Cai Qiufeng

Effects of processing methods on the immunoreactivity of silver carp parvalbumin

Parvalbumin(PV)is the major allergen in fish products,which could induce serious IgE-mediated food anaphylaxis when sensitive people encountered with it.A variety of clinical symptoms including angioedema,respiratory symptoms,gastrointestinal symptoms,and even death could be caused after ingestion or contact with fish or fish products or even just inhalation of fish cooking vapors.Besides monitoring the allergens in foods,reducing allergenicity of fish products is another important way to decrease the incidence of fish allergy.Therefore,the purpose of this study was to analyze the change of immunoreactivity and digestion stability of parvalbumin after different food processings.Four processing methods including surimi preparation,roast,fried,high pressured methods were evaluated in this study.Tricine-SDS-PAGE and Western-blotting were used to determine the variation of contents,immunoreactivity and digested stability of parvalbumin in the processed products.The results showed that,though surimi prepartion,roast,and fried processing could increase the gastric stability,high pressure processing could effectively reduce the content and immunoreactivity of parvalbumin in fish products.And the significance of these findings remains to be established.

Purification and polyclonal antibody preparation of parvalbumin from silver carp(Hypophthalmichthy molitrix) skeletal muscle

Parvalbumin is the major allergen of fish species. Investigation into this protein is beneficial not only to detecting allergen,but also to producing aquatic products with low allergenicity. In the present study,parvalbumin from the skeletal muscle of silver carp (Hypophthalmichthy molitrix) was first purified to homogeneity by homogenization,centrifugation,heat treatment extraction and gel filtration chromatography on Superdex 75. Purified parvalbumin revealed three protein bands with molecular mass of 12,14 and 24 ku,respectively,as detected by Tricine-SDS-PAGE under non-reducing conditions while under reducing conditions,only a band corresponding to 12 ku was detected. Western-blotting using anti-frog parvalbumin monoclonal antibody (PARV-19) positively reacted with the three protein bands of 12,14 and 24 ku,suggesting they are different forms of parvalbumin. A polyclonal rabbit anti-silver carp parvalbumin antibody was prepared and immunoglobulin G (IgG) was purified by Protein A Sepharose affinity column. Dot-blot revealed that the antibody reacted with parvalbumin even after dilution to 1/51200,suggesting its higher titer. Western-blotting analysis indicated that parvalbumins from four fishes (common carp,silver carp,crucian carp,sea bream) can all be detected by the polyclonal antibody specifically.

Studies on the immunoreactivity of the major allergen tropomyosin in mantis shrimp (Squilla oratoria).

Crustacean is one of the eight kinds of allergen sources in coastal areas,which causes the IgE-mediated hypersensitive reactions with clinical manifestations including urticaria,angioedema,asthma,and even fatal anaphylaxis. Tropomyosin (TM) is the major allergen of decapod crustaceans with highly conserved amino acid sequences. The purpose of this study is to confirm whether TM is a major cross-reactive allergen in mantis shrimp (Squilla oratoria) which is taxonomically distinct from decapods and largely consumed as a delicacy in China. Quantification of TM in different species of crustaceans was also conducted. Muscle sample from mantis shrimp was homogenized with phosphate buffer to prepare heated extracts and separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A protein band with molecular mass of about 36 ku was detected by IgE-immunoblotting by all of the sera with crustacean allergy,suggesting it is the major allergen of mantis shrimp. This protein was further purified to homogeneity by acetone powder preparation,isoelectric point precipitation,ammonium sulfate fractionation (40%-60% saturation),and heat treatment. Purified protein was demonstrated to be TM by Western blot using polyclonal antibody against TM from Chinese mitten crab (Eriocheir sinensis). Heated extracts from other crustaceans including mud crab (Scylla serrata),Pacific white shrimp (Penaeus vannamei) and short necked clam (Venerupis variegata) were also prepared,and the cross-reactivity of TM from mantis shrimp with TMs from these crustaceans was confirmed by inhibition immunoblotting using polyclonal antibody against TM and inhibition ELISA using sera with crustacean allergy. Quantification by ELISA using polyclonal antibody against TM revealed that TM content in mantis shrimp muscle is much lower than that in Pacific white shrimp and mud crab muscle,which was about 45 times lower in mantis shrimp muscle than that in Pacific white shrimp muscle. However,its allergenicity seems equivalent to other decapod crustaceans as showed by inhibition ELISA using sera with crustacean allergy. This may be due to the degradation of TM in mantis shrimp by its endogenous serine proteinases and cathepsins which destroyed the IgG-binding epitope but not the IgE-binding epitopes. In conclusion,this study demonstrated that allergenicity of mantis shrimp is almost equivalent to decapods and TM is the major allergen in mantis shrimp with high cross-reactivity to decapod crustaceans.