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Results from high altitude studies in humans and controlled animal experiments suggest that hypoxia exposure induces alterations in gut microbiota composition, which may in turn affect host metabolism. However, well-controlled studies investigating the effects of normobaric hypoxia exposure on gut microbiota composition in humans are lacking. The aim of this study was to explore the impact of mild intermittent hypoxia (MIH) exposure on gut microbiota composition in men with overweight and/or obesity. We performed a randomised, single-blind crossover study, in which participants were exposed to MIH (FiO2: 15%, 3×2 h per day) and normoxia (FiO2: 21%) for seven consecutive days. Following the MIH and normoxia exposure regimens, faecal samples were collected for determination of faecal microbiota composition using 16S rRNA gene-amplicon sequencing in the morning of day 8. Paired faecal samples were available for five individuals. Furthermore, tissue-specific insulin sensitivity was determined using the gold-standard two-step hyperinsulinemic-euglycemic clamp. MIH did not affect microbial alpha and beta-diversity but reduced the relative abundance of Christensenellaceae and Clostridiaceae bacterial families. MIH significantly increased the abundances of obligate anaerobic bacterial genera including Fusicatenibacter, Butyricicoccus and Holdemania, whilst reducing Christensenellaceae R-7 group and Clostridium sensu stricto 1, although these findings were not statistically significant after correction for multiple testing. Furthermore, MIH-induced alterations in abundances of several genera were associated with changes in metabolic parameters such as adipose and peripheral insulin sensitivity, plasma levels of insulin, fatty acids, triacylglycerol and lactate, and substrate oxidation. In conclusion, we demonstrate for the first time that MIH exposure induces modest effects on faecal microbiota composition in humans, shifting several bacterial families and genera towards higher abundances of anaerobic butyrate-producing bacteria. Moreover, MIH-induced effects on faecal microbial composition were associated with parameters related to glucose and lipid homeostasis, supporting a link between MIH-induced alterations in faecal microbiota composition and host metabolism. The study was registered at the Netherlands Trial Register: NL7120/NTR7325.

Background: Methods and approaches that can be used in food and nutrition research are changing at a faster pace than ever. Whereas animal methods are mostly known for their use in food safety analysis (see Part I), they also play in important role in proof-of-concept and mechanistic studies of products, as well as studying potency, efficacy, and tolerance of foods and food ingredients. Members of the International Life Sciences Institute (ILSI) Europe have formed an expert group to review possibilities, opportunities, and challenges for the potential use of alternative testing strategies in nutrition research and regulatory requirements, supporting the 3Rs principle of Replacement, Reduction, Refinement of animal research, which can ultimately be used in support of regulatory submissions for pre-market authorisation. Scope and approach: For the different areas of food for specific groups and health claims, the acceptability of nonanimal approaches is evaluated in comparison to legislative requirements in Europe. The alternative approaches considered cover emerging tools and methodologies such as organoids, organs-on-a-chip or human in vitro gastrointestinal simulators. Conclusions: In nutrition research, there has been a long tradition of following a certain experimental trajectory for grounding scientific hypotheses starting from in vitro data moving on to in vivo verification in a preferred animal model and finally proving this in a human setting. From a regulatory perspective there is no specific requirement for animal experimentation that justifies the use of the majority of animal experiments in the assessment of nutritional content and value of food products. However, animal data are mostly considered as the standard, and guidance for alternative approaches that would be accepted is lacking. It is therefore important to further build evidence and offer validation for the adequacy of already existing in vitro tools to ensure their suitability for substantiating dose levels and further planning clinical trials. What are we waiting for?

Antibiotic exposure disturbs the developing infant gut microbiota. The capacity of the gut microbiota to recover from this disturbance (resilience) depends on the type of antibiotic. In this study, infant gut microbiota was exposed to a combination of amoxicillin and clavulanate (amoxicillin/clavulanate) in an in vitro colon model (TIM-2) with fecal-derived microbiota from 1-month-old (1-M; a mixed-taxa community type) as well as 3-month-old (3-M; Bifidobacterium dominated community type) breastfed infants. We investigated the effect of two common infant prebiotics, 2′-fucosyllactose (2’-FL) or galacto-oligosaccharides (GOS), on the resilience of infant gut microbiota to amoxicillin/clavulanate-induced changes in microbiota composition and activity. Amoxicillin/clavulanate treatment decreased alpha diversity and induced a temporary shift of microbiota to a community dominated by enterobacteria. Moreover, antibiotic treatment increased succinate and lactate in both 1- and 3-M colon models, while decreasing the production of short-chain (SCFA) and branched-chain fatty acids (BFCA). The prebiotic effect on the microbiota recovery depended on the fermenting capacity of antibiotic-exposed microbiota. In the 1-M colon model, the supplementation of 2’-FL supported the recovery of microbiota and restored the production of propionate and butyrate. In the 3-M colon model, GOS supplementation supported the recovery of microbiota and increased the production of acetate and butyrate.

The rise of antibiotic resistance poses a global problem. To avoid this, alternative therapeutic options should be explored, e.g. lytic bacteriophage therapy. Well-designed and described research on effectivity of oral bacte-riophage therapy is lacking, therefore the aim of this study was to determine whether the in vitro model of the colon (TIM-2) could be used to investigate the survival and efficacy of therapeutic bacteriophages. For this, an antibiotic-resistant (CmR) E. coli DH5 alpha(pGK11) was used in combination with a corresponding bacteriophage. For the survival study, the TIM-2 model was inoculated with the microbiota of healthy individuals and a standard feeding (SIEM) was fed over the course of the 72 h experiment. To test the bacteriophage, different interventions were carried out. Survival of bacteriophages and bacteria was followed by plating of the lumen samples at different time points 0, 2, 4, 8, 24, 48, and 72 h. In addition, the stability of the bacterial community was determined with the use of 16S rRNA sequencing. Results showed that the phage titers could be decreased by activity from the commensal microbiota. Levels of the phage host (here E.coli) were decreased in the in-terventions with the phage shot. Multiple shots did not seem to be more effective than a single shot. At the same time, the bacterial community was not disturbed and remained stable throughout the experiment, which is in stark contrast to treatment with antibiotics. Mechanistic studies such as this one are required to optimize efficacy of phage therapy.

Background Gut bacteria-derived short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA) are considered to have beneficial metabolic, anti-inflammatory as well as anti-carcinogenic effects. Previous preclinical studies indicated bidirectional interactions between gut bacteria and the chemotherapeutic capecitabine or its metabolite 5-FU. This study investigated the effect of three cycles of capecitabine on fecal SCFA and BCFA levels and their associations with tumor response, nutritional status, physical performance, chemotherapy-induced toxicity, systemic inflammation and bacterial abundances in patients with colorectal cancer (CRC). Methods Forty-four patients with metastatic or unresectable CRC, scheduled for treatment with capecitabine (± bevacizumab), were prospectively enrolled. Patients collected a fecal sample and completed a questionnaire before (T1), during (T2) and after (T3) three cycles of capecitabine. Tumor response (CT/MRI scans), nutritional status (MUST score), physical performance (Karnofsky Performance Score) and chemotherapy-induced toxicity (CTCAE) were recorded. Additional data on clinical characteristics, treatment regimen, medical history and blood inflammatory parameters were collected. Fecal SCFA and BCFA concentrations were determined by gas chromatography–mass spectrometry (GC–MS). Gut microbiota composition was assessed using 16S rRNA amplicon sequencing. Results Fecal levels of the SCFA valerate and caproate decreased significantly during three cycles of capecitabine. Furthermore, baseline levels of the BCFA iso-butyrate were associated with tumor response. Nutritional status, physical performance and chemotherapy-induced toxicity were not significantly associated with SCFA or BCFA. Baseline SCFA correlated positively with blood neutrophil counts. At all time points, we identified associations between SCFA and BCFA and the relative abundance of bacterial taxa on family level. Conclusions The present study provided first indications for a potential role of SCFA and BCFA during capecitabine treatment as well as implications for further research. Trial registration The current study was registered in the Dutch Trial Register (NTR6957) on 17/01/2018 and can be consulted via the International Clinical Trial Registry Platform (ICTRP).

ScopeWestern type of diets are characterized by high animal protein intake and are associated with various chronic inflammatory diseases. With a higher protein consumption, excess undigested protein will reach the colon and be subsequently metabolized by gut microbiota. Depending on the type of protein, fermentation in the colon generates different metabolites with varying biological effects. This study aims to compare the impact of protein fermentation products from different sources on gut health. Methods and resultsThree high protein diets (vital wheat gluten [VWG], lentil, or casein) are submitted to the in vitro model of colon. Fermentation of excess lentil protein for 72 h results in highest production of short-chain fatty acids and lowest production of branched-chain fatty acids. Exposure of Caco-2 monolayers or Caco-2 monolayers co-cultured with THP-1 macrophages to luminal extracts of fermented lentil protein results in less cytotoxicity of Caco-2 monolayers and less damage to barrier integrity, when compared to VWG and casein. Lowest induction of interleukin-6 is observed in THP-1 macrophages after treatment with lentil luminal extracts, which is identified to be regulated by aryl hydrocarbon receptor signaling. ConclusionThe findings indicate that protein sources affect the health effects of high protein diet in the gut.

Buttermilk (BM), the by-product of butter making, is similar to skim milk (SM) composition. However, it is currently undervalued in dairy processing because it is responsible for texture defects (e.g., crumbliness, decreased firmness) in cheese and yogurt. One possible way of improving the incorporation of BM into dairy products is by the use of technological pretreatments such as membrane filtration and homogenization. The study aimed at characterizing the effect of preconcentration by reverse osmosis (RO) and single-pass ultra-high-pressure homogenization (UHPH) on the composition and microstructure of sweet BM to modify its techno-functional properties (e.g., protein gel formation, syneresis, firmness). The BM and RO BM were treated at 0, 15, 150, and 300 MPa. Pressure-treated and control BM and RO BM were ultracentrifuged to fractionate them into the following 3 fractions: a supernatant soluble fraction (top layer), a colloidal fraction consisting of a cloudy layer (middle layer), and a high-density pellet (bottom layer). Compositional changes in the soluble fraction [lipid, phospholipid (PL), protein, and salt], as well as its protein profile by PAGE analysis, were determined. Modifications in particle size distribution upon UHPH were monitored by laser diffraction in the presence and absence of sodium citrate to dissociate the casein (CN) micelles. Microstructural changes in pressure-treated and non-pressure-treated BM and RO BM particles were monitored by confocal laser scanning microscopy. Particle size analysis showed that UHPH treatment significantly decreased the size of the milk fat globule membrane fragments in BM and RO BM. Also, pressure treatment at 300 MPa led to a significant increase in the recovery of total lipids, CN, calcium, and phosphate in the BM soluble fraction (top layer) following ultracentrifugation. However, PL were primarily concentrated in the pellet cloud (middle layer), located above the pellet in BM concentrated by RO. In contrast, PL were evenly distributed between soluble and colloidal phases of BM. This study provides insight into the modifications of sweet BM constituents induced by RO and UHPH from a compositional and structural perspective.

BACKGROUND: Despite the great range of health-beneficial activities associated with dietary polyphenols, their influence on gut ecology remains poorly understood. Only a few studies have examined the impact of black chokeberry polyphenols present in different matrices on human gut microbiota, and in fact none have examined encapsulated black chokeberry polyphenols. OBJECTIVE: The objective of this study was to evaluate the effect of black chokeberry polyphenols in pulp, extract and encapsulate (in a maltodextrin:gum Arabic polymer system) on human gut microbiota and fecal short-chain and branched-chain fatty acids (SCFA and BCFA, respectively). METHODS: The effect of black chokeberry polyphenols on gut microbiota was tested in a validated, dynamic in vitro model of the colon (TIM-2) for 24 h by applying five different interventions (Pulp, Extract, Encapsulate, Encapsulate control, SIEM) to the standardized microbiota from five healthy donors. RESULTS: We observed that the fermentation of black chokeberry polyphenols in the in vitro colon model resulted in shifts in the standardized microbiota and differentiation in the extent of the production of SCFA and BCFAs. Synergy between maltodextrin+gum Arabic+polyphenols resulted in an increase in the relative abundances of some health-promoting taxa and decrease in the disease related taxa Alistipes. Encapsulation increased the SCFA production and decreased the BCFA production in the lumen. CONCLUSIONS: Although encapsulation of polyphenols may provide a robust way for their protection during their transit along the upper gastrointestinal tract, their effect on the gut microbiota should be further investigated both by using different coating materials and with in vivo studies.

Most studies of the microbiota in the human gut focus on the bacterial part, but increasing information shows that intestinal fungi are also important for maintaining health. This can be either by directly influencing the host or by indirectly influencing the gut bacteria that link to host health. Studies of fungal communities in large cohorts are scarce; therefore, this study aims at obtaining more insight into the mycobiome of healthy individuals and how this mycobiome interacts with the bacterial component of the microbiome. For this purpose, ITS2 and 16S rRNA gene amplicon sequencing was performed on fecal samples from 163 individuals which were available from two separate studies to analyze the fungal and bacterial microbiome, respectively, as well as the cross-kingdom interactions. The results showed a much lower fungal, as compared to bacterial, diversity. Ascomycota and Basidiomycota were the dominant fungal phyla across all the samples, but levels varied enormously between individuals. The ten most abundant fungal genera were Saccharomyces, Candida, Dipodascus, Aureobasidium, Penicillium, Hanseniaspora, Agaricus, Debaryomyces, Aspergillus, and Pichia, and here also extensive inter-individual variation was observed. Correlations were made between bacteria and fungi, and only positive correlations were observed. One of the correlations was between Malassezia restricta and the genus Bacteroides, which have both been previously described as alleviated in IBD. Most of the other correlations found were with fungi that are not known as gut colonizers but originate from food and the environment. To further investigate the importance of the observed correlations found, more research is needed to discriminate between gut colonizers and transient species.

Aqueous biphasic systems (ABSs) based on sodium polyacrylate (NaPA), ethylene oxide/propylene oxide (EO/PO) polymers, and (EO)x-(PO)y-(EO)x triblock copolymers were prepared and applied aiming at continuous fructooligosaccharide (FOS) production and separation. EO/PO hydrophilicity/hydrophobicity balance had a significant effect on ABS formation. To develop an integrated process including the continuous enzymatic (levansucrase) production of FOSs and their purification while improving the production yield by further glucose separation, the potential of these novel polymer-based ABSs as alternative platforms was investigated. They were used for the partitioning of different carbohydrates (FOS, sucrose, d-fructose, and d-glucose) and levansucrase. Results revealed a highly polymer-dependent partition of carbohydrates and a poorly dependent one of the enzymes. Changing EO/PO and copolymers, FOS was purified with high yields (72.94-100.0%). Using polypropylene glycol 400 + NaPA 8000-based ABS, the FOS was precipitated in the interphase and separated from the other components. Pluronic PE-6800 + NaPA 8000 was identified as the best ABS for FOS continuous production and in situ purification, while minimizing levansucrase inhibition by d-glucose. This system allowed selective partition of FOSs and d-glucose toward the top phase and that of levansucrase and its substrates toward the bottom one. COnductor-like Screening MOdel for Real Solvent (COSMO-RS) suggested that ABS formation may have been due to NaPA and polymer/copolymer competition to form hydrogen bonds with water molecules. Moreover, the partition of FOSs and sugar may have been the result of a subtle balance between hydrogen bonding of sugar and polymer/copolymer and electrostatic misfit of solute with NaPA. Finally, two integrated processes were proposed to be applied with real FOS extracts obtained by chemical or enzymatic hydrolysis of inulin or by transfructosylation of concentrated sucrose solutions using bacterial levansucrases. published

BackgroundShort chain fatty acids (SCFA) are increasingly recognized for their potential ability to alleviate obesity-associated chronic low-grade inflammation and disturbed energy homeostasis. Evidence suggests that an increase in circulating SCFA might be necessary to induce beneficial alterations in energy metabolism.ObjectiveTo compare the bioaccessibility of two different SCFA-enriched triglycerides: Akovita SCT (butyrate and hexanoate esterified with long chain fatty acids) and tributyrin/caproin (solely butyrate and hexanoate) and investigate whether the SCFA from orally administrated Akovita SCT reach the circulation and affect postprandial metabolism in men with overweight/obesity.MethodsThe site, speed, and amount of SCFA release from Akovita SCT and tributyrin/caproin were assessed in a validated In vitro Model of the stomach and small intestine (TIM-1). Subsequently, a double-blind placebo-controlled randomized crossover study was conducted at Maastricht University with fourteen men with overweight/obesity (BMI 25–35 kg/m2) of which twelve men finished all testdays and were included for analysis. The participants received a liquid high fat mixed meal test containing either a low (650 mg), medium (1,325 mg), or high dose (2,000 mg) of Akovita SCT or a placebo (sunflower oil) in randomized order. Blood was sampled at baseline and after ingestion for 6 h for the primary outcome plasma butyrate and hexanoate concentration. Secondary outcomes included hydrogen breath, appetite, gastrointestinal complaints, circulating glucagon-like peptide 1, free fatty acids, glucose, triglycerides, insulin, and cytokines concentrations.ResultsIn TIM-1, tributyrin/caproin was rapidly cleaved in the gastric compartment whereas the release of SCFA from Akovita SCT occurred predominantly in the small intestine. In vivo, all doses were well-tolerated. The medium dose increased (P < 0.05) and the high dose tended to increase (P < 0.10) postprandial circulating butyrate and both doses increased circulating hexanoate (P < 0.05) compared to placebo. Nevertheless, Akovita SCT supplementation did not affect any secondary outcomes compared to placebo.ConclusionEsterifying SCFA-enriched triglycerides with long chain fatty acids delayed SCFA release from the glycerol backbone. Akovita SCT increased postprandial circulating butyrate and hexanoate without changing metabolic parameters in men with overweight/obesity. Future randomized clinical trials should investigate whether long-term Akovita SCT supplementation can aid in the treatment or prevention of metabolic disorders.Clinical trial registrationwww.ClinicalTrials.gov, identifier: NCT04662411.

The prevalence of intestinal parasitic infection remains high in developing countries, especially because of geographic and socio-demographic factors. This study aimed to evaluate intestinal parasitic infection, as well as its risk factors, among children aged 36–45 months in a rural area (North Kodi) and an urban area (Kupang) of East Nusa Tenggara, Indonesia. Anthropometry, socio-demographic factors and personal hygiene practices were assessed. A total of 214 children participated in the study, and 200 stool samples were collected for intestinal parasite examination. Approximately 30.5% (61/200) of the children were infected with one or more intestinal parasites (67.2%; 41/61 being mono-parasitic infections and 32.8%; 20/61 being poly-parasitic infections). A total of 85 intestinal parasites were detected, consisting of 35.3% (30/85) protozoa and 64.7% (55/85) helminths. The predominant protozoa were Giardia lamblia (43%; 13/30) and Blastocystis spp. (33.3%; 10/30), whereas the predominant helminths were Trichuris trichiura (50.9%; 28/55) and Ascaris lumbricoides (43.6%; 24/55). Moreover, intestinal parasitic infection was associated with rural area (OR 4.5; 95%CI 2.3–8.6); the absence of treatment with deworming drugs (OR 2.56; 95%CI 1.3–5.0); sanitation facilities without a septic tank (OR 4.3; 95%CI 2.1–8.5); unclean water as a source of drinking water (OR 4.67; 95%CI 2.4–9.4); no handwashing practice after defecation (OR 3.2; 95%CI 1.4–7.3); and stunted children (OR 4.4; 95%CI 2.3–8.3). In conclusion, poly-parasitic infections were common in this study. Poor personal hygiene practice and sanitation factors contributed to the high prevalence of intestinal parasitic infection in 36–45-month-old children in East Nusa Tenggara, Indonesia.

This study's objective was to investigate the effects of dietary Se (in the form of selenomethionine) on the antioxidant activity and selenoprotein gene expressions in layer breeder roosters. One hundred and eighty, 36-wk-old Jingfen layer breeder roosters were randomly allocated to one of 5 dietary treatments (0, 0.25, 0.5, 1, or 2 mg/kg Se) for 6 wk on a corn-soybean meal-based diet. Antioxidant parameters and selenoprotein gene expressions were assessed at the end of the experiment. The results showed that Se supplementation significantly increased the activity of T-SOD, CAT, GSH-Px, and superoxide anion scavenging ability in plasma (P ≤ 0.05), and activities of T-SOD, CAT, GSH-Px, superoxide anion scavenging ability, and hydroxyl radical scavenging ability in the liver, kidney, and testis (P < 0.05). Moreover, MDA levels were significantly reduced in plasma, liver, kidney, and testis (P < 0.01), compared to the control group. Furthermore, the dietary administration of Se significantly increased TrxR2 and GPx4 mRNA levels in kidney and testis, and ID1 mRNA levels in liver and kidney. Most of the antioxidant parameters and selenoprotein-related gene expressions significantly increased, and MDA significantly decreased at dietary supplementation with 0.5 mg/kg Se. Whereas a higher dose of Se level (1 or 2 mg/kg) inhibited the activities of some of the antioxidant enzymes and selenoprotein-related gene expressions in selected tissues. In conclusion, dietary Se supplementation with 0.5 mg/kg significantly improved roosters' antioxidant status and selenoprotein-related gene expression in liver, kidney, and testis, while higher doses led to inhibit these; dietary Se might increase reproductive performance by enhancing their antioxidant status in roosters.

This clinical study explored the associations between the intestinal microbiota, chemotherapy toxicity, and treatment response in postmenopausal oestrogen receptor positive breast cancer patients.Oestrogen receptor positive postmenopausal breast cancer patients were prospectively enroled in a multicentre cohort study and treated with 4 cycles of (neo)adjuvant adriamycin, cyclophosphamide (AC) followed by 4 cycles of docetaxel (D). Patients collected a faecal sample and completed a questionnaire before treatment, during AC, during D, and after completing AC-D. Chemotherapy toxicity and tumour response were determined. Intestinal microbiota was analysed by amplicon sequencing of the 16 S rRNA V4 gene-region. In total, 44 patients, including 18 neoadjuvant patients, were included, and 153 faecal samples were collected before AC-D (n = 44), during AC (n = 43), during D (n = 29), and after AC-D treatment (n = 37), 28 participants provided all four samples. In the whole group, observed species richness reduced during treatment (p = 0.042). The abundance of Proteobacteria, unclassified Enterobacterales, Lactobacillus, Ruminococcaceae NK4A214 group, Marvinbryantia, Christensenellaceae R7 group, and Ruminococcaceae UCG-005 changed significantly over time. Patients with any grade diarrhoea during docetaxel treatment had a significantly lower observed species richness compared to patients without diarrhoea. In the small group neoadjuvant treated patients, pathologic response was unrelated to baseline intestinal microbiota richness, diversity and composition. While the baseline microbiota was not predictive for pathologic response in a rather small group of neoadjuvant treated patients in our study, subsequent shifts in microbial richness, as well as the abundance of specific bacterial taxa, were observed during AC-D treatment in the whole group and the neoadjuvant group.

The gut microbiota has been indicated to play a crucial role in health and disease. Apart from changes in composition between healthy individuals and those with a disease or disorder, it has become clear that also microbial activity is important for health. For instance, butyrate has been proven to be beneficial for health, because, amongst others, it is a substrate for the colonocytes, and modulates the host’s immune system and metabolism. Here, we studied the effect of a blend of three mushrooms (Ganoderma lucidum GL AM P-38, Grifola frondosa GF AM P36 and Pleurotus ostreatus PO AM-GP37)) on gut microbiota composition and activity in a validated, dynamic, computer-controlled in vitro model of the colon (TIM-2). Predigested mushroom blend at three doses (0.5, 1.0 and 1.5 g/day of ingested mushroom blend) was fed to a pooled microbiota of healthy adults for 72 h, and samples were taken every day for microbiota composition (sequencing of amplicons of the V3-V4 region of the 16S rRNA gene) and activity (short-chain fatty acid (SCFA) production). The butyrate producing genera Lachnospiraceae UCG-004, Lachnoclostridium, Ruminococcaceae UCG-002 and Ruminococcaceae NK4A214-group are all dose-dependently increased when the mushroom blend was fed. Entirely in line with the increase of these butyrate-producers, the cumulative amount of butyrate also dose-dependently increased, to roughly twice the amount compared to the control (medium without mushroom blend) on the high-dose mushroom blend. Butyrate proportionally made up 53.1% of the total SCFA upon feeding the high-dose mushroom blend, compared to 27% on the control medium. In conclusion, the (polysaccharides in the) mushroom blend led to substantial increase in butyrate by the gut microbiota. These results warrant future mechanistic research on the mushroom blend, as butyrate is considered to be one of the microbial metabolites that contributes to health, by increasing barrier function and modulating inflammation.

The aim of the current study was to investigate whether degradation of rapeseed meal (RSM) by a swine gut microbiota consortium was improved by modifying RSM by treatment with cellulase (CELL), two pectinases (PECT), or alkaline (ALK) compared to untreated RSM and to assess whether microbiota composition and activity changed. The predicted relative abundances of carbohydrate digestion and absorption, glycolysis, pentose phosphate pathway, and pyruvate metabolism were significantly increased upon CELL and ALK feeding, and CELL and ALK also exhibited increased total short-chain fatty acid (SCFA) production compared to CON. Megasphaera, Prevotella, and Desulfovibrio were significantly positively correlated with SCFA production. Findings were validated in ileal cannulated pigs, which showed that CELL and ALK increased fiber degradation of RSM. In conclusion, CELL and ALK rather than PECT1 or PECT2 increased fiber degradation in RSM, and this information could guide feed additive strategies to improve efficiency and productivity in the swine industry.

The aim of the research was to compare the survival of a blend of five probiotic strains (2 bifidobacteria and 3 lactobacilli) in a capsule within capsule (Duocap®) containing Ahiflower®oil, as compared to the strains in the powder (with or without Ahiflower oil), or the strains when present in the inner capsule only. This was tested in a validated, dynamic in vitro model of the stomach and small intestine (TIM-1), simulating human adults. Experiments were performed both in the gastric compartment of the model, as well as in the complete system (stomach + small intestine). Survival of the strains after transit through the gastric compartment in the Duocap capsule was higher by about a factor of 1.5 compared to the other 3 variables. In these gastric experiments, the Ahiflower oil did not seem to have an additional benefit, in the sense that it did not increase survival over the strains alone. After transit through the complete gastrointestinal tract survival was approximately 2-fold higher for the strains within the Duocap capsule, compared to the strains within the inner capsule or the powder. In these experiments, Ahiflower oil did have an additional benefit. The survival of the strains in the combination of powder with Ahiflower oil showed a similar survival as that of the Duocap, although in the first few hours of the experiments survival of both species lagged behind, and only caught up at the end of the test. In conclusion, the developed capsule-in-capsule technology increased the amount of viable cells in the upper gastrointestinal tract, mainly due to the presence of the polyunsaturated fatty acids contained in the outer capsule, which particularly protected the blend of probiotics in the small intestine.

High- and low-acetylated galactoglucomannooligosaccharides (GGMOS_Ac and GGMOS, respectively) were assayed as substrates in the TIM-2 in vitro colon model using, as inoculum, fecal microbiota from the elderly. The effects on the microbiota and their activity were also compared to a standard ileal efflux medium (SIEM). GGMOS resulted in higher organic acid productions and higher short-chain fatty acids/total organic acid molar ratios. Although comparable Actinobacteria abundances were observed with both substrates, GGMOS fermentation led to higher Firmicutes/Bacteroidetes ratios and lower Proteobacteria percentages than GGMOS_Ac. No differences were found concerning the percentages of beneficial genus such as Blautia, Faecalibacterium, Coprococcus, or Bifidobacterium. However, higher bacterial diversities and numbers of genera such as Oscillospira and Lachnospira were found with GGMOS_Ac. This suggests that GGMOS would be more suitable substrates for the elderly, even though GGMOS_Ac promoted positive effects that support the interest of further research using these oligosaccharides as "carriers" of desired substituents.

Intestinal organoids are advanced cellular models, which are widely used in mammalian studies to mimic and study in vivo intestinal function and host-pathogen interactions. Growth factors WNT3 and RSPO1 are crucial for the growth of intestinal organoids. Chicken intestinal organoids are currently cultured with mammalian WNT3a and RSPO1, however, maintaining their longevity has shown to be challenging. Based on the limited homology between mammalian and avian RSPO1, we expect that chicken-derived factors are required for the organoid cultures. We examined the effect of chicken-specific WNT3 and RSPO1 in combination with Prostaglandin E2 and a Forkhead box O1-inhibitor on the development of chicken intestinal organoids. Isolated crypts from embryonic tissue of laying hens were successfully cultured for 15 passages in the presence of chicken-derived WNT3 and RSPO1, whereas no growth was observed when mammalian factors were used. Furthermore, stem cells maintained their ability to differentiate into goblets, enterocytes, and enteroendocrine cells for the 3D and 2D structures. The chicken intestinal organoids can be an important tool to study responses of the intestinal epithelium to pathogens, drugs or feed additives.

Peptide hormones play a crucial role in body weight and glucose homeostasis. In this study, we aimed to explore this association and recruited 43 obese and 31 age- and sex-matched lean participants. We assessed their body mass index (BMI), waist circumference (WC), waist-to-height ratio (WtHR), percentage body fat (PBF), fasting blood levels of peptide hormones (GLP-1, GLP-2, insulin, leptin, ghrelin, CCK, and PYY), fasting blood sugar (FBS), and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). We tested the associations between peptide hormones and markers of obesity and insulin resistance (IR) by using the Independent-Samples t-test and Mann-Whitney U test, partial correlation, and logistic regression. FBS, insulin, HOMA-IR, GLP-1, GLP-2, and leptin were significantly higher in the obese group; ghrelin and CCK were significantly higher in lean participants, and no difference was seen for PYY. Controlling for BMI, GLP-1 was positively correlated with WtHR, while ghrelin was inversely correlated with WtHR. GLP-1 was correlated with HOMA-IR. GLP-1 was associated with obesity and IR markers in the regression model. Our results show that obese and lean adults display significant differences in plasma peptide hormone levels. GLP-1 levels were independently associated with markers of obesity and IR. Restoring the appetite hormone balance in obesity may represent a potential therapeutic target.

The third year with the corona virus daily on our retina has started. We've seen many variants of the virus. All very infectious, some apparently more life-threatening than others. Apparently, because I think it is hard to compare the number of cases during the first waves, when people were not yet vaccinated, to the current omicron wave, where clever people have had several vaccine shots. In some countries already up to 4. No matter what antivaxers say about this, all credible scientific data shows that these shots protect against the virus and although they don't stop you from getting sick, you get less sick. Most people that still end up in hospital are those that refused to get a vaccine, or are somehow compromised, e.g. because of a non-functioning immune system. Yet, still some people that have had their vaccine shots end up in the hospital. And although for antivaxers this is the ultimate proof that vaccines don't work, this of course is rubbish... (I usually use stronger phrases, but this gets printed black on white, if you know what I mean)... It would be pertinent to study why the virus breaks through the immune protection of these unfortunate people. Some believe it may be because of the microbiota. Several studies have shown a role of the gut microbiota composition in disease severity, together with vitamin D, cholesterol and other factors. Last year, it was a hype to write a `review' on gut microbiota and the effect on COVID-19, and also the board of Beneficial Microbes has received several submissions of so-called reviews on the topic. However, all of these were rejected, as they were mere speculations about how the gut microbiota might affect virus infection and diseases severity, without any data whatsoever. However, there are now some good studies out there that have shown that a proper gut microbiota may indeed influence disease severity, recently reviewed (Kim, 2021). And in small studies, certain taxa (like Bifidobacterium adolescentis) and microbial pathways (like carbohydrate metabolism) have been correlated to higher neutralising antibodies against the virus (Ng et al., 2022). Very little cause-and-effect evidence still. However, as I stated last year, it may not be too surprising for the knowledgeable reader that the microbiota plays a role in COVID, as the microbiota plays a role in everything that can be wrong with us! We just need to understand the mechanisms better to be able to make use of the microbiota for better disease outcome and perhaps even treatment.

The gut microbiota has been shown in recent years to be involved in the development and severity of type 2 diabetes (T2D). The aim of the present study was to test the effect of a 2-week functional food intervention on the gut microbiota composition in prediabetic individuals. A randomized double-blind, cross-over trial was conducted on prediabetic subjects. Fifteen volunteers were provided products made of: (i) 50% taro flour + 50% wheat flour; (ii) these products and the probiotic L. plantarum IS-10506; or (iii) these products with beetroot adsorbed for a period of 2 weeks with 2 weeks wash-out in between. Stool and blood samples were taken at each baseline and after each of the interventions. The gut microbiota composition was evaluated by sequencing the V3–V4 region of the 16S rRNA gene and anthropometric measures were recorded. The total weight loss over the entire period ranged from 0.5 to 11 kg. The next-generation sequencing showed a highly personalized microbiota composition. In the principal coordinate analyses, the samples of each individual clustered closer together than the samples of each treatment. For six individuals, the samples clustered closely together, indicating a stable microbiota. For nine individuals, the microbiota was less resilient and, depending on the intervention, the beta-diversity transiently differed greatly only to return to the composition close to the baseline during the wash-out. The statistical analyses showed that 202 of the total 304 taxa were significantly different between the participants. Only Butyricimonas could be correlated with taro ingestion. The results of the study show that the highly variable interindividual variation observed in the gut microbiota of the participants clouded any gut microbiota modulation that might be present due to the functional food interventions.

Infusions of the short-chain fatty acid (SCFA) acetate in the distal colon improved metabolic parameters in men. Here, we hypothesized that combining rapidly and slowly fermentable fibers will enhance distal colonic acetate production and improve metabolic health. In vitro cultivation studies in a validated model of the colon were used to identify fiber mixtures that yielded high distal colonic acetate production. Subsequently, in two randomized crossover studies, lean and prediabetic overweight/obese men were included. In one study, participants received supplements of either long-chain inulin+resistant starch (INU+RS), INU or maltodextrin (PLA) the day prior to a clinical investigation day (CID). The second trial studied beta glucan+RS (BG+RS) versus BG and PLA. During each CID, breath hydrogen, indirect calorimetry, plasma metabolites/hormones were assessed during fasting and postprandial conditions. Additionally, fecal microbiota composition and SCFA were determined. In prediabetic men, INU+RS increased plasma acetate compared to INU or PLA (P < .05), but did not affect metabolic parameters. In lean men, INU+RS increased breath hydrogen and fasting plasma butyrate, which was accompanied by increased energy expenditure, carbohydrate oxidation and PYY and decreased postprandial glucose concentrations (all P < .05) compared to PLA. BG+RS increased plasma butyrate compared to PLA (P < .05) in prediabetic individuals, but did not affect other fermentation/metabolic markers in both phenotypes. Fiber-induced shifts in fecal microbiota were individual-specific and more pronounced with INU+RS versus BG+RS. Administration of INU+RS (not BG+RS) the day prior to investigation improved metabolic parameters in lean but not in prediabetic individuals, demonstrating that effects were phenotype- and fiber-specific. Further research should study whether longer-term supplementation periods are required to elicit beneficial metabolic health in prediabetic individuals. Trial registration numbers: Clinical trial No. NCT03711383 (Inulin study) and Clinical trial No. NCT03714646 (Beta glucan study).

Simple Summary Besides the already known factors that increase the risk of breast cancer, like hormonal treatment, heredity, and obesity, growing evidence exists that intestinal microbiota can influence breast cancer carcinogenesis. Current clinical information into the role of the intestinal microbiota in breast cancer patients is limited. This study aimed to see whether there are differences in intestinal microbiota richness, diversity, and composition between oestrogen receptor positive breast cancer patients and controls. We concluded that the intestinal microbiota richness, diversity, and composition were not different between breast cancer patients and postmenopausal controls. An increased relative abundance of Dialister and Veillonellaceae was observed in breast cancer patients scheduled for adjuvant treatment, which might be caused by a relative decrease in other bacteria due to surgery associated factors rather than an absolute increase. For future studies, we strongly advise a more homogeneous group of breast cancer patients of preferably treatment-naive patients. Abstract Background: Previous preclinical and clinical research has investigated the role of intestinal microbiota in carcinogenesis. Growing evidence exists that intestinal microbiota can influence breast cancer carcinogenesis. However, the role of intestinal microbiota in breast cancer needs to be further investigated. This study aimed to identify the microbiota differences between postmenopausal breast cancer patients and controls. Patients and methods: This prospective cohort study compared the intestinal microbiota richness, diversity, and composition in postmenopausal histologically proven ER+/HER2- breast cancer patients and postmenopausal controls. Patients scheduled for (neo)adjuvant adriamycin, cyclophosphamide (AC), and docetaxel (D), or endocrine therapy (tamoxifen) were prospectively enrolled in a multicentre cohort study in the Netherlands. Patients collected a faecal sample and completed a questionnaire before starting systemic cancer treatment. Controls, enrolled from the National Dutch Breast Cancer Screening Programme, also collected a faecal sample and completed a questionnaire. Intestinal microbiota was analysed by amplicon sequencing of the 16S rRNA V4 gene region. Results: In total, 81 postmenopausal ER+/HER2- breast cancer patients and 67 postmenopausal controls were included, resulting in 148 faecal samples. Observed species richness, Shannon index, and overall microbial community structure were not significantly different between breast cancer patients and controls. There was a significant difference in overall microbial community structure between breast cancer patients scheduled for adjuvant treatment, neoadjuvant treatment, and controls at the phylum (p = 0.042) and genus levels (p = 0.015). Dialister (p = 0.001) and its corresponding family Veillonellaceae (p = 0.001) were higher in patients scheduled for adjuvant treatment, compared to patients scheduled for neoadjuvant treatment. Additional sensitivity analysis to correct for the potential confounding effect of prophylactic antibiotic use, indicated no differences in microbial community structure between patients scheduled for neoadjuvant systemic treatment, adjuvant systemic treatment, and controls at the phylum (p = 0.471) and genus levels (p = 0.124). Conclusions: Intestinal microbiota richness, diversity, and composition are not different between postmenopausal breast cancer patients and controls. The increased relative abundance of Dialister and Veillonellaceae was observed in breast cancer patients scheduled for adjuvant treatment, which might be caused by a relative decrease in other bacteria due to prophylactic antibiotic administration rather than an absolute increase.

The complex microbial community found in the human gut consist of members of multiple kingdoms, among which are bacteria and fungi. Microbiome research mainly focuses on the bacterial part of the microbiota, thereby neglecting interactions that can take place between bacteria and fungi. With the rise of sequencing techniques, the possibilities to study cross-kingdom relationships has expanded. In this study, fungal-bacterial relationships were investigated using the complex, dynamic computer-controlled in vitro model of the colon (TIM-2). Interactions were investigated by disruption of either the bacterial or fungal community by the addition of antibiotics or antifungals to TIM-2, respectively, compared to a control without antimicrobials. The microbial community was analyzed with the use of next generation sequencing of the ITS2 region and the 16S rRNA. Moreover, the production of SCFAs was followed during the interventions. Correlations between fungi and bacteria were calculated to investigate possible cross-kingdom interactions. The experiments showed that no significant differences in alpha-diversity were observed between the treatments with antibiotics and fungicide. For beta-diversity, it could be observed that samples treated with antibiotics clustered together, whereas the samples from the other treatments were more different. Taxonomic classification was done for both bacteria and fungi, but no big shifts were observed after treatments. At the level of individual genera, bacterial genus Akkermansia was shown to be increased after fungicide treatment. SCFAs levels were lowered in samples treated with antifungals. Spearman correlations suggested that cross-kingdom interactions are present in the human gut, and that fungi and bacteria can influence each other. Further research is required to gain more insights in these interactions and their molecular nature and to determine the clinical relevance.

Most research on the human microbiome focuses on the bacterial component, and this has led to a lack of information about the fungal component (mycobiota) and how this can influence human health, e.g., by modulation through the diet. The validated, dynamic computer-controlled model of the colon (TIM-2) is an in vitro model to study the microbiome and how this is influenced by interventions such as diet. In this study, it was used to the study the gut fungal-community. This was done in combination with next-generation sequencing of the ITS2 region for fungi and 16S rRNA for bacteria. Different dietary interventions (control diet (SIEM), high-carbohydrate, high-protein, glucose as a carbon source) were performed, to see if diet could shape the mycobiome. The mycobiome was investigated after the adaptation period, and throughout the intervention period which lasted 72 h, and samples were taken every 24 h. The fungal community showed low diversity and a greater variability when compared to bacteria. The mycobiome was affected most in the first hours of the adaptation period. Taxonomic classification showed that at the phylum-level Ascomycota and Basidiomycota dominated, while Agaricus, Aspergillus, Candida, Penicillum, Malassezia, Saccharomyces, Aureobasidium, Mycosphaerella, Mucor and Clavispora were the most abundant genera. During the intervention period, it was shown that the change of diet could influence the diversity. Clustering of samples for different time points was analyzed using Bray–Curtis dissimilarities. Samples of t0 clustered together, and samples of all other time points clustered together. The Bray–Curtis-dissimilarity analysis also showed that for the different dietary interventions, samples treated with glucose clustered together and were different from the other groups (p < 0.05, PERMANOVA). Taxonomic classification showed that the genera Alternaria, Thanatephorus, Candida and Dekkera differentially changed for the various diet groups (p < 0.05, Kruskal–Wallis). These results show that the mycobiota could be modelled in TIM-2; however, the low diversity and high variability make studying fungal, as compared to bacterial, communities, much more challenging. Future research should focus on the optimization of the stability of the fungal community to increase the strength of the results.

The effect of a Citrus Fruit Extract high in the polyphenols hesperidin and naringin (CFE) on modulation of the composition and activity of the gut microbiota was tested in a validated, dynamic in vitro model of the colon (TIM-2). CFE was provided at two doses (250 and 350 mg/day) for 3 days. CFE led to a dose-dependent increase in Roseburia, Eubacterium ramulus, and Bacteroides eggerthii. There was a shift in production of short-chain fatty acids, where acetate production increased on CFE, while butyrate decreased. In overweight and obesity, acetate has been shown to increase fat oxidation when produced in the distal gut, and stimulate secretion of appetite-suppressive neuropeptides. Thus, the data in the in vitro model point towards mechanisms underlying the effects of the polyphenols in CFE with respect to modulation of the gut microbiota, both in composition and activity. These results should be confirmed in a clinical trial.

The speed of starch-digestion defines peak blood-glucose concentrations. Slow digestion is beneficial for diabetic individuals. To investigate the effects on blood-glucose it is important to be able to predict the amount of digestible starch. Therefore, the aim of this study was to compare the digestibility of different starches in a validatedin-vitromodel of the upper gastrointestinal (GI) tract. Digestion was performed in theTNO dynamic, computer-controlledin-vitromodel of the upper GI-tract (TIM-1). Release of glucose was measured over time. Products tested were taro-flour, native-, and modified taro-starch. The latter two were also tested with beet-juice adsorbed. These were compared to wheat-flour and a glucose-solution. Modified taro-starch showed a similar glucose-bioaccessibility as wheat-flour (81-83%), while the other products tested had a lower bioaccessibility (60-75%). Adsorption of beet-juice affected digestibility of the modified taro-starch, but not the native-starch. Taro-based products can be used to lower plasma glucose concentrations in diabetic individuals.

The aim of the study was to investigate the prebiotic potential of xylo-oligosaccharides (XOS) from sugarcane in a validated, dynamic, computer-controlled in vitro model of the colon (TIM-2) simulating human adults. In two sets of experiments, each with a different microbiota, 3 different doses of XOS were tested at 1.0 g/day, 1.5 g/day and 3.0 g/day. The in vitro model was run for 72 h, and at the start and subsequently every 24 h samples were taken and analysed for short-chain fatty acids (SCFA) and gut microbiota composition. SCFA were analysed using ion chromatography, whereas microbiota composition was analysed using sequencing of the V3-V4 region of the 16S rRNA gene. XOS showed a similar SCFA production per gram of substrate as the control medium, including butyrate, which is considered to be important for gut health. In both sets of experiments XOS showed a consistent dose-dependent increase in abundance over time of the genus Bifidobacterium, and within that of the species B. adolescentis and an unidentified species (labelled ‘sp.1’). The results show the potential prebiotic effect of XOS from sugarcane, by its capacity to generate butyrate and increase the health-beneficial bifidobacteria.

Diet-related modulation of gut microbiota and its metabolic activity represents an intriguing research context, particularly in the case of disorders related to imbalances in gut microbial communities. We here explored the effects of Bacillus coagulans GBI-30, 6086 (BC30), β-glucans, and innovative whole-grain pastas, with or without these functional ingredients, on gut microbiota from three groups of children, presenting different susceptibility to type 1 diabetes, by using the well-controlled TNO in vitro model of the proximal colon (TIM-2). Short- and branched-chain fatty acids production and microbiota composition were assessed by means of gas chromatography and 16S rRNA gene profiling, respectively. In most cases, in vitro dietary interventions caused microbiota-dependent modulations as a result of intergroup variability, but also specific changes in microbial groups were shared between the three microbiotas, highlighting specific diet-microbial taxa connections.

The objective of the present study was to compare the effect of dairy matrices (fermented milk and ice cream) and a dietary supplement containing the same amounts of inulin and the same viable counts of Lactobacillus acidophilus and Bifidobacterium animalis on the survival of probiotics when submitted to simulated digestion. The survival was evaluated by plate counts in MRS agar, the metabolic status by flow cytometry and the cell morphology by scanning electronic microscopy. The percentages of live cells observed by flow cytometry and by plate counts were higher in the fermented milk when compared with the other products (p

There are scarce data on plant sterols (PS) and gut microbiota relationship. The purpose of this study is to compare the interaction between PS and gut microbiota through in vitro colonic fermentation studies using a validated system (TIM-2) with a PS-enriched dose (similar to 2 g/day) from two sources (food PS-source ingredient and commercial standard) using microbiota from lean and obese populations. Fecal sterols, short chain fatty acids (SCFA) and microbiota composition were determined by GC/MS, IEC, and 16S-sequencing, respectively.PS-feeding decreased coprostanol and ethylcoprostanol concentration and increased the production of acetate and butyrate (mainly with lean microbiota). In addition, the PS-enrichment dose increased the proportion of some genera from phylum Firmicutes with lean and obese microbiota.The results obtained suggest that the gut microbiota preferably use PS as a substrate. In addition, PS-enrichment dose had no effect on the production of SCFA but modified the microbial profile of lean and obese populations.

Background and Objectives. The gut microbiota has been shown to be involved in the development and severity of type 2 diabetes (T2D). The aim of the present study was to test the effect of potential functional food ingredients, alone or in combination, on the gut microbiota composition in diabetic rats in a pilot study of 1 week of feeding. Methods. In a pilot study to modulate the composition of the gut microbiota, (i) native taro starch, (ii) modified taro starch, (iii) beet juice, (iv) psicose, (v) the probiotic L. plantarum IS-10506, (vi) native starch combined with beet juice, (vii) native starch to which beet juice was adsorbed, (viii) modified starch combined with beet juice, and (ix) modified starch to which beet juice was adsorbed were fed to rats in which T2D was induced with streptozotocin (STZ). After one week, the composition of the gut microbiota was evaluated by sequencing the PCR-amplified V3-V4 region of the 16S rRNA gene. Results and Conclusions. The next-generation sequencing showed that 13 microbial taxa of the gut microbiota were significantly different between groups, depending on the treatment. The results of this pilot study will be used to design a 4-week intervention study in STZ-induced T2D rats to determine the best functional food for counteracting T2D, including their effects on satiety hormones. This should ultimately lead to the development of functional foods for prediabetic and diabetic individuals.

Abstract The aim of this study was to evaluate the effects of soy-based beverages manufactured with water-soluble soy extract, containing probiotic strains (Lactobacillus acidophilus LA-5 and Bifidobacterium longum BB-46) and/or acerola by-product (ABP) on pooled faecal microbiota obtained from lean and obese donors. Four fermented soy beverages (FSs) (“placebo” (FS-Pla), probiotic (FS-Pro), prebiotic (FS-Pre), and synbiotic (FS-Syn)) were subjected to in vitro digestion, followed by inoculation in the TIM-2 system, a dynamic in vitro model that mimics the conditions of the human colon. Short- and branched-chain fatty acids (SCFA and BCFA) and microbiota composition were determined. Upon colonic fermentation in the presence of the different FSs formulations, acetic and lactic acid production was higher than the control treatment for faecal microbiota from lean individuals (FMLI). Additionally, SCFA production by the FMLI was higher than for the faecal microbiota from obese individuals (FMOI). Bifidobacterium spp. and Lactobacillus spp. populations increased during simulated colonic fermentation in the presence of FS-Syn in the FMLI and FMOI. FS formulations also changed the composition of the FMOI, resulting in a profile more similar to the FMLI. The changes in the composition and the increase in SCFA production observed for the FMLI and FMOI during these in vitro fermentations suggest a potential modulation effect of these microbiotas by the consumption of functional FSs. Graphical abstract Key points • Soy beverages increased Bifidobacterium abundance in microbiota from obese individuals. • The synbiotic beverage increased Bifidobacterium abundance in microbiota from lean individuals. • The synbiotic beverage changed the microbiota from obese individuals, approaching the lean profiles.

The present work aimed at understanding gut microbiota bioconversion of phenolic compounds (PC) and organic acids in predigested Hibiscus sabdariffa (Hb) calyces and the mixture of Hb and Agave (Agave tequilana Weber) fructans (AF). With this purpose, dried Hb and Hb/AF were predigested with enzymatic treatment, and then fermented in a dynamic in vitro model of the human colon (TIM-2). After HPLC-ESI-QToF-MS analysis of samples taken at 0, 24, 48 and 72 h of fermentation, it was observed that hydroxycinnamic acids, flavanols, flavonols, and anthocyanins were mainly transformed into derivatives of hydroxyphenylpropionic, hydroxyphenylacetic and hydroxybenzoic acids. Moreover, organic acids, such as hydroxycitric and hibiscus acids, were formed along with unidentified lactones and reduced compounds. Interestingly, no differences were observed between microbial-derived metabolites formed after the fermentation of Hb and Hb/AF. In conclusion, colonic fermentation of polyphenol-rich Hb yields a wide range of microbial phenolic metabolites with potential effects on health., RMB and SGSA acknowledge to Science and Technology for Development Program (CYTED) through the Ibero-American Network of Underutilized Autonomous Foods (ALSUB-CYTED, 118RT0543). SGSA acknowledges CONACYT-Mexico for the sabbatical grant 260935. The Spanish Ministry of Science, Innovation and Universities (Project AGL2015-69986-R) is also acknowledged. This research has been made possible with the support of the Dutch Province of Limburg.

Two of the most important Mexican plant-foods are tomato (Solanum lycopersicum L.) and husk tomato (Physalis ixocarpa Brot.). In this study three objectives were followed: i) to evaluate the bioaccessible phenolic compounds (PC) in T and HT during upper gastrointestinal digestion, ii) to in vitro ferment the indigestible fractions of the samples to evaluate the short-chain fatty acids (SCFA) production, iii) the microbial metabolites, bioconverted PC and volatile organic compounds (VOCs) generated during the fermentation. Vanillic acid was the most bioaccessible PC and after 48 h, 3-hydroxyphenylacetic acid was the most abundant microbial metabolite identified in both samples. The identification of VOCs belonging to terpenes (and derivatives) group in T and HT can be product of the microbial metabolism of carotenoids. The study shows new knowledge of the in vitro intestinal digestion and fermentation of T and HT final compounds with biological potential which should be evaluated in further studies.

At the start of 2020 we were thrilled to have reached 10 years of Beneficial Microbes! Little did we know that soon after Europe and the rest of the world (with Asia already earlier) would be in lock-down due to COVID-19. It has been a strange year. And now, at the start of 2021, the excitement of having a vaccine is tempered by the fact that everywhere mutants of the virus pop up. Although this was likely to occur, as also the influenza virus keeps mutating, it means that at the moment it is unclear as to whether the current situation of lock-downs and social distancing will remain for a longer period than we had anticipated and hoped for at the end of 2020 when it became clear that several vaccines were efficacious. Some studies have shown a role of the gut microbiota composition in disease severity, together with vitamin D, cholesterol and other factors. It was a hype to write a 'review' on gut microbiota and the effect on COVID-19, and also the board of Beneficial Microbes has received several submissions of so-called reviews on the topic. However, all of them were rejected, as they were mere speculations about how the gut microbiota might affect virus infection and disease severity, without any data whatsoever. However, there are some good studies out there that have shown that a proper gut microbiota may indeed influence disease severity, such as recently reviewed by Kim (2021). All in all, this may not be too surprising for the knowledgeable reader, as they would know that the microbiota plays a role in everything that can be wrong with us!

Resumen del póster presentado a la 1st International E-Conference on Antioxidants in Health and Disease, celebrada del 1 al 15 de diciembre de 2020., Mango byproducts are a relevant source of phenolic compounds that have already been shown to display a multitud e of health beneficial properties. The biological activity of a compound is determined by its absorption and metabolism. Considering the low bioavailability of polyphenols at small intestinal level, it is important to study the biotransformation of mango peel polyphenols bythe gut microbiota to determine the chemical structure of the potential bioactive microbial·derived metabolites. Thus, gut microbiota bioconversion of polyphenols in predigested mango 'Ataulfo' peel was studied using a validated, dynamic in vitro human colon model (TIM·2) with faecal microbial inoculum. Driel peels were predigested with enzymatic treatment, followed by TIM·2 fermentation (72h). Samples were taken at O, 24, 48 and 72h and a nelyzed by HPLC·QToF. Derivatives of hydroxyphenylpropionic, hydroxyphenylacetic and hydroxybenzoic acids, as well as pyrogallol were the main polyphenols identified. These metabolites might derive from the biotrasnsformation of flavonoids (flavanols and flavonols), gel lates and gallotannins in mango peel. Despite the high content of ellagic acid, low amounts were detected in TIM·2 samples due to trasnsformation mainly into urolithins A and C. Xanthone and benzophenone derivatives, specific to mango, remained after the colonic biotransformation, contrary to flavonoids, which completely disappeared. In conclusion, microbiel derived metabolites, especially xanthone and benzophenone derivatives, are partially stable after colonic fermentation, and thus have the potential to contribute to mango peel bioactivity.

We recently showed that the gut microbiota composition of stunted children was different from that of children with normal nutritional status. Here, we compared immune status and fecal microbial metabolite concentrations between stunted and normal children, and we correlated macronutrient intake (including energy), metabolites and immune status to microbiota composition. The results show that macronutrient intake was lower in stunted children for all components, but after correction for multiple comparison significant only for energy and fat. Only TGF-β was significantly different between stunted children and children of normal nutritional status after correction for multiple comparisons. TNF-alpha, IL-10, lipopolysaccharide binding protein in serum and secretory IgA in feces were not significantly different. Strikingly, all the individual short-chain and branched-chain fatty acids were higher in fecal samples of stunted children (significant for acetate, valerate and total SCFA). These metabolites correlated with a number of different microbial taxa, but due to extensive cross-feeding between microbes, did not show a specific pattern. However, the energy-loss due to higher excretion in stunted children of these metabolites, which can be used as substrate for the host, is striking. Several microbial taxa also correlated to the intake of macronutrients (including dietary fibre) and energy. Eisenbergiella positively correlated with all macronutrients, while an uncharacterized genus within the Succinivibrionaceae family negatively correlated with all macronutrients. These, and the other correlations observed, may provide indication on how to modulate the gut microbiota of stunted children such that their growth lag can be corrected. Trail registered at https://clinicaltrials.gov/ct2/show/NCT04698759.

Husk tomato (Physalis ixocarpa Brot. ex. Horm) is mainly used in the preparation of many Mexican sauces due to its unique and slightly acidic flavor, both in raw and cooked forms. These sauces also usually contain Serrano hot pepper (Capsicum annuum L), onion (Allium cepa L.), garlic (Allium sativum L.), coriander (Coriandrum sativum L.) and salt. Mexican sauces are a pre-Hispanic staple food, yet there is scarce knowledge on the phenolic compounds (PC) that reach the colon bound to the indigestible fraction (IF) after intestinal digestion. Thus, the aim of the present work was to evaluate the indigestible fraction of two types of Mexican sauces made with cooked and raw husk tomato: cooked green sauce (CGS) and raw green sauce (RGS). IF of CGS and RGS were fermented in the in vitro model of the human colon (TIM-2) to investigate the PC bioconversion by the gut microbiota after 24, 48 and 72 h. PC of the original sauces and their predigested fractions, as well as the formed metabolites were identified and monitored by HPLC-ESI-QToF-MS. Cooking husk tomato significantly increased the total indigestible fraction (TIF), mainly due to its insoluble indigestible fraction (IIF), and diminished PC. Flavonoids (flavonols and flavones) were the most abundant phenolic group in digested sauces followed by capsaicinoids (a characteristic group derived from hot pepper), hydroxycinnamic acids, and hydroxybenzoic acids. The metabolites 3-(ρ-hydroxyphenyl) propionic acid, 3-(3-hydroxyphenyl) propionic acid and 4-hydroxyphenylacetic acid were the most abundant colonic metabolites identified, which are thought to be derived from the biotransformation of flavonoids and hydroxycinnamates. These results are the first obtained on in vitro colonic fermentation of Mexican sauces and should be considered in future studies on the health effects related to consuming this staple food., The Spanish Ministry of Science, Innovation and Universities (Project AGL2015-69986-R) is also acknowledged. Alicia Paulina Cárdenas Castro thanks CONACYT-Mexico for the financial support, grant registration number: 662703 and Koen Venema acknowledges the support of the Dutch Province of Limburg.

The aim of current study was to investigate in an in vitro study how enzymatic and chemical pretreated rapeseed meal (RSM) influences the fiber fermentation and microbial community in the swine large intestine. RSM was processed enzymatically by a cellulase (CELL), two pectinases (PECT), or chemically by an alkaline (ALK) treatment. 16S rRNA gene sequencing data was performed to evaluate changes in the gut microbiota composition, whereas short-chain fatty acid (SCFA) production (ion-chromatography) and non-starch polysaccharides (NSP) composition (using monoclonal antibodies; mAbs) were used to assess fiber degradation. The results showed that ALK, CELL, PECT1, and PECT2 changed microbial community composition, increased the predicted abundance of microbial fiber-degrading enzymes and pathways, and increased acetic acid, propionic acid, butyric acid, and total SCFA production. The increased microbial genera positively correlated with SCFA production. Monoclonal antibody analyses showed that the cell wall polysaccharide structures of RSM shifted after ALK, CELL, PECT1, and PECT2 treatment. The degradation of NSP during the fermentation period was dynamic, and not continuous based on the epitope recognition by mAbs. This study provides the first detailed analysis of changes in the swine intestinal microbiota due to RSM modified by ALK, CELL, PECT1, and PECT2, which altered the microbial community structure, shifted the predicted functional metagenomic profile and subsequently increased total SCFA production. Our findings that ALK, CELL, PECT1, and PECT2 increased fiber degradability in RSM could help guide feed additive strategies to improve efficiency and productivity in swine industry. The current study gave insight into how enzymatic treatment of feed can alter microbial communities, which provides good opportunity to develop novel carbohydrase treatments, particularly in swine feed.

The aim of the study was to investigate the effect of untreated and processed rapeseed meal (RSM) on fibre degradability by pig gut microbiota and the adaptation of the microbiota to the substrate, by using the Swine Large Intestine in vitro Model (SLIM). A standardized swine gut microbiota was fed for 48 h with pre-digested RSM which was processed enzymatically by a cellulase (CELL), two pectinases (PECT), or chemically by an alkaline (ALK) treatment. Amplicons of the V3-V4 region of the 16S rRNA gene were sequenced to evaluate the gut microbiota composition, whereas short chain fatty acids (SCFA) were measured to assess fibre degradation. Adaptive gPCA showed that CELL and ALK had larger effects on the microbiota composition than PECT1 and PECT2, and all substrates had larger effects than CON. The relative abundance of family Prevotellaceae was significantly higher in CELL treatment compared to others. Regardless of the treatments (including CON), the relative abundance of Dorea, Allisonella, and FamilyXIIIUCG_001 (in the order of Clostridiales) were significantly increased after 24 h, and Parabacteroides, Mogibacterium, Intestinimonas, Oscillibacter, RuminococcaceaeUCG_009, Acidaminococcus, Sutterella, and Citrobacter were significantly higher in abundance at time point 48 compared to the earlier time points. Prevotella 9 had significant positive correlations with propionic and valeric acid, and Mogibacterium positively correlated with acetic and caproic acid. There was no significant difference in SCFA production between untreated and processed RSM. Overall, degradability in the processed RSM was not improved compared to CON during the RSM adaptation period. However, the significantly different microbes detected among treatments, and the bacteria considerably correlating with SCFA production might be important findings to determine strategies to shorten the fibre adaptation period of the microbiota, in order to increase feed efficiency in the animal, and particularly in pig production.

Protein- and sugar-rich food products processed at high temperatures contain large amounts of dietary advanced glycation endproducts (dAGEs). Our earlier studies have shown that specifically protein-bound dAGEs induce a pro-inflammatory reaction in human macrophage-like cells. To what extent these protein-bound dAGEs survive the human gastrointestinal (GI) tract is still unclear. In this study we analysed gastric and small intestinal digestion of dAGEs using the validated, standardised TNO in vitro gastroIntestinal digestion model (TIM-1), a dynamic in vitro model which mimics the upper human GI tract. This model takes multiple parameters into account, such as: dynamic pH curves, peristaltic mixing, addition of bile and pancreatic digestive enzymes, and passive absorption. Samples of different digested food products were collected at different time points after (i) only gastric digestion and (ii) after both gastric plus small intestinal digestion. Samples were analysed for dAGEs using UPLC-MS/MS for the lysine derived Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL), and the arginine derived methylglyoxal-derived hydroimidazolone-1 (MG-H1), and glyoxal-derived hydroimidazolone-1 (G-H1). All AGEs were quantified in their protein-bound and free form. The results of this in vitro study show that protein-bound dAGEs survive gastrointestinal digestion and are additionally formed during small intestinal digestion. In ginger biscuits, the presence MG-H1 in the GI tract increased with more than 400%. This also indicates that dAGEs enter the human GI tract with potential pro-inflammatory characteristics.

The aim of this study was to assess the germination, survival and metabolic activity of the probiotic Bacillus coagulans GBI-30, 6086 [GanedenBC30] (BC30) in a dynamic, computer controlled in vitro model of the gastrointestinal (GI) tract, simulating human adults. Experiments were performed in the presence of a meal to maximise germination, due to the presence of germination-triggers. Both an upper GI tract (stomach and small intestine; TIM-1) and a colon model (TIM-2) were used, where material exiting TIM-1 was added to TIM-2. Spores of BC30 were introduced in the gastric compartment of TIM-1 and samples were taken immediately after the pylorus. Moreover, for 6 h, every hour the ileal efflux was collected and a subsample was plated for viable counts (spores and germinated cells). The remainder of the sample was fed to TIM-2, and after 24 h another sample was taken and tested for viable counts. In addition, samples were taken from the dialysates of the model and analysed using LC-MS/MS to determine bacterial metabolites and digestion products. Survival after transit through the gastric compartment was high (97%) and most cells were still in the spore form (76%). Survival after transit through TIM-1 was on average 51%, meaning that on average half of the orally provided spores was found back as cfu on the agar plates. Of these on average 93% were germinated cells and only 7% were spores. 24 h after the start of the experiments germination had increased in TIM-2 to 97% vegetative cells, and only 3% spores. No further loss of viability was observed in TIM-2. In terms of metabolic activity, increased levels of amino acids, dipeptides and citric acid cycle metabolites were found compared to experiments in the absence of BC30. In conclusion, BC30 spores germinate to a large extent (>90%) in the presence of germination triggers in the small intestine in a model that closely mimics the physiological conditions of human adults. Of the oral dose, as much as half of the cells survived transit through the upper GI tract, and based on the metabolite profile, these cells were metabolically active. Either these cells or the enzymes released from the dead cells aided in digestion of the meal. These insights help explain some of the observations in previous experiments, and support the understanding of the mechanism of action of the probiotic BC30.

The digestive system of the chicken plays an important role in metabolism, immunity, and chicken health and production performance. The chicken ceca harbor a diverse microbial community and play a crucial role in the microbial fermentation and production of energy-rich short-chain fatty acids (SCFA). For humans, dogs, and piglets in vitro digestive system models have been developed and are used to study the microbiota composition and metabolism after intervention studies. For chickens, most research on the cecal microbiota has been performed in in vivo experiments or in static in vitro models that may not accurately resemble the in vivo situations. This paper introduces an optimized digestive system model that simulates the conditions in the ceca of the chicken, i.e., the Chicken ALIMEntary tRact mOdel-2 (CALIMERO-2). The system is based on the well-validated TNO in vitro model of the colon-2 (TIM-2) and is the first dynamic in vitro digestion model for chickens species. To validate this model, the pH, temperature, and different types of microbial feeding were compared and analyzed, to best mimic the conditions in the chicken ceca. The bacterial composition, as well as the metabolite production at 72 h, showed no significant difference between the different microbial feedings. Moreover, we compared the CALIMERO-2 digestive samples to the original inoculum and found some significant shifts in bacterial composition after the fermentation started. Over time the bacterial diversity increased and became more similar to the original inoculum. We can conclude that CALIMERO-2 is reproducible and can be used as a digestive system model for the chicken ceca, in which the microbial composition and activity can be maintained and shows similar results to the in vivo cecum. CALIMERO-2 can be used to study effects on composition and activity of the chicken cecum microbiota in response to in-feed interventions.

Several validated dynamic in vitro models of the colon have been developed for humans, but there is no dynamic in vitro fermentation model for pigs. This study was conducted to modify the human, dynamic, computer-controlled TNO in vitro model of the colon (TIM-2) for pigs and investigate effects of different starch sources and polysaccharides on swine microbiota structure, ecological network, predictive functional profile, and short-chain fatty acids production. Our study showed that three different types of starch or two polysaccharides greatly impacted microbiota composition. Co-occurrence network analysis indicated that microbiota fed with different sources of starch changed the network topological properties. Functional profiles were predicted to vary significantly among the three starch treatments, and the original pig faecal inoculum was more similar to maize starch treatment. On the other hand, compared with maize starch and arabinoxylans (AX), the microbial composition of the original inoculum was more similar when AX-XG (arabinoxylans and xyloglucan) were added, and the functional profile of the original inoculum also clustered with AX-XG. The cumulative production of acetic, propionic, and butyric acid on maize starch were significantly higher than those on potato starch and wheat starch, while only the amount of acetic acid was significant higher on AX-XG than that on AX. In conclusion, supplementation of maize starch as the starch source together with AX and XG, leads to the bacteria being more stable in the in vitro model and closer to the original inoculum and microbial function compared to potato starch, wheat starch and AX. A maize basal diet may improve energy absorption in the large intestine in growing pigs.