The Effect Of Ultrasound-assisted Glycation On The Allergenicity Of α-lactalbumin And The Development Of Hypoallergenic Protein

Cow’s milk（CM）is rich in nutrients and easy to be absorbed by the human body.It not only provides energy for our bodies but also meets our nutritional needs.CMα-lactalbumin（α-LA）is the main whey protein,but it is also one of the earliest allergens that young children are exposed to.For people who are allergic to CM,it seems to be the best choice to completely refuse to drink CM,but the nutrition CM provides is indispensable to people,especially the essential amino acids it contains.If remove CM from the diet of patients with CMPA,it is bound to cause its nutritional imbalance.Therefore,it is urgent to develop new technologies to reduce the allergenicity of CM.Previous studies have shown that glycation can reduce the allergenicity of ultrasound pretreatment ofα-LA,but there are few studies on the effect of different hexose on the structure and allergenicity of ultrasound-pretreated ofα-LA.The relationship between the allergenicity and digestion characteristics of ultrasound-assisted glycatedα-LA during in vitro simulated digestion also needs to be further studied.Therefore,in this paper,α-LA was used as the research object,andα-LA was modified by ultrasound in coordination with different reducing sugars.The structures of glycatedα-LA and its digested products were characterized by high resolution mass spectrometry,SDS-PAGE,circular dichroism,fluorescence and infrared spectroscopy.Indirect competitive ELISA method was used to investigate the effect of ultrasound combined with glycation on the relationship betweenα-LA allergenicity and digestive properties,with the binding capacity of IgE and IgG as indicators.The allergenicity of glycatedα-LA and its digestive products was analyzed.The preparation process of hypoallergenic whey protein was optimized by single factor experiment and orthogonal experiment.The conclusions are as follows:（1）The effects of different reducing sugars（glucose,allose,mannose）on the structure and allergenicity ofα-LA pretreated by ultrasound wave were studied using the binding ability of IgE and IgG as indexes.The results show that:the molecular weight increased and the free amino content ofα-LA decreased due to glycation.The sequence of hexose glycation activity was D-allose>D-mannose>D-glucose.Glycation affectsα-LA secondary structure,α-helix structure decreases andβ-folding increases.The spherical structure ofα-LA was gradually expanded by glycation,andα-LA was unfolded,which resulted in the decrease of surface hydrophobicity,fluorescence intensity,and the redshift ofλ_max.The conformational modification ofα-LA by D-allose was the strongest.The IgG and IgE binding ability ofα-LA was reduced by glycation,and ultrasound pretreatment promoted the reduction,among which D-allose had the best effect on reducing the allergenicity ofα-LA.（2）The allergenicity ofα-LA modified by ultrasound-assisted glycation decreased significantly during simulated digestion in vitro.Because D-allose glycation sites are close to linear allergy epitopes,there are only 9 glycation sites,and the number ofα-LA glycation sites increased to 10 after ultrasound pretreatment,thus blocking the recognition of linear allergy epitopes by IgG/IgE antibodies.In addition,the hydrolysis sites of digestive enzymes are just on their epitopes,and the cleavage of digestive enzymes destroys the allergenicity epitopes,so the allergenicity of glycatedα-LA and its digests is reduced.The hydrolysis degree ofα-LA modified by ultrasound-assisted glycation was decreased during the simulated digestion in vitro.This was because the acquisition of monosaccharides affected the cleavage of peptide bonds near the glycation site by digestive enzymes,thus reducing the digestibility of glycationα-LA.In addition,the tertiary structure ofα-LA changed with the digestion process,exposing more fluorescent chromophores,increasing the intensity of fluorescence,and gradually increasing the polarity of the microenvironment where the fluorophore resides.（3）Ultrasound-assisted glycation reaction can reduce the binding ability of WPI to IgE and thus reduce its potential allergenicity,but the inhibition degree obtained by adjusting the intensity of various conditions is limited.The optimal conditions obtained through the single factor test and orthogonal test are as follows:the ultrasound power was 130 W,the ultrasound time was 15 min,the glycation reaction temperature was 65℃,and the reaction time was 3 h.Based on the best process parameters to do a verification test,the results showed that the OD450 value of WPI-N was 0.207,and the OD450 value of glycated WPI was 1.064,that is,the IgE inhibition rate was 26.376%,indicating that the effect of reducing allergenicity was the best.