Effect Of Culture Conditions On Lactiplantibacillus Plantarum LIP-1 Freeze-drying Survival Rate And The Internal Mechanism Of Action

Improving the freeze-drying survival rate of strains by changing the medium conditions has become a research hotspot,but the research in the selection of culture conditions and related mechanisms is not comprehensive.In this research,we took Lactiplantibacillus plantarum LIP-1 as an example,studied the effect of changing the culture conditions on the freeze-drying survival rate of the strain,and described in detail the changes of physiological response and metabolic regulation when the culture conditions were changed by adopting the subcellular damage identification process and bioinformatics data,so as to explore the internal mechanism underlying the effect of different culture conditions on the freeze-drying survival rate of the strain.The main research results are as follows:1.The internal mechanism underlying the effect of adjusting buffer salt composition and initial p H on freeze-drying survival rate of the strain（1）Compared to the disodium hydrogen phosphate/citric acid buffer salt system,adding 0.12 mol/L sodium acetate/dipotassium hydrogen phosphate/sodium citrate buffer salt to the optimized culture medium could significantly improve the freeze-drying survival rate of four strains of L.plantarum（P<0.05）,which was related to the regulation of potassium ion in buffer salt on cell membrane fatty acid composition and biofilm formation.Potassium ions in buffer salts could upregulate the trk A and lys R gene in the cell membrane fatty acid regulatory gene cluster at the transcriptional level,as well as the expression of the ACC and FAB families of fatty acid metabolism synthesis genes at the transcriptional and protein levels.By doing so,the fatty acid carbon chain was extended and the unsaturated fatty acids content was promoted,therefore the integrity and compactness of the cell membrane was improved,which could reduce the damage of cell membrane.At the same time,the potassium ions in the buffer salt could upregulate the expression of the lux S gene in the Lux S/Autoinducer-2（AI-2）at the transcriptional level,as well as the expression of biofilm synthesis gene cys E and the accumulation of biofilm,and eventually improved the freeze-drying survival rate.（2）Compared with the initial p H 7.4 group,the initial p H 6.8 group could improve the freeze-drying survival rate of the L.plantarum LIP-1（P<0.05）significantly.It was related to the regulation of cell membrane,stress protein,and surface protein by the strains.In the p H6.8 group,the strain grew rapidly and produced more acid,resulting in the upregulation of the expression of transcripts and protein of citrate lyase,ACC family and FAB family,leading to the promotion of the relative content of cyclopropane and unsaturated fatty acids,which could sustain the integrity and compactness of the cell membrane and reduce the damage of cell membrane.At the same time,with the acid stress,the expression of cold stress proteins was increased,which improved the freeze-drying resistance of the strain.In addition,the acid stress also promoted the expression of transcripts and protein of carb,URA4 and PYR family,which enhanced the transformation from glutamine to uracil,therefore drove the up-regulated expression of UPRT at the transcription and protein levels,increased the accumulation of surface protein,enhanced the integrity of cell wall,and eventually improved the freeze-drying survival rate of the strain.2.The effect of adding trace substances to culture medium on the freeze-drying survival rate of strains and its internal mechanism（1）In terms of amino acids,adding 0.05 mol/L cysteine and 0.10 mol/L glutamate to MRS medium could significantly improve the freeze-drying survival rate of the strain（P<0.05）.By exploring its internal mechanism,we have found that:The strain reduced the damage of DNA,cell membrane and cell wall by metabolizing cysteine,and then improved the freeze-drying resistance of the strain.The strain could convert L-cysteine into pyruvic acid and NH₃,and NH₃helps to increase the intracellular p H and reduce the damage of DNA.At the same time,the metabolized L-cysteine could produce glutathione with well antioxidant activity,which could reduce the oxidation of long-chain unsaturated fatty acid and Cyclopropene fatty acid,and thus reducing the damage of cell membrane.L-cysteine could also upregulate the expression of srt A gene,enhance the anchoring of surface proteins on the cell wall for its enhanced integrity,and eventually improve the freeze-drying survival rate of the strain.The strain reduced the damage of cell membrane and cell wall by metabolizing glutamate and therefore improved the freeze-drying resistance of the strain.The glutamate enables the strain to grow rapidly and produce more acid.With the acid stress,the expression of membrane protein regulatory genes sec G and Dna K was promoted,the anchoring of cell membrane of Dna K protein was enhanced as well,which increased the integrity of the cell membrane.At the same time,with the acid stress,the relative content of long-chain unsaturated fatty acids and cyclopropane fatty acids in the cell membrane was increased,which could maintain the fluidity and compactness of the cell membrane of the strain and reduce the damage of cell membrane.In addition,the glutamate could upregulate the expression of the mur L,mur D,and van Y genes and accumulate peptidoglycan to increase cell wall integrity,and eventually improve the freeze-drying survival rate.（2）In terms of the bases,adding 0.15 mol/L guanine and 0.70 mol/L thymine to the MRS medium could significantly improve the freeze-drying survival rate of the strain（P<0.05）.By exploring its internal mechanism,we have found that:The strain could reduce the damage of DNA and cell membrane and promote biofilm synthesis by metabolizing guanine,so as to improve the freeze-drying resistance of the strain.The metabolized guanine could provide raw materials for the synthesis of DNA and reduce the damage of DNA.At the same time,guanine enables the strain to grow rapidly and produce more acid.With the acid stress,the synthesis of signal molecule pp Gpp was increased,leading to the upregulation of the expression of cyclopropane fatty acid gene cfs and the synthesis of cyclopropane fatty acid.In addition,the acid stress forced the strain to increase the synthesis of unsaturated fatty acids to maintain the fluidity and compactness of cell membrane,and reduce the damage of cell membrane.The increase of signal molecule pp Gpp could promote the expression of bip A and pga C genes and the formation of biofilm,and eventually improved the freeze-drying survival rate of the strain.The strain could reduce the damage of cell wall,DNA and cell membrane by metabolizing thymine,and promote the synthesis of biofilm,thereby improving the freeze-drying resistance of the strain.Thymine,as a raw material for the synthesis of peptidoglycan,could improve the integrity of the cell wall.The metabolized thymine could promote the accumulation of serine,which could be further decomposed into pyruvate and acetylserine.Decomposition to pyruvate promoted the synthesis of NH₃and ATP,and then improve the intracellular p H of the strain and reduced the damage of DNA.The ATP could also promote the upregulation of acyl-Co A thioesterase activity and the accumulation of long-chain unsaturated fatty acids and cyclopropane fatty acids to maintain the fluidity and compactness of cell membrane.The transformation of acetylserine from serine also increased the expression of cys E gene and the formation of biofilm,and eventually improved the freeze-drying survival rate of the strain.In this research,we analyzed the effect on the freeze-drying survival rate of the strain by changing culture conditions,and explored the intrinsic influence mechanism of subcellular structural damage in combination with multi-omics.This research revealed the diverse regulation of freeze-drying resistance of the strain under different culture conditions,and laid an theoretical foundation and data support for the development and utilization of freeze-drying lactic acid bacteria.