Preparation of Soybean Peptide and Its Effects of Immune Function on Mice
|School||Guangdong Ocean University|
|Course||Animal Nutrition and Feed Science|
|Keywords||soybean peptide protease liquid fermentation immune function mice|
The purpose of this study was to carve out a low cost production route of soybeanpeptide, through screening strains from the natural environment that suitable for the liquidfermentation of soybean meal, and the fermentation technology of soybean peptide wasoptimized to improve the degree of protein hydrolysis and increase the low molecularpeptide of the fermented liquid.98samples were collected from the natural environment, growing plants and animalintestinal tract of Zhanjiang.117Bacillus strains that produced proteinase were isolatedfrom the samples by the flat transparent circle method, and there were11strains’ diameterratios of the transparent circle and colony （H/C） were more than5.0; The proteinaseactivities of the11Bacillus strains’ fermented liquid were measured by Folin method, andthe y-6strain’ s protease production was the most powerful, its proteinase activity of thefermented liquid reached to2852IU/mL when in48hour; the y-6strain was identified asBacillus amyloliquefaciens through the morphological and dyeing characteristics’observation. Physiological and biochemical tests and16S rDNA gene sequence analysis,and temporarily named Bacillus amyloliquefaciens strain y-6.Bacillus amyloliquefaciens strain y-6was inoculated into the GYP medium with4%,through oscillatory fermentation in32℃,180r/min for6,12,18,24,30,36,42and48hours, then4000r/min centrifuged for20min, and the supernatant was the measured crudeenzyme; The enzyme activity of the protease, amylase, cellulose enzyme, chitosan enzyme,pectinase of the crude enzyme was measured respectively. The results showed that, theactivity of the protease, amylase, cellulose enzyme and chitosan enzyme was1072.3IU/mL,3.75IU/mL,8.35IU/mL,565.9IU/mL respectively after48hours, there was noobvious pectinase activity. The Protease properties of the fermentation broth was studied,The results showed that, the protease was neutral proteinase, the enzyme activity washighest when the pH value was7.07.5, and its most suitable temperature was55℃, ithad good temperature stability which the temperature belowed40℃; This enzyme did nottolerate acidic environment，and it was deactivated below pH5.0, it could not detectenzyme activity below pH3.0when kept for1hour; Ions Ca2+, Zn2+and Tween-80hadcertain activation function to the enzyme, while ions Mn2+、Fe3+, Cu2+and EDTA hadcertain inhibition to it. The experimental design was arranged and the experiment data was analyzed with theMinitab version15.1software, The technology of soybean peptide preparation that theBacillus amyloliquefaciens strain y-6fermented low temperature soybean meal was studied;The main effects and interaction of factor pH, inoculated quantity, shaker rotate speed,fermentation temperature and volume of medium were studied by Plackett Burman design,and three important influence factors of inoculated quantity, temperature and shaker rotatespeed were determined;15response surface analysis tests were designed by Box-Behnkencenter combination, and parameters optimum selection of the three factors of inoculatedquantity, temperature and shaker rotate speed were further conducted; The optimizedparameters were: inoculated quantity6.3%, fermentation temperature39.89℃, shakerrotate speed187r/min. The test results and the regression model predicted a goodcoincidence, this suggested that, the establishment of the regression model was reliable,and the soybean peptide conversion could be accurately predicted; The soybean peptideconversion increased from63.7%to78.15%after the fermentation parameteroptimization.120mice were randomly allotted into5lots, each with4groups, each group was tenmice; The control group was provided with the basal diet, the there treatment groups werefed with basal diet supplemented with Homemade soybean peptide at the level of1%,2%,4%respectively, the immune organ index, serum lysozyme, phagocytosis of macrophagusand serum hemolysin were measured after21days; The results showed that, the soybeanpeptide could significantly increase the immune organs index, enhance the phagocyticactivity of the peritoneal macrophage, stimulate the production of mice’s chicken antierythrocyte antibody, but the serum lysozyme activity had no significant difference.The result indicated that it was completely feasible to perpetrate soybean peptide withthe high yield protease Bacillus to ferment low temperature soybean meal, and it had ahigh content of soybean peptide, it could also enhanced the mice’s immune function.