PaPer 148 Ital. J. Food Sci., vol. 28 - 2016 Keywords: removal, peracetic acid, cutting boards, Escherichia coli EFFECTIVENESS OF SANITIZING AGENTS IN INACTIVATING ESCHERICHIA COLI (ATCC 25922) IN FOOD CUTTING BOARD SURFACES. REmOVAl E. COLI USING DIFFERENT SANITIZERS CEZAR AUGUSTO BElTRAmE, EDUARDA BOFF mARTElO, RAíZA DE AlmEIDA mESqUITA, IEDA ROTTAVA, JUlIANA BARBOSA, ClARICE STEFFENS*, GECIANE TONIAZZO, EUNICE VAlDUGA and ROGéRIO lUIS CANSIAN Department of Food Engineering, URI, Erechim, Av. 7 de Setembro, 1621, CEP 99700-000, Erechim, RS, Brazil, *Corresponding author: Tel. 550xx543520-9000, email: claristeffens@yahoo.com.br AbstrAct the objective of this study was to investigate Escherichia coli adhesion on new and used poly- ethylene cutting board surface and evaluate it’s removal using different sanitizer (peracetic acid, chlorhexidine, sodium hypochlorite and organic acids). results indicated that the number of ad- herent cells increased with time in both surfaces evaluated. Evaluating the sanitizer action, 0.5% peracetic acid was more effective in removal E. coli than chlorhexidine and organic acids at same concentration in both surfaces. Peracetic acid and sodium hypochlorite also showed effectiveness at concentrations of 0.2% and 0.5% on new surfaces, respectively. 0.8% of chlorhexidine and 2.0% of organic acids showed similar effectiveness in the removal E. coli on new and used surfaces, re- spectively. these results suggest that peracetic acid is considerable promise sanitizer for applica- tion in surfaces of the food processing industry. mailto:claristeffens%40yahoo.com.br?subject= Ital. J. Food Sci., vol. 28 - 2016 149 INtrODUctION E. coli is a gram-negative bacteria that pre- sent surface layer organizations of the type fimbriae, exopolysaccharides (EPs) or flagel- la, that favor the adherence to materials or host cell surfaces motility and pathogenici- ty. Food can become contaminated with E. coli when animals are slaughtered or pro- cessed, even if precautions are taken and also when it is handled by a person infect- ed with E. coli, or from cross-contamination (bEUMEr and KUsUMANINGrUM, 2003). Food residues left on food processing or handling equipment may provide a niche of microor - ganisms that can rapidly grow. the growth of pathogenic bacteria can result in cross- contamination from food processing surfac- es such as cutting boards to food products (MONtVILLE et al., 2012). In the food industry, good manufacturing, hygienic production and regular cleaning and disinfection procedures are very important, since food safety and quality are determined by the efficacy of sanitizer agents (KrOLAsIK et al., 2010). bacteria have the ability to ad- here to any surface including, but not limited to, glass, stainless steel, polypropylene, rub- ber and wood (cOQUEt et al., 2002; tEIXEI- rA et al., 2008). to prevent bacterial attach- ment on surfaces the choosing an appropri- ate sanitizer is very important for achieving a satisfactory end result in microbiological indexes. Many sanitizers have been broadly used across many industries to reduce path- ogenic bacterial contamination in food prod- ucts or on kitchen utensils, because these compounds have been shown to effective- ly inactivate foodborne pathogenic bacteria (cAbEÇA et al., 2012; FrANK, 2003; rOssONI and GAYLArDE, 2000). therefore, more stud- ies into the bactericidal properties of sani- tizers at different concentrations and con- tact times are required to define the correct application. Many researchers have examined materi- als employed in manufacturing of foods con- tact surfaces such as stainless steel (cAbEÇA et al., 2012; FrANK, 2003; KrOLAsIK et al., 2010; rOssONI and GAYLArDE, 2000; rYU and bEU- cHAt, 2005), but few reports bacterial remov- al on commercial polyethylene cutting boards used in industrial food preparation have been published to date. the objective of this study was to evaluate the E. coli adhesion on new and used cutting board surfaces and removal with different sanitizers used in food industry (peracetic acid, chlorhex- idine, sodium hypochlorite and organic acids). For each sanitizer tested, different concentra- tions were evaluated over 72 h, determining the sanitizer’s effectiveness on new and used poly- ethylene cutting boards. MAtErIAL AND MEtHODs Surface material the food processing surfaces evaluated in this study was new and used polyethylene cutting board, white high-density polyethylene (HDPE plastic). the boards were obtained from cutting room of a slaughter unit, where the used sur- faces had around of 45 days of handle. surfac- es materials with 1.0 cm x 1.0 cm plates were cut, cleaned by brushing employing liquid deter- gent and water, and rinsed with distilled water. they were immersed in 70% ethanol, for 1 h, to fat removal, and again rinsed with distilled wa- ter and air dried. the surfaces were exposed to ultraviolet light 254 nm for 1 h to sanitize them, as described by PArIZZI (1999), before deposi- tion of any bacterial cultures. Adhesion of Escherichia coli on food processing surfaces bacterial strains were obtained from seattle, UsA, 1946 (American type culture collection; rockville, MD, UsA). For the study of adherence was used an E. coli (Atcc 25922) strain, grown previously in Luria bertani broth - Lb (tryptone 10.0 g L-1, yeast extract 5.0 g L-1, Nacl 5.0 g L-1) and incubated at 35ºc (±2) for 24 h. E. coli was chosen as indicator organism, commonly pre- sent in industrial food plants. the cleaned surfaces were immersed, at 25ºc, in Erlenmeyer containing 100 mL of Lb supple- mented with a suspension of bacterial cells in or- der to obtain a count of 103 cFU mL-1. the ster- ilized surface, for each time, was immersed in these Erlenmeyer with sterilized forceps and in- cubated at 35°c in Lb broth. the quantities of adhered cells per square centimeter were eval- uated for 72 h of contact time (0.1, 1, 3, 6, 12, 24, 48, and 72 h) on new and used surface. the initial time (0 h) corresponds to the analysis per- formed immediately after the immersion of the surfaces in the Erlenmeyer containing the medi- um culture and the bacterial suspension. tripli- cates were performed for each treatment. After the incubation, the surfaces were with- drawn from the bacterial suspension E. coli and transferred to tubes, containing 10 mL of peptone water 0.1% (p/v) for 1 min, to remove planktonic cells. subsequently, immersed in tubes containing 5 mL of the same diluent so- lution and vortex for 1 min, to remove sessile cells (PArIZZI, 1999). the contact areas were swabbed and the adhered microorganisms in the swabs were transferred to tubes, contain- ing 10 mL of peptone water 0.1% (p/v) sterilized at 121°c, for 15 min. the tube was stirred us- ing a vortex for 10 s to release the bacteria from the swab. Next, 1 mL of solution was carefully plated on Lb agar, incubated at 35-37°c for 24 h, to colony counting. 150 Ital. J. Food Sci., vol. 28 - 2016 Efficiency of different sanitizers against Escherichia coli on food processing surfaces the sanitizers used in this study were cho- sen to represent those used in the food indus- try. the following sanitizers were used: peracet- ic acid 15% (Johnson Diversey, são Paulo-sP, brazil), chlorohexidine 20% (AD Foods Industry Ltda, Laguna-sc, brazil), sodium hypochlorite 10% (csM chemical Products Ltda, chapecó- sc, brazil) and organic acids (formulated with lactic acid-30%, citric acid-3%, ascorbic acid- 3%, and salts of fatty acids-7% in water). For each sanitizer, different concentrations (0, 0.2, 0.5, 0.8 and 2.0%) were investigated for 10 min of exposure, to evaluate their efficiency in re- moval the adhered cells. these agents were di- luted in sterilized distilled water according to the supplier’s instructions. After this treatment, the surfaces were immersed (separately) in 10 mL of sterilized water, for 1 min and repeated twice to removal the excess of sanitizer. the counts of bacterial adhesion and inacti- vation by sanitizers were carried out using swab on cutting boards, evaluated through the stand- ard plate count method. then, plated on Lb agar, incubated at 35-37°c for 24 h to colony count- ing. All determinations were performed in tripli- cate and the results expressed in terms of mean values (PArIZZI et al., 2004). Statistical analysis Descriptive analyses, including the mean value and variability (standard deviation) and graphic displays were performed. results ob- tained in experimental design described previ- ously were performed considering a 95% confi- dence level (p<0.05) by the tukey’s test, using the software statistica 8.0 (statsoft Inc®, UsA). rEsULts E. coli adhesion in food processing surfaces Fig. 1 show the number of E. coli adhered on new and used cutting board surfaces with dif- ferent contact times. Numbers of E. coli were es- timated and expressed as log 10 colony forming units per cm2 (log cFU cm-2). A fast adhesion of E. coli on both surfaces studied were observed for up to 12h, becoming constant after 24h on used surfaces, when the maximum population reached (6.92 log cFU cm-2). A significant dif- ference (p<0.05) was observed in the intensi- ty of adhesion between the surfaces until 24h. Effect of different sanitizers for inactivating Escherichia coli Figs. 2, 3, 4 and 5 show the data’s of inacti- vation E. coli on new and used cutting boards Fig. 1 - counts of E. coli on new and used cutting board sur- faces without the presence of sanitizers, over 72 h of con- tact time. bars represent the standard errors of the mean from triplicate experiments and * simbolize significant dif- ferece (p<0.05). Fig. 2 - the efficacy of different concentrations of peracet- ic acid (0, 0.2, 0.5, 0.8 and 2.0%) on the reduction of E. coli on: (a) new and (b) used cutting board surfaces, over 72 h. bars represent the standard errors of the mean from trip- licate experiments. a b Ital. J. Food Sci., vol. 28 - 2016 151 sanitizer (2.0%) was effective until 1h of contact (Fig. 3 b), and reduce around 2.5 log cFU cm-2 of cells after 72 h. In this way, chlorhexidine sani- tization had a better effect on removal attached cell on new surfaces. For 0.8% chlorhexidine was observed com- pletely E. coli removed on new boards with 1h of contact, but not was effective on the used boards. consequently, the lower concentrations investigated (0.2 and 0.5%) not show complete inactivation. On used cutting board, all concen- trations of sanitizer studied not inactive bacte- ria after 1 h of contact. According to the suppliers, also organic acids are suggested in a concentration of 0.5%. In this way, this concentration showed efficiency only for 10 min, on both surfaces evaluated. Higher concentrations, 0.8 and 2.0%, were effective for removing E. coli up to 1 and 3 h of contact on new surfaces, respectively (Fig. 4a). the results also indicated that the amount of adherent cells reduced 2.4 log with 2.0% organic acid and was efficient for 1h on used surfaces (Fig. 4b). this Fig. 3 - the efficacy of different concentrations of chlorhex- idine (0, 0.2, 0.5, 0.8 and 2.0%) on the reduction of E. coli on: (a) new and (b) used cutting board surfaces, over 72 h. bars represent the standard errors of the mean from trip- licate experiments. Fig. 4 - the efficacy of different concentrations of organic acid (0, 0.2, 0.5, 0.8 and 2.0%) on the reduction of E. coli on: (a) new and (b) used cutting board surfaces, over 72 h. bars represent the standard errors of the mean from trip- licate experiments. with different concentrations (0, 0.2, 0.5, 0.8 and 2.0%) of peracetic acid, chlorhexidine, or- ganic acid and sodium hypochlorite sanitizers, respectively, over 72 h of contact time. Fig. 2 a and b demonstrates that the bacteria exhibited a significant decrease in the survival rate of viable cells after treatment with peracet- ic acid. the concentration of 0.5% peracetic acid indicated by the supplier was completely effec- tive for inactivating E. coli at all times investigat- ed on new surfaces, while 0.2% peracetic acid was effective for up to 6 h, and able to reduce the number of adhered cells of 4.4 and 5.0 log for 48 and 72 h, respectively (Fig. 2a). In Fig. 3 a is possible to observe that only the highest concentration of chlorhexidine (2.0%) was completely effective for inactivating E. coli on new surfaces, for 72 h. In used surfaces this a b a b 152 Ital. J. Food Sci., vol. 28 - 2016 low efficiency of organic acids can be explained by the fact that the compounds are in a disso- ciated form at the product application moment and dilute the sanitizer, so a higher dissociation leads to lower efficiency (bELtrAME et al., 2012). Fig. 5 demonstrates the efficiency of sodium hypochlorite against E. coli. the concentration (0.5%) indicated by the supplier was able to re- move bacteria cells, at all exposure times, on new surface (Fig 5a). On the other hand, to ob- tain the same effect, on the used surface, a con- centration of 2.0% was required (Fig 5b). Effectiveness correlation between different sanitizers the sanitation in food surfaces, including cut- ting boards is critical for the control of micro- bial contamination of foods and is a significant concern of food preparation and processing in- dustries and public health agencies. In this way, to compare the efficacy of sanitizers (peracetic acid, chlorhexidine, sodium hypochlorite and organic acids) used in the food industries was evaluated a concentration of 0.5%, after 3 h of contact, on new and used surfaces for E. coli re- moval (Fig. 6). the concentration of 0.5% corre- spond the minimum recommended by the sup- plier and 3 h of contact is the maximum time (practiced by the food industry) for disinfecting surfaces used. comparing the sanitizers, the peracetic acid was completely effective in removing E. coli on new and used surfaces (p<0.05), as well as for sodium hypochlorite only new surfaces. It was found that chlorhexidine and organic acids ex- hibit reductions on new and used cutting boards (Fig. 6), without significant difference between the sanitizers (p>0.05), but less effectively than other sanitizers evaluated in this work (p<0.05). DIscUssION the differences of adhesion on cutting boards could be due microbiological, physical and chemical parameters related to the polyethyl- ene. Particularly, in this study can be verify that the used surfaces have higher counts until 24 h (Fig. 1), possibly due to the surface character- istics, which visually present more cracks and wear by 45 days of use in the slaughter unit. the surface topography has been widely stud- ied, since microorganisms adhere more easi- ly in fissures or cracks, and can resist clean- ing and disinfecting procedures (HILbErt et al., 2003; PArIZZI et al., 2004). thus, macroscopic and microscopic characteristics are crucial for microbial adhesion, reflected in the food con- tamination by spoilage or pathogenic microor- ganisms (VADILLO-rODrÍGUEZ et al., 2004). Af- ter 48 h the number of adherent cells remained constant over time in both surfaces. this was Fig. 5 - the efficacy of different concentrations of sodium hypochlorite (0, 0.2, 0.5, 0.8 and 2.0%) on the reduction of E. coli on: (a) new and (b) used cutting board surfaces, over 72 h. bars represent the standard errors of the mean from triplicate experiments. Fig. 6 - the efficacy of different sanitizers (concentration of 0.5%), over 3 h, on the reduction E. coli from new and used cutting board surfaces. Means (± standard deviations) fol- lowed by the same letters represents no significant difference at 5% level (tukey’s test) between the sanitizers and surfaces. a b Ital. J. Food Sci., vol. 28 - 2016 153 also observed in surface reaches saturation lev- el with greater numbers of planktonic cells and not result in greater number of adherent cells (HOOD and ZOttOLA, 1997). the results of bacteria removal demonstrate that from 48 h of contact (in used surface), even with concentrations 4 times superior than rec- ommended by suppliers, peracetic acid was not effective. this suggests that the attachment in- crease during the contact time. similar results was found by other researcher (ADEtUNJI and IsOLA, 2011). MILLEr et al. (1996) evaluated the potential of water for removal E. coli 0157:H7 from polyethylene cutting boards, and the micro- organism was incubated for 0 to 30 h, at 37°c, to determine their inhibitory potential. the au- thors observed an increase in bacteria cells on the boards during the first 30 min of contact, and the water removed 2.3 log cFU cm-2 from the surface. cAbEÇA et al. (2012) carried out a study of disinfection on stainless steel surfaces, using biguanide and peracetic acid, and verified that they were able to reduce E. coli cells adhered of 2.2 and 2.1 log cFU cm-2 for 10 min, respective- ly, with a concentration of 0.5% (w/v). In the pre- sent work was possible reduce 3.5 log cFU cm-2 after for 3 h, at the same concentration of per- acetic acid on new and used polyethylene cut- ting boards. Peracetic acid disinfectant activity is based on the release of active oxygen. It disrupts the chemiosmotic function of the lipoprotein cy- toplasmic membrane and transports through dislocation or rupture of cell walls. It may also be effective on outer membrane lipoproteins, fa- cilitating action against Gram-negative bacteria. Intracellular peracetic acid can also oxidize es- sential enzymes. thus, vital biochemical path- ways, transported through the membrane and intracellular solute levels of are damaged, and alterations in the DNA molecule (KItIs, 2004). In this study, all concentrations of chlorhex- idine not were effective for the removal of bacte- ria after 1 h. this low activity may be due mech- anism action, rapid absorption of bacterial cells, resulting in several cytological modifications that affect permeability and optical properties. stud- ies have shown that chlorhexidine reacts with the cell from lipophobic groups, causing a diso- rientation of the lipoprotein membrane and gen- erating a change in osmotic barrier function (KU- DAVIDANAGE et al., 2009). chlorhexidine is a cat- ionic molecule with a wide antimicrobial spec- trum against both Gram-positive and Gram-neg- ative bacteria (MOHAMMADI and AbbOtt, 2009). this group of biguanides differs from other cati- onic biocides that interact only superficially with the lipid bilayer altering fluidity through cati- on displacement and head group bridging (GIL- bErt and MOOrE, 2005). In a study performed by HOUArI and DI MArtINO (2007) the authors verified that chlorhexidine diacetate (Fluka) was able to inhibit the biofilm formation of different bacteria such as E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus ep- idermidis at conventional in-use concentrations. second Patel (2005), the bacteria resistance to antimicrobial agents begins at the attach- ment phase and increase with the biofilm age. Although, bacteria in biofilms are surrounded by an extracellular matrix that might physically re- strict the diffusion of antimicrobial agents, this does not seem to be a predominant mechanism of biofilm-associated antimicrobial resistance. Another indication of high counts are the sur- face roughness and hydrophobicity that can sig- nificantly affect the attachment, formation plac- es for microorganism’s accommodation and per- manent adhesion. MOVAssAGH et al. (2010), showed counts of 7.69 log UFc cm-2 for E. coli O 111 on polyethylene surfaces. second the au- thors, bacteria encountered in food processing environments can be very hardy and difficult to remove. bacterial attachment and subsequent survival involved interactions between a bacte- rial cell, surface and surrounding microenvi- ronment. the removal bacteria by sodium hypochlorite can be associated with water forms hypochlor- ous acid, which contains active chlorine (a strong oxidizing agent). chlorine exerts its antibacteri- al action by irreversible oxidation of a sulfhydryl group of essential enzymes to microorganisms, disabling metabolic functions of the bacterial cell (POGGIO et al., 2012). sodium hypochlorite may also have a deleterious effect on the bacte- rial DNA, involving the formation of chlorinated derivatives of nucleotide bases. Furthermore, it has been reported that sodium hypochlorite can induce disruption of the bacterial membrane (Mc DONNEL and rUssEL, 1999). Organic acids have an inhibitory action in the undissociated form, from 100 to 600 times great- er than the dissociated form. Undissociated or- ganic acid can permeate the cell membrane by diffusion and release protons in the cytoplasm of the cell. the influx of protons induces acidifi- cation of the cytoplasm and dissipates the mem- brane proton potential (KItKO et al., 2009). this inhibits the transport mechanism for the sub- strate, energy generation and synthesis of mac- romolecules (stOPFOrtH et al., 2003). cONcLUsIONs In both surfaces studied it was observed a fast adhesion of E. coli and present lower counts in new surface when compared with used. the bio- film formed on used polyethylene cutting boards reduces significantly the action of sanitizers. Among the sanitizers evaluated, peracetic acid was the most efficient for reducing E. coli counts. On the new cutting boards concentration of 0.5% peracetic acid was effective in eliminating E. coli adhesion during 72 h evaluated and un- 154 Ital. J. Food Sci., vol. 28 - 2016 til 1 h in used surface. Hypochlorite, chlorhex- idine and organic acids demonstrated similar ef- fects until 1h, reducing the total adhesion with 0.8 and 2.0% on new and used cutting boards, respectively, although 2.0% sodium hypochlo- rite has been effective for total removal until 72 h. the order of efficacy in removing E. coli was as follows: peracetic acid, sodium hypochlorite, chlorhexidine and organic acids. the results of the study showed the impor- tance of hygiene procedures on surfaces that come into contact with food. 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