llb, Gary C. Smitha

Salmonella in all treatments. Bacterial populations decreased below the detection limit ( 0.4 log CFU/cm2) as early as 7 h

International Journal of Food Microbioagar media. The results indicated that acid adaptation may not cause increased resistance of Salmonella to the microbial hurdles

involved in jerky processing and that use of modified marinades in manufacturing jerky may improve the effectiveness of

drying in inactivating Salmonella.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Salmonella; Beef; Jerky; Drying; Storage; Marination; Acid adaptation

1. Introduction Because it is nutritious (high in protein and iron, low

in fat), shelf-stable (0.751.0 moisture protein ratio),during drying or remained detectable even after 60 days of storage, depending on acid adaptation, pre-drying treatment, andaCenter for Red Meat Safety, Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523-1171, USAbDepartment of Food Science and Nutrition, Colorado State University, Fort Collins, CO 80523, USA

Received 12 March 2002; received in revised form 1 November 2002; accepted 8 February 2003

Abstract

This study evaluated the influence of pre-drying marinade treatments on inactivation of acid-adapted or nonadapted

Salmonella on beef jerky during preparation, drying and storage. The inoculated (five-strain composite, 6.0 log CFU/cm2) slices

were subjected to the following marinades (24 h, 4 jC) prior to drying at 60 jC for 10 h and aerobic storage at 25 jC for 60days: (1) no marinade, control (C), (2) traditional marinade (TM), (3) double amount of TM modified with added 1.2% sodium

lactate, 9% acetic acid, and 68% soy sauce with 5% ethanol (MM), (4) dipping into 5% acetic acid and then TM (AATM), and

(5) dipping into 1% Tween 20 and then into 5% acetic acid, followed by TM (TWTM). Bacterial survivors were determined on

tryptic soy agar with 0.1% pyruvate and xylose lysine tergitol 4 (XLT4) agar. Results indicated that drying reduced bacterial

populations in the order of pre-drying treatments TWTM (4.86.0 log CFU/cm2)zAATMzMM>TMzC (2.65.0 log CFU/cm2). Nonadapted Salmonella were significantly (P< 0.05) more resistant to inactivation during drying than acid-adaptedPatricia A. KendaEffect of acid adaptation on inactivation of Salmonella during

drying and storage of beef jerky treated with marinades

Mehmet Calicioglua, John N. Sofosa,*, John Samelisa,Historically, jerky is among the oldest of meat

products that are preserved by salting and drying.

0168-1605/$ - see front matter D 2003 Elsevier Science B.V. All rights re

doi:10.1016/S0168-1605(03)00107-7

* Corresponding author. Tel.: +1-970-491-7703; fax: +1-970-

491-0278.

E-mail address: John.Sofos@colostate.edu (J.N. Sofos).www.elsevier.com/locate/ijfoodmicro

logy 89 (2003) 5165and known as microbiologically safe ( < 0.70 aw),

jerky is in high demand as a snack food and widely

available to consumers in convenience stores in North

America. Today, jerky is produced by consumers at

home as well as by industrial establishments. Con-

sequently, numerous recipes for making jerky are

available and are based on using meat from several

served.

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 516552species, e.g., beef, poultry, game animals, variable

preparation procedures of meat, e.g., thick or thin

slices, different marination techniques, e.g., various

ingredients, volume, time and temperature, and varia-

tions in drying processes, e.g., oven, food dehydrator

and smokehouse, and drying temperature (high vs.

low temperature). Such a large diversity in process

parameters may cause variation in effectiveness of

drying for inactivating pathogenic bacteria that may

be present on the raw material (Centers for Disease

Control and Prevention (CDC), 1995; Keene et al.,

1997). For example, Eidson et al. (2000) reported that

eight foodborne disease outbreaks occurred due to

consumption of contaminated beef jerky (six Salmo-

nella and two Staphylococcus aureus) between 1966

and 1995 in the state of New Mexico alone. A recent

report (Levine et al., 2001) by the Food Safety and

Inspection Service of the United States Department of

Agriculture (FSIS/USDA) indicated that for the period

1990 to 1999, cumulative prevalence of Salmonella in

jerky produced in the US federally inspected plants

was 0.31%.

Efficacy of the jerky-making process in inactivat-

ing foodborne pathogens has been evaluated by a

number of researchers (Holley, 1985a,b; Harrison

and Harrison, 1996; Harrison et al., 1997, 1998,

2001; Keene et al., 1997; Faith et al., 1998; Albright,

2000). Results of these studies are somewhat contra-

dictory in making recommendations to consumers

with respect to drying temperature and time. For

example, Harrison and Harrison (1996) reported that

a traditional jerky-preparation process, such as dry-

ing at 60 jC for 10 h, was sufficient to deliver a 5-log reduction of Escherichia coli O157:H7, Salmo-

nella typhimurium and Listeria monocytogenes in

marinated whole-muscle beef jerky. In contrast,

Keene et al. (1997) reported that drying at V 63jC was not a reliable method for eliminating E. coliO157:H7 in marinated (pH 4.2) whole-muscle ven-

ison jerky.

In response to the Salmonella and E. coli

O157:H7 outbreaks linked to jerky (CDC, 1995;

Keene et al., 1997), USDA/FSIS (1998) has sug-

gested cooking meat to 71.1 jC before drying toeliminate the risk of pathogens. Harrison et al. (2001)

tested the effectiveness of pre-drying heating of

inoculated beef strips at 71.1 jC, boiling in tradi-

tional marinade, post-drying heating at 57.4 jC for10 min, and traditional marinade for reducing num-

bers of foodborne pathogens during drying at 60 jCfor 10 h. The authors reported that all preparation

procedures yielded equal to or greater than 5.8, 3.9

and 4.6 log reductions of E. coli O157:H7, L. mono-

cytogenes and Salmonella, respectively, as measured

on selective agar media. Pre-heating of meat and/or

drying of jerky at high temperatures for extended

periods of time may result in a product that differs in

texture from traditional jerky and, thus, it may be of

reduced consumer acceptability. Use of chemical

intervention strategies as pre-drying treatments, how-

ever, has not been studied adequately (Albright,

2000). Such interventions can be a viable option to

avoid severe heat treatments while they may further

provide residual antimicrobial effects during storage.

These chemicals may include organic acids or salts

(e.g., acetic acid, lactates), ethanol, and food grade

surfactants (e.g., polysorbates). Gould (2001) empha-

sized the importance of designing new food preser-

vation methods/technologies to inactivate bacteria

while avoiding severe treatments that may change

desired characteristics of the food, since most of the

classical food preservation methods (e.g., chilling,

freezing, drying) slow down or inhibit growth of

bacteria rather than inactivating them. Leistner

(2000) indicated that preservation methods/technolo-

gies that simultaneously or sequentially expose bac-

teria to multiple stress factors (hurdles) lead bacterial

cells to metabolic exhaustion or homeostatic disturb-

ance, followed by cellular death.

Acid adaptation of Salmonella has been reported to

enhance its survival in acidic foods as well as to

increase cross-protection to other types of stresses

such as heat (Leyer and Johnson, 1992, 1993; Tsai

and Ingham, 1997; Wilde et al., 2000; Casadei et al.,

2001; Mazzotta, 2001). Another concern is that

stressed pathogens may exhibit lower infectious doses

and, thus, be of increased virulence, because it has

been hypothesized that populations of enteric bacteria

that are resistant to acid may not be reduced signifi-

cantly by the acidity of the gastric environment to

eventually cause infection (Gorden and Small, 1993).

Therefore, the objective of the present study was to

evaluate the effectiveness of several chemical-based

pre-drying treatments (modified marinades) on inacti-

vation of acid-adapted or nonadapted Salmonella cellsduring drying and storage of whole-muscle beef jerky.

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 5165 532. Materials and methods

2.1. Bacterial strains used and preparation of

inoculum

A five-strain composite inoculum of S. typhimu-

rium (ST) was used for inoculating beef slices. These

strains were SF530 (UK1), R-4 (Copenhagen,

DT104), R-5 (Copenhagen, DT104), ATCC700408,

and ATCC14028. Each strain was propagated (35 jC,24 h) and maintained on tryptic soy agar (TSA, Difco

Laboratories, Sparks, MD) slants at 4 jC. Strains weresubcultured monthly. The cultures were activated by

transferring a loopful of each strain into 9 ml of tryptic

soy broth (TSB, Difco) and incubating at 35 jC for 24h. A 0.1 ml portion of each culture was then transferred

into 9-ml tubes of glucose-free TSB for nonacid-

adapted cells and in glucose-free TSB with 1% added

glucose (Sigma, St. Louis, MO) for acid-adapted cells

(Wilde et al., 2000). After incubation at 35 jC for 2224 h, individual cultures were combined in a sterile

oak-ridge tube prior to centrifuging at 6000 rpm

(2900 g) (Eppendorf, model 5402) for 15 min at21 jC. The resulting pellet was washed once with0.1% phosphate buffered saline (PBS, Sigma) to

remove residual organic material, recentrifuged, and

then resuspended in PBS to a final volume of 100 ml.

The average level of inoculum was 7.5 log CFU/ml.

Supernatant pH values were approximately 5.1 and 7.1

for acid-adapted and nonadapted cultures, respectively.

2.2. Preparation of meat slices

Vacuum packaged and frozen ( 18 jC) beefinside rounds ( < 3 months) were purchased from the

Colorado State University Meat Science Laboratory

(Fort Collins, Colorado). Following thawing at 4 jCovernight, inside rounds were sliced at 0.6 cm thick-

ness using a food slicer (model 610, Hobart, Troy,

OH) and cut into pieces of 8.7 4.0 cm using a plastictemplate and knife. Approximately 100 slices (2.2 kg)

of meat were vacuum packaged and kept frozen at

18 jC until used (13 weeks).

2.3. Inoculation procedure

Frozen beef slices were thawed at 4 jC for 24 h

and placed on plastic trays that were covered withaluminum foil. In a laminar-flow hood, 0.5 ml of the

Salmonella inoculum was placed on the upper surface

of each slice and spread onto the entire surface area

using a sterile bent glass rod. Bacteria were allowed to

attach to the meat surface for 15 min at ambient

temperature. The beef slices were then flipped over

and the other side was inoculated following the same

procedure. The resulting level of inoculum was ap-

proximately 6.0 log CFU/cm2.

2.4. Pre-drying treatment description

Pre-drying treatments included: (i) control, no

treatment (C), (ii) marination with traditional mari-

nade (TM) (pH 4.3), (iii) increased (double the

amount) marination with modified marinade (MM)

(pH 3.0), (iv) 10-min immersion in 5% acetic acid

solution (pH 2.5), followed by TM (AATM), and (v)

sequential 15-min immersion in 1% Tween 20 solu-

tion (polyoxyethylene-20-sorbitan monolaurate) (pH

6.6), then 10-min immersion in 5% acetic acid sol-

ution (pH 2.5), followed by marination with TM

(TWTM).

2.5. Pre-drying treatment preparation and application

The TM was prepared for 1.0 kg of meat (Andress

and Harrison, 1999) as follows: 60 ml soy sauce

(Kikkoman Foods, Walworth, WI), 15 ml Worcester-

shire sauce (Heinz, Pittsburgh, PA), 0.6 g black

pepper (Heller Seasoning and Ingredients, Chicago,

IL), 1.25 g garlic powder (Excalibur Seasoning,

Pekin, IL), 1.5 g onion powder (Excalibur), and

4.35 g old hickory smoked salt (Tone Brothers,

Ankeny, IA). In the present study, 34 ml of this

marinade was spread manually onto 450 g inoculated

beef slices to cover the entire surface area using

flame-sterilized forceps. Approximately 30 ml of the

marinade remained on the meat slices.

The modified marinade was prepared for 1.0 kg

of meat as follows: 120 ml of milder soy sauce

(Kikkoman) containing approximately 4.75.0%

ethanol as preservative, 30 ml of Worcestershire

sauce, 0.6 g black pepper, 1.25 g garlic powder,

1.5 g onion powder, 4.35 g smoke-flavored salt, 3.6

ml food grade sodium-L-lactate of a 60% preparation

(Purac, Lincolnshire, IL), and 16 ml of glacial aceticacid (Mallinckrodt Baker, Paris, KY) to adjust the

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 516554pH to 3.0. A 77 ml portion of this marinade solution

was spread onto 450 g of beef, and mixed to cover

the surfaces of the meat slices. Approximately 60 ml

of the MM (double that of traditional marinade)

remained on the meat as determined by difference.

For treatment (iv) (AATM), meat slices were dipped

at ambient temperature for 10 min into a 5% (v/v)

acetic acid solution prepared using glacial acetic acid

(Mallinckrodt Baker) (450 ml per 450 g of meat) in

a glass container. These slices were drained for 2

min to remove excessive fluid using an empty de-

hydrator tray. Slices were then placed on a tray

covered with aluminum foil and marinated with

traditional marinade in the same manner described

for TM. For treatment (v) (TWTM), meat slices were

dipped into 1% (v/v) Tween 20 (Fisher Scientific,

Fair Lawn, NJ) solution (450 ml per 450 g of meat)

[for 15 min at ambient temperature]. Slices were

drained for 2 min, before marination as for (iv)

(AATM). Tween 20 (polysorbate 20, or polyoxy-

ethylene-20-sorbitan monolaurate) is permitted to be

used in food as an adjuvant (Anonymous, 2001). The

black pepper, garlic and onion powder had been

irradiated by their manufacturers. Following each

treatment, the slices on the trays were covered with

aluminum foil and held at 4 jC for 24 h prior todrying.

2.6. Drying

Treated and refrigerated (24 h) meat slices were

dried at 60 jC for 10 h in American Harvest Garden-master dehydrators (model FD-1000, Nesco, Chaska,

MN). The dehydrators were cylindrical in shape and

consisted of a base unit and three drying trays. The

dehydrator base unit generated hot air, which venti-

lated upward through the sides and a hole in the

middle of the trays. The target temperature was based

on the air temperature measurement taken from the

middle hole of the dehydrator. The dehydrators with

empty trays were preheated for approximately 20 min

to 60 jC (140 jF). The empty trays were thenreplaced with other trays pre-loaded with meat slices.

During drying, temperature of the dehydrator air,

through the middle hole, and the surface temperature

of meat slices on each of the bottom, middle, and top

trays were monitored using thermocouples (Type Kbeaded probes, Pico Technology Cambridge, UnitedKingdom) and real-time data-recording software (Pico

Technology). After drying, the jerky strips were held

in the dehydrators overnight to allow the moisture

level in the jerky slices to equilibrate, and then placed

into 24-oz Whirl-Pak sterile plastic bags (Nasco, Fort

Atkinson, WI) for storage at ambient temperature

(25F 1 jC).

2.7. Analysis

Two samples (1 slice per sample) per treatment

were aseptically transferred into sterile plastic bags

(Nasco) at each sampling interval. These intervals

included after inoculation, and 0 [24 h after (4 jC)inoculation and marination)], 4, 7 and 10 h during

drying for each treatment, and days 15, 30 and 60

during storage. A 25 ml portion of 0.1% sterile

buffered peptone water (BPW) (Difco) was added to

sample bags prior to pummeling for 2 min at ambient

temperature. Serial decimal dilutions were made using

9-ml BPW tubes and 0.1 ml portions were surface

plated onto each of duplicate plates of each agar

medium. Bacteria were enumerated using tryptic soy

agar (Difco) with 0.1% sodium pyruvate (Fisher

Scientific) (TSAP) (Leyer and Johnson, 1992), and

xyloselysinetergitol 4 (XLT4) (Difco) agar. All

plates were incubated at 35 jC for 48 h. The enumer-ation detection limit was 0.4 log CFU/cm2. Lowercounts on selective media were due to injury. When

numbers of bacteria dropped below the detection limit

by direct plating, enrichment of samples was done.

Briefly, the remaining portion of the pummeled sam-

ple with 25 ml BPW was incubated within the sample

bag at 35 jC for 24 h. Aliquots of 0.5 ml and 0.1 mlwere transferred to 10 ml tetrathionate (TT) broth

(Difco) and Rappaport Vassiliadis (RV) broth (Difco),

respectively, and incubated at 42 jC for 24 h. Sus-pensions were then streak plated onto brilliant green

sulfa (BGS) agar (Difco) and XLT4 agar and incu-

bated at 35 jC for 2448 h for characteristic (black)colonies of Salmonella.

In addition to microbiological analyses, pH and

water activity (aw) of beef jerky slices were deter-

mined at the same sampling intervals. The pH was

measured from samples used for microbiological

analysis (25 ml BPW added and pummeled for 2

min) using a digital pH meter (Accumet 50; FisherScientific, Houston, TX) with a glass pH electrode

(Hanna Instruments, Ann Arbor, MI). The aw of a

beef slice was determined using standardized meth-

ods as described in the 16th edition of Association

of Official Analytical Chemists method 978.18

(Mulvaney, 1998). One jerky slice was cut into

small pieces to fit into the plastic container and

measurement was done using a water activity meter

(Model D2100, Rotronic Instrument, Huntington,

NY).

2.8. Statistical analysis

Two independent replicates of the study were

conducted. Microbiological data were converted to

log CFU/cm2 and evaluated using a 2 2 5 5 2[(acid adaptation numbers of replicates pre-dryingtreatments drying (sampling) times agar media,respectively)] factorial design. Data were analyzed

by analysis of variance for main (fixed) effects (acid

adaptation, pre-drying treatment, drying time, and

was used for all statistical analyses. Storage data were

analyzed separately. Mean counts of surviving bacte-

rial populations between 0 and 7 h during drying were

used to determine D-values by calculating the inverse

of the slope of the linear regression line. Standard

deviations of the pH and water activity data were

calculated.

3. Results

Statistical analysis of the microbial data revealed

that the main effects of pre-drying treatment, acid

adaptation, drying time and agar media, and the

interactions of acid adaptation pre-drying treat-ment drying time and pre-drying treatment dryingdrying time agar media were significant (P < 0.05),as listed from the most effective to the least. However,

data from each agar medium are presented separately

because the sizable volume of data did not allow

emper

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 5165 55agar media) and four-way interactions between acid

adaptation, pre-drying treatment, drying time, and

agar media using the Statistical Analysis System

(version 6.1, SAS Institute, Cary, NC). Least squares

means were separated using Fishers least significance

difference test (LSD) using the general linear models

(GLM) procedure of SAS. A significance level of 0.05

Fig. 1. Mean (n= 4) temperatures of dehydrator air (middle hole air tusing a home-type food dehydrator.ature) and the surface of beef slices during drying at 60 jC for 10 hpresentation in a single figure.

3.1. Temperature

Changes in dehydrator air temperature and meat

surface temperatures during drying are shown in Fig.

1. After loading the preheated dehydrator with meat

slices, air temperature decreased from 60 to 40 jC atthe beginning of drying, and then gradually

increased back to 60 jC within approximately 4 h.Surface temperatures of meat from all trays reached

60F 2 jC within approximately 6 h. At the end ofdrying, temperatures of meat surfaces decreased to

room temperatures (ca. 2530 jC) within approx-imately 1 h.

Fig. 2. Survival of bacteria during preparation (marination at 4 jC, 24 h) and drying (60 jC, 10 h) of beef jerky inoculated with nonacid-des, a

h and

(1.2

acid

ic aci

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 516556adapted (A) and acid-adapted (B) Salmonella and treated with marina

pre-drying treatment or marinade prior to refrigeration at 4 jC for 24jC for 24 h, and then dried; MM, marinated with modified marinadeheld at 4 jC for 24 h, and then dried; AATM, dipped into 5% aceticdipped into 1% Tween 20 (pH 6.6) for 15 min, and then 5% acetinoculation; 0: after marination. Results are meanF standard deviation, ns determined on tryptic soy agar with 0.1% pyruvate (TSAP). C, no

drying; TM, marinated with traditional marinade (pH 4.3), held at 4

% lactate, 9% acetic acid, and soy sauce with 5% ethanol) (pH.3.0),

solution (pH 2.5) for 10 min, and then marinated with TM; TWTM,

d solution for 10 min followed by marination with TM. AI: after= 4.

3.2. Effect of agar media

Numbers of bacteria recovered on XLT4 in the

products inoculated with nonadapted culture were

significantly (P < 0.05) lower than those recovered

on TSAP in MM, AATM, and TWTM treatments after

4 h of drying, in all treatments after 710 h of drying

(Figs. 2 and 3), and during the entire storage period

(except for TWTM on 60 days) (Figs. 4 and 5). In the

products inoculated with acid-adapted cultures, sig-

nificantly (P < 0.05) lower counts of bacteria were

recovered on XLT4 than those on TSAP in MM,

AATM, and TWTM before drying (0 h), in all treat-

ments after 4 h of drying, in C and TM after 7 and 10

h of drying (Figs. 2 and 3) and up to 30 days during

storage (Figs. 4 and 5). MM, AATM, and TWTM

treatments became more destructive on acid-adapted

Salmonella after 7 h of drying since the difference

between the two agar media was not significant

(P>0.05). These results suggest that pre-drying treat-

ments MM, AATM and TWTM resulted in faster and

greater levels of cellular injury in Salmonella during

h) an

s det

marination. Results are meanF standard deviation, n= 4.

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 5165 57Fig. 3. Survival of bacteria during preparation (marination at 4 jC, 24(A) and acid-adapted (B) Salmonella and treated with marinades, a

treatments were as described in Fig. 2. AI: after inoculation; 0: afterd drying (60 jC, 10 h) of beef jerky inoculated with nonacid-adaptedermined on xylose lysine tergitol 4 agar (XLT4) (B). Pre-drying

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 516558drying, as estimated by the differences between non-

selective (TSAP) and selective (XLT4) agar media.

This is particularly true for acid-adapted cell popula-

tions in treatments C and MM.

3.3. Effect of pre-drying treatments

There was no significant (P>0.05) reduction in the

initial numbers of bacteria as determined by TSAP in

Fig. 4. Fate of surviving bacteria during aerobic storage at 25 jC for 60 day(B) Salmonella after treating with marinades and drying at 60 jC for 10 h,drying treatments were as described in Fig. 2. AI: after inoculation; 0: afteany treatment, irrespective of acid adaptation (00.9

log) (Figs. 2 and 3). However, regardless of acid

adaptation, initial bacterial numbers as determined

by XLT4 were significantly (P < 0.05) reduced after

application of MM and TWTM and holding of meat

slices at 4 jC for 24 h (0.92.1 log) (Figs. 2 and 3).Counts on AATM were significantly ( P < 0.05)

reduced in products inoculated with the acid-adapted

culture only (1.9 log) (Fig. 3).

s on beef jerky inoculated with nonacid-adapted (A) and acid-adapted

as determined on tryptic soy agar with 0.1% pyruvate (TSAP). Pre-

r marination. Results are meanF standard deviation, n= 4.

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 5165 593.4. Effect of drying

Regardless of acid adaptation or the recovery

media used, initial bacterial counts were significantly

(P < 0.05) reduced in all treatments after 4 h of drying

and the largest reductions caused by each treatment

were observed during this period (Figs. 2 and 3).

Bacterial populations declined further between 4 and

7 h of drying in all treatments. In contrast to declines

Fig. 5. Fate of surviving bacteria during aerobic storage at 25 jC for 60 daySalmonella after treating with marinades and drying at 60 jC for 10 h, atreatments were as described in Fig. 2. AI: after inoculation; 0: after maridetected for the first 4 h of drying, reductions between

4 and 7 h of drying were not significant (P>0.05) in

all treatments. Populations of acid-adapted bacteria

dropped below the detection limit ( 0.4 log CFU/cm2) on XLT4 in MM, AATM and TWTM products

after 7 h of drying (Fig. 3), and remained undetectable

by direct plating during storage (Fig. 5). No signifi-

cant reduction in bacterial populations was found in

any treatment inoculated with either acid-adapted or

s on beef jerky inoculated nonacid-adapted (A) and acid-adapted (B)

s determined on xylose lysine tergitol 4 agar (XLT4). Pre-drying

nation. Results are meanF standard deviation, n= 4.

nonadapted cultures between 7 and 10 h of drying. In

general, MM, AATM, and TWTM resulted in signifi-

cantly lower bacterial populations as determined with

both TSAP and XLT4 than the C and TM at 4, 7 and

10 h of drying, for both culture types (Figs. 2 and 3).

Populations of previously acid-adapted bacteria, as

determined by use of both agar media (Fig. 3) were

significantly (P < 0.05) lower within each treatment at

4, 7, and 10 h of drying compared to those of non-

adapted cultures (Fig. 2). Significantly (P < 0.05)

lower numbers of acid-adapted bacteria were recov-

ered even before drying (0 h) in MM, AATM, and

TWTM treatments (Fig. 3). These results indicate that

acid adaptation may sensitize Salmonella to the pro-

cessing conditions described in the present study.

Indeed, total log reductions in the numbers of bacteria

in the products inoculated with nonadapted Salmo-

nella at the end of 10 h of drying, as determined on

nonselective (TSAP) and selective (XLT4) media

were 2.63.1 for control, 3.03.1 for TM, 4.44.9

for MM, 4.14.9 for AATM and 4.85.6 for TWTM.

The corresponding numbers in products inoculated

with acid-adapted cells, which were 3.35.0 for con-

trol, 3.85.0 for TM, 6.16.0 for MM, 5.56.0 for

AATM, and 6.06.0 for TWTM clearly indicate a

negative influence of acid adaptation on Salmonella

survival during jerky processing, and in accordance to

the marinade treatment applied.

Irrespective of acid adaptation, MM, AATM and

TWTM treatments resulted in smaller D-values than

did the C and TM treatments, indicating faster inacti-

vation of bacteria during drying (Figs. 2 and 3). D-

values in products inoculated with acid-adapted cells

were smaller than in products inoculated with non-

adapted cells, indicating a possible increase in suscept-

ibility of acid-adapted cells to hot-air drying for 7 h.

3.5. Effect of storage

With the exception of treatment C, which had more

survivors than other treatments after drying, and

TWTM at 60 days, no treatment resulted in significant

reduction of the populations of nonadapted, surviving-

bacteria determined using TSAP (Fig. 4) during the

60-day storage period. On XLT4 (Fig. 5), however,

significant declines occurred in products at 15 days

nocul

10 h

d

jC f24 h

, and

bient

ic aci

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 516560Table 1

Mean pH values [(n= 4, (standard deviation)] of beef jerky slices i

various pre-drying marination treatments before drying at 60 jC for

Steps Time Inoculated with acid-adapted Salmonella

Ca TMb MMc AATM

Processing 0 h 5.56

(0.18)

5.37

(0.12)

4.34

(0.09)

4.35

(0.05)

4 h 5.75

(0.21)

5.41

(0.14)

4.68

(0.05)

4.71

(0.17)

7 h 5.81

(0.30)

5.73

(0.18)

4.69

(0.08)

4.74

(0.20)

10 h 5.87

(0.33)

5.80

(0.30)

4.84

(0.14)

4.79

(0.20)

Storage Day 15 5.86

(0.30)

5.81

(0.21)

4.89

(0.08)

4.83

(0.21)

Day 30 5.91

(0.19)

5.88

(0.17)

4.94

(0.09)

4.83

(0.09)

Day 60 5.91

(0.14)

5.90

(0.15)

4.98

(0.06)

4.89

(0.03)

a No pre-drying treatment or marinade prior to refrigeration at 4b Marinated with traditional marinade (pH 4.3), held at 4 jC forc Marinated with modified marinade (1.2% lactate, 9% acetic acid

dried.d Dipped into 5% acetic acid solution (pH 2.5) for 10 min at am

marinade.e Dipped into 1% Tween 20 (pH 6.6) for 15 min, and then 5% acetwith traditional marinade.ated with acid-adapted or nonadapted Salmonella and subjected to

and storage at 25 jC for 60 days

Inoculated with nonacid-adapted Salmonella

TWTMe C TM MM AATM TWTM

4.36

(0.06)

5.59

(0.21)

5.38

(0.22)

4.55

(0.12)

4.73

(0.11)

4.63

(0.14)

4.55

(0.10)

5.75

(0.08)

5.52

(0.01)

4.76

(0.14)

4.79

(0.09)

4.91

(0.05)

4.53

(0.05)

5.70

(0.08)

5.56

(0.03)

4.81

(0.10)

4.89

(0.10)

4.93

(0.07)

4.71

(0.21)

5.61

(0.13)

5.49

(0.18)

4.76

(0.21)

4.85

(0.19)

4.86

(0.23)

4.78

(0.21)

5.53

(0.08)

5.55

(0.10)

4.71

(0.17)

4.84

(0.11)

4.82

(0.15)

4.77

(0.10)

5.32

(0.27)

5.55

(0.03)

4.79

(0.10)

4.80

(0.05)

4.95

(0.15)

4.89

(0.04)

5.64

(0.03)

5.59

(0.10)

4.93

(0.12)

4.95

(0.07)

5.00

(0.08)

or 24 h and drying.

, and then dried.

soy sauce with 5% ethanol) (pH 3.0), held at 4 jC for 24 h, and then

temperature, drained for 2 min, and then marinated with traditional

d solution for 10 min at ambient temperature, followed by marination

(MM, TWTM) or 30 days (C, TM, AATM). At the end

of 60 days of storage, bacteria were still detectable in

all treatments by direct plating on TSAP. Therefore,

when the products were inoculated with nonadapted

cultures, complete elimination of the pathogen was not

achieved by any treatment within 60-day storage. In

contrast, populations of surviving bacteria in products

inoculated with acid-adapted cultures rapidly declined

within all treatments as determined on both agar media

(Figs. 4 and 5). Counts were below the detection limit

in MM, AATM, and TWTM treatments during the

entire storage period, and in C and TM by 60 days.

Salmonella populations were completely eliminated

(enrichment negative) by 15 days in MM and TWTM,

and by 30 days in AATM products, whereas viable

cells were recovered from C and TM products by

enrichment after 60 days of storage.

3.6. pH and aw

Table 1 shows pH values of each treatment

throughout the experiment. The pH values of products

from all treatments slightly increased during drying

and storage. Water activity of all products decreased

rapidly during drying, falling below bacterial growth

permitting ( < 0.84) values after 7 h of drying (Table

2). There were no clear differences in aw reductions

among individual treatments during drying and no

notable difference was found among treatments dur-

ing storage. The ultimate aw of the finished products

varied between 0.600 and 0.700. Fluctuations in awbetween sampling times were probably due to varia-

tion among slices.

4. Discussion

Results of the present study indicate that inactiva-

tion of Salmonella during drying of beef jerky is

affected by the culture history and by the type of

pre-drying treatment. Although drying reduced bacte-

rial populations in all treatments, use of MM, AATM,

and TWTM resulted in faster and greater levels of

reduction under the conditions described in this study.

Increasing the efficacy of food preservation meth-

ods, such as drying to inactivate bacteria, may be

ocula

10 h

d

)

)

)

) (0.022) (0.127) (0.018) (0.029) (0.095) (0.038)

)

0.687 0.674 0.554 0.667 0.608 0.631

)

)

jC fo24 h

, and

bient

ic aci

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 5165 61Table 2

Mean aw values (n= 4, (standard deviation)) of beef jerky slices in

various pre-drying marination treatments before drying at 60 jC for

Steps Time Inoculated with acid-adapted Salmonella

Ca TMb MMc AATM

Processing 0 h 0.961

(0.011)

0.958

(0.011)

0.952

(0.008)

0.941

(0.006

4 h 0.898

(0.016)

0.829

(0.048)

0.765

(0.012)

0.854

(0.030

7 h 0.806

(0.037)

0.732

(0.035)

0.724

(0.030)

0.730

(0.012

10 h 0.663

(0.032)

0.637

(0.095)

0.577

(0.010)

0.728

(0.010

Storage Day 15 0.664

(0.019)

0.640

(0.017)

0.554

(0.040)

0.710

(0.035

Day 30 0.649

(0.005)

0.641

(0.011)

0.578

(0.004)

0.601

(0.016

Day 60 0.652

(0.003)

0.639

(0.023)

0.606

(0.008)

0.621

(0.030

a No pre-drying treatment or marinade prior to refrigeration at 4b Marinated with traditional marinade (pH 4.3), held at 4 jC forc Marinated with modified marinade (1.2% lactate, 9% acetic acid

dried.d Dipped into 5% acetic acid solution (pH 2.5) for 10 min at am

marinade.e Dipped into 1% Tween 20 (pH 6.6) for 15 min, and then 5% acetwith traditional marinade.(0.016) (0.040) (0.030) (0.021) (0.024) (0.027)

0.665

(0.021)

0.670

(0.021)

0.600

(0.020)

0.678

(0.032)

0.636

(0.013)

0.682

(0.005)

0.645

(0.007)

0.665

(0.028)

0.693

(0.011)

0.674

(0.022)

0.637

(0.019)

0.654

(0.040)

r 24 h and drying.

, and then dried.

soy sauce with 5% ethanol) (pH 3.0), held at 4 jC for 24 h, and then

temperature, drained for 2 min, and then marinated with traditional

d solution for 10 min at ambient temperature, followed by marinationted with acid-adapted or nonadapted Salmonella and subjected to

and storage at 25 jC for 60 days

Inoculated with nonacid-adapted Salmonella

TWTMe C TM MM AATM TWTM

0.954

(0.003)

0.963

(0.004)

0.949

(0.005)

0.950

(0.005)

0.955

(0.004)

0.959

(0.003)

0.856

(0.037)

0.890

(0.033)

0.776

(0.154)

0.866

(0.016)

0.900

(0.009)

0.893

(0.046)

0.719

(0.006)

0.811

(0.018)

0.832

(0.028)

0.547

(0.095)

0.708

(0.111)

0.755

(0.017)

0.692 0.664 0.528 0.671 0.628 0.547

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 516562achieved by use of two approaches. The first could

include increasing the severity of existing processing

parameters (e.g., higher temperature, longer time),

which is not desirable because it can adversely affect

the quality of the food (Gould, 2001). The second

approach could be to render bacteria more vulnerable

to the effects of existing parameters. This approach

can be accomplished by designing hurdles that can

reduce the ability of bacteria to resist processing

parameters, including drying temperature. These hur-

dles may target bacterial attachment and/or homeo-

stasis (Leistner, 2000). Treatments MM, AATM and

TWTM, used in the present study, may be considered

applications of this approach. For example, Casadei et

al. (2001) reported that heat sensitivity of S. typhimu-

rium increased 100-fold in liquid media when the pH

dropped from 7.0 to 3.0 and/or in the presence of up

to 10% ethanol. Similarly, Jordan et al. (1999) and

Barker and Park (2001) reported that combinations of

organic acids or organic acid salts (e.g., 50 mmol

lactate), ethanol (5%), and low pH (3.0, HCl) pro-

vided significant reductions of stationary phase cells

of E. coli O157:H7 and L. monocytogenes in liquid

media (e.g., up to 5 logs in 4 min), suggesting cellular

death by disruption of pH homeostasis and by leading

to changes in gene expression and enzyme activity.

These findings were adopted in the present study in

order to design a relevantly modified jerky pre-drying

treatment (MM) by using commercially available soy

sauce containing approximately 5% ethanol, and add-

ing 2% of a 60% solution of sodium lactate, and 9%

acetic acid to TM. The volume of marinade solution

was also increased by using double the volume of soy

sauce and Worcestershire sauce to deliver a higher

marinade solution amount per slice than was used in

the treatment TM. The MM used in the present study

was significantly more effective in reducing the pop-

ulations of Salmonella cells on beef slices during

drying than were TM and C treatment, regardless of

acid adaptation. These findings indicated that combi-

nations of lactate, ethanol and acetic acid may have a

potential for use in destruction of pathogenic bacteria

in processed foods.

It is known that bacterial cells attached to the

surface of a product such as meat become more

resistant to stress factors such as heat than nonat-

tached cells (Humphrey et al., 1997). Another studyshowed that pre-spraying of beef carcasses inoculatedwith high levels of E. coli 0157:H7, with 5% Tween

20 followed by spraying with 2% lactic acid resulted

in significantly higher reduction of the pathogen

compared to spraying with lactic acid alone or water

(Calicioglu et al., 2002). It has been speculated that

Tween 20 might loosen or prevent cellular attachment

on the meat surface via its surfactant and hydrophobic

effects, thus making cells more vulnerable to the

effect of subsequent acid exposure. This proposed

carcass decontamination method was applied as a

pre-drying treatment in the present study (TWTM)

prior to acetic acid dip and marinating with a tradi-

tional recipe. Tween 20 (polysorbate 20, or polyoxy-

ethylene-20-sorbitan monolaurate) is permitted in

food as an adjuvant under US food legislation (Anon-

ymous, 2001). In the present study, TWTM was

slightly more effective than AATM but differences

were not significant (P>0.05). Both treatments were

significantly (P < 0.05) more effective at inactivating

Salmonella than C and TM.

Survival of the pathogen during drying was not

significantly (P>0.05) different between C and TM

jerky in the present study, indicating no additional

antimicrobial effect from TM. Although TM contains

Worcestershire sauce, it was not acidic enough (pH

4.3) to cause an appreciable reduction in the pH of the

TM product compared to the control product, given

the limited volume used per beef slice. Other research-

ers studied the effectiveness of TM in reducing

numbers of Salmonella and other pathogens. Harrison

and Harrison (1996) reported that drying of tradition-

ally marinated whole-muscle jerky at 60 jC for 10 hwas sufficient to deliver >5.0 log units reduction of E.

coli O157:H7, L. monocytogenes, and S. typhimurium,

as determined with selective agar media (bismuth

sulfite agar for Salmonella). A more comprehensive

study with E. coli O157:H7, L. monocytogenes, and

Salmonella, was carried out by Harrison et al. (2001).

The effectiveness of traditional marinade, oven-heat-

ing at 71 jC before drying, boiling in marinade priorto drying, and post-drying heating of traditionally

marinated jerky at 57.3 jC for 10 min in reducingnumbers of the bacteria was compared with whole-

muscle beef jerky during drying at 60 jC for 10 h. Atthe end of drying, total reductions from all treatments

were equal or greater than 5.8, 3.9, and 4.6 log units

for E. coli O157:H7, L. monocytogenes, and Salmo-nella, respectively, even with traditional marinade. In

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 5165 63the present study, although effectiveness of TM and

other treatments is greatly increased on acid-adapted

cultures, C and TM resulted in lower bacterial reduc-

tions compared to MM, AATM and TWTM during

drying and, thus, appeared to cause more rapid decline

of survivors during storage. While MM, AATM and

TWTM caused immediate bacterial injury followed

by death, C and TM caused a delayed injury resulting

in longer survival of bacteria. Unlike heat-based

interventions, which have immediate effect, modified

marinades described in this study (MM, AATM,

TWTM) may provide residual antimicrobial effect

against possible post-drying contamination.

Numbers of the pathogen were continually reduced

in all treatments until 4 or 7 h of drying and then

followed a much slower reduction, or remained

unchanged. This decelerated reduction has been

described as a tailing effect (Shadbolt et al., 2001).

In the present study, the tailing effect was seen in

almost all treatments, but to extents that were depend-

ent upon acid adaptation, pre-drying treatment and agar

media. The causes of tailing or its implications to food

safety have not been completely elucidated. Humphe-

son et al. (1998) investigated mechanisms of tailing

with Salmonella enteritidis PT4 in liquid media during

exposure to different temperatures. Their results indi-

cated that tailing was more evident at higher temper-

atures (e.g., 60 jC) than at lower temperatures (e.g., 49jC), and that tailing was reduced in the presence ofchloramphenicol, indicating that production of heat-

shock proteins may be responsible for cell protection

and the tailing (Humpheson et al., 1998). Similar

results have been reported for E. coli by Shadbolt et

al. (2001) who indicated that cells synthesize an intra-

cellular de novo protein under low aw conditions which

results in the tailing effect. The tailing effect observed

in this study, however, may have also been due to

product case hardening, potential effects of high alti-

tude, and the low humidity conditions of Colorado

(Albright, 2000). Survival characteristics of bacteria in

the tail part of the inactivation curve may raise the

question whether these bacteria become more resistant

to adverse conditions such as high acid compared to

initial bacterial populations, and thus increase their

hazardous potential. Although not evaluated in this

study, Buchanan et al. (1994) investigated nonthermal

inactivation of L. monocytogenes and reported that thetailing effect was not due to presence of amore resistantsubpopulation. Their results revealed that inactivation

rates and tailing of the pathogen in liquid media as

affected by pH and sublethal temperatures (up to 40

jC) were independent of initial population density.Nevertheless, the potential implications of tailing on

food safety and food preservation require additional

study.

There is evidence that acid adaptation of Salmo-

nella may enhance its survival in acidic foods as well

as increase cross-protection to other type of stresses

(Leyer and Johnson, 1992, 1993; Tsai and Ingham,

1997; Wilde et al., 2000; Casadei et al., 2001; Maz-

zotta, 2001). In the present study, however, the

opposite occurred. Acid-adapted cells were more

vulnerable to the secondary stress factors (drying)

alone or in combination with mild acidic environment

(marination) than nonadapted Salmonella. Similar

results were reported for Salmonella and other patho-

gens by other researchers (Dickson and Kunduru,

1995; Leyer and Johnson, 1997; Ryu et al., 1999;

Casadei et al., 2001). For example, Dickson and

Kunduru (1995) reported that acid-adapted Salmo-

nella, as compared to nonadapted Salmonella, had

equal or greater sensitivity to organic acid rinses on

the beef tissue. Likewise, Ryu et al. (1999) reported

that there was no significant difference in the survival

of acid-adapted, nonadapted and acid-shocked cells of

E. coli O157:H7 in dried beef powder during storage.

This difference might be the result of use of multiple

hurdles in the meat drying process, such as high acid,

temperature and aw, as well as the differences in acid

adaptation methods. It might be speculated that the

level of cross-protection from acid adaptation is

dependent on presence and severity of other stress

factors as a function of time. It seems that acid (e.g.,

pHV 4.8), heat (e.g., z 60 jC) and low aw (e.g.,< 0.800) may provide combinations of destructive

conditions that can overcome any cross-protection

provided by acid adaptation.

In conclusion, results of the present study indicated

that acid-adapted cells of Salmonella did not exhibit

any increased resistance to the drying process com-

pared to that of nonadapted cells. Our results also

revealed that using food grade chemicals as pre-

drying treatments (MM, AATM, and TWTM)

improved the effectiveness of the meat-drying process

for inactivating Salmonella compared to the processused in traditional jerky making. The effects of these

(GPO), Washington, DC, pp. 5259 (April).

Barker, C., Park, S.F., 2001. Sensitization of Listeria monocyto-

Casadei, M.A., Ingram, R., Hitchings, E., Archer, J., Gaze, J.E.,

M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 5165642001. Heat resistance of Bacillus cereus, Salmonella typhimui-

rum, and Lactobacillus delbrueckii in relation to pH and etha-

nol. Int. J. Food Microbiol. 63, 125134.

Centers for Disease Control and Prevention (CDC), 1995. Outbreak

of salmonellosis associated with beef jerkyNew Mexico.

Morb. Mort. Wkly. Rep. 44, 785788.

Dickson, J.S., Kunduru, M.R., 1995. Resistance of acid-adapted

Salmonellae to organic acid rinses on beef. J. Food Prot. 58,

973976.

Eidson, M., Sewell, C.M., Graves, G., Olson, R., 2000. Beef jerky

gastroenteritis outbreaks. J. Environ. Health 62, 913.genes to low pH, organic acids and osmotic stress by ethanol.

Appl. Environ. Microbiol. 67, 15941600.

Buchanan, R.L., Golden, M.H., Whiting, R.L., Philips, J.G., Smith,

J.L., 1994. Non-thermal inactivation models for Listeria mono-

cytogenes. J. Food Sci. 59, 179188.

Calicioglu, M., Kaspar, C.W., Buege, D.R., Luchansky, J.B., 2002.

Effectiveness of spraying with Tween 20 and lactic acid to

decontaminate inoculated Escherichia coli O157:H7 and indig-

enous E. coli biotype I on beef. J. Food Prot. 65, 2632.treatments on sensory attributes of the final product

are being evaluated in another study. Based on the

findings of the present study, use of MM, AATM or

TWTM in the jerky-making process and storing it for

15 to 30 days at ambient temperature, or use of TM

and at least 60 days of storage prior to consumption

may provide at least a 5.0 log reduction in Salmonella

count.

Acknowledgements

This project was funded by USDA-CSREES

National Integrated Food Safety Initiative and by the

Colorado Agricultural Experiment Station. We thank

Excalibur Seasoning, and Heller Seasoning and

Ingredients, for providing irradiated spices.

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M. Calicioglu et al. / International Journal of Food Microbiology 89 (2003) 5165 65

Effect of acid adaptation on inactivation of Salmonella during drying and storage of beef jerky treated with marinadesIntroductionMaterials and methodsBacterial strains used and preparation of inoculumPreparation of meat slicesInoculation procedurePre-drying treatment descriptionPre-drying treatment preparation and applicationDryingAnalysisStatistical analysis

ResultsTemperatureEffect of agar mediaEffect of pre-drying treatmentsEffect of dryingEffect of storagepH and aw

DiscussionAcknowledgementsReferences