Purification And Application Of Antifungal Metabolites From Lactobacillus

In this study,Lactobacillus plantarum ALAC-4 and ALAC-6 as the research object,which has the antifungal characteristics.The metabolites of ALAC-4 and ALAC-6 were purified and characterized and were used as biological preservatives in the production of soy sauce.The results were showed as follows:?1?The metabolites of Lactobacillus plantarum ALAC-4 and ALAC-6were purified by ammonium sulfate precipitation,gel filtration chromatography and reversed-phase high performance liquid chromatography,and after ultrafiltration centrifuge tube concentration,and obtained the antifungal proteins NS3-1 and SDZ3-1.The rate of antifungal of NS3-1 and SDZ3-1 was 59.88%and 61.76%,respectively.The protein content of NS3-1and SDZ3-1 was 51.24 g/m L and 56.61 g/m L respectively,which determined by Bradford method.Both NS3-1 and SDZ3-1 have reached electrophoretic purity,which were detected by electrophoresis with SDS polyacrylamide.The relative molecular mass of NS3-1 is about 31.04 ku,and the relative molecular mass of SDZ3-1 is about 37.37 ku.We did mass spectrometry analysis to NS3-1 and SDZ3-1 by MALDI-TOF/TOF^{T M} method and retrieved the database of Lactobacillus protein by Mascot search software,and the results showed that NS3-1 and SDZ3-1 were unknown proteins.?2?By studying the antifungal properties of NS3 and SDZ3,it is found that NS3 has antifungal activity between p H3.0-6.0 and SDZ3 has antifungal activity between p H3.0-5.0.The NS3 and SDZ3 have good heat stability.The antifungal activity of NS3 is not affected by ultraviolet radiation,and the inhibition of ultraviolet radiation on SDZ3's antifungal activity.Methanol,ethanol,isopropanol and acetonitrile inhibit the inhibition of NS3 and SDZ3to varying den grees.Na⁺,K⁺,Ca²⁺and Zn²⁺promoted the inhibition of NS3to different degrees.Fe²⁺had little effect on the antifungal activity of NS3.Na⁺,Ca²⁺and Fe²⁺inhibited the inhibition of SDZ3 to varying degrees.K⁺and Zn²⁺had little effect on the inhibition of SDZ3.Tween-20,Tween-80 and SDS promoted the inhibition of NS3 and SDZ3 to varying degrees.Glycerol had little effect on the inhibition of NS3 and SDZ3.Both NS3 and SDZ3 have good salt tolerance.The minimum inhibitory concentration of NS3 was 13.66?g/m L,the antifungal rate was 91.50%,the minimum inhibitory concentration of SDZ3 was 35.76?g/m L,and the antifungal rate was 90.17%.?3?Lactobacillus plantarum ALAC-4 and ALAC-6 have a wide range of fungal inhibition,and can grow in salt concentration of 2%-14%.The soy sauce can reach the standard of marketed soy sauce in the aspects of sensory evaluation and physicochemical indexes,which were produced by soy sauce produced by the addition of strains and the soy sauce produced by the soy sauce which are not added to the soy sauce.During the antifungal test,two methods were adopted:1:Adding Lactobacillus plantarum ALAC-4 and ALAC-6 to the production of soy sauce.After the soy sauce was successfully brewed,it was diluted three times,and the total number of fungi and yeast was determined for five days.2:After adding the pure protein NS3 and SDZ3 to the homemade soy sauce,it was diluted three times,and the total number of fungi and yeast was determined for five days.The total number of molds and yeasts measured by the two methods was lower than that of the three times diluted marketed soy sauce.It is indicated that the addition of Lactobacillus plantarum to the production process of soy sauce or the addition of the pure protein products to the soy sauce can inhibit the growth of the fungus and prolong the shelf life of the soy sauce.