Allergenicity assessment of ß-lactoglobulin ferulic acid-glucose conjugates.

We prepared the ß-lactoglobulin (BLG)-ferulic acid (FA)-glucose (Glu) conjugates by alkaline method and Maillard reaction to assess the allergenicity. FA and Glu can form a ternary covalent conjugate with BLG, as evidenced by the shortening of SEC retention time, upward migration of SDS-PAGE protein bands, considerable decrease in free amino and sulfhydryl content, and changes in multistructure. BLG-Glu-FA conjugates weakly bound to immunoglobulin E in allergic sera was weak, reduced interleukin 4 and tumor necrosis factor α levels in RBL-2H3 cells and histamin and interleukin 6 secretion levels in KU812 cells, and inhibited the nuclear factor-κB signaling pathway. In vivo experiments showed that the conjugates regulated T-cell homeostasis in mouse splenic and mesenteric lymphocytes and attenuated splenic and duodenal immune injury. Therefore, the conjugates of BLG with FA combined with Glu altered the epitope structure and exhibited low allergenicity.

Gut Microbiome-Serum Metabolism Revealed the Allergenicity of Ferulic Acid Combined with Glucose-Modified ß-Lactoglobulin.

A novel strategy combining ferulic acid and glucose was proposed to reduce ß-lactoglobulin (BLG) allergenicity and investigate whether the reduction in allergenicity was associated with gut microbiome and serum metabolism. As a result, the multistructure of BLG changed, and the modified BLG decreased significantly the contents of IgE, IgG, IgG1, and mMCP-1 in serum, improved the diversity and structural composition of gut microbiota, and increased the content of short-chain fatty acids (SCFAs) in allergic mice. Meanwhile, allergic mice induced by BLG affected arachidonic acid, tryptophan, and other metabolic pathways in serum, the modified BLG inhibited the production of metabolites in arachidonic acid metabolism pathway and significantly increased tryptophan metabolites, and this contribution helps in reducing BLG allergenicity. Overall, reduced allergenicity of BLG after ferulic acid was combined with glucose modification by regulating gut microbiota, the metabolic pathways of arachidonic acid and tryptophan. The results may offer new thoughts alleviating the allergy risk of allergenic proteins.

Simulated in vitro gastrointestinal digestion of ß-lactoglobulin treated by ultrasound: Detection of peptides profile and the antioxidant activity.

The influence of ultrasonic pretreatment on the release and antioxidant activity of potential antioxidant peptides after in-vitro simulated gastrointestinal digestion of ß-lactoglobulin (BLG) were measured by HPLC-MS/MS, chemical and cellular-based assays. The gastrointestinal digest was fractionated into four fractions by Sephadex G-25 gel filtration column, and fractions showed a considerable ABTS·+ scavenging ability. The fraction with the strongest antioxidant activity was produced by ultrasonicated BLG after gastrointestinal digestion, which relies on ultrasonic-promoted proteolysis to produce many small-molecule antioxidant peptides. The best active fraction has better cellular antioxidant activity and protection of H2O2-induced oxidative HepG2 cell model, which significantly increases the activities of antioxidant enzyme, and is concentration-dependent. HPLC-MS/MS analysis showed that there were more potential antioxidant peptides in the best active fraction. This research will provide a basis for the further application of ultrasonic in dairy products, which can promote the release of more potential antioxidant peptides-derived from gastrointestinal digestion.

A comparative study on the allergenic potential of ß-lactoglobulin conjugated to glucose, caffeic acid and caffeoyl glucopyranose.

This work intends to perform a comparative study on the allergenic potential of ß-lactoglobulin (BLG)-glucose, BLG-caffeic acid and BLG-caffeoyl glucopyranose conjugates. The modifications changed the molecular weight and multi-structure of BLG and destroyed the allergenic epitope, which resulted in a decrease in the IgE binding level and the release ability of histamine and IL-6 in KU812 cells. Compared with BLG, the conjugates reduced the serum levels of IgG, IgE, ß-Hex and IL-4 in vivo, while increasing the level of interferon-γ, which caused an imbalance of Th1/Th2 immune response. Meanwhile, these conjugates not only increased the relative abundance of allergy-related gut flora, such as Lachnospiraceae, norank_o_Clostridia_UCG-014, Erysipelotrichaceae, Turicibacter and Lachnospiraceae_NK4A136_group, but also improved the level of short-chain fatty acids (SCFAs). Caffeoyl glucopyranose with a large molecular weight and long carbon chains exerted a great influence on the allergy-related gut flora and SCFAs. Therefore, the changes in the Th1/Th2 balance and SCFA level produced by the allergy-related gut flora were responsible for reducing the potential allergy of BLG.

Molecular structure, IgE binding capacity and gut microbiota of ovalbumin conjugated to fructose and galactose:A comparative study.

Ovalbumin (OVA) was modified by fructose (Fru) and galactose (Gal) to study the structure, IgG/IgE binding capacity and effects on human intestinal microbiota of the conjugated products. Compared with OVA-Fru, OVA-Gal has a lower IgG/IgE binding capacity. The reduction of OVA is not only associated with the glycation of R84, K92, K206, K263, K322 and R381 in the linear epitopes, but also with conformational epitope changes, manifested as secondary and tertiary structural changes caused by Gal glycation. In addition, OVA-Gal could alter the structure and abundance of gut microbiota at phylum, family, and genus levels and restore the abundance of bacteria associated with allergenicity, such as Barnesiella, Christensenellaceae_R-7_group, and Collinsela, thereby reducing allergic reactions. These results indicate that OVA-Gal glycation can reduce the IgE binding capacity of OVA and change the structure of human intestinal microbiota. Therefore, Gal glycation may be a potential method to reduce protein allergenicity.

Simulated in vitro digestion of α-lactalbumin modified by phosphorylation: Detection of digestive products and allergenicity.

The effects of phosphorylation on the allergenicity of bovine α-lactalbumin (BLA) and digestive products were studied in vitro digestion. Two components with different molecular weight and conformation were obtained from natural and phosphorylated BLA. In vivo and in vitro assessment of allergenicity showed that phosphorylation prior to digestion significantly decreased the IgE/IgG binding capacity and allergic response in KU812 cells, and reduced the levels of IgG, IgE, IL-4 and histamine, with an increase in IFN-γ levels in mouse serum, depending on the changes in BLA structures, producing numerous small peptides. There were four phosphorylated sites (S22, T29, S47 and S70) in the high molecular weight components of phosphorylated BLA after digestion. These phosphorylated sites could mask the linear epitopes of digestive products, resulting in reduced allergic activity. Phosphorylation prior to digestion of dairy products can reduce the risk of anaphylaxis in patients with milk allergy to some extent.

Mechanism of Reduction in Allergenicity and Altered Human Intestinal Microbiota of Digested ß-Lactoglobulin Modified by Ultrasonic Pretreatment Combined with Glycation.

The effects of ultrasound combined with glycation (UCG) on the allergenicity and human microbial community of ß-Lg during in vitro digestion were studied by ELISA, cell experiments, and 16S rRNA high-throughput sequencing. UCG modification and subsequent digestion significantly reduced allergenicity. The decrease in the allergenicity of ß-Lg depended not only on the low digestibility of glycated ß-Lg, which led to the decrease of some peptides with complete immunogenicity, but also the masking effect of glycation on allergen epitopes of ß-Lg. Meanwhile, UCG modification and subsequent digestion could alter the structures of intestinal microbiota and the community abundance at phylum, family, and genus levels, such as Bacteroidota, Fusobacteriota, Enterobacteriaceae, Bacteroidaceae, Ruminococcaceae, Bacteroides, and Faecalibacterium. These results show that simulated in vitro digestion of modified ß-Lg reduces allergenicity and alters human intestinal microbiota, which could provide a theoretical basis for studying the relationship between intestinal dysbiosis and cow's milk allergy.

Investigation into predominant peptide and potential allergenicity of ultrasonicated ß-lactoglobulin digestion products.

The effect of ultrasonicated ß-lactoglobulin on the allergenic potential of predominant peptide was studied in vitro digestion. Gastrointestinal (GI) digestion of ultrasonicated ß-lg was fractionated into four fractions, which have different molecular weight and allergenic potentials. The lowest allergenicity of fraction was produced by ultrasonicated ß-Lg after GI digestion, depending on the changes in the structure of ß-Lg by ultrasonic and the promotion of its proteolysis, resulting in the production of numerous small peptides with significantly reduced IgE activity and basophil histamine release. Mass spectrometry analysis showed that ultrasonic can promote the further hydrolysis of large intermediate peptides, Y42, L54, L57/L58, L95, L104/F105, L122 were target residues that became more available to protease by the pretreatment of ultrasonic, thus have a smaller molecular weight with reduced allergenic potential. Ultrasonic processing of milk products alone could reduce the risk of an allergenic reaction in milk allergy patients to some extent.

Improved antitumor activity and IgE/IgG-binding ability of α-Lactalbumin/ß-lactoglobulin induced by ultrasonication prior to binding with oleic acid.

Bovine α-lactalbumin (α-La)/ß-lactoglobulin (ß-Lg) was pretreated through ultrasonic treatment and subsequently binding with oleic acid (OA) by heat treatment. And, the antitumor activity, IgE/IgG-binding ability, and structural modifications were investigated. After α-La/ß-Lg were treated by ultrasonic prior to binding with OA, the treated α-La/ß-Lg showed high antitumor activity and IgE/IgG-binding ability, and significantly affected the structural modifications, which reflected by the reduction in α-helix content, the increase of molecular weight, intrinsic fluorescence intensity, and surface hydrophobicity. Molecular docking studies indicated that OA bound to α-La/ß-Lg by hydrogen bonds and hydrophobic interaction. Therefore, ultrasonic prior to binding with OA could improve antitumor activity and IgE/IgG-binding ability of α-La/ß-Lg as a result of structural modifications. And, ultrasonic prior to binding with fatty acid processing of milk products alone may increase the antitumor activity, this change may enhance the risk of an allergenic reaction in milk allergy patients to some extent. PRACTICAL APPLICATIONS: Fatty acids, natural ligands associated with the bovine milk proteins, and milk protein-fatty acid complex has a variety of functional applications in the food industry. This study revealed that antitumor activity, IgE/IgG-binding ability, and structural modifications of α-La/ß-Lg induced by ultrasonic prior to binding with oleic acid. It will be beneficial to understand the mechanism of the functional changes of protein. Ultrasonic prior to binding with oleic acid will be more likely to develop a practical technology to improve the functional characteristics of milk protein and design the optimal nutritional performance of milk food.

Glycation of ß-lactoglobulin combined by sonication pretreatment reduce its allergenic potential.

ß-lactoglobulin (ß-Lg) was treated through different ultrasonic power and subsequently glycated with galactose to investigate its structural changes and immunological properties, and then evaluated by high-resolution mass spectrometry, enzyme-linked immunosorbent assay and basophil histamine release test. Ultrasonication combined with glycation (UCG) modification significantly reduced the IgE/IgG-binding capacity, and the release of ß-hexosaminidase, histamine and interleukin-6, accompanied with changes in the secondary and tertiary structures. The decrease in the allergenicity of ß-Lg depended not only on the glycation of K47, 60, 83, 91 and 135 within the linear epitopes, but also on the denaturation of conformational epitopes, which was supported by the glycation-induced alterations of the secondary and tertiary structures. This study confirmed that UCG modification is a promising method for decreasing the allergenic potential of allergic proteins, which is likely to develop a practical technology to produce hypo-allergenic milk.

Influence of ultrasonic pretreatment on the structure, antioxidant and IgG/IgE binding activity of ß-lactoglobulin during digestion in vitro.

The influence of ultrasonic pretreatment on the structure, antioxidant activity, and IgG/IgE binding activity of ß-lactoglobulin (ß-Lg) during digestion in vitro were studied by spectroscopy, chromatography and ELISA. After ß-Lg was treated by ultrasonic prior to digestion in vitro, the treated ß-Lg showed high intrinsic fluorescence emission, more of the hydrolytic products and high antioxidant activity, which depend on the proteolysis promoted by ultrasonic, and produced many small-molecule antioxidant peptides. Native ß-Lg was resistant to gastric digestion and retained its allergenicity. However, the allergenicity of ultrasonicated ß-Lg after gastric digestion was increased due to ultrasonic promotes the production of peptides with intact structure and immunogenicity. Subsequent gastrointestinal digestion, ultrasonicated ß-Lg has a diametrically opposite results because the increase of small peptides with the decreasing of immunogenicity. Therefore, the structural changes of ß-Lg by ultrasonic and gastrointestinal digestion were responsible for improving the antioxidant activity and reducing the IgG/IgE binding activity.

The mechanism of the reduction in allergenic reactivity of bovine α-lactalbumin induced by glycation, phosphorylation and acetylation.

Bovine α-lactalbumin (α-Lac) allergy is a common health problem. This study assesses the allergenic reactivity and the structural properties of α-Lac after protein modification (glycation, phosphorylation and acetylation) by ELISA, cells experiment and high-resolution mass spectrometry. Three modified methods significantly reduced the IgE/IgG-binding capacity, and the release of histamine and interleukin-6, and changed the conformational structure of α-Lac. α-Lac was glycated at K13, K16, K94, K98, and K108, phosphorylated at Y18, S22, Y103, and S112, and acetylated at K13, T33, S34, T38, S47, K62, S69, S70, K108, and K114, respectively, leading to masking the linear epitopes of α-Lac. Therefore, the decrease of allergenic reactivity of α-Lac induced by glycation, phosphorylation and acetylation depends upon not only the shielding effect of their modified sites, but also the change of conformational structure. This study confirmed that protein modification was a promising method for decreasing the allergenic reactivity of allergic proteins.

Observation of the structural changes of α-lactalbumin induced by ultrasonic prior to glycated modification.

Bovine α-lactalbumin (BLA) was treated by ultrasonic at 150 W/cm2 for different times and subsequently glycated with mannose by dry-heating. Molecular weight, intrinsic fluorescence spectra, glycation sites and degree of modified BLA were observed. The proteinaceous high molecular weight components were formed after ultrasonic prior to glycated modification, while the conformational changes were obvious. Prior to ultrasonic pretreatment, K62, K114, and K122 of BLA were identified. After treated by ultrasound at 150 W/cm2 for 5, 10, 15, and 20 min, the sites were increased to four, four, five, and five, respectively. All glycated sites of modified BLA exhibited a higher degree of substitution per peptide (DSP) values compared to native BLA. Ultrasonic at 150 W/cm2 for 20 min revealed the most significant change in the BLA structure. Therefore, conformational changes, the intensified glycation site, and DSP value were responsible for the structural changes of BLA. Practical applications BLA is suitable as an ingredient for infant nutrition in food, and has immune-modulating, antioxidant, antibacterial, and antitumor activity etc. This study revealed that the structural changes of BLA induced by ultrasonic prior to glycated modification. It will be beneficial to understand the mechanism of the functional changes of modified BLA. Ultrasonic prior to glycated modification will be more likely to develop a practical technology to modify protein in the food industry, and improve the functional characteristics of food, such as produce hypo-allergenic cow's milk in future.

The accumulation, histopathology, and intestinal microorganism effects of waterborne cadmium on Carassius auratus gibelio.

Cadmium (Cd) is known to be a potentially toxic heavy metals to the fish health and growth. Carassius auratus gibelio (C. a. gibelio) specimens were exposed to waterborne Cd (0, 0.05, 0.10, 0.15, and 0.20 mg/L CdCl2) for 14 days. Cd accumulation, liver and intestine histopathology, and intestinal microorganism were investigated in the present study. The results indicated that Cd accumulation in the gill, liver, intestine, and muscle gradually decreased as Cd concentration increased. The gill accumulated higher amounts of Cd than other tissues. The histopathology of liver and intestine underwent changes with different Cd concentrations, including hepatocyte hypertrophy, aggregation of blood cells, sinusoids, lipidosis, necrosis of hepatic tissues, the erosion of villi, necrosis in the mucosal layer, the appearance of vacuoles in the lamina propria, hyperplasia, and swelling of goblet cells. Moreover, the core gut microbiota existed in the intestinal microorganism and did not change as Cd concentration increased. However, the diversity of intestinal microorganism was significantly reduced compared with that of the control sample. The present results indicated that C. a. gibelio exposed to Cd suffered toxicity, and Cd could affect the biodiversity of the intestinal microbiota of C. a. gibelio.

Ultrasonic Pretreatment Combined with Dry-State Glycation Reduced the Immunoglobulin E/Immunoglobulin G-Binding Ability of α-Lactalbumin Revealed by High-Resolution Mass Spectrometry.

Bovine α-lactalbumin (α-LA) is one of major food allergens in cow's milk. The present work sought to research the effects of ultrasonic pretreatment combined with dry heating-induced glycation between α-LA and galactose on the immunoglobulin E (IgE)/immunoglobulin G (IgG)-binding ability and glycation extent of α-LA, determined by inhibition enzyme-linked immunosorbent assay and high-resolution mass spectrometry, respectively. The IgE/IgG-binding ability of glycated α-LA was significantly decreased as a result of ultrasonic pretreatment, while the average molecular weight, incorporation ratio (IR) value, location and number of glycation sites, and degree of substitution per peptide (DSP) value were elevated. When the mixtures of α-LA and galactose were pretreated by ultrasonication at 150 W/cm2, glycated α-LA possesses seven glycation sites, the highest IR and DSP values, and the lowest IgE/IgG-binding ability. Therefore, the decrease in the IgE/IgG-binding ability of α-LA depends upon not only the shielding effect of the linear epitope found to be caused by the glycation of K13, K16, K58, K93, and K98 sites but also the intensified glycation extent, which reflected in the increase of the IR value, the number of glycation sites, and the DSP value. Moreover, allergenic proteins and monosaccharides pretreated by ultrasonication and then followed by dry-state glycation were revealed as a promising way of achieving lower allergenicity of proteins in food processing.

Improved Antioxidant Activity and Glycation of α-Lactalbumin after Ultrasonic Pretreatment Revealed by High-Resolution Mass Spectrometry.

High-resolution mass spectrometry was performed to investigate the relationship between bovine α-lactalbumin (α-LA) subjected to ultrasonication and glycation treatment with respect to antioxidant activity. After α-LA was pretreated by ultrasonication combined with glycation, the treated α-LA showed low intrinsic fluorescence emission and high antioxidant activity at increased ultrasonic power levels. Prior to ultrasonic pretreatment, three glycated sites were identified, whereas the number of glycation sites was increased to four, four, five, and six after ultrasonic power at 60, 90, 120, and 150 W/cm2, respectively, for 15 min. Thus, no obvious difference was found among the glycation sites at the ultrasonic power of 60 and 90 W/cm2. The average degree of substitution per peptide molecule of α-LA was used to evaluate the glycation level per glycation site. All the samples pretreated by ultrasonication exhibited a higher glycation level compared with the untreated samples. Ultrasonic power at 150 W/cm2 showed the most highly enhanced glycation extent and antioxidant activity. Therefore, the intensified glycation extent and the conformational changes of protein were responsible for the increase of antioxidant activity of α-LA. Moreover, high-resolution mass spectrometry is an efficient technique to understand the mechanism of the improved antioxidant activity.