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Food Microbiology 31 (2012) 167e172

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Food Microbiology

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Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other winebacteria

Lorena Díez a, Beatriz Rojo-Bezares c, Myriam Zarazaga b, Juan M. Rodríguez d, Carmen Torres b,c,Fernanda Ruiz-Larrea a,*

aUniversity of La Rioja, Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Departamento de Agricultura y Alimentación,Av. Madre de Dios 51, 26006 Logroño, SpainbUniversidad de La Rioja, Área de Bioquímica y Biología Molecular, Departamento de Agricultura y Alimentación, Av. Madre de Dios, 51, 26006 Logroño, SpaincArea de Microbiología Molecular, Centro de Investigación Biomédica de La Rioja, Logroño, SpaindDepartamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, 28040 Madrid, Spain

a r t i c l e i n f o

Article history:Received 10 May 2011Received in revised form28 January 2012Accepted 13 March 2012Available online 20 March 2012

Keywords:Pediocin PA-1Oenococcus oeniBacterial growth inhibitionWine

* Corresponding author. Tel.: þ34 941 299749; fax:E-mail address: fernanda.ruiz@unirioja.es (F. Ruiz-

0740-0020/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.fm.2012.03.006

a b s t r a c t

Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficientlywell characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditionalantimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage.In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide andethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; inaddition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grapemust is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocinPA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted moresensitive to pediocin PA-1 (IC50 ¼ 19 ng/ml) than the other LAB species (IC50 ¼ 312 ng/ml). Cooperativeinhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth.Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce thebacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative inwinemaking.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

During the winemaking process, a plethora of interactionsoccurs among the microorganisms present in the fermenting must:yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB).When any of these microorganisms grows in an uncontrolledmanner, spoilage of wine occurs and this spoilage can happen atmultiple stages of the vinification. Sulphur dioxide is the traditionalantimicrobial agent used during the winemaking process to controlbacterial growth and wine spoilage. This chemical preservative canbe used in combination with other antimicrobial agents, and thus,the use of lysozyme (resolution Oeno 10/97) or dimethyl dicar-bonate (resolution Oeno 5/2001) is approved by the InternationalOrganisation of Vine and Wine (OIV) for bacterial growth controlduring winemaking (Bartowsky, 2009).

þ34 941 299721.Larrea).

All rights reserved.

Bacteriocins are ribosomally-synthesized antimicrobialpeptides produced by certain bacteria, which thus inhibit thegrowth of other competing bacteria. Bacteriocins can be classifiedinto four groups (Nes et al., 2007; Montalbán-López et al., 2011),being pediocin PA-1 the most extensively studied bacteriocin of theclass IIa, the so-called “pediocin PA-1 family” or “pediocin PA-1-like” bacteriocins, characterized by the N-terminal region thatharbours the particularly well conserved “pediocin” motif(YGNGVXCXK) (Rodríguez et al., 2002). Pediococcus acidilacticistrains have been largely used as fermentation culture starters fora wide range of food products, which include vegetable (e.g.sauerkraut), meat (e.g. sausages) (Papagianni and Anastasiadou,2009) and dairy products (e.g. cheese) (Drider et al., 2006) and,in fact, pediocin PA-1 producing P. acidilactici have been inadver-tently or empirically used for many years as starter cultures ina variety of food fermentations. Currently, pediocin PA-1 producingcultures and pediocin PA-1 containing fermentates have foundnumerous applications in food industry either to control themicrobial succession during fermentation, or to inhibit the growth

Table 1Bacteria and yeast strains included in this study.

Microorganismsa

(number of strains)Species Sourceb Number of

strains

LAB (n ¼ 53) Lactobacillus plantarum Wine 26Lactobacillus hilgardii Wine 2Lactobacillus sp Wine 1Leuconostoc mesenteroides Wine 1Pediococcus acidilactici Wine 1Pediococcus acidilactici J347-29 Fermented

sausage1

AAB (n ¼ 18) Pediococccus parvulus Wine 2Pediococcus pentosaceus Wine 1Oenococcus oeni Wine 19Acetobacter pasteurianus Wine 2Acetobacter orleanensis Wine 6Gluconobacter oxydans Wine 8

CECT 360 1Yeast (n ¼ 16) Gluconobacter oxydans

subsp. suboxydansWine 1

Brettanomyces bruxellensis CECT 1451 1Brettanomyces sp Wine 1Candida ishiwadae Must 1Candida pulcherrima AF 1Hansenula anomala AF 1Kloeckera apiculata AF 1Kluyveromyces dobzhanski Must 1Metschnikowia pulcherrima CECT 1691 1Picchia membranefaciens CECT 1115 1Rhodotorula rubra AF 1Saccharomyces cerevisiae Wine 1Saccharomyces ellipsoideus AF 1Saccharomycodes ludwigii AF 1Schizosaccharomyces pombe Must 1Zygoascus hellenicus Must 1Zygosaccharomyces veronae CECT 1679 1

a LAB: lactic acid bacteria; AAB: acetic acid bacteria.b CECT :Spanish Culture Type Collection; AF: alcoholic fermentation.

L. Díez et al. / Food Microbiology 31 (2012) 167e172168

of spoilage microorganisms during storage (Drider et al., 2006).Pediocin PA-1, either alone or in combination with other preser-vation technologies, extends the hurdles to the growth of spoilagebacteria in a number of fermented foods, and offers the advantageof being active at acid pH and acting synergistically with othercompounds, such as lactate or organic acids (Drider et al., 2006).Moreover, no adverse effects related to any possible toxicity ofpediocin have been shown to date. Most probably, the peptidenature of bacteriocins, which makes them highly susceptible todigestive enzymes, allows their rapid inactivation and degradationin the gastro-intestinal tract (Delves-Broughton, 2011).

A previous work of our group showed the activity of nisin,a group I LAB bacteriocin, in inhibiting the growth of a number ofLAB of wine origin, alone and in combination with sulphur dioxide(Rojo-Bezares et al., 2007), revealing the potential of such antimi-crobial peptides as tools for wine microbiological control. In thiscontext, the objectives of this study were, on one hand, to inves-tigate the possibility of controlling bacterial growth during wine-making and storage by the use of a pediocin PA-1 containingfermentate produced by a P. acidilactici strain; on the other hand, toevaluate the effect of ethanol and grape must on the production ofpediocin PA-1 by a P. acidilactici producer strain.

2. Materials and methods

2.1. Bacteria and yeast strains

The following microorganisms were used in this study: 53 LAB(19 Oenococcus, 29 Lactobacillus, 3 Pediococcus, 1 Leuconostoc andP. acidilactici J347-29), 18 AAB strains (11 Gluconobacter, 7 Aceto-bacter) and 16 yeast strains (Table 1). Most of the strains wereisolated formwine and belong to the microbial culture collection ofthe University of La Rioja. P. acidilacitici J347-29 29 is a naturalisogenic variant of the pediocin PA-1 producing strain P. acidilactici347 (Rodríguez et al., 1997) and harbours the complete pediocin PA-1 operon (pedABCD).

2.2. Culture and growth conditions

All the LAB strains, except those belonging to the speciesOenococcus oeni, were cultivated for 48 h onto MRS agar plates(Scharlau Chemie S.A., Barcelona, Spain) at 30 �C in an air atmo-sphere containing 5% CO2. O. oeni strains were cultivated in thesame culture medium for 72e96 h at 30 �C under anaerobicconditions (anaerobic system BR038B, Oxoid Ltd., Basingstoke,England) (7e10% final CO2 concentration). AAB were cultivated for48 h onto manitol agar plates, composed of n-manitol (25 g/l;Panreac Quimica S.A., Barcelona, Spain), yeast extract (5 g/l;Scharlau Chemie S. A), peptone (3 g/l; Difco, Becton, Dickinson andCo., Le Pont de Claix, France) and agar (20 g/l; Difco). Yeasts werecultivated for 48 h onto YPD agar plates, containing yeast extract(10 g/l; Scharlau Chemie), peptone (20 g/l; Difco), glucose (20 g/l;Panreac Quimica S A), and agar (20 g/l; Difco).

P. acidilacitici J347-29 was incubated in MRS broth (Oxoid) at20 �C for 48 h with vigorous shaking. Samples were taken atdifferent times and tested for growth inhibitory activity against theindicator strain Pediococcus pentosaceus E 11, using the microtiterand diffusion assays described below. Cell growth was followed byoptical density at 660 nm, as well as by colony counting onto MRSagar plates.

2.3. Growth inhibitory activity

The inhibitory activity of pediocin PA-1 (SigmaeAldrich QuímicaS. A, Madrid, Spain) was determined by calculating the minimal

inhibitory concentration in the microtiter dilution assay (Rojo-Bezares et al., 2006) with the following modifications. MRS brothwas used for LAB, mannitol broth was used for AAB and YPD brothfor yeasts. Microtiter plates were incubated at 30 �C for 24 h for fastgrowing bacteria (AAB and LAB except O. oeni) and 72 h for slowgrowing bacteria (O. oeni), after which bacterial growth wasmeasured byoptical density at 655 nm in amicrotiter reader (Model45, Bio-Rad Laboratories, Hercules, CA). Inhibitory concentration(IC) was defined as the smallest amount of antimicrobial agentneeded to inhibit more than 50% the growth of a given microor-ganism after incubation. Minimal bactericide concentration (MBC)was determined as the minimal concentration of the antimicrobialagent that killed more than 99.9% of the initial inoculum afterincubation from48 to 72 h ontoMRS agar plates. IC50was defined asthe antimicrobial agent concentration needed to inhibit 50% of thetested microorganisms. Controls were included in all the assays.

Pediocin PA-1 activity was tested in the concentration range625e0.33 ng/ml (double dilutions). Potassium metabisulphite(Sepsa-Enartis, San Martino, Italy) was tested in the concentrationrange 12,800e6.25 mg/ml (double dilutions). Absolute ethanol(Panreac) was diluted in sterile water to obtain final concentrationsof 4, 6, 8, 10, 12, 14, 16, 18 and 20% (vol/vol). These antimicrobialagents were tested against the LAB, AAB and yeast collection andtheir corresponding MBC and IC50 values were determined.

In experiments that combined two antimicrobial agents, thesubinhibitory concentrations of ethanol were: 6% for LAB andyeasts, and 3% for AAB. Subinhibitory concentrations of pediocinPA-1 were: 10 ng/ml for O. oeni and 156 ng/ml for the remainingLAB.

The spot on the lawn method described by Rojo-Bezares et al.(2006) was used to test the activity of P. acidilactici J347-29

Table 2Values of inhibitory concentrations (IC) andminimal bactericide concentrations (MBC) of the tested antimicrobial agents in the series of lactic acid bacteria, acetic acid bacteriaand yeast included in this study.

Antimicrobial agents Microorganism Number of strains IC (ng/ml) MBC (ng/ml)

Range IC50 Range MBC50

Metabisulphite LABa 28 50e12,800 400 400e12,800 1600O.oeni 19 50e400 200 100e1600 400AAB 18 50e1600 400 12.50e>12,800 1600Yeasts 16 1600e12,800 12,800 >12,800 >12,800

Metabisulphite þ ethanolb LABa 28 12.5e800 25 12.50e12,800 100O.oeni 19 50e1600 100 200e3200 400AAB 18 12.5e1600 25 12.50e1600 25Yeasts 16 25e12,800 3200 25e>12,800 >12,800

Ethanolc LABa 28 12e20% 14% ND NDO.oeni 19 6e16% 8% ND NDAAB 18 4e10% 6% ND NDYeasts 16 4e20% 12% ND ND

Pediocin LABa 28 <0.33e>625 312.50 >625 >625O.oeni 19 <0.33e>625 19 4.80e>625 78AAB 18 >625 >625 >625 >625Yeasts 16 >625 >625 >625 >625

Pediocin þ ethanolb LABa 28 <0.33e>625 78.12 >625 >625O.oeni 19 1.22e>625 9.73 4.88e>625 >625AAB 18 >625 >625 >625 >625Yeasts 16 >625 >625 >625 >625

Metabisulphite þ pediocind LABa 28 <6.25e400 100 200e800 400O.oeni 19 12.50e200 100 50e800 200AAB 18 ND ND ND NDYeasts 16 ND ND ND ND

ND: not determined.a LAB: lactic acid bacteria except O. oeni; AAB: acetic acid bacteria.b Ethanol: subinhibitory concentrations of ethanol were as follows: 6% for LABa, O. oeni and yeasts, and 3% for AAB.c Ethanol : ethanol concentration is expressed as % (vol/vol).d Pediocin: subinhibitory concentrations of pediocin were as follows : 156 ng/ml for LABa and 10 ng/ml for O. oeni.

L. Díez et al. / Food Microbiology 31 (2012) 167e172 169

against the collection of oenological LAB strains. The activity of cell-free supernatants of cultures of P. acidilactici J347-29 was alsotested by an agar-well diffusion test (Rojo-Bezares et al., 2006)against the indicator strain P. pentosaceus E11. Antimicrobialactivity was detected by the apparition of an inhibition halo of thegrowth of the indicator strain.

2.4. Production of pediocin PA-1 by P. acidilactici J347-29

The production of pediocin PA-1 by P. acidilactici J347-29 wasstudied when it was grown in presence of ethanol (0, 2, 4 and 8%),

Fig. 1. Inhibitory concentration (IC) values of metabisulphite against O. oeni strains, -

grape must (0, 20, 40, 60, 80 and 100%), and with combinations ofboth agents in the culture broth (20, 40, 60, 80 and 100% grapemustwith 2% or 4% ethanol). Initial cell concentrations of 107 cfu/mlwereincubated at 20 �C under vigorous shaking in 50 ml broth culturesfor up to 48 h. Samples (1 ml) were taken at different times todetermine cell growth and pediocin PA-1 production by the agardiffusion test and themicrotiter assaywith P. pentosaceus E11 as theindicator strain. Antimicrobial activity measured by the microtiterassay (activity units, a.u.) was defined as the maximal fold-dilutionof the antimicrobial agent that inhibited more than 50% the growthof the bacteria strain after incubation.

in absence, , in presence of a subinhibitory concentration of pediocin (10 ng/ml).

Fig. 2. Inhibitory concentration (IC) values of metabisulphite against LAB strains except O. oeni,-in absence,, in presence of a subinhibitory concentration of pediocin (159 ng/ml).

L. Díez et al. / Food Microbiology 31 (2012) 167e172170

3. Results and discussion

3.1. Pediocin PA-1 antimicrobial activity

Pediocin PA-1 was an efficient antimicrobial agent against ourcollection of wine LAB, with an IC50 value of 19 ng/ml for O. oeni and312 ng/ml for the other wine LAB species (Table 2). O. oeni wasclearly the most susceptible species to pediocin, similarly to theformerly reported effect of nisin on wine LAB (Rojo-Bezares et al.,2007). Pediocin inhibitory effect was slightly enhanced in thepresence of low concentrations of ethanol (6%), and thus, IC50values decreased one or two orders of dilution for LAB in thepresence of ethanol (Table 2). The effect of pediocin PA-1 wasbacteriostatic except for a number of O. oeni strains that did notsurvive after pediocin treatment (42% of the assayed O. oeni strains,data not shown). O. oeni species is characterised by its high resis-tance to ethanol and to acid pH (G-Alegría et al., 2004); neverthe-less, this species seems to be more sensitive to both nisin (Rojo-Bezares et al., 2007) and pediocin (this work) than other LABspecies. Both bacteriocins act on the membrane surface formingpores and provoking cell death by loss of metabolites to theextracellular medium and subsequent cellular lysis (Cotter et al.,

Fig. 3. Antimicrobial activity of P. acidilactici J347-29 supernatants and cell growth [optical2%; O 4%; B 8%. Antimicrobial activity: dd; cell growth (OD660): -----.

2005). It is well-known that O. oeni adjusts its membrane fluidityto the presence of ethanol in the culture broth (Da Silveira et al.,2003) and, thus, prevents metabolite leakage and increases itsresistance to ethanol; however, the overall membrane composition,rather than its fluidity, has been reported to determine bacterialsusceptibility to both nisin and pediocin PA-1 (Bennik et al., 1997).Thus, it could be hypothesised that these different mechanisms ofaction previously reported by other authors for ethanol and forbacteriocins could account for the different susceptibility of O. oenito ethanol and to both bacteriocins nisin and pediocin.

Our results showed that AAB and yeast were not inhibited bypediocin PA-1 in the studied concentration range (0.33e625 ng/ml)either in absence or in presence of ethanol (Table 2). These resultsare in accordance with previous reports showing that pediocin PA-1 is active mostly against Gram-positive bacteria (Le Blay et al.,2007). In contrast, nisin was previously reported to posses someantimicrobial activity against AAB (Rojo-Bezares et al., 2007).

3.2. Combined effect of metabisulphite and pediocin

Our results showed a cooperative antimicrobial effect of ped-iocin and metabisulfite against our LAB strain collection.

density at 660 nm (OD660)] obtained after incubation in presence of ethanol: A 0%; -

Fig. 4. Antimicrobial activity of P. acidilactici J347-29 supernatants obtained after incubation in presence of grape must: A 0%, - 20%, O 40%, B 60%, C 80% and , 100%.

L. Díez et al. / Food Microbiology 31 (2012) 167e172 171

Metabisulphite IC50 value against O. oeni decreased 2-fold inpresence of a subinhibitory concentration of pediocin (10 ng/ml)(Table 2 and Fig. 1); and 4-fold against the other LAB species inpresence of a subinhibitory concentration of pediocin (156 ng/ml)(Table 2 and Fig. 2). These results are similar to those previouslyreported by our group for nisin, which decreased metabisulphiteminimal inhibitory concentration against O. oeni by 2-fold when itwas added at the subinhibitory concentration of 10 ng/ml (Rojo-Bezares et al., 2007), and by 8-fold against other wine LABspecies (Rojo-Bezares et al., 2007). These cooperative effects ofeither pediocin PA-1 or nisin in combinationwithmetabisulphite ininhibiting LAB growth could have important applications in thefood and beverage industries and could help to decrease the levelsof metabisulphite that are currently used to prevent bacterialgrowth.

3.3. Antimicrobial activity of pediocin PA-1 produced byP. acidilactici J347-29 under oenological conditions

The antimicrobial activity of P. acidilactici J347-29 was deter-mined by the spot on the lawn method against a collection ofoenological LAB and AAB strains, and showed a good correlationwith the activity determined by the microtiter assay. Sixteen out of19 O. oeni tested strains (84%) and 23 out of 29 strains of the otherLAB species (79%) were inhibited by P. acidilactici J347-29, whichgenerated a clear inhibition halo. Negative results were obtainedwith AAB strains. These results corroborated the activity of ped-iocin PA-1 (SigmaeAldrich) shown in Table 2 and suggested thatP. acidilactici J347-29 could be an appropriate strain to obtain

0

Fig. 5. Antimicrobial activity of P. acidilactici J347-29 supernatants obtained afterincubation of the pediocin producing strain in presence of combinations of ethanol andgrape must. Antimicrobial activity was measured by the agar diffusion method asdescribed in Methods section. , 0% ethanol, - 2% ethanol, F 4% ethanol.

pediocin PA-1-enriched fermentates. A previous study reportedengineered Saccharomyces cerevisiae yeast strains that were able toproduce pediocin as they expressed the pediocin PA-1 operon fromP. acidilactici strain PAC1-0 and showed the anti-Listeria activity ofthe transgenic yeast strains (Schoeman et al., 1999). In our case, weexamined the response of P. acidilactici J347-29 to the presence ofethanol and grape must in the culture broth. P. acidilactici J347-29did not show pediocin PA-1 activity when 8% ethanol waspresent in the culture broth, whereas pediocin PA-1 activity wasdetected in the supernatant earlier when 4% ethanol was present inthe culture broth than in absence of ethanol in the culture broth(Fig. 3). P. acidilactici J347-29 grew in presence of 2, 4 and 8%ethanol, reaching high bacterial populations in presence of 2 and 4%ethanol (94e88% of the population reached in absence of ethanol)(Fig. 3). P. acidilactici J347-29 was able to grow in presence of up to80% must in the culture broth (Fig. 4) and, in fact, it reached similarbacterial populations to those obtained under control conditionswithout grape must (data not shown); in addition, production ofpediocin PA-1 increased, obtaining maximal values with 60% grapemust in the culture broth (Fig. 4). P. acidilactici J347-29 was not ableto grow in 100% grape must, probably due to the lack of assimilablenitrogen-containing molecules (<40mg/l) and vitamins needed forbacterial growth. When binary combinations of ethanol and grapemust were assayed results (Fig. 5) showed that maximal pediocinactivity was obtained in the supernatant of the culture broth whenP. acidilactici J347-29 was grown in presence either of 20% grapemust and 2e4% ethanol, or 40e60% grape must and 2% ethanol

Table 3Growth inhibition of LAB and AAB strains by the pediocin producer P. acidilacticistrain J347-29 determined by the spot on the lawn method.

Indicator bacteria(Number of isolates)

Growth inhibition produced byP. acidilactici strain J347-29

þ þ/� e

Oenococcus oeni (19) 15 1 3Lactobacillus plantarum (17) 12 e 5Leuconostoc mesenteroides (1) 1 e e

Lactobacillus paracasei (2) 1 e 1Pediococcus pentosaceus (3) 3 e e

Pediococcus parvulus (2) 2 e e

Pediococcus acidilactici (1) 1 e e

Lactobacillus hilgardii (2) 2 e e

Lactobacillus brevis (1) 1 e e

Acetic acid bacteria (16) e e 16

Bacterial growth inhibition: þ.Weak growth inhibition: þ/�.No growth inhibition: �.

L. Díez et al. / Food Microbiology 31 (2012) 167e172172

(Fig. 5). These results indicated that pediocin production was notimpeded by the presence of ethanol or grape must in the fermen-tation broth, and that the effect of pediocin inhibition of oeno-logical LAB strains (Table 3) could be extrapolated to oenologicalconditions with presence of ethanol (2e4%) and grape must in thefermenting broth, althoughmore studies are needed to confirm thishypothesis. To our knowledge this is the first non-oenological strainreported to produce a well-known bacteriocin such as pediocin PA-1 under oenological conditions or in the presence of ethanol andgrape juice. The bacteriocin nisin had been reported to be an effi-cient inhibitor of oenological LAB strains (Rojo-Bezares et al., 2007),but to date no nisin producer has been shown to grow in wine. Theoccurrence of Pediococcus species during grape must fermentationsand in wines had been largely reported (Beneduce et al., 2004;Pfannebeckera and Fröhlicha, 2008). They have been generallyconsidered as undesirable in winemaking because most reportsfocused on species such as Pediococccus parvulus, Pediococcusdamnosus, P. pentosaceus, which spoil wines because of theproduction of metabolic compounds, such as acetic acid, diacetyland acetoin, or exoplysaccharides, that alter wine sensorial prop-erties (Walling et al., 2005). In contrast, these spoiling character-istics have never been associated to P. acidilactici. Our resultssuggest that pediocin containing fermentates with the appropriateoenological composition could offer efficient antibacterial activityfor microbiological control of musts and that a natural pediocin PA-1 producer could act as a biological agent for microbiologicalcontrol during winemaking and to inhibit the growth of LABspoiling strains in the wine and brewing industries.

Acknowledgements

This research was financially supported by grants AGL2007-60504 and AGL2010-15466 of the Spanish Ministry of Science andInnovation MICINN and FEDER of the European Community. LorenaDiez was a contractual technician supported by grants AGL2007-60504 and CENIT-2008/1002 of the MICINN-CDTI.

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