Effect of the storage temperature on the quality of eggs inoculated with Salmonella Enteritidis onto shell

Abstract

This study explored the temperature-dependent effect on the growth characteristics of Salmonella Enteritidis (SE) on eggshell toward identifying an appropriate storage temperature for unwashed eggs in an actual distribution environment. Among the test storage temperatures (10 °C, 25 °C, and 35 °C), 25 °C was determined to be an appropriate storage temperature, with no effect of changing temperature on the control of SE on eggshell. Regarding the effect of the temperature on egg quality, the quality indicators of egg such as Haugh unit, yolk index, albumin index, and albumin pH were significantly maintained. These results indicated that unwashed eggs should be distributed at 25 °C for SE control, and the storage temperature should be below 10 °C from at least day 4 onward after the start of distribution to maintain egg quality. This study will assist for safety management of unwashed egg in an actual distribution environment.

Introduction

Salmonellosis caused by Salmonella spp. infection induces acute symptoms, such as fever, abdominal pain, nausea, diarrhea, and vomiting (WHO, 2023). According to the Ministry of Food and Drug Safety (MFDS), Salmonella spp. was the most common foodborne pathogen (26%) from 2018 to 2022 (MFDS, 2023). Additionally, the Centers for Disease Control and Prevention (CDC) estimated that non-typhoidal Salmonella ranked second as the cause of annually acquired foodborne illnesses (11%), first as the cause of hospitalizations (35%), and first as the cause of foodborne pathogen-associated deaths (28%) in the United States (CDC, 2023).

Among the Salmonella serotypes, S. enterica serovar Enteritidis (SE) is the predominant cause of foodborne salmonellosis and frequently contaminates eggs and egg products (EFSA and ECDC, 2018). Regarding the contamination scenarios of SE on eggs, one possible route is that SE directly contaminates the contents and shell of eggs before oviposition (vertical transmission). Another possible route is SE penetration of the eggshell following (horizontal transmission) attachment of the bacteria to the eggshell surface (Zhang et al., 2011). It is unclear which route is the major cause of egg contamination by SE, but Martelli and Davies (2012) stated that the majority of egg contamination by SE was not in contents but on eggshells in 1989–2009. Horizontal transmission can also occur during distribution or cooking due to cross-contamination following the detachment of pathogens from eggshell (Chousalkar et al., 2021). Therefore, reducing pathogens on eggshells could be an important consideration for preventing cross-contamination of foods.

The Haugh unit (HU), yolk index, albumen index, and pH of yolk and albumen are common measures of egg quality (Kim et al., 2014; Lee et al., 2014). Especially, HU is widely used in the egg industry (MAFRA, 2023; USDA, 2023). During storage, the internal quality of egg deteriorates due to the loss of carbon dioxide through the eggshell pores, causing the pH of the albumen to increase (Huopalahti et al., 2007). Ovomucin is a glycoprotein responsible for the gel-like properties of albumen. Weakening of the interaction between ovomucin and lysozyme at alkaline pH during storage causes albumen thinning, which directly affects the HU and albumen index (Liu et al., 2018; Shan et al., 2020; Wang et al., 2012).

Stringent control of the temperature of transportation and storage is required to minimize the growth of foodborne pathogens on eggs and maintain egg quality (Chousalkar et al., 2018). In the Republic of Korea, both washed, and unwashed eggs are allowed to be distributed, and the storage temperature is regulated at 0–10 °C and 1–35 °C, respectively. In the United States, eggs must be distributed after washing and stored below 7 °C within 36 h after laying.

A number of studies have investigated the relationship between temperature and growth characteristics of SE on eggs (Kingsbury et al., 2019; Messens et al., 2006; Park et al., 2015). Unwashed eggs can be distributed at various temperatures (Lee and Hong, 2005; Lee et al., 2004). Bovill et al. (2000) and Huang et al. (2019) investigated the growth characteristics of Salmonella spp. on various food matrices as a function of storage temperature. However, the assessment of survival characteristics of Salmonella spp. on eggshell, which is the most frequent vehicle of foodborne outbreaks, and the quality deterioration of eggs with temperature changes is not sufficient.

Thus, we assessed the effect of temperature alteration on SE survival on eggshell and egg quality maintenance to provide valuable scientific evidence for the safety management of unwashed eggs in the actual distribution environment.

Materials and methods

Preparation of unwashed eggs and inoculation of SE

The unwashed eggs were purchased from a local layer farm in the Republic of Korea (JH egg farm, Seoun-myeon, Anseong-si, Gyeonggi-do, Republic of Korea). The inoculation of SE on eggshell followed the protocol of Park et al. (2015) with minor modifications. SE (ATCC 13076) was pre-incubated in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD, USA) for 24 h at 37 °C, and then the supernatant was discarded after centrifugation of the culture at 5000×g, 4  °C for 5 min. The remaining cells were washed and resuspended with 10 mL of phosphate buffer (PBS; LPS Solution, Daedeok-gu, Daejeon, Republic of Korea). Twenty microliters of 8 log CFU/mL of SE were inoculated at 10 random spots on the eggshell, and then the inoculated eggs were stored in a biosafety cabinet for 1 h before incubation under each condition.

Incubation conditions

To investigate an appropriate storage temperature for SE control on eggshell, SE-inoculated eggs were incubated at three different temperatures (10 °C, 25 °C, and 35 °C). Additionally, the SE-inoculated eggs were incubated under changing temperature conditions to confirm the effect of the storage temperature on the control of SE on eggshell as well as on the maintenance of the egg quality.

Determination of SE

To assess the growth characteristics of SE on eggshell, each egg sample was placed in a sample bag (1020W; 3 M, St. Paul, MN, USA) containing 10 mL of peptone buffer (BPW; Oxoid, Hampshire, UK), which was pre-heated at 35 °C and gently rubbed over the eggs for 1 min. Subsequently, the rinsate was plated onto xylose lysine deoxycholate (XLD; Difco, Becton Dickinson, Sparks, MD, USA) plates and incubated at 37 °C for 24 h. After incubation, typical colonies were counted, and the remaining rinsate was enriched at 37 °C for 24 h. When no colonies were formed on XLD, 0.1 mL of the enriched culture was added to 10 mL of Rappaport − Vassiliadis R10 broth (RVB; Difco, Becton Dickinson, Sparks, MD, USA) and enriched at 41.5 °C for 24 h. Enriched RVB was streaked on XLD plates using a loop. The XLD plates were incubated at 37 °C for 24 h and examined for SE. To allow log transformation of data when no SE colonies were detected in the rinsate but SE was detected following enrichment, a count of 5 CFU/egg was assigned. When there were no colonies by both direct plating and enrichment, a count of 1 CFU/egg was assigned.

Quality assessment

The quality assessment was conducted according to Kim et al. (2014) with minor modifications. The widths (mm) of thick albumen and yolk were measured with vernier calipers (CD-30PSX; Takatsu, Mitutoyo, Kawasaki, Japan), and the heights (mm) of thick albumen and yolk were measured with a tripod micrometer (Digital Haugh Tester; Orka Food Technology, West Bountiful, UT, USA). HU was calculated by Eq. (1).

$$\mathrm{HU}=100\log (H+7.57-{1.7W}^{0.37})$$ 1

where H is the height of the thick albumen, and W is the weight of the egg.

The yolk index and albumen index were calculated by Eq. (2) and (3), respectively.

$$\mathrm{Yolk}\mathrm{index}=\frac{\mathrm{Yolk}\mathrm{height}}{\mathrm{Yolk}\mathrm{width}}$$ 2

$$\mathrm{Albumen}\mathrm{index}=\frac{\mathrm{Thick}\mathrm{albumen}\mathrm{height}}{\mathrm{Thick}\mathrm{albumen}\mathrm{width}}$$ 3

The albumen and yolk of each egg were separated and homogenized, and the pH of the albumen and yolk were measured using a pH meter (S210; Mettler Toledo, Greifensee, Zürich, Switzerland).

Statistical analysis

All data were analyzed using the SPSS software package (SPSS 26.0; IBM Corp., New York, NY, USA). One-way ANOVA was used to determine the effect of each storage time and condition on SE, HU, yolk index, albumen index, and the pH of yolk and albumen. Duncan's multiple range test was used to determine differences in mean values.

Results and discussion

Effect of temperature on growth of SE on eggshells

The growth characteristics of SE on eggshell during storage at three different temperatures (10 °C, 25 °C, and 35 °C) for 21 days are provided in Table 1. SE was not detected on 3 days after inoculation (DAI) at 25 °C and on 5 DAI at 35 °C among the tested temperatures. By contrast, SE survived on the eggshell for 21 days at 10 °C. From the data, 25 °C was determined to be an appropriate storage temperature for unwashed eggs for safety management against SE. Therefore, we conducted further studies to investigate the effect of altering the storage temperature on the control of SE and the quality of eggs. With respect to the relationship between storage temperature and SE survival on eggshell, Messens et al. (2006) reported that the SE population on eggshell decreased more rapidly as the temperature increased from 15 to 25 °C. Similarly, in the current study, SE was detected on the eggshell at 10 °C during all test days, whereas SE was not detected at 25 °C from 3 DAI.

Table 1.

Effect of storage temperature on growth of SE on eggshells

Temperature ( °C )	Mean ± SEM log CFU/egg1 during storage (day)
	0	1	3	5	7	14	21
35	4.20 ± 0.26a	0.70 ± 0.00b	0.47 ± 0.23b	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c
25	4.20 ± 0.26a	3.12 ± 0.08b	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c
10	4.20 ± 0.26a	3.55 ± 0.09ab	2.98 ± 0.13bc	2.50 ± 0.45c	2.56 ± 0.18c	2.39 ± 0.16 cd	1.77 ± 0.06d

Means in the same row followed by different letters are significantly different (p < 0.05)

¹To allow log transformation of data when no SE colonies were detected following direct plating, but SE was detected after enrichment, a count of 5 CFU/egg was assigned. When there were no colonies by both direct plating and enrichment, a count of 1 CFU/egg was assigned

To investigate the survival of SE inoculated on eggshell under storage temperature conditions, the eggs were incubated in the absence and presence of changing temperature conditions after an initial temperature of 25 °C, which was determined to be an appropriate storage temperature for unwashed eggs, as mentioned above. As shown in Table 2, SE was not detected 3 DAI when the temperature was continuously maintained at 25 °C. SE was also detected before 3 DAI although the temperature was changed to 10 °C or 35 °C. These results indicated that altering the storage temperature from 25 °C to 10 or 35 °C was not effective for controlling SE on eggshell. Bovill et al. (2000; 2001) reported that temperature fluctuation did not affect the growth of Salmonella in pasteurized milk, chicken liver pâté, and minced chicken. However, observed that temperature changes affected the growth of SE on fresh-cut cantaloupe. Without temperature changes, the SE population was maintained at the initial level (3 log CFU/g) at 4 °C for 7 days and increased significantly at 8 and 12 °C. When the cantaloupe was initially exposed to room temperature (25 °C) for 4 h before storage at 4 °C, the number of SE colonies increased by 0.6 log CFU/g, whereas the growth of SE was not significant at 4 °C for 7 days in the absence of a temperature change. Huang et al. (2019) reported that changing temperature did not affect the growth of Salmonella on cut cantaloupe, honeydew, watermelon, and radish. On the other hand, the growth of Salmonella on fresh-cut pineapple was affected by storage temperature. It seems that the different growth characterization of Salmonella by storage temperature on various foods is caused by physico-chemical properties of matrices. Li et al. (2020) reported that the patterns and morphology of attached bacteria are associated with roughness of matrices, and the numbers of attached five Salmonella cells were significantly correlated with the surface roughness for all five S. enterica strains.

Table 2.

Effect of changing the storage temperature on SE on eggshells

Duration of 25 °C storage (day)	Altered temp. ( °C )	Mean ± SEM log CFU/egg1 during storage (day)
		0	1	2	3	4	5	6	7
7	-	4.16 ± 0.10a	1.34 ± 0.27b	0.12 ± 0.12c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.66 ± 0.66bc	0.00 ± 0.00c
1	35	4.16 ± 0.10a	1.34 ± 0.27b	0.23 ± 0.15c	0.00 ± 0.00c	0.00 ± 0.00c	0.12 ± 0.12c	0.00 ± 0.00c	0.00 ± 0.00c
	10	4.16 ± 0.10a	1.34 ± 0.27b	0.40 ± 0.18b	1.47 ± 0.60b	0.96 ± 0.43b	0.40 ± 0.18b	0.70 ± 0.32b	0.61 ± 0.31b
2	35	4.16 ± 0.10a	1.34 ± 0.27b	0.12 ± 0.12c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c
	10	4.16 ± 0.10a	1.34 ± 0.27b	0.12 ± 0.12c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.38 ± 0.38c
3	35	4.16 ± 0.10a	1.34 ± 0.27b	0.12 ± 0.12c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c
	10	4.16 ± 0.10a	1.34 ± 0.27b	0.12 ± 0.12c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c	0.00 ± 0.00c

Means in the same row followed by different letters are significantly different (p < 0.05)

¹To allow log transformation of data when no SE colonies were detected following direct plating, but SE was detected after enrichment, a count of 5 CFU/egg was assigned. When there were no colonies by both direct plating and enrichment, a count of 1 CFU/egg was assigned

Effect of changing storage temperature on HU

To confirm whether the storage temperature affected the quality of eggs inoculated with SE onto the shell, we measured the HU, as a representative quality indicator of eggs, in the presence and absence of changing temperature conditions. When eggs inoculated with SE onto shell were maintained at 25 °C for 7 days, the HU decreased significantly by around 18.7% on day 7 (Fig. 1). Meanwhile, when the storage temperature was lowered to 10 °C on 3 DAI and 4 DAI, the HU decreased by around 8.4% and 10.2% on day 7, respectively. Similar to our findings, Samli et al. (2005) reported that the HU of eggs decreased by around 16.5%, 41.2%, and 55.6% at 5 °C, 21 °C, and 29 °C on day 10, respectively. It seems that decreasing HU caused by the deterioration of thick albumen by liquefaction in high temperature. Liu et al. (2016) reported that albumen liquefaction by the increased pH and weakened interaction between ovomucin-lysozyme resulted decreasing HU, significantly. Likewise, Raji et al. (2009) found that the HU of eggs stored at 40 °C decreased by 64.4%, which was twice that observed during storage at 32 °C (32.2%).

Fig. 1.

Effect of temperature alteration during 25 °C storage on HU of eggs. Data represent mean ± SEM

Effect of changing storage temperature on yolk index and albumen index

The effect of altering storage temperature on the yolk index is presented in Fig. 2. When the eggs were continuously stored at 25 °C, the yolk index decreased significantly by around 22.9% (from 0.48 ± 0.01 to 0.37 ± 0.01) on day 7. However, when the initial storage temperature of 25°C was lowered to 10 °C on day 3 and day 4, the yolk index decreased by around 8.3% (0.44 ± 0.01) and 10.4% (0.43 ± 0.01) on day 7, respectively. Jones et al. (2018) reported that the yolk index was not significantly changed at 4 °C for 6 weeks but decreased significantly by 45% at 22 °C. The albumen index showed a significant 46.7% decrease (from 0.15 ± 0.01 to 0.08 ± 0.01) when the temperature was continuously maintained at 25 °C (Fig. 3). However, it decreased by 26.7% when the initial storage temperature of 25 °C was lowered to 10 °C on day 3 (0.11 ± 0.01) and day 4 (0.11 ± 0.01), respectively. In previous studies on the relationship between storage temperature and albumin index, the height and width of albumen were significantly affected by the temperature, and the albumen index decreased with increasing temperature (Keener et al., 2006; Lee et al., 2014; Samli et al., 2005). Furthermore, Ha et al. (2002) reported that the albumen index decreased by around 45.6%, 53.1%, 67.2%, and 72.7% at 15 °C, 20 °C, 25 °C, and 30 °C on day 10 of storage, respectively. Additionally, we monitored the pH of yolk and albumen with storage temperature. From the data (Fig. 4A and B), altering the storage temperature did not affect the yolk pH. Conversely, when the initial storage temperature of 25 °C was lowered to 10 °C on day 3 and day 4, the pH of albumen was significantly maintained the initial level compared to continuous storage at 25 °C. Several publications reported that the storage temperature did not significantly affect the yolk pH, whereas the albumen pH increased significantly with increasing temperature (Choi et al., 2017; Kim et al., 2014; Lee et al., 2016). This difference was attributed to the lower release of carbon dioxide from yolk compared to albumen.

Fig. 2.

Effect of temperature alteration during 25 °C storage on yolk index of eggs. Data represent mean ± SEM

Fig. 3.

Effect of temperature alteration during 25 °C storage on albumen index of eggs. Data represent mean ± SEM

Fig. 4.

Effect of temperature alteration during 25 °C storage on pH of yolk (A) and albumen (B). Data represent mean ± SEM

Declarations

Conflict of interest

The authors declare no conflicts of interest.