BacteriocinBacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are typically considered to be narrow spectrum antibiotics, though this has been debated [1] They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. Bacteriocins were first discovered by A. Gratia in 1925.[2][3] He was involved in the process of searching for ways to kill bacteria, which also resulted in the development of antibiotics and the discovery of bacteriophage, all within a span of a few years. He called his first discovery a colicine because it killed E. coli.

Classification of bacteriocins

Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. There are several large categories of bacteriocin which are only phenomenologically related. These include the bacteriocins from gram-positive bacteria, the colicins [4], the microcins, and the bacteriocins from Archaea. The bacteriocins from E. coli are called colicins (formerly called 'colicines,' meaning 'coli killers'). They are the longest studied bacteriocins. They are a diverse group of bacteriocins and do not include all the bacteriocins produced by E. coli. For example the bacteriocins produced by Staphylococcus warneri, are called as warnerin [5] or warnericin. In fact, one of the oldest known so-called colicins was called colicin V and is now know as microcin V. It is much smaller and produced and secreted in a different manner than the classic colicins. The bacteriocins of lactic acid-fermenting bacteria are called lantibiotics. This naming system is problematic for a number of reasons. First, naming bacteriocins by what they putatively kill would be more accurate if their killing spectrum were contiguous with genus or species designations. The bacteriocins frequently possess spectra that exceed the bounds of their named taxa and almost never kill the majority of the taxa for which they are named. Further, the original naming is generally derived not from the sensitive strain the bacteriocin kills, but instead the organism that produces the bacteriocin. This makes the use of this naming system a problematic basis for theory; thus the alternative classification systems.

Methods of classification

Alternative methods of classification include: method of killing (pore forming, dnase, nuclease, murein production inhibition, etc), genetics (large plasmids, small plasmids, chromosomal), molecular weight and chemistry (large protein, polypeptide, with/without sugar moiety, containing atypical amino acids like lanthionine) and method of production (ribosomal, post ribosomal modifications, non-ribosomal).

One method of classification fits the bacteriocins into Class I, Class IIa/b/c, and Class III. [6]

Class I bacteriocins

The class I bacteriocins are small peptide inhibitors and include nisin.

Class II bacteriocins

The class II bacteriocins are small heat-stable proteins. The class IIa bacteriocins (pediocin-like bacteriocins) are the largest subgroup and contain an N-terminal consensus sequence -Tyr-Gly-Asn-Gly-Val-Xaa-Cys across this group. The C-terminal is responsible for species-specific activity, causing cell-leakage by permeabilizing the target cell wall. Class IIa bacteriocins have a large potential for use in food preservation as well medical applications, due to their strong anitlisterial activity, and broad range of activity. The class IIb bacteriocins (two-peptide bacteriocins) require two different peptides for activity. Other bacteriocins can be grouped together as Class IIc (circular bacteriocins). These have a

wide range of effects on membrane permeability, cell wall formation and pheromone actions of target cells.

Class III bacteriocins

Large, heat-labile protein bacteriocins.

Abstract

A total of 220 strains of LAB isolated from 32 samples of traditional fermented food from Senegal were screened for bacteriocin production. Two bacteriocin producers, Lactococcus lactis subsp. lactis and Enterococcus faecium, were identified from 12 bacteriocin-producing isolates on the basis of phenotypic analyses and 16S rDNA sequence. Both bacteriocins produced by new isolates show antimicrobial activity against Listeria monocytogenes and Bacillus coagulans whereas only that produced by Lactococcus lactis has an activity against Bacillus cereus. Bacteriocin-producing Lactococcus lactis strains were found in a variety of traditional foods indicating a high potential of growth of this strain in variable ecological complex environment. Partial 16S rDNA of the two bacteriocin producers obtained in this study has been registered to Genbank databases under the accession number AY971748 for Lactococcus lactis subsp. lactis (named CWBI-B1410) and AY971749 for Enterococcus faecium (named CWBI-B1411). The new bacteriocin-producing Lactococcus lactis subsp. lactis strain has been selected for identification and application of the bacteriocin to food preservation.

Keywords : bacteriocin, food preservation, lactic acid bacteria

1 . Introduction

Lactic acid bacteria (LAB) are the biological basis for the production of a great multitude of fermented foods (Lasagno et al., 2002). The most important contribution of these bacteria to fermented products is to preserve the nutritive qualities of the raw material and inhibit the growth of spoilage and pathogenic bacteria (Matilla-Sandholm et al., 1999). This inhibition may be due to the production of many metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, diacetyl and bacteriocins (Ennahar et al., 2000 ; Lasagno et al., 2002). Some bacteriocins kill only bacteria belonging to the same species as producer whereas other bacteriocins kill a broad range of Gram positive bacteria (Conventry et al., 1997 ; Ennahar et al., 2000 ; Mc Auliffe et al., 2001 ; Garneau et al., 2002). The incorporation of these compounds as biopreservative ingredient into model food has been shown to be effective in the control of pathogenic and spoilage micro-organisms (O'Sullivan et al., 2002). They have attracted considerable interest in recent years and several works have focused on the isolation and development of new strains of bacteriocin-producing bacteria. The detection rate of bac+ strains from LAB isolates can be as low as 0.2% and therefore needs a large number of isolates from food sources (Conventry et al., 1997).

The preservation of foods by lactic fermentation has a long history of use in Africa. The multitude of products can be an appropriate ecological habitat for holding wild strains of LAB capable of producing bacteriocin. The aim of the current study was to select bacteriocin-producing LAB from such products in order to use these proteinaceous inhibitors to improve the microbial quality and safety of foods.

2. Materials and methods2.1. Culture and media

MRS1 agar (MRS with 0.1% glucose, and 50 µg/ml of cycloheximide) and M17m agar (0.5% glucose and 50 µg/ml cycloheximide) were utilised for isolation of bacteria from food sources. Different food-borne pathogens and Gram+ bacteria including Escherichia coli, Salmonella infantis, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes and Lactobacillus curvatus from CWBI

collection were used as sensitive. Strains of Lactobacillus curvatus are found to be the most suitable indicator for the quantification of antimicrobial effects of all bacteriocins investigated in both agar and broth system (Conventry et al., 1997).

2.2. Food sources

Strains were isolated from a total of 32 samples of traditional foods from Senegal (Table 1).

2.3. Detection of antimicrobial activity

Lactic acid bacteria were isolated from samples, by direct plating on MRS1 or M17m. A 10% (w/v) food sample in diluent [0.1% (w/v) peptone] was homogenized and 10-fold serially diluted. Plates of serial dilution in MRS1 and M17m media were incubated anaerobically (BD, BBL Campypak microaeroplilic systems Envelopes, Sparks, USA) for 48 h at 30°C. Plates providing a total of 300 colonies were overlaid with a set of 6 indicators and incubated at 37°C for 12 h. Colonies producing zones of growth inhibition in the indicator lawn were isolated from within the agar, inoculated into broth media (MRS1 or M17m) and incubated for 24 h at 30°C. Culture supernatant was prepared as follows: an overnight culture of each isolate was centrifuged at 8,000 r.p.m. The resulting supernatant was neutralized (pH 6.5) with NaOH 5N, sterilized by filtering with acrodisc (pore size 0.22 µm) and assayed for the presence of an inhibitor in the broth following the Agar well diffusion assay (WDA) technique (Barefoot et al., 1983) as follows. Molten agar was first seeded with indicator organism (110 µl of overnight culture per 20 ml of agar) in sterile Petri dishes, and after solidification, dried for 15 min. under flow hood. Wells of uniform diameter (6 mm) were bored in the agar. Aliquots (60 µl) of the cell-free supernatant (CFS) were dispensed in wells, and plates were incubated overnight at 37°C. Inhibition of growth was determined by an area of inhibition surrounding each agar well.

2.4. Bacteriocin assay

To confirm the bacteriocin effect, catalase (65 UI/ml) was added to CFS and the technique was repeated. The effect of various enzymes and heat treatment of CFS activity were also investigated. Units and MIC (Minimum inhibitory concentration): the activity present in the neutralized (pH 6.5) cell-free supernatant of producing cultures was determined by twofold serial dilution of the supernatant in sterile phosphate buffer pH 6 (Barefoot et al., 1983). Activity units per milliliter (AU/ml) were determined as the inverse of the last dilution at which growth inhibition was still detectable following the agar WDA. To determine the effects of enzymatic treatments, samples (180 µl of twice the minimum inhibitory concentration corresponding to the supernatant from the cell-free culture) were incubated with 20 µl portion of following enzyme solutions, P3911 (16.6 UI/ml), type XIV (7.9 UI/ml), type XVIII (0.66 UI/ml), proteinase K (59.2 UI/ml), chymotrypsin (700 UI/ml) at 37°C for 1 h 30 min. (Jack et al., 1996) and the residual activity was measured following the WDA. Positive controls were incubated with 20 µl of 50 mM phosphate buffer (pH 6.5). To determine the effect of temperature and pH on the stability of the inhibitor sample corresponding to a dilution of ¼ of neutralized cell-free supernatant

(pH 6.5) in 50 mM phosphate buffer at pH 5, 7 and 9, and aliquots of each were subsequently heated to 60, 70, 80, 90, 100, 110 and 121°C for 10 min. (Ryan et al., 1996). The remaining activity was assayed and compared to activity at each pH prior to heat treatment.

2.5. Bacterial identification

Selected isolates were examined microscopically for cellular morphology and Gram stain phenotype. Catalase activity was tested by spotting colonies with 3% hydrogen peroxide. Fermentation of different sugars was determined by API 50 CHL (Biomerieux). PCR was used to amplify the 16S rRNA gene of bacteriocin-producing strains. The 16S rDNA sequence was determined by direct sequencing. Total DNA was isolated by using Wizard genomic DNA purification kit (Promega, Madison, USA). Primers used for PCR and DNA sequencing are presented in table 2. The PCR amplification was performed with the primer pair SPO/SP6 targeted against regions of 16S rDNA (Ventura et al., 2001). Amplification of DNA was performed in a Mastercycler personal thermal cycler (Eppendorf). PCR conditions included a hot start at 96°C (5 min.), 25 cycles consisting of hybridation at 50°C (1 min), polymerisation at 72°C (2 min.), denaturation at 96°C (1 min) and a final extension at 72°C (10 min.). PCR products were resolved by electrophoresis in 1% (w/v) agarose gel and visualized by ethidium bromide (1 µl/10 ml) staining. 16S rDNA PCR amplicons were purified following the microcon YM-100 kit (Bedford, MA, USA) and sequenced using the Big Dye Terminator V3.0 kit as specified by the supplier with primers described in table 2 while automated sequencing of both strands of the PCR products was done on an ABI 3100 automated gene sequencer (ABI, Forster, USA). The sequences obtained (350–500 bp) were then assembled in silico (Vector NTI) using overlapping zones between the various sequences to form the contiguous sequence. Phylogenetic analysis was realised by an alignment of sequence consensus of the 16S rDNA genes collected in an international database (Genebank). The results were then expressed in percentage of homology between the submitted sequence and the sequences resulting from the database.

3. Results and discussion3.1. Detection of antimicrobial activity and bacteriocin assay

A total of about 70000 colonies isolated from food samples were examined for detection of antibacterial activity against a set of 6 indicators. A total of 340 (0.6% detection rate) colonies producing a growth inhibition area in the indicator lawn were recorded. Staphylococcus aureus demonstrated the highest detection rate among indicator bacteria. Two hundred and twenty colonies which displayed antibacterial activity against the indicator lawn were randomly isolated and purified; 20 of these strains produced antibacterial activity in the neutralized cell-free supernatant, whereas only 12 confirmed the activity when the CFS was treated with catalase (65 UI/ml). The activity was either completely or partially inactivated by proteolytic enzymes (Figure 1) but was resistant to heat (Figure 2). These results

demonstrated that antimicrobial compounds produced by our 12 isolates were heat stable protein or peptide indicating bacteriocin-like substances. Four food samples (12.5% incidence rate) yielded bacteriocin-producing strains. The distribution of bacteriocin producers in food samples is presented in table 2. Our observations could be correlated with similar studies reported these last years. Garver et al. (1993) have reported 13% of products yielding bacteriocin-producers by direct plating and 21% by enrichment while Conventry et al. (1997) obtained 43% by direct plating and 46% by enrichment. Lasagno et al. (2002) identified two bacteriocin-producers among 206 isolates selected on the basis of the inhibition of Lactobacillus plantarum by the CFS using the WDA. Four of the twelve bacteriocin-producing isolates obtained (designated CWBI-B1410, CWBI-B1411, CWBI-B1427 and CWBI-B1428) were selected for further study on the basis of their food source, indicator used for detection, activity against seven indicators and stability of activity upon repeated subcultures.

3.2. Inhibitory spectra

The sensivity of 33 bacterial strains from different genera to the bacteriocin-like substances produced by the four selected isolates are presented in table 3. Neutralised cell-free supernatant from CWBI-B1410, CWBI-B1427 and CWBI-B1428 isolates demonstrated similar spectra of activity broader than the one produced by the CWBI-1411 isolate. This one showed a reasonably diverse spectrum. All bacteriocins produced by the four isolates showed antimicrobial activity against Bacillus coagulans involved in food spoilage, and the food-poisoning bacterium Listeria monocytogenes, whereas only bacteriocins produced by CWBI-B1410, CWBI-B1427 and CWBI-B1428 isolates inhibited Bacillus cereus.

3.3. Bacteriocin activity

Activity units per ml (AU/ml) of bacteriocins was determined following WDA assay and presented in table 4. Bacillus coagulans showed the more sensivity to bacteriocins. The level of activity against this

strain, (104 to 105 AU/ml), can be correlated with similar studies reported these last years. Flynn et al. (2002) have reported 106 AU/ml, using a bacteriocin produced by Lactobacillus salivarius subsp. salivarius, whereas Garcia et al. (2003) reported 105 AU/ml with enterocin EJ97 produced by Enterococcus feacalis EJ97. Bacteriocins produced by CWBI-B1410, CWBI-B1427 and CWBI-B1428 isolates also demonstrated an activity of 103 AU/ml against Pediococcus pentosaseus whereas that produced by CWBI-B1411 is more active against Listeria monocytogenes (data not shown). CWBI-B1410 showed the highest production of bacteriocin.

3.4. Identification of bacteriocin-producers

Bacteriocin-producing isolates were catalase negative, and Gram positif cocci (CWBI-B1410, CWBI-B1427 and CWBI-B1428) or ovoid (CWBI-B1411). Fermentation of different carbohydrates was performed using the API 50 CHL system (API Biomerieux). 22 reactions (sugar fermentation) were determined and 6 of them provided a mean of discriminating them (Table 5). Identification made by the API database correlation indicated that all the four strains were Lactococcus lactis subsp. lactis. However, a low percentage of similarity (91%) was obtained for CWBI-B1411 (Table 6).

Nucleotide sequences of 16S rDNA of the four bacteriocin-producing isolates were carried out to confirm or infirm biochemical species identification. The determined 16S rDNA sequences of isolates were compared directly with the Genebank database. A high level of similarity of 16S ribosomal DNA nucleotide sequences (99% of matches) of CWBI-B1410, CWBI-B1427 and CWBI-B1428 strains was observed with the sequences of Lactococcus lactis subps. lactis strains whereas the sequence of CWBI-B1411 strain matched best with that of Enterococcus faecium strains (Table 6). The closest matches for CWBI-B1411 strain and Enterococcus faecium were different from the identification determined by API method (Table 6). However, we prioritized genetic identification because of it accordance with morphology analysis, and the low percentage of similarity obtained by biochemical analysis which could be due to the inadequacy of API CHL 50 for well identification of Enterococcus strains. Partial 16S rDNA of the two bacteriocin-producing isolates Lactococcus lactis subsp. lactis (CWBI-B1410) and Enterococcus faecium (CWBI-B1411) have been registered to Genebank databases respectively under the accession numbers AY971748 and AY971749. Due to the high amount of bacteriocin produced in the culture supernatant, the Lactococcus lactis subsp. lactis strain (named CWBI-B1410) was selected for further investigations.

4. Conclusion

Traditional fermented foods from Senegal provide an appropriate ecological habitat for wild bacteriocin-producing LAB. Bacteriocin producers mainly belong to Lactococcus lactis subsp. lactis group. Bacteriocin-producing Lactococcus lactis strains were found in a variety of fermented products indicating a high potential of growth of this strain in different ecological complex environment. The produced bacteriocins showed a broad spectrum of activity including spoilage microorganisms and pathogens associated with food such as Bacillus coagulans, Listeria monocytogenes and Bacillus cereus. These results show the potential usefulness of these bacteriocins justifying a more in depth investigation for their identification and application as food biopreservatives.

Production, Purification, Stability and Efficacyof Bacteriocin from Isolates of Natural Lactic AcidFermentation of VegetablesVinod Kumar Joshi1*, Somesh Sharma1 and Neerja S. Rana21Fermentation Technology Laboratory, Department of Postharvest Technology2Department of Vegetable Crops, Dr. Y. S. Parmar University of Horticulture and Forestry,Nauni, 173230 Solan (H.P.), IndiaReceived: November 11, 2005Accepted: March 14, 2006SummaryThe antimicrobial activity of partially purified bacteriocin produced during naturallactic acid fermentation of carrot, radish and cucumber was assessed and characterized.Out of ten strains, the isolated strain CA 44 of Lactobacillus genus from carrot fermentationproduced bacteriocin with maximum antimicrobial activity against Escherichia coli, Staphylococcusaureus and Bacillus cereus, though it was more effective against E. coli than others.Bacteriocin was stable at up to 100 °C but its activity declined compared to that at 68 °C

and was completely lost at 121 °C. The maximum antimicrobial activity was retained withinthe pH range of 4–5, but it was adversely affected by the addition of papain. Bacteriocinwas also effective against B. cereus in different fruit products (pulp, juice and wine) indicatingits potential application as a biopreservative in fruit products.Key words: antimicrobial, bacteriocin, lactic acid fermentation, Lactobacillus, Staphylococcus,Bacillus cereus, E. coli, pathogenic microorganism, stability, biopreservativeIntroductionPreservation of vegetables by lactic acid fermentationis an ancient practice involving lactic acid bacteria(LAB), which predominantly produce lactic acid besidescertain compounds such as bacteriocin, which has antimicrobialactivity against other groups of microorganisms.The antimicrobial activity of bacteriocins producedby LAB has been detected in foods such as dairy products,meats, barley, sourdough, red wine, fermented vegetables,etc. (1–5). Therefore, the strains of lactic acidbacteria have also potential to act as a biopreservative ornatural food preservative (6–8). The bacteriocins producedinhibited food spoilage and pathogenic bacteriasuch as Staphylococcus aureus, Escherichia coli, Bacillus cereus,B. subtilis, Listeria monocytogenes and Clostridium perfringenswhich are recalcitrant to traditional food preservationmethod (9). The use of bacteriocins or the microorganismsthat produce them is attractive to the food industryin the face of increasing consumer demand fornatural products and the growing concern about foodbornediseases. It has also necessitated the need to exploitthe biologically derived antimicrobial substancesproduced by LAB. It is not clear if any bacteriocin isproduced in the vegetables fermented by LAB in naturalor inoculated fermentation. The bacteriocin produced bythe strains isolated from naturally fermented vegetableshas neither been characterized nor checked for its efficacyin various food products. Therefore, keeping in viewthe above objectives the present investigations were carriedout and the results obtained are discussed here.Materials and MethodsFermented vegetablesVegetables (carrot, radish and cucumber) procuredfrom the markets were washed, peeled and grated/sliced.V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006) 435*Corresponding author; Fax: ++91 (0)1792 252 242; E-mail: vkjoshipht@rediffmail.comThe grated carrot and radish were fermented with drysalt 2 % (by mass) at 27 °C, whereas sliced cucumberswere fermented in 3 % (by mass per volume) brine at 32°C. Predominant microflora were isolated from thesesamples.Pathogenic bacterial culturesStandard bacterial cultures, viz. Escherichia coli (0165),Staphylococcus aureus (B-43-5) and Bacillus cereus procuredfrom Central Research Institute (CRI), Kasauli, were usedin bacteriocin screening procedures and all the cultureswere maintained as per the recommended practices.Isolation and identification of bacteriocinproducing bacteriaThe bacteriocin producers from naturally fermentedcarrot, radish and cucumber were isolated by pour platemethod technique as per the conventional method (10)using MRS agar. After incubation for 24–48 h at 32 °C,typical colonies were isolated and purified. The isolateswere differentiated on the basis of their morphological,cultural and physiological characteristics such as oxidasetest, utilization of citrate as a sole carbon source and catalasetest (10,11), and accordingly were tentatively identifiedup to the genus level (12).Screening of isolates for antimicrobial activityAntimicrobial activity of the bacterial isolates againstall the pathogenic microorganisms was determined bywell diffusion method (13–16) under aerobic conditions.

Agar plates were inoculated with 100 mL of each targetmicroorganism after growing them in a broth and dilutingappropriately. Wells (3 mm) were cut into the platesand 100 mL of cell-free culture supernatant fluid of theisolated strain was placed into each well. The inhibitoryactivity against E. coli was tested on EMB agar whereasStaphylococcus aureus and Bacillus cereus were tested onnutrient agar. Plates were kept at cool temperature for 2 hand then incubated at 37 °C for 24 h. The antimicrobialactivity was determined by measuring the diameter ofthe inhibition zone around the wells. The bacterial isolateshowing the widest zone of inhibition against the targetmicroorganism was selected for further studies.Partial purification of bacteriocinIsolated strain having maximum antimicrobial zonewas grown in MRS broth at 37 °C for 24 h. After incubation,the broth was centrifuged at 5000 × g for 10 minand the cells were separated out. Supernatant was usedas a crude bacteriocin. Different concentrations of ammoniumsulphate were added to the supernatant. Afterstirring on a magnetic stirrer, it was kept undisturbed at4 °C overnight. Precipitates formed were collected by centrifugationat 10 000 × g for 10 min and redissolved in 20mmol sodium phosphate buffer with pH=6.0. Inhibitionzone of different fractions was recorded in comparisonwith the crude bacteriocin.Characterization of bacteriocinHeat stabilityA volume of 5 mL of bacteriocin in different testtubes was overlaid with paraffin oil to prevent evaporationand then heated at 68 and 100 °C for 10 and 20 min,respectively, and at 121 °C for 15 min under pressure.The heat-treated bacteriocin samples were then assayedfor antimicrobial activity as described earlier.Effect of pHA 5-mL aliquot of partially purified bacteriocin wastaken in test tubes and the pH values of the contentswere adjusted to 2–9 individually, using either dilutedNaOH or HCl (1 M NaOH or 1 M HCl solution). Afterallowing the samples to stand at room temperature for 2 hthe activity was assayed as described earlier.Effect of proteolytic enzyme (papain)A 5-mL aliquot of bacteriocin preparation was takenin test tubes and treated with papain (100 TU) 1 mg/mLat pH=7. The test tubes with and without the enzyme(control) were incubated for 2 h at 37 °C and heated for3 min at 100 °C to denature the enzyme. Both the controland the samples were assayed for antimicrobial activityby using well diffusion method.Determination of preservative effect of bacteriocinThe food products, viz. juice (apple), pulp (apricot)and prepasteurized wine (plum) were sterilized and inoculatedwith Bacillus cereus at 108 CFU/mL. Initial count ofinoculated samples was recorded and bacteriocin supernatantat a concentration of 0.05 to 0.5 % was added. After24 and 72 h, the plate count was recorded and comparedwith the control (without bacteriocin).Results and DiscussionBased on morphological and biochemical tests, all theisolates were identified as belonging to lactic acid bacteria(LAB) group except RA33, which was identified asyeast. The isolate CA44 (giving maximum antimicrobialactivity) was Gram-positive, rod shaped, negative forcatalase and peroxidase test, having circular and whitecolonies on the MRS media. The strain was also positivefor galactose, arabinose, mannitol, sorbitol, sucrose, glucose,trehalose, lactose, raffinose and negative for maltose,citrate and arginine test. Isolate CA44 from carrotproduced the maximum inhibition zone against all thetested microorganisms and was maximum against E. coli.

The best conditions for bacteriocin production by Lactobacillusplantarum in batch fermentation were the saltconcentration ranging from 2.3 to 2.5 % and temperatureranging from 22–27 °C (17). Lactobacillus plantarum strainisolated from fermented carrots which produced bacteriocinwith antibacterial activity against Staphylococcusaureus and spheroplasts of Gram-negative bacteria (18)and Lactococcus lactis ssp. cremoris was also isolated fromradish fermentation (1).An increase in antimicrobial activity after partial purificationof crude bacteriocin by ammonium sulphate precipitationtook place (Fig. 1). The fraction with the highestbacteriocin activity was precipitated with 20–30 %436 V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006)(by mass per volume) ammonium sulphate. The antimicrobialactivity (in terms of inhibition zone diameter) increasedfrom 12 to 23 mm. There was 1.91-fold increasein the partially purified bacteriocin activity than that ofcrude bacteriocin. Earlier, the inhibitory activity of bacteriocinisolated from malted barley was precipitated fromcell free supernatant using 40 % ammonium sulphate saturation,and resuspended in 2 mmol sodium phosphatebuffer, pH=6.0 and purified using chromatography (19).Partially purified bacteriocin was found to be stableat 68 °C for up to 20 min. At 100 °C for 10 min it couldretain 55 % of antimicrobial activity, while at the sametemperature for 20 min, only 28 % of activity could beretained (Table 1). However, after incubation for 15 minat 121 °C, the complete loss of activity took place. Comparedto the earlier reports on bacteriocin, residual activitywas lower in our study than reported earlier (20).Furthermore, since tolerance of bacteriocin to heat isknown to depend on the stage of purification, pH, presenceof culture medium, other protective components,etc. that might have influenced the antimicrobial activityin our findings too. The heat stability of bacteriocin discussedhere indicates that it could be used as biopreservativein combination with thermal processing to preservethe food products. Furthermore, when comparativelylow temperature is employed for processing comparedto high temperature being used at present, the retentionof nutrients would be higher. However, more studies onthese aspects are needed.The partially purified bacteriocin showed maximumactivity against the target microorganisms at pH=5.0(Fig. 2), but after pH=5.0 the activity of the bacteriocingradually but continuously decreased. At pH=9.0, theantimicrobial activity was drastically reduced to morethan 2.5 times that of the control. Thus, the bacteriocinwas found active over a wide pH range with the highestactivity at low pH range of 4–5. Earlier, the bacteriocinproduced by a newly isolated Bacillus species strain 8Awas found active over a pH range of 5–8 but was inactivatedwhen incubated outside these limits (9). Anotherbacteriocin produced by Lactococcus lactis D53 and 23 wasactive over a wide pH range with the highest activityshown at low pH range of 3–5 (13), as was the case withthe bacteriocin from Pediococcus sp. (21). Bacteriocin activitywas completely lost when treated with proteolyticenzyme (papain), which is in agreement with the earlierreport (22). The bacteriocin pediocin ACH from Pedicoccusacidilacti was sensitive to proteolytic enzymes and wascompletely inactivated by several proteolytic enzymes(22,23). The stability of bacteriocin to different conditionsreflects that such compounds can withstand theconditions normally encountered in food processing, sowould remain effective during processing.The partially purified bacteriocin from isolate CA44was also tested for preservative effect against B. cereus(Table 2), and clearly the preservative effect in juice, wine

and pulp increased with the increase in the concentrationof bacteriocin. Maximum reduction of Bacillus cereuspopulation of 92 % was observed in wine followed byjuice (87 %) and pulp (63 %) at a concentration of 0.5 %.V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006) 4370510152025Crude 0–20 20–30 30–40 40–50 50–60 60–70 70–80Inhibition zone diameter/mmbacteriocinm/V(ammonium sulphate)/%Fig. 1. Increase in antimicrobial activity of bacteriocin fromLactobacillus sp. isolate (CA44) using ammonium sulphate fractionationTable 1. Effect of temperature on antimicrobial activity of partially purified bacteriocin from isolated Lactobacillus sp. (CA44)Temperature/°C t/minInhibition zone diameter/mmE. coli B. cereus S. aureus68 10 23 (100) 19 (100) 20 (95)20 22 (95) 19 (100) 20 (95)100 10 15 (65.21) 13 (68.42) 11 (55)20 10 (43.47) 9 (47.36) 6 (28.57)121 15 0 0 0Control (without heat treatment) – 23 19 21Values in parentheses represent retention of antimicrobial activity (in %)5.07.09.011.013.015.017.019.021.023.025.0Inhibitionzone diameter/mmE. col iB. cereusS. aureuspHControl 2 3 4 5 6 7 8 9Fig. 2. Effect of pH on antimicrobial activity of partially purifiedbacteriocin from Lactobacillus sp. isolate (CA44)However, in control (without bacteriocin), no reductionwas observed in the count of B. cereus. The results (Fig.3) further revealed that microbial count drastically decreasedin wine and the same pattern was followed injuice too. In pulp, only a concentration of bacteriocinabove 0.2 % drastically decreased the microbial count.Highest antimicrobial activity of bacteriocin against thetarget microorganism in wine could partly be attributedto inhibitory effect of ethanol. Briefly, the results indicatethat bacteriocin possessed several desirable characteristicsof a biopreservative.ConclusionThe study revealed that bacteriocin from Lactobacillussp. isolated from natural lactic acid fermentation ofvegetables possesses a wide spectrum of inhibitory activityagainst Escherichia coli, Staphylococcus aureus andBacillus cereus. Therefore, it has a potential for applicationas a biopreservative in different food products assuch or in combination with other preservation methods.Since lactic acid fermentation is employed mostlyfor development of products, especially for flavour andtaste of the fermented products, the production of bacteriocinin such products assumes more significance asbiopreservative apart from imparting probiotic effect tothe product.

Screening and application of bacteriocin-producing thermotolerantlactic acid bacteriaJ. Nakayama1, S. Nitisinprasert2, T. Zendo1, P. Wilaipun3, A. Swetwiwathana4, W. Noonpakdee5,W. Malaphan6, H. Matsusaki7, K. Doi1, K. Sonomoto1, 8

1Faculty of Agriculture, Graduate School, Kyushu Univ., 2Faculty of Agro-Industry, Kasetsart Univ., 3Facultyof Fisheries, Kasetsart Univ., 4Faculty of Agricultural Technology, King Mongkut’s Institute of TechnologyLadkrabang (KMITL), 5Faculty of Science, Mahidol Univ., 6Faculty of Science, Kasetsart Univ., 7Faculty ofEnvironmental and Symbiotic Sciences, Prefectural Univ. of Kumamoto, 8Bio-Architecture Center, KyushuUniv.Objective: Bacteriocins produced by lactic acid bacteria (LAB) have attracted special interestsfrom the aspect of their potential use as safe and natural antimicrobials which can be applied asfood preservatives, fine chemicals or post-antibiotic pharmaceuticals. Since bacteriocins arerelatively small peptides whose molecular weights are ranging from two thousands to seventhousands in most cases, they are generally thermostable and heat tolerant. This feature would givean advantage for the practical use in food and other industries. On the other hand, LAB aregenerally mesophilic and their bacteriocin biosyntheses are even more susceptible to hightemperature than primary metabolism. This would be disadvantageous when bacteriocin-producingLAB are applied into natural fermented food as a starter culture or into animals as probiotics.Especially in tropical countries, this would be more serious. To overcome this problem, wescreened thermotolerant LAB (TLAB) which can efficiently produce bacteriocin at more than 40oCfrom Thai natural resources.Results and DiscussionScreening: To aim bacteriocin-producing TLAB, we have performed a large scale screening on ourLAB collections. LAB strains have been isolated from Thai traditional fermented foods such asPla-ra (fermented fish) and Nham (fermented meat) or other Thai natural resources such as silageand chicken intestine. TLAB were evaluated according to the growth at temperatures ranging from40oC to 50oC. Bacteriocin activity and spectrum was examined by a conventional plate assay withtwelve indicator strains including genera of Bacillus, Micrococcus, Listeria, Lactobacillus,Enterococcus, Leuconostoc, Pediococcus, and Lactococcus. Antimicrobial activity againstgram-negative bacteria is occasionally examined by using Escherichia coli and Salmonella sp.After the screening under normal temperature, we examined bacteriocin productivity attemperatures higher than 40oC.During the course of screening, we found a number of bacteriocin-producing LAB which couldgrow well at higher than 40oC but failed to produce bacteriocin at the same conditions. However,some strains listed in Table 1 showed a significant bacteriocin activity at the temperatures higherthan 40oC. Especially, it is noticeable that Lactobacillus reuteri KUB-AC5, Pediococcuspentosaceus TISTR 536 and Pediococcus acidilactici KUB-L0026 showed significant bacteriocinactivity even at 50oC.As a result, more than ten bacteriocin-producing TLAB strains belonging to four genera,Enterococcus, Lactobacillus, Lactococcus and Pediococcus, were found with a variety ofantimicrobial spectra. It should be noted that Lactobacillus reuteri KUB-AC5 isolated fromchicken intestine showed antimicrobial activity against both gram-negative and gram-positivebacteria. Pediococcus acidilactici KUB-L0026 also showed antimicrobial activity againstgram-negative bacteria and Bacillus cereus but not against LAB tested.Peptide purification and structural analysis: As a result of purification, some strains were foundto produce more than one peptides having bacteriocin activity. Enterococcus faecium NKR-5-3isolated from Pla-ra produced at least four peptides having bacteriocin activity. One of them wasidentified as a known bacteriocin, brochocin A, while the other three were found to have novelamino acid sequences. Enterococcus faecium KU-B5 isolated from sugar apple produced threepeptides with bacteriocin activity and two were identified as known bacteriocin, enterocin A and B,while the other one named enterocin X was found to have a novel amino acid sequence.Lactobacillus salivarius AC21 isolated from chicken intestine also produced two peptides namedSalAα and SalAβ, which were suggested to link with each other by disulfide bridge. SalAα isidentical to known bacteriocin Abp118α while SalAβ has a new amino acid sequence. Thistwo-peptide bacteriocin was named salivacin K21.Application: Pediococcus pentosaceus TISTR 536 isolated from Nham produces knownbacteriocin pediocin PA-1 which shows strong anti-liesteria activity. TISTR 536 was applied as astarter culture for Nham and compared to nisin Z-producing strain. TISTR 536 exhibited higherproductivity of both lactic acid and bacteriocin, and its pediocin PA-1 produced during thefermentation period was more stable than nisin Z produced under the same condition. TISTR 536applied to Nham showed strong antagonism against Salmonella anatum which is commonlycontaminated Salmonella species. This effect was significantly higher than that by non-bacteriocinproducingcontrol strain, suggesting a synergism between lactic acid and pediocin PA-1. This data

suggests the potential use of TISTR 536 as a starter for Salmonella-free Nham production.The other bacteriocin-producing TLAB found in this project would be also applicable for certainpurposes. Lactobacillus reuteri KUB-AC5 could be applied as probiotics with anti-Salmonellaactivity for broiller chicken. E. faecium NKR-5-3 could be applied as a starter for Pla-ra. E.faecalis K-4 could be applied as a starter culture for silage. Feeding back of bacteriocin-producingTLAB to original source would be good way for the biocontrol of natural microflora.References: (1) S. Nitisinprasert, N. Nilphai et al., Screening and identification of effective thermotolerantlactic acid bacteria producing antimicrobial activity against Escherichia coli and Salmonella sp. resistant toantibiotics. Kasetsart Journal (Nat. Sci.). 34, 387-400 (2000). (2) P. Wilaipun and T. Zendo et al., Thetwo-synergistic peptide bacteriocin produced by Enterococcus faecium NKR-5-3 isolated from Thaifermented fish (Plara). ScienceAsia. 30, 115-122 (2004). (3) A. Swetwiwathana, N. Lotong et al., Maturationof Nham - a Thai fermented meat product. Fleisch Wirtschaft International. 22, 46-49 (2007).Table. 1 Bacteriocin-producing thermotolerant LAB found in this projectProducer strain SourceToleranttemp.(ºC)*BacteriocinAntimicrobialspectrum**Main researcherE. faecium NKR-5-3 Pla-ra 40 Peptide A, B, C, D Broad P. WilaipunE. faecalis NKR-4-1 Pla-ra 40Two-peptidelantibioticBroad P. WilaipunE. raffinosus KU822 Ornament fish 40 Peptide A, B Moderate P. WilaipunLb. plantarum PMU33 Somfak 45 Plantaricin W Broad W. NoonpakdeeLc. Lactis WNC20 Nham 40 Nisin Z Nisin type W. NoonpakdeeLb. fermentum onil2 Nham 40 Peptide 1256da Narrow W. NoonpakdeeP. pentosaceus WNK19 Nham 40 Pediocin PA-1 Class IIa type W. NoonpakdeeLc. Lactis KU-T1 Tofu’s whey 41 Unknown Narrow W. MalaphanE. faecium KU-B5 Sugar apple 41-43 Enterocin A, B, X Class IIa type W. MalaphanLb. reuteri KUB-AC5 Chicken intestine 50 Peptide KAC5 E. coli, Salmonella S. NitisinprasertLb. salivarius AC21 Chicken intestine 45 Salivacin K21 Broad H. MatsusakiP. pentosaceus TISTR 536 Nham 50 Pediocin PA-1 Class IIa type A. SwetwiwathanaLc. lactis N100 and N190 Nham 40 Nisin Z Nisin type A. SwetwiwathanaP. acidilactici KUB-L0026 Silage 50 Peptide KPA26B. cereus, E.. coli,SalmonellaS. NitisinprasertE. faecalis K-4 Silage 45 Enterocin SE-K4 Class IIa type K. Doi* Highest temperature at which bacteriocin was produced.** Activity in culture supernatant. Nisin type, showing strong activity against wide range of Gram-positive bacteria;Class IIa type, showing strong activity especially against Listeria sp.

Determine maximum antimicrobial activity of bacteriocins.

Microbial Update International • June, 2007 •

Preserving vegetables using lactic acid fermentation involves lactic acid bacteria (LAB) that predominantly produce lactic acid in addition to bacteriocins. The antimicrobial activity of bacteriocins produced by LAB has been detected in

foods, such as dairy products, meats, barley, sourdough, red wine and fermented vegetables.

Researchers indicate that strains of LAB have potential to act as a biopreservative or natural food preservative. The bacteriocins are able to inhibit spoilage and pathogenic bacteria, such as S. aureus, E. coli, B. cereus, B. subtilis, L.

monocytogenes and C. perfringens. These bacteria are not responsive to traditional food preservation methods.

The use of bacteriocins or the microorganisms that produce them is attractive to the food industry in the face of increasing consumer demand for natural products and the concerns over foodborne diseases. It has also led to the need

to exploit the biologically derived antimicrobial substances produced by LAB.

It is not clear if any bacteriocin is produced in vegetables fermented by LAB in natural or inoculated fermentation. The bacteriocin produced by strains isolated from naturally fermented vegetables has neither been characterized nor

checked for efficacy in various food products. So, Indian scientists decided to characterize the antimicrobial activity of partially purified bacteriocin produced during the natural lactic acid fermentation of carrot, radish and cucumber.

Out of 10 strains, the isolated strain CA 44 of the Lactobacillus genus from carrot fermentation produced a bacteriocin that had strong antimicrobial activity against E. coli, S. aureus and B. cereus, although it was more effective against E. coli than other bacteria. The bacteriocin was stable up to 100 C, but its activity had declined compared to bacteriocin at

68 C. Its stability was completely lost at 121 C.

The maximum antimicrobial activity was retained within a pH range of 4 to 5. But this activity was adversely affected by the addition of papain, a cysteine protease present in papaya, and which is useful in tenderizing meat and other

proteins. Bacteriocins were also effective against B. cereus in different fruit products--pulp, juice and wine--indicating their potential application as biopreservatives in these types of products.