Part IV Safety and quality issues 10.1 Introduction All food products are susceptible to deterioration in their quality during storage. Chilled foods in particular are highly perishable and the time during which the quality is maintained at a consumer acceptable standard can be termed the shelf- life. The definition of shelf-life has been given by several authors as the time between the production and packaging of the product and the point it becomes unacceptable under defined environmental conditions (Ellis 1994) or the time at which it is considered unsuitable for consumption (Singh 1994). The end of a product’s shelf-life will be due to deleterious changes to quality caused by biological, chemical, biochemical and physiochemical means, or by food safety concerns due to the growth of food pathogens which may not necessarily cause any changes in product quality. There are few reference books available which give lists of shelf-lives for chilled foods as the shelf-life of each specific product is unique and based on the particular recipe, raw ingredients and manufacturing and storage conditions used. If there are any changes to these, then the shelf-life will be liable to change (see Section 10.2). Whilst there is some guidance available in the literature for chilled foods (Ellis 1994) and MAP foods (Day 1992) the shelf-life of products should be defined scientifically during product development following the procedures outlined in this chapter. The rationale for arriving at a particular shelf-life will undoubtedly encompass safety, quality and commercial decisions. It is unlikely that all of these will be in agreement and the safety of the product must always assume the highest priority. There are, however, many commercial and marketing pressures to consider which will put some constraints on whether the shelf-life obtained from microbiological 10 Shelf-life determination and challenge testing G. Betts and L. Everis, Campden and Chorleywood Food Research Association evaluation is acceptable from a commercial viewpoint. For example, how does the shelf-life compare with that of similar competitors’ products? Does the shelf- life provide sufficient time for the sale of a significant proportion of the product within the shelf-life, thereby minimising ‘end-of-shelf-life’ stock disposal? Is the shelf-life long enough to suit weekly shopping, which is the way that most chilled foods are purchased (Evans et al 1991). If there is a commercially viable minimum shelf-life, then this needs to be considered at the product development stage and the recipe altered accordingly. Another constraint for chilled food manufacturers is the rapid expansion of the chilled foods market. Over the past ten years there has been considerable development in the number and types of products available (Dennis and Stringer 2000). There are approximately 7,458 new products per year, of which 3,616 are chilled (CCFRA 1999). The chilled foods market in 1997 was ￡5.1 billion (Anon. 1998). Such an active market requires rapid development of new product formats and ingredient combinations with short launch times. Traditionally, the safety and quality of new products would have been evaluated solely by the use of laboratory studies which are time consuming and expensive. Predictive mathematical modelling techniques are now available and are gaining increasing use in the development of new products. Their use in shelf-life determination will be discussed later in Section 10.3. In addition to commercial pressures for extensive and rapid product development there is a consumer pressure for fresh tasting products with less salt and preservatives which require minimum preparation (Gibson and Hocking 1997). These requirements have the potential to increase the growth of food spoilage organisms and pathogens and thus decrease the likely shelf-life attainable under chilled storage conditions. Such product changes mean that new combinations of ingredients and preservative factors need to be used to maximise shelf-life. This will also be discussed throughout this chapter. Determination of the shelf-life of a product is decided by a combination of safety requirements, quality and marketing issues and customer demands. Arriving at the correct shelf-life is essential for product success. This chapter illustrates how this can be achieved. 10.2 Factors affecting shelf-life 10.2.1 Product considerations Before a new product can be developed there are a number of fundamental considerations to be made which will affect the shelf-life likely to be achieved. Product description The first step to take is to decide what essential product characteristics are required; for example, is it a dairy product or a tomato-based product? Is it to be a homogeneous sauce or will it contain particulate matter? The generic product type will give an initial indication of the microorganisms likely to be of concern 260 Chilled foods to the product and thus the shelf-life likely to be achieved. For example, non- acidic dairy products are more susceptible to rapid growth of microorganisms and are likely to have a shorter shelf-life than, for example, acidic products which are more inhibitory to growth. For pasteurised chilled products, the heat treatment required may also be different based on these essential product characteristics (Gaze and Betts 1992) and the heat processing requirements can be decided at this stage. Product packaging The desired product packaging format needs to be considered. If the product is to be pasteurised in-pack, then provided that there is no post-process contamination, there should be fewer vegetative spoilage organisms or pathogens in the product and the attainable shelf-life should be relatively long. Bacterial spores will remain present in the product but these grow relatively slowly at chill temperatures (see Chapter 7). If the product is assembled after cooking, then there is a greater risk of contamination with microorganisms even in good hygienic conditions. The shelf-life of these products is likely to be shorter than those for in-pack pasteurised products. The gaseous atmosphere of the packaging needs to be considered. If the product is packed under normal atmospheric conditions then aerobic spoilage organisms such as Pseudomonas species can grow and rapidly spoil chilled products held at C605oC. If, however, modified atmosphere conditions are used which exclude oxygen, then spoilage will be by facultative or strict anaerobes which grow more slowly under good chill conditions (see Chapter 7). Preservatives Is the product to be marketed as preservative free or low salt? Removing such ingredients will allow more rapid growth of microorganisms and thus reduce the shelf-life unless additional preservative factors are included, e.g. addition of lemon juice. The effect of removing traditional preservatives should be considered at this early stage and a list of alternatives investigated. Shelf-life constraints It may be the case that there is a minimum shelf-life that must be achieved to make the product commercially viable, e.g. ten days to allow storage over a weekend period. This must be defined at the beginning of the shelf-life determination in order that the product formulation and packaging specifications chosen are likely to achieve the minimum desired shelf-life. Often products have a perceived maximum shelf-life by the consumer which is shorter than that which can be achieved in reality. For example, Evans (1998) reviewed the mean actual and perceived shelf-lives of a range of chilled products and found that for pa?te′ the actual storage life was over 10 days whilst the perceived storage life was only 4 days. Having formulated the specific product and packaging characteristics, it is likely that a target shelf-life will be derived based on past experience. This Shelf-life determination and challenge testing 261 should then be confirmed by a scientific approach outlined in Section 10.4. Once the shelf-life has been determined there are a number of factors which will affect shelf-life as described below. These also need to be considered at the product development stage to ensure that they are under control during routine production of the product. 10.2.2 Raw materials The raw materials used in the preparation of a product will influence the biochemistry and microbiology of the finished product. In order to achieve a consistent shelf-life, the quality of the raw materials needs to be standardised and the attributes most likely to affect product shelf-life should be laid down in specifications. Variations in the quality of raw ingredients can lead to variations in the final product which may affect product shelf-life. Variations in raw material can occur for a number of reasons: natural variation, variety change, a change of supplier, seasonal availability or pre-processing applied to raw materials. The manufacture of coleslaw provides an example of where shelf-life can be influenced by the seasonal availability of freshly harvested cabbage which has a low yeast count, whereas cabbage from cold storage has a higher yeast count. Use of cabbage from cold stores results in coleslaw with a markedly shorter shelf-life owing to the higher starting levels of yeast introduced via the raw ingredients. If an ingredient for a raw product does not meet an agreed specification, e.g. for levels of microorganisms, it is still possible to use the ingredient for a different purpose, e.g. to be added to a product before cooking, provided there is no compromise to food safety. The likely consequences of using higher levels of organisms can be evaluated using predictive models (Section 10.3). Tolerance limits for those ingredients that exert a key preservative effect in the final product, such as the percentage of salt, need to be established during the development of the product or in challenge testing (see Section 10.6) and be stated in the product process and formulation specifications. Any variability in the levels of these ingredients due to inaccuracy in weighing ingredients during routine production will affect the shelf-life achieved. Ingredients which are crucial to product safety or stability during the assigned shelf-life should be identified using product hazard analysis (Leaper 1997) and the levels of these ingredients must be controlled during routine production. For example, for chilled MAP foods a salt level of 3.5% in the aqueous phase can be a key controlling factor for these foods and the salt level must be monitored for each batch of product manufactured (Betts 1996). Product formulation can be used to overcome natural variability of critical factors in raw materials and thereby reduce the variation of the final product. The pH is one of the most important factors affecting the degree of heat processing required to achieve sterilisation. In tomatoes there is a variability of acidity between cultivars. Product formulation can be used to overcome this variability either by blending high and low acidity cultivars or by the addition of 262 Chilled foods permitted organic acids. Again, it is crucial that where pH is used as a key preservative factor, the pH of each batch of product should be monitored to ensure that the target level is achieved. 10.2.3 Assembly of product In complex and multi-component products, contact between components may result in migration of flavourings, colourings, moisture or oil from one component to another. This may limit shelf-life due to quality changes, e.g. in multi-layered trifles where the visual appearance is impaired by the migration of colouring components from one layer to another, and in fruit pies where migration of moisture from the filling to the pastry crust leads to a loss of texture. Alternatively, migration brings together substrates for chemical reactions, and it is the products of these reactions which influence product shelf-life. Pizza toppings containing unblanched green peppers may be susceptible to rancid off-flavours as a result of the enzyme lipoxygenase in the green peppers coming into contact with fatty acid substrates in the pizza base or in the cheese. The way in which multi-component products are assembled can have major effects on the microbiological safety of the product. If components which are stable due to low A w or low pH are placed in contact with components which are inherently unstable due to a high A w or high pH, there will be a layer formed between the two components where the A w and pH is now suitable for microbial growth. Any microorganisms in the stable component could grow in this layer. Migration or contact between components can be limited to extend shelf-life and needs to be considered during product development. Where migration and contact are detrimental to product quality, the use of edible films or packaging in separate compartments could be considered. 10.2.4 Processing Processing encompasses a wide range of treatments that may be given to food from simply chopping or washing, (e.g. ready-to-eat chilled salads), to heat processing, acidification, addition of preservatives, fermentation or salting. Processing exerts a considerable effect on the microflora, chemical, biochemical and sensory properties of a food. In some cases, the purpose of food processing may be primarily intended to achieve the desired characteristics of the product, e.g. fermented salads, and can result in changes that extend the shelf-life by reducing pH. In other cases, processing may be specifically selected to influence shelf-life limiting factors, e.g. heat processing to inactivate microorganisms and deleterious food enzymes. If a process is used to achieve product shelf-life, there should be an awareness that small changes in the processing conditions can have a marked effect on shelf-life. Any stages of product manufacture which are essential for the safe shelf-life of the product should be identified during a hazard analysis and suitably controlled during routine production. Shelf-life determination and challenge testing 263 10.2.5 Hygiene Poor hygienic control during preparation, processing and packaging can result in a high level of organisms being introduced into the product; for example, poor cleaning of meat slicing equipment results in an increase in the microbiological count. This may have adverse effects on the safety and quality of the product, which would affect shelf-life. The shelf-life for a particular product can only reflect the range of variables that were included in the testing procedures. Consistent hygienic control during subsequent production runs is essential if product with the assigned shelf-life is to be produced (see Chapters 13 and 14). 10.2.6 Packaging Packaging prevents contamination by microorganisms, protects against physical damage and can be used to isolate the product from adverse environmental factors such as light, atmospheric oxygen or humidity. Packaging materials can be used to filter out light of specific wavelengths to prevent or reduce photocatalysed reactions that would result in oxidation or nutrient degradation. Products sensitive to oxidation by atmospheric oxygen can be protected by selection of packaging materials with the appropriate barrier properties to oxygen ingress and, similarly, selection of barrier properties with respect to moisture can be used to prevent the product from drying out or retain the humidity within the pack. Modified atmosphere packaging extends the shelf-life of meats and vegetables either by directly affecting the critical quality factor (as in the case of fresh red meat, where the bright red colour is maintained by a high oxygen atmosphere), or by influencing the rate at which reactions leading to adverse quality changes proceed. In addition, active packaging, e.g. O 2 /CO 2 /H 2 O absorbers, or ethanol emitters may extend shelf-life (see Chapter 6). Where the atmosphere within the package is modified, i.e. is not the normal atmospheric composition, then the effects on microorganisms should be considered. Excluding oxygen from packs will prevent the normal growth of spoilage organisms of air-packed chilled foods, i.e. Pseudomonas spp. Therefore, foods may become unsafe due to growth of anaerobic pathogens such as C. botulinum (Anon. 1992), but these products may appear to be organoleptically stable (Betts 1996). 10.2.7 Storage and distribution The conditions applied to the final product during storage and distribution can have a marked effect on shelf-life. Temperature, lighting and humidity influence which microorganisms grow, which biochemical reactions and physical changes take place and the rate they occur. To make an assessment of the shelf-life of the final product, the conditions that the product is likely to encounter and the effects that they will have on the product need to be known or determined. Limited numbers of time-temperature surveys of chilled products during storage, 264 Chilled foods transportation, retailing and handling in the home have been performed, some of which have been summarised by B?gh-S?resen and Olsson (1990). Currently, the UK Food Safety Temperature Regulations (Food Safety (Temperature Control) Regulations 1995 SI No. 2200) allow a maximum of 8oC during distribution and retail display of chilled products. It is important that this is considered when the shelf-life of products is determined, as any assessments done at lower temperatures will be affected by use of a higher temperature (see Section 10.4). 10.2.8 Consumer handling Consumer handling of chilled products can affect the quality and the safety of the product. Factors such as the time taken to carry the product home, consumer perceptions of chilled foods and domestic storage conditions need to be taken into consideration when setting up the time and temperature regime to be used in storage trials. This is perhaps the part of the chill chain that is most variable and over which the manufacturer has least influence and control. A survey of consumer handling of chilled foods in the UK (Evans 1998) indicated that most consumers shopped at least once a week for quantities of chilled foods. In the majority of cases, transport to the home was by car or foot (83%), taking an average of 43 minutes to get food from the retail store into the home refrigerator. The temperature of the foods generally ranged from 4–20oC. Domestic refrigerator temperatures were found to have an overall mean temperature of 6oC with a range of C01oC to +11oC. On average, only 30% of refrigerators were operating below 5oC. Of the refrigerators included in the survey, 7.3% were running at average temperatures of greater than 9oC, though positional temperature differences, particularly in fridge-freezers and larder freezers, indicate this figure to be higher if product is stored in the top of these refriger- ators. Evans (1998) has also provided additional data on consumer practices. Whilst such information gives an indication of the temperatures and times that are likely to be needed in shelf-life trials to simulate consumer handling, there is still the decision to be made with respect to what is a reasonable worst case to use. If the ‘worst case’ temperatures and holding times that have been recorded were used in shelf-life tests, few of the products currently in the market-place would achieve the target shelf-life. The manufacturer has to estimate where ‘reasonable’ abuse ends and ‘unreasonable’ abuse begins. Consumer handling of products may not be as intended or envisaged by the manufacturer. Many chilled products are purchased on the basis of the ‘fresh image’, but then frozen at home. Opening and partial use of vacuum or modified atmosphere packaged products invalidates the shelf-life information with respect to the remaining product. A survey of consumer perceptions of the shelf-life of chilled foods has indicated that whilst consumers believe that most chilled foods should be stored for two days or less, in reality the same householders were storing certain chilled foods for considerably longer periods of time (Evans et al. 1991). Shelf-life determination and challenge testing 265 10.2.9 Legislative requirements Legislative responsibilities for the chilled food manufacturer are described fully in Chapter 2. The main legislative restrictions for chilled foods in relation to shelf-life are the distribution and storage temperatures which can be used. In the UK, chilled foods can be stored at temperatures up to 8oC and this should be taken into consideration when defining the shelf-life during product develop- ment. It may be possible that a product would be able to be stored at lower temperatures throughout much of its shelf-life during retail distribution and storage, however, with the maximum temperature specification of 8oC, it is possible that on occasion a batch of the product would be stored at 8oC throughout its shelf-life and therefore it should be able to withstand this time and temperature regime whilst maintaining product quality and safety. In addition, if the chilled product is to be exported to other EU countries, there will be different chilled temperature restrictions. These will also need to be considered during product development. There are many different requirements for chilled products throughout the EU and a working document, first draft of a proposal for a European parliament and council regulation on the hygiene of foodstuffs, is being circulated which would harmonise temperature regulations throughout the EU (Anon. 1997). With respect to MAP chilled foods, there are guidelines which restrict the shelf-life of these products to ten days or less at chill temperatures of C208oC, unless specific controlling factors are in place to minimise the potential for growth of psychrotrophic C. botulinum (Anon. 1992, Betts 1996). Any deviations from these guidelines should be made only after scientific evidence that the alternative preservation systems in the products will prevent the growth of, or toxin production by, C. botulinum. 10.2.10 Effects of intrinsic/extrinsic factors The factors discussed above, namely the type and source of ingredients and the subsequent processing and packaging, will influence the types and levels of microorganisms that will be present, and the chemical and biochemical reactions that can occur, in the final product. The ability of organisms to grow or cause problems, or for chemical reactions to proceed in the final product, will be dependent on the properties of the final product, i.e. pH, A w (known as intrinsic factors), and on the external factors that the final product encounters, such as temperature (known as extrinsic factors). Intrinsic factors include: ? water activity (A w ) (available water) ? pH/total acidity ? type of acid ? preservatives, including salt and spices ? nutrients ? natural microflora ? redox potential (Eh) 266 Chilled foods ? available oxygen ? natural biochemistry (enzymes, chemical reactants). Extrinsic factors include: ? heat treatment (processing, cooking or reheating of the food prior to consumption) ? headspace gas composition ? temperature throughout storage and distribution ? relative humidity (Rh) ? light (UV and IR). Table 10.1 gives examples of minimum growth conditions for pathogenic and spoilage organisms of concern to chilled foods. It must be stressed that such Table 10.1 Minimum growth conditions for microorganisms which may be associated with chilled food Type of microorganism Minimum pH Minimum A w Anaerobic Minimum for growth for growth growth a growth temp b (oC) Pathogens (c) Salmonella 4.0 0.94 Yes 7 Staphylococcus aureus 4.0 0.83 Yes 6 (4.5 for toxin) (0.90 for toxin) (10 for toxin) Bacillus cereus 4.4 0.91 Yes C604 (psychrotrophic) Clostridium botulinum 4.6 0.93 Yes 10 proteolytic A, B, F Non-proteolytic B, E, F 5.0 0.97 Yes 3.3 Listeria monocytogenes 4.3 0.92 Yes 0 Escherichia coli 4.4 0.95 Yes 7.0 Vibrio parahaemolyticus 4.8 0.94 Yes 5 Yersinia enterocolitica 4.2 0.96 Yes C02 E. coli O157 4.5 0.95 Yes C06.5 Spoilage organisms (d) Pseudomonas 5.5 0.97 No C600 Enterobacter aerogenes 4.4 0.94 Yes 2 Lactic acid bacteria 3.8 0.94 Yes 4 Micrococci 5.6 0.9 No 4 Yeasts 1–5 0.8 Yes C05 Moulds C602.0 0.6 No C600 Notes: The table lists various species and indicates approximate growth and survival limits with the various factors acting alone. Interactions between factors are likely to considerably alter these values. a For example, in vacuum pack. b Minimum growth temperatures are for growth in typical neutral pH, high water activity, chilled foods. c Data for pathogens taken from Anon. 1997. d Data for spoilage organisms taken from Brown 1991. Shelf-life determination and challenge testing 267 values are typical values only and are dependent on the particular strain of organisms used in the study and the storage conditions used. Such data should only be used as an indication of the microorganisms likely to grow in a product and should be supported by thorough scientific evaluation. 10.3 Modelling shelf-life As discussed previously, one of the most important factors in determination of product shelf-life is the potential for the growth of microorganisms. There is therefore a requirement to test new chilled products to assess the potential for growth of pathogens or spoilage organisms and thus define the period of time during which the product is considered acceptable for its intended use. Traditionally, the only objective approach to this has been extensive laboratory studies of pilot scale production of the product (see Section 10.4). This approach is still the best way to do final testing of the product, but it is very time consuming and costly during early product development stages. With the vast number of new products reaching the shelves every week, an alternative approach is required to aid shelf-life determination. A technique gaining increasing application in chilled food production is predictive models which can be powerful tools at all stages of product development and manufacture. Chapter 7 describes the types of models available for microorganisms; their use in shelf-life determination is elaborated upon here. Three examples of the use of models are given below. 1. During product conception/development. To give an initial indication of major problems, e.g. to take an extreme situation, if a neutral pH product with a 20-day shelf-life is envisaged but pathogen models show that L. monocytogenes, B. cereus and Salmonella could all grow within two days then it will be apparent at the earliest point that the product will not be feasible. 2. During Product Formulation. For example, having decided on a product type which is likely to be safe and stable for the desired shelf-life, then models can help in final product recipe formulation. There may be three different combinations of salt and pH which will give the same shelf-life at a given temperature, e.g: pH salt % (w/v) 6.4 0.8 6.2 0.7 6.0 0.4 However, one of these may have a better taste in terms of consumer preference. The effect of these different recipes on microorganisms can be assessed without the requirement for laboratory trials at this stage. 268 Chilled foods 3. In Setting Specifications. There are very few microbiological specifications for the chilled food manufacturer; however, two very useful publications on final product specifications are PHLS (1996) and IFST (1999) which can help in deciding microbiological specifications. These two documents take different approaches. For example, the PHLS document gives levels of different microorganisms which are acceptable at the end of the shelf-life, i.e. at point of consumption. If a chilled foods manufacturer wanted to use these speci- fications to ensure end-of-life microbiological levels were acceptable, then predictive models can be used to give an indication of the levels of micro- organisms which could be present at the beginning of life for any product formulation to achieve the end-of-life specifications. If, for example, the rec- ommended level of TVC or Enterobacteriaceae at end-of-life was 10 6 cfu per gram, then predictive models could be used as shown in the examples below. Example 1 Enterobacteriaceae pH 5.5, salt 5.25 % (w/v), 15oC Initial level Intended Predicted level Time to 10 6 per gram shelf-life @ 5 days (days) 10 5 days 1.39C210 4 13.5 100 5 days 2.39C210 5 9 10 3 5 days 2.75C210 6 6 10 4 5 days 1.5C210 7 3 As can be seen from the above, in order to achieve a shelf-life of five days, with an end specification of no more than 10 6 cfu/g, the initial level of Enterobacteriaceae should be no more than 10 3 cfu/g. The IFST document gives maximum levels of microorganisms which should be present after manufacture of product. Predictive models can be used to give an indication of the likely increase in these levels during different chilled storage regimes. Example 2 Enterobacteriaceae pH 5.5, salt 2% (w/v) Temperature Initial level Time to 10 6 (days) (manufacture specification) (end specification) 3oC 100 33 5oC 100 17 8oC 100 7 10oC 100 4 As can be seen, if after manufacture the level is 100 cfu/g, the temperature of storage can have a huge effect on the likely shelf-life, e.g. at 3oC it would be 33 days compared to just seven days at 8oC. Shelf-life determination and challenge testing 269 Most predictive model systems will give information which can be used to help in defining the shelf-life of a product. Data will be available as one or all of the following: ? lag time, i.e. time before an increase in numbers occurs ? generation time, i.e. time taken for each cell to double ? growth curve which will show the lag time, exponential (or fastest) growth phase and stationary phase of growth ? time taken to reach a target level of cells, or conversely ? numbers of cells present after a pre-determined time. Currently, most microbial predictive models are based on the growth of one species or genus of organism or, in some cases, a group of related organisms, e.g. lactic acid bacteria, Enterobacteriaceae. Where more than one organism is a cause for concern, predictions need to be obtained from the relevant models and interpreted by suitably trained personnel. Example 3 This shows how different spoilage models can be used to assess the likely shelf- life of a chilled meat product. ? Organisms of concern Lactic acid bacteria Enterobacteriaceae ? End-of-life determined when: Lactics @ 10 6 per gram Enterobacteriaceae @ 10 6 per gram ? Product formulation pH = 5.5 A w = 0.983 % Salt = 3 Organism Initial level Time to 10 6 Shelf-life (cfu/g) days days Lactic acid bacteria 100 9 9 Enterobacteriaceae 100 7 7 As can be seen, the Enterobacteriaceae will be limiting to the shelf-life because the Enterobacteriaceae reach a level of 10 6 cfu/g within seven days whereas the lactics do not reach this level until the ninth day. 10.4 Determination of product shelf-life In order to assess the shelf-life of a new or existing product it is usual to conduct microbiological analyses of the final product for typical spoilage organisms and 270 Chilled foods pathogens during storage at specified times and temperatures. As this may require the use of contract services it can be expensive and it is therefore important that the shelf-life trials are done at the right stage during product development. A useful approach to shelf-life determination has been described by Brown (1991), where a three-staged approach is taken, i.e. Phase I Pilot Scale Phase II Pre-production Phase III Full Production. 10.4.1 Shelf-life considerations at pilot scale At this stage, it is likely that the product is a new idea or marketing concept and will need to be produced on a small scale in the development kitchen in order to assess the likelihood of product success in terms of flavour, colour, texture and eating quality. Many of the details about the product are unlikely to be known, so only a preliminary consideration of product shelf-life is possible (Fig. 10.1). Initially, the product can be described on a theoretical basis by listing the likely ingredients, processing and packaging properties, and storage require- ments. This listing can be used to evaluate the critical properties of the product with respect to shelf-life and to highlight likely changes in quality, possibilities of microbiological spoilage and any potential food safety problems. Ingredients identified as likely to contain food-poisoning organisms and spoilage bacteria can be monitored at all stages in the preparation of the product. Changes in the numbers of organisms due to each particular operation can then be noted and used when assessing risks associated with the product, and should suggest, or help in assessing, the most important changes to monitor during storage trials. For example, cooking may reduce the numbers of organisms, whereas a holding period or the introduction of a particular ingredient may increase the numbers. It is important even at the pilot plant scale to consider the likely hazards associated with the product using an approach such as Hazard Analysis of Critical Control Points (e.g. Leaper 1997). Using the data collected in the paper exercise on the ingredients, processing and packaging, an assessment should be made of the potential for contamination with food-poisoning organisms and their growth during storage. If there is a possibility of the product containing infectious pathogens (e.g. Listeria or Salmonella) or toxin producers such as Staphylococcus aureus, Bacillus cereus or the Clostridia, then their potential for harm will need to be evaluated in later shelf-life trials. Initial indications of the likely growth of spoilage organisms or pathogens can be gained from predictive models (Section 10.3). Shelf-life assessments made on product produced at the kitchen scale are limited to some extent, as it is inevitable that, during the development of a chilled product, the product and processes will change several times. Moreover, the conditions are unlikely to equate to those of a ‘real’ production situation. The Shelf-life determination and challenge testing 271 quality of the raw materials and standards of handling and hygiene will differ from full scale production and the size of the ‘batch’ will be considerably smaller, resulting in less variability in the final product. Despite these limitations, data indicative of product shelf-life can be gained from observations of products made on the kitchen scale, realistically portioned, packed and stored at 8oC (unless knowledge to the contrary of the storage conditions for the product is available) for at least their target shelf-life, or a shorter period if the changes become acceptable. The material produced on a kitchen or pilot scale should not be subject to extensive sensory or microbiological examination, but each of the changes Fig. 10.1. Pilot-scale shelf-life determination (adapted from Brown 1991). 272 Chilled foods contributing to quality should be identified and described. However, before the product can be tasted it must be shown to be microbiologically safe. The overall objective is to identify and describe product acceptability and the extent of sensory change during storage. The type of observation needed is that which will be useful to describe the characteristics of the product that lead to the product becoming unacceptable to the consumer. In many instances there is likely to be a particular quality change, a ‘critical quality parameter’, that limits shelf-life. By combining the background data with that collected from actual storage trials of the product, it should be possible to gain an overall impression of whether the product is likely to meet its target shelf-life. At this stage of testing, the assessors will normally have been closely involved with development of the product. If the product does not achieve the quality desired, they should be able to decide whether changes in processing, packaging or formulation can be used to achieve the desired quality, and whether they require re-testing on the kitchen scale or whether such changes could be adopted for the pre-production run. If the quality changes are unacceptable then the product or processing concept will need to be reconsidered with a view to overcoming these problems. Again, the use of predictive models may help to decide whether changes in product composition are likely to affect microbial shelf-life. 10.4.2 Shelf-life considerations at pre-production scale (Fig. 10.2). The objectives of pre-production are to scale up production, to provide and confirm product, process and formulation specifications, and to ensure that the product produced is viable (meets the marketing brief, including the target shelf- life). Pre-production runs are undertaken on either pilot plant equipment or, preferably, by the production of batches on full scale equipment. When the development of the process is complete, details should be documented in accordance with the requirements of any quality system in place, e.g. HACCP. The product specification will need to address raw materials, product formulation, assembly of the product, processing, hygiene and cleaning, and packaging. Deviation from these specifications may have an impact on product shelf-life, therefore shelf-life is only applicable to product produced under these defined conditions. Any modifications to any aspect of production should be recorded. Shelf-life testing should be most extensive at the pre-production stage. To set up shelf-life tests, the storage conditions, a sampling protocol, and the analyses to be performed need to be defined. Storage conditions Storage conditions should be chosen carefully to match or represent real conditions to which the product is likely to be exposed during its life. The key storage condition for chilled food is the time–temperature regime. Both time and temperature must be specified, covering the temperature range encountered and the times spent in distribution, retail cabinets and in consumer use, including Shelf-life determination and challenge testing 273 transport from store to home and storage in domestic refrigerators. The sequence in which holding times and temperatures are applied requires careful consideration. A product purchased ‘early’ in its shelf-life may spend a larger proportion of the total shelf-life in a domestic refrigerator at temperatures which are generally higher than legislation permits in retail outlets (Rose et al. 1990). Similarly, the impact of conditions encountered during transportation from store to home may differ depending upon where it occurs in the shelf-life. Fig. 10.2 Pre-production runs (adapted from Brown 1991). 274 Chilled foods It is therefore difficult to determine every combination of times and temperatures likely to be experienced by a product and a decision must be taken on what combinations will be representative of normal conditions. It has been suggested, on the basis of experience within the food industry, (Brown 1991), that holding the product at not less than 8oC with a hold at 22oC for four hours at the earliest possible sale time, provides a reasonable default from which to begin. As experience of the product and product performance increases, standard shelf-life testing conditions can then be reviewed. It is important to remember that the shelf-life of a product will be true only for the batch of product and the temperatures tested. If the product achieves the described shelf-life when tested at 8oC, it will undoubtedly achieve a longer shelf-life at lower temperatures, e.g. 5oC. Conversely, if shelf-life trials were done at 5oC and the product was held at higher temperature, it is likely that the shelf-life will be shorter than anticipated. A final consideration before deciding time and temperatures is whether they meet customer requirements. Many retailers of chilled product have built up specific shelf-life testing requirements on the basis of knowledge of their own distribution and storage facilities. It is advisable to ensure that any tests considered will meet these requirements before starting shelf-life studies. Sampling times Once the time and temperature regime has been established, sufficient product must be held under these conditions to allow several units of product to be assessed on more than one sampling occasion. The extent and frequency of sampling is very dependent on previous experience of the product or similar products, and the stability of the product. It is undesirable to use fewer than three replicates for each sampling time but preferable to use five as this will allow greater statistical analysis of the data. The number and frequency of sampling occasions is dependent on the target shelf-life of the product. Sampling at the beginning of shelf-life (day 0), at the target shelf-life, and on at least three occasions in between is suggested. For a short shelf-life product (2–5 days) this may result in samples being taken daily, and less frequently for a longer shelf-life product. It is advisable to take additional samples beyond the target shelf-life to monitor the margin of safety and/or quality. Key parameters to monitor in shelf-life tests should have been identified as a result of the observations made at the kitchen or pilot scale stage, and through HACCP assessments. To determine the extent of change with time, sensory assessments (Chapter 12), microbiological analyses and chemical analyses (Chapters 8 and 9) will be needed. In normal production, there will be some variability of most key parameters. It is important that the relevant extremes of these key parameters are included in shelf-life tests to ensure that the shelf-life is applicable to all the extremes of product that may be produced. To ensure such product is available it may be appropriate to make a pre-production batch that represents the extreme situation for shelf-life testing. In some cases, either the presence of a microorganism Shelf-life determination and challenge testing 275 potentially critical to the shelf-life of a product cannot be guaranteed, or the level of contamination with it is likely to be subject to considerable variability. Shelf-life determination may then be best approached by deliberately adding the organism to the product in challenge testing studies. If such an approach is adopted, an awareness of the limitations is essential for interpretation of these tests (see Section 10.5). Shelf-life tests should ideally be repeated on more than one pre-production run to test day-to-day variation and to provide a level of confidence and experience of the product. The number of sampling occasions may be reduced based on data from the first trials. A route for interpreting the information collected in shelf-life trials is illustrated in Figure 10.2. Safety should always be of prime importance; therefore, the first limit to establish is the maximum safe shelf-life of the product. The main reasons for chilled foods becoming unsafe are the growth of pathogens or toxins produced by microorganisms if present. Controls and monitoring procedures used in routine HACCP analysis should minimise the likelihood of the presence of pathogens. Secondly, the shelf-life tests should enable the maximum quality shelf-life to be defined. Establishing the criteria of importance, and defining the lowest acceptable standard is a matter of manufacturer policy. Many companies use a mix of ‘brand image’, price, market share and customer complaint level to set and confirm the final end of shelf-life quality standards. Changes in appearance, texture and flavour will occur as a result of the chemical or biochemical reactions, changes in physical structure, and the growth of spoilage microorganisms. These may be measured in terms of microbial count, the value of a specific quality-related factor such as the peroxide value for oxidised fats, or by sensory evaluation. Changes in quality will reach a point where the product no longer achieves the standards laid out in the marketing brief. This time interval defines the quality shelf-life. Once the shelf-life has been determined in terms of safety and quality, these can be compared with the target shelf-life. Ideally, both should exceed the target shelf-life. The shelf-life should be set as the safety or quality limit, whichever is the shorter, though it is always preferable that shelf-life is limited by quality rather than safety, as in most cases changes in quality can be discerned by smell, taste or appearance, whereas such changes cannot be relied upon to indicate safety limits. Results from challenge tests should also be reviewed in order to assess the safety of the product. As scaling up from pre-production to full scale production may cause some changes, it is suggested that the maximum shelf-life is reduced to provide an additional safety margin. If the safe shelf-life is less than the target, then either the product needs to be reconsidered or the viability of marketing the product with a shorter shelf-life needs to be assessed. 10.4.3 Shelf-life at full production scale (Fig. 10.3) The objective of full-scale production is to produce product for sale. Shelf-life testing at this stage is to confirm the determinations made at the pre-production 276 Chilled foods stage. The same key parameters should be monitored in shelf-life tests, but special care should be taken to test the full range of variation that is produced, particularly if this is greater than at the pre-production stage. If the shelf-life differs from that of pre-production runs, then it is necessary to reconsider both pre-production and full production runs to identify the cause. Shelf-life tests should, ideally, be performed on product produced during the first three full production runs. As experience of the product increases and Fig. 10.3 Full-scale production flow chart (adapted from Brown 1991). Shelf-life determination and challenge testing 277 feedback via consumer complaints becomes available, the shelf-life can be adjusted accordingly. It is inevitable that the ingredients, the process or distribution of the product will change in time. Although each small change may, by itself, seem irrelevant to the shelf-life of the product, taken together these changes may have a marked effect. It is important that those responsible for shelf-life testing be informed of even apparently small changes, and that periodically the whole process be reviewed and confirmatory tests performed if necessary. 10.5 Maximising shelf-life There are a number of steps that can be taken to increase the shelf-life of a product whilst maintaining product safety. These are described below. 10.5.1 Product formulation changes Minor changes in the composition of a product may be sufficient to prevent or delay the growth of spoilage organisms and thus increase the period of time before it is unacceptable for use. This is particularly the case where the factor being changed, e.g. salt, is limiting to growth. There is currently a trend to reduce the levels of added salt in ready-to-eat products. Where this is the case, a minor change in the acidity of the product may achieve a similar effect in increasing shelf-life. The example below illustrates this point. With no salt present, this product would be limited to a shelf-life of just five days, but with 2% salt the shelf-life can be increased. Example Enterobacteriaceae pH 5.50 Temperature 8oC Salt (% w / v ) Count @ 5 days 5 1.1C210 2 4 2.89C210 2 3 5.78C210 3 2 6.8C210 5 1 3.1C210 7 0.5 8.8C210 7 10.5.2 Use of new technologies The most effective way to inactivate pathogens, spoilage organisms and enzymes is to apply a heat process. However, this may cause various changes to the flavour of a product and may detract from the fresh taste. There is the 278 Chilled foods potential for using alternative cold-processing systems for products which will inactivate microorganisms and in some cases enzymes, but will not cause heat damage to the product. Examples of such technologies include the use of high pressure and ultrasound (see Section 10.7) (Mermelstein, 1998). 10.5.3 Storage temperatures This is one of the most effective ways for all chilled foods manufacturers to maximise the shelf-life of a product. If final products and raw materials could be stored at 2oC instead of 5oC and retail storage was done at 5oC instead of 8oC this could markedly increase the shelf-life. This is illustrated in Table 10.2 for a ready-meal product stored at various on-site and retail temperatures. 10.5.4 Use of modified atmosphere Chapter 7 discusses different packaging formats available for chilled products. With regard to the microbiological shelf-life, the use of increased levels of carbon dioxide and exclusion of oxygen can markedly increase shelf-life (Betts 1996). It must be stressed, however, that the use of MAP for chilled products may restrict the shelf-life to ten days unless it can be shown to be able to prevent the growth of C. botulinum (see Section 10.2). 10.6 Challenge testing There is often some confusion surrounding the differences between shelf-life determination and challenge testing. During the development of new chilled products there are two different aspects which need to be considered: Table 10.2 Levels of Pseudomonas (Ps) present after each storage period from an initial level of 10 cfu/g On-site storage for 2 days Retail display for 6 days Likely Temp Level of Ps Temp Final level of shelf-life after 2 days Ps (cfu/g) (days) (cfu/g) 2oC163oC 7.8C210 5 9.0 8oC 7.3C210 8 5.5 3oC21oC 1.0C210 6 9.0 8oC 7.5C210 8 5.5 5oC543oC 2.3C210 6 8.5 8oC 8.7C210 8 5.0 Notes: Product characteristics: Chilled ready-meal; Salt – 0.8% w/v; pH – 6.2; Target life – 8 days; End of life – Pseudomonas @10 7 /g (starting level 10/g) Shelf-life determination and challenge testing 279 1. Is the product safe and stable during normal production and storage conditions and for how long, i.e. what is its shelf-life? 2. Is the product likely to be safe and stable during its shelf-life if it became contaminated with food pathogens and spoilage organisms, i.e. are the product formulation and storage conditions inherently safe with respect to the inoculated organisms? In the first case stated above, only the microorganisms that are naturally present in the batch of product will be present and able to grow during storage. Ideally, under good manufacturing conditions and using a HACCP approach to product manufacture, there will be minimal chance of food pathogens, e.g. Salmonella, being present in the product. Therefore, during shelf-life determination there may be an absence of food pathogens and the product may be considered to be safe during the assigned shelf-life. However, it is possible that on occasions during routine production, the product may be contaminated with food pathogens or different spoilage organisms not present in the batch used for shelf-life determination. If this occurred, then the manufacturer would need to know if the product was likely to remain safe and stable during storage. This ‘what if’ scenario is the rationale behind the use of challenge testing trials. With the requirement under the Food Safety (UK) Regulations to show ‘due diligence’ with respect to the safety of foods, many food manufacturers are doing inoculated challenge tests on new and existing products. Microbiological challenge testing is the laboratory simulation of what can happen to a product during manufacture, distribution and subsequent storage. This involves inoculation of the product with relevant microorganisms and holding of the product under a range of controlled environmental conditions to assess the risk of food poisoning or to establish product stability in the case of food spoilage organisms. Notermans et al. (1993) describes four stages to challenge testing as (1.) an appropriate experimental design; (2.) an inoculation procedure; (3.) a test procedure; and (4.) interpretation of results. Important aspects of these stages are described further below. 1. Design of challenge test There are three main ways in which a challenge test can be applied to new or existing chilled products: 1. to determine the safety of the product 2. to determine the potential for spoilage of the product 3. to assess the stability of new recipes or reformulated products (Anon. 1987). Safety is the prime consideration when producing food for commercial sale and it must be ensured that the product represents a minimum hazard to the consumer. Microbiological challenge testing should be done if the microorganism of concern is suspected to be present in low numbers (Notermans and in’t Veld 280 Chilled foods 1994) or has the potential to be contained in raw material or introduced into the product at some stage during production and distribution. With respect to assessing the safety of a chilled product with regard to food pathogens, the organisms likely to be a hazard for the product and necessitate challenge testing, should be identified during routine hazard analysis, e.g. HACCP. Use of this approach to identify organisms of concern is described by Notermans and in’t Veld, (1994). Having identified which organisms are to be tested, it is necessary to consider what particular aspects of the product and storage conditions are to be challenged. There are a variety of factors involved in the overall preservation of a food; these are the same intrinsic factors or extrinsic factors which are considered when determining shelf-life. In challenge testing, variation of each of these factors in turn enables those factors effective in preserving a food to be determined. Challenge tests can therefore be done to evaluate different formulations of the food. They can also be done to assess the effect of storing the product under a variety of controlled storage conditions. These should include conditions to simulate abuses which may be encountered during distribution and consumer handling after purchase. Processing and packaging conditions may be key elements in the shelf-life of chilled foods, and must not be ignored in challenge tests. Another important aspect to consider during design of a challenge test is the number of samples to be evaluated at each sampling point to allow sufficient data to be obtained for statistical analysis. In addition, the number of sampling times needs to be defined. As a starting point, the regime used for shelf-life deter- mination can be used, i.e. there should be a minimum of five sampling times, one at the beginning, one at the end and three spaced throughout the total time period. On each occasion, there should be a minimum of three and ideally five or even ten samples analysed in order to gain confidence in the reproducibility of the results. It is not unusual for levels of microorganisms to stay constant, and even decrease over a period of hours, days or weeks before beginning to increase (Curiale 1998). Sufficient sampling times should be done throughout the trial to ensure that any initial decrease in microbial levels is not taken to be the end point of the trial and so subsequent growth following this is missed. Each experiment needs to be adequately controlled to ensure that the results obtained are meaningful. These should include positive controls in which the organisms under test are known to grow, (i.e. in laboratory media), as well as negative controls in which growth is not expected to occur. Suitable controls may be a standard product of known shelf-life, uninoculated samples, products stored under standard extrinsic conditions. Controls provide a measure against which change may be judged but also serve as indicators of reproducibility between experiments. Challenge testing is neither a quick nor a simple matter. It is usually used if other methods of ensuring safety or stability need to be supplemented or are thought inadequate in the circumstances of the particular foodstuff. At the outset, it is important to define clearly the reasons for undertaking the challenge Shelf-life determination and challenge testing 281 test and the aims of the experiments. It must then be decided whether a challenge test will meet those objectives. Once it has been determined that a challenge test is to be undertaken, it is necessary to determine the most effective way of satisfying the objectives. This involves consideration of the type of product to be tested, the factors believed to contribute to its stability and the conditions to which the product might reasonably be expected to be subjected to during its normal life. Experiments should then be carefully designed to elucidate the preservative mechanisms operating in the product and the tolerances of these factors without causing failure of stability or safety. 2. Inoculation procedures The choice of organisms for use in challenge tests is very important as they must provide a realistic challenge to the product. If the organisms chosen were particularly resistant to a preservative used in the product, then they may grow whereas other resistant strains would not. Conversely, if the organisms used are very sensitive to the antimicrobials used then they would fail to grow and the product would appear to be safe or stable when in fact it may have failed the challenge test if more realistic organisms were chosen. The cultures chosen should ideally have been isolated from a food source similar to the product under test, or conditioned by growing them in a product sample, or laboratory media with similar characteristics (Notermans and in’t Veld, 1994). Cultures from recognised culture collections, e.g. National Collection of Industrial and Marine Bacteria, are preferable as they allow full traceability for comparison between different challenge tests but should be checked to ensure that they behave in a similar manner to freshly isolated strains. It is preferable to use a cocktail of two or more strains of each microorganism in order to provide a greater challenge to the product. Other important considerations are the size, e.g. 0.1ml and method of inoculation used. In terms of inoculum size, a minimal amount should be used so that the characteristics of the product, such as A w , are not affected. Anon. (1987) details inoculation procedures for liquid, dry and intermediate moisture products. The number of organisms per millilitre must be realistic. The levels must be high enough to be easily detected, e.g. minimum level of 100 cells per gram of product, however, they should not be so high that they easily overcome the preservation capability of the product. Conducting a challenge test with an inoculum size of 10 6 cells per gram is unrealistic and is likely to lead to product failure. 3. Interpretation of results Before the experiment begins it is important to define the criteria of acceptability of the product. The criteria used will vary depending on whether the system under consideration is intrinsically stable or unstable, and whether safety or spoilage is being assessed. For intrinsically stable systems the end- point of a challenge test will be defined by microbial growth or undesirable organoleptic changes after a given period of time. For intrinsically unstable 282 Chilled foods systems there may be a variety of end-points, including growth to a specified number of organisms per unit weight of food, change of lag phase or generation time, or organoleptic spoilage. In the case of safety testing, the end-point may be the start of growth pathogens, the growth of pathogens to a specified number, the number of organisms per unit weight of food or toxin production. If the specified end-point is reached earlier than anticipated then the product should be reformulated or the processing and storage conditions adjusted accordingly. If the product survives the challenge test, i.e. the test criteria are not reached during the desired shelf-life, then the product can be considered suitable in relation to the specific conditions tested. If there are any changes, however small, to the product formulation, processing, storage, distribution or retail conditions then the results of the challenge test can no longer be considered to be reliable. A worked example of how challenge testing procedures can be applied to a chilled pasta product is discussed by Anon. (1987). 10.7 Future trends This chapter has outlined current thinking with respect to shelf-life determina- tion with particular emphasis on microbial safety and quality. Although this process may be considered an extensive approach, as confidence in setting shelf- life increases, this will enable new approaches to be assessed and utilised. One of the major areas to assist in shelf-life determination is predictive microbiological modelling. In particular, the development of food sector specific models will increase in the future. Models have been developed for fish products available from the Ministry of Fisheries, Technical University at Denmark (Seafood Spoilage Predictor on the internet – http://www.dfu.min.dk/micro/ssp/ help/usingssp.htm) and are currently in progress for cured meat products. Such models will enable an evaluation to be made of the interactive effects of a mixed microbial flora typically found in these products on food spoilage. There will be an increasing use of alternative technologies to improve the quality and/or shelf- life of chilled foods. Currently, high pressure is already used for chilled pa?te′s, orange juice and ethnic dishes such as salsa (Mermelstein 1998). As such technology becomes more widely used, the equipment costs will be reduced and the usage may increase. The chilled food market seems set to increase sales over the next few years and accurate, effective shelf-life determination will be one of the keys to its success. 10.8 References ANON, (1987). Microbiological Challenge Testing. Campden Food & Drink Research Association Technical Manual No. 20. Shelf-life determination and challenge testing 283 ANON, (1992) Advisory Committee on the Microbiological Safety of Food (ACMSF). Report on Vacuum Packaging and Associated Processes. HMSO London. ANON, (1997) Harmonisation of Safety Criteria for Minimally Processed Foods. Inventory Report. FAIR Concerted Action FAIR CT96-1020. ANON, (1998) Chilled Foods Market Report Plus. Keynote Publications. 1998. BETTS G D, (1996) A Code of Practice for the Manufacture of Vacuum and Modified Atmosphere Packaged Chilled Foods. CCFRA Guideline No. 11. CCFRA, Chipping, Campden, UK. B?GH-SORENSEN L and OLSSON P, (1990) The chill chain. In: Gormley, T. R. (ed.) Chilled Foods. The state of the art. Elsevier Applied Science, pp. 245. BROWN H M, (1991) Evaluation of the Shelf Life for Chilled Foods. Campden Food & Drink Research Association Technical Manual No. 28. CCFRA, (1999) Product Intelligence Dept. CURIALE M S, (1998) Limiting growth: microbial shelf-life testing. Food Product Design 7 (11) 72–83. DAY B F P D, (1992) Guidelines for the Good Manufacturing and Handling of Modified Atmosphere Packed Food Products. CCFRA Technical Manual No. 34, Chipping Campden, UK. DENNIS C and STRINGER M, (2000) Introduction: the chilled foods market. In: Chilled Foods: a comprehensive guide, 2nd Edition. Eds. M Stringer and C Dennis. Woodhead Publishing Ltd., Cambridge, pp. 1–16. ELLIS M J, (1994) The methodology of shelf-life determination. In: Shelf-life Evaluation of Foods. Eds. C.M.D Man and A.A. Jones. Blackie Academic and Professional. EVANS J A, STANTON J I, RUSSELL S L and JAMES S J, (1991) Consumer handling of chilled foods: A survey of time and temperature conditions. MAFF Publications, London, pp. 102. EVANS J A, (1998) Consumer perceptions and practice in the handling of chilled foods. In: Sous vide and cook-chill processing for the food industry. Ed. S. Ghazala. Aspen Publishers Inc., Maryland. GAZE J E and BETTS G D, (1992) Food Pasteurisation Treatments. CCFRA Technical Manual No. 27. CCFRA, Chipping Campden, UK. GIBSON A M and HOCKING A D, (1997) Advances in the predictive modelling of fungal growth in food Trends in Food Science and Technology, 8 353–8. IFST, (1999) Development and use of microbiological criteria for foods. IFST Booklet. ISBN 0 905367 16 2. LEAPER S, (1997) HACCP: a practical guide (second edition). CCFRA Technical Manual No. 38, Chipping Campden, UK. MERMELSTEIN N H, (1998) High pressure processing begins, Food Technology, 2 (6) 104–6. NOTERMANS S and IN’T VELD P, WIJTZES T and MEAD G C, (1993) A user’s guide to microbial challenge testing for ensuring the safety and stability of food products, Food Microbiology, 10 145–57. NOTERMANS S and IN’T VELD P, (1994) Microbiological challenge testing for 284 Chilled foods ensuring safety of food products, International Journal of Food Micro- biology, 24 33–39. PHLS, (1996) Microbiological guidelines for some ready to eat foods sampled at the point of sale, PHLS Microbiology Digest, 13 (1) 41–43. ROSE S A, STEADMAN S and BRUNSKILL R, (1990) A temperature survey of domestic refrigerators. CCFRA Technical Memoranda 577. SINGH, (1994) Scientific principles of shelf-life evaluation. In: Shelf-life Evaluation of Foods. Eds. C M D Man and A A Jones. Blackie Academic and Professional. Shelf-life determination and challenge testing 285