LWT - Food Science and Technology 78 (2017) 63e69

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LWT - Food Science and Technology

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Microbial contamination of tofu in Korea and growth characteristics of

Bacillus cereus isolates in Tofu
Da-Young Lee a, Ki-Hyun Kwon b, Changhoon Chai c, Se-Wook Oh a, *
a
Department of Food and Nutrition, Kookmin University, Seoul 02707, Republic of Korea
b
Korea Food Research Institute, Gyeonggi 13539, Republic of Korea
c
Division of Applied Science, Kangwon National University, Chuncheon 24341, Republic of Korea

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

Article history: This study investigated the microbial contamination of commercial tofu from local supermarkets in
Received 19 July 2016 Seoul, Korea. Growth modeling of Bacillus cereus isolated from spoiled tofu was used to determine the
Received in revised form appropriate and temperature for safe storage of tofu. During the monitoring of 100 commercial tofu
24 November 2016
products, pathogenic bacteria (B. cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella spp.,
Accepted 27 November 2016
Available online 28 November 2016
and enterohemorrhagic Escherichia coli O157:H7 [EHEC]) were not detected. Aerobic bacteria were iso-
lated from 32 of 100 tofu samples because of different sterilization and packaging methods. To isolate the
dominant microorganism involved in tofu putrefaction, tofu was intentionally spoiled by storage at 30
C
Keywords:
Bacterial contamination
for 24 h. After spoilage, the most abundant colonies were harvested and conrmed as B. cereus by
Tofu analyzing their 16 S ribosomal RNA (rRNA) sequences and fatty acid compositions. To investigate growth
Spoilage bacteria properties on tofu, isolated B. cereus was cultured and inoculated on tofu. The growth of B. cereus at
Identication different storage temperatures (15
C, 20
C, 25
C, and 30
C) was analyzed. As a result, the appropriate
Growth model storage time of tofu stored at 5
C, 10
C, and 15
C was determined to be 9.99, 4.17, and 2.08 days,
respectively.
2016 Elsevier Ltd. All rights reserved.

1. Introduction Biagio, & Sandra, 2016).

Tofu, a popular soy-based food in East Asian countries, is an
inexpensive source of high-quality protein (Pontecorvo & Bourne,
1978; Pre
stamo & Fontecha, 2007). Tofu contains approximately
6.0%e8.4% protein and 79%e87% water, and has a near-neutral pH
(5.2e6.2) (Kovats, Doyle, & Tanaka, 1984). It has been reported that
tofu is easily contaminated by microorganisms, even when stored
in refrigerated conditions (Dotsom, Frank, & Cavaletto, 1977).
Spoiled tofu has a sour taste and an unpleasant smell due to bac-
terial growth. Therefore, tofu is a perishable product with a very
short shelf life compared to other soybean products. The preser-
vation of tofu is inuenced by environmental factors such as bac-
terial load, storage temperature, air composition, and
manufacturing process (Angeles & Marth, 1971; Kang, Lee, & Oh,
1998). Prevention of microbial spoilage of tofu is considered an
important research topic to ensure food safety (Diana, Maurice,
Claudio, Luciana, & Elisabetta, 2013; Franca, Giovanna, Anita,
* Corresponding author.
E-mail address: swoh@kookmin.ac.kr (S.-W. Oh).
Estimation of growth characteristics by predictive models using
mathematical equations can be used to estimate the growth, sur-
vival, and inactivation of microbes by using mathematical equa-
tions (Juneja et al., 2007; Rodri
guez, Alcal,

a, Gimeno, & Cosano,
2000; SantAna, Franco, & Schaffner, 2012). Primary models
describe changes in microbial counts or other microbial responses
with time. Sigmoidal models, such as modied Gompertz equa-
tions, and logistic and Baranyi models are widely used for tting
observed microbial growth data and predicting microbial growth
rates (Gibson, Bratchell, & Roberts, 1988). Secondary models
describe the responses of primary models to changes in environ-
mental conditions such as temperature, pH, or water activity.
Among these, temperature is the major factor affecting bacterial
growth that is likely to show variation during food processing and
storage (Hiroshi & Satoshi, 2005 ); therefore, a number of predictive
models of bacterial growth in food have been developed to model
the effect of temperature changes. Several studies have been re-
ported for predictive models of Bacillus cereus growth in broth and
foods (Heo, Lee, & Ha, 2009; Lanciotti, Sinigaglia, Gardini, Vannini,
& Guerzoni, 2001; Sutherland, Aherne, & Beaumont, 1996; Olmez &

http://dx.doi.org/10.1016/j.lwt.2016.11.081
0023-6438/ 2016 Elsevier Ltd. All rights reserved.
64 D.-Y. Lee et al. / LWT - Food Science and Technology 78 (2017) 63e69
Aran, 2005). However, to date, there have been no reports of pre-
dictive models to simulate the growth of B. cereus in tofu.
The objectives of the present study were the following: (1) to
evaluate the microbial contamination of tofu in Korea; (2) to isolate
and identify the dominant bacteria in spoiled tofu; and (3) to es-
timate the growth characteristics by using growth models of arti-
cially inoculated B. cereus isolated from spoiled tofu.
2. Materials and methods
2.1. Tofu
From May to August in 2014, 100 packages of tofu were pur-
chased from different local supermarkets in Seoul, Korea from May
to August, and transported to the laboratory at 4
C, where im-
mediate microbiological analysis was performed. Aerobic bacteria,
Escherichia coli and pathogenic bacteria were analyzed according to
Food and Drug Administration protocols in the Bacteriological
Analytical Manual.
2.2. Enumeration of aerobic bacteria, Escherichia coli, coliforms,
Staphylococcus aureus, and B. cereus
First, 10 g of tofu was homogenized with 90 mL of 0.85% sterile
saline (8.5 g/L of sodium chloride; SigmaeAldrich) for 2 min using a
stomacher (Laboratory Blender Stomacher 400; Seward, London,
UK). Then, 1 mL of the homogenate was serially diluted tenfold with
9 mL 0.85% sterile saline (8.5 g/L of sodium chloride; Sigma-
eAldrich), and 1 mL of the diluted solution was spread onto each
selective media. Aerobic bacteria, E. coli, and coliforms were
counted using a Petrilm aerobic count and an E. coli/coliform
count (3M, St Paul, MN, USA). BairdeParker agar base (Oxoid)
supplemented with egg yolketellurite emulsion was used as the
selective media for S. aureus. Mannitol egg yolk polymyxin agar
base (Oxoid) supplemented with egg yolk emulsion was used as the
selective media for B. cereus. The plates were incubated at
30
Ce37
C for 24e48 h and colonies with specic shape and color
were selected and conrmed by polymerase chain reaction (PCR).
2.3. Microbiological analysis of Listeria monocytogenes, Salmonella
spp., and E. coli O157:H7
Listeria enrichment broth (Oxoid) was used as enrichment broth
for L. monocytogenes. Oxford agar (Oxoid) containing a Listeria se-
lective supplement was used as selective agar and incubated at
30
C for 24e48 h. To detect Salmonella spp., 0.2% sterile peptone
water and RappaporteVassiliadis Broth (Oxoid) was used as
enrichment broth. Xyloseelysineedeoxycholate agar (Oxoid) was
used as the selective agar and incubated at 37
C for 24 h. To detect
E. coli O157:H7, modied tryptone soya broth (Oxoid) containing
novobiocin (10 mg/L; SigmaeAldrich) was used as the enrichment
broth. Telluriteecexime sorbitol MacConkey agar (Oxoid) and
tryptone bile X-glucuronide agar (Oxoid) were used as selective
agars and incubated at 37
C for 24 h. Colonies with specic shape
and color were selected and conrmed by PCR.
2.4. Isolation of spoilage bacteria
Dominant spoilage bacteria were isolated from spoiled tofu
incubated at 37
C for 24 h. A 25 g sample of spoiled tofu was ho-
mogenized with 225 mL of 0.85% sterile saline in a sterile sample
bag (Whirl-pak, 19  30 cm; Nasco, Fort Atkinson, WI, USA), fol-
lowed by homogenization for 2 min using a stomacher. Serial di-
lutions were spread onto tryptone soya agar (TSA, Oxoid) and
incubated at 37
C for 24 h. Two types of dominant colonies
differing in their morphological appearance were isolated.
2.5. Identication of spoilage bacteria
Genomic DNA was isolated using a 5% boiling resin (Chelex 100
Resin; Bio-Rad, Hercules, CA, USA). The extracted genomic DNA was
amplied for 16 S ribonucleic acid RNA (rRNA) gene sequencing by
PCR using EF-Taq DNA polymerase (Solgent) and the universal
primers, 27 F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492 R (50 -
GGTTACCTTGTTACGACTT-30 ). PCRs were run in a 9700 PCR System
(Applied Biosystems, Carlsbad, CA, USA) using the following con-
ditions: 15 min at 95
C; 30 cycles of 20 s at 95
C, 40 s at 50
C,
90 s at 72
C; 5 min at 72
C. Sequencing of the 16 S rDNA gene was
performed using an ABI PRISM 3730XL DNA analyzer (Applied
Biosystems). To determine potentially homologous sequences, the
sequencing results were subjected to a GenBank BLASTN search
(http://www.ncibi.nlm.nih.gov/BLAST) and aligned using Phrap
(http://www.phrap.org).
2.6. Analysis of cellular fatty acid composition
Cellular fatty acid composition of isolated spoilage bacteria was
analyzed according to the standard protocol of the Microbial
Identication System (MIDI; Microbial II, Inc., Newark, Delaware,
USA) (Miller, 1982). The cell extracts were analyzed with a 6890 gas
chromatograph (Agilent Technologies, Santa Clara, California, USA)
equipped with a 30 m  320 mm cross-linked methyl siloxane
column (HP-1). The cellular fatty acid composition proles were
analyzed using Microbial Identication System software (MIDI).
The prole of each test strain was identied by comparison with the
commercial database provided with the Sherlock MIS version 6.2
(Yang, Vauterin, Vancanneyt, Swings, & Kersters, 1993).
2.7. Bacterial growth experiment and modeling
Tofu was cut into approximately 15  15  40-mm pieces,
transferred to a sterile sample bag (Nasco, USA), and articially
inoculated with culture cocktails combined with two isolated
bacteria species to obtain approximately 3 log colony-forming units
(CFU)/mL. Each inoculated tofu piece was stored at 15, 20, 25, or
30
C, and, during incubation, a portion of the tofu was removed for
analysis of bacterial growth by counting colonies on TSA plates. The
results were converted to a log scale.
Growth curves were tted to a modied Gompertz equation (Eq.
(1)) and the kinetic variables of growth, such as lag time (LT) and
maximum specic growth rate (SGR), were determined (Gibson
et al., 1988) using GraphPad Prism V4.0 (GraphPad Software, San
Diego, CA, USA).
   
SGR
Y N0 C$exp  exp 2:718$ $LT  T 1 ;
C
(1)
where Y is the log viable number of bacteria, N0 is the log initial
number of bacteria, C is the difference between initial and nal
bacterial numbers as Nmax/N0, SGR is the maximum specic growth
rate, LT is the time of delay before growth, and T is the sampling
time.
A secondary model describes the effects of temperature on the
variables of a primary model such as LT and maximum SGR.
Exponential decay (Eq. (2)) and exponential growth (Eq. (3)) were
used to analyze the effects of temperature on LT and SGR using
SigmaPlot software v. 12.0 (Systat Software Inc., Richmond, CA,
USA).
 D.-Y. Lee et al. / LWT - Food Science and Technology 78 (2017) 63e69 65

Table 1
Population distribution of aerobic bacteria from 100 tofu samples.

Prevalence Aerobic bacteria population range (log CFU/g) Mean SD (log CFU/g)

Negative Positive 0e1 1e2 2e3 3e4 4e5 5e6

68/100 32/100 4/32 9/32 6/32 4/32 7/32 2/32 3.73 1.63
(68%) (32%) (12.5%) (28.1%) (18.8%) (12.5%) (21.9%) (6.3%)

Abbreviations:CFU, colony-forming unit; SD, standard deviation.

Table 2 detected in 32 of 100 samples because of the different sterilization

Prevalence of pathogenic bacteria and Escherichia coli in 100 tofu processes and packaging methods, and the average bacterial count
samples.
was 3.73 log CFU/g. Aerobic bacterial counts were distributed as
Bacteria Prevalence follows: 0e1 log CFU/g 4 (12.5%), 1e2 log CFU/g 9 (28.1%), 2e3
Escherichia coli N.D. a log CFU/g 6 (18.8%), 3e4 log CFU/g 4 (12.5%), 4e5 log CFU/g 7
Coliforms N.D. (21.9%), and 5e6 log CFU/g 2 (6.3%) (Table 1). The microbial
Bacillus cereus N.D. quality of tofu is affected by the hygiene condition of the processing
Staphylococcus aureus N.D.
environment. The packaged tofu was set into an anaerobic state by
E. coli O157:H7 N.D.
Listeria monocytogenes N.D. soaking in lling water and had been heated in a manufacturing
Salmonella spp. N.D. process for coagulation of soybean protein. In addition, there may
a
Not detected (N. D.).
be an additional heat-treatment process during the nishing step,
depending on the manufacturer. This additional step aims to
decrease the microbial ora through sterilization. For that reason,
aerobic bacteria would not have been detected for most of the
LT aLT $expbLT $T; (2) sterilized tofu samples. Pathogenic bacteria and E. coli were not
detected in any of the tested tofu (Table 2). However, some reports
where T is temperature, and aLT and bLT are the regression constants. of pathogenic bacteria detected in tofu exist; coliform bacteria
(Rehberger, Wilson, & Glatz, 1984) and various pathogenic bacteria,
SGR SGR0 aSGR $expbSGR $T; (3)
including Pseudomonas spp., E. coli, Enterococcus spp., lactic acid
where T is temperature, and SGR0, aSGR, and bSGR are the regression bacteria, B. cereus, Staphylococcus spp., Salmonella spp., Yersinia
constants. spp., and Cronobacter sakazakii (Ananchaipattana et al., 2012).
Shelf life, dened as the time taking to reach 6 log CFU/g, was In another report, Enterobacteriaceae, Enterococci, Staphylococci,
estimated using a modied Gompertz equation (Eq. (1)). Nmax and and yeast were detected in tofu, and the total aerobic mesophilic
N0 should be set for 5
C and 10
C, and N0 is 1 log CFU/g. A logistic count was 105 CFU/g, which is higher than the result of the present
function (Eq. (4)) was used to calculate the Nmax value required to study (Ashena, 1994).
estimate shelf life. The SGR and LT values calculated by the sec- Quantitative microbial analysis performed in the present study
ondary model at 5
C, 10
C, and 15
C were used for estimating the did not involve an enrichment step; therefore, S. aureus and B. ce-
shelf life. reus will not be detected when present at low concentrations such
as below the detection limit (log 1 CFU/g). In addition, B. cereus may
am not be detected if it is present in the spore state, which survives the
Nmax Nmax0   bm ; (4)
heat-treatment step during the manufacturing process.
1 T
T0
3.2. Isolation and identication of spoilage bacteria
where T is the temperature, and Nmax0, am, and bm are the regres-
sion constants. Dominant spoilage bacteria were isolated from spoiled tofu that
was incubated at 37
C for 24 h. Two strains of spoilage bacteria
were isolated from spoiled tofu according to the distribution ratio
3. Results and discussion and colony shape. The T-1 strain, which was the dominant spoilage
bacteria, was identied on the basis of its 16 S rRNA gene similarity
3.1. Microbial contamination of tofu to B. cereus (98.4% similarity with B. cereus HN-Beilezhu 1, GenBank
accession number JQ917438). The T-2 strain, which was the second-
In the present study, the presence of aerobic bacteria, E. coli, and dominant population, was also identied on the basis of 16 S rRNA
six different pathogenic bacteria (coliforms, S. aureus, L. mono- gene similarity to B. cereus (98.1% similarity with B. cereus Bc,
cytogenes, Salmonella spp., B. cereus, and E. coli O157:H7) in 100 GenBank accession number KC814644) (Table 3). The spoilage
commercial tofu samples were examined. Aerobic bacteria were bacteria identied in the present study were somewhat different

Table 3
Taxonomic details of dominant bacteria in spoiled tofu as identied on the basis of the 16 S rRNA gene partial sequence.

Isolated bacteria Accession no. Bacterial strain names showing % similarity with isolates

T-1 JQ917438 Bacillus cereus strain HN-Beihezhu1 (98.4%)

KM224528 Bacillus sp. XQW6 (98.3%)
JQ308572 Bacillus cereus strain OPP5 (98.3%)
T-2 KC814644 Bacillus cereus strain Bc (98.1%)
FJ174643 Bacillus thuringiensis strain 130YG3 (98%)
KF648904 Bacillus sp. PVL04 (97.4%)
66 D.-Y. Lee et al. / LWT - Food Science and Technology 78 (2017) 63e69

Table 4
Major fatty acid proles of isolated spoilage bacteria.

Compounds Fatty acid composition (%)

T-1 T-2

Saturated fatty acids

12:0 0.24 0.36
13:0 e 0.09
14:0 3.37 2.87
16:0 2.99 4.16
17:0 0.27 0.13
18:0 0.35
Unsaturated fatty acids
15:1 u5c 0.37 0.36
16:1 u7c alcohol 0.58 0.60
16:1 u11c 0.14 0.18
17:1 u7c 0.11
18:1 u9c 0.13
Branched fatty acids
11:0 iso e 0.14
12:0 iso 0.60 0.96
13:0 iso 9.83 12.23
13:0 anteiso 0.98 1.41
Fig. 1. Growth of spoilage bacteria in tofu at different temperatures and prediction
14:0 iso 4.32 4.66
curves according to the modied Gompertz equation. Predicted value by the modied
15:0 iso 37.41 30.96
Gompertz equation (d) and observed value at 15
C (C), 20
C (B), 25
C (;), and
15:0 anteiso 4.54 4.52
30
C ().
16:0 iso 4.56 5.45
17:1 iso u10c 1.98 1.95
17:1 iso u5c 5.78 5.35
17:1 anteiso A 0.99 0.88 (7.10%, and 8.74%, respectively). Branched fatty acids, i.e., 15:0 iso
17:0 iso 7.10 8.74 (41.94%), 13:0 iso (7.26%), and 17:0 iso (9.38%), were reported as the
17:0 anteiso 1.15 1.46 major fatty acids in 27 strains of B. cereus (Teska et al., 2003). In
18:0 iso 0.15
another report, 77 strains of B. cereus isolated from raw milk,
19:0 iso 0.12
Hydroxy chicken, cereals, and meat contained 15:0 iso (30.25%), 16:0 iso
15:0 2OH 0.84 0.59 (11.23%), 13:0 iso (8.85%), and 17:0 iso (9.20%) as the major fatty
17:0 iso 3OH 0.11 acids (Dikbas, 2010). Therefore, T-1 and T-2 were conrmed as B.
Summed featuresa
cereus.
Summed feature 2 3.78 3.35
Summed feature 3 8.24 7.64

Summed feature 2 contained one or more of the following fatty acids: 14:0 3OH and/
or 16:1 iso I. Summed feature 3 contained one or more of the following fatty acids:
16:1 u6c and/or 16:1 u7c.
a
Summed features represent groups of two or three fatty acids that could not be
separated by gas-liquid chromatography with the Microbial Identication (MIDI)
system.
from those from previous reports. Acinetobacter calcoaceticus var.
anitratus and Klebsiella pneumoniae subgroup pneumoniae were the
major spoilage bacteria isolated from tofu after 2 days of storage at
room temperature (20
Ce25
C) (Shin, 2006). Bacillus sp., B. meg-
aterium, Enterobacter sakazakii, and four types of B. cereus were
isolated from tofu after 2 days of storage at 37
C (No, Park, Hwang,
& Meyers, 2002). These different results might originate from the
different microbial contamination of raw materials, as well as from
contamination occurring during the manufacturing process. In
Chinese fermented tofu (sofu), B. cereus, B. subtilis, and Clostridium
perfringens were isolated, leading researchers to conclude that B.
cereus is a potential health hazard (Han, Beumer, Rombouts, &
Nout, 2001).
3.3. Fatty acid composition of the isolated spoilage bacteria
3.4. Growth characteristics of spoilage bacteria
Growth characteristics of spoilage bacteria were observed ac-
cording to predictive growth modeling. The aim of predictive
growth modeling is to use mathematical models to provide a
quantitative estimate of microbial growth in foods (Te &
Zwietering, 1999; Zhou, Fu, Li, Cheng, & Liang, 2009). The growth
of spoilage bacteria in tofu and the predicted curves according to a
modied Gompertz equation at different temperatures (15
C,
20
C, 25
C, and 30
C) are presented in Fig. 1. Table 5 summarizes
the LT and SGR values, obtained using a modied Gompertz equa-
tion, for spoilage bacteria on tofu that was stored at 15
C, 20
C,
25
C, or 30
C. The LT values of the spoilage bacteria on tofu stored
at 15
C, 20
C, 25
C, or 30
C were calculated to be 9.1, 6.9, 4.9, and
2.9 h, respectively, and the SGR values were 0.113, 0.327, 0.536, and
1.134, respectively. It can be observed that with increasing storage
temperature, LT values decreased and SGR values increased. The
goodness of t of the primary model was evaluated using the co-
efcient of determination (R2). The R2 values ranged between 0.98
Table 5
Lag time (LT) and maximum specic growth rate (SGR) of spoilage bacteria in tofu at
different temperatures (modied Gompertz equation).

The fatty acid compositions of the T-1 and T-2 strains were Temperature Growth parameters Regression analysis
determined and the results are presented in Table 4. T-1 and T-2 (
C)
Lag time, Maximum specic growth rate, R2 RMSE Bf
were identied by comparison with the database (Teska, Coyne, LT (h) SGR (log CFU/g/h)
Ezzell, Allan, & Redus, 2003) using the Microbial Identication 15 9.1 0.113 0.98 0.30 1.009
System (MIDI; Microbial ID, Inc., Newark, Delaware, U.S.A.). The T-1 20 6.9 0.327 0.99 0.22 1.002
and T-2 strains contained the highest relative amounts of the 25 4.9 0.536 1.00 0.15 1.001
branched fatty acid 15:0 iso (37.41% and 30.96%, respectively) fol- 30 2.9 1.134 0.99 0.22 1.001

lowed by 13:0 iso (9.83% and 12.23%, respectively), and 17:0 iso Abbreviations:CFU, colony-forming unit; RMSE, root-mean-square error.
 D.-Y. Lee et al. / LWT - Food Science and Technology 78 (2017) 63e69 67

Table 6
Lag times (LT) and maximum specic growth rates (SGRs) of spoilage bacteria in tofu (exponential equation).

Variables Equations Statistical indices

R2 RMSE Bf

Lag time, LT 25:941e; al indicesriT 0.9822 0.001 1.016

LT (h)
Maximum SGR 0:0141 0:01951FUt 0:1356 T 0.9937 0.003 1.019
specic
growth rate,
SGR
[(log CFU/g)/h]

Abbreviations: CFU, colony-forming unit; RMSE, root-mean-square error.

and 1.00. The RMSE value from modied Gompertz modeling at Table 7
15
C, 20
C, 25
C, and 30
C was 0.15e0.30. The RMSE is a mea- Observed and predicted lag time (LT) and maximum specic growth rate (SGR) of
spoilage bacteria in tofu.
surement of the variability between the predicted and actual
observed values for a particular variable, and a lower value in- Temperature Lag time, LT (h) Maximum specic growth
dicates that the model is a good representation of the experiment (
C) rate,
SGR [(log CFU/g)/h]
data (Sutherland et al., 1996). In addition, the bias factor (Bf) values
at the same temperature were 1.009, 1.002, 1.001, and 1.001, Observed Predicted Observed Predicted

respectively. Bf indicates whether, on average, the predicted values 15 9.1 9.3 0.113 0.135
are either higher or lower than the observed values (Hwang & 20 6.9 6.6 0.327 0.280
25 4.9 4.7 0.536 0.564
Marmer, 2007). A Bf in the range of 0.90e1.05 is considered good,
30 2.9 3.33 1.134 1.125
a Bf of either 0.70e0.90 or 1.06e1.15 is considered acceptable, and a
Bf of either <0.7 or >0.5 is considered unacceptable. Therefore, Abbreviations:CFU, colony-forming unit.

these results suggest that the predicted curves generated in the

present study show good ts for the growth of B. cereus isolates on
values for LT and SGR in the secondary models were 1.016 and 1.019,
tofu at different temperatures (15
C, 20
C, 25
C, and 30
C). In
respectively, which are within the acceptable range. Table 7 shows
addition, the results indicate that a modied Gompertz model is a
the observed and predicted RE values for LT and SGR (Delignette-
useful tool for predicting the growth of B. cereus isolates on tofu.
Muller, Rosso, & Flandrois, 1995). The observed LT values were
Secondary models were developed for the LT and SGR to predict
9.1, 6.9, 4.9, and 2.9, and the predicted values were 9.3, 6.6, 4.7, and
the effects of temperature on bacterial growth. Indices derived
3.3 at 15
C, 20
C, 25
C, and 30
C, respectively. The observed SGR
from exponential decay and growth equations are shown in Table 6,
values were 0.113, 0.327, 0.536, and 1.134, and the predicted SGR
and Fig. 2 shows the regression curve of LT and SGR according to an
values were 0.135, 0.280, 0.564, and 1.125 at 15
C, 20
C, 25
C, and
exponential equation. The exponential equations for LT and SGR
30
C, respectively.
were LT 25.941$exp (0.0684 T) and SGR 0.0141 0.0195 exp
The shelf life of tofu was calculated using a modied Gompertz
(0.1356 T), respectively. To evaluate the secondary model, deter-
equation. First, Nmax was calculated using a logistics equation.
mination of coefcient (R2), RMSE, and bias factor (Bf) values were
Second, the initial level of spoilage bacteria (N0) was set at 2 log
calculated (Table 6). The data obtained from tting the quadratic
CFU/g and the maximum population level was set at 6 log CFU/g.
exponential equation showed high correlation coefcients
Finally, LT and SGR values were obtained for each specic temper-
(R2  0.98) for LT and SGR in all test conditions. RMSE values for LT
ature from the secondary model. For tofu that is normally stored at
and SGR were all within the acceptable range (>0.003). The Bf
refrigeration temperature, the shelf life was calculated for both
refrigeration temperature (5
C and 10
C) and room temperature
(15
C). The results showed that the safe storage times for tofu at 5,
10, and 15
C were 9.99, 4.17, and 2.08 days, respectively (Table 8
and Fig. 3), indicating that a low storage temperature is impor-
tant for extending the shelf life of tofu.
The storage temperature is also important: a shelf life of 9.99
days at 5
C is decreased to 4.17 days at 10
C, indicating that shelf
life increases by about 5 days when tofu is stored at 5
C (Fig. 3A and
B). The estimation of growth characteristics by mathematical
modeling could therefore be a very useful tool for manufacturers,
allowing them to identify the important variables that determine
the shelf life of their product. Furthermore, it would allow

Table 8
Predicted shelf life of articially inoculated tofu estimated by growth
modeling.

Temperature (
C) Predicted shelf life (days)

5 9.99
Fig. 2. Regression curve of lag time (LT) and maximum specic growth rate (SGR).
10 4.17
Predicted value by exponential equation (d) and observed value of lag time (C) and
15 2.08
maximum specic growth rate (B).
68 D.-Y. Lee et al. / LWT - Food Science and Technology 78 (2017) 63e69
Fig. 3. Estimation of the shelf life of articially inoculated tofu with Bacillus cereus
isolates at 5
C (A), 10
C (B), and 15
C (C).
manufacturers to study the consequences of decreasing the tem-
perature within the distribution chain, taking into consideration
the effect on shelf life and hence the economic impact.
4. Conclusions
In the present study, microbial contamination of commercial
tofu was analyzed. E. coli and pathogenic bacteria, including B. ce-
reus, S. aureus, L. monocytogenes, Salmonella spp., and E. coli
O157:H7 were not detected in the 100 tofu samples tested. How-
ever, aerobic bacteria were isolated in 32% of the tested tofu sam-
ples, with an average contamination level of 3.73 log CFU/g. To
estimate the shelf life of tofu, dominant spoilage bacteria were
isolated from spoiled tofu and a growth model was developed. Two
dominant spoilage bacterial strains, T-1 and T-2, were isolated and
conrmed as B. cereus by 16 S rRNA sequence and fatty acid
composition. Growth models of the spoilage bacteria on tofu were
determined at four different temperatures. A modied Gompertz
equation provided the optimal t as a primary growth model, and
secondary models of the LT and SGR were derived from exponential
decay and growth equations, respectively. Bacterial growth is the
main cause of tofu spoilage and the primary growth model suggests
that temperature is the most important factor for bacterial growth
in tofu. The shelf life of tofu when stored at 5
C, 10
C, and 15
C
was calculated as 9.99, 4.17, and 2.08 days, respectively, to guar-
antee microbiological safety. In summary, estimation of growth
characteristic by predictive modeling was used to determine the
shelf life of tofu after it was articially inoculated with spoilage
bacteria.
Acknowledgments
This study was supported by a program of High Value-added
Food Technology Development of Korea Institute of Planning and
Evaluation for Technology in Food, Agriculture, Forestry and Fish-
eries (IPET) funded by the Ministry of Agriculture, Food and Rural
Affairs (grand No. 313032-03-2-HD020).
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