Effects Of Ultrasound-assisted Glycation On ?-Lg Allergenicity During Simulated Digestion In Vitro

Food allergy can cause an abnormal response of immune system,which in turn triggers a series of clinical symptomsafter the human body ingesting food allergens,including skin,gastrointestinal and respiratory tract.Cow's milk(CM),have been widely used as the primary source of nutrition for humans,especially for infants.CM is one of the eight major allergens,which is mainly due to the presence of?-lactoglobulin(?-Lg)or other milk proteins,in this regard,the percent of causing cow's milk allergy(CMA)being 82%from?-Lg.At present,there is no effective treatment for CMA.Strictly avoiding the intake of allergens is the only and most effective method,but which will inevitably lead to nutritional deficiency.Therefore,it is urgent to develop new technologies to reduce the allergenicity of CM.Previous studies have shown that ultrasound-assisted glycation(UAG)modification can greatly reduce the allergenicity of?-Lg.However,the allergenicity and epitopes are still unknown after UAG modified?-Lg enters the human body for digestion and absorption.In this paper,?-Lg was used as the object to study the ultrasound-assisted glycation(UAG)on?-Lg allergenicity.The IgG/IgE binding property was as an indicator to evaluate the allergenicity of?-Lg by the method of indirect competitive ELISA and the response surface method was used to optimize the conditions for UAG to reduce the allergenicity of?-Lg.The method of spectroscopy,mass spectrometry was used to study the the effect of UAG on the structure and explain the mechanism of reducing the allergenicity of?-Lg from the molecular level.The changes of digestion characteristics,structure and allergenicity were analyzed through simulated digestion in vitro ELISA,and KU812 cell model.The conclusions are as follows:(1)The conditions for UAG modification reduce the allergenicity of?-Lg were optimized through the response surface experiment,and the structural changes were studyed.The optimum program(i.e.,ultrasonic time,weight ratio,and ultrasonic power of 15 min,1:1,and 300 W,respectively)was determined by response surface methodology(RSM).The results of spectroscopy and mass spectroscopy showed that UAG significantly changed the secondary structure of?-Lg,causing the reduction of free amino group,surface hydrophobicity,fluorescence intensity,and red shift of the maximum emission wavelength(?_max).The pretreatment of ultrasound increased the glycation sites of?-Lg,and the DSP value of glycated peptides was significantly increased.?-Lg without ultrasonic pretreatment had 16 glycation sites,after ultrasonic pretreatment had 19 glycation sites.It indicated that the decrease of?-Lg allergenicity was due to the masking of linear epitopes and the damaged of conformational epitopes.(2)The method of indirect competitive ELISA,scanning electron microscopy(SEM),dynamic light scattering(DLS)and mass spectrometry were used to study the UAG on changes in digestive properties and allergenicity of?-Lg during digestion in vitro.The results showed that the allergenicity and the degree of hydrolysis of UAG modified?-Lg(?-Lg-Lac-300)had a reduced during gastrointestinal digestion.Glycation limited?-Lg to form a smaller particle structure during gastric digestion,then aggregated during intestinal digestion.The fluorescence intensity first increased and then decreased,and the?max appeared red shift.The results of mass spectrometry showed that the glycation sites of?-Lg-Lac-300 were reduced by 4 after gastric digestion,and there was no changed after intestinal digestion.It indicated that UAG modification can effectively reduce the allergenicity of?-Lg during digestion.(3)The cytokines were detected to evaluate the allergenicity of digestion products of UAG modified?-Lg by using the KU812 cell model.The results showed that the degranulation ability of KU812 cells activated by digestion product of?-Lg-Lac-300 was reduced gradually during the digestion,and the contents of cytokines(such as:histamine,TNF-?,IL-4,IL-6,IL-10,etc.)also decreased successively,it was always lower than native?-Lg.Therefore,UAG modification reduced the allergenicity of?-Lg during the digestion,but the final digestion product still has allergenicity.