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AMERICAN JOURNAL OF FOOD AND NUTRITION Print: ISSN 2157-0167, Online: ISSN 2157-1317, doi:10.5251/ajfn.2016.6.3.91.100 © 2016, ScienceHuβ, http://www.scihub.org/AJF Effect of Natural Fermentation on the Vitamins, Amino Acids and Protein Quality Indices of Sorghum-Based Complementary Foods Sengev, I. A*., Ariahu, C. C. and Gernah, D. I. Department of Food Science and Technology, University of Agriculture, P.M.B. 2373, Makurdi, Benue State, Nigeria * Corresponding author: Sengev, I. A. Department of Food Science and Technology, University of Agriculture, P.M.B. 2373, Makurdi, Benue State, Nigeria, E–mail: [email protected] Tel: +2348035020615 ABSTRACT This study investigated the effect of natural fermentation on the vitamins, amino acid and the quality of protein of sorghum-based complementary foods. Samples were formulated based on 16% protein to satisfy the nutrient requirement of preschool children to obtain non-fermented sorghum/mango mesocarp/crayfish (NFSMF), non-fermented sorghum/fluted pumpkin leaf/fish (NFSPF), fermented sorghum/ mango mesocarp/crayfish (FSMF) and fermented sorghum/ fluted pumpkin leaf /crayfish (FSPF). The vitamins, amino acids and growth study parameters were examined using standard procedures. Vitamin A (193.50 - 322.10μ/100g), B12 (0.48 – 0.54mg/100g) and D (0.42 – 0.93mg/100g) increased significantly (p< 0.05) with fermentation, both for samples containing mango mesocarp and fluted pumpkin powders while retinol equivalent decreased with fermentation. All the samples met the essential amino acids requirement as recommended by WHO/FAO/UNU pattern 2007. All the essential amino acids increased significantly with fermentation. NFSMF and NFSPF exhibited higher growth response when fed to rats compared to FSMF and FSPF. Sample NFSMF gave better protein efficiency ratio (PER) (2.48) and net protein ratio (NPR) (4.12) followed by NFSPF, FSMF and FSPF. Among all the samples, NFSMF gave the best PER result. Key words: Sorghum, Vitamins, Crayfish, Mango, amino acids INTRODUCTION According to the recent FAO database, the total number of undernourished people in the world, almost 794.6 million, is still unacceptably high, and eradication of hunger remains a major global challenge (FAO/IFAD/WFP, 2015). Hence, the search for alternative food ingredients remains of utmost importance. In Nigeria, traditional complementary foods are usually produced from staple cereals and legumes prepared either individually or as composite gruels (Walker, 1990). Cereal grains are considered to be one of the most important sources of dietary proteins, carbohydrates, vitamins, minerals and fibre for people in developing countries. Complementary feeding period is the time when malnutrition starts in many infants, contributing significantly to the high prevalence of malnutrition in children less than 5 years of age worldwide (Daelmans and Saadeh, 2003). The introduction of complementary food in Nigeria usually starts between age 4 and 6 years; and usually involves the use of a semi-liquid porridge prepared locally by the mother from staple cereals or tubers (Bentley et al., 1991), (Nout, 1993), (Guptill et al., 1993). Combination of common cereals, which are deficient in lysine but have sufficient amount of sulphur-containing amino acids, with inexpensive animal protein sources like crayfish that are rich in lysine can be used to improve the nutritive value of a food product. Sorghum variety (Sorghum bicolor [L.] Moench) is one of the most important staple foods in low-income and high-income countries ( Shehab et al., 2010). The nutrient composition of sorghum indicates that it is a good source of energy, proteins, carbohydrates, vitamins and minerals including the trace elements, particularly iron and zinc, except calcium. Sorghum grain contains minerals such as phosphorus, potassium and magnesium in varying quantities (Dicko et al., 2006). Wet processing including soaking, germination and fermentation leads to a reduction in phytic acid and increases of the minerals solubility in foods and could thus improve bioavailability of minerals in cereals and legumes (Afify et al., 2011a). The low digestibility is due to protein-protein, protein-carbohydrate, protein (poly) 91 Amer. J. Food & Nutr., 2016, 6(3): 91-100 o phenol and carbohydrate- (poly) phenol interactions (Knudsen et al., 1988). The choice of crayfish is based on the fact that it has high protein in range of 60 to 70% (Obizoba, 1985) and its inclusion in sorghum-based foods would improve on the protein quality. Mango pulp is reported to be rich in micronutrients (Belly, 2006) and fluted pumpkin was reported to be rich in amino acids and has haematinic properties (Fasuyi, 2006), (Dina et al., 2006). FAO/WHO/UNICEF (1971) emphasised the use of local foods formulated at home and guided by the following principles: (i) high nutritional value to supplement breastfeeding, (ii) acceptability, (iii) low price, and (iv) use of local food materials (Dewey and Brown, 2003), (Pelto et al., 2003). Therefore, the aim of this study was to develop and evaluate the nutritional properties of the complementary foods using sorghum, crayfish, mango mesocarp and fluted pumpkin powders. MATERIALS AND METHODS dried in hot air oven at 70 C for 12h and milled into fine flour of 0.5mm instead of 0.212mm. Preparation of Mango Mesocarp Powder: The method of Sengev et al. (2010) was adopted with modification. Five kilograms of partially ripe mango fruits, chul kpev (a local variety) (pH = 3.8, Brix = 7.0, Refractive Index = 1.34) were sorted, washed, peeled and the mesocarp was manually sliced to an average thickness of 2.5mm. The slices were spread on a tray covered with aluminum foil and oven-dried at 70 o 1 C for 24h to a moisture content of about 10%. The slices were milled using a disc attrition mill (Model: AII Asiko, Nigeria) and sieved through a 0.5mm sieve to obtain mango mesocarp flour (MMF). Preparation of Crayfish Powder: The method of Onuorah and Akinjede (2004) was adopted with modification. About 1kg of crayfish was washed to remove extraneous materials, sundried for 12h at relative humidity of 65%. The sample was milled using hammer mill (Model: Brook Crompton Series 2000, England) and sieved through 0.5mm laboratory test sieve instead of 0.6mm to obtain crayfish flour. Sample Procurement: About 10kg of red sorghum grains [Sorghum bicolor, (L) Moench] and 5kg of semi ripe mango fruits (a local variety) (Mangifera indica) popularly known as Wua nyian and Chul kpev in Tiv respectively, 1kg of crayfish (Procambarus clarkii) and fluted pumpkin leaves (Telferia accidentalis) each were sourced from a local market in Makurdi, Benue State. Vitamin pre-mix (Vitalyte) was also sourced from a pharmacy store in Makurdi. These materials were transported to the Department of Food Science and Technology, University of Agriculture, Makurdi for processing prior to product formulation and subsequent analysis. Male wistar strain weanling albino rats (4 weeks old) were sourced from National Institute of Trypanasomiasis Research, Vom, Plateau State. Preparation of Sorghum Flour: About 10kg of sorghum was dehulled using rice huller (Model: Navin, Madras) and washed with tap water and sun dried for 12h at average relative humidity of 65%. The dehulled grains were milled using attrition mill (Model: Asiko AII). The flour was sieved using a laboratory test sieve of 0.5mm aperture. Solid State Fermented of Sorghum Flour: Fermentation of sorghum flour was carried out using the method of Sengev et al. (2010) with modification. The milled sorghum flour was divided into two equal parts. One part was mixed with tap water in the ratio o of 2:1w/v and allowed to ferment for 48h at 30 1 C, relative humidity of 65% in none air-tight covered plastic tray. At the end of the fermentation, a pH of 3.80 was recorded. The fermented sorghum was Preparation of Fluted Pumpkin Leaf Powder: The flute pumpkin leaves were washed with tap water, steam blanched for 3 sec. and dried under the shade to constant weight. The dried leaves were milled using attrition mill (Model: AII Asiko, Nigeria) and screen through a 0.5mm sieve to obtain fluted pumpkin leaf flour. Blend formulation: The blends were formulated based on protein and mass balance equations. Sorghum, crayfish, mango mesocarp and fluted pumpkin leaf powders were blended based on 16% protein level as shown in Table 1. Feeding trial Studies: Feeding trial studies were conducted using 21 weanling male albino rats (Wistar strain), that were clinically healthy (average weight of 90.10g), using a modification of the method described by Rasaco (2002). A complete randomized design (CRD) was used in which the rats were initially weighed to the nearest 0.1g at the start of each feeding trial and allocated into the seven cages based on weight equivalent with 3 rats per cage. The cages were placed on ceramic tiles to permit collection of faeces. The six diets formulated according to Pellet and Young (1980) along with the basal diet (Table 2) were evaluated. The diets were administered to the rats based on their body weight (ie 10g food per 100g body weight) and water adlibitum for 28 days. The total food intake of the rats was determined by recording the food left after daily 92 Amer. J. Food & Nutr., 2016, 6(3): 91-100 intake. Daily weight gain was obtained by weighing all the rats individually. Protein consumption was calculated from the food intake. All faeces collected in the last 10 days of feeding were pooled together, dried, weighed and milled into fine powder for analysis. amino acid values reported were the averages of two determinations. Norleucine was the internal standard. Statistical Analysis: The data generated were statistically analysed using analysis of variance (ANOVA). Means were tested for significant difference using Duncan’s Multiple Range Test (DMRT) (Wahua, 1999). Significance level was accepted at p < 0.05. RESULTS Vitamin Content and Retinol Equivalent of Sorghum-Based Complementary Foods: The results of vitamin concentration and retinol equivalent of sorghum-based complementary foods are presented in Table 3. The results revealed that significant difference (p<0.05) existed between the samples. Retinol equivalent ranged from 120.70 – 202.60 RE, Vitamin A ranged from 193.50 – 322.10 μg/100g, Vitamin C ranged from 11.30 – 12.96 mg/100g, Vitamin B1 ranged from 0.87 – 0.94 mg/100g, Vitamin B6 ranged from 0.43 – 0.55 mg/100g, Vitamin B9 ranged from 0.13 – 0.14mg/100g, Vitamin B12 ranged from 0.48 – 0.53 mg/100g and Vitamin D ranged from 0.42 – 0.93 mg/100g for NFSMF, NFSPF, FSMF and FSPF. Amino Acid Composition of Sorghum–Based Complementary Foods: The essential and non essential amino acid compositions of sorghum crayfish based complementary foods are presented in Table 4 and 5 respectively. The essential amino acids were compared with 2007 WHO/FAO/UNU report. For the essential amino acids, lysine ranged from 49.40 to 58.80mg/g protein, threonine ranged from 36.20 to 44.61mg/g protein, valine ranged from 35.15 to 44.20mg/g protein, methionine values ranged from 19.45 to 24.79mg/g protein. Others include isoleucine which ranged from 27.45 to 35.05mg/g protein, leucine ranged from 69.03 to 78.40mg/g protein and phenylalanine ranged from 39.55 to 45.89mg/g protein. Tryptophan ranged from 1.62 to 2.06mg/g protein. The essential amino acids met the WHO/FAO/UNU 2007 pattern. Protein Quality Evaluation: Net Feed intake and body weight achieve were employed to determine growth study parameters such as protein efficiency ratio (PER), relative protein efficiency ratio (R-PER), net protein ratio (NPR) and relative net protein ratio (R-NPR). Others include feed conversion factors such as feed conversion ratio (FCR) and feed conversion efficiency (FCE) following the procedure described by Shiriki et al. (2015). Analyses Vitamin Analysis: Vitamin A, B1, B6, B9, B12 and D were evaluated using HPLC (Model: BLC-10/11, BUCK Scientific, USA) techniques as described by AOAC (2005). For each sample, 3.0g was mixed with 5mL of n-hexane and 20mL of HPLC grade water. The mixture was homogenized at 12000 rpm and centrifuged (3500 x g) for 30 min. followed by sequential filtration through whatman No 1 filter paper and 0.45μm membrane. Then 15μL of the supernatant was injected into the HPLC equipped with a UV detector set at 254nm. The peaks of the vitamins in the samples were calculated in relation to the peaks of standard vitamins. Determination of Vitamin C Using Direct Tittration Method: Vitamin C content was determined according to Suntornsuk et al. (2002) by direct titration with iodine solution (Potassium iodide + Iodine). Ten gram (10g) of the flour was mixed with 100mL of distilled water in 250mL beaker and allowed to stand for 30min. The extract (40mL) was transferred into a 250mL volumetric flask and 2 drops of 0.5% starch indicator solution was added. The mixture was titrated with 0.0057M solution of iodine previously standardized with ascorbic acid solution. Vita min C (mg / 100 g ) Vita min C concentration 100 x Volume of extract 1 For the non-essential amino acids, histidine ranged from 18.00 to 19.56 mg/g protein, arginine ranged from 60.80 to 75.36 mg/g protein, aspartic acid ranged from 77.47 to 88.45 mg/g protein, serine ranged from 30.06 to 38.92 mg/g protein, glutamic acid ranged from 84.97 to 97.86 mg/g protein, proline ranged from 24.92 to 34.62 mg/g protein. Others include glycine which ranged from 38.83 to 48.96 mg/g protein, alanine ranged from 29.87 to 34.77 mg/g protein, cystine ranged from 11.95 to 15.77 mg/g protein and tyrosine ranged from 22.21 to 29.79 mg/g protein. Amino Acid Analysis : Amino acid analysis was by ion exchange chromatography (IEC) [FAO/WHO, 1991] using the Technicon Sequential Multisample (TSM) Amino Acid Analyser (Technicon Instruments Corporation, New York). The period of analysis was 76 min for each sample. The gas flow rate was 0.50 mL/min at 60°C with reproducibility consistent within ±3%. The net height of each peak produced by the chart recorder of the TSM (each representing an amino acid) was measured and calculated. The 93 Amer. J. Food & Nutr., 2016, 6(3): 91-100 Table 1: Formulation of Blends Blend Ingredient mix (g/100g) Sorghum Mango Mesocarp Powder Fluted Pumpkin Leaf Powder Crayfish Powder NFSMF 91.06 0.17 - 8.77 NFSPF 91.04 - 0.19 8.77 FSMF 91.06 0.17 - 8.77 FSPF 91.04 - 0.19 8.77 Key: NFSMF= Non-Fermented Sorghum + Mango Mesocarp + Crayfish, NFSPF= Non-Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, FSMF = Fermented Sorghum + Mango Mesocarp + Crayfish, FSPF =Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish. Table 2: Formulation of Experimental Diets Ingredient Formulated Diet NFSMF NFSPF FSMF FSPF WSF Basal Diet Milk Blend 57.78 56.06 58.52 57.28 Corn Starch 22.22 23.94 21.48 22.72 80.00 43.32 Milk Powder 36.68 Sorghum Flour 80.00 Vegetable Oil 10 10 10 10 10 10 10 Rice Husk 5 5 5 5 5 5 5 Salt 4 4 4 4 4 4 4 Vitalyte 1 1 1 1 1 1 1 Key: NFSMF= Non-Fermented Sorghum + Mango Mesocarp + Crayfish, NFSPF= Non-Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, FSMF = Fermented Sorghum + Mango Mesocarp + Crayfish, FSPF =Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, WSF = Whole Sorghum Flour Table 3: Effect of Natural Fermentation on the Vitamin Content of Sorghum-Based Complementary Foods Sample *Retinol Vitamin A Vitamin C Vitamin B1 Vitamin B6 Vitamin B9 Vitamin B12 Vitamin D Equivalent (μg/100g) (mg/100g) (mg/100g) (mg/100g) (mg/100g) (mg/100g) (mg/100g) a c ab b d b c c NFSMF 202.60±0.39 224.80±0.27 12.46±0.01 0.87±0.03 0.43±0.00 0.13±0.02 0.51±0.01 0.61±0.01 b a a b c b b b FSMF 169.60±0.27 322.10±0.21 12.96±0.20 0.88±0.00 0.48±0.00 0.13±0.00 0.53±0.00 0.77±0.00 c d b a a a d d NFSPF 124.30±0.37 193.50±0.03 11.30±0.41 0.94±0.00 0.55±0.00 0.14±0.04 0.48±0.01 0.42±0.00 d b ab b b a a a FSPF 120.70±0.32 306.60±0.46 11.70±0.82 0.89±0.00 0.53±0.00 0.14±0.01 0.54±0.00 0.93±0.00 -3 RDA 400 400 25 0.6 0.6 0.2 1.2x10 0.02 LSD0.05 0.94 0.61 1.30 0.03 0.01 0.01 0.01 0.01 Values are mean ± Standard deviation of three determinations. Means in the same column not followed by the same superscript are significantly different (p < 0.05) Key: NFSMF= Non-Fermented Sorghum + Mango Mesocarp + Crayfish, NFSPF= Non-Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, FSMF = Fermented Sorghum + Mango Mesocarp + Crayfish, FSPF = Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, *Conversion factor based on 6μg/100g β-Carotene: 1 Retinol Equivalent, RDA = Recommended Daily Dietary Allowance from IOM (2005) 94 Amer. J. Food & Nutr., 2016, 6(3): 91-100 Growth Response of Rat Fed with SorghumBased Complementary Food: The results of growth response of rats fed with sorghum and crayfishbased complementary foods for 28 days are shown in Figure 1. In the rat bioassay, all rats survived to the end of the observation and gained positive body weight except for zero protein group. The test diets fed to the animals include NFSMF, NFSPF, FSMF and FSPF. Others include WSF as control, basal diet as non-protein diet and milk as the standard or reference diet. The increase in body weight was reported as 52.82g (40.56%) for NFSMF, 44.20g (35.16%) for NFSPF, 35.57g (28.03%) for FSMF and 14.35 (14.66%) for FSPF. Others include 5.95g (6.22%) for WSF 53.50g (39.41%) for milk while the basal diet recorded -35.03 (40.45%) reduction in body weight during the 28-day feeding trial. blends could be ascribed to the contributions from mango mesocarp and fluted pumpkin leaf. Thiamin is responsible for the metabolism of carbohydrates and branched-chain amino acids. The results of this study did not show any evidence of increase in thiamin during fermentation as reported by Kazanas and Field (1981) probably due to short fermentation time. Dicko et al. (2006) again reported 0.35mg/100g thiamin for sorghum grain lower than values reported in this study. The vitamin B6 (Pyridoxine) in the blends was lower but compared favourably with the report of Duodu et al. (2003) and Dicko et al. (2006) who reported 0.50mg/100g pyridoxine. Vitamin B6 is responsible the metabolism of amino acids and glycogen in the body. The low values could be attributed to the dehulling which removed the pericap, aleurone layer and the germ leading to reduction vitamin B6 concentration. The vitamin B6 concentration in the samples fell slightly below the recommended dietary allowance of 0.6mg/day as reported by IOM (2005). The vitamin B9 (Folic acid) did not show significant difference between the fermented and non-fermented samples. However, the blends provided at least 65% of the RDA (IOM, 2005). Folic acid is important for many metabolic reactions, in the biosynthesis of DNA and RNA and the inter-conversions of amino acids. Also, folate possesses antioxidant competence that protects the genome by preventing free radical attack (LeBlanc et al., 2011). Protein Quality Evaluation of Sorghum-CrayfishBased Complementary Foods: The growth study parameters of rats fed for 28 days with sorghumcrayfish-based complementary foods and the reference diet (Standard) are presented in Table 5. The food conversion ratio (FCR) ranged from 3.80 to 30.15, food efficiency conversion (FCE) ranged from 3.33 to 26.34%. Similarly, the protein efficiency ratio (PER) for the test diets ranged from 0.47 to 2.68, the relative protein efficiency ratio (R-PER) ranged from 0.44 to 2.50, net protein ratio (NPR) ranged from 2.74 to 4.44 while relative net protein utilization (R-NPR) ranged from 2.48 to 4.02. The vitamin B12 concentration of the blends increased significantly (p<0.05) with fermentation. The increase in vitamin B12 could be attributed to activities of microorganisms during solid state fermentation of sorghum flour. Murdock and Fields (1984) reported that the levels of vitamin B12 and folic acid were increased by lactic acid fermentation of maize. Mustafa et al. (2013) also reported the production of vitamin B12 by Klebsiella pneumonia during solid state fermentation of agricultural waste. The abundance of vitamin B12 implies that prospective consumers would not suffer from pernicious anaemia. Vitamin B12 is required for the metabolism of fatty acids, amino acids, nucleic acids and carbohydrates (QuesadaChanto et al., 1994). DISCUSSION Vitamin Content and Retinol Equivalent of Sorghum-Based Complementary Foods: The retinol (RE) content of the blends decreased significantly (p<0.05) with fermentation while the vitamin A (retinol) content increased. It is postulated that the observed increase in vitamin A may be ascribed to its synthesis by microorganism during fermentation. The summation of retinol activity (REA) i.e RE+vitamin A met the RDA recommended by IOM (2005) for all the samples except for NFSPF which delivered 317.80 μg/100g (79.45%) REA. The occurrence of vitamin A in the blend may be ascribed to the inclusion of crayfish in the diets. Hamdi (2011) reported 810μg/100g vitamin A in Procambarus clarkii . The inclusion of fluted pumpkin and mango mesocarp powders increased vitamin C concentration in the blends. The vitamin C concentration in the blends was higher than the value reported for plain sorghum. Dicko et al. (2006) reported <0.001mg/100g vitamin C for plain sorghum. The higher values of vitamin B1 (thiamin) in the Fermentation significantly (p<0.05) increased the concentration of vitamin D. The high amount of vitamin D in the blends would meet the high demand of the vitamin for skeletal growth in infant. 95 Amer. J. Food & Nutr., 2016, 6(3): 91-100 Fig. 1: Effect of natural fermentation on the growth response of rats fed with sorghum-based complementary foods Key: NFSMF= Non-Fermented Sorghum + Mango Mesocarp + Crayfish, NFSPF= Non-Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, FSMF = Fermented Sorghum + Mango Mesocarp + Crayfish, FSPF =Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, WSF = Whole Sorghum Flour 96 Amer. J. Food & Nutr., 2016, 6(3): 91-100 Table 4: Essential Amino Acid Composition of Sorghum--Based Complementary Foods Essential Amino Acid WHO/FAO/UNU Sample LSD0.05 (mg/g protein) Pattern* 2007 NFSMF FSMF NFSPF FSPF Lysine 39 49.40±1.13b 57.10±1.27a 52.10±1.13b 58.80±0.42a 2.90 Threonine 20 36.20±1.27c 39.60±0.99b 41.45±0.92b 44.61±0.68a 2.74 Valine 33 35.15±1.20c 41.00±0.28b 40.25±0.92b 44.20±1.56a 3.04 Methionine 19 19.45±0.21d 21.46±0.08c 22.81±0.12b 24.79±0.59a 0.89 Isoleucine 25 27.45±0.21d 31.05±0.35b 29.08±0.75c 35.05±0.21a 1.22 Leucine 49 71.17±0.25b 72.46±0.08b 69.03±0.94c 78.40±0.01a 1.36 Phenylalanine 34 39.55±1.07c 41.82±0.74b 42.77±0.79b 45.89±0.40a 2.18 Tryptophan 5 1.62±0.08c 1.81±0.03b 1.66±0.04c 2.06±0.01a 0.13 Values are mean ± Standard deviation of two determinations. Means in the same row not followed by the same superscript are significantly different (p < 0.05). Key: NFSMF= Non-Fermented Sorghum + Mango Mesocarp + Crayfish, NFSPF= Non-Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, FSMF = Fermented Sorghum + Mango Mesocarp + Crayfish, FSPF =Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, LSD = Least Significant Difference, *Recalculated amino acid scores from 2007 WHO/FAO/UNU report. Table 5: Effect of Fermentation on Non-Essential Amino Acid Composition of Sorghum-Based Complementary Foods Amino Acid Sample LSD0.05 (mg/g protein) NFSMF FSMF NFSPF FSPF Histidine 18.00±0.43a 18.36±0.21a 18.34±0.80a 19.56±0.81a 1.71 Arginine 69.80±0.07b 75.36±0.16a 74.56±1.20a 75.15±0.74a 1.97 Aspartic acid 77.47±0.04c 75.62±0.71d 84.85±0.21b 88.45±0.77a 1.49 Serine 30.06±0.38d 33.67±1.20c 36.10±0.71b 38.92±0.04a 2.01 Glutamic acid 84.97±0.24c 93.28±0.23b 86.50±1.70c 97.86±0.63a 2.56 Proline 24.92±0.54c 32.67±0.76ab 31.45±1.34b 34.62±0.73a 2.49 c c b a Glycine 38.83±1.52 40.61±0.88 44.45±0.92 48.96±1.19 3.21 Alanine 29.87±0.38c 32.93±0.59ab 31.75±1.63bc 34.77±0.66a 2.63 Cystine 12.12±0.32b 11.95±1.98b 14.75±0.21ab 15.77±0.93a 3.08 Tyrosine 22.21±0.02c 23.93±0.26b 26.60±1.41a 29.79±0.71d 2.22 Values are mean ± Standard deviation of two determinations. Means in the same row not followed by the same superscript are significantly different (p < 0.05) Key: NFSMF= Non-Fermented Sorghum + Mango Mesocarp + Crayfish, NFSPF= Non-Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, FSMF = Fermented Sorghum + Mango Mesocarp + Crayfish, FSPF =Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, LSD = Least Significant Difference. Table 6: Effect of Natural Fermentation on Protein Quality of Sorghum-Based Complementary Foods Parameter Experimental Diet LSD0.05 NFSMF FSMF NFSPF FSPF WSF Milk Basal FCR 4.33±0.02e 6.35±0.01c 5.11±0.07d 13.74±0.54b 30.15±0.25a 3.80±0.05e -3.18±0.01f 1.54 b d c e f a FCE 23.10±0.11 15.65±0.14 19.58±0.29 7.29±0.29 3.33±0.04 26.34±0.38 -31.45±0.13g 0.53 PER 2.48±0.02b 1.70±0.02d 2.06±0.04c 0.81±0.03e 0.47±0.00f 2.68±0.05a -23.83±0.16g 0.16 b d c e f a g R-PER 2.31±0.02 1.58±0.002 1.92±0.04 0.75±0.03 0.44±0.00 2.50±0.05 -22.20±0.14 0.14 NPR 4.12±0.03b 3.40±0.02d 3.68±0.06c 2.74±0.04e 3.26±0.00d 4.44±0.07a 0.20±0.16f 0.16 R-NPR 3.73±0.03b 3.08±0.01d 3.33±0.05c 2.48±0.04e 2.95±0.03d 4.02±0.07a 0.18±0.13f 0.15 Values are mean ± Standard deviation of two determinations. Means in the same row not followed by the same superscript are significantly different (p < 0.05). Key: NFSMF= Non-Fermented Sorghum + Mango Mesocarp + Crayfish, NFSPF= Non-Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, FSMF = Fermented Sorghum + Mango Mesocarp + Crayfish, FSPF = Fermented Sorghum + Fluted Pumpkin Leaf + Crayfish, WSF = Whole Sorghum Flour, FCR = Food Conversion Ratio, FCE = Food Conversion Efficiency, PER = Protein Efficiency Ratio, R-PER = Relative Protein Efficiency Ratio, NPR = Net Protein Ratio, R-NPR = Relative Net Protein Ratio. 97 Amer. J. Food & Nutr., 2016, 6(3): 91-100 Amino Acid Composition of Sorghum-Based Complementary Foods: The essential amino acid composition of the blends met the amino acid pattern by WHO/FAO/UNU (2007). The amino acid contents of non-fermented samples were less than their corresponding fermented samples. This could be due to the synthesis of some essential amino acids during solid state fermentation. The tryptophan concentration of the blends increased significantly (p<0.05) with fermentation. This could also be attributed to the synthesis of some amino acids by microorganisms during solid state fermentation (SSF). The increase in amino acids in the fermented product shows that the carbohydrate utilization is closely proportional to protein production during solid substrate fermentation. Singh et al. (1990) reported that single cell protein produced by Aspergillus niger contained 30.4% crude protein and had an essential amino acid profile featuring a high lysine content and appreciable amounts of methionine and tryptophan, and 12.9% fat, which comprised of all the essential fatty acids. with fermented sorghum meal had a mean body weight loss of -2.8g; whereas those fed diets containing non-fermented sorghum meal had a mean body weight gain of 13.8 g. The rats fed on FSPF and WSF could not show any marked increase in weight during the feeding period of 28 days. Protein Quality of Sorghum-Based Complementary Foods: The values obtained for feed conversion efficiency (FCE) were a reflection of the feed conversion ratio (FCR) values since FCE is the reciprocal of FCR expressed as a percentage. The highest feed conversion efficiency was observed for the reference diet (Milk) followed by NFSMF>NFSPF>FSMF>FSPF in that order. The FCE decreased with increase in food consumption while the FCR increased with decrease in food consumption during rat bioassay. FCR and FCE are important calculations for growth which can be used to determine if the diets are being used as efficiently as possible (Goddard, 1996). In this study, the FCR of 4.33 to 5.11 and FCE of 23.10 to 19.58 for NFSMF and NFSPF respectively indicated good growth. The lower value of FCR is indicative of the better utilization of the diet. Protein Efficiency Ratio (PER) indicates the relationship between weight gain in the test animals and the corresponding protein intake. The PER (ability of protein to support growth) of NFSMF (2.48) compared well with the PER of milk diet (2.68). This observation agreed with the findings of Okoye (1992) who reported that the PER of casein was 2.50. This observation demonstrated the positive contribution of crayfish to the test diet. Protein Advisory Group, PAG (1971) recommended a PER value of 2.10 for complementary foods. Net protein ratio (NPR) and relative NPR indices accounted for the maintenance requirement of the weanling rat. It relates the weight changes in the animals fed the test diets to those fed the control diet (Shiriki et al., 2015). The non-essential amino acids also followed the same pattern as NFSMF had lower values compared to FSMF in most cases. The amino acid content of FSPF was slightly higher than NFSPF except for aspartic acid and cystine. The reduction in amino acids with fermentation may be due to its utilization for growth and production of enzymes and other organic compounds by the microorganisms during SSF. Reduction in the concentration of some amino acid of the blends during solid state fermentation was in agreement with the findings of Madgi (2011) who observed a reduction in amino acid content of pearl millet after 24h fermentation. Growth Response of Rats Fed with Sorghumbased Complementary Foods: The results of the growth response of rats fed with the test diets, NFSMF, NFSPF, FSMF and FSPF as presented in Figure 1 revealed that NFSMF competed keenly with the standard diet (milk) followed by NFSPF and FSMF with FSPF recording the least growth performance. The encouraging performance of the non fermented diets could be partly due to absence of flovour and taste imparted by fermentation leading to increased consumption and subsequent increase in weight. 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