Accepted Manuscript

Use of Time Temperature Integrators in food safety management

Konstantinos P. Koutsoumanis, Maria Gougouli

PII: S0924-2244(15)00050-3
DOI: 10.1016/j.tifs.2015.02.008
Reference: TIFS 1631

To appear in: Trends in Food Science & Technology

Please cite this article as: Koutsoumanis, K.P., Gougouli, M., Use of Time Temperature Integrators in
food safety management, Trends in Food Science & Technology (2015), doi: 10.1016/j.tifs.2015.02.008.

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 ACCEPTED MANUSCRIPT

1 Use of Time Temperature Integrators in food safety management

3 Konstantinos P. Koutsoumanis* and Maria Gougouli

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5 Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology,

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6 School of Agriculture, Faculty of Agriculture, Forestry and Natural Environment, Aristotle

7 University of Thessaloniki, Thessaloniki 54124, Greece.

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9 * Corresponding author: Konstantinos P. Koutsoumanis, Aristotle University of Thessaloniki,

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Department of Food Science and Technology, School of Agriculture, Thessaloniki, Greece 541
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11 24. Phone: +30 2310991647, Fax: +30 2310991647, e-mail: kkoutsou@agro.auth.gr

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13 ABSTRACT

14 Distribution, retail and domestic storage of food are considered as the weaker links in a food

15 safety management system. Conditions during the above stages of the food chain are out of

16 manufacturer’s direct control and often deviate from specifications. This lack of control may

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17 result to increased microbial growth and negate the efforts made in improving and maintaining

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18 food safety at the earlier stages of the chain via Hazard Analysis and Critical Control Points

19 system, Good Manufacturing and Hygiene Practices. Ideally, a cost-effective way to individually

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20 monitor the temperature conditions of food products throughout distribution, retail and domestic

21 storage, indicating their real time-temperature history, would be an effective safety management

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tool. Time Temperature Integrators (TTIs) could potentially fulfill the above requirements.
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23 Despite the extensive research on the application of TTIs for monitoring food spoilage and

24 quality, however, limited information is available for their use in food safety management. The
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25 objective of the present study is to present the state of the art and application scheme of TTIs in
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26 food safety management. The effectiveness of TTIs in improving food safety is demonstrated
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27 through a case study for Listeria monocytogenes in Ready-to-Eat foods. We show that a

28 microbial TTI can assure a maximum limit in the growth of the pathogen from production to
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29 consumption time by informing the consumers when this limit is exceeded in a product unit. The

30 latter limit, which we call Growth Tolerance Criterion, can be considered as a Performance
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31 Criterion for the growth of L. monocytogenes during distribution and storage. The applicability
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32 of the microbial TTI in reducing consumer exposure to L. monocytogenes is also presented using

33 a probabilistic approach.

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35 Keywords: Time Temperature Integrators, Listeria monocytogenes, Risk Management

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36 Introduction

37 Systematic management of food safety includes raw material selection and control of

38 conditions during processing, distribution, retail and domestic storage. However, the latter are

39 the weaker links of the food supply chain. The conditions during transportation and at the retail

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40 level are out of manufacturer’s direct control and often deviate from specifications. In addition,

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41 temperature control is completely lacking during transportation from the retail stores to

42 consumer refrigerators and during domestic storage until the time of preparation and

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43 consumption. Available quantitative evidence from studies and surveys at distribution, retail and

44 domestic level illustrates the magnitude of the problem. Likar and Jevsnik (1996) studied the

45
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cold chain at retail level and reported that although the storage conditions were properly labelled
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46 on the packages of the products inspected, in most of the cases the measured temperatures

47 differed from the required ones, even for up to 10 oC. Koutsoumanis, Pavlis, Nychas and
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48 Xanthiakos (2010) reported retail storage temperature in Greece ranging from 0 to 11.7 oC with a
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49 mean of 4.98 oC, median 5.44 oC and a standard deviation of 2.90 oC. At a domestic level, the
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50 results of nine survey studies conducted in UK, France, Ireland and Greece (Anonymous, 2007;

51 Breen et al., 2006; Evans, Stanton, Russell, & James, 1991; Flynn, Blair, & McDowell, 1992; ,
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52 Johnson et al., 1998; Kennedy et al., 2005; Laguerre, & Flick, 2004; Rose, Steadman, &

53 Brunskill, 1990; Taoukis, Giannakourou, Koutsoumanis, & Bakalis, 2005; Worsfold, & Griffith,
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54 1997), with a total of 1171 consumer refrigerators tested, showed a weighted mean temperature
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55 of 6.4 o C with 64.1% of the refrigerators operating at temperature >5 oC.

56 Currently, much of the government and regulatory authority effort to reduce the risk posed

57 by foodborne pathogens has focused on the application of effective systems, such as Hazard

58 Analysis and Critical Control Points (HACCP) within the food production elements of the food

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59 chain. The lack of control during distribution, retail and domestic storage of foods, however, can

60 often negate much of the effort made in improving and maintaining food safety at the earlier

61 stages of the chain. Indeed, epidemiological data show that a significant number of foodborne

62 disease cases can be attributed to storage temperature abuse (WHO, 1996). In the case of Listeria

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63 monocytogenes, a pathogen able to grow at refrigeration storage, the available risk assessment

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64 models in Ready-To-Eat (RTE) foods predict that nearly all cases of listeriosis result from the

65 consumption of high numbers of the pathogen (FAO/WHO, 2004). These models have stressed

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66 the great impact of the growth between manufacture and consumption on the rates of listeriosis

67 and indicated that storage time and temperature during distribution, retail and domestic storage

68 are among the most important risk factors.

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69 Based on the above analysis, effective food safety management systems should rely on
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70 prevention through monitoring, recording and controlling of critical parameters during the entire

71 product’s life cycle, which includes the post-processing phase and extends to the time of use by
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72 the final consumer. Such systems require continuous monitoring and control of storage
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73 conditions, mainly of time and temperature. Time Temperature Integrators (TTIs) allow such

74 control to product unit level. TTIs can show an easily measurable, time and temperature
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75 dependent change that cumulatively reflects the time-temperature history of the food product.

76 State of the art development and studies on TTIs have shown the reliability and effectiveness of
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77 TTIs as tools for monitoring and optimizing spoilage of certain chilled foods (Giannakourou,

78 Koutsoumanis, Nychas, & Taoukis, 2001, 2005; Koutsoumanis, Giannakourou, Taoukis, &

79 Nychas, 2002; Taoukis, Koutsoumanis, & Nychas, 1999; Vaikousi, Biliaderis, & Koutsoumanis,

80 2009). The current TTI technology and the scientific approach with regards to quantitative study

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81 of safety risk in foods allow the undertaking of the next important step i.e. the application of

82 TTIs to manage safety risks of foods.

83 The objective of this study was to present the state of the art in TTIs application and

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84 demonstrate their use as food safety management tools. The methodology of TTIs’ application,

85 as a mitigation strategy to reduce safety risk, is presented through a case study for L.

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86 monocytogenes in ready-to-eat (RTE) foods using a probabilistic approach, which takes into

87 account the variability of factors affecting the concentration of the pathogen at the time of

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88 consumption. The results of the study show the effectiveness of the TTI in monitoring the growth

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89 of L. monocytogenes during distribution and storage and reducing consumer exposure to the
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90 pathogen.

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92 Time Temperature Indicators

93 A Time-Temperature Integrator or Indicator (TTI) can be defined as a simple, inexpensive

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94 device that can show an easily measurable, time-temperature dependent change that reflects the
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95 full or partial temperature history of a food product to which it is attached. Based on reliable

96 models of food microbiology and shelf life and the kinetics of TTI response, the effect of
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97 temperature can be monitored, recorded and translated from production to the consumer’s table.
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98 The principle of TTI operation is an irreversible change usually expressed as a visible response,
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99 in the form of a mechanical deformation, color development or color movement. The rate of

100 change is temperature dependent, increasing at higher temperatures similarly to most reactions.

101 The visible response, thus, gives a cumulative indication on the storage conditions that the TTI

102 has been exposed to. The extent to which this response corresponds to a real time-temperature

103 history depends on the type of the indicator and the physicochemical principles of its operation.

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104 Indicators can, therefore, be classified according to the kind of their functionality and the

105 information they convey. Different classifications and terminology have been proposed, partly

106 reflecting the evolution of the indicators (Byrne, 1976; Schoen, & Byrne, 1972; Koutsoumanis,

107 & Taoukis, 2005; Singh, & Wells, 1985; Taoukis, Fu, & Labuza, 1991; Taoukis, 2001).

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108 Taoukis and Labuza (2003) classified TTIs based on their functions into the following three

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109 categories:

110 1) Critical temperature indicators (CTI), which show exposure above (or below) a reference

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111 temperature.

112 2) Critical temperature/time integrators (CTTI), which show a response that reflects the

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cumulative time-temperature exposure above a reference critical temperature.
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114 3) Time temperature integrators or indicators (TTI), which give a continuous, temperature

115 dependent response throughout the product’s history.

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116 Another classification is based on the principle of TTI operation, which includes
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117 mechanical, chemical, electrochemical, enzymatic, microbiological or photochemical reactions.

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118 In the last twenty years various types of TTI based on the above principles have been the focus

119 of both scientific and industrial trials.

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120 The 3M MonitorMark® (3M Co., St Paul, Minnesota) (Manske, 1976) is diffusion-based

121 indicator label and rely on the color change of an oxidable chemical system controlled by
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122 temperature-dependent permeation through a film. One of the first significant applications of TTI
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123 was the use of this indicator by the World Health Organization (WHO) to monitor refrigerated

124 vaccine shipments. The Monitor Mark® Temperature Monitor and Freshness Check, based on

125 diffusion of proprietary polymer materials (Arens et al., 1997) was also developed by the same

126 manufacturer.

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127 The CheckPoint®TTI (VITSAB A. B., Malmö, Sweden) is a simple enzymatic TTI. It is

128 based on a color change caused by a pH decrease, which is the result of a controlled enzymatic

129 hydrolysis of a lipid substrate (Agerhem, & Nilsson, 1981; Blixt, Tornmarck, Juhlin, Salenstedt,

130 & Tiru, 1977). Hydrolysis of the substrate causes acid release and the pH drop is translated in a

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131 color change of the pH indicator from deep green to bright yellow. These TTI systems are

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132 claimed to have a long shelf life, if kept chilled before activation.

133 Temptime Freshness Monitor® and Fresh-Check® integrators (Temptime Indicator, Paris,

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134 France) (previously Lifelines) are based on a solid state polymerisation reaction (Fields, &

135 Prusik, 1983; Patel, Preziosi, & Baughman, 1976; Patel, & Yee, 1980). Given that these

136
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integrators are active before use, and more specifically from the time of production, they have to
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137 be stored under deep frozen, where the change is very slow. Traceo®, Traceo® Restauration and

138 eO® are three types of commercial microbial TTIs developed by Cryolog (CRYOLOG S.A.,
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139 Quimper, France), which are based on the growth and metabolic activity of patent microbial
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140 strains (http://www.cryolog.com/en/). Before use, these TTIs are stored frozen to prevent the
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141 bacterial growth, while activation is obtained simply by defrosting them for a few minutes at

142 room temperature. Once they are put on the food, the temperature-dependent growth of the TTI
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143 microorganisms causes a pH drop in the tags leading to an irreversible color change of the

144 medium chromatic indicator (Ellouze et al., 2008).

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145 The OnVuTM TTI (Ciba Specialty Chemicals & Freshpoint, SW) is a newly introduced
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146 solid state reaction TTI. It is based on photosensitive compounds, organic pigments e.g.,

147 benzylpyridines, that change color with time at rates determined by temperature. It is activated

148 by UV light. A filter is then added over the label to prevent it from being recharged. The blue

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149 inner heart changes to white as a function of time and temperature. The system can be applied as

150 a label or printed directly onto the package.

151 A recently full-history TTI which launched in the market was Keep-it® fresh (Keep-it

152 Technologies) (Keep-it Technologies, 2014; Skjervold, Salbu, Heyerdahl, & Lien, 2007). This

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153 system is based on a chemical reaction. In particular, it comprises an immobilized reactant (e.g.,

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154 Fe3+), and a mobile reactant (i.e. ferrocyanide), initially contained in separate compartments, and

155 separated by a sealing. The TTI is activated by removing the sealing between the compartments

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156 whereby the mobile reactant in a time–temperature dependent way is brought into contact with

157 the immobilized reactant resulting in a visually detectable reaction signal.

158
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In addition to the above commercial TTIs, a number of TTI prototypes have been developed
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159 in laboratory trials (Galagan, & Su, 2008; Kuswandi et al., 2012; Pereira, de Arruda, & Stefani,

160 2015; Rani, & Abraham, 2006; Vaikousi, Biliaderis, & Koutsoumanis, 2008; Wanihsuksombat,
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161 Hongtrakul, & Suppakul, 2010; Yan et al., 2008). More recently, Zabala et al. (2015) proposed a
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162 new manufacturing procedure based on roll-to-roll printing techniques of microbial TTIs.
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163 In general, for the successful application of TTIs for monitoring food quality, a requirement

164 that the TTI response matches the quality loss of the food must be fulfilled (Koutsoumanis, &
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165 Taoukis, 2005). Thus, it is necessary that the temperature dependence of the TTI response to be

166 similar to that of the food quality loss and, the TTI end point to coincide with the end of
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167 product’s shelf life. In this context, the applicability of a particular TTI as a quality indicator
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168 requires a systematic kinetic study of both the food product deterioration and the TTI response

169 (Taoukis, 2001; Taoukis, & Labuza, 2003). The kinetic modeling approach and the methodology

170 for applying TTI in food quality monitoring have been described in detail by Taoukis (2001) and

171 have been applied for the optimization of the chill chain management of fish and meat products

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172 in many studies (Giannakourou et al., 2001, 2005; Koutsoumanis, Taoukis, & Nychas, 2005;

173 Taoukis et al., 1999; Vaikousi et al., 2009). In addition to the above mentioned studies, TTIs

174 have been applied to evaluate the quality of various food products including frozen vegetables

175 (Giannakourou, & Taoukis, 2002, 2003), dairy products (Fu, Taoukis, & Labuza, 1991), meat

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176 and poultry (Labuza, & Fu, 1995; Smolander, Alakomi, Ritvanen, Vainionpää, & Ahvenainen,

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177 2004), fresh sea food (Mendoza et al., 2004) and fresh mushrooms (Bobelyn, Hertog, & Nicolaï,

178 2006).

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179 Despite the extensive research on the application of TTIs for monitoring food spoilage and

180 quality, limited information are available for their use in food safety management. Koutsoumanis

181
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et al. (2005) presented the principles of a novel chill chain management policy, coded “Safety
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182 Monitoring and Assurance System” (SMAS) for the optimisation of the distribution of chilled

183 food products within the chill chain. In this system, a new approach based on actual risk
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184 evaluation at important points of the chill chain is used in order to promote products to the next
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185 stage of distribution. This evaluation based on product’s time-temperature history, variation in
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186 product’s characteristics and the use of predictive models for the growth of food pathogens,

187 allows to give priority to products in such a way that risk at consumption time is minimized. The
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188 authors of the latter study stressed the potential use of TTIs in SMAS to evaluate the time-

189 temperature history of the product units. Ellouze and Augustin (2010) studied the applicability of
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190 biological TTIs as quality and safety indicators for meat products. However, the TTIs in the
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191 latter study were design based on product quality/spoilage. In general, the framework for TTI

192 design and application in food safety management has not been clearly defined.

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194 TTIs as food safety management tools

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195 The risk-based food safety management is an important step in improving food safety based

196 on science by linking safety requirements and criteria to the public health problems that they are

197 designed to address. Advances in Quantitative Microbiological Risk Assessment, resulted to a

198 more systematic relation between the performance of a control measure or a series of control

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199 measures to the level of control needed to manage a food safety risk. The concepts of the

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200 Appropriate Level of Protection (ALOP) and the Food Safety Objective (FSO) have been

201 proposed (FAO/WHO, 2004; ICMSF, 2002) to establish a link between public health outcomes

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202 and metrics in the food chain. The ALOP, introduced in the Agreement on Sanitary and

203 Phytosanitary Measures (SPS Agreement) of the World Trade Organization (WTO) is defined as:

204
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“the level of protection deemed appropriate by the Member establishing a sanitary or
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205 phytosanitary measure to protect human, animal and plant life or health within its territory” and

206 represents a country's currently achieved public health status in relation to food safety and
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207 (WTO, 1995). At a later stage, ICMSF (2002) introduced the FSO in order to translate the ALOP
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208 into a benchmark in the food chain that could be communicated and managed by the food
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209 industry. The FSO is defined as “the maximum frequency and/or concentration of a hazard in a

210 food at the time of consumption that provides or contributes to the ALOP” (CAC, 2004).
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211 Performance Objective (PO) and Performance Criterion (PC) are additional metrics created

212 to complement the ALOP and FSO (Gorris, 2005). PO, which is the maximum frequency and/or
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213 concentration of a hazard in a food at a specified step in the food chain before the time of
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214 consumption that provides or contributes to an FSO or ALOP as applicable, and PC, which is the

215 effect in frequency and/or concentration of a hazard in a food that must be achieved by the

216 application of one or more control measures to provide or contribute to a PO or an FSO (CAC,

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217 2004), are means for operational food safety management at food chain stages before the time of

218 consumption.

219 When establishing performance criteria consideration must be given to the initial level of a

220 hazard and changes occurring during production, distribution, storage, preparation and use of a

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221 product. By integrating the changes in a hazard from the initial level (H0), minus the sum of the

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222 reductions (R), plus the sum of increase (I), one arrives at a concentration/prevalence that at

223 consumption time must be lower than a FSO as expressed in the following equation (ICMSF,

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224 2002):

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226 H0–ΣR+ΣI≤FSO
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227 where: H0=Initial level of the hazard, ΣR =Total (cumulative) reduction of the hazard, ΣI =Total
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228 (cumulative) increase of the hazard, FSO, H0, R and I are expressed in log10 units.

229 In most cases, the factor ΣI of equation (1) depends heavily on the growth of the pathogens
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230 during distribution, retail and domestic storage. Temperature abuses that often occur during the
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231 chill chain may result to increased microbial growth and nullify the efforts made for controlling

232 the rest parameters H0 and ΣR via HACCP, Good Manufacturing and Hygiene Practices. Thus,
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233 setting a PC for the growth during distribution and storage would be of great importance.
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234 Applying a control measure to comply with the PC for this part of the chain, however, is not an
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235 easy task since, as it has been stressed previously, conditions during transportation, retail and,

236 especially, domestic level vary significantly and are out of manufacturer’s direct control.

237 Ideally, a cost-effective way to individually monitor the temperature conditions of food

238 products throughout distribution, retail and domestic storage indicating their real time-

239 temperature history would be an effective safety management tool. TTIs could potentially fulfill

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240 the above characteristics. The application scheme of TTI in food safety management is presented

241 in Fig. 1. There two main requirements in applying TTI as a control measure for compliance to a

242 PC related to the growth of pathogens during distribution and storage. First, the temperature

243 dependence of the TTI response must be similar to the temperature dependence of pathogen’s

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244 growth. Second, the end point of the TTI must coincide with the time required for a total growth

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245 of the pathogen that equals to the level set by the PC. Assuming that the above requirements are

246 met, the TTI can be used as an end point indicator attached to individual product units, which is

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247 readable by the food managers and/or consumers, providing information about their safety risk

248 status.

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Based on the application scheme presented in Fig. 1, TTIs could be also used to assure
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250 compliance with regulated food safety criteria. Among the new microbiological criteria that have

251 been incorporated in the EU Regulation 2073/2005 of particular interest are those concerning L.
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252 monocytogenes in RTE foods as, for certain food categories, they no longer require zero
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253 tolerance, but rather specify a maximum allowable concentration limit of 100 CFU/g or ml
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254 (European Commission, 2005). For RTE foods that are able to support the growth of L.

255 monocytogenes, the new Regulation demands the absence of the pathogen (in 25 g) “before the
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256 food has left the immediate control of the food business operator, who has produced it”, but

257 allows for up to 100 CFU/g for “products placed on the market during their shelf-life”.
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258 Assuming that the absence of the pathogens in 25g in the former stage of the chain is met (<-1.4
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259 log CFU/g), a maximum growth of 3.4 log CFU/g during distribution and storage would assure

260 compliance with the limit of 100 CFU/g (Koutsoumanis and Angelidis, 2007) and, thus, can be

261 used as a PC in TTI application.

262

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263 Case study: Application of a TTI for L. monocytogens control in RTE foods

264 To demonstrate the applicability of TTIs as a food safety management tool a case study for

265 L. monocytogenes in RTE foods was investigated. In a previous study (Vaikousi et al., 2008) we

266 developed a microbial TTI prototype, based on the growth and metabolic activity of a

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267 Lactobacillus sakei strain. In this particular TTI system, an irreversible color change of a

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268 chemical indicator from red to yellow progressively occurs due to the pH decline, as a result of

269 Lb. sakei growth and metabolism of a selected growth medium. Furthermore, experiments

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270 conducted in the latter study on the effect of inoculum level showed a negative linear

271 relationship between the level of Lb. sakei inoculated in the system medium and the end point of

272
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the TTI (time at which a distinct visual color change from red to final yellow is observed). The
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273 above relationship provides the ability of easily adjusting the TTI end point by using an

274 appropriate inoculum level of Lb. sakei. Herein, the applicability of the microbial TTI to monitor
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275 the growth of L. monocytogenes during distribution and storage of pate was evaluated. Growth
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276 data of L. monocytogenes in meat pate (pH 6.3, 2% NaCl, 103 ppm nitrite) from Combase
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277 (http://www.combase.cc/, source: Food Standards Agency funded data generated at Leatherhead

278 Food Research Associations, UK) were compared with the growth of Lb. sakei in the TTI system
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279 and the color response of the TTI. In addition, the effectiveness of the TTI in reducing consumer

280 exposure to the pathogen was demonstrated. The initial contamination level of the L.
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281 monocytogenes in pate was described with a Normal distribution with mean=-9.0 log CFU/g,
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282 SD=3.5 (Gallagher, Ebel, & Kause, 2003), and a minimum value of -2.3 log CFU/g assuming

283 pate packages of 200g. For the growth of the pathogen, a physiological state h0=1 and maximum

284 population density MPD=8.5 log CFU/g was assumed. The storage temperature (oC) and the

285 storage time until consumption (days) of pate were described with a Normal (4.98, 2.93) and a

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286 Normal (7, 1.5) distribution, respectively, based on literature data (Koutsoumanis et al., 2010).

287 The concentration of the pathogen in pate and the color of the TTI attached to the product at the

288 time of consumption were estimated based on the Arrhenius model using Monte Carlo

289 simulation.

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290 Growth of L. monocytogenes in pate was very close to the growth of Lb. sakei in the TTI

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291 (Fig. 2). As a result, the temperature dependence of the pathogen’s growth in pate was similar to

292 that of the TTI’s end point, which corresponds to Lb. sakei population of approximately 107-108

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293 CFU/ml. This can be seen from the parallel position of the respective Arrhenius plots in Fig. 3

294 (R² = 0.974). In particular, the activation energies of L. monocytogenes growth in pate and the

295
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reverse of the TTI end point were 131.9 and 129.3 kJ/mole, respectively. At this point, it should
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296 be noted that the observed variability at 0°C=273°K (0 value at x-axis, Fig. 3) can be attributed

297 to the fact that this temperature is very close to the minimum temperature for L. monocytogenes
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298 growth.
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299 The initial Lb. sakei level was adjusted at 105 CFU/ml. With this initial level, the end point
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300 of the TTI is reached when it is exposed to time-temperature conditions allowing 3 logs CFU/g

301 growth of L. monocytogenes in pate. An example for 12 oC is shown in Fig. 4. Based on the
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302 above, the use of the TTI can assure a maximum limit in the growth of the pathogen from

303 production to consumption time by informing the consumer when this limit is exceeded in a
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304 product unit. In fact, the latter limit, which we call Growth Tolerance Criterion (GTC), can be
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305 considered as a PC for the growth of L. monocytogenes during distribution and storage. It needs

306 to be noted that without the use of the TTI, meeting a PC related to microbial growth during

307 distribution and storage is almost impossible due to the increased variability of the chill chain

308 conditions and the lack of control in domestic storage. The use of the TTI, however, can provide

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309 an indication that cumulatively reflects the time-temperature history of the food down to a

310 product unit level and inform the consumer when the performance criterion is not met.

311 The concentration of the pathogen and the color of the TTI at the time of consumption were

312 estimated based on the Arrhenius models (Fig. 3) using Monte Carlo simulation in order to take

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313 into account the variability of initial concentration of the pathogen, storage time and storage

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314 temperature. Fig. 5 presents the predicted concentration L. monocytogenes in contaminated pate

315 packages in relation to the time distance between the TTI end point and the time to consumption.

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316 In the x-axis of the latter figure, negative values indicate packages in which the TTI end point

317 has been reached before consumption. As expected the concentration of the pathogen in these

318
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packages at the time of consumption is significantly higher compared to packages in which the
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319 TTI end point has not been reached. This can be attributed to the fact that in general these

320 packages have been exposed to more abusive time-temperature conditions as indicated by the
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321 TTI response.

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322 The use of the TTI designed to provide a GTC of 3 logs CFU/g resulted in a significant
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323 reduction of consumer exposure to L. monocytogenes (Fig. 6). Assuming that packages in which

324 the TTI end point has been reached before consumption are discarded, the percentage of
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325 consumed contaminated packages with L. monocytogenes concentrations above 102 CFU/g

326 (safety criterion based on EU regulation 2073/2005) is being reduced from 8.4% to 0.36% with
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327 the use of TTI. The predicted percentage of packages in which the TTI end point has been
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328 reached before consumption was 10.6%. It needs to be noted that this percentage depends on

329 both the time-temperature conditions of the chill chain, the shelf life of the product and the

330 chosen GTC level. For example, if a better control of storage temperature during distribution and

331 storage, a shorter shelf life or an increase of the GTC level was being applied, the result would

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332 have been a significant reduction of the packages in which TTI end point is reached before

333 consumption.

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335 Conclusions and Future Challenges

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336 The present study demonstrates the methodology and the benefits of TTIs application in

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337 food safety management. The TTIs can be adjusted in order to indicate the compliance to a

338 selected GTC. The GTC can be considered as a PC for the growth of pathogenic organisms

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339 during distribution and storage and it can be defined according to the respective FSO. A case

340 study for L. monocytogenes in RTE foods showed that TTI application can significantly decrease

341
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consumer exposure to the pathogen and reduce the safety risk. It needs to be noted that such TTI
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342 application may lead to the rejection of product units which are not contaminated with the

343 pathogen. This cost should be taken into account in the risk management decision for TTI
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344 application. However, even in the latter case it should be mentioned that the time temperature
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345 conditions that lead to TTI colour change usually allows extened growth of spoilage bacteria and
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346 thus product units which are rejected by the TTI are more likely to be spoiled. Further advances

347 in TTIs technology can extend their applicability in food safety management. A new idea under
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348 investigation in the research project acknowledged in this work is related to a double TTI that

349 can be applied to non RTE foods which are exposed to a cooking step prior consumption. Such
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350 system should be based on the combination of two different TTIs, i.e. one for distribution and
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351 storage and a second for consumer cooking. By combining the two TTIs in one system, the

352 indication on the cumulative time-temperature history during storage from the first TTI can be

353 used by the second one to set a performance criterion determining the time-temperature

354 conditions during cooking. In this way, for products exposed to temperature abuse during chill

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355 chain the double TTI will inform the consumers about the need for more effective cooking

356 providing a visual indication for the time required to be cooked.

357

358 Acknowledgements

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359 This research has been co-financed by the European Union (European Social Fund - ESF)

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360 and Greek national funds through the Operational Program "Education and Lifelong Learning"

361 of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALES:

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362 Reinforcement of the interdisciplinary and/or inter-institutional research and innovation.

363

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525

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526 Figure Captions

527 Fig. 1. Schematic representation of TTI application in food safety management. (TTIend point: time

528 at which a distinct visual color change is observed, Teff: effective temperature, EA: Activation

529 energy, tPg=PC:time required for a total growth of the pathogen equal to the level set by the

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530 performance criterion (PC)).

531 Fig. 2. Comparison between growth of Listeria monocytogenes in pate and Lactobacillus sakei in

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532 the microbial TTI system (Vaikousi et al., 2008) at 0 (red square symbols), 4-5 (blue circle

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533 symbols) and 10 (orange triangle symbols) oC.

534 Fig. 3. Arrhenius plots for Listeria monocytogenes growth rate (µmax) in pate and microbial TTI

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535 end point. The reference temperature (Tref) was set to 273°K.
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536 Fig. 4. Relation between growth of Listeria monocytogenes growth in pate at 12 oC and response

537 of the TTI system adjusted to provide a Growth Tolerance Criterion of 3 logs CFU/g. ∆Ε
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538 represents the index of total color change (Vaikousi et al., 2008).

539 Fig. 5. Monte Carlo simulation (5000 iterations) results for Listeria monocytogenes
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540 concentration in contaminated pate packages in relation to the distance between TTI end point
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541 and time to consumption. Negative values in the x-axis indicate packages in which the TTI end
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542 point has been reached before consumption.

543 Fig. 6. Effectiveness of TTI application on consumer exposure to Listeria monocytogenes from
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544 pate consumption.

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TTI end point TTI response Teff ( TTI ) = ∫ T (t ) dt

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15.0 Storage T emperature Profile

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TTI end point 7.0 E A(TTI )

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Highilights

• The study demonstrates TTIs application in food safety management

• TTIs can indicate the compliance to a selected Growth Tolerance Criterion
(GTC)
• GTC is a Performance Criterion PC for pathogen’s growth
• The effectiveness of TTIs’ application is presented through a case study

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• TTI can significantly decrease consumer exposure to the pathogen

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