Effect And Mechanism Of Pulsed Electric Fields And Ultrasound Combined With Glycation On The Allergenicity Of Proteins

Food allergy is an abnormal immune response in digestive system or whole body induced by a certain food or food additive.It includes IgE-induced and non-IgE-induced immune responses.Food allergy could cause gastrointestinal disorder,urticarial,asthma and even allergic shock.The incidence of food allergy has been increasing in recent years.Preliminary statistics show that 2.5%-5%adults and 6%-8%children are allergic to food.Therefore,reducing the allergenicity of food is the key problem that need to be solved.Meanwhile,it is significant to reduce the incidence of food allergy and improve the safety of processed food by seeking food processing technology and studying the mechanism of reducing food allergy.This study assessed the main allergens in hen eggs and bovine milk,ovalbumin(OVA)and β-lactoglobulin(β-Lg).The effect of pulsed electric fields(PEF)and ultrasound combined with glycation on the structure and allergenicity of OVA and βLg was investigated by using immunologic,spectroscopic,mass spectroscopic and proteomic methods.The mechanism of reduced allergenicity of β-Lg induced by PEF and ultrasound combined with glycation was explored at macro and molecular level.The main results are as following:(1)The effect of PEF treatment on allergenicity and structure of OVA was investigated by using immunologic and spectroscopic methods.The changes in allergenicity of OVA induced by PEF treatment were evaluated by IgG and IgE binding abilities using the rabbits’ antisera and egg allergy patients’ sera obtained by animal assay.The structural changes of OVA were determined by conventional spectroscopy.The results showed that the antisera from rabbits were of high efficiency and specificity.When OVA were treated at low electric field intensity(≤ 25 kV/cm)or for short time(≤60 μs),the secondary structure was nearly retained while the IgG and IgE binding capacities gradually increased due to the partial unfolding of OVA.This was reflected in the increase in free SH content,surface hydrophobicity and UV absorption at 280 nm.However,However,when OVA were treated at high electric field intensity(≥ 30 kV/cm)or for long time(≥ 120μs),the IgG and IgE binding abilities significantly reduced due to the refolding and aggregation of OVA.This was also reflected in the decrease in free SH content,surface hydrophobicity,UV absorption and α-helix content.(2)The influence of ultrasound treatment on allergenicity and structure of OVA was studied by using immunologic and spectroscopic methods.The results indicated that ultrasound treatment significantly enhanced the allergenicity of OVA.The secondary structure of OVA almost retained after ultrasound treatment at 200 W.The free sulphydryl content,surface hydrophobicity,UV absorption at 280 nm were gradually increased when the power was improved from 400 to 600 W,implying the occurrence of molecule unfolding.When the power was 800 W,the binding ability was slightly decreased associated with the aggregation of OVA molecule.The free sulphydryl content,surface hydrophobicity,UV absorption at 280 nm and α-helix content were decreased while βsheet content was increased.(3)The effect of ultrasound pretreatment combined with glycation on the allergenicity and structure of OVA was investigated by using immunologic and spectroscopic methods.The results suggested that ultrasound pretreatment promoted the increase of molecular weight of glycated OVA and glycation after ultrasound pretreatment significantly reduced the IgG and IgE binding abilities of OVA.It significantly changed the secondary and tertiary structure of OVA,reflecting in the increase of α-helix content and UV absorption at 280 nm,and decrease of β-sheet content,surface hydrophobicity and fluorescence emission intensity with red-shift of λmax.(4)The influence of ultrasound pretreatment combined with glycation on the oxidation activity,emulsifiability and foamability of OVA and the changes in allergenicity of OVA during in vitro digestion were studied.The oxidation activity was evaluated by measuring reducing power,DPPH radical-scavenging activity and TEAC.The results showed that ultrasound pretreatment combined with glycation significantly increased the oxidation activity,emulsifiability and foamability of OVA.After gastric and intestinal digestion,the allergenicity of OVA induced by ultrasound pretreatment combined with glycation always remained at a very low level.It indicated that ultrasound pretreatment combined with glycation could be a good method to decrease the allergenicity of OVA.(5)There are a lot of post-translational modification sites in OVA molecule.It may make OVA isomers have different allergenicity.Therefore,OVA was isolated and purified by isoelectric precipitation,ammonium sulfate precipitation and size exclusion chromatography,and the OVA isomer were identified by ion exchange chromatographymass spectrometry(IXC-MS).The results showed that 172 OVA isoforms were identified while 84 ovomucoid(OVM)isoforms were characterized.Moreover,the PTMs sites and composition of saccharide chain at N292 have significant influence on the pI of OVA isoforms.It provides the basis for subsequent study about the effect of PTMs on the allergenicity of OVA isoforms.(6)The mechanism of ultrasound pretreatment combined with dry-state glycation reduced the allergenicity of β-Lg was explored at macro and molecular level by using immunologic,spectroscopic,mass spectroscopic and proteomic methods.The results implied that glycation with namnose after ultrasound pretreatment significantly reduced the allergenicity of β-Lg by the modification of linear epitopes through glycation and the destruction of conformational epitopes through changing tertiary structure of β-Lg.Besides,ultrasound pretreatment promoted the decrease in allergenicity of β-Lg by increasing glycation sites and extent.(7)The mechanism of PEF pretreatment combined with glycation decreased the allergenicity of β-Lg was investigated at macro and molecular level by using immunologic,spectroscopic,mass spectroscopic and proteomic methods.The results showed that PEF pretreatment combined with glycation significantly reduced the allergenicity of β-Lg.It dramatically changed the tertiary structure of β-Lg,reflecting in the increase of UV absorption at 280 nm and decrease of surface hydrophobicity and intrinsic fluorescence intensity with a red shift of λ max.Moreover,the results indicated that PEF pretreatment at 25 kV/cm for 60 μs promoted the reduction of allergenicity by increasing glycation sites and extent of β-Lg,whereas single PEF treatment under the same condition markedly enhanced the allergenicity by partially unfolding β-Lg.