Thermal Inactivation of Escherichia coli O157:H7

in Strawberry Puree and Its Effect on

Anthocyanins and Color
Hsin-Yun Hsu, Lihan Huang, and James Swi-Bea Wu
Raw whole strawberries, if contaminated with pathogens, such as Escherichia coli O157:H7, must be pasteurized
prior to consumption. Therefore, the objective of this research was to investigate the thermal inactivation kinetics of E.
coli O157:H7 in strawberry puree (SP), and evaluate the changes in anthocyanins and color, and the survival of yeasts
and molds (YM) after thermal processing. Inoculated with a 5-strain cocktail, fresh SP, with or without added sugar (20
and 40 Brix), was heated at 50, 52, 54, 57.5, 60, and 62.5 C to determine the thermal resistance of E. coli O157:H7.
In raw SP, the average D-values of E. coli O157:H7 were 909.1, 454.6, 212.8, 46.1, and 20.2 s at 50, 52, 54, 57.5,
and 60 C, respectively, with a z-value of 5.9 C. While linearly decreasing with temperature, the log D-values of E.
coli O157:H7 increased slightly with sugar concentration. The log degradation rates of anthocyanins increased linearly
with temperature, but decreased slightly with sugar concentrations. These results suggest that sugar may provide some
protection to both E. coli O157: H7 and anthocyanins in SP. The browning index was not affected by heating at 50
and 52 C at low sugar concentrations, but increased by an average of 1.28%, 2.21%, and 10.1% per min when SP was
exposed to heating at 54, 57.5, and 60 C, respectively. YM was also inactivated by heating. This study demonstrated
that properly designed thermal processes can effectively inactivate E. coli O157:H7 and YM in contaminated SP, while
minimizing the changes in anthocyanins and color.

Abstract:

M: Food Microbiology
& Safety

Keywords: anthocyanins, Escherichia coli O157:H7, strawberry, thermal processing

This study investigated the thermal inactivation kinetics of E. coli O157:H7 in strawberry puree
and its effect of degradation of anthocyanins and changes in color. The results of this work may be used to develop and
optimize thermal processes that minimize the degradation of anthocyanins during pasteurization of strawberry puree.

Practical Application:

Introduction
Over the past 2 decades, the consumption of fresh produce has
greatly increased due to consumers preference for healthier, minimally processed foods (Olaimat and Holley 2012). During the
same period, however, the outbreaks of foodborne illnesses associated with fresh produce have also increased (Warriner and others
2009). For fresh produce, microbial contamination can occur at
any stage during growth, harvest, processing, storage, and transportation (Olaimat and Holley 2012). Outbreaks of Escherichia coli
O157:H7 have been associated with a range of foods, including ground beef, raw milk, and contaminated water, apple cider,
and yogurt (Linton and others 1999). Recently, fresh strawberries
from a farm in Oregon were implicated in an outbreak of E. coli
O157:H7, which caused at least 15 sicknesses, including 1 death
(FDA 2011). This outbreak was the first recorded E. coli O157:H7
infections linking to fresh strawberries (CDC Foodborne Outbreak Online Database 2011).
MS 20130895 Submitted 7/1/2013, Accepted 11/7/2013. Authors Hsu and Wu
are with Inst. of Food Science and Technology, National Taiwan Univ., Taipei, 106,
Taiwan. Author Huang is with the Eastern Regional Research Center, Agricultural
Research Service, U.S. Dept. of Agriculture, Wyndmoor, PA, 19038, U.S.A. Direct
inquiries to author Huang (E-mail: lihan.huang@ars.usda.gov).
Mention of trade names or commercial products in this publication is solely
for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Dept. of Agriculture.

M74

Journal of Food Science r Vol. 79, Nr. 1, 2014

Strawberry is a juicy, soft-textured berry with an attractive

bright red color, and usually consumed as a fresh fruit or processed
into frozen berries or puree. Once contaminated with foodborne
pathogens, such as E. coli O157:H7, strawberries cannot be easily
decontaminated for fresh consumption. Strawberry puree (SP),
on the other hand, is widely used as an intermediate ingredient in
a variety of products, such as yoghurt, jams or jellies, smoothies,
snacks, and drinks (Huang and others 2013). For SP, an attractive
red color is an important quality attribute (Saenz and others 1993).
Anthocyanins impart a characteristic reddish color to strawberries
and have antioxidant properties that may play a key role in the prevention of diseases, including cancer, diabetes, and neuronal and
cardiovascular illnesses (Konczak and Zhang 2004). Many factors,
including temperature, sugars, pH, light, oxygen, enzymes, ascorbic acid, sulfite salts, metal ions, and copigments, may influence
the anthocyanins stability (Wang and Xu 2007). Sucrose at 40%
concentration was reported to stabilize the red color of anthocyanins in strawberry (Wrolstad and others 1990). During thermal
processing, heat increases temperature and,therefore, may promote
the degradation of anthocyanins in SP, resulting in changes in
color (browning), nutritional components, and flavor (Zabetakis
and others 2000; Patras and others 2009). Therefore, the objective
of this study was to investigate the inactivation kinetics of E.
coli o157:H7 in fresh SP and the effect of thermal processing
on anthocyanins and color changes after exposure to elevated
temperatures.

C 2013 Institute of Food Technologists

R
Journal of Food Science 
No claim to original US government works
doi: 10.1111/1750-3841.12327

Further reproduction without permission is prohibited

Pasteurization of strawberry puree . . .

Table 1The D-values (s) and z-values ( C) of E. coli O157:H7 in different sugar concentrations of SP.

50
52
54
57.5
60
62.5
z-value ( C)

8 Brix

R2

0.991
0.994
0.993
0.949
0.986

909.1
454.6
212.8
46.1
20.2

43.7
12.9
25.9
9.7
4.7

5.9 0.3

0.998

Materials and Methods

Bacterial cultures and preparation
Five rifampicin-resistant strains of E. coli O157:H7, including
ATCC 43888, 43889, 43890, 45756, and 11082, were obtained
from the stock culture collection of the Eastern Regional Research Center of USDA Agricultural Research Service, located at
Wyndmoor, Pa. The rifampicin-resistant strains of E. coli O157:H7
were used to differentiate the bacteria from numerous background
bacteria in the fresh sample (Huang 2010).
One night before experiment, a loopful of each strain was
individually transferred to 10 mL brain heart infusion broth
(BD/Difco, Sparks, Md., U.S.A.) supplemented with 100 g/mL
rifampicin (Sigma, R35015G, Sigma Aldrich Co., Mo., U.S.A.)
and held at 37 C in an orbital shaker (approximately 100 rpm)
for 20 h.

20 Brix
1008.0
499.2
243.9
68.5
23.9

45.4
14.0
17.8
3.7
3.9

6.2 0.1

R2
0.982
0.978
0.977
0.931
0.943
0.999

40 Brix
1428.6
588.2
333.3
117.7
37.5
18.7
6.6

71.8
15.8
24.2
11.1
5.3
2.5
0.2

R2
0.963
0.975
0.978
0.927
0.939
0.905
0.997

Each culture was harvested by centrifugation (2400 g for

15 min at 4 C), washed twice with 10 mL 0.1% peptone water
(PW, BD/Difco), recentrifuged, and resuspended in 5 mL PW.
A cocktail of the bacteria was formed by combining and mixing
the washed cultures. The cocktail, containing approximately 109
CFU/mL of E. coli O157:H7, was immediately used to inoculate
SP samples. Fresh cultures were prepared for each experiment.

Sample preparation and inoculation

Fresh strawberries were purchased from a local supermarket 1 d
before experiments and stored at 4 C. After washing with running tap water, the strawberries were cut into smaller pieces using
a sterile French fry cutter (GPC-3664, Progressive International
Co., Wash., U.S.A.), and then placed in filter bags (Whirl-Pak, 207
mL., 95 mm 180 mm 0.08 mm, NASCO-Fort Atkinson,
Fort Atkinson, Wis., U.S.A.). The sugar concentration of fresh

Figure 1Survival curves of E. coli O157:H7 in SP with different sugar concentrations. (A) 8 Brix; (B) 20 Brix; and (C) 40 Brix.

Vol. 79, Nr. 1, 2014 r Journal of Food Science M75

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Temperature ( C)

Pasteurization of strawberry puree . . .

strawberry was approximately 8 Brix. To evaluate the effect of
sugar on thermal inactivation of E. coli O157:H7 and degradation
of anthocyanins, sucrose (Sigma, S85015KG) was added to adjust
to the sugar concentrations to 20 and 40 Brix. Each bag, containing 5 0.5 g of cut strawberries, was inoculated 0.1 mL bacterial
cocktail. After inoculation, the strawberry samples were mixed
and crushed in the filter bags to attain fresh SP, pressed to form
a thin layer (<1 mm) in the filter bags, and then vacuum-sealed
(approximately 15 mBar) to evacuate residual air.

M: Food Microbiology
& Safety

Changes in total anthocyanins content (TAC) and color

TAC. The TAC in the sample was determined using the pHdifferential method described by Lee and others (2005). A 2-buffer
system was used, including potassium chloride buffer at pH 1.0
(0.025 M) and sodium acetate buffer at pH 4.5 (0.4 M).
To measure TAC, a 0.1 mL aliquot of the SP filtrate was transferred to a 10 mL volumetric flask. The sample was adjusted to a
final volume of 10 mL with the buffers, and kept in dark at ambient
temperature for 30 min. The absorbance at 510 and 700 nm was
then measured. TAC was calculated by measuring the absorbance
of pelargonidin-3-glucoside, which is the major anthocyanins in
Thermal resistance of E. coli O157:H7 in SP
The inoculated samples were subjected to submersion heating strawberry (Eq. 1):
in a circulating hot water bath (RTE 17, NESLAB, Thermo Fisher
 mg  A MW DF 1000
Scientific Inc., Mass., U.S.A.) maintained at 50, 52, 54, 57.5, 60,
T AC
=
(1)

L
l
and 62.5 C, respectively. Samples were fully submerged under hot
water, with heating time ranging from 10 to 5760 s, depending
where A = (A510 A700)pH1.0 (A510 A700)pH4.5 ; MW
on temperature. The thermal treatment was terminated by imme(molecular weight): 433.2 g/mol for pelargonidin-3-glucoside;
diately placing the heat-treated samples into an iced water bath.
DF: dilution factor; l: the path length in cm; : 22400, molar
extinction coefficient in L/mol/cm for pelargonidin-3-glucoside.
Recovery of surviving E. coli O157:H7
The coefficient 1000 is the conversion factor from g to mg.
E. coli O157:H7 was recovered from heat-treated samples by
Browning index (Bi). The absorption spectrum of each SP
adding 10 mL of 0.1% cold PW to each sample bag. The samples filtrate was scanned over the wavelength range between 400 and
were then mixed for 2 min in a mechanical stomacher (Model 700 nm using a UV-visible spectrophotometer (S2150UV, Unico,
Bag Mixer 100W, Interscience Co., France). The filtrate, after N.J., U.S.A.). The Bi of each sample was calculated as the ratio of
proper decimal dilutions, was plated onto Tryptic Soy agar (TSA, A420 to A520 nm (Tsai and Ou 1996). Bi was used to describe
BD/Difco) plates containing 100 g/mL rifampicin. The plates the browning during thermal processing (Sadilova and others
were maintained at room temperature for 2 h to allow heat-injured 2009).
cells to resuscitate, and then incubated at 37 C for 24 h. With
Measurement of sugar concentration and pH-value in
rifampicin in TSA plates, only E. coli O157:H7 cells were re- SP. The concentration of sugar ( Brix) was measured at room
covered without the need to use a selective medium, which can temperature using a refractometer (HR-032 ATC, AFAB Entercause damages to heat-injured cells, while eliminating background prises, Fla., U.S.A.). The pH-value was measured using a pH meter
microorganisms in the samples.
(Model 420A, Orion Inc., Mass., U.S.A).

Figure 2The combined effect of temperature and sugar concentration in SP on the thermal death time of E. coli O157:H7. Each data point is the mean
of 3 D-value measurements.

M76 Journal of Food Science r Vol. 79, Nr. 1, 2014

Pasteurization of strawberry puree . . .

Results and Discussion

Data analysis and modeling

The thermal inactivation experiments were repeated at least
3 times for each timetemperature combination and all of the
plate counts of E. coli O157:H7 and physicochemical analyses
were conducted in triplicates (n = 3) in each experiment. The
YM tests were conducted in duplicate. The number of microbial
colonies was converted to logarithm (base 10) of colony forming
units per gram (log CFU/g).
The D-values of E. coli O157:H7 in SP were calculated from
the survival curves, which were constructed by plotting the log
bacterial counts against heating time for each temperature. Linear
regression was used to correlate the log counts of E. coli O157:H7
to heating time. The D-values were calculated from the linear
portion of each survival curve. The z-value of E. coli O157:H7
was calculated from the inverse of the slope of the linear regression curve between the log D-values and heating temperatures.
R
The D- and z-values were analyzed using the Microsoft Excel
spreadsheet.
Multiple linear regression, conducted using R (http://
www.r-project.org/), was used to analyze the relative effects of

Thermal inactivation of E. coli O157:H7

Figure 1 shows the isothermal survival curves of E. coli O157:H7
in SP with different levels of sugar (8, 20, and 40 Brix, respectively) at temperatures between 50 and 62.5 C. The inactivation
of E. coli O157:H7 followed the first-order kinetics, as the log
counts of E. coli O157:H7 decreased linearly with time under
each heating conditions (Figure 1). Table 1 lists the average Dvalues of E. coli O157:H7 in SP. The z-values of E. coli O157:H7
are 5.9, 6.2, and 6.6 C in SP with 8, 20, and 40 Brix, respectively.
In this study, both heating temperature and sugar concentration
affected the thermal resistance of E. coli O157:H7 in SP (Table 1),
and the z-value of E. coli O157:H7 increased gradually with the
sugar concentration.
The z-values of E. coli O157:H7 on single-strength apple,
orange, and white grape juice were reported at 5.6, 4.8, and
5.3 C, respectively (Mazzotta 2001). In apple juice, the z-values of
3 strains of E. coli O157:H7, N-4163, N-4170, and N-4173, were
determined as 4.9, 5.4, and 5.5 C (Enache and others 2011).
Therefore, the z-value of the E. coli O157:H7 on SP obtained in
this study is very close to those high acid fruit juice (pH value =
3.33.9) values reported in the literature.

Figure 3Retention of total anthocyanins in SP with different sugar concentrations. (A) 8 Brix; (B) 20 Brix; and (C) 40 Brix.

Vol. 79, Nr. 1, 2014 r Journal of Food Science M77

M: Food Microbiology
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temperature and sugar concentration on thermal death time of

E.coli O157:H7 and degradation of anthocyanins in SP during
heating.

Survival of yeasts and molds (YM). The filtrates of heattreated samples, after proper dilution, were plated onto Dichloran
Rose Bengal Chlortetracyclin agar (BD/Difco) plates to enumerate YM. The plates were wrapped with aluminum foil to provide
darkness and incubated at 25 C for 5 d.

Pasteurization of strawberry puree . . .

Comparing with the D- and z-values obtained in singlestrength and concentrated white grape juice (58 Brix) reported
by Mazzotta (2001) and Enache and others (2006), it appears that
the thermal resistance E. coli O157:H7 is much higher for the
pathogens in concentrated juice than in single-strength juice. For
example, the D60 C for E. coli O157:H7 was 2.41 min in white
grape juice concentrate and only 0.7 min in single-strength juice,
and z-values were 9.2 C in concentrate and only 5.3 C in singlestrength juice.
Figure 2 further illustrates the combined effect of heating temperature and sugar concentration on survival of E. coli O157:H7
in SP. Generally, the log D-values decreased linearly with temperature, but increased linearly with the sugar concentration. This
relationship can be described by Eq. 2, and Table 3 lists the coefficients for this equation. Eq. 2 separates the relative effect of each
factor (temperature and sugar concentration) on its contribution
to the thermal death time. Based on this equation, the relative
contribution of temperature, or the z-value, can be calculated
from the inverse of the slope of T (1/b) in Eq. 2. According to
Table 2, the average apparent z-value of E. coli O157:H7 is 6.3 C
in SP. For each degree in the increase of Brix, the log D-value is
increased by 0.78%.
log (D) = a + b T + c Sug a r

Table 2Combined effect of temperature and sugar concentration on the thermal death time of E. coli O157:H7 in SP (R2 =
0.994).
Parameter

Estimate

Standard error

t-value

Pr(>|t|)

a
b
c

10.74
0.158
7.77e-3

0.19
0.003
1.0e-3

57.77
46.41
7.72

< 2.0e-16
7.85e-16
3.27e-06

Table 3Combined effect of temperature and sugar concentration on degradation of anthocyanins in SP during heating
(log (K An th oc yanin) = a + b T + c Su g a r , R2 = 0.986).
Parameter

Estimate

Standard error

t-value

Pr(>|t|)

a
b
c

12.78
0.170
5.23e-3

0.33
0.006
1.0e-3

38.66
28.36
3.176

5.78e-14
2.29e-12
7.98e-03

M: Food Microbiology
& Safety

Changes in pH, total anthocyanins, and Bi

The sugar concentration, pH-value, and TAC in the raw SP
were found to be 8 Brix, 3.7 mg/L, and 32.5 mg/L, respectively.
The sugar concentration and pH-value of SP did not change
significantly in all the thermal treatments. However, the color
(2) of SP experienced slight changes after thermal processing.

Figure 4Effect of temperature and sugar concentration on the degradation rate of anthocyanins in SP during heating.

M78 Journal of Food Science r Vol. 79, Nr. 1, 2014

Pasteurization of strawberry puree . . .

C
=1K
C0

Ant ho c ya ni n

(3)

During heating, the anthocyanins may degrade to colorless

derivatives, which are subsequently converted to insoluble brown
pigments. Higher heating temperatures and longer heating times
will lead to undesirable anthocyanins degradation and browning
(Wang and Xu 2007). Color change and anthocyanins content was

highly correlated (Patras and others 2011). The changes in the Bi

of SP during heating were affected by heating time, temperature,
and to a lesser extent, by sugar concentration (Figure 5). At 50 C,
Bi was not affected by heating for up to 4320 s in all samples, regardless of the sugar concentration (Figure 5A). At 52 C, heating
for up to 2160 s did not affect the Bi of SP with 8 and 20 Brix, but
caused Bi to increase in SP with 40 Brix. At temperature above
54 C, heating caused the Bi of SP to increase with temperature
at all sugar levels (Figure 5B and 5C). The difference in sugar
concentration in SP did not appear to affect the development of
color. By linear regression between Bi and heating time, it was
estimated that the Bi of SP was increased by 1.28%, 2.21%, and
10.1% per min in all samples when SP was exposed to heating at
54, 57.5, and 60 C, respectively.

The survival of YM
In untreated SP, the YM count (YMC) was between 2.9 and
3.4 log CFU/g (Table 4). Under the sampling plans, no survivors
of YM were recovered in samples heat-treated at 50 and 52 C.
Some survivors were recovered at temperatures above 54 C. It
appears that sugar also protected YM in SP, as more YM were
recovered under the same temperature and with the same heating time at T > 54 C. At 60 C, the D-value of YM was 50,
58.8, and 64.5 s in SP with 8, 20, and 40 Brix, respectively. At
57.5 C, the D-value of YM was 83.3 and 111.1 s in SP with 20 and
40 Brix, respectively. Since no survivors of YM were recovered
from samples heat treated at 50, 52, and 54 C, it was not possible
to determine the D-values of YM at these heating temperatures.
However, it was possible to roughly estimate the D-values at these

Figure 5Effect of temperature and heating time on Bi of SP.

Vol. 79, Nr. 1, 2014 r Journal of Food Science M79

M: Food Microbiology
& Safety

Figure 3 shows the relative degradation of anthocyanins (C/C0 )

in SP after thermal processing. Previous studies revealed that thermal degradation of anthocyanins followed the first-order reaction kinetics in strawberry juice and concentrates (Garzon and
Wrolstad 2002). In this study, however, it appears that the degradation of anthocyanins in SP follows the zero-order reaction under
constant temperature conditions, which was manifested by the linear decrease in C/C0 (Figure 3) and can be described by a simple
equation (Eq. 3). The rate of degradation (K_Anthocyanins ) in anthocyanins (C/C0 ) was affected by both temperature and sugar
concentration (Figure 4), and the logarithm of K_Anthocyanins is a
linear function of temperature and sugar concentration (Table 3).
According to Table 3, the logarithm of K_Anthocyanins increased linearly with temperature, but decreased in the same manner with
the sugar concentration during heating. This result indicates that
heating caused degradation of anthocyanins, while increased sugar
concentration provided some protective effect. Saccharides are effective for the stabilization of anthocyanins in foods (Wrolstad and
others 1990; Watanabe and others 2011), and the negative value
of c in Table 3 shows the protective effect.

Pasteurization of strawberry puree . . .

Table 4Survival counts of yeasts and molds in SP with different study also investigated the effect of sugar concentration on survival
sugar concentrations.
of E. coli O157:H7, anthocyanins degradation, and browning. The
Temperature ( C)
50

52

54

57.5

M: Food Microbiology
& Safety

60

Time (s)

8 o Brix

20 o Brix

40 o Brix

0
720
1440
2160
2880
3600
4320
0
360
720
1080
1440
1800
2160
0
180
360
540
720
900
1080
0
50
100
150
200
250
300
0
20
40
60
80
100
120

2.9
<1.5a
<1.5
<1.5
<1.5
<1.5
<1.5
2.9
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
2.9
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
2.9
2.3
<1.5
<1.5
<1.5
<1.5
<1.5
2.9
2.3
2.1
<1.5
<1.5
<1.5
<1.5

3.1
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
3.1
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
3.1
1.7
<1.5
<1.5
<1.5
<1.5
<1.5
3.1
2.5
1.9
<1.5
<1.5
<1.5
<1.5
3.1
2.5
2.4
2.0
<1.5
<1.5
<1.5

3.4
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
3.4
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
3.4
2.4
<1.5
<1.5
<1.5
<1.5
<1.5
3.4
3.1
2.5
<1.5
<1.5
<1.5
<1.5
3.4
2.8
2.7
2.4
<1.5
<1.5
<1.5

The detection limit is 1.5-log CFU/g in current study.

Microbial counts are the means of duplicated experiments.

temperatures by combining the D-values of all sugar levels available at 57.5 and 60 C. Using the combined D-values (all sugar
levels) at 57.5 and 60 C, the estimated z-value for YM in SP
was 11.2 C. Using the averaged D-value at 60 C as a reference
(57.8 s), the approximated D-value of YM was 452, 299, and 198 s
at 50, 52, and 54 C, respectively. These D-values were not accurate, but only provided rough estimations of the thermal resistance
of YM at these temperatures. Since the sampling intervals were
720, 360, and 180 s at 50, 52, and 54 C, they were probably longer
than 1 decimal reduction time (1D) at each heating temperature.
Since the initial YMC was approximately 3 log CFU/g in raw
SP, the YM counts were indeed reduced to below the detection
level after the first sampling point at 50, 52, and 54 C. Based
on the results listed in Table 4, the thermal processing conditions
used in this study were effective in eliminating the YM naturally
occurring in SP.

Conclusion
Contaminated strawberries are difficult to decontaminate for
direct human consumption. Thermal processing can be used to
eliminate E. coli O157:H7. The data from this study established
the thermal inactivation kinetics of E. coli O157:H7 in SP and
demonstrated the effect of heating on the degradation of anthocyanins and development of color in thermally processed SP. This

M80 Journal of Food Science r Vol. 79, Nr. 1, 2014

results of this study suggested that the thermal resistance of E. coli

O157:H7, anthocyanins, and Bi were affected by both temperature
and sugar concentration. The log D-values of E. coli O157:H7 decreased linearly with temperature, but slightly increased with sugar
concentration. For anthocyanins, the log of degradation rate increased linearly with temperature, but decreased slightly with sugar
concentration. The Bi was not affected by heating at 50 and 52
C at low sugar concentrations, but increased by approximately
1.28%, 2.21%, and 10.1% per min when SP was exposed to heating at 54, 57.5, and 60 C, respectively. The YM were reduced
to below detection levels after heating. The results of this study
may be used for establishing critical limits for thermal processing to eliminate E. coli O157:H7 in SP, while minimizing the
degradation of anthocyanins and development of color.

Acknowledgment
This project was supported by a USDA/NIFA grant (2011
6800320096) and National Science Council of Taiwan.

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