Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

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A standardised static in vitro digestion method suitable for food – an international consensus

Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S. A. S. Nagar,Mohali, Punjab-160 062, India, Institute of Biochemistry, Faculty of Medicine, Polytechnic University, Via Ranieri 67, IT-60100 Ancona, Italy,Green Biotechnology Research Group, The Special Division for Human Life Technology, National Institute of Advanced Industrial Science andTechnology, 1-8-31 Midorigaoka, Ikeda, Osaka-563-8577, Japan, and Department of Medicinal Chemistry & Natural Products,The Hebrew University of Jerusalem, School of Pharmacy-Faculty of Medicine, Jerusalem 91120, IsraelReceived March 2, 2004

Chitosan chemistry and pharmaceutical perspectives.

1: Magnetic Particles: Preparation, Properties and Applications: M. Ozaki. 2: Maghemite (gamma-Fe2O3): A Versatile Magnetic Colloidal Material C.J. Serna, M.P. Morales. 3: Dynamics of Adsorption and Oxidation of Organic Molecules on Illuminated Titanium Dioxide Particles Immersed in Water M.A. Blesa, R.J. Candal, S.A. Bilmes. 4: Colloidal Aggregation in Two-Dimensions A. Moncho-Jorda, F. Martinez-Lopez, M.A. Cabrerizo-Vilchez, R. Hidalgo Alvarez, M. Quesada-PMerez. 5: Kinetics of Particle and Protein Adsorption Z. Adamczyk.

Surface and Colloid Science

Industrial applications of microbial lipases

The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function

Unusually versatile substrate specificity is shown by lipases. Not only do they hydrolyze triacylglycerols-for example, in the stomach and intestine during digestion of dietary fat-and various synthetic esters and amides, but their high stability in organic solvents permits their use in transesterification reactions and ester synthesis as well. Reactions based on lipase catalysis usually proceed with high regio- and enantioselectivity. Thus, the Ca2+ antagonist diltiazem (1) was obtained with lipase from Serratia marcescens. Over 30 lipases have been cloned in the last few years. Since the tertiary structure of 12 lipases is known, there are presently significant efforts to improve this class of enzymes by protein engineering techniques, in view of their use in detergents and other fields of industrial application.

Lipases: Interfacial Enzymes with Attractive Applications.

‘Interfacial activation’ of lipases: facts and artifacts

The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).

Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography.

The growing interest in the mechanisms controlling membrane-bound enzymes has incited a number of biochemists to study in more detail certain aspects of heterogeneous catalysis. In the biological membrane, "immobilized" enzymes act on water-soluble or water-insoluble substrates. Because of difficulties encountered in the purification of these proteins, our knowledge of their kinetic behavior is still very scanty. This explains why most experimental approaches used so far deal with model systems based on the combination either of (a) an immobilized enzyme + soluble substrate or (b) a soluble enzyme + insoluble substrate. The former system ami its kinetic implications have been discussed in reviews by McLaren & Packer ( 1) and Thomas (2, 2a), as well as in the proceedings of a symposium devoted to this subject (3). In this review, we limit ourselves to studies based on approach b. It will come as no surprise that most of the work reported so far deals with lipolysis. Theoretically, heterogeneous catalysis might be studied also using polyamino acids, polynucIeotides, or polysaccharides as substrates in insoluble form. These compounds are of biological interest and soluble enzymes hydrolyzing them are available in a pure state. Some progress has been made using mono­ molecular surface films of proteins; these polymers are more difficult to manage in vitro than lipids. The naturally occurring (phospho) lipids are important building stones of the biological membranes. They are water insoluble and spontaneously form lipid-water interfaces such as monomolecular films, bilayers, emulsions, liposomes, or micelles. In addition, a number of soluble enzymes that play an important role in biological events, such as digestion, membrane and cell fusion, lipid biosynthesis, pinocytoses, etc., are known and have been isolated in pure form. The fact that it is the

Robert Verger

Papers

Lipases: Interfacial Enzymes with Attractive Applications.

‘Interfacial activation’ of lipases: facts and artifacts

Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography.

Interfacial Enzyme Kinetics of Lipolysis

Action of phospholipase A at interfaces.