The fate of Salmonella enteritidis PT4 in home-made mayonnaise prepared with citric acid

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1 Letters in Applied Microbiology 1999, 28, The fate of Salmonella enteritidis PT4 in home-made mayonnaise prepared with citric acid R. Xiong, G. Xie and A.S. Edmondson Food Research Group, Leeds Metropolitan University, Leeds, UK 1886/98: received and accepted 14 October 1998 R. XIONG, G. XIE AND A.S. EDMONDSON The fate of Salmonella enteritidis PT4 in home-made mayonnaise prepared with citric acid solution (citric acid concentration of 4 98% (w/v)) was investigated. It was found that ph of mayonnaise is closely related to the ratio of egg yolk to citric acid, and the inactivation rate of the micro-organisms increases as the ratio decreases and/or incubation temperature increases. To achieve Salm. enteritidis PT4-free home-made mayonnaise prepared with pure lemon juice (citric acid concentration 5% (w/v)), it is recommended that the ph should be 3 30 or below, or, in practice, at least 20 ml pure lemon juice per fresh egg yolk should be used. For the use of ml pure lemon juice per egg yolk, the product should be held at 22 C or over for at least 72 h and for the use of over 35 ml pure lemon juice per egg yolk, for at least 48 h before consumption or refrigeration. INTRODUCTION There have been many reported outbreaks of food poisoning caused by Salmonella associated with the consumption of home-made mayonnaise (Radford and Board 1993). In these outbreaks, raw eggs have been identified as a major vehicle of infection. The fate of Salmonella in mayonnaise prepared with acetic acid (vinegar) has been extensively studied and its safety has been clearly established. To maintain mayonnaise Salmonella free, the ph should be 4 1 or less and the product should be held at C for at least 72 h unless pasteurized eggs are used (Smittle 1977). Perales and Garcia (1990) suggested that to prevent salmonellosis transmission by homemade mayonnaise, vinegar should be used as the sole acidulant (ph ) and the product should be stored in a warm place for some hours or days before consumption. In their review, Radford and Board (1993) recommended that mayonnaise should be prepared with vinegar to a ph of 4 1 or less and that the product should be held for at least 24 h at C before refrigeration. Xiong et al. (1998) proposed a new method to link the ph of mayonnaise to the ingredients; they recommended that mayonnaise should contain at least 20 ml (white wine) vinegar per fresh egg yolk and that the product should be held in a warm place for at least 1 d, or preferably 3 d, before consumption or refrigeration. In contrast, few studies have focused on the fate of Sal- Correspondence to: R. Xiong, Food Research Group, Leeds Metropolitan University, Leeds, LS1 3HE, UK ( R.Xiong@lmu.ac.uk). monella in mayonnaise prepared with citric acid (lemon juice) and its safety has not been clearly established (Radford and Board 1993; Membre et al. 1997). At equivalent ph values, vinegar used in mayonnaise was more bactericidal than lemon juice, and the use of vinegar was advised by Perales and Garcia (1990). Xiong et al. (1998) found that for mayonnaise prepared with vinegar, the ratio of egg yolk to vinegar can be used as a safety indicator, which is easier to use than ph measurement. The ratio of egg yolk to citric acid solution may also be used as a safety indicator for mayonnaise prepared with citric acid. The aims of this study were to investigate the fate of Salm. enteritidis PT4 in home-made mayonnaise prepared with citric acid and the relationship between the safety of mayonnaise and the ratio of egg yolk to citric acid solution. MATERIALS AND METHODS Measurement of acidity and citric acid concentration Titratable citric acidity was measured as follows: (i) 10 g sample was diluted to 100 ml solution with distilled water; (ii) 20 ml of the solution was titrated against 0 1 mol l 1 sodium hydroxide using phenolphthalein as indicator; (iii) three readings were averaged out; (iv) the concentration (% (w/v)) of citric acid in the sample was calculated. Two bottles of commercial pure lemon juice were purchased from a local supermarket. The first was made from 1999 The Society for Applied Microbiology

2 SALMONELLA IN CITRIC ACID MAYONNAISE 37 the natural juice of eight lemons with added preservative (sodium metabisulphate) and had the cloudy appearance of squeezed lemon juice. The second was made from natural lemon juice, sodium metabisulphate and vitamin C and had a clear appearance. The concentration of citric acid in these two bottles of lemon juice was measured and the average values were 4 98 and 5 99% (w/v), respectively. The citric acid concentration of 4 98% was used throughout this study because, except for the preservative, it is squeezed lemon juice (ph and citric acid concentration % (w/v)) which is widely used in mayonnaise recipes. All citric acid solutions for mayonnaise were made up using Citric Acid Anhydrous (Fisher Scientific UK Limited, UK) and distilled water. Mayonnaise and ph measurement The mayonnaise was prepared using the following recipe: 20 g egg yolk, 250 ml pure sunflower oil, 2 g salt, 1 5 g mustard, 1 g sugar, 0 5 g white pepper and different amounts of the citric acid solution (10, 15, 20, 25, 30, 35 ml). Mayonnaise was prepared as follows: (i) all ingredients except the oil were mixed with an electronic hand-mixer and (ii) oil was poured steadily while blending at high speed. After preparation, the mayonnaise was stored overnight at room temperature. ph was measured at room temperature using a Philips PW 9420 ph meter (Pye Unicam, Cambridge, UK). Test organism and growth condition A loopful of Salmonella enteritidis PT4 (the strain had been isolated by the Central Public Health Laboratories, Colindale, following an outbreak of food poisoning involving mayonnaise) from a Nutrient Agar slope at room temperature was inoculated into 10 ml 0 1% peptone (Oxoid) containing 10% egg yolk emulsion (Oxoid). The culture was incubated statically at 37 C for 24 h. Experimentation showed that this produced a stationary phase. The culture was then added to the mayonnaise at the rate of 1 ml 100 g 1 to give approximately 10 6 g 1 organisms, and blended with a Colworth stomacher (Seward, London, UK) for 2 min. The contaminated mayonnaise products were immediately stored at C and C, respectively. Microbiological analysis For viable counts, the samples were taken at time intervals after contamination. At each time interval, 10 g contaminated mayonnaise were weighed into a stomacher bag, 90 ml Buffered Peptone Water (BPW) (Oxoid) added and the mixture homogenized for 1 min with a Colworth stomacher. Serial 10-fold dilutions in 0 1% peptone diluent were plated onto XLD agar (Oxoid) using the surface drop method (Roberts et al. 1995). The XLD agar plates were incubated at 37 C for at least 24 h and the viable colonies then enumerated. For presence/absence tests, a 25 g mayonnaise sample was added to 225 ml BPW in a sterile container at each time interval. The mixture was swirled well and incubated at 37 C for 24 h. After incubation, 0 1 ml of the mixture or enrichment was added to 4 ml Rappaport-Vassiliadis (RV) R10 broth (Difco) in prepared indirect RABIT tubes (Rapid Automated Impedance Technique; Don Witley Scientific, Shipley, UK). The RABIT tubes were placed into a RABIT incubator block and incubated at 42 C for 18 h (Donaghy and Madden 1993). All the experiments in this study were performed in duplicate. RESULTS AND DISCUSSION The fate of Salm. enteritidis PT4 in mayonnaise prepared with citric acid solution is illustrated in Fig. 1. The microorganism did not grow in any of the tested samples because the ph values were all below 4 05, which is the minimum ph required for the growth of Salmonella in citric acid solution (Chung and Geopfert 1970). Comparing Fig. 1a and 1b, it can be seen that the inactivation of Salm. enteritidis PT4 at 22 C was more rapid than at 5 C. At an egg yolk to citric acid ratio of 1 33 (the ratio of egg yolk to citric acid is defined as the ratio of the weights of these two ingredients), for example, the time for the number of surviving micro-organisms to be reduced to the undetectable level (83 cfu g 1 )at 22 C was 24 h, while at 5 C it was 48 h. The results of the presence/absence tests (Table 1) also show that the microorganism was still detectable in all the tested samples stored at 5 C after 168 h, while at 22 C no micro-organisms were detected in the same time duration. This confirms that storage of mayonnaise at a lower temperature protects Salmonella to some extent against the antimicrobial effects of the acids used (Perales and Garcia 1990). However, the shelf-life of mayonnaise stored at 22 C is much shorter than at 5 C. All mayonnaise samples stored at 22 C were spoiled by moulds in 6 10 d, while no visible moulds were observed after 1 month when they were stored at 5 C. Figure 1(a and b) also shows that the inactivation rates of Salm. enteritidis PT4 increase as the ratio of egg yolk to citric acid decreases. At the ratios of 2 0, 1 33, 1 00, 0 80, 0 67 and 0 57, the micro-organism was inactivated to undetectable level at 22 C within 48, 24, 7, 4, 2 and 1 h, respectively. It is apparent from Table 1 that when the ratio was greater than 1 00, the micro-organism was present in all the samples after 72 h incubation whatever the storage temperature used, while it was not detected at 22 C after 72 h with a ratio of 1 00 or less, and after 48 h with a ratio of 0 57 or less. The smaller the ratio, the shorter the survival time period for Salm. enteritidis PT4. The reason for this may be that as the citric acid concentration increases (Table 1), the ph falls and this, in

3 38 R. XIONG ET AL. Fig. 1 The behaviour of Salmonella enteritidis PT4 at different ratios of egg yolk to citric acid at 5 C (a) or 22 C (b): ratio of 2 0 (); ratio of 1 33 (r); ratio of 1 0 (ž); ratio of 0 8 (Ž); ratio of 0 67 () and ratio of 0 57 (R) Table 1 Relationships between the ratio of egg yolk to citric acid, ph, citric acid concentration and microbiological safety of mayonnaise Ratio of Citric acid egg Presence/absence of Salmonella enteritidis PT4 at concentration yolk to ph in mayonnaise citric Expt 1 Expt 2 5 C 22 C (% w/v) acid ph used ph used 24 h 48 h 72 h 168 h 24 h 48 h 72 h 96 h 168 h Expt 1 Expt / ND* / ND / / ND ND / ND ND * ND, not done. /, one positive result from two experiments. turn, results in more undissociated acid molecules in the mayonnaise. The more undissociated acid molecules that are present, the more rapid the inactivation (Smittle 1977; Radford and Board 1993). In this study, the ph of mayonnaise was measured immediately after preparation (data not shown) and just before testing (Table 1). The ph of mayonnaise drops as the ratio decreases. The relationship between them can be described by the following equation: ph r r 2 (R ) where ph is the ph of mayonnaise measured just before testing, and r is the ratio of egg yolk to citric acid. The high correlation coefficient (R ) indicates that the ph of

4 SALMONELLA IN CITRIC ACID MAYONNAISE 39 mayonnaise is closely related to the ratio of egg yolk to citric acid. When the ratio was equal to or less than 1 00, the ph was 3 32 or less and no viable cells were detected at 22 C after 72 h incubation. This implies that the ph of 3 32 or less is critical for the safety of mayonnaise prepared with citric acid and stored at room temperature. The ph of 3 32 is much lower than the ph of 4 0 or 4 1 recommended for mayonnaise prepared with acetic acid and stored at C. The ph of mayonnaise is an intrinsic safety indicator, but it is usually impossible for caterers and domestic cooks to use because of the lack of facilities with which to measure ph in the kitchen. To avoid ph measurement, the use of the ratio of egg yolk to acetic or citric acid was proposed by Xiong et al. (1998). In practice, egg yolk is usually used by number not by weight. The weight of an egg yolk varies with size. On average, weights of a small, medium and large size egg yolk are 13, 15 and 17 g, respectively (Griswold 1973). The heaviest egg yolk (large size) used by Xiong et al. (1998) was g. For convenience and safety, it has been assumed that the heaviest weight of the yolk for a large, medium or small size of egg is 20 g (25 g for the yolk of a jumbo egg ( 70 g)). Therefore, the ratio of egg yolk to citric acid can easily be converted into the amount of citric acid solution per egg yolk (20 g). For example, the ratios of 1 00, 0 80, 0 67 and 0 57 can be converted to 20, 25, 30 and 35 ml citric acid solution per egg yolk, respectively. The ratio of egg yolk to acid (vinegar or lemon juice) can also be used to assess the microbiological safety of a mayonnaise recipe (Xiong et al. 1998). In this study, it was found that when the recipes used less than 20 ml citric acid solution per egg yolk, the Salmonella survived for more than 96 h at 22 C, while the micro-organism was inactivated at 22 C within 72 h when at least 20 ml citric acid per egg yolk was used. This suggests that a mayonnaise recipe using less than 20 ml citric acid solution per egg yolk can be considered unsafe unless pasteurized eggs are used. However, many mayonnaise recipes use only 15 ml or less lemon juice per egg yolk (Ellison 1982; Day-Lewis and Heathcote 1983; Ceserani et al. 1988; Madison 1996). If raw eggs are used for these recipes, there would be a very high risk of food poisoning. If unsafe mayonnaise recipes can be eliminated, outbreaks of food poisoning attributed to home-made mayonnaise may be reduced significantly or even eliminated. The ratio makes it possible to set a safety criterion for mayonnaise recipes. To maintain Salm. enteritidis PT4-free mayonnaise prepared with pure lemon juice (citric acid concentration 5% (w/v)), it is recommended that the ph should be 3 30 or below, or in practice, at least 20 ml pure lemon juice per egg yolk should be used. After preparation, when using ml pure lemon juice per egg yolk, the product should be held at 22 C or over for at least 72 h and when using over 35 ml pure lemon juice per egg yolk, for at least 48 h before consumption or refrigeration. This recommendation is made with regard to the advice of the Chief Medical Officer that eating raw eggs should be avoided and that for vulnerable people such as the elderly, the sick, babies and pregnant women, pasteurized eggs should be used (Department of Health 1998). Several researchers have reported the reduced recovery of stressed Salmonella on selective agars (Janssen and Busta 1973; Strantz and Zottola 1993). As XLD agar is a selective agar, it might not adequately support resuscitation and eventual colony formation by citric acid-injured cells. It indicates that further research on the ability of citric acid-injured cells to grow on XLD agar is needed. However, the use of XLD agar does not affect the results of the presence/absence tests from which the recommendation is derived. ACKNOWLEDGEMENTS The authors thank Chris Boothby, Russ Fisher, Mary Rolf and K. Watson for technical support. REFERENCES Ceserani, V., Kinton, R. and Foskett, D. (1988) Contemporary Cookery. London: Edward Arnold. Chung, K.C. and Geopfert, J.M. (1970) Growth of Salmonella at low ph. Journal of Food Science 35, Day-Lewis, T. and Heathcote, T. (1983) The Englishwoman s Kitchen. London: Chatto & Windus. Department of Health (1998) Expert advice repeated on Salmonella and raw eggs. Available from deptgdh/coi0316e.ok (accessed October 5, 1998). Donaghy, J.A. and Madden, R.H. (1993) Detection of Salmonella in animal protein by Rappaport-Vassiliadis broth using indirect impediometry. International Journal of Food Microbiology 17, Ellison, J.A. (1982) Traditional British Cookery. London: Ward Lock Limited. Griswold, R.M. (1973) The Experimental Study of Foods. London: Constable London. Janssen, D.W. and Busta, F.F. (1973) Injury and repair of several Salmonella serotypes after freezing and thawing in milk salads. Journal of Milk and Food Technology 36, Madison, D. (1996) Homemade mayonnaise. Available from: ³ (Accessed January 5, 1997). Membre, J., Majchrzak, M. and Jolly, I. (1997) Effects of temperature, ph, glucose, and citric acid on the inactivation of Salmonella typhimurium in reduced calorie mayonnaise. Journal of Food Protection 60, Perales, I. and Garcia, M.I. (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, Radford, S.A. and Board, R.G. (1993) Review: fate of pathogens in home-made mayonnaise and related products. Food Microbiology 10,

5 40 R. XIONG ET AL. Roberts, D., Hooper, W. and Greenwood, M. (1995) Practical Food Microbiology 2nd edn. London: Public Health Laboratory Service. Smittle, R.B. (1977) Microbiology of mayonnaise and salad dressing: a review. Journal of Food Protection 40, Strantz, A.A. and Zottola, E.A. (1993) A modified plating technique for the recovery and enumeration of stressed Salmonella typhimurium Hf. Journal of Food Protection 52, Xiong, R., Xie, G. and Edmondson, A.S. (1998) Microbiological safety indicator for mayonnaise recipes: ratio of egg yolk to white wine vinegar. In Culinary Arts and Sciences II: Global and National Perspectives ed. Edwards, J.S.A. and Lee-Ross, D. pp Bournemouth: Worshipful Company of Cooks Centre for Culinary Research.