The pattern of peptides released from dairy and egg proteins is highly dependent on the simulated digestion scenario

Evaluating the gastrointestinal (GI) fate of proteins is part of the assessment to determine whether proteins are safe to consume. In vitro digestion tests are often used for screening purposes in the evaluation of potential allergenicity. However, the current pepsin resistant test used by the European Food Safety Authority, only corresponds to fasted gastric conditions representative of a late phase adult stomach. In addition, these tests are performed on isolated proteins and the effect of the food matrix and processing are not systematically considered. The aim of this research is to compare three different static in vitro GI scenarios that are physiologically relevant. Namely, an infant, early phase (fed state) adult and late phase (fasted state) adult model. These protocols are applied to well-characterised isolated dairy (β-lactoglobulin and β-casein) and egg (lysozyme and ovalbumin) proteins and the impact of food matrix/processing on their proteolysis is also investigated. A combination of SDS-PAGE, LC-MS/MS and spectrometric assay was used for the evaluation of the proteolysis. Results highlight differences across the three GI scenarios whether on isolated proteins or within food matrices. The infant model led to incomplete digestion, leaving intact egg proteins, either isolated or in the food matrix, and intact β-lactoglobulin in the milk. In addition, peptides greater than 9 amino acids were found throughout the intestinal phase for all proteins studied, regardless of the scenario. This reinforces the difficulty of linking protein digestibility to potential allergenicity because many other factors are involved that need further investigation

Can dynamic in vitro digestion systems mimic the physiological reality?

During the last decade, there has been a growing interest in understanding the fate of food during digestion in the gastrointestinal tract in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore simple static in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments but these models are quite basic and hardly recreate the complexity of the digestive tract. In contrast, dynamic models that allow pH regulation, flow of the food and injection in real time of digestive enzymes in the different compartments of the gastrointestinal tract are more promising to accurately mimic the digestive process. Most of the systems developed so far have been compared for their performances to in vivo data obtained on animals and/or humans. The objective of this article is to review the validation towards in vivo data of some of the dynamic digestion systems currently available in order to determine what aspects of food digestion they are able to mimic. Eight dynamic digestion systems are presented as well as their validation towards in vivo data. Advantages and limits of each simulator is discussed. This is the result of a cooperative international effort made by some of the scientists involved in Infogest, an international network on food digestion

Prématurité de la mise bas chez la truie et signification du poids à la naissance du porcelet

International audienc

Report on EFSA project OC/EFSA/GMO/2017/01 “In vitro protein digestibility” (Allergestion)

The primary objective of the assessment of novel proteins is to evaluate whether they are safe to consume, including potential allergenicity. As part of a suite of assessments, the in vitro digestion of protein has been seen as a useful exercise. Thus, in line with the guidance offered by the EFSA GMO Panel we are using an early phase and a late phase gastric simulation as well as a simulation of the infant gastric compartment, all followed by intestinal phases. These digestion scenarios were used with a panel of 10 proteins from plant and animal origin that were proteins with distinct allergenic potential. The results from the SDS‐PAGE and densitometry show significant and mainly expected differences between the different digestion scenarios. The milk proteins were fully digested in the intestinal phase but the BLG was largely resistant to pepsin. In contrast, the egg proteins showed significant persistence except under late phase conditions. For the plant proteins, KTI and ConA were largely resistant to all conditions whereas LIP and AP were only resistant to infant conditions. Similarly, Ara h 1 showed some resistance to infant gastric conditions. The LC‐MS analysis of peptides was able to highlight a number of clusters where differences were seen between the digestion scenarios and these could in some cases be mapped onto the primary sequence and where relevant compared with known allergenic epitopes. Under the different digestion scenarios, we were able to show significant differences in the persistence of peptides larger than 9 amino acids and significant overlap of abundant peptides from early phase intestinal digestion and known epitopes for a number of proteins. Although, linking these differences to immunological responses (epitope mapping) still seems to be quite challenging, there are clear differences between scenarios and strong potential for improved risk assessment

Static in vitro digestion tests to assess allergenic risk of novel proteins

IntroductionEvaluating the gastrointestinal fate of proteins is paramount to assess whether they are safe toconsume. The resistance of proteins to digestion may play a role in determining their allergenicpotential since incomplete digestion may cause undesired immune responses via sensitisation inthe duodenum. In order to evaluate the digestibility of proteins, in vitro protocols seem appropriatewhen ethical constraints hinder in vivo studies. The current in vitro digestion model used by theEuropean Food Safety Authority (EFSA Journal 2017, 15, 4862) to assess allergenicity of proteinsis the pepsin resistant test. This uses harsh gastric conditions of acidity and enzyme concentrationthat would mimic the end of gastric emptying (late phase) or fasted state in human adults. A morerealistic approach considering other relevant conditions in healthy adults (early phase or fed state),or impaired digestion (e.g. infants) may provide useful information on how the combined effect of pHand enzyme concentration affects protein digestibility.ObjectiveOur current project contracted by EFSA aims looking at the combination of three different static invitro digestion models to evaluate the resistance of proteins to digestion by gastrointestinal enzymesthat could sensitise and eventually trigger an allergic reaction. The main objective is the developmentand validation of a robust in vitro digestion protocol for purified proteins consistently reproducibleacross different laboratories.MethodologyComparison of an infant, early phase adult, and late phase adult model is being applied on a panelof allergen/non-allergen proteins from animal and vegetal origin. The proteolysis rate and extent isdetermined with SDS-PAGE and LCMS in order to detect persistent intact protein and hydrolysisproducts larger 9 amino acids.Main findingsDifferences in the kinetics of proteolysis may be found across models for proteins that are not pepsinresistant.ConclusionThis project highlights the importance of a multi-test protocol to assess protein digestibility

Manufacture of Whey Protein Hydrolysates Using Plant Enzymes: Effect of Processing Conditions and Simulated Gastrointestinal Digestion on Angiotensin-I-Converting Enzyme (ACE) Inhibitory Activity

Hydrolysis of proteins leads to the release of bioactive peptides with positive impact on human health. Peptides exhibiting antihypertensive properties (i.e., inhibition of angiotensin-I-converting enzyme) are commonly found in whey protein hydrolysates made with enzymes of animal, plant or microbial origin. However, bioactive properties can be influenced by processing conditions and gastrointestinal digestion. In this study, we evaluated the impact of three plant enzymes (papain, bromelain and ficin) in the manufacture of whey protein hydrolysates with varying level of pH, enzyme-to-substrate ratio and time of hydrolysis, based on a central composite design, to determine the degree of hydrolysis and antihypertensive properties. Hydrolysates made on laboratory scales showed great variation in the type of enzyme used, their concentrations and the pH level of hydrolysis. However, low degrees of hydrolysis in papain and bromelain treatments were associated with increased antihypertensive properties, when compared to ficin. Simulated gastrointestinal digestion performed for selected hydrolysates showed an increase in antihypertensive properties of hydrolysates made with papain and bromelain, which was probably caused by further release of peptides. Several peptides with reported antihypertensive properties were found in all treatments. These results suggest plant enzymes used in this study can be suitable candidates to develop ingredients with bioactive properties

Static in vitro digestion tests to assess allergenic risk of novel proteins

IntroductionEvaluating the gastrointestinal fate of proteins is paramount to assess whether they are safe toconsume. The resistance of proteins to digestion may play a role in determining their allergenicpotential since incomplete digestion may cause undesired immune responses via sensitisation inthe duodenum. In order to evaluate the digestibility of proteins, in vitro protocols seem appropriatewhen ethical constraints hinder in vivo studies. The current in vitro digestion model used by theEuropean Food Safety Authority (EFSA Journal 2017, 15, 4862) to assess allergenicity of proteinsis the pepsin resistant test. This uses harsh gastric conditions of acidity and enzyme concentrationthat would mimic the end of gastric emptying (late phase) or fasted state in human adults. A morerealistic approach considering other relevant conditions in healthy adults (early phase or fed state),or impaired digestion (e.g. infants) may provide useful information on how the combined effect of pHand enzyme concentration affects protein digestibility.ObjectiveOur current project contracted by EFSA aims looking at the combination of three different static invitro digestion models to evaluate the resistance of proteins to digestion by gastrointestinal enzymesthat could sensitise and eventually trigger an allergic reaction. The main objective is the developmentand validation of a robust in vitro digestion protocol for purified proteins consistently reproducibleacross different laboratories.MethodologyComparison of an infant, early phase adult, and late phase adult model is being applied on a panelof allergen/non-allergen proteins from animal and vegetal origin. The proteolysis rate and extent isdetermined with SDS-PAGE and LCMS in order to detect persistent intact protein and hydrolysisproducts larger 9 amino acids.Main findingsDifferences in the kinetics of proteolysis may be found across models for proteins that are not pepsinresistant.ConclusionThis project highlights the importance of a multi-test protocol to assess protein digestibility

Static in vitro digestion tests to assess allergenic risk of novel proteins

IntroductionEvaluating the gastrointestinal fate of proteins is paramount to assess whether they are safe toconsume. The resistance of proteins to digestion may play a role in determining their allergenicpotential since incomplete digestion may cause undesired immune responses via sensitisation inthe duodenum. In order to evaluate the digestibility of proteins, in vitro protocols seem appropriatewhen ethical constraints hinder in vivo studies. The current in vitro digestion model used by theEuropean Food Safety Authority (EFSA Journal 2017, 15, 4862) to assess allergenicity of proteinsis the pepsin resistant test. This uses harsh gastric conditions of acidity and enzyme concentrationthat would mimic the end of gastric emptying (late phase) or fasted state in human adults. A morerealistic approach considering other relevant conditions in healthy adults (early phase or fed state),or impaired digestion (e.g. infants) may provide useful information on how the combined effect of pHand enzyme concentration affects protein digestibility.ObjectiveOur current project contracted by EFSA aims looking at the combination of three different static invitro digestion models to evaluate the resistance of proteins to digestion by gastrointestinal enzymesthat could sensitise and eventually trigger an allergic reaction. The main objective is the developmentand validation of a robust in vitro digestion protocol for purified proteins consistently reproducibleacross different laboratories.MethodologyComparison of an infant, early phase adult, and late phase adult model is being applied on a panelof allergen/non-allergen proteins from animal and vegetal origin. The proteolysis rate and extent isdetermined with SDS-PAGE and LCMS in order to detect persistent intact protein and hydrolysisproducts larger 9 amino acids.Main findingsDifferences in the kinetics of proteolysis may be found across models for proteins that are not pepsinresistant.ConclusionThis project highlights the importance of a multi-test protocol to assess protein digestibility

Structural characterization of heat-induced protein aggregates in model infant milk formulas

International audienceHeat treatments induce structural modifications of bovine milk proteins. In this study, we aimed to investigate how these structural modifications are affected by the whey protein profile of infant milk formulas (IMFs) and heat treatment parameters. Three model IMFs (1.3% of proteins; caseinwhey protein ratio 4060) differing by the whey protein profile (13.37 α-lactalbumin (α-LA)β-lactoglobulin (β-LG), 13.261.80 α-LAβ-LGlactoferrin (LF) or 10.130.40 α-LAβ-LGLF), were heated at 67.5 °C or 80 °C to reach an akin whey protein denaturation extent of 65%. Protein structures were analyzed by asymmetrical flow field-flow fractionation coupled with multiangle light scattering and differential refractometer, transmission electron microscopy and electrophoresis. The unheated IMFs were used as reference. The results showed that LF addition in IMFs induced partial casein micelle disintegration before heating. In the absence of added LF, the heat-denatured whey proteins either formed soluble whey protein aggregates or casein micelle-bound whey protein aggregates. The latter were favored at the expense of soluble aggregates in the heated IMFs with the LF content increase and the concomitant β-LG content decrease. Consequently, the casein micelle structure was strongly dependent on the β-LG and LF amounts in IMFs and on the heating temperature. In the IMFs containing β-LG and LF and at temperature greater than the β-LG denaturation temperature, the casein micelles exhibited filamentous appendages on its surface after heating. Below the denaturation temperature of β-LG and in IMFs containing trace β-LG amount, the heated casein micelles were perfectly spherical with a smooth surface. © 2020 Elsevier Lt