Different Sources Of Lactic Acid Bacteria In Fermented Food Preparation And Regeneration Molecular Classification And Plasma System Research

Lactic acid bacteria (LAB) is the world-recognized probiotics. It is a hotspot in lactobacillus industrial research to screened and identified Enshine lactobacillus from fermented products and improves it. Due to thick compact cell wall of gram-positive bacterium, to which category LAB belongs to, the transformation of LAB is always using electroporation. However, there are many factors which can affect or even stop the transformation, On the other hand, the protoplast transformation system has a longer operation but it were reliable. Normally, as long as the lactobacillus can form protoplast and it can regenerate in specific medium, the transformation can realize with the presence of PEG. So the technical key in protoplast transformation system of LAB is the preparation and regeneration of protoplast.This thesis takes the fermented food as materials, such as market yogurt, sour vegetables, Cantonese sausage and fermented sausage.145strains of lactic acid bacteria were screened by isolated using MRS plate dilution. Its main biochemical characteristics have been initially recognized by motility, gas production test, KOH text, catalase, and acid production test and other biological experiments.(1) RS-PCR Typing techniques will make145strains lactobacillus amplification. After analysis and classification,145strains lactobacillus were divided into A, B, C, D, E, F. G, H, I, J, K, L, M, N, O, P, Q, in all17subtypes. Among them,44strains of lactic acid bacteria, which have been isolated and obtained from yogurt, exist A, B, and D altogether three different subtypes.32strains of lactic acid bacteria, which have been isolated and obtained from sour vegetables, exist C, D, E, F, and G five different subtypes.35strains of lactic acid bacteria isolated from Cantonese sausage exist H, J, O, and Q four different subtypes.34strains of lactic bacteria isolated from fermented sausage exist H, I, J, K, L, M, N, P and Q nine subtypes. It demonstrates lactic acid bacteria strains from fermented sausage possess favorable polymorphism. And the same subtypes existing in the different fermented food samples also show the universality of lactic acid bacteria distribution.(2) The analysis of diversity can show that Shannon’s Information index, in0.2573-0.5983between, average0.3930; for Nei’s gene diversity is located in between0.1327-0.4082, Nei’s gene diversity for0.2365average; Reflect lactobacillus abundant genetic diversity.(3) Screening lactic acid bacteria strain of different subtypes strains to conduct16SrRNA gene sequencing analysis. It shows that type A is strains Lactobacillus acidophilus; type B, type D are Lactobacillus casei; type E, G, H, K, O, and Q are Lactococcus lactis; type C,F, J, L, M, and P are Enterococcus faecalis:type I is Lactococcus garvieae. The subtypes of strains belong to can be found:the main lactic acid bacteria in yogurt are Lactobacillus acidophilus and Lactobacillus casei. The main bacterial strains in sour vegetables, Cantonese sausage, and fermented sausage are Lactococcus Lactis and Enterococcus faecalis. The percentage of lactic acid bacteria stains that Lactococcus lactis occupies in the three kinds of fermented food are59.4%、76.4％、38%respectively. The percentage of lactic bacteria strains that Enterococcus faecalis occupies in the three kinds of fermented food are25%、20.5％、55.9%.Based on these results, one strain of Lactobacillus casei YF-2,is selected and studied. The conditions for Lactobacillus casei YF-2and Lactobacillus rhamnosus LV108protoplast preparation and regeneration were studied. Some parameters including growth phase, enzyme concentration, cell treatment, osmotic stabilizer, medium composition and so on were optimized. The results showed that:(1) When prepare protoplast, adding certain amount of glycin into the culture medium can increase the formation rate of the protoplast. Study finds that in the process of cultivating YF-2, by adding1.2%glycin, the formation rate of protoplast can be incerased by about20%. While for LV108, adding2%glycin into the culture medium can increase the formation rate of protoplast by28%.(2) Different strains need different kinds and amount of enzymes. This study shows that Lactobacillus casei FY-2are harder to get protoplast than Lactobacillus rhamnosus. Lysozyme alone cannot make YF-2to release protoplast. Only by mixing with mutanolysin can generate protoplast of high production. When there is Mutanolysin of25ug/ml, the time needed to prepare protoplast will decrease as the concentration of lysozyme increases. However, if the concentration of lysozyme is too high, it will affect the regeneration of protoplast to a great extent. While for Lactobacillus rhamnosus LV-108, by using lysozyme of50mg/ml, protoplast of high quality can be prepared.(3)For Lactobacillus casei YF-2the5%inoculation amount to MRS medium containing1.2%glycin cultivated to5-6hour. The cultures were digested with25mg/mL lysozyme and25μg/mL mutanolysin at37℃with shaking one times per15min for90min, and LPB (adjusting pH to8.0) was used as osmotic pressure stabilizer. For the regeneration of protoplast, the RM1medium was more efficient. The peak of regeneration frequency is33.5%. For Lactobacillus rhamnosus LV108the5%inoculation amount to MRS medium containing2%glycin cultivated to5-6hour. The cultures were digested with50mg/mL lysozyme at37℃with shaking one times per15min for75min, and LPB (adjusting pH to8.0) was used as osmotic pressure stabilizer. For the regeneration of protoplast, the RM1medium was more efficient. The peak of,the regeneration frequency in RM1was20.4%.