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.

Dietary supplementation with 1-kestose induces altered locomotor activity and increased striatal dopamine levels with a change in gut microbiota in male mice.

1-Kestose (KES), a dietary fiber and prebiotic carbohydrate, benefits various physiological functions. This study aimed to examine whether diets supplemented with KES over three consecutive generations could significantly affect some host physiological aspects, including behavioral phenotypes and gut microbial ecology. Mice that received KES-supplemented diets for three generations demonstrated increased activity compared with those fed diets lacking KES. Furthermore, the KES group showed increased striatal dopamine (DA) and serotonin (5-HT) levels. The observed increase in DA levels within the striatum was positively correlated with locomotor activity in the KES group but not in the control (CON) group. The α-diversities were significantly lower in the KES group compared to the CON group. The three-dimensional principal coordinate analysis revealed a substantial distinction between the KES and CON groups across each generation. At the genus level, most gut microbiota genera exhibited lower abundances in the KES group than in the CON group, except for Bifidobacteria and Akkermansia. Spearman's rank-order analysis indicated significant negative correlations between the striatal DA levels and α-diversity values. These findings suggest that prolonged supplementation with KES may stimulate increased locomotor activity along with elevated striatal DA levels, which are potentially associated with KES-induced alterations in the gut microbiota.

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.

A Double-Blind, Randomized, Placebo-Controlled Trial of the Effect of 1-Kestose on Defecation Habits in Constipated Kindergarten Children: A Pilot Study.

Constipation is common in children and can significantly affect quality of life. Prebiotics are reportedly helpful for constipation in adults, but few studies have examined their use in young children. In this study, the effect of 1-kestose (kestose), which has excellent bifidobacterial growth properties, on constipation in kindergarten children (n = 11) was compared with that of maltose (n = 12) in a randomized, double-blind study. Three grams of kestose per day for 8 weeks did not affect stool properties, but significantly increased the number of defecations per week (Median; 3 → 4 times/week, p = 0.017, effect size = 0.53). A significant decrease in Intestinibacter, a trend toward increased bifidobacteria, and a trend toward decreased Clostridium sensu stricto were observed after kestose ingestion, while concentrations of short-chain fatty acids in stools were unchanged.

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.

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.

Consumption of indigestible saccharides and administration of Bifidobacterium pseudolongum reduce mucosal serotonin in murine colonic mucosa.

SCFA increase serotonin (5-hydroxytryptamine, 5-HT) synthesis and content in the colon in vitro and ex vivo, but little is known in vivo. We tested whether dietary indigestible saccharides, utilised as a substrate to produce SCFA by gut microbiota, would increase colonic 5-HT content in mice. Male C57BL/6J mice were fed a purified diet and water supplemented with 4 % (w/v) 1-kestose (KES) for 2 weeks. Colonic 5-HT content and enterochromaffin (EC) cell numbers were lower in mice supplemented with KES than those without supplementation, while monoamine oxidase A activity and mRNA levels of tryptophan hydroxylase 1 (Tph1), chromogranin A (Chga), Slc6a4 and monoamine oxidase A (Maoa) genes in the colonic mucosa, serum 5-HT concentration and total 5-HT content in the colonic contents did not differ between groups. Caecal acetate concentration and Bifidobacterium pseudolongum population were higher in KES-supplemented mice. Similar trends were observed in mice supplemented with other indigestible saccharides, that is, fructo-oligosaccharides, inulin and raffinose. Intragastric administration of live B. pseudolongum (108 colony-forming units/d) for 2 weeks reduced colonic 5-HT content and EC cell numbers. These results suggest that changes in synthesis, reuptake, catabolism and overflow of 5-HT in the colonic mucosa are not involved in the reduction of colonic 5-HT content by dietary indigestible saccharides in mice. We propose that gut microbes including B. pseudolongum could contribute to the reduction of 5-HT content in the colonic mucosa via diminishing EC cells.

Supplementation of 1-Kestose Modulates the Gut Microbiota Composition to Ameliorate Glucose Metabolism in Obesity-Prone Hosts.

Insulin resistance leads to the onset of medical conditions such as type 2 diabetes, and its development is associated with the alteration in the gut microbiota. Although it has been demonstrated that supplementation with prebiotics modulates the gut microbiota, limited evidence is available for effects of prebiotics on insulin resistance, especially for humans. We investigated the prebiotic effect of 1-kestose supplementation on fasting insulin concentration in obesity-prone humans and rats. In the preliminary study using rats, the hyperinsulinemia induced by high-fat diet was suppressed by intake of water with 2% (w/v) 1-kestose. In the clinical study using obese-prone volunteers, the fasting serum insulin level was significantly reduced from 6.5 µU/mL (95% CI, 5.5-7.6) to 5.3 (4.6-6.0) by the 12-week intervention with supplementation of 10 g 1-kestose/day, whereas it was not changed by the intervention with placebo (6.2 µU/mL (5.4-7.1) and 6.5 (5.5-7.6) before and after intervention, respectively). The relative abundance of fecal Bifidobacterium was significantly increased to 0.3244 (SD, 0.1526) in 1-kestose-supplemented participants compared to that in control participants (0.1971 (0.1158)). These results suggest that prebiotic intervention using 1-kestose may potentially ameliorate insulin resistance in overweight humans via the modulation of the gut microbiota. UMIN 000028824.

Increase in muscle mass associated with the prebiotic effects of 1-kestose in super-elderly patients with sarcopenia.

Sarcopenia causes functional disorders and decreases the quality of life. Thus, it has attracted substantial attention in the aging modern world. Dysbiosis of the intestinal microbiota is associated with sarcopenia; however, it remains unclear whether prebiotics change the microbiota composition and result in the subsequent recovery of muscle atrophy in elderly patients with sarcopenia. This study aimed to assess the effects of prebiotics in super-elderly patients with sarcopenia. We analyzed the effects of 1-kestose on the changes in the intestinal microbiota and body composition using a next-generation sequencer and a multi-frequency bioimpedance analysis device. The Bifidobacterium longum population was significantly increased in the intestine after 1-kestose administration. In addition, in all six patients after 12 weeks of 1-kestose administration, the skeletal muscle mass index was greater, and the body fat percentage was lower. This is the first study to show that administration of a prebiotic increased the population of B. longum in the intestinal microbiota and caused recovery of muscle atrophy in super-elderly patients with sarcopenia.

Gut commensals suppress interleukin-2 production through microRNA-200/BCL11B and microRNA-200/ETS-1 axes in lamina propria leukocytes of murine large intestine.

The role of microRNAs (miRNAs) in how microbiota influence the host intestinal immune system is not fully understood. We compared the expression profiles of miRNAs and mRNAs in lamina propria leukocytes (LPL) in the large intestines of germ-free (GF) and specific pathogen-free (SPF) mice. Microarray analysis revealed different expression profiles of miRNAs and mRNAs between GF and SPF mice. Quantitative real time-PCR (qRT-PCR) showed that the level of miR-200 family members was significantly higher in SPF mice than in GF mice. In silico prediction followed by qRT-PCR suggested that Bcl11b, Ets1, Gbp7, Stat5b, and Zeb1 genes were downregulated by the miR-200 family. Western blotting revealed that the expression of BCL11B and ETS-1, but not ZEB1, in large intestinal LPL was significantly lower in SPF mice than in GF mice. Interleukin (IL)-2 production in cultured LPL upon stimulation with phorbol 12-myristate 13-acetate and ionomycin for 24 h was significantly lower in SPF mice than in GF mice. Conventionalization of GF mice substantially recapitulated SPF mice in terms of the expression of miR-200 family members and their target genes and IL-2 production in large intestinal LPL. Considering that BCL11B and ETS-1 reportedly function as transcription factors to activate the Il2 gene, we propose that the presence of gut commensals suppresses IL-2 production in large intestinal LPL, at least in part through post-transcriptional downregulation of Bcl11b and Ets1 genes by miR-200 family members.

1-Kestose supplementation mitigates the progressive deterioration of glucose metabolism in type 2 diabetes OLETF rats.

The fructooligosaccharide 1-kestose cannot be hydrolyzed by gastrointestinal enzymes, and is instead fermented by the gut microbiota. Previous studies suggest that 1-kestose promotes increases in butyrate concentrations in vitro and in the ceca of rats. Low levels of butyrate-producing microbiota are frequently observed in the gut of patients and experimental animals with type 2 diabetes (T2D). However, little is known about the role of 1-kestose in increasing the butyrate-producing microbiota and improving the metabolic conditions in type 2 diabetic animals. Here, we demonstrate that supplementation with 1-kestose suppressed the development of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, possibly through improved glucose tolerance. We showed that the cecal contents of rats fed 1-kestose were high in butyrate and harbored a higher proportion of the butyrate-producing genus Anaerostipes compared to rats fed a control diet. These findings illustrate how 1-kestose modifications to the gut microbiota impact glucose metabolism of T2D, and provide a potential preventative strategy to control glucose metabolism associated with dysregulated insulin secretion.

Intestinal microbiota transplantation reveals the role of microbiota in dietary regulation of RegIIIß and RegIIIγ expression in mouse intestine.

RegIIIß and RegIIIγ are antimicrobial peptides expressed in intestinal epithelial cells. Expression of these peptides is reportedly decreased by high-fat diet (HFD) and increased by indigestible oligosaccharides in mice. Clearly, these dietary regimens change the structure of intestinal microbiota. We employed an intestinal microbiota transplantation (IMT) to test whether diet-induced changes in the expression of these peptides are mediated by gut microbiota. C57BL/6J mice were fed either a normal-fat diet (NFD), a HFD, or a NFD supplemented with or without 1-kestose (KES), an indigestible oligosaccharide. Ileal RegIIIß and RegIIIγ mRNA levels were lower in mice receiving IMT from HFD-fed mice than in those receiving NFD-fed mice and higher in mice receiving IMT from KES-supplemented mice than in those receiving the mice without KES supplementation. Western blot analysis showed that serum RegIIIß levels changed in parallel with the ileal mRNA levels. We propose that HFD- and KES-induced changes in the ileal RegIIIß and RegIIIγ expression and in the circulating RegIIIß levels are mediated, at least in part, by intestinal microbiota.

Reply to Comment on Watanabe, A.; Kadota, Y.; Yokoyama, H.; Tsuruda, S.; Kamio, R.; Tochio, T.; Shimomura, Y.; Kitaura, Y. Experimental Determination of the Threshold Dose for Bifidogenic Activity of Dietary 1-Kestose in Rats. Foods 2020, 9, 4.

The manuscript entitled "Comment on Experimental Determination of the Threshold Dose for Bifidogenic Activity of Dietary 1-Kestose in Rats" by Shen et al [...].

Experimental Determination of the Threshold Dose for Bifidogenic Activity of Dietary 1-Kestose in Rats.

1-Kestose is a non-digestible oligosaccharide consisting of glucose linked to two fructose units. While 1-kestose is not digested in the small intestine of mammals, it is fermented in the ceca and colon, where the growth of bifidobacteria is promoted. In the present study, we assessed the threshold dose of dietary 1-kestose that increased cecal bifidobacterial levels in rats. Rats were fed experimental diets containing 0% to 0.3% 1-kestose for four weeks. The levels of the genus Bifidobacterium and total gut bacteria were significantly increased in cecal samples of rats fed the 0.3% 1-kestose diet. Further, a significant correlation between the dose of 1-kestose and the levels of cecal Bifidobacterium and total gut bacteria was observed. The minimum dose of dietary 1-kestose to induce significant bifidogenic activity in rats was 0.3% by weight in the diet.

Feeding of 1-Kestose Induces Glutathione-S-Transferase Expression in Mouse Liver.

Functional food ingredients, including prebiotics, have been increasingly developed for human health. The improvement of the human intestinal environment is one of their main targets. Fructooligosaccarides (FOS) are oligosaccharide fructans that are well studied and commercialized prebiotics. 1-Kestose, one of the components of FOS, is considered to be a key prebiotic component in FOS. However, to our knowledge, no studies have been reported on the physiological efficacy of 1-Kestose regarding its anti-oxidative activity. In the present study, we examined the effects of dietary 1-Kestose on gene expression of antioxidative enzymes in the liver, kidney and epididymal adipose tissue of mice by quantitative RT-PCR (qRT-PCR). We demonstrated that a 1-Kestose-rich diet increased mRNA and enzymatic activity levels of glutathione-S-transferase (GST) in mouse liver. These results suggest the possibility that dietary 1-Kestose as a prebiotic may enhance antioxidative activity in mice.

1-Kestose, the Smallest Fructooligosaccharide Component, Which Efficiently Stimulates Faecalibacterium prausnitzii as Well as Bifidobacteria in Humans.

The concept of prebiotics was established more than 30 years ago. While the prebiotic concept has now expanded thus includes non-carbohydrate substances and diverse categories other than foods, fructooligosaccharides (FOS) have still predominantly been used as pebiotics, because the effects of FOS exclusively act through the enrichment of Bifidobacterium and Lactobacillus spp., which have been classified as beneficial intestinal commensals so far. Now the commercially available FOS products are synthetic mixture of several kinds of FOS components including 1-kestose (GF2), nystose (GF3) and GF4. In our previous studies, superiority of 1-kestose to the longer-chain FOS components such as nystose with regard to bifidogenic activity was clearly demonstrated. Recently, a broader range of beneficial bacteria including butyrate-producing indigenous bacteria have been recognized and expected to be new probiotic strains. Among them, resident Faecalibacterium prausnitzii is a butyrate producer with a significant anti-inflammatory effect thus expected to be useful as a next-generation probiotic. However, this bacterium is extremely oxygen-sensitive thus can be difficult to grow industrially. On the other hand, we have clearly demonstrated a significant prebiotic effect of 1-kestose, which is the smallest component of FOS, on F. prausnitzii in the gut of humans. These findings suggest that 1-kestose has impressive potential as a new prebiotic targeting F. prausnitzii, a next-generation probiotic strain, as well as bifidobacteria.

Ca2+-dependent inhibition of branched-chain α-ketoacid dehydrogenase kinase by thiamine pyrophosphate.

Catabolism of the branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) is regulated by the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which in turn is regulated by phosphorylation catalyzed by BCKDH kinase (BDK). Thiamine pyrophosphate (TPP) is required as a coenzyme for the E1 component of the BCKDH complex and can also bring about activation of the complex by inhibiting BDK. The present study shows that free Ca2+ in the physiological range greatly increases the sensitivity of BDK to inhibition by TPP (IC50 of 2.5 µM in the presence of 1 µM free Ca2+). This novel mechanism may be responsible for the stimulation of BCAA oxidation by conditions that increase mitochondrial free Ca2+ levels, e.g. in skeletal muscle during exercise.

Endurance performance and energy metabolism during exercise in mice with a muscle-specific defect in the control of branched-chain amino acid catabolism.

It is known that the catabolism of branched-chain amino acids (BCAAs) in skeletal muscle is suppressed under normal and sedentary conditions but is promoted by exercise. BCAA catabolism in muscle tissues is regulated by the branched-chain α-keto acid (BCKA) dehydrogenase complex, which is inactivated by phosphorylation by BCKA dehydrogenase kinase (BDK). In the present study, we used muscle-specific BDK deficient mice (BDK-mKO mice) to examine the effect of uncontrolled BCAA catabolism on endurance exercise performance and skeletal muscle energy metabolism. Untrained control and BDK-mKO mice showed the same performance; however, the endurance performance enhanced by 2 weeks of running training was somewhat, but significantly less in BDK-mKO mice than in control mice. Skeletal muscle of BDK-mKO mice had low levels of glycogen. Metabolome analysis showed that BCAA catabolism was greatly enhanced in the muscle of BDK-mKO mice and produced branched-chain acyl-carnitine, which induced perturbation of energy metabolism in the muscle. These results suggest that the tight regulation of BCAA catabolism in muscles is important for homeostasis of muscle energy metabolism and, at least in part, for adaptation to exercise training.