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.

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.

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