traditional fermented foods - apjor.comapjor.com/files/1378398165.pdf · traditional fermented...

September 2013, Volume: I, Issue: IX

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CHARACTERIZATION OF BACTERIOCINS FROM THE ISOLATES OF

TRADITIONAL FERMENTED FOODS

Mr.Sekar.K.V, Associate Professor, Department of Microbiology, The Oxford College of

Science, HSRLayout,Bangalore 560 102

Dr.Bharathi S, Vice Principal & Head, Department of Microbiology, The Oxford College of


Mr.Yogesh B J, Associate Professor, Department of Microbiology, The Oxford College of


ABSTRACT

Bacteriocins are proteinaceous compounds that inhibit the growth of their closely related

species. Some of them are inhibitory towards spoilage and food borne pathogenic bacteria. This

study was performed for partial characterization of bacteriocins produced by Lactic Acid

Bacteria isolated from traditionally used fermented foods. Lactic acid bacteria were isolated

from pickle, idly batter, curd and butter. All the isolates were found to be Gram positive rods and

identified as Lactobacillus by various biochemical tests. Antimicrobial activity was found in

Lactic acid bacteria isolated from pickle, idly batter, and curd. Crude bacteriocin was extracted

from different LAB and tested against indicator organisms such as Escherichia coli,

Staphylococcus, Bacillus cereus , and Listeria monocytogenes. All the three bacteriocins showed

almost similar effect on the indicator organisms. All the bacteriocins were resistant up to 121oC

for 10 mins and sensitive to alkaline pH. Proteinaceous nature of bacteriocins was confirmed

with proteinase K. Bacteriocins were produced in de Man Rogosa Sharpe broth by batch

fermentation and partially purified by centrifugation, ultra filtration, ammonium sulphate

precipitation and finally extracted with solvent. Partially purified bacteriocins were separated by

SDS-PAGE and it was found that these were 6kDa proteins. The characterized bacteriocins are

good antibacterial candidates to prevent spoilage of stored food; further characterization can

lead to commercialization of bacteriocin as food preservatives.

Key words: Bacteriocin, Fermented foods, Lactic acid bacteria

Running title: Characterization of bacteriocins of lactic acid bacteria

Abbreviations: LAB, Lactic Acid Bacteria; LABC, Lactic Acid Bacteria from curd; LABI,

Lactic Acid Bacteria from; LABP, Lactic Acid Bacteria from Pickle; MHA, Mueller Hinton

Agar ; MRS, de Man Rogosa Sharpe; SDS PAGE, Sodium Dodecyl Sulphate Poly Acrylamide

Gel Electrophoresis.


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INTRODUCTION

In food production it is crucial that suitable actions are taken to guarantee the safety and stability

during the food shelf life. In particular, the current trends adopted by modern consumers and

food legislations, have made this aim a great challenge for the food industry (Brul and Coote

1999). Now a days, the consumer requires foods of high quality, without chemical preservatives,

safe and possessing long shelf life. Concomitantly, the food legislations have restricted the use of

some chemical preservatives in different foods (Brul and Coote 1999). These have caused many

problems for the food industry.

Currently there are discussions concerning the new antimicrobial agents which can be applied in

food conservation systems. These agents could be used through the combination of various under

lethal treatments and could provide the necessary protection to the food against pathogen and

spoilage microorganisms (Peck 1997).

Similarly, a new perspective of food conservation emphasizes the application of the named

“natural antimicrobial system”, which could use the synergistic action of several elements unlike

antibiotics which were once highly effective become ineffective as bacteria mutate and develop a

resistance to them. It includes the animal, plant and microbial products having antimicrobial

properties jointly with physical nature, packaging procedures, manufacture procedures and the

storage food procedures. These actions in an associated use could produce a synergistic effect to

propitiate an unfavorable environment to the microbial survival (Gould 1995).

Searching new technologies to be applied for food conservation, the use of substances produced

by microorganisms deserves prominence. Among these substances, bacteriocins deserve special

attention (Daw and Falkiner 1996). These molecules are included in naturally occurring

preservatives class and biological molecules of low molecular weight (Brull and Coote 1999).

Lactic acid bacteria are traditionally used as starter cultures for the fermentation of foods and

beverages because of their contribution to flavour and aroma development and to spoilage

retardation (Gilliland 1986). The preservative effect is mainly due to the acidic conditions that

these bacteria create in food during their development, but they are capable of producing and

excreting inhibitory substances other than lactic acid and acetic acid. These include hydrogen

peroxide, ethanol, diacetyl, carbon di oxide, bacteriocin or antibiotic like substances (De Vuyst

and Vandamme 1994 a). They differ from usual antibiotics in at least two ways in that they are

ribosomally synthesized while antibiotics are generally secondary metabolites (Rodriguez et al.

2002) and they have relatively narrow inhibiting spectra as they are only lethal to bacteria

closely related to the producer strain (Riley and Wertz 2002). Although bacteriocins may be

produced by Gram-positive and Gram-negative bacteria, those from lactic acid bacteria (LAB)

are of particular interest due to their potential use in the food industry as natural safe food

preservatives (Cleveland, Montville, Nes, and Chikindas 2001; O’Sullivan, Ross, and Hill 2002).

Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Bifidobacterium, and Propionibacterium

used in the food fermentation are admittedly bacteriocin producers. Many of these bacteriocins

have their different characteristics studied, which is necessary before their rational use as


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biopreservatives (Cleveland et al. 2001). Bacteriocins are proteinaceous antimicrobial

compounds that inhibit Gram-positive bacteria, particularly closely related species

(Klaenhammer 1988; De Vuyst and Vandamme 1994 b). Some of them are inhibitory towards

spoilage and food borne pathogenic bacteria including Bacillus sp, Clostridium sp,

Staphylococcus sp, Listeria sp etc.

Therefore bacteriocins of LAB are of particular interest because of their existing and potential

applications as natural preservatives in foods (Holzapfel et al. 1995; Delves-Broughton et al,

1996; Stiles, 1996) might broaden the application range of bacteriocins from LAB in the food

industry (Yurong Gao et al. 2010) and as genetic markers in food grade cloning and expression

systems (Allison and Klaenhammer 1996; Platteeuw 1996). In terms of safety, bacteriocins from

LAB have attracted more interest than those from other resources, because most of LAB are

related to fermented foods (Osmanagaoglu 2007).

MATERIALS AND METHODS

Culture media used were obtained from Himedia, India, while all chemicals were purchased

from SD Fine Chemicals, India and proteolytic enzymes, molecular weight markers were from

Bangalore Genei Pvt Ltd, India.

The following samples, curd, pickle, idli-batter and butter were used for the isolation of LAB and

the test organisms used were Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes

and Escherichia coli.

Screening for bacteriocin producers

The samples were inoculated in MRS broth for enrichment of resident lactic acid bacteria at

370C for 24 hrs and enriched broth cultures were grown on MRS agar and it was propagated

twice in MRS broth at 30oC before use (de Man et al. 1960).

The pathogenic indicator strains were maintained on nutrient agar slants and were sub cultured in

Luria Bertani broth for their optimal growth (Sambrook et al 1989). These were serially diluted

in sterile water blank up to the dilution of 10-5

and then diluted cultures were poured on sterile

MRS agar plates and the plates were incubated at 37oC for 16 hrs till colonies appeared. The

plates were then over laid with MRS soft agar inoculated with lactic acid bacteria and incubated

at 37oC for 24 hrs and observed for zone of inhibition.

Characterization of bacteriocin producers

The bacterial strains that were selected as potential bacteriocin producer were subjected to

morphological, cultural and biochemical characterization, antimicrobial sensitivity testing.

Assay for bacteriocin activity

Bacteriocin activity of LAB isolated from various fermented food samples was tested by agar

well diffusion method. For this, the culture supernatants were collected and made to react with

the indicator organisms on Mueller Hinton agar plates.

Effect of temperature


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To determine the stability of the bacteriocin, supernatant of LAB culture was heated at various

temperatures by keeping time as a constant factor and residual bacteriocin activity was detected

against indicator organisms.

Effect of pH

Sensitivity of bacteriocin towards various pH conditions was tested by adjusting the pH of

culture supernatant in various ranges from 4 to 8 at room temperature for a defined period and

the activity was determined against indicator organisms.

Effect of proteolytic substances

In this process the culture supernatants were tested with proteinase K at the concentration of

1mg/ml and incubated at 37oC for 2 hrs and the activity was assayed.

Production and purification of bacteriocin

All the bacteriocin producers were effectively subjected to grow in de Man Rogosa Sharpe broth

by batch fermentation. After 12 hrs incubation the fermented broth was stabilized at room

temperature for 1 hr then the broth culture was subjected for cell mass recovery by centrifugation

for 10 mins at 12000 rpm.

Ultra filtration

The supernatant containing bacteriocin was further subjected to ultra filtration process and this

was accomplished by minimizing the pore size by treating the ordinary filter paper which is

having the pore size more than 1µm with gelatin.

Ammonium sulphate precipitation

To concentrate the filtrates which are obtained after ultra filtration process, they were

precipitated with ammonium sulphate (40% saturation and held over night at 7oC with gentle

stirring. After overnight incubation the samples were centrifuged at 8000rpm for 15 mins and the

surface pellicles and the bottom pellets were independently recovered and resuspended in 2ml of

50mM phosphate buffer.

Solvent recovery

The concentrated bacteriocins were again recovered by solvent extraction method. To the

concentrated bacteriocins 15 volumes of a mixture of chloroform/methanol (2/1, V/V) was added

and kept at 4oC for one hr. The precipitate formed was centrifuged at 10000rpm for 30 mins and

pellets were collected and resuspended in 3ml of ultra pure water.

SDS PAGE

One dimensional polyacrylamide gel electrophoresis was performed under denaturing conditions

according to Laemmli (1970).

RESULTS AND DISCUSSION

Enrichment, isolation & screening for bacteriocin producers

Lactic acid bacteria were enriched and isolated from 4 different samples and screened for their

antimicrobial activity against the indicator organisms. Out of the four samples, Pickle, Curd and

Idly batter showed the action against the indicator organisms and Butter did not show any action

against indicator organisms.


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Characterization of bacteriocin producers

The organisms which were identified as potential bacteriocin producers were designated as

LABP, LABI, LABC obtained from Pickle, Idly batter and curd respectively. All the isolates

from these three different samples belonged to Gram positive rods and were identified as

Lactobacillus sp.

Assay for bacteriocin activity Culture free supernatants were obtained from LAB isolated from the various fermented food samples and the Bacteriocin activity was tested by Agar Well Diffusion Method (Schillinger and Lucke 1989). All the three isolates were resistant to their own bacteriocin but showed little sensitivity to antimicrobial substances of other isolates, this certainly proved that all the three isolates are not identical and are different. It has already been reported by Evandroleite de Souza et al, 2005 about colicin producing cells (E.coli) where 30% of the E.coli natural population produces colicins to which only 70% of strains were resistant.

Fig 1: Effect of Bacteriocin on Listeria monocytogenes

Fig 2: Effect of Bacteriocin on Staphylococcus aureus


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Fig 3: Effect of Bacteriocin on E.coli

Figure 4: Effect of Bacteriocin from LABP against Indicator Organisms

Figure 5: Effect of Bacteriocin from LABI against Indicator Organisms


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Figure 6: Effect of Bacteriocin from LABC against Indicator Organisms

Effect of temperature

Bacteriocin activity was tested for all the three samples at 60oC, 70

oC, 80

oC, 100

oC and 121

oC

for 10mins and all the samples were found to be resistant. Most of the known bacteriocins

produced by LAB are heat stable and low molecular mass peptide bacteriocin (M.Zamfir et al

1999).

Effect of pH

Bacteriocin activity was tested for all the three samples at pH 4, 5, 6, 7 and 8, at 28oC for 4 hrs

and all the samples were found to be resistant. Alkaline pH was not used for extraction also in

regards to bacteriocins inactivation at alkaline pH, as reported by R Yang et al 1992.

Effect of proteolytic substances

When the culture supernatants were tested with proteinase K at the concentration of 1mg/ml at

37oC for 2hrs, the activity of the bacteriocin was completely lost, by this the isolated bacteriocins

were identified as proteins.

Production and purification of bacteriocin

The potential bacteriocin producers were grown in MRS broth for the mass production of

bacteriocins. After 12hrs incubation, broth cultures were centrifuged and culture supernatants

were obtained for further purification. The culture supernatants were then filtered and

precipitated with ammonium sulphate. The white precipitate formed was further purified by

solvent extraction method. The partially purified bacteriocins were subjected to SDS PAGE to

find out the molecular weight of the proteins. All the bacteriocins, which were extracted from the

different samples, were having 6kDa protein (Fig 3). The purification methods followed gave

purified bacteriocins which constituted of one protein band and purity level can be expected to

be more than 95% as similar reports by D.Guyonnet et al 2000, who used HPLC and mass

spectrometry analysis to conform the purity level.


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Figure 7: Separation of Bacteriocin from LAB in SDS- PAGE

CONCLUSION

The application of bacteriocins as biopreservatives for food matrices started approximately

20 years ago. In these years, a lot of studies have focused on the inhibition of spoilage bacteria

vehiculated with foods and beverages by bacteriocins and their application appeared as a good

alternative to chemical compounds and antibiotics. Whether deliberately added or produced in

situ, bacteriocins have been found to play a defining role in the control of undesirable flora, as

well as in the establishment of beneficial bacterial populations. However, the effect of

bacteriocins, bacteriocinogenic strains or their combinations would not alleviate the practical

food safety issues associated with a large variety of foods, e.g. they may be efficient only in a

narrow pH range, which excludes their utilization in many food products. Thus, a single

bacteriocin-based technique could fit with a single food matrix and its application should be

tested on a “product by product” basis. Furthermore, it can be concluded that, in addition to the

traditional hurdle technology represented by low temperature and vacuum packaging or MAP,

the exploitation of bacteriocinogenic cultures, as well as their pure bacteriocins holds a great

potential for extension of shelf-life and improvement of microbiological safety of vegetable raw

materials and final products.

ACKNOWLEDGEMENTS The authors are indebted to Principal and the Management of The oxford college of science,

Bangalore, India.


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