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Research Article Microbiology

International Journal of Pharma and Bio Sciences ISSN

0975-6299

PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN FROM

FERMENTED VEGETABLES

S. HEMALATHA*1 AND K. LOGESHWARI

2

1Department of Biotechnology, Vels University, Chennai, Tamil Nadu, India

2Department of Plant Biology and Plant Biotechnology, Meenakshi College for Women, Chennai,

Tamil Nadu, India

ABSTRACT

Bacillus sp., Leuconostoc mesenteroides, Vagococcus sp., and Pediococcus sp. were isolated from fermented vegetables. All the four isolates produced bacteriocin and they have showed antibacterial activity against E. coli, B. subtilis and K. pneumoniae. Bacteriocin was stable at various temperature and pH. But it was sensitive to enzymes such as α amylase, lipase and pepsin and also affected by high percentage of salt concentration. SDS-PAGE analysis have shown peptide < 35 KDa. KEYWORDS: bio preservative, bacteriocin, pH, temperature, enzymes, antimicrobial

*Corresponding author

S. HEMALATHA

Department of Biotechnology, Vels University, Chennai, Tamil Nadu, India

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INTRODUCTION

Bacteriocins are ribosomally synthesized, extracellularly released low-molecular-mass peptides or proteins which have a bactericidal or bacteriostatic effect on other (usually closed related) species1, 2. They were first discovered by Gratia in 1925. Bacteriocin production has been found in numerous species of bacteria, among which, due to their ‘‘Generally Recognized as Safe’’ (GRAS) status, bacteriocins produced by lactic acid bacteria (LAB) have attracted great interest in terms of food safety 3,4, 5. Bacteriocin from lactic acid fermentation of carrot, radish and cucumber had antimicrobial activity. Bacteriocin was also effective against B. cereus in different fruit products (pulp, juice and wine) indicating its potential application as a bio preservative in fruit products6. The potential of using bacteriocins of lactic acid bacteria, primarily used as bio preservatives, represents a perspective, alternative antimicrobial strategy for continuously increasing problem with antibiotic resistance7. Until now, approaches to seek improved food safety have relied on the search for more efficient chemical preservatives or on the application of more drastic physical treatments (e.g. high temperatures). Nevertheless, these types of solutions have many drawbacks: the proven toxicity of many of the commonest chemical preservatives (e.g. nitrites), the alteration of the organoleptic and nutritional properties of foods. There is a growing consumer demand for processed food products containing lower levels or no chemical preservatives, leading to indigenous research studies in the field of screening bacteriocin as food preservatives. Therefore, the present study was investigated on the application of bacteriocin from the fermented vegetables as food preservatives.

MATERIALS AND METHODS

Fermentation of Vegetables Vegetables (carrot, avocado, mango, lemon and cucumber) procured from the local markets in Chennai were washed, peeled and

grated. The grated vegetables were fermented with dry salt 2 % (by mass) at 32°C8 .

Isolation of Bacteria The bacteriocin producers from naturally fermented lemon, mango and carrot were isolated by pour plate method using MRS agar9. After incubation for 24–48 h at 32°C, typical colonies were isolated and purified. Identification of Bacteria The bacterial isolates were identified on the basis of their morphological, cultural and physiological and biochemical characteristics as outlined in Bergey’s Manual of determinative Bacteriology10. Pathogenic Bacterial Cultures Standard bacterial cultures such as Escherichia coli (MTCC 553), Bacillus subtilis (MTCC 121) and Klebsiella pneumoniae (MTCC 890) procured from Marina lab, Chennai were used in bacteriocin screening procedures. Bacteriocin Assay11

Antimicrobial activity of the bacterial isolates against all the pathogenic microorganisms was determined by well diffusion method under aerobic conditions. Agar plates were inoculated with 100 µl of each target microorganism after growing them in a broth and diluting appropriately. Wells (3 mm) were cut into the plates and 50 µl of cell-free culture supernatant fluid of the isolated strain was placed into each well. Ampicillin was used as the control. The inhibitory activity against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae was tested on nutrient agar. Plates were kept incubated at 37 °C for 24 h. The antimicrobial activity was determined by measuring the diameter of the inhibition zone around the wells. The bacterial isolate showing the widest zone of inhibition against the target microorganism was selected for further studies. Of them, two colonies from lemon and one from carrot and mango were studied.

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Purification of Bacteriocin8

Isolated strain having maximum antimicrobial zone was grown in MRS broth at 37 °C for 24 h. After incubation, the broth was centrifuged at 5000 × g for 10 min and the cells were separated out. Different concentrations of ammonium sulphate were added to the supernatant. After stirring on a magnetic stirrer, it was kept undisturbed at 4 °C overnight. Precipitates formed were collected by centrifugation at 10, 000 × g for 10 min and redissolved in 0.5M potassium phosphate buffer with pH 7.0 and dialysed against the same 0.5 M potassium phosphate buffer with pH 7.0. Characterization of Bacteriocin (i) Effect of Temperature8

To determine the effect of temperature, a volume of 5 ml of bacteriocin in different test tubes was overlaid with paraffin oil to prevent evaporation and then heated at 40ºC, 60ºC, 80ºC, 100ºC and 121°C for 15 minutes. The heat-treated bacteriocin samples were then assayed for antimicrobial activity .

(ii) Effect of pH8

A 5ml aliquot of bacteriocin was taken in test tubes and the pH values of the contents were adjusted to 4 – 8 individually, using either 1 N NaOH or 1 N HCl solution. After allowing the samples to stand at room temperature for 2 hours the antinmicrobial activity was assayed . (iii) Effect of Enzymes8

The sensitivity of the active substance to enzymes was tested on bacteriocin incubated at 30ºC and were treated for 2 hours with 0.1 mg/ml with the following enzymes: α- amylase, RNase, pepsin and lipase (all from Hi Media Laboratory Pvt. Ltd. India). The bacteriocin treated with enzymes was then assayed for antimicrobial activity . (iv) Effect of Salt Concentrations (Nacl2)

12

The effect of salt tolerance was tested by using different concentrations of sodium chloride 5%,

10%, 15% and 20%. The tubes containing bacteriocin and sodium chloride were assayed for antimicrobial activity . Determination of Preservative Effect of Bacteriocin8 The fruit juices, viz. apple, mango, pomegranate was sterilized and inoculated

with E. coil. 200 l of bacteriocin was added to

it. 50µl of sample was spread plate on nutrient agar plates. After 24 hours, the plate count was recorded and compared with the control. The reduction of population percentage was calculated as follows: Reduction of population percentage = Reduction in microbial count / Total microbial count X 100 Protein estimation13 and Molecular Weight Determination in SDS-PAGE14

Protein concentration was determined using Bovine serum albumin as standard. The amount of protein present in the sample was expressed in mg/ml. SDS PAGE was performed by using 12% resolving gel (pH 8.8) and 5% stacking gel (pH6.8) in Tris–glycine buffer (8.3). Polyacrylamide gel was stained with Coomassie Brilliant Blue. After staining, the molecular weight of protein samples was determined using molecular weight marker.

RESULTS

Isolation and Identification of Bacteria Bacteria were isolated from vegetables such as lemon, mango and carrot by using pour plate method9 and they were identified by the morphology, physical characterization and biochemical test such as catalase, oxidase, indole, nitrate reduction, sugar fermentation as outlined in the Bergey’s manual of determinative Bacteriology10 and the bacteria were identified as Bacillus sp., Leuconostoc mesenteroides, Vagococcus sp. and Pediococcus sp. (Figure 1).

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Bacteria isolated from fermented vegetables

Bacillus sp. Leuconostoc mesenteroides

Vagococcus sp. Pediococcus sp.

Figure 1

Pure cultures of bacteria maintained on Nutrient agar Bacteriocin Assay Antimicrobial activity of the bacteriocin against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae was determined by well diffusion method under aerobic conditions. The inhibitory activity was shown in Graph 1

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Graph 1 Antimicrobial activity of Bacteriocin against pathogenic organisms

Characterization of Bacteriocin (i) Effect of temperatures on antibacterial activity of bacteriocin Bacteriocin retained its activity up to 100°C for 50% and its activity reduced drastically when heated at 121°C for 15 minutes as shown in Graph 2.

Graph 2 Antimicrobial activity of bacteriocin against pathogenic

microorganisms at different temperature

(ii) Effect of pH on the antibacterial activity of bacteriocin Bacteriocin was active in a wide range of pH (4–8). Bacteriocin showed maximum activity against the test microorganisms at pH 7.0 as shown in Graph 3.

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Graph 3 Antimicrobial activity of bacteriocin against pathogenic organisms at different pH

(iii) Effect of enzymes on antibacterial activity of bacteriocin Bacteriocin produced by the test isolates was tested for their sensitivity to various enzymes. The antimicrobial activity was lost after treatment with the proteolytic enzyme pepsin whereas treatment with amylase, lipase, RNase did not affect its activity as shown in Graph 4.

Graph 4 Antimicrobial activity of bacteriocin against pathogenic organisms on different enzymes

(iv) Effect of salt concentrations (NaCl2) on antibacterial activity of bacteriocin Purified bacteriocin was assessed for its sensitivity to different salt concentrations. Bacteriocin retained its activity when incubated with 5% sodium chloride and there is gradual decrease in its activity with the increasing percentage of sodium chloride as shown in Graph 5.

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Graph 5 Antimicrobial activity of bacteriocin against pathogenic

organisms at different salt concentration

Determination of Preservative Effect of Bacteriocin The preservative effect of bacteriocin in the fruit juices was shown in the Table 1. There was reduction in the microbial population after the addition of bacteriocin.

Table 1 Preservative effect of bacteriocin produced by bacterial isolates from

fermented vegetables on different fruit juices against E. coli

Bacteriocin Bacteriocin %

Apple

Mango

Pomegranate

Preservative effect %

Control - 0 0 0

Bacillus sp.

0.2 42 73

59

Leuconostoc mesenteroides

0.2 74 71 73

Vagococcus sp.

0.2 73 76 59

Pediococcus sp. 0.2 55 58 56

Estimation of Protein and SDS PAGE The amount of protein is 0.05-0.08 mg/ml present in all the isolates .The qualitative analysis of protein was determined by SDS-PAGE as shown in Figure 2.

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Figure 2 Protein profiling of bacteriocin isolated from the micro

organisms of fermented vegetables by SDS - PAGE

Lane 1 - Bacillus sp. Lane 2 - Bacillus sp. Lane 3 - Leuconostoc mesenteroides Lane 4 - Vagococcus sp. Lane 5 - Pediococcus sp. Lane 6 - Pediococcus sp. Lane 7 - Protein marker

DISCUSSION

In the present study, bacteriocin producing microorganisms were isolated from fermented vegetables such as mango, lemon and carrot. They were oxidase,methyl red , indole , nitrate reduction and citrate utilisation were negative and positive for glucose fermentation and based on these biochemical tests and morphological characterisation were identified as Bacillus sp, Leuconostoc mesentroides, Vagococcus sp and Pediococcus sp.( Figure 1). The isolated organisms were tested for antibacterial activity against three pathogens such as B. subtilis, E. coli, K. Pneumoniae . The highest inhibitory activity was demonstrated by Leuconostoc mesenteroides against E. coli, Bacillus subtilis and K. pneumoniae, while the least activity was demonstrated by Pediococcus sp. (Graph 1). Therefore, the inhibitory activity of bacterial isolates is an indication of possession of antibacterial activity. Results revealed the presence of bacteriocin in the test organisms. It is also an indication that the bacteria can be used as probiotics and biopreservative. Bacteriocin produced by the test isolates was heat stable. The bacteriocin produced by Bacillus sp was most heat stable as there was maximum zone of inhibition (7mm) against

Bacillus subtilis at 60°C ( Graph 2). There was no activity exerted by Vagococcus sp and Pediococcus sp at 121°C.Loss of activity after heat treatment at 121°C is due to long exposure to heat15. The activity of bacteriocin was also pH dependent. The highest antibacterial activity was seen in neutral pH ranges so it could be used in acidic range of foods16. Bacillus sp, Leuconostoc mesenteroides and Vagococcus sp have shown maximum zone of inhibition (7mm) against B. subtilis at pH 7 (Graph 3). Pathogenic test organisms were sensitive to bacteriocin treated with amylase, RNase and lipase comparing to proteolytic enzyme pepsin because of the presence of glycosylated and lipid moities in the bacteriocin. The antimicrobial activity was lost or unstable after treatment with the proteolytic enzyme. This is because the proteolytic enzyme, pepsin failed to modify the antimicrobial activity of the bacteriocin, which is not unusual and might be the presence of unusual amino acids in the bacteriocin structure, or cyclic N-and/or C- terminally blocked peptides17. Bacteriocin with amylase, RNase have shown maximum zone of inhibition against E. coli, B. subtilis and K. Pneumoniae (Graph 4) .The effect of salt concentration on

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antibacterial activity of bacteriocin was also studied. The bacteriocin have shown maximum zone of inhibition (5mm) against B. subtilis and K. pneumoniae at 5% of sodium chloride ( Graph 5). Increase in the concentration of sodium chloride decreases the antibacterial activity. Protein profiling of bacteriocin isolated from the micro organisms of fermented vegetables was determined by SDS PAGE. Bacteriocin from Bacillus sp, Leuconostoc mesenteroides and Vagococcus sp have shown two distinct bands corresponding to the molecular weight of 55 KDa and <14.4 KDa. But bacteriocin from Pediococcus sp. has also shown four bands, among them two distinct bands corresponding to the molecular weight of 55 KDa and <14.4 KDa. But they have also shown high molecular weight bands corresponding to 97.4 KDa and 66 KDa (Figure 2). Bacteriocin of 66 KDa from fermented food has been reported18. Therefore, bacteriocin characterized in the present study have shown molecular weight ranging for high class bacteriocin to low class bacteriocin. High molecular weight may be due to their

appearance as aggregates in native state. Hence it should be further purified to ensure its activity against food borne pathogen in food industry. The bacteriocin from the isolates were also tested for preservative effect against E. coli and maximum reduction of E. coli population of 76% was observed in mango juice followed by apple juice(74%) at a concentration of 0.2% ( Table 1). Preservative effect can be increased with the increase in the bacteriocin concentration. These results indicate that bacteriocin possessed desired characteristics of biopreservative. However in control (without bacteriocin), no reduction was observed in the count of E. coli.

CONCLUSION

The stability of bacteriocin to different conditions reflects that such compounds can withstand the conditions normally encountered in food processing, so would remain effective during processing. Bacteriocin can be used in industrial applications as biopreservatives.

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