Preparation of Hypoallergenic Kamaboko: Removal of Parvalbumin and Collagen from Fish Meat by Water-Bleaching, Mechanical Grindi

日本調理科学会誌 Vol.Preparation of Hypoallergenic 50，No. 4，141～150（2017） Kamaboko from〔Original Fish Meat paper〕 Preparation of Hypoallergenic Kamaboko: Removal of Parvalbumin and Collagen from Fish Meat by Water-bleaching, Mechanical Grinding and Extraction with Potassium Chloride Kaori Kurata*1§ Masaya Itoh*2,3 Masahiro Matsumiya*2 Akira Dobashi*1 Yasuharu Itagaki*4,5 Kazuo Shiomi*6 Marine products account for 40% of the dietary animal protein consumed in Japan. For people with seafood allergies that prevent the consumption of any seafood, production of hypoallergenic processed seafood products is essential. The major allergens in fish meat are parvalbumin（PA） and collagen. Here, the allergenicity of eight kinds of commercial fish meat cake, known as kamaboko, was assessed using enzyme-linked immunosorbent assay. Boiled and steamed kamaboko products were found to be less allergenic than the raw fish meat. We removed PA and collagen from fish meat by water-bleaching and mechanical grinding, respectively and produced a starting material that is abundant in actmyosin, an ingredient important in the gelation of kamaboko. Hypoallergenic kamaboko was then prepared by adding salt to the fish meat and steaming. Keyword：food allergy, fish allergy, kamaboko, water-bleaching, mechanical grinding fibers （Muntener et al., 1995）. The main allergen isolated INTRODUCTION from cod （Elsayed and Aas, 1971; Elsayed and Apold, The number of people suffering from food allergies has 1983）, salmon （Lindstrom et al., 1996）, Jack mackerel been increasing in recent years（Sampson, 2004; Seheila et （Shiomi et al., 1998）, carp （Bugajska-Schretter et al., al., 2006）. Many types of foods can cause allergies, includ- 1999）, and Alaskan pollack（Van et al., 2004） was identi- ing eggs, milk, rice, soybeans, peanuts, fruit, and seafood. fied as a 12-kDa PA. PA is also known to show antigenic Seafood allergies have a major impact throughout the cross-reactivity between species（Bernhisel-Broadbent et world（Crespo et al., 1995; Iikura et al., 1999; Taylor et al., al., 1992; Pascual et al., 1992; Hilger et al., 2004; Van et 2004; Schiere et al., 2004; Rance et al., 2005）, particularly al., 2005）. in Japan where large quantities of seafood are consumed We previously identified Japanese patients with fish and marine products account for 40% of dietary animal allergies who reacted to another higher molecular weight protein. Thus, as nutrition is a major concern for people protein but not to PA. This protein was later identified as with allergies restricting them from consuming any sea- collagen, another major allergen in fish（Shiomi et al., food（Aldámiz-Echevarría et al., 2008）, the production of 1999; Hamada et al., 2001）. Collagen is a component in the hypoallergenic processed seafood products is essential. protein fraction of muscle tissue. It is insoluble in cold The major allergens in fish meat are parvalbumin （PA） water but dissolves easily in acetic acid and hot water. It and collagen. PA is a sarcoplasmic protein with a globular is composed of a 120-kDa α-chain and 240-kDa β-chain. structure that is extremely soluble in water（Aas and Allergenic molecules can be removed from fish prod- Elsayed, 1975）. The muscles of fish and amphibians con- ucts through chemical and physical processing. Water- tain relatively large quantities of PA （Gerday, 1982）, bleaching of fish meat results in the elution of water-solu- which plays an important role in the relaxation of muscle ble PA. In contrast, collagen, which is water-insoluble, cannot be removed by water-bleaching but is instead 1 * Tokyo University of Pharmacy and Life Sciences mechanically removed by grinding, a refining process that *2 Nihon University *3 Present Affiliation: Megmilk Snow Brand Co., Ltd. removes the skin and muscle. *4 Kanagawa Prefectural Institute of Public Health Actomyosin（AM） is one of the constituent proteins of *5 Present affiliation: Hokkaido Bunkyo University *6 Tokyo University of Marine Science and Technology myofibrils and is extracted from fish meat in potassium § Inquiry Tokyo University of Pharmacy and Life Sciences chloride（KCl） solution. Allergenicity to AM has not been 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan reported in people who are allergic to fish products. AM TEL 042（676）3095 FAX042（670）7067 E-mail：[email protected] mixtures can form a gel structure when treated with salt （141） 15 日本調理科学会誌 Vol. 50 No. 4（2017） followed by heating; this processing gives kamaboko its kamaboko product. Artificial colors and commonly used rich elasticity. We attempted to obtain a fraction that has food additives such as starch are not recorded in Table 1. abundant AM but lower PA or collagen in order to make Fish that are commonly used for the production of com- hypoallergenic kamabokos. In this study, this fraction was mercial kamaboko were obtained, and the edible portions termed the AM fraction. were separated from the whole fish and stored at -20℃. There are various processed seafood products in Japan. White croakers（ Pennahia argentata） caught in the East In this study, we focused on kamaboko as a traditional China Sea was supplied by Suzuhiro Co. Ltd.（Odawara, Japanese processed seafood product. Fish paste products Japan）. Alaskan pollack（ Gadus chalcogrammus）, classi- such as kamaboko are mainly produced by subjecting the fied as cod fish, was purchased from a fish supplier in fish paste （calledsurimi in Japanese）, consisting of mus- Kushiro. Japanese anchovy（ Engraulis japonicus）, classi- cle protein, blood, and fat, to a water-bleaching process in fied as blue fish, along with sardine and horse mackerel order to remove the components that hinder gelling of the （species unknown）, was provided by Kanagawa Prefec- product. Kamaboko products are classified into three tural Fisheries Technology Center（Miura, Japan）. types based on the production process: steamed, boiled, In addition, the frozen surimi of Alaskan Pollack that and fried. Typically, white fishes such as white croaker was processed at-sea immediately after harvest was pur- and cod, and occasionally, blue fishes such as sardine and chased from a fish supplier in Kushiro. This at-sea pro- Horse mackerel are used. We assessed the allergenicity of cessed surimi is of the highest quality grade, SA. commercial kamaboko products and found that steamed Serum collected from four subjects reactive to PA and kamaboko represents a potentially important hypoaller- three subjects reactive to collagen was respectively pooled genic food product. for the determination of immunoglobulin E（IgE） reactiv- We evaluated methods for removing collagen, either by ity（allergenicity） using enzyme-linked immunosorbent mechanical meat grinding or by a process that exploits assay（ELISA）. Pooled serum samples of these two aller- the differences in solubility between AM and collagen. genicity types were used because most cases of food aller- Based on our findings, we developed a novel method for gies are identified in children and sera is scarce. preparing hypoallergenic kamaboko with reduced levels of Development of a novel method to remove allergens PA and collagen from commercially available frozen PA is easily removed from fish meat due to its high surimi utilizing the addition of salt to the AM fraction fol- water-solubility. However, a process for the removal of lowed by steaming. collagen had not yet been developed. Thus, we established the method followed by water-bleaching, grinding, MATERIALS AND METHODS extracting with 0.6M KCl, dialysis, and salting out, and Materials investigated its potential to produce hypoallergenic Eight kinds of commercially available kamaboko were kamaboko with reduced levels of PA and collagen. purchased at a local supermarket in Kanagawa, Japan. AM, the gelation of which gives kamaboko its charac- Among these products, four were steamed（1-4）, two teristic gel strength, is highly soluble in aqueous solutions were boiled（1, 2）, and two were fried（1, 2）. Table 1 containing neutral salts such as KCl and NaCl. Thus, when shows the fish ingredients used in the production of each fish meat is extracted using a 0.6 M KCl aqueous solution, Table 1. Raw fish indredients in commercial kamaboko Raw fish ingredisnts Product White Cod Horse Sardine Other croaker fish mackerel Steamed-1 ＋＋－－ Steamed-2 －＋－－ Goldenthread, misc. Steamed-3 ＋－－－ Steamed-4 －＋－－ Misc. Boiled-1 ＋－－－ Misc. Boiled-2 －＋＋＋ Mackerel, Atka mackerel Fried-1 ＋－＋－ Tuna, misc. Fried-2 －－－＋ Glowbelly, Red mullet, misc. ＋, present; －, not present; misc., minor amounts of other fish products 16 （142） Preparation of Hypoallergenic Kamaboko from Fish Meat collagen cannot be extracted. Moreover, the protein mix- After water-bleaching, the meat was ground with a ture obtained by lyophilization of the supernatant likely commercial meat grinder（OMC-12; Ohmichi Industrial contains trace amounts of PA not removed by the water- Co. Ltd., Japan） according to the manufacturer’s instruc- bleaching process. Various proteins other than PA can be tions. The samples were ground using plates with succes- recovered from the solution by salting-out and subsequent sively smaller mesh diameters of 9.6, 6.4, 3.2 and 1.9 washing. mm. Determination of IgE reactivity of kamaboko and fresh For determination of IgG reactivity（antigenicity） using fish meat using fluorescent ELISA ELISA, a standard curve was generated with purified PA Samples of purchased kamaboko and the edible portions extracted from Pacific cod（ Gadus microcephalus） and of fish meat were homogenized for 1 min in a 5-fold vol- purified collagen extracted from Chum salmon（ Oncho- ume of 10 mM phosphate buffer （PB）（pH 7.0） or 0.5 M rhychus keta）. acetic acid. Each mixture was centrifuged（3,000 × g, 4 Determination of IgG reactivity using ELISA ℃, 20 min） and the supernatant was filtered through a Samples were homogenized for 1 min with a 5-fold vol- 5-μm syringe filter to obtain the fish extracts for evalua- ume of 10 mM PB（pH 7.0） or 0.5 M acetic acid.

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