The Oxidative Stress Resistance And Aerobic Growth Of Lactic Acid Bacteria

The effects of oxygen to lactic acid bacteria (LAB) are flexible and divided into two aspects:even though recognized as aerotolerant bacteria, lactic acid bacteria are severely damaged by Reactive Oxygen Species (ROS), e.g. Superoxide radical (O2), Hydrgen peroxide (H2O2), and Hydroxyl free radical (HO). The Reactive Oxygen Species are major factors in mortality and aging, and preventing these killing effects is important for bacteria. On the other hand, while growing under aerobic conditions, the metabolism of some LAB may greatly change. The final biomass, fermentation products are often distinctly different from fermentation under static growing. More specially, some LAB undergo respiration when they are cultured in the presence of oxygen and heme (or heme and menaquinone). While undergoing respiration, the biomass may be greatly improved, the toxicity of ROS may be reduced, and survival of cells may be extended. In this paper, we studied the mechanisms of the oxidative stress resistance in several strains of LAB, and showed the influence of aerobic growth and respiration on lactobacilli.1. The effect of aerobic condition on the metabolism of Lb. plantarumLb. plantarum is one of the few LAB with large genome, which contributes to its complex metabolism and application in many areas such as food fermentation and transformation of organic substances, etc. The dissolved oxygen has both beneficial and harmful effects on the metabolism of Lb. plantarum, which is discussed in this dissertation in detail. The biomass of Lb. plantarum in the aerobic condition was higher than that in the static condition, with a1.8higher value of OD600-But the viability of cells under the aerobic condition was undermined soon after the logarithmic phase, and the viable cells were less than104CFU/mL after24hours. Then the consuming of glucose and accumulation of lactic acid and acetic acid in the culture were compared in aerobic and static conditions. The glucose in the culture was consumed in9hours in both conditions. In the aerobic condition, but not the static condition, there was a remarkable transformation of lactic acid to acetic acid, accompanied by yielding additional ATP, which agreed with the difference between biomass in the two conditions. What was more, a large amount of H2O2was accumulated in the culture through the NADH oxidase and pyruvate oxidase in the aerobic condition, which lead to the low viability of cells after12hours’agitation. Thus it is of significance to explore how to apply aerobic metabolism in Lb. plantarum to gain both high biomass and cell viability.2. The key roles of lactic acid in the oxidative stress resistanceWhile aerobic growing in rich glucose medium, Lb. plantarum produced high level of H2O2and lactic acid. The great amount of H2O2accumulation was thought to be the main effects of cell death after cells enter into stationary state. Under aerobic growth conditions, Lb. plantarum cells could reach significantly higher OD600values but rapidly died then. Compared to anaerobic growth conditions, part of lactic acid was transformed to acetic acid and additional energy was gained after glucose exhausted. In this process, high level of hydrogen peroxide may be accumulated as a side product in glucose rich medium. If hydrogen peroxide couldn’t be removed from cultures immediately, the survival of cells decreased rapidly at an exponential rate within a few hours. Evidences were presented that lactic acid plays crucial roles in the hydrogen peroxide lethal damage. Compared to individual hydrogen peroxide or lactic acid challenge, the viability of Lb. plantarum cells under cooperative hydrogen peroxide and lactic acid challenge was significantly lower.[Fe"’] in the culture was also important in this process, while Iron chelators including diethylenetriamine pentaacetic acid (DTPA), O-phenanthroline (O-phe) could improve the survival of Lb. plantarum LA1cells under cooperative hydrogen peroxide and lactic acid challenge. To our knowledge,[Fe2+] and lactic acid in the cultures was related to Hydrogen peroxide toxicity via’Fenton reaction’directly or indirectly. The conflict between energy gained and high concentration of hydrogen peroxide generated in the process of lactic acid and acetic acid transformation was the reason why Lb. plantarum rapidly suicide after reached a higher cell density. Cells could be well protected with the addition of exogenous catalase or the activation of its endogenous catalase by heme, especially the later. Scavenging hydrogen peroxide efficiently made harvesting robust Lb. plantarum cells with a greater density possibly.3. The protection to Lb. plantarum with the endogenetic catalase gene katAs H2O2is accumulated to higher concentration, cells are killed gradually and all the processes described above will be stop consequently. This problem can be solved by heme or exogenous catalase (e.g. bovine liver catalase) supplemented to cultures. While cells growing under catalase or heme permitted aerobic conditions, H2O2is scavenged rapidly, higher cell viability is maintained, more lactic acid is transformed to acetic acid, and the final OD600values are slightly higher compared to aerobic growth. The activity of exogenous catalase would be affected by several environmental effects especially the pH, and loss after a long period of cultivation. The activation of endogenous catalase by heme is more useful for Lb. plantarum maintaining viability during aerobic growth.4. The effect of respiration to Lb. plantarum and Lb. brevisLb. plantarum and Lb. brevis have the similar non-flexible and non-redundant respiration chain which needs dehydrogenases, quinones, and cytochrome oxidase bd. Heme is the essential factor for cytochrome oxidase bd, and can’t be produced by the lactobacilli. As Lb. plantarum and Lb. brevis also lack quinones, heme and menanquinone should be provided to operate this chain. While oxygen was need for the GFP fluorescence, the weaker fluorescence signals were corresponding with the low level of oxygen in cells under respiration. Compared with aerobic growth, after the glucose in medium was exhausted, the cells under respiration transformed the lactic acid to acetic acid more quickly. Comparison with Lactococcus lactis, the respiration of Lb. plantarum was different in many aspects. Even though more ATP was yielded though the lactic acid-acetic acid transformation, more H2O2was also accumulated by Lb. plantarum. There were no significant difference between the great biomass yield of Lb. plantarum under aerobic growth and respiration. While cell survival was high with heme and menaquinone addition, the similar effect was also observed if only heme present. If heme present, the maintenance of cell survival of Lb. plantarum was due to the endogenesis catalase, not the respiration. As the metabolism was verified between Lb. plantarum and Lb. brevis, the results of respiration were different. In conclusion, as more biomass could be gained and the survival could be extended, the respiration was beneficial to Lb. brevis.5. Enhancing the oxidative resistance of lactobacillus co-cultured with BacillusSome of Bacillus strains are regard as probiotic microorganisms and widely used in health care, stock and poultry raising. In this paper, the protective effects of B. subtilis168to lactobacillus in the intestinal tract are researched.lt is traditionally considered that the germination and growth of Bacillus spores in the intestinal tract consume a large amount of oxygen which create anerobic environment for the growth of lactic acid bacteria (LAB).In this study, Lb. plantarum LA and Lb. brevis ATCC367were co-cultured with B. subtilis168aerobically in vitro, the cell survival of Lactobacillus species was significantly improved than that of the strains along, especially for the Lb. plantarum strains with10times higher colony-forming units (CFU)/ml in the storage for two days at room temperature. Meanwhile, H2O2concentration in the co-cultured cultures was undetectable comparison with that of the cultures of Lb. plantarum with the level of mM. When Lb. plantarum LA was cultivated in the medium supplemented with the autolysate of B. subtilus168, the similar elimination of H2O2and improved vital maintenance were obtained. Moreover, By means of detecting catalase activity of LA. plantarum LA, determining amounts of H2O2produced by Lb. plantarum LA and measuring DNA integrity of Lb. plantarum LA, we indicated that heme released by autolysed cells of it, which activated the catalase produced by Lb. plantarum LA, contributed to removing of H2O2in the environment. Reduction of oxidative stress helped to maintain DNA integrity of Lb. plantarum LA. Consequently, Lb. plantarum LA benefit from B. subtilis to improve cell viability. Our study provided a supplement to the current mechanisms for probiotic effects of Bacillus.6. High level expression of catalase in Streptococcus thermophilus and the protection to Lactobacillus bulgaricusStreptococcus thermophilus is an important bacterium of lactic acid bacteria widely used in the dairy fermented products, in particular for the yoghurt manufacture in combination with Lactobacillus delbrueckii. In industrial processes, both of those strains often suffer from H2O2damage which causes growth ceasing due to the toxicity. Here, a heme-dependent catalase gene (katE) was isolated from Lactobaillus brevis ATCC367, and transferred into S. thermophilus ST5. The functional expression was observed in S. thermophilus ST5by the determination of the O2formation from H2O2and Native-PAGE analysis. The catalase activity of the recombinant S. thermophilus ST5/pB6KatE reached6.3μmol H2O2min/108CFU which was close to that of the native strain Lb. brevis ATCC367. The expression of KatE in S. thermophilus ST5conferred enhanced oxidative stress resistance, as measured by the increase the viable cells of the exponential and stationary phase at52-and143-fold higher after exposure to6mM or10mM H2O2for1h, respectively. The cell survival ratio was also increased obviously under aerobic conditions. Furthermore, the expressed catalase KatE in S. thermophilus ST5was also beneficial to the partner of Lb. delbrueckii subsp. bugaricus ATCC11842in milk. The survival ratios of the strain Lb. delbrueckii subsp. bugaricus ATCC11842after exposure to6mM or10mM H2O2were increased18-and127-fold at exponential and stationary phase, respectively. Those results showed that the expression of catalase in S. thermophilus could eliminate H2O2from the mixed cultures, thereby protect both S. thermophilus and Lb. delbrueckii from the deleterious effects.