Effect Of Fermentation Temperature On The Molecular Mechanism Of Bacterial Activity Of Lacticaseibacillus Paracasei PC-01 Before And After Freeze-drying

Lacticaseibacillus paracasei PC-01 is a lactic acid bacteria with good prebiotic characteristics isolated from sour yak milk in Longren Township,Dangxiong County,Lhasa,Tibet.It is a commonly used starter for the production of dairy products,cereal products and other products.It has a wide application prospect and economic value.Many studies have shown that in the process of high-density fermentation,different culture temperatures will have a significant impact on the activity of fermentation strains before and after freeze-drying,but there are few deep-seated mechanisms of significant differences in the activity of fermentation strains affected by different culture temperatures.Therefore,it is of great significance for the development and utilization of Lacticaseibacillus paracasei PC-01 to reveal the influence mechanism of internal protein and phenotype of the strain on the activity of Lacticaseibacillus paracasei PC-01 under different high-density fermentation temperatures.In this study,Lacticaseibacillus paracasei PC-01 was selected as the research object.By setting different culture temperatures of 32.5℃and 37℃,Lacticaseibacillus paracasei PC-01 was highly fermented and freeze-dried.During the fermentation,the number of live bacteria and OD value were measured and the growth curve was made.Based on this,the key time points of different growth periods were selected,Quantitative determination of TMT protein was carried out on samples taken from key time points and after freeze-drying of bacteria by using Tandem Mass Tags(TMT)technology.KEGG function annotation and enrichment analysis were carried out on the identified differential proteins.At the same time,the key enzyme activity,cell membrane fatty acid,cell membrane fluidity,cell membrane hydrophobicity and cell membrane integrity of samples before and after freeze-drying were measured.The morphology of freeze-dried bacteria was observed by scanning electron microscope,The experimental results show that:(1)After high-density fermentation,the number of viable bacteria before and after freeze-drying in the 32.5℃experimental group was significantly higher than that in the37℃experimental group;(2)Before and after freeze-drying,the activities of Na~+-K~+-ATPase and Lactate Dehydrogenase(LDH)in the 32.5℃experimental group were higher than those in the37℃experimental group;The fluidity,hydrophobicity and integrity of cell membrane in the 32.5℃experimental group were higher than those in the 37℃experimental group.The ratio of unsaturated fatty acid to saturated fatty acid in the 32.5℃experimental group was significantly higher than that in the 37℃experimental group;After freeze-drying,the degree of cell shrinkage in the 32.5℃experimental group was less than that in the 37℃experimental group;(3)Taking the 37℃experimental group as the control group,162 proteins were differentially expressed in the 32.5℃experimental group before freeze-drying,of which29 proteins were significantly up-regulated(FC≥1.5,P<0.05)and 133 proteins were significantly down regulated(FC≤0.83,P<0.05);After freeze-drying,195 proteins were differentially expressed in the 32.5℃experimental group,of which 18 proteins were significantly up-regulated(FC≥1.5,P<0.05)and 177 proteins were significantly down regulated(FC≤0.83,P<0.05).After analyzing the action mechanism of different proteins between the two experimental groups by KEGG database,it was found that the main metabolic pathways affecting the cell activity of the two experimental groups during high-density fermentation were membrane transport and carbohydrate metabolism,and the protein with the largest difference multiple was component s of ECF transporter,whose function was related to membrane transport;After freeze-drying,the significantly different proteins are mainly concentrated in pyruvate metabolism,fructose and mannose metabolism,purine metabolism and other metabolic pathways.The protein with the largest difference is aldehyde ketone reductase,which is mainly involved in the synthesis and metabolism of fatty acids.The purpose of this study is to analyze the influence mechanism of different high-density fermentation temperature on the activity of Lacticaseibacillus paracasei PC-01before and after freeze-drying,so as to provide theoretical reference for the industrial production of probiotic products.