Screening, Identification And Optimization Of Fermentation Conditions For Bacterial Strain Producing Lactobacillin

Lactobacillin is the protein with biological activity arising from lactic acid bacteria, which is added to foods to inhibit spoilage organisms and pathogens.At present, the G- bacteria can't be inhibited by Nisin which is widely used in food industry.So it is necessary for us to find a lactobacillin-producing lactic acid bacteria which can inhibit both G＋and G- bacterium.In this paper, lactobacillin-producing bacterium is screened from the traditional fermented food (such as milk, mustard, pickled garlic, pickled beans) in Hefei. First of all, through the enrichment of skim milk culture medium and the use of color reaction and Gram staining, biochemical analysis, such as paper chromatography technology, screening of lactic acid bacteria have been nine strains. And then five kinds of bacterium such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli. Aspergillus niger, Saccharomyces cerevisiae, were used as indicator. Oxford cup with agar diffusion method for advanced screening, and the method of being excluded by experimental acid, hydrogen peroxide finalized the selection of lactic acid bacteria--D8 strain,which could inhibit the both G＋and G- bacterium growth. And the result of degradation with papain and trypsin prove that the lactobacillin which D8 strain produced is protein. Whereas the result of the test of gene stability show that the gene of D8 strain is stable.Through detection of D8 strain's appearance, biochemical characteristics and 16S rDNA gene sequence homology analysis, the it was identified as Lactobacillus plantarumThe optimal medium for the D8 strain producing lactobacillus best were as follows:beef extract 2%, yeast extract 0.75%, sucrose 2.5%, triammonium citrate 0.2%, sodium acetate 0.5%, K2HPO40.4%, MgSO4·7H2O 0.058%, MnSO4-4H2O 0.025%.The optimum fermentation conditions were:fermentation temperature 35℃, initial pH value 6.0, and adding 2% tween-80. In these conditions the diameter of bacteriostatic ring can reach 3.15 cm. Based on the experimental data of batch fermentation, the kinetic model of cell growth, lactobacillin production and substrate consumption were proposed. The Logistic model for growth, the Luedeking-Piretequation equation for lactobacillin production and a simple equation for sucrose consumption were constructed. Results showed that the production of lactobacillin was partly growth-associated.Immobilization of D8 strain is also studied in this paper. Results showed that the immobilization of D8 strain to produce the lactobacillin is possible. The optimal factors for the immobilization of D8 strain are:alginic acid 5%, cell concentration 3 %, CaCl2 1.5%. The cell concentration of immobilized D8 strain is 2.5 X 108 cfu/mL. The optimum fermentation conditions of the immobilized D8 strain are:fermentation temperature 40℃, initial pH value 7.0, and without adding tween-80.The optimization of fermentation conditions for D8 strain producing lactobacillin and the study on kinetic model and immobilization lay the foundation for the further investigation.