Optimization Of Fermentation Medium Of Lactobacillus Crispatus And Preliminary Isolation,Purification And Identification Of Their Antibacterial Substances

Candida vaginitis(VVC)is characterized by high incidence,and high recurrence,and is usually treated with antifungal drugs such as fluconazole and clotrimazole.But the female vagina is a dynamically balanced microecosystem that colonize the dominant Lactobacilli species and opportunistic pathogens.Antifungal drugs not only kill pathogenic bacteria but also affect the normal growth of Lactobacillus,fail to fundamentally restore the vaginal microecological balance,thus increasing the susceptibility of the vagina and leading to frequent recurrence of VVC.Lactobacillus crispatus is an endogenous vaginal bacterium that can secrete metabolites with bacteriostatic activity to resist the growth and colonization of pathogenic bacteria.This metabolite can theoretically be used as a potential drug to rebuild the microecological balance,reduce the generation of drug resistance and reduce the recurrence rate of VVC.Therefore,we plan to conduct preliminary separation,purification and identification of antibacterial substances,in order to build the foundations for the future research.In this study,the single factor experiment,Plackett-Burman design and Box-Behnken Design were used to optimize the fermentation medium composition and culture conditions of Lactobacillus crispatus to improve the production of antibacterial substances.The results were as follows:whey powder 20 g/L,glucose 4 g/L,dipotassium hydrogen phosphate 0.75g/L,initial p H 8.1,inoculation amount 4%,cultured at 37℃ for 32 h,the yield of antibacterial substances increased from 96.0 U/m L(MRS medium)to 166.2 U/ m L,increased by 42.2%.At the same time,the cost of medium is reduced.Experiments on protease sensitivity,acid and base stability and heat stability showed that the bacteriostatic substances might be glycoproteins or glycopeptides with good acid and base resistance and heat stability.The specific activity of antibacterial substances initially purified by centrifugation,ammonium sulfate precipitation,hydrophobic column chromatography,dialysis and other methods increased from 218.6 U/mg to 700.7 U/mg,an increase of3.2 times.Preliminary identification was carried out by phenol-sulfuric acid method,biuret reaction,UV,FTIR,HPLC and SDS-PAGE.The results showed that the antibacterial substance was a glycoprotein with a small amount of aromatic amino acids and a molecular weight of about 55 kD.