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The FAO recommends the digestible indispensable amino acid score (DIAAS) as the measure for protein quality, for which the true ileal digestibility needs to be assessed in humans or pigs. However, due to high costs and ethical concerns, the FAO strongly encourages as well the development of validated in vitro methods, which complement the in vivo experiments. Method: Recently, an in vitro workflow, based on the validated static INFOGEST protocol, was developed and compared towards in vivo data. In parallel to the validation with in vivo data, the repeatability and reproducibility of the in vitro protocol were tested in an international ring trial (RT) with the aim to establish an international ISO standard method within the International Dairy Federation (IDF). Five different dairy products (skim milk powder, whole milk powder, whey protein isolate, yoghurt, and cheese) were analyzed in 32 different laboratories from 18 different countries, across 4 continents. Results: in vitro protein digestibilities based on Nitrogen, free R-NH2, and total amino acids as well as DIAAS values were calculated and compared to in vivo data, where available. Conclusion: The in vitro method is suited for quantification of digestibility and will be further implemented to other food matrices.

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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.

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Highlights • Tissue microstructure controlled rate and extent of in vitro lipolysis in almonds. • Lipolysis methods using pH-stat and GC analysis were in good agreement. • Increasing lipid bioaccessibility led to increased levels of digestibility. • Almond cell walls restrict lipid release, thus hindering digestion kinetics., Although almonds have a high lipid content, their consumption is associated with reduced risk of cardiovascular disease. One explanation for this paradox could be limited bioaccessibility of almond lipids due to the cell wall matrix acting as a physical barrier to digestion in the upper gastrointestinal tract. We aimed to measure the rate and extent of lipolysis in an in vitro duodenum digestion model, using raw and roasted almond materials with potentially different degrees of bioaccessibility. The results revealed that a decrease in particle size led to an increased rate and extent of lipolysis. Particle size had a crucial impact on lipid bioaccessibility, since it is an indicator of the proportion of ruptured cells in the almond tissue. Separated almond cells with intact cell walls showed the lowest levels of digestibility. This study underlines the importance of the cell wall for modulating lipid uptake and hence the positive health benefits underlying almond consumption.

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Background: The particle size and structure of masticated almonds have a significant impact on nutrient release (bioaccessibility) and digestion kinetics. Objectives: The goals of this study were to quantify the effects of mastication on the bioaccessibility of intracellular lipid of almond tissue and examine microstructural characteristics of masticated almonds. Design: In a randomized, subject-blind, crossover trial, 17 healthy subjects chewed natural almonds (NAs) or roasted almonds (RAs) in 4 separate mastication sessions. Particle size distributions (PSDs) of the expectorated boluses were measured by using mechanical sieving and laser diffraction (primary outcome). The microstructure of masticated almonds, including the structural integrity of the cell walls (i.e., dietary fiber), was examined with microscopy. Lipid bioaccessibility was predicted by using a theoretical model, based on almond particle size and cell dimensions, and then compared with empirically derived release data. Results: Intersubject variations (n = 15; 2 subjects withdrew) in PSDs of both NA and RA samples were small (e.g., laser diffraction; CV: 12% and 9%, respectively). Significant differences in PSDs were found between these 2 almond forms (P < 0.05). A small proportion of lipid was released from ruptured cells on fractured surfaces of masticated particles, as predicted by using the mathematical model (8.5% and 11.3% for NAs and RAs, respectively). This low percentage of lipid bioaccessibility is attributable to the high proportion (35–40%) of large particles (>500 μm) in masticated almonds. Microstructural examination of the almonds indicated that most intracellular lipid remained undisturbed in intact cells after mastication. No adverse events were recorded. Conclusions: Following mastication, most of the almond cells remained intact with lipid encapsulated by cell walls. Thus, most of the lipid in masticated almonds is not immediately bioaccessible and remains unavailable for early stages of digestion. The lipid encapsulation mechanism provides a convincing explanation for why almonds have a low metabolizable energy content and an attenuated impact on postprandial lipemia. This trial was registered at isrctn.org as ISRCTN58438021.

Oat mixed-linkage b-glucan has been shown to lower fasting blood cholesterol concentrations due notably to an increase in digesta viscosity in the proximal gut. To exert its action, the polysaccharide has to be released from the food matrix and hydrated. The dissolution kinetics of b-glucan from Three oat materials, varying in their structure, composition and degree of processing, was investigated by incubating the oats at 37◦C over multiple time points (up to 72 h). The samples were analysed for b-glucan content, weight-average molecular weight and rheological behaviour. Regardless of the materials studied and the processing applied, the solubilisation of b-glucan was not complete. Mechanical and hydrothermal processing led to differences in the viscosity flow curves of the recovered solutions, With the presence of particulates having a marked effect. This study revealed that the structure and processing methods applied to oat materials resulted in varied and complex rheological properties, especially when particulates are present.

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