Bifidogenic Growth Stimulator Research Articles

Selective bioelectronic sensing of pharmacologically relevant quinones using extracellular electron transfer in Lactiplantibacillus plantarum

Redox-active small molecules containing quinone functional groups play important roles as pharmaceuticals, but can be toxic if overdosed. Despite the need for a fast and quantitative method to detect quinone and its derivatives, current sensing strategies are often slow and struggle to differentiate between structural analogs. Leveraging the discovery that microorganisms use certain quinones to perform extracellular electron transfer (EET), we investigated the use of Lactiplantibacillus plantarum as a whole-cell bioelectronic sensor to selectively sense quinone analogs. By tailoring the native EET pathway in L. plantarum, we enabled quantitative quinone sensing of 1,4-dihydroxy-2-naphthoic acid (DHNA) - a gut bifidogenic growth stimulator. We found that L. plantarum could respond to environmental DHNA within seconds, producing concentration-dependent electrical signals. This sensing capacity was robust in different assay media and allowed for continuous monitoring of DHNA concentrations. In a simulated gut environment containing a mixed pool of quinone derivatives, this tailored EET pathway can selectively sense pharmacologically relevant quinone analogs, such as DHNA and menadione, amongst other structurally similar quinone derivatives. We also developed a multivariate model to describe the mechanism behind this selectivity and found a predictable correlation between quinone physiochemical properties and the corresponding electrical signals. Our work presents a new concept to selectively sense quinone using whole-cell bioelectronic sensors and opens the possibility of using probiotic L. plantarum for bioelectronic applications in human health.

DETERMINATION OF THE LYOPHILIZED EXOPOLYSACCHARIDES (L-EPS) OF LACTOBACILLUS BACTERIA BIFIDOGENIC GROWTH STIMULATOR EFFECT

Bu çalışmada, Ankara ilinin farklı bölgelerinden temin edilen serbest dolaşan köy tavuklarının gastrointestinal sisteminden Lactobacillus cinsine ait 119 bakteri izole edilmiştir. Yüksek ekzopolisakkarit (EPS) üretim kapasitesine sahip 11 izolat seçilmiştir. Seçilen izolatların biyokimyasal ve moleküler tanımlamaları gerçekleştirilmiş ve tanımlama sonuçlarına göre 6 izolatın Lactobacillus salivarius, 2 izolatın Lactobacillus agilis, 2 izolatın Lactobacillus reuteri ve 1 izolatın da Lactobacillus saerimneri olduğu tespit edilmiştir. Yüksek EPS üreten 3 suşun (L. salivarius ZDM2132, BİS312 ve BİS722) EPS’leri kültür ortamından izole edilerek liyofilize edilmiştir. Bifidobacterium gallinarum ATCC 33777 suşunun, L. salivarius ZDM2132, BİS312 ve BİS722 bakterilerden elde edilen liyofilize EPS’yi (l-EPS) fermente edebilme kapasitesi ve bifidobakterilerin gelişimini düzenleyici (BGD) etkileri ticari bir prebiyotik olan inülin ile karşılaştırılmıştır. l-EPS’lerin B. gallinarum tarafından fermente edildiği ve inülinden daha iyi bir BGD etkisi gösterdiği gözlemlenmiştir.

Assessment of anti-biofilm activity and bifidogenic growth stimulator (BGS) effect of lyophilized exopolysaccharides (l-EPSs) from Lactobacilli strains

ABSTRACTIn the present study, a total of used six lactic acid bacterial strains that isolated from children’s feces and milk products. This study investigated the exopolysacch aride production of these strains. In the culture broth, highest exopolysaccharide production capacity was detected in GD-11, LB-69, and B-3 strains. Monosaccharide compositions of the exopolysaccharides that were purified and lyophilized in the culture medium were investigated and dominant monomer was found to be mannose in all strains. The biofilm-forming capacity of 13 pathogen bacteria was determined and four strains with the highest biofilm-forming capacity were selected as test bacteria for anti-biofilm studies. Different concentrations of l-exopolysaccharides of three lactic acid bacteria were tested for anti-biofilm effect. Highest anti-biofilm effects were exhibited by the L. fermentum LB-69 (90%) against B. cereus RSKK 863. In the statistical analyses performed using the available data, a strong correlation was found between the amount of mannose present in l-exopolysaccharides and the anti-biofilm activity exhibited by l-exopolysaccharides (p < 0.01). In this study, it was concluded that the l-exopolysaccharides of the L. fermentum LB-69 strain could be used as efficient agents with both bifidogenic growth stimulator effect and anti-biofilm effect. If these agents are taken via oral route, it would be possible to provide beneficial effects for the host by increasing the development of bifidobacteria present in the gastrointestinal tract as well as to prevent and treat the diseases by hindering the biofilm formation by harmful bacteria.

Effects of a Formula Containing Two Types of Prebiotics, Bifidogenic Growth Stimulator and Galacto-oligosaccharide, and Fermented Milk Products on Intestinal Microbiota and Antibody Response to Influenza Vaccine in Elderly Patients: A Randomized Controlled Trial

We investigated the effect of a formula containing two different prebiotics (bifidogenic growth stimulator and galacto-oligosaccharide) and fermented milk products on intestinal microbiota and antibody responses to an influenza vaccine in enterally fed elderly in-patients. Patients were administered either formula containing prebiotics and fermented milk products (group F: n = 12, 79.9 ± 9.5 years old) or standard formula (group C: n = 12, 80.7 ± 10.1 years old) via percutaneous endoscopic gastrostomy during a 14-week intervention period. Subjects were immunized with an influenza vaccine (A/H1N1, A/H3N2, and B) at week 4 of the intervention. Blood biochemical indices, intestinal bacteria populations and antibody titers were analyzed. Bifidobacterium counts increased significantly in group F compared with group C. The enhanced antibody titers against A/H1N1 were maintained in group F for a longer period compared with group C. The titers against A/H3N2 were unchanged between both groups, and those against B were significantly lower in group F than in group C, although few subjects had seroprotective titers against A/H3N2 and B. These results suggest that administration of the formula containing prebiotics and fermented milk products may maintain antibody titers for longer periods through the improvement of intestinal microbiota.

A Novel Lactobacillus casei LP1 Producing 1,4-Dihydroxy-2-Naphthoic Acid, a Bifidogenic Growth Stimulator.

1,4-Dihydroxy-2-naphthoic acid (DHNA) is a bifidogenic growth stimulator (BGS) and could be a functional food ingredient since bifidobacteria are beneficial for human health. For that reason, lactic acid bacteria producing DHNA have been screened. A lactic acid bacterium LP1 strain isolated from a natural cheese was confirmed to produce DHNA, analyzed by a HPLC method. The strain was identified as Lactobacillus casei by 16S rRNA gene sequence analysis. The cell-free supernatant of fermented whey produced by L. casei LP1 presented the BGS activity for three bifidobacterial strains such as Bifidobacterium longum subsp. infantis KCTC 3127, Bifidobacterium bifidum KCTC 3202, and Bifidobacterium breve KCTC 3220 which were human-originated. To the best of our knowledge, a L. casei strain which can produce DHNA was firstly identified in this study.

Enhanced vaccination effect against influenza by prebiotics in elderly patients receiving enteral nutrition.

We investigated the effect of prebiotics on the immunological response after influenza vaccination in enterally fed elderly individuals. The intervention group was given an enteral formula containing lactic acid bacteria-fermented milk products. In addition, two different types of other prebiotics, galacto-oligosaccharide and bifidogenic growth stimulator, were also given. The two prebiotics improved intestinal microbiota differently. In a control group, a standard formula without prebiotics was given. An enteral formula with (intervention group [F]) or without (control group [C]) prebiotics was given through percutaneous endoscopic gastrostomy to elderly participants for 10 weeks. Influenza vaccine was inoculated at week 4. Nutritional and biochemical indices, intestinal micro bacteria and immunological indices were analyzed. The Bifidobacterium count in groups F and C at week 0 was 6.4 ± 1.9 and 6.6 ± 3.0 (log10 [count/g feces]), respectively. Although the count in group C decreased at week 10, the count in group F increased. The Bacteroides count in group F increased from 10.7 ± 0.9 to 11.4 ± 0.5, but decreased in group C from 11.2 ± 0.2 to 10.7 ± 0.4. Although the enhanced titers of H1N1, H3N2 and B antigens against the vaccine decreased thereafter in group C, these enhanced titers in group F could be maintained. Our findings suggest that prebiotics affect the intestinal microbiota and might maintain the antibody titers in elderly individuals.

Detection of 1,4-dihydroxy-2-naphthoic Acid from commercial makgeolli products.

To support beneficial effects of makgeolli for human health, we investigated for the presence of 1,4-dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator (BGS), from commercial makgeolli products. Among eleven makgeolli products (A∼K), four showed positive peaks for DHNA in high performance liquid chromatography analysis. Makgeolli product A in particular contained the highest concentration of DHNA (0.44 ppm), as confirmed by liquid chromatography-mass spectrometry. Furthermore, BGS activity of the makgeolli product A was higher than those of products in which DHNA was not detected. These results indicate that makgeolli can be a good source for DHNA and that DHNA-enriched makgeolli could be developed by modifying manufacturing procedures and controlling its microbiota.

Leuconostoc mesenteroides CJNU 0147 and Lactobacillus casei CJNU 0588 Improve Growth of a Bifidobacterium lactis Strain in Co-cultures

Previous studies have confirmed that fermented whey produced by Leuconostoc mesenteroides CJNU 0147 or Lactobacillus casei CJNU 0588 display bifidogenic growth stimulator (BGS) activity. The present study sought to determine if the strain itself can improve the growth of bifidobacteria in co-cultures. In reinforced clostridial medium (RCM), both strains stimulated the growth of a Bifidobacterium lactis strain during the exponential phase and also stimulated the growth during almost all growth phases in whey broth. Fermented whey containing viable Leu. mesenteroides CJNU 0147 and L. casei CJNU 0588 cells maintained viability of the B. lactis strain stored at 10℃ in MRS broth. Viable cell count of the B. lactis strain without the fermented whey was decreased to 5.6 log cfu/mL after 15 days, whereas that of the strain with the fermented whey was slightly increased to 7.1 log cfu/mL as compared with initial viable cell count of 6.9 log cfu/mL.

Optimal production of fermented whey presenting bifidogenic growth stimulator activity

Various culture conditions for the production of fermented whey presenting bifidogenic growth stimulator (BGS) activity were evaluated using Leuconostoc mesenteroides CJNU 0147 and Lactobacillus casei CJNU 0588. The BGS activity of fermented whey produced with mixed culture of Leu. mesenteroides CJNU 0147 and L. Casei CJNU 0588 was higher than those of single cultures. The optimal temperature for the production of the fermented whey was 20°C. The anaerobic culture conditions via nitrogen gas supply had no influence on the BGS activity of fermented whey. The BGS activity of the heat-treated fermented whey samples was slightly decreased by 7.63, 11.66, and 15.12% at 80, 100, and 121°C, respectively for 15 min. Pilot-scale (75 L) fermented whey was produced using the 2 freeze-dried cell powders of CJNU 0147 and CJNU 0588 and spray-dried. The spray-dried fermented whey presented BGS activity, indicating it can be used as a functional food material.

The Bifidogenic Growth Stimulator Inhibits the Growth and Respiration of Helicobacter pylori

Triple therapy with amoxicillin, clarithromycin, and a proton-pump inhibitor is a common therapeutic strategy for the eradication of Helicobacter pylori (H. pylori). However, frequent appearance of clarithromycin-resistant strains is a therapeutic challenge. While various quinones are known to specifically inhibit the growth of H. pylori, the quinone 1,4-dihydroxy-2-naphthoic acid (DHNA) produced by Propionibacterium has strong stimulating effect on Bifidobacterium. We were interested to see whether DHNA could inhibit the growth of H. pylori in in vitro or in vivo experimental setting. The minimum inhibitory concentration (MIC) of DHNA was determined by the agar dilution method. The inhibitory action of DHNA on the respiratory activity was measured by using an oxygen electrode. Germ-free mice infected with H. pylori were given DHNA in free drinking water containing 100 μg/mL for 7 days. DHNA inhibited H. pylori growth at low MIC values, 1.6-3.2 μg/mL. Likewise, DHNA inhibited clinical isolates of H. pylori, resistant to clarithromycin. However, DHNA did not inhibit other Gram negative or anaerobic bacteria in the normal flora of the human intestine. Both H. pylori cellular respiration and adenosine 5'-triphosphate (ATP) generation were dose-dependently inhibited by DHNA. Similarly, the culture filtrates of propionibacterial strains inhibited the growth of H. pylori, and oral administration of DHNA could eradicate H. pylori in the infected germ-free mice. The bifidogenic growth stimulator DHNA specifically inhibited the growth of H. pylori including clarithromycin-resistant strains in vitro and its colonization activity in vivo. The bactericidal activity of DHNA was via inhibition of cellular respiration. These actions of DHNA may have clinical relevance in the eradication of H. pylori.

Production of extracellular bifidogenic growth stimulator (BGS) from Propionibacterium shermanii using a bioreactor system with a microfiltration module and an on-line controller for lactic acid concentration

Production of a bifidogenic growth stimulator (BGS) by Propionibacterium freudenreichii subsp. shermanii (Propionibacterium shermanii) using lactic acid as a carbon source was investigated using different cultivation methods. When a continuous bioreactor system with a filtration device was used at a dilution rate of 0.075 h(-1), the average BGS concentration was 2.4 mg/l, which corresponds to a BGS productivity per cultivation time of 1.8 x 10(-1) mg x l(-1) x h(-1). The BGS productivity per cultivation time in continuous cultivation with filtration was 1.9-fold that (9.4 x 10(-2) mg x l(-1).h(-1)) in a conventional batch cultivation. In fed-batch cultivation with feed-back control using an on-line lactic acid controller with a lactic acid biosensor, it was possible to prevent substrate inhibition by maintaining the lactic acid concentration in culture broth low at 3.3 g/l, and an enhanced BGS production (31 mg/l) was successfully attained. The BGS productivity per cultivation time (2.1x10(-1) mg x l(-1) x h(-1)) in the fed-batch cultivation with feed-back control was 2.2-fold that in the conventional batch cultivation. A new bioreactor system was developed by coupling a continuous bioreactor system with a filtration device to an on-line lactic acid controller. Using the new bioreactor system, we produced BGS continuously at a high level of 47 mg/l. The BGS productivities per cultivation time (3.5 mg.l(-1) x h(-1)) and the total volume of medium used (1.7 x 10(-1) mg x l(-1) x h(-1)) obtained in the new bioreactor system were 37-fold and 2.1-fold those in the conventional batch cultivation, respectively. These results described above clearly demonstrate the positive effects of both the continuous filtration for removal of metabolites (propionic and acetic acids) inhibitory to cell growth and feed-back control of lactic acid concentration in the culture broth on BGS production by P. shermanii. This paper is the first report on BGS production by the propionic acid bacterium using lactic acid as a carbon source.

Relationships between quality parameters and content of glycerol galactoside and porphyra-334 in dried laver nori Porphyra yezoensis

Glycerol galactoside (GG; floridoside + isofloridoside) and porphyra-334 (P-334) are contained in nori (Susabinori Porphyra yezoensis and Asakusanori Porphyra tenera). Glycerol galactoside has been found to have bifidogenic growth stimulator activity and P-334 is known to have ultraviolet-absorbing activity in the UVA region of sunlight. These substances have, respectively, potential for application to pre-biotic foods and in cosmetics as a sunscren.. In the present study, to investigate the relationships between GG and P-334 contents and the quality of nori, we measured the GG and P-334 contents with other components (total protein, chlorophyll-a, β-carotene and phycobillins) that are related to the quality of nori samples produced from different production areas and with different qualities. We found that the GG content was closely negatively correlated with the contents of other components, whereas P-334 was positively correlated with the other components. From these results, it is suggested that low-quality nori is a potential source of GG, and as a source for P-334, scraps of nori produced during nori processing should be suitable.

Production of Bifidogenic Growth Stimulator by Co-cultivation of Propionibacterium shermanii and Lactobacillus Strain Using Lactose as a Carbon Source

Production of a bifidogenic growth stimulator (BGS) by propionibacterial strains was investigated using lactic acid as a carbon source. The maximum concentration (1.9 mg/L) of BGS was obtained in the mono-cultivation of Propionibacterium shermanii using 20 g/L of lactic acid. The co-cultivation, in which lactose was gradually converted only to lactic acid by a homo-fermentative lactic acid bacterium and subsequently the lactic acid was used as a carbon source for BGS production by P. shermanii, resulted in the successful BGS production. The BGS concentration obtained in the co-cultivations of P. shermanii and Lactobacillus casei or P. shermanii and Lactobacillus bulgaricus was 1.8 or 6.4 mg/L, respectively.

Improved Analytical Precision of 1,4-Dihydroxy-2-naphthoic Acid by High Performance Liquid Chromatography Using Dithiothreitol as Mobile Phase Additive

1,4-dihydroxyl-2-naphtoic acid (DHNA) is a bifidogenic growth stimulator from Propionibacterium freudenreichii. According to a method described earlier, the peak area of DHNA measured by a high-performance liquid chromatography (HPLC) gradually increases with repeated analyses of the same sample. We hypothesized that the oxidizability of DHNA was the cause of poor precision. Therefore, we attempted to develop an improved method using dl-dithiothreitol (DTT) as a mobile phase additive. A DHNA standard solution (5 µg/ml) was analyzed five times in a row by either the original or the improved method. The relative standard deviation (R.S.D.) of the peak area was 37.0% and 1.6%, respectively. The linearity of the improved method was confirmed in the range of 0.25-10 µg/ml (R2 = 0.9998). These data indicate that the addition of DTT to the mobile phase improves precision of the analysis of DHNA by HPLC.

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.

Bifidogenic growth stimulator for the treatment of active ulcerative colitis: a pilot study

Objectives Experimental studies have shown that luminal antigens are involved in chronic intestinal inflammatory disorders. Bifidogenic growth stimulator (BGS) is a prebiotic preparation produced by Propionibacterium freudenreichii isolated from Swiss cheese. Previously BGS was shown to act in the colon as a growth stimulator of Bifidobacteria. This study investigated the efficacy and safety of BGS in the treatment of ulcerative colitis. Methods Twelve patients with mildly to moderately active ulcerative colitis received orally 4.5 g of BGS daily for 4 wk in an open-label treatment protocol while the baseline anti-inflammatory therapy was continued. The response to treatment was evaluated clinically and endoscopically. Concentrations of short-chain fatty acids and the composition of commensal bacteria, including Bifidobacteria, Enterobacteria and Bacteroides species, were studied in stool samples. Results Patients showed improvement in their clinical activity index scores, with a significant decrease in the score from 7.4 ± 2.8 to 4.7 ± 1.5 (mean ± standard error of the mean, P < 0.01). The endoscopic index score decreased from 4.4 ± 1.7 to 2.8 ± 1.8 ( P < 0.05) with treatment. Patients showed an increase in stool butyrate concentrations after BGS treatment ( P < 0.05). There were no significant changes in stool levels of bacteria as a result of BGS treatment. No side effects related to BGS were observed. Conclusions Oral BGS therapy may represent a non-toxic way to treat ulcerative colitis. However, controlled studies are needed to demonstrate its efficacy in the treatment of this disorder.

経管栄養摂取重度要介護高齢者に対するプロピオン酸菌による新規ビフィズス菌増殖促進物質を含有する乳清発酵物の糞便細菌, 腐敗産物並びに便通・便性に及ぼす効果

経管栄養摂取重度要介護高齢者に対するプロピオン酸菌による新規ビフィズス菌増殖促進物質を含有する乳清発酵物の糞便細菌, 腐敗産物並びに便通・便性に及ぼす効果

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.

A Novel Bifidogenic Growth Stimulator Produced by Propionibacterium freudenreichii

A novel bifidogenic growth stimulator (BGS) was present in the cell-free filtrate and in methanol extract fraction of the starter (Propionibacterium freudenreichii) cells for the manufacture of Swiss-type cheese. After purification of the BGS isolated from the lyophilized cells, the mass (217.037) was determined by high-resolution electron impact mass spectrometry (MS) and liquid chromatography-MS spectra. Various experimental analyses indicated that the chemical structure of the BGS was 2-amino-3-carboxy-1, 4-naphthoquinone (ACNQ). We examined the activity of the P. freudenreichii ET culture containing the BGS on a gut bacterial composition using an anaerobic continuous culture system and found that the BGS seems to enhance the selective utilization of oligosaccharides by bifidobacteria. Furthermore, a placebo-controlled study of the effects of BGS on fecal flora and stool frequency was carried out in healthy human subjects. A drink with the sterilized ET-3 culture was administered once a day for 7 days. Bifidobacterium percentage in the fecal flora and stool frequency were significantly increased by administration of the P. freudenreichii culture. The ACNQ exhibited growth stimulation of bifidobacteria at an extremely low concentration and enhanced the activities of NADH oxidase and NADH peroxidase in bifidobacteria. It was revealed that ACNQ works as a good electron acceptor of NAD (P)H diaphorase. The ACNQred was easily auto-oxidized and also acted as a better electron donor of NAD (P) H peroxidase. These ACNQ-mediated reactions seem to play roles in NAD (P) +-regeneration processes and seem to be responsible for the growth stimulation of bifidobacteria.