I General introduction

I General introduction

Foods based on oils and fats represent a large proportion of the energy intake in the diet of consumers in most of the world. Nutritional advice is to limit the amount of fat in the overall diet, in particular of saturated fat. As a result, the past decades have shown a reduction in the per capita consumption of oil- and fat-based foods in developed countries and a relative shift to low-fat/low-calorie products. Fats and oils can be attacked by various fat-splitting microorganisms if the conditions for growth are favorable, e.g. temperature, moisture, availability of low-molecular weight nutrients. Enzymes produced by contaminating lipolytic flora can hydrolyze the fat to yield free fatty acids and trigger fatty acid oxidation. At the same time, fats and oils can protect microorganisms so that they may survive for quite some time (Troller and Christian, 1978; Hersom and Hulland, 1980; Gaze, 1985). This would present

a hazard in particular if the organisms were infectious pathogens. Most oil- and fat-based foods contain a certain amount of moisture and non-fat nutrients. Their physical structure is a very important parameter. The products may exist either as a fat-continuous system (i.e. yellow-fat-spreads such as butter and margarine or other dairy and non-dairy spreads and reduced fat spreads) or as a water-continuous system (i.e. mayonnaise, salad dressings, and other water- continuous spreads). This has a strong impact on the microbiological stability of the food. In water-in-oil products, such as margarine, the water is present as well-dispersed fine droplets throughout the fat phase. The inability of microorganisms to move between droplets is a major intrinsic preservation factor. Fat can act as a barrier to microbial growth and for this reason fat-continuous systems are usually much more stable than water-continuous systems. A small category of oil- and fat-based products is characterized by extremely low water contents (e.g. butter oil, ghee, vanaspati, cocoa butter substitutes, and cooking oils) that, generally, limits microbial growth but not necessarily excludes growth under extreme conditions.

Presently, water-in-oil emulsions exist in the market with fat levels ranging from 20% to 80%, whereas products with fat levels as low as 3% have been successfully introduced recently in the USA, UK, and The Netherlands (van Zijl and Klapwijk, 2000). Oil-in-water emulsions do occur also in a considerable range of fat contents. The preservation properties of the wide range of different oil- and fat-based products that are currently safely marketed differ substantially. Product innovations such as inclusion of spices and fresh herbs in water-in-oil products may affect the product structure and, possibly, the microbiological load.

The composition of butter is subject to stringent regulations and for this reason has not changed much over the years, albeit that new types of butter-making processes have been introduced. Reduced fat variants of butter have appeared in the market, in most cases containing 40% butterfat. Products based on mixtures of butter–butter fat and vegetable fat blends (m´elanges) have been developed that can be full-fat, medium-fat, or low-fat (Madsen, 1990). Typical butter or margarine manufacturing processes may produce them.

There are no indications that industrially produced oil- and fat-based foods play a significant role in food-borne disease (Delamarre and Batt, 1999; Michels and Koning, 2000; Smittle, 2000; van Zijl and Klapwijk, 2000). However, most of the products are vulnerable to spoilage microorganisms (i.e. acid- tolerant types). The increased attention to hygiene during manufacturing (Mostert and Lelieveld, 2000), the quality of raw materials, and the pasteurization conditions applied have all contributed significantly to product and process designs with a very good safety record. The implementation of HACCP for

481 assuring proper production is essential. Continued attention for safety and quality remains necessary,

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however, especially with respect to product innovations. An incident with Listeria contaminated butter occurred in Finland due to poor factory hygiene (Lyytik¨ainen et al., 1999, 2000). In an outbreak due to Escherichia coli O157:H7 in the United States in 1993, for which epidemiological evidence implicated bulk mayonnaise as a vehicle of transmission (Anonymous, 1993a), the organism was found to be more tolerant of acidic conditions than either Salmonella spp. or Listeria monocytogenes, which were the organisms previously considered as the main hazards. Several studies subsequently showed that pathogens such as Listeria spp., Salmonella spp., and E. coli O157:H7 may survive certain conditions under which their acid tolerance is strengthened by exposure to, among other factors, non-lethal acid levels (Leyer and Johnson, 1993; Leyer et al., 1995; Grahan et al., 1996; Duffy et al., 2000; Smith, 2003). Such stress-hardening conditions should be avoided but, in order to do this, knowledge of how ecological conditions in a food product or the production environment impact on the growth–survival of pathogens is needed. The occurrence of stress-hardened pathogens, which may survive in acidic foods and passage through the stomach (pH 1.0–3.0), or which may be less well inactivated by pasteurization, emphasizes the need to assess potential survival of these organisms using acid-adapted cultures when possible.

Keeping up-to-date with information on growth capabilities of pathogenic microorganisms is an essential exercise for food professionals. Changes of manufacturing or marketing practices and launch of product innovations need to be accompanied by a safe product and process design as well as proper practical implementation and control thereof, as is further detailed out in this chapter.

II Mayonnaise and dressings

A Definitions Mayonnaise. Mayonnaise can be a very well-defined product with specific levels of oil (min. 52%),

egg yolk (min. 6%), salt (min. 1%), total acid (min. 0.75%), and pH (max. 4.5) fixed in local regulations (Michels and Koning, 2000). The types of oil used are mainly soybean, rapeseed, and sunflower oil and sometimes cottonseed and olive oil. Low-oil mayonnaise products have come on the market since 1980s. This has led to specific labeling of the fat content and a clear indication on the label that the product is a low-fat or reduced-calorie mayonnaise (or similar wording). Although there is legislation to specify the composition of real mayonnaise in many countries, this is less so for low-oil mayonnaise, dressings, and emulsified sauces.

The Codex Alimentarius Regional European Standard for Mayonnaise defines mayonnaise as

a condiment sauce obtained by emulsifying edible vegetable oil(s) in an aqueous phase consisting of vinegar, the oil-in-water emulsion being stabilized by hen’s egg yolk (FAO/WHO, 1989). This Codex Standard recognizes the following optional ingredients: egg white, egg products, sugar, salt, condiments, herbs, spices, fruits and vegetables including fruit juice and vegetable juice, mustard and dairy products. As acidifying agents, the use of acetic, citric, lactic, malic, and tartaric acids and the salts thereof are allowed. Benzoic acid and sorbic acid and the salts thereof are allowed as preservatives. Other additives may include stabilizers, antioxidants, colors, and flavors (together with a flavor enhancer such as monosodium glutamate). The Standard further stipulates that the total fat content of mayonnaise (from oil and egg yolk) should be 78.5% (w/w) and the technically pure egg yolk content should not be less than 6%. The Association of the Mayonnaise and Condiment Sauce Industry of the EEC adopted

a total fat content of minimum 70% (w/w) and a minimal egg yolk content of 5% (w/w) (CIMSCEE, 1991). According to the US standards of identity for mayonnaise, the vegetable oil content must be at least 65%, the pH may range from about 3.6 to 4.0 with acetic as the predominant acid representing

MICROORGANISMS IN FOODS 6

0.29–0.5% of the total product (US-DHEW, 1975a). The aqueous phase should contain 9–12% salt and 7–10% of sugar.

Low-fat or reduced-calorie mayonnaises, salad dressings, salad creams, and other emulsified products made with oil, emulsifier, and vinegar, typically have a lower fat content than mayonnaise. Salad dressings will have a more fluid consistency to make the product “pourable”, while most mayonnaises are “spoonable”. They normally contain egg yolk although some are made with dairy-based emulsifiers.

Dressings. Salad dressings are defined in the United States by the FDA as emulsified semi-solid foods prepared from vegetable oils, vinegar, lemon juice, and/or lime juice, egg yolk-containing ingredients, and a cooked or partially cooked starchy paste (US-DHEW, 1975b). The finished product contains no less than 30% of edible vegetable oil and has the equivalent of 4% liquid egg yolk (US-FDA, 1993). The pH is 3.2–3.9 and acetic acid makes up 0.9–1.2% of the total product. The aqueous phase contains 3–4% of salt and 20–30% of sugar (Smittle, 1977).

There are few compositional specifications for dressings or other emulsified sauces (meat and fish sauces). They generally contain less oil than mayonnaise and salad dressings, but all are oil-in-water emulsions in which the emulsifying agent usually is egg yolk. Because of the presence of vinegar or other weak acids in most mayonnaise, dressings, and emulsified sauces, their pH is low, which has a significant influence on the microbiological stability. Low-calorie or low-sodium formulations are more susceptible to spoilage than the traditional products.

B Important properties In Europe, the pH of mayonnaise is typically between 3.0 and 4.2, with 4.5 as the highest value permitted

(this is the legal maximum in Denmark). The percentage salt or sugar is not fixed by regulations, but is mostly between 1% and 12% of the aqueous phase. The level of acetic acid in the aqueous phase is typically between 0.8% and 3.0%. Dressings typically have a smaller fat phase than mayonnaise and a starch phase, which helps to give the required consistency. Due to the larger aqueous phase, in which acid and salt are diluted, they are more vulnerable than mayonnaise to microbial spoilage. Typically, the acetic acid content of dressings is 0.5–1.5% of the aqueous phase and the pH 1.0–4.2. Levels of salt (1–4%) and sugar (1–30%) in the aqueous phase contribute little to microbiological stability.

No clear distinction exists worldwide between mayonnaise, low-fat mayonnaise, salad dressings and other emulsified products made with oil, emulsifier and vinegar. They normally contain egg yolk, although some are made with dairy-based emulsifiers or contain no oil and are not emulsified and are two-phase systems like vinaigrettes. This large group of dressings can be “pourable” or “spoonable” (depending on starch content).

Mayonnaise, dressings, and other emulsified sauces can be categorized into inherently stable or unstable products on the basis of their aqueous phase composition and shelf-life.

Stable products. These products are stable at ambient temperatures, not sensitive to spoilage, whether open or closed, because of an aqueous phase composition that inhibits growth of all relevant spoilage organisms, in particular acetic acid-tolerant lactobacilli, yeasts, and molds. Their closed shelf-life will

be 6 months to 1 year, limited for organoleptic reasons only. After opening, the sauces are not sensitive to spoilage and they can be kept at ambient temperatures or chilled for as long as their quality remains acceptable.

Unstable products. These products allow (slow to rapid) growth of lactobacilli and/or yeasts. Ingre- dients used should be selected to minimize the initial contamination; good process design and hygienic

483 practices can eliminate or prevent (re-)contamination during manufacturing. Depending on the specific

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properties of the preservation system, products may have a relatively long ambient shelf-life in the closed jar (e.g. 6 months to 1 year). However, recontamination during consumer-use restricts the shelf-life of the product after opening to a few weeks (ambient) or months (refrigerated). Where products have a minimal built-in preservation, closed shelf-life may be restricted to a few weeks.

C Methods of processing and preservation The production of low-acid mayonnaise, salad dressings, and thousand island dressings is similar to

that of mayonnaise. However, some of the former products will have a (cooked) starch phase to give the required consistency to the product while others are simply made by incorporating a starch phase into the mayonnaise phase, resulting in a low-oil product. Manufacturing can be by using either batch or continuous processes (Lopez, 1987).

In a batch process (Figure 11.1), liquid (salted) egg yolk is mixed with an acid water phase consisting of vinegar, spices, flavor, salt and/or sugar, and (optional) a (cooked) starch phase. Oil is added under intensive mixing and the resulting coarse emulsion is usually passed through a colloid mill to achieve the small oil droplets (predominantly 5–10 µm) required for a good consistency. Consistency of “spoonable” mayonnaise is expressed in Steven’s value, measured by recording the resistance in gram of a measuring gauge pressed into the product at a fixed speed. Steven’s values typically are between 50 and 200 g. For liquid products, the Bostwick value is used, which is the run length in cm of the product when released in a measuring tray and measured within 30 s. Typical values are 5–10 cm. The inclusion of (cooked) starch is optional and depends largely on the oil content of the mayonnaise. The freshly made mayonnaise is pumped to a holding vessel, and packaged in glass jars, tubs, buckets, or other containers for consumer/professional use. In a batch manufacturing process of a dressing containing particulates, the particulates can be mixed after the colloid mill with the dressing base. An alternative route to

Figure 11.1 Layout of a batch dressings production line.

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Figure 11.2 Layout of a continuous mayonnaise production line.

preventing product damage is to omit the colloid mill and to introduce the particulates in the first mixer after preparation of the dressing base.

In a continuous process (Figure 11.2), a set of proportioning pumps combines the right amounts of liquid egg, oil, water phase (with vinegar) and cooked starch phase (optional), which are emulsified in an emulsifying cylinder and finally passed through a colloid mill. To ensure the safety of the starch phase, the pH is usually adjusted with vinegar to below pH 4.5 to control any pathogen hazard. When particulates are present, the starch phase can be added (through an in line mixer) directly to the final product buffer vessel.

Whatever process is used, the processing lines should be free of relevant microbial contaminants at the start of the process and the use of hygienically well-designed process and packaging equipment is key to the production of such vulnerable products (EHEDG, 2003; 3-A, 2003). Equipment traditionally used for the manufacture of mayonnaise and dressings has not been easy to clean. However, increasingly more types of hygienic mixers, colloid mills, valves, pumps, and filling machines become available that can be cleaned-in-place (CIP). Their use is recommended as this allows the best control over cleaning and disinfection. Proper manual cleaning is usually still required for mills, fillers, etc., that are difficult to clean by CIP alone (e.g. irregular surfaces). For chilled products hygienic processing, clean (decon- taminated) ingredients and proper refrigerated storage are necessary to obtain the desired shelf-life.

In the manufacturing of a microbiologically stable mayonnaise, there is normally no technological need to apply a heat process unless elimination of enzymes is required from ingredients like vinegar or spices. These enzymes could break down the starch, if present. The aqueous phase containing ingredients such as herbs or mustard, which could contain pathogenic or spoilage microorganisms, may

be pasteurized before mixing. Also liquid egg yolk or other egg products should be pasteurized (e.g. by the supplier) before use in the manufacturing process. The overall effect of the manufacturing process on the initial microbial load of the ingredients is negligible when a cold process design is used. The various emulsified products have a continuous water phase and the microbial load is not affected by the presence of oil droplets. The safety and stability characteristics of mayonnaise and dressings are dictated primarily by the low pH (range in use is pH 3.0–4.5) and the preservative effect of acetic acid (added as vinegar) or lactic acid. In mayonnaise, salt and to a much lesser extent sugar can reduce the wateractivity and this helps to inhibit spoilage

485 organisms. Due to the limited water content, the salt-in-brine in the aqueous phase can be as high as

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12%, resulting in a water activity of about 0.92. A preservative such as sorbic acid can be used where legally permitted, but a major proportion of the acid will dissolve in the oil phase leaving only 40–60% in the active undissociated form in the aqueous phase to protect against yeast (and lactobacilli). When benzoic acid is used, an even larger proportion of the preservative dissolves in the fat phase, which makes it even less effective.

The paper by Michels and Koning (2000) provides details on product and process design of different product types, including procedures and calculations to evaluate the effect of organic acids–preservatives and overall product stability. Some investigations on the inactivation of infectious pathogens in regular and reduced-calorie (“lite”) mayonnaises or yellow-fat spreads, challenging commercially manufactured products, show a protective effect of high fat levels to the survival of pathogens whereas storage at higher temperatures increases inactivation (Hathcox et al., 1995; Holliday et al., 2003). Others have reported increased survival of pathogens at refrigeration temperature as compared to room temperature (Weagant et al., 1994). Good knowledge of the exact chemical and physical composition of the products tested is essential in the interpretation of the results. Whereas challenge tests are commonly performed with rather high numbers of pathogens inoculated onto products, tests evaluating low-level inoculation or natural contamination of a pathogen may be the realistic scenarios. Leuschner and Boughtflower (2001) described a reproducible laboratory-scale procedure for preparation of mayonnaise containing low levels (10–1000 cfu/g in the final product) of Salmonella Enteritidis. Systems like these, which simulate a mayonnaise that is naturally contaminated at a low-level, can be used for the validation of the stability and safety of innovative formulations or to test products prepared using new preservation methods. Predictive modeling has been used to simulate pathogen inactivation rates as a function of product formulation and environmental conditions (Membr´e et al., 1997), as have novel approaches such as neural networks (Xiong et al., 2002).

D Microbial spoilage and pathogens Mayonnaise and dressings products are often produced using a cold process design, and microbial

growth is only controlled by the specific properties of their formulation. These properties (i.e. low pH, presence of acetic acid, etc.) restrict potential problems to certain acid-tolerant microorganisms.

Initial microflora. The microbial load of mayonnaise, dressings, and emulsified sauces comes from the various ingredients and from contamination during processing and packaging. Typical components that can carry spoilage microorganisms are mustard, pickles, dry vegetables and herbs, and blue cheese. Water, refined oil, vinegar, and pasteurized egg are normally free of relevant contamination when they are handled according to Good Manufacturing Practices (GMP) conditions based on the General Principles of Food Hygiene (CAC, 2001a).

r The refined oils used are normally free of microbial contamination as a result of the refining process, which involves steaming at a temperature well above 100 ◦

C. The oil has a very low-moisture content of <0.1% and will thus not allow microbial growth. r When egg is used, it is mainly as pasteurized liquid egg yolk preserved with salt (8–11%) or with salt and potassium sorbate (e.g. 92% egg yolk, 7% salt, and 1% sorbate). With unpasteurized egg preparations, there is a risk of contamination with S. Enteritidis and its use is advised against. European Commission (EC) regulations require pasteurized eggs to be distributed and stored at a temperature of ≤4 ◦

C and Salmonella spp. absent in five samples of 25 g. Aerobic Plate Count (APC) should be ≤ 10 5 cfu/g and coliforms ≤100 cfu/g. Commercial pasteurized liquid egg products often have counts

MICROORGANISMS IN FOODS 6

well below these values. The APC values commonly are a few hundred to a few thousand bacteria per g of product. The same holds for coliforms, which often are absent in 0.1 g.

r Vinegar can be obtained through fermentation of different raw materials (e.g. alcohol, wine, malt, cider). The acetic-acid content typically is about 8–11% and thus vinegar is usually not sensitive

to spoilage. Artisanal vinegars with low acetic-acid levels occasionally carry spoilage organisms. Vinegar is often pasteurized to eliminate enzyme activity originating from the fermentation process. This also ensures the absence of the rare but extremely acetic acid-tolerant mold Moniliella acetoabu- tans and the acetic acid-resistant Lactobacillus acetotolerans (able to multiply in the presence of 9–11% acetic acid at pH 5.0; Entani et al., 1986).

r With mustard, 1.8–2.5% acetic acid typically contained in its formulation prevents survival of infec- tious pathogens, although high levels of acetic acid-tolerant lactobacilli are known to occur. Yeasts

are normally not found because of the antimycotic activity of allylisothiocyanate present in many mustard types.

r Herbs and spices are likely to be contaminated with spoilage organisms or pathogens such as Salmonella spp. and Escherichia coli O157:H7. Industrial experience (Michels and Koning, 2000) is

that the incidence rate of Salmonella spp. in raw spices and herbs is about 1%. r The use of dairy ingredients in emulsified products is quite common, but in most cases these are

pasteurized by the producer. Fermented products, e.g. yoghurt and cheese, will have a high load of lactic acid bacteria, and yeasts also may be present. Blue cheeses require special attention due the presence of high levels of acetic acid-tolerant molds and lactobacilli. With soft cheeses in particular, there is a possibility for the presence of Listeria monocytogenes.

r Citric acid and acids other than acetic acid or vinegar may be used in emulsified products. Lactic acid typically consists of a 50% or 80% solution of dl-lactic acid with no significant microbial load.

Citric acid commonly is used as acid or as concentrated citric juice (30 ◦ Brix) with a low pH of ± 3.1. Spoilage is prevented by pasteurization of the concentrate (or preservation with sulfite) to eliminate spoilage yeasts and molds. Other weak (e.g. malic) or strong (e.g. phosphoric) acids that may be used are free of relevant contamination.

r Starch is often used in emulsified products and it can be either “natural”, requiring cooking to 85–90 ◦ C to set the starch, or “instant”, in which case the starch does not require any cooking. The microbial load

of natural and instant starches typically is very low, with only a few species of Bacillus and Clostridium present and no pathogens. However, certain processed starches can contain Salmonella spp.

r Common ingredients such as sugar, salt, and preservatives like sorbic and benzoic acid or the salts thereof normally have a very low microbial load. In sugar produced in small industrial operations,

osmotolerant yeasts such as Zygosaccharomyces rouxii or Z. bailii may occur that can cause spoilage of acetic acid-containing emulsified products.

r All products should be made with potable water, which is free of pathogens and acetic acid-tolerant microorganisms.

Spoilage. Microbial spoilage is mainly caused by a small group of acid-tolerant yeasts and lactobacilli. Spoilage by molds is rare because most molds have a limited tolerance to acetic acid (Smittle and Flowers, 1982).

Yeasts. Only yeasts that are resistant to acetic acid are likely to present a spoilage problem to mayonnaise and dressings. Well-known species are Z. bailii and Pichia membranaefaciens, which can grow in the presence of ±3% acetic acid (Thomas and Davenport, 1985). The name of the latter yeast indicates that it grows as a film on the surface of a medium. In practice growth is only observed when sufficient oxygen is present (Smittle and Flowers, 1982). These two species are probably responsible for the majority of the spoilage incidents caused by yeasts. Other species occasionally observed are Z. rouxii,

487 Saccharomyces cerevisiae, and Candida magnolia. Yeasts may cause spoilage by gas formation or by

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growing as brownish colonies on the surface of mayonnaise, which may appear as small oil droplets. Sometimes growth is limited to ± 10 4 cfu/g, probably because of oxygen depletion.

Lactobacilli. Lb. fructivorans has been reported as the main lactobacillus species causing spoilage of mayonnaise-type products (Smittle and Flowers, 1982). Others report that Lb. plantarum and Lb. buchneri are the organisms most frequently isolated from spoiled products in Europe, while Lb. fructivorans is less commonly isolated (Michels and Koning, 2000). Lactobacilli occasionally grow to very high numbers without causing evident spoilage. Development of heterofermentative lactobacilli will result in visible spoilage due to gas formation and will cause a decrease in pH.

Molds. The majority of molds cannot grow in the presence of ≥0.5% acetic acid. Because jars are steam capped and oxygen is consumed due to oil oxidation, only a little oxygen is generally available which also limits mould growth. Growth of a Geotrichum spp. on the surface of mayonnaise has been observed in jars with faulty seals. Although spoilage by molds is very rare, some acid-tolerant types have been reported. Tuynenburg Muys (1971) refers to M. acetoabutans, found in acetic acid preserves, which is unique in its ability to multiply in the presence of 8–9% acetic acid. Reference is also made to Monascus ruber and Penicillium glaucumi, able to grow in the presence of ≥1% acetic acid; Pen. roqueforti, present in blue cheese, also belongs to this group.

Pathogens. Mayonnaise and dressings with final pH values above 4.1 offer a potential risk of food poisoning by Salmonella spp. or other infectious pathogens (E. coli O157:H7; L. monocytogenes), either

because strains of these pathogens can be particularly acid-tolerant or because the acidulant type and its final concentration may not be adequate to kill such pathogens. Even worse, the product properties may be insufficient to prevent multiplication of infectious pathogens or of Staphylococcus aureus. This is not a concern with properly formulated industrial products, for which die-off of pathogens of concern has been assured as part of the product design, but it can be for home-made products. Addition of commercial mayonnaise/dressing to salads is considered to retard growth of pathogens and to reduce concern (Smittle, 1977; Doyle et al., 1982). Smittle (2000) reviewed the microbiological safety of mayonnaise, salad dressings and sauces produced in the United States focusing on the death and survival of food-borne pathogens in relation to the product formulation. Through detailed statistical analysis of literature data, this study provided support of the remarkable safety record of these products when commercially produced keeping to safe product formulations, implementation of GMP and HACCP systems. Proper food handling and storage practices were also found to be key in ensuring the safety of mayonnaise-based delicatessen foods in food-service operations. As reported by Bornemeier et al. (2003), temperature conditions and product characteristics of typical deli-salads sold in several grocery- stores were conducive of growth of pathogens.

Salmonella. Nearly all incidents involving Salmonella spp. were caused by mayonnaise made at home, in restaurants, or in institutional kitchens. The general use of pasteurized egg yolk, an adequate

level of vinegar (typically giving more than 1% acetic acid in the water phase) and a pH below 4.5, has prevented food-borne illness from industrially produced mayonnaise during the past 40 years.

A major outbreak with 10000 cases in Denmark in 1955 led to a Danish regulation that the pH of mayonnaise should be below 4.5. Thereafter, there were two more Danish incidents with Salmonella-contaminated mayonnaise (S. Typhimurium biotype 17 in both cases) from large man- ufacturers. In the first case, the pH of the mayonnaise was 5.1 and the count of Salmonella spp. was

1.8 × 10 5 cfu/g at the time of analysis, 4 days after the meal (Petersen, 1964). The second outbreak had 41 cases and two fatalities. The mayonnaise was made with raw eggs and had a pH of 6.0; 2 days

MICROORGANISMS IN FOODS 6

after the event the Salmonella spp. count of the mayonnaise was 6 × 10 6 cfu/g (Meyer and Oxhøj, 1964). In 1976, a serious salmonellosis outbreak occurred among passengers on four outgoing and re- turn flights from Las Palmas (Spain). Approximately 500 passengers were ill, with six fatalities. S. Typhimurium phage type 96 was isolated from mayonnaise (pH not mentioned) used on the in- criminated flights and from a food handler involved in the preparation of the mayonnaise (Davies and Wahba, 1976).

Salmonella Enteritidis has been associated to several cases of food poisoning due to home-made or restaurant-made mayonnaise in the USA, the UK, Argentina, and many other countries (Anonymous, 1988; St Louis et al., 1988; Eiguer et al., 1990). This serovar of Salmonella was responsible for 78% of all outbreaks of known aetiology in Spain (Perales and Audicana, 1988). In a serious US outbreak, 404 of 965 persons at risk (mean age 64.2 year) in a New York City hospital became ill and 9 people died (median age 77.5 years); the source of the incident was hospital-prepared mayonnaise made with raw eggs; contamination with S. Enteritidis was traced back to a farm corporation (Telzak et al., 1990).

The use of raw egg for mayonnaise contributed to an outbreak at a wedding reception in 1992, in which 81 guests and 11 catering staff became ill due to S. Enteritidis (Chandrakumer, 1995). In 1995, in Uruguay 600 cases of salmonellosis were traced to sandwiches that were made with small batches of mayonnaise. The source of the contamination was S. Enteritidis from unpasteurized eggs (Anonymous, 1995).

Use of vinegar for acidification of mayonnaise to pH 5.0 will prevent multiplication of S. Enteritidis in home-made mayonnaise, but the pathogen can survive for a few days at 20 ◦

C when citric acid or a low level of acetic acid is used as acidulant (Perales and Garc´ıa, 1990; Kurihara et al., 1994; Lock and Board, 1994, 1995). In home-made mayonnaise with 0.1% of acetic acid (0.85% acetic acid in the water phase), S. Enteritidis survived for 5–6 days at 30 ◦

C or 30 ◦

C, but for only 1 day in commercial mayonnaise with 2.26% acetic acid in the water phase. Refrigerated storage of a home-made mayonnaise at 10 ◦ C gave less than a factor three reduction in 9 days, while Salmonella spp. were eliminated from the

commercial mayonnaise (5 log 10 reduction) in 3–6 days.

Lock and Board (1994) studied 24 varieties of commercial mayonnaise with pH values of 2.6 to 4.8 and various types of acids and found rapid inactivation of S. enteritidis PT 4 (inoculated at 2.5 10 4 /g) in all cases; in eight samples, Salmonella spp. could not be recovered after 48 h from products stored at 20 ◦

C, whereas Salmonella spp. were undetectable after 20 min in a fat-free mayonnaise with pH 2.6. At 4 ◦

C, Salmonella spp. generally survived longer. Interestingly, not all incidents were caused by S. Enteritidis. There is a low incidence of S.Typhimurium in eggs. In the UK, 120 of 700 people reported gastrointestinal illness after eating in a large metropolitan building (Mitchell et al., 1989). Salmonella Typhimurium phage type 49 was isolated from a tartare sauce made from the mayonnaise remaining from the meal. The mayonnaise was made from fresh eggs, oil, and vinegar. The same Salmonella type was isolated from the patients and from bird droppings at the farm that supplied the eggs. Further incidents attributed to S.Typhimurium are reported in the review by Radford and Board (1993).

The concentrations of acetic acid used in commercially produced mayonnaise cause rapid inactivation of salmonellae and L. monocytogenes, thus reducing the risk of any low-level pathogen (re)contamination of pasteurized egg. In typical American reduced-calorie mayonnaise, Salmonella spp. and L. monocy- togenes were inactivated in 3 days in products with a pH below 4.1 and 0.7% acetic acid in the aqueous phase (Glass and Doyle, 1991). In this way, chance introduction of contamination would still result in a product that is safe before it reaches the consumer. Adequate inactivation is less likely for the acid-resistant E. coli O157:H7, which survived for 7 days in commercial mayonnaise (Glass et al.,

1993). Inoculation of 6.5 × 10 3 E. coli O157:H7 in the commercial mayonnaise implicated in the 1993

489 outbreak showed survival for over 8 days at 20 ◦

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C (Zhao and Doyle, 1994). Rapid inactivation of Salmonella spp. was found for salad dressing (pH 3.2–3.3; ± 1% acetic acid; inactivation within 1–6 h) or mayonnaise (pH 3.8-4.0; ± 0.5% acetic acid; inactivation within 1–18 h) kept at ambient temperature or at 37 ◦ C.

C and over 34 days at 5 ◦

Another factor that can influence the death rate of salmonellae in mayonnaise is the type of oil used. Extra virgin olive oil, known to contain high levels of phenolic compounds, has been shown to contribute to rapid inactivation in home-made mayonnaise (Radford et al., 199l; ICMSF, 1996, pp. 242–3).

The FDA has established requirements for pH and acetic acid in mayonnaises and dressings con- taining raw egg preparations (US-FDA, 1990, 1994), meant to ensure inactivation of Salmonella spp. by holding the product for 72 h before it is made available to consumers. This regulation considers that raw eggs or raw egg yolk containing ingredients may be used when the final pH is not above 4.1 and the acidity of the aqueous phase, expressed as acetic acid, is not less than 1.4% (US-FDA, 1990). However, due to Salmonella outbreaks associated with raw eggs, this last option is rarely used in the USA or Europe.

Staphylococcus aureus. The survival and possible growth of Staph. aureus in mayonnaise has been studied extensively (Smittle, 1977). Due to the low pH of these products and the presence of acetic acid, the organism is unable to grow and is normally of no significance for mayonnaise and dressings. In one of the Danish incidents, however, contamination with 1 million of δ-toxin-producing staphylococci was observed in Salmonella-contaminated mayonnaise with an elevated pH of 6.0 (Meyer and Oxhøj, 1964). The possible growth of Staph. aureus and toxin production in home-made mayonnaise was studied in detail by Gomez-Lucia et al. (1987, 1990). Toxin was found only in mayonnaise acidified with vinegar with a pH ≥5.0; at pH 4.5, no toxin was formed.

Listeria monocytogenes. Contamination of raw egg yolk with L. monocytogenes has been reported and the behavior of the pathogen has been studied in reduced-calorie mayonnaise (Leasor and Foegeding, 1990); with 0.7% of acetic acid in the aqueous phase, L. monocytogenes did not grow at 23.9 ◦

C and was inactivated by a factor of 10 4 within 3 days (Glass and Doyle, 1991). In four commercial mayonnaise products with pH 3.3–3.9, Listeria inactivation at 26.6 ◦

C was directly related to aqueous phase acetic acid concentrations; at 2.2% acetic acid, the pathogen was reduced by a factor of 10 8 in 72 h and at 0.67% acetic acid in 192 h (Erickson and Jenkins, 1991).

Escherichia coli O157:H7. Mayonnaise was epidemiologically implicated as the vehicle for trans- mission of E. coli O157:H7 in a 1993 outbreak of food-borne disease in Oregon, USA. In this outbreak,

62 cases were traced to the consumption of contaminated ranch and blue cheese dressings and contam- inated seafood salad. The evidence available suggested that the mayonnaise (pH 3.9) was contaminated by tainted meat by the retailer. The bulk mayonnaise as delivered was not suspect (Anonymous, 1993b). Erickson et al. (1995) did not find the pathogen in pasteurized egg yolk or the wet processing en- vironment of three plants processing mayonnaise or other emulsified sauces. The primary source for enterohaemorrhagic E. coli (EHEC) appears to be cattle. Meat and milk from cattle have been directly linked to many outbreaks, but the Oregon outbreak has shown that recontamination of mayonnaise and dressings with this pathogen may lead to unexpected outbreaks. There is another report of a possible outbreak due to mayonnaise contaminated with a non-motile E. coli O101, where 300 people became ill in Eastern Germany in 1988 (B¨ulte, 1995).

The incidents stimulated research on E. coli O157:H7 in mayonnaise, with a focus on its survival in acid environments. E. coli O157:H7 has been shown to be unusually resistant to acid pH as suggested

MICROORGANISMS IN FOODS 6

by an outbreak associated with consumption of apple cider (Besser et al., 1993) and by apple cider inoculated with E. coli O157:H7 (Zhao et al., 1993). E. coli O157:H7 survived much better than

a control strain of E. coli at low pH and numbers remained unchanged after 24 h of incubation in Trypticase Soy Broth at pH 3 and 4 (Miller and Kaspar, 1994). The pathogen may also be more tolerant of some organic acids or other antimicrobials as it has been found to survive well in acid foods and beverages (Duffy et al., 2000; Koodie and Dhople, 2001; Mayerhauser, 2001). In commercial regular mayonnaise and reduced-calorie mayonnaise dressing, E. coli O157:H7 survived 7 days at

C, indicating that it survives better than Salmonella spp. and L. monocytogenes (Glass et al., 1993). Weagant et al. (1994) studied the survival of three strains of E. coli O157:H7 obtained from the

mayonnaise implicated in the Oregon outbreak, using an inoculum size of 10 8 cfu/g in a mayonnaise product with pH 3.65, and noted that the pathogens died off quite rapidly at ambient temperature.

Survivors were found for a maximum of 72 h at 25 ◦

C, whereas they could be recovered for up to

C. Four different sauces were then made from this mayonnaise and challenge tested. Survival of one EHEC strain in thousand island dressing (pH 3.76) was about 35 days at 5 ◦ C; in seafood sauce (pH 4.38) and blue cheese dressing (pH 4.44), the numbers of survivors were about 500 times higher at that point in time. In a fourth, mayonnaise-mustard sauce (pH 3.68), the inactivation at 5 ◦ C was very rapid as no survival was observed after 5 days. Similar findings were reported for two strains

35 days at 7 ◦

of E. coli O157:H7 inoculated into a commercial mayonnaise (pH 3.91) at a level >10 6 cfu/g, where no survival was observed after 96 h at 22 ◦

C (Raghubeer et al., 1995). Zhao and Doyle (1994) inoculated

6.5 × 10 3 E. coli O157:H7 in the commercial mayonnaise implicated in the 1993 Oregon outbreak (pH 3.6–3.8 and 0.37% titratable acid) and found survival for 8–21 days at 20 ◦

C and 34–55 days at 5 ◦ C. In a ranch salad dressing with pH 4.51 kept at 4 ◦

C, E. coli O157:H7 survived better (over 17 days) than strains of generic E. coli and Enterobacter aerogenes that were used as references (no survivors found after 14 days). The reference strains did not survive for 4 days in a mayonnaise prod- uct with a composition meeting the FDA requirements for pH and acetic acid in products contain- ing raw egg (US-FDA, 1994). Rapid inactivation of EHEC was observed in commercial mayonnaise with pH ≤4.0 by Erickson et al. (1995). The authors concluded that intact packages of commer- cial mayonnaise and mayonnaise dressings pose negligible EHEC contamination and health hazard risks.

In most studies on antimicrobial activity of weak acids done with E. coli O157:H7 and other bacterial pathogens, acetic acid proved more inhibitory than lactic acid, with minimal inhibition by citric acid (Smittle, 1977; Conner et al., 1990; Conner and Kotrola, 1995). In Brain Heart Infusion broth at pH

C, but in All Purpose Tween broth acidified with acetic acid to the same pH no growth was observed in 70 days (Davies et al., 1992). Survival and growth at refrigeration temperatures may be dependent on the composition of the bacteriological medium, and consequently on the food formulation (Kauppi et al., 1996).

5.0 acidified with lactic acid, visible growth of E. coli O157:H7 was evident in 2–3 days at 25 ◦

Other pathogens. Food-borne pathogens like Clostridium botulinum, Cl. perfringens, and Bacillus cereus are unable to grow in mayonnaise and dressings at a pH ≤4.5 and are thus of no signifi- cance (Michels and Koning, 2000). An outbreak in the UK caused by B. cereus (and Staph. aureus) has been reported, where the pH of the product involved may have been above 4.6 (Radford and Board, 1993). Campylobacter jejuni has been observed in egg yolk but the organism is not heat- resistant, is unable to grow below 30 ◦

C and is likely to be inactivated rapidly in the presence of acetic acid.

OIL- AND FAT-BASED FOODS