[Construction of gut microbial culture banks: advances, methods and perspectives].

Recently, the gut microbiota-based live biotherapeutics (LBPs) development, the interaction between gut microbial species and the host, and the mining of new antimicrobial peptides, enzymes and metabolic pathway have received increasing attention. Culturing gut microbial species is therefore of great importance. This review systemically compared the construction advances of gut microbial culture banks and also analyzed the differences of methods used by research groups to give insight into the construction and enrichment of gut microbial resources. Presently, the gut microbial culture banks have included more than 1 000 bacterial species, belonging to 12 phyla, 22 classes, 39 orders, 96 families, and 358 genera. Among these, Firmicutes, Proteobacteria, Bacteroidota, and Actinomycetota exhibited the greatest diversities at the species level. The sequencing data showed that there are more than 2 000 species inhibited in the human gut. Therefore, the cultured gut microbial species are far from saturation. In terms of the construction method, the stool samples were pre-treated with ethanol or directly spread and cultured in the non-selective nutritional rich medium (represented by Gifu anaerobic medium) to obtain single colony. Then single colony was further purified. Generally, a simplified isolation and culture method is sufficient to obtain the most common and important intestinal bacterial species, such as Bifidobacteria-Lactobacillus, Akkermansia muciniphila, Faecalibacterium prausnitzii, Prevotella and S24-7 family strains. Finally, microbial resources with great diversities at the strain level are required for further functional research and product development. Samples covering hosts with distinct physiological status, diets or regions are necessary.

Effects of Alkaline Hydrogen Peroxide and Cellulase Modifications on the Physicochemical and Functional Properties of Forsythia suspensa Dietary Fiber.

Besides active substances, Forsythia suspensa is rich in dietary fiber (DF), but it is often wasted or discarded and not put to good use. In order to improve the function of Forsythia DF, it was modified using alkaline hydrogen peroxide (AHP) and cellulase (EM). Compared to the control DF (ODF), the DF modified using AHP (AHDF) and EM (EMDF) had a looser microstructure, lower crystallinity, and higher oil holding capacity (OHC) and cation exchange capacity (CEC). The AHP treatment significantly increased the water holding capacity (WHC) and water swelling ability (WSA) of the DF, while the EM treatment achieved just the opposite. Moreover, the functional properties of AHDF and EMDF, including their cholesterol adsorption capacity (CAC), nitrite ion adsorption capacity (NAC), glucose adsorption capacity (GAC), glucose dialysis retardation index (GDRI), α-amylase inhibitory activity, and DPPH radical scavenging activity, were far better than those of ODF. Together, the results revealed that AHP and EM modifications could effectively improve or enhance the physicochemical and functional properties of Forsythia suspensa DF.

Species- or genus-dependent immunostimulatory effects of gut-derived potential probiotics.

The immune regulatory effects of probiotics have been widely recognized to be strain-specific. However it is unknown if there is a species- or genus-dependent manner. In this study, we use an in vitro mesenteric lymph node (MLN) model to systematically evaluate the immunostimulatory effects of gut-derived potential probiotics. The results exhibit an obvious species or genus consensus immune response pattern. RNA-seq shows that T cell-dependent B cell activation and antibody responses may be inherent to this model. Of the five tested genera, Akkermansia spp. and Clostridium butyrium directly activate the immune response in vitro, as indicated by the secretion of interleukin-10. Bifidobacterium spp. and Bacteroides spp. activate immune response with the help of stimuli (anti-CD3 and anti-CD28 antibodies). Lactobacillus spp. blunt the immune response with or without stimuli. Further investigations show that the cell surface protein of A. muciniphila AH39, which may serve as a T cell receptor cognate antigen, might evoke an in vitro immune activation. In vivo, oral administration of A. muciniphila AH39 influences the proportion of T regulatory cells (Tregs) in MLNs and the spleen under homeostasis in both specific pathogen-free and germ-free mice. All these findings indicate the distinct effects of different genera or species of potential gut-derived probiotics on intestinal and systemic immunity.

Protective effects of Bacteroides fragilis against lipopolysaccharide-induced systemic inflammation and their potential functional genes.

Bacteroides fragilis, one of the potential next-generation probiotics, has been demonstrated to alleviate inflammation-associated diseases. In this study, we compare the anti-inflammatory effects of six Bacteroides fragilis strains on systemic inflammation and link their strain-specific characteristics, both physiologically and genetically, to their function. A lipopolysaccharide (LPS)-induced systemic inflammation model in mice was used as an in vivo model to compare the effects of different B. fragilis strains. Short-chain fatty acids (SCFAs) were measured by gas chromatography-mass spectrometry (GC-MS). The in vitro immunomodulatory properties were evaluated in LPS-stimulating RAW264.7 cell lines. Orthologous gene clusters were compared using OrthoVenn2. The results indicate a strain-specific in vitro anti-inflammatory effect. Effective strains induce higher colon SCFAs in vivo and interleukin-10 (IL-10) production in vitro. Comparative genomic analysis showed that the SCFA-inducing strains possess three genes relating to carbohydrate metabolism (GH2, GH35 families) and binding and transportation (SusD), all of which are associated with niche fitness and expansion. IL-10-inducing strains share a highly similar gene, wbjE, which may result in a distinct O-antigen structure of LPS and influence their immunomodulatory properties. B. fragilis is strain-specific against LPS-induced systemic inflammation in mice. The beneficial effects of a specific strain may be attributed to its SCFA and IL-10 inducing abilities. Strain-specific potential genes can be excavated to link these differences.

Characteristics of an In Vitro Mesenteric Lymph Node Cell Suspension Model and Its Possible Association with In Vivo Functional Evaluation.

In a previous study, we uncovered three immune-responsive patterns of gut microbes using an in vitro mesenteric lymph node cell suspension model, abbreviated as the MLN model hereafter. We used Akkermansia muciniphila and Clostridium butyricum as the first group directly inducing an immune response, Bifidobacterium sp. and Bacteroides sp. as the second group evoking an immune response with the help of stimuli (anti-CD3 and anti-CD28 antibodies), and Lactobacillus sp. as the third group blunting the immune response with or without stimuli. Our group previously clarified the immune-activation characteristics of A. muciniphila and linked its in vivo immune induction effect in GF and SPF mice under homeostasis. In the present study, we supplemented the characteristics of C. butyricum and B. bifidum in the in vitro MLN model and addressed the specific elements of the model. Finally, we used an in vivo TNBS-challenge model to show the functional differences between these species with different response patterns in vitro. The results showed that C. butyricum and B. bifidum evoked an immune response in vitro in a dose-dependent and strain-unique manner. Although TLR2, rather than TLR4, is indispensable for immune activation in the present in vitro model, it may not involve interaction between TLR2 and bacterial ligands. Like the PBMC model, the present in vitro MLN model is highly dependent on cell resources and should be given more attention when used to conduct a quantitative comparison. Finally, a mixture of two strong immunogenic strains, A. muciniphila and C. butyricum, significantly increased the mortality of TNBS-challenged (2,4,6-trinitrobenzene sulfonic acid, TNBS) mice, indicating a possible link between the in vitro MLN model and in vivo functional evaluation. However, more evidence is needed to clarify the associations and underlying mechanisms.

A new Illumina MiSeq high-throughput sequencing-based method for evaluating the composition of the Bacteroides community in the intestine using the rpsD gene sequence.

Bacteroides is a bacterial genus that is known to closely interact with the host. The potential role of this genus is associated with its ecological status and distribution in the intestine. However, the current 16S V3-V4 region sequencing method can only detect the abundance of this genus, revealing a need for a novel sequencing method that can elucidate the composition of Bacteroides in the human gut microbiota. In this study, a core gene, rpsD, was selected as a template for the design of a Bacteroides-specific primer set. We used this primer set to develop a novel assay based on the Illumina MiSeq sequencing platform that enabled an accurate assessment of the Bacteroides compositions in complex samples. Known amounts of genomic DNA from 10 Bacteroides species were mixed with a complex sample and used to evaluate the performance and detection limit of our assay. The results were highly consistent with those of direct sequencing with a low Bacteroides DNA detection threshold (0.01 ng), supporting the reliability of our assay. In addition, the assay could detect all the known Bacteroides species within the faecal sample. In summary, we provide a sensitive and specific approach to determining the Bacteroides species in complex samples.

Beneficial effect of GABA-rich fermented milk on insomnia involving regulation of gut microbiota.

Insomnia is a common health problem in modern societies. GABA, an inhibitory neurotransmitter, can promote relaxation and reduce anxiety. In this study, milk was fermented with Lactobacillus brevis DL1-11, a strain with high GABA-producing capacity. The potential beneficial effects of this fermented milk on anxiety and sleep quality were evaluated in animal experiments. Sixty mice were divided into control, non-GABA fermented milk (NGFM), low-dose GABA fermented milk (LGFM, 8.83 mg/kg.bw), medium-dose GABA fermented milk (MGFM, 16.67 mg/kg.bw), high-dose GABA fermented milk (HGFM, 33.33 mg/kg.bw) and diazepam groups. The results of open field test and elevated plus-maze test indicated decreases in anxiety behavior after oral HGFM administration. Moreover, mice in the HGFM group exhibited a significantly prolonged sleep time after an intraperitoneal injection of sodium pentobarbital and a shortened sleep latency after an intraperitoneal injection of sodium barbital. These results indicate a beneficial effect of HGFM on sleep. Additionally, significant increases in the relative abundances of Ruminococcus, Adlercreutzia and Allobaculum and the levels of some short-chain fatty acids (SCFAs), such as butyric acid, were observed in the HGFM group. The results suggest that GABA-fermented milk may improve sleep and the protective pathways may involve in regulation of gut microbiota and increase of SCFAs level.

Influence of oral administration of Akkermansia muciniphila on the tissue distribution and gut microbiota composition of acute and chronic cadmium exposure mice.

Cadmium (Cd) contamination is a serious food safety problem. Acute and chronic Cd exposure changes the gut microbiota composition and damages the gut barrier function. Akkermansia muciniphila (AKK), a promising candidate for the next-generation probiotics, has been reported to protect the mucus layer in the colon and significantly decrease the effects of Cd exposure in mice. Thus, the mice model was adopted to investigate the influence of oral administration of AKK on the toxic distribution and changes of gut microbiota composition caused by acute and chronic Cd exposure. In both acute and chronic Cd exposure experiments, 40 mice were divided into four groups (normal group, AKK group, Cd group and Cd plus AKK group). The Cd contents in feces and tissues were measured by a flame or graphite furnace atomic absorption spectrophotometer and gut microbiota composition was determined through 16S rRNA gene sequencing. The results showed that the gavage of AKK could not reduce the accumulation of Cd in the liver and kidney. The oral administration of AKK showed a certain influence on the gut microbiota composition of acute Cd exposure mice and limited influence on that of chronic Cd exposure mice. These results indicate the failure of AKK, as a potential protective probiotic, to reduce Cd toxicity. However, the gavage of AKK did have an influence on the gut microbiota composition of normal mice, especially on some genera in the Clostridiales order. Besides, when considering AKK's probiotic potential and its effects on host health and disease, we should take into consideration its influence on the gut microbiota composition and micro-environment.

A next generation probiotic, Akkermansia muciniphila.

Akkermansia muciniphila, a symbiotic bacterium of the mucus layer, can utilize mucin as its sole carbon, nitrogen, and energy source. As an abundant resident in the intestinal tract of humans and animals, the probiotic effects of A. muciniphila including metabolic modulation, immune regulation and gut health protection, have been widely investigated. Various diseases such as metabolic syndromes and auto-immnue diseases have been reported to be associated with the disturbance of the abundance of A. muciniphila. In this review, we describe the biological characterization of A. muciniphia, the factors that influence its colonization of the intestinal tract; and discuss the current state of our knowledge on its role in host health and disease.

Oral Supplementation of Lead-Intolerant Intestinal Microbes Protects Against Lead (Pb) Toxicity in Mice.

Oral exposure to the heavy metal lead (Pb) causes various dysfunctions in animals. However, the influence of gut bacteria on Pb absorption, bioaccumulation, and excretion is largely unknown. In this study, we use a mouse model to investigate the relationship between gut microbiota, Pb-intolerant intestinal microbes and Pb toxicity. First, mice were treated with a broad-spectrum antibiotic cocktail to deplete their gut microbiota, and were then acutely and orally exposed to Pb at 1304 mg/kg for 3 days. Compared to the control mice, antibiotic-treated mice had increased Pb concentrations in the blood and primary organs and decreased Pb fecal concentrations, suggesting that gut microbiota limited the Pb burden that developed from acute oral Pb exposure. Next, three Pb-intolerant gut microbes, Akkermansia muciniphila, Faecalibacterium prausnitzii, and Oscillibacter ruminantium, were orally administered to mice, and their effects against Pb toxicity were evaluated. F. prausnitzii treatment significantly promoted the fecal Pb excretion and reduced Pb concentrations in blood (from 152.70 ± 25.62 µg/dL to 92.20 ± 24.33 µg/dL) and primary tissues. Supplementation with O. ruminantium significantly decreased Pb concentrations in blood (from 152.70 ± 25.62 µg/dL to 104.60 ± 29.85 µg/dL) and kidney (from 7.30 ± 1.08 µg/g to 5.64 ± 0.79 µg/g). Treatment with F. prausnitzii and O. ruminantium also upregulated tight junction (TJ) protein expression and the production of short-chain fatty acids by colonic microbiota, and showed protective effects against liver and kidney toxicity. These results indicate the potential for reducing Pb toxicity by the modulation of gut microbiota.

Effects of subchronic oral toxic metal exposure on the intestinal microbiota of mice.

Oral exposure to toxic metals such as cadmium (Cd), lead (Pb), copper (Cu) and aluminum (Al) can induce various adverse health effects in humans and animals. However, the effects of these metals on the gut microbiota have received limited attention. The present study demonstrated that long-term toxic metal exposure altered the intestinal microbiota of mice in a metal-specific and time-dependent manner. Subchronic oral Cu exposure for eight weeks caused a profound decline in gut microbial diversity in mice, whereas no significant changes were observed in groups treated with other metals. Cd exposure significantly increased the relative abundances of organisms from the genera Alistipes and Odoribacter and caused marked decreases in Mollicutes and unclassified Ruminococcaceae. Pb exposure significantly decreased the abundances of eight genera: unclassified and uncultured Ruminococcaceae, unclassified Lachnospiraceae, Ruminiclostridium_9, Rikenellaceae_RC9_gut_group, Oscillibacter, Anaerotruncus and Lachnoclostridium. Cu exposure affected abundances of the genera Alistipes, Bacteroides, Ruminococcaceae_UCG-014, Allobaculum, Mollicutes_RF9_norank, Rikenellaceae_RC9_gut_group, Ruminococcaceae_unclassified and Turicibacter. Al exposure increased the abundance of Odoribacter and decreased that of Anaerotruncus. Exposure to any metal for eight weeks significantly decreased the abundance of Akkermansia. These results provide a new understanding regarding the role of toxic metals in the pathogenesis of intestinal and systemic disorders in the host within the gut microbiota framework.

The effect of dipping pretreatment on ochratoxin A accumulation in sultanas and currants.

The dipping pretreatment on the occurrence of ochratoxin A (OTA) in sultanas and currants was investigated. Grape samples were divided into two groups before a drying process. One group was dipped with potassium carbonate-ethyl oleate, and the other group was left without treatment (the control). OTA were detected using solid-phase extraction clean-up and a high-performance liquid chromatography fluorescence detector. Results showed that OTA content was below the limit of detection in the fresh grapes of both varieties. OTA were not detected in dried vine fruits before storage. However, a real naturally environmental storage revealed that the dipping pretreated samples were contaminated with OTA earlier and to a greater extent than the control. The OTA concentration in the treated and control samples after one-year storage was 0.22 and 0.19 µg/kg for sultanas and 0.34 and 0.21 µg/kg for currants, respectively. These results indicated that dipping pretreatment might increase the toxin contamination and safety risk.

Chemical composition, antibacterial activity, and mechanism of action of the essential oil from Amomum kravanh.

Amomum kravanh is widely cultivated and used as a culinary spice. In this work, the chemical composition of the essential oil obtained by hydrodistillation of A. kravanh fruits was analyzed by gas chromatography-mass spectrometry, and 34 components were identified. 1,8-Cineole (68.42%) was found to be the major component, followed by α-pinene (5.71%), α-terpinene (2.63%), and ß-pinene (2.41%). The results of antibacterial tests showed that the sensitivities to the essential oil of different foodborne pathogens tested were different based on the Oxford cup method, MIC, and MBC assays, and the essential oil exhibited the best antibacterial activity against Bacillus subtilis, a gram-positive bacterium, and Escherichia coli, a gram-negative bacterium. Growth in the presence of Amomum kravanh at the MIC, as measured by monitoring optical density over time, demonstrated that the essential oil was bacteriostatic after 12 h to both B. subtilis and E. coli. Observations of cell membrane permeability, cell constituent release assay, and transmission electron microscopy indicated that this essential oil may disrupt the cell wall and cell membrane permeability, leading to leakage of intracellular constituents in both B. subtilis and E. coli.

Chemical composition and antibacterial activity of the essential oil from green huajiao (Zanthoxylum schinifolium) against selected foodborne pathogens.

Green huajiao, which is the ripe pericarp of the fruit of Zanthoxylum schinifolium Sieb. et Zucc, is widely consumed in Asia as a spice. In this work, the chemical composition of the essential oil from green huajiao was analyzed by gas chromatography (GC) and GC/mass spectrometry (MS), and the majority of components were identified. Linalool (28.2%), limonene (13.2%), and sabinene (12.1%) were found to be the major components. The antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the essential oil were evaluated against selected bacteria, including food-borne pathogens. The results showed that the sensitivities to the essential oil were different for different bacteria tested, and the susceptibility of Gram-positive bacteria tested was observed to be greater than that of Gram-negative bacteria. The antibacterial activity of the essential oil was particularly strong against Staphylococcus epidermidis , with MIC and MBC values of 2.5 and 5.0 mg/mL, respectively. A postcontact effect assay also confirmed the essential oil had a significant effect on the growth rate of surviving S. epidermidis . The antibacterial activity of the essential oil from green huajiao may be due to the increase in permeability of cell membranes, and the leakage of intracellular constituents, on the basis of the cell constituents' release assay and electron microscopy observations.