FOOD

Food Microbiology 22 (200

iafri

ira

ty of

4; acc

Pediococci are part of the non-starter lactic acid bacteria, LAB, contributing towards cheese ripening. This study was aimed at

Pediococci are Gram-positive lactic acid bacteria,

1990; Beresford, 2003).

style cheese produced under less commercialized condi-

identied Pediococcus acidilactici and Pediococcus pen-tosaceus as the constituent species in cheese (Garvie,1984, 1986; Litopoulou-Tzanetaki et al., 1989b; Tzane-

ARTICLE IN PRESS

Corresponding author. Tel.: +27-12-420-3209; fax: +271-420-

takis and Litopoulou-Tzanetaki, 1992; Gerasi et al.,2003).

0740-0020/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fm.2004.08.001

2839.

E-mail address: elna.buys@up.ac.za (E.M. Buys).LAB, that are used as starters in the industrialfermentation of meat and vegetables. In bacterialripened cheese pediococci may occur as part of thenon-starter lactic acid bacteria (NSLAB) that contributetowards cheese ripening (Dacre, 1958a; Litopoulou-Tzanetaki et al., 1989a). The role of pediococci in cheeseripening has not been fully explained; instead pediococciwork in synergy to affect the typical cheese avour(Franklin and Sharpe, 1963; Peterson and Marshall,

tions (Cogan et al., 1997). The highest counts of107108 cfu/g have been isolated at different stages, 2,4, 6 and 12 weeks of Cheddar ripening (Dacre, 1958a, b;Elliott and Mulligan, 1968; Litopoulou-Tzanetaki et al.,1989a, b). Among artisan cheese, pediococci has beenisolated throughout ripening (Cogan et al., 1997;Bouton et al., 1998; Prodromou et al., 2001; Gerasi etal., 2003). Characterization of the pediococci isolatesfrom both commercial and farm-style cheese hasstyle cheese. Logarithmic counts of LAB ranged from 6.9 to 9.4 cfu/g. Microscopic examination identied 110 (18%) of 606 isolates

as presumptive pediococci distributed among farm-style cheese (pasteurized Gouda, young and matured; un-pasteurized aged

Bouquet, aged and matured Gouda), in numbers of 33, 21, 28, 12 and 16, respectively. Pediococci were absent in commercial

Cheddar cheese. Characterization of pediococci identied 49 Pediococcus acidilactici and 61 Pediococcus pentosaceus isolates. Fifty-

two isolates from both species, 27 (24%) P. acidilactici and 25 (23%) P. pentosaceus, inhibited Lactococcus lactis NCDO 176

through the action of pediocins. Among these isolates, seven (6%) P. acidilactici and six (7%) P. pentosaceus inhibited Bacillus

cereus ATCC 1178 while 17 (15%) P. acidilactici and 20 (18%) P. pentosaceus inhibited Listeria monocytogenes ATCC 7644.

Inhibition levels were variable against L. monocytogenes ATCC 7644 and low against B. cereus ATCC 1178. Both Pediococcus

species showed similar inhibition patterns; however, more isolates of P. pentosaceus inhibited L. lactis NCDO 176 and L.

monocytogenes ATCC 7644 compared to P. acidilactici.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Pediococci; P. pentosaceus; P. acidilactici; Pediocin; Farm-style cheese

1. Introduction Pediococci have been isolated from both commercialCheddar cheese (Dacre, 1958a, b) and artisan or farm-isolating, characterizing and evaluating the antimicrobial activity of Pediococcus species occurring among commercial and farm-Characterization and antimicrobisolated from South A

Obert. Z. Gur

Department of Food Science, Universi

Received 1 April 200

AbstractMICROBIOLOGY5) 159168

l activity of Pediococcus speciescan farm-style cheese

, E.M. Buys

Pretoria, Pretoria 0002, South Africa

epted 1 August 2004

www.elsevier.com/locate/yfmic

Some strains of Pediococcus species produce anti-microbial peptides that inhibit closely related LAB andGram-positive spoilage and pathogenic bacteria (Klaen-hammer, 1993; Ennahar et al., 2000a). These bacter-iocins are designated as pediocins and they exert highantimicrobial activity against Listeria species. As aresult, pediocins are characterized as class IIa bacter-iocins (Ennahar et al., 2000b).Consumer concerns over the potential health or adverse

effects associated with chemically synthesized preservativeshave stimulated interest in the use of natural metabolitesfrom LAB like bacteriocins (Roller, 1995; Cleveland et al.,2001). The present study was aimed at identifying andcharacterizing pediococci among the NSLAB from SouthAfrican farm-style cheese and determining pediocin

2.2. Farm-style cheese

cheese fair, Table 2 (ARC; Irene; South Africa). Farm-style cheeses were kept under refrigeration temperatureof 472 1C for 1 month before analyses.

2.3. Chemical analysis

Farm-style cheeses were subjected to chemical analy-sis, salt, pH and moisture. All analyses were done intriplicates; the mean and standard deviation were notedfor all samples. Moisture, NaCl and pH were deter-mined as described by Bradley et al. (1992).

2.4. Microbial analysis

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ology

SA

ene,

ene,

cil

O.Z. Gurira, E.M. Buys / Food Microbiology 22 (2005) 159168160Pasteurized and un-pasteurized cheese samples, 50 g,of farm-style cheeses produced from various small-scalefarmers were obtained from the annual ANPI farm-style

Table 1

Bacterial strains and media used in this study

Organism Source

P. acidilactici ST1 Institute of Wine Biotechn

University of Stellenbosch,

P. pentosaceus isolates Farm-style cheese (ARC Ir

South Africa)

P. acidilactici isolates Farm-style cheese (ARC Ir

South Africa)

Lactococcus lactis subsp. diacetilactis

NCDO 176

Agriculture Research Coun

(ARC), SA

Listeria monocytogenes ATCC 7644 ARC

Bacillus cereus ATCC 1178 ARCproduction among isolates of Pediococcus species as wellas their activity against food pathogens, Bacillus cereusand Listeria monocytogenes.

2. Material and methods

2.1. Commercial cheddar cheese

Single herd commercial Cheddar cheese samples, 500 g,from the Animal Nutrition and Animal Products Institute(ANPI) of the Agriculture and Research Council (ARC,Irene, South Africa), at different ripening stages, 1 day, 1,2, and 3 months (m) were used for the isolation ofpediococci. These samples were stored and ripened at atemperature of 1015 1C and a relative humidity of 85% inthe department of Food Science (Pretoria, South Africa)to give sample 15 days, 1.5 and 2.5 months.OIT=Optimum incubation temperature.2.4.1. Bacterial cultures

A pure culture of pediocin producing P. acidilacticiST1 was obtained from the department of Wine andBiotechnology (University of Stellenbosch, Stellen-bosch, South Africa) and used as the positive controlfor morphological and physiological identication ofpediococci isolates from farm-style cheese as well as inthe antimicrobial assay. Other bacterial strains availableamong the laboratory cultures, their source, incubationtemperature and growth media are presented in Table 1.

2.5. Isolation and characterization of Pediococcusspecies from commercial cheddar and farm-style cheese

2.5.1. Isolation of non-starter lactic acid bacteria from

commercial cheese

Samples of cheese, 10 g, were aseptically grated andemulsied in sterile 2% (w/v) tri-sodium citrate. Serialdilutions were made in peptone saline water. Serialdilutions of the samples were inoculated and spreadplated on duplicate plates of MRS agar. Plates wereincubated at a temperature of 30 1C for 5 days in a gas-pack anaerobic atmosphere (Oxoid, New Hampshire,UK). Total LAB counts were determined and the meanof the duplicate plates was noted. The method of Jordan

Media OIT (1C)

, MRS broth (de Man Rogosa &

Sharpe 1960)

30

MRS broth 30

MRS broth 30

MRS broth 30

Tryptone Soya broth (Biolab,

Midrand)

30

BrainHeart Infusion broth (Biolab,

Midrand, SA)

37

the diameter of the resulting zone of inhibition was

ARTICLE IN PRESSd Micand Cogan (1993) was followed with modications; 20colonies were randomly selected from the highestdilution with a colony count of 30300 cfu per plate.These colonies were inoculated in MRS broth andmicroscopically examined after growth at 30 1C for 24 h.Cultures were puried by streak plating before identi-cation. Gram-positive catalase negative cocci occurringas pair, tetrads, short chains and irregular clusters werestored as presumptive pediococci isolates in TryptoneSoy Broth 0.6% agar (w/v) in 2ml Eppendorf tubes at atemperature of 18 1C for 2 months.

2.5.2. Isolation of non-starter lactic acid bacteria from

farm-style cheese

The same procedure as that for commercial cheese wasused to isolate LAB from the farm-style cheese. However,all colonies occurring on the highest dilution within a rangeof 50100 cfu per plate were picked and characterized.

2.6. Identification of isolates

Physiological identication under different growthparameters (temperature, pH and NaCl) of presumptivepediococci isolates was done according to the somemethods described by Garvie (1986) in the BergeysManual. Prior to tests the presumptive pediococci isolateswere propagated twice in MRS broth at 30 1C for 24h.Growth characteristics were monitored daily at 40 and

50 1C in tubes of MRS broth over a 3-day period.Tolerance of the isolates to salt was assessed after 3 days ofincubation at concentrations of 4% and 6.5% (w/v) NaClin MRS broth. Growth at pH 4.2 and 7.5 was tested usingMRS broth adjusted aseptically with 1N HCl and 1NNaOH and results were noted after 3 days at 30 1C. P.acidilactici ST1 was used as the positive control.

2.7. Antimicrobial activity of crude pediocin extracts

from Pediococcus species isolated from farm-style cheese

Three methods, the disc assay by Kim et al. (1995),with modication from El-Adway (2001), the agar spotmethod as described by Con et al. (2001) and the overlaymethod as described by Ray and Miller (2000), werecompared in determining the antimicrobial activity ofthe crude pediocin extract (CPE) produced from P.acidilactici ST1 grown for 48 h at 30 1C and centrifugedat 3000g for 15min at 4 1C (Skytta et al., 1993). In allassays MRS broth was used as a negative control andtreated identically as the pediococci culture used in theantimicrobial assays. Since only one test assay was usedthis is the assay that will be described.The inhibitory activity of the CPE from P. acidilactici

ST1 was determined on solid agar media againstLactococcus lactis subsp. diacetilactis NCDO 176 byadding 1.5% (w/v) agar to MRS broth media using the

O.Z. Gurira, E.M. Buys / Foodisc assay technique (El-Adawy, 2001) as follows: Themeasured in mm as the distance from the edge of thepaper disc to the edge of the clearing zone. Clear zonesextending for 0.5mm or more were considered aspositive for inhibition (Litopoulou-Tzanetaki et al.,1989b; El-Adawy, 2001).

2.8. Antimicrobial activity of isolates from Pediococcusspecies against B: cereus ATCC 1178 andL: monocytogenes ATCC 7644

The agar disc diffusion technique (I) was used for thedetermination of the antimicrobial activity of the CPEproduced from isolates of both Pediococcus speciesagainst B. cereus ATCC 1178 and L. monocytogenesATCC 7644. Modications to the agar disc methodincluded the neutralization of the cell-free supernatant,CPE, from the 48 h culture broth. The pH of thesupernatant was adjusted to 6.5 using 10M NaOHbefore the immersion of the paper discs.The test micro-organism B. cereus ATCC 1178 or L.

monocytogenes ATCC 7644 was propagated twice andthen grown for 1824 h in 10ml of the appropriategrowth media (Table 1). The turbidity of the culturebroth was compared with the McFarland tubes to givean estimate of the bacterial population (Harrigan, 1998).A sample of 1ml of 1 106 cfu/ml was transferred andspread plated on the appropriate media and allowed todry. Paper discs previously immersed in the CPE for30min were aseptically transferred on the agar platesand allowed to dry before being incubated at theappropriate temperature for the growth of each micro-organism for 24 h. A positive control, P. acidilactici ST1,and a negative control, MRS broth, were treated in asimilar manner as the isolates of Pediococcus species.The zones of inhibition occurring around the paper discswere compared after 24 h incubation.

3. Results and discussion

3.1. Isolation and characterization of Pediococcusspecies from commercial Cheddar cheese and farm-style

cheese

3.1.1. Commercial cheese

Logarithmic counts of LAB at the different ripeningpediococci culture was grown for 48 h at 30 1C andcentrifuged at 3000g for 15min at 4 1C to give the CPE(Skytta et al., 1993). A sterile lter paper disc (WhitmanAA, 13.0mm diameter, Merck, Midrand, SA) wasdipped into the CPE for 30min, and then applied onMRS agar plates and overlaid with MRS soft agar 0.6%(w/v) agar seeded with 1 106 cfu/ml of L. lactis NCDO176. The plates were incubated overnight at 30 1C and

robiology 22 (2005) 159168 161stages of the commercial Cheddar cheese (data not

shown) were similar ranging from 7.73 log10 at 1 day to8.16 log10 at 1 month, followed by a log reduction to7.46 log10 at 1.5 months and an increase to 8.53 log10 atthe end of 3 months. Morphological characterization ofthe 140 LAB isolates identied Gram-positive rods aspresumptive lactobacilli and Gram-positive chains aspresumptive streptococci. Gram-positive cocci occur-ring in pairs, tetrads or clusters were absent henceconrming the absence of pediococci. Several reportshave noted the absence of pediococci among theNSLAB in commercial Cheddar cheese (Jordan andCogan, 1993; Lues and Botha, 1999). The absence ofpediococci may be attributed to either high or good

ized Bouquet (RAB) as well as in aged and maturedGouda (RAG and RMG) (Table 3).Isolates of pediococci occurred in relatively higher

numbers in pasteurized farm-style cheese compared toun-pasteurized cheese (Table 3). PYG and PMGpossessed 22 and 21 isolates while RMG and RAGhad 16 and 12 isolates, respectively. However, RAB had28 isolates. The higher number of pediococci isolates inpasteurized cheese may be attributed to the survival andproliferation of thermo-tolerant pediococci isolates(Elliott and Mulligan, 1968; Litopoulou-Tzanetaki etal., 1989a; Grappin and Beuvier, 1997). In un-pasteur-ized cheese, fermentation of the milk is dependent on the

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es (w7

n-pas

MB

90702170.470.570.570

a; PM

AG: r

O.Z. Gurira, E.M. Buys / Food Microbiology 22 (2005) 159168162microbial quality of cheese milk used in the productionof commercial Cheddar cheese as well as the hygienicprotocols during production and ripening. These con-ditions eliminate contaminant NSLAB like pediococci(Litopoulou-Tzanetaki et al., 1989a; Grappin andBeuvier, 1997; Williams and Banks, 1997).

3.1.2. Farm-style cheese

3.1.2.1. Chemical properties of cheese and log counts of

NSLAB. The logarithmic counts and physiochemicalproperties of the eight farm-style cheeses are presentedin Table 2. Logarithmic counts of LAB counts amongthe cheese varied and ranged between 6.9 log10 in RMBto 9.4 log 10 in RGG. The pH of cheeses ranged from4.98 in RGG to 5.66 in PYG cheese. Moisture contentwas lowest in PMP 19.6% while in other cheese itranged from 32.4% to 44.0%. The salt content rangedfrom 1.5% to 5.5% and the salt-in-moisture ratio washigh in PMP and RAB 13.5% and 14.5%, respectively.

3.1.2.2. Characterization and distribution of isolates of

pediococci among farm-style cheese. Among the farm-style cheese, a total of 110, 18%, of the LAB isolateswere Gram-positive cocci occurring in pairs, tetrads andclusters. These isolates were morphologically identiedas presumptive pediococci (Dacre, 1958a, b; Elliott andMulligan, 1968). The isolates were distributed inpasteurized Gouda (PYG and PMG) and un-pasteur-

Table 2

Logarithmic count of lactic acid bacteria (LAB) and chemical properti

Characteristic Pasteurized cheesea U

PMG PYG PMP R

LAB 8.0270.09 7.9170.09 7.5770.06 6.PH 5.4970.08 5.6670.29 5.0970.18 5.H2O (%) 35.470.37 42.770.27 19.670.80 32NaCl (%) 1.570 2.370.14 3.170.06 5S: Mc (%) 4.270.04 5.070.00 13.570.00 14

aPMG: pasteurized matured Gouda; PYG: pasteurized young GoudbRMB: raw milk matured Bouquet; RAB: raw milk aged Barbond; R

goat Gouda.

cS:M=Salt-in-moisture %: NaCl %/(NaCl %+moisture %).spontaneous LAB occurring in cheese (Cogan et al.,1997). Occurrence of pediococci among un-pasteurizedcheese may be attributed to the persistence of pediococciamong the adventitious LAB. However, diversity in theLAB may competitively reduce the number of pedio-cocci isolates RMG and RAG compared to PYG andPMG (Prodromou et al., 2001).Pediococci isolates were absent in PMP and in both

RMB as well as RGG (Table 3). The absence ofpediococci in PMP and RMB may be attributed to thephysio-chemical properties of these two cheeses. Theinhibitory effect of the high salt-in-moisture content inPMP and RMB of 13.5% and 14.3% (w/v) (Table 2),respectively, may account for the absence of pediococciin PMP and RAB (Fox et al., 1998; Crow et al., 2002;Gerasi et al., 2003).While no clear physio-chemical explanation may be

given for the absence of pediococci in RGG, it ispossible that the high LAB count of 9.40 log10 (Table 2)could have an inuence on the occurrence of pediococci.Possibly, the unavailability of a readily fermentablesubstrate, competition for nutrients as well as theproduction of antimicrobial substances by other LABmay limit or inhibit the growth of pediococci in RGG(Fleming et al., 1975; Garvie, 1986; Vandenbergh, 1993).Pediococci isolates from farm-style cheeses were

characterized into two species, P. acidilactici and P.pentosaceus. Differentiation of the two species was based

SD) of South African Farm-style cheese (n 8)

teurized cheeseb

RAB RAG RMG RGG

.08 7.0770.52 7.9270.07 7.1270.16 9.4070.141

.02 5.2970.02 5.4670.12 5.4370.03 4.9870.05

.27 35.370.48 37.070.61 36.170.51 44.070.24

.23 3.870.04 2.170.04 1.470.16 2.170.00

.01 9.670.00 5.470.0 3.670.00 4.670.00

P: pasteurized matured Parmesan.

aw milk aged Gouda; RMG: raw milk natured Gouda; RGG: raw milk

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Un

RM

0

0

a; PM

AG: r

ntage

l of t

d Micon the growth of P. acidilactici and P. pentosaceus at 40and 50 1C, respectively, where P. pentosaceus failed togrow at 50 1C. These reports are similar to workreported from goat milk and cheese as well as in otherdairy products where both species constituted part ofthe NSLAB (Garvie, 1984; Litopoulou-Tzanetaki et al.,1989b).Isolates from both Pediococcus species occurred

among ve farm-styles cheeses in variable numbers.More isolates were characterized as P. pentosaceuscompared to P. acidilactici in PYG, PMG and RAG(Table 3). P. pentosaceus occurs more frequently and inhigher numbers in milk and among dairy productscompared to P. acidilactici (Garvie, 1984; Tzanetakisand Litopoulou-Tzanetaki, 1989, 1992; Gerasi et al.,

Table 3

Occurrence and distribution of Pediococcus species in farm-style cheese

Characteristics Pasteurized cheesea

PMG PYG PMP

Pediococci

isolates 21 33 0

% pediococcic 19 30 0

P. acidilactici 6d (29%)e 12 (40%)

P. pentosaceus 15 (71%) 21 (60%)

aPMG: pasteurized matured Gouda; PYG: pasteurized young GoudbRMB: raw milk matured Bouquet; RAB: raw milk aged Barbond; R

goat Gouda.cPercentage of presumptive pediococci isolates per cheese as a percedNumber of the respective species occurring in the cheese.ePercentage of the speciesoccurring in the respective cheese as a tota

O.Z. Gurira, E.M. Buys / Foo2003). Although isolates of P. acidilactici were present inthis study, few reports have characterized this speciesamong pediococci isolates from cheese (Tzanetakis andLitopoulou-Tzanetaki, 1992; Boubekri and Ohta, 1996).Simultaneous occurrence of isolates from both P.pentosaceus and P. acidilactici among the cheeses maybe attributed to the close characteristics relationshipshared by the two species (Garvie, 1986; Simpson andTaguchi, 1998; Raccach, 1999).

3.2. Antimicrobial activity of crude pediocin extracts

from Pediococcus species isolated from South Africanfarm-style cheese

3.2.1. Evaluation of antimicrobial techniques

In order to select the most appropriate agar diffusiontechnique in determining and screening inhibitionpotential of the CPE from pediococci isolates againstthe indicator and food pathogens, three procedures wereevaluated. Clear zones of inhibition were producedusing I whilst II and III produced fuzzy zones (data notshown). Based on these results, technique I was chosenas the most appropriate method for antimicrobialdetermination. In antimicrobial assays it is recom-mended to measure circular zones as positive forinhibition and neglect any unclear or vague zones(Piddock, 1990; Ray and Miller, 2000).

3.3. Antimicrobial activity of the crude pediocin extract

produced by pediococci isolates from farm-style cheese

against L: lactis NCDO 176

A total of 52, 47%, among the 110 isolates from bothPediococcus species exerted antagonism against L. lactisNCDO 176 (Tzanetakis and Litopoulou-Tzanetaki,1989). Among these isolates 27, 24%, were from P.

-pasteurized cheeseb

B RAB RAG RMG RGG

28 12 16 0

26 11 14 0

24 (86%) 5 (42%) 14 (88%)

4 (14%) 7 (58%) 2 (12%)

P: pasteurized matured Gouda.

aw milk aged Gouda; RMG: raw milk natured Gouda; RGG: raw milk

of the total of the presumptive pediococci isolates.

he number of pediococci in the respective cheese.

robiology 22 (2005) 159168 163acidilactici (Table 4) and 25, 23%, were P. pentosaceus(Table 5). Antagonism from both P. pentosaceus and P.acidilactici isolates has been reported among goat milk,Feta and Kaseri cheese as a result of the production ofantimicrobial substances (Tzanetakis and Litopoulou-Tzanetaki, 1989; Litopoulou-Tzanetaki et al., 1989b).Pediococci exert antagonism against other micro-

organisms primarily through the production of lacticacid, in addition some strains within Pediococcus speciesproduce antimicrobial peptides known as bacteriocins,pediocins (Daeschel, 1989). The use of an acid-tolerantL. lactis, NCDO 176, in the present study eliminatesinhibition due to lactic acid production thereby attribut-ing the inhibition of the indicator micro-organism topediocins produced by some isolates of the twoPediococcus species (Ray and Miller, 2000).Isolates of P. acidilactici (Table 4) and P. pentosaceus

(Table 5) exerted comparable antagonism at mediuminhibition, with 14 and 17, respectively, while fourshowed high inhibition against L. lactis NCDO 176. Thesimilarity in the antagonism shown by the isolates from

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acidil

Lo

0.9

0.6

0.9

0.8

0.5

0.5

0.9

1.0

1.0

d MicTable 4

Antimicrobial activity of the crude pediocin extract from strains of P.

Level of inhibitiona

Strain No inhibition Strain

4 40

5 49

8 50

10 52

11 54

12 65

14 92

42 120

44 121

45

O.Z. Gurira, E.M. Buys / Foo164both Pediococcus species may be attributed to thesimilarity in the inhibitory spectrum of pediocinsproduced from both species (Ray and Daechel, 1994;Ray and Miller, 2000). Antimicrobial spectrum of bothP. acidilactici and P. pentosaceus has been reported toinclude L. Lactis strains among the sensitive indicator topediocins (Eijsink et al., 1998; Ray and Miller, 2000).Isolates from the two Pediococcus species 34, 31%, P.

acidilactici (Table 4) and 24, 22%, P. pentosaceus (Table5) did not exert antagonism against L. lactis NCDO 176.The absence of pediocin production among pediococciisolates from cheeses was attributed to the absence ofthe plasmid that encodes for pediocin production(Daeschel and Klaenhammer, 1985; Litopoulou-Tzane-taki et al., 1989a).

47

53

55

58

59

60

61

63

66

67

87

96

106

113

114

115

116

119

123

124

126

127

137

138

Total strains 34 9

% of strains 31 8

Mean zone size 0.8

0.54: No inhibition, 0.51.0mm: low inhibition, 1.12.0mm: medium inhibaMean of three trials.actici (n 61) against L. lactis NCDO 176

w Strain Medium Strain High

3 1.6 2 2.2

6 1.9 72 2.3

7 1.1 76 2.5

9 2.0 78 2.3

62 1.8

74 1.3

75 1.1

77 1.8

79 1.8

80 1.1

robiology 22 (2005) 1591683.4. Antimicrobial activity of isolates from Pediococcusspecies against food pathogens, B: cereus ATCC 1178and L: monocytogenes ATCC 7644

A similar number of isolates 27 and 25 from P.acidilactici and P. pentosaceus, respectively, showedantagonism against L. lactis NCDO 176. Among theseisolates, those exerting inhibition zones of 1mm and lesswere not used for the subsequent antimicrobial assays.Thirty-nine isolates; 21 P. pentosaceus and 18 P.acidilactici were used for subsequent assay.The cell-free supernatant, CPE, of isolates from both

Pediococcus species showed low inhibition against B.cereus ATCC 1178 (Table 6) and variable antagonismagainst L. monocytogenes ATCC 7644 (Table 7).

81 1.5

83 1.3

84 1.9

140 2.0

14 4

12 4

1.6 2.3

ition, 2.13.5mm: high inhibition.

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pento

Lo

0.9

0.9

0.9

0.8

d MicTable 5

Antimicrobial activity of the crude pediocin extract from strains of P.

Level of inhibitiona

Strain No inhibition Strain

15 29

17 37

18 40

20 91

23

25

26

27

28

30

32

33

34

35

36

39

64

88

89

93

O.Z. Gurira, E.M. Buys / FooAgainst B. cereus ATCC 1178, 12 isolates, 12%, fromboth Pediococcus species showed low antagonism whileP. pentosaceus ST38, 1%, showed medium inhibition.The inhibitory spectrum of pediocins from bothPediococcus species has been reported to inhibit B.cereus (Piva and Headon, 1994; Elegado et al., 1997; El-Adawy, 2001). However, no reports have drawncomparisons between the inhibitory activities of strainsfrom either Pediococcus species against B. cereus (Rayand Daechel, 1994). The low activity exerted by isolatesfrom both Pediococcus species against B. cereus ATCC1178 can be attributed to the differences in thesensitivity of the strains of the B. cereus used in thedifferent assays (Raccach and Geshell, 1993; Meghrouset al., 1999; Ray and Miller, 2000). Low inhibition of B.cereus ATCC 1178 may be further attributed tovariation in strains susceptibility as determined by theorigin of the pathogenic strain. While more reports areyet to conrm, strains of Pediococcus species isolatedfrom meat have been reported to exert greater antagon-ism against bacterial strains that are prevalent in meatproducts (Nieto-Lozano et al., 2002). Presumably,strains of B. cereus isolated from cheese may be more

99

103

117

131

Total strains 24.0 4.0

% of strains 22 4

Mean zone size 0.0 0.9

0.54: No inhibition, 0.51.0mm: low inhibition, 1.12.0mm: medium inhibaMean of three trials.saceus (n 49) against L. lactis NCDO 176

w Strain Medium Strain High

13 1.9 1 2.5

16 1.3 19 2.1

22 1.1 21 2.5

24 1.3 94 2.5

31 1.5

38 1.3

41 1.6

43 1.8

56 1.3

73 1.3

85 1.4

90 1.1

95 1.5

97 1.1

98 2

100 1.1

101 1.1

robiology 22 (2005) 159168 165susceptible to pediocin producing pediococci isolatesfrom cheese.The antimicrobial activity of strains of pediococci was

higher against L. monocytogenes ATCC 7644 (Table 7),33%, compared to B. cereus ATCC 7644 (Table 6),13%, as more strains of Pediococcus species inhibited L.monocytogenes ATCC 7644. The higher antimicrobialactivity exerted by isolates of Pediococcus species maybe attributed to the high susceptibility of some strains ofL. monocytogenes to pediocins (Eijsink et al., 1998;Montville and Chen, 1998; Song and Richard, 1997;Ray and Miller, 2000). Susceptibility of L. monocyto-genes may be attributed to the differences in the lipidcomposition between the two pathogens (Ennahar et al.,2000b).

3.5. Comparison of the antimicrobial activity of isolates

Pediococcus species

A comparable number of isolates of both Pediococcusspecies exerted inhibitory activity against L. lactisNCDO 176 (Tables 4 and 5) and L. monocytogenesATCC 7644 (Table 7) at all three inhibitory levels.

17.0 4.0

15 4

1.4 2.4

ition, 2.13.5mm: high inhibition.

ARTICLE IN PRESS

39) o

P.

Str

d MicTable 6

Antimicrobial activity of the crude pediocin extracts from strains (n

Level of inhibitiona

P. acidilactici

Strain No inhibition Strain Low

O.Z. Gurira, E.M. Buys / Foo166Likewise, a similar number of isolates showed lowinhibition against B. cereus ATCC 1178 (Table 6).Contrary to these ndings, the comparison of theantimicrobial activity has been reported to differ amongstrains from different Pediococcus spp. Similarities in theantimicrobial activity were reported for strain from thesame species rather than across the species (Skytta et al.,

Table 7

Antimicrobial activity of the crude pediocin extracts from strains (n 39) of

Level of inhibitiona

P. acidilactici

Strain No inhibition Strain Low Strain Medium Strain H

80 6 0.5 2 1.3 9 2

74 0.5 3 1.3 62 3

75 1 7 1.5 77 2

140 0.5 72 1.8 79 3

76 1.3 81 2

78 1.3

83 1.7

84 1.5

Total strains 1 4 8 5

% of strains 1 4 7 4

Mean zone size 0.6 1.5 2

0.5mm4: No inhibition, 0.51.0mm: low inhibition, 1.12.0mm: medium iaMean of three trials.

2 3 0.7

6 9 0.5 1

7 72 0.5 1

62 78 0.6 1

74 79 1 2

75 81 0.9 2

76 140 0.5 3

77 4

80 5

83 7

84 9

9

9

9

10

Total strains 11 7 1

% of strains 11 6 1

Mean zone size 0.7

0.5mm4: No inhibition, 0.51.0mm: low inhibition, 1.12.0mm: medium iaMean of three trials.f P. acidilactici and P. pentosaceus against B. cereus ATCC 1178

pentosaceus

ain No inhibition Strain Low Strain Medium

robiology 22 (2005) 1591681993). Among all strains of P. acidilactici and moststrains of the P. pentosaceus, none showed a similar levelof inhibition across all three test micro-organisms.However, P. pentosaceus ST38 exerted medium inhibi-tion against all three micro-organisms. Differences inthe inhibition pattern or susceptibility of the threegenera may be attributed to the variation in the

P. acidilactici and P. pentosaceus against L. monocytogenes ATCC 7644

P. pentosaceus

igh Strain No inhibition Strain Low Strain Medium Strain High

.4 73 22 0.5 1 1.3 13 2.5

.3 24 0.5 16 1.3 56 2.5

.2 31 0.5 19 1.5 100 2.2

.8 41 1 21 1.5 101 2.8

.1 38 1.1

43 1.3

85 1.8

90 1.3

94 1.2

95 1.7

97 1.6

98 1.4

1 4 12 4

1 4 10 4

.8 0.6 1.4 2.5

nhibition, 2.13.5mm: high inhibition.

1 21 0.9 38 1.2

3 38 1.2

6 43 0.9

9 85 0.8

2 101 0.5

4

1

1

6

3

0

4

5

8

0

5 5 1

4 6 1

0.9 1.2

nhibition, 2.13.5mm: high inhibition.

higher inhibition than P. acidilactici.

Bacteriocin: safe, natural antimicrobials for food preservation. Int.

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Characterization and antimicrobial activity of Pediococcus species isolated from South African farm-style cheeseIntroductionMaterial and methodsCommercial cheddar cheeseFarm-style cheeseChemical analysisMicrobial analysisBacterial cultures

Isolation and characterization of Pediococcus species from commercial cheddar and farm-style cheeseIsolation of non-starter lactic acid bacteria from commercial cheeseIsolation of non-starter lactic acid bacteria from farm-style cheese

Identification of isolatesAntimicrobial activity of crude pediocin extracts from Pediococcus species isolated from farm-style cheeseAntimicrobial activity of isolates from Pediococcus species against B. .5em cereus ATCC 1178 and L. .5em monocytogenes ATCC 7644

Results and discussionIsolation and characterization of Pediococcus species from commercial Cheddar cheese and farm-style cheeseCommercial cheeseFarm-style cheeseChemical properties of cheese and log counts of NSLABCharacterization and distribution of isolates of pediococci among farm-style cheese

Antimicrobial activity of crude pediocin extracts from Pediococcus species isolated from South African farm-style cheeseEvaluation of antimicrobial techniques

Antimicrobial activity of the crude pediocin extract produced by pediococci isolates from farm-style cheese against L. .5em lactis NCDO 176Antimicrobial activity of isolates from Pediococcus species against food pathogens, B. .5em cereus ATCC 1178 and L. .5em monocytogenes ATCC 7644Comparison of the antimicrobial activity of isolates Pediococcus species

ConclusionReferences