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- MATERIALS AND METHODS
The objective of this study was to investigate the probiotic properties of the fermented vegetable derived lactic acid bacterium, L. plantarum. L. plantarum 10hk2 showed antibacterial activity against pathogenic bacteria and immunomodulating effects on murine macrophage cell lines. RAW 264.7 cells stimulated with viable cells of this probiotic strain increased the amounts of pro-inflammatory mediators such as IL-1β, IL-6 and TNF-α, as well as the anti-inflammatory mediator, IL-10. ICR mice fed with viable cells of L. plantarum 10hk2 had reduced numbers of enteric Salmonella and Shigella species in comparison to controls from 2 weeks after supplementation, and this effect was observed for up to 4 weeks. The findings of this study suggest that this specific lactic acid bacterial strain, which is derived from vegetable fermentation, holds great promise for use in probiotics and as a food additive since it can reduce the number of some pathogenic bacteria through production of lactic acids.
It is well known that lactic acid bacteria are beneficial to host health because they inhibit the growth of harmful microorganisms. In particular, ingestion of certain members of the Lactobacillus genus is believed to have beneficial effects on the health of the host (1, 2). Probiotic strains may be able to maintain a metabolically active state during gastrointestinal passage. In other words, the viability of probiotics could be crucial for improvement of the balance of intestinal bacterial flora (3). Additionally viable bacteria have been reported to provide greater stimulation of intestinal immunity in hosts than nonviable bacteria (4). Furthermore, infectious diseases in general are still a major problem, intestinal infectious diseases caused by pathogenic or opportunistic microorganisms in particular posing a more serious problem because overuse of antibiotics has induced antibiotic-resistant bacteria. Among harmful and opportunistic bacteria, Escherichia and Salmonella together with Shigella species are the most serious pathogens (3, 5).
Macrophages are tissue-based phagocytes that play a central role in initiating the first line of defense in host immunity. Activated macrophages, which typically become activated in the presence of microbial components, phagocytose micro-organisms, secrete pro-inflammatory cytokines and NO, and hence may regulate the host's immunity (6, 7). Several studies have demonstrated that viable or heat-killed cells of Lactobacilli and Bifidobacterium species, as well as their cell components, are capable of stimulating production of NO, IL-6 and TNF-α in macrophage cell lines (8–10). Some investigators have found that the effects on induction of cytokines are greater in viable bacteria than in heat-killed cells (11, 12). In response to internal or external invasion, these cytokines contribute to the defense mechanisms of the host, while over-expression of these pro-inflammatory cytokines may induce immunopathological disorders (11). Therefore, a balance between pro- and anti-inflammatory cytokines is important for host immunity (9, 13).
Anti-inflammatory IL-10 is known to block induction of synthesis of TNF-α, IL-1β and IL-6 by mouse peritoneal macrophages (14, 15). IL-10 also stimulates B cells and augments antibody production in activated B cells (16, 17). Studies on the anti-inflammatory effects of lactic acid bacteria have focused solely on heat-killed forms of these bacterial cells (9, 13, 18). Interestingly, it has previously been demonstrated by others that, in RAW 264.7 cells, LPS of Gram-negative bacteria can induce not only production of pro-inflammatory cytokines, such as TNF-α and IL-6, but also production of anti-inflammatory molecules such as IL-10 (11, 12).
In this study, the biochemical characteristics of L. plantarum 10hk2 were examined with the aim of assessing its potential use as a probiotic. In addition, its probiotic effects when mice were fed with viable cells were assessed. Another purpose of the study was to determine the immune enhancing effects of viable cells in comparison to heat-killed cells of this strain on pro- and anti-inflammatory cytokine induction, using the macrophage cell line RAW 264.7.
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- MATERIALS AND METHODS
Based on its tolerance to acid, heat and freezing, the L. plantarum 10hk2 isolated in this study holds promise as a potentially beneficial probiotic strain. The rate of recovery of this strain after freeze drying was quite different from that of L. plantarum ATCC 14917. After freeze drying under the same culture and freezing conditions (including similar initial cell numbers of 1.1 and 1.5 × 109 CFU/g, respectively, data not shown), the numbers of viable L. plantarum 10hk2 and L. plantarum ATCC 14917 were 3.0 × 1012 CFU/g and 1.7 × 1011 CFU/g, respectively. The viability of lactic acid bacteria after lyophilization seems to be species-specific. Many factors which influence the rate of survival after freeze-drying, such as bacterial species, cell density, use of a lyoprotectant, drying medium and freezing rate, have been reported (27, 28). In conclusion, L. plantarum 10hk2 was found to be a less stress sensitive species than L. plantarum ATCC 14917.
In our study, each mouse consumed 4 to 5 grams of the experimental diet per day on average (data not shown), accordingly the amount of lactic acid bacteria consumed was calculated to be at least 1.0 × 108 CFU/g/day. Supplementation of probiotics results in an excessive number of lactic acid bacteria relative to the number of pathogenic bacteria in the intestinal microflora; thus, this change in balance of intestinal flora may result in effective antibacterial activity that could inhibit the in vivo growth of enteric bacteria. The relative proportions of enteric and Salmonella and Shigella species had decreased at the end of two and three weeks of supplementation from 14% to 6% and 6% to less than 1%, respectively. For probiotic applications in chickens, dried bacterial cells of the Lactobacillus strains were mixed with commercial feed to achieve a desired bacterial count of 106 CFU/g of the feed. This treatment resulted in stable lactobacilli counts and reduced the numbers of Enterobacteriaceae in the intestine (29). This probiotic strain is believed to pass safely from the stomach to the intestine, and could therefore inhibit the growth of enteric bacteria such as E. coli, Salmonella and Shigella species. The present results could be explained by the ability of L. plantarum 10hk2 to produce huge amounts of lactic acid compared to the commercial strain, L. plantarum ATCC 14917. In vitro, the antibacterial activity of the cultural supernatant of L. plantarum 10hk2 against S. paratyphi and S. flexneri was found to be stronger than that of L. plantarum ATCC 14917. One plausible reason for this having occurred is the huge amounts of lactic acid that are produced by L. plantarum 10hk2. Some previous studies have also indicated that the inhibitory action of lactic acid bacteria is due to their production of lactic acid (30, 31). In addition, the dry weight of the feces was measured and found to be significantly less in the L. plantarum 10hk2 group (over 30% less) relative to the control group (data not shown). This observation suggests that improved dietary digestion is achieved by adding lactic acid bacteria to the feed.
An increase in the percent of CD19+ cells in the spleens of the healthier mice which had been injected with an external antigen (SRBC) and fed with L. plantarum 10hk2 may be explained by the reduction in the number of harmful enteric bacteria and altered balance of the intestinal microflora. In other words, the ability of the viable L. plantarum 10hk2 to induce pro-inflammatory mediators such as NO, IL-6 and TNF-α and anti-inflammatory IL-10 may have resulted in induction of immune responses, resulting in healthier hosts. This finding is in accordance with the fact that the Lactobacillus strains were demonstrated to have strain-specific effects on B- and T cell proliferation. Also L. acidophilus treatment has been found to enhance B cell response with external LPS stimulation (32). Furthermore, TNF-α production is enhanced by exposure to lactic acid bacteria (12, 16), and therefore the effectiveness of B cells would, at least in part, have been enhanced. In a more recent review, a response regulator of B lymphocytes, CD19 deficiency, was found to inhibit infiltration of neutrophils and macrophages, and to decrease expression of cytokines such as IL-6 and TGF-β in mice. CD19, a regulator of B cell response, has also been demonstrated to regulate IL-6 expression in wound healing tissues in mice (33).
In this study, we compared the ability of viable and heat-killed cells of L. plantarum 10hk2 to induce production of pro- and anti-inflammatory responses in RAW 264.7 cells. The ability of this strain to induce release of these cytokines was reduced after viable cells had been subjected to heat treatment. These results are consistent with a recent study which examined Lactococcus strains (18), and several other previous studies (8–12). In regards to the release of anti-inflammatory cytokines in J774.1 cells, viable cells of L. casei have been found to stimulate greater amounts of TGF-β than do heat-killed bacteria (12). However, this study has demonstrated that viable cells of L. plantarum 10hk2 are able to simultaneously induce both pro- and anti-inflammatory mediators. A previous study found that, in human peripheral blood mononuclear cells stimulated with live L. rhamnosus and L. acidophilus, production of both pro- and anti-inflammatory cytokines, such as IL-6, TNF-α and IL-10, was induced in amounts even greater than those produced by LPS (11).
In conclusion, it was found in this study that L. plantarum 10hk2 supplementation enhances the health of mice by reducing the number of harmful enteric bacteria in the intestines, these changes being due to production of huge amounts of lactic acids and to the immune-enhancing effects of L. plantarum 10hk2. The present study also demonstrates that viable cells of this strain can induce both pro- and anti-inflammatory mediators, which helps to maintain a balance between these two adverse types of mediator. Based on these results, it appears that this probiotic strain may be a good candidate for a food additive to keep organisms healthier.