Background/Objectives: Animal studies have shown that food additives may adversely affect the gut microbiome. However, the effect of food additives on the microbiome in adults with Crohn's disease (CD) remains less explored. This study investigated the impact of food additives on gut microbiome and fibre fermentation capacity in adults with CD and healthy controls (HCs) using in vitro faecal fermentations. Methods: Faeces from 6 HCs and 6 patients with CD in clinical remission (Harvey Bradshaw Index < 5) were used for in vitro fermentation of a fibre mix with one of 12 food additives (calcium propionate, carboxymethylcellulose, carrageenan kappa, cinnamaldehyde, maltodextrin, polysorbate-80, potassium sorbate, sodium benzoate, sodium sulphite, titanium dioxide, turmeric, and xanthan gum). Short-chain fatty acids (SCFAs) were measured using gas chromatography, the microbiome was profiled with 16S rRNA amplicon sequencing and total bacterial load was measured with qPCR. Results: Maltodextrin increased acetate production in both groups. In HCs, turmeric increased acetate and butyrate production, sodium sulphite reduced acetate production, and maltodextrin reduced butyrate production. Microbiome Shannon α-diversity increased with titanium dioxide (both groups), and with carrageenan kappa only in patients with CD. In both groups, the addition of maltodextrin and polysorbate-80 induced significant shifts in microbiome structure (β-diversity). Significant shifts were seen with maltodextrin (HC: R2 = 6.8%, p = 0.001; CD: R2 = 5.1%, p = 0.004) and sodium sulphite (HC: R2 = 6.9%, p = 0.001). Maltodextrin significantly decreased the estimated absolute abundance of Escherichia-Shigella in patients CD; sodium benzoate, potassium sorbate, and calcium propionate did so in HCs. Faecalibacterium decreased in the presence of polysorbate-80 in the HC and CD groups, as well as in the presence of maltodextrin in the CD group. Total bacterial load decreased with polysorbate-80, potassium sorbate, maltodextrin and calcium propionate in both groups. Xanthan gum decreased total bacterial load in HCs. Conclusion: Certain food additives significantly affected fibre fermentation capacity and microbiome structure, with only modest differences observed according to participants' health status.

Approximately 5-7% of dietary fatty acids escape absorption in the small intestine and reach the colon (1) . How these fatty acids interact with the gut microbiota remains poorly understood. The aim of this study was to determine how medium chain fatty acids (MCFA), long chain saturated (LCSFA), and unsaturated fatty acids (UFA) modulate short-chain fatty acid (SCFA) production by human gut microbiota in vitro, in the presence and absence of fermentable mixed fibre (MF). Anaerobic 24 h batch culture fermentations (pH 7, 37oC) were inoculated individually with faecal slurries from 12 healthy adults (6 males; mean (SD) age: 34.7 (7) years; BMI: 26 (4.2) kg/m 2 ). Cultures were performed for each individual fatty acid [MCFA: caprylic (C8:0), capric (C10:0), lauric (C12:0); LCSFA: myristic (C14:0), palmitic (C16:0), stearic (C18:0); UFA: oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and eicosapentaenoic acid (EPA) (C20:5)], both in the presence or absence of MF (inulin, apple pectin, alpha cellulose and high resistant maize starch). Acetate, propionate and butyrate were quantified using gas chromatography with flame ionisation detector. Paired T-test or Wilcoxon signed-rank test were performed to compare fatty acids + MF with MF alone, and fatty acids alone with fibre-free basal medium. Data are presented as median and interquartile range (IQR). This study was approved by the University of Glasgow Ethics Committee (Ref: 200240004). In the presence of MF, MCFA significantly reduced total SCFA production compared with MF alone (38.4 (IQR 8) µmol/ml): caprylic acid 9.4 (IQR 5.1) µmol/ml, capric acid 11.3 (IQR 4.2) µmol/ml, and lauric acid 15 (IQR 5.7) µmol/ml (all P<0.002). Significant reductions were also found with these treatments for acetate, propionate and butyrate (P<0.02). LCSFA+MF did not change SCFA production relative to MF alone. In the presence of MF, total SCFA decreased with linoleic acid 31.3 (IQR 22.4) µmol/ml, linolenic acid 9.1 (IQR 21.1) µmol/ml and EPA 30.8 (IQR 15.9) µmol/ml compared with MF alone 38.4 (IQR 8) µmol/ml (all P<0.005), but not with oleic acid 35.3 (IQR 6.7) µmol/ml. Acetate and butyrate were also significantly reduced in these treatments (P<0.003). Notably, propionate increased with both oleic acid 7.4 (IQR 5.4) µmol/ml and EPA 11.1 (IQR 13.4) µmol/ml compared with MF alone 5.4 (IQR 2.6) µmol/ml (P<0.002 and P=0.034, respectively). Under fibre-free conditions, broadly similar patterns were observed, with MCFA causing the greatest reductions in SCFAs, UFA showing moderate decreases, and LCSFA showing no change in SCFAs compared with the no-fibre control. These findings suggest that the modifying effect of dietary fatty acids on gut microbial activity in vitro depends on their chain length and degree of unsaturation. Dietary intervention studies are needed to confirm these effects and to explore the implications to metabolic health.

BACKGROUND AND AIMS:Serum low-density lipoprotein (LDL)-cholesterol varies in response to the isonergetic replacement of saturated (SFA) with unsaturated fats (UFA), as reported in our previous screening study, 'Reading, Imperial, Surrey, Saturated fat Cholesterol Intervention-1' (RISSCI-1). In the present study, we aimed to determine if differences in postprandial absorption and metabolic handling of SFA could explain variability in responsiveness of serum LDL-cholesterol to dietary SFA intake. METHODS:Male participants classified according to their fasting LDL-cholesterol response to the replacement of SFA with UFA in our screening study (RISSCI-1) as higher (HR, n = 19) or lower responders (LR, n = 17), were placed on two, 4-week isoenergetic diets (higher-SFA/lower-UFA followed by lower-SFA/higher-UFA). After each diet, [U-13C]palmitic acid was given in the first of two sequential meals to trace postprandial saturated fat absorption. RESULTS:Repeating the previous dietary regime in the same participants resulted in similar inter-individual variation in LDL-cholesterol response, with greater reductions in lipid ratios (total cholesterol:high density lipoprotein (HDL) and LDL-cholesterol:HDL-cholesterol) and largest LDL-1-subfraction in the HR versus the LR group (p < 0.0001 for all). There was no difference in the absorption of SFA between groups but a significantly greater recovery of [U-13C]palmitic acid-triacylglycerol in the postprandial period in LR relative to the HR group after SFA replacement with UFA was observed (p = 0.019). CONCLUSIONS:Dietary SFA absorption did not explain individual variation in serum LDL-cholesterol response to SFA replacement with UFA. Exploratory findings of a difference in postprandial [U-13C]palmitic acid-triacylglycerol warrants further investigation.

BACKGROUND & AIMS:Studies in-vitro and in animals propose that vitamins and minerals can alter the human gut microbiome. Human trials replicating these findings are scarce or used micronutrient supplementation in supraphysiological doses. We explored the effect of multivitamin/multimineral (MVMM) supplementation on the gut microbiome, and how participant's background diet can modify this effect. METHODS:In a randomised, controlled, cross-over study, 28 healthy adults (68 % female, mean [SD], 33 [13] years]) received either a MVMM supplement containing dosages close to the UK dietary recommendations for 23 micronutrients (intervention period) or no supplement (control period), for 10-days, separated by a 15-day washout period. Participants' diet was recorded and replicated between the two periods. Faecal microbiota composition was profiled using 16S rRNA sequencing, metabolome with 1H NMR, short chain fatty acids (SCFAs) with gas chromatography, and sulphide concentration with colorimetry. Associations between background diet and microbiome responses during MVMM supplementation were explored using machine learning. RESULTS:Following MVMM supplementation, the absolute abundance of Lachnoclostridium and UCG_005 taxa were lower, while the concentrations of total SCFAs, specifically propionate and butyrate, and total sulphide were higher than the control period. Compared to baseline, MVMM supplementation decreased the absolute abundance of Desulfobacterota, Actinobacteriota, Bifidobacteriaceae, Erysipelatoclostridiaceae and Veillonellaceae, and increased the concentration of propionate. Background dietary intakes of saturated fat and total carbohydrates related with the MVMM-induced effect of decreasing Desulfobacterota. Likewise, the intakes of vitamins B2, B12, and E, along with iron, related with the effect of MVMM on decreasing Erysipelatoclostridiaceae. No changes were observed within the control period. CONCLUSION:In healthy adults, short-term MVMM supplementation influences the gut microbiota composition and diet-originating microbial metabolites and potentially host health. REGISTRATION:This trial was registered at clinicaltrials.gov as NCT05371704.

BACKGROUND:Serum low density lipoprotein (LDL) cholesterol shows marked interindividual variation in response to the replacement of saturated fatty acids (SFAs) with unsaturated fatty acids (UFAs). OBJECTIVES:To demonstrate the efficacy of United Kingdom guidelines for exchanging dietary SFAs for UFAs, to reduce serum LDL cholesterol and other cardiovascular disease (CVD) risk factors, and to identify determinants of the variability in LDL cholesterol response. METHODS:Healthy males (n = 109, mean ± SD age 48 ± 11 y; BMI 25.1 ± 3.3 kg/m2), consumed a higher-SFA/lower-UFA diet for 4 wk, followed by an isoenergetic, lower-SFA/higher-UFA diet for 4 wk (achieved intakes SFA:UFA as % total energy 19.1:14.8 and 8.9:24.5, respectively). Serum LDL cholesterol, CVD risk markers, peripheral blood mononuclear cell (PBMC) gene expression, and dietary intakes were assessed at baseline and the end of each diet. RESULTS:Transition from a higher-SFA/lower-UFA to a lower-SFA/higher-UFA diet significantly reduced fasting blood lipids: LDL cholesterol (-0.50 mmol/L; 95% confidence interval [CI]: -0.58, -0.42), high-density lipoprotein (HDL) cholesterol (-0.11 mmol/L; 95% CI: -0.14, -0.08), and total cholesterol (TC) (-0.65 mmol/L; 95% CI:-0.75, -0.55). The dietary exchange also reduced apolipoprotein (apo)B, TC:HDL cholesterol ratio, non-HDL cholesterol, E-selectin (P < 0.0001), and LDL subfraction composition (cholesterol [LDL-I and LDL-II], apoB100 [LDL-I and LDL-II], and TAG [LDL-II]) (P < 0.01). There was also an increase in plasma biomarkers of cholesterol intestinal absorption (β-sitosterol, campesterol, cholestanol), and synthesis (desmosterol) (P < 0.0001) and fold change in PBMC LDL-receptor mRNA expression relative to the higher-SFA/lower-UFA diet (P = 0.035). Marked interindividual variation in the change in serum LDL cholesterol response (-1.39 to +0.77 mmol/L) to this dietary exchange was observed, with 33.7% of this variation explained by serum LDL cholesterol before the lower-SFA/higher-UFA diet and reduction in dietary SFA intake (adjusted R2 27% and 6.7%, respectively). APOE genotype was unrelated to serum LDL cholesterol response to SFA. CONCLUSIONS:These findings support the efficacy of United Kingdom SFA dietary guidelines for the overall lowering of serum LDL cholesterol but showed marked variation in LDL cholesterol response. Further identification of the determinants of this variation will facilitate targeting and increasing the efficacy of these guidelines. The RISSCI-1 study was registered with ClinicalTrials.Gov (No. NCT03270527).

UK guidelines recommend dietary saturated fatty acids (SFAs) should not exceed 10% total energy (%TE) for cardiovascular disease prevention, with benefits observed when SFAs are replaced with unsaturated fatty acids (UFAs). This study aimed to assess the efficacy of a dietary exchange model using commercially available foods to replace SFAs with UFAs. Healthy men (n = 109, age 48, SD 11 year) recruited to the Reading, Imperial, Surrey, Saturated fat Cholesterol Intervention-1 (RISSCI-1) study (ClinicalTrials.Gov n°NCT03270527) followed two sequential 4-week isoenergetic moderate-fat (34%TE) diets: high-SFA (18%TE SFAs, 16%TE UFAs) and low-SFA (10%TE SFAs, 24%TE UFAs). Dietary intakes were assessed using 4-day weighed diet diaries. Nutrient intakes were analysed using paired t-tests, fasting plasma phospholipid fatty acid (PL-FA) profiles and dietary patterns were analysed using orthogonal partial least square discriminant analyses. Participants exchanged 10.2%TE (SD 4.1) SFAs for 9.7%TE (SD 3.9) UFAs between the high and low-SFA diets, reaching target intakes with minimal effect on other nutrients or energy intakes. Analyses of dietary patterns confirmed successful incorporation of recommended foods from commercially available sources (e.g. dairy products, snacks, oils, and fats), without affecting participants’ overall dietary intakes. Analyses of plasma PL-FAs indicated good compliance to the dietary intervention and foods of varying SFA content. RISSCI-1 dietary exchange model successfully replaced dietary SFAs with UFAs in free-living healthy men using commercially available foods, and without altering their dietary patterns. Further intervention studies are required to confirm utility and feasibility of such food-based dietary fat replacement models at a population level.

Purpose Validated biomarkers of food intake (BFIs) have recently been suggested as a useful tool to assess adherence to dietary guidelines or compliance in human dietary interventions. Although many new candidate biomarkers have emerged in the last decades for different foods from metabolic profiling studies, the number of comprehensively validated biomarkers of food intake is limited. Apples are among the most frequently consumed fruits and a rich source of polyphenols and fibers, an important mediator for their health-protective properties. Methods Using an untargeted metabolomics approach, we aimed to identify biomarkers of long-term apple intake and explore how apples impact on the human plasma and urine metabolite profiles. Forty mildly hypercholesterolemic volunteers consumed two whole apples or a sugar and energy-matched control beverage, daily for 8 weeks in a randomized, controlled, crossover intervention study. The metabolome in plasma and urine samples was analyzed via untargeted metabolomics. Results We found 61 urine and 9 plasma metabolites being statistically significant after the whole apple intake compared to the control beverage, including several polyphenol metabolites that could be used as BFIs. Furthermore, we identified several endogenous indole and phenylacetyl-glutamine microbial metabolites significantly increasing in urine after apple consumption. The multiomic dataset allowed exploration of the correlations between metabolites modulated significantly by the dietary intervention and fecal microbiota species at genus level, showing interesting interactions between Granulicatella genus and phenyl-acetic acid metabolites. Phloretin glucuronide and phloretin glucuronide sulfate appeared promising biomarkers of apple intake; however, robustness, reliability and stability data are needed for full BFI validation. Conclusion The identified apple BFIs can be used in future studies to assess compliance and to explore their health effects after apple intake. Moreover, the identification of polyphenol microbial metabolites suggests that apple consumption mediates significant gut microbial metabolic activity which should be further explored.

BACKGROUND:Apples are rich in bioactive polyphenols and fiber. Evidence suggests that consumption of apples or their bioactive components is associated with beneficial effects on lipid metabolism and other markers of cardiovascular disease (CVD). However, adequately powered randomized controlled trials are necessary to confirm these data and explore the mechanisms. OBJECTIVE:We aimed to determine the effects of apple consumption on circulating lipids, vascular function, and other CVD risk markers. METHODS:The trial was a randomized, controlled, crossover, intervention study. Healthy mildly hypercholesterolemic volunteers (23 women, 17 men), with a mean ± SD BMI 25.3 ± 3.7 kg/m2 and age 51 ± 11 y, consumed 2 apples/d [Renetta Canada, rich in proanthocyanidins (PAs)] or a sugar- and energy-matched apple control beverage (CB) for 8 wk each, separated by a 4-wk washout period. Fasted blood was collected before and after each treatment. Serum lipids, glucose, insulin, bile acids, and endothelial and inflammation biomarkers were measured, in addition to microvascular reactivity, using laser Doppler imaging with iontophoresis, and arterial stiffness, using pulse wave analysis. RESULTS:Whole apple (WA) consumption decreased serum total (WA: 5.89 mmol/L; CB: 6.11 mmol/L; P = 0.006) and LDL cholesterol (WA: 3.72 mmol/L; CB: 3.86 mmol/L; P = 0.031), triacylglycerol (WA: 1.17 mmol/L; CB: 1.30 mmol/L; P = 0.021), and intercellular cell adhesion molecule-1 (WA: 153.9 ng/mL; CB: 159.4 ng/mL; P = 0.028), and increased serum uric acid (WA: 341.4 μmol/L; CB: 330 μmol/L; P = 0.020) compared with the CB. The response to endothelium-dependent microvascular vasodilation was greater after the apples [WA: 853 perfusion units (PU), CB: 760 PU; P = 0.037] than after the CB. Apples had no effect on blood pressure or other CVD markers. CONCLUSIONS:These data support beneficial hypocholesterolemic and vascular effects of the daily consumption of PA-rich apples by mildly hypercholesterolemic individuals.This trial was registered at clinicaltrials.gov as NCT01988389.

AbstractApples are a rich source of polyphenols and fiber. Proanthocyanidins (PAs), the largest polyphenolic class in apples, can reach the colon almost intact where they interact with the gut microbiota producing simple phenolic acids. These metabolites have the potential to modulate gut microbiota composition and activity and impact on host physiology. A randomized, controlled, crossover, dietary intervention study was performed to determine the broad effects of whole apple intake on fecal gut microbiota composition and activity. Forty heathy mildly hypercholesterolemic volunteers (23 women, 17 men), with a mean BMI (± SD) 25.3 ± 3.7 kg/m2 and age 51 ± 11 years, consumed 2 apples/day (Renetta Canada, rich in PAs), or a sugar matched control apple beverage, for 8 weeks separated by a 4-week washout period in a random order. Fecal and 24-h urine samples were collected before and after each treatment. The broad effects of apple intake on fecal gut microbiota composition were explored by the high throughput sequencing (HTS) of 16S rRNA gene lllumina MiSeq sequencing (V3-V4 region). Sequencing data analysis was performed using the Quantitative Insight Into Microbial Ecology (QIIME) open-source pipeline version 1.9.1. Specific bacterial groups were also enumerated using the quantitative Fluorescence In Situ Hybridization (FISH). Furthermore, the potential formation of microbial polyphenol metabolites, after apple intake, was explored in urine using Liquid Chromatography (LC) High-Resolution Mass Spectrometry (HRMS) metabolomics. Preliminary analysis showed no changes in gut microbiota abundances measured by Illumina MiSeq, after correction for multiple testing. Apple intake significantly decreased Enterobacteriaceae population (P = 0.04) compared to the control beverage, as determined with FISH. Twenty-four polyphenol microbial metabolites were identified in higher concentrations in the apple group (P < 0.05) compared to the control, including valerolactones, valeric and phenolic acids. In conclusion, preliminary data suggest that the daily intake of 2 Renetta Canada apples significantly decreased Enterobacteriaceae population, a family known for its pathogenic members, in healthy mildly hypercholesterolemic subjects. Moreover, several polyphenol microbial metabolites were identified, suggesting that microbial activity is crucial and a prerequisite for the absorption of apple polyphenols, producing active metabolites with potential health benefits.

Apples are a rich source of polyphenols and fiber. A major proportion of apple polyphenols escape absorption in the small intestine and together with non-digestible polysaccharides reach the colon, where they can serve as substrates for bacterial fermentation. Animal studies suggest a synergistic interaction between apple polyphenols and the soluble fiber pectin; however, the effects of whole apples on human gut microbiota are less extensively studied. Three commercial apple varieties—Renetta Canada, Golden Delicious and Pink Lady—were digested and fermented in vitro using a batch culture colonic model (pH 5.5–6.0, 37 °C) inoculated with feces from three healthy donors. Inulin and cellulose were used as a readily and a poorly fermentable plant fiber, respectively. Fecal microbiota composition was measured by 16S rRNA gene Illumina MiSeq sequencing (V3-V4 region) and Fluorescence in Situ Hybridization. Short chain fatty acids (SCFAs) and polyphenol microbial metabolites were determined. The three apple varieties significantly changed bacterial diversity, increased Actinobacteria relative abundance, acetate, propionate and total SCFAs (p < 0.05). Renetta Canada and Golden Delicious significantly decreased Bacteroidetes abundance and increased Proteobacteria proportion and bifidobacteria population (p < 0.05). Renetta Canada also increased Faecalibacterium prausnitzii, butyrate levels and polyphenol microbial metabolites (p < 0.05). Together, these data suggest that apples, particularly Renetta Canada, can induce substantial changes in microbiota composition and metabolic activity in vitro, which could be associated with potential benefits to human health. Human intervention studies are necessary to confirm these data and potential beneficial effects.

There is now considerable scientific evidence that a diet rich in fruits and vegetables can improve human health and protect against chronic diseases. However, it is not clear whether different fruits and vegetables have distinct beneficial effects. Apples are among the most frequently consumed fruits and a rich source of polyphenols and fiber. A major proportion of the bioactive components in apples, including the high molecular weight polyphenols, escape absorption in the upper gastrointestinal tract and reach the large intestine relatively intact. There, they can be converted by the colonic microbiota to bioavailable and biologically active compounds with systemic effects, in addition to modulating microbial composition. Epidemiological studies have identified associations between frequent apple consumption and reduced risk of chronic diseases such as cardiovascular disease. Human and animal intervention studies demonstrate beneficial effects on lipid metabolism, vascular function and inflammation but only a few studies have attempted to link these mechanistically with the gut microbiota. This review will focus on the reciprocal interaction between apple components and the gut microbiota, the potential link to cardiovascular health and the possible mechanisms of action.

Evidence suggests that a high intake of fruits and vegetables may decrease the risk of chronic diseases, including cardiovascular disease, and that components such as polyphenols and fiber may play a key role in reducing disease risk factors. Apples are among the most frequently consumed fruits in the world and an important source of polyphenols and fiber. Their consumption has been associated with beneficial lipid changes, delayed glucose response, antioxidative and antiinflammatory properties, reduced blood pressure and effects on gut microbiota composition. However, many of these effects are not well explored and the specific mechanisms are unclear. This chapter will describe human intervention studies, animal and in vitro studies investigating the impact of apples and apple components on cardiometabolic disease risk factors and possible mechanisms linked to the gut microbiota.

Apolipoprotein E (APOE) genotype is believed to play an important role in cardiovascular risk. APOE4 carriers have been associated with higher blood lipid levels and a more pro-inflammatory state compared with APOE3/E3 individuals. Although dietary fat composition has been considered to modulate the inflammatory state in humans, very little is known about how APOE genotype can impact on this response. In a follow-up to the main SATgenε study, we aimed to explore the effects of APOE genotype, as well as, dietary fat manipulation on ex vivo cytokine production. Blood samples were collected from a subset of SATgenε participants (n=52/88), prospectively recruited according to APOE genotype (n=26 E3/E3 and n=26 E3/E4) after low-fat (LF), high saturated fat (HSF) and HSF with 3.45g docosahexaenoic acid (DHA) dietary periods (each diet eight weeks in duration assigned in the same order) for the measurement of ex vivo cytokine production using whole blood culture (WBC). Concentrations of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha were measured in WBC supernatant samples after stimulation for 24h with either 0.05 or 1μg/ml of bacterial lipopolysaccharide (LPS). Cytokine levels were not influenced by genotype, whereas, dietary fat manipulation had a significant impact on TNF-α and IL-10 production; TNF-α concentration was higher after consumption of the HSF diet compared with baseline and the LF diet (P<0.05), whereas, IL-10 concentration was higher after the LF diet compared with baseline (P<0.05). In conclusion, our study has revealed the amount and type of dietary fat can significantly modulate the production of TNF-α and IL-10 by ex vivo LPS-stimulated WBC samples obtained from normolipidaemic subjects.