Development Of Method For Evaluating Citrus Allergenicity And The Mechanism Of Adverse Food Reaction Induced By Satasuma Mandarin

Citrus is popular because of its unique flavor and is one of the most important fruits in the world.Although citrus is rich in various healthy functional compounds,some people still suffer adverse food reactions(Shanghuo,allergies,etc.)when eating citrus.And this sensory experience has gradually become one of the indicators for people to evaluate fruit quality.However,due to the weak research foundation,high requirement for both research facilities and interdisciplinary thinking,the progress on adverse food reactions caused by intake of citrus has been backward.With the rapid development of pomology,molecular biology,analytical chemistry,medical science and the integration of disciplines,this traditional scientific problem of Shanghuo caused by the intake of satsuma has revived.Based on this current situation,a method for screening low allergenic citrus germplasm resources has been established to select low allergenic citrus varieties,so as to provide edible citrus for special populations with allergies.Afterwards the sensitization of different citrus varieties is evaluated and the mechanism of allergen proteins accumulation is explored.The reasons of adverse reactions in human body is also explained from the perspective of citrus allergy.Finally,through the established epidemiological research platform,combined with the previous citrus allergen proceedings,the mechanism of adverse reactions on human beings caused by the intake of citrus fruits is proposed.The main results are as follows:1.The effects of tissue type,harvest maturity and genetic factors on the expression of genes that related to citrus fruit allergies remain poorly understood.In the present study,a multiplex real-time PCR assay was developed to monitor the expression of citrus allergen genes individually with the advantages of much fewer sample requirements and simultaneously multiple target genes detection.Gene specific primer pairs and Taqman probes of three citrus allergen genes Cit s 1.01,Cit s 2.01,Cit s 3.01 and the house-keeping gene ?-actin were designed based on gene sequence differences.The PCR results showed that differential expression patterns were found during the ripening process.The expression levels of Cit s 3.01 were much higher than those of Cit s 1.01 and Cit s 2.01 in both peel and pulp tissues among ten citrus cultivars.Data suggested that Kao Phuang Pummelo could be safely consumed with a potential low risk in allergenicity.2.The inherent allergens of citrus fruits,such as Cit s 1,Cit s 2,Cit s 3 can causeallergic reactions.A better understanding of the genetic factors(cultivar to cultivar)affecting the allergenic potential of citrus fruits would be beneficial for further identification of hypoallergenic genotypes.In the present study,an immunoblotting quantification approach was adopted to assess the potential allergenicity of 21 citrus cultivars,including nine subgroups(tangerine,satsuma,orange,pummelo,grapefruit,lemon,kumquat,tangor and tangelo).To prepare highly sensitive and specific rabbit polyclonal antibodies,antigenicity of purified rCit s 1.01,rCit s 2.01 and rCit s 3.01 peptides were enhanced with high epitope density in a single protein molecule.The data integration of three citrus allergen quantifications demonstrated that the four pummelo cultivars(Kao Phuang Pummelo,Wanbai Pummelo,Shatian Pummelo,and Guanxi Pummelo)were potentially hypoallergenic,compared with other 8 subgroups.Moreover,the immunological analyses with sera of allergic subjects revealed that Shatian Pummelo and Guanxi Pummelo showed the lowest immunoreactivity in 8 representative citrus cultivars.These potential hypoallergenic genotypes are of great significance to not only allergic consumers but also citrus breeders in the genetic improvement of hypoallergenic citrus as breeding resources.3.Excessive consumption of Satsuma mandarin(Citrus unshiu)to induce an adverse food reaction characterized by itching or a sore throat,gum swelling and mouth ulcer,which is referred to as Shang huo in traditional Chinese medicine which affects a large segment of the population.In this study,we defined such a symptom as a Satsuma-induced syndrome(SIS)and applied a standard questionnaire to quantitatively evaluate SIS by recruiting volunteers for Satsuma mandarin consumption in a double-blind placebo-controlled manner.The results showed that SIS was characterized with symptoms affecting the oral cavity syndrome,gastrointestinal tract,respiratory tract and skin either individually or in combination.72.7% participants with Satsuma challenge suffer SIS after three days,but only 30% participants in the placebo group.Based on the analysis of the hemogram,cytokines,IgE,and IgG,SIS was defined as an immune-mediated food adverse reaction,which could induce low-grade inflammation.The plasmic metabolomic profiles indicated that 15-Hete and 12-Hete were the vital inflammatory metabolites associated with SIS,and PC(20:5(5Z,8Z,11 Z,14Z,17Z)/16:1(9Z))might be the potential biomarker of SIS symptom.In summary,a low allergenic citrus germplasm screening method was established and applied for the identification of a grapefruit citrus as a potential hypoallergenic citrusvariety.At the same time,it was found that the citrus allergen was not induced by the intake of satsuma.In addition,through metabolomics and data analysis of blood samples from people participating in the double-blind placebo-controlled food challenge test,it was indicated that satsuma induced low-grade inflammation in the human body by the arachidonic acid metabolic pathway.Nowadays,with the development of scientific concepts in citrus fruits quality,nutrition and health protection,and the emerging of noval research technologies,the research on adverse food reaction caused by citrus intake in this study is an important supplement to improve the quality of citrus fruits and can guide people to consume citrus healthily and safely.