Chapter 1

INTRODUCTION
Main important factor which effect human health is Diet. Meat is an important constituent
of a well-balanced and healthy diet due to its nutritional status. Meat is a good source of
fatty acids, iron, high biological value protein, vitamin B complex, selenium, phosphorus
and zinc. Some epidemiological data has shown a link between its consumption and
increased risk of cardiovascular, metabolic diseases and several forms of cancers (Pereira
and Vicente, 2013). The World Health Organization (WHO) has determined that the dietary
factors account for all cancers in Western countries is at least 30 percent and in developing
countries is up to 20 percent.

The entire hint that meat potency to cause cancer came from numerous large European
populations. Studies which have been conducted back in early 1990s shows that the
vegetarian peoples were almost on half risk as possible to develop cancer in relation to meat
eaters, when monitoring for other lifestyle and diet factors (Thorogood et al., 1994; Grant,
2014). Numerous hypotheses have been advanced to clarify the link between cancer risk
and meat consumption. According to study conducted at World Cancer Research Fund
(WCRF, 2011) there were 30 to 50 increased risk with colorectal cancer when red and
processed meat products consumption is highest. Exclusively with regard to processed meat
product consumption, investigators in the EPIC study discovered an 11 percent bigger risk
of dying from cancer with the eating of 50 grams per day (Rohrmann et al., 2013). First,
meat is devoid of antioxidants, fiber and Phytochemicals, and many other supportive
nutrients that contain properties which are protective against cancers (Nieto et al., 2010).

Furthermore, meat also comprises on saturated fat, animal protein, and in exceptional cases
some carcinogenic composites e.g. such as polycyclic aromatic hydrocarbons (PAHs) and
heterocyclic amines (HCAs) formed in the cooking and processing of meat. If meat is
cooked on elevated temperatures HCAs formed, and in burning of organic substances PAHs
formed are said to rise in cancer risk. Moreover, if meat and other animal based products
contain higher fat content may increase in hormone production, thus this situation elevated
the risk of cancers which are hormone-related such as prostate cancer and breast. Moreover,
salt-preserved and smoked preserved meat products contain (NOCs) N-nitroso compounds
that are possibly carcinogenic to humans (Santarelli et al., 2008). Especially heamoglobin
the iron containing compound causes the production of N-nitroso compounds in processed

1
meat products (Cross et al., 2003). Meat products preserved with nitrites contains these
compounds N-nitroso compounds are mainly having association with risk of cancer (Loh
et al., 2011). The work of Anderson (2012) at the University of Minnesota followed
individuals for a nine-year period and identified over 60% cases of the pancreatic cancer in
those who frequently consumed burned or charred meat. While another factor associated
with processed meat products is that they are high in sodium content as they are mostly
preserved with the salt studies shows the direct link between the high blood pressure and
high sodium containing diet (Johnson and Nguyen, 2001; He and Macgregor, 2002).

Both processed meat and red meat products contribute in the risk of heart disease because
of the sodium and saturated fat content According to the study of European Prospective
Investigation into Cancer and Nutrition (EPIC), which had 448,568 women and men,
researchers revealed a strong correlation among risk of death from CVD by consuming
processed meat products. Thirty percent increased risk of death found from CVD in those
individuals who consumed processed meat products more than 160 grams per day,
compared with those who consumed 10 to 20 grams per day (Rohrmann et al., 2013). Most
big cities in the Pakistan have witnessed a surge in the popularity of char-grilled meat and
meat products (Malik, 2013).

Color oxidation and Lipid oxidation are main reasons of quality deterioration in the meat
products throughout storage; estimate price of the industry is over $700 million annually.
The most instances oxidation is Lipid oxidation, it is a free radical chain reaction which
can be termed as initiation, propagation, and termination processes (Yogesh et al., 2012).
It is a primary mechanism which deteriorates the quality such as development of off-flavor,
rancidity, degradation in texture, changing of color in meat and its products during storage,
which renders them and made meat unfit for human consumption. These reactions take
meat towards discoloration, off-flavor and in the loss of nutritive value, eventually
declining consumer confidence in the product. Intake of such food products which contain
lipid constituents which are oxidized can modify proteins, DNA, tumor initiation and
membrane structure in biological system (Porter et al., 1995; Muik et al., 2005). High
oxidative constancy of muscle-based product is vital when trying to delay or avoid the
creation of warmed-over flavor or rotten products (Young et al., 2003) and to raise the
suitability of meat and meat based products. Improved oxidative stability of raw product is
well thought-out valuable for both the processing industry and the end users.

2
In the vegetable and fruit industry, the procedures of preparation and processing may lead
to 1/3rd of the product being rejected, due to growing manufacture of food plant processing;
disposal of by-products signifies a rising difficult since the material of plant is typically
prone to microbial decay, therefore restrictive further exploitation. Waste of fruit has gone
into one of the major sources of (MSW) municipal solid wastes, and this is an ever harder
environmental problem. At the present time, two main methods to trash MSW are
incineration and landfill. Though, unsuitable controlling of landfill will outcome in releases
of carbon dioxide and methane and burning involves the subsequent releases and formation
of pollutants and secondary trashes such as furans, acid gases, dioxins as well as
particulates, which can pose serious health and environmental risks. For these causes, there
is a crucial necessity to hunt for resource and value-added practice for fruit wastes (Qdais
et al., 2010; Buekens et al., 1998).

On the other hand, residues of Fruit could be cheap and freely accessible resources of
compounds which are bioactive and use in the pharmaceutical and food industries. Diverse
antioxidant potency is present in Different types of fruit residues and the deviation is very
huge. In addition, the essential bioactive compounds are quantified and recognized, are
cyanidin 3 glucoside, catechin, kaempferol, epicatechin, gallic acid, homogentisic acid, and
chlorogenic acid are widely present in certain residues of fruit (Deng et al., 2012). In
previous work, the antioxidant strength and the amount of phenolic compounds were
noticed to be high in some fruits peels which indicates that fruit deposits have the ability to
be consumed as a source of bioactive compounds e.g.as natural anti-oxidants (Ajila et al.,
2007; Okonogi et al., 2007). By-products of vegetables and fruits are also good supply of
micro-nutrients which are organic in nature; such as poly-phenolics, carotenoids, vitamin
C, tocopherols and other bioactive compounds, these compounds can be utilized as food
fortification and dietary supplements purposes (Schieber et al., 2001; O'Shea et al., 2012;
Jansen et al., 2013).

A sharp responsiveness of customers nowadays is on a relationship between health and

diet, prevention of disease has nurtured growth in the enlargement of ‘functional’ food
products (Ohata and Arihara, 2010). Due to possible health hazards of some artificial
antioxidants growing attention has been paid to recognize natural antioxidants which are
possibly more economically feasible and effective antioxidants. One of Most significant
category of natural antioxidant is phenolic compound which is our concern, and much

3
validation is extracted on the potency of antioxidant and prevention of diseases (Scalbert
et al., 2005; Fresco et al., 2006). Absolutely, functional foods symbolize an innovative,
important, and fast growing share of the complete food market. Epidemiological work has
pointed out that intake of functional foods imparts in health benefits, for example it reduced
the risk of stroke and coronary heart disease, as well as certain cancer types (Williamson,
2009). However, available locally and internationally scientific literature does not provide
information that consumption of functional meat products supplemented with fruits and
vegetables Phytochemicals are beneficial for health or not. Additionally, research on
interactions and stability of Phyto-chemicals with meat elements throughout processing
process and storage need to be initiated. Therefore, the supplementation study of fruits and
vegetables Phytochemicals in meat products may provide information regarding
Phytochemicals action to prevent the formation of potential carcinogens in meat products.
In particular, the focus should be on the amounts of fruits and vegetables Phytochemicals
that will not cause any major problems for acceptability of functional meat products.

Ultrasound is an emerging technology that was established to reduce processing, exploit

quality and to ensure food product safety. Ultrasound is useful to impart progressive
properties in processing of food e.g. food preservation, mass transfer improvement, thermal
treatments assistance and analysis of food and texture manipulation (Knorr et al., 2011). A
chief application of ultrasound is in extraction process on a variety of food components (for
example, herbal, oil, protein, polysaccharides) and as well as on bioactive constituents (for
example, antioxidants) based on animal and plant resources (Vilkhu et al., 2008).
Cavitation is an action of ultrasound technology, which creates micro bubbles and high
shear forces which boosts erosion of surface; mass transfer and fragmentation resulting in
fast rate of extraction and higher yield of extracted materials. The main advantages of
ultrasound technology have minimum result on extractable materials, organic solvents
prevention, as their action also works in the solvents of GRAS, it reduces extraction time,
which can possibly boost the withdrawal of heat sensitive food and bioactive components
at lower temperatures during processing and possibly in large scale industries (Awad et al.,
2012).

The extracted Phytochemicals can be used in meat to further enhancing the meat
acceptability and improving the functional stability during storage. Therefore, it can be the
most viable approach to extract the Phytochemicals from fruit and vegetable waste using

4
the innovative technology. The present study will be undertaken to achieve the following
objectives:
 Drawing out of bio-active composites from vegetable and fruit byproducts by using
ultrasound technology
 Develop a range of functional/healthier un-cooked and fried fish meat products
using extracted bioactive compounds
 Characterization of functional fish meat products for oxidative stability and
consumer acceptability at different storage intervals

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Chapter 2
REVIEW OF LITERATURE
2.1 Meat and composition

Meat can be defined as “it is a part of skeletal muscle, which is consumable, of an animal
which was healthy at slaughtering time”. In developing countries meat is commonly used
as a protein source. People consume meat by cooking or by processing into many forms to
escape related spoilage (Olaoye et al., 2010). Meat is composed of 4 components which
include protein, water, carbohydrate, lipids. It’s also composed of many miner components
such as enzymes, vitamins, flavors compounds and pigments. By combining the all major
and minor components they give meat its texture, structure, color, flavor and nutritive
value. But, due to its particular chemical and biological nature meat endures advanced
deterioration from slaughtering until consumption (Olaoye, 2011).

Meat and its products are usually accepted as good sources of biological high value
proteins, minerals, fat soluble vitamins, bioactive compounds and trace elements. Meat is
an excellent source of niacin, vitamin B6 and B12, zinc, iron, phosphorus, riboflavin, long-
chain omega-3 fatty acids, selenium, pantothenic acid, and vitamin D. meat is also a source
of a variety of bioactive substances e.g. carnitine, taurine, ubiquinone, glutathione,
carnosine, creatine and endogenous antioxidants (Williams, 2007).

Common lean red meat is low in fat content and it is reasonable in cholesterol level; it is
rich in many essential vitamins, protein and in minerals. Although the nutritional
composition of this meat will vary to some extent according to feeding routine, season,
breed, and meat cut. Meat has a very defined composition as the it provides the healthy
nutrition to human generally it provides the vitamins, minerals, and all three energy
yielding nutrients. Nadine (2007) said in one of his work on red meat about the content of
fatty acids, amino acids, cholesterol, vitamin B6 and selected minerals in cuts of meat of
different animal species. Cholesterol content in all cuts of raw meat was in the range if
10g/100g. fat content in the majority of raw meat cuts was less than 10g/100g. Composition
of fatty acids was variable within and between species. Proportion of SFA (saturated fatty
acids) ranged from 30% to 52%. There was highest level of Fe and Zn in lamb and beef.
Concentration of Vitamin B-6 in raw meat ranged from 0.32-0.80mg/100g. Meat is an only
source of micronutrients in form of higher bio-availability.

6
Chicken (broiler) is a good protein source, chicken contain less fat and it contain fewer
saturated fat as compare to beef. Moreover, protein is best source essential amino acids.
Composition of chicken is Water 74.6, Ash 1.0, Protein 12.1, Lipid 11.1, Fiber 0.0 and
Carbohydrates 1.2. Chicken also provides vitamins B12 and B6, zinc, phosphorus and iron.
A food which comes from animal source delivers a variety of micro-nutrients which are
tough to obtain in acceptable quantity from plant sources. A research was conducted by the
Nutrition Collaboration Research Support Program (NCRSP) acknowledged six micro
nutrients, which were low in primary plant based diets of school children which are such
as: zinc, calcium, iron, riboflavin, vitamin b-12, vitamin A (Jorge, 2010).

Marine animals are rich source of protein, low in fat and contain other nutrients which are
positively recognized to health. Selected micronutrients are rich in marine animals than
plants and mammalian meats. Fish chemical composition generally varies among species
as well as individuals and some other factors such as: sex, age, season and environment.
Fish is only a protein source which contains all essential amino acids. Protein and lipids are
the main component of fish foods but on the other hand fish contains low carbohydrates
level (<0.5%). Fish is a great and good source of valuable vitamins, minerals and
micronutrients (Zulem, 2014).

2.2 Oxidation of meat and health risks

Latest works supports the theory that oxidation of protein is responsible for the lessened
functional properties of proteins (myofibrillar) and the deterioration or defect of the quality
in fish, poultry and frozen meat e.g. texture changes, loss of redness, loss of water holding
capacity (Xia et al., 2009; Soyer et al., 2010; Estévez et al., 2011; Filgueras et al., 2011;
Utrera and Estévez, 2014; Utrera et al., 2012; Huang et al., 2013; Timm-Heinrich et al.,
2013). Fish and meat products are extremely perishable due to the richness of proteins
(Lucera et al., 2012). They undergo rapid post-mortem changes, mostly proteolysis and
lipolysis which takes product towards sensory deterioration and the risks of food-borne
illnesses becomes high (Casaburi et al., 2011). Main cause of deterioration in pre-cooked
meat and its products is lipid oxidation. Synthetic antioxidants like (EDTA), (BHA)
butylated hydroxyl anisole and (BHT) butylated hydroxyl toluene have effectively been
used to decrease lipid oxidation in meat. But, these antioxidants are synthetic that’s why
these foodstuffs suffer from a negative image from consumers (Yu-Yue et al., 2013).

7
Meat is a perishable product which gives a favorable growth environment for many
microorganisms. Meat is also vulnerable to rancidity due to enzymatic and chemical
activities. Breakdown of nutrients such as protein, fat and carbohydrate of meat causes
development of off flavor, slim formation and off odor which makes meat unpleasant for
human consumption. That’s why it is essential to regulate or control the meat spoilage to
maintain its texture, flavor, nutritional value and to increase its shelf life (Dave and Ghaly,
2011). Two factors play an important role in the meat deterioration: (1) pre slaughter
handling (2) post slaughter handling. When an animal is bare to pre slaughter stress, due
to this pH of meat changes to lower to higher level of pH scale depending upon the lactic
acid production and glycogen content of animal’s muscle decreased breakdown of glycogen
in animal muscle produced lactic acid via anaerobic glycolytic pathway. Due to high pH
ranges from 6.4-6.8 result in dry, firm and dark meat. Throughout processing and storage
of meat there are three mechanisms for spoilage of meat and its products. A) Autolytic
enzymatic spoilage. (B) Microbial spoilage. (C) Lipid oxidation (Miller, 2002). A number
of hypotheses are associated with the disease caused by the consumption of such rancid
meat and a meat product includes cardio vascular diseases, Cancers of several types,
Diabetes. Meat and its products quality is decreased by the lipid per oxidation.

Lipid peroxidation causes by the reactive oxygen species, which leads to severe health
problems (Bou et al., 2007). Lipids oxidation is responsible for reduction in nutritional
quality. When microbial contamination happens with lipid oxidation it changes taste of
meat. This can cause financial loss e.g. food poisoning and meat spoilage. Many tactics are
present to reserve their quality and safety e.g. chemical preservatives (Van et al., 2014),
super-chilling (Cyprian et al., 2013), high hydrostatic pressure (Feng et al., 2014), active
packaging (Barbosa-Pereira et al., 2014), natural preservatives such as chitosan (Darmadji
and Izumimoto, 1994), citrus by-products (Viuda-Martos et al., 2009) and nano particles
(Xie et al., 2011). Prolong refrigeration of meat and its product can lead to poor quality due
to lipid peroxidation. Oxidation of lipids occurs in those fats that are high in unsaturated
fatty acids (components of phospholipids). Lipid oxidation can be stops with the help of
supplementation of vitamin E to the animal (Lauridsen et al., 1997). Protein and lipid
oxidation are two major problems related to meat quality during storage and these problem
leads meat to off flavor development and rancidity. Antioxidants are being used to decrease
oxidation process and increase shelf life (Lauridsen et al., 1997). Uses of those agents
which have both properties, anti-microbial and anti-oxidant are helpful in preserving meat

8
or extending shelf life. Researchers have shown that lipid oxidation can be delayed or
inhibit by synthetic or natural food additives e.g. anti-oxidants (Sallam et al., 2004).
Compounds which prevent or delay oxidation process by ending the initiation of Oxidation
are called antioxidants. Antioxidants are of two types synthetic and natural. (BHT)
Butylated Hydroxy toluene and (BHA) Butylated Hydroxy anisole included in synthetic
antioxidants. Both of them are restricted these days, because they have carcinogenic
property. Dietary antioxidants can control lipid oxidation (Gladine et al., 2007). Those
antioxidants which are derived from plant based are called natural antioxidants e.g.
phenolic compounds, vitmins and flavonoids (El-Ghorab et al., 2012).

2.3 Phytochemicals

Phytochemicals is a term generally used for a diverse range of bioactive entities present in
plants. These phyto-constituents are responsible for providing color, flavor and natural
defensive system against pests however, these secondary metabolites are not essential for
growth and developments however, confer some health beneficial aspects against
pathogens and pests (Chede, 2013). The phytochemical profiling of these moieties avails
the presence of these bioactive compounds in fruits by providing the hint about the quantity
and characteristics of these bioactivities of plant material. These screening tests also assist
as preliminary step in many researches related protocols as isolation, purification and
utilization of inherent plant based bioactive compounds for pharmaceutics, Nutraceutics,
medicines and agro based industrial use (Mathew et al., 2012). These phytochemicals, used
up by fresh fruits or their byproducts, have been recommended to provide an inclusive
variability of biological roles including antioxidant, anti-mutagenicity, anti-aging, anti-
carcinogenicity and anti-inflammation to human health (Azar and Ahmad, 2015).

The compounds that are capable of preventing or inhibiting rancidity in food systems are
antioxidants. In order to counter the oxidation problems antioxidants that synthesized from
natural sources are widely used as food ingredients. While people have awareness about
adverse effects of synthetic compounds on health, consumer preference has greatly shifted
towards the natural ingredients. So now a days need to explore the natural antioxidants
including phenolic acid, carotenoids, tocopherols and vitamin C but the polyphenols are
most the important in this regard. Sources of these are fruits and vegetables, their products
correlated with reduced incidence of cardiovascular and chronic diseases and of certain
cancers (Horubala, 1999; Borowska, 2003).

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Natural antioxidants include the most important group named as plant phenolics, these are
present in almost all plants consist mostly of flavonoids, anthocyanins, phenolic acids, and
their glycosides. Tocopherols, resins or lignans are less polar substances which may be
added into oils and fats which increase their stability on heating and storage. Stabilization
of dispersions of food can be done by using more polar phenolics. Particularly, high amount
of phenolics found in tea leaves, roasted coffee, spices and cocoa beans. Efforts to increase
the level of antioxidant in the blood stream were not effective because most of phenolics
are deactivated immediately after or before the reabsorption via intestine wall. The need of
antioxidants in vivo would be lower if the intake of polyunsaturated fatty acid esters would
be kept sufficient, but not excessive (Pokorny, 2001).

Phenolic antioxidants interact with free lipidperoxy or lipidoxy free radicals, formed in
result of lipid oxidation, hence stopping their further self-breakdown (Frankel, 2005). Lipid
auto oxidation inhibition is essential not only in foodstuffs during their heating or storage,
but also to reduce lipids oxidation after ingestion and absorption via the intestinal wall.

2.3.1 Phytochemicals and antioxidants in fruits and vegetables waste

Antioxidant is very effective in delaying process of oxidation at a low concentration

(Gordon, 2001). Chances of many chronic diseases can be reduced by taking high dietary
antioxidants. e.g. cancers, cataracts and heart diseases (Vaya and Aviram, 2001). A
wide range of antioxidants are mutually vital for the elimination of free radicals to
defend the foodstuff from adverse properties of reactive oxygen species (ROS).
Particular enzymes and also cellular molecules which are non-enzymatic involve in the
purification of ROS. There are two types of antioxidants; Enzymatic and Non-Enzymatic
antioxidants (Jakus, 2000).

Enzyme act as major primary intracellular endogenous antioxidant defenses method. This
enzymatic anti-oxidant system includes catalase and superoxide dismutase (Yang et al.,
1999). Three iso-forms have been found of SODs (Superoxide dismutases). It shows a
vibrant part in scavenging O2 generated via electron transport chain, endoplasmic
reticulum and cytosolic oxidases. The extracellular superoxide dismutase is a tetrameric
protein found in the extra cellular space. It also catalyzes the dismutation of superoxide
into H2O2 (Ivanova and Zinoviev, 2000). Catalase is located in mitochondria and a large

10
tetrameric protein which removes hydrogen per oxide by catalyzing its transformation
into water (Krinsky, 1993).

Antioxidants which are Non-enzymatic can be further categorized into two groups:
exogenous and endogenous antioxidants. Transition metal binding proteins are the
foremost extracellular endogenous antioxidants which can found in human plasma; are
hepatoglobin, albumin and transferrin. They react with transition metals and hence
control or regulate the manufacture of metal-catalyzed free radicals. Ceruloplasmin and
Albumin are the copper ions isolates. Hepatoglobin reacts with haemoglobin but ferritin
and transferrin react with free iron (lvanova and Zinoviev, 2000). Uric acids, Lipoic,
ubiquinone, glutathione and bilirubin are non-protein endogenous antioxidants and have
the ability to prevent the oxidation reaction by hunting free radicals (Shahidi, 1997).

Many efficient exogenous antioxidants have usually of dietary origin. Best known
antioxidants are vitamins e.g. tocopherol, ascorbic acid, carotenoids, polyphenols and
quinones. These molecules can hinder the oxidative reactions by hunting free radicals.
Vitamin C has numerous antioxidant properties. It also scavenges others certain non-
radicals such as HOCl as well as free radicals (Packer and Cadenas, 2002). Phenolics
consists of all types antioxidants and common examples of antioxidants are (BHT)
butylated hydroxy toluene, (BHA) butylated hydroxyanisole, (TBHQ) tertiary butyl
hydroquinone, propyl and dodecyl gallates, octyl (Sherwin, 1989). Example of commonly
antioxidant used in food is butylated hydroxyanisole (BHA). There are two isomers that
collectively form BHA i-e 2-tert-butyl-4-hydroxyanisole (2-BHA, 10%) and 3-tertbutyl- 4-
hydroxanisole (3-BHA, 90%). Solubility of BHA are maximum in fats and oils (Madhavi
et al., 1996). There is no change occur in color, flavor after adding BHA and its efficiency
increased when it is added with other antioxidants e.g BHT or TBHQ. It is found increase
in effectiveness of BHA when we use BHA with dodecyl gallate than it is used single in
margarine (Coulter, 1988).

Mainly it is used in cereal products i-e foods having low fats. When 0.02% BHA is used in
pastry and cracker, stability of these are found 27-33 respectively (Madhavi et al., 1996).
21-22 days’ stability was founded in both products when used at 0.01% level. Levels of use
in bakery range from 0.01-0.04%. Properties of butylated hydroxytoluene are white
crystalline solid, odor is faint phenolic, and solubility in fats and oils is very good and also
used in food products. There is no change occur in odor, color and flavor when added in

11
food products. It can be used with other antioxidants like PG and TBHQ (Adegoke et al.,
1998). Its behavior with citric acid, BHA, and TBHQ is synergistic. With breakfast dry
cereals, snack foods, baked goods and vegetable oils, it used as antioxidant (Madhavi et al.,
1996). Maximum dose of Butylated Hydroxy Tolune is used in cake up to 200 mg/kg
(Coulter, 1988). When auto-oxidation of fats and oils occur, antioxidants are degraded
(Madhavi et al., 1996).

To inhibit oxidation in vegetable oils TBHQ is found very functional. The activities of
antioxidant are due to two hydroxyl groups present in antioxidants. Its stability is a
maximum at high temperature. There is no chance of complex formation with iron or
copper. BHA and BHT are equal or less active as compared to TBHQ. With fats and oils it
gives no color and odor. It shows synergistic behavior with other antioxidants and chelators
(Sherwin, 1989). Intake of antioxidants in the form of polyphenols in our body is through
diet and mostly fruits, vegetables, dry legumes, olives, cereals, coffee and tea are the main
sources of these phenols. Polyphenols act as antioxidants by reducing oxidations that occur
at cellular level and thus minimize the chances of various diseases like cancer,
neurodegenerative, cardiovascular and osteoporoses. Phenolic acids are of two types:
benzoic acid and cinnamic derivatives. Derivatives of benzoic acid e.g. Gallic Acid and
protocatechuic acid are very rare present in some type of plants that are consumed; therefore
these are not taken yet nutritional importance. These are present in lesser percentage in
plants except some berries, which have up to 270 mg/kg fresh weight (Shahidi et al., 1995).
Different types of antioxidants obtained from plant sources have been explored and are
used to inhibit oxidation (Pokorny, 2001). These are obtained from plants in form of
extracts and are founded useful by protecting the effect of free radicals which lead to
deterioration of food products and lipid oxidation. These are considered strong as compared
to synthetics (Oktay et al., 2003).

There are six classes of flavonoids on the basis of structures i-e flavones, isoflavones,
anthocyanidins, flavonols, flavanones and flavanols (Peterson and Johanna, 1998).
Quercetins are important type of flavonols. Onions are the richest source of flavonols (up
to 1.2g per kg fresh wt) and some other sources are leeks, curly kale, broccoli and
blueberries. In some extent these are also present in red wine and tea. Commonly these are
present in glycosylated nature. There are 5 and 10 different glycosides are present fruits
(Macheix et al., 1990). Mostly skin and leaves of plants i-e tissues present outside contain

12
flavonols because their synthesis occurs with the help of light (Price et al., 1995). Luteolin
and apigenin are major constituents of flavonols. Parsley and celery are only identified
edible sources up to now. Some kinds of flavones are also found in wheat and millet
(Sartelet et al., 1996). Large contents of methoxylated flavones are observed in citrus peel
(in mandarin up to 6.5 g/L) (Shahidi and Naczk, 1995).

Some amounts of flavones are present in mint and tomatoes which are taken as human
foods but in citrus these are present in higher concentrations. Glycosylation are present in
flavones by a disaccharide at carbon no. 7, bitter taste and no flavor would be in presence
of neohesperidose and rutinose respectively. 200-600mg hesperidin and 15-85mg narirutin
in one liter are observed in citrus juice. One glass of citrus juice would contain 40-10mg
flavanone glycosides. Higher flavones contents are present in white spongy part i-e albedo
and membranes that seprate the juice segments and it is found that one glass orange juice
is five time lesser flavones contents than that of whole fruit (Tomas-Barberan and Clifford,
2000).

Carotenes and xanthophylls are two classes of carotenoids. Hydrocarbons and oxygen in
different types of groups like keto, epoxy, hydroxyl or methoxyl are present in carotenes
and xanthophylls respectively. Typical type of carotene and xanthophylls are lycopene and
lutein, or xanthin respectively. Monocyclic (carotene), bicyclic (carotene), or acyclic
(lycopene) are different types of structural forms of carotenoids. It is observed that
conjugated types of double bonds are present in carotenoids and plant tissues contain all
carotenoids in Trans form. Carotenoids have much importance due to resistance against
different diseases e.g. muscular degeneration, cataracts and atherosclerosis. Functionality
of carotenoids depends upon the type and number of functional groups present in structure
of molecules and conjugated double bonds (Beutner et al., 2000). Products are not oxidized
by carotenoids, these are physical quenched. There is controversy about hydrogen donation
and oxygen quenching activities of carotenoids. There are two units of phenylpropane
present in lignans. Linseed contains largest contents of lignans. Some other sources of
lignans are cereals, fruits and some vegetables but these all contains one thousand times
less quantity of lignans as linseed (Adlercreutz and Mazur, 1997). Microflora present in
intestine metabolizes the lignans into enterodiol and enterolactone. When 2 units of lignans
are breakdown into its units that are actually present in very low quantities, ingested in our
body and do not move toward metabolites of urine and plasma. So, these natures of lignans

13
are not source of enterodiol and entero lactone (Heinonen et al., 2001). It had been tried to
find precursors of these units by fermenting food in vitro with microflora. By different kind
of experiments, it has been confirmed that linseed contains largest contents and other all
having low quantities.

Human diet contains stilbenes in very low quantity. Wine contains one type of stilbenes in
very low quantity i.e. resveratrol that shows effects as ant carcinogenic and this was highly
studied by scientists (Pezzuto and Park, 2002). Due to presence in very low quantity of
resveratrol in diet, it shows its effectiveness even at its normal intake. Tannins are present
in plants and important constituents of phenolic compound that behave like as antioxidants.
Substantial accumulation of tannins, occur in all types of parts e-g leaves, seeds, fruits and
vegetables. There is resemblance between galls produced in plants by insect attack and
production of tannins in increased quantity. Tannic acid is present in 12% in leaves on dry
basis of sumac. Mostly plant tissues e.g. leaves and fruits contain vegetable tannins
(Waterman and Mole, 1994). Structure of Gallic acid is very important for tannins that
could be hydrolyzed and such kind of tannins is produced by metabolism (secondary).
These hydrolyzed tannins are found in ester form such as gallo-tannins (Haslam, 1998).
Leuco anthocyanidins were same as tannins that were condensed. phenolic flavan-3-ol
(catechin) nucleus is the basic unit of structure in this category. Oligomers contain 2 to 10
or more units of polymers and catechin (Waterman and Mole, 1994).

Over-all, plant and fruits residues are known to enclose an extensive variation of
phytochemicals, e.g. carotenoid, vitamins and polyphenols. Up to 5000 different
phytochemicals have been recognized in vegetables, grains and fruits (Liu, 2003).
Saponins, tannins, flavonoids and alkaloids are present in fruits, checked by analysis of
phytochemicals (Thomas et al., 2008). Fruits are full of antioxidants, particularly
epicatechin, vitamin C and proanthocyanidins (Thomas et al., 2008). Therefore, natural
antioxidants present in vegetables and fruits and can hunt free radicals and be responsible
for oxidative constancy to many food stuffs together with meat products which are high in
fat content (Yogesh et al., 2012). Lately, phenolic compounds are abundantly present in
fruit extracts which were also added to meat to delay protein or lipid oxidation and
discoloration. These fruit extracts contain phenolics of pomegranate juice, extract of white
grapes (Jongberg et al., 2011) and strawberry extracts, arbutus berry, blackberry and
hawthorn (Ganhão et al., 2010).

14
Phenolic compounds of bayberry pomaces were evaluated for the antioxidant properties.
The key anthocyanin was cyanidin-3-O-glucoside (3.0–6.2 g/kg) on dry weight and the key
flavonol was quercetin-3-O-glucoside (296.2–907.9 mg/kg) on dry weight. Myricetin and
Quercetin were also detecting in the pomaces of bayberry, myricetin deoxyhexoside and
quercetin deoxyhexoside and were cautiously identified. The leading phenolic acids were
protocatechuic acid (29.5–57.2 mg/kg on dry weight basis) and gallic acid (102.9–241.7
mg/kg on dry weight basis). Phenolic acids such as vanillic, caffeic, p-hydroxybenzoic,
ferulic acids and pcoumaric were also found in the pomaces od bayberry (Zhou et al., 2009).

Extracts of different four citrus based herbal products were examined on the basis of their
antioxidant activities. This showed the significant activities of all the products. By this
experiment one thing also revealed that these products have not much reducing strength
(Su et al., 2008). Rose has been used to extract Gallic acid which is also a phenolic
compound and which has been demonstrated to exhibit significant antioxidant properties.
This compound has been shown to reduce lipid oxidation and too retard the aging process
in experimental mice Vitamin E, tea catechins, ascorbic acid and rosemary extracts are only
some of the extensively researched natural antioxidants (Mccarthy et al., 2001). Baratto et
al. (2003) suggested that frequent feeding of vegetables and fruits is related with a lower
hazard of cardiovascular disease and cancer. Worldwide countless plants have showed also
a powerful hunting activity to counter free radicals and antioxidant activity. This
antioxidant ability can be explored in food manufacturing industry by means of plants as
origin of antioxidants to prevent the rancidity and oxidation of lipids. In actual point, in
modern years, study has concentrated on therapeutic plants to withdraw low-cost. Natural
antioxidants which can substitute artificial additives like (BHT) butylated hydroxytoluene
and (BHA) butylated hydroxyanisole that possibly can be toxic and even carcinogenic.
Now-a-days there is a tough trend towards separating organic antioxidants from natural
bases as alternatives so a primary field of solicitation of herbs is the security of animals and
their foodstuffs against oxidation (Haak et al., 2006). Latest examinations in the arena of
antioxidants have focused on naturally occurring particles to please consumer worries
above toxicity and safety of food additives. Polyphenols are interesting between natural
antioxidants, since they are extensively spread in plants and display several antioxidant
properties (Giannenas et al., 2005).

15
2.3.2 Phytochemicals and antioxidants in banana peel and cabbage leaves

Banana is broadly grown tropical fruit cultivated in many countries cultivated in 130
countries. Eatable bananas are derived from australimusa and eumusa sequences which
have different origins from same genus. Most edible bananas are either resulting from Musa
accuminata. Three common species of Musa (M. cavendishii, M. paradisiaca, M.
sapientum) are commonly grownup in the world. M. cavendishii are known as dessert
banana, is sweeter and not as much of starchy. M. sapientum is known as true banana and
eaten as raw when it is fully mature (Valmayor et al., 1999; Robinson et al., 1996).

Botanically banana is a type of a berry fruit and it is consumable. Banana is produced by

many herbaceous flowering plant and these plants related to the Musa genus. It is a lost
coast and ideal food in world. Almost all new seedless eatable bananas obtain via two wild
species –Musa balbisiana and Musa acuminata. Banana is useful as antioxidant,
insecticidal, color absorber and in preparation of various functional foods such as wine,
alcohol, biogas and cattle feed. Therefore, the main point of view of this review article is;
explore the nutraceutical effects of banana to cure different diseases (Mohpatra et al.,
2010). Banana is produce round about 16% in world and it is a second largest produce fruit.
India has produce 27 % of banana in the world. Banana is easily eatable and digestible and
highly nutritious fruit then others. Banana is digested in 105min. potassium and calcium
found abundantly in banana and it is nadir in sodium content. Bananas help in the body's
maintenance of calcium, nitrogen, and phosphorus, which work to form healthy and
redeveloped tissues. Banana is common for odor, texture and easy to eat and peel (Robinson
et al., 1996). Banana is used against many diseases. Banana is used to cure intestinal
disorders like ulcer. Banana is one from the few fruits that ulcer patients can easily and
safely consume. Bananas also neutralize acidity of stomach or gastric juices that reduces
the ulcer irritation by covering the lining of stomach. Banana is not only relieved painful
ulcer and gastric or intestinal disorders but also support to healing in burns and wound.
Banana leaves are also use in burns and wound. Banana is also beneficial for constipation,
diarrhea, and arthritis and in treatment of anemia (Wall et al., 2006).

The peel of banana is a rich source of crude fat 3.8-11 (Emaga et al., 2008), crude protein
6-9%, total dietary fiber 43.2–49.70%, starch 3%, polyunsaturated fatty acids such as
linoleic acid, alpha linolenic acid and essential amino acids e.g. phenylalanine, valine,
threonine, leucine and other micronutrients such as Ca, Mg, P, K (Emaga et al., 2007). All

16
essential amino acids except lysine are advanced than FAO standard. Decrease in starch
and hemicelluloses and slightly increase in protein and lipid content and increase in
resolvable sugar involves in ripening or maturation of fruits. Endogenous enzymes, the
breakdown of hemicellulose and starch can explain the increased soluble sugar content
(Emaga et al., 2007). For removal of banana oils (amyl acetate) skins can also be used that
can be used for food essence. Pectin (10-20%), hemicellulose (6.4-9.4%), cellulose (7.6-
9.6%) and lignin (6-10%) are also good source of banana peel. Pectin remove by peel of
banana also has rhamnose, glucose, arabinose, galactose and xylose (Emaga et al., 2008).
As compared to pulp higher concentration of micronutrients such as Fe and Zn were found
in banana peel. So for cattle and poultry peel might be a good feed material (Emagart et al.,
2008; Adeniji et al., 2008; Dormond et al., 1998). In wine (Faturoti et al., 2006), production
of ethanol (Tewari et al., 1986), as base material for pectin removal and used as a substrate
for the production of biogas (Ilori et al., 2007) banana peel can also be used. Ash of banana
peel can also be used as alkali for soap manufacture and can use as fertilizers for banana
plants (Udosen and Enang, 2000).

Almost 500 above polyphenols have been discovered in food and beverages and they are
the richest bioactive compound (Neveu et al., 2010; Faller and Failho, 2010). Polyphenols
are abundantly found in the banana peel and pulp with majority of antioxidants.
Polyphenols are secondary metabolites with extremely diverse chemical structure (Faller
and Fialho, 2010; Manach et al., 2004; Shahidi and Naczk, 2004). Polyphenols imparts a
main part in plant’s inborn defense system mechanism. Difference in growing condition,
adulthood state, storing capacity, cultivar and handling results differ meaningfully in the
content present in plant (Jaffery et al., 2003; Faller and Fialho, 2010). Uncooked banana
paste contain epicatechin, gallic acid, catechin, epigallocatechin, and prodelphinidin dimer
(Harnly et al., 2006; Del et al., 2003; Arts et al., 2000; Pascual-Teresa et al., 2000). Bananas
peel rich in dopamine and catecholamine (Gonzalez-Montelongo et al., 2010). Bananas
have high antioxidant activity and have many health benefits it has been receiving an
increases and considerable interest from nutritionist, scientist and consumers (Neveu et al.,
2010). It helps in the inhibition of type 2 diabetes, cancer and cardiovascular diseases.
These plant metabolites have been implicated (Arts and Hollman, 2005; Scalbert et al.,
2005).

17
Researchers have been conducted on banana peel (Musa acuminata) which indicates that it
is a potential cause of bioactive complexes e.g. polyphenols and flavonoids. Due to their
high free radical scavenging activity polyphenols and flavonoids got vast range of
medicinal property (Singhal and Ratra, 2013). Phytochemical compounds found
abundantly in banana peel, mainly antioxidants. Expected quantity of phenolic compound
in banana (Musa acuminata) peel varies from 0.90 to 3.0/100g (Someya et al., 2002).
Brassica oleracea var. capitata scientific name of Cabbage is a vegetable whose
consumption is possibly of countless effect as it is used as food in many counties. Almost
the entire world and constitutes a chief component of diet of Central European. Cabbage
goes to cruciferous vegetables which are exposed to be more powerfully related with the
protection of cancer with respect to the consumption of vegetable in general (Finley, 2005).
Crucifers contains anti-carcinogenic properties which are of most often recognized to
(GLS) glucosinolates and produces from the breakdown of indolesi and (ITC) iso-
thiocyanates. These vegetables contain anti-oxidative potential and has drawn
comparatively slight attention so far. Still, among them cabbage “crucifers” also comprise
on anti-oxidative compounds (Kusznierewicz et al., 2008) which includes flavonoids
(Hertog et al., 1992).

There is a bigger interest in the likely health encouraging properties of phenolics (Brassica
oleracea var capitata) and (B. oleracea var italica) scientific name of broccoli, having great
nutritional status besides hold organo-sulfur phyto-chemicals that add to their antioxidant
action, which might have anti-carcinogenic effects (Jeffery and Jarrell, 2001). Other
antioxidant compounds which are available in high conc. in cruciferous vegetables include
the flavonoids for example kaempferol and quercetin (Formica and Regelson, 1995).
Vegetables which belong to brassicaceae family are great source of health promoting
elements, which helps to lower the risk of diseases. They possess anti-cancerous
glucosinolates as well as antioxidants of both hydrophobic phase (vitamin E and
carotenoids) and hydrophilic phase (polyphenols and vitamin C) which can help to
neutralize oxidative reactions and quench free radicals. Major antioxidants in brassicaceae
vegetable is phenolic compounds present with ascorbic acid, mean while only 20% of lipid
soluble antioxidants are responsible for total antiradical capacity (Podsedek, 2007). Data
has been analyzed on Brussels sprout, brassica species, red cabbage and broccoli and results
show that all of them considered having most efficient antiradical system (Podsedek, 2007).
The latter contain antho-cyanins which have high antioxidant activity (Proteggente et al.,

18
2002; Singh et al., 2006; Can and Altiokka, 2007). Total level of phenolic substance in
freshly harvested cabbage was range between (31.4-288.3) and 43.3-248.8 mg 100 g-1. The
lower most content of these constituents was noticed in white cabbage, the highest in red
‘Langedijker’ and ‘Kissendrup’ cultivars. A significant in consistency in red cabbage was
also observed (Maria et al., 2010).

2.4 Ultrasound assisted extraction

It is the first significant process to segregate the natural bioactive compounds from plants
and materials, known as extraction. There are many different extraction methods have been
advanced for the withdrawal of natural oils and bioactive compounds through microwave
assisted extraction, expelling extraction process, solvent extraction, and supercritical fluid
extraction (SFE) and so on (Wang and Weller, 2006). Each of these processes has some
shortcomings with them, such as huge investment is needed in supercritical fluid extraction
(SFE), squat output in expelling method, and additional quantity is required in solvent
extraction and in microwave assisted extraction aqueous stage is required to attain the
various targeted compounds in pecuniary circumstances (Samaram et al., 2013). A novelty
through Ultrasound-assisted extraction is introduced for the extraction of oil and bioactive
compounds from various sources. By the use of ultrasound power generates extra vibration
in target compounds are extracted in fluid solvent phase from solid materials.
Consequently, higher production in small removal (extraction) time alongside the
consumption of squat solvent amount is the highly significant pros of this method.
Additionally, this process of extraction has exceptional usage with polar and non-polar
solvents at different temperatures (Samaram et al., 2015).

2.5 Development of functional meat

In these days, much awareness has been seen in the consumers towards the use of artificial
spices in food industry and has enhanced the demand for natural food preservatives in the
industry ((European Food Safety Authority, 2010). A research study conducted by (Su et
al., 2007) stated that antioxidants in the diet have constructive effects on human body and
health they guard the imperative cellular parts such as lipids, membrane, proteins, and DNA
from oversensitive oxygen species. Many synthetic antioxidants have been rejected by the
consumers due to carcinogenetic effect (Altmann et al., 1986). Butylated hydroxyl anisol
(BHA) and butylated hydroxyl toluene (BHT) have been implicated in the children care,
(ADHD) Deficit Hyperactivity Disorder (Feingold, 1982). Presently, the concern in natural
19
occurring antioxidants has been improved as they are well-thought-out to be inoffensive
than the artificial antioxidants, and have larger application prospective for consumer’s
palatability, acceptability, shelf-life and stability of products of meat (Kang et al., 2008;
Naveena et al., 2008; Park and Kim, 2008).Throughout new decades the consumers,
regulatory authorities and food industries have advanced an important attention in
functional foods due to their latent human health benefits above and over their elementary
nutritional worth (Viuda Martos et al., 2013). Numerous works have exposed sign of
removal of synthetic food additives can perfect the behavior disorder (Konikowska et al.,
2012). Lately a negative movement about the potential health hazards by consumption of
meat occurred (Verma and Banerjee, 2010; Biswas et al., 2011) and understanding the
association between health and nutrition has caused in the expansion of idea of functional
foods comprising on meat products (Bhat and Bhat, 2011). Application of extracts of
natural plant as a substitute to synthetic or chemical antioxidants and antimicrobials to
regulate food borne diseases, suppressing oxidation of lipids and thus prolonging the
quality and shelf life of food produces is a growing claim in the industry of Food. By-
products of vegetables and fruits have been accepted as an imperative source for an
extensive selection of Phytochemicals and non-digestible constituents that in combination,
or independently, might act as synergistically to donate to the health and nutritional aids to
food products e.g. meat products. In General, the antioxidant ability of meat is actually low
and can be enhance by the accumulation of flavonoids in meat throughout processing in the
form of parts of plants which are ironic in extracts or their flavonoids.

2.6 Health benefits of functional meat

“A food that beneficially affects one or more target functions in the body beyond adequate
nutritional effects in a way that is relevant to either an improved state of health and well-
being and /or reduction of risk of disease” (Yadav, 2013). Back in two thousand years,
Hippocrates stated that “Let food be your Medicine and Medicine be your food”. Later
from long time a pure connection was between the nutritional health status and food which
people ate. There is also a saying in Ayurveda “when diet is wrong, medicine is of no use,
when diet is correct medicine is of no need. The original idea after functional food is to
decrease the occurrence of long-lasting diseases by reducing the intake of habitually
consumed foods (Vasconcellos, 2001). Recent studies show that antioxidants are being
used to preserve raw meat as well as cooked meat. Karre et al. (2013) examined the effect

20
of anti-oxidants which have been extracted by several plant extracts and fruit juices on
poultry and meat. (Shah et al., 2014) have been studied on oxidative strength of meat via
defensive role of many plant extracts; same as Kumar et al. (2015). In human bodies,
enzymes also include in antioxidant defense mechanism e.g. (glutathione peroxidases,
superoxide dismutase, and catalase), copper binding extra-cellular proteins and iron e.g.
(transferrin, albumin, haptoglobin, lactoferrin, and ceruloplasmin), vitamins which are anti-
oxidants e.g. (vitaminE, β-carotene and vitamin C), and further cellular complexes e.g.
glutathione, quinones, bilirubin and uric acid (Krinsky, 1992).

Natural anti-oxidants have proven efficiency in the form of a blend of active compounds,
leaves, a powder and pure extract, of original seeds, etc. all of them are being used to reduce
flavor deterioration and lipid oxidation in products of meat, which has motivated a vast
attention in the meat industry to discover non-traditional food element plans. The
responsiveness towards natural anti-oxidants is delicate by the new global development to
progressively point out artificial food additives which have conventionally been used in the
food chain. Current and latent natural anti-oxidant practical have been applied to meat to
enhance shelf life security have been focused on plant based complexes; their objectives
mainly on fresh meat (Karre et al., 2013; Kumar et al., 2015; Shah et al., 2014). To one side
from enhancing organoleptic qualities and shelf-life, the usage of nutraceuticals which are
plant derived and rich in anti-oxidant phenolics and flavonoids, which might let meat
processors to produce innovative products with enriched nutritional health aids e.g.
combination of ingredients of vegetable food, such as walnut and rice bran extracts which
are ironic in vitamin B, polyphenols and vitamin E, and into substitute of sausage of
vegetable oil, re-structured beef and also other treated meats to improve the anti-oxidative
stability, textural properties and nutritional value of products (Alvarez et al., 2012; Alvarez
et al., 2011; Cofrades et al., 2004; Jiménez-Colmenero et al., 2010). A study has been
conducted, whose results tells us enhancement in texture, color content and vitamin A of
beef patties by the addition of the cooked sweet potatoes and carrots (Saleh and Ahmed,
1998). Similarly, Csapo et al. (2006) made a range of meat products by adding lutein to
supplement the content of pro-vitamin A, thus stimulating the health of eye. Likewise,
apple pomace is rich in polyphenol remainder of apple juice, which can be a significant
basis of nutraceuticals which contains anti-oxidants for processed meat (Hyson, 2011;
Rather et al., 2015). Existence of dietary-fibers in vegetable and fruit elements imparts extra
aids to products of meat. Antioxidant compounds which are extracted from plants have

21
therapeutic functions when incorporated into processing of meat. Role of these therapeutic
anti-oxidants in stimulating health and hindering numerous physical sicknesses and
pathological circumstances have been well documented.

Intake of meat and its produces which are ironic in natural anti-oxidants has been presented
to strengthen the endogenous efficiency of antioxidant beside ROS-induced degenerative
diseases, oxidative stress, and tissue damage. Although natural occurring phenolic
antioxidants have overall helpfulness in helping the complete health, the defense of the
gastro-intestinal track’s health appears to be utmost noticeable because absorption is not
needed (Halliwell et al., 2005). Dissimilar antioxidants which are non- protein contain the
narrow part of stabilizing the free-radicals, destroying radical propagation. Peptides of
antioxidant may utilize former biological functionalities e.g. anticancer, anti-hyperten,
immune modulatory, opioid activities and antimicrobial (Mine et al., 2010). Therefore,
peptides of antioxidant which occurs naturally derived from the hydrolysis of protein are
nowadays deliberated as innovative and imminent food ingredients to support human
health.

22
Chapter 3

MATERIALS AND METHODS

3.1 Procurement of raw materials

Fruit and vegetable by-products (banana peel and cabbage leaves) were collected from local
fruits and vegetables processing industries in Faisalabad, Pakistan. The selected raw
materials were cleaned to remove the adhered dirt, dust and other foreign debri s.

3.2 Extraction of total polyphenols contents (TPC) from banana peel and cabbage
leaves

Pre dried the banana peel and cabbage leaves powder and weight (100±0.1g) using the
electronic weighing balance (Model Kern 440-35N) for each treatment. A 3-level five
factor Box-Behnken Design was used to study the effect of extraction/sonication
temperature (˚C), amplitude level, water/meal ratio, extraction/sonication time (minutes)
and pH conditions for maximum yield of total polyphenols from dried fruits and vegetable
samples. The coded design of real experiments is given in Table 3.1. The pH of the solution
was monitored continuously and was adjusted by 0.2mol/L NaOH and HCl, respectively
while the temperature of the aqueous system was controlled within ±1.5˚C.

Table 3.1: Coded and actual levels of independent variables for optimization of total
polyphenols yield as determined by Box-Behnken design

Independent variables Coded levels

-1 0 1
Extraction temperature (˚C) 40 50 60
Amplitude level (%) 30 60 90
Water/Meal ratio 20 30 40
Sonication time (minutes) 20 40 60
pH 4 6 8

The extracted TPC solutions were separated and then dried in a hot air oven at 50°C. Yield
of TPC was calculated as percentage of dried polyphenol extract.

23
Table 3.2: Treatment combinations for extraction of TPC from dried banana and cabbage
leaves powder as determined by Box-Behnken design

Independent variables
Extraction
Extraction Extraction
run Amplitude Water/meal Extraction
temperature time
level (%) ratio pH
(˚C) (Minutes)
1 50 (0) 60 (0) 40 (1) 40 (0) 8 (1)
2 60 (1) 60 (0) 30 (0) 40 (0) 8 (1)
3 50 (0) 60 (0) 20 (-1) 40 (0) 4 (-1)
4 50 (0) 30 (-1) 30 (0) 40 (0) 4 (-1)
5 40 (-1) 30 (-1) 30(0) 40 (-1) 6 (0)
6 50 (0) 30 (-1) 30 (0) 60 (1) 6 (0)
7 50 (0) 60 (0) 30 (0) 20 (-1) 4 (-1)
8 50 (0) 30 (-1) 20 (-1) 40 (0) 6 (0)
9 40 (-1) 60 (0) 30 (0) 40 (0) 8 (1)
10 50 (0) 60 (0) 30 (0) 40 (0) 6 (0)
11 40 (-1) 60 (0) 30 (0) 60 (1) 6 (0)
12 50 (0) 60 (0) 40 (1) 60 (1) 6 (0)
13 60 (1) 30 (-1) 30 (0) 40 (0) 6 (0)
14 60 (1) 60 (0) 40 (1) 40 (0) 6 (0)
15 50 (0) 30 (-1) 30 (0) 40 (0) 8 (1)
16 60 (1) 60 (0) 30 (0) 20 (-1) 6 (0)
17 50 (0) 60 (0) 40 (1) 20 (-1) 6 (0)
18 50 (0) 90 (1) 30 (0) 60 (1) 6 (0)
19 50 (0) 60 (0) 30 (0) 60 (1) 4 (-1)
20 50 (0) 60 (0) 20 (-1) 40 (0) 8 (1)
21 40 (-1) 60 (0) 40 (1) 40 (0) 6 (0)
22 50 (0) 60 (0) 30 (0) 40 (0) 6 (0)
23 50 (0) 30 (-1) 30 (0) 20 (-1) 6 (0)
24 50 (0) 60 (0) 20 (-1) 20 (-1) 6 (0)
25 60 (1) 90 (1) 30 (0) 40 (0) 6 (0)
26 50 (0) 60 (0) 30 (0) 60 (1) 8(-1)
27 50 (0) 30 (-1) 40 (1) 40 (0) 6 (0)
28 50 (0) 90 (1) 30 (0) 20 (-1) 6 (0)
29 50 (0) 90 (1) 30 (0) 40 (0) 6 (0)
30 50 (0) 60 (0) 20 (-1) 40 (0) 6 (0)
31 50 (0) 90 (1) 20 (-1) 60 (1) 6 (0)
32 50 (0) 60 (0) 30 (0) 40 (0) 6 (0)
33 40 (-1) 60 (0) 20 (-1) 40 (0) 6 (0)
34 60 (1) 60 (0) 30 (0) 60 (1) 6 (0)
35 60 (1) 60 (0) 30 (0) 40 (0) 4 (-1)
36 40 (-1) 90 (1) 30 (0) 40 (0) 6 (0)
37 40 (-1) 60 (0) 30 (0) 20 (-1) 6 (0)
38 50 (0) 60 (0) 30 (0) 40 (0) 6 (0)

24
39 50 (0) 90 (1) 30 (0) 40 (0) 8 (1)
40 40 (-1) 60 (0) 30 (0) 40 (0) 4 (-1)
41 50 (0) 60 () 40 (1) 40 (0) 4 (-1)
42 50 (0) 60 (0) 30 (0) 40 (0) 6 (0)
43 60 (1) 60 (0) 20 (-1) 40 (0) 6 (0)
44 50 (0) 90 (1) 40 (1) 40 (0) 6 (0)
45 50 (0) 60 (0) 30 (0) 20 (-1) 8 (1)
46 50 (0) 90 (1) 30 (0) 40 (0) 4 (-1)

3.3 Analysis of fruit and vegetable phytochemical extracts

The phytochemical extracts were analyzed for chemical composition.

3.3.1 Total polyphenols

Total polyphenols was measured by using Folin-Ciocalteu method following the protocol
of (Singleton et al., 1999). For the intention, 50 µL extract was added to 250 µL of Folin-
Ciocalteu reagent with 750 µL of 20% sodium carbonate solution and the volume was made
upto 5 mL with distilled water. After 2 hours, absorbance was recorded at 765 nm with
UV/Visible Spectrophotometer against control.

3.3.2 Determination of cyanogenic glycosides

The cyanogenic contents in extract samples was estimated by alkaline titration
according to the method outlined in AOAC (1990) Method No. 26.115. 20 g of extract
was taken in kjeldhal flask following the addition of 200 mL distilled water and was
slowly mixed the sample. The sealed flask was given rest (3h) to get proper hydrolysis
process of the mixture. Then, distillation process was carried out by connecting the flask
to a vapor distilling apparatus and distillate was collected in a flask containing 20 mL
of 2.5% NaOH solution until a marked volume. 8 mL of 6 M NH 4OH and 2 mL of 5%
KI solution was transferred into the distillate solution before titration against 0.02 M
AgNO3 by microburette. The used volume of AgNO 3 was noted for each individual
extract sample.

3.3.3 Estimation of tannin

For tannins, 0.5 g of the sample extract and 100 mL of distilled water was added in a
conical flask, gently boiling for 1 hour and was filtered using Whatman No. 44 filter
paper. The filtrate was diluted to 100 mL and then cooled. For the greenish blue color
development, 50 mL aliquot was put into each flask. This was followed by 5 mL Folin–

25
Dennis reagent, 10 mL of saturated sodium carbonate solution and was diluted to 100
mL with distilled water. After thorough mixing, the flasks was allowed to stand in a
water bath at 250 °C for 20 min and the optical density was measured at 700 nm through
spectrophotometer. Distilled water was used as blank regarding the calibration curve.
Standard tannic acid solution will be prepared from which a standard curve was drawn
(absorbance versus concentration in mg/cm 3). From this curve, the concentrations for
each sample were used for the tannin content calculation (Nwinuka et al., 2005).

3.4 Functional meat product development and analysis

Natural fruit and vegetable phytochemical extracts was supplemented at different
concentration to fish meat for preparation of different meat products i.e. patties, balls/pops
and finger/sticks. The partial/parfrying of the products was carried out to determine the
lipid stability. Fried meat products were vacuum sealed in plastic bags and then were stored
at refrigerator and −18 °C in a freezer for a storage period of 60-days. The moisture content
also the oxidative stability of oils extracted from fish meat products was assessed by
measuring peroxide value (AOCS 1998 Method No. Cd 8‒53) and free fatty acid value
(AOCS 1998 Method No. Ca 5a‒40), respectively.

3.5 Sensory evaluation of meat products

Experienced and untrained assessors carried out the sensory analysis of meat product
samples according to the instructions given by Meilgaard et al. (2007). Each judge gave the
written informed consent after explanation of risks and benefits of participation prior to the
study. The panelists were provided informative instructions and brief definitions of
attributes such as color, odor and overall acceptability. Samples were presented to
participants in sensory booths under white lighting. The order of presentation was balanced
to avoid carry-over effects. Each panelist received the samples assigned with random three‒
digit code numbers. Each panelist was asked to list their preference on a 9‒cm comparison
line (1 = dislike extremely to 9 = like extremely). The sensory analysis was performed at
different storage intervals for experimental treatments.

3.6 Statistical analysis

The behavior of the Box-Behnken Model was explained by the following the quadratic
Equation. The data of TPC yield obtained for each treatment was subjected to statistical
analysis to determine the level of significance by using the software package Statistic 8.1
according to the method described by Montgomery (2008). The average of the three runs

26
was reported as the measured value with standard deviation. The Duncan’s multiple range
(DMR) test was used to estimate the level of significance that existed among the mean
values. The sample analysis for storage stability and consumer acceptability was carried
out in triplicate and the significant differences were calculated among means at a
probability level of 5%.

27
Chapter 4

RESULTS AND DISCUSSION

4.1 Optimal extraction of total polyphenols from banana peel and cabbage leaves

A 3-level five factors Box-Behnken design was used to study the effect of extraction
temperature (˚C), amplitude power level, water/meal ratio, extraction time (minutes) and
extraction pH conditions for maximum yield of total polyphenols from banana peel and
cabbage leaves. The total number of experimental runs for each banana peel and cabbage
leaves was 46 as determined by the Box-Behnken design. The experimental response
regarding total polyphenol content (TPC) yield in banana peel and cabbage leaves samples
as a result of different extraction treatments have been depicted in Table 4.1. The percent
values of TPC yield from banana peel and cabbage leaves samples ranged from a minimum
value of 15.55±0.13% to a maximum value of 24.4±0.17% for banana peel and from
minimum value of 9.8±0.12% to a maximum value of 19.8±0.15%. The results revealed
that extraction conditions significantly affect the TPC yield from banana peel and cabbage
leaves. There is very limited published data that provides an information or support to the
TPC yield from banana peel and cabbage leaves. The extraction run point 25 (extraction
temperature, 60˚C; amplitude/sonication level, 90; extraction pH, 6; water/meal ratio, 30
and extraction time, 40 minutes showed maximum TPC yield for banana peel (24.4±0.17%)
and cabbage leaves (19.8±0.15%), respectively and the lowest TPC yield 15.55±0.13% and
9.8±0.12% was for extraction run 4 (extraction temperature, 50˚C; extraction pH, 4;
water/meal ratio, 30; amplitude level 30 and extraction time, 40 minutes) of banana peel
and extraction run 5 (extraction temperature, 40˚C; extraction pH, 6; water/meal ratio, 30;
amplitude level 30 and extraction time, 40 minutes) of cabbage leaves, respectively.

The optimized parameters for the extraction method of TPC were: aqueous extraction with
ratio 1:20 (water:meal) for both cabbage leaves and banana peel extraction time 40 minutes
with constant stirring at 60°C and sonication with high intensity probe ultrasound. The
optimal sonication conditions among all tested were 30% of maximum ultrasonic power
for 40 minutes. These parameters were optimized taking into account the necessity of
ultrasonic waves to eliminate interaction between the TPC and the other peel fractions but
minimizing the ultrasonic power and time of treatment to avoid the raising of sample
temperature caused by the ultrasound application. The temperature of the sample after 40

28
min of sonication ranged from 60 to 65°C, this temperature did not exceed in any case. TPC
yield obtained from banana peel (24.4±0.17%) and cabbage leaves (15.55±0.13%) was
similar to findings of earlier reported studies in literature. Although extraction yields
obtained were similar or higher to those described in the literature, former methods of
extraction were more aggressive than the proposed in the present work.

4.1.1 Fitting the experimental model

The predicted values of TPC yields were calculated using regression model and were
compared with experimental values to assess the validity of second-order polynomial
response model. The predicted values of TPC yields were located within the range of
experimental values (Table 4.1). The computed values for degree of correlation (0.9947),
coefficient of variation (2.32%), adjusted coefficients of determination (0.9850) and
adequate precision (30.772) indicate the adequacy and reliability of the applied second-
order polynomial response model. The degree of correlation value for TPC yield was close
to 1, which indicated that the second-order polynomial response model explained about
99.47% of the variability observed in the present study. The coefficient of variation less
than 5% indicates that the model is reproducible (Wang et al., 2006). The mathematical
second order polynomial response model found after fitting the function to the experimental
data sometimes does not satisfactorily describe the effect of independent variable whether
significant or non-significant. Thus, the model fitted was evaluated to know the effects of
independent variables as linear, interaction and quadratic coefficients using the analysis of
variance (ANOVA). The results indicated that TPC yield was significantly affected by the
model effects (Table 4.2, 4.3) banana peel and cabbage leaves, respectively. The linear and
quadratic effects model effects were observed more significant as compared to interaction
(p≤0.01). The order of model effects observed during TPC yield from dried banana peel
and cabbage leaves was linear >quadratic >interaction. The results further substantiated
that the linear coefficients (extraction temperature, extraction pH, amplitude level and
extraction time) (p≤0.01) and quadratic term coefficient (extraction pH × extraction pH)
(water/meal ratio × water/meal ratio) (p≤0.01) were most significant.

29
 Table 4.1: Percent values of TPC yield as determined by the Box-Behnken design

Independent variables Total polyphenols

extract yield (%)
Extraction
Extraction Extraction Cabbage Banana
run Amplitude Water/meal Extraction
temperature time leaves Peels
level (%) ratio pH
(˚C) (Minutes) (R1) (R2)
1 50 (0) 60 (0) 40 (1) 40 (0) 8 (1) 18.5 23
2 60 (1) 60 (0) 30 (0) 40 (0) 8 (1) 19.8 24.3
3 50 (0) 60 (0) 20 (-1) 40 (0) 4 (-1) 12.95 17.45
4 50 (0) 30 (-1) 30 (0 ) 40 (0) 4 (-1) 11.05 15.55
5 40 (-1) 30 (-1) 30(0) 40 (-1) 6 (0) 9.8 14.3
6 50 (0) 30 (-1) 30 (0) 60 (1) 6 (0) 13.6 18.1
7 50 (0) 60 (0) 30 (0) 20 (-1) 4 (-1) 12.85 17.35
8 50 (0) 30 (-1) 20 (-1) 40 (0) 6 (0) 11.7 16.2
9 40 (-1) 60 (0) 30 (0) 40 (0) 8 (1) 14.8 19.3
10 50 (0) 60 (0) 30 (0) 40 (0) 6 (0) 14.8 19.3
11 40 (-1) 60 (0) 30 (0) 60 (1) 6 (0) 13.6 18.1
12 50 (0) 60 (0) 40 (1) 60 (1) 6 (0) 17.3 21.8
13 60 (1) 30 (-1) 30 (0) 40 (0) 6 (0) 14.8 19.3
14 60 (1) 60 (0) 40 (1) 40 (0) 6 (0) 18.5 23
15 50 (0) 30 (-1) 30 (0) 40 (0) 8 (1) 14.8 19.3
16 60 (1) 60 (0) 30 (0) 20 (-1) 6 (0) 16.6 21.1
17 50 (0) 60 (0) 40 (1) 20 (-1) 6 (0) 15.3 19.8
18 50 (0) 90 (1) 30 (0) 60 (1) 6 (0) 18.6 23.1
19 50 (0) 60 (0) 30 (0) 60 (1) 4 (-1) 14.85 19.35
20 50 (0) 60 (0) 20 (-1) 40 (0) 8 (1) 16.7 21.2
21 40 (-1) 60 (0) 40 (1) 40 (0) 6 (0) 13.5 18
22 50 (0) 60 (0) 30 (0) 40 (0) 6 (0) 14.8 19.3
23 50 (0) 30 (-1) 30 (0) 20 (-1) 6 (0) 11.6 16.1
24 50 (0) 60 (0) 20 (-1) 20 (-1) 6 (0) 13.5 18
25 60 (1) 90 (1) 30 (0) 40 (0) 6 (0) 19.8 24.3
26 50 (0) 60 (0) 30 (0) 60 (1) 8(-1) 18.6 23.1
27 50 (0) 30 (-1) 40 (1) 40 (0) 6 (0) 13.5 18
28 50 (0) 90 (1) 30 (0) 20 (-1) 6 (0) 16.6 21.1
29 50 (0) 90 (1) 30 (0) 40 (0) 6 (0) 14.8 19.3
30 50 (0) 60 (0) 20 (-1) 40 (0) 6 (0) 16.7 21.2
31 50 (0) 90 (1) 20 (-1) 60 (1) 6 (0) 15.5 20
32 50 (0) 60 (0) 30 (0) 40 (0) 6 (0) 14.8 19.3
33 40 (-1) 60 (0) 20 (-1) 40 (0) 6 (0) 11.7 16.2
34 60 (1) 60 (0) 30 (0) 60 (1) 6 (0) 18.6 23.1
35 60 (1) 60 (0) 30 (0) 40 (0) 4 (-1) 16.05 20.55
36 40 (-1) 90 (1) 30 (0) 40 (0) 6 (0) 14.8 19.3
37 40 (-1) 60 (0) 30 (0) 20 (-1) 6 (0) 12 17
38 50 (0) 60 (0) 30 (0) 40 (0) 6 (0) 14.8 19.3

30
39 50 (0) 90 (1) 30 (0) 40 (0) 8 (1) 19.8 24.3
40 40 (-1) 60 (0) 30 (0) 40 (0) 4 (-1) 11.05 15.55
41 50 (0) 60 () 40 (1) 40 (0) 4 (-1) 14.75 19.25
42 50 (0) 60 (0) 30 (0) 40 (0) 6 (0) 14.8 19.3
43 60 (1) 60 (0) 20 (-1) 40 (0) 6 (0) 16.7 21.2
44 50 (0) 90 (1) 40 (1) 40 (0) 6 (0) 10.5 23
45 50 (0) 60 (0) 30 (0) 20 (-1) 8 (1) 16.6 21.1
46 50 (0) 90 (1) 30 (0) 40 (0) 4 (-1) 16.05 20.55

31
Table 4.2: ANOVAs of the predicted second-order polynomial model for TPC yield of
banana peel

df F value p value
Source of variation
Intercept 20 442.02*** ˂0.0001
A:Extraction temperature 1 33065.09*** ˂0.0001
Linear B:Amplitude Level 1 3095.98*** ˂0.0001
C:Water/meal ratio 1 401.24*** ˂0.0001
D:Sonication time 1 401.24*** ˂0.0001
E:pH 1 1741.49*** ˂0.0001
AB 1 0.000 1.0000
AC 1 0.000 1.0000
AD 1 0.31 1.5829
AE 1 0.000 1.0000
BC 1 0.000 1.0000
Interaction BD 1 0.000 1.0000
BE 1 0.000 1.0000
CD 1 0.000 1.0000
CE 1 0.000 1.0000
DE 1 0.000 1.0000
A2 1 2.70 < 0.1127
B2 1 0.30 < 0.5886
Quadratic C2 1 19.21 < 0.0002
D2 1 43.23 < 0.0001
E2 1 94.59 < 0.0001
Residual 25 - -
Lack of Fit 20 - -
Pure Error 5 - -
Cor. Total 45 - -

32
Table 4.3: ANOVAs of the predicted second-order polynomial model for TPC yield of
cabbage leaves

df F value p value
Source of Variation
Intercept 20 4482.57*** < 0.0001
A:Extraction temperature 1 30628.13*** < 0.0001
Linear B:Amplitude Level 1 31250.00*** < 0.0001
C:Water/meal ratio 1 4050.00*** < 0.0001
D:Sonication time 1 4753.13*** < 0.0001
E:pH 1 17578.13*** < 0.0001
AB 1 0.000 1.0000
AC 1 0.000 1.0000
AD 1 12.50 0.0016
AE 1 0.000 1.0000
BC 1 0.000 1.0000
Interaction BD 1 0.000 1.0000
BE 1 0.000 1.0000
CD 1 0.000 1.0000
CE 1 0.000 1.0000
DE 1 0.000 1.0000
A2 1 1.70 0.2036
B2 1 0.19 0.6672
Quadratic C2 1 232.01 < 0.0001
D2 1 288.07 < 0.0001
E2 1 1037.12 < 0.0001
Residual 25 - -
Lack of Fit 20 - -
Pure Error 5 - -
Cor. Total 45 - -

33
4.1.2 Single factor analysis

The effect of extraction temperature (˚C), amplitude level (%), water/meal ratio, extraction
time (mints) and pH conditions on TPC yield was analyzed by standardizing the levels from
–1 to +1 for each factor. The increased in extraction temperature at specific level resulted
in higher percentage of TPC yield when amplitude level, pH, water/meal ratio and
extraction/sonication time (mints) conditions were set at mean values i.e. 90, 6, 30, and 40
minutes, respectively for both banana peel and cabbage leaves. It is also evident from the
Table 4.2, 4.3 that the extraction temperature was the most significant process variable
which significantly affected the TPC yield. A different effect of water/meal ratio on the
percentage of TPC yield was observed by setting the extraction temperature (˚C), amplitude
level (%), pH and extraction time (mints) conditions at center point. The significant
production of TPC was obtained at initial points of water/meal ratio.

4.1.3 Analysis of mutual interaction effect

The ultimate objective of employing response surface methodology in this study was to
find out the significant effects of parameters viz., extraction temperature, amplitude level,
water/meal ratio, pH and extraction/sonication time to find out the optimum conditions for
TPC yields. The conditions and mutual interaction terms which determine the rate of
maximum TPC yields are difficult to optimize and cannot be manipulated directly from the
response model. The optimum extraction conditions for the maximum TPC yields were
obtained by varying two independent parameters and fixing the other three variables at the
coded zero level. Response surface for the mutual interactions effects of extraction
temperature (oC) and amplitude level (%) on the yield of TPC (%) at water/meal ratio 30,
sonication time 40 minutes and pH 6. The extraction temperature, 60˚C;
amplitude/sonication level, 90; extraction pH, 6; water/meal ratio, 30 and extraction time,
40 minutes showed maximum TPC yield for banana peel (24.4±0.17%) and cabbage leaves
(19.8±0.15%), respectively and the lowest TPC yield (15.55±0.13%, 9.8±0.12%) was at
extraction temperature, 50˚C; extraction pH, 4; water/meal ratio, 30; amplitude level 30
and extraction time, 40 minutes of banana peel and extraction yield of TPC at temperature,
40˚C; extraction pH, 6; water/meal ratio, 30; amplitude level 30 and extraction time, 40
minutes for cabbage leaves, respectively. The results revealed that extraction conditions
significantly affect the TPC yields from banana peel and cabbage leaves.

34
The surface plot for chosen model variables shown in Fig. 4.1 and 4.2 illustrates
relationship for the mutual interaction effect of extraction temperature (oC) and amplitude
level (%) on the yield of TPC (%) when water:meal ratio 30, sonication time minutes 40
and pH 6 was set at medium level. The results indicated that increase of extraction
temperature higher than mean level at low water/meal ratio led to significant yield of total
phenolic content. The high water/meal ratio in combination with high extraction
temperature showed reduction effect on TPC yield.

Fig 4.3 and 4.4 shows the response surface for the mutual interaction effect of extraction
temperature (oC) and water:meal ratio on the yield of TPC (%) when amplitude level 60,
sonication time minutes 40 and pH 6 was set at medium level. This figure indicated that at
lowest temperature as 40oC and water:meal ratio 20 leads to lowest quantity of TPC yield.
At the middle values as 50oC and water:meal ratio 30 result was different. But with the
highest values as 60oC and water:meal ratio 40, the high TPC value was obtained. Figure
4.5 and 4.6 indicates the interaction observed between extraction temperature (oC) and
sonication time (minutes) on the yield of total polyphenols extract (%). At lower value of
extraction temperature (oC) and sonication time (minutes), the low yield of the TPC was
obtained. Optimum value was near the medium level which gave better results of TPC
yield. Figure 4.7 and 4.8 showed interactions observed between extraction temperature (oC)
and pH. At higher value of extraction temperature (oC) and higher value of pH ratio, the
higher yield of the TPC was noted. Similar trend was also recorded in Fig. 4.9 to 4.20. The
results showed the temperature was major effective factor, whereas other had slight less
effects. It has also been cited that increase in TPC yield is due to the strong effect of
extraction time–temperature on the mass transfer rate of the water soluble polyphenols in
the cell wall. However, when extraction temperature and time were kept constant, increase
in water:meal ratio was the reason for an exponential increase in the yield. This is due to
the availability of more liquid which increases the driving force of polyphenols out of the
meal. The researchers showed that the use of the higher temperature is unpractical, because
it effects on decreasing of TPC yield of the extract. It was also unpractical to use the meal
to water ratio more than optimum, because under these conditions the yield of solids
increases not much, but dilution of the system occurs, that reduces its functional properties
during further application in food technology.

35
Figure 4.1: Response surface for the mutual interaction effect of extraction temperature
(oC) and amplitude level (%) on the yield of total polyphenols extract (%) in cabbage leaves
at: water/meal ratio 30, sonication time 40 minutes and pH 6.

Figure 4.2: Response surface for the mutual interaction effect of extraction temperature
(oC) and amplitude level (%) on the yield of total polyphenols extract (%) in banana peel
at: water/meal ratio 30, sonication time 40 minutes and pH 6.

36
Figure 4.3: Response surface for the mutual interaction effect of extraction temperature
(oC) and water:meal ratio on the yield of total polyphenols extract (%) in cabbage leaves
at: amplitude level 60%, sonication time 40 minutes and pH 6.

Figure 4.4: Response surface for the mutual interaction effect of extraction temperature
(oC) and water:meal ratio on the yield of total polyphenols extract (%) in banana peel at:
amplitude level 60%, sonication time 40 minutes and pH 6.

37
Figure 4.5: Response surface for the mutual interaction effect of extraction temperature
(oC) and sonication time (minutes) on the yield of total polyphenols extract (%) in cabbage
leaves at: amplitude level 60%, water/meal ratio 30 and pH 6.

Figure 4.6: Response surface for the mutual interaction effect of extraction temperature
(oC) and sonication time (minutes) on the yield of total polyphenols extract (%) in banana
peel at: amplitude level 60%, water/meal ratio 30 and pH 6.

38
Figure 4.7: Response surface for the mutual interaction effect of extraction temperature
(oC) and pH on the yield of total polyphenols extract (%) in cabbage leaves at: amplitude
level 60%, water/meal ratio 30 and sonication time 40 minutes.

Figure 4.8: Response surface for the mutual interaction effect of extraction temperature
(oC) and pH on the yield of total polyphenols extract (%) in banana peel at: amplitude level
60%, water/meal ratio 30 and sonication time 40 minutes.

39
Figure 4.9: Response surface for the mutual interaction effect of amplitude level (%) and
water/meal ratio on the yield of total polyphenols extract (%) in cabbage leaves at:
extraction temperature 50oC, sonication time 40 minutes and pH 6.

Figure 4.10: Response surface for the mutual interaction effect of amplitude level (%) and
water/meal ratio on the yield of total polyphenols extract (%) in banana peel at: extraction
temperature 50oC, sonication time 40 minutes and pH 6.

40
Figure 4.11: Response surface for the mutual interaction effect of amplitude level (%) and
sonication time (minutes) on the yield of total polyphenols extract (%) in cabbage leaves
at: extraction temperature 50oC, water/meal ratio 30 and pH 6.

Figure 4.12: Response surface for the mutual interaction effect of amplitude level (%) and
sonication time (minutes) on the yield of total polyphenols extract (%) in banana peel at:
extraction temperature 50oC, water/meal ratio 30 and pH 6.

41
Figure 4.13: Response surface for the mutual interaction effect of amplitude level (%) and
pH on the yield of total polyphenols extract (%) in cabbage leaves at: extraction temperature
50oC, water/meal ratio 30 and sonication time 40 minutes.

Figure 4.14: Response surface for the mutual interaction effect of amplitude level (%) and
pH on the yield of total polyphenols extract (%) in banana peel at: extraction temperature
50oC, water/meal ratio 30 and sonication time 40 minutes.

42
Figure 4.15: Response surface for the mutual interaction effect of water/meal ratio and
sonication time (minutes) on the yield of total polyphenols extract (%) in cabbage leaves
at: extraction temperature 50oC, amplitude level 60% and pH 6.

Figure 4.16: Response surface for the mutual interaction effect of water/meal ratio and
sonication time (minutes) on the yield of total polyphenols extract (%) in banana peel at:
extraction temperature 50oC, amplitude level 60% and pH 6.

43
Figure 4.17: Response surface for the mutual interaction effect of water/meal ratio and pH
on the yield of total polyphenols extract (%) in cabbage leaves at: extraction temperature
50oC, amplitude level 60% and sonication time 40 minutes.

Figure 4.18: Response surface for the mutual interaction effect of water/meal ratio and pH
on the yield of total polyphenols extract (%) in banana peel at: extraction temperature 50oC,
amplitude level 60% and sonication time 40 minutes.

44
Figure 4.19: Response surface for the mutual interaction effect of sonication time
(minutes) and pH on the yield of total polyphenols extract (%) in cabbage leaves at:
extraction temperature 50oC, amplitude level 60% and water/meal ratio 30.

Figure 4.20: Response surface for the mutual interaction effect of sonication time
(minutes) and pH on the yield of total polyphenols extract (%) in banana peel at: extraction
temperature 50oC, amplitude level 60% and water/meal ratio 30.

45
4.1.4 Chemical characterization of total polyphenols extract

The banana peels and cabbage leaves contain up to 9.5% dry matter, ash 11%, 8 percent
crude protein, ether extract 6% and 4.8 percent total phenolics which were very close to the
findings of Bakshi and Wadhwa (2013). The cyanogenic compounds (1.44-1.47±0.14) and
tannins (6.55-7.90±0.22) mostly present in the waste were found responsible for the
astringent taste (Emaga et al., 2007).

4.2 Functional meat product development and analysis

Natural fruit and vegetable phytochemical extracts was supplemented at different

concentration (0.5%, 1%, 1.5%) to fish meat for preparation of different meat products i.e.
patties, balls/pops and finger/sticks. The partial/parfrying of the products was carried out
to determine the lipid stability.

4.2.1 Peroxide values of functional meat products

Table 4.4 to 4.9 presents the mean values of peroxides in different TPC supplemented fish
products stored at temperatures 4˚C and -18˚C. Peroxide values (PV) of all the treatments
increased at the end of second interval then decreased at the end of last storage interval. PV
values of all treatments were higher and significantly different at the beginning and the end
of the storage period. The decrease of the PV at the end of the storage may occur owing to
decomposition of hydro peroxides into secondary oxidation products (Vanitha et al., 2015).
A similar trend was also observed by the Yerlikaya et al. (2005) during the refrigerated
studies of fish patties from anchovy and Ucak et al. (2011). The values of peroxide value,
free fatty acid, thiobarbutric acid and total volatile base nitrogen in fish burgers at the end
of storage increased significantly determined as 4.98 (±0.22) meqO2/kg of fat, 0.94 (±0.01)
% of oleic acid, 0.58 (± 0.02) mg MA/kg of sample and 4.78 (±0.02) mg/100 g of sample,
respectively (Vanitha et al. 2015). Several lipid oxidation indices were assessed to follow
up the development of oxidation in frozen state. Peroxide value and thiobarbituric acid
reactive substances showed primary and secondary oxidation, respectively (Losada et al.,
2007). When the peroxide value exceeded 10 meq oxygen/ kg fat of fish meat, the fish meat
is then considered unfit for human consumption or refused (Khidhir et al., 2013).

Decrease of PV could be attributed to different rates of lipid oxidation (Arvanitoyannis et

al., 2005). Lipids in cooked meat could be more easily oxidized than those in raw meat

46
(Widayaka et al., 2001). The chemical spoilage associated with fish during storage is
mainly due to fish lipid degradation (auto-oxidation). In general, fish have high degree of
unsaturated lipids than other food commodities. Fish lipids are subjected to two main
changes, lipolysis and auto-oxidation. The main reactants in these processes involves
atmospheric oxygen and fish unsaturated lipids, leading to the formation of hydroperoxides,
associated with tasteless, flavor and accompanied by brown yellow discoloration of the fish
tissue. Upon further degradation of hydroperoxides is the formation of strong rancid flavors
e.g. aldehydes and ketones, usually associated with spoilt fatty fish species (Khidhir et al.,
2013). The increase of lipid content than the lipid oxidations resulting from action of
lipolytic enzymes (Lipases and phospholipases) that fish phospholipids undergo
degradation to produce hydroperoxides, aldehydes and ketones which are responsible for
the development of oxidative rancidity (Khidhir et al., 2013).

Oxidation of highly unsaturated lipids is other factor which highly related to the production
of off-flavors and odors and also as influencing, protein denaturation and texture changes
(Orak and Kayisoglu, 2008). The most common cause of oil deterioration is rancidity and
the most common cause of rancidity in oils and fats is oxidation. Oxidation of the oil, in
oily fish, gives rise to rancid odors and flavors; these can limit the storage life of such
species more quickly than the protein changes that govern the extractable protein value. An
important stage in the oxidation is the reaction of oxygen with the unsaturated fatty acid
molecules to form hydroperoxides; the amount of these can be used as a measure of the
extent of oxidation in the early stages. The peroxide test is a measure of the formation of
hydroperoxides. An increase in the PV is most useful as an index of the earlier stages of
oxidation; as oxidation proceeds and peroxides are degraded the PV can start to fall
(Yerlikaya et al., 2005).

4.2.2 Moisture content of functional meat products

Table 4.10 to 4.15 presents the mean values of moisture in different TPC supplemented fish
products stored at temperatures 4˚C and -18˚C. Moisture content of fish products at
refrigerated temperature was found in parallel to the studies of Vanitha et al. (2005). While
the moisture content of fish products at deep storage was found in parallel to the studies of
Mahmoudzadeh et al. (2009). Moisture content increased in all treatments at the end of
research trial (Mahmoudzadeh et al., 2010) which is in close to the present findings.

47
Table 4.4: Peroxide value (meqO2 /kg) of patties treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 1.36±0.15g 2.30±0.18c 3.10±0.20a 2.80±0.19b 1.36±0.15g 1.45±0.16e 1.56±0.17d 1.40±0.16f
0.5% 1.35±0.15f 1.86±0.20a 1.63±0.19b 1.51±0.18c 1.35±0.15f 1.39±0.16e 1.42±0.17d 1.42±0.17d
e c a
1% 1.34±0.16 1.38±0.18 1.58±0.20 1.49±0.19b 1.34±0.16e 1.36±0.17d 1.38±0.18c 1.37±0.17c
1.5% 1.33±0.17d 1.36±0.18c 1.44±0.19a 1.40±0.19b 1.33±0.17d 1.34±0.17d 1.36±0.18c 1.36±0.0c
a-g
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.5: Peroxide value (meqO2 /kg) of meat balls treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
g c a
Control 1.34±0.14 2.10±0.18 2.90±0.20 2.60±0.19b 1.34±0.14g 1.43±0.16e 1.54±0.17d 1.38±0.15f
0.5% 1.33±0.15f 1.84±0.20a 1.61±0.19b 1.49±0.18c 1.33±0.15f 1.37±0.16e 1.40±0.17d 1.40±0.17d
1% 1.32±0.17d 1.36±0.18c 1.56±0.20a 1.47±0.19b 1.32±0.17d 1.34±0.18c 1.36±0.18c 1.35±0.18c
d c a
1.5% 1.31±0.12 1.34±0.13 1.42±0.15 1.38±0.14b 1.31±0.12d 1.32±0.12d 1.34±0.13c 1.34±0.13c
a-g
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.6: Peroxide value (meqO2 /kg) of sticks treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
g c a
Control 1.33±0.13 2.10±0.17 2.80±0.20 2.50±0.19b 1.33±0.13g 1.42±0.15e 1.53±0.16d 1.37±0.14f
e a b
0.5% 1.32±0.16 1.83±0.20 1.60±0.19 1.48±0.18c 1.32±0.16e 1.36±0.17d 1.39±0.17d 1.39±0.17d
1% 1.31±0.11f 1.35±0.14c 1.55±0.16a 1.46±0.15b 1.31±0.12e 1.33±0.13d 1.35±0.14c 1.34±0.14c
d c a
1.5% 1.30±0.12 1.33±0.13 1.41±0.15 1.37±0.14b 1.30±0.12d 1.31±0.12d 1.33±0.13c 1.33±0.13c
a-g
Means with different superscript within a row differs significantly (p≤0.05)

48
Table 4.7: Peroxide value (meqO2 /kg) of patties treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 1.37±0.13g 2.40±0.18c 3.20±0.20a 2.80±0.19b 1.37±0.13g 1.46±0.14f 1.57±0.17d 1.51±0.16e
0.5% 1.36±0.15f 1.87±0.20a 1.64±0.19b 1.51±0.18c 1.36±0.15f 1.40±0.16e 1.43±0.17d 1.43±0.16e
e c a
1% 1.35±0.13 1.39±0.15 1.59±0.17 1.49±0.16b 1.35±0.13e 1.37±0.14d 1.39±0.15c 1.38±0.14d
1.5% 1.34±0.11d 1.37±0.12c 1.45±0.13b 1.51±0.14a 1.34±0.11d 1.35±0.11d 1.37±0.12c 1.37±0.12c
a-g
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.8: Peroxide value (meqO2 /kg) of meat balls treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 1.35±0.14g 2.02±0.18c 3.10±0.20a 2.07±0.19b 1.35±0.14g 1.44±0.16e 1.55±0.17d 1.39±0.15f
0.5% 1.34±0.15f 1.85±0.20a 1.62±0.19b 1.50±0.18c 1.34±0.15f 1.38±0.16e 1.41±0.17d 1.41±0.17d
e c a
1% 1.33±0.12 1.37±0.14 1.57±0.15 1.48±0.14b 1.33±0.12e 1.35±0.13d 1.37±0.14c 1.36±0.14c
1.5% 1.32±0.12d 1.35±0.13c 1.43±0.15a 1.39±0.14b 1.32±0.12d 1.33±0.12d 1.35±0.13c 1.35±0.13c
a-g
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.9: Peroxide value (meqO2 /kg) of sticks treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 1.34±0.15g 2.10±0.18c 2.90±0.20a 2.60±0.19b 1.34±0.15g 1.42±0.16e 1.54±0.17d 1.38±0.15f
0.5% 1.33±0.15f 1.84±0.20a 1.61±0.19b 1.49±0.18c 1.33±0.15f 1.37±0.16e 1.40±0.17d 1.40±0.17d
e c a
1% 1.32±0.12 1.36±0.14 1.56±0.15 1.47±0.14b 1.32±0.12e 1.34±0.13d 1.36±0.14c 1.35±0.14c
1.5% 1.31±0.13d 1.340.14c 1.42±0.15a 1.38±0.14b 1.31±0.13d 1.32±0.13d 1.340.14c 1.340.14c
a-g
Means with different superscript within a row differs significantly (p≤0.05)

49
Table 4.10: Moisture content (%) of fish patties treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
b c d
Control 66.55±0.19 65.60±0.18 65.17±0.17 64.24±0.16e 66.55±0.19b 66.54±0.19b 66.67±0.20a 66.68±0.20a
0.5% 66.18±0.19b 65.22±0.18c 65.10±0.17d 64.16±0.16e 66.18±0.19b 66.17±0.19b 66.28±0.20a 66.29±0.20a
1% 66.11±±0.19b 65.15±0.18c 65.08±0.17d 64.11±0.16e 66.11±0.19b 66.10±0.19b 66.20±0.20a 66.21±0.20a
1.5% 66.06±0.19b 65.10±0.18c 65.02±0.17d 64.06±0.16e 66.06±0.19b 66.05±0.19b 66.15±0.20a 66.16±0.20a
a-e
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.11: Moisture content (%) of fish meat balls treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
b c d
Control 66.53±0.19 65.58±0.18 65.15±0.17 64.22±0.16e 66.53±0.19b 66.52±0.19b 66.65±0.20a 66.66±0.20a
b c d
0.5% 66.16±0.19 65.20±0.18 65.08±0.17 64.14±0.16e 66.16±0.19b 66.15±0.19b 66.26±0.20a 66.27±0.20a
1% 66.09±0.19b 65.13±0.18c 65.06±0.17d 64.09±0.16e 66.09±0.19b 66.08±0.19b 66.18±0.20a 66.19±0.20a
1.5% 66.04±0.19b 65.08±0.18c 65.00±0.17d 64.04±0.16e 66.04±0.19b 66.03±0.19b 66.13±0.20a 66.14±0.20a
a-e
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.12: Moisture content (%) of fish sticks treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
b c d
Control 66.54±0.19 65.59±0.18 65.16±0.17 64.23±0.16e 66.54±0.19b 66.51±0.19b 66.66±0.20a 66.67±0.20a
b c d
0.5% 66.17±0.19 65.21±0.18 65.09±0.17 64.15±0.16e 66.17±0.19b 66.16±0.19b 66.27±0.20a 66.28±0.20a
1% 66.10±0.19b 65.14±0.18c 65.07±0.17d 64.10±0.16e 66.10±0.19b 66.09±0.19b 66.19±0.20a 66.20±0.20a
1.5% 66.05±0.19b 65.09±0.18c 65.01±0.17d 64.05±0.16e 66.05±0.19b 66.04±0.19b 66.14±0.20a 66.15±0.20a
a-e
Means with different superscript within a row differs significantly (p≤0.05)

50
Table 4.13: Moisture content (%) of fish patties treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 66.56±0.19b 65.60±0.18c 65.18±0.17d 64.25±0.16e 66.56±0.19b 66.55±0.19b 66.68±0.20a 66.69±0.20a
0.5% 66.19±0.19b 65.22±0.18c 66.01±0.17d 64.17±0.16e 66.19±0.19b 66.18±0.19b 66.29±0.20a 66.30±0.20a
1% 66.11±0.19b 65.15±0.18c 65.09±0.17d 64.12±0.16e 66.12±0.19b 67.01±0.19b 66.21±0.20a 66.22±0.20a
1.5% 66.06±0.19b 65.02±0.18c 65.03±0.17d 64.07±0.16e 66.07±0.19b 66.06±0.19b 66.16±0.20a 66.17±0.20a
a-e
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.14: Moisture content (%) of fish meat balls treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 66.54±0.19b 65.59±0.18c 65.16±0.17d 64.23±0.16e 66.54±0.19b 66.53±0.19b 66.66±0.20a 66.67±0.20a
0.5% 66.17±0.19b 65.21±0.18c 65.09±0.17d 64.15±0.16e 66.17±0.19b 66.16±0.19b 66.27±0.20a 66.28±0.20a
1% 66.10±0.19b 65.14±0.18c 65.07±0.17d 64.10±0.16e 66.10±0.19b 66.09±0.19b 66.19±0.20a 66.20±0.20a
1.5% 66.05±0.19b 65.09±0.18c 65.01±0.17d 64.05±0.16e 66.05±0.19b 66.04±0.19b 66.14±0.20a 66.15±0.20a
a-e
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.15: Moisture content (%) of fish sticks treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
b c d
Control 66.55±0.19 65.60±0.18 65.17±0.17 64.24±0.16e 66.55±0.19b 66.52±0.19b 66.67±0.20a 66.68±0.20a
0.5% 66.18±0.19b 65.22±0.18c 65.10±0.17d 64.16±0.16e 66.18±0.19b 66.17±0.19b 66.28±0.20a 66.29±0.20a
1% 66.11±0.19b 65.15±0.18c 65.08±0.17d 64.11±0.16e 66.11±0.19b 66.10±0.19b 66.20±0.20a 66.21±0.20a
1.5% 66.06±0.19b 65.10±0.18c 65.02±0.17d 64.06±0.16e 66.06±0.19b 66.05±0.19b 66.15±0.20a 66.16±0.20a
a-e
Means with different superscript within a row differs significantly (p≤0.05)

51
4.2.3 Free fatty acid values of functional meat products

Table 4.16 to 4.21 presents the mean values of FFA in different TPC supplemented fish
products stored at temperatures 4˚C and -18˚C. Free fatty acid of fish products at different
storage days was found in parallel to the studies of Vanitha et al. (2005). A similar trend
was also observed by the Yerlikaya et al. (2005) during the refrigerated studies of fish
patties from anchovy and Ucak et al. (2011). Genuine FFA contents of lipids from both
fillets and mince increased in a similar fashion during storage at -18˚C, while concomitantly
their P levels decreased (de Koning and Mol, 1991). When fish based products are frozen
and cold stored, unfavorable changes take place in the texture and appearance.
Simultaneously, free fatty acids are formed from the lipids. These changes have been
attributed to enzymic reactions which take place at a rate governed by the temperature of
frozen storage. The FFA generated during storage originated from the phospholipids, the
neutral lipids or both. This is in contrast to the mince lipids where the contributions from
both phospholipids and neutral lipids to FFA formation could be calculated (de Koning and
Mol, 1991).

Increase in FFA results from the enzymatic hydrolysis of esterified lipids (Hwang and
Regenstein, 1993). Free fatty acids (FFA) content has been used to establish the grade of
deterioration. Lipid (glycerol-fatty acids esters) present in the fish muscle undergoes
hydrolysis, resulting in the release of fatty acids. Due to lipid hydrolysis, FFA accumulates
in the tissue during frozen storage, especially at high temperatures around -10 to -20°C.
Slow freezing rates or fluctuating storage temperatures may result in the lysis of lysosomes
and thereby increased activity of some endogenous lipases resulting in increased rates of
FFA accumulation (Khidhir et al., 2013).

52
Table 4.16: Free fatty acids (% of Oleic acid) of fish patties treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 0.22±0.15f 0.32±0.17d 0.55±0.19b 0.64±0.20a 0.22±0.15f 0.28±0.16e 0.33±0.17d 0.36±0.18c
0.5% 0.21±0.15f 0.31±0.17d 0.51±0.19b 0.58±0.20a 0.21±0.15f 0.27±0.16e 0.29±0.17d 0.32±0.18c
f d b
1% 0.21±0.15 0.30±0.17 0.48±0.19 0.52±0.20a 0.21±0.15f 0.27±0.16e 0.28±0.17d 0.30±0.18c
1.5% 0.20±0.15f 0.29±0.17d 0.42±0.19b 0.50±0.20a 0.20±0.15f 0.25±0.16e 0.27±0.17d 0.29±0.18c
a-f
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.17: Free fatty acids (% of Oleic acid) of fish meat balls treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 0.20±0.15f 0.30±0.17d 0.53±0.19b 0.62±0.20a 0.20±0.15f 0.26±0.16e 0.32±0.17d 0.34±0.18c
0.5% 0.19±0.15f 0.29±0.17d 0.49±0.19b 0.56±0.20a 0.19±0.15f 0.25±0.16e 0.27±0.17d 0.30±0.18c
f d b
1% 0.21±0.15 0.28±0.17 0.46±0.19 0.50±0.20a 0.19±0.15f 0.24±0.16e 0.26±0.17d 0.28±0.18c
1.5% 0.18±0.15f 0.26±0.17d 0.40±0.19b 0.48±0.20a 0.18±0.15f 0.23±0.16e 0.25±0.17d 0.27±0.18c
a-f
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.18: Free fatty acids (% of Oleic acid) of fish sticks treated with banana peel extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 0.21±0.15f 0.31±0.17d 0.54±0.19b 0.63±0.20a 0.21±0.15f 0.27±0.16e 0.33±0.17d 0.35±0.18c
0.5% 0.20±0.15f 0.30±0.17d 0.50±0.19b 0.57±0.20a 0.20±0.15f 0.26±0.16e 0.28±0.17d 0.31±0.18c
f d b
1% 0.22±0.15 0.29±0.17 0.47±0.19 0.51±0.20a 0.20±0.15f 0.25±0.16e 0.27±0.17d 0.29±0.18c
1.5% 0.19±0.15f 0.27±0.17d 0.41±0.19b 0.49±0.20a 0.19±0.15f 0.24±0.16e 0.26±0.17d 0.28±0.18c
a-f
Means with different superscript within a row differs significantly (p≤0.05)

53
Table 4.19: Free fatty acids (% of Oleic acid) of fish patties treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 0.23±0.15f 0.33±0.17d 0.56±0.19b 0.65±0.20a 0.23±0.15f 0.29±0.16e 0.34±0.17d 0.37±0.18c
0.5% 0.22±0.15f 0.32±0.17d 0.52±0.19b 0.59±0.20a 0.22±0.15f 0.28±0.16e 0.30±0.17d 0.33±0.18c
f d b
1% 0.22±0.15 0.31±0.17 0.49±0.19 0.53±0.20a 0.22±0.15f 0.28±0.16e 0.29±0.17d 0.31±0.18c
1.5% 0.21±0.15f 0.30±0.17d 0.43±0.19b 0.51±0.20a 0.21±0.15f 0.26±0.16e 0.28±0.17d 0.30±0.18c
a-f
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.20: Free fatty acids (% of Oleic acid) of fish meat balls treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 0.21±0.15f 0.31±0.17d 0.54±0.19b 0.63±0.20a 0.21±0.15f 0.27±0.16e 0.33±0.17d 0.35±0.18c
0.5% 0.20±0.15f 0.30±0.17d 0.50±0.19b 0.57±0.20a 0.20±0.15f 0.26±0.16e 0.28±0.17d 0.31±0.18c
f d b
1% 0.22±0.15 0.29±0.17 0.47±0.19 0.51±0.20a 0.20±0.15f 0.25±0.16e 0.27±0.17d 0.29±0.18c
1.5% 0.19±0.15f 0.27±0.17d 0.41±0.19b 0.49±0.20a 0.19±0.15f 0.24±0.16e 0.26±0.17d 0.28±0.18c
a-f
Means with different superscript within a row differs significantly (p≤0.05)

Table 4.21: Free fatty acids (% of Oleic acid) of fish sticks treated with cabbage leaves extracts
4˚C -18˚C
Treatment 0 3 6 9 0 15 30 60
Control 0.22±0.15f 0.32±0.17d 0.55±0.19b 0.64±0.20a 0.22±0.15f 0.28±0.16e 0.34±0.17d 0.36±0.18c
0.5% 0.21±0.15f 0.31±0.17d 0.51±0.19b 0.58±0.20a 0.21±0.15f 0.27±0.16e 0.29±0.17d 0.32±0.18c
f d b
1% 0.23±0.15 0.30±0.17 0.48±0.19 0.52±0.20a 0.21±0.15f 0.26±0.16e 0.28±0.17d 0.30±0.18c
1.5% 0.20±0.15f 0.28±0.17d 0.42±0.19b 0.50±0.20a 0.20±0.15f 0.25±0.16e 0.27±0.17d 0.29±0.18c
a-f
Means with different superscript within a row differs significantly (p≤0.05)

54
4.3 Sensory evaluation of functional meat products

Sensory evaluation is an important tool in a product development. Acceptance of a food

product depends upon the consumer’s perception of the color, taste, texture, flavor and
overall acceptability into overall impression of quality. Although chemical, physical and
microbiological tests are employed to check the quality of a food product, but these tests
can not provide such kind of information whether consumer will accept it or not. The
meatballs prepared, deep fried and then subjected to sensory evaluation by panelist from
parent department for different attributes viz., color, taste and overall acceptability and
scores were recorded using a nine point hedonic scale after every 15 days to assess the
liking and disliking of the panelists. Sensory scores of fish products are given in tables 4.22
to 4.57 while it shows a significant difference in sensory score at refrigeration temperatures
where sensory scores of fish patties decreased significantly (p<0.05) throughout at
refrigeration storage. Whereas the sensory scored at the -18ºc shows the good sensory
characteristics relatively. Acceptable at end of 2nd month with treatment 0.5% results were
in parallel to findings of Mahmoudzadeh et al. (2009). When fish based products are frozen
and cold stored, unfavorable changes take place in the texture and appearance. Both FFA
and peroxides have been implicated as the cause of decrease in sensory scores (de Koning
and Mol, 1991). The appearance, texture, odor and flavor of the fish burger started
decreasing at a significant level of 1% level (Vanitha et al., 2015). Yerlikaya et al. (2005)
reported the limit of acceptability for anchovy patties in refrigerated storage for 6 days.
Turhan et al. (2001) determined the shelf life of refrigerated raw anchovy (E. encrasicholus)
patties for 6 days at 4 °C. The above mentioned studies showed the very similar results with
the present study. According to Orak and Kayisoglu (2008) the decrease in the values of
sensory analyses was faster than chemical changes during frozen storage. Appearance of
off flavor may be attributed to WOF (Warm Over Flavor). WOF is usually associated with
reheated meats and includes odors and flavors commonly described as stale or rancid also
interaction of ketones, aldehydes, alcohols, hydrocarbons, acids, and epoxides with
proteins may be produced off-colors (Thanonkaew et al., 2006). Sensory scores decreased
during the storage of anchovy patties at the storage temperature of 4°C and Anchovy patties
were consumable up to 6 days (Yerlikaya et al. 2005). Overall, protein and lipid oxidation
can account for the toughened texture, off-flavor and unappealing odor of frozen stored sea
foods (Thanonkaew et al., 2006).

55
Table 4.22: Color evaluation of meat balls treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
a b c d
Control 8.5±0.9 6.5±0.8 5.3±0.7 4.2±0.6 3.9±0.5e
0.5% 8.5±0.9a 6.9±0.8b 5.9±0.7c 4.9±0.6d 4.1±0.5e
1% 8.1±0.9a 6.8±0.8b 5.8±0.7c 4.8±0.6d 4.0±0.5e
a b c d
1.5% 6.5±0.9 5.9±0.8 5.3±0.7 4.1±0.6 3.7±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.23: Flavor evaluation of meat balls treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
a b c d
Control 8.5±0.9 6.4±0.8 5.2±0.7 4.1±0.6 3.8±0.5e
a b c d
0.5% 8.5±0.9 6.8±0.8 5.8±0.7 4.8±0.6 4.1±0.5e
1% 8.1±0.9a 6.7±0.8b 5.7±0.7c 4.7±0.6d 4.0±0.5e
1.5% 6.1±0.9a 5.8±0.8b 5.2±0.7c 4.0±0.6d 3.6±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.24: Overall acceptability of meat balls treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
Control 8.5±0.9a 6.4±0.8b 5.2±0.7c 4.1±0.6d 3.8±0.5e
0.5% 8.5±0.9a 6.8±0.8b 5.8±0.7c 4.8±0.6d 4.1±0.5e
a b c
1% 8.1±0.9 6.7±0.8 5.7±0.7 4.7±0.6d 4.0±0.5e
1.5% 6.1±0.9a 5.8±0.8b 5.2±0.7c 4.0±0.6d 3.6±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.25: Color evaluation of meat balls treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
a b c
Control 8.5±0.5 8.1±0.4 7.3±0.3 6.9±0.2d 6.4±0.1e
a b c
0.5% 8.5±0.5 8.3±0.4 7.9±0.3 7.5±0.2d 7.1±0.1e
1% 8.1±0.5a 7.9±0.4b 7.5±0.3c 7.1±0.2d 6.9±0.1e
a b c
1.5% 6.3±0.5 6.1±0.4 5.4±0.3 5.1±0.2d 4.4±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.26: Flavor evaluation of meat balls treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
Control 8.5±0.5a 8.2±0.4b 7.2±0.3c 6.7±0.2d 6.4±0.1e
0.5% 8.5±0.5a 8.4±0.4b 7.8±0.3c 7.5±0.2d 7.2±0.1e
1% 8.1±0.5a 7.8±0.4b 7.6±0.3c 7.3±0.2d 6.8±0.1e
1.5% 6.1±0.5a 6.1±0.4b 5.5±0.3c 5.1±0.2d 4.3±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.27: Overall acceptability of meat balls treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
Control 8.5±0.5a 8.1±0.4b 7.2±0.3c 6.6±0.2d 6.4±0.1e
0.5% 8.5±0.5a 8.4±0.4b 7.8±0.3c 7.6±0.2d 7.2±0.1e
a b c d
1% 8.1±0.5 7.9±0.4 7.6±0.3 7.2±0.2 6.8±0.1e
1.5% 6.2±0.5a 6.1±0.4b 5.4±0.3c 4.9±0.2d 4.3±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)

56
Table 4.28: Color evaluation of patties treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
Control 7.8±0.9a 6.8±0.8b 5.2±0.7c 4.8±0.6d 4.5±0.5e
0.5% 7.8±0.9a 7.2±0.8b 5.5±0.7c 5.3±0.6d 4.9±0.5e
a b c
1% 7.7±0.9 7.0±0.8 5.4±0.7 5.2±0.6d 4.7±0.5e
1.5% 7.5±0.9a 6.9±0.8b 5.2±0.7c 5.0±0.6d 3.9±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.29: Flavor evaluation of patties treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
Control 7.8±0.9a 6.7±0.8b 5.0±0.7c 4.6±0.6d 3.9±0.5e
0.5% 7.8±0.9a 7.1±0.8b 5.4±0.7c 5.1±0.6d 4.6±0.5e
a b c d
1% 7.7±0.9 6.9±0.8 5.3±0.7 5.0±0.6 4.6±0.5e
1.5% 7.1±0.9a 6.8±0.8b 5.1±0.7c 4.9±0.6d 3.7±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.30: Overall acceptability of patties treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
a b c
Control 7.8±0.9 6.8±0.8 5.5±0.7 4.6±0.6d 3.9±0.5e
a b c
0.5% 7.8±0.9 7.0±0.8 5.6±0.7 4.9±0.6d 4.3±0.5e
1% 7.7±0.9a 6.8±0.8b 5.4±0.7c 4.8±0.6d 4.1±0.5e
a b c
1.5% 7.4±0.9 5.8±0.8 5.0±0.7 4.6±0.6d 3.9±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.31: Color evaluation of patties treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
Control 8.4±0.5a 7.2±0.4b 7.1±0.3c 6.8±0.2d 6.4±0.1e
0.5% 8.4±0.5a 7.4±0.4b 7.2±0.3c 7.1±0.2d 6.8±0.1e
a b c d
1% 8.2±0.5 7.3±0.4 7.1±0.3 6.8±0.2 6.7±0.1e
1.5% 7.8±0.5a 7.1±0.4b 6.8±0.3c 6.1±0.2d 5.4±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.32: Flavor evaluation of patties treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
Control 8.4±0.5a 7.4±0.4b 7.2±0.3c 6.9±0.2d 6.6±0.1e
0.5% 8.4±0.5a 7.6±0.4b 7.4±0.3c 7.2±0.2d 6.9±0.1e
1% 8.2±0.5a 7.5±0.4b 7.4±0.3c 6.9±0.2d 6.9±0.1e
a b c
1.5% 7.8±0.5 7.2±0.4 6.9±0.3 6.2±0.2d 5.6±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.33: Overall acceptability of patties treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
a b c
Control 8.4±0.5 7.3±0.4 7.1±0.3 6.9±0.2d 6.5±0.1e
a b c
0.5% 8.4±0.5 7.5±0.4 7.3±0.3 7.1±0.2d 6.9±0.1e
1% 8.2±0.5a 7.4±0.4b 7.2±0.3c 6.9±0.2d 6.8±0.1e
a b c
1.5% 7.8±0.5 7.1±0.4 6.9±0.3 6.1±0.2d 5.5±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)

57
Table 4.34: Color evaluation of sticks treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
Control 8.4±0.9a 6.4±0.8b 5.2±0.7c 4.1±0.6d 3.8±0.5e
0.5% 8.4±0.9a 6.8±0.8b 5.8±0.7c 4.8±0.6d 4.0±0.5e
a b c d
1% 8.1±0.9 6.7±0.8 5.7±0.7 4.7±0.6 3.9±0.5e
1.5% 6.4±0.9a 5.8±0.8b 5.2±0.7c 4.0±0.6d 3.6±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.35: Flavor evaluation of sticks treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
Control 8.4±0.9a 6.3±0.8b 5.1±0.7c 4.0±0.6d 3.7±0.5e
0.5% 8.4±0.9a 6.7±0.8b 5.7±0.7c 4.7±0.6d 4.0±0.5e
a b c
1% 8.1±0.9 6.6±0.8 5.8±0.7 4.6±0.6d 3.9±0.5e
1.5% 6.4±0.9a 5.7±0.8b 5.1±0.7c 3.9±0.6d 3.5±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.36: Overall acceptability of sticks treated with banana peel extract (at 4˚C)
Treatment 0 15 30 45 60
a b c d
Control 8.4±0.9 6.3±0.8 5.1±0.7 4.0±0.6 3.7±0.5e
a b c d
0.5% 8.4±0.9 6.7±0.8 5.7±0.7 4.7±0.6 4.0±0.5e
1% 8.1±0.9a 6.6±0.8b 5.6±0.7c 4.6±0.6d 3.9±0.5e
a b c d
1.5% 6.4±0.9 5.7±0.8 5.1±0.7 3.9±0.6 3.5±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.37: Color evaluation of sticks treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
Control 8.5±0.5a 8.1±0.4b 7.3±0.3c 6.9±0.2d 6.4±0.1e
0.5% 8.5±0.5a 8.3±0.4b 7.9±0.3c 7.5±0.2d 7.1±0.1e
a b c
1% 8.1±0.5 7.9±0.4 7.5±0.3 7.1±0.2d 6.9±0.1e
1.5% 6.3±0.5a 6.1±0.4b 5.4±0.3c 5.1±0.2d 4.4±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.38: Flavor evaluation of sticks treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
Control 8.4±0.5a 8.1±0.4b 7.1±0.3c 6.6±0.2d 6.3±0.1e
0.5% 8.4±0.5a 8.3±0.4b 7.7±0.3c 7.4±0.2d 7.1±0.1e
a b c
1% 8.1±0.5 7.7±0.4 7.5±0.3 7.2±0.2d 6.7±0.1e
1.5% 6.4±0.5a 6.0±0.4b 5.4±0.3c 5.0±0.2d 4.2±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.39: Overall acceptability of sticks treated with banana peel extract (at -18˚C)
Treatment 0 15 30 45 60
a b c
Control 8.4±0.5 8.0±0.4 7.1±0.3 6.5±0.2d 6.3±0.1e
a b c
0.5% 8.4±0.5 8.3±0.4 7.7±0.3 7.5±0.2d 7.1±0.1e
1% 8.1±0.5a 7.8±0.4b 7.5±0.3c 7.1±0.2d 6.7±0.1e
a b c
1.5% 6.4±0.5 6.0±0.4 5.3±0.3 4.8±0.2d 4.2±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)

58
Table 4.40: Color evaluation of meat balls treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
Control 7.4±0.9a 5.4±0.8b 4.2±0.7c 3.1±0.6d 3.3±0.5e
0.5% 7.4±0.9a 5.8±0.8b 4.8±0.7c 3.8±0.6d 3.9±0.5e
a b c d
1% 7.1±0.9 5.7±0.8 4.7±0.7 3.8±0.6 3.7±0.5e
1.5% 5.4±0.9a 4.8±0.8b 4.2±0.7c 3.6±0.6d 3.1±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.41: Flavor evaluation of meat balls treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
Control 7.4±0.9a 5.3±0.8b 4.1±0.7c 3.8±0.6d 3.5±0.5e
0.5% 7.4±0.9a 5.7±0.8b 4.7±0.7c 4.0±0.6d 3.7±0.5e
a b c d
1% 7.1±0.9 5.6±0.8 4.6±0.7 3.9±0.6 3.6±0.5e
1.5% 5.0±0.9a 4.7±0.8b 4.1±0.7c 3.7±0.6d 3.1±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.42: Overall acceptability of meat balls treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
a b c
Control 7.4±0.9 5.3±0.8 4.1±0.7 4.0±0.6d 3.7±0.5e
a b c
0.5% 7.4±0.9 5.7±0.8 4.7±0.7 4.7±0.6d 3.9±0.5e
1% 7.1±0.9a 5.6±0.8b 4.6±0.7c 4.6±0.6d 3.8±0.5e
a b c
1.5% 5.0±0.9 4.7±0.8 4.1±0.7 3.9±0.6d 3.2±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.43: Color evaluation of meat balls treated with cabbage leaves extract (at -18˚C)
Treatment 0 15 30 45 60
Control 7.4±0.5a 7.0±0.4b 6.2±0.3c 5.8±0.2d 5.3±0.1e
0.5% 7.4±0.5a 7.2±0.4b 6.8±0.3c 6.4±0.2d 6.0±0.1e
a b c
1% 7.1±0.5 6.8±0.4 6.4±0.3 6.0±0.2d 5.8±0.1e
1.5% 5.2±0.5a 5.0±0.4b 4.3±0.3c 4.0±0.2d 3.3±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.44: Flavor evaluation of meat balls treated with cabbage leaves extract (at -18˚C)
Treatment 0 15 30 45 60
Control 7.4±0.5a 7.1±0.4b 6.1±0.3c 5.6±0.2d 5.3±0.1e
0.5% 7.4±0.5a 7.3±0.4b 6.7±0.3c 6.4±0.2d 6.1±0.1e
a b c
1% 7.1±0.5 6.7±0.4 6.5±0.3 6.2±0.2d 5.7±0.1e
1.5% 5.1±0.5a 5.0±0.4b 4.4±0.3c 4.1±0.2d 3.2±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.45: Overall acceptability of meat balls treated with cabbage leaves extract (at -
18˚C)
Treatment 0 15 30 45 60
Control 7.4±0.5a 7.0±0.4b 6.1±0.3c 5.5±0.2d 5.3±0.1e
0.5% 7.4±0.5a 7.3±0.4b 6.7±0.3c 6.5±0.2d 6.1±0.1e
1% 7.1±0.5a 6.8±0.4b 6.5±0.3c 6.1±0.2d 5.7±0.1e
1.5% 5.1±0.5a 5.0±0.4b 4.3±0.3c 3.8±0.2d 3.2±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)

59
Table 4.46: Color evaluation of patties treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
Control 6.4±0.9a 5.7±0.8b 4.1±0.7c 3.7±0.6d 3.4±0.5e
0.5% 6.7±0.9a 6.1±0.8b 4.4±0.7c 4.2±0.6d 3.8±0.5e
a b c d
1% 6.6±0.9 6.9±0.8 4.3±0.7 4.1±0.6 3.6±0.5e
1.5% 6.4±0.9a 5.8±0.8b 4.1±0.7c 4.0±0.6d 3.0±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.47: Flavor evaluation of patties treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
Control 6.7±0.9a 5.7±0.8b 4.0±0.7c 3.5±0.6d 3.1±0.5e
0.5% 6.7±0.9a 6.0±0.8b 4.3±0.7c 3.0±0.6d 3.6±0.5e
a b c d
1% 6.6±0.9 5.8±0.8 4.2±0.7 4.0±0.6 3.5±0.5e
1.5% 6.0±0.9a 5.7±0.8b 4.0±0.7c 3.8±0.6d 3.0±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.48: Overall acceptability of patties treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
a b c
Control 6.7±0.9 5.7±0.8 4.4±0.7 3.5±0.6d 3.1±0.5e
a b c
0.5% 6.7±0.9 6.9±0.8 4.5±0.7 3.8±0.6d 3.3±0.5e
1% 6.6±0.9a 5.7±0.8b 4.3±0.7c 3.7±0.6d 3.1±0.5e
a b c
1.5% 6.3±0.9 4.7±0.8 4.4±0.7 3.5±0.6d 3.0±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.49: Color evaluation of patties treated with cabbage leaves extract (at -18˚C)
Treatment 0 15 30 45 60
Control 7.3±0.5a 6.1±0.4b 5.9±0.3c 5.7±0.2d 5.3±0.1e
0.5% 7.3±0.5a 6.3±0.4b 6.1±0.3c 6.0±0.2d 5.7±0.1e
a b c d
1% 7.1±0.5 6.2±0.4 6.0±0.3 5.7±0.2 5.6±0.1e
1.5% 6.7±0.5a 6.0±0.4b 5.7±0.3c 5.0±0.2d 4.3±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.50: Flavor evaluation of patties treated with cabbage leaves extract (at -18˚C)
Treatment 0 15 30 45 60
Control 7.3±0.5a 6.3±0.4b 6.1±0.3c 5.8±0.2d 5.5±0.1e
0.5% 7.3±0.5a 6.5±0.4b 6.3±0.3c 6.1±0.2d 5.8±0.1e
a b c d
1% 7.1±0.5 6.4±0.4 6.3±0.3 5.8±0.2 5.8±0.1e
1.5% 6.7±0.5a 6.1±0.4b 5.8±0.3c 5.1±0.2d 4.5±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.51: Overall acceptability of patties treated with cabbage leaves extract (at -18˚C)
Treatment 0 15 30 45 60
a b c d
Control 7.3±0.5 6.2±0.4 6.0±0.3 5.8±0.2 5.4±0.1e
a b c d
0.5% 7.3±0.5 6.4±0.4 6.2±0.3 6.0±0.2 5.8±0.1e
1% 7.1±0.5a 6.3±0.4b 6.1±0.3c 5.8±0.2d 5.7±0.1e
a b c d
1.5% 6.7±0.5 6.0±0.4 5.8±0.3 5.0±0.2 4.4±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)

60
Table 4.52: Color evaluation of sticks treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
Control 7.3±0.9a 5.3±0.8b 4.1±0.7c 3.5±0.6d 2.8±0.5e
0.5% 7.3±0.9a 5.7±0.8b 4.7±0.7c 3.7±0.6d 3.1±0.5e
a b c d
1% 7.1±0.9 5.6±0.8 4.6±0.7 3.6±0.6 3.0±0.5e
1.5% 5.3±0.9a 4.7±0.8b 4.1±0.7c 3.4±0.6d 2.9±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.53: Flavor evaluation of sticks treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
a b c d
Control 7.3±0.9 5.2±0.8 4.9±0.7 3.5±0.6 3.0±0.5e
0.5% 7.3±0.9a 5.6±0.8b 4.6±0.7c 3.7±0.6d 3.2±0.5e
1% 7.1±0.9a 5.5±0.8b 4.5±0.7c 3.6±0.6d 3.1±0.5e
a b c d
1.5% 6.4±0.9 5.7±0.8 5.1±0.7 3.4±0.6 2.9±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.54: Overall acceptability of sticks treated with cabbage leaves extract (at 4˚C)
Treatment 0 15 30 45 60
a b c d
Control 7.3±0.9 5.2±0.8 4.0±0.7 3.5±0.6 3.0±0.5e
a b c d
0.5% 7.3±0.9 5.6±0.8 4.6±0.7 3.6±0.6 3.1±0.5e
1% 7.1±0.9a 5.5±0.8b 4.5±0.7c 3.5±0.6d 3.0±0.5e
1.5% 5.3±0.9a 4.6±0.8b 4.0±0.7c 3.4±0.6d 2.9±0.5e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.55: Color evaluation of sticks treated with cabbage leaves extract (at -18˚C)
Treatment 0 15 30 45 60
Control 7.4±0.5a 7.0±0.4b 6.2±0.3c 5.8±0.2d 5.3±0.1e
0.5% 7.4±0.5a 7.2±0.4b 6.8±0.3c 6.4±0.2d 6.0±0.1e
a b c
1% 7.1±0.5 6.8±0.4 6.4±0.3 6.0±0.2d 5.8±0.1e
1.5% 5.2±0.5a 5.0±0.4b 4.3±0.3c 4.0±0.2d 3.3±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.56: Flavor evaluation of sticks treated with cabbage leaves extract (at -18˚C)
Treatment 0 15 30 45 60
a b c
Control 7.3±0.5 7.0±0.4 6.0±0.3 5.5±0.2d 6.2±0.1e
0.5% 7.3±0.5a 7.2±0.4b 6.6±0.3c 6.3±0.2d 6.0±0.1e
1% 7.1±0.5a 6.6±0.4b 6.4±0.3c 6.1±0.2d 5.6±0.1e
a b c
1.5% 5.4±0.5 5.9±0.4 5.3±0.3 4.9±0.2d 3.1±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)
Table 4.57: Overall acceptability of sticks treated with cabbage leaves extract (at -18˚C)
Treatment 0 15 30 45 60
a b c
Control 7.3±0.5 7.0±0.4 6.0±0.3 5.4±0.2d 5.2±0.1e
a b c
0.5% 7.3±0.5 7.2±0.4 6.6±0.3 6.4±0.2d 6.0±0.1e
1% 7.1±0.5a 6.7±0.4b 6.4±0.3c 6.0±0.2d 5.6±0.1e
1.5% 5.3±0.5a 5.0±0.4b 4.3±0.3c 3.7±0.2d 3.2±0.1e
a-e
Means with different superscript within a row differs significantly (p≤0.05)

61
Chapter 5

SUMMARY

Diet is an important factor affecting human health and well-being. Meat is an important
component of a healthy and well balanced diet due to its nutritional richness. However,
meat is devoid of fiber, antioxidants and phytochemicals, and other helpful nutrients that
have shown to be protective against different diseases. Lipid and color oxidation are major
causes of quality deterioration in meat products during storage, costing the industry over
$700 million annually. These reactions lead to off-flavor, discoloration, and loss of nutritive
value, ultimately decreasing consumer confidence in the product. Intake of such food
products with oxidized lipid constituents can modify DNA, proteins, membrane structure
and tumor initiation in biological system. In the fruit and vegetable industry, the preparation
and processing procedures can lead to one third of the product being discarded. Due to
increasing production of plant food processing, by-products disposal represents a growing
problem since the plant material is usually prone to microbial spoilage, thus limiting further
exploitation. On the other hand, fruits and vegetables by-products are also promising
sources of organic micronutrients such as carotenoids, polyphenolics, tocopherols, vitamin
C, and other bioactive compounds which can be used as dietary supplements and food
fortification purposes.

A heightened awareness of consumers nowadays on the relationship between diet and

health/disease prevention has fostered growth in the development of ‘functional’ food
products. Epidemiological studies have pointed out that consumption of functional foods
imparts health benefits, e.g. reduced risk of coronary heart disease and stroke, as well as
certain types of cancer. However, available locally and internationally scientific literature
does not provide information that consumption of functional meat products supplemented
with fruits and vegetables phytochemicals are beneficial for health or not. Furthermore,
investigations on stability and interactions of phytochemicals with meat constituents during
processing and storage need to be initiated. Therefore, the supplementation study of fruits
and vegetables phytochemicals in meat products may provide information regarding
phytochemicals action to prevent the formation of potential carcinogens in meat products.
In particular, the focus should be on the amounts of fruits and vegetables phytochemicals
that will not cause any major problems for acceptability of functional meat products.

62
Ultrasound is one of the emerging technologies that was developed to minimize processing,
maximize quality and ensure the safety of food products. Ultrasound is applied to impart
positive effects in food processing such as improvement in mass transfer, food preservation,
assistance of thermal treatments and manipulation of texture and food analysis. A major
application of ultrasound is for facilitating the extraction process of a variety of food
components (e.g., herbal, oil, protein, polysaccharides) as well as bioactive ingredients (e.g.
antioxidants) from plant and animal resources. The major advantages of ultrasound are
minimum effect on extractable materials, avoidance of organic solvents as its action also
works in GRAS solvents, reduction in extraction time, which can potentially enhance the
extraction of heat sensitive bioactive and food components at lower processing
temperatures and potentially in large industrial scales.

The extracted phytochemicals can be used in meat to further enhancing the meat
acceptability and improving the functional stability during storage. Therefore, it can be the
most viable approach to extract the phytochemicals from fruit and vegetable waste using
the innovative technology. The present study was undertaken to achieve the following
objectives:

 Extraction of bioactive compounds from fruit and vegetable waste/by-products by

using ultrasound technology
 Develop a range of functional/healthier un-cooked and fried fish meat products
using extracted bioactive compounds
 Characterization of functional fish meat products for oxidative stability and
consumer acceptability at different storage intervals

Pre dried the banana peel and cabbage leaves powder and weight (100±0.1g) using the
electronic weighing balance (Model Kern 440-35N) for each treatment. A 3-level five
factor Box-Behnken Design was used to study the effect of extraction/sonication
temperature (˚C), amplitude level, water/meal ratio, extraction/sonication time (minutes)
and pH conditions for maximum yield of total polyphenols from dried fruits and vegetable
samples. The pH of the solution was monitored continuously and was adjusted by 0.2mol/L
NaOH and HCl, respectively while the temperature of the aqueous system was controlled
within ±1.5˚C. Natural fruit and vegetable phytochemical extracts was supplemented at
different concentration to fish meat for preparation of different meat products i.e. patties,
balls/pops and finger/sticks. The partial/parfrying of the products was carried out to

63
determine the lipid stability. Fried meat products were vacuum sealed in plastic bags and
then were stored at refrigerator and -18 °C in a freezer for a storage period of 60-days.
Experienced and untrained assessors carried out the sensory analysis of meat product
samples. The behavior of the Box-Behnken Model was explained by the following the
quadratic Equation. The data of TPC yield obtained for each treatment was subjected to
statistical analysis to determine the level of significance by using the software package.
The sample analysis for storage stability and consumer acceptability was carried out in
triplicate and the significant differences were calculated among means at a probability level
of 5%.

A 3-level five factors Box-Behnken design was used to study the effect of extraction
temperature (˚C), amplitude power level,water/meal ratio, extraction time (minutes) and
extraction pH conditions for maximum yield of total polyphenols from banana peel and
cabbage leaves.The total number of experimental runs for each banana peel and cabbage
leaves was 46 as determined by the Box-Behnken design. The percent values of TPC yield
from banana peel and cabbage leaves samples ranged from a minimum value of
15.55±0.13% to a maximum value of 24.4±0.17% for banana peel and from minimum value
of 9.8±0.12% to a maximum value of 19.8±0.15%. The results revealed that extraction
conditions significantly affect the TPC yield from banana peel and cabbage leaves. There
is very limited published data that provides an information or support to the TPC yield from
banana peel and cabbage leaves. The extraction run point 25 (extraction temperature, 60˚C;
amplitude/sonication level, 90; extraction pH, 6; water/meal ratio, 30 and extraction time,
40 minutes showed maximum TPC yield for banana peel (24.4±0.17%) and cabbage leaves
(19.8±0.15%), respectively and the lowest TPC yield 15.55±0.13% and 9.8±0.12%was for
extraction run 4 (extraction temperature, 50˚C; extraction pH, 4; water/meal ratio, 30;
amplitude level 30 and extraction time, 40 minutes) of banana peel and extraction run 5
(extraction temperature, 40˚C; extraction pH, 6; water/meal ratio, 30; amplitude level 30
and extraction time, 40 minutes) of cabbage leaves, respectively.

The optimized parameters for the extraction method of TPC were: aqueous extraction with
ratio 1:20 (water:meal) for both cabbage leaves and banana peel extraction time 40 minutes
with constant stirring at 60°C and sonication with high intensity probe ultrasound. The
optimal sonication conditions among all tested were 30% of maximum ultrasonic power
for 40 minutes. These parameters were optimized taking into account the necessity of

64
ultrasonic waves to eliminate interaction between the TPC and the other peel fractions but
minimizing the ultrasonic power and time of treatment to avoid the raising of sample
temperature caused by the ultrasound application. The temperature of the sample after 40
min of sonication ranged from 60 to 65°C, this temperature did not exceed in any case. TPC
yield obtained from banana peel (24.4±0.17%) and cabbage leaves (15.55±0.13%) was
similar to findings of earlier reported studies in literature. Although extraction yields
obtained were similar or higher to those described in the literature, former methods of
extraction were more aggressive than the proposed in the present work.

The predicted values of TPC yields were calculated using regression model and were
compared with experimental values to assess the validity of second-order polynomial
response model. The predicted values of TPC yields were located within the range of
experimental values. The computed values for degree of correlation (0.9947), coefficient
of variation (2.32%), adjusted coefficients of determination (0.9850) and adequate
precision (30.772) indicate the adequacy and reliability of the applied second-order
polynomial response model. The degree of correlation value for TPC yield was close to 1,
which indicated that the second-order polynomial response model explained about 99.47%
of the variability observed in the present study. The coefficient of variation less than 5%
indicates that the model is reproducible. The mathematical second order polynomial
response model found after fitting the function to the experimental data sometimes does
not satisfactorily describe the effect of independent variable whether significant or non-
significant. Thus, the model fitted was evaluated to know the effects of independent
variables as linear, interaction and quadratic coefficients using the analysis of variance
(ANOVA). The results indicated that TPC yield was significantly affected by the model
effects banana peel and cabbage leaves, respectively. The linear and quadratic effects
model effects were observed more significant as compared to interaction (p≤0.01). The
order of model effects observed during TPC yield from dried banana peel and cabbage
leaves was linear >quadratic >interaction. The results further substantiated that the linear
coefficients (extraction temperature, extraction pH, amplitude level and extraction time)
(p≤0.01) and quadratic term coefficient (extraction pH × extraction pH) (water/meal ratio
× water/meal ratio) (p≤0.01) were most significant.

The effect of extraction temperature (˚C), amplitude level (%), water/meal ratio, extraction
time (mints) and pH conditions on TPC yield was analyzed by standardizing the levels from

65
–1 to +1 for each factor. The increased in extraction temperature at specific level resulted
in higher percentage of TPC yield when amplitude level, pH, water/meal ratio and
extraction/sonication time (mints) conditions were set at mean values i.e. 90, 6, 30, and 40
minutes, respectively for both banana peel and cabbage leaves. The extraction temperature
was the most significant process variable which significantly affected the TPC yield. A
different effect of water/meal ratio on the percentage of TPC yield was observed by setting
the extraction temperature (˚C), amplitude level (%), pH and extraction time (mints)
conditions at center point. The significant production of TPC was obtained at initial points
of water/meal ratio.

The ultimate objective of employing response surface methodology in this study was to
find out the significant effects of parameters viz., extraction temperature, amplitude level,
water/meal ratio, pH and extraction/sonication time to find out the optimum conditions for
TPC yields. The conditions and mutual interaction terms which determine the rate of
maximum TPC yields are difficult to optimize and cannot be manipulated directly from the
response model. The optimum extraction conditions for the maximum TPC yields were
obtained by varying two independent parameters and fixing the other three variables at the
coded zero level. Response surface for the mutual interactions effects of extraction
temperature (oC) and amplitude level (%) on the yield of TPC (%) at water/meal ratio 30,
sonication time 40 minutes and pH 6. The extraction temperature, 60˚C;
amplitude/sonication level, 90; extraction pH, 6; water/meal ratio, 30 and extraction time,
40 minutes showed maximum TPC yield for banana peel (24.4±0.17%) and cabbage leaves
(19.8±0.15%), respectively and the lowest TPC yield (15.55±0.13%, 9.8±0.12%) was at
extraction temperature, 50˚C; extraction pH, 4; water/meal ratio, 30; amplitude level 30
and extraction time, 40 minutes of banana peel and extraction yield of TPC at temperature,
40˚C; extraction pH, 6; water/meal ratio, 30; amplitude level 30 and extraction time, 40
minutes for cabbage leaves, respectively. The results revealed that extraction conditions
significantly affect the TPC yields from banana peel and cabbage leaves.

The surface plot for chosen model variables illustrates relationship for the mutual
interaction effect of extraction temperature (oC) and amplitude level (%) on the yield of
TPC (%) when water:meal ratio 30, sonication time minutes 40 and pH 6 was set at medium
level. The results indicated that increase of extraction temperature higher than mean level
at low water/meal ratio led to significant yield of total phenolic content. The high
water/meal ratio in combination with high extraction temperature showed reduction effect
66
on TPC yield. The response surface was drawn for the mutual interaction effect of
extraction temperature (oC) and water:meal ratio on the yield of TPC (%) when amplitude
level 60, sonication time minutes 40 and pH 6 was set at medium level. This indicated that
at lowest temperature as 40oC and water:meal ratio 20 leads to lowest quantity of TPC
yield. At the middle values as 50oC and water:meal ratio30 result was different. But with
the highest values as 60oC and water:meal ratio 40, the high TPC value was obtained. The
interaction was observed between extraction temperature (oC) and sonication time
(minutes) on the yield of total polyphenols extract (%). At lower value of extraction
temperature (oC) and sonication time (minutes), the low yield of the TPC was obtained.
Optimum value was near the medium level which gave better results of TPC yield. The
interactions was observed between extraction temperature (oC) and pH. At higher value of
extraction temperature (oC) and higher value of pH ratio, the higher yield of the TPC was
noted. The results showed the temperature was major effective factor, whereas other had
slight less effects. It has also been cited that increase in TPC yield is due to the strong effect
of extraction time–temperature on the mass transfer rate of the water soluble polyphenols
in the cell wall. However, when extraction temperature and time were kept constant,
increase in water:meal ratio was the reason for an exponential increase in the yield.

Natural fruit and vegetable phytochemical extracts was supplemented at different

concentration (0.5%, 1%, 1.5%) to fish meat for preparation of different meat products i.e.
patties, balls/pops and finger/sticks. Peroxide values (PV) of all the treatments increased at
the end of second interval then decreased at the end of last storage interval. PV values of
all treatments were higher and significantly different at the beginning and the end of the
storage period. The decrease of the PV at the end of the storage may occur owing to
decomposition of hydro peroxides into secondary oxidation products. Moisture content
increased in all treatments at the end of research trial. Similar trend was found for free fatty
acids. Fish products shows a significant difference in sensory score at refrigeration
temperatures where sensory scores of fish patties decreased significantly (p<0.05)
throughout at refrigeration storage. Whereas the sensory scored at the -18ºC shows the good
sensory characteristics relatively.

67
 Conclusions and Recommendations

A 3-level five factors Box-Behnken design was used to study the effect of extraction
temperature (˚C), amplitude power level,water/meal ratio, extraction time (minutes) and
extraction pH conditions for maximum yield of total polyphenols from banana peel and
cabbage leaves.The total number of experimental runs for each banana peel and cabbage
leaves was 46 as determined by the Box-Behnken design. The percent values of TPC yield
from banana peel and cabbage leaves samples ranged from a minimum value of
15.55±0.13% to a maximum value of 24.4±0.17% for banana peel and from minimum value
of 9.8±0.12% to a maximum value of 19.8±0.15%. Natural fruit and vegetable
phytochemical extracts was supplemented at different concentration (0.5%, 1%, 1.5%) to
fish meat for preparation of different meat products i.e. patties, balls/pops and finger/sticks.
Peroxide values and FFA of all the treatments increased at the end of second interval then
decreased at the end of last storage interval. Moisture content increased in all treatments at
the end of research trial. Fish products shows a significant difference in sensory score at
refrigeration temperatures where sensory scores of fish patties decreased significantly
throughout at refrigeration storage. Whereas the sensory scored at the -18ºC shows the good
sensory characteristics relatively.

It can be concluded that the several fruit and vegetable byproducts from food processing
industries meet the criteria of antioxidant definition. They are certain to be an excellent
source of natural antioxidants if used as high-quality ingredients in functional foods or
dietary supplements. The applications of plant food byproducts will definitely bring about
added value to both, the industry and the consumer. The industry can benefit from
economic incomes and the consumer from the excellent nutritional value of these materials
with potential health claims.

68
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