Lactosylation Of Proteins Research Articles

Microwave-assisted solid-phase synthesis of lactosylated peptides for food analytical application

The lactosylation of whey proteins affects their properties, especially their allergenicity and nutritional value, which matters in infant feeding. The quantification of lactosylated peptides requires analytically pure standards which are not commercially available. Herein, we proposed a fast, simple, and efficient protocol for the synthesis of lactosylated peptides on solid support based on microwave-assisted synthesis combined with boronate affinity chromatography utilizing the functionalized resin developed in our research group. We have used our method to synthesize identified modified peptides derived from β-lactoglobulin in tryptic digestion. Thus, the standard peptides contain the dabcyl moiety for determination of the amount in a sample and a cleavable linker to obtain tryptic analogs of β-lactoglobulin. Moreover, for the first time, we applied the functionalized resin to enrich the sample of human serum albumin lactosylated in vitro, into lactosylated peptides.

The effect of heat treatment on the lactosylation of milk proteins

Protein lactosylation is a significant modification that occurs during the heat treatment of dairy products, causing changes in proteins' physical-chemical and nutritional properties. Knowledge of the detailed lactosylation information on milk proteins under various heat treatments is important for selecting appropriate thermo-processing and identifying markers to monitor heat load in dairy products. In the present study, we used proteomics techniques to investigate lactosylated proteins under different heating temperatures. We observed a total of 123 lactosylated lysines in 65 proteins, with lactosylation even occurring in raw milk. The number of lactosylated lysines and proteins increased moderately at 75°C to 130°C, but dramatically at 140°C. We found that 6 out of 10, 9 out of 16, 6 out of 12, and 5 out of 15 lysine residues in κ-casein, β-lactoglobulin, α-lactalbumin, and αS1-casein, respectively, were lactosylated under the applied heating treatment. Moreover, different lactosylation states of individual lysines and proteins can indicate the intensity of heating processes. Lactosylation of K14 in β-lactoglobulin could distinguish pasteurized and UHT milk, while lactosylation of lactotransferrin can reflect moderate heat treatment of products.

Functional and physiochemical properties of the yoghurt modified by heat lactosylation and microbial transglutaminase cross-linking of milk proteins.

This study aimed to recognize the effect of Maillard reaction (MR) on the functional properties of milk proteins and the physiochemical, textural, and sensory properties of yoghurt. Heating at 100°C for 2h increased the carbohydrate ratio in caseins, whey proteins, and total milk proteins from 2.83%, 1.93%, and 1.8% to 4.15%, 3.58%, and 5.32%, respectively. Solubility of the lactosylated caseins, whey proteins, and total milk proteins is increased at low pH values compared to that of the control caseins, whey proteins, and total milk proteins. Lactosylation at 70 and 100°C increased the emulsion activity index (EAI) of caseins at all pH values, especially at pH below 6, and this increment was higher for casein samples treated at 100°C. Foam volume of whey proteins and total milk proteins also increased for samples lactosylated at 100°C compared to control samples. The combination of heating and microbial transglutaminase (MTGase) had a synergistic and enhancing effect on the pH values of yoghurt samples, especially in yoghurt samples produced by whole milk protein compared to control samples. Viscosity and hardness of yoghurt samples were enhanced by heat lactosylation, MTGase treatment, and also storage for 21 days at 7 ± 1°C.

Effects of feed composition, protein denaturation and storage of milk serum protein/lactose powders on lactosylation

SummaryDuring whey powder production, the feed is subjected to several heat treatments which can cause lactosylation of proteins. In this study, lactosylation of whey proteins was evaluated in spray‐dried powders before and after storage by varying the native protein fraction as well as the serum protein/lactose ratio in the powders. The lactosylation of native α‐lactalbumin and β‐lactoglobulin in the powders before storage was not affected to a large extent by the protein denaturation or if the feed had been heat treated in a high or low lactose environment. After storage (relative humidity of 23.5%, 30 °C, 25 days), the kinetic of lactosylation tended to increase with increasing native protein fraction and bulk protein content in the powders. An explanation could be that proteins dissolved in the lactose glassy structure might have a lower reactivity, while proteins present in the protein glassy structure with dissolved lactose may display higher lactosylation reactivity.

Thermally-Induced Lactosylation of Whey Proteins: Identification and Synthesis of Lactosylated β-lactoglobulin Epitope.

The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, β-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a β-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

Changes in proteins, physical stability and structure in directly heated UHT milk during storage at different temperatures

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.

Protein lactosylation in UHT milk during storage measured by Liquid Chromatography–Mass Spectrometry and quantification of furosine

The initial stage of the Maillard reaction, protein lactosylation, occurs during heat treatment of milk and continues during subsequent storage. We compared the initial lactosylation as well as the rate of lactosylation of milk proteins during storage in UHT milk subjected to direct or indirect heat treatment using liquid chromatography (LC) coupled with electrospray injection mass spectrometry (ESI‐MS). Furosine content was used as an overall marker to allow for a quantitative correlation of lactosylation measured by LC‐ESI‐MS in the UHT milks. Protein lactosylation increased during the storage period of 6 months at 20 °C. Both the initial extent and the rate of lactosylation positively correlated with the number of lysine residues in the different proteins. An exponential or linear correlation with furosine concentration could be established for major and minor lactosylated proteins, respectively.

Quantification of lactosylation of whey proteins in stored milk powder using multiple reaction monitoring

Lactosylation in stored milk powder was quantified by multiple reaction monitoring (MRM), a mass spectrometry-based quantification method. The MRM method was developed from a knowledge of peptide fragmentation. The neutral losses of 162Da (cleavage of galactose) and 216Da (the formation of furylium ion) which were representative of lactosylated peptides were specifically selected as MRM transitions. Quantification of lactosylated protein was based on the peak areas of these fragmentation ions. The MRM results showed an increase in peak areas of the two transition fragments from tryptic digests of whey proteins in stored milk protein concentrate powder. A good correlation between the MRM and furosine results indicated that MRM based on tryptic digests of whole products was a feasible method for quantification of modified milk proteins.

Proteomic Analysis of Temperature-Dependent Changes in Stored UHT Milk

Molecular changes in milk proteins during storage of UHT-treated milk have been investigated using two-dimensional electrophoresis (2-DE) coupled to MALDI-TOF mass spectrometry. UHT-treated samples were stored at three different temperatures, 4 °C, 28 °C, and 40 °C, for two months. Three main changes could be observed on 2-DE gels following storage. They were (1) the appearance of diffuse staining regions above the position of the monomeric caseins caused by nondisulfide cross-linking of α and β-caseins; (2) the appearance of additional acidic forms of proteins, predominantly of α(S1)-casein, caused by deamidation; and (3) the appearance of "stacked spots" caused by lactosylation of whey proteins. The extent of the changes increased with increased storage temperature. Mass spectrometric analysis of in-gel tryptic digests showed that the cross-linked proteins were dominated by α(S1)-casein, but a heterogeneous population of cross-linked forms with α(S2)-casein and β-casein was also observed. Tandem MS analysis was used to confirm deamidation of N(129) in α(S1)-casein. MS analysis of the stacked spots revealed lactosylation of 9/15 lysines in β-lactoglobulin and 8/12 lysines in α-lactalbumin. More extensive analysis will be required to confirm the nature of the cross-links and additional deamidation sites in α(S1)-casein as the highly phosphorylated nature of the caseins makes them challenging prospects for MS analysis.

Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures

The Maillard reaction between lactose and proteins occurs during thermal treatment of milk and lactosylated β-lactoglobulin, α-lactalbumin and caseins have widely been used to monitor the quality of dairy products. We recently demonstrated that a number of other whey milk proteins essential for nutrient delivery, defense against bacteria/virus and cellular proliferation become lactosylated during milk processing. The extent of their modification is associated with the harshness of product manufacturing. Since fat globule proteins are also highly important for the health-beneficial properties of milk, an evaluation of their lactosylation is crucial for a complete understanding of aliment nutritional characteristics. This is more important when milk is the unique dietary source, as in the infant diet. To this purpose, a sequential proteomic procedure involving an optimized milk fat globule (MFG) preparation/electrophoretic resolution, shot-gun analysis of gel portions for protein identification, selective trapping of lactosylated peptides by phenylboronate chromatography and their analysis by nanoLC-ESI-electron transfer dissociation (ETD) tandem MS was used for systematic characterization of fat globule proteins in milk samples subjected to various manufacturing procedures. Significant MFG protein compositional changes were observed between samples, highlighting the progressive adsorption of caseins and whey proteins on the fat globule surface as result of the technological process used. A significant lactosylation of MFG proteins was observed in ultra-high temperature sterilized and powdered for infant nutrition milk preparations, which well paralleled with the harshness of thermal treatment. Globally, this study allowed the identification of novel 157 non-redundant modification sites and 35 MFG proteins never reported so far as being lactosylated, in addition to the 153 ones ascertained here as present on other 21 MFG-adsorbed proteins whose nature was already characterized. Novel MFG proteins include components involved in nutrient delivery, defense response against pathogens and cellular proliferation/differentiation. Nutritional, biological and toxicological consequences of these findings are here discussed, highlighting their possible impact on children's diet.

Correlation between lactosylation and denaturation of major whey proteins: an investigation by liquid chromatography–electrospray ionization mass spectrometry

The Maillard-reaction-induced lactosylation of the major whey proteins, alpha-lactalbumin (alpha-La) and beta-lactoglobulins (beta-Lg) A and B, occurring upon heating at 70, 80 and 90 degrees C for 1 to 5 h in the presence of lactose excess, was studied by HPLC coupled to electrospray ionization single and tandem mass spectrometry (HPLC-ESI-MS, MS/MS). The presence of significant amounts of mono and bi-lactosylated forms of the three proteins and their increase with heating temperature and time were assessed from MS data. Evidences for a concomitant, significant denaturation, involving partial tertiary structure unfolding, were also obtained in the case of beta-lactoglobulins. A subsequent ESI-MS and MS/MS investigation on the tryptic digests of heated protein solutions exhibiting high percentages of mono and bi-lactosylated forms provided information on lactosylation sites. In particular, the latter were identified both on tryptic and on aspecific peptides, whose unusual relevance (compared to similar studies) was found to be due mainly to heat-induced protein degradation, occurring before protein digestion with trypsin. Among lactosylation sites identified only on tryptic peptides, i.e., those reasonably related to intact protein lactosylation, two lysines residues were found for alpha-La, both located in accessible regions of its tertiary structure. In the case of beta-Lg, besides three sites common to variants A and B (leucine 1, lysines 70, and 75), lysine 69 was found to be lactosylated only in variant B. Its proximity to a critical region of beta-Lg tertiary structure suggests that the difference between the two variants could be ascribed to a different evolution of their conformation upon heating.

Evaluation of the thermal history of bovine milk from the lactosylation of whey proteins: an investigation by liquid chromatography–electrospray ionization mass spectrometry

Reversed-phase high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (RP-HPLC-ESI-MS) has been used for analysis of the native and lactosylated forms of the main whey proteins, alpha-lactalbumin and beta-lactoglobulins A and B, in commercial bovine milk samples after different thermal treatment (pasteurisation and ultra high-temperature, UHT, treatment), of different lipid content, and of different brands, to find markers of the thermal history of the milk. A new quantification strategy was developed, based on peak-area integration after multiple ion current extraction and considering all the ions detectable in the multi-charge ESI mass spectrum for each type of protein. Validation of the procedure for native forms was first accomplished by calibration with model solutions. Linearity was always good. Sensitivity was different for alpha-lactalbumin and beta-lactoglobulins; the signal was stronger for the latter with only a slight difference between variants A and B of beta-lactoglobulins. Application of the quantification approach to pasteurised and UHT milk samples showed that the distributions of the three proteins and of their three main forms (native, and mono and bi-lactosylated) in whey extracts can be used as statistically robust discriminatory properties for recognition of commercial thermal treatment of milk.

Investigation of the Lactosylation of Whey Proteins by Liquid Chromatography−Mass Spectrometry

Heat treatment of milk induces a reaction between the milk proteins and lactose, resulting in lactosylated protein species. The lactosylation of the two major whey proteins alpha-lactalbumin and beta-lactoglobulin was investigated by reversed phase liquid chromatography-mass spectrometry (LC-MS). Three sample series, consisting of aqueous model solutions of each whey protein separately and in mixture and whole milk, were heated for different time periods, and the progression of the lactosylation reaction was monitored. The observed degrees of lactosylation and the reaction kinetics showed that the lactosylation of beta-lactoglobulin was not influenced by the presence of other components, whereas the lactosylation of alpha-lactalbumin was enhanced in whole milk compared to the aqueous model systems. An in-depth evaluation of the LC-MS data yielded information regarding changes of physicochemical properties of the whey proteins upon lactosylation. Whereas retention time shifts indicated changes in hydrophobicity for both alpha-lactalbumin and beta-lactoglobulin, changes in the charge state distribution denoting conformational alterations were observed only for beta-lactoglobulin. The analysis of different liquid and solid milk products showed that the lactosylation patterns of the whey proteins can be used as indicators for the extent of heat treatment.

Isolation and Characterization of Copolymers of β-Lactoglobulin, α-Lactalbumin, κ-Casein, and αs1-Casein Generated by Pressurization and Thermal Treatment of Raw Milk

Raw skim milk was submitted to high pressure (300 to 600MPa) and temperature (4 to 70°C) treatments for 2 or 5min. The combined effects of pressure and temperature on milk proteins induced structural changes and polymer and copolymer formation characterized by anion-exchange and size-exclusion fast protein liquid chromatography and electrophoretic techniques. Approximately half of the β-lactoglobulin formed polymers, and the other half formed large copolymers, mainly with κ-casein, α-lactalbumin via intermolecular disulfide bond exchange, and αs1-casein via physicochemical interactions, in proportions of 1.0:0.7:0.3:0.1, respectively. Minor whey proteins (serum albumin, immunoglobulins, and lactoferrin) also participated in the formation of the copolymers but to a lesser extent. Two populations of the copolymers were found with apparent molecular masses ranging from 440 to 2000 kDa for the first and more than 2000 kDa for the second. On the contrary, for heated milks the aggregation kinetics obtained by combination of high pressure and thermal treatment were very fast, as no intermediates such as dimers and small size oligomers were observed after pressurization, whatever the temperature studied. Lactosylation of proteins as well as proteolysis were very limited. A β-casein amino-terminal peptide of 22 kDa was specifically recovered in milk samples treated under the more drastic conditions (500 MPa/55°C per 5min and 600 MPa/70°C per 5min) and might have been generated by neutral proteases such as elastase released from somatic cells present in milk. No casein was released from the micelle whatever the combination of high pressure and temperature studied.

Lactosylation of milk proteins during the manufacture and storage of skim milk powders

Abstract Extensive lactosylation of milk proteins in standard skim milk powder dried against air between 185 and 90°C (inlet and outlet temperatures of the air) was detected by capillary electrophoresis. Optimisation of the drying conditions included keeping the outlet temperature low (preferably

The Carbohydrate Portion of Milk Glycoproteins

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.