Levels of lipid-derived gut microbial metabolites differ among plant matrices in an in vitro model of colon fermentation.

This study explored differences in microbial lipid metabolites among sunflower seeds, soybeans, and walnuts. The matrices were subjected to in vitro digestion and colonic fermentation. Defatted digested materials and fiber/phenolics extracted therefrom were added to sunflower oil (SO) and also fermented. Targeted and untargeted lipidomics were employed to monitor and tentatively identify linoleic acid (LA) metabolites. Walnut fermentation produced the highest free fatty acids (FFAs), LA, and conjugated LAs (CLAs). Defatted digested walnuts added to SO boosted FFAs and CLAs production; the addition of fibre boosted CLAs, whereas the addition of phenolics only increased 9e,11z-CLA and 10e,12z-CLA. Several di-/tri-hydroxy-C18-FAs, reported as microbial LA metabolites for the first time, were annotated. Permutational multivariate analysis of variance indicated significant impacts of food matrix presence and type on lipidomics and C18-FAs. Our findings highlight how the food matrices affect CLA production from dietary lipids, emphasizing the role of food context in microbial lipid metabolism.

Tempeh fermentation improves the nutritional and functional characteristics of Jack beans (Canavalia ensiformis (L.) DC).

The effect of two processing methods of Jack beans (i.e. cooked bean (CB) and cooked tempeh (CT)) on the in vitro digestibility of protein and starch, as well as the production of short chain fatty acids (SCFAs), γ-aminobutyric acid (GABA), and tryptophan (Trp) metabolites after in vitro colonic fermentation, was investigated. CT was obtained by fungal fermentation after cooking under acidic conditions. CT had significantly higher protein, lower digestible starch, lower total fiber, higher free phenolic compounds, and higher ash content compared to CB. CT exhibited better in vitro protein digestibility than CB and less glucose release during in vitro digestion than CB. A comparable concentration of total SCFAs and GABA was produced after in vitro fermentation of CB and CT, but CB produced more indole than CT, resulting in higher amounts of total Trp metabolites. In summary, our findings show that tempeh fermentation improves the nutritional quality of Jack beans and describe the impact of fermentation on the digestibility of nutrients and the formation of metabolites during colonic fermentation.

The effects of carrot shape and oral processing behaviour on bolus properties and ß-carotene bioaccessibility of raw carrots.

The aim of this study was to investigate the effects of carrot shape (cube vs. julienne) and oral processing behaviour, specifically chewing time, on bolus properties and bioaccessibility of ß-carotene in raw carrots. Participants (n = 20) consumed raw carrot cubes (15 × 15 × 15 mm, 4.2 g/bite) and raw carrot julienne (2 × 3 × 90 mm, 4.2 g/bite) with normal (cube: 20 s/bite; julienne: 28 s/bite) and short (cube: 10 s/bite; julienne: 14 s/bite) chewing time. Expectorated boli were collected and characterized for number and mean area of carrot bolus particles. The proportion of easily extractable ß-carotene of the carrot bolus was taken as an approximate indicator of the potentially bioaccessible ß-carotene. Longer chewing time resulted in significantly more and smaller carrot bolus particles, larger particle surface area (p < 0.01) and higher proportion of easily extractable ß-carotene than shorter chewing of raw carrots of both shapes (Cube_Normal vs. Cube_Short: 29 ± 7 % vs. 23 ± 7 %; Julienne_Normal vs. Julienne Short: 31 ± 8 % vs. 26 ± 6 %, p < 0.05). Carrot shape significantly influenced number and size of bolus particles (p < 0.01) with carrot julienne generating more and smaller carrot bolus particles than carrot cubes. These differences in bolus properties between carrot julienne and cubes did not influence the proportion of easily extractable ß-carotene (p > 0.05). We conclude that differences in oral processing behaviour and the corresponding differences in bolus properties produce only modest differences in ß-carotene bioaccessibility of raw carrots regardless of carrot shape.

Consuming almonds with chocolate or lettuce influences oral processing behaviour, bolus properties and consequently predicted lipid release from almonds.

Lipids in almonds are naturally encapsulated by cell walls which may reduce the actual metabolizable energy content of almonds. Oral processing increases the accessibility of lipids to digestive enzymes by grinding the almond matrix. This study aimed to investigate the effect of adding accompanying foods (chocolate and iceberg lettuce) to almonds on oral processing behaviour, bolus properties and predicted lipid release. Natural chewing times of four almond model foods including one almond (1.3 g), four almonds (4.6 g), one almond with chocolate (4.3 g) and one almond with iceberg lettuce (4.3 g) were collected from n = 59 participants in duplicate. Expectorated boli at the moment of swallowing were characterized for number and mean area of almond bolus particles. Predicted lipid bioaccessibility was estimated using a previously validated model. At similar bite size (4.3-4.6 g), the addition of chocolate and iceberg lettuce to almonds significantly decreased (p < 0.05) chewing time and significantly increased (p < 0.05) eating rate compared to consumption of almonds alone. Almond bolus particle sizes were similar for almonds consumed alone (one and four almonds) and with chocolate, while consuming almonds with lettuce generated significantly fewer and larger almond bolus particles (p < 0.05). Predicted lipid bioaccessibility of almonds consumed with iceberg lettuce was significantly lower (p < 0.05) than for almonds consumed alone (one and four almonds) and almonds consumed with chocolate. Eating rate correlated significantly and positively with the mean area of bolus particles and significantly and negatively with predicted lipid release. In conclusion, combining almonds with other foods such as chocolate and lettuce influences oral processing behaviour and bolus properties and consequently predicted lipid bioaccessibility of almonds, highlighting the impact of food matrix and consumption context on these aspects.

Proximate composition, microstructure, and protein and starch digestibility of seven collections of Jack bean (Canavalia ensiformis) with different optimal cooking times.

Because of its high protein content, Jack bean (Canavalia ensiformis) is a promising alternative protein source. However, the utilization of Jack bean is limited due to the long cooking time to achieve palatable softness. We hypothesize that the cooking time may influence protein and starch digestibility. In this study, we characterized seven Jack bean collections with different optimal cooking times in terms of their proximate composition, microstructure and protein and starch digestibility. Kidney bean was included as a reference for microstructure and protein and starch digestibility. Proximate composition showed that Jack bean collections have a protein content ranging from 28.8 to 39.3%, a starch content ranging from 31 to 41%, a fiber content from 15.4 to 24.6%, and a concanavalin A content in the range 35-51 mg/g dry cotyledon. Particle sizes ranging between 125 and 250 µm were chosen as a representative sample of the whole bean to characterize microstructure and digestibility of the seven collections. Confocal laser microscopy (CLSM) revealed that Jack bean cells have an oval shape and contain starch granules embedded in a protein matrix similar to kidney bean cells. The diameter of Jack bean cells was measured by image analysis of CLSM micrographs and ranged from 103 to 123 µm, while the diameter of starch granules was 31-38 µm, comparatively larger than that of the kidney bean starch granules. Isolated intact cells were used to determine the starch and protein digestibility in the Jack beans collections. The digestion kinetics of starch followed a logistic model, whereas the digestion kinetics of protein followed a fractional conversion model. We found no correlation between optimal cooking time and kinetic parameters of protein and starch digestibility, implying that optimal cooking time is not predictive of protein and starch digestibility. In addition, we tested the effect of reduced cooking times on protein and starch digestibility on one Jack bean collection. The result showed that reducing cooking time significantly reduces starch digestibility, but not protein digestibility. The present study contributes to our understanding of the effect of food processing on protein and starch digestibility in legumes.

Impact of High-Fiber or High-Protein Diet on the Capacity of Human Gut Microbiota To Produce Tryptophan Catabolites.

This study investigated the effect of high-fiber-low-protein (HF) and high-protein-low-fiber (HP) diets on microbial catabolism of tryptophan in the proximal colon (PC) and distal colon(DC) compartments of the Simulator of the Human Intestinal Microbial Ecosystem. The microbiota in PC and DC was dominated by Bacteroidetes and Firmicutes, in which Bacteroidetes were more abundant in DC (∼60% versus 50%) and Firmicutes were more abundant in PC (∼40% versus 25%). Most of the tryptophan catabolites were determined at a higher concentration in PC samples than in DC samples, but the overall concentration of tryptophan catabolites was over 10-fold higher in DC samples than that in PC samples. Interestingly, indole-3-propionic acid and oxindole were only identified in DC samples. A two-week dietary intervention by the HF diet enriched the abundance of Firmicutes in PC, whereas the HP diet enriched the abundance of Proteobacteria. Compared to the HP diet, the HF diet favored the microbial production of indole-3-acetic acid, indole-3-lactic acid, indole-3-aldehyde, and indole-3-propionic acid in both PC and DC compartments. To conclude, these findings increase the understanding of the effect of diets on the microbial production of tryptophan catabolites in the colon.

Effect of whole foods on the microbial production of tryptophan-derived aryl hydrocarbon receptor agonists in growing pigs.

Effects of whole foods on the microbial production of tryptophan-derived aryl hydrocarbon receptor (AhR) ligands in the intestine were investigated in a pig model. Ileal digesta and faeces of pigs after feeding of eighteen different foods were analyzed. Indole, indole-3-propionic acid, indole-3-acetic acid, indole-3-lactic acid, kynurenine, tryptamine, and indole-3-aldehyde were identified in ileal digesta, which were also identified in faeces but at higher concentrations except indole-3-lactic acid, together with skatole, oxindole, serotonin, and indoleacrylic acid. The panel of tryptophan catabolites in ileal digesta and faeces varied across different foods. Eggs induced the highest overall concentration of catabolites in ileal digesta dominated by indole. Amaranth induced the highest overall concentration of catabolites in faeces dominated by skatole. Using a reporter cell line, we observed many faecal samples but not ileal samples retained AhR activity. Collectively, these findings contribute to food selection targeting AhR ligands production from dietary tryptophan in the intestine.

Distinct effects of fiber and colon segment on microbiota-derived indoles and short-chain fatty acids.

Effects of pectin, inulin, and their combination on the production of microbiota-derived indoles and short-chain fatty acids (SCFAs) from different colon segments were investigated in a batch system inoculated with microbiota from proximal colon (PC) and distal colon (DC) compartments of the Simulator of Human Intestinal Microbial Ecosystem. Bacteria from DC compartment had a higher abundance of Firmicutes and a stronger capacity to produce indoles and SCFAs than bacteria from PC compartment. Fiber supplementation significantly increased the production of SCFAs, indole-3-propionic acid, and indole-3-lactic acid, but decreased the production of oxindole, tryptamine, and serotonin. Pectin specifically promoted the production of indole-3-acetic acid and indole-3-aldehyde. Interestingly, supplementation of pectin or inulin increased the relative abundance of Bacteroidetes whereas supplementation of a mixture of two fibers decreased it. Overall, these results suggest that fiber supplementation and colon segment affect the composition of gut microbiota and the microbial catabolism of tryptophan.

In vitro gastrointestinal bioaccessibility and colonic fermentation of lignans from fresh, fermented, and germinated flaxseed.

This research assessed the influence of fermentation and germination as well as of particle size on lignan bioaccessibility from flaxseed by simulated in vitro gastrointestinal digestion. In vitro simulated colonic fermentation was used to study lignan release and its conversion into enterolignans. In addition, tea was included as a representative sample to investigate the stability of lignans in the gastrointestinal tract. Only secoisolariciresinol (SECO) was detected in flaxseed samples. SECO bioaccessibility in fermented flaxseed was highest among all matrices but limited to ≈1% (P < 0.001). Lignan bioaccessibility was significantly influenced by particle size too (P < 0.001 for both). In the colon, fermented flaxseed produced the highest SECO release among all flaxseed samples (≈65%), and the highest conversion of enterolignan (≈1.0%), whereas the conversion of lignans in tea brew was relatively high (≈15%). Lignan conversion varies greatly among donors due to inter-individual differences in microbiota activity. Food fermentation could be a viable strategy for increasing lignan release and conversion to enterolignan.

Monitoring the effect of cell wall integrity in modulating the starch digestibility of durum wheat during different steps of bread making.

Reduction of starch digestibility in starchy foods is beneficial for lowering the risks for major non-communicable diseases. Preserving cell integrity is known to delay starch digestibility in flour but its effect in bread is not clear. In this study, the effect of increasing particle size on in vitro starch digestibility of durum wheat flour, dough, and bread was investigated. Cell integrity was retained during bread processing for medium (1000 µm-1800 µm), and large (>1800 µm) flour, whereas in small one cell walls were mostly damaged (<350 µm). In vitro starch digestibility of flour decreased increasing particle size, but no difference was found in dough. In bread, instead, a modest decrease of starch digestibility for the bread made by large particle was observed, likely due to its dense structure. In conclusion, a high particle size could limit starch digestibility in durum wheat flour but not in bread.

Influence of oral processing behaviour and bolus properties of brown rice and chickpeas on in vitro starch digestion and postprandial glycaemic response.

PURPOSE: Oral processing behaviour may contribute to individual differences in glycaemic response to foods, especially in plant tissue where chewing behaviour can modulate release of starch from the cellular matrix. The aim of this study was to assess the impact of chewing time of two starch based foods (brown rice and chickpeas) on bolus properties, in vitro starch digestion and postprandial glycaemic excursion in healthy subjects. METHODS: In a cross-over trial participants (n = 26) consumed two carbohydrates-identical test meals (brown rice: 233 g; chickpeas: 323 g) with either long (brown rice: 41 s/bite; chickpeas: 37 s/bite) or short (brown rice: 23 s/bite; chickpeas: 20 s/bite) chewing time in duplicate while glycaemic responses were monitored using a continuous glucose monitoring device. Expectorated boli were collected, then bolus properties (number, mean area, saliva amylase activity) and in vitro starch digestion were determined. RESULTS: Longer chewing resulted in significantly (p < 0.05) more and smaller bolus particles, higher bolus saliva uptake and higher in vitro degree of intestinal starch hydrolysis (DH_Schewing time%) than shorter chewing for both foods (brown rice: DH_S%23 s = 84 ± 4% and DH_%S41s = 90 ± 6%; chickpeas: DH_S%20 s = 27 ± 3% and DH_%S37s = 34 ± 5%, p < 0.001). No significant effect of chewing time on glycaemic response (iAUC) (p > 0.05) was found for both meals. Brown rice showed significantly and considerably higher in vitro degree of intestinal starch hydrolysis and glycaemic response (iAUC) than chickpeas regardless of chewing time. No significant correlations were observed between bolus properties and in vitro starch hydrolysis or glycaemic response (p > 0.05). CONCLUSION: Differences in the innate structure of starch based foods (brown rice compared to chickpeas) have a larger effect on postprandial glucose response than differences in mastication behaviour although oral processing behaviour showed consistent effects on bolus properties and in vitro starch digestion. Trial registration ClinicalTrials.gov identifier: NCT04648397 (First posted: December 1, 2020).

Lentil Protein and Tannic Acid Interaction Limits in Vitro Peptic Hydrolysis and Alters Peptidomic Profiles of the Proteins.

In this study, the nature of lentil protein-tannic acid (LPTA) interaction and its effect on in vitro pepsin digestion were investigated. LPTA mixtures containing 1% w/v LP and 0.001-0.5% TA were prepared and characterized in terms of particle size, thermal properties, and secondary and tertiary structures. A 20-fold increase in particle size was observed in LPTA0.5% compared to LP control (without TA), indicating aggregation. Static quenching of tryptophan residues within the protein hydrophobic folds was observed. Increasing TA levels also enhanced protein thermal stability. Over 50% reduction in free amino groups of LPTA 0.5%, relative to LP, was observed after pepsin digestion. Cleavage specificity of pepsin and peptidomic profile of LP were modified by the presence of TA in LPTA 0.5%. This study showed that 0.5% w/v TA induced protein aggregation and reduced LP digestibility by hindering the accessibility of pepsin to the protein network, thus modifying the profile of released peptides.

Tryptophan Supplementation Increases the Production of Microbial-Derived AhR Agonists in an In Vitro Simulator of Intestinal Microbial Ecosystem.

The aryl hydrocarbon receptor (AhR) plays an important role in intestinal homeostasis, and some microbial metabolites of tryptophan are known AhR agonists. In this study, we assessed the impact of tryptophan supplementation on the formation of tryptophan metabolites, AhR activation, and microbiota composition in the simulator of the human intestinal microbial ecosystem (SHIME). AhR activation, microbial composition, and tryptophan metabolites were compared during high tryptophan supplementation (4 g/L tryptophan), control, and wash-out periods. During tryptophan supplementation, the concentration of several tryptophan metabolites was increased compared to the control and wash-out period, but AhR activation by fermenter supernatant was significantly decreased. This was due to the higher levels of tryptophan, which was found to be an antagonist of AhR signaling. Tryptophan supplementation induced most microbial changes in the transverse colon including increased relative abundance of lactobacillus. We conclude that tryptophan supplementation leads to increased formation of AhR agonists in the colon.

Insights into gut microbiota metabolism of dietary lipids: the case of linoleic acid.

It has been recognized that, next to dietary fibre and proteins, gut microbiota can metabolize lipids producing bioactive metabolites. However, the metabolism of dietary lipids by human gut microbiota has been poorly explored so far. This study aimed to examine the change in lipids, particularly linoleic acid (LA), induced by the chemical form of lipids and the presence of the plant matrix. Short-chain fatty acid (SCFA) production was monitored to get an insight into microbial activity. Free LA, glyceryl trilinoleate and soybean oil as well as digested intact (DS) and broken (BS) soybean cells were subjected to in vitro fermentation using human faecal inoculums. Confocal microscopy was used to visualize the soybean cell integrity. Three LA metabolites, including two conjugated fatty acids (CLAs, 9z,11e and 9e,11e) and 12hydroxy, 9z C18:1, were identified and monitored. Free LA addition improved the LA metabolite production but reduced SCFA concentrations compared to trilinoleate and soybean oil. Breaking cell integrity had impacts on CLA, hydroxy C18:1 and SCFA production and free fatty acid release within the first 24 h of fermentation, but this effect vanished with time. In contrast, soybean oil only increased free LA release and hydroxy C18:1 production. The content of several FAs decreased during fermentation suggesting a substantial conversion in microbial metabolites. Besides, LA metabolites were also identified in the fermentation pellets suggesting the incorporation of microbial FA metabolites into bacterial cells. This study expands our understanding of microbial metabolism of dietary lipids with a special emphasis on the role of food- and diet-related factors.

ß-Glucan Interaction with Lentil (Lens culinaris) and Yellow Pea (Pisum sativum) Proteins Suppresses Their In Vitro Digestibility.

In this study, ß-glucan interaction with lentil and yellow pea proteins and the effect on in vitro protein digestibility were investigated. Proteins were mixed with ß-glucan at mass ratios of 1:0.5, 1:1, and 1:2. The interaction between ß-glucan and the proteins was demonstrated by the decrease in transmittance and surface charge and the increase in particle size of the complexes. Bright-field microscopy showed the formation of aggregates between the biopolymers, although increased molecular size was not observed by discontinuous native polyacrylamide gel electrophoresis. Fluorescence microscopy indicated that ß-glucan formed aggregates with lentil proteins, while the interaction with yellow pea proteins appeared as distinct phases of protein within the ß-glucan network. The in vitro protein digestibility of lentil and pea protein decreased by 27.3 and 34.5%, respectively, in the presence of a ß-glucan mass ratio of 1:2. The findings confirm the possibility to modulate protein digestibility by changing the physical characteristics of a food matrix.

Dry-heat processing at different conditions impact the nutritional composition and in vitro starch and protein digestibility of immature rice-based products.

Immature grain represents a precious nutritional source in many rural Africa areas. To optimize processing of immature rice into pepeta (a traditional rice-flakes produced from immature rice grains), immature rice (TXD306 variety) harvested at 18 and 26 days after 50% heading were processed in the laboratory under different soaking (0 and 12 h) and roasting temperature (80, 100 and 120 °C) regimes. Riboflavin, nicotinic acid, nicotinamide and iron concentration increased with severity of roasting temperature, while thiamine has an opposite trend. Heating promoted the transformation of insoluble into soluble dietary fiber, increased lipid digestibility decreasing protein one, which showed the highest value when rice was roasted at 100 °C. Soaking before roasting significantly increased moisture and iron content while slightly increased riboflavin, nicotinic acid and nicotinamide when compared to unsoaked products. Among roasted products, starch digestibility increased with roasting temperature. Microstructure analysis indicated a complete loss of cell wall integrity in cooked rice, determining a complete starch and protein digestion while this is delayed in raw rice and roasted products. We concluded that roasting at 100 °C is the optimum temperature to produce pepeta of the highest protein digestibility and low starch digestibility. Soaking before roasting at 120 °C is best when retaining micronutrients is considered.

In vitro colonic fermentation of red kidney beans depends on cotyledon cells integrity and microbiota adaptation.

In the present study we investigated the effect of cellular integrity on microbial utilization of proteins and carbohydrates by gut microbiota. Cotyledon cells from red kidney beans with different levels of structural integrity were fermented in-vitro by microbial communities previously adapted to the conditions of ascending, transverse and descending colon. The effect of bacterial adaptation to substrate was also assessed by using microbiota exposed to a diet rich in bean cells. Microscopy analyses indicate that cell integrity was maintained during fermentation. The amount of gas generated and the rate of total gas production was higher in broken cells compared to intact cells which suggest a faster and more extensive utilization of nutrients when cell wall is broken. A significantly higher butyric and propionic acid level was detected in broken cells at the end of the fermentation. Moreover, adapted bacterial communities were more efficient in fermenting bean cells where higher amounts of butyrate were produced in all colon regions independently of sample integrity. Bacterial communities of the distal colon appeared to be the most efficient in carbohydrate and protein fermentation as witnessed by the higher levels of gas, and short chain fatty acids. It was also found that cell integrity and adaptation to bean cells modulate the hierarchy of nutrient utilization, with non-starch polysaccharides preferred over starch and proteins by microbiota exposed to bean cells. Our results demonstrated that structural aspects of foods, such as cell integrity in plant tissues, may modulate nutrients utilization by gut microbiota.

Substrate-Driven Differences in Tryptophan Catabolism by Gut Microbiota and Aryl Hydrocarbon Receptor Activation.

SCOPE: This study aims to investigate the effect of tryptophan sources on tryptophan catabolism by gut microbiota and the aryl hydrocarbon receptor (AhR) activation. METHODS AND RESULTS: Four substrates (free tryptophan, soybean protein, single and clustered soybean cells) containing an equimolar amount of tryptophan, but with a different bioaccessibility are studied using in vitro batch fermentation. Tryptophan catabolites are identified by LC-MS/MS. AhR activity is measured by HepG2-Lucia AhR reporter cells. The total amount of tryptophan-derived catabolites increases with decreasing level of substrate complexity. Indole is the major catabolite produced from tryptophan and it is the most abundant in the free tryptophan fermentation. Indole-3-acetic acid and indole-3-aldehyde are abundantly generated in the soybean protein fermentation. The soybean cell fermentation produced high concentrations of tryptamine. Interestingly, large amounts of short-chain fatty acids (SCFAs) are also found in the soybean cell and protein fermentation. Both tryptophan-derived catabolites and SCFAs are able to increase AhR reporter activity over time in all four groups. CONCLUSION: This study illustrates that bacterial catabolism of tryptophan and resulting AhR activation in the gut is modulated by the food matrix, suggesting a role for food design to improve gut health.

Inhibition of α-glucosidases by tea polyphenols in rat intestinal extract and Caco-2 cells grown on Transwell.

Inhibition of maltase, sucrase, isomaltase and glucoamylase activity by acarbose, epigallocatechin gallate, epicatechin gallate and four polyphenol-rich tea extract from white, green, oolong, black tea, were investigated by using rat intestinal enzymes and human Caco-2 cells. Regarding rat intestinal enzyme mixture, all four tea extracts were very effective in inhibiting maltase and glucoamylase activity, but only white tea extract inhibited sucrase and isomaltase activity and the inhibition was limited. Mixed-type inhibition on rat maltase activity was observed. Tea extracts in combination with acarbose, produced a synergistic inhibitory effect on rat maltase activity. Caco-2 cells experiments were conducted in Transwells. Green tea extract and epigallocatechin gallate show dose-dependent inhibition on human sucrase activity, but no inhibition on rat sucrase activity. The opposite was observed on maltase activity. The results highlighted the different response in the two investigated model systems and show that tea polyphenols are good inhibitors for α-glucosidase activity.