Proposal TTI

Development of a Time temperature indicator to monitor the real
shelf life of perishable food products for consumer’s safety and
awareness
Outline of the problem:
Expectation of customers towards foods with improved sensory quality, increased
nutritional properties and extended shelf life has led to various improvements in packaging of the
processed food products. Nearly ¾ of all food related illness are the result of poor temperature
control. The temperature variations in a food product can lead to changes in product quality and
safety. Since temperature determines the storage parameter of a processed food product,
therefore monitoring and controlling of temperature is of great importance as it is directly related
to the safety and quality aspects of the food. Consumption of spoiled foods will apparently lead
to a lot of health consequences. Perishables deteriorate in relatively short period of time. For
every 10°C increase above the optimum temperature, the shelf-life of delicate fruit and
vegetables is halved. Optimum temperature range for the growth of pathogens is from 5°C to
57°C. This range is called as danger zone. So, in order for a food to be free from spoilage, it must
be maintained below 4°C. Prolonged exposure to extreme or elevated temperature can force
ripening of fruit, cause dangerous spoilage of foods such as seafood. In some condition, such as
heating pre-prepared food, it is essential to know that a critical temperature has been exceeded.
Besides the microbial growth and possible food borne illness, the high temperature (above 21°C)
accelerates physical changes and chemical reactions promoting deterioration on foods (Karel,
1984). Gregory (1996) reported that vitamins and aroma are affected under temperature above 50
°C and luminosity conditions during storage, transport and commercialization.
An intelligent package is one that can monitor the quality and safety condition of a food
product and provide early warning to the consumer or food manufacturer. Moreover, customer
security assurance is of primary importance. Normally the safety of a chilled ready to eat food is
answered by “Best by” date in the labeling. But that does not explain whether the food product
has been exposed to elevated temperature during storage or transportation. This can be monitored
using a Time Temperature Indicator (TTI).

Current Status:
The applicability of TTIs has been evaluated for various perishable foods including
chilled fish (Tinker et al., 1985; Taoukis et al., 1999), dairy products (Chen and Zall, 1987;
Grisius et al., 1987; Shellhammer and Singh, 1991), meat and poultry (Labuza and Fu, 1995),
frozen fruit and vegetables (Singh and Wells, 1987; Giannakourou and Taoukis, 2002), frozen
meats (Rodriguez and Zaritzki, 1983; Singh and Wells, 1985; Yoon et al., 1994), and to estimate
the remaining shelf life of food products (Fu, Petros, & Theodore, 1999; Rice, 1989; Sherlock,
Fu, & Taoukis, 1991; Taoukis and Labuza, 1989; Taoukis et al., 1999; Taoukis, 2001), A TTI
developed based on a fadable ink, which disappear in a defined time period (Galagan., Y, 2008).
An amylase type time temperature indicator was developed based on the reaction between starch
and amylase (Sun Yan, 2008)
Based on the mechanism, there are three different types of TTIs are commercially
available. (1) Polymerization - Fresh-Check®
by LifeLines Technology, Inc. is based on
polymerisation reaction of diacetylenic monomers. As polymerisation takes place, the colour of a
‘‘bulls-eye’’ patterned indicator changes gradually. (2) Enzymatic or chemical reaction -
Checkpoint®
developed by Vitsab, based on an enzymatic reaction causing pH-change in the
reaction mixture, is activated by breaking the seal between a solution containing lipolytic
enzyme and its lipid substrate. The reaction is visualised with a pH dye included in the system,
the colour of the dye is changed from green to yellow as the pH changes during the reaction. (3)
Diffusion - 3M MonitorMark®
USA, has launched a TTI which is diffusion based indicator label
and is on the color change of an oxidable chemical system controlled by temperature-dependent
permeation through a film. The process is activated by a blue-dyed fatty acid ester diffusing
along a wick. A viscoelastic material migrates into a diffusely light reflective porous matrix at a
temperature dependent rate. MonitorMark has two versions of TTI, one for monitoring
distribution, for industrial use and other, providing consumer information, the smart label
(Kuswandi et al., 2011). Timestrips® (Timestrip UK Limited, UK are smart labels for monitor
how long a product has been open or how long it has been in use. The label is automatically
activated when the consumer opens the packaging or it can be supplied as an external label that
consumers can manually activate when they first use a product (Selman, 1995; Kuswandi et al.,
2011). (eO)® TTI, is based on a time-temperature depended pH change caused by controlled
microbial growth selected strains of lactic acid bacteria that is expressed to colour change

through suitable pH indicators. Once they attached to the food, in case of temperature
fluctuation, or when the product reaches its use by date, the temperature-dependent growth of the
TTI microorganisms causes a pH drop in the tags leading to an irreversible color change of the
indicator which becomes red (Ellouze et al., 2008; Taoukis, 2008).
Although, lot of literature available on TTI based on various mechanisms, cost and
complexity of the device makes it difficult for practical application. This work focuses on
development of an economical, efficient and simple TTI, based on chemical reaction that can
have large application in the market.
Importance of the work:
The role of temperature on the spoilage rate in foods, as it is for any chemical reaction, is
well known. The temperature not only affects the rate of spoilage reactions, which involve both
bacterial and autolytic enzymes, but it also affects the rate at which bacteria multiply. For
example, generation time for Pseudomonas fragi, a common fish-spoilage bacterium, is about 12
hours at 32°F; it is only about 2 hours at 55°F (Duncan and Nickerson 1961). Storage
temperature can also influence which type of disease develops. For example in potatoes the fungi
that causes dry rot can grow rapidly at lower temperatures (15 -25°C) whereas the bacteria that
cause the soft smelly rot, only grows rapidly at warmer temperatures (> 25°C).Thus, Storage
temperature is very important to guarantee microbial quality and safety of perishable foods.
Temperature fluctuation during storage and distribution of food will lead to spoilage of food and
thus by reduction in shelf life and quality of the food product. Consumption of spoiled food
product leads to various health hazardous like Stomach cramps, Fever, Headache, Nausea,
Vomiting, Diarrhea and may also result in fatal end. So it is essential to understand and educate
customers about the time temperature history of the food product. This can be achieved by
attaching a TTI on the external surface of the food product, which will undergo colour change
with varying temperature to which the food is being subjected.

Objectives:
 To develop a Time temperature indicator (TTI) that changes colour with respect to the
change in temperature of food product, thus by understanding the temperature history of
the food product for food safety.
 To optimize TTI for few selected perishable food product.
 To monitor the real shelf life of a perishable food product using TTI.
Technical Program:
Designing of TTI:
TTI can be optimized into a label form that can be attached externally on the food
product to monitor the time temperature history of the product. Label can be designed in such a
way that it contains two compartments. These two compartments can be separated by a physical
barrier to avoid contact between activator and the gel matrix. Removing physically the barrier
between two compartments will results in activation of the TTI and helps to monitor the time
temperature history of the food product to which it is being attached.
TTI response:
To identify the TTI response, storage experiments to be conducted at the same isothermal
and non-isothermal conditions as those applied for the perishable food product. For six different
temperature profiles the continuous colour change of the reaction window of TTIs will be
measured instrumentally at regular time intervals using a colour measuring instrument until the
end point colour will be reached.
Change in colour of TTI:

The change in colour of TTI will be measured using a colour analyzer. The L*, a*, b*
chroma system, which uses the corresponding value of total color difference (∆E) as dynamic
parameters, will be used to analyze the dynamic change in the indicator’s color. According to the
indicator kinetics characterized by Taoukis and Labuza (1989), the total color difference value X
= ∆E of the indicator could be expressed in terms of a response function as follows:
F(X) = kt
Where, k stands for the rate constant of the reaction that is correlated with temperature, and t
stands for the storage time. By plotting a curve between the response function of total color
difference F(X) and time, a straight line could be obtained, and the k of different storage
temperatures could be calculated from the slope. Taking the logarithm on both sides of the
Arrhenius function:
lg k = lg A
By plotting a curve between lg k and 1/T, a straight line will be obtained. The activation energy
could be calculated from the slope, and A from the intercept directly.
Storage Tests:
The sets of TTI and food products will be stored at various temperatures using incubators.
The samples will be periodically taken to measure the TTI response and food during regular
intervals. All samples will be analysed in triplicates.
Determination of microbiological quality:
The growth of total aerobic bacteria and psychrotrophic bacteria in the selected food
product will be measured at five constant temperatures: 2, 5, 7, 12, and 15°C. Samples will be
drawn at predetermined intervals according to the storage temperature conditions. The lag times
will be determined graphically and the growth rate constant will be calculated through linear of
the exponential phase of the growth curve.

Samples of food product will be aseptically weighed, added to 1/4 strength Ringer's
solution, and will be homogenized in a stomacher for 60 seconds at room temperature. Decimal
serial dilutions in quarter strength Ringer's solution will be prepared and duplicate 1 ml or 0.1 ml
samples of appropriate dilutions will be poured or spread on the surface of the appropriate media
in petri dishes for enumeration of Total viable count (TVC). All plates will be examined visually
for typical colony types and morphological characteristics that were associated with each growth
medium.
Sensory Evaluation:
The typical sensory analysis for selected food product will be followed during the storage
experiments at different temperatures. The evaluation will be performed by a trained sensory
panel (8–10 people) using QDA on 15 point scale (Stone, H. & Sidel, 1993). The sensory
qualities will be judged under fluorescent light in a booth room with individual boxes, rating
from 1 (excellent fresh) to 10 (extremely deteriorated). The cut-off score was fixed at 7.5. The
sample will be considered as unacceptable when a mean score above five is reached.
Calibration curves of TTI response vs. quality changes:
TTI is an indicator to show the food quality changes during storage or distribution. So
there should be a fixed relationship between TTI response and food qualities, otherwise TTI
would not be a reliable indicator. The data points of TTI response could directly be correlated
with those of the food qualities by eliminating the storage time, a common independent variable,
from both relationships. The lines will be estimated by using the kinetic equations for TTI
response and food product quality.

Year-wise plan of work:
First year:
1. Development of time temperature indicator using various pH indicators.
2. Optimization of the pH dyes for the development of TTI
3. Optimizing parameters of TTI for specific perishable food products.
Second Year:
1. Designing of TTI into a label form
2. Application of the indicator with perishable food products and accessing their shelf
life.
Third Year:
1. Studies on relationship between the color change of TTI and quality characteristics of
the food product.
2. Presentation of the work in conferences.
3. PhD thesis preparation

References
 Chen, J.H. & Zall, R.R., (1987). Packaged milk, cream and cottage cheese can be
monitored for freshness using polymer indicator labels. Dairy Food Sanit. 7, 402–404.
 Duncan, D. W., Jr. & J. T. R. Nickerson. (1961). Effect or environmental and
physiological conditions on the exponential phase growlh or Pseudomonasfragi (ATCC
4973). In Proc. Low Temp. Microbiol. Symp., p. 253-262. Campbell Soup Co., Camden,
N.J.
 Ellouze, M., Pichaud, M., Bonaiti, C., Coroller, I., Couvert, O., Thuault, D. & Vaillant,
R., (2008) Modelling pH evolution and lactic acid production in the growth medium of a
lactic acid bacterium: application to set a biological TTI. International Journal of Food
Microbiology, Vol. 128, p. 101–107. ISSN 0168-1605.
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