The Study On Physiological Damage And Protection Strategies Of Lactobacillus Plantarum During Freeze-drying

In this study,12 Lactobacillus plantarum strains were used as the research object.At 18?,24?,30? and 37?,the phylogenetic tree,the growth curve,cell membrane fatty acid composition and survival rate after freeze-drying were detected and analysed to determin the potential correlations between these physicochemical properties by the Pearson correlation coefficient,especially the correlation between fatty acid and survival rate after freeze-drying.At the same time,screen three strains with obvious differences.And then selected two fermentation temperatures with large differences in the fatty acid related to survival rate after freeze-drying.Next,determined the glass transition temperature,collapse temperature,cell membrane properties and activities of key enzymes after freeze-drying of the three strains by using sucrose,sorbitol and PBS as protective medium in order to find differences in physiological damage by fatty acid differences,strain differences,and protective media differences during freeze-drying.Finally,combined with the physiological damage mechanism,the freeze-drying survival rates were improved by different protection strategies,including the addition of fatty acids in the fermentition process,the replacement of different protective agents,the combination of protectants,and the optimization of freeze-drying process.The main conclusions were as follows:Firstly,the results of phylogenetic tree showed the 12 L.plantarum strains examed were different from each other.The results of growth curve of the 12 L.plantarum strains examed at 18?,24?,30? and 37? showed,when the fermentation temperature was decreased from 37? to 18?,the specific growrh rate was decreased from 0.12-0.25OD₆₀₀/h to 0.01-0.04 OD₆₀₀/h;and the length of lag phase was extended form 2.64-5.00h to 6.41-13.75 h.The results of fatty acid composition analysis showed the 4 main FAs were identified as hexadecanoic acid?C16:0?,octadecanoic acid?C18:0?,octadecenoic acid?C18:1?,and nonadecenoic acid?C19:1?.Tetradecanoic acid?C14:0?,hexadecenoic acid?C16:1?,octadecatrienoic acid?C18:2?were also detected,at low relative concentrations.When the fermentation temperature was decreased from 37? to 18?,the ratio of unsaturated fatty acid to saturated fatty acid?U/S?increased from 0.42-0.79to 0.66-2.05.That is,fatty acids are changed in an unsaturated direction.The results of survival rates after freeze-drying showed that both sucrose and sorbitol had significant protective effects compared to PBS.Sucrose has the best protective effect,and the survival rates after freeze of L.plantarum were improved by 10-77%,the survival rates after dry were increased by 20-82%,and the survival rates after freeze-drying were increased by 30-78%.Pearson correlation coefficient showed that the survival rate after freeze-drying was positively correlated with the relative content of C18:1 and U/S.The U/S and the relative amounts of C18:1 appear to be the main parameters relevant to the freeze-drying resistance of L.plantarum.Lastly,three strains with large differences in the16S rRNA gene,growth rate,lag time,fatty acid composition,and survival after freeze-drying were selected:AR113,AR307 and WCFS1.18? and 37? with large difference of the relative content of C18:1 were used as the fermentation temperature.And PBS,sorbitol and sucrose were used as protectants for mechanism analysis.The results showed that in terms of protective agent,compared with PBS,sucrose mainly improved the freeze-drying resistance of L.plantarum by increasing the glass transition temperature,collapse temperature,cell membrane integrity and fluidity,activities of LDH and ATP enzyme.While sorbitol mainly through increasing activities of LDH and ATP enzyme;the difference in the freeze-drying resistance between strains was mainly due to cell membrane integrity and fluidity,activities LDH and ATP enzyme.Protection strategies were adopted and the mechanisms were reversed.The results were as follows:the addition of C18:1 resulted in an increase in cell membrane fluidity,activities of LDH and ATP enzyme,thereby increasing survival rate after freeze-drying by 6-62%.By replacing the protectants,it was found that the protective effect of trehalose and sucrose was more remarkable than mannose in the small molecule sugar,and the survival rates after freeze-drying were improved to 40-80%.Sorbitol and mannitol have significantly lower effects than sugars,and the survival rates after freeze-drying were both less than 15%.The macromolecular sugars were all good,which can significantly improve the freeze-drying survival rate to 7-74%.Since the effects were not satisfactory,the protectants were combined and the freezing temperature was optimized in the presence of the optimal multiple protectants.The results of the optimal protectants,freezing temperature and survival rate of the three strains were as followed:AR113,soybean polysaccharide+trehalose,-20?,and 91.0%,respectively;AR307,soybean polysaccharide+sucrose+mannitol,-40?,and 81.0%,repectively;WCFS1,soybean polysaccharide+trehalose,-196?,94.4%,repectively.Through mechanism reversal,it was proved that the multiple protectants can improve survival rate after freeze-drying by increasing the glass transition temperature and collapse temperature,enhanceing the cell membrane integrity and fluidity,and increasing the activities of ATP and PK enzyme.