Sky Journal of Food Science Vol. 6(4), pp. 041 - 047, October 2017 Available online http://www.skyjournals.org/SJFS ©2017 Sky Journals Full-Length Research Paper Evaluation of the anti-nutritional properties and amino acid profile of soaked and germinated Kpaakpa (Hildegardia barteri) seed flour using response surface methodology Anyadioha, Josephat Ikechukwu1, Okoli, Eric Chigozie2, Attaugwu, Roseline Nwabugwu2, 1 Department of Food Science and Technology, Madonna University, Nigeria, Akpugo Campus. Department of Food Science and Technology, Ebonyi State University, Abakaliki. 2 Accepted 29 September 2017 The effects of soaking and germination on the anti-nutritional composition and amino acid profile of the Kpaakpa (Hildegardia barteri) seed flour were investigated. The seeds were soaked for 12, 24 and 36 h and allowed to germinate for 2, 4 and 6 days respectively. The germinated seeds were dried milled into flour and analyzed for anti-nutrient composition and amino acid profile. Results were statistically analyzed and fitted into a second-order polynomial equation. A face-centred response surface method using three level-two factors full factorial central composite design was employed to optimize the process parameters. The anti-nutrient composition was generally reduced by both soaking and germination. The multiple regression model developed showed that trypsin inhibitor was minimized at 36 h soaking and 5.7 days germination with the desirability of 0.9999 which showed good interaction between the variables. The amino acid profile showed that soaking and germination enhanced nutritionally useful essential amino acids including sulphur-containing amino acids such as methionine and cysteine when compared with the control sample. Keywords: Soaking, germination, Hildegardia barteri, anti-nutrients, amino acids. INTRODUCTION Legumes refer to edible seeds of leguminous plants belonging to the Leguminosae family. Generally, legumes are the main plant source of proteins in a human diet containing 17-25% except for soybeans which contain 40% protein. They are also rich in dietary fibre, carbohydrates, lipids and minerals (Tharanathan and Mahadevamma, 2003). Minor compounds of legumes are polyphenols and bioactive peptides (Rocio Campos-Vega et al., 2009). Kpaakpa seed plant is a tropical leguminous plant in the family of Steruliacea which is grown mostly in the semi-arid forests with other plants. The plant grows from Ivory Coast to Nigeria. It is a less utilized legume that contains 17.5% crude protein, 37.5% crude fat, 2.8% ash and *Corresponding author. E-mail: ikechukwuanyadioha@gmail.com. Tel.:+2348157644992. 6.5% crude fibre (Ogunsina et al., 2010). Studies on the fatty acid characterization and profile have been carried out. The study showed that H. barteri oil contains an almost equal amount of myristic, palmitic, stearic and linolenic acids which is quite uncommon among all oilseeds. Palmitic acid was the major fatty acid, up to 29.4% followed by stearic acid. This is similar to that of palm kernel oil which has 82% saturated fatty acids and 18% unsaturated acids (Akinoso et al., 2006) The seeds are consumed in West Africa as raw or roasted nuts and have a flavour resembling peanuts. The kernel is eaten raw or roasted or used as condiments in traditional food preparation(Inglett et al., 1973). In Nigeria, the seeds of Kpaakpa (H. barteri) are consumed in few rural communities in Ebonyi and Enugu states respectively. Despite this high nutrient content of kpaakpa seeds, their utilization is impaired by some inherent constraints such as the presence of several anti-nutrients and 42 Sky J. Food Sci. toxicants and also due to lack of literature on the influence of processing treatment on these anti-nutritional factors. Nutritionally, any substance which may interfere with normal growth, reproduction or health when consumed regularly in the amount existing in the normal component of diet should be considered harmful and therefore toxic (Liener, 1994, Apata et al., 1997). The full nutritional benefits of Kpaakpa (H. barteri) can only be realized if the food commodity is subjected to various forms of processing treatment to reduce to a tolerable level the adverse effects of anti-nutritional factors and enhance the amino acid profile. This study is, therefore, designed to investigate the effect of processing treatments on the anti-nutrient composition and amino acid profile of the seed using a face-centred central composite design. These will provide the basis for their exploitation of food. and soaked as described above. After the soaking process, the seeds were spread inside the germination bags and placed in a jute bag which has previously been soaked with water and covered also with the jute bag. These samples were allowed to germinate for 2, 4 and 6 days respectively. After the germination, the seeds were oven-dried at a temperature of 50°C for 48 h. After the oven drying, the seeds were dehulled, milled, sieved and packaged in an airtight plastic container and stored in the refrigerator before analysis. Experimental design This experiment was designed using Minitab software version 14.0. It is a face centred central composite design that has two major factors: soaking time (x1) and germination (x2) where each factor has three levels (32) given a total of nine runs as shown in Table 1. MATERIALS AND METHODS Sample sourcing Anti-nutrient composition of the flour The H. barteri seeds were hand-picked around the trees at the Independent Layout Area of Enugu metropolis and Ezzaa Local Government Area of Ebonyi State. The anti-nutrient composition of each sample of the flour was determined as follows: Determination of trypsin inhibitor and haemagglutinin was carried out according to the spectrophotometric method described by Armfield (1985). Tannin and phytic acid were determined by the Follin-Dennis spectrophotometric method of Onwuka (2005) while the oxalic acid content of each sample was determined using the titration method of Onwuka (2005). The amino acid profile of the samples was determined by the method of Na (1986). Preparation of raw H. barteri seed flour The outer covers of the seeds were removed, winnowed and oven-dried at a temperature of 50°C for 24 h. The dried seeds were manually dehulled, milled into flour using Panasonic blending machine and sieved through a 500-micron mesh sieve. The milled and sieved flour was packaged in an airtight container and stored in a refrigerator before analysis. packaged in an airtight container and stored in a refrigerator before analysis. Preparation of soaked H. barteri seed flour Two hundred grams each of the oven-dried seeds were weighed and put in germination bags and soaked in clean water for 12, 24 and 36 h respectively. Soaking allowed the seeds to imbibe water, loosen the seeds and destroy some anti-nutritional components that are naturally inherent in legumes. After soaking, the seeds were drained off and H. barteri seeds were oven-dried at a set temperature of 50°C for 48 h. The dried seeds were dehulled, milled, sieved into flour and packaged in an airtight container and stored in the refrigerator for compositional analysis. Preparation of germinated H. barteri seed flour Two hundred grams of H. barteri seeds were weighed Statistical analysis All the responses were determined in triplicates. Data were analyzed statistically using a statistical software package for social science (SPSS version 17.0 for windows, SPSS Inc. Illinois, USA). Mean separation was carried out using Least Significant Difference (LSD) at p > 0.05. Experimental data generated from the anti-nutrients properties were further analyzed using Minitab software (version 14.0). The above analysis involved fitting data into the simple second-order polynomial model equation for the theoretical prediction of the response variables. The model equation is represented as below: 2 2 y = a + b1x1 + b2x2+ b11x 1+ b22x 2 + b12x1x2 +e (1) Where, y = the responses, a = constant regression coefficient b1, b2 = linear coefficients of the independent variables b12 = coefficient of the interaction, x1, x2 = independent variables, x12 = the interaction, b11& b22 are quadratic regression coefficient terms, e = error Anyadioha et al. 43 Table 1. Coded experimental design. RUN 1 2 3 4 5 6 7 8 9 A -1 0 -1 0 1 0 1 1 -1 B 1 -1 -1 0 0 1 1 -1 0 A= soaking time: -1= 12 h, 0 = 4 h, +1 = 36 h, B= Germination time: -1 = 2 days, 0 = 4 days, +1 = 6 days Factors: 2, Replicates: 1 Base run: 9, Total runs: 9 Base, blocks: 1, Total blocks: 1 Two-Cube factorial: Full factorial Cube points: 4 Center points in a cube: 1 Axial point: 4 Center points in axial:0, Alpha: associated with the observation of y. RESULTS AND DISCUSSION Phytic acid There was a significant (p < 0.05) reduction of phytic acid composition between the ingeminated and the germinated samples ranging from 78 mg/100g to 234 mg/100g as germination time increased. The reductions were comparable to the results reported for other germinated legumes such as African oil bean (Enujiugha, 2003) and were due to an increase in phytase enzyme activity as reported by (Reddy et al., 2002) in Faba beans. This was confirmed by the regression analysis performed on the phytate data as shown in Table 3a and 3b. The result showed that none of the variables was significant (p<0.05) but from the regression table, the quadratic effect of germination indicated that there was a reduction in phytate as germination time increased. Analysis of variance shown in Table 3b indicated that phytate data could not be fitted into a regression model going by the low R2 of 0.624 and low F value of 0.99 and p-value of 0.837. Tannin Soaking and germination reduced tannin level significantly (p<0.05) owing to the leaching of soluble tannin compounds during soaking and the presence of polyphenol oxidase and its hydrolysis during the sprouting process (Torres et al., 2007). This was confirmed by the data on Tannin obtained from the regression coefficient which showed that both linear and quadratic effects of soaking time were highly significant (p<0.05) following the F- values of 141.16, 13.13 and p– values of 0.001 and 0.033 respectively. The R2 and R2adj. Of 0.99 and 0.974 further showed the extent of the goodness of fit of the data to both the linear and quadratic model. The experimental equation developed for Tannin is given by: Tannin=1.0911- 0.6916x1+ 0.4783x1 2 (2) Trypsin inhibitor The result in Table 2 showed a significant decrease in trypsin inhibitor in Kpaakpa seed flour and this agreed with the report of Frias et al. (1995) who reported a reduction of trypsin inhibitor in lentils. The trypsin inhibitor data were also fitted into the regression analysis model for prediction purposes. The regression result revealed that the main effect of soaking and germination was significant as an increase in the two variables brought about a significant reduction in trypsin inhibitors. Similarly, the quadratic effect of soaking time was significant (p < 0.05). The interaction effect of soaking and germination time had negative effects on trypsin inhibitors but was not statistically significant (p < 0.05). The model showed a good fit with the R2 of 0.998 and R2 adj. of 0.996 for trypsin inhibitor. This agreed with the report of Hannika (1982), Okpala and Okoli (2012). A good fit model R2 should be at least 0.75. Hence, the equation given below could be applied in predicting Trypsin inhibitors in food systems. 2 TI= 317.132 - 96.427x1 - 147.795x2+69.637x1 (3) 44 Sky J. Food Sci. Table 2.Effect of processing soaking and germination on the anti-nutrients (mg/100g). Responses Haemagg Phytate RHBT a 1.49 + 0.02 234.25 a +3.0 S12 ab 1.86 +0.05 acd 150 + 0.7 S12G2 d 0.8 + 0.05 ef 88.17 +2.13 S12G4 ef 0.4 +0 .07 ef 98.12 +6.71 S12G6 f 0.41 + 0.07 ef 78.47 + 0.63 S24 bc 1.22 +0.04 a 216 + 1.04 S24G2 d 0.9 + 0 a 0.553 b + 0.06 b 2.88 + 0.38 b 784 + 7.34 0.442c +0.049 0.432 e + 0.035 b 2.33 + 0.10 bd 0.414 + 0.016 d 1.66 + 0.50 ef 0.532 c + 0.04 e 2.30 +0.36 0.432 + 0.04 353.33 + 3.53 65.6 +6.56 c 479.6 +4.79 Oxalate 0.572 +0.088 Tannin 3.44 + 0.28 Trypsin Inhibitor 893 + 8.93 a a b 2.89 + 0.02 bd d 638 + 6.38 474 + 7.74 e ac 193 + 1.93 S24G4 e 0.6 + 0.08 ef 78 + 1.26 S24G6 a 0.41 + 0.01 e 93..65 f + 5.7 bd 0.410 + 0.04 ef 0406 + 0.08 1.87 + 0.12 1.05 + 0.01 0.45 + 0.23 328.67 + 3.28 158.80 + 1.58 c c c d f f f S36 c 1.1 + 0.01 bc 209 + 1.69 0.470 b + 0.070 03.14 ab + 0.05 623.3 e 3+ 6.23 S36G2 d 0.83 + 0.11 acd 117 + 1.4 S36G4 ef 0.54 + 0.12 de 92.24 f + 1.03 cd 0.421 f + 0.20 0.59 + 0.21 d 0.85 + 0.17 0.49 + 0.07 c 288.0 + 2.8 e 128.97 + 1.28 0.432 e + 0.03 470 + 4.7 ed ef S36G6 a 0.37 + 0.09 f 94.46 + 2.2 ef 0.410 + 0.17 f + Values are means std deviations of Triplicate samples. Means with different superscripts within the Sam raw are significantly different from each other (p<0.05) Key: RHBF – Raw Hildegardiabarteri flour, S12 – Soaked Hildegardiabarteri for 12 h S12G2 – Soaked for 12 h Germinated 2 days. S12G4 Soaked 12 h Germinated 4 days S12G6 – Soaked 12 h Germinated 6 days. S24 – Soaked for 24 h. S24 G2 – Soaked 24 h Germinated for 2 days S24 G4 – Soaked for 24 h germinated for 4 days. S 36 – Soaked for 36 h S36 G2 – Soaked for 36 h germinate 2 days, S36 G4 – Soaked for 36 h germinated for 4days. S36 G6 – Soaked for 36 h germinated for 6 days, Haemagglu – haemagglutinin, Trypsin Inhib – trypsin Inhibitor Table 3. Amino acid profile (mg/100g). RHBF S12 S12G2 S12G4 Isoleu Leucin 1333.1±0.014 1933.33±0.007 b 1278.5±0.14 d 1888.53±0.02 c 1295.9±.69 b 1844.43±0.007 a 1288.3±0214 c 1778.83±0.02 d Lysine S24 S24 G4 1277.43±0.007 c 1255.63±0.01 f a 1956.33±0.02 a 1955.63±0.014 1666.63±0.007 f 1888.43±.014 c 1977.13±0.02 1884.33±0.02 1933.33±0.02 d b b 1855.53±0.14 Methion 350.1±0.014 b 362.22611±0.014 a 35822±0.007 b 3848±0.007 355.5602±0.01 a c 355.02±0.78c Cystine 365.64±0.007 377.77±0.014 a 368.67±0.02 b 366.12±0.007 c 3578.57±0.014 e 345.47±0.02 f Phenylal 1666.33±0.014 f 1774.43±0.007 d 1888.23±0.014 a 1865.33±0.02 b 1789.63±0.14 c 1699.93±0.01 e Tyrosine 1002.03±0.02 a 1010.33±0.02 a 1035.23±0.014 a 1066.35±0.03 a 1012.23±0.02a 1003.13±0.02 a Threonine 1255.03±0.014 a 1188.23±0.02 c 1077.23±0.02 e 1205.23±0.02 b 1122.23±0.01d 1204.73±0.6 d Tryptophan 435.36±0.007 a 422.26±0.007 c 418.27±0.01 e 430.14±0.02 b 408.56±.007f 421.14±0.02 d Valine 1555.20±0.02 a 1399.20±0.02 b 1266.20±0.03 d 1312.10±0.02 c 1220.0±0.02e 1204.10±0.02f Arginine 2222.03±0.014 a 2144.3±0.19 d 2155.50±0.02 c 2220.3±0.03 b 2144.30±0.04d 2044.13±0.023 c Histidine 830.26±0..007 c 844.24±0.02 a 818.26±0.007 822.03±.01 e b 833.31±.02b 844.24±.02 a Alanine 1224.20±.02 1199.30±.03 1895.5±0.2 c 1966.30±.03 a 1887.50±.033 d 1933.3±.01 b Aspartic 3555.2±.02 a 3244.3±.05 3144.10±.02 c 3025.30±.01 d 2023.3±.02 f 2144.2±.02 e Glutamic 3666.30±.05 b 3674.40±.03 a 3585.0±.04 c 3474.20±.03 d 3333.3±.01 Glycine 1553.3±.01 d 1775.8±.02 a 1688.20±.03 b 1599.4±.02 1477.4±.01 c Proline 1885.2± 0.00a 1777.95±.035 d e f b 1800.2±.02c a 1766.3±.02e Soyabean Seed (raw) 200 330.3 1841.0±.02 f 3444.3±.02 e 1550.0±.01 e f b 1755.53±.01 f Values are mean ± standard deviation (P< 0.05) values with different superscripts along the row are significantly different (P<0.05). Key: RHBF -Raw Hildegardiabarteri flour S12-Soaked for 12 h S12G2 Soaked for 12 h and germinated for 2days S12G4 Soaked for 12 h and germinated for 4days S24G4 Soaked for 24 h and germinated for 4days S36G4 Soaked for 36 h and germinated for 4days f Anyadioha et al. 45 response surface plot for trypsin inhibitor 600 trzpsin inhibitor 400 200 1 0 -1 0 A B -1 1 Figure 1.Response surface trypsin inhibitor. plot for Figure 2.Optimization graph for trypsin inhibitor. The response surface plot that explained the influence of soaking and germination time on trypsin inhibitors is shown in Figure 1. Haemagglutinin The analysis of variance and face centred central composite design regression coefficients for haemagglutinin showed that the main effect of germination time had a significant negative influence on haemagglutinin level with F - value and p-value of 40 and 0.007, respectively. This implied that as the germination time progressed, the level of haemagglutinin decreased. The combined influence of soaking and germination resulted to a reduction in haemagglutinin level although this reduction was not significant. The experimental data of haemagglutinin was fitted into a linear model with R2 0.969 and R2 adj. 0.915 thus indicating model adequacy. The predictive model equation for haemagglutinin reads thus: Haemagglutinin = 0.5655+0.022333X1 (4) Oxalate Results in Table 2 showed that after germinating, 72% of the original value of the oxalates in the control sample is retained. This was evidenced by the regression analysis performed on this data. The main effect of soaking and germination times were significant A and B having p values of 0.03 and 0.003 respectively, showing an antagonistic effect on oxalates. However, the quadratic effect of soaking time was also significant (p = 0.02). The goodness of fit of the mathematical regression model was checked by the determination of the correlation coefficient (R2). The value of R2 (0.975) for equation 8 indicated that the sample variation of 97.5% for oxalates was attributed to the soaking and germination treatment given to the samples and that only 2.5% of the total variation could not be explained by the model. The model obtained for oxalate is given below: 2 Oxalate = 0.414 - 5.333x1- 13.833x2+ 10.333x1 (5) Optimization To minimize trypsin inhibitor level, the optimal D of 0.75821, y=150.04 curr36 h soaking and 5.7 days germination with the desirability of 0.9999 (Figure 2). The amino acid profile of the flour was analyzed and the results showed that the protein contained nutritionally useful quantities of most of the essential amino acids (lysine, tryptophan, threonine, etc) including sulphur – 46 Sky J. Food Sci. containing amino acids such as methionine and cysteine. Soaking and germination improved the nutritional quality of these essential amino acids (Isoleucine, methionine, lysine, etc) Lysine increased significantly during this process ranging from 1666.63 mg/100 g in RHBF to 1977.13 mg/100 g in S12G2. All the soaked and germinated samples appreciated their level of lysine. The high level of Lysine in the protein composition of Kpaakpa seed flour is in line with the composition of all grain legumes which are good sources of lysine (FAO/WHO/UNU 1985). An average grain legume contains 1.5 g/100 g and so provides a useful supplement to most staple foods, such as maize. Methionine This is one of the least concentrated amino acids in H. barteri protein after tryptophan as is expected in legumes (350.1 mg/100 g) (13). These values, however, improved with soaking and germination as reported in samples S12 (362.226 mg/100 g), S12G2 (358.226 mg/100 g), S12G4 (384.8mg/100 g), S24G4 (355.56 and 355.02mg/100 g) respectively. Methionine is nutritionally important as its sufficiency in protein enhances the supply of cysteine in the body. Cysteine There was a general increase in cysteine levels of all the treated samples when compared with the control sample except sample S12G4 which had 345.47 mg/100 g. Sample S12 had the highest level of cysteine (327.77 mg/100 g) while S12G4 had the lowest (345.47 mg/100 g). Samples S12G4 and S24 had the least reduction as germination progressed. Threonine The difference between RHBF and S12G4 was significant as the level decreased from 1255.23 mg/100 g to 1035.23 mg/100 g respectively. This value appreciated in S12G4 (1205.23) and S24G4 (1204.73 mg/100 g) indicating that the increase was due to processing which led to changes in amino acid profile. There was a significant decrease in the threonine level of S12 (1188.28), S12G4 (1077.23) S24 (1122.23) when compared with the raw sample which had 1255.23 mg/100 g. However, samples S12G4 and S24G4 appreciated slightly during soaking and germination. Tryptophan This is the least concentrated essential amino acid in the protein of Kpaakpa seed flour. The raw Kpaakpa flour had the highest value of 435.36 mg/100 g as against the sample soaked for 12h (S12) which had the lowest value of 408.56 mg/100 g. The rest of the processed sample showed a downward trend as the duration of soaking and germination increased. Despite this reduction, the retained level of tryptophan meets the FAO/WHO/UNU (1985) amino acid composition of protein in common legumes. The value of 435 mg/100 g for the control sample compares favourably well with the tryptophan value of 164 mg/100 g in soybean. Conclusion This research work has shown that soaking and germination could be used to improve the nutritional value of Kpaakpa seed. It was found from this study that soaking and germination reduced anti-nutritional factors in Kpaakpa seed which implied enhanced bioavailability of such minerals like phosphorus, iron, zinc and calcium. Response optimization of trypsin inhibitor was performed using numerical optimization. The optimized parameter showed a significant interaction between soaking and germination which resulted in a significant reduction in anti-nutrient levels. The amino acid analysis performed on the flour showed that the protein contained nutritionally useful quantities of most of the essential amino acids including sulphur-containing amino acids such as methionine and cysteine. REFERENCES AkinosoR, Igbeka JC, Olayanju TMA,Bankole LK (2006). Effects of storage on the quality of palm kernel and sesame oils.Proc. 7th Int. Conf. and 28thAnnual General Meeting, November 6-10, Zaria, Nigeria. Apata DF (1997).Trypsin inhibitor and other antinutritional factors in tropical legume seeds. Trop. Sci., 37: 52 59. ArmfieldSO, Ismond MAH, Murray EO (1985). The fate of antinutritional fact during the preparation of faba bean protein isolate using micellization technique. Can Inst of Food SciInnol. J., 18137-143 Enujiugha VN, Badejo AA, Iyiola SO,Oluwamukomi MO (2003).Effect of germination on the nutritional and functional properties of African oil bean (PentaclethramacrophyllaBenth) seed flour.Food, Agriculture and Environment, 1(3&4), 72-75. FAO/WHO/UNU (1985).Energy and Protein Requirements.Technical Report Series 724, Geneva Switzerland cited by Oyaerekua, M.A and Adeyeye EI (2011).The Amino Acid Profile of the Brain and eyes of African giant pouch rat (Cricetomysgambianus).Agriculture and Biology J. North America. 2(2): 368-375. Frias J,Diaz-Pollan C, Hedley CL, Vidal Valverde C (1995).Evolution of trypsin inhibitor activity during germination of lentils. J. of Agric. and Food Chem., 43: 2231-2234. Hannika RG(1982). Use of Response surface methodology in sensory Evaluation. Food Technol., 36: 96-101. Inglett GE, Cavias JF, Spencer GF (1973). Seed Composition of Hildegardia Barteri.Eco. Bot., 27(1),128-130. Liener IE (1994).Implications of the anti-nutritional component in soybean food Crit. Rev FoodSci.Nutr., Na AH, Shin MS, Jhon DY, Hong YH (1986). Studies on the changes in free amino acids of Yellow Corvina(Pseudosciaenamanchurica) during Gulbi processing. J. Korean physical properties of Kariya seeds. Int. Agrophys., 25, 97-100I Ogunsina BS, Olaoye IO, Adegbenjo AO, Babawale BD (2011).Nutritional and physical properties of Kariya seeds. Int. Agrophys., 25, 97-100I Okpala LC, Okoli EC (2012). Development of cookies made with cocoyam, Anyadioha et al. fermented sorghum and germinated pigeon pea flour blends Using response surface methodology J. Food Sci. Technol. DOI: 10.1007/s 13197-0120749-1 OnwukaGI (2005). Food Analysis and Instrumentation theory and practice.Naphtali Publisher Ltd Lagos p. 56-62. Reddy NR (2002).Food phytates.Boca Raton FL USA: CRC Press p. 2551. Torres A, Frias Vidal–Valverde C(2007).Germinated Cajanuscajan seeds as ingredients products: Chemical, biological and sensory evaluation. Food Chemistry (101):202-211. 47 Tharanathan RN, Mahadevamma S(2003). Grain legumes-a boon to human nutrition.Trends in Food Sci. and Technol., 14: 501-518. Rocio Campos-Vega, GuadalupeLoarca-Piña and Dave Oomah B.(2009).Minor components of pulses and their potential impact on human health. Food Res. Int., 43: 461- 482.