Luria-Bertani Research Articles

In vitro evaluation of antimicrobial and antioxidant activities of algal extracts

The aim of this study was to evaluate the antioxidant capacity, the antimicrobial proprieties of algae Ascophyllum nodosum and Schizochytrium spp. against one of major swine enteric pathogen Escherichia coli O138 by broth macro-dilution method in Luria–Bertani (LB) medium. The antimicrobial effect of the algal extracts at supplementation of 0.12%, 0.06% and 0.03% (v/v) on E. coli O138, genetically characterised by PCR, was evaluated by following the bacterial growth. The antioxidant activity was determined by the ABTS Radical Cation Decolorisation Assay. In particular, the log10 E. coli used as control resulted significantly higher than 0.12% at 3 hours (8.82 ± 0.07 and 8.18 ± 0.07 log10 cells/mL, respectively; p<.01) suggesting an inhibitory activity related to the dose. No effect activity was observed with Schizochytrium spp. against E. coli growth. A. nodosum and Schizochytrium spp. exhibited antioxidant capacity (p<.05). The combination of them (1:1) exhibited antioxidant activity suggesting a synergistic effect (p<.05). The different proprieties of algal species that can modulate the O138 E. coli growth, one of the major pathogen of swine species, together with the antioxidant capacity, make them a promising functional feed additive to improve the gut health, therefore further studies are needed to confirm these activities in vivo. Highlights The aim of the study was to evaluate the antimicrobial and antioxidant proprieties of two species of algae: Ascophyllum nodosum and Schizochytrium spp. Ascophyllum nodosum revealed antimicrobial effect against Escherichia coli O138. Both algae exhibited antioxidant capacity also with a synergistic effect.

High-Throughput Preparation of Antibacterial Polymers from Natural Product Derivatives via the Hantzsch Reaction.

SummaryThe Hantzsch and free-radical polymerization reactions were combined in a one-pot high-throughput (HTP) system to simultaneously prepare 30 unique polymers in parallel. Six aldehydes derived from natural products were used as the starting materials to rapidly prepare the library of 30 poly(1,4-dihydropyridines). From this library, HTP evaluation methods led to the identification of an antibacterial polymer. Mechanistic studies revealed that the dihydropyridine group in the polymer side-chain structure plays an important role in resisting bacterial attachment to the polymer surface, thus leading to the antibacterial function of this polymer. This research demonstrates the value of multicomponent reactions (MCRs) in interdisciplinary fields by discovering functional polymers for possible practical applications. It also provides insights to further developing new functional polymers using MCRs and HTP methods with important implications in organic chemistry, polymer chemistry, and materials science.

Optimization of medium composition for propagation of recombinant Escherichia coli

AbstractRecombinant protein production in heterologous hosts often seems a simpler and more effective way than its production by natural producer. The secretion of recombinant protein inEscherichia colihas many advantages comparing to than in insect or mammalian cells. The important factor for high-level recombinant protein production is the sufficient amount ofE. colibiomass. Therefore, the aim of this study was to optimize the composition of propagation medium resulting in the maximum biomass yield of recombinantE. colias the part of fermentation strategy for neuraminidase (NA) production. Three independent variables including glucose, asparagine and phosphate concentrations, and four dependent variables, such as biomass yield, residual concentrations of glucose or asparagine and pH of the propagation medium after fermentation, were chosen to the optimization by Response Surface Methodology (RSM). The optimal conditions for the maximum biomass yield expressed as dry cell weight (DCW) (16.57±0.55 g DCW.L−1) were as follows: glucose concentration of 39.37 mM, asparagine concentration of 62.68 mM and phosphate concentration of 14.80 mM. For this model, the predicted values for the responses are close to the experimental values. The yield of desired pET15b-neu plasmid fromE. colicells cultivated in optimized propagation medium was almost 23 % higher than in commonly used Luria-Bertani (LB) medium suggesting that asparagine may be involved in the induction of plasmid amplification.

New source of cellulase production using a metagenomic technique

The cellulase enzymes with high effectiveness under conditions agreeable to the industrial processes necessities are one of the keys for the successful development of chemical and drug synthesis. The soil metagenome is an affluent source for the discovery of new natural products. The objective of the current study was to identify the isolated functional gene(s) of the cellulase enzyme by using metagenomics. The plan of work composed of collection of different soil samples, isolation of total DNA, fragmentation, cloning, and expression of the isolated gene(s) in the suitable host microorganism. The total genomic DNA was extracted using a kit (QIAGEN), and then digested by different restriction enzymes BamHI. The digested fragments ranging from ~300-5000 bp were ligated, cloned into pUC19 vector, and then transformed into Escherichia coli DH5α. The resulting clones were screened as cellulase producers using a qualitative method. The positive clones which showed hydrolysis on the plate were screened once more in Luria-Bertani (LB) medium. The plasmids were isolated and then tested using universal primer (M13), to detect the fragment size and sequence for the Polymerase Chain Reaction (PCR) products. This study establishes an effortless and professional method for cloning of recent cellulase genes through ecological metagenomes. In the outlook, the metagenomic guide approachs may be functional to the elevated selection of novel cellulase from the environment.

English

Heat shock proteins (HSPs) are a family of proteins that are produced by all living organisms in response to exposure to stressful condition. The present research aims to study the physiology of Bacillus spp. (SUBB01) under aeration in diverse culture media and temperature at 47, 48, 49, 50, 52, 53 and 54°C. Bacterial growth was measured through enumeration of the viable and culturable growing cells that are capable of producing the colony-forming units (CFUs) on Luria–Bertani (LB) and nutrient agar (NA) plates for 24 to 48 h. The work also focused on the repercussion of Bacillus spp. against excessive temperatures (37, 45 and 50°C) in minimal media under shaking condition. Isolated microbes were demonstrated under a light microscope to observe their cellular morphology, shape and organization. There was high-temperature shock up to 50°C in both LB, NA, and agar media, with the presence of a demanding defense mechanism against heat shock in these bacterial cells. However, further molecular studies on the genetic makeup of such stress responses as well as the growth retrieval mechanisms of Bacillus spp. through the exogenous organic factors would be very important. Key words: Heat stress, Bacillus spp. (SUBB01), viable cells, minimal media, bacterial growth.

Water microbial disinfection via supported nAg/Kaolin in a fixed-bed reactor configuration

In this paper we have investigated and demonstrated the antimicrobial capabilities of nano-silver-4 wt%-kaolin (nAg-4-Kn) composite supported on borosilicate glass beads (BGB). Tests have been conducted in a fixed bed reactor on effluent from the secondary clarifier of a conventional wastewater treatment plant (WWTP) in Abu Dhabi, United Arab Emirates (UAE). The prepared BGB with immobilized nAg-4-Kn (nAg-4-Kn/BGB) were characterized using the RAMAN spectroscopy, TEM equipped with EDX and Focus Ion Beam Scanning Electron Microscope (FIB-SEM) techniques. The rate of disinfection was assessed through Luria-Bertani (LB)-agar plate cell counting technique. The results showed complete disinfection after few hours, which was preserved even after several days in repeated runs. The nAg-4-Kn/BGB was reused, demonstrating that the immobilization of nAg-4-Kn was stably achieved, and the activity and integrity of the composites on the BGB were preserved. First order disinfection kinetic constants were estimated to be 2.76 cm h−1 and 2.56 cm h−1 in two consecutive runs. Analyses of the beads after the experiments showed minor losses of nAg from the kaolin matrix thereby corroborating reusability of these materials.

Polyurethane Degrading Bacteria Isolated from Decayed Teak Wood (Tectona grandis Linn. f.)

Polyurethane is a class of polymers characterized by their substantial tensile strength and high melting points which enables them to be extremely durable. Despite its xenobiotic origin, polyure-thane has been found susceptible to biodegradation by microorgan-isms. The main object of this research was to investigate polyurethane degradation by the bacterium isolated from decayed teak wood (Tec-tona grandis Linn. f.). Polyurethane degrading bacteria were isolated using selective medium in the form of LB (Luria Bertani) with the ad-dition of 0.3% Impranil. Bacterial biodegradation activities shown by the changes of polyurethane structure analyzed by FT-IR spectrosco-py. The bacterial identification was carried out based on observations on the morphological characteristics of the colonies, cell morphology and sequences of 16S rRNA encoding genes. One of the potential iso-late that successfully isolated was K9, which demonstrates the disap-pearance of the 1735/cm peak of the characteristic function urethane in the FT-IR analysis. Analysis of the 16S rRNA encoding gene showed that the potential isolate having 98% similarity index to Bacillus safen-sis strain FO-36b. The activity shown by the isolate suggests that the bacteria could be a promising agent for polyurethane degradation.

Characterization and antifungal activity of the yellow pigment produced by a Bacillus sp. DBS4 isolated from the lichen Dirinaria agealita.

This study emphasis the production of yellow pigment from endolichenic Bacillus sp. isolated from the lichen Dirinaria aegialita (Afzel. ex Ach.) B.J. Moore. Yellow pigment-producing twenty different strains were investigated. The hyperactive pigment-producing bacterial strain was identified as Bacillus gibsonii based on 99 % sequence similarity. Maximum bacterial pigment production appeared in Luria Bertani medium. Methanol extraction of the pigment and its partial purification using TLC was carried out. Furthermore, isolated pigments were characterized using UV-visible spectroscopy, FTIR spectroscopy, and GC-MS results related to the possibility of the carotenoid occurrence. The pigment also exhibited efficient antifungal activity against selected fungal pathogens of economic importance. Likewise, the pigment extract evaluated for the total antioxidant potential using Phosphomolybdenum and Ferric reducing antioxidant power assay and the results represented in Ascorbic Acid Equivalent (AAE)- 21.45 ± 1.212 mg/mL. The SC50 of the pigment extract found to be 75.125 ± 0.18 µg/ml determined by the ABTS assay.

Low-molecular-weight chitosan relieves enterotoxigenic Escherichia coli-induced growth retardation in weaned pigs

The objective of the present study was to evaluate the effects of low-molecular-weight chitosan (LMWC) on the growth performance, immune responses and intestinal health of weaned pigs challenged by enterotoxigenic Escherichia coli (ETEC). A total of 32 weaned pigs were randomly allocated to four treatments: non-challenged (fed with basal diet), ETEC-challenged (fed with basal diet) and ETEC-challenged plus 50 or 100 mg/kg LMWC supplementation, respectively. After 11 days feeding, the non-challenged pigs were infused with sterilised Luria–Bertani culture, while the remaining pigs were infused with 2.6 × 1011 colony-forming units of ETEC. At 3 days post-challenge, all pigs were administered d-xylose at 0.1 g/kg body weight. One hour later, blood samples were obtained, and the pigs then euthanised to collect intestinal samples. Data showed that only 100 mg/kg LMWC supplementation attenuated (P < 0.05) the average daily gain reduction caused by ETEC. Furthermore, besides the decreased (P < 0.05) serum tumour necrosis factor-α and immunoglobulin (Ig) G concentrations detected in ETEC-challenged pigs supplemented with LMWC at 50 or 100 mg/kg, the higher dose (100 mg/kg) also decreased (P < 0.05) the serum IgM concentration and increased (P < 0.05) the villus height and villus height-to-crypt depth ratio in both the jejunum and ileum, and the sucrase activity in the ileal mucosa. Moreover, LMWC supplementation (50 or 100 mg/kg) in ETEC-challenged pigs elevated (P < 0.05) the mRNA levels of jejunal mucosal peptide transporter 1 and ileal mucosal peptide transporter 1, divalent metal transporter 1 and zinc transporter 1, and decreased (P < 0.05) the ileal and caecal E. coli abundances, while 100 mg/kg LMWC additionally elevated (P < 0.05) the ileal Bacillus abundance, and caecal and colonic Bifidobacterium abundances. These results suggest that LMWC helps alleviate ETEC-induced growth retardation in weaned pigs, which could be associated with the inhibition of the immune responses and improved intestinal health.

A test strip platform based on a whole-cell microbial biosensor for simultaneous on-site detection of total inorganic mercury pollutants in cosmetics without the need for predigestion

Mercury pollutants such as mercuric chloride (HgCl2), mercurous chloride (Hg2Cl2) and mercuric ammonium chloride (Hg(NH2)Cl) are often found in cosmetics. Previous attempts at the on-site detection of mercury were hindered by the complicated and dangerous pretreatment procedure of converting various forms of mercury to Hg (II) ions. In this study, a test strip platform was developed based on a whole-cell microbial biosensor for the simultaneous detection of soluble and insoluble inorganic mercury pollutants in cosmetics without the need for predigestion. The genetic circuits with constitutively expressed MerR as sensor proteins and inducible red fluorescent protein (RFP) as the reporter were introduced into Escherichia coli to construct the mercury detection biosensor. The RFP fluorescence intensity of this biosensor showed a excellent linear relationship (R2=0.9848) with the Hg (II) concentration ranging from 50nM to 10μM in Luria-Bertani (LB) broth. Further research indicated that this biosensor could respond not only to Hg (II) ions but also to insoluble Hg2Cl2 and Hg2Cl2. The transcriptomic results confirmed the mercury conversion ability of the whole-cell biosensor from a gene expression perspective. This biosensor was embedded on filter paper to form a test strip, which could be used to determine whether the total inorganic mercury pollutants in cosmetics exceeded 1mg/kg. Therefore, this strip provided a low cost, easy-to-use, and instrument-independent method for the detection of mercury pollution in cosmetics, while this study revealed the unique advantages of microbial biosensors in the automatic bioconversion of targets.

The Potential Applications of Bacillus sp. and Pseudomonas sp. Strains with Antimicrobial Activity against Phytopathogens, in Waste Oils and the Bioremediation of Hydrocarbons

Biodegradation is one of the primary mechanisms for the elimination of petroleum and other hydrocarbon pollutants from the environment. This study presents the results obtained with two newly isolated microorganisms and their potential applications in bioremediation, agriculture, and industrial fields. Twenty-five strains of microorganisms were isolated from plant materials and were subject to a selection process on the basis of antimicrobial activity. Two bacterial strains, respectively Bacillus mycoides (Bm) and Pseudomonas putida (B1), were selected for further experiments, based on the largest inhibition zones against the phytopathogens Erwinia carotovora and Xanthomonas campestris. The production of biosurfactants and enzymes was evaluated in specific media. In order to assess the production of biosurfactants, submerged bioprocesses were carried out on Yeast Malt Peptone Glucose (YMPG), M44, Luria-Bertani (LB), and King B media (KB); the supernatants were used to form emulsions with heptane, octane, and sunflower oil, and the emulsifying indices were determined.

A Label-Free, Non-Intrusive, and Rapid Monitoring of Bacterial Growth on Solid Medium Using Microwave Biosensor.

Microwave resonator sensors are attractive for their contactless and label-free capability of monitoring bacterial growth in liquid media. This paper outlines a new label-free microwave biosensor based on a pair of planar split ring resonators for non-invasive monitoring of bacterial growth on a solid agar media. The sensor is comprised of two split ring resonators with slightly different resonant frequencies for differential operation. The transmission coefficient (S21) of the sensor is considered as the sensor's response with a designed and measured quality factor above 200 to ensure a high-resolution operation of the biosensor. Two resonant frequencies of 1.95 and 2.11 GHz represent the sensing signal and the reference signal, respectively. The developed sensor demonstrates high performance in monitoring the growth dynamics of Escherichia coli (E. coli) on Luria-Bertani (LB) agar with 4 mm thickness. The sensor's resonant amplitude response demonstrated 0.5 dB variation corresponding to the bacterial growth over 48 hours when bacteria were spread on LB agar starting with initial OD600 = 1.5. Moreover, 0.6 dB change in the sensor's response was observed over 96 hours of bacterial growth starting with an initial OD600 = 1.17 spotted on LB agar. The measured results fit well to the curves created using Richards' bacterial growth model, showing the strength of the sensor as a potential candidate for use in predictive food microbiology systems.

Isolation and Characterization of a Novel Diethylstilbestrol-Degrading Bacillus subtilis JF and Biochemical Degradation Metabolite Analysis.

Diethylstilbestrol (DES) can adversely affect the immune system of developing fetuses or even elicit toxic responses such as nerve toxicity and genotoxicity in human beings, thereby warranting methods to remove DES from the environments. The present study characterized a novel DES-degrading Bacillus subtilis JF and analyzed the degradation metabolites. The strain was collected at the China General Microbiological Culture Collection Center (Collection number: CGMCC 7950). The environmental effects, such as DES concentrations, pH levels, and temperature, on the strain’s degradation ability were tested. Degradation metabolites of DES by strain JF were analyzed via high performance liquid chromatography (HPLC) and liquid-chromatography time of flight mass spectrometry (LC-TOF-MS). Results indicated that B. subtilis JF can effectively degrade DES within a concentration of 25–200 mg/L. Increasing pH levels (pH > 7) are reported to increase the degradation rate of DES by the strain. The optimal temperature for strain JF to degrade DES was identified as 45°C. In this study, 4, 4′-hexene estrogen quinones (DESQ) and DES-4-semiquinone were speculated as two degradation metabolites of DES, and both can be completely degraded by strain JF. A slight reduction of DES in the blank system [DES cultured in Luria-Bertani (LB) medium without strain JF] was observed in this study. The reduction trend in the blank system only occurred during the first few days (about 4 days) and was considerably lesser than the decomposition and transformation effect of DES via strain JF. Furthermore, the metabolite DESQ could not be further decomposed in blank LB medium without strain JF. All the results demonstrate that complete degradation of DES in the fermentation broth occurs due to the function of strain JF rather than organic decomposition. In conclusion, the high efficiency of degradation and the potential to degrade DES completely indicates that strain JF has potential for the bioremediation of DES-contaminated environments (soil, river, and so on) and fermented foods.

Rapid and solitary production of mono-rhamnolipid biosurfactant and biofilm inhibiting pyocyanin by a taxonomic outlier Pseudomonas aeruginosa strain CR1

Biosurfactant - Rhamnolipids (RLs) and antibacterial toxin - pyocyanin (PYO) produced by Pseudomonas aeruginosa strains have great potential for biotechnological applications. Generally, RLs are produced as a mixture of di-rhamnolipids (di-RLs) and mono-rhamnolipids (mono-RLs). Mono-RLs possess superior emulsification and antimicrobial properties and are costlier than di-RLs. In this study, a taxonomic outlier P. aeruginosa strain CR1 isolated from rhizosphere soil was explored for mono-RLs and PYO production. Phylogenetically strain CR1 resembles avirulent outlier P. aeruginosa strain ATCC9027, lacks archetypical virulence genes and harbors unique pathways for the synthesis of solely mono-RLs and PYO. Strain CR1 produced RL biosurfactant which efficiently emulsified hydrocarbons, showed hemolysis and inhibited Bacillus subtilis. At 37 °C, strain CR1 exclusively produced 21.77 g L−1 and 19.22 g L−1 rhamnolipid in glycerol amended Luria Bertani (LB) medium and basal medium amended with rice bran oil, respectively after 54 h growth. Besides RL production was unaffected under varying nitrogen sources. Structural characterization using FTIR, TLC, and LC–MS confirmed that strain CR1 exclusively produced mono-RLs, majorly dominated by Rha-C10-C10, Rha-C10-C8, and CH3-Rha-C12:2-C10:1. The compound was stable over a wide pH range (4–12), salinity (25%) and 100 °C indicating its applicability under harsh environmental conditions. In addition, strain CR1 produced 4.5 μg mL−1 PYO, which could efficiently inhibit biofilm formation by Bacillus species. The environmental outlier strain CR1 can be used for the industrial production of biotechnologically important mono-RLs and PYO.

Evaluation of the Strain Bacillus amyloliquefaciens YP6 in Phoxim Degradation via Transcriptomic Data and Product Analysis.

Phoxim, a type of organophosphorus pesticide (OP), is widely used in both agriculture and fisheries. The persistence of phoxim has caused serious environmental pollution problems. In this study, Bacillus amyloliquefaciens YP6 (YP6), which is capable of promoting plant growth and degrading broad-spectrum OPs, was used to study phoxim degradation. Different culture media were applied to evaluate the growth and phoxim degradation of YP6. YP6 can grow rapidly and degrade phoxim efficiently in Luria–Bertani broth (LB broth) medium. Furthermore, it can also utilize phoxim as the sole phosphorus source in a mineral salt medium. Response surface methodology was performed to optimize the degradation conditions of phoxim by YP6 in LB broth medium. The optimum biodegradation conditions were 40 °C, pH 7.20, and an inoculum size of 4.17% (v/v). The phoxim metabolites, O,O-diethylthiophosphoric ester, phoxom, and α-cyanobenzylideneaminooxy phosphonic acid, were confirmed by liquid chromatography–mass spectrometry. Meanwhile, transcriptome analysis and qRT-PCR were performed to give insight into the phoxim-stress response at the transcriptome level. The hydrolase-, oxidase-, and NADPH-cytochrome P450 reductase-encoding genes were significantly upregulated for phoxim hydrolysis, sulfoxidation, and o-dealkylation. Furthermore, the phoxim biodegradation pathways by YP6 were proposed, for the first time, based on transcriptomic data and product analysis.

A simple protocol for expression of isotope-labeled proteins in Escherichia coli grown in shaker flasks at high cell density.

Protein expression in E. coli grown in shaker flasks is a routine and pivotal tool in many research laboratories. To maximize protein yields, cells are normally induced in the middle of the linear growth phase, typically at an OD600 of ≤ 1 for cells grown in Luria-Bertani (LB) medium at 37°C. We recently showed that the E. coli linear growth phase can be extended to higher cell density when cells are cultured under less than optimal conditions such as in minimal medium and/or at lower temperatures. Maximizing the yield of protein per unit volume of culture is important for reducing the costs, especially when isotopically labeling is required. Here, we present a modified minimal medium and a simple protocol that can increase the protein yield up to fourfold in a pH-stabilized LB medium and up to sevenfold in a modified M9+ medium (M9++). When M9++ medium coupled with the high density (OD600 ~ 6) cell growth protocol are used to express uniformly 15N- or 15N/13C-labeled proteins, the amount of 15NH4Cl and 13C6-glucose for a given cell mass is reduced by 50% and ~ 65%, respectively, relative to the traditional low density (OD600 ~ 1) cell growth protocol with M9 medium; the inclusion of 0.1% LB in the minimal medium permits a reduction in the concentration of both the trace element solution and MgCl2, which can cause precipitation. Mass data indicate that inclusion of 0.1% LB does not significantly affect the isotope enrichment level.

Growth optimization of Bacillus subtilis 11A isolated from Indonesian native chicken (Gallus domesticus) for bacteriocin production

This study aimed to obtain the optimal growth of Bacillus subtilis 11A isolated from Indonesian native chicken (Gallus domesticus). The growth optimization included pH medium conditions (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5) and different incubation temperatures (31, 33, 35, 37, 39, 41°C). Isolate was incubated for 12 hours using Luria Bertani broth. Optical density was observed using spectrophotometer (λ600) at 0 and 12 hours of incubations. Bacteriocin activity against pathogens Staphylococcus aureus, Salmonella typhimurium and Escherichia coli was determined using a well-diffused method. Data was statistically analyzed using a one-way analysis of variance (ANOVA) followed with Duncan’s New Multiple Range Test (DMRT) to distinguish the treatment means. The results showed that the optimum pH for bacterial growth was at pH 6.5. Optimum temperature for bacterial growth was at 39°C. The highest bacteriocin activity of B. subtilis 11A against indicator pathogens was resulted at stationary phase (1906.14, 2179.79, 2343.07 AU/ml). It can be concluded that B. subtilis 11A isolated from Indonesian native chicken grew optimally for bacteriocin production at pH 6.5 and temperature 39°C. The highest bacteriocin production and activity produced by Bacillus subtilis 11A was at stationary phase.

Potensi Mikroorganisme Resisten dari Pantai Timur Surabaya Sebagai Biosorben Seng

East coast of Surabaya is one of region in Indonesia which contaminated by heavy metals, such as zinc. Zinc is one of essential heavy metals for organisms but be dangerous for human if comsumed in high concentration. So, it is important to to solve the problem to reduce the concentration of zinc in east coast of Surabaya through bioremediation using microorganism. Zinc resistant microorganism could be isolated from zinc contaminated region and be used for bioremediation agent because some microorganism developed resistance mechanism to zinc by bioaccumulation or biosorption. The aims of this study are to isolate and characterize zinc resistant microorganism from east coast of Surabaya. The most resistant microorganism will be observed for the growth in medium supplemented with zinc and without zinc. The potency of the chosen isolate to biosorb of zinc was also analyzed. Water sample from east coast of Surabaya was serries dilluted and innoculated on Luria Bertani Agar medium using spread plated method. Microbial resistance to zinc was analyzed by grown the cell on medium containing various concentration of zinc to determine Minimum Inhibitory Concentration (MIC) value of each microbial isolate. Cell turbidity will be observed in the chosen isolate using spectrophotometer in wave length of 600 nm to get the growth curve. The potency of the chosen isolate to biosorb of zinc was analyzed using atomic absorption spectrophotometer. From this study, six bacteria and three yeast have been isolated from east coast of Surabaya, designated as PmbZn1, PmbZn2, PmbZn3, PmbZn4, PmbZn5, PmbZn6, PmbZn7, PmbZn8, dan PmbZn9. All isolates could grow on medium containing 8 mM ZnSO4. The most resistant bacterial was isolate PmbZn5 with the MIC of 9 mM ZnSO4. The potency of the isolate to biosorb of zinc was 94,79 and 93,57% in medium containing 4 mM and 7 mM ZnSO4, respectively.Key words: Bacteria, biosorb, resistant, yeast, zinc.

KLONING GEN carB Salmonella typhi MENGGUNAKAN VEKTOR EKSPRESI pET-16b dan SEL INANG Escherichia coli JM109

The effort of cloning the Lister strain of Salmonella typhi (NCTC 786, BCC 712) carB gene using pET-16b expression vector and E. coli JM109 as host cell has been done. The carB gene and the pET-16b expression vector were both prepared from their recombinant plasmid digested using BamHI and NdeI as restriction enzymes. The pG-carB-11-ST recombinant plasmid was isolated from Escherichia coli XL10(pG-carB-11-ST) and the pET-carA-ST recombinant plasmid was from Escherichia coli DH5α(pET-carA-ST). After being ligated (in a ratio of vector:gene of 1:3, 1:5 and 1:6) in the presence of T4 Bacteriofage DNA Ligase, the ligation mixture was used to transform Escherichia coli JM109 cells and plated out onto Luria Bertani medium containing ampicillin. An amount of 369 produced colonies were screened for the presence of the appropriate recombinant plasmid using combination of plasmid miniprep and agarose gel electrophoresis. None of the recombinant plasmids being suspected to carry the Salmonella typhi carB gene. It is suggested to repeat cloning process using E. coli JM109 or using E. coli XL10 as host cell which were known to have large cloning efficiency and can be used for large plasmids up to 25 kb.

Antibacterial Interactions of Colloid Nanosilver with Eugenol and Food Ingredients

This study was conducted to investigate antibacterial properties of the colloidal silver nanoparticles (SNPs) and eugenol, alone and in combination, on Staphylococcus aureus and Salmonella Typhimurium and their interactions with food constituents (fat, protein, and carbohydrate). We examined antibacterial activities of SNPs and eugenol in Luria-Bertani (LB) broth and 1.5 and 3% fat ultrahigh-temperature (UHT) milk. MICs of eugenol and SNPs (particle size of 31.3 nm) were also investigated in the presence of sunflower oil, meat extract, and starch at concentrations of 2, 5, and 10% to examine the interactions between food constituents and antimicrobial agents. MICs and MBCs of eugenol and SNPs for both bacteria were at 2,500 and 25 μg/mL, respectively. Combinations of the two substances had additive and synergistic effects on Salmonella Typhimurium and S. aureus, respectively. Both compounds had bactericidal activity. In food matrices, results indicated that eugenol only in sunflower oil at 5 and 10% concentrations had significant antibacterial activity. A similar result was achieved for SNPs with 10% meat extract. In LB broth, eugenol at 2,500 and 5,000 μg/mL achieved 6-log reductions in the microbial population of both bacteria after 3 h, while SNPs achieved the same effect after 9 h. In UHT milk with 1.5% fat, eugenol at 5,000 μg/mL and SNPs at 25 μg/mL achieved 6-log reductions in bacterial populations after 24 h. Thus, the antimicrobial activity of both eugenol and SNPs depended on the medium in which the experiment was conducted, and the combination of both antimicrobial agents increased the antimicrobial effect.