Identifikasi dan karakterisasi Laktoferin susu kuda sebagai imunomodulator

NURLIYANI, Promotor Prof.Dr.Ir. Mochamad Adnan, M.Sc

2006 | Disertasi | S3 Ilmu Pertanian (Ilmu Pangan)

Fungsi bioaktif laktoferin susu kuda sejauh ini belum banyak diungkap, terma suk fungsi imunomodulatornya. Tujuan penelitian ini, yaitu untuk menentukan tipe glikan laktoferin susu kuda terkait dengan fungsi imunomodulatornya, dan mengevaluasi laktoferin susu kuda dan laktoferin susu sapi dalam respon imun humoral dan seluler. Laktoferin susu kuda dipurifikasi pada kolom penukar ion. Tipe glikan dianalisis dengan lektin spesifik. Pengujian respon imun humoral (respon IgA dan IgG serum dan cairan intestinal, serta sel-sel penghasil IgA) dan seluler (respon proliferatif, kadar IL-2 dan IL-4 dan aktivitas fagositosis), menggunakan mencit betina Balb/c umur 6-8 minggu yang diberi laktoferin susu kuda secara oral dengan dosis 0,0; 0,1; 0,5 dan 1,0 mg/ekor/hari selama 28 hari. Pengambilan sampel untuk uji respon imun humoral dan seluler, dilakukan pada hari ke 7; 14; 21; dan 28. Analisis IgA dan IgG, IL-2 dan IL-4 dengan metode ELISA. Analisis imunohistokimia dilakukan untuk mengetahui sel-sel penghasil IgA. Pengujian proliferasi limfosit dengan MTT, dan pengujian fagositosis in vitro digunakan partikel lateks. Hasil penelitian menunjukkan glikan laktoferin susu kuda bereaksi dengan lektin WGA bukan dengan Con-A. Respon IgG dan IgA serum mencit perlakuan laktoferin susu kuda 1,0 mg mencapai maksimum pada hari ke 21, sedangkan laktoferin susu sapi belum memberikan respon sampai hari ke 28. Respon subklas IgG1 dan IgG2b serum mencit perlakuan laktoferin susu kuda dosis 0,5 mg mulai hari ke 14 sampai ke 28 lebih besar daripada kontrol. Respon IgA dan IgG cairan intestinal perlakuan laktoferin susu kuda pada hari ke 21, lebih tinggi daripada perlakuan laktoferin susu sapi dan kontrol. Sel-sel positif penghasil IgA di jaringan intestinal lebih tinggi pada perlakuan laktoferin susu kuda dibanding kontrol. Indeks stimulasi limfosit perlak uan laktoferin susu kuda pada hari ke 14 lebih tinggi daripada kontrol dan perlakuan laktoferin susu sapi, tetapi indeks stimulasi pada hari ke 28 perlakuan laktoferin susu sapi lebih tinggi daripada perlakuan laktoferin susu kuda dan kontrol. Kadar IL-2, IL-4 kultur limfosit, dan IL-4 serum perlakuan laktoferin susu kuda lebih tinggi dibanding kontrol, sedangkan IL-2 serum hampir semuanya tidak terdeteksi. Jumlah sel fagosit pada mencit perlakuan laktoferin susu kuda lebih tinggi daripada kontrol. Kesimpulan penelitian ini adalah glikan laktoferin susu kuda merupakan tipe kompleks yang berpengaruh pada proliferasi limfosit terhadap mitogen. Laktoferin susu kuda dapat berperan sebagai imunomodulator dalam peningkatan respon imun humoral dan seluler. Respon imun mencit yang diiduksi laktoferin susu kuda lebih cepat daripada laktoferin susu sapi.

The bioactive function of horse milk lactoferrin has not been studied, particularly the horse milk lactoferrin as immunomodulator. The aims of this study were to determine the glycan type of horse milk lactoferrin related to its immunomodulatory function, and evaluate the horse and bovine milk lactoferrin as immunomodulator in humoral and cellular immune responses. Horse milk lactoferrin was purified on ion exchange chromatography. The glycan type was analyzed with specific lectin. Female Balb/c mice 6-8 weeks old, fed 0.0 (control); 0.1, 0.5 and 1.0 mg/mice/day of horse milk lactoferrin orally, in an assay period of 28 day. The humoral immune response (serum and intestinal fluids IgA and IgG, IgA producing cells in intestinal tissue), and cellular immune response (lymphocyte proliferation, levels of IL-2 and IL-4, and phagocytosis activity), were assayed on the 7th, 14th, 21st and 28th day. Immunoglobulin A and IgG, IL-2 and IL-4 in serum and lymphocyte culture were detected with ELISA, IgA producing cells was analyzed with IHC method, lymphocyte proliferation was assayed with MTT, and in vitro phagocytosis of peritoneal macrophages were assayed with latex beads. The results showed that glycan of horse milk lactoferrin reacted with WGA, but not with Con-A. On the day of 21st, responses of serum IgG and IgA of mice fed horse milk lactoferrin 1.0 mg were maximum, whereas mice fed bovine milk lactoferrin have not been showed these response on the 21 st -28th day. Responses of intestinal fluids IgA and IgG of mice fed horse milk lactoferrin were higher than mice fed bovine milk lactoferrin and control, on the 21st day. Responses of subclasses IgG1 and IgG2b of mice fed horse milk lactoferrin, were higher than the control, on the day of 14th – 28th. The IgA producing cells in the intestinal tissue of mice fed horse milk lactoferrin were higher than the control. Stimulation index of mice lymphocyte fed horse milk lactoferrin was higher than the control and bovine milk lactoferrin on the day of 14th, whereas stimulation index on the 28th day of mice lymphocyte fed bovine milk lactoferrin was higher than horse milk lactoferrin and the control. Level of IL-2, IL-4 in lymphocyte culture, and IL-4 in serum of mice fed horse milk lactoferrin, were higher than control. Serum IL-2 was undetectable. The phagocyte cells of mice fed horse milk lactoferrin was higher than bovine milk lactoferrin and the control. In conclusion, horse milk lactoferrin glycan was complex type that effect proliferative respon to mitogen. Horse milk lactoferrin can serve as immunomodulator in increasing humoral and cellular immune responses. The immune response that was induced by horse milk lactoferrin was faster than bovine milk lactoferrin.

Kata Kunci : Laktoferin Susu Kuda,Imunomodulator