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Isolation and Characterization of Biotechnologically Potent Micrococcus luteus Strain From Environment.

Byline: Ali Akbar Uzma Sitara Imran Ali Niaz Muhammad and Shabir Ahmad Khan

Abstract

This study was conducted to isolate antagonistic and biotechnologically potent bacteria from environment. Micrococcus luteus was the only bacterium found producing antimicrobial metabolites and exhibited good probiotic characteristics. Micrococcus luteus showed antibacterial activity against foodborne pathogens Salmonella typhimurium (16-20 mm) Listeria monocytogenes (11-15 mm) and Escherichia coli (5-10 mm). The bacterium was also found active against Klebsiella pneumoniae and Staphylococcus aureus. It was found producing industrially important enzymes including protease lipase and phytase confirmed by screening with specific substrate supplementations. The bacterium was able to survive in 2% bile salt and 4% NaCl with good probiotic potential. The DPPH radical scavenging activity of the Micrococcus luteus was found 79.610.02% while the total phenolic content was 200g/mL equivalent to gallic acid.

Keywords: Probiotics industrial enzymes protease lipase phytase food safety Micrococcus luteus Klebsiella pneumoniae Staphylococcus aureus.

INTRODUCTION

Infectious diseases related to bacterial pathogens remains a big problem in developed and developing countries (Mirnejad et al. 2013). The increasing antibacterial drug resistance to commonly in-use antibiotics is enhancing the severity of infections (Jamalifar et al. 2011) and posing substantial burden over health and economy along with increase in mortality and morbidity (Tanvir et al. 2012; Akbar and Anal 2011). The bacteria with probiotics and antagonistic properties are proved capable of reducing the microbial load in food and showed in-vitro and in-vivo activities against pathogens (Akbar and Anal 2014a; Mirnejad et al. 2013).

Alternative sources for pathogens control

such as probiotics antioxidant antimicrobial plant

materials and nanoparticles etc are getting appraisal due to its wide range of acceptability for human use (Akbar and Anal 2014b; Abd El-Rhman et al.

2009). Exploring new and existing microbial resources for its novel uses are of utmost important (Malik et al. 2013; Akbar and Anal 2011). Some groups of microbes such as lactic acid bacteria and Bifedobacterium are known having: probiotic capability application in food safety and food enrichment are repeatedly reported (Olorunfemi et al. 2006) whereas member of other groups such as Leuconostoc and yeast are also important due to its human friendly activities (Ibrahem 2013). Bacteria belonging to pathogenic and opportunistic pathogenic groups rarely known as probiotics such as Pseudomonas and Micrococcus spp. are also reported with its antibacterial capability against some other bacteria (Abd El-Rhman et al. 2009).

The Micrococcus spp. is commonly known as

normal skin microflora and its antibacterial activity has rarely been reported against different pathogens. This bacterium is ubiquitous in nature and can be

easily found in natural environments (Willey et al.

2008). It can degrade hydrocarbons (Zhuang 2003) toxic organic pollutants and can tolerate the presence of metals (Sandrin and Maier 2003). Its property of precipitating gold by crystallizing it on their surface can be used for gold enrichment in low concentration gold ores (Marakushev 1991). This organism can be exploited for its abilities of producing technologically important compounds and processing for industrial applications. The data regarding antibacterial probiotic activities and industrially important metabolites production of this bacterium is limited and it need further explorations. Determination of such activities from an environmental isolates of Micrococcus luteus is the focus of this study. The objective of this study was to isolate environmental bacteria with antibacterial capability against some target bacteria and to characterize and identify its possible role for the welfare of human being in term of its probiotic capability or production of industrially valuable compounds.

MATERIALS AND METHODS

Isolation and identification

Environmental sampling of open (agriculture

fields and garden) and closed (room kitchen and laboratory) environments was conducted for isolation of bacterial species with antimicrobial activity against pathogenic bacteria. The colonies with similar morphology on the sampling plate were considered as duplicate and only prominent and isolated colonies were sub-cultured for identification.

Sampling plates (50) each containing media deMan Rogosa and Sharpe (MRS) agar (Himedia India) and nutrient agar (Merck Germany) were exposed randomly to the environment for 1 min at

10 different places in different time interval in order

to obtain the environmental bacteria. The plates were observed for the presence of visible and distinguished bacterial colonies after 24-48 h aerobic incubation at 37oC. The fungal colonies were ignored and only bacterial colonies were sub- cultured on MRS agar for antagonistic activity determination. The bacteria with antibacterial activity were identified with the help of Gram's

staining colonies characteristics catalase oxidase coagulase reactions bacitracin susceptibility and biochemical profiling using API kits (Biomerieux France).

Antibacterial activity determination

The antibacterial activity of the selected

isolates was evaluated with the help of three different methods including agar well diffusion spot on lawn and supernatant mix methods following (Akbar and Anal 2014a).

In agar well diffusion method MRS broth cultured (24 h) with selected bacteria were centrifuged at 8000 rpm for 20 min the supernatant were filtered (0.2 m pore diameter Minisart Germany) sterilized and 100 L were poured in 6 mm wells previously made on Muller Hinton agar (Merck Germany) seeded with the target bacteria and incubated at 37oC for 16-24 h.

In spot on lawn method 5 L of the selected

bacteria were spotted on the surface of MRS agar and incubated at 37oC for 24 h. A sterile semisolid nutrient agar (0.7 % agar w/v) seeded with the target bacteria were poured over the surface of visible colonies of selected bacteria in MRS agar and incubated at 37oC for 16-24 h. Clear zone around the colonies were observed and recorded.

In supernatant mix method an equal amount of filtered sterile supernatant from 24 h MRS culture was mixed with a double strength nutrient broth. Target bacteria (106 CFU/mL) were inoculated to the test tubes. Nutrient broth containing test bacteria but no supernatant was used as positive control. The tubes were incubated at 37oC for 24 h and the growth of target bacteria in the tubes was determined with the help of standard plate count and optical density measurement at OD600 using spectrophotometer.

Determination of total phenolic content

Total phenolic contents were determined following the methodology used by Arora and Chandra (2010). Test samples (0.5 mL) were mixed with 0.2 mL of FolinCiocalteu (FC) reagent and incubated at room temperature. After 10 min an amount (0.6 mL) of 20% (w/v) sodium carbonate was added to the tubes. The reaction mixture was incubated at 40oC for 30 min. The optical density

was determined at absorbance 765 nm for phenolic activity. All the tubes were properly protected from light by covering with aluminium foil. Gallic acid was used as standard.

Determination of DPPH radical-scavenging activity The antioxidant activity against 2 2- diphenyl-1-picrylhydrazyl (DPPH) radical was carried out by mixing DPPH methanol solution (0.025g DPPH/1000 mL methanol) with 0.5 mL of supernatant (24 h culture) and kept for 30 min in dark place at room temperature. The absorbance was measured at 517 nm. Methanol was used as a blank while DPPH methanol solution without sample was used as control. Following formula was used for scavenging activity calculation (Pianpumepong and

Noomhorm 2010).

Scavenging activity (%) = [1-(absorbance of sample/absorbance of control)] x 100

Determination of probiotics characteristic

The bacteria with a broad spectrum of

antibacterial activity in all three type of antibacterial test were further selected for its probiotic properties and biotechnologically important products screening. The probiotics characteristics of the bacteria were determined by exposing it to different growth conditions following the methodology of (Pianpumepong and Noomhorm (2010) and Kaushik et al. (2009) with slight modifications.

Acidic and alkaline pH tolerance

Fresh culture of test bacteria 2 L (107-108

CFU/mL) was inoculated to MRS broth with a pH adjusted at 2.0 3.0 5.0 8.0 and 10 priorly with 2 M HCl / NaOH and incubated at 37oC for 24 h. Same bacteria in normal MRS broth were used as a control. Bacterial growth was confirmed with the help of visible observation and standard plate counting.

Salt tolerance

Fresh culture 2 L of test bacteria (107-108

CFU/mL) was introduced to the MRS agar and broth supplemented with 4 6 and 8 % of salt (NaCl) and incubated at 37 oC for 24 h. Normal MRS broth and agar inoculated with same bacteria was used as

control. Visible growth in MRS agar and broth was observed for results documentation.

Bile salt tolerance

Fresh culture 2 L (107-108 CFU/mL) was introduced to the MRS agar and broth supplemented

with 0.3 1.0 and 2.0% of bile salt (Oxgall) (Biomark India) and incubated at 37oC for 24h. Normal MRS broth and agar inoculated with same bacteria was used as control. Visible growth in MRS

agar and broth was observed for results documentation.

Growth on different temperature

Growth ability of the test bacteria in a range of temperature (08 25 35 40 and 44oC) was observed by inoculating it to MRS agar and broth and incubated at 37oC for 24 h.

Determination of hydrophobicity

Fresh culture of test bacteria was centrifuged at 8000 rpm for 20 min and washed twice with sterile normal saline. The cells suspension was measured for optical density (OD600) (A0). Part of suspension (3 mL) were mixed with 1 mL of toluene and blended for 2 min left standing for 15-20 min to get two separate phases. The optical density (OD600) of the lower aqueous phase (A) was measured. Percentage of cell surface hydrophobicity (% H) was calculated by using the following equation (Pianpumepong and Noomhorm 2010). Equation

Screening of enzyme production (proteases lipases amylases and phytase) Agar supplemented with specific substrate was used for the screening of industrially important enzymes in this study. Methods by Ali et al. (2014) were followed with slight modifications.

Nutrient and MRS agar supplemented with 10% skim milk (autoclaved at 115oC for 20 min) was used for the screening of proteases. The bacteria were inoculated to the surface of plates and the plates were observed for clear zone around the bacterial colonies after incubation at 37oC for 24-48 h.

Tween agar (peptone 10 g calcium chloride hydrate 0.1 g sodium chloride 5 g tween-80 10 g agar 15 g in 1 liter distilled water. pH 7.0-7.4 sterilized at 115oC for 20 min) and Tributyrin agar base (Himedia) supplemented with 1% trybutyrin was used for lipases screening. The agars were inoculated and incubated at 37oC for 24-72 h to observe clear zone around the colonies.

Starch agar (soluble starch 1% in nutrient and MRS agar) was used for the screening of amylase production. The bacteria was inoculated to the Petri dishes and incubated at 37oC for 24-48 h. The plates were stained with iodine and clear zones were observed around the colonies.

The phytase screening medium (PSM) (Glucose 1.5% (NH4)2SO4 0.5% KCl 0.05% MgSO4.7H2O 0.01% NaCl 0.01% CaCl2. 2H2O 0.01% FeSO4 0.001% MnSO4 0.001% sodium phytate 0.5% agar 1.5% pH 6.5) was used for the screening of phytase enzyme. The bacteria was inoculated to the plates and incubated at 37oC for

24-72 h. Clear zone around the colonies were observed after incubation for phytase production (Singh et al. 2013).

RESULTS

Twelve different species of bacteria including Gram-negative and Gram-positive were isolated from environment. Out of all 12 bacteria only one bacterium identified as M. luteus was found to have antibacterial activity against a group of foodborne pathogens. The bacteria were initially identified with the help of its morphology; colonies characteristics biochemical tests using different media bacitracin test and API Staph ID 32 kit for biochemical profiling. The isolated bacterium (M. luteus) was found highly active against the target

pathogens including Salmonella typhimurium Escherichia coli Listeria monocytogenes and showed mild activity against Staphylococcus aureus and Klebsiella pneumoniae (Fig. 1). No activity was found against Enterococcus faecalis (Table I). The antibacterial activity was confirmed by three different methods including agar well spot on lawn

and supernatant mix method. It was observed that M. luteus was more active against Gram-negative as compared to Gram-positive bacteria. Increased

antibacterial activity of M. luteus was observed on MRS agar as compared to nutrient agar. No extensive acidification of nutritional media was observed only a slight decrease of pH equal to 5 in MRS media were noted at 24 h incubation.

Table I.- Antibacterial activity of Micrococcus luteus against different bacteria.

Target bacteria###Agar well###Spot on lawn

###method###method

Salmonella typhimurium###++++###++++

Escherichia coli###++###++

Listeria monocytogenes###+++###+++

Staphylococcus aureus###+###+

Klebsiella pneumoniae###+###+

Enterococcus faecalis###-ve###-ve

During the probiotic characteristic analysis it was observed that isolated M. luteus can grow in bile salt (2%) and NaCl 4% no growth in increasing salt concentrations (6 and 8%) were observed the bacteria was unable to grow at 8 and 45oC and in high acidic (pH 3) or alkaline (pH 8) environment. Growth was only observed in medium with pH 5 (Table II). The hydrophobicity of the bacteria to hydrocarbon was found 120.19%. The metabolites from M. luteus showed 79.610.02% DPPH scavenging activity and total phenolic contents of

200g/mL equivalent to Gallic acid (Table II). In the enzyme screening phase the bacterium was found producing protease phytase and lipase with inability of amylase production (Fig. 2). Enzyme production was confirmed by observing clear zone around colonies. The bacteria showed good sign of phytase and lipase production after 72 h incubation.

Table II.- Different characteristic of Micrococcus luteus isolated from environment.

No###Test###Results

1###Salt tolerance

###4%###+ve

###6%###-ve

###8%###-ve

2###Bile salt tolerance###

###0.3%###+ve

###0.5%###+ve

###1.0%###+ve

###2.0%###+ve

3###Temperature tolerance

###8o C###-ve

###25oC###+ve

###30oC###+ve

###40oC###+ve

###45oC###-ve

4###pH tolerance###

###2###-ve

###3###-ve

###5###+ve

###8###-ve

###10###-ve

5###Protease production###+ve

6###Amylase production###-ve

7###Lipase production###+ve

8###Phytase production###+ve

9###Polyphenolic activity###200g/mL

10###% DPPH activity###79.61 0.02%

11###% Hydrophobicity###12 0.19 %

DISCUSSION

Micrococcus is obligate aerobic Gram- positive cocci with the ability to survive in diverse and stress conditions. It was first isolated by Alexander Fleming in 1929 as Micrococcus lysodeikticus (Umadevi and Krishnaveni 2013). Data regarding the antibacterial activity of M. luteus is limited and no data regarding its antioxidant and polyphenolic activity are available. Sharma et al. (2012) isolated the Micrococcus spp. from soil with

promising antibacterial activity against Bacillus subtilis Staphylococcus aureus Escherichia coli Shigella dysenteriae Shigella sonnei Shigella shiga Salmonella typhi and Klebsiella Pneumonia. They also reported xylanases from the same species.

In this study it was noted that M. luteus can produce antibacterial and antioxidant compounds in simple nutritional media (nutrient agar/broth).

Increased antibacterial activity was observed when bacteria was grown in enriched media (MRS). Due to its low acidification activity in the media this organism can be used against foodborne pathogens and product enrichment where near to neutral pH is desirable for its quality and physical behaviors such as milk products.

Abd El-Rhman et al. (2009) reported that M. luteus isolated from gonads and intestine of Nile tilapia showed antimicrobial activity against Aeromonas hydrophila and was found with good probiotic abilities for enhancing the growth of Nile tilapia when utilized as functional diet. Chabrillon et al. (2005) reported good probiotic abilities of the genus Micrococcus isolated from sea bream in term of antagonism toward Vibrio harveyi and its adherence and competitive exclusion abilities. Irianto and Austin (2003) successfully used (in- vitro) M. luteus as a probiotic in Rainbow trout Oncorhynchus mykiss farming against A. salmonicida infection. Sugita et al. (1998) reported the antibacterial activity of M. luteus from fish intestine against Vibrio vulnificus. Micrococcus was supplemented as diet in fish having the ability to reduce the pathogenic microbial flora and enhancing food adsorption in the intestine (Abd El-Rhman et al. 2009). Umadevi and Krishnaveni (2013) reported the antibacterial activity of M. lutues isolated from sea water against S. aureus Klebsiella sp. and Pseudomonas.

The antioxidant compounds enhance antimicrobial activity due to its toxicity against bacterial cells (Pianpumepong and Noomhorm

2010). Microorganisms may provide easier way for production of such antioxidants compared to other natural sources such as plants. Extensive studies are needed to explore the possible beneficial role of this bacterium for the welfare of human beings. Yuen et al. (2009) reported efficient proteolytic and lipolytic activities of M. luteus isolated from fermented fish sauce. Our results are in agreement with their study. Lawrence et al. (1967) and Joseph et al. (2011) reported lipases production from Micrococcus spp. Phytase are used as food supplement to facilitate the phytate utilization in poultry for phosphate availability which is an important growth enhancement component in poultry production (Saima et al. 2014). Tomova et al. (2013) reported

protease lipase and phytase production from M. luteus isolated from old cave in Bulgaria. Results of our study are similar with their findings. The M. lutues isolated in this study was found biotechnologically important as it was producing protease lipase and phytase. It also showed promising results in term of probiotic capabilities antibacterial and antioxidant metabolites production.

CONCLUSIONS

It was found in this study that M. luteus isolated from environment are capable of producing biotechnologically important metabolites. It also produced antimicrobial compounds and was found antagonistic to a group of foodborne pathogens. The bacteria can grow on minimal media with no extra efforts. Thus its use in industrial production of different important metabolites and as a selective probiotic in facilitation of fish and animal farming can be of low cost and result oriented. The bacteria showed promising results in term of its resistance to bile salt and antibacterial activity. This bacterium needs more attention for its exploration to use for human benefits.

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Author:Uzma, Ali Akbar; Ali, Sitara Imran; Muhammad, Niaz; Khan, Shabir Ahmad
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9PAKI
Date:Aug 31, 2014
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