The Allergenicity Changes Of Ultrasound Assistance Luteolin Treated β-lactoglobulin And Its Impact On Human Intestinal Flora

Milk is rich in protein and is an important nutritional supplement;however,the presence of many allergens in milk causes clinical adverse reactions in the skin,gastrointestinal tract and respiratory tract when ingested by some people.How to reduce the allergenicity of milk is an urgent problem to be solved at home and abroad.This paper focuses on the main allergens in milk β-Lactoglobulin(Bovine β-Lactoglobulin BLG)was treated with ultrasound assisted luteolin(LUT).Firstly,its structure and allergenicity changes were analyzed using spectroscopy,enzyme linked immunosorbent assay(ELISA),and KU812 cell degranulation test;Then,the processed BLG was subjected to simulated digestion in vitro to analyze the structure and allergenicity changes of the digested product;Finally,in vitro fermentation technology,chromatography technology,and 16 S rRNA gene amplification sequencing technology were used to determine changes in short chain fatty acids(SCFAs)and human intestinal flora,analyze the impact of digestive products on human intestinal flora and SCFAs,and elaborate the correlation between allergenicity and intestinal flora SCFAs.The following conclusions were drawn:(1)The effects of ultrasound assisted LUT on the structure and allergenicity of BLG were studied using Fourier transform infrared spectroscopy,circular dichroism spectroscopy,ELISA,and KU812 cell degranulation techniques.The results showed that the main interaction between BLG and LUT before and after ultrasound treatment was hydrophobic.Ultrasound assisted LUT treatment significantly changed the secondary and tertiary structures of BLG,and decreased the IgE(Immunoglobulin E)binding ability of BLG,as well as the release ability of interleukin-6(IL-6),interleukin-4(IL-4),and histamine(HIS)in KU812 cells.Therefore,ultrasound assisted LUT treatment changes the conformational epitope of BLG and reduces its allergenicity.Secondary ultrasound assisted LUT treatment has the best desensitization effect on BLG.(2)The changes in the structure and allergenicity of BLG digestion products treated with ultrasound assisted LUT were studied using in vitro simulated digestion,high performance liquid chromatography,and cell experiments.The results showed that the hydrolysis degree of BLG treated with ultrasound-assisted LUT was lower than that of untreated BLG,resulting in a more compact structure of the digestion product,which was characterized by a decrease in the intrinsic fluorescence intensity of the digestion product,a significant decrease in the content of irregular curling,and a significant increase in the content of β-sheet.After ultrasound assisted LUT treatment,the IgE binding ability of the hydrolysate of BLG and the release ability of IL-6 and HIS in KU812 cells decreased.The structure of ultrasound assisted LUT treated BLG after digestion,is relatively compact,and LUT can cover the allergic epitopes of BLG and reduce its allergenicity,among which,the allergenicity of BLG treated with secondary ultrasound assisted LUT after digestion is the lowest.(3)Techniques such as in vitro digestion,16 S rRNA gene amplification sequencing technology,and chromatography were used to study the effects of BLG digestion products treated with secondary ultrasound assisted LUT on human intestinal flora and SCFAs content,and to analyze the correlation between IL-6,HIS,and SCFAs and intestinal flora.The results showed that the BLG digestion products after ultrasound assisted LUT treatment had a certain impact on the structure of human intestinal flora.At the genus classification level,the natural BLG group significantly reduced the abundance of Rombousia,Bacteroides,Megamonas,Collinsella,and Bifidobacterium,and the content of acetic acid,propionic acid,and butyric acid significantly decreased.The bacterial community structure and relative abundance of the BLG group treated with secondary ultrasound assisted LUT were similar to those of the control group,manifested in restoring the reduced relative abundance of Bacteroides,Megamonas,and Collinsella in the BLG group,and significantly increasing the content of acetic acid,propionic acid,and butyric acid.In addition,SCFAs were significantly positively correlated with the relative abundance of Bacteroides,Megamonas,and Collinsella(P< 0.05),while HIS and IL-6 were negatively correlated with the relative abundance of acetic acid,propionic acid,and butyric acid producing Bacteroides,Megamonas,and Collinsella.That is,the BLG digestion products treated with secondary ultrasound assisted LUT will affect the structures of Bacteroides,Megamonas,and Collinsella,producing more acetic acid,propionic acid,and butyric acid,which may reduce the secretion of HIS and IL-6,thereby reducing the allergenicity of BLG.