Optimization Of Culture Medium For Lactobacillus Helveticus, Isolation And Application Of Surface Layer Proteins

S-layer proteins represent the outmost component of many bacteria and archaea.Each S-layer protein is composed of a single protein or glycoprotein, scarcely of twoor more protein species, and forms crystalline arrays. The S-layer protein subunits arenon-covalently linked to each other as well as to the supporting cell wall, and can bedisintegrated into monomers by high concentration of hydrogen bond-disruptingagents such as urea，lithium chloride or guanidine hydrochloride. The content ofnon-polar amino acids of the S-layer sequences is relatively high, resulting in theirhydrophobic property. That is the reason of their ability of self-assemble,whichmeans isolated S-layer subunits can recrystallize into regular arrays in suspension,air-liquid interfaces, on solid supports, or lipid films and liposomes once thedisrupting agent used for their isolation has been removed.Lactic acid bacterial which can colonize human intestinal tract is beneficial tohuman body. They can improve or adjust intestinal microbial flora balance.Metabolites of them also have various benefits, such as reducing gut pH value,inhibiting spoilage bacterial growth and weakening their ability to produce poison,preventing constipations，helping digest, enhancing immunity. It has been reportedthat colonization of Lactic acid bacterial that is believed to be the first step forbenefits is related to surface layer proteins.On the basis of ability to self-assemble and be adhesive to intestinal tract, theobjective of the paper was to study whether the S-layer can improve properties ofdrug delivery carrier-alginate microspheres. That is to say when the S-layer assembledon the surface of alginate microspheres, the properties of sustained release andadhesion to tissues of microspheres were better or not. We chose Lactobacillushelveticus for isolation of S-layer proteins. First, we optimized the extraction methodsof the S-layer by two ways:（1）optimize the culture medium of Lactobacillushelveticus, for the purpose of increasing the amount of bacterial;（2）compare the extraction methods of different denaturing agents in different concentrations anddifferent pH values,in order to make purity and amount of S-layer proteins reach agood level. Then we studied the adsorption effect of S-layer proteins by fluorescencemicroscopy,scanning electron microscopy, and SDS-PAGE.In the experiment of optimization of culture medium, glucose, peptone, ammon-ium citrate tribasic were the three critical medium components among others by thePlackett-Burman design. The steepest ascent method was used to access the optimalregion of the medium composition. After using Box-Behnken design and response su-rface methodology, the optimal medium composition was achieved（g/L）: peptone,10;yeast extract,5;sodium acetate,6.25; Tween80,1.25; K2HPO₄,2.5; beef extract,8.96；glucose,9.47; ammonium citrate tribasic,3; MgSO₄·7H₂O,0.2; MnSO₄·H₂O,0.0625.In the experiment of comparisons among different extract methods, production ofS-layer proteins was the lowest by6M,8M,9M urea. Although the production ofprotein by2M,4M,6M guanidine hydrochloride and5M lithium chloride is aboutequal, the purity was better extracted by lithium chloride. So we chose （pH=2）5Mlithium chloride for protein extraction.Alginate microspheres absorption onto S-layer proteins was confirmed byfluorescence microscopy, scanning electron microscopy, and SDS-PAGE. The processtook place very quickly, so we presumed that the absorption process relied onelectrostatic force.