Bacterial Inactivation by Common Food Industry Sanitizers

Abstract

The efficacy of peracetic acid (0.05%, 0.5%, and 1%), sodium hypochlorite (0.2%, 0.6%, and 1%), and benzalkonium chloride (0.3%, 1.15%, and 2%) was evaluated against Staphylococcus aureus (ATCC 6538), Salmonella enterica serovar Typhimurium, (ATCC 14028), Enterococcus hirae (ATCC 8043), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 9027), and Listeria monocytogenes (ATCC 35152) using stainless steel discs, following European Committee for Standardization (CEN) guidelines. According to CEN, a sanitizer must achieve at least a 5 Log₁₀ CFU reduction to be considered effective. Peracetic acid at 1% demonstrated the highest inactivation capacity, reducing all tested strains by more than 7 Log₁₀ CFU/mL. P. aeruginosa (ATCC 9027) showed high tolerance to sodium hypochlorite and benzalkonium chloride, with reductions below 2 Log₁₀ CFU/mL even at maximum concentrations. Both sodium hypochlorite and benzalkonium chloride, at their highest tested concentrations, effectively inactivated S. aureus, S. typhimurium, E. hirae, L. monocytogenes, and E. coli, achieving reductions greater than 7 Log₁₀ CFU/mL. Overall, sanitizers were effective only at intermediate or maximum concentrations recommended by the manufacturers, suggesting that minimum label concentrations should be avoided to ensure microbiological control.

1. Introduction

According to the Codex Alimentarius, food safety refers to the assurance that food does not pose a risk to consumers. This means that food should not cause adverse health effects when prepared and consumed as intended. Therefore, it is essential that food products are free from hazards that may compromise consumer health [1], including microbial contamination. Pathogenic and spoilage bacteria can spread throughout food processing environments via various contact surfaces—such as cutting boards, knives, and processing equipment—which may serve as reservoirs or vectors for these microorganisms [2]. Additionally, certain pathogenic microorganisms, such as Salmonella spp. and Listeria monocytogenes, possess the ability to form biofilms. This characteristic enhances their resistance to cleaning and sanitation procedures, increasing their likelihood of persisting in food processing environments. As a result, these sites can become continuous sources of contamination for food products [3,4].

Food industries must implement continuous and mandatory measures to minimize contamination risks throughout the entire production process, especially in areas and on surfaces where raw materials are handled. The effectiveness of these measures is closely tied to the quality of sanitation programs, including the methods and products employed. This is critical because residues generated during processing serve as key nutrient sources for bacterial growth [2,3,4,5]. Surface and equipment disinfection is typically carried out using chemical sanitizers, which can be classified based on their mode of action into oxidizing agents, surfactants, and iodophors. For example, peracetic acid and halogenated compounds are classified as oxidizing agents, whereas benzalkonium chloride—a quaternary ammonium compound—is a type of surfactant [6].

When selecting sanitizing agents for use on food contact surfaces, it is essential to evaluate their efficacy in reducing microbial contamination under specific operational conditions. Other critical factors include cost-effectiveness, ease of application, residue persistence, rinsing requirements, potential for irritation or toxicity, and compatibility with water availability at the site [7]. Moreover, it is well established that microbial sensitivity varies significantly among different species and strains, depending on the class and concentration of the sanitizer used [8,9]. The indiscriminate or improper use of sanitizing agents in food production may also exert selective pressure, promoting the emergence and persistence of resistant bacterial species. To address this concern, continuous monitoring is vital, including the identification of bacterial populations across the production chain and the assessment of their resistance profiles. Such efforts are crucial for detecting and controlling the spread of multi-resistant isolates [10].

Therefore, this study aimed to evaluate the efficacy of commonly used sanitizers in the food industry (peracetic acid, sodium hypochlorite, and benzalkonium chloride), at different concentrations, against pathogenic and/or spoilage bacteria of relevance to this sector. The tested organisms included Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Enterococcus hirae (ATCC 8043), Salmonella enterica serovar Typhimurium (ATCC 14028), and Listeria monocytogenes (ATCC 35152).

2. Materials and Methods

2.1. Efficacy of Different Sanitizers Used in the Food Industry

Three different chemical sanitizers were chosen for convenience among the sanitizing principles with authorized use in the food industry [11] and commercially available in Brazil (

Table 1. Sanitizers allowed for use in the food industry, recommended concentrations, and respective neutralizers.

Sanitizers	Active Principle	Recommended Concentration	Neutralizing
Peracetic acid	Peracetic acid 14%; hydrogen peroxide 23%; glacial acetic acid	0.05–1%	Nutrient broth with 0.6% sodium thiosulfate
Sodium hypochlorite	Sodium hypochlorite 8% active chlorine	0.2–1%	Nutrient broth with 0.5% Tween
Benzalkonium chloride	Alkyl dimethyl benzyl ammonium chloride 22%	0.3–2%	Nutrient broth with 0.6% sodium thiosulfate

) were evaluated. The minimum and maximum values tested were those recommended by the manufacturers on the disinfectant labels. Additionally, an intermediate concentration was calculated from the average of these values (Table 1).

To ensure that the effect of each sanitizer does not last for a longer time than recommended, a neutralization step was carried out. In this work, specific neutralizers were previously tested and used for each active ingredient, as recommended by Jaenisch et al. [12] (Table 1).

2.2. Bacterial Strains and Maintenance of Reference Microorganisms

In this work, six strains were used: S. aureus (ATCC 6538), P. aeruginosa (ATCC 9027), E. coli (ATCC 9027), Enterococcus hirae (ATCC 8043), Salmonella typhimurium (ATCC 14028), and Listeria monocytogenes (ATCC 35152), all provided by the Fiocruz Foundation.

Ampoules containing lyophilized microorganisms were reconstituted with 1.0 mL of Trypticase Soy Broth (TSB). This suspension was diluted in 5.0 mL of the same broth and stirred until a homogeneous suspension was obtained. Briefly, 0.5 mL of the bacterial suspension was inoculated into Petri dishes containing Trypticase Soy Agar (TSA) medium, generating a confluent growth on the medium surface. The plates were incubated for 24 h at 36 ± 1 °C.

To the medium with confluent growth, 10 mL of cryoprotective solution (meat extract, pancreatic digest of casein, and glycerol) was added, scraping the surface to obtain a cell suspension. This bacterial suspension was aliquoted and frozen. The work culture was obtained from the preserved suspension.

2.3. Bacterial Suspension of Work

Using a 10 μL disposable loop, 8 scopes of the preserved bacterial suspension were transferred to each tube containing an inclined TSA medium, incubated for 24 h at 37 °C. After growth, 0.1% peptone water was added to achieve a bacterial suspension with a concentration of 10⁹ CFU/mL, visually estimated by a McFarland standard scale.

2.4. Evaluation of the Bactericidal Activity of Commercial Sanitizers

As support for the microorganisms, 2 cm diameter 304 stainless steel discs (TSM laser^®, Santa Maria, Brazil) were used. The tests were carried out according to the methodology recommended by the European Standardization Committee (CEN) [13] for tests on a non-porous surface to evaluate the bactericidal and/or fungicidal activity of chemical disinfectants used in food, industrial, domestic, and institutional settings, with adaptations made regarding the strains of recommended microorganisms due to availability constraints. The test flowchart is illustrated in Figure 1.

According to the CEN standard, antimicrobial activity is defined by the difference in bacterial counts, expressed in logarithmic units (Log₁₀ CFU), between a positive control (unexposed population) and the population exposed to the disinfectant. This value reflects the number of bacterial cells inactivated during the specified contact time of 15 min. To be considered effective, a sanitizer must achieve a reduction of at least 5 Log₁₀ CFU in the bacterial population [13].

In this study, the efficacy of sanitizers was evaluated by counting colonies in Petri dishes containing TSA after 24 h incubation at 37 °C. As carriers, stainless steel 304 discs with 2 cm in diameter previously treated and sanitized were used, according to the recommendations of the standard [13].

Briefly, the evaluation of the efficacy of the sanitizers was carried out by the contamination of five discs with 50 μL of the bacterial suspension adjusted to 10⁹ CFU/mL, according to a McFarland standard scale, followed by the addition of 0.05% of reconstituted skimmed milk powder (Elegê^®, Rio Grande, Rio Grande do Sul, Brazil), which was used as an interfering substance in the action of the sanitizing product, simulating the presence of organic matter in the manufacturing environment. For each bacterial strain evaluated, three discs were used to assess the bactericidal activity of the disinfectant, while two additional discs served as positive controls (unexposed to the disinfectant). Following inoculation, all discs—both treatment and control—were incubated in a bacteriological oven at 37 °C for 40 min to facilitate drying and fixation of bacterial cells, thereby halting bacterial multiplication. Subsequently, the discs were left at room temperature for an additional 20 min to allow for temperature equilibration.

To the actual test, 100 μL of the test product was added in three different concentrations [peracetic acid (0.05%, 0.5%, and 1%); sodium hypochlorite (0.2%, 0.6%, and 1%); benzalkonium chloride (0.3%, 1.15%, and 2%)] to the contaminated and dry discs. To evaluate the positive control, the sanitizer was replaced by 100 μL of sterile water. After 15 min of contact of the product with the contaminated surface of the discs, they were immersed in 10 mL of specific neutralizing solution for each sanitizer and 5 g of glass beads. After 5 min of neutralization, a 1 mL aliquot was removed, making serial dilutions [13].

2.5. Plating and Counting Bacteria

Plating was performed using the pour plate method in triplicate in TSA medium, incubated at 37 °C for 24 h. The effectiveness of each sanitizer was assessed by the difference between the number of colony-forming units of bacteria recovered from the positive controls and that of the discs exposed to the sanitizer. From the growth in the plates with TSA medium, the counting was performed, and the results are expressed in Log₁₀ CFU/mL.

3. Results and Discussion

In general, this study revealed variations in the efficacy of the different concentrations of the same sanitizer, differences in the effectiveness of various sanitizers against the same microorganism, and variability in the sensitivity of the evaluated strains to the tested sanitizers (Figure 2, Figure 3 and Figure 4).

Figure 2 presents the results of bacterial population inactivation following 15 min of exposure to peracetic acid at concentrations recommended by the manufacturer. Although the McFarland standard was intended to yield an initial microbial concentration of approximately 9 Log₁₀ CFU, the actual counts observed in the control samples ranged from 7.22 to 7.83 Log₁₀ CFU. These observed values were used as the baseline for calculating the efficacy of the sanitizing agents.

It can be inferred that peracetic acid was ineffective at eliminating the tested bacteria when applied at the minimum recommended concentration (0.05%). However, the intermediate concentration proved effective against all bacteria except Pseudomonas aeruginosa (ATCC 9027), while the highest concentration completely inactivated all tested bacteria. Notably, peracetic acid was the only sanitizer among those tested to be effective against P. aeruginosa, albeit only at the highest recommended concentration. Peracetic acid is believed to act similarly to other oxidizing agents by reacting with cellular proteins and enzymes [6], and its antimicrobial activity increases at elevated temperatures (40 °C) [14]. A high tolerance of P. aeruginosa to quaternary ammonium sanitizers, especially when forming biofilms, has been previously reported [15], suggesting that peracetic acid may be a viable alternative for controlling this species in food processing environments.

Similarly, Lee et al. [16] demonstrated that 2% peracetic acid reduced bacterial populations of E. coli, S. aureus, S. Typhimurium, P. aeruginosa, and E. hirae by 7 Log₁₀ CFU in a suspension test after 5 min of exposure. In studies evaluating peracetic acid’s effect on biofilms of Salmonella spp. and S. aureus on stainless steel surfaces, the lowest concentration tested (0.001%) inactivated 3.3 Log₁₀ CFU of S. aureus and reduced Salmonella spp. by 5.88 Log₁₀ CFU [17]. Barbosa et al. [18] reported a 3.57 Log₁₀ CFU reduction in E. coli strains on stainless steel knives following 10 min exposure to 0.2% peracetic acid. Additionally, biofilms formed by three isolates of Listeria monocytogenes on stainless steel were almost completely inactivated (~100%) after 3 min exposure to 0.5% peracetic acid [19]. Figure 3 illustrates bacterial sensitivity to sodium hypochlorite after 15 min of exposure at concentrations of 0.2%, 0.6%, and 1%. Similar to peracetic acid, the lowest recommended concentration was ineffective against the tested strains. Sodium hypochlorite was effective at intermediate and maximum concentrations against all species except P. aeruginosa (ATCC 9027). Chlorine exerts antimicrobial effects through multiple mechanisms, including altering membrane permeability, reducing bacterial cell size, and oxidizing surface proteins. Moreover, hypochlorous acid, the active form of sodium hypochlorite, is more effective in slightly acidic environments [6].

Likewise, Riazi and Matthews [20] analyzed the reduction in microorganisms in stainless steel discs and found that the highest concentration (0.05%) of sodium hypochlorite tested was effective against S. aureus, S. enteridis, and L. monocytogenes bacteria, except P. aeruginosa, which was reduced by only 2 Log₁₀ CFU. Rosado et al. [21] demonstrated the effectiveness of 0.01% sodium hypochlorite for 10 min of exposure against biofilms of Enterococcus faecalis and Enterococcus faecium, reducing by 1.40 and 2.74 Log₁₀ CFU, respectively.

Despite the ineffectiveness of sodium hypochlorite in all concentrations tested against P. aeruginosa (ATCC 9027), other studies have shown significant reductions in the population of this species by this agent. Kohler et al. [22] verified the reduction of 5 Log₁₀ CFU of P. aeruginosa using 0.04% sodium hypochlorite for 15 min in surface tests with stainless steel carriers in the presence of organic load (bovine serum albumin). Stainless steel discs containing P. aeruginosa were disinfected with sodium hypochlorite (1.31%) for 4 min, and an average reduction of 8.75 Log₁₀ CFU was observed [23].

Benzalkonium chloride, a quaternary ammonium compound, exhibited the greatest variation in effectiveness among the sanitizers evaluated (Figure 4). Notably, Enterococcus hirae (ATCC 8043) and Listeria monocytogenes (ATCC 35152) were the most sensitive microorganisms, being completely inactivated at the lowest concentration recommended on the product label (0.3%). In contrast, Salmonella typhimurium (ATCC 14028) showed tolerance to this sanitizer at both intermediate and minimum concentrations, while Pseudomonas aeruginosa (ATCC 9027) tolerated even the highest benzalkonium chloride concentration tested. At this maximum concentration, all other tested microorganisms were fully inactivated. Quaternary ammonium compounds are positively charged surfactants that readily bind to the negatively charged surfaces of most microbes, disrupting cell walls and membranes after prolonged contact [6]. Furthermore, their antimicrobial activity increases at lower temperatures (around 10 °C) [14], making their use practical and economical, especially when applied with well water.

Poimenidou et al. [24] analyzed the resistance of Listeria monocytogenes biofilms on stainless steel surfaces to quaternary ammonium compounds, reporting an average reduction of 3.9 Log₁₀ CFU. Similarly, Du et al. [25] demonstrated that an isopropyl alcohol/quaternary ammonium disinfectant (0.02%) could achieve a 4-log reduction in Salmonella after 10–15 min, even in the presence of substantial organic load. The effectiveness of disinfectants is influenced by the type and amount of organic material present. For example, Iñiguez-Moreno et al. [17] found that quaternary ammonium-based sanitizers (400 mg/mL) were more effective in the presence of 10% meat extract, whereas peracetic acid-based sanitizers (200 mg/mL) showed enhanced activity in the presence of 10% egg yolk and whole milk. In the same study, reductions of 1.21 Log₁₀ CFU were observed for Pseudomonas aeruginosa on stainless steel surfaces after 30 min, while L. monocytogenes, Escherichia coli, Salmonella Enteritidis, and S. typhimurium exhibited reductions of up to 6 Log₁₀ CFU.

Although quaternary ammonium compounds have a broad spectrum of activity, they are generally less effective against Gram-negative bacteria [26], which may explain the reduced efficacy of the sanitizer against S. typhimurium (ATCC 14028) and P. aeruginosa (ATCC 9027) in our experiments.

Among the isolates tested, P. aeruginosa (ATCC 9027) demonstrated notably high tolerance to various sanitizing agents under the conditions evaluated. This phenomenon has been documented in other studies [27,28], including reports of P. aeruginosa and Pseudomonas cepacia survival in iodine-based solutions and quaternary ammonium compounds [28]. Differences in the composition of lipopolysaccharides (LPSs) and an increased content of Mg²⁺ ions in P. aeruginosa strengthen LPS interactions, enhancing the bacterial outer membrane’s integrity. Additionally, the presence of low-efficiency porins limits molecular diffusion into the cell [27], factors that likely contribute to the observed variations in sanitizer susceptibility.

The definition of resistance in the context of sanitizing agents differs from that used for antimicrobial agents such as antibiotics. The term acquired resistance is more appropriate here and generally refers to non-plasmid-mediated adaptations that arise when bacterial populations are exposed to gradually increasing concentrations of biocidal compounds. This form of resistance is often linked to apparent tolerance resulting from exposure to sublethal or ineffective sanitizer concentrations, rather than from true genetic or physiological adaptations of the microorganisms [29].

The study conducted by Bernardi et al. [30], which evaluated sanitizers against spoilage fungi isolated from meat and dairy products, revealed a discrepancy between the concentrations recommended by manufacturers and those actually used in the food industry. Peracetic acid was the most commonly used agent (45%), followed by biguanide compounds (25%), with similar usage patterns observed in both cheese and meat industries. However, the concentrations applied were often near the minimum levels indicated on product labels, potentially compromising microbiological efficacy. Consequently, the recurrent use of sublethal doses, coupled with limited knowledge of effective concentrations, may facilitate the persistence of microorganisms in industrial environments.

This observed tolerance underscores the need to reevaluate sanitizer concentrations for bacterial control as well. Furthermore, the efficacy of sanitizers can vary widely among species and strains, emphasizing the importance of isolating microorganisms directly from industrial settings and assessing their susceptibility to disinfectants.

4. Conclusions

Peracetic acid, at concentrations recommended on the product label, was the only sanitizer effective against all bacterial strains evaluated (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus hirae, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes). This agent represents the best alternative for the food industry, especially in settings with high microbiological diversity or processes requiring broad-spectrum antimicrobial activity.

Sodium hypochlorite demonstrated satisfactory efficacy against most tested microorganisms, except the spoilage bacterium P. aeruginosa. Thus, it may be a viable option when the primary targets are pathogens such as Salmonella spp., E. coli, or S. aureus, provided that P. aeruginosa is not a significant concern.

Quaternary ammonium, in the form of benzalkonium chloride, showed high efficacy against E. hirae and L. monocytogenes even at the lowest recommended concentration, making it suitable for environments where these pathogens are prioritized and where lower chemical aggressiveness on surfaces is desired.

Overall, the data suggest that the minimum concentrations recommended by manufacturers may be insufficient to ensure effective microbiological control in food industry settings. Therefore, a risk-based microbiological approach is recommended, taking into account the resident microbiota, the sanitizer’s properties, and application conditions to optimize sanitation efficacy.