Co-administration of the prebiotic 1-kestose and the paraprobiotic Lactiplantibacillus plantarum FM8 in magellanic penguins promotes the activity of intestinal Lactobacillaceae and reduces the plc gene levels encoding Clostridium perfringens toxin.

Despite the well-known potential health benefits of prebiotics and non-viable probiotics (paraprobiotics) in various animal species, research regarding their use in penguins is scarce. Our study aimed to investigate the impact of a combined administration of prebiotics and paraprobiotics (referred to here as "parasynbiotics") on the gut microbiome and overall health of Magellanic penguins (Spheniscus magellanicus). The parasynbiotics consisted of 1-kestose, which is a fructooligosaccharide comprising sucrose and fructose, and heat-killed Lactiplantibacillus plantarum FM8, isolated from pickled vegetables. It was administered to eight penguins aged <3 years (Young-group) and nine penguins aged >17 years (Adult-group) for 8 weeks. Results from 16S rRNA sequencing revealed that compared to baseline, parasynbiotic administration significantly decreased the relative abundance of intestinal Clostridiaceae_222000 in both groups and significantly increased that of Lactobacillaceae in the Young-group. Quantitative real-time polymerase chain reaction revealed a significant decrease in the plc gene levels encoding alpha-toxin of Clostridium perfringens in the Young-group after parasynbiotic administration (P=0.0078). In the Young-group, parasynbiotic administration significantly increased the plasma levels of total alpha-globulin (P=0.0234), which is associated with inflammatory responses. Furthermore, exposure of dendritic cells to heat-killed L. plantarum FM8 promoted the secretion of interleukin 10, a major anti-inflammatory cytokine. Overall, parasynbiotic administration enhanced the activity of gut Lactobacillaceae, decreased the levels of C. perfringens and its toxin encoding plc gene, and reduced inflammatory response in penguins. These results provide novel insights into the potential benefits of parasynbiotics for improving penguin health.

Effects of 1-kestose on microbiome changes caused by vonoprazan: a randomized, double-blind, placebo-controlled pilot study.

BACKGROUND AND AIM: Potassium-competitive acid blockers more strongly suppress the gastric acid barrier than proton pump inhibitors and cause dysbiosis. However, preventive measures in this regard have not been established. We aimed to evaluate whether 1-kestose, a known prebiotic, was effective at alleviating dysbiosis caused by potassium-competitive acid blockers. METHODS: Patients scheduled to undergo endoscopic resection for superficial gastroduodenal tumors were enrolled and randomized 1:1 to receive either 1-kestose or placebo. All patients were started on potassium-competitive acid blocker (vonoprazan 20 mg/day) and took 1-kestose 10 g/day or placebo (maltose) 5 g/day for 8 weeks. The primary outcome was the effect of 1-kestose on potassium-competitive acid blocker-induced alterations in the microbiome. The fecal microbiome was analyzed before and after potassium-competitive acid blocker treatment via MiSeq (16S rRNA gene V3-V4 region). RESULTS: Forty patients were enrolled, and 16 in each group were analyzed. In the placebo group, the Simpson index, an alpha diversity, was significantly decreased and relative abundance of Streptococcus was significantly increased by 1.9-fold. In the kestose group, the Simpson index did not change significantly and relative abundance of Streptococcus increased 1.3-fold, but this was not a significant change. In both groups, no adverse events occurred, ulcers were well healed, and pretreatment and posttreatment short-chain fatty acid levels did not differ. CONCLUSIONS: The potassium-competitive acid blocker caused dysbiosis in the placebo group; this effect was prevented by 1-kestose. Thus, 1-kestose may be useful in dysbiosis treatment.

Oral administration of Brevibacterium linens from washed cheese increases the proportions of short-chain fatty acid-producing bacteria and lactobacilli in the gut microbiota of mice.

Brevibacterium linens (B. linens) is a dairy microorganism used in the production of washed cheese. However, there has been little research on B. linens, especially regarding its effects in vivo. Herein, we report the morphological characteristics of B. linens, such as its two-phase growth and V- and Y-shaped bodies. We also report that oral administration of B. linens increased the diversity of the gut microbiota and promoted the growth of lactobacilli and short-chain fatty acid-producing bacteria, such as Lachnospiraceae and Muribaculaceae. These findings suggest that the ingestion of B. linens may have beneficial effects in humans and animals.

Combined oral intake of short and long fructans alters the gut microbiota in food allergy model mice and contributes to food allergy prevention.

BACKGROUND: It has become clear that the intestinal microbiota plays a role in food allergies. The objective of this study was to assess the food allergy-preventive effects of combined intake of a short fructan (1-kestose [Kes]) and a long fructan (inulin ([Inu]) in an ovalbumin (OVA)-induced food allergy mouse model. RESULTS: Oral administration of fructans lowered the allergenic symptom score and alleviated the decreases in rectal temperature and total IgA levels and increases in OVA-specific IgE and IgA levels induced by high-dose OVA challenge, and in particular, combined intake of Kes and Inu significantly suppressed the changes in all these parameters. The expression of the pro-inflammatory cytokine IL-4, which was increased in the allergy model group, was significantly suppressed by fructan administration, and the expression of the anti-inflammatory cytokine IL-10 was significantly increased upon Kes administration. 16 S rRNA amplicon sequencing of the gut microbiota and beta diversity analysis revealed that fructan administration may induce gut microbiota resistance to food allergy sensitization, rather than returning the gut microbiota to a non-sensitized state. The relative abundances of the genera Parabacteroides B 862,066 and Alloprevotella, which were significantly reduced by food allergy sensitization, were restored by fructan administration. In Parabacteroides, the relative abundances of Parabacteroides distasonis, Parabacteroides goldsteinii, and their fructan-degrading glycoside hydrolase family 32 gene copy numbers were increased upon Kes or Inu administration. The concentrations of short-chain fatty acids (acetate and propionate) and lactate were increased by fructan administration, especially significantly in the Kes + Inu, Kes, and Inu-fed (Inu, Kes + Inu) groups. CONCLUSION: Combined intake of Kes and Inu suppressed allergy scores more effectively than single intake, suggesting that Kes and Inu have different allergy-preventive mechanisms. This indicates that the combined intake of these short and long fructans may have an allergy-preventive benefit.

Incidence and clinical relevance of postoperative diarrhea after minimally invasive gastrectomy for gastric cancer: a single institution retrospective study of 1476 patients.

PURPOSE: Postoperative diarrhea (PD) remains one of the significant complications. Only a few studies focused on PD after minimally invasive surgery. We aimed to investigate PD after minimally invasive gastrectomy for gastric cancer. METHODS: A total of 1476 consecutive patients with gastric cancer undergoing laparoscopic or robotic gastrectomy between 2009 and 2019 at our institution were retrospectively reviewed. PD was defined as continuous diarrhea for ≥ 2 days, positive stool culture, or positive clostridial antigen test. The incidence, causes, and related clinical factors were analyzed. RESULTS: Of the 1476 patients, the median age was 69 years. Laparoscopic and robotic approaches were performed in 1072 (72.6%) and 404 (27.4%), respectively. Postoperative complications with Clavien-Dindo classification grade of ≥ IIIa occurred in 108 (7.4%) patients. PD occurred in 89 (6.0%) patients. Of the 89 patients with PD, Clostridium difficile, enteropathogenic Escherichia coli, and methicillin-resistant Staphylococcus aureus were detected in 24 (27.0%), 16 (33.3%), and 7 (14.6%) patients, respectively. Multivariate analysis revealed that age ≥ 75 years (OR 1.62, 95% CI [1.02-2.60], p = 0.042) and postoperative complications (OR 6.04, 95% CI [3.54-10.32], p < 0.001) were independent risk factors for PD. In patients without complications, TG (OR 1.88) and age of ≥ 75 years(OR 1.71) were determined as independent risk factors. CONCLUSION: The incidence of PD following minimally invasive gastrectomy for gastric cancer was 6.0%. Older age and TG were obvious risk factors in such a surgery, with the latter being a significant risk even in the absence of complications.

Kestose Increases the Relative Abundance of Faecalibacterium spp. and Nominally Increases Cow Milk Tolerant Dose in Children with Cow's Milk Allergy - Preliminary Results.

A single-arm study was conducted with 10 children aged 2-12 years with severe cow's milk allergy (CMA) requiring complete allergen elimination. Subjects were administered kestose, a prebiotic, at 1 or 2 g/day for 12 weeks. Results of a subsequent oral food challenge (OFC) showed a statistically significant increase in the total dose of cow's milk ingestion (1.6 ml vs. 2.7 ml, p = 0.041). However, the overall evaluation of the OFC results, TS/Pro (total score of Anaphylaxis Scoring Aichi (ASCA)/cumulative dose of protein), showed no statistically significant improvement, although the values were nominally improved in seven out of 10 subjects. The 16S rDNA analysis of fecal samples collected from the subjects revealed a statistically significant increase in the proportion of Faecalibacterium spp. (3.8 % vs. 6.8%, p = 0.013), a type of intestinal bacterium that has been reported to be associated with food allergy. However, no statistically significant correlation was found between Faecalibacterium spp. abundance and the results of the OFC.

Erythritol alters gene transcriptome signatures, cell growth, and biofilm formation in Staphylococcus pseudintermedius.

BACKGROUND: Erythritol was found to inhibit the growth of microorganisms. The present study aimed to demonstrate the growth inhibition of Staphylococcus pseudintermedius by erythritol and to define the changes in gene transcription signatures induced by erythritol. Changes in the gene transcription profiles were analysed by RNA sequencing and quantitative reverse transcription PCR. Gene ontology analysis was performed to assign functional descriptions to the genes. RESULTS: Erythritol inhibited S. pseudintermedius growth in a dose-dependent manner. We then performed a transcriptome analysis of S. pseudintermedius with and without 5% (w/w) erythritol exposure to validate the mechanism of growth inhibition. We revealed that erythritol induced up-regulation of three genes (ptsG, ppdK, and ppdkR) that are related to the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Glucose supplementation restored the up-regulation of the PTS-related genes in response to erythritol. In addition, erythritol down-regulated eleven genes that are located in a single pur-operon and inhibited biofilm formation of S. pseudintermedius. CONCLUSIONS: These findings indicated that erythritol antagonistically inhibits PTS-mediated glucose uptake, thereby exerting a growth inhibitory effect on S. pseudintermedius. Moreover, erythritol inhibits the 'de novo' IMP biosynthetic pathway that may contribute to biofilm synthesis in S. pseudintermedius.

Characterization and alteration of product specificity of Beijerinckia indica subsp. indica ß-fructosyltransferase.

The trisaccharide 1-kestose, a major constituent of fructooligosaccharide, has strong prebiotic effects. We used high-performance liquid chromatography and 1H nuclear magnetic resonance spectroscopy to show that BiBftA, a ß-fructosyltransferase belonging to glycoside hydrolase family 68, from Beijerinckia indica subsp. indica catalyzes transfructosylation of sucrose to produce mostly 1-kestose and levan polysaccharides. We substituted His395 and Phe473 in BiBftA with Arg and Tyr, respectively, and analyzed the reactions of the mutant enzymes with 180 g/L sucrose. The ratio of the molar concentrations of glucose and 1-kestose in the reaction mixture with wild-type BiBftA was 100:8.1, whereas that in the reaction mixture with the variant H395R/F473Y was 100:45.5, indicating that H395R/F473Y predominantly accumulated 1-kestose from sucrose. The X-ray crystal structure of H395R/F473Y suggests that its catalytic pocket is unfavorable for binding of sucrose while favorable for transfructosylation.

Impact of Gut Microbiota on Host Aggression: Potential Applications for Therapeutic Interventions Early in Development.

Aggression in the animal kingdom is a necessary component of life; however, certain forms of aggression, especially in humans, are pathological behaviors that are detrimental to society. Animal models have been used to study a number of factors, including brain morphology, neuropeptides, alcohol consumption, and early life circumstances, to unravel the mechanisms underlying aggression. These animal models have shown validity as experimental models. Moreover, recent studies using mouse, dog, hamster, and drosophila models have indicated that aggression may be affected by the "microbiota-gut-brain axis." Disturbing the gut microbiota of pregnant animals increases aggression in their offspring. In addition, behavioral analyses using germ-free mice have shown that manipulating the intestinal microbiota during early development suppresses aggression. These studies suggest that treating the host gut microbiota during early development is critical. However, few clinical studies have investigated gut-microbiota-targeted treatments with aggression as a primary endpoint. This review aims to clarify the effects of gut microbiota on aggression and discusses the therapeutic potential of regulating human aggression by intervening in gut microbiota.

Consumption of 1-Kestose Upregulates MicroRNA-200 and -192/215 Families in Lamina Propria Leukocytes of the Murine Large Intestine.

By comparing germ-free mice and specific pathogen-free mice, we recently demonstrated that the presence of gut commensals upregulates microRNA-200 family members in lamina propria leukocytes (LPL) of the murine large intestine. The present study tested whether the consumption of 1-kestose (KES), an indigestible oligosaccharide that alters gut microbiota composition, influences the microRNA expression in the LPL. Supplementation of KES (4%) in drinking water for 2 wk increased the levels of miR-182-5p, -205-5p, -290a-5p, miR-200 family members (miR-141-3p, -200a-3p, -200b-3p, -200c-3p, and -429-3p) as well as miR-192/215 family members (miR-192-5p, -194-5p, and -215-5p) as determined by microarray analysis in large intestinal LPL of C57BL/6 mice. Quantitative reverse transcription-PCR further confirmed the increase in miR-192-5p, -194-5p, -200a-3p, -200b-3p, -200c-3p, -205-5p, and 215-5p. KES consumption significantly increased Bifidobacterium pseudolongum in the cecal contents. In a separate experiment, intragastric administration of B. pseudolongum (109 CFU/d) for 7 d increased the levels of miR-182-5p, -194-5p, and -200a-3p and tended to increase the levels of miR-200b-3p, -215-5p, and -429-3p. These results suggest that dietary KES influences miRNA expression in the large intestinal LPL, which may be associated with the increased population of B. pseudolongum.

In children with cow's milk allergy, 1-kestose affects the gut microbiota and reaction threshold.

BACKGROUND: Interventions targeting the gut microbiota for treating food allergy (FA) have been gaining much attention. Although several studies have examined the effects of probiotics, few have verified the effects of prebiotic intervention on FA in humans. METHODS: We conducted a preliminary open-label, parallel-group comparison trial in children diagnosed with severe cow's milk allergy (CMA) who were instructed to ingest baked milk (BM; bread or cookies) daily. The subjects either received or did not receive the prebiotic 1-kestose (kestose) daily for 6 months. CMA symptoms and the threshold dose for milk protein were evaluated by oral food challenge with heated milk or BM. Blood and fecal samples were also collected for investigations of the antigen-specific immunoglobulin (Ig) E levels and microbiota composition. RESULTS: Kestose treatment significantly increased the threshold dose for milk protein, and decreased the milk- and casein-specific IgE levels in serum. In those treated with kestose, the abundance of Fusicatenibacter spp. significantly increased in the feces, and a significant inverse correlation was seen between the abundance of Fusicatenibacter spp. and the milk- and casein-specific IgE levels. CONCLUSION: Kestose treatment induced some tolerance to milk protein via changes in the gut microbiota composition in children with FA. IMPACT: A 6-month treatment with the prebiotic kestose increased the threshold dose for milk protein, and decreased the serum levels of milk- and casein-specific IgE in children diagnosed with cow's milk allergy. The kestose treatment increased the abundance of Fusicatenibacter spp. in the gut, which was inversely correlated with the antigen-specific IgE levels. This is the first study to demonstrate that a prebiotic intervention induced some tolerance to an allergen in children with food allergy.

Efficacy of 1-kestose supplementation in patients with mild to moderate ulcerative colitis: A randomised, double-blind, placebo-controlled pilot study.

BACKGROUND: Ulcerative colitis involves an excessive immune response to intestinal bacteria. Whether administering prebiotic 1-kestose is effective for active ulcerative colitis remains controversial. AIMS: This randomised, double-blind, placebo-controlled pilot trial investigated the efficacy of 1-kestose against active ulcerative colitis. METHODS: Forty patients with mild to moderate active ulcerative colitis were randomly treated with 1-kestose (N = 20) or placebo (maltose, N = 20) orally for 8 weeks in addition to the standard treatment. The Lichtiger clinical activity index and Ulcerative Colitis Endoscopic Index of Severity were determined. Faecal samples were analysed to evaluate the gut microbiome and metabolites. RESULTS: The clinical activity index at week 8 was significantly lower in the 1-kestose group than in the placebo group (3.8 ± 2.7 vs. 5.6 ± 2.1, p = 0.026). Clinical remission and response rates were higher in the 1-kestose group than in the placebo group (remission: 55% vs. 20%, p = 0.048; response: 60% vs. 25%, p = 0.054). The Ulcerative Colitis Endoscopic Index of Severity at week 8 was not significantly different (2.8 ± 1.6 vs. 3.5 ± 1.6, p = 0.145). Faecal analysis showed significantly reduced alpha-diversity in the 1-kestose group, with a decreased relative abundance of several bacteria, including Ruminococcus gnavus group. The short-chain fatty acid levels were not significantly different between the groups. The incidence of adverse events was comparable between the groups. DISCUSSION: Oral 1-kestose is well tolerated and provides clinical improvement for patients with mild to moderate ulcerative colitis through modulation of the gut microbiome.

Current Status of the Diagnosis of Early-Stage Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) can be treated with surgery, chemotherapy, and radiotherapy. Despite medical progress in each field in recent years, it is still insufficient for managing PDAC, and at present, the only curative treatment is surgery. A typical pancreatic cancer is relatively easy to diagnose with imaging. However, it is often not recommended for surgical treatment at the time of diagnosis due to metastatic spread beyond the pancreas. Even if it is operable, it often recurs during postoperative follow-up. In the case of PDAC with a diameter of 10 mm or less, the 5-year survival rate is as good as 80% or more, and the best index for curative treatment is tumor size. The early detection of pancreatic cancer with a diameter of less than 10 mm or carcinoma in situ is critical. Here, we provide an overview of the current status of diagnostic imaging features and genetic tests for the accurate diagnosis of early-stage PDAC.

Changes in intestinal bacteria and imbalances of metabolites induced in the intestines of pancreatic ductal adenocarcinoma patients in a Japanese population: a preliminary result.

BACKGROUND: The relationship between pancreatic ductal adenocarcinoma (PDAC) and the intestinal environment is not fully understood. The purpose of this study was to elucidate the characteristics of the intestinal environment in PDAC. METHODS: We performed a case-control study of 5 Japanese patients with unresectable PDAC located in the body or tail (PDAC-bt). The number of patients analyzed was limited for this preliminary study. We included 68 healthy subjects, herein control, of pre-printed study in the preliminary study. 16S rRNA amplicon sequencing and metabolomic analysis were performed using fecal samples from the subjects. RESULTS: There was no difference in the Shannon index and Principal Coordinate Analysis between PDAC-bt and the control. However, a significant increase in oral-associated bacteria (Actinomyces, Streptococcus, Veillonella, Lactobacillus) was observed. A significant decrease of Anaerostipes was demonstrated in the feces of PDAC-bt compared with the control. The intestinal propionic acid and deoxycholic acid were significantly lower in PDAC-bt compared with the control. CONCLUSIONS: We showed that the intestinal environment of PDAC-bt is characterized by an increase in oral-associated bacteria and an imbalance of metabolites but without changes in alpha and beta diversity of the gut microbiota profiles.Clinical Trial Registration: www.umin.ac.jp, UMIN 000041974, 000023675, 000023970.

Erythritol alters phosphotransferase gene expression and inhibits the in vitro growth of Staphylococcus coagulans isolated from canines with pyoderma.

Staphylococcus coagulans (SC) belongs to a group of coagulase-positive staphylococci occasionally isolated from the skin lesions of dogs with pyoderma. We recently revealed that erythritol, a sugar alcohol, inhibited the growth of SC strain JCM7470. This study investigated the molecular mechanisms involved in this growth inhibition of JCM7470 by erythritol, and determine whether erythritol inhibits the growth of SC isolated from the skin of dogs with pyoderma. Comprehensive analysis of the gene expression of JCM7470 in the presence of erythritol revealed that erythritol upregulated the expression of glcB and ptsG genes, both of which encode phosphotransferase system (PTS) glucoside- and glucose-specific permease C, B, and A domains (EIICBA), respectively, associated with sugar uptake. Moreover, erythritol suppressed in vitro growth of all 27 SC strains isolated from the skin lesions of canine pyoderma, including 13 mecA gene-positive and 14 mecA gene-negative strains. Finally, the growth inhibition of the SC clinical isolates by erythritol was restored by the addition of glucose. In summary, we revealed that erythritol promotes PTS gene expression and suppresses the in vitro growth of SC clinical isolates from dogs with pyoderma. Restoration of the erythritol-induced growth inhibition by glucose suggested that glucose starvation may contribute to the growth inhibition of SC.

1-Kestose Supplementation Increases Levels of a 5α-Reductase Gene, a Key Isoallolithocholic Acid Biosynthetic Gene, in the Intestinal Microbiota.

1-Kestose (kestose) is the smallest fructooligosaccharide component and shows a particularly high prebiotic function. Both kestose and the bile acid metabolite isoallolithocholic acid (isoalloLCA) are known to be beneficial for human health, especially in terms of immune homeostasis in the gastrointestinal system; however, the effect of kestose on the levels of microbial isoalloLCA producers remains to be clarified. IsoalloLCA is known to be produced by several members of the phylum Bacteroidota that carry the 5α-reductase (5AR) gene, a key isoalloLCA biosynthetic gene. Thus, we designed a specific primer set to detect the 5AR gene based on the consensus sequences of the genes from several isoalloLCA producers. Using real-time quantitative PCR with this primer set and fecal DNA samples, we compared the 5AR gene level (5ar-level) in the intestinal microbiota of a kestose-supplemented group (n=20) and a placebo group (n=16) before and after intake for 12 wk. The 5ar-level was significantly increased in the kestose-supplemented group (p=0.015), but not in the placebo group (p=0.379), indicating that kestose supplementation increased the 5ar-level in human intestinal microbiota. Our findings suggest that targeting functional gene levels could potentially be used to predict and understand the beneficial prebiotic effects associated with changes in gut microbiota.

Extracellular fructooligosaccharide degradation in Anaerostipes hadrus for co-metabolism with non-fructooligosaccharide utilizers.

Butyrate producing bacteria are one of the major components of the human gut microbiota. Their major metabolite, butyrate, has several beneficial properties for host health. Fructooligosaccharides (FOSs) are well documented prebiotics and are hydrolyzed by intracellular glycoside hydrolase family 32 (GH32) enzyme in several butyrate producers, whereas butyrate producers Anaerostipes hadrus and Anaerostipes butyraticus possess extracellular GH32 enzymes. The present study characterized the extracellular GH32 enzymes in the organisms to consider possible cross-feeding of FOSs with other microbes. Culture supernatant of A. hadrus actively hydrolyzed kestose and nystose, i.e., degrees of polymerization 3 and 4 FOSs, respectively, whereas that of A. butyraticus did not hydrolyzed. When co-cultured with Lacticaseibacillus rhamnosus GG in the presence of nystose, which was negative for growth on the FOSs but positive for growth on FOS degradants, A. hadrus promoted the growth of L. rhamnosus GG, but A. butyraticus did not. The observed negative results in A. butyraticus would be due to the presence of a stop codon in the gene encoding extracellular GH32. Genomic analysis revealed that A. hadrus conserved a single extracellular GH32 enzyme at the species level. The enzyme was phylogenetically distinguished into two groups, but the two groups shared similar FOS degradation properties. The results obtained here suggested that A. hadrus is active for extracellular degradation of FOSs and provides its degradants to other microbes. This study provides a basis of knowledge to understand how ingested FOSs are co-metabolized in gut microbiota.

Ribotype-dependent growth inhibition and promotion by erythritol in Cutibacterium acnes.

BACKGROUND: The close balance between Cutibacterium acnes and the skin flora, particularly between C. acnes phylotypes, has been suggested to play an important role in the onset of acne. C. acnes has been classified into ribotypes (RTs) based on polymorphisms in its 16S rRNA sequence, with RT4 and RT5 being associated with the onset of acne and RT6 with healthy skin. AIMS: The present study investigated the impact of erythritol on the growth of C. acnes strains classified into different RTs and attempted to elucidate the molecular mechanisms underlying its effects. METHODS: Culturing tests were performed on several RTs of C. acnes with or without erythritol. A transcriptional analysis of HM554 (RT6) and HM514 (RT5) was also conducted. RESULTS: The growth of RT2 and RT6, RTs associated with healthy skin, was significantly promoted in a medium containing 10% (W/W) erythritol, whereas that of RT1, RT3, RT4, RT5, and RT8, RTs associated with the development of acne, was inhibited. A RNA-seq analysis of HM554 showed that the expression of six genes (EIGs) potentially involved in carbohydrate metabolism was strongly induced by the presence of 10% erythritol (Log2 fold change >2.0 and p-value <0.05). A comparative expression analysis by qPCR revealed that EIGs other than g3pD were strongly induced by erythritol in HM514, similar to HM554, whereas g3pD was only slightly induced. CONCLUSION: Erythritol inhibited the growth of RTs associated with acne and promoted that of RTs associated with healthy skin. The enzyme encoded by g3pD may play an important role in the metabolism of erythritol and the dissolution of its growth inhibitory effects on C. acnes.

Enzymatic and structural characterization of ß-fructofuranosidase from the honeybee gut bacterium Frischella perrara.

Fructooligosaccharide is a mixture of mostly the trisaccharide 1-kestose (GF2), tetrasaccharide nystose (GF3), and fructosyl nystose (GF4). Enzymes that hydrolyze GF3 may be useful for preparing GF2 from the fructooligosaccharide mixture. A ß-fructofuranosidase belonging to glycoside hydrolase family 32 (GH32) from the honeybee gut bacterium Frischella perrara (FperFFase) was expressed in Escherichia coli and purified. The time course of the hydrolysis of 60 mM sucrose, GF2, and GF3 by FperFFase was analyzed, showing that the hydrolytic activity of FperFFase for trisaccharide GF2 was lower than those for disaccharide sucrose and tetrasaccharide GF3. The crystal structure of FperFFase and its structure in complex with fructose were determined. FperFFase was found to be structurally homologous to bifidobacterial ß-fructofuranosidases even though bifidobacterial enzymes preferably hydrolyze GF2 and the amino acid residues interacting with fructose at subsite - 1 are mostly conserved between them. A proline residue was inserted between Asp298 and Ser299 using site-directed mutagenesis, and the activity of the variant 298P299 was measured. The ratio of activities for 60 mM GF2/GF3 by wild-type FperFFase was 35.5%, while that of 298P299 was 23.6%, indicating that the structure of the loop comprising Trp297-Asp298-Ser299 correlated with the substrate preference of FperFFase. The crystal structure also shows that a loop consisting of residues 117-127 is likely to contribute to the substrate binding of FperFFase. The results obtained herein suggest that FperFFase is potentially useful for the manufacture of GF2. KEY POINTS: • Frischella ß-fructofuranosidase hydrolyzed nystose more efficiently than 1-kestose. • Trp297-Asp298-Ser299 was shown to be correlated with the substrate preference. • Loop consisting of residues 117-127 appears to contribute to the substrate binding.

The Effects of Enteral Nutrition on the Intestinal Environment in Patients in a Persistent Vegetative State.

Enteral nutrition (EN) is a rational approach to providing nutritional intake via the intestines in patients who are unable to tolerate parenteral nutrition. We conducted a preliminary study to investigate the effects of EN on the intestinal environment in 10 patients in a persistent vegetative state (PVS) (n = 5 each in the EN and EN with probiotics; Clostridium butyricum MIYAIRI 588) groups compared with 10 healthy controls. The results of 16S amplicon sequencing of the intestinal microbiota showed that EN led to dysbiosis with a decrease in α-diversity and an obvious change in ß-diversity. A particularly significant decrease was seen in useful intestinal bacteria such as Bifidobacterium and butyrate-producing bacteria. Analysis of intestinal metabolites also supported these results, showing significant decreases in butyric and pyruvic acid after EN. Although C. butyricumMIYAIRI 588 improved some intestinal metabolites that were decreased after EN, it did not improve the dysbiosis of the intestinal microbiota. These findings indicate that EN causes dysbiosis of the intestinal microbiota and an imbalance in some intestinal metabolites in patients in a PVS. Moreover, although C. butyricumMIYAIRI 588 improved the imbalance of some intestinal metabolites after EN, it did not prevent dysbiosis of the intestinal microbiota.