Removal of Mixed-Species Biofilms Developed on Food Contact Surfaces with a Mixture of Enzymes and Chemical Agents

Sanicip Bio Control (SBC) is a novel product developed in Mexico for biofilms’ removal. The aims of this study were to evaluate (i) the removal of mixed-species biofilms by enzymatic (protease and α-amylase, 180 MWU/g) and chemical treatments (30 mL/L SBC, and 200 mg/L peracetic acid, PAA) and (ii) their effectiveness against planktonic cells. Mixed-species biofilms were developed on stainless steel (SS) and polypropylene B (PP) in whole milk (WM), tryptic soy broth (TSB) with meat extract (TSB+ME), and TSB with chicken egg yolk (TSB+EY) to simulate the food processing environment. On SS, all biofilms were removed after treatments, except the enzymatic treatment that only reduced 1–2 log10 CFU/cm2, whereas on PP, the reductions ranged between 0.59 and 5.21 log10 CFU/cm2, being the biofilms developed in TSB+EY being resistant to the cleaning and disinfecting process. Higher reductions in microbial load on PP were reached using enzymes, SBC, and PAA. The employed planktonic cells were markedly more sensitive to PAA and SBC than were the sessile cells. In conclusion, biofilm removal from SS can be achieved with SBC, enzymes, or PAA. It is important to note that the biofilm removal was strongly affected by the food contact surfaces (FCSs) and surrounding media.


Introduction
Biofilms are growing communities of microorganisms adhered to a surface and were embedded in self-produced extracellular polymeric substances (EPS) [1]. The type and amount of EPS are strain-dependent and vary with the environmental conditions in which biofilms are formed. Nevertheless, the general composition of EPS includes polysaccharides, proteins, lipids, and extracellular DNA [1,2]. Biofilm development confers advantages to microbial cells, such as physical resistance to refrigeration, heat, desiccation, acidity, and salinity; mechanical resistance to liquid streams in pipelines; and chemical protection against antimicrobials and disinfectants [3,4]. Otherwise, biofilms cause corrosion in equipment, biofouling in water systems, and post-process contamination contributing to food spoilage. Recently, biofilms have been associated with the generation of foodborne diseases [5].
Almost 800 foodborne disease outbreaks are reported every year in the USA, causing approximately 15,000 foodborne illnesses, 800 hospitalizations, and 20 deaths [6]. The National Institutes of Health estimated that over 65% of microbial diseases are related to biofilm formation [7]. Listeria monocytogenes, Salmonella, and Shiga toxin-producing Escherichia coli are related to 82% of all hospitalizations and deaths in the USA. Another important biofilm-former microorganism commonly implicated in foodborne diseases is Bacillus cereus (2%) [6].

Biofilm Removal on SS
To evaluate biofilm removal, mixed-species biofilms were developed on SS and PP coupons in three culture media. A mixture of proteolytic and amylolytic enzymes, and SBC were used for biofilm removal, and PAA was applied as a disinfectant ( Figure 2). In biofilms developed on SS, the cellular densities ranged from 6.46 to 6.67 log 10 (Table 1). Moreover, we analyzed the population before and after the treatments. In this regard, differences in the initial count between each species were observed (p ≤ 0.05; Table 1). In addition, all treatments (except the enzymatic) reached over 6 log 10 CFU/cm 2 of microorganism reduction in the mixed-species biofilms on SS. After enzymatic treatments, the counts of biofilms developed in tryptic soy broth (TSB) with 100 mL/L chicken egg yolk (TSB+EY) and whole milk (WM) were not different to those of their control (p > 0.05; Figure 3). However, the cell density of P. aeruginosa in biofilms developed in TSB+EY was reduced after the enzymatic treatment (p ≤ 0.05). The same occurred with E. coli and L. monocytogenes in WM (p ≤ 0.05, Table 1).

Biofilm Removal on PP
The initial biofilm counts were higher on PP (~7.49 log 10 CFU/cm 2 ) than on SS (p ≤ 0.05). Furthermore, the biofilms showed greater resistance on PP compared to on SS, with reductions between 0.59 and 5.21 log 10 CFU/cm 2 (p ≤ 0.05; Figure 3). However, significant differences were observed in the initial counts of each microorganism (p ≤ 0.05). On PP, the low reductions were obtained with enzymatic treatments (p > 0.05, Figure 3). Moreover, E. coli and L. monocytogenes were recovered of biofilms developed in TSB with meat extract (TSB+ME) after treatments with SBC with or without the previous enzymes' application ( Table 2).  Even when the initial counts of Salmonella and P. aeruginosa were similar (p > 0.05) in biofilms developed in TSB+ME and TSB+EY; Salmonella loads were higher than those of P. aeruginosa after all treatments (p ≤ 0.05). Moreover, these microorganisms were recovered after all treatments applied on PP, with cellular densities between 2.37 and 4.63 Log 10 CFU/cm 2 .
In general, the microorganisms in the biofilms developed on PP were more resistant to removal and disinfection treatments than in the other residues or onto SS. L. monocytogenes were not recovered on biofilms developed in WM after treatments with PAA. Moreover, B. cereus was not quantified before treatments in biofilms developed in WM, and in most cases, was fully reduced after the removal and disinfection process ( Table 2).

Epifluorescent Microscopy and SEM Analyses
Representative micrographs of mixed-species biofilms developed on SS in the different culture media were obtained by SEM and epifluorescence microscopy ( Figure 4). In concordance with the counting plate technique before removal treatments, metabolically active cells were observed by epifluorescent microscopy. Furthermore, EPS and food residues were observed ( Figure 4A-C) and confirmed by SEM ( Figure 4D-I). After the removal and disinfection process of biofilms developed on SS, metabolically active cells were not observed by epifluorescence microscopy, except on the coupons with enzymatic treatment. However, through SEM, some bacterial cells and residues of EPS were observed, particularly after treatments with enzymes ( Figure 5). Otherwise, cells and EPS were detected on PP after all treatments. Nevertheless, the biofilms were considerably removed in comparison to the images obtained before the treatments (Figure 4). After enzymes use, the microorganisms were easily observed due to EPS removal ( Figure 5).

Discussion
Biofilms contribute to pathogen spread and food contamination, cause damage to food processing equipment, and increase antimicrobial resistance, representing significant losses to the public and private sectors [5,20]. In this study, we assessed the effect of different treatments against planktonic and mixed-species biofilms developed under conditions that simulate a food processing environment. The assessed products reached reductions of >5 log 10 CFU/mL against planktonic cells. A reduction of 5 log 10 CFU/mL is the minimum to consider a disinfectant as effective [21,22]. An inappropriate cleaning process can leave up to 100 g/L of organic matter [23]; hence, microbicidal tests were also carried out in the presence of food residues. The efficacy of PAA was reduced in egg yolk and meat extract due to the fact that proteins and fats affect the availability of the oxidant agents [24]. Considering the time exposition used in this research (30 s), the reduction obtained (99.99%) was higher than that in other studies. For example, the products Suma Tab D4 and Suma Bac D10 (quaternary ammonium compounds, 240 and 740 mg/L, respectively) reduced 5 log 10 of L. monocytogenes after 5 min in whole milk [23].
Biofilms are the main bacterial lifestyle in food processing environments, and sessile microorganisms are more resistant than are planktonic cells. Therefore, we evaluated the effect of different treatments on the removal of mixed-species biofilms developed in different culture media (tryptic soy broth (TSB) with chicken egg yolk (TSB+EY), and with meat extract (TSB+ME), and WM). SDW treatments (included as controls), showed significant reductions in P. aeruginosa, S. enteritidis, and S. typhimurium. SDW can cause cellular lysis, due to solutes absence, and it can also dissolve simple sugars, mineral salts [25], and some cellulose structures [26]. The use of chemical agents for biofilm control in food environments is not always effective; therefore, their efficacy should be improved by the combination of biological agents and physical methods.
Enzymes represent a great alternative for biofilm removal [27]. Biofilms in the food processing environment are composed of multiple microorganisms, resulting in an EPS with a heterogenic composition [28]. Recently, it has been demonstrated that polysaccharides in biofilms developed by Gram-negative bacteria, such as alginic acid, are the main component of the EPS matrix [13]. For example, the EPS matrix of S. Typhimurium is mainly composed of aggregative fimbriae and extracellular polysaccharides (cellulose) [29]. In contrast, proteins are the main compound in biofilms of Gram-positive bacteria [3]; however, they also produce polysaccharides as well as dextran [30]. Therefore, it is recommended to use a mixture of enzymes, because these molecules have specific activity [31]. In this study, the removal of mixed-species biofilms was evaluated using a mixture of alkaline protease and α-amylase. On SS surfaces, biofilm removal ranged between 93.4 and 96.3%. The low removal of biofilms developed in TSB+EY on PP (12.2%) was attributed to the high content of lipids in the egg yolk, which were not decomposed by the enzymes applied [32]. Ripolles-Avila et al. [3] achieved a removal of~2.3 log CFU/cm 2 of S. Typhimurium on SS 304 with a mix of enzymes (protease, lipase, and amylase), which is in agreement with the findings in this study.
Kumari and Sarkar [33] used a serine protease, resulting in a complete reduction in B. cereus biofilms (4.08 log 10 CFU/cm 2 ) developed in skim milk. The difference between this report and our results could be explained by the low cell density in the biofilms; moreover, after 24 h of incubation, the EPS matrix is not mature [19,34]. The EPS matrix is an important component of biofilms and represents more than 90% of the total mass of these structures [35]. The EPS matrix is the first resistance mechanism of the microorganisms in the biofilms against chemical and physical agents and environmental conditions. EPS components can react with the disinfectant molecules, protecting the microorganisms in the biofilm [36]. Recently, it has been reported that the application of an enzymatic cleaner (1 h at 50 • C) reduced 79.72% of S. enterica biofilm [28]. Nonetheless, to achieve this reduction, the samples were exposed for a least 1 h at 50 • C; this procedure is not viable for real conditions on an open surface in the food industry, a fact that was not considered in that report. Our study, however, was designed considering the application of the removal process on open surfaces in that environment.
To improve biofilm removal, enzymatic and chemical treatments were applied. With the combination of these treatments, microorganisms were not recovered from biofilms developed on SS. On PP, the reductions with the enzymatic and chemical treatments ranged from 3.06 to 4.76 log 10 CFU/cm 2 (Figure 3), and these results were greater than those reported in other studies [36]. The selection of the type of FCS is vital in the food processing environment. The high biofilm removal on SS is related to its hydrophilic nature, the presence of metallic ions on the surface [37], the germicidal activity of the quaternary ammonium compounds, and the organic acids in the SCB. In previous research, it was demonstrated that PAA at 3500 mg/L kills the cells in biofilms of Staphylococcus aureus without removing them [15]. Moreover, lower reductions were obtained on PP, because hydrophobic surfaces increase cell aggregation and biofilm development [15,19]. The aqueous solution has minimal contact with the surface of PP; even SBC has quaternary ammonium compounds and surfactants that decrease the superficial tension of water, facilitating biofilm removal [27]. These compounds and the organic acids in their formulation promoted biofilm removal from SS.
Salmonella and P. aeruginosa were recovered in great amounts after the removal treatments. This is related to their high counts before the treatment; moreover, it has been demonstrated that the biofilm formation by Salmonella is favored in the presence of other bacteria such as Pseudomonas sp. and Bacillus sp. [38]. In addition, it was reported that Salmonella biofilms were more sensitive to disinfectants when they were developed on SS than on PP [15]. Microorganism aggregation within a three-dimensional structure can provide protection against biocides activity [39]. Almeida et al. [39] observed that two well-defined layers exist in tri-species biofilms, on the surface of E. coli and in the deep mixed regions of L. monocytogenes and S. enterica. This can explain the absence of E. coli after the application of all treatments.
Some studies have reported a higher resistance in sessile than in planktonic microorganisms to antimicrobials [40,41]. In line with this, the PAA reduced 2-3 log 10 CFU/mL more in the assays with planktonic cells than in biofilms developed on PP treated with SBC or enzymes and then PAA. In addition to the EPS matrix, the presence of catalase in the microorganisms could play a role in peracetic acid decomposition. Unfortunately, the resistance mechanisms involved in mixed-species biofilms are not entirely clear [42].
Biofilms are complex structures composed of multilayers of microorganisms, EPS, and water channels [43]. The microorganisms in the biofilms are in different states: metabolically active, metabolically inactive, and dead cells. Therefore, it is important to use more than one technique for biofilm studies. Nowadays, it is difficult to use epifluorescence microscopy as a counting technique for cells in biofilms, because bacteria in a biofilm usually develop layers and residues such as TSB and EY emit strong auto-fluorescence [16,44]. However, it is possible to observe surviving cells after decontamination treatments, even in those treatments where it is not possible to achieve their expression in culture media, either by the detection limit of the technique or by the metabolic state of the bacteria (sub-lethally damaged cells or non-cultivable but metabolically active cells) [42]. SEM enables observing the architecture of the biofilm, without distinguishing living or dead cells [45]. Hence, for biofilm studies, complementary techniques should be used.
Disinfectant effectiveness on biofilms varies depending on disinfectant characteristics; type of surface; microorganisms in the biofilm; and other factors such as exposure time and temperature [15,41]. Furthermore, interspecies interactions generated within the biofilms have an effect on the dynamics and resistance within the biofilm [38]. Moreover, food residues rich in proteins, lipids, and carbohydrates decrease disinfectant effectiveness, thereby increasing bacteria survival and encouraging cross-contamination due to the increase in bacterial persistence on FCSs [5,38]. Currently, FCS coating, enzymatic disruption, quorum sensing inhibition, biosurfactants, bacteriophages, bacteriocins, essential oils, furanone derivates, high hydrostatic pressure, non-thermal plasma, ultrasound, and photocatalysis have been proposed for biofilm control [9,46]; however, these communities still represent a considerable challenge to food industries and scientists.

Bacterial Strains
The microorganisms used to biofilm formation were E. coli ATCC 11303, S. Typhimurium ATCC 14028, S. Enteritidis ATCC 13076, P. aeruginosa ATCC 15442, L. monocytogenes ATCC 19111, and B. cereus ATCC 14579 (vegetative stage). Before utilization, the microorganisms were incubated individually in TSB (Becton Dickinson Bioxon, Le Pont de Claix, France) at 37 • C for 24 h in aerobic and static conditions to yield a final concentration of 10 7 CFU/mL.

Chemical and Enzymatic Agents
The assessed products were Sanicip Bio Control (active product obtained of the mixture of SBC 1 and SBC 2, National Sanitation Foundation (NSF) numbers 155919 and 155920, respectively) and Sanicip PAA (peracetic acid, PAA; 200 mg/L, NSF number 144381) (CIP & GROUP, Tlajomulco de Zuñiga, Mexico). Deterzyme 520/180 is a mixture of alkaline protease and α-amylase produced by Bacillus licheniformis and Bacillus subtilis, respectively (ENMEX, Tlalnepantla, Mexico), which was used to the assessment of biofilm removal.

Microbicidal Activity against Planktonic Cells
Bactericidal efficacy assays were performed according to AOAC Official Method 960.09 09 [21] with the products SBC (8 mL/L) and Sanicip PAA (200 mg/L). The concentrations used are approved for hard surfaces [23].

Biofilm Development and Quantification
Mixed-species biofilms were developed in three culture media: TSB with 100 mL/L chicken egg yolk, (TSB+EY), TSB with 100 g/L meat extract (TSB+ME), and WM. Briefly, each coupon was individually introduced into a new polypropylene tube (15 mL Centrifuge tube, Corning CentriStar, New York, NY, USA) containing 5 mL of the corresponding culture media and was inoculated with 25 µL of each bacterial species (1 × 10 6 CFU/mL). S. Typhimurium was used in biofilms developed in TSB+ME and WM and S. Enteritidis was inoculated in TSB+EY. The tubes with the coupons were incubated at 25 • C for 120 h. After this period, the coupons were removed from the tube, immersed into a new fresh medium and inoculated with the same microorganisms (1 × 10 6 CFU/mL), and incubated for 120 h. At the end of the incubation period, the coupons were removed from the tube using sterile forceps, rinsed by vortexing (150 rpm/10 s) in 5 mL of Dulbecco's phosphatebuffered saline (PBS; Sigma-Aldrich, St Louis, MO, USA). The conventional plate counting on tryptic soy agar with lactose (10 g/L; Sigma-Aldrich, St. Louis, MO, USA) and phenol red (0.1 g/L; Hycel, Zapopan, Jalisco, Mexico) was realized. For quantification of E. coli and B. cereus in multispecies biofilms, cefsulodin (50 µg/mL; Sigma-Aldrich, St. Louis, MO, USA) and polymyxin B (70 µg/mL; Sigma-Aldrich, St. Louis, MO, USA) were added to culture media, respectively. Petri dishes were incubated at 37 • C for 24 h. Colonies of E. coli and L. monocytogenes were yellow due to lactose fermentation; the other microorganism colonies were colorless. Salmonella and P. aeruginosa were distinguished using the oxidase test [19]. Each quantification was carried out in triplicate. Controls without microorganisms were included for the determination of contamination.

Removal and Disinfection Treatment Procedures
At the end of the incubation period, the coupons were removed from the tubes using sterile forceps and rinsed as above. Then, treatments to remove biofilms with SBC 30 mL/L or with a mixture of alkaline protease and α-amylase produced by Bacillus licheniformis and Bacillus subtilis, respectively (Deterzyme 520/80; ENMEX, Tlalnepantla, Mexico), were applied according to

Scanning Electron Microscopy (SEM) Analysis
Before and after removal and disinfection treatments, coupons of each material (SS and PP) were rinsed with PBS as above, then was immersed in 20 mL/L glutaraldehyde (DermoDex, Tlalpan, CDMX, Mexico) for 2 h at 4 • C to fix the biofilm. After serial dehydration in ethanol (30,50,60,70,90, and 95 mL/100 mL) for 10 min each at 4 • C, every coupon was rinsed (three 10 min rinses) in absolute ethanol [48]. Samples were dried and were coated with gold for 30 s [49]. Biofilms were observed under a TESCAN Mira 3 LMU Model field emission scanning electron microscope (Brno-Kohoutovice, Czech Republic).

Statistical Analysis
All of the experiments were performed in triplicate; the statistical analysis was carried out using ANOVA; the percentages data were arcsine square root transformed. The variances were examined by the least significant difference (LDS) test in the software Statgraphics Centurion XVI.I (Statpoint Technologies, Inc., Warrenton, VA, USA).

Conclusions
The resistance of mixed-species biofilms developed by E. coli, S. typhimurium, S. enteritidis, P. aeruginosa, L. monocytogenes, and B. cereus under conditions that simulate the dairy, meat, and egg processing industries was strongly affected by the type of FCS and surrounding media. The use of Sanicip Bio Control and enzymes plus Sanicip PAA were effective in removing the biofilms developed on SS. Hence, efforts should be conducted to prevent cell aggregation, promote the use of hydrophilic materials such as stainless steel, and use protocols of cleaning and disinfection based on the use of biological and chemical agents. Moreover, enzymatic agents are a great alternative to biofilm control in the food industry, establishing their use according to the type of food residues, potential microorganisms in the biofilm, and optimum temperature to maximize their activity. These results can contribute to applying novel approaches for controlling biofilms in food processing environments, improving food safety and quality.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding authors upon request.