Antimicrobial study of synthesized ferulate esters was successfully carried out against both Gram-positive (Bacillus subtilis, Staphylococcus epidermidis and Staphylococcus aureus) and Gram-negative (Salmonella typhimurium, Klabsiella Pneumoniae and Escherichia coli) bacteria. Screening by agar diffusion test was employed for preliminary features followed by Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) assays. Ferulate esters demonstrated anti-bacterial activity with diameter of inhibition zones ranged from 8 to 16 mm for S. typhimurium, E. coli, S. epidermidis and S. aureus. For MIC assay, the minimum concentration of ferulate esters against E. coli was 50% followed by S. typhimurium (33.3%), S. epidermidis (20%) and S. aureus (20%). While for MBC assay, ferulate esters only showed bacteriostatic effect. Therefore, this research proved that the ferulate esters possess moderate antibacterial inhibition against both types of bacteria.

Extracellular proteins secreted by diverse bacteria with broad spectrum of antimicrobial activity can be used as an alternative treatment against antibiotic resistant and spoilage microorganisms. However, the extracellular proteins produced by bacteria are affected by environmental stress factors and growth conditions. Therefore, antimicrobial activity of extracellular proteins secreted by Bacillus subtilis ATCC21332 in the presence of Cymbopogon flexuosus as stress inducer and cultivated in different growth conditions was evaluated against four selected strains of Gram-positive and Gram-negative bacteria, including Bacillus cereus, Escherichia coli, Shigella sonnei and Staphylococcus epidermidis by agar-well diffusion test and microdilution assay. Agar-well diffusion test was carried out to identify the antimicrobial susceptibility, while microdilution assay was done to determine the inhibition and bactericidal effects. B. subtilis ATCC21332 cells were treated with a low concentration (0.01 MIC) of C. flexuosus essential oil and cultured in various growth conditions such as different pH media (pH 6, pH 7 and pH 8) ornutrient sources, including 1% (w/w) of carbon (glucose, sucrose and starch), 1% (w/w) of nitrogen (casein, gelatin and urea) and 1% (w/w) of inorganic salt (sodium nitrate, calcium chloride and sodium dihydrogen phosphate). After 72 h of fermentation at 30°C, the secreted extracellular proteins were then extracted and analysed for antimicrobial activity. Results showed that the extracellular proteins secreted by stress induced B. subtilis ATCC21332 and cultivated in media with 1% of sucrose exhibited antimicrobial activity against two bacterial strains which are S. epidermidis and B. cereus with 8.0 ± 0.8 mm and 6.0 ± 0.0 mm of inhibition zone respectively. Meanwhile, the extracellular proteins extracted from bacteria cultured in each different media supplemented either with 1% of casein, 1 % of sodium nitrate and 1% of calcium chloride only showed antimicrobial activity against B. cereus with inhibition zone of 7.5 ± 0.5 mm, 8.0 ± 1.0 mm and 9.0 ± 0.0 mm respectively. Although the extracellular proteins indicated inhibitory effect against B. cereus and S. epidermidis, however the proteins only exhibited bactericidal effect towards B. cereus. Keywords: Antimicrobial Activity, Extracellular Proteins, Bacillus subtilis ATCC21332, Cymbopogon flexuosus Essential Oil.

Protein production by bacteria might be increased in stressful conditions such as in the presence of antimicrobial agents. Many studies proved that antibiotics or antimicrobial agents at low concentration are able to activate or repress gene transcription process in bacteria. However, there are still few studies on potential of natural antimicrobial compounds such as Cymbopogon essential oils acting as specific chemical signal that can trigger biological functions of bacteria. Therefore, this study aims to explore the potential of natural antimicrobial compound (Cymbopogon flexuosus and Cymbopogon nardus) at low concentration in regulating proteins production by Lactobacillus plantarum ATCC8014. The bacteria cells of L. plantarum ATCC8014 are exposed to Cymbopogon essential oils at low concentration in fermentation process for 48 hours at 37°C. SDS-PAGE analysis showed that a new intracellular protein with approximate size of 40 kDa was produced by L. plantarum ATCC8014 after being enhanced with C. nardus essential oil. Besides, the intracellular proteins, each with approximate size of 85 kDa, 45 kDa and 28 kDa synthesized by L. plantarum ATCC8014 prior to inducing with C. nardus or C. flexuosus were expressed differently. Some of the intracellular proteins were highly expressed and some of the proteins were repressed based on the intensity of protein bands appeared. Hence, L. plantarum ATCC8014 in the presence of Cymbopogon essential oils at low concentration could regulate the intracellular proteins production. The isolated protein also showed antimicrobial activity against selected Gram-positive and Gram-negative bacteria.

Several studies have reported that sub inhibitory concentrations of antibiotics or antimicrobials are capable to modulate bacteria transcription processes. Therefore, the bacteria might have introduced new proteins in mild stress surroundings like in the presence of antimicrobial agents at low concentrations. However, there are limited elucidations about unexpected ability of natural antimicrobial compounds to become a signaling agent capable to induce biological functions in bacteria at low concentrations. Thus, this present study aims to e'xplore the proteins production by Lactobacillus plantarum ATCC 8014 in the presence of Allium sativum at sub-minimal inhibitory concentration (sub-MIC). The Minimum Inhibition Concentration (MIC) of A. sativum against L. plantarum was 33.33% from microdilution assay. L. plantarum cells were treated with A. sativum at sub-MIC (0.05 x MIC) in the fermentation process. Three new protein bands (approximate size of 97.83 kD, 53.56 kD and 46.71 kD) were detected by SDS-PAGE profile for the treated bacteria. LC-MSIMS analysis identified 11 possible proteins from the three protein bands expressed in mild stress condition. The proteins showed antimicrobial activity toward several Gram-positive and I Gram-negative bacteria. Hence, L. plantarum ATCC 8014 in mild stress condition with the presence of 0.05 x MIC A. sativum could regulate bioactive proteins production.

A novel and simple dual lipases system was effectively developed in attaining high performance of ferulate esters by transesterification between ethyl ferulate and olive oil. Enzyme screening revealed 1: 9 w/w of Novozym 435-Lipozyme RMIM to be the most efficient lipases ratio for the reaction synthesis. Response surface methodology (RSM) based on four-factor-five-level central composite design (CCD) was used to study interactive effects of reaction factors: time, lipase dosage, ratio substrates and temperature. A high percent conversion of 94.03 % was achieved under the optimum conditions of 12 h, 87 mg lipases, 1: 2 ratio substrates and 60 áµ’C, which compared well with the maximum predicted value of 94.83 %.

Antimicrobials agents at low concentration could stress out bacteria by inducing proteins production via regulating the transcription and translation process. The interruption of bacterial quorum sensing (QS), or cell-to-cell communication is known to have the potential to weaken the bacterial pathogenicity by inhibiting their communication. This study aims to explore the potential of synthetic antimicrobial compound, ethyl pentanoate in stimulating proteins production by Bacillus subtilis ATCC11774 as well as to determine the anti-QS activity of microbial proteins produced. The bacterial cells were exposed to 0.01 MIC of ethyl pentanoate in fermentation process at 37°C for 48 h and 72 h respectively. The proteins produced were further isolated and analyzed by using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Results showed that a new extracellular protein with approximate size of 15 kDa was produced by B. subtilis ATCC11774 after being treated with ethyl pentanoate at 37°C for 48 h and 72h. Despites of new protein band production, there was a deletion of protein band with approximate size of 18 kDa on protein synthesized at 72 h of fermentation. Whilst, the anti-QS activity of microbial proteins produced by B. subtilis at 37°C for 48 h and 72 h was determined by agar- wells diffusion assay, resulting “halo” inhibition zone ranged from 10.00 ± 1.00 to 10.33 ± 0.56 in diameter. Therefore, B. subtilis ATCC11774 in the presence of ethyl pentanoate at 0.01 MIC could regulate the extracellular protein production and expression. The isolated protein also exhibited anti-QS activity.

Blood clotting is an emerging problem that seriously threatens the health of human beings such as strokes and heart attacks. For the treatment, anti-coagulants are used in which it could prevent and interrupt the process involved in the formation of blood clots. Microbial proteases have now been attracted since it is cost effective and no side effects than typical anti-coagulant agents. The protease enzymes were discovered from many microorganisms including Bacillus sp. Previous study reported that B. subtilis could produce a serine protease, known as Bacillopeptidase F with anti-coagulant activity. However, study on anti-coagulant activity of microbial protein secreted by B. subtilis after being induced with Cymbopogonflexuosus essential oil and cultured at difference pH media is still limited. In this present study, B. subtilis ATCC21332 cells were treated with a low concentration (0.01 MIC) of C. flexuosus essential oil and cultured in difference pH media (pH 6, pH 7 and pH 8) at 30°C for 72 h of fermentation. The extracellular proteins were then extracted and precipitated by using 80% of ammonium sulfate before being further identified using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE) and tested for anti-coagulant activity. SDS-PAGE analysis exhibited a proteins profile with a band in approximate size of 30 kDa was appeared for the treated bacteria with C. flexuosus essential oil and cultivated at three difference pH of media. The extracellular proteins secreted by B. subtilis ATCC21332 after being treated with 0.01 MIC of C. flexuosus essential oil and cultivated either at pH 6 or pH 7 or pH 8 could also prevent blood clotting. However, the extracellular proteins produced by B. subtilis ATCC21332 without inducing with 0.01 MIC of C. flexuosus essential oil only exhibited anti-coagulant activity when the bacterial cells were cultured at pH 7. As a conclusion, B. subtilis ATCC21332 can be enhanced to produce anti-coagulant enzymes after being treated with low concentration of C. flexuosus essential oil and cultivated in natural pH media or even in slightly basic or slightly acidic environment. Further study should be done to purify, identify and analyze the anti-coagulant enzymes from B. subtilis ATCC21332. Keywords: Cymbopogonflexuosus Essential Oil, pH effect, Anti-Coagulants Activity, Extracellular Proteins, Bacillus subtilis ATCC21332.

Purpose of study: Bacteria can naturally produce pigments that can be useful for various applications as they possess antimicrobial metabolites among other numerous benefits towards the human health. This study was carried out to identify the species of marine bacterial isolates PMA, PM3C1 and PM5C1 exhibiting yellow, orange and green colors respectively. Methodology: The current study is using Polymerase Chain Reaction (PCR) amplification and sequence analysis of their 16S rRNA gene. The stability of pigments extracted from the bacterial samples was also analyzed against different temperature and light conditions. Main Findings: Sequence alignment using BLAST revealed that the yellow, orange, and green-pigmented bacteria have 84% similarity with Staphylococcus aureus, 85% similarity with Exiguobacterium profundum and 95% similarity with Pseudomonas aeruginosa respectively. The green pigment showed major changes in color following exposure to sunlight and fluorescent light, and when incubated at 24°C and 50°C. Exposure to direct sunlight also results in the reduction of color for the yellow and orange extracts, while no effect was observed for both pigments under fluorescent light. Incubation at 50°C results in the reduction of the orange color, while the yellow pigment was observed to be unaffected suggesting its stability at high temperature. Implications: Natural pigments production can provide many advantages including reduction of pollution generation, ease of disposal and other benefits to the human health.

The effects of important reaction parameters for enhancing flavour esters formation through lipase-catalyzed reaction were investigated in this study. Various commercial immobilized lipases were used to catalyze the esterification reaction between short-chain fatty acids and alcohols to produce flavour esters which are nonyl caprylate and ethyl valerate which differ in chain length of esters. These synthetic flavour esters with fruity notes are widely used in food, cosmetic and pharmaceutical industries. The effect of various reaction parameters was optimized to obtain a high yield of flavour esters. A maximum percentage for nonyl caprylate with conversion of flavour esters more than 90 % in a solvent-free system was successfully obtained under the following conditions: reaction time (RT), 5 hours; reaction temperature, 40 °C; amount of immobilized lipase, 25 % w/w of total substrate and shaking speed 200 rpm. Compared to ethyl valerate, a maximum percentage conversion of flavour ester more than 80 % in solvent free system was successfully obtained under following conditions: reaction time (RT), 45 minutes; reaction temperature, 40 °C; amount of immobilized lipase, 15 % w/w of total substrate and shaking speed 200 rpm. Comparison between these two ester showed that the chain length give an effect to optimize the reaction condition in esterification reaction.

Oleyl oleate was synthesized by green process of lipase-catalyzed esterification reaction between oleic acid and oleyl alcohol. Dual enzymes system consisting of Novozym 435 and Lipozyme TL IM were used due to its specificity towards an ester bond. The reaction was optimized by statistical approach of RSM with four important parameters such as reaction time, reaction temperature, amount of enzymes and molar ratio of substrates. 97.52 % of oleyl oleate was achieved at the most optimum condition (58.67 min, 59.68 °C, 0.37 g enzymes and 2.88 molar ratio of substrates). Regression analysis shows all parameters were significant (Prob P < 0.05) except for the reaction temperature. Keywords—Enzymatic esterification, Dual Enzymes System, Oleyl oleate, Response surface methodology.

Stressful environment especially in the presence of antimicrobial agents could increase the levels of protein produced by bacteria. Therefore, this study aims to explore the potential of natural antimicrobial compound, Allium sativum at low concentration in producing protein by Bacillus subtilis ATCC11774. The bacterial cells were exposed to 0.01 MIC of A. sativum in fermentation process at 30 °C and 37 °C for 12 h, 24 h, 48 h and 72 h. Analysis by Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (SDS-PAGE) showed that new intracellular proteins with approximate size of ~13 kDa and ~38 kDa, each was produced by B. subtilis after being treated with A. Sativum at 37 °C for 24 h and at 30 °C for 72 h respectively. Besides, new extracellular proteins approximately ~20 kDa and ~36 kDa in size, each was synthesized by B. subtilis prior to treatment with A. sativum at 37 °C for 48 h and 72 h subsequently. However, the proteins were expressed differently in which some were highly expressed, while some were repressed based on the intensity of protein bands appeared. Thus, protein production by B. subtilis ATCC11774 could be enhanced in the presence of 0.01MIC of A. sativum.

Previous studies have established that subinhibitory concentrations of antibiotics or antimicrobials are potent modulators of transcription process in bacterial cells. Hence, the bacteria might be introduced new proteins in mild stress environments like in the presence of antimicrobial agents at low concentrations. Although, there are still limited studies on the potential of antimicrobials at low doses play as a signaling agent that capable to modulate biological functions in bacteria. Therefore, this study aims to explore proteins production by Lactobacillus plantarum ATCC 8014 during stress which is in the presence of ethyl pentanoate at sub-minimal inhibitory concentration (sub-MIC). The Minimum Inhibition Concentration (MIC) of ethyl pentanoate against L. plantarum is 14.29% and was performed by microdilution assay. L. plantarum cells were treated with ethyl pentanoate at sub-MIC (0.05 x MIC) in the fermentation process. Two new protein bands (approximate size of 46.51 kD and 6.91 kD) were detected for the treated bacteria showed by SDS-PAGE profile. Of the two bands, eight possible proteins were identified by LC-MS/MS analysis. Thus, L. plantarum ATCC 8014 capable to produce new proteins in mild stress condition with the presence of 0.05 x MIC ethyl pentanoate. Futhermore, the isolated microbial proteins exhibit antimicrobial activity against several Gram-positive and Gram-negative bacteria. Copyright © WJSTR, all rights reserved.

Many studies have reported that the primary activity of most inhibitors of bacterial function is to modulate transcription processes at much lower concentrations than that required for antibiosis. Therefore, the bacteria might be produced and secreted more proteins in the mild stress surroundings (e.g. in the presence of low doses of antimicrobial agents) than in the normal environment. However, not much is known about unexpected ability of natural antimicrobial compounds at low concentration to become a signaling agent that capable to modulate biological functions in bacteria. Thus, this study aims to explore the potential of natural antimicrobial compound (Allium sativum) at sub-minimal inhibitory concentration (sub-MIC) in regulating proteins production by Bacillus subtilis ATCC 21332. The Minimum Inhibition Concentration (MIC) of A. sativum on B. subtilis resulting 14.29% was determined by microdilution assay. The bacteria cells were further exposed to A. sativum at sub-MIC (0.05 x MIC) in fermentation process. SDS-PAGE profile showed that two protein bands with approximate size of 51.36 kD and 9.74 kD were produced for the bacteria treated with A. sativum. LC-MS/MS analysis identified six possible proteins from the two bands expressed in mild stress condition. The proteins exhibited antimicrobial activity towards several Gram-positive and Gram-negative bacteria. Hence, B. subtilis ATCC 21332 in mild stress condition with the presence of 0.05 x MIC A. sativum could regulate bioactive proteins production. Copyright © AJBCPS, all rights reserved. Keywords: Bacillus subtilis ATCC 21332, Allium sativum, proteins, sub-MIC, antimicrobial agent, transcription

Bacterial intercellular communication, or quorum sensing (QS), controls the pathogenesis of many medically important organisms. Anti-QS compounds have the ability to attenuate bacterial pathogenicity. The current quest for new antimicrobials aimed at discovering non-toxic inhibitors of QS from natural sources which can be used for the treatment of bacterial infections in human. The objective of this research is to study the anti-QS potential in some of local ulam commonly found in Malaysia. In this study, seven types of local ulam namely; Parkia speciosa, Cosmos cardatus, Centella asiatica, Manihot esculenta leaf sprigs, Psophocarpus tetragonolobus, Polygonum minus and Oenanthe javanica were tested on the anti-QS potentials in fresh (edible or macerated) forms and methanol extracts via biomonitor strain Chromabacterium violaceum ATCC 12472. This biomonitor strain has an ability to produce a purple pigment (violacein) under QS-control. The results exhibited the wide variation in the anti-QS activities on selected local ulam in fresh and methanol extract forms. The highest anti-QS activity was recorded by P. minus and C. asiatica extracts as the lowest of minimum QS inhibition concentration value (7.81 mg/ml) was indicated by both extracts respectively. This study introduces not only a new mode of action and possible validation for traditional plant use, but also a potentially new therapeutic direction for the treatment of bacterial infections.

Euphorbia hirta is an annual broad-leaved herb and widely used as traditional medicine to treat various ailments. This herb was tested on the anti quorum sensing (anti-QS) potentials in fresh (edible or macerated) forms and acetone extracts via biomonitor strain Chromabacterium violaceum (ATCC 12472). The biomonitor strain has an ability to produce purple pigment (violacein) under QS-control. The different parts of E. hirta extracts were then subjected to preliminary phytochemical screening using standard procedures and finally analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). Preliminary screening on fresh parts of this herb revealed that leaves exhibited the highest anti-QS activities towards C. violaceum. The results also exhibited the wide variation in the anti-QS activities on whole plants, flowers, stems, leaves and roots of E. hirta from acetone extraction. The highest anti-QS activities were recorded by leaves and flowers extracts as the lowest of minimum QS inhibition concentration values (1.8906 mg/ml) were indicated by both extracts respectively. Phytochemical screening of E. hirta extracts revealed the presence of carbohydrates, lipids, protein, flavonoids, alkaloids, saponins, resins, steroids, acidic compounds, tannins, glycosides, phenols and terpenoids. The quantitave phytochemical assays via GC-MS indicated that this herb rich with fatty acids, terpenoids and phenolic compounds.