Evolution-aided improvement of the acid tolerance of Levilactobacillus brevis and its application in sourdough fermentation.

Levilactobacillus brevis is crucial in food fermentation, particularly in sourdough production. However, the cultivation of L. brevis faces a challenge with accumulation of lactic acid, a major inhibitor. We aimed to increase the acid tolerance of L. brevis, an industrial strain for sourdough fermentation. We used the adaptive laboratory evolution (ALE) to obtain lactic acid tolerant strains. The evolved strain's fermentation and metabolite profiles, alongside sensory evaluation, were compared with the parental strain by using various analytical techniques. The ALE approach increased lactic acid tolerance in the evolved strain showing an increased growth rate by 1.1 and 1.9 times higher than the parental strain at pH 4.1 and 6.5, respectively. Comprehensive analyses demonstrated its potential application in sourdough fermentation, promising reduced downstream costs. The evolved strain, free from genetically modified organisms concerns, has great potential for industrial use by exhibiting enhanced growth in acidic conditions without affecting consumers' bread preferences.

The immunomodulatory effects of 3-monochloro-1,2-propanediol on murine splenocyte and peritoneal macrophage function in vitro.

3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. MCPD has been reported genotoxic in vitro, and reproductive toxicity and carcinogenicity in rats. To evaluate the immunomodulatory effect of MCPD on murine splenocyte and macrophage in vitro, we investigated splenocyte blastogenesis by concanavalin A (Con A), anti-CD3, and lipopolyssacharide (LPS), the production of cytokines from splenocyte, and the activity of mouse peritoneal macrophages. There was a significant decrease in lymphocyte blastogenesis to Con A or anti-CD3 at subtoxic dose of MCPD. A significant decrease in splenocyte blastogenesis to LPS was also observed. The production level of interferon (IFN)-gamma on splenocyte culture with Con A was significantly reduced at the higher concentration than 1.0mM of MCPD. The levels of interleukin (IL)-4 and IL-10 were also decreased at high concentrations of MCPD. There was a significant decrease in production of nitric oxide (NO) by peritoneal macrophages treated with MCPD. MCPD also inhibits tumor necrosis factor (TNF)-alpha production of stimulated macrophages. These results indicate that MCPD might be able to reduce the functionality of lymphocytes and peritoneal macrophages in vitro.

Evaluation of the potential immunotoxicity of 3-monochloro-1,2-propanediol in Balb/c mice II. Effect on thymic subset, delayed-type hypersensitivity, mixed-lymphocyte reaction, and peritoneal macrophage activity.

3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. To evaluate the immunotoxicity of MCPD, we investigated its effect on the thymic subset, delayed-type hypersensitivity, mixed-lymphocyte reaction and peritoneal macrophage activity. MCPD was administered by gavage for 14 days at 0, 25, 50, and 100 mg/kg/day to female Balb/c mice. The thymic subsets and annexin-V positive cells in thymic cells were quantified by flow cytometry. Mixed-lymphocyte reaction, delayed-type hypersensitivity and peritoneal macrophage activity were assessed. The mixed-lymphocyte reaction and delayed-type hypersensitivity were not significantly changed. However, there were significant increases in the apoptosis of mice treated with high dose of MCPD compared to the vehicle control. A significant decrease in the CD4+CD8+ thymic subset of mice treated with high dose of MCPD was observed. The activity of peritoneal macrophage was significantly reduced in high dose group. These results indicate that MCPD could modulate the immune function in Balb/c mice.

Immunotoxic effect of beta-chlorolactic acid on murine splenocyte and peritoneal macrophage function in vitro.

Beta-chlorolactic acid is a major intermediate of 3-monochloro-1,2-propanediol (MCPD) in mammalian species, which a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. beta-Chlorolactic acid has not been studied on immunotoxicity. To evaluate the immunomodulatory effect of beta-chlorolactic acid on murine splenocyte and macrophage in vitro, we investigated splenocyte blastogenesis by concanavalin A (Con A), anti-CD3 and lipopolysaccharide (LPS), the production of cytokines from splenocyte, and the activity of mouse peritoneal macrophages. beta-Chlorolactic acid suppressed significantly splenic blastogenesis to Con A or anti-CD3 from 8.5 to 54.7% at doses comprised between 200 and 800 microM. beta-Chlorolactic acid also suppressed significantly splenic blastogeneis to LPS from 8.5 to 71.5% at doses comprised between 200 and 800 microM. The production level of interferon (IFN)-g on splenocyte culture with Con A was significantly reduced from 21.5 to 51.4% at the higher concentration than 100 microM of beta-chlorolactic acid. The levels of interleukin (IL)-2 and IL-4 were also decreased 22.6-58.4 and 10.2-36.6%, respectively, at high concentrations of beta-chlorolactic acid. There was a significant decrease from 6.1 to 40.8% in the production of nitric oxide (NO) by peritoneal macrophages treated with 400-1000 microuM beta-chlorolactic acid. These results indicate that beta-chlorolactic acid might be able to induce immunotoxic effect on immune response of lymphocytes and peritoneal macrophages in vitro.

Bisphenol A-induced downregulation of murine macrophage activities in vitro and ex vivo.

Bisphenol A (BPA) is known to have detrimental effects on the reproductive system, but the toxicity of BPA on immune responses has not been systematically investigated. We investigated the effects of BPA exposure on the activities of murine peritoneal macrophages through evaluation of BPA-induced alteration of nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) synthesis, and expression of co-stimulatory molecules B7. Macrophages were examined ex vivo from mice orally treated with various doses of BPA for 5 consecutive days per week for 4 weeks followed by culture for 2 or 4 days in the presence of lipopolysaccharides (LPS). Macrophages from naive mice were also stimulated with LPS ± BPA for 2 or 4 days. NO production was decreased with the in vitro exposure to 1, 10 and 100µM BPA. NO production was lower in the BPA-exposed mice than the control mice with all doses. In vitro, BPA suppressed TNF-α secretion with significant reduction at 10 and 100µM BPA. Similar findings were observed with the macrophages from the BPA-exposed mice. This study provides the substantial evidence on BPA-induced alteration in macrophage activity.

Evaluation of the potential immunotoxicity of 3-monochloro-1,2-propanediol in Balb/c mice. I. Effect on antibody forming cell, mitogen-stimulated lymphocyte proliferation, splenic subset, and natural killer cell activity.

3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. MCPD has been reported genotoxic in vitro, and reproductive toxicity and carcinogenicity in rats. However, no previous studies have investigated MCPD-induced alterations in the immune system. In the present study, MCPD was administered by gavage for 14 days at 0, 25, 50, and 100 mg/kg per day to female Balb/c mice. The antibody-mediated immune response to sheep red blood cells (SRBC) was assessed using the antibody-forming cell (AFC) assay, and splenic cell phenotypes were quantified by flow cytometry. Hematological and histopathological changes were assessed. Mitogen-stimulated spleen lymphocyte proliferation and natural killer (NK) cell activity were evaluated. The T-lymphocyte blastogenesis by concanavalin A (Con A) or anti-CD3 and B-lymphocyte blastogenesis by lipopolysaccharide (LPS) were not significantly changed. There were no significant changes in the hematological and histopathological findings of MCPD-treated mice. However, the significant decrease in thymus weight was observed in 100 mg dose group, even though that did not change body weight gain. The cellularities of spleen and thymus were significantly reduced in high-dose group. Exposure to high dose of MCPD decreased the AFC response to SRBC in mice. There was a significant decrease in NK cell activity of mice treated with high dose of MCPD. These results indicate that MCPD could modulate the immune function in Balb/c mice.

Evaluation of lymphocyte subpopulations in draining lymph node cells following allergen and irritant.

The murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for the assessment of the contact sensitization potential. However, there is a need to develop a non-radioisotopic endpoint for the LLNA, because of the radioisotopic method's requiring the use of special facilities. In this study, we investigated to evaluate the lymphocyte subpopulations in the lymph node cells following allergen and irritant treatment. Female Balb/c mice were treated by the topical application on the dorsum of both ears with sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI), and α-hexylcinnamaldehyde (HCA), and an irritant, sodium lauryl sulfate (SLS), once daily for three consecutive days. The lymph node (LN) cells were harvested 72h after the final treatment. Phenotypic analysis of lymphocytes subsets was performed with a flow cytometry. The allergens DNCB, TDI, and HCA and an irritant, SLS increased cell number compared to the vehicle. Mice were treated with DNCB, HCA, and TDI showed a preferential increase in the percentage of B220+CD40+ cells compared with vehicle and irritant-treated mice. There was an increase in B220+CD86+ cells of mice treated with DNCB, TDI, and HCA, but no significant increases were observed in mice treated with SLS. Mice were treated with DNCB and TDI showed an increase in the percentage of B220+CD23+ cells compared with vehicle and irritant-treated mice. These results suggest that analysis of B cell activation marker, CD40 on B cells may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice.