Effect of physical parameters in enhancing prodigiosin production and anti-MRSA activity of marine bacterium, Serratia marcescens IBRL USM84

Aims@#Marine bacteria have been reported to produce potential natural pigment with pharmaceutical properties and their growth can be manipulated in the laboratory to increase pigment production and their antimicrobial activity. Hence, this study aimed to enhance the prodigiosin production in Serratia marcescens IBRL USM84 by improving physical conditions.@*Methodology and results@#The quantification of the pigment produced by S. marcescens IBRL USM84, bacterial cell growth, and its antibacterial activity in the broth medium were determined using a spectrophotometry method. Meanwhile, the antibacterial effect of red pigment on MRSA cells was observed under a scanning electron microscope (SEM). This marine isolate produced the highest yield of prodigiosin (6.95 μg/mL) when cultivated in marine broth with the addition of 0.2% of agar, 25 °C incubation temperature, initial medium pH of 7, 150 rpm of agitation speed for 48 h of cultivation time under light illumination. There was an increment of 151.81% in prodigiosin production after enhancement compared to before the enhancement of cultural conditions. SEM observations revealed that severe damage to the cell’s morphologies was exposed to red pigment as indicated by the formation of small dents, which led to completely collapse and eventually, cell death.@*Conclusion, significance and impact of study@#A positive correlation between pigment production and antibacterial activity was observed in the present study. The results supported the fact that marine bacteria are a reservoir of various pigments with antimicrobial properties. Also, the pigment production by S. marcescens and its antibacterial activity were significantly influenced by physical parameters.

In vitro antibacterial activity of endophytic fungus Aspergillus flavus IBRL-C8 from Senna siamea against Gram-bacteria

Aims@#To evaluate the antibacterial efficacy of ethyl acetate extract of Aspergillus flavus IBRL-C8 against Gram-positive and Gram-negative bacteria.@*Methodology and results@#In this experiment, an endophytic fungus which identified as A. flavus IBRL-C8 was extracted using ethyl acetate and methanol, from Senna siamea, prior to in vitro antibacterial test on eight Gram-bacteria. The results were significantly more enunciated to the ethyl acetate extract since the Gram-bacteria signified 9.0 to 20.0 mm of inhibition zones on Muller Hinton Agar (MHA) during disc diffusion assay. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extract were ranged from 125-1000 µg/mL and 125-2000 µg/mL, respectively. Time-kill assay depicted the ethyl acetate extract of A. flavus IBRL-C8 exceptionally retarded methicillin-resistant Staphylococcus aureus (MRSA) and also manifested extended antibacterial activity. The maximum reduction in cell numbers occurred at 2MIC concentration (250 µg/mL) during the interval time of 16 h. The malformations noticed from microscopic observations where the transformation of structural annihilation from regular spherical morphology to non-spherical shape with an irregular surface and also disruption around the cell membrane when the MRSA treated with ethyl acetate extract of A. flavus IBRL-C8. @*Conclusion, significance and impact of study@#This study proposed the ethyl acetate extract of A. flavus IBRL-C8 as a potential antibacterial agent against MRSA infection, which can be useful in pharmaceutical application.

Ceratobasidium ramicola IBRLCM127, an endophytic fungus isolated from Curcuma mangga Valeton & Zijp with strong anti-candidal activity

Ceratobasidium ramicola IBRLCM127, an endophytic fungus isolated from the rhizome of the local medicinal plantCurcuma mangga Valeton & Zijp, was found to possess significant anti-candidal activity. This fungal endophyte wascultivated in submerged fermentation system using yeast sucrose medium supplemented with host plant water extractand cultivated at 25°C, agitated at 120 rpm for 12 days. The ethyl acetate was used as a solvent to extract compoundsin the fermentative broth. The fungal ethyl acetate extract demonstrated significant inhibitory zones toward cells ofCandida albicans with minimum inhibitory concentration (MIC) value of 2.5 mg/ml, of which exerting yeastocidaleffect. The time–kill study conducted at three distinct ethyl acetate concentrations (half MIC, MIC, and 2 MICvalues) revealed that the growth of C. albicans cells was concentration-dependent. Yeastostatic activity was shownat lower concentration and yeastocidal activity was shown at higher concentration. The structural degeneration of theC. albicans cells after treated with ethyl acetate extract was observed under the scanning and transmission electronmicroscopes and the results exhibited various cell deformities including severe damage of the cell extracellularly andintracellularly which led to cell death beyond repair, thus suggesting that the extract could be a potential antifungalagent.

The potential use of prodigiosin as a shrimp feed additive and its dynamic influence on the shrimp gut microbial community – an in vitro gut model

Aims@#A simple in vitro model system was applied in this study assessing the dynamics of the microbial community associated with the shrimp gut system to understand the changes that influence dietary variables. @*Methodology and results@#The diversity and abundance of microbiome were monitored within two different treatment slurries inoculated with shrimp faecal samples as to mimic the effect of diet manipulation, and 16S rRNA gene of MiSeq Illumina-based sequencing was applied. The different diets tested were a commercial standard diet and a prodigiosin added diet. There was very clear separation between the commercial standard diet and prodigiosin added diet as revealed by the total viable counts (TVC) and sequencing data. It suggested that the microbial community of the shrimp gut system exhibited a dynamic response with the treatments and allochthonous bacterial present. The prodigiosin added diet was clearly separated from the commercial standard diet serving as a potential shrimp feed additive. The sequencing data analysis showed that members of the genera Vibrio, Shigella and Photobacterium became predominant on the commercial standard diet treatment. The prodigiosin-added diet treatments indicated an abundance of members of the genera Micrococcus, Arthrobacter, and Shigella. @*Conclusion, significance and impact of study@#In vitro model system-based testing of diets could be a useful method to determine the potential effect of diet manipulation on shrimp gut system microbiome members.

Production, characterization and antibacterial activity of prodigiosin pigment produced by Pseudoalteromonas rubra BF1A IBRL associated with a marine macroalgae Enteromorpha sp

Aims@#Marine bacteria are a great source of natural pigments, which can be used as colouring agent in food, textile, cosmetics and aquaculture industry to overcome the drawbacks poses by the synthetic pigments. The aim of the study is to identify the potential bio pigment producer, determine the antimicrobial susceptibilities, and characterize the pigment produced. @*Methodology and results@#In this study, the surface attached marine bacteria isolated from the surface of seaweed, Enteromorpha sp. has been identified as Pseudoalteromonas rubra BF1A IBRL through the molecular identification step. This species produced intracellular and extracellular red pigment with antibacterial activity. The susceptible bacteria include Bacillus subtilis, Bacillus cereus, Methicillin Resistant Staphylococcus aureus, Staphylococcus aureus and also Acinetobacter anitratus with inhibition zone ranges from 7.33 to 10.33 mm, whereas Minimum inhibitory concentration (MIC) values ranges from 0.055 to 8.88 mg/mL. The UV/vis analysis indicated that the maximal absorbance of ISO and DE pigment extract were at 531 and 534 nm, respectively. Based on the antimicrobial activity, the extracellular extract poses greater antibacterial activity, thus was selected as the potential pigment extract and were further evaluated. The Thin Layer Chromatography (TLC) profile of the DE extract showed one major band under visible light ((Rf = 0.87) and the bioautography analysis of the pigmented band showed positive activity against both Gram-positive and Gram-negative bacteria. The pigment in DE extract was identified as prodigiosin based on the spectroscopic properties, presumptive test and HPLC analysis. @*Conclusion, significance and impact of study@#This study highlights the dual benefits of the P. rubra BF1A IBRL pigment extract, which exhibited both tinctorial and pharmacology benefits, thus it can be act as colouring agent with own preservative value in food, textile, or cosmetics industries.

Assessment of cultivation parameters influencing the growth, pigment production and anti-MRSA activity of Pseudoalteromonas rubra BF1A IBRL isolated from Malaysian marine environment

Aims@#Pigments are coloured substances that exhibit important characteristics to many industries including food, textile, cosmetics, food, pharmaceutical and also aquaculture industry. Naturally derived pigments from marine bacteria do not only exhibit the tinctorial property but are also known to possess broad range of antimicrobial activities. From the industrial point of view, the necessity to obtain suitable culture conditions for maximum yield of cell growth and pigment production is of utmost importance. @*Methodology and results@#The effect of cultural conditions, including light, pH, temperature, agitation speed and size of inoculum on bioactivity of an epiphytic marine bacteria, Pseudoalteromonas rubra BF1A IBRL was studied using shake flask technology. The antimicrobial activity was determined using the Lorian method. As a result, prodigiosin pigment extract obtained from P. rubra BF1A IBRL showed inhibitory activity against the MRSA strain. Pseudoalteromonas rubra BF1A IBRL produced the highest level of prodigiosin and anti-MRSA activity (P<0.05) in Marine broth at initial pH of 7.6 incubated at dark condition at temperature of 26 °C, agitation speed of 120 rpm and 2% (v/v) (1 × 106 CFU/mL) of inoculums size. @*Conclusion, significance and impact of study@#A high correlation between pigmentation and antibacterial activity were observed anticipating that the pigment has its own antibacterial properties. The above findings supported the fact that epiphytic marine bacteria were fruitful source for pigmented bioactive compounds, and the physical parameters had significantly influence of the pigment production.

Assessment of cultivation parameters influencing the growth, pigment production and anti-MRSA activity of Pseudoalteromonas rubra BF1A IBRL isolated from Malaysian marine environment

Aims@#Pigments are coloured substances that exhibit important characteristics to many industries including food, textile, cosmetics, food, pharmaceutical and also aquaculture industry. Naturally derived pigments from marine bacteria do not only exhibit the tinctorial property but are also known to possess broad range of antimicrobial activities. From the industrial point of view, the necessity to obtain suitable culture conditions for maximum yield of cell growth and pigment production is of utmost importance. @*Methodology and results@#The effect of cultural conditions, including light, pH, temperature, agitation speed and size of inoculum on bioactivity of an epiphytic marine bacteria, Pseudoalteromonas rubra BF1A IBRL was studied using shake flask technology. The antimicrobial activity was determined using the Lorian method. As a result, prodigiosin pigment extract obtained from P. rubra BF1A IBRL showed inhibitory activity against the MRSA strain. Pseudoalteromonas rubra BF1A IBRL produced the highest level of prodigiosin and anti-MRSA activity (P<0.05) in Marine broth at initial pH of 7.6 incubated at dark condition at temperature of 26 °C, agitation speed of 120 rpm and 2% (v/v) (1 × 106 CFU/mL) of inoculums size. @*Conclusion, significance and impact of study@#A high correlation between pigmentation and antibacterial activity were observed anticipating that the pigment has its own antibacterial properties. The above findings supported the fact that epiphytic marine bacteria were fruitful source for pigmented bioactive compounds, and the physical parameters had significantly influence of the pigment production.

Antimicrobial activity of Aspergillus sp. IBRL MP15 CCL, an endophytic fungus isolated from Swietenia macrophylla leaf against human pathogens

@#Aims: Endophytes are microorganisms residing in the living tissues of the host plant and may contribute to their hostplant by producing a plethora of bioactive compounds that provide survival value to the plant. This study aimed toevaluate the antimicrobial activity of Aspergillus sp. IBRL MP15 CCL, an endophytic fungus isolated from Swieteniamacrophylla leaf.Methodology and results: The antimicrobial activity was evaluated with disc diffusion and a colorimetric brothmicrodilution test against 15 organisms comprising of 4 Gram-positive bacteria and 4 Gram-negative bacteria, 4 fungiand 3 yeast. On disc diffusion assay, the fungal extract was shown to inhibit the growth of 7 test bacteria and 3 testyeast. The antibacterial activity was more pronounced with extract from fungal culture with host plant extractsupplementation with significantly larger inhibition zones on all susceptible test microorganisms. The minimal inhibitoryconcentration of the extract ranged from 250 to 4000 μg/mL indicating different level of susceptibility of the testedpathogens against the fungal extract. The killing kinetic study shows that antimicrobial activity of the fungal extract isconcentration dependent and it can act as bactericidal at higher concentration.Conclusion, significance and impact of study: The findings of this study suggest that Aspergillus sp. IBRL MP15CCL can be a promising source of antimicrobial agent to be further studied and developed

Enhancement of cellulolytic enzymes and xylanase production via classical mutational techniques under solid-state fermentation condition

Aims: High cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass (LB). The present study aims at developing a local cellulolytic fungal strain through random mutagenesis coupled with the feasibility of solid-state fermentation (SSF) by utilizing agricultural wastes such as oil palm frond (OPF) as the substrate. Methodology and results: Out of 95 wild isolates tested, native fungal strain Aspergillus niger, designated DWA8 was isolated as the top enzymatic secretor. For quantitative enzyme analysis, SSF was conducted using 1x106 spore/mL inoculated onto 5 g of ground OPF, incubated at room temperature for 7 days, with 70% moisture content and an initial medium pH of 7. Random mutagenesis has always been tempting in the enhancement of enzyme production. In this work, the compounded treatment of microwave, ultraviolet (UVC) and Ethyl Methanesulfonate (EMS) have generated an Aspergillus niger MUE3.06 mutant with an overall increase of 114% in CMCase activity, approximately 70% in FPase and Xylanase activity respectively compared with the parental DWA8 strain. Thus this finding is capable to be fully developed as an established mutational scheme to create highly productive filamentous fungus in a cheap, simple and sustainable way. Conclusion, significance and impact of study: It was the first attempt to explore the combine effect of the three popular mutagens upon cellulases and xylanases. It is believed that more diversified of mutagen types induce more diversified mutation pattern (with instructive planning), which is very desirable in creating new enzymes with novel abilities.

In vitro antimicrobial activities of methanolic extract from marine alga Enteromorpha intestinalis

OBJECTIVE: To extract the bioactive compound from Enteromorpha intestinalis (E. intestinalis) and determine its in vitro antimicrobial activity. METHODS: E. intestinalis was extracted by methanol and subjected to antimicrobial screening. The antimicrobial activity was studied by using disc diffusion and broth dilution method. The effect of the extract on the growth profile of the bacterial was also examined via time-kill assay. Microscopy observations using SEM was done to determine the major alterations in the microstructure of methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: The results showed methanolic extract of E. intestinalis exhibited a favourable antimicrobial activity against tested bacteria with produced inhibition zone ranging from 8.0 to 19.0 mm. However, all the tested fungi and yeast were resistant to the extract treatment. Time kill assay suggested that methanolic extract of E. intestinalis had completely inhibited MRSA growth and also exhibited prolonged antibacterial activity. The main abnormalities noted from the microscopic observations were the structural deterioration in the normal morphology and complete collapsed of the bacteria cells after 36 h of treatment. CONCLUSIONS: The significant antibacterial activity shown by crude extract suggested its potential against MRSA infection. The extract may have potential to develop as antibacterial agent in pharmaceutical use.

Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7 cell line model using comet assay

Objective:To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta (E. hirta) in MCF-7 cell line model using comet assay. Methods: The cytotoxicity of E. hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E. hirta was assessed by using Comet assay. Results: Both toxicity tests exhibited significant toxicity result. In the comet assay, the E. hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage. The extract of E. hirta showed significant toxicity against brine shrimp with an LC50 value of 620.382 μg/mL (24 h). Comparison with positive control potassium dichromate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity. Conclusion:The present work confirmed the cytotoxicity and genotoxicity of E. hirta. However, the observed toxicity of E. hirta extracts needs to be confirmed in additional studies.

Characterization of cellulase, hemicellulase and lipase and its use in deinking of laser printed paper

Aims: It is recognized that laser printed paper are difficult to deink using conventional method. This had lead to the suggestion of enzymatic approach to overcome the problem encountered by commonly employed deinking techniques. The present study aimed to investigate 7 commercially available enzymes for their suitability use in deinking of laser printed paper. Methodology and results: 3 cellulases, hemicellulases, xylanase and 2 lipases were used in enzymatic deinking of laser-printed wastepaper. Cellulase A “Amano”3 (C), Hemicellulase (H) and lipase (L) were selected for used in deinking because they possess either highest activity or broad pH stability compared to others enzymes. Different combination of enzymes was carried out to evaluate their effectiveness in deinking process. CH enzymes sequence was determined to be the most effective sequence in toner removal with 1.90% of brightness increment. However, only 0.95% of brightness increment was gained by enzyme sequence L. Highest deinking efficiency obtained was not proportional to the highest total reducing sugar produced. Conclusion, significance and impact of study: Enzyme (cellulase and hemicellulase) can be used to de-ink laserprinted wastepaper, which are difficult to be deinked by conventional chemical deinking process. Thus, enzyme deinking has high possibility as alternative method to current chemical deinking process which is not environmental friendly.

Pharmacological screening of methanolic extract of Ixora species

Objective:To investigate the antimicrobial activity of methanolic extracts of different parts of Ixora species. Methods:Antimicrobial activity was carried out using disc diffusion assay against fungi, gram-positive and gram-negative bacteria. Results:All methanolic extracts of different parts of Ixora species showed a broad-spectrum of antibacterial and antiyeast activities, which inhibited the growth of at least one bacterium or yeast. There was no remarkable difference between different Ixora species observed in this study. Conclusions:The significant antimicrobial activity shown by this Ixora species suggests its potential against infections caused by pathogens. The extract may be developed as an antimicrobial agent.

Mannanase production by Aspergillus niger USM F4 via solid substrate fermentation in a shallow tray using palm kernel cake as a substrate

Aims: A local fungal isolate, Aspergillus niger USM F4 produced high level of mannanase activity when cultivated in a shallow tray system (45 x 40 x 7 cm3) using palm kernel cake (PKC), an easily available cheap agricultural waste which are found abundantly in Malaysia. Methodology and Results: A range of 0.25 to 1.5 cm bed heights were investigated in tracking in the most suitable condition and maximum production of mannanase. The highest mannanase production of 918.68 U/g substrate was obtained on the fifth day of cultivation after using all the optimised cultural conditions that consisted of 400 g of PKC that equivalent to 0.50 cm of substrate thickness with the particle size of ≤ 0.5 mm, moisture content of 80% (w/w) with the addition of 2% (w/w) molasses as a carbon source and 4% (w/w) ammonium nitrate as a nitrogen source, inoculums size of 1x107 spores/ml, with once at every 24 h of mixing frequency and cultivation temperature at room temperature 30±2 °C. Conclusion, significance and impact of study: The results obtained from this study showed that a shallow tray system was suitable to be used for getting highest enzyme production in SSF. Besides using a bigger volume of substrate, the correct substrate bed height is also important.