Perubahan Profil Berat Molekul dan Alergenisitas Shellfish Akibat Proses Pengolahan dengan Panas

Abstract

Shellfish merupakan salah satu kelompok pangan hasil laut yang diketahui menjadi sumber alergi bagi sebagian orang dan dikenali memberikan dampak yang nyata bagi kasus alergi di dunia. Shellfish dapat menyebabkan alergi pada orang atopik karena adanya kandungan protein alergen yang mampu berikatan dengan salah satu mediator penyebab reaksi hipersensitif yaitu IgE. Beberapa alergen yang terdapat pada kelompok shellfish yang telah diketahui adalah tropomiosin, arginin kinase, myosin light chain 2, sarcoplasmic calcium binding protein (SCBP), dan troponin 1. Konsentrasi protein alergen dari masing-masing spesies shellfish bervariasi. Bioaktivitas suatu protein, termasuk alergen dapat berubah karena adanya perubahan struktur molekul penyusunnya. Perubahan struktur dapat disebabkan proses pengolahan yang melibatkan panas, tekanan, fermentasi, dan interaksi dengan bahan lainnya. Proses pengolahan berpeluang dalam mengubah maupun merusak struktur epitop yang dimiliki oleh protein alergen sehingga kemampuannya dalam berikatan dengan IgE menjadi menurun bahkan hilang. Perubahan ini mempunyai pengaruh yang potensial terhadap perubahan alergenisitas suatu protein alergen. Proses pengolahan yang umum dilakukan terhadap produk hasil laut antara lain adalah perebusan, penggorengan dan pengalengan. Meskipun alergen dari berbagai jenis shellfish di dunia telah diidentifikasi, akan tetapi masih sedikit sekali studi mengenai pengaruh pengolahan terhadap profil ekstrak protein berbagai jenis shellfish yang ada di Indonesia serta sifat alergenisitasnya. Tujuan dari penelitian ini adalah mengidentifikasi keberadaan berbagai jenis protein alergen pada beberapa jenis shellfish yang banyak menyebabkan kejadian alergi di Indonesia dan perubahan reaktivitasnya akibat proses pengolahan dengan panas dan tekanan. Penelitian ini terbagi menjadi tiga tahap. Tahap pertama dilakukan untuk mendapatkan ekstrak protein 4 jenis shellfish (udang vaname, kepiting bakau, kerang hijau, dan cumi-cumi) yang banyak menyebabkan alergi dan banyak dikonsumsi baik segar maupun yang diolah dengan 3 jenis pengolahan (perebusan, penggorengan, dan autoklaf). Tahap kedua adalah karakterisasi ekstrak protein yang diperoleh meliputi analisis konsentrasi protein terlarut dan profil berat molekul protein. Tahap ketiga adalah pengujian efek pengolahan penggorengan, perebusan, dan autoklaf terhadap alergenisitas protein shellfish. Pengujian alergenisitas protein shellfish selama proses pengolahan secara kualitatif menggunakan immunoblotting dan kuantitatif menggunakan ELISA. Hasil penelitian menunjukkan bahwa proses pengolahan berpengaruh terhadap perubahan konsentrasi komposisi gizi makro keempat jenis shellfish. Proses pengolahan berpengaruh terhadap penurunan kadar protein shellfish segar secara berfluktuatif, sedangkan pengolahan juga menurunkan solubilitas protein keempat jenis shellfish secara signifikan. Berdasarkan hasil uji elektroforesis SDS-PAGE ditemukan pita protein diduga alergen dengan berat molekul 33-38 kDa yang diduga sebagai alergen tropomiosin, 40 kDa diduga sebagai arginine kinase, dan 20 kDa diduga sebagai myosin light chain. Proses pengolahan mereduksi jumlah pita protein shellfish segar dalam rentang BM 10-260 kDa, bahkan proses autoklaf mereduksi pita protein pada rentang BM tersebut secara keseluruhan pada keempat jenis shellfish. Hasil uji reaktivitas IgE menunjukkan bahwa proses pengolahan autoklaf menurunkan alergenisitas udang vaname secara signifikan, secara berfluktuatif pada kerang hijau dan cumi-cumi, namun tidak menurunkan reaktivitas IgE terhadap ekstrak kepiting bakau. Hal ini terjadi karena dalam proses pengolahan kepiting dilakukan tanpa memisahkan daging dari cangkangnya dan hanya dibuka cangkang penutup karapasnya. Meski demikian, proses autoklaf tidak menghilangkan alergenisitas shellfish secara keseluruhan diduga karena masih terdapat epitop linier yang bersifat reaktif dengan berat molekul yang kecil. Adapun proses penggorengan justru meningkatkan reaktivitas IgE pada kepiting bakau dan cumi-cumi. Hasil immunoblotting masih menunjukkan adanya pita protein reaktif dengan berat molekul 43 kDa pada keempat jenis shellfish yang diberi perlakuan perebusan, sedangkan pada proses autoklaf tidak ditemui pita protein yang reaktif seperti pada hasil SDS PAGE. Secara keseluruhan, proses autoklaf merupakan proses yang paling potensial digunakan dalam menurunkan alergenisitas udang vaname dan berpeluang digunakan dalam menghasilkan produk pangan hipoalergenik.

Shellfish is one of the seafood groups that was known to be a source of allergies for some people and have a real effect on allergy cases in the world. Shellfish can cause allergies in atopic people due to the presence of allergen proteins that are able to bind to one of the mediators that cause hypersensitivity reactions, namely IgE. Some of the known allergens in shellfish groups are tropomyosin, arginine kinase, myosin light chain 2, sarcoplasmic calcium binding protein (SCBP), and troponin 1. The allergen protein concentration of each shellfish species varies. The bioactivity of a protein, including allergens, can change due to changes in its structure. Structural changes can be caused by a food processing that involves heat, pressure, fermentation, and interactions with other materials. The food processing has the opportunity to change or damage the epitope structure of the allergen protein which the ability to bind to IgE decreases or even disappears. These changes have a potential effect on changes in the allergenicity of an allergen protein. Common processing for marine products include boiling, frying and autoclaving. Although allergens from various types of shellfish in the world have been identified, there are still very few studies on the effect of processing on the protein extract profiles of various types of shellfish in Indonesia and their allergenicity. The purpose of this study was to identify the presence of various types of protein allergens in several types of shellfish which cause a lot of allergies in Indonesia and changes in their reactivity due to heat and pressure processing. This research divided into three stages. The first stage was carried out to obtain protein extracts of four types of shellfish (white leg shrimp, mud crab, green mussle, and squid) which cause a lot of allergies and are mostly produced both fresh and processed with three types of processing (boiling, frying, and autoclaving). The second stage was the characterization of the protein extract. The third stage was analyze the effect of frying, boiling, and autoclaving processing on shellfish protein allergenicity. Analysis of the effect of processing on shellfish protein allergenicity qualitatively conducted by immunoblotting and quantitatively by ELISA. The results showed that the processing had a fluctuative effect on macro nutrients compotition especially on decreasing the protein content of fresh shellfish, while the processing also significantly decreased the protein solubility of the four shellfish species. Based on the results of the SDS-PAGE electrophoresis test, it was found that protein bands suspected to be allergens with a molecular weight of 33-38 kDa were suspected to be tropomyosin allergen, 40 kDa were suspected to be arginine kinase, and 20 kDa were suspected to be myosin light chain. The processing reduces the number of protein bands of fresh shellfish in the range of BM 10-260 kDa, even the autoclaving reduces protein bands in the range of BM as a whole in the four types of shellfish. The results of the IgE reactivity test showed that the autoclave process significantly reduced the allergenicity of vannamei shrimp, fluctuative on green mussel, and squid, but did not significantly reduce the IgE reactivity of mud crabs. This happens because in the crab processing was done without separating the meat from the shell and only opening the shell of the carapace. However, the autoclave process did not completely eliminate shellfish allergenicity, presumably because there were still reactive linier epitopes with smaller molecular weights. The frying process actually increases IgE reactivity in mud crab and squid. The results of immunoblotting showed the presence of reactive protein bands with a molecular weight of 43 kDa in the four types of shellfish treated with boiling, while in the autoclave process no reactive protein bands were found as in the SDS PAGE results. Overall, the autoclave process is the most potential process in reducing vanname shrimp allergenicity and has the potential to be used in producing hypoallergenic food.