Effect Of Nitrogen Source On Lyophilization Tolerance And Fermentation Viability Of Lactobacillus Bulgaricus B61-3

Lactobacillus bulgaricus is the core fermentation strain in fermented milk,and its lyophilized powder ferment is the main form of industrial application.However,the freeze-drying process can cause damage to the bacterium,reduce the strain survival and fermentation viability,and degrade the fermentative performance of the fermentate.Studies have shown that the nitrogen source of the medium is an important factor in the preparation of freeze-dried fermenters,but the effect of nitrogen source on the fermentation viability of Lactobacillus bulgaricus freeze-dried powder is not completely clear.In this study,we analyzed the effect of nitrogen source on freeze-drying tolerance and fermentation vigor of lyophilized powder of Lactobacillus bulgaricus B61-3,and investigated the mechanism of the effect of nitrogen source on fermentation vigor of the strain.This study provides some basis for the development and application of autonomous fermenters.The results of the study are as follows:Lactobacillus bulgaricus B61-3 was used as a research object to screen the types of nitrogen sources and their additions that could improve the freeze-drying resistance of the bacterium based on 16 commonly used industrial fermentation nitrogen sources.The results showed that 3.0% of bovine bone peptone significantly increased the freeze-drying survival rate of B61-3 strain from 9.68% to 18.90%,and further found that the average fermentation viability of lyophilized powder prepared with bovine bone peptone as the nitrogen source increased by 22.15% compared with the control group,and the fermentation end point time was shortened by 1 h.The electron microscope observation showed that the fermenting organisms in the control group exhibited long and The electron microscope observation showed that the fermentative organisms of the control group showed long and irregular curly shape,while the organism morphology showed a short rod shape with smooth surface when bovine bone peptone was used as the nitrogen source.The length-to-diameter and area-to-volume ratios were significantly reduced,which may be related to the improved lyophilization survival and lyophilized powder fermentation viability of the strains.In order to analyze the mechanism of bovine bone peptone to improve the fermentation viability of the strain,we firstly analyzed the utilization of amino acids in different nitrogen sources,and the results showed that the amino acid content of bovine bone peptone was significantly higher than that of the control group,and the amino acid verification test confirmed the best effect of phenylalanine,which significantly increased the number of viable bacteria in culture and lyophilization survival rate,and the lyophilization survival rate increased from 8.43% to 16.02%.The freeze-drying process led to a significant decrease in the activity of fermentation key enzymes such as lactate dehydrogenase,β-galactosidase and ATPase,while the intracellular activities of all the above enzymes were significantly higher than the control group and the extracellular β-galactosidase activity was lower after freeze-drying in the bovine bone peptone cultured strain.The gene expression results showed that the relative gene expression of cysteine ABC transporter protein permease YxdL,amino acid ABC transporter ATP binding protein YxdM and membrane formation related gene YfhO in the biofilm synthesis pathway of the strain cultured in bovine bone peptone were significantly up-regulated(p< 0.05),confirming that bovine bone peptone regulates cell membrane synthesis and protein transport related genes,improves cell membrane integrity and maintains enzyme activity,thus promoting the fermentation vigor of the strain.