Study On Improving The Lyophilization Survival Rate Of Lactobacillus Sp.by Regulating The Composition Of Cell Membrane

Lactobacillus is a kind of probiotics with the functions of maintaining intestinal flora homeostasis,improving immunity,antagonizing bacteria and so on.In order to make Lactobacillus play an ideal probiotics effect,sufficient number and vitality of Lactobacillus must be ensured.Freeze-drying and adding suitable lyophilized protectant is the most common preparation method of Lactobacillus.However,the protective effect of lyophilized protectant is strain specific,and the protective effect of the same protectant on different species of Lactobacillus is greatly different,and it is time-consuming and laborious to screen and optimize the protective agent for each species of Lactobacillus.Therefore,in this paper,the methods of adjusting cell membrane composition of Lactobacillus to improve stress resistance were studied,investigated the high freeze-dried survival cell membrane fatty acid composition characteristics of Lactobacillus,The effects of various methods of regulating cell membrane composition on the survival rate of lyophilization stability were investigated,It has guiding significance for the preparation of high activity Lactobacillus powder.The main research contents and results are as follows:First,twenty-four strains of Lactobacillus from different species and genera were cultured under the same culture conditions,and the bacterial sludge was collected.The vacuum freeze-drying was carried out under the same lyophilized protective agent and the same lyophilized technological conditions.At the same time,the same bacterial sludge was used to analysis,the morphology and size were counted by microscopy,the level of capsular polysaccharide was detected by anthrone-sulfuric acid method,and the fatty acid composition of cell membrane was extracted and detected by GC-MS.In order to explore the characteristics of fatty acid composition of cell membrane of Lactobacillus with high lyophilized survival rate,the differences of survival rate and membrane fatty acid composition of Lactobacillus with low capsular polysaccharide level and similar morphology and size were compared.The results showed that the survival rates of different species of Lactobacillus were significantly different under the same freeze-drying conditions(33.29%-82.47%),and the strains with higher survival rates had the characteristics of higher cell membrane fatty acid unsaturation and shorter average chain length.Then,Lactobacillus plantarum N8 was selected as the research object to study the changes of membrane fatty acid composition and lyophilized survival rate after various environmental stress treatments.The results showed that the cell membrane unsaturation,the proportion of cyclopropane fatty acids and the composition of average acyl chain length of Lactobacillus plantarum N8 showed regular and small changes,and the lyophilized survival rate only increased or decreased slightly after various environmental stress treatments.In addition,initial viable bacteria count and storage stability of Lactobacillus plantarum N8decreased after stress treatment.Next,use different exogenous fatty acids and tween adjusted the cell membrane of Lactobacillus plantarum N8,experiments show that after adding foreign long chain fatty acids Lactobacillus plantarum N8 unable to grow normally,unable to use those fatty acids to get adjustment of Lactobacillus cell membranes,and only the addition of oleic acid,Tween 80 and Tween 20 did not affect the normal growth of Lactobacillus plantarum N8.The changes of fatty acid composition in cell membrane of Lactobacillus were detected by adding these substances and the survival rate of lyophilized Lactobacillus was calculated.The results showed that the fatty acid unsaturated rate of Lactobacillus plantarum N8 cell membrane increased from 58.64±0.46%to 79.15±0.56%,and the survival rate increased from 55.40±3.45%to 64.84±1.05%.Adding oleic acid increased the unsaturation and the survival rate increased slightly.Adding Tween 20 shortened the mean chain length of membrane fatty acids and slightly increased the lyophilized survival rate.Afterwards,using substances that regulate the composition of bacterial cell membranes,such as beta-carotene,a polyisoprene structure,vitamin K₁,and squalene;melatonin,an amino acid derivative adsorbed on the surface of cell membrane to regulate membrane fluidity;cyclodextrin,vitamin E and other substances that can enter the cell membrane and regulate it.By adding these substances,the change of survival rate and cell membrane fatty acid were detected.The results showed that these regulating substances had different effects on the lyophilized survival rate of Lactobacillus plantarum N8.Adding?-carotene and melatonin significantly increased the lyophilized survival rate of Lactobacillus plantarum N8 from53.97±1.46%to 68.53±1.79%and 72.67±2.32%respectively.After cultured with?-carotene and lyophilized,the survival rate of the bacterial powder was 51.30±1.83%and the viable bacteria count was 3.65±0.11×10¹¹CFU·g^-1 after stored at 37?for 2 months.Finally,the combination of the regulation methods that effectively improve the lyophilized survival rate of Lactobacillus plantarum N8 was used to study whether combined use could enhance the regulation effect and further improve the lyophilized survival rate of Lactobacillus.The results showed that after adding 2mg/m L Tween 80 and 500?g/m L?-carotene and 500?g/m L melatonin,Lyophilized survival rate of Lactobacillus plantarum N8increased from 53.97±1.46%to 86.05±1.27%.The lyophilized survival rate of Lactobacillus of other species was significantly improved after cultured with these substances.