Milena Corredig

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

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.

The Structure of the Casein Micelle of Milk and Its Changes During Processing

The majority of the protein in cows milk is contained in the particles known as casein micelles. This review describes the main structural features of these particles and the different models that have been used to define the interior structures. The reactions of the micelles during processing operations are described in terms of the structural models.

The mechanisms of the heat-induced interaction of whey proteins with casein micelles in milk

Abstract The heat-induced interactions between whey proteins and casein micelles were investigated by defining the final product of the reaction when milk was heated at temperatures up to 90°C. By looking at the changes of the interactions in skim milk and in resuspended casein micelles, to which different amounts of whey protein had been added, information on the mechanisms that determine the heat-induced protein–protein interactions in milk was derived. The ratio of α -lactalbumin and β -lactoglobulin to κ -casein and the ratio of α -lactalbumin to β -lactoglobulin found in the micellar pellet were used as indices of these heat-induced reactions occurring in milk. The results suggested that at these low temperature (70–90°C) with batch heating conditions, whey proteins form soluble complexes which act as intermediates in the heat-induced association of α -lactalbumin and β -lactoglobulin with the micelles. The presence of β -lactoglobulin was necessary for any association of whey protein with casein micelles to occur; furthermore, the amount of β -lactoglobulin found in the micellar pellet after heating seemed to be limited by a discrete number of binding sites available on the micelles.

Effect of temperature and pH on the interactions of whey proteins with casein micelles in skim milk

Abstract Skim milk was heated at temperatures in the range 75–90 °C, at pH values of 6.8, 6.2 and 5.8, The amounts of α-lactalbumin and β-lactoglobulin which interacted with the casein micelles during heat treatment were quantified by SDS-polyacrylamide gel electrophoresis of the micellar fractions isolated by ultra-centrifugation. Both α-lactalbumin and β-lactoglobulin appeared to interact similarly with casein micelles at temperatures up to 85 °C. The amount of whey protein complexed with micelles increased with time, reaching plateau values that, at the highest temperatures, were comparable with the quantity present in the original skim milk. In general, faster reaction of the whey proteins with the micelles was found at lower pH and higher temperatures. The rates and extent of the reaction changed also when additional α-lactalbumin and β-lactoglobulin isolates were added to milk before heating. The reaction between α-lactalbumin and casein micelles depended to a relatively small extent upon environmental variations (pH and temperature), while β-lactoglobulin interactions were more affected, so that a more complex behaviour may be attributed to the latter protein.

Effect of different heat treatments on the strong binding interactions between whey proteins and milk fat globules in whole milk

The heat-induced binding of whey proteins to milk fat globule membranes in whole milk was investigated by quantitative electrophoresis and laser scanning densitometry. Both α-lactalbumin and β-lactoglobulin bound to the surfaces of fat globules when milk was heated in a water bath in the temperature range 65–85 °C. The interaction behaviour of α-lactalbumin did not seem to change with temperature, and the total amount of protein bound was ∼ 0·2 mg/g fat contained in the cream. The quantity of βlactoglobulin interacting with the milk fat globules increased with temperature from 02 to 0·7 mg/g fat between 65° and 85 °C. Even in whole milk heated at batch pasteurization temperatures (60–65 °C), α-lactalbumin and β-lactoglobulin were found attached to the fat globules. The interactions of the whey proteins with intact fat globule membranes were also investigated in milk heated in an industrial system (a pilot scale UHT and high temperature short time module), and the results were compared with those from the laboratory treatment (simple batch heating). The binding of the whey proteins to fat globules differed between milk heated by UHT using indirect steam heating or direct steam injection (DSI). However, the surface load in milk treated by DSI was not comparable to that of milk treated by batch heating or indirect steam heating, because of the changes in fat globule size and membrane composition caused by the DSI process.

Heating of milk alters the binding of curcumin to casein micelles. A fluorescence spectroscopy study

Curcumin, a polyphenolic compound present in turmeric, is a hydrophobic molecule that has been shown to bind to casein micelles. The present work tested the hypothesis that surface changes in the casein micelles caused by heat-induced interactions with the whey proteins would affect the binding of curcumin. Binding was quantified by direct and tryptophan quenching fluorescence spectroscopy. Curcumin binds to the hydrophobic moieties of the casein proteins, with a 10nm blue shift in its fluorescence emission peak, and causes quenching of the intrinsic fluorescence spectra of the proteins. The fluorescence intensity of curcumin increased after heating of milk at 80°C for 10min; a similar trend in the binding constants was also observed with casein micelles separated from the soluble proteins by centrifugation. There was an increase in the non-specific interactions with heating milk at 80°C for 10min, both in milk as well as in casein micelles separated from the serum proteins. The increased capacity of milk proteins to bind curcumin after heat treatment can be attributed to whey protein denaturation, as whey proteins bind to the surface of casein micelles with heating.

Impact of interfacial composition on emulsion digestion and rate of lipid hydrolysis using different in vitro digestion models.

A sequential in vitro model of digestion was used to investigate the changes in the physicochemical properties of emulsions during gastrointestinal transit. Oil-in-water emulsions were prepared with whey protein isolate (WPI) or soy protein isolate (SPI) at the same protein concentration (1.5%). Despite pepsinolysis of both proteins during the gastric phase, emulsions stabilized with WPI were more stable compared to those prepared with SPI. For both emulsions, the size of the oil droplets, which plays a critical role in lipid digestion, was extensively altered during the duodenal phase due to the presence of bile salts (BS) and phospholipids (PL). As shown by ζ-potential measurements, the results suggested the displacement of both proteins from the interface by BS; however, the displacement was much faster for the WPI-emulsions. The change in interfacial composition of the oil droplets was significantly affected by inclusion of PL and phospholipase A(2) (PLA(2)) in the in vitro digestion model. The interfacial activity of pancreatic triglyceride lipase (PTL) was markedly affected in the presence of the surface-active compounds present in the digestive fluids, including BS, PL, colipase (COL) and PLA(2). A higher percentage of lipid hydrolysis was obtained in the presence of COL and PLA(2) than with BS alone or mixed BS-PL. SPI-emulsions consistently showed a higher degree of lipolysis compared to the WPI-emulsions regardless of the in vitro digestion model used. The results support the conclusion that the interfacial composition of the original emulsion plays a major role in determining the extent of lipolysis.

Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion

Wild blueberries are rich in polyphenols and have several potential health benefits. Understanding the factors that affect the bioaccessibility and bioavailability of polyphenols is important for evaluating their biological significance and efficacy as functional food ingredients. Since the bioavailability of polyphenols such as anthocyanins is generally low, it has been proposed that metabolites resulting during colonic fermentation may be the components that exert health benefits. In this study, an in vitro gastrointestinal model comprising sequential chemostat fermentation steps that simulate digestive conditions in the stomach, small intestine and colon was used to investigate the breakdown of blueberry polyphenols. The catabolic products were isolated and biological effects tested using a normal human colonic epithelial cell line (CRL 1790) and a human colorectal cancer cell line (HT 29). The results showed a high stability of total polyphenols and anthocyanins during simulated gastric digestion step with approximately 93% and 99% of recovery, respectively. Intestinal digestion decreased polyphenol- and anthocyanin- contents by 49% and 15%, respectively, by comparison to the non-digested samples. During chemostat fermentation that simulates colonic digestion, the complex polyphenol mixture was degraded to a limited number of phenolic compounds such as syringic, cinnamic, caffeic, and protocatechuic acids. Only acetylated anthocyanins were detected in low amounts after chemostat fermentation. The catabolites showed lowered antioxidant activity and cell growth inhibition potential. Results suggest that colonic fermentation may alter the biological activity of blueberry polyphenols.

Effect of dynamic high pressure homogenization on the aggregation state of soy protein.

Although soy proteins are often employed as functional ingredients in oil-water emulsions, very little is known about the aggregation state of the proteins in solution and whether any changes occur to soy protein dispersions during homogenization. The effect of dynamic high pressure homogenization on the aggregation state of the proteins was investigated using microdifferential scanning calorimetry and high performance size exclusion chromatography coupled with multiangle laser light scattering. Soy protein isolates as well as glycinin and beta-conglycinin fractions were prepared from defatted soy flakes and redispersed in 50 mM sodium phosphate buffer at pH 7.4. The dispersions were then subjected to homogenization at two different pressures, 26 and 65 MPa. The results demonstrated that dynamic high pressure homogenization causes changes in the supramolecular structure of the soy proteins. Both beta-conglycinin and glycinin samples had an increased temperature of denaturation after homogenization. The chromatographic elution profile showed a reduction in the aggregate concentration with homogenization pressure for beta-conglycinin and an increase in the size of the soluble aggregates for glycinin and soy protein isolate.

Surface adsorption alters the susceptibility of whey proteins to pepsin-digestion.

An in vitro digestion model mimicking the gastric phase of the human gastrointestinal tract coupled with SDS-PAGE and MALDI-TOF mass spectroscopy was employed to study the hydrolysis profiles of whey proteins in solution and adsorbed at the oil-water interface. The objective of this work was to understand the differences in hydrolysis behaviour of whey protein isolates once adsorbed at the interface, and comparisons were carried out with pure beta-lactoglobulin and alpha-lactalbumin fractions. In solution, while beta-lactoglobulin appeared to be resistant to enzymatic treatment, alpha-lactalbumin was fully degraded. Adsorption of both proteins at the oil-water interface affected their conformational structure and susceptibility to peptic hydrolysis. Adsorbed beta-lactoglobulin was hydrolyzed into small polypeptides and in contrast, the resistance of alpha-lactalbumin to pepsin increased upon adsorption at the interface. In addition, changes in the particle size distribution of the droplets during pepsin hydrolysis mainly depended on the original protein concentration. The results suggested that exchanges occur at the interface between adsorbed and non-adsorbed protein, that is to say that either some protein desorb from the interface and does not fully recover its structure in solution, or that hydrolysis of the protein at the interface induces further adsorption and hydrolysis of the protein in solution. These mechanisms have important implications in the digestibility of the proteins.