Degree Of Glycosylation Research Articles

Improvement of quercetin bioaccessibility by whey protein isolate/D-tagatose conjugates: Effect on the structural characterization through simultaneous rheological and FTIR techniques

To improve the performance of whey protein isolates (WPI) as an encapsulation agent in the food industry, we investigated the formation mechanism of WPI and D-tagatose (DT) conjugates using simultaneous rheological and Fourier-Transform infrared spectroscopy (FTIR) techniques, and analysed its effects in encapsulating quercetin (Que). The degree of glycosylation of the conjugates increased to 18 % after heating treatment, whereas particle size and potential decreased to 64 nm and −39 mV, with WPI/D10 showing the lowest value. Simultaneous rheological and Fourier transform infrared analyses showed that the structure of the WPI/DT complexes changed, and the gel properties were much better than those of WPI. Several new peaks appeared at 2961, 1525, 1450, 1392, and 1236 cm-1, indicating that adding DT affected the structure of WPI. DT promoted secondary structural changes in WPI by increasing the degree of hydrocarbon chain, O-H group vibration, and C-O stretching. The WPI/DT conjugates increased the solubility of Que to 60.74 % and ABTS clearance to 73.98 %. The study may offer a theoretical foundation for using WPI/DT-encapsulated Que in food industry.

Glycosylated gelatin prepared based on electron beam irradiation and its physicochemical properties

The influence of electron beam irradiation (EBI) treatment on the modification of gelatin-galactose glycosylation was thoroughly examined. The results of the degree of grafting and browning revealed that EBI triggered the glycosylation reaction of gelatin. The degree of glycosylation exhibited a gradual increase with the rising irradiation dose, reaching a maximum of 25 kGy. Moreover, the irradiation process opened up gelatin's internal structure, exposing its hydrophobic groups. This exposure led to an enhancement in sample surface hydrophobicity. The fluorescence intensity at the maximum emission wavelength of the fluorescence spectra decreased; Fourier infrared spectroscopy demonstrated a new absorption peak at 1074 cm−1 for the glycosylation product. These findings substantiate that gelatin formed a new product through covalent bonding with galactose. Glycosylation boosted the emulsification stability of gelatin from 1.92 min to 10.42 min and improved its emulsification and rheological properties. These outcomes affirm that EBI can effectively induce the glycosylation reaction of gelatin, thereby enhancing its functional properties. In addition, EBI has the potential to supplant the conventional heating glycosylation method. This study lays a solid theoretical foundation for the future application of glycosylation and gelatin.

Effect of Mono- and Polysaccharide on the Structure and Property of Soy Protein Isolate during Maillard Reaction.

As a protein extracted from soybeans, soy protein isolate (SPI) may undergo the Maillard reaction (MR) with co-existing saccharides during the processing of soy-containing foods, potentially altering its structural and functional properties. This work aimed to investigate the effect of mono- and polysaccharides on the structure and functional properties of SPI during MR. The study found that compared to oat β-glucan, the reaction rate between SPI and D-galactose was faster, leading to a higher degree of glycosylation in the SPI-galactose conjugate. D-galactose and oat β-glucan showed different influences on the secondary structure of SPI and the microenvironment of its hydrophobic amino acids. These structural variations subsequently impact a variety of the properties of the SPI conjugates. The SPI-galactose conjugate exhibited superior solubility, surface hydrophobicity, and viscosity. Meanwhile, the SPI-galactose conjugate possessed better emulsifying stability, capability to produce foam, and stability of foam than the SPI-β-glucan conjugate. Interestingly, the SPI-β-glucan conjugate, despite its lower viscosity, showed stronger hypoglycemic activity, potentially due to the inherent activity of oat β-glucan. The SPI-galactose conjugate exhibited superior antioxidant properties due to its higher content of hydroxyl groups on its molecules. These results showed that the type of saccharides had significant influences on the SPI during MR.

Changes in whey protein produced by different sterilization processes and lactose content: Effects on glycosylation degree and whey protein structure

In this study, the interaction patterns between whey protein and lactose were investigated after different ratios of whey protein and lactose (1:2.5; 1:3.0; 1:3.5; 1:4.0; 1:4.5) were treated with two typical sterilization process parameters in powdered products (68 °C, 20 min; 72 °C, 15 min) and two typical sterilization process parameters in liquid products (121 °C, 10 min and 121 °C, 15 min). It was found that the degree of the Maillard reaction of whey protein-lactose solution increased with increasing lactose content, increasing sterilization temperature, or extending sterilization time, which was accompanied by changes in the protein structure of whey protein. In addition, glycosylation accelerates the decrease in free sulfhydryl content, decreasing surface hydrophobicity and decreasing fluorescence intensity. These findings highlight the advantages of designing protein-carbohydrate formulations with a high ratio of whey protein-lactose (1:2.5) to meet the requirements of the product standards for preterm/low-birth-weight infant formulas. When comparing only the sterilization processes of powder and liquid products, the pasteurization process of powder products significantly reduced the degree of the Maillard reaction caused by heat treatment and reduced protein denaturation.

Effect of electron beam irradiation on glycosylation reaction and structural characterization of whey isolate protein.

Whey protein isolate (WPI) is a high-quality animal protein resource. The modification of WPI through physical, chemical and biological methods can substantially improve the functional properties of proteins. This study investigated the effect of electron beam irradiation (EBI) on the modification of WPI-xylose glycosylation. The degree of grafting and browning revealed that EBI promoted WPI glycosylation. The maximum emission wavelength of intrinsic fluorescence was red-shifted and the fluorescence intensity was reduced, suggesting that irradiation induced the unfolding of the WPI structure, thereby promoting glycosylation. Fourier-transformed infrared spectroscopy revealed that the covalent binding of the conjugates occurred on the introduction of the hydrophilic groups, resulting in decreased surface hydrophobicity. When compared with conventional wet-heat glycosylation, irradiation-assisted glycosylation improved the emulsifying activity of WPIfrom 179.76 ± 0.83 to 277.83 ± 1.44 m2 g-1, and the emulsifying and rheological properties improved. These results confirmed that EBI can increase the degree of WPI glycosylation and improve the functional properties of proteins, thereby laying a theoretical foundation for the further application of WPI. © 2024 Society of Chemical Industry.

Sodium-dependent phosphate transporter NaPi2b as a candidate for targeted therapy: features of structure, function, and expression

The sodium-dependent phosphate transporter NaPi2b is an integral membrane protein of the SLC34 phosphate transporter family and is an attractive target for precision therapy of several human diseases. Together with other members of this family, the NaPi2b transporter is involved in maintaining phosphate homeostasis in the mammalian body. The NaPi2b transporter gene (SLC34A2) has a broad expression pattern in healthy tissues, including small intestinal epithelial cells, where NaPi2b plays a major role in the absorption of dietary phosphate. NaPi2b transports one divalentorthophosphoric acid residue into cells along with three sodium ions. NaPi2b transport is regulated by dietary phosphate, pH, hormones, and vitamins including vitamin D, estrogen, glucocorticoids, and epidermal growth factor. The NaPi2b transporter exists in two isoforms – 689 and 690 amino acid residues. The molecular weight of NaPi2b depends on the degree of glycosylation and varies from 70 to 100 kDa. According to various sources, the transporter has from 6 to 12 transmembrane domains, 2 co-transport domains, a large extracellular localization domain, as well as N- and C-terminal domains that face the inside of the cell. Impaired NaPi2b function leads to the development of several diseases, including pulmonary alveolar microlithiasis and hyperphosphatemia, and pulmonary alveolar microlithiasis is known to be associated with mutations in the SLC34A2 gene encoding NaPi2b. High levels of NaPi2b have been found in several malignant tumors, including ovary, lung, breast, thyroid, colon, bladder, liver, stomach, kidney, and in gliomas. The tumor-specific conformation of the large extracellular domain of the NaPi2b transporter, mutations, and features of expression of the transporter gene in normal and pathological conditions show that NaPi2b is a promising target for the development of highly selective targeted drugs against it for the treatment of cancer and metabolic disorders.

Comparison of sea buckthorn fruit oil nanoemulsions stabilized by protein-polysaccharide conjugates prepared using β-glucan from various sources

To understand the influence of β-glucans structure on the emulsifying properties of protein-polysaccharide conjugates, sodium caseinate (NaCas) was utilized to form glycosylation conjugates with varying degrees of glycosylation (10.68–17.50%) using three β-glucans from bacteria, yeast, and oats. This process induced alterations in the secondary structure of protein. The nanoemulsions prepared with the glycosylated conjugates exhibited superior stability compared to those formulated solely with NaCas, particularly under conditions of drastic pH fluctuations and extended storage periods. The nanoemulsion prepared with the NaCas-Salecan conjugate demonstrated exceptional stability at pH 4 and 6, or storage for 20 days. Additionally, it significantly attenuated the oxidation of unsaturated fatty acids and exhibited the lowest levels of aggregation, flocculation, and free fatty acid release rate during in vitro digestion. This study suggested the potential of the NaCas-Salecan conjugates in enhancing the stability of nanoemulsions and facilitating the colorectal-targeted delivery of sea buckthorn fruit oil.

Effect of casein genetic variants and glycosylation on bovine milk foaming properties

The effects of κ‐casein (κ‐CN) and β‐casein (β‐CN) genetic variant and κ‐CN glycosylation degree (GD, low or high) on interfacial and foaming properties of bovine skim milk were investigated. No significant effect was measured for milks with different ĸ‐CN and β‐CN genetic variants. However, milks of higher GD exhibited lower surface tension, enhanced foamability and differences in secondary protein structure compared to lower GD skim milks. Glycan attachment is believed to affect surface activity and the spread and packing of protein at the foam bubble liquid–air interface, leading to differences in foaming performance.

Development of a biotechnology process for the production of a novel hyperglycosylated long-acting recombinant bovine follicle-stimulating hormone.

Follicle-stimulating hormone (FSH) is an important protein used for bovine ovarian hyperstimulation in multiple ovulation and embryo transfer technology (MOET). Several attempts to produce bovine FSH (bFSH) in recombinant systems have been reported, nonetheless, up to date, the most commonly used products are partially purified preparations derived from porcine or ovine (pFSH or oFSH) pituitaries. Here we describe the development of a biotechnology process to produce a novel, hyperglycosylated, long-acting recombinant bFSH (LA-rbFSH) by fusing copies of a highly O-glycosylated peptide. LA-rbFSH and a nonmodified version (rbFSH) were produced in suspension CHO cell cultures and purified by IMAC with high purity levels (>99%). LA-rbFSH presented a higher glycosylation degree and sialic acid content than rbFSH. It also demonstrated a notable improvement in pharmacokinetic properties after administration to rats, including a higher concentration in plasma and a significant (seven-fold) reduction in apparent clearance (CLapp). In addition, the in vivo specific bioactivity of LA-rbFSH in rats was 2.4-fold higher compared to rbFSH. These results postulate this new molecule as an attractive substitute for commercially available porcine pituitary-derived products.

Glucosamine and Silibinin Alter Cartilage Homeostasis through Glycosylation and Cellular Stresses in Human Chondrocyte Cells.

Osteoarthritis is more prevalent than any other form of arthritis and is characterized by the progressive mechanical deterioration of joints. Glucosamine, an amino monosaccharide, has been used for over fifty years as a dietary supplement to alleviate osteoarthritis-related discomfort. Silibinin, extracted from milk thistle, modifies the degree of glycosylation of target proteins, making it an essential component in the treatment of various diseases. In this study, we aimed to investigate the functional roles of glucosamine and silibinin in cartilage homeostasis using the TC28a2 cell line. Western blots showed that glucosamine suppressed the N-glycosylation of the gp130, EGFR, and N-cadherin proteins. Furthermore, both glucosamine and silibinin differentially decreased and increased target proteins such as gp130, Snail, and KLF4 in TC28a2 cells. We observed that both compounds dose-dependently induced the proliferation of TC28a2 cells. Our MitoSOX and DCFH-DA dye data showed that 1 µM glucosamine suppressed mitochondrial reactive oxygen species (ROS) generation and induced cytosol ROS generation, whereas silibinin induced both mitochondrial and cytosol ROS generation in TC28a2 cells. Our JC-1 data showed that glucosamine increased red aggregates, resulting in an increase in the red/green fluorescence intensity ratio, while all the tested silibinin concentrations increased the green monomers, resulting in decreases in the red/green ratio. We observed increasing subG1 and S populations and decreasing G1 and G2/M populations with increasing amounts of glucosamine, while increasing amounts of silibinin led to increases in subG1, S, and G2/M populations and decreases in G1 populations in TC28a2 cells. MTT data showed that both glucosamine and silibinin induced cytotoxicity in TC28a2 cells in a dose-dependent manner. Regarding endoplasmic reticulum stress, both compounds induced the expression of CHOP and increased the level of p-eIF2α/eIF2α. With respect to O-GlcNAcylation status, glucosamine and silibinin both reduced the levels of O-GlcNAc transferase and hypoxia-inducible factor 1 alpha. Furthermore, we examined proteins and mRNAs related to these processes. In summary, our findings demonstrated that these compounds differentially modulated cellular proliferation, mitochondrial and cytosol ROS generation, the mitochondrial membrane potential, the cell cycle profile, and autophagy. Therefore, we conclude that glucosamine and silibinin not only mediate glycosylation modifications but also regulate cellular processes in human chondrocytes.

Serum Clusterin Concentration and Its Glycosylation Changes as Potential New Diagnostic Markers of SARS-CoV-2 Infection and Recovery Process.

COVID-19 is an infectious disease caused by the SARS-CoV-2 virus. Glycoprotein clusterin (CLU) has many functions such as phagocyte recruitment, complement system inhibition, apoptosis inhibition, hormone and lipid transport, as well as in the immune response. The study aimed to assess the changes in CLU concentrations and the profile and degree of CLU glycosylation between patients with severe COVID-19, convalescents, and healthy subjects (control). The profile and degree of serum CLU N-glycosylation were analyzed using lectin-ELISA with specific lectins. CLU concentrations were significantly lower and relative reactivities of CLU glycans with SNA (Sambucus nigra agglutinin) were significantly higher in severe COVID-19 patients in comparison to convalescents and the control group. The relative reactivities of CLU glycans with MAA (Maackia amurensis agglutinin), together with relative reactivity with LCA (Lens culinaris agglutinin), were also significantly higher in patients with severe COVID-19 than in convalescents and the control group, but they also significantly differed between convalescents and control. The development of acute inflammation in the course of severe COVID-19 is associated with a decrease in CLU concentration, accompanied by an increase in the expression of α2,3-linked sialic acid, and core fucose. Both of these parameters can be included as useful glycomarkers differentiating patients with severe COVID-19 from convalescents and the control group, as well as convalescents and healthy subjects.

Conjugation of Soybean Proteins 7S/11S Isolate with Glucose/Fructose in Gels through Wet-Heating Maillard Reaction.

Conjugation with glucose (G) and fructose (F) via the Maillard reaction under the wet-heating condition is a natural and non-toxic method of improving the technological functions of 7S/11S proteins in different kinds of gels. It may be used as an affordable supply of emulsifiers and an excellent encapsulating matrix for gels. This study aimed to create a glucose/fructose-conjugated 7S/11S soy protein via the Maillard reaction. The conjugation was confirmed by determining the SDS-PAGE profile and circular dichroism spectra. In addition, these conjugates were comprehensively characterized in terms of grafting degree, browning degree, sulfhydryl content, surface hydrophobicity (H0), and differential scanning calorimetry (DSC) through various reaction times (0, 24, 48, and 72 h) to evaluate their ability to be used in food gels. The functional characteristics of the 7S/11S isolate-G/F conjugate formed at 70 °C, with a high degree of glycosylation and browning, were superior to those obtained at other reaction times. The SDS-PAGE profile indicated that the conjugation between the 7S and 11S proteins and carbohydrate sources of G and F through the Maillard reaction occurred. Secondary structural results revealed that covalent interactions with G and F affected the secondary structural components of 7S/11S proteins, leading to increased random coils. When exposed to moist heating conditions, G and F have significant potential for protein alteration through the Maillard reaction. The results of this study may provide new insights into protein modification and establish the theoretical basis for the therapeutic application of both G and F conjugation with soy proteins in different food matrixes and gels.

Impact of Protein Nanoparticle Shape on the Immunogenicity of Antimicrobial Glycoconjugate Vaccines.

Protein self-assembling nanoparticles (NPs) can be used as carriers for antigen delivery to increase vaccine immunogenicity. NPs mimic the majority of invading pathogens, inducing a robust adaptive immune response and long-lasting protective immunity. In this context, we investigated the potential of NPs of different sizes and shapes-ring-, rod-like, and spherical particles-as carriers for bacterial oligosaccharides by evaluating in murine models the role of these parameters on the immune response. Oligosaccharides from Neisseria meningitidis type W capsular polysaccharide were conjugated to ring-shape or nanotubes of engineered Pseudomonas aeruginosa Hemolysin-corregulated protein 1 (Hcp1cc) and to spherical Helicobacter pylori ferritin. Glycoconjugated NPs were characterized using advanced technologies such as High-Performance Liquid Chromatography (HPLC), Asymmetric Flow-Field Flow fractionation (AF4), and Transmission electron microscopy (TEM) to verify their correct assembly, dimensions, and glycosylation degrees. Our results showed that spherical ferritin was able to induce the highest immune response in mice against the saccharide antigen compared to the other glycoconjugate NPs, with increased bactericidal activity compared to benchmark MenW-CRM197. We conclude that shape is a key attribute over size to be considered for glycoconjugate vaccine development.

The conformational dynamics of Hepatitis C Virus E2 glycoprotein with the increasing number of N-glycosylation unraveled by molecular dynamics simulations

The Hepatitis C Virus (HCV), responsible for causing hepatitis and a significant contributor to liver disorders, presents a challenge for treatment due to its high genetic variability. Despite efforts, there is still no effective medication available for this virus. One of the promising targets for drug development involves targeting glycoprotein E2. However, our understanding of the dynamic behavior of E2 and its associated glycans remains limited. In this study, we investigated the dynamic characteristics of E2 with varying degrees of glycosylation using all-atom molecular dynamics simulations. We also explored glycan’s interactions with the protein and among themselves. An overall increase in correlation between the vital protein regions was observed with an increase in glycan number. The protein dynamics is followed by the analysis of glycan dynamics, where the flexibility of the individual glycans was analyzed in their free and bound state, which revealed a decrease in their fluctuation in some cases. Furthermore, we generated the free energy landscape of individual N-glycan linkages in both free and bound states and observed both increases and decreases in flexibility, which can be attributed to the formation and breakage of hydrogen bonds with amino acids. Finally, we found that for a high glycosylation system, glycans interact with glycoprotein and form hydrogen bonds among themselves. Moreover, the hydrogen bond profiles of a given glycan can vary when influenced by other glycans. In summary, our study provides valuable insights into the dynamics of the core region of HCV E2 glycoprotein and its associated glycans. Communicated by Ramaswamy H. Sarma

Effect of composition, casein genetic variants and glycosylation degree on bovine milk whipping properties

This study investigated the individual and combined effects of ĸ-Casein (ĸ-CN; AA, AB, BB), β-Casein (β-CN; A1A1, A1A2, A2A2) and high and low ratios of glycosylated ĸ-CN to total ĸ-CN, referred to as the glycosylation degree (GD), on bovine cream whipping properties. The genetic variants of individual cows were identified using reversed-phase high-performance liquid chromatography (RP-HPLC) and verified through liquid chromatography–mass spectrometry (LC-MS). A previously discovered relationship between days-in-milk and GD was validated and used to obtain high and low GD milk. Whipped creams were created through the mechanical agitation of fat standardised cream from milk of different ĸ-CN, β-CN, and GD combinations, and whipping properties (the ability to whip, overrun, whipping time and firmness) were evaluated. No significant correlation was measured in whipping properties for cream samples from milks with different ĸ-CN and β-CN genetic variants. However, 80 % of samples exhibiting good whipping properties (i.e., the production of a stiffened peak) were from milk with low GD suggesting a correlation between whipping properties and levels of glycosylation. Moreover, cream separated from skim milk of larger casein micelle size showed superior whipping properties with shorter whipping times (<5 min), and higher firmness and overrun. Milk fat globule (MFG) size, on the other hand, did not affect whipping properties. Results indicate that the GD of κ-CN and casein micelle size may play a role in MFG adsorption at the protein and air interface of air bubbles formed during whipping; hence, they govern the dynamics of fat network formation and influencing whipping properties.

Characterization and emulsifying ability evaluation of whey protein-pectin conjugates formed by glycosylation

Glycosylation is a method that enhances the functional properties of proteins by covalently attaching sugars to them. This study aimed at preparing three conjugates (WP-HG, WP-SBP, and WP-RGI) by dry heating method to research the influence of different pectin structures on the functional properties of WP and characterize properties and structures of these conjugates. The research results manifested that the degree of glycosylation (DG) of HG, SBP and RGI were 13.13 % ± 0.07 %, 23.27 % ± 0.3 % and 36.39 % ± 0.3 % respectively, suggesting that the increase of the number of branch chains promoted the glycosylation reaction. The formation of the conjugate was identified by the FT-IR spectroscopy technique. And SEM showed that WP could covalently bind to pectin, resulting in a smoother and denser surface of the conjugates. The circular dichroism analysis exhibited that the glycosylation reaction altered the secondary structure of WP and decreased the α-Helix content. This structural change in the protein spatial conformation led to a decrease in the hydrophobicity of protein surface. But the addition of pectin further regulated the hydrophilic-hydrophobic ratio on the surface of the protein, thus improving the emulsification properties of WP. In addition, the glycosylation could improve the stability of the emulsion, giving it a smaller droplet size, higher Zeta-potential and more stable properties. In a word, this study pointed out the direction for the application of different pectin structures in the development of functional properties of glycosylation products in food ingredients.

Influence of biotic and abiotic factors on prymnesin profiles in three strains of Prymnesium parvum

Harmful algal blooms of Prymnesium parvum have resulted in significant fish kill events globally. These haptophytes produce the ichthyotoxic prymnesins, large polyethers categorized into A-, B- and C-types based on their carbon backbones with several analogs varying by the degree of glycosylation, chlorination, and double bonds. However, the influence of various biotic or abiotic factors on prymnesin profiles remains unknown. We investigated the influence of growth phase, nitrogen availability, light intensity, and salinity on prymnesin profiles of three P. parvum strains (UTEX-2797, K-0374 and PPSR01). Strains were selected based on their chemotypic expression of the three prymnesin backbones. Results demonstrated that different growth conditions led to strain-specific changes in prymnesin profiles. In the stationary phase, increased glycosylation was observed compared to the exponential growth phase for all strains. Additionally, at early stationary phase the tri-chlorinated analogs represented >90 % of all prymnesins of P. parvum strains UTEX-2797 and PPSR01 when cultured at salinity of 10 psu. By gaining an understanding of the effects of biotic and abiotic factors, culture conditions could be modified to promote specific prymnesin profiles to support the development of analytical reference materials. Furthermore, evaluating prymnesin profiles in combination with toxicity assays will provide insights into prymnesin biosynthesis, mode of action and toxicity.

New findings in the metabolism of the saffron apocarotenoids, crocins and crocetin, by the human gut microbiota.

The main constituents of saffron are the apocarotenoids crocins and crocetin, present in the stigmas. Numerous healthy properties, especially those related to the effects on the central nervous system, have been attributed to these compounds but the metabolites responsible for these effects are still unknown. Previous evidences in animal models suggest a role for the gut microbiota in the pharmacokinetics and the neuroprotective effects of these compounds. However, the interaction between these apocarotenoids and the gut microbiota has been poorly studied. In this article, we have thoroughly investigated the batch fermentation of crocin-1 and crocetin (10 μM) with human fecal samples of two donors at different incubation times (0-240 h) using a metabolomic approach. We corroborated a rapid transformation of crocin-1 which looses the glucose molecules through de-glycosylation reactions until its complete transformation into crocetin in 6 hours. A group of intermediate crocins with different degrees of glycosylation were detected in a very short time. Crocetin was further metabolized and new microbial metabolites produced by double-bond reduction and demethylation reactions were identified for the first time: dihydro and tetrahydro crocetins and di-demethyl crocetin. In addition, we detected changes in the levels of the short chain fatty acids valeric acid and hexanoic acid suggesting further structural modifications of crocetin or changes in the catabolic production of these compounds. This research is a pioneering study of the action of the human gut microbiota on the saffron apocarotenoids and goes one step further towards the discovery of metabolites potentially involved in the benefits of saffron.

Quantitation of MUC5AC and MUC5B by Stable Isotope Labeling Mass Spectrometry.

Mucins MUC5AC and MUC5B are large glycoproteins that play an essential role in the innate defense of epithelial surfaces and their quantitation in biological samples would be informative about the health status of the tissue/samples they are derived from. However, they are difficult to study and quantify with traditional methods such as ELISA and western blot, due to their size, heterogeneity, and high degree of glycosylation. We successfully implemented a stable isotope labeling mass spectrometry approach for absolute quantification of mucin macromolecules. Here, in detail, we describe this accurate and sensitive liquid chromatography and mass spectrometry (LC-MS) method applied for both MUC5AC and MUC5B quantification in diverse and complex biological samples.

High-temperature glycosylation of saccharides to modify molecular conformation of egg white protein and its effect on the stability of high internal phase emulsions

This paper investigates the freeze–thaw stability of oil-in-water emulsions stabilized by high-temperature wet heating glycosylation products. Glucose (Glu), D-fructose (Fru), xylose (Xyl), maltodextrin (MD), oligofructose (FO), and oligomeric isomaltulose (IMO) were chosen as sugar sources for the glycosylation reaction with egg white proteins (EWPs) at 120 °C to prepare the GEWPs. The study reveals that the type of sugar significantly influences the Maillard reactions with EWPs. The degree of glycosylation was highest in the Xyl group with the greatest reducing capacity and lowest in the MD, FO, and IMO groups. High-temperature wet glycosylation treatment induced changes in the secondary and tertiary structures of EWP. Elevated temperature exposed hydrophobic groups within the protein, while covalent binding of hydrophilic carbohydrates via the Maillard reaction decreased the protein's H0 value. Improved foaming and emulsifying properties were attributed to the increase in α-helix content, disulfide bond formation, and reduced surface tension. Emulsions prepared from GEWPs exhibited higher apparent viscosity and G' compared to those from natural EWPs, with the GEWP/Xyl group showing the highest values. After freeze–thaw treatment, the GEWP/Fru and GEWP/FO groups demonstrated superior stability and reduced freezing point, along with minimal microstructural alterations. These findings underscore the importance of sugar type in the stability of high internal phase emulsions (HIPEs) stabilized by GEWPs, indicating that a tailored Maillard reaction can yield stabilizers with exceptional freeze–thaw stability for emulsions.