INFOGEST Protocol Research Articles

In Vivo and In Vitro Comparison of the DPP-IV Inhibitory Potential of Food Proteins from Different Origins after Gastrointestinal Digestion.

Dipeptidyl-peptidase IV (DPP-IV) plays an essential role in glucose metabolism by inactivating incretins. In this context, food-protein-derived DPP-IV inhibitors are promising glycemic regulators which may act by preventing the onset of type 2 diabetes in personalized nutrition. In this study, the DPP-IV-inhibitory potential of seven proteins from diverse origins was compared for the first time in vitro and in vivo in rat plasma after the intestinal barrier (IB) passage of the indigested proteins. The DPP-IV-inhibitory potentials of bovine hemoglobin, caseins, chicken ovalbumin, fish gelatin, and pea proteins were determined in rat plasma thirty minutes after oral administration. In parallel, these proteins, together with bovine whey and gluten proteins, were digested using the harmonized INFOGEST protocol adapted for proteins. The DPP-IV half-maximal inhibitory concentration (IC50) was determined in situ using Caco-2 cells. The DPP-IV-inhibitory activity was also measured after IB passage using a Caco2/HT29-MTX mixed-cell model. The peptide profiles were analyzed using reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC-MS/MS) with MS data bioinformatics management, and the IC50 of the identified peptides was predicted in silico. The in vitro and in vivo DPP-IV-inhibitory activity of the proteins differed according to their origin. Vegetable proteins and hemoglobin yielded the highest DPP-IV-inhibitory activity in vivo. However, no correlation was found between the in vivo and in vitro results. This may be partially explained by the differences between the peptidome analysis and the in silico predictions, as well as the study complexity.

Addressing criticalities in the INFOGEST static in vitro digestion protocol for oleogel analysis

The interest on the digestive fate of oleogels, i.e., substitutes for solid fats rich in liquid oil, have pushed researchers to use the widely adopted INFOGEST protocol for static in vitro digestion. However, this protocol was originally designed to simulate the digestibility of conventional foods and to accommodate the large fraction of oil in oleogels, researchers have deliberately modified the INFOGEST protocol, inadvertently leading to results difficult to be compared.In this study, we highlighted possible problems that may arise during oleogel simulated digestion such as under- or overestimation of oleogel lipolysis. The effect of oleogel amount, oleogelator type and concentration, and shear applied during digestion on the rate and extent of oleogel digestion was studied. The release of fatty acids during the application of INFOGEST protocol was monitored using the pH-stat method and compared to those analyzed by HPLC-ELSD. Oleogels’ structural information was obtained using brightfield, polarized, and fluorescence microscopy, and DSC. We determined that lipolysis of ethylcellulose oleogels follow the “interaction with enzymes and bile salts” pattern, whereas that of wax oleogels follow the “disintegration of oleogel and interaction with enzymes and bile salts”. We also observed that the chemical composition of wax, crystal morphology, and crystal distribution do not alter the lipolysis of oil entrapped inside the wax crystals. We finally recommended a few minimal but fundamental modifications to the INFOGEST protocol to achieve more reliable results from the static in vitro digestion of oleogels and possibly other lipid-based systems.

Fungal digestive enzymes promote macronutrient hydrolysis in the INFOGEST static in vitro simulation of digestion

The objective of this study was to test the hydrolytic efficacy of 6 fungal enzymes in the INFOGEST static in vitro simulation of gastrointestinal (GI) digestion. First, the INFOGEST protocol was adapted for testing of exogenous enzymes. Second, a dose-response study of 3 individual fungal proteases, a lipase, and an amylase with glucoamylase demonstrated improved dietary protein, lipid, and carbohydrate hydrolysis, respectively, from an oral nutritional supplement (ONS) under simulated gastric or GI conditions, compared to pepsin and pancreatin-based control conditions. Third, a combination of the 6 enzymes (BC-006) improved macronutrient digestion, including enhanced release of individual amino acids from ONS and mixed meal substrates. Finally, we validated digestive models of aging and proton pump inhibitor (PPI) use, and showed that BC-006 improved gastric digestion under these compromised digestive conditions. The INFOGEST static simulation is a feasible tool to rapidly screen and profile exogenous enzymes for digestive efficacy in vitro.

Static in vitro digestion methods for assessing essential minerals in processed meat products

The consumption of processed meat products has increased due to its convenient preparation and characteristics related to taste and consumer acceptance. In this study, we evaluated the content and bioaccessibility of iron (Fe), zinc (Zn), calcium (Ca) and magnesium (Mg) in processed pork and beef products by dialysis assay. In addition, different assays of static in vitro digestion (traditional solubility and INFOGEST) were applied to verify the results on the bioaccessibility of these essential elements. The average content of essential elements (expressed in mg/100 g) were 0.78 ± 0.24 and 1.51 ± 0.51 for Fe; 1.79 ± 0.55 and 3.57 ± 1.42 for Zn; 13.24 ± 9.99 and 20.29 ± 10.45 for Ca and 19.43 ± 2.78 and 26.15 ± 7.33 for Mg, in pork and beef products, respectively. However, we observed high variation between brands and batches. The dialysis assay presented variations in bioaccessibility for all essential elements evaluated in the samples. The results for dialyzable Fe were lower than expected (in vivo assays), probably due to the mechanism simulated in dialysis. This assay was considered inadequate for the simulation of in vitro digestion for meat samples that contain heme-Fe. When comparing the different static in vitro digestion assays employed, the dialysis test showed the lowest bioaccessibility percentages for all essential elements evaluated, when compared with the other in vitro digestion methods. The highest bioaccessible fractions were obtained by applying the INFOGEST protocol. We found that according to the in vitro digestion simulation assay used, the results for bioaccessible percentage displayed high variation. These observations highlight the importance of the assay type used for this purpose. These results did not indicate which is the better assay, but demonstrated the differences of each and their relevance for estimating bioaccessibility of essential elements in meat products.

Characterization and Influence of Static In Vitro Digestion on Bioaccessibility of Bioactive Polyphenols from an Olive Leaf Extract.

Olive leaves, one of the most abundant olive production by-products, have shown incredible potential for their characteristic bioactive compound composition, with unique compounds such as the polyphenol oleuropein. In order to evaluate the bioaccessibility of bioactive compounds present in an olive leaf extract, samples were submitted to an in vitro digestion process following INFOGEST protocol, and qualitative and quantitative characterization of the original extract and digestive samples at different times were carried out using HPLC-ESI-TOF-MS. The analyzed extract presented an abundance of phenolic compounds, such as secoiridoids, with oleuropein being the main identified compound. The in vitro digestion process showed an effect on the phenolic profile of the extract, with a lower recovery in the gastric phase and an increase at the beginning of the intestinal phase. Most of the studied compounds showed high bioaccessibility at the end of the digestion, with oleuropein, ligstroside, and quercetin-3-O-galactoside being among the ones with higher value. These findings show the potential for future use of olive leaf polyphenols. However, further research is needed in order to evaluate the absorption, delivery, and interaction of these compounds with the colon.

Bioaccessibility and Oxidative Stability of Omega-3 Fatty Acids in Supplements, Sardines and Enriched Eggs Studied Using a Static In Vitro Gastrointestinal Model.

Modern dietary habits have created the need for the design and production of functional foods enriched in bioactive compounds for a healthy lifestyle. However, the fate of many of these bioactive compounds in the human gastrointestinal (GI) tract has not been thoroughly investigated. Thus, in the present study, the bioaccessibility of omega-3 fatty acids was examined. To that end, different foods and supplements underwent simulated digestion following the INFOGEST protocol. The selected samples were foods rich in omega-3 fatty acids both in free and bound form—i.e., dietary fish oil supplements, heat-treated fish, and eggs enriched with omega-3 fatty acids. The oxidation of polyunsaturated fatty acids (PUFAs) was measured at each stage of the digestion process using peroxide value (PV) and TBARS and by quantifying individual omega-3 fatty acids using a gas chromatograph with flame ionization detector (GC-FID). The final bioaccessibility values of omega-3 fatty acids were determined. Changes in the quantity of mono-saturated fatty acids (MUFAs) and saturated fatty acids (SFAs) were recorded as well. The results indicated a profound oxidation of omega-3 fatty acids, giving rise to both primary and secondary oxidation products. Additionally, stomach conditions seemed to exert the most significant effect on the oxidation of PUFAs during digestion, significantly decreasing their bioaccessibility. The oxidation rate of each fatty acid was found to be strongly correlated with its initial concentration. Finally, the oxidation pattern was found to be different for each matrix and emulsified lipids seemed to be better protected than non-emulsified lipids. It is concluded that digestion has a profound negative effect on omega-3 bioaccessibility and therefore there is a need for improved protective mechanisms.

A guide for the evaluation of in vitro bioaccessibility of carotenoids.

Ongoing efforts to improve the nutritional content of carotenoids in food sources to solve global health problems must also consider bioaccessibility and bioavailability of carotenoids. Carotenoid bioaccessibility encompasses carotenoid release from the food matrix, solubilization in lipid emulsion droplets, and transfer to mixed micelles for further absorption into the intestine. Bioavailability refers to the fraction of carotenoids which enters into circulation. To understand the mechanisms controlling bioaccessibility, in vitro digestion methods have been developed and are a useful approach to overcome some of the disadvantages associated with in vivo protocols. The INFOGEST protocol comprises three phases (oral, gastric and intestinal) to simulate adult human digestion of foods, as an international consensus for the conditions of static in vitro digestion. The last steps in any digestion protocol involve isolation of the target food compounds and subsequent analysis, steps not included in the INFOGEST protocol because they may differ depending on specific physical-chemical characteristics and quantification analysis of the target analyte. In this chapter, we describe the INFOGEST protocol which has been adapted for the analysis of carotenoid bioaccessibility, including determination and calculation of enzyme activities according to INFOGEST guidelines, removal of undigested material, collection of mixed micelles containing carotenoids, extraction, storage and subsequent identification and quantification by liquid chromatography analysis of both free and acylated carotenoids.

Comparing nutritional and digestibility aspects of sustainable proteins using the INFOGEST digestion protocol

• Most of the alternative proteins have a good protein hydrolysis and digestibility. • An in vitro DIAAS calculation method based on INFOGEST protocol was developed. • Whey, potato, blood plasma and yeast protein concentrates have a high IVDIAAS. • Food products with complex matrix and low protein content require further optimisation of the method. We studied several sustainable alternative protein concentrates and a whey protein concentrate as reference, to determine their protein composition, digestibility and quality using the harmonized INFOGEST static protocol. The proteins concentrates were analyzed to determine their conversion factor, degree of hydrolysis, true ileal digestibility, in vitro digestible indispensable amino acid score (IVDIAAS) and total absorbable amino acids and total essential amino acids. The results revealed that whey, blood plasma concentrate and yeast protein concentrate have a high mean true ileal indispensable amino acid in vitro digestibility (91.1–85.8%), closely followed by corn, pea, potato and proteins derived from lesser meal worms (ranging between 82.5 and 77.9%). Mycoprotein gave a much lower score in the mass balance, so that its other results could not be interpreted as being reliable. Whey, potato, blood plasma and yeast protein concentrates ranked highest in IVDIAAS (between 119 and 97.2), followed by lesser meal worm and pea (between 73.8 and 57.8) with corn protein concentrate having the lowest IVDIAAS due to underrepresentation of lysine. The method and data presented in this paper can form a start for further applying the INFOGEST in vitro digestion protocol to evaluate protein quality.

Effect of agitation and added cholesterol esterase on bioaccessibility of phytosterols in a standardized in vitro digestion model

There is great interest in reaching a consensus method for in vitro digestion of foods. The COST INFOGEST network of researchers has developed a standardized protocol, which is appropriate for basic digestibility studies, but may be insufficient for more complex studies, such as micronutrient bioaccessibility. In the present study, the bioaccessibility of phytosterols (PS) was evaluated using the standardized and modified versions of the in vitro digestion protocol. The INFOGEST protocol was used without modification for 2% solutions of either free phytosterols (FPS) or esterified phytosterols (EPS) in soybean oil. The effect of two variables which have yet to be explored using the INFOGEST protocol was subsequently evaluated. Those variables are: (1) changing the mode of agitation from orbital shaking to head over heels (HOH) rotation and (2) adding pancreatic cholesterol esterase (CE) during the intestinal phase. Bioaccessibility of FPS was greater than that of EPS, and HOH rotation caused a further increase in bioaccessibility of FPS but not EPS. Addition of CE caused a slight, but significant improvement in bioaccessibility of EPS, though hydrolysis was incomplete. Our findings highlight a need for further evaluation of the standardized in vitro digestion protocol, especially for complex studies such as lipid bioaccessibility.

A Case Study of the Response of Immunogenic Gluten Peptides to Sourdough Proteolysis

Celiac disease is activated by digestion-resistant gluten peptides that contain immunogenic epitopes. Sourdough fermentation is a potential strategy to reduce the concentration of these peptides within food. However, we currently know little about the effect of partial sourdough fermentation on immunogenic gluten. This study examined the effect of a single sourdough culture (representative of those that the public may consume) on the digestion of immunogenic gluten peptides. Sourdough bread was digested via the INFOGEST protocol. Throughout digestion, quantitative and discovery mass spectrometry were used to model the kinetic release profile of key immunogenic peptides and profile novel peptides, while ELISA probed the gluten’s allergenicity. Macrostructural studies were also undertaken. Sourdough fermentation altered the protein structure, in vitro digestibility, and immunogenic peptide release profile. Interestingly, sourdough fermentation did not decrease the total immunogenic peptide concentration but altered the in vitro digestion profile of select immunogenic peptides. This work demonstrates that partial sourdough fermentation can alter immunogenic gluten digestion, and is the first study to examine the in vitro kinetic profile of immunogenic gluten peptides from sourdough bread.

Lipid digestibility and bioaccessibility of a high dairy fat meal is altered when consumed with whole apples: Investigations using static and dynamic in vitro digestion models

It remains unclear how consuming whole apples, as a food matrix containing pectin and polyphenols, influences lipid digestibility and bioaccessibility from dairy-based meals. Pectin and polyphenols modulate lipid digestion in vitro, but their functionality can be altered by gastric pH, which is buffered by co-consuming dairy products. In the present study, a dairy meal containing 3 wt% protein and 10 wt% fat was digested with and without apples using static (i.e., adapted from INFOGEST standardized static model) and dynamic (TIM-1) in vitro methods to investigate the structure, digestibility, and bioaccessibility of milk fat from emulsion droplets in the presence of consumed apples. Furthermore, in the static digestion, the initial gastric pH was adjusted to 3.0 (i.e., acidic digestion, as INFOGEST protocol suggested) or remained unadjusted (pH = 6.5, i.e., neutral digestion, buffered by the dairy meal) to explore the effects of gastric acidity. Emulsion droplet structure and lipid digestibility in the static digestion were primarily influenced by gastric pH [mean lipolysis rate = 99.32 % and 83.95 % (p < 0.05) for the digestions with neutral and acidic pH in the gastric phase, respectively], but not the presence of apple. However, co-consumption of apples reduced the lipid bioaccessibility measured by TIM-1 [mean bioaccessibility = 65.44 % and 98.92 % (p < 0.05) for the digestions with and without apples, respectively]. Discrepancies in the bioaccessibility of individual fatty acids between the static and TIM-1 models were observed, which may be attributed to variations in the experimental parameters, e.g., chyme transit and enzyme concentration. Overall, this study shows the potential to preferentially modify lipid digestibility and bioaccessibility in high-fat meals by co-consuming apples. It also cautions attention to the digestive conditions of in vitro simulation studies when making comparisons to in vivo conditions.

In vitro digestion of polysaccharides: InfoGest protocol and use of small intestinal extract from rat

Starch, dextran, pectin and modified citrus pectin were subjected to intestinal digestion following InfoGest protocol and a rat small intestine extract (RSIE) treatment. Gastric stage did not show any modification in the structure of the carbohydrates, except for modified pectin. Regarding intestinal phases, starch was hydrolyzed by different ways, resulting in a complementary behavior between InfoGest and RSIE. Contrarily, digestion of dextran was only observed using RSIE. Similar situation occurred in the case of pectins with RSIE, obtaining a partial hydrolysis, especially in the modified citrus pectin. However, citrus pectin was the less prone to hydrolysis by enzymes. The results demonstrated that InfoGest method underestimates the significance of the carbohydrates hydrolysis at the small intestine, thus indicating that RSIE is a very reliable and useful method for a more realistic study of polysaccharides digestion.

Insights on the intestinal absorption of chlorophyll series from microalgae

The bioaccessibility and subsequent uptake by Caco-2 human intestinal cells of chlorophyll pigments from Scenedesmus obliquus were determined for the first time. In order to evaluate the impact of different types of the matrix on bioaccessibility of chlorophyll from microalgae, three different products were evaluated: isolated chlorophyll extract (ICE); wet ultrasonicated biomass (WUB); and whole dried biomass (WDB). The samples were submitted to in vitro digestion model according to the INFOGEST protocol, and Caco-2 cells determined the intestinal uptake. Chlorophyll pigments were determined by HPLC-PDA-MS/MS. A total of ten chlorophyll pigments (8,318.48 µg g−1) were separated in S. obliquus biomass, with chlorophyll a (3,507.76 µg g−1) and pheophytin a' (1,598.09 µg g−1) the major ones. After in vitro digestion, all tested products showed bioaccessible chlorophylls. However, the total bioaccessibility results were as follows: ICE (33.45%), WUB (2.65%), WDB (0.33%). Five compounds were bioaccessible in ICE, three in WUB, and one in WDB. The hydroxypheophytin a showed the highest bioaccessibility (212%) in ICE, while pheophytin a' in WUB (11%) and WDB (2%). As a result, bioavailability estimates of ICE using the Caco-2 cell showed hydroxypheophytin a (102.53%), followed by pheophytin a' (64.69%) as the chlorophyll pigments most abundant in intestinal cells. In summary, from a nutritional perspective, these three types of the matrix (WDB, WUB, and ICE) influence the promotion of chlorophyll bioaccessibility. In this way, the data suggest that chlorophylls bioaccessibility from ICE is greater than that in WDB and WUB. Therefore, ICE should be considered a product that provides bioavailable chlorophyll and could be the best choice, such as ingredients in the development of functional foods chlorophyll-based.

Bioaccessibility and intestinal uptake of carotenoids from microalgae Scenedesmus obliquus

This study aimed to investigate the carotenoids bioaccessibility of the whole dried biomass (WDB), wet ultrasonicated biomass (WUB), and isolated carotenoid extract (ICE) from Scenedesmus obliquus using an in vitro digestion model. Also, the intestinal uptake of ICE was investigated. The samples were submitted to in vitro digestion model according to the INFOGEST protocol, and Caco-2 cells determined the intestinal uptake. Carotenoids were determined by HPLC-PDA-MS/MS. The results showed that the bioaccessibility was improved according to the type of product (ICE > WUB > WDB). The all-trans-canthaxanthin (99.07%), all-trans-α-carotene (48.94%), and all-trans-β-cryptoxanthin (31.87%) were the most bioaccessible compounds in ICE, WUB, and WDB, respectively. Due to the ICE showed the best bioaccessibility, it was considered the most efficient condition and subjected to uptake by intestinal cells. ICE uptake demonstrated that the most abundant carotenoids in Caco-2 cells were all-trans-β-crocoxanthin (17.34%), followed by all-trans-echinenone (7.55%). In contrast, all-trans-lutein (0.88%) was the least abundant in cells. The xanthophylls were uptake in greater proportion than carotenes. Finally, the results showed the significant influence of the product nature in promoting the bioaccessibility of microalgae carotenes and xanthophylls.

In Vitro Assessment of the Impact of Industrial Processes on the Gastrointestinal Digestion of Milk Protein Matrices Using the INFOGEST Protocol.

The goal of this study was to determine the impact of industrial processes on the digestion of six milk protein matrices using the harmonized INFOGEST in vitro static digestion protocol. First, this method was optimized to simple protein matrices using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) to compare the intestinal protein hydrolysis obtained with increasing quantities of pancreatin. Similar results were achieved with the originally required pancreatin amount (trypsin activity of 100 U.mL−1) and with a quantity of pancreatin equivalent to a trypsin activity of 27.3 U.mL−1, which was thus used to perform the in vitro digestion of the milk matrices. Molecular weight profiles, peptide heterogeneity from LC-MS/MS data, calcium, free amino acid, and peptide concentrations were determined in the gastric and intestinal phases to compare the milk protein digests. Results showed that the industrial process affected not only the protein distribution of the matrices but also most likely the protein structures. Indeed, differences arose in terms of peptide populations generated when the caseins were reticulated or when their calcium concentrations were reduced.

Bio-Based Nanoparticles as a Carrier of β-Carotene: Production, Characterisation and In Vitro Gastrointestinal Digestion.

β-carotene loaded bio-based nanoparticles (NPs) were produced by the solvent-displacement method using two polymers: zein and ethylcellulose. The production of NPs was optimised through an experimental design and characterised in terms of average size and polydispersity index. The processing conditions that allowed to obtain NPs (<100 nm) were used for β-carotene encapsulation. Then β-carotene loaded NPs were characterised in terms of zeta potential and encapsulation efficiency. Transmission electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction analysis were performed for further morphological and chemical characterisation. In the end, a static in vitro digestion following the INFOGEST protocol was performed and the bioaccessibility of β-carotene encapsulated in both NPs was determined. Results show that the best conditions for a size-controlled production with a narrow size distribution are lower polymer concentrations and higher antisolvent concentrations. The encapsulation of β-carotene in ethylcellulose NPs resulted in nanoparticles with a mean average size of 60 ± 9 nm and encapsulation efficiency of 74 ± 2%. β-carotene loaded zein-based NPs resulted in a mean size of 83 ± 8 nm and encapsulation efficiency of 93 ± 4%. Results obtained from the in vitro digestion showed that β-carotene bioaccessibility when encapsulated in zein NPs is 37 ± 1%, which is higher than the value of 8.3 ± 0.1% obtained for the ethylcellulose NPs.

Bioaccessibility and cellular uptake by Caco-2 cells of carotenoids and chlorophylls from orange peels: A comparison between conventional and ionic liquid mediated extractions

Native extracts from orange peels were obtained by a conventional method using acetone and, an alternative method using ionic liquid (1-butyl-3-methylimidazolium chloride ([C4mim]Cl)). The bioaccessibilities and cellular uptakes of carotenoids, esters and chlorophylls were evaluated, since the influence of esterification on bioaccessibility and bioavailability is not well established. For this, the extracts were emulsified, submitted to in vitro simulated digestion model according to the INFOGEST protocol, followed by uptake by Caco-2 cells. Compounds were separated, identified and quantified by HPLC-PDA-MS/MS. After digestion, 22.0% and 26.2% of the total carotenoids and 45.9% and 68.7% of the chlorophylls were bioaccessible from the acetone and [C4mim]Cl extracts, respectively. The bioaccessibilities of xanthophylls and carotenes were significantly higher than those of the mono- and diesters. The uptake by Caco-2 cells varied from 130.2 to 131.9 ng/mg cell protein for total carotenoids and from 243.8 to 234.2 ng/mg cell protein for chlorophylls in the acetone and [C4mim]Cl extracts, respectively. In general, xanthophylls and esters were better absorbed than carotenes.

Heat treatment of β-lactoglobulin affects its digestion and translocation in the upper digestive tract.

Heat treatment is a commonly applied unit operation in the processing of β-lactoglobulin containing products. This does, however, influence its structure and thereby impacts its activity and digestibility. We describe how various heat-treatments of β-lactoglobulin change the digestibility using a modified version of the current consensus INFOGEST protocol. Additionally, protein was investigated for its translocation over the intestinal epithelial barrier, which would bring them in contact with immune cells. The extent of gastric digestibility was higher when the protein structure was more modified, while the influence of glycation with lactose was limited. Translocation studies of protein across Caco-2 cell monolayers showed a lower translocation rate of protein heated in solution compared to the others. Our study indicates that structural modifications after different heat-treatments of β-lactoglobulin increase in particular gastric digestibility and the translocation efficiency across intestinal epithelial cells.

Stability of antibacterial and coccidiostat drugs on chicken meat burgers upon cooking and in vitro digestion

The impact of culinary practices – oven or microwave cooking combined with herbs and/or beer – on antibacterial and coccidiostat drugs stability and bioaccessibility in chicken meat was evaluated. Fourteen compounds from 6 classes (β-lactams, tetracyclines, sulfonamides, fluoroquinolones, macrolides, and coccidiostats) were monitored after cooking and in vitro digestion (INFOGEST protocol) at two fortification levels. Depending on their reduction, the presence of transformation products derived from cooking or digestion was investigated.In general, compounds were stable during cooking except amoxicillin, chlortetracycline and tylosin (reductions > 50%). Molecular rearrangement and dechlorination reactions are the most probable transformations derived from cooking. Adding herbs/beer does not benefit their reductions. During in vitro digestion, maximum bioaccessibilities of 60% were observed for all quantified compounds. As drugs and bile salts interact, increasing the absorption of lipophilic drugs, their bioaccessibility predictions must not be based only on the determination of their free form using LC–MS/MS.

Protein digestion of different protein sources using the INFOGEST static digestion model.

In vitro digestion systems are valuable tools for understanding and monitoring the complex behavior of food degradation during digestion, thus proving to be good candidates for replacing in vivo assays. The aim of the present work was to study protein hydrolysis in a selection of different protein sources using the harmonized INFOGEST static protocol: three isolated proteins (collagen, zein, and whey protein) and five foods (sorghum flour, wheat bran cereals, peanuts, black beans, and pigeon peas). The proteins of all the substrates were analyzed by SDS-PAGE and HPLC-MS/MS. Individual amino acid composition was analyzed by high-performance liquid chromatography (HPLC). EAA/NEAA (essential amino acids/ nonessential amino acids) ratios in the substrates from low to high were as follows: wheat bran cereals, peanuts, collagen, zein, whey protein, sorghum, pigeon peas, and black beans. The results revealed sorghum, whey protein, and zein as good sources of BCAA. In all substrates, no intact protein from the substrates was visually detected by SDS-PAGE after the intestinal phase of in vitro digestion with the INFOGEST protocol. However, digestion-resistant peptides were detected in all substrates after the intestinal digestion phase. Protein hydrolysis was high in whey protein isolate and pigeon pea and low for wheat bran cereals and bovine collagen.