Indian Journal of Experimental Biology Vol. 41 , June 2003 , pp. 609-613

Detection and characterization of enterocins from Enterococcus sp.

Randhir Singh* * & s R Garg*

Department of Veterinary Public Health , CCS Haryana Agricultural University, Hisar, 125 004, India

Received 22 OClOber 2002; revised 26 February 2003

Examination of 90 isolates of Enterococcus sp. revealed production of enterocin by two isolates of E. faecalis which was inhibitory to Listeria monocytogenes. Two isolates of E. gallinarum produced enterocin active against Staphylococcus aureus. None of the isolates antagonized Salmonella enteritidis. The enterocins of E. faecalis isolates were inactivated by 0.­chymotrypsin but not by trypsin and papain , while those of E. gallinarum were resistant to all the three enzymes. Enterocins produced by all 4 strains were resistant to heating at 60°C for 30 min and 80°C for 10 min, but sensitive to 121 °C for 15 min. At 100°C for 10 min , two enteroc ins, one each of E.faecalis and E. gallinarum were inactivated, while the remaining two retained the bactericidal activity .

Keywords: Enterocin, Enterocinogeni c ac tivity, Enterococcus sp., Listeria, Salmonella

A considerable attention is being given to the bacteriocin-like activity of bacteria particularly lactic acid bacteria (LAB) many of which are food grade organisms. Among these, the microorganisms of the genus Enterococcus have been repolted to produce bacteriocins, termed enterocins, showing antagonism against a variety of bacteria. It has been suggested that E. faecium can be used synergistically along with other LAB as starter culture because of its ability to inhibit Listeria in cheese' . In a similar finding, inhibitory effects of enterocins have been demonstrated against Staphylococcus aureus in skimmed milk and yoghurt and also against Listeria monocytogenes in yoghurt rnilk2

. Preservative effect of enterocin in meat and meat products has also been reported3

.4 . Stability of enterocins over a wide range of temperature and pH adds to their potential as food preservativess.

In India, reports regarding isolation of indigenous strains of enterocinogenic organisms are not available. The present investigation was carried out to study enterocinogenic activity among the indigenous strains of Enterococcus against L. monocytogenes, S. au reus and Salmonella enteritidis and to characterize enterocins.

Materials and Methods Enterococcus isolates - Ninety isolates of differ­

ent species of Enterococcus isolated from raw milk,

*Correspondent author: E-mail: srgarg @hau.nic. in **Present address: Assistant Professor, Department of Veterinary Epidemiology and Preventi ve Medicine, College of Veterinary Sciences, Punjab Agricultural University, Ludhi ana 141 004, India

chicken meat, chevon, fish, sewage and faecal matter of pig, buffalo and man were used in the investiga­tions. These included 50 isolates of E. faecalis, 9 of E. gallinarum, 17 of E. raffinosus and 7 each of E. pseudoavium and E. solitarius.

Indicator organism - Two isolates each of Listeria monocytogenes, Staphylococcus aureus and Salmo-

. nella enteritidis were used as indicator organisms. Strains MTCC 1143 (NCTC 11994) and MTCC 657 (NCTC 7973) of L. monocytogenes were obtained from the Institute of Microbial Technology, Chandigarh, India. The isolates of S. aureus and Salmonella enteri­tidis were taken from the laboratory culture collection.

Preliminary screening of isolates for antagonistic activity - The isolates were screened for antagonistic activity on indicator organisms on tryptose soya agar (TSA) supplemented with 0.6% yeast extract (TSA YE) by deferred antagonism method6 with slight modification. For this, 5 j..ll of overnight broth cultures of the test isolates of Enterococcus sp. were spotted on the surface of TSA YE plate. The plate was then incubated for 18-20 hr at 37°C to allow the colony development. A thin layer of 3-4 ml semisolid TSA YE (containing 0.7% agar w/v) inoculated with 106 cells of indicator organism was overlaid on the surface of the incubated plate taking care not to dis­turb the spots of test isolates. After solidification of the medium, the plate was incubated again at 3rC for 18-20 hr and examined for zone of inhibition of growth of indicator organism around the spots. Any clear zone around the test spots was considered an­tagonistic activity on the indicator organism.

610 INDIAN J EXP BIOL, JUNE 2003

Detection of enterocinogenic activity of the isolates­T he isolates which showed inhibition of indicator or­ganisms were reexam ined to exclude any inhibitory activity due to lact ic ac id production, hydrogen perox­ide production and bacteriophages.

Exclusion of inhibition by lactic acid - The iso­lates were retested fo r inhibition of indicator organ­isms by the same method as mentioned above except that TSA YE did not contain dextrose to prevent lactic ac id production in the culture medi um 7 .

Exclusion of lytic bacteriophages-To rul e o ut bacteriophage activity , the isolates were retested by deferred antagon ism method exp lained above except that thin layer of semisolid medium contai ning indica­tor organ ism was plated on the opposite side of the test spots6

. In this, after the growth of test culture spots, the agar was loosened from the edge of the petri dish with a sterile spatu la and the medium was in­verted into the lid , exposing the bottom surface of the agar. The exposed surface was then seeded with the indicator organi sm, incubated and examined as men­tioned earli er. Presence of inhibitory zone was con­sidered due to diffusible e nterocin.

Exclusion of inhibition due to hydrogen peroxide ­For negating the inhibitory activity due to hydrogen peroxide, cell- free culture fi ltrates (CCFs) of the iso­lates were treated with the enzyme peroxidase (SRL, [ndia 10 mg/m l) and then exam ined for inhibitory ac­tivity by the well-diffusion method8 with s light modi­ficat io n as mentioned below.

Preparation of cell-free culture filtrates (CCFs)­Each enterocin producing strain was grown in 100 ml tryptose soya broth (TSB) supplemented with 0.6% yeast extract and I % Tween 80 at 37°C for 18-24 hr. The culture was centrifuged at 5000 g for 20 min at 4°e. T he pH of the supernatant flu id was then ad­justed to 7.0 with I N NaOH and it was filtered through a 0.22 11m membrane filter. CCF was concen­trated lO-fold by ultrafiltration at room temperature through an amicon PM 10 membrane (molecular weigh t cut-off 10,000). Unconcentrated and concen ­trated CCFs were e ither examined immediately for antimicrobial activ ity or stored at -20°e.

Examination of CCF for antimicrobial activity ­Well diffusion method8 was used. Semisolid TSA YE plates (0.7 % agar) were overl aid with 3-4 ml semi ­so lid TSA YE seeded with 106 ce lls of indicator organ­ism. Wells (7 mm diam) were cut in these agar plates asept ica lly and their bottoms were sea led. The wells were loaded with 100 111 of concentrated and non­concentrated CCFs of the potential enteroc in producer

strains. The pl ates were placed at 4 °C for 4 hr to al­low diffusion of CCFs into the agar. The plates were then incubated at 37°C for 15-20 hr. Inhib ition of in­dicator organism was detected by a c lear zone around the we ll .

Sensitivity of enterocin to enzymes-Test CCFs were examined for sens iti vity to a-chymotryps in , pa­pain and trypsin (SRL, Indi a I mg/ml of CCF)Y. En­zyme solutions (10 mg/ml) in Sorensen's phosphate buffer were used for treatment. Buffer of pH 8.0 was used fo r a-chymotrypsin and trypsin and that of pH 6.4 for papain. Enzyme solution (0 .1 ml) was added to I ml of concentrated CCF and the mixture was incu­bated at 37°C fo r I hr. The residual anti mi crobial ac­tivity was determined by we ll diffusion assay as de­scribed earlier. TSB and untreated CCFs were used as controls.

Sensitivity of enterocin to heat - For determining the sensitivity of CCF to heat, glass test tubes contain­ing I ml al iquots of concentrated CCFs were exposed to 60°C for 30 min, 80°C fo r 10 mi n, 100°C for 10 min and 12 1°C for 15 min in a water bath or auto­clave. The tubes were immediate ly cooled down after exposure. CCFs were examined for antimicrobial ac­tivity by well diffusion assay as described earlier. Un­treated CCFs were used as control.

Results and Discussion Enterocinogenic activity against L. monocytogenes ­

On preliminary screeni ng of the isolates for antago­ni sm, among 90 isolates of Enterococcus sp., 33 and two isolates caused inhibition of L. monocytogenes and S. aureus, respectively. None of the iso lates acted agai nst Salmonella enteritidis.

On further examination of 33 antilisteric isolates of Enterococcus sp. se lected o n preliminary screening, onl y 9 isolates caused inhibition of the indi cator L. monocytogenes strai ns in tryptose soya agar without dextrose. Thi s suggested that the inhibitory activity of these was not due to lactic ac id production because there was practically no sig nifi cant reduction in pH of the culture medium. T hese 9 isolates included 8 iso­lates of E. faecalis of meat, fish and milk origin and one of E. solitarius iso lated from fish. The remaining 24 isolates, which fa iled to inhibit L. monocytogenes on the culture medium without dextrose, were not examined further. These were considered to cause inhibitio n of indicator organism due to lactic acid production and decline in pH of the culture med ium.

On subsequent exam ination of the above men­tioned 8 isolates of E. faeca lis and one of E. solitarius

SINGH & GARG: DETECTION & CHARACTERIZATION OF ENTEROCINS 6 11

for lytic bacteriophages activity on L. monocytogenes, only two isolates of E. faecalis, one each from chicken meat and raw milk, revealed that their inhibi­tory activity was not due to lytic bacteriophages.

Both the isolates further evidenced the absence of hydrogen peroxide activity in the cu lture medium. Consequently , these two isolates, E. faecalis C9 and E. faecalis M3, were considered to demonstrate enet­erocin activity on L. monocytogenes MTCC 1143 and MTCC 657 .

Eneterocin activity in cell-free culture filtrates­CCFs of E. faecalis isolates C9 and M3 showed inhi­bition of L. monocytogenes strains by well diffusion assay. However, the zones of inhibition were narrow (8-9 mm including well of 7 mm in di am) which be­came appreciable only after 10-fold concentration of the filtrates.

During the present investigations, thus, two isolates of E. faecalis di splayed antilisteric b acteriocinogenic activity. During the recent years, several strains of E.

" faecium and E. faecalis with antil isteric activity have been isolated and described by different workers

,o.12.

However, the present work reports similar property of the indigenous strains.

Enterocinogenic activity againstS. aweus­Examination of the two isolates of E. gallina rum S5 and S6 of sewage origin selected during the prelimi­nary screening showed that the antagonism was due to enterocins in both the cases . However, antili steric E. faecalis C9 and M3 isolates did not show activity against S. aureus.

Enterocin activity in celljree culture filtrates­CCFs of E. gallina rum S5 and E. gallina rum S6 after lO-fo ld concentration revealed enterocjn activity against S. aureus. Their zones of inhibition measured

12 and 11 mm including the well of 7 mm diam. Inhi­bition of S. aureus by enterococcal bacteriocin has also been reported previousl /. 13. 14.

During the present investigations, it was observed that though a large proportion of Enterococcus iso­l ~tes were inhibitory to L. monocytogenes but in many

'cases inhibition was probably due to other factors like, lactic acid production , lytic bacteriophages and hydrogen peroxide production in the culture medium. Therefore, it becomes essential to eliminate the role of such factors while examining enterocin production by the isolates.

The present studies revealed narrow spectrum of acti vity of enterocins as the inhibition was restricted to L. monocytogenes and S. aureus by different sets of isolates . Moreover, none of the isolates was acti ve against Salmonella enteritidis. In many earlier studies9

. ' 5, it has been observed that enterocins and other bacteriocins generally have narrow spectrum of actlVlty. Further, bacteriocins of Gram-positive bacteria have been reported normall y not to be active aga inst Gram-negative bacteria l5

. However, contrary to these observations, some workers have recorded broad spectrum activity of enterocins. Laukova et al. 16

have reported E. faecalis V24 enterocin to be antagoni stic to Enterobacter cloacae, E. asburiae, Proteus spp., Salmonella spp., Acinetobacter lwoffi, Y. enterocolitica and L. monocytogenes. Laukova et al. 17 have also found enterocin of E. faecium CCM 423 1 active against Gram-positive bacteria like streptococci, enterococci and staphylococci and Gram-negative bacteria like E. coli. Cell-free supernatant fluids of large number of strains of E. faeca lis (7117), E. faecium (511 0) and E. durans (411 0) have been shown to exhibit antagoni stic activity

Table I - Nature of enteroc ins of £. faecalis and E. galiinarul1l

Treatment

Enzyme treatment a -chymot ryps in

Papain Tryps in

Heat treatment o

60 C for 30 min o

80 C for 10 min o

100 C for 10 min o

12 1 C for 15 min

£. faeca lis C9

+ +

+

+

(+) ac ti vity re tained; (-) ac ti vi ty lost

Retention o f activity afte r treatment E. faecalis M3 £. gallinarum S5

+ +

+

+

+

+ + +

+

+

+

£. gallina rum S6

+ + +

+

+

612 INDIAN J EXP BIOL, JUNE 2003

Figs 1-2 - (I) - Effect of enzyme treatment on E. faecalis M3 enterocin [A- a -chymotrypsin, B- papain, C- uninoculated tryptose soya broth, 0- trypsin, E-untreated culture filtrate). (2) - Effect of enzyme treatment on E. gallinarum 55 enterocin [A- a-chymotrypsin, B­papain, C- trypsin, 0- peroxidase, E- uninoculated tryptose soya broth, F- untreated culture filtrate).

against Gram-negative organisms Vibrio cholerae 01 and non-01 18

.

Characteristics of enterocins - Effect of different enzymes and heat treatments on enterocin activity in the lO-fold concentrated CCFs of E. faecalis C9, E. faecalis M3, E. gallina rum S5, and E. gallinarum S6 isolates has been depicted in Table 1.

All the four enterocins retained activity after treat­ment with papain and trypsin. However, treatment with a-chymotrypsin resulted in loss of enterocin activity of E. faecalis, but not of E. gallina rum (Figs 1,2).

As far as their heat stability was concerned, all the four enterocins were resistant to heat treatment at 60DC for 30 min and at 80DC for 10 min, but were sensitive at 121 DC for 15 min . Exposure at 100DC for 10 min showed variable results. Enterocins of E. fae­calis M3 and E. gallinarum S5 (Fig. 3) were resistant, while those of E. faecalis C9 and E. gallina rum M3 were sensitive to such treatment.

Heat stability of enterocin at 60DC for 30 min and 80DC for 10 min was similar to the results on entero­cin V24 by Laukova et al. 13 and enterocin NA by Olasupo et al. 19

• Variation in the stability at 100DC for 10 min, similar to the present observations, have been recorded earlier I8,20.21 .

Difference in the properties of enterocins produced by 4 isolates suggested that they were different from

Fig. 3 - Effect of heat trea tment on E. gallina rum 55 enterocin [A- 60°C for 30 min, B- 80°C for 10 min, C- 100°C for 10 min ,

0- 121 °C for 15 min, E- untreated culture filtrate].

each other. While the loss of acti vity of E. faecalis enterocins due to the action of a-chymotrypsin indicated their proteinaceous nature, this could not be established in case of g ' gallinarum probably due to

I

use of only three proteolytic enzymes in the present investigations. However, Jennes et a/. 21 have reported

SINGH & GARG: DETECTION & CHARACTERIZATION OF ENTEROCINS 613

inactivation of enterocin of E. gallina rum by proteolytic enzymes.

Several earlier workers21.22 have reported enterocins

as pure proteins, but some have reported these to be glycoproteins2o

,23 . The insensitivity of enterocins to proteolytic enzymes and their heat stabi lity in the pre­sent studies suggested that these might be protein complexes .

Thus, the present investigatio ns led to the detection of 4 enterocinogenic strains of Enterococcus sp. and showed promising antagonism against two important foodborne pathogens, L. monocytogenes and S. aureus, which are major threat to public health worldwide. Some workers have shown the potential of bacteriocins as effective biopreservatives in foods such as cheese I , mile, meat and meat products4

• The isolates reported in the present study may also have good potential in controlling spoil age and pathogenic bacteria in foods which needs further exploration .

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