Amino acid content , fatty acid content and anti nutritional factor of seeds of new hybrid varieties of Echinochloa frumentacea ( Sanwa ) minor millets

Cereals are the staple diet of most of the world’s population. The millets are very important staple food in the rural parts of India. Millets can secure India’s food and farming in future because it is amazing in their nutrition contents. Echinochloa frumentacea (Sanwa) millet is good source of energy and provide protein, fatty acid, minerals, vitamins, dietary fibre and polypheonals. Proteins present in various foods differ in their nutritive value on account of the difference in the amino acid contents. The amino acid content, fatty acid content (TSFA and TUFA) and anti nutritional factor ranged from 0.0008 to 0.522%, 24.2 to 26.0%, 73.5 to 75.4% and 0.301 to 0.302, 0.0202 to 0.0204 g/100 g and 31.95 mg/100 g respectively. No cyanide content and haemagglutinin activity were found. Nutritionally the seeds of E. frumentacea variety DFM-1 and HR-374 are rich in aspartic acid (essential amino acid) content and total unsaturated fatty acid content.


INTRODUCTION
Millets are a group of cereal species crops or grain like food that has been used by large group of people in rural, tribal and hilly areas in Asia and Africa (Ravindra et al., 2008;Anonymous, 2006;Rao et al., 2011;Odoemalam and Osu, 2009).Millet is a cereal crop plant belonging to different genera but all within the grass family, Poaceae and subfamily Panicoideae (FAO, 1972;1991).As the minor millets are consumed by the poor, they guard them against food and nutritional insecurity imposed by various agronomic, socio economic and political factors.Minor millets can thus act as a shield against nutritional deficiency disorders and provide nutritional security.These grains will be used for traditional as well as novel foods (Vanithasri et al., 2012).All of them are small seeded grasses having high capability of resistance to extreme environmental conditions in which major cereals fail to give substantial yields (Amadou et al., 2013;McDonoug et al., 2000;Black et al., 2006;Ahmed et al., 2013).Echinochloa frumentacea minor millets are high energy, nutritious foods comparable to other major cereals and some of them are even better with regard to protein and mineral content (Fe, Ca, Mn, Mg).Fat is one of the major nutrients which provide energy, promote body growth, maintain and repair body tissue, promote reproduction and lactation and regulate body process.Fats are carriers of fat soluble vitamins.Dietary fat must also provide essential fatty acids (EFA) which are the functional components of membrane lipids and have other important metabolic function.Fats are made up of fatty acids which include saturated fatty acids like palmitic and stearic, monounsaturated fatty acids (MUFA) like oleic and polyunsaturated fatty acid (PUFA) likes linoleic acid and linolenic acid (Singhai and Shrivastava, 2002;Nagraj, 1995).Lipids are relatively minor constituents in cereal grains; however, they contribute significantly to diet as a source of invisible fat and essential fatty acid (Achaya, 1986(Achaya, , 1987)).Nevertheless E. frumentacea represents a good source of essential amino acid and essential fatty acid (linoleic acid and linolenic acid).However, it must be pointed out that, E. frumentacea also contains some anti-nutritional factors which inhibits proteolytic and amylolytic enzymes, limits mineral, protein and starch digestibility and makes poor human bioavailability of proteins.
This study was therefore conducted to assess the levels of amino acid content, fatty acid content and antinutrinational factors in the seeds of BMVL-29 and BMVL-172 variety of E. frumentacea for awareness and exploitation.

MATERIALS AND METHODS
In the present study two new hybrid, authentic, healthy and matured seeds of minor millets viz., E. frumentacea (variety BMVL-29 and BMVL-172), under investigation were procured from Agriculture Research Station of Jawaharlal Nehru krishi Vishwavidyalaya, Dindori (M.P.) and were studied for their amino acid, fatty acid and antinutritional factors.

Amino acid analysis
The amino acid composition of seeds of hybrid variety BMVL-29 and BMVL-172 of E. frumentacea was analyzed by using liquid chromatography mass spectroscopy (LC-MS).

Solvent extraction and sample preparation
Solvent extraction was done by Soxhlet apparatus and stock solution was prepared by dissolving 10 mg of each amino acid in 100 ml of diluents (acetonitrile/formaic acid) and it was properly shaken.Working standard solution of 1 mg/L was prepared by this stock solution.

LC-MS analysis
LC-MS analysis of sample was done by using C18 column (Brava Amino 5 µ, 4.6 × 250 mm).Column temperature was maintained at 40°C. 10 µl of sample was injected for 10 min, 0.1% Formic acid in water and 0.1% Formic acid in acetonitrile (95+5) were used as mobile phase and its flow rate was 0.8 ml/min.Ionization of sample component were performed on electron spin resonance (ESR) mode (70 eV).

Fatty acid analysis
The hybrid variety , of E. frumentacea seeds were studied for their fatty acid composition by gas chromatography.Powdered sample of experimental seeds were subjected to solvent extraction in Soxhlet apparatus for 20 h, using petroleum ether (40 to 60°C) as solvent.Lipids were then estimated gravimetrically by the method of Colowick and Kaplan (1957).Methyl esters of the lipids were prepared by the method of Chowdhary et al. (1984) and analysed by gas liquid chromatogram (GLC).Gas chromatograms were recorded using flame ionization detector (FID) with split ratio 1:50.

Antinutritional factors
The seeds of E. frumentacea variety BMVL-29 and BMVL-172 were studied for their tannin content, oxalate content, trypsin inhibitor activity, cyanide content and haemagglutinin activity.Cyanide and tannin contents of seeds were determined by the method of AOAC (1970).The total oxalate content in the form of oxalic acid was determined by using the method of Talpatra et al. (1948).Trypsin inhibitor activity was determined according to the method as described by Kakade et al. (1969) with certain modifications by Gupta and Deodhar (1975).Haemagglutinin activity was determined by the method as given by Liener (1955).

RESULTS AND DISCUSSION
The results of amino acid composition of seed protein of E. frumentacea variety BMVL-29 and BMVL-172 are given in the Table 1.The seeds of E. frumentacea variety BMVL-29 and BMVL-172 were found to have highest amount of Aspartic acid (0.522%), whereas Lysine content was reported 0.047 and 0.046% in E. frumentacea variety BMVL-29 and BMVL-172 respectively.In both the variety of E. frumentacea other amino acid in the decreasing order were glutamic, methionine, L-omithine HCl, alanine, arginine HCl, DL-Tryptophan, serine, glycine, proline = valine, threonine, tyrosine, phenylalanine, leucine = L-Hydroxyproline>isoleucine.
From the perusal of the data it appears that both the varieties of E. frumentacea minor millets seeds are lacking in Cystine, Histidine, 2-Aminobutaric and Lcysteine amino acids.It has been found that the amount of aspartic acid was maximum while the quantity of isoleucine was minimum but methionine levels of these variety of minor millets was more than the amount present in cereal grains.Methionine is of special importance to animals as a therapeutic and nutritional factor.It protects animals against liver injuries by chloroform, industrial halogenated fumes, and protein deficient diets and prevents the great loss of body nitrogen in the case of fractures, burns and surgical operations (Crocker and Barton, 1952).However, the amino acid composition of seed protein of both the variety (BMVL-29 and BMVL-172) of E. frumentacea under study was found to be in general accordance with reported values (FAO, 1970;Glew et al., 2008;Hui, 1996).Tables 2 and 3 showed the variation of fatty acid content of hybrid E. frumentacea variety BMVL-29 and BMVL-172.The saturated fatty acid, Caprylic acid was found to be highest (2.1%) in variety E. frumentacea BMVL-29 and lowest (1.1%) in variety E. frumentacea BMVL-172.The Palmitic acid content was reported higher (17.1%) in variety Echinochloa frumentacea BMVL-172 and lower in the variety E. frumentacea BMVL-29.The percentage of Stearic acid was found maximum (6.1%) in the variety E. frumentacea BMVL -172 and minimum (5.0%) in the variety E. frumentacea BMVL-29.The Arachidic acid was found to be maximum (1.1%) in the variety E. frumentaca BMVL-172, while minimum in the variety E. frumentacea BMVL-29.The variety E. frumentacea BMVL-172 has maximum (0.4%) Bahenic acid content while minimum (0.3%) in the variety of E. frumentacea BMVL-29.The total saturated fatty acid (TSFA) content was to be greater (26.0%) in the variety of E. frumentacea BMVL-172 than the variety E. frumentacea BMVL-29(24.2%).
The unsaturated fatty acid, E. frumentacea variety BMVL-172 contain maximum amount (29.5%) of Oleic acid (MUFA).Whereas the Linoleic acid was found to be highest (46.9%) in the variety E. frumentacea BMVL -29.The Linolenic acid and Ecosenoic acid content was found to be greater (1.0 and 0.5% respectively) in the variety E. frumentacea BMVL-29 than the variety E. frumentacea BMVL-172 (0.7 and 0.4% respectively).The variety E. frumentacea BMVL-29 contain minimum value of mono unsaturated fatty acid (MUFA) and maximum value of polyunsaturated fatty acid (PUFA).Total unsaturated fatty acid (TUFA) content was found to be highest (75.4%) in the variety E. frumentacea BMVL-29 and lowest (73.5%) in the variety E. frumentacea BMVL-172.E. frumentacea variety BMVL -29 was found to be superior to the variety E. frumentacea BMVL-172 under investigation.It contains highest content of linoleic acid (46.9%).The amount of linoleic acid in millet oil is higher in comparison with most other types of vegetable oils (Ravindra et al., 2008).The linoleic acid is one of the most important polyunsaturated fatty acid in human food, because of its prevention of distinct heart vascular disease (Boelhouwer, 1983).This acid is most important essential fatty acid required for growth, physiological function and maintenance, which cannot be synthesized by the human body and one, has to depend on dietary source for their adequate supply.The body metabolizes linoleic and linolenic acid into arachiodonic acid and docosahexaenoic acid (DHA) respectively which are essential to the normal development of central nervous system (Brich et al., 2007;Jacobson et al., 2008).Various developmental problems including attentiondeficit/hyperactivity disorder (ADHD) in children have been linked to biological deficiencies in polyunsaturated fatty acids.Additionally, there is evidence that symptoms may be reduced with PUFA supplementation (Sinn and Bryan, 2007).
The result of anti nutritional factors of the E. frumentacea varieties are shown in Table 4.The tannin content of different varieties of E. frumentacea ranged from 0.301 to 0.302 g/100 g.These values are lower than the earlier findings of Pasala and Bjorn (1989) and are well below the fatal dose (Sarjekar and Shrivastava, 1994).The total oxalate content (in terms of oxalic acid) was found to be maximum (0.0204 g/100 g) in the variety of E. frumentacea BMVL-172 while it was minimum (0.0202 g/100 g) in the variety of E. frumentacea BMVL-29.
The Trypsin inhibitor activity was found to be maximum (31.95 mg/100 g) in the variety E. frumentacea BMVL-29.However, no trypsin inhibitor activity was reported in the variety of E. frumentacea BMVL-172.No Cyanide content and haemagglutinin activity were found in the varieties of E. frumentacea under study.The value of anti nutritional factors reported in study was lies within the leather dose.These anti nutritional factors may be reduced by simple soaking, heating and germination or fermentation.It is now established that phytates, polyphenols and tannins can contribute to antioxidant activity of the millet foods, which is an important factor in health, aging and metabolic diseases (Bravo, 1998).

Conclusion
Cereals and millets constitute a major component of diet consumed in developing countries like India.E. frumentacea (Sanwa) millets are the staple food for millions of poor people in the world.The seed of E. frumentacea (variety BMVL-29 and BMVL-172) millets contain significant quantities of essential amino acids particularly the sulphur containing amino acid (methionine), essential fatty acids (PUFA and MUFA) and leather amount of antinutritional factor.It will be a useful and economical source of protein provided that some legume or milk is consumed along with these minor millet, that is, suitable for good nutritional supplementation.Variations in the various constituents of the E. frumentacea millets seeds have been attributed to variety, conditions, fertilizer treatments and climatic conditions.Most of the anti nutritional factors are heat-labile and since only humans consume millets after cooking, it would not constitute any major health hazard.It can be concluded that nutritional benefit of.E. frumentacea minor millets can be enhanced when all type of processing treatments employed at domestic levels were effective in reducing the biological active factors and therefore could be used to enhance better quality in food materials.Compared to rice and wheat, minor millets contain little high amount of anti nutritional factors.But these anti nutritional factors are plant based phyto chemicals that possess therapeutic qualities and hence are recommended by doctors for various diseases.Diabetics need to control their blood sugar, hypertension as well as cholesterol level and that is why doctors recommend minor millets for these problems.
*ND-not detected, * The values given in the tables are the mean of the triplicate values obtained.