Amino acid composition of wild yam ( Dioscorea spp . )

Tubers of wild yam (Dioscorea alata, D. bulbifera var. vera, D. esculenta, D. oppositifolia var. oppositifolia, D. oppositifolia var. dukhumensis, D. pentaphylla var. pentaphylla, D. spicata, D. tomentosa and D. wallichi) were analyzed for its amino acids composition. Asparatic acid and glutamic acid were found to be predominant among all the investigated Dioscorea species which ranged from 5.21 to 9.36 and 3.20 to 8.12 g/100 g protein respectively. The essential amino acids such as isoleucine, phenylalanine tyrosine, histidine, isoleucine and valine were found to be more or less higher than FAO/WHO (1991) requirement pattern. Thus, the present investigation demonstrated that the Dioscorea spp. can act as a good source of amino acid.


Introduction
Since time immemorial in the tropics and subtropics, roots and tubers are the most important food crops (Behera et al., 2009).In developing countries, the nutritional value of roots and tubers lie in their potential ability to provide one of the cheapest sources of dietary energy in the form of carbohydrates (Ugwu, 2009).The genus Dioscorea (Yam) belongs to the family Dioscoreaceae.It comprises 35-400 species (Caddick et al., 2002) and is distributed throughout the tropic and subtropic regions especially in West Africa, parts of Central America and the Caribbean, the Pacific islands and Southern Asia.Dioscorea has been suggested to have nutritional superiority when compared within the tropical root crops.They are reported as good sources of essential dietary nutrients (Bhandari et al., 2003;Shanthakumari et al., 2008;Maneenoon et al., 2008;Arinathan et al., 2009;Shajeela et al., 2011).
These wild yams make an important contribution to the diets of the tribal people of India.The tubers are found with a high amount of protein, good properties of essential amino acids and appeared as a fairly good source of many dietary minerals.However, their wide utilization is limited due to the presence of some toxic and anti-nutritional factors.In India, the cooked wild tubers are known to be consumed by the Palliyar and Kanikkar tribe (Arinathan et al., 2007;Shanthakumari et al., 2008;Shajeela et al., 2011) living in South-Eastern slope of Western Ghats, Tamilnadu.The information on the subject of the chemical and nutritional content of wild edible tuber is not enough (Babu et al., 1990;Nair and Nair, 1992;Rajyalakshmi and Geervani, 1994;Shanthakumari et al., 2008;Alozie et al., 2009;Anatharaman et al., 2009;Mohan and Kalidass, 2010;Shajeela et al., 2011).In spite of their importance as a food source, to the best of our knowledge, there are no published studies on the amino acid composition of wild yam tubers consumed by Palliyars and Kanikkars tribes, South-Eastern slopes of Western Ghats, Tamilnadu and also information on the amino acid composition of wild yam is scarce.The present study was therefore initiated to evaluate the amino acid profile of Dioscorea spp.for the first time.

Materials and methods
Plant sample: Nine samples of wild yam tubers (D. alata, D. bulbifera var. vera, D. esculanta, D. oppositifolia, var. oppositifolia, D. oppositifolia var. dukhumensis, D. pentaphylla var. pentaphylla, D. spicata, D. tomentosa and D. wallichi) grown in sandy loam soil consumed by the tribals Kanikkars/Palliyars were collected using multistage sampling technique in three consecutive rainy seasons during August and January 2016 from the South Eastern slopes of Western Ghats, Virudhunagar district, Madurai District, Kanyakumari district, Tirunelveli district in Tamilnadu.The plant specimens were identified with the help of local flora and authenticated by Botanical Survey of India (Southern Circle), Coimbatore.

Amino acid analysis
The total protein was extracted by a method of Basha et al. (1976).By precipitation with cold 20% trichloroacetic acid (TCA) the extracted proteins were purified.In an evacuated sealed tube a protein sample of 30 mg was hydrolysed by 6N HCl (5mL), which was kept in a hot air oven at 100°C for 24 hrs.The sealed tube was broken.After the addition of de-ionized water, the acid was removed completely by repeated flash evaporation.Dilution was effected by means of citrate buffer pH 2.2 to such an extent that the solution contained 0.5 mg protein ml.The solution was passed through a Millipore filter (0.45iM) and by using an automated pre-column (OPA) the solution was derivatized with O-phthaldialdehyde.Amino acids were analysed by a reverse-phase HPLC (method L 7400, HITACHI, Japan) fitted with a Denali Cig 5 micron column (4.6 X 150 mm).The flow rate was 1 ml min with fluorescence detector.Using the Liddelle and Saville (1959) method, the cystine content of protein sample was obtained separately.For the determination of tryptophan content of proteins, aliquots containing known amounts of proteins were dispersed into glass ampoules together 1 ml 5M NaOH.The ampoules were flame sealed and incubated at 110°C for the 18 hrs.Using the method of Spies and Chambers, (1949) as modified by Rama Rao et al. (1974) the tryptophan contents of the alkaline hydrolysates were determined colorimetrically.The contents of the different amino acids were expressed as 100 g proteins and were compared with FAO / WHO (1991) reference pattern.The essential amino acid score was estimated as follows: Essential amino acid score = grams amino acid in l00 g of total protein/ grams of essential amino acid in 100g of FAO, WHO (1991) reference pattern x 100.

Results and discussion
The amino acid composition of the Dioscorea species is summarized in Table 1.Glutamic and aspartic acid was the most abundant amino acid with values ranging from 5.21 to 9.36 and 3.20 to 8.12 g/100 g protein respectively.Among the species identified D. oppositifolia var.dukhumensis contain a higher amount of glutamic acid and similarly, D. alata contain a higher amount of aspartic acid.These values were comparable with D. bulbifera, D. deltoidea, D. versicolor, D. triphylla (Bhandari et al., 2003), D. rotundata (Ogunlade et al., 2011) and D. dumenforum (Alozie et al., 2009).
Aspartic and glutamic acids are dispensable amino acids since most of the dispensable amino acids derive their alpha-amino groups from glutamic acid which, in turn, has alpha ketoglutamic acid and ammonia as its precursors.Equally, aspartic acid is a precursor of such essential amino acid as asparagine, methionine, threonine and lysine (Berg et al., 1992).In the presenting investigation of Dioscorea species the sulphur containing amino acid (cystine and methionine) was the most lining amino acid followed by tryptophan and lysine which agreed with the report of Osagie (1992).
As per the FAO/WHO (1991) requirement pattern the following eleven amino acid threonine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and tryptophan have been registered as essential amino acids.Generally, in most of the present investigation, Dioscorea species deficiency of the essential amino acids via, threonine, valine, sulphur containing amino acids leucine, lysine and tryptophan are noted when compared with FAO/WHO (1991) requirement pattern (Table 2).In the presently investigated Dioscorea species the essential amino acids threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine and histidine are found to be lower when compared with early reports, the tubers of Manihot utilissima and Dioscorea alata (Ciacco and D' Appolonia, 1978) and Dioscorea dermentrum varieties (Alozie et al., 2009).
Table 3 shows the essential amino acid (EAA) score of Dioscorea species herbs studied.The results show that among the Dioscorea species tribes studied, D. wallichi was lowest the EAA score.The sulphur contain amino acids (Met + Cys) and lysine turned out to be the most limiting all species with EAA score ranging between 44.0-63.2 and 21.37 to 50.69 respectively.Sulphur containing amino acids were the limiting one in all analysed wild yam tubers.These results agree well with the study results reported by Splittstoesser et al. (1973) and Bhandani et al. (2003).In general, the essential amino acids such as phenylalanine, tyrosine, histidine, isoleucine and valine in Dioscorea species tuber have a high essential amino acid score, which implies that essential amino acids present in these tubers have high biological value.physiological effects in consumers.Alkaloids give protection to plants from a variety of herbivores and some of them possess important pharmacological activity such as analgesic, antibacterial and antibiotic.In conclusion, the findings of this study suggest that the wild Dioscorea species are rich certain amino acid and non-essential amino acids.However, these Dioscorea species could be used as alternatives to cereals in human and animal nutrition or in livestock industries since they compare favourably in their amino acids compositions.

Table 2 .
Essential amino acid (EEA) composition of Dioscorea species compared to the FAO/WHO reference pattern

Table 3 .
Essential amino acid (EEA) score of Dioscorea species a-FAO/WHO 1991 reference pattern