Effect Of In Vitro Gastrointestinal Digestion On Allergens Of Aquatic Products

The incidence of food allergy created an increasing problem and is considered as a major concern of food safety. Among the 8 major sources of food allergens, two species (crustaceans and fish) are blong to sea food. Although many researches on the mechanism of food allergens sensitizing hypersensitivity reactions have been carried out, the mechanisms by which food allergens sensitize an individual and the stability of food allergen in alimentary tract remains unclear. Most of them are thought to sensitize via the gastrointestinal (GI) tract. In consequence, it is generally regarded that an allergen must endure degradation in the GI tract. Stability to digestion has been proposed as one of the indication to assess the allergenic potential of transgenic foods. However, recently, the validity of digestion stability as a criterion for protein allergenicity assessment has encountered some criticism. Thus, further study is needed to evaluate the stability of allergens to digestion.Crustaceans such as mud crab (Scylla serrata), Chinese mitten crab (Eriocheir sinensis), grass prawn (Penaeus monodon) and white-leg shrimp (Penaeus vannamei) are regarded as a delicacy for its higher meat content and ??ition. These crustaceans have a promising fishery industry in China and even over the world. Crucian carp (Carassius auratus) and silver carp (Hypophthalmichthys molitrix) also have a promising freshwater fishery industry and possess an important economical value. Because of the increasing consumption of such kinds of sea foods, the report about occurrence of hypersensitive reactions is also extending. However, researches that systemically study the in vitro gastrointestinal digestion on sea food are scarce. In the present study, the digestive stability of the major crustacean allergen (tropomyosin, TM) and fish allergen (parvalbumin, Pav), factors that may affect the digestion result and the effect of gastrointestinal digestion on allergenicity of allergens were all analyzed for the development of hypoallergenic seafood products in the future.First, standard simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared based on the United States Pharmacopeia. The stability of allergenic protein tropomyosin and parvalbumin to SGF and SIF was analyzed, respectively. The results were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. In SGF system, crustacean allergen tropomyosin was relatively resistant to pepsin, while fish allergen parvalbumin was rapidly degraded within a short period of time. In SIF, TM can be decomposed to different degrees, resulting several fragments, while parvalbumin was stable in the SIF, hardly to be decomposed.Next, digestion of crustaceas myofibrillar proteins and fish muscle proteins by SGF and SIF were also carried out. The stability of allergenic protein and other proteins was compared and tested, respectively. In SGF, myosin heavy chain (MHC) and actin was relatively more rapidly decomposed than TM; in SIF, crab actin is more stable than TM, while shrimp actin is more degradable than TM. The results showed that food allergens are not necessarily more resistant to digestion than other proteins. To clarify the identity of the degraded protein fragments, Western blot using specific polyclonal antibodies against Actin, TM and Pav were carried out. The results also suggested the degradation pattern of allergen by SGF and SIF was almost not affected by the presence of other proteins.Considering the real physiological condition of human, the pepsin/protein ratios and pH were studied to reflect the influence to stability of allergens. It is identified that the apparent susceptibility of allergens to proteolysis is dependent on the pepsin to allergen ratio. TM possess??elatively higher stability, even the pepsin to allergen ratio reached 10/1, some stable degraded fragments could still be identified. At pH≥5, the digestion of allergenic proteins by SGF was completely suppressed.Finally, IgE immunoblotting and inhibition ELISA were conducted to detect the IgE-binding epitopes in the digestion products and the allergenicity of TM by proteinases. Results showed that compared with undigested sample, the allergenicity of all digested samples was reduced to different degrees, especially by trypsin. As for crab, chymotrypsin is more effective in reducing allergencity of TM than trypsin, while for shrimp, both enzymes revealed similar and faint effects. These results suggested that proteinase treatment is an appropriate way in reducing TM allergenicity and is helpful for future development of hypoallergenic seafood products.