Evaluation of Rhamnolipid and Surfactin to Reduce the Adhesion and Remove Bioﬁlms of Individual and Mixed Cultures of Food Pathogenic Bacteria

Food Control 25 (2012) 441e447

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Food Control

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Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove bioﬁlms of individual and mixed cultures of food pathogenic bacteria

Milene Zezzi do Valle Gomes, Marcia Nitschke*

Depto. Físico-Química, Instituto de Química de São Carlos (IQSC) e USP, Av. Trabalhador São Carlense, 400, Caixa Postal 780, São Carlos, SP, CEP 13560-970, Brazil

article info abstract

Article history: Biofilms represent a great concern for food industry, since they can be a source of persistent contami- Received 18 August 2011 nation leading to food spoilage and to the transmission of diseases. To avoid the adhesion of bacteria and Received in revised form the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants, 16 November 2011 microbial compounds that can modify the physicochemical properties of surfaces changing bacterial Accepted 22 November 2011 interactions and consequently adhesion. Different concentrations of the biosurfactants, surfactin from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated to reduce the adhesion Keywords: and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Bacterial adhesion Biofilms Staphylococcus aureus, Listeria monocytogenes and Salmonella Enteritidis were studied using polystyrene Biosurfactants as the model surface. The pre-conditioning with surfactin 0.25% reduced by 42.0% the adhesion of Rhamnolipids L. monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1.0% reduced by 57.8% Surfactin adhesion of L. monocytogenes and by 67.8% adhesion of S. aureus to polystyrene.Biosurfactants were less effective to avoid adhesion of mixed cultures of the bacteria when compared with individual cultures. After 2 h contact with surfactin at 0.1% concentration, the pre-formed biofilms of S. aureus were reduced by 63.7%, L. monocytogenesby 95.9%, S. Enteritidis by 35.5% and the mixed culture biofilm by 58.5%. The rhamnolipids at 0.25% concentration removed 58.5% the biofilm of S. aureus, 26.5% of L. monocytogenes, 23.0% of S. Enteritidis and 24.0% the mixed culture after 2 h contact. In general, the increase in View metadata, citation and similar papers at core.ac.uk brought to you by CORE concentration of biosurfactants and in the time of contact decreased biofilm removal percentage. These results suggest that surfactin and rhamnolipids canprovided be explored by Elsevier to- Publisher control Connector the attachment and to disrupt biofilms of individual and mixed cultures of the food-borne pathogens. Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license.

1. Introduction Biofilm formation can be controlled by cleaning strategies using chemical and physical methods however, bacteria living in biofilms The attachment of bacteria to surfaces and the consequent are more resistant to disinfectants and more difficult to remove biofilm formation has serious implications in food, environmental mechanically when compared to planktonic forms (Simões et al., and medical fields. The occurrence of biofilm in food processing 2010). Surfactants are the chemical products usually utilized for environments can lead to spoilage and transmission of diseases cleaning food contact surfaces. representing a risk for the health of consumers and a cause of In last years biosurfactants (BS), surface-active compounds of economical losses to industry (Simões, Simões, & Vieira, 2010). microbial origin, have been attracted attention due to their low Listeria monocytogenes, Salmonella Enteritidis and Staphylo- toxicity and high biodegradability when compared to synthetic coccus aureus are examples of food-borne pathogenic bacteria able surfactants (Banat et al., 2010; Nitschke et al., 2005). Bacterial to form biofilms in a variety of surfaces commonly used in food derived biosurfactants exhibit a variety of chemical structures and environment such as stainless steel, plastics, rubber and glass properties useful for industrial purposes. Surfactin, a lipopeptide (Marques et al., 2007; Meylheuc, van Oss, & Bellon-Fontaine, 2001; produced by Bacillus subtilis and rhamnolipid, a glycolipid from Oliveira, Oliveira, Teixeira, Azeredo, & Oliveira, 2007; Stepanovic, Pseudomonas aeruginosa are the best known biosurfactants. These Cirkovic, Ranin, & Svabic-Vlahovic, 2004). molecules present strong surface activity, emulsion forming ability and has shown antimicrobial properties (Nitschke, Costa, & Contiero, 2010; Ongena & Jacques, 2008). * Corresponding author. Tel.: þ55(0) 16 33738101; fax: þ55(0) 16 33739952. Previous studies has shown that adsorption of biosurfactants to E-mail address: [email protected] (M. Nitschke). a solid surface can modify its hydrophobicity affecting the adhesion

0956-7135 Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license. doi:10.1016/j.foodcont.2011.11.025 442 M. Zezzi do Valle Gomes, M. Nitschke / Food Control 25 (2012) 441e447 process and consequently the biofilm formation. A 0.1% surfactin polystyrene surface: rhamnolipids 0.25%, 0.5% and 1.0% (w/v) solution was reported to reduce significantly the adhesion of aqueous solutions and surfactin 0.1%, 0.25% and 0.5% (w/v) aqueous L. monocytogenes and E. sakazakii to stainless steel and poly- solutions. A 96 well polystyrene microplate was filled with 200 mlof propylene (Nitschke et al., 2009). The pre-conditioning of poly- each concentration of biosurfactant. After 24 h at room tempera- styrene with 0.1% solution of surfactin reduces by 84% the adhesion ture, solution was removed and the wells were gently washed 3 of L. monocytogenes ATCC 7644 whereas rhamnolipids at 0.75% times with 200 ml of sterile distilled water. Control wells were filled reduced the adhesion of L. monocytogenes ATCC 15313 by 82% with 200 ml of water. (Araújo, Lins, Santa Anna, Nitschke, & Freire, 2011). The anti- adhesive properties of biosurfactants shows to be dependant on 2.6. Adhesion test the surface and microorganism involved as well as the temperature, and the type and concentration of the surfactant (Shakerifard, The wells of the polystyrene microplate pre-conditioned with Gancel, Jacques, & Faille, 2009; Zeraik & Nitschke, 2010). Most biosurfactants were filled with 180 ml of TSYE broth, inoculated studies regarding anti-adhesive properties of biosurfactants were with 20 ml of bacterial suspension (of pure or mixed cultures) and conducted using pure cultures of microorganisms and in the incubated at 35 C for different time intervals. Cell suspension was absence of culture medium however, it is known that mixed removed, and wells washed with water, fixed with 200 mlof fi cultures are predominantly found in bio lms and that the presence methanol for 15 min and stained for 15 min with crystal violet 1.0% ’ of nutrients can affect the adhesion. Irie, O Toole, and Yuk (2005) (w/v). The wells were washed and filled with 200 ml of acetic acid fi reported that rhamnolipids can disperse bio lms of the respira- 33% (v/v). The adhesion kinetics of adhered cells was estimated by tory pathogen Bordetella bronchiseptica, suggesting another reading the optical density at 630 nm (Mireles, Toguchi, & Harshey, potential use for biosurfactants. The present work evaluates the 2001) using a microplate reader (TP Reader e Thermoplate). effect of the pre-conditioning of polystyrene surfaces with surfactin and rhamnolipid on the adhesion of pure and mixed growing 2.7. Disruption of pre-formed biofilms cultures of food pathogenic bacteria. The potential of the biosurfactants to disrupt pre-formed biofilms of such bacteria was also Biofilm formation by L. monocytogenes, S. aureus and Salmonella investigated. Enteritidis was performed as individual cultures and mixed cultures. The 96 well polystyrene microplate was filled with 180 ml 2. Materials and methods of TSYE broth, inoculated with 20 ml of individual or mixed bacterial suspension and incubated at 35 C for 24 h. Cell suspension was 2.1. Bacterial strains removed and 180 ml of fresh TSYE broth and inoculum were added and incubated for more 24 h. After 48 h of growth the cell L. monocytogenes ATCC 19112, S. aureus ATCC 25923 and suspension was removed and the formed biofilm were added of Salmonella enterica Enteritidis PNCQ0301 were stored at - 20 Con 200 ml of surfactin (0.1% and 0.5% concentrations) or rhamnoli- trypticase soy broth (TSB) supplemented with 20% glycerol (v/v). pids(0.25% and 1.0% concentrations) solutions. The effectiveness of biosurfactants was verified after 2 h, 6 h and 12 h of contact with 2.2. Medium and culture conditions the biofilms. The wells were washed three times with 200 mlof distillated water and the adhered cells were quantified as described Bacterial strains were cultivated in trypticase soy broth (TSB) or above. agar (TSA) supplemented with 6 g l 1 (w/v) of yeast extract (TSYE) and incubated at 35 C for 24 h. 2.8. Contact angle measurements 2.3. Preparation of bacterial suspension Polystyrene samples of 3 cm2 were cleaned as described by Bacteria from stock medium were inoculated on a TSYE agar Zeraik and Nitschke (2010). The samples were conditioned with the plate at 35 C for 24 h. Nine milliliters of sterile distilled water were biosurfactants for 24 h at room temperature, washed with 10 ml of added to the culture and then scraped from the plate. The dense water and left to dry in a desiccator for 24 h. As control, samples of bacterial suspension obtained was adjusted to have 109 CFU ml 1 polystyrene were immersed in distillated water for 24 h. The and used to perform the adhesion tests. To prepared mixed contact angle of water was assessed by the sessile drop technique at m cultures, 1 ml of each standardized individual cultures were pooled 20 C using a drop volume of 4 l on a Contact Angle System (CAM in a sterile glass tube. 200- KSV).

2.4. Biosurfactants 2.9. Physicochemical characterization of cell surfaces

Rhamnolipids were produced by P. aeruginosa LBI using soybean To evaluate hydrophobic/hydrophilic nature of bacterial cells oil as carbon source as described by Nitschke et al. (2010). Rham- the Microbial Adhesion to Solvents Test (MATS) was applied nolipids presented a surface tension of 30.8 mN m 1, purity of 60%, (Bellon-Fontaine, Rault, & van Oss, 1996). Four solvents were used: and critical micelle concentration (CMC) of 92.4 mg l 1. chloroform (acid/electron acceptor), hexadecane (nonpolar), ethyl Surfactin were produced by B. subtilis using cassava wastewater acetate (basic/electron donor) and decane (nonpolar). A bacterial as substrate as previously described (Nitschke et al., 2004). Sur- suspension containing approximately 109 CFU ml 1 prepared in factin presented a surface tension of 26.9 mN m 1, purity of 78%, 2.4 ml of NaCl 0.15 mol l 1 was mixed in vortex with 0.4 ml of each and CMC of 33 mg l 1. solvent for 2 min. After 15 min the phases was completely sepa- rated and the optical density of aqueous phase was measured at 2.5. Surface conditioning 400 nm (Genesys 10 UV - Thermo Scientiﬁc). The adhesion percentage to each solvent was calculated by the equation: (1 A/ Based on previous work (Nitschke et al., 2009) we select three A0) 100, where A0 is the absorbance of the bacterial suspension concentrations of each biosurfactant for the pre-conditioning of before mixing and A is the absorbance after mixing. M. Zezzi do Valle Gomes, M. Nitschke / Food Control 25 (2012) 441e447 443

2.10. Scanning electron microscopy (SEM) a 0.7 Bioﬁlms of L. monocytogenes were established in polystyrene samples of 1 cm2 as described above (item 3.7). After 48 h, the 0.6 samples were washed three times with 2 ml of distillated water and added of 2 ml of surfactin 0.1%. Control samples were added of 0.5 distilled water. After 2 h contact with the biosurfactant the samples )mn03 were rinsing with 6 ml of distilled water and the dehydration 0.4 procedure was carried out with increasing concentrations of 6(DO ethanol:water as described by Zeraik and Nitschke (2010). The 0.3 samples were maintained desiccated until gold sputtering and visualized by scanning electron microscope (LEO 440 - Zeiss) 0.2 operating at 20 kV. 0.1 2.11. Statistics 0.0 Results were expressed as the mean of at least three indepen- 0 1020304050 Time (h) dent replicates. Error bars represent the standard error. The data were analyzed by ANOVA, and the means were compared using the b 0.7 Tukey test with 5% probability.

0.6 3. Results and discussion 0.5

3.1. Adhesion kinetics on conditioned surfaces )mn036(D 0.4 Preliminary tests were conducted with the three concentrations of each biosurfactant to assess the adhesion of L. monocytogenes, 0.3

S. aureus and S. Enteritidis during 48 h of growth. The pre- O conditioning of the surface with rhamnolipids or surfactin was 0.2 able to reduce the adhesion of L. monocytogenes in all concentrations tested (data not show). However, it was observed that the 0.1 concentration of 1.0% of rhamnolipids and 0.25% of surfactin were 0.0 the most effective reducing the adhesion of bacteria by 57.8% and 0 1020304050 42.0% respectively during the 48 h of incubation. The comparison of Time (h) both treatments and control is shown in Fig. 1a. The increase in 0.4 c concentration shows different behavior for each biosurfactant; for rhamnolipids the increase in concentration also increases the anti- adhesive activity whereas for surfactin we observed a signiﬁcant 0.3 reduction on the adhesion only when the concentration was increased from 0.1% to 0.25% but the increase from 0.25% to 0.50 % was not signiﬁcant (data not shown). Rodrigues, Banat, van der Mei, )mn0 Teixeira, and Oliveira (2006) also observed that a high concentra- 0.2 3

tion of rhamnolipid was needed to achieve anti-adhesive activity 6(D on silicone rubber suggesting that it can be related with the lost of adsorbed molecules in the washing process since the rhamnolipids O interact with the surface by weak forces. Rivardo, Turner, Allegrone, 0.1 Ceri, and Martinotti (2009) observed that an increase in concentration of lipopeptides from Bacillus spp. above 100 mgml1 reduced their anti-adhesive properties against S. aureus. 0.0 The adhesion of S. aureus was reduced with the treatment of the 0 1020304050 surface with rhamnolipids (Fig. 1b). The concentration of 1.0% was Time (h) able to reduce the adhesion by 67.8% during 48 h of growth. The surfactin, on the other hand, increased the adhesion of S. aureus so; Fig. 1. Adhesion kinetic of (a) L. monocytogenes, (b) S. aureus and (c) S. Enteritidis on 6 this biosurfactant is not indicated as anti-adhesive agent of this polystyrene surface of microtitre plates pre-conditioned with surfactin 0.25% ( ), rhamnolipids 1.0% (B) and control (-). The data represent the average of at least three pathogen on polystyrene. The pre-conditioning of surface with independent replicates standard error. surfactin 0.25% reduced the adhesion of Salmonella Enteritidis by 42.3% during 48 h of incubation. The rhamnolipid did not show a signiﬁcant reduction on adhesion even at the highest concen- effective to reduce the adhesion of mixed culture of L. monocytogenes tration tested (1.0%). The average reduction on adhesion during the and Salmonella Enteritidis, however, the treatment with rhamnoli- 48 h was not statistically signiﬁcant relating to control (Fig. 1c). pids 1.0% reduces the adhesion of mixed culture of L. monocytogenes Based on the best results obtained for the pure cultures we eval- and S. aureus to 44.5% in the interval of 9e24 h of cultivation. These uate the effect of biosurfactants on adhesion of mixed cultures of results suggest that the biosurfactants are less effective against the L. monocytogenes and S. aureus using rhamnolipids 1.0% and mixed cultures when comparing to the pure cultures of the bacteria. L. monocytogenes and S. Enteritidis for surfactin 0.25%. Fig. 2 shows To explain the behavior observed we have performed MATS test and that the treatment of the surface with surfactin 0.25% was not contact angle measurements. 444 M. Zezzi do Valle Gomes, M. Nitschke / Food Control 25 (2012) 441e447

L. monocytogenes and S. aureus. The hydrophobicity and electrical 0.4 a charge of bacterial surface are the main physicochemical forces involved in microbial adhesion to solid surfaces (Goulter, Gentle, & Dykes, 2009). The hydrophobicity is related to hydrophobic components (lipids) present on cell wall whereas the polar char- 0.3 acter of cell surface is attributed to carboxylate, phosphate and amino groups as well to exopolysaccharides. To adhesion occurs,

)mn )mn attractant forces (electrostatic, van der Waals and Lewis acidebase 0.2 interactions) between cell and solid surface must be strongest than 0 36( DO 36( repulsive forces (Briandet, Herry, & Bellon-Fontaine, 2001; Hood & Zottola, 1995).

0.1 3.3. Contact angle measurements

The polystyrene is a hydrophobic surface once it presented 0.0 a water contact angle of 84.7 . The contact angle measurements 0 10 20 30 40 50 (Table 2) shows that the pre-conditioning with biosurfactants Time (h) reduces the hydrophobicity of the solid surface, especially surfactin b 0.25% and rhamnolipids 1.0%, which were the concentrations that we 0.4 have observed the highest reduction on the adhesion of the susceptible bacteria. The increase in rhamnolipid concentration was proportional to the decrease in hydrophobicity and the 1.0% 0.3 concentration rendering the polystyrene surface a strongly hydro-

)mn 036( DO 036( )mn philic nature (Table 2). The conditioning of surface with biosurfactants modiﬁes its hydrophobic character since the adsorption 0.2 of these amphiphilic molecules may be orientated accordingly to the nature of surface and environment involved. The nonpolar moiety of surfactant interacts with hydrophobic surfaces while polar moiety is 0.1 exposed to the aqueous environment resulting on a reduction in the hydrophobicity of solid surface (Shakerifard et al., 2009).

0.0 3.4. Effect of biosurfactants on bacterial adhesion 0 10 20 30 40 50 Time (h) Surfactin is a cyclic heptapeptide that is considered an anionic surfactant due to aspartic and glutamic acid residues that are Fig. 2. Adhesion kinetic of mixed culture of (a) L. monocytogenes þ S. Enteritidis and (b) L. monocytogenes þ S. aureus on polystyrene surface of microtitre plates pre- negatively charged at neutral pH (Shen, Lin, Thomas, Taylor, & conditioned with surfactin 0.25% (6) or rhamnolipids 1.0% (B) and control (-). The Penfold, 2011). Rhamnolipids are also anionic surfactants owing data represent the average of at least three independent replicates standard error. to the presence of carboxyl and rhamnosyl groups (Ishigami, Gama, Fumiyoshi, & Choi, 1993). The biosurfactants turn the polystyrene less hydrophobic probably as a consequence of their orientation at 3.2. Microbial adhesion to solvents test the surface exposing the negatively charged groups. The adhesion of L. monocytogenes was reduced after the pre- fi The af nity of bacteria for each solvent is showed in Table 1. conditioning of polystyrene with both biosurfactants. The anti- fi L. monocytogenes have higher af nity for chloroform compared adhesive effect could be related to the electrostatic repulsion with hexadecane indicating a predominance of basic character in between the negative charges of bacterial surface and the nega- fi cell surface. S. aureus presented high af nity for chloroform and tively charged polystyrene (after the treatment) and to a reduction hexadecane indicating high hydrophobicity and a predominance of on hydrophobic interactions. The increase on adhesion of S. aureus fi basic properties. S. Enteritidis showed low af nity for all solvents on polystyrene pre-conditioned with surfactin showed not to be tested, been characterized as a weak acid surface. The mixed related with the interactions between negative charges of bacteria fi culture of L. monocytogenes and S. Enteritidis showed low af nity and surface, once it should result in repulsion. This fact could be for all solvents tested, in the same way as the mixed culture of attributed to changes in surface charges during growth due to

Table 2 Table 1 Water contact angle measurements of polystyrene surface pre- Percentage affinity of bacterial cells to solvents used in MATS test. conditioned withdifferent concentrations of the biosurfactants. Microrganisms % affinity toa Treatment Contact angle ()b Decane Hexadecane Ethyl acetate Chloroform Controla 84.77 1.50 L. monocytogenes 33.3 4.9 24.0 8.0 24.1 4.2 54.3 0.6 Surfactin 0.1% 66.16 2.11 S. aureus 33.2 8.3 93.7 5.1 9.2 1.1 97.8 1.7 Surfactin 0.25% 62.47 1.23 S. Enteritidis 12.4 1.3 2.4 1.6 27.1 4.2 0 Surfactin 0.5% 64.51 0.17 L. monocytogenes 12.2 1.7 20.6 2.1 18.9 3.5 20.6 4.3 Rhamnolipid 0.25% 72.47 2.95 þ S. aureus Rhamnolipid 0.5% 64.19 2.18 L. monocytogenes 7.0 1.8 14.1 1.3 29.1 0.3 10.7 2.8 Rhamnolipid 1.0% 11.45 2.20 þ S. Enteritidis a Surface conditioned with distilled water. a mean of three independent replicates standard deviation. b Mean of 9 drops standard deviation. M. Zezzi do Valle Gomes, M. Nitschke / Food Control 25 (2012) 441e447 445 modifications in the chemical composition of the cell wall in Table 3 response to environmental conditions, or to an excretion of acid Removal of bacterial biofilms formed on samples polystyrene surface after different contact times with surfactin solutions. metabolites that neutralizes the negative charges of glutamic and aspartic acid residues (Shakerifard et al., 2009) decreasing the Microrganism Treatment % removal repulsion of surfactin and exposing the hydrophobic aminoacid 2 h 6 h 12 h moieties therefore increasing the hydrophobic interactions and A A A S. aureus Surfactin 0.1% 63.7a 54.3a 47.9a A A A favoring the adhesion. Fowler, Stacy, and Blackwell (2008) sug- Surfactin 0.5% 57.3a 62.4a 37.9b A A A gested that cyclic lipopeptides could increase the adherence of L. monocytogenes Surfactin 0.1% 95.9a 97.4a 99.9a B B B Surfactin 0.5% 85.2a 86.6a 90.9a S. aureus since they are analogous to quorum sensing autoinducers A A A S. Enteritidis Surfactin 0.1% 35.3a 27.6a 20.7b that stimulate the adhesion of Staphylococci on surfaces. The B A A Surfactin 0.5% 12.8a 27.0b 10.9a A A A reduction on adhesion of S. aureus after the pre-conditioning with Mixed culture Surfactin 0.1% 58.9a 42.6b 45.6b A A A rhamnolipid 1.0% can be related with the strong reduction of Surfactin 0.5% 54.0a 31.7b 39.5b hydrophobicity of polystyrene combined with charge repulsion. Each bacteria and the mixed culture were evaluated individually. For each column Salmonella Enteritidis presented low acid properties, so it was ex- and bacteria, the values followed by the same uppercase letter do not differ pected an increase in the adhesion after the conditioning of the significantly (P < 0.05). For each line and bacteria, the values followed by the same fi < surface with the biosurfactants due to attractive interaction lowercase letter do not differ signi cantly (P 0.05). between positive charges of the bacteria and the negatively charged rhamnolipids. Surfactin 0.1% disrupts 58.9% of biofilm after 2 h of surface. In fact, it was observed for rhamnolipids but not for sur- treatment whereas rhamnolipids at 1.0% concentration disrupt factin. The reduction on the adhesion of S. Enteritidis on poly- 31.2% of biofilm after 6 h contact (Tables 3 and 4). styrene pre-conditioned with surfactin suggests that other factors, Fig. 3 illustrates the effect of surfactin treatment on such as the presence of cell appendages or the production of exo- L. monocytogenes biofilms established on polystyrene samples. It polysaccharides must be involved with the adhesion ability of this shows that L. monocytogenes are enclosed by EPM (extracellular microorganism as also observed by Oliveira et al. (2007). Differ- polymeric material) and also the presence of pores and cavities ences on molecular aggregation and orientation of the bio- representing a mature biofilm. In Fig. 3(b) it is possible to observe surfactants as well as on the hydrophobicity of polystyrene the noteworthy reduction on the cells and EPM after 2 h of contact (Table 2) may have contributed to the behavior observed. with surfactin 0.1%. The pre-conditioning with surfactin 0.25% did not decrease In order for surfactants to be effective in removing biofilms, they significantly the adhesion of the mixed culture of L. monocytogenes would have to penetrate into the interface between the solid and S. Enteritidis during the whole experiment (Fig. 2a). This mixed substrate and the biofilm so they could adsorb at the interface and culture exhibit weak acid properties and also adhere weakly to reduce the interfacial tension. Consequently, the attractive inter- nonpolar solvents and so, positively charged groups could interact actions between the bacterial surfaces and the solid surface may be with negative charges in the surface leading to the increase in decreased, which would ease lead to the removal of the film adhesion. L. monocytogenes and S. aureus mixed culture also pre- (McLandsborough, Rodriguez, Pérez-Conesa, & Weiss, 2006). sented weak polar and nonpolar character and the pre- In general the increase in concentration of biosurfactants did not conditioning with rhamnolipids 1.0% reduces the adhesion during increase significantly the removal of biofilms. The results also the interval 9e24 h of incubation (Fig. 2b). The adhesion rate of showed that in general, short periods of contact with both surfac- each bacteria present in mixed culture can be different so, the effect tants were more effective to disrupt biofilms than long contact observed may be due to the anti-adhesive activity against an times. The effect of biosurfactant concentration on the disruption of individual microrganism. Moreover, the presence of one genus can biofilms was also investigated by Dusane, Nancharaiah, Zinjarde, influence the adhesion of a second one as observed by Rieu, and Venugopalan (2010) who reported that 100 mM rhamnoli- Lemaître, Guzzo, and Piveteau (2008) which have found that pids was able to disrupt 93% of the biofilm of Bacillus pumilus while when in mixed cultures, S. aureus produced a peptide that at concentrations below 0.4 mM removal was not observed. promotes the adhesion of L. monocytogenes to stainless steel. Impressive increase on L. monocytogenes biofilm after 12 h contact with rhamnolipid 1.0% suggests that the biosurfactant 3.5. Disruption of pre-formed biofilms stimulates the cell growth; it could be used as a nutrient so, long contact times would permit enzymatic adaptation to the new In order to assess the potential of biosurfactants to remove biofilms, pure and mixed culture of the pathogens were treated with surfactin and rhamnolipid and the effect of concentration and Table 4 fi time of contact with the surfactants was evaluated. The bio lm of Removal of bacterial biofilms formed on samples polystyrene surface after different L. monocytogenes was efficiently disrupted by surfactin, at 0.1% contact times with rhamnolipid solutions. concentration 95.9% of the biofilm was removed after 2 h contact Microrganism Treatment % removal (Table 3). The treatment with rhamnolipids 0.25% removed about 26.5% of the biofilm after 2 h contact and the increase in concen- 2h 6h 12h A A A fi S. aureus Rhamnolipid 0.25% 58.5a 52.8a 32.5b tration of rhamnolipids to 1.0% increases the formation of bio lm B A B Rhamnolipid 1.0% 46.53a 46.9a 19.1b reaching 144% after 12 h (Table 4). The biofilm formed by S. aureus A A A L. monocytogenes Rhamnolipid 0.25% 26.5a 1.7b 12.7a was susceptible to the treatment with both biosurfactants. Results B A B Rhamnolipid 1.0% 7.6a 21.7a 144.8b A A A described in Tables 3 and 4 show that the time of 2 h was the most S. Enteritidis Rhamnolipd 0.25% 23.1a 30.9b 16.3c B A A effective for the removal of biofilms, rhamnolipids 0.25% and sur- Rhamnolipid 1.0% 13.5a 28.9b 20.9a A A A factin 0.1% disrupting 58.5% and 63.7% respectively. The biofilm of S. Mixed culture Rhamnolipid 0.25% 24.0a 16.4b 16.5b Rhamnolipid 1.0% 31.2A 30.9B 18.6A Enteritidis was the less susceptible to the treatments. The best a a b result was observed for surfactin 0.1% that disrupted 35.3% after 2 h Each bacteria and the mixed culture were evaluated individually. For each column and bacteria, the values followed by the same uppercase letter do not differ and rhamnolipids 0.25% that disrupted 30.9% of the biofilm in 6 h of significantly (P < 0.05). For each line and bacteria, the values followed by the same contact (Tables 3 and 4). The mixed culture containing the three lowercase letter do not differ significantly (P < 0.05). Negative values indicate an bacteria was removed more efficiently by surfactin than by increase on adhesion. 446 M. Zezzi do Valle Gomes, M. Nitschke / Food Control 25 (2012) 441e447

also showed potential as agents to disrupt pre-formed biofilms of the food pathogens furthermore surfactin was more efficient than rhamnolipids. The time of contact and the concentration of biosurfactant have influenced the ability to disrupt the biofilms. Further studies should be conducted to evaluate the effect of pH, ionic force, temperature and nutrients on the hydrophobicity of the surfaces pre-conditioned with biosurfactants as well as their effect on biosurfactant molecular aggregation. The biofilm eradication should be assessed with older biofilms, low concentrations of biosurfactants and short contact times.

Acknowledgments

Authors would like to thank to FAPESP for the grant and to CAPES for the scholarship.

References

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