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BIOSEPARASI, METODE HANDAL UNTUK EKSTRAKSI GASSERICIN A YANG DIPRODUKSI OLEH Lactobacillus gasseri LA39

Joni Kusnadi1,Tri Ardyati2, Ella Saparianti1

1Jurusan THP, Fakultas Tekn. Pertanian, Universitas Brawijaya 2Jurusan Biologi, Fakultas MIPA, Universitas Brawijaya

Telah dipresentasikan pada Seminar Nasional Stem Cell dan Hasil-hasil Penelitian Ilmu Hayati, LSIH Malang 10 Oktober 2009

ABSTRAK

Bakteriosin adalah protein antibakteri yang diproduksi oleh bakteri asam laktat,

karena mampu menghambat spesies lain, bakteriosin dapat digunakan sebagai biopreservatif untuk mengontrol bakteri patogen dan pembusuk. Untuk mempelajari sifat-sifat antibakteri, sifat kimia dan untuk menentukan efektivitasnya pada sistem pangan, diperlukan protein bakteriosin dalam jumlah besar dengan konsentrasi yang tinggi. Sekarang ini, sebagaian besar metoda purifikasi bakteriosin banyak mengandalkan presipitasi menggunakan ammonium sulfat dan presipitat kimia lainnya. Penggunaan metoda ini selain mahal juga diyakini tidak menghasilkan produk yang baik karena banyak protein lain dari medium bisa terpresipitasi, rendemennya tidak begitu tinggi serta kemungkinan terikutnya residu bahan kimia yang digunakan.

Sel bakteri penghasil bakteriosin yang telah dimatikan dan abu sekam padi yang banyak mengandung silika dan karbon keduanya adalah bahan alami yang mempunyai kemampuan mengadsorpsi bakteriosin. Kemampuan bahan alami ini selanjutnya akan digunakan sebagai dasar bagi prinsip purifikasi bakteriosin yang dikenal sebagai bioseparasi.

Proses adsorpsi dan desorpsi bakteriosin oleh bahan alami ini ternyata sangat tergantung pada pH medium dan faktor-faktor lainnya. Oleh karena itu pada penelitian ini dipelajari berbagai kondisi yang mempengaruhi proses adsorpsi dan desorpsi bakteriosin seperti pH, suhu, kecepatan agitasi, waktu dan kecepatan sentrifugasi.

Hasil penelitian menunjukkan bahwa ternyata proses adsorpsi dan desorpsi gassericin A seperti beberapa bakteriosin lainnya, terutama sangat tergantung pada pH medium. Adsorpsi maksimum dan kisaran pH optimum untuk sel yang berbeda adalah sebagai berikut: menggunakan sel pemroduksi bakteriosin dan sel bakteri indikator, 87,5%. Hasil konfirmasi dengan menggunakan SDS-PAGE dan in situ activity assay menunjukkan bahwa ekstraksi protein bakteriosin menggunakan metode ini tingkat kemurniannya relatif tinggi, dengan berat molekul yang sesuai yaitu 3800 Da.. Metoda bioseparasi menggunakan sel bakteri menggunakan prinsip adsorpsi-desorpsi ini merupakan metoda purifikasi yang sederhana untuk memproduksi bakteriosin dari bakteri asam laktat dalam jumlah besar untuk dapat digunakan sebagai biopreservatif pangan. Kata kunci: Bioseparasi, gassericin A, adsorpsi-desorpsi

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SUMMARY

Bacteriocins is an antibacterial protein mostly produced by lactic acid bacteria, and due to its capability to inhibit other species, bacteriocin has a potential as biopreservatives in order to control the growth of pathogenic and � utrefactive bacteria. To study their chemical and antibacterial properties and their effectiveness in food system, it is necessary to obtain relatively large quantities of these peptides in pure and concentrated form. Currently, most methods rely on ammonium sulphate precipitation and other chemical methods. However, there is general agreement that it does not yield a good product because many other proteins from the medium can also be precipitated and the yield is not very high.

It is known that , in general, cells of a bacteriocin-producing bacterial strain and rice hull ash which rich in carbon and silica are natural material which can adsorb bacteriocin. Furthermore, their capability to adsorp bacteriocin will be used as a basic principle for bacteriocin purification so called bioseparation.

Adsorption and desorption process using these natural material are mostly pH dependent and depend on other factors. Therefore, in this research various condition influencing adsorption and desorption of bacateriocin eg. pH, tempereature, centrifugation speed etc were studied.

The result of this experiment showed that adsorption-desorption process of gassericin A, similar to other bacteriocins were pH dependent. The maximum adsorption and optimal pH range for different cells were as follows: bacteriocin producing-cells and indicator cells, 87,5% at pH 6.5. Confirmation using SDS-PAGE and in situ activity assay showed that the purity of bacteriocin extracted using this method is ralatively high with relative molecular weight is 3800 Da. Bioseparation method using bacterial cell based on adsorption-desorption principle is simple and can be used to produce large quantity of bacteriocins from lactic acid bacteria to be used as food biopreservatives. Keywords: Bioseparation, gassericin A, adsorption-desorption

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DAFTAR PUSTAKA

Bhunia,A.K., M.C. Johnson, and B. Ray, 1987, Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulfate –polyacrilamide gel

electrophoresis. J. Industrial microbiol 2:319-322.

Bhunia,A.K., M.C. Johnson, and B. Ray, 1988. Purification, charac-terization and microbial spectrum of a bacteriocin produced by Pediococcus acidilactici J. Applied Bacteriology. 65: 261-268

Bhunia,A.K., M.C. Johnson, B. Ray, and N. Kalchayanand, 1991. Mode of action of pediocin AcH from Pediococcus acidilactici H on sensitive bacterial strain, J. appl. Bacteriol. 70:25-33.

Biswas, S.R., P. Ray, M. C. Johnson, and B. Ray, 1991. Influence of growth conditions on the production of a bacteriocin , pediocin AcH, by Pediococcus acidilactici strain. Appl. Environ. Microbiol. 57(4):1265-1267.

Bukhtiyarova, M.R., R. Yang and B. Ray, 1994. Analysis of pediocin-AcH gene cluster from plasmid pSMB74 and its expression in pediocin-negative Pediococcus acidilactici strain. Appl. Environ. Microbiol. 60:3405-3408.

Cheeseman, G.C. and N.J. Berridge, 1968 Observation on molecular weeight and chemical composition of nisin A. Biochem., J. 71:185-195.

Coventry, M.J., J. B. Gordon, M. Alexander, M.W. Hickey, and J. Wan 1996. A food grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomic calcium silicate. Appl. Environ. Microbiol., 62:1764-1769.

Daba, H., S. Pandian, J.J. Gosselin, R.E. Simard, J. Huang, and C. Laeroix, 1991. Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides. Appl. Environ. Microbiol. 57:3450-3455.

Einarsson, H. and H.L. Lauzon, 1995, Biopreservation of Brined Shrimp (Pandalus borealis) by bacteriocin from Lactic Acid Bacteria. Appl. Environ. Microbiol. 61:6669-675.

Guyonnet, D., C. Fremaux, Y.Cenatiempo, and J.M.Berjeaud, 2000, Method for rapid purification of class IIa bacteriocins and comparison of their activities, Appl. Environ. Microbiol., 66: 1744-1748.

Inoue,H., H. Nojima, and H. Okayama, 1990, High efficiency transformation of Eschericia coli with plasmid, Gene 96:23-28.

James, M.E., N.M. Ahmed, and M.G. Johnson, 1996, Identification of two bacteriocins producing bacteria from garlic and ginger root active against food borne pathogens, abst. 140-11, p 31 In Abstract of IFT Annual meeting , New Orleans, L.A.

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Janes, M.E., R. Nannapaneni , A. Proctor, and M.G. Johnson, 1998, Rice hull ash and silicic acid as adsorbents for concentration of bacteriocins, Appl. Environ. Microbiol., 64: 4403-4409.

Jimenez-Diaz, R., J.I. Ruiz-Barba and D.P. Catchcart, 1995. Purification and partial amino acid sequence of plantaricin S, a bacteriocin produced by Lactobacillus plantarum LPCO 10, the activity of which depend on the complementary action of two peptides. Appl. Environ. Microbiol. 61:4459-4463.

Joosten, H.M.I.J., and M. Nunez, 1995, Absorption of nisin and enterocin in polypropylene and glass surfaces and its prevention by Tween 80, Lett. Appl. Microbiol, 21:389-392.

Kanatani, K., K. Yoshida, T. Tahara, H. Miura, M. Sakamoto, and M. Oshimura, 1991, . Isolation and characterization of plasmid DNA in Lactobacillus acidophilus, Agric. Biol. Chem. 55:2051-2056.

Kanatani, K., Oshimura, M. and Sano, K. 1995. Isolation and characterization of acidocin A and cloning of the bacteriocin gene from Lactobacillus acidophilus. Appl. Environ. Microbiol., 61:1061-1067.

Kim, W.J., B. Ray, and M.C. Johnson, 1992, Plasmid transfer by conjugation and electroforation in Pediococcus acidilactici , J. Appl. Bacteriol., 72, 201.

Kojic, M., J. Svircevic, A. Benina and L. Topisirovic, 1991, Bacteriocin-producing strain of Lactococcus lactis subsp. diacetilactis S50. Appl. Environ. Microbiol., 57: 1835-1837.

Klaenhammer, T.R. 1988. Bacteriocin from Lactic Acid Bacteria. Biochimie 70:337-349.

Kusnadi,.J. 1999. Produksi dan uji aktivitas antibakteri bakteriosin isolat Pediococcus sp.24 serta identifikasinya. Thesis Program Magister.

Kusnadi, J., S. Margino, E.S. Rahayu, 1999, Optimasi produksi bakteriosin oleh Pediococcus sp.24 dan aktivitas antibakterinya. Prosiding Seminar Nasional Pangan. PAU Pangan dan Gizi UGM Yogyakarta.

Kusnadi.J., K. Sano, M. Otani, S. Rahayu, and S. Margino, 2000, Comparison of several methods for purification of bacteriocin produced by Leuconostoc SM22. Science.

Leroy, F. and L. De Vuyst, 1999, The presence of salt and a curing agent reduces

bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation, Appl. Environ. Microbiol., 65:5350-5356.

Lewus, C.B., S. Sun, T.J. Montville, 1992. Production of an amylase-sensitive bacteriocin by an atypical Leuconostoc paramesenteroides strain. Appl. Environ. Microbiol., 61:1061-1067.

Proctor, A., and S. Palaniappan, 1989, Soy oil lutein adsorption by rice hull ash. J. Am. Oil Chem. Soc., 66:1618-1621.

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Proctor, A., and S. Palaniappan, 1990, Adsorption of soy oil free fatty acids by rice hull ash. J. Am. Oil Chem. Soc., 67:15-17.

Proctor, A., C. Adhikari, G.D. Blyholder, 1995, Mode of oleic acid adsorption on rice hull ash cristobalite, J. Am. Oil Chem. Soc., 72:331-335.

Ray, S.K., M.C. Johnson, and B. Ray, 1989, Bacteriocin plasmid of Pediococcus acidilactici , J. Ind. Microbiol., 4:163

Ray, S.K., W.J. Kim, M.C. Johnson, and B. Ray, 1989, Conjugal transfer of a plasmid encoding bacteriocin production and immunity in Pediococcus acidilactici H., J. Appl. Bacteriol., 66, 393

Sahl, H.G., 1985. Influence of staphyloccin-like peptide Pep5 on membrane potential of bacterial cells and cytoplasmic membrane vesicles. J. Bacteriol. 152:833-836.

Sahl, H.G, 1991. Pore formation in bacterial membranes by cationic lanthibiotics p.347-358 dalam G. Jung and H.G. Sahl (eds.). Nisin and novel lanthibiotic, Escom Publishers, Leiden, The Netherland.

Schagger, H., and G. Von Jagow. 1987. Tricine sodium dodecyl sulfate polyacrilamide gel electrophoresis for the separation of proteins in the range of 1 to 100 kDA. Anal. Biochem. 166:368-379.

Tagg, H.G., A.S. Dajani, and A.S. Wannamaker, L.W., 1976. Bacteriocin of gram positive bacteria, Bacteriol. Rev. 40:722-756.

Tantrakulshiri, J., N.Jeyashoke, and K. Krisanangkura, 1997, Utilization of Rice hull ash as a support material for immobilization of Candida cylindracea lipase, J. Am. Oil Chem. Soc., 74:173-175.

White, R.J. and A. Hurst, 1968. The location of nisin in the producer organism Streptococcus lactis, J. Gen. Microbiol 53:171-179.

Yang,R., M.C. Johnson and B. Ray. 1992. Novel method to extract large amounts of bacteriocins from lactic acid bacteria. J. Food Microbiology. 11: 281-291.