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.

Direct labeling of milk cells without centrifugation for counting total and differential somatic cells using flow cytometry.

Somatic cell count (SCC) in milk is an essential indicator for defining and managing udder health. However, analyzing differential SCC (dSCC) can be helpful in determining the type or evolution stage of mastitis. A high abundance of polymorphonuclear cells (PMN) is associated with acute mastitis; however, the status of a chronic disease is less well characterized. A method capable of analyzing SCC and dSCC can prove to be a helpful tool for monitoring the status of evolution of mastitis disease in a better way. Therefore, a new direct-flow cytometry method was developed to count and differentiate somatic cells in milk without the steps of centrifugation or washing, avoiding variabilities that occur due to enrichment or loss of specific cell types. In this new method, SCC is analyzed using the method of DNA staining with Hoechst stain, whereas dSCC are analyzed using specific antibodies targeting 2 main cell types associated with mastitis: PMN cells and antigen-presenting cells, which are associated with innate and adaptive immunity. Equivalent SCC values were obtained between the new method and the routine ISO 13366-2 method in a comparison of 240 raw milk samples. Furthermore, dSCC results were confirmed by microscopy after May-Gründwald-Giemsa staining in 165 quarter milk samples from healthy and diseased cows. The method was verified with fluorescence microscopy on the 2 targeted cell types and in raw milk samples. The newly developed method is independent of any instrument and can be further designed to differentiate other cell types and animal species by selecting appropriate antibodies.

A dose-response strategy reveals differences between normal-weight and obese men in their metabolic and inflammatory responses to a high-fat meal.

A dose-response strategy may not only allow investigation of the impact of foods and nutrients on human health but may also reveal differences in the response of individuals to food ingestion based on their metabolic health status. In a randomized crossover study, we challenged 19 normal-weight (BMI: 20-25 kg/m(2)) and 18 obese (BMI: >30 kg/m(2)) men with 500, 1000, and 1500 kcal of a high-fat (HF) meal (60.5% energy from fat). Blood was taken at baseline and up to 6 h postprandially and analyzed for a range of metabolic, inflammatory, and hormonal variables, including plasma glucose, lipids, and C-reactive protein and serum insulin, glucagon-like peptide-1, interleukin-6 (IL-6), and endotoxin. Insulin was the only variable that could differentiate the postprandial response of normal-weight and obese participants at each of the 3 caloric doses. A significant response of the inflammatory marker IL-6 was only observed in the obese group after ingestion of the HF meal containing 1500 kcal [net incremental AUC (iAUC) = 22.9 ± 6.8 pg/mL × 6 h, P = 0.002]. Furthermore, the net iAUC for triglycerides significantly increased from the 1000 to the 1500 kcal meal in the obese group (5.0 ± 0.5 mmol/L × 6 h vs. 6.0 ± 0.5 mmol/L × 6 h; P = 0.015) but not in the normal-weight group (4.3 ± 0.5 mmol/L × 6 h vs. 4.8 ± 0.5 mmol/L × 6 h; P = 0.31). We propose that caloric dose-response studies may contribute to a better understanding of the metabolic impact of food on the human organism. This study was registered at clinicaltrials.gov as NCT01446068.

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.

In vitro DIAAS of Swiss soybean cultivars using the INFOGEST model: Increase in protein quality from soybean to soymilk and tofu.

To support the transition towards more sustainable and healthy diets, viable alternatives to foods of animal origin need to be identified. Many plant-based protein sources are currently marketed with claims of minimal environmental impact, but very limited consideration has been given to their protein quality and bioavailable mineral content considering the fact that animal-based foods are typically the primary source of both in Western diets. In this study, traditionally consumed soy foods (cooked soybeans, soymilk, tofu) from different Swiss soybean cultivars were nutritionally characterized and the in vitro digestibility of individual amino acids and total protein were assessed using an in vitro model based on the static INFOGEST protocol; the protein quality was evaluated using the in vitro digestible indispensable amino acid score (DIAAS). The results reveal an increase in total protein in vitro digestibility across the traditional soy food production value chain: 52.1-62.7% for cooked soybeans, 84.1-90.6% for soymilk, and 94.9-98.4% for tofu. Protein quality, determined using the recommended amino acid pattern for 0.5-3 years old, was "low" (no claim) for cooked soybeans (DIAAS < 60), while soymilk (DIAAS = 78-88) and tofu products (DIAAS = 79-91) were of similar "good" protein quality, with considerably higher DIAAS values than those of cooked soybeans (P < 0.001). The iron and zinc contents in soy foods were substantial, but high molar ratios of phytic acid (PA) to iron (PA/Fe; >8) and PA to zinc (PA/Zn; >15) indicate a possible strong inhibition of iron and zinc bioavailability. Based on the DIAAS results, soymilk and tofu would be suitable plant-based alternatives to animal-based foods, while future efforts should focus on optimizing soybean preparation to overcome the negative effects of the plant tissue matrix as well as processing steps to reduce mineral absorption inhibiting substances.

Comparison of in vitro digestibility and DIAAR between vegan and meat burgers before and after grilling.

Plant-based meat alternatives of high quality and digestibility could be a way to reduce meat consumption and, consequently, the environmental impact. However, little is known about their nutritional characteristics and digestion behaviour. Therefore, in the present study, the protein quality of beef burgers, known as excellent source of protein, was compared with the protein quality of two highly transformed veggie burgers, based on soy or pea-faba proteins, respectively. The different burgers were digested according to the INFOGEST in vitro digestion protocol. After digestion, total protein digestibility was determined, either based on total nitrogen (Kjeldahl) analysis, or after acid hydrolysis based on total amino groups (o-phthalaldehyde method) or total amino acids (TAA; by HPLC). The digestibility of individual amino acids was also determined, and the digestible indispensable amino acid score (DIAAS) was calculated based on in vitro digestibility. The impact of texturising and grilling on in vitro protein digestibility and the digestible indispensable amino acid ratio (DIAAR) was evaluated at the level of the ingredients and the finished products. As expected, the grilled beef burger had the highest in vitro DIAAS values (Leu 124 %), and grilled soy protein-based burger reached in vitro DIAAS values that could be rated as good (soy burger, SAA 94 %) protein source, according to the Food and Agriculture Organization. The texturing process did not significantly affect the total protein digestibility of the ingredients. However, grilling led to a decrease in digestibility and DIAAR of the pea-faba burger (P < 0.05), which was not observed in the soy burger, but led to an increase in DIAAR in the beef burger (P < 0.005).

Mealworm larvae (Tenebrio molitor) and crickets (Acheta domesticus) show high total protein in vitro digestibility and can provide good-to-excellent protein quality as determined by in vitro DIAAS.

Edible insects, such as mealworms (Tenebrio molitor larvae; TM) and crickets (Acheta domesticus; AD), are a sustainable, protein-dense novel food with a favorable amino acid profile, which might be an alternative to animal proteins. To assess the protein quality of TM and AD, we assessed the digestible indispensable amino acid scores (DIAAS), considering individual amino acids and their ileal amino acid digestibility, using an in vitro model based on the INFOGEST digestion protocol. In addition, we evaluated if various processing and food preparation steps influenced the in vitro digestibility of individual amino acids and the in vitro DIAAS values of TM and AD and compared them to chicken breast as a reference of excellent protein quality. The total protein in vitro digestibility ranged from 91 to 99% for TM and from 79 to 93% for AD and was negatively affected by oven-drying and, to a lesser extent, by chitin-reduction. The in vitro DIAAS values were 113, 89, and 92 for chicken, blanched TM, and blanched AD, respectively, when considering the indispensable amino acid (IAA) requirements of young children between 6 months and 3 years. Across different processing and food preparation methods, the in vitro DIAAS values ranged between 59 and 89 for TM and between 40 and 92 for AD, with the lowest values found in chitin-reduced insects. Due to their similarities to chicken regarding protein composition, total protein in vitro digestibility, and in vitro DIAAS values, TM and AD might be an alternative to traditional animal proteins, provided that suitable processing and food preparation methods are applied. Our in vitro DIAAS results suggest that TM and AD can thus be considered good-quality protein sources for children older than 6 months. The DIAAS calculations are currently based on crude protein (total nitrogen × 6.25), resulting in an overestimation of insect protein content, and leading to an underestimation and potential misclassification of protein quality. The in vitro model applied in this study is a valuable tool for product development to optimize the protein quality of edible insects. Further studies are required to assess the in vivo DIAAS of insects in humans.

In vitro digestibility of dietary proteins and in vitro DIAAS analytical workflow based on the INFOGEST static protocol and its validation with in vivo data.

The FAO recommends the digestible indispensable amino acid score (DIAAS) to determine protein quality in foods, preferably tested in vivo. Here, the INFOGESTin vitrodigestion protocol was applied and supplemented with an analytical workflow allowing the assessment of protein digestibility and DIAAS calculation. The protocol was applied to selected samples WPI, zein, collagen, black beans, pigeon peas, All-Bran®, and peanuts. The total protein digestibility, digestibility of individual amino acids (AA), and DIAAS values were established and compared with in vivo data for the same substrates. Total protein digestibility (total Nitrogen, r = 0.7, P < 0.05; primary amines (OPA), r = 0.6, P < 0.02; total AA, r = 0.6, P < 0.02) and digestibility of individual AA (r = 0.6, P < 0.0001) were in good agreement, between in vitro and in vivo, with a mean difference of 1.2 %. In vitro DIAAS was highly correlated with DIAAS obtained from in vivo true ileal digestibility values (r = 0.96, R2 = 0.89, P < 0.0001) with a mean difference of 0.1 %.

Elucidation of the Bovine Intramammary Bacteriome and Resistome from healthy cows of Swiss dairy farms in the Canton Tessin.

Healthy, untreated cows of nine dairy herds from the Swiss Canton Tessin were analyzed three times within one year to identify the most abundant species of the intramammary bacteriome. Aseptically collected milk samples were cultured and bacteria identified using MALDI-TOF. Of 256 cows analyzed, 96% were bacteriologically positive and 80% of the 1,024 quarters were positive for at least one bacterial species. 84.5% of the quarters were healthy with somatic cell counts (SCC) < 200,000 cells/mL, whereas 15.5% of the quarters showed a subclinical mastitis (SCC ≥ 200,000 cells/mL). We could assign 1,288 isolates to 104 different bacterial species including 23 predominant species. Non-aureus staphylococci and mammaliicocci (NASM) were most prevalent (14 different species; 73.5% quarters). Staphylococcus xylosus and Mammaliicoccus sciuri accounted for 74.7% of all NASM isolates. To describe the intramammary resistome, 350 isolates of the predominant species were selected and subjected to short-read whole genome sequencing (WGS) and phenotypic antibiotic resistance profiling. While complete genomes of eight type strains were available, the remaining 15 were de novo assembled with long reads as a resource for the community. The 23 complete genomes served for reference-based assembly of the Illumina WGS data. Both chromosomes and mobile genetic elements were examined for antibiotic resistance genes (ARGs) using in-house and online software tools. ARGs were then correlated with phenotypic antibiotic resistance data from minimum inhibitory concentration (MIC). Phenotypic and genomic antimicrobial resistance was isolate-specific. Resistance to clindamycin and oxacillin was most frequently observed (65 and 30%) in Staphylococcus xylosus but could not be linked to chromosomal or plasmid-borne ARGs. However, in several cases, the observed antimicrobial resistance could be explained by the presence of mobile genetic elements like tetK carried on small plasmids. This represents a possible mechanism of transfer between non-pathogenic bacteria and pathogens of the mammary gland within and between herds. The-to our knowledge-most extensive bacteriome reported and the first attempt to link it with the resistome promise to profoundly affect veterinary bacteriology in the future and are highly relevant in a One Health context, in particular for mastitis, the treatment of which still heavily relies on antibiotics.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Validation of an in vitro digestive system for studying macronutrient decomposition in humans.

The digestive process transforms nutrients and bioactive compounds contained in food to physiologically active compounds. In vitro digestion systems have proven to be powerful tools for understanding and monitoring the complex transformation processes that take place during digestion. Moreover, the investigation of the physiological effects of certain nutrients demands an in vitro digestive process that is close to human physiology. In this study, human digestion was simulated with a 3-step in vitro process that was validated in depth by choosing pasteurized milk as an example of a complex food matrix. The evolution and decomposition of the macronutrients was followed over the entire digestive process to the level of intestinal enterocyte action, using protein and peptide analysis by SDS-PAGE, reversed-phase HPLC, size exclusion HPLC, and liquid chromatography-MS. The mean peptide size after in vitro digestion of pasteurized milk was 5-6 amino acids (AA). Interestingly, mostly essential AA (93.6%) were released during in vitro milk digestion, a significantly different relative distribution compared to the total essential AA concentration of bovine milk (44.5%). All TG were degraded to FFA and monoacylglycerols. Herein, we present a human in vitro digestion model validated for its ability to degrade the macronutrients of dairy products comparable to physiological ranges. It is suited to be used in combination with a human intestinal cell culture system, allowing ex vivo bioavailability measurements and assessment of the bioactive properties of food components.

The NutriChip project--translating technology into nutritional knowledge.

Advances in food transformation have dramatically increased the diversity of products on the market and, consequently, exposed consumers to a complex spectrum of bioactive nutrients whose potential risks and benefits have mostly not been confidently demonstrated. Therefore, tools are needed to efficiently screen products for selected physiological properties before they enter the market. NutriChip is an interdisciplinary modular project funded by the Swiss programme Nano-Tera, which groups scientists from several areas of research with the aim of developing analytical strategies that will enable functional screening of foods. The project focuses on postprandial inflammatory stress, which potentially contributes to the development of chronic inflammatory diseases. The first module of the NutriChip project is composed of three in vitro biochemical steps that mimic the digestion process, intestinal absorption, and subsequent modulation of immune cells by the bioavailable nutrients. The second module is a miniaturised form of the first module (gut-on-a-chip) that integrates a microfluidic-based cell co-culture system and super-resolution imaging technologies to provide a physiologically relevant fluid flow environment and allows sensitive real-time analysis of the products screened in vitro. The third module aims at validating the in vitro screening model by assessing the nutritional properties of selected food products in humans. Because of the immunomodulatory properties of milk as well as its amenability to technological transformation, dairy products have been selected as model foods. The NutriChip project reflects the opening of food and nutrition sciences to state-of-the-art technologies, a key step in the translation of transdisciplinary knowledge into nutritional advice.

Comparison of nutritional composition between plant-based drinks and cow's milk.

The high decline in liquid milk consumption in Western countries has been compensated by the increased consumption of processed dairy products and the rapidly increasing number of new plant-based beverages constantly introduced in the market, advertised as milk substitutes and placed on shelves near milk products. To provide better understanding about the nutritional value of these drinks compared with cow's milk, 27 plant-based drinks of 8 different species and two milk samples were purchased from two big retailers in Switzerland, and their composition regarding protein, carbohydrate, fat, vitamin, and mineral contents and residue load [glyphosate, aminomethylphosphonic acid (AMPA), and arsenic] was analyzed quantitatively and qualitatively. Energy and nutrient intakes were calculated and compared with the dietary reference values for Germany, Austria and Switzerland (D-A-CH). In addition, the digestible indispensable amino acid score (DIAAS) was calculated to estimate the quality of the proteins. Milk contained more energy; fat; carbohydrate; vitamins C, B2, B12, and A; biotin; pantothenic acid; calcium; phosphorus; and iodine than most plant-based drinks. Soy drinks provided slightly more protein and markedly more vitamins B1 and B6, folic acid, and vitamins E and D2 (with supplemented vitamin D2) and K1, magnesium, manganese, iron, and copper than milk and the other plant-based drinks. However, with the exception of cow's milk and soy drinks, which had > 3% protein, most milk alternatives contained ≤ 1% protein; therefore, they cannot be considered good protein sources. In regard to protein quality, milk was outstanding compared with all plant-based drinks and exhibited higher calculated DIAASs. Our results show that the analyzed plant-based drinks are not real alternatives to milk in terms of nutrient composition, even if the actual fortification is taken into account. Improved fortification is still an issue and can be optimized using the most bioavailable and soluble derivatives. Complete replacement of milk with plant-based drinks without adjusting the overall diet can lead to deficiencies of certain important nutrients in the long term.

The use of alkaline phosphatase and possible alternative testing to verify pasteurisation of raw milk, colostrum, dairy and colostrum-based products.

Pasteurisation of raw milk, colostrum, dairy or colostrum-based products must be achieved using at least 72°C for 15 s, at least 63°C for 30 min or any equivalent combination, such that the alkaline phosphatase (ALP) test immediately after such treatment gives a negative result. For cows' milk, a negative result is when the measured activity is ≤ 350 milliunits of enzyme activity per litre (mU/L) using the ISO standard 11816-1. The use and limitations of an ALP test and possible alternative methods for verifying pasteurisation of those products from other animal species (in particular sheep and goats) were evaluated. The current limitations of ALP testing of bovine products also apply. ALP activity in raw ovine milk appears to be about three times higher and in caprine milk about five times lower than in bovine milk and is highly variable between breeds. It is influenced by season, lactation stage and fat content. Assuming a similar pathogen inactivation rate to cows' milk and based on the available data, there is 95-99% probability (extremely likely) that pasteurised goat milk and pasteurised sheep milk would have an ALP activity below a limit of 300 and 500 mU/L, respectively. The main alternative methods currently used are temperature monitoring using data loggers (which cannot detect other process failures such as cracked or leaking plates) and the enumeration of Enterobacteriaceae (which is not suitable for pasteurisation verification but is relevant for hygiene monitoring). The inactivation of certain enzymes other than ALP may be more suitable for the verification of pasteurisation but requires further study. Secondary products of heat treatment are not suitable as pasteurisation markers due to the high temperatures needed for their production. More research is needed to facilitate a definitive conclusion on the applicability of changes in native whey proteins as pasteurisation markers.

Higher microbial diversity in raw than in pasteurized milk Raclette-type cheese enhances peptide and metabolite diversity after in vitro digestion.

Numerous bacteria are responsible for hydrolysis of proteins during cheese ripening. The raw milk flora is a major source of bacterial variety, starter cultures are needed for successful acidification of the cheese and proteolytic strains like Lactobacillus helveticus, are added for flavor improvement or acceleration of ripening processes. To study the impact of higher bacterial diversity in cheese on protein hydrolysis during simulated human digestion, Raclette-type cheeses were produced from raw or heat treated milk, with or without proteolytic L. helveticus and ripened for 120 days. Kinetic processes were studied with a dynamic (DIDGI®) in vitro protocol and endpoints with the static INFOGEST in vitro digestion protocol, allowing a comparison of the two in vitro protocols at the level of gastric and intestinal endpoints. Both digestion protocols resulted in comparable peptide patterns after intestinal digestion and higher microbial diversity in cheeses led to a more diverse peptidome after simulated digestion.

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.

A standardised semi-dynamic in vitro digestion method suitable for food - an international consensus.

The link between food and human health is increasingly a topic of interest. One avenue of study has been to assess food disintegration and interactions within the gastrointestinal tract. In vitro digestion models have been widely used to overcome the constrictions associated with in vivo methodology. The COST Action INFOGEST developed an international, harmonised protocol for static simulation of digestion in the upper gastrointestinal tract of adults. This protocol is widely used; however, it is restricted to providing end-point assessment without considering the possible structural changes. On the other hand, there are dynamic models that provide more physiologically relevant data but are expensive and difficult to access. There is a gap between these models. The method outlined in this article provides an intermediate model; it builds upon the harmonised static model and now includes crucial kinetic aspects associated with the gastric phase of digestion, including gradual acidification, fluid and enzyme secretion and emptying. This paper provides guidance and standardised recommendations of a physiologically relevant semi-dynamic in vitro simulation of upper gastrointestinal tract digestion, with particular focus on the gastric phase. Adaptations of this model have already been used to provide kinetic data on nutrient digestion and structural changes during the gastric phase that impact on nutrient absorption. Moreover, it provides a simple tool that can be used in a wide range of laboratories.

Digestion of milk proteins: Comparing static and dynamic in vitro digestion systems with in vivo data.

In the frame of the COST action INFOGEST, a static in vitro digestion protocol has been elaborated aiming at the improvement of data comparability by harmonizing the experimental conditions. The success in harmonization was confirmed with inter-laboratory trials using skim milk powder as a standardized model food. Moreover, the physiological relevance of the gastric and intestinal endpoints of the static digestion protocol was demonstrated in a pig in vivo trial, with the same skim milk powder and samples collected from different sections of the digestive tract, as well as in a human study with from jejunal effluents. In vivo, digestion is a dynamic process influenced by peristalsis and by the gradual secretion of enzymes and juices and the dwell time of the food. To mimic these physiological mechanisms, dynamic in vitro digestion protocols are widely used. Until now, the differences of protein hydrolysis taking place during dynamic and static in vitro digestion have not been investigated. In this study, the gradual hydrolysis of the main milk proteins present in skim milk powder was digested with the dynamic DIDGI®-system using adult digestion protocol and the static harmonized INFOGEST method. Protein hydrolysis was analyzed by gel electrophoresis, peptide patterns were measured with mass spectrometry, and free amino acids with high pressure liquid chromatography. The peptide patterns at the gastric and intestinal endpoints of in vitro digestion showed a good approximation to the in vivo results from pigs. Moreover, gradual peptide generation was comparable in both in vitro digestion conditions. However, the dynamic protocol reflected the physiological situation better at the level of free amino acid release. Nonetheless, in both in vitro digestion protocols, absorption of free amino acids is not simulated, and they are therefore limited in reflecting the in vivo situation at this level.

The relevance of a digestibility evaluation in the allergenicity risk assessment of novel proteins. Opinion of a joint initiative of COST action ImpARAS and COST action INFOGEST.

The current allergenicity assessment of novel proteins is based on the EFSA GMO guidance. Recently, EFSA launched a new guidance document on allergenicity assessment of GM plants (2017). This document describes, amongst other topics, the new scientific and regulatory developments on in vitro protein digestibility tests. The EFSA GMO Panel stated that for in vitro protein digestibility tests, additional investigations are needed before any additional recommendation in the form of guidance can be provided. To this end, an interim phase is considered necessary to evaluate the revisions to the in vitro gastrointestinal digestion test, proposed by EFSA. This prompted the establishment of a joint workshop through two COST Action networks: COST Action ImpARAS and COST Acton INFOGEST. In 2017, a workshop was organised to discuss the relevance of digestion in allergenicity risk assessment and how to potentially improve the current methods and readouts. The outcome of the workshop is that there is no rationale for a clear readout that is predictive for allergenicity and we suggest to omit the digestion test from the allergenicity assessment strategy for now, and put an effort into filling the knowledge gaps as summarized in this paper first.

INFOGEST static in vitro simulation of gastrointestinal food digestion.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.