The Mechanism Of Low-Frequency Ultrasonic Combined With Glycation Modification To Reduce The Allergenicity Of β-Lactoglobulin

Cow milk is rich in a variety of protein,fats,carbohydrates,vitamins and minerals,It is a nutritious food with excellent functional properties.As a food base,it is widely used in infant milk powder.However,due to the allergenic properties of cow’s milk,it has brought harm to the health and life of allergic patients.Therefore,reducing the allergenicity of milk is a key issue that needs to be resolved urgently.In this paper,the main allergen of milk,β-lactoglobulin（β-Lg）,was studied by spectroscopy and mass spectrometry.The effects of low-frequency ultrasonic combined with glycation modification on the structure ofβ-Lg were studied from the aspects of intact protein and digestive products.ELISA,histamine release and animal model were used to study the effects of low-frequency ultrasonic combined with glycation modification on the structure ofβ-Lg.The main conclusions of this paper as follows:（1）The effects of low-frequency ultrasound on the structure,antioxidant activity and Ig G/Ig E binding ability ofβ-Lg digestion products were studied by spectroscopy,chromatography and ELISA.Low-frequency ultrasonic promoted the hydrolysis ofβ-Lg and produced more digested products.After in vitro gastrointestinal digestion,the intrinsic fluorescence intensity and antioxidant activity were increased.Nativeβ-Lg was resistant to gastric digestion and retained its allergenicity.Low-frequency ultrasonic promoted the production of peptides（strong immunogenicity）in digested products,and the Ig G/Ig E binding ability increases after gastric digestion.In the subsequent digestion of the gastrointestinal tract,ultrasonic promotes the production of more small molecule peptides and reduces the Ig G/Ig E binding ability.Low-frequency ultrasonicβ-Lg digestion products have increased antioxidant activity and decreased Ig G/Ig E binding capacity.（2）The effect of low-frequency ultrasonic on the antioxidant capacity ofβ-Lg digestion products were measured by mass spectrometry and cell experiments.The results were as follows.Four fractions（F1,F2,F3,F4）were separated by Sephadex G-25 gel filtration column（G-25）,and each fraction was showed a considerable ABTS·⁺scavenging ability.Low-frequency ultrasonic promotes the hydrolysis ofβ-Lg,and after digestion,the F4 fraction with the strongest antioxidant activity was obtained.Cell experiment results showed that the F4 fraction increases the activities of superoxide dismutase,catalase,and glutathione peroxidase,and is concentration dependent.It can have good cellular antioxidant activity against H₂O₂-induced oxidative Hep G2 cell model.Low-frequency ultrasonic pretreatment greatly improves the antioxidant capacity of F4 fraction.（3）The effect of low-frequency ultrasonic on the allergenic potential and linear epitopes ofβ-Lg digestion products was studied.GI digestion of ultrasonicatedβ-lg was fractionated into four fractions（F1,F2,F3,F4）by Sephadex G-25 gel filtration column,and the fractions have different allergenic potentials.The low-frequency ultrasonic treatment ofβ-Lg has the lowest allergenicity after digestion in the gastrointestinal tract,which depends on the changes in the structure ofβ-Lg by low-frequency ultrasonic and the promotion ofβ-Lg hydrolysis,thereby producing a large number of small molecule peptides,the Ig E activity and basophil histamine release were significantly reduced.HPLC-MS/MS analysis shows that low-frequency ultrasound reduces the content of immunogenic peptides inβ-Lg digestion products.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,and thus have a smaller molecular weight.Low-frequency ultrasonic reduces the allergenicity ofβ-Lg digestion products.（4）β-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.Low-frequency ultrasonic combined with glycation modification significantly reduced the Ig E/Ig G-binding capacity,and the release ofβ-hexosaminidase,histamine and interleukin-6.The decrease in the allergenicity ofβ-Lg depended not only on the glycation of Lys47、Lys60、Lys83、Lys91 and Lys135 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 low-frequency ultrasonic combined with glycation modification is a promising method for decreasing the allergenic potential of allergic proteins.（5）The effects of low-frequency ultrasonic combined with glycation modification on the structure,allergenicity and human microbial community ofβ-Lg digestion products were studied by spectroscopy,chromatography,enzyme-linked immunosorbent assay and basophil histamine release test.Low-frequency ultrasonic combined with glycation modification significantly reduced the Ig G/Ig E binding activity,and the release ofβ-hexosaminidase,histamine and interleukin-6,accompanied with changes in the structures of intestinal flora.The decrease in the allergenicity ofβ-Lg depended not only on the low digestibility of glycatedβ-Lg,which led to the decrease of some peptides with complete immunogenicity,but also the glycation has a masking effect on allergen epitopes ofβ-Lg.Meanwhile,low-frequency ultrasonic combined with glycation modification can change the structural of intestinal flora,such as Bacteroidota,Fusobacteriota,Enterobacteriaceae to Bacteroidaceae,Ruminococcaceae,Bacteroides and Faecalibacterium.This result indicated that low-frequency ultrasonic combined with glycation changes the structure ofβ-Lg digested products,reduces the Ig G/Ig E binding capacity and affects the structure of the human intestinal microflora.（6）To explore the effect of low-frequency ultrasonic combined with glycation modifiedβ-Lg on the allergenicity of mice through in vivo experiments in mice.Using ultrasonic combined with glycation modifiedβ-Lg sensitized mice to measure the serum immune factor levels in mice,it can be concluded that low-frequency ultrasonic combined with glycation modification can significantly reduce the mice’s spleen index and allergic symptoms scores,the content of Ig E in the mouse serum was greatly reduced,increased the secretion level of IFN-γ（Th1）and reduced the secretion level of IL-4（Th2）.The results of 16S r RNA high-throughput sequencing showed thatβ-Lg modified by low-frequency ultrasonic combined with glycation did not change the diversity of the intestinal flora of mice,but it could affect the structure of the intestinal flora.At the phylum level,decrease the Bacteroidetes and increase the relative abundance of Firmicutes;at the family level,increase the relative abundance of Lactobacillaceae.The results show that low-frequency ultrasonic combined with glycation modifiedβ-Lg can significantly affect the immune symptoms of mice and the structure of the intestinal flora.