The Effect of Food Matrix Taken with Probiotics on the Survival of Commercial Probiotics in Simulation of Gastrointestinal Digestion.

The adequate survival of probiotics in the harsh environment of the gastrointestinal (GI) tract plays a crucial role in the expression of their functional properties. The aim of the present study was to evaluate the survival of commercial probiotics during digestion using a standardised INFOGEST 2.0 model extended with three food matrices simulating three scenarios for the consumption of probiotics: on an empty stomach, with juice, or with food (porridge). All eight products matched the bacterial content stated on the label. After simulated digestion, we observed an average decrease in viability of 1.6 log10 colony forming units (CFU) when the product was co-digested with water, a 2.5 log10 CFU decrease in the presence of juice, and a 1.2 log10 CFU decrease in the presence of porridge. The survival rate of the probiotics was statistically higher in the test samples with porridge (91.8%) than in those with juice (79.0%). For two products, the number of lactobacilli and bifidobacteria strains after digestion was less than <3 × 105 CFU, which can be considered insufficient. The present study has shown that the survival of probiotic strains during GI passage depends not only on their ability to withstand these harsh conditions but may also be influenced by the manufacturing process and by the foods consumed together with the probiotics.

Changes of polyphenols and their antioxidant activities in non-pigmented, red and black rice during in vitro digestion.

Polyphenols, a major bioactive constituent in rice grain, require processing and digestion before being absorbed by human body. Free and bound phenolics, flavonoids and their antioxidant activities in non-pigmented, red and black rice after cooking and INFOGEST digestions of oral, gastric and intestinal phases were investigated. It showed that cooking caused great losses of polyphenols and antioxidant activity. Free ferulic, isoferulic and p-coumaric acid in most rice were highest at intestinal phase (p < 0.05). Bound ferulic acid in three colored rice, bound p-coumaric acid in black rice and catechin in red rice were higher at oral and/or gastric phase. After cooking, total flavonoids of non-pigmented and pigmented rice were highest at intestinal and gastric phase, respectively. Cyanidin-3-O-glucoside, peonidin-3-O-glucoside and quercetin peaked at intestinal phase in black rice. It suggested that black rice has a greater potential to be used in meal balance and functional product development.

Influence of InVitro Digestion on Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Activity of Plant-Protein Hydrolysates Obtained from Agro-Industrial By-Products.

This study investigates the production of protein hydrolysates with dipeptidyl peptidase-IV (DPP-IV) inhibitory activity from agro-industrial by-products, namely olive seed, sunflower seed, rapeseed, and lupin meals, as well as from two plant protein isolates such as pea and potato. Furthermore, the effect of simulated gastrointestinal digestion on the DPP-IV inhibitory activity of all the hydrolysates was evaluated. Overall, the lowest values of IC50 (1.02 ± 0.09 - 1.24 ± 0.19 mg protein/mL) were observed for the hydrolysates with a high proportion of short-chain [< 1 kDa] peptides (i.e., olive seed, sunflower seed, and lupin) or high content of proline (i.e., rapeseed). Contrarily, the IC50 of the pea and potato hydrolysates was significantly higher (1.50 ± 0.13 - 1.93 ± 0.13 mg protein/mL). In vitro digestion led to an increase in peptides <1 kDa for almost all hydrolysates (except olive and sunflower seed meals), which was noticeable for rapeseed, pea, and potato hydrolysates. Digestion did not significantly modify the DPP-IV inhibitory activity of olive, sunflower, rapeseed, and potato hydrolysates, whereas a significant decrease in IC50 value was obtained for pea hydrolysate and a significant increase in IC50 was obtained for lupin hydrolysate. Thus, this work shows the potential of agro-industrial by-products for the production of protein hydrolysates exhibiting DPP-IV inhibition.

Probiotic Encapsulation: Bead Design Improves Bacterial Performance during In Vitro Digestion (Part 2: Operational Conditions of Vibrational Technology).

The development of functional foods is a viable alternative for the prevention of numerous diseases. However, the food industry faces significant challenges in producing functional foods based on probiotics due to their high sensitivity to various processing and gastrointestinal tract conditions. This study aimed to evaluate the effect of the operational conditions during the extrusion encapsulation process using vibrating technology on the viability of Lactobacillus fermentum K73, a lactic acid bacterium with hypocholesterolemia probiotic potential. An optimal experimental design approach was employed to produce sweet whey-sodium alginate (SW-SA) beads with high bacterial content and good morphological characteristics. In this study, the effects of frequency, voltage, and pumping rate were optimized for a 300 µm nozzle. The microspheres were characterized using RAMAN spectroscopy, scanning electron microscopy, and confocal laser scanning microscopy. The optimal conditions for bead production were found: 70 Hz, 250 V, and 20 mL/min with a final cell count of 8.43 Log10 (CFU/mL). The mean particle diameter was 620 ± 5.3 µm, and the experimental encapsulation yield was 94.3 ± 0.8%. The INFOGEST model was used to evaluate the survival of probiotic beads under gastrointestinal tract conditions. Upon exposure to in vitro conditions of oral, gastric, and intestinal phases, the encapsulated viability of L. fermentum was 7.6 Log10 (CFU/mL) using the optimal encapsulation parameters, which significantly improved the survival of probiotic bacteria during both the encapsulation process and under gastrointestinal conditions compared to free cells.

Evaluation of the nutritional quality of yeast protein in comparison to animal and plant proteins using growing rats and INFOGEST model.

Yeast, identified as a microorganism, boasts a considerable protein content, positioning yeast protein as a highly promising alternative in the quest for sustainable protein sources. The primary aim of this study is to evaluate the protein quality of yeast protein and compare it with animal proteins (whey concentrate/isolate proteins) and plant proteins (soy, wheat, pea proteins). Notably, yeast protein exhibits the highest ratio of indispensable/dispensable amino acids (IAAs/DAAs, 0.91). However, in both in vivo and in vitro digestion experiments, yeast protein demonstrated lower true protein digestibility (TPD) and true ileal digestibility (TID) compared to other proteins. Despite this, the yeast protein's amino acid score (AAS, 1.37 for >3 years), protein digestibility-corrected amino acid score (PDCAAS, 100 % for >3 years), and digestibility-corrected amino acid score (DIAAS, 82.42 % for >3 years) of yeast protein surpassed those of plant proteins, yet remained lower than animal proteins primarily due to its lower digestibility.

Differential Digestive Stability of Food-Derived microRNAs: The Case of miR-30c-5p and miR-92a-3p in Polyfloral Honey.

Dietary microRNAs (miRs) represent a new area in food science. Although they have been found in many foods, including honey, more research is needed about their stability and fate during digestion. Hence, this study aimed to analyze the digestive stability of two selected miRs in honey. We extracted miR-92a-3p and miR-30c-5p from pasteurized and unpasteurized forms of polyfloral honey using two different methods and kits: a column-based manual method and a phenol-free semi-automated magnetic-bead-based method. The latter option was used for the subsequent analysis of samples according to the INFOGEST static in vitro digestion protocol. Also, the honey samples were examined for exosome-like particles using dynamic light scattering. Although the expression levels of both miRs were significantly lower following intestinal digestion, we found a difference in the resilience of the miRs to gastrointestinal conditions, with miR-30c-5p being relatively stable compared to miR-92a-3p following digestion, regardless of the honey's pasteurization treatment. Moreover, there was marked heterogeneity in the extracellular vesicle profile of the pasteurized sample. We identified the presence of two broadly conserved miRs in honey: miR-92a-3p and miR-30c-5p. Despite honey exhibiting high digestibility, miR-92a-3p was less resilient than miR-30c-5p, demonstrating considerable resistance under gastrointestinal conditions. Although further research is needed, the results obtained from this study may represent a starting point for utilizing honey as a source of exogenous miRNAs for preventive strategies and more "natural" treatments.

Bioaccessibility of bioactive compounds from Pereskia aculeata and their cellular antioxidant effect.

Ora-pro-nobis (Pereskia aculeata) is a Cactaceae plant with edible leaves and fruits whose extracts are consumed to promote health, albeit bioactive compounds' bioaccessibility was still not assessed. To address this, ora-pro-nobis fruits (FE) and leaf extracts (LE) were subjected to in vitro digestion to better understand how this process impacts the bioactivities of the extracts. The study investigated the composition of the extracts, their cytotoxicity, and their chemical, plasmatic, and cellular antioxidant capacity. The results revealed that total polyphenolics were about 70% bioaccessible in LE and FE, with phenylalanine being the most bioaccessible essential amino acid in leaves (42.7%) and fruits (83.6%). The samples' antioxidant activity (CUPRAC) was reduced by 25%. LE demonstrated antioxidant activity against human plasma oxidation and haemolysis (21.8%), but digestion mitigated these activities. FE diminished haemolysis (47.0%) and presented cytotoxicity (IC50 = 1086 µg/mL) to HUVEC cells, but these properties were lost following digestion. Ultimately, digestion partially degraded the samples' bioactive compounds, diminishing their cellular protection against oxidative stress.

Lipolysis and Sterol Stability and Bioaccessibility of Wholemeal Rye Bread Enriched with Plant Sterols Subjected to Adult and Elderly Digestion Conditions.

This study evaluated the impact of different digestion conditions (adult and senior) on lipolysis and bioaccessibility of plant sterols (PS) and phytosterol oxidation products (POPs) in PS-enriched wholemeal rye bread. Under adult digestion conditions, the addition of gastric lipase (GL) reduced lipolysis products (by 6.1% for free fatty acids and 11.7% for monoacylglycerols) and the bioaccessibility of PS by 6.7%, compared to the control. In digestion with both GL and cholesterol esterase (CE), these reductions were 12.9, 20.1, and 11.3%, respectively. Both modifications (GL and GL + CE) increased the bioaccessibility of POPs by 4.5-4.0%. When simulating the elderly digestion, the modified gastric and intestinal phases did not alter PS bioaccessibility but decreased POPs bioaccessibility by 21.8% compared to control, along with reduced lipolysis. Incorporating GL and CE thus approached physiological conditions and influenced lipid digestion. Elderly simulated digestion conditions resulted in a positive outcome by maintaining PS bioaccessibility while reducing potentially harmful POPs.

Coupling of cold atmospheric plasma treatment with ultrasound-assisted extraction for enhanced recovery of bioactive compounds from cornelian cherry pomace.

Cornelian cherry pomace is produced during the production of juice from this traditional superfood. Due to its high nutritive value, the by-product can be utilized as a source of bioactive compounds. The present study aimed to develop a sustainable methodology for the recovery of bioactive compounds based on the combination of atmospheric cold plasma (CAP) with ultrasound assisted extraction. The pomace was treated with cold plasma under different conditions. Cyclodextrin was used as green extraction enhancer due to its capacity to develop inclusion complexes with bioactive compounds. CAP pretreatment before extraction appeared to enhance the recovery of the target compounds. GC-MS analysis and in vitro digestion analysis conducted in order to evaluate the composition and the protentional bioavailability of the bioactive compounds. CHEMICALS COMPOUNDS: ß-cyclodextrin (PubChem CID: 444041), DPPH free radical (PubChem CID: 2735032), Trolox (PubChem CID: 40634), sodium carbonate (PubChem CID: 10340), gallic acid (PubChem CID: 370) potassium chloride (PubChem CID: 4873), sodium acetate (PubChem CID: 517045), loganic acid (PubChem CID: 89640), pyridine (PubChem CID: 1049, BSTFA(PubChem CID: 94358), potassium chloride (PubChem CID: 4873), ammonium carbonate (PubChem CID: 517111), calcium chloride dehydrate (PubChem CID: 24844), potassium dihydrogen phosphate (PubChem CID: 516951), magnesium chloride hexahydrate (PubChem CID: 24644), sodium hydrogen carbonate (PubChem CID: 516892), sodium chloride (PubChem CID: 5234).

Challenges of the Application of In Vitro Digestion for Nanomaterials Safety Assessment.

Considering the increase in the production and use of nanomaterials (NM) in food/feed and food contact materials, novel strategies for efficient and sustainable hazard characterization, especially in the early stages of NM development, have been proposed. Some of these strategies encompass the utilization of in vitro simulated digestion prior to cytotoxic and genotoxic assessment. This entails exposing NM to fluids that replicate the three successive phases of digestion: oral, gastric, and intestinal. Subsequently, the resulting digestion products are added to models of intestinal cells to conduct toxicological assays, analyzing multiple endpoints. Nonetheless, exposure of intestinal cells to the digested products may induce cytotoxicity effects, thereby posing a challenge to this strategy. The aim of this work was to describe the challenges encountered with the in vitro digestion INFOGEST 2.0 protocol when using the digestion product in toxicological studies of NM, and the adjustments implemented to enable its use in subsequent in vitro biological assays with intestinal cell models. The adaptation of the digestion fluids, in particular the reduction of the final bile concentration, resulted in a reduced toxic impact of digestion products.

Current advances for in vitro protein digestibility.

Protein is an essential macronutrient in our diet, source of nitrogen and essential amino acids, but the biological utilization of dietary protein depends on its digestibility and the absorption of amino acids and peptides in the gastrointestinal tract. The methods to define the amount and the quality of protein to meet human nutritional needs, such as the Digestible Indispensable Amino Acid Score (DIAAS), require the use of animal models or human studies. These in vivo methods are the reference in protein quality evaluation, but they are expensive and long-lasting procedures with significant ethical restrictions. Therefore, the development of rapid, reproducible and in vitro digestion methods validated with in vivo data is an old demand. This review describes the challenges of the in vitro digestion methods in the evaluation of the protein nutritional quality. In addition to the technical difficulties to simulate the complex and adaptable processes of digestion and absorption, these methods are affected by similar limitations as the in vivo procedures, i.e., analytical techniques to accurately determine bioavailable amino acids and the contribution of the endogenous nitrogen. The in vitro methods used for the evaluation of protein digestibility, with special attention on those showing comparative data, are revised, emphasizing their pros and cons. The internationally harmonized digestion protocol proposed by the INFOGEST network is being adapted to evaluate protein and amino acid digestibility. The inter-laboratory reproducibility of this protocol was demonstrated for dairy products. The in vivo/in vitro comparability results obtained to date with this protocol for several plant and animal sources are promising, but it requires an extensive validation with a wider range of foods and substrates with known in vivo digestibility. These in vitro methods will probably not be applicable to all foods, and therefore, it is important to identify their limitations, not to elude their use, but to apply them within the limits, by using the appropriate standards and references, and always as a complementary tool to in vivo tests to reduce their number.

Solubility, (micro)structure, and in vitro digestion of pea protein dispersions as affected by high pressure homogenization and environmental conditions.

In this work, dispersions were prepared with commercial pea protein isolate (PPI) and subjected to different (i) high pressure homogenization (HPH) intensities (0 - 200 MPa) (room temperature, pH 7) or (ii) environmental conditions (60 °C, pH 7 or pH 12) to generate dispersions with distinct protein molecular and microstructural characteristics, impacting protein solubility. Besides, protein digestion was analyzed following the static INFOGEST in vitro digestion protocol. Generally, increasing pressure of the homogenization treatment was linked with decreasing particle sizes and enhanced protein digestion. More specifically, the dispersion that did not undergo HPH (0 MPa) as well as the dispersion treated at 60 °C, pH 7, had highly similar microstructures, consisting of large irregular particles (10 - 500 µm) with shell-like structures, and exhibited low solubility (around 15 % and 28 %, respectively), which resulted in limited proteolysis (35 % and 42 %, respectively). In contrast, the dispersion subjected to HPH at 100 MPa and the dispersion treated at 60 °C, pH 12 also had similar microstructures with small and homogeneous particles (<1 µm), and exhibited relatively good solubility (54 % and 31 %, respectively), which led to enhanced protein digestion levels (87 % and 74 %, respectively). This study highlights the potential of food processing on macronutrient (micro)structure and further gastrointestinal stability and functionality.

Comparison of protein digestibility of human milk and infant formula using the INFOGEST method under infant digestion conditions.

Many improvements have been made to bring infant formula (IF) closer to human milk (HM) regarding its nutritional and biological properties. Nevertheless, the protein components of HM and IF are still different, which may affect their digestibility. This study aimed to evaluate and compare the protein digestibility of HM and IF using the infant INFOGEST digestion method. Pooled HM and a commercial IF were subjected to the infant INFOGEST method, which simulates the physiological digestion conditions of infants, with multiple directions, i.e. the curd state, gel images of SDS-PAGE, molecular weight distribution, free amino acid concentrations and in vitro protein digestion rate. HM underwent proteolysis before digestion and tended to have a higher protein digestion rate with finer curds during gastric digestion, than the IF. However, multifaceted analyses showed that the protein digestibility of HM and IF was not significantly different after gastrointestinal digestion. In conclusion, the infant INFOGEST method showed that the digestibility of HM and IF proteins differed to some extent before digestion and after gastric digestion, but not at the end of gastrointestinal digestion. The findings of this study will contribute to the refinement of IF with better protein digestibility in infant stomach.

Assessment of in vitro digestibility and postdigestion peptide release of mare milk in relation to different management systems and lactation stages.

Mare milk has a unique protein composition that makes it a preferred option for adult and infant nutrition. Several functional properties have been attributed to this milk, but with little current evidence. In fact, knowledge on mare milk composition is still limited. In particular, studies addressing the performance of mare milk proteins during human gastrointestinal digestion are scarce, which limits the understanding of mare milk nutritional quality and functionality. For this reason, the present study describes the digestibility of mare milk proteins and the release of peptides as affected by management and lactation stage, factors which are known to affect milk composition. Mare milk samples from 3 different farms collected during 6 mo of lactation (n = 54) were subjected to a static in vitro gastrointestinal model to measure peptide release and protein digestibility. In the present study, a detailed description of protein and individual amino acid behavior during the digestion process was given. For the first time, digestion of the 2 equine ß-lactoglobulin isoforms (I and II) was described individually. In addition, it was found that lactation stage and management system can significantly affect protein digestibility and peptide release during gastrointestinal digestion of mare milk. Presumably, differences in the composition of mare milk influence the protein structure and enzyme accessibility, which might have an impact on digestion behavior. Although no specific bioactive peptides were identified, several precursors of previously described bioactive peptides were found. These findings could support the idea of mare milk as a food with added value.

Single versus multiple metabolite quantification of in vitro starch digestion: A comparison for the case of pulse cotyledon cells.

Different quantification methods for in vitro amylolysis were compared for individual chickpea and lentil cotyledon cells (ICC) as a relevant case study. For the first time, much-applied spectrophotometric methods relying on the quantification of certain functional groups (i.e., DNS, GOPOD) were compared to chromatographic quantification of starch metabolites (HPLC-ELSD). The estimated rate constant and linked initial rates of amylolysis were highly correlated for DNS, GOPOD, and HPLC-ELSD. However, absolute amylolysis levels depended on the applied method and sample-specific metabolite formation patterns. Multiresponse modelling was employed to further investigate HPLC-ELSD metabolite formation patterns. This delivered insight into the relative importance of different amylolysis reactions during in vitro digestion of pulse ICC, proving that maltotriose and maltose formation determined the overall amylolysis rate in this case. Multiresponse reaction rate constants of maltotriose and maltose formation were highly correlated to single response amylolysis rate constants (and initial rates) obtained for all three quantification methods.

Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

SCOPE: Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). METHODS AND RESULTS: Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p < 0.05). Enzyme digestion, compared to bile treatment (I60 + bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p < 0.05). Nanoparticle tracking analysis shows a significant reduction (p < 0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. CONCLUSION: Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

Recent progress in the preparation of bioactive peptides using simulated gastrointestinal digestion processes.

Bioactive peptides (BAPs) represent a unique class of peptides known for their extensive physiological functions and their role in enhancing human health. In recent decades, owing to their notable biological attributes such as antioxidant, antihypertensive, antidiabetic, and anti-inflammatory activities, BAPs have received considerable attention. Simulated gastrointestinal digestion (SGD) is a technique designed to mimic physiological conditions by adjusting factors such as digestive enzymes and their concentrations, pH levels, digestion duration, and salt content. Initially established for analyzing the gastrointestinal processing of foods or their constituents, SGD has recently become a preferred method for generating BAPs. The BAPs produced via SGD often exhibit superior biological activity and stability compared with those of BAPs prepared via other methods. This review offers a comprehensive examination of the recent advancements in BAP production from foods via SGD, addressing the challenges of the method and outlining prospective directions for further investigation.

High-Moisture Extrusion of a Dietary Protein Blend Impairs In Vitro Digestion and Delays In Vivo Postprandial Plasma Amino Acid Availability in Humans.

BACKGROUND: Industrial processing can alter the structural complexity of dietary proteins and, potentially, their digestion and absorption upon ingestion. High-moisture extrusion (HME), a common processing method used to produce meat alternative products, affects in vitro digestion, but human data are lacking. We hypothesized that HME of a mycoprotein/pea protein blend would impair in vitro digestion and in vivo postprandial plasma amino acid availability. METHODS: In Study A, 9 healthy volunteers completed 2 experimental trials in a randomized, double-blind, crossover design. Participants consumed a beverage containing 25 g protein from a "dry" blend (CON) of mycoprotein/pea protein (39%/61%) or an HME content-matched blend (EXT). Arterialized venous blood samples were collected in the postabsorptive state and regularly over a 5-h postprandial period to assess plasma amino acid concentrations. In Study B, in vitro digestibility of the 2 beverages were assessed using bicinchoninic acid assay and optical fluorescence microscopy at baseline and during and following gastric and intestinal digestion using the INFOGEST model of digestion. RESULTS: Protein ingestion increased plasma total, essential (EAA), and branched-chain amino acid (BCAA) concentrations (time effect, P < 0.0001) but more rapidly and to a greater magnitude in the CON compared with the EXT condition (condition × time interaction, P < 0.0001). This resulted in greater plasma availability of EAA and BCAA concentrations during the early postprandial period (0-150 min). These data were corroborated by the in vitro approach, which showed greater protein availability in the CON (2150 ± 129 mg/mL) compared with the EXT (590 ± 41 mg/mL) condition during the gastric phase. Fluorescence microscopy revealed clear structural differences between the 2 conditions. CONCLUSIONS: These data demonstrate that HME delays in vivo plasma amino acid availability following ingestion of a mycoprotein/pea protein blend. This is likely due to impaired gastric phase digestion as a result of HME-induced aggregate formation in the pea protein. This trial was registered at clinicaltrials.gov as NCT05584358.

In vitro simulated study of macronutrient digestion in complex food using digestive enzyme supplement.

Digestive enzymes secreted by the body are vital for digestion and nutrient absorption. Enzyme supplements are commonly used to support them in achieving optimal digestion. Herein, the efficacy of digestive enzyme supplement (DigeSEB Super) in digestion of complex food was assessed using INFOGEST simulated static and modified semi-dynamic in vitro digestion models. Digestive enzyme supplement was found to assist the endogenous digestive enzymes to disintegrate the food matrix. Hence, it reduced the viscosity of the gastric digesta by 2.75 fold (p = 0.04) compared to the control digestion (only endogenous digestive enzymes) during the first hour of digestion. Similarly, enzyme supplement showed statistically higher release of reducing sugars in the gastric digestion (p ≤ 0.05) indicating improved digestion of carbohydrates. Further, digestion of proteins and fats was also improved in the presence of enzyme supplement. The kinetic aspects of the semi-dynamic model (transient nature of gastric secretions and gradual acidification) was found to alter the macronutrient digestion compared to the static digestion. Thus, semi-dynamic model should be preferred for the in vitro studies. Overall, current study demonstrated the potential of a digestive enzyme supplement to improve digestion by aiding digestive action of the endogenous enzymes.

Enhancing the nutritional and bioactive benefits of faba bean flour by combining preprocessing and thermoplastic extrusion: A comprehensive study on digestion-resistant peptides.

This research assessed how three preprocessing techniques [soaking (S), soaking and reconstitution (SR), and soaking and dehulling (SD)] impact the protein digestibility and bioactivity of faba bean flours when combined with thermoplastic extrusion. Samples were compared against a control (C) of extruded faba bean flour without preprocessing. Applying preprocessing techniques followed by extrusion diminished antinutrient levels while enhancing protein hydrolysis and in vitro bioactivity in higher extent compared to C. Specifically, SD combined with extrusion was the most effective, achieving an 80% rate of protein hydrolysis and uniquely promoting the release of gastric digestion-resistant proteins (50-70 kDa). It also resulted in the highest release of small peptides (<3kDa, 22.51%) and free amino acids (15.50%) during intestinal digestion. Moreover, while all preprocessing techniques increased antioxidant (ABTS radical-scavenging), antidiabetic, and anti-hypertensive activities, SD extruded flour displayed the highest levels of dipeptidyl peptidase inhibition (DPP-IVi, IC50=13.20 µg/mL), pancreatic α-amylase inhibition (IC50=8.59 mg/mL), and angiotensin I-converting enzyme inhibition (ACEi, IC50=1.71 mg protein/mL). As a result, it was selected for further peptide and in silico bioactive analysis. A total of 24 bioactive peptides were identified in intestinal digests from SD extruded flour, all with potential DPP-IVi and ACEi activities, and six were also predicted as antioxidant peptides. VIPAGYPVAIK and GLTETWNPNHPEL were highlighted as resistant bioactive peptides with the highest antidiabetic and antioxidant potential. Our findings demonstrated that combining preprocessing (particularly SD) and thermoplastic extrusion enhances protein digestibility in faba beans and promotes the release of beneficial bioactive peptides in the intestine.