Single exposure of food-derived polyethylene and polystyrene microplastics profoundly affects gut microbiome in an in vitro colon model.

Microplastics (MPs) are widespread contaminants highly persistent in the environment and present in matrices to which humans are extensively exposed, including food and beverages. MP ingestion occurs in adults and children and is becoming an emerging public health issue. The gastrointestinal system is the most exposed to MP contamination, which can alter its physiology starting from changes in the microbiome. This study investigates by an omic approach the impact of a single intake of a mixture of polyethylene (PE) and polystyrene (PS) MPs on the ecology and metabolic activity of the colon microbiota of healthy volunteers, in an in vitro intestinal model. PE and PS MPs were pooled together in a homogeneous mix, digested with the INFOGEST system, and fermented with MICODE (multi-unit in vitro colon model) at loads that by literature correspond to the possible intake of food-derived MPs of a single meal. Results demonstrated that MPs induced an opportunistic bacteria overgrowth (Enterobacteriaceae, Desulfovibrio spp., Clostridium group I and Atopobium - Collinsella group) and a contextual reduction on abundances of all the beneficial taxa analyzed, with the sole exception of Lactobacillales. This microbiota shift was consistent with the changes recorded in the bacterial metabolic activity.

Impact of cooking methods of red-skinned onion on metabolic transformation of phenolic compounds and gut microbiota changes.

Herein, we investigated the stability and bioaccessibility of phenolics in differently cooked red-skinned onion (RSO) and consequently their impact on the gut microbiota and metabolism of phenolics. In fact, the different processes used to cook vegetables can modify and re-arrange the molecular profiles of bioactive compounds, such as phenolics in phenolic-rich vegetables, such as RSO. Fried and grilled RSO were compared to raw RSO and a blank control and subjected to oro-gastro-intestinal digestion and subsequent colonic fermentation. For upper gut digestion, the INFOGEST protocol was used, and for lower gut fermentation, a short-term batch model, namely, MICODE (multi-unit in vitro colon gut model), was employed. During the process, phenolic compound profile (through high-resolution mass spectrometry) and colon microbiomics (qPCR of 14 core taxa) analyses were performed. According to the results, the degradation driven by the colon microbiota of RSO flavonols resulted in the accumulation of three main metabolites, i.e., 3-(3'-hydroxyphenyl)propanoic acid, 3-(3'-hydroxyphenyl)acetic acid and 3-(3',4'-dihydroxyphenyl)acetic acid. Also, colonic fermentation of raw onions resulted in a substantial increase in beneficial taxa, which was larger compared to the heat-treated onions, particularly Lactobacillales and beneficial clostridia. Also, a higher level of inhibition of opportunistic bacteria was seen for the raw onion samples, namely, Clostridium perfringens group and Escherichia coli. Thus, our results showed that RSO, and especially the raw one, is an excellent dietary source of flavonols that are strongly metabolized by gut bacteria and can positively modulate the gut microbiota. Although additional in vivo studies are necessary, this work is one of the first to explore how RSO processed with different cooking methods can differently impact the phenolic metabolism and microbiota composition in the large intestine of humans, fine-tuning the antioxidant nature of foods.

Beneficial metabolic transformations and prebiotic potential of hemp bran and its alcalase hydrolysate, after colonic fermentation in a gut model.

Hemp seed bran (HB) is an industrial food byproduct that is generally discarded. Knowledge on the functional capabilities of HB is limited and it is not known the impact of HB on human colon microbiota, where vegetable fibers are metabolized. In this work, we investigated in depth the prebiotic potential of HB and HB protein extract hydrolyzed by alcalase (HBPA) in comparison to fructooligosaccharides (FOS) after human distal colonic fermentation using MICODE (multi-unit in vitro colon gut model). During the 24 h of fermentation, metabolomics (SPME GC/MS) and microbiomics (MiSeq and qPCR) analyses were performed. The results indicated that HBPA on a colonic fermentation had a higher prebiotic index than HB (p < 0.05), and slightly lower to that of FOS (p > 0.05). This feature was described and explained as HBPA colonic fermentation produces beneficial organic fatty acids (e.g. Pentanoic and Hexanoic acids); reduces detrimental phenol derivates (e.g. p-Cresol); produces bioactives VOCs (e.g. Acetophenone or 4-Terpineol); increases beneficial bacteria (e.g. 1.76 fold and 2.07 fold more of Bifidobacterium bifidum and Bacteroides fragilis, respectively) and limits opportunistic bacteria (e.g. 3.04 fold and 2.07 fold less of Bilophila wadsworthia and Desulfovibrio, respectively). Our study evidenced the prebiotic role of HB and HBPA, and within the principles of OneHealth it valorizes a byproduct from the queen plant of sustainable crops as a food supplement.

Maternal amoxicillin affects piglets colon microbiota: microbial ecology and metabolomics in a gut model.

The first weeks of life represent a crucial stage for microbial colonization of the piglets' gastrointestinal tract. Newborns' microbiota is unstable and easily subject to changes under stimuli or insults. Nonetheless, the administration of antibiotics to the sow is still considered as common practice in intensive farming for pathological conditions in the postpartum. Therefore, transfer of antibiotic residues through milk may occurs, affecting the piglets' colon microbiota. In this study, we aimed to extend the knowledge on antibiotic transfer through milk, employing an in vitro dedicated piglet colon model (MICODE-Multi Unit In vitro Colon Model). The authors' focus was set on the shifts of the piglets' microbiota composition microbiomics (16S r-DNA MiSeq and qPCR-quantitative polymerase chain reaction) and on the production of microbial metabolites (SPME GC/MS-solid phase micro-extraction gas chromatography/mass spectrometry) in response to milk with different concentrations of amoxicillin. The results showed an effective influence of amoxicillin in piglets' microbiota and metabolites production; however, without altering the overall biodiversity. The scenario is that of a limitation of pathogens and opportunistic taxa, e.g., Staphylococcaceae and Enterobacteriaceae, but also a limitation of commensal dominant Lactobacillaceae, a reduction in commensal Ruminococcaceae and a depletion in beneficial Bifidobactericeae. Lastly, an incremental growth of resistant species, such as Enterococcaceae or Clostridiaceae, was observed. To the authors' knowledge, this study is the first evaluating the impact of antibiotic residues towards the piglets' colon microbiota in an in vitro model, opening the way to include such approach in a pipeline of experiments where a reduced number of animals for testing is employed. KEY POINTS: • Piglet colon model to study antibiotic transfer through milk. • MICODE resulted a robust and versatile in vitro gut model. • Towards the "3Rs" Principles to replace, reduce and refine the use of animals used for scientific purposes (Directive 2010/63/UE).

Red Beetroot Fermentation with Different Microbial Consortia to Develop Foods with Improved Aromatic Features.

The European culinary culture relies on a wide range of fermented products of plant origin, produced mostly through spontaneous fermentation. Unfortunately, this kind of fermentations is difficult to standardize. Therefore, the use of commercial starter cultures is becoming common to achieve more stable, reproducible, and predictable results. Among plant-based fermentation processes, that of the red beet (Beta vulgaris L. var. conditiva) is scarcely described in the scientific literature. In this work, we compared different types of fermentation methods of beetroot and evaluated the processes' micro-biological, physico-chemical, structural, and volatilome features. A multi-variate analysis was used to match the production of specific VOCs to each starter and to define the correlations between the process variables and volatilome. Overall, the results showed a successful lactic acid fermentation. The analysis of the volatilome clearly discriminated the metabolic profiles of the different fermentations. Among them, the sample fermented with the mixture was the one with the most complex and diversified volatilome. Furthermore, samples did not appear softened after fermentation. Although this work had its weaknesses, such as the limited number of samples and variety, it may pave the way for the standardization of artisanal fermentation procedures of red beetroot in order to improve the quality and safety of the derived food products.

Roasting and frying modulate the phenolic profile of dark purple eggplant and differently change the colon microbiota and phenolic metabolites after in vitro digestion and fermentation in a gut model.

The way of cooking vegetables could differently affect the phenolic profiles of foods and their impact on human colon microbiota. In this work, we investigated the stability and bioaccessibility as well as the impact and fate of dark purple eggplant (DPE) phenolic compounds in the gut microbiota after grilling or frying in comparison to the raw one. After cooking, DPE underwent a gastro-intestinal digestion along with a proximal colon fermentation using the short-term batch model MICODE (multi-unit in vitro colon gut model). During the process, the phenolic compounds profiles (through high-resolution mass spectrometry) and microbiomics (qPCR of 14 core taxa) analyses were performed. Results showed that thermal treatments increased the amount of extractable phenolic compounds as well as their bioaccessibility. The highest gastro-intestinal release was observed in fried DPE (2468.46 ± 13.64 µmol/100 g), followed by grilled DPE (1007. 96 ± 12.84 µmol/100 g) and raw DPE (900.93 ± 10.56 µmol/100 g). Mass spectrometry analysis confirmed that colonic bacteria were able to metabolize DPE phenolic compounds mainly to 3-(3'-hydroxyphenyl)propanoic acid. Furthermore, results indicated that frying was better than grilling in terms of fostering more the growth of beneficial bacterial taxa and limiting that of opportunistic taxa. For example, fried DPE determined an increase in abundance of Bifidobacteriaceae Lactobacillales of 2.66 and 3.80 times. This work is one of the first exploring how cooking methods can affect the phenolic composition of DPE and differently impact on the colon microbiota tuning and modifying the food functionalities.

Looking for peptides from rice starch processing by-product: Bioreactor production, anti-tyrosinase and anti-inflammatory activity, and in silico putative taste assessment.

One of the major challenges for the modern society, is the development of a sustainable economy also aiming at the valorization of agro-industrial by-products in conjunction with at a significant reduction of generated residues from farm to retail. In this context, the present study demonstrates a biotechnological approach to yield bioactive peptides from a protein fraction obtained as a by-product of the rice starch production. Enzymatic hydrolysis, with the commercial proteases Alcalase and Protamex, were optimized in bioreactor up to 2 L of volume. The two best digestates, selected with respect to peptide release and extract antioxidant capacity, were further fractionated (cut-offs of 10, 5, and 1 kDa) via cross-flow filtration. Amino acid composition indicated that most of the fractions showed positive nutritional characteristics, but a putative bitter taste. A fraction obtained with Alcalase enzyme (retentate 8 kDa) exerted anti-inflammatory potential, while the smaller molecular weight fractions (retentate 1-5 kDa and permeate < 1 kDa) were more active in tyrosinase inhibition. The latter were further sub-fractionated by size-exclusion chromatography. From the 15 most anti-tyrosinase sub-fractions, 365 peptide sequences were identified via liquid chromatography coupled with high resolution mass spectrometry. The present data support the possible exploitation of bioactive peptide from rice starch by-product as ingredients into food, nutraceutical, pharmaceutical, and cosmetic formulations.

K-Clique Multiomics Framework: A Novel Protocol to Decipher the Role of Gut Microbiota Communities in Nutritional Intervention Trials.

The availability of omics data providing information from different layers of complex biological processes that link nutrition to human health would benefit from the development of integrated approaches combining holistically individual omics data, including those associated with the microbiota that impacts the metabolisation and bioavailability of food components. Microbiota must be considered as a set of populations of interconnected consortia, with compensatory capacities to adapt to different nutritional intake. To study the consortium nature of the microbiome, we must rely on specially designed data analysis tools. The purpose of this work is to propose the construction of a general correlation network-based explorative tool, suitable for nutritional clinical trials, by integrating omics data from faecal microbial taxa, stool metabolome (1H NMR spectra) and GC-MS for stool volatilome. The presented approach exploits a descriptive paradigm necessary for a true multiomics integration of data, which is a powerful tool to investigate the complex physiological effects of nutritional interventions.

The Exploitation of a Hempseed Byproduct to Produce Flavorings and Healthy Food Ingredients by a Fermentation Process.

Following the One Health principles in food science, the challenge to valorize byproducts from the industrial sector is open. Hemp (Cannabis sativa subsp. sativa) is considered an important icon of sustainability and as an alternative food source. Hemp seed bran, in particular, is a byproduct of industrial hemp seed processing, which is not yet valorized. The success, and a wider market diffusion of hemp seed for food applications, is hindered by its unpleasant taste, which is produced by certain compounds that generally overwhelm the pleasant bouquet of the fresh product. This research concerns the exploration of hemp seed bran through fermentation using beneficial lactobacilli, focusing on the sensorial and bioactive traits of the products when they are subjected to bacterial transformation. By studying of the aromatic profile formation during the fermentation process the aim was to modulate it in order to reduce off-odors without affecting the presence of healthy volatile organic compounds (VOCs). Applying multivariate analyses, it was possible to target the contribution of processing parameters to the generation of flavoring and bioactive compounds. To conclude, the fermentation process proposed was able to reduce unpleasant VOCs, whilst at the same time keeping the healthy ones, and it also improved nutritional quality, depending on time and bacterial starters. The fermentation proposed was a sustainable biotechnological approach that fitted perfectly with the valorization of hemp byproducts from the perspective of a green-oriented industrial process that avoids synthetic masking agents.

Multiunit In Vitro Colon Model for the Evaluation of Prebiotic Potential of a Fiber Plus D-Limonene Food Supplement.

The search for new fiber supplements that can claim to be "prebiotic" is expanding fast, as the role of prebiotics and intestinal microbiota in well-being has been well established. This work explored the prebiotic potential of a novel fiber plus D-Limonene supplement (FLS) in comparison to fructooligosaccharides (FOS) over distal colonic fermentation with the in vitro model MICODE (multi-unit in vitro colon gut model). During fermentation, volatilome characterization and core microbiota quantifications were performed, then correlations among volatiles and microbes were interpreted. The results indicated that FLS generated positive effects on the host gut model, determining: (i) eubiosis; (ii) increased abundance of beneficial bacteria, as Bifidobacteriaceae; (iii) production of beneficial compounds, as n-Decanoic acid; (iv) reduction in detrimental bacteria, as Enterobaceteriaceae; (v) reduction in detrimental compounds, as skatole. The approach that we followed permitted us to describe the prebiotic potential of FLS and its ability to steadily maintain the metabolism of colon microbiota over time. This aspect is two-faced and should be investigated further because if a fast microbial turnover and production of beneficial compounds is a hallmark of a prebiotic, the ability to reduce microbiota changes and to reduce imbalances in the productions of microbial metabolites could be an added value to FLS. In fact, it has been recently demonstrated that these aspects could serve as an adjuvant in metabolic disorders and cognitive decline.

Effect of formulations and fermentation processes on volatile organic compounds and prebiotic potential of gluten-free bread fortified by spirulina (Arthrospira platensis).

Gluten free (GF) foods, designed and marketed for the needs of people who are unable to metabolize gluten, in recent years have aroused growing interest that has led to the conquest of important market segments, with a strongly growing trend. Given the low protein content of standard GF flours, it is particularly important to fortify GF foods, and to study the effect that this process exerts on functional and sensorial characteristics. In this work, fortification of GF bakery goods was done with the addition of Arthrospira platensis (spirulina) flour. Two different dough formulations (with and without fortification) were fermented by four different processes, including spontaneous, single strains and sourdough starters. The baked products were then subjected to "consumer's tests". During the process, fermentation performances, prebiotic activity, and the VOC (Volatile Organic Compound) profiles were analyzed and compared through robust multivariate statistics. The results obtained evidenced that fortification led to a product with more abundant (medium organic acids) and exclusive bioactives (thymol, borneol, and nicotinic acid), which were correlated to the prebiotic activity of spirulina breads. This work, for the first time indicates that spirulina can be used to fortify GF bakery, improving also its functional potential.

Antioxidant and Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides Obtained from Alcalase Protein Hydrolysate Fractions of Hemp (Cannabis sativa L.) Bran.

Proteins from hemp bran (HPB), a byproduct of the hemp seed food-processing chain, were chemically extracted, hydrolyzed by Alcalase, and separated by membrane ultrafiltration into four fractions (MW <1, 1-3, 3-5, and >5 kDa). The antioxidant and antihypertensive properties of the initial extract and the fractions were evaluated by in vitro assays for their ability to scavenge radical species, bind with metal ions, reduce ferric ions, and inhibit angiotensin-converting enzyme (ACE) activity. Bioactive peptides were identified by high-resolution mass spectrometry and sequence comparison with BIOPEP and BioPep DB databases. The hydrolysate was strongly antioxidant and ACE-inhibiting; the most bioactive peptides were further concentrated by ultrafiltration. Of the 239 peptides identified, 47 (12 antioxidant and 35 ACE-inhibitory) exhibited structural features correlated with the specific bioactivity. These results highlight the promise of hydrolysate and size-based HPB fractions as natural functional ingredients for the food or pharmaceutical industry.

Plant Volatiles of Lettuce and Chicory Cultivated in Aquaponics Are Associated to Their Microbial Community.

In this work, an aquaponic cultivation system for Lactuca sativa (L.) and Chicorium intybus (L.) was compared to a hydroponic one, focusing on the main microbial populations related to food safety and their volatile compounds (VOCs), concluding with Spearman correlations among the microbes and VOCs. Different sections of both systems were sampled at the end of the commercial development of the plants. Plants cultivated in aquaponics were in general more contaminated than those from hydroponics, while for the cultivation waters a higher contamination of the hydroponics than aquaponics system was unexpectedly observed. Furthermore, the chicory exhibited higher levels of all microbial groups compared to lettuce grown under the same cultivation system. The results obtained also showed correlations between the distribution of some VOCs and microbial groups in the phyllosphere, while some examples of positive correlations between 2-nonanone (a positive phytostimulant compound) and anaerobic bacilli of the rhizosphere in lettuce were reported. So far, multivariate analysis of VOCs was able to discriminate on the basis of varieties but not on the cultivation systems. In conclusion, the microbial characteristics of the two ecosystems depended both on plant variety and cultivation method but further studies will need to deeply investigate the variables influencing the microbial quality of vegetable foods obtained by aquaponics. On the other hand, the analysis of the VOCs was more related to the microbial community of each plant variety considered, whatever the cultivation system. In precision agriculture, metabolomics may represent an opportunity to study the holobiome and through it the interactions between plants and their microbial populations, to possibly provide for a tool to assess the microbiological quality of vegetable foods obtained by aquaponic systems.

Colonic In Vitro Model Assessment of the Prebiotic Potential of Bread Fortified with Polyphenols Rich Olive Fiber.

The use of olive pomace could represent an innovative and low-cost strategy to formulate healthier and value-added foods, and bakery products are good candidates for enrichment. In this work, we explored the prebiotic potential of bread enriched with Polyphenol Rich Fiber (PRF), a defatted olive pomace byproduct previously studied in the European Project H2020 EcoProlive. To this aim, after in vitro digestion, the PRF-enriched bread, its standard control, and fructo-oligosaccharides (FOS) underwent distal colonic fermentation using the in vitro colon model MICODE (multi-unit colon gut model). Sampling was done prior, over and after 24 h of fermentation, then metabolomic analysis by Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (SPME GCMS), 16S-rDNA genomic sequencing of colonic microbiota by MiSeq, and absolute quantification of main bacterial species by qPCR were performed. The results indicated that PRF-enriched bread generated positive effects on the host gut model: (i) surge in eubiosis; (ii) increased abundance of beneficial bacterial groups, such as Bifidobacteriaceae and Lactobacillales; (iii) production of certain bioactive metabolites, such as low organic fatty acids; (iv) reduction in detrimental compounds, such as skatole. Our study not only evidenced the prebiotic role of PRF-enriched bread, thereby paving the road for further use of olive by-products, but also highlighted the potential of the in vitro gut model MICODE in the critical evaluation of functionality of food prototypes as modulators of the gut microbiota.

Volatilome changes during probiotic fermentation of combined soy and rice drinks.

Plant-based drinks as a substitute for animal milk consumption are crucial products in the food industry. Soy and rice drinks are the most successful milk substitutes but are low in fiber and protein contents, respectively, whilst being rich in sugars. Generally, an improvement is foreseen; thus, apart from supplement addition, a natural occurring strategy is functionalizing the drinks by beneficial bacteria fermentation. The aim of this work is to develop novel plant-based drinks assessing different mixtures of soy and rice milks fermented with single or multi-strain probiotics (Lactobacillus fermentum, L. plantarum, L. helveticus, Bifidobacterium bifidum, and B. longum). The drinks were characterized to study bacterial performances, by means of culture-dependent and -independent techniques, and their volatilome, by means of solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis. Through multivariate analysis, these features were investigated and correlated to define accurate descriptors of the produced functional drinks. The results showed that combined drinks and multi-strain fermentation generated higher-value products. For example, combined drinks in comparison with single ones had a lower amount of toxic 2-acetyl-3,5-dimethylfuran and higher abundances of desirable compounds such as 2-butanone, 3-hydroxy and butanoic acid. Multivariate analysis of volatile metabolites and physiological parameters could offer a novel approach to assess the quality of functional plant-based drinks and result in a decisional tool for industrial applications.

Gluten free sourdough bread enriched with cricket flour for protein fortification: Antioxidant improvement and Volatilome characterization.

Insects represent a novel source of edible high nutritional value proteins which are gaining increasing interest as an alternative to traditional animal foods. In this work, cricket flour was used to produce gluten-free sourdough breads, suitable for celiac people and "source of proteins". The doughs were fermented by different methods and pH and microbial growth, volatile compounds, protein profile, and antioxidant activity, before and after baking, were analyzed and compared to standard gluten-free doughs. The results showed that cricket-enriched doughs and the standard had similar fermentation processes. Cricket enrichment conferred to the breads a typical flavoring profile, characterized by a unique bouquet of volatile compounds, made by nonanoic acid, 2,4-nonadienal (E,E), 1-hexanol, 1-heptanol, and 3-octen-2-one, expressed in different amounts depending on the type of inoculum. Finally, antioxidant activities were significantly enhanced in cricket breads, indicating that cricket powder provides to bakery gluten-free goods high nutritional value proteins and antioxidant properties.

Microbial Fermentation of Industrial Rice-Starch Byproduct as Valuable Source of Peptide Fractions with Health-Related Activity.

The rice-starch processing industry produces large amounts of a protein-rich byproducts during the conversion of broken rice to powder and crystal starch. Given the poor protein solubility, this material is currently discarded or used as animal feed. To fully exploit rice's nutritional properties and reduce this waste, a biotechnological approach was adopted, inducing fermentation with selected microorganisms capable of converting the substrate into peptide fractions with health-related bioactivity. Lactic acid bacteria were preferred to other microorganisms for their safety, efficient proteolytic system, and adaptability to different environments. Peptide fractions with different molecular weight ranges were recovered from the fermented substrate by means of cross-flow membrane filtration. The fractions displayed in vitro antioxidant, antihypertensive, and anti-tyrosinase activities as well as cell-based anti-inflammatory and anti-aging effects. In the future, the peptide fractions isolated from this rice byproduct could be directly exploited as health-promoting functional foods, dietary supplements, and pharmaceutical preparations. The suggested biotechnological process harnessing microbial bioconversion may represent a potential solution for many different protein-containing substrates currently treated as byproducts (or worse, waste) by the food industry.

Intestinal fermentation in vitro models to study food-induced gut microbiota shift: an updated review.

In vitro gut fermentation models were firstly introduced in nutrition and applied microbiology research back in the 1990s. These models have improved greatly during time, mainly over the resemblance to the complexity of digestion stages, the replication of experimental conditions, the multitude of ecological parameters to assay. The state of the science is that the most competitive models shall include a complex gut microbiota, small working volumes, distinct interconnected compartments and rigorous bio-chemical and ecological settings, controlled by a computer, as well as a free-hands accessibility, not to contaminate the mock microbiota. These models are a useful tool to study the impact of a given diet compound, e.g. prebiotics, on the human gut microbiota. The principal application is to focus on the shift of the core microbial groups and selected species together with their metabolites, assaying their diversity, richness and abundance in the community over time. Besides, it is possible to study how a compound is digested, which metabolic pathways are triggered, and the type and quantity of microbial metabolites produced. Further prospective should focus on challenges with pathogens as well as on ecology of gut syndromes. In this minireview an updated presentation of the most used intestinal models is presented, basing on their concept, technical features, as well as on research applications.

Functional, nutritional, antioxidant, sensory properties and comparative peptidomic profile of faba bean (Vicia faba, L.) seed protein hydrolysates and fortified apple juice.

Enzymatic hydrolysis of plant-derived proteins can improve their quality by offering opportunities for food applications. In this study, three proteolytic enzymes (pepsin, trypsin, Alcalase®) were used, alone or combined, to produce faba bean protein hydrolysates (PHs). Their functional, nutritional and antioxidant properties were evaluated, and the peptidomic profile was assessed by LC-MS/MS. Hydrolysis improved solubility of faba proteins at acidic and neutral pH, and their antioxidant properties. Peptidomic analysis identified 2031 peptides in the different PHs. Among them, 9 showed 100% homology with previously known antioxidant peptides and several others had antioxidant motifs in their sequences. Sensory data analysis showed that after addition of PHs to apple juice, no significant differences were perceived between control and some of the PHs. This study demonstrates that enzymatic hydrolysis enhances the functional and antioxidant properties of faba bean proteins. Specifically, hydrolysates can be used as functional food ingredients to produce fortified beverages.