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Safety and Modulatory Effects of Humanized Galacto-Oligosaccharides on the Gut Microbiome.

ABSTRACT: Complex dietary carbohydrate structures including β(1-4) galacto-oligosaccharides (GOS) are resistant to digestion in the upper gastrointestinal (GI) tract and arrive intact to the colon where they benefit the host by selectively stimulating microbial growth. Studies have reported the beneficial impact of GOS (alone or in combination with other prebiotics) by serving as metabolic substrates for modulating the assembly of the infant gut microbiome while reducing GI infections. N-Acetyl-D-lactosamine (LacNAc, Galβ1,4GlcNAc) is found in breast milk as a free disaccharide. This compound is also found as a component of human milk oligosaccharides (HMOs), which have repeating and variably branched lactose and/or LacNAc units, often attached to sialic acid and fucose monosaccharides. Human glycosyl-hydrolases do not degrade most HMOs, indicating that these structures have evolved as natural prebiotics to drive the proper assembly of the infant healthy gut microbiota. Here, we sought to develop a novel enzymatic method for generating LacNAc-enriched GOS, which we refer to as humanized GOS (hGOS). We showed that the membrane-bound β-hexosyl transferase (rBHT) from Hamamotoa (Sporobolomyces) singularis was able to generate GOS and hGOS from lactose and N-Acetyl-glucosamine (GlcNAc). The enzyme catalyzed the regio-selective, repeated addition of galactose from lactose to GlcNAc forming the β-galactosyl linkage at the 4-position of the GlcNAc and at the 1-position of D-galactose generating, in addition to GOS, LacNAc, and Galactosyl-LacNAc trisaccharides which were produced by two sequential transgalactosylations. Humanized GOS is chemically distinct from HMOs, and its effects in vivo have yet to be determined. Thus, we evaluated its safety and demonstrated the prebiotic's ability to modulate the gut microbiome in 6-week-old C57BL/6J mice. Longitudinal analysis of gut microbiome composition of stool samples collected from mice fed a diet containing hGOS for 5 weeks showed a transient reduction in alpha diversity. Differences in microbiome community composition mostly within the Firmicutes phylum were observed between hGOS and GOS, compared to control-fed animals. In sum, our study demonstrated the biological synthesis of hGOS, and signaled its safety and ability to modulate the gut microbiome in vivo, promoting the growth of beneficial microorganisms, including Bifidobacterium and Akkermansia.

SUBMITTER: Arnold JW

PROVIDER: S-EPMC8058378 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPrebiotic galacto-oligosaccharides (GOS) have an extensively demonstrated beneficial impact on intestinal health. In this study, we determined the impact of GOS diets on hallmarks of gut aging: microbiome dysbiosis, inflammation, and intestinal barrier defects ("leaky gut"). We also evaluated if short-term GOS feeding influenced how the aging gut responded to antibiotic challenges in a mouse model of Clostridioides difficile infection. Finally, we assessed if colonic organoids could reproduce the GOS responder-non-responder phenotypes observed in vivo.ResultsOld animals had a distinct microbiome characterized by increased ratios of non-saccharolytic versus saccharolytic bacteria and, correspondingly, a lower abundance of β-galactosidases compared to young animals. GOS reduced the overall diversity, increased the abundance of specific saccharolytic bacteria (species of Bacteroides and Lactobacillus), increased the abundance of β-galactosidases in young and old animals, and increased the non-saccharolytic organisms; however, a robust, homogeneous bifidogenic effect was not observed. GOS reduced age-associated increased intestinal permeability and increased MUC2 expression and mucus thickness in old mice. Clyndamicin reduced the abundance Bifidobacterium while increasing Akkermansia, Clostridium, Coprococcus, Bacillus, Bacteroides, and Ruminococcus in old mice. The antibiotics were more impactful than GOS on modulating serum markers of inflammation. Higher serum levels of IL-17 and IL-6 were observed in control and GOS diets in the antibiotic groups, and within those groups, levels of IL-6 were higher in the GOS groups, regardless of age, and higher in the old compared to young animals in the control diet groups. RTqPCR revealed significantly increased gene expression of TNFα in distal colon tissue of old mice, which was decreased by the GOS diet. Colon transcriptomics analysis of mice fed GOS showed increased expression of genes involved in small-molecule metabolic processes and specifically the respirasome in old animals, which could indicate an increased oxidative metabolism and energetic efficiency. In young mice, GOS induced the expression of binding-related genes. The galectin gene Lgals1, a β-galactosyl-binding lectin that bridges molecules by their sugar moieties and is an important modulator of the immune response, and the PI3K-Akt and ECM-receptor interaction pathways were also induced in young mice. Stools from mice exhibiting variable bifidogenic response to GOS injected into colon organoids in the presence of prebiotics reproduced the response and non-response phenotypes observed in vivo suggesting that the composition and functionality of the microbiota are the main contributors to the phenotype.ConclusionsDietary GOS modulated homeostasis of the aging gut by promoting changes in microbiome composition and host gene expression, which was translated into decreased intestinal permeability and increased mucus production. Age was a determining factor on how prebiotics impacted the microbiome and expression of intestinal epithelial cells, especially apparent from the induction of galectin-1 in young but not old mice. Video abstract.

Project description:BackgroundFructo-oligosaccharides (FOS), inulin, and galacto-oligosaccharides (GOS) are widely recognized prebiotics that profoundly affect the intestinal microbiota, including stimulation of bifidobacteria and lactobacilli, and are reported to elicit several health benefits. The combination of dietary FOS and inulin with calcium phosphate was reported to stimulate commensal Lactobacillus populations and protect the host against pathogenic Enterobacteriaceae, but little is known about the effects of GOS in diets with a different level of calcium phosphate.MethodsWe investigated the microbiome changes elicited by dietary supplementation with GOS or inulin using diets with high (100 mmol/kg) and low (30 mmol/kg) calcium phosphate levels in adult Wistar rats. Rats were acclimatized to the respective experimental diets for 14 days, after which fecal material was collected, DNA was extracted from fecal material, and the V3‑V4 region of the bacterial 16S rRNA gene was amplified with PCR, followed by microbial composition analysis. In tandem, the organic acid profiles of the fecal material were analyzed.ResultsFeeding rats non-supplemented (no prebiotic-added) diets revealed that diets rich in calcium phosphate favored members of the Firmicutes and increased fecal lactic, succinic, acetic, propionic, and butyric acid levels. In contrast, relatively low dietary calcium phosphate levels promoted the abundance of mucin degrading genera like Akkermansia and Bacteroides, and resulted in increased fecal propionic acid levels and modest increases in lactic and butyric acid levels. Irrespective of the calcium phosphate levels, supplementation with GOS or inulin strongly stimulated Bifidobacterium, while only high calcium phosphate diets increased the endogenous Faecalibaculum populations.ConclusionsDespite the prebiotic's substantial difference in chemical structure, sugar composition, oligomer size, and the microbial degradation pathway involved in their utilization, inulin and GOS modulated the gut microbiota very similarly, in a manner that strongly depended on the dietary calcium phosphate level. Therefore, our study implies that the collection of detailed diet information including micronutrient balance is necessary to correctly assess diet-driven microbiota analysis. Video Abstract.

Project description:BackgroundMost research on galacto-oligosaccharides (GOS) has mainly focused on their prebiotic effects on the hindgut, but their beneficial effects on the small intestine (SI) have received little attention. Since jejunum is the important place to digest and absorb nutrients efficiently, optimal maturation of the jejunum is necessary for maintaining the high growth rate in the neonate. Therefore, this study investigates the effect of the early intervention with GOS on the intestinal development of the jejunum.MethodsA total of 6 litters of neonatal piglets (10 piglets per litter; Duroc × Landrace × Large White) with an average birth weight of 1.55 ± 0.05 kg received 1 of 2 treatments based on their assignment to either the control (CON) group or the GOS (GOS) group in each litter. Piglets in the GOS group were orally administrated 10 mL of a GOS solution (reaching 1 g GOS/kg body weight) per day from the age of 1 to 7 d; the piglets in the CON group were treated with the same dose of physiological saline. All piglets were weaned on d 21. On d 8 and 21 of the experimental trial, 1 pig per group from each of the 6 litters was euthanized.ResultsThe early intervention with GOS increased the average daily gains in the third week (P < 0.05). Decreased crypt depth was also observed in the jejunum of the piglets on d 21 (P < 0.05). The early intervention with GOS increased the jejunal lactase activity on d 8, maltase activity and sucrase activity on d 21 (P < 0.05). In addition, the early intervention with GOS also facilitated the mRNA expression of Sodium glucose co-transporter 1 (SGLT1) on d 8 and the mRNA expression of Glucose transporter type 2 (GLUT2) on d 21 (P < 0.05). It was further determined that GOS up-regulated the mRNA expression of preproglucagon (GCG), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor (EGF). GOS also up-regulated the protein expression of glucagon-like peptide-2 (GLP-2) and EGF in the jejunum of the piglets. Furthermore, it was also found that GOS enhanced the protein expression of ZO-1 and occludin on d 8 (P < 0.05), as well as increased the mRNA expression of TGF-β and decrease the mRNA expression of IL-12 (P < 0.05).ConclusionsThese results indicate that GOS have a positive effect on piglet growth performance in addition to decreasing the crypt depth and enhancing functional development in jejunum of suckling piglets.

Project description:Salmonella is estimated to cause one million foodborne illnesses in the United States every year. Salmonella-contaminated poultry products are one of the major sources of salmonellosis. Given the critical role of the gut microbiota in Salmonella transmission, a manipulation of the chicken intestinal microenvironment could prevent animal colonization by the pathogen. In Salmonella, the global regulator gene fnr (fumarate nitrate reduction) regulates anaerobic metabolism and is essential for adapting to the gut environment. This study tested the hypothesis that an attenuated Fnr mutant of Salmonella enterica serovar Typhimurium (attST) or prebiotic galacto-oligosaccharides (GOS) could improve resistance to wild-type Salmonella via modifications to the structure of the chicken gut microbiome. Intestinal samples from a total of 273 animals were collected weekly for 9 weeks to evaluate the impact of attST or prebiotic supplementation on microbial species of the cecum, duodenum, jejunum, and ileum. We next analyzed changes to the gut microbiome induced by challenging the animals with a wild-type Salmonella serovar 4,[5],12:r:- (Nalr) strain and determined the clearance rate of the virulent strain in the treated and control groups. Both GOS and the attenuated Salmonella strain modified the gut microbiome but elicited alterations of different taxonomic groups. The attST produced significant increases of Alistipes and undefined Lactobacillus, while GOS increased Christensenellaceae and Lactobacillus reuteri The microbiome structural changes induced by both treatments resulted in a faster clearance after a Salmonella challenge.IMPORTANCE With an average annual incidence of 13.1 cases/100,000 individuals, salmonellosis has been deemed a nationally notifiable condition in the United States by the Centers for Disease Control and Prevention (CDC). Earlier studies demonstrated that Salmonella is transmitted by a subset of animals (supershedders). The supershedder phenotype can be induced by antibiotics, ascertaining an essential role for the gut microbiota in Salmonella transmission. Consequently, modulation of the gut microbiota and modification of the intestinal microenvironment could assist in preventing animal colonization by the pathogen. Our study demonstrated that a manipulation of the chicken gut microbiota by the administration of an attenuated Salmonella strain or prebiotic galacto-oligosaccharides (GOS) can promote resistance to Salmonella colonization via increases of beneficial microorganisms that translate into a less hospitable gut microenvironment.

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Safety and Modulatory Effects of Humanized Galacto-Oligosaccharides on the Gut Microbiome.

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