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. 2024 Jun 22;103(9):104012. doi: 10.1016/j.psj.2024.104012

Effect of combined electrolyzed reduced water and slightly acidic electrolyzed water spraying on the control of Salmonella, eggshell quality, and shelf life of eggs during storage

Mingming Tu *, Yao Zang *, Qingnan Mo *, Xingyun Yuan *, Dengqun Shu *, Guosheng Zhang †, Jie Hu *, Yanjiao Li *, Renxin Liu *, Shan Bing *, Yitian Zang *,1
PMCID: PMC11279722  PMID: 38986359

Abstract

Slightly acidic electrolyzed water (SAEW) is a safe and effective disinfectant, but its sterilizing efficiency is compromised by organic matter on the egg surface. Electrolyzed reduced water (ERW) is a harmless cleaner with a decontamination effect on a variety of surfaces and can be used to remove organic matter. This study assesses the effectiveness of a combination of ERW and SAEW in eliminating Salmonella and manure mixture from egg surfaces, as well as its impact on egg quality during storage. The results show that ERW (74.14%) was more effective than deionized water (DW, 64.69%) and SAEW (70.20%) (P < 0.05) in removing manure from egg surfaces. The damage to the cuticle of eggshell treated with ERW for 28 s was similar to that of DW (P > 0.05) and less than that of SAEW (P < 0.05). Spraying ERW for 10 s followed by SAEW for 18 s (ERW + SAEW) completely removed Salmonella from the egg surface, with no bacteria detected in the residual wash solution. Additionally, ERW + SAEW demonstrated superior preservation of egg quality during storage at 25℃ than the control and ERW single treatment (P < 0.05). Moreover, ERW + SAEW resulted in less weight loss compared to SAEW single treatment (P < 0.05). In conclusion, the sequential use of ERW and SAEW appears to be a promising approach for sterilizing eggs. It not only removes organic matter and Salmonella from the egg surface but also improves the preservation quality of the egg at 25 ℃.

Key words: slightly acidic electrolyzed water, electrolyzed reduced water, egg; storage

INTRODUCTION

Eggs are widely consumed around the world due to their high protein content (Caner and Cansiz, 2007; Chousalkar et al., 2013). However, eggs can be contaminated with many types of microorganisms, such as Salmonella enteritidis (S. enteritidis) or Escherichia coli (E. coli), which can penetrate the eggshell, thus presenting a risk of the transmission of food-borne diseases to consumers (Ni et al., 2014; Zang et al., 2019; Sheng et al., 2021). Therefore, washing eggs before their sale is considered a necessary process in many countries (Chousalkar et al., 2013; Sheng et al., 2021). Yet, some studies have shown that traditional chemical cleaners are associated with drug residues and cause public health safety hazards (Cao et al., 2009; Sheng et al., 2021). In addition, most cleaning and disinfecting agents are readily inactivated by organic matter (e.g., chicken manure) that adheres to the surface of the eggshell (Davies and Breslin, 2003). Accordingly, it is particularly important to develop a new environmentally friendly cleaning and disinfecting agent to remove bacteria and feces from the surface of eggs.

Slightly acidic electrolyzed water (SAEW) with a near-neutral pH is considered one of the most promising disinfectants for eggs due to its high antimicrobial efficacy and low impact on human health (Cao et al., 2009; Ni et al., 2014; Sheng et al., 2021). For instance, Cao et al. (2009) found SAEW as an effective egg surface sanitizer for Salmonella. Our previous study found that SAEW was not only effective in the elimination of S. enteritidis and E. coli on eggs but could also be useful in the preservation of fresh egg quality during storage (Zang et al., 2019). Nevertheless, organic material, such as manure and feathers, on eggs may significantly reduce the bactericidal effect of SAEW (Zang et al., 2015).

Electrolyzed reduced water (ERW) with high pH (10.0–11.5), high dissolved hydrogen, and low ORP (-800 to -900 mV), is produced by the electrolysis of a dilute (ca. 0.1%) sodium chloride (NaCl) solution using an electrolysis apparatus. In recent years, ERW has gained popularity as a cleaning agent due to its reported decontamination effects on various surfaces, including cutting boards and kitchen utensils, which is important for food safety (Belay et al., 2021; Khalid et al., 2023). Bialka et al. (2004) have reported that ERW can be used to remove the soil that may be present in the eggs. Jiménez-Pichardo et al. (2016) have also reported that ERW shows an excess of hydroxyl ions that can participate in the formation of more symmetric and weak hydrogen bonds and show a high capacity to dissolve mineral deposits and disrupt biofilms extracellular matrix. Several studies have shown that ERW has a strong rinsing effect on surfaces due to the presence of sodium hydroxide (NaOH) and hydrogen (H2). NaOH saponifies oils, producing water-soluble sodium fatty acids and glycerol, and H2 enhances this reaction by surrounding and separating the organic elements with its small size, large contact area, and hydrophobic properties (Huang et al., 2008; Jiménez-Pichardo et al., 2016).

Accordingly, a combination of ERW and SAEW may be more effective: the ERW is used to remove the fecal contamination from the egg surface and is followed by the SAEW for disinfection (ERW + SAEW). Many studies have reported the synergistic effect of using two types of electrolytic water (Koseki et al., 2004; Ni et al., 2016; Iram et al., 2021). Koseki et al. (2004) earlier found that treating lettuce with mildly heated ERW for 5 min, followed by washing with chilled acidic electrolyzed water for 1 or 5 min, reduced the number of pathogens on lettuce by 3 to 4 log10 CFU/g. Ni et al. (2016) suggested that the method of cleaning with ERW followed by disinfection with SAEW significantly removes bacteria from the surface of vehicles transporting live pigs and is superior to separate treatments with ERW and SAEW. However, there are no reports of using this combined method to sterilize eggs. Hence, there is a need to investigate the inactivation effect of ERW + SAEW on bacteria on the surface of eggs.

In addition, studies have reported that using disinfectants or detergents may remove the cuticle that protects the egg (Liu et al., 2016; Sheng et al., 2021; Yuan et al., 2022). The cuticle is present from the beginning of the egg and protects the egg not only against microbial invasion but also against the escape of water and CO2 during storage, thus maintaining the quality of the egg during storage (Sheng et al., 2021). Therefore, as well as studying the effect of ERW + SAEW on the removal of bacteria from the egg surface, its effect on the egg surface shell and egg quality during storage needs to be further investigated.

As a result, the overall objectives of this study were to evaluate the removal effects of different treatments on egg surface contaminants, the removal effect of different treatments on a mixture of S. enteritidis and manures on the surface of eggs, the effects of different treatments on eggshell quality of eggs, the effects of combined ERW and SAEW treatment on egg quality in storage.

MATERIALS AND METHODS

Preparation of Samples

Eggs weighing between 55 to 59 g each were obtained from a local poultry farm (Nanchang, China). These eggs were commercial eggs from 40-wk-old Hy-Line Gray hens, and the experiment was conducted during the spring season. The eggs were transported to the laboratory within 5 h of being laid. Upon arrival, the eggs were visually inspected and any with cracks were discarded.

Preparation of Treatment Solution

ERW with a pH of 11.21 ± 0.01 and oxidation-reduction potential (ORP) of -886 ± 5 was produced using a generator (prepared in-house by the Animal Production Laboratory of Jiangxi Agricultural University) at a voltage of 150 V, to electrolyze a solution of NaCl (1 g /L) for 1.5 h. Deionized water (DW) with a pH of 6.51 ± 0.01 and an ORP of 382 ± 5 was produced using a water purifier (TST-UPB-10, Shijiazhuang TST Equipment Co., Ltd., Shijiazhuang China). The SAEW at available chlorine concentration (ACC) of 25 mg/L was selected as 25 mg/L based on Yuan et al. (2021), and was obtained from a non-membrane generator (V9, Zhongshan Lady Li Electrical Co. Ltd., Guangdong China). Before each experiment, the pH and ORP of the treatment solutions were measured by the pH meter (HM-30R, Shanghai Leichi Co., Ltd., Shanghai China) and an ORP meter (SX-630, Shanghai Shanxin Co., Ltd., Shanghai China).

Preparation of Bacterial Cultures and Manure Mixtures

S. enteritidis was chosen as the contaminant for this study because it is commonly used as an indicator of bacteria in egg disinfection (Bialka et al., 2004., Zang et al., 2019). The strain of S. enteritidis used (BNCC-103134, Bena Culture Collection Center, Henan, China) was hydrated according to the manufacturer's instructions. To validate the viable counts, 0.1 mL 10-fold serial dilutions of broth cultures were plated onto sterile tryptic soy agar supplemented with 0.6 percent yeast extract (TSA-YE, CVCC, Beijing, China) and incubated at 37 ℃ for 24 h. The culture was then used in further research.

A 6% liquid manure solution (as described by Zang et al., 2015) was prepared by mixing 30 g of dried chicken manure obtained from a Hyland Grey commercial eggs farm with 500 ml of sterile deionized water. The solution was then inactivated by autoclaving (LDZX-50KBS, Shanghai Shenan Co., Ltd., Shanghai, China). After shaking, the manure solution was combined with equal portions of the prepared cultures to obtain contaminated cultures with populations of approximately 8 log10 CFU/mL and a liquid manure solution concentration of 3%.

Egg Surface Manure Removal Test

A one-way experimental design was used in this section to compare the effectiveness of DW, SAEW, and ERW spray treatments for removing chicken manure from egg surfaces. The eggs were first aspirated with a syringe to remove all contents, and only the shells were used in the studies to prevent interference with the results caused by the evaporation of water from the eggs. See below for details.

The eggs were first washed with tap water and drilled at the bottom (3–4 mm diameter), then the contents were slowly sucked out of the holes with a syringe, washed again with tap water, and air dried under a biosafety hood. The glue (FM580-310A, Bairui Runxing Technology Development Co., Ltd, Beijing, China) was then used to seal the empty eggs. Finally, the empty egg, with only the shell left, was prepared for the next step. The egg contamination process was then carried out using the artificial contamination method as Bing et al. (2019) previously reported. Each empty egg was immersed in a sterile solution of poultry litter (3% concentration) for 10 min. The eggs were then air-dried in a biosafety hood at 25℃ for 60 min and weighed. After contamination, the empty eggs were divided into the following 3 groups, each with 25 eggs: the DW, SAEW and ERW groups. Empty eggs were treated as described above and sprayed with DW, SAEW and ERW for 21 s, respectively. After treatment, the empty eggs were air-dried in a biosafety hood at 25℃ for 60 min. Finally, the air-dried empty eggs were weighed and recorded again. The "egg surface manure removal rate" refers to the percentage of manure removed from the eggshell surface after treatment compared to the initial weight of contamination. It is calculated using the following formula:

Eggsurfacemanureremovalrate=M1−M2M1*100%

Where M1 stood for the weight of the infected egg. M2 stood for the weight of the sprayed egg.

Egg Surface Bacterial and Manure Mixture Removal Test

The decontamination efficacies of DW, SAEW, ERW and a combination of ERW and SAEW (ERW + SAEW) on eggs contaminated with a mixture of S. enteritidis and chicken manure were compared. The freshly deposited eggs were sterilized, contaminated, and treated in 4 distinct methods in this experiment.

The eggs were first washed with tap water and immersed in a commercial chlorine-based sanitizer (Beijing Zhenhe Medical Technology Co., Ltd., Beijing, China) for 10 min at an ACC of 30 mg/L, then washed with sterilized deionized water and air-dried under a biosafety hood (YJ-VS, Wuxi Instrument Manufacturing Co., Ltd., Jiangsu, China). Egg contamination was then performed using the artificial contamination method outlined above. The final concentrations of S. enteritidis inoculated onto the egg surface averaged approximately 6.26 ± 0.18 log10 CFU/egg.

The eggs were contaminated and divided into five groups of ten eggs each: control, DW, SAEW, ERW, and ERW + SAEW groups. The DW, SAEW, and ERW groups were sprayed with sterilized DW, SAEW and ERW for 28 s (References to Yuan et al. 2021). For the ERW + SAEW group, eggs were sprayed with ERW for 10 s followed by SAEW for 18 s. A manual compression sprayer (SX-CS5C, Ichibashita Holdings Ltd., Zhejiang, China) with a spray flow of 0.18 L/min and a spray pressure of 100 KPA was used in this test. The egg samples were placed into a sterile plastic bag containing 50 ml sterile phosphate buffer saline at pH 7.4 (HB8507, Qingdao Hope Bio-Technology Ltd., Qingdao, China) and vigorously shaken for 1 min after being sprayed. The viable bacterial population was serially diluted with sterile 0.1% buffered peptone water (HB4084, Qingdao Hope Bio-Technology Ltd., Qingdao, China). A volume of 0.1 ml of each S. enteritidis dilution in triplicate on TSA plates and incubated at 37 ℃ for 24 h. The microbial count was expressed as log10 CFU/egg. The untreated and contaminated eggs were used as controls.

Furthermore, The number of surviving bacteria in the residual wash solution was determined. Use sterilized plastic tubs to collect the rinsed residual liquid from underneath the eggs. Microbiological cultures were counted using the method described above after treatment. The microbial count was expressed as log10 CFU/mL.

Eggshell Quality Measurement Test

This section aims to compare the effects of different treatments on eggshells, focusing primarily on the eggshell cuticle staining test, scanning electron microscope observation, and eggshell strength measurement.

Cuticle Staining. To evaluate the effect of spraying SAEW, ERW, and DW on the cuticle of eggs, the cuticle was measured by the dyeing method. The cuticle of the eggs was measured following the protocol by Medina-Gudiño et al. (2020). In this test, 20 freshly laid eggs were divided into 4 groups: the untreated group (control group), ERW sprayed group, SAEW sprayed group and DW sprayed group. Eggs (all fresh eggs, without washing treatment) were sprayed with SAEW, ERW, or DW for 28 s, respectively. After treatment, all eggs were air-dried and stained with 0.1% trypan blue solution (G-clone Biotechnology Co., Ld., Beijing, China) for 1 min, then washed with sterilized DW for 3 s, and air-dried for 60 min. Color determination was performed on 5 random zones per egg using a spectrophotometer (YS3060, Shenzhen Shanenshi Co., Ltd., Guangdong, China). The color space (CIELAB) detection includes L (for lightness, 100 indicates white and 0 means black), a (magenta to green), and b (yellow to blue). The color difference (ΔEab) can be expressed as a single numerical value, defined by the following formula:

ΔEab=[(ΔL)2+(Δa)2+(Δb)2]

Where ΔL represents the difference in lightness. Δa represents the difference in the magenta to green axis. Δb represents the difference in the yellow to blue axis. A higher ΔEab denotes a higher staining affinity and hence more cuticle coverage.

Scanning Electron Microscopy of Eggshell

In this experiment, 20 newly laid eggs were divided into 4 groups: control, DW, ERW and SAEW group. In the ERW, SAEW and DW groups, the eggs were sprayed with ERW, SAEW or DW for 28 s. All eggs were air-dried and then trimmed into 1 cm2 pieces using forceps. The outer surface of the egg was then fixed on a sample table. After sputtering the samples with platinum, the surface morphology of the eggshell was observed using a scanning electron microscope (SEM) instrument (Regulus 8100; Nippon Electron Corporation, Tokyo, Japan) operating at 15 kV.

Eggshell Strength. In this study, 20 freshly deposited eggs were separated into 4 groups: control, DW, ERW and SAEW group. In the ERW, SAEW and DW groups, the eggs were sprayed with ERW, SAEW or DW for 28 s. To test shell strength, all eggs were air-dried and placed, blunt end up and tip-down on an eggshell strength analyzer (KQ-1A, Beijing Tenovo International Co., Ltd., Beijing, China).

Storage Test

The storage test was conducted to examine the effect of different treatments on egg quality (Haugh unit, weight loss, yolk index and albumen pH) during storage at 25 ℃ for 7 wk.

Eggs were divided into the following five groups, with 80 eggs per group: control (no treatment), SAEW treatment group (SAEW, 25 mg/L), ERW treatment group and ERW + SAEW treatment group. For the ERW and SAEW groups, eggs were sprayed with ERW or SAEW (25 mg/L) for 28 s. Meanwhile, eggs in the ERW + SAEW treatment group, eggs were sprayed with ERW for 10 s followed by SAEW for 18 s at ACC of 25 mg/L. After treatment, all eggs were air-dried, placed in open plastic egg trays with their small ends down, and stored under ambient conditions at 25 ℃ and 60% relative humidity. All eggs were marked and weighed to determine their initial weight. The Haugh unit (HU), weight loss, yolk index and albumen pH were measured weekly using 10 randomly selected eggs from each group.

Haugh Unit. The albumen height was measured using a digital caliper (EQ-1A, Mitutoyo Co., Kawasaki-shi, Japan). The HU was calculated as described by Caner and Yüceer (2015).

Weight Loss. The eggs were weighed using an analytical balance (ATY-124; Shimadzu, Japan) sensitive to 0.01g, and the weight loss was calculated following Caner and Yüceer (2015).

Yolk Index. The digital caliper was used to measure the diameter of the yolk, while the height of the albumen and yolk was measured using a tripod micrometer (NFN381; Nanjing Mingao Instruments and Equipment Co. Ltd, Nanjing, China). The yolk index was determined following the method described by Caner and Yüceer (2015).

Albumen pH. The egg albumen was isolated, homogenized, and placed in a beaker. pH values were measured using a pH meter (HM-30R, Shanghai Leica Co., Ltd., Shanghai, China).

Statistical Analysis

The means of all parameters were measured in triplicate. Statistical analysis was performed using Origin version 9.0 (OriginLab, Northampton, MA). Tukey's test was used to assess the differences among effects (P < 0.05). Additionally, a one-way analysis of variance (ANOVA) followed by Tukey's test was conducted to compare the means of multiple groups. Third-degree polynomial regression models were employed to describe the changes in egg quality during storage. The selection of polynomial regression models was based on the goodness of fit, as determined by the R2 coefficients, with higher R2 values indicating better model fit.

RESULT AND DISCUSSION

The Removal Effects of Different Treatments on Egg Surface Contaminants

Sometimes the surface of eggs can be contaminated with organic matter, such as chicken manure, which can easily cause bacterial infections. At the same time, the presence of organic matter also reduces the effectiveness of subsequent disinfection. It is thus necessary to compare the effectiveness of different treatments in removing contaminants from the surface of eggs. The effectiveness of different treatments of spray treatment for the removal of chicken manure from egg surface is shown in Figure 1. As can be seen in Figure 1, the removal of contaminants by ERW (74.14%) was significantly higher (P < 0.05) than that by DW treatment (64.69%). The results indicated that ERW was more effective than DW in removing contaminants from the egg surface. Many studies have similar results (Kim et al., 2005; Huang et al., 2008). In 2005, Kim et al.(2005) found a similar result that the cecal material on the dorsal area of chicken carcasses was significantly reduced by pre-spraying with ERW compared to DW treatment (P < 0.05). In 2008, Huang et al. (2008) reported that ERW has a good cleaning function due to the presence of NaOH and a lot of active hydrogen. The NaOH can be saponified with oil to produce water-soluble sodium fatty acids and glycerol, while H2 can enhance reactions by surrounding and separating organic elements through its small size, large contact area and hydrophobic nature. In 2016, Ni et al. (2016) reported that ERW could remove a mixture of dust and feces from the surface of vehicles transporting live pigs. In 2023, Khalid et al. (2023) reported that ERW effectively removes grease residues from equipment surfaces. These studies demonstrate that ERW is a cleaning and decontaminating agent with great potential.

Figure 1.

Figure 1

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The effectiveness of different treatments of spray treatment for the removal of chicken manure from egg surface. Different letters significant differences (P < 0.05). DW: deionized water. ERW: electrolyzed reduced water. SAEW: slightly acidic electrolyzed water.

Moreover, no significant difference between ERW and SAEW in removing chicken manure from the surface of eggs was observed, while no significant difference was found between SAEW and DW (P = 0.06). Nonetheless, it can be seen that SAEW also removes chicken fecal liquid from the surface of eggs. Among the various types of electrolyzed water, SAEW has gained attention for its efficient bactericidal ability. In this study, we found that SAEW also has some removal capacity for manure, despite its low efficiency. The pH value affects the effective chlorine content in chlorine formulations. SAEW has a neutral pH range of 5.0 to 6.5 and its primary component is hypochlorous acid (HClO). HClO is a strong oxidizing agent, and it may react with some of the oils present in the chicken manure solution in a redox reaction, thereby removing them. This reaction could also be the reason why the bactericidal effect of SAEW can be significantly reduced by the interference of organic matter.

The Removal Effect of Different Treatments on S. enteritidis on The Surface of Eggs

The Removal Effect of Different Treatments on S. enteritidis on The Surface of Eggs in The Presence of Chicken Manure.Figure 2 shows that ERW is significantly more effective than DW (P < 0.05) in removing bacteria from the egg surface, which may be due to the decontaminating effect of ERW. Besides, ERW may also have a bactericidal effect, although this is limited. Similar results have been reported in other studies. Koseki et al. (2004) discovered that soaking raw lettuce in ERW for 5 min significantly reduced E. coli O157:H7 and Salmonella by about 1.8 log10 CFU/g compared to the DW treatment (P < 0.05). Iram et al. (2021) concluded that ERW has some antimicrobial effects, although it has not been studied as extensively as other acidic electrolytic waters in terms of antimicrobial activity. Naturally, research on ERW has focused on antioxidant stress and gut modification due to its richness in H2. Moreover, there was no significant difference between the SAEW-treated group and the ERW + SAEW-treated groups in terms of complete removal of Salmonella from the egg surface at ACC of 25 mg/L for 28 s (P > 0.05), and both treatments were significantly superior to the DW and ERW treatments (P < 0.05).

Figure 2.

Figure 2

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The removal of Salmonella counts from egg surface by different treatments of spray treatment. DW: eggs were sprayed deionized water for 28 s. ERW: eggs were sprayed electrolyzed reduced water for 28 s. DW + SAEW: eggs were sprayed deionized water for 10 s followed by SAEW for 18 s. SAEW: eggs were sprayed slightly acidic electrolyzed water for 28 s. ERW + SAEW: eggs were sprayed electrolyzed reduced water for 10 s followed by SAEW for 18 s. Different letters significant differences (P < 0.05).

Studies have reported combining two types of electrolyzed water for cleaning and disinfection purposes (Koseki et al., 2004; Ni et al., 2016; Iram et al., 2021). Ni et al.(2016) have used ERW washing followed by SAEW disinfection to inactivate bacteria in vehicles transporting pigs. In another work, Ni et al. (2014) used this method to remove bacteria from the surface of eggs. Their study reported that the combination of the two electrolyzed waters was significantly superior to the separate treatment of the two electrolyzed waters (P < 0.05). This result can be caused by the presence of more organic matter on the surface of objects such as vehicles or eggs. Organic matter reacts with the HClO in SAEW, which in turn significantly reduces the sterilizing effect of SAEW (Bing et al., 2019). Therefore, the combined treatment method results in a higher decontamination effect of the object, as ERW removes organic matter from its surface, enhancing the sterilization effect of SAEW.

Contrary to their findings, our results did not demonstrate a significant difference between the ERW + SAEW and SAEW alone groups (P > 0.05), which differs from the outcomes of the previous study. This could be due to two reasons. Firstly, we used spraying instead of dipping to disinfect the eggs. The force generated by spraying quickly washes away the organic matter on the surface of the eggs, reducing its impact on the disinfection effect. Secondly, the fact that SAEW can react with organic matter proves that SAEW also has some effect in removing contaminants. This is supported by our aforementioned results (Yuan et al., 2022). Over the past 20 y, there have been many reports on the effective antimicrobial properties of SAEW (Bialka et al., 2004; Cao et al., 2009). As early as 2004, Bialka et al. (2004) reported that acidic electrolyzed water (AEW) caused damage to eggshells while effectively inactivating bacteria on the surface of eggs. Then, in 2009, Cao et al. (2009) started to use SAEW, which has a nearly neutral pH and low corrosiveness, to disinfect Salmonella on the surface of eggs and found that the inactivation of bacteria on the surface of eggs by SAEW was better than that by NaClO and equal to that by AEW (P < 0.05). However, it was also reported that egg surface disinfection by SAEW could damage the shell membrane on the surface of eggs, which in turn affected the weight loss rate of eggs during storage (Sheng et al., 2021). Besides, the damage to the egg surface can also affect the strength of the shell, weakening its protective effect on the egg and leading to the invasion of bacteria, posing a food safety risk (Yuan et al., 2022). Hence, an assessment of the impact of egg sterilization on eggshell quality is essential.

The Number of Surviving Bacteria in The Residual Wash Solution From Different Treatments. The removal of bacteria from the surface of an egg does not necessarily mean that the bacteria have been inactivated. There is a risk that the bacteria may be washed into the waste liquid with the wash solution, which could pose a threat to environmental safety. Table 1 shows a difference in the number of viable bacteria in the residual wash between the DW and ERW treatment groups (P < 0.05), indicating that ERW had a bactericidal effect. Suzuki et al. (2021) discovered that the culturability of Salmonella decreased by 1.9 log10 after 30 seconds of ERW (pH = 12.5) treatment. However, it is worth noting that both the DW and ERW groups had a viable count greater than 2 log10 CFU/mL in the residual wash. This suggests that the use of a single wash, even with ERW, still poses a risk of bacterial transmission. However, both treatments with SAEW (ERW + SAEW and SAEW groups) in Table 1 showed no viable bacteria in the residual solution, confirming the effectiveness of SAEW. This also demonstrates the necessity of sterilizing eggs to reduce the risk of bacterial transmission.

Table 1.

The number of surviving bacteria in the residual wash solution from different treatments.

Treatment DW1 ERW2 SAEW3 ERW + SAEW4
Surviving bacteria
(log10 CFU/mL)		 2.88 ± 0.29A 2.35 ± 0.47A No detection No detection
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1

DW: deionized water.

2

ERW: electrolyzed reduced water.

3

SAEW: Slightly acidic electrolyzed water.

4

ERW + SAEW: Spraying ERW followed by SAEW.

A

Identical superscript letters indicate no significant difference (P > 0.05).

The Effects of Different Treatments on Eggshell Quality of Eggs

Effect of Different Treatments on Eggshell Strength.Figure 3A illustrates the effect of the different treatments on eggshell strength. It can be seen from Figure 3A that none of the DW, ERW and SAEW treatment groups had any effect on the strength of the eggs compared to the control group (no treatment) (P > 0.05). This result implies that the short duration of spray washing by DW, ERW and SAEW may not affect the eggshell strength.

Figure 3.

Figure 3

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(A) The effect of the different treatments on eggshell strength. (B) Cuticle staining. (C) Cuticle assessment of eggs by scanning electron microscopy. DW: eggs were sprayed deionized water for 28 s. CT: untreated eggs. ERW: eggs were sprayed electrolyzed reduced water for 28 s. SAEW: eggs were sprayed slightly acidic electrolyzed water for 28 s. Cuticle staining and cuticle assessment of eggs by scanning electron microscopy.

Effect of Different Treatments on The Cuticle of Eggs. The cuticle, which covers the outside of the eggshell, consists of hydroxyapatite crystals, polysaccharides, lipids and glycoproteins (Liu et al., 2016). It seals the pores of the egg and increases the mechanical strength of the shell (Gole et al., 2014; Liu et al., 2016). The cuticle staining method is commonly employed to assess the impact of disinfectants on eggshell cuticles (Bialka et al., 2004). Variations in the cuticle are reflected in the ΔEab values' variability. In their study, Bialka et al. (2004) utilized this method to investigate the effect of AEW (pH = 2.7) on eggshell cuticles, demonstrating a significant impact (P < 0.05). Following staining, CIELAB parameters were measured to quantify color differences between the CT, DW, ERW and SAEW groups. As shown in Figure 3B, untreated eggs had a uniform blue color, whereas ERW and DW-treated eggs had a lighter blue color respectively. Table 2 showed that there were significantly differences in L, b and ΔEab values were observed between the CT group (39.43, -16.86, 60.54) and ERW (53.69, -5.33, 44.21) and DW (53.47, -5.35, 44.47) groups (P < 0.05). Leleu et al. (2011) have reported that a larger value of ΔEab is indicative of a higher level of cuticle coverage. Thus, our results suggest that DW and ERW spray treatments caused damage to the stratum corneum. Furthermore, no significant difference was observed between DW and ERW groups in L, a, b, and ΔEab values (Table 2) (P > 0.05). Bialka et al. (2004) have a similar result, they reported that the ERW (pH = 11.4) treatment may significantly hurt the cuticle of the eggs (P < 0.05). However, Leleu et al. (2011) have a different result. They showed that the cleansing process does not damage the stratum corneum, even when combined with brushing and spraying. As a result, it is necessary to adopt electron microscopy methods to make further studies on whether ERW and DW spray treatments affect the eggshell cuticle.

Table 2.

The color difference values of eggshell dyeing of eggs with different treatments.

Treatment L1 a2 b3 ∆Eab4
SAEW 67.45 ± 0.86C 7.85 ± 0.47C 15.19 ± 1.04C 34.39 ± 0.85C
ERW 53.69 ± 0.86A -3.98 ± 0.59A -5.33 ± 0.62A 44.21 ± 0.74B
DW 53.47 ± 1.12A -4.08 ± 0.53A -5.35 ± 0.49A 44.47 ± 1.01B
CT 39.43 ± 0.75B -5.83 ± 1.09B -16.86 ± 0.65B 60.54 ± 0.39A
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1

L: Lightness from black to white, ranging from 0 to 100.

2

a: Negative a corresponds to green, positive a corresponds to red.

3

b: Negative b corresponds to blue, positive b corresponds to yellow.

4

∆Eab: Represent the color difference.

A, B, C

Means within a column without a common superscript are significantly different (P < 0.05). Identical superscript letters indicate no significant difference (P > 0.05).

In addition, a statistical difference (P < 0.05) in all 4 parameters (L, a, b, and ΔEab) between control eggs (39.43, -5.83, -16.86, and 60.54) and eggs sprayed with SAEW (67.45, 7.85, 15.19, and 34.39) can be seen in Table 2. Besides, Figure 3B shows a very clear color contrast. The results of lower ΔEab values for eggs in the SAEW group indicate changes in the egg cuticle after SAEW application. Many studies report cuticle damage after egg sterilization with chemicals such as sodium carbonate and sodium hypochlorite, as we have observed (Liu et al., 2016; Sheng et al., 2021; Yuan et al., 2022). Our previous study also found that SAEW significantly affects the cuticle of eggs, causing damage and cracking of the cuticle, and even risking re-infestation of the eggs with bacteria. It was also found that the probability of bacterial contamination of eggs increased by about 37% when eggs were re-contaminated after SAEW rinsing (Yuan et al., 2022). Yet, Liu et al. (2022) had a different result, where they found that SAEW spray disinfection did not have any effect on the eggshell cuticle by the same staining method. In any case, these different speculations can be resolved by scanning electron microscopy of eggshells.

Electron Microscopy of Eggshell Surfaces After Different Treatments. After the first assessor had assigned scores to the cuticle of each egg, a second assessment by viewing a series of SEM images (Figure 3C) was carried out to investigate if the cuticle was destroyed by different spraying treatments. As can be seen from Figure 3C, the surface of eggs in the control group was rough and uneven with fewer cracks, which may be caused by the irregular distribution of the cuticle (Liu et al., 2016). However, it can be noted that there are larger and more cracks on the surface of the eggs in the DW, ERW, and SAEW treatment groups. This result also confirmed our above statement that the DW, ERW, and SAEW spraying treatment may destroy the cuticle of eggs. Notably, compared to the other treatment groups, more cracks appeared on the surface of the eggshells after SAEW spraying. Similar findings of small cracks in unwashed eggs and more cracks in SAEW-treated eggs were reported by Yuan et al. (2022). In severe cases, too many cracks may not only increase the risk of bacterial invasion but also leave the egg unprotected during storage, allowing large amounts of moisture to escape through cracks or air holes in the shell, which in turn can lead to a reduction in egg quality during storage. For this reason, there is also a need for additional studies on the effects of different treatments on the quality of the eggs during the storage period.

Effect of Combined ERW and SAEW Treatment on Egg Quality in Storage

HU.Figure 4A shows the HU of the eggs treated in different ways during the 7 wk of storage at 25℃. For all treatments, HU decreased during the storage period (P < 0.05). This is consistent with previous findings (Yüceer et al., 2016; Zang et al., 2019). The decrease in the HU value is due to a decrease in the height of the thick proteins, which are hydrolyzed and thinned out during storage (Yüceer et al., 2016; Zang et al., 2019). After 7 wk of storage, the HU values of the Control, ERW, SAEW, and ERW + SAEW groups were 23.16, 29.16, 34.80, and 35.49, respectively. According to the FAO (2003) guidelines, eggs from the SAEW and ERW + SAEW groups are still classified in quality class B (HU = 59–31). Furthermore, the HU of eggs in the ERW group differed significantly from that of the control group at 4 wk (P < 0.05), indicating that egg washing helps prevent a decrease in HU during storage. Numerous studies have highlighted the efficacy of egg washing in reducing microbial contamination on the eggshell surface and curbing microbial metabolic activity resulting from microbial penetration through the eggshell (Yüceer et al., 2016; Zang et al., 2019). This microbial metabolism accelerates the hydrolysis of thick proteins, thereby potentially lowering the HU. Our results corroborate these findings, indicating a substantial decontamination effect of ERW on the egg surface, which may account for the higher HU observed in the ERW group compared to the untreated egg group. It is noteworthy that by the 5th wk of storage, the HU value of the Control group had already dropped below 40. Furthermore, according to the results depicted in Figure 4A, it is evident that in terms of HU, eggs treated with ERW + SAEW and SAEW exhibited an extended shelf life of almost 2 wk.

Figure 4.

Figure 4

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Polynomials of third degree regression(Haugh unit, albumen pH, yolk index, weight loss) were used. Changes in (A) Haugh unit, (B) albumen pH, (C) yolk index, weight loss. CT: untreated eggs. SAEW:eggs were sprayed slightly acidic electrolyzed water for 28 s. ERW: eggs were sprayed electrolyzed reduced water for 28 s. ERW + SAEW: eggs were sprayed electrolyzed reduced water for 10 s followed by SAEW for 18 s.

Figure 4A shows that the HU of eggs from the ERW + SAEW and SAEW groups was better than those in the control group at wk 3 and in the ERW group at wk 5 (P < 0.05). Additionally, there was no significant difference between the ERW + SAEW group and the SAEW group throughout the storage period (P > 0.05). Similarly, several studies have reported the ability of SAEW to inactivate bacteria on the surface of eggs, thereby preventing a decrease in egg HU (Zang et al., 2019; Sheng et al., 2021). Several studies have shown that SAEW inactivates bacteria on the surface of eggs more effectively than NaClO (equivalent to AEW) (Cao et al., 2009; Zang et al., 2019), and therefore SAEW prevents the loss of HU during storage, with HU values of 40.8 for control eggs and 55.3 for SAEW-treated eggs after 30 d of storage (Zang et al., 2019). Other studies have also shown that improved sterilization of eggs has a positive effect on the preservation of HU during storage. Yüceer et al. (2016) showed that ozone slowed the rate of HU decline (P < 0.05) and that function increased with an increase in ozone concentration. Hu et al. (2021) found that after irradiation with blue light, which has a significant bactericidal effect, the HU of eggs decreased from an initial 86.27 to 49.28 after 4 wk, while the HU of the control group decreased more rapidly, from 86.27 to 33.54.

In addition, a polynomial equation was used to regress the change in HU values over time. Studies have shown that changes in HU during storage can be accurately reflected by a polynomial regression equation (Drabik et al., 2021; Yuan et al., 2022). The coefficient of determination (R2) of the fitted equation in this study was found to be greater than 0.98 (P < 0.01), indicating a valid fit. The equation demonstrates that the coefficients of the parameter X2 (where x represents time in weeks) that have a positive effect on the Y-value (HU-value) of the pairing formula follow the order of magnitude ERW + SAEW (2.943) > SAEW (2.370) > ERW (1.224) > CT(1.215). This finding also reflects the effect of different treatments on the HU value of eggs during storage.

Albumen pH. The albumen pH of the protein is often used as an indicator of the freshness of the egg (Caner and Yüceer, 2015). Figure 4B shows that the albumin pH of all groups increased with storage time. After 7 wk of storage at 25℃, the albumin pH of the control group was 9.46, the ERW group was 9.40, the SAEW group was 9.23 and the ERW + SAEW group was 9.27. This result is consistent with previous studies (Caner and Yüceer, 2015; Zang et al., 2019). This increase is caused by the release of CO2 from the eggs as storage time increases, leading to a rise in pH (Caner and Yüceer, 2015; Zang et al., 2019). Furthermore, as the pH level of the albumin in eggs increases, the interaction force between the ovalbumin-enzyme complex weakens, resulting in a decrease in albumin concentration. This decrease in concentration leads to a decrease in viscosity and a watery appearance, ultimately resulting in a decrease in the HU value (Wang et al., 2022). This finding is consistent with our previous observation of a reduced HU score. Additionally, there is a difference between the eggs in the ERW group (9.132)and those in the CT group (9.234) at wk 3, which can be attributed to the cleaning and decontamination effect of ERW. During the storage period, the protein pH of eggs from the ERW + SAEW and SAEW groups differed significantly from that of the CT group in wk 3 and from that of the ERW group in wk 4. This result was consistent with the variation of HU values.

Polynomial regression equations were also used to model the changes in albumin pH in each group and the coefficient of determination (R2 > 0.98) and significance tests (P < 0.01)of the equations proved that the equations were valid. In the equation formulations of the groups, the coefficients of X (storage time in weeks), which was significantly and positively correlated with the Y-value (protein pH), were ranked in order of magnitude: CT (0.2929) > ERW (0.2427) > SAEW (0.1918) > ERW + SAEW (0.1774).

Yolk Index. The yolk index, which reflects yolk quality and egg freshness, was obtained by measuring the yolk height and width. Figure 4C illustrates the deterioration in yolk quality during storage. After 7 wk of storage at 25 ℃, the control group had a yolk index value of 0.188, while the ERW, SAEW, and ERW + SAEW groups had values of 0.209, 0.229, and 0.230, respectively. Research has shown that eggs stored for extended periods may be susceptible to microbial invasion, which can cause the hydrolysis of albumin and the penetration and diffusion of its water content (Wang et al., 2022). This process weakens the lecithin membrane, ultimately leading to a reduction in total solids and liquefaction of the yolk (Wang et al., 2022). The initial yolk index value in this study was 0.466. After 3 wk, differences were observed among the ERW + SAEW group (0.375), the SAEW group (0.327), the control group (0.333), and the ERW group (0.337) (P < 0.05). This finding suggests that sterilization of the egg surface prevents a decrease in the yolk index, which is supported by other studies (Yüceer et al., 2016; Sheng et al., 2021). Moreover, Fig. 4C also shows the polynomial regression equation for the yolk index, with a coefficient of determination (R2) of 0.989, indicating its validity (P < 0.01). In this equation, the coefficients in front of X (storage time in weeks), which has the most significant effect on the decrease in the Y value (Yolk index), are ranked in descending order as ERW + SAEW (-0.0367) > SAEW (-0.0395) > ERW (-0.0530) > CT(-0.0535), consistent with the ranking of the HU values above.

Weight Loss. Figure 4D displays the weight loss of the control, ERW, SAEW and ERW + SAEW over a 7-wk storage period at 25℃. The weight loss of the eggs increased in all groups during the storage period. Similar results have been reported by several researchers (Zang et al., 2019; Sheng et al., 2021; Wang et al., 2022). Previous studies have reported that weight loss in stored eggs is caused by the escape of moisture and CO2 through the eggshell pore, and the presence of microorganisms can accelerate this process (Sheng et al., 2021; Wang et al., 2022). Therefore, cleaning or inactivating microorganisms can help maintain egg weight during storage. After 7 wk of storage, the control group exhibited the highest weight loss at 10.98%. In comparison, the ERW, SAEW, and ERW + SAEW groups experienced weight losses of 9.75, 9.29, and 8.47%, respectively. The ERW + SAEW group showed differences from the other groups by wk 5 (P < 0.05). By wk 6, the SAEW group also exhibited differences from the control group (P < 0.05), and by wk 7, ERW showed differences from the control group (P < 0.05). It is worth noting that, unlike other indicators of egg quality during storage, the ERW + SAEW group exhibited differences from the SAEW group in terms of weight loss (P < 0.05). The greater weight loss observed in the SAEW group compared to the ERW + SAEW group may be attributed to the fact that the SAEW group caused more damage to the shell cuticle on the surface of the eggshell. Although the ERW + SAEW group included SAEW treatment, the duration of its SAEW treatment (18 s) was shorter than that of the SAEW-treated group (28 s). The cuticle on the eggshell serves to prevent the escape of water and CO2 through its pores. However, this study suggests that SAEW causes more damage to the shell cuticle than ERW, which may result in greater water and CO2 loss during storage. Furthermore, the equation obtained by fitting the polynomial regression (R2 > 0.98, P < 0.01) reveals that the coefficients in front of X (where X represents the storage time in weeks) have a negative effect on the Y-value (rate of weight loss), and are ranked in ascending order as CT (2.182) > ERW (2.147) > SAEW (1.689) > ERW + SAEW (1.654).

CONCLUSIONS

ERW exceeded DW in removing chicken manures from the surface of eggs (P < 0.05), and both ERW + SAEW and SAEW alone treatments were able to completely remove Salmonella from the surface of eggs contaminated with chicken manures at an ACC of 25 mg/L for 28 s. Additionally, the short duration of spray washing by DW, ERW, and SAEW may not affect the eggshell strength. Besides, the residual wash of DW and ERW groups had a viable count greater than 2 log10 CFU/mL, while no viable count was found in SAEW and ERW + SAEW groups. However, the SAEW spray treatments resulted in more significant damage to the cuticle of eggshells, as evidenced by lower ΔEab values and scanning electron microscopy, compared to the ERW spray treatments (P < 0.05). Meanwhile, when evaluating the effect of different treatments on the quality of eggs during storage, it was found that both the ERW + SAEW and SAEW groups were effective in preventing the decrease of HU and Yolk index and the increase of albumen pH due to better decontamination and bacterial inactivation. Nevertheless, the weight loss rate of SAEW was significantly higher than that of ERW + SAEW due to the impact of SAEW on the surface cuticle of the eggshell (P < 0.05), which allowed more water and CO2 to escape. Taken together, the combination of ERW and SAEW can not only compensate for the deficiencies of the live bacteria in the residual solution of ERW, but also reduce the effect of SAEW on the eggshells, allowing the advantages of each treatment to be fully exploited. Overall, the combination of ERW surface contamination removal followed by SAEW disinfection effectively eliminates bacteria from egg surfaces, reducing the risk of bacterial transmission from residual liquid after rinsing. In addition, this method improves egg storage quality at 25℃.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial support from the Natural Science Foundation of Jiangxi Province (20224BAB205034) and the Technology System of the Modern Agricultural Poultry Industry of Jiangxi (JXARS).

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