Title of Invention

"A PROCESS FOR THE PREPARATION OF POLYPHENOLS FROM FINGER MILLET"

Abstract

A process for preparation of polyphenols from finger millets A process for preparation of polyphenols from finger millets. The novelty of the process is that it makes use of the by-product of the millet processing industry and the polyphenols isolated could be used an independent entity or could be used as part of the neutral or active materials for enhancing the shelf-life of the food products or to improve the gastrointestinal health of the human subjects or as pharamaconutrient. It could also be applied to other food materials to improve their shelf-life and quality attributes. The process steps are: mixing the finger millet powder in a solvent consisting of 0.5 - 3 parts mineral acid and 99.5 - 97 part of methanol followed by refluxing the above said mixture, concentrating the extract at 30-50°C and 200-400 mbar vacuum by evaporating the methanol, neutralizing the concentrate by known methods, and drying the above obtained polyphenol rich precipitate to get pure polyphenol.

Full Text

This invention relates to a process for preparation of polyphenols from finger millets Polyphenols are a class of phytochemicals produced by plants as protective agents against external attack. Cereal polyphenols are edible constituents and are largely concentrated at the pericarp or seed coat of the majority of the cereals (Reed JD., Journal of Agriculture and Food Chemistry, 35, 461-464, 1987). Polyphenols are known to possess antioxidant, anti-microbial and antiviral properties and hence provide several health benefits (Konowalchuk j and Spiers, Journal of Food Science, 41, 1013-1017, 1976). Finger millet is one of the important minor cereals used in Indian subcontinent and also in some of the African countries. It is a small seeded grain with brick red to dark colored seed coat. The seed coat comprises 12-18% of the kernel and is fully edible. The coloring matter of the seed coat is largely due to pigments containing several phenolic compounds. Although the seed coat is a rich source of proteins, minerals and dietary fiber, it affects the aesthetic quality of the millet food by imparting dark and unattractive color and also non-chewy texture. Because of this, the non-traditional millet consumers do not relish millet foods, although, the nutritional and health benefits of the millets are appreciated by one and all. In view of this, efforts have been made to separate the major portion of the seed coat from the endosperm matter, not only from native but also from processed millet such as malted and parboiled millet. The seed coat forms the by-product during preparation of refined millet flours, refined malt flour and also decortication of the millet. In recent years, processing of millet, especially, malting of finger millet is gaining momentum and the seed coat from the malted millet is available to the tune of about 5 metric tons per day. The seed coat from the millet decortication and preparation of refined flour also add to the availability of the seed coat. Most of the seed coat obtained in these industries is mainly utilized in feed formulation and as a constituent of compost to a limited extent. The millet exhibits extremely good storage life and can be stored for years under normal conditions without substantial qualitative and quantitative loss in its quality. The good shelf-life of the millet seeds is generally attributed to the phytochemicals, especially, the polyphenol constituents of the seed coat matter. The anti-microbial property of the polyphenols could be one of the reasons for good storage qualities of the millet. The millet food is known for its hypoglycemic properties and this special quality may be contributed by the seed coat components such as dietary fibre and polyphenols which are known to bind to the digestive enzymes and slow down the carbohydrate digestibility (Goldstein Jl & swain T., Phytochemistry, 4, 185-192, 1965; Tamir M & Alumot E., Journal of Science and Food Agriculture, 20, 199-202,1969). It has also been recorded that the incidence of gastrointestinal disorders with special reference to duodenal ulcer is rare among regular millet eating population (Tovey F.I., Tropical and Geographical Medicine, 24, 107-117,1972)). Although, these benefits were known traditionally no efforts have been made to identify the components of the millet seed coat contributing towards its pharmaco-nutritional characteristics to the best of our knowledge. Isolation of phytochemicals including polyphenols is being carried out for various food and allied uses but the drawbacks of hitherto known processes are that, majority of the processes confine to vegetables and medicinal plants (Burns RE., Agron. Journal, 63, 511,1997; Maxson E., & Rooney LW., Cereal Chemistry, 49, 719,1972; Jones WT., Sbroadhurst RB.. & Lyttleton JW., Phytochemistry, 15, 1407, 1976; Leung J.Fenton TW, Mueller MM, & Clandnin DR, Journal of Food Science, 44, 1313, 1979) and not to cereals in general and millets in particular. However, few reports pertaining to cereals such as sorghum, millets and barely confine mainly to assaying of polyphenols, that too following the extraction by cold acidic methanol. The drawbacks of such methods is that, only a part of the phenolics could be extracted. Yet another drawback of hitherto known process is that most of the methodologies utilize whole seeds and not the seed coat matter alone and as a result the volume of solvent needed for extraction is much higher than that for seed coat alone. Besides, the residual part of the cereals may not be edible. In view of this, the main object of the present invention is to isolate the phytochemicals rich in polyphenols by suitable solvent systems. Another object of the present invention is to concentrate the polyphenols from the isolated phytochemicals. Yet another object of the present invention is to examine and evaluate the antimicrobial properties of the isolated phytochemicals enriched with polyphenols. Accordingly a process for preparation of polyphenols from finger millets which comprises; a) mixing the finger millet powder having less than 200 micro particle size in a solvent consisting of 0.5 - 3 parts mineral acid and 99.5 - 97 part of methanol followed by refluxing the above said mixture till the extract becomes colorless, b) concentrating the extract at 30-50°C and 200-400 mbar vacuum by evaporating the methanol, c) neutralizing the concentrate by known methods as described herein, d) drying the above obtained polyphenol rich precipitate to get pure polyphenol. Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that, the variations, which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described therein, will become apparent to those skilled in the art from the pre - going description. Such modifications are intended to fall within the scope of the invention and appended claims. The following examples are given by way of illustration of the invention and therefore should not be construed to limit the scope of the present invention. Example 1 The seed coat of the native finger millet (1 kg) was pulverised to particle size less than 250 microns. The pulverized seed coat (100g) was mixed with 1L acidic methanol, containing concentrated hydrochloric acid and methanol in the ratio of 1:99 (v/v). The contents were refluxed using a boiling water bath for 30min. The extract was filtered using Whatman.No 1 filter paper and the residue was again refluxed with fresh 1 L of solvent for 30 min and the extract was filtered. The process was repeated 2 more times and the extracts were pooled together and concentrated at 40°C under 360 mbar vacuum till the methanol was fully evaporated. The concentrate was neutralized by saturated sodium bicarbonate solution to neutral pH and the contents were centrifuged to separate the supernatant from the precipitated salts. The supernatant and the extract were assayed for their polyphenol content (Folin - Dennis method, Swain T., & Hillis., Journal of Science and Food Agriculture, 10, 1959)). Subsequently, the extract was freeze-dried and its polyphenols content assayed. The polyphenol contents of seed coat from malted and hydrothermally treated millet were also extracted following the method detailed for native millet, and their polyphenol contents were assayed and tannic acid equivalent. Source of seed coat Polyphenol contents (g/100g) Native millet 1.10 Malted millet 0.70 Hydrothermally treated millet 0.85 Example 2 The seed coat of the native finger millet (1 kg) was pulverised to particle size less than 250 microns. The pulverized seed coat (100g) was mixed with 1L acidic methanol, containing concentrated hydrochloric acid and methanol in the ratio of 1:99 (v/v). The contents were refluxed using a boiling water bath for 30min. The extract was filtered using Whatman.No. 1 filter paper and the residue was again refluxed with fresh 1 L of solvent for 30 min and the extract was filtered. The process was repeated 2 more times and the extracts were pooled together and concentrated at 40°C under 360 mbar vacuum till the methanol was fully evaporated. The concentrate was neutralized by saturated sodium bicarbonate solution to neutral pH and the contents were centrifuged to separate the supernatant from the precipitated salts. The supernatant and the extract were assayed for their polyphenol content (Folin - Dennis method, Swain T., & Hillis., Journal of Science and Food Agriculture, 10, 1959)). Subsequently, the extract was freeze-dried and its polyphenols content assayed. The polyphenol^ sample was dissolved in double distilled water to prepare a solution containing 200 microgram in 20 microlitre water. The individual phenolic components of the solution were analyzed by HPLC on a reverse phase C-18 ODS (25cm * 4.6mm, 5nm) column, Shimadzu SCL 6A system controller, UV-VIS spectrophotometer detector, with methanol / 0.1%TFA in water as the eluent at a flow rate of 1ml/min with monochromatic detection at 295nm. The gradient program employed for the analysis is as follows. (Table Removed) The HPLC profile showed thirteen constituents with retention times of 2.89, 3 52, 4.87, 5.88, 9.50, 10.2, 13.9, 14.16, 15.3, 18.9, 19.33, 21.13, 21.67, 24.51min. Example 3 The seed coat of the native finger millet (1 kg) was pulverised to particle size less than 250 microns. The pulverized seed coat (100g) was mixed with 1L acidic methanol, containing concentrated hydrochloric acid and methanol in the ratio of 1:99 (v/v). The contents were refluxed using a boiling water bath for 30min. The extract was filtered using Whatman.No. 1 filter paper and the residue was again refluxed with fresh 1 L of solvent for 30 min and the extract was filtered. The process was repeated 2 more times and the extracts were pooled together and concentrated at 40°C under 360 mbar vacuum till the methanol was fully evaporated. The concentrate was neutralized by saturated sodium bicarbonate solution to neutral pH and the contents were centrifuged to separate the supernatant from the precipitated salts. The supernatant and the extract were assayed for their polyphenol content (Folin - Dennis method, Swain T., & Hillis., Journal of Science and Food Agriculture, 10, 1959). The polyphenols material was dissolved in double distilled water to contain 100 microgram per ml water and 5 microlite solution was injected and the solution was used for isolation of individual phenolics by preparative HPLC. Preparative HPLC was carried out on a C-18 (prep-ODS shim pack, 20 * 250mm) preparative column, Shimadzu SCL10A system controller, diode array detector, using an isocratic elution with methanol / 0.1%TFA (85:15) and at a flow rate of 10ml/min. Five major constituent fractions were collected (retention time interval of about 40min) with retention times of 5.57, 6.40, 7.16, 11.70, 28.27min. The total polyphenols extracted from the native millet seed coat and also the individual phenolics isolated by the preparative HPLC were examined for their antimicrobial activity. The anti-microbial activity was assessed against the food borne pathogens belonging to both gram +ve and gram -ve bacteria. The assessment of anti-microbial activity was by agar well diffusion assay technique. Freshly subcultured standard indicator microorganisms were individually incorporated into plate count agar (PCA) plates by pour plating method. Wells were cut at uniform distance in the agar plates and known quantities of various water-soluble extracts were placed inside each well and allowed to diffuse. The plates were then incubated at 37°C for 24 h. The anti-microbial activity of the extract was assessed by measuring the zone of inhibition surrounding each well. The food borne pathogens, namely, Bacillus cereus, Staphylococcus aureus, Listeria, monocytogenes, Escheritia coli, Yersinia enterocolitica and Helicobacter pylori were included as indicator microorganism. (Table Removed) Among the various phenolics isolated following preperative HPLC, fraction No. 2 and fraction No. 5 showed 16 mm and 11 mm microbial inhibitory zones respectively. The main advantages of the present invention are that, it utilizes the phytochemical rich material namely seed coat matter, which happens to be the by-product of millet processing industry. The process makes use of the simple technique of refluxing the pulverised seed coat with acidic methanol (1% HCI in methanol), which extracts almost all the polyphenol contents of the millet seed coat and which on neutralization doesn't form any toxic or health hazardous residues. The method permits precipitating out non-polyphenolic phytochemicals from the extract by simple adjustment of the acidity of the extract using any of the alkaline solutions, although, the total extract in dry powdery form or polyphenol rich material in solution form could be used for the antimicrobial activity. The other advantage of the process is that it can be applied to the seed coat matter from native, malted and hydrothermally treated or such other processed material which will be available at competitive price. The extract or solvent systems consisting of mineral acids and methanol are priced low and are readily available. Concentration of the extract including its preparation in dry form does not affect the polyphenol contents of the extract. The novelty of the process is that it makes use of the by-product of the millet processing industry and the polyphenols isolated could be used an independent entity or could be used as part of the neutral or active materials for enhancing the shelf-life of the food products or to improve the gastrointestinal health of the human subjects or as pharamaconutrient. It could also be applied to other food materials to improve their shelf-life and quality attributes. FLOW CHART EXTRACTION OF POLYPHENOLS (Figure Removed) FROM FINGER MILLET SEED COAT FLOW CHART EXTRACTION OF POLYPHENOLS FROM FINGER MILLET SEED COAT We claim; 1. A process for preparation of polyphenols from finger millets which comprises; a) mixing the finger millet powder having less than 200 micro particle size in a solvent consisting of 0.5 - 3 parts mineral acid and 99.5 - 97 part of methanol followed by refluxing the above said mixture till the extract becomes colorless, b) concentrating the extract at 30-50°C and 200-400 mbar vacuum by evaporating the methanol, c) neutralizing the concentrate by known methods as described herein, d) drying the above obtained polyphenol rich precipitate to get pure polyphenol. 2. A process for preparation of polyphenols from finger millets, substantially as herein described with reference to the examples.

Documents:

539-DEL-2003-Abstract-(02-01-2009).pdf

539-del-2003-abstract.pdf

539-DEL-2003-Claims-(02-01-2009).pdf

539-del-2003-claims.pdf

539-DEL-2003-Correspondence-Others-(02-01-2009).pdf

539-del-2003-correspondence-others.pdf

539-del-2003-correspondence-po.pdf

539-DEL-2003-Description (Complete)-(02-01-2009).pdf

539-del-2003-description(complete).pdf

539-del-2003-Drawings.pdf

539-DEL-2003-Form-1-(02-01-2009).pdf

539-del-2003-form-1.pdf

539-del-2003-form-18.pdf

539-DEL-2003-Form-2-(02-01-2009).pdf

539-del-2003-form-2.pdf

539-DEL-2003-Form-3-(02-01-2009).pdf

539-del-2003-form-3.pdf