Journal of Food Engineering 113 (2012) 471–478

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Journal of Food Engineering

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Selection of an optimum pH-indicator for developing lactic acid bacteria-based

time–temperature integrators (TTI)
Min Jung Kim, Seung Won Jung, Hye Ri Park, Seung Ju Lee ⇑
Department of Food Science and Technology, Dongguk University-Seoul, 26 Pil-Dong 3-ga, Jung-Gu, Seoul 100-715, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Among all time–temperature integrator (TTI) types, microbial-based TTIs prevail since their response is
Received 21 October 2011 directly related to food spoilage. For the development of lactic acid bacteria-based TTIs, optimum pH-
Received in revised form 26 May 2012 indicators were chosen as follows. Five kinds of potential pH-indicators and four species of lactic acid
Accepted 27 June 2012
bacteria were used to create microbial-based TTIs. The qualifying standards for pH-indicator selection
Available online 13 July 2012
were defined as (1) the effect of microorganism inhibition, (2) the simplicity of mechanistic interpreta-
tion of the color development reaction, and (3) intensity of color development. In particular, the reaction
Keywords:
of TTI color development could be enhanced from non zero-order to zero-order. Overall, the optimum
Time–temperature integrator (TTI)
Microbial-based TTI
pH-indicator for the microbial TTIs was bromocresol green, followed by bromocresol purple, bromophe-
pH-indicator nol blue, chlorophenol red, and congo red, in descending rank order. In conclusion, the choice of pH-indi-
Color development cators was found to be selective to assure effective color development of TTI.
Lactic acid bacteria Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Minnesota) is a diffusion based TTI. An acidic substance that dif-

Time–temperature integrators (TTIs) are defined as cost-effec-
tive, simple, and user-friendly devices to easily monitor the tem-
perature conditions of food throughout storage and distribution,
and are therefore applied to various food products (Wanihsuksom-
bat et al., 2010). Consumers can easily check the quality of food
using TTIs, which are usually expressed as a visible response of col-
or development or color movement that matches or correlates to
the shelf life of a food stuff at a target temperature (Taoukis
et al., 1999; Kerry et al., 2006). TTIs are already commercially avail-
able in many countries (Riva et al., 2001), and new types of TTIs
have recently been developed (Yan et al., 2008; Vaikousi et al.,
2008, 2009; Wanihsuksombat et al., 2010). However, rigorous
studies on TTI components such as color indicators are lacking.
TTI color changes through various mechanisms. The newly
introduced OnVuTM TTI (Ciba Specialty Chemicals & Freshpoint,
SW) is a solid state reaction TTI. Photosensitive compounds such
as benzylpyridines are activated and colored by exposure to low
wavelength light. This activated state (dark blue) changes to a col-
orless state (Tsironi et al., 2008, 2011). As a polymer type TTI, the
Fresh-check TTI (Temptime Corp., Morris Plains, NJ, USA) is based
on the polymerization reaction of diacetylenic monomers that
change into a highly darkened polymer (Riva et al., 2001; Hong
and Park, 2000; Nuin et al., 2008). The TT SensorTM TTI (Avery
dennison Corp., USA) and 3 M Monitor Mark (3 M Co., St Paul,
⇑ Corresponding author. Tel./fax: +82 2 2260 3372.
E-mail address: Lseungju@dongguk.edu (S.J. Lee).
fuses into the indicator label causes a color change from yellow
to bright pink, or a viscoelastic material progressively diffuses into
a light-reflective microporous matrix and the indicator changes
color from light to dark gray/black (Mendoza et al., 2004; Shimoni
et al., 2001; Taoukis, 2008). The color development mechanism of
enzymatic and microbial TTIs is dependent on pH decrease. The
checkpoint TTI (VITSAB A.B., Malmo, Sweden) is an enzymatic TTI
showing a pH dependent color change from deep green to bright
yellow due to the enzymatic hydrolysis of a lipid substrate
(Giannakourou and Taoukis, 2002, 2003; Giannakourou et al.,
2005; Lee and Lee, 2008; Taoukis et al., 1999; Bobelyn et al.,
2006; Kerry et al., 2006). The TRACE and eO TTI (CRYOLOG, Gentil-
ly, France) is a microbial TTI that has an irreversible color change
due to a pH decrease as a result of microbial growth and metabo-
lism of the growth medium (Ellouze et al., 2008; Vaikousi et al.,
2009). Microbial TTIs prevail among all others since their response
is closely related to microbial food spoilage (Ellouze et al., 2008).
The visible color change of a TTI can be determined using a
pH-indicator. A pH-indicator is a halochromoic compound that is
usually a weak acid or weak base. The color change of pH-indica-
tors depends on the activity of hydronium ions (H3O+) and hydro-
gen ions (H+). Because of their dramatic color change, these are
frequently employed in analytic chemistry, biology experiments,
and recently, smart packaging sensors or indicators (Kerry et al.,
2006; Courbat et al., 2009). There are many indicators with pH
transition ranges and some are employed for the detection of
microorganisms. BCP plate count agar (Eiken chemical Co., Japan),
used for the detection of lactic acid bacteria, includes bromocresol

0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2012.06.018
472 M.J. Kim et al. / Journal of Food Engineering 113 (2012) 471–478

green. Darukaradhya et al. (2006), Miura et al. (2004), and Vinde- Table 1
rola and Reinheimer (2000) also utilized bromocresol green for Selected pH indicators.

detection of lactic acid by microorganisms in foods. Some lactic pH indicator Color at low pH pH transition Color at high color
acid bacteria can be detected using bromocresol purple (Williams range
et al., 2001; Adan and Tan, 2007). Chlorophenol red is used in Bromocresol green Yellow 3.8–5.4 Blue
microbial TTIs to monitor the spoilage of modified atmosphere Bromocresol Yellow 5.2–6.8 Purple
packaged minced meat (Vaikousi et al., 2009). A mixture of bro- purple
Bromophenol blue Yellow 3.0–4.6 Blue/violet
mothymol blue and methyl red is used in lactic acid-based TTIs Chlorophenol red Yellow 4.8–6.4 Red
for various color changes (Wanihsuksombat et al., 2010). In this Congo red Blue 3.0–5.2 Blue/violet
manner, some indicators are used to determine pH change along
with organic acids; however, studies examining the selection of
optimum indicators are lacking.
were filled with 100 ll of MRS broth medium. Wells in column 1
In general, to perform mathematical modeling to predict food
were filled with 200 ll of pH indicator standard. Twofold serial
qualities from TTI color, one or more quality indicators of food
dilutions were made with 100 ll MRS broth in columns 1–10 by
are selected and evaluated. This quality loss indicator of food can
use of the standard technique. Column 11 was left free from pH
be chemical (development of off-flavor or off-colors from oxidation
indicators as an organism control and column 12 as a medium con-
or other chemical reactions, loss of an active component like a
trol. Inoculated medium, 100 ll, was added to columns 1–11, dou-
nutrient, cosmetic or therapeutic ingredient), biological (microbial
bling the volume in each well. Uninoculated medium (100 ll) was
growth, enzymatic deterioration), and physical (texture loss, phase
added to column 12. Each MIC determination was run in triplicate.
separation) (Taoukis, 2008). On the other hand, the quality indica-
Plates were sealed with a plastic cover, incubated microaerobically
tors of TTIs are only expressed with color values that can be de-
at 37 °C for 24 h. The visible color change was used to indicate the
scribed by different variables. Based on the imaginary positive
presence of uninhibited bacterial growth (color change) or inhibi-
primaries X, Y, and Z, the CIE system for color is described by Y,
tion (no color change) of bacterial growth in each well. The lowest
x, and y, and in the Hunter Lab system, color is defined as L⁄, a⁄,
concentration that inhibited the bacterial growth after incubation
and b⁄. Furthermore, L⁄, a⁄, and b⁄ color values are used in the CIE-
was taken as the MIC of pH indicators.
LAB system, and additional terms such as hue (h), chroma value
(C), and total color change (DE) are utilized (Macdougall, 2002).
The color change of TTI devices under time–temperature history 2.2. Preparation of pH-indicator mixture solutions
is represented by using color values such as the chroma value
(Giannakourou and Taoukis, 2002, 2003; Giannakourou et al., Because powdered indicators have low solubility in MRS broth,
2005; Lee and Lee, 2008; Taoukis et al., 1999) and total color differ- pH-indicator solutions were first prepared according to the Korea
ence (Tsironi et al., 2011; Wanihsuksombat et al., 2010; Vaikousi Industrial Standard Methods (KS M 0015: 2008) as shown in
et al., 2009). Moreover, additional color values are used, such as Table 2.
reflectance of light (Bobelyn et al., 2006), lightness (Shimoni Then, 100 mL of the indicator solutions was mixed with 900 mL
et al., 2001), and the yellow/blue opponent co-ordinate (Nuin et of MRS broth (55 g/L). The mixing proportion was determined by
al., 2008), to define a TTI’s color. For the mechanistic interpretation referring to commercial BCP plate count agar containing 0.04 g/L
of TTI kinetics, a (pseudo) zero order reaction is mainly used of Bromocresol green (Eiken chemical Co., Japan). All pH-indicator
(Mendoza et al., 2004). mixture solutions were prepared in this way.
The objective of this study was to select an optimum pH-indica-
tor for microbial TTIs. First, we determined the pH range caused by 2.3. Preparation of lactic acid bacteria-based TTIs with various pH-
the growth of lactic acid bacteria used in the microbial TTIs and po- indicators
tential pH-indicators that had similar transition ranges were se-
lected and prepared. Then, the qualifying standards for selection Lactic acid bacteria produce acids, sour off-flavors, and odors
were defined as follows: inhibition of microbial growth, degree depending on their growth, and are identified as spoilage microor-
of color development, and mechanistic interpretation. Finally, the ganisms (Cayre et al., 2003). In related studies of microbial-based
best pH indicator that satisfied the above standards was selected. TTIs, these microorganisms were used to monitor food products
(Vaikousi et al., 2008, 2009). In this study, the four kinds of lactic
acid bacteria mentioned previously were used. These strains were
2. Materials and methods maintained in MRS broth containing 20% glycerol at 70 °C. The
frozen stock cultures were sub-cultured twice in MRS broth incu-
2.1. Selection of pH-indicators bated at 37 °C before the organisms were used in the experiments
(Muthukumarasamy and Holley, 2006; Brachkova et al., 2010). The
The selection of indicators was based on their pH transition cultured microorganisms were inoculated into the pH-indicator
range. The initial pH of de Man, Rogasa, and Sharpe (MRS) broth mixture solutions to produce lactic acid bacteria-based TTIs in
(Difco, Detroit, MI, USA) is 7.0 ± 0.2, and it was reduced to around liquid and were stored under the isothermal condition of 37 °C in
4.0 ± 0.3 with the growth of the lactic acid bacteria (12 h, 37 °C). incubators (SH-75B Biofree Co., Ansan, Korea).
Thus any indicators that included even part of the range of pH
4.0–7.0 were selected, and are shown in Table 1. Table 2
The KS-standard methods of preparing pH indicator solutions.
A serial dilution technique using 96 well micro plates was used
to obtain minimal inhibitory concentration (MIC) values of the pH Indicator Method of preparation
indicators against lactic acid bacteria (LAB). Bromocresol green 0.04 g + 20 ml Ethyl alcohol (95 v/v%) + 80 ml D.W.*
In this study, the lactic acid bacteria, Lactobacillus amylovorus Bromocresol purple 0.05 g + 20 ml Ethyl alcohol (95 v/v%) + 80 ml D.W.
KCCM 40431, Lactobacillus pontis KCCM 41635, Lactococcus lactis Bromophenol blue 0.10 g + 20 ml Ethyl alcohol (95 v/v%) + 80 ml D.W.
Chlorophenol red 0.10 g + 20 ml Ethyl alcohol (95 v/v%) + 80 ml D.W.
subsp. lactis KCCM 32406, and Lactococcus plantarum KCCM
Congo red 0.10 g + 100 ml D.W.
40701 were purchased from the Korean Culture Center of Microor-
*
ganisms (KCCM). The wells in columns 2–12 of the micro plates D.W.: Distilled water.
 M.J. Kim et al. / Journal of Food Engineering 113 (2012) 471–478 473

Table 3
Inhibition activity of pH indicators against lactic acid bacteria.

pH indicator Concentration (mg/mL) MIC*** (mg/mL)

100 50 25 12.5 6.25 3.125 1.5625 0.7813 0.3907 0.1954
Bromocresol green LBA* **   + + + + + + + 12.5 < MIC < 25
LBP    + + + + + + +
LCL    + + + + + + +
LCP    + + + + + + +
Bromocresol purple LBA   + + + + + + + + 25 < MIC < 50
LBP   + + + + + + + +
LCL   + + + + + + + +
LCP   + + + + + + + +
Bromophenol blue LBA    + + + + + + + 12.5 < MIC < 25
LBP    + + + + + + +
LCL    + + + + + + +
LCP    + + + + + + +
Chlorophenol red LBA    + + + + + + + 12.5 < MIC < 25
LBP    + + + + + + +
LCL    + + + + + + +
LCP    + + + + + + +
Congo red LBA  + + + + + + + + + 50 < MIC < 100
LBP  + + + + + + + + +
LCL  + + + + + + + + +
LCP  + + + + + + + + +
*
LBA: Lactobacillus amylovorus KCCM 40431, LBP: Lactobacillus pontis KCCM 41635, LCL: Lactococcuslactis subsp. lactis KCCM 32406, and LCP: Lactococcus plantarum KCCM
40701.
**
+: Color change and : No color change.
***
MIC: Minimal inhibitory concentration.

2.4. pH and titratable acidity (TA) measurement 6 drops of 0.5% phenolphthalein (w/v ethanol) to the sample and
titrated with 0.1 N NaOH until a faint pink color persists. Titratable
pH was measured using a pH-meter (S20 SevenEasy™ pH, Met- acidity was expressed as percentage of lactic acid, determined
tler-Toledo International Inc., Seoul, Korea) for 10 min at room using Eq. (1).
temperature. The titratable acidity was assessed by the titration
0:1 N NaOH required  0:1 N NaOH factor  0:009
method of Nwafor and Ikenebomeh (2009) using 0.1 N NaOH. Lactic acidð%Þ ¼
Weight of sample
Approximately, 10 g of the culture fluid was diluted with approxi-
 100 ð1Þ
mately 20 ml of sterilized distilled water before titration. Added 5–

Fig. 1. The changes in pH and titratable acidity (TA) of TTIs with the pH indicator, bromocresol green, and four different lactic acid bacteria, stored at 37 °C. (A): Lactobacillus
amylovorous KCCM 40431, (B): Lactobacillus pontis KCCM 41635, (C): Lactococcus lactis subsp. lactis KCCM 32406, (D): Lactococcus plantarum KCCM 40701. Solid line: pH,
dotted line: TA.
474 M.J. Kim et al. / Journal of Food Engineering 113 (2012) 471–478

Fig. 2. Evolution of the total color change (DE, left section) and color response function (F(X), right section) upon storage of the TTIs at 37 °C. (A): Bromocresol green, (B):
bromocresol purple, (C): bromophenol blue, (D): chlorophenol red, (E): congo red. X: Lactobacillus amylovorous KCCM 4043, : Lactobacillus pontis KCCM 41635, N: Lactococcus
lactis subsp. lactis KCCM 32406 and j: Lactococcus plantarum KCCM 40701.
 M.J. Kim et al. / Journal of Food Engineering 113 (2012) 471–478
2.5. Color measurements and kinetic parameters of the TTIs
The color changes of the TTIs were measured with a chroma
meter (Chroma meter CR-300, Konica Minolta Sensing Inc., Tokyo,
Japan) and expressed as the index of total color change, DE.
 lactic acid production is also not linear (Vereecken and Van Impe,
DE ¼ ½ðDL Þ2 þ ðDa Þ2 þ ðDb Þ2 1=2
⁄ ⁄ ⁄
where CIE color DL , Da and Db are lightness, redness–greenness,
and yellowness–blueness differences between samples and targets,
respectively. Prior to measuring the color, the TTIs were centrifuged
(Combi-514R, Hanil Science Ins., Seoul, Korea 679g, 4 °C and
10 min) due to the color interference of the microorganisms.
According to the TTI color response kinetics characterized by Taou-
kis and Labuza (1989), the total color change value X = DE of the pH
indicator mixture solution can be expressed in terms of a response
function as follows:
FðXÞ ¼ ½lnð1=1  XÞ1=2 ¼ kt
where k stands for the reaction rate constant and t stands for the
storage time. By plotting a curve between the color response value
F(X) and time, a regression line could be obtained, and the k of dif-
ferent cultivation temperatures could be calculated from the slop.
Taking the logarithm on both sides of the Arrhenius function:
Ea
ln k ¼ þ ln A
2:303RT
By plotting a curve between ln k and 1/T, a regression line was
obtained. The activation energy (Ea) could be calculated from the
slope, and A from the intercept directly.
3. Results and discussion
3.1. Inhibition effect on microbial growth
The inhibition activity of pH-indicators with micro-well dilu-
tion assay was present in Table 3. The minimal inhibitory concen-
tration (MIC) of pH-indicators ranged from 12.5 to 100 mg/mL. The
bromocresol green, bromophenol blue, and chlorophenol red
exhibited the strongest antibacterial activity against selected
microorganism at concentrations of 12.5–25 mg/mL. Antibacterial
activity was least for congo red at concentrations of 50–100 mg/
mL and greatest for bromocresol purple at concentrations of
25–50 mg/mL. Concentration of pH indicators used in this study
was a state of lower than MIC of pH indicators.
The experimental data for changes in the level of pH (decrease)
and the titratable acidity of the TTIs with bromocresol green during
12 h of storage at 37 °C are shown in Fig. 1. Even though there were
some differences in pH and TA levels among the lactic acid bacte-
ria, the entire pH range was similar, from 3.8 up to 7.0. This pH
range was found to coincide with that from MRS broth containing
lactic acid bacteria and no pH-indicator stored for 12 h at 37 °C.
Also, for the TTIs with the other pH-indicators, the pH and TA
change profiles showed a similar tendency as in Fig. 1 for the TTI
Table 4
The reaction rates (k) and coefficients of determination (R2) of the TTIs.
Sample Lactobacillus amylovorus KCCM Lactobacillus pontis KCCM
40431 41635
k R2 k R2
Bromocresol green 0.251 0.9798 0.188
Bromocresol purple 0.250 0.8671 0.216
Bromophenol blue 0.141 0.9995 0.134
Chlorophenol red 0.134 0.9555 0.104
Congo red 0.115 0.9732 0.137
475
with bromocresol green. Thus, the selected pH-indicators could
be used for microbial TTIs.
In general, the shape of microbial growth with time is not a sim-
ple linear model. Because lactic acid production by lactic acid bac-
teria is proportional to the concentration of microbes, the model of
ð2Þ 2002; Poschet et al., 2005; Ellouze et al., 2008). This is why the
pH decrease by lactic acid bacteria with time is not linear, as
shown in Fig. 1.
3.2. TTI color response kinetics
In the TTI implement system, to apply a TTI response to food the
function of the TTI response should first be defined (Giannakourou
et al., 2005). The kinetics of the color evolution of the TTIs was ex-
pressed as change in DE and F(X), as shown in Fig. 2.
ð3Þ As shown in the left section of Fig. 2, bromocresol green and
bromocresol purple showed the greatest color change (DE value
is nearly 80), followed by bromophenol blue and chlorophenol
red (DE value is nearly 50), and congo red (DE value is nearly
30). The color development of the TTIs basically depended on the
pH indicator’s transition range. Because the pH range of the TTIs
(3.8–7.0) included the transition ranges of bromocresol green (pH
3.8–5.4) and bromocresol purple (5.2–6.8), the color change of
ð4Þ the TTIs could fully cover the ranges of the two pH-indicators. In
contrast, congo red had a low transition range (3.0–5.2), that was
not included in 3.8–7.0, and therefore, the color change of cong
red was not complete and the TTI containing congo red showed a
low DE value. Even though the transition range of chlorophenol
red was entirely comprised in 3.8–7.0, the DE value was not high.
This may be because the original color of the MRS broth affected
the TTI color as well.
The results of converting DE to F(X) are shown in the right sec-
tion of Fig. 2. These color response functions, F(X), were found to be
more linear. Generally, the response function of a TTI is regarded as
a (pseudo) zero order reaction of which mechanistic interpretation
is mainly used (Mendoza et al., 2004). The reaction rate, k, of the
color response was determined by linear regression analysis (Table
4).
The rate constants of bromocresol green and bromocresol pur-
ple, which had large color changes, were high. Congo red had a rel-
atively lower reaction rate compared to these two. Overall, the
coefficients of determination (R2) were high enough to meet the
zero-order reaction in descending order of bromocresol green, bro-
mophenol blue, bromocresol purple, chlorophenol red, and congo
red. The zero-order reaction of a color response, indicating that
the color response change is linear, is known to be mainly used
for TTI products. However, this notion conflicts with the fact that
the pH decrease or titratable acidity increase by lactic acid bacteria
with time was not linear (Fig. 1), resulting in a color response
change that should also be not linear. This disagreement implies
there were nonlinear interactions between the pH-indicators and
lactic acid contents. This nonlinear relationship could compensate
Lactococcus lactis subsp. lactis KCCM Lactococcus plantarum KCCM
32406 40701
k R2 k R2
0.9966 0.222 0.9889 0.222 0.9874
0.9681 0.237 0.9719 0.263 0.9869
0.9687 0.135 0.9866 0.148 0.9879
0.9230 0.126 0.9130 0.125 0.9176
0.9732 0.098 0.7953 0.112 0.8890
476 M.J. Kim et al. / Journal of Food Engineering 113 (2012) 471–478

for a nonlinear pH change, resulting in a linear relationship be-

tween color response and storage time. These topics will be dis-
cussed more in the latter part.

3.3. Identification of the color development mechanism of microbial-

based TTIs

The original assumption is that lactic acid is produced by the

Fig. 3. The total color change of indicators with pH decrease by using 1.0 M lactic
acid solution, : Bromocresol green, j: bromocresol purple, X: bromophenol blue,
N: chlorophenol red and d: congo red.
microorganisms in a TTI, and then the acid reacts with pH-indica-
tors resulting in color development. But it is unclear whether the
relationships between the acid and color development according
to pH-indicators are linear, concave, or convex.
Fig. 3 shows that there were DE changes for each indicator solu-
tion with a decrease in pH by using 1.0 M lactic acid increasingly
(Ellouze et al., 2008). The pH-indicator solutions prepared accord-
ing to Table 2 had various pH levels. But once they were added to
the MRS broth, the pH converged to 6.35 ± 0.005 for all indicators.
It seems that the pH level of the MRS broth did not change by the
addition of the indicator solutions. Therefore, the starting pH level

Fig. 4. The comparison of total color change between estimated and experimental data with time (Lactococcus plantarum KCCM 40701). (A): Bromocresol green, (B):
bromocresol purple, (C): bromophenol blue, (D): chlorophenol red, and (E): congo red. Solid line: estimated color value and dotted line: experimental color value.
 M.J. Kim et al. / Journal of Food Engineering 113 (2012) 471–478 477

would be fixed and not affected by the kinds of indicators, which
may be desirable for TTI performance. Bromocresol green showed
the biggest change in DE. The final DE values of bromocresol green,
bromocresol purple, bromophenol blue, chlorophenol red, and con-
go red were 97.71, 79.94, 79.80, 70.43, and 49.48, respectively, en-
ough to be observed even visually. There were some patterns in
their color change profiles (Fig. 3) as follows: (1) linear type, (2)
convex type, and (3) concave type. In the case of bromocresol
green, the increase rate of DE was nearly constant as pH dropped;
therefore, it belonged to linear form. Bromocersol purple appeared
to be of convex form. Its color changed rapidly at the initial stage,
and then the color change rate was gradually low. Its color change
range was 5.2–6.8. The others including chlorophenol red, bromo-
phenol blue, and congo red appeared to be of concave type. Their
color change rates increased as pH decreased.
The DE change profiles of the indicator mixture solutions were
not the same as those of the TTIs in Fig. 2. This is because Fig. 2 and
Fig. 3 have different x-axis variables such as pH and time, respec-
tively. The pH change with time was not linear as shown in
Fig. 1. Combining Fig. 1 and Fig. 3, it would be expected to corre-
spond to Fig. 2. Substituting pH in Fig. 3 with the corresponding
time in Fig. 1, the solid lines (estimates) in Fig. 4 could be obtained,
compared to the dot lines (experimental data) from Fig. 2. Fig. 4
shows that there is very high agreement between the estimated
and experimental data. This indicates that the DE response
patterns of TTIs (Fig. 2) were simply due to the combination of
the patterns of lactic acid or pH changes (Fig. 1) and the DE re-
sponse patterns of pH-indicators (Fig. 3). The important finding
here is that although the lactic acid production of a TTI was not
in zero-order reaction, if the pH-indicator was properly chosen,
the color response of TTI could be in zero-order reaction.
3.4. Selection of optimum pH-indicator
For the development of microbial-based TTIs, the standards for
selecting qualified pH-indicators were defined as follows: (1) the
effect of microorganism inhibition, (2) the simplicity of mechanis-
tic interpretation of the color response function (an agreement
with zero-order reaction), and (3) magnitude of color development
(DE).
As show in Table 5, consequently the bromocresol green was
found to be the best indicator for microbial-based TTIs, followed
by bromocresol purple, bromophenol blue, chlorophenol red, and
congo red, in descending rank order.
3.5. Arrhenius analysis of the microbial-based TTIs
The bromocresol green was used to find the activation energy
(Ea) of the microbial-based TTIs. In accordance with Eq. (4), the acti-
vation energy could be calculated by plotting a curve between ln k
and 1/T. The Ea values of LBA (Lactobacillus amylovorus) based TTIs,
LBP (Lactobacillus pontis) based TTIs, LCL (Lactococcuslactis subsp.

Table 5
The standards and results for the selection of an optimum pH indicator.

Indicator Lactic acid bacteria Inhibition* F(X)** DE***

Bromocresol green Lactobacillus amylovorus KCCM 40431 – Good Good
Lactobacillus pontis KCCM 41635 – Good Good
Lactococcus lactis subsp. lactis KCCM 32406 – Good Good
Lactococcus plantarum KCCM 40701 – Good Good
Bromocresol purple Lactobacillus amylovorus KCCM 40431 – Good Good
Lactobacillus pontis KCCM 41635 – Normal Good
Lactococcus lactis subsp. lactis KCCM 32406 – Normal Good
Lactococcus plantarum KCCM 40701 – Bad Good
Bromophenol blue Lactobacillus amylovorus KCCM 40431 – Good Normal
Lactobacillus pontis KCCM 41635 – Normal Normal
Lactococcus lactis subsp. lactis KCCM 32406 – Good Normal
Lactococcus plantarum KCCM 40701 – Good Normal
Chlorophenol red Lactobacillus amylovorus KCCM 40431 – Bad Normal
Lactobacillus pontis KCCM 41635 – Bad Normal
Lactococcus lactis subsp. lactis KCCM 32406 – Bad Normal
Lactococcus plantarum KCCM 40701 – Normal Normal
Congo red Lactobacillus amylovorus KCCM 40431 – Bad Bad
Lactobacillus pontis KCCM 41635 – Normal Bad
Lactococcus lactis subsp. lactis KCCM 32406 – Bad Bad
Lactococcus plantarum KCCM 40701 – Normal Bad
*
Insignificant.
**
Agreements with zero-order reaction: good, R2 > 0.98; normal, 0.98 > R2 > 0.93 and bad, 0.93 > R2.
***
Magnitude of color development: good, DE (final experimental value) > 70; normal, 70 > DE > 40 and bad, 40 > DE.

Table 6
The kinetic and Arrhenius parameters (k, reaction rate and Ea, activation energy) and coefficients of determination (R2) of the bromocresol green-LAB-based TTIs.

Temperature (°C) Microbial-based TTIs

LBA-TTIs* LBP-TTIs LCL-TTIs LCP-TTIs
k R2 Ea (kJ/mol) k R2 Ea (kJ/mol) k R2 Ea (kJ/mol) k R2 Ea (kJ/mol)
15 0.0057 0.9798 105.24 0.0054 0.9788 116.35 0.0045 0.9845 109.40 0.0045 0.9812 103.88
20 0.0129 0.9844 0.0121 0.9845 0.0081 0.9739 0.0137 0.9745
25 0.0334 0.9897 0.0298 0.9776 0.0383 0.9908 0.0324 0.9622
30 0.0874 0.9940 0.1036 0.9940 0.0981 0.9911 0.0954 0.9985
37 0.1772 0.9735 0.1918 0.9757 0.1987 0.9615 0.1850 0.9707
*
LBA-TTIs, Lactobacillus amylovorus based TTIs; LBP-TTIs, Lactobacillus pontis based TTIs; LCL-TTIs, Lactococcuslactis subsp. lactis based TTIs and LCP-TTis, Lactococcus
plantarum based TTIs.
478 M.J. Kim et al. / Journal of Food Engineering 113 (2012) 471–478
lactis) based TTIs, and LCP (Lactococcus plantarum) based TTIs were
105.24, 116.35, 109.40 and 103.88 kJ/mol, respectively (Table 6).
These values were the range obtained by Vaikousi et al. (2009). La-
buza (1982) previously reported that typical Ea values for enzy-
matic loss, hydrolysis, lipid oxidation, nutrient loss, and microbial
growth were 41.84–62.76, 62.76, 41.84–104.60, 83.68–125.52,
and 83.68–125.52 kJ/mol, respectively. Generally, the difference
in Ea between a food product and TTI must be close to ±20 kJ/mol
to apply TTI to food products. Therefore, LBA-TTI, LBP-TTI, LCL-
TTI, and LCP-TTI seem to be applicable in the food products.
4. Conclusion
The color development of TTIs is important for their application
in food products. Therefore, in this study, we selected an optimum
pH-indicator for the development of lactic acid bacteria-based TTIs.
For the evaluation of color development by TTIs, we should consider
not only the original color of the pH-indicator, but also other vari-
ables such as the color of the microbial medium (TTI base) and some
color interference caused by the microorganisms. It was interest-
ingly found that the color reaction orders of lactic acid bacteria-
based TTI altered with different pH-indicators, and could be en-
hanced from non zero-order to zero-order. Actually, the zero-order
reaction depends on reaction time, not substrate concentrations, so
the interpretation of color response becomes more effective to con-
trol and design TTI products. Therefore, this aspect could be one
more factor that should also be taken into account for the evalua-
tion of color development by TTIs. To conclude, the choice of pH-
indicators for developing lactic acid bacteria-based TTIs was found
to be selective to assure effective color development according to
time–temperature history. This finding will allow for other types
of TTIs containing pH-indicators, such as acid dependent TTIs.
Acknowledgements
This research was supported by the Agriculture Research Center
(ARC, 710003-03-1-SB110) program of the Ministry for Food, Agri-
culture, Forestry and Fisheries, Korea.
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