MINI PROJECT

A PROJECT REPORT
On
Physio-Chemical Properties of Black Rice

in partial fulfilment for the award of the degree of

BACHELOR OF ENGINEERING

IN

FOOD TECHNOLOGY

Submitted By:
Shubhendu Shandilya (22BFT10010)
Harshit Parashar (22BFT10040)
Anya Shukla (22BFT10042)

Divyanshi (22BFT10044)
Pratik Kumar Singh (22BFT10048)

Under the Guidance of

Dr. Davinder Pal Singh

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 Supervisor

BONAFIDE CERTIFICATE

Certified that this project report “Physio-Chemical Properties of Black Rice ” is the
bonafide work of “Shubhendu Shandilya, Harshit Parashar, Anya Shukla, Divyanshi,
Pratik Kumar Singh who carried out the project work under my/our supervision.

SIGNATURE SIGNATURE

Dr. Davinder Pal Singh Prof. Dr. Naveen Dwivedi

Associate Professor Head of Department

Department of Biotechnology Engineering and Food Technology

Submitted for the project viva-voice examination held on__________________

INTERNAL EXAMINAR EXTERNAL EXAMINAR

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 ACKNOWLEDGEMENT

It is our immense pleasure to express sincere gratitude to our project

supervisor Dr. Davinder Pal Singh, Associate Professor, Department of
Biotechnology and Food Technology engineering, Chandigarh University, Gharuan
whose guidance, encouragement, suggestions, cooperation and patience throughout
the course of work helped us in this project. We are thankful to all the teachers, who
have contributed their unique experiences to add the worth and value to our work.
We are grateful to the Department of Food Technology Engineering for giving us
such an opportunity to showcase our skills and sharpen our creative mindsets. Last
but not least, we are thankful to our friends and family members who have given us
valuable insights and reviews and have provided us immense support.

Date: 05 August, 2024

Shubhendu Shandilya

Harshit Parashar

Anya Shukla

Divyanshi

Pratik Kumar

iii
 Table Content

Sr. no. Content Page no.

1. Introduction 1-3
2. Literature Review 4-13
3. Objective 14-15
4. Material and Method 16-19
5. Result and Discussion 20-32
6. Reference 33-34

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 Abstract
Black rice, often called "forbidden rice," is recognized for its exceptional nutritional profile and
numerous health benefits. This report synthesizes evidence from 25 key studies, highlighting the health-
promoting properties and physicochemical characteristics of black rice. Key analyses focus on moisture
content, fat content, ash content, and particle size distribution to understand its nutritional and functional
[Link] levels of anthocyanins, phenolics, flavonoids, and other bioactive compounds in
black rice provide significant antioxidant, anti-inflammatory, anti-cancer, anti-obesity, anti-
diabetic, neuroprotective, and cardiovascular benefits. Black rice extract effectively suppresses
pro-inflammatory mediators and cytokines, reduces oxidative stress, improves lipid profiles, and
helps manage chronic diseases such as cancer, cardiovascular disorders, diabetes, and
neurodegenerative [Link] characterization shows that black rice contains 12-
14% moisture, 2-3% fat, and 1-2% ash content. The particle size distribution indicates a diverse
range suitable for various preparations and nutritional [Link] findings support the notion
that black rice is exceptionally healthy due to its rich bioactive content, making it valuable
nutritionally and therapeutically. This report advocates for increased consumption of black rice as
a functional food for better health and well-being. Further research is needed to optimize
cultivation, processing, and storage techniques to maximize the bioavailability of its bioactive
compounds and enhance its health benefits.

साराांश

काले चावल, जिन्हें अक्सर "निषिद्ध चावल" कहा िाता है , अपिे उत्कृष्ट पोिण प्रोफाइल और कई

स्वास््य लाभों के ललए मान्यता प्राप्त है । यह ररपोटट 25 प्रमख

ु अध्ययिों के साक्ष्यों का संकलि करती है ,

िो काले चावल के स्वास््य को बढावा दे िे वाले गुणों और भौनतक-रासायनिक षवशेिताओं पर प्रकाश डालती

है । प्रमुख षवश्लेिण िमी की मात्रा, वसा की मात्रा, राख की मात्रा, और कण आकार षवतरण पर केंद्रित हैं

ताकक इसके पोिण और कायाटत्मक गुणों को समझा िा सके।काले चावल में उच्च स्तर के एंथोसायनिि,

फेिोललक्स, फ्लेवोिोइड्स और अन्य िैव-सकिय यौगगक महत्वपूणट एंटीऑक्सीडेंट, षवरोधी-भड़काऊ, कैं सर रोधी,

मोटापा रोधी, मधुमेह रोधी, न्यूरोप्रोटे जक्टव, और हृदय संबंधी लाभ प्रदाि करते हैं। काले चावल का अकट

प्रभावी रूप से भड़काऊ मध्यस्थों और साइटोककन्स को दबाता है , ऑक्सीडेद्रटव तिाव को कम करता है ,

v
ललषपड प्रोफाइल में सुधार करता है , और कैं सर, हृदय रोग, मधुमेह और तंत्रत्रका संबध
ं ी रोगों िैसी पुरािी

बीमाररयों को प्रबंगधत करिे में मदद करता है ।भौनतक-रासायनिक षवशेिताओं के अिुसार, काले चावल में

12-14% िमी, 2-3% वसा, और 1-2% राख की मात्रा होती है । कण आकार षवतरण षवलभन्ि प्रकार की तैयारी

और पोिण अिुप्रयोगों के ललए उपयुक्त षवषवधता को इंगगत करता है ।ये निष्किट इस धारणा का समथटि

करते हैं कक काले चावल अपिे समद्

ृ ध िैव-सकिय सामग्री के कारण अत्यगधक स्वस्थ हैं, िो इसे पोिण

और गचककत्सा दोिों दृजष्टकोणों से मूल्यवाि बिाते हैं। यह ररपोटट बेहतर स्वास््य और कल्याण के ललए

काले चावल के एक कायाटत्मक भोिि के रूप में बढते सेवि की वकालत करती है । इसके िैव -सकिय

यौगगकों की िैवउपलब्धता को अगधकतम करिे और इसके स्वास््य लाभों को बढािे के ललए खेती, प्रसंस्करण

और भंडारण तकिीकों को अिक

ु ू ललत करिे के ललए आगे के शोध की आवश्यकता है ।

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 INTRODUCTION
Black rice, often called the "forbidden rice," has an excellent nutritional profile and a wide
range of proven health benefits (Hu et al., 2017). This ancient grain, traditionally reserved only for
the Chinese royal family, has gained increasing popularity worldwide due to growing awareness
of its exceptional nutritional value and therapeutic potential (Zhang et al., 2010). This report
synthesizes evidence from 25 key scientific studies to provide a comprehensive understanding of
the rich health-promoting properties and physicochemical characteristics of black rice (Chen et al.,
2006). The main analyses focused on evaluating the moisture content, fat content, ash content, and
particle size distribution of black rice, with the aim of gaining deeper insights into its nutritional
composition and functional attributes (Ryu et al., 1998). Black rice is unique from most other rice
varieties in that it contains high levels of anthocyanins, a class of powerful antioxidants from the
flavonoid family, which give it its distinctive deep purple to black coloration. (Kim et al., 2013).
These bioactive compounds contribute to the grain's remarkable health benefits, ranging from anti-
inflammatory and antioxidant effects to anti-cancer, anti-obesity, and neuroprotective activities
(Limtrakul et al., 2015).

Asian countries have farmed and consumed black rice for thousands of years (Hirunpanich
et al., 2005). Its supposed origin site is China, India, and Southeast Asia; it was for the elite and
royalty only, a way of earning its name, "forbidden rice" (Zhang et al., 2010). Such is the historical
importance assigned to black rice, which is deeply rooted in the cultures of these places: rare but
for the health-giving characteristics associated with it. Historically, the cultivation of black rice
spread beyond Asia, driven by increasing awareness of its nutritional value and how it acts upon
health promotion, leading to its increased popularity worldwide (Kim et al., 2013).

Black rice has been a nutritionally variant rice among others. It is well known to contain
proteins, essential amino acids, B vitamins, and vitamin E, as well as minerals like iron, zinc, and
magnesium (Wang & Zhang, 2017). Dietarily, black rice is also sufficiently rich in dietary fiber,
which greatly aids digestive health as well as weight management (Hu et al., 2017). High
anthocyanins and other phenolic compounds that are present in black rice constitute strong
antioxidants, which are needed to neutralize free radicals as well as inhibiting oxidative stress
(Chen et al., 2006).

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 Researchers reported several health impacts identified with black rice. Their antioxidant
properties prevent diseases, reduce inflammation, and maintain cardiovascular health (Chen et al.,
2006). The anticancer properties of anthocyanins have been expressed in terms of their ability to
inhibit cell proliferation and stimulate apoptosis of cancer cells (Ryu et al., 1998). Black rice has
a low glycemic index that may improve blood sugar and thereby lower the propensity for
developing type-2 diabetes (Wang & Zhang, 2017). It is also endowed with an anti-inflammatory
attribute, which makes it a good medicine for diseases involving inflammation in the body, such
as arthritis and inflammatory bowel disease (Jang & Xu, 2009).

The moisture content of black rice ranges between 12% and 14%, which is acceptable to
ensure there is no quality loss during storage and processing (Ryu et al., 1998). Sufficient moisture
helps to minimize microbial action and enzymatic activities that may lead to spoilage. Proper
moisture content helps to keep the rice stable, thus retaining its nutritional and sensory qualities
over time (Hu et al., 2017). Moreover, it causes an effect on the cooking characteristics of black
rice, including its texture and palatability, thus affecting its quality (Kim et al., 2013).

Black rice contains between 2% and 3% fat. Although it is much lower in fat than other
cereals, the percentage amount of essential fatty acids and fat-soluble vitamins it contributes to
nutrient intake is very important. It adds energy content to black rice and aids in fat-soluble nutrient
absorption, such as vitamins A, D, E, and K. The fatty acids composition, that is, the essential
polyunsaturated fatty acids, is also advantageous for cardiovascular health (Wang & Zhang, 2017).

The ash content of black rice, between 1% to 2%, gives an index of the mineral content in
the grain, which contains the essential elements: iron, zinc, magnesium, and calcium (Chen et al.,
2006). These minerals play crucial roles in various physiological processes, including oxygen
transport, functioning of the immune system, bone health, and enzymatic activity (Xu et al., 2017).
Ash content refers to the total mineral residue left after all the organic matter of the rice is burned;
it therefore indicated its nutritional quality and potential health benefit (Hirunpanich et al., 2005).

It provides information from the sieve analysis about the particle size distribution of black
rice and, hence, milling quality and its usability in different food uses (Kim et al., 2013). Sieve
analysis is composed of the passing of black rice grains through a set of sieves with various mesh
sizes to separate the grains by size. This size distribution then influences the cooking nature,
texture, and mouthfeel of the rice. For instance, the bigger particles take longer times to cook and

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have a chewy characteristic; however, the smaller ones take little time to cook and softer (Zhang
et al., 2010). This knowledge of particle size distribution allows designing processing methods that
can enhance the desired attributes of black rice in food products (Wang & Zhang, 2017).

These physicochemical properties in black rice, such as moisture content, fat content, ash content,
and size distribution, have been found quite crucial to determine the quality of nutrition, storage
stability, and other applications of food. Optimizing such a detailed understanding would lead us
to maximize the health benefits and utilization in food and pharmaceutical products. Further
studies need to also examine these properties at the subsequent stages of enriching the value of
black rice as a functional food (Hu et al., 2017).

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 Literature Review

Limtrakul et al. 2015 studied the anti-inflammatory potential of a black rice extract rich
in anthocyanins and other antioxidant compounds. The researchers found that the black rice extract
was able to significantly suppress inflammatory processes in cell culture experiments. Specifically,
the extract inhibited the production of key inflammatory mediators like nitric oxide, as well as pro-
inflammatory cytokines such as TNF-alpha and IL-6. Further analysis revealed that the extract
interfered with important signaling pathways that regulate inflammation, namely the MAPK and
NF-κB pathways. Phytochemical testing showed the extract contained high levels of beneficial
compounds like phenolics, flavonoids, and anthocyanins, which likely contributed to its anti-
inflammatory effects. In summary, this study demonstrated the black rice extract has promising
therapeutic potential for treating inflammation-related conditions, especially chronic diseases like
cancer. Based on these promising results, the researchers conclude that black rice shows promise
as a natural anti-inflammatory agent that could be useful for preventing or managing inflammatory
disorders.

Kaneda et al. (2006) studied the antioxidant potential of extracts obtained from black
rice bran (Kaneda et al., 2006). The researchers identified and quantified the key antioxidative
compounds in the extracts, and evaluated their ability to scavenge reactive oxygen species (ROS).
They found that the major antioxidants in black rice bran were cyanidin-3-glucoside and cyanidin,
which exhibited significantly higher ROS-scavenging activity compared to other polyphenols
present. The extraction was performed using 50% ethanol, and the total polyphenol content was
determined using the Folin-Dennis method. Active compound identification was done through
thin-layer chromatography and HPLC/LC-MS analysis, which confirmed high levels of ferulic
acid, cyanidin-3-glucoside, and cyanidin. The researchers also observed that trace metals like
manganese and iron influenced the antioxidant activity of the black rice bran extracts, and the
choice of solvent system impacted the antioxidative properties as well. Overall, the results showed
that black rice bran extracts had potent antioxidant properties due to their rich polyphenol content.
Based on these findings, the researchers conclude that black rice bran is a promising natural source
of antioxidants that could be leveraged in health foods and cosmetic products to help alleviate
oxidative stress-related disorders.

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 Thanuja and Parimalavalli (2018) researched the health-promoting features of black rice,
belonging to the Oryza sativa L. species. The researchers found that black rice is becoming an
increasingly popular functional food due to its high nutritional value and abundance of bioactive
[Link] to white rice, black rice was shown to contain higher levels of proteins,
vitamins, minerals, and essential amino acids. Notably, black rice was found to be rich in
antioxidants, phenolics, flavonoids, and anthocyanins, which contribute to its impressive health-
promoting [Link] to the study, the high antioxidant content of black rice plays a
crucial role in preventing chronic diseases, improving heart health, and reducing atherosclerosis.
These antioxidants can neutralize free radicals that otherwise cause inflammatory processes and
[Link], the researchers reported that black rice is abundant in dietary fiber, which is
beneficial for gut health, aids digestion, and supports weight management. The fiber in black rice
also helps prevent plaque formation in arteries, thereby minimizing the risk of heart attacks,
strokes, and high blood [Link] just eliminating cancer risk factors, the researchers found
that the phytochemicals and antioxidants in black rice can also protect against DNA damage during
carcinogenesis and inhibit the proliferation of cancer cells. Furthermore, black rice was shown to
enhance liver function, reduce blood sugar and cholesterol levels, and support proper brain
function, potentially lowering the risk of neurodegenerative diseases like Alzheimer's and
[Link] summary, the findings indicate that black rice is superior to white and brown rice in
terms of its nutrient profile, fiber content, and antioxidant potential, making it a highly valuable
functional food with broad health benefits.

Elkot et al. (2022) investigated the functional, physicochemical, rheological,

microbiological, and sensory properties of the synbiotic ice cream produced with camel milk, black
rice powder, and Lactobacillus acidophilus LA-5. The research work aimed at designing synbiotic
camel milk ice cream, and the survival chance of the probiotic bacteria during storage was taken
into consideration. Black rice powder was incorporated at various levels of its place with skim
milk powder. The following results were obtained by the researchers: Addition of black rice
powder to ice cream formulations increases the overrun of the ice cream, increases viscosity, and
enhances meltdown resistance but lowers the freezing point of the product. The best formulations
for sensory evaluation were in 25% and 50% black rice powder. The physicochemical properties

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of the ice cream were also analyzed. This indicated a rise in the ash and carbohydrate contents with
increasing levels of black rice powder, thereby highly improving the texture of the ice cream.
Microbiological analysis showed that frozen storage at -18°C for 60 days maintained the viability
of probiotic culture, with the highest counts recorded in the samples containing 75% black rice
powder. Sensory scores were significantly higher for products containing 25% and 50% black rice
powder, implying that those formulations are more acceptable to the consumers. The work of this
paper concluded that black rice powder has value as a material for improving the quality and
functionality of synbiotic camel milk ice cream primarily because of its beneficial impacts on
probiotic survival and sensory attributes.

Lang et al. (2019) studied the influence of drying temperature and long-term storage on
phenolic compounds in black rice. This study aimed at identifying the effects that these factors
have on the phenolic content, particularly free phenolics, flavonoids, and anthocyanins. The results
indicate that elevated drying temperatures decreased free phenolics, flavonoids, and anthocyanins
but increased extractability of bound phenolics within temperatures of 60°C and 80°C, during
which some polymerization ensued. In the present work, ferulic, caffeic, p-coumaric, and gallic
acids were identified as the most thermally unstable of the phenolic compounds. Nitrogen
atmosphere storage resulted in the best preservation of phenolics. It included drying and storage
of black rice from Brazil, measurement of its color, phenolic content, and flavonoids by different
methods. It was noticed that the higher the temperature for drying was, the higher the luminosity
and chroma values were, which are connected with the degradation of anthocyanin. The study
established significant interaction effects between drying temperature, storage conditions, and
time. In relation to retention of phenolic content, however, drying at temperatures lower than 60°C
was preferred. Discussed topics included several aspects concerning nonthermal stabilization of
anthocyanins, processing effects on phenolics, and cooking and storage effects on antioxidant
activities. The study concluded that phenolic compounds in black rice can play the role of a defense
mechanism in food products against lipid oxidation, further highlighting the necessity of proper
drying and storage conditions to preserve the nutritional and functional properties of black rice.

Finocchiaro et al. (2010) have studied the biodiversity of flavonoid content in rice
caryopsis using the simultaneous accumulation of anthocyanins and proanthocyanidins in a black-
grained genotype. The scientists proved that pigmented rice varieties, including black and red rice,

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contain a higher total antioxidant capacity and the contents of polyphenols in comparison to white
rice. From all black rice genotypes, high TAC and polyphenol were shown in Artemide. The
amount of antioxidants within the pigmented rice varieties was assessed by using a modified
Prussian blue assay coupled with the Trolox equivalent antioxidant capacity method. Results
showed that TAC levels were significantly higher in black rice than in white rice with a very high
TAC value of 94.81 mmol Trolox/kg for the black cultivar Venere. Higher proanthocyanidins
contents were reported to be found in the black-grained samples, whereas total polyphenol content
was found to be richer in pigmented rice varieties. The presence of anthocyanins and
proanthocyanidins in the black rice cultivar Artemide appeared as a possible synergistic effect,
leading to many health benefits. Researchers concluded that these rice varieties with increased
flavonoid content should be selected or bred to be better nutraceuticals for rice. The research
explained the cardiovascular health benefit and antioxidant activities of pigmented rice as potential
sources of nutraceuticals.

Chen et al. (2006): Chen and colleagues evaluated anti-metastatic activities of

anthocyanins isolated from black rice, peonidin 3-glucoside and cyanidin 3-glucoside. The study
investigated molecular mechanisms involving cancer cell invasion and motility inhibition by these
anthocyanins. The investigators determined that peonidin 3-glucoside and cyanidin 3-glucoside
inhibited the invasion and motility of several lines of cancer cells: SKHep-1 cells. We also found
that anthocyanins induced a significant downregulation of MMP-9 and u-PA, which are the
enzymes degrading the extracellular matrix during metastasis of tumor cells. Furthermore, those
anthocyanins inhibited DNA-binding activity and nuclear translocation of transcription factors AP-
1 and NF-κB, which also reduce the further invasiveness of cancer cells. In vivo, the anthocyanins
proved to inhibit tumor growth in nude mice through xenografting experiments. Results of this
nature could be used to point out that anthocyanins from black rice are pertinent candidates for
chemo preventive agents. The research could further add to existing knowledge on how natural
compounds, such as anthocyanins, can be utilized in the prevention and therapy of cancer through
the interaction with cellular pathways that lead to the progression of cancer.

This study, by Pang et al. in 2018, aimed at the investigation of antioxidant activity and
phenolic content of rice whole grains and bran, placing particular emphasis on bound phenolics in
different varieties of rice. Bound phenolics and their contribution to the overall antioxidant

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capacity of rice were also determined, along with their potential health benefits. They studied the
bound phenolics of rice bran across various types of rice, both pigmented and non-pigmented.
From this study, they were able to conclude that black rice bran contains very high antioxidant
activity, presumed to be due to a rich content of phenolic compounds. Processing methods could
be associated with substantial effects during the release and bioavailability of such phenolic
compounds. The study concluded that rice, in particular, its bran, can be an important dietary
source of antioxidants, hence taking along health benefits related to the prevention of oxidative
stress-related diseases. Results also acknowledge that Black rice is a functional food with added
health benefits resulting from its high antioxidant capacity.

Ryu et al. (1998): Ryu and his group experimented with several cultivars of black rice to
measure the concentration of anthocyanin pigments through HPLC. Here, they were focused to
obtain accurate measurement of the concentration and composition of the anthocyanin pigments
present in black rice. This experiment ensured that black rice samples were separated into their
several anthocyanin pigments present there using the HPLC. This study identifies enormous
differences in anthocyanins between black rice varieties, with some varieties having higher
anthocyanin, which is useful and healthy for its antioxidant activity. The nutrition and functionality
of anthocyanins in black rice were emphasized based on the contribution of health benefits in black
rice. The high anthocyanin in the black rice has significant scientific research to back up the use
of the latter as a functional food or nutraceutical, considering that it is associated with health
benefits.

Kushwaha (2016). The author Kushwaha researched the nutritional value and health
benefits of black rice mainly about its high fiber, antioxidants, vitamins B and E, iron, and
anthocyanins. Black rice, an ancient name for which was "forbidden rice," was known for its
health-promoting effects. A comparison study carried out on black rice at different altitudes in
Nepal clearly revealed more anthocyanin with high altitude, which might be due to better
environmental conditions at that place. More levels of anthocyanin and longer maturation periods
along with higher grain yields have been observed in black rice grown at higher altitudes compared
with other rice varieties grown at lower altitudes. In addition, the calcium and iron content in black
rice were significantly higher than in other rice varieties. The health benefits of this grain, such as
lowering the atherosclerotic plaque or improving cholesterol levels, were attributed mainly to

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black rice anthocyanins. Therefore, black rice was proved to be a useful nutrient addition in the
diet with high antioxidant content and useful for the improvement of heart health and the reduction
of risk of cancer development.

Zhang et al. (2018): Zhang and other authors studied the antioxidant and anti-inflammatory
effects of anthocyanins from black rice. In the current study, authors have indicated that such
anthocyanins could significantly reduce oxidative stress and inflammation in both in vitro and in
vivo models. The analysis of cell pathways revealed that anthocyanins from black rice suppressed
the activation of NF-κB and MAPKs, central regulators of inflammatory responses. Diet
supplementation of experimental animals with black rice anthocyanins increased antioxidant
enzyme activities while reducing pro-inflammatory cytokines levels. The outcomes suggest
antioxidants in the diet with high anti-inflammatory activities via anthocyanins found in black rice,
where it can possibly provide good health benefits in the prevention and control of chronic diseases
caused by oxidative stress and inflammation.

Hu et al. (2017) conducted a rat model to observe the cardiovascular effects of black rice.
The test was to measure how black rice affects the lipid profiles and the progression of
atherosclerosis. The analyses exhibited marked improvement in lipid profiles after black rice
treatment, such as reduced total cholesterol, LDL cholesterol, and triglycerides, with increased
levels of high-density lipoprotein (HDL) cholesterol. Additionally, black rice intervention resulted
in decreased atherosclerotic plaques formed inside the arterial walls, possibly due to its high
anthocyanin content whereby the impact is presumed to be through antioxidant and anti-
inflammatory effects. In sum, results from this study provide evidence to incorporate black rice in
nutrition for cardiovascular health and thus promise a promising role in developing related
preventive or therapeutic interventions in cardiovascular diseases.

Wang et al. (2016) investigated the effects of black rice extract on obesity and associated
metabolic disorders in a high-fat diet-induced obese mouse model. They want to know if it will
reduce obesity and beneficially impact metabolic health. The results obtained indicated reduced
body weight gain, adipose tissue mass and serum lipid levels in the obese mice after
supplementation with the black rice extract. Besides reducing body weight gain, it improved the
insulin sensitivity and the level of glucose tolerance, suggesting that it may offer a number of
metabolic health benefits. The scientists attributed these effects to high anthocyanin content in

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black rice, showing strong antioxidant and anti-inflammatory activity. It concluded that black rice
extract could well be a promising dietary intervention for obesity and metabolic disorders and
provided insights into potential mechanisms of action.

Xu et al. (2017) the anti-diabetic potential of black rice anthocyanins in a mouse model of
diabetes. The authors tried to elucidate the possible role of black rice anthocyanins in enhancing
glucose metabolism and sensitivity to insulin. The anthocyanins of black rice resulted in a
significant reduction of fasting blood glucose and increased insulin sensitivity in diabetic mice,
along with improving pancreatic β-cell functions and reducing oxidative stress and inflammation
markers in the liver and adipose tissue. Findings in such studies may be seen as useful for black
rice anthocyanins as a food for diabetic control and prevention of complications disease, thereby
affirming their potential as a functional food ingredient with anti-diabetic properties.

Liu et al. (2018) examined how black rice anthocyanins abated cognitive decline and
neuroinflammation in the model of Alzheimer's disease. The present study attempts to assess if
black rice anthocyanins can alleviate cognitive impairment and neuroinflammation in Alzheimer's
disease. It was found that treatment with this anthocyanin improved the cognitive functions of the
mice with Alzheimer's disease. Behavioral studies indicated improved memory and learning
capabilities in the mice. The treatment also alleviated neuroinflammatory markers and oxidative
stress within the brain, suggesting neuroprotection. Black rice anthocyanins have the potential to
be regarded as a promising nutritional intervention for Alzheimer's disease, considering potential
benefits to cognitive health and neuroprotection.

Choi et al. (2018) studied the anticancer activity of black rice anthocyanins through the use
of a colorectal cancer model in which they applied the latter with the objective of evaluating
whether black rice anthocyanins can inhibit proliferation of cancer cells and promote tumor
growth. The researchers found that anthocyanins from black rice strongly inhibit the proliferation
of colorectal cancer cells and induce programmed cell death through activation of caspase-3 and
caspase-9. The study further showed that in a mouse xenograft model, anthocyanins profoundly
suppressed tumor growth. The molecular mechanisms by which black rice anthocyanins could
exert anti-cancer effects may lie in their modulation of crucial signalling pathways under
regulation of apoptosis and cell cycle. In summary, the results indicate the use of black rice

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anthocyanin as a potential natural anti-cancer agent that could serve to prevent or even treat
colorectal cancer.

Kim et al. 2019 studied the effects of black rice anthocyanins on skin health and
photoaging. Here, the aim was to establish whether black rice anthocyanins could have protective
effects on the skin from UV-induced damage and thus improve general skin health. Black rice
anthocyanins were also demonstrated to possess profound anti-photoaging activities, including
significant reduction in the wrinkle formation caused by UV irradiation and improvement of both
skin elasticity and hydration in a mouse model. Anthocyanins increased the expression of collagen
and elastin in the skin and indicated its potential use in health and anti-aging applications for skin.
This led to the conclusion that black rice anthocyanins can be considered to offer natural protection
against UV-induced damage to contribute to healthier skin and youthful skin.

Lee et al. 2017 investigated the black rice extract's ability to induce anti-inflammatory
effects in an inflammatory bowel disease mouse model. Here, the study aimed to assess if the black
rice extract could attenuate inflammation within the intestinal area and ultimately enhance gut
health. The researchers observed that black rice extract greatly decreased markers of intestinal
inflammation, which include TNF-α, IL-1β, and IL-6 in the IBD mice. It was demonstrated that
the extract would improve the gut barrier functionality and decrease oxidative stress in intestinal
tissue, thereby making this extract beneficial for gut health. In addition, the study concluded that
the black rice extract changed the gut microbiota composition with higher growth of beneficial
bacteria, with concomitant suppression of pathogenic bacteria. At this point, it can be said that
black rice extract has potential as a promising dietary intervention in the management of
inflammatory bowel diseases and to enhance gut health in general.

Kong et al. (2016) have studied the preventive effects of black rice anthocyanins on mouse
lung cancer cells. This study is conducted to explore the prevention of diet-induced initiation and
progression of lung cancer by dietary anthocyanins from black rice. In this study, marked reduction
of number and size of lung tumors was observed in mice. The black rice anthocyanins were shown
to induce apoptosis through the intrinsic apoptosis pathway by upregulation of Bax and
downregulation of the Bcl-2 proteins of lung cancer cells. This should also have been in addition
to inhibiting the activation of NF-κB and reducing the expression of COX-2 and iNOS, which

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contribute to the mechanisms of inflammation and tumor progression. Thus, black rice
anthocyanins have significant chemopreventive activities against lung cancer.

Sun et al. (2017) investigated the anti-obesity activity of black rice extract in a high-fat
diet-induced obese mouse. In this study, the scientists tried to find out whether black rice extract
can inhibit obesity and enhance metabolic wellbeing. Black rice extract significantly reduced body
weight gain, adiposity, and serum lipid levels in obese mouse. Moreover, it elevated insulin
sensitivity and glucose metabolism also. These benefits were due to a high anthocyanin content in
black rice, which has strong antioxidant and anti-inflammatory properties. Black rice extract was
shown to be a promising dietary intervention against the development and progression of obesity
and related metabolic disorders.

Tang et al. 2018 examined the preventive effects of black rice anthocyanins on the livers
of a model afflicted with non-alcoholic fatty liver disease (NAFLD). The study determined the
efficacy of black rice anthocyanins in lowering liver steatosis and inflammation. The researchers
demonstrated that black rice anthocyanins caused a significant decrease in liver fat accumulation,
inflammation, and oxidative stress in NAFLD mice. This was through AMP-activated protein
kinase, which is activated in the control process of lipid metabolism and increases the reduction of
liver steatosis accumulation. Further, black rice anthocyanins also inhibited the expression of pro-
inflammatory cytokines and markers of fibrosis; hence, they may have a positive role in the
prevention and therapy of NAFLD.

Chen et al. (2017) explored the neuroprotective effects of black rice anthocyanins in a
mouse model of Parkinson's disease. They wanted to know if black rice anthocyanins could inhibit
the death of dopaminergic neurons as well as ameliorate motor dysfunction. Research revealed
that black rice anthocyanins dramatically rescued the loss of dopaminergic neurons and restored
most of the motor functions in Parkinson's mice. Meanwhile, black rice anthocyanins reduced
oxidative stress and neuroinflammation in the central nervous system. The authors hypothesized
that the neuroprotective activity of black rice anthocyanins was associated with activating the
Nrf2-ARE pathway, increasing antioxidant and detoxifying enzymes. These results indicate that
black rice anthocyanins could be a promising dietary intervention for neuroprotection in
Parkinson's disease.

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 Gao et al. (2018) studied the antidiabetic activity of the extract of black rice in a model of
type 2 diabetes in mice. Here, they aimed to determine whether black rice extract can improve
glycemic control and restoration of lost insulin sensitivity. The black rice extract showed
significant reduction of fasting blood glucose levels, improvement of insulin sensitivity, and
increased glucose tolerance in the diabetic mice. It also decreased oxidative stress and
inflammation in the liver and adipose tissue. The researchers surmised that these effects were due
to the considerable antioxidant and anti-inflammatory potentials of black rice, attributing this
largely to its high anthocyanin content. The findings, therefore, suggest that black rice extract may
be useful in the management of type 2 diabetes and the prevention of its complications.

Wang et al. (2019) investigated the potential of black rice anthocyanins to alleviate
cardiovascular disturbances in a rat model of hypertension. The objective was to investigate
whether black rice anthocyanins exert an antihypertensive effect by modifying blood pressure and
improving vascular function. Black rice anthocyanins have been shown to markedly lower blood
pressure and improve endothelial function in hypertensive rats. These anthocyanins further
reduced oxidative stress and inflammation in vascular tissue, which suggested protection on the
cardiovascular level. The authors concluded by saying that black rice anthocyanins could play an
important role as a component in diets aimed at the management of hypertension and improvement
of cardiovascular health.

Zhao et al. 2018 examined the anti-inflammatory and antioxidant effects of anthocyanins
from black rice in an inflammatory arthritis model. The study intended to investigate the protective
activity of anthocyanins from black rice against joint inflammation and oxidative impairment. The
findings of the researchers demonstrated that anthocyanins obtained from black rice markedly
suppressed inflammation, pain, and swelling of arthritic mice. The levels of pro-inflammatory
cytokines and oxidative stress markers in the joint tissue were also reduced by anthocyanins. Based
on the study, black rice anthocyanins may inhibit the NF-κB and MAPK signaling pathways to
exert anti-inflammatory and antioxidant properties. These results showed the potential of using
black rice anthocyanins as a natural therapeutic agent for inflammatory arthritis.

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 Objectives of the Project

The primary objective of this project is to conduct a comprehensive analysis of the

physicochemical properties and health benefits of black rice (Oryza sativa L.), also known as
"forbidden rice," and to develop a new product utilizing its unique attributes. The specific
objectives include:

1. Nutritional Analysis:

o To evaluate the nutritional composition of black rice, including its content of

proteins, essential amino acids, vitamins (especially B vitamins and vitamin E), and
minerals such as iron, zinc, and magnesium.

o To determine the levels of dietary fiber and bioactive compounds, particularly

anthocyanins, phenolics, and flavonoids, that contribute to its health benefits.

2. Health Benefits:

o To review and synthesize existing research on the health-promoting properties of

black rice, focusing on its antioxidant, anti-inflammatory, anti-cancer, anti-obesity,
anti-diabetic, neuroprotective, and cardiovascular effects.

o To analyze how black rice can help in the prevention and management of chronic
diseases such as cancer, cardiovascular disorders, diabetes, and neurodegenerative
diseases.

3. Physicochemical Properties:

o To characterize the physicochemical properties of black rice, including moisture

content, fat content, ash content, and particle size distribution.

o To understand how these properties affect the nutritional quality, storage stability,
and culinary applications of black rice.

4. Product Development:

o To utilize the unique nutritional and functional properties of black rice in the
creation of a new product that leverages its health benefits.

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 o To develop muffins using black rice as a primary ingredient, ensuring they meet
consumer expectations for taste, texture, and nutritional value.

o To design and test the new black rice muffins, assessing their market potential,
consumer acceptance, and potential health claims.

o To evaluate the nutritional profile of the black rice muffins, including their protein,
fiber, vitamin, and mineral content, as well as their levels of bioactive compounds.

5. Promoting Black Rice as a Functional Food:

o To advocate for the increased consumption of black rice as a functional food that
promotes better health and well-being.

o To provide recommendations for incorporating black rice into various dietary

patterns and culinary applications based on its nutritional and health benefits.

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 Material and Method

Physio-Chemical Properties of Black Rice:

Black Rice Samples Black rice samples were obtained from a local market. All analyses were
conducted in triplicate.

• Moisture Content Moisture content was determined using a standard oven-drying method.
Approximately 5 grams of black rice sample was weighed and placed in a pre-weighed
petri dish. The sample was then dried in an oven at 60°C until a constant weight was
achieved. The moisture content was calculated as the percentage loss in weight of the
original sample.

Moisture Content (%) = (Initial Weight - Final Weight) / Initial Weight × 100

• Fat Content Fat content was measured using the Soxhlet extraction method. Around 2
grams of finely ground black rice sample was weighed into a cellulose extraction thimble.
The thimble was then placed in a Soxhlet extractor, and n-Hexane was used as the solvent.
The extraction was carried out for 6 hours. The extracted fat was dried and weighed, and
the fat content was calculated as a percentage of the initial sample weight.

Fat Content (%) = (Weight of Extracted Fat / Initial Weight) × 100

• Sieve Analysis Particle size distribution of the black rice samples was determined by sieve
analysis. Approximately 100 grams of black rice was placed on the top of a stack of
standard sieves number 30,44,60,86,100 The sieve stack was mechanically shaken for 10
minutes, and the weight of the material retained on each sieve was recorded.

The particle size distribution was calculated as the percentage of the total sample weight

retained on each sieve.

Percentage Retained = (Weight Retained on Sieve / Total Weight) × 100

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 • Anthocyanin Extraction from Black Rice: Three different rice were used for anthocyanin
extraction:

Polished white rice (Oryza sativa L. indica)

Rice powder (Oryza sativa L. indica)

Black rice (Oryza sativa L. indica)

All rice samples were obtained from local grocery stores and stored at room temperature
before extraction.

Extraction Procedure

The anthocyanin extraction procedure was the same for all three rice sources:

1. 10 g of the rice material (whole polished rice, rice powder, or black rice) was combined
with 100 mL of 95% ethanol in a beaker.
2. The mixture was stirred continuously for 1 hour at room temperature.
3. The ethanolic extract was filtered through the Whatman No. 1 filter paper.
4. Inclusion of the absorbance measurement at 480 nm using a spectrophotometer.

Anthocyanin content = A×MW×DF×1000/e×l

A= absorbance

MW= molecular weight

DF= dilution factor

e= molar extinction cofficent

L= path length

Path length= 1cm

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 Molar extinction cofficent is 26,900 L/[Link].

MW= 449.2g/mol

Product (Muffins):

Muffin Ingredients

The muffins were prepared using the following ingredients in varying ratios:

• Black rice
• Whole wheat flour
• Sugar, Baking Powder, Flavour, Milk

Muffin Preparation

For each muffin, 10 grams of the black rice and whole wheat flour mixture was used, with the
black rice comprising 0.5%, 1%, 2%, or 2.5% of the total flour mixture.

The specific steps for muffin preparation were:

1. The required amounts of black rice and whole wheat flour were weighed out to achieve the
desired ratios (0.5%, 1%, 2%, 2.5% black rice).
2. The black rice and whole wheat flour were combined and mixed thoroughly.
3. The remaining muffin ingredients were combined and mixed according to a standard
muffin recipe.
4. 10 grams of the black rice-whole wheat flour mixture was added to the muffin batter and
gently incorporated.
5. The muffin batter was divided into a greased muffin moulds, filling each cup
approximately 3/4 full.

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 6. The muffins were baked at 356°F (180°C) for 25-30 minutes until a toothpick inserted in
the center came out clean.
7. The muffins were allowed to cool for 5 minutes before removing them from the moulds.

This process was repeated to create muffins with each of the 4 different black rice ratios (0.5%,
1%, 2%, 2.5%).

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 Result and Discussion

Physicochemical Properties of Black Rice:

Moisture Content:

Calculations:

where:

• Wi = Initial weight of the sample

• Wd = Weight of the dried sample

• Empty Petri Plates Weights (g):

o Plate 1: 44.358
o Plate 2: 43.038
o Plate 3: 39.447
• Weights of Sample + Petri Plates Before Drying (g):
o Plate 1: 49.358
o Plate 2: 48.038
o Plate 3: 44.447
• Weights of Sample + Petri Plates After Drying (g):
o Plate 1: 48.875
o Plate 2: 47.605
o Plate 3: 43.910

Calculations:

1. Initial Weight of Sample :

o Plate 1: 49.358−44.358=5.00049.358 - 44.358 = 5.00049.358−44.358=5.000
grams

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 o Plate 2: 48.038−43.038=5.00048.038 - 43.038 = 5.00048.038−43.038=5.000
grams
o Plate 3: 44.447−39.447=5.00044.447 - 39.447 = 5.00044.447−39.447=5.000
grams
2. Final Weight of Sample After Drying :
o Plate 1: 48.875−44.358=4.51748.875 - 44.358 = 4.51748.875−44.358=4.517
grams
o Plate 2: 47.605−43.038=4.56747.605 - 43.038 = 4.56747.605−43.038=4.567
grams
o Plate 3: 43.910−39.447=4.46343.910 - 39.447 = 4.46343.910−39.447=4.463
grams

The moisture content of the black rice samples was found to be 9.69%, which is an acceptable
level to ensure quality and stability during storage and processing. This moisture content helps
minimize microbial growth and enzymatic activities that could lead to spoilage, thus retaining the
nutritional and sensory qualities of the black rice.

Fat Content:

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 The following weights for the fat content analysis in triplicates:

Initial Weight of Sample (g): 2.0 grams for each replicate

Weights of Extracted Fat (g):

o Replicate 1: 0.052 grams

o Replicate 2: 0.055 grams
o Replicate 3: 0.050 grams

Calculation

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The black rice samples were found to contain 2.62% fat content. While this is lower than many
other cereal grains, the fatty acid composition of black rice, particularly the essential
polyunsaturated fatty acids, contributes significantly to its cardiovascular health benefits.

Ash Content:

the following weights for the ash content analysis in triplicates:

• Initial Weight of Sample (g): 3.0 grams for each replicate

• Weights of Ash (g):
o Replicate 1: 0.062 grams
o Replicate 2: 0.059 grams
o Replicate 3: 0.065 grams

The ash content of the black rice samples ranged from 1-2%, indicating a good mineral content,
including essential elements like iron, zinc, magnesium, and calcium. These minerals play crucial

23 | P a g e
roles in various physiological processes, such as oxygen transport, immune function, bone health,
and enzymatic activity.

Sieve analysis:

Table: Sieve Analysis of Black Rice

Sieve Number Sieve Size (µm) Weight Retained (g) Percentage

Retained (%)
30 600 20.0 20.0
44 355 30.0 30.0
60 250 25.0 25.0
86 180 15.0 15.0
100 150 8.0 8.0
Pan - 2.0 2.0
Total 100.0 100.0

The percentages are calculated using the formula:

The sieve analysis revealed a diverse range of particle sizes in the black rice samples. This size
distribution influences the cooking characteristics, texture, and mouthfeel of the rice, with

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 larger particles taking longer to cook and having a chewier texture, while smaller particles
cook faster and have a softer mouthfeel. This information can be used to optimize processing
methods and target specific applications for the black rice.

Summary of Physicochemical Properties:

Property Replicate 1 Replicate 2 Replicate 3 Replicate 4

Moisture 9.66 8.66 10.74 9.69
Content
Fat Content 2.6 2.75 2.5 2.62
Ash Content 2.07 1.97 2.17 2.07

Anthocyanin Content Analysis:

The anthocyanin content in black rice was measured by assessing the absorbance of extracts at
480 nm using a spectrophotometer. The results for the different types of black rice were as
follows:

• Black Rice Powder: Absorbance at 480 nm = 2.199

• Polished Black Rice: Absorbance at 480 nm = 0.659
• Normal Black Rice: Absorbance at 480 nm = 0.720

Using the following formula, the anthocyanin content was calculated:

where:

• A = Absorbance at 480 nm
• MW= Molecular weight of the anthocyanin (cyanidin-3-glucoside, MW = 449.2 g/mol)
• DF = Dilution factor

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 • ϵϵ = Molar extinction coefficient (26,900 L/mol·cm for cyanidin-3-glucoside)
• l= Path length of the cuvette (1 cm)

Calculations:

1. Black Rice Powder:

• Absorbance (A) = 2.199
• Molecular weight (MW) = 449.2 g/mol
• Dilution factor (DF) = 1 (assuming no dilution)
• Molar extinction coefficient (ϵ) = 26,900 L/mol·cm
• Path length (l) = 1 cm

2. Polished Black Rice:

• Absorbance (A) = 0.659

• Molecular weight (MW) = 449.2 g/mol
• Dilution factor (DF) = 1 (assuming no dilution)
• Molar extinction coefficient (ϵ) = 26,900 L/mol·cm
• Path length (l) = 1 cm

3. Normal Black Rice:

• Absorbance (A) = 0.720

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 • Molecular weight (MW) = 449.2 g/mol
• Dilution factor (DF) = 1 (assuming no dilution)
• Molar extinction coefficient (ϵ) = 26,900 L/mol·cm
• Path length (l) = 1 cm

Sample Type Absorbance (480 Anthocyanin Content (mg/L)

nm)
Black Rice Powder 2.199 36.47
Polished Black Rice 0.659 11.02
Normal Black Rice 0.720 12.02

These results indicate that black rice powder has the highest anthocyanin content, followed by
normal black rice and polished black rice. The significantly higher anthocyanin content in black
rice powder suggests that it retains more of these beneficial compounds compared to the processed
forms. This finding underscores the importance of minimal processing to preserve the health-
promoting properties of black rice.

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 Product (Muffins):

0.5% Black Rice

2.5% Black Rice

2% Black Rice 1% Black Rice

Sensory Evaluation For Muffins

A sensory evaluation of the developed black rice muffins was conducted using a 9-point hedonic
scale, where 1 = Dislike Extremely and 9 = Like Extremely. The panelists included:
• Dr. Davinder Pal Singh
• Dr. Mohit Singla
• Harshit Parashar
• Bhawanjot Singh
Table: Sensory Evaluation of Black Rice Muffins
Panelist Appearance Texture Flavor Overall Acceptability

Dr. Davinder Pal Sing 7 7 8 8

Dr. Mohit Singla 7 7 7 7
Harshit Parashar 8 7 9 9

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Bhawanjot Singh 7 8 8 8
Pranav Pnakj Sood 8 7 7 7

Appearance:
• The muffins received high scores for appearance, with an average score of 8.0. The deep
purple color imparted by the black rice flour was visually appealing to the panelists.
Texture:
• The texture of the muffins was rated slightly lower, with an average score of 7.0. While the
muffins were generally well-received, some panelists noted that the texture could be
improved for a more consistent mouthfeel.
Flavor:
• Flavor received an average score of 7.8. The unique taste of black rice was appreciated,
though a few panelists suggested that the flavor could be enhanced with additional
ingredients to balance the earthiness of the black rice.
Overall Acceptability:
• The overall acceptability of the black rice muffins averaged at 7.8. This indicates that the
product is generally liked and has potential for commercial success, though minor
adjustments could further improve its sensory attributes.
The sensory evaluation of black rice muffins indicates a positive reception from the
panelists, particularly in terms of appearance and flavor. The results suggest that with minor
improvements in texture, the product can achieve higher overall acceptability. The
incorporation of black rice flour not only enhances the nutritional profile of the muffins but
also provides a unique and appealing product for health-conscious consumers.

Conclusion:
The comprehensive analysis of black rice (Oryza sativa L.) in this project highlighted its
exceptional nutritional and functional properties, confirming its status as a superfood with
significant health benefits. The physicochemical properties evaluated included moisture
content, fat content, ash content, and particle size distribution, along with the anthocyanin
content. These analyses provided valuable insights into the nutritional quality and potential
health benefits of black rice.

1. Physicochemical Properties:

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 o Moisture Content: The moisture content of black rice was found to be 9.69%.
Maintaining this moisture level is crucial for preserving the quality of black rice
during storage and processing, preventing microbial growth and enzymatic
reactions that could lead to spoilage.

o Fat Content: The fat content was determined to be 2.62%, which, although lower
compared to other cereals, plays a crucial role in providing essential fatty acids and
fat-soluble vitamins. The lipids contribute to the overall energy content and aid in
the absorption of fat-soluble nutrients.

o Ash Content: The ash content was found to be 2.07%, indicating a rich mineral
composition including iron, zinc, magnesium, and calcium. These minerals are
essential for various physiological functions, such as oxygen transport, immune
function, bone health, and enzyme activity.

o Particle Size Distribution: The sieve analysis showed a diverse particle size
distribution, indicating the suitability of black rice for various culinary applications.
This distribution affects the cooking characteristics, texture, and mouthfeel of the
rice, making it a versatile ingredient.

2. Anthocyanin Content:

o The anthocyanin content was highest in black rice powder (36.74 mg/L), followed
by normal black rice (12.02 mg/L) and polished black rice (11.02 mg/L).
Anthocyanins are potent antioxidants with significant health benefits, including
anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, neuroprotective, and
cardiovascular effects.

3. Product Development:

o Black Rice Muffins: Utilizing the unique properties of black rice, muffins were
developed as a health-promoting food product. The sensory evaluation of the
muffins was conducted using a 9-point hedonic scale, with panelists Dr. Davinder
Pal Singh, Dr. Mohit Singla, Harshit Parashar, and Bhawanjot Singh, Pranav Pankaj
Sood. The results were as follows:

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 ▪ Appearance: Average score of 8.0, indicating a visually appealing product
due to the deep purple color imparted by the black rice flour.

▪ Texture: Average score of 7.0, with feedback suggesting that minor

improvements could enhance consistency.

▪ Flavor: Average score of 7.8, reflecting a generally favorable response,

though some panelists suggested flavor enhancements.

▪ Overall Acceptability: Average score of 7.8, demonstrating that the product

is well-liked and has commercial potential.

The findings of this project underscore the significant potential of black rice as a functional
food ingredient. Its rich bioactive content and unique nutritional properties make it not only
valuable nutritionally but also therapeutically. The development of black rice muffins
showcases the practical application of black rice in creating health-promoting food
products that cater to health-conscious consumers.

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