PEER-REVIEWED ARTICLE Frederick Breidt,*1 Kathryn Kay,2

Food Protection Trends, Vol 34, No. 3, p.132-138

Jason Osborne,3 Barbara Ingham4
International Association for Food Protection and Fletcher Arritt2
6200 Aurora Ave., Suite 200W, Des Moines, IA 50322-2864
*1
USDA-ARS Food Science Research Unit, 322 Schaub
Hall, Box 7624, North Carolina State University, Raleigh,
NC 27695, USA
Dept. of Food, Bioprocessing and Nutrition Sciences,
2

400 Sullivan Drive, North Carolina State University,

Raleigh, NC 27695, USA
Dept. of Statistics, 2311 Stinson Drive, Box 8203, North
3

Carolina State University, Raleigh, NC 27695, USA

Dept. of Food Science, 1605 Linden Drive, University
4

of Wisconsin, Madison, WI 53706, USA

Mention of a trademark or proprietary product does not
constitute a guarantee or warranty of the product by
the U. S. Department of Agriculture or North Carolina
Agricultural Research Service, nor does it imply approval
to the exclusion of other products that may be suitable.

Thermal Processing of Acidified Foods

with pH 4.1 to pH 4.6

ABSTRACT INTRODUCTION

S A
helf-stable acidified foods with a pH at or below ll processors of acidified and low-acid canned
4.6 must be processed to achieve a 5-log foods must register with the Food and Drug
reduction for vegetative bacterial pathogens. Administration (FDA) and file scheduled processes
Published research does not exist to adequately for each product that they manufacture. These process
support the Food and Drug Administration process filings must be supported by research that substantiates
filings for products with pH 4.1–4.6 or to define critical the lethality of the scheduled process for microbial
limits for acid and acidified foods with pH values in this pathogens. For acidified foods with a pH of 4.1 or lower,
range. Using a non-inhibitory vegetable-based medium, we research has defined heat processing conditions that
developed models and data for the thermal destruction ensure a 5-log reduction of the pathogens of concern,
of acid-resistant vegetative microbial pathogens, Escherichia coli O157:H7, Salmonella enterica and
including 5-strain cocktails of Escherichia coli O157:H7, Listeria monocytogenes (3, 5). These data identify the
Salmonella enterica, and Listeria monocytogenes in holding time at the recommended temperature for the
acidified foods with pH values of 4.1 to 4.6. Under the “cold spot” in the product container, as defined by a
experimental conditions, Listeria monocytogenes was process authority. However, no published peer-reviewed
the most heat- and acid-resistant pathogen. A z-value of documented research exists to support FDA process
16.7°F, an F-value (at 160°F) of 5.6 min, and a table of filings for processing acidified food products with pH
recommended processing conditions were estimated from 4.1–4.6. This lack of documentation is challenging to
the thermal processing data. This work addresses a lack all areas of the industry but especially so for small food
of documentation that is challenging to all areas of the processors that lack the resources to support challenge
industry, especially small processors. studies conducted by private laboratories.

* Corresponding author: Phone: +1 919.513.0186; Fax: +1 919.513.0180; E-mail: [email protected]

132  Food Protection Trends May/June

To conduct studies on the safety of acidified food, laboratory hard to quantify and may vary with cultivar, growing season,
conditions should be established that allow equal or greater weather, and other factors. Therefore, CJ was chosen to
survival of the target vegetative microbial pathogens, represent a “worst-case” condition for the survival of bacterial
compared with survival under the conditions of the relevant pathogens in thermal processing studies. Results of studies
food products. For this reason, previous studies of thermal that use CJ medium can be viewed as applicable to a variety
processing or acid killing of vegetative pathogens in acidified of acidified foods. Research with acetic acid and gluconic acid
vegetables were done with cucumber juice medium (CJ, (as a non-inhibitory buffer) in CJ medium (2–4) has been
to be described later) or cucumbers in a low-salt (2% or used to support process filings for a variety of acidified food
less) brine as a medium representative of various acidified products that may or may not contain cucumbers, including
vegetable products (1–4). Cucumbers do not contain flavorings, syrups, dressings, toppings, sauces, salsas, and
compounds that are known to be inhibitors of microbial others
survival or growth, but cucumbers do contain amino acids, (F. Arritt and B. Ingham, personal communication).
sugars, and other compounds that may aid in the survival The objective of this research was to define thermal
of bacterial pathogens (1, 7). Many other vegetables or processing conditions that will ensure a 5-log reduction of
ingredients typically present in acidified vegetable products, vegetative bacterial pathogens in acidified food products
including cabbage, peppers, garlic, horseradish and others, that have pH values above 4.1 and below 4.6. Acetic acid
contain antimicrobial compounds (8, 9). However, use of is perhaps the most common organic acid used in acidified
media containing these natural antimicrobials for pathogen foods, particularly acidified vegetables. Acetic acid was
reduction studies with acidified foods is problematic, because therefore included in CJ for thermal processing studies.
the active concentration of these natural antimicrobials is Gluconic acid, which has been shown to function as a non-

TABLE 1. Bacterial strains

Strain ID Strain Name Previous IDa Food Origin

B0195 Listeria monocytogenes SRCC 529 Pepperoni
B0196 Listeria monocytogenes SRCC1791 Yogurt
B0197 Listeria monocytogenes SRCC 1506 Ice cream
B0198 Listeria monocytogenes SRCC 1838 Cabbage
B0199 Listeria monocytogenes SRCC 2075 Diced coleslaw
B0200 Escherichia coli O157:H7 ATCC 43888 Laboratory strain
B0201 Escherichia coli O157:H7 SRCC 1675 Apple cider outbreak
B0202 Escherichia coli O157:H7 SRCC 1486 Salami outbreak
B0203 Escherichia coli O157:H7 SRCC 2061 Ground beef
B0204 Escherichia coli O157:H7 SRCC 1941 Pork
B0206 Salmonellab Braenderup SRCC 1093 10% salted yolk
B0207 Salmonella Cerro SRCC 400 Cheese powder
B0208 Salmonella Enteritidis SRCC 1434 Ice cream
B0209 Salmonella Newport SRCC 551 Broccoli with cheese
B0210 Salmonella Typhimurium SRCC 1846 Liquid egg

SRCC strains obtained from Silliker, Inc., Chicago, IL; ATCC, American Type Culture Collection, Manassas, VA; ID,
a

identification
b
Salmonella enterica strains with the serotype (non-italicized)

foodprotection.org Food Protection Trends   133

 99 FIGURE 1. Thermal death time data
for E. coli O157:H7 in 100 mM (0.6%)
acetic acid, pH 4.6, at 133°F (56°C).
88 Three independent replications of the
S urv iv ors (L og C F U/ml)

data are shown (triangles) for the log

CFU/ml survivors vs. time (min). The
77
Survivors (log CFU/ml)

fitted Weibull model for these data is also

shown (solid line).
66

55

44

33

22
00 20
20 40
40 60
60 80
80 100
100 120
120 140
140
TTime
ime (min)
(min)

inhibitory buffer in CJ in studies of E. coli O157:H7 and L. monocytogens and S. enterica strain cocktails, and Luria
related strains of this species under acid conditions (1), agar for plating E. coli O157:H7 cocktails, were obtained
was also tested in place of acetic acid with E. coli O157:H7 from BD Biosciences (BD Biosciences, San Jose, CA). All
to determine the effect of pH on the effectiveness of heat chemicals used in the study were obtained from Fisher
processing in the absence of an inhibitory organic acid effect Scientific (Itasca, IL) unless otherwise specified.
on bacterial survival.
Thermal processing
MATERIALS AND METHODS Bacteria used in this experiment consisted of cocktails of
Media five strains each of three foodborne pathogens, as shown in

T he CJ medium used to determine 5-log reduction

values for target pathogens was prepared from brined
cucumbers as previously described (2). Size 2A pickling
Table 1. Bacteria were grown statically at 37°C for 16 h in 5
ml tryptic soy broth (TSB, Difco, Becton Dickinson, Sparks,
MD, for Listeria) or Luria broth (LB, Becton Dickinson,
cucumbers of mixed varieties (about 2 cm in diameter) for Salmonella and E. coli) modified to contain 1% glucose
were obtained from a commercial processor. Approximately to induce acid tolerance, as previously described (1, 3, 6).
640 g of cucumbers (Cucumis sativus) were placed in a The five cultures of each species were each grown separately,
1.4-liter jar with 640 ml of brine. The brine contained 2% harvested by centrifugation (3,000 × g, 10 min, 10°C, Sorvall
NaCl and 100 mM (0.6%) acetic acid, with the balance Superspeed Centrifuge, SS-34 rotor, DuPont Instruments,
sterile H2O, at pH 4.6 after equilibration. For some E. coli Newton CT), re-suspended in 0.5 ml sterile saline (0.85%
O157:H7 experiments, the acetic acid was replaced with 20 NaCl) and combined into a single-pathogen inoculum
mM (0.4%) gluconic acid. The pH was adjusted by adding cocktail. A 1.5 ml aliquot was added to 150 ml of CJ medium
predetermined amounts of HCl, based on a titration of the in a 300 ml water-jacketed fermentation flask, to give a final
blended cucumbers. The brined cucumbers were pasteurized cell concentration of approximately 108 CFU/ml. Prior to
at 165°F (74°C); internal temperature at the cold spot in inoculation, flasks were equilibrated at a temperature of 133°
the jar) for 15 min in a steam-heated waterbath and left at to 151°F (56° to 66°C, to be indicated later), with magnetic
room temperature (23° + 2°C) for 1 week or longer to allow stirring, using a heating-cooling waterbath (Neslab RTE-
equilibration of sugars, amino acids, and other compounds 111, Newington, NH). CJ temperature was confirmed using
between the brine and cucumbers. Following equilibration, sterile type ‘T’ thermocouples inserted into the medium,
the brine was removed from the jars as needed and aseptically and recorded with a data-logging apparatus (Omega 3000,
transferred to double walled sterilized fermentation jars for Omega Engineering, Inc., Stamford, CT). The CJ in the
thermal processing studies. Tryptic soy agar, used for plating flasks was inoculated through a rubber septum, using a

134  Food Protection Trends May/June

 TABLE 2. Estimated 5-log reduction times

Pathogen Name Temp°C Temp°F 5LR a Std. Err.b

56 132.8 126.10 5.30

58 136.4 88.79 4.23

60 140 95.74 4.32
Escherichia coli O157:H7
62 143.6 56.00 1.82
64 147.2 24.06 1.07
66 150.8 11.71 0.64
56 132.8 150.73 7.74
60 140 87.46 4.77
Salmonella enterica
64 147.2 24.55 1.14
66 150.8 10.55 0.50
56 132.8 156.70 8.66
60 140 125.31 8.64
Listeria monocytogenes
64 147.2 28.75 1.46
66 150.8 14.32 0.86

a
Estimated 5-log reduction time in minutes
b
Standard error for the 5-log reduction time

3 ml syringe. A nitrogen gas blanket was maintained nonlinear regression model for time-temperature survival data
over the liquid using filtered (0.2 um) industrial grade (5-log reduction values, 5D) was fit, assuming normally
N2 gas (≤20 ppm O2; Air Products, Raleigh, NC). At distributed errors with constant variance and mean function:
indicated time intervals, depending on temperature,
1 ml of the cell suspension was withdrawn through the log10 (S(τ)) = No − 5(τ /t*)β
septum and 0.1 ml was immediately cooled by dilution
into 0.9 ml of pH 7.0 MOPS (3-[N-morpholino] with No = the initial cell count, τ representing the
propanesulfonic acid, Sigma-Aldrich Chemical Co., St. observation times, t* the 5-log reduction time, and β a
Louis, MO) at room temperature (23 + 2°C), followed shape parameter for the Weibull curve. Linear regression
by serial dilution in sterile saline (0.85% NaCl) and was used for estimating the z-value, using the log10 5-D
plating on tryptic soy agar or Luria agar, using an values estimated for each temperature, as previously
automated spiral plater (Autoplate 4000, Advanced described (3, 5). A reference temperature of 160°F (71.1°C)
Instruments, Norwood, MA). Plates were incubated was used for F-values. Curve fitting was done with custom
at 37°C for 24 h prior to determination of log CFU/ Matlab algorithms or SAS software (SAS Institute, Cary,
ml with an automated plate reader (QCount, Advanced NC), using proc NLIN for the Weibull model.
Instruments). Three or more independent replications
of each experiment were carried out. RESULTS

Modeling
To determine D- and z-values, a combination of Weibull and
T hermal death-time curves were generated for
the 5-strain single-pathogen cocktails of E. coli
O157:H7, S. enterica, and L. monocytogenes. A typical
linear models was used, as previously described (3). A Weibull survival curve (for E. coli O157:H7 in CJ with acetic acid

foodprotection.org Food Protection Trends   135

 2.4
2.4 FIGURE 2. The log 10 5-log
reduction times for cocktails of
E. coli O157:H7 at pH 4.6 for
2.2
2.2 131°F to 151°F (56–66°C) in
0.6% acetic acid (Triangle up),
min.)

2.0 E. coli O157:H7 in gluconic

2.0 acid buffer (Triangle down),
min.)

S. enterica in 0.6% acetic acid

1.8
1.8 (circles), and L. monocytogenes
og1010

in 0.6% acetic acid (diamonds)

Time(L(log

are shown. The dashed line was

1.6
1.6 determined by linear regression
for the entire data set, with an
T ime

1.4 R 2 = 0.90; the solid line was

1.4 based on the L. monocytogenes
regression (R 2 = 0.91) with F 160
1.2
1.2 increased to 5.6.

1.0
1.0
132
132 135
135 138
138 141
141 144
144 147
147 150 150 153153

(o F )
Temperature (ºF)
T emperature

TABLE 3. Table of z and F values at pH 4.6

Pathogen Name z-val (°F)a Rsqb F160c

Escherichia coli O157:H7d 17.4 0.91 4.44

Salmonella enterica 15.6 0.96 3.34

Listeria monocytogenes 16.7 0.91 4.89

Combinede 17.1 0.90 4.30

a
Estimated z-value in °F
b
R-squared value for the regression line used to estimate the z-value
c
The processing time at 160°F in min
d
Combined data for acetic acid and gluconic acid buffer experiments
e
Combined data for all experiments

at 56°C, 133°F) is shown in Fig. 1. The 5-log reduction S. enterica or E. coli O157:H7, depending on temperature
times for E. coli O157:H7, S. enterica, and L. monocytogenes (Table 2).
in CJ with acetic acid at pH 4.6 are presented in Table 2. For data on each pathogen, as well as for the combined
The results for thermal death experiments for E. coli in CJ data on all species tested, z-values based on the 5-log
with gluconic acid showed that the 5-log reduction times reduction times were estimated (Table 3). E. coli O157:H7
were equal to or less than the 5-log reduction times for each had the highest z-value (17.4°F) with a standard error (SE)
temperature tested with acetic acid (data not shown). For of 2.0°F. The z-values for S. enterica and L. monocytogenes
all temperatures and treatments, L. monocytogenes was the were 15.6°F (1.4) and 16.7°F (2.1), respectively (SE in
most heat and acid-resistant organism tested, followed by parentheses). Figure 2 shows the z-value plot for the

136  Food Protection Trends May/June

 TABLE 4. Recommended processing conditions for acidified foods (pH 4.1 to 4.6) to
achieve a 5-log pathogen reduction

Temp°C Temp°F 5LR (min)a Temp°C Temp°F 5LR (min)

60.6 141 77.8 71.7 161 4.9

61.1 142 67.7 72.2 162 4.3

61.7 143 59.0 72.8 163 3.7

62.2 144 51.4 73.3 164 3.2

62.8 145 44.8 73.9 165 2.8

63.3 146 39.0 74.4 166 2.5

63.9 147 34.0 75.0 167 2.1

64.4 148 29.6 75.6 168 1.9

65.0 149 25.8 76.1 169 1.6

65.6 150 22.4 76.7 170 1.4

66.1 151 19.5 77.2 171 1.2

66.7 152 17.0 77.8 172 1.1

67.2 153 14.8 78.3 173 0.9

67.8 154 12.9 78.9 174 0.8

68.3 155 11.2 79.4 175 0.7

68.9 156 9.8 80.0 176 0.6

69.4 157 8.5 80.6 177 0.5

70.0 158 7.4 81.1 178 0.5

70.6 159 6.5 81.7 179 0.4

71.1 160 5.6 82.2 180 0.4

Recommended processing time (5-log reduction time) in min

a

combined data set (z = 17.1°F) as well as the z-value line O157:H7, 3.3 min for S. enterica, and 4.9 min for
for recommended processing conditions, which had a L. monocytogenes (Table 3).
z-value of 16.7°F and an F-value at 160°F (F160) of 5.6
min. The processing time at a reference temperature of DISCUSSION
71°C (F160) was estimated from observed data based on
the average 5-log reduction time being 4.4 min for E. coli R esults from previous studies of non-thermal processing
conditions for acidified foods showed that E. coli

foodprotection.org Food Protection Trends   137

O157:H7 was significantly more acid resistant than 16.7°F and an F160 of 5.6 min is shown as the solid line
S. enterica or L. monocytogenes at 10°C (50°F) in cold-fill- in Fig. 2. Based on these z- and F-values, a table of
hold acidified food products at pH 3.3 to 3.8 (3, 5). Data recommended processing conditions was generated in
on previous thermal processing at pH 4.1 showed that E. coli the range of temperatures typically used for commercial
O157:H7 and L. monocytogenes had similar heat and acid processing of acidified vegetables (Table 4).
resistance (3). By analysis of the estimated z and F values Previously, five times the standard error estimate was added
from the current study, it was found that E. coli O157:H7 to the 5-log reduction times as an arbitrary safety factor for
was more heat resistant than S. enterica and L. monocytogenes recommended thermal processes (3). For recommended
at temperatures above 74°C (166°F), but less resistant thermal processing times and temperatures for acidified
than L. monocytogenes below that temperature. Below 74°C foods using the data in Table 4, the authors suggest that
(166°F), L. monocytogenes was more heat resistant than cells a competent process authority should be consulted to
of the other two species. The physiological basis for this determine if any additional safety factor is needed.
difference is unclear. To derive a single set of parameters that
will ensure safety under all processing conditions, the F160 ACKNOWLEDGMENTS
for L. monocytogenes (which had a z-value of 16.7°F) was
increased from 4.8 min (as shown in Table 3) to 5.6 min. The
resulting thermal processing recommendation, a z-value of
T he authors acknowledge Ms. Sandra Parker for excellent
editorial assistance. This work was supported in part by a
U.S. Department of Agriculture (USDA) National Integrated
16.7°F and a F160 of 5.6 min, allowed achievement of a 5-log Food Safety Initiative grant (2011-51110-31019), Pickle
reduction of all strains tested for any temperature in the range Packers International, Inc. (Washington, D.C.), and USDA
tested and used for the recommended processing times and Cooperative State Research, Education, and Extension
temperatures. A linear model, corresponding to a z-value of Service project 2006-01240.

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138  Food Protection Trends May/June