INTERNATIONAL JOURNAL OF Kumar Y et al.; Phytochemical profile and neutraceutical value of amla fruits

MODERN PHARMACEUTICAL RESEARCH

Volume 1, Issue 4

ISSN 2319 – 5878

Research Article

AN INVESTIGATION INTO PHYTOCHEMICAL PROFILE AND NUTRACEUTICAL VALUE OF AMLA (EMBLICA OFFICINALIS) FRUITS Nikhil K Sachan1, Sudhir Singh Gangwar2, Ranjana Sharma2, Yatindra Kumar2* 1

University Institute of Pharmacy, C.S.J.M. University, Kanpur – 208 024 Uttar Pradesh

2

Dept. of Pharmacy, G.S.V.M. Govt. Medical College, Kanpur – 208 002 Uttar Pradesh

Received 12 Feb 2013; Revised 20 Feb 2013; Accepted 25 Feb 2013

ABSTRACT Recently, nutraceuticals and medicinal plants have become the focus of current medical research. The fruit or fruit pulp of Emblica officinalis is a reputed drug of Ayurvedic, Unani, Siddha and Homoeopathic systems of medicine and believed to increase defense against diseases. The fruit primarily contained tannins, alkaloids, phenolic compounds, amino acids, carbohydrates and other compounds especially the essential nutrients. The vitamin C content in Amla varies from 200-900mg /100g from batch to batch. The comprised investigation explored physico-chemical, phyto-chemical and nutraceutical profile of this versatile and reputed ethnomedicinal fruit through basic scientific research to establish the rational scientific foundation of its utility as rejuvenating food and tonic. Key words: Indian gooseberry, food supplement, medicinal foods, emblic myrobalan, healthcare

INTRODUCTION Fruits are amongst the first food items known to human beings. Fruits, whether fresh or dried, have always formed a part of the staple diet of human beings [1]. The reason for this is that they are rich in nutrients and provide some of the essential minerals, vitamins, and the like, to our body. Apart from that, they also help in curing a number of diseases. Amla consist of fress or dried fruits of Emblica officinalis; it is one of the important herbal drugs used traditionally both as a medicine and as a tonic to build up lost vitality and vigor [2]. In Unani medicine, it is described as a tonic for heart and brain. According to the two main classic texts on Ayurved, Charak Samhita and Sushrut Samhita, Amalaki is regarded as “the best among rejuvenative herbs”, “useful in relieving cough and skin disease” and “the best among the sour fruits”

[3]

. Amla grows in tropical and subtropical parts of India, China, Indonesia,

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and the Malay Peninsula [4, 5]. The fruit is spherical pale yellow with six vertical furrows. The average weight of the fruit is 40-50g. The fruits of Amla are widely used in the Ayurvedic preparations and are believed to increase defense against diseases. Fresh fruit is refrigerant, diuretic and laxative. Fruit is also carminative and stomachic. Dried fruit is sour and astringent. It is an important dietary source of vitamin C, amino acids, and minerals. Amla is presently underutilized fruit, but has enormous potential in the world market. It is almost entirely unknown in the world market and needs to be popularized. The export of amla fruits from India, reported to be about 500 tons. It is being exported under the category of Ayurvedic and Unani herbs [6]. Its medicinal and nutritional properties and culinary uses need to be highlighted. A nutrient is defined as a chemical whose absence from diet for a long enough time results in a specific change in health. We need nutrients for normal body growth and development, for maintaining cells and tissues, for fuel to do physical and metabolic processes. Foods provide six major classes of nutrients viz., carbohydrates, lipids, proteins, minerals, vitamins and water. The first five are called essential nutrients. Carbohydrates, protein and fats are called macronutrients because our bodies need large quantities of them. The science of nutrition helps us to improve our food choices by identifying the amounts of nutrients we need, the best food sources of those nutrients we need, and other components in foods that may be helpful or harmful. Learning about nutrition will help us make better food choices, improve our health, reduce the risk of many chronic diseases and increase our longevity. The term nutraceutical is a portmanteau word coined with “nutrition” and “pharmaceutical” which refers to a foodstuff (as a fortified food or dietary supplement) that provides health benefits. The American Neutraceutical Association defines it as ‘a nutraceutical, is any substance that is a food or a part of a food and provides medical or health benefits, including the prevention and treatment of disease. Such products may range from isolated nutrients, dietary supplements and specific diets to genetically engineered designer foods, herbal products, and processed foods such as cereals, soups and beverages’. As Indians are becoming conscious about their health and look, the market of dietary supplements and sport nutrition products is expected to grow faster. The neutraceutical market has been growing at compound annual growth rate (CAGR) of 20 percent for the last three years. The Indian nutraceutical market is estimated to grow to $2731 million to 2016 at a CAGR of 13 percent. The Indian pharmaceutical market is dominated by the pharmaceuticals and Fast Moving Consumer Goods (FMCG) giants. The Indian nutraceutical market was $1180 in the year 2011

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[7]

. The

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dietary supplements constitutes largest category accounting for 64% of nutraceutical market driven primarily by pharmaceutical sector in the form of vitamins and mineral supplements. It is thus becoming a logical progression for many food companies to enter into nutraceutical market. The trend towards preventive healthcare have been observed with many pharmaceutical companies including Novartis, GlaxoSmithKline and Cadila Healthcare etc which have diversified into the production of nutraceuticals. Then there are fast moving companies like Cadbury India and Dabur that have had the presence in the market with a slew of supplements and additives. On the end of the spectrum companies that specialize in ayurvedic / herbal healthcare products like the Himalaya Drug Company

[8]

. The Big global

players in which are always on lookout for ways to diversify their products line, have set up the functional food or nutraceutical divisions; the pharmaceutical companies are also now adopting the nutraceuticals and recent trends is convergence of food manufacturing companies with pharmaceuticals to implement the research strategy. The evidence based investigation concept called positivism play a greater role in the pharmaceutical and nutraceutical research. In fact the humans, throughout the history, have been with noticeable concern in the healthcare, though the concepts themselves took a very long time to develop into a body of knowledge. A logical approach to the study of drugs and their activities is the recognition of the basic principles behind the biochemical events leading to drug actions. Nowadays, an increasing amount of insight into the behavior of drugs at the macromolecular level has been developed and there is a lot of direct and indirect evidence supporting these biochemical postulations of drug action. However, in the so-called pre-scientific period, natural products have a history of therapy in the form of folk remedies, but little of today’s drug therapy is based on these remedies. Some of the natural products currently used, either as such or as derivatives may often be used originally for other purposes, such as arrow poisons, part of religious or other rituals, and even cosmetics. An intensive and extensive study of the naturally occurring molecules identified as ‘therapeutically active’ is desired urgently to come out with new therapeutic entities. Similarly, recognising the merits of a wider usage of certain fruits and botanicals have triggered their scientific validation towards intended nutraceutical utility. Therefore these products, after having proven their ability of being fortified food supplements towards preventive healthcare, under the light of modern scientific principles, can be formulated to regulated markets, and simultaneously can boost the sale in domestic market convincing the educated consumers who are the major buyers of these products.

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MATERIALS AND METHODS Collection and Processing of the Materials The fresh fruits of Amla were purchased from open market at district Kanpur of Uttar Pradesh state. The fruits were processed to remove the fruit pulp for preliminary physicochemical analysis and isolated pulp was homogenized and passed through a strainer for further subject to spray drying obtaining dry powder. Fresh fruit pulp was analysed for pH, moisture content, titratable acidity, ash and total water-insoluble solids according to Association of Official Analytical Chemists (A.O.A.C.)

[9]

. For the spray drying, the

homogenized juice was evaporated to about 50% with a rotary vacuum evaporator and mixed with maltodextrin (5% w/v of initial juice) followed by spray drying using spray dryer at 200°C inlet temperature and 150°C outlet temperature with optimized aspiration speed to dry the sample. The powder was stored in an airtight container and kept in desiccators till further use. Moisture Content Determination The moisture content of the sample was estimated using the Karl-Fischer titration apparatus using standard KFR reagent. Minerals and Crude Fat Estimation The determination of minerals and crude content of the samples were determined as per AOAC (1997) procedures; whereas minerals (P and Fe) were estimated as per the AOAC (1990) procedures. Calcium, Sodium and Magnesium were estimated using the flame photometry. Total Phenolic Content TPC was determined using the Folin-Ciocalteu’s reagent using the method as per Lako et al. (2008)

[10]

. Samples of 0.3 ml were introduced into test tubes followed by 1.5 ml of Folin-

Ciocalteu’s reagent (diluted 10 times with water) and 1.2 ml of sodium carbonate (7.5% w/v). The tubes were vortexed, covered with parafilm and allowed to stand for 30 min. Absorption at 765 nm was measured. If the sample absorbance exceeded 1, the sample was appropriately diluted to give a reading of less than 1. Total phenol contents were expressed in gallic acid equivalents (mg per 100 gram fresh fruit). The gallic acid standard line has the equation y = 0.0111x - 0.0148 (R2 = 0.9998), where y is absorbance at 765 nm and x is concentration of gallic acid in mg/L. All Absorptions were measured in three replicates.

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Detection of Carbohydrates Extracts were dissolved separately and were tested with Molisch reagent, Fehling’s reagent, Bendict solution, and Barfoed’s test for detection of carbohydrates [11]. a) Molisch’s test: To the filtrate, added few drops of alcoholic alpha nepthol and 2ml of concentrated sulfuric acid slowly through the side of test tube; presence of carbohydrate produce a violet colour ring at the junction of two layers. b) Fehling’s test: A little fraction of filtrate treated with Fehlin’s solution I & II and then heated on a water bath. A brick red precipitate is indicator for reducing sugars. c) Bendict’s test: Small quantity of filtrate treated with equal quantities of Bendict’s reagent, heated subsequently on a water bath result to formation of a brown precipitate in presence of reducing sugars. d) Barfoed’s test: The different extracts were treated with Barfoed’s reagent. Monosaccharides, if present, produce a brick red precipitate. Detection of Glycosides Glycosides were confirmed by subjecting the acid hydrolysed extract to Legal’s test, Borntrager test and Libermann-Burchard’s test [11]. a) Legal’s test: Hydrolysate was dissolved in pyridine and sodium nitro-prusside solution, added sodium hydroxide; a colour change result in presence of glycosides. b) Borntrager’s test: A few milliliters of hydrolysate treated with chloroform, decanted off chloroform layer, added equal quantity of dilute ammonium solution. A pink colour is produced in ammonical layer in presence of glycosides. c) Libermann-Burchard’s test: Hydrolysate treated with chloroform, to this added Libermann-burchard reagent; a colour change result in presence of glycosides. Detection of Fixed oil and Fats [11, 12] a) Spot Test: A small quantity of sampler was separately pressed between two filter papers and was observed for appearance of oil stain on paper which will indicate presence of fixed oils or fats. b) Saponification test: Few drops of 0.5N potassium hydroxixe along with one or two drops of phenolphthalein were added to various extracts, heated on a water bath for 12 hours. Saponification or no saponification indicates the presence or absence of oil and fats.

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Detection of Protein & Amino acids The spay dried extract was subject to Million’s test reagent, Biuret test reagent, and Ninhydrin test reagent, presence of amino acids and proteins is indicated by production of red, violet and blue colour respectively[11]. Detection of Phenolic compounds & Tannins All the dry extracts were dissolved in minimum amount of water, filtered and subject to Ferric chloride test, Gelatin test. Filtrate on addition of few drips of ferric chloride produce a violet colour precipitate in presence of tannins. A white precipitate is resulted in presence of tannins on addition of 1ml 1% solution of gelatin to the filtrate[11-13]. Table 1: Preliminary Macroscopic and Physical Examination of E. officinalis Fruit Shape

Globular

Size of Fruits

3.8cm (Average)

Fruit Weight

41.24 – 52.48g

Colour of Fruits

Greenish Yellow

Sp. Features

Segmented through 6 ridges

Pulp Weight

91% of Fruit Weight

pH

3.1 – 3.4

Seeds

Six

Odour

Astringent & Sour

Taste

Characteristic

(sweet, sour, pungent,

bitter and astringent)

Off-white Cream Colour to

Spray Dried Powder

Brownish Yellow Water Soluble Solids

≥28 % w/w

Foreign Matter

< 1 % w/w

Moisture Content

8.1 % w/w

Ash Value

< 8 % w/w

Acid Insoluble Ash

< 1.1 % w/w

Organic Extractives

28 – 30 % w/w

Sand / Silica

Absent

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Detection of Phytosterols Small quantity of the dry extracts dissolved in about 5ml of the chloroform and subjected to Salkowski’s test and Libermann-Burchard’s test [11-13]. a) Salkowski’s test: one ml of the chloroform solution, prepared as above was added with few drops of concentrated sulfuric acid; green colour is the indicative of phytosterols. b) Libermann-Burchard’s test: The chloroform solution, prepared as above was treated with few drops of concentrated sulfuric acid followed by one ml of acetic anhydride. Presence of phytosterols is confirmed by the production of a bluish green colour. Detection of Alkaloids Small fractions of solvent free extracts were separately stirred with a milliliters of dilute hydrochloric acid and filtered, the filtrate is tested with Mayer’s reagent, Wagner’s reagent, Hanger’s reagent, Dragendroffs reagent to confirm the presence or absence of alkaloids as indicated by production of cream, reddish brown, yellow or brown colour respectively with these reagents in presence of alkaloidal substances [11, 13]. Detection of Flavonoids Shinoda’s test – Small quantity of the extract was dissolved in alcohol, to those pieces of magnesium followed by concentrated hydrochloric acid was added drop wise and heated. Appearance of magenta color shows the presence of flavonoids[12,13]. Table: 2: Composition of fruit pulp of Emblica officinalis Composition

Percentage

Moisture

81.1%

Protein

0.5%

Fat

0.1 %

Mineral Matter

0.7%

Carbohydrate

14.2%

Fibre

3.4%

Calcium

0.05

Phosphorus

0.02

Iron

1.2 mg/100g

Nicotinic Acid

0.2 mg/100g

Vitamin C

600mg/100g

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Detection of Saponins Foam test – The extract was diluted with 20 ml of distilled water and it was agitated in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam shows the presence of saponins[12]. Detection of Gums and Mucilage Small quantity of the extracts were added separately to 25 ml of absolute alcohol with constant stirring and filtered. The precipitate was dried in air and examined for its swelling properties. No swelling was observed indicates the absence of gums and mucilages. Precipitate tested for the swelling and presence and carbohydrate[11]. Estimation of Ascorbic Acid The determination of ascorbic acid content was performed using the method described in Indian Pharmacopoeia. Sample solution equivalent to 0.2 mg ascorbic acid/ml was prepared in water containing 3% (w/v) metaphosphoric acid. It was titrated against standard 2, 6 dichlorophenol 8ndophenols (2,6 DCIP) solution of 0.5 mg/ml concentration until the pink color developed completely. The operation was repeated with a blank (IP, 1966) [14].

RESULTS AND DISCUSSION The fresh fruits of Amla were of about three centimeter average length and about 3.8 cm in diameter. All the fruits were smooth segmented into six almost equal segments with formed ridges extending from apex to base; the fruits were ranging from 21.24 to 32.48 gram in weight (Fig. 1). The preliminary macroscopic properties and observations of physical examination of fresh fruits and spray dried juice/extract are tabulated in Table 1. The fruits of Emblica officinalis are rich in tannins. The fruits have 28% of the total tannins distributed in the whole plant. The Amla is reported to contain two hydrolysable tannins Emblicanin A and B, which have antioxidant properties, one on hydrolysis gives gallic acid, ellagic acid and glucose wherein the other gives ellagic acid and glucose. Emblica fruit is a rich source of vitamin C (ascorbic acid); a small fruit contains more vitamin C than a large fruit. The fruit also contains considerably higher concentration of most minerals calcium, phosphorus, iron and nicotinic acid along with amino acids (Table 2). The tests for aminoacids have been identified positive; it is reported to have variety of amino acids viz. glutamic acid, proline, aspartic acid, alanine, and lysine etc.

Qualitative chemical

examination of the dried fruit powder of Emblica officinalis revealed the presence or absence

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of various plant constituents. The observations were recorded in + (present) or – (absent) and are summarized in the Table 3.

Table: 3: Phytochemical Screening of Emblica officinalis Constituents Tests/Reagents Alkaloids o Hager’s Reagent o Wagner’s Reagent o Mayer’s Test o Dragendorff Test Carbohydrate o Molisch’s Reagent o Fehling Solution o Benedict’s Reagent o Iodine Test o Borntrager Glycosides o Legal’s Test o Liberman Burchard's Test Fixed Oils and Fats o Saponification Test o Spot Test Proteins and Amino Acids o Millon’s Reagent o Biuret Test o Nin-hydrin Test Phenolic Compounds o Neutral FeCl3 o Gelatin Test Saponins o Foam Test Phytosterols o Liberman Test o Salkowski test Gums and Mucilages

Observations + + + + + + + + + + + − − − + + + + − − + +

Amla is one of the most celebrated herbs in the Indian traditional medicine system, and is becoming increasingly well known for its unusually high levels of Vitamin C, which is resistant to storage and heat damage due to cooking. This fruit is native to India as it is self

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evident from the English name ‘Indian Gooseberry’. It has found its use in various herbal products for medicinal and nutritional value. Eating the fruits and vegetables" is one of the tried and true recommendations for a healthy diet. The edible portion of most types of fruits contains 75-95% of water. Fruits usually are low in calories and with the exception of avocados and olives, contain practically contain no fat. More than 900 different phytochemicals have been identified as components of food, and many more phytochemicals continue to be discovered today. It is estimated that there may be more than 100 different phytochemicals in just one serving of vegetables/fruit bite. These popularly known as antioxidants can scavenge or mop off the harmful free radicals produced in the body. Free radicals damage cellular membranes, proteins and DNA and cells and

produce

a

range

of

diseases in body which could be prevented with such fruits consumption.

Phenols,

flavonoids, anthocyanins and carotenoids are some of the important antioxidant found in fruits and

vegetables. The

Figure 1: Fresh Fruits of Emblica officinalis

study has explored nutrient and non-nutrient components of E. ofiicinalis. As the results revealed that the Amla has important nutritional factors contained with minerals, amino acids and vitamin. Its proven nutraceutical potential will serve the popularity of fruit in the world wide educated population. The vitamin C in Amla is remarkably stableeven after prolonged cold storage or cooking, probably due to the presence of phenolic compounds tannins and phytophenols which retarded the oxidation of ascorbic acid present in the fruit. Amla has been and remains a product of substantial interest in India and south asia. It is particularly noteworthy that amla is reported as an essential component in rasayanas such as triphala and chyawanprash to assist the body attain a state of improved nutrition that prevents aging and enhances longevity. Indian

Herbals Research and Supply Co. Ltd, furnished information submitted by GRAS Associated to USFDA on behalf of Natreon Inc for consideration of Amla products under ‘Generally Regarded as Safe’ Category and exemption from premarketing approval for its products

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[15]

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Many of investigations and traditional literature has been cited for the purpose. In fact this will help the Amla fruit to get potential market abroad in addition to India. So the present investigation will lay supportive base for the nutraceutical utility of Indian Gooseberry in international market.

CONCLUSIONS Amla, is an important medicinal plant of India. Its fruits have potent antioxidant activity due to the presence of tannoids, tannins, vitamin C and flavonoids. It has been used as rejuvenating herb. The observations in present investigation indicated that the emblic fruit is highly nutritious. With the ease of long distance transportation with high degree of stable Vitamin C in storage and thermal treatments, it servs as the packets of vitamin C with wrapped with essential minerals amino acids and other vital nutrients.

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Nayak, Priyanka, Tandon, Dileep Kumar and Bhatt, Devendra Kumar. Study on Changes of Nnutritional and Organoleptic Quality of Flavored Candy Prepared from Aonla (Emblica officinalis G.) During Storage. International Journal of Nutrition and Metabolism. 2012, Vol. 4, 7, pp. 100 - 106.

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Krishnaveni M, Mirunalini S. Therapeutic Potential of Phyllanthus emblica (amla): the Ayurvedic Wonder. J Basic Clin Physiol Pharmacol. 2010, Vol. 21, 1, pp. 93 105.

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Patel, S.S. and Goyal, R.K. Emblica officinalis Geart.: A Comprehensive Review on Phytochemistry, Pharmacology and Ethnomedicinal Uses. Research Journal of Medicinal Plant. 2012, Vol. 6, pp. 06 - 16.

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Golechha, Mahaveer, Bhatia, Jagriti and Arya, Dharmveer Singh. Studies on effects of Emblica officinalis (Amla) on oxidative stress and cholinergic function in scopolamine induced amnesia in mice. J. Environ. Biol. 2012, Vol. 33, pp. 95 - 100.

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Srivasuki, K.P. Nutritional and Healthcare Benifits of Amla. J. Pharmacognosy. 2012, Vol. 3, 2, pp. 147 - 151.

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Pathak, R.K. Status Report on Genetic Resources of Indian Gooseberry – Aonla (Emblica officinalis Gaertn.) in South and Southeast Asia. Bhag Mal, V. Ramanatha Rao and R.K. Arora [Editor] IPGRI Office for South Asia, New Delhi, India [Online] 2003.[Cited:Feb.15,2013.] http://www.agroecommerce.com/lms/download/fruits/aonla/profile_aonla.pdf.

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Kaur, Ravindra, George, Priya and Iyer, Nimisha. The Indian Nutraceutical market valued at $1480 Million in 2011 to grow to $2731 Million in 2016 – Finds Frost & Sullivan. North America, Mumbai, India : Frost Sullivan, June 12, 2012. Available through:http://www.frost.com/sublib/display-press-release.do?Src=RSS&id= 261666318.

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A.O.A.C., S. Williams. [Editor] Official Methods of Analysis of the Association of Official Analytical Chemists. Washington DC : Association of Official Analytical Chemists, 1984, p. 1141.

10. Lako, J., Trenerry, V.C. and Rochfort, S. Routine analytical methods for use in South Pacific regional laboratories. 3, 2008, International Food Research Journal, Vol. 15, pp. 313 - 323. 11. Sachan, Nikhil K. and Kumar, Yatindra. Phytochemical Investigation of Ficus racemosa Bark - An Ethnomedicinal Plant. 2, 2010, Vol. 1, pp. 80 - 84. 12. Gupta, Shailesh and Kohli, Seema. Phytochemical screening of Sarcostigmma acidum W. & Ar. 3, 2010, Vol. 1, pp. 170 - 173. ISSN 0976-7126. 13.

Kalaiarasan, , A. and John, Ahmed S. Phytochemical screening and Antibacterial activity of Sida cordifolia L. (Malvaceae) leaf extract. 2, 2010, Vol. 1, pp. 94 - 98.

14. I.P. Indian Pharmacopoeia. New Delhi : Ministry of Health, Controller of Publications, Govt. of India, 1966. 15. McQuate, Robert S., Kraska, Richard C. and Bidlack, Wayne R. GRAS Assessment Notification - Emblica officinalis Extract. Food Usage Conditions for General Recognition of Safety. Jacklight Lane, Bend OR : GRAS Associates, LLC, 2009. pp. 01 - 153, FDA GRAS Evaluation Report - GRN 295.

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