Effect of Different Conditions of Citric Acid and Acetic Acid Decontamination against Listeria Monocytogenes in Lettuce

Wan Norhafizah Wan Azman and John Yew Huat Tang*

Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin (Besut Campus), 22200 Besut, Terengganu, Malaysia.

Received | February 22, 2024; Accepted | August 06, 2024; Published | October 07, 2024

*Correspondence | John Yew Huat Tang, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin; Email: [email protected]

Citation | Azman, W.N.W. and J.Y.H. Tang. 2024. Effect of different conditions of citric acid and acetic acid decontamination against Listeria monocytogenes in lettuce. Sarhad Journal of Agriculture, 40(Special issue 1): 22-27.

DOI | https://dx.doi.org/10.17582/journal.sja/2024/40/s1.22.27

Keywords | Acetic acid, Citric acid, Listeria monocytogenes, Lettuce, Time, Concentration

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The pathogenic bacteria that causes listeriosis in humans is called Listeria monocytogenes. Being facultative anaerobic, the bacteria may live in both oxygen-rich and oxygen-deficient environment. It represents an important pathogen because L. monocytogenes can tolerate osmotic stress and persist at temperatures below freezing (Rhoades et al., 2009). Food contaminated with L. monocytogenes can cause listeriosis, a potentially fatal infection. It is a significant public health hazard in the US, where an estimated 1,600 individuals get critically ill from it every year, leading to 260 deaths, according to the US Centers for Disease Control and Prevention (CDC) (Rogalla and Bomar, 2023).

According to Oliver et al. (2005) and Rahimi et al. (2010), listeriosis is linked to consumption of tainted milk, soft or semi-soft cheese, undercooked, ready to eat foods, and unwashed raw fruits and vegetables. The condition primarily affects elderly people, pregnant women, infant, and adults with compromised immune systems. People who are not in these groups are less likely to become ill from it, but they can be impacted on occasion. According to the CDC, infected people may have fever, muscle aches, and gastrointestinal symptoms such as nausea or diarrhea.

Fruits and vegetables are a key part of a healthy diet, which is why there is a global push to boost their consumption (FAO/WHO, 2005). Globally, people are becoming more health conscious these days, and they tend to eat more veggies. Given that vegetables are frequently eaten in their least processed state, the public would be concerned if L. monocytogenes was found in them (Dogbe, 2010). Foods classified as minimally processed are those that are produced, consumed raw, and have not undergone any formal processing or treatment to lower the levels of harmful bacteria, their spores, or their toxins. The purpose of this study is to examine the effects of different citric acid and acetic acid concentrations, application methods, and time duration on decontamination against L. monocytogenes on lettuce.

Materials and Methods

Preparation of vegetables

The selected vegetable lettuce is purchased from the local hypermarket. Damaged layer, leaves and the lettuce core lettuce were removed and discarded. The selected leaves were then cut into circular pieces with a diameter of 4 cm each using sterile scalpel. The leaves were then placed in plastic Petri dish.

Preparation of L. monocytogenes inoculums

The strain of L. monocytogenes (ATCC 13932) was grown in the 10 ml tryptic soy broth (Merck, Germany) for 24 h at 30oC. The culture was poured into the eppendorf tube and centrifuged using the centrifuging machine (5000 rpm, 10 min, Eppendorf Centrifuge 5418). Cell pellets was resuspended in saline solution, and measured the optical density using the spectrophotometer with wavelength of 600 nm. The L. monocytogenes inoculum was set at absorbance 1.949 equals to approximately 6.0×109 cfu/ml.

L. monocytogenes inoculated on the salad vegetables

A 0.1 ml of the washed bacterial culture of concentration 109 cfu/ml was inoculated onto the upper surface of the lettuce. The inoculum of L. monocytogenes was spot-inoculated in 6-8 droplets and then spread around using the pipette tip for the entire surface to be covered as evenly as possible. While spreading the inoculums, the cut edge of the vegetables was avoided. Then, the inoculum was allowed for contact with the vegetables for 24 h and kept at 4oC.

Decontamination of vegetables

The decontamination methods that were conducted are:

1. Dipping the lettuce into the acetic acid solution with different concentration and time (without agitation)
2. Dipping the lettuce into the citric acid solution with different concentration and time (without agitation)
3. Dipping the lettuce in the acetic acid solution with different concentration and time (with agitation)
4. Dipping the lettuce in the citric acid solution with different concentration and time (with agitation)

Using the samples that are incubated earlier, the decontamination experiments was carried out to investigate the effect of the different decontamination methods on the reduction of the L. monocytogenes.

The acetic acid solution and citric acid solution of concentration 0.5%, 1.0%, 1.5% and 2.0% v/v was prepared. The pH of the two solutions with different concentration was measured using the pH meter (CRISON micropH2001). Then, the samples that are incubated earlier were treated with the different solution of different concentration, time and physical application. The vegetable was immersed in the solution with different parameters in the beaker. For the treatment with agitation, the vegetable was immersed in the solution and agitated with the magnetic stirrer hot plate. Lastly, the vegetables samples were drained and placed in sterile bag that contain saline solution to dilute the remaining acid residue on the vegetables.

Enumeration of L. monocytogenes

The vegetables samples were transferred into the stomacher bag containing 50 ml saline solution and homogenized for 30s by mixing the solution with samples. The serial dilutions were done using the homogenized samples (0.1 ml) until 10-4 cfu/ml. For the enumeration of L. monocytogenes, the samples from the serial dilution were spread on the surface of the PALCAM agar (Merck, Germany). The plates were incubated at 30oC for 48 hours. The colony counts will be transformed to log cfu/cm2 for the vegetable samples. While the uninoculated samples were also stored under the same condition (30oC and 48 h) and was analyzed for the presence of the L. monocytogenes.

Data analysis

The microbial counts were transformed to the logarithms before calculating the means and standard deviations. The population densities are using log cfu/cm2 for the vegetables sample. Thus, the population densities of L. monocytogenes at the end result were compared to determine the effectiveness of the decontamination methods. The data from the analysis were evaluated using the Tukey’s post-hoc test (SPSS). The significance level for all tests is 0.05.

Results and Discussion

Kim et al. (2011) stated that lettuce is one of the most widely consumed vegetables that undergoes minimal processing, meaning it is typically consumed raw. It is challenging to guarantee the safety and quality elements of the vegetables throughout production because the minimally processed vegetables are not subjected to harsh preservation methods (Kondo et al., 2006).

Table 1 shows the outcome of the study on the impact of time and citric acid solution concentration on the survival of L. monocytogenes inoculation on salad vegetable surfaces. L. monocytogenes was not detected in all the uninoculated lettuce samples.

The L. monocytogenes colonies were counted 24 h after being incubated at 30°C in the incubator. An inoculum size of 6.48 log cfu/cm2 of L. monocytogenes were recovered on the PALCAM agar. Sterile distilled water without citric acid serve as control throughout the study. Table 1 summarized the result of citric acid as decontamination agent for lettuce artificially contaminated with L. monocytogenes. Physical force of agitation have shown effectively reduce the numbers of L. monocytogenes even without the use of citric acid. Decontamination with agitation in sterile water showed significant reduction of L. monocytogenes to 4.55 log cfu/cm2 and 4.24 log cfu/cm2 after 15 min and 30 min, respectively.

 

Table 1: The effect of the different concentration of citric acid (0, 0.5, 1.0, 1.5, and 2.0%), time (0, 15, and 30 min) and the method (agitate, without agitate) on lettuce inoculated with L. monocytogenes (log cfu/cm2).

Concentration (%)	Time
	0 min	15 min	30 min
Agitation
0.0	6.48±0.00aA	4.55±0.04bA	4.24±0.23bA
0.5	6.48±0.00aA	2.95±0.92bB	0.00±0.00cB
1.0	6.48±0.00aA	0.00±0.00bC	0.00±0.00bB
1.5	6.48±0.00aA	0.00±0.00bC	0.00±0.00bB
2.0	6.48±0.00aA	0.00±0.00bC	0.00±0.00bB
Without agitation
0.0	6.48±0.00aA	5.94±0.33abA	5.72±0.00bA
0.5	6.48±0.00aA	5.70±0.61abA	4.83±0.59bAB
1.0	6.48±0.00aA	4.95±0.95abB	4.29±1.32bB
1.5	6.48±0.00aA	0.00±0.00bC	0.00±0.00bC
2.0	6.48±0.00aA	0.00±0.00bC	0.00±0.00bC

 

a, b, c different small letters within row indicate significant difference (p<0.05). A, B, C different capital letters within column indicate significant difference (p<0.05).

 

Fifteen minutes decontamination duration capable of removing all the pathogen with citric acid with concentration 1.0% and above. Concentration of 0.5% citric acid reduce the numbers to 2.95 log cfu/cm2. Longer treatment duration (30 min) proved to increase the effectiveness in removing L. monocytogenes from lettuce with citric acid (0.5%) significantly remove all the inoculated L. monocytogenes. Soaking lettuce without agitation, L. monocytogenes was significantly reduced after 30 mins in control, 0.5, and 1.0% of citric acid to 5.72, 4.83 and 4.29 log CFU/cm2, respectively. Citric acid concentration of 1.5 and 2.0% significantly reduced the number of L. monocytogenes after 15 mins to no detectable level. Based on the result, the agitation plays an important role in reducing the numbers of L. monocytogenes at a higher number and shorter time.

The effects of acetic acid treatment on the inactivation of L. monocytogenes on the lettuce are shown in the Table 2. The number of L. monocytogenes was reduced significantly after the samples are treated with 0.5% acetic acid for 15 min with agitation. Increasing the treatment time from 15 to 30 min did not show any significant (p< 0.05) reduction in the number of L. monocytogenes. Increasing acetic acid concentration to 1.0, 1.5 and 2.0% significantly reduced the numbers to non-detectable level after 15 mins of treatment.

 

Table 2: The effect of the different concentration of citric acid (0%, 0.5%, 1.0%, 1.5%, 2.0%), time (0, 15, and 30 min) and the method (agitate, without agitate) on lettuce inoculated with L. monocytogenes (log cfu/cm2).

Concentration (%)	Time
	0 min	15 min	30 min
Agitation
0.0	6.41±0.08aA	4.47±0.66bA	4.32±0.17bA
0.5	6.41±0.08aA	0.00±0.00cB	0.00±0.00cB
1.0	6.41±0.08aA	0.00±0.00cB	0.00±0.00cB
1.5	6.41±0.08aA	0.00±0.00bB	0.00±0.00bB
2.0	6.41±0.08aA	0.00±0.00bB	0.00±0.00bB
Without agitation
0.0	6.41±0.08aA	6.68±0.66aA	6.35±0.11aA
0.5	6.41±0.08aA	4.37±0.59bB	4.30±0.28bB
1.0	6.41±0.08aA	4.48±0.30bB	4.39±0.16bB
1.5	6.41±0.08aA	5.84±0.07bB	4.77±1.09bB
2.0	6.41±0.08aA	0.00±0.00bC	0.00±0.00bC

 

a, b, c different small letters within row indicate significant difference (p<0.05). A, B, C different capital letters within column indicate significant difference (p<0.05).

 

Treatment without agitation showed no significantly reduction in control samples after 15 and 30 mins. However, significantly reduction was seen with 15 mins of treatment with 0.5, 1.0, 1.5 and 2.0% acetic acids with 4.37, 4.48 and 5.84 log CFU/cm2, respectively. Increasing the treatment duration to 30 mins did not significantly reduced the L. monocytogenes numbers. Acetic acid with 2.0% reduced the L. monocytogenes to no detectable level.

According to Tirpanalan et al. (2011), organic acid is useful in lowering the number of L. monocytogenes because acetic and citric acids have antimicrobial qualities that can lower the pH of the solution and, through the dissociation of hydrogen ions, lower the internal cellular pH of the organism. The disruption of the cell’s ability to maintain the internal cellular pH can have an impact on the permeability of the membrane and substrate transport (Betts and Elveris, 2005). Beuchat and WHO (2002) reported that washing fruits and vegetables surfaces with organic acid may help lower the amount of microorganisms present. It has been demonstrated that simple procedures such as rubbing lemons, which contain citric acid, on sliced fruits can be effective to either eliminate or significantly slow the growth of bacteria. Other study also reported similar finding of lemon juice application to the surface of jicama and papaya that have been contaminated with Salmonella typhi reduces the numbers compared to the control group temporarily because the numbers increased after several hours (Escartin et al., 1989). Another study found that treating salad greens with up to 1500 ppm of citric acid had no effect on the growth of bacteria over the four days that they were stored at 10oC (Shapiro and Holder, 1960).

Organoleptic changes may occur for vegetables treated with organic acids as shown by Akbas and Ïlmez (2009) that acetic acid change lettuce organoleptic properties at concentration greater than 1.0%. However, it is unclear whether citric acid can alter the organoleptic qualities of the vegetables. Thus, it is preferable to attain the greatest degree of microbial population decrease at the lowest concentration of organic acids with shortest treatment duration as a precaution to preserve the vegetables quality and safety. Therefore, the physical application (agitation) can be employed to increase the efficacy of the organic acid decontamination treatment.

The lipophilic qualities of the organic acids such as citric acid, acetic acid, malic acid, tartaric acid, sorbic acid, and lactic acid allow them to permeate the pathogens plasma membrane and acidify the inside of the cells (Booth et al., 1989). Although organic acids are categorized as weak acids but they are capbale of impeding microbial development more effectively than strong acids. Organic acids are generally recognised as safe (GRAS) for use in food treatments regardless the types of food thus making them safe for use by household to lower the risk of microbiological infection.

Conclusions and Recommendations

To sum up, organic acids like acetic and citric acid can effectively act as a decontamination agent against L. monocytogenes in lettuce to guarantee safe ingestion. In order to effectively eradicate L. monocytogenes from lettuce, two crucial elements are the concentration of acid and the physical application while washing the vegetables.

Acknowledgements

This research was supported by Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin.

Novelty Statement

The current research work investigates effect of organic acids to remove L. monocytogenes from lettuce to ensure safe consumption of raw lettuce.

Author’s Contribution

Wan Norhafizah Wan Azman and John Yew Huat Tang: Designed the study.

Wan Norhafizah Wan Azman: Performed the experiments and collected the data.

All the authors read and approved the final manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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