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"Klebsiella sp. GMD08 is one of the bacteria that has the capability to dissolve insoluble inorganic phosphate into soluble phosphate ion through their organic acid production. Transposon is a genetic element agent usually used to generate mutant through mutagenesis. Thus it can be used to identify the genetic functions involved in those phosphate solubilizing mechanisms. This research was conducted to identify the genes of Klebsiella sp. GMD08 involved in phosphate solubilization through sequence detection obtained from a hyper-solubilizing phosphate mutant library. Mutation was conducted by inserting mini-Tn5 transposon hosted in Escherichia coli S17-1/λpir [pBSL202] into Klebsiella sp. GMD08 chromosome by the filter mating conjugation method. Trans conjugant mutant candidates were then qualitatively and quantitatively analyzed for their solubilizing ability to dissolve tricalcium phosphate [Ca3(PO4)2] using pikovskaya medium. The organic acid characteristics of transconjugant mutants were detected using High-performance liquid chromatography (HPLC). Meanwhile, suspected genes involved in phosphate solubilizing were detected using the sequencing method obtained from the transposon insertion result. Nucleotide Basic Local Alignment Search Tool (nucleotide BLAST) was used to identify the nucleotide base sequence similarity with the database. The results showed that PB116 and PB122 were the two main transconjugant mutants obtained from transposon mutagenesis which had higher tricalcium phosphate dissolving ability. Gluconic acid was the main organic acid produced by Klebsiella sp. GMD08 phosphate solubilizing mechanism. Moreover, arginine repressor (ArgR) and malate dehydrogenase gene (mdh) coding gene were involved in Klebsiella sp. GMD08 phosphate solubilizing mechanism."

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Pemanfaatan bakteri telah banyak dilakukan pada banyak aspek kehidupan manusia dan menghasilkan dampak yang positif. Salah satu pemanfaatannya adalah asam laktat dan bakteriosin yang diekskresikan dari bakteri Streptococcus macedonicus. Bakteri Streptococcus macedonicus MBF10-2 adalah salah satu galur dari bakteri asam laktat yang diketahui memiliki potensi aktivitas antimikroba terhadap beberapa bakteri Gram positif. Aktivitas antimikroba ini diharapkan dapat dikembangkan sebagai produk perawatan kulit. Dalam penelitian ini digunakan medium berbasis nabati yaitu de Man, Rogose, dan Sharpe (MRS) Vegitone untuk menjamin kehalalan dari proses dan produk akhir. Oleh karena itu, penelitian ini bertujuan untuk memperoleh kondisi optimum perolehan massa sel terbanyak selama fermentasi pada medium de Man, Rogose, Sharpe (MRS) Vegitone pada skala yang diperbesar untuk meningkatkan produksi lisat. Optimasi kondisi optimum perolehan massa sel dilakukan dengan melakukan pengkulturan sel secara bertahap hingga skala besar pada fermentor. Optimasi pelisisan sel dilakukan dengan cara menggunakan metode ultrasonikasi dan gabungan dari ultrasonikasi dengan penambahan lisozim (enzimatik) dengan kondisi pada pH 7 dan 8 dengan pengulangan 20x dan 40x. Pengujian untuk mengkonfirmasi bahwa bakteri sudah terlisis dengan baik yaitu menggunakan pewarnaan Gram dan pengujian MTT Assay. Hasil percobaan menunjukkan bahwa perolehan massa sel yang didapat pada medium MRS Vegitone adalah 7.987 gram, dan perolehan massa sel pada medium MRS standar adalah 8.7013 gram. Hasil dari optimasi lisis menunjukkan bahwa metode gabungan ultrasonikasi dan enzimatik dengan kondisi pH 8 dan pengulangan 40x memberikan hasil yang lebih baik, dibuktikan dengan pengujian perwarnaan Gram yang menunjukkan bahwa sel yang terlisis paling banyak dan rendemen hasil freeze dry sebesar 5.7267%. Dari pengujian MTT Assay juga menunjukkan bahwa sel telah terlisis dengan baik. Dapat disimpulkan bahwa waktu inkubasi optimum medium MRS Vegitone adalah 16 jam dengan efisiensi jumlah massa sel pada medium MRS Vegitone adalah 8,21% lebih sedikit jika dibandingkan dengan medium MRS Standar dan kondisi lisis yang optimum adalah dengan metode gabungan ultrasonikasi dan enzimatik dengan kondisi pH 8 dan pengulangan 40x dengan perolehan rendemen hasil freeze dry sebesar 5.7267%.

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The use of bacteria has been done in many aspects of human life and has a positive impact. Several of the potential substance are lactic acid and bacteriocin. One of the example is Streptococcus macedonicus. Streptococcus macedonicus MBF10-2 is one of the strains of lactic acid bacteria that have antimicrobial activity against several Gram-positive bacteria. This antimicrobial activity is expected to be developed as a skin care product. In this study, vegetable-based medium was used, namely de Man, Rogose, and Sharpe (MRS) Vegitone to ensure halalness of the process and final product. Therefore, this study aims to obtain the optimum conditions for obtaining the most cell mass gain during fermentation in the de Man, Rogose, and Sharpe (MRS) Vegitone on a scale that is enlarged to increase lysate production. Optimization of the optimum conditions for cell mass gain was done by culturing cells gradually to a large scale in fermentor. Optimization of cell lysis is done by using ultrasonication method and a combination of ultrasonication with the addition of lysozyme (enzymatic) with conditions at pH 7 and 8 with repetitions of 20 times and 40 times. Tests to confirm that the bacteria has been properly destroyed, that is, using Gram staining and MTT assay. The results showed that the cell mass gain obtained in the MRS Vegitone medium was 7.987 grams and the cell mass gain obtained in Standard MRS medium was 8.7013 grams. The results of lysis optimization showed that the combined method of ultrasonication and enzymatic with condition at pH 8 with repetitions of 40 times gave better results, proven by the Gram staining test which showed that the most cells are destroyed and the freeze dry yield was 5.7267 %. In MTT assay also shows that the cell has been properly destroyed. It can be concluded that the optimum incubation time of MRS Vegitone medium is 16 hours with the efficiency of cell mass in the MRS Vegitone medium was 8.21% less when compared to the standard MRS medium and the optimum lysis condition is the combined of ultrasonication and enzymatic method with pH 8 and 40 times repetition with the yield of freeze dry is 5.7267%.

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"ABSTRAKKulit memiliki beragam mikrobiota yang bersifat komensal maupun patogen yang berkontribusi terhadap kesehatan. Penentuan dan identifikasi mikrobiota yang terdapat di kulit kini menjadi topik riset yang menarik. Bakteri kulit tersebut dapat dieksplorasi menjadi sumber zat aktif yang berpotensi dalam pengembangan farmasetika kosmetik maupun kesehatan kulit sebagai proteksi kulit terhadap bakteri patogen. Penelitian sebelumnya telah berhasil mengisolasi dan mengarakterisasi komposisi mikrobiota bakteri dari sampel kulit pria dan wanita Indonesia dewasa muda. Tujuan dari penelitian ini adalah memperoleh komposisi optimum galur-galur bakteri terpilih tersebut dalam bentuk koktail bakteri yang telah dioptimasi kondisi campuran dan waktu inkubasi bersamanya dari penelitian sebelumnya. Analisis kemampuan setiap galur bakteri untuk bertahan hidup dalam suatu populasi bersama dilakukan dengan menggunakan pengamatan visual konvensional Deferred Growth Inhibition Assay (DGIA) termodifikasi, maupun secara molekuler berbasis asam nukleat Real-Time qPCR. Antar galur-galur bakteri memiliki potensi saling menginhibisi saat dikultur bersama,sehingga waktu pengulturan bersama terbaik hasil penelitian sebelumnya yaitu 2 dan 4 jam dipilih dalam penelitian ini untuk optimasi konsentrasi masing-masing galur. Hasil real time q-PCR dengan primer rancangan unik yang dipilih terhadap 3 jenis variasi komposisi,yang didukung pula oleh hasil DGIA, menunjukkan bahwa komposisi yang terbaik dalam hal kesetaraan pertumbuhan sel adalah pada komposisi 2 yaitu Micrococcus luteus MBF05-19J : Bacillus subtilis MBF10-19J : Staphylococcus warneri MBF02-19J : Staphylococcus hominis MBF12-19J sebesar 1 : 1: 0,5 : 0,5 dengan waktu inkubasi 2 jam, dan komposisi 3 yaitu Micrococcus luteus MBF05-19J : Bacillus subtilis MBF10-19J : Staphylococcus warneri MBF02-19J : Staphylococcus hominis MBF12-19Jsebesar 1,5 : 1: 0,5 : 0,5 dengan waktu inkubasi 4 jam.

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ABSTRACT

The skin has a variety of commensal and pathogenic microbiota that contribute to health. The determination and identification of skin microbiome have become an interesting research topic. These skin bacteria can be explored as a potential source of active substances in the development of cosmetics pharmaceuticals as skin protection against pathogenic bacteria. Previous studies have succeeded in isolating and characterizing the composition of bacterial microbiota from skin samples from Indonesian men and women in young adults. The aim of this study was to obtain the optimum composition of the selected bacterial strain in the form of a bacterial cocktail that had been optimized for mixed conditions and incubation time with it from previous studies. Analysis of the ability of each strain to survive in a shared population is carried out using conventional visual observations of modified Deferred Growth Inhibition Assay (DGIA), as well as molecularly nucleic acids based using Real-Time q-PCR. Each bacterial strain has the potential to inhibit each other when cultured together, so that the best time from the previous research results, 2 and 4 hours, was chosen in this study to optimize the concentration of each strain. Real-Time q-PCR results with a unique primer design selected for 3 types of composition variation, supported by the results of DGIA, show that the best composition in terms of equality of cell growth is in composition 2 namely Micrococcus luteus MBF05-19J: Bacillus subtilis MBF10-19J: Staphylococcus warneri MBF02-19J: Staphylococcus hominis MBF12-19J for 1: 1: 0.5: 0.5 with incubation time of 2 hours, and composition 3 namely Micrococcus luteus MBF05-19J: Bacillus subtilis MBF10-19J: Staphylococcus warneri MBF02-19J: Staphylococcus hominis MBF12-19J 1.5: 1: 0.5: 0.5 with incubation time 4 hours."