Amino acid profile and protein quality in tuber and leaf of Coccnia abyssinica (Lam.) (Cogn.) accessions of Ethiopia

Abstract The protein content and amino acid profile of Anchote (Coccinia abyssinica) leaves and tubers were determined from ten different accessions taken from Debre Zeit Agricultural Research Center, Ethiopia. Crude protein content was determined by Kjeldahl method and amino acid profile was analyzed using performic acid oxidation and acid hydrolysis by ninhydrin‐derivatized analysis with amino acid analyzer. Crude protein content of Anchote tuber ranged from 10.70% ± 0.26% to 13.72% ± 0.10%, whereas the crude protein content in leaves were ranged between 30.38 ± 0.01% (“240407‐1”) and 35.42 ± 0.05% (“223109‐1”). Total amino acid content ranged from 45.12 to 62.89 and 67.31 to 75.69 g/100 g protein for tuber and leaf samples, respectively. The mean values of essential, conditionally essential and nonessential amino acids were 37.22 & 36.79%; 28.62 & 24.10%; and 34.16 & 39.11% for tubers and leaves, respectively. Arginine in tubers and glutamic acid in leaves ranked the highest of all amino acids; while the least dominant essential amino acid was methionine in both parts. Among the essential amino acids, leucine was dominant in all accessions tested with values ranged from 3.12 to 5.32 g/100 g protein in tubers and from 5.15 to 5.65 g/100 g protein in leaves. In general, the average amino acid content was higher in the leaves (71.08 g/100 g protein) compared to the tubers (51.11 g/100 g protein). The nutritional quality of Coccinia abyssinica leaves and tubers range as follows: total essential amino acids (TEAA)/ total amino acids (TAA) (37.57 & 36.82%), TEAA/total non‐essential amino acids (TNEAA) ratio (0.60 & 0.58), The predicted protein efficiency ratio (P‐PER) (1.22 & 1.80), Essential amino acid index (EAAI) (35.28 & 53.93%), Predicted biological value (P‐BV) (26.76 & 47.09%), Nutritional index (4.11 & 17.71%), and Amino acid score (73 & 108) for tuber and leaf sample, respectively. A significant variability was observed in protein and amino acid profile among accessions and plant parts, and the leaf part were found to be richer in protein content and associated nutritional quality.

principally categorized under root and tuber crops (Holstein, 2012). Its newly growing leaves along with the tendrils are also used as nutritious vegetable served after being cooked (Abera, 1995). The tuber is prepared in different ways for consumption; cooked and served with a fermented spice prepared from coriander (Coriandrum sativum), sweet basil (Ocimum basilium), ginger (Zingiber officinale), garlic (Allium sativum) and salt, and also prepare as a soup after drying and grinding into powder (Habtamu & Kelbessa, 1997). It is also cooked for special occasions and holydays in sliced form and pounded after mixing with plenty of butter and spices (Abera, 1995;Asfaw, 1997;Habtamu & Kelbessa, 1997). The crop has appreciable nutritional composition mainly of protein and calcium (Habtamu, Fekadu, & Gullelat, 2013;Habtamu & Kelbessa, 1997).
Anchote grows in wide environmental conditions from drier to cooler regions of Western and South Western region of Ethiopia (Endashaw, 2007). This makes the crop to be a potential food security crop. However, Anchote did not get adequate attention in terms of improving its productivity, and hence it has remained as one of underutilized crops in Ethiopia. So far, there has been little effort made to undertake varietal development to identify suitable cultivars with different desirable traits adaptable to the different agro-ecological zone of Ethiopia, which makes its use to be limited to specific regions.
Research output on Anchote especially on its nutritional value is very limited and lack of scientific information on this crop is a common problem (Daba, Derebew, Wesene, & Waktole, 2012;Tilahun, Sentayehu, Amsalu, & Weyessa, 2014). The scanty information about the nutrition content including amino acid profile on the available Anchote accessions coupled with lack of awareness about the crop itself still makes it untapped. Information on the amino acid profile of the C. abyssinica accessions grown in Ethiopia is not available. Therefore, this study was conducted to evaluate the amino acid profiles and protein quality of tuber and leaf parts of five ex situ conserved accessions of Ethiopia.

| Sample preparation
Anchote tuber and leaf samples were harvested from Debre Zeit Agricultural Research Center experimental field from November 2011 to January 2012. Three healthy tubers from each accession were washed, peeled, and sliced using knife into small pieces and mixed thoroughly in order to prepare 400 g of samples which were placed in a paper bag and dried to a constant weight in a hot air oven (DHG-9055A, Memment Germany) set at about 105°C. To prepare the leaf samples, 200 g of newly growing tips of leaves were cleaned and chopped into small pieces and oven dried at 70°C to a constant weight. The oven dried leaf and tuber samples were then milled to fine powder using an electrical miller (FW 100, Yusung Industrial Ltd, China). The powder was sieved using 0.425 mm mesh size. Finally, the dried powder samples were put into paper bags and packed with airtight polyethylene bags to store it in a refrigerator at 4°C until further analysis.

| Crude protein determination
Crude protein content was estimated by the Kjeldhal method according to AOAC, (2000) using the official method 979.09. Accurately weighed 0.5 g sample was digested with a known quantity of concentrated H 2 SO 4 (Sigma-Aldrich, USA) in the Kjeltec digestion apparatus (Gerhardt vapodest, Germany). The digested material was distilled after the addition of alkali. The released ammonia was collected in 4% boric acid Kjeltec Automatic Distilling Unit. The resultant boric acid contained the ammonia released from the digested material, and then titrated with 0.1N hydrochloric acid (HCl) (Sigma-Aldrich, USA). The protein content was determined by multiplying the nitrogen content by a factor of 6.25.
Norvalene was used as an internal standard to normalize the recovery of each amino acid from injection to injection. The method was calibrated over the range of 0.08%-22.7% for each amino acid.
Tryptophan (Trp) was not analyzed for the reason that acid hydrolysis results complete destruction of tryptophan and requires an alternative hydrolysis procedure for accurate quantification (Wathelet, 1999).

| Evaluation of protein quality
Nutritional qualities of the protein in the leaf and tuber samples of Anchote were determined based on the obtained amino acid profiles.
The parameters determined were as follows: The proportion of total essential amino acids (TEAA) to the total amino acids (TAA) of the protein was calculated using the method of Chavan, McKenzie, and Shahidi (2001).
Amino acid score of the essential amino acid composition was calculated according to Chavan et al. (2001).
Where: n = number of essential amino acids, a, b …..j = represent the concentration of essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine,) in the tested sample and av, bv…..jv = content of the same amino acids in standard protein (%) (egg or casein), respectively.
The predicted protein efficiency ratio (P-PER) calculated by the regression equations as cited by Mune et al. (2011).
The nutritional index was calculated according to Ijarotimi and Keshinro (2013).

| Amino acid composition
Proteins are composed of different amino acids and hence the nutritional quality of a protein determined by the content, proportion, and availability of its amino acids (Becker, 2007). The result for amino acid profile of five Anchote accessions selected based on their protein content is presented in Table 2.
The amino acids profile of Anchote tuber showed that Arg (6.50-9.52 g/100 g protein) was the highest, while Met (0.30-0.40 g/100 g protein) was the least in concentration for four accessions, "223097", "223087-1", "223085", and "223090-1". Whereas, in accession "NJ" Asp (7.42 g/100 g protein) was the highest and Pro was the least (0.60 g/100 g protein) in concentration. In Anchote leaf, Glu (7.87-10.47 g/100 g protein) scored the highest value except in accession "KICHI" where Asp (9.35 g/100 g protein) was the most abundant, Leu was the dominant essential amino acid in all Anchote accessions ranged from 3.12 to 5.32 g/100 g protein for tuber and from 5.15 to 5.65 g/100 g protein for leaf. Accession "NJ" in tuber and "240407-1" in leaf were recorded the highest Leu content. Met was T A B L E 2 Amino acid composition in selected five accessions of Anchote tuber and leaf powder (g/100 g protein dry weight basis) the least in concentration among all essential amino acids in both tuber and leaf part, which was in agreement with germplasm accessions of Dioscorea species (Babu et al., 2007) and sweet potato cultivars (Van Hal, 2000). Arg was the most abundant amino acid among conditionally essential amino acids of all accessions in tuber part and in one of the accession evaluated for leaf part ("KICHI") with values ranging from 6.28 to 9.52 g/100 g protein. Gly was the highest amino acid in leaf of Anchote for the rest of accessions. With regard to nonessential amino acids Glu was dominantly found in tuber (5.23-5. 62 g/100 g protein) and leaf (7.87-10.47 g/100 g protein) with the exception of accession "223090-1" and "NJ" in tuber, and "KICHI" in leaf revealed Asp the highest of all nonessential amino acid. These results are comparable with most vegetable protein (El-Adawy, Rahma,  (Akubugwo et al., 2007;Aremu, Olaofe, & Akintayo, 2006;Hassan & Umar, 2006).
The total amino acid (TAA) content of Anchote ranged from 45.12 to 62.89 g/100 g protein in tuber and from 67.31 to 75.69 g/100 g protein in leaf. The amino acid content was higher in leaf (71.08 g/100 g protein) compared to the tuber (51.11 g/100 g protein). This could relate to the highest crude protein content that was recorded for leaf (35.42%) compared to the tuber (13.72%). This observation is agreed with the report that states leaves and vines of sweet potato were high in total amino acids than the tubers (Kenyon, Anandajayasekeram, Ochieng, & Ave, 2006).
A balanced or high-quality protein contains essential amino acids in ratios commensurate with human needs. This can be determined by comparing the amino acid contents of various proteins with the FAO reference pattern. The FAO reference pattern based on the essential amino acid requirements of young children (1-2 years) is considered the preferred reference protein (Cheftel, Cuq, & Lorient, 1985). Thus, the average proportions of the essential amino acid profile of Anchote tuber and leaf were compared with the (WHO, 2007) reference pattern for the preferred age group as shown in Table 3.
All the essential amino acids were found in both tuber and leaf of Anchote except tryptophan (Trp), which was not determined in this study. Met and His were found in limited amount for tuber and leaf part, and this limitation might be explained by two possible reasons; they might be denaturized during analysis or their values are very limited in Anchote. The low availability of Met is in accordance with the previous studies (Montagnac et al., 2009;Van Hal, 2000). To compensate this limitation in Anchote, additional consumption of animal or plant proteins such as milk, egg, lentils, and pulses are highly recommended (Andini, Yoshida, & Ohsawa, 2013).
Essential amino acids Ile, 3.70; Thr, 3.46; sulfur containing amino acids (SAAs) 4.20; and Aromatic amino acids (AAAs), 4.73 g/100 g protein in leaf, and Ile,3.14 and Thr, 3.06 g/100 g protein in tuber of Anchote were higher than the reference standards (WHO, 2007) (Ile 3.10;Thr, 2.70; SAAs, 2.60 and AAAs, 4.60 g/100 g protein). These results suggests that Anchote can be exploited for those essential amino acids which are found in adequate amount in either of its edible part to enhance protein quality especially when preparing weaning/ complimentary food products.

| Protein quality
The nutritional quality of a food protein depends on the kinds and amounts of amino acids it contains, and represents a measure of the efficiency with which the body can utilize the protein (Chawanje, Barbeau, & Grün, 2001). The protein quality of Anchote tuber and leaf were determined based on their amino acid profile and presented in Table 4. In Anchote leaf, the content of SAAs (Met + Cys) was 4.20 g/100 g protein and in its tuber, it was 2.07 g/100 g protein.
Whereas, the P-BV of the leaf was higher than that of beach pea protein isolates (36.5%-40.13%), raw popcorn flour (36.45%), flour blends made from fermented popcorn-bambara groundnut (32.69%) and fermented popcorn-African locust bean-bambara groundnut (39.94%) (Chavan et al., 2001;Ijarotimi & Keshinro, 2011. The P-BV obtained from Anchote leaf was in agreement with the suggested biological value (45%) for plant-based proteins (Ogundele et al., 2012). The nutritional index for Anchote tuber was 4.11%, whereas for the leaf part it was 17.71%. Anchote leaf nutritional index was higher than formulated complementary food (5.98%-12.73%) of plant-based protein (Ijarotimi & Keshinro, 2013). The amino acid score is the ratio of the amino acid content in the sample protein to the content of the same amino acid in the requirement pattern. The amino acid score of Anchote tuber (73) was lower when compared to beach pea protein isolates (108-110), whereas the content in Anchote leaf (108) had a similarity with this report (Chavan et al., 2001).

| CONCLUSION
The study investigated the protein content, amino acid profile, and nutritional quality of leaf and tuber samples from different Anchote accessions. The leaf sample was ranked best compared to the tuber sample in crude protein and amino acid content as well as protein quality. Anchote can be exploited for those essential amino acids (Leu, Ile, Thr, SAAs, and AAAs) which are found in adequate amount in either of its edible part to enhance protein quality especially when preparing plant-based weaning/complimentary food products. The dominant essential amino acid was Leu in all Anchote accessions and accession "NJ" in tuber and accession "240407-1" in leaf was recorded the highest Leu content. Met and His were found in limited amount in both tuber and leaf part. The amino acid composition also varies among accessions in both tuber and leaf samples. Therefore, through selection and hybridization of protein-rich accessions it can be possible to overcome low level of protein. Moreover, genetic modification can be applied to improve the availability and quality of protein.
T A B L E 4 Estimated nutritional quality of protein for Anchote tuber and leaf samples based on their amino acid profile