Screening,Characteristic And Application Of Lactobacillus With Antifungal Activity

Lactic acid bacteria are widely existed in natural environment.Studies have shown that some lactic acid bacteria can produce antifungal metabolites to inhibit the growth of fungi,and their application to food preservation or feed storage has important practical value.In order to establish a screening system of lactic acid bacteria with antifungal activity,the sensitivity,simplicity and feasibility of 96-well plate method,oxford cup agar diffusion method,punch method and dual-culture overlay assay method were compared.Lactic acid bacteria with broad-spectrum antifungal activity was screened out from 1158 strains of lactic acid bacteria preserved in the laboratory.Then,the fermentation characteristics and antifungal effect of the lactic acid bacteria were studied,and the antifungal active substances produced by lactic acid bacteria were further explored.The strain with excellent antifungal activity was applied to alfalfa silage to investigate their impact on microbial diversity and feed fermentation quality in the silage process;it was applied to the production of steamed buns to study its impact on the shelf life of steamed buns.The main results are as follows:(1)Antifungal activity on 9 strains of lactic acid bacteria were conducted by 96-well plate method,oxford cup agar diffusion method,punch method and dual-culture overlay assay method.The results showed that the 96-well plate method and dualculture overlay assay method could show the antifungal activity of two strains of lactic acid bacteria(ZZUA 530 and ZZUA 537)against Aspergillus niger.Compared to 96-well plate method,the dual-culture overlay assay method could show the difference of antifungal activity of ZZUA 530 and ZZUA 537 against A.niger.Therefore,a highthroughput screening system with 96-well plate as the primary screening method and dual-culture overlay assay method as the rescreening method was established.(2)Three strains of lactic acid bacteria with high inhibition on A.niger were screened out from 1158 strains by 96-well plate method,and they are ZZUA 493,ZZUA 570 and ZZUA 606 respectively.Among them,ZZUA 493 which has strong inhibitory activity to A.niger,Aspergillus oryzae,Trichoderma longibrachiatum and Aspergillus flavus was screened out by double-layer plate scribing method.It was identified as Lactobacillus plantarum by physiological and biochemical experiments and 16 S r DNA sequence analysis.Furthermore,ZZUA 493 had a strong antifungal activity on Staphylococcus aureus,Bacillus subtilis,Micrococcus luteus,Pseudomonas aeruginosa,Escherichia coli and Listeria monocytogenes.(3)The growth of Fusarium graminearum could be inhibited by adding 10-fold concentrated fermentation supernatant of ZZUA 493 to the culture medium,and the growth of F.graminearum could not be inhibited by adjusting the p H of the concentrated fermentation supernatant to 6.0.The antifungal activity of the fermentation supernatant of ZZUA 493 against A.niger was studied by 96-well plate method,and the results showed that the antifungal activity was not affected by heat treatment,protease and catalase,but decreased significantly when the p H of the fermentation supernatant was adjusted to 6.0.According to the above results,it can be inferred that acidic substances play an important role in the antifungal activity of ZZUA 493.(4)The alfalfa silage results showed that compared with the natural silage,the inoculation of ZZUA 493 could significantly reduce the p H of silage,increase the content of lactic acid and acetic acid;compared with the inoculated commercial strain YX,the inoculated ZZUA 493 could significantly reduce the content of E.coli and aerobic bacteria at 30 d of silage;compared to the control group,the content of ammonia nitrogen in silage at 60 d of inoculated with ZZUA 493 for 60 d decreased significantly.The results showed that the fermentation quality of alfalfa silage could be improved by adding ZZUA 493.(5)Compared with the control group without adding lactic acid bacteria,adding ZZUA 493 to the production of steamed buns could make the steamed buns have an acidic flavor;the results of steamed buns storage experiment showed that the number of fungi on the surface of steamed buns fermented by ZZUA 493 was significant lower than the control group,which indicated that the addition of lactic acid bacteria ZZUA 493 could extend the shelf life of steamed buns.