Screening Of Protease-producing Bacterium In Facultative Anaerobic Conditions And Study On Its Application For Fermenting Soybean Meal

In order to improve the quality of fermented soybean meal(SBM), one bacterialstrain that produced protease both under anaerobic and aerobic conditions wasisolated and identified as Bacillus methylotrophicus NCU507. However, thefermented SBM produced an unpleasant flavor which could be relieved byco-fermentation with Lactobacillus plantarum NCU116. In the comparison ofmonoxenie fermentation and mixed fermentation, the presence of L. plantarumNCU116led to a shorter stable time of NCU507(from48h to24h). In addition,crude protein content and titratable acidity of mixed fermentation were higher thanthose of monoxenie fermentation.This paper conducted the following studies:(1) Research on technology of breeding special bacteria fermented SBM.Protein-rich samples like fermented blank bean and fermented soybean were collectedfor isolation of bacterial strains. They were suspended, diluted and plated on caseinmedium. Microorganisms showing transparent cycle in both aerobic and anaerobicenvironments were picked up for16S rRNA analysis. The obtained16S rRNA genesequence was blasted and deposited in genbank. Phylogenetic trees(NJ) wereconstructed by MEGA5.2. Based on16S rRNA sequence analysis, NCU507showed100%identity to B. methylotrophicus (EU194897). Meanwhile, NCU507was foundto show the shortest distance with B. methylotrophicus (EU194897) in phylogenetictree(NJ). It was thus determined as B. methylotrophicus NCU507.(2) Cytotoxicity assay of B. methylotrophicus NCU507. Firstly, the growthcurve of B.methylotrophicus NCU507was measured. In0~8h, the numbers of B.meth--ylotrophicus NCU507increased rapidly to9.1×107, then in8~20h, thenumbers were increased slowly and reached to4.8×108; At40h, the nubers werereduced and downed to1.8×107. Then, B. methylotrophicus NCU507(108cfu/mL、107cfu/mL and106cfu/mL) were applied on Caco-2monolayers for24h and thecytotoxicity was evaluated by measurement of LDH release and microscopicobservation. The result show that the amount of LDH release in the supernatant of Caco-2cultured in the absence or presence of the bacteria was unchanged, indicatingthat B. methylotrophicus NCU507didn’t cause cell lysis.(3) The optimal conditions for protease activity and stability was pH7.0and40℃,the enzymic activity was activated by Mn2+(4).Research on B. methylotrophicus NCU507solid state fermentation ofsoybean meal. On the basis of single-factor experiments, the optimization of ferm--entation time was96h, temperature was37℃，inoculum was4%and the material towater ratio was1:1. Response surface analysis showed that the optimization offermentation time was85h，temperature was36.8℃, material to water ratio was1:1.2.For the dynamic analysis of soybean meal fermentation, Bacterial quantityexhibited a stable stage at about8.85logCFU/g during the first48h. It then decreasedsignificantly (P<0.05, from8.83to6.66logCFU/g) in48~96h. The relative contentof crude protein increased from47.68%to52.90%. Content of small peptide went upquickly in the first36h, then followed a slower increase (from21.40%to28.13%).The titratable acidity of monoxenie fermentation maintained at about0.49%(w/w)in the initial24h, then decreased significantly (from0.48%to0.15%, w/w) in24~72h. From72to96h, titratable acidity declined to and remained at0%(w/w).(5) Research on mixed solid state fermentation of soybean meal. On the basisof single-factor experiments, the optimization of fermentation inoculation ratio was1:4, inoculum was9%, time was96h, the material to water ratio was1:1. Responsesurface analysis showed that the optimization of inoculation ratio was1:5, inoculumwas7.5%, material to water ratio was1:1.2.For the dynamic analysis of soybean meal fermentation, the presence ofNCU116in the mixed fermentation showed that the stable time of NCU507wasshortened (from48h to24h). At first, the counts of NCU507were remained at9.58log CFU/g, then decreased significantly (from9.72to4.72log CFU/g) in24~96h.However, the counts of NCU116kept stable at8.83log CFU/g in a longer time until48h, then decreased (from8.89to6.00logCFU/g. The relative content of crudeprotein during mixed fermentation increased from47.68%to57.08%(w/w). Thecontent of small peptide increased significantly (from0.30%to22.55%) in the first 48h, then the rising speed was slower in48~96h. At the end, the content reached to26.78%. A fast increase in titratable acidity of mixed fermentation (from the initial0.60%to1.73%, w/w) occurred during the first12h, and then decreased significantly(from1.73%to0.75%, w/w) in12~48h followed by a stable period in48~96h.