Industrial Stress And Genetic Stability Of Lactobacillus Bulgaricus With Low-Postacidification Ability

Lactic acid bacteria (LB A) are a group of bactercia which can ferment sugar into lactate and widely used in food industry and health care.However, wild strains often do not harbor various fine characteritics which are necessary for application in industry production. So the traditional breeding mehtods such as mutagenesis breeding and protoplasts fusion are needed in breeding strains with fine characteristics. The postacidification of yoghurt is mainly ascribed to the proceeding lactose fermentation of Lactobacillus delbrueckii subsp.bulgaricus and the postacidification will lead to the over acid taste and low sensory property of yoghurt, moreover the shelf life of yoghurt is also shortened. Thus, it’s very important to obtain the Lactobacillus delbrueckii subsp.bulgaricus with low-postacidification ability by mutafenesis breeding and the genetic stability assay of the mutant strains, metabolism dynamics of the H+-ATPase-defective mutant strains.There are several experiments in this study:mutagenesis breeding of H+-ATPase-defective strain, the influnce of acid stress and cold stress to the growing and H+-ATPase activity of parent and mutant strains, genetic stability assay of the mutant strains, metabolism dynamics of the H+-ATPase-defective mutant strains,comparison of H+-ATPase expression between parent and mutant strains and development of the low postacidification yoghurt.First of all, one H+-ATPase-defective mutants named as KLDS1.9201-11was obtained by mutagenesis breeding of Lactobacillus delbrueckii subsp.bulgaricus KLDS1.9201acquired from DICC (Dairy Industry Culture Collection, Key Laboratory of Dairy Science) which could served as fine yoghurt starter strain. The growth and pH of KLDS1.9201and KLDS1.9201-11were measured and found that the growth rate of the KLDS1.9201-11was obvious lower than KLDS1.9201, and in the growing process, the pH value is significantly higher than KLDS1.9201,in another word, the ability of acid production of KLDS1.9201-11is weak.Total RNA were extracter from log phase and stable phase of KLDS1.9201and KLDS1.9201-11by TIANGEN DP430bacterial RNA extraction Kit respectively, then they were reverse transcripted into cDNA after treated with DNaseI (RNase-free). The cDNA in the log phase and stable phase of H+-ATPase of KLDS1.9201and KLDS1.9201-11were determined by Real-Time quantitative PCR with the16s rDNA as the internal control gene.Result indicated that there were significant difference between the expression of H+-ATPase of KLDS1.9201and KLDS1.9201-11(p<0.05). In the log phase, the expression amount of H+-ATPase of KLDS1.9201-11was twenty-four percent of that of KLDS1.9201, in the stable phase,the expression amount of H+-ATPase of KLDS1.9201-11was thirty-three percent of that of KLDS1.9201.The survival rate of KLDS1.9201and KLDS1.9201-11were significantly decreased after acid stress, the number of viable cells were reduced by86.98%and99.98%,the H+-ATPase activity were decreased by13.33%and21.15%, KLDS1.9201-11showed a stronger acid sensitivity. After cold stress, the viable cell count of KLDS1.9201and KLDS1.9201-11decreased by76.71%and4.88%, the H+-ATPase activity of the parent strain decreased by4.27%, the H+-ATPase activity of the mutant increased by29.71%. The results indicating that the cold adaptability of the mutant strain is better than that of the parent strain, which is conducive to the improvement of the number of viable cells in the production process of the DVS yogurt starter.The genome DNA of KLDS1.9201-11was extracted by TIANamp Bacteria DNA Kit from the first generation to the eighth generation, which was used for RAPD analysis by using five kinds of primers screened out. DNA fingerprinting analysis results of RAPD analysis of the mutant strains showed that, the DNA fingerprint of mutant strain KLDS1.9201-11did not change basically after serial passaged by8times,moreover,the number of random amplified bands were same and brightness were similar, namely, genomic DNA of KLDS1.9201-11has a high degree of homology, which was not change with the loss of acidification capacity.Bacteria solution’s metabolites of KLDS1.9201and KLDS1.9201-11were analyzed by high-performance liquid chromatography method from the first generation to the tenth generation, the metabolism product were glucose and lactic acid. The results show that the glucose metabolic rate of KLDS1.9201is always higher than that of KLDS1.9201-11and the lactic acid production is higher, too. And KLDS1.9201-11can stably inherited to the eighth generation, it began to reverse mutation from the nine generations.Low-postacidification yoghurt was fermented with co-culture of Streptococcus thermophilus KLD3.9210with Lactobacillus delbrueckii subsp.bulgaricus KLDS1.9201-11and KLDS1.9201respectively. Sensory perception demonstrated that yoghurt fermented with KLDS1.9201-11had similar taste, texture and flavor with that fermented with KLDS1.9201. After21days storage at4℃, pH of yoghurt fermented with KLDS1.9201-11was4.13, and the titration acidity was930T, while the pH of yoghurt fermented with KLDS1.9201was3.94, and the titration acidity was1140T.