Study On The Allergenicity Of Peanut Protein Ara H2 And Development Of Hypoallergenic Products

Peanuts,as one of the few characteristic agricultural products with advantages in trade,consumption,and yield among China’s grain and oil crops,are an important source of high-quality oil and protein supplementation for the body.However,peanuts have been recognized by the Food and Agriculture Organization of the United Nations as one of the eight major allergic foods.Ara h2,as the main allergen in peanuts,can cause allergic symptoms involved in the skin,mouth,and digestive tract.Therefore,reducing or even eliminating the allergenicity of peanut allergens has become an urgent scientific problem in the world.Glycation is a safe and effective processing technique that alters the structure and functional properties of proteins while further affecting their allergenicity.However,the effects of different reducing sugar glycation on the structure,glycation degree and allergenicity of Ara h2,as well as the correlation among them,are unknown.Moreover,there are currently no relevant reports on the mechanism of the allergenicity changes of glycated Ara h2 based on high-resolution mass spectrometry technology at the molecular level.Therefore,the paper aim to study the influence of five reducing sugars(ribose,galactose,glucose,fructose and lactose)on the structure,glycation degree and allergenicity of glycated Ara h2.The structural changes of Ara h2 glycated with different reducing sugars were studied based on spectroscopy technology;Indirect competitive ELISA and cell model were used to analyze the allergenicity changes of glycated Ara h2;The glycation sites were identified by high resolution mass spectrometry technology;The mechanism of the decreased allergenicity of Ara h2 was explored from the molecular level;Finally,the optimization conditions of the preparation process of hypoallergenic peanut protein powder were explored.The main research conclusions are as follows:(1)The effect of five reducing sugars modification on the structure of Ara h2 was studied.Ribose,galactose,glucose,fructose and lactose glycated Ara h2 are denoted as Ara h2-Rib,Ara h2-Gal,Ara h2-Glu,Ara h2-Fru and Ara h2-Lac,respectively.The results showed that the molecular weight of Ara h2 increased after glycation with the five reducing sugars,and the molecular weight of Ara h2-Rib was larger.It was found that five reducing sugar modifications caused changes in the secondary and tertiary structures of Ara h2 by ultraviolet spectroscopy,fluorescence spectroscopy,circular binary spectroscopy and infrared spectroscopy.Among them,the increase of UV absorption value,the decrease of fluorescence intensity and the the highest α-helix content were occurred in Ara h2-Rib.(2)The effects of five reducing sugars modification on the allergenicity of Ara h2 were studied,and the mechanism was analyzed based on high resolution mass spectrometry.The results showed that the Ig G/Ig E binding ability of other glycated Ara h2 samples significantly reduced(P<0.05)except Ara h2-Lac.Among these samples,Ara h2-Rib had the lowest Ig G/Ig E binding rates and cytokine release level,and galactose as an aldohexose,Ara h2-Gal had lower allergenicity than Ara h2-Fru.14,9,7,7 and 3 glycation sites were identified in Ara h2-Rib,Ara h2-Gal,Ara h2-Glu,Ara h2-Fru and Ara h2-Lac,respectively,and K52,R115 and R143 were only found in Ara h2-Rib.Mechanism analysis showed that 10 glycation sites with high DSP values in Ara h2-Rib were located in Ig E linear epitopes of Ara h2,which caused epitopes to be masked,resulting in the lowest allergenicity of Ara h2-Rib compared to other Ara h2 glycation samples.The allergenicity of Ara h2-Gal was lower than that of Ara h2-Fru might be due to more glycation sites located in the Ig E linear epitopes of Ara h2.(3)Single factor and orthogonal test were used to optimize the process of hypoallergenic peanut protein powder.The results showed that when the mass ratio of peanut protein to ribose was 1:1,the reaction time was 2.5 h,the p H of sample solution was 7.0 and the reaction temperature was 70℃,the desensitization effect of peanut protein powder was the best.When the binding rate of natural peanut protein was82.80%,the Ig E binding rate of glycated peanut protein powder was 18.15%,which was close to the optimal result in orthogonal test.