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Food Control 22 (2011) 1404e1407

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Food Control

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Lactobacillus sakei 1 and its influence adhesion of monocytogenes on stainless steel surface

Lizziane K. Winkelströter, Bruna C. Gomes, Marta R.S. Thomaz, Vanessa M. Souza, Elaine C.P. De Martinis*

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n, 14040-903 Ribeirão Preto, São Paulo, Brazil

article info abstract

Article history: is of particular concern for the food industry due to its psychrotolerant and Received 31 August 2010 ubiquitous nature. In this work, the ability of L. monocytogenes culturable cells to adhere to stainless steel Received in revised form coupons was studied in co-culture with the bacteriocin-producing food isolate sakei 1as 11 February 2011 well as in the presence of the cell-free neutralized supernatant of L. sakei 1 (CFSN-S1) containing sakacin Accepted 22 February 2011 1. Results were compared with counts obtained using a non bacteriocin-producing strain (L. sakei ATCC 15521) and its bacteriocin free supernatant (CFSN-SA). Culturable adherent L. monocytogenes and lac- Keywords: tobacilli cells were enumerated respectively on PALCAM and MRS agars at 3-h intervals for up to 12 h and Listeria monocytogenes Lactobacillus sakei after 24 and 48 h of incubation. Bacteriocin activity was evaluated by critical dilution method. After 6 h of Adhesion incubation, the number of adhered L. monocytogenes cells in pure culture increased from 3.8 to 5.3 log 2 Biofilms CFU/cm (48h). Co-culture with L. sakei 1 decreased the number of adhered L. monocytogenes cells 2 (P < 0.001) during all sampling times with counts lower than 3.0 log CFU/cm . The CFNS-S1 also led to a significant and similar reduction in culturable adhered L. monocytogenes counts for up to 24 h of incubation, however after 48 h of incubation, re-growth of L. monocytogenes number of adhered cells was observed, likely due to lack of competition for nutrients. L. sakei ATCC 15521 or its supernatant (CFNS-SA) did not reduce the number of adhered L. monocytogenes cells on stainless steel surface and from 6 h of incubation, listerial counts were between 4.3 and 4.5 log CFU/cm2. These results indicate that L. sakei 1 and its bacteriocin sakacin 1 may be useful to inhibit early stages of L. monocytogenes adherence to abiotic surface. Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license.

1. Introduction products mainly due to its ability to produce acid, which prevent the growth of a great variety of pathogens (Champomier-Verges, Lactic acid (LAB) constitute a group of Gram-positive, Chaillou, Cornet, & Zagorec, 2001; McLeod et al., 2008; Najjari, non-motile, microorganisms widespread in nature and adapted to Ouzari, Boudabous, & Zagorec, 2008; Ringo & Gatesoupe, 1998). grow under different environmental conditions (Castellano, The preservative ability of LAB in foods is attributed mainly to Holzapfel, & Vignolo, 2004; McLeod, Nyquist, Snipen, Naterstad, & production of antimicrobial metabolites such as organic acids, Axelsson, 2008; Ringo & Gatesoupe, 1998). In general, the metab- hydrogen peroxide and bacteriocins (Castellano et al., 2008; Jones, olism of LAB leads to an improvement of sensorial attributes of Zagorec, Brightwell, & Tagg, 2009; McLeod et al., 2008). Bacterio- dairy, vegetable, fish and meat products and some strains may even cins are ribosomally synthesized peptides produced and secreted inhibit growth of spoilage and pathogenic microorganisms, by both Gram-negative and Gram-positive bacteria that display contributing for safety and shelf-life extension of those products antimicrobial properties against closely related bacteria (Garneau, (Castellano, Belfiore, Fadda, & Vignolo, 2008; Castellano et al., 2004; Martin, & Vederas, 2002; Sit & Vederas, 2008). Guerrieri et al., 2009; Hammami, Rhouma, Jaouadi, Rebai, & Nesme, Several studies have focused on the effect of bacteriocins against 2009; Lopez et al., 2007; Minei, Gomes, Ratti, D’Angelis, & De foodborne pathogens with emphasis on L. monocytogenes, a psy- Martinis, 2008). Specifically, Lactobacillus sakei presents tolerance chrotolerant and ubiquitous microorganism responsible for to low temperatures and high salt concentrations, playing impor- sporadic cases and outbreaks of foodborne infections involving tant role in fermentation and/or preservation of fresh meat and fish mainly immunocompromised individuals, the young, the elderly and pregnant women (Castellano et al., 2004; De Martinis, Públio, Santarosa, & Freitas, 2001; Farber & Peterkin, 1991; Hu et al., * Corresponding author. Tel.: þ55 16 3602 4267; fax: þ55 16 3602 4725. 2008; Jones et al., 2009; Leriche, Chassaing, & Carpentier, 1999). E-mail address: [email protected] (E.C.P. De Martinis). L. monocytogenes is able to adhere and form biofilm on food-contact

0956-7135 Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license. doi:10.1016/j.foodcont.2011.02.021 L.K. Winkelströter et al. / Food Control 22 (2011) 1404e1407 1405 surfaces such as polystyrene, glass and stainless steel (Di 2.2.2. Pure culture studies þ Bonaventura et al., 2008; Dunne, 2002; Leriche et al., 1999). Cells Overnight cultures of L. monocytogenes, L. sakei 1 bac and adhered in biofilms are a major problem because they can be L. sakei ATCC 15521 bac were prepared and 4 ml was individually a source of persistent contamination of materials or foodstuffs inoculated (ca. 108 cells/ml for each L. monocytogenes, L. sakei 1 and coming into contact with them, contributing for the spread of ATCC 15521) in 800 ml of tryptic soy broth (Oxoid, UK) supple- spoilage or pathogenic bacteria throughout food production lines mented with 6 g l 1 of yeast extract (Oxoid, UK) e TSBYE, as (Ammor, Tauveron, Dufour, & Chevallier, 2006; Di Bonaventura appropriate. Inoculated broths were transferred to the system et al., 2008; Dunne, 2002; Garcia-Almendarez, Cann, Martin, described in Section 2.2.1 and incubated at 25 C for up to 48 h Guerrero-Legarreta, & Regalado, 2008; Houdt & Michiels, 2010). under constant agitation at 15 rpm, reached with a magnetic stirrer To avoid risks due to bacterial contamination of food supplies, (Fisatom, São Paulo, Brazil). bacteriocins of LAB may be useful and they can be incorporated into products in different ways. Bacteriocin-producing cultures may be 2.2.3. Estimating bacterial adhesion in biofilms formed in vitro added directly to the food or purified preparations can be used, After 3, 6, 9, 12, 24 and 48 h of incubation a single coupon was Ò such as nisin, commercialized as Nisaplin (Danisco, Denmark). sampled and rinsed with 20 ml of phosphate buffered saline (PBS, Food-grade fermented substrates (milk or whey) are other options pH 7.4) to remove non-adhered cells whereas the adhered cells for incorporating biopreservatives in foods and, ALTAÔ 2341 (Kerry were removed manually by scrubbing the upper surface of the Biosciences, Ireland) is a preparation obtained from a pediocin PA-1 coupon with a sterile cotton swab ca. 100 times. To estimate producing-strain (Deegan, Cotter, Hill, & Ross, 2006; Gálvez, adhesion, the swabs were transferred to tubes containing 10 ml of Abriouel, López, & Omar, 2007; Sobrino-López & Martín-Belloso, PBS, vortexed for about 1 min and serial dilutions were prepared 2008). and 100 ml of each dilution were surface plated on tryptic soy agar The possibility of controlling pathogens in biofilms with bacte- (Oxoid, UK) supplemented with 6g l 1 of yeast extract (Oxoid, UK) - riocins has been suggested by some researchers but the mecha- TSAYE, for L. monocytogenes and MRS agar (Oxoid, UK) for lacto- nisms involved in the action of these biopreservative agents in the (Bagge, Hjelm, Johansen, Huber, & Gram, 2001; Chae & bacterial community of a biofilm needs further investigation Schraft, 2000; Minei et al., 2008). (Ammor et al., 2006; Guerrieri et al., 2009; Kumar, Parvathi, George, Krohne, & Karunasagar, 2009; Leriche et al., 1999; Minei et al., 2.2.4. Co-inoculation studies 2008; Zhao, Doyle, & Zhao, 2004). Evaluation of biofilm formation in co-culture was also carried out The objective of the present work was to evaluate the action of a in a system as described in Section 2.2.1, using the following the food isolate Lactobacillus sakei 1 and its bacteriocin on viable conditions: (i) L. monocytogenes (4 ml of 108 cell/ml), (ii) cells of L. monocytogenes adhered in biofilms formed in vitro. L. monocytogenes (4 ml of 108 cell/ml) plus L. sakei 1(4mlof108 cell/ ml) and (iii) L. monocytogenes (4 ml of 108 cell/ml) plus L. sakei ATCC 15521 (4 ml of 108 cell/ml). The systems were incubated at 25 C for up to 48 h with constant agitation (15 rpm). Bacterial populations 2. Materials and methods adhered to stainless steel coupons were determined after 3, 6, 9, 12, 24 and 48 h (Section 2.2.3) by surface plating serially diluted 2.1. Bacterial strains and culture conditions suspensions of adhered cells removed from stainless steel on PAL- CAM agar (Oxoid, UK) for enumeration of L. monocytogenes and on L. monocytogenes ATCC 19115 and Lactobacillus sakei ATCC 15521 MRS agar (Oxoid, UK) for L. sakei. (bac , non bacteriocin-producing) were used in the present study. þ The bacteriocin produced by L. sakei 1 was quantified by the Bacteriocin-producing L. sakei 1 (bac ) was previously isolated critical dilution assay based on the work by Mayr-Harting, Hedges, from pork sausage (De Martinis et al., 2001) and the bacteriocin and Berkeley (1972) and Martinez and De Martinis (2005). Briefly, produced was characterized by D’Angelis, Polizello, Nonato, BHI agar plates were overlaid with semi-solid BHI (BHI plus 0.8% of Spadaro, and De Martinis (2009) and named sakacin 1. Stock bacteriological agar, Oxoid) inoculated with 105e106 cells/ml of cultures were kept at 80 C in Brain Heart infusion broth (BHI, L. monocytogenes, used as indicator microorganism. Serial dilutions Oxoid, Basingstoke, UK) for L. monocytogenes and De Man, Rogosa of the CFSN-S1 were prepared in 10 mM phosphate buffer (pH 7.0), and Sharpe broth (MRS, Oxoid, UK) for lactobacilli, both added of and 10 mL aliquot of each dilution was spotted on surface of semi- 20% (v/v) glycerol (Synth, Brazil). Working cultures were made by solid BHI seeded with L. monocytogenes. The plate was incubated at inoculation (1%, v/v) in appropriate culture media, followed by 37 C for 24 h and observed for the highest dilution with inhibition 24 h-incubation at 37 C for listeria and 25 C for lactobacilli. halo of L. monocytogenes growth. The results were reported as arbitrary units per ml (AU/ml).

2.2. In vitro biofilm formation studies 2.2.5. Studies with supernatant of LAB cultures The neutralized cell-free supernatant of L. sakei 1 containing non 2.2.1. System for in vitro biofilm formation purified sakacin 1 was obtained by inoculating 1 ml of overnight Stainless steel coupons were pre-treated according to methods culture of L. sakei ca. 108 cell/ml in 100 ml of MRS broth and incu- previously described (Marsh, Luo, & Wang, 2003; Minei et al., bating at 25 C for 24 h. The grown broth culture was centrifuged at 2008). Briefly, coupons were cleaned with neutral Extran solution 4 Cat6720g for 25 min (Sorvall RC Plus, Du Pont, USA), pH was (Merck, Darmstadt, Germany), boiled in 1% (w/v) sodium lauryl adjusted to 7.0 and it was filter-sterilized through 0.22 mm pore-size sulphate (Vetec, Brasil) for 5 min, rinsed with 100% isopropilic membrane (Millipore, São Paulo, Brazil). The filtrate obtained from alcohol (J. T. Baker, Mexico) for 5 min, and washed with distilled L. sakei 1 was referred as “cell-free neutralized supernatant of water for 5 min. The coupons were dried at room temperature, and L. sakei 1” (CFNS-S1) and the titer of the bacteriocin sakacin 1 was six coupons were clamped vertically in a sterile circular support, determined as described by Mayr-Harting et al. (1972). A similar placed in a beaker and covered with aluminum foil. The system for preparation was obtained from L. sakei ATCC 15521 (CFNS-SA) biofilm formation was sterilized in autoclave at 121 C for 15 min and used as negative control of spent culture medium without prior to inoculation (Minei et al., 2008). bacteriocin. 1406 L.K. Winkelströter et al. / Food Control 22 (2011) 1404e1407

CFNS-1 and CFNS-SA were separately added to TSBYE, in the Guerrieri et al. (2009) used a small-scale model to evaluate the proportion of 50% (v/v), for a final volume of 800 ml in the system antilisterial activity in biofilms using bacteriocin-producers (Lacto- as described in Section 2.2.1. These preparations were inoculated bacillus plantarum 35d, Enterococcus casseliflavus IM 416K1) in with 4 ml of overnight culture of L. monocytogenes (ca. 108 cell/ml) comparison with two non bacteriocin-producers (L. plantarum 396/ and experiments of biofilm formation were carried out as described 1, E. faecalis JH2-2). Those authors observed a strain-specific inhi- for pure and co-cultures, except that non-selective medium bition of adhesion to abiotic surface and higher inhibition was (TSAYE) was used for enumeration of L. monocytogenes. caused by bacteriocin-producing strains. In the present work, bacteriocin quantification revealed inhibi- 2.3. Statistical analysis tory activity varying from 2400 to 4267 AU/ml in co-cultures and the titer was 6400 AU/ml in CFNS-S1. Results were calculated as CFU of adherent microorganisms per The results from experiments with CFNS-S1 from L. sakei 1on cm2 and expressed as mean (standard deviation) of three inde- biofilm formation by L. monocytogenes on stainless steel coupons is pendent replicates. Comparisons among treatments were per- also shown in Fig. 1 and a significant reduction of counts of listeria formed using one-way analysis of variance with a significance level adhered cells (P < 0.05) was observed for up to 24 h of incubation. of P < 0.05 and the Student-Newman-Keuls test with P < 0.05 was However, after 48 h of incubation, re-growth of L. monocytogenes applied to evaluate significant differences. Analyses were per- adhered to stainless steel coupons was observed (4.8 log CFU/cm2) formed using the software package Sigma Stat 3.1 (Systat Software, likely due to lack of competition for nutrients or selection of Inc., USA). bacteriocin-tolerant strains. The CFNS-SA did not inhibit adherence by L. monocytogenes cells and after 3 h of incubation population 2 3. Results and discussion already reached 3.0 to 5.1 log CFU/cm . After 48 h of incubation, there was no significant difference in The application of LAB cultures or even their metabolites to counts of culturable adhered L. monocytogenes in the presence of improve safety of foods in processing environments is promising L. sakei 1orL. sakei ATCC 15521, suggesting that at that time, the and the potential of bacteriocins produced by different microor- inhibitory effect observed for L. sakei was due to unspecific mech- ganisms to prevent biofilm formation by L. monocytogenes has been anisms (such as nutrient competition and acid production) and shown by several authors (Deegan et al., 2006; Garcia-Almendarez could not be attributed solely to the action of bacteriocin (Alves, et al., 2008; Guerrieri et al., 2009; Leriche et al., 1999; Minei et al., Martinez, Lavrador, & De Martinis, 2006; Hammami et al., 2009; 2008; Zhao et al., 2004). Minei et al., 2008). In this work, it was observed that pure cultures of L. monocytogenes, L. sakei 1 and L. sakei ATCC 15521 adhered to 4. Conclusions stainless steel coupons since early stages of incubation. For all 3 2 culture conditions, after 9h of incubation there was >10 CFU/cm The results obtained in the present study indicate that L. sakei 1 adhered to coupons and at the end of experiments (48 h) the pop- and its bacteriocin sakacin 1 may be useful to inhibit early stages of 2 ulations were respectively 5.3, 3.1 and 3.8 log CFU/cm . The results L. monocytogenes adherence on stainless steel surfaces, encour- obtained with co-inoculation studies (Fig. 1) showed that pop- aging more detailed studies to evaluate the specific mechanisms ulation of culturable L. monocytogenes adhered cells were higher involved with these processes. than 103 CFU/cm2 in the presence of L. sakei ATCC 15521, while lis- terial adherence in the presence of the bacteriocin-producing Acknowledgments L. sakei 1was<103 CFU/cm2 at all sampling times, indicating a significant inhibition (P < 0.05) in comparison with the number of The authors thank The São Paulo State Research Foundation cells adhered when L. monocytogenes was cultivated alone. (FAPESP, Processes # 2007/56720-0, 2006/05574-1 and 2007/ 56365-6) for financial support.

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