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. 2024 Dec 4;34(6):1507–1516. doi: 10.1007/s10068-024-01751-5

Comparative evaluation of sanitation strategies against Listeria monocytogenes on food-contact surfaces in enoki mushroom (Flammulina velutipes) processing facilities

Kyung Min Park 1, Su-Bin Lee 1, Hyobeen Chae 1, Injun Hwang 1, Se-Ri Kim 2, Hyun Dong Lee 3, Song-Yi Choi 1,✉
PMCID: PMC11914709  PMID: 40110399

Abstract

We investigated strategies to reduce Listeria monocytogenes contamination on food contact surfaces during enoki mushroom processing. Chemical disinfectants and thermal treatments were evaluated on conveyor belts, stainless steel, plastic surfaces, and Velcro strips. Without organic matter, chemical disinfectants effectively reduced L. monocytogenes, with stainless steel showing the highest susceptibility. Organic matter decreased disinfectant efficacy, but sodium hypochlorite remained most effective on stainless steel. Peracetic acid was more effective on conveyor belts and plastic surfaces than on stainless steel. Combining peracetic acid with dry heating synergistically reduced L. monocytogenes on Velcro strips. Moist heat at 70 °C alone was insufficient, but when combined with hot air drying, it effectively reduced L. monocytogenes on Velcro strips. Our findings emphasize the importance of surface-specific strategies combining chemical disinfection and thermal treatment for eliminating L. monocytogenes in mushroom processing environments.

Keywords: Enoki mushroom, Listeria monocytogenes, Chemical disinfectant, Heat treatment, Synergism

Introduction

Listeria monocytogenes is a foodborne pathogen that causes invasive listeriosis, a serious infection that potentially leads to meningitis, sepsis, encephalitis, spontaneous abortions, and stillbirths. People at a high risk of invasive listeriosis include individuals ≥ 65 years old, pregnant women, newborns, and immunocompromised individuals (Charlier et al., 2017). L. monocytogenes causes relatively few foodborne disease outbreaks in the United States, accounting for less than 1% of cases annually. However, it has a high case fatality rate of approximately 20% (CDC, 2024). Fresh-produce farms are encouraged to implement rigorous cleaning and sanitization processes as good agricultural practices to minimize the risk of L. monocytogenes contamination of their products. Despite its relatively low incidence, L. monocytogenes is associated with a high mortality rate, highlighting the need to effectively control this pathogen.

Flammulina velutipes, commonly known as enoki or winter mushroom, is a long, thin, and white mushroom species. L. monocytogenes is isolated from various types of edible mushrooms in North America and China (Chen et al., 2018). Interestingly, enoki mushrooms were recalled in Canada due to the possible presence of L. monocytogenes in 2019 (Government of Canada, 2024). Consumption of L. monocytogenes-contaminated enoki mushrooms is associated with multistate human listeriosis cases, affecting 30 patients and causing four deaths over the past three years in the United States (CDC, 2024). Recent outbreaks and product recalls highlight the need for stringent food safety measures and enhanced monitoring to mitigate the risks associated with L. monocytogenes contamination during enoki mushroom production and distribution.

Most large-scale mushroom production companies and farms use bottle cultivation. Generally, cultivating enoki mushrooms involves substrate preparation, spawn inoculation, mycelium infiltration, mycelium scraping, collaring, and harvest. Substrate for enoki mushroom includes hardwood sawdust and a supplement, generally wheat bran through other options are used (Sharma and Kumar, 2011). The substrate is loaded in bottles and is the sterilized with autoclaving. After thermal processing and cooling, spawn is inoculated onto the substrate, and the bottles are moved into growing rooms. Mycelia are allowed to infiltrate fully through the substrate and the complete infiltrated surface is scraped to clear away mycelial growth. After the stem grows 1–2 cm at the top of the bottle under low temperature (8 °C), plastic sheaths are wrapped around the bottle to form a cylindrical shape to suppress the oxygen level and elongate the matured basidiocarps between 14 to 18 days (Harith et al., 2014). The moist, low temperature, and nutrient-rich environment required for enoki mushroom growth provides favorable conditions for the survival and proliferation of L. monocytogenes, a hardy pathogen capable of persisting in food-processing environments.

Hygienic practices for mushroom cultivation are crucial to prevent cross-contamination and biological hazards. The selection of sanitization techniques depends on the surface material, moisture content, and presence of organic matter (Midelet & Carpentier, 2002). Hygienic practices using appropriate disinfectants and procedures are essential to prevent the spread of L. monocytogenes in mushroom production plants. Chemical disinfectants, such as sodium hypochlorite and peracetic acid, are commonly used to control foodborne pathogens, but their efficacy is limited, especially against L. monocytogenes (Bang et al., 2017). Chlorine-based disinfectants can react with organic materials, which may be decrease its antimicrobial activity (Gómez-López et al., 2017). This issue is particularly concerning when it comes to washing step for products or food contact surfaces during food processing, as the wash solutions often contain organic materials such as soil, damaged produce exudates, and plant debris. Most studies regarding the antimicrobial efficacy of disinfectant have been performed under clean condition without organic materials. However, significant difference may occur when the experiment is carried out under the presence of organic materials such as mushroom debris or exudates. Thus, the information on operation parameters (sanitizer type, treatment time, and organic materials) is critical to establish the performance of sanitizing process for food contact surfaces.

Heat treatment is a promising alternative or complementary approach owing to its strong antimicrobial activity (Bang et al., 2017). A common treatment for sanitizing food contact surfaces is to ensure that the temperature of all surfaces reaches at least 77 °C, or 71 °C for a minimum of 30 s (UFPA, 2013). Most research has shown that thermal treatment using hot water or steam is recommended to reduce or eliminate potential pathogenic microorganisms (Brooks and Flint, 2008; Meireles et al., 2016; Yang et al., 2017). In contrast, there are few data on the effects of dry heat on pathogen inactivation. Valdramidis et al. reported that thermal inactivation of pathogens using dry heat. Escherichia coli populations were reduced by 2 logs after 2 h at 80 °C (Valdramidis et al., 2005). Lindsay et al. also confirmed that at least 4-log reductions in Listeria innocua inoculated onto food contact surface showed with air dry treatment at 65 °C for 7 h (Lindsay et al., 2013). However, no studies have investigated the combination efficacy of heat treatment and sanitizers such as sodium hypochlorite or peracetic acid for reducing L. monocytogenes on food contact surfaces. Therefore, the objective of this study was to compare the effectiveness of individual treatments (sanitizers and heat) and the combination of heat and sanitizers for reducing L. monocytogenes on food contact surfaces. Additionally, this study confirmed the impact of organic materials on sanitization efficacy to identify the most effective microbial control methods. To achieve this, mushroom extract medium was used as a critical organic material that could directly obtained during the enoki mushroom processing.

Materials and methods

Bacterial strains and growth conditions

Five strains of L. monocytogenes were used to compare the effectiveness of various sanitizing strategies on different food contact surfaces. These strains were isolated from plastic wrappers collected from four enoki mushroom cultivation factories in Korea between 2020 and 2021 according to the Food Code of Korea (2020). The presumptive colonies cultured individually on tryptic soy agar (Difco Laboratories, Detroit, MI, USA) for 24 h at 37 °C were identified as L. monocytogenes using a commercial real-time PCR detection kit (Genelix L. monocytogenes real-time PCR detection kit; Sanigen, Anyang, Korea). The identified L. monocytogenes strains were cultured in tryptic soy broth (TSB; Difco Laboratories) for 24 h at 37 °C, mixed with glycerol to a final concentration of 25%, and stored at –80 °C.

Preparation of the mushroom extract medium

To confirm the impact of organic matter on sanitizing efficacy, mushroom extract medium was used as a critical organic material. Sterilized mushroom substrate was provided from mushroom cultivation industry in Korea. 100 g of mushroom substrate was extracted by grinding performed for 5 min with a pestle and mortar with 100 mL of sterilized distilled water. The mixture was then stored for 4 h to release of mushroom constituents at room temperature. The solid materials were filtered out using centrifuge for 30 min at 5000 × g to remove large particulate debris. The extract was first filtered through a 0.45-µm filter (Merck Millipore, Ltd., Cork, Ireland) for primary filtration and then through a 0.22-µm filter (Merck Millipore, Ltd.) for secondary filtration to sterilize. The resulting extract was diluted to 2% extract broth with distilled water and used to prepare a mixed strain of L. monocytogenes.

Preparation of chemical disinfectants

Based on the maximum permissible concentration for each product outlined by the Food Additive Codex (2022) for equipment and utensil sanitizers, three sanitizers were prepared: sodium hypochlorite (200 mg/L; Sigma-Aldrich, St. Louis, MO, USA), peracetic acid (200 mg/L; Sigma-Aldrich), and chlorine dioxide (200 mg/L; Epiclean, Sungchan, Seoul, Korea). Slightly acidic electrolyzed water (SAEW) was produced by injecting 0.1% NaCl (Duchsan, Gwangju, Korea) into a non-membrane-electrolyzed water generator (HBS 3000; HanBio, Incheon, Korea) and applying a current of 60A, resulting in a pH of 5.8. Four sanitizers were freshly prepared immediately before use to maximize the sanitization efficacy. A chlorine meter (HI 95771; Chlorine DPD, LaMotte, MD, USA) was used to measure the chlorine concentrations in the sodium hypochlorite solution, SAEW, and chlorine dioxide solution, according to the manufacturer’s instructions. Peracetic acid concentration was measured using a peracetic acid test strip (Sigma-Aldrich).

Food contact surface selection and preparation of test coupons

American Iron and Steel Institute (AISI) 304 stainless steel and conveyor belt (polyurethane) coupons (2 × 3.5 cm) were cut from larger units of the material. Bottle wrappers collected from an enoki mushroom processing facility were divided into two components (Fig. 1): plastic parts (polypropylene, PP) and Velcro strips (Nylon) and cut into 2 × 3.5 cm coupons. The stainless steel coupons were cleaned with 70% ethanol and autoclaved at 121 °C for 15 min. The conveyor belt, plastic surface, and Velcro strip coupons were immersed in 100% methanol for 1 h at room temperature (approximately 22 °C), washed twice with distilled water, and cleaned with 70% ethanol for 1 h at room temperature. All coupons were dried in a biosafety cabinet overnight. Before L. monocytogenes inoculation, each coupon was placed in an individual Petri dish and exposed to UV light for 4 h to ensure no L. monocytogenes contamination.

Fig. 1.

Fig. 1

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Bottle wrapper used in Enoki mushroom farms

Bacterial cocktail preparation and inoculation on food contact surfaces

Each strain was inoculated into 10 mL of TSB and incubated for 24 h at 37 °C to obtain fully active cultures. To create five strain cocktails, equivalent volumes of each overnight-grown strain were combined in a 50 mL centrifuge tube. The cocktail was centrifuged at 4500 rpm for 15 min, and the supernatant was removed. To compared the antimicrobial effects depending on the presence of organic material, the cell pellet was resuspended in 1 mL of phosphate-buffered saline (Difco Laboratories) or mushroom extract medium, and the cell density was adjusted to 6–7 log CFU/mL (OD600 > 1.0) before use. Then, 200 µL of the diluted strain suspension was uniformly inoculated onto the food contact surfaces and allowed to dry for 2 h to facilitate L. monocytogenes attachment.

Chemical disinfectant treatment for food contact surfaces

Four types of surfaces such as stainless steel, conveyor belt, plastic part and Velcro strip of mushroom bottle wrapper inoculated with L. monocytogenes were dipped in 30 mL of each disinfectant solution at room temperature for 5, 10, 30, and 60 min. After dipping for each treatment time, the coupons were placed in 30 mL of D/E neutralizing broth (Difco Laboratories) for 2 min to neutralization. Next, 1 mL of the neutralized solution was serially diluted tenfold and plated on PALCAM agar (Difco Laboratories) to enumerate the surviving L. monocytogenes and plate colonies were counted after 48 ± 2 h at 35 °C. Negative controls were obtained by placing the food contact surfaces without bacterial cells and distilled water treatment for the surfaces inoculated with L. monocytogenes was used as the positive control.

Combination treatment with chemical disinfectants and dry sanitization for velcro strip coupon

L. monocytogenes inoculated on Velcro strip were treated with 50 and 100 ppm concentration of sodium hypochlorite and peracetic acid for 10 min, respectively. After treatment, the Velcro strip coupons were removed using sterile forceps, transferred to sterile Petri dishes, and dried at 50, 60, 70, and 80 °C for 2 h in a convection oven (VS-1202D4; Vision Bionex, Bucheon, Korea). The temperature was measured using a thermocouple data logger (PT100; Testo, Lenzkirch, Germany). The viable cell counts were determined using the method described above.

Combination treatment with wet and dry sanitization for velcro strip coupon

Autoclave heating was evaluated as a wet heating treatment. Tested temperatures ranged from 50 to 80 °C and duration of treatment varied from 15 to 90 min. After wet heating treatment for each duration time, the Velcro strips were subjected to dry sanitization at 50, 60, 70, and 80 °C for 2 h. The temperature was measured using a thermocouple data logger (PT100; Testo, Lenzkirch, Germany).

Data analyses

The mean number of colonies per mL was converted to log CFU/coupon, and the means were calculated. Statistical analyses were conducted using one-way analysis of variance (ANOVA) and Tukey’s honest significant difference test to determine any significant differences (p < 0.05) among the sanitization treatments. ANOVA and Tukey’s analysis was performed using the SPSS version 26 software (IBM SPSS Statistics). Each data was derived from 5-plicates measurements.

Results and discussions

Efficacy of chemical disinfectants against L. monocytogenes contamination on food contact surfaces

In the present study, the changes in the sanitizing efficacy by organic matter were examined by inoculating L. monocytogenes on food contact surfaces, both in the absence (using PBS) and presence of organic material (using mushroom extract medium). When L. monocytogenes cells were exposed to disinfectants without organic material, a significant reduction in their population was observed on all food contact surfaces: conveyor belts, stainless steel, and plastic wrappers (Table 1). Distilled water treatment did not lead to significant changes in microbial counts on the tested surfaces. However, sodium hypochlorite, peracetic acid, and chlorine dioxide effectively reduced the bacterial load by > 5 log CFU/coupon within 5 min on the all tested surfaces. Whereas a greater than 5-log reduction of L. monocytogenes with treatment of SAEW was achieved after 10 min. Remarkably, no growth of L. monocytogenes was detected after 30 min of exposure to sodium hypochlorite, peracetic acid, SAEW, and chlorine dioxide without organic matter such as mushroom exudates. Notably, stainless steel surfaces demonstrated the greatest reduction in bacterial counts with sodium hypochlorite treatment. As expected, the results of this study showed all disinfectants were highly effective in clearing the surfaces. The similar result was described by Byun et al. who reported that sodium hypochlorite achieved up to 5 log CFU/cm2 reduction after 5 min of contact time (Byun et al., 2021).

Table 1.

Populations of surviving Listeria monocytogenes on conveyor belts, stainless steel, Velcro strips, and plastic surfaces of bottle wrapper after treatment with 200 mg/L of sodium hypochlorite, peracetic acid, slightly acidic electrolyzed water, and chlorine dioxide in the absence of organic matter. Treatments were applied for 5, 10, 30, and 60 min in the absence of organic matters. (unit: logCFU/coupon)

Conveyor belt Stainless steel Plastic wrapper Velcro strip
Distilled water
0 min 6.31 ± 0.58 6.20 ± 0.67b 8.82 ± 0.58b 7.85 ± 1.22c
5 min 7.05 ± 0.43 7.64 ± 0.25 8.07 ± 0.42 8.26 ± 0.88
10 min 6.72 ± 0.07 7.56 ± 0.34 8.00 ± 0.10 7.82 ± 0.24
30 min 7.19 ± 0.06 7.05 ± 0.22 7.95 ± 0.07 7.55 ± 0.33
60 min 6.99 ± 0.29 7.22 ± 0.59 7.50 ± 0.29 7.15 ± 0.10
Sodium hypochlorite
0 min 6.31 ± 0.58c* 6.20 ± 0.67b 8.82 ± 0.58b 7.85 ± 1.22c
5 min n.da** n.da n.da 1.32 ± 1.28b
10 min n.da n.da n.da 1.16 ± 0.94ab
30 min n.da n.da n.da n.da
60 min 0.82 ± 1.43b n.da n.da n.da
Peracetic acid
0 min 6.31 ± 0.58b 6.20 ± 0.67c 8.82 ± 0.58b 7.85 ± 1.22b
5 min n.da 1.51 ± 1.61b n.da n.da
10 min n.da n.da n.da n.da
30 min n.da n.da n.da n.da
60 min n.da n.da n.da n.da
Slightly acidic electrolyzed water
0 min 6.31 ± 0.58b 6.20 ± 0.67c 8.82 ± 0.58b 7.85 ± 1.22c
5 min n.da 3.47 ± 0.48b n.da 1.09 ± 0.22b
10 min n.da n.da n.da n.da
30 min n.da n.da n.da n.da
60 min n.da n.da n.da n.da
Chlorine dioxide
0 min 6.31 ± 0.58b 6.20 ± 0.67d 8.82 ± 0.58b 7.85 ± 1.22b
5 min n.da 1.16 ± 2.01c n.da n.da
10 min n.da 0.83 ± 1.43b n.da n.da
30 min n.da n.da n.da n.da
60 min n.da n.da n.da n.da
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*a−dMean values with different uppercase letters within the same column denote significant (p < 0.05) differences

**n.d, not detected

The presence of organic matter, such as mushroom debris, compromised the efficacy of disinfectants across different food contact surface materials. As a result in Fig. 2, sodium hypochlorite exhibited good performance on stainless steel surfaces, achieving up to 5 log CFU/coupon reduction in L. monocytogenes populations after 5 min treatment on 200 mg/L concentration. This highlights the potent antimicrobial activity of sodium hypochlorite against pathogens on stainless steel surfaces, even in the presence of organic matter. Chlorine-based disinfectants are strong oxidizing agents, and their activity is the result of destroying cell membranes, oxidizing respiratory components, inhibiting protein synthesis, and hindering the biosynthesis of damaged DNA (Marriott et al., 2006). However, previous studies have shown that organic matters from food or plant debris can reduce the sanitization efficacy of chlorine-based sanitizers (Jo et al., 2018).

Fig. 2.

Fig. 2

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Efficacy of chemical disinfectants (200 mg/L) against Listeria monocytogenes on four food-contact surfaces in the presence of organic matter. (A) Sodium hypochlorite; (B) Peracetic acid; (C) Slightly acidic electrolyzed water; (D) Chlorine dioxide. Surface coupons were treated with disinfectants for 5, 10, 30, and 60 min. Surviving bacteria were enumerated and expressed as means ± SD (n = 5). Bars with different letters indicate significant differences (p < 0.05). n.d: not detected

Inactivation of foodborne pathogens by chlorine-based disinfectants was generally decreased with increasing the organic material concentrations. Our results showed that organic material, such as mushroom exudates, negatively influenced the sanitization efficacy of chlorine-based disinfectants compared to conditions without organic matter. However, significant reductions in L. monocytogenes were observed, varying across different types of food contact surfaces in the presence of organic matter. These results suggest that the type of surface is probable to be a significant factor influencing the effectiveness of chlorine-based disinfectants in eliminating foodborne pathogens on food contact surfaces. Peracetic acid was the most effective disinfectant on tested food contact surfaces, achieving enhanced bacterial reduction with increasing treatment time. Notably, after 30 min of exposure, L. monocytogenes was undetectable on the conveyor belt, plastic wrapper, and Velcro strip coupons treated with 200 mg/L peracetic acid. Whereas peracetic acid did not completely inactivate L. monocytogenes on stainless steel surfaces until after 60 min of treatment. These results were unexpected, since the high efficacy of peracetic acid on food contact surfaces has previously been reported (Small et al., 2007) and the antimicrobial activity of peracetic acid is relatively unaffected by organic matter compared to other oxidizing biocides (Kitis, 2004). Lee et al. observed that treatment with peracetic acid was able to inactivate the cells of L. monocytogenes on stainless steel and polystyrene surfaces (Lee et al., 2016). The similar result was described by Tote et al. who reported that peracetic acid eliminated viable S. aureus and Pseudomonas aeruginosa on the stainless steel surfaces after only 1 min (Tote et al., 2010). An opposite result was shown by Rossoni et al. who reported that peracetic acid was less effective than sodium hypochlorite in elimination the adherent cells of foodborne pathogens on stainless steel (Rossoni and Gaylarde, 2000). A similar trend was shown in a study conducted by Tomičić et al. where sodium hypochlorite was more effective than peracetic acid reducing the bacterial adherence against L. monocytogenes and Escherichia coli on stainless steel (Tomicic et al., 2023). Factors that influence the effectiveness of antimicrobial agents include surface hydrophobicity, electrostatic charging, roughness, and the presence of pores. The relationship between surface hydrophobicity and antimicrobial activity by disinfectants is not clear. However, the surface of stainless steel is considered hydrophobic (Yuk et al., 2006; Bartz and Schneider, 2006; Wu et al., 2018) and peracetic acid is generally hydrophilic solution. As a result, antimicrobial activity by peracetic acid against foodborne pathogens on hydrophobic stainless steel surfaces may be less effective compared to other hydrophilic surfaces (Vandekinderen et al., 2009).

For SAEW, a greater than 5-log reduction of L. monocytogenes was achieved within 30 min on the plastic wrapper surface. The count of viable cells after SAEW treatment for 60 min reveals that this disinfectant was the least effective in eliminating L. monocytogenes on conveyor belt (6.21 log CFU/coupon), following stainless steel (5.98 log CFU/coupon) and Velcro strip (4.45 log CFU/coupon). Similar results were observed with chlorine dioxide treatment. Compared with the initial cell level (8.47 log CFU/coupon) on plastic wrapper, the levels of L. monocytogenes were significantly reduced to < 1 log CFU/coupon after treatments with chlorine dioxide after 30 min. In addition, the cells inoculated on conveyor belt and plastic wrapper had counts below the detection limit of 1.0 log CFU/coupon after 60 min. The present study indicated that organic matter such as mushroom extract medium impaired efficacies of all sanitizers against L. monocytogenes on food contact surfaces, which is consist with the finding on polystyrene surface (Korany et al., 2018). In agreement with our findings, protein and fat residues on stainless steel reduced the efficacies of chlorine dioxide, hydrogen peroxide, and acidic electrolyzed water against L. monocytogenes (Vandekinderen et al., 2009; Moretro et al., 2019). The present results show that sodium hypochlorite was the most effective against L. monocytogenes on stainless steel surfaces and peracetic acid exhibited a 5-log reduction of viable cells on conveyor belt, plastic wrapper, and Velcro strips surface within 30 min in the presence of organic matter. As a result, we suggest applying the bacteria control strategies after the complete removal of residual debris to obtain the powerful antimicrobial activity, particularly in conveyor belt and plastic wrapper.

Efficacy of disinfectant and dry heat treatment against L. monocytogenes contamination on velcro strips

The development of control strategies to prevent L. monocytogenes contamination on plastic wrapper is crucial since it was placed around the bottle to form a cylindrical shape with mushroom for up to 20 days. Plastic wrappers typically comprise two components: a plastic surface and a Velcro strip that allows the two ends to adhere together securely. In our study, Velcro strips exhibited more resistant to disinfectants compared to plastic surfaces of plastic wrapper. As a result, the Velcro strip surfaces poses a greater challenge in eliminating L. monocytogenes compared to the plastic portion. Therefore, this study was conducted to identify the optimal microbial reduction technology for controlling Listeria contamination on food contact surfaces, specifically targeting Velcro strip of bottle wrapper.

Table 2 shows the result of antimicrobial activity against L. monocytogenes on the Velcro strip surfaces before and after treatment with distilled water, sodium hypochlorite (50 or 100 mg/mL), or peracetic acid (50 or 100 mg/mL) plus drying at 50, 60, 70, and 80 °C for 2 h. The initial population of L. monocytogenes on Velcro strip surfaces was 7.67 log CFU/coupon. After treatment of Velcro strips with distilled water for 10 min, the population reduced to 6.55 log CFU/coupon. When exposed to temperatures of 50, 60, and 70 °C after washing with distilled water, the viable cells of L. monocytogenes were reduced by 2.46, 2.67, and 2.49 log CFU/coupon compared to initial count (7.67 log CFU/coupon), respectively. Although rinsing with distilled water alone reduced by 1 log CFU/coupon against L. monocytogenes on the Velcro strip surface, dry heat treatment at 80 °C after rinsing with distilled water exerted synergistic effects, significantly reducing the attached L. monocytogenes population.

Table 2.

Synergistic effects of combined disinfectant and drying treatment on L. monocytogenes survival on Velcro strips of plastic wrapper. (unit: logCFU/coupon)

Disinfectant Concentration
(mg/mL)		 Initial count Count after treatment Viable cell count at air drying temperature
50 ℃ 60 ℃ 70 ℃ 80 ℃
Distilled water 7.67 ± 0.07d 6.55 ± 0.06c 5.21 ± 0.60b 5.00 ± 0.37b 5.18 ± 0.09b n.da
Sodium hypochlorite 50 2.82 ± 0.12b 3.55 ± 1.02c 1.08 ± 1.28ab n.da n.da
100 4.13 ± 0.19b 2.70 ± 0.46ab 0.92 ± 1.60a n.da n.da
Peracetic acid 50 4.38 ± 0.10b 0.87 ± 1.50a n.da n.da n.da
100 4.61 ± 0.25c 1.08 ± 1.23b n.da n.da n.da
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a−cMean values with different uppercase letters within the same row denote significant (p < 0.05) differences

**n.d, not detected

The combination treatment of disinfectants and dry heat was also more effective than disinfectants alone for elimination of L. monocytogenes on Velcro strip surfaces. When Velcro strips were treated with sodium hypochlorite at 50 and 100 mg/L for 10 min, the population of L. monocytogenes decreased significantly, by 3.54 and 4.06 log CFU/coupon, respectively. During drying for 2 h at 50 and 60 °C, L. monocytogenes cells on Velcro strip surfaces that had been treated with sodium hypochlorite at 50 and 100 mg/mL showed a significant decreasing trend but the viable cells were not fully eliminated on Velcro strip surfaces. Complete inactivation occurred at up to 70 °C. The effectiveness of disinfection treatment combining peracetic acid and dry heat indicated that the use of peracetic acid, even at low concentration, showed notable effectiveness in reducing L. monocytogenes on Velcro strip surfaces. At 50 °C after peracetic treatment with 50 or 100 mg/mL, L. monocytogenes exhibited a surviving cell concentration of 0.87 and 1.08 log CFU/coupon, respectively. Notably, exposure to temperature ≥ 60 °C after peracetic acid treatment at both concentrations led to complete elimination of L. monocytogenes on the Velcro strip surface. These finding highlight the potential of combining dry heat treatment with chemical disinfectants, particularly peracetic acid, for enhanced control of L. monocytogenes on challenging surfaces like Velcro strip surfaces in mushroom processing line. A study by Ruiz-Llacsahuanga et al. (2022) reported the 5 log reduction of Listeria innocua, a surrogate for L. monocytogenes, on stainless steel and plastic surfaces after thermal treatment with peracetic acid. Other researcher (Ohman et al., 2024) also demonstrated that peracetic acid treatment resulted in a greater reduction in L. monocytogenes compared to the use of quaternary ammonium compounds (QAC) and dry heat treatment after cleaning with chemical disinfectants was substantially more effective than cleaning alone on food contact surfaces (Sovljanski et al., 2023). The combination of sanitizers and heat was effective to remove attached Salmonella and L. monocytogenes on stainless steel and rubber (Carballo and Araujo, 2012). The present study demonstrated a high temperature air drying of 80 °C after rinsing with tap water for 10 min may be exhibit a synergistic effect for the reduction of attached L. monocytogenes on plastic wrapper. However, the surface material such as bottle wrapper is heated to 80 °C for 2 h, which deteriorated the plastic surface and melted the adhesive bond between the Velcro fabric and the plastic surface. While high-temperature above 80 °C shows excellent results in lab scale, it may not be practically suitable for plastic wrappers used in enoki mushroom production facilities.

Efficacy of wet and dry heat treatment against L. monocytogenes contamination on Velcro strips

As shown in Table 3, wet heat treatment at 80 °C was more effective than that at 70 °C in eliminating L. monocytogenes. L. monocytogenes count was 3.79 log CFU/coupon after wet heating at 70 °C for 15 min, but L. monocytogenes was not detected after wet heating at 80 °C for same time, indicating the enhanced antimicrobial activity of wet heat treatment at high temperatures against L. monocytogenes on Velcro strips. Moist heat is more effective than dry heat against Listeria. Crandall et al. reported that a 5-log reduction of L. innocua inoculated on stainless steel for deli slicers was observed when coupons were treated with moist heat at 82 °C for 3 h (Crandall et al., 2012). Whereas, L. monocytogenes on stainless steel, Polytetrafluoroethylene (PTFE), and rubber could survive a dry heat treatment of 85 °C for 15 min (Crandall et al., 2012). The superheated water at 125 °C was more effective than other cleaning methods for removing the pathogen cells and biofilm from food contact surfaces (Wirtanen et al., 1996) and hot water (90 to 100 °C) applied for 15 s to polyvinyl chloride caps with attached L. monocytogenes produced greater than a 6log-reduction (Berrang et al., 2014). Although wet heating at 60 and 70 °C did not completely eliminate L. monocytogenes, dry heating at temperatures ≥ 60 °C effectively reduced the L. monocytogenes count below the detection limit (< 1.0 log CFU/coupon). The wet heating to 75 °C resulted in sufficient level of reduction (5-log) on L. monocytogenes strains (Zhou et al., 2019) and wet heating at up to 75 °C reported similar results on Salmonella strains (Newkirk et al., 2018). In this study, wet heat treatment at 70 °C alone is insufficient to fully inactivate attached L. monocytogenes, combining it with a subsequent drying step at temperatures exceeding 60 °C could effectively reduce the pathogen levels below the detection limit. The effectiveness of combining wet heat and dry heat treatment against foodborne pathogens on food contact surfaces has not yet been fully investigated. It has also been observed that moist heat not only directly kills L. monocytogenes, but also induces heat shock response, which has been shown to continuously reduce the organism’s survivability (Lin et al., 2012). It seems likely that L. monocytogenes can be completely eliminated when food contact surfaces are fully dried, and implementing a drying step after cleaning can be utilized as an important microbial control strategy in enoki mushroom production facilities (Ohman et al., 2024).

Table 3.

Synergistic effects of combined wet heating and air drying on L. monocytogenes survival on Velcro strips of plastic wrapper. (unit: logCFU/coupon)

Wet heat temperature Treatment time Count after
wet heat treatment		 Viable cell count at air drying temperature
60 ℃ 70 ℃
Initial count: 8.81 ± 0.07
60 ℃ 15 min 3.79 ± 0.44b n.d n.d
30 min 3.75 ± 0.52b n.d n.d
60 min n.da n.d n.d
90 min n.da n.d n.d
70 ℃ 15 min 3.79 ± 0.41b n.d n.d
30 min 0.14 ± 0.53a n.d n.d
45 min n.da n.d n.d
60 min n.da n.d n.d
90 min n.da n.d n.d
80 ℃ 15 min n.d n.d n.d
30 min n.d n.d n.d
45 min n.d n.d n.d
60 min n.d n.d n.d
90 min n.d n.d n.d
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a−cMean values with different uppercase letters within the same column denote significant (p < 0.05) differences

**n.d, not detected

In conclusion, this study evaluated the efficacies of chemical disinfectants and thermal treatments, alone and in combination, against L. monocytogenes contamination on various enoki mushroom contact surfaces. Sodium hypochlorite, peracetic acid, SAEW, and chlorine dioxide reduced the L. monocytogenes count by approximately 6 log CFU/coupon after 10 min without organic matter. The greatest reduction was observed on stainless steel, with sodium hypochlorite reaching a count of 5 log CFU/coupon within 5 min. Although the presence of organic matter decreased the disinfectant efficacy, sodium hypochlorite remained effective on stainless steel. Peracetic acid was most effective on conveyor belts and plastic surfaces. Velcro strip surfaces were more resistant to disinfectants; however, combination of peracetic acid and dry heat treatment at ≥ 60 °C completely eliminated L. monocytogenes. The wet heat at 80 °C or the combination of wet heat at 70 °C plus dry heat at 60 °C also effectively reduced the contamination on the Velcro strips. These results suggested that utilizing residue heat above 80 °C from autoclaving for mushroom substrate could be an effective method for controlling L. monocytogenes. This approach may offer a practical and energy-efficient solution for Listeria control on plastic wrapper in enoki mushroom processing environment.

Acknowledgements

This research was supported by the grants (RS-2024-00230820) from the Rural Development Administration

Funding

This study was funded by Rural Development Administration, RS-2024-00230820.

Declarations

Conflict of interest

The authors declare no conflicts of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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