The characterization of bacteriocins produced by Lactobacillus plantarum strains isolated from traditional fermented foods in Indonesia and the detection of its plantaricin-encoding genes

Lactobacillus plantarum is widely found in either anaerobic plant matter or fermented foods, and it has been recognized as producing antimicrobial bacteriocins. This study aimed to characterize the antimicrobial bacteriocins of L. plantarum and detect its genes that encode plantaricins. Samples were isolated from traditional fermented foods from Indonesia. Antimicrobial activity was evaluated using the agar diffusion assay procedure. The titration method applied the maximum amounts of lactic acid at 1054 mg/mL and hydrogen peroxide at 3.85 mg/mL. Based on the results, the supernatant of the L. plantarum strains appeared to have a broad spectrum of antimicrobial activity against pathogens, which would be active at pH 2.0–12.0 and stable temperature. In addition, almost all of the L. plantarum strains contained plantaricin-encoding genes (e.g. plnA, plnF,plnJK, and plnW), which were grouped into one cluster as indicated by phylogenetic analysis. Therefore, this study discovered clear evidence of the potential of some L. plantarum strains to act as antimicrobial agents

Identification of Antibiotic-Resistance Genes from Lactic Acid Bacteria in Indonesian Fermented Foods

Lactic acid bacteria (LAB) are known to have an important role in food fermentation and are thought to have health-promoting abilities such as probiotic properties. In this study, LAB were isolated from Indonesian fermented foods such as dadih (fermented buffalo milk), tempoyak (fermented durian), bekasam (fermented meat), and tape ketan (fermented glutinous rice). Those isolates were investigated for their resistance to two antibiotics: chloramphenicol and erythromycin. Recent efforts in food science have sought to identify genetic markers for antibiotic resistance within LAB strains, so that these genes can be selected for genetic modification. Such research is presently being directed toward the development of food-grade vectors (plasmid). The aim of this study is to screen LAB isolated from Indonesian traditional fermented foods, for chloramphenicol and erythromycin resistance. In this study, a total of 120 LAB samples were taken from traditional Indonesia fermented foods, and were tested for resistance to chloramphenicol and erythromycin. The results show that three LAB strains remained resistant to doses of up to 5 μg/mL chloramphenicol, while the LAB strain Lactobacillus plantarum showed resistance to the antibiotic erythromycin up to a concentration of 15 μg/mL

Antibacterial Activity of Extracellular Protease Isolated From an Algicolous Fungus Xylaria psidii KT30 Against Gram-Positive Bacteria

Infectious diseases became more serious problem for public health in recent years. Although existing antibacterial drugs have been relatively effective, they do not rule out the emergence of resistance to the drug. Therefore, the intensive exploration of new bioactive compounds from natural, especially peptide compounds began in recent decades in order-handling infection. This study aimed to isolate, purify and test the potential application of Xylaria psidii KT30 extracellular protease as antibacterial agent against Gram-positive bacteria. X. psidii KT30, a marine fungus isolated from red seaweed Kappaphycus alvarezii showed antibacterial activity against Bacillus subtilisand Staphylococcus aureus. Antibacterial compounds of this fungus were predicted as a group of proteases. Extracellular protease exhibited an optimum activity when potato dextrose broth was used as cultivation medium. Furthermore, the highest activity of these proteases was found on fungal extract after day 15 of cultivation with value of 2.33 ± 0.19 U/mL. The partial purification of proteases using G-75 column chromatography resulted in 2 groups of fractions and showed protease activity based on zymogram assay. The extracellular proteases obtained from those fractions have 3 patterns of molecular mass based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis which are 56.62, 89.12, 162.18 kDa

ANTIBACTERIAL ACTIVITY ASSAY OF MANGROVE EXTRACTS AGAINST SALMONELLA TYPHI AND LISTERIA MONOCYTOGENES

The antibacterial activities of mangrove species, Avicennia marina, Sonneratia caseolaris (collected from Teluk Payo, Banyuasin, South Sumatera), Ceriops tagal, Rizhopora apiculata, and Sonneratia alba (collected from Sadai, South Bangka) were screened against Salmonella typhi and Listeria monocytogenes by agar disk diffusion assays. Extractions were conducted using organic solvents (methanol, ethyl acetate, and acetone, subsequently). Most of the extracts tested showed potential antibacterial activity against both pathogens. The methanol extracts of the bark from S. alba and the fruit from A. marina showed particularly large inhibition zones (15 mm) against S. typhi. The acetone extract of S. alba leaves showed the largest inhibition zone (14 mm) when tested against L. Monocy-togenes. Further partial purifications of selected extracts which showed strong inhibition were performed by silica gel column chromatography using various eluent compositions with different polarities. The third fraction of methanol extract from S.alba leaves eluted with chloroform:methanol (1:5) resulted in a remarkably large inhibition zone (23 mm) against S. typhi. The third and seventh fractions of acetone extract from S. alba leaves eluted with ethyl acetate:methanol (7:3) resulted in a large inhibition zones (15 mm) against L. monocytogenes. In addition, the sixth fraction of methanol extract from A. marina fruit eluted with chloroform : methanol (6:4) resulted in the largest inhibition zone (17 mm) against L. monocytogenes. These results indicated that mangrove extracts could be developed as potential biomaterials for biopharmaceutical as well as biopreservation industries.Keywords: antibacterial activity, mangrove, column chromatograph

ISOLASI DAN IDENTIFIKASI AWAL SENYAWA INHIBITOR RNA HELIKASE VIRUS HEPATITIS C DARI EKSTRAK BUAH MANGROVE Avicennia marina (Forsk.) Vierh

Hepatitis C virus is the cause of hepatitis C disease which has high virulence. Recent therapy using combination of ribavirin and alpha interferon has short efficiency (< 80%). Thus, the discovery of new drug is needed. Antiviral drugs can be discovered through molecular target therapy by finding the inhibitor of RNA helicase that play role in viral replication. Inhibitor can be derived from chemical compound produced by mangrove. The aim of this research was to isolate the active compound groups from fruit of  Avicennia marina (Forsk) which had inhibitory activity against RNA helicase. Inhibitory activity was measured by releasing of phosphate inorganic in  colorimetric ATPase assay. Crude extract was fractionated using gel filtration chromatography with methanol in chloroform solvent. The result showed that fraction 2 has the highest inhibitory activity i.e. 81.78%. Phytochemical test of crude extract indicated positive result for flavonoids, alkaloids, steroid dan triterpenoid, tannin and saponins. Moreover, high performance liquid chromatography (HPLC) analysis showed absorption peak with the high abundance at the retention time of  7.250; 18.983 and 20.050 minute at 216 nm, 247 nm and 263 nm, respectively. According to the results of phytochemical, TLC, and HPLC analyses, inhibitor compound from fruit of  A. marina (Forsk) was suggested it belongs to flavonoids.Key words: Avicennia marina, flavonoid, hepatitis C virus, RNA helicas

APLIKASI MARKA MOLEKULER PADA BUAH DAN BIJI KOPI ASAL KALIMANTAN TIMUR

Two quantitative traits, cherry and green bean characters are the important phenotypic selection in coffee breeding practice. The important well-known character from coffee markets is cherry and bean size. In this study, 43 genotypes of coffee were collected from four districts in East Kalimantan i.e. Kutai Kertanegara, Kutai Timur, Berau dan Paser Utara. The objective of this study was to identify cherry and green bean character using quantitative trait locus (QTL) molecular marker, genetic variation from developed alleles, cluster analysis and association analysis of molecular marker, and phenotype observation. Based on polymorphic information content (PIC) of primers used in this study, the genetic variation was low. Based on cluster analysis, two major groups were identified. The first group corresponds to Arabika that consisted of 3 districts, Kutai Timur, Berau and Paser Utara. The second group correspond to Robusta mostly from Kutai Kertanegara.Significant association of primer markers M480 and M312 with QTL has suggested that they can be used as specific primers linked to size of cherry and green bean.Furthermore,they were potential marker assisted breeding in coffee breeding program

Karakteristik Fisik dan Kimia Gelatin dari Tulang Ikan Patin dengan Pre-Treatment Asam Sitrat

Penelitian ini bertujuan untuk melakukan ekstraksi gelatin dari tulang ikan patin menggunakan asam sitrat dan menganalisis karakteristik fisiko-kimianya. Ekstraksi gelatin melalui dua tahap yaitu tahap pre-treatment dengan asam sitrat dan ekstraksi utama dengan aquadest. Tahap pre-treatment dilakukan dengan variasi waktu yakni 24, 36, 48 dan 56 jam. Tahapan ekstraksi utama dilakukan pada suhu 45, 55, 65, dan 75°C. Hasil ekstraksi dilanjutkan dengan analisis keberadaan protein gelatin dengan metode SDS-PAGE. Analisis fisiko-kimia gelatin meliputi derajat keasaman (pH), rendemen, kekuatan gel, profil tekstur, viskositas, kadar air, kadar abu, kadar protein dan kadar lemak juga dilakukan. Hasil SDS-PGE gelatin perlakuan terbaik (yakni dengan pre-treatment 48 jam dan ekstraksi utama pada suhu 75°C) diketahui memiliki bobot molekul 162 kDa. Gelatin hasil ekstraksi terbaik memiliki rendemen sebesar 6,14%. Gelatin tulang ikan patin memiliki pH 4,46, kekuatan gel 364,19 bloom, daya kunyah sebesar 261,76 g dan viskositas 3,83 cP. Kadar proksimat gelatin tulang ikan patin meliputi kadar air, kadar abu, kadar protein dan kadar lemak masing-masing 7,72; 0,38; 58,70, dan 2,79%. Hasil penelitian juga menunjukan bahwa semakin tinggi suhu ekstraksi dan lamanya proses pre-treatment yang digunakan, maka bobot molekul protein menjadi semakin tinggi pula. Kesimpulannya, pre-treatment asam sitrat selama 48 jam dan ekstraksi utama pada suhu 75°C telah berhasil menunjukkan hasil yang paling baik dan dapat terkarakterisasi sifat fisik serta kimianya.Physical and Chemical Characteristics of Gelatin from Pangasius Catfish Bone with Pre-Treatment of Citric AcidThis research aimed to extract gelatin from Pangasius catfish bone using citric acid and then analyse its physicochemical characteristics. The extraction of gelatin was done in two stages soaking i.e pre-treatment with citric acid and main extraction by aquadest. Pre-treatment was done by soaking bones at 24, 36, 48 and 56 h, then the main extraction was done by soaked leached bones in temperature 45, 55, 65, and 75°C. The gelatin from catfish bone then was analyzed the presence of protein by SDS-PAGE and its physico-chemical including pH, yield, gel strength, texture profile, viscosity, moisture content, ash content, protein content, and fat content. Based on SDS-PAGE, fish bone gelatin had molecular weight of 162 kDa. The best extraction treatment (pre-treatment 48 h and main extraction at 75°C) produced 6.14% of gelatin yield. Gelatin of pangasius catfish bone had pH 4.46, gel strength of 353.76 bloom, chewing power of 261.76 g, and viscosity of 3.83 cP. Water, ash, protein and fat content was 7.72, 0.38, 58.70, and 2.79%, respectively. The higher extraction temperature and the longer time for pre-treatment process, the higher molecule weight of protein. As conclusion, the best treatment that was found in the sample with the pre-treatment at 48 h and the main extraction at 75˚C, was successfully characterized on its physicochemicals.•|•|•|

Isolation of DNA Aptamers for Enteropathogenic Escherichia coli (EPEC) Detection using Bacterial-SELEX Approach

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative pathogenic bacterium that causes diarrheal disease, especially in infants and children. Aptamers are short chain oligonucleotides that have high affinity, specificity, and selectivity to their targets, which have potential to be developed as a method for diagnosing pathogens. In this study, aptamer was isolated through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method using whole cells bacteria (Bacterial-SELEX) for recognizing pathogenic E. coli EPEC K1.1 which was isolated from children with diarrhea in Indonesia. Ten rounds of bacterial-SELEX procedure were conducted with modification conditions by using Top10, DH5a E. coli cells, Listeria monocytogenes, and Lactobacillus plantarum S34 as counter-selections. The selection process was started with a pool of ssDNA random library consisting of a random base with 40-nucleotides long flanked with fixed primers sequence for aptamer amplification purpose. Short single-stranded DNA amplification was done by symmetric and asymmetric PCR. The highly enriched oligonucleotide pools (pooled 8, 9, and 10) were cloned and the resulting ssDNA aptamers were identified by Sanger DNA sequencing. Finally, twelve aptamers with unique sequences and various secondary structures including G-quadruplex sequence motif within aptamers were obtained as candidates specific aptamer for detection and capturing of EPEC K1.1

Dipeptidyl Peptidase IV (DPP-IV) Inhibitory Activity of Ultrafiltration and Gel Filtration Fraction of Gelatin Hydrolyaste Derived from Bone of Fish for Antidiabetes

Bioactive peptides have been investigated largely for many biofunctional properties. The aim of this research was to determine the inhibitory activity of ultrafiltration (UF) and gel filtration (GF) fractions of gelatin derived from bone of Pangasius catfish against dipeptidyl peptidase IV (DPP-IV). Previous studies have shown that gelatin from skins of salmon, hake, halibut, milkfish, tilapia and bone of pangasius catfish have the activity in DPP-IV inhibition. While, as inhibitor, most of previous and recent studies shown that separation and fractination of gelatin hydrolysate increase their activity. This research was conducted in three stages including gelatin hydrolysates fractination by ultrafiltration (UF), UF fraction loaded into gel filtration (GF) and DPP-IV inhibition measurement. The fish bone gelatin was hydrolyzed using flavourzyme at three enzyme/substrate ratios (E/S of 3%, 6% and 9%) with incubation times 4, 6, and 8 h. Then, the hydrolysates fractioned by ultrafiltration with 3 kDa cutoff membrane continued with Superfine G-25 sephadex column (65 cm x 3 cm, flow rate 1 mL/min). The result shown that both group of fractions i.e UF and GF have inhibitory activity regarding their capacity to inhibit DPP-IV.  The UF fraction &gt;3 kDa derived from 9% (E/S) ratio for hdyrolysis were superb as DPP-IV inhibitor than other fractions, the highest one also has bioactivity higher than other previous fish gelatin fractions.

PENGARUH MUTAGEN ULTRAVIOLET DAN ETIL METAN SULFONAT TERHADAP KADAR PENISILIN G PADA Penicillium chrysogenum

ABSTRACTPenicillium chrysogenum is one of the fungi which known to produce penicillin G antibiotics. The current superior strain P. chrysogenum producing high penicillin G has gone through various stages from the wild type isolates. The wild type isolates of P. chrysogenum from Indonesia also have great potential to be developed. Therefore, this study aimed to determine the changes of penicillin G levels from mutated P. chrysogenum isolate with both ultraviolet (UV) radiation and chemical mutagens ethyl methane sulfonate (EMS) compared to the wild type isolates. Random mutation through UV radiation, EMS chemical mutagen and their combination was carried out on wild type isolates of P. chrysogenum. Antibacterial activity was tested against Escherichia coli and Bacillus subtilis. The level of penicillin G produced was detected using HPLC with the Penicillin G as the standard. The results showed that the treatment of UV, EMS mutations and their combination can increase the antibacterial activity as well as levels of penicillin G than the wild type. Mutant C5-4.10 isolate resulted from the combination UV and EMS had the best antibacterial activity and produced penicillin G. level 2.9 times compared to the wild type.Keywords: antibiotic, HPLC, mutation, Penicillium chrysogenum, penicillin GABSTRAKPenicillium chrysogenum adalah salah satu kapang yang diketahui memproduksi antibiotik penisilin G. Strain P. chrysogenum unggul penghasil penisilin G tinggi yang saat ini tersedia merupakan galur yang telah melalui berbagai tahapan pemuliaan dari wild type-nya. Isolat P. chrysogenum asal Indonesia juga memiliki potensi besar untuk dikembangkan. Oleh karena itu, penelitian ini bertujuan untuk mengetahui perubahan kadar penisilin G dari isolat P. chrysogenum yang telah dimutasikan menggunakan radiasi sinar ultraviolet (UV) dan mutagen kimiawi etil metan sulfonat (EMS) dibandingkan dengan wild type nya. Mutasi acak menggunakan radiasi sinar UV, mutagen kimiawi EMS dan kombinasi keduanya dilakukan pada isolat P. chrysogenum. Uji aktivitas antibakteri dilakukan terhadap Escherichia coli dan Bacillus subtilis. Kadar penisilin G yang diproduksi dideteksi menggunakan HPLC dan dibandingkan dengan standar Penicillin G. Hasil penelitian menunjukkan bahwa mutasi menggunakan sinar UV, EMS dan kombinasi keduanya dapat meningkatkan aktivitas antibakteri serta kadar penisilin G dibandingkan dengan wild type nya. Isolat mutan C5-4.10 hasil mutasi kombinasi sinar UV dan EMS memiliki aktivitas antibakteri terbaik dan kadar penisilin G sebesar 2,9 kali lipat dibandingkan dengan wild type nya.Kata kunci: antibiotik, HPLC, mutasi, Penicillium chrysogenum, penisilin