Lebensm.-Wiss. u.-Technol., 35, 284–288 (2002)

New Procedure for the Detection of Lactic Acid Bacteriain Vegetables Producing Antibacterial Substances

Rosario Gomez, Marina Munoz, Begona de Ancos and M. Pilar Cano*

R. Gomez: Ciudad Universitaria s/n, Department of Dairy Foods Science & Technology,Instituto del Frıo-C.S.I.C., 28040, Madrid (Spain)

M. Munoz, B. de Ancos, M. Pilar Cano: Ciudad Universitaria s/n, Department of Plant Foods Science &Technology, Instituto del Frıo-C.S.I.C., 28040, Madrid (Spain)

(Received July 24, 2001; accepted December 6, 2001)

There is an increasing interest in antibacterial-producing lactic acid bacteria (LAB) as protective cultures on ready-to-usevegetables. A new procedure for detection of active strains has been developed, which consisted of anaerobic incubation (30 1C, 2 d)of juice of ‘iceberg lettuce’ on MRS agar or lactose-bromcresol purple (LBP) agar followed by overlaying with the indicator strain insoft agar (7.5 g/L). After overnight incubation at appropriate temperature, the plates were examined for clear inhibition zone aroundthe colonies. The developed procedure permitted the detection of active LAB in 3 d compared with several weeks for the traditionalprocedure. The use of LBP agar in conjunction with incubation of plates at 8 1C may give additional information about the nonacidiccharacteristic of antibacterial substances secreted, and also about the psychrotrophic behavior of producer strains, which could be ofinterest as biocontrol cultures to improve the safety of ready-to-use vegetables commonly stored under refrigeration.

r 2002 Published by Elsevier Science Ltd.

Keywords: lactic acid bacteria; vegetables; antibacterial activity

Introduction

Ready-to-use vegetables salads generally contain few orno antimicrobial additives, and rely on refrigeration asthe main preservation factor. However, psychrotrophicpathogens have been isolated from a wide variety of rawor processed vegetables, in the absence of sensorydefects. Vegetable products have been implicated in atleast two outbreaks of listeriosis (Breidt and Flemming,1997). A variety of other pathogens (Nguyen and Carlin,1994) has also been found on fruit and vegetableproducts, including Salmonella and Shigella spp., enter-opathogenic strains of Escherichia coli, Aeromonashydrophila, Yersinia enterocolitica, and Staphylococcusaureus. These pathogens grow and cause disease depend-ing on the type of product, conditions of storage (time,temperature, atmosphere), and competitive microflora.Thus, there is a particular interest to introduce additionalsafety measures for these products (Brackett, 1992).Efficient inhibition of the growth of pathogens byantibacterial-producing lactic acid bacteria (LAB)on ready-to-use vegetables may be achieved by strainsthat grow and secrete these compounds under refrigera-

*To whom correspondence should be addressed.E-mail: pcano@if.csic.es

0023-6438/02/$35.00r 2002 Published by Elsevier Science Ltd. All art

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tion conditions (Bennik et al., 1999). Lactic acid bacteriacompete with other microbes by modifying the micro-environment by their metabolic end products (Niku-Paavola et al., 1999). Beside lactic acid, bacteriocins(Bennik et al., 1999) and non-proteinaceous lowmolecular mass compounds (LMMC) are produced byLAB in foods and contribute to the antimicrobial effectsof added LAB cultures. Benzoic acid, methylhydantoinand mevalonic acid lactone contribute to the micro-bicidal action of Lactobacillus plantarum (Niku-Paavolaet al., 1999), and reuterin and pyroglutamic acid to thoseof L. reuteri (Talarico and Dobrogsz, 1989) and L. casei(Huttunen et al., 1995), respectively. According to theauthors, these biologically active substances differ frombacteriocins since LMMCs display a wide spectrum ofactivity against both Gram-positive and Gram-negativebacteria and fungi.On the other hand, the ecological adaptation of addedLAB is an important factor for their effectivenessas biocontrol cultures, and their isolation from thesame vegetables on which they will be further usedas inhibitory agent could be interesting. However,vegetables generally harbor low numbers of LAB,and the detection of LAB-producing antimicrobialproperties in vegetables by the traditional procedureis difficult and time consuming because it requires

doi:10.1006/fstl.2001.0858icles available online at http://www.idealibrary.com on

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the isolation, purification and evaluation of greatnumber of strains.The present report describes the successful developmentof a simple, direct plating procedure for the detectionof antimicrobial-producing LAB in ‘iceberg lettuce’obtained at the retail market level, which is commonlyused as major ingredient in ready-to-use mixed salads.

Materials and Methods

Juice preparation and microbiological analysisThe antibacterial-producing strains were isolated fromjuices of lettuce cv. Iceberg purchased from retailmarkets in Madrid, Spain. Juices were made fromlettuce immediately after purchasing, as follows: lettucewas washed thoroughly with water, mixed with steriledistilled water (1:1), crushed in a domestic juice maker(Osterizer Blender 4094), and centrifuged in a Sorvallmodel RC-5B refrigerated superspeed centrifuge at8000 rev/min for 30min at 4 1C. The lettuce supernatantfluid was then filtered through a number 4 qualitativepaper filters (Whatman International Ltd, Maidstone,England) to obtain the fresh juice, which was stored for8 d at 8 1C. Experiments were replicated three timesusing juices obtained from different lettuces. Thesamples were removed at time 0 (day of juice prepara-tion) and at 2-d intervals up to 8 d of storage at 8 1C foranalysis. Dilutions of samples were plated in duplicateon different media for the counting of the followingmicroorganisms: total aerobic mesophilic and psychro-trophic bacteria, respectively, on tryptone soya agar(TSA) (Oxoid, Unipath Ltd, Basingstoke, U.K.) in-cubated at 30 and 8 1C for 2 and 10 d, and lactic acidbacteria (LAB) on MRS (Oxoid) incubated underanaerobic conditions at 30 1C for 48 h. The lactose-bromcresol purple (LBP) agar, previously reported byMcKay et al. (1970), was also evaluated for thedetection of LAB. The medium consisted of tryptone(20 g), yeast extract (5 g), lactose (10 g), gelatin (2.5 g),agar (11 g) (Oxoid), NaCl (0.4 g), sodium acetate (1.5 g),bromcresol purple (0.02 g) (Merck, Madrid, Spain) anddistilled water (1,000mL).

Detection of strains producing antibacterial substancesThe developed procedure consisted of anaerobicincubation of appropriate serial decimal dilutions ofthe lettuce juice on growth media, and further detectionof inhibitory strains against indicator strain as follows.MRS (Oxoid) and LBP were used as growth media.After incubation of plates at 30 and 8 1C until bacterialgrowth was evident (2 and 10 d, respectively), theplates were overlaid with 10mL of log phase cells ofLactococcus lactis IFPL 359 (C106 cfu/mL) in soft agar(7mg/mL). The plates were then incubated for 24 h at30 1C, and subsequently examined for clear inhibitionzones around the colonies. When the LBP agar wasused, transparent and purple color inhibition zones weredetected, while in MRS agar only transparent halos

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were found. Inhibition was scored positive if the zonewas �5mm.The antagonistic activity of the strains selected by theprocedure described above was confirmed by using theagar spot test and agar cell-free supernatant diffusion(ASD) assay previously described by Casla et al. (1996).For the ASD assay, the supernatant fluid from culturetest strains in MRS was filtered through 0.22 mm poresize filter (Micro Filtration Systems, Sierra CourtDublin, CA, U.S.A.), adjusted to pH 6.0 with 5mol/LNaOH, treated with 1mg/mL catalase at 25 1C for30min, and 1mg/mL proteinase K (Sigma ChemicalCo., St. Louis, MO, U.S.A), and later spotted (5 mL) induplicate over the plates inoculated with the indicatorstrains.

Bacterial strains and culture conditionsThe colonies showing inhibition halos by the developedprocedure described above were picked from countableplates, grown in MRS broth (Oxoid) at 30 1C for 24 h,purified, and maintained as frozen stock cultures at�80 1C in MRS broth with 25% (w/v) glycerol untilthey were required. A preliminary characterization ofpurified isolates was performed by the usual physiolo-gical test for classification of LAB. The criteria foridentification of selected strains have been describedelsewhere (Casla et al., 1996). Carbohydrate fermenta-tion patterns (Api 50 CH; Biomerieux S.A., Marcy-l’Etoile, France) were also used.The target strains used to check the bacterial inhibitoryactivity in vitro were Listeria monocytogenes CECT4032, S. aureus CECT 435, A. hydrophila CECT 4226,E. coli CECT 515, Salmonella typhimurium CECT 443,Enterobacter cloacae CECT 4502, Citrobacter freundiiCECT 401T, Erwinia carotovora CECT 225T. All thesetarget strains were obtained from Spanish Type CultureCollection (Valencia, Spain). Furthermore, an addi-tional LAB, L. lactis IFPL 359 (kindly provided by theDepartment of Dairy Products, Instituto del Frıo(CSIC), Madrid), was included in this study for itssensivity to a wide range of bacteriocins from lactic acidbacteria (Casla et al., 1996).All the target microorganisms were grown aerobically intryptone soya broth (TSB Oxoid), except L. lactis IFPL359 which was grown in M17 broth (Oxoid) with glucose(5mg/mL) as carbon source (Vegarud et al., 1983).Incubation temperatures were 30 1C for 24 h for allmicroorganisms, except S. typhimurium which wasgrown at 37 1C for the same time.

Results and Discussion

Juices of ‘iceberg lettuce’ were used to isolate potentialproducer strains of antibacterial substances. The micro-biological characteristics of the juices during therefrigerated storage show a rather high initial load oftotal mesophilic bacteria increasing about 3 log cfu/mLduring the chilled incubation (Fig. 1). Psychrotro-phic bacteria were the main component of the total

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Fig. 1 Bacterial growth kinetics in lettuce juice incubated for 8 d at 8 1C. [(&), Total aerobic mesophilic bacteria; (*),psychrotrophic bacteria; (~), lactic acid bacteria. Values are the mean and standard error; experiments that were run in triplicateand analysis were performed in duplicate]

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microflora, since similar counts were obtained afterincubating in TSA at 8 1C and 30 1C. The lactic acidbacteria (LAB) also reached maximum levels after thefirst 4 d of chilled incubation, but never exceeded 5.6 logcfu/mL. Figure 1 also shows that there was a persistentsignificant difference between total viable counts andLAB counts even towards the end of incubation.Nguyen-The and Carlin (1994) also reported lownumbers of LAB in fresh vegetables. As a result ofmicrobial growth, the pH values decreased from 6.4(70.03) to 5.8 (70.07) during the chilled storage period.

Detection of bacteria with inhibitory activityThe traditional screening of antibacterial-producinglactic acid bacteria (LAB) is generally time-consuming,since the procedure involves the procedure of randomlypicking of colonies developed on MRS agar underanaerobic incubation, purification, and evaluation usingagar spot test and well-diffusion assay of a large numberof strains, of which many do not have antimicrobialactivity.In the present work, the detection of antibacterial-producing strains in lettuce juice was done directly onlactose-bromcresol purple (LBP) agar, by overlayingLBP plates on which the colonies had been grown withthe indicator strain L. lactis IFPL 359. The LBP agarhad been previously reported by McKay et al. (1979) fordifferentiating Lactococcus parenteral and derivativestrains with reduced proteolytic activity (prt-) andcapacity to metabolize lactose (lac-). Lactose was chosenas carbon source in the medium since in bacteriapotentially present in lettuce, its utilization is largelyrestricted to LAB species, while most enterobacteria donot metabolize this sugar (Brenner et al., 1984).Bromcresol purple was used with the aim to differentiateacid-producing strains from those secreting other anti-bacterial substances. For comparative purposes, theMRS agar was also used to grow the LAB of juices.Anaerobic incubation of plates was used to minimizefalse-positive reactions due to hydrogen peroxide(Casla et al., 1996; Vescovo et al., 1996). Furthermore,

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anaerobic conditions allow the growth of LAB, andinhibit aerobic bacteria species associated with theproduct such as Pseudomonas spp. (Palleroni, 1984).No significant differences in viable counts were foundbetween both media, which indicated the suitability ofthe LBP medium for the growth of LAB. Two kinds ofantibacterial-producing strains grown in the LBP agarplates were differentiated, depending on the color ofinhibition halos produced after growing the indicatorstrain. Purple halos were ascribed to inhibitory sub-stances secreted by strains which do not lower pH of themedium, while those that are transparent may be due toacids such as lactic acid. Such differentiation would notbe possible with MRS agar.Since cultures that produce nonacidic antibacterialsubstances have interesting applications in nonfermen-ted foods (Breidt and Fleming, 1997; Bennik et al.,1999), including ready-to-use vegetables, only thecolonies showing purple haloes on LBP plates wereselected by the procedure described above. However,on MRS plates all colonies showing inhibition haloswere selected because it was not possible to differentiatethem. A total of 54 and 17 strains grown at 30 1Con MRS and LBP agar, respectively, exhibited inhibi-tory activity against the indicator strain, L. lactis IFPL359. The antagonistic activity of the 71 isolates wasconfirmed using the agar spot test. The 17 isolates fromLBP agar plates incubated at 30 1C and a total of 5 outof 54 strains detected on MRS secreted nonacidicantibacterial substances, which was demonstrated bytesting the cell-free supernatants of cultures grown inMRS broth by the ASD technique. These results suggestthat the number of inhibitory strains may be over-estimated by the agar spot test, a method commonlyused when large numbers of strains have to be screened.Some antibacterial substances of Lactobacillus spp. havebeen described which are produced exclusively whencultures are grown on agar medium (Barefoot andKlaenhammer, 1983; Garriga et al., 1993).The 22 strains that produced antibacterial substancesother than acids were also evaluated for their potentialuse as biopreservative agents for ready-to-use vegetables

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Table 1 Inhibitory spectrum of lactic acid bacteria strains isolated from lettuce juice

Lactic acid bacteria strains

Indicator strains IFPV 5 IFPV 6 IFPV 7 IFPV 8 IFPV 9 IFPV 10 IFPV 14 IFPV 15 IFPV 16 IFPV 17

Lactococcus lactisIFPL 359

25 18 20 25 20 15 10 10 10 10

Listeria monocytogenesCECT 4032

20 11 10 17 NI NI NI NI NI NI

Staphylococcus aureusCECT 435

5 5 5 5 NI NI 6 6 5 NI

Erwinia carotovoraCECT 225T

6 5 NI 5 5 5 6 5 6 NI

Salmonella typhimuriumCECT 443

10 5 6 6 NI 5 12 6 8 5

Citrobacter freundiiCECT 401T

8 6 6 6 NI NI NI NI NI NI

Note: IFPL, Instituto del Frıo Productos Lacteos Culture Collection (Madrid, Spain); IFPV, Instituto del Frıo ProductosVegetales Culture Collection (Madrid, Spain); CECT, Coleccion Espanola de Cultivos Tipo (Valencia, Spain); NI, no inhibition;The data represent the diameter of the inhibition zone (in mm) after using the agar cell-free supernatant difussion (ASD) test.

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commonly stored at low temperature, using the ASDtest. A total of 7 out of 17 strains previously detected inLBP agar also grown and produced antibacterialsubstances other than acids in MRS broth at 8 1C.However, only one strain out of five detected in MRSsecreted these compounds under refrigerated conditions.In addition, to test the suitability of the developedprocedure for the direct detection of LAB producingnonacidic antibacterial substances at 8 1C, the bacterialflora of juice was grown on LBP agar for 10 d at thistemperature and then overlaid with the indicator strainin soft agar. Only two strains showing activity grewunder these conditions, which secreted nonacidic sub-stances as it was confirmed later using the ASDtechnique. Physiological assays and carbohydrate fer-mentation indicate that the ten strains secreting anti-bacterial substances other than acids at 8 1C wereLactobacillus plantarum and Lb. rhamnosus. The strainswere considered suitable as biopreservation agents onrefrigerated vegetables, and they were also tested usingthe ASD test against a wide range of Gram-positive andGram-negative bacteria to check their inhibition spec-trum. Treatment of active supernatant fluids withcatalase, NaOH and proteinase K did not modify theinitial inhibitory titre, suggesting that hydrogen per-oxide, acids and proteinaceous substances such asbacteriocins did not account for the observed inhibition.None of the tested strains displayed inhibitory activityagainst E. cloacae, A. hydrophila and E. coli. As Table 1shows, the spectrum of inhibition varied among theisolates. Inhibition activity was demonstrated by teststrains against S. typhimurium and E. carotovora, withthe exceptions for IFPV 9, IFPV 17 and IFPV 7. L.monocytogenes, S. aureus and C. freundii were inhibitedby IFPV 5, IFPV 6, IFPV 7 and IFPV 8. S. aureus wasalso inhibited by IFPV 14, IFPV 15 and IFPV 16.Inhibition halos were also evident for all cultures grownin MRS broth at low temperature, although concentra-tion of the supernatants was necessary to obtain asimilar inhibitory titre compared with supernatants ofcultures grown at 30 1C.

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In conclusion, the results indicate that the developedprocedure effectively allowed for screening of antibac-terial-producing strains by direct plating of the lettucejuices on agar media, incubation at 30 1C and furtheroverlaying with a indicator strain in soft agar. It waspossible to detect antibacterial-producing LAB in 3 d,independent of the growth medium used, compared withseveral weeks for the traditional procedure. The use ofLBP agar in conjunction with incubation of plates at8 1C may give additional information on the nonacidiccharacteristics of antibacterial substances secreted, andalso on the psychrotrophic behavior of producer strains.Strains with such characteristics could be of interest asbiocontrol cultures to improve the safety of ready-to-usevegetables commonly stored under refrigeration.Further research is needed to identify compoundsproduced by LAB, that are responsible for the inhibitionof target microorganisms, and to test whether theselected strains or their metabolites are effective indynamic biological systems such as vegetables. This typeof work is in progress in our laboratory.

Acknowledgements

This work was supported by the projects ALI98-0820-Eand ALI-99-1211 of the Comision Interministerial deCiencia y Tecnologıa in Spain.

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