Studies On The Aerobic Metabolism Of Lactobacilli And Mechanisms For Probiotic Effects Of Bacillus Subtilis

Although the classification of lactic acid bacteria (LAB) is based on their ability of fermenting glucose to produce lactate under anaerobic or microaerobic conditions, many LAB could grow and metabolize lactate under the aerobic condition. Lactobacilli are typical LAB and widely used in fermented foods, feeds and probiotic cultures, as a result of their organoleptic and probiotic qualities, their capacity to produce antimicrobial substances and their ease of use in fermentation processes. Lactobacilli display different metabolism characteristics under the aerobic condition. It is of great improtance to take full advantage of their aerobic metabolism to obatain high biomass and matain viability of cells, especially in the production process.Bacillus subtilis is a kind of probiotic widely used in the aquatic and stock breeding. The administration of spores of B. subtilis to animals could markedly increase the counts of viable lactobacilli in the gastrointestinal tract and feces, but the related mechanisms are not well clarified.This thesis focued on the aerobic metabolism of Lb. plantarum and Lb. brevis, the mechanisms for probiotic effects of B, subtilis based on the aerobic metabolism of lactobacilli, and the exploration of process innovation of mixed microbial ecological agents by co-culturing lactobacilli with B. subtilis. The results were decribed as follows:1. Studies on the fermentation and aerobic metabolism of Lb. plantarum and the oxidative stressLb. plantarum is widely used in fermented foods, feeds and probiotic cultures. This theis studied the metabolism characteristics of Lb. plantarum LAI under the following four conditions:①unaerobic condition;②aerobic condition;③respiratory condition (aerobic condition plus exogenous hematin and vitamin K.2, which completed the electron transport chain);④catalase protected condition (aerobic condition plus exogenous hematin, which activated the catalase of Lb. plantaum). The characteristics included:①biomass (OD600),②biability of cells (CFU/mL);③metabolites including lactate, actate and ethanol;④oxidative stress (the concentration of H2O2in the culture).The participation of oxygen in the metabolism exerted both beneficial and harmful effects on Lb. plantarum LAI. The aerobic metabolism could bring an increase of biomass from3.5to5.0, while producing as as high as3to4mM H2O2leading to the rapid death of cells after the highest biomass. The main end metabolites of glucose fermentation of Lb. plantarum LA1was lactate, which changed to lactate and a great quantity of actate when oxygen was available. When its catalase was activated by exogenous hematin, the cells with high vitality could transform lactate to actate completely, generationg extra ATP and more biomass. In addition, the reports on the aerobic respiration of Lb. plantarum are limited. Our studies indicated that although aerobic respiration improved the aerobic metabolism of Lb. plantarum, it produced little improvement of biomass, which had no difference with other aerobic conditions.2. Studies on the fermentation and aerobic metabolism of Lb. brevisAs an important fermentation strain, Lb. brevis is always used in the production of Cheddar cheese and play an important role in the maturity of cheese. We detected the metabolism characteristics of Lb. brevis ATCC367under four conditions, as listed above. The participation of oxygen in the metabolism substantially benefited Lb. brevis ATCC367, by improving its biomass to4.1(aerobic condition) or5.5(respiratory condition) with no H2O2accumulation detected. The main end metabolite of glucose fermentation of Lb. brevis ATCC367was lactate, accompanied by CO2and a little ethnol. In the aerobic condition, the end metabolites also changed to the mixture of lactate and actate, and then actate completely, generationg extra ATP and the remarkable increase of biomass. There is no reports on the aerobic respiration of Lb. brevis. Our results indicated that aerobic respiration notablely improved the aerobic metabolism of Lb. brevis, leading to much rapider metabolites transformation and higher biomass than any other conditions.3. The transformation from Iactate to acetate in the aerobic metabolism of Lb. brevisThere is a transformation from Iactate to acetate in the aerobic metabolism of both Lb. plantarum and Lb. brevis. This transformation in Lb. plantarum is fully understood, which is through the Iactate oxidase (LOX), pyruvate oxidase (POX) and acetate kinase (AK). The toxic H2O2is produced by pyruvate oxidase in this pathway. There was no H2O2accumulation detected in the transformation in Lb. brevis, and the detailed pathway was unclear. We constructed the pox single-crossover mutant and pdh single-crossover mutant of Lb. brevis ATCC367. The fermented metabolism of the two mutants was similar to that of the wild type. But conferring the aerobic metabolism, the biomass of the pdh single-crossover mutant was much less than that of the wild type, accompanied by more Iactate, less acetate and no transformantion from Iactate to acetate. There was no difference of the aerobic metabolism between the pox single-crossover mutant and the wild type. Thus the dominant role of the pyruvate dehydrogenase (PDH) in the aerobic metabolism of Lb. brevis was clarified. In addition, real time RT-PCR indicated the up-regulated expression of pdh and pox in the aerobic condition, especially when glucose was exhauted. The results demonstrated the positive regulation of oxygen and the negative regulation of glucose on PDH and POX synthesis. This thesis clarified the ATP-yielding pathway from Iactate to acetate in the aerobic metabolism of Lb. brevis, which provided a theoretical basis for the high density fermentation and its resistance against the oxidative stress.4. The beneficial effects of Bacillus subtilis on lactobacilli against oxidative stressB. subtilis is widely used in probiotic products. It is traditionally considered that B.subtilis consumes oxygen in the gut, thus creating a favorable environment for the growth of another kind of probiotics, lactobacilli. But oxygen in the gut is inevitable, and some Lactobacillus species can use oxygen to obtain improved biomass while producing toxic H2O2. We detected the autolysis of B. subtilis in the simulated intestinal fluid, and demonstrated the released heme by HPLC analysis. When Lb. plantarum LA1was co-cultured with B. subtilis aerobically for2days, the viable cell counts of it was109times higher than that of the free-living aerobic culture. When the cell lysate of B. subtilis was added to the medium, the catalase of Lb. plcmiarum was actived, and high cell viability and high DNA integrity were obtained, with no H2O2accumulation detected. It was indicated that both the catalases in vital cells of B. subtilis and the heme released by autolysed cells contributed to the resistance against oxidative stress of lactobacilli. Our study provided a supplement to the current mechanisms for probiotic effects of B. subtilis.5. The innovation of mixed microbial ecological agents by co-culturing lactobacilli with B. subtilisTraditionally, the mixed microbial ecological agents are processed by mixing the sole culture of latobacilli and B.subtilis. Some Lactobacillus species are able to use oxygen to obtain improved biomass while producing toxic H2O2; B. subtilis have a perfect system for resisting the oxidative stress, for example, producing heme and catalase, which are inexistent in lactobacilli. Therefore, it could be of many advantages to co-culture latobacilli with B.subtilis. In order to obtain a high bacterial protein content, high viable cells of lactobacilli and high spores of B. subtilis, we optimized the growth state of seed, the inoculation quantity and the inoculation proportion, and got the best condition of co-culture in the MRS medium (0.5%glucose), that was:1.5%Lb. casei BL23plus10%B.subtilis168, and the incubation time was24hours;1.0%Lb. plantanim LAI plus10%B.subtilis168, and the incubation time was18hours. There are still many possibilities of improvement in applying co-culture to the industry. This thesis contributed to the exploration of process innovation of mixed microbial ecological agents by co-culturing lactobacilli with B. subtilis, which provided a possibility of cost saving in industry.