Characterization of Antimicrobial Activities of Pediococcus pentosaceus Vtcc-B-601

Article history: Received on: 10/04/2014 Revised on: 06/05/2014 Accepted on: 19/05/2014 Available online: 27/05/2014 In this study, Pediococcus pentosaceus VTCC-B-601 was investigated and characterized for bacteriocin production. The antimicrobial activities were produced strongly at the late exponential phase (5x10 CFU/ml), corresponding to the activity of cephalosporin (13.3μg/ml) against Salmonella typhimurium ATCC 19430, Pseudomonas aeruginosa ATCC 27853, Staphylococus aureus ATCC 25923, and Micrococcus luteus ATCC 10240. The bacteriocin activity was reduced after proteinase K treatment while the activity was still stable in high temperature. This work supplied a Pediococcus pentosaceus bacteriocin identification that was useful in food preservation, clinical use, and agriculture.


INTRODUCTION
Pediococcus pentosaceus, a gram positive bacteria, belongs to a group of homofermentative lactic acid bacteria (LAB), is the best-known major member of probiotic bacteria (Gibson and Fuller, 2000;Rolfe, 2000), which can prevent cardiovascular diseases, prevent harmful pathogens from accessing the gastrointestinal mucosa (Rinkinen et al., 2003;Duggan et al., 2002) and provoke immune reaction (Ouwehand et al., 2003;Vinderola et al., 2005).Bacteriocins of Pediococcus are small, heat stable and non-lanthionine containing peptides belonging to the class II which are biologically active proteins demonstrating a bactericidal mode of action in foodborne pathogens, including Bacillus cereus, Clostridium perfringens, Listeria species and S. aureus (Jiménez et al., 1993;Klaenhammer, 1988;Jack and Tagg, 1995;Tagg et al., 1976;Carminati et al., 1989;Spellberg, 2008).Antibiotic-resistant pathogens have been becoming more and more severe, resulting in the potential epidemic threatening human life.The ratio of pathogens resisted to available antibiotics was extremely high and has been tending to increase rapidly.

In
Vietnam, Streptococcus pneumonia, a very common cause of respiratory infection had the highest prevalence of penicillin-resistant (71.4%) and erythromycin-resistant (92.1%) of the 11 countries in the Asian network for surveillance of resistant pathogens, 75% of pneumococci was resistant to three or more classes of antibiotic (Martys, 1982).
Obviously, pathogens have been resisting to available antibiotics by mutations or genetic material changes with a very high rate, which progressed faster than the development of new antibiotic invention.
In addition, some available antibiotics have side-effects on consumer (Martys, 1982).In fact, chloramphenicol has effects on the performance of bone marrow, resulting in negative effects on blood cell production.Besides, macrolide can cause vomit, diarrhea, and ototoxicity.
In food industry, food formulated with chemical preservatives or antibiotics for longer shelf-life influences on human health.Besides, using antibiotic in aquaculture for long time will cause many risks of potential epidemics.Therefore, development of the natural antimicrobial agents is a great interest for the application in food preservation, agriculture, clinical use, and environmental science (Cleveland et al., 2001).For the above reasons, detection and characterization of bacteriocin in Pedio pentosaceus (VTCC-B-601) are necessary.

Bacteriocin assay
Antimicrobial effects were tested on pathogens by the agar -well -diffusion assay as described by Toba (Toba et al., 1991).The tested microorganisms were grown for 18 to 24h in 10 ml LB medium, then dilute to 7.10 10 CFU/ml.The 150 µl of cellfree filtrates was added into each well in the 50 µl pathogen-spread plate.The plates were incubated for 12 h at 37 o C. The clear zone around each well showing antimicrobial activity was measured and analyzed.

Thermostability test
Cell-free filtrate was treated with temperature.Two ml of cell-free filtrate were poured into test tubes and capped tightly, then treated at 70 o C, 80 o C, 90 o C, 100 o C for 30 minutes and 121 o C for 15 minutes.These tubes were cooled and tested for antimicrobial activity (Maja et al., 2010).

Test for proteinase K sensitivity
Five ml of cell-free filtrate were added with proteinase K to get final concentration at 100µg/ml, 500µg/ml and 1000µg/ml (Rajaram et al., 2009).The reaction was incubated at 37 o C for 2h before antimicrobial test.

Detection of antimicrobial activities of Pediococcus pentosaceus
Cell-free-supernatant was harvested at early exponential phase (OD = 0.5, 10h), late exponential phase (OD = 1.39, 20h), stationary phase (OD = 1.35, 36h), and dead phase (OD = 0.5, 49h) (Figure 1).Supernatant of Pediococcus pentosaceus was collected after centrifugation at 12000 g for 15 min, then the clear supernatant was sterilized by filtration (0.45µm).The cell-free filtrates were used for antimicrobial assay and further characterization.The activities of antimicrobial compounds considerably varied along with the changes of growth phases of Pediococcus pentosaceus VTCC-B-601 on Salmonella typhimurium ATCC 19430, Pseudomonas aeruginosa ATCC 27853, Staphylococus aureus ATCC 25923, and Micrococcus luteus ATCC 10240.Remarkably, the average inhibition zone value is 13.08 mm in early exponential phase, 19.58 mm in late exponential phase, 18.41 mm in stationary phase, and 16.66 mm in dead phase (Figure 2, Table 1).Micrococcus luteus ATCC 10240.These pathogens were chosen as indicator strains because of their prevalence in environment and the high resistance to many kinds of available antibiotics.Particularly, Salmonella typhimurium, a gram negative bacteria cause gastroenteritis and diarrhea in human and animal, resisted to streptomycin, ampicillin, rifampicin, nalidixic acid, chloramphenicol, and trimethoprin.Pseudomonas aeruginosa resisted to βlactam, carbapenems, aminoglycoside, and fluoroquinolones.
Therefore, the success of this study may be useful in the contribution to the resolving of antibiotic-resistant microorganism as well as applying this bacteriocin extensively in aquaculture, clinical treatment, food preservation.The study exploited the antimicrobial activity of Pediococus pentosaceus bacteriocin originated in Vietnam in order to extend the antimicrobial agent sources in Vietnam and worldwide on commonly highly resistant pathogens.
Clearly, bacteriocin production was different in the different growth phases.At the early exponential phase, bacteria growth was not enough to produce high amount of bacteriocin.At the late exponential phase, the antimicrobial activity was highest because of the highest growth.
At stationary and dead phase, the antimicrobial activity trended to decrease moderately.The decreasing of antimicrobial activity in stationary and dead phases may be due to the degradation by proteinase in culture.Therefore, it was appropriate to harvest Pediococus pentosaceus (VTCC-B-601) cell-freesupernatant at the late exponential phase for strong antimicrobial activities.

Bacteriocin characterization
The cell-free-filtrate was treated with proteinase K at 100μg/ml, 500μg/ml and 1000μg/ml.The results in figure 4 showed the antimicrobial activity test on Micrococcus luteus ATCC 10240.The antimicrobial activities strongly reduced in cases of proteinase K used at 100μg/ml, 500μg/ml and 1000μg/ml were (figure 4).The inhibition zone nearly disappeared around the sampled wells.Results from proteinase K treatment proved that antimicrobial activities in cell free-supernatant of this bacteria was due to proteinaceous bacteriocin, not H 2 O 2 or other antimicrobial agents (Mohankumar , 2011).As seeing in table 2 and figure 3, there was no reduction in antimicrobial activities of bacteriocin in all temperature treatments from 70 o C to 100 o C, even autoclaved at 121 o C for 15 minutes.Micrococcus luteus was used as an illustration for the antimicrobial activities of heated bacteriocin.Consequently, the bacteriocin produced by Pediococus pentosaceus (VTCC-B-601) was stable as even heating at 100 o C for 30 minutes or autoclaving at 121 o C for 15 minutes.With this properties, Pediococus pentosaceus bacteriocin could be used as the improved food preservative.

Fig. 3 :
Fig. 3: Antimicrobial activities of bacteriocin after temperature-treatment on Micrococcus luteus at 70 o C, 80 o C, 90 o C, 100 o C. The MRS medium was used as control (C).

Table . 1
: The statistic analysis of antimicrobial activity of Pedio pentosaceus (VTCC-B-601) cell free supernatant at different growth phases on indicator strains.

Table . 2
: The statistic analysis of antimicrobial activity of Pedio pentosaceus (VTCC-B-601) cell-free-supernatant after treatment with different temperature levels.