diet, especially to tone


up our digestive system and for providing vigour. Regular use
of vegetables, supplies many of the essential health building
and protective substances, such as vitamins and minerals.
India ranks second in vegetable production. Therefore,the
abundant production of vegetables during the season, results
in a glut in the market. Consequently, a large quantity of
vegetables gets spoiled resulting in a considerable loss of
natural resources. Therefore, preservation can be the only tool
to save the wastage of vegetables and make them available in
off-season.
Fermentation is one of the oldest forms of food preservation
in the world. Earlier, the term Fermentation was used for the
production of alcoholic beverages like wine but it encompasses
all the foods made by the application of microorganisms
including lactic acid bacteria (LAB) or their enzymes. Lactic
acid fermentation can be one of the methods to preserve these
vegetables and to provide variety to the diet in the form of
diversified products and is employed throughout the world, in
conjunction with chemical preservation, using salt and acid.
Until the development of curing and freezing during the past
180 years, lactic fermentation (LA) was one of the most
important method for preserving foods (Joshi et al., 2011) In
consistence with the changing life style of consumers attitude
towards food products with fresh and convenience food, lactic
acid fermentation provides food with diversified taste and
flavour characteristics.

Plate 1: Fermented vegetables

materials. Lactic acid fermentation is used for commercial bulk


storage, to increase the availability of seasonal vegetables
and to obtain desired sensory product quality (Plate 1.). Lactic
acid fermentation removes fermentable sugars, prevents growth
of pathogenic microorganisms, stabilizes the product and
enhances the flavour (Fleming et al., 1983). Although most
vegetables can be lactic acid fermented, cabbage, olives garlic,
red beet, gourd, mixture of cabbage, carrot, onion, red beet
and cucumber are the vegetables that are fermented
commercially in large volumes for human consumption
(Fleming, 1982; Karovicova and Kohajdova, 2005; Sharma,
et al., 2011). These vegetable contain sugars and nutritionally
adequate substances as substrate for the growth of lactic acid
bacteria and other organisms. Recently, lactic acid fermentation
has been used in vegetables such as lupins, peas, lentils,
cassava, carrot and radish to achieve technological or
nutritional advantages (Kim et al., 1987 and Buckenhuskes,
2001). Besides lactic acid fermentation of vegetable juices,
applied as a preservation method for the production of finished
and half-finished products, is again being ranked as an
important technology and it is being further investigated
because of the growing amount of raw materials processed in
this way in the food industry. In Korea, fermented vegetables
known as kimchi is an almost ubiquitous accompaniment to
meals (Adams and Moss, 1996; Ha Jae-Ho et al., 1989). Chinese
cabbage or radish is generally used as a source vegetable for
kimchi, adding various ingredients such as red pepper, garlic,

The Chinese were the first to ferment the vegetables and there
is evidence to show that they had prepared such vegetables
at the time of building the great wall of China (Pederson, 1971).
Today, because of the development of efficient heat sterilizing
and refrigeration systems, lactic acid fermentation, to some
extent has lost its significance as a food preservation method
in industrialized countries, but is being used as a taste
diversification tool. It is extensively used in developing
countries, but is gaining importance more in developed
countries as a food with therapeutic value. It is true especially
for vegetables, where either natural fermentation or
fermentation by pure culture of lactic acid bacteria is practiced
even in the developed countries (Pederson, 1971; Fleming and
Mcfeeters, 1981; Anderson et al., 1990).
Role and advantages of Vegetables Fermentation
Fermented foods including fruits and Vegetables play an
important role in providing food security, enhancing livelihoods
and improving the nutrition and social well being of millions of
people around the world, particularly the marginalized and
vulnerable groups (Table 1). This is achieved through improved
food preservation that combines salting to selectively control
the microorganisms and fermentation to stabilize the treated
2

A Panorama of lactic acid bacterial fermentation of vegetables

onion, ginger, jeotkal (salted and fermented sea foods) (Hawer


et al., 1988). Lactic acid fermentation reduces the pungency of
radish kimchi (Kim and Rhee, 1993). It was once the only
method for successful preservation of cucumbers pickling in
brine. It increases the range of raw materials that can be used
to produce edible food products and removing anti- nutritional
factors to make food safe- to- eat. It is definitely a cheap and
energy efficient means of preserving perishable raw materials

Approximately, 30% of women consume less than their daily


requirements of energy and at least 40% of women world-wide
suffer from iron-deficiency anaemia. Fermentation enhances
the nutritional value of a food product though increased
vitamin levels and improves digestibility, especially of some
legumes, enriches products with desirable microbial metabolites
e.g. lactic acid or amino acids.
There are many traditional beliefs about the medicinal
properties of fermented food products. The Fur ethnic group
in Sudan strongly believe that the consumption of fermented
foods protects them from diseases (Dirar, 1992). Koumiss (a
fermented milk product in Russia) has been used to treat
tuberculosis. Pulque (a fermented fruit sap) is felt to have
medicinal properties in Mexico. The beneficial health effects
of lactic acid bacteria on the intestinal flora are well documented
(Ottogalli and Galli, 1997). Substances in fermented foods have
been found to have a protective effect against the development
of cancer (Frohlich et al, 1997). A study in Tanzania has shown
that children fed with fermented gruels had a 33% lower
incidence of diarrhoea than those fed unfermented gruels,
owing to the inhibition of pathogenic bacteria by lactic acid
forming bacteria (Svanberg, 1992).

Table 1: Role of vegetable fermentation

Improving food security


Increasing income and employment
Improving nutrition
Providing Medicinal benefits
Creates new products for new markets
Develop characteristic sensory properties

There are several options for preserving fresh vegetables


including drying, freezing, canning and pickling. However,
many of these are inappropriate for use on the small-scale in
developing countries. Freezing of vegetables is not
economically viable at the small-scale. Fermentation requires
very little sophisticated equipment, either to carry out the
fermentation or for subsequent storage of the fermented
product. Many fermented foods have played a very significant
role in preserving food to enhance food security. About 60%
of the fermented foods of Sudan are famine or survival foods.
Gundruk is a very important food product in Nepal ensuring
food security for many Nepali communities especially in remote
areas. It is served as a side dish with the main meal and is also
used as an appetiser in the bland, starchy diet.

Technology of Vegetable Fermentation


In vegetables, lactic acid fermentation is commonly employed
for the effective utilization of vegetables and providing
diversified taste and flavoured vegetable products. The
fermentation in vegetables is usually carried out by a class of
bacteria called Lactic acid bacteria.
Lactic Acid Bacteria
The lactic acid bacteria are a group of Gram positive bacteria,
non-respiring, non-spore forming, cocci or rods, which produce
lactic acid as the major end product of the fermentation of
carbohydrates. They are the most important bacteria in
desirable food fermentations, being responsible for the
fermentation of sour dough bread, sorghum beer, all fermented
milks, cassava (to produce gari and fufu) and the most pickled
(fermented) vegetables. Historically, bacteria from the genera
Lactobacillus, Leuconostoc, Pediococcus and Streptococcus
are the main species involved. Several more have been
identified (Table 2), but they play a minor role in lactic
fermentation.

Many vegetables contain naturally occurring toxins and antinutritional compounds. These can be removed or detoxified
by the action of micro-organisms during fermentation. For
instance the fermentation process that produces the Sudanese
product Kawal removes the toxins from the leaves of Cassia
obtusifolia and fermentation is an important step in ensuring
that cassava is safe to eat. The production of fermented
vegetable products provides income and employment to
millions of people around the world.
The optimum health and nutrition of individuals is dependent
upon a regular supply of food and a balanced diet. When diets
are sub-optimal, the individuals capacity for work and
achievements are greatly reduced. The most vulnerable groups
are women, children and weaning infants. Availability of food,
dietary restrictions and taboos, misconceptions, limited time
available for feeding or eating contribute to create a group of
individuals who are nutritionally disadvantaged.

Lactic acid bacteria carry out their reactions - the conversion


of carbohydrate to lactic acid plus carbon dioxide and other
organic acids - without the need for oxygen. They are described
as micro-aerophilic as they do not utilise oxygen. Because of
this, they do not cause drastic changes in the composition of
the food. Some of the menber of lactic acid family are homo-

Joshi and Sharma

fermentative, i.e. they only produce lactic acid, while others


are hetero-fermentative and produce lactic acid plus other
volatile compounds and small amounts of alcohol.
Lactobacillus acidophilus, L. bulgaricus, L. plantarum, L.
caret, L. pentoaceticus, L brevis and L. thermophilus are
examples of lactic acid-producing bacteria involved in food
fermentations. All species of lactic acid bacteria have their
own particular reactions and niches, but overall, L. plantarum
a homo-fermenter produces high acidity in all vegetable
fermentations and play the major role. All lactic acid producers
are non-motile gram positive rods that need complex
carbohydrate substrates as a source of energy. The lactic acid
they produce is effective in inhibiting the growth of other
bacteria that may decompose or spoil the food. Because the
whole group are referred to as lactic acid bacteria it might
appear that the reactions they carry out are very simple, with
the production of one substrate. However,the lactic acid
bacteria are a diverse group of organisms with a diverse
metabolic capacity. This diversity makes them very adaptable
to a range of conditions and is largely responsible for their
success in acid food fermentations. Despite their complexity,
the whole basis of lactic acid fermentation centres on the ability
of lactic acid bacteria to produce acid, which then inhibits the
growth of other non-desirable organisms. Species of the genera
Streptococcus and Leuconostoc produce the least acid. Next
are the heterofermentative species of Lactobacillus which
produce intermediate amounts of acid, followed by the
Pediococcus and lastly, the homofermenters of the
Lactobacillus species, which produce most of the acid.
Homofermenters, convert sugars primarily to lactic acid, while
heterofermenters produce about 50% lactic acid plus 25% acetic
acid and ethyl alcohol and 25% carbon dioxide. The other
compounds are important as they impart particular tastes and
aromas to the final product. Leuconostoc mesenteroides is a

bacterium associated with the sauerkraut and pickle


fermentations. This organism initiates the desirable lactic acid
fermentation in these products. It differs from other lactic acid
species in that it can tolerate fairly high concentrations of salt
and sugar (up to 50% sugar). L. mesenteroides initiates growth
in vegetables more rapidly over a range of temperatures and
salt concentrations than any other lactic acid bacteria. It
produces carbon dioxide and acids which rapidly lower the pH
and inhibit the development of undesirable micro-organisms.
The carbon dioxide produced replaces the oxygen, making the
environment anaerobic and suitable for the growth of
subsequent species of lactobacillus. Removal of oxygen also
helps to preserve the colour of vegetables and stabilises any
ascorbic acid that maybe present. Organisms from the Gram
positive Propionibacteriaceae family are responsible for the
flavour and texture of some fermented foods, especially Swiss
cheese, where they are responsible for the formation of eyes
or holes in the cheese. These bacteria break down lactic acid
into acetic, propionic acids and carbon dioxide.
Lactic Acid Fermentation
The pathways of lactic acid production differ for the two
types of fermentation i.e., the homofermenters and the
heterofermenters. Homofermenters produce mainly lactic acid,
via the glycolytic (EmbdenMeyerhof) pathway).
Heterofermenters produce lactic acid plus appreciable amounts
of ethanol, acetate and carbon dioxide, via the 6phosphoglucanate/phosphoketolase pathway. The glycolytic
pathway is used by all lactic acid bacteria except Leuconostoc,
group III Lactobacilli, oenococci and weissellas. Normal
conditions required for this pathway are excess sugar and
limited oxygen. Axelsson et al (1998) gives an in-depth account
of the biochemical pathways for both homo- and heterofermenters.

Table 2: Major lactic acid bacteria in fermented plant or vegetable products


Homofermenter

Facultative homofermenter

Obligate heterofermenter

Enterococcus faecium
Enterococcus faecalis
Lactobacillus acidophilus
Lactobacillus lactis
Lactobacillus delbrueckii
Lactobacillus salivarius
Pediococcus pentocacus
Streptococcus thermophilus
Pediococcus acidilactici
Pedicoccus damnosus

Lactobacillus bavaricus
Lactobacillus casei
Lactobacillus coryniformis
Lactobacillus curvatus
Lactobacillus plantarum
Lactobacillus sake

Lactobacillus brevis
Lactobacillus buchneri
Lactobacillus cellobiosus
Lactobacillus confuses
Lactobacillus coprophilus
Lactobacillus fermentatum
Lactobacillus sanfrancisco
Leuconostoc dextranicum
Leuconostoc mesenteroides
Leuconostoc paramesenteroides

(Source: Beuchat 1995; Sharma et.al., 2011).

A Panorama of lactic acid bacterial fermentation of vegetables

Factors efffecting vegetables Fermentation

development of lactic flora, which generally requires very


special condition to proliferate. It was reported that lactic acid
bacteria depends essentially on the plant sugars for growth
(Montet, et.al., 1999). For some vegetables with low nutrient
content, such as turnip and cucumber, the addition of sugar
promotes bacterial growth, thereby accelerating fermentation
as jaggery is added during lactic acid fermentation of sweet
turnip (Anand and Das, 1971). Adding 1-2 percent sucrose or
jiggery increased lactic acid production during fermentation.
Spices are also added to most of the lactic acid fermented
vegetables to improve the flavour of finished product, besides
these spices have an antimicrobial effect, which means that
they have a selective role in the development of bacteria during
fermentation (Laencina et.al., 1985). Mustard seeds are also of
interest as they contain volatile aromatic compounds with
antibacterial and antifungal properties. Adding 6-10 percent
of mustard seed powder to turnip and 6 percent salt mixture
increased lactic acid levels during fermentation (Anand and
Das, 1971). Sethi, (1990) recommended addition of 3 % salt, 1.0
% mustard, 0.015% sodium benzoate and 0.01% potassium
metabisulphite to 1:1 carrot water mixture for making black
carrot beverage called Kanji.

Microorganisms
As described earlier ,lactic acid bacteria (LAB) responsible for
vegetable fermentation. Micro-organisms vary in their optimal
pH requirements for growth. Most bacteria favour conditions
with a near neutral pH . The varied pH requirements of different
groups of micro-organisms is used to effect fermented foods
where successions of micro-organisms take over from each
other as the pH of the environment changes. Certain bacteria
are acid tolerant and will survive at reduced pH levels. Notable
acid-tolerant bacteria include the Lactobacillus and
Streptococcus species, which play a role in the fermentation
of vegetable products. Oxygen requirements vary from species
to species. Many researchers have studied the changing
pattern of microflora involved in the fermentation of kimchi
along with the taste and odour produced during fermentation.
Major microflora isolated from kimchi were; Lactobacillus
plantarum, Lactobacillus brevis, Streptococcus faecalis,
Leuconostoc mesentroides, Pediococcus cerevisae and
Achromobacter. Recently, most of the fermented vegetable
juices are manufactured by lactoferment Process through
the use of pure starter cultures (Buckenhuskes, 1993)

Water activity

Temperature

In general, bacteria require a fairly high water activity (0.9 or


higher) to survive. There are a few species which can tolerate
water activities lower than this, but usually the yeasts and
fungi will predominate on foods with a lower water activity.

Different bacteria can tolerate different temperatures, which


provides enormous scope for a range of fermentations. While
most bacteria have a temperature optimum of between 20 to
30C, there are some (the thermophiles) which prefer higher
temperatures (50 to 55C) and those with colder temperature
optima (15 to 20C). Most lactic acid bacteria work best at
temperatures of 18 to 22C. The Leuconostoc species which
initiate fermentation have an optimum of 18 to 22C.
Temperatures above 22C, favour the lactobacillus species.

Hydrogen ion concentration (pH)


The optimum pH for most bacteria is near the neutral point (pH
7.0). Certain bacteria are acid tolerant and will survive at reduced
pH levels. Notable acid-tolerant bacteria include the
Lactobacillus and Streptococcus species, which play a vital
role in the fermentation of dairy and vegetable products.

Additives
Oxygen availability

Salt concentration

Some of the fermentative bacteria are anaerobes, while others


require oxygen for their metabolic activities. Some, lactobacilli
in particular, are microaerophilic i.e they grow in the presence
of reduced amounts of atmospheric oxygen. In aerobic
fermentations, the amount of oxygen present is one of the
limiting factors. It determines the type and amount of biological
product obtained, the amount of substrate consumed and the
energy released from the reaction.

Lactic acid bacteria tolerate high salt concentrations. The salt


tolerance gives them an advantage over other less tolerant
species and allows the lactic acid fermenters to begin metabolism,
which produces acid, that further inhibits the growth of nondesirable organisms. Leuconostoc is noted for its high salt
tolerance and for this reason, initiates the majority of lactic acid
fermentations. Majority of vegetables are fermented in the salt
concentration range of 2-5 per cent (Table 3).
Nutrients

Production Technology and Preservation of Fermented


Vegetables

Some of ingredients, added more or less empirically to lactic


acid acid fermented vegetables, seem to enhance the

Lactic acid fermentations are carried out under three basic

Joshi and Sharma

Table 3: Salt concentration used during lactic acid fermentation of vegetables


Vegetable

Salt concentration (% wt)

Type of salting

2-3
3
5-8
4-7
1.5-32.52.5

Dry salt
Dry salt
Brine
Brine
BrineDry saltDry salt

2-5
5.3
7.5-8.5
2.2
2.5

Brine
Brine
Brine
Dry salt
Dry salt

CabbagesSauerkraut
Korean Kimchi
Cucumbers
Green olives
Carrots Raddish

Black olives
Over-night Dill pickles
Genuine Dill pickles
Sauerrruben (turnip)
Pickled leafy vegetables

types of condition dry salted, brined and non-salted. Salting


provides a suitable environment for lactic acid bacteria to grow
which impart the acid flavour to the vegetable. Sauerkraut is
one example of an acid fermentation of vegetables. The name
sauerkraut literally translates as acid cabbage. The sauerkraut
process can be applied to any other suitable type of vegetable
product. Because of the importance of this product in the
German diet, the process has received substantial research in
order to commercialize and standardize production. Table 4
summarises the fermented vegetables developed along with
the microorganisms involved.

References
Fleming, 1982
Steinkraus et al., 1983
Fleming, 1982
Fleming, 1982
Niketic-Aleksic et al., 1973
Joshi, et. al., 2008
Sharma and Joshi, 2007
Battcock and Azam-Ali, 1998
Frazier and Westhoff, 1998
Frazier and Westhoff, 1998
Frazier and Westhoff, 1998
Frazier and Westhoff, 1998

vegetable to expell the juice, which contains fermentable sugars


and other nutrients suitable for microbial activity. The first
micro-organisms to start acting are the gas-producing cocci
(L. mesenteroides). These microbes produce acids. When the
acidity reaches 0.25 to 0.3% (calculated as lactic acid), these
bacteria slow down and begin to die off, although their enzymes
continue to function. The activity initiated by the L.
mesenteroides is continued by the lactobacilli (L. plantarum
and L. Cucumeris) until an acidity level of 1.5 to 2% is attained.
The high salt concentration and low temperature inhibit these
bacteria to some extent. Finally, L. pentoaceticus continues
the fermentation, bringing the acidity to 2 to 2.5% thus,
completing the fermentation. The end products of a normal
fermentation are lactic acid along with smaller amounts of acetic
and propionic acids, a mixture of gases of which carbon dioxide

The fermentation process


Vegetables are washed and shredded and placed in a jar and
salt is added (Figure 2). Mechanical pressure is applied to the

Table 4: Fermented vegetables developed along with the microorganisms involved.


Fermented Vegetables

Microorganisms involved

Reference

Fermented fruit juices


Fermented vegetable juices
Pickled leafy vegetables
Pak-Gard-Dong

Lactobacillus casei
Lactobacillus strains

Karovicova and Kohajdova et al., 1986


Sethi, 1990

Lactobacillus brevis,Pediococcus
cerevisiaeLactobacillus plantarum
Lactobacillus plantarum,Pediococcus
pentosaceus
Lactobacillus plantarum,Lactobacillus
brevis,Streptococcus faecalis,Leuconostoc
mesenteroides,Pediococcus pentosaceus
Lactobacillus plantarum,Lactobacillus
brevis,Bacillus coagulans
Pediococcus pentosaceus
Bacillus subtilis,Lactobacillus plantarum

Boonlong, 1986;Joshi et al., 2003

Gundruk
Bossam kimchi (Pickled cabbage kimchi)

Sunki (Otaki turnip)

Kawal (Sickle pod plant)

Karki, 1986
Joshi et al., 2003

Makayama, 1957

Battcock and Azam-Ali, 1998

A Panorama of lactic acid bacterial fermentation of vegetables

Use of starter cultures


To produce vegetables of consistent quality, starter cultures
(similar to those used in the dairy industry) have been
recommended. Not only do the starter cultures ensure
consistency between batches, they speed up the fermentation
process as there is no time lag while the relevant microflora
colonise the sample. Because the starter cultures used are
acidic, they also inhibit the undesirable micro-organisms. It is
possible to add starters traditionally used for milk fermentation,
such as Streptococcus lactis, without any adverse effect on
final quality. Because these organisms only survive for a short
time (long enough to initiate the acidification process) in the
fermenting medium, they do not disturb the natural sequence
of micro-organisms. On the other hand, if Leuconostoc
mesenteroides is added in the early stages, it gives a good
flavour to the final product, but alters the sequence of
subsequent bacterial growth and results in a product that is
incompletely fermented. Joshi et al. (2007) reported sequential
culture fermentation for production of fermented radish and
carrot by using cultures of Streptococcus lactis, Pediococcus
cerevisiae, Lactobacillus plantarum at the rate of 2 % each.
The cultures were added in sequence to the shredded carrots
after 24, 48 and 72 h of fermentation, respectively (Sharma,
2005; Joshi, et al., 2007)

Figure 2: Natural carrot fermentation

is the principal gas, small amounts of alcohol and a mixture of


aromatic esters. The acids, in combination with alcohol form
esters, which contribute to the characteristic flavour of
fermented vegetable. The acidity helps to control the growth
of spoilage and putrefactive organisms and contributes to the
extended shelf life of the product. Besides acidity other
metabolites are also formed which have inhibitory effect on
other competing bacteria and pathogenic microorganisms
(Table 5). Changes in the sequence of desirable bacteria, or
indeed the presence of undesirable bacteria, alter the taste
and quality of the product.

It is possible to use the juice from a previous fermentation as


a starter culture for subsequent fermentations. The efficacy of
using old juice depends largely on the types of organisms
present in the juice and its acidity.

Table 5: Metabolites of lactic bacteria which may be inhibitory to other pathogenic and food spoilage organisms
Product

Main target organisms

Organic acids
Lactic acid
Putrefactive and Gram-negative bacteria, some fungi
Acetic acid
Putrefactive bacteria, clostridia, some yeasts and some fungi
Hydrogen peroxide
Pathogens and spoilage organisms, especially in protein rich foods
Enzymes
Lactoperoxidase system
Pathogens and spoilage causing bacteria (milk and diary products) with hydrogen peroxide
Lysozyme
(by recombinant DNA)
Undesired Gram-positive bacteria
Low molecular weight metabolites
Reuterin
Wide spectrum of bacteria, yeasts and molds
Diacetyl
Gram-negative bacteria
Fatty acids
Different bacteria
Nisin
Some LAB and Gram-positive bacteria, notably endospore-formers
Other
Gram-positive bacteria, inhibitory spectrum according to producer strain and bacteriocin type
Source: Breidt & Fleming, 1997, Joshi et al., 2006

Joshi and Sharma

Non- salted, lactic acid fermented vegetables

from fermented mushroom was prepared (Joshi et al., 1996).


The sauce has a typical mushroom flavour though lactic, acetic
acid and other ingredients used in sauce preparation also
imparted their respective tastes and aromas to the product.
The original texture of unfermented mushroom was spongy
and the use of lactic acid fermentation brought the consistency
of mushroom sauce to the desirable level besides improved
flavour. Different varieties of radish have been evaluated for
lactic acid fermentaton (Sharma et al., 2011). While response
surface methodology has been employed to optimize the
nutrients and stimulators needed for this fermentation (Joshi
et al., 2012).

Some vegetables are fermented by lactic acid bacteria, without


the prior addition of salt or brine. Examples of non-salted
products include gundruk (consumed in Nepal), sinki and other
wilted fermented leaves. The detoxification of cassava through
fermentation includes an acid fermentation, during which time
the cyanogenic glycosides are hydrolysed to liberate the toxic
cyanide gas. The fermentation process relies on the rapid
colonisation of the food by lactic acid producing bacteria,
which lowers the pH and makes the environment unsuitable
for the growth of spoilage organisms. Oxygen is also excluded
as the Lactobacilli favour an anaerobic atmosphere. Restriction
of oxygen ensures that yeasts do not grow. For the production
of sinki, fresh radish roots are harvested, washed and wilted
by sun-drying for one to two days. They are then shredded,
re-washed and packed tightly into an earthenware or glass jar,
which is sealed and left to ferment. The optimum fermentation
time is twelve days at 30C. Sinki fermentation is initiated by
L. fermentum and L. brevis, followed by L. plantarum. During
fermentation the pH drops from 6.7 to 3.3. After fermentation,
the radish substrate is sun-dried to a moisture level of about
21%. For consumption, sinki is rinsed in water for two minutes,
squeezed to remove the excess water and fried with salt, tomato,
onion and green chilli. The fried mixture is then boiled in rice
water and served hot as soup along with the main meal (Joshi
and Thakur, 2000).

Preservative Action in Fermented Vegetables


Lactic acid bacteria are inhibitory to many other
microorganisms when they are cultured together (Adams, 1990)
and this the basis of the extended shelf-life and improved
microbiological safety of lactic-fermented foods. Lactobacillus
species can produce a variety of metabolites that are inhibitory
to competing bacteria, including psychrotrophic pathogens.
Antagonistic behavior of lactic acid bacteria may be either
due to their major metabolic end products or via production of
low molecular weight compounds. The low molecular weight
substances elaborated by LAB, capable of exhibiting
antagonism are termed as bacteriocins. These are proteins in
nature, which exert a bactericidal mode of action on susceptible
bacteria (Tagg et al., 1976). Lactic acid bacteria (LAB) are
capable of producing inhibitory substances in smaller amounts
as discussed earlier. Bacteriocin or other substances elaborated
by LAB are listed in Table 6 while the bacteriocin produced
and their antimicrobial spectrum is shown in Table 7.

Lactic fermented Vegetable products


Preparation of fermented foods using LAB has been suggested
to improve their acceptability. A special fermented vegetable
beverage traditionally known as Kanji has been prepared from
crimson coloured carrots supplemented with salt and mustard
powder (Sethi, 1990). Lactic acid fermentation is employed to
prepare various products such as fermented cucumber extract,
fermented carrot, radish and cucumber based appetizer, sauce,
RTS chutney, sauce and other products (Sharma, 2011, Joshi
and Sharma, 2009; Joshi and Sharma, 2010; Joshi et al.,
2011a&b)) .

Consumer awareness against the hazardous and ill effect nature


of synthetic chemicals used for preservation of food products
and development of the process for the production of
convenient food or semi-processed products forced the
research workers to focus on the development of natural,
stable, antibiotic and safe substances, such as bacteriocin as
an antimicrobial agent or biopreservative. It can be used
directly as a pure compound in unfermented processed
products or by the use of lactic acid bacteria that produced
bacteriocin as a starter culture in fermented fruits and
vegetables like cabbage, cauliflower, olives, cucumber and
peppers (Lucke and Earnshaw, 1991). If food is going to be
fermented by LAB, the use of bacteriocin-producing starter
culture provide added value to the product. The presence of a
nisin producer among the strains used to make cheedar cheese
provides enough nisin to increase the shelf-life of pasteurized

Lactic acid fermentation is used to stabilize beverages by


acidification. The process renders the lactose more digestible
for people with lactose intolerance. Traditional East-African
beverages such as Muratina (Sauce tree) or
mnazi(fermented coconut palm) have also been studied. Only
Lactobacillii were isolated from Muratina and only
Streptococcii from Mnazi.
A method for extension of postharvest life of mushroom by
inducing lactic acid fermentation was developed and sauce

A Panorama of lactic acid bacterial fermentation of vegetables

processed cheese made from it from 14-87 days at 220C (Roberts


and Zottola, 1993).

(Bhunia et al., 1998). The use of pure starter culture (LAB) has
been reported for the inhibition of spoilage organism
Pseudomonas fluorescens and Staphylococcus typhimurium
(Raccach et al., 1979). The growth of Staphylococcus aureus
is also inhibited when grown with Pediococcus cerevisiae and
Lactobacillus plantarum. A wide variety of raw foods are
preserved by LAB fermentation including milk, meat, fruit and
vegetables (Daeschel, 1989). Reduction of pH and removal of
a large amount of carbohydrates by fermentation are the primary
preserving actions that these bacteria provide, though, LAB
are capable of producing inhibitory substances that are
antagonistic towards other microorganisms as described earlier
(Joshi and Thakur, 2000; Battcock and Azam-Ali, 1998).

Table 6: Bacteriocins or bacteriocin-like substances elaborated by


lactic acid bacteria
Producing microorganism

Bacteriocin or bacteriocin
like substances

Lactobacillus plantarum

Plantacin B, Plantaricin C,
Plantacin N
Nisin, Lacticin, Lactococcin
G
Diplococcin, Lactococcin
Pediocin AcH, Pediocin
P-A-1
Mesenterocin 5
Lactocidin, Acidophilin
Caseicin

Lactococcus lactis sub sp. lactis


Lactococcus lactis sub sp. cremoris
Pediococcus acidilactici
Leuconostoc mesenteroides UL5
Lactobacillus acidophilus
Lactobacillus caseii

Table 7: Bacteriocin producing microorganisms and their inhibitory


spectrum
Microorganism

Bacteriocin
Inhibitory Spectrum

Bacillus subtilis

Subtilin Gram +ve


bacteria
Nisin Many gram
+ve bacteria
Lacticin-481 Lactic
acid bacteria
(LAB)and
Clostridium
Pediocin PA1
Some LAB, Listeria
Sakacin A Some
LAB, Listeria
Mesenterocin 5
Some LAB, Listeria
Microgard Gramve
bacteria, some yeasts
and moulds
Sakacin P E. coli,
Lactic acid bacteria

Source: Sarkar and Misra, 2001

Some bacteria possess the genetic capability to synthesize


bacteriocin. Nisin (a polypeptide antibacterial substance
produced by lactic acid bacterium, Lactococcus lactis formally
Streptococcus lactis, during fermentation) is one of the earliest
known bacteriocin, showing antimicrobial activity against a
range of Gram-positive bacteria, particularly spore forming
(Broughton, 1990). A bacteriocin producing strain Lactococcus
lactis sub sp. cremoris R was isolated from radish and
designated bacteriocin as Lactococcin R (Yildirim and Johnson,
1998). It was active against many food borne pathogenic and
food spoilage bacteria such as Clostridium, Staphylococcus,
Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus,
Leuconostoc, Sterptococcus and Pediococcus spp. but was
inactive against Gram-negative bacteria. Reuterin a broad
spectrum antimicrobial agent active against certain bacteria,
yeasts and fungi (Axellson et al., 1998), was produced by
heterofermentative organism Lactobacillus ruterii. Reuterin
may have application in the preservation of food stuffs by
reducing pathogenic and spoilage causing organisms
(Daeschel, 1989). The pediococci used as a starter culture in
the vegetable and meat fermentation have been of much recent
investigations with regard to their bacteriocin producing ability
although, pediococci was known to produce a range of
bacteriocins and other metabolites with broad spectrum
antibacterial activity (Skytta et al., 1993). Pediocins produced
by Pediococcus spp. can inhibit a wide range of pathogens
including Listeria monocytogenes and the Gram-negative
Psedomonas fluorescens (Singhal and Kulkarni, 1999). But
pedicin AcH produced by Pediococcus acidilactici has been
shown to have antibacterial activity against Staphylococcus
aureus, Listeria monocytogenes and Clostridium perfringes

Lactococcus lactis sub sp. lactis


Lactococcus lactis sub sp. lactis CNRZ

Pediococcus acidilactici
Lactobacillus sake
Leuconostoc mesenteroides UL5
Propionibacterium shermanni

Lactobacillus sakei

Source: Barnby-Smith, 1992; Vaughan et al., 2001

Healthful effects of lactic bacterial fermented Products


Probiotic effect
One of the reasons for the increasing interest in fermented
foods is their ability to promote the functions of the human
digestive system in a number of positive ways. As early as
1900, Metchnikoff pointed out the use of fermented milks in
the diet for prevention of certain diseases of the
gastrointestinal tract and promotion of healthy day-to-day life.
Since then, a number of studies have shown that the fermented
food products do have a positive effect on health status in
many ways. A fermented food product or live microbial food
9

Joshi and Sharma

supplement, which has beneficial effects on the host by


improving intestinal microbial balance is generally understood
to have probiotic effect (Fuller, 1989).

lactobacilli, their enzymes or the metabolic products present


in the fermented food product may act as antigens, activating
production of antibodies.

Flatulence reducing activity

Future Trends

The lactic acid fermented products do have flatulence reducing


effect. During fermentation of the beans for preparation of
temphe, the trypsin inhibitor is inactivated and the amount of
several oligosaccharides which usually cause flatulence are
significantly reduced (Hesseltine, 1983). Bean flour inoculated
with Lactobacillus and fermented with 20% moisture content,
showed a reduction of the stachyose content (DuszkiewiczReinhard et al., 1994)

Vegetable fermentation employing Lactic acid bacteria is one


of the oldest forms of food preservation technology in the
world is strongly linked to culture and tradition, especially in
rural households and village communities. It is a relatively
efficient, low energy preservation process, which increases
the shelflife and decreases the need for refrigeration or other
forms of food preservation technology. Thus, a highly
appropriate technique for use in developing countries and
remote areas where access to sophisticated equipment is
limited. It is therefore, essential to increase the knowledge and
understanding of the methods of preparation, in order to
improve the efficiency of fermentation, especially the traditional
processes. The potential areas for improvement of lactic acid
fermented products are: better understanding of fermented
products; refining the process; creating a supportive
environment for production of fermented food products;
development of new starter cultures. With many of the
fermented products knowledge of the processes involved is
poor. It is likely that the basic principles apply across the world,
but production conditions vary enormously from region-toregion, giving rise to numerous variations of the basic
fermented product. It is not the intention or the desire to
standardise the process and thereby lose this huge diversity,
rather it is to harness the tremendous potential these methods
have to contribute to increasing not only the quantity, but
quality of food available to the worlds population. If the
processes are to be refined, with a view to production on a
larger scale, it is essential to have a scientific understanding
of the fermentation processes. This can be developed by the
isolation and characterization of the essential micro-organisms
involved; determination of the role of external factors in
fermentations and the effects of these on the metabolism of
micro-organisms; investigation on the effects of pre-treatments
of raw materials on the fermentation process; identification of
the options for further processing and how these affect the
taste and texture of the product; biotechnologies need to be
developed which are affordable by the poor, since it is they
who are likely to benefit most by improvements in the traditional
processes and product incorporate objective methods of
process control and to standardize quality of the final products.
Fermented foods should be recognised as part of each
countries heritage and culture and efforts are made to preserve
the methods of production. More research has to be done to
ascertain the factor or combination of factors responsible for
the positive consequence of fermented diets. Consumers need

Hypercholesteremic effect
Hepner et al. (1979) reported hypercholesteremic effect of
yoghurt in human subjects receiving a one-week dietary
supplement. Studies on supplementation of infant formulation
with Lactobacillus acidophilus showed that the serum
cholesterol in infants was reduced from 147 mg/ml to 119 mg/
100 ml (Harrison and Peat, 1975). The ability of yoghurt to
lower the cholesterol in serum by controlled human trials had
also been reported (Poppel and Schafsma 1996). Possible role
of LAB in lowering cholesterol concentration and various
mechanisms by which it may be possible has also been
discussed (Haberer et al., 1997).
Anticarcinogenic effect
Apart from this, there are interesting data on anticarcinogenic
effect of fermented foods showing a potential role of lactobacilli
in reducing or eliminating procarcinogens and carcinogens
from the alimentary canal (Shahani, 1983; Mital and Garg, 1995).
Mutagenicity
The fermentation of foods is also reported to reduce the
mutagenicity of foods by degrading the mutagenic substances
during the process. Lactic acid bacteria isolated from dadih, a
traditional Indonesian fermented milk, were found to be able
to bind mutagens and inhibit mutagenic nitrosamines. Milk
fermented with Lactobacillus acidophilus LA-2 was
demonstrated to suppress faecal mutagenicity in the human
intestine.
Immune system
Some LAB present in fermented milk products, are found to
play an important role in the immune system of the host after
colonisation in the gut (De Simone, 1986). The mechanism of
this effect is not clearly known, but it is speculated that the

10

A Panorama of lactic acid bacterial fermentation of vegetables

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to be made aware of the numerous benefits of fermented foods


and their prejudices against fermented foods, especially those
traditionally produced at the home scale are dispelled.

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12

Intl. J. of Food. Ferment. Technol. 2(1): 13-18, June, 2012

Research Paper

Application of thermostable -amylase in Streptomyces


erumpens liquefaction of cassava bagasse
for production of lactic acid
Ramesh C. Ray* and Shaktimay Kar

Microbiology Laboratory, Regional Center of Central Tuber Crops Research Institute, Bhubaneswar, India
*Email: rc_rayctcri@rediffmail.com
Paper no: 32

Received: 14 Jan 2012

Received in revised form: 19 April 2012

Accepted: 19 May 2012

Abstract
Application of thermostable - amylase (crude and purified) produced by Streptomyces erumpens on
liquefaction of cassava bagasse into sugars for further conversion to lactic acid (LA), was compared
with that of a commercial enzyme Termamyl (Novozyme, Denmark). By applying crude S. erumpens
amylase (10%, v/v), 21.89 g LA was achieved per 100g bagasse at 96h of incubation, which was 44.01%
less than LA yield obtained with Termamyl (2%, v/v). However, when partially purified S. erumpens
amylase was applied at 5% (v/v) concentration or Termamyl at 2% (v/v) concentration to liquefy
cassava bagasse, the LA yield was similar, i.e., 38.45 LA/100g bagasse. The purified form of S. erumpens
amylase was found as effective as commercial Termamyl enzyme in liquefying cassava bagasse to
sugars for further conversion to LA.
2012 New Delhi Publishers. All rights reserved

Keywords: - amylase, cassava bagasse, Lactic acid, Streptomyces erumpens

Lactic acid (LA) is a versatile organic acid having various


applications in food industry such as acidulant,in pickling as
a, flavour and preservative (Oh et al., 2005). LA can be obtained
on an industrial scale either by microbial fermentation or
chemical synthesis. In recent years, the fermentation approach
has become more successful because of the increasing market
demand for naturally produced LA. Of the 80,000 tonnes of LA
produced world wide every year, about 90% are made by
microbial fermentation employing LA bacteria (LAB) (Kadam
et al., 2006). LAB (Lactobacillus spp., those include L.
acidophilus, L. plantarum, L. casei, L. gasseri, etc) are
traditionally fastidious microorganisms and have complex
nutritional requirements due to their limited ability to
biosynthesize B-vitamins and amino acids (Fitzpatrick and O
Keeffe, 2001). Generally, LA is produced using costly substrate
such as cane or beet sugar as the carbon source. Thus, a low

cost of feed stock is very much desirable to reduce the


production cost by replacing sugar with starchy agricultural
and forest residues (John et al., 2005, 2006, 2007). Starch is the
most abundant carbon compound in the world, which can be
hydrolyzed to fermentable sugars by acid and enzyme
hydrolysis. Among the agro-industrial residues, cassava
bagasse, one of the major solid waste released during extraction
of starch from cassava (Manihot esculenta Crantz) proved to
be an alternative ideal bio-resource for production of various
end products such as enzymes (Kar and Ray, 2008; Kar et al.,
2011), organic acids (Jyothi et al., 2005) and ethanol (Ray et
al., 2008), because of its high starch content (55-65 % on dry
weight basis) (Ray et al., 2008).
Amylases constitutes one of the important group of enzymes
that are widely used in starch processing industries. Among
the starch hydrolyzing enzymes, thermostable -amylase is of

Ray and Kar

utmost significance (Haki and Rakshit, 2003) as it breaks only


-1, 4 glucosidic bonding in starch (Tonkova, 2006). Further,
the enzyme possesses high temperature stability that can
overcome high viscosity and mass transfer problems commonly
associated with starch slurry and facilitate higher conversion
of starch into fermentable sugars.

[soluble starch, 1.0%; beef extract, 1.0%; yeast extract, 0.2%;


MgSO4, 0.1%, glycerol, 0.02% and 100 ml double distilled H2O],
by transferring a loop full of organism from a fresh culture and
incubated at 50C for 24h at 120 rpm in an orbital incubator.
Enzyme sources

But many lactobacilli are unable to degrade starch as they


either do not produce or produce in less quantity of starch
degrading enzymes such as -amylase, pullulanase and
glucoamylase (Gupta et al., 2003). Thus, in order to utilize the
starchy residues such as cassava bagasse, it is necessary to
add starch-degrading enzymes to the fermentation medium to
catalyze conversion of starch in to sugars. However step
invariably escalates the production cost. To overcome this
problem, an indigenously produced thermostable -amylase
from an actinomycete, Streptomyces erumpens MTCC 7317
was effectively demonstrated to degrade both soluble and
cassava starch into sugar in our previous studies (Kar et al.,
2009; Kar et al., 2010).

Commercial enzymes
Termamyl is a thermostable (>90C) - amylase enzyme and
Dextrozyme (>45C) is a fungal amyloglucosidase
(glucoamylase) enzyme. The enzymes were procured from Arun
and Co. Mumbai, India.
S. erumpens enzyme
Crude -amylase was prepared from S. erumpens as follows;.
Sterile SB medium (50 ml) taken in 250 ml Erlenmeyer flasks (in
triplicate) were inoculated with 2% inoculum and agitated at
120 rpm in an incubator shaker (Remi Pvt Ltd, Mumbai, India)
at 50C. After 36h, the culture broths from the flasks were
pooled and centrifuged at 8000 rpm in a refrigerated centrifuge
(Model C-24, Remi Pvt. Ltd, Mumbai, India) for 20 min at 4C.
The clear cell free supernatant was used as the crude enzyme.

The present investigation was carried out to study further the


application of (1) crude and (2) partially purified -amylase
from S. erumpens on LA production using cassava bagasse as
the substrate. The LA yield and conversion efficiency of S.
erumpens -amylase was also compared with that of a
commercial amylase (Termamyl).

Partially purified -amylase was prepared as follows; A total


of 100 ml of culture filtrate (crude enzyme) was brought to 60%
ammonium sulphate saturation at 4C in an ice bath for
overnight was carried out. Precipitated protein was collected
by centrifugation at 8000 rpm at 4C for 20 min and dissolved
in minimum volume of phosphate buffer (0.1M, pH 6.0). Enzyme
solution was dialyzed against the same buffer for 24h at 4C
with continuous stirring and three changes of the same buffer.
The dialyzed enzyme solution was used as partially purified
enzyme.

Materials and methods


Chemicals
All the chemicals used were of analytical reagent grade and
were procured from M/s Merck Specialties,Mumbai, India.
Microbial strains and inoculum preparation

Cassava bagasse

Lactobacillus plantarum MTCC 1407 was used for the


production of LA. The culture was obtained from.The Institute
of Microbial Technology, Chandigarh, India and was
maintained on Mann Rogassa Sharpe (MRS) agar slants at
4C. The bacterial inoculum was prepared in 250 ml Erlenmeyer
flask containing 100 ml of MRS liquid medium by transferring
one loop full of organism (L. plantarum) from stock culture
and incubated at 35C for 48h at 120 rpm in an orbital incubator
shaker (Remi Pvt. Ltd., Mumbai, India).

Cassava bagasse [(g/100g dry residue); moisture, 11.2; starch,


63.0; crude fibre, 10.8; crude protein, 0.9 and total ash, 1.2]
(Ray et al., 2008) was used as the substrate for LA production.
Cassava bagasse was collected during starch extraction from
cassava using the mobile starch extraction plant, developed
by our institute (Edison et al., 2006). Because of its high water
and starch content, the residue was de-watered, sun dried for
6-8 days and then was ovendried at 80 C for 24h to prevent
microbial deterioration and stored in air-tight container, until
required.

Streptomyces erumpens MTCC 7317 was previously isolated


from a brick kiln soil and it possessed thermostable -amylase
activity. The optimum enzyme activity was obtained at pH,
6.0-7.0 and temperature, 50C (Kar and Ray, 2008). This organism
also produces thermostable pectinase (Kar and Ray, 2011) and
amylopullulanase (Kar et al., 2011). S. erumpens inoculum was
prepared in soluble starch-beef extract (SB) broth (pH 7.0)

LA production
The starch slurry was prepared in 500 ml Erlenmeyer flask by
mixing cassava bagasse (25g) with 250 ml of distilled water. It

14

Application of thermostable -amylase in Streptomyces erumpens liquefaction of cassava bagasse for production of lactic acid

was liquefied either with Termamyl (2%, v/v) or with different


levels of S. erumpens crude -amylase (2, 5 and 10%, v/v) at
50C for 1h. The liquefied broth was subsequently cooled
down to 45oC and then, saccharified with amyloglucosidase
(AMG, 2%, v/w) by incubating for 24h at 45C in an incubator.
After saccharification process was over, the volume of the
broth was adjusted to 500 ml with distilled water and to it,
constituents of MRS liquid medium except glucose (carbon
source) [(gl-1): peptone, 10.0; beef extract,10.0; yeast extract,
5.0; Na2HPO4, 2.0; sodium acetate, 5.0; tri-ammonium citrate,
2.0; MgSO4, 0.2; MnSO4,0.2; CaCO3, 4.0; Tween 80, 0.1ml and
pH adjusted to 6.8] were added in appropriate proportions,
and autoclaved at 15 lb pressure for 20 min. Then, the flasks
were cooled to room temperature (30 20C) and were inoculated
with 2% L. plantarum inoculum, and incubated at 35C for 96h

in an incubator shaker at 120 rpm. Triplicate flasks were


maintained for the experiment. At interval of 24h, culture broth
from individual flask was taken out and centrifuged at 8000
rpm in a refrigerated centrifuge for 20 min at 4C. The clear
supernatant was used for LA estimation. The flow- chart for
LA production from cassava bagasse is shown in Figure 1.
In another set of experiment, in lieu of crude enzyme, partially
purified S. erumpens -amylase (2, 5 and 10%) was used for
liquefaction of cassava bagasse, and the remaining steps were
the same as described in Figure 1 for production of LA.
Triplicate flasks were maintained for this experiment. LA yield
from cassava bagasse was calculated as follows:
LA yield (%) = LA produced
x 100
Starch present in cassava bagasse

Figure 1: Flow chart for lactic acid production from cassava bagasse

15

Ray and Kar

Starch conversion efficiency (%) was calculated as follows;

was proportional to the applied enzyme concentration as well


as duration of incubation (fermentation). Maximum LA
production (21.89g LA/100g bagasse) was achieved by
application of 10% crude amylase at the end of 96 h incubation,
showing 36.5% yield (assuming 63% starch present in 100g
bagasse) and 31.5% starch conversion efficiency. When the
performance of crude enzyme was compared with Termamyl,
the production of LA and conversion efficiency were 44.01%
and 43.2% less in crude enzyme(10%) than that of Termamyl.
This was due to the formation of less fermentable sugar (17 g
sugar) from the hydrolysis of 100g cassava bagasse by crude
enzyme in comparison to that of Termamyl (30 g sugar/100 g
bagasse). To obtain highest LA production, a key factor is to
release more available sugar into the medium for fermentation.
In this context, purified S. erumpens -amylase was applied in
the next experiment in order to increase the starch hydrolysis
efficiency.

Starch conversion efficiency (%):


Amount of starch converted to LA
x 100
Initial starch present in cassava bagasse
Biochemical analysis
LA content was estimated by the method described by Amerine
and Ough (1984) using UV-Vis spectrophotometer (Model no
CE 7250, Cecil Instrument, UK) and expressed as g/100g. The
reducing sugar content in fermented broth (before and after
fermentation) was estimated using Nelsons method
(Mahadevan and Sridhar, 1998). Enzyme activities [S. erumpens
(crude and partially purified) amylase and Termamyl] were
measured by incubating 0.2 ml of either S. erumpens amylase
or Termamyl with 0.5 ml of 0.2% soluble starch in 0.1M
phosphate buffer (pH 6.0) at 50C for 30 min. Reducing sugars
formed were measured by Nelsons method (Mahadevan and
Sridhar, 1998). One unit of enzyme activity was defined as the
amount of enzyme which produced 1m/min of reducing sugar
with glucose as the standard under the conditions described.

The crude extract of a-amylase was purified with 60%


ammonium sulphate precipitation, which contained 2.5 mg/ml
protein and showed a specific activity of 136.0 Units/ml protein.
After dialysis, specific activity increased to 408.0 Units/mg
protein with yield of 24.24% and 3.0 fold purification (Table 1).

Statistical analysis

When partially purified S. erumpens amylase (PSEA) at 2, 5


and 10% concentration were applied to cassava bagasse with
AMG at a constant rate (2%, v/w), it gave 66-94% more
fermentable sugars (20, 29 and 33 g sugar/100g bagasse,
respectively) in comparison to crude enzyme (Figure. 3).
Highest LA production (42.5 g/100 g bagasse) was observed
with 70.8% yield after 96h of incubation by the application of
10% Partially -amylase.John et al. (2007) reported LA
production (54.0 g/100 g cassava bagasse) obtained at 60h
with the application of 8.3% commercial enzyme (-amylase).
Likewise, the optimum LA production for Lactobacillus
delbrueckii and L. plantarum were found to be 32.0 and 46.0
g/100g, respectively using alfalfa fibre as the carbohydrate
source, whereas with that of soya fibre, it was 44.0 g/100 g
(Hassan et al., 2001). Further, in our study, 5% PSEA and 2%
Termamyl showed no significant difference (Fishers LSD test
p< 0.05%) on fermentable sugar (29 g/100 g) after enzymatic
hydrolysis and subsequently, on LA production (38.45 g LA).
Likewise, by the application of 5% partially enzyme, the starch

The data of enzyme production were analyzed using one way


ANOVA. Where significant difference in ANOVA (p< 0.05) was
detected the Fishers Least Significant Difference (LSD)
multiple comparison test was applied to compare the factor
level difference. The analyses were performed using MSTATC (Version 2.0, Michigan State University, Michigan, USA).
Results and discussion
Cassava bagasse was hydrolyzed by three sets of thermostable
amylases: crude, partially purified enzyme (from S.
erumpens) and Termamyl and the hydrolysates were used as
the sole carbon source for LA production. In the first
experiment, by applying 2, 5 and 10% of crude amylase on
cassava bagasse, 10, 15 and 17 g reducing sugars /100g
bagasse were produced (showing 15.0, 22.7 and 25.7%
conversion efficiency respectively), which were further
converted to LA by L. plantarum (Figure 2). LA production

Table 1: Partial purification of amylase for liquefaction of cassava bagasse


Purification steps
Culture filtrate (crude)
Ammonium sulphate precipitation
After dialysis

Total
volume (ml)

Total enzyme
activity (Units)

Total
protein (mg)

Yield
(%)

Specific activity
(Units/mg protein)

Fold of
purification

100
15
16

34000
6747
8241.6

250
24.8
20.2

100
19.8
24.24

136
272
408

0
2.0
3.0

16

Application of thermostable -amylase in Streptomyces erumpens liquefaction of cassava bagasse for production of lactic acid

Figure 2: Lactic acid production and residual sugars from cassava


bagasse with application of S. erumpens crude amylase (2, 5 and
10%) and Termamyl (2%)

Figure 3: Lactic acid production and residual sugars from cassava


bagasse with application of S. erumpens purified amylase (2, 5 and
10%) and Termamyl (2%)

to LA conversion efficiency (54.0%) was similar with that of


2% Termamyl (55.0%). However, a higher starch to LA
conversion ratio of 61.1% was obtained in case of 10% PSEA.

as it produces 94% more LA from cassava bagasse. Likewise,


the conversion efficiency and LA yield using 5% purified
amylase was found to be similar to that of 2% Termamyl. Hence,
the indigenous application of PSEA for conversion of starch
in cassava bagasse have potential to save considerable amount
of LA production cost.

Generally, LA bacteria are deficient in cellulolytic and amylolytic


characters necessitating the prior hydrolysis of cellulosic and
starchy wastes for their better utilization. Enzymatic hydrolysis
is preferred to acid hydrolysis for this purpose (John et al.,
2007). Woiciechowski et al. (2002) reported 97.3% conversion
of starch into sugar in cassava bagasse by treatment with
Termamyl and AMG. Simultaneous saccharification and lactic
acid production is done with the addition of amylolytic enzymes
(Termamyl and AMG) into starch and inoculated with L.
delbrueckii (Anuradha et al., 1999). The yield of LA was 45%.
Co-immobilization of starch-degrading organisms like
Aspergillus awamori and lactic acid-producing bacteria
Streptococcus lactis was also done for the simultaneous
saccharification and lactic acid production (Kurusava et
al.,1988); however, the yield of LA was poor. Further, almost
all commercial amylases available in market are in purified form;
the purification results in removal of impurities from the enzyme
thereby enhancing its catalyzing activity. In our findings, the
PSEA (5%) was found equally efficient as commercial
-amylase (Termamyl, 2%) in converting starch in cassava
bagasse into sugar.

Reference
Amerine ,M.A. and Ough, C. 1984. Methods for Analysis of Musts
and Wines, New York, Wiley- Inter Science Publication, John
Wiley and Sons, pp. 447.
Anuradha, R., Suresh, A.K. and Venkatesh, K.V. 1999. Simultaneous
saccharification and fermentation of starch to lactic acid.
Process Biochem. 35: 367375.
Edison, S., Anantharaman, M. and Srinivas, T. 2006. Status of cassava
in India an overall view. Tech Bull Ser, 46, Pp, 79, Central
Tuber Crops Research Institute, Thiruvanathapuram, India,
Fitzpatrick, J.J. and O_Keeffe, U. 2001. Influence of whey protein
hydrolyzate addition to whey permeate batch fermentations
for producing lactic acid. Process Biochem. 37: 183186.
Gupta, R., Gigras, P., Mohapatra, H., Goswami, V.K. and Chauhan,
B. 2003. Microbial - amylases: a biotechnological
perspective. Process Biochem. 38: 1599-1616.
Haki ,G.D. and Rakshit, S.K. (2003). Developments in industrially
important thermostable enzymes. Biores. Technol. 89: 17-34.
Hassan, K.S., Moldes, B., Richard, G.K. and Richard, J.S. 2001.
Lactic acid production by simultaneous saccharification and
fermentation of alfalfa fiber. J. Biosci. Bioeng. 92: 518-523.
John, R.P., Nampoothiri ,K.M. and Pandey, A. 2006. Solid state
fermentation for L- lactic acid production from agro wastes

Conclusion
It was concluded that application of partially purified S.
erumpens -amylase was more effective than crude amylase
17

Ray and Kar

using Lactobacillus delbrueckii. Process Biochem. 41: 759763.


John, R.P., Nampoothiri, M.K. and Pandey, A. 2007. Production of
lactic acid from biomass: an overview on process
developments and future perspectives. Appl.Microbiol.
Biotechnol., 74: 524-534.
John, R.P., Nampoothiri, K.M., Nair, A.S. and Pandey, A. 2005. L
(+)-lactic acid production using Lactobacillus casei in solid
state fermentation. Biotechnol. Lett., 27: 1685-1688.
Jyothi, A.N., Sasikiran, K., Nambisan, B. and Balagopalan, C. 2005.
Optimization of glutamic acid production from starch factory
residue using Brevibacterium divaricatum. Process Biochem.,
40: 3576-3579.
Kadam, S.R., Patil, S.S., Bastawde, K.B., Khire, J.M. and Gokhale,
D.V. 2006. Strain improvement of Lactobacillus delbrueckii
NCIM 2365 for lactic acid production. Process Biochem. 41:
120126.
Kar, S. and Ray, R.C. 2011. Purification, characterization and
application of thermostable exo-polygalacturonase from
streptomyces erumpens MTCC 7317. J. Food Biochem, 35:
133-147.
Kar, S., Datta, T.K. and Ray, R.C. 2010. Optimization of
thermostable - amylase production by Streptomyces
erumpens MTCC 7317 in solid-state fermentation using
cassava fibrous residue. Braz. Arch. Biol. Technol. 53: 301309.
Kar, S. and Ray, R.C. 2008. Partial characterization and optimization
of extracellular thermostable Ca2+ inhibited -amylase
production by Streptomyces erumpens MTCC 7317. J. Sci.
Ind. Res. 66: 252- 255.
Kar, S., Ray, R.C. and Mohapatra, U.B. 2012. Purification,

characterization and application of thermostable


amylopullulanase from Streptomyces erumpens MTCC 7317
under submerged fermentation. Ann. Microbiol. 62:931-937.
Kar, S., Swain, M.R. and Ray, R.C. 2009. Statistical optimization of
alpha-amylase production with immobilized cells of
Streptomyces erumpens MTCC 7317 in Luffa cylindrica L.
sponge discs. Appl. Biochem. Biotechnol. 152; 177188.
Kurusava, H, Ishikawa, H, and Tanaka, H 1988. L-lactic acid
production from starch by co- immobilized mixed culture
system of Aspergilus awamori and Streptococcus lactis.
Biotechnol. Bioeng. 31: 183187.
Mahadevan. A. and Sridhar, R. 1998. Methods in Physiological Plant
Pathology, 5th edn. Madras, Sivakami Publication.
Oh. H., Wee. Y.J., Yun. J.S., Han. S.H., Jung. S. and Ryu. H.W. 2005.
Lactic acid production from agricultural resources as cheap
raw materials. Biores. Technol., 96: 1492-1498.
Ray. R.C., Mohapatra. S., Panda. S. and Kar. S. 2008. Solid substrate
fermentation of cassava fibrous residue for production of
- amylase, lactic acid and ethanol. J. Envron. Biol., 29: 111115.
Tonkova, A. 2006. Microbial starch converting enzymes of the amylase family. In: Ray R.C. and O.P. Wards (eds.), Microbial
Biotechnology in Horticulture Volume I. Enfield, NH, Science
Publishers, pp. 421- 472. .
Varadarajan, S. and Miller. D.J. 1999. Catalytic upgrading of
fermentation-derived organic acids. Biotechnol. Prog. 15: 845
854.
Woiciechowski, A.L., Nitsche, S., Pandey, A. and Socool, C.R. 2002.
Acid and enzymatic hydrolysis to recover reducing sugars
from cassava bagasse: an economic study. Braz. Archi. Biol.
Technol., 45:393-400.

18

Intl. J. of Food. Ferment. Technol. 2(1): 19-25-, June, 2012

Research Paper

Changes in phytochemicals during fermentation


of wine grapes
A. K. Sharma*, S.V. Navale, S.N. Aute, G.S. Karibasappa, D.P. Oulkar and P. G. Adsule

National Research Centre for Grapes, Pune, Maharashtra, India


*

Email: sharmadrajay@gmail.com

Paper no: 33

Received: 12 Jan,2012 Received in revised form: 14 April, 2012

Accepted: 19 May, 2012

Abstract
Changes in various phytoemicals during fermentation of grape cultivars for red wine (Cabernet
Sauvignon and Zinfandel) and white wine (Sauvignon Blanc and Charark-4; a cross of Chardonnay
and Arkavati) were determined. The fermentation was performed in food grade plastic vessels, using
a commercial wine yeast strain Premier Cuveeat 201C. The data were recorded on total phenols,
anthocyanin,Ferric ion reducing antioxidant power (FRAP), DPPH assay and individual phenolic
content and amino acids. At the initial stage of fermentation, total phenols were higher in Zinfandel
than in Cabernet Sauvignon, but increased to 4-folds at the completion of fermentation. Higher levels
of total phenols and antioxidant activities were noted in red wines than in white ones. Free radical
scavenging activity (DPPH) was higher in Cabernet Sauvignon than in Zinfandel. However,resveratrol
content was more than twice in Zinfandel wine than in Cabernet Sauvignon wine. White wines
showed low levels of phenolic compounds and some phenolics viz.; epicatechingallate, resveratrol
and kaempferol were found absent. During the process of fermentation quantities of various amino
acids were changed.Alanine, serine, lysine and vaniline amino acids were found absent at beginning
but on the completion of fermentation presence of these amino acids was confirmed and quantified.
2012 New Delhi Publishers. All rights reserved

Keywords: Wine, phytochemicals, phenols, resveratrol, amino acids

The quality of wine is decided by consumer preference.The


most important factor that contributes to wine character is the
kind of grapes used, while the fermentation has been recognized
to be essential for the quality and stability of wine. Red wine is
the result of must fermentation and extraction of various
compounds from the pulp, seeds and skin. Grape skin
fermentation helps in the extraction of colour compounds
relevant to the wine structure, its body, bouquet and aroma
perception, besides the extraction of various substances from
polyphenolic to nitrogen compounds, polysaccharides,
pectins, mineral substances, pyrazine, terpenes, etc. (Klenar,
et al., 2004). Usually, large amounts of phenolic compounds,
mostly the flavonoids as well as their byproducts are present
in grapes at high concentrations (Kanneret, et al., 1994), but

their presence is affected by number of factors including grape


variety, skin colour, sun exposure, vinification technique and
aging (Price et al., 1995, McDonald et al., 1998; Burns et al.,
2001 and Pakhale et al., 2007). The phenolics profile of wine is
not the same as those of fresh grapes because of significant
changes occurring during winemaking process from the berry
crushing stage, to subsequent fermentation and wine aging
(Meyer 1997). The phenolic compounds, anthocyanins of
grapes and their antioxidant activities have been extensively
studied and a strong correlation exits between antioxidant
capicity and total phenols (Rrice-tvans 1995; Kalt et al., 1999;
Netzel et al., 2003) and represents about 2050% of the total
nitrogen in the must. The amino acid profile of wine can be
used to differentiate various wines according to vine variety,

Sharma et al.

geographical origin and year of production (Huang and Ough.


1991; Hollendet et al., 1995). Proline makes the majority of the
free amino acids in wine and its largest amount is found in
fresh grapes. The present investigation was conducted to
study the changes in phytochemicals and antioxidant activities
of wine grapes during process of winemaking.

Physico-chemical analysis
The collected samples were analyzed following standard
operating procedures. Total phenols were quantified by
following the procedure of Singleton and Rossi (1965).
Content of anthocyanins in samples was estimated by using
method of Fulekiand Francis (1968). Antioxidant activities were
measured by Ferric ion reducing antioxidant power (FRAP),
and Free radical scavenging activity (DPPH assay). In
measuring of FRAP method of Benzie and Strain (1996) was
adopted and in case of DPPH assay method of Arnouset al.
(2001) was followed. LC-MS/MS (Agilent Technologies with
series hyphenated to API 4000 Qtrap (ABS Sciex) mass
spectrometer equipped with electrospray ionization (ESI+)
probe 1200) was used for quantification of individual phenolic
content in samples. For amino acids, the HPLC (Perkin Elmer
200 series) of column G8 (50 x 4.6 mm ID x 1.8 m) was used,
with mobile phase A: 0.1 % pentadecaflurooctonoic acid and
0.1 % formic acid in Water: Methanol (90:10), B: 0.1% formic
acid in water: methanol (10:90). Calibration curves were
obtained by plotting the peak areas against different
concentration of amino acids and poly phenolic compounds.

Materials and methods


Raw material
The study was carried out during cropping season of 2009 at
the National Research Center for Grapes, Pune. The bunches
of grape cultivars for red wine (Cabernet Sauvignon and
Zinfandel) and white wine (Sauvignon Blanc and Charark-4; a
cross of Chardonnay and Arkavati) were harvested when Total
Soluble Solids (TSS) content reached around 20 B. The berries
were destemmed manually and crushed to extract juice. To
suppress the development of natural microflora 100 ppm
potassium metabisulpite (KMS) was added to the must/juice
which was kept at 0 C for overnight.
Fermentation

Results and discussion

A commercial wine yeast strain Premier Cuvee was used for


fermentation. The must/juice was fermented in food grade
plastic vessels which were kept at 201C. After recording of
initial observations, samples were taken during fermentation
on 2nd, 3rd, 6th and on 11th day when fermentation was completed
and skin separation was observed. During the fermentation
process, the must of red grapes was punched and fermenting
white juice was mixed two times every day. The samples were
collected and each sample was replicated three times for
analysis of various parameters. The generated data were
analyzed as per CRD using SPSS program.

Significant differences were noted in total phenols content of


different samples at various stages of fermentation of wine
grape varieties except Sauvignon Blanc (Table 1) where same
value i.e. 230 mg/ was recorded on 3rd and 6th day. Total phenols
were recorded higher in red varieties than white ones. Values
of 3760 and 1670 mg/L were recorded in Cabernet Sauvignon
and Zinfandel, respectively while in Sauvignon Blanc and
Charak-4, these values were 220 and 370 mg/L, respectively on
11th day. Zinfandel recorded initially higher concentration of
total phenols (1050 mg/L) than Cabernet Sauvignon (950 mg/
L) but experienced only slight increase during fermentation.

Table 1: Changes in total phenols and anthocyanin content at different intervals during fermentation process of wine grapes.
Days

Total Phenols (mg/L)


Red
CabernetSauvignon

Initial
2
3
6
11
SEM
CD at1%

950
1770
1990
3880
3760
5.362
16.16

Anthocyanin (mg/L)
White

Red

Zinfandel Sauvignon Blanc Charark-4


1050
1330
1740
1850
1670
3.162
9.53

330
260
230
230
220
2.828
8.52

652
680
545
393
370
6.251
18.84

20

White

CabernetSauvignon

Zinfandel

1070
8330
14330
19080
15030
2.236
6.74

3400
7940
10210
12350
8340
3.162
9.53

Sauvignon Blanc Charark-4


-

Changes in phytochemicals during fermentation of wine grapes

On the other hand, in both the white varieties, total phenols


were significantly declined as the fermentation progressed. In
general, concentration of phenolics rises during grape
fermentation. But results of present study were different in
case of white grapes. During the fermentation of white wine
grapes decrease of gallic and protocatechuic acids was noticed
by Budic-Leto and Lovric (2002).In wine, there are two groups
of phenolic acids; hydroxybenzoic acids and hydroxycinnamic
acids (Cabrita et al. 2008). Tian et al. (2009) found that the
evolution of hydrobenzoic acids during the fermentation of
dry wine, semi-sweet wine and ice wine. Higher concentrations
of total hydroxybenzoic acids were noted in all the wines. The
concentrations of total phenols in all varieties were different.
In case of Cabernet Sauvignon about 4 times and in Zinfandel
only 0.6 time total phenols were increased during fermentation.
These results are confirmatory to those of Sulc and Lachman
(2005) who also found significant differences in total phenols
among the varieties. Red wines contained on an average 2
times more total phenols in comparison with grape must at the
beginning of winemaking. White and red wines differed
significantly in total phenols content during vinification
process. The red and white wines differ not only in final
contents of phenolics, but also in their extreme increase in red
wines during fermentation.The phenolic compounds in wine
range from relatively simple compounds to complex tannintype substances with antioxidant activity. Wine phenolic
composition is conditioned by the grape used and by the
winemaking processes that determine their extraction into the
must and their further stability in wine. Grape phenolics depend
on the variety and other factors that affect berry development
such as soil, geographical location and weather
conditions.Wine samples of Cabernet Sauvignon showed
higher levels of Catechin (117mg/L) and Epicatechin (59.8 mg/
L) and similar trend was observed in Zinfandel. Lower levels
of Kaempferol (0.316 mg/L), Resveratrol (0.503 mg/L) and p
Coumaric acid (0.521 mg/L) were recorded in wine of Cabernet
Sauvignon. On the other hand, Kaempferol in Zinfandel has
minimum quantity i.e. 0.139 mg/L and Epicatechin Gallate (0.386
mg/L). More than double the quantity of Resveratrol was
found in Zinfandel wine than Cabernet Sauvignon wine (Table
2). White wines showed very low concentration of different
phenolic. Epicatechin Gallate, Resveratrol and Kaempferol were
absent in white wines from Sauvignon Blanc and Charark-4.
pCoumaric acid was absent from Sauvignon Blanc wine
however, only 0.807 mg/l was noted in wine of Charark-4. The
wines of Sauvignon Blanc contained lower concentration of
the phenolic compounds as well as total phenolics than wines
from Charark-4. Content of total and individual phenols were
found to be characteristic of the grape variety. Red wines
contained higher total as well as individual phenols than white.
Phenolic compounds have long been considered to be the

basic components of wines and over 200 compounds have


been identified. Resveratrol is mainly contained in the skins of
grapes (Schmandke, 2002) due to this reason resveratrol was
absent from white wines.Flavonols, such as quercetin,
kaempferol, and myricetin, are localized in grape skin and occur
in glycosidic forms. They exist in trace amount in white wines
(Singleton, 1992). Our findings are also matched with these
results.
The anthocyanin contents were higher in Cabernet Sauvignon
than in Zinfandel at all levels of fermentation and remained
absent in white varieties (Table 1). Maximum concentration of

Figure 1: Total ion chromatograph of 100 ng/mL calibration standard (A), red wine (B), white wine (C)

21

Sharma et al.

anthocyanins in both the varieties was recorded on 6th day


this finding is same as recorded by Sims and Bates (1994). who
observed the maximum rate of anthocyanins extraction between
days 4 and 6 of fermentation of crushed grapes.The increase
in anthocyanin concentrations, recorded in given time intervals,
was closely correlated with the production of alcohol, which
was the most important factor of maceration of colouring
matters. Balik (2006) recorded maximum concentrations of
anthocyanins one day earlier than those of alcohol. The
anthocyanins are localized in the skin tissues of most grape
cultivars; fermentation and maceration have a profound effect
on the amount of anthocyanins present in the final wine. An
extreme example of this would be the separation of the solid
parts of the grape berry from the juice with little or no maceration
resulting in a wine with little or no red colour (Kennedy, 2008).

sample in comparison to the initial value in Cabernet


Sauvignon while more than four times increase was noted in
Zinfandel. However, higher FRAP values were noted in
Cabernet Sauvignon at initial and final fermentation than
Zinfandel. Very low FRAP values were noted from both the
white varieties and declining trend was noted. The red wine
samples showed higher free radical scavenging activities than
white varieties as estimated by DPPH assay. The antioxidant
values were increased with advancement of fermentation in
both the red varieties. Wines made from Cabernet Sauvignon
recorded with higher antioxidant as compared to Zinfandel.
The FRAP and DPPH values were higher in red wine varieties
than white. Katalini et al. (2008) also noted that the red wines
have greater reducting power capacity than white wines.
Significant differences in reducing power capacity of some
red wines can be related to differences in flavonoid content,
especially anthocyanins which represent a numerous flavonoid
sub-group of very efficient free radical scavengers with

Ferric ion reducing antioxidant power (FRAP) values (Table 3)


were registered more than double on the last day (11th day)

Table 3: Changes in antioxidant property during fermentation process at the interval of days
FRAP (mg/l)
Red

White

CabernetSauvignon
Initial
2
3
6
11
SEM
CD at1%

DPPH (mM)
Red

Zinfandel Sauvignon Blanc Charark-4

290
280
340
320
660
5.477
16.51

130
380
510
600
570
10.624
32.02

0.33
0.32
0.19
0.28
0.26
0.010
0.03

CabernetSauvignon

1.7
2.3
1.5
1.1
1.3
0.028
0.09

White
Zinfandel Sauvignon Blanc

1.24
1.51
1.46
2.10
2.29
0.054
0.16

1.19
1.23
1.60
1.76
1.90
0.053
0.16

0.39
0.42
0.35
1.13
0.36
0.019
0.06

Charark-4
0.60
0.74
0.61
0.52
0.63
0.079
NS

Table 4: Instrument parameters for the individual phenolic compounds.


ESI+

Sr.no. Name of compound RT (min)


Q1
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

Epicatechingallate
Caffeicacid
Epicatechin
p-Coumaricacid
Quicetrin
Syringicacid
Vanillicacid
Quercetin
Resvaretrol
Catechin
Kaempferol

7.87
7.42
7.14
7.86
8.24
7.29
7.21
9.63
9.24
6.84
7.46

[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+

443
181
291
165
449
199
169
303.0
229.0
291.0
287.0

Quantifier

Qualifier

Q3

DP

CE

CXP

Q3

CE

CXP

123.0
89.1
123.0
147.0
303.2
155.0
125
153.0
135
165.0
165.0

51
26
50
56
45
56
55
55
29
38
53

19
43
23
16
15
14
14
45
22
17
40

5
6
5
6
9
7
5
5
6
7
2

273
135
139,165
119 , 91
129, 85
123, 77
93
137, 69
107, 119
139,123
121

12
27
20 ,18
25, 35
22, 35
18,38
21
45,80
30,55
22
50

5
8
6
5,3
6
5,2
3
5,1
4,5
7, 2
5

* Q1- Precursor ion, Q3- product ion, DP- declusturing potential, CE- collision energy, CXP- collision exit potential

22

Changes in phytochemicals during fermentation of wine grapes

Table 2: Concentration (mg/L) of different phenolic compounds in wines


Phenolic compounds

Concentration (mg/L)
Red

Epicatechingallate
Caffeic acid
Epicatechin
p- Coumaric acid
Quicetrin hydrate
Rutin hydrate
Syringic acid
Quercetin
Resveratrol
Catechin
Kaempfero
Total Phenolics

White

Cabernet Sauvignon

Zinfandel

Sauvignon Blanc

Charark-4

1.10
2.14
59.80
0.52
9.10
7.98
5.27
7.08
0.50
117.00
0.316
4160.00

0.39
2.47
59.30
2.65
1.65
1.97
5.29
2.71
1.08
71.40
0.14
1050.00

0.00
0.35
0.11
0.00
0.60
0.24
0.16
0.11
0.00
0.07
0.00
20.00

0.00
0.33
1.54
0.81
4.07
0.22
0.29
0.18
0.00
3.51
0.00
50.00

Table 5: Changes in quantity of amino acids (mg/l) during fermentation of wine grapes
Amino acid

Red wine grapes


Cabernet Sauvignon
st

Arginine
Aspartic Acid
Glutamic Acid
Alanine
Histidine
Hydroxyproline
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Serine
Tryptophan
Tyrosine
Vaniline

th

White wine grapes


Zinfandel

st

Sauvignon Blanc
th

th

Charak-4

1 day

12 day

1 day

12 day

1 day

12 day

1 day

12th day

113
0
0
0
5.08
15.5
1.36
9.53
1.53
4.17
0
448
0
0
1.37
0

79.9
5.7
23
94.5
36.9
32.4
32.7
30.2
0
31.3
9.06
1150
13.5
0.285
11.4
10.5

702
21.8
70.9
242
35.4
5.33
15.1
0
1.58
9.37
26.9
421
81.3
6.19
25
30

80.1
26.1
41.6
54.5
12.9
8.22
31.9
33.4
12.5
8.19
13.1
462
19.2
0
24.2
21.8

34.4
0.274
0.105
0.94
2.97
10.3
0.367
0
0
0.831
0
940
4.39
0
1.05
0.646

80.4
29
30.1
58.3
8.74
20.2
25.8
29.2
11.4
10.9
9.22
928
21.1
0.946
20.1
18.3

37.3
2.49
0.203
15.3
3.39
9.93
0
0
1.55
2.61
0.156
200
0
0.063
3.37
0

126
69.4
67
189
16.5
18.2
107
64.3
29.3
52.7
26.8
733
39.2
0
55.4
55.3

confirmed excellent antioxidant properties. The value of FRAP


and DPPH increased during course of fermentation. However,
DPPH and FRAP values in Charark-4 and Sauvignon Blanc
were low and declined non-significantly during winemaking
process.

st

st

grapes (Sauvignon Blanc and Charak-4). The concentration of


glutamic acid and aspartic acid was decreased at final level of
fermentation of Cabernet Sauvignon but the concentration of
glutamic acid, Histidine, Ornithine, Phenylalanine, Tryptophan,
Tyrosine, and Vaniline was declined in Zinfandel. The
quantities of Leucine and Lysine amino acids were increased
at final level of fermentation of Cabernet Sauvignon and Charak4. Amino acids in wine originate from various sources such as
indigenous compounds in grapes, metabolites by yeast during
growth phase exerted by living yeast or released by proteolysis
during the autolysis of dead yeast or by enzymatic degradation

The concentrations of amino acids in both red and white wines


were measured at initial and final fermentations. The
concentrations of different amino acids were changed
dramatically during the fermentation process (Table 5). Arginine
was decreased in both the red wine grapes (Cabernet
Sauvignon and Zinfandel) but was increased in white wine

23

Sharma et al.

characteristics of aged red wines. Journ Agri Food Chem.,


49:5736-5742.
Balk, J. 2006. Dynamics of changes in total anthocyanins during the
fermentative maceration of grapes.HortSci (Prague) 33:103107.
Budic-Leto, I. and Lovric, T. 2002. Identification of phenolic acids
and changes in their content during fermentation and ageing
of white wines Posip and Rukatac. Food Technol Biotechnol,
40 (3):221-225.
Burns, J., Gardner, P.T., Matthews, D., Duthie, G.G., Lean, M.E.J.,
Crozier, A. 2001. Extraction of Phenolics and Changes in
Antioxidant Activity of Red Wines during Vinification.
JourAgric Food Chem., 49:5797-5808.
Cabrita, M.J., Torres, M., Palma, V., Alves, E., Pato, R., Costa
Feritas AM 2008.Impact of malolactic fermentation on low
molecular weight phenolic compounds. Talanta, 74:12811286.
Cataldi, T.R.I., Nardiello, D. 2003. Determination of Free Prolin and
Monosaccharides in wine samples by High- perfofmance
anion- exchange chromatography with pulsed amperometric
detection (HPAEC-PAD). Jour Agri. Food Chem., 51:37373742.
Fuleki, T., and Francis, J. 1968. Quantitative methods for
anthocyanins.1. Extraction and determination of total
anthocyanin in cranberries.J. Food Sci., 33:72-78.
Haung Z, Ough CS 1991 Amino Acid profiles of commercial Grape
juices and wines. Amer Jour Enol Viti., 42:261-267.
Holland, M.V., Bernreuther, A., and Reniero, F. 1995. The use of
amino acid as a fingerprint for the monitoring of European
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Belton, P. S., Delgadillo, I., Gil, A. M., and Webb, G. A.
(Eds.), The Royal Society of Chemistry, Cambridge, UK.
Kanner J, Frankel E, Granit R, German B, and Kinsella E 1994
Natural antioxidants in grapes and wines. J. Agri. Food Chem.,
42: 6469.
Katalini. V., Ljubenkov, I., Pezo, I., Generali, I., Strievi, O., Milo,
M., Modun D,and Boban M 2008 Free resveratrol monomers
in varietal red and white wines from Dalmatia (Croatia).Period
Biolo 110: 7783.
Kennedy, J.A. 2008. Grape and wine phenolics: Observations and
recent findings.Cien InvAgr., 35: 107-120.
Klenar, I., Berovic, M., and Wondra, M. 2004. Phenolic Compounds
in Cabernet Sauvignon and Merlot Wines. Food Technol
Biotechnol., 42:11-17.
Mauricio, J.C., Valero, E., Milln, C., and Ortega, J.M. 2001.Changes
in Nitrogen Compounds in Must and Wine during
Fermentation and Biological Aging by Flor Yeasts. J Agric
Food Chem,49 (7):33103315. DOI: 10.1021/jf010005v
McDonald, M.S., Hughes, M., Burns, J., Lean, M.E.J., Matthews
D. and Crozier, A. 1998. Survey of the free and conjugated
myricetin and quercetin content of red wines of different
geographical origins. JAgric Food Chem 46: 368375.
Meyer, A.S., Yi, O.S., Pearson, D.A., Waterhouse, A.L., and Frankel,
E.N. 1997. Inhibition of human low density lipoprotein
oxidation in relation to composition of phenolic antioxidants
in grapes (Vitisvinifera). J Agric Food Chem., 45:1638-1643.

of grape protein (Cataldiet al. 2003). Yeast and bacteria use


these amino acids to grow and ferment the must and can release
some of these amino acids back into the wine after fermentation.
In general, in white wines average levels of free amino acids
was higher and more variable than those of the red musts.
Proline makes the majority of the free amino acids in wine and
is the largest amount found in fresh grapes. Arginine is an
important nutrient for yeast growth (Ough. 2009). But during
fermentation of white grapes, quantity of arginine was
increased. During fermentation, yeasts assimilate between 1
and 2 g/L of amino acids but towards the end of fermentation,
yeasts excrete significant but variable amounts of different
amino acids.The pH values lower than 3.5 have been found to
inhibit arginine consumption (Terrade and Mira, 2006). The
pH of white wines was less than 3.5 that might resulted have
increases of arginine content. Finally, at the end of alcoholic
fermentation, a few hundred mg/L of amino acids remain;
proline generally represents half. Our results are similar to the
findings of other authors. Proline is major amino acid in all
wine grape varieties studied. Tryptophan disappeared in the
winemaking process of Zinfandel and Charak-4 and very low
quantity of this amino acid was generated in Cabernet
Sauvignon and Sauvignon Blanc. During the fermentation
process, some amino acids were excreted in variable
amount.The yeast strains release some amino acids including
L-threonine, L-tryptophan, L-cysteine, and L-methionine during
grape fermentation (Mauricio, et al. 2001). However, the content
of amino acid in grape must or wine were affected by various
factors like variety, yeast strain, stage of fermentation process
etc.
From this study it may be concluded that the total phenols
and anthocyanin content increased in red wine grape varieties
along with advancement of fermentation. Maximum
anthocyanin content was recorded on sixth day of
fermentation. Red wines contained higher total as well as
individual phenols than white. The presence of important
phenolic compounds viz.; resveratrol and kaempferol was
found in red wines only where the grape skin was fermented.
The total phonels have significance as an antioxidant
compound
Acknowledgements
Authors are thankful to Dr. K. Banerjee (I/C National Referral
Laboratory) for extending use of LCMS/MS and HPLC for
analysis of wine samples.
References
Arnous, A., Makris, D.P., Kefalas, P. 2001. Effect of principal
polyphenolic components in relation to antioxidant

24

Changes in phytochemicals during fermentation of wine grapes

Ough, C.S. 2009. Acids and Amino Acid in Grapes and Wines.Modern
Methods of Plant Analysis Wine Analysis, pp 98-121
Linskens, H. F., and Jackson, J. F. (Eds.) Springer.
Pakhale, S.S., Karibasappa, G.S., Ramchandani, A.G., Bhushan, B.,
and Sharma A 2007. Scavenging effect of Indian grape
polyphenols on 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical by electron spin resonance spectrometry. Indian Jour
Experi Biol., 45: 968-973.
Price, S.F., Breen, P.J., Valladao, M. and Watson, B.T. 1995. Cluster
sun exposure and quercetin in Pinot noir grapes and wines.
Amer. J. Enol. Vitic., 46:187194.
Schmandke, H. 2002. Resveratrol and piceid in grapes and soybeans
and products made from them. Ernhrungs-Umsch 49: 349
352.
Sims, C.A. and Bates, R.P. 1994. Effects of skin fermentation time
on the phenols, anthocyanins, ellagic acid sediment, and
sensory characteristics of a red Vitisrotundifoliawine.
American Journal Enol Vitic 45:56-62.

Singleton, V.L. 1992. Tannins and the qualities of wines.Plant


Polyphenols- Synthesis, Properties, and Significance ,pp
859880. Hemingway, R.W. (Ed.), Plenum Press.
Singleton, V.L., and Rossi, J.A. 1965. Colorimetric of total phenolics
with phosphomolibdic phosphotungstic acid reagent.Amer
Journal Enol Vitic,16:144 -158.
ulc, M.,and Lachman, J. 2005. Phenolics and antioxidant activity
of wines during the winemaking process.http://old.af
.mendelu.cz/mendelnet2005/articles/tech/sulc.pdf Accessed
on 11/08/2009.
Terrade, N., and Mira de Ordua, R. 2006. Impact of winemaking
practices onarginine and citrulline metabolism during and after
malolactic fermentation. J. Appl. Microbiol. 101:406-411.
Tian, R.R., Pan, Q.H., Zhan, J.C., Li, J.M., Wan, S.B., Zhang, Q.H.,
and Huang WD 2009. Comparison of phenolic acids and
Flavan-3-ols during wine fermentation of grapes with different
harvest times. Molec,14:827-838.

25

Intl. J. of Food. Ferment. Technol. 2(3): 27-36-, June, 2012

Research paper

Cloning and expression of rec-pediocin CP2 in


Escherichia coli using OmpA and TAP gene
fusion approach
Balvir Kumar*, P. P. Balgir and B. Kaur
Department of Biotechnology, Punjabi University, Patiala, Punjab, India
*

Email: balvirkumarbt@gmail.com, balvirkumar@yahoo.co.in

Paper no: 34 Received: 26 Jan, 2012

Received in revised form: 17 April,2012

Accepted: 19 May,2012

Abstract
Study was aimed at cloning, expression and characterization of rec-pediocin CP2 in Escherichia coli
BL21(DE3) using ompA and tap gene fusion expression approach. pET32(b)-pedA vector containing
an inframe fusion of sequences encoding E. coli ompA secretion signal, two tandem affinity purification
tags and rec-pediocin synthetic gene was introduced into a pediocin CP2 resistant strain of E. coli
BL21(DE3). Rec-protein was accumulated in periplasmic fraction as well as inclusion bodies of
transformed E. coli cultures and was extracted by cell lysis. Crude fusion protein did not show any
biological activity. Purification of rec-pediocin involved two tandem affinity chromatographic steps
based on 6XHis-tag and Strep-tag. In between these, enterokinase cleavage was carried out. After
digestion, rec-pediocin displayed a very strong bactericidal activity against Listeria monocytogenes.
The process based on T7-driven pET expression system and tandem affinity chromatography resulted
in approximately 10 times higher yield of rec-pediocin (in terms of specific activity) as compared to the
native P. acidilactici MTCC5101.
2012 New Delhi Publishers. All rights reserved

Keywords: Rec-pediocin, Pediococcus acidilactici, Pediocin CP2, Heterologous expression,


Cloning, Bacteriocin
Bacteriocins of lactic acid bacteria have long been used for
fermentation and preservation of meat and milk (Gillor et al.,
2008). Pediocins are prone to proteolytic digestion in the
gastrointestinal tract and seem to be non-toxic and lack any
potential antigenicity/toxicity in animals (Bhunia et al., 1990;
Guerra et al., 2001; Kheadr et al., 2010). Native hosts suffer
from low fermentation yields and poor recovery due to in vitro
interactions of bacteriocins with other proteins (Daba et al.,
1994; Elegado et al., 1997; Stein et al., 2003; Beaulieu et al.,
2007). Keeping this in view, a large number of heterologous
systems have been developed for controlled expression of
pediocins including Bifidobacterium longum (Moon et al.,
2005), Enterococcus faecalis (Somkuti and Steinberg, 2003),
Escherichia coli (Halami and Chandrashekar, 2007; Liu et al.,
2011), Lactococcus lactis (Halami and Prakash, 2008), L. sakei
(Johnsen et al., 2005), Streptococcus thermophilus (Somkuti

and Steinberg, 2003), Saccharomyces cerevisiae (Schoeman


et al., 1999) and Pichia pastoris (Beaulieu et al., 1999). Recpediocins were secreted out in the culture medium by attaching
specialized secretary signals such as Bifidobacterial -amylase
signal peptide (Moon et al., 2005), lactococcin A secretory
apparatus (Arques et al., 2008) and yeast mating factor -1S
signal peptide (Schoeman et al., 1999). Attachment of highly
specific affinity tags facilitated their detection by affinity
purification, immuno-fluorescence, immuno-precipitation and
Western blotting. Many reports indicated decrease in viability
of recombinant cells (Miller et al., 1998), low expression levels
(Horn et al., 1998; 1999), loss of recombinant plasmids from
expression host (Coderre and Somkuti, 1999) and aggregation
of rec-pediocin with other host proteins (Beaulieu et al., 2007).
Expression of inactive rec-pediocin in inclusion bodies
following over expression in the recombinant host (Halami

Kumar et al.

and Chandrashekar, 2007; Liu et al., 2011) further complicated


the purification as additional steps of isolation, purification
and refolding are needed.

DH5 MTCC1652, E. coli BL21(DE3) MTCC1679 and Listeria


monocytogenes MTCC657 were procured from MTCC,
Chandigarh, India. E. coli cultures were revived and maintained
in LB medium (containing tryptone 10g/l, yeast extract 5g/l,
NaCl 10g/l) at 37C for 24h. L. monocytogenes MTCC657 was
maintained in Brain Heart Infusion broth (Himedia) at 37C. P.
acidilactici LB42 procured from Prof. R. K. Malik (NDRI, Karnal,
India) was grown in MRS medium at 37C and maintained as
glycerol stocks. E. coli expression vector pET32(b) was kindly
provided by Prof. R.K. Jethi, Ambala College of Engineering
and Applied Research, Ambala Cantt., India.

Pediocin CP2 is a class IIa bacteriocin produced by


Pediococcus acidilactici MTCC 5101 (Kaur and Balgir, 2004;
Balgir et al., 2010). It has a great commercial importance owing
to its remarkable heat stability (121oC for 15min), activity over
a wide pH range (2.5 to 9.5), higher specificity and
effectiveness at very low concentrations. Pediocin production
in P. acidilactici MTCC 5101 has been linked to a 8.9 kb plasmid
pCP289 (Kaur and Balgir, 2007). It inhibits growth of Aspergillus
flavus, Clostridium sporogenes, Enterococcus faecalis,
Escherichia coli, Lactobacillus brevis, L. bulgaricus,
Leuconostoc mesenteroides, Listeria monocytogenes,
Micrococcus flavus, Neisseria mucosa, Pediococcus
acidilactici, P. pento-saceus, Pseudomonas putida, P.
aeruginosa, Salmonella typhimurium, Staphylococcus albus,
S. aureus, Streptococcus mutans and S. pyogenes. It kills
sensitive bacterial strains in a bactericidal manner and
susceptible fungal cultures by inhibiting their sporulation (Kaur
and Balgir, 2008; Kumar et al., 2011). Here, we report high level
expression of rec-pediocin in E. coli BL21(DE3) followed by
tandem affinity purification and glycine/-mercaptoethanol
(-ME) mediated in vitro refolding of rec-pediocin CP2. This
method provides approximately 10 times higher specific activity
of rec-pediocin than native pediocin CP2, produced in a short
time to be used for biophysical and in vitro therapeutic studies.

Designing synthetic rec-pediocin fusion gene construct


Pediocin CP2 gene cluster was designed according to codon
usage of E. coli using various bioinformatics tools of Sequence
Manipulation Suite version 2 available online (Stothard, 2000).
Novagens pET32(b) vector was selected for expression of
rec-pediocin in E. coli BL21(DE3). To facilitate periplasmic
expression, a 22 amino acid long secretion signal of outer
membrane protease (OmpA) of E. coli BL21(DE3) was
incorporated at N-terminus of rec-protein. To enhance recovery
of rec-protein from cell lysates, two affinity tags were added in
tandem (TAP-tandem affinity purification technique). Two
proteolytic cleavage sites were added downstream of each
affinity tag to facilitate their selective removal. Pediocin
sequence was manipulated to enhance antimicrobial range,
production and secretion of rec-pediocin in heterologous host
(Tominaga and Hatakeyama, 2007). Figure 1 comparatively
illustrates sequence features of the designed rec-pediocin
fusion protein and native pediocin CP2. Parental pediocin CP2
sequence (KYYGNGVTCGKHSC) was changed to
TKYYGNGVSCTKSGC. The designed 94 amino acid long
sequence of rec-pediocin was then reverse translated using
SMS reverse translate tool. Finally, KpnI site and SalI restriction
sites were added 5 and 3 and it was further polished using

Materials and methods


Procurement and maintenance of cultures
P. acidilactici MTCC5101 was grown in de Man, Ragosa and
Sharpe medium (MRs) (Lactobacillus Heteroferm Screen Broth,
Himedia) containing 0.1% Tween-80 and pH6.5 at 37C. E. coli

Figure 1: Comparison of sequence features of rec-pediocin fusion protein and native pediocin CP2

28

Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach

oligo analysis tool of Generunner. Sequence features of


designed fusion gene construct are shown in Figure 2. Pediocin
gene construct has been synthesized by a German based
company (GENART). It was supplied in the lyophilized powder
form which was reconstituted and multiplied in recombinant
E. coli DH5 after its transformation.

Screening and selection of recombinant E. coli BL21


(DE3)-pedA
Transformed E. coli BL21(DE3)-pedA was spread on Luria Agar
plates containing 100mg/ml ampicillin and incubated at 37C
for 24h. Around 100 isolates were picked up, multiplied by
repeated subculturing and maintained as glycerol stocks.
Isolates were then screened for recombinant pET32(b)-pedA
by comparative plasmid profiling and PCR assay. A sequence
specific pair of primers constituting a forward primer
5TGGCGCTGGCGGGCTTTG3 and a reverse primer
5CTGATGGCCGCCGGTCG3 was used to amplify a 239bp
fragment of rec-pediocin gene construct. PCR reactions were
set up using 1ng template DNA, 250pM each of forward and
reverse primer, 2U iTaq DNA polymerase (Invitrogen), 1X iTaq
Buffer, 10M MgCl2, and 50M dNTPs. GENART carrier
plasmid and native pET32(b) were incorporated as positive
and negative controls respectively. Their reaction mixtures
contained 1U Vent DNA polymerase (Invitrogen) and 1X Vent
DNA polymerase buffer in addition to standard components.
PCR was carried out in Techne thermal cycler in four segments.
Segment I consisted of initial denaturation at 95C for 3 min
followed by segment II having 30 cycles of denaturation at
95C for 1 min, annealing at 51C for 1 min and synthesis at
72C for 1 min. Segment III included a final extension of 5 min
at 72C and PCR products were kept at 4C for 5 min during
segment IV. PCR products were analyzed on 1.2% agarose gels
in the presence of Novagens molecular weight marker. Final
confirmation of insert DNA was carried out by sequencing
239bp amplicon outsourced at Bangalore Genei.

Isolation and analysis of plasmid DNA


Expression vector pET32(b) and GENART carrier plasmid were
isolated using mdi plasmid DNA miniprep kit. Isolated plasmids
were analyzed on 0.8% agarose gels containing 5g/ml ethidium
bromide. Electrophoresis was carried out in 1X TBE buffer
(containing 0.089M Tris, 0.089M Boric acid and 0.002M EDTA,
pH8.3) at 50V for 1h. Gels were finally visualized on UVtransilluminator.
Construction of recombinant pET32(b)-pedA
Sequential double digestion with KpnI and SalI was carried
out for linearization of vector and isolation of synthetic
pediocin gene construct. Digestions were performed using
100g plasmid DNA, 50U KpnI and SalI, 1X restriction enzyme
buffers and 1X BSA at 37C for 3 to 4h. Reactions were stopped
by keeping reaction vials at 65C for 20 min. Separated
restriction fragments, bearing rec-pediocin fusion gene
construct (300bp) and linearized pET vector (5843bp), were
extracted from agarose gels using Bangalore Genei gel
extraction kit. Ligation reaction was performed using 1 unit T4
DNA ligase per g of restricted DNA and IX cohesive end
ligation buffer. 5g recombinant pET32(b)-pedA vector was
used to transform E. coli DH5 (primary storage host) and E.
coli BL21(DE3) (expression host). Cultures were multiplied in
LB medium containing ampicillin (100g/ml).

Expression and purification of rec-pediocin fusion protein


Expression of rec-pediocin was induced in early log phase

Figure 2: Sequence and features of rec-pediocin fusion gene construct

29

Kumar et al.

10 min and centrifuged at 10,000Xg at 4oC for 10min. Supernatant


was separated very carefully to remove all the liquid, because
residual EDTA might interfere with binding to the metal affinity
resin. Pellet was suspended very gently in 0.8 ml ice cold 5mM
MgSO4 and stirred slowly for 10min on ice. Shocked cells of
recombinant E. coli appeared round instead of rod shaped
under the light microscope. Finally, cells were separated by
centrifugation at 10,000Xg at 4oC for 10min and supernatant
was collected in a new vial and stored at 4oC.

cultures (3-4h) of E. coli BL21(DE3) by adding 1mM IPTG.


Cultures were allowed to grow for additional 4h after induction
at 37C. E. coli BL21(DE3) transformed with native pET32(b)
was incorporated in the study as a negative control. Cells
were harvested by centrifugation at 6000g, washed with 0.9%
NaCl and resuspended in 50mM Tris (pH7.5) containing 2mM
EDTA and 0.1% Triton X100. Resuspended cells were freeze
thawed twice and disrupted by sonication for 1min (Halami
and Chandrashekar, 2007). Inclusion bodies (IBs) were
precipitated by centrifugation, washed with 2M urea and
resuspended in 6M urea, 1mM EDTA and 50mM Tris-HCl,
pH8.0. The urea solublized IBs were resuspended slowly in
refolding buffer consisting of 50mM Tris (pH7.5), 50mM NaCl,
1mM EDTA, 5mM -mercaptoethanol, 5mM immidazole and
1M glycine and stirred for 18-20h at room temperature that
would assist in protein refolding and solublization (Eisenmesser
et al., 2000). His-tag protein purification column (Bangalore
Genei) was charged with refolding buffer containing 5mM
immidazole and solublized IBs were loaded onto it. Unbound
proteins were washed in 50mM Tris (pH7.5), 50mM NaCl, 1mM
EDTA, 5mM -mercaptoethanol, 20mM immidazole and 1M
glycine. Nickel bound poly-his tagged rec-pediocin fusion
protein was eluted in 50mM Tris (pH7.5), 50mM NaCl, 1mM
EDTA, 5mM -mercaptoethanol, 0.5M immidazole and 1M
glycine.

Biophysical characterization of recombinant pediocin CP2


Analysis of the urea lysates, periplasmic extracts, poly-His
tagged rec-pediocin fusion protein and digested rec-pediocin
was carried out on 20% SDS-polyacrylamide gels in presence
of medium range protein molecular weight marker (Bangalore
Genei) run at 100V for 90 min. Biophysical analysis of unfolded
and refolded rec-pediocin CP2 was carried out by semipreparative C-18 reverse phase HPLC using the protocol
described by Elegado and others (1997). Gradient elution was
performed using solvent B (99.9% acetonitrile with 0.1% TFA)
against solvent A (water with 0.1% TFA) at a flow rate of 1.5
ml/min (Halami and Chandrashekar, 2007). All the protein peaks
were collected manually, concentrated by vacuum evaporation
and subjected to bioassay to identify the rec-pediocin peak.
UV absorption spectrum (from 190 to 350nm) of the purified
rec-pediocin was also compared with that of rec-pediocin
fusion protein.

Fusion protein was digested with recombinant human


enterokinase in a reaction mixture containing 1mg His-tagged
fusion protein, 5 units enterokinase, 1X enterokinase cleavage
buffer, and 1X enterokinase dilution buffer at 20C for 16h.
Enzyme was inactivated by keeping the reaction mixture at
65oC for 20min. Digested fusion protein was further separated
on 500l Strep-tactin affinity resin. Resin was packed under
gravity flow in a polypropylene syringe plugged with glass
wool. It was equilibrated using 2 volumes of 1X wash buffer
(containing 1M Tris-HCl, pH8.0, 1.5M NaCl, 10mM EDTA).
Rec-protein was loaded and washed five times each with 1
volume 1X wash buffer. Flow throughs and washings were
collected in separate vials to check for presence of rec-protein.
N-terminal TAP fragment was eluted from the resin 6 times
each with 0.5 volume 1X elution buffer (containing 1M TrisHCl, pH8.0, 1.5M NaCl, 10mM EDTA, 25mM Desthiobiotin).
Elution samples were collected and OD was checked at 280nm.
Strep-tactin resin was regenerated using regeneration buffer
(comprising of 1M Tris-HCl, pH8.0, 1.5M NaCl, 10mM EDTA,
10mM HABA).

Preparation of native pediocin CP2 of P. acidilactici


MTCC5101
Native pediocin CP2 preparation was prepared by growing the
P. acidilactici MTCC5101 in MRS broth at 37C for 24h. It was
further purified by Adsorption-Desorption method described
previously by Yang and others (1992).
Pediocin CP2 activity assay and protein estimation
Bacteriocin activity assay was performed against indicators L.
monocytogenes MTCC657 and P. acidilactici LB42 as per
standard method (Bhunia et al., 1988). Bacteriocin activity was
calculated as arbitrary unit (AU) and expressed as AU/ml.
Protein estimation was carried out by measuring OD at 280nm
using BSA as a standard.
Results

Periplasmic expression was checked in induced cell cultures


which were harvested by centrifugation at 6500Xg for 15min
at 4oC and resuspended thoroughly in 0.8ml sucrose lysis buffer
(30mM Tris-HCl, 20% sucrose, pH8.0) and 1.6l of 0.5M EDTA,
pH8.0. Suspension was stirred slowly at room temperature for

Designing synthetic rec-pediocin gene construct


Rec-pediocin CP2 fusion peptide carries 5 point mutations
including Thr1, Ser9, Thr11, Ser13 and Gly14 in N-terminal region
as compared to native pediocin CP2 produced by P. acidilactici

30

Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach

Construction of recombinant pET32(b)-pedA vector

MTCC5101, pediocin PA-1 produced by P. acidilactici PAC1.0


and pediocin AcH produced by P. acidilactici AcH vide
GenBank accession numbers ACS70931.1, P29430.2 and
AAA98337 respectively. Periplasmic expression and
purification of rec-protein was facilitated by ompA secretion
signal of E. coli, and tandem affinity tags. E. coli codon choice
and Novagens pET32(b) further were maximized expression
of rec-pediocin CP2 in heterologous system.

pET32(b)-pedA of 6143bp was constructed using sequences


of native pET32(b) and pediocin synthetic fusion gene
construct. A 300bp fragment of synthetic pediocin gene
construct was ligated to 5843bp fragment of double digested
pET32(b) (Figure 3). E. coli DH5 and E. coli BL21(DE3) were
transformed with 6.1 kb recombinant vector and multiplied in
LB medium containing ampicillin.

The theoretical molecular weight of rec-pediocin CP2 was


predicted as 9.87 kDa with a pI of 8.67 (using Compute pI/Mw
and TagIdent tools), which was approximately three times
higher than the native pediocin CP2. Sequence manipulation
suite tool predicted its Test Code value as 1.063 which is
more than 0.95 for a protein coding sequence (Fickett, 1982).
ProtParam tool (Expasy proteomics server) estimated half-life
of recombinant pediocin CP2 as 30h (in mammalian
reticulocytes, in vitro), >20h (in yeast, in vivo), >10h (in
E. coli, in vivo). The instability index (II) of 10.12 classified it
as stable. Simple Modular Architecture Research Tool
(SMART) identified a bacteriocin domain in the designed
construct with a significant e-value.

Screening and selection of recombinant E. coli BL21(DE3)


Plasmids were extracted from transformed cultures and
analyzed on 1.2% agarose gels. Most of the isolates indicated
presence of 6.1kb plasmids which were subjected to PCR. A
PCR product of 239 base pairs further confirmed the presence
of rec-pediocin fusion gene construct in them (Figure 4).
Sequence analysis of the amplified products also confirmed
presence of original insert DNA i.e. rec-pediocin synthetic gene
in correct orientation.
Expression and purification of rec-pediocin CP2
Rec-protein was purified by TAP technology where a sequential

Figure 3: Analysis of double digested plasmids in 2% agarose gel.


Lane 1, native pET32(b), lane 2, double digested pET32(b), lane 3,
medium range DNA ruler (Bangalore Genei), lane 4, double digested
GENART plasmid with pediocin fusion gene construct and lane 5,
undigested gene carrier plasmid

Figure 4: Analysis of PCR products on 1.2% agarose gel. Lane 1,


0.05 to 10kb DNA ruler (Novagen) and lane 2, 239bp PCR product
from recombinant pET32(b)-pedA

31

Kumar et al.

offered approximately 10 times higher yields of rec-pediocin


(9,00,000 AU/mg specific activity) as a result of over-expression
in T7-driven system and a very high purification level achieved
through tandem affinity purification with an intermediate
proteolytic digestion. Recovery of rec-protein form urea cell
lysates was 675% and 29.4 fold purity was displayed by TAP
purification. Fractions containing rec-pediocin fusion protein
obtained from the 1st metal affinity matrix were collected and
activity assay was performed against L. monocytogenes and
P. acidilactici LB42. No activity was observed in any of the
elution fraction against tested indicators. They were digested
with enterokinase and N-terminal fusion tags were separated
from rec-pediocin using strep-tactin affinity purification (Figure
7) that improved purity level without interfering with final yield
of the product. Rec-pediocin recovered after digestion of fusion
protein with enterokinase successfully inhibited growth of
indicator strain in a disc diffusion bioassay (Figure 8). Antimicrobial activity of rec-pediocin was also comparatively higher
(128AU for 103 cfu/ml indicator) than native pediocin CP2
(400AU for 103 cfu/ml indicator) when tested against L.
monocytogenes. Thus, it indicated that engineered protein
displayed lower MIC value against tested indicator strain as

metal affinity and strep-tactin affinity chromatographies were


carried out. A 9.87 kDa rec-protein was eluted from His-tag
affinity matrix with 0.5 M immidazole as indicated in Figure 5.
Over expression of rec-protein was observed in periplasmic
fraction as well as IBs (Figure 6). However, induced cells were
unable to synthesize rec-protein as evidenced by lack of
protein band of the expected size (Figure 5). Upon overexpression in heterologous systems, rec-protein accumulated
in IBs of E. coli as a result of reducing conditions of the
cytosol. Cells were disrupted, and IBs were harvested to extract
rec-protein. IBs were subsequently washed and resolublized
for proper folding of rec-proteins (Kane and Hartley, 1991;
Mukhopadhyay, 1997).
Subsequenly, native pediocin CP2 obtained using adsorptiondesorption approach from Pediococcal culture broth and recpediocin extracted from recombinant E. coli using tandem
affinity purification approaches are compared in Table 1. Native
Pediococcal host produced pediocin CP2 with a specific
activity of 30,612 AU/mg that increased to 95000 AU/mg after
purification by adsorption-desorption method. There was 3.1
fold increase in specific activity of pediocin CP2 with only
31.66% recovery. On the other hand recombinant system

Figure 5: Analysis of rec-protein on silver stained polyacrylamide


gel. Lane 1, His-tag purified rec-pediocin fusion protein, lane 2, urea
lysate of E. coli BL21(DE3) transformed with native pET32 (b),
lanes 3 and 5, urea lysates from recombinant isolates and lane 4,
protein molecular weight marker (Bangalore Genei)

Figure 6: SDS-PAGE analysis of periplasmic extract from hyper


expressing E. coli. Lane 1, periplasmic fraction from induced cells,
lane 2, protein molecular weight marker (Bangalore Genei) and lanes 3
and 4, Ni-NTA purified rec-pediocin fusion protein

32

Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach

Table 1: Purification of native and rec-pediocin using different approaches


Bacteriocin Sample

Total Volume
(ml)

Cell free supernatant


Native pediocin CP2
(Adsorption-Desorption method)
Culture broth
Rec-pediocin
(Tandem affinity purification)
a

Total Protein
(mg)

Total Bacteriocin
Activity
(AU)

Specific
Activity
(AU/mg)

Folds of
Purity

% Recovery

400
10

392
40

1,20,00,000
38,00,000

30,612 a
95,000 a

3.103

100
31.66

400
10

ND
90

Nil
8,10,00,000

ND
9,00,000 a

29.4

675

Data is an average of two experimental sets

Figure 7: Gel analysis (20% SDS-PAGE) of rec-pediocin purified by


TAP technology. Lane 1, strep-tactin purified rec-pediocin, lane 2,
protein molecular weight marker (Bangalore Genei), lane 3, His-tag
purified rec-pediocin fusion protein and lane 4, fusion product digested with enterokinase

Figure 8: Bacteriocin disc assay plate showing inhibition of


indicator bacteria around bacteriocin discs numbered 1 to 3
and 6 to 10
from pure rec-pediocin preparation. Refolding of rec-pediocin
to its active confirmation in refolding buffer was confirmed in
two separate experiments including growth inhibition of L.
monocytogenes and P. acidilactici LB42 in a disc diffusion
bioassay and a complete loss of activity in undigested sample.
The MIC values of engineered pediocin bearing five point
mutations were lower than native pediocin CP2. UV absorption
spectrum of the purified rec-pediocin was also compared with
that of rec-pediocin fusion protein. Absorbance maximum of
the recombinant pediocin was observed at 195 nm with a
shoulder at around 220 nm as compared to 205 nm for rec-

reported previously (Tominaga and Hatakeyama, 2007).


Biophysical characterization of rec-pediocin CP2
In a semi-perperative RP-HPLC, refolded rec-pediocin was
eluted as a single major peak at 3.036 min with a shoulder at
3.202 min as compared to unfolded rec-pediocin that showed
late elution at 3.25 min (Figure 9). Minor peaks at 2.559 and
2.702 min indicated contamination of rec-pediocin with TAP
fragment. Another cycle of Strep-tactin affinity purification
was therefore incorporated to remove protein contaminants
33

Kumar et al.

expression is controlled by lacI repressor protein. It is deficient


in lon protease and ompT an outer membrane protease that
can degrade expressed proteins during purification (Grodberg
and Dunn, 1988). By adjusting concentration of IPTG,
expression can be regulated from very low up to robust, fully
induced levels. Additionally, the heterologous expression of
class IIa pediocins in E. coli as fusion proteins will improve
protein solublization and avoid possible toxic effects on the
host cell (Quadri et al., 1997; Moon et al., 2006; Beaulieu et al.,
2007).
This bioprocess offered approximately 10 times higher yields
achieved collectively through PT7 based pET32(b)-pedA and
tandem affinity purification. Fusion protein itself did not show
any biological activity, but upon cleavage by an enterokinase,
biologically active pediocin PA-1 was obtained. As it was
expected, rec-pediocin displayed 3.125 times more antimicrobial
activity than its native counterpart. This confirms the findings
of Tominaga and Hatakeyama (2007) that pediocins with
multiamino acid substitutions prove more useful in terms of
their final yields and bacteriocin activity against food spoilage
organisms. A PT5 based pQE32 has been previously used for
over-expression of pediocin F of P. acidilactici F in E. coli
(Osmanagaoglu et al., 2000). Similarly, Beaulieu et al. (2007)
cloned and expressed thioredoxin-pediocin PA-1 fusion protein
in E. coli. Moon et al. (2006) coexpressed pedA with His-tagged
DHFR using vector pQE40PED in E. coli M15. Recombinants
displayed very high pediocin activity upon overexpression
with IPTG and subsequently fusion protein was purified by
Ni-NTA metal affinity chromatography. Native pediocins were
recovered from their fusion partners by enzymatic digestion.

Figure 9: Reverse phase chromatograms of A) rec-pediocin fusion


protein and B) pure rec-pediocin

pediocin fusion protein (Figure 10). The difference may be


attributed to the N-terminal TAP fragment.
Discussion

Many reports suggested that the refolding buffer containing


5-15 mM -mercaptoethanol, and 1M glycine facilitates
renaturation of proteins, allows correct formation of disulfide
bonds and reduces protein aggregation during refolding
process (Rogl et al., 1998; de Bernardez Clark, 2001; Halami
and Chandrashekar, 2007; Tominaga and Hatakeyama, 2007).
In accordance with earlier reports, present strategy using
recombinant pET32(b)-pedA and E. coli BL21(DE3) allowed
copious accumulation of recombinant protein in IBs of
transformed cells and slow dilution of urea solubilized IBs in a
refolding buffer consisting of -ME along with glycine assisted
in proper folding of the rec-pediocin in its proper secondary
structure.

Figure 10: UV absorption spectra of rec-pediocin and its fusion


protein

E. coli BL21(DE3) was selected for achieving high level


production of rec-proteins as extensive workout has already
been done to study regulation of gene expression and to
achieve biological activity of rec-proteins (Makrides, 1996). In
addition, a variety of highly specific fusion partners are
available that facilitate detection of recombinant proteins by
affinity purification, immuno-fluorescence, immunoprecipitation, Western blotting (Kumar et al., 2011). In
presence of selection pressure, E. coli cells usually maintain a
very high copy number (approximately 40 /cell) of pET vector
and its derivatives that carry a strong T7 phage promoter whose

Spectroscopic analysis is a widely exploited method to study


refolding of rec-proteins. Previously, Patra et al. (2000) had
used this technique to study refolding of recombinant human
growth hormone. Similarly, Halami and Chandrashekar (2007)
compared UV absorption spectra of native and recombinant
pediocin PA-1 to study refolding of rec-protein. Their UV
absorption spectra were almost identical except for a shoulder
34

Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach

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coli outer membrane protease that cleaves T7 RNA
polymerase during purification. J. Bacteriol., 170: 1245-1253.
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affecting production of two bacteriocins from lactic acid
bacteria on whey. Int. J. Food Microbiol., 70: 267-281.
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purification and refolding of an anti-listerial peptide produced
by Pediococcus acidilactici K7. Electron J. Biotechnol., 10(4).
Doi:10.2225.
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immunity genes from Pediococcus acidilactici K7 using pAMJ
shuttle vector into Lactococcus lactis MG1363. Ind. J.
Biotechnol., 7(4): 550-553.
Horn, N., Martinez, M.I., Martinez, J.M., Hernandez, P.E., Gasson,
M.J., Rodrguez, J.M. and Dodd, H.M. 1998. Production of
pediocin PA-1 by Lactococcus lactis using the lactococcin A
secretory apparatus. Appl. Environ. Microbiol., 64: 818-823.
Horn, N., Martinez, M.I., Martinez, J.M., Hernandez, P.E., Gasson,
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production of pediocin PA-1 and co-production of nisin and
pediocin PA-1 by Lactococcus lactis. Appl. Environ.

at 220 nm in case of recombinant pediocin which was attributed


to the N-terminal extention of pre-peptide sequence and tag
fragment. Our results support these observations as
biophysical changes in rec-pediocin and its refolding can be
easily studied by a simple spectroscopic analysis.
Conclusion
Pediocins have attracted health conscious society because of
their remarkable stability, broader antimicrobial spectrum,
higher specificity and effectiveness in very low concentrations.
Pediocin CP2 has a great potential to contribute in food, health
and pharmaceutical industry. Large scale production of
pediocin CP2 using a fastidious strain of P. acidilacitici
MTCC5101 is highly uneconomical and its purification using
conventional methods are cumbersome and time consuming
too. In recent years, a great deal of diverse expression systems
were exploited for cloning, expression and purification of
pediocins at laboratory scale but data are lacking for industrial
pro-cesses. Hence, it necessitated for the development of low
cost, industrially viable and con-tinuous system in order to
exploit this natural bioactive compound in food and
pharmaceutical industry. A bioprocess is now in place where
rec-pediocin fusion protein can be expressed in large quantity
using a cheaper culture medium, recovered from IBs, solubilized
and refolded to its biologically active conformation and purified
easily by TAP purification described in the study. The
preliminary characterization that has been done with recpediocin has revealed its several desirable properties as in
vivo antimicrobial agent. What remains unexplored is to use
this knowledge for formulating novel therapeutics and study
their suitability as in vivo antimicrobial agents.
Acknowledgement
Authors acknowledge the UGC, New Delhi, India, for providing
financial assistance in the form of Rajiv Gandhi National
Fellowship to Mr. Balvir Kumar.
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36

Intl. J. of Food. Ferment. Technol. 2(1): 37-41, June, 2012

Research paper

Effect of growth conditions on extracellular -amylase


production by Bacillus thuringiensis
V.R. Tembhurkar1*, M.K. Bannatwala2, S.S. Udare3 and M.G. Kalyankar4

1*

Department of Microbiology, ASC College, Jalna, India


Department of Medical Microbiology, Greater Kailash Hospital, Indore, India
3
Department of Biotechnology, MGM College, Aurangabad, India
4
Deptepartment of Biotechnology, University of Houston, Clear Lake, USA
2

Email: vrtembhurkar@gmail.com.

Paper no: 35

Received: 19 December 2011

Received in revised form: 14 April 2012

Accepted: 17 May 2012

Abstract
Influence of few growth conditions and media components on alpha amylase production by Bacillus
thuringiensis in lab scale batch process were determined. B. thuringiensis produced maximum amylase
after 72hrs of incubation. The optimum temperature and pH for amylase production were found to be
31oC and 7, respectively. The optimized media composed of [g/l] 10.0g Rice flour; 5.0 Peptone; CaSO4
4.0; MgSO4 2.0; NaCl 1.0; FeSO4 0.5 and pH 7. The alpha amylase produced was working optimally at
60oC, pH 5, [S] 20 mg/mL, [E] 2.0 mL. Many of these parameters are in compliance with the insecticidal
crystal protein production by B. thuringiensis. Therefore, the process can be used for simultaneous
production of alpha amylase and bioinsecticide in a single fermentation cycle.
2012 New Delhi Publishers. All rights reserved

Keywords: Bacillus thuringiensis, Alpha amylase, Insecticide Crystal proteins,growth

Bacillus thuringiensis (Bt) is a popular microbial insecticide


extensively applied in agriculture and forestry for controlling
insect pests (Ninfa and Rosas-Garca, 2009). These insecticidal
proteins are products of cry genes whose expression peaks in
stationary phase of growth. These insecticidal proteins generally
accumulate in the mother cell compartment (intracellular) to form
a crystal inclusion (ICP) that can account for 20 to 30% of the
dry weight of the sporulated cells (Schnep et al., 1998). The ICP
of Bt has been reported to be active against certain insect
species among the orders Lepidoptera, Diptera, Coleoptera.
Hymenoptera, Homoptera, Orthoptera, Mallophaga and against
nematodes, mites, and protozoa (Feitelson et al., 1992; Beegle
and Yamamoto, 1992; Feitelson, 1993). Bt is already a useful
alternative or supplement to synthetic chemical pesticide
application in commercial agriculture, forest management, and
mosquito control (Schnep et al., 1998).

Although Bt is well known for bioinsecticide production it is


also a potential producer of alpha amylase. A few strains of Bt
do produce and secrete several isozymes of alpha amylases
(Maity et al., 2011). But alpha amylase production parameters
of Bt are largely unexplored thus, its use for large scale
production of alpha amylase is still obscure. Microbial amylases
have completely replaced chemical hydrolysis of starch in starch
processing industries (Vidyalakshmi et al., 2009). Both ICP
and alpha amylase are of immense commercial importance. The
previous one in intracellular and later is secreted out of the
cell, additionally their synthesis is independent. The objective
of this work is to optimize growth conditions and media
designing for maximum production of alpha amylase by Bt. If
the results match with ICP production parameters, this will
allow simultaneous production of alpha amylase and ICP in
single fermentation cycle. Such Simultaneous fermentation

Tembhurkar et al.

of two products will be a cost effective option for their large


scale production. The results obtained are discussed in this
communication.

determine effect of temperature, the reacting mixture was


incubated at different temperatures range 30 to 100 oC for 15min
followed by sugar liberated estimated by DNS method (Bernfeld,
1955). For pH optimization, buffers of different pH were used;
acetate buffer for pH 4 & 5; phosphate buffer for pH 6, 7 & 8;
glycine/NaOH buffer of pH 9. Effect of substrate concentration
was analyzed using 1 to 10% starch. 0.1 to 2.0 ml of enzyme
was used to study the effect of enzyme concentration. Optimum
reaction time was determined by measuring reducing sugar
produced after every 5min for 60min. Values of kinetic
constants were estimated by plotting Lineweaver-Burk double
reciprocal plot of 1/[S] Vs. 1/[V].

Materials and methods


Test Culture: Bacillus thuringiensis ATCC 10752 was used in
present studies and its amylase production ability was
confirmed by plating on starch agar composed of [g/l] 5.0g
Beef extract; 0.5 g NaCl; 1.0 g Starch, 2.0g Agar agar, pH 7.
After 48hr of incubation, starch hydrolysis was detected by
reacting with Lugals iodine (Harley and Prescott, 2002).
Enzyme assay: Alpha amylase activity was measured in terms
of hydrolysis product, the reducing sugars released by enzyme
action. The reducing sugars released were measured by
Bernfeld method that uses 3,5-di-nitrosalicylic acid as coupling
agent (Bernfeld, 1955). One enzyme unit (U/ml) of alpha
amylase is the amount of enzyme required to liberate 1mole
of product (Dey et al., 2003).

Result and discussion


Optimization of growth conditions: Alpha amylase production
by Bt reached to maximum level on third day of incubation after
which amylase concentration progressively decreased till fifth
day of incubation (Table 1). The amylase production was 10
fold higher at 31oC than at other temperatures tested (Table 2).
Deviation of initial media pH from neutral pH (7) negatively
affected alpha amylase production (Table 3). According to
findings of Yezza et al. (2005) entomotoxicity of Bt reached peak
at 72hrs of incubation . This observation is in agreement with
findings of Vidyalakshmi et al (Vidyalakshmi et al., 2009). Ozkan
et al. (2003). concluded in his study of toxin production by B.
thuringiensis HD500 that the highest concentrations of Cry4Ba
was produced at 25C, while the optimum temperature for
production of Cry11Aa was 30C (Ozkan et al., 2003). Khanh
Dang Vu et al.(2009) also reported a pH value of 7 was optimum
for bioinsecticide production. Abdel-Hameed et al., (1991) stated
that if the pH of a culture medium is not between the range 6.57.5, sporulation and -endotoxin formation could be adversely
affected (Khanh Dang Vu et al., 2009; Abdel-Hameed et al.,
1991).

Optimization of growth conditions: Alpha amylase production


with respect to incubation time, incubation temperature and
media pH was estimated in independent batch experiments.
The composition of fermentation medium used was [g/l] 5.0 g
Beef extract; 0.5 g NaCl; 1.0 g Starch, pH 7. The fementation
medium was inoculated with 1% inoculum. Inoculum was
prepared in one step of 48hr incubation in media of composition
similar to the method used for fermentation medium. In time
course experiment, amylase activity was assayed after every
24hrs till activity was dropped after constant rise. For
temperature optimization the fermentation medium flasks were
incubated at different temperatures viz. 28C, 31C, 34C and
37C, 41C. Similarly, for pH optimization, media pH was
adjusted in the range of 4 to 9 using 0.1N NaOH/HCl.
Media formulation: Effect of carbon source, nitrogen source
and micronutrient was determined. For carbon source
optimization media composition was [g/L] 10.0g Carbon source;
5.0 Beef extract; pH 7. Carbon sources used were rice flour,
wheat flour, corn flour, potato and pure starch. Following this,
nitrogen source was optimized in media composed of [g/L]
10.0g Optimum carbon source; 5.0 Nitrogen source; pH 7. Yeast
extract, tryptone, beef extract, peptone, urea, ammonium nitrate
and ammonium sulphate were tested as nitrogen sources.
Lastly effect of micronutrients (Na+, Mg2+, Ca2+, Fe2+) at various
concentrations (0.05, 0.1, 0.2, 0.4%) on amylase production
was analyzed. Production media used contained [g/L] 10.0g
Optimum carbon source; 5.0 Optimum nitrogen source; Salt to
be tested (NaCl/MgSO4/CaSO4/FeSO4) and pH 7.

Table 1: Time course of amylase production.


Incubation Time (hr)

Amylase activity (U/mL)

24
48
72
96
120

22.77
15.92
112.14
88.96
16.66

Table 2: Effect of incubation temperature on amylase production.


Incubation Temperature (oC)
28
31
34
37
41

Enzyme characterization: The optimum temperature, pH,


substrate concentration, enzyme concentration, reaction time
and kinetic constants (Km and V max) were determined. To
38

Amylase activity (U/mL)


7.40
122.2
11.11
7.40
7.40

Effect of growth conditions on extracellular -amylase production by Bacillus thuringiensis

Table 3: Effect of initial media pH on amylase production.


pH

Amylase activity (U/mL)

4
5
6
7
8
9

7.40
11.11
14.81
122.2
14.81
14.81

Media formulation: Four carbon sources were tested as


substitute for pure starch. The values presented in table 4
show that all the four carban sources enhanced amylase
production than pure starch. It has been reported that corn
starch was better than pure starch for stimulation of amylase
production (Ajayi et al., 2006). Amylase production was better
when peptone was used as nitrogen source compared to other
organic nitrogen sources (Table 5). In case of inorganic
nitrogen sources, nitrate was better than ammonium sulphate
for stimulation of amylase production. The support for this
observation is the report of Dharani Aiyer (Dharani Aiyer, 2004).
But we found that in all the nitrogen sources tested urea was
found to be the best. In the presence of urea, amylase yield
was almost 2.34 times when nitrate was used as a source of
nitrogen and 1.45 when peptone was used as nitrogen source.
Earlier starch medium had been used successfully for
production ICP by Bt and their finding was that starch based
medium give higher yield of endotoxin compared to yield
obtained in glucose based defined medium (Ming Chang et
al., 2008). Effect of metal ions on amylase production by Bt
was studied. Four concentrations of metal salts were used.
The results (Table 6) show that Na+ stimulated amylase
production when 0.1% NaCl was added in fermentation medium.
MgSO4 was required at 0.2% concentration. Increasing
concentration of CaSO4 linearly increased amylase production.
FeSO4 at 0.05% concentration was acting as stimulant but
higher concentration was toxic to amylase production. It has
been reported that Ca++ and Mg++ stimulate amylase activity
whereas Fe++ was inhibitor of amylase production (Elif Sarikaya
et al., 2000). Sikdar et al. (1991) identified Fe++ one of the
requirements for the production of ICP. It was proved that
Mg++ was essential for the synthesis of the ICP as the level of
crystal protein synthesis was almost zero when Mg was
omitted from the medium (Ozkan et al., 2003). But Ca++ had no
effect on toxin production. Whereas their studies with B.
thuringiensis var. israelensis HD500 showed that Fe ++
negatively influenced the synthesis of the ICP and Mg and Ca
favoured the toxin production. It has also been reported that
Mg, Ca, Fe and Na were required for toxin production by Bt
(Fernando Hercos Valicente et al., 2010).

Table 4: Optimization of carbon source.


Carbon Source

Amylase activity (U/mL)

Starch (Pure)
Rice flour
Wheat Flour
Corn Flour
Potato

90.72
104.79
102.67
103.12
101.61

Table 5: Optimization of nitrogen source.


Nitrogen Source

Amylase activity (U/mL)

Yeast Extract
Tryptone
Beef extract
Peptone
Urea
Ammonium Nitrate
Ammonium Sulfate

66.66
96.29
77.77
107.40
155.55
66.66
48.14

Table 6: Effect of metal ions on amylase production.


Metal Ion
Na+

Mg++

Ca++

Fe++

Concentration (%)

Amylase activity (%)*

0.05
0.1
0.2
0.4
0.05
0.1
0.2
0.4
0.05
0.1
0.2
0.4
0.05
0.1

92
116
72
48
80
76
100
48
72
140
124
176
116
68

Enzyme characterization: Effect of various parameters on alpha


amylase activity was studied. The enzyme activity was
maximum at 60oC (13.17 U/mL) after which activity of alpha
amylase was reduced (Figure 1). At 90oC the amylase activity
was very poor (1.03 U/mL). The optimum pH was 5 (Figure 2),
at this pH amylase activity was 17.97 U/mL. Enzyme activity
was the highest when 20 mg/ml substrate concentration was
used (Figure 3) and with increase in enzyme concentration
amylase activity also increased (Figure 4). Effect of reaction
time (Figure 5) showed that amylase activity reached a peak at

* Note: In table 6 percent amylase activity in presence of metal salts


with respect to amylase activity in absence of metal salts is presented.
The activity higher than 100% indicates stimulation of amylase
activity. And values less than 100% indicate suppression of amylase
activity.

39

Tembhurkar et al.

Figure 1: Effect of temperature on amylase activity

Figure 2: Effect of pH on amylase activity

Figure 3: Effect of substrate concentration on amylase activity

Figure 4: Effect of enzyme concentration on amylase activity

Figure 5: Effect of reaction time on amylase activity.

Figure 6: Lineweaver Burk plot for determination of Vmax and Km

40

Effect of growth conditions on extracellular -amylase production by Bacillus thuringiensis

25th min. (48.14 U/mL) after that activity reduced slightly and
remained stable up to 60min. Lee et al., characterized alpha
amylase from 19 serotypes of B. thuringiensis (Lee et al., 1988).
They found amylase worked optimally in temperature range 55
to 60 oC and optimum pH range was 6.7 to 7.2. Kinetic constants
for Bt alpha amylase were determined by plotting Lineweaver
Burk double reciprocal plot (Figure 6). The values obtained for
Vmax and Km were 666.67 U/ml and 3.49 mg/mL respectively.
These values are fairly high than reported by Elif Demirkan
(2011). It was found Km and Vmax of B. subtilis amylase were
1.08 mg/ml and 100 U/ml, respectively. After random
mutagenesis he could isolate a strain that produced amylase
with higher Km (1.43 mg/ml) and Vmax (151 U/ml).

Biotechnol. 3(10): 519-522.


Elif Demirkan. 2011. Production, purification and characterization
of -amylase by Bacillus subtilis and its mutant derivates.
Turk. J. Biol. 35: 705-712.
Elif Sarkaya. and Velittin Gurgun. 2000. Increase of the a-amylase
yield by some Bacillus strains. Turk. J. Biol. 24: 299308.
Feitelson, J.S. 1993. The Bacillus thuringiensis family tree in
Advanced engineered pesticides. New York, USA, pp 63-71.
Feitelson, J.S, Payne, J. and Kim, L. 1992. Bacillus thuringiensis:
insects and beyond. Biotechnology. 10: 271275.
Harley, J.P.; Prescott, L.M. 2002. Carbohydrates III: Starch
Hydrolysis in Laboratory exercises in microbiology. McGraw
Hill, pp 139-140.
Khanh Dang Vu; Tyagi, R.D.,Valero, J.R. and Surampalli, R.Y. 2009.
Impact of different pH control agents on biopesticidal activity
of Bacillus thuringiensis during the fermentation of starch
industry wastewater. Bioprocess. Biosyst. Eng. 32: 511519.
Kuppusamy, M. and Balaraman, K. 1990. Extra cellular hydrolytic
enzyme secretion in Bacillus thuringiensis H14 & B.
sphaericus & their significance in media design. Indian. J.
Med. Res. 91: 149-50
Lee, K.J.; Park, D.W.; Lee, H.H. and Lee, Y.J. 1988. Examination of
production of alpha amylase by Bacillus thuringiensis, 19
serotypes. Kor. J. Appl. Microbiol. Bioeng. 16(5): 348-351.
Maity, C.; Samanta, S.; Halder, S.K.; Das, P.K.; Pati, B.R.; Jana, M.
and Mondal, K.C. 2011. Isozymes of -amylases from
Newly Isolated Bacillus thuringiensis CKB19: Production
from Immobilized cells. Biotechnol. Bioproc. E. 16: 312-319.
Ming Chang; Shun-Gui Zhou; Na Lu and Jin-Ren Ni 2008. Starch
processing wastewater as a new medium for production of
Bacillus thuringiensis. World. J. Microbiol. Biotechnol. 24:
441447.
Ninfa, M. and Rosas-Garca 2009. Biopesticide Production from
Bacillus thuringiensis: An Environmentally Friendly
Alternative. Recent. Pat. Biotechnol. 3: 28-36.
Ozkan, M.; Dilek, F.B.; Yetis, U. and Ozcengiz, G. 2003. Nutritional
and cultural parameters influencing antidipteran deltaendotoxin production. Res. Microbiol. 154(1), 49-53.
Schnep, E.; Crickmore, N., Van Rie J., Lereclus, D., Baum, J.,
Feitelson, J., Zeigler, D.R. and Dean, D.H. 1998. Bacillus
thuringiensis and its pesticidal crystal proteins. Microbiol.
Mol. Biol. Rev. 62(3): 775806.
Sikdar, D.P., Majumdar, M.K. and Majumdar, S.K. 1991. Effect of
Minerals on the production of the delta-endotoxin by Bacillus
thuringiensis subps. israelensis. Biotechnol. Lett. 13:511-514.
Vidyalakshmi, R., Paranthaman, R. and Indhumathi, J. 2009. Amylase
Production on Submerged Fermentation by Bacillus spp.
World. J. Chem. 4(1): 89-91.
Valicente, F.H., Edmar de Souza Tuelher; Santos Leite, M.I.; Freire,
F.L. and Viera, C.M. 2010. Production of Bacillus thuringiensis
biopesticide using commercial lab medium and agricultural
by- products as nutrient sources. Braz. J. Maize. Sorg. 9(1):
1-11.
Yezza, A., Tyagi, R.D., Vale, J.R. and Surampalli, R.Y. 2005.
Production of Bacillus thuringiensis based biopesticides in
batch and fed batch cultures using wastewater sludge as a
raw material. J. Chem. Technol. Biotec. 80: 502510.

Conclusion
Bacillus thuringiensis ATCC 10752 is potential producer of alpha
amylase. Studies of lab scale batch process indicate optimum
fermentation time was 72hrs and optimum temperature was 31oC.
The optimized media composed of [g/l] 10.0g Rice flour; 5.0 Peptone;
CaSO4 4.0; MgSO4 2.0; NaCl 1.0; FeSO4 0.5; pH 7. The alpha amylase
produced was partially characterized. The enzyme worked efficiently
at 60 oC, pH 5. The alpha amylase activity was maximum when
substrate concentration was kept 20 mg/mL. With increase in enzyme
concentration upto 2.0mL the enzyme activity linearly increased.
The optimum reaction time was 25 min. The values of Vmax and Km
were 666.67 U/ml and 3.49 mg/mL respectively. Most of the optimum
production parameters matched with the production parameters of
ICP by B. thuringiensis previously reported by other workers. Thus
B. thuringiensis can be used for production of alpha amylase and
ICP simultaneously in single fermentation cycle.

Acknowledgement
We express our gratitude to MGMs Institute of Biosciences and
Technology, Aurangabad for providing all the necessary financial and
technical support.

References
Abdul-Hameed, A., Carlberg, G. and El-Tayeb, O.M. 1991. Studies
on the Bacillus thuringiensis H-14 strains isolated in EgyptIV Characterization of fermentation conditions for endotoxin
prodtucion. World. J. Microbiol. Biotechnology. 7: 231-236.
Ajayi, A.O. and Fagade, O.E. 2006. Growth pattern and structural
nature of amylases produced by some Bacillus species in
starchy substrates. Afr. J. Biotechnol. 5(5): 440-444.
Beegle, C.C. and Yamamoto, T. 1992. History of Bacillus
thuringiensis Berliner research and development. Can.
Entomol. 124:587616.
Bernfeld, P. 1955. Amylases, alpha and beta in: Methods in
Enzymology. Academic Press, USA, pp 49-158.
Dey, G.; Singh, B. and Banerjee, R. 2003. Immobilization of -amylase
produced by Bacillus circulans GRS 313. Braz. Arch. Biol.
Technol. 46(2): 167-176.
Dharani Aiyer, P.V. 2004. Effect of C:N ratio on alpha amylase
production by Bacillus licheniformis SPT 27. Afr. J.
41

Intl. J. of Food. Ferment. Technol. 2(1): 43-48, June, 2012

Research paper

Design of cascade membrane filtration process for


clarification of whey proteins and membrane fouling
Pranav Kaushik Pidatala* and Senthil R. Kumar

Downstream Processing Lab, Department of Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, India
*Email: pranavkaushik.p@gmail.com
Paper no: 36

Received: 29 April ,2012

Received in revised form: 17 April, 2012

Accepted: 14 May, 2012

Abstract
Whey protein isolates are processed by membrane separation techniques - Micro-filtration, Nanofiltration and Ultra-filtration. Proteins, Glyco-macropeptide, Lacto-albumin, Lacto-globulin and Bovine
Serum Albumin are collected by employment of 10, 30, 50 and 100 kilo-Daltons (kDa) ultra-filtration
membranes, respectively based mainly on protein molecular weights. Pressure drop, trans-membrane
pressure, flux and feed volume were optimized for lab scale. Volume and concentration values for all
streams were calculated for micro-filtration, nano-filtration and 4 ultra-filtration membranes and
satisfactory yield values were obtained. Through scale-up criteria, a cascade design has been proposed
for pilot scale whose pressure values fall within the range of standard pressure values. Fouling
studies were carried out in lab scale by calculating fouling and membrane resistances for all membranes
and effect of anti-fouling chemical agents SDS and NaOH on the membrane cleaning has been
analyzed. The study has given on overall idea about the design corresponding to 6 membranes
arranged in series to clarify 4 proteins from milk.
2012 New Delhi Publishers. All rights reserved

Keywords: Whey Proteins, Membrane Filtration, Flux, Scale-up, Membrane Fouling

Whey is liquid portion of the milk left out in cheese industry


after milk is treated with rennet. In cheese manufacturing
industry, it is a by-product, released as dairy waste water into
the surrounding environment. The dischangecreates
ecological imbalance by altering the biochemical oxygen
demand (BOD) value of nearby water bodies which in turn
affecting the aquatic life. By processing the whey at industry
level, pure proteins are obtained and served as food
supplements and biological oxygen demand value can also be
controlled. At industrial level, the problem of protein
clarification is addressed with the purification process taking
place under the action of certain buffering systems that
maintain the particular pH conditions that correspond to the
iso-electric points of the desired proteins. This does ensures
separation of the protein but the recovery of the proteins at
industrial scale from the buffer systems becomes difficult

because separation of proteins is done by precipitation


methods does buffers. The proteins get precipitated, but
require chemicals to bring back to their native states. At
industrial level, it is very expensive to use the chemicals for
purifying the proteins. Beside, a few proteins are sensitive to
precipitation methods. So, a need arises to design a much
simpler method of purification.
Microfiltration 1kDa Nano-filtration 10kDa Ultra-filtration

30kDa Ultra-filtration 50kDa Ultra-filtration 100kDa


Ultra-filtration
The above mentioned series is a cascade mode filtration
process where, at each junction, each of the 4 desired proteins
are isolated. Particles are separated on the basis of their
molecular size with the use of pressure and specially designed
semi-permeable membranes. This process at the industry level

Pidatala and Kumar

involves handling of bulk volumes of fluid and requires a large


number of filtration procedures. The current process attempts
to come up with an innovative cascade mode by implementing
only mechanical methods.
From Figure 1, Composition of Milk Solids: 1 ml = 1.03 gm. So,
in 1 liter, solids = (13% of 1litre * 1.03) = 133.9gm. Proteins: 27
*133.9 = 36.15gm. Whey proteins: 0.198*36.15 = 7.158 gm.

Figure 2: Pump connected to membrane cartridge ends and pressure


transducers for protein separation

Figure 1: Composition of Milk

Materials and Methods


The whole methodology involved the clarification of whey
proteins into separates fractions and then, subsequent design
studies of the membranes involved, in cascade mode. Buffalo
milk was boiled. Then fat, casein calcium and phosphorous
were removed. The left-out product was passed through 6
membrane filters arranged in series to clarify proteins from
other impurities.
A. Pretreatment of Whey
One liter of buffalo milk was boiled for 15 minutes at 50 0C and
centrifuged for 6000rpm for 12 minutes to remove fat from the
top layer. RPM Centrifuge was be maintained below 8000rpm
by the shearing force to ovoid alternation of the native protein
conformation centrifuge will not alter the native protein
confirmations. Then HCl was added to precipitate casein.
Figure 3: Cascade Membrane Filtration Process

Minerals Calcium and Phosphorous were removed by


precipitation in the form of salt i.e., Calcium Phosphate. The
whey was sent into the membrane filters for clarification.

Cascade Mode: MF
NF 1kDa
UF 10kDa UF 30kDa
UF 50kDa UF 100kDa

B. Membrane Filtration Process in Cascade Mode (Figure 2)

The proteins were confirmed qualitatively and quantitatively;


and yield values are calculated.

Each of the samples, after the respective filtration, was subjected


to subsequent filtration process. This sequential process is
termed as cascade membrane filtration as shown in Figure 3.

Scale up Criteria: Scale up is linear as per the real time industry


level pilot plants.

The methodology applied in the Figure 3, is depicted as


sequential representation shown below:

Ratio of Feed Volume to Membrane Area and Permeate Flux


values for pilot and lab scales are constant and Pilot scale

44

Design of cascade membrane filtration process for clarification of whey proteins and membrane fouling

Pressure Drop = 3 * (Lab scale Pressure Drop). These were


scaled up from lab scale results to pilot plant scale.

The results obtained and summarized in Table 6.

C. Fouling of Membranes

Optimization of parameters gives an insight on deciding the


optimum conditions for effective and efficient purification of
the proteins. Rotational speed of the pump was varied and
different time values were noted. Lower speed values reduced
protein yield and higher speed values denatured the proteins.
So for each membrane separation process, the average speed
value was taken and flux values were calculated. These flux
values were found to be within the standard flux range values.
Pressure transducers were fixed to inlet and outlet ends of
membrane cartridge and pressures at these points are noted
down. As permeate is open to atmosphere, permeate pressure
is taken as atmospheric pressure. For each membrane, pressure
and flux values were optimized.

B. Optimization

Due to continuous filtration and prolonged usage of


membranes, the pores are blocked and thick layer of feed
particles get deposited on the inner surface of membrane. Thus
decreasing the efficiency of the membrane decreases and
evaluationaly the recovery of permeates. As the protein
concentration in the feed solution increases, the maximum
achievable flux decreases. Fouling effect should be controlled
as it decreases the efficiency of protein purification on industrial
scale. The flux equation for diffusion of solvent through the
membrane is
Flux = P * (TMP) where, Permeability, P is reciprocal of product
of viscosity and membrane resistance. For proteins with
concentration less than 1 % (grams of whey proteins per 100
ml of milk), above equation is valid. The fouling resistance can
be calculated by:
Flux= Tans-membrane Pressure/ (Permeate Viscosity*(Fouling
resistance + Membrane Resistance)

1.

TMP = 0.5 * (Pin + Pout) P permeate

2.

Pressure Drop = (Pin Pout)

3.

Flux = Volume of permeate collected / (Membrane area *


Collection Time)

Permeate Yield = Permeate Recovery * (Concentration of


permeate/Concentration of feed)

Hence TMP, permeate viscosity and flux values are to be known


for calculating the resistance.

Only for NF 1kDa, instead of permeate; retentate values are


used as the proteins exist in retentate. Permeate Yield is the
design parameter which measures the extent of protein purified.

Results and discussion


A. Pre-treatment of whey
Table 1: Volume statistics for Pre-treatment of whey
Sl. no
1.
2.
3.
4.
5.
6.
7.

Volumes used

Amount (ml)

Initial Raw milk


Defatted milk
2N HCl for precipitation of casein
After casein removal from whey
Calcium chloride of 1.2 g/lt
NaOH of 6N
Final Volume of the Purified Whey

Comments

1,000
738
22
563
32
25
608

From Murrah breed buffalo


Separated 262ml fat
Total 760 ml product
Separated 197 ml casein
Total 595 ml product
Total 620 ml product
Ca3 (PO4)2 12ml removed

Table 2: Flux, TMP and Pressure Drop values for whey


Sl no
Type
mode) in bar

Whey (Cascade)
Time(sec)

Pressure (Whey - cascade

Flux(lt/sq.mt-hr)

1
MF
166
67.77
2
NF 1 kDa
1328
8.47
3
UF 10 kDa
744
18.25
4
UF 30 kDa
667
18.78
5
UF 50 kDa
621
18.12
6
UF 100 kDa
133
52.05
C. Design of Cascade Membrane Separation Process
45

Pin

Pout

TMP

Pressure Drop

2.347
2.443
2.312
2.320
2.250
2.250

1.297
1.313
1.122
1.130
1.020
0.980

0.822
0.878
0.782
0.725
0.635
0.615

1.05
1.13
1.06
1.19
1.23
1.27

Pidatala and Kumar

D. Proposing values for pilot plant using Scale-up criteria:


MF Membrane
1.

lab= 1.05 bar; so pilot = 3.15 bar lab should be (1 to


3.5 bars) & pilot should be (1 to 7 bars). So pressure
drop is satisfied.

(Volume of feed/ Area) for pilot scale => (Volume of feed


/ Area) for lab scale = 608/16 sq.cm

Inference from Tabular Values

= 380lt/sq.mt. As volume of feed for pilot scale is 500 lt


(Basis); Area => (500/380) = 1.315sq.mt
2.

Initially, pure water was sent into the membranes to note the
flux values. Then whey was sent into the membranes to check
flux values. Again, water was sent and flux values were noted.
Water flux values were decreased when both experiments were
compared. Due to the continuous purification process, fouling
phenomena occurred. Hence, the flux value decreased. The
fouling phenomena were measured in terms of fouling and
membrane resistance values. Membrane cleaning was
performed to observe alteration in flux values and reduction of

(Volumetric Flow Rate/ Area) of permeate for pilot scale


= 67.78 ml/sq.mt-sec
So volumetric flow rate, Q per => 67.78*1.315 = 89.18lt/
hr

3.

Pilot scale Pressure Drop =3*(Lab scale Pressure Drop).

Table 3: Volume & Concentration values for feed, retentate & permeate of all 6 membranes
Type

MF-F

MF-R

MF-P / NF-F

NF-P

NF-R /UF 10-F

UF 10-P

UF 10-R /UF 30-F

204
1.25

198.8
1.50

Volume (ml)
608
103.9
504.1
101.3
402.8
Concentration(g/l)
0.75
0.50
0.75
0.75
0.75
MF- Microfiltration, NF- Nanofiltration, UF- Ultrafiltration, F- Feed, P- Permeate, R- Retentate
Type
Volume (ml)
Concentration (g/l)

UF 30-P

UF 30R/UF 50-F

UF 50-P

UF 50R/UF 100-F

UF 100-P

UF 100-R

121
1.625

77.8
2.75

47.2
3.00

30.6
0.75

26.7
0.50

3.9
0.25

Basis for pilot plant scale was taken as 500 liters as for linear scale up, there should be an initial basis
value as per the real time industry requirement.
Table 4: Permeate Recovery and Yield values
Membrane

Flu
x(lt/sq.mt-hr)

TMP
(bar)

Pressure
Drop(bar)

Permeate
Recovery

Yield
Permeate

Lab Scale
Volume-ml

Pilot Scale
Volume-lt

MF(0.1um)
NF 1kDa
UF 10kDa
UF 30kDa
UF 50kDa
UF 100kDa

67.77
8.47
18.25
18.78
18.12
52.05

0.822
0.878
0.782
0.725
0.635
0.615

1.05
1.13
1.06
1.19
1.23
1.27

79.76
79.91
50.746
60.865
60.668
87.25

79.76
79.91
84.57
65.73
73.054
58.166

608
504.1
402.8
198.8
77.8
30.6

500
414.550
331.250
163.486
63.980
25.164

Permeate Recovery = (Volume of permeate / Volume of feed) * 100


Table 5: Proposed Pilot Scale Values
Sl no
1.
2.
3.

Parameters

MF

NF

UF 10

UF 30

UF 50

UF100

Pilot plant area (mt^2)


Volumetric flow rate(lt/hr)
Pressure drop, (bar)

1.32
89.18
3.15

1.32
11.14
3.39

1.32
24.01
3.18

1.32
24.7
3.57

1.32
23.84
3.69

2.14
68.48
3.8

Based on the equations, the pilot plant values were calculated.


E. Fouling of membranes

46

Design of cascade membrane filtration process for clarification of whey proteins and membrane fouling

Table 6: Membrane Fouling studies Flux and resistance values for whey and water
a) Before Fouling with i).Pure water and ii).Whey
Sl. no

Pure water

MF

NF

UF 10

UF 30

UF 50

UF 100

Time for 5 ml permeate rise(sec)


Flux (lt/sq.mt-hr)

40
281.25

1121
10.04

618
18.2

596
18.88

468
24.04

101
68.54

679
16.57

668
16.84

623
18.06

141
49.09

After whey filtration is done for about 3 hours, the membranes were tested for fouling phenomena.
b) After Fouling with i).Pure water and ii).Whey
1.
Time for 5 ml permeate rise(sec)
51
1206
635
2.
Flux (lt/sq.mt-hr)
220.59
9.33
17.72

743
15.14

529
21.27

138
50.16

Again membranes were treated with whey to calculate the resistances.


1.
Time for 5 ml permeate rise(sec)
172
2.
Flux (lt/sq.mt-hr)
65.41

692
16.26

648
17.36

189
36.63

1.
2.

After cleaning with water for 20 minutes, whey is sent into the filtration cartridges.
1.
Time for 5 ml permeate rise(sec)
158
1305
2.
Flux (lt/sq.mt-hr)
71.2
8.62

1425
7.89

685
16.43

the flux values near to the initial values indicating that the
membranes were washed properly and pores were cleared.
These flux values are inversely proportional to total resistance
of the membranes. So, membrane resistance and fouling
resistance for each membrane were found out as other
parameters were known on lab scale. Using the correlations,
the pilot plant fouling resistance for each membrane was found
out. Usually pilot plant scale operations either go for WaterBack Washing or Air Flushing for the reduction of fouling as
use of reagents is very expensive and these reagents cannot
be reusable on pilot scale.

fouling. Due to continuous filtration of NaOH and SDS reagents,


the membrane pores were unblocked and fouling was reduced
to minimum.
By finding out flux and TMP values, the membrane resistance
and fouling resistance values for all 6 membranes were
calculated. In order to prevent fouling, chemical agents like
NaOH, SDS are circulated through membranes to clear the pores.
As per the cleaning process, NaOH is circulated for 2 hours
and then pure water flux is calculated.
Fouling of all membranes was studied in lab scale by estimating
pure water flux, whey flux in cascade mode, fouling water flux
(after protein separation). The decrement in the water flux had
shown that fouling took place. Cleaning of membranes restores

Conclusion
Whey proteins have a high nutritional value. They are

c) Membrane resistance and fouling resistance values in sq.mt/lt units


Sl no
1.
2.
3.

Whey

MF

NF

UF 10

UF 30

UF 50

UF 100

Permeate viscosity(milli. Pa-sec)


Membrane Resistance(* 10^6)
Fouling Resistance

1.01095
11.44768
10,09,416

1.01095
10.109716
8,72,687.9

1.01825
4.64071
2,75,888

1.02373
4.20543
1,31,987

1.0438
3.38443
1,14,394

1.0073
1.24776
4,29,859

NF

UF 10

UF 30

UF 50

UF 100

672
16.74

502
22.41

124
55.83

601
18.72

483
23.29

112
61.81

Table 7: Cleaning of membranes


Sl no
1.
2.
1.
2.

Water

MF

Time for 5 ml permeate rise (sec)


46
1262
628
Flux (It/sq.mt-hr)
244.56
8.91
17.1
Then SDS was circulated for 2 hours and again pure water flux was calculated.
Time for 5 ml permeate rise (sec)
42
1191
621
Flux (lt/sq.mt-hr)
267.86
9.45
18.12

47

Pidatala and Kumar

Hinrichs, J. 2004. Fractionation of whey proteins by ultra-high


pressure, Bull. Int, Dairy Federal. 389.24.
Jonsson, A.S. Tragardh, (1990). Ultrafiltration applications Desalination, 77-78:135179.
Kirk, D.E., Montgomery, M.W. and Kortekaas, M.G., 1983.
Clarification of pear juice by hollow fiber ultrafiltration. J.
Food Sci., 48:1663-1666.
M. Carmen Alm ecija, Rub en Ib a nez, Antonio Guadix, Emilia M.
Guadix. Effect of pH on fractionation of whey proteins with
a ceramic ultrafiltration membrane.
Marshal, K.R. 1982. Industrial Isolation of Milk Proteins, Whey
Protein Development in Dairy Chemistry. Applied Science
Publisher, London.
Marshall, A.D., Munro, P.A. and Tragardh, G. 1993. The effect of
protein fouling in microfiltration and ultrafiltration on
permeate flux, protein retention and selectivity: a literature
review, Desalination, 91: 65108.
Maubois, J. L. and Ollivier, G. 1997. Extraction of Milk Proteins.
Food Proteins and Their Applications. New York Publishers,
p 579.
Mehra, R. and Kelly, P.M. 2004.Whey protein fractionation using
cascade membrane filtration. International Dairy Federation
Bulletin: Advances in Fractionation and Separation:
Processes for Novel Dairy Applications, 389:40-44.
O. Wallberg, 2003. Fractionation and concentration of whey proteins
by ultrafiltration. J. Chem Engg.
Pongsathon Limsawat, Suwattana Pruksasri. 2010. Separation of
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Quinones H.J., and Phillips, 2007. Influence of Protein
Standardization by Ultra-filtration on the viscosity, color
and sensory properties of skim milk. J. Dairy Sci., 80:31423151.
Scopes R.K., 1993. Protein Purification, Principles & Practice.
SpringerVerlag, NY.
Van R. Reis, and A. Zydney, 2007. Bio-process membrane
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Van Reis, R., Gadam, S., Frautschy, L.N., Orlando, S., Goodrich,
E.M., Saksena, S., Zydney, A.L. 1997. High Performance
Tangential Flow Filtration, J. Bioengg. 56:71-82.
Zeman, L.J. and Zydney, A.L. 1996. Microfiltration and
Ultrafiltration: Principles and Application, Marcel Dekker,
New York Publications.
Zydney, A.L. 1998. Protein separations using membrane filtration:
new opportunities for whey fractionation, Int. Dairy J. 8
243.

biologically essential molecules which provide instant energy


when consumed and hence, a major commercial interest at the
industrial level. Preparation of low fat milk, low sodium milk
and low potassium milk using ultra filter and nano filter
membranes, recovery of whey proteins in cheese production
by membrane separation are few applications of the present
work. The milk protein constituents of whey are clarified into
different fractions and their respective percentage yields have
been estimated by quantitative studies - concentration values
and permeate yields. The purification process is cheaper,
economical and produces efficient results. Design of a
simplified cascade membrane filtration process has been
studied and the results have been proposed for a pilot scale
plant. Subsequent fouling studies were conducted to calculate
the membrane and fouling resistances. Cleaning procedures
were carried out to find out the flux changes and efficiency of
membranes. Over all, the work gives a clear idea about lab
scale cascade membrane filtration process of hollow fiber
module in clarifying of milk whey proteins.
References
Alomirah, H.F., Alli. I. 2004. Separation and characterization of
lactoglobulin and lactalbumin from whey and whey protein
preparations, Int. Dairy J. 14 411.
Bottomley, R.C., 1989. Process for obtaining concentrates having
high -lactalbumin content from whey Eur. Patent Appl. EP
311 283 A2.
Carol Ann Patterson., 2005. Membrane Processing: State of The Art
Technology, The Pathfinders Research Limited, National
Research Council, Canada.
Cheryan, M 1986. Ultrafiltration Handbook, Technomic
Publication, Lancaster.
Cowan, S. and S. Ritchie 2007. PES membrane for whey protein
separation. Separation Science and Technology, 42:2405-2418.
Esther W.Y.Li, Yoshinori Mine, Comparision of chromatographic
Profile of Glyco-macropeptide from cheese whey isolated
using different methods, J. Food Science.
Field, R.W., Wu, D., Howell, J.A.. Gupta, B.B. 1995. Critical flux
concept for microfiltration fouling. Journal of Membrane
Science 100 259272.
Helicon. 2003. Protein Concentration and Dia-filtration by Tangential
Flow Filtration. In issue special of Millipore Corporation,
Billerica.

48

Intl. J. of Food. Ferment. Technol. 2(1): 49-56, June, 2012

Research paper

Microbiological and biochemical characterization of


Seera: A traditional fermented
food of Himachal Pradesh
Savitri*1, N. Thakur1, D. Kumar2 and T.C. Bhalla1

Department of Biotechnology, Himachal Pradesh University, Summer hill, Shimla, Himachal Pradesh, India
Shoolini University of Biotechnology and Management Sciences, Bhajol, Solan, Himachal Pradesh, India

*Email: savvy2000in@yahoo.com
Paper no: 37

Received: 26 April, 2012

Received in revised form: 17 May, 2012

Accepted: 19 June, 2012

Abstract
Seera is a nutritious, easily digestible traditional fermented food made from wheat grains in Bilaspur,
Kangra, Hamirpur, Mandi and Kullu districts of Himachal Pradesh, India. Samples during seera
fermentation were analysed for various microbiological and biochemical parameters. The microflora
isolated from seera mainly comprised of Saccharomyces cerevisiae, Cryptococcus laurentii and
Torulospora delbrueckii among yeasts and Lactobacillus amylovorus, Cellulomonas sp.,
Staphylococcus sciuri, Weisella cibaria, Bacillus sp., Leuconostoc sp. and Enterobacter sakazakii
among bacteria. Protein content decreased from 14.9% on day 1 to 8.2% on 5th day of fermentation.
However, final product obtained had higher protein content i.e. 10.4%. Total sugars decreased with
the time of fermentation.Seera had 11.9 mg reducing sugars/g dry matter. Starch content decreased
initially from 72.1 to 57.0 % (w/w) on dry weight basis from first to fourth day. After steeping and
drying of seera, the starch content increased to 87.4% (w/w) on dry weight basis. Amylase and
protease activity increased with the fermentation up to 4th day, then it started decreasing and very low
amylase activity was recorded in the final product. A significant increase in thiamin, riboflavin, nicotinic
acid and cyanocobalamin was observed during fermentation of seera. The level of essential amino
acids especially methionine, phenylalanine, threonine, lysine and leucine also increased during seera
fermentation.
2012 New Delhi Publishers. All rights reserved

Keywords: Fermented foods, Seera, Himachal Pradesh, Traditional.

Indigenous fermented foods such as bread, cheese and wine


have been prepared and consumed since antiquity and some
of these products have become an integral part of culture and
tradition (Battcock and Azam-Ali, 1998). The high nutritive
value, sensory characteristics and easy digestibility made the
fermented foods an integral part of diet of people almost every
part of the world (Sankaran, 1998). Fermented foods generally
preserve pleasant flavor, aroma, texture, enhanced nutritive
values and good keeping quality under ambient conditions

(Law et al., 2011). Fermented cereals and pulses along with


millets have been the staple food of people of Asian and African
sub-continent since the time immemorial (Narashimahan and
Rajalakshmi, 1999) and the cereal foods are the most dominant
group of fermented foods consumed in India. Diversity of
fermented foods in Asia is directly related to food culture of
each and every community, and also availability of raw materials
(Tamang, 2011).

Savitri et al.

addition of any inoculum. This was kept at 250C for natural


fermentation to take place. On fourth day water was decanted
and the grains were washed with tap water. The softened wheat
grains were gently mashed and fermented overnight. Wheat
bran was removed by filtration through muslin cloth and starch
was allowed to settle down. Water was decanted off and solids
settled at the bottom of the vessel were dried to form seera.
During fermentation of seera, the samples were drawn every
day during six days and were analysed for microbiological and
biochemical aspects.

In Himachal Pradesh, a number of traditional fermented foods


and beverages are popular which are unique in comparison to
other parts of the country. Bhatooru, chilra, seera, siddu,
gulgule, marchu, sepubari, pickles (of local fruits and
vegetables) and fermented beverages (kinnauri, chhang, sura,
behmii, etc.) are some of the indigenous fermented products
of Himachal Pradesh (Savitri and Bhalla, 2007). Seera also called
Nishasta is a traditional fermented food prepared in Bilaspur,
Kangra, Hamirpur, Mandi, Shimla and Kullu districts of
Himachal Pradesh. It is a starch based food made by soaking,
crushing and fermenting wheat grains used to prepare sweet
dish/snack generally served in breakfast or for people during
religious fast. It holds special significance to the village people
of Himachal Pradesh where during drought seera is offered to
the God of water for rain. Seera is also recommended for people
suffering from jaundice/ hepatitis and to the post-natal
women.Similar starchy food tapai made form cassava is very
popular and is used to prepare sweet delicacies in Malaysia
(Law et al., 2011).Village based cooperatives in Himachal
Pradesh market seera in small packets (Plate I) and thus it also
serves as a source of revenue for many rural people.

Microbial profile during the fermentation


One gram of soaked wheat sample was withdrawn at an interval
of 24 h and was plated on Nutrient agar, Czapek Malt agar,
Lactobacillus Selection Agar Base and Czapek Dox Agar plates
for isolation of bacteria, yeasts, lactic acid bacteria and fungi.
The number of colonies of bacteria, yeasts and moulds that
appeared on plates were counted and expressed as log cfu
(colony forming units) g-1 of the sample. The microorganisms
isolated were identified at and submitted to Microbial Type
Culture Collection and Gene Bank (MTCC), Chandigarh.

In the traditional method of preparation of seera, wheat grains


are soaked in water for 2-3 days to allow natural fermentation.
After fermentation, grains are ground and steeping is done to
allow the starch grains and some proteins to settle down, bran
is removed. The settled solids are then,sun dried and the dried
material is called seeraAlthough seera is a popular traditional
fermented food of Himachal Pradesh, there is no information
about its microbiological and biochemical aspects. So, the
present investigation was carried out to evaluate the
microbiological and biochemical properties of seera and the
results are discussed in this communication.

Biochemical analysis
Samples during seera fermentation were analysed for various
biochemical parameters viz., total acidity by Amerine et al.
(1980) while pH was measured by using digital pH meter (MAC).
Total proteins were estimated by using the methods of Lowry
et al. (1951) and total carbohydrates were estimated by phenol
sulphuric acid method (Dubois et al., 1956). Reducing sugars
were estimated by DNSA method given by Miller (1959). Starch
was estimated according to Hedge and Hofreiter (1962). The
amount of different enzymes during the course of seera
fermentation was also studied. Protease activity was assayed
by the method given by Manachini et al. (1988) and amylase
activity of the samples during seera fermentation was assayed
according to Bernfield (1955).

Materials and methods


Chemicals
All the chemicals used were of analytical grade and were
procured from Hi-media, sd-fine, Loba Chemie and
Merck.Wheat grains for preparation of seera were purchased
from local market of Shimla, Himachal Pradesh.For HPLC,
Lichrosorb RP-18 (5 M) and for gas chromatography,
Chromosorb WHP 15% SE-30, 1/8x 2 m column were used.

Vitamin and amino acid analysis


Vitamins in the samples during seera preparation were analysed
according to najdrova et al. (2004). For assay of water soluble
B-vitamins the samples were filtered through 0.45 pore size
filters. The mobile phase comprised of acetonitrile: HPLC water
(75:25) and 0.1% orthophosphoric acid. Samples (5 l) of the
solution of water-soluble vitamins were injected in to the HPLC
column. Identification of vitamins was made by comparing
their retention times and UV spectra with those of standards.

Preparation of seera
The details about seera and the traditional method of seera
preparation were studied by visiting various places in Himachal
Pradesh and by active interaction with local people.. To make
seera under laboratory conditions, 200g of wheat grains were
cleaned thoroughly and soaked in 300ml tap water without the

For amino acid analysis sample was first hydrolysed (Schilling


et al., 1996)and then derivatization (Hsek, 1991)was done.
The derivatised amino acids were then analysed using GC.

50

Microbiological and biochemical characterization of Seera

Gas chromatographic analysis was carried out on a


NetelChromatograph GC (MICHRO-9100)equipped with
Chromosorb WHP 15% SE-30 column coupled with Flame
Ionization Detector. The GC was operated at the oven
temperature 170-295C, injector temperature 170-280C, ramp
rate 5oC and carrier flow (nitrogen) 5 ml/min.

found to produce antimicrobial products that lead to safe and


long storang of foods (Corganet al., 2007; Kalui et al., 2009;
Parvezet al., 2006; Steinkraus, 2002). The presence of
Enterobactersakazakiiin case of seera fermentation is not a
matter of concern as its population starts declining with the
fall in pH and finally, it disappears (Savitri, 2007).

Results and discussion

Chemical and biochemical analysis of seera


The pH and titrable acidity of seera prepared by soaking wheat
grains in water was estimated and the results are presented in
Figure 1. There was a sharp decrease in initial pH from first to
second day. This decline in pH was observed up to 4th day and
after that pH again increased because of washing given to the
samples. The low pH supports the growth of various yeast
and lactic acid bacteria important in food fermentation. With
the decrease in pH, total acidity increased from 0.009% initially
to 0.45% in the final product (Table 2). The main factors
regulating acidification and changes in pH are the amount of

Microbiological analysis of seera


In order to study microflora during seera fermentation, the
samples were periodically drawn at interval of 24 h for 6 days
and were analysed for viable counts.
During fermentation
The microbial counts increased substantially (Figure 1).The
microflora isolated from seera comprised of yeasts mainly
Saccharomyces cerevisiae, Cryptococcus laurentii and
Torulospora delbrueckii and bacteria including Lactobacillus
amylovorus, Cellulomonas sp., Staphylococcus sciuri,
Weisella cibaria, Bacillus sp., Leuconostocsp. and
Enterobacter sakazakii (Table 1). Out of these, Saccharomyces
cerevisiae and Lactobacillus sp. persisted in the final
product.Lactic acid bacteria have been reported as the most
important group of microorganisms involved in spontaneous
or natural fermentation of foods (Kalui et al. 2010). Bacillus
sp., Enterobacter sakazaki and Staphylococcus sciuri were
also present in early stages of fermentation probably originated
from air or water used for steeping of wheat.
Increase in total bacterial count was found to be higher as
compared to that of yeast count. Bacterial fermentation during
soaking is found to be important for the acidification.
Lactobacillus and Leuconostoc sp. predominate the microflora
as the fermentation progressed. Similar profile of lactic acid
bacteria, aerobic mesophiles and enterobacteriaceae, which
constituted the primary microflora of pozol (prepared by
soaking of maize) have been reported by Wacher et al. (1993).
The lactic acid bacteria present in fermented foods have been

Figure 1: Changes in pH, titrable acidity and total count during seera
fermentation

Table 1: Microbial population during seera fermentation


Time Predominant microorganisms
(days)
1.
2.
3.
4.
5.
6.

Bacillus sp.,Enterobactersakazakii, Cellulomonas sp., Saccharomyces cerevisiae, Staphylococcus sciuri


Leuconostoc sp., Lactobacillus amylovorus, S. cerevisiae, Cryptococcus laurentii, Torulspora delbrueckii, Staphylococcus sciuri
Leuconostoc sp., L. amylovorus, S. cerevisiae, C. laurentii, T. delbrueckii, Weisella cibaria
Leuconostoc sp., L. amylovorus, S. cerevisiae
S. cerevisiae, Leuconostoc sp., Lactobacillus sp.
(Seera) Lactobacillus sp., S. cerevisiae

51

Savitri et al.

Table 2: Biochemical analysis of seera


Parameters

Values*

pH
Titrable acidity (% of dry matter)
Total count (log cfu/g dry matter)
Protein (mg/g of dry matter)
Total sugars (% of dry matter)
Reducing sugars (mg/g of dry matter)
Starch (% of dry matter)
Amylase** (U/g of dry matter)
Protease***(U/g of dry matter)

3.450.055
0.440.006
5.39
10.40.20
89.00.43
11.90.53
87.41.51
3.60.36
1.020.05

* Values are mean SD of three observations


** One unit of amylase activity was defined as the amount of enzyme required to release one g of maltose/mg of substrate/min under the
assay conditions
***One unit of protease activity was defined as the amount of enzyme required to release one g of tyrosine/mg of substrate /min under the
assay conditions.
nd

dry matter to 16.3 mg/g dry matter in 24 h. After 2 day of


fermentation, the level of reducing sugars started decreasing
till day 4 and again increased on 5th day (Figure 2). Increase in
sugar content in the final product was either due to
concentration of reducing sugars by drying of seera or due to
the activity of amylolytic enzymes (Table 2).Mosha and
Svanberg (1983) have reported the hydrolysis of starch and
oligosaccharides present in the substrates of fermentation
resulting in an increase in reducing sugars due to the activity
of amylases present in cereal grains or secreted by
microorganisms. The decrease in reducing sugars with
prolonged fermentation was attributed to their utilization by
fermenting microflora as observed by Daeschel et al. (1987).

fermentable carbohydrates.Acidification of wheat based


substrate during food fermentation is an essential requirement
for the prevention of harmful microorganisms and the
associated risks of poisoning and spoilage (Katina, 2005).

The change in total titrable acidity from 0.072% at 0 h to 0.25%


after 96 h was reported in fermentation of fufu i.e. a cassava
based fermented food of Nigeria (Oyewole, 1990). Similar rise
in acidity, decrease in pH, increased solubility and digestibility
have also been reported during the preparation of Indian warri,
oncom, tempeh, gari, pozol, miso, mahewas, kenkey and idli
(Soni and Sandhu, 1990b; Beuchat, 1983; Soni et al., 2001).
The level of protein decreased from 14.9-8.2 mg/g of dry matter
from first to fifth day of fermentation (Figure 2). This may be
due to the increase in the activity of proteolytic enzymes with
the progress of fermentation. However, the higher protein
content in seera as compared to the 5th day of fermentation
was due to the concentration of various components during
its drying (Table 2).Niba (2003) reported that controlled
fermentation can be applied for the production of
physiologically beneficial components i.e. for increasing
amount of proteins.
With the progress of fermentation, the level of total sugars
decreased from 74.8% to 67.5% (Figure 2). The decrease may
be due to the utilization of sugars by the fermenting
microorganisms. The sugars are rapidly metabolized to acids,
ethanol, biomass, carbon dioxide and other metabolites required
for the growth of microorganisms with concomitant decrease
in total sugars during fermentation (Mensah, 1997). However,
higher total sugar content in the final product may be due to
its concentration during drying (Table 2) of Seera.

Figure 2: Change in total protein, total sugars, reducing sugars and


starch during seera fermentation

The level of reducing sugars doubled initially from 8.3 mg/g

52

Microbiological and biochemical characterization of Seera

active at low pH i.e. below 5.5. After 3rd day, protease activity
started decreasing and finally the activity was 1.02 U/g on dry
weight basis (Table 2). Decrease in activity may be due to the
disappearance of microorganisms responsible for the
production of protease. The degree of proteolysis and the
amount of acids and volatile compounds formed depend both
on the kinds of microorganisms present in the starter and on
the process parameters. Lactic acid bacteria possessing
proteolytic and amylolytic properties have been considered
to be most effective in delaying staling in bread (Corsetti et
al., 1998).

The starch content during fermentation of seera was decreased


initially from 72.1-57.0 % (w/w) from 1-4th day (Figure 2). After
steeping and drying of seera, the starch content increased to
87.4% (w/w) on dry weight basis (Table 2). The decrease in the
starch content initially may be due to the degradation of starch
during fermentation by the action of amylases.Giraud et al.
(1994) reported the degradation of cassava starch by the
amylolytic activities of Lactobacillus plantarum during
fermentation of raw cassava.
Amylase and protease activity
As shown in Figure 3, the amylase activity increased with the
increase in fermentation up to 4th day and then, it starts
decreasing. A very low amylase activity was observed in the
final product (Table 2). Similar trend in amylase activity was
reported in fermentation of dosa, and Punjabi warri (Soni and
Sandhu, 1999). Increase in amylase activity might have been
contributed by fermentative microorganisms and then due to
decline in pH, the activity decreased. Low pH has also been
shown to slow down the amylase activity of malt (Abegaz et
al. 2002)

Vitamin and amino acid content of seera


The vitamin content of seera and wheat grains were analyzed
by HPLC (Figure 4) and results are presented in Table 3. A
significant increase in thiamine, riboflavin, nicotinic acid and
cyanocobalamin was observed during fermentation of seera.
Pyridoxine (0.079 mg/g of dry matter) was observed in seera
which was absent in wheat grains taken at the start of the
fermentation. The rise in the level of various vitamins especially
thiamine and riboflavin appears to be due to increase in
microflora and yeasts, most of which have the ability to produce
vitamins from simple precursors.Fermentations, which involve
yeasts and particularly those in which the yeasts are consumed
along with the substrate, tend to be enriched in the watersoluble B vitamins (Steinkraus, 1998). The changes in thiamin,
riboflavin and niacin content in case of idli fermented with
pure cultures than with natural microflora has also been

During initial 24 h, there was a very small increase in protease


activity (0.71-1.25U/gon dry weight basis); however, the
activity increased drastically i.e. 7.37 U/g (on dry weight basis)
on 3rd day (Figure 3). This increase in activity may be due to
the decrease in pH and activation of protease at low pH.Cereal
proteases have been shown to be generally active at low
pH.Katina (2005) reported that protease in sourdough are

Figure 4: HPLC chromatogram of vitamins content of 1) wheat grains


and 2) Seera (Thiamine- RT=2.08 min, pyridoxine- RT=2.23 min,
nicotinic acid- RT=2.24 min, cyanocobalamin- RT=2.34 min, riboflavin- RT=3.02 min)

Figure 3: Amylase and protease profile during seera fermentation

53

Savitri et al.

Table 3: Vitamin and amino acid content in wheat grains and seera
*

Vitamins and amino acids (mg/g dry matter)

Thiamine (per 100 g)


Riboflavin
Nicotinic acid
Pyridoxine
Cyanocobalamin
Methionine
Phenylalanine
Threonine
Lysine
Leucine

Wheat

0.620.03
0.0030.0002
0.0490.003
ND
0.0060.0008
2.70.26
2.80.43
1.70.062
1.10.08
4.10.09

Seera

3.70.17
0.1290.005
0.560.04
0.0790.004
0.330.04
8.40.3
12.21.2
2.40.26
1.40.026
4.20.36

*Values are mean SD of three observations


ND= Not Detected

activities (Figure 5).

observed earlier (Steinkraus, 1998). Rajalakshmi and Vanaja


(1967) had also reported that thiamin and riboflavin content
increased during idli fermentation. Akolkar (1977) has also
observed an increase in thiamine, riboflavin and niacin content
during the idlifermentation.

El-Tinay et al. (1979) have observed a significant increase in


threonine and methionine level during kisra (sorghum product)
fermentation. During tempeh fermentation (soaking of
soybeans in water), a fivefold increase in free amino acid
content has been observed by Hering et al. (1991).

Thefermentation brought about an increase in essential amino


acid content (Table 3). A significant increase in methionine
(2.7-8.4 mg/g) and phenylalanine (2.8 to 12.2 mg/g) was
observed. However, threonine (1.7 to 2.4 mg/g), lysine (1.1 to
1.4 mg/g) and leucine (4.1 to 4.2 mg/g) exhibited marginal
increase in seera as compared to the substrate (wheat grains).
The increase in these amino acids in seera fermentation is an
indication of proteins hydrolysis caused by the activities of
proteolytic enzymes as well as the addition of such amino
acids by the fermentative microbes due to their metabolic

Conclusion
The present study has demonstrated for the first time that
during soaking of wheat grains, a mixed microbial fermentation
of complex ecology develops. This fermentation adds quality
to seera by way of enhancing its protein content, vitamin and
essential amino acids as compared to the control.While the
Western world can afford to enrich its foods with synthetic
vitamins, the developing world must rely upon biological

Figure 5: GC chromatogram of amino acids content of 1) wheat grains and 2) Seera (Peaks 1- chloroform, 2,3- leucine, lysine and threonine,
4- methionine and 5-phenylalanine)

54

Microbiological and biochemical characterization of Seera

Plate I: Dried seera and seera packed in small packets

enrichment for its vitamins and essential amino acids. As seera


is biologically enriched with vitamins and amino acids during
fermentation so it forms a good source of nutrition to the
people who consume it.

Sci., 90: 4005-4021.


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Acknowledgement
One the author (Savitri) gratefully acknowledges Council of
Scientific and Industrial Research (CSIR), New Delhi, India for
financial support in the form of Junior Research Fellowship
and Senior Research Fellowship.
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56

Intl. J. of Food. Ferment. Technol. 2(1): 57-61, June, 2012

Research paper

Preparation and evaluation of Mahua


(Bassia latifolia) Vermouth
Preeti Yadav1*, Neelima Garg2 and Deepa Dwivedi3

Microbiology Lab, Division of Postharvest Management, Lucknow, India


Division of Postharvest Management Central Institute for Subtropical Horticulture, Lucknow, India
3
Department of Applied Plant Sciences, Babasaheb Bhimrao Ambekar, University, Lucknow, India
2

Email: priti_june10@rediffmail.com

Paper no: 38

Received: 19 Jan, 2012

Received in revised form: 14 April, 2012

Accepted: 17 May, 2012

Abstract
Mahua is used for preparation of distilled liquors since time immemorial. Vermouth was prepared from
young mahua wine after its fortification and flavouring with traditional Indian herbs, viz. black pepper,
cinnamon, clove, cumin, fenugreek, large cardamom, nutmeg, fennel and Indian Cassia. Initially, mahua
wine had 6.00 B TSS, 0.63 % acidity, 2.11 mg/L 100 ml ascorbic acid, 0.09 % tannins, 0.19 % reducing
sugars and 10.5 % alcohol. The fortified mahua vermouth after one year of ageing had 6.40 B TSS, 0.57
% acidity, 1.19 mg/L 100 ml ascorbic acid, 0.11 % tannins, 0.27 % reducing sugars and 18.4% alcohol.
The vermouth was organoleptically more acceptable than the mahua wine. Phenolics, viz. gallic acid,
caffeic acid, pcoumaric acid and kaempferol as well as the flavanols, (+)-catechins and (-)-epi-catechin
were detected in both the mahua wine and vermouth.
2012 New Delhi Publishers. All rights reserved

Keywords: Mahua, Vermouth, Bassia latifolia, Saccharomyces cerevisiae, Aromatic herbs,spices

Mahua (Bassia latifolia) the Kalpavriksha, is the most valued


tree among tribal communities; whose every part is of service
to the people of Central India. Its flower has been used for
ethanol fermentation. Fruit pulp is a good source of sugar,
whereas dry husk makes a good source of absolute alcohol.
Earlier wine had been prepared from mahua flowers (Yadav et
al., 2009). However, one limiting factor with mahua wine is its
burnt starchy flavour. Vermouth is basically, a fortified wine
flavored with aromatic herbs and spices including, cardamom,
cinnamon, chamomile, cloves and marjoram (Pilon, 1954). Even
ginger, citrus peel, cloves, corriander may be used for this
purpose (Jarczyk and Wzorek, 1977). Onkarayya (1985)
reported mango vermouth having high acceptability. Joshi et
al. (1991) prepared plum vermouth while Attri et al. (1993)
developed sand pear vermouth and reported that addition of
spices/herbal extract increased the total phenols, aldehyde

and ester contents. Sweet apple vermouth with 15 % ethanol


was reported by Joshi and Sandhu, (2000). Flavour of wine is
of utmost significance and is one of the quality parameter of
its evaluation. To mask the burnt starchy flavor of mahua wine
and improve its acceptability, mahua vermouth was prepared.
It was matured and evaluated at different intervals for various
physico-chemical, biochemical and sensory quality
characteristic and the results have been discussed in this paper.
Materials and methods
Mahua flower collection
In the month of April, mahua flowers from Rehmankhera area
were collected in morning hours on polythene sheets (20m X
20m) laid down under the trees, filled in clean polythene bag
and brought to lab under hygienic conditions.

Yadav et al.

Juice extraction

concentration in the vermouth was determined


spectrophotometrically by the method of Caputi et al. (1968).
Phenolics in mahua vermouth were analyzed by high
performance liquid chromatography as described by Basha et
al. (2004) with slight modification. Microbial count was carried
out as per method of Speck (1985).

Mahua flowers were washed thoroughly with tap water.


Diseased, rotten and damaged flowers were sorted out, the
clean and undamaged flowers were crushed in a fruit mill,
pressed with hydraulic press and the juice (from all the parts
of the flower) was collected in clean stainless steel utensils.

Statistical Analysis

Yeast culture

The data recorded during the course of investigation were


subjected to statistical analysis as per the method of Analysis
of Variance (ANOVA) with two factors in Completely
Randomised Design (CRD). All determinations were run in
triplicate and values were averaged. The standard deviation
(SD) was also calculated. The significant differences on the
mean value were compared by the least significant difference
(LSD) test at a significance level of 0.01. The data were subjected
to statistical analysis using Statistical Package for Agricultural
Workers developed by O.P. Sheoran of CCSHAU, Hisar.

The yeast culture Saccharomyces cerevisiae (var.


ellipsoideus), used in the present investigation, was obtained
from the culture collection of microbiology laboratory of CISH,
Lucknow. The culture was maintained on Yeast Extract Peptone
Dextrose (YEPD) Agar slants which was re-cultured every
month.
Mahua base wine
Mahua base wine was prepared as per the method described
by Yadav et al. (2009).

Sensory Analysis

Mahua vermouth

The sensory analysis of mahua vermouth was conducted by a


panel of semi-skilled judges (Amerine, 1980). The attributes
considered on a 10 point scale for colour (20%), clarity (20%),
aroma (10%), taste (10%), astringency (10%), freedom from
acetic acid (10%), sweetness (10%) and body (10%). The overall
rating was obtained by calculating the average of the scores.

1000 ml of mahua base wine was distilled and different spice


and herbs (Table 1) were added to distillate which were kept
for extraction for two days at 20oC. The content was filtered
and added @ 10% to the base wine, mixed well and kept for
aging for two months. The vermouth was transferred to glass
bottles; crown corked, pasteurized and kept under ambient
conditions for further aging. The entire process of mahua wine
and vermouth preparation is shown in Figure 1.

Results and discussion


Biochemical analysis of mahua flower juice is shown in Table
2. It shows that the juice is rich in sugar and may be utilized for
production of wine. No microbial growth could be observed in
mahua vermouth after one year of aging. The vermouth
prepared by mahua had 18.4 % alcohol, 1.26 mg/100g tannin
content compared to the base wine where it was 10.50 %; 0.09
%, (Table 3). Non-significant changes were observed in
biochemical parameters of mahua vermouth (Table 4) during
maturation. It had TSS of 6.40 B, acidity 0.57 %, ascorbic acid

Physicochemical Analysis
Vermouth was analyzed for various biochemical and sensory
parameters at 3 months interval. Total soluble solids (TSS)
content of vermouth were recorded using hand refractometer
(Erma, Japan). The titratable acidity, tannins, ascorbic acid,
non enzymatic browning was determined using the methods
described by Ranganna (1986). The reducing sugar was
determined using the methods of AOAC (1985). Ethanol
Table 1: Spices and herbs used in mahua vermouth preparation
Common name

Gram/ litre mahua distillate

Black pepper
Cinnamon
Clove
Cumin
Fenugreek
Large cardamom
Nutmeg
Fennel
Indian Cassia

15.5
9.2
7.0
12.8
6.0
6.5
8.2
50
8

58

Botanical name

Part used

Piper nigrum L.
Cinnamonum zeylanicum Beryn.
Syzygium aromaticum L.
Cumis cyminum L.
Zingiber officinale Rosc.
Amomum subulatum Roxb.
Myristica fragrans Houth.
Foeniculum vulgare
Cinnamomum tamala

Fruit
Bark
Fruit
Seeds
Dried roots
Seeds
Seeds
Seed
Leafs

Preparation and evaluation of Mahua (Bassia latifolia) Vermouth

mg/100g) as well as the flavanols (+)-catechins (35.31 mg/100g)


and (-)-epi-catechin (39.51 mg/100g) (Table 5). Attri et al. (1993)
reported that addition of herbs/spices improves the phenolic
content of vermouth. Goldberg et al. (1998) have reported high
concentrations of (+)-catechin and (-)-epicatechin in red
Burgundy and Canadian wines. Arts et al. (2000) reported the
presence of (+)-catechin, (-)-epicatechin, (+)-gallocatechin, (-)
-epigallocatechin, (-)-epicatechin gallate and (-)epigallocatechin gallate in 8 types of black tea, 18 types of red
and white wines, apple juice, grape juice, iced tea, beer,
chocolate milk, and coffee using HPLC. Sirohi et al. (2005)
developed nutritionally rich and therapeutically value added
whey based mango herbal beverage. Heinonen et al. (1998)
reported that berry and fruit wines contain phenolic
compounds, some of which are capable of protecting lipids
against oxidation also in a hydrophobic lipid system.
Table 2: Biochemical composition of mahua flower juice
Parameters

Mahua juice

T.S.S (0B)
Acidity (%)
Ascorbic Acid (mg/100ml)
Tannins (%)
Reducing sugar (g %)

13.0
0.11
3.15
0.11
1.04

Table 3: Biochemical and nutritional characteristics of mahua wine


Figure. 1: Flow sheet for the preparation of mahua flower wine and
vermouth

Parameters

Mahua wine

T.S.S (0B)
Acidity (%)
Volatile acidity (%)
Ascorbic acid (mg/100ml)
Tannins (%)
Reducing Sugar (%)
Alcohol (%)
NEB

1.19 mg 100-1 ml at the time of bottling which decreased


marginally during storage while the reducing and total sugars
increased from a level of 0.27 % to 0.33 %, respectively. There
are reports of loss of ascorbic acid and antioxidants during
storage of fruit wines (Patras et al., 2009). The phenolic
compounds reflected through HPLC in mahua vermouth
included gallic acid (122.59 mg/100g), caffeic acid (10.76 mg/
100g), pcoumaric acid (4.18 mg/100g) and kaempferol (282.16

6.0
0.63
0.07
2.11
0.09
0.19
10.50
0.021

Table 4: Biochemical changes in mahua vermouth during one year of maturation


Storage
periods
0
3
6
9
12

SO2
(ppm)
107
105
103
100
97

TSSb
(0B)

6.2
6.2
6.4
6.4
6.4

Acidityc
Ascorbic
(%) acidd (mg/100g)
0.59
0.58
0.58
0.57
0.57

1.26
1.24
1.23
1.20
1.19

Tanninse
sugarg (g %)

Reducing
sugarg (%)

Total
sugarf(g %)

Alcoholh
(%)

Non-enzymatici
browning

0.32
0.29
0.28
0.20
0.11

0.16
0.17
0.19
0.23
0.27

0.39
0.37
0.35
0.33
0.33

18.40
18.40
18.40
18.40
18.40

0.020
0.029
0.035
0.038
0.040

Data followed by different letters for each column are significantly different by statistical (p > 0.01).
Statistical analysis due to storage periods: a 0.001; b 0.001; c non-significant; d 0.001; e 0.001; f non-significant; g 0.001; h non-significant; i
0.002

59

Yadav et al.

Table 5: Changes in phenolics compoundsb during storage of mahua vermouth


Storage Source of variationa
periods
Total phenols
0
3
6
9
12
a

494.5410.28a
475.3119.23a
436.2039.11a
417.1719.03a
409.457.72a

Gallic acid

(+)-Catechin

(-)-Epi-catechin

Caffeic acid

p-coumaric acid

122.5925.76b
112.4513.76ab
107.3818.67ab
99.1914.98ab
92.8724.74ab

35.31 1.45b
32.561.65b
30.851.78b
30.192.98b
28.752.57a

39.516.98a
35.266.34ab
32.834.96ab
29.744.77ab
29.184.29b

10.763.06a
11.982.95b
13.112.25b
16.871.98b
18.351.59b

4.180.95a
5.351.45b
5.871.34b
5.991.75b
6.181.11b

Data followed by different letters for each column are significantly different by LSD
Values are expressed as mg/100gram standard error (n=33).

kaempferol
282.1649.4a
281.1946.4b
280.9753.1b
280.9151.9b
280.5749.6b

test (p<00.1).

Vermouth is an aromatized wine having added sugar, roots,


herbs, spices and flowers (Fessler, 1971). It contains ethyl
alcohol, sugars, acid, tannins, aldehyde, esters, amino acids,
vitamins, anthocyanins, fatty acids and minor constituents
like flavouring compounds (Joshi, 1997; Joshi et al., 1999).
The additives dont boost the alcohol content, but they do
sculpt the flavor of the wine. Fessler (1971) has also

recommended use of herbs, roots and spices for development


of flavoured wines having distinctive character. Vermouth can
have alcohol content between 15 % and 19 % by volume (Joshi
and Sandhu, 2000). Since mahua flower have burnt starchy
flavours, herbs were used to mask the same and replace with
sweet spicy flavor.

Figure 2: Sensory evaluation of mahua wine and vermouth during storage

60

Preparation and evaluation of Mahua (Bassia latifolia) Vermouth

Heinonen, I.M., Lehtonen, P.J. and Hopia A.I. 1998. Antioxidant


activity of berry and fruit wines and liquor, Food Chem,
46:25-31.
Jarczyk, A. and Wzorek, W. 1977. Fruit and Honey. Wines, In:
Alcoholic Beverages, AH, Rose, (ed), Academic Press,
London, pp. 387-421.
Joshi, V.K. 1997. Fruit wines, 2nd End. Directorate of Extension
Education, Dr. YS Parmar University of Horticulture and
Fortry, Solan (HP), India.p255
Joshi, V.K. and Sandhu, D.K. 2000. Influence of Ethanol
Concentration, Addition of Spices Extract and Level of
Sweetness on Physico-chemical Characteristics and Sensory
Quality of Apple Vermouth, Brazillian Arch Biol Technol,
43(5):537-545.
Joshi, V.K., Attri, B.L. and Mahajan, B.V.C. 1991. Production and
Evaluation of vermouth from plum fruits, J Food Sci Technol,
28:38-141.
Onkarayya H, 1985. Mango vermouth A new alcoholic beverage,
Indian Food Packer, 39(1):40-45.
Patras, K., Brunton, N.P., Pieve S.D. and Butler, F. 2009. Impact of
high pressure processing on total antioxidant activity, phenolic,
ascorbic acid, anthocyanin content and colour of strawberry
and blackberry purees, Innovative Food Science and
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Pilon, J.F. 1954. Production of Vermouth, Am JEnol Vitic, 5:30-46.
Sirohi, D., Patel, S., Choudhary, P.L. and Sahu, C. 2005. Studies on
preparation and storage of whey-based mango herbal pudina
(Mentha arvensis) beverage, Journal of Food Science and
Technology, 42(2):157-161
Yadav, P., Garg, N. and Diwedi, D.H. 2009. Standardization of pretreatment conditions for mahua wine preparation, J.
Ecofriendly Agric., 4(1):88-92.
Yadav, P., Garg, N. and Dwivedi, D.H. 2009. Effect of location of
cultivar, fermentation temperature and additives on the
physico-chemical and sensory qualities on mahua (Madhuca
indica J.F. Gmel.) wine preparation, Natural Product
Radiance, 8:406-418.

The sensory results clearly reflected improvement in its aroma


and acceptability during storage (Figure 2).
Conclusions
Mahua vermouth, after one year of aging, was found to be
superior to the base wine based on physio-chemical and
sensory qualities. Aging further improved its acceptability. A
variety of phenolic compounds found in the Mahua wine has
added its value as an antioxidant rich beverage.
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V.L. and Webb, A.D. 1980. The Technology of Wine making,
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Arts, I.C.W., Putte, B. and Hollman, P.C.H. 2000, Catechin contents
of food commonly consumed in the Netherlands, 2. Tea, Wine,
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differences in the phenolics compounds of muscadine and
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3(10):523-528.
Fessler, J.H. 1971. Erythorbic Acid and Ascorbic Acid as Antioxidants
in Bottled wines, Am. J. Enol. Vitic, 12:1:20-24.
Goldberg, D.M., Karumanchiri, A.T.S. and Soleas, G.J. 1998.
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61

-amylase
from
Endomyces
fibuliger an indigenous yeast isolate of Western Himalayas
Intl. J. of Food. Ferment.
Technol.production
2(1): 63-69,
June,
2012

Research paper

-amylase production from Endomyces fibuliger an


indigenous yeast isolate of Western Himalayas
Keshani Bhushan, Anamika Jain, O.P. Sharma, B. Singh and S.S. Kanwar*

Department of Microbiology, Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India
Regional Station, Indian Veterinary Research Institute, Palampur, Himachal Pradesh, India
* Department of Microbiology, Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India
2

Email: sskanwar1956@gmail.com

Paper no: 39

Received: 26 April, 2012 Received in revised form: 17 May, 2012

Accepted: 19 June, 2012

Abstract
Amylase production, its purification, characterization and optimization have been studied in Endomyces
fibuliger. The enzyme was purified to about 13-fold by using gel permeation chromatography (Sephadex
G-25, Sephadex G-100). The purity of enzyme was checked by the use of Native-PAGE and SDS-PAGE.
The molecular weight of enzyme was 55KD with the presence of two polypeptide units. Enzyme was
most active at pH 7.0, temperature 30 C with optimum incubation time of 30 minutes. The maximum
activity of the enzyme was at 2 % (w/v) soluble starch concentration. The enzyme was para-constitutive
in nature. The parameters for amylase production were optimized by Response Surface Methodology
(RSM) . The best carbon source was soluble starch, the optimum pH values were 5.0 and 8.0, and the
optimum temperatures were 30 C and 37 C. Ca+ and Mg+ had enhancing effect on its production.
2012 New Delhi Publishers. All rights reserved

Keywords: Endomyces fibuliger, Amylase, Sephadex, Starch, Response surface methodology

from bacteria and filamentous fungi is available but little is


known on the amylases of yeasts despite the fact that a number
of them can grow on starch. Yeasts have received considerable
attention on account of their importance in brewing and baking
industries. One of the major applications is their ability to
produce a variety of industrially important enzymes which have
applications in food, fermentation, textile and paper industry
(Walsh, 2002).

Starch degrading enzymes (amylases) are the most important


microbial enzymes and are of great significance in food industry.
Now-a-days, the microbial amylases are considered
indispensable in meeting the industrial demand of enzymes
(Pandey et al., 2000). Amylases constitute a class of industrial
enzymes having approximately 25 per cent of the enzyme market
(Rao et al., 1998). The amylase family of enzymes has been
well characterized through the study of various
microorganisms. Two major classes of amylases identified
among the microorganisms, are alpha-amylase (endo-1,4alpha-D-glucan glucohydrolase, EC-3.2.1.1), and glucoamylase
(exo-1,4-alpha-D-glucan glucanohydrolase, EC-3.2.1.3). In
addition, beta-amylase (-1,4-glucan maltohydrolase, EC3.2.1.2), which is generally of plant origin, has also been
reported from a few microbial sources.
A considerable amount of information pertaining to amylases

63

In food industry (brewing and baking), starch as a raw material


needs pretreatment for liquefaction and saccharification for
its conversion. Common alcohol producing yeast,
Saccharomyces cerevisiae lacks the enzyme amylase, which
is needed to hydrolyze starch into simple monomeric units
(Banerjee et al., 1988). Thus, more attention has been paid
towards amylase producing yeasts especially of food origin.
Amylolytic yeasts are potentially valuable in utilization of

Bhushan et al.

starchy substances for direct conversion into single cell protein


(SCP) or ethanol (Soni et al. 1991). Now-a-days, recombinant
yeast strains are also produced for better yield of the amylases
(Galdino et al., 2011). Yeasts are known to produce at least two
type of amylases i.e. alpha-amylase (E.C. 3.2.1.1) and
glucoamylase (E.C. 3.2.1.3) which differ from each other with
respect to their mechanism of action causing starch liquefaction
and saccharification, respectively (DeMot and Verachtert,
1985). The assay methods to determine these amylases also
differ which help to differentiate between different type of
amylases.

The medium was inoculated with 1% inoculum grown in potato


dextrose broth (PDB). The medium was then, incubated at 27C
for five days. After incubation, culture broth was filtered and
centrifuged at 8000g for 15 minutes at 4C. The supernatant
obtained was then, used as the crude enzyme for extracellular
amylolytic activity. The enzyme was assayed by measuring
the increase in reducing sugars by complex formation with
dinitrosalicylic acid (Miller, 1950). However, this assay method
did not work here due to interference by the reducing sugars
in the enzyme extract leading to high blank values. Therefore,
amylase assay was done by the starch-iodine method (Xiao et
al., 2006) which is specific to -amylase. One unit of amylase
activity is defined as the disappearance of an average of 1mg
of iodine binding starch material per minute in the assay reaction
(Vandyk and Caldwell, 1956). Protein content was determined
at 280nm by using spectrophotometric method.

It is well documented that extracellular amylase production by


microbes is greatly influenced by media components, especially
carbon and nitrogen sources, minerals and physical factors
such as pH, temperature, agitation, dissolved oxygen and
inoculum density (Gigras et al., 2002). Response surface
methodology (RSM) is an experimental strategy for seeking
the optimum conditions for a multivariate system (He et al.,
2004). Application of RSM has gained attention of researchers
for optimizing media components and process parameters
(Vohra and Satyanarayana, 2002).

Enzyme purification and characterization


The purification was carried out first by freezing the crude
enzyme extract and lyophilizing it at -60 C in a freeze dryer
(Edwards EF4 Modulyo). The lyophilized protein was dissolved
in acetate buffer (0.2M, pH 5.2) and applied to a sephadex G-25
column. The protein was eluted by applying the same buffer.
Twenty fractions of 5ml each were collected and pooled on the
basis of results of enzyme activity. The fractions with maximum
activity were lyophilized and again purified by sephadex G100 superfine column.

Forty-three yeast isolates derived from various fermented foods,


alcoholic beverages and traditional inocula of Western
Himalayas, and characterized by using traditional and molecular techniques (Pathania et al., 2010), were investigated in the
present study for the production of amylases. The best strain
was employed to produce amylases and important parameters
for production were optimized by Response surface methodology (RSM).

The protein on electrophoretic separation showed two bands.


Sodium dodecyl sulphate- polyacrylamide gel electrophoresis
(SDS-PAGE) was used for the determination of molecular weight
of the desired amylolytic enzyme with glyceraldehydes 3phosphate dehydrogenase (36KD), ovalbumin (45KD),
glutamic dehydrogenase (55KD), fructose-6-phosphate kinase
(84KD), phosphorylase b (97KD) and galactosidase (116KD)
as molecular weight markers and native polyacrylamide gel
electrophoresis (Native PAGE) was used to determine the
number of proteins present in the sample. PAGE was performed
on 1.5mm and 0.75mm thick plate with a current of 30-40 volts.
The gel, buffer and samples were prepared according to the
procedure of Laemmli (1970). Staining of SDS-PAGE was done
by coomassie blue stain and Native-PAGE was stained by silver
stain.

Materials and methods


Organism, growth conditions and enzyme production
Forty-three yeast isolates derived from various fermented foods
were screened for amylolytic activity. Starch hydrolysis test
(Shaw et al., 1995) was performed in order to identify the
amylolytic strains among the available isolates. Enzyme activity
was checked on starch agar medium. The surface of starch
agar plate was point inoculated and incubated at 28C for 72
hours. The growth obtained was flooded with 5-10 ml of iodine
solution for twenty minutes. The presence of clear zone shows
amylase production. The most active strain, Endomyces
fibuliger was then, used for further study. This strain was
isolated from dhaeli used as traditional inoculum by the local
people of Lahaul and Spiti (Himachal Pradesh) and was
molecularly characterized as reported earlier (Pathania et al.,
2010). The organism was grown at 28C on PDA. Enzyme
production was carried out in a 250 ml Erlenmeyer flask
containing standard production medium (Kwan et al., 1993).

The purified enzyme was further characterized for activity at


varying temperature (10C-80C), pH (4-9), substrate
concentration (0.5-4g/100ml) and incubation time.
Optimization by applying response surface methodology
(RSM)
The chemically defined standard medium (Kwan et al., 1993)

64

-amylase production from Endomyces fibuliger an indigenous yeast isolate of Western Himalayas

was first optimized by one variable at a time (OVAT) approach


for alpha-amylase production. This medium was used for
further optimization by applying RSM of central composite
design (CCD). The levels of four independent variables (starch
concentration (A), temperature (B), pH (C) and divalent ion
concentration (D) chosen for this study were optimized by
RSM. Each factor in the design was studied at five different
levels (Table 1). A set of 30 experiments was performed. Each
experiment was carried out in triplicates. The full experimental
plan with respect to their values in actual and coded form is
listed in Table 2. Upon completion of experiments, the average
of alpha-amylase production was taken as the dependent
variable or response (Y).

Statistical analysis and modeling


The data obtained from RSM on alpha-amylase production
was subjected to the analysis of variance (ANOVA). The
results of RSM were used to fit a polynomial equation (1), as it
represents the behavior of such a system more appropriately,
Y=0+1A+2B+3C+4D+11A+22B+33C+44D
+12AB +13AC+14AD+23 BC+24BD+34CD

Where y is response variable, 0 is intercept, 1, 2, 3 and 4


are linear coefficients, 11, 22, 33 and 44 are squared
coefficients, 12, 13, 14, 23, 24 and 34 are interaction
coefficients and A,B,C,D,A, B, C, D, AB, AC, AD, BC, BD
and CD are level of independent variables.

Table 1: Range of values for the RSM


Independent variables
Starch (%)
Temperature(C)
pH
CaCl2 (mg)

-2
2
25
4
4

-1
4
30
5
6

0
5
31
6
8

+1
6
37
8
12

(1)

Statistical significance of the model equation was determined


by Fischers test value, and the proportion of variance explained
by the model was given by the multiple coefficient of

+2
8
43
10
16

Table 2: Experimental design and results of CCD of RSM


Experiment No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Starch
Temperature
concentration(%) (A)
(C) (B)
4
2
2
8
5
8
8
8
5
6
2
2
8
2
5
6
5
5
5
8
8
5
5
5
5
5
5
2
2
5

25
37
25
25
30
37
37
37
43
30
25
37
37
37
25
31
30
31
31
25
25
37
30
37
31
31
31
37
25
30

pH
(C)

CaCl2
(mg) (D)

8.0
3.0
4.0
4.0
5.0
8.0
5.0
8.0
6.0
6.0
4.0
4.0
4.0
4.0
6.0
6.0
6.0
10
6.0
8.0
8.0
8.0
6.0
4.0
6.0
6.0
4.0
8.0
8.0
6.0

12
4.0
12
12
12
12
16
4.0
8.0
8.0
12
4.0
12
12
8.0
8.0
6.0
8.0
12
12
4.0
4.0
12
4.0
8.0
16
12
12
4.0
8.0

65

Predicted

Activity(U/mg)
Experimental

0.1
0.56
0.006
-0.067
0.53
0.91
1.13
1.01
-0.033
0.67
0.006
-0.014
0.098
-0.007
0.33
0.7
0.7
0.027
0.74
0.47
0.56
0.76
0.71
-0.037
0.67
0.99
0.21
0.36
0.29
6.64

0.039
0.366
0.0
0.0
1.133
0.9228
1.1263
0.9684
0.0
0.8043
0.0
0.0
0.0
0.0
0.0
0.8043
0.803
0.0
0.796
0.497
0.544
0.92
0.788
0.0
0.0
0.8
0.0
0.437
0.489
0.8

Bhushan et al.

Table 3: Summary of purification process


Purification step

Volume of
extract (ml)

Protein
(mg)

Total activity
(units)

Specific activity
(U/mg)

Recovery
(%)

Purification fold

Crude extract
Sephadex G-25
Sephadex G-100

100
40
25

159
2.8
1.03

95
18.47
7.8

.8545
6.59
7.57

100
19.2
8.2

1
11.04
13

Table 4: Anova for alpha-amylase production


Source

Sum of square

Degree of freedom

Mean square

F-value

P-value

Model
Error
Total

3.9
0
5.1

14
1
29

0.28
0

3.48

0.0112

R=0.7645
Adjusted R=0.5447
Predicted R=0.3713
Adequate precision=5.996

determination, R squared (R) value. Design Expert (Ver 7.0,


STATEASE. Inc., Minneapolis, USA) was used in this
investigation.

obtained was 0.8545U/mg. The total protein in the crude enzyme


extract was found to be 1.59mg/ml.
Purification of enzyme

Results and discussion

During purification process, the culture supernatant was


concentrated (34 fold) and low molecular weight compounds
were removed from the enzyme extract by chromatography on
sephadex G-25 gel filtration (Figure.1). The amylolytic enzyme
was further purified by sephadex G-100 gel filtration. A 13-fold
purification of amylolytic enzyme was achieved with a yield of
8.2% of the original activity (Figure. 2). The enzyme preparation
obtained after sephadex G-100 column had a specific activity
of 7.57.

Enzyme production and assay methods


The production of extracellular alpha-amylase from Endomyces
fibuliger was first checked on starch agar medium and 1cm
zone of clearance was observed. In order to obtain sufficient
amount of enzyme, standard medium designed by Kwan et al.
(1993) was used. The enzyme assays were done in the native
state, heat inactivated state, after dialysis and after autoclaving
using DNS method. The enzyme showed activity both in
inactivated state as well as after stopping the reaction. These
results indicated that the enzyme was probably heat stable. To
check the accuracy of the DNS method in the present study,
salivary amylase was used as a test case. Salivary amylase did
not show measureable activity after stopping the reaction.
These results suggest that the enzyme assay by the DNS
method was not correct probably due to the interference by
reducing sugars existing in the enzyme extract. Thus, it was
necessary to look for an alternate method where such type of
interference could be avoided. So, the starch-iodine method
was used which is based on assay for the substrate and not
the product of enzyme reaction. This study was also carried
out with salivary amylase and the results were found to be in
accordance with that of yeast amylase. Therefore, starch iodine
method was found to be more appropriate in detecting the
amylolytic activity in the present study. The specific activity

The pooled fractions from gel permeation chromatography


developed using coomassie brilliant blue (CBB) staining
indicated the presence of two bands. These two bands indicate
polypeptide subunits in the enzyme fraction. The molecular
mass established by SDS-PAGE developed using CBB was
found to be 55KD. To detect the low concentration of proteins
the pooled fractions from GPC were developed using silver
staining. Native-PAGE indicated the presence of two bands
showing two polypeptide units. One of them was the major
band while other one was minor.
Characterization of enzyme
The purified enzyme of Endomyces fibuliger was tested for its
activity on soluble starch at its different concentrations and
the maximum activity of amylolytic enzyme was noticed at 2 %
(w/v) soluble starch concentration. The optimum incubation
time was found to be 30 min. The enzyme was most stable at
66

-amylase production from Endomyces fibuliger an indigenous yeast isolate of Western Himalayas

Figure 1: Elution profile of Endomyces fibuliger amylase on Sephadex


G-25 column (2.5 12 cm bed size). The concentrated crude extract
containing 159 mg protein was loaded on the column and eluted
isocratically with 0.2 M acetate buffer pH 5.2. Fractions (5.0 ml each)
were collected and assayed for amylolytic activity. Elution for proteins was monitored as absorbance at 280 nm. Amylase activity is
expressed as Units/mg.

Figure 3: 3-D surface diagram of amylase production shows on zaxis against the effect of temperature and pH plottted on x-axis and yaxis, respectively.

pH 7.0 with 3.084U/mg of activity. The optimum temperature


for enzyme activity was 30C. It was active over a wide range
of temperature i.e. from 20C to 50C after which a sudden
decline in activity was observed.
Response surface methodology for optimization of
parameters
By one variable at a time approach, the medium was optimized
for enzyme production. Carbon sources used for amylase
production were amylum, sorbose, arabinose, maltodextrin,
dextrose, sucrose, xylose and soluble starch. The organism
showed enzyme production with all carbon sources but the
maximum enzyme production was obtained with soluble starch.
Since, enzyme production was seen in glucose containing
medium, although less than starch, it appeared that the enzyme
had para-constitutive nature. Enzyme production was checked
with different divalent ions: Ni, Mn, Cu, Ca, Zn, Ba, Fe, Sr and
Mg. All ions showed inhibition to enzyme production except
Ca and Mg ions. Ca was found to be more effective than Mg
but the presence of both ions was required for better enzyme
production. Ca produces stabilizing effect during enzyme
production and hence, favours the process (Kar and Ray, 2008).
Similarly, temperature (25oC-43oC) and pH (4-10) ranges were
studied for enzyme activity. The results of CCD experiments
for studying the effect of four independent variables are

Figure 2: Elution profile of Endomyces fibuliger amylase on Sephadex


G-100 column. Sample: Pooled fraction (2.8 mg protein) from Sephadex
G-25 column chromatography (Figure 1) positive for amylase activity. Elution was with 0.2M acetate buffer pH 5.2. Fractions (5.0 ml
each) were collected and assayed for amylolytic activity.

67

Bhushan et al.

presented along with the mean predicted and observed


responses in Table 4. The regression equations obtained after
ANOVA gave the kind of alpha-amylase production as a
function of the initial values of starch concentration,
temperature, pH and divalent ion concentration.

experimental and predicted values for alpha-amylase


production. The adjusted R corrects the R value for the sample
size and for the number of terms in the model. If there are many
terms in the model and the sample size is not very large, the
adjusted R may be noticeably smaller than the R.The model
F-value of 3.48 and values of probability > F (<0.05) indicated
that the model terms are significant. For alpha-amylase
production C, B and C are significant model terms.

The final response equation that represented a suitable model


for alpha-amylase production is given below:
Y=0.67+0.093A+0.011B+0.28C-0.021D+ 0.024A-0.23 B
0.30C+0.091D+0.045AB+0.11AC+0.026AD + 0.069
BC-0.001BD-0.052CD
(2)

Response surface was generated by plotting the response


(alpha-amylase production) on the z-axis against any two
independent variables while keeping the other independent
variables at their O levels. Figure 3 depicts three-dimensional
diagram and a contour plot of calculated response surface
from the interaction between temperature and pH while keeping
all other variables at their O level. At specific temperature
and pH combinations i.e. 30 C and 5.0; 37 C and 8.0; 37 C
and 5.0, better enzyme production was seen. Starch
concentration and temperature had linear relationship (Figure
5). Enzyme production was found to be independent of the
divalent ion concentration effect but at least 4 mg of divalent
ion concentration is required for better enzyme production
(Figure 4). Thus, fourth parameter shows very little interaction
to other parameters.

Where Y is enzyme production, A is starch concentration (%),


B is temperature ( oC), C is pH and D is divalent ion
concentration (mg).
The coefficient of determination (R) was calculated as 0.7645
for alpha-amylase production, indicating that the statistical
model can explain 76.45% of variability in the response. The R
value is always between 0 and 1. The closer the R is to 1.0, the
stronger the model and the better it predicts the response
(Haaland, 1989). The purpose of statistical analysis is to
determine the experimental factors, which generate signals that
are large in comparison to the noise. Adequate precision
measures signal to noise ratio. An adequate precision of 7.404
for alpha-amylase production was recorded. The predicted R
of 0.3713 is in reasonable agreement with the adjusted R of
0.5447. This indicated the good agreement between the

The mathematical model generated during RSM


implementation was validated by conducting check point
studies. The experimentally obtained data were compared with

Figure 5: Interaction graph showing the effect of interactions of


starch concentration and temperature on amylase production.

Figure 4: Graph showing the effect of starch concentration (x-axis)


and CaCl2 concentration (y-axis) on amylase production

68

-amylase production from Endomyces fibuliger an indigenous yeast isolate of Western Himalayas

Kar, S. and Ray, R. 2008. Statistical optimization of -amylase


production by Streptomyces erumpens MTCC 7317 cells in
calcium alginate beads using response surface methodology.
Polish Jour Microbiol, 57: 49-57.
Kwan, S.H., So, K.H., Chan, K.Y. and Cheng, S.C. 1993. Production
of thermotolerant alpha amylases by Bacillus circulans. World
Jour Microbiol Biotechnol, 9: 50-52.
Laemmli, U.K. 1970. Cleavage of structural protein during the
assembly of the head of bacteriophage T4. Nature, 227: 680685.
Miller, G.L. 1950. Use of Dinitrosalicylic acid reagent for
determination of reducing sugar. Anal Chem, 32: 426-428.
Pandey A, Nigam P, Soccal RC, Soccal TV, Singh D and Mohan R.
2000. Review: Advances in microbial amylases. Biotechnol
Appl Biochemistry, 31: 135-152.
Pathania, N., Kanwar, S.S., Jhang, T., Koundal, K.R. and Sharma,
T.R. 2010. Application of different molecular techniques for
deciphering genetic diversity among yeast isolates of
traditional fermented food products of Western Himalayas.
World Journ Microbiol Biotechnol 26:1539-1547.
Rao, M.B., Tanksale, A.M., Gathe, M.S. and Despande, V.V. 1998.
Molecular and Biotechnological aspects of microbial
proteases. Microbiol Molecul Biolog Reviews, 62: 597-635.
Shaw, F.J., Lin, P.F., Chen, C.S. and Chen, C.H. 1995. Purification
and characterization properties of an extracellular alpha
amylase from Thermus sp. Botanical Bulletin of Academia
Sinica, 36: 195-200.
Soni, S.K., Boparai, P.K. and Sandhu, I.K. 1991. Amylase production
by Lipomyces starkeyi. Indian Journ Microbiol, 31: 285-289.
Vandyk, J.W. and Caldwell, M.L. 1956. A study of the kinetics of waxy
maize starch by crystalline pancreatic amylase from swine. Journ
Americ Chemic Socie, 78: 3345-3350.
Vohra, A. and Satyanarayana, T. 2002. Statistical optimization of
the medium components by response surface methodology
to enhance phytase production by Pichia anomala. Process
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Walsh. 2002. Protein Biochemistry and Biotechnology. 547p. John
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activities. Analytic Biochemis, 351:146-148.

the predicted one and the prediction error was calculated. The
observed and the predicted values obtained were quite close
(Table 2) and therefore, showed good predictability of the
model.
Conclusion
Endomyces fibuliger is an excellent producer of alpha-amylase
enzyme, which is para-constitutive in nature. Organism showed
enzyme production in both alkaline and acidic conditions.
Enzyme is active at a wide range of temperature. So, it can play
an important role in various industrial processes viz. brewing,
baking, distillery etc. The indigenous yeast isolate can also be
directly supplied to the local people by co-immobilizing it with
alcohol producing strains for cereal based beverages.
References
Banerjee, M., Debnath, S. and Majumdar, S.K. 1988. Production of
alcohol from starch by direct fermentation. Biotechnology
Bioengineering, 32:831-834.
De Mot, R. and Verachtert, H. 1985. Purification and characterization
of the extracellular amylolytic enzymes from the yeast
Filobasidium capsuligenum. Appl Environmental Microbiol,
50: 1474-1482.
Galdino, A.S., Silva, R.N, Lottermann, M.T., Alvares, A.C.M.,
Moraes LMP, Torres FAG, de Freitas SM, and Ulhoa CJ.
2011. Biochemical and Structural Characterization of Amy1:
An Alpha-Amylase from Cryptococcus flavus Expressed in
Saccharomyces cerevisiae. Enzyme Research, doi:10.4061/
2011/157294.
Gigras, P., Sahai, V. and Gupta, R. 2002. Statistical media optimization
and production of its alpha-amylase from Aspergillus oryzae
in a bioreactor. Curr Microbiol, 45: 203-208.
Haaland, P.D. 1989. Statistical problem solving. In Experimental
Design in Biotechnology ed. Haaland, PD pp. 1-18. New
York and Basel: Marcel Dekker, Inc.
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Clostridium butyricum. Lett Applied Microbiol, 39: 363-368.

69

Intl. J. of Food. Ferment. Technol. 2(1): 71-79, June, 2012

Research paper

Statistical screening of media components for the


production of arginine deiminase by
Weissella confusa GR7
Baljinder Kaur* and Rajinder Kaur
Department of Biotechnology, Punjabi University, Patiala, Punjab, India
*

Email: baljinderbt@hotmail.com

Paper no: 40

Received: 17 Jan, 2012

Received in revised form: 14 April,2012

Accepted: 17 May, 2012

Abstract
A lactic acid bacterial strain capable of metabolizing arginine via arginine deiminase pathway was
isolated from lassi, an Indian fermented food. On the basis of biochemical analysis it was identified as
Weissella confusa. Response surface methodology (RSM) was used to optimize media components
such as carbon and nitrogen sources, minerals, inducers and metal ions for enhancing its specific
arginine deiminase activity. A Placket-Burman design was applied to identify significant factors affecting
enzyme production in Weissella confusa GR7. Central composite rotatable design was applied further
to optimize the concentrations of sucrose, copper, magnesium, manganese and arginine for maximizing
arginine deiminase activity. Statistical design has suggested a Two Factor Interaction model which
was validated experimentally where it showed 10 fold increases in specific arginine deiminase activity
over basal medium. By solving the regression equation and analyzing the response surface cartons,
optimal concentration of various components was determined as 2.5g/l sucrose, 5g/l fructose, 10g/l
tryptone, 5mM MgSO4, 5mM MnSO4, 25mM arginine, 5mM Copper, 50mg/ml CTAB and 50mM Tween
80. The model was found satisfactory as the coefficient of determination was 0.83. Results indicate an
improved arginine deiminase production in statistically optimized medium using response surface
methodology.
2012 New Delhi Publishers. All rights reserved

Keywords: Arginine deiminase, Weissella confusa, Central composite rotatable design, Response
surface methodology, Lactic acid, Bacteria

L-Arginine is a semi-essential amino acid that plays a key role


in cellular metabolism. It not only serves as a building block of
proteins and as a precursor of polyamines, but also as a source
of carbon, nitrogen, and energy through a variety of metabolic
pathways in bacteria. Microorganisms catabolize arginine
mainly through arginase pathway, ADI pathway, arginine
succinyltransferase (AST) pathway and arginine transaminase/
oxidase/dehydrogenase pathway. The ADI pathway was
established in lactic acid bacteria which were described in

strains belonging to genera of Enterococcus, Lactobacillus,


Leuconostoc, Oenococcus, Streptococcus and Weissella (Crow
and Thomas, 1982; Zuniga et al., 2002; Liu et al., 2003; Ammor
and Mayo 2007; Spano et al., 2007; Yu et al., 2010).
Weissella species are Gram-positive, non-spore-forming,
heterofermentative, non-motile, and irregular or coccoid rod
shaped organisms. Members of the genus Weissella have been
isolated from a variety of sources, such as fresh vegetables,
fermented cabbage, fermented foods, and fermented silage,

Kaur and Kaur

meat or meat products and soil (Niven et al., 1957; Milbourne,


1983; Dellaglio et al., 1984; Dellaglio and Torriani, 1986; Collins
et al., 1993; Magnusson et al., 2002; Katina et al., 2009).
Weisella confusa has been detected in sugar cane, carrot juice
and occasionally, in raw milk and sewage (Hammes & Vogel,
1995). Several strains of Weissella have been exploited for
production of exopolysaccharides and dextran from wheat
sourdough baking (Katina et al., 2009). Weissella kimchii
PL9023 is a well established probiotic that can be utilized as a
food source (probiotic) (Lee, 2005). The RSM, formerly known
as Box-Wilson methodology, is the most widely used statistical
technique to evaluate relationship between a set of controllable
experimental factors and observed results (Kennedy and
Krouse, 1999; Lee and Chen, 1997; Liu et al., 2003). RSM is
capable of finding optimum set of experimental factors that
produce maximum or minimum value of response and represent
the direct and interactive effects of process variables through
two dimensional and three dimensional graphs.

activity was based on standard method of Angelis et al. (2002).


Assay mixture consisted of 150l of 50 mM arginine, 2.3ml of
50 mM acetate buffer (pH 5.5), 50 l of cell wall or cytoplasm
preparation, and 3.6 l of sodium azide (0.05%). Controls
without substrate and without enzyme were included. After
incubation at 37oC for 1h, the reaction was stopped by adding
0.5ml solution of 2N HCl and precipitated protein was removed
by centrifugation. Citrulline content in CFS was determined
by Archibalds method (1944). One enzyme unit was defined
as the amount of enzyme required to catalyze formation of
1mol citrulline per min. One milliliter of supernatant was added
to 1.5ml of an acid mixture of H3PO4-H2SO4 (3/1 v/v) and 250 l
of diacetyl monoxime (1.5% 2, 3 butanadiona monoxime) in
10% (v/v) methanol, mixed and boiled in dark for 30 min. After
cooling for 10 min, the absorbance at 460 nm was measured.
Finally, specific activity was calculated as international enzyme
units present per mg (IU/mg) of protein.
Experimental designs

The present study was aimed to statistically screen media


components that increase ADI activity of Weissella species.
There are no reports on the optimization of media components
for ADI enzyme production from Weissella species using RSM.
Media optimization was carried out in two steps: (i) PlackettBurman design used for selection of most influential media
components, (ii) CCRD design was used for further
optimization to enhance ADI activity using influential process
variables.

In present study, MAM broth was used as basal medium and


effect of various carbons, nitrogen sources, minerals, inducers
and metal ions, was studied for enhancing ADI activity of W.
confusa GR7. RSM was adopted for improving enzyme activity
using Design Expert Software version 8.0.2 trial statistical
software (State-Ease Inc., Minneaopolis, MN, USA). PlackettBurman design is an efficient tool, especially when a large
number of variables are involved, for screening variables that
are able to determine the influence of various factors with
minimal number of experiments, which is time saving and
maintain convincing information on each component (Krishnan
et al., 1998; Abdel-Fattah et al., 2005). It is a saturated
orthogonal design work at two-levels, and can be constructed
on the basis of fractional replication of a full factorial design.
Total number of experiments to be carried out in PlackettBurman design are n+1, where n is the number of variables.
Each variable was represented at two levels, upper and lower
denoted by high (+) and low (-) respectively (Plackett and
Burman, 1946). Here, a total of eleven factors or variables were
screened in twelve trials. 11 variables namely fructose, sucrose,
tryptone, meat extract, MnSO4, MgSO4, copper, arginine, CTAB,
tween 80, CO2 were screened as shown in Table1 whereas,
phosphate buffer, pH-6, temperature at 37oC and incubation
time of 24 h were kept constant. Effect of each variable is
quantified as difference between the average of measurements
made at the high and the average of measurements made at the
low level of that factor. It is possible to calculate effect of each
variable on ADI activity following equation no. 1.

Materials and methods


Bacterial strain and culture medium used
A lactic acid bacterial (LAB) isolate Weissella confusa GR7
(phenotypic identification by MTTC, Chandigarh) isolated
from dairy sample collected from local market was used in the
present study. MAM medium consisting of tryptone-10g;
yeast extract-5g; arginine-3g; KH2PO4-0.5g; MgSO4-0.2g;
MnSO4-0.05g; Tween80-1ml; glucose-5g; Agar-2g; H2O1000ml, (pH 6.0) was used for enzyme production. Optical
density of the inoculums was adjusted to 1.0 and 1% (v/v)
inoculum was used. Cultures were incubated at 37oC for 24 h
and sub-cultured thrice in MAM broth, before subjecting to
ADI assay.
Enzyme assay
To determine enzyme activity, 24h old cultures were centrifuged
at 8,000 rpm for 10min. Cell free supernatant (CFS) was assessed
for extracellular protein and enzyme activity. For assaying
intracellular ADI activity, cell pellet was resuspended in lysis
buffer. Total protein was estimated by measuring absorption
at 280nm using standard curve of BSA. The assay of ADI

E (Xi) = (2 ( Pi+ - Pi-) / N.....................................................(1)


Where E (Xi) is the concentration effect of tested variables;
Pi+ and Pi- represent ADI activities obtained in various trials,
72

Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7

where the variable (Xi) measured was present at the high or


low level, respectively, and N is the number of trials
(experiments) performed.

contribution towards enzyme activity. The experimental results


of Plackett-Burman design were fitted in central composite
rotatable design (CCRD) of experiments to be employed to
optimize concentration of most contributing factors for ADI
activity (Table2). Fructose (5g/l), CTAB (50mM), tween 80
(50mM), phosphate buffer (50mM), pH-6, temperature 37C
and incubation time of 24 h were constant factors as were
influencing ADI activity, therefore kept at specific value during
CCRD design. Sucrose (5g/l), copper (10mM), MgSO4 (10mM),
MnSO 4 (10mM) and arginine (50mM) concentrations
significantly enhanced ADI activity in W. confusa GR7.

Standard error (SE) of the concentration effect was the square


root of the variance of an effect, and the significance level
(P-value) of the effect of each constituent was determined
using students t-test as given by the equation no. 2;
t (xi)=E(xi)/SE........................................................................(2)
Where E (xi) is the effect of variable Xi
This model does not describe the interaction among factors
and it is used for preliminary screening and evaluates important
factors that influence the response. From the regression
analysis of the variables, the factors having a significant effect
on ADI activity were further optimized by central composite
rotatable design (CCRD). Total 50 experiments were employed
in CCRD to estimate curvature and interaction effects of
selected five variables i.e. sucrose (A), copper (B), MgSO4 (C),
MnSO4 (D) and arginine (E). All experiments were carried out
at 37oC and pH-6 for 24 h. Fructose (5g/l), CTAB (50mM), tween
80 (50mM) and phosphate buffer (50mM) were kept as constant
factors as they are known to influence ADI activity.

Results so obtained were fed into Design-Expert software and


analyzed using analysis of variance (ANOVA) as appropriate
to the experimental design used. Based on the CCRD, the
experimental levels of specific ADI activity under each set of
condition were determined and compared with the
corresponding predicted levels (Table 2). The maximum
experimental value for ADI specific activity was 2.8081 IU/mg,
while RSM predicted response as 1.76 IU/mg. The close
correlation between the experimental and predicted data
indicates the appropriateness of the experimental design. The
quality of the model can also be checked using various criteria.
The calculated regression equation for the optimization of
media constituents assessed the specific activity (Y) as a
function of these variables. Multiple regression analysis of
the experimental data was carried out and statistical equation
was generated that gives ADI enzyme activity according to
equation no. 3 as shown below:

Results and discussion


When grown in unoptimized modified MAM broth, Weissella
confusa GR7 showed 0.27 IU/mg of specific ADI activity.
Specific ADI activity was further optimized using two statistical
tools (Plackett-Burman design and CCRD). The PlackettBurman design screens important variables that may affect
production of this therapeutic protein i.e ADI, but does not
consider the interactive effects among the variables as in CCRD.
In CCRD, each selected variable is studied at five different
levels along with other variables, and therefore, interactions
among these variables at different levels could be studied. In
order to enhance enzyme activity, experiments were designed
to optimize media constituents using Plackett- Burman
experimental design. Table 1 presents PlackettBurman design
for 11 culture variables and their corresponding response in
terms of specific enzyme activity. When the concentration effect
value (E (Xi)) of the tested variable was positive, the influence
of the variable was greater at the high concentration tested,
and when negative, the influence of the variable was greater at
low concentration. The variation in ADI specific activity in
different sets ranged from -0.0138 to 2.0455 IU/mg, reiterating
the importance of selection and identification of important
factors. On the basis of their percent contribution, fructose,
sucrose, MgSO4, CTAB, tween 80 and copper are proved to be
very effective for maximizing enzyme activity from this lactic
acid bacterial isolate. Other variables had only a minor

Y = +0.68+0.12*A-0.15*B-0.011*C-0.11
*D+0.11*E-0.17*A*B+0.046*A*C0.18*A*D+0.12*A*E+0.051*B*C+0.19*B*D
-0.11*B*E-0.010*C*D+0.064*C*E-0.18*D*E........................(3)
Where, Y is the specific enzyme activity and A, B, C, D, E is
coded values of sucrose, copper, magnesium, manganese, and
arginine respectively.
Table 3 shows ANOVA results for the RSM Two Factor
Interaction (2FI) for response Y. According to the present model
A, B, D, E, AB, AD, AE, BD, BE and DE are significant model
terms.2FI model was found to be the best fit model for the
specific enzyme activity with the highest F-value when
compared to other models (Table 4). ANOVA for ADI specific
activity (Y, IU/mg) indicated the F-value to be 11.10, which
implies the model to be significant. Model terms having values
of Prob > F less than 0.0500 indicate model terms are
significant, whereas those greater than 0.10 are insignificant.
The Lack of Fit F-value of 138.35 implies the Lack of Fit is
significant. There is only a 0.01% chance that a Lack of Fit Fvalue this large could occur due to noise. ANOVA indicated
the R2 value of 0.8305 for response Y. This ensured a good

73

Fructose)
(g/l

0
5
0
5
5
0
0
5
0
5
5
0

Run

1
2
3
4
5
6
7
8
9
10
11
12

5
0
5
0
5
0
0
5
0
0
5
5

0
0
10
10
0
0
0
0
10
10
10
10

10
0
10
10
0
0
10
10
0
10
0
0

10
10
0
10
0
0
0
10
10
0
0
10

Sucrose Meat ExtractTryptone CuSO


(mM)
(g/l)
(g/l)
(g/l)
4

0
0
0
0
10
0
10
10
10
10
0
10

MgSO
(mM)
4

Table 1: Experimental run and results obtained in Plackett-Burman design

10
10
0
0
0
0
10
0
0
10
10
10

MnSO
(mM)
4

50
50
50
0
50
0
0
0
50
50
0
0

CTAB
(mg/ml)

50
0
0
50
50
0
50
0
50
0
50
0

0
50
50
0
0
0
50
50
50
0
50
0

Tween-80 Arginine
(mM)
(mM)

0
5
5
5
5
0
5
0
0
0
0
5

%
CO2

0.43
1.62
0.4
0.27
2.04
-0.01
0.58
0.38
0.44
0.15
0.2
0.25

Specific
activity
Experimental
value

0.57
0.57
0.57
0.57
0.57
0.57
0.57
0.57
0.57
0.57
0.57
0.57

ADI
(IU/mg)
Predicted
value

Kaur and Kaur

74

Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7

Table 2: Experimental run and results obtained in CCRD design


Run

Sucrose
(g/l)

CuSO4
(mM)

MgSO4
(mM)

MnSO4
(mM)

Arginine
(mM)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50

5
5
0
2.5
5
8.4
2.5
2.5
5
0
2.5
2.5
2.5
0
5
5
0
0
0
-3.4
0
5
2.5
5
5
0
5
0
5
2.5
5
0
5
2.5
0
0
0
2.5
5
0
2.5
2.5
2.5
5
2.5
2.5
2.5
0
5
0

10
0
10
5
0
5
-6.9
5
10
10
5
5
5
0
10
10
10
10
0
5
10
0
5
0
0
0
0
0
0
5
0
0
10
5
10
10
0
5
10
10
5
5
5
10
5
16.9
5
0
10
0

0
0
10
5
10
5
5
-6.9
10
0
5
5
5
10
10
0
0
0
0
5
10
0
5
10
0
10
10
10
0
16.9
10
0
0
5
10
10
0
5
0
0
5
5
5
10
5
5
5
0
10
10

0
0
10
5
0
5
5
5
0
0
5
-6.9
5
0
10
10
0
10
10
5
0
10
5
0
10
0
10
10
0
5
10
0
10
5
0
10
10
5
0
10
16.9
5
5
0
5
5
5
0
10
10

0
50
50
25
50
25
25
25
50
50
84.5
25
25
50
0
0
0
0
50
25
50
0
25
0
50
0
0
0
0
25
50
50
50
25
0
0
0
25
50
50
25
25
25
0
-34.5
25
25
0
50
50

75

Specific ADI activity (IU/mg)


Experimental Value
Predicted Value
0.34
2.33
0.7
0.79
2.81
0.76
1.05
0.88
0.93
0.47
0.47
0.77
0.77
0.71
0.52
0.52
0.32
0.72
0.51
0.51
0.52
0.51
0.78
0.78
0.59
0.31
0.42
0.33
0.98
0.51
0.66
0.73
0.21
0.73
0.29
0.83
0.67
0.74
0.57
0.53
0.56
0.74
0.77
0.32
0.51
0.31
0.78
0.57
0.55
0.28

0.68
0.28
0.97
0.56
1.76
1.03
1.04
0.68
0.71
2.17
2.05
0.44
0.17
0.48
0.31
1.05
0.45
1.16
0.57
0.95
0.39
0.24
0.9
069
0.31
0.39
0.41
0.4
0.68
0.58
0.33
0.68
0.71
1.02
0.21
0.68
0.32
0.83
0.42
0.43
0.68
1.21
0.44
0.25
0.96
0.68
0.32
0.65
0.93
0.68

Kaur and Kaur

Table 3: One way ANOVA analysis of Response Surface designs


Source

Sum of squares

Model
A-Sucrose
B-Copper
C-Magnesium
D-Manganese
E-Arginine
AB
AC
AD
AE
BC
BD
BE
CD
CE
DE
Residual
Lack of Fit
Pure Error
Cor Total

7.87
0.61
1.02
5.42E-03
0.56
0.48
0.91
0.067
1.03
0.47
0.082
1.14
0.37
3.39E-03
0.13
0.99
1.61
1.6
3.01E-03
9.48

Degree of freedom
15
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
34
27
7
49

Mean squares

F- value

p-value
prob > F

0.52
0.61
1.02
5.42E-03
0.56
0.48
0.91
0.067
1.03
0.47
0.082
1.14
0.37
3.39E-03
0.13
0.99
0.047
0.059
4.29E-04

11.1
12.87
21.51
0.11
11.9
10.18
19.25
1.43
21.71
9.88
1.74
24.2
7.89
0.072
2.8
21

< 0.0001
0.001
< 0.0001
0.737
0.0015
0.003
0.0001
0.2407
< 0.0001
0.0035
0.1964
< 0.0001
0.0082
0.7903
0.1033
< 0.0001

significant

138.35

< 0.0001

significant

Coefficient of determination (R2) = 0.8305


Table 4: Statistical summary of model
Source

Sum of Squares

Mean vs Total
Linear vs Mean
2FI vs Linear
Quadratic vs 2FI
Cubic vs Quadratic
Residual
Total

23.07
2.67
5.2
0.13
1.34
0.14
32.54

Degree of freedom
1
5
10
5
15
14
50

Mean Square
23.07
0.53
0.52
0.026
0.089
9.93E-03
0.65

correlation between observed and expected values of the 2FI


model indicated that this model could explain 83% response
variability. The adequate precision which measures the signal
to noise ratio of 16.229 indicates an adequate signal. A ratio
greater than 4 is desirable.

F-value

p-value

Prob > F

3.46
11
0.5
9

0.0101
< 0.0001
0.7734
< 0.0001

Linear vs Mean
Suggested
Aliased

MgSO4, 5.0 mM MnSO4. Results indicated that concentration


of sucrose in the culture medium had profound effect on specific
activity of arginine deiminase (Figure 1a to 1c ). CCRD employed
has signified that sucrose at concentration of 2.5 g/l was
optimal for maximum enzyme activity. Similar effect was
observed in case of arginine which was used as an inducer of
ADI activity. As its concentration is raised there is concomitant
increase in specific ADI activity (Figure 1a, 1d and 1e). Results
indicated a central value of arginine as 25 mM for optimal
enzyme production. Presence of CuSO4 and MnSO4 caused
marginal increase in ADI activity as compared to other two
components (Figure 1b to 1e).

The three-dimensional response surface plots based on


interactions between the variables showed an increase in
specific ADI activity as the concentration of each variable
reached an optimum level, beyond which a decline was
observed (Figure 1a to 1e). 3D response surface plots were
obtained by plotting response i.e specific ADI activity (IU/
mg) on the Z-axis against any two variables on X and Y-axis
while keeping other three variables at their central values, were
2.50g/l sucrose, 25 mM arginine, 5.0 mM CuSO4, 5.0 mM

Weissella confusa GR7 showed 0.27 IU/mg specific ADI activity


in unoptimized MAM broth, which was raised to 2.8081 IU/mg
76

Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7

(a)

Figure1: Three-dimensional response cartons showing effects and interaction of (a) arginine (mM) and sucrose (g/l), (b) copper sulphate (mM)
and sucrose (g/l), (c) manganese sulphate (mM) and sucrose (g/l), (d) arginine (mM) and manganese sulphate (mM) and (e) arginine (mM) and
copper sulphate (mM) on specific ADI activity of W. confusa GR7.

77

Kaur and Kaur

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in RSM optimized media (run 5). It was improved by optimizing


concentration of arginine, sucrose, copper, manganese
sulphate and magnesium sulphate concentration. Arginine
influenced ADI activity as it acts as an inducer for ADI activity
as previously reported in Streptococcus lactis, Lactobacillus
sanfrancisciscensis CB1, Weissella koreensisMSI-3, Weissella
koreensis MSI-14, Lactobacillus buchneri CUC-3 (Crow and
Thomas, 1982; Mira de Ordua et al., 2000; Angelis et al.,
2002; Yu et al., 2010). The nature and concentration of sugars
also affect ADI activity as reported earlier in Lactobacillus
sanfrancisciscensis CB1 and did not grow in MAM medium
with arginine as sole carbon source (Angelis et al., 2002). In
Streptococcus lactis ADI activity was increased 5- to- 10 fold
in galactose grown cultures as compared to glucose or lactose
grown cultures (Crow and Thomas, 1982). Higher concentration
of sugars may repress ADI activity as reported in Lactobacillus
sake (Montel et al., 1987). In our case the effect of sugars,
inorganic nutrients and surfactants on the activity of ADI was
investigated and results showed maximum specific activity at
concentration of 2.5g/l sucrose, 5mM MgSO4, 5mM Copper,
and 50mg/ml CTAB. Similar, findings are also reported in a
Enterococcus faecalis NJ402 isolate (Li et al., 2005). Based on
the results obtained, mediums consisting of sucrose (2.5g/l),
fructose (5g/l), tryptone (10g/l), MgSO4 (5mM), MnSO4 (5mM),
arginine (25mM), CuSO4 (5mM), CTAB (50mg/ml) and tween
80 (50mM), pH 6, at 37 oC for 24 h was optimized.
Conclusion
Sequential statistical strategies, Placket- Burman design
followed by CCRD were used successfully to find out optimum
values of the significant factors to achieve maximum ADI
activity in W.confusa GR7. The experimental result (2.8081IU/
mg) in a medium optimized for ADI production was in close
agreement with the predicted value of 2FI model (1.76 IU/mg)
and hence, the model was successfully validated. The specific
enzyme activity showed about 10 fold increases over the basal
medium. Further, it is important to discover newer lactic acid
bacterial strain via ADI pathway that produces therapeutic
enzyme of importance in healthcare industry.
Acknowledgement
Authors acknowledge UGC, New Delhi for funding Maulana
Azad National Fellowship for Minority Students to Mrs.
Rajinder Kaur (vide letter no.F.40-116(M/S)/2009(SA-III/
MANF).
References
Abdel-Fattah, Y.R., Saeed, H.M., Gohar, Y.M. and El-Baz, M.A.
2005. Improved production of Pseudomonas aeruginosa
uricase by optimization of process parameters through

78

Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7

soli sp. nov., a lactic acid bacterium isolated from soil. Int. J.
Syst. Evol. Microbiol., 52:831-834.
Milbourne, K. 1983. Thermal tolerance of Lactobacillus viridescens
in ham. Meat Sci. 9: 113-119.
Mira de Ordua, R., Liu, S.Q., Patchett, M.L. and Pilone, G.J. 2000.
Ethyl carbamate precursor citrulline formation from arginine
degradation by malolactic wine lactic acid bacteria. FEMS
Microbiol. Lett., 183:31-35.
Montel, M.C. and Champomier, M.C. 1987. Arginine catabolism in
Lactobacillus sake isolated from meat. Appl. Environ.
Microbiol., 53:2683-2685.
Niven, C.F., JR., and Evans, J.B. 1957. Lactobacillus viridescens
nov. sp., a heterofermentative species that produces a green
discolouration of cured meat pigments. J. Bacteriol., 73:758759.

Plackett, R.L. and Burman, J.P. 1946. The design of optimum


multifactorial experiments. Biometrika., 33:305-325.
Spano, G. Massa, S. Arena, M.E. Manca de Nadra, M.C. 2007.
Arginine metabolism in wine Lactobacillus plantarum: in vitro
activities of the enzymes arginine deiminase (ADI) and
ornithine transcarbamilase (OTCase). Ann. Microbiol., 57:6770.
Yu, J.J. and Oh, S.H. 2010. Isolation and characterization of lactic
acid bacteria strains with producing capacity from natural
sea salt. J. Microbiol., 48:467-472.
Zuniga, M. Miralles, M.C. and Perez-Martinez,G. 2002. The product
of arcR, the sixth gene of the arc Operon of Lactobacillus
sakei, is essential for expression of the arginine deiminase
pathway. Appl. Environ. Microbiol., 68: 6051-6058.

79

Intl. J. of Food. Ferment. Technol. 2(1): 81-86, June, 2012

Research paper

Effect of nitrogen source on the fermentation behaviour


of musts and quality of wine from two Cvs. of pineapple
B.L. Attri

Central Institute of Temperate Horticulture, Regional Station, Mukteshwar - 263 138 (Uttarakhand), India
*Email: attribl_cith@rediffmail.com
Paper no: 41

Received: 19 Jan, 2012

Received in revised form: 25 April, 2012

Accepted: 19 May, 2012

Abstract
The composition of the musts prepared from the pineapple fruits cvs. Kew and Queen by amelioration
of TSS by addition of KMS (100ppm), with and without DAHP (0.1%) as nitrogen source was found
to vary significantly. The fermentation rate of the pineapple musts having nitrogen source, carried out
by the yeast Saccharomyces cerevisiae var. ellipsoideus was comparatively faster than those without
DAHP and over a period of 10 days. After six months of storage the physico-chemical characteristics
of the pineapple wines revealed significantly higher alcohol (10.56 and 11.44 %) in the treatments
having nitrogen source with better fermentation efficiency of yeast (48 and 52%). The acidity and
volatile acidity of these treatments was significantly higher. The pineapple wines having more alcohol
were found to have lower aldehydes (112.78 and 117.47 mg/litre) whereas total esters (94.65 and 104.77
mg/litre) and phenolic contents (148.70 and 156.54 mg/litre) were found to be higher. From the sensory
quality point of view, the wine prepared from cv. Queen was preferred to cv Kew. The costs of the final
products were Rs. 35.38 and Rs. Rs. 31.82 per 650 ml bottle in cv. Kew and Queen, respectively.
2012 New Delhi Publishers. All rights reserved

Keywords: Keywords: Pineapple nitrogen source, must, fermentation, wine, phenolic content,
Saccharomyces cerevisiae, sensory

Introduction
The pineapple (Ananas comosus (L.) Merr. Syn. Ananas sativus
Schult.f) originated from Paraguay belongs to family
Bromeliaceae (Bertoni 1919). It is one of the most important
and wanted tropical fruits because of good source of vitamin
A, B, C and calcium. Apart from its various products viz., slices,
juice, squash, jam, and mixed fruit jam, other by-products are
alcohol, calcium citrate, citric acid and vinegar. The dried waste
after juice extraction is valuable cattle feed. It is being cultivated
in different countries like Hawaiian Islands, Philippines,
Malaysia, Thailand, Brazil, Ghanna, Kenya, Mexico, Taiwan,
S. Africa, Australia and India. It was introduced in India in

1548 and is coming up well with a total area of about 25,000 ha


in North- Eastern states viz., Assam, Tripura, and Meghalaya,
W. Bengal, Kerala, Tamilnadu, Goa, Karnataka and Orissa (Sen,
1985). The warm and humid tropical climatic conditions of
Andaman and Nicobar Islands, situated between 100 31 and
130 42 North latitude and 920 14 and 940 16 East longitude in
the Bay of Bengal, are congenial for its cultivation. Although,
the fruits is used as fresh as well as processed into various
forms but glut production is high, producers do not get
remunerative returns and are forced to sell their produce at
very low prices. The composition as well as the nutritional
status of pineapple fruits grown in these islands is at par with
the fruits being produced elsewhere in other parts of the

Attri

country. Apart from other products that can be processed into


alcoholic beverages from the extracted juice which is a good
alternative that in turn will increase the socio-economic status
of local farmers. The Kew and Queen cultivars of pineapple
are performing well in these islands. The area and production
of pineapple fruits is expected to increase substantially because
of adoption of year round production technology (Singh, 1996).
Different products prepared from pineapple have been well
documented (Anon. 1985) but information on pineapple wine
preparation is meager. Therefore, the present investigations
were carried out to utilize the pineapple fruits of these cultivars
grown in Andaman and Nicobar Islands for wine production.

at room temperature for ageing/maturation upto six months.


The flow diagram for the preparation of pineapple wines has
been illustrated in Figure 1.
Analysis of juice and wine

Materials and methods


Collection of pineapple fruits
The physiologically matured fruits of pineapple cvs. Kew and
Queen were harvested from the research farm of Central
Agricultural Research Institute, Port Blair . The harvested fruits
were allowed to ripen in the laboratory at a room temperature
of 25-300 C and after getting the required colour of the skin of
the fruits, these were analysed for various physical characters.
Extraction of juice
The fully ripened fruits of both the pineapple cvs. were peeled
manually with steel knife and after cutting into small pieces
and removing the central core, the juice was extracted by
passing the pineapple pieces through a juicer. The extracted
juice was filtered through a muslin cloth followed by heating
upto 85-90o C for 5 minutes in a steel container.
Preparation of must
To prepare pineapple must, the total soluble solids (TSS) of
the juice were ameliorated to 220 B with 70 per cent sugar syrup.
The prepared must was filled in glass containers having 100
ppm potassium metabisulphite (KMS). Further, di-ammonium
hydrogen phosphate (DAHP) @ 0.1% was added as a nitrogen
source in two treatments (T1 and T3) whereas other two (T2
and T4) musts were devoid of any nitrogen source.
Fermentation and maturation

Figure 1: Flow diagram for the preparation of pineapple wine

An active culture of yeast Saccharomyces cerevisiae var.


ellipsoideus was added @ 5% (v/v) containing approximately
104-105 cells/ ml in the glass containers having must, equipped
with air locks at a temperature of 221oC. Samples of the
fermenting musts were taken periodically to determine the
changes in total soluble solids (oB). The fermentation rate (fall
of TSS/24hour) was recorded till the TSS became stable. The
pineapple wine thus prepared was siphoned, filtered and stored

The different physico-chemical characteristics i.e., Total


Soluble Solids (0Brix), titratable acidity (% malic acid), pH of
extracted juice and moisture (%) of the fruit were measured by
standard methods described by Ranganna (1986) whereas
ascorbic acid (mg/100g) and carotenoid contents (g/100g)
were estimated as per the methods of AOAC (1975).
After storage, different treatments of pineapple wine were

82

Effect of nitrogen source on the fermentation behaviour of musts and quality of wine

analyzed for various physico-chemical characteristics and


overall sensory quality. The TSS, acidity, pH and ascorbic
acid were recorded as described earlier. The alcohol (%) was
estimated by a standard colorimetric methods given by Caputi
et al (1968), volatile acidity (% Acetic acid) by the methods of
Pilone et al (1972) whereas aldehydes and total esters were
determined according to the methods described by Amerine
and Ough (1979) and Libraty (1961) respectively. The total
phenols were estimated by the method of Singleton and Rossi
(1965).

yeast cells which has also been reported earlier by Joshi et al.
(1991). The fermentation rate however declined towards the
completion of fermentation which may be due to more alcohol
production inhibiting the fermentation efficiency of the yeast
(Attri et al 1994). The fermentation efficiency of the yeast was
found 48 and 52 per cent in T1 and T-3 (nitrogen source) as
compared to 45 and 50 per cent in T2 and T-4 (without nitrogen
source) which has been depicted in Fig. 3. The higher efficiency
may be attributed to the addition of nitrogen source for the
yeast during fermentation.
Table 1: Composition of different cultivars of pineapple

Sensory analysis

Characters

For overall sensory quality, a panel of 10 judges was asked to


give score out of 20,keeping in view the colour, appearance,
aroma and taste of the wines.

Length (cm)
Breadth (cm)
Weight (g)
Volume (ml)
Specific gravity (w/v)
Juice (%)
Waste (%)
T.S.S. (0B)
Acidity (% MA)
Brix acid ratio
Ascorbic acid (mg/100g)
Carotenoids (g/100g)
Moisture (%)
Dry matter (%)

Statistical analysis
The available data for physico-chemical characteristics of
different pineapple musts and wines were analysed in a
Completely Randomized Block Design (CRBD) as described
by Panse and Sukhatme (1989).
Cost of production of pineapple wine
The cost of production of pineapple wines from both the cvs.
Kew and Queen was also estimated keeping in view the present
rates of various items used in the fermentation and thereafter.
The overhead charges as well as additional charges on
fermentation were also added along with sales tax and profit of
the producer.

\Cultivars
Kew

Queen

15.700.040
12.500.040
1060.008.16
1099.007.55
0.9640.0008
56.000.489
44.000.489
15.500.081
0.9380.0016
16.520.057
39.130.257
38.990.608
78.830.048
21.170.048

10.500.092
9.800.092
680.004.08
707.001.53
0.9620.0002
52.000.408
48.000.408
12.000.081
1.0450.0008
11.480.069
45.000.326
58.000.347
76.9800.122
23.100.122

The physico-chemical characteristics of the wines revealed


significant differences in all the treatments. The TSS of the
wines prepared by adding DAHP was lower as compared to
those without it. It might be due to higher utilization of the
sugars by yeast during fermentation. The acidity (%MA) of
all the pineapple wines del to increased during fermentation.
Attri (2009) reported that initial sugar concentration increased
the acidity in the cashew apple wine. The pH of the wines were
found in corrobation with acidity. Similarly, the ascorbic acid
which was 39.13 and 45.00 mg/100g in extracted juice of both
the pineapple cvs. was reduced to 15.40 and 19.50 mg/100g,
respectively in T2 and T-4 whereas 18.70 and 21.76 mg/100g,
respectively in T1 and T-3. The reduction may be attributed
due to the loss during boiling of the extracted juice. The alcohol
production was significantly better in the treatments having N
source than others which may be due to the more utilization of
the sugars during fermentation. Likewise acidity, the volatile
acidity (%AA) was also recorded significantly higher in the
wines with more alcohol content.

Results and discussion


The composition of both the cvs. of pineapple (Table 1) was
comparable to those which are being cultivated elsewhere in
the country. The length, breadth, weight, volume, juice (%),
TSS, brix acid ratio and moisture (%) of the fruits recorded was
comparatively more in Kew than Queen cultivar whereas the
acidity (%), ascorbic acid, carotenoid contents and dry matter
were higher in the latter than the former. The difference in the
physico-chemical characteristics of the pineapple cultivars
may be due to their inherent genetic characteristics. The
composition of the musts of two cvs. with and without nitrogen
source (DAHP) was found to be different appreciably (Table
2). The variation may be attributed to the initial composition of
the pineapple fruit juice. The fermentation of the musts was
very fast and the TSS became stable just after 10 days (Figure
2). It is apperent that the fermentation was comparatively faster
in the treatments having DAHP than those without DAHP.
The higher fermentation rate in the musts having nitrogen
source may be due to fast growth and multiplication of the

The other constituents viz., aldehydes, total esters and


phenols varied significantly among the wines of both cultivars

83

Attri

Table 2: Physico-chemical characteristics of pineapple musts


Cultivars

Treatments

Kew

Characteristics of musts
TSS (oB)

Acidity (%MA)

22.0
22.0
22.0
22.0
NS

0.850
0.875
1.010
1.014
0.004

T1
T2
T3
T4
-

Queen
CD=0.05

p H Ascorbic acid (mg/100g) Carotenoids (g/100g)


4.12
4.08
3.98
3.92
0.046

34.35
35.50
41.20
42.45
0.465

27.65
29.57
43.87
45.28
0.254

T1=Must with 0.1% DAHP, T2= Must without DAHP, T3= Must with 0.1% DAHP, T4 =Must without DAHP

Figure 2: Fermentation rate of different treatments of pineapple


must

Figure 3: Comparison of fermentation efficiency of two musts by


yeasts

(Table 3). The aldehydes contents were recorded significantly


lower 112.78 and 117.47 mg/litre in T1 and T-3 having more
alcohol as compared to 119.45 and 123.86 mg/litre in T2 and T-4
respectively in Kew and Queen. The total esters were
significantly better (94.65 and 104.77 mg/litre) in T1 and T-3
than T2 and T-4 (86.97 and 101.20 mg/litre). Likewise total esters,
total phenolic contents were also recorded higher in T1 and
T-3 (148.70 and 156.54 mg/litre) than T2 and T-4 (141.63 and

151.87 mg/litre). The results are in confirmation with those of


Attri (2009) in cashew apple wine where higher alcohol
production have reduced aldehydes and increased total esters
and phenolic constituents.
In the overall sensory quality, the pineapple wine from cv.
Queen had an edge over than that of Kew (Figure 4) which
may be due to better colour of the final product because of
initial colour of juice.

Table 3 : Physico-chemical characteristics of pineapple wines


Cultivars Treatments

Kew
Queen
CD=0.05

T1
T2
T3
T4

Characteristics of wines
TSS
(o B)

Acidity
(%MA)

4.8
5.0
4.6
5.0
0.399

1.012
0.975
1.045
1.020
0.0023

pH
Ascorbic acid
(mg/100g)
(% v/v)
4.00
4.06
3.89
3.94
0.113

18.70
15.40
21.76
19.50
0.118

84

Alcohol
acidity
(%AA)

Volatile
(mg/litre)

Aldehydes
(mg/ litre)

10.56
9.90
11.44
11.00
0.143

0.009
0.007
0.010
0.008
0.012

112.78
119.45
117.47
123.86
0.330

Total esters
Total
(mg/litre) phenols
94.65
86.97
104.77
101.20
0.173

148.70
141.63
156.54
151.87
0.131

Effect of nitrogen source on the fermentation behaviour of musts and quality of wine

Table 4: Cost of production of pineapple wine


S. No. Material required

Kew
Quantity

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.

Queen

Rate (Rs.)

Amount (Rs.)

Quantity

Rate (Rs.)

Amount (Rs.)

Pineapple fruit
100kg
Transportation
Juice extraction
2 mandays
Sugar
4.0 kg
Potassium metabisulphite @ 100 ppm
DAHP @ 0.1%
65.0g
Glass bottles (650 ml)
100
Crown corks
100
Total cost
Overhead charges @20%
Additional charges @ 15% on fermentation
Excise duty
Grand total
Sales tax @20%
Profit @ 20%
-

10.00/kg
60.00/day
18.00/kg
6.5g
3.00/bottle
0.25/cork
3.00/bottle
-

100 kg
2 mandays
5.0 kg
15.00
65.0g
100
100
240.00
-

8.00/kg
60.00/day
18.00/kg
6.5g
3.00/bottle
0.25/cork
3.00/bottle
-

Sale price/bottle

1000.00
50.00
120.00
72.00
15.00
300.00
25.00
1597.00
320.00
300.00
2457.00
491.00
590.00
3538.00
35.38

800.00
50.00
120.00
90.00
15.00
15.00
300.00
25.00
1415.00
283.00
212.00
300.00
2210.00
442.00
530.00
3182.00
31.82

Conclusion
The wine made by the addition of nitrogen sources had better
fermentability than those without it. The physico-chemical and
sensory qualities of both the wines were acceptable but the
product from cv. Queen was preferred. The cost of production
of pineapple wine was also preferable. It can be concluded
that there is an ample scope for the utilization of pineapple
fruits for production of wine which are good in nutrients and
available throughout the year in the islands by the method
described here.
References
Amerine MA and Ough CS. 1979. Wine and Must Analysis, 2nd Edn.
John Wiley and Sons, New York.
Anonymous. 1985. Home Scale Processing and Preservation of Fruits
and Vegetables. 7th Edn. CFTRI, Mysore. Pp: 28-29.
AOAC. 1975. Official Methods of Analysis. Association of Official
Analytical Chemists. 12th Edn. Washington, D.C.
Attri BL, Lal BB and Joshi VK. 1994. Technology for the preparation
of sand pear vermouth. Indian Food Packer, 48(1):39-45.
Attri BL. 2009. Effect of initial sugar concentration on the physicochemical characteristics and sensory qualities of cashew apple
wine. Natural Product Radiance, 8(4):374-379.
Bertoni MS. 1919. Ancient Paraguay, Series II, 4: 250-332.
Caputi A, Ucda Jr M and Brown T. 1968. Spectrophotometric
determination of ethanol in wine. Am. J. Enol. Vitic., 19:160165.
Joshi VK, Attri BL, Gupta JK and Chopra SK. 1990. Comparative
fermentation behaviour, physico-chemical characteristics and

Figure 4: Overall sensory quality of different pineapple wines


(maximum score = 20, NS = Nitrogen source)

Further, for developing any product, the most important factor


is its cost of production. It has been calculated on lab scale
(Table 4) which indicated that the product can be sold at a
reasonable cost with good profit margin. The cost of production
calculated for the product (Rs. 35.38 and 31.82 for Kew and
Queen pineapple wine) is quite reasonable for any low alcoholic
beverage available in the market. The product prepared from
the pineapple fruits is comparable to other fruit wines described
by Joshi et al (1990) and Attri et al (1994).

85

Attri

sensory qualities of fruit honey wines. Indian J. Hort., 47


(1): 49-54.
Joshi VK, Sharma PC and Attri BL. 1991. A note on deacidification
activity of Schizosaccharomyces pombe in plum must of
variable compositions. J. Applied Bacteriol, 70: 385-390.
Libraty V. 1961. Ester determination and their application in wine.
M.Sc. Thesis. University of California, Davis.
Panse VG and Sukhatme PV. 1989. Statistical Methods for
Agricultural Workers. ICAR Publication, N. Delhi.
Pilone GJ, Rankine BC and Hatcher CJ. 1972. Evaluation of an
improved method of measuring volatile acid in wine. Brewing
Spirit Rev., 91:62-66.

Ranganna S. 1986. Handbook of Analysis and Quality Control for


Fruit and Vegetable Products. 2nd Edn. Tata McGraw Hill
Pub. Co. Ltd., N. Delhi.
Sen SK. 1985. Pineapple, In Fruits of India: Tropical and Sub Tropical,
edited by T.K. Bose and S.K. Mitra. Nayaprakash Publishers,
Calcutta. pp: 296-319.
Singh DB. 1996. Year round pineapple production. The Hindu, Dec.,
26, 1996. pp. 28.
Singleton VL and Rossi JA Jr. 1965. Colorimetetry of total phenolics
with phosphomolybdic-phosphotungustic acid reagents.
Am.J. Enol.Vitic., 16:144-158.

86

Intl. J. of Food. Ferment. Technol. 2(1): 87-91, June, 2012

Research note

Effect of Solid state fermentation and yeast species on


composition of apple pomace: Application of PCA
V.K. Joshi1* and D.K. Sandhu2

Department of Food Science and Technology Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal
Pradesh,India
2
Department of Microbiology,Guru Nanak Dev University,Amritsar,Punjab,India
*

Email: v.k.joshipht@rediffmail.com

Paper no: 42

Received: 19 Jan, 2012

Received in revised form: 14 April, 2012

Accepted: 21 May, 2012

Abstract
Apple pomace (AP) fermented powders were made after solid state fermentation with different yeasts
(Saccharomyces cerevisiae,Candida utilis and Torula utilis) and after removal of ethanol was made
into powders. Application of Principal Component Analysis (PCA) to the compositional data clearly
separated the unfermented apple powders from the fermented apple fomace powders. The PCA separated
the fermented apple pomace from unfermented based upon the parameters selected viz. total sugars,
TSS, vitamin C, crude proteins, moisture, crude fat and soluble protein. The summary of Eigenanalysis
clearly shows that the first two PCs accounted for most of the variations found in the composition of
samples. Out of the two PCs, the PC 1 was defined by TSS, vitamin-C, crude fat and titratable acidity
while PC 2 contrasted the treatment based upon the total sugar and crude proteins. However, yellow
colour and moisture content were less related as shown by their intensities. The unfermented AP was
distinct for total sugar and TSS while fermented apple pomace samples were specific in vitamin-C,
crude protein, crude fat and soluble protein. Based on the mineral analysis, the PCA failed to differentiate
between unfermented and fermented APP when treatment and attributes (vector loadings) were plotted
together. Ferrous defined the PC 1 strongly while as shown by their intensities K, Cu, Na and Ca, were
weekly correlated. The PC-2 was defined by Zn and Mn contents.
2012 New Delhi Publishers. All rights reserved

Keywords: Solid state fermentation, PCA, Apple pomace, Protein, Minerals, Yeast

Apple pomace (AP) - a byproduct of apple juice processing


industry, being highly biodegradable, poses a problem for its
disposal. It is a good source of sugar, acid and mineral besides
the fibre content (Smock and Neubert, 1950; Downing, 1989;
Joshi et al. 2009) and its disposal into the environment is a
loss of precious resource. Utilization of the pomace using
fermentation technology for concomitant production of animal
feed and ethanol could be one of the promising alternatives
available especially due to its low protein contents making it a
poor animal feed supplement (Hang, 1988). Microbial

fermentations had been used previously for the production of


protein enriched feed and food products from high
carbohydrate raw materials by direct solid state fermentation
(SSF) of cassava meal with a food fungus, Aspergillus niger
(Raimbault and Alazard, 1980). Recently, efforts have also been
made to use AP as a substrate for the production of ethanol,
biogas, citric acid and edible fibres (Hang, 1987 Joshi et al.
2009). Hang (1988) also reported that using SSF with food
yeast Candida utilis, nutritive value of apple pomace could
be increased considerably.

Joshi and Sandhu

Solid substrate or state fermentation (SSF) refers to any process


in which the substrate is a solid and there is no freely available
water. It has been used for centuries, in the preparation of a
variety of food products such as meso, soysauce, tofu and
tempeh (Hessltine, 1972). Solid state fermentation has also
been applied for ethanol production. (Hang et al., 1982). Gupta
et al. (1990) conducted SSF of apple pomace for ethanol
production. Ghanum (1992) cultivated mixed cultures of
Trichoderma and yeasts, and a combination of T.reesei and
Kluyveromyces maxiams offered a maximum yield of 51 per
cent and efficiently converted leaf pulp into proteins (39.4%).
But there is a limited information available on the effect of
solid state fermentation on the physico-chemical characteristics
of apple pomace fermented with different yeasts and dried
into powder after removal of ethanol and analysis the data
using maltivariete analysis tool. The results of studies carried
out on the aspects of the application of PCA for analysis of
data obtained from the solid state fermentation of apple pomace
have been discussed in this communication.

Principal Components (first three), correlation coefficients,


matrix and eigen -vectors. The analysis was performed without
rotation as per the soft wase. The interpretation of data from
PCA was made by plotting Principal Components I vs 2 or 1 vs
3 and attributes loading as vectors along with treatments
(species), simultaneously.
Results and discussion
Physico- chemical characteristics
The results show that there was a considerable reduction in
the total sugar content in the FAPPs (fermented apple pomace
powders), though these still contained residual sugar in
sufficient amount. Compared to the control, the FAPP contained
significantly less TSS and no difference due to the yeasts
type could be recorded. The fermented and dried apple pomace
contained significantly more acid content than the dried and
unfermented apple pomace. The fermented pomace made by
fermentation with different yeasts contained similar amount of
total sugar. Compared to the CP content of AP, the FAPPs
contained almost 3 times more crude proteins CP. Among the
three yeasts tried, Candida gave the highest crude protein.
Increase in the protein content by fermentation is highly
desirable as apple pomace has been described as low protein
and high carbohydrates food having low palatability and
digestibility in rumen (Sharma et al., 1986; Hang, 1988). But,
there was only a slight increase in the soluble proteins of the
FAPPs compared to the APP without fermentation. A significant
increase (1.5 to 2.0 fold) in crude fat in the fermented apple
pomace powder (FAPPs) as compared to the unfermented
(APP) also took place. Similar to these observations, production
of fat by oleaginous yeasts including Candida curvata has
been described earlier (Ratledge, 1989; Holdsworth and
Ratledge, 1991).

Materials and methods


Solid State fermentation
Using the optimized conditions, solid state fermentations of
apple pomace with three yeasts were carried out as reported
earlier (Joshi and Sandhu, 1996). After the completion of
respective fermentations, samples for ethanol estimation were
removed and the remaining fermented pomace was extracted
with water and the extract was distilled. The left-over material
was dried at 60lC in a dryer followed by grinding into powder.
The powder was packed in polyethylene bags of 200 gauge
and sealed by a pouch sealing machine.Analysis of dried
fermented apple pomace for various physico-chemical
characteristics viz., TSS, total sugar, pH, crude proteins, soluble
proteins, crude fat, vitamin C, mineral, total acid, rehydration
ratio etc. were carried out as reported earlier (Joshi and Sandhu,
1996). Parameters like total ash, calorific values, crude fibres of
apple pomace powders were got analysed from the Central
Food Technological Research Institute, Mysore (India) while
the remaining parameters were analysed in the departmental
laboratories of the then Postharvest Technology Department,
UHF, Nauni, Solan, (H.P.) India.

Nutritionally important vitamin-C showed 1.5 to 2.0 fold


increase in the fermented apple pomace compared to the
unfermented one. According to Lee and Mattick (1989) though
average ascorbic acid content of apple is about 5 mg/100 g,
the peel contained more ascorbic acid than pulp and it could
have been the reason for higher vitamin-C content of dried AP
as it contains a considerable amount of peel. There was a
significant increase in the crude fibres of AP after fermentation
apparently due to the increase by drying due to loss of water.
The increase is desirable as the crude fibres, constitute an
important part of the nutritionally balanced diet. There were
significant differences in the total ash content of the
unfermented and fermented apple pomace. The increase in
total ash is clearly the contribution made by drying where loss
of water resulted in comparative increase in various
components in the dried matter.

Principal Component analysis


The means of Physico chemical Characteristics were used
for Principal Components Analysis (PCA) as per the
instructions given for this computer package, PCA. BAS
(Ludwig and Reynolds, 1988). Various treatments and the
compositional values constituted the data. The data of
chemical characteristics were analysed for significance of
variation by CRD. The output was obtained in the form of

88

Effect of Solid state fermentation and yeast species on composition of apple pomace: Application of PCA

were specific in vitamin-C, crude protein, crude fat, soluble


protein. Out of the selected parameters, total sugar, crude
proteins, vitamin-C and TSS were related and separated the
fermented apple pomace from non-fermented. Out of these
parameters, total sugars and TSS were highly correlated with
the unfermented apple pomace while crude proteins and
vitamin-C were related with fermented apple pomace powders.

Mineral composition of the fermented and unfermented AP


shows that K content increased significantly in all the fermented
samples compared to the unfermented. There was no
difference in the K contents of the dried apple pomace fermented
by different yeasts as was the case of Na. Magnesium content
decreased in the fermented APP while Ca levels were
comparable. Copper concentration registered an increase in to
the fermentated powder. The increase in Zn and Mn was 1.5
and 3 to 5 fold respectively. The enhancement of Fe content in
the fermented powder was more than 2 fold. The increase in
the mineral content could be attributed to the effect of drying.

Based on mineral analysis, the PCA could not differentiate


between unfermented and fermented APP (Figure 2) when
treatment and attributes (vector loadings) were plotted. As
per the Kaiser criterion, the PC accounting for the highest
variation was PC-1 with Eigenvector value of more than 1.
Ferrous defined the PC 1 strongly while as shown by their
intensity K, Cu, Na and Ca, were weekly correlated. Along the
first PC, Fe contrasted the fermented poamce by Candida utilis
and Torula utilis from unfermented and that fermented by
Saccharomyces. The PC-2 was defined by Zn and Mn contents.

Principal component analysis


The basic chemical data of fermented and unfermented apple
pomace were analysed by PCA. The projection of the yeasts
and the vector loading (chemical parameters) is shown in
Figure 1. The PCA separated the fermented apple pomace from
unfermented clearly based upon the parameters selected. The
summary of Eigenanalysis (Table 1) clearly shows that the
first two PCs accounted for most of the variations found in the
samples. Out of the two PCs, the PC 1 was defined by TSS,
vitamin-C, crude fat and titratable acidity while PC 2 contrasted
the treatment based upon the total sugar and crude proteins.
However, yellow colour and moisture were less related as
shown by their intensities. The unfermented AP was distinct
for total sugar, TSS while fermented apple pomace samples

To sum up, in the SSF of apple pomace, compared to the


unfermented, the apple pomace powders after fermentation
with different yeasts were found to be enriched with several
nutritionally important components. The application of PCA
technique have proved to be effective in interpreting the data
such as that obtained from solid state fermentation of apple
pomace and have clearly charaterized and separated fermented
apple pomace from fermented.

Figure 1: Projection of physico-chemical analysis data of apple pomace powders obtained with or without fermentation by different yeasts
in planes defined by principle component 1 and 2 (1) = unfermented apple pomace, Apple pomace fermented by (2)= Sacchamyces,(3)=Candida,
(4)= Torula
89

Joshi and Sandhu

Table 1: Principal Components Analysis output of data of physico-chemical parameters of apple pomace fermented by different yeasts
Eigen values
(a) All parameters except minerals (12 attributes)
1 = 3.486
2 = 0.502
3 = 0.011
4 = 0.001
(b) All minerals (8 attributes)
1 = 3.998
2 = 0.001
3 = 0.00
4 = 0.00

Per cent of trace

Accumulated & trace

87.2
12.5
0.3
0.0

87.2
99.7
100.0
100.0

100.00
0.00
0.00
0.00

100.0
100.0
100.0
100.0

Figure 2: Projection of mineral analysis data of apple pomace powders obtained with or withot fermentation by different yeasts in planes
defined by principal component 1 and 2 (1) Unfermeted apple pomace, Apple pomace fermented by (2) = Saccharomyces, (3) = Candida,
(4) = Torula

90

Effect of Solid state fermentation and yeast species on composition of apple pomace: Application of PCA

Joshi, V.K. and Sandhu, D.K. 1994. Solid state fermentation of apple
pomace for production of ethanol and animal feed. In Solid
State Fermentation. Ashok Pandey, Eastern Wiley Ltd., New
Delhi, pp. 93-98.
Joshi, V.K.and Sandhu, D.K. 1996. Composition of the distillates,
from the solid state fermentation of apple pomace by different
yeasts. Nat. Acad. Sci. letter,49(1),1-13
Joshi, V.K. and Sandhu, D.K. 1996. Preparation and evaluation
of animal feed using solid state fermentation of apple
pomace. Bioresource Technol., 56:251-255.
Joshi, V.K. and Rana S. Neerja. 2009. Microbial Technology for the
Production of Value Added Products from Apple Pomace,
Agriculturally Impotant Microorganisms (Vol.II),
(Eds.George, G.K., D.K. Arora, T.P.R., A.K.Srivatava
Academic World (13) pp.271-286.
Kaur, K. 1989. Microbial transformation of apple pomace to recover
industrial Products. M.Sc. (Hons.) Thesis, Punjab University,
Chandigarh.
Keunedy, M.J. 1994. Apple pomace and Kiwifruit : Processing
options. Australasian Biotechnol. 4:43-49.
Lee, C.Y. and Mattick, L.R. 1989. Composition and Nutritive value
of apple; products. In: Processed Products. (C.D.L.
Downing, ed). AVI Novanstrand Reinhold, New York, pp.
303-322.
Lammel, S.A., Heimsch, R.C. and Edwards, L.L., 1979. Optimizing
the continous production of Candidta utilis and
Saccharomyces fibuliger on potato processing waste water.
Appl. Environ. Microbiol, 37:227-232.
Ngadi, M.O. and Corrota, L.R. 1992. Solid state fermentation of
apple pomace as affected by moisture and bioreactor mixing
speed. J. Fd. Sci., 57(3):667-670.
Ranganna, S. 1986. Hand book of Analysis and Quality Control for
Fruit and Vegetable Products. 2nd Edn. Tata McGraw Hill
Publishing Co., Ltd. New Delhi.
Ratledge, C. 1989. Microbiol Technology of Lipids : Lipid Technol.
1:34-39.

References
AOAC 1980. Association of Official Analytical Chemists. Official
Methods of Analysis. ed. Hortwitz. W. 13th edn, Washington.
D.C.
Downing, D.L. 1989. Processed Apple Products. AVI Van Nostrand
Reinhold, Newyork. p. 433.
Fontenot, J.P., Bovard K.P., Oltzen, R.R., Rumsey T.S. and Driode,
B.M. 1977. Supplementation of apple pomace with nonprotein nitrogen for gestating cows. J. Anim. Sci.,
46:513-522.
Ghildyal, N.P., Lonsane, B.L., Sreekantiah, K.R. and
Sreenivasamurthy, V. 1985. Economics of submerged and
solids state fermentation for the production of
amyloglucosidase. J. Food Sci. Technol. 22:171-176.
Gupta, L.K., Pathak, G. and Tiwari, R.P. 1990. Effect of nutrition
variables on solid state alcoholic fermentation of apple pomace
by yeasts. J. Sci. Food. Agric. 50:55-62.
Hang, Y.D. and Walter, R.H. 1989. Treatment and utilization of
apple processing waste.In; Processed Apple Products, ed.
Downing D.L. AVI Van Nostrand Reinhold, New York,
365-376.
Hang, Y.D., Lee, C.Y. and Woodams, E.F. 1982. A solid state
fermentation system for production of ethanol from apple
pomace. J. Food Sci. 47:1851-1852.
Hang, Y.D. and Woodams, E.F. 1984. Apple pomace : A potential
substrate for citric acid production by Aspergillus niger.
Biotechnol Lett. 6:763-764.
Hang, Y.D. 1988. Improvement of the nutritional value of apple
pomace by fermentation. Nutrition Reports International,
38(1): 207-209.
Holdsworth, J.E. and Ratledge, C. 1991. Triglyceride synthesis in
the oleaginous yeast Candida curvata D. Lipids, 26(2):111118.
Joshi, C. and Joshi, V.K. 1990. Food Processing Waste Management
Technology, Need for an integrated approach. Indian Food
Packer, 44: 56-67.

91

Intl. J. of Food. Ferment. Technol. 2(1): 93-97, June, 2012

Research note

Processing potential of newly introduced Amla cultivars


grown in lower Himalayan Region of Himachal Pradesh
Manisha Kaushal1* and Shashi K. Sharma2

1*

Department of Food Science and Technology, Nauni, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan,
Himachal Pradesh,India
2
Institute of Biotechnology and Environmental Science, Neri, Hamirpur, Dr YS Parmar University of Horticulture and Forestry,
Nauni, Solan, Himachal Pradesh,India
*

Email: manishapht@gmail.com

Paper no: 43

Received: 20 March ,2012 Received in revised form: 19 April ,2012

Accepted: 16 June, 2012

Abstract
Varietal performance studies of wild and introduced variety of Amla were conducted at Regional
Horticultural and Forestry Research Station, Bhota, Himachal Pradesh. The selections of amla (NA-6,
NA-7 and NA-10) were made from Faizabad (UP) and introduced at Research station Bhota, Hamirpur
and at farmers field during the year 2002 to evaluate their performance. It was inferred that cultivar
NA-7 and NA-10 have outperformed the traditional local strain for almost all the characteristics studied.
These cultivars were having higher survival rate, shorter juvenile phase, better tree canopy and yield.
The fruit of Local (wild) variety was found smaller than the introduced variety like NA-7 and NA-10. In
terms of chemical characteristics, the berries of local cultivars had comparatively higher soluble solids
(11.36oB) and ascorbic acid (275.7 mg/100g) than NA-7 and NA-10 varieties. However, on the basis of
antioxidant potential NA-7 had higher phenol content (21.02 g/100g) as compared to the local and NA10 cultivars. The fruits of local variety were used to prepare pickle while NA-7 and NA-10 were
employed to prepare preserve.
2012 New Delhi Publishers. All rights reserved

Keywords: Amla, Introduced, Desi, Performance studies, Pickle, Preserve

The Amla/Aonla or Indian Gooseberry (Emblica officinalis


Gaertn., family Euphorbiaceae) is a minor sub-tropical tree
indigenous to Indian sub-continent. Owing to its hardy nature,
it has been successfully grown in dry and marginal regions
extending from the base of the Himalayas to Sri Lanka and
from Malacca to South China. Added to it, the plants and
fruits of amla are regarded as sacred in Hindu mythology.
Probably, it is the only fruit recognized by the Ayurvedic
System of Medicines for complete sound health. Being one of
the richest sources of vitamin C and several active tannoid
principles (Emblicannin A, Emblicannin B, Punigluconin and
Pedunculagin), it possess expectorant, purgative, spasmolytic,
anti-bacterial, hypoglycemic properties (Rastogi 1993, Rao et

al. 1985, Jamwal et al 1959, Jayshri and Jolly 1993).


Lower Himalayan region of Himachal Pradesh has
characteristic features of skewed rainfall distribution and
sloppy terrains which render the region vulnerable to drought
like situations during most part of the year. Under such an
agro-ecological situation, the status of commercial fruit crops
like mango, litchi etc is quite weak. Amla performs well in the
region but its plantations of local strains are sporadic. During
the recent past a number of varieties have been introduced in
the region which is also giving outstanding performance with
respect to yield and quality of the fruit. These introduced
cultivars are producing an average yield of more than 100 Kg/

Kaushal and Sharma

tree/year. But, because of high acidic and astringent nature of


amla fruits, very little quantity of the harvest is utilized for
fresh consumption and most of the crop is wasted. Thus, amla
has not been able to make significant contribution to the social
economic upliftment of the region despite of its high yield and
nutritional potential. The present studies were therefore,
designed for evaluating the high yielding varieties of amla for
various physico-chemical properties and thereby, working out
their potential for value addition and income generation for
the rural livelihood.

temperature away from direct sunlight. Preparation of pickle


involves the steps outlined in Figure 2.
Selection of fruit

Washing and blanching

Sun drying (3-4hrs) to remove surface water

Add spices, oil & mix well

Keep in sun for few days

Materials and methods

The varietal performance studies were conducted at Regional


Horticultural and Forestry Research Station, Bhota, Hamirpur
(Himachal Pradesh) on different introduced and locally grown
strains of amla. Data were recorded for 10 year old plantation
of NA-7, NA-10 cultivars and Local strains of aonla on their
survival, tree size, juvenile period and average yield during
their 9th and 10th year of growth. The study on physico- chemical
evaluation and value addition potential of these cultivars was
conducted at the Department of Food Science and Technology,
Dr Y S Parmar University of Horticulture and Forestry, Nauni,
Solan during 2010-11 and 2011-12.

Store amla pickle at room temperature

Amla pickle
Figure 2: Flow diagram of amla/aonla pickle preparation

For the preparation of aonla candy, mature fruit were washed,


pricked and dipped in 2 percent salt solution for 24 hours. The
fruit were thoroughly washed and blanched in boiling water
for 5 minutes and steeped in 50 Brix syrup solution for 24
hours. The next day steeping was done in 60 Brix for 24 hours.
Again steeping was done in 70 Brix for 72 hours. Excess syrup
was drained. The fruit were dried to 15% moisture content
and coated with powdered sugar/pectin. Packaging was done
in polythene pouches (400 gauge).

For physical parameters, the fruit were evaluated for berry


colour, weight, pressure, size and pulp:stone ratio. The chemical
characteristics such as moisture, total soluble solids (TSS),
titratable acidity, sugars, ascorbic acid, carotenoids, phenols,
crude fibre and ash were estimated as per the standard
procedure described by Ranganna (1986) and AOAC (1995).
For value addition studies, the products like preserve and
pickle were prepared as per the recipe of Lal et al. (1959).Small
size amla fruit (Local/Desi) which are not suitable for
preparation of preserves and other confectionary items, were
utilized for pickle making. The procedure for amla preserve
preparation (Figure 1) involves washing and selecting firm
and sound amla fruit followed by dipping in 2% common salt
solution until the green fruit changes to a creamish green colour,
with replacement of the brine solution on alternate days. The
fruit were thoroughly washed, pricked with a stainless steel
pricker and then, blanched in boiling water for 4 to 5 minutes.
Sugar equal to the weight of fruits was sprinkled over the fruit
and kept overnight. The next day one boiling was given to the
whole mass and the syrup was then drained out. The syrup
was thoroughly boiled and concentrated by adding more sugar
to 54-55Brix strength and mixed with fruit. The following day
the fruit were taken out and syrup is concentrated to 75Brix
by adding sugar and boiling. Amla fruit were added back and
allowed to stand in syrup for couple of days. When the oBrix
of the syrup stabilized at around 70, the preserve was packed
in clean, sterilized, dry glass jars and stored at ambient room

Amla preserve: Various steps involved in the preparation of amla


preserve are depicted in Figure 2.
Selection of fruit

Washing and dipping in salt (2%) for 2-3 days

Pricking, blanching in boiling water (4-5 minutes)

Sugar equal to weight of amla sprinkled on fruit dipping and


boiling in sugar solution by raising the TSS on alternate days till
final TSS reaches 70o B

Dipping of amla segments in sugar syrup

Storage in jars at cool and dry place


Figure 1: Flow diagram of alma preserve preparation

94

Processing potential of newly introduced Amla cultivars grown in lower Himalayan Region of Himachal Pradesh

Plate 1: Amla cultivars (Local, NA-7 and NA-10)

Plate 2a: Amla pickle (Local variety)

Plate 2b: Amla preserve (Introduced variety)

Results and discussion


traditional Local strain for almost all the characteristics studied.
These cultivars were having higher survival rate, shorter
juvenile phase, better tree anchorage and yield as well. The
findings are contrary to those of Rao and Subramanyam (2009)
who evaluated ten year old plantations of different aonla
cultivars and reported better performance in NA-10 under red
sandy loam conditions of Andhra Pradesh. But, the present
findings are in close proximity with those of Patil et al. (2010)
who evaluated eight varieties of Regional Fruit Research
Station, Katol, Maharastra and reported that variety NA-7 gave
the highest fruit yield, followed by Kanchan and Chakaiya.
Regional variability in performance of different cultivars of
aonla has also been described by Pathak (2003).

Performance studies
The performance data (pooled averages) of different amla
cultivars presented in Table 1 revealed that survival per cent
was recorded highest for local strains though statistically it
was at par with cultivar NA-10 (71%). The length of juvenile
period (years) was observed almost similar in all the cultivars.
As far as tree growth was concerned the tree height was
recorded highest for NA-7 which was significantly greater than
the local strains. The height of NA-10 was statistically at par
with NA-7. Tree spread was almost similar among the different
cultivars. Per tree yield data indicates that highest yield was
obtained for cultivar NA -7 which was significantly higher
than that of local. From the results presented it can easily be
inferred that cultivar NA-7 and NA-10 have out-performed the

On the basis of the results the two cultivars (NA-7, NA-10)


were taken further for evaluation and product development.

95

Kaushal and Sharma

Table 1: Performance of introduced amla cultivars under low hill region of Himachal Pradesh (Pooled data for tree size and yield for two
years)
Cultivar
NA-7
NA-10
Local
CD(0.05)

Survival
(%)

Juvenile
Period (years)

Height

Tree Size (m)


Spread

Yield
(kg)

5
71
72
7.2

3
3
5
NS

4.75
4.40
3.3
1.33

4.92
4.63
3.90
NS

92
83
52
18.7

Physical characteristics
The comparative study of three cultivars of amla viz., NA-7,
NA 10 and Local showed that the fruits were round, ribbed
and pale green. The average fruit weight and seed weight varies
from 6.58 to 33.8g and 0.94 to 2.70g respectively (Plate 1). The
different components of amla fruit accounted for 6.07-14.26
per cent peel, 69.77-85.49 per cent pulp and 7.10- 15.12 per cent
seed. The maximum and minimum fruit weight was attained by
NA-10 (33.8g) and Local (6.58g), while the maximum seed weight
by NA-7 (2.70g) with a minimum of 0.94 g by Local variety. Out
of the three varieties, the skin of NA-7 cultivar was glossy and
shiny with colour parameters of Yellow Green 144C as read
from Horticulture Colour Charts, while the other two cultivars
were having Yellow Green 145A colour. The size parameters of
three cultivars showed that the Local, NA-7 and NA-10 had a
length (mm) of 21.8, 36.44 and 33.47 with a diameter measuring
22.19, 38.08 and 38.47 mm respectively. Thus, the average size
of NA-7 aonla cultivar was found larger than the other two (
Table 2). The data were in conformity with those obtained by
Goyal et al (2008), Mishra (2009) and Kalra (1988) for different
amla cultivars.

(Emblica officinalis)
Local

NA-7

NA-10

Moisture (%)
Total solids (%)
TSS (oB)
Titratable acidity
(% citric acid)
Brix/acid ratio
Sugars (%)
Reducing
Total
Ascorbic acid (mg/100g)
Total phenols (g/100g)
Fibre (%)
Juice recovery (%)

84.6
15.4
11.36
2.73

82.2
17.8
9.8
2.97

86.18
13.82
9.15
2.17

4.16

3.29

4.21

4.76
5.54
275.7
20.63
2.70
40.0

3.17
4.54
205.0
21.02
3.83
42.0

3.21
5.12
200.3
20.99
3.41
44.0

Chemical characteristics
Data pertaining to the chemical composition of amla cultivars
are presented in Table 3 indicate that moisture content in amla
berries (Local, NA-7 and NA-10) ranged between 82.2 to 86.18
per cent with a maximum in NA-10 and minimum in NA-7
cultivars. In all the three cultivars of amla, the total soluble
solids (oB) and titratable acidity (% citric acid) lied between
9.15 to 11.36 and 2.17 to 2.97 respectively, where Local and
NA-7 cultivar had highest TSS and acidity respectively. The
brix/acid ratio was maximum in NA-10 (4.21) and minimum in
NA-7 (3.29) cultivars. As reported earlier, the amla fruit is
regarded as a rich source of ascorbic acid besides other vital
nutrients. The level of vitamin C in amla berries varied from
200.3 to 275.7mg/100g with the maximum in Local cultivar.
Besides vitamins, the amla fruit also contained good proportion
of total phenols, in different cultivars; their respective values
were recorded as 20.63, 21.09 and 20.99 g/100g for Local, NA7 and NA-10, respectively. The fibre content in the amla
cultivars was found maximum in NA-7 (3.83%) with a minimum
in Local (2.70%). Thus, the berries of amla were regarded as a
fruit containing good proportion of vitamin C and phenols

Table 2: Physical analysis of different varieties of Amla (Emblica


officinalis)
Parameters
Local
NA-7
Weight (g)
6.58
32.8
Size (mm)
Length (V)
21.8
36.44
Breadth (H)
22.19
38.08
Berry Quotient (V/H) 0.96
0.95
Peel wt (%)
14.26
6.07
Pulp wt (%)
69.77
85.49
Seed wt (%)
15.12
7.10
Stone:pulp ratio
1:5.2
1:12.5
Colour
Yellow Green Yellow
145A
Green 144C
Green & Glossy
Pressure (lbs/in2)
19.5

Parameters

NA-10
33.8
33.47
38.47
0.86
10.89
77.32
11.77
1:9.2
Yellow
Green 145C
27.0

Table 3: Chemical analysis of different varieties of Amla

96

Processing potential of newly introduced Amla cultivars grown in lower Himalayan Region of Himachal Pradesh

besides other components which can be used for value addition


(Kumar et al. 2006).

References
AOAC. 1995. Official Methods of Analysis 16th edn, Association of
Official Analytical Chemists, Washington, DC.
Goyal, R.K., Patil, R.T., Kingsly, A.R.P., Walia, H., and Kumar, P.
2008. Status of postharvest technology of aonla in India- a
review. American Journal of Food Technology. 3 (1):13-23
Jamwal, K.S., Sharma, I.P.,and Chopra, L. 1959. Pharmacological
investigations on the fruits of Emblica officinalis. J. Sci.
Ind.Res. 18: 180-181.
Jayshri, S, and Jolly, C.I. 1993. Phytochemical , antibacterial and
pharmacological investigations on Monordica chiranlia and
Emblica officinalis. Ind. J. Pharm. Sci. 1: 6-13.
Kalra, C.L. 1988.The chemistry and technology of Amla (Phyllanthus
emblica) - a resume. Indian Food Packer. 42(4): 67-82.
Kumar, G. S., Nayaka, H., Dharmesh, S. M. and Salimath, P. V. 2006.
Free and bound phenolic antioxidants in amla (Emblica
officinalis) and turmeric (Curcuma longa). Journal of Food
Composition and Analysis. 19: 446-452.
Lal, G, Siddappa, G.S. and Tandon, G.L. 1959. Preservation of Fruits
and Vegetables, Indian Council of Agriculture Research, New
Delhi, p 483.
Mishra, P., Srivastava, V., Verma, D., Chauhan, O. P. and Rai, G. K.
2009. Physico-chemical properties of Chakiya variety of
Amla (Emblica officinalis) and effect of different dehydration
methods on quality of powder. African Journal of Food
Science. 3(10): 303-306.
Pathak, R. K. 2003. Status Report on Genetic Resources of Indian
Gooseberry - Aonla (Emblica officinalis Gaertn.) in South
and Southeast Asia. IPGRI Office for South Asia National
Agriculture Science Centre (NASC). 99p.
Patil, S. R., Suryawanshi, A. B., and Phad, G. N. 2010. Performance
of some aonla (Emblica officinalis Gaertn) cultivars under
Vidarbha condition of Maharashtra. International Journal of
Plant Sciences, 5 (1): 36-37.
Rao, K. D. and Subramanyam, K. 2009. Growth and yield performance
of aonla varieties under scarce rainfall zone. Agric. Sci. Digest,
29 (2): 45-47.
Ranganna, S. 1986. Handbook of Analysis and Quality Control for
Fruit and Vegetable Products, 2nd edn, Tata McGraw Hill
Pub. Co. Ltd. New Delhi. p 1110.
Rao, T.S., Kumari, K.K., Netaji, B., and Subhokta, P.K. 1985.
Ayurveda Siddha. J Res. 6:213-224.
Rastogi, R.P. 1993. Compendium of Indian Medicinal plants, CDRI,
Lucknow and ID, New Delhi 1: 530.

Processed Products
Preserve: On the basis of sensory evaluation, it was observed
that amla preserve scored 7.0, 7.5 and 7.5 for colour, flavour
and body characteristics respectively (Plate 2a). On the basis
of taste, the sample scored 8.0 thus emphasizing its overall
acceptability (Table 4).
Amla pickle: To improve the texture of the fruit and also to
remove astringency, brining is important in pickling. Sensory
evaluation of the amla pickle showed that the pickle scored 7.0
for colour and flavour on the basis of 9 point Hedonic Rating
(Table 4). However, the taste perception of the product scored
7.85 out of 9 (Plate 2b). Thus, on the basis of overall
acceptability, amla pickle prepared by using local cultivars
highly acceptable (7.93) apart from meeting the specifications
laid down in FPO (1955).
Table 4: Sensory evaluation of processed amla products
S.No
1.
2.
3.
4.
5.

Attributes*
Colour
Flavour
Body
Taste
Overall Acceptability

Pickle

Preserve

7.00.2
7.00.15
7.450.08
7.850.10
7.930.10

7.00.10
7.50.18
7.50.11
8.00.12
8.00.15

*on 9 point Hedonic Scale


Conclusion
It has been concluded from the studies that newly introduced
cultivars (NA-7 and NA-10) aonla has great yield potential for
quality fruit production (in terms of fruit colour, appearance,
weight, length, and diameter the fruits, and other physicochemical features) under the lower Himalayan region.

97

Intl. J. of Food. Ferment. Technol. 2(1): 99-101, June, 2012

Research note

Evaluation of the stability of plum anthocyanin


powder in RTS based model solution
M. Preema Devi2, V.K. Joshi2 and Y. Indrani Devi1

Department of Postharvest technology of Hort. Crops, Bidhan Chandra Krishi Viswavidyalaya,West Bengal,India
Department of Food Science and Technology, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal
Pradesh,India
2

Email: v.k.joshipht@rediffmail.com

Paper no: 44

Received: 15 Jan, 2012

Received in revised form: 19 April, 2012

Accepted: 14 May, 2012

Abstract
Colour of a foodstuff is one of the most important elements determining its acceptance besides
enhancing its delicacy. Storage stability of the dried plum anthocyanin powder with 300B maltodextrin
as a carrier in RTS beverage model solution showed more effect of temperature at 350C than at 00C.
There were less changes in dark light condition than in day and UV light. The change in colour was
rapid in the first 1 month than in the later period of storage. By the use of plum pomace and with the
above optimized conditions, crude anthocyanin pigments can be produced and used in processed
food.
2012 New Delhi Publishers. All rights reserved

Keywords: Plum, anthocyanin, RTS, Model solution

Colour of a food is not a label flavour type, but it also provides


information on its quality and condition. In many food systems,
colour acts as an indicator of condition of food such as fitness
to eat. Because of consumer awareness and concern for healthful
and fully balanced food, now-a-days people have developed
increasing interest in food colourants of natural origin.
Anthocyanins are water-soluble pigments which exist in the
cell sap. The total amount of anthocyanin in plum ranges from
44.1 to 231.29 mg as cyanidin 3-glucoside per 100g fresh tissue
(Casal et al 2002) The waste of the plum after processing is
generally thrown out by the processing industries which contain
sufficient quantity of anthocyanin pigment. It can be used for
extraction of biocolour. The anthocyanins in general are very
sensitive to different factors including storage conditions and
therefore, how the plum anthocyanins would behave is not
known. Before commercializing these biocolours the evaluation
of their stability in model solution is very important keeping

these in view,anthrocyanins were extracted and their stability


in RTS based model solution was determined and reported
here.
Materials and methods
Anthocyanins from plum pomace were extracted using 50%
ethanol and 0.2% citric acid, which was further concentrated
upto 10 fold. Spray drying of the concentrate was done using
30 0B maltodextrin as a carrier. Concentration of anthocyanin
powder was optimized separately. RTS beverage (model
solution) were prepared as per the FPO specifications(Devi
2008). Each treatment was analysed for TSS, total sugar,
titratable acid, colour measurement, tintometer and pH after
every one month for 3 months of storage and changes
occurring were recorded. Analysis was carried as per the
standard method reported elsewhere (Ranganna , 1986) The
storage conditions were 0 0C Day Light, 35 0C Day Light, 35 oC

100

T6 :350C Dark

T5 :250C Dark

T4 :00C Dark

T3 :350C UV

T2 :350C Day

T1 :00CDay

Times (Months)

Treatments

TSS

14.0 14.1

14.0 14.1

14.0 14.2

14.0 13.9

14.0 13.9

14.0 14.2

0.70

0.70

0.40

0.70

0.70

1.40

pH
3

3.15 3.22

L
%
3 change

2.20 78.50 73.4 2.90


0

%
chang
e

3.30
12.8 0.30
0.30 0.29
12.70 0

3.30
12.8 0.30
0.30 0.29
12.70 0

3.30
12.9 0.50
0.30 0.29
12.70 0

3.15 3.20

3.15 3.20

3.15 3.21

1.60 78.50 73.6 2.80


0

1.60 78.50 73.6 2.80


0

1.90 78.50 73.3 3.00


0

10.00
2.20 78.50 73.9 2.50
12.8 0.30
3.15 3.22
0.30 0.27
12.70 0
0

10.00
2.80 78.50 73.7 2.50
12.8 0.30
0
3.15 3.24
0.30 0.27
12.70 0

6.60
12.9 0.50
0.30 0.28
12.70 0

% Total sugar %
%
Acidity
change 0
3 change
3 change 0
a

7.70 6.90 10.40 19.00 16.4 13.70 3.60 1.30 63.90 3.20 4.03
0

7.70 7.00 9.00 19.00 16.3 14.20 3.60 1.80 50.00 3.20 3.99
0

7.70 7.40 3.90 19.00 15.8 16.80 3.60 1.80 50.00 3.20 3.92
0

7.70 5.70 26.00 19.00 16.6 12.60 3.60 1.00 72.20 3.20 4.10
0

7.70 6.60 14.30 19.00 16.5 13.20 3.60 1.10 69.40 3.20 4.10
0

26.00

24.70

22.50

28.00

28.00

23.40

%
b
% Yellow unit % change
Red unit
%
3
3 change 0
3 change 0
3 change 0

7.70 7.30 7.80 19.00 16.0 15.80 3.60 1.70 52.70 3.20 3.95
0

Table 1: Per cent change in various physico-chemical and colour characteristics of the RTS based model solution after three months of storage

Devi et al.

Evaluation of the stability of plum anthocyanin powder in RTS based model solution

U.V. Light, 0 0C Dark Light, 25 0C Dark Light, 35 0C Dark Light.


The RTS was packed in a 30 ml bottle.The concentration of the
plum anthocyanin powder used was 4% which was
standardized in earlier experiment. Three replications were
made for each treatment. The data were analysed statistically
by CRD as per the precribed method (Cochran and Cox, 1963)

month than the later periods.Thus,the studies need to be


directed so that the anthrocyanin after extraction and
concentration remain stable after their addition to the food
products. The pigment have to withstand the drastic
conditions of food processing,if they are to be used in
processing.

Results and discussion

Conclusion

Physico-chemical characteristics
There was a slight increase in the TSS, total sugar and pH
during the different storage conditions and storage interval in
RTS based model solution. However, the titratable acidity
showed a slight decrease during different storage conditions
and storage intervals. Although, statistically no significant
differences were observed in this parameters as the variation
were too narrow that almost a constant TSS, total sugar,
titratable acidity and pH were observed along the experiment.
These findings are in accordance with those of Viguera et.al.
(1998). Attri (2004) also conducted stroage study of microbial
Pigment and reported that there were no significant changes
in these parameters in RTS beverage prepared with microbial
pegments during storage at 20, 30 and 370C.
There was a significant change in the colour measurement of
the RTS based model solutions in different light and
temperature conditions as well as during storage for 3 months
interval, which is also in line with the findings of Attri (2004).
The a value which gives the depth of redness was found to
be decreasing as the storage interval increases. These findings
are in agreement with those of Pilando et. al. (1985) who
reported that anthocyanins, the red pigments in raspberry and
other fruits, degrade and polymerized easily with passage of
time with time. These findings are further in conformity with
Timberlake and Bridle (1976) who studied in model system,
found that mixtures of anthocyanins and catechin during
storage gradually lost colour in the red region of the spectrum
while increasing in the brown region.According to Skrede
(1985) day light storage of syrups lowered half-life of Hunter
a values by 10-30% as compared with dark storage which is
in line with the observations recorded in our study.
It is also clear from the values given in table 1 that changes in
TSS and total sugar during storage was not even 1%.It is
desirable from food processing point of view. However, it is
very much evident that the changes in acidity, pH, L, a, b
values of colour measurement and the red and yellow units of
tintometer colour unit were very drastic. The fading of colour
was very much apperent during 3 month of storage. Similarly,
effect of storage in light was greater than in lower temperature
and in dark conditions.The contrasting effect of storage
revealed the occurrence of faster changes during the first one

The storage studies have clearly shown that high temperature


of 35 oC, storage in Day and UV light adversely affected the
biocolour. The visual light proved to be more destructive than
Dark .In brief, storage studies reflected that the products were
stable and the pigment did not induce any biochemical change.
As anthocyanin is water soluble, the plum anthocyanin can
be used commercially in food products where water is the
main solvent through more studies on stability would be
needed.
References
Attri, D.,2004. Production and evaluation of microbial colours using
apple pomace. Dr Y S Parmar University of Horticulture and
Forestry, Nauni, Solan, HP, India.
Casal, B.A.C., Byrne D.H., Zevallos L.C. and Okie W. R., 2002.
Total phenolic and anthocyanin content in red fleshed peaches
and plums Acta Horticulturae. 5:592-589.
Cochran,W.G. and Cox,G.M., 1963. Experiment Designs.14th ed.
P613. Asia Publishing House,Bombay
Pilando, L. S., Wrolstad, R. E. and Heatherbell, D. A.,1985. Influence
of fruit composition, maturity and mold contamination on
the colour and appearance of strawberry wine. Jour Food
Science. 50: 1121-1125.
Skrede, S., 1985 Color quality of blackcurrant syrups during storage
evaluated by hunter L, a, b values. Journ Food Science. 50:
514-525.
Timberlake, C. F. and Bridle, P.,1976 Interactions between
anthocyanins, phenolic compounds and acetaldehyde and
their significance in red wines. Amer Jour Enol Viticult. 27:
97-99.
Viguera, C. G., Zafrilla P., Artes F., Romero F., Abellan P. and Barberan
F. A. T.,1998. Colour and anthocyanin stability of red
raspberry jam. 78: 565-573.
Joshi, V.K.,Mutum Preema Devi and Attri,Devender,2011. Bicolour:
Chemistry,Production,Safety and Market Potential.In:Food
Biotechnology:Priciples and Practices.Joshi,V.K. and Singh,
R.S.(Eds),I.K Publishers,New Delhi. Pp 641-689
Joshi,V.K.,Devender,Attri, B,Anju and Bhushan,Shashi. 2003.
Microbial Pigment. Indian Journ Biotechnol 2: 363-369
Ranganna S. 1986.Handbook of Analysis of Quality Control for
fruit and Vegetable Products,2nd Edn. Tata Mcgraw Hill Publ.
Co, New Delhi
Devi Mutum Preema,2008.Refinement of extraction method and
evauation of anthrocyanins from plum.M.Sc Thesis,Dr.
Y.S.Parmar
University
of
Horticulture
and
Forestry,Nauni,Solan, H.P 1997
101

Book Review

Bio-processing of Foods
ASIA TECH PUBLISHERS INC, NEW DELHI
This book is based on the proceedings of the conference on New Horizons in Bo-Processing of Foods organized by Department
of Food Engineering and Technology held at Sant Longowal Institute of Engineering and Technology from 25th -26th February
2011. The selected papers that were presented in th conference have been compiled and edited in the form of book. It provides
an in-depth understanding on different topics that are of great relevance to food and fermentation technology. This indispensible
outcome is the result of combined efforts of different academic credentials in the area of food technology and biotechnology. To
make the concept more clear, the contents of this treatise have been divided into following sections:

Section I: Bio-processes for Value added Products

Section II: Bio-Safety and Quality system in Food Processes

Section III: Bio-Preservation and Bio -Packaging of food products

Section IV: Bio-processing and Bio- management of Agro industrial waste

The each sections comprises of many chapters. The chapters clearly demonstrate the recent developments and advances made
in the field of food Industry and applications of biotechnology especially its latest innovations in food processing. The
prominent topics covered in the book are

Potential application of lactic acid bacteria in functional food

Genetically modified food

Importance and Nutitive value,Sole present status and future strategies in Fruit wines in India

The production of organic acids and enzymes using solid-state fermentation

Bio preservation: A novel approach to food preservation.

PlumRTS : Screening cultivars

Biosensors in food processing

Nutritive value and importance of fruit wines

Storage quality of Olive oil

Enzymatic preparation of high Fructose syrup from Inulin.

Biotechnological tools :potential in food quality and safety

Nowadays bio technology plays an important role in the food industry as integral part of processing. This field for food
applications has expanded rapidly, particularly in the last two decades, aiming to optimize processing parameters, design novel
functional foods, alternative applications for several agricultural products and/or improve the safety, health impact and the
quality of foods. During the last years, much research has focused on the improvement of enzyme behaviour in the conditions
in which they were to be used, and especially on the increase of their thermal and operational stability. This book attempts to
present and update account of the most recent efforts and technologies to manipulate and improve the versatility and effectiveness
of enzyme and biotechnological interventions in food technology. In this regard, the use of food enzymes have been covered
in the three chapters of this volume.
The text is supported by a number of clear, informative diagrams for better understanding. The book is highly useful for postgraduate students and researchers of food technology, biotechnology, applied biology, microbiology and biochemical engineering.
It presents the basic and applied aspects from all the possible facets to serve as a text-cum-reference book. The book includes
numerous informative diagrams supporting the text. The book provides information about the latest research and advances on
the topics biotechnology in food processing which would be an asset to all the readers.
Dr ( Mrs) Neerja Rana
Assistant Professor
Department of Basic Sciences
Dr Y.S. Parmar University of Horticulture and Forestry Nauni Solan(HP)

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