Gerd E. Vegarud

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.

Mineral-binding milk proteins and peptides; occurrence, biochemical and technological characteristics.

Minerals and trace elements in cows milk occur as inorganic ions and salts or form complexes with proteins and peptides, carbohydrates, fats and small molecules. The main mineral binder or chelators of calcium are the caseins, alphas1-casein, alphas2-casein, beta-casein and kappa-casein, but also whey proteins and lactoferrin bind specific minerals like calcium, magnesium, zinc, iron, sodium and potassium. Less documented is the binding of trace elements. Peptides obtained by in vitro or in vivo hydrolysis act as mineral trappers through specific and non-specific binding sites. They may then function as carriers, chelators, of various minerals and thus enhance or inhibit bioavailability. Peptides from milk proteins have found interesting new applications in the food industry as products with improved functionality or as ingredients of dietary products, or used in pharmaceutical industry. Fortification of foods with minerals in a low concentration has for a long time been used in some countries to overcome mineral deficiency, which is an increasing problem in humans. These types of foods are being used to create a new generation of super foods in the industry today.

Importance of casein micelle size and milk composition for milk gelation.

The economic output of the dairy industry is to a great extent dependent on the processing of milk into other milk-based products such as cheese. The yield and quality of cheese are dependent on both the composition and technological properties of milk. The objective of this study was to evaluate the importance and effects of casein (CN) micelle size and milk composition on milk gelation characteristics in order to evaluate the possibilities for enhancing gelation properties through breeding. Milk was collected on 4 sampling occasions at the farm level in winter and summer from dairy cows with high genetic merit, classified as elite dairy cows, of the Swedish Red and Swedish Holstein breeds. Comparisons were made with milk from a Swedish Red herd, a Swedish Holstein herd, and a Swedish dairy processor. Properties of CN micelles, such as their native and rennet-induced CN micelle size and their zeta-potential, were analyzed by photon correlation spectroscopy, and rennet-induced gelation characteristics, including gel strength, gelation time, and frequency sweeps, were determined. Milk parameters of the protein, lipid, and carbohydrate profiles as well as minerals were used to obtain correlations with native CN micelle size and gelation characteristics. Milk pH and protein, CN, and lactose contents were found to affect milk gelation. Smaller native CN micelles were shown to form stronger gels when poorly coagulating milk was excluded from the correlation analysis. In addition, milk pH correlated positively, whereas Mg and K correlated negatively with native CN micellar size. The milk from the elite dairy cows was shown to have good gelation characteristics. Furthermore, genetic progress in relation to CN micelle size was found for these cows as a correlated response to selection for the Swedish breeding objective if optimizing for milk gelation characteristics. The results indicate that selection for smaller native CN micelles and lower milk pH through breeding would enhance gelation properties and may thus improve the initial step in the processing of cheese.

Size of native and heated casein micelles, content of protein and minerals in milk from Norwegian Red Cattle—effect of milk protein polymorphism and different feeding regimes

Abstract Milk samples of 59 cows of the Norwegian Red Cattle breed receiving three different supplementary concentrates, were analysed for genotypes of caseins and whey proteins, the content of different milk salts (Ca 2+ , Ca, Mg and citrate), the content of total protein, casein and whey protein and the mean micellar size of native and heated casein micelles. The genotype of α s1 -casein had a statistically significant effect on the content of protein and casein, and the content of whey protein and the casein number were significantly influenced by different feeding regimes, and the content of citrate. The mean size of native and heated casein micelles was significantly influenced by the feeding regimes, genotype of α s1 -casein (native mean size only) and κ -casein, pH and the content of casein, whey protein and casein number. The heat-induced changes in mean micellar size were significantly affected by the calcium ion activity which accounted for approximately 40% of the total variation.

Different digestion of caprine whey proteins by human and porcine gastrointestinal enzymes

The objective of the present study was twofold: first to compare the degradation patterns of caprine whey proteins digested with either human digestive juices (gastric or duodenal) or commercial porcine enzymes (pepsin or pancreatic enzymes) and second to observe the effect of gastric pH on digestion. An in vitro two-step assay was performed at 37 degrees C to simulate digestion in the stomach (pH 2, 4 or 6) and the duodenum (pH 8). The whey proteins were degraded more efficiently by porcine pepsin than by human gastric juice at all pH values. Irrespective of the enzyme source, gastric digestion at pH 2 followed by duodenal digestion resulted in the most efficient degradation. Lactoferrin, serum albumin and the Ig heavy chains were highly degraded with less than 6 % remaining after digestion. About 15, 56 and 50 % Ig light chains, beta-lactoglobulin (beta-LG) and alpha-lactalbumin remained intact, respectively, when digested with porcine enzymes compared with 25, 74 and 81 % with human digestive juices. For comparison, purified bovine beta-LG was digested and the peptide profiles obtained were compared with those of the caprine beta-LG in the digested whey. The bovine beta-LG seemed to be more extensively cleaved than the caprine beta-LG in the whey. Commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities. This could lead to conflicting results when comparing human in vivo protein digestion with digestion using purified enzymes of non-human species. Consequently the use of human digestive juices might be preferred.

Survival of lactic acid bacteria from fermented milks in an in vitro digestion model exploiting sequential incubation in human gastric and duodenum juice

In the present study, the survival of 9 lactic acid bacteria (5 Lactococcus strains, 3 Lactobacillus strains, and 1 strain of Enterococcus hirae), was investigated in vitro under conditions similar to human digestion using human gastric and duodenal juices. The tolerance of the bacteria was also tested with traditional methods using acidic conditions and bile salts. The strains were subjected to a model digestive system comprising sequential incubation in human gastric and duodenal juices, in a 2-step digestion assay at 37°C, simulating the human upper gastrointestinal tract with human gastric juices at pH 2.5 and human duodenal juices at pH 7. The bacterial strains were tested either as washed cells from culture media or in fermented milk. The initial in vitro testing in acid and bile salts showed that Lactobacillus strains and the E. hirae strain displayed a significantly higher acid tolerance than the lactococci. The lactobacilli and the Enterococcus numbers increased, whereas the lactococci decreased at least 1 log during the bile salt treatment. The Lactobacillus strains showed the highest survival rate in the model digestive system when washed bacterial cultures were used with a minor log reduction, whereas the lactococci numbers were reduced by at least log 4. However, when using fermented milks in the model digestion system it was demonstrated that the Enterococcus strain and 2 strains of Lactococcus lactis ssp. cremoris benefited significantly from the presence of the fermented milk as food matrix, with log numbers >log 7 and 5, respectively, after digestion of the fermented milk. The analyses reported comprise a comprehensive in vitro testing regimen suitable for evaluation of the survival of candidate probiotic bacteria in human digestion as an initial prescreen to clinical trials.

Identification of lactoferrin peptides generated by digestion with human gastrointestinal enzymes

Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17-41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is digested by nonhuman enzymes. The degradation of LF in the GI tract should be taken into consideration when health effects are proposed, because LF has now been approved by the European Food Safety Authority as a dietary supplement in food products.

In vitro studies of the digestion of caprine whey proteins by human gastric and duodenal juice and the effects on selected microorganisms

The in vitro digestion of caprine whey proteins was investigated by a two-step degradation assay, using human gastric juice (HGJ) at pH 2.5 and human duodenal juice (HDJ) at pH 7.5. Different protein and peptide profiles were observed after the first (HGJ) and second (HDJ) enzymatic degradation. The minor whey proteins serum albumin, lactoferrin and Ig were rapidly degraded by HGJ, while alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG) were more resistant and survived both 30 and 45 min of the enzymatic treatment. Further digestion with HDJ still showed intact beta-LG, and the main part of alpha-LA also remained unchanged. The protein degradation by HGJ and HDJ was also compared with treatment by commercial enzymes, by using pepsin at pH 2.5, and a mixture of trypsin and chymotrypsin at pH 7.5. The two methods resulted in different caprine protein and peptide profiles. The digests after treatment with HGJ and HDJ were screened for antibacterial effects on some selected microorganisms, Escherichia coli, Bacillus cereus, Lactobacillus rhamnosus GG and Streptococcus mutans. Active growing cells of E. coli were inhibited by the digestion products from caprine whey obtained after treatment with HGJ and HDJ. Cells of B. cereus were inhibited only by whey proteins obtained after reaction with HGJ, while the products after further degradation with HDJ demonstrated no significant effect. Screenings performed on cells of Lb. rhamnosus GG and S. mutans all showed no signs of inhibition.

Angiotensin I-Converting Enzyme (ACE) Inhibitory Activity and ACE Inhibitory Peptides of Salmon (Salmo salar) Protein Hydrolysates Obtained by Human and Porcine Gastrointestinal Enzymes

The objectives of the present study were two-fold: first, to detect whether salmon protein fractions possess angiotensin I-converting enzyme (ACE) inhibitory properties and whether salmon proteins can release ACE inhibitory peptides during a sequential in vitro hydrolysis (with commercial porcine enzymes) and ex vivo digestion (with human gastrointestinal enzymes). Secondly, to evaluate the ACE inhibitory activity of generated hydrolysates. A two-step ex vivo and in vitro model digestion was performed to simulate the human digestion process. Salmon proteins were degraded more efficiently by porcine enzymes than by human gastrointestinal juices and sarcoplasmic proteins were digested/hydrolyzed more easily than myofibrillar proteins. The ex vivo digested myofibrillar and sarcoplasmic duodenal samples showed IC50 values (concentration required to decrease the ACE activity by 50%) of 1.06 and 2.16 mg/mL, respectively. The in vitro hydrolyzed myofibrillar and sarcoplasmic samples showed IC50 values of 0.91 and 1.04 mg/mL, respectively. Based on the results of in silico studies, it was possible to identify 9 peptides of the ex vivo hydrolysates and 7 peptides of the in vitro hydrolysates of salmon proteins of 11 selected peptides. In both types of salmon hydrolysates, ACE-inhibitory peptides IW, IY, TVY and VW were identified. In the in vitro salmon protein hydrolysates an ACE-inhibitory peptides VPW and VY were also detected, while ACE-inhibitory peptides ALPHA, IVY and IWHHT were identified in the hydrolysates generated with ex vivo digestion. In our studies, we documented ACE inhibitory in vitro effects of salmon protein hydrolysates obtained by human and as well as porcine gastrointestinal enzymes.

Genetic variants of Norwegian goats milk composition, micellar size and renneting properties

Abstract A survey has been conducted of the allele frequency of genetic variants of various caseins, mainly α s1 -casein, in herds of Norwegian goats located in north, west and southeast Norway. In addition, identifying the variants, milk composition, micellar size and renneting properties of the milks have been determined.