Introduction

Microbiological contamination may occur at various points along the food chain, including production, processing, storage, distribution, and preparation. Spoilage microorganisms may result in the loss of nutritional and sensory properties of food products, such as discoloration, unpleasant odor, and off-flavor (Odeyemi et al., 2020). It is estimated that a significant portion of the total fruits and vegetables is wasted each year worldwide due to the microbial and biochemical spoilage of food (Alegbeleye et al., 2022), which causes immense economic losses for both producers and retailers. In addition, foodborne pathogens have become a serious public health threat in the last few years. The most common pathogenic microorganisms include Esherichia coli, L. monocytogenes, Bacillus cereus, Clostridium botulinum, Staphylococcus aureus, Campylobacter jejuni, and Salmonella spp. (Bintsis 2017). As noted by the World Bank, foodborne disease causes total economic loss of up to $95.2 billion within low- and middle-income countries, and the annual cost of curing foodborne illnesses is estimated at $15 billion (James and Segovia, 2020). Therefore, microbial contamination is a challenging and significant issue for the food industry.

In recent years, various processing techniques have been applied to ensure the microbiological quality and safety of food products. Thermal sterilization has been one of the most widely utilized methods to achieve long-term shelf stability of food products by inactivating microorganisms and endogenous enzymes (Dong et al., 2021; Gavahian et al., 2020; Wu et al., 2020). However, conventional thermal processing operations also lead to the loss of heat-sensitive nutrients (such as vitamins and phenolic antioxidants) and the generation of potential toxic compounds such as acrylamide, furan, and acrolein, resulting in lowered nutritional value and sensory quality of food products (van Boekel et al., 2010).

In recent years, with an increase of consumers’ demands for fresh and high-quality foods, nonthermal processing techniques, mainly pulsed electric fields, ultrasound, high hydrostatic pressure, cold plasma, and high-pressure carbon dioxide, have drawn considerable attention within the food industry (Anbarasan et al., 2022; Niveditha et al., 2021; Sruthi et al., 2022; Wu et al., 2020). Plasma-activated water (PAW), also known as plasma-treated water (PTW), is obtained by nonthermal plasma discharge over or in distilled water (Xiang et al., 2020). PAW exhibits excellent antimicrobial activity against various bacteria, yeast, mold, and viruses (Thirumdas et al., 2018). Because of its antibacterial efficacy and eco-friendly nature, PAW has been successfully applied to improve the microbial safety and quality of various food products, such as fruits, vegetables, meat and fish products, edible mushrooms, fish, eggs, and cereal products (Liao et al., 2019; Rahman et al., 2022; Xiang et al., 2019b). It has been reported that the antibacterial efficacy of PAW is influenced by the plasma discharge type, working gas, plasma activation time, and treatment time (Shaw et al., 2018; Xiang et al., 2018; Xu et al., 2016). In addition, certain environmental factors also affect the antimicrobial properties of PAW, such as organic matter (Xiang et al., 2019a) and temperature (Shen et al., 2016). Shen et al. (2016) discovered that the antibacterial activity of PAW against S. aureus decreased remarkably with increasing storage temperature (−80, −20, 4, and 25 °C). Similar findings were also observed by Tsoukou et al. (2020), who determined that storing PAW and plasma-activated saline at −80 or −150°C provided a better means to retain their bactericidal activity than higher temperatures (−16°C, 4°C, and room temperature) over long-term storage.

The above data suggested that temperature may affect the antimicrobial efficacy of PAW. However, to the best of our knowledge, the changes in the antibacterial and physicochemical properties of PAW after heating at different temperatures have not been well examined. Therefore, the current work aimed to investigate the influences of heating (30–80°C) on the antibacterial effects and physicochemical properties of PAW. In addition, the antimicrobial efficacy of PAW in combination with mild heating (40, 50, and 60°C) against L. monocytogenes and S. typhimurium was also investigated.

Materials and methods

Bacterial strains and chemicals

L. monocytogenes strain American Type Culture Collection (ATCC) 15313 used in this study was obtained from the ATCC. S. typhimurium strain China Center of Industrial Culture Collection (CICC) 21484 was purchased from the CICC (Beijing, China). Tryptic soy agar (TSA) and tryptic soy broth (TSB) were provided by Aobox Biotechnology Co., Ltd. (Beijing, China).

Cultivation of strains

L. monocytogenes (Gram-positive) and S. typhimurium (Gram-negative) were individually grown on TSA plates for 24 h at 37°C. One single colony of each strain was inoculated into 50 mL of TSB and incubated at 37°C overnight with constant shaking (120 rpm). Then, the cells were harvested by centrifugation at 5000 × g for 10 min at 4°C and washed thrice with sterile sodium chloride solution (0.85%, w/v). The cell pellets were then resuspended in 30 mL of sterile saline (0.85% NaCl) at a final density of approximately 9 log₁₀ CFU/mL.

Preparation of PAW

In this work, PAW was prepared with an atmospheric pressure plasma jet (APPJ) system based on gliding arc discharge in air (Xiang et al., 2019b). The distance between the atmospheric-pressure plasma jet nozzle and the water surface was 5 mm. Compressed air (approximately 0.18 MPa) was selected as the working gas at a flow rate of 30 L/min at the jet outlet. Every 200 mL of sterile distilled water (SDW) was activated by plasma (discharge power 750 W) for 60 s to acquire PAW.

Influences of heating on the antibacterial efficacy of PAW

As shown in Figure 1, an aliquot of PAW (1.0 mL) was put into microcentrifuge tubes, which were incubated in a shaking water bath for 10 min at different temperatures (30, 40, 50, 60, 70, and 80°C). After incubation, the PAW samples were quickly cooled to room temperature (Xiang et al., 2020). Thereafter, 100 μL of each bacterial suspension was violently mixed with 900 μL of the obtained PAW and then incubated for 8 min at ambient temperature (Xiang et al., 2019a). Thereafter, the obtained mixtures were 10-fold serially diluted with sterile saline solution (0.85% NaCl). Then 100 μL of the appropriate dilution was spread plated onto TSA plates. Colonies on the plates were counted after incubation at 37°C for 24 h and the results were expressed as log₁₀ CFU/mL. All of the tests were performed in triplicate.

Influences of heating on the physicochemical properties of PAW

An aliquot of PAW (1.0 mL) was put into the tubes and incubated in a shaking water bath for 10 min at different temperatures (30, 40, 50, 60, 70, and 80°C). Thereafter, the PAW samples were quickly cooled to room temperature and the main physicochemical properties of PAW were measured.

pH

The pH levels of PAW samples were measured using a SevenGo Duo pH Meter (Mettler-Toledo, Switzerland).

Oxidation reduction potential (ORP)

The ORP value was determined using a 501 rechargeable ORP composite electrode (INESA Scientific Instrument Co., Ltd., Shanghai, China) connected to a SevenGo Duo pH Meter.

Electrical conductivity

The electrical conductivity was measured using a conductivity meter (INESA Scientific Instrument Co., Ltd., Shanghai, China).

H₂O₂

The H₂O₂ concentrations of PAW samples were measured using a colorimetric detection kit (D799773-0050) according to the manufacturer’s protocol (Sangon Biotech Shanghai Co., Ltd., Shanghai, China).

NO₃⁻

The NO₃⁻ content of PAW samples was determined by ultraviolet absorption spectrometry at a wavelength of 220 nm (Shen et al., 2016).

NO₂⁻

The NO₂⁻ contents of PAW samples were measured with a colorimetric assay kit (D799311-0050, Sangon Biotech Shanghai Co., Ltd., Shanghai, China) according to the manufacturer’s instructions.

Antibacterial activity of PAW combined with mild heating

L. monocytogenes and S. typhimurium were selected to investigate the bactericidal efficacy of PAW in combination with mild heating. For PAW treatment alone, 900 μL of fresh PAW was mixed with 100 μL of bacterial suspension and incubated at 25°C for 4 min in a shaking water bath. For mild heating treatment alone, 900 μL of sterile saline solution was mixed with 100 μL of bacterial suspension and then incubated in a shaking water bath at 40, 50, and 60°C for 4 min, respectively. For the combined treatments, 900 μL of PAW was blended with 100 μL of bacterial suspension and then incubated in a shaking water bath at 40, 50, and 60°C for 4 min, respectively. Then, the mixtures were immediately cooled to room temperature and the bacterial viability was evaluated with the previously described plating method. The SDW-treated cells served as the control.

Statistical analysis

All analyses were performed in at least triplicate. Data are presented as the mean ± standard deviation (SD) and were analyzed using SPSS version 24.0 for Windows (IBM SPSS Inc., Chicago, IL, USA). Statistically significant differences were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple-range test with a level of P < 0.05.

Results and discussion

Influence of heating treatment on the inactivation efficacy of PAW

The influences of mild heating on the inactivation efficacy of PAW against L. monocytogenes and S. typhimurium are illustrated in Figure 2. The initial populations of L. monocytogenes and S. typhimurium vegetative cells were 9.21 log₁₀ CFU/mL and 9.28 log₁₀ CFU/mL, respectively. After the treatment with PAW preheated at 30°C, the population of L. monocytogenes decreased significantly by 1.54 log₁₀ CFU/mL (P < 0.05). The heating treatment (40–80°C for 10 min) caused a significant decrease in the antimicrobial activity of PAW against L. monocytogenes (Figure 2A). After the treatments of PAW preheated at 40, 50, 60, 70, and 80°C, the populations of L. monocytogenes were markedly reduced by 1.40-, 1.17-, 1.20-, 1.02-, and 0.85-log values, respectively, which were lower than that of PAW preheated at 30°C (P < 0.05). A similar trend was also observed for S. typhimurium (Figure 2B). These data show that the heating treatment at 40–80°C for 10 min causes a significant decrease in the inactivation efficacy of PAW against L. monocytogenes and S. typhimurium. These data are consistent with previous findings of Shen et al. (2016) and Tsoukou et al. (2020) that PAW exhibits higher antibacterial activity when stored at low temperature.

Effect of heating treatment on the pH values of PAW

The influences of heating treatment on the antimicrobial activity of PAW may be attributed to the changes in the physicochemical properties of PAW. The acidification of plasma-treated liquid has been previously reported, and acidic pH is considered to play a crucial role in the bactericidal action of PAW (Thirumdas et al., 2018). As seen in Figure 3A, the pH value of PAW was 3.17 after plasma activation for 60 s, which was significantly (P < 0.05) lower than the 6.20 of SDW. The acidification of plasma-treated solution is mostly attributed to the formation of NO₃⁻, NO₂⁻, and peroxynitrites from reactive nitrogen species (RNS) during plasma discharge (Liao et al., 2018; Thirumdas et al., 2018). After preheating at 30°C for 10 min, the pH value of PAW increased to 3.23. As shown in Figure 3A, there were no significant changes in the pH values of PAW samples after the heating treatment at 30–80°C for 10 min (P > 0.05). A similar finding has been reported in our previous study (Xiang et al., 2020), in which the pH values of PAW remained essentially stable after mild heating at 25–55°C for 30 min.

Influence of heating treatment on the ORP values of PAW

ORP is a measure of the ability of a solution to oxidize or reduce another substance and is related to the concentration of oxidizers as well as their activity and strength (Thirumdas et al., 2018). As presented in Figure 3B, after plasma activation for 60 s, the ORP value of SDW was 298.00 mV, and the ORP value of PAW was 571.83 mV (P < 0.05). Similar findings were reported by Joshi et al. (2018) that the ORP of PAW was 534.52 mV, which was significantly higher than the ORP of distilled water of 376.54 mV. According to previous reports, the higher ORP value of PAW is associated with the production of reactive species and plays a crucial role in the antibacterial action of PAW (Thirumdas et al., 2018). Thus, ORP serves to be a good indicator for the efficacy of PAW and its antimicrobial capacity (Joshi et al., 2018). As shown in Figure 3B, no significant changes in the ORP values of PAW were observed after heating at 30–80°C for 10 min (P > 0.05). Similar findings have been reported in our previous study (Xiang et al., 2020) and elsewhere (Shen et al., 2016). For example, there was no obvious change in the ORP of PAW samples stored at −80, −20, 4, or 25°C for up to 30 days (Shen et al., 2016).

Influence of heating treatment on the electrical conductivity of PAW

Electrical conductivity is commonly used to assess the ability of a liquid to conduct an electrical current and is directly related to the concentration of ions in the solution. As shown in Figure 3C, the electrical conductivity of PAW was 409.00 μS/cm after plasma activation for 60 s, which was significantly higher than the 4.22 μS/cm of SDW (P < 0.05). The increase in the conductivity of PAW may be attributed to the formation of active ions in water during plasma activation (Joshi et al., 2018; Suwal et al., 2019). Similarly, Sergeichev et al. (2021) also found a significant increase in the electrical conductivity of water after microwave discharge plasma exposure. Preheating at 30 or 40°C for 10 min caused no significant changes in the electrical conductivity of PAW (P > 0.05). However, an increase in the electrical conductivity of PAW was noticed after the heating treatment at 40–80°C for 10 min (Figure 3C). The electrical conductivity of PAW was increased after heating treatment at 40–80°C for 10 min (P < 0.05). In addition to the amount and composition of ionic species, the temperature also affects the electrical conductivity of a solution (Zhang et al., 2020b). At elevated temperatures, the ionization and the mobility speeds of the ions in solution may be accelerated, leading to an increase in their conductivity. Further investigations are still needed to understand the role of electrical conductivity in the antibacterial efficacy of PAW.

Effect of heating treatment on reactive species levels of PAW

Various reactive oxygen and nitrogen species (RONS) are generated in solutions during plasma activation, including H₂O₂, nitrite, nitrate, ozone, hydroxyl radicals, and superoxide anions (Thirumdas et al., 2018). As depicted in Table 1, the concentration of H₂O₂ in PAW was 38.13 μmol/L after 60 s of plasma activation, which was in agreement with previous studies (Sergeichev et al., 2021; Shen et al., 2016). Shen et al. (2016) observed that the H₂O₂ concentrations of PAW stored at −80 °C did not vary significantly over 30 days. In contrast, the H₂O₂ levels in PAW samples decreased to 6.0 μmol/L from approximately 24.4 μmol/L after 30-day storage at −20, 4, and 25°C (Shen et al., 2016). H₂O₂, a long-lived powerful oxidizing agent, is thought to be involved in the antimicrobial action of PAW. As one of the main reactive oxygen species in PAW, H₂O₂ is thought to be mainly generated by the following chemical reactions at the gas/liquid interface (Thirumdas et al., 2018).

H₂O + e⁻→ H• + •OH + e⁻ (1)

H₂O + e⁻→ H⁺ + •OH + 2e⁻ (2)

•OH + •OH → H₂O₂ (3)

As displayed in Table 1, the H₂O₂ levels of PAW decreased following heating treatment at 30–80°C for 10 min, which was in accordance with the decreasing trend of bactericidal activity (Figure 2). The concentration of H₂O₂ decreased from 38.13 to 29.00 μmol/L as the heating temperature increased from 30 to 80°C. As a reactive molecule, H₂O₂ is unstable and decomposes exothermally into water and oxygen gas by the following reaction:

2 H₂O₂(aq) → 2 H₂O(l) + O₂(g) (4)

Heating, light, and catalysts can accelerate this decomposition process (Anikin et al., 2022). Therefore, it can be speculated that the decrease in the bactericidal efficacy of PAW may be related to the demonstration of H₂O₂ accelerated by the heating treatment.

RNS, including NO₃⁻, NO₂⁻, and peroxynitrites, are also thought to play significant roles in the antimicrobial properties of PAW (Schnabel et al., 2014; Thirumdas et al., 2018). As shown in Table 1, the NO₃⁻ and NO₂⁻ levels of PAW increased to 921.18 and 812.29 μmol/L, respectively, after plasma treatment for 60 s. NO₃⁻ and NO₂⁻ in PAW are generated by the dissolution of nitrogen oxides formed by gas-phase reactions of N₂ and O₂ or H₂O in the air plasma (Pavlovich et al., 2014).

N₂(g) + O₂(g) → 2NO(g) (5)

2NO(g) + O₂(g) → 2NO₂(g) (6)

NO(g) + O₃(g) → NO₂(g) + O₂(g) (7)

2NO₂(g) + H₂O(l) → NO₂⁻ + NO₃⁻ + 2H⁺ (8)

NO₂⁻ + H₂O₂ + H⁺ → NO₃⁻ + H₂O + H⁺ (9)

The heating treatment also caused remarkable changes in the levels of NO₃⁻ and NO₂⁻ in PAW. After incubation at indicated temperature (30–80°C) for 10 min, the NO₃⁻ amounts in PAW increased remarkably in a temperature-dependent manner (Table 1). As the heating temperature increased from 30 to 80°C, the content of NO₃⁻ increased from 932.00 to 1135.29 μmol/L. Similar findings were reported in previous studies, which showed that the NO₃⁻ concentration of PAW increased in a temperature-dependent manner following mild heating at 25–55°C for 30 min (Xiang et al., 2020). In addition, Shen et al. (2016) reported that the NO₃⁻ in PAW was more suitable during storage at higher temperatures, with 25°C > 4°C > −20°C > −80°C obtained in descending order for the NO₃⁻ contents in PAW. In contrast with the changing tendency of NO₃^-, the levels of NO₂⁻ in PAW decreased remarkably with increasing heating temperatures from 30 to 80°C (Table 1). The significant changes in the NO₂⁻ and NO₃⁻ levels may contribute to the decrease in the antibacterial activity of PAW (Figure 2).

In this work, only the contents of long half-life reactive species (H₂O₂, NO₃⁻, and NO₂⁻) in PAW were measured after heating treatment. Many highly reactive species with short lifetimes may also be involved in the antimicrobial action of PAW. Therefore, in future studies, more attention should be paid to the stability of the short half-life of reactive species in PAW during heating treatment or storage.

Synergistic antibacterial efficacy of PAW with mild heating against bacteria

The antibacterial activity of PAW combined with mild heating (40, 50, and 60°C) against L. monocytogenes and S. typhimurium was also investigated. As exhibited in Figure 4, L. monocytogenes and S. typhimurium decreased by 0.83- and 1.29-log values, respectively, after PAW treatment alone at 25°C for 4 min. The population of L. monocytogenes decreased by 0.01, 0.30, and 2.06 log₁₀ CFU/mL following the mild heat treatments at 40, 50, and 60°C for 4 min, respectively. Although mild heating decreased the antibacterial capacity of PAW (Figure 2), the combined treatment of PAW and mild heating showed a greater antibacterial effect against L. monocytogenes than any other single treatment. As indicated in Figure 4A, the populations of L. monocytogenes were reduced by 3.55, 6.61, and 7.83 log₁₀ CFU/mL, followed by the PAW treatment combined with mild heating at 40, 50, and 60°C for 4 min, respectively. The enhanced antibacterial efficacy of PAW combined with mild heating was also observed for S. typhimurium (Figure 4B). Similarly, the populations of S. typhimurium cells were reduced by 4.70 and 7.35 log₁₀ CFU/mL, respectively, after PAW treatment combined with mild heating at 40 and 50°C for 4 min. When treated with PAW at 60°C, the population of S. typhimurium was reduced from approximately 9.35 log₁₀ CFU/mL to an undetectable level (Figure 4B). Similar findings were also reported in previous studies (Bai et al., 2020; Choi et al., 2019; Liao et al., 2020; Zhang et al., 2020a). For instance, PAW exhibited greater antimicrobial activity against B. cereus spores at higher temperatures (Bai et al., 2020). Zhang et al. (2020a) also found that Saccharomyces cerevisiae decreased by 4.40-log after the synergistic combination of PAW and mild heat at 50°C for 6 min, which was significantly higher than the individual treatments of PAW at 25°C (0.27-log) or mild heat at 50°C for 6 min (1.92-log).

Although preheating at 40–80°C for 10 min caused a significant decrease in the inactivation efficacy of PAW, PAW combined with mild heating (40, 50, and 60°C) exhibited enhanced antibacterial activity against L. monocytogenes and S. typhimurium (Figure 4). Zhang et al. (2020a) found that the synergistic antifungal activity of PAW combined with mild heat against S. cerevisiae might be attributed to membrane damage, oxidative stress, and disruption of mitochondrial membrane potential. Liao et al. (2020) also observed the disruption of external structure and leakage of intracellular components in B. cereus spores after the combination treatment of PAW and mild heat. In the future, the mechanisms underlying the synergistic antimicrobial action of PAW and mild heating should be investigated with transcriptomic, proteomic, and metabolomic approaches. The practical application of PAW and mild heating has been reported in previous studies. For instance, the sequential application of PAW and mild heat was more effective in the inactivation of the naturally contaminating microorganisms as well as L. monocytogenes and S. aureus inoculated into salted kimchi cabbage (Choi et al., 2019). The combined treatment did not deteriorate the quality of salted Chinese cabbage, including the salinity, acidity, moisture content, and reducing sugar content. Similar findings were also reported by Liao et al. (2020) who found that PAW combined with mild heat (40 and 55°C) exhibited stronger inactivation efficacy against B. cereus spores in rice without adverse effects on the texture and sensory qualities of the rice after cooking. More attention should be paid to the application of this method in other perishable foods, such as fruits and vegetables. In addition, the influences of PAW combined with mild heat on the nutritional and sensory qualities of foods also need to be assessed in more detail.

Conclusion

In summary, heating treatment at 40–80°C for 10 min caused a significant decrease in the antibacterial efficacy of PAW against L. monocytogenes and S. typhimurium. Heating treatment caused significant changes in the physicochemical properties of PAW, mainly including electrical conductivity, H₂O₂, NO₂⁻, and NO₃⁻, which may contribute to a reduction in its antibacterial effect. These data suggest that temperature is one of the important factors affecting the antibacterial activity of PAW. Interestingly, however, PAW combined with mild heating (40–60°C for 4 min) exhibited enhanced antibacterial activity against L. monocytogenes and S. typhimurium. The results in this work provide a basis for the practical application of PAW in the food industry. In future studies, more attention should be paid to the application of this method to various foods and its influences on the nutritional and sensory qualities of foods. In addition, more research is needed to reveal the mechanisms underlying the synergistic antimicrobial action of PAW and mild heat.