| Lactobacillus helveticus, is a lactic-acid producing, rod-shaped bacterium of the genus Lactobacillus. Many researchs shows that the strains belonging to the L helveticus species have health-promoting properties such as preventing gastrointestinal infections, enhancing protection against pathogens, modulating the host immune responses, and affecting the composition of the host intestinal microbiota. L. helveticus cell surface layers (S-layers) are monomolecular arrays composed of a single protein or glycoprotein species. The wealth of information available on the structure, chemistry, genetics and assembly of S-layers revealed a broad spectrum of applications in microcarriers, diagnostic devices, biosensors, vaccines, as well as targeting and delivery systems.In this study, the Fed-Batch fermentation experiments are conducted to establish optimum culture medium for L. helveticus fermentation based on the formulation of MRS medium in the first. The effects of carbon and nitrogen sources, and carbon-to-nitrogen ratio, on the growth of L. helveticus is examined by single factor tests. Secondly, the Fermentation conditions such as temperature, inoculum size, fermentation pH are optimized for maximum production cell biomass of L. helveticus. Finally, the S-layer protein extraction conditions are optimized. Through a series of exploration and optimization, the number of viable cells in the fermentation and the amount of S-layer protein extraction significantly increased. The main conclusions of this study are as follows:1.Applying single factor experiment to optimize L. helveticus main composition of culture medium in the shake flask fermentation, we find yeast powder and glucose are significantly better than the other carbon and nitrogen source; and adjustment of the ratio between the carbon and nitrogen element to achieve better effect:the enrichment effect of NaAC,K2HPO3 and Na2PO3 as the only buffer salt in culture medium are better than others. Then orthogonal experiment of three kinds of buffer salt is executed to optimize the medium composition, optimal medium composition as:yeast extract 14.1 g/L, NaAC 8.12g/L, Tween 80 1.0g/L,K2HPO3 18.61g/L,Na2PO3 9.94g/L. beef extract 10g/L, glucose 15g/L,MgSO4·7H2O 0.2g/L,MnSO4·H2O 0.05g/L. The number of viable cells reach to 9.4×108CFU/mL. that is 2.7 times higher than MRS.2.Firstly, on the basis of optimal medium to determine the optimal fermentation conditions in shake flask by single factor experiment, the conditions as:fermentation temperature 37 ℃. inoculum 5%, standing fermentation. On this basis we use the 10L fermenter to optimize fermentation condition and the fermentation condition are temperature 37℃, inoculum 5%, anaerobic fermentation and pH=6.8. By optimizing the fermentation conditions, the number of viable cells reach to 1.4×109CFU/mL in the 10L fermenter, which is 4-fold compared to control group.3. Through the pre-experiment results, we found that different cell harvesting times have a significant influence on the amount of the S-layer protein extraction. We study the effect of different harvested time12 h,16 h,20 h and 24 h on the S-layers protein purity and quality. As a result, when 20 hours of fermentation we harvest cells can obtain the maximum extraction amount of the S-layer protein. That is 1.48mg per gram of wet cells. |
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