International Journal of Food Microbiology 73 (2002) 395 407 www.elsevier.

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Predictive microbiology: towards the interface and beyond

T.A. McMeekin *, J. Olley, D.A. Ratkowsky, T. Ross
School of Agricultural Science, University of Tasmania, PO Box 252-54, Hobart, Tasmania 7001, Australia Received 21 May 2001; accepted 9 August 2001

Abstract This review considers the concept and history of predictive microbiology and explores aspects of the modelling process including kinetic and probability modelling approaches. The journey traces the route from reproducible responses observed under close to optimal conditions for growth, through recognition and description of the increased variability in responses as conditions become progressively less favourable for growth, to defining combinations of factors at which growth ceases (the growth/no growth interface). Death kinetics patterns are presented which form a basis on which to begin the development of nonthermal death models. This will require incorporation of phenotypic, adaptive responses and may be influenced by factors such as the sequence in which environmental constraints are applied. A recurrent theme is that probability (stochastic) approaches are required to complement or replace kinetic models as the growth/no growth interface is approached and microorganisms adopt a survival rather than growth mode. Attention is also drawn to the interfaces of predictive microbiology with microbial physiology, information technology and food safety initiatives such as HACCP and risk assessment. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Predictive microbiology; History and process; Growth/no growth interface; Microbial physiology; IT and food safety

1. Introduction Methods of food preservation such as salting, drying and fermentation have been carried out for thousands of years representing an empirical approach to the control of microbial populations in food. These processes continue to this day amongst indigenous populations, and, one suspects, with traditional products produced in more sophisticated societies. Early examples of the application of scientific principles to food preservation include Pasteurs work
* Corresponding author. Tel.: +61-362-262636; fax: +61-362262642. E-mail address: Tom.McMeekin@utas.edu.au (T.A. McMeekin).

on the specificity of undesirable fermentations in wine and the supply of lactic starter cultures by Hansens in Denmark at the end of the 19th century. However, while much of the fermentation industry (probably because of large-scale production and the influence of chemical engineers) has adopted quantitative approaches, large parts of food microbiology have remained essentially qualitative or at best semiquantitative. Thus shake and plate techniques allow enumeration to within F 0.5 log, with minimum levels of detection as high as 100 cfu/g. Furthermore, most probable number techniques often have very wide confidence limits and enrichment procedures allow the presence (not necessarily the absence) of a particular organism to be recorded in a sample. The sample, of course, may be totally inadequate to

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 6 6 3 - 8

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provide a true representation of the prevalence (probability of occurrence) of the organism in the product lot, let alone a numerical indication of its density. This qualitative/semiquantitative state of affairs will continue to impede the progress of food microbiology as a discipline that seeks to understand microbial behaviour in foods and thus provide the scientific basis upon which the food industry can supply safe and wholesome food. The situation is encapsulated by a quotation from the renowned physicist Lord Kelvin: When you can measure what you are speaking about and express it in numbers you know something about it; but when you cannot measure it, when you cannot express it in numbers, your knowledge is of a meagre and unsatisfactory kind. In defence of food (and probably other) microbiologists, deriving the quantitative laws of physics that govern the natural world was an exercise not unduly complicated by variability inherent in biological systems and uncertainty about the presence and potential of particular microorganisms. In many senses, the uncertainty factor is increasing in a world characterised by condensation, stratification and mobility of the human population. We are experiencing an unprecedented rate of change as a result of scientific and technological advances. Adaptation to and exploitation of change is a primary characteristic of microorganisms that, because of their small size, speed of reproduction, phenotypic plasticity and genetic promiscuity, colonise almost every conceivable habitat on earth. Thus, it is not surprising that we are faced with the emergence and reemergence of foodborne microbial pathogens (Lederberg, 1997). Strategies to deal with these threats range from reactive measures in which resources are mobilised rapidly to address critical knowledge gaps to longer term strategic measures. This longer term research is needed to improve our ability to respond quickly to new microbial threats and assist us to be more proactive at anticipating and preventing emergence (Buchanan, 1997). Important elements of a proactive approach are the accumulation of quantitative information on microbial behaviour in foods (predictive microbiology) and an increased understanding of microbial physiology (McMeekin et al., 1997). Predictive microbiology (the quantitative microbial ecology of foods) has, after a considerable gestation

period, emerged strongly as an essential element of modern food microbiology. This contribution will consider the development of predictive microbiology with particular reference to interfaces. There are several connotations of interface that not only describe boundaries of scientific interest such as the growth/no growth interface but also interfaces between disciplines that have led to significant conceptual and technological advances.

2. Concept and history of predictive microbiology The concept of predictive microbiology is that a detailed knowledge of microbial responses to environmental conditions enables objective evaluation of the effect of processing, distribution and storage operations on the microbiological safety and quality of foods. It involves the accumulation of knowledge on microbial behaviour in foods and its distillation into mathematical models. Application of this condensed knowledge is through devices that store and match the information with the environmental conditions experienced by microorganisms in foods. These provide cost-effective surrogates for traditional microbiological testing to estimate shelf life and safety and, when properly constructed and applied, predictive models may be viewed as potentially the ultimate rapid method. As indicated in the Introduction, methods for the preservation of foods have been practised for thousands of years and with the passage of time, many preservation methods have been characterised and their scientific basis determined. An early example of a predictive model is found in the thermal processing of foods where a heat process sufficient to destroy 1012 spores of Clostridium botulinum type A is used. The process is characterised by a predictive model developed by Esty and Meyer (1922) and despite potential complications of shoulders and tails, the efficacy of the process is widely accepted by the canning industry. In large part, this arises from the magnitude of the safety factor built into the model. Heat processes have also been determined to ensure the thermal destruction of nonspore-forming organisms such as milk pasteurisation protocols for Mycobacterium tuberculosis and more recently for

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Salmonella in roast beef and psychrotropic pathogens in sous-vide products. Where protocols are selected to minimise processing and the safety factor is reduced, more rigorous selection and validation of models is required. In the case of thermal processes, oversimplifying description of the response on the basis of log linear kinetics may be inappropriate and the effect of shoulders and tails requires consideration. When nonthermal constraints are proposed to reduce numbers of low infective dose pathogens through several log cycles, such as in meat fermentations, an exact description of pathogen behaviour including variability in response, must be incorporated in the model if it is to be used with certainty. While the botulinum cook may have been the first predictive model (albeit only recently recognised as such) to find widespread utility in the food industry, reference to the potential use of predictive microbiology to describe microbial growth can be found in the 1930s when Scott (1937) wrote: A knowledge of the rates of growth of certain microorganisms at different temperatures is essential to studies of the spoilage of chilled beef. Having these data it should be possible to predict the relative influence on spoilage exerted by the various organisms at each storage temperature. Further, it would be possible to predict the possible extent of the changes in populations that various organisms may undergo during the initial cooling off of the sides of beef in the meatworks when the meat surfaces are frequently at temperatures very favourable to microbial proliferation. Clearly, Scott understood the potential to use accumulated kinetic data on microbial growth responses to predict the shelf life and safety of foods. Despite being unable to realise its full potential due to lack of computing power, it is salient to note that Scotts work on the effects of temperature, water activity and CO2 concentration enabled shipments of nonfrozen beef carcasses and quarters from the antipodes to the UKan early example of the Hurdle Concept in action. The literature remained relatively silent on predictive microbiology until the 1960s and 1970s when manuscripts began to appear addressing food spoilage and food poisoning problems. The former issues were investigated using kinetic models (Spencer and Baines, 1964; Nixon, 1971; Olley and Ratkowsky,

1973a,b). An important theme in any scientific discipline is the recognition of overarching similarities as a first step in the development of a mechanistic understanding of the process involved. In investigating the microbial spoilage of fish, Olley and Ratkowsky (1973a,b) recognised the fundamental similarity of the response to temperature of many spoilage processes and proposed a universal spoilage curve. From this curve, these authors conceived the relative rate concept that has become the keystone in the application of predictive models and a forerunner to variants such as the gamma concept (Zwietering et al., 1996). The second area of research in predictive microbiology in the 1970s that dealt with prevention of botulism and other microbial intoxications was based on probability models (Genigeorgis, 1981; Roberts et al., 1981). The 1980s saw a marked increase in interest in predictive microbiology as a result of major food poisoning outbreaks and consequent public (and political) awareness of the requirement for a safe and wholesome food supply. Both traditional pathogens and foods (Salmonella in eggs) and emerging pathogens (Listeria monocytogenes) with unusual characteristics (psychrotrophy) contributed to the prioritisation of food safety research by governments in the USA, UK, other EU countries and Australia and New Zealand. Through the 1980s and a large part of the 1990s, kinetic modelling approaches dominated the predictive microbiology scene, but more recently, a return to probability modelling has been evident. This trend can be attributed to the following. (i) Recognition that variability in response time (generation time and lag phase duration) estimates are not normally distributed but are usually described by a gamma or even inverse Gaussian distribution where response time variance is proportional to the square or the cube of the mean response time (Ratkowsky et al., 1996). (ii) Emergence of dangerous pathogens (particularly Escherichia coli 0157:H7) with very low infective doses where the knowledge base required description of conditions to prevent their proliferation or which lead to their inactivation. (iii) Increased awareness of stochastic approaches as a result of quantitative microbial risk assessment studies.

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3. The modelling process Predictive microbiology is concerned with the accumulation and synthesis of knowledge. Ideally, all published, or otherwise archived knowledge on microbial behaviour in foods should be accessible to and accessed by researchers wishing to confirm or advance the state of knowledge. Clearly, this is not always (perhaps not often) the case and numerous instances can be cited where old papers were not located in literature searches, for example, square root-type temperature dependence models (Ratkowsky et al., 1982, 1983) were originally dek (1926). proposed as a power function by Belehra This oversight was subsequently corrected with a dek in the special tribute to Professor Jan Belehra Preface to the monograph Predictive Microbiology: Theory and Application (McMeekin et al., 1993). Researchers and funding agencies should be aware, despite the limitations of some publications, of the cost benefits available from judicious use of published work rather than total reliance on de novo generation of data (Ross, 1999a). Optimum benefit is derived when the retrieved knowledge is thoroughly and systematically analysed to exclude that which is patently erroneous or has been misinterpreted. Lack of critical evaluation during the review process or when citing references may lead to legitimising concepts and procedures that will subsequently inhibit the acceptance of predictive microbiology as an effective and reliable procedure to judge the microbial safety and quality of foods. Ross et al. (2000) in considering the theory and philosophy of mathematical modelling drew attention to the competing claims of empirical and mechanistic models. The former are pragmatic in nature and describe the data in a useful mathematical relationship. On the other hand, mechanistic models are derived from a theoretical basis, provide interpretation of the response observed in terms of the underlying mechanisms and are more amenable to refinement as knowledge of the system increases. Among the models commonly used in predictive microbiology, none are purely mechanistic, many have some underlying basis and some are simply curve-fitting exercises that at the extreme are unique to the data used to generate the model.

During the kinetic modelling boom of the 1980s, two major modelling approaches were used. (i) Models based on the sequential determination of the effect of individual factors or growth rates, for example, a square root or Arrhenius model for temperature dependence to which terms for water activity, pH etc. were added. Characteristically, the experimental methods involved close interval determinations for each environmental factor tested. (ii) Polynomial models based on response surface methodology where experiments usually involved simultaneous determination of the effects of several factors on microbial behaviour. The selection of the variables on the response surface was often determined by a central composite experimental design. This suffered from an inability to determine adequately the effect of sufficient factor combinations across the entire multidimensional surface under consideration, particularly at the edges. While both approaches are empirical, proponents of the former argue that they contain parameters with biological relevance, whereas response surface, polynomial models represent a black box. In these, biological significance is hidden and cross product and other terms that are needed to describe responses may make the model a unique description of the data set used for its generation. Recent trends indicating increased use of computational neural networks advance the black box approach and may inhibit the search for mechanistic and biologically relevant models. Ross et al. (2000) also commented on aspects of practical model building including the range of characteristics investigated (growth, death, survival, toxin formation) and the variables modelled that often include temperature, water activity, pH, nitrite concentration and gaseous atmosphere, and on occasions, organic acid or other preservative concentrations. The sequential process adopted in modelling usually consists of developing a primary model to determine the magnitude of the responses of interest such as maximum specific growth rate, lag phase duration, time to reach a specified level (cell numbers or metabolites) or death rate. A secondary model is then constructed to show the dependence of these factors on environmental conditions, and on occasions, the algorithm is incorporated into computer software packages to generate a tertiary model.

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At various stages within national predictive microbiology projects, for example, the UK MAFF Food Micromodel Program, quality assurance committees were established to prescribe the minimum standards for model development and validation. Several authors have also defined experimental protocols (e.g. McMeekin et al., 1993). Perhaps the occasion of the 3rd International Conference on Predictive Microbiology should reactivate the need to develop minimum experimental standards. Bacterial taxonomists prescribe the minimum amount of experimental data required to describe a new bacterial species. A similar set of guidelines by experts in predictive modelling may allow reviewers and editors to examine more closely journal submissions purporting to contain predictive models.

4. Towards the growth/no growth interface Monod (1949) in his classic review The Growth of Bacterial Cultures stated that The growth of bacterial cultures, despite the immense complexity of the phenomena to which it testifies, generally obeys relatively simple laws which make it possible to define certain quantitative characteristics of the growth cycle, essentially the three growth constants: total growth (G), exponential growth rate (R) and growth lag (L). That these definitions are not purely arbitrary and do correspond to physiologically distinct elements of the growth cycle is shown by the fact that, under appropriately chosen conditions, the value of any one of the three constraints may change widely without the other two being significantly altered. The accuracy, the ease, the reproducibility of bacterial growth constant determinations is remarkable and probably unparalleled so far as biological quantitative characteristic are concerned. Such an opinion from a Nobel Laureate would have provided encouragement for early proponents of predictive microbiology for whom a basic premise was that the responses of microbial populations to environmental factors are reproducible. Clearly, without reproducible responses it would not be possible, from past observations, to predict future behaviour. However, Monod (1949) was primarily concerned with defining the general shape of the bacterial growth curve (a primary model) for organisms growing under

well-controlled laboratory conditions including studies of diauxie in which a series of growth phases could be induced. His review did not extend to analysing the effect of environmental factors on the magnitude of the three variables (secondary models) although this possibility was foreshadowed: Under certain specific conditions quantitative interpretations of the primary effect of the agent studied may even be possible. As we are now aware from predictive modelling studies, growth rates under conditions that permit rapid population development are remarkably reproducible. We are also aware that estimates of lag phase duration show greater variability and that as a population experiences progressively harsher conditions, response times become longer and variability increases markedly. Recognition of this variability and its characterisation by a particular distribution was an important step in the development of predictive modelling (Ratkowsky et al., 1996). These observations are entirely consistent with the general rule that biological processes display variability that must be characterised if the ecology of organisms in any environment is to be understood. Thus, as a microbial population moves progressively towards conditions that will eventually preclude growth (the growth/no growth interface), the ability of kinetic models to provide an accurate description becomes increasingly limited. An appropriate strategy in this circumstance is to select a response time consistent with the severity of the microbial hazard and estimate the probability that the population will respond more quickly than the selected level (McMeekin et al., 1993).

5. At the growth/no growth interface A consistent observation from many studies in predictive microbiology is that there is a minimum finite rate of growth beyond which population development does not occur even with markedly extended periods of incubation. This boundary may be set by a single factor such as temperature or a combination of factors, for example, temperature, water activity, pH etc. Generally, when more than one factor constrains population development, the absolute level of each factor required to prevent growth is lessened. This is the essence of the Hurdle Concept advocated for many years by Professor Leistner and his colleagues

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at the Federal Centre for Meat Research in Kulmbach, Germany (Leistner, 1978, 1992). The Hurdle Concept seeks to determine the minimum set of conditions to prevent the growth of pathogens, or better still, to cause inactivation. The goal is to produce foods that can remain stable and safe (even without refrigeration) and are acceptable organoleptically and nutritionally due to the mild processes applied. A commonly used analogy, introduced by Dr. M. Cole, to describe that set of minimal processing conditions is the Food Safety Cliff. The cliff edge represents those sets of product formulations beyond which foods are potentially unsafe and the top of the cliff represents the set of conditions that just prevent pathogenic microorganisms from growing. At a long distance from the cliff edge, there is great certainty that the food will be safe as pathogens have been eliminated or are unable to grow. That confidence decreases markedly as the cliff edge is approached. Conditions distant from the cliff edge represent highly processed foods, those nearer the edge are more natural products including minimally processed foods. Thus, knowledge of the position of the cliff edge can be used to design foods that just prevent microbial growth. This knowledge will be invaluable in optimising the amount and stringency of processing so that the aesthetic changes to the quality of the food are minimised. Further, while the Hurdle Concept is widely accepted as a food preservation strategy, its potential has not been fully realised as it is a largely qualitative concept, the application of which is often empirical. The intelligent selection of hurdles in terms of the number required, the intensity of each and the sequence of application to achieve a specified outcome provides significant potential to approach the edge of the food safety cliff with certainty. There are many possible physiological explanations to explain the cessation of growth including denaturation of ribosomes, membrane lipid phase changes, and energy diversion to deal with environmental insults to the point where insufficient energy remains to fuel biosynthetic processes (Knochel and Gould, 1995). While the prospect of a new generation of mild but effective processing techniques will require a thorough understanding of microbial physiology, considerable advances can be achieved by modelling the growth/no growth interface. In effect,

this type of modelling quantifies the hurdle concept. Early attempts to define nongrowth conditions were based on + or observations (Christian and Waltho, 1962) with some of these studies used to develop probability models for growth. A more systematic approach to interface modelling was reported by Ratkowsky and Ross (1995) to predict the growth/ no growth interface for Shigella flexneri as affected by temperature, pH, water activity and nitrite concentration. The procedure involved modifying a growth rate model by taking the logarithm of both sides of the equation and replacing the left-hand side with a logit term (logit p), where p is the probability of growth occurring. This or similar approaches were subsequently used by Presser et al. (1998) for E. coli, Bolton and Frank (1999) for L. monocytogenes, Ross (1999a) for Klebsiella oxytoca, Salter et al. (2000) for E. coli and Tiengunoon et al. (in press) for L. monocytogenes.

6. The lag phase and fluctuating conditions The growth/no growth interface represents a boundary at which the growth rate is zero and the lag phase is infinite. Probably more than any other factor, accurate determination of the lag phase has created problems for predictive microbiologists. Indeed in many practical applications of predictive models such as the hygienic assessment of meat processing operations (e.g. Gill et al., 1991), the lag phase is ignored. The great difficulty is that cells contaminating a food product range in physiological competence from those that are actively dividing, to those that display a physiological lag phase, to those that are damaged and require repair before resolving lag, to those that have entered a state of suspended animation (viable but nonculturable), to those that are dead. Further modelling complications may arise when fluctuations in environmental conditions are of sufficient magnitude or rapidity to induce a population that has resolved its lag phase to once again enter one of the lag states listed above. The duration of the phase is affected by factors such as the identity and phenotype of the bacterium (Buchanan and Cygnarowicz, 1990), inoculum size (Baranyi and Roberts, 1994), the physiological history

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of the population (McMeekin et al., 1993) and by changes in the physiochemical environment such as temperature (Zwietering et al., 1994), pH, water activity and nutrient availability (Buchanan and Cygnarowicz, 1990). While studying the effect of temperature shifts and fluctuations on growth rate, several authors have noted effects on the lag phase (Walker et al., 1990; Fu et al., 1991; Duh and Schaffner, 1993; Blackburn et al., 1999). It has often and Davies, 1994; Membre been observed that lag time is inversely proportional to the maximum specific growth rate (Smith, 1985; McMeekin et al., 1993; Baranyi and Roberts, 1994). The time required to resolve the growth lag depends on the growth rate of the organism, which is dictated by the growth environment. Lag time duration has often been considered erratic and evaluation of predictive models has shown that lag times are less reliably predicted than generation times. This has usually been attributed to the effect of the prior history of cells on the duration of the lag time. Robinson et al. (1998) formalised a concept of the lag time as being dictated by two elements: (i) the amount of work required of the cell to adjust to a new environment and/or repair injury due to the shift to the new environment, and (ii) the rate at which those repairs and adjustments can be made. The latter is presumed to respond to the environment in the same way, relatively, that generation time does (i.e. if the environment causes the generation time to double, the lag time will also double). In effect, physiological lag times before exponential growth commences reduce the potential growth of an organism during a given period of time. The potential exists to force an organism into a long lag by manipulation of mild environmental change. More severe environmental changes may lead to cell injury or even death. In situations characterised by variability and uncertainty, the development of good mechanistic models is impossible and of good empirical kinetic models improbable. As was the case with kinetic growth models near the growth/no growth interface, the adoption of a stochastic or probability has proved to be an effective option (Ross, 1999a). This work demonstrated that the apparent variability in lag phase duration can be reduced by introducing the concept of

relative lag times (RLTs) or generation time equivalents, that is, the ratio of lag time to generation time. Using this approach, a common pattern of distribution of RLTs for a wide range of species across a wide range of conditions has emerged. The introduction of the stochastic modelling approach to describe lag phase duration will have a profound effect on the application of predictive microbiology allowing operators to move away from the worst-case scenario. We also foreshadow that the stochastic procedures now widely used in microbial risk assessments will find considerable utility in the description of specific food-processing operations.

7. Beyond the growth/no growth interface Beyond the growth/no growth interface, rapid death induced by thermal energy or irradiation has been relatively well characterised. However, patterns of nonthermal death are less well established and, as was the case with growth kinetics, slow rates of decline are likely to display considerable variability. The general pattern observed when death is induced by low water activity conditions is a rapid phase followed a more gradual phase of decline. Generally, the magnitude of the rapid phase increases with the severity of the challenge, but thereafter, the rate of decline proceeds at approximately the same rate. Under many circumstances, complete extinction of the population is not achieved. With low pHinduced death, a third more rapid decline phase is observed that may be attributed to energy depletion as a result of proton pumping to maintain cytoplasmic pH homeostasis. The importance of energy status is also seen in markedly different responses to the sequence of water activity and pH challenges (Shadbolt et al., 2001). Reduced water activity followed by reduced pH leads to a gradual decline, whereas the reverse sequence causes rapid death on application of the second hurdle. Again this may be explained by postulating that the initial acid challenge depletes the cells energy reserves to the point where it is unable to deal with a subsequent water activity challenge. Conversely, the less energetically demanding water activity constraint (Krist et al, 1998a) when applied first allows the cell

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to retain sufficient energy reserves to deal more effectively with a subsequent acid challenge.

8. The interface between predictive microbiology and microbial physiology Monod (1949) was also aware that the quantitative description of microbial population behaviour (ecology) was inextricably linked to the underlying physiology of the cell when he wrote: There is little doubt that as a further advances are made towards a more integrated picture of cell physiology, the determination of growth constants will have a much greater place in the arsenal of microbiology. Further he counselled The fallacy of considering certain naive mechanistic schemes, however, as appropriate interpretations of unknown, complex phenomena should be avoided. The former statement reflects the development of predictive microbiology in which the patterns of responses observed provide clues to underlying physiological events in the cell. As an example, consider a typical Arrhenius plot of bacterial growth. Theory predicts that a plot of the natural logarithm of growth rate versus the reciprocal of absolute temperature will yield a linear relationship, the slope of which is the apparent energy of the reaction. While this is true for simple chemical reactions, complex biological phenomena such as microbial growth display continually downward sloping curves. On occasions, these were interpreted as representing two linear regions with different activation energies (Mohr and Krawiec, 1980) where it is tempting to suggest that the discontinuity indicates a major physiological change (e.g. membrane lipid phase change) in the cell: perhaps an example of Monods fallacy of a naive mechanistic scheme. What can be deduced from a typical Arrhenius plot for bacterial growth is that there is a normal physiological range where Arrhenius kinetics provide a reasonable description of the observed response and where a constant activation energy is appropriate. Beyond that, region activation energy estimates change continually: in the high-temperature region due to irreversible denaturation and in the low-temperature region due to reversible denaturation of macromolecules.

An alternative description of the effect of temperature on microbial growth rates was provided by dek (1926) and revived by Ratkowsky et al. Belehra (1982) as the square root model. In this model, a square root transformation of data is preferred to a logarithmic transformation resulting in a linear response that is extrapolated to the theoretical minimum temperature for growth (Tmin), that is, where the regression line intersects the temperature axis of a square root plot. dek (1930) was dismissive of the applicaBelehra tion of chemical kinetics to biological processes when he wrote: The problem of temperature coefficients in biology was initiated by chemists and has suffered from the beginning from this circumstance. Attempts to apply chemical temperature velocity formulae (the Q10 rule and the Vant Hoff Arrhenius law) to biological processes failed because some of the temperature constants used in chemistry (Q10, m) can be said not to hold good in biological reactions. Neverthe dek-type kinetics provide a good fit less, while Belehra to data, a square root plot is perhaps less informative of the changing energy demands outside the normal physiological range that can be deduced from an Arrhenius plot. Observed patterns of response to lowered water activity and pH also provide clues to the mechanisms by which these hurdles affect the microbial cell. As an example, the constants of the microbial growth curve respond consistently to decreasing water activity levels viz.: the growth rate constant decreases, lag phase duration increases and cell yield remains constant until near the limiting level for growth. At this point, a marked reduction in yield is observed. When the information contained in the primary model is transformed into a secondary model, the optimum water activity is revealed together with a linear response to decreasing water activity in the suboptimal range (e.g. see Troller and Christian, 1978 for S. aureus and Krist et al., 1998a for E. coli). When experiments are carried out with different humectants, specific solute effects are also noted (Troller and Christian, 1978). Further, as water activity conditions become progressively harsher, a common observation is that the minimum temperature for growth increases (e.g. McMeekin et al., 1987). This observation could be explained by invoking an energy diversion hypothesis

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(Csonka, 1989; Knochel and Gould, 1995). That is, energy required to deal with the water activity hurdle is unavailable to overcome the concurrent temperature barrier to growth, and as a result, the minimum temperature for growth must increase. While superficially attractive, this explanation is not consistent with the primary water activity/cell yield response that indicates that over a wide water activity range, all available substrate is converted into approximately the same amount of biomass (Krist et al., 1998a). A similar observation is made for temperature/cell yield responses, and for both constraints, the growth rate constant declines sequentially as temperature and water activity decrease in the suboptimal region. Conversely, at lowered pH levels, cell yield declines progressively but the growth rate constant is maintained across a wide pH range (Krist et al., 1998a). This response reflects the well-documented requirement for cells to maintain a constant internal pH consistent with efficient operation of enzymes and the need to expend energy pumping protons from the cytoplasm. The mechanism of microbial response to low-water activity stress involves the synthesis or accumulation of compatible solutes. These compounds stabilise enzyme structure and maintain them in an active configuration. Compatible solutes are also known to confer protective effects against low-temperature stress (Ko et al., 1994), an observation consistent with the similar yield and growth rate constant responses observed at reduced temperatures and at reduced water activities. While Krist et al. (1998a) discounted major energy diversion as a growth-limiting mechanism at lowered water activity, the concept of a critical activation energy was proposed (Krist et al., 1998b). This hypothesis was derived from observations on E. coli grown without water activity limitation (0.997) and under stressful conditions (0.977) with and without the compatible solute, glycine betaine. The normal physiological temperature range was reduced by appropriately 50% at aw = 0.977 compared with aw = 0.997 but by approximately 25% at aw = 0.977 in the presence of glycine betaine. The observed minimum temperature for growth at aw = 0.977 was 25.8 C from which a critical activation energy of 178 kJ/mol was computed using the Arrhenius-based ther-

modynamic model of Ross (1999b). Applying this critical activation energy value to the other conditions, a minimum growth temperature of 12.1 C was calculated at aw = 0.997 and 17.8 C at aw = 0.977 plus glycine betaine. Both values were close to ( f 1 C) of the observed minimum temperature. Above we described a method to model the growth/no growth interface that is characterised by a sharp delineation between growth and no growth conditions. A physiological explanation for this observation may be embodied in the concept of a critical activation energy for growth. In the case of water activity and temperature hurdles, this may reflect the degree of enzyme unfolding that is ameliorated in the presence of compatible solutes. It would therefore be interesting to compare the stability of selected enzymes at the same critical activation energy achieved by various water activity/temperature combinations in the presence/absence of compatible solutes. Furthermore, the critical activation energy hypothesis should be extended by measurement of the ATP expenditure required to maintain internal pH homeostasis. When considering survival and death kinetics, it is important also to take into account the physiological state of the organism and adaptive responses that enhance resistance to unfavourable conditions. The phenomenon of acid habituation provides a good example of the phenotypic plasticity of microorganisms. Brown et al. (1997) provided, in part, a physiological explanation for increased acid tolerance in response to mild stress by characterising an increase in the level of cyclopropane fatty acids in the cell membrane. Production of these compounds is energetically expensive (three ATPs per mole synthesised) and membrane composition and acid tolerance return to approximately that of unstressed cells when the acid constraint is removed. While an increase in cyclopropane fatty acids per se may not be responsible directly for increased acid tolerance, it is of interest to note that in acidophiles such as Thiobacillus, > 60% of membrane lipid fatty acids are of the cyclopropane variety (Levin, 1971). In keeping with the interface theme of this paper, it is clear that the bacterial cell membrane is a crucial interface between the cell and the surrounding environment. In relation to growth at reduced pH levels,

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active transport of protons across the membrane is essential and for survival rearrangement of membrane fatty acid composition appears to have a central role. Advances in instrumentation that can monitor membrane-related events in real time offer the prospect of increased understanding of the physiological processes that underlie the patterns of growth and death embodied in predictive models. Examples include fluorescence ratio imaging techniques that allow real time observation of changes in internal pH in response to environmental insults (Siegumfeldt et al., 2000) and the MIFEk System that provides direct measurement of ion fluxes across cell membranes. Originally designed for use with plants cells (Shabala et al., 1997; Shabala, 2000), the MIFEk System has recently been adapted for use with large microbial cells (Shabala et al., 2001b) and microbial films deposited or grown on glass surfaces (Shabala et al., 2001a). When used in tandem, fluorescence ratio imaging and the MIFEk System will provide a powerful combination to assess the efficacy of protocols to disturb intracellular homeostasis and the role of membrane transport systems in maintaining homeostasis. Together, the techniques have the potential to provide information that will be the basis of a new generation of mild but effective food preservation procedures. The speed at which information on the efficacy of antimicrobial combinations will accumulate will be measured in hours rather than days or weeks required using conventional microbiology. As an example, using the MIFEk System, we have demonstrated in 2 3 h that proton efflux from a Thraustochytrid sp. ceases at 8 C. Collapse of this essential physiological process presumably correlates with crystallisation of the cell membrane and effectively indicates the minimum temperature for growth of the organism. By contrast, temperature gradient incubator experiments to determine the minimum temperature for growth can continue for up to 90 days.

9. The interface with information technology Although Scott (1937) had devised the concept of predictive microbiology, in reality, the development of predictive models was constrained until the advent of the computer and information technology age. Sim-

ilarly, the application of predictive models is largely through the use of information technology. Importantly, this resource allows the continual accumulation of knowledge and, as a consequence, should lead to development of better models and greater scope for their application. As an example, consider the work of Gill et al. (e.g. Gill et al., 1991) on modelling the hygienic efficacy of meat processing operations. Gills original model was based only on the temperature response of E. coli using a limited data set. Application using the Delphik temperature logging system relied solely on temperature history information and ignored other effects (such as water activity) known to limit microbial growth on meat carcasses during chilling. This is a typical worst-case scenario but nevertheless led to a useful concept, the process hygiene index (PHI), based on the potential growth of E. coli at the slowest cooling point of the carcass. The criteria for the threeclass sampling plan devised as a decision support system for the PHI were derived on the basis of cooling regimes known to produce meat of adequate hygienic quality. From this beginning, Gill and his colleagues (Gill et al., 1991) in New Zealand and Canada developed PHI recommendations for spraychilled carcasses, hot boned product, offal handling and the transport and distribution of meat. Models for E. coli growth developed subsequently are based on much more extensive data sets and include the effect of water activity, pH and lactate concentration (Presser et al., 1997). This emphasises that knowledge is cumulative, can be stored, retrieved and interpreted and provides greater precision in describing the quantitative microbial ecology of foods. The accumulation of knowledge is also the cornerstone of quantitative microbial risk assessment. This procedure has been trialed as a measure of the hygienic equivalence of foods in international trade and involves hazard identification, exposure assessment, dose response assessment and risk characterisation. To date, most microbial risk assessments have been big picture attempts to quantify the risk of disease arising from certain microorganism/food combinations (Cassin et al., 1998). While these may be valuable in a comparative sense and indicate where knowledge is lacking, they are always characterised by variability and uncertainty. The latter category

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places a particular constraint on achieving a definitive outcome from a quantitative risk assessment. If uncertainty is removed (and variability minimised), the stochastic approaches embodied in risk assessment techniques should have the potential to characterise the microbiological consequences of a food-processing operation. To test this hypothesis, we selected production of fresh salmon fillets and attempted to define those factors mainly responsible for limiting shelf life (Rasmussen et al., 2001). Characterisation of the harvesting and processing operations allowed development of a process risk model to quantify the risk that the stated shelf life will not be achieved. Bacterial numbers in water and ice and on fish and contact surfaces were collected over a period of 9 months and fitted to distribution functions. The model constructed using Analytica 2.0 predicted mean ice slurry water counts of log 3.36/ml (observed 3.35/ml), fish surface contamination levels to be log 3.31/ml (observed log 3.23/ml). The average predicted shelf life at 4 C was 6.5 days (observed 6.2 days). An importance analysis carried out on the model using Analytica demonstrated that storage temperature had a much greater influence on shelf life than contamination levels. This demonstrates clearly that when sufficient information is available and uncertainty is eliminated, a stochastic approach can provide an accurate microbial profile of a specific processing operation. We predict that stochastic modelling packages such as @ Risk and Analytica will be used increasingly to characterise food-processing operations and to suggest effective strategies to achieve food safety and/or food quality objectives.

developing food safety objectives but cannot occur effectively without quantitative information. Predictive microbiology assists the formulation of HACCP plans by identifying hazards and critical control points and in specifying limits and corrective action (Miles and Ross, 1999). With QMRA, predictive models have a particular role as a cost-effective means to provide the exposure assessment information, a critical element in risk assessment.

11. Conclusions Microbial food safety is of concern to industry, government and the population at large with each group anticipating the provision of a safe and wholesome food supply as a basic tenet of a developed society. It is clear that predictive microbiology has a major role to play in meeting this aspiration and that already it has become an essential element of modern food microbiology. This status has been achieved by continually improving our understanding of the quantitative microbial ecology of foods and by developing interfaces with other disciplines to apply the knowledge contained in predictive models. Its utility will be further enhanced when predictive microbiology is recognised as an effective rapid method. We foreshadow that consolidation of existing and development of new interfaces will lead to acceptance of predictive microbiology as a mature subdiscipline of microbiology. In particular, alignment of quantitative knowledge of microbial behaviour in foods with understanding of underlying physiological processes offers the prospect of a new generation of mild, but effective, food preservation procedures.

10. The interface with food safety initiatives Predictive microbiology through interfaces with many other disciplines has emerged as a paradigm of modern food microbiology. It provides a scientific basis to underpin the HACCP concept and quantitative microbial risk assessment. A dynamic interaction exists between HACCP (the tool by which safety is built into food-processing operations) and risk assessment (a measure of the effectiveness of HACCP on other safety assurance programs). This interaction may be facilitated by Acknowledgements The authors thank the many postgraduate students and colleagues for contributions to predictive microbiology research at the University of Tasmania over a period of 25 years. We particularly acknowledge the continuing financial support of Meat and Livestock Australia and assistance from the Department of Industry Science and Resources to attend the 3rd International Conference on Predictive Modelling in

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T.A. McMeekin et al. / International Journal of Food Microbiology 73 (2002) 395407 Krist, K., Ross, T., McMeekin, T.A., 1998a. Final optical density and growth rate: effects of temperature and NaCl differs from acidity. Int. J. Food Microbiol. 43, 195 204. Krist, K., Ross, T., Olley, J., 1998b. A mechanism for the limitation of microbial growth by temperature and water activity. Proc. Poster Sessions, ISOPOW 7, May June 1998, Helsinki, Finland. Hakapaino Oy, Helsinki, pp. 37 41. Lederberg, J., 1997. Infectious disease as an evolutionary paradigm. Emerging Infect. Dis. 3, 417 423. Leistner, L., 1978. Hurdle effect and energy saving. In: Downey, W.K. (Ed.), Food Quality and Nutrition. Applied Science Publ., London, pp. 553 557. Leistner, L., 1992. Food Preservation by combined methods. Food Res. Int. 25, 151 158. Levin, R.A., 1971. Fatty acids of Thiobacillus thioxidans. J. Bacteriol. 108, 992 995. McMeekin, T.A., Chandler, R.E., Doe, P.E., Garland, C.D., Olley, J., Putro, S., Ratkowsky, D.A., 1987. Model for the combined effect of temperature and water activity on the growth rate of Staphylococcus xylosus. J. Appl. Bacteriol. 62, 543 550. McMeekin, T.A., Olley, J., Ross, T., Ratkowsky, D.A., 1993. Predictive Microbiology: Theory and Application. Research Studies Press, Taunton, UK. McMeekin, T.A., Brown, J.L., Krist, K., Miles, D., Nuemeyer, K., Nichols, D.S., Olley, J., Presser, K., Ratkowsky, D.A., Ross, T., Salter, M., Soontranon, S., 1997. Quantitative microbiology: a basis for food safety. Emerging Infect. Dis. 3, 541 549. , J.-M., Ross, T., McMeekin, T.A., 1999. Behaviour of Membre Listeria monocytogenes under combined chilling processes. Lett. Appl. Microbiol. 28, 216 220. Miles, D.W., Ross, T., 1999. Identifying and quantifying risks in the food production chain. Food Aust. 51, 298 303. Mohr, P.W., Krawiec, S., 1980. Temperature characteristics and Arrhenius plots for nominal psychrophiles, mesophiles and thermophiles. J. Gen. Microbiol. 121, 311 317. Monod, J., 1949. The growth of bacterial cultures. Annu. Rev. Microbiol. 3, 371 394. Nixon, P.A., 1971. Temperature integration as a means of assessing storage conditions. Report on Quality in Fish Products, Seminar No. 3. Fishing Industry Board, Wellington, New Zealand, pp. 34 44. Olley, J., Ratkowsky, D.A., 1973a. Temperature function integration and its importance in storage and distribution of flesh foods above the freezing point. Food Technol. Aust. 25, 66 73. Olley, J., Ratkowsky, D.A., 1973b. The role of temperature integration in monitoring fish spoilage. Food Technol. N. Z. 8, 147 153. Presser, K., Ratkowsky, D.A., Ross, T., 1997. Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration. Appl. Environ. Microbiol. 63, 2355 2360. Presser, K., Ross, T., Ratkowsky, D.A., 1998. Modelling the growth limits (growth/no growth) interface of Escherichia coli as a function of pH, lactic acid and temperature. Appl. Environ. Microbiol. 64, 1733 1779. Rasmussen, S.K.J., Ross, T., Olley, J., McMeekin, T.A., 2001. A process risk model for the shelf life of Atlantic salmon fillets. Int. J. Food Microbiol., in press.

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