5 

10 

15 

20 

25 

30 

35 

40 

1 APPLICATION OF NOVEL HURDLE TECHNOLOGIES TO MEAT CARCASS 

2 TRIMMINGS FOR REDUCTION OF PATHOGENS 

3 

4 

6 A Final Report to 

7 

8 USDA, FSIS, OPPD 

9 

Dr. David Zeitz 

11 Room 4803, South Building 

12 1400 Independence Ave., SW 

13 Washington, DC 20250-3700 

14 

USDA-FSIS Non-Assistance Cooperative Agreement # FSIS-C-14-2004 

16 

17 

18 

19 

From 

21 

22 Drs. Jimmy T. Keeton1

, Steve Ricke2, Robin Anderson3, Douglas Miller1, and Njongmeta Nenge 

23 Lynda Azefor4 (Ph.D. Candidate) 

24 

26 1Department of Animal Science, Texas A&M University, College Station, TX 77843 

27 2

 Department Food Science, University of Arkansas, AR 72704 

28 3USDA-ARS, Southern Plains Agricultural Research Center, Food and Feed Safety Research 

29 Unit, College Station, TX 77845

4

 Food Science, Texas A & M University, College Station, TX 77845-2472 

31 

32 

33 

34 

36 

37 

38 January 31, 2006 

39 

41 

42 Key words: Pathogen interventions, Salmonella Typhimurium, E. coli O157:H7 and Listeria 

43 monocytogenes, beef carcass 

1 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

EXECUTIVE SUMMARY 

More people are concerned about the safety of the food they consume than at any other 

time in history. Food-borne diseases are attributed largely to in-home contamination, but food 

processors bear the greatest responsibility for food safety. They are required by the United 

States Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) or Food and 

Drug Administration (FDA) to implement strict measures to reduce or eliminate any potential 

pathogen or hazard that might be introduced to the food during harvest, processing and handling. 

One of the largest challenges in the meat industry is keeping the product safe and free 

from contamination (Keeton and Harris 2004). Small meat plants face a particular challenge due 

to their limited personnel and financial resources. Salmonella Typhimurium (ST), Escherichia 

coli O157:H7 (EC) and Listeria monocytogenes (LM) are among the pathogens most commonly 

associated with contamination of meat and poultry products. These organisms pose a serious 

problem for decontamination of carcasses and/or ready-to-eat (RTE) products due to their ability 

to survive and grow in extreme and stringent conditions such as low pH or refrigeration 

temperatures. Contamination can be a problem, especially in small plants, since many small 

processors deal with both raw (e.g. refrigerated beef trim) and processed product (fully-cooked 

RTE items) in close proximity, thus increasing the risk of cross-contamination unless proper 

safety measures are implemented and strict control measures enforced. The use of organic acids 

in sprays for carcass decontamination and the effective reduction of pathogenic bacteria have 

been adopted by meat processors industry wide. Treatments designed for use in combination or 

in sequence, that are cost effective as pathogen reduction interventions for small meat processing 

plants, have not been validated. 

2 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

The objectives of this study were to evaluate undeveloped and cost effective pathogen 

interventions using a multiple hurdle approach suitable for small meat plant operations in an 

effort to further reduce contamination and growth of pathogens, specifically Salmonella spp., 

E.coli O157:H7 and Listeria monocytogenes. Pre-rigor, warm beef rounds were surface 

inoculated with a three-pathogen cocktail of rifampicn-mutant strains of Salmonella 

Typhimurium, E. coli O157:H7 and Listeria monocytogenes. After inoculation, the rounds were 

left at room temperature for 5 to10 minutes to allow for bacterial attachment, and then sprayed 

for 15 to 20 sec in a fixed pressure, self-contained spray cabinet. Six decontamination solutions 

were used: 1:4 acidified calcium sulfate (ACS, Safe2O RTE01®, Mionix Corporation, Rocklin, 

CA) : water, lactic acid (LA 2.5% L-lactic acid, Purac America, Inc., Lincolnshire, IL), 100 μL 

(100 ppm) epsilon-polylysine (EPL, Save-ory® PL-25, Chisso, Corporation, Tokyo, Japan), 1:4 

ACS RTE01 : water + 100 μL (100 ppm) ε-polylysine, (EPL), 100 μL (100 ppm) EPL, and 

sterile distilled water (W), and tested for effectiveness. All treatments, once diluted to 

appropriate concentration, were placed in stainless steel containers and heated to 50-55°C in a 

water bath. The solutions were then applied using a customized pressurized spray system with 

nozzles that delivered a specific volume (14 ml/sec) of the treatment solutions. The experiments 

were replicated three times. All recoveries of inoculated pathogens were done on aseptically 

sectioned 50 cm2

 portions. A designated portion was removed from each sample and combined 

separately with 20 ml of sterile PBS in stomacher bags. Counts of rifampicin-resistant E. coli 

O157:H7, Salmonella Typhimurium and Listeria monocytogenes were determined by plating 

appropriate dilutions onto pre-poured plates of a selective-differential medium, LSPR and 

Modified Oxford Agar base (MOX), respectively. 

3 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

Our results showed that a sequential application of warm (55°C) ACS followed by EPL at 

constant pressure for 15 to 20 sec to pre-rigor beef rounds significantly reduced inoculated levels 

of Salmonella Typhimurium, E. coli O157:H7 and Listeria monocytogenes with an extended 

effect over seven storage days. This combination was more effective than single treatments of 

ACS, LA, EPL or W alone. LM appeared to be more sensitive to the initial decontamination 

treatments on day 0 when compared to ST and EC, but unlike ST and EC did not show further 

reductions over a 7 day refrigerated storage period. This may have been due in part to LM’s 

increased tolerance to cold temperatures or later becoming resistant to EPL during storage. The 

separate modes of action of ACS and EPL might contribute to their synergistic effectiveness for 

inhibiting the growth of pathogens. 

From the observations in this study, it appears that a sequential application of ACS + 

EPL can be a better strategy for pathogen reduction in small meat plants than a single 

decontamination treatment, and could also provide a more ‘fail-safe’ pathogen reduction 

strategy. 

Further experiments are currently being performed to investigate the sequence of addition 

of ACS and EPL, the time interval between the applications (5 to 15 min) on pathogen reduction 

and the effects of varying the concentrations of each component. It is also worth investigating the 

antimicrobial effect of LA and EPL when applied in sequence. Additional work is needed to 

optimize our application techniques for reducing pathogens in small meat and poultry processing 

operations. When the treatment sequences have been optimized, the procedures will be tested in 

small meat plants in the College Station area. 

4 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

ABSTRACT 

The use of organic acids as beef carcass decontaminants in the meat and poultry industry 

is well documented. However, there is limited information on the use of these acids in a multiple 

hurdle fashion for small meat and poultry plants to reduce contamination and growth of 

pathogens, specifically Salmonella Typhimurium (ST), E. coli O157:H7 (EC) and Listeria 

monocytogenes (LM). This study evaluated the effectiveness of the application of warm 

solutions of 1:4 acidified calcium sulfate (ACS) RTE01:water, 2.5% lactic acid (LA), 1:4 ACS 

RTE01:water + 100 μL (100 ppm) epsilon-polylysine (EPL), 100 μL (100 ppm) EPL, and sterile 

distilled water (W) for reducing Salmonella Typhimurium, E. coli O157:H7 and Listeria 

monocytogenes on the surface of fresh, pre-rigor beef rounds secured from a local abattoir. All 

treatments were applied for 15 to 20 sec at 50-55°C under a constant pressure of 137.9 kPa (20 

psi) to deliver 0.2082 L/15 sec (14 ml/sec). ACS followed immediately with EPL significantly 

reduced levels of inoculated ST, EC and LM with mean log reductions of 2.26, 1.49 and 2.38, 

respectively, over a 7 day refrigerated storage period. ACS + EPL was more effective than single 

treatments of ACS, LA, EPL or W alone. Single treatments of ACS, LA and EPL were more 

effective than water alone for reducing ST and EC, but only LA was effective against LM. 

Warm water was the least effective for reducing pathogens. LM appeared to be more sensitive to 

the initial decontamination treatments on day 0 when compared to ST and EC, but unlike ST and 

EC did not show further reductions over a 7 day refrigerated storage period. Further studies are 

required to test the effects sequential application on the efficacy of the treatments and to 

determine if sequence of treatment application, duration of the treatment or frequency of 

exposure have an effect on the acquisition rates of antimicrobial resistance and virulence of the 

pathogens. 

5 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

INTRODUCTION 

Despite advances in technology and medical sciences, food-borne illnesses continue to be 

a serious public health problem and of significant concern to the food industry. The Centers for 

Disease Control and Prevention, one of the 13 operating components of the Department of 

Health and Human services, has estimated that there are 76 million cases of food-borne illness 

annually, resulting in 325,000 hospitalizations and 5,000 deaths (Mead and others 1999). 

Pathogen reduction strategies have been implemented by the regulatory agencies and the food 

industry to maintain consumer confidence at a time when consumers are increasingly concerned 

about the safety of what they eat. Agro-terrorism also is of concern due to the hazards that could 

exist if intervention measures are not put in place to detect, reduce or eliminate pathogens 

introduced intentionally into the food supply. 

Meat and poultry are among the top five items implicated in food-borne illness 

outbreaks. Pathogen-contaminated meat and poultry are thought to cause at least 2.5 million 

illnesses and 1,000 deaths every year (Frenzen and others 2000). As a result, several approaches 

have been developed to decontaminate meat and poultry products during the harvesting process 

(Mermelstein 2001; Connor 2001; Huffman 2002; White 2002). Some of these methods include 

cold and hot water rinses; steam pasteurization or steam vacuum treatment; carcass trimming; a 

variety of chemical rinses including chlorine/chlorine dioxide, ozonated or electrolyzed water, 

trisodium phosphate, acidified calcium sulfate, and organic acid rinses (such as lactic acid) with 

or without surfactants. Antimicrobial compounds may also be added to many ready-to-eat (RTE) 

products including sodium or potassium lactate, sodium diacetate, sodium citrate and a variety of 

antioxidant compounds that also exhibit antimicrobial properties including various spices or their 

6 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

extracts (e.g. rosemary extract), fruit preparations (e.g. dried plums) or synthetic antioxidants. 

Most of these individually will provide a 0.5 to 3 log reduction in pathogens, with water rinses 

being the least effective. A time lag between treatment of the carcass and fabrication into cuts or 

trimmings also can allow bacterial attachment to occur (biofilm formation) which decreases the 

effectiveness of most washing procedures. For these reasons, the immediate application post 

harvest of a combination of more than one intervention treatment to carcasses often has been 

found to produce a greater antimicrobial effect than any single treatment, often working in a 

synergistic manner. The latter has been referred to as hurdle technology. Combined hurdles have 

been found not only to enhance pathogen reduction, but also serve to improve the quality of 

meat/poultry resulting in more shelf-stable products. 

The United States Department of Agriculture-Food Safety Inspection Service (USDAFSIS) issued a final regulation on July 25, 1996 (USDA-FSIS 1996) establishing pathogen 

reduction requirements applicable to meat establishments. These were designed to reduce the 

occurrence and numbers of pathogens in or on meat and poultry products, thus reducing the risk 

of food-borne disease affecting millions of people (Mead and others 1999). The principal source 

of transmission of microbes such as E. coli O157:H7, Salmonella spp., Campylobacter, 

Staphylococcus aureus and others are from the hides of animals arriving at processing plants or 

carcasses that become cross-contaminated with intestinal contents during processing (USDAFSIS, 2004). For ready-to-eat (RTE) products, cross-contamination or recontamination by 

pathogens in the processing plant (e.g. human handling, contaminated processing equipment) is 

generally the major concern (Borch and Arinder, 2002). 

Recontamination of cooked products can result in a more serious problem for 

decontamination than untreated raw products, especially for spore forming microbes like 

7 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

Clostridium or cold-tolerant, psychrotrophic bacteria such as Listeria monocytogenes, because of 

a lack of competing microflora (e.g. lactic acid bacteria). Listeriosis acquired from the 

consumption of RTE products represents a serious public health concern because of the high 

mortality rates associated with the illness. However, contamination of raw materials (e.g. 

refrigerated beef trimmings) by Listeria also can be a problem, especially in small plants. Since 

many small processors deal with both raw and processed products, often in close proximity, this 

increases the risks of cross-contamination unless proper safety measures are implemented and 

strictly controlled. 

The regulation entitled “Pathogen Reduction: Hazard Analysis and Critical Control Point 

(HACCP) systems,” has four basic components, one of which is that all establishments develop 

and implement a system of preventive controls to improve product safety. Federal regulations 

(CFR 2002; USDA-FSIS 1993) now specify the minimum thermal processing requirements for 

cooked meat/poultry products as well as establishing ‘zero tolerance’ for contamination of beef 

products. In response to demands from consumers (CAST 2004) and government regulations for 

safer meat products, a wide range of studies testing possible interventions have been conducted, 

particularly in the last ten years. 

The most commonly used chemical decontamination methods are rinses containing 

chlorine, chlorine dioxide, acidified sodium chlorite, electrolyzed water, ozone, trisodium 

phosphate (TSP) and cetylpyridinium chloride (CPC). The latter compound has been evaluated 

in several studies (Ransom and others 2003; Pohlman and others 2002 a, b; Huffman 2002) and 

was just recently approved for food use by the USDA-FSIS. The ‘gaseous’ antimicrobials 

(chlorine, chlorine dioxide, ozone, acidified sodium chlorite which generates an oxy-halogen) are 

usually applied as an aqueous solution and generally have resulted in a 2 to 4 log reduction of 

8 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

pathogens depending on concentration, temperature of application and contact time. However, 

the suppression tends to be transient, providing no extended bactericidal/bacteristatic effects after 

treatment. The primary reason for this effect is that these compounds are readily reactive with 

organic compounds, thus quickly removing them from solution and/or negating further action 

against bacterial cells. TSP on the other hand is an alkaline salt solution that can leave residual 

reactive hydroxyl radicals in the treated products and suppress further growth. It has been found 

to improve the color of meat products, but the treatment also generates large amounts of alkaline 

phosphates, which can be environmentally harsh and create a problem for disposal. The use of 

organic acids as a carcass washing intervention has been a particularly active area of study, with 

the most commonly used acids being lactic and acetic acid (Dorsa and others 1998; Castillo and 

others 1998 a, b, 1999, 2000, 2001 a, b; Mermelstein 2001, Huffman 2002; Pohlman and others 

2002 a, b; Ransom and others 2003). Both lactic and acetic acid are generally recognized as safe 

(GRAS) by the U.S Food and Drug Administration. Lactic acid and acetic acid tend to offer the 

best residual efficacy for suppressing further pathogen proliferation during long-term refrigerated 

storage in which the meat is subsequently ground (Pohlman and others 2002 a, b; Castillo 2001 

a, b; Dorsa and others 1998). The rinse concentrations used are usually 2 to 5% and both acids 

are most effective if applied immediately after hot water washes (95°C) or as heated solutions 

(usually ~55°C) on hot, pre-rigor carcasses. While such applications are both effective, high heat 

treated products can acquire an undesirable color, loss of ground emulsion stability and increased 

acidic flavor if the residue is too high. 

Acidified sodium chloride (ASC) has shown increased antibacterial effects when 

combined with lactic acids. Castillo and others (1998 b) investigated the effectiveness of ASC 

solutions activated with phosphoric or lactic acid for reducing pathogens on inoculated beef 

9 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

carcass surfaces and found a reduction in pathogen numbers though still within countable limits. 

Acidified calcium sulfate (ACS) (sold by Mionix Corp., Rocklin, CA under the trade name 

Safe2O®) has been found to be very effective as a beef and poultry carcass washing agent 

(Huffman 2002; Dickens and others 2004) as well as a rinsing agent for RTE meats with 

considerable residual listericidal/listeristatic activity (Nuñez de Gonzalez and others 2004). It is a 

GRAS ingredient for use on food products and consists of a complex blend of sulfuric acid, 

calcium sulfate, calcium hydroxide, and an organic acid (e.g. lactic acid) adjusted to a final pH of 

~1.5. According to Mionix, ACS plus organic acids disable the proton pumps in bacterial 

membranes and act as a metabolic inhibitor, thus attacking bacteria in a different fashion than 

organic acids alone (e.g. lactic acid). However, only a few studies have shown its potential 

residual antimicrobial effects. This promising intervention demands additional study and 

evaluation, especially as a component of hurdle technology. In addition, epsilon polylysine 

(EPL) has been found to enhance the antimicrobial effects of ACS especially against lactic acid 

spoilage bacteria (Hiraki 2002). Epsilon polylysine is also GRAS and is thought to act in a 

different manner than ACS by causing disruption of the bacterial cell surface (Yoshida and 

others 2002). EPL concentrations of 0.02% and 0.04% have been shown to have antimicrobial 

activity against E.coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes, with 

Salmonella Typhimurium being the most sensitive of all three pathogens (Geornaras and Sofos 

2005). When combined with other antimicrobials EPL has enhanced antimicrobial activity. The 

potential use of these combined treatments merits further evaluation in food systems and 

especially because of their ease of application for small plant operators. 

Against this background, the present study was aimed at evaluating relatively 

undeveloped and cost effective pathogen reduction interventions at multiple levels for the small 

10 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

meat plant as hurdles to reduce contamination and growth of pathogens, specifically Salmonella 

Typhimurium, E. coli O157:H7, and Listeria monocytogenes. This study used rinses consisting 

of the application of a 1:4 acidified calcium sulfate (ACS) RTE01:water, 2.5% lactic acid (LA), 

1:4 ACS RTE01:water + 100 μL (100 ppm) ε-polylysine (EPL), 100 μL (100 ppm) EPL and 

sterile distilled water (W) as a control. All treatments were applied hot (50-55o

C) using a selfcontained spray cabinet, on pre-rigor beef rounds destined for use as ground beef or trimmings to 

be incorporated into further processed products (e.g. raw and cooked sausages). One or two 

washes on carcasses/trimmings in succession, in a hurdle fashion, were proposed such that initial 

bactericidal agents also may provide residual bacteriostatic effects to improve antimicrobial 

efficacy while minimizing undesirable effects on meat quality. The long term objectives were to 

develop interventions that will benefit the small processor by enhancing safety, being simple to 

apply, cost-effective and increasing shelf-life by reducing spoilage bacteria in addition to 

pathogens. The specific objectives were to: 

1) Evaluate warm water (control), lactic acid, acidified calcium sulfate and/or epsilonpolylysine as beef carcass/trimmings decontamination agents that are applied as a warm rinse 

following standard washing procedures. This was done initially in a model system using minimal 

application procedures to reduce the time and cost of the intervention for small plant. Different 

concentrations of the treatment were examined to determine optimal reduction of Salmonella 

Typhimurium, E.coli O157:H7 and Listeria monocytogenes. 

2) Determine the residual antimicrobial effects of ACS with and without EPL over time 

on carcasses/trimmings and selected raw products. 

3) Evaluate the efficacy of the best intervention identified above in two to three small 

local processing plants and to determine its efficacy as part of the HACCP plan. 

11 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

4) Evaluate the bactericidal/bacteriostatic efficacy of the best treatment on a cooked 

ready-to-eat (RTE) product contaminated with a Listeria cocktail. Due to time limitations, this 

objective was not covered, but it merits further study. 

MATERIALS AND METHODS 

Decontamination Procedures 

Six decontamination solutions were used: 1:4 acidified calcium sulfate (ACS, Safe2O 

RTE01®, Mionix Corporation, Rocklin, CA):water, lactic acid (LA 2.5% L-lactic acid, Purac 

America, Inc., Lincolnshire, IL), 100 μL (100 ppm) epsilon-polylysine (EPL, Save-ory® PL-25, 

Chisso, Corporation, Tokyo, Japan), 1:4 ACS RTE01:water + 100 μL (100 ppm) ε-polylysine, 

(EPL), 100 μL (100 ppm) EPL, and sterile distilled water (W), and tested for effectiveness. All 

treatments, once diluted to appropriate concentration, were placed in stainless steel containers 

and heated to 50-55°C in a water bath. Treatment solutions were applied using a pressurized 

spray system with nozzles that delivered a specific volume (14 ml/sec) of the treatment solutions 

(Figures 1 and 2). 

Spray Cabinet 

A custom-built isolation spray cabinet designed by CHAD Corporation was used to apply 

treatment solutions (Figures 1 and 2). Capture and containment of all spray, residue, pathogens 

and meat particles was accomplished with biohazard autoclave bags. Internal decontamination of 

the spray cabinet was performed using 20% hypochlorite (Chlorox®) in a removable reservoir 

attached to a cleaning/decontamination nozzle through the lid. After each treatment, the cabinet 

was rinsed with hot water which was collected in a biohazard receptacle and sterilized before 

discarding. This step was necessary to prevent any possible contact of ACS and chlorine. The 

12 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

cabinet was then disinfected using Chlorox®. External cabinet surfaces were swabbed with a 

70% alcohol (ethanol) solution to insure decontamination of the stainless steel cabinet. All runoff treatment solutions from the cabinet were collected in a Biohazard® bag and autoclaved, 

except for treatments containing ACS that were decontaminated by adding a 1:2 ratio of ACS to 

the soiled solution. In all procedures, care was taken to avoid contact of ACS and chlorine which 

are incompatible. 

Calculation of Treatment Volume 

Two nozzles were situated inside the cabinet near the top and bottom of the cylindrical 

spray chamber, through which treatments were delivered (Nozzle specification: H 1/8 VVSS 

° 65015 Nozzle with a spray angle of 65 ). At a spraying pressure of 137.9 kPa (20 psi), the flow 

° was 0.4164L/min (0.11 GPM) per nozzle, resulting in a spray angle of 51 /nozzle. Solution 

delivery was 0.2082 L/15 sec as shown in the following calculations: 

1US Gal = 3.785 Liters 

1L = 0.2642 Gal 

0.11 Gal x 3.785 L/gal = 0.4164 L/min 

2 nozzles x 0.4164 L/min ÷ 60 sec/min x 15 sec 

0.01388 L/min/60sec/min x 15 sec = 0.2082 L/15 sec or 14 ml/sec 

After inoculation, individual beef round samples were sprayed for 15-20 seconds while 

rotating at constant rate of ~5 revolutions/15 sec in a uniform spray stream. Three, 50 cm2 

portions of each pre-rigor beef round were then excised from the skin side surface using sterile 

stainless steel forceps and a scapel. Two portions were stored at 4°C in separate sterile ZipLock® bags and marked for analysis at 2 and 7 days post harvest, respectively. The third portion 

was immediately placed in a stomacher bag into which 20 ml of sterile phosphate buffered saline 

13 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

(PBS) solution was added and the contents pummeled in a stomacher for one minute to dislodge 

pathogens before examination. 

Collection of Beef Rounds 

Fresh, pre-rigor beef round samples were secured at a local abattoir by excising a 15 x 25 

x 5 cm surface portion of muscle from the dorsal side of a wholesale round immediately after 

skinning and prior to evisceration of market weight steer/heifer carcasses. Samples were taken 

randomly and immediately placed in sterile plastic bags, stored in an insulated container and 

transported to the isolation laboratory at the USDA-ARS Southern Plains Agricultural Research 

Center. Inoculations were initiated approximately 2.5 hours post sampling and treatments 

randomized for each replication. Inoculations were applied to a fixed surface area 15 x 10 cm2

 as 

described below. 

Bacterial Strain and Inoculum Preparation 

To negate the effect of indigenous microorganisms and/or pathogens that could reside 

on the beef rounds at harvest, rifampicin-resistance mutants were used for inoculation. 

Rifampicin-resistance mutants were derived from parent strains of Listeria monocytogenes strain 

Scott A (serotype 4b) obtained from Dr. J. F. Frank, University of Georgia. E. coli O157:H7 

strain ATTC 43895, and Salmonella Typhimurium strain NVSL 95-1776 (kindly provided by Dr. 

R. Anderson of USDA-ARS Food and Food Safety Unit, College Station Texas) were used to 

inoculate pre-rigor beef rounds in this study. The selected mutants were maintained on tryptic 

soy agar slants at 4°C. 

Rifampicin-resistant strains of Salmonella Typhimurium, E.coli O157:H7 and Listeria 

monocytogenes were resuscitated on three consecutive days using tryptic soy broth. A 12-hour 

culture of the pathogens was used to prepare a cocktail of inoculum. The procedure was as 

14 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

follows: two consecutive transfers into tryptic soy broth were carried out and incubated at 35°C 

for 24 hours. Twelve-hour cultures were diluted 100 fold and plated on TSA to determine initial 

cell numbers. Equal volumes of the 12-hour culture of each pathogen strain were centrifuged and 

the cells re-suspended in 5 ml of PBS, combined to form a cocktail and diluted 100 fold using 

PBS to provide an inoculation medium of 6.5 CFU/ml of PBS for each pathogen. 

Selection Media 

PBS was used as a recovery medium. A selective differential medium (lactose-sulfitephenol red-rifampicin agar, LSPR) was prepared as described by Castillo and others (1998 b) 

with slight modifications. The medium simultaneously enumerates rifampicin-resistant mutants 

Salmonella Typhimurium and E.coli O157:H7. The lactose-sulfite-phenol red-rifampicin agar 

used in this experiment consisted of the following ingredients per liter: tryptic soy agar (TSA, 

Difco, Detroit, MI) 40 g, yeast extract (Difco) 3 g, beef extract (Difco) 3 g, lactose (EMI 

Industries, Inc., Gibbstown, NJ) 5 g, sodium sulfite (MCB Reagents, Cincinnati, OH) 2.5 g, 

ferrous sulfate ( MCB Reagents) 0.3 g, phenol red (Fisher Scientific, Fair Lawn, NJ) and 25 mg, 

rifampicin (Sigma Chemical) 0.1 g. Phenol red was dissolved in 2 ml 0.1N NaOH before adding 

to the medium. The medium without rifampicin was autoclaved at 121°C for 15 min and cooled 

to 50°C. Rifampicin was dissolved in 5 ml methanol, filter-sterilized, and added to the sterile 

medium prior to pouring into Petri plates. The medium did not contain the 0.1 g cycloheximide 

contained in the original formulation. Rifampicin-resistance E.coli O157:H7 produced yellow 

colonies on the medium, whereas the rifampicin-resistant Salmonella Typhimurium developed 

colonies with a black center surrounded by a pink halo. TET and RV were used for Salmonella 

Typhimurium enrichment and UVM and Fraser broth were used for Listeria monocytogenes 

enrichment as prescribed by USDA-FSIS (2002). 

15 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

Rehydrated commercial Modified Oxford Agar base (Oxoid) was used for recovery of 

Listeria monocytogenes. The medium without supplement was autoclaved at 121°C for 15 min, 

cooled to 50o

C and then, one vial of the supplement dissolved in 10 ml of sterile distilled water 

was added per liter before dispensing into Petri plates. Listeria monocytogenes detection is based 

on the hydrolysis of esculin to 6, 7-dihydroxycoumarin (esculetin) and its reaction with ferric 

ions in the medium; hence Listeria’s presence is identified by a dark (blackened) zone due to the 

esculin hydrolysis usually within 24 hours. 

Sample Excision and Inoculum Recovery 

A 15 x 10 cm2 section was outlined on the surface of a freshly harvested beef round using 

a sterile template. A 2 ml aliquot of the pathogen cocktail (6.8 CFU) was inoculated onto each 

template area and the inoculum spread uniformly with a sterile swab to yield an initial count of 

6.35 logs on a 50 cm2

 area. The inoculated beef round was set aside for 10 min at room 

temperature to allow for bacterial adhesion to the meat surface. Individual beef round samples 

were then sprayed in a sealed, stainless steel cabinet (manufactured by CHAD Corporation) for 

15-20 sec using heated treatment solutions (50-55°C) as previously described. Data are reported 

as the mean log10 reductions of each respective organism from an initial inoculation level of 6.35 

CFU/microorganism, recovered from a surface area of 50 cm2

. Mean log reductions were 

calculated by subtracting the mean log CFU/50 cm2

 recovered after treatment and storage as 

described earlier, from the initial log CFU/50 cm2

 inoculum counts before treatment: [Log 6.35 

CFU/50 cm2

 of each organism in the cocktail before treatment – mean log CFU/50 cm2/organism 

recovered post treatment = Mean log reduction] . 

16 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

-

Experimental Design and Statistical Analysis 

The experiment was a 5 X 3 factorial design where five decontamination treatments (ACS, 

LA, ACS+EPL, EPL and W) and three storage days (0, 2 and 7) were used as main factors. The 

effects of decontamination treatments and storage days and their interaction were tested by the 

General Linear Procedure (GLM) using SPSS (Statistical Package for Social Science) for 

Windows release 13.0. The least significant difference (LSD) pair-wise multiple comparison test 

was used to compare the mean differences when the effects of the treatments were significant 

(p<0.05). 

RESULTS AND DISCUSSION 

Preliminary Trials 

Preliminary trials were conducted with the antimicrobial treatments to establish 

inoculation and recovery procedures, determine the most appropriate method of treatment 

application (i.e., combine LA with EPL in the same solution or apply separately and 

sequentially), establish spray cabinet pressure conditions to deliver a specified volume of liquid, 

and to develop decontamination procedures for the spray cabinet to ensure complete inactivation 

and containment of pathogens. Preliminary mean log reductions for ST, EC and LM by storage 

day are presented in Tables 1-3, respectively. Reductions shown are somewhat indicative of 

treatment effects on the respective pathogens, but not definitive since adjustments were being 

made in the experimental procedures. Although these means cannot be analyzed statistically, 

they are presented for information purposes and were used to assist in the selection of subsequent 

treatment combinations. In the preliminary trials, EPL was added directly to a 2.5% LA solution, 

17 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

but additional information provided by the distributor of EPL indicated that direct contact with 

the acid might decrease its antimicrobial efficacy, which was consistent with our observations. 

Numeric values shown in Tables 1-3 are the mean log reductions from the initial counts 

of each pathogen (log 6.35 CFU/50cm2

) that was inoculated onto pre-rigor beef rounds as a 

cocktail, and then treated with a hot (50-55o

C) antimicrobial spray. 

Salmonella Typhimurium (ST) 

 Table 1 shows the mean log reductions of ST due to treatment solutions. On storage day 

0, an initial application of ACS and LA showed a mean log reduction of 1.6 and 1.1 logs, 

respectively. LA combined with EPL (0.7) and EPL (0.7) alone had numerically lower reduction 

values than ACS and LA alone, but were comparable to water (0.6) for reducing ST counts. 

Comparable reductions of 2.9 and 2.7 logs on storage days 2 and 7 were noted for ACS and LA, 

respectively. The hot water treatment had comparable reductions of 2.1 and 2.6. LA + EPL 

reductions were 1.2 and 1.1 logs on days 2 and 7 while, EPL reductions were 1.1 and 1.4, 

respectively. These preliminary results for ST were not consistent with the replicated data 

obtained later. Even in the preliminary trials, it is interesting to note that larger reductions in ST 

seemed to occur after 2 and 7 days storage at 4o

C. 

Escherichia coli O157:H7 (EC) 

In Table 2, an initial application of ACS reduced EC by 1.8 log while LA showed a mean 

reduction of 1.1 log followed by W (0.9) and LA+ EPL (0.7). EPL alone had a mean log 

reduction of 0.2. On storage day 2, ACS had the highest mean log reduction (3.1) followed by 

LA + EPL (2.4). Likewise, LA, EPL and W had greater reductions in EC on days 2 when 

compared to day 0. The same trends were noted on day 7 as compared to day 2, with slightly 

lower reductions for LA + EPL and EPL and higher reductions for W. All treatments, to varying 

18 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

degrees, appeared to inhibit the growth of E.coli O157: H7 over a 7 day refrigerated storage 

period. 

Listeria monocytogenes (LM) 

 Table 3 shows that the largest mean log reductions in LM tended to occur with the initial 

ACS treatment (3.5) followed by the application of LA + EPL (2.0). All treatments, except ACS, 

appeared to offer greater suppression of growth on days 2 and/ or 7 than at day 0. It is interesting 

to note that LA had the greatest numeric reduction (4.1) on day 7 while ACS had less reduction 

than on days 0 and 2 for most treatments. 

Overall, mean log counts in Tables 1-3 seemed to show increased antimicrobial 

effectiveness over 7 days of refrigerated storage. However, these were only preliminary results. 

Because of the adjustments being made in testing conditions during the preliminary trials, the 

changes in procedures may have influenced the log counts and these data may not truly reflect 

treatment effects. It was determined at this stage, however, that the sequence of application for 

EPL in combination with ACS or LA could be important. Because of the larger initial bacterial 

reductions with ACS, it was decided to apply ACS and EPL sprays to pre-rigor beef rounds in a 

sequential manner (ACS + EPL) in three replicated experiments that followed the preliminary 

trials. Application of ACS, followed by EPL at different time intervals (data not shown), have 

also shown reductions in Salmonella Typhimurium, E.coli O157:H7 and Listeria 

monocytogenes. 

Results of Decontamination Treatments 

Salmonella Typhimurium (ST) 

19 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

The analysis of variance of the log reduction data for Salmonella Typhimurium showed 

that the interaction between decontamination treatments and storage days was significant 

(p<0.05). Due to the significant interaction between the main factors, a plot of the treatment 

means and LSD procedure were used to compare all treatment means instead of testing and 

comparing the individual levels of each factor (Table 4). As shown in Figure 3 and Table 4, the 

effectiveness of treatments varied depending on the storage day. Initial mean log reductions of 

ST were not different among the spray treatments initially on day 0, but with slight reductions 

from the initial inoculum level. By storage day 2, the ACS + EPL treatment had reduced ST by 

2.0 log from the initial inoculum level, while ACS, LA and EPL tended to show reductions, but 

these were not significantly different from the W treatment. W was used as a control to 

demonstrate the effect of warm water alone and to give a basis of comparison to the other 

treatments. On storage day 7, ACS + EPL had reduced ST by 4.38 log while the other treatments, 

except W, reduced ST by > 1 log. W tended to have the same level of ST on day 7 as at the time 

of initial inoculation. Treatment with LA alone was consistent with other studies in which 2.5% 

lactic acid has been shown to be effective in reducing pathogens on cold beef carcass surfaces 

and in ground beef produced from the carcass trimmings (Castillo and others 2001). In general, 

all decontamination treatments, except W, caused a reduction in ST counts after 7 days of 

refrigerated storage with the ACS + EPL combination being the most effective antimicrobial 

treatment against ST. EPL concentrations of 0.02% and 0.04% have been shown to have 

antimicrobial activity against E.coli O157:H7, Salmonella Typhimurium, and Listeria 

monocytogenes, with Salmonella Typhimurium being the most sensitive of all three pathogens 

(Geornaras and Sofos 2005). Based on these results and those of Geornaras and Sofos (2005), 

EPL appears to have enhanced antimicrobial activity in combination with acidic antimicrobials. 

20 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

-

Escherichia coli O157:H7 (EC) 

The analysis of variance of the log reduction data for Escherichia coli O157:H7 

demonstrated that the interaction between decontamination treatments and storage days was not 

significant. As a result, the data were pooled across treatments and across storage days and 

reported as main effect means in Tables 7 and 8. Mean log reductions in E.coli O157:H7 

segregated by treatment and storage are presented in Table 5 for information only. Table 7 shows 

the independent effect (p<0.05) of decontamination treatments and Table 8 the effect of storage 

days on mean log reductions of EC. Least significant difference (LSD) comparisons of 

decontamination treatments over all storage days indicated that ACS + EPL was the most 

effective treatment with an overall reduction in EC of 1.49 log. ACS, LA and EPL reductions 

were approximately half (0.89, 0.88 and 0.65 log, respectively) the ACS + EPL treatment, but 

significantly more effective than W alone (0.32 log). The effects of ACS, LA and EPL were not 

significantly different from one another in reducing EC, though all treatments were effective to 

varying degrees for reducing EC below the initial inoculation level of 6.35 CFU/50 cm2

. 

Reductions in EC after spraying with warm (50-55oC) decontamination solutions for 15 

to 20 seconds on storage day 0 (0.49 log) were (p<0.05) less than the reductions observed for 

days 2 and 7 (0.92 and 1.14 log, respectively). Overall, mean log reductions for all treatments 

were greater after the second day. The ACS + EPL combination was the most effective 

antimicrobial treatment against EC while warm water showed the least reduction. 

Listeria monocytogenes (LM) 

The analysis of variance of the mean log reductions for Listeria monocytogenes 

demonstrated that interaction between decontamination treatments and storage days was not 

significant. As a result, the data were pooled across treatments and across storage days and 

21 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

-

reported as main effect means in Tables 7 and 8. Mean log reductions in Listeria monocytogenes 

after spraying with warm (50-55°C) decontamination treatment solutions for 15 to 20 seconds 

are presented in Table 6 for information only. The effects of decontamination treatments over 

storage (Table 7) were significant while storage alone (Table 8) did not affect mean log 

reductions of LM (p> 0.05). Least significant difference (LSD) comparisons of decontamination 

treatments in Table 7 showed ACS + EPL to cause the greatest reduction (2.38 log) in LM. A 

similar result when ACS was used in combination with EPL has been shown by Geornaras and 

Sofos (2005). LA (1.54 log) was more effective at reducing LM than W alone (0.78 log), but 

ACS (1.48 log) and EPL (1.36) were not different from the W control. LA treatment was not 

different from ACS or EPL treatments. Reductions in LM were not different across storage 

(Table 8) and ranged from 1.39 to 1.61 log. 

Previous studies in our laboratory have demonstrated the antimicrobial effectiveness of 

ACS (bactericidal effect) and LA (bacteriostatic effect) on LM inoculated onto the surface of 

frankfurters and stored at 4.5o

C (Nuñez de Gonzalez and others 2004). In this study, the greatest 

mean log reductions in ST, EC and LM on pre-rigor beef rounds were obtained when ACS was 

applied followed immediately by EPL. ST showed the greatest susceptibility to the ACS + EPL 

treatment over storage followed by EC. LM appeared to be more sensitive to the initial 

decontamination treatments on day 0 when compared to ST and EC, but unlike ST and EC did 

not show further reductions over a 7 day refrigerated storage period. This may have been due in 

part to LM’s increased tolerance to cold temperatures. In addition, Delihas and others (1995) 

have shown some microorganisms to be highly susceptible to EPL in vitro on the first day of 

treatment, but later becoming resistant to EPL during storage. Other studies have shown that 

22 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

some microorganisms possess EPL-degrading enzymes, which make them resistant to EPL or 

compounds containing EPL (Kito and others 2002). 

The separate modes of action of ACS and EPL might contribute to their synergistic 

effectiveness for inhibiting the growth of pathogens. ACS, which contains lactic acid, are 

believed to disable the proton pumps in bacterial membranes and act as a metabolic inhibitors, 

thus attacking bacteria in a different fashion than organic acids alone (e.g. lactic acid). EPL is 

thought to act in a different manner than ACS by causing disruption of the bacterial cell surface 

(Hiraki 2002). The differences in sensitivity of gram-positive and gram-negative organisms to 

EPL is not fully understood, but it is speculated that the types and/or number of cell surface 

receptors or proteases secreted by an organism contribute to its sensitivity as well as differences 

in their respective cell envelope make up (Delihas and others 1995). 

The enhanced antimicrobial activity of EPL against Salmonella Typhimurium and E. coli 

O157:H7 when used in combination with sodium acetate or acetic acid has been demonstrated by 

Geornaras and Sofos (2005). In this study, ST, EC and LM were more sensitive to ACS + EPL 

than EPL alone. Although Geornaras and Sofos (2005) used different treatment combinations of 

EPL, they reported ST grown in vitro to have more resistance to EPL, than EC or LM. 

Preliminary results in our laboratory showed the efficacy of EPL to be reduced when combined 

with LA and used as a decontamination spray for pre-rigor beef rounds. 

Castillo and others (2001) have shown 4% LA at 55o

C and sprayed for 30 sec or 4% LA 

at 65o

C and sprayed for 15 sec or 30 sec to consistently result in undectable levels of E. coli 

O157: H7 on chilled beef carcasses. In the present study, 2.5% LA showed reductions of ST, EC 

and LM, but to a lesser extent than the higher concentrations of LA. Castillo and others (1998) 

also reported mean reductions in E. coli O157:H7 and Salmonella Typhimurium of 3.7 and 3.8 

23 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

log, respectively, when a hot water spray (95o

C) was used for beef carcass decontamination. 

However, warm (55°C) water application to beef rounds in this study showed only minimal 

reductions in pathogens. While high level (5%) applications of LA and hot water (95°C) are both 

effective, treated products can acquire an undesirable color, loss of ground emulsion stability and 

increased acidic flavor if the residue is too high. 

CONCLUSIONS 

A sequential warm (55°C) spray application of ACS followed by EPL at constant 

pressure for 15 to 20 sec reduced ST, EC and LM inoculated on the skin-side surface of pre-rigor 

beef rounds more effectively than a single treatment of ACS, LA, EPL or W alone. This 

confirms the fact that multiple interventions can be a better strategy for pathogen reduction in the 

small processing plants than single treatments and could also provide a more ‘fail-safe’ pathogen 

reduction strategy for small meat and poultry processing plants. Based on observations in this 

study, it appears that the combination of antimicrobial agents that express different modes of 

action for suppressing pathogen growth, and the sequential application of different 

decontamination sprays (e.g. ACS + EPL) are significant factors for obtaining greater reductions 

in pathogen numbers on beef carcasses at slaughter. Further experiments are currently being 

performed to investigate the sequence of addition of ACS and EPL, the time interval between the 

applications (5 to 15 min) on pathogen reduction and the effects of varying the concentrations of 

each component. It is also worth investigating the antimicrobial effect of LA and EPL when 

applied in sequence. Additional work is needed to optimize our application techniques for 

reducing pathogens in small meat and poultry processing operations. When the treatment 

24 
1 sequences have been optimized, the procedures will be tested in small meat plants in the College 

2 Station area. 

25 
1 

2 

3 

4 Figure 1. 

5 

6 

7 

8 

A custom built isolation spray cabinet designed by CHAD Corporation, Showing containment 

chamber, three stainless steel reservoirs for application of treatment, White plastic receptacle for 

application of cleaning solutions, and pressure gauges. 

9 

10 

11 

12 

13 

14 

15 

26 
1 

2 

3 Figure 2. 

4 A custom built isolation spray cabinet designed by CHAD Corporation, showing rotating 

5 stainless steel hooks for suspension of beef rounds 

27 
1 Table 1. Mean log reductions of Salmonella Typhimurium after spraying with warm (50-55°C) 

2 decontamination treatment solutions for 15 to 20 seconds. 

3 

Mean Log Reductionsb 

Storage Day at 4o

C 

Treatmenta 0 2 7 

ACS 1.6 2.9 2.7 

LA 1.1 2.9 2.7 

LA+EPL 0.7 1.2 1.1 

EPL 0.7 1.1 1.4 

W 0.6 2.1 2.6 

4 a

1:4 acidified calcium sulfate (ACS) RTE01:water ; 2.5% lactic acid (LA), 2.5% lactic acid (LA)+ 100 μL (100 

5 ppm) ε-polylysine, (EPL) , 100 μL (100 ppm) EPL and sterile distilled water (W). 

6 b Mean Log reductions = (Log of CFU/50 cm2 of initial inoculum counts before treatment) – (Log CFU/50cm2 after 

7 treatment). Log CFU/50 cm2 of each organism in the cocktail before inoculation was 6.35. 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 Table 2. Mean log reductions of E. coli O157:H7 after spraying with warm (50-55°C) 

20 decontamination treatment solutions for 15 to 20 seconds. 

28 
1 

Mean Log Reductionsb 

Storage Day 

Treatmenta 0 2 3 

ACS 1.8 3.1 2.7 

LA 1.1 1.9 2.0 

LA+EPL 0.7 2.4 1.8 

EPL 0.2 2.1 1.3 

W 0.9 1.9 2.5 

2 a

1:4 acidified calcium sulfate (ACS) RTE01:water ; 2.5% lactic acid (LA), 2.5% lactic acid (LA)+ 100 μL (100 

3 ppm) ε-polylysine, (EPL) , 100 μL (100 ppm) EPL and sterile distilled water (W). 

4 b Mean Log reductions = (Log of CFU/50 cm2 of initial inoculum counts before treatment) – (Log CFU/50cm2 after 

5 treatment). Log CFU/50 cm2 of each organism in the cocktail before inoculation was 6.35. 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 Table 3. Mean log reductions of Listeria monocytogenes after spraying with warm (50-55°C) 

20 decontamination treatment solutions for 15 to 20 seconds. 

29 
1 

Mean Log Reductionsb 

Storage Day 

Treatmenta 0 2 7 

ACS 3.5 3.2 2.4 

LA 1.6 2.4 4.1 

LA+EPL 2.0 2.4 2.7 

EPL 1.3 2.4 2.1 

W 1.6 1.5 2.1 

2 a1:4 acidified calcium sulfate (ACS) RTE01:water ; 2.5% lactic acid (LA), 2.5% lactic acid (LA)+ 100 μL (100 

3 ppm) ε-polylysine, (EPL) , 100 μL (100 ppm) EPL and sterile distilled water (W). 

4 b Mean Log reductions = (Log of CFU/50 cm2 of initial inoculum counts before treatment) – (Log CFU/50cm2 after 

5 treatment). Log CFU/50 cm2 of each organism in the cocktail before inoculation was 6.35. 

6 

7 

8 

9 

10 

11 

12 

13 

14 

30 
0 

Mean Log Reduction of ST 

5.00 

4.00 

3.00 

2.00 

1.00 

0.00 

0 2 

Decontamination 

Treatments 

A 

L 

AP 

P 

W 

7 

1 

Storage Days 

2 

3 

4 

Fig 3. Mean log cell reduction of Salmonella Typhimurium after spraying with warm (50 

to 55 oC) decontamination treatment solutions for 15 to 20 seconds. 

5 

6 

7 

8 

9 

10 

11 

31 
1 Table 4. Mean log reductions of Salmonella Typhimurium after spraying with warm (50-55°C) 

2 decontamination treatment solutions for 15 to 20 seconds. 

3 

Mean Log Reductionsb

 ± SD 

Storage Day 

Treatmenta 0 2 7 

ACS 0.45ABC ± 0.17 0.95ABC ± 0.17 1.1BCD ± 0.44 

LA 0.55ABC ± 0.26 0.78ABC ± 0.64 1.32CD ± 0.32 

ACS+EPL 0.45ABC ± 0.00 2.00D ± 0.53 4.38E

 ± 1.9 

EPL 0.28AB ± 0.55 0.85ABC ± 0.17 1.0BCD ±0 .11 

W 0.18AB ± 0.29 0.08A ± 0.40 0.08A ± 0.23 

4 

5 

6 

7 

8 

9 

10 

a

 1:4 acidified calcium sulfate (ACS) RTE01: water, 2.5% lactic acid (LA), 1:4 ACS RTE01: water +100 μL (100 

ppm) ε - polylysine, (EPL), 100 μL (100 ppm) EPL, and sterile distilled water (W). b

 Mean Log reductions = ( Log CFU/50cm2 of initial inoculum counts before treatment) – (Log CFU/50cm2 

after treatment). Log CFU/50cm2 of each organism in the cocktail before inoculation was 6.35 

ABCDE Different superscript letters indicate that means are different (p<0.05) (LSD). 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

32 
1 Table 5. Mean log reductions of E.coli O157:H7 after spraying with warm (50-55°C) 

2 decontamination treatment solutions for 15 to 20 seconds. 

3 

Mean Log Reductionsb

± SD 

Storage Day 

Treatmenta 0 2 7 

ACS 0.65 ± 0.10 0.95 ± 0.17 1.11 ± 0.42 

LA 0.68 ± 0.25 0.65 ± 0.40 1.32 ± 0.30 

ACS+EPL 0.88 ± 0.35 2.00 ± 0.47 1.62 ± 0.35 

EPL 0.05 ± 0.52 0.85 ± 0.17 1.10 ± 0.1 

W 0.18 ± 0.06 0.15 ± 0.36 0.62 ± 0.37 

4 

5 

6 

7 

8 

9 

a

1:4 acidified calcium sulfate (ACS) RTE01; 2.5% lactic acid (LA), 1:4 ACS RTE01 + 100 μL (100 ppm) εpolylysine (EPL), 100 μL (100 ppm) EPL and sterile distilled water (W). b

 Mean Log reductions = (Log of CFU/50cm2 of initial inoculum counts before treatment) – (log CFU/50cm2 after 

treatment). Log CFU/50cm2 of each organism in the cocktail before treatment was 6.35. 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

33 
1 Table 6. Mean log reductions of Listeria monocytogenes after spraying with warm (50-55°C) 

2 decontamination treatment solutions for 15 to 20 seconds. 

3 

Mean Log Reductionsb

± SD 

Storage Day 

Treatmenta 0 2 7 

ACS 1.25 ± 0.53 1.72 ± 0.49 1.48 ± 0.55 

LA 1.55 ± 0.44 1.15 ± 0.26 1.92 ± 0.31 

ACS+EPL 2.28 ± 0.59 2.58 ± 2.10 2.28 ± 1.37 

EPL 0.98 ± 0.68 1.65 ± 0.36 1.45 ± 0.35 

W 0.88 ± 0.49 0.55 ± 0.20 0.92 ± 0.58 

4 

5 

6 

7 

a

1:4 acidified calcium sulfate (ACS) RTE01; 2.5% lactic acid (LA), 1:4 ACS RTE01 + 100 μL (100 ppm) εpolylysine (EPL), 100 μL (100 ppm) EPL and sterile distilled water (W). b

 Mean Log reductions = (Log of CFU/50cm2 of initial inoculum counts before treatment) – (log CFU/50cm2 after 

treatment). Log CFU/50cm2 of each organism in the cocktail before treatment was 6.35. 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

34 
1 Table 7. Analysis of Variance results for log cell reductionsb of, E.coli O157:H7and Listeria 

2 monocytogenes recovered from beef after spraying with warm (50-55°C) decontamination 

3 treatment solutions for 15 to 20 seconds. 

4 

Antimicrobial Treatmenta 

ACS LA ACS+EPL EPL W STD Error 

E.coli O157:H7 0.89 B 0.88 B 1.49C

 0.65 B 0.32 A 0.11 

Listeria monocytogenes 1.48AB 1.54 CB 2.38D 1.36 AB 0.78 A 0.26 

5 a

 1:4 acidified calcium sulfate (ACS) RTE01: water, 2.5% lactic acid (LA), 1:4 ACS RTE01: water + 100 μL (100 

6 ppm) ε - polylysine, (EPL), 100 μL (100 ppm) EPL, and sterile distilled water (W). 

7 b Mean Log reductions = (Log CFU/50cm2 of initial inoculum counts before treatment) – (Log CFU/50cm2 after 

8 treatment). Log CFU/ 50cm2 of each organism in the cocktail before inoculation was 6.35. 

9 ABCDMeans in same row with different superscript letters are significant (p<0.05) (LSD). 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

24 

25 

26 

35 
1 Table 8. Analysis of variance for log cell reductionsa (log CFU/cm2

) of E. coli O157:H7, and 

2 Listeria monocytogenes recovered from beef rounds after spraying with warm (50-55°C) 

3 decontamination treatments for 15 to 20 seconds at storage days 0 (immediately after treatment) 

4 2 and 7 at 4oC . 

5 

Storage days at 4oCb 

0 2 7 STD Error 

E.coli O157:H7 0.49 A 0.92 B 1.14B

 0.08 

Listeria monocytogenes 1.39A 1.53 A 1.61A 0.20 

6 

7 

8 

9 

a Mean Log reduction = (Log CFU/50cm2

 of initial inoculum counts before treatment) – (log CFU/50cm2 after 

treatment). Log CFU/50cm2 of each organism in the cocktail before treatment was 6.35. ABMeans in same row with different superscript letters are significant (p< 0.05) (LSD). 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 ACKNOWLEDGEMENTS 

21 The research was supported by grants from USDA-FSIS Non-Assistance Cooperative Agreement 

22 #FSIS-C-14-2004. The authors are thankful to Texas Agricultural Experiment Station for their 

23 support and the Fulbright program for sponsoring N. L. A. Njongmeta. 

36 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

-

REFERENCES 

Anderson MER, Marshall RT, Stringer WC, Naumann HD. 1977. Combined and individual 

effects washing and sanitizing on bacterial counts of meat - a model system. J Food Prot 40: 

668-670. 

Anderson MER, Marshall RT. 1989. Interaction of concentration and temperature of acetic acid 

solution on reduction of various species of microorganisms on beef surfaces. J Food Prot 52: 

312-315. 

Borch E, Arinder P. 2002. Bacteriological safety issues in red meat and ready to eat products, as 

well as control measures. Meat Sci 62: 381 390. 

CAST Issue Paper. 2004. Intervention strategies for the microbial safety of foods of animal 

origin. 25 (Jan) 1-24. 

Castillo A, Lucia LM, Roberson DB, Stevenson TH, Mercado I, Acuff GR. 2000. Lactic acid 

sprays reduced bacterial pathogens on cold beef carcass surface and in subsequently 

produced Ground beef. J Food Prot 64 (1):58-62. 

Castillo A, Lucia LM, Kemp GK, Acuff GR. 1998. Reduction of Escherichia coli O157:H7 and 

Salmonella Typhimurium on beef carcasses surface using acidified sodium chlorite. J Food 

Prot 62 (6):580-584. 

Castillo A, Lucia LM, Goodson KJ, Savell JW, Acuff GR. 1998. Use of hot water for beef 

carcass decontamination. J Food Prot 61 (1):19-25. 

Castillo A, Lucia LM, Mercado I, Acuff GR. 2001. In-plant evaluation of a lactic acid treatment 

for reduction of bacteria on chilled beef carcasses. J Food Prot 64 (5):738-740. 

37 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

Castillo A, Hardin MD, Acuff G, Dickson J. 2001. Reduction of microbial contaminants on 

carcasses. In: Junenja VK, Sofos JN Editors. Control of Food-borne Microorganisms. New 

York: Marcel Dekker. p 351-382. 

Connor D. 2001. Poultry-borne pathogens: plant consideration. In Sams AR editor. Poultry Meat 

Processing. New York: CRC Press. p 138-156. 

Delihas N, Riley LW, Loo W, Berkowitz J, Poltoratskaia N. 1995. High sensitivity of 

Mycobacterium species to the bactericidal activity by polylysine. FEMS Microbiol Letters 

132:233-237. 

Dickson JS, Anderson ME. 1992. Microbiological decontamination of food animal carcasses by 

washing and sanitizing systems: a review. J Food Prot 55:133-140. 

Dickens JA, Ingram KD, Hinton A Jr. 2004. Effects of applying Safe2O poultry wash to broiler 

wings on shelf-life, Listeria monocytogenes, Pseudomonads, Staphylococcus species, and 

psychrotrophic bacteria levels after three, seven, and ten days of storage. Poul Sci 83:1047-

1050. 

Dorsa WJ, Cutter CN, Siragusa GR. 1998. Long-term effect of alkaline, organic acid, or hot 

water washes on the microbial profile of refrigerated beef contaminated with bacterial 

pathogens after washing. J Food Prot 61:300-306. 

Ellebracht EA, Castillo A, Lucia LM, Miller RK, Acuff GR. 1999. Reduction of pathogens using 

hot water and lactic acid on beef trimmings. J Food Sci 64 (6):1094-1099. 

Frenzen PD, Majchrowicz A, Buzby JC, Imhoff B and the FoodNet Work Group. 2000. 

Consumer acceptance of irradiated meat and poultry products. Agricultural Information Bull. 

757. US Department of Agriculture, Economic Research Service, Washington, DC. 

38 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

Geornaras I, Sofos JN. 2005. Activity of ε-polylysine against Escherichia coli O157:H7, 

Salmonella Typhimurium, and Listeria monocytogenes. J Food Sci 70 (9):M404-M408. 

Huffman RD. 2002. Current and future technologies for the decontamination of carcasses and 

fresh meat. Meat Sci 62: 285-294. 

Kito M, Onji Y, Yoshida T, Nagasawa T. 2002. Occurrence of ε-poly-L-lysine-degrading 

enzyme in ε-poly-L-lysine-tolerant Sphingobactrium multivorum OJ10: purification and 

characterization. FEMS Microbiol letters 207:147-151. 

Keeton TJ, Harris KB. 2004. The hazard analysis and critical control point system and 

importance of verification. In: Beier RC, Pillai SD, Phillips TD, Ed. Ziprin RL, Associate Ed. 

Preharvest and Postharvest Food Safety: Contemporary Issues and Future Directions. 

Blackwell Publishing. P 257-269. 

Mermelstein HN. 2001. Sanitizing meat. Food Technol 55(3):64-68. 

Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV. 1999. 

Food related illness and death in the United States. Emerg Infect Dis 5:607-625. 

Nuñez de Gonzalez MT, Keeton JT, Acuff GR, Ringer LJ, Lucia LM. 2004. Effectiveness of 

acidic calcium sulfate with propionic and lactic acid and lactates as post-processing dipping 

solutions to control Listeria monocytogenes on frankfurters with or without potassium lactate 

and stored vacuum packaged at 4.5 degrees C. J Food Prot 64 (5):915-921. 

Pohlman FW, Stivarius MR, McElyea KS, Johnson ZB Johnson MG. 2002a. The effects of 

ozone, chlorine dioxide, cetylpyridium chloride and trisodium phosphate as multiple 

antimicrobial interventions on ground beef. Meat Sci 61:307-313. 

39 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

Pohlman FW, Stivarius MR, McElyea KS, Johnson ZB Johnson MG. 2002b. Reduction of 

microorganisms in ground beef using multiple intervention technology. Meat Sci 61: 315­

322. 

Ransom JR, Belk KE, Sofos JN, Smith GC. 2003. Treatment of beef hides with cetylpyridinium 

chloride solution to reduce contamination before slaughter of beef cattle. Final Report to the 

National Cattlemen’s Beef Association, Centennial, CO from the Center for Red Meat 

Safety, Department of Animal Sciences, Colorado State University, Fort Collins, CO p 6. 

 SPSS Version 13.0. 2004. Statistical Package for the Social Sciences, Chicago, IL. 

USDA-FSIS. 1993. Immediate actions: cattle clean meat program, FSIS correlation packet, 

interview guidelines for inspectors. United States Department of Agriculture, Washington, 

DC. 

USDA-FSIS 1996. Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) 

Systems. Department of Agriculture. Federal Register, July 25, 1996, Vol 61, Number 144, 

38805. Office of the Federal Register, National Archives and Records Administration. 

USDA-FSIS 1999. Appendix A. Compliance guidelines for meeting lethality performance 

standards for certain meat and poultry products. 

USDA-FSIS. 2002. Media and reagents. Office of Public Health and Science, 950 College 

Station Road, Athens, GA 30605. MLG Appendix 1.02:13-45. 

U.S. Department of Agriculture Food Safety Inspection Service. 2002. Pathogen Reduction; 

Hazard Analysis and Critical Control Point (HACCP) Systems. Title 9 Code of Federal 

Regulations, Parts 304, 308, 310, 320, 327, 381, 416 and 417. U.S. Government Printing 

Office, Washington, D.C. 

40 
1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

USDA-FSIS. 2004. Salmonalla incidence in broilers. Available at: 

http://www.fsis.usda.gov/OPPDE/animalprod/Publications. 

White R 2002. USDA takes aim at a deadly microbe. Food Qual 9(6):6-7. 

Yoshida T, Hikari J, Nagasawa. 2002. ε-Poly-L-Lysine. Polyamides and complex proteinaceous 

materials I. Biopolymer. (7):107-121. 

Hiraki J. 2002. ε-Polylysine; its development and utilization. Fine Chem 29:18-25. 

Hiraki J, Ichikawa T, Ninomiya S, Seki H, Kimura S, Yanagimoto Y, Barnett JW. 2003. Use of 

AMME studies to confirm the safety of ε-polylysine as preservative in food. Reg Toxicol 

Pharmacol 37:328-40. 

41