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Biofilm formation on stainless steel as a function of time and temperature and control through sanitizers
Accepted Manuscript Biofilm formation on stainless steel as a function of time and temperature and control through sanitizers Marcília Rosado de Castro, Meg da Silva Fernandes, Dirce Yorika Kabuki, Arnaldo Yoshiteru Kuaye PII:
S0958-6946(16)30360-0
DOI:
10.1016/j.idairyj.2016.12.005
Reference:
INDA 4122
To appear in:
International Dairy Journal
Received Date: 15 August 2016 Revised Date:
10 December 2016
Accepted Date: 10 December 2016
Please cite this article as: Rosado de Castro, M., da Silva Fernandes, M., Kabuki, D.Y., Kuaye, A.Y., Biofilm formation on stainless steel as a function of time and temperature and control through sanitizers, International Dairy Journal (2017), doi: 10.1016/j.idairyj.2016.12.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Biofilm formation on stainless steel as a function of time and temperature and
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control through sanitizers
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Marcília Rosado de Castroa, Meg da Silva Fernandesa*, Dirce Yorika Kabukib, Arnaldo
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Yoshiteru Kuayea
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Campinas (UNICAMP), Rua Monteiro Lobato 80, Cidade Universitária Zeferino Vaz,
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Campinas, SP, Brazil, CEP 13083-862
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Department of Food Technology and b Department of Food Science, University of
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*Corresponding author. Tel.: +55 19 35214011
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E-mail address: [email protected]
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ABSTRACT
29 Enterococcus spp. contamination was screened from a Minas Frescal cheese processing
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line. Biofilm formation of Enterococcus faecium and Enterococcus faecalis isolates was
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evaluated and the effect of sanitization procedures in the control of these biofilms was
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investigated. Enterococcus spp. were detected in raw milk, milk machine, door handle,
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floor, drain, thermometer, and Minas Frescal cheese. Biofilm formation on stainless
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steel was modelled as a function of time (0, 1.2, 4, 6.8, and 8 days) and temperature (7,
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13, 27, 41, and 47 °C) using response surface methodology. The model showed that E.
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faecium biofilms were formed from 1 to 8 days at 12 to 47 °C, while E. faecalis
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biofilms were formed from 1 to 8 days at 10 to 43 °C. None of the sanitizers (sodium
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hypochlorite 100 mg L-1, peracetic acid 300 mg L-1, and chlorhexidine digluconate 400
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mg L-1) was able to completely eliminate the biofilms.
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1.
Introduction
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Minas Frescal cheese is the most consumed cheese in Brazil (ABIQ, 2014) and is considered the only genuine national cheese. The simple production technology
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attracts the interest of small and large factories. Minas Frescal is defined as a fresh
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cheese, produced with pasteurised whole and semi-slimmed milk by enzymatic
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coagulation (chymosin). It has a high moisture content (average 55%), 17–19% fat,
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1.5% salt, pH range of 5.0 to 5.3, and is lightly pressed or not (Peresi et al., 2001).
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These characteristics allied to the processing conditions favour the proliferation of
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various bacteria, including Enterococcus.
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The prevalence of Enterococcus in cheese may be associated with the ability of this genus to resist pasteurisation and cooling temperatures, and a wide range of pH and
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salinity (Giraffa, 2003). The presence of Enterococcus in food is controversial, because
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although they are responsible for desirable technological characteristics in some foods,
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such as rennet cheese, they can cause foodborne diseases due to their pathogenicity
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(Foulquié-Moreno, Sarantinopoulos, Tsakalidou, & De Vuyst, 2006). Enterococcus spp.
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strains show biochemical and biotechnological properties, such as proteolytic, lipolytic,
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esterolytic activities, besides the use of citrate, which promotes texture and typical taste
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in several types of cheeses such as Manchego, Mozzarella, Monte Veronese, Fontina,
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Caprino, Serra, Venaco and Comte (Giraffa, 2003). However, proteolysis and lipolysis
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are undesirable in Minas Frescal cheese, once the enzymes produced during
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microorganism growth cause degradation of fat and protein, change- aroma and flavour,
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reduce shelf life of the products, and decrease the production yield (Sørhaug &
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Stepaniak, 1997). In addition, Enterococcus are considered opportunistic nosocomial
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pathogens that cause human infections especially in immune-compromised individuals.
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enterococci isolated from food (Cariolato, Andrighetto, & Lombardi, 2008; Fernandes
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et al., 2015a; Gomes et al., 2008; Kasimoglu-Dogru, Gencay, & Ayaz, 2010). Great
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concern over the presence of enterococci in the food is due to transfer of virulence
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genes among strains (Eaton & Gasson, 2001). This gene transfer capability can occur in
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the gastrointestinal tract of humans due to consumption of food contaminated with
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enterococci through bacterial conjugation (Çitak, Yucel, & Orhan, 2004; Gelsomino,
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Vancanneyt, Condon, Swings, & Cogan, 2001).
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One of the main reasons accounting for the existence of enterococci in dairy products is their ability to adhere and form biofilms on food contact surfaces
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(Fernandes, Kabuki, & Kuaye, 2015b; Jahan & Holley, 2014). Biofilms are complex
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and structured microbial communities, surrounded by a matrix of extracellular
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polymeric substances (EPS), adhered to each other and/or to a surface or interface
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(Costerton, Lewandowski, Caldwell, Korber, & Lappin-Socott, 1995), where the
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microorganisms exhibit distinct phenotypes, metabolism, physiology and gene
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transcription. These biofilms are a major focus of contamination affecting food quality
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and safety (Simões, Simões, & Vieira, 2010). Therefore, it is of foremost importance to
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gain insights on factors influencing Enterococcus spp. biofilm to dairy processing
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surfaces.
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One of the strategies to control biofilm formation is the application of effective
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sanitation procedures by combined use of detergents and sanitizers. The sanitizers are
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responsible for the reduction of spoilage microorganisms and elimination of pathogens
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to safe levels. Sodium hypochlorite and peracetic acid are sanitizers widely used in the
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food industry. Chlorhexidine is probably the most widely used biocide in antiseptic
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products, in particular in handwashing and oral products but also as a disinfectant and
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preservative (McDonnell & Russell, 1999). It is noteworthy that the effectiveness of
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sanitizers is usually assessed by laboratory tests (suspension and use-dilution tests),
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without considering the production of exopolysaccharides, compounds mainly
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responsible for the protection of microbial biofilms against the action of sanitizers.
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The objective of this study was to evaluate the ability of E. faecium and E.
faecalis isolated from a Minas Frescal cheese processing line to form biofilms, using
response surface methodology as a function of time and temperature. The efficiency of
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sanitizers to control these biofilms was also investigated.
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2.
Material and methods
2.1.
Sampling
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A total of 156 samples of raw and pasteurised milk, whey, cheese curd,
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equipment and utensil surfaces, environmental air, and Minas Frescal cheeses were
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collected on two visits in August and October 2010 in a cheese processing line in a
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dairy industry in São Paulo State - Brazil.
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2.1.1. Raw material and food samples
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At each visit were collected: four samples of raw milk type A (1000 mL), one
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pasteurised milk (1000 mL), three curd (350 g) and three whey (1000 mL). In addition,
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10 samples (400 g each) of the packaged cheese from the same batch were sampled.
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From these, 5 samples were analysed on the same day of collection, and the other five
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units were analysed on the last day of the shelf life as reported on the label (15 days).
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The cheeses analysed after 15 days were stored under refrigeration at 4 °C.
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2.1.2. Environmental surfaces and air A total of 47 samples per visit from the surfaces of equipment, utensils, and facilities were analysed. Samples were collected after routine cleaning process. Depending on the surface characteristics, two collection methods were used:
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sponge contact method or swab contact technique. The surface characteristic is
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dependent of accessibility and physical design surface configuration (curve, plane) of
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the utensils or/and equipment. The sponge contact method consists of a sterilised
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cellulose sponge (Nasco, Wisconsin, USA) pre-moistened in 20 mL of 0.1% peptone
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water containing 0.5% sodium thiosulphate, according to the recommendation of
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American Public Health Association - APHA (Evancho, Sveum, Moberg, & Frank,
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2001). Surfaces evaluated by sponge method were: stainless steel table, shelf, sealing
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machine, door handle, hose, floors, and walls (milking room, raw milk reception room,
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pasteurisation room, cheese processing room, cold chamber, and cold room at 9 °C for
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storage of utensils used in cheese production), drains (cheese processing room and
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utensils storage room), squeegee, cheese processing tanks, raw milk tank, and utensils
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(moulds, knife to cut the curd, milk stirrer, and thermometer) used in cheese processing.
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The swab technique was used on the milking machine surface, as previously reported
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(Evancho et al., 2001).
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The areas were defined using sterile disposable 50 or 100 cm-2 moulds,
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according to the equipment and utensils configuration. The samples were packed in
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cooler with ice for transport to the Hygiene and Legislation Laboratory, and the
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analyses were carried out within 24 h after collection.
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Environmental air samples were collected in different places of the factory (milking room, raw milk reception, pasteurisation room, cheese production room, cold
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chamber, and cold room at 9 °C). Air sampling was performed using the sedimentation
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method, according to APHA (Evancho et al., 2001).
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2.2.
Isolation and identification of Enterococcus spp.
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The raw material and food samples (25 g or 25 mL) were diluted 1:10 in 0.1% peptone water (Difco, Becton, Dickinson and Company, Sparks, USA), plated on
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Kenner Faecal (KF) Streptococcus Agar (Difco) supplemented with 1% of 2,3,5-
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triphenyl-tetrazolium chloride – TTC (Vetec Química, Rio de Janeiro, Brazil) and
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incubated at 45 °C for 48 h (Hartman, Deibel, & Sieverding, 2001). The environmental
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surface samples were subjected to serial decimal dilution in 0.1% peptone water, plated
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on KF agar and incubated at 45 °C for 48h. The biochemical analyses (catalase
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production, growth at 10 °C and 45 °C, growth in BHI broth containing 6.5% NaCl,
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growth in BHI broth at pH 9.6, and growth on bile-esculin agar) for the identification of
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the genus Enterococcus were performed according to methodology specified by APHA
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(Hartman et al., 2001).
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Genetic identification at species level was done by polymerase chain reaction (PCR). Genomic DNA from pure cultures was extracted according to Furrer, Candrian,
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Hoefelein, and Luethy (1991). The PCR reaction was carried out as previously
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described (Dutka-Malen, Evers, & Courvalin, 1995).
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2.3.
Evaluation of proteolytic and lipolytic presence in Enterococcus spp.
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All isolates of the genus Enterococcus were screened for protease (Hartman et al., 2001) and lipase enzymes (Frank & Yousef, 2004).
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The protease enzyme was evaluated in PCA (Difco) added with 1% reconstituted skim milk, followed by incubation at 28 °C for 72 h. Confirmation was
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obtained by the formation of a translucent halo around the colonies after addition of 0.2
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mL of 10% acetic acid for 1 min. Bacillus cereus NCTC 1143 was used as positive
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control.
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The lipase enzyme was evaluated in Spirit Blue Agar (Difco) added with lipase reagent, followed by incubation at 32 °C for 48 h. Confirmation was obtained by the
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formation of a light colour and/or an intense blue colour halo around the colony.
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Staphylococcus aureus ATCC 6539 was used as positive control.
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2.4.
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Assessment of biofilm formation
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2.4.1. Central composite rotational design
To evaluate the biofilm formation, a central composite rotational design (CCRD)
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with two factors (time of contact and exposure temperature) was performed according to
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the methodology proposed by Rodrigues and Iemma (2005). Three replicates of each test were performed at 7; 13; 27; 41; and 47 °C, and
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times of contact 0, 1.2; 4; 6.8; and 8 days. Time zero corresponded to the test carried out
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immediately after immersion of the coupons into the vial containing milk and the
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bacterial suspension. Table 1 shows the relationship between temperature and time of
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contact for all tests.
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The responses (cfu cm-2) were adjusted by using the quadratic polynomial
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regression model (log cfu cm-2 = b0+ b1T + b2 T2 + b3t + b4t2 + b5Txt), where b0 to b5
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correspond the coefficients of the model, T is temperature, and t is the time of contact.
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Two experimental designs were performed: the first consisting of a mix with
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These strains were isolated in this study and selected from different samples. The strains
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were selected for possess the ability to produce lipases and proteases and present
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pathogenic profile for carry virulence and antibiotic resistance genes (these analyses
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were performed in these strains as described by Fernandes et al., 2015a) (Table 2).
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2.4.2. Biofilm formation
Biofilm formation was evaluated using AISI 304#4 stainless steel coupons
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(0.366 µm roughness, 10 mm × 10 mm × 1 mm). Before use, the coupons were
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sanitized (Fernandes et al., 2015b), suspended by a polyamide yarn, packed in glass
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vial, and sterilised at 121 °C for 15 min.
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Whole UHT milk (Jussara, Franca, Brazil) was used as culture medium for the biofilms formation, which was previously evaluated for aerobic counts (Laird, Gambrel-
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Lenarz, Scher, Graham, & Reddy, 2004) and spores (Frank & Yousef, 2004). The
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counts were below the detection limit for aerobic counts and spores method.
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For each experiment, each bacterial strain was separately inoculated in brain heart infusion (BHI) broth (Difco) at 35 °C for 24 h. Then, 1 mL of each activated
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bacterial strain was added in a sterile flask and vortexed (2800 rpm) for 1 min. Serial
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decimal dilutions were performed until the suspension reached a concentration of
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approximately 1×104 cfu of microorganisms per mL.
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For all tests, immediately after culture inoculation, counts from UHT milk were
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performed on plate count agar (PCA) (Difco) incubated at 35 °C for 48 h as a control
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test. The pool of microorganisms (1×106 cfu mL-1) was inoculated in milk in sterile
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flasks (100 mL) to reach approximately 1× 104 cfu mL-1, containing 4 suspended
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coupons. The flasks were incubated at different temperatures (7, 13, 27, 41, and 47 °C).
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inoculated with the same cultures used initially. The period of 48 h for replacement of
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culture medium in containers was determined according to the current raw milk quality
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regulation established by Federal Brazilian Inspection Service - the maximum time
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between milking and receiving the milk in the establishment where it will be processed
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(Brasil, 2002). The method was reported by Fernandes, Fujimoto, Schneid, Kabuki, and
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Kuaye (2014).
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The discarded milk was subjected to bacteria counts in PCA (Difco) incubated at 35 °C for 48 h, and was compared with the population adhered to stainless steel
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coupons. The biofilm formation on the coupons was evaluated by plate counting
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technique for all experiments (Fernandes et al., 2015b). In such technique, at each time
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and temperature of contact, two coupons were separately immersed into 10 mL of 0,1%
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peptone water for 1 min to remove the planktonic cells. Then, each coupon was
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immersed in 5 mL of the same solution and vortexed (2800 rpm) for 2 min to remove
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the sessile cells (Fernandes et al., 2015b). The resulting solution was serially diluted in
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0.1% peptone water and plated onto PCA agar (Difco), and the plates were incubated at
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35 °C for 48 h.
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2.5.
Statistical Analysis
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The statistical analyses were performed using the STATISTICA 7.0 software
(Statsoft, Tulsa, USA).
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2.6.
Experimental verification of the model fitting
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The same experimental designs (I: five E. faecalis strains; II: five E. faecium strains) were conducted in three replicates under conditions not evaluated before (after 3
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days of contact at 25 °C) to test the efficiency of the model. Coupons preparation and
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incubation, and the determination of biofilm formation were performed as described in
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Section 2.4.2.
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2.7.
Efficiency of sanitizers for removal biofilms
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For this assay, sodium hypochlorite solution 100 mg L-1 total chlorine pH 9.4 (Super Candida Indústria Anhembi SA, Osasco, Brazil), peracetic acid 300 mg L-1 pH
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2.8 (Divosan Forte, Johnson Diversey, Sintra, Portugal), and chlorhexidine digluconate
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400 mg L-1 pH 6.4 (Neobiodine, Neobrax Ltda, Barretos, Brazil) were used, according
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to the instructions of the manufacturers.
Three replicates were performed for each treatment and in each replicate two
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coupons were used for each sanitation step and two for the control. The control coupons
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did not receive sanitizer, and their counts were used to calculate the number of decimal
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reductions due to the sanitization step. The biofilm formation was analysed according to
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the method described in Section 2.4, and the combination of temperature and time of
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contact was determined according to the optimum point of the experimental design (3.5
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days of contact at 25 °C), for the two experimental designs (I: five E. faecalis strains; II:
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five E. faecium strains).
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After incubation, the coupons were rinsed in 0.1% peptone water for removal of
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planktonic cells, and then immersed in 10 mL of each sanitizing solution for 10 min at
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25 °C. To inactivate the sanitizing effect, the coupons were transferred to 10 mL of 1%
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sodium thiosulphate or 0.5% Tween 80 for 10 min, for sodium hypochlorite/peracetic
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neutralising solution, and immersed in 5 mL of 0.1% peptone solution and vortexed for
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two minutes to remove the sessile cells (Fernandes et al., 2015b). For the control
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coupons, the same procedures were repeated except the steps of sanitization and
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neutralisation. The adhered cells were enumerated in PCA incubated at 35 °C for 48 h.
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3.
Results and discussion
3.1.
Enterococcus spp. in Minas Frescal cheese processing plant
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The average Enterococcus spp. count in raw milk was 2.74 log cfu mL-1 (Table
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3), which is relatively low when compared with those observed by other authors in
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Brazil (Fernandes et al., 2015a; Tebaldi, Oliveira, Boari, & Piccoli, 2008). In fact, the
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good manufacture conditions such as mechanical milking, maintaining and storage of
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raw milk at 4 °C, and cheese production in the same place of milking justified these
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results.
Enterococcus spp. counts in pasteurised milk were <1 log cfu mL-1,
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demonstrating the effectiveness of the thermal process to reduce the level of this
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microorganism, despite it does not always result in complete elimination of these
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bacteria in milk and dairy products (Foulquié-Moreno et al., 2006).
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Enterococcus spp. were detected in surfaces of milking machine, door handles,
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floor, and thermometer (Table 3). The milking machine had the highest counts, with
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mean values of 5 log cfu cm-2. Although it is located outside the processing
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environment, its hygienic-sanitary control is critical, thus it represents a focus of
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Enterococcus contamination in raw milk.
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The door handles of the cold chamber and utensils storage room (9 °C) had
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counts of up to 3.8 log cfu cm-2, representing a major cross-contamination, because they
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are near to the cheese processing room, with constant contact with handlers.
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In the first sampling, the presence of Enterococcus spp. was also detected in the thermometer used to control the process. Even though, it was found a relatively low
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count (1.74 log cfu per unit), this fact can be considered a risk due to the contact with
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pasteurised milk at danger temperature zone that can allow bacteria growth.
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Enterococcus spp. has been detected in the air from milking room and milk reception
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room (1 cfu per plate), but it was not detected in the air of the processing room.
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Enterococcus spp. are persistent micro-organisms of the environment, including
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airborne. According to Muzslay, Moore, Turton, and Wilson (2013), unrecognised
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colonisation and/or the aerosolisation of enterococci together with inadequate cleaning
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can lead to heavy, widespread, and persistent environmental contamination.
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Enterococcus spp. strains were detected in only 20% (4/20) of the cheese
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samples (Table 3). Among the positive samples, three samples was analysed on the day
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of collection, and one sample after 15 days of storage at 4 °C, with counts ranging from
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<1 log cfu g-1 to 4.62 log cfu g-1. Thus, the samples with positive results for
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Enterococcus spp. probably were contaminated individually after processing, through
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cross-contamination by equipment and utensils, as formerly mentioned.
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Of the 155 isolates confirmed as Enterococcus genus by biochemical tests 76%
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(120/155) were identified as E. faecalis and 24% (35/155) as E. faecium by PCR. E.
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faecalis was the predominant species observed in the raw milk (76%, 85/112), Minas
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Frescal cheese (100%, 12/12), and processing environment (75%, 23/31). The
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prevalence of these two species has been reported for different types of cheese
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(Devriese, Pot, Damme, Kersters, & Haesebrouck, 1995; Fernandes et al., 2015a;
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Gomes et al., 2008).
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3.2.
Lipolytic and proteolytic presence in Enterococcus spp.
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Of the 155 Enterococcus isolates from cheese processing, 52% (81/155) were
positive for proteases and lipases. From raw milk samples, the proteolytic activity was
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observed in 17 strains of E. faecium isolates, 47 strains of E. faecalis, while 13 strains
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of E. faecium and 50 strains of E. faecalis showed lipolytic activity. The production of
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these enzymes may impact negatively on the processed milk, since proteases act on
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casein, causing bitter in dairy products and lipases confer the rancid taste of milk by the
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release of short-chain fatty acids (Chen, Daniel, & Coolbear, 2003). Enterococcus
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producers of proteases and lipases were observed for the different samples (raw milk,
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environmental samples, and Minas fresh cheese). The production of these enzymes is
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dependent on each strain, due to the great genetic diversity among the strains, and only
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a few are responsible for producing these enzymes (Nörnberg, Tondo, & Brandelli,
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2009).
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3.3.
Modelling of biofilm formation by Enterococcus faecium and Enterococcus
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faecalis on stainless steel
A response surface approach has been applied to demonstrate the influence of
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contact time and temperature and their interactions on the biofilm formation of
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Enterococcus spp. on stainless steel surface. All statistical analyses were performed at a
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5% significant level (p<0.05). However, due to the large variability involving
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microorganisms, the parameters were considered significant at p-values lower than 10%
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(p<0.1) (Table 4) as described by Rodrigues and Iemma (2005). Thus, only the term
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associated with the interaction (temperature × time) was not significant for both species
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evaluated.
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The analysis of variance for the experiment 1 (E. faecium) indicated that the
model was significant (p<0.05), with Fcalculated value 2.65 times greater than the Ftabulated
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and coefficient of determination (R2) of 0.88, which validated the model, demonstrating
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that it fits well to the experimental data (Table 5).
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The model was also significant (p<0.05) for the experiment 2 (E. faecalis). The Fcalculated was 1.54 times greater than the Ftabulated and the coefficient of determination
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(R2) was 0.82 (Table 5). With these results, mathematical models have been developed
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with the encoded variables that describe the process of biofilm formation of E. faecium
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(Equation 1) and E. faecalis (Equation 2) as a function of time of contact (t) and
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temperature (T) (equations for the time intervals 0–8 days and temperatures from 7 to
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47 °C).
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Log cfu cm-2 = 7.35146 + 1.19592T – 1.70483T2 + 1.71043t – 1.30893t2
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The predicted values for the biofilm formation of E. faecium and E. faecalis on
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stainless steel under different times and temperature conditions in food industries can be
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determined from Equations 1 and 2.
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The lower temperature used in this study was 7 °C, since the target bacteria are able to grow at refrigeration temperatures. The intermediate temperatures were
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considered as those that allow microorganism growth, and the highest temperature was
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determined according to the bacteria growth range. It is noteworthy that analysed
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temperatures (7–47 °C) are often found in dairy factories.
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Table 6 shows the influence of contact time and temperature on the biofilm formation (sessile cells) by E. faecium (Experiment 1) and E. faecalis (Experiment 2).
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In addition, Table 6 shows the counts of these microorganisms (planktonic cells) in milk
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after 2 days of incubation for each temperature, for comparison.
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The lowest E. faecium and E. faecalis sessile cells counts (1.08 log cfu cm-2 and
376
1.24 log cfu cm-2, respectively) were observed after 4 days of contact at 7 °C, while the
377
highest counts were observed after 4 days of contact at 27 °C for E. faecium and E.
378
faecalis (6.90 log cfu cm-2 and 7.81 log cfu cm-2, respectively) (Table 6). After 8 days at
379
27 °C, E. faecium and E. faecalis sessile cells counts declined to 6.13 log and 6.96 log
380
cfu cm-2, respectively, which can represent the phase of detachment of the biofilm
381
fragments, and can lead to food contamination. It is important to note that all isolates
382
exhibited virulence genes and resistance to various antibiotics (Table 2). In addition,
383
these biofilm fragments can colonise other regions, resulting in new biofilms (Simões et
384
al., 2010).
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In this study, milk was previously inoculated with approximately 2 log cfu mL-1 of E. faecium or E. faecalis planktonic cells. Both Enterococcus species were able to
387
multiply in milk at different temperatures (7 to 47 °C, Table 6). The lowest E. faecium
388
(3.25 log cfu mL-1) and E. faecalis (3.52 log cfu mL-1) counts were observed at 7 °C,
389
while the highest E. faecium (9.20 log cfu mL-1) and E. faecalis counts (8.75 log cfu
390
mL-1) were observed at 27 °C. One of the aspects that influence the biofilm formation is
391
the concentration of microorganisms in the medium. The higher population of
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microorganisms in milk, greater the biofilm formation on stainless steel (Peña et al.,
393
2014).
394
Analysis of response surfaces and contour curves generated by the model (Fig. 1) allow checking the optimum region for biofilm formation. E. faecium isolates
396
showed an optimum range to form biofilms from 3.5 to 7.2 days and 25.5 to 47 °C (Fig.
397
1A). For E. faecalis, this range was 3.5 days to 8 days of contact, from 21 to 43 °C (Fig.
398
1B).
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Enterococcus spp. have the ability to multiply in a wide temperature range (5–50
400
°C), with optimum temperatures between 35 and 43 °C (Fisher & Phillips, 2009). In this
401
study, the optimum temperatures for biofilm formation were between 21 and 47 °C,
402
close to the ideal for this microorganism. These results are in agreement with Meira,
403
Barbosa, Athayde, Siqueira-Junior, and Souza (2012) who observed more intense
404
biofilm formation at the optimal growth temperature of microorganisms.
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405
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It is noteworthy that the ideal temperature range for biofilm formation of E. faecium and E. faecalis is encountered in Minas cheese manufacture. The refrigerated
407
pasteurised milk is pumped into the processing tank and heated to 35–37 °C and then
408
rennet is added to promote the coagulation of milk. During cheese processing, this
409
temperature range remains for about 3 h, enough time for microorganism growth, with
410
possible biofilm formation, compromising the food safety.
412
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3.4.
Experimental verification of the models
413 414
A condition that has not been evaluated in the central composite design was chosen to
415
verify if the models found are able to describe the actual results. The temperature of 25
416
°C was chosen, since most samples were collected at this temperature. The time of 3.5
17
ACCEPTED MANUSCRIPT days was chosen because it is an intermediate time not used in the construction of the
418
model. The experiment 1 (E. faecium) showed a deviation 0.23 cfu cm-2 log (3.8%),
419
with predicted value of 5.81 log cfu cm-2 and experimental value of 6.04 log cfu cm-2.
420
Experiment 2 (E. faecalis) had predicted value (6.36 log cfu cm-2) higher than the
421
experimental value (6.01 log cfu cm-2).
422 423
3.5.
Assessment of the effectiveness of sanitizers in control of biofilms
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In this study, we have investigated the efficacy of sodium hypochlorite (100 mg L-1), peracetic acid (300 mg L-1), and chlorhexidine digluconate (400 mg L-1) against
427
biofilm formed by E. faecium and E. faecalis on stainless steel surface. As shown in
428
Table 7, the sanitizers in the concentrations recommended by the manufacturers reduced
429
the cells count of E. faecium and E. faecalis biofilms. Sodium hypochlorite reduced
430
more the number of cells of E. faecium biofilms (reduction of 2.74 log cfu cm-2) than
431
peracetic acid and chlorhexidine digluconate (reduction of 1.57 and 1.68 log cfu cm-2,
432
respectively). For the cell count of E. faecalis biofilms (6.01 cfu cm-2), peracetic acid
433
presented better performance (reduction of 3.18 log cfu cm-2) than sodium hypochlorite
434
and chlorhexidine digluconate (reduction of 1.40 and 1.72 log cfu cm-2, respectively).
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Biofilms are formed by aggregates of microorganism cells, EPS matrix, and
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organic matter from food. This structure hinders the action of sanitizers, thus the
437
surviving microorganisms can develop rapidly, especially in the presence of residues,
438
and contaminate the processed food (Simões et al., 2010). Therefore, bacteria removal
439
from contact surfaces can only be achieved by adopting combined actions, such as the
440
application of detergent prior to the sanitizing agent or a product that combines a
441
detergent with a sanitizer (Fernandes et al., 2015b). Fernandes et al. (2015a,b) showed
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ACCEPTED MANUSCRIPT that mono-species biofilm (E. faecium and E. faecalis) and multi-species biofilm (with
443
Listeria monocytogenes) were resistant to different sanitizers, with biguanide being the
444
less efficient sanitizer. In these studies, peracetic acid was the most efficient sanitizer, as
445
also evidenced in other studies (Meira et al., 2012; Park et al., 2012). The high
446
efficiency of peracetic acid to microbial biofilms is due to its ability of not reacting with
447
organic matter, besides being a strong oxidant. Organic and inorganic matter can
448
inactivate compounds based on chlorine and chlorhexidine. Therefore, they are often
449
unable to eliminate microbial biofilms (Bridier, Briandet, Thomas, & Dubois-
450
Brissonnet, 2011).
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4.
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Dissemination of Enterococcus was observed in raw milk, environmental samples, and Minas Frescal cheese. The milking machine stood out with the highest
456
Enterococcus counts, along with door handles, which showed persistent contamination
457
during the collections.
The results of response surface methodology showed that E. faecium and E.
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faecalis were able to form biofilms on stainless steel surface at times and temperatures
460
ranging from 1 to 8 days of contact and 12 to 47 °C for the former, and 1 to 8 days of
461
contact and 10 to 43 °C for the latter; these are temperatures commonly used throughout
462
the Minas cheese processing. Therefore, the possible presence of these bacteria in
463
environments or surfaces under abusive conditions of temperature and time, like the
464
ones used in this study, should be prevented in Frescal Minas cheese processing aimed
465
at food safety.
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In addition, none of the tested sanitizers has completely eliminated the biofilms, evidencing the difficulty of surfaces’ sanitization after the biofilm formation.
468 469
Acknowledgements
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470 This work was supported by the Conselho Nacional de Desenvolvimento
472
Científico e Tecnológico (CNPq) – Process n.140334/2009-2. The authors are indebted
473
to Fundação André Tosello for donating the Enterococcus faecalis ATCC 7080.
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Figure legend
Fig. 1. Surface response and contour curves as a function of exposure temperature and time of contact for biofilm formation of Enterococcus faecium (A) and Enterococcus faecalis (B) on
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stainless steel.
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Table 1
Central composite rotational design for the relationship between the variables exposure temperature and
1 2 3 4 5 6 7 8 9 10 11
Temperature ( °C) 13 13 41 41 7 47 27 27 27 27 27
2 -1 +1 -1 +1 0 0 -1.41 +1.41 0 0 0
Time (days) 1.2 6.8 1.2 6.8 4 4 0 8 4 4 4
Variable 1, exposure temperature; Variable 2, time of contact; -1, lower level; 0, central point; +1,
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higher level; -1.41 and +1.41, axial points.
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Variable 1 -1 -1 +1 +1 -1.41 +1.41 0 0 0 0 0
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Test
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time of contact. a
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Table 2
Origin of Enterococcus faecium and Enterococcus faecalis strains used in the evaluation of biofilm formation. a
E. faecium E84 E. faecium E106 E. faecium E113
Virulence genes +
Door handle (cold chamber) Raw milk Floor (cheese room)
ERI, GEN, STREP, TET RA, TET ERI, STREP, TET ERI, GEN, RA, STREP, TET ERI, RA
esp, efaA, ace, vanB
Door handle (cheese processing room) Floor (milking room)
E. faecium E120 Experiment 2 E. faecalis E2 E. faecalis E15
Raw milk Minas Frescal cheese Thermometer
E. faecalis E110
Door handle (cheese processing room) Milking machine
GEN, STREP ERI, GEN, RA, STREP, TET GEN, STREP, TET ERI
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E. faecalis E40
E. faecalis E94
a
Antibiotic resistance
gelE, esp, efaA, ace, vanB esp, vanB
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Experiment 1 E. faecium E42
Source
esp, ace
esp, ace, vanB
esp, vanB gelE, esp, efaA, ace, vanB
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Microorganism
GEN, STREP, TET
gelE, esp, efaA, vanB
esp, efaA, ace gelE, esp, efaA, ace, vanB
All isolates were positive for proteolytic and lipolytic activity. Abbreviations are: AMP, ampicillin (10
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µg); CLO, chloramphenicol (30 µg); ERI, erythromycin (15 µg); GEN, gentamicin (120 µg); NOR, norfloxacin (10 µg); RA, rifampicin (5 µg); STREP, streptomycin (300 µg); TEC, teicoplanin (30 µg);
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TET, tetracycline (30 µg); VAN, vancomycin (30 µg).
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Table 3
Enterococcus spp. counts in the raw material, processing environment, and Minas Frescal cheese. Source
Raw milk reception
Environment air (log cfu per plate) Milking machine (log cfu per unit) Wall (log cfu cm-2) Floor (log cfu cm-2) Environment air (log cfu per plate) Wall (log cfu cm-2) Floor (log cfu cm-2) Raw milk reception tank 1 (log cfu cm-2) Raw milk reception tank 2 (log cfu cm-2) Raw milk 1 (log cfu mL-1) Raw milk 2 (log cfu mL-1)
2nd <1 5.41 <1 <1 <1 <1 <1 <1 <1 4.61 1.57
<1 <1 <1 <1
<1 <1 <1 <1
<1 <1 2.07 <1 <1
<1 <1 2.20 <1 <1
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Milking room
Collection 1st <1 4.89 <1 <1 1 <1 <1 <1 <1 2.32 2.49
Environment air (log cfu per plate) Wall (log cfu cm-2) Floor (log cfu cm-2) Pasteurised milk (log cfu mL-1)
Cold chamber
Environment air (log cfu per plate) Shelf (log cfu cm-2) Door handle (log cfu per unit) Wall (log cfu cm-2) Floor (log cfu cm-2)
Cold room at 9 °C for storage of utensils used in cheese production
Environment air (log cfu per plate) Moulds (log cfu per unit) Knife to cut the curd (log cfu cm-2) Door handle (log cfu per unit) Milk stirrer (log cfu per unit) Wall (log cfu cm-2) Floor (log cfu cm-2) Squeegee (log cfu cm-2)
<1 <1 <1 3.53 <1 <1 <1 <1
<1 <1 <1 3.81 <1 <1 <1 <1
Cheese processing room
Environment air (beginning) (log cfu per plate) Environment air (middle) (log cfu per plate) Environment air (end) (log cfu per plate) Whey drainer (log cfu cm-2) Sealing machine (log cfu cm-2) Hose (log cfu cm-2) Stainless steel table (log cfu cm-2) Wall (log cfu cm-2) Floor (log cfu cm-2) Drain (external surface) (log cfu cm-2) Drain (internal surface) (log cfu cm-2) Processing tank (log cfu cm-2) Thermometer (log cfu per unit) Cheese whey (log cfu mL-1) Cheese curd (log cfu g-1) Cheese (collection day) 1 (log cfu g-1) Cheese (collection day) 2 (log cfu g-1) Cheese (collection day) 3 (log cfu g-1) Cheese (collection day) 4 (log cfu g-1) Cheese (collection day) 5 (log cfu g-1) Cheese (after storage) 1 (log cfu g-1) Cheese (after storage) 2 (log cfu g-1) Cheese (after storage) 3 (log cfu g-1) Cheese (after storage) 4 (log cfu g-1) Cheese (after storage) 5 (log cfu g-1)
<1 <1 <1 <1 <1 <1 <1 <1 <1 <1 2.99 <1 1.74 <1 <1 4.46 4.62 <1 <1 3.30 <1 <1 4.56 <1 <1
<1 <1 <1 <1 <1 <1 <1 <1 2.50 <1 4.97 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
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Pasteurisation room
ACCEPTED MANUSCRIPT Table 4 Regression coefficients for biofilm formation (log cfu cm-2) of E. faecium and E. faecalis. Enterococcus faecalis Regression p-value coefficient 7.35146 0.000346 1.19592 0.070893 -1.70483 0.039127 1.71043 0.019847 -1.30893 0.089989 -0.57000 0.475001
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Average Temperature Temperature2 Time Time2 Temperature × time
Enterococcus faecium Regression p-value coefficient 6.82147 0.000120 1.69864 0.007229 -1.28326 0.039551 1.51911 0.011327 -1.49452 0.023429 0.55500 0.358462
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Factors
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Table 5
Analysis of variance (ANOVA) for biofilm formation of E. faecium and E. faecalis on stainless steel. a Parameter Regression Error Total
Sum of squares 58.33 7.26 65.59
Df 4 6 10
Mean square 14.58 1.21 6.59
Fcalculated 12.04
p-value 0.0049
E. faecalis
Regression Error Total
56.95 12.20 69.15
4 6 10
14.23 2.03 6.91
7.00
0.019
Df, degrees of freedom. E. faecium: % explained variation (R2) = 88.93, Ftabulated 4;6;0,05 = 4.53; E. faecalis: % explained variation (R2) = 82.35, Ftabulated 4;6;0,05 = 4.53.
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Strain E. faecium
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Table 6
E. faecium and E. faecalis sessile cell counts on stainless steel surface as a function of time of contact and temperature and planktonic cell counts in UHT milk after 2 days of incubation. a
41
47
a
Planktonic cell counts E. faecium E. faecalis 3.25 ± 0.15 3.52 ± 0.27 ND ND ND ND 8.12 ± 0.06 8.06 ± 0.08 ND ND ND ND 9.20 ± 0.43 8.75 ± 0.21 ND ND ND ND ND ND ND ND ND ND 8.37 ± 0.24 8.72 ± 0.17 ND ND 8.37 ± 0.13 8.28 ± 0.25 ND ND
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27
Sessile cell counts E. faecium E. faecalis ND ND 1.08 ± 0.07 1.24 ± 0.25 1.58 ± 0.06 2.72 ± 0.13 ND ND 4.61 ± 0.17 5.91 ± 0.26 0.26 ± 0.24 0.49 ± 0.43 ND ND 6.90 ± 0.14 7.08 ± 0.19 6.69 ± 0.03 7.19 ± 0.19 6.89 ± 0.14 7.81 ± 0.13 6.13 ± 0.63 6.96 ± 0.26 5.89 ± 0.09 5.89 ± 0.12 ND ND 6.70 ± 0.12 7.22 ± 0.33 ND ND 6.15 ± 0.28 4.53 ± 0.99
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Time (days) 2 4 1.2 2 6.8 0 2 4 4 4 8 1.2 2 6.8 2 4
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Values (log cfu cm-2 for sessile cell counts, log cfu mL-1 for planktonic cell counts) are the average of
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Table 7
Reduction of E. faecium and E. faecalis after biofilm formation using different sanitizers. a Initial counts
E. faecium* E. faecalis**
6.04 ± 0.34 6.01 ± 0.36
Peracetic acid 300 mg L-1 1.57b.B ± 0.17 3.18a.A ± 0.87
Sodium hypochlorite 100 mg L-1 2.74a.A ± 0.47 1.40b.B ± 0.33
Chlorhexidine digluconate 400 mg L-1 1.68b.A ± 0.08 1.72b.A ± 0.01
Reduction was after biofilm formation at 25 °C for 3.5 days, and after 10 min contact using different
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Microorganism
sanitizers. Values are means (log cfu cm-2) ± standard deviation; means in the same row followed by the same lowercase superscript letter and means in the same column followed by the same uppercase
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superscript letter are not significantly different by the Tukey’s test (p ≥ 0.05).
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Figure 1.
S0958-6946(16)30360-0
DOI:
10.1016/j.idairyj.2016.12.005
Reference:
INDA 4122
To appear in:
International Dairy Journal
Received Date: 15 August 2016 Revised Date:
10 December 2016
Accepted Date: 10 December 2016
Please cite this article as: Rosado de Castro, M., da Silva Fernandes, M., Kabuki, D.Y., Kuaye, A.Y., Biofilm formation on stainless steel as a function of time and temperature and control through sanitizers, International Dairy Journal (2017), doi: 10.1016/j.idairyj.2016.12.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Biofilm formation on stainless steel as a function of time and temperature and
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control through sanitizers
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Marcília Rosado de Castroa, Meg da Silva Fernandesa*, Dirce Yorika Kabukib, Arnaldo
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Yoshiteru Kuayea
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Campinas (UNICAMP), Rua Monteiro Lobato 80, Cidade Universitária Zeferino Vaz,
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Campinas, SP, Brazil, CEP 13083-862
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Department of Food Technology and b Department of Food Science, University of
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*Corresponding author. Tel.: +55 19 35214011
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E-mail address: [email protected]
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______________________________________________________________________
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ABSTRACT
29 Enterococcus spp. contamination was screened from a Minas Frescal cheese processing
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line. Biofilm formation of Enterococcus faecium and Enterococcus faecalis isolates was
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evaluated and the effect of sanitization procedures in the control of these biofilms was
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investigated. Enterococcus spp. were detected in raw milk, milk machine, door handle,
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floor, drain, thermometer, and Minas Frescal cheese. Biofilm formation on stainless
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steel was modelled as a function of time (0, 1.2, 4, 6.8, and 8 days) and temperature (7,
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13, 27, 41, and 47 °C) using response surface methodology. The model showed that E.
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faecium biofilms were formed from 1 to 8 days at 12 to 47 °C, while E. faecalis
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biofilms were formed from 1 to 8 days at 10 to 43 °C. None of the sanitizers (sodium
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hypochlorite 100 mg L-1, peracetic acid 300 mg L-1, and chlorhexidine digluconate 400
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mg L-1) was able to completely eliminate the biofilms.
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1.
Introduction
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Minas Frescal cheese is the most consumed cheese in Brazil (ABIQ, 2014) and is considered the only genuine national cheese. The simple production technology
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attracts the interest of small and large factories. Minas Frescal is defined as a fresh
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cheese, produced with pasteurised whole and semi-slimmed milk by enzymatic
50
coagulation (chymosin). It has a high moisture content (average 55%), 17–19% fat,
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1.5% salt, pH range of 5.0 to 5.3, and is lightly pressed or not (Peresi et al., 2001).
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These characteristics allied to the processing conditions favour the proliferation of
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various bacteria, including Enterococcus.
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The prevalence of Enterococcus in cheese may be associated with the ability of this genus to resist pasteurisation and cooling temperatures, and a wide range of pH and
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salinity (Giraffa, 2003). The presence of Enterococcus in food is controversial, because
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although they are responsible for desirable technological characteristics in some foods,
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such as rennet cheese, they can cause foodborne diseases due to their pathogenicity
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(Foulquié-Moreno, Sarantinopoulos, Tsakalidou, & De Vuyst, 2006). Enterococcus spp.
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strains show biochemical and biotechnological properties, such as proteolytic, lipolytic,
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esterolytic activities, besides the use of citrate, which promotes texture and typical taste
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in several types of cheeses such as Manchego, Mozzarella, Monte Veronese, Fontina,
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Caprino, Serra, Venaco and Comte (Giraffa, 2003). However, proteolysis and lipolysis
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are undesirable in Minas Frescal cheese, once the enzymes produced during
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microorganism growth cause degradation of fat and protein, change- aroma and flavour,
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reduce shelf life of the products, and decrease the production yield (Sørhaug &
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Stepaniak, 1997). In addition, Enterococcus are considered opportunistic nosocomial
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pathogens that cause human infections especially in immune-compromised individuals.
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enterococci isolated from food (Cariolato, Andrighetto, & Lombardi, 2008; Fernandes
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et al., 2015a; Gomes et al., 2008; Kasimoglu-Dogru, Gencay, & Ayaz, 2010). Great
72
concern over the presence of enterococci in the food is due to transfer of virulence
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genes among strains (Eaton & Gasson, 2001). This gene transfer capability can occur in
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the gastrointestinal tract of humans due to consumption of food contaminated with
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enterococci through bacterial conjugation (Çitak, Yucel, & Orhan, 2004; Gelsomino,
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Vancanneyt, Condon, Swings, & Cogan, 2001).
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One of the main reasons accounting for the existence of enterococci in dairy products is their ability to adhere and form biofilms on food contact surfaces
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(Fernandes, Kabuki, & Kuaye, 2015b; Jahan & Holley, 2014). Biofilms are complex
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and structured microbial communities, surrounded by a matrix of extracellular
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polymeric substances (EPS), adhered to each other and/or to a surface or interface
82
(Costerton, Lewandowski, Caldwell, Korber, & Lappin-Socott, 1995), where the
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microorganisms exhibit distinct phenotypes, metabolism, physiology and gene
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transcription. These biofilms are a major focus of contamination affecting food quality
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and safety (Simões, Simões, & Vieira, 2010). Therefore, it is of foremost importance to
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gain insights on factors influencing Enterococcus spp. biofilm to dairy processing
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surfaces.
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One of the strategies to control biofilm formation is the application of effective
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sanitation procedures by combined use of detergents and sanitizers. The sanitizers are
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responsible for the reduction of spoilage microorganisms and elimination of pathogens
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to safe levels. Sodium hypochlorite and peracetic acid are sanitizers widely used in the
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food industry. Chlorhexidine is probably the most widely used biocide in antiseptic
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products, in particular in handwashing and oral products but also as a disinfectant and
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preservative (McDonnell & Russell, 1999). It is noteworthy that the effectiveness of
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sanitizers is usually assessed by laboratory tests (suspension and use-dilution tests),
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without considering the production of exopolysaccharides, compounds mainly
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responsible for the protection of microbial biofilms against the action of sanitizers.
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The objective of this study was to evaluate the ability of E. faecium and E.
faecalis isolated from a Minas Frescal cheese processing line to form biofilms, using
response surface methodology as a function of time and temperature. The efficiency of
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sanitizers to control these biofilms was also investigated.
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2.
Material and methods
2.1.
Sampling
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A total of 156 samples of raw and pasteurised milk, whey, cheese curd,
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equipment and utensil surfaces, environmental air, and Minas Frescal cheeses were
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collected on two visits in August and October 2010 in a cheese processing line in a
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dairy industry in São Paulo State - Brazil.
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2.1.1. Raw material and food samples
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At each visit were collected: four samples of raw milk type A (1000 mL), one
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pasteurised milk (1000 mL), three curd (350 g) and three whey (1000 mL). In addition,
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10 samples (400 g each) of the packaged cheese from the same batch were sampled.
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From these, 5 samples were analysed on the same day of collection, and the other five
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units were analysed on the last day of the shelf life as reported on the label (15 days).
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The cheeses analysed after 15 days were stored under refrigeration at 4 °C.
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2.1.2. Environmental surfaces and air A total of 47 samples per visit from the surfaces of equipment, utensils, and facilities were analysed. Samples were collected after routine cleaning process. Depending on the surface characteristics, two collection methods were used:
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sponge contact method or swab contact technique. The surface characteristic is
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dependent of accessibility and physical design surface configuration (curve, plane) of
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the utensils or/and equipment. The sponge contact method consists of a sterilised
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cellulose sponge (Nasco, Wisconsin, USA) pre-moistened in 20 mL of 0.1% peptone
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water containing 0.5% sodium thiosulphate, according to the recommendation of
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American Public Health Association - APHA (Evancho, Sveum, Moberg, & Frank,
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2001). Surfaces evaluated by sponge method were: stainless steel table, shelf, sealing
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machine, door handle, hose, floors, and walls (milking room, raw milk reception room,
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pasteurisation room, cheese processing room, cold chamber, and cold room at 9 °C for
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storage of utensils used in cheese production), drains (cheese processing room and
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utensils storage room), squeegee, cheese processing tanks, raw milk tank, and utensils
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(moulds, knife to cut the curd, milk stirrer, and thermometer) used in cheese processing.
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The swab technique was used on the milking machine surface, as previously reported
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(Evancho et al., 2001).
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The areas were defined using sterile disposable 50 or 100 cm-2 moulds,
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according to the equipment and utensils configuration. The samples were packed in
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cooler with ice for transport to the Hygiene and Legislation Laboratory, and the
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analyses were carried out within 24 h after collection.
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Environmental air samples were collected in different places of the factory (milking room, raw milk reception, pasteurisation room, cheese production room, cold
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chamber, and cold room at 9 °C). Air sampling was performed using the sedimentation
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method, according to APHA (Evancho et al., 2001).
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2.2.
Isolation and identification of Enterococcus spp.
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The raw material and food samples (25 g or 25 mL) were diluted 1:10 in 0.1% peptone water (Difco, Becton, Dickinson and Company, Sparks, USA), plated on
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Kenner Faecal (KF) Streptococcus Agar (Difco) supplemented with 1% of 2,3,5-
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triphenyl-tetrazolium chloride – TTC (Vetec Química, Rio de Janeiro, Brazil) and
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incubated at 45 °C for 48 h (Hartman, Deibel, & Sieverding, 2001). The environmental
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surface samples were subjected to serial decimal dilution in 0.1% peptone water, plated
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on KF agar and incubated at 45 °C for 48h. The biochemical analyses (catalase
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production, growth at 10 °C and 45 °C, growth in BHI broth containing 6.5% NaCl,
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growth in BHI broth at pH 9.6, and growth on bile-esculin agar) for the identification of
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the genus Enterococcus were performed according to methodology specified by APHA
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(Hartman et al., 2001).
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Genetic identification at species level was done by polymerase chain reaction (PCR). Genomic DNA from pure cultures was extracted according to Furrer, Candrian,
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Hoefelein, and Luethy (1991). The PCR reaction was carried out as previously
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described (Dutka-Malen, Evers, & Courvalin, 1995).
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2.3.
Evaluation of proteolytic and lipolytic presence in Enterococcus spp.
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All isolates of the genus Enterococcus were screened for protease (Hartman et al., 2001) and lipase enzymes (Frank & Yousef, 2004).
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The protease enzyme was evaluated in PCA (Difco) added with 1% reconstituted skim milk, followed by incubation at 28 °C for 72 h. Confirmation was
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obtained by the formation of a translucent halo around the colonies after addition of 0.2
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mL of 10% acetic acid for 1 min. Bacillus cereus NCTC 1143 was used as positive
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control.
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The lipase enzyme was evaluated in Spirit Blue Agar (Difco) added with lipase reagent, followed by incubation at 32 °C for 48 h. Confirmation was obtained by the
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formation of a light colour and/or an intense blue colour halo around the colony.
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Staphylococcus aureus ATCC 6539 was used as positive control.
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2.4.
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Assessment of biofilm formation
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2.4.1. Central composite rotational design
To evaluate the biofilm formation, a central composite rotational design (CCRD)
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with two factors (time of contact and exposure temperature) was performed according to
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the methodology proposed by Rodrigues and Iemma (2005). Three replicates of each test were performed at 7; 13; 27; 41; and 47 °C, and
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times of contact 0, 1.2; 4; 6.8; and 8 days. Time zero corresponded to the test carried out
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immediately after immersion of the coupons into the vial containing milk and the
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bacterial suspension. Table 1 shows the relationship between temperature and time of
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contact for all tests.
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The responses (cfu cm-2) were adjusted by using the quadratic polynomial
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regression model (log cfu cm-2 = b0+ b1T + b2 T2 + b3t + b4t2 + b5Txt), where b0 to b5
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correspond the coefficients of the model, T is temperature, and t is the time of contact.
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Two experimental designs were performed: the first consisting of a mix with
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These strains were isolated in this study and selected from different samples. The strains
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were selected for possess the ability to produce lipases and proteases and present
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pathogenic profile for carry virulence and antibiotic resistance genes (these analyses
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were performed in these strains as described by Fernandes et al., 2015a) (Table 2).
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2.4.2. Biofilm formation
Biofilm formation was evaluated using AISI 304#4 stainless steel coupons
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(0.366 µm roughness, 10 mm × 10 mm × 1 mm). Before use, the coupons were
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sanitized (Fernandes et al., 2015b), suspended by a polyamide yarn, packed in glass
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vial, and sterilised at 121 °C for 15 min.
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Whole UHT milk (Jussara, Franca, Brazil) was used as culture medium for the biofilms formation, which was previously evaluated for aerobic counts (Laird, Gambrel-
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Lenarz, Scher, Graham, & Reddy, 2004) and spores (Frank & Yousef, 2004). The
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counts were below the detection limit for aerobic counts and spores method.
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For each experiment, each bacterial strain was separately inoculated in brain heart infusion (BHI) broth (Difco) at 35 °C for 24 h. Then, 1 mL of each activated
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bacterial strain was added in a sterile flask and vortexed (2800 rpm) for 1 min. Serial
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decimal dilutions were performed until the suspension reached a concentration of
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approximately 1×104 cfu of microorganisms per mL.
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For all tests, immediately after culture inoculation, counts from UHT milk were
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performed on plate count agar (PCA) (Difco) incubated at 35 °C for 48 h as a control
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test. The pool of microorganisms (1×106 cfu mL-1) was inoculated in milk in sterile
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flasks (100 mL) to reach approximately 1× 104 cfu mL-1, containing 4 suspended
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coupons. The flasks were incubated at different temperatures (7, 13, 27, 41, and 47 °C).
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inoculated with the same cultures used initially. The period of 48 h for replacement of
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culture medium in containers was determined according to the current raw milk quality
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regulation established by Federal Brazilian Inspection Service - the maximum time
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between milking and receiving the milk in the establishment where it will be processed
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(Brasil, 2002). The method was reported by Fernandes, Fujimoto, Schneid, Kabuki, and
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Kuaye (2014).
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coupons. The biofilm formation on the coupons was evaluated by plate counting
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technique for all experiments (Fernandes et al., 2015b). In such technique, at each time
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and temperature of contact, two coupons were separately immersed into 10 mL of 0,1%
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peptone water for 1 min to remove the planktonic cells. Then, each coupon was
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immersed in 5 mL of the same solution and vortexed (2800 rpm) for 2 min to remove
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the sessile cells (Fernandes et al., 2015b). The resulting solution was serially diluted in
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0.1% peptone water and plated onto PCA agar (Difco), and the plates were incubated at
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35 °C for 48 h.
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2.5.
Statistical Analysis
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The statistical analyses were performed using the STATISTICA 7.0 software
(Statsoft, Tulsa, USA).
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2.6.
Experimental verification of the model fitting
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The same experimental designs (I: five E. faecalis strains; II: five E. faecium strains) were conducted in three replicates under conditions not evaluated before (after 3
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days of contact at 25 °C) to test the efficiency of the model. Coupons preparation and
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incubation, and the determination of biofilm formation were performed as described in
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Section 2.4.2.
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2.7.
Efficiency of sanitizers for removal biofilms
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For this assay, sodium hypochlorite solution 100 mg L-1 total chlorine pH 9.4 (Super Candida Indústria Anhembi SA, Osasco, Brazil), peracetic acid 300 mg L-1 pH
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2.8 (Divosan Forte, Johnson Diversey, Sintra, Portugal), and chlorhexidine digluconate
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400 mg L-1 pH 6.4 (Neobiodine, Neobrax Ltda, Barretos, Brazil) were used, according
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to the instructions of the manufacturers.
Three replicates were performed for each treatment and in each replicate two
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coupons were used for each sanitation step and two for the control. The control coupons
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did not receive sanitizer, and their counts were used to calculate the number of decimal
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reductions due to the sanitization step. The biofilm formation was analysed according to
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the method described in Section 2.4, and the combination of temperature and time of
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contact was determined according to the optimum point of the experimental design (3.5
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days of contact at 25 °C), for the two experimental designs (I: five E. faecalis strains; II:
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five E. faecium strains).
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After incubation, the coupons were rinsed in 0.1% peptone water for removal of
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planktonic cells, and then immersed in 10 mL of each sanitizing solution for 10 min at
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25 °C. To inactivate the sanitizing effect, the coupons were transferred to 10 mL of 1%
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sodium thiosulphate or 0.5% Tween 80 for 10 min, for sodium hypochlorite/peracetic
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neutralising solution, and immersed in 5 mL of 0.1% peptone solution and vortexed for
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two minutes to remove the sessile cells (Fernandes et al., 2015b). For the control
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coupons, the same procedures were repeated except the steps of sanitization and
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neutralisation. The adhered cells were enumerated in PCA incubated at 35 °C for 48 h.
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3.
Results and discussion
3.1.
Enterococcus spp. in Minas Frescal cheese processing plant
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The average Enterococcus spp. count in raw milk was 2.74 log cfu mL-1 (Table
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3), which is relatively low when compared with those observed by other authors in
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Brazil (Fernandes et al., 2015a; Tebaldi, Oliveira, Boari, & Piccoli, 2008). In fact, the
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good manufacture conditions such as mechanical milking, maintaining and storage of
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raw milk at 4 °C, and cheese production in the same place of milking justified these
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results.
Enterococcus spp. counts in pasteurised milk were <1 log cfu mL-1,
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demonstrating the effectiveness of the thermal process to reduce the level of this
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microorganism, despite it does not always result in complete elimination of these
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bacteria in milk and dairy products (Foulquié-Moreno et al., 2006).
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Enterococcus spp. were detected in surfaces of milking machine, door handles,
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floor, and thermometer (Table 3). The milking machine had the highest counts, with
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mean values of 5 log cfu cm-2. Although it is located outside the processing
292
environment, its hygienic-sanitary control is critical, thus it represents a focus of
293
Enterococcus contamination in raw milk.
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The door handles of the cold chamber and utensils storage room (9 °C) had
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counts of up to 3.8 log cfu cm-2, representing a major cross-contamination, because they
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are near to the cheese processing room, with constant contact with handlers.
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In the first sampling, the presence of Enterococcus spp. was also detected in the thermometer used to control the process. Even though, it was found a relatively low
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count (1.74 log cfu per unit), this fact can be considered a risk due to the contact with
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pasteurised milk at danger temperature zone that can allow bacteria growth.
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Enterococcus spp. has been detected in the air from milking room and milk reception
302
room (1 cfu per plate), but it was not detected in the air of the processing room.
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Enterococcus spp. are persistent micro-organisms of the environment, including
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airborne. According to Muzslay, Moore, Turton, and Wilson (2013), unrecognised
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colonisation and/or the aerosolisation of enterococci together with inadequate cleaning
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can lead to heavy, widespread, and persistent environmental contamination.
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Enterococcus spp. strains were detected in only 20% (4/20) of the cheese
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samples (Table 3). Among the positive samples, three samples was analysed on the day
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of collection, and one sample after 15 days of storage at 4 °C, with counts ranging from
310
<1 log cfu g-1 to 4.62 log cfu g-1. Thus, the samples with positive results for
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Enterococcus spp. probably were contaminated individually after processing, through
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cross-contamination by equipment and utensils, as formerly mentioned.
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Of the 155 isolates confirmed as Enterococcus genus by biochemical tests 76%
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(120/155) were identified as E. faecalis and 24% (35/155) as E. faecium by PCR. E.
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faecalis was the predominant species observed in the raw milk (76%, 85/112), Minas
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Frescal cheese (100%, 12/12), and processing environment (75%, 23/31). The
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prevalence of these two species has been reported for different types of cheese
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(Devriese, Pot, Damme, Kersters, & Haesebrouck, 1995; Fernandes et al., 2015a;
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Gomes et al., 2008).
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3.2.
Lipolytic and proteolytic presence in Enterococcus spp.
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Of the 155 Enterococcus isolates from cheese processing, 52% (81/155) were
positive for proteases and lipases. From raw milk samples, the proteolytic activity was
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observed in 17 strains of E. faecium isolates, 47 strains of E. faecalis, while 13 strains
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of E. faecium and 50 strains of E. faecalis showed lipolytic activity. The production of
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these enzymes may impact negatively on the processed milk, since proteases act on
328
casein, causing bitter in dairy products and lipases confer the rancid taste of milk by the
329
release of short-chain fatty acids (Chen, Daniel, & Coolbear, 2003). Enterococcus
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producers of proteases and lipases were observed for the different samples (raw milk,
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environmental samples, and Minas fresh cheese). The production of these enzymes is
332
dependent on each strain, due to the great genetic diversity among the strains, and only
333
a few are responsible for producing these enzymes (Nörnberg, Tondo, & Brandelli,
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2009).
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3.3.
Modelling of biofilm formation by Enterococcus faecium and Enterococcus
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faecalis on stainless steel
A response surface approach has been applied to demonstrate the influence of
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contact time and temperature and their interactions on the biofilm formation of
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Enterococcus spp. on stainless steel surface. All statistical analyses were performed at a
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5% significant level (p<0.05). However, due to the large variability involving
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microorganisms, the parameters were considered significant at p-values lower than 10%
344
(p<0.1) (Table 4) as described by Rodrigues and Iemma (2005). Thus, only the term
345
associated with the interaction (temperature × time) was not significant for both species
346
evaluated.
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The analysis of variance for the experiment 1 (E. faecium) indicated that the
model was significant (p<0.05), with Fcalculated value 2.65 times greater than the Ftabulated
349
and coefficient of determination (R2) of 0.88, which validated the model, demonstrating
350
that it fits well to the experimental data (Table 5).
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The model was also significant (p<0.05) for the experiment 2 (E. faecalis). The Fcalculated was 1.54 times greater than the Ftabulated and the coefficient of determination
353
(R2) was 0.82 (Table 5). With these results, mathematical models have been developed
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with the encoded variables that describe the process of biofilm formation of E. faecium
355
(Equation 1) and E. faecalis (Equation 2) as a function of time of contact (t) and
356
temperature (T) (equations for the time intervals 0–8 days and temperatures from 7 to
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47 °C).
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Log cfu cm-2 = 7.35146 + 1.19592T – 1.70483T2 + 1.71043t – 1.30893t2
(2)
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The predicted values for the biofilm formation of E. faecium and E. faecalis on
364
stainless steel under different times and temperature conditions in food industries can be
365
determined from Equations 1 and 2.
366 367
The lower temperature used in this study was 7 °C, since the target bacteria are able to grow at refrigeration temperatures. The intermediate temperatures were
15
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considered as those that allow microorganism growth, and the highest temperature was
369
determined according to the bacteria growth range. It is noteworthy that analysed
370
temperatures (7–47 °C) are often found in dairy factories.
371
Table 6 shows the influence of contact time and temperature on the biofilm formation (sessile cells) by E. faecium (Experiment 1) and E. faecalis (Experiment 2).
373
In addition, Table 6 shows the counts of these microorganisms (planktonic cells) in milk
374
after 2 days of incubation for each temperature, for comparison.
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The lowest E. faecium and E. faecalis sessile cells counts (1.08 log cfu cm-2 and
376
1.24 log cfu cm-2, respectively) were observed after 4 days of contact at 7 °C, while the
377
highest counts were observed after 4 days of contact at 27 °C for E. faecium and E.
378
faecalis (6.90 log cfu cm-2 and 7.81 log cfu cm-2, respectively) (Table 6). After 8 days at
379
27 °C, E. faecium and E. faecalis sessile cells counts declined to 6.13 log and 6.96 log
380
cfu cm-2, respectively, which can represent the phase of detachment of the biofilm
381
fragments, and can lead to food contamination. It is important to note that all isolates
382
exhibited virulence genes and resistance to various antibiotics (Table 2). In addition,
383
these biofilm fragments can colonise other regions, resulting in new biofilms (Simões et
384
al., 2010).
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In this study, milk was previously inoculated with approximately 2 log cfu mL-1 of E. faecium or E. faecalis planktonic cells. Both Enterococcus species were able to
387
multiply in milk at different temperatures (7 to 47 °C, Table 6). The lowest E. faecium
388
(3.25 log cfu mL-1) and E. faecalis (3.52 log cfu mL-1) counts were observed at 7 °C,
389
while the highest E. faecium (9.20 log cfu mL-1) and E. faecalis counts (8.75 log cfu
390
mL-1) were observed at 27 °C. One of the aspects that influence the biofilm formation is
391
the concentration of microorganisms in the medium. The higher population of
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microorganisms in milk, greater the biofilm formation on stainless steel (Peña et al.,
393
2014).
394
Analysis of response surfaces and contour curves generated by the model (Fig. 1) allow checking the optimum region for biofilm formation. E. faecium isolates
396
showed an optimum range to form biofilms from 3.5 to 7.2 days and 25.5 to 47 °C (Fig.
397
1A). For E. faecalis, this range was 3.5 days to 8 days of contact, from 21 to 43 °C (Fig.
398
1B).
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Enterococcus spp. have the ability to multiply in a wide temperature range (5–50
400
°C), with optimum temperatures between 35 and 43 °C (Fisher & Phillips, 2009). In this
401
study, the optimum temperatures for biofilm formation were between 21 and 47 °C,
402
close to the ideal for this microorganism. These results are in agreement with Meira,
403
Barbosa, Athayde, Siqueira-Junior, and Souza (2012) who observed more intense
404
biofilm formation at the optimal growth temperature of microorganisms.
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405
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It is noteworthy that the ideal temperature range for biofilm formation of E. faecium and E. faecalis is encountered in Minas cheese manufacture. The refrigerated
407
pasteurised milk is pumped into the processing tank and heated to 35–37 °C and then
408
rennet is added to promote the coagulation of milk. During cheese processing, this
409
temperature range remains for about 3 h, enough time for microorganism growth, with
410
possible biofilm formation, compromising the food safety.
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3.4.
Experimental verification of the models
413 414
A condition that has not been evaluated in the central composite design was chosen to
415
verify if the models found are able to describe the actual results. The temperature of 25
416
°C was chosen, since most samples were collected at this temperature. The time of 3.5
17
ACCEPTED MANUSCRIPT days was chosen because it is an intermediate time not used in the construction of the
418
model. The experiment 1 (E. faecium) showed a deviation 0.23 cfu cm-2 log (3.8%),
419
with predicted value of 5.81 log cfu cm-2 and experimental value of 6.04 log cfu cm-2.
420
Experiment 2 (E. faecalis) had predicted value (6.36 log cfu cm-2) higher than the
421
experimental value (6.01 log cfu cm-2).
422 423
3.5.
Assessment of the effectiveness of sanitizers in control of biofilms
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In this study, we have investigated the efficacy of sodium hypochlorite (100 mg L-1), peracetic acid (300 mg L-1), and chlorhexidine digluconate (400 mg L-1) against
427
biofilm formed by E. faecium and E. faecalis on stainless steel surface. As shown in
428
Table 7, the sanitizers in the concentrations recommended by the manufacturers reduced
429
the cells count of E. faecium and E. faecalis biofilms. Sodium hypochlorite reduced
430
more the number of cells of E. faecium biofilms (reduction of 2.74 log cfu cm-2) than
431
peracetic acid and chlorhexidine digluconate (reduction of 1.57 and 1.68 log cfu cm-2,
432
respectively). For the cell count of E. faecalis biofilms (6.01 cfu cm-2), peracetic acid
433
presented better performance (reduction of 3.18 log cfu cm-2) than sodium hypochlorite
434
and chlorhexidine digluconate (reduction of 1.40 and 1.72 log cfu cm-2, respectively).
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Biofilms are formed by aggregates of microorganism cells, EPS matrix, and
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organic matter from food. This structure hinders the action of sanitizers, thus the
437
surviving microorganisms can develop rapidly, especially in the presence of residues,
438
and contaminate the processed food (Simões et al., 2010). Therefore, bacteria removal
439
from contact surfaces can only be achieved by adopting combined actions, such as the
440
application of detergent prior to the sanitizing agent or a product that combines a
441
detergent with a sanitizer (Fernandes et al., 2015b). Fernandes et al. (2015a,b) showed
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ACCEPTED MANUSCRIPT that mono-species biofilm (E. faecium and E. faecalis) and multi-species biofilm (with
443
Listeria monocytogenes) were resistant to different sanitizers, with biguanide being the
444
less efficient sanitizer. In these studies, peracetic acid was the most efficient sanitizer, as
445
also evidenced in other studies (Meira et al., 2012; Park et al., 2012). The high
446
efficiency of peracetic acid to microbial biofilms is due to its ability of not reacting with
447
organic matter, besides being a strong oxidant. Organic and inorganic matter can
448
inactivate compounds based on chlorine and chlorhexidine. Therefore, they are often
449
unable to eliminate microbial biofilms (Bridier, Briandet, Thomas, & Dubois-
450
Brissonnet, 2011).
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4.
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Dissemination of Enterococcus was observed in raw milk, environmental samples, and Minas Frescal cheese. The milking machine stood out with the highest
456
Enterococcus counts, along with door handles, which showed persistent contamination
457
during the collections.
The results of response surface methodology showed that E. faecium and E.
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faecalis were able to form biofilms on stainless steel surface at times and temperatures
460
ranging from 1 to 8 days of contact and 12 to 47 °C for the former, and 1 to 8 days of
461
contact and 10 to 43 °C for the latter; these are temperatures commonly used throughout
462
the Minas cheese processing. Therefore, the possible presence of these bacteria in
463
environments or surfaces under abusive conditions of temperature and time, like the
464
ones used in this study, should be prevented in Frescal Minas cheese processing aimed
465
at food safety.
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In addition, none of the tested sanitizers has completely eliminated the biofilms, evidencing the difficulty of surfaces’ sanitization after the biofilm formation.
468 469
Acknowledgements
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470 This work was supported by the Conselho Nacional de Desenvolvimento
472
Científico e Tecnológico (CNPq) – Process n.140334/2009-2. The authors are indebted
473
to Fundação André Tosello for donating the Enterococcus faecalis ATCC 7080.
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Figure legend
Fig. 1. Surface response and contour curves as a function of exposure temperature and time of contact for biofilm formation of Enterococcus faecium (A) and Enterococcus faecalis (B) on
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stainless steel.
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Table 1
Central composite rotational design for the relationship between the variables exposure temperature and
1 2 3 4 5 6 7 8 9 10 11
Temperature ( °C) 13 13 41 41 7 47 27 27 27 27 27
2 -1 +1 -1 +1 0 0 -1.41 +1.41 0 0 0
Time (days) 1.2 6.8 1.2 6.8 4 4 0 8 4 4 4
Variable 1, exposure temperature; Variable 2, time of contact; -1, lower level; 0, central point; +1,
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higher level; -1.41 and +1.41, axial points.
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Variable 1 -1 -1 +1 +1 -1.41 +1.41 0 0 0 0 0
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time of contact. a
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Table 2
Origin of Enterococcus faecium and Enterococcus faecalis strains used in the evaluation of biofilm formation. a
E. faecium E84 E. faecium E106 E. faecium E113
Virulence genes +
Door handle (cold chamber) Raw milk Floor (cheese room)
ERI, GEN, STREP, TET RA, TET ERI, STREP, TET ERI, GEN, RA, STREP, TET ERI, RA
esp, efaA, ace, vanB
Door handle (cheese processing room) Floor (milking room)
E. faecium E120 Experiment 2 E. faecalis E2 E. faecalis E15
Raw milk Minas Frescal cheese Thermometer
E. faecalis E110
Door handle (cheese processing room) Milking machine
GEN, STREP ERI, GEN, RA, STREP, TET GEN, STREP, TET ERI
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E. faecalis E40
E. faecalis E94
a
Antibiotic resistance
gelE, esp, efaA, ace, vanB esp, vanB
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Experiment 1 E. faecium E42
Source
esp, ace
esp, ace, vanB
esp, vanB gelE, esp, efaA, ace, vanB
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Microorganism
GEN, STREP, TET
gelE, esp, efaA, vanB
esp, efaA, ace gelE, esp, efaA, ace, vanB
All isolates were positive for proteolytic and lipolytic activity. Abbreviations are: AMP, ampicillin (10
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µg); CLO, chloramphenicol (30 µg); ERI, erythromycin (15 µg); GEN, gentamicin (120 µg); NOR, norfloxacin (10 µg); RA, rifampicin (5 µg); STREP, streptomycin (300 µg); TEC, teicoplanin (30 µg);
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TET, tetracycline (30 µg); VAN, vancomycin (30 µg).
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Table 3
Enterococcus spp. counts in the raw material, processing environment, and Minas Frescal cheese. Source
Raw milk reception
Environment air (log cfu per plate) Milking machine (log cfu per unit) Wall (log cfu cm-2) Floor (log cfu cm-2) Environment air (log cfu per plate) Wall (log cfu cm-2) Floor (log cfu cm-2) Raw milk reception tank 1 (log cfu cm-2) Raw milk reception tank 2 (log cfu cm-2) Raw milk 1 (log cfu mL-1) Raw milk 2 (log cfu mL-1)
2nd <1 5.41 <1 <1 <1 <1 <1 <1 <1 4.61 1.57
<1 <1 <1 <1
<1 <1 <1 <1
<1 <1 2.07 <1 <1
<1 <1 2.20 <1 <1
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Milking room
Collection 1st <1 4.89 <1 <1 1 <1 <1 <1 <1 2.32 2.49
Environment air (log cfu per plate) Wall (log cfu cm-2) Floor (log cfu cm-2) Pasteurised milk (log cfu mL-1)
Cold chamber
Environment air (log cfu per plate) Shelf (log cfu cm-2) Door handle (log cfu per unit) Wall (log cfu cm-2) Floor (log cfu cm-2)
Cold room at 9 °C for storage of utensils used in cheese production
Environment air (log cfu per plate) Moulds (log cfu per unit) Knife to cut the curd (log cfu cm-2) Door handle (log cfu per unit) Milk stirrer (log cfu per unit) Wall (log cfu cm-2) Floor (log cfu cm-2) Squeegee (log cfu cm-2)
<1 <1 <1 3.53 <1 <1 <1 <1
<1 <1 <1 3.81 <1 <1 <1 <1
Cheese processing room
Environment air (beginning) (log cfu per plate) Environment air (middle) (log cfu per plate) Environment air (end) (log cfu per plate) Whey drainer (log cfu cm-2) Sealing machine (log cfu cm-2) Hose (log cfu cm-2) Stainless steel table (log cfu cm-2) Wall (log cfu cm-2) Floor (log cfu cm-2) Drain (external surface) (log cfu cm-2) Drain (internal surface) (log cfu cm-2) Processing tank (log cfu cm-2) Thermometer (log cfu per unit) Cheese whey (log cfu mL-1) Cheese curd (log cfu g-1) Cheese (collection day) 1 (log cfu g-1) Cheese (collection day) 2 (log cfu g-1) Cheese (collection day) 3 (log cfu g-1) Cheese (collection day) 4 (log cfu g-1) Cheese (collection day) 5 (log cfu g-1) Cheese (after storage) 1 (log cfu g-1) Cheese (after storage) 2 (log cfu g-1) Cheese (after storage) 3 (log cfu g-1) Cheese (after storage) 4 (log cfu g-1) Cheese (after storage) 5 (log cfu g-1)
<1 <1 <1 <1 <1 <1 <1 <1 <1 <1 2.99 <1 1.74 <1 <1 4.46 4.62 <1 <1 3.30 <1 <1 4.56 <1 <1
<1 <1 <1 <1 <1 <1 <1 <1 2.50 <1 4.97 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
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Pasteurisation room
ACCEPTED MANUSCRIPT Table 4 Regression coefficients for biofilm formation (log cfu cm-2) of E. faecium and E. faecalis. Enterococcus faecalis Regression p-value coefficient 7.35146 0.000346 1.19592 0.070893 -1.70483 0.039127 1.71043 0.019847 -1.30893 0.089989 -0.57000 0.475001
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Average Temperature Temperature2 Time Time2 Temperature × time
Enterococcus faecium Regression p-value coefficient 6.82147 0.000120 1.69864 0.007229 -1.28326 0.039551 1.51911 0.011327 -1.49452 0.023429 0.55500 0.358462
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Factors
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Table 5
Analysis of variance (ANOVA) for biofilm formation of E. faecium and E. faecalis on stainless steel. a Parameter Regression Error Total
Sum of squares 58.33 7.26 65.59
Df 4 6 10
Mean square 14.58 1.21 6.59
Fcalculated 12.04
p-value 0.0049
E. faecalis
Regression Error Total
56.95 12.20 69.15
4 6 10
14.23 2.03 6.91
7.00
0.019
Df, degrees of freedom. E. faecium: % explained variation (R2) = 88.93, Ftabulated 4;6;0,05 = 4.53; E. faecalis: % explained variation (R2) = 82.35, Ftabulated 4;6;0,05 = 4.53.
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Strain E. faecium
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Table 6
E. faecium and E. faecalis sessile cell counts on stainless steel surface as a function of time of contact and temperature and planktonic cell counts in UHT milk after 2 days of incubation. a
41
47
a
Planktonic cell counts E. faecium E. faecalis 3.25 ± 0.15 3.52 ± 0.27 ND ND ND ND 8.12 ± 0.06 8.06 ± 0.08 ND ND ND ND 9.20 ± 0.43 8.75 ± 0.21 ND ND ND ND ND ND ND ND ND ND 8.37 ± 0.24 8.72 ± 0.17 ND ND 8.37 ± 0.13 8.28 ± 0.25 ND ND
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27
Sessile cell counts E. faecium E. faecalis ND ND 1.08 ± 0.07 1.24 ± 0.25 1.58 ± 0.06 2.72 ± 0.13 ND ND 4.61 ± 0.17 5.91 ± 0.26 0.26 ± 0.24 0.49 ± 0.43 ND ND 6.90 ± 0.14 7.08 ± 0.19 6.69 ± 0.03 7.19 ± 0.19 6.89 ± 0.14 7.81 ± 0.13 6.13 ± 0.63 6.96 ± 0.26 5.89 ± 0.09 5.89 ± 0.12 ND ND 6.70 ± 0.12 7.22 ± 0.33 ND ND 6.15 ± 0.28 4.53 ± 0.99
SC
13
Time (days) 2 4 1.2 2 6.8 0 2 4 4 4 8 1.2 2 6.8 2 4
M AN U
Temperature (°C) 7
Values (log cfu cm-2 for sessile cell counts, log cfu mL-1 for planktonic cell counts) are the average of
AC C
EP
TE D
three repetitions ± standard deviation; ND, not determined.
ACCEPTED MANUSCRIPT
Table 7
Reduction of E. faecium and E. faecalis after biofilm formation using different sanitizers. a Initial counts
E. faecium* E. faecalis**
6.04 ± 0.34 6.01 ± 0.36
Peracetic acid 300 mg L-1 1.57b.B ± 0.17 3.18a.A ± 0.87
Sodium hypochlorite 100 mg L-1 2.74a.A ± 0.47 1.40b.B ± 0.33
Chlorhexidine digluconate 400 mg L-1 1.68b.A ± 0.08 1.72b.A ± 0.01
Reduction was after biofilm formation at 25 °C for 3.5 days, and after 10 min contact using different
RI PT
a
Microorganism
sanitizers. Values are means (log cfu cm-2) ± standard deviation; means in the same row followed by the same lowercase superscript letter and means in the same column followed by the same uppercase
AC C
EP
TE D
M AN U
SC
superscript letter are not significantly different by the Tukey’s test (p ≥ 0.05).
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
A
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
B
Figure 1.