q2-metnet: QIIME2 package to analyze 16S rRNA data via high-quality metabolic reconstructions of the human gut microbiota.

MOTIVATION: 16S rRNA gene sequencing is the most frequent approach for the characterization of the human gut microbiota. Despite different efforts in the literature, the inference of functional and metabolic interpretations from 16S rRNA gene sequencing data is still a challenging task. High-quality metabolic reconstructions of the human gut microbiota, such as AGORA and AGREDA, constitute a curated resource to improve functional inference from 16S rRNA data, but they are not typically integrated into standard bioinformatics tools. RESULTS: Here, we present q2-metnet, a QIIME2 plugin that enables the contextualization of 16S rRNA gene sequencing data into AGORA and AGREDA. In particular, based on relative abundances of taxa, q2-metnet determines normalized activity scores for the reactions and subsystems involved in the selected metabolic reconstruction. Using these scores, q2-metnet allows the user to conduct differential activity analysis for reactions and subsystems, as well as exploratory analysis using PCA and hierarchical clustering. We apply q2-metnet to a dataset from our group that involves 16S rRNA data from stool samples from lean, allergic to cow's milk, obese and celiac children, and the Belgian Flemish Gut Flora Project cohort, which includes faecal 16S rRNA data from obese and normal-weight adult individuals. In the first case, q2-metnet outperforms existing algorithms in separating different clinical conditions based on predicted pathway abundances and subsystem scores. In the second case, q2-metnet complements competing approaches in predicting functional alterations in the gut microbiota of obese individuals. Overall, q2-metnet constitutes a powerful bioinformatics tool to provide metabolic context to 16S rRNA data from the human gut microbiota. AVAILABILITY: Python code of q2-metnet is available in https://github.com/PlanesLab/q2-metnet and https://figshare.com/articles/dataset/q2-metnet_package/26180446. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Extending PROXIMAL to predict degradation pathways of phenolic compounds in the human gut microbiota.

Despite significant advances in reconstructing genome-scale metabolic networks, the understanding of cellular metabolism remains incomplete for many organisms. A promising approach for elucidating cellular metabolism is analysing the full scope of enzyme promiscuity, which exploits the capacity of enzymes to bind to non-annotated substrates and generate novel reactions. To guide time-consuming costly experimentation, different computational methods have been proposed for exploring enzyme promiscuity. One relevant algorithm is PROXIMAL, which strongly relies on KEGG to define generic reaction rules and link specific molecular substructures with associated chemical transformations. Here, we present a completely new pipeline, PROXIMAL2, which overcomes the dependency on KEGG data. In addition, PROXIMAL2 introduces two relevant improvements with respect to the former version: i) correct treatment of multi-step reactions and ii) tracking of electric charges in the transformations. We compare PROXIMAL and PROXIMAL2 in recovering annotated products from substrates in KEGG reactions, finding a highly significant improvement in the level of accuracy. We then applied PROXIMAL2 to predict degradation reactions of phenolic compounds in the human gut microbiota. The results were compared to RetroPath RL, a different and relevant enzyme promiscuity method. We found a significant overlap between these two methods but also complementary results, which open new research directions into this relevant question in nutrition.

BN-BacArena: Bayesian network extension of BacArena for the dynamic simulation of microbial communities.

MOTIVATION: Simulating gut microbial dynamics is extremely challenging. Several computational tools, notably the widely used BacArena, enable modeling of dynamic changes in the microbial environment. These methods, however, do not comprehensively account for microbe-microbe stimulant or inhibitory effects or for nutrient-microbe inhibitory effects, typically observed in different compounds present in the daily diet. RESULTS: Here, we present BN-BacArena, an extension of BacArena consisting on the incorporation within the native computational framework of a Bayesian network model that accounts for microbe-microbe and nutrient-microbe interactions. Using in vitro experiments, 16S rRNA gene sequencing data and nutritional composition of 55 foods, the output Bayesian network showed 23 significant nutrient-bacteria interactions, suggesting the importance of compounds such as polyols, ascorbic acid, polyphenols and other phytochemicals, and 40 bacteria-bacteria significant relationships. With test data, BN-BacArena demonstrates a statistically significant improvement over BacArena to predict the time-dependent relative abundance of bacterial species involved in the gut microbiota upon different nutritional interventions. As a result, BN-BacArena opens new avenues for the dynamic modeling and simulation of the human gut microbiota metabolism. AVAILABILITY AND IMPLEMENTATION: MATLAB and R code are available in https://github.com/PlanesLab/BN-BacArena.

Human gut microbiota fermentation of cooked eggplant, garlic, and onion supports distinct microbial communities.

Heating and cooking vegetables not only enhances their palatability but also modifies their chemical structure, which in turn might affect their fermentation by resident gut microbes. Three commonly consumed vegetables that are known to undergo chemical browning, also known as Maillard reaction, during cooking - eggplant, garlic, and onion - were each fried, grilled, or roasted. The cooked vegetables were then subjected to an in vitro digestion-fermentation process aimed to simulate the passage of food through the human oro-gastro-intestinal tract. In the last step, the undigested fractions of these foods were anaerobically fermented by the complex human gut microbiota. We assessed the structure of microbial communities maintained on each cooked vegetable by high-throughput 16S rRNA gene amplicon sequencing, measured the levels of furosine, a chemical marker of the Maillard browning reaction, by HPLC, and determined the antioxidant capacities in all samples with ABTS and FRAP methods. Overall, vegetable type had the largest, statistically significant, effect on the microbiota structure followed by the cooking method. Onion fermentation supported a more beneficial community including an expansion of Bifidobacterium members and inhibition of Enterobacteriaceae. Fermentation of cooked garlic promoted Faecalibacterium growth. Among cooking methods, roasting led to a much higher ratio of beneficial-to-detrimental microbes in comparison with grilling and frying, possibly due to the exclusion of any cooking oil in the cooking process.

Cultivar and Harvest Time of Almonds Affect Their Antioxidant and Nutritional Profile through Gut Microbiota Modifications.

Almonds are a rich source of beneficial compounds for human health. In this work, we assessed the influence of almond cultivars and harvest time on their morphological (length, width and thickness) and nutritional (ash, moisture, proteins) profiles. We also evaluated the impact of an in vitro digestion and fermentation process on almonds' antioxidant and phenolic content, as well as their support of gut microbiota community and functionality, including the production of short-chain fatty acids (SCFAs), lactic and succinic acids. The length, width, and thickness of almonds varied significantly among cultivars, with the latter two parameters also exhibiting significant changes over time. Moisture content decreased with maturity, while protein and ash increased significantly. Total antioxidant capacity released by almonds after digestion and fermentation had different trends depending on the antioxidant capacity method used. The fermentation step contributed more to the antioxidant capacity than the digestion step. Both cultivar and harvest time exerted a significant influence on the concentration of certain phenolic compounds, although the total content remained unaffected. Similarly, fecal microbiota modulation depended on the cultivar and maturity stage, with the Guara cultivar and late maturity showing the largest effects. Cultivar type also exerted a significant impact on the concentration of SCFAs, with the Guara cultivar displaying the highest total SCFAs concentration. Thus, we conclude that cultivar and harvest time are key factors in shaping the morphological and nutritional composition of almonds. In addition, taking into account all the results obtained, the Guara variety has the best nutritional profile.

Development of a food composition database of different food contaminants CONT11 and estimation of dietary exposure in children of southern Spain.

Increasing food security is one of the Sustainable Development Goals. One of the main risks in food is the increase in food contaminants. Processing methods, such as the addition of additives or heat treatment, influence contaminant generation and increase their levels in food. The aim of the present study was to create a database using a methodology similar to that of food composition databases but with a focus on potential food contaminants. CONT11 collects information on 11 contaminants: hydroxymethyl-2-furfural, pyrraline, Amadori compounds, furosine, acrylamide, furan, polycyclic aromatic hydrocarbons, benzopyrene, nitrates, nitrites and nitrosamines. This is collected for more than 220 foods obtained from 35 different data sources. A food frequency questionnaire validated for use with children was used to validate the database. Contaminant intake and exposure in 114 children aged 10-11 years were estimated. Outcomes were within the range of values described by other studies, confirming the usefulness of CONT11. This database will allow nutrition researchers to go a step further in assessing dietary exposure to some food components and the association of this with disease, whilst also informing strategies to reduce exposure.

Total Zn of foods and bioaccesible fractions in the small and large intestine after in vitro digestion and fermentation with fecal material of healthy adults and children: Influence of culinary techniques.

The healthy status of human beings is associated with an appropriate nutritional status in Zn, which must firstly be bioavailable. We measured the total Zn amount and its bioaccesibility in raw foods and after cooking by common culinary techniques. These foods were submitted to an in vitro digestion and fermentation with faecal inocula from healthy adults and children to evaluate Zn bioaccesibility in the small and large intestine. Mean total Zn amount provided by foods was 8.080 µg/g. Zn amount released from food in the small intestine was significantly different among several food groups and lower in raw vegetal foods compared to cooked ones (frying, roasting and grilling; p < 0.05); the same behaviour was found in the large intestine for healthy children. Zn bioaccesibility in the large intestine varied statistically according to the subjects' idiosyncrasies, and was higher in healthy children (p < 0.05) probably due to growth demands and different composition of the colonic microbiota. In healthy adults and children, the bioaccesible fractions were 33.0 ± 20.4 % for the small intestine, 16.4 ± 22.0 and 59.6 ± 29.9% for the large one, and the non-bioaccessible ones 50.6 ± 19.9 and 7.4 ± 9.1%, respectively.

Effect of in vitro digestion and fermentation on antioxidant capacity of weight loss foods and Maillard reaction products content.

Cereal snacks and meal replacement shakes are gaining popularity as part of a low-calorie diet. However, some concerns have been risen in relation to their nutrient content and industrial processing. Here we analyzed 74 products, including cereal bars, cereal cakes and meal replacement shakes. We measured furosine and 5-hydroxymethyl-furfural (HMF) due to their relation with industrial processing, mainly thermal treatment, as well as antioxidant capacity after in vitro digestion-fermentation. Most of the products reported a high sugar content, including also large concentrations of HMF and furosine. Small differences were found on antioxidant capacity, although chocolate addition tended to increase the antioxidant power of products. According to our results, antioxidant capacity released after fermentation is higher, which points out to the importance of gut microbes in releasing potentially bioactive compounds. Additionally, we have found alarmingly high concentrations of furosine and HMF, which calls to research into new technologies for food processing to minimize their generation.

Stance4Health Nutritional APP: A Path to Personalized Smart Nutrition.

Access to good nutritional health is one of the principal objectives of current society. Several e-services offer dietary advice. However, multifactorial and more individualized nutritional recommendations should be developed to recommend healthy menus according to the specific user's needs. In this article, we present and validate a personalized nutrition system based on an application (APP) for smart devices with the capacity to offer an adaptable menu to the user. The APP was developed following a structured recommendation generation scheme, where the characteristics of the menus of 20 users were evaluated. Specific menus were generated for each user based on their preferences and nutritional requirements. These menus were evaluated by comparing their nutritional content versus the nutrient composition retrieved from dietary records. The generated menus showed great similarity to those obtained from the user dietary records. Furthermore, the generated menus showed less variability in micronutrient amounts and higher concentrations than the menus from the user records. The macronutrient deviations were also corrected in the generated menus, offering a better adaptation to the users. The presented system is a good tool for the generation of menus that are adapted to the user characteristics and a starting point to nutritional interventions.

Effects of different foods and cooking methods on the gut microbiota: an in vitro approach.

To support personalized diets targeting the gut microbiota, we employed an in vitro digestion-fermentation model and 16S rRNA gene sequencing to analyze the microbiota growing on representative foods of the Mediterranean and Western diets, as well as the influence of cooking methods. Plant- and animal-derived foods had significantly different impacts on the abundances of bacterial taxa. Animal and vegetable fats, fish and dairy products led to increases in many taxa, mainly within the Lachnospiraceae. In particular, fats favored increases in the beneficial bacteria Faecalibacterium, Blautia, and Roseburia. However, butter, as well as gouda cheese and fish, also resulted in the increase of Lachnoclostridium, associated to several diseases. Frying and boiling produced the most distinct effects on the microbiota, with members of the Lachnospiraceae and Ruminococcaceae responding the most to the cooking method employed. Nevertheless, cooking effects were highly individualized and food-dependent, challenging the investigation of their role in personalized diets.

Relationship of Thermal Treatment and Antioxidant Capacity in Cooked Foods.

Most of the foods we eat undergo a cooking process before they are eaten. During such a process, the non-enzymatic browning occurs, which generates compounds such as furosine, 5-hydroxymethylfurfural (HMF) and furfural. These are considered markers of cookedness and can therefore be used as quality indicators. In this work, we study the production of these compounds in different foods (both of plant and animal origin) that are cooked with different techniques. Additionally, we investigate correlations between the production of these markers of cookedness and the antioxidant capacity produced after in vitro digestion and fermentation. We observe that, in general, cereals and vegetables are more thermally damaged. Toasting and frying produce the highest concentrations of Maillard compounds whereas boiling the lowest. Furosine content shows a significant positive correlation with in vitro digestion data in fried foods, and with fermentation in roasted foods. Furfural content shows a significant positive correlation with in vitro digestion results in roasted foods, specifically in the Folin-Ciocalteu method.

The Intake of Antioxidant Capacity of Children Depends on Their Health Status.

The gastrointestinal digestion of food and further gut microbial activity render a myriad of different molecules that could be responsible for the biological activities that are classically assigned to their parent compounds. This has been previously shown for some phytochemicals whose antioxidant capacity was either increased or decreased after being metabolized by gut microbes. Whether a global antioxidant capacity that is extracted from food is determined by the gut microbial community structure is still not well described. In the present study, we in vitro digested and fermented 48 different foods that were submitted to different culinary treatments using the stools of lean children, obese children, celiac children and children with an allergy to cow's milk proteins. Their antioxidant capacities were assessed with the DPPH and FRAP assays, and the percentage that each food contributed to their daily antioxidant intake as well as their antioxidant capacity by portion size was inferred. Overall, cereals, fruits and vegetables displayed a higher contribution to their daily antioxidant intake, while tubers, fish and meat exhibited a higher antioxidant capacity by serving size. The food that was fermented in the lean children's and those children that were allergic to cow's milk protein's fecal material, showed a higher antioxidant capacity, which could imply that there is a larger role of the gut microbiota in this area.

Dietary Melanoidins from Biscuits and Bread Crust Alter the Structure and Short-Chain Fatty Acid Production of Human Gut Microbiota.

Melanoidins are the products of the Maillard reaction between carbonyl and amino groups of macromolecules and are readily formed in foods, especially during heat treatment. In this study we utilized the three-stage Human Gut Simulator system to assess the effect of providing melanoidins extracted from either biscuits or bread crust to the human gut microbiota. Addition of melanoidins to the growth medium led to statistically significant alterations in the microbial community composition, and it increased short-chain fatty acid and antioxidant production by the microbiota. The magnitude of these changes was much higher for cultures grown with biscuit melanoidins. Several lines of evidence indicate that such differences between these melanoidin sources might be due to the presence of lipid components in biscuit melanoidin structures. Because melanoidins are largely not degraded by human gastrointestinal enzymes, they provide an additional source of microbiota-accessible nutrients to our gut microbes.

The Gut Microbiota of Obese Children Releases Lower Antioxidant Capacity from Food than That of Lean Children.

The prevalence of obesity has been increasing in children over the last few decades, becoming a concern for health professionals and governments. Gut microbial community structure in obese people have been found to differ from that of lean subjects for some taxa which could result in different production of microbial metabolites. The aim of the present work was to study whether the gut microbiota from obese children extracts a different concentration of antioxidant capacity than the gut microbiota from lean children. For this purpose, different foods were in vitro digested and in vitro fermented using fecal material from obese and lean children. FRAP, DPPH and Folin-Ciocalteu methods were used to measure the antioxidant capacity released during digestion and fermentation. Overall, when using lean gut microbiota, antioxidant capacity released was higher when measured via DPPH and FRAP. Moreover, according to DPPH results, lean gut microbiota could potentially release more antioxidant power from vegetables than from animal products, while obese gut microbiota did the opposite. On the contrary, with the FRAP method obese gut microbiota released higher levels of antioxidant power from plant products than from animal products, but the final antioxidant capacity was still lower than that released by lean gut microbiota. Therefore, these results reflect that the total antioxidant capacity of foods is influenced by the gut microbiota, although whether that antioxidant capacity is released from plant or animal products can be slightly influenced by the method used for analysis.

Prediction of degradation pathways of phenolic compounds in the human gut microbiota through enzyme promiscuity methods.

The relevance of phenolic compounds in the human diet has increased in recent years, particularly due to their role as natural antioxidants and chemopreventive agents in different diseases. In the human body, phenolic compounds are mainly metabolized by the gut microbiota; however, their metabolism is not well represented in public databases and existing reconstructions. In a previous work, using different sources of knowledge, bioinformatic and modelling tools, we developed AGREDA, an extended metabolic network more amenable to analyze the interaction of the human gut microbiota with diet. Despite the substantial improvement achieved by AGREDA, it was not sufficient to represent the diverse metabolic space of phenolic compounds. In this article, we make use of an enzyme promiscuity approach to complete further the metabolism of phenolic compounds in the human gut microbiota. In particular, we apply RetroPath RL, a previously developed approach based on Monte Carlo Tree Search strategy reinforcement learning, in order to predict the degradation pathways of compounds present in Phenol-Explorer, the largest database of phenolic compounds in the literature. Reactions predicted by RetroPath RL were integrated with AGREDA, leading to a more complete version of the human gut microbiota metabolic network. We assess the impact of our improvements in the metabolic processing of various foods, finding previously undetected connections with output microbial metabolites. By means of untargeted metabolomics data, we present in vitro experimental validation for output microbial metabolites released in the fermentation of lentils with feces of children representing different clinical conditions.

The Stance4Health Project: Evaluating a Smart Personalised Nutrition Service for Gut Microbiota Modulation in Normal- and Overweight Adults and Children with Obesity, Gluten-Related Disorders or Allergy/Intolerance to Cow's Milk.

Unhealthy diets represent a major risk for the pathogenesis of metabolic and chronic inflammatory diseases. Improving the quality of diet is important to prevent chronic diseases, and diet-induced modifications of the gut microbiota (GM) community likely play an important role. The EU-funded Stance4Health project aims at performing a randomized clinical trial based on a nutritional intervention program in the context of normal weight and overweight adults as well as children with obesity and gluten-related disorders or allergy/intolerance to cow's milk. The trial will evaluate the efficacy of a Smart Personalised Nutrition (SPN) service in modifying GM composition and metabolic function and improving consumer empowerment through technology adoption.

Growth of Bifidobacterium species is inhibited by free fatty acids and bile salts but not by glycerides.

High-fat diets have been associated with lower gut and fecal abundances of genus Bifidobacterium. Here, we investigated whether commonly consumed dietary free fatty acids have any detrimental effect on the growth of B. adolescentis, B. bifidum, and B. longum. We found that the presence of free fatty acids in the medium inhibits the growth of Bifidobacterium species to a varying degree, with capric (C10:0), oleic (C18:1), and linoleic (C18:2) acids displaying the largest effect. In comparison, free fatty acids did not affect the growth of Escherichia coli. When fats were added as a mixture of mono- and diacylglycerols, the inhibitory effect on Bifidobacterium growth was abolished.

Evaluating the effects of a standardized polyphenol mixture extracted from poplar-type propolis on healthy and diseased human gut microbiota.

INTRODUCTION: A large body of evidence suggests that propolis exerts antioxidant, anti-inflammatory, and antimicrobial activities, mostly ascribed to its polyphenol content. Growing evidence suggests that propolis could modulate gut microbiota exerting a positive impact on several pathological conditions. The aim of this study was to determine the in vitro impact of a poplar-type propolis extract with a standardized polyphenol content, on the composition and functionality of gut microbiota obtained from fecal material of five different donors (healthy adults, and healthy, obese, celiac, and food allergic children). METHODS: The standardized polyphenol mixture was submitted to a simulated in vitro digestion-fermentation process, designed to mimic natural digestion in the human oral, gastric, and intestinal chambers. The antioxidant profile of propolis before and after the digestion-fermentation process was determined. 16 S rRNA amplicon next-generation sequencing (NGS) was used to test the effects on the gut microbiota of propolis extract. The profile of the short-chain fatty acids (SCFA) produced by the microbiota was also investigated through a chromatographic method coupled with UV detection. RESULTS: In vitro digestion and fermentation induced a decrease in the antioxidant profile of propolis (i.e., decrease of total polyphenol content, antiradical and reducing activities). Propolis fermentation exhibited a modulatory effect on gut microbiota composition and functionality of healthy and diseased subjects increasing the concentration of SCFA. CONCLUSIONS: Overall, these data suggest that propolis might contribute to gut health and could be a candidate for further studies in view of its use as a prebiotic ingredient.

Development of an Unified Food Composition Database for the European Project "Stance4Health".

The European Commission funded project Stance4Health (S4H) aims to develop a complete personalised nutrition service. In order to succeed, sources of information on nutritional composition and other characteristics of foods need to be as comprehensive as possible. Food composition tables or databases (FCT/FCDB) are the most commonly used tools for this purpose. The aim of this study is to describe the harmonisation efforts carried out to obtain the Stance4Health FCDB. A total of 10 FCT/FCDB were selected from different countries and organizations. Data were classified using FoodEx2 and INFOODS tagnames to harmonise the information. Hazard analysis and critical control points analysis was applied as the quality control method. Data were processed by spreadsheets and MySQL. S4H's FCDB is composed of 880 elements, including nutrients and bioactive compounds. A total of 2648 unified foods were used to complete the missing values of the national FCDB used. Recipes and dishes were estimated following EuroFIR standards via linked tables. S4H's FCDB will be part of the smartphone app developed in the framework of the Stance4Health European project, which will be used in different personalized nutrition intervention studies. S4H FCDB has great perspectives, being one of the most complete in terms of number of harmonized foods, nutrients and bioactive compounds included.

Effect of roasting conditions on cocoa bioactivity and gut microbiota modulation.

Cocoa is a highly consumed food with beneficial effects on human health. Cocoa roasting has an important influence on its sensory and nutritional characteristics; therefore, roasting could also play a role in cocoa bioactivity. Thus, the aim of this paper is to unravel the effect of cocoa roasting conditions on its antioxidant capacity and modifications of gut microbiota after in vitro digestion-fermentation. HMF and furfural, chemical markers of non-enzymatic browning, were analyzed in unroasted and roasted cocoa powder at different temperatures, as well as different chocolates. The antioxidant capacity decreased with roasting, most probably due to the loss of phenolic compounds during heating. In the case of the evaluated chocolates, the antioxidant capacity was 2-3 times higher in the fermented fraction. On the other hand, HMF and furfural content increased during roasting due to increasing temperatures. Moreover, unroasted and roasted cocoa powder have different effects on gut microbial communities. Roasted cocoa favored butyrate production, whereas unroasted cocoa favored acetate and propionate production in a significant manner. In addition, unroasted and roasted cocoa produced significantly different gut microbial communities in terms of composition. Although many bacteria were affected, Veillonella and Faecalibacterium were some of the most discriminant ones; whereas the former is a propionate producer, the latter is a butyrate producer that has also been linked to positive effects on the inflammatory health of the gut and the immune system. Therefore, unroasted and roasted cocoa (regardless of the roasting temperature) promote different bacteria and a different SCFA production.