Combined effect of various salt concentrations and lactic acid bacteria fermentation on the survival of Escherichia coli O157:H7 and Listeria monocytogenes in white kimchi at different temperatures

Abstract

This study was conducted to investigate the effect of lactic acid fermentation and salt on the survival of Escherichia coli O157:H7 and Listeria monocytogenes in white kimchi containing various salt concentrations during storage at 4 and 15 °C. The survivals of pathogens during fermentation differed depending on salt concentrations and storage temperature. The survival of pathogens in kimchi containing 3% salt was higher than that in kimchi containing 1 and 2% salt, which may be related to the fact that lactic acid bacteria remained constant throughout the initial stage of fermentation. Thus, there was a lower reduction in the pH of kimchi containing 3% salt regardless of storage temperature. These protective effects may result from a gradual reduction in pH; however, the mechanisms are not clearly understood. Therefore, further investigations are needed to explain the mechanism by which lactic acid fermentation and salt in kimchi affect the growth of foodborne pathogens.

Introduction

Microbial food safety is a major safety concern in food industry since the presence of foodborne pathogens remains a cause of human foodborne illness and deaths worldwide. Especially, Escherichia coli O157:H7 (EC), Listeria monocytogenes (LM) and Salmonella spp. are the major foodborne pathogens that contaminate acidic foods; in particular, outbreaks of EC, have been responsible for the consumption of acidic foods (Lee et al., 2010; Topalcengiz and Danyluk, 2017).

Kimchi is a major traditional Korean fermented food made by mixing salted napa cabbage, garlic, ginger, green onion, and salted seafood, which is then fermented. It is considered a healthy food because of the anti-cancer, anti-inflammatory, blood cholesterol-reducing, and immune-boosting effects imparted by lactic acid bacteria (LAB) (Kwak et al., 2014). Furthermore, kimchi is generally recognized as safe because of its acidic pH and high salinity. The pH and salinity of commercial kimchi are from 3.8 to 4.2 and 2.5%, respectively (Inatsu et al., 2004; Patra et al., 2016). However, recently, it has been reported that several foodborne outbreaks, especially outbreaks of E. coli and coliform, were associated with kimchi consumption in South Korea. According to a school food service survey from 2010 to 2012, 50% of foodborne disease outbreaks occurred at schools in Gyeonggi-do Province may have been due to kimchi consumption (Kim et al., 2014). In 2012, 1642 children at seven schools in Incheon suffered from foodborne disease after eating kimchi contaminated by E. coli O169, which resulted from kimchi that had been processed after insufficient ripening periods (Cho et al., 2014). Additionally, outbreaks of E. coli O6 in 2013 and 2014 were significantly related with kimchi consumption (Shin et al., 2016). In 2014, E. coli O6 outbreaks affected 1022 students at 10 schools in Incheon province and associated with insufficiently fermented young radish kimchi (Shin et al., 2016). These suggested that large outbreaks may be highly attributed to short ripening period and this led to less acidic kimchi, which increased the hazard of incomplete microbial inhibition. Moreover, salted napa cabbage is the primary source of E. coli, and thus, it should be taken into account the inhibition of EC, which is one of the most harmful forms of E. coli and causes severe illness (Kim et al., 2015).

Salt is commonly used as an addictive to enhance the flavor of food. High salt concentrations can prevent the bacterial growth by lowering the water activity, thereby improving shelf-life of foods (Alide et al., 2020). Organic acids are also mainly used as food preservatives because of their antimicrobial effects (Ricke, 2003). Among them, lactic acid is widely applied in the food industry and lactic acid produced by LAB is considered key factors for inhibiting the growth of pathogens in kimchi. Combined treatment of acid and salt is widely employed in acidic foods, such as cheese and fermented meat and vegetables, due to antimicrobial effects and improvements in sensory quality (Rowe and Kirk, 1999). However, several studies have demonstrated that a combination of salt and lactic acid results in protective effects on foodborne pathogens. For example, Bae et al. (2017) observed an antagonistic effect of salt and lactic acid on EC. Yoon et al. (2014) found that the survival of Shigella flexneri in cucumber puree was significantly increased when treated with lactic acid and 3, 6, or 9% NaCl. Similarly, the addition of NaCl protected E. coli strains when challenged in media acidified with lactic acid (Casey and Codon, 2002). These findings have cast doubt on the protective effects of lactic acid and salt on EC in kimchi.

Although a number of studies reported the antagonistic effect of lactic acid and salt against EC, no study has investigated the relationship between the effects of lactic acid and salt on EC in kimchi. Kimchi is pickled in salt and then lactic acid can be produced by LAB as kimchi fermentation went on, thereby affecting the survival of foodborne pathogens in accordance with the effects of the combined treatment of lactic acid and salt. Especially, salt concentration and storage temperature affected the production of lactic acid in kimchi caused by LAB and therefore these factors may concern the combined effect of lactic acid and salt. Thus, in this study, we evaluated the combined effects of lactic acid produced by LAB fermentation and salt as well as the effects of different storage temperatures (15 and 4 °C) on the survival of EC (gram-negative) and LM (gram-positive) in white kimchi.

Materials and methods

Bacterial strains

EC ATCC 35150 and LM ATCC 19115 were individually cultured in 5 mL tryptic soy broth (TSB; Difco, Sparks, MD, USA) and TSB with 0.6% yeast extract (Difco), respectively, at 37 °C for 24 h. The cultures were centrifuged at 10,000 × g for 5 min, and the supernatant was decanted, and the pellet was resuspended in buffered peptone water (Difco). All cultures were maintained as frozen stocks at − 70 °C. Activated cultures were inoculated in their appropriate media for 24 h prior to experimental use.

Vegetable inoculation

Napa cabbage (Brassica rapa subsp. pekinensis) was cut into pieces sized approximately 3 × 3 cm, and the prepared culture of EC or LM was diluted to 10⁹ CFU/mL in 3 L distilled water. Prepared napa cabbage was immersed in 3 L of culture of EC or LM suspension for 20 min at room temperature (22 ± 2 °C), and then dried in a laminar flow biosafety hood for 2 h with the fan running.

White kimchi preparation and treatment

White kimchi was prepared according to the typical method of the Rural Development Administration, Republic of Korea. Inoculated napa cabbage was soaked in a 10, 20, and 30% brine solution for about 2 h and then drained. Garlic (Allium sativum), ginger (Zingiber officinale), Chinese radish (Raphanus sativus), and green onion (Allium schoenoprasum) were trimmed, washed, and transversely cut into 2–3 cm-long pieces. The brined napa cabbage was converted into white kimchi by adding the following prepared ingredients: 10 g of garlic, 5 g of ginger, 100 g of Chinese radish, 100 g of green onion, and 45 mL of fermented anchovy juice per 3 kg of brined napa cabbage. All components were thoroughly mixed and then brine (0, 3, and 6%) was added in 10, 20, and 30% brined napa cabbage kimchi to a final salt concentration of 1, 2, and 3%, respectively. The prepared white kimchi was left to ferment at 4 or 15 °C prior to bacterial enumeration.

Bacterial enumeration

To enumerate the pathogens and LAB at each temperature after different storage periods, the inoculated white kimchi samples (10 g) were diluted with 20 mL sterile 0.2% peptone water (PW; Difco) in sterile plastic stomacher bags and homogenized with a stomacher (BagMixer® 400, Interscience, Saint-Nom-la-Breteche, France) for 2 min. The diluted sample was serially diluted by 10-fold with sterile 0.2% PW, and 0.1 mL of the sample or diluent was plated onto MacConkey sorbitol agar (Difco), Oxford agar base (Difco) supplemented with Modified Oxford Antimicrobic Supplement (Difco), and Rogosa and Sharpe broth (Difco) for enumerating EC, LM, and LAB, respectively. The plates were incubated at 37 °C for 24 h, and then typical colony characteristics of the microorganisms were assessed. All experiments were repeated three times with duplicate plates.

Chemical analysis

The pH of the prepared white kimchi was determined using a pH meter (pHi 510, Beckman Coulter Inc., Brea, CA, USA) using homogenized samples diluted 1:1 with distilled water. The salinity of white kimchi before and after fermentation was determined using a portable salt meter (TDH/Salt-meter, Daeyoon Scale Industrial Co., Ltd., Seoul, Korea). The water activity of white kimchi was determined using a water activity meter (LabMASTER-a_w, Novasina, Lachen, Switzerland).

Statistical analysis

Before analysis, the mean of duplicate plates from three replicates was converted to log₁₀ CFU/g. Analysis of variance (ANOVA) for a completely randomized design was conducted using SAS (Version 9.4. SAS Institute, Cary, NC, USA). When the effect was significant (p < 0.05), means were separated with Duncan’s multiple range test.

Results and discussion

Effect of salt concentration on the survival of foodborne pathogens in white kimchi during fermentation at 15 °C

The survival of EC or LM and LAB in white kimchi containing various salt concentrations stored at 15 °C is shown in Figs. 1 and 2, respectively. Before storage, the initial levels of EC and LM in white kimchi containing various salt concentrations was approximately 4–5 log₁₀ CFU/g. As shown in Fig. 1A, B, the viability of EC in white kimchi containing 1 and 2% salt increased to 6.28 and 5.71 log₁₀ CFU/g, respectively, with a similar survival pattern in LAB after 2 days; subsequently, the survival of EC exhibited a marked decline to 2.26 and 1.87 log₁₀ CFU/g, respectively, at the end of fermentation. Similarly, LM in white kimchi containing 1 and 2% salt exhibited enhanced survival of 5.11 and 5.21 log₁₀ CFU/g, respectively, after 2–3 days and then decreased until the end of fermentation (Fig. 2A, B). In contrast, the populations of LM in white kimchi containing 3% salt increased from 4.04 to 5.10 log₁₀ CFU/g over 10 days of storage (Fig. 2C). Similarly, the viability of EC was higher than that found in white kimchi containing 1 and 2% salt at the end of storage period, suggesting that the inhibitory effect against pathogens was reduced with increasing salt concentrations.

Fig. 1.

Survival of EC ATCC 35150 and LAB in white kimchi containing various salt concentrations during storage at 15 °C. (A) 1% salt (B) 2% salt (C) 3% salt

Fig. 2.

Survival of LM ATCC 19115 and LAB in white kimchi containing various salt concentrations during storage at 15 °C. (A) 1% salt (B) 2% salt (C) 3% salt

In the presence of 1 and 2% salt, the populations of LAB exhibited a rapid increase during 3–5 days of storage and remained constant, reaching approximately 8 log₁₀ CFU/g after 10 days. However, in samples containing 3% salt, LAB grew slowly to the same level over 7 days of storage and remained constant until the end of storage (Figs. 1C and 2C). The populations of LAB in white kimchi inoculated with EC were > 2 log₁₀ CFU/g higher than that of LAB in kimchi inoculated with LM at the initial stage of fermentation; however, differences between these populations were not observed at the end of fermentation. In the present study, the populations of foodborne pathogens slightly increased in the presence of 1 and 2% salt at an early stage. These results imply that an increase in the populations of foodborne pathogens in samples containing 1 and 2% salt during storage for 2–3 days at 15 °C may be associated with a low salt concentration and insufficient growth of LAB at the initial stage of fermentation because LAB can sufficiently grow after a few days of fermentation, whereas 3% salt was adequate to control the number of foodborne pathogens regardless of the populations of LAB.

Effect of salt concentration on the survival of foodborne pathogens in white kimchi during fermentation at 4 °C

Storage at refrigerated temperature leads to the deceleration of kimchi fermentation. Accordingly, the populations of foodborne pathogens were rapidly reduced as the temperature increased. Generally, a similar trend between Figs. 3 and 4 was observed. Populations of LAB in white kimchi increased as the salt concentration increased. The populations of LAB in samples containing 1 and 2% salt increased to approximately 5 log₁₀ CFU/g over 7 days and then was maintained up to 28 days, while the populations of LAB in samples containing 3% salt did not change greatly for 7 days and then started to increase steadily after 7 days up to approximately 8 log₁₀ CFU/g. On the contrary, lower salt concentrations showed stronger inhibitory effects against both EC and LM, which coincided with the results at 15 °C. Thus, inhibitory effects in lower salt concentrations were attributed to the rapid increase in LAB population at the initial stage of fermentation. However, contrasting results between 15 °C and 4 °C were obtained, representing that the populations of foodborne pathogens did not increase at the initial stage of fermentation at 4 °C but instead remained, which may be due to the low temperature.

Fig. 3.

Survival of EC ATCC 35150 and LAB in white kimchi containing various salt concentrations during storage at 4 °C. (A) 1% salt (B) 2% salt (C) 3% salt

Fig. 4.

Survival of LM ATCC 19115 and LAB in white kimchi containing various salt concentrations during storage at 4 °C. (A) 1% salt (B) 2% salt (C) 3% salt

Overall, our results exhibited the enhanced survival of EC and LM in white kimchi containing 3% salt stored at 15 °C and 4 °C compared to that of pathogens in samples containing 1 or 2% salt. For example, the survival of both EC and LM in kimchi containing 1 and 2% salt declined from 3.33–4.31 to 1.29–1.90 log₁₀ CFU/g throughout the experimental period (Figs. 3A, B and 4A, B). In contrast, at 3% salt, the numbers of EC decreased by 1 log₁₀ CFU/g and that of LM slightly increased by 0.5 log₁₀ CFU/g (Figs. 3C and 4C). Similarly, the presence of 3% salt enhanced the survival of EC when treated with organic acids and this antagonistic effect on E. coli may result from a stress regulator known as RpoS, which plays a significant role in controlling the stress response to a treatment of organic acid and salt (Bae et al., 2017). However, two pathogens were more resistant to stress in samples containing 3% salt, especially under storage at 4 °C, indicating that there was no inhibition of pathogens by the end of the fermentation. The rapid decrease in pH at the initial stage of fermentation significantly affects the reduction in coliform levels (Song et al., 2019). Based on these findings, a rapid decrease in pH may also affect the decrease in EC and LM. Thus, the effect of pH on the survival of foodborne pathogens in various salt concentrations was investigated for subsequent experiments.

pH

It is generally known that the higher the storage temperature, the faster is the metabolism of LAB and probiotics, which leads to a reduction in pH (Jesus et al., 2016). Figure 5 represented the changes in pH during the fermentation of white kimchi containing various salt concentrations. The pH of white kimchi decreased more rapidly when it was soaked in lower salt concentrations, suggesting that the number of surviving pathogens was greater in white kimchi containing higher salt concentrations than in the kimchi containing lower salt concentrations. In this study, the results revealed that higher storage temperature reduces the pH rapidly, which can be explained by the fact that high temperatures of kimchi fermentation lead to a rapid reduction in pH compared to the results at low temperatures (Choi et al., 2018). In particular, the differences in pH between kimchi containing 3% salt and 1 or 2% salt were greater at 4 °C (Fig. 5C, D) than at 15 °C (Fig. 5A, B) during fermentation, which resulted in the highest survival of either EC or LM during the fermentation of white kimchi containing 3% salt stored at 4 °C. Interestingly, in the presence of 1 and 2% salt, the changes in pH were not associated with the number of LAB, but were associated with salt concentrations.

Fig. 5.

The pH of white kimchi containing various salt concentrations during storage at 15 °C and 4 °C. (A) EC, 15 °C (B) LM, 15 °C (C) EC, 4 °C (D) LM, 4 °C

Jordan and Davies (2001) reported that when acid-stressed EC cells were exposed to NaCl, their growth and recovery were enhanced because NaCl improved their ability to regulate intracellular pH. These results could also be closely related to the fact that a higher salt concentration, rather than lower salt concentrations, results in a higher pH in white kimchi. Cheigh et al. (1994) found that changes in pH are related to the fermentation characteristics and ripening period of fermented vegetables. Additionally, when the pH is reduced to less than 4.0 during lactic acid-based fermentation of vegetables, the growth of pathogens is limited (Savard and Champagne, 2017). This implies that the pH of fermented vegetables may be an important factor in the survival of pathogens. Similar results have been reported in previous studies, which found that a low-salt environment caused acceleration of the fermentation rate and pH reduction in fermented vegetables, such as kimchi and paocai, compared to a high-salt environment; therefore, microbial safety can be achieved in fermented vegetable using a low salt concentration (Park and Kim, 1991; Zhang et al., 2016).

Previous studies demonstrated that the survival of LM showed no significant differences between 0 and 7 days of fermentation in 60% of commercial Korean kimchi samples (Inatsu et al., 2004), which is consistent with our results. Furthermore, E. coli and Salmonella can grow if kimchi is not sufficiently fermented (Choi et al., 2018). This may be because insufficient fermentation can result in a pH greater than 4.0. To meet the standards of fermented food, the pH of the food product must be lower than 4.6 and a final pH of 4.1 or less is ideal (Eifert et al., 2020). This may explain why the populations of foodborne pathogens was not sufficiently decreased in samples containing 3% salt in this study. Therefore, sufficient fermentation (pH ≤ 4.1) is recommended for the microbial safety of kimchi, and it is important to reduce contamination during kimchi processing, such as washing materials with a sanitizing agent, cleaning food contact surfaces, and fast ripening. Taken together, to reduce the microbial hazard, reducing the sodium content of kimchi is important during ripening of kimchi at either 15 °C or 4 °C. During fermentation at 15 °C, it is recommended that kimchi should be fermented for at least 1 week. Unfortunately, longer fermentation period is needed when kimchi is fermented at 4 °C. Thus, if kimchi needs to be stored for long period of time, it should be fermented at a refrigerated temperature and high salt concentration is not recommended because refrigerated temperature inhibited or delayed the ripening of kimchi. Further investigation is also needed to determine the fermentation conditions of kimchi for microbial safety.

In conclusion, combined treatment with acid and salt is a type of hurdle technology commonly used to prevent foodborne pathogens in the food industry. However, our results revealed that this combination did not sufficiently inhibit the numbers of EC and LM in kimchi containing 3% salt stored at refrigeration temperature. This protective effect may be due to a gradual decrease in pH in the presence of 3% salt during the initial stage of fermentation and this pH value may be affected by salt concentrations. These findings are very advantageous for the kimchi industry to clarify which conditions cause the highest survival of EC and LM in kimchi.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.