Propionibacterium Freudenreichii ET-3 Research Articles

Anti-osteoporosis effects of 1,4-dihydroxy-2-naphthoic acid in ovariectomized mice with increasing of bone density

Objective1,4-Dihydroxy-2-naphthoic acid (DHNA) is the main component of the metabolic products after fermentation by Propionibacterium freudenreichii ET-3. In this study, DHNA, vitamin K2 (VK2), and risedronate (Ris) were administered to ovariectomized (OVX) mice to ascertain their suppressive effects on bone mass reduction. MethodsFifty mice were divided into five groups: Sham, OVX, DHNA, VK2, and Ris groups. The drugs were administered daily for 8 weeks. Subsequently, blood was collected from the orbital venous plexus. The bilateral femurs and L5 lumbar vertebra were excised. To determine the effects of the drugs, we performed histomorphometric analyses of the left femur and L5 by micro-CT, biochemical analyses of serum samples, and histological analyses of the right femur. ResultsThe results for the total bone mineral density, bone volume density, trabecular thickness, trabecular number, trabecular separation, and histological analyses showed that bone resorption was improved in the DHNA and Ris groups compared with the OVX group. Furthermore, the biochemical analyses revealed that the production of inflammatory cytokines was suppressed in the DHNA and Ris groups. ConclusionsThe present findings show that DHNA suppresses the bone mass reduction in OVX mice, suggesting that it could be an alternative treatment for postmenopausal osteoporosis.

Identification of a probiotic bacteria‐derived activator of the aryl hydrocarbon receptor that inhibits colitis

The aryl hydrocarbon receptor (AhR) recognizes environmental xenobiotics and is originally thought to be involved in the metabolism (detoxification) of the substances. Recently, AhR is highlighted as an important regulator of inflammation. Notably, accumulating evidence suggests that activation of the AhR suppresses inflammatory bowel diseases (IBDs). Therefore, non-toxic AhR activators become attractive drug candidates for IBD. This study identified 1,4-dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2) abundantly produced by Propionibacterium freudenreichii ET-3 isolated from Swiss-type cheese, as an AhR activator. DHNA activated the AhR pathway in human intestinal epithelial cell line Caco2 cells and in the mouse intestine. Oral treatment of mice with DHNA induced anti-microbial proteins RegIIIβ and γ in the intestine, altered intestinal microbial flora and inhibited dextran sodium sulfate (DSS)-induced colitis, which recapitulated the phenotypes of AhR activation in the gut. As DHNA is commercially available in Japan as a prebiotic supplement without severe adverse effects, DHNA or its derivatives might become a promising drug candidate for IBD via AhR activation. The results also implicate that intestinal AhR might act not only as a sensor for xenobiotics in diet and water but also for commensal bacterial activity because DHNA is a precursor of vitamin K2 produced by vitamin K2-synthesizing commensal bacteria as well as propionic bacteria. Hence, DHNA might be a key bacterial metabolite in the host-microbe interaction to maintain intestinal microbial ecosystem.

Safety evaluation of Propionibacterium freudenreichii ET-3 culture

Propionibacterium freudenreichii ET-3 culture, a cell-free product of whey fermentation using P. freudenreichii ET-3 (7025), has been shown to promote the growth of Bifidobacteria through the action of 1,4-dihydroxy-2-naphthoic acid (DHNA), and therefore, has potential use in the food and supplement industries. Although currently used as a food ingredient in Japan, the safety of this novel ingredient has not been previously evaluated through traditional toxicity testing. Therefore, here we report the results of standard toxicological testing performed on P. freudenreichii ET-3 culture. In a 4-week oral toxicity study, administration of 6000 mg/kg body weight/day P. freudenreichii ET-3 culture was without compound-related adverse effects on clinical signs, body weights, food consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and gross and microscopic findings in male and female Sprague–Dawley rats. Furthermore, in vitro mutagenicity testing demonstrated that P. freudenreichii ET-3 culture was non-mutagenic in the bacterial reverse mutation assay using a standard battery of bacterial strains ( Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2 uvrA) and non-clastogenic in Chinese hamster lung cells in the mammalian chromosome aberration test. Together, the results of these studies support the safety of P. freudenreichii ET-3 culture for use in foods for human consumption.

Safety of high doses of Propionibacterium freudenreichii ET-3 culture in healthy adult subjects

Propionibacterium freudenreichii ET-3 (7025) culture, a cell-free product of whey fermentation by P. freudenreichii ET-3, has been shown to promote the growth of Bifidobacteria through the action of 1,4-dihydroxy-2-naphthoic acid (DHNA). Here we report the results of two clinical studies designed to evaluate the safety of high doses of P. freudenreichii ET-3 culture medium. Study 1 had a randomized, double-blind, crossover design. Ten healthy male and four healthy female subjects received 45 tablets of either P. freudenreichii ET-3 culture medium (total daily intake of 3 g solid content and 283.5 μg of DHNA; active group) or placebo (unfermented product) during two 1-week supplementation periods separated by a 4-week washout period. In Study 2, 11 healthy men took four tablets of P. freudenreichii ET-3 culture medium per day (total daily intake of 0.267 g solid content and 22.5 μg of DHNA) for a period of 13 weeks. In both studies, hematological, clinical chemistry, and urinary parameters were measured before and after each supplementation period and gastrointestinal symptoms were assessed by questionnaire. In Study 1, there were no statistically significant differences between placebo and active supplementation periods in any measured parameter and the incidence of gastrointestinal symptoms were similar between groups. In Study 2, total protein, white blood cell count, hemoglobin, and mean corpuscular hemoglobin concentration decreased significantly from baseline and mean corpuscular volume and urine pH increased from baseline. The changes in hematological parameters were deemed not to be due to P. freudenreichii ET-3 culture medium supplementation given that all parameters remained within normal ranges and were not consistent with any clinically meaningful effect.

Fermentation Metabolites fromLactobacillus gasseriandPropionibacterium freudenreichiiExert Bacteriocidal Effects in Mice

Lactobacillus gasseri OLL 2716 promotes the elimination of Helicobacter pylori and is utilized in yogurts that are specifically labeled as health foods. On the other hand, milk whey fermented by Propionibacterium freudenreichii ET-3, which increases the numbers of Bifidobacterium, is effective for intestinal disorders. We previously demonstrated that oral administration of L. gasseri and P. freudenreichii fermentation metabolites (LP-FM) improved calf intestinal microflora and reduced the incidence of diarrhea. However, the detailed immunological mechanisms responsible for these effects remain to be fully understood. In this study, we investigated whether LP-FM stimulates the innate immune response and promotes the elimination of Listeria monocytogenes in mice. The C57BL/6 female mice that were treated with LP-FM or L. gasseri fermentation metabolites alone for 4 weeks had more peripheral white blood cells than the untreated control mice. In particular, LP-FM-treated mice had higher CD4- and CD8-positive T-cell counts. The levels of reactive oxygen and nitrogen species produced by peritoneal macrophages were also higher in LP-FM-treated mice. Furthermore, LP-FM-treated mice that were infected with L. monocytogenes exhibited significant enhancement of the elimination of Listeria from the spleen and the liver in comparison with untreated control mice infected with Listeria. The activation of innate immunity by LP-FM was increased by the combination of fermentation metabolites from P. freudenreichii. These results suggest that LP-FM, which contains metabolites from L. gasseri and P. freudenreichii, stimulates the function of the innate immune system, thereby significantly promoting the elimination of L. monocytogenes in mice.

Influences of Prebio Support<sup>TM</sup> (Mixture of Fermented Products of <i>Lactobacillus gasseri</i> OLL2716 and <i>Propionibacterium</i> <i>freudenreichii</i> ET-3) on the Composition and Metabolic Activity of Fecal Microbiota in Calves

The influence of Prebio Support™ (PS), which is a mixture of fermented products of Lactobacillus gasseri OLL2716 and Propionibacterium freudenreichii ET-3, on the fecal microbiota and fecal metabolites in calves were investigated. During the intake of PS, the number of bifidobacteria was significantly higher (p<0.05), and the fecal water content (p<0.05) and fecal ammonia (p<0.05) were significantly lower in the PS intake group than in the control group. Furthermore, fecal concentrations of sulfide tended to decrease and short-chain fatty acids (acetic, butyric, and propionic acids) tended to increase through the intake of PS. The numbers of other fecal bacteria and the fecal pH of the PS intake group did not differ from those of the control group. The fecal condition, such as hardness, in calves given PS was better than that of the control group. These findings indicate that PS intake effectively improves the fecal environment, and there is a possibility of it alleviating clinical symptoms.

Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption

The main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 is 1,4-dihydroxy-2-naphthoic acid (DHNA), which has a naphthoquinone skeleton, as in vitamin K2. This study showed that DHNA improved bone mass reduction with osteoporosis model mice caused by FK506. Growth of the intestinal bacterium Lactobacillus bifidus is specifically facilitated by DHNA. The present study used osteoporosis model mice to investigate the effects of DHNA on bone remodeling. FK506, an immunosuppressant, was used to prepare osteoporosis model mice. Thirty mice were divided into three groups: FK group, FK+DHNA group, and control group. In the FK group, FK506 was administered to induce bone mass reduction. In the FK-DHNA group, FK506 and DHNA were administered concurrently to observe improvements in bone mass reduction. To ascertain systemic and local effects of DHNA, we investigated systemic pathological changes in colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments. Compared to the FK group without DHNA, colon damage and kidney dysfunction were milder for FK+DHNA group, and production of inflammatory cytokines (interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha) was more suppressed. Furthermore, compared to the group without DHNA, histological analyses and radiography showed that bone resorption was suppressed for the DHNA group. Culture experiments using osteoclasts from murine bone marrow showed osteoclast suppression for the DHNA group compared to the group without DHNA. These results show that DHNA has some effects for improving bone mass reduction caused by FK506.

Electrochemical regulation of the end‐product profile in Propionibacterium freudenreichii ET‐3 with an endogenous mediator

The end-product profile of the glucose fermentation by Propionibacterium freudenreichii ET-3 changed on an electrochemical treatment, in which the culture vessel was filled with a carbon felt anode. Acetate and propionate were produced as final end products in a molar ratio of 2:3 without any electrochemical treatments at the point of the consumption of lactate as an intermediate of the glucose fermentation. The ratio was changed to 1:1 at the point of the lactate consumption by the electrochemical incubation at an electrode potential of 0.4 V versus Ag|AgCl for 100 h. During further electrochemical incubation, propionate was oxidized to acetate as a final end-product in the microbe-containing anode chamber. 1,4-Dihydroxy-2-naphthoic acid produced by P. freudenreichii ET-3 itself would receive electrons from the metabolic pathway and serve as an electron transfer mediator from the microbial cells to the electrode.

Characteristic of milk whey culture with Propionibacterium freudenreichii ET-3 and its application to the inflammatory bowel disease therapy

After the screening of microorganism culture, the culture of Propionibacterium freudenreichii ET-3 in the milk whey (milk whey culture) was found to stimulate the growth of our own Bifidobacteria in the colon but not the growth of other microorganisms. One of the active substances was identified as 1,4-dihydroxy-2-naphthoic acid (DHNA).In healthy volunteers, the ingestion containing milk whey culture significantly increased the population of Bifidobacteria to total fecal bacterium. In the TNBS-induced colitis model of rats, milk whey culture significantly accelerated the healing of the colitis in a dose-dependent manner. It has been reported that DHNA inhibited the lymphocyte infiltration through reduction of MAdCAM-1 in DSS colitis model of mice and that the ingestion of milk whey culture was effective in the treatment of ulcerative colitis in human pilot study. These findings suggest that milk whey culture is a useful prebiotic for the therapy of inflammatory bowel disease.

Production of extracellular bifidogenic growth stimulator by anaerobic and aerobic cultivations of several propionibacterial strains

Production of a bifidogenic growth stimulator (BGS) by propionic acid bacteria was investigated under anaerobic and aerobic culture conditions. To measure the concentration of extracellular BGS produced by propionic acid bacteria, we evaluated the effects of bioassay conditions using Bifidobacterium longum as a test microorganism on the formation of a growth-stimulation zone. The diameter of the growth-stimulation zone was significantly affected by both the component concentrations and the pH of a bioassay medium. The optimum component concentrations and pH of a bioassay medium were one-half of the normal values and 8.5, respectively. Using the bioassay method, we can measure the concentration of BGS produced by propionic acid bacteria ranging in concentrations from 0.1 microg/l to 1 mg/l using 1,4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as standards. Of six dairy propionic acid bacterial strains tested, the four strains (Propionibacterium freudenreichii ET-3, P. shermanii PZ-3, P. acidipropionici JCM 6432, and P. jensenii JCM 6433) produced BGS at a concentration range of 4-23 mg/l under the anaerobic culture conditions. Analysis of high performance liquid chromatography (HPLC) showed that more than 70% of total BGS produced in supernatant samples was DHNA and no ACNQ was produced by the strains. The effect of oxygen supply on BGS production was investigated for the four BGS-producing strains. The aerobic conditions exerted in positive effects on BGS production by only P. acidipropionici JCM 6432. The concentration of BGS obtained in the aerobic cultivation of P. acidipropionici JCM 6432 was 1.3-fold than that in anaerobic cultivation. Different properties (BGS production as well as cell growth and glucose metabolism) occurring in response to the aerobic conditions were observed, depending on the propionic acid bacterial strain used. This paper is the first report on BGS production by propionibacterial strains except for P. freudenreichii.

Maximum Production of 1,4-Dihydroxy-2-Naphthoic Acid by Fed-Batch and Anaerobic/Aerobic Culture of Propionibacterium freudenreichii ET-3

Abstract Maximum production strategy of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch and anaerobic/aerobic culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and the cell produces DHNA and MK in the molar ratio of 3:2 by consuming lactose in skim milk powder medium under normal anaerobic condition. We found that in anaerobic fed-batch culture, which limited lactose concentration, the cell stopped producing MK. As a result, DHNA concentration in fed-batch culture increased as lactose feeding rate decreased, and the maximum DHNA concentration in fed-batch culture was 0.40 mM, which is 3.3-fold that in anaerobic batch culture. However, propionate accumulation inhibited and then stopped DHNA production in anaerobic fed-batch culture. Our previous study showed that the cell could consume propionate under aerobic condition and this knowledge based on metabolic flux distribution for DHNA production was applied here. The cell produced DHNA continuously by switching from anaerobic condition to aerobic condition after lactose feeding. DHNA concentration in the optimal condition was 0.51 mM, which is 4.2-fold that in anaerobic batch culture.

Optimal aerobic cultivation method for 1,4-dihydroxy-2-naphthoic acid production by Propionibacterium freudenreichii ET-3

To investigate the effects of oxygen supply on Propionibacterium freudenreichii ET-3 metabolism and 1,4-dihydroxy-2-naphthoic acid (DHNA) production in detail, the strain was cultured by switching from anaerobic condition to aerobic condition at 72 h (termed anaerobic-aerobic switching culture hereafter) employing different oxygen transfer rates (OTRs) in the range of 0.08-0.90 mg/(l.h). It was found that a 0.08 mg/(l.h) OTR could not change the metabolism or improve the DHNA production of P. freudenreichii ET-3. When the OTR was in the range of 0.23-0.66 mg/(l.h), propionate, which inhibits DHNA production significantly, was consumed during the aerobic phase. Final DHNA concentration increased to 0.22 mM, irrespective of OTR. When the OTR was 0.90 mg/(l.h), a sudden increase in dissolved oxygen (DO) concentration during the aerobic phase resulted in a sudden decrease in DHNA concentration. To attenuate the stresses caused by propionate and oxygen exposure, we designed an optimal cultivation in which the anaerobic and aerobic phases were repeated three times alternately. As a result, propionate concentration was maintained below the level that inhibits DHNA production, and no DO concentration was detected throughout the culture. The final DHNA concentration in this culture was 0.33 mM, which is 2.7-fold that in the anaerobic culture and 1.5-fold that in the anaerobic-aerobic switching culture.

Aerobic culture of Propionibacterium freudenreichii ET-3 can increase production ratio of 1,4-dihydroxy-2-naphthoic acid to menaquinone

This is the first report on the production of both 1,4-dihydroxy-2-naphthoic acid (DHNA) and menaquinone by Propionibacterium freudenreichii ET-3. DHNA can be a stimulator of bifidogenic growth, and menaquinone has important roles in blood coagulation and bone metabolism. During anaerobic culture, DHNA and menaquinone concentrations reached 0.18 mM and 0.12 mM, respectively. The molar ratio between these products was approximately 3:2, which was not affected by culture pH and temperature over the ranges of 6.0-7.0 and 31-35 degrees C, respectively. As for organic acid, propionate and acetate accumulated at concentrations of 0.3 M and 0.15 M, respectively, and the propionate accumulation particularly inhibited further production of DHNA. To improve DHNA production, we switched from anaerobic condition to aerobic condition during the culture when lactose was depleted. DHNA concentration continued to increase even after lactose exhaustion, reaching 0.24 mM. In contrast to DHNA production, menaquinone production stopped after the switch to aerobic condition. The total molar production of DHNA and menaquinone was 0.3 mM irrespective of aerobic culture and anaerobic-aerobic switching culture. Therefore, the anaerobic-aerobic switching culture could increase the production ratio of DHNA to menaquinone. The DHNA concentration obtained from the anaerobic-aerobic switching culture was 1.3-fold higher than that in the anaerobic culture, because P. freudenreichii ET-3 utilized propionate accumulated in the medium via the reversed methylmalonyl CoA pathway under aerobic condition. The culture method proposed in this study could be applicable to industrial-scale fermentation using 1000 l of media, by which 0.23 mM DHNA was produced.

Milk Whey Culture With Propionibacterium freudenreichii ET-3 Is Effective on the Colitis Induced by 2,4,6-Trinitrobenzene Sulfonic Acid in Rats

This study aimed to evaluate whether milk whey culture with Propinibacterium freudenreichii ET-3 (milk whey culture), which has been reported to have Bifidogenic activity, is effective on the colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rats. For the induction of colitis, the colon was clamped and 0.1 M TNBS in 35% ethanol was injected into the luminal side of the clamped portion under pentobarbital anesthesia. From the next day of colitis induction, milk whey culture was administered orally at doses of 1 and 3 g/kg, twice a day for 9 days. On the 10th day, rats were sacrificed and ulcer size was measured. Milk whey culture significantly accelerated the healing of the colitis in a dose-dependent manner, but culture medium did not. To clarify the active substance, the effects of propionic acid and acetic acid contained in milk whey culture was tested. Sodium propionate significantly accelerated the healing of TNBS-induced colitis, but sodium acetate did not. The above results show that milk whey culture may become a useful prebiotic for the therapy of inflammatory bowel disease and that propionic acid may be one of the active substances contained in milk whey culture.

Isolation and identification of a new bifidogenic growth stimulator produced by Propionibacterium freudenreichii ET-3.

We have found a new growth stimulator for bifidobacteria in the culture broth of Propionibacterium freudenreichii ET-3. The bifidogenic growth stimulator (BGS) was purified by Diaion HP-20 column chromatography and preparative HPLC. Spectroscopic methods including 1H-NMR, UV, and LC-ESI-MS experiments indicated that the chemical structure of the bifidogenic growth stimulator was 1,4-dihydroxy-2-naphthoic acid (DHNA). Approximately 10 mg/L of DHNA was found to be produced in the culture broth of P. freudenreichii ET-3.

Effect of Ingested Culture of Propionibacterium freudenreichii ET-3 on Fecal Microflora and Stool Frequency in Healthy Females

The effect of the ingested culture of Propionibacterium freudenreichii ET-3 that produced bifidogenic growth stimulator (BGS) on stool frequency and quantity was investigated in 41 young healthy women in a randomized double-blind crossover study. The fecal samples of 7 of these 41 subjects were analyzed to study the modification of intestinal microflora by BGS. The results were compared with the effects of an ingested placebo tablet. The ratio of bifidobacteria population (%) to total bacteria population during the BGS-tablet intake period was significantly increased (p < 0.05) in comparison with that during the placebo tablet intake period. There were no significant differences in fecal pH and water content during the experimental periods. However, the putrefactive metabolites, namely, fecal indole and skatole concentration, of the BGS-tablet intake period significantly decreased (p < 0.05) in comparison with those of the placebo tablet period. The stool quantity of the BGS-tablet intake period was slightly higher (p = 0.083) than that of the nonintake period. Moreover, the stool frequency of the BGS-tablet intake period chosen from those originally with lower frequency (≤ 4 times/week) was significantly higher (p < 0.05) than that of the placebo tablet intake period. These results show that the ingestion of P. freudenreichii ET-3 culture increases the number of defecations of constipated females, indicating an improvement of intestinal microflora.

Effects of Culture-Powder of Propionibacterium freudenreichii ET-3 on Fecal Microflora of Normal Adults

The effect of a culture powder of Propionibacterium freudenreichii ET-3 cells containing a bifidogenic growth stimulator (BGS) on fecal flora was studied in twelve healthy subjects with a mean age of 42.6 (range, 27-56 years). One gram of BGS powder containing 17.4 U of BGS activity was administered for 14 days, three times a day after meals. A significantly higher number of bifidobacteria in fecal samples was observed during the intake period of the BGS powder. The frequency of occurrence of the bifidobacteria also increased from 92% to 100%. These results indicate that the BGS acts in the intestine as a growth stimulator of bifidobacteria when the BGS powder is administered.