Development of culture methods capable of culturing a wide range of predominant species of intestinal bacteria.

In recent years, with the development of non-cultivation approaches, it has become evident that intestinal bacteria have a significant impact on human health. However, because one-third of the genes cannot be annotated, it is difficult to elucidate the function of all intestinal bacteria by in silico analysis, and it is necessary to study the intestinal bacteria by culturing them. In addition, various media recommended for each individual bacterium have been used for culturing intestinal bacteria; however, the preparation of each medium is complex. To simultaneously culture many bacteria and compare bacterial phenotypes under the same conditions, a medium capable of culturing a wide range of bacteria is needed. In this study, we developed GAM + blood medium (GB medium), which consists of Gifu anaerobic medium containing 5% (v/v) horse blood; it is easy to prepare and it allowed the successful cultivation of 85% of the available predominant species in the human intestinal microbiota.

Isolation of the high polyamine-producing bacterium Staphylococcus epidermidis FB146 from fermented foods and identification of polyamine-related genes.

It has been reported that the intake of polyamines contributes to the extension of healthy life span in animals. Fermented foods contain high concentrations of polyamines thought to be derived from fermentation bacteria. This suggests that bacteria that produce high levels of polyamines could be isolated from fermented foods and utilized as a source of polyamines for human nutrition. In this study, Staphylococcus epidermidis FB146 was isolated from miso, a Japanese fermented bean paste, and found to have a high concentration of putrescine in its culture supernatant (452 µM). We analyzed the presence of polyamines in the culture supernatants and cells of the type strains of 21 representative Staphylococcus species in addition to S. epidermidis FB146, and only S. epidermidis FB146 showed high putrescine productivity. Furthermore, whole-genome sequencing of S. epidermidis FB146 was performed, and the ornithine decarboxylase gene (odc), which is involved in putrescine synthesis, and the putrescine:ornithine antiporter gene (potE), which is thought to contribute to the release of putrescine into the culture supernatant, were present on plasmid DNA harbored by S. epidermidis FB146.

Gut bacterial aromatic amine production: aromatic amino acid decarboxylase and its effects on peripheral serotonin production.

Colonic luminal aromatic amines have been historically considered to be derived from dietary source, especially fermented foods; however, recent studies indicate that the gut microbiota serves as an alternative source of these amines. Herein, we show that five prominent genera of Firmicutes (Blautia, Clostridium, Enterococcus, Ruminococcus, and Tyzzerella) have the ability to abundantly produce aromatic amines through the action of aromatic amino acid decarboxylase (AADC). In vitro cultivation of human fecal samples revealed that a significant positive correlation between aadc copy number of Ruminococcus gnavus and phenylethylamine (PEA) production. Furthermore, using genetically engineered Enterococcus faecalis-colonized BALB/cCrSlc mouse model, we showed that the gut bacterial aadc stimulates the production of colonic serotonin, which is reportedly involved in osteoporosis and irritable bowel syndrome. Finally, we showed that human AADC inhibitors carbidopa and benserazide inhibit PEA production in En. faecalis.

Putrescine Production by Latilactobacillus curvatus KP 3-4 Isolated from Fermented Foods.

Polyamines are aliphatic hydrocarbons with terminal amino groups and are essential for biological activities. It has been reported that polyamines have health-promoting effects in animals, such as the extension of lifespan by polyamine intake. The identification of a high polyamine-producing bacterium from foods could lead to the development of a novel probiotic candidate. We aimed to identify high polyamine-producing bacteria from food, and isolated and collected bacteria from vegetables and fermented foods produced in Japan. We successfully acquired Latilactobacillus curvatus KP 3-4 isolated from Kabura-zushi as a putrescine producing lactic acid bacteria. Comparing the polyamine synthesis capability of L. curvatus KP 3-4 with that of typical probiotic lactic acid bacteria and L. curvatus strains available from the Japan Collection of Microorganisms, it was found that only L. curvatus KP 3-4 was capable of exporting high levels of putrescine into the culture supernatant. The enhancement of putrescine production by the addition of ornithine, and whole-genome analysis of L. curvatus KP 3-4, suggest that putrescine is synthesized via ornithine decarboxylase. The administration of L. curvatus KP 3-4 to germ-free mice increased the concentration of putrescine in the feces.

Next-generation prebiotic promotes selective growth of bifidobacteria, suppressing Clostridioides difficile.

Certain existing prebiotics meant to facilitate the growth of beneficial bacteria in the intestine also promote the growth of other prominent bacteria. Therefore, the growth-promoting effects of ß-galactosides on intestinal bacteria were analyzed. Galactosyl-ß1,4-l-rhamnose (Gal-ß1,4-Rha) selectively promoted the growth of Bifidobacterium. Bifidobacterium longum subsp. longum 105-A (JCM 31944) has multiple solute-binding proteins belonging to ATP-binding cassette transporters for sugars. Each strain in the library of 11 B. longum subsp. longum mutants, in which each gene of the solute-binding protein was disrupted, was cultured in a medium containing Gal-ß1,4-Rha as the sole carbon source, and only the BL105A_0502 gene-disruption mutant showed delayed and reduced growth compared to the wild-type strain. BL105A_0502 homolog is highly conserved in bifidobacteria. In a Gal-ß1,4-Rha-containing medium, Bifidobacterium longum subsp. infantis JCM 1222T, which possesses BLIJ_2090, a homologous protein to BL105A_0502, suppressed the growth of enteric pathogen Clostridioides difficile, whereas the BLIJ_2090 gene-disrupted mutant did not. In vivo, administration of B. infantis and Gal-ß1,4-Rha alleviated C. difficile infection-related weight loss in mice. We have successfully screened Gal-ß1,4-Rha as a next-generation prebiotic candidate that specifically promotes the growth of beneficial bacteria without promoting the growth of prominent bacteria and pathogens.

Bifidobacterium response to lactulose ingestion in the gut relies on a solute-binding protein-dependent ABC transporter.

This study aims to understand the mechanistic basis underlying the response of Bifidobacterium to lactulose ingestion in guts of healthy Japanese subjects, with specific focus on a lactulose transporter. An in vitro assay using mutant strains of Bifidobacterium longum subsp. longum 105-A shows that a solute-binding protein with locus tag number BL105A_0502 (termed LT-SBP) is primarily involved in lactulose uptake. By quantifying faecal abundance of LT-SBP orthologues, which is defined by phylogenetic analysis, we find that subjects with 107 to 109 copies of the genes per gram of faeces before lactulose ingestion show a marked increase in Bifidobacterium after ingestion, suggesting the presence of thresholds between responders and non-responders to lactulose. These results help predict the prebiotics-responder and non-responder status and provide an insight into clinical interventions that test the efficacy of prebiotics.

Health-Promoting Effects of Dietary Polyamines.

The purpose of this paper is to summarize the latest information on the various aspects of polyamines and their health benefits. In recent years, attempts to treat cancer by reducing elevated polyamines levels in cancer cells have been made, with some advancing to clinical trials. However, it has been reported since 2009 that polyamines extend the healthy life span of animals by inducing autophagy, protecting the kidneys and liver, improving cognitive function, and inhibiting the progression of heart diseases. As such, there is conflicting information regarding the relationship between polyamines and health. However, attempts to treat cancer by decreasing intracellular polyamines levels are a coping strategy to suppress the proliferation-promoting effects of polyamines, and a consensus is being reached that polyamine intake does not induce cancer in healthy individuals. To provide further scientific evidence for the health-promoting effects of polyamines, large-scale clinical studies involving multiple groups are expected in the future. It is also important to promote basic research on polyamine intake in animals, including elucidation of the polyamine balance between food, intestinal bacteria, and biosynthesis.

Improved effects of okara atomized by a water jet system on α-amylase inhibition and butyrate production by Roseburia intestinalis.

Improving the physicochemical properties of okara for various applications in foods is of great importance. Here, okara and microcrystalline cellulose (MCC) were atomized using a water jet (WJ) system. The WJ-treated okara and MCC dispersed homogeneously in water, and their median sizes in particle size distribution were 6.6 µm and 9.5 µm, respectively. The dispersions of WJ-treated okara and MCC showed high apparent viscosity and shear thinning behavior. Moreover, the inhibition of α-amylase activities by WJ-treated okara was more effective than that by untreated MCC and cellulose. Furthermore, the production of short-chain fatty acids by 32 dominant species of human gut microbes was determined. An increase in butyrate production by Roseburia intestinalis was observed in the presence of WJ-treated okara, but not in untreated okara or WJ-treated MCC. These results demonstrate that WJ system can be used on okara to increase inhibited α-amylase activities and butyrate production by gut microbiota.

Development of a new chromogenic method for putrescine quantification using coupling reactions involving putrescine oxidase.

Quantification of polyamines, including putrescine, is generally performed using high-performance liquid chromatography (HPLC) or gas chromatography. However, these methods are time-consuming because of sample derivatization and analytical reagent preparation. In this study, we developed a simple and high-throughput putrescine quantification method on a 96-well microtiter plate using putrescine oxidase from Rhodococcus erythropolis NCIMB 11540, peroxidase, 4-aminoantipyrine, and N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt. The developed method (named as PuO-POD-4AA-TOPS method) was applicable to bacterial culture supernatants. Furthermore, putrescine concentrations determined by the developed method roughly corresponded to the concentrations determined by HPLC.

Measuring the Antimicrobial Activity of Lauric Acid against Various Bacteria in Human Gut Microbiota Using a New Method.

Lauric acid (LA) has a broad spectrum of anti-microbiological activities against enveloped viruses and various bacteria, and might be useful to protect against microbial infection and control the balance and distribution of bacteria in human gut microbiota. It is not necessarily more difficult to measure antimicrobial activity the traditional way, but it is, however, more laborious. In the present study, we developed a new method to measure the antimicrobial activity of LA in multiple samples with a microplate reader. A "test complex" (TC) was produced consisting of 100 µL of agar medium with LA in the bottom layer and 300 µL of broth in the top layer in 96-well deep-well microplates. Afterward, analysis of the broth in the top layer showed that the antimicrobial activity was the same as that of the "control complex," (CC) which consisted of 100 µL of agar medium in the bottom layer and 300 µL of broth with LA in the top layer. Furthermore, evaluation of the antimicrobial effect of the TC when using a microplate reader was the same as that with the use of the colony counting method. The colony counting method has confirmed that the antimicrobial activity of LA when bacteria are inoculated into the broth was equivalent between CC and TC, and we validated this by correlating the number of bacteria with absorbance. In addition, the broth itself in TC was transparent enough that the turbidity of broth can be used as an index of the number of bacteria, which enabled the use of a microplate reader for multiple samples. For human gut microbes, LA was shown to have low antimicrobial activity against commensal lactic acid bacteria, but high antimicrobial activity against pathogenic Bacteroides and Clostridium, suggesting that LA might modulate intestinal health, as confirmed by the proposed method.