M. M. Rahman
Institute of Food Science & Technology, Bangladesh Council of Scientific & Industrial Research Dhaka-1205, Bangladesh
M. M. Rahman*
Institute of Food Science & Technology, Bangladesh Council of Scientific & Industrial Research Dhaka-1205, Bangladesh
U. F. Shahjadee
Institute of Food Science & Technology, Bangladesh Council of Scientific & Industrial Research Dhaka-1205, Bangladesh
A. Z. Rupa
Institute of Food Science & Technology, Bangladesh Council of Scientific & Industrial Research Dhaka-1205, Bangladesh
M. N. Hossain
Institute of Food Science & Technology, Bangladesh Council of Scientific & Industrial Research Dhaka-1205, Bangladesh
Germinated soy flour; Nutritional quality; Aflatoxins, Microbiological quality
Institute of Food Science & Technology, Bangladesh Council of Scientific & Industrial Research Dhaka-1205, Bangladesh
Quality and Nutrition
Collection of Soybean seed Soybean seeds were collected from Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur. For experimental analysis, 20 seeds of each kind were placed on water soaked filter paper in sterilized Petri dish. Distilled water was added daily to the experimental seeds. Completely Randomized Design (CRD) was followed for placing the Petri dishes on the laboratory bench. The proximate analysis, amino acids content, minerals and enzyme activities were evaluated of raw and germinated soy flour. Preparation of germinated Soy Flour- Processing of soy bean seeds The soybean seeds were soaked in 0.5% aqueous calcium hypochlorite solution for 2.0 min and then washed 7-8 times with distilled water to remove hypochlorite residues from the seed surfaces. After surface sterilization, the soybean seeds were soaked in distilled water in a sterilized bowl for 16-18 hours. Then 1.0 kg seeds were placed on sterilized wet cotton cloth in a sterilized tray (4ft x 2ft) in dark condition for germination at room temperature. During the germination, 100ml distilled water was sprayed on soybean seeds to support the germination process and the room temperature was recorded as 19°C. After germination the grains were boiled for one hour to remove tripsin inhibitor and then dried up to 10% moisture content using solar/mechanical drier. The dried soybean were devegetated and ground to make germinated soy flour. Proximate analysis The proximate analysis including protein, fat, carbohydrate, ash, fiber, moisture and mineral contents of raw and germinated soy flour was evaluated. Protein content was determined using Macro-Kjeldahl method. (Ranganna,1979). The carbohydrate fat, ash, fibre, moisture, and mineral contents (calcium, phosphorus and iron) of soybeans were determined by the method as described in "A Manual of Laboratory Techniques" (Anonymous, 1976). Enzyme activity assay Raw and germinated soy flour (0.5g) was separately grinded in a mortar with cold 0.1M phosphate buffer with respective pH (amylase & invertase at pH 6.7 and protease at pH 5.5) and finally crushed into paste using a homogenizer. The temperature was maintained at 4oC by putting ice in the outer chamber of the homogenizer. The suspension was then filtered through few layers of cheese cloth in cold room. The filtrate was collected and clarified further by centrifugation in a refrigerated centrifuge at 10,000 rpm for 15 min at 4oC. Amylase Amylase activity was assayed by the method as described (Jayaraman, 1981). Starch solution (1.0%) was used as substrate (1.0g in 100 ml of 0.1M phosphate buffer, pH 6.7). The amylase activity was measured by estimating the release of maltose calculated from the standard curve prepared with maltose. One unit of amylase activity was defined as the amount required for liberating 1.0 mg of maltose in 15 min at 37oC. Invertase Invertase activity was assayed by the method as described (Mahadevan and Sridhar, 1982) using sucrose as substrate. The invertase activity was measured by estimating the release of glucose calculated from the standard curve prepared with glucose. One unit of invertase activity was defined as the amount required for liberating 1.0 mg of glucose in 15 min at 37oC. Protease Protease activity was assayed by the method as described (Mahadevan and Sridhar, 1982) using milk protein casein as substrate. The protease activity was measured by estimating the release of leucine calculated from the standard curve prepared with leucine. One unit of protease activity was defined as the amount required for liberating 1.0 mg of Leucine in 30 min at 37oC. Amino acids analysis Amino acid composition of germinated and raw soy flour was determined by using an amino acid analyzer (Model No: 228-39015-38; Shimadzu, Japan), able to determine fourteen amino acids (Anonymous, 1993). Sample (0.5g) soy flour was pasted with 50ml 6N HCl by mortar pestle, filter and filtrate was hydrolyzed for 22-24 hours in a hydrolyzing apparatus. After hydrolyzing HCl was removed from filtrate with distill water for 3-4 times by evaporation in a water bath. After completing the evaporation, the stock solution was prepared and mark up to 25ml in a volumetric flask by using 0.1N HCl. This stock solution was used for the determination of amino acids. Microbiological analysis- Total viable count Test sample and microbial media were prepared according to standard operating procedure. The sample suspension and decimal dilutions were prepared following international guidelines (ISO 6887). The test sample 1.0 gram were mixed thoroughly in plate count agar and poured into the petri dishes and kept in the biosafety cabinet at room temperature to solidify. The inoculated petri dishes were incubated upright position in the incubator at 30 °C ± 1 °C for 72 h ± 3 h. All the analysis was done in duplicate and the results was expressed in CFU/g according to the ISO 7218:1996 methods. Coliforms and E. coli Test sample, initial suspension and sufficient number of dilutions were made following the standard method (ISO 6887). The diluted and non-diluted samples were poured in screw cap tube containing double and single strength Lauryl sulfate tryptose broth, EC broth and Brilliant green lactose bile broth separately and incubated at 30 °C or 37 °C for 24 h or 48 h. Three tubes of double and single strength liquid selective enrichment medium are inoculated with a specified quantity of the test sample if the initial product is liquid, or with a specified quantity of an initial suspension The most probable number of coliforms per millilitre or per gram of sample (i.e. the MPN) is calculated from the number of tubes in the new series showing gas formation (ISO 4831:2006). A table for determination of most probable numbers is used (ISO 7218, ISO 7251:2005). Yeast and molds The Dichloran Rose Bengal Chloramphenicol (DRBC) and Dichloran 18% Glycerol (DG18) agar media were prepared according to instruction. Serial dilutions of the sample were prepared using peptone solution (ISO 6887). The petri dishes were prepared and inoculated using spread plate method. For high water activity foods (aw > 0.95), Dichloran Rose Bengal Chloramphenicol (DRBC) agar was used and for reduced water activity foods (aw < 0.95), Dichloran 18% Glycerol (DG18) agar was used. The DRBC plates were incubated upright position at 25 ±1°C for 5 days and DG18 agar plates were incubated for 7 days. Confirmation of individual presumptive yeast colonies was done by microscopic examination, as some bacteria are capable of growth on DRBC agar (ISO 21527-1, 2: 2008). The result was expressed according to international methods (ISO 7218:1996). Salmonella Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. (ISO 6579:2002, MOD). Pre-enrichment in non-selective liquid medium, Enrichment in selective liquid media, selective agar media, biochemical media and serological reagents were prepared following the international method (ISO 6579:2002, MOD). Test sample diluted in peptone water was inoculated in preenrichment in non-selective liquid medium and incubated at 37 °C ± 1 °C for 18 h ± 2 h. Enrichment in selective liquid media Rappaport-Vassiliadis medium with soy (RVS broth) and Muller-Kauffmann tetrathionate/novobiocin broth (MKTT broth) are inoculated with the culture incubated at appropriate temperature for 24 h ± 3 h. Then from enrichment medium the culture were plated onto XLD agar plate for the isolation of lactose-positive Salmonella and Salmonella typhi and Salmonella paratyphi strains. Statistical analysis Each experiment was replicated three times. Reported data represented in the tables are the mean values ± SD obtained from three individual experiments. Data were subjected to analysis of variance using the Microsoft Excel program (Redmond, Washington DC, USA.).
Bangladesh J. Sci. Ind. Res. 51(3), 167-174, 2016
Journal