Beneficial effect of mildly pasteurized whey protein on intestinal integrity and innate defense in preterm and near-term piglets

Background. The human digestive tract is structurally mature at birth, yet maturation of gut functions such as digestion and mucosal barrier continues for the next 1–2 years. Human milk and infant milk formulas (IMF) seem to impact maturation of these gut functions differently, which is at least partially related to high temperature processing of IMF causing loss of bioactive proteins and formation of advanced glycation end products (AGEs). Both loss of protein bioactivity and formation of AGEs depend on heating temperature and time. The aim of this study was to investigate the impact of mildly pasteurized whey protein concentrate (MP-WPC) compared to extensively heated WPC (EH-WPC) on gut maturation in a piglet model hypersensitive to enteral nutrition. Methods. WPC was obtained by cold filtration and mildly pasteurized (73 °C, 30 s) or extensively heat treated (73 °C, 30 s + 80 °C, 6 min). Preterm (~90% gestation) and near-term piglets (~96% gestation) received enteral nutrition based on MP-WPC or EH-WPC for five days. Macroscopic and histologic lesions in the gastro-intestinal tract were evaluated and intestinal responses were further assessed by RT-qPCR, immunohistochemistry and enzyme activity analysis. Results. A diet based on MP-WPC limited epithelial intestinal damage and improved colonic integrity compared to EH-WPC. MP-WPC dampened colonic IL1-β, IL-8 and TNF-α expression and lowered T-cell influx in both preterm and near-term piglets. Anti-microbial defense as measured by neutrophil influx in the colon was only observed in near-term piglets, correlated with histological damage and was reduced by MP-WPC. Moreover, MP-WPC stimulated iALP activity in the colonic epithelium and increased differentiation into enteroendocrine cells compared to EH-WPC. Conclusions. Compared to extensively heated WPC, a formula based on mildly pasteurized WPC limits gut inflammation and stimulates gut maturation in preterm and near-term piglets and might therefore also be beneficial for preterm and (near) term infants.</p

Protein digestion kinetics, a proxy for postprandial amino acid responses

Protein in human nutrition delivers essential amino acids (AA) and nitrogen-containing molecules, which are required for maintenance and growth. Current protein quality definitions, consider how well the source AA composition matches a reference and its total digestibility in the gastrointestinal tract. However, the kinetics of availability, reflected in plasma AA peak concentrations after ingestion, are usually not considered, while this is an important determinant of the use of the AA building blocks for tissue protein synthesis or oxidation. Another clinical outcome related to dietary protein intake is the amount of protein reaching the colon, which can negatively influence gut heath. Key determinants of postprandial plasma (pp) AA peak concentrations and colonic protein flow are the physiological, physical, and chemical processes that occur in the stomach and small intestine, including gastric emptying rate, protein hydrolysis kinetics and total protein digestibility. In turn, these are influenced by the characteristics of the ingested food, such as protein composition, product processing and product matrix. The overall research question addressed in this thesis was: How do food characteristics impact overall protein digestion, and how is this reflected in postprandial plasma (pp) AA peak concentration and colonic protein flow? The specific aims were to: gain insights in the relative importance of gastric emptying and protein hydrolysis kinetics in determining the postprandial AA peak concentration, differentiate the individual effects of the mentioned food characteristics on gastrointestinal processes and assess and improve correlation between in vitro and in vivo studies. The work combined in vitro and in vivo digestion studies on milk proteins in humans and piglets to advance the ability to formulate products with an improved protein quality tailored to the specific needs of vulnerable populations as for instance infants or elderly people.The effect of product matrix on the pp AA concentrations of whey protein and casein was investigated in healthy elderly. Furthermore, to better understand the relative importance of gastric emptying and protein hydrolysis, in vitro investigations in a newly developed two-step semi-dynamic model of the gastrointestinal tract simulating elderly conditions were undertaken. Addition of extra carbohydrates and lipids lowered the pp AA concentrations of whey protein and casein in healthy elderly. These differences appeared to be mediated by the gastrointestinal behavior of the products.The effect of whey protein heat induced denaturation, as well as the effect of changing native milk protein composition by incorporation of non-clotting casein, on pp AA concentrations was studied in neonatal piglets. In addition, to gain insights in the role of protein hydrolysis, gastric emptying measurement and pharmacokinetic modelling of pp AA concentrations was performed. Changing native whey protein composition by inclusion of non-clotting casein increased pp AA concentrations, but whey protein heat induced denaturation did not. Both interventions did not affect gastric emptying. The differences induced by protein composition were partly explained by the difference in AA composition, but more likely differences in protein hydrolysis and absorption kinetics. To further elucidate the mechanisms of effect, protein hydrolysis and absorbable product release kinetics and mechanisms as affected by milk protein composition or denaturation were investigated in vitro models of the gastrointestinal tract simulating infant conditions. Both whey protein denaturation and β-casein inclusion increased the rate of intact protein loss without affecting absorbable product release during. The results suggested that intermediate digestion product characteristics are important in relation to pp AA responses, as it was found that the correlation between in vitro digestion measures and in vivo pp AA concentrations was greatly improved when intermediate digestion products were taken into considerationThe kinetics of protein hydrolysis and absorbable product release of human milk (HM) and cow’s milk based infant formula (IF) were analyzed in detail in an in vitro two-step semi-dynamic model of the gastrointestinal tract simulating infant conditions. IF gastrointestinal protein hydrolysis and absorbable product release was found to be faster than HM, possibly due to the presence of digestion resistant proteins in HM, which offers directions to bring IF closer to HM.The effect of IF protein composition and matrix on protein digestibility and colonic protein flow was investigated in ileal cannulated piglets. Fermented formula had a significantly higher apparent ileal crude protein digestibility than standard and hydrolyzed formula, and displayed lower ileal proteolytic activity than standard formula, possibly via a mechanistic pathway that involves a different physiological response.Overall, it was concluded that not only gastric emptying rate, but also protein hydrolysis kinetics determine the pp AA peak concentration. Regarding the individual effects of food characteristics, firstly, it was concluded that protein composition is a major determinant of both gastric emptying and protein hydrolysis kinetics and is therefore a key means to further optimize pp AA peak concentration of nutritional solutions to meet the specific requirements of infants and elderly. Secondly, heat induced protein denaturation only limitedly affects gastric emptying, protein hydrolysis kinetics, and pp AA concentrations. However, the impact of heat induced denaturation on intermediate digestion product characteristics is large, which could potentially infer a change in&nbsp; bioactivity relevant for health. Lastly, protein product matrix’s main effects are on gastric emptying via physical and physiological feedback mechanisms, but protein hydrolysis can also be affected. Concerning the correlation of in vitro and in vivo: in vitro protein digestion product release that includes intermediate peptides (<5 kDa)&nbsp; correlates with in vivo postprandial AA peak concentrations better than does absorbable digestion product (Free AA + di- and tripeptides) release only. Recommendations for improvement of milk products to optimize protein quality tailored to the needs of elderly and infants thus include specific changes in protein composition

Attention modulation by proportion congruency: the asymmetrical list shifting effect

Proportion congruency effects represent hallmark phenomena in current theorizing about cognitive control. This is based on the notion that proportion congruency determines the relative levels of attention to relevant and irrelevant information in conflict tasks. However, little empirical evidence exists that uniquely supports such an attention modulation account; moreover, a rivaling account was recently proposed that attributes the effect of proportion congruency to mere contingency learning. In the present study, the influences of shifts in list-wide (Experiment 1) or item-specific (Experiment 2) proportion congruency were investigated. As predicted by attention modulation but not by contingency learning; strong asymmetries were observed in such shifting: An increase in the proportion of congruent trials had only limited impact on the size of the congruency effect when participants were initially trained with a mostly incongruent list, but the impact was substantial for an equivalent increase of incongruent trials when participants were initially trained with a mostly congruent list. This asymmetrical list shifting effect directly supports attention modulation by proportion congruency manipulations and as such provides a novel tool for exploring cognitive control. Implications of our findings for existing theories of cognitive control are discussed

Gastrointestinal Protein Hydrolysis Kinetics : Opportunities for Further Infant Formula Improvement

The postprandial plasma essential amino acid (AA) peak concentrations of infant formula (IF) are higher than those of human milk (HM) in infants. In addition, several HM proteins have been recovered intact in infant stool and appeared digestion resistant in vitro. We, therefore, hypothesized that gastrointestinal protein hydrolysis of IF is faster than HM and leads to accelerated absorbable digestion product release. HM and IF protein hydrolysis kinetics were compared in a two-step semi-dynamic in vitro infant digestion model, and the time course of degree of protein hydrolysis (DH), loss of intact protein, and release of free AA and peptides was evaluated. Gastric DH increase was similar for IF and HM, but the rate of intestinal DH increase was 1.6 times higher for IF than HM. Intact protein loss in IF was higher than HM from 120 min gastric phase until 60 min intestinal phase. Intestinal phase total digestion product (free AA + peptides <5 kDa) concentrations increased ~2.5 times faster in IF than HM. IF gastrointestinal protein hydrolysis and absorbable product release are faster than HM, possibly due to the presence of digestion-resistant proteins in HM. This might present an opportunity to further improve IF bringing it closer to HM

Assessment of milk protein digestion kinetics: Effects of denaturation by heat and protein type used

Knowledge about how molecular properties of proteins affect their digestion kinetics is crucial to understand protein postprandial plasma amino acid (AA) responses. Previously it was found that a native whey protein isolate (NWPI) and heat denatured whey protein isolate (DWPI) elicit comparable postprandial plasma AA peak concentrations in neonatal piglets, while a protein base ingredient for infant formula (PBI, a β-casein-native whey protein mixture) caused a 39% higher peak AA concentration than NWPI. We hypothesized that both whey protein denaturation by heat as well as changing protein composition by including β-casein, increases the rate of intact protein loss, and that changing the protein composition (by including β-casein), but not whey protein denaturation, yields a faster absorbable product release. Therefore NWPI (91% native), DWPI (91% denatured) and PBI hydrolysis was investigated in a semi-dynamic in vitro digestion model (SIM). NWPI and DWPI hydrolysis were also compared in a dynamic digestion model with dialysis (TIM-1) to exclude potential product inhibition effects that may occur in a closed vessel digestion model as SIM. In both models, the degree of hydrolysis (DH), loss of intact protein, and release of absorbable products (SIM: <0.5 kDa peptides and free AA, TIM-1: bioaccessible AA) were monitored. Additionally, in SIM, intermediate product amounts and their characteristics were determined. DWPI showed considerably faster intact protein loss, but similar DH and absorbable product release kinetics compared with NWPI in both models. Furthermore, more, relatively large, intermediate products were released from DWPI than from NWPI. PBI showed increased intact protein loss, similar DH, and absorbable product release kinetics, but more, relatively small, intermediate products than NWPI. In conclusion, both whey protein denaturation and β-casein inclusion increased the rate of intact protein loss without affecting absorbable product release during in vitro digestion. Our results suggest that intermediate digestion product characteristics are important in relation to postprandial AA responses

INFOGEST inter-laboratory recommendations for assaying gastric and pancreatic lipases activities prior to in vitro digestion studies

In vitro digestion studies often use animal digestive enzyme extracts as substitutes of human gastric and pancreatic secretions. Pancreatin from porcine origin is thus commonly used to provide relevant pancreatic enzymes such as proteases, amylase and lipase. Rabbit gastric extracts (RGE) have been recently introduced to provide gastric lipase in addition to pepsin. Before preparing simulated gastric and pancreatic extracts with targeted enzyme activities as described in in vitro digestion protocols, it is important to determine the activities of enzyme preparations using validated methods. The purpose of this inter-laboratory study within the INFOGEST network was to test the repeatability and reproducibility of lipase assays using the pH-stat technique for measuring the activities of gastric and pancreatic lipases from various sources. Twenty-one laboratories having different pH-stat devices received the same protocol with identical batches of RGE and two pancreatin sources. Lipase assays were performed using tributyrin as a substrate and three different amounts (50, 100 and 200 mu g) of each enzyme preparation. The repeatability results within individual laboratories were satisfactory with coefficients of variation (CVs) ranging from 4 to 8% regardless of the enzyme amount tested. However, the inter laboratory variability was high (CV > 15%) compared to existing standards for bioanalytical assays. We identified and weighted the contributions to inter-laboratory variability of several parameters associated with the various pH-stat equipment used in this study (e.g. reaction vessel volume and shape, stirring mode and rate, burette volume for the automated delivery of sodium hydroxide). Based on this, we established recommendations for improving the reproducibility of lipase assays using the pH-stat technique. Defining accurate and complete recommendations on how to correctly quantify activity levels of enzyme preparations is a gateway to promising comparison of in vitro data obtained from different laboratories following the same in vitro digestion protocol.European Commission COST Action [FA1005 INFOGEST]; INRAE; 'Healthy and Safe Food System (KB37) ' knowledge base program of the Dutch Ministry of Agriculture, Nature and Food Quality (LNV) [KB-37-001-007]The authors thank the European Commission COST Action FA1005 INFOGEST and INRAE for financing the meetings and conferences that enable the 21 laboratories to network and organise the work presented in this article. We also acknowledge the 'Healthy and Safe Food System (KB37) ' knowledge base program of the Dutch Ministry of Agriculture, Nature and Food Quality (LNV) , with grant number KB-37-001-007 for funding of this work performed at Wageningen Food & Biobased Research. We are grateful to Dr Sawsan Amara (CEO of Lipolytech SA., Marseille, France) for her generous gift of RGE and to Drs Jan Ludemann, Anja Jensen, Olaf Friedrich and Claus Middelberg from Nordmark Arzneimittel GmbH & Co. KG (Uetersen, Germany) for their generous gift of Nordmark pancreatin. Finally, we thank Drs Maria Fatima Cabral and Judite Costa from iBET for their technical support and discussion

Sterilization in a liquid of a specific starch makes it slowly digestible in vitro and low glycemic in rats

Diabetics are recommended to eat a balanced diet containing normal amounts of carbohydrates, preferably those with a low glycemic index. For solid foods, this can be achieved by choosing whole-grain, fiber-rich products. For (sterilized) liquid products, such as meal replacers, the choices for carbohydrate sources are restricted due to technological limitations. Starches usually have a high glycemic index after sterilization in liquids, whereas low glycemic sugars and sugar replacers can only be used in limited amounts. Using an in vitro digestion assay, we identified a resistant starch (RS) source [modified high amylose starch (mHAS)] that might enable the production of a sterilized liquid product with a low glycemic index. Heating mHAS for 4-5 min in liquid increased the slowly digestible starch (SDS) fraction at the expense of the RS portion. The effect was temperature dependent and reached its maximum above 120 degrees C. Heating at 130 degrees C significantly reduced the RS fraction from 49 to 22%. The product remained stable for at least several months when stored at 4 degrees C. To investigate whether a higher SDS fraction would result in a lower postprandial glycemic response, the sterilized mHAS solution was compared with rapidly digestible maltodextrin. Male Wistar rats received an i.g. bolus of 2.0 g available carbohydrate/kg body weight. Ingestion of heat-treated mHAS resulted in a significant attenuation of the postprandial plasma glucose and insulin responses compared with maltodextrin. mHAS appears to be a starch source which, after sterilization in a liquid product, acquires slow-release properties. The long-term stability of mHAS solutions indicates that this may provide a suitable carbohydrate source for low glycemic index liquid products for inclusion in a diabetes-specific diet

Postprandial Amino Acid Kinetics of Milk Protein Mixtures are Affected by Composition, but Not Denaturation, in Neonatal Piglets

Background: Multiple studies have indicated that formula-fed infants show a different growth trajectory compared with breastfed infants. The observed growth rates are suggested to be linked to higher postprandial levels of branched chain amino acids (BCAAs) and insulin related to differences in protein quality. Objective: We evaluated the effects of milk protein denaturation and milk protein composition on postprandial plasma and hormone concentrations. Methods: Neonatal piglets were bolus-fed randomly, in an incomplete crossover design, 2 of 3 milk protein solutions: Native whey protein isolate (NWPI), denatured whey protein isolate (DWPI), or protein base ingredient, comprising whey and casein (PBI). Postprandial plasma amino acids (AAs), insulin, glucagon-like peptide 1, glucose, and paracetamol concentrations were assayed. Plasma responses were fitted with a model of first-order absorption with linear elimination. Results: DWPI (91% denatured protein) compared with NWPI (91% native protein) showed lower essential amino acids (EAAs) (∼10%) and BCAA (13-19%) concentrations in the first 30-60 min. However, total amino acid (TAA) concentration per time-point and area under the curve (AUC), as well as EAA and BCAA AUC were not different. PBI induced a ∼30% lower postprandial insulin spike than NWPI, yet plasma TAA concentration at several time-points and AUC was higher in PBI than in NWPI. The TAA rate constant for absorption (ka) was twofold higher in PBI than in NWPI. Plasma BCAA levels from 60 to 180 min and AUC were higher in PBI than in NWPI. Plasma EAA concentrations and AUCs in PBI and NWPI were not different. Conclusions: Denaturation of WPI had a minimal effect on postprandial plasma AA concentration. The differences between PBI and NWPI were partly explained by the difference in AA composition, but more likely differences in protein digestion and absorption kinetics. We conclude that modifying protein composition, but not denaturation, of milk protein solutions impacts the postprandial amino acid availability in neonatal piglets.</p

INFOGEST Inter-Laboratory Recommendations for Assaying Gastric and Pancreatic Lipases Activities Prior to In Vitro Digestion Studies

In vitro digestion studies often use animal digestive enzyme extracts as substitutes of human gastric and pancreatic secretions. Pancreatin from porcine origin is thus commonly used to provide relevant pancreatic enzymes such as proteases, amylase and lipase. Rabbit gastric extracts (RGE) have been recently introduced to provide gastric lipase in addition to pepsin. Before preparing simulated gastric and pancreatic extracts with targeted enzyme activities as described in in vitro digestion protocols, it is important to determine the activities of enzyme preparations using validated methods. The purpose of this inter-laboratory study within the INFOGEST network was to test the repeatability and reproducibility of lipase assays using the pH-stat technique for measuring the activities of gastric and pancreatic lipases from various sources. Twenty-one laboratories having different pH-stat devices received the same protocol with identical batches of RGE and two pancreatin sources. Lipase assays were performed using tributyrin as a substrate and three different amounts (50, 100 and 200 mu g) of each enzyme preparation. The repeatability results within individual laboratories were satisfactory with coefficients of variation (CVs) ranging from 4 to 8% regardless of the enzyme amount tested. However, the inter laboratory variability was high (CV \u3e 15%) compared to existing standards for bioanalytical assays. We identified and weighted the contributions to inter-laboratory variability of several parameters associated with the various pH-stat equipment used in this study (e.g. reaction vessel volume and shape, stirring mode and rate, burette volume for the automated delivery of sodium hydroxide). Based on this, we established recommendations for improving the reproducibility of lipase assays using the pH-stat technique. Defining accurate and complete recommendations on how to correctly quantify activity levels of enzyme preparations is a gateway to promising comparison of in vitro data obtained from different laboratories following the same in vitro digestion protocol