Potensi Hidrolisat Protein Belalang Sawah (Oxya chinensis) sebagai Produk Hipoalergenik dan Agen Antiinflamasi

Abstract

Belalang sawah (Oxya chinensis) merupakan salah satu sumber protein alternatif yang prospektif dikembangkan sebagai pangan masa depan karena kandungan proteinnya tinggi, profil asam aminonya lengkap, dan ketersediaannya melimpah di berbagai wilayah Indonesia. Namun demikian, pemanfaatannya masih terkendala oleh dua masalah utama, yaitu (i) neofobia terhadap bentuk serangga utuh, dan (ii) adanya risiko alergi akibat kandungan protein alergen. Pengolahan belalang melalui hidrolisis enzimatis dapat menjadi solusi kedua kendala tersebut, yaitu dengan mengolahnya menjadi hidrolisat protein yang berpotensi sebagai pangan olahan intermediat yang bersifat hipoalergenik dan sekaligus memiliki potensi biofungsional. Penelitian ini bertujuan untuk: (1) memperoleh metode isolasi dan hidrolisis protein belalang yang paling efektif melalui systematic literature review (SLR); (2) mengevaluasi proses isolasi protein serta menganalisis karakteristik kimia belalang mentah, tepung, dan isolat protein belalang sawah; (3) menganalisis pengaruh hidrolisis enzimatis terhadap alergenisitas protein menggunakan serum penderita alergi serta mengidentifikasi protein/peptida alergeniknya; dan (4) mengevaluasi aktivitas antiinflamasi hidrolisat protein belalang secara in vitro serta mengidentifikasi senyawa peptida bioaktifnya. Penelitian ini dilaksanakan dalam empat tahap. Tahap pertama berupa systematic literature review (SLR) untuk menetapkan metode isolasi dan kondisi hidrolisis protein serangga yang paling efektif. Tahap kedua adalah isolasi protein dengan variasi pH pengendapan untuk menentukan titik isoelektrik serta karakterisasi kimia belalang mentah, tepung belalang, dan isolat protein belalang. Tahap ketiga yaitu proses hidrolisis enzimatis dengan enzim alkalase 1-5% E/S, kemudian dilakukan analisis alergenisitasnya melalui uji ELISA dengan kit krustasea, elektroforesis dengan SDS-PAGE, dan immunoblotting menggunakan serum penderita alergi mengonsumsi belalang, serta mengidentifikasi peptida alergenik dengan kromatografi cair-spektrometri massa resolusi tinggi (LC-HRMS). Tahap keempat mengevaluasi aktivitas antiinflamasi hidrolisat protein belalang sawah secara in vitro pada sel makrofag RAW 264.7 yang diinduksi lipopolisakarida (LPS), didahului fraksinasi peptida dengan membran ultrafiltrasi dan diakhiri identifikasi peptida bioaktif menggunakan LC-HRMS. Berdasarkan hasil SLR, isolasi protein serangga paling efektif dilakukan melalui ekstraksi pada kondisi basa yang dilanjutkan dengan presipitasi pada titik isoelektrik, serta didukung proses penghilangan lemak untuk meningkatkan kemurnian protein. Proses hidrolisis paling efektif menggunakan enzim alkalase yang mampu menghasilkan derajat hidrolisis tinggi, sedangkan ultrafiltrasi ditetapkan sebagai metode fraksinasi peptida. Kajian tersebut juga menunjukkan bahwa hidrolisat protein serangga berpotensi memiliki aktivitas antioksidan dan antiinflamasi. Temuan ini menjadi dasar metodologis bagi pelaksanaan tahap penelitian selanjutnya. Hasil isolasi protein menunjukkan bahwa isolat protein belalang sawah yang diekstraksi pada pH 10,5 dengan suhu 40°C selama 60 menit memiliki kadar protein 92,78% basis kering (bk), meningkat dari 69,66% bk pada tepung belalang atau setara dengan peningkatan relatif sekitar 33%. Kondisi ekstraksi pada suhu 40°C menghasilkan rendemen yang lebih tinggi dibandingkan ekstraksi pada suhu kamar. Titik isoelektrik protein belalang sawah berada pada pH 4,0. Proses isolasi juga memperkaya profil asam amino, terutama kelompok asam amino hidrofobik, rantai cabang, dan bermuatan yang berperan penting dalam nilai gizi dan bioaktivitas. Proses hidrolisis enzimatis pada isolat protein belalang sawah menggunakan alkalase pada konsentrasi 1–5% E/S menghasilkan derajat hidrolisis 69,83–74,48% yang meningkat seiring naiknya konsentrasi enzim. Hidrolisis enzimatis dengan rasio enzim alkalase 1-5% E/S terbukti efektif menurunkan alergenisitas sebesar 98,33-99,19% dari sampel isolat protein. Profil elektroforesis menunjukkan hilangnya pita protein berukuran sedang hingga besar pada hidrolisat, termasuk degradasi pita pada kisaran bobot molekul tropomiosin. Hasil analisis immunoblotting menunjukkan bahwa serum penderita alergi reaktif terhadap belalang mentah dan tepung belalang, tetapi kehilangan reaktivitas sepenuhnya terhadap seluruh sampel hidrolisat. Temuan ini menegaskan bahwa hidrolisis enzimatis merupakan langkah kunci untuk menghasilkan produk yang hipoalergenik. Analisis LC-HRMS dan bioinformatika telah mengidentifikasi empat kandidat peptida alergenik dari protein belalang sawah berbobot molekul 14-17 kDa. Hidrolisat protein belalang sawah juga menunjukkan potensi sebagai agen antiinflamasi. Fraksinasi menghasilkan tiga fraksi berdasarkan ukuran molekul, dan rentang konsentrasi 12,5–50 µg/mL terbukti aman terhadap sel. Ketiga fraksi menurunkan produksi oksida nitrat secara bergantung dosis. Pada model pra-inkubasi dengan fraksi berukuran kecil (< 3 kDa) menunjukkan penghambatan oksida nitrat (NO) tertinggi yaitu 70,52% yang merepresentasikan skenario konsumsi preventif. Pada model ko-inkubasi menunjukkan penghambatan NO tertinggi sebesar 40,96% pada fraksi 3-10 kDa. Analisis LC-HRMS terhadap fraksi tersebut mengidentifikasi empat kandidat sekuens peptida yang diprediksi bersifat antiinflamasi. Kebaruan yang dihasilkan dari penelitian ini adalah: (1) diperolehnya hidrolisat protein belalang sawah yang berpotensi dikembangkan sebagai pangan hipoalergenik yang dibuktikan dengan adanya penurunan imunoreaktivitas, (2) teridentifikasi empat kandidat peptida yang berasal dari protein alergen belalang sawah; (3) hidrolisat protein belalang sawah memiliki aktivitas antiinflamasi in vitro yang dibuktikan dengan adanya kemampuan penghambatan oksida nitrat (NO) pada sel makrofag RAW 264.7 dengan model ko-inkubasi dan pra-inkubasi; dan (4) teridentifikasi empat kandidat peptida bioaktif antiinflamasi pada hidrolisat protein belalang sawah. Hasil ini menunjukkan bahwa hidrolisis enzimatis berperan ganda, yaitu menurunkan pengenalan epitop oleh IgE sekaligus melepaskan peptida bioaktif dari struktur protein induknya. Secara praktis, hidrolisat protein belalang sawah berpotensi dikembangkan sebagai bahan pangan hipoalergenik dan agen antiinflamasi, sehingga mendukung pemanfaatan belalang sebagai sumber protein alternatif yang berkelanjutan.

The rice field grasshopper (Oxya chinensis) is a promising alternative protein source for future food production due to its high protein content, complete amino acid profile, and abundant availability across various regions of Indonesia. However, their utilization is still hampered by two main issues: (i) neophobia toward whole insects, and (ii) the risk of allergies due to the presence of allergenic proteins. Processing grasshoppers through enzymatic hydrolysis can address both of these challenges by converting them into protein hydrolysates that have the potential to serve as hypoallergenic intermediate food products with biofunctional properties. This study aimed to identify the most effective methods for grasshopper protein isolation and enzymatic hydrolysis through a systematic literature review (SLR); to evaluate the protein isolation process and analyze the chemical characteristics of raw grasshoppers, grasshopper flour, and protein isolate from the rice grasshopper; to investigate the effect of enzymatic hydrolysis on protein immunoreactivity using sera from allergic patients and to identify the allergenic proteins/peptides; and to evaluate the in vitro anti-inflammatory activity of grasshopper protein hydrolysates and identify their bioactive peptide compounds. The study was conducted in four stages. The first stage involved a systematic literature review (SLR) to determine the most effective methods for isolating and hydrolyzing insect proteins. The second stage involved protein isolation using varying precipitation pH levels to determine the isoelectric point, as well as the chemical characterization of raw grasshoppers, grasshopper meal, and grasshopper protein isolates. The third stage involved enzymatic hydrolysis using 1–5% E/S alkalase, followed by analysis of allergenicity via ELISA using a crustacean test kit, SDS-PAGE electrophoresis, and immunoblotting using serum from patients allergic to grasshoppers, as well as the identification of allergenic peptides using LC-HRMS. The fourth stage evaluates the in vitro anti-inflammatory activity of the hydrolysate on lipopolysaccharide-induced RAW 264.7 macrophages, preceded by peptide fractionation using an ultrafiltration membrane and followed by the identification of bioactive peptides using LC-HRMS. Based on the SLR results, insect protein isolation was most effectively performed through extraction under alkaline conditions followed by precipitation at the isoelectric point, supported by a defatting process to enhance protein purity. The hydrolysis process was most effective using alkalase enzyme, which is capable of producing a high degree of hydrolysis, whereas ultrafiltration was established as the peptide fractionation method. The review also indicated that insect protein hydrolysates potentially possess antioxidant and anti-inflammatory activities. These findings served as the methodological basis for the implementation of the subsequent research stages. The protein isolation results showed that the rice field grasshopper protein isolate, extracted at pH 10.5 and 40°C for 60 minutes, had a protein content of 92.78% (db), up from 69.66% db in grasshopper flour—equivalent to a relative increase of approximately 33%. Extraction at 40°C yielded a higher protein yield compared to extraction at room temperature. The isoelectric point of the rice-field grasshopper protein was at pH 4.0. The isolation process also enriched the amino acid profile, particularly the hydrophobic, branched-chain, and charged amino acid groups that play important roles in nutritional value and bioactivity. Enzymatic hydrolysis of rice field grasshopper protein isolates using alkalase at concentrations of 1–5% (w/v) resulted in a degree of hydrolysis of 69.83–74.48%, which increased as the enzyme concentration rose. Enzymatic hydrolysis with an alcalase enzyme ratio of 1-5% E/S has been proven to be effective in reducing allergenicity by 98.33-99.19% of protein isolate samples. The electrophoresis profile showed the disappearance of medium- to large-sized protein bands in the hydrolysate, including degradation of the band within the molecular-weight range of tropomyosin. Immunoblotting analysis showed that the sera of allergy sufferers reacted with raw grasshopper and grasshopper flour but completely lost their reactivity toward all hydrolysate samples. These findings confirm that enzymatic hydrolysis, and not merely flouring, is the key step in producing a hypoallergenic grasshopper-based product. LC-HRMS analysis combined with bioinformatics identified four candidate allergenic peptides in the rice-field grasshopper. Hydrolysates of rice field grasshopper protein also show potential as anti-inflammatory agents. Fractionation yielded three fractions based on molecular weight, and a concentration range of 12.5–50 µg/mL was found to be safe for cells. All three fractions reduced nitric oxide production in a dose-dependent manner. In the pre-incubation model with the small-molecule-weight fraction (< 3 kDa), the highest inhibition of nitric oxide (NO) was observed at 70.52%, representing a preventive consumption scenario. In the co-incubation model, the highest NO inhibition of 40.96% was observed in the 3–10 kDa fraction. LC-HRMS analysis of this fraction identified four candidate peptide sequences predicted to have anti-inflammatory properties. The novel findings of this study are: (1) the production of a rice field grasshopper protein hydrolysate with potential for development as a hypoallergenic food, as evidenced by a reduction in immunoreactivity; (2) the identification of four peptide candidates derived from rice field grasshopper allergenic proteins; (3) the rice field grasshopper protein hydrolysate exhibits in vitro anti-inflammatory activity, as evidenced by its ability to inhibit nitric oxide (NO) in RAW 264.7 macrophages using co-incubation and pre-incubation models; and (4) the identification of four candidate bioactive anti-inflammatory peptides in the rice field grasshopper protein hydrolysate. These results indicate that enzymatic hydrolysis plays a dual role: it reduces epitope recognition by IgE while simultaneously releasing bioactive peptides from the parent protein structure. In practical terms, rice field grasshopper protein hydrolysate has the potential to be developed as a hypoallergenic food ingredient and an anti-inflammatory agent, thereby supporting the use of grasshoppers as a sustainable alternative protein source.