Degree Of Glycation Research Articles

Protein fingerprinting and quantification of β-casein variants by ultra-performance liquid chromatography–high-resolution mass spectrometry

Infant formulations are constantly evolving as novel protein ingredients are added to make them more closely mimic the protein profile of human milk; however, precise analytical methods for characterizing and quantifying the major milk proteins in such formulations are currently lacking. This article describes an ultra-performance liquid chromatography-high-resolution mass spectrometry method for intact proteins that can efficiently detect, identify, and characterize the major milk proteins and their proteoforms (phosphorylation status, degree of glycation, genetic variants among others) in ingredients and final products, with an emphasis on detecting and quantifying specific genetic variants of β-casein in infant formulas. Method sensitivity allows detection of β-casein A1 in A2-based infant formulas with a limit of detection of 2% (grams of β-casein A1 per 100 g of total β-casein). Protein glycation affects signal intensity in a linear fashion, which permits proteins to be quantified from their mass spectrometry signals after correction according to their measured glycation index. The method was validated for the quantification of β-casein in infant formulas. Repeatability ranged from 2 to 3% and intermediate reproducibility from 5 to 9%. Calculated β-casein amounts ranged between 77 and 110% of the values based on formulations and published protein profiles for milk. Altogether, this method can be used for general fingerprinting as well as specific characterization and quantification of individual major milk proteins in dairy-based ingredients and products.

Mitigation of isoquercitrin on β-lactoglobulin glycation: Insight into the mechanisms by mass spectrometry and interaction analysis

Formation of advanced glycation end products (AGEs) on foods imposes threats to human health after intaking. This research firstly evaluated the inhibition of isoquercitrin on β-lactoglobulin (β-Lg) glycation, the mechanisms were elucidated by fluorescence spectroscopy, Orbitrap MSn and molecular docking. Fluorescence spectra indicated that isoquercitrin effectively alleviated the formation of AGEs, it could stabilize the conformation structure of glycated β-Lg (G-β-Lg), change the micro-environment in the vicinity of chromophores. SDS-PAGE analysis revealed the suppressed cross-linking of G-β-Lg induced by isoquercitrin. The number of glycation site detected on G-β-Lg was reduced from ten to eight after the addition of isoquercitrin, and the relative glycation degree of substitution of per site (RGDSP) of most glycation sites were also greatly decreased. As indicated by intermolecular interaction, isoquercitrin quenched the fluorescence of β-Lg via a static mechanism, and their combination is an endothermic processing mainly derived by hydrophobic interaction, hydrogen bonds, and van der Waals forces. Isoquercitrin interacted with β-Lg to form an equimolar complex, and one hydrogen bond was formed between isoquercitrin and Lys69 (4.96 Å). Above results proved that isoquercitrin can be a promising anti-glycation agent used in food system to prevent the formation of harmful glycation products.

Application of Mass Spectrometry in Analysis of Non-Enzymatic Glycation Proteins in Diabetic Blood

Abstract Diabetes mellitus (DM) is a metabolic disorder disease characterized by hyperglycemia. Long-term physiological hyperglycemia will aggravate the degree of non-enzymatic glycation (NEG) proteins. Because the abnormal changes of non-enzymatic process can cause a series of pathological variation, NEG proteins have attracted more and more attention. Mass spectrometry (MS) is an indispensable and powerful analytical tool for the qualitative and quantitative analysis of proteins due to its ultra-high sensitivity, excellent detection limits, and multi-component simultaneous analysis, and has been applied to the identification and qualification of NEG proteins. Herein, the principle of NEG proteins and the application of MS in the identification and quantitation of NEG plasma proteins and hemoglobin (Hb) in blood are reviewed. The forecast in the analysis of NEG proteins based on MS is also provided.

Physicochemical, structural and sensory properties of smooth hound autolysates-sugar conjugates formed using a glycosylation reaction

The conjugation of smooth hound viscera autolysates (SHVA) and glucose (Gluc) through the Maillard reaction (MR) was studied. The degree of glycation, free amino groups, color parameters, structural (infrared and scanning electron microscopy analyses) and antioxidant properties of the resulting MR products (MRP) were studied. The degree of glycation reached 87%, at the end of the reaction, suggesting the effectiveness of the MR with heating. In addition, the MR improved the sensory attributes of the MRP, particularly, by reducing the unfavorable bitter taste. Structural changes were observed in the infra-red spectra of the final conjugates, compared to the un-conjugated ones. These changes were consistent with the antioxidant activities of the different samples. It was found that the antioxidant capacity of the MRP derived from two different autolysates increased with heating time. Thus, the combination of SHVA and Gluc led to the production of bioactive products with different structural and enhanced biological characteristics.

Mechanisms of isoquercitrin attenuates ovalbumin glycation: Investigation by spectroscopy, spectrometry and molecular docking

This research firstly investigated the inhibitory effect of isoquercitrin (ISQ) on Ovalbumin (OVA) glycation. The mechanism was elucidated through the interaction between OVA and ISQ, and changes in glycation sites and degree of each site as deduced by spectroscopy, spectrometry and molecular docking. ISQ significantly inhibited OVA glycation by attenuating the conformational change induced by glycation. It quenched the fluorescence of Trp via static mechanism, and exposed Trp residues to a more hydrophobic surroundings. Formation of OVA-ISQ complex was a endothermic processing driven by hydrophobic interactions, van der Waals forces and hydrogen bonds. LC-Orbitrap-MS/MS revealed that ISQ altered the location of glycation and alleviated the glycation degree of most sites. Molecular docking results indicated that ISQ inserted into the hydrophobic pocket of OVA with six hydrogen bonds and one π-π stacking formed between ISQ and the amino acid residues of OVA, leading to the altered glycation activity of some sites.

Functional and structural properties of spirulina phycocyanin modified by ultra-high-pressure composite glycation

Phycocyanin (PC), a plant-based protein with interesting biological activity, is rarely directly applied in the food industry because it has structural and functional limitations. This study combined ultra-high-pressure (UHP) treatment with glycation to improve PC functionality and explored resulting structural changes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, scanning electron microscopy, Fourier-transform infrared spectroscopy, circular dichroism, and UV–visible spectroscopy. The UHP treatment obviously improved the speed and degree of glycation and the composite-modified PC (CM-PC) showed high solubility and good emulsifying and foaming performance. Scanning electron microscopy images showed the CM-PC surface was loose and fluffy. Gel electrophoresis, Fourier-transform infrared spectroscopy, and circular dichroism results demonstrated that the content of α-helix decreased from 78.1% in PC to 26.6% in CM-PC, and hydroxyl groups were introduced. UV–visible spectroscopy showed that the mechanism of composite modification involved stretching of the PC and promotion of binding with sugars.

Influence of Human Serum Albumin Glycation on the Binding Affinities for Natural Flavonoids

AbstractIncreasing the degree of glycation in diabetes could affect the ability of plasma proteins in binding to small molecules and active compounds. In this study, the influence of glycation of Human serum albumin (HSA) on the binding affinities for six dietary flavonoids was investigated by fluorescence spectra. Glycated HSA was prepared through incubation with glucose and characterized by several methods to confirm the glycation. It was found that the level of glycation increased with the increasing incubation time. The glycation of HSA increased the binding affinities for flavonoids by 1.40 to 48.42 times, which indicates that modifications caused by the glycation may have different influences on the interactions of flavonoids with HSA at separate binding sites on this protein. These results are valuable for understanding the influence of diabetes on the metabolism of flavonoids and other bioactive small molecules in human body.

Glycation-induced structural modification of myofibrillar protein and its relation to emulsifying properties

In the present study, myofibrillar protein (MP) and dextran (DX) conjugates were prepared via glycation in a liquid solution. The effects of DX molecular weight on structural and emulsifying properties of conjugates compared with those of MP were investigated. The glycation was confirmed by evaluating modification degree of glycation (DG), electrophoresis and Fourier transform infrared spectroscopy. As DX molecular weight increased, the Ca2+-ATPase activity decreased markedly due to modification of MP, and the formation of MP-DX conjugates reduced surface hydrophobicity owing to the unfolding behaviour of MP and covalent attachment of DX. In addition, combined analysis of turbidity and reactive sulfhydryl contents indicated that MP may undergo a conformation unfolding and become looser and more flexible upon glycation. Glycation was able to increase the emulsifying activity and emulsion stability of MP, and MP-DX 70 showed the best emulsifying properties. A good correlation between reactive sulfhydryl contents and emulsifying properties was obtained, and the correlation coefficients were 0.930 for emulsifying activity and 0.961 for emulsion stability. Meanwhile, there was a negative correlation between surface hydrophobicity and emulsifying properties with correlation coefficients of −0.965 and −0.895, respectively. Overall, glycation could alter the structural characteristics and enhance the emulsifying properties of MP.

Glycated Beef Protein Hydrolysates as Sources of Bitter Taste Modifiers

Being averse to bitter taste is a common phenomenon for humans and other animals, which requires the pharmaceutical and food industries to source compounds that can block bitterness intensity and increase consumer acceptability. In this work, beef protein alcalase hydrolysates (BPAH) and chymotrypsin hydrolysates (BPCH) were reacted with glucose to initiate Maillard reactions that led to the formation of glycated or advanced glycation end products (AGEs), BPAH-AGEs and BPCH-AGEs, respectively. The degree of glycation was higher for the BPAH-AGEs (47–55%) than the BPCH-AGEs (30–38%). Analysis by an electronic tongue instrument showed that BPAH-AGEs and BPCH-AGEs had bitterness scores that were significantly (p < 0.05) less than quinine. The addition of BPAH-AGEs or BPCH-AGEs to quinine led to significant (p < 0.05) reductions (up to 38%) in bitterness intensity of quinine. The use of 3% hydrolysate to react with glucose yielded glycated peptides with a stronger ability to reduce quinine bitterness than when 1% was used. Calcium release from HEK293T cells stably expressing the T2R4 human bitter taste receptor was significantly (p < 0.05) attenuated by BPAH-AGEs (up to 96%) and BPCH-AGEs (up to 92%) when compared to the BPAH (62%) and BPCH (3%) or quinine (0%). We concluded that BPAH-AGEs and BPCH-AGEs may be used as bitter taste blockers to formulate better tasting foods.

Molecular forces and gelling properties of heat-induced gel from egg white protein glycated with isomalto-oligosaccharide

Glycation with fructo-oligosaccharide (FO) and isomalto-oligosaccharide (IMO) was employed to improve the heat-induced egg white protein (EWP) gels. Results showed that EWP glycated with IMO exhibited a greater degree of glycation (DG) and better gelling properties than those of FO-EWP conjugates. The electrophoresis and liquid chromatography mass spectrometry (LC-MS) indicated that IMO was successfully covalently cross-linked with EWP. Fourier transform infrared spectroscopy (FTIR) showed that the glycation increased the percentages of intramolecular and intermolecular β-sheets of EWP and lowered the contents of 310-helical structure. The rheological test indicated that the gel-forming temperature of EWP was brought forward with the increase of glycation time. Furthermore, the glycation could induce the formation of EWP gel characterized by the higher strength of hydrogen bonds and hydrophobic interactions and lower strength of disulfide bonds. The changes of intermolecular forces resulted in a fine network structure of EWP gel, which significantly enhanced the fracture strength, bound water relaxation proportion and water-holding capacity (WHC) of the gel. A positive correlation existed in the WHC and gel fracture strength. This study proved that glycation with IMO efficiently improved the gelling properties of EWP.

Insight into the stabilization mechanism of emulsions stabilized by Maillard conjugates: Protein hydrolysates-dextrin with different degree of polymerization

The physical and oxidation stability of emulsions stabilized by conjugates prepared by Maillard reaction between whey protein hydrolysates (WPH) and linear dextrin (LD) with different degree of polymerization (DP) were investigated. In accordance with the gradient ethanol precipitation, LD1 (DP = 23.78), LD2 (DP = 47.62) and LD3 (DP = 64.96) were prepared. WPH-LD conjugates were obtained by Maillard reaction in dry-heating condition and the degree of glycation (DG) of conjugates increased with the increasing content of LD. Structural analysis (fluorescence spectra and circular dichroism) revealed that the secondary structure of WPH changed after glycation and the conjugates had less α-helix content than unmodified WPH. Meanwhile, the greater DPPH radical scavenging ability and iron chelating activity of conjugates were demonstrated compared with unmodified WPH. Conjugates containing LD with low and middle DP could form Pickering emulsions due to the aggregation of LD with short chains. The superior storage stability, oxidative stability and physical stability against environment stress of emulsions stabilized by conjugates, enhanced with increasing DP of LD in conjugates, were attributed to the combined action of stabilization of particle emulsifiers and greater steric hindrance (occupied the dominant position) provided by conjugates. Maillard reaction between LD and WPH was efficient to produce conjugates with high potential use as emulsifying agents to stabilize emulsions with excellent physical and oxidative stability.

Improving the emulsion freeze-thaw stability of soy protein hydrolysate-dextran conjugates

The processing parameters of Maillard reaction products (SPH-D) from soy protein hydrolysates (SPH) and dextran (D) were assessed according to the degree of glycation, browning index and creaming index to improve the freeze-thaw stability in an oil-in-water emulsion. SPH-D conjugates were produced through the wet method under optimal conditions for each factor at a mass ratio of 2:3, SPH concentration of 40 g/L, temperature of 85 °C and dispersion in a phosphate buffer (pH 8) incubated for 1 h. Such conditions reported the lowest creaming index and the best freeze-thaw stability. The emulsion prepared from SPH-D was reduced by 48%, 40% and 48% after each freeze-thaw cycle. Meanwhile, a freeze-thaw stability reduction was also observed with an excessive Maillard reaction. Additionally, the confocal laser scanning microscopy analysis indicated that the optimum SPH-D emulsion was more stable to the freeze-thaw treatment and could be used as an effective emulsifier in frozen foods.

Application of preheating treatment in up- and down-regulating the glycation process of dietary proteins

Heating is the most commonly used treatment in food industry. This work reported the influence of preheating treatment (PT, 98 °C) on the following glycation behavior of bovine serum albumin (BSA), β-lactoglobulin (β-Lg) and β-casein (β-CN) at 90 °C. The PT was shown to induce both the unfolding and aggregation of proteins, which changed their subsequent glycation kinetics based on measurements of free amino group content and UV absorbance at 294 and 420 nm. Ten minutes of PT unfolded the globular structures of BSA and β-Lg and exposed more glycation sites, which largely up-regulated their glycation degrees. By contrast, 40 min of PT induced more severe aggregation in BSA and β-Lg during PT and the subsequent glycation process, which hid a portion of glycation sites and down-regulated their glycation degree. The glycation behavior of β-CN was almost not affected by PT, possibly due to its intrinsic disordered structure and high thermal stability. Proteomics analysis showed that PT resulted in changes in the number of glycated structures and preference of lysine residues to be glycated. These results suggested PT as an effective way in regulating the glycation behavior of dietary proteins, depending on PT duration and structure of treated protein.

Changes on the Structural and Physicochemical Properties of Conjugates Prepared by the Maillard Reaction of Black Bean Protein Isolates and Glucose with Ultrasound Pretreatment.

The conjugates of black bean protein isolate (BBPI) and glucose (G) were prepared via the wet heating Maillard reaction with ultrasound pretreatment. The physicochemical properties of UBBPI-G conjugates prepared by ultrasound pretreatment Maillard reaction had been compared with classical Maillard reaction (BBPI-G). The reaction rate between BBPI and glucose was speeded up by ultrasound pretreatment. A degree of glycation (DG) of 20.49 was achieved by 2 h treatment for UBBPI-G, whereas 5 h was required using the classical heating. SDS-PAGE patterns revealed that the BBPI-G conjugates with higher molecular weight were formed after glycosylation. The results of secondary structure analysis suggested that the α-helix and β-sheet content of UBBPI-G were lower than that of BBPI-G. In addition, UBBPI-G conjugates had exhibited bathochromic shift compared with BBPI by fluorescence spectroscopy analysis. Finally, UBBPI-G achieved higher level of surface hydrophobicity, solubility, emulsification property and antioxidant activity than BBPI and BBPI-G (classical Maillard reaction).

Time effect on structural and functional properties of whey protein isolate-gum acacia conjugates prepared via Maillard reaction.

The functional properties of whey protein isolate (WPI) are sensitive to pH, ionic strength, and temperature. This prevents its application in various food systems and processing technologies. The conjugation of proteins with polysaccharides via the Maillard reaction is an efficient method to improve the functionality of proteins. The purpose of this work was to conjugate gum acacia (GA) with WPI via the dry-heating Maillard reaction and to investigate the effect of reaction time on the physicochemical and functional properties of WPI-GA conjugates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance size exclusion chromatography confirmed the formation of higher molecular weight conjugates. The degrees of glycation for WPI-GA conjugates incubated for 1, 3, 5, and 7days were 28.14%, 44.98%, 49.50%, and 51.20%, respectively. The glycation reaction reduced the surface hydrophobicity and fluorescence intensity of WPI significantly (P < 0.05). Functional properties of the conjugates, such as solubility, stability against heat-induced insolubility, and emulsion properties were all superior to the control WPI. However, a reaction time longer than a day resulted in a high degree of browning and decreased the functionality of the conjugate significantly (P < 0.05). The results indicated that conjugation of WPI with GA can be a promising way to enhance its functional properties. However, the reaction time suitable for producing conjugates with superior functional properties was not necessarily the highest glycation degree that could be reached. © 2019 Society of Chemical Industry.

Influence of Protein Hydrolysis on the Freeze-thaw Stability of Emulsions Prepared with Soy Protein - Dextran Conjugates.

Protein hydrolysis on the freeze-thaw stability of emulsions prepared with soy protein - dextran conjugates were investigated. Soy protein isolate-dextran (SPI-D) and soy protein hydrolysates-dextran (SPH-D) conjugates with different degree of hydrolysis (DH) were formed by Maillard reaction. The formation of protein-polysaccharide conjugates between SPI/SPH and dextran molecules was confirmed by SDS-PAGE; this finding was consistent with the degree of glycation and the browning index. The freeze-thaw emulsion stability was investigated. The results confirmed that the SPH3-D (DH at 3%) emulsion with 3% DH of SPI exhibited the lowest creaming index after experiencing 1, 2, and 3 freeze-thaw cycles , with results of 7.69%, 20.74% and 31.30%, respectively. The SPH3-D emulsion had a significantly lower average particle size, which was reduced by 48.28% compared to the SPI-D emulsion. Meanwhile, the SPH3-D solution had low interfacial tension. The confocal laser scanning microscopy analysis indicated that the SPH3-D emulsions were strongly stable against the freeze-thaw treatment and could be used as effective emulsifiers in frozen foods.

Study of Aberrant Modifications in Peptides as a Test Bench to Investigate the Immunological Response to Non-Enzymatic Glycation

A side effect of diabetes is formation of glycated proteins and, from them, production of advanced early glycation end products that could determine aberrant immune responses at the systemic level. We investigated a relevant aberrant post-translational modification (PTM) in diabetes based on synthetic peptides modified on the lysine side chain residues with 1-deoxyfructopyranosyl moiety as a possible modification related to glycation. The PTM peptides were used as molecular probes for detection of possible specific autoantibodies developed by diabetic patients. The PDC-E2(167-186) sequence from the pyruvate dehydrogenase complex was selected and tested as a candidate peptide for antibody detection. The structure-based designed type I’ β-turn CSF114 peptide was also used as a synthetic scaffold. Twenty-seven consecutive type 1 diabetic patients and 29 healthy controls were recruited for the study. In principle, the ‘chemical reverse approach’, based on the use of patient sera to screen the synthetic modified peptides, leads to the identification of specific probes able to characterize highly specific autoantibodies as disease biomarkers of autoimmune disorders. Quite surprisingly, both peptides modified with the (1-deoxyfructosyl)-lysine did not lead to significant results. Both IgG and IgM differences between the two populations were not significant. These data can be rationalized considering that i) IgGs in diabetic subjects exhibit a high degree of glycation, leading to decreased functionality; ii) IgGs in diabetic subjects exhibit a privileged response vs proteins containing advanced glycation products (e.g., methylglyoxal, glyoxal, glucosone, hydroimidazolone, dihydroxyimidazolidine) and only a minor one with respect to (1-deoxyfructosyl)-lysine.

Association between hemoglobin glycation index and cardiometabolic risk factors in Korean pediatric nondiabetic population.

PurposeThe hemoglobin glycation index (HGI) represents the degree of nonenzymatic glycation and has been positively associated with cardiometabolic risk factors (CMRFs) and cardiovascular disease in adults. This study aimed to investigate the association between HGI, components of metabolic syndrome (MS), and alanine aminotransferase (ALT) in a pediatric nondiabetic population.MethodsData from 3,885 subjects aged 10–18 years from the Korea National Health and Nutrition Examination Survey (2011–2016) were included. HGI was defined as subtraction of predicted glycated hemoglobin (HbA1c) from measured HbA1c. Participants were divided into 3 groups according to HGI tertile. Components of MS (abdominal obesity, fasting glucose, triglycerides, high-density lipoprotein cholesterol, and blood pressure), and proportion of MS, CMRF clustering (≥2 of MS components), and elevated ALT were compared among the groups.ResultsBody mass index (BMI) z-score, obesity, total cholesterol, ALT, abdominal obesity, elevated triglycerides, and CMRF clustering showed increasing HGI trends from lower-to-higher tertiles. Multiple logistic regression analysis showed the upper HGI tertile was associated with elevated triglycerides (odds ratio, 1.65; 95% confidence interval, 1.18–2.30). Multiple linear regression analysis showed HGI level was significantly associated with BMI z-score, HbA1c, triglycerides, and ALT. When stratified by sex, age group, and BMI category, overweight/obese subjects showed linear HGI trends for presence of CMRF clustering and ALT elevation.ConclusionsHGI was associated with CMRFs in a Korean pediatric population. High HGI might be an independent risk factor for CMRF clustering and ALT elevation in overweight/obese youth. Further studies are required to establish the clinical relevance of HGI for cardiometabolic health in youth.

Application of Glycation in Regulating the Heat-Induced Nanoparticles of Egg White Protein.

Due to the poor thermal stability of egg white protein (EWP), important challenges remain regarding preparation of nanoparticles for EWP above the denaturation temperature at neutral conditions. In this study, nanoparticles were fabricated from conjugates of EWP and isomalto-oligosaccharide (IMO) after heating at 90 °C for 30 min. Meanwhile, the effects of protein concentration, temperature, pH, ionic strength and degree of glycation (DG) on the formation of nanoparticles from IMO-EWP were investigated. To further reveal the formation mechanism of the nanoparticles, structures, thermal denaturation properties and surface properties were compared between EWP and IMO-EWP conjugates. Furthermore, the emulsifying activity index (EAI) and the emulsifying stability index (ESI) of nanoparticles were determined. The results indicated that glycation enhanced thermal stability and net surface charge of EWP due to changes in the EWP structure. The thermal aggregation of EWP was inhibited significantly by glycation, and enhanced with a higher degree of glycation. Meanwhile, the nanoparticles (<200 nm in size) were obtained at pH 3.0, 7.0 and 9.0 in the presence of NaCl. The increased thermal stability and surface net negative charge after glycation contributed to the inhibition. The EAI and ESI of nanoparticles were increased nearly 3-fold and 2-fold respectively, as compared to unheated EWP.

Understanding glycation kinetics of individual peptides in protein hydrolysates

Protein hydrolysates contain peptides with different lengths, type (α/ε-) and number of amino groups; these properties might influence the peptide glycation kinetics during the Maillard reaction. To identify the effects of peptide properties and hydrolysate composition on glycation kinetics, the glycation kinetics of individual peptides in hydrolysates was followed using quantitative peptide analysis. α-Lactalbumin was hydrolysed and glycated with d-glucose (0–8 h, 50 °C, dry heating with 65% humidity). The hydrolysates (degree of hydrolysis 2, 4, 6, and 8%) contained 25 unique peptides, ranging from 2 to 123 AAs with 0–12 lysine(s). The glycation rate constant (kg) and the maximum average degree of glycation (DG_Pav,max) of peptides were independent of the hydrolysate composition. The maximum DG of α-NH2 and ε-NH2 groups was 12.8% and 60.0%, respectively. With this information, the DG_Pav,max of individual peptides [9–59% for peptides with 0–2 lysine(s)] could be predicted.