The Study On Screening, Identification And The Active Site Of The Flocculating Activity Strains

In this paper,27flocculation activity strains were isolated from sour liquidby dilution coated plate method. At last,8highly efficient and stableflocculation activity strains were re-screened. Those strains were identified asLactobacillus paracasei, based on their colony character, physiologicalcharacteristics, as well as16S rDNA sequence homology comparison andreferencing Bergey’s Manual of Systematic Bacteriology.Four significant factors have selected from the basal medium by thesingle-factor and Plackett-Burman experimental design, which includingglucose, sodium acetate, magnesium sulfate, yeast extract.Using theresponse surface design to optimize and analysis these four factors. finally,the optimal concentrations of the response factors were obtained, Theoptimum fermentation medium were determined as follows（g/L）: glucose20g,sodium acetate5.85g, MgSO₄·7H₂O0.378g, yeast extract9.15g.Based on single-factor experiment, four response factors including initialpH, inoculation, cultivating temperature and cultivating time were select asresponse factors and flocculation rate was used as response value, thenfigure out the relationship between factors and flocculation rates withBox-Behnken central composite experiment. Finally build the quadraticregression equation was Y1=-103.602+5.166X₁+5.848X₂+8.312X₃+0.0580X₄-1.028X₁²-0.14X₁X₂+0.263X₁X₃+0.0185X₁X₄-0.141X₂²-0.097X₂X₃-0.003X₂X₄-0.166X₃²-0.002X₃X₄-0.0006X₄²and get the correlation coefficient R²=0.9550.By analyzing the significant impact and the interaction of each factor, Theoptimum culture conditions were determined as follows: initial pH of6.0,inoculation of8%, cultivating temperature of27℃, cultivating time of48h.Under this culture conditions, the theoretical value of the flocculation rate was48.72%and the verified value was48.65%.The flocculation activity of the strain’s fermentation medium, the cell precipitate suspensions and bacterial extracellular polymers were measuredto determine the active site of the bacterial, and it was showed that the activesite is the bacterial itself. The next step was extracted bacterial surfaceproteins, cell wall, protoplast then determination of the flocculation rate, theresult showed that the active site of flocculation activity strains is thesurface-layer protein.