A mechanistic kinetic model for lipid oxidation in Tween 20-stabilized O/W emulsions.

Models predicting lipid oxidation in oil-in-water (O/W) emulsions are a requirement for developing effective antioxidant solutions. Existing models do, however, not include explicit equations that account for composition and structural features of O/W emulsions. To bridge this gap, a mechanistic kinetic model for lipid oxidation in emulsions is presented, describing the emulsion as a one-dimensional three phase (headspace, water, and oil) system. Variation in oil droplet sizes, overall surface area of oil/water interface, oxidation of emulsifiers, and the presence of catalytic transition metals were accounted for. For adequate predictions, the overall surface area of oil/water interface needs to be determined from the droplet size distribution obtained by dynamic and static light scattering (DLS, SLS). The kinetic model predicted well the formation of oxidation products in both mono- and polydisperse emulsions, with and without presence of catalytic transition metals.

Adverse drug events caused by three high-risk drug-drug interactions in patients admitted to intensive care units: A multicentre retrospective observational study.

AIMS: Knowledge about adverse drug events caused by drug-drug interactions (DDI-ADEs) is limited. We aimed to provide detailed insights about DDI-ADEs related to three frequent, high-risk potential DDIs (pDDIs) in the critical care setting: pDDIs with international normalized ratio increase (INR+ ) potential, pDDIs with acute kidney injury (AKI) potential, and pDDIs with QTc prolongation potential. METHODS: We extracted routinely collected retrospective data from electronic health records of intensive care units (ICUs) patients (≥18 years), admitted to ten hospitals in the Netherlands between January 2010 and September 2019. We used computerized triggers (e-triggers) to preselect patients with potential DDI-ADEs. Between September 2020 and October 2021, clinical experts conducted a retrospective manual patient chart review on a subset of preselected patients, and assessed causality, severity, preventability, and contribution to ICU length of stay of DDI-ADEs using internationally prevailing standards. RESULTS: In total 85 422 patients with ≥1 pDDI were included. Of these patients, 32 820 (38.4%) have been exposed to one of the three pDDIs. In the exposed group, 1141 (3.5%) patients were preselected using e-triggers. Of 237 patients (21%) assessed, 155 (65.4%) experienced an actual DDI-ADE; 52.9% had severity level of serious or higher, 75.5% were preventable, and 19.3% contributed to a longer ICU length of stay. The positive predictive value was the highest for DDI-INR+ e-trigger (0.76), followed by DDI-AKI e-trigger (0.57). CONCLUSION: The highly preventable nature and severity of DDI-ADEs, calls for action to optimize ICU patient safety. Use of e-triggers proved to be a promising preselection strategy.

Influence of heat treatments on the functionality of soy protein hydrolysates in animal cell cultures.

Soy protein hydrolysates enhance integral viable cell density (IVCD) and recombinant protein production (Immunoglobulin, IgG) in cell cultures, but their functionality varies from batch-to-batch. This is undesirable since it affects both quantity and characteristics of the recombinant proteins. It is hypothesized that the variability of hydrolysates is due to variations in meal and hydrolysate processing treatments. To study this, hydrolysates were produced from meals heated at 121 °C/0-120 min. The heating decreased free amino acid and reducing monosaccharide contents in meals (0.72-0.27% and 3.3-2.6%) and hydrolysates (14.7-7.1% and 16.9-7.9%). Dry heating introduced large variation in the IVCD ((115-316%), but additional heating in suspension reduced it (131-159%). The decrease in IVCD variation corresponded with decreased variation in carboxymethyl-lysine (CML) and lysinoalanine (LAL) contents. Thus, meal and hydrolysate processing induced substantial variation in hydrolysate functionality. It is therefore critical to establish strict process controls for meal and hydrolysate production to ensure consistency.

Effect of Pea Legumin-to-Vicilin Ratio on the Protein Emulsifying Properties: Explanation in Terms of Protein Molecular and Interfacial Properties.

In isolates from different pea cultivars, the legumin-to-vicilin (L:V) ratio is known to vary from 66:33 to 10:90 (w/w). In this study, the effect of variations in the L:V ratio on the pea protein emulsifying properties (emulsion droplet size (d3,2) vs protein concentration (Cp)) at pH 7.0 was investigated using a purified pea legumin (PLFsol) and pea vicilin fraction (PVFsol). Despite a different Γmax,theo, the interfacial properties at the oil-water interface and the emulsifying properties were similar for PLFsol and PVFsol. Hence, the L:V ratio did not affect the pea protein emulsifying properties. Further, PLFsol and PVFsol were less efficient than whey protein isolate (WPIsol) in stabilizing the emulsion droplets against coalescence. This was explained by their larger radius and thus slower diffusion. For this reason, the difference in diffusion rate was added as a parameter to the surface coverage model. With this addition, the surface coverage model described the d3,2 versus Cp of the pea protein samples well.

The path of proteolysis by bovine chymotrypsin.

Chymotrypsin is one of the major proteases in intestinal protein digestion. Observations about the type of bonds that are hydrolysed (specificity and preference) were in the past derived from the peptide composition after digestion or hydrolysis rates of synthetic peptides. In this study, the path of hydrolysis by bovine chymotrypsin, i.e formation and degradation of peptides, were described for α-lactalbumin, ß-lactoglobulin and ß-casein. The peptide compositions, determined with UPLC-PDA-MS at different time points were used to determine the digestion kinetics for individual cleavage sites. It was evaluated how statements on (secondary) specificity from literature were reflected in the release kinetics of peptides. ß-Lactoglobulin reached the highest degree of hydrolysis (10.9 ± 0.1 %) and was hydrolysed fastest (28 ± 1 mMpeptide bonds/s/mMenzyme), regardless of its globular (tertiary) structure. Chymotrypsin showed a preference towards aromatic amino acids, methionine and leucine, but was also tolerant to other amino acids. For the cleavage sites within this preference, Ì´73% of the cleavage sites were hydrolysed with high or intermediate selectivity. For the missed cleavages within the preference, 45 % was explained by hindrance of proline, which affected hydrolysis only when in positions P3, P1' or P2'. No clear indication (based on primary structure) was found to explain the other missed cleavages. A few cleavage sites were hydrolysed extremely efficient in α-lactalbumin (F9, F31, W104) and ß-casein (W143, L163, F190). This study gave unique and quantitative insight in peptide formation and degradation by chymotrypsin in the digestion of proteins. The approach used showed potential to explore the path of hydrolysis for other proteases with less defined specificity.

Variations in foam collapse and thin film stability with constant interfacial and bulk properties.

The stability of foams is commonly linked to the interfacial properties of the proteins and other surfactants used. This study aimed to use these relationships to explain differences in foam stability observed among similar beer samples from different breweries. The foam stability was different for each sample (Nibem foam stability ranged from 206 to 300 s), but ranking was similar for all three foaming methods used, thus independent of the method, gas, etc. Differences in foam stability were dominated by differences in coalescence, as illustrated by the correlation with the stability of single bubbles and thin liquid films. The differences in coalescence stability could not be explained by the measured interfacial properties (e.g. surface pressure, adsorption rate, dilatational modulus and surface shear viscosity), or the bulk properties (concentration, pH, ionic strength, viscosity), since they were similar for all samples. The drainage rates and disjoining pressure isotherms measured in thin liquid films were also similar for all samples, further limiting the options to explain the differences in foam stability using known arguments. The differences in coalescence stability of the thin films was shown to depend on the liquid in between the adsorbed layers of the thin film, using a modified capillary cell to exchange this liquid (to a buffer, or one of the other samples). This illustrates the need to review our current understanding and to develop new methods both for experimental study and theoretical description, to better understand foam stability in the future.

Towards absolute quantification of protein genetic variants in Pisum sativum extracts.

In recent years, several studies have used proteomics approaches to characterize genetic variant profiles of agricultural raw materials. In such studies, the challenge is the quantification of the individual protein variants. In this study a novel UPLC-PDA-MS method with absolute and label-free UV-based peptide quantification was applied to quantify the genetic variants of legumin, vicilin and albumins in pea extracts. The aim was to investigate the applicability of this method and to identify challenges in determining protein concentration from the measured peptide concentrations. Analysis of the protein mass balance showed significant losses of proteins in extraction (37%) and of peptides in further sample preparation (69%). The challenge in calculating the extractable individual protein concentrations was how to deal with these insoluble peptides. The quantification approach using average amino acid concentrations in each position of the sequence showed most reproducible results and allowed comparison of the genetic protein composition of 8 different cultivars. The extractable protein composition (µM/µM) was remarkably similar for all cultivar extracts and consisted of legumins A1 (12.8 ± 1.2%), A2 (1.1 ± 0.4%), B (9.9 ± 1.6%), J (7.5 ± 1.0%) and K (10.3 ± 2.1%), vicilin (15.2 ± 1.7%), provicilin (15.7 ± 2.5%), convicilin (9.8 ± 0.8%), albumin A1 (7.4 ± 2.0%), albumin 2 (10.0 ± 1.5%) and protease inhibitor (0.4 ± 0.4%).

The effect of structured medication review followed by face-to-face feedback to prescribers on adverse drug events recognition and prevention in older inpatients - a multicenter interrupted time series study.

BACKGROUND: The effectiveness of interventions to improve medication safety in older inpatients is unclear, given a paucity of properly designed intervention studies applying clinically relevant endpoints such as hospital-acquired preventable Adverse Drug Events (pADEs) and unrecognized Adverse Drug Events (uADEs). Therefore, we conducted a quality improvement study and used hospital-acquired pADEs and uADEs as main outcomes to assess the effect of an intervention aimed to improve medication safety in older inpatients. METHOD: The study followed an interrupted time series design and consisted of three equally spaced sampling points during baseline and during intervention measurements. Each sampling point included between 80 to 90 patients. A total of 500 inpatients ≥65 years and admitted to internal medicine wards of three Dutch hospitals were included. An expert team retrospectively identified and assessed ADEs via a structured patient chart review. The findings from baseline measurement and meetings with the internal medicine and hospital pharmacy staff were used to design the intervention. The intervention consisted of a structured medication review by hospital pharmacists, followed by face-to-face feedback to prescribers, on average 3 days per week. RESULTS: The rate of hospital-acquired pADEs per 100 hospitalizations was reduced by 50.6% (difference 16.8, 95% confidence interval (CI): 9.0 to 24.6, P <  0.001), serious hospital-acquired pADEs by 62.7% (difference 12.8, 95% CI: 6.4 to 19.2, P <  0.001), and uADEs by 51.8% (difference 11.2, 95% CI: 4.4 to 18.0, P <  0.001). Additional analyses confirmed the robustness of the intervention effect, but residual bias cannot be excluded. CONCLUSIONS: The intervention significantly decreased the overall and serious hospital-acquired pADE occurrence in older inpatients, and significantly improved overall ADE recognition by prescribers. TRIAL REGISTRATION: International Standard Randomized Controlled Trial Number Register, trial registration number: ISRCTN64974377 , registration date (date assigned): 07/02/2011.

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 ß-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 ß-casein, increases the rate of intact protein loss, and that changing the protein composition (by including ß-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 ß-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.

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.

Hydrophobicity Enhances the Formation of Protein-Stabilized Foams.

Screening proteins for their potential use in foam applications is very laborious and time consuming. It would be beneficial if the foam properties could be predicted based on their molecular properties, but this is currently not possible. For protein-stabilized emulsions, a model was recently introduced to predict the emulsion properties from the protein molecular properties. Since the fundamental mechanisms for foam and emulsion formation are very similar, it is of interest to determine whether the link to molecular properties defined in that model is also applicable to foams. This study aims to link the exposed hydrophobicity with the foam ability and foam stability, using lysozyme variants with altered hydrophobicity, obtained from controlled heat treatment (77 °C for 0-120 min). To establish this link, the molecular characteristics, interfacial properties, and foam ability and stability (at different concentrations) were analysed. The increasing hydrophobicity resulted in an increased adsorption rate constant, and for concentrations in the protein-poor regime, the increasing hydrophobicity enhanced foam ability (i.e., interfacial area created). At higher relative exposed hydrophobicity (i.e., ~2-5 times higher than native lysozyme), the adsorption rate constant and foam ability became independent of hydrophobicity. The foam stability (i.e., foam collapse) was affected by the initial foam structure. In the protein-rich regime-with nearly identical foam structure-the hydrophobicity did not affect the foam stability. The link between exposed hydrophobicity and foam ability confirms the similarity between protein-stabilized foams and emulsions, and thereby indicates that the model proposed for emulsions can be used to predict foam properties in the future.

A method to identify and quantify the complete peptide composition in protein hydrolysates.

Automated approaches from proteomics are used to characterise peptides for food applications and in protein digests. Peptide annotations and confidence in these annotations are then based on the fragment spectra. Low reproducibility in repeat analyses has been reported even for annotations with high confidence. When analysing protein hydrolysates (in food) it is important to determine criteria that yield highly reproducible annotations. This study provides a structured approach to determine these criteria. Tryptic hydrolysates of α-lactalbumin, ß-lactoglobulin and ß-casein were analysed manually and automatically, using an UPLC-PDA-MS method for untargeted identification and absolute label-free quantification of peptides. A lock mass with two components was introduced resulting in an average mass error of 1 ppm. Processing filters were set to ensure reliable annotations based on MS/MS fragmentation, while maintaining maximum amount of information. Peptides in the individual hydrolysates with an MS intensity above the limit of annotation represented 99% of total MS intensity and were 100% consistently annotated between four replicates. Amino acid and peptide sequence coverages for the individual protein hydrolysates were 99-100% and 89-95%, respectively. Mixing the hydrolysates resulted in a loss of 11% of the peptide annotations above the LOA and lower reproducibility (97%) for the remaining annotations, as well as more co-eluting peptides. Calculated concentrations of co-eluting peptides in mixed hydrolysates varied 37 ± 21% from the value for single hydrolysates. The proposed approach allows complete description of peptide composition with highly repeatable annotations and quantification of peptides even in mixed hydrolysates.

Evaluation of PBN spin-trapped radicals as early markers of lipid oxidation in mayonnaise.

Quality deterioration of mayonnaise is caused by lipid oxidation, mediated by radical reactions. Assessment of radicals would enable early lipid oxidation assessment and generate mechanistic insights. To monitor short-lived lipid-radicals, N-tert-butyl-α-phenylnitrone (PBN), a spin-trap, is commonly used. In this study, the fate of PBN-adducts and their impact on lipid oxidation mechanisms in mayonnaise were investigated. The main signals detected by Electron Spin Resonance (ESR) were attributed to L-radicals attached to 2-methyl-2-nitrosopropane (MNP), one of three degradation products of the PBN-peroxy-adduct. The second degradation product, benzaldehyde, was detected with Nuclear Magnetic Resonance (1H NMR), in line with MNP-L adduct generation. For the third class of degradation products, LO-radicals, their scission products were detected with 1H NMR and indicated that LO-radicals have a major impact on downstream oxidation pathways. This precludes mechanistical studies in presence of PBN. Degradation products of PBN-adducts can, however, be used for early assessment of antioxidants efficacy in oil-in-water emulsions.

Urinary excretion of advanced glycation end products in dogs and cats.

The present study was conducted with privately owned dogs and cats to investigate whether a relationship exists between the dietary AGEs and the urinary excretion of AGEs, as indication of possible effective absorption of those compounds in the intestinal tract of pet carnivores. For this purpose, data were collected from both raw fed and dry processed food (DPF) fed to dogs and cats, through spot urine sampling and questionnaires. Raw pet food (RF, low in AGE diets) was fed as a primary food source to 29 dogs and DPF to 28 dogs. Cats were categorized into 3 groups, which were RF (n = 15), DPF (n = 14) and dry and wet processed pet food (DWF, n = 25). Urinary-free carboxymethyllysine (CML), carboxyethyllysine (CEL) and lysinoalanine (LAL) were analysed using ultrahigh-performance liquid chromatography (UHPLC)-mass spectrometry, and were standardized for variable urine concentration by expressing the AGE concentrations as a ratio to urine creatinine (Ucr) concentration (µg/µmol Ucr). Urinary excretion of CML, CEL and LAL in dogs fed with DPF was 2.03, 2.14 and 3 times higher compared to dogs fed with RF (p < .005). Similar to the dogs, a significant difference in CML:Ucr, CEL:Ucr and LAL:Ucr between the three diet groups was observed in cats (p-overall < 0.005, ANOVA), in which the RF fed group excreted less AGEs than the other groups. Linear regression coefficients and SE of CML:Ucr, CEL:Ucr and LAL:Ucr showed that body weight and neuter status were significantly correlated with CML and CEL excretion, but not to LAL excretion. Our results revealed a significant correlation between dietary AGEs and urinary excretion of free CML, CEL and LAL, and also showed that endogenous formation of these AGEs occurs in both dogs and cats under physiological conditions.

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 (k a) 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.

31 P NMR assessment of the phosvitin-iron complex in mayonnaise.

Lipid oxidation is the main reason for the limited shelf life of mayonnaise. One of the main catalysts of this process is iron, which is introduced in its ferric (Fe(III)) form via phosvitin, an egg yolk phosphoprotein rich in phosphoserines. The binding of Fe(III) to phosvitin and its ability to establish a redox couple with Fe(II) is believed to determine the oxidation rate of unsaturated lipids. In this work, a 31 P NMR based method was developed to quantify loading of phosvitin with Fe(III) and its reductive release. Both features could be quantified in model phosvitin solutions by exploiting the paramagnetic broadening of 31 P NMR signal of phosphoserine residues by Fe(III). This method was then successfully applied to quantify the phosvitin-Fe(III) loading in mayonnaise water phase by liquid NMR, whereas 31 P NMR MAS could only provide a qualitative measure. The 31 P NMR method showed a direct relation between loading of the Fe(III)-phosvitin complex and lipid oxidation.

Maillard induced aggregation of individual milk proteins and interactions involved.

The aggregation of α-lactalbumin, ß-lactoglobulin and ß-casein after heating in dry state was studied in absence and presence of saccharides. In absence of saccharides, differences were observed in the extent of aggregation. Differences between the proteins were mostly due to differences in covalent aggregation. The presence of glucose during the heat treatment of milk proteins significantly increased the extent of aggregation, and decreased differences between proteins. α-Lactalbumin was selected as a model protein for the study of cross-links formed after heat treatment. In the presence of saccharides, these cross-links were found to consist of 36% of disulphide bridges (compared to >75% in the absence of glucose), followed by other cross-links such as lanthionine. Larger saccharides led to a decrease in Maillard induced aggregation; maltotriose actually even inhibited the formation of α-lactalbumin aggregates.

Apparent ileal digestibility of Maillard reaction products in growing pigs.

The absorption of Maillard reaction products (MRP) from dietary origin has been linked to the occurrence of chronic diseases. The aim of the present study was to determine the effects of toasting time of rapeseed meal (RSM) and the processing method of the diets (pelleting and extrusion) that included RSM on the apparent ileal digestibility (AID) of total lysine, fructosyl-lysine (FL), carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), lanthionine (LAN) and lysinoalanine (LAL) in growing pigs. The study consisted of a 2×3 factorial design with toasting time of RSM (60, 120 min) and diet processing method (mash, pelleted, extruded) as factors. Fifty growing pigs were individually fed one of the experimental diets for 4.5 consecutive days. Following euthanasia, samples of digesta were collected from the terminal 1.5 m of the small intestine. Increasing the toasting time of RSM increased the contents of FL, CML and CEL, whereas the additional effects of the diet processing methods were relatively small. Lysinoalanine and lanthionine were not detected in the diets; therefore, digestibility of these compounds could not be determined. The contents of FL, CML and CEL in the ileal chyme were positively correlated to their contents in the diets. The AID of the MRP from thermally-treated RSM were overall low and were not related to their contents in the diets. The AID of FL ranged between -8.5 and 19.1%, whilst AID of CML and CEL ranged from -0.2 to 18.3 and 3.6 to 30%, respectively. In conclusion, thermal treatments have clear effects on the contents of MRP in the diets. These compounds have relatively low digestibility in growing pigs.

Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and Human Trypsins.

Based on trypsin specificity (for lysines and arginines), trypsins from different sources are expected to hydrolyze a given protein to the same theoretical maximum degree of hydrolysis (DHmax,theo). This is in contrast with experiments. Using α-lactalbumin and ß-casein, this study aims to reveal if the differences among experimental DHmax (DHmax,exp) by bovine, porcine, and human trypsins are due to their secondary specificity. Peptide analysis showed that ∼78% of all the cleavage sites were efficiently hydrolyzed by porcine trypsin, and ∼47 and ∼53% were efficiently hydrolyzed by bovine and human trypsins, respectively. These differences were explained by the enzyme secondary specificity, that is, their sensitivities to the amino acids around the cleavage sites. The DHmax predictions based on the secondary specificity were 4 times closer to the DHmax,exp than the predictions based on trypsin specificity alone (DHmax,theo). Proposed preliminary relations between binding sites and trypsin secondary specificity allow DHmax,exp estimations of tryptic hydrolysis of other proteins.

Maillard induced glycation behaviour of individual milk proteins.

This paper set out to differentiate the Maillard induced glycation reactivity of individual milk proteins using different saccharides under well-defined reaction conditions. α-Lactalbumin, ß-lactoglobulin and ß-casein were incubated with mono-, di- and trisaccharides in the dry state under standardised buffered conditions and glycation was expressed relative to the available reactive groups per protein (DG). Protein reactivity, described by the DGmax and initial speed of glycation (v), followed the same order for each protein-saccharide incubation: α-lactalbumin > ß-lactoglobulin ≫ ß-casein. Glycation of whey proteins by different monosaccharides was double that of ß-casein. Differences in DG between whey proteins and ß-casein decreased with increased saccharide size. A two-fold difference was found for glycation in the presence of the dimers lactose and maltose for ß-casein but not for the whey proteins. The percentage of glycated lysines increased with increased lysine to protein size ratio.