Karakterisasi Polyphasic Bakteri Asam Laktat asal Produk Susu Kuda Sumbawa Terfermentasi dan Potensinya sebagai Penghasil Bakteriosin

Mulyawati, Alifia Issabella and Yoga Dwi Jatmiko, S.Si, M.App.Sc, Ph.D. and Tri Ardyati, M.Agr., Ph.D. (2019) Karakterisasi Polyphasic Bakteri Asam Laktat asal Produk Susu Kuda Sumbawa Terfermentasi dan Potensinya sebagai Penghasil Bakteriosin. Magister thesis, Universitas Brawijaya.

Abstract

Produk fermentasi tradisional dari susu kuda Sumbawa merupakan salah satu produk unggulan yang berpotensi sebagai habitat bakteri asam laktat (BAL) penghasil bakteriosin. Bakteriosin merupakan protein yang memiliki aktivitas antimikroba. Penelitian bertujuan untuk menganalisis keragaman BAL dari produk susu kuda Sumbawa terfermentasi berdasarkan karakter polyphasic, mengevaluasi spektrum aktivitas bakteriosin yang dihasilkan oleh bakteri tersebut, mengidentifikasi spesies bakteri penghasil bakteriosin unggul berdasarkan sekuen 16S rDNA, dan mengevaluasi potensi bakteri penghasil bakteriosin tersebut dalam meningkatkan keamanan pangan produk fermentasi susu kuda Sumbawa. Sampel produk susu kuda Sumbawa terfermentasi diperoleh dari Kabupaten Bima, Dompu, dan Sumbawa, Provinsi Nusa Tenggara Barat, Indonesia. Bakteri diisolasi dari sampel dengan metode dilusi bertingkat dan ditumbuhkan pada media MRS dan M17 agar mengandung 1 % CaCO3 menurut metode spread plate. Kandungan proksimat dan pH masing-masing sampel susu diukur. Karakter fenotip BAL yang diamati meliputi morfologi koloni dan sel serta aktivitas enzim katalase. Karakter genotip ditentukan dengan metode PCR-RFLP menggunakan dua pasang primer dengan target ITS dan spacer region 5,8S. Kromosomal DNA diisolasi menggunakan metode heat-treatment. Amplikon dari spacer region 5,8S digunakan sebagai template untuk analisis RFLP menggunakan dua enzim restriksi HaeIII dan HindIII. Pola pita DNA pada elektroforesis gel agarosa digunakan untuk mengelompokkan isolat-isolat BAL menggunakan dendogram dengan pendekatan polyphasic dan algoritma Neigbor-Joining. Setiap kelompok dipilih satu isolat representatif untuk diidentifikasi berdasarkan sekuen 16S rDNA. Isolat-isolat BAL yang berpotensi sebagai penghasil bakteriosin diseleksi berdasarkan aktivitas antibakteri crude cells terhadap Staphylococcus aureus ATCC 6538 dan Escherichia coli ATCC 25922 dengan metode disc-diffusion. Cell-free supernatant (CFS) dengan pH netral dari isolat BAL yang terseleksi diuji kemampuan penghambatannya terhadap 10 bakteri patogen dengan metode disc-diffusion untuk mengetahui kisaran/spektrum aktivitas antibakteri. Isolat BAL dengan spektrum antibakteri luas dikonfirmasi dengan perlakuan proteinase-K. Bakteriosin dimurnikan secara parsial menggunakan teknik presipitasi ammonium sulfate 80 % dilanjutkan dialisis untuk mendapatkan partially purified bacteriocin (PPB). Aktivitas bakteriosin pada setiap tahap pemurnian diuji dengan metode disc-diffusion. Berat molekul presipitat bakteriosin dan PPB diestimasi menggunakan metode SDS-PAGE. Aktivitas bakteriosin dari pita protein hasil SDS-PAGE dikonfirmasi dengan metode soft-agar overlay. Densitas BAL tertinggi pada media MRS diperoleh dari sampel susu kuda asal Kabupaten Bima 1,43 × 104 CFU/mL dan pada media M17 tertinggi dari sampel Sumbawa

English Abstract

Traditional fermented products from Sumbawa mare's milk have potential as the habitat of bacteriocin-producing lactic acid bacteria (LAB). Bacteriocin is a protein that has antimicrobial activity. The aims of this study were to analyze the diversity of LAB isolated from fermented mare's milk products based on polyphasic characters, to evaluate the spectrum of bacteriocin activity produced by LAB, to identify the best bacteriocin- producing bacteria strains based on 16S rDNA sequences, and to evaluate its potency in improving the safety of fermented mare’s milk products from Sumbawa. The samples used were fermented mare's milk products collected from Bima, Dompu, and Sumbawa Regencies, West Nusa Tenggara Province, Indonesia. Bacteria were isolated from the sample by serial dilution method and grown on MRS and M17 agar containing 1% CaCO3 using the spread plate method. The proximate content and pH of milk samples was also measured. The characterization of LAB diversity was conducted based on the phenotypic characters, i.e., the morphology of colonies and cells, and the activity of the catalase enzyme. Furthermore, LAB isolates were characterized based on genotypic characters using the PCR-RFLP method. Two pairs of primer targeting ITS and 5.8S spacer regions from gDNA used for PCR. The gDNA was isolated using the heat-treatment method. The amplicon of the 5.8S spacer region was used as the PCR-RFLP template using two restriction enzymes, namely HaeIII and HindIII. DNA bands from PCR-RFLP were visualized using agarose gel electrophoresis method. The DNA bands were made for clustering the LAB isolates obtained. One representative isolate of each cluster was chosen for sequencing based on 16S rDNA. The LAB were selected based on antibacterial activity produced by the crude cells against Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 25922 using the disc-diffusion method. The antibacterial activity spectrum of bacteriocins produced by the selected isolates was tested by the disc-diffusion method using cell-free supernatant pH 6.5 against 10 strains of pathogenic bacteria. The LAB isolates with broad-spectrum were selected and further confirmed with proteinase-K to inactivate antibacterial activity indicating that the inhibition ability was derived from the protein. The bacteriocin was then partially purified using 80% ammonium sulfate precipitation to obtain precipitated bacteriocin and continued with dialysis to obtain partially purified bacteriocin (PPB). The bacteriocin activity was assayed at each purification step using the disc-diffusion method. The molecular weight of proteins from precipitated bacteriocins and PPB was estimated using the SDS-¬PAGE method. In addition, the bacteriocin activity of protein band from the result of SDS-PAGE was confirmed with the soft-agar overlay method

Other obstract

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Item Type:	Thesis (Magister)
Identification Number:	TES/637.1/MUL/k/2019/041902169
Uncontrolled Keywords:	-
Subjects:	600 Technology (Applied sciences) > 637 Processing dairy and related products > 637.1 Milk processing
Divisions:	S2/S3 > Magister Biologi, Fakultas MIPA
Depositing User:	yulia Chasanah
Date Deposited:	31 Aug 2022 02:46
Last Modified:	31 Aug 2022 02:48
URI:	http://repository.ub.ac.id/id/eprint/193832

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