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FE 412

[Methods of Research for FE]

Development and Evaluation of Unpolished
Black Rice (Oryza sativa L.) in Producing Kefir
A topic proposal presented to the Faculty of Chemical and Food Engineering Department
of Batangas State University Main II, in partial fulfillment of the requirements for the
subject, Methods of Research for Food Engineering, for the degree of Bachelor of
Science in Food Engineering

Submitted to:

Engr. Marielli Katherine C. Untalan

[College Instructor]

Submitted by:

Antenor, Mary Anne C.

Comita, Christine Joy
Maullon, Meredith S.

December 6, 2017


Trends in food and beverages may change over time but one thing remains the same; to

produce a food that will give people good health and address timely medical issues.

Functional foodsor foods with health benefits beyond basic nutritionare becoming a

subject of high interest and high demand. The IFIC (International Food Information

Council) Foundations 2016 Food and Health Survey found that nearly half of consumers

said that weight loss/management is a health benefit they are interested in getting from

foods. About one-third of Americans listed increased energy, cardiovascular health,

healthy aging, or digestive health. On an article from Leatherhead Food Research,

the Global functional food market is forecast to reach 54B USD, an increase of 25%

compared to the last available data from 2013. Leatherhead research carried out for the

report indicates that although less than a quarter (23%) of UK consumers are inclined to

believe the health claims typically made by functional foods, 1/3 are of the opinion that

these types of product can play a major role in helping to combat some of the health

problems facing society today. Another trend that was discussed in different seminars

internationally is the purple power and use of ancient grains. There is rising interest for

deep purple plants that is generally known for high antioxidant content such as acai berry,

blueberries and speciallyblack rice.

Also called the Forbidden rice and Obama rice, the aromatic black rice, which is

found in Asia, is rich in phytochemicals that fights cancer. As suggested by its name, it is

believed that black rice was first cultivated in China, especially for the Emperor, whose

food is forbidden among the royal subjects. Organic farming advocates are now pitching

calls for the massive cultivation of colored rice, particularly black rice, hoping to make a
crack at the global organic food market by supplying unique rice varieties from the


In the Philippine folklore, it is believed that black rice is the food of demigods.

Recently, black rice was introduced in the local market and because of the demand, the

cultivation of black rice is starting to gain ground in Luzon. There are five

differently-colored rice varieties other than white brown, red, pink, violet and black

which are being cultivated in the Philippines. However, these indigenous rice varieties are

rarely sold in the market, as they are usually consumed by the farmers themselves.

Black rice beats the nutritional benefits of brown and red rice combined. Rich in

fibre, it comes loaded with antioxidants, phytonutrients, phytochemicals, Vitamin E,

protein, iron, and other nutrients. It is believed to be beneficial for the liver, kidney and

stomach. It has high content of anthocyanin, which help in preventing risks of cancer. Its

low sugar and glycemic content makes it an extremely desirable dietary option for heart

patients, diabetics as well as for those with high blood pressure.

Aside from utilization of such amazing crops, another trend in food and beverages

today is the growing demand for plan-based food products. As plant-based milks, meat

alternatives and vegan offerings have rapidly moved into the mainstream, consumers are

looking for innovative options to take the inherent benefits of plants into their daily lives

The most popular fermented milk product is yogurt but interestingly, another kind

of food/ beverage which is Kefir. Kefirs tart and refreshing flavor is similar to a

drinking-style yogurt, but it contains beneficial yeast as well as friendly probiotic

bacteria found in yogurt. The naturally occurring bacteria and yeast in kefir combine
symbiotically to give superior health benefits when consumed regularly. It is loaded with

valuable vitamins and minerals and contains easily digestible complete proteins.

With these nutritional facts and lack of utilization of produced black rice in the

Philippines, the researchers came up with the idea of producing kefir from black rice.


The study aims to produce kefir from black rice. Specifically, it sought to answer the

1. What formulation of black rice will be acceptable based on sensory evaluation?

2. Is there a significant difference upon varying the concentrations of black rice milk
with constant amount of kefir grains in terms of its physicochemical properties?

3. What are the properties of the best formulation of Kefir in terms of the following

a. Physicochemical properties

a.1. Acidity

a.2. Alcohol content

a.3. Total solids

a.4. Phenolic content

b. Microbial analysis

4. What is the percentage of the best formulation of Kefir in terms of its anthocyanin


There is no significant difference upon varying the concentrations of black rice milk

with constant amount of kefir grains in terms of its physicochemical properties.


The results of the study can be means for the promotion of Black rice Kefir in the

market. Hence, it is believed that the results of this study would be a great help to

Filipinos engaged in the beverage industry.

This study would also encourage local farmers and manufacturers to propagate not

only commercially used Black rice but also varieties of less commercial value that can

yield added earnings for farmers.

To the university, the study could serve as potential source of income when the

product output is released in the market.

This study aims to produce Black rice kefir. The knowledge and discoveries in the

study will enhance on what is known and what is to be learned by the faculty and

students. Ultimately, this study will be very helpful to the future researchers who will

conduct related studies about Black rice and its properties in terms of the production of



This study will focus on developing Black Rice Kefir from unpolished black rice.

Black rice will be bought in Mindoro considering that there are farmers producing black


Formulation and analysis of the physicochemical properties such as acidity, alcohol

content, total solids, and phenolic content will be done at BSU while the microbiological
properties like bacteria, yeast, viruses, fungi and parasites will be done in an accredited


The sensory evaluation will be held at Batangas State University Alangilan Campus.

The aroma, taste, texture, color, and over- all acceptability of the Black Rice Kefir will be

determined by 50 respondents from Food Engineering Department. Given the three the

different samples, respondents were tasked to evaluate the sensory properties using 9-

point Hedonic Scale rating wherein 9- Like Extremely is the highest and 1- Dislike

Extremely is the lowest.

The shelf life and packaging of the Kefir are not included in this study but will focus

only on the production and analysis of Kefir.



Black Rice Kefir Product Kefir with known

Grains Development Physicochemical
Properties and
Kefir Grains Physicochemical Microbial
Testing Analysis

Microbial Analysis


Acidity- the quality, state, or degree of being acid. It is one of the

physicochemical properties of kefir.

Alcohol content- the amount of alcohol that is present in a substance. Another

physicochemical property that determine the alcohol present in


(https://www.collinsdictionary.com/dictionary/english/alcohol -con


Anthocyanin- any of various soluble glycoside pigments producing blue to red

coloring in flowers and plants.


Antioxidants-a substance (such as beta-carotene or vitamin C) that inhibits

oxidation or reactions promoted by oxygen, peroxides, or free

radicals. (https://www.merriam-webster.com/dictionary/antioxidant)

Black Rice- is a range of rice types of the species Oryza sativa L., some of which are

glutinous rice. It is one of the main recipes in making the product.


Fermentation- is a metabolic process that consumes sugar in the absence of oxygen.

The main processinmaking the product.


FODMAP (Fermentable Oligosaccharides Disaccharides Monosaccharides and

Polyols)- collection of short chain carbohydrates and sugar alcohols found in food

naturally or as food additives (fodmapfriendly.com)

Kefir- is a fermented milk drink. It is the product that is to be produced in this study.

Physicochemical properties- of or relating to chemistry that deals with the

physicochemical properties of substances.

( https://www.merriam-webster.com/dictionary/physicochemical )

Phytochemicals- chemical compounds produced by plants, generally to help them

thrive or thwart competitors, predators, or pathogens. Phytochemical

content of the various rice types were divided into several groups such as

carotenoids, phenolics, alkaloids, nitrogen and organosulfur containing

compounds. Phenolic compounds were sub-grouped as phenolic acids,

flavonoids, coumarins and tannins. (Choi et al., 2007b)

Probiotic- are defined as microorganisms that are believed to provide health benefits

when consumed. The term probiotic is currently used to name ingested

microorganisms associated with benefits for humans and animals.


Rice Milk- is a grain milk made from rice. It is mostly made from brown rice and

commonly unsweetened. ( en.wikipedia.org/wiki/Rice_milk)

Total solid- is a measurement that includes the combination of total dissolved solids

and total suspended solids. One of the physicochemical properties of kefir.



This chapter deals with the research literature and studies that provided information

to support and enhances the present study.


Black Rice (Oryza sativa linn.)

Black rice (BlR) is especially rich in anthocyanin pigments, phytochemicals, protein

and vitamins. China cultivates the most BlR followed by Sri Lanka, Indonesia, India,

Philippines etc. Thailand occupies the ninth position when it comes to BlR cultivation. BlR

is known for its antioxidant properties (Ichikawa et al., 2011; Sompong et al., 2011). The

antioxidants are crucial for memory enhancement and strengthening of the immune system.

In a study (Antiallergic Activities of Pigmented Rice Bran Extracts in Cell Assays) that the

pigments of colored rice bran inhibit allergic reactions in vitro. The prevention of

cancer-cell invasion property of peonidin, peonidin 3-glucoside, cyanidin 3-glucoside, and

other major anthocyanins of black rice has been reported by (Chen et al., 2006; Ichikawa et

al., 2001) also reported that BlR are efficient, and two fold stronger, with respect to

antioxidant activities of blueberries. Black rice is cultivated in different parts of the country:

around 20 hectares in Tarlac; 15 hectares in Bulacan, and 5 hectares in Nueva Ecija. It is

also cultivated in some parts of Baler, Aurora, Isabela, Mindoro, and Bataan. Black rice

grains are shown in Figure 1:

Figure 1. Black Rice Grains

Physico-chemical Properties and ProximateComposition of Black rice

In a studyof six different rice varieties marketed in Penang, Malaysia (locally grown

and imported) were evaluated for proximate composition, physiochemical properties and

cooking qualities. Overall, Black rice variety had the highest protein content (8.16%)

with lowest fat content (0.07%). Between the various rice varieties investigated, thousand

kernel weight varied between 16.97-19.43 g, length/breadth (l/b) ratio was between

2.09-3.75, while bulk density varied between 0.81-0.86 g/ml. Amylose content was highest

(27.71%) in white rice (local, medium grain type) with lowest recorded for brown rice

variety (3.36%). Results obtained in this study are useful for preparation of novel rice

based food products, based on the individual requirements. Another importance of this

study is to analyze how different varieties will improve health based on their differing

properties and composition. The table below shows the results on the study of

Physicochemical properties and Proximate composition of 6 varieties of rice in Malaysia:

Table 1 and 2: Physicochemical properties and Proximate Composition of 6 rice

varieties in Malaysia

Rice Milk

Rice milk is made from milled rice and water and is the least allergenic of all alternative

milk products, making it a good option for those with lactose or nut allergies. Rice milk is

the third most popular nondairy alternative next to soy milk and almond milk. Drinking

rice milk supplies many essential nutrients via the bran and germ of this whole grain, but

there are also some setbacks compared to other milk alternatives. Rice milk contains

more carbohydrates than cow's milk. However, unlike cow's milk, rice milk doesn't

contain lactose or cholesterol. This makes it healthy for your heart as well as safe for

those who are lactose intolerant. One cup of rice milk contains about 140 calories,

compared to 185 calories in a cup of whole cow's milk, and 97 calories in a cup of

skimmed cow's milk. Rice milk contains about three grams of fat per cup, while whole
cow's milk contains about 11 grams per cup. Rice milk is also a tasty alternative for

vegans and vegetarians who are allergic to soy. Rice milk is first and foremost

lactose-free. It is for this reason it has become one of the go-to beverage of individuals

who suffer from cow milk protein (CMP) sensitivity. Meanwhile, a study has found that

children with CMP allergy tolerate it better than cow or soy milk. (Tolerance to a rice

hydrolysate formula in children allergic to cows milk and soy Fiocchi A., Travaini M.)

Rice milk appears to be better absorbed by infants stomachs, too, as was shown in yet

another study. (www.ncbi.nlm.nih.gov/pubmed24724771.com)

Rice milk is a superior source of B vitamins, magnesium, niacin, phosphorus, and

manganese as well. Not only that as this drink is packed with carbohydrates, making it an

excellent energizing beverage. Rice milk is FODMAP diet-friendly, too, so individuals

with irritable bowel syndrome may be able to drink this beverage.

1. Pro: Great Alternative

Rice milk is the most hypoallergenic of all milk products. People with lactose intolerance

or casein allergy cannot have dairy, and those with soy or nuts allergies cannot drink soy

or almond milk. Rice milk contains a generous supply of balanced nutrition for those who

are not able to tolerate other milk alternatives.

2. Pro: No Saturated Fat of Cholesterol

Rice milk has the least amount of fat compared to all other milk alternatives. There is

only 1 gram of fat per cup of rice milk and all of the fat is unsaturated. Rice milk is also

cholesterol free and therefore it works well for people with dietary restrictions on fat and

3. Pro: Good Source of B Vitamins

Rice is naturally high in all B vitamins which are essential to your metabolism,

circulation and nerve function. Some of these vitamins may be lost during rice milk

processing, but most of these nutrients are made up by the fortified products.

4. Pro: Promote Cardiovascular Health

There are plenty of heart healthy nutrients in the rice bran, and these nutrients are also

contained in rice milk. The unsaturated fat comes from rice bran oil, which can lower

blood cholesterol. Niacin and vitamin B6 are also effective for this purpose. Rice milk is

an excellent source of magnesium, which helps control blood pressure. Iron and copper

increases red blood cell production, giving you better oxygenation and vitality.

5. Lots of Antioxidants

Rice milk contains more manganese and selenium than any other alternatives. These are

powerful antioxidants that help protect from all kinds of infections and cancers. Drinking

rice milk can also give the immune system a boost.

Kefir from rice milk

For the lactose intolerant, kefirs abundance of beneficial yeast and bacteria provide

lactase, an enzyme which consumes most of the lactose left after the culturing process.

Kefir can be made from any type of milk, cow, goat or sheep, coconut, rice or soy.

Although it is slightly mucous forming, the mucous has a clean quality to it that creates

ideal conditions in the digestive tract for the colonization of friendly bacteria. Advantages

of rice milk kefir: (1)Boosts Immunity (2) Potentially Fights Cancer (3) Supports
Digestion and Combats IBS (Irritable Bowel Syndrome) (4) Improves Lactose

Intolerance Symptoms.

6. Kefir has the added benefits of probiotics like those in yogurt. Probiotics are

known as friendly bacteria that can ease IBS symptoms such as bloating and digestive

distress in some people.

Optimum saccharification conditions of black rice

A study of fermented milk product developed based on simultaneous fermentation of

reconstituted milk and black rice hydrolysate. The optimum saccharification conditions

of black rice hydrolysate were found to be 60 , 200 U/g and 7 h for temperature,

enzyme loading and time,respectively. By using one-factor-at-a-time approach and

orthogonal array design, the optimum fermentation conditions were determined as 5%,

20%, 3%, 40 and 4 h for sucrose concentration, black rice hydrolysate concentration,

inoculum size, temperature and time, respectively. The resulting product had good quality

and great potential for further development.

Change in pH and total soluble solids

The study found out that the change in pH and total soluble solid of probiotic germinated

black rice juice significantly (p 0.05) decreased at 72-h fermentation (5.670.12 to

2.86 0.04 and 7.00 0.00 to 6.40 0.00 brix at 0-h and 72-h fermentations,

respectively). On the other hand, the amount of titratable acidity expressed as lactic acid

and the viable cell count significantly (p0.05) increased at 72-h fermentation (0.11
0.06 to 0.430.06 (% lactic acid) and 3.60 x 106 to 2.75 x 108 CFU/ml at 0-h and 72-h

fermentations, respectively). Interestingly, the amount of gamma-Amino Butyric Acid

(GABA) had a significant difference (p0.05) twice as high as that of the control group

(0.25 0.01 and 0.13 0.01 mg/100g, respectively). In addition, the free radical

scavenging capacity assayed by DPPH method also showed that the IC50 values were

significantly (p0.05) higher than the control (147.710.96 and 202.551.24 mg/ml,

respectively). After 4 weeks of cold storage at 4 C, the viable cell counts of lactic acid

bacteria reduced to 1.37 x 106 CFU/ml. In conclusion, fermented germinated native black

rice juice could be served as a healthy beverage for vegans and people who are allergic to

cow milk products.

Black rice milk as alternative in goat milk in producing kefir

This study were to evaluate the properties of kefir prepared with a combination of

goat milk and black rice extract and its influence on the improvement of beta-cells in

streptozotocin-nicotinamide (STZ-NA)-induced diabetic rats. Subtitution of goat milk

with black rice extract in kefir fermentation could decrease their pH and increase in

radical-scavenging activity. Inulin addition able to increase (p<0.05) in

radical-scavenging activity of kefir. Alcohol and total phenolic contents of kefir increased

(p<0.05) after 7 d of storage, while the pH decreased. To improve insulin-producing

pancreatic beta-cells in diabetic rats, it was required at least 2.0 ml dose of kefir
combination from goat milk and black rice extract to achieve similar effect to

glibenclamide as antidiabetic agent.



This chapter deals with the methods of research. It describes the research methods,

sensory evaluation process, physicochemical properties evaluation and the statistical

methods used.


This study utilized experimental method in order to produce Black Rice Kefir. The

methods was used to determine the physicochemical properties as well as the microbial

flora of the Kefir.

Preparation of rice milk from UBR

Unpolished Black rice (Oryza Sativa Linn.) milk was extracted from a black rice

from Mindoro. UBR will be rinsed then soaked in water with different rice to water

ratios; 1:5, 1:10, 1:15 in terms of grams. The rice will be drained without throwing out

the water. In a blender, combine rice and 100 grams or water. All rice milk can be

strained using cheesecloth

Fermentation of rice milk

Fermentation experiments will proceed after the rice milk is heated at 47 degrees C.

for every 6 cups of rice milk, cup kefir grain will be added. The kefir will be fermented

for 48-72 hours at 30 degrees C. Flavorings can be added after.

Chemical and Microbiological Analyses

Samples will be taken at 24-h intervals for chemical and microbiological analyses.

The pH will measured with a pH meter. Total acidity, expressed as percent lactic acid,

will be determined by titrating with 0.02 N NaOH to pH of 8.2. Total soluble solid will

be analyzed by hand refractometer. Viable cell counts (CFU/ml) were evaluated by the

standard plate method with lactobacilli MRS medium after a 48-h inoculation at 30 C.

Sensory Evaluation

The black rice kefir for 3- day fermentation at 30 C were sensory evaluation and

compared with each 3 samples. by 50 untrained panellists (staff and students) from the

The panelists evaluated the sample using a nine-point hedonic scales ranging from 1

(extremely dislike) to 9 (extremely like). Each panelist evaluated the samples for color,

flavor,btaste, texture, and overall acceptability.

Physicochemical analysis

The best formulation with regards to the result of sensory evaluation will be tested

for physicochemical properties including anthocyanin content by an accredited

Statistical Analysis

Analysis of Variance (ANOVA) will be used to analyze the data gathered. It will be

used in order to determine if there is a significant difference with the sensory evaluation

of the various formulations. The statistical treatment will also be used to determine which

of the formulation have attained the highest rating among the panelist.


GUO Fei-xiang, HAN Qing-qing, GU Rui-xia*, LU Mao-lin (Jiangsu Province Key

Laboratory of Dairy Biotechnology and Safety Control, College of Food Science and

Engineering, Yangzhou University, Yangzhou 225127,China) Development of a

Fermented Milk Product from Reconstituted Milk and Black Rice Hydrolysate

N. Mongkontanawat. Bacteria Fermentation of Germinated Native Black Rice Milk

Mixture by Probiotic Lactic Acid

Abou-Dobara MI, Ismail MM, Refaat NM (2016) Chemical Composition, Sensory

Evaluation and Starter Activity in Cow, Soy, Peanut and Rice Milk

Oktay Yerlikaya. Usage of rice milk in probiotic yogurt production

Nurliyani Nurliyani, Ahmad Hamim Sadewa, Sunarti Kefir Properties Prepared with

Goat Milk and Black Rice (Oryza sativa L.) Extract and its Influence on the Improvement

of Pancreatic beta-Cells in Diabetic Rats

Chen, P. N., Kuo, W. H., Chiang, C. L., Chiou, H. L., Hsieh, Y. S., & Chu, S. C. (2006).
Black rice anthocyanins inhibit cancer cells invasion via repressions of MMPs and u-PA
expression. Chemico-Biological Interactions, 163(3), 218-229.

Choi, S. P., Kang, M. Y., Koh, H. J., Nam, S. H., & Friedman, M. (2007a). Antiallergic
activities of pigmented rice bran extracts in cell assays. Journal of Food Science, 72(9),

Figure1 Black rice grains http://www.palawan-philippines.com/philippines-products.html