Study Of The Mechanism Of Probiotics Alleviating Tropomyosin-induced Food Allergy Based On Gut Microbiota-host Metabolism Pathway

Food allergy is a global food safety problem,and seafood allergy has been increasing in the last 2 decades affecting nearly 2.4%of allergic patients.Tropomyosin(TM)is the major antigen of White shrimp(Litopenaeus vannamei)which can provoke IgE-mediated hypersensitivity reactions.Previous studies indicated that bifidobacteria and lactic acid bacteria can inhibit inflammation and alleviate food allergy by gut microflora reconstruction,intestinal metabolism and mucosal immune response regulation.In the present study,we first purified TM from Litopenaeus vannamei by ammonium sulfate fractionation precipitation and isoelectric precipitation.TM was identified by LC-MS and quantified by BCA.To establish the TM allergy and probiotic therapy mouse model,Balb/c mice were sensitized and challenged by intraperitoneal(IP)injection of TM and treated by orally administrating of Bifidobacterium longum subsp.or Bacillus coagulans 09.712 for 20 consecutive days from day14 to 35.Mice body weight changes,anaphylaxis response and diarrhea were evaluated,serum IgE and hiatamine were determined and cytokine profile of spleen cells were analyzed.Mucosal mast cells and goblet cells were detected by Histological analysis.Eosinophils and CD4+ T cells were identified by immunohistochemistry.Apoptotic cells were observed by TUNEL.The expression levels of mRNA in small intestine,large intestine and spleen were also detected to explore the mechanism.Metabolites in fecal samples were extracted and analyzed by gas chromatography and time-of-flight mass spectrometry(GC-TOF-MS).Fecal microbiota composition was analyzed by 16S rRNA metagenomic sequencing.The main findings are as follows:1.TM could be purified effectively by adjusting the extraction buffer to pH 6.5 to 7.5 and then adding ammonium sulfate to approximately 30%saturation.The protein was identified as Lit v 1 tropomyosin(Litopenaeus vannamei)with the molecular weight of 32.8 kD and isoelectric point of 4.75 by liquid chromatography-mass spectrometry.The concentration of TM was about 43 ?g/?L determined by a BCA Protein Assay Kit.2.Female and male Balb/c mice were sensitized and challenged by intraperitoneal(IP)injection of TM with different dosage.Results showed that female Balb/c mice had severer allergic symptoms and body weight losses.Serum IgE can only be determined in female mice and the level of serum HIS were higher in female mice.Th2 type cytokines(IL-4)were higher in female mice than that in male mice,and Thl type cytokines(IL-10,IFN-?)were lower.All these results showed that high-dosage TM-allergy female mouse model was successfully established.3.Therapeutic group were orally administered with Bifidobacterium longum subsp.or Bacillus coagulans 09.712 for 20 consecutive days from day 14 to 35.The allergic symptoms were alleviated,serum levels of IgE and histamine as well as Th2 cytokines were decreased,while Thl cytokines were increased,indicating that these two probiotics can alleviate TM induced allergy.4.Histological.changes showed that intestinal lesions in TM-sensitized mice were presented,mainly manifested in the disorder of intestinal villus,with some intestinal villus even shedding into the intestinal lumen.Furthermore,ulceration can be seen in certain position.Allergy related cells such as goblet cells,mast cells,CD4+ T cells increased and eosinophil infiltrated,and increased apoptosis were also observed.Histological changes mainly present in small intestine rather than big intestine.These allergy-induced intestinal lesions can be relieved by Bifidobacterium longum subsp.and Bacillus coagulans 09.712 administration.5.Metabolites of mice intestinal contents were analyzed by GC-TOF-MS.Analysis indicated that Bifidobacterium longum subsp.and Bacillus coagulans 09.712 can alleviated TM-induced seafood allergy by influencing arginine metabolism pathway,which mainly involves glutamine,alanine,aspartic acid and glutamic acid metabolism.6.16S amplicon sequencing shows that intestinal microflora changes might be associated with TM-induced food allergy.Lactobacillaceae and Bacteroidales decreased significantly while Lachnospiraceae and Desulfovibrionaceae increased.Bifidobacterium longum subsp.and Bacillus coagulans 9.712 can regulate intestinal flora structure to alleviate TM-induced food allergy.In conclusion,TM may induce intestinal mucosal immune response by altering the intestinal flora and its metabolic pathway,disrupting Th1/Th2 balance and skewing to Th2 polarization.These results provide a favorable theoretical basis for the mechanism study of TM-induced food allergy and therapeutic effect of probiotics,offer potential target for the prevention and treatment of food allergy.