359

Journal of Food Protection, Vol. 74, No. 3, 2011, Pages 359–364

doi:10.4315/0362-028X.JFP-10-294
Copyright G, International Association for Food Protection

Effects of Sodium Citrate plus Sodium Diacetate and Buffered

Vinegar on Escherichia coli O157:H7 and Psychrotrophic Bacteria
in Brine-Injected Beef
AMUDHAN PONRAJAN,1,2 MARK A. HARRISON,1 JACOB R. SEGERS,2 BRADLEY K. LOWE,2 RUSSELL O. MCKEITH,2
T. DEAN PRINGLE,2 KARINA G. MARTINO,1 JAKE H. MULLIGAN,1 AND ALEXANDER M. STELZLENI2*

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1Department of Food Science and Technology and 2Department of Animal and Dairy Sciences, University of Georgia, Athens, Georgia 30602, USA

MS 10-294: Received 15 July 2010/Accepted 4 December 2010

ABSTRACT
The objective of this research was to examine the effects of sodium citrate plus sodium diacetate or buffered vinegar on
Escherichia coli O157:H7 and psychrotrophic bacteria when incorporated in brine solutions for injected beef. Two experiments
were conducted in which 30 top rounds and 30 top sirloins were injected (110%) to contain (i) 0.5% sodium chloride and 0.4%
sodium tripolyphosphate as the control (CNT); (ii) CNT with a 1% solution of 80% sodium citrate plus 20% sodium diacetate
(SCzD); or (iii) CNT with 2% buffered vinegar (VIN) in the final product. For the E. coli challenge, muscles were surface
inoculated to target 6 log CFU/cm2. After injection and 10 days of storage in a vacuum package (4uC), one half of each muscle
was sampled raw and the other half was cooked to an internal temperature of 60uC with a 12-min hold. For raw samples, a
significant reduction of 0.6 and 1.0 log CFU/g of E. coli O157:H7 was observed in both SCzD- and VIN-injected top rounds
and sirloins, respectively. All cooked samples were E. coli O157:H7 negative. For psychrotrophic analysis, subprimals were
injected and vacuum packaged for 10 days at 0 ¡ 1uC. After 10 days of storage, steaks were fabricated and placed in aerobic
display (4 ¡ 1uC) for 1, 7, 14, and 21 days. Psychrotrophic organism growth was restricted in SCzD and VIN samples when
compared with CNT on all days except day 1. Sodium citrate plus sodium diacetate or buffered vinegar may improve the safety
and shelf life of multineedle brine-injected beef.

Escherichia coli O157:H7 was first identified as a
foodborne pathogen in 1982, when it was associated with
two outbreaks of hemorrhagic colitis caused by the
consumption of undercooked frozen ground beef patties
(3, 7, 9). A 1993 multistate outbreak of E. coli O157:H7
associated with hamburgers at a national fast-food chain
increased the awareness about this pathogen (2). In
response, the U.S. Department of Agriculture, Food Safety
Inspection Service (USDA-FSIS) declared E. coli O157:H7
an adulterant in ground beef in October 1994 under the
Federal Meat Inspection Act (4, 16). In January 1999, the
USDA-FSIS further clarified that E. coli O157:H7 was also
considered an adulterant in nonintact and whole muscle,
nonintact beef (8, 15). However, three outbreaks of E. coli
O157:H7 in August 2000, June 2003, and August 2004
were associated with whole muscle, nonintact beef products.
In 2005, the USDA-FSIS mandated that processors
producing whole muscle, nonintact beef products must
reassess their hazard analysis and critical control point
plans, specifically addressing the biological hazard E. coli
O157:H7 (17).
E. coli O157:H7 adulteration of beef subprimals is
primarily from surface contamination during the harvesting
* Author for correspondence. Tel: 706-583-0398; Fax: 706-542-0399;
E-mail: astelz@uga.edu.
process. Sporing (13) has shown that when subprimals were
contaminated with pathogens such as E. coli (K-12), the
pathogen could be internalized during multineedle injection.
Current research has focused on a two-step injection process
to reduce or eliminate E. coli contamination, in which the
subprimal undergoes a surface decontamination step
followed by multineedle injection (4, 6). Little research
has been conducted to streamline this process by examining
the efficacy of including antimicrobials directly in the brine
solution (11, 19, 20). Furthermore, thermal processing has
been proven to be an effective method of reducing or
eliminating internalized pathogens in whole muscle, non-
intact beef in experimental settings (10, 13). However, the
thermal destruction of internalized pathogens needs to be
evaluated under simulated commercial conditions for roast
beef products. Therefore, the objectives of this research
were to examine the efficacy of sodium citrate plus sodium
diacetate or buffered vinegar in the brine solution (i) against
E. coli O157:H7 in beef top rounds and sirloins and (ii) on
psychrotrophic bacterial growth for beef steaks after
extended aerobic storage.
MATERIALS AND METHODS
Experiment 1: E. coli O157:H7 cultures. Four strains of E.
coli O157:H7 (ATCC 43888, human fecal isolate; E0122, cattle
isolate; K3995, spinach isolate; and F4546, alfalfa sprout outbreak
360 PONRAJAN ET AL.
isolate) were used for this study. E. coli O157:H7 strains were
activated by three consecutive transfers from fresh stock cultures
(280uC) into 9 ml of tryptic soy broth (Difco, BD, Sparks, MD)
containing 50 mg/ml of ampicillin (ampicillin sodium salt, Sigma-
Aldrich, St. Louis, MO) at 24 ¡ 2 h intervals incubated at 37 ¡
2uC. On the day of the experiment, 1 ml of the overnight culture
from each strain was transferred into 9 ml of fresh tryptic soy broth
plus ampicillin. Three-milliliter samples from each strain were
pooled together to form the initial inoculum cocktail (12 ml)
containing approximately 107 CFU/ml.
Experiment 1: meat procurement and inoculation.
Institutional Meat Purchase Specifications 169A beef top rounds
(30) and 184B beef top sirloins (30) from cull cows were obtained
(FPL Food, LLC, Augusta, GA) and transported (0 ¡ 2uC) to the
University of Georgia Meat Science and Technology Center
(Athens). On the day of the experiment, 10 top rounds or top
sirloins were placed in sterile stainless steel pans on sterile stainless
steel wire racks inside a biological safety cabinet (Class II, Type
A2, NuAire, Plymouth, MN).
To calculate the inoculation level, overnight cultures were
enumerated to determine the number of E. coli O157:H7 colonies
for each strain, and then calculations were made to determine how
many dilutions were needed and how many microliters of the
master inoculum were required to target 6.0 log CFU/cm2 for each
surface area. The calculated amount of E. coli O157:H7 was spot
inoculated on the lean surface of the muscle. Top rounds were
inoculated on three surfaces (two 8 by 8 cm and one 5 by 5 cm).
Top sirloins were inoculated on three surfaces (two 6 by 6 cm and
one 5 by 5 cm). Sterile aluminum templates were used as an
inoculation guide, and the corners of the designated area were
marked using dye (GL #80 Purple Brite, Great Lakes Meat
Branding Ink, Koch Supplies Inc., Kansas City, MO). The
inoculated beef was allowed to rest for 30 min, allowing the E.
coli O157:H7 to attach. To calculate the initial E. coli O157:H7
population on the inoculated beef surface, a portion (5 by 5 cm) of
each muscle was aseptically excised using sterile scalpel handles
and blades prior to enhancement.
Experiment 1: subprimal injection and storage. Inoculated
subprimals were injected through the lean face with one of three
solutions: 0.5% sodium chloride and 0.4% sodium tripolypho-
sphate as a control (CNT); CNT with a 1% solution of 80%
sodium citrate plus 20% sodium diacetate (SCzD) (IONAL LC,
WTI Inc., Jefferson, GA), or CNT with 2% buffered vinegar (VIN)
(MOstatin V, WTI Inc.) in the final product. A recirculating
multineedle injector with 21 needles (4-mm), operating at 41
strokes per min at 130 kPa (Injectamatic PI21, Koch Equipment
LLC, Kansas City, MO) was calibrated using noninoculated
muscles to deliver a 10% pickup. Brine solutions were made 12 h
prior to injection and mixed for 4 h. Immediately prior to use, the
brine solution was remixed to ensure a homogenous solution. Prior
to injection of treatment subprimals, two of the calibration
subprimals were surface inoculated (6.0 log CFU/cm2) to
intentionally contaminate the brine and needles to ensure that all
treatment subprimals were exposed to similar conditions. Samples
were collected from the brine solution: after mixing, after
contamination with calibration muscles, after injection of the fifth
subprimal, and after injection of the 10th subprimal. Brine pH was
also measured before injection. Additionally, the pH and weight of
the subprimals were measured before and after injection. After
injection, samples were vacuum packaged (30 to 50 ml of O2/m2/
24 h; 101,325 Pa; 23uC; B-620 series, Cryovac Sealed Air
Corporation, Duncan, SC) using a A300/16 (Multivac, Cryovac
J. Food Prot., Vol. 74, No. 3
Sealed Air Corporation) vacuum packager and stored in incubators
(LR5 TempGuard, MedRep, Inc., Newnan, GA) at 4 ¡ 2uC for
10 days to simulate transportation and storage.
Experiment 1: subprimal sampling. After 10 days of
storage, subprimals were removed from their vacuum package and
placed in sterile stainless steel pans. The pH and weight of the
samples were recorded. The subprimals were split into two halves,
with each top round half having one inoculation site (top round, 8
by 8 cm; top sirloin, 6 by 6 cm). One half of each muscle was
sampled raw; the other half was cooked to an internal temperature
of 60uC (with a 12-min hold) following an eight-step commercial
roast beef cycle using a commercial smokehouse (Alkar model
450, Alkar-RapidPak, Inc., Lodi, WI) and USDA-FSIS guidelines
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(14) for a 6.5 to 7.0 log reduction of Salmonella spp.
Sampling was conducted by turning the subprimal upside
down and aseptically removing a thin layer (0.2 to 0.3 cm) from
the bottom surface. A sterile stainless steel block template (5 by
5 cm) was used to create an impression on the shaved surface. The
subprimal was then cut along the impression of the template using
a sterile knife for each cut. The excised sample was placed on a
sterile cutting board and divided into three sections based on height
(top, middle, and bottom). Each section was aseptically separated
from the sample block. Sample sections were then placed into
stomacher bags. The cooked roast beef was sampled as early as
possible after thermal processing to prevent residual cooking.
Experiment 1: E. coli O157:H7 analysis. To analyze
surface inoculation counts, the samples (5 by 5 cm) were placed
into sterile stomacher bags with 225 ml of 0.1% peptone (Difco,
BD). The samples from each third of the raw and cooked beef were
weighed and placed in stomacher bags with 200 ml of E. coli
medium (EC medium), modified (Difco, BD) containing novobi-
ocin antimicrobic supplement (Difco, BD). All samples were
stomached (Stomacher 400 circulator, Seward Laboratory Systems
Inc., Bohemia, NY) for 2 min at 230 rpm. Serial dilutions were
made using 9 ml of 0.1% peptone. The brine samples were
analyzed using serial dilutions as above and were spiral plated
(Autoplate 4000, Spiral Biotech, Norwood, MA) onto tryptic soy
agar (BD, Franklin Lakes, NJ) containing 50 mg/ml of ampicillin
(Sigma-Aldrich). Plates were incubated at 37 ¡ 2uC for 24 ¡ 2 h
and enumerated using a colony counter (Reichert Analytical
Instruments, Depew, NY).
When E. coli O157:H7 was below the detection limit (1.6 |
101 CFU/g for 50 g of sample) the samples in EC medium were
enriched and incubated at 37 ¡ 2uC for 24 ¡ 2 h. After 24 h, 10 ml
of the EC medium from each sample was streaked onto tryptic soy
agar plus ampicillin plates and incubated an additional 24 h at 37
¡ 2uC. Suspected colonies were confirmed for E. coli O157 using
latex agglutination rapid detection kits (Dryspot E. coli O157 Latex
Kit, Oxoid Limited, Hampshire, UK).
Experiment 2: psychrotrophic bacteria. Similar procedures
for meat procurement and subprimal injection were followed as in
experiment 1, with the following changes. The subprimals were not
inoculated, and injections were conducted at the University of
Georgia Meat Science and Technology Center (Athens). After
injection, subprimals were vacuum packaged (30 to 50 ml of O2/
m2/24 h; 101,325 Pa; 23uC; B-620 series, Cryovac Sealed Air
Corporation) using a Type 800 vacuum packager (Original
Henkelman Vacuum Systems, Henkelman BV, The Netherlands)
and stored (0 ¡ 2uC) for 10 days. All equipment was thoroughly
cleaned between each treatment to avoid mixing treatment
solutions.
J. Food Prot., Vol. 74, No. 3 E. COLI AND PSYCHROTROPHIC BACTERIA INHIBITION IN BRINE-INJECTED BEEF 361

TABLE 1. Least square means of beef top round and top sirloin percent pickup, purge, and pH a
Brine solutionb:

Characteristic CNT SCzD VIN

Top rounds
Pickup, % 11.40 ¡ 0.55 10.28 ¡ 0.81 11.96 ¡ 0.62
Purge, % 1.76 ¡ 0.12 B 2.89 ¡ 0.27 A 2.84 ¡ 0.39 AB
pH, before injection 5.71 ¡ 0.03 Z 5.72 ¡ 0.05 Z 5.70 ¡ 0.04 Z
pH, after injection 6.11 ¡ 0.10 AY 5.79 ¡ 0.04 B YZ 5.92 ¡ 0.05 AB Y
pH, after 10 days 5.71 ¡ 0.05 BZ 5.89 ¡ 0.05 AY 5.83 ¡ 0.05 AB Y

Top sirloins
Pickup, % 11.44 ¡ 0.55 10.26 ¡ 0.54 11.09 ¡ 0.64
Purge, % 1.60 ¡ 0.11 2.59 ¡ 0.22 2.42 ¡ 0.26

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B A A
pH, before injection 5.62 ¡ 0.02 BZ 5.80 ¡ 0.03 AZ 5.82 ¡ 0.02 AZ
pH, after injection 5.88 ¡ 0.06 Y 5.88 ¡ 0.04 Z 6.00 ¡ 0.05 Y
pH, after 10 days 5.97 ¡ 0.04 AY 5.79 ¡ 0.04 BZ 5.74 ¡ 0.04 BZ

a
Values are means ¡ standard errors. Means within a row with different letters (A and B) differ (P , 0.05); means within a column,
subprimal, and trait with different letters (Y and Z) differ (P , 0.05).
b
CNT, 0.5% sodium chloride and 0.4% sodium tripolyphosphate; SCzD, CNT with a 1% solution containing 80% sodium citrate plus
20% sodium diacetate; VIN, CNT with 2% buffered vinegar in the final product.

Experiment 2: steak fabrication and sampling. After
10 days of storage, each subprimal was fabricated into four steaks
ca. 2.54 cm thick. The steaks were randomly placed on absorbent
pads (Dri-Loc AC-40, Cryovac Sealed Air Corporation) in
polystyrene trays (Cryovac Sealed Air Corporation) and wrapped
with an oxygen-permeable polyvinylchloride overwrap (O2 trans-
mission ~ 23,250 ml/m2/24 h, 72 gauge; Pro Pack Group, Oakland,
NJ). Steaks were stored at 4 ¡ 2uC with lighting to simulate a retail
display (960 lux) environment and were sampled for psychrotrophic
organisms on days 1, 7, 14, and 21. To sample each steak, a section
of surface area (5 by 5 cm) was aseptically excised to a depth of
0.2 cm. Samples were placed in sterile labeled stomacher bags.
Experiment 2: psychrotrophic organism analysis. Two
hundred twenty-five milliliters of 0.1% peptone was added to each
stomacher bag containing the sample (5 by 5 cm). The sample was
then stomached (Stomacher 400 circulator, Seward Laboratory
Systems Inc.) for 2 min at 230 rpm. Serial dilutions were made for
all samples using 9 ml of 0.1% peptone. The dilutions were then
spiral plated (Autoplate 4000, Spiral Biotech) onto plate count
agar, and the plates were incubated at 4 ¡ 2uC for 7 days. Plates
were counted using a colony counter (Reichert Analytical
Instruments).
Statistical analysis. For experiment 1, the data for treatment
effects on percent pickup, purge, pH, and E. coli O157:H7 counts
were analyzed as a completely randomized split-plot design with
the individual subprimals defined as the experimental unit and the
observational unit. Subprimal was nested within replication, and
subprimal within treatment was considered the random variable.
The E. coli O157:H7 counts from each third of the uncooked and
the cooked halves were converted to log CFU per gram. For
experiment 2, psychrotrophic counts were analyzed as a
completely randomized split-plot with subprimal as the whole
plot and steak randomly assigned to day of display as the split-
plot. The count from each sample was converted to log CFU per
square centimeter. Data were analyzed using the SAS v.9.1 system
(Proc Mixed; SAS Institute, Cary, NC). Means were separated
using the PDIFF option in LSMEANS with Tukey adjustment.
When an interaction was present, data were reanalyzed with the
BY statement. Differences among means were considered signi-
ficant at a # 0.05.
RESULTS AND DISCUSSION
Experiment 1: E. coli O157:H7 challenge. There was
no difference (P . 0.05) for percent brine pickup among
CNT, SCzD, or VIN for top rounds or top sirloins
(Table 1). The percent pickup averaged 11.21% for top
rounds and 10.93% for top sirloins. Percent purge after
10 days of vacuum storage exhibited a similar trend for top
rounds and top sirloins. Subprimals that received CNT had
1.13 (P , 0.001) and 0.99% (P , 0.05) less purge than
SCzD samples for top rounds and top sirloins, respective-
ly. However, purge loss was similar (P . 0.05) for CNT
compared with VIN and SCzD compared with VIN in top
rounds and top sirloins. Top round pH prior to injection was
similar (P . 0.05) among treatments. Immediately after
injection, CNT pH was higher (P , 0.05) than SCzD, and
VIN was similar (P , 0.05) to both CNT and SCzD
subprimals. After 10 days of storage, CNT pH was lower (P
, 0.05) than SCzD, and VIN was intermediate (P . 0.05)
to both treatments. There was an immediate pH increase (P
, 0.01) for CNT- and VIN-injected samples. However,
after 10 days of storage, CNT pH decreased (P , 0.01) to
preinjected levels, whereas SCzD pH continued to increase
(P , 0.05). Initial pH for top sirloins was lower (P , 0.05)
for CNT than SCzD and VIN. However, after enhance-
ment, CNT and VIN pH increased (P , 0.05), and all three
treatments were similar (P . 0.05). After 10 days of
vacuum storage, VIN pH decreased (P , 0.05) but was still
similar (P . 0.05) to SCzD. Top sirloin pH from CNT
continued to numerically increase and was greater (P ,
0.05) than SCzD- and VIN-enhanced samples after 10 days
of storage. In the current study, pH (initial, after
enhancement, or after 10 days of storage) did not influence
(covariate analysis) E. coli O157:H7. Currently there is no
literature describing how changes in meat pH after brine
362 PONRAJAN ET AL.
TABLE 2. Least square means for surface E. coli O157:H7 inoculation level and E. coli O157:H7 recovered from the top, middle, and
bottom thirds of enhanced raw beef top rounds and top sirloins a
Location CNT
Top rounds
Inoculation level (log CFU/cm2) 6.40 ¡ 0.06
E. coli O157:H7 (log CFU/g)
Top 1/3 5.46 ¡ 0.04 AY
Middle 1/3 4.67 ¡ 0.17 AZ
Bottom 1/3 4.47 ¡ 0.25 AZ
Top sirloins
Inoculation level (log CFU/cm2) 6.72 ¡ 0.07
E. coli O157:H7 (log CFU/g)
Top 1/3 5.98 ¡ 0.12 AY
Middle 1/3 5.51 ¡ 0.15 AZ
Bottom 1/3 5.31 ¡ 0.16 AZ
a
Values are means ¡ standard errors. Means within a row with different letters (A and B) differ (P , 0.05); means within a column,
subprimal, and third with different letters (Y and Z) differ (P , 0.05).
b
CNT, 0.5% sodium chloride and 0.4% sodium tripolyphosphate; SCzD, CNT with a 1% solution containing 80% sodium citrate plus
20% sodium diacetate; VIN, CNT with 2% buffered vinegar in the final product.
injection with antimicrobials influences the effectiveness of
the antimicrobials.
During the injection of top rounds and top sirloins no E.
coli O157:H7 was detected in the brine solution prior to
calibration. The E. coli O157:H7 level in the brine was
between 4.1 and 4.6 log CFU/ml for all treatments
immediately after injecting the inoculated calibration
subprimals, and it continued to increase between 4.7 and
5.2 log CFU/ml after injection of the 5th and 10th
subprimals for both top rounds and top sirloins.
Top round E. coli O157:H7 counts from the top third of
CNT were greater (P , 0.05) than SCzD and VIN samples by
0.40 and 0.31 log CFU/g, respectively (Table 2). However, there
was no difference (P . 0.05) between SCzD and VIN. In the
middle and bottom third of top rounds, CNT had greater mean E.
coli O157:H7 counts compared with SCzD and VIN (P ,
0.05). There was a mean difference of 0.66 and 0.63 log CFU/g
between the CNT versus SCzD and CNT versus VIN samples,
respectively. Injected top sirloin E. coli O157:H7 counts
followed a similar trend as top round samples (Table 2). CNT
top sirloin subprimals had more E. coli O157:H7 in the top third
when compared with VIN (P , 0.01), but there was no
difference between CNT and SCzD or SCzD and VIN
samples (P . 0.05). In the middle and bottom third, CNT
exhibited a greater mean E. coli O157:H7 count compared with
SCzD or VIN (P , 0.05). There was a mean difference of 1.02
and 1.06 log CFU/g between CNT versus SCzD and CNT
versus VIN, respectively. Among the three treatments, the top
third layer had greater E. coli O157:H7 counts compared with the
middle and bottom third layers (P , 0.05). E. coli O157:H7 was
not detected in any of the thirds from the cooked beef top rounds
or top sirloins (data not shown; detection limit 1.20 log CFU/g).
Experiment 2: retail display challenge. Extended
aerobic storage of top rounds showed that psychrotrophic
J. Food Prot., Vol. 74, No. 3
Brine solutionb:
SCzD VIN
6.38 ¡ 0.05 6.40 ¡ 0.05
5.06 ¡ 0.12 BY 5.15 ¡ 0.10 BY
3.74 ¡ 0.13 BZ 3.82 ¡ 0.11 BZ
3.82 ¡ 0.09 BZ 3.75 ¡ 0.09 BZ
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6.56 ¡ 0.04 6.56 ¡ 0.07
5.68 ¡ 0.16 AB Y 5.39 ¡ 0.12 BY
4.07 ¡ 0.09 BZ 4.15 ¡ 0.14 BZ
4.00 ¡ 0.12 BZ 4.07 ¡ 0.12 BZ
organisms increased (P , 0.05) until day 14 for CNT and
until day 21 for SCzD and VIN samples (Table 3). Steaks
enhanced with only sodium chloride and sodium tripolyphos-
phate had higher levels of psychrotrophic organisms on days
7, 14, and 21 compared with SCzD and VIN (P , 0.05).
There was a mean difference in psychrotrophic populations of
2.36 and 2.18 log CFU/cm2 between CNT and SCzD or
CNT and VIN steaks, respectively, across all days. Steaks
from SCzD had fewer (P , 0.05) psychrotrophic organisms
than VIN on day 14 (P , 0.01), but there was no difference
(P . 0.05) between SCzD and VIN on days 7 or 21. Similar
to top rounds, extended aerobic storage of enhanced top
sirloin steaks resulted in psychrotrophic bacteria increasing
until day 14 for CNT and until day 21 for SCzD and VIN
samples. Top sirloin CNT steaks had a higher level of
psychrotrophic organisms on all days of aerobic storage
compared with SCzD and VIN (P , 0.05). There was a
mean difference in psychrotrophic populations of 1.88 and
2.33 log CFU/cm2 between CNT and SCzD or CNT and
VIN for all days. Psychrotrophic counts for VIN were lower
than SCzD steaks on day 7 (P , 0.01), but there was no
difference (P . 0.05) on the other sampling days.
Beef destined for brine injection that is contaminated
with E. coli O157:H7 could pass contamination to the line,
needles, and brine, which could further contaminate
subsequent cuts. Once the brine solution becomes contam-
inated, pathogens are not only translocated from the surface,
but may be injected directly into the deep tissues. The
inclusion of antimicrobial agents in brine solutions could be
an effective method to decrease the risk of pathogen
transmission from cut to cut. Wicklund et al. (20) reported
that, when using recirculated brines with inoculated strip
steaks, steaks injected with fresh brine had greater counts
than steaks injected with recycled solutions. The study
revealed that each steak subjected to a particular injection
J. Food Prot., Vol. 74, No. 3 E. COLI AND PSYCHROTROPHIC BACTERIA INHIBITION IN BRINE-INJECTED BEEF 363

TABLE 3. Least square means for psychrotrophic bacteria for reduction of E. coli K-12. The results from the current
enhanced steaks from beef top rounds and top sirloins over 21 days study using a 1% solution of sodium citrate (80%) plus
of aerobic storage a diacetate (20%) or buffered vinegar (2%) are similar to
Brine solutionb: those from previous research.
Storage The USDA-FSIS recommends cooking roast beef for
day CNT SCzD VIN
12 min after the center temperature of the roast has reached
Top rounds 60uC to achieve a 6.5 to 7.0 log reduction of Salmonella
1 1.87 ¡ 0.15 Z 1.74 ¡ 0.09 Z 1.75 ¡ 0.08 Z
(14). The results from this research indicate that following
7 6.58 ¡ 0.10 AY 3.34 ¡ 0.14 BY 3.54 ¡ 0.13 BY USDA-FSIS guidelines for cook and hold times are also
14 9.47 ¡ 0.12 AX 5.76 ¡ 0.09 CX 6.20 ¡ 0.08 BX effective at eliminating E. coli O157:H7. In addition, Gill
21 9.63 ¡ 0.12 AX 7.27 ¡ 0.23 BW 7.33 ¡ 0.15 BW and McGinnis (5) cooked retail whole muscle, nonintact
Top sirloins beef from 63 to 68uC and reported that cooking whole
1 3.88 ¡ 0.17 2.65 ¡ 0.21 2.70 ¡ 0.11 muscle, nonintact beef to medium rare would be adequate

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AZ BZ
7 8.20 ¡ 0.10 AY 5.46 ¡ 0.23 BY 4.42 ¡ 0.26
14 9.46 ¡ 0.10 AX 7.66 ¡ 0.25 BX 7.02 ¡ 0.22
21 9.79 ¡ 0.05 AX 8.04 ¡ 0.18 BW 7.85 ¡ 0.09
a 2.6 to 4.2 log CFU/g reduction when the meat was cooked
Values are means ¡ standard errors presented in log CFU per
square centimeter. Means within a row with different letters
(A through C) differ (P , 0.05); means within a column and
subprimal with different letters (W through Z) differ (P , 0.05).
b
CNT, 0.5% sodium chloride and 0.4% sodium tripolyphosphate;
SCzD, CNT with a 1% solution containing 80% sodium citrate
plus 20% sodium diacetate; VIN, CNT with 2% buffered vinegar
in the final product.
treatment had received a different level of E. coli
contamination. In the current study the needles and brine
were intentionally contaminated prior to the initiation of the
study to ensure that each subprimal received the same
treatment. Furthermore, the inclusion of sodium citrate plus
sodium diacetate or buffered vinegar in the brine solution
did not inhibit E. coli O157:H7 in the brine solution when
compared with the control brine.
Although the exact composition of MOstatin V is not
known (18), the active ingredient of buffered vinegar is
acetic acid. The effects of acetic acid and citric acid, the
main ingredient of IONAL LC, have been shown to be
effective against E. coli O157:H7 (1). Typically, the main
method for inactivation of pathogens on brine-injected beef
has been to apply an antimicrobial to the surface of beef cuts
prior to injection. Surface decontamination has been shown
to be successful in reducing the E. coli O157:H7 load on
inoculated beef prior to brine injection by .1.0 log CFU/
100 cm2 (6). However, it was reported that 73 of 76 brine-
injected samples still had internal positive results for E. coli
O157:H7 (6). Evaluating the effectiveness of surface
decontamination on E. coli O157:H7–inoculated beef,
Echeverry et al. (4) reported that 1.0 to 3.0 log CFU/g E.
coli O157:H7 were transferred internally. Although signif-
icant reductions of E. coli O157:H7 were reported, this
research identifies a potential need for antimicrobials to
reach internal tissues. Furthermore, Paulson et al. (11)
reported that including 3.0% sodium lactate plus 0.25%
diacetate in a 0.3% salt and 0.3% phosphate brine solution
provided a greater coliform reduction in brine-injected strip
steaks than sodium lactate alone. Similarly, Wicklund et al.
(19) reported that sodium lactate or a combination of
sodium lactate and diacetate when incorporated in brines for
inoculated strip steaks resulted in 1 to 2 log CFU/g
BZ
CY for reducing aerobic bacteria. Similarly, Luchansky et al.
BX (10), using a commercial gas grill to cook E. coli O157:H7–
BW inoculated whole muscle, nonintact beef steaks, reported a
between 48.9 and 60uC. The current study supports that
cooking brine-injected roast beef to 60uC with a 12-min
hold under commercial conditions is an effective method for
eliminating internalized E. coli O157:H7.
Sallam (12) reported that dipping fresh salmon slices in
a 2.5% aqueous solution of sodium acetate, sodium lactate,
or sodium citrate could extend aerobic storage time by up to
42%, with sodium acetate having the greatest effect
followed by sodium lactate and sodium citrate. It was
reported that the control samples had 1.92 to 3.26 log more
psychrotrophic bacteria than the treated samples at the end
of the shelf-life study. These results are comparable to the
current study, where sodium citrate plus sodium diacetate
and buffered vinegar restricted the growth of psychrotrophic
bacteria over extended aerobic storage. Although aerobic
storage of steaks for 21 days is not done, these data show
that SCzD and VIN are effective at reducing the outgrowth
of psychrotrophic organisms in brine-injected top round and
top sirloin steaks.
This study indicates that sodium citrate plus sodium
diacetate or buffered vinegar can be included in enhance-
ment solutions for whole muscle, nonintact beef top rounds
and top sirloins as a hurdle against E. coli O157:H7. The use
of these antimicrobials in the brine solution will also control
the growth of psychrotrophic organisms during aerobic
storage of brine-enhanced steaks. Additionally, thermal
processing of injected beef top round and top sirloin
subprimals for 12 min after reaching 60uC internally was
effective at eliminating internalized E. coli O157:H7.
ACKNOWLEDGMENTS
The Georgia Food Processing Advisory Council funded this research
in cooperation with World Technology Ingredients Inc. and FPL Foods
LLC. Li Ma and Larry Beuchat, Center for Food Safety, University of
Georgia, Griffin, supported this project by providing the E. coli O157:H7
cultures.
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