Effects Of Amino Acids On Freeze-drying Resistance Of Lactiplantibacillus Plantarum LIP-1 And Its Internal Mechanism

At present,there are few reports on the addition of amino acids to MRS medium to improve the freeze-drying survival rate of probiotics,and the internal mechanism of action is still unclear.In order to reduce the damage caused by freeze-drying to the strains,exogenous amino acids were added to the MRS medium to study the effects of different kinds of amino acids on the freeze-dried survival rate of four Lactiplantibacillus plantarum strains.And using Lactiplantibacillus plantarum LIP-1 as a model strain,the mechanism of action was explored.The results of the study showed that compared with the MRS blank control group without amino acids,adding 0.04 g/L aspartic acid and 0.06 g/L methionine to the MRS medium had no significant effect on the viable cell number of the four Lactiplantibacillus plantarum strains(P>0.05),but the freeze-drying survival rate was significantly improved(P<0.05).Adding 0.04 g/L glycine to MRS medium had no significant effect on the viable cell number of four Lactiplantibacillus plantarum strains(P>0.05),but could significantly improve the freeze-drying survival rate of Lactiplantibacillus plantarum N1 and Lactiplantibacillus plantarum LIP-1(P<0.05).Using Lactiplantibacillus plantarum LIP-1 as a model strain,the internal mechanism of three amino acids to improve the freeze-drying survival rate of the strain was explored,and it was found that:(1)Compared with the MRS blank control group,Lactiplantibacillus plantarum LIP-1 by metabolizing methionine aspartic acid,the content of peptidoglycan in the cell wall increased by 88.66μg/m L,which enhanced the resistance of the cell wall.The content of long-chain unsaturated fatty acids in the cell membrane increased,of which cyclopropane fatty acid increased by 2.36%,and the unsaturated/saturated fatty acid ratio(U/S)increased by 0.49,maintaining better cell membrane integrity.Intracellular p H increased by 0.62,reducing DNA damage.The activities of lactate dehydrogenase and Na~+K~+-ATPase were increased by 4.95 U/g and 4.02 U/g respectively,which reduced the damage of key enzymes.(2)Compared with the MRS blank control group,Lactiplantibacillus plantarum LIP-1 by metabolizing methionine,the scavenging rate of hydroxyl radicals by Lactiplantibacillus plantarum LIP-1 increased by 6.28%,DPPH free radical scavenging rate increased by 24.64%and the intracellular reactive oxygen species level decreased by 33.12%.The improvement of the antioxidant capacity of the strain reduces the oxidation of unsaturated fatty acids in the cell membrane fatty acid of the strain,and maintains a better cell membrane integrity.The content of intracellular lactate dehydrogenase increased by10.85 U/g,and the content of intracellular ATP increased by 0.04μmol/10~9cell,which promoted the strain to generate more biofilm and enhanced the resistance of the strain to freeze-drying.Intracellular p H increased by 0.34,reducing DNA damage.The activity of Na~+K~+-ATPase increased by 3.04 U/g,which reduced the damage of key enzymes.(3)Compared with the MRS blank control group,Lactiplantibacillus plantarum LIP-1 by metabolizing glycine,the content of peptidoglycan in the cell wall increased by33.45μg/m L,maintaining the integrity of the cell wall.Cyclopropane fatty acid in cell membrane increased by 0.67%,U/S increased by 0.2,maintaining better cell membrane integrity.Intracellular p H increased by 0.4,reducing DNA damage.Lactate dehydrogenase activity and Na~+K~+-ATPase activity were increased by 1.59 U/g and 5 U/g,respectively,reducing the damage of key enzymes.The above results showed that adding aspartic acid and methionine to MRS medium could improve the freeze-dried survival rate of Lactiplantibacillus plantarum,but there were strain differences in adding glycine.By metabolizing amino acids,strains could change the composition of cell walls and cell membranes,thereby reducing the damage to them by freeze-drying.By increasing the intracellular p H of the strain,the DNA damage of the strain by freeze-drying was reduced.The strains could also reduce the damage caused by freeze-drying by increasing the production of biofilm,improving the antioxidant capacity of the strains and protecting the key enzyme activities,thereby improving the freeze-drying survival rate of the strains.This study provided a new idea for improving the anti-freeze-drying activity of the strain by changing the medium composition.