Egg safety: Salmonella culturability in acidified raw egg-based foods

Egg safety: Salmonella culturability in acidified raw egg-based foods

Words by Dr Andrea R McWhorter

Raw or lightly cooked eggcontaining foods are frequently identified as sources of Salmonella during outbreak investigations of human gastroenteritis cases.1,2,3 Despite health and safety recommendations, raw egg-based foods continue to be linked with salmonellosis outbreaks and remain a public health issue.

Between 2001 and 2011, 36% of the total number of Australian Salmonella outbreaks were attributed to the consumption of contaminated raw egg-based foods.3 Mayonnaise and mayonnaise-like sauces (aioli, Bánh mì butter) are among the most common egg-based foods linked with salmonellosis.4

Over the past 30 years, egg safety research has demonstrated that there are several factors contributing to the persistence of Salmonella in raw egg-based food items. Storage temperature and acidification of raw egg-based sauces have been highlighted as the most important parameters in controlling Salmonella culturability.5,6,7,8,9,10 Consequently, many food safety recommendations regarding the use of raw eggs have been based on these outcomes.

In Australia, health and safety regulators recommend that raw egg-based foods should be prepared using fresh eggs, acidified to pH ≤ 4.2, and stored at 5°C for no more than 24 hours.11,12 pH ≤ 4.0 inhibits Salmonella culturability after a short incubation.5,6,7,8,9

Counter to safe food recommendations, however, Salmonella culturability is reduced faster at temperatures ≥ 25°C.

Following four to six hours of incubation at room temperature (23-25°C), Salmonella are not culturable from acidified mayonnaise preparations.5,6,7,9,10,13 At 5°C, however, bacteria remain culturable for longer

periods.5,6,7,9,10,13

Recipe variation is another factor that could contribute to Salmonella persistence in raw egg-based foods.10 Many recipes call for the use of different egg components such as yolk only, whole egg, or a combination of both. Whole eggbased sauce preparations have recently been shown to support culturable Salmonella significantly longer compared with yolk only preparations.10 The pH of egg white is higher (8-9) than yolk (6-8)14 which may interfere with the acidification of sauce preparations.

Over time, egg quality changes which can also affect the physical properties of raw egg containing sauces. As an egg ages, carbon dioxide is lost through the egg shell pores leading to an increase in pH for both the yolk and egg white.15 Additionally, egg white contains bactericidal proteins, such as lysozyme and ovotransferin, which also decrease in activity over time.15 Thus, for raw egg-based sauces, the use of fresh eggs should be strongly recommended.

Many mayonnaise recipes also vary in the type and quantity of recommended food acids. The effectiveness of two common food acids, lemon juice and white vinegar, added singly or in a combination of different ratios, has recently been investigated.10

Acidified sauces limit Salmonella culturability regardless of acid type or combination used as long as the sauce pH was ≤ 4.0.5,7,8,10,13,16

Yolk - food acid ratios (as defined by mass) have also been proposed to serve as guidelines for sauce acidification. A ratio of 1.0 produces a pH of approximately 3.5 and reduces culturable bacteria after a few hours of incubation.8,16

In McWhorter, Sexton et al. (2020)10 and McWhorter, Khan et al. (2021),17 the yolk:acid ratio for laboratory sauce experiments was consistently 1.78, which yielded a pH of 3.8.

A comparison of commercially prepared sauces found that yolk:food acid ratios ranged between 0.22 and 5.33, depending on preparation. Sauces with lower ratios were more successful at reducing Salmonella culturability.10 Egg yolk mass, however, can vary significantly and is linked with the age and breed of layer hen.18 Additional research is necessary to establish more broadly applicable egg:food acid ratios that consider yolk mass variability and the use of egg white.

Salmonella culturability from raw egg-based foods is also linked with the initial bacterial load. Acidified mayonnaise preparations (pH 3.5) inoculated with a high Salmonella load (109 CFU/g) supported culturable bacteria for eight hours at 25°C and 60 hours at 5°C, longer compared with preparations containing 106 CFU/g bacteria and stored under the same conditions (Figure 1).17 These results highlight the importance of minimising Salmonella in the egg supply chain as well as maximising good kitchen hygiene behaviours that prevent cross contamination and bacterial amplification.

To date, the disease potential for Salmonella in acidified raw egg-based foods has not been well characterised. A recent study investigated the effect of storage temperature and acidification stress on bacterial virulence. A decline in bacterial culturability was linked with a reduction in both in vitro and in vivo bacterial virulence and viability.17

Bacterial motility was significantly reduced in acidified mayonnaise compared with bacteria grown to stationary phase.

Salmonella motility is thought to contribute to virulence by enabling bacteria to come into proximity with host intestinal epithelial cells,

Figure 1. Culturability of Salmonella is affected by temperature, pH, and bacterial load. Data are presented as mean log10 transformed CFU/gram mayonnaise ± the standard error of the mean. Culturability of bacteria was tested over a 96-hour period in mayonnaise preparations at pH 3.5, 4.0, 4.5, or 5.0 and stored at 5°C (A) and 25°C (B). Mayonnaise (pH 3.5) inoculated with 109 CFU/gram and stored at 5°C or 25°C exhibited culturable bacteria (C).

facilitating invasion.19,20 In the same study, Salmonella also exhibited reduced invasiveness into cultured intestinal epithelial cells following isolation from mayonnaise. It has been hypothesised that in acidic environments Salmonella reduces the expression of flagellar proteins and this may be linked with a reduction in cell invasion.

Salmonella in acidified mayonnaise preparations stored for 12 hours at either 5°C or 25°C has also been shown to have reduced virulence in mice.17 To date there have been no other studies investigating the in vivo virulence of Salmonella in acidified raw egg-based sauces.

Non-motile Salmonella, however, are known to have reduced virulence in mouse models.19,21 During in vivo infection, bacteria would experience additional stress associated with passage through the gastrointestinal system of mice.22 Combined with prior acid exposure in mayonnaise, the harsh host environment would present further challenges for injured bacterial cells. Therefore, where possible, acidification of foods containing raw eggs should be recommended.

Worldwide, food safety authorities generally do not recommend the use of raw eggs in food. A significant body of egg safety research has demonstrated that acidified raw egg-based sauces, when stored at room temperature for at least four hours, significantly reduces the culturable form of Salmonella and that immediate storage at 5°C may facilitate persistence of viable bacteria.

Heat treating egg components has also been shown to reduce the risk of viable Salmonella and is the recommended practice in the USA.23 Future research is needed to determine whether sauce acidification and heat treatment followed by cold storage further minimises the culturability of Salmonella in acidified raw egg-based sauces.

References

1. Threlfall, E. J. et al. (2014). “Egg-borne infections of humans with Salmonella: not only an S. enteritidis problem.” World’s Poultry Science

Journal 70(1): 15-26. 2. Glass, K. E. et al. (2016). “Bayesian source attribution of salmonellosis in South Australia.”

Risk analysis 36(3): 561-570. 3. Moffatt, C.R.M.. et al. (2016). “Salmonella

Typhimurium and outbreaks of egg-associated disease in Australia, 2001 to 2011.” Foodborne pathogens and disease 13(7): 379-385. 4. Ford, L. et al. (2018). “The Epidemiology of

Salmonella enterica outbreaks in Australia, 20012016.” Frontiers in Sustainable Food Systems 2: 86. 5. Perales, I. and M. Garcia (1990). “The influence of pH and temperature on the behaviour of Salmonella Enteritidis phage type 4 in home-made mayonnaise.” Letters in applied microbiology 10(1): 19-22. 6. Erickson, J., P. and P. Jenkins (1991).

“Comparative Salmonella spp. and Listeria monocytogenes inactivation rates in four commercial mayonnaise products.” Journal of food protection 54(12): 913-916. 7. Lock, J. and R. Board (1995). “The fate of

Salmonella Enteritidis PT4 in home-made mayonnaise prepared from artificially inoculated eggs.” Food Microbiology 12: 181-186. 8. Xiong, R. et al. (1999). “The fate of Salmonella

Enteritidis PT4 in home-made mayonnaise prepared with citric acid.” Letters in applied microbiology 28(1): 36-40. 9. Keerthirathne, T. P. et al. (2019). “The Combined

Effect of pH and Temperature on the Survival of Salmonella enterica Serovar Typhimurium and Implications for the Preparation of Raw Egg

Mayonnaise.” Pathogens 8(4): 218. 10. McWhorter, A. R. et al. (2020). “The effects of varied food acid ratios and egg components on

Salmonella Typhimurium culturability from raw egg-based sauces.” Food Microbiology: 103555. 11. Anonymous. (2012). “Egg Food Safety.” https://www.sahealth.sa.gov.au/wps/wcm/

connect/public+content/sa+health+internet/ public+health/food+safety+for+businesses/ food+safety+issues/egg+food+safety. 12. Anonymous. (2016). “Food safety guildelines for the preparation of raw egg products” https://www.foodauthority.nsw.gov.au/sites/ default/files/_Documents/retailfactsheets/ safe_preparation_of_raw_egg_products.pdf. 13. Lock, J. and R. Board (1994). “The fate of

Salmonella enteritidis PT4 in deliberately infected commercial mayonnaise.” Food microbiology 11(6): 499-504. 14. Fromm, D. (1966). “The influence of ambient pH on moisture content and yolk index of the hen’s yolk.” Poultry Science 45(2): 374-379. 15. Guyot, N. and S. Réhault-Godbert (2017).

Understanding the natural antibacterial defences of egg white and their regulation Florence

Baron, INRA–Agrocampus Ouest, France.

Achieving sustainable production of eggs

Volume 1, Burleigh Dodds Science Publishing: 181-214. 16. Zhu, J. et al. (2012). “Survival of Salmonella in home-style mayonnaise and acid solutions as affected by acidulant type and preservatives.”

Journal of food protection 75(3): 465-471. 17. McWhorter, A. R. et al. (2021). “Acidification and extended storage at room temperature of mayonnaise reduce Salmonella Typhimurium virulence and viability.” Food Research

International 141: 110117. 18. Silversides, F. and K. Budgell (2004). “The relationships among measures of egg albumen height, pH, and whipping volume.” Poultry

Science 83(10): 1619-1623. 19. Jones, B. D. et al. (1992). “Invasion by Salmonella typhimurium is affected by the direction of flagellar rotation.” Infection and Immunity 60(6): 2475-2480. 20.Saini, S. et al. (2011). “Continuous control of flagellar gene expression by the -28–FlgM regulatory circuit in Salmonella enterica.”

Molecular microbiology 79(1): 264-278. 21. Haiko, J. and B. Westerlund-Wikström (2013).

“The role of the bacterial flagellum in adhesion and virulence.” Biology 2(4): 1242-1267. 22. Palmer, A. D. and J. M. Slauch (2017).

“Mechanisms of Salmonella pathogenesis in animal models.” Human and Ecological Risk

Assessment: An International Journal 23(8): 1877-1892. 23. Anonymous. (2019). “Shell eggs from farm to table.” https://www.fsis.usda.gov/food-safety/ safe-food-handling-and-preparation/eggs/shelleggs-farm-table

Dr Andrea McWhorter is a postdoctoral research fellow in the School of Animal and Veterinary Sciences at the University of Adelaide. f