| The raw materials of fruits and vegetables can be fermented by lactic acid bacteria to improve the flavor of the product and strengthen the nutritional value.However,many fruit materials such as oranges,lemons and green plums have high citric acid content,which significantly inhibits the growth,reproduction and fermentation characteristics of lactic acid bacteria.Therefore,it is particularly important to screen out a strain with strong tolerance and excellent fermentation performance.In this thesis,firstly,three strains of lactic acid bacteria belonging to Lactobacillus plantarum and Lactobacillus rhamnosus with better fermentation performance,tested previously among Lactobacillus strains stored in our lab,were used to compare the citric acid tolerant in MRS medium and using different fruit and vegetable juices as fermentation medium.The starting strains with better acid performance underwent adaptive evolution.The cell morphology and cell membrane composition of the cell after evolving was analyzed.Comparative genome analysis was conducted in order to explain the citric acid tolerance mechanism.The evolved strain with improve citric acid tolerance was used in the fermentation of fruits and vegetables,and a two-bacterial sequential inoculation fermentation process has been developed for the fruits and vegetables with multiple dominant organic acids.The main conclusions of this study are as follows:(1)Using the 3 strains of Lactobacilluspreviously characterized,the cell growth was compared in MRS medium and twelve typical fruit and vegetable juices added with different types and concentrations of organic acids.It is found that there are significant differences in acid stress between the types of organic acids and different strains,while Lactobacillus plantarum D(LP-D),Lactobacillus plantarum F(LP-F)and Lactobacillus rhamnosus S(LR-S)showed better tolerance against Malic acid,citric acid and tartaric acid respectively.LP-D was selected as the starting strain for adaptive evolution to obtain a citric acid tolerant strain.(2)The laboratory adaptive evolution strategy and the fully automatic high-throughput microbial droplet culture instrument(MMC)strategies were used for strain domestication.The laboratory adaptive evolution strategy has the advantage of larger cell density range monitered,and therefore,the strain evolved by this strategy showed better citric acid resistance.After 378 generations of domestication,the OD of the strains cultured at 1.5%citric acid increased by 1.9-3.1 times,and the accumulation of lactic acid increased by 1.6-2.6times compared with that of MMC evolution.Therefore,laboratory adaptive evolution is used for the rest of domestication.(3)During the 180 days of laboratory adaptive evolution(1260 generations),the tollerence of the strain gradually improved.The OD of the original strain reached 1.85 at 1%citric acid,and did not grow at all at 2% citric acid concentration.While the OD of the LPD-1260 reached 2.68 at 2% citric acid concentration,and the accumulation of lactic acid reached 15.9 g·L-1,while the starting strain LP-D does not grow at all at this citric acid concentration.LPD-1260 were cultured continuously without citric acid pressure and stored at-20? for XX months,and the improved tolerance of LPD-1260 was still maintained,indicating the genetic stability of the stress resistance.(4)Microscopic examination and scanning electron microscopic examination of the domesticated strains showed that the domesticated strains LPD-840 and LPD-1260 had individual offspring with longer cell length,and in LPD-840 culture,the length increased to about 4-5 times compared with the original ones,and it was about is about 7-8 times for LPD-1260.Microscopic examination of the strains before and after domestication was carried out every 4h of fermentation.In the microscopic examination,no LP-D was found to have longer cell length,while part of the LPD-1260 cells showed the longer cell length at 8h,12 h,16h and 20 h,which indicated that changes in cell morphology was not related to the growth period.The strains of different domesticated generations were analyzed by flow cytometry,and it was found that with the increase of domesticated generations,thelength of cells increased,and the number of elongated ones in all cells increased as well.The average length of a single cell of LPD-1260 is 3.3 times longer than that of LP-D,and the length of elongated cells is 6.04 times longer than that of LP-D,and the proportion of longer cells accounts for17.2% of all cells.The flow cytometer further sorted short and long subgroup cells.It was found that the cells in long subgroup had better citric acid tolerance compared with that of the short subgroup.The cell length of LPD-1260 cultured at different citric acid concentrations were analyzed by flow cytometry,and it was found that high citric acid would induce the length of the evolved cells.(5)The cell membrane of bacteria is the first defensive barrier against external environmental stress.Lactic acid bacteria can change the fatty acid composition in their cell membrane according to changes in external growth conditions to reduce the damage to cells caused by environmental stress.Therefore,we determined the types and contents of fatty acids in the cell membrane of the strains before and after adaptive evolution under different citric acid concentrations,and found C16:0 and C18:0 accounted for the majority of fatty acids,reaching 90%-98%.The C18:0 of the domesticated strain was lower than that of the original strain.With the increase of the evolving generation,the fatty acid composition changed,and the types and contents of the fatty acids were significantly different under varied citric acid concentrations.At the same concentration of citric acid,as the evolving generation increases,unsaturated fatty acid and carbon chain length increased accordingly.(6)Through the mutation detection of whole genome resequencing,it was found that LPD-840 had 106 SNPs and 61 Indels,and LPD-1260 had 105 SNPs and 55 Indels.After COG annotation,most of the mutated genes related to carbohydrate transport and metabolism,amino acid transport and metabolism,cell wall/membrane/envelope biogenesis and transcription;after KEGG annotation,the number of mutated genes mostly related to metabolic pathways,amino acid biosynthesis and secondary metabolites and biosynthesis.Among them,it was discovered thatthere is a mutation in nucleotide blocking protein,which is related to the functions of cell cycle control,cell division and chromosome partitioning after the annotation,and it has been reported that the nucleotide blocking protein function when bacteria face environmental stress,and this protein protect chromosomes by reducing cell division.Therefore,the changes in cell morphology of strains after domestication are likely to be related to the mutation of this gene.(7)Different fruit and vegetable juices were fermentated using obtained stress-resistant strains.For substrate with high citric acid as the main organic acid,such as green plum juice,lemon juice and passion fruit juice,LPD-1260 showed a significant improved fermentation efficacy;for fruits and vegetables with more complex organic acids(with more than one dominant organic acids)a two-bacteria sequential inoculation fermentation process was established with the evolved citric acid resistant strain LPD-1260 as one of the strains.Due to the collaboration of the strains,which has varied tolerances,the sequential inoculation fermentation has significant advantages over other processes. |
PDF Full Text Request