Metabolic Activity Of Gut Microbiota Research Articles

Metabolic potential of gut microbiota in helminth infections as a way to achieve bronchial asthma control

The aim of the review was to analyze modern experimental studies and clinical trials aimed at assessing metabolic activity of gut microbiota in bronchial asthma (BA) and helminth infections.Being one of the most common chronic heterogeneous respiratory diseases, bronchial asthma secures its place among global health problems of great socioeconomic importance. In recent years, a lot of data has been accumulated indicating that the state of gut microbiota is an important factor determining the state of human health and affecting immune mechanisms underlying the development of allergic diseases in childhood. Dysbiosis of gut microbiota is due not only to changes in its composition, but also to disturbances in its metabolism. In accordance with the “gut – lung axis” concept, maintaining healthy gut microbiota and correcting its disorders, including strategies aimed at activating synthesis of short-chain fatty acids in the intestine, may become a new way to prevent and treat chronic respiratory diseases in childhood. In turn, experimental and epidemiological studies have shown the immunomodulatory activity of helminths. It is assumed that their impact on the composition and function of gut microbiota is one of the mechanisms by which helminths influence the immune response of the host and the course of BA.

Phytate Hydrolysate Differently Modulates the Immune Response of Human Healthy and Cancer Colonocytes to Intestinal Bacteria.

(1) Phytic acid (PA) is a component of cereal seeds and legumes, therefore its consumption is much higher in a vegan and vegetarian diet compared to a conventional diet. The diet is the main driver of metabolic activity of gut microbiota, therefore, the ability to degrade phytates by the microbiota of vegans significantly exceeds that of the gut microbiota of omnivores. The aim of the study was to investigate the early phase of the immune response of colonocytes treated with an enzymatic hydrolysate of phytic acid (hPA120) and gut bacteria. (2) Cell lines derived from healthy (NCM460D) and cancer (HCT116) colonic tissue and fecal bacteria from vegan (V) and omnivorous (O) donors were investigated. Fecal bacteria were grown in mucin and phytic acid supplemented medium. Cultured bacteria (BM) were loaded onto colonocytes alone (V BM and O BM) or in combination with the phytate hydrolysate (V BM + hPA120 and O BM + hPA120). After a treatment of 2 h, bacterial adhesion, secretion of cytokines, and the expression of genes and proteins important for immune response were determined. (3) All bacteria-treated colonocytes increased the expression of IL8 compared to controls. The significant increase of the secreted IL-8 (p < 0.01) in both cell lines was observed for O BM and O BM + hPA120. The increase of TNF, IL-1β, and IL-10 secretion in healthy colonocytes (V BM alone and with hPA120 treatments; p < 0.05) and for TNF and IL-10 in cancer cells (treatments except O BM + hPA120 and V BM, respectively; p > 0.05) were stated. A comparison of solely the effect of hPA120 on bacteria-treated colonocytes (BM vs. BM + hPA120) showed that hPA120 decreased expression of NFkB1 and TNFR (p < 0.001) in healthy colonocytes. In cancer colonocytes, the expression of TLR4 and IL1R increased after BM + hPA120 treatment, whereas the secretion of IL-8 and MYD88 and TNFR expression decreased (p < 0.01). (4) The investigated hPA120 showed a differentiated modulatory activity on the immune response of healthy and cancer human colonocytes. Especially when analyzed independently on the gut bacteria origin, it reduced the proinflammatory response of HCT116 cells to gut bacteria, while being neutral for the bacteria-treated healthy colonocytes.

Probiotic Intervention in the Treatment of Diabetes Mellitus: A Review

Diabetes is a noncommunicable lifestyle condition that impacts millions of individuals worldwide. Diabetes is a physiological illness that affects several different organs in the human body. Several studies have found a direct relationship between gut microbiota and diabetes control. Probiotic intervention in the treatment of diabetes mellitus has been the center of focus in the current scenario. Alteration in composition and metabolic activity of gut microbiota significantly contributes to human health. However, the key mechanism of gut microbiota in the inhibition of diabetes is not fully understood. This review discusses the effect of probiotics on diabetes and the role of gut microbiota. It emphasizes on the pharmacological effects of probiotics on diabetic symptoms like glycemic response, hypercholesterolemia, hypertension, as well as gestational diabetes.

UPLC-Q-TOF/MS based fecal metabolomics reveals the potential anti-diabetic effect of Xiexin Decoction on T2DM rats

Xiexin Decoction (XXD), a traditional Chinese medicine prescription composed of Rhei rhizome (RR), Scutellaria radix (SR) and Coptidis rhizome (CR), has been used to cure diabetes in clinical practices for thousands of years, but its mechanism is not clear. Our previous study indicated that XXD could significantly ameliorate the symptom of type 2 diabetes mellitus (T2DM) rats by shifting the composition of gut microbiota. However, the effect of XXD on the metabolic activity of gut microbiota is not clarified. In this study, the underlying mechanism of XXD on the amelioration of T2DM was explored by fecal metabolic profiling analysis based on ultra performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC-Q-TOF/MS). The disordered metabolic profiles in T2DM rats were notably improved by XXD. Ten potential biomarkers, which were mainly involved in arachidonic acid metabolism, amino acid metabolism, bile acid metabolism, glycolysis and gluconeogenesis, were identified. Furthermore, these metabolites were closely related to SCFAs-producing and anti-inflammatory gut microflora. After XXD intervention, these biomarkers restored to the normal level at some extent. This study not only revealed potential biomarkers and related pathways in T2DM rats affected by XXD, but also provided a novel insight to uncover how traditional herb medicines worked from fecal metabolomics.

Состав кишечной микробиоты как фактор риска развития ожирения у детей

Nowadays obesity resulting from abnormal or excessive fat deposits in a body has become a true epidemic. Risk factors that cause the disease include improper lifestyle, hereditary predisposition, as well as metabolic activity of gut microbiota. Research works performed over the last decades indicate that microbes that create colonies in human intestines play a significant role in maintaining proper metabolism. There is a correlation between disorders in gut microbiota structure and immune disorders, elevated susceptibility to infections, and obesity. There is more and more evidence that gut microbiota and its overall bacterial genome exert their influence on nutrients assimilation and regulate energy metabolism and fat accumulation. Certain differences were detected in microbiota gut structure in children and adults with obesity and people with proper body mass index. Delivery and feeding are among key factors influencing gut microbiota formation in a child. Thus, research results indicate that natural birth, as opposed to cesarean section, can prevent obesity occurrence in a child. Breast-feeding also makes a substantial contribution into development of an infant since breast milk is balanced food that provides optimal metabolism in an infant’s body and helps creating proper gut microbiota. At the same time, according to data obtained via numerous research works, artificial feeding can be related to obesity occurrence in future. Ways to fight obesity include medication therapy, dietary nutrition, physical activity as well as bariatric surgery; the latter is nowadays considered to be the most efficient procedure on the matter. Reduction in body mass via influencing gut microbiota is a promising trend in research in the sphere. Despite there are objective data on benign effects produced by probiotics and prebiotics on gut microbiota, experts haven’t been able to reach agreement on their efficiency yet.

Gut microbiota as risk factor causing obesity in children

Nowadays obesity resulting from abnormal or excessive fat deposits in a body has become a true epidemic. Risk factors that cause the disease include improper lifestyle, hereditary predisposition, as well as metabolic activity of gut microbiota. Research works performed over the last decades indicate that microbes that create colonies in human intestines play a significant role in maintaining proper metabolism. There is a correlation between disorders in gut microbiota structure and immune disorders, elevated susceptibility to infections, and obesity. There is more and more evidence that gut microbiota and its overall bacterial genome exert their influence on nutrients assimilation and regulate energy metabolism and fat accumulation. Certain differences were detected in microbiota gut structure in children and adults with obesity and people with proper body mass index. Delivery and feeding are among key factors influencing gut microbiota formation in a child. Thus, research results indicate that natural birth, as opposed to cesarean section, can prevent obesity occurrence in a child. Breast-feeding also makes a substantial contribution into development of an infant since breast milk is balanced food that provides optimal metabolism in an infant’s body and helps creating proper gut microbiota. At the same time, according to data obtained via numerous research works, artificial feeding can be related to obesity occurrence in future. Ways to fight obesity include medication therapy, dietary nutrition, physical activity as well as bariatric surgery; the latter is nowadays considered to be the most efficient procedure on the matter. Reduction in body mass via influencing gut microbiota is a promising trend in research in the sphere. Despite there are objective data on benign effects produced by probiotics and prebiotics on gut microbiota, experts haven’t been able to reach agreement on their efficiency yet.

Metabolic activity of gut microbiota in children during their first year of life which had undergone partial bowel resection

Background. Studying the metabolic activity of gut microbiota has diagnostic value as it allows determination of the extent of microbiota perturbation, which is an important indicator of the post-operative gastrointestinal tract function.Objective. The study aimed at investigating the metabolic activity of gut microbiota in children during their first year of life which had undergone partial bowel resection.Methods. The spectrum of fecal short chain fatty acids (SCFA) was assessed using gas-liquid chromatography (GLC). Also, a comparative analysis of clinical and anamnestic data in children of the first year of life which had undergone partial bowel resection versus apparently healthy children was carried out.Results. Dynamic follow-up of children which had undergone bowel surgery revealed a reduction in acetic acid (С2) fecal level and increased propionic acid (С3) and oleic acid (С4) levels by the end of the first year of life. An age-adjusted comparative estimate has shown that the markers of anaerobic microflora, specifically, the level of propionic acid, are also high at the age of 7–12 months. A comparative analysis of metabolic activity of gut microbiota in children which had undergone bowel resection revealed lower fecal levels of oleic acid and higher total fatty acid content as compared to healthy children.Conclusion. Children which had undergone bowel resection, by the end of first year of life, have higher anaerobic and lower aerobic activity of gut microflora. A comparative analysis of metabolic activity of gut microflora in children which had undergone surgical treatment versus children of health groups I and II demonstrated the absence of significant differences between obligate (С2) and anaerobic (С3, anaerobic index [AI]) bacteria. However, lower fecal С4 values have been found in post-operative children during their second half-year of life, which may attest to persistent inflammation of the gut. Total fecal acid content throughout the entire follow-up period was higher in children which had undergone bowel resection, which reflects the activation of gut microbiota characterized by its species diversity.

Физическое развитие и характеристика метаболической активности микробиоты у детей первого года жизни, перенесших резекцию кишечника

Aim: to assess the rate of growth and metabolic activity of gut microbiota in infants who experienced bowel resection within the first year of life and received specialized nutrition with extensive hydrolyzed whey proteins with medium-chain triglycerides and nucleotides. Patients and Methods: prospective cohort study was conducted in two groups of infants. The study group included 37 infants with partial bowel resection within the first months of life. The control group included 75 healthy infants. Postoperatively, study group children were bottle-fed and received an infant formula (extensive hydrolized whey proteins with medium-chain triglycerides) till the age of 12 months. Some control group children were breast-fed while control group children on a mixed or formula feeding. In all children, antenatal and perinatal anamnesis and physical development within the first year of life were assessed. Additionally, the amounts of short-chain fatty acids in the stool were evaluated. Results: the rate of growth in infants who experienced bowel surgery was similar to that of healthy infants within the first year of life. By the age of 12 months, steady increase in the proportion of infants with average weight (SD -1 to +1) and significant reduction in the proportion of infants with low weight (SD -1 to -2) were reported. A group of overweight infants (SD +1 to +2) emerged since the age of 6 months. The specifics of metabolic activity of gut microbiota in children after surgery were a significant increase in propionic acid amount in the stool and a trend to an increase in anaerobic index by the age of 12 months. Conclusions: this growth pattern illustrates an adequate nutrition during the recovery of bowel functional activity and its ability to absorb and to uptake nutrients. KEYWORDS: infants, specialized nutrition, physical development rate, bowel metabolic activity. FOR CITATION: Vakhlova I.V., Fedotova G.V., Boronina L.G., Samatova E.V. Physical development and of gut microbiota in infants who experienced bowel resection within the first year of life. Russian Journal of Woman and Child Health. 2021;4(2):155–161. DOI: 10.32364/2618- 8430-2021-4-2-155-161.

Effect of Diet on the Gut Microbiota Associated with Obesity.

Obesity is abnormal or excessive fat accumulation that is associated with progression of metabolic diseases including type 2 diabetes mellitus, cardiovascular disease, nonalcoholic fatty liver disease, and cancer. Gut microbiota (GM) have received much attention as essential factors in development and progression of obesity. The diversity, composition, and metabolic activity of GM are closely associated with nutrient intake and dietary pattern. Scientific evidence supports the idea that dietary pattern directly changes the GM profile; therefore, diet is a crucial component related to interactions between GM and obesity progression. A literature review showed that dietary factors such as probiotics, prebiotics, fat, fatty acids, and fiber dramatically alter the GM profile related to obesity. Furthermore, different dietary patterns result in different GM composition and activity that can contribute to amelioration of obesity.

Epigenetic Regulation at the Interplay Between Gut Microbiota and Host Metabolism.

Gut microbiota communities have coevolved for millions of years in a symbiotic relationship with their mammalian hosts. Elucidating and understanding the molecular mechanisms by which microbiota interacts with its host and how this contributes to the homeostasis of the host is crucial. One of these molecular relationships is the so-called chemical crosstalk between microbiota and host metabolisms, including the poorly explored epigenetic regulation of host tissues by the metabolic activity of gut microbiota in response to changes in diet. DNA methylation and histone modifications are epigenetic marks partly regulated by enzymes such as methylases and acetylases, whose activity depend on host and microbiota metabolites that act as substrates and cofactors for these reactions. However, providing a complete mechanistic description of the regulatory interactions between both metabolisms and the impact on the expression of host genes through an epigenetic modulation, remains elusive. This article presents our perspective on how metabolomic, metagenomic, transcriptomic, and epigenomic data can be used to investigate the “microbiota–nutrient metabolism–epigenetics axis.” We also discuss the implications and opportunities this knowledge may have for basic and applied science, such as the impact on the way we structure future research, understand, and prevent diseases like type 2 diabetes or obesity.

A Road Less Traveled: Trafficking Hydrogen Sulfide and B12

Similar in their very low intracellular abundance, the metabolism of hydrogen sulfide (H2S) and vitamin B12 intersect at the sulfur mobility in PPD network where B12 serves as a cofactor, and H2S spews from enzymes with relaxed specificity. While only two mammalian enzymes require B12 for activity, cells maintain a regiment of chaperones to tailor and escort B12 from its point of entry into the cell to its targets. During this inter‐compartmental voyage, B12 is translocated between active sites in transient protein‐protein complexes, which exhibit the artful use of coordination chemistry. Structural enzymology studies on trafficking proteins are providing molecular insights into the cellular strategies for moving this portly organometallic cofactor. While the metabolic activity of gut microbiota represents a limited source of B12, it is a substantial source of H2S and necessitates adaptations by host cells to routine exposure to this respiratory toxin. Our studies are revealing the metabolic reprogramming that is part of the stress response to H2S in colon epithelial cells. Thus, although low in abundance, B12 and H2S have profound impacts on cellular biochemistry.Support or Funding InformationNational Institutes of Health (DK45776, HL58984, GM112455)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lactobacillus reuteri improves gut barrier function and affects diurnal variation of the gut microbiota in mice fed a high-fat diet.

Lactobacillus reuteri FN041 is a secretory IgA-targeted Lactobacillus strain from human breast milk that has probiotic potential. The aim of this study was to test whether FN041 can alleviate dyslipidaemia and mucosal-barrier damage caused by a high-fat diet (HFD) and whether it can affect diurnal variation of the intestinal microbiota. C57BL/6 mice were fed either a normal chow diet or high-fat diet (HFD) for 7 weeks and were treated with either PBS as a control or L. reuteri FN041 for 4 weeks. Our results showed that FN041 treatment significantly attenuated HFD-induced weight gain (P < 0.01), accumulation of testicular fat, an increase in locomotor activity during the active phase (P < 0.01), triglyceridaemia, hypercholesterolaemia (P < 0.05), liver Fas overexpression, and Srebp1c mRNA expression inhibition. Moreover, FN041 treatment improved intestinal epithelial barrier function and induced a daily oscillation-dependent change in short-chain fatty acid production by the gut microbiota. A deeper understanding of the molecular pathways participating in intestinal barrier and microbiota modifications, and changes to lipid metabolism under the influence of FN041, will have important implications by potentially opening new horizons for the development of relevant foods to prevent metabolic disorders and unrelated intestinal diseases.

A Probe-Enabled Approach for the Selective Isolation and Characterization of Functionally Active Subpopulations in the Gut Microbiome

Commensal microorganisms in the mammalian gut play important roles in host health and physiology, but a central challenge remains in achieving a detailed mechanistic understanding of specific microbial contributions to host biochemistry. New function-based approaches are needed that analyze gut microbial function at the molecular level by coupling detection and measurements of in situ biochemical activity with identification of the responsible microbes and enzymes. We developed a platform employing β-glucuronidase selective activity-based probes to detect, isolate, and identify microbial subpopulations in the gut responsible for this xenobiotic metabolism. We find that metabolic activity of gut microbiota can be plastic and that between individuals and during perturbation, phylogenetically disparate populations can provide β-glucuronidase activity. Our work links biochemical activity with molecular-scale resolution without relying on genomic inference.

A critical review of the role of milk and other dairy products in the development of obesity in children and adolescents.

Existing reviews suggest that milk and other dairy products do not play a role in the development of obesity in childhood, but they do make an important contribution to children's nutrient intake. It is thus curious that public health advice on the consumption of dairy products for children is often perceived as unclear. The present review aimed to provide an overview of the totality of the evidence on the association between milk and other dairy products, and obesity and indicators of adiposity, in children. Our search identified forty-three cross-sectional studies, thirty-one longitudinal cohort studies and twenty randomised controlled trials. We found that milk and other dairy products are consistently found to be not associated, or inversely associated, with obesity and indicators of adiposity in children. Adjustment for energy intake tended to change inverse associations to neutral. Also, we found little evidence to suggest that the relationship varied by type of milk or dairy product, or age of the children, although there was a dearth of evidence for young children. Only nine of the ninety-four studies found a positive association between milk and other dairy products and body fatness. There may be some plausible mechanisms underlying the effect of milk and other dairy products on adiposity that influence energy and fat balance, possibly through fat absorption, appetite or metabolic activity of gut microbiota. In conclusion, there is little evidence to support a concern to limit the consumption of milk and other dairy products for children on the grounds that they may promote obesity.

Metabolic adaptation of colonic microbiota to galactooligosaccharides: a proof-of-concept-study.

Prebiotics have been shown to reduce abdominal symptoms in patients with functional gut disorders, despite that they are fermented by colonic bacteria and may induce gas-related symptoms. To investigate changes in the metabolic activity of gut microbiota induced by a recognised prebiotic. Healthy subjects (n = 20) were given a prebiotic (2.8 g/day HOST-G904, HOST Therabiomics, Jersey, Channel Islands) for 3 weeks. During 3-day periods immediately before, at the beginning and at the end of the administration subjects were put on a standard diet (low fibre diet supplemented with one portion of high fibre foods) and the following outcomes were measured: (i) number of daytime gas evacuations for 2 days by means of an event marker; (ii) volume of gas evacuated via a rectal tube during 4 h after a test meal; and (iii) microbiota composition by faecal Illumina MiSeq sequencing. At the beginning of administration, HOST-G904 significantly increased the number of daily anal gas evacuations (18 ± 2 vs. 12 ± 1 pre-administration; P < 0.001) and the volume of gas evacuated after the test meal (236 ± 23 mL vs. 160 ± 17 mL pre-administration; P = 0.006). However, after 3 weeks of administration, these effects diminished (11 ± 2 daily evacuations, 169 ± 23 mL gas evacuation). At day 21, relative abundance of butyrate producers (Lachnospiraceae) correlated inversely with the volume of gas evacuated (r = -0.52; P = 0.02). The availability of substrates induces an adaptation of the colonic microbiota activity in bacterial metabolism, which produces less gas and associated issues. Clinical trials.gov NCT02618239.

Reduced metabolic activity of gut microbiota by antibiotics can potentiate the antithrombotic effect of aspirin

In this study, we investigated the effects of antibiotics on the pharmacological effects of aspirin. The antithrombotic activity of aspirin was evaluated after antibiotic treatment using tail bleeding assay. The pyrosequencing analysis and selective medium culture assay were performed to investigate the alterations in gut microbiota. In addition, the in vitro metabolism assay with fecal suspension and in vivo pharmacokinetic experiments with antibiotic treatment were conducted. Ampicillin treatment significantly prolonged the bleeding time in aspirin-dosed rats. Oral administration of ampicillin significantly reduced gut microbial aspirin-metabolizing activity by 67.0% in rats. Furthermore, systemic exposure to aspirin and its primary metabolite (M1) was significantly increased in ampicillin-treated rats. The results from the pyrosequencing and selective medium culture with rat fecal samples revealed that ampicillin treatment led to the changes of the amounts and composition profile of gut microbiota. These findings suggest that co-administration of antibiotics can modulate the metabolism and pharmacokinetics of aspirin via suppression of metabolic activity of gut microbiota, which could potentiate the therapeutic potency of aspirin.

Dietary Casein and Soy Protein Isolate Modulate the Effects of Raffinose and Fructooligosaccharides on the Composition and Fermentation of Gut Microbiota in Rats.

Although diet has an important influence on the composition of gut microbiota, the impact of dietary protein sources has only been studied to a minor extent. In this study, we examined the influence of different dietary protein sources regarding the effects of prebiotic oligosaccharides on the composition and metabolic activity of gut microbiota. Thirty female rats were fed casein and soy protein isolate with cellulose, raffinose (RAF), and fructooligosaccharides (FOS). Microbiota composition was examined by real-time qPCR and denaturing gradient gel electrophoresis. Dietary protein source affected cecum microbiota; acetic acid concentration and Lactobacillus spp. populations were greater with soy protein than with casein. Prebiotic oligosaccharides had distinctive effects on gut microbiota; RAF increased the acetic acid concentration and Bifidobacterium spp. populations, and FOS increased the butyric acid concentration regardless of the dietary protein. Likewise, Bifidobacterium sp., Collinsella sp., and Lactobacillus sp. were detected in microbiota of the rats fed RAF, and Bacteroides sp., Roseburia sp., and Blautia sp. were seen in microbiota of the rats fed FOS. Interactions between dietary proteins and prebiotic oligosaccharides were observed with Clostridium perfringens group populations and cecum IgA concentration. RAF and FOS decreased C. perfringens group populations in casein-fed rats, and the combination of soy protein and RAF substantially increased cecum IgA concentration. These results indicate that dietary proteins can differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, depending on the type of carbohydrate polymers involved.

Modulating Composition and Metabolic Activity of the Gut Microbiota in IBD Patients.

The healthy intestine represents a remarkable interface where sterile host tissues come in contact with gut microbiota, in a balanced state of homeostasis. The imbalance of gut homeostasis is associated with the onset of many severe pathological conditions, such as inflammatory bowel disease (IBD), a chronic gastrointestinal disorder increasing in incidence and severely influencing affected individuals. Despite the recent development of next generation sequencing and bioinformatics, the current scientific knowledge of specific triggers and diagnostic markers to improve interventional approaches in IBD is still scarce. In this review we present and discuss currently available and emerging therapeutic options in modulating composition and metabolic activity of gut microbiota in patients affected by IBD. Therapeutic approaches at the microbiota level, such as dietary interventions alone or with probiotics, prebiotics and synbiotics, administration of antibiotics, performing fecal microbiota transplantation (FMT) and the use of nematodes, all represent a promising opportunities towards establishing and maintaining of well-being as well as improving underlying IBD symptoms.

Metabolic activity of gut microbiota and xenobiotics

The intestine habitat is the natural collection of symbiotic microorganisms. The bacterial population enables many permanent metabolic activities in this environment. Inside the intestine of mammals there are an extended genome of millions of bacterial genes named microbiome. In recent years, there has been an increased interest of scientists to discover the place and the role of bio-ecological content and modulation of gut microbiota in a host organism using prebiotics, probiotics and synbiotics, which may have a great benefit for human health.

Repeated cooking and freezing of whole wheat flour increases resistant starch with beneficial impacts on in vitro fecal fermentation properties

Resistant starch (RS) has shown benefits to gastrointestinal health, but it is present in only small amounts in most grain-based foods. The purpose of this study was to increase RS in whole wheat flour to improve its potential health benefits. Zero to 7 cycles of cooking (20 min, boiling water) and freezing (−18 °C, 23 h) of whole wheat flour in water (1:15 %w/v) were performed. Increasing cooking–freezing cycles increased RS from 1.03 to 8.07% during in vitro starch digestion. During in vitro fecal fermentation, increasing cooking–freezing cycles increased short chain fatty acids, mainly propionate. Increases in butyrate were also noted during the first 8 h of fermentation. All flours resulted in significant increases in Bifidobacterium of >0.5 log during fermentation compared to baseline. Thus, even modest increases in the RS content of whole wheat flour modulated the metabolic activity of gut microbiota to increase production of beneficial metabolites.