Synergistic bactericidal effect and mechanism of X-ray irradiation and citric acid combination against food-borne pathogens on spinach leaves

Highlights

• X-ray and citric acid combination showed a synergistic bactericidal effect against two major pathogens on spinach leaves.

• Cell membrane damage and ROS generation were the main bactericidal mechanism.

• The combined treatment did not result in adverse changes in quality of spinach.

Abstract

In this study, we investigated the antimicrobial activity of the X-ray irradiation and citric acid (CA) combination against Escherichia coli O157:H7 and Listeria monocytogenes on the surface of spinach leaves and elucidated the mechanisms underlying their synergistic interaction. Upon treatment with 0.3 kGy X-ray irradiation and 1% CA combination, the cell counts of E. coli O157:H7 and L. monocytogenes reduced by 4.23 and 3.69 log CFU/mL on spinach leaves, respectively. The synergistic reduction in the cell counts of E. coli O157:H7 and L. monocytogenes by the combination treatment was 0.95 and 1.14 log units, respectively. The X-ray and CA combination exerts its antimicrobial effect by damaging the bacterial cell membrane and enhancing the generation of intracellular reactive oxygen species in the pathogens. The enhanced bactericidal effect of the combination treatment may not be due to the loss of intracellular enzyme activity. We also evaluated the effect of the combination treatment on the quality attributes of spinach leaves. The combination treatment did not result in adverse changes in color and texture of spinach leaves. These results demonstrate the potential of citric acid and X-ray irradiation combination for decontaminating foodborne pathogens on fresh produce.

Introduction

Fresh produce is a rich source of dietary essential vitamins and minerals. The World Health Organization (WHO), Food and Agricultural Organization (FAO), and the United States Department of Agriculture (USDA) encourage the consumption of fruits and vegetables to decrease the risk of developing cancer and cardiovascular diseases (Allende et al., 2006). However, the consumption of raw fresh produce, particularly leafy green vegetables, is associated with risk of infection from foodborne pathogens (Lynch et al., 2009). The most common pathogenic microorganisms associated with fresh produce include Escherichia coli O157:H7 and Listeria monocytogenes (Lee et al., 2004). Globally, E. coli O157:H7 is a public health risk due to its low infective dose, ability to affect all age groups, and capability to produce Shiga toxins that cause hemolytic uremic syndrome in humans (Hilborn et al., 1999). In 2006, a multi-state outbreak of E. coli O157:H7 infection in USA caused due to consumption of contaminated green leaf spinach resulted in 205 confirmed cases of illness, 31 cases of hemolytic-uremic syndrome, and three deaths (CDC, 2006). One of the most dangerous pathogenic bacteria associated with the fresh produce is L. monocytogenes. This pathogen can grow on food products even after the product is subjected to antimicrobial processing and packaging procedures, such as refrigeration, controlled atmosphere packaging, and chlorine treatment (Mintier and Foley, 2006). Furthermore, L. monocytogenes can cause meningitis, encephalitis, or septicemia in immuno-compromised patients, pregnant women, infants, and the elderly (Back et al., 2014). Therefore, it is important to control microbial contamination in fresh produce to maintain microbiological safety and product quality.

Commercially, sodium hypochlorite is commonly used for sanitizing fresh foods. However, chlorine-based disinfectants do not efficiently reduce the microbial load in leafy green vegetables. A total chlorine concentration of 100–600 ppm in wash water can only achieve a 1–2 log CFU/g reduction of bacteria on the surface of food product (Cossu et al., 2018). The limited efficacy of chlorine is mainly due to the reaction between chlorine and organic materials that leak from the surface of the fresh produce, which rapidly reduces the chlorine concentration (Cossu et al., 2018). Hence, chlorine is added frequently to maintain an effective residual free chlorine level in the washing tank (Mukhopadhyay et al., 2019). However, the recurrent application of chlorine results in the generation of potentially harmful byproducts, such as chloramines and trihalomethanes (Mahmoud et al., 2010). Therefore, there is a need to develop a safe and sustainable decontamination strategy as an alternative to chlorine or other chemical sanitizers.

Food-grade natural antimicrobials such as, organic acids have potential applications in food decontamination (Oliveira et al., 2017). Among the organic acids, citric acid (CA) is generally recognized as safe (GRAS) for use as a food ingredient and is often used to inactivate surface pathogens of fruits and vegetables (Chen et al., 2016). Huang and Chen (2011) reported that CA inhibits the growth of E. coli O157:H7, L. monocytogenes, and Salmonella spp. on the surface of fresh produce. The antimicrobial effect of CA has been attributed to several factors, including membrane disruption, inhibition of essential metabolic reactions, stress on intracellular pH homeostasis, and the accumulation of toxic anions (Brul and Coote, 1999). The major advantage of organic acids, such as CA as a disinfectant for food products is an increased consumer acceptance. Additionally, the pathogens have a lower probability of developing antimicrobial resistance against these food-grade chemicals compared to the standard sanitizers (Oliveira et al., 2017). Moreover, organic acids are effective within a wide temperature range and are not affected by water hardness (Sagong et al., 2011). However, organic acids have limited efficacy when compared to conventional antimicrobials, such as antibiotics, chlorine, and nitrates (Oliveira et al., 2017). CA treatment has been reported to achieve about 1-log pathogen reduction (Chen et al., 2016; Huang and Chen, 2011). Thus, there is a need to develop novel strategies that can integrate organic acid treatment with other physical treatments to enhance the antimicrobial activity against foodborne pathogens.

Ionizing radiations, such as gamma-rays, electron beams, and X-rays, have potential antimicrobial application without affecting the quality of leafy green vegetables (Mahmoud et al., 2010). Among the ionizing radiations, X-ray has several advantages over other ionizing irradiations used in the food industry. X-ray has a higher penetration power than electron beams. Additionally, X-ray is not associated with harmful radioactive emissions, such as gamma rays that are associated with Cobalt-60 or Cesium-137 (Mahmoud, 2009a; Park and Ha, 2019). X-ray irradiation at a dose of 10 kGy can be safely used to reduce the pathogenic microorganism load (WHO, 1981). Several studies have demonstrated the antimicrobial efficacy of X-ray against pathogenic microbes in various foods, including spinach leaves (Mahmoud, 2009a, 2009b; Mahmoud et al., 2010). However, there are no studies investigating the antimicrobial efficacy of combination treatment with X-rays and organic acid against pathogenic microorganisms on fresh produce.

The objectives of this study were to evaluate the synergistic antimicrobial activity of combination treatment with X-ray irradiation and CA against E. coli O157:H7 and L. monocytogenes on spinach surface, and to determine the effect of this combination treatment on the spinach quality. Additionally, we investigated the potential mechanism underlying the synergistic action of this combination.