***Salmonella* spp. and *Listeria monocytogenes* Risk Assessment for
Production and Cooking of Blade Tenderized Beef Steaks**

*Principal Investigators:*

> James L. Marsden, Ph.D. <jmarsden@oznet.ksu.edu>
>
> Randall K. Phebus, Ph.D. <rphebus@oznet.ksu.edu>
>
> Harshavardhan Thippareddi, Ph.D. <hthippar@oznet.ksu.edu>
>
> Curtis L. Kastner, Ph. D. <ckastner@oznet.ksu.edu>
>
> James B. Gosch, GRA <jgosch@oznet.ksu.edu>

*Submitted to :*

> National Cattlemen\'s Beef Association

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Department of Animal Sciences and Industry

Kansas State University

Manhattan, KS 66506

## ABSTRACT

Thermal destruction of *Salmonella* spp. and *L. monocytogenes* in blade
tenderized steaks cooked to target end point temperatures of 120, 130,
140, 150, 160, and 170°F on a natural gas commercial grill was
evaluated. Top sirloin subprimals were inoculated with five strain
cocktails of *Salmonella* spp. and *L. monocytogenes*, separately to a
level of ca. 10^7^ cfu/cm^2^ on the top surface. Steaks of varying
thicknesses (i.e. weights) were cut from each of the subprimals and
cooked to one of the six endpoint temperatures. After removal from the
oven, steaks were immersed in an ice bath to halt thermal lethality, and
cooled to a temperature below 100F before sampling. Although differences
(p0.05) were observed in *Salmonella* spp. reductions on cooking steaks
obtained from blade tenderized and non-tenderized subprimals, cooking to
target endpoint temperatures of 130°F resulted in similar reductions in
*Salmonella* spp. A target internal temperature of 120F produced greater
(p0.05) log reductions (CFU/g) in non-tenderized (NT) vs. tenderized (T)
steaks (3.8 log reduction vs. 2.7 log reduction). At 130F, T and NT
steaks produced 4.0 and 4.4 log reductions, respectively, which was not
significantly different, likely due to high standard deviations in
bacterial counts, especially in low weight steaks. Cooking of steaks
obtained from tenderized and non-tenderized subprimals showed
differences (p0.05) in *L. monocytogenes* reductions when cooked to
target internal end point temperatures of 120, 130, 140, 150 and 160 °F,
but not 170°F. Complete destruction of *L. monocytogenes* was observed
in steaks cooked to 170°F target temperature. It was observed that even
after immersion in an ice bath, internal temperatures continued to rise
above the target temperatures by as much as 11F (in 5 oz steaks). In
food service application, where steaks would not be rapidly cooled in
this manner, an additional margin of safety would be incorporated into
the cooking process due to this natural temperature rise after removal
from the grill. At cooking temperatures ranging from rare (130°F) to
well done (170°F), there were no differences in *Salmonella* spp.
between intact and non-intact steaks using a natural gas commercial
grill, although differences in *L. monocytogenes* reductions were
observed. Both intact and non-intact steaks are safe for consumers when
cooked to the endpoint temperatures 140°Fby the natural gas commercial
grill method.

**INTRODUCTION**

The meat industry must provide consistent quality and uniformity in its
products to remain competitive. The quality characteristic that has
received primary focus is tenderness. The primary technologies used to
ensure tenderness and retail cut uniformity are blade tenderization and
restructuring; technologies very effective and critical to the economic
well being of the meat industry. Restructured meat products that result
from the fibrinogen technology offer advantages to the industry and
consumers. High quality products can be created from lower-cost muscle
pieces. Precise control over composition, shape, and portioning are
advantages of restructuring.

Blade tenderization is one of the most effective methods of meat
tenderization, particularly for cuts higher in connective tissue. It
also can significantly reduce the variability in tenderness regardless
of the muscle. Even though the generic microbiological quality of blade
tenderized muscle has been shown to be equivalent to non-tenderized
controls on a per gram basis (Boyd et al., 1978), bacteria may be
translocated into the muscle. Therefore, research is required to
quantify this bacterial relocation, identify critical control points to
minimize the risk for pathogenic contamination, and define effective
cooking schedules for resultant products.

In November 1997, the National Advisory Committee on Microbiological
Criteria for Foods (NACMCF), Meat and Poultry Subcommittee stated that
*Due to the low probability of pathogenic organisms being present in or
migrating from the external surface to the interior of beef muscle, cuts
of intact muscle (steaks) should be safe if the external surfaces are
exposed to temperatures sufficient to effect a cooked color change.*
However, if meat is restructured so that original external surfaces are
repositioned to the interior, as in restructured (fibrinogen) steaks,
that interior will become contaminated. Additionally, if the surface of
an intact muscle or muscle system is violated by mechanical
tenderization (blade tenderization), contamination may be carried from
the surface to the interior of the cut. The NACMCF Meat and Poultry
Subcommittee also stated in 1997 that there is a lack of scientific data
to address the hazards associated with those processes that may cause
translocation of pathogens to the interior of meat cuts. Because of the
widespread use of these two technologies and the potential food safety
risks they may pose, the recently formed Beef Industry Food Safety
Council (BIFSCO) identified this research as a priority for the beef
industry. This industry group facilitates input from producers, packers,
processors, distributors, restaurants, and food retailers in an effort
to identify and implement workable *E. coli* O157:H7 controls in the
beef industry.

Kansas State University has successfully performed a similar project
\"*Escherichia coli* O157:H7 Risk Assessment for Production and Cooking
of Blade Tenderized and Fibrinogen Process Beef Steaks\" and presented
results to USDA-FSIS during the public meeting held in Washington, D.C.
on March 8, 1999. Results indicate that the blade tenderization process
transfers 3-4% of surface contamination to the interior of the muscle.
Cooking studies indicated that a target internal temperature of 120F
produced greater (p0.05) log reductions (CFU/g) in non-tenderized (NT)
vs. tenderized (T) steaks (5.2 log reduction vs. 3.2 log reduction),
showing that blade tenderized steaks presented an increased risk only
when cooked to very low internal temperatures. However, at endpoint
temperatures of 140, 150, 160 and 170F, log reductions were greater than
6 logs in both T and NT steaks, which represented virtually complete
destruction. At cooking temperatures ranging from rare (130°F) to well
done (170°F), there were no differences in *E. coli* O157:H7 between
intact and non-intact steaks using the oven broiling method. It was
concluded that both intact and non-intact steaks are safe for consumers
when cooked to the endpoint temperatures 140°F by the oven broiling
method. Although this study evaluated the risk of *E. coli* O157:H7,
other foodborne pathogens such as *Salmonella* and *Listeria
monocytogenes* have not been considered. It is possible that the
translocation of these pathogens could be different compared to *E.
coli* O157:H7 and further, the cooking protocols required for
inactivation of *Salmonella* and *Listeria monocytogenes* could be
different.

**Therefore, the blade tenderization technology requires research to
determine the process and preparation protocols required to ensure the
safety of these effective and widely used technologies**. Unlike ground
beef which is now almost exclusively cooked to 160F by food service
establishments as a result of USDA and state health department
recommendations, mechanically tenderized beef steaks are often perceived
as whole muscle cuts and are, thus, prepared to customer orders of rare
to medium doneness, which may pose a health risk to consumers. By
validating the process and cooking protocols against *Salmonella* spp.
and *Listeria monocytogenes*, suppliers and food service will be able to
effectively establish standard cooking recommendations for food service
HACCP programs for preparation of these products.

**OBJECTIVE**

The objective of the study is to determine the effectiveness of rare to
well done cooking temperatures on reducing *Salmonella* spp. and
*Listeria monocytogenes* populations contaminating the interior of meat
cuts as a result of blade tenderization of artificially contaminated
beef sub-primal surfaces.

**MATERIALS AND METHODS**

**[Preparation and Cooking of Blade Tenderized Steaks]{.underline}**:
Top sirloins (loin, beef top sirloin butt, center cut, boneless, cap
off; IMPS 184B) were surface inoculated (non-fat side) to target
contamination level of ca. 10^7^ *Salmonella* spp. and *Listeria
monocytogenes* /cm^2^. After an attachment period of 2 hrs, one set of
top sirloins were blade tenderized (fat side down) using two passes
through a Ross tenderizer (Fig. 1) with the units conveyor moving 1.25
inches (31.75 mm) forward and 0.5 inches (12.7 mm) laterally between
each blade cycle. The 448 blades (Fig. 2) of the tenderizer produce 32
incisions per square inch with each passage through the unit (Figs. 3
and 4). The unit was thoroughly sanitized and disinfected between each
subprimal processed. Matching top sirloins were inoculated, sliced, and
cooked but did not receive the blade tenderization treatment. Tenderized
and control subprimals were fabricated using North American Meat
Processors guidelines. A cutting guide was used to uniformly cut steaks
of 0.5, 0.9 or 1.25 inch target thicknesses. Steaks were aseptically
trimmed on the non-inoculated fat surface to target weights of 5, 8 and
12 oz (142, 226 and 340 g 5 g), for respective thicknesses.

Steaks were held at 4C until cooking (ca. 1 hr). A Type T thermocouple
was inserted into the geometric center of each steak through the side of
the steak. Steaks were cooked on both sides to a specific end-point
temperature (uncooked, 120, 130, 140, 150, 160, and 170F) on a
commercial gas grill with 400 °F surface temperature. A sanitized weight
(1 lb) was placed top of each steak when the steak was placed on the
grill and a new sanitized weight was placed after the steak was flipped
upon reaching the mid point of the cooking process to prevent curling.
After reaching the target end-point, the steak was immediately removed
from the grill, placed in oven cook bags and submerged in an ice bath.
Temperature of the steaks was monitored until the steaks cooled to
100°F. A center slice of each steak was then blended to produce a
uniform sample and 25 g portion of the blended sample was stomached with
50 mL of peptone water (0.1%, PW) for 2 min. The samples were
microbiologically analyzed by plating on selective and non-selective
repair media for each organism. *Salmonella* spp. and *Listeria
monocytogenes* survival on non-tenderized control steaks was compared to
survival in tenderized steaks.

***[Salmonella spp. and Listeria monocytogenes Cultures:]{.underline}***
A five strain cocktail of *Salmonella* spp. and *Listeria monocytogenes*
was used in all investigations. Cultures were grown in tryptic soy broth
at 35C for 18 h to stationary phase, centrifuged and cell pellets from
all five strains resuspended in 0.1% peptone water to establish a
cocktail inoculum. The cocktail of each organism was mist inoculated on
the top sirloins to obtain a surface inoculum of ca. 10^7^ CFU/cm^2^.

**[*Enumeration of* *Salmonella spp. and Listeria
monocytogenes:*]{.underline}** Surviving *Salmonella* spp. and *Listeria
monocytogenes* populations were enumerated by direct plating on Xylose
Lysine De-oxycholate Agar (XLD) and modified Xylose Lysine De-oxycholate
Agar (mXLD). Samples were plated on Modified Oxford Agar (MOX) and
Tryptose Phosphate Agar (TPA) to recover the injured cells and estimate
the rate of injury. Each subprimal (blade tenderization study) used in
the study was mist inoculated with the 5 strain inoculum of each
pathogen, separately. After 2 hr attachment, 40 cm^2^ of inoculated meat
surface (ca. 2 mm depth) was excised and plated to confirm initial
surface inoculation levels (CFU/cm^2^).

After cooking to specified internal temperatures, a 2 in. wide strip
(obtained parallel to the path of the blade penetration) of each steak
was placed in a Waring blender jar, diluted 1:5 with 0.1% chilled
peptone water, homogenized for 1 min, and plated to enumerate surviving
*Salmonella* spp. and *Listeria monocytogenes* levels. Bacterial
population reductions due to cooking was determined based on a
comparison to inoculated but uncooked control steaks receiving the same
processing treatment. In cases where the specific cooking
time/temperature reduced the inoculated *Salmonella* spp. and *Listeria
monocytogenes* populations to below the detection level of direct
plating, samples were enriched according to modified USDA protocols.
Three replications of the experiment were performed with duplicate
steaks being cooked and analyzed for each cooking treatment within
replications.

***[Experimental Design:]{.underline}*** A 2 x 3 x 7 factorial
experimental design was used for the study, with two organisms
(*Salmonella* spp., and *L. monocytogenes*), three weight ranges (5, 8,
and 12 oz), and seven target endpoint temperatures (uncooked, 120, 130,
140, 150, 160, and 170 °F) as parameters. Three replications were
performed and the results were statistically analyzed using general
linear models procedures using SAS analytical software.

**RESULTS AND DISCUSSION**

Statistical analysis of all main effects (treatment, weights, and
temperature) and all possible combinations of interaction, with
bacterial reduction as the dependent variable for *Salmonella* spp.
reductions, revealed a significance (p0.05) for the main effects and an
interaction between the treatment (tenderized vs. non-tenderized) and
temperature and weight and temperature. For all temperatures (treatment
and thickness interaction), the *Salmonella* spp. reductions were
greater in non-tenderized steaks (4.42 log CFU/g) compared to tenderized
steaks (3.63 log CFU/g) for 5 oz steaks and the *Salmonella* spp.
reductions were similar for 8 and 12 oz steaks (p\>0.05) (Fig. 5).
Further, *Salmonella* spp. reductions during cooking of tenderized and
non-tenderized steaks were greater (p0.05) in non-tenderized steaks
(3.76 log CFU/g) compared to tenderized steaks (2.73 log CFU/g) at 120
°F, and the treatments had similar *Salmonella* spp. reductions
(p\>0.05) at 130, 140, 150, 160 and 170 °F. This finding indicates that
although tenderization of subprimals destined for further fabrication
into steaks results in survival of larger populations of *Salmonella*
spp. at 120 °F, tenderization did not result in survival of the pathogen
when the steaks were cooked to internal temperatures of 130 °F (rare),
the normal recommended cooking temperatures for beef steaks.

The *Salmonella* spp. reductions obtained in this study were lower than
the *E. coli* O157:H7 reductions reported by Sporing (1999) cooking to
the same endpoint temperatures of 120, 130, 140, 150, 160 and 170°F.
This probably was due to the use of a commercial grill using natural gas
compared to the oven broiler used by Sporing (1999), which results in
uniform surface temperatures compared to a commercial grill.

Cooking of steaks obtained from tenderized and non-tenderized top
sirloins inoculated with *L. monocytogenes* revealed differences for
main effects (treatment, weights and temperature) and no interaction
between the parameters. *L. monocytogenes* reductions were greater
(p0.05) in non-tenderized steaks compared to tenderized steaks except
for steaks cooked to 170°F, where complete reductions in *L.
monocytogenes* populations were observed for both the tenderized and
non-tenderized steaks (Fig. 6). The results for *L. monocytogenes*
reductions were different from *Salmonella* spp. and *E. coli* O157:H7
reductions from tenderized and non-tenderized steaks due to higher
surface inoculum achieved with *L. monocytogenes* due to modification in
the experimental procedure. This was achieved by re-suspending the *L.
monocytogenes* cell pellet in smaller volume of the PW compared to
*Salmonella* spp. and *E. coli* O157:H7 to achieve higher surface
inoculum level on the top sirloin surface. Further, reductions obtained
for *L. monocytogenes* were lower compared to the *Salmonella* spp. and
*E. coli* O157:H7 for each of the target temperatures, probably due to
higher thermal tolerance of *L. monocytogenes*. Although reductions
achieved on cooking tenderized steaks obtained from subprimals
inoculated with *L. monocytogenes*, the probability of high surface
contamination of subprimals with *L. monocytogenes* is very low.

Johnston et al. (1978) reported that salmonellae survived on both the
surface and in the core of mechanically tenderized roasts oven cooked to
an internal temperature of 130F. The authors hypothesized that the
presence of viable salmonellae on surface could be either due to purging
of the cells from the center or survival on the surface. Johnston et al.
(1978) concluded that tenderized roasts cooked to 130F may be a public
health problem. The reductions obtained at this target end point cooking
temperature were 3.44 and 4.03 log CFU/g in the present study for steaks
obtained from tenderized and non-tenderized sub-primals respectively,
and may not be sufficient to result in a 6.5 log reductions as required
by the proposed performance standards for *Salmonella* spp. in beef
products. However, the risk of these high levels of *Salmonella* spp.
during production of raw steaks is relatively low due to the minimal
exposure of the meat surfaces of the primals used for the steaks and the
use of intervention treatments used in the slaughter operations that
further reduce the *Salmonella* spp. contamination levels on the
carcasses.

Johnston et al. (1978) did not evaluate the effect of cooking
inoculated, non-tenderized roasts to 130F. Further, the authors used
beef roasts (5 lb) in the experiment, whereas the present study
evaluated the microbial reductions that could be achieved in steaks
processed from the roasts (subprimals) to more accurately resemble
restaurant operations. In addition, use of a larger mass of meat (5 lb)
and heating to an internal temperature of 130F would have resulted in a
further increase in temperature, higher than what we observed in our
study, resulting in higher lethalities and reductions in salmonellae.

The experimental design of this study incorporated the use of an ice
bath to rapidly halt thermal lethality in the steaks after removal from
the broiler, to provide more accurate information concerning thermal
destruction achieved at the identified target internal temperatures. It
is important to note that even after transfer to the ice bath, internal
temperatures of the steaks continued to rise above these target
temperatures, by as much as 11F (Figs. 7 and 8). Temperature increases
tended to be larger at the low target temperatures, and for 5 oz.
Steaks, but no significant differences were observed within treatments
or steak weights. In food service applications, the finished product
would not be cooled as in this scientific study, and internal
temperatures would likely rise to higher endpoints and be maintained for
longer periods of time, thereby resulting in increased margin of safety
in regard to pathogen destruction.

## SUMMARY AND CONCLUSIONS

Results show that there is no difference in risks between intact
(non-tenderized) and non-intact (tenderized) steaks at cooking
temperatures ranging from rare (130F) to well done (170F) for
*Salmonella* spp. However, differences in *Listeria monocytogenes*
reductions between steaks obtained from tenderized and non-tenderized
subprimals were observed on cooking to target temperatures of up to
160°F, and not 170°F. Sporing (1999) recommended a target endpoint
temperature of 140°F for cooking both tenderized and non-tenderized
steaks based on extensive studies with *E. coli* O157:H7 to reduce the
risk of this pathogen in steaks obtained from tenderized subprimals. The
present study shows that same target temperature would reduce the risk
of *Salmonella* spp and *L. monocytogenes* in steaks obtained from blade
tenderized subprimals. Baseline studies performed by USDA-FSIS indicates
very low contamination levels of these pathogens on beef carcasses and
concentrations of these pathogens would be even lower on cuts obtained
from interior muscles such as those used for fabricating the steaks.
Thus, both intact and non-intact steaks are safe for consumers, when
cooked to the internal temperatures of rare to well done using a
commercial grill.

In this study, we intentionally inoculated beef cuts with high levels of
*Salmonella* spp. and *Listeria monocytogenes* in order to quantify the
effect of mechanical tenderization on the translocation of bacteria from
the surface of the beef cuts into interior of the muscle. The levels of
contamination used do not reflect levels that are likely to be present.
In actual practice, the source point of contamination for these
pathogens is at the carcass level and contamination is prevented or
reduced through application of numerous processing steps, including
validated anti-microbial technologies and enforcement of USDA-FSIS's
zero tolerance policy for physical defects. The potential of
contamination is further reduced by the removal of the carcass surface
by trimming before mechanical tenderization.

A target internal temperature of 140F appears to provide the necessary
thermal destruction required to virtually eliminate *Salmonella* spp.
and *L. monocytogenes* risk. By including validated antimicrobial
intervention strategies in the slaughter and fabrication processes to
improve initial microbial quality of products destined for blade
tenderization, the likelihood of pathogenic contamination is further
decreased, thus decreasing the level of process lethality required
during cooking of tenderized cuts.

## REFERENCES

Johnston, R.W., M.E. Harris, and A.B. Moran. 1977. Effect of mechanical
tenderization on beef rounds inoculated with salmonellae. J. Food Safety
1:201-209.

SAS, 1990. Statistical Analysis System. User's guide. 4^th^ Ed. SAS
Institute, Inc., Cary, NC.

Sporing, S. B. 1999. *Escherichia coli* O157:H7 risk assessment for
production and cooking of blade tenderized beef steaks. M. S. Thesis.
Kansas State University, Manhattan, KS.

**LEGEND TO FIGURES:**

Fig. 1. Photograph of Ross TC700M Blade Tenderization unit.

Fig. 2. Photograph of blade head (produces 32 penetrations/in^2^) for
the Ross TC700M Blade Tenderization unit.

Fig. 3. Schematic of blade tenderization process, step 1 (prior to
penetration).

Fig. 4. Schematic of blade tenderization process, step 2 (during
penetration).

Fig. 5. Reductions in *Salmonella* spp. in tenderized and non-tenderized
steaks cooked to various endpoint temperatures.

Fig. 6. Reductions in *Listeria monocytogenes* in tenderized and
non-tenderized steaks cooked to various endpoint temperatures.

Fig. 7. Maximum internal temperatures reached in 5, 8 and 12 oz
tenderized and non-tenderized steaks cooked to six target endpoint
temperatures during cooking of steaks inoculated with *Salmonella* spp.

Fig. 8. Maximum internal temperatures reached in 5, 8 and 12 oz
tenderized and non-tenderized steaks cooked to six target endpoint
temperatures during cooking of steaks inoculated with *Listeria
monocytogenes*.

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