International Journal of Food Microbiology 140 (2010) 61–75

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International Journal of Food Microbiology

journal homepage: www. e l s ev i e r. c om / l o c a t e / i j fo o dm i c r o

Evaluation of fermentation, drying, and/or high pressure processing on viability of

Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Trichinella

spiralis in raw pork and Genoa salami☆

Anna C.S. Porto-Fett a,1, Jeffrey E. Call a, Bradley E. Shoyer a, Dolores E. Hill b, Claudette Pshebniski c,

George J. Cocoma d

, John B. Luchansky a,

⁎

a U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Microbial Food Safety Research Unit, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA b Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, 10300 Baltimore Avenue, Beltsville, MD, 20705, USA c Santa Maria Foods ULC, Brampton, ON, L6T 5M4, CA d Professional Resource Organization, 1405 Irvine Drive, Edmond, OK, 73003, USA

article info

Article history:

Received 18 December 2009

Received in revised form 3 February 2010

Accepted 9 February 2010

Keywords:

Listeria monocytogenes

Escherichia coli O157:H7

Salmonella spp.

Trichinella spiralis

Pork

High pressure processing

☆ Portions of this research were presented at the Annual Meeting of the International

Association for Food Protection, Dallas, TX, July 12 to 15, 2009 (Call et al., 2009; PortoFett et al., 2009). Mention of trade names or commercial products in this publication is

solely for the purpose of providing specific information and does not imply

recommendation or endorsement by the U.S. Department of Agriculture.

⁎ Corresponding author. Tel.: +1 215 233 6676; fax: +1 215 233 6581.

E-mail address: john.luchansky@ars.usda.gov (J.B. Luchansky).

Present address: Oser Technologies, P.O. Box 62, Blacksville, WV, 26521, USA.

0168-1605/$ – see front matter. Published by Elsevier B.V.

doi:10.1016/j.ijfoodmicro.2010.02.008

abstract

We evaluated the effectiveness of fermentation, drying, and high pressure processing (HPP) to inactivate

Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Trichinella spiralis in Genoa salami

produced with trichinae-infected pork. In addition, we evaluated the effectiveness of using HPP to inactivate

T. spiralis larvae in pig masseter tissue. In part A, Genoa salami batter (about 2.3 log larvae/g) prepared with

trichinae-infected pork was separately spiked with a five-strain cocktail of each microbial pathogen (about

7.0 log CFU/g) and subsequently fermented at 20 °C and about 90 to 95% RH for 6 h and then at 27 °C and

about 90 to 95% RH for 26 h before being dried at 20 °C and about 65 to 75% RH for 40 h and then at 17 °C and

about 65 to 75% RH to/for: A) 25 d (65 mm casing), B) a target aw of 0.92 (65 mm casing), C) 35 d (105 mm

casing), or D) a target aw of 0.94 (105 mm casing). Inactivation of L. monocytogenes, E. coli O157:H7, and

Salmonella spp. after fermentation and drying ranged from about 1.1 to 1.3, about 1.1 to 2.2, and about 4.2 to

4.8 log CFU/g, respectively. After drying, three replicate salami samples in each of two trials for each

treatment were subjected to HPP. Pressurization at 600 MPa or at 483 MPa for 1 to 12 min reduced pathogen

numbers by an additional 1.6 to ≥5.0 (L. monocytogenes), 4.7 to ≥5.8 (E. coli O157:H7), and 1.9 to 2.4

(Salmonella)log CFU/g. After storage for 28 d at 4 °C, L. monocytogenes levels decreased by up to an additional

3.0 log CFU/g, whereas an additional decrease of up to about 1.1 and 1.7 log CFU/g was observed for E. coli

O157:H7 and Salmonella, respectively. In contrast, in each of three trials, T. spiralis was inactivated (about

2.3 log larvae/g) in Genoa salami by all treatments of fermentation and drying as confirmed by both

microscopy and mouse bioassays. In part B, in each of two trials, a 10-g portion (2 replicates per treatment)

of infected pig masseter muscle (about 3.4 log larvae/g) were pressurized at 483 and 600 MPa for 0.5 to

5 min. T. spiralis was inactivated in pig masseter by all treatments of HPP as confirmed by both microscopy

and mouse bioassays. Thus, fermentation and drying and/or HPP of contaminated Genoa salami or pork are

effective for inactivating L. monocytogenes, E. coli O157:H7, Salmonella spp., and/or T. spiralis larvae. These

data validate that HPP can be used as an alternate to curing for trichinae control and as a post-process

intervention to meet performance standards and/or compliance guidelines for the three microbial pathogens

evaluated herein.

Published by Elsevier B.V.

1. Introduction

Although a concerted effort by both the meat industry and

regulatory agencies has appreciably reduced the incidence of Listeria

monocytogenes and Salmonella spp. in fermented sausage, the USDA/

FSIS reported the prevalence of these pathogens in fermented meats

at about 1.4% (10 of 698 samples) and 3.3% (27 of 830 samples),

respectively, in federally inspected plants between 1997 and 1999

(Levine et al., 2001). Likewise, from 1995 to 1999, 3445 regulatory

samples of dry and semi-dry sausages tested negative for Escherichia 1
62 A.C.S. Porto-Fett et al. / International Journal of Food Microbiology 140 (2010) 61–75

coli O157:H7 (Levine et al., 2001). To further insure the safety of

fermented meats, the USDA/FSIS established performance standards

that require manufacturers of ready-to-eat (RTE) sausage to validate

the lethality of their processes. For dry and semi-dry fermented meats,

as detailed by Nickelson et al. (1996), the USDA/FSIS accepts the

following five options for control of pathogens: i) utilize heat as

detailed in 9 CFR 318.17, ii) use a validated 5D inactivation treatment,

iii) conduct a hold-and-test program for finished product, iv) propose

other strategies to achieve 5D inactivation, and iv) conduct raw

batter-testing and deliver 2D inactivation (see also, Reed, 1995 and

USDA-FSIS, 2003a). The USDA/FSIS performance standards also

require processors of red meat and poultry products to achieve a

6.5-log (beef and pork) or a 7.0-log (chicken and turkey) reduction for

Salmonella, as well as maintain a “zero tolerance” for L. monocytogenes

in cooked and RTE meats (USDA-FSIS, 2001b; USDA-FSIS, 2003a).

With further regard to L. monocytogenes, manufacturers of RTE red

meat and poultry products are also required to include a post-process

lethality step, or to include a post-process intervention to preclude

outgrowth of this pathogen during refrigerated shelf life, or to deliver

both lethality and inhibition to achieve the so called Alternative 1, 2,

or 3 status (USDA-FSIS, 2001b; USDA-FSIS, 2003a).

Genoa salami, a dry sausage traditionally manufactured using raw

pork, salt, and spices, relies on a decrease of pH and water activity

(aw) during fermentation and drying for both its quality and safety

attributes. The USDA standard of identity for Genoa requires it to

have a moisture-to-protein ratio (M:Pr) value of ≤2.3:1.0 (USDAFSIS, 2005). To be categorized as shelf-stable, in association with

other hurdles such as salt and curing agents, Genoa salami must

attain: i) a pH of pH 5.2 and an aw of b0.95, or ii) only a pH of bpH 5.0,

or iii) only an aw of b0.91 (http://www.fsis.usda.gov/PDF/FSRE_SS_7Principles.pdf). Several investigators have evaluated the lethality

of processes and/or have developed post-process interventions for

microbial pathogens in fermented sausages (Calicioglu et al., 2001;

Ihnot et al., 1998; Faith et al., 1998; Nightingale et al., 2006; PortoFett et al., 2008). Among the many biological, chemical, and physical

interventions used to control and/or eliminate foodborne pathogens

in fermented RTE meat products are the addition of bacteriocins and/

or of food-grade antimicrobials to the formulation, and/or the

application of high pressure processing (HPP), hydrodynamic

pressure processing (HDP), heat, or E-beam irradiation to the

finished product (Cabeza et al., 2009; Deumier and Collignan, 2003;

Hinkens et al. 1996; Marcos et al., 2005). However, relatively little is

known about the effectiveness of HPP when used in combination

with fermentation and drying to inactivate targeted microbial

pathogens such as L. monocytogenes, E. coli O157:H7, or Salmonella

spp. in Genoa salami. Thus, one objective of the present study was to

evaluate modifications to a traditional/standard protocol for processing Genoa salami, alone and in combination with HPP, for efficacy

towards the above mentioned microbial pathogens while enhancing

product quality.

Trichinellosis remains one of the most significant zoonotic foodborne diseases worldwide (Dorny et al., 2009;Gottstein et al., 2009). Itis

commonly associated with the ingestion of raw or uncooked pork that

becomes infected with the encysted larvae of Trichinella spiralis

(Gamble, 1996; Mitreva and Jasmer, 2006). Modifications, including

implementation of Good Agricultural Practices in swine production,

have dramatically decreased the prevalence of trichinae in conventionally raised swine to virtually non-detectable levels in the United States

over the past 60 years (Gamble et al., 1998; Gamble et al., 1999; Pyburn

et al., 2005). Moreover, when present, levels of this parasite in pork are

quite low, ranging from 0.003 to 1000 larvae/g (Doby, 1987; Gamble et

al., 1999). Thus, pigs continue to harbor this parasite, albeit at reduced

prevalence and levels than in the previous six decades and, therefore,

may serve as a vector for its transmission to humans in the absence of

Good Agricultural Practices, Good Manufacturing Practices, and/or

Hazard Analysis Critical Control Point standards.

Although sporadic in occurrence, trichinellosis in humans still

occurs in the U.S., usually from non-pork sources such as from feasting

on improperly prepared game meats (Kennedy et al., 2009). However,

the historical association of this parasite with swine is primarily

responsible for Federal control measures that must be strictly

followed during further processing of RTE pork products in the U.S.

and in Canada (Gamble and Bush, 1999). More specifically, the control

of T. spiralis in pork must be accomplished by mandatory carcass

inspection of pigs at slaughter or by post-slaughter processing

interventions, namely heating, freezing, and/or curing of products

containing pork (USDA-FSIS, 2001a). In fact, prescribed conditions for

the time and temperature of heating and/or freezing of pork are used

as a trichinae intervention for all parts of the muscle tissue of pork or

products containing pork tissue. Curing is also used as a trichinae

intervention for products containing pork, including Genoa salami.

However, the effectiveness of curing to eliminate T. spiralis larvae

depends on a combination of various processing parameters and on

the product formulation, specifically on the temperature and time of

fermentation/drying and the salt level, respectively. Therefore, curing

alone, as a post-slaughter intervention, is not recommended by the

International Commission on Trichinellosis (ICT) as a stand alone

method to inactivate T. spiralis larvae in products made with raw pork

(Gamble et al., 2000). Additional/alternative processing technologies

for trichinae control are considered on a case-by-case basis by USDA/

FSIS using criteria described in more detail elsewhere (USDA-FSIS,

2001a).

High pressure processing (HPP) has received considerable

attention as a post-processing intervention to eliminate pathogens

in RTE products, especially products that might be altered by other

interventions, notably by thermal treatment. Although numerous

studies have been conducted to determine the effect of HPP on

microbial pathogens directly in foods, including L. monocytogenes,

E. coli O157:H7, and Salmonella (Hayman et al., 2004; Jofré et al.,

2008; Marcos et al., 2005; Morales et al., 2006, Murano et al., 1999;

Simpson and Gilmour, 1997), few have been conducted on foodborne

parasites such as T. spiralis (Ohnishi et al., 1992; Ohnishi et al., 1994;

Gamble et al., 1998; Lindsay et al., 2005). In the present study, we

evaluated the effect of fermentation/drying on viability of T. spiralis

in Genoa salami. We also evaluated HPP for efficacy towards

T. spiralis larvae in trichinae-infected pig masseter muscle as an

alternate to curing for trichinae control. We also validated the

integrated lethality of a post-process HPP treatment in combination

with a traditional protocol for production of Genoa salami towards

L. monocytogenes, E. coli O157:H7, and Salmonella to confirm that

existing performance standards and/or compliance guidelines were

being met.

2. Material and methods

2.1. Microbial strains and preparation of inocula

The multi-strain cocktails (Table 1) of L. monocytogenes, E. coli

O157:H7, and Salmonella spp. were confirmed, cultured, combined,

and/or maintained as described previously (Porto et al., 2002; PortoFett et al., 2008). The nematode, T. spiralis (Table 1), was maintained

by successive passage in female Sprague–Dawley rats (Covance,

Denver, PA). Every 60 d the infected rats were killed using CO2 gas,

and viable larvae were collected by artificial digestion from the

muscle tissue and inoculated into rats by oral lavage as described

below (Gamble, 1996). The commercial starter cultures (Kerry BioScience, Rochester, MN) used in the production of the Genoa salami,

that being Bactoferm HPS (Pediococcus acidilactici), Saga 75

(P. pentosaceous), and Saga 444 (Kocuria varians), were maintained

and prepared according to the instructions provided by the

manufacturer.
A.C.S. Porto-Fett et al. / International Journal of Food Microbiology 140 (2010) 61–75 63

Table 1

Strains of L. monocytogenes, E. coli O157:H7, Salmonella, and T. spiralis used in this study.

Microbial strains Strain # Other designation Source Typea Ref.

L. monocytogenes

E. coli

S. typhimurium

S. typhimurium

S. copenhagen

S. typhimurium

S. typhimurium

T. spiralis

MFS2

MFS102

MFS104

MFS105

MFS110

FSIS OB070361

JBL2139

JBL2347

JBL1411

MFS-248

MFS-330

8457

FSIS OB060362

JBL3270

T1

H7776

ScottA

LM-101 M

F6854

C7927

SLH21788

EC505B

380-94

H3380

Environmental isolate from a pork processing plant

Frankfurter isolate, 1998 BilMar outbreak

Clinical isolate, 1983 Massachusetts pasteurized milk outbreak

Beef and pork sausage isolate

Turkey frankfurter isolate

Ground beef isolate

Clinical isolate, 1991 Massachusetts apple cider outbreak

Clinical isolate, 1994 Wisconsin daycare outbreak

Beef isolate

Salami outbreak

Hog carcass isolate

Hog carcass isolate

Pork isolate

Clinical isolate associated with pork sausage

Clinical isolate

Pig isolate

1/2a

4b

4b

4b

1/2a

O157:H7

O157:H7

O157:H7

O157:H7

O157:H7

DT104

Porto et al. (2002)

CDC (1998)

Linnan et al. (1988)

Glass and Doyle (1989)

Schwartz et al. (1988)

USDA-FSIS (2007)

Besser et al. (1993)

Proctor (1996)

Calicioglu et al. (2001)

Anonymous (1995)

Wonderling et al. (2003)

Wonderling et al. (2003)

Juneja et al. (2001)

USDA-FSIS Outbreaks and Special

Projects Laboratory, Athens, GA

Briggs and Fratamico (1999)

Gamble (1996)

a Serotypes for L. monocytogenes and E. coli strains; Phage types for Salmonella strains.

2.2. Recovery of T. spiralis by artificial digestion

As described by Gamble (1996), a 10-g sample of pork, pig

masseter tissue, or Genoa salami and/or an entire rat or mouse was

transferred to a blender cup (Model MB-101; Homeland Housewares,

Pacoima, CA) previously sanitized with a quaternary ammoniumbased germicide, multipurpose detergent (Misty® Biodet ND32;

Amrep, Marietta, GA) and containing 50 mL of artificial digestion

fluid (about pH 2.0), that being 1% pepsin (wt/vol; Fisher Scientific,

Rochester, NY) and 1% hydrochloric acid (vol/vol; Mallinckrodt

Chemicals, Phillipsburg, NJ). After blending for eight-second intervals

a total of three successive times, an additional 250 mL of digestion

fluid and a magnetic stir bar were added to each sample, and the

contents were vigorously mixed for up to 1 h in an incubator while

maintained at 47 °C. Next, samples were filtered through a stainlesssteel #45 size sieve (355 μm; U.S. Standard Sieve, Thomas Scientific,

Swedesboro, NJ) to remove undigested debris and then allowed to

settle for about 1 h at room temperature. The sediment was washed

with sterile distilled water and allowed to resettle until the

supernatant was clear. Samples were then fed to mice or examined

under a stereo microscope as detailed below.

2.3. Inoculation of pigs with Trichinella larvae and harvesting of infected

pork meat

For each of three trials, to establish infection, two, three-monthold, castrated male pigs (Yorkshire X Duroc crossbreed) were fed only

one gelatin capsule (#13; Torpac Inc., Fairfield, NJ) using a 2 3/8"

metal balling gun (Model #4000; Ideal Instruments/Neogen Inc.,

Lexington, KY) containing about 10,000 T. spiralis muscle larvae

suspended in tap water. Infective larvae used for the inoculum were

recovered from the above mentioned Sprague–Dawley rats by

artificial digestion. Inoculated pigs were housed in separate pens

and were quarantined from non-infected pigs in accordance with the

Animal Welfare Act, Guide for the Care and Use of Laboratory Animals

(http://www.nap.edu) and with the approval of the USDA/ARS

Beltsville Area Institutional Animal Care and Use Committee (BAACUC) under protocol #08-044. The pigs were fed about 2 lbs of a 16%

protein-based feed (Purina Growena; Land O'Lakes Purina Feed, LLC,

Glenview, IL) per day, with water available ad libitum. After about

35 d, pigs were humanely euthanized by chemical anaesthesia in

accordance with methods provided in the Guide for the Care and Use

of Laboratory Animals. The triceps, picnics, hams, neck, rump, and

loins were hand trimmed of excess fat and connective tissue with an

alcohol-sterilized sterile butchers' knife to yield a total of about 50 to

70 lbs of meat from each animal. The resulting large chunks/portions

of infected meat were cut into smaller pieces and transported on ice to

the USDA/ARS Eastern Regional Research Center (ERRC; Wyndmoor,

PA) where it was placed into sterile nylon-polyethylene bags (Prime

Source Packaging Ltd., Houston, TX), vacuum-sealed to 950 mbar

(Ultravac-500; Bunzl/Koch, Kansas City, MO), and then stored at 4 °C

for up to 7 d. Whole masseter muscle was separately harvested from

each pig and transported on ice to the ERRC where it was also

vacuum-sealed and then stored at 4 °C for up to 7 d. Larval burdens in

harvested pork and masseter samples were determined as described

below within 3 d following slaughter/harvest. Storage at 4 °C has little

effect, if any, on viability of trichinae larvae. More specifically, Kotula

et al. (1990) reported 100% survival of T. spiralis after storage for 135 d

at −3.9 °C and Hill et al. (2009) reported survival for 106 h at −6.6 °C

in pork meat within bags from each the air was expelled just prior to

heat sealing.

2.4. Inoculation of batter and processing of Genoa

As described in more detail below, salami was prepared with the

assistance of, and in collaboration with, a commercial processor using

their standard make protocol and with adherence to the guidelines in

9 CFR 318.10 (USDA-FSIS, 2001a) for “curing” of sausage as delineated

under method 6. In brief, approximately 200 lbs (about 90 kg) of raw

salami batter was prepared in each of six batches. In three of these six

batches, salami was prepared using trichinae-infected pork [35 lbs or

13.6 kg (17.6%)], but without the masseter tissue, obtained as

described above, along with boneless, skinless pork picnics

[103.6 lbs or 49.4 kg (52.3% of total weight)] and pork trim [59.4 lbs

or 27 kg (30% of total weight)] that were purchased fresh from a local

wholesaler. Regardless, for the three batches of salami that did not

contain T. spiralis larvae, pork picnics were substituted for the infected

pork. This blend of pork meat was mixed using a sanitized mixer/

grinder (Model 4346; Hobart Corporation, Troy, OH) to achieve a ratio

of about 80% lean meat to 20% fat. The blend was subsequently coarse

ground through a grinding plate with 3/8" holes (Model 4346;

Hobart), and then stored overnight at 4 °C, for convenience, in large

(100 lbs capacity), covered, high-density polyethylene containers

(Bunzl/Koch). The mixer/grinder was sanitized with a quaternary

ammonium-based germicide, multipurpose detergent (Misty® Biodet

ND32), rinsed thoroughly for 3 min with hot water (87.8 °C), and then

allowed to air dry at room temperature for up to 1 h. The coarse

ground pork was re-loaded into the sanitized mixer/grinder, and the

following dry ingredients and spices were added: high-grade sodium

chloride (2.9%; The Canadian Salt Company Ldt., Pointe Claire, QC,

Canada), dextrose (1%; Tate & Lyle of Nealanders International Inc.,

Mississauga, ON, Canada), white pepper (0.08%; Wiberg Canada
64 A.C.S. Porto-Fett et al. / International Journal of Food Microbiology 140 (2010) 61–75

Corporation, Oakville, ON, Canada), sodium ascorbate (0.05%; H & A

Industrial Inc., Toronto, ON, Canada), garlic powder (0.02%; Wiberg),

sodium nitrate (0.015%; Wiberg), and sodium nitrite (0.005%;

Wiberg). The blended batter was then mixed for about 1 min before

about 31 g of Bactoferm HPS, about 10.4 g of Saga 75, and about 8.5 g

of Saga 444 collectively diluted with a total of 15 mL of sterile water

(final concentration of about 6.0 log CFU/g of batter) and 900 mL of

either the L. monocytogenes, E. coli O157:H7, or Salmonella spp.

pathogen cocktails (final concentration of about 7.0 log CFU/g of

batter) were added to the batter. Mixing was continued for an

additional 2 min, and then the inoculated salami batter was fine

ground through a 3/16" grinding plate (Model 4346; Hobart) and

stuffed into either about 65 mm (about 2.5 in.) diameter× about

160 mm (about 6.3 in.) length or about 105 mm (about 4.0 in.)

diameter × about 200 mm (about 8.0 in.) length cellulose-based

casings (Naturin R2; Weinham, Germany) using either a stainless

steel table-top manual piston stuffer (24 lbs capacity; Model FD9051200; F. Dick, Esslingen, Germany) or a floor-type, hydraulicdriven piston stuffer (50 lbs capacity; Model SC-50, Koch Equipment,

Kansas City, MO). After stuffing, the salami chubs were hand tied with

twine, transferred to a temperature- and humidity-controlled walk-in

incubator (EJS Systems Inc., Changrin Falls, OH) with an air flow of 1.0

to 1.5 m/s, and hung vertically on racks so that the individual chubs

were not touching. The relative humidity (RH) and air temperature

were controlled and measured using the Dynamist 2000 System (EJS)

and Partlow MRC5000 chart recorder (EJS). The salami was held/

fermented at 20 °C and about 90 to 95% RH for 6 h, and then at 27 °C

and about 90 to 95% RH for 26 h. Next, the chubs were dried at 20 °C

and about 65 to 75% RH for 40 h, and then at 17 °C and about 65 to 75%

RH for/to: A) 25 d (65 mm casing), B) a target aw of 0.92 (65 mm

casing), C) 35 d (105 mm casing), or D) a target aw of 0.94 (105 mm

casing). Treatments A and C are the standard/traditional make

protocols practiced by our cooperating processor for small and large

casing sizes, respectively. The end point for drying used for treatments

A and C were determined as prescribed in 9 CFR 318.10 for salami

prepared with added salt of less than 2.9 lbs per hundredweight and a

casing diameter at stuff of either 65 mm or 105 mm. The drying times

for treatments B and D were selected based on a target aw value of

0.92 or 0.94, respectively, to yield the best quality product in terms of

taste and texture, in combination with high pressure processing

(HPP) for trichinae control. Following fermentation and drying, the

chubs were treated by HPP and subsequently stored at 4 °C as

described below.

2.5. Pressurization of Genoa salami and pig masseter muscle

i) HPP of Genoa: After fermentation and drying, but prior to

refrigerated storage, large (105 mm casing) and small (65 mm casing)

diameter chubs or portions therefrom were separately transferred to

nylon-polyethylene bags (Prime Source Packaging) and vacuumsealed to 950 mbar. Each chub was then transferred to a second

nylon-polyethylene bag and vacuum-sealed a second time to

950 mbar. Salami chubs were then shipped overnight on ice to either

Gridpath Inc. (Stoney Creek, ON, Canada), Virginia Polytechnic

Institute and State University (VPI; Blacksburg, VA), or the National

Center for Food Safety and Technology (NCFST; Summit Argo, IL) for

treatment by HPP. The VPI and NCFST are equipped with a 35-liter

capacity high pressure processing unit (Model Quintus Food Press

35L-600; Avure Technologies, Kent, WA). This unit can generate up to

600 MPa (87,000 psi) at temperatures between 40° and 95 °F. Gridpath is equipped with a commercial 55-liter capacity high pressure

processing unit (Model 6000/55; NC Hyperbaric, Burgos, Spain).

Salami chubs were pressurized at 600 MPa (87,000 psi) for 0, 1, 2, 3, 5,

or 7 min or at 483 MPa (70,000 psi) for 0, 5, 7, 10, or 12 min. For

salami treated with HPP, the average come-up-times (CUT) were

131.4 s (±16.3 s) for 87,000 psi and 114.7 s (± 19.0 s) for 70,000 psi.

The average initial temperatures of the water in the pressure vessel

were 66.9 °F (± 4.04 °F) for 87,000 psi and 66.7 °F (±3.84 °F) for

70,000 psi. For each pressure level, the above mentioned values for

CUT and initial temperature are the average of 96 measurements. Each

measurement was a separate run in the HPP unit, that being

pressurization of three chubs for a given pressure level and exposure

time for each pathogen in each of two trials. The pressure release

times were instantaneous. The temperature range attained after

pressurizing to 87,000 psi was 83° to 98 °F and was 74° to 91 °F after

pressurizing to 70,000 psi. The pressure-treated salami was placed on

ice and shipped via overnight courier to ERRC for microbiological

analyses. Relative to the levels of pressure applied, 600 MPa for 3 min

was tested because it has been approved as a technology for treating

meats and is being used by the industry to process RTE whole muscle

and sliced meats to enhance quality, extend shelf-life, and inactivate

pathogens, particularly L. monocytogenes (Avure). The lower pressure

level, 483 MPa, was selected to evaluate if it would be lethal for the

target pathogens, but less disruptive to the product. ii) HPP of masseter

muscle: In each of two trials, two 10-g portions of tissue were

aseptically harvested from random areas of masseter muscle (about

3.4 log larvae/g) that were previously removed from trichinaeinfected pigs as described above. The excised masseter tissue was

separately vacuum packaged and then shipped overnight on ice to VPI

for HPP or was pressure treated at ERRC (2-liter capacity; Model 2L,

Avure). The Model 2L unit can generate up to 690 MPa (100,000 psi)

at temperatures ranging from 50° to 194 °F. The samples of masseter

tissue were subjected to 600 MPa (87,000 psi) for 0.5, 1, 1.5, or 3 min,

or to 483 MPa (70,000 psi) for 0, 1, 2, 3, or 5 min. For masseter muscle

treated with HPP, the average CUT were 122.5 s (±1 s) for 87,000 psi

and 101.3 s (±3.8 s) for 70,000 psi. The average initial temperatures

of the water in the pressure vessel were 69.0 °F (±0 °F) for 87,000 psi

and 77.3 °F (±2.6 °F) for 70,000 psi. The above mentioned values for

CUT and initial temperature are the average of 96 measurements. Each

measurement was a separate run in the HPP unit, that being

pressurization of for a given pressure level and exposure time for a

single trial. The pressure release times were instantaneous. The

temperature range attained after pressurizing to 87,000 psi was 81° to

86 °F and was 85° to 88 °F after pressurizing to 70,000 psi. The Avure

2L does not record the water temperature inside the vessel during

come up and pressurization. However, because the pressure applied

was the same as for the units used at VPI and GridPath we assume that

the same increase in temperature was achieved on the Avure 2L unit.

In addition, for each exposure time we programmed the Avure 2L unit

to achieve the target pressurization level within a 5-min period. After

treatment, the samples were shipped via overnight courier on ice to

ERRC and/or processed within 24 h to screen for viable larvae as

described below.

2.6. Storage of Genoa salami

Following HPP treatment, chubs previously inoculated with L.

monocytogenes, E. coli O157:H7, or Salmonella spp. were removed

from the bags, placed on Styrofoam trays (1012S, Genpak, Glens Falls,

NY), and sliced/sectioned with the aid of an ethanol-sterilized knife.

Each slice/section (about 120 g for 65 mm casing or about 430 g for

105 mm casing) was separately placed into nylon-polyethylene bags

(Prime Source Packaging) and vacuum sealed. Samples were

incubated at 4 °C for 0, 7, 14, and 28 d. It was not possible to test

salami for viable larvae of T. spiralis during refrigerated storage, since

prior treatment by fermentation and drying were lethal for T. spiralis.

2.7. Recovery of L. monocytogenes, E. coli O157:H7, Salmonella spp., and

indigenous flora from Genoa salami

In each of two trials, for each diameter of salami, fermentation/

drying condition, HPP treatment, and storage time, a total of three
A.C.S. Porto-Fett et al. / International Journal of Food Microbiology 140 (2010) 61–75 65

salami samples were analyzed for the presence and/or levels of either

L. monocytogenes, E. coli O157:H7, or Salmonella spp. Single 25 g

portions from each sample were transferred into sterile filtered

stomacher bags (K & R Technologies, Frederick, MD) containing 25 ml

of 0.1% sterile peptone water and stomached for 2 min (Stomacher

400; Seward, Cincinnati, OH). The resulting slurry, with or without

prior dilution in 0.1% sterile peptone water, was surface-plated (100,

250, or 500 μL) onto Modified Oxford agar (MOX; Difco, Becton,

Dickinson and Company, Franklin Lakes, NJ) for enumeration of L.

monocytogenes, onto MacConkey sorbitol (SMAC; Difco) for enumeration of E. coli O157:H7, or onto Rappaport-Vassiliadis agar (RV;

Difco) for enumeration of Salmonella spp. Typical colonies of each

pathogen were counted after aerobic incubation of these plates at

37 °C for either 48 h (MOX) or 24 h (SMAC and RV). When Salmonella

spp., E. coli O157:H7, or L. monocytogenes numbers decreased to below

detection (≤0.3, ≤0.3, and ≤1.0 log CFU/g, respectively) by direct

plating, the presence or absence of each pathogen was determined by

selective enrichment essentially as described (Hinkens et al., 1996;

Ihnot et al., 1998; Cook, 1999). Likewise, at selected intervals after

stuffing and after drying, total aerobic plate counts (TPC) were

enumerated by spread plating the resulting slurry, with or without

prior dilution in 0.1% sterile peptone water, onto BHI agar plates and

incubating for 72 h at 30 °C. For enumeration of total lactic acid

bacteria (LAB), the slurry was spread-plated, with or without prior

dilution in sterile 0.1% peptone water, onto Mann, Rogosa and Sharp

(MRS; Difco) agar plates and incubated anaerobically (10.1% carbon

dioxide, 4.38% hydrogen and balance nitrogen; Bactron IV Anaerobic/

Environmental Chamber, Sheldon Manufacturing Inc., Cornelius, OR)

for 48 h at 37 °C.

2.8. Recovery of T. spiralis from Genoa salami and pig masseter

For Genoa salami, in each of three trials, for each diameter of

salami, fermentation/drying treatment, and HPP treatment, a total of

two 10-g salami samples were analyzed for the presence of viable T.

spiralis larvae via microscopy and/or bioassay. Likewise, for pig

masseter muscle, in each of two trials, a total of two, 10-g masseter

tissue samples were analyzed to determine the effect of HPP on the

viability of T. spiralis larvae. After digestion of each test sample as

described above, a 0.1- to 1-ml portion was examined via microscopy

(Model MZFLIII; Leica Microsystems, Wetzlar, Germany) at 30× to

50× magnification to enumerate and determine the presence of

viable/non-viable larvae by visual inspection. Alternatively, digested

samples were orally gavaged into mice for determining infectivity

(see below). i) Infectivity assay in mice. All samples were tested for the

presence of infective larvae by orally gavaging 100 μL of the digested

samples into female Swiss-Webster mice (Taconic, Albany, NY) using

a 1-mL syringe (Becton-Dickinson) and a 20-gauge, 1.5" length oral

gavage needle (Cadence Science, Lake Success, NY) essentially as

described by Hill et al. (2007). Approximately 30 d post-infection,

mice were euthanized by CO2 asphyxiation and then skinned and

eviscerated in accordance with the Guide for Animal Care and Use of

Laboratory Animals. Eviscerated and skinned mice were transferred to

sterile nylon-polyethylene bags and placed at 4 °C for up to 3 d. Each

mouse was separately digested in total as described above to

subsequently examine microscopically for the presence of larvae in

the muscle tissue.

2.9. Physical–chemical analyses of Genoa salami

For each trial and for each treatment, the aw of one chub/portion

per treatment was measured at each sampling interval by placing a

nominal amount (about 3 g) of salami into a sampling cup and

analyzing it using an electronic water activity meter (Aqualab Model

Series 3; Decagon Devices, Pullman, WA) according to the manufacturer's instructions. For pH measurements, a 25-g portion of a chub

was fi 

peptone water and then macerated for 2 min. For each trial and for

each pathogen, the pH of one chub/portion per treatment was

measured after fermentation using a model 6000P pH/temperature

electrode and a model 5500 pH meter (Daigger, Vernon Hills, IL). The

proximate composition of Genoa salami was determined in one of the

six trials using a 100-g composite sample comprised of the three

salami portions taken directly after fermentation/drying (N= 1 trial,

n= 3 salami portions per trial). Proximate composition analyses were

performed according to methods approved and described by the

Association of Official Analytical Chemists (McNeal, 1990) as

conducted by a commercial testing laboratory.

2.10. Statistical analyses

Data were analyzed using version 9.1.3 of the SAS statistical

package (SAS Institute Inc., Cary, NC). When the three pathogens

tested were inoculated into Genoa salami batter, analysis of variance

(ANOVA) was performed to evaluate the effects of fermentation,

drying, and HPP on pathogen reduction over time. When microbial

numbers decreased to below detection (≤0.3,≤0.3, and ≤1.0 log CFU/g,

respectively), a value of zero was used for determination of the

arithmetic mean. The individual means and standard deviations of

pathogen viability in Genoa salami for each treatment were determined

from the average of three samples at each sampling interval for each of

the two trials. Differences in the proximate composition among

treatments A, B, C, and D were also evaluated using ANOVA. Mean

separations were performed using the Bonferroni LSD method.

3. Results

3.1. Levels of indigenous flora

Direct plating and/or enrichment of the six batches of skinless pork

picnics and pork trim before inoculation revealed the absence of any

indigenous L. monocytogenes, E. coli O157:H7, or Salmonella spp. by

both direct plating (≤1.0 log CFU/g) and by enrichment (data not

shown). The average initial levels of TPC and LAB in batter after

stuffing were 7.75±0.11 log CFU/g and 7.56±0.12 log CFU/g,

respectively. The average levels of TPC in Genoa salami after drying

for treatments A, B, C, and D were 9.07±0.23, 8.32± 0.70, 8.85± 0.08,

and 8.49± 0.70 log CFU/g, respectively, whereas the average levels of

LAB in Genoa salami after drying for treatments A, B, C, and D were

8.72± 0.62, 8.67± 0.36, 8.56± 0.77, and 9.11± 0.14 log CFU/g, respectively. Essentially there were no appreciable differences in the

initial and in the final levels of TPC and LAB among treatments A, B, C,

and D. Also, there were no appreciable differences in the levels of TPC

and LAB for a given treatment.

3.2. Proximate composition of Genoa salami

Results from proximate composition analyses (Table 2) did not

show significant (PN0.05) differences in the levels of carbohydrates,

fat, nitrite, or acidity among treatments A, B, C, and D. Regarding the

levels of ash, moisture, protein, M:Pr ratio, salt, aw, and pH, proximate

composition analyses revealed only subtle differences (Pb0.05) for a

given chemical parameter among treatments A, B, C, and D. Our data

confirmed that, with the exception of treatment D, treatments A, B,

and C were in compliance with the USDA/FSIS guidelines for the M:

Pr≤2.3:1.0 to be designated as Genoa salami. However, all treatments

achieved the combined pH (pHb5.2) and aw (aw 0.95) parameters/

values to be designated by USDA/FSIS as shelf-stable (http://www.

fsis.usda.gov/PDF/FSRE_SS_7Principles.pdf). The average initial pH of

the salami batter before stuffing was pH 6.04± 0.02 (Table 3). No

statistical differences (PN0.05) in pH values were observed among

treatments after fermentation and drying. After fermentation and

transferred to a ltered stomacher bag containing 25 ml of 0.1%
66 A.C.S. Porto-Fett et al. / International Journal of Food Microbiology 140 (2010) 61–75

Table 2

Proximate composition of raw batter and Genoa salami.

Analyses Batter1 Genoa

A

salami treatments2

B C D

Ash (g/100 g)

Carbohydrates (g/100

Fat (g/100 g)

Moisture (g/100 g)

Protein (g/100 g)

M:Pr

Salt (g/100 g)

Nitrite

Acidity (%)

Water activity

pH

g)

3.74

1.05

19.48

59.30

16.43

3.61:1

3.01

1.33

0.66

0.961

5.80

±

±

±

±

±

±

±

±

±

±

±

0.07d

0.45a

2.73a

2.20a

0.72d

0.04a

0.06d

0.52a

0.01b

0.007a

0.08a

6.07

0.10

27.90

37.81

28.43

1.33:1

4.88

1.0

2.31

0.884

4.56

±

±

±

±

±

±

±

±

±

±

±

0.08a

0.0a

0.52a

0.50c

0.30a

0.03d

0.04a

0.00a

0.04a

0.009c

0.14b

4.85

0.71

26.73

44.58

23.64

1.57:1

3.94

1.0

2.13

0.926

4.65

±

±

±

±

±

±

±

±

±

±

±

0.26b,c

0.67a

4.25a

6.35b,c

1.81b

0.20d

0.13c

0.00a

0.30a

0.011c

0.21b

5.22

0.32

24.14

46.89

23.51

1.99:1

4.23

1.0

2.01

0.920

4.64

±

±

±

±

±

±

±

±

±

±

±

0.03b

0.31a

1.69a

1.26b,c

0.01b

0.04c

0.01b

0.00a

0.10a

0.008c

0.09b

4.68

0.93

21.56

53.03

19.82

2.68:1

3.75

1.5

1.84

0.940

4.66

±

±

±

±

±

±

±

±

±

±

±

0.01c

0.08a

0.98a

1.22a,b

0.30c

0.11b

0.00c

0.70a

0.03a

0.005a,b

0.09b

1 Proximate composition analyses conducted in Genoa salami batter were determined in each of the six trials performed using

batter portions taken directly before fermentation/drying (N=6 trial, n=2 batter portions per trial). 2 Proximate composition of Genoa salami was determined in one of the six trials using a 100-g composite sample comprised

fermentation/drying (N=1 trial, n= 3 salami portions per trial).

a 100-g composite sample comprised of the two

of the three salami portions taken directly after

drying, the pH of the salami decreased to pH 4.56± 0.14, 4.65± 0.21,

4.64± 0.04, and 4.66± 0.09 for treatments A, B, C, and D, respectively.

The average initial aw of the salami batter before stuffing was aw

0.961± 0.007 (Table 4); however, after fermentation and drying, the

average aw for treatments A, B, and C were significantly (Pb0.05) lower

than for treatment D. The average aw ofthe resulting salami generated by

treatments A, B, C, and D was aw 0.884±0.009, 0.926±0.011, 0.920±

0.008, and 0.940±0.005, respectively. For treatments C and D, the target

aw values of 0.92 and 0.94 were achieved after 14±3 d and 22±2 d of

drying, respectively. After HPP and subsequent storage at 4 °C for 28 d,

the aw decreased to about aw 0.875, 0.919, 0.913, and 0.930 for

treatments A, B, C, and D, respectively.

3.3. Viability of L. monocytogenes, E. coli O157:H7, and Salmonella spp.

in Genoa salami after fermentation and drying

For Genoa salami stuffed into either a 65-mm or a 105-mm diameter

casing there wereno significant differences (PN0.05) in the comparative

viability of L. monocytogenes, E. coli O157:H7, or Salmonella after

fermentation (Tables 5, 6, and 7, respectively). After drying, for a

given pathogen there were no significant (Pb0.05) differences in viability between treatments A and B and between treatments C and D. For

comparisons among the three pathogens, after drying, there were

significant (Pb0.05) differences in viability of L. monocytogenes and

E. coli O157:H7 compared to viability of Salmonella spp. for a given

treatment. Results showed that Salmonella spp. decreased to a greater

extent after drying for all treatments in Genoa salami than either

L. monocytogenes or E. coli O157:H7 for both casing sizes tested. Total

reductions after fermentation and drying of Genoa salami ranged from

about 1.1 to 1.3, about 1.1 to 2.2, and about 4.2 to 4.8 log CFU/g for

L. monocytogenes, E. coli O157:H7, and Salmonella spp., respectively.

3.4. Viability of L. monocytogenes, E. coli O157:H7, and Salmonella spp.

in Genoa salami after HPP and storage

Storage of Genoa salami without prior HPP treatment had

comparatively little effect on L. monocytogenes (Table 5) and E. coli

O157:H7 (Table 6), whereas levels of Salmonella spp. (Table 7) decreased by as much as about 1.5 log CFU/g at 4 °C within 28 d. In

contrast, HPP had a significant effect (Pb0.05) on the extent of inactivation of L. monocytogenes, E. coli O157:H7, and Salmonella spp. in

Genoa salami compared to salami that were not treated with high

pressure (Tables 5, 6, and 7, respectively). Regardless of exposure time

for HPP, when salami was pressurized at 600 MPa there were significant

(Pb0.05) differences in viability of L. monocytogenes between treatments A and B or between treatments C and D. However, regardless of

the duration of the application of high pressure, when salami was

pressurized at 483 MPa after fermentation/drying, no statistical

(PN0.05) differences were observed in viability of L. monocytogenes

between treatments A and B or between treatments C and D. Regardless

ofthe amount of pressure or the time of exposure,there were significant

(Pb0.05) differences in viability of L. monocytogenes between treatments A and B or between treatments C and D after 28 d of refrigerated

storage. Likewise, regardless of pressure level, or time of exposure, or

length of storage, there were no statistical differences (PN0.05) in the

extent of inactivation of Salmonella spp. or E. coli O157:H7 in Genoa

salami between treatments A and B or between treatments C and D. For

control samples of salami that were not treated via HPP, regardless of

the conditions for drying, there were significant (Pb0.05) differences in

viability of L. monocytogenes and E. coli O157:H7 compared to viability of

Salmonella spp. when Genoa salami were subsequently stored 4 °C for

up 28 d; more cells of L. monocytogenes or E. coli O157:H7 survived

fermentation and drying than did cells of Salmonella spp.

Following pressurization at 600 MPa for 1 to 5 min or at 483 MPa for

5 to 12 min, numbers of L. monocytogenes were reduced by an additional

1.6 to ≥5.0 log CFU/g compared to their levels after fermentation and

drying. However, regardless of the pressure or time of exposure, during

28 d of storage at 4 °C, L. monocytogenes levels decreased by up to an

additional 3.0 log CFU/g in treatments A, B, and D, whereas levels of the

pathogen increased by 0.4 to 2.2 log CFU/g in treatment C. When salami

was not treated via HPP, numbers of L. monocytogenes were reduced by

up to an additional 0.4 log CFU/g during storage at 4 °C for up to 28 d.

With regard to E. coli O157:H7 and Salmonella spp., regardless of the

amount of pressure or the time of exposure, it was only possible to

recover these pathogens via enrichment. Nonetheless, following

pressurization and storage, E. coli O157:H7 numbers were reduced by

an additional ≥4.7 to ≥5.8 log CFU/g, whereas numbers of Salmonella

spp. were reduced by an additional ≥1.9 to ≥2.4 log CFU/g compared to

levels present after fermentation and drying. When salami was not

treated with pressure, numbers of E. coli O157:H7 and Salmonella spp.

were reduced by up to an additional about 1.1 and 1.7 log CFU/g,

respectively. Our results established that it was possible to achieve a

total reduction of about 3.6 to 6.1 log CFU/g of L. monocytogenes, about

≥6.2 log CFU/g of E. coli O157:H7, and about ≥6.6 log CFU/g of

Salmonella spp. by fermentation and drying in combination with HPP.

Collectively, these results established the effectiveness of HPP as a postprocess treatment to inactivate L. monocytogenes, E. coli O157:H7, and

Salmonella spp. in Genoa salami.

3.5. Viability of T. spiralis in Genoa salami after fermentation, drying,

and HPP

Regardless of the processing conditions, fermentation and drying

alone were sufficient for reducing the initial load (about 2.3 log
Table 3Average of pH values following processing, pressurization, and storage of Genoa salami.

Processing step: Casing diameter Casing di

65 mm 105 mm

Battera 5.80±0.08 5.80±0.0

Fermentationa 4.58±0.12 4.58±0.1

Drying to targetb

Time or aw

A) 25 days

4.56±0.14

B) aw 0.92

4.65±0.21

C) 35 days

4.64±0.04

HPP at 600 MPa for

Storage at 4 °C for:

(min)c:

7 days

14 days

28 days

0

4.63

(0.10)

4.65

(0.22)

4.71

(0.23)

4.73

(0.25)

1

4.59

(0.18)

4.79

(0.16)

4.92

(0.06)

4.95

(0.04)

2

4.71

(0.01)

4.83

(0.03)

4.89

(0.01)

4.96

(0.04)

3

4.72

(0.03)

4.79

(0.12)

4.78

(0.19)

4.69

(0.19)

5

4.73

(0.03)

4.72

(0.26)

4.78

(0.23)

4.93

(0.06)

0

4.76

(0.14)

4.62

(0.18)

4.64

(0.15)

4.67

(0.18)

1

4.87

(0.04)

4.71

(0.11)

4.82

(0.05)

4.84

(0.02)

2

4.77

(0.11)

4.79

(0.08)

4.77

(0.04)

4.84

(0.01)

3

4.87

(0.01)

4.66

(0.18)

4.70

(0.13)

4.68

(0.17)

5

4.86

(0.03)

4.82

(0.08)

4.60

(0.21)

4.85

(0.03)

0

4.64

(0.04)

4.63

(0.21)

4.65

(0.18)

4.69

(0.23)

1

4.57

(0.14)

4.70

(0.15)

4.76

(0.11)

4.78

(0.11)

2

4.63

(0.06)

4.80

(0.13)

4.79

(0.01)

4.86

(0.16)

3

4.67

(0.04)

4.67

(0.22)

4.70

(0.16)

4.68

(0.19)

5

4.69

(0.06)

4.82

(0.13)

4.61

(0.16)

4.85

(0.13)

HPP at 483 MPa for

Storage at 4 °C for:

(min)c:

7 days

14 days

28 days

0

4.63

(0.10)

4.65

(0.22)

4.71

(0.23)

4.73

(0.25)

5

4.65

(0.23)

4.77

(0.17)

4.74

(0.19)

4.85

(0.06)

7

4.89

(0.01)

4.90

(0.05)

4.78

(0.31)

4.92

(0.02)

10

4.86

(0.06)

4.81

(0.03)

4.67

(0.21)

4.71

(0.17)

12

4.79

(0.11)

4.69

(0.18)

4.89

(0.03)

4.84

(0.16)

0

4.76

(0.14)

4.62

(0.18)

4.64

(0.15)

4.67

(0.18)

5

4.70

(0.01)

4.72

(0.10)

4.75

(0.01)

4.84

(0.00)

7

4.71

(0.01)

4.85

(0.16)

4.76

(0.0)

4.83

(0.01)

10

4.73

(0.02)

4.77

(0.04)

4.70

(0.12)

4.71

(0.24)

12

4.68

(0.11)

4.66

(0.16)

4.79

(0.03)

4.81

(0.12)

0

4.64

(0.04)

4.63

(0.21)

4.65

(0.18)

4.69

(0.23)

5

4.65

(0.13)

4.71

(0.14)

4.68

(0.11)

4.75

(0.08)

7

4.72

(0.14)

4.77

(0.09)

4.75

(0.01)

4.84

(0.23)

10

4.71

(0.15)

4.80

(0.14)

4.62

(0.14)

4.68

(0.20)

12

4.71

(0.07)

4.69

(0.17)

4.75

(0.11)

4.75

(0.14)

abc

Mean

Mean

Mean

of

of

of

six trials (±standard deviation) (N=6 trials, n=3 samples per trial).

six trials (±standard deviation) (N=6 trials, n=1 sample per trial).

two to four trials (±standard deviation) (N=2 to 4 trials, n=1 sample per trial).
Table 4Average of aw values following processing, pressurization, and storage of Genoa salami.

Processing step: Casing diameter Casing dia

65 mm 105 mm

Battera 0.961 (0.007) 0.961 (0.0

Fermentationb 0.959 (0.003) 0.959 (0.0Drying to targetb A) 25 days B) aw 0.92 C) 35 days 

Time or aw 0.884 (0.009)

HPP at 600 MPa for (min)b: 0 1 2 3 5 0 1

0.926 (0.011)

2 3 5 0 1

0.920 (0.008)

2 3 5 0 

0.884 0.885 0.883 0.887 0.881 0.926 0.927 0.926 0.926 0.929 0.920 0.917 0.921 0.923 0.921 0

(0.009) (0.007) (0.010) (0.008) (0.009) (0.011) (0.010) (0.011) (0.008) (0.009) (0.008) (0.009) (0.009) (0.006) (0.006) (

Storage at 4 °C for: 7 days 0.879 0.879 0.883 0.887 0.887 0.924 0.926 0.926 0.927 0.924 0.913 0.916 0.916 0.914 0.915 0

(0.010) (0.011) (0.009) (0.009) (0.010) (0.011) (0.008) (0.008) (0.009) (0.010) (0.009) (0.006) (0.008) (0.008) (0.008) (

14 days 0.876 0.876 0.874 0.878 0.879 0.922 0.922 0.922 0.923 0.923 0.915 0.911 0.912 0.916 0.912 0

(0.014) (0.008) (0.016) (0.011) (0.010) (0.009) (0.013) (0.013) (0.011) (0.010) (0.010) (0.010) (0.014) (0.011) (0.010) (

28 days 0.877 0.870 0.872 0.876 0.872 0.922 0.919 0.919 0.918 0.917 0.913 0.911 0.915 0.916 0.906 0

(0.010) (0.019) (0.014) (0.010) (0.013)

HPP at 483 MPa for (min)b: 0 5 7 10 12

(0.009)

0

(0.010)

5

(0.010)

7

(0.010)

10

(0.013)

12

(0.006)

0

(0.007)

5

(0.005)

7

(0.005)

10

(0.006)

12

(

0

0.884 0.885) 0.886 0.886 0.885 0.926 0.927 0.926 0.928 0.927 0.920 0.921 0.924 0.923 0.921 0

(0.009) (0.011) (0.011) (0.011) (0.011) (0.011) (0.011) (0.011) (0.011) (0.011) (0.008) (0.003) (0.007) (0.006) (0.005) (

Storage at 4 °C for: 7 days 0.879 0.876 0.880 0.879 0.881 0.924 0.926 0.926 0.923 0.928 0.913 0.912 0.917 0.911 0.914 0

(0.010) (0.011) (0.007) (0.015) (0.008) (0.011) (0.011) (0.007) (0.015) (0.008) (0.009) (0.010) (0.006) (0.008) (0.008) (

14 days 0.876 0.873 0.875 0.878 0.879 0.922 0.925 0.921 0.925 0.923 0.915 0.912 0.912 0.912 0.913 0

(0.014) (0.009) (0.009) (0.008) (0.012) (0.009) (0.009) (0.009) (0.008) (0.012) (0.010) (0.011) (0.012) (0.008) (0.007) (

28 days 0.877 0.880 0.877 0.877 0.875 0.922 0.920 0.921 0.919 0.921 0.913 0.912 0.907 0.912 0.912 0

(0.010) (0.008) (0.008) (0.008) (0.012) (0.009) (0.008) (0.008) (0.008) (0.012) (0.006) (0.005) (0.007) (0.007) (0.004) (

a Mean of two trials (±standard deviation) (N=2 trials, n=2 samples per trial).b Mean of six trials (±standard deviation) (N=6 trials, n=3 samples per trial).
Table 5Recovery of L. monocytogenes (log CFU/g) following processing, pressurization, and storage of Genoa salami.

Processing step: Casing diameter Casing di

65 mm 105 mm

Batter 7.14a (0.17) 7.14 (0.1

Fermentation 6.44 (0.41) 6.41 (0.3

Drying to target time or aw A) 25 days B) aw 0.92 C) 35 days 

5.84 (0.22) 6.08 (0.12) 6.02 (0.09) 

HPP at 600 MPa for (min): 0 1 2 3 5 0 1 2 3 5 0 1 2 3 5 

5.84 4.22 4.14 3.29 3.46 6.08 3.81 3.23 2.82 2.14 6.02 1.80 1.90 ≤1.0 ≤1.0 

(0.22) (0.43) (0.40) (1.65) (0.57) (0.12) (0.54) (0.61) (0.92) (1.08) (0.09) (1.56) (1.25) (0.0) (0.0) 

[3/6]b [3/6] 

Storage at 4 °C for: 7 days 5.59 3.72 3.23 3.18 2.60 6.14 2.36 2.79 2.36 2.36 5.73 2.50 1.77 1.60 1.46 

(0.24) (0.19) (0.40) (0.42) (0.59) (0.18) (1.20) (0.94) (0.88) (0.88) (0.24) (0.79) (0.81) (1.10) (0.79) 

14 days 5.75 3.57 3.20 2.23 2.40 6.09 2.68 1.28 1.34 1.56 5.92 2.56 1.62 1.49 ≤1.0 

(0.28) (0.46) (0.71) (0.65) (1.03) (0.26) (0.26) (1.03) (0.68) (0.20) (0.14) (0.63) (1.14) (1.26) (0.45) 

[2/4] [1/1] [1/1] [1/1] [4/4] 

28 days 5.47 3.09 3.08 3.00 2.59 5.81 1.71 ≤1.0 ≤1.0 ≤1.0 5.59 3.02 2.34 2.40 2.26 

(0.27) (0.71) (0.54) (0.61) (0.60) (0.26) (0.58) (0.0) (0.0) (0.0) (0.43) (0.32) (0.93) (1.09) (1.36) 

[5/5] [4/4] [5/5] [1/1] 

HPP at 483 MPa for (min): 0 5 7 10 12 0 5 7 10 12 0 5 7 10 12 

5.84 4.02 3.15 4.29 3.64 6.08 3.96 3.07 3.35 3.09 6.02 2.34 2.01 ≤1.0 1.15 

(0.22) (0.22) (1.48) (0.20) (0.73) (0.12) (0.52) (0.47) (0.16) (0.41) (0.09) (1.67) (1.53) (0.0) (1.18) 

[1/1] [1/1] [3/6] [1/4] 

Storage at 4 °C for: 7 days 5.59 3.48 3.48 3.31 3.30 6.14 3.30 2.75 2.77 2.16 5.73 2.59 2.28 2.21 1.71 

(0.24) (0.58) (0.36) (0.33) (0.58) (0.18) (0.39) (0.71) (0.41) (0.39) (0.24) (0.36) (1.0) (0.74) (1.02) 

[1/1] 

14 days 5.75 3.63 3.41 2.98 3.33 6.09 2.84 2.60 1.42 1.50 5.92 2.06 1.73 1.33 1.37 

(0.28) (0.53) (0.70) (1.13) (0.74) (0.26) (0.94) (0.13) (0.82) (0.79) (0.14) (1.20) (0.82) (0.85) (1.06) 

[1/1] [1/1] [1/1] [2/2] 

28 days 5.47 3.10 3.10 3.52 3.08 5.81 1.02 ≤1.0 ≤1.0 ≤1.0 5.59 2.56 2.55 2.83 2.52 

(0.27) (0.46) (0.25) (0.33) (0.60) (0.26) (0.68) (0.0) (0.0) (0.0) (0.43) (0.40) (0.86) (0.55) (0.54) 

[2/2] [2/2] [3/6] 

a Log CFU/g average of 6 samples (±standard deviation) (N=2 trials, n=3 replicates; detection limit by direct plating=≤1.0 log CFU/g).b Values in brackets are numbers of samples negative by enrichment from among the total number of samples that were enriched.
Table 6Recovery of E. coli O157:H7 (log CFU/g) following processing, pressurization, and storage of Genoa salami.

Processing step: Casing diameter Casing di

65 mm 105 mm

Batter 7.20a±0.15 7.20±0.

Fermentation 6.46±0.14 6.60±0.

Drying to target A) 25 days B) aw 0.92 C) 35 days 

time or aw 5.03±0.35 6.03±0.27 5.59±0.81 

HPP at 600 MPa for (min): 0 1 2 3 5 0 1 2 3 5 0 1 2 3 5 

5.03 ≤0.30 ≤0.30 ≤0.30 ≤0.30 6.03 ≤0.30 ≤0.30 ≤0.30 ≤0.30 5.59 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.35) (0.0) (0.0) (0.0) (0.0) (0.27) (0.0) (0.0) (0.0) (0.0) (0.81) (0.0) (0.0) (0.0) (0.0) 

[1/4]b [2/5] [5/6] [6/6] [3/4] [6/6] [6/6] [6/6] [5/6] [5/6] [6/6] [6/6] 

Storage at 4 °C for: 7 days 4.77 ≤0.30 ≤0.30 ≤0.30 ≤0.30 6.17 ≤0.30 ≤0.30 ≤0.30 ≤0.30 5.24 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.08) (0.0) (0.0) (0.0) (0.0) (0.21) (0.0) (0.0) (0.0) (0.0) (0.02) (0.0) (0.0) (0.0) (0.0) 

[5/5] [6/6] [6/6] [6/6] [4/4] [6/6] [6/6] [6/6] [5/6] [6/6] [5/6] [6/6] 

14 days 4.49 ≤0.30 ≤0.30 ≤0.30 ≤0.30 5.98 ≤0.30 ≤0.30 ≤0.30 ≤0.30 4.82 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.29) (0.0) (0.0) (0.0) (0.0) (0.34) (0.0) (0.0) (0.0) (0.0) (0.34) (0.0) (0.0) (0.0) (0.0) 

[3/4] [3/4] [3/5] [5/6] [5/6] [6/6] [6/6] [6/6] [4/6] [5/6] [5/6] [6/6] 

28 days 4.20 ≤0.30 ≤0.30 ≤0.30 ≤0.30 5.92 ≤0.30 ≤0.30 ≤0.30 ≤0.30 4.47 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.38) (0.0) (0.0) (0.0) (0.0) (0.16) (0.0) (0.0) (0.0) (0.0) (0.72) (0.0) (0.0) (0.0) (0.0) 

[1/4] [0/5] [2/6] [3/6] [5/5] [6/6] [6/6] [4/6] [6/6] [6/6] [6/6] [6/6] 

HPP at 483 MPa for (min): 0 5 7 10 12 0 5 7 10 12 0 5 7 10 12 

5.03 ≤0.30 ≤0.30 ≤0.30 ≤0.30 6.03 ≤0.30 ≤0.30 ≤0.30 ≤0.30 5.59 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.35) (0.0) (0.0) (0.0) (0.0) (0.27) (0.0) (0.0) (0.0) (0.0) (0.81) (0.0) (0.0) (0.0) (0.0) 

[0/2] [1/3] [5/6] [5/6] [6/6] [6/6] [6/6] [6/6] [6/6] [5/5] [6/6] [6/6] 

Storage at 4 °C for: 7 days 4.77 ≤0.30 ≤0.30 ≤0.30 ≤0.30 6.17 ≤0.30 ≤0.30 ≤0.30 ≤0.30 5.24 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.08) (0.0) (0.0) (0.0) (0.0) (0.21) (0.0) (0.0) (0.0) (0.0) (0.02) (0.0) (0.0) (0.0) (0.0) 

[6/6] [6/6] [6/6] [6/6] [5/5] [5/5] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] 

14 days 4.49 ≤0.30 ≤0.30 ≤0.30 ≤0.30 5.98 ≤0.30 ≤0.30 ≤0.30 ≤0.30 4.82 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.29) (0.0) (0.0) (0.0) (0.0) (0.34) (0.0) (0.0) (0.0) (0.0) (0.34) (0.0) (0.0) (0.0) (0.0) 

[3/5] [3/3] [4/6] [4/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] 

28 days 4.20 ≤0.30 ≤0.30 ≤0.30 ≤0.30 5.92 ≤0.30 ≤0.30 ≤0.30 ≤0.30 4.47 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.38) (0.0) (0.0) (0.0) (0.0) (0.16) (0.0) (0.0) (0.0) (0.0) (0.72) (0.0) (0.0) (0.0) (0.0) 

[3/3] [3/5] [4/6] [5/6] [5/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] 

a Log CFU/g average of 6 samples (±standard deviation) (N=2 trials, n=3 replicates; detection limit by direct plating=≤0.3 log CFU/g).b Values in brackets are numbers of samples negative by enrichment from among the total number of samples that were enriched.
Table 7Recovery of Salmonella (log CFU/g) following processing, pressurization, and storage.

Processing step: Casing diameter Casing d

65 mm 105 mm

Batter 6.95a (0.13) 6.95 (0.1

Fermentation 5.61 (0.13) 5.91(0.13

Drying to target A) 25 days B) aw 0.92 C) 35 days 

time or aw 2.50 (0.23) 2.21 (0.52) 2.18 (0.65) 

HPP at 600 MPa for (min): 0 1 2 3 5 0 1 2 3 5 0 1 2 3 5 

2.50 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.21 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.18 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.23) (0.0) (0.0) (0.0) (0.0) (0.52) (0.0) (0.0) (0.0) (0.0) (0.65) (0.0) (0.0) (0.0) (0.0) 

[5/5]b [4/5] [4/6] [6/6] [5/6] [5/6] [0/4] [4/5] [5/5] [6/6] [6/6] [6/6] 

Storage at 4 °C for: 7 days 1.70 ≤0.30 ≤0.30 ≤0.30 ≤0.30 1.66 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.76 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.58) (0.0) (0.0) (0.0) (0.0) (0.90) (0.0) (0.0) (0.0) (0.0) (0.40) (0.0) (0.0) (0.0) (0.0) 

[4/5] [5/6] [5/6] [5/6] [6/6] [5/5] [6/6] [6/6] [5/6] [6/6] [6/6] [6/6] 

14 days 1.88 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.64 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.09 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.31) (0.0) (0.0) (0.0) (0.0) (0.75) (0.0) (0.0) (0.0) (0.0) (1.1) (0.0) (0.0) (0.0) (0.0) 

[6/6] [6/6] [5/6] [6/6] [6/6] [5/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] 

28 days 1.09 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.45 ≤0.30 ≤0.30 ≤0.30 ≤0.30 1.07 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.53) (0.0) (0.0) (0.0) (0.0) (0.50) (0.0) (0.0) (0.0) (0.0) (0.31) (0.0) (0.0) (0.0) (0.0) 

[6/6] [6/6] [6/6] [6/6] [5/6] [6/6] [6/6] [6/6] [6/6] [6/6] [5/6] [6/6] 

HPP at 483 MPa for (min): 0 5 7 10 12 0 5 7 10 12 0 5 7 10 12 

2.50 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.21 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.18 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.23) (0.0) (0.0) (0.0) (0.0) (0.52) (0.0) (0.0) (0.0) (0.0) (0.65) (0.0) (0.0) (0.0) (0.0) 

[4/6] [5/5] [4/6] [5/6] [4/4] [3/4] [4/4] [5/5] [6/6] [6/6] [6/6] [6/6] 

Storage at 4 °C for: 7 days 1.70 ≤0.30 ≤0.30 ≤0.30 ≤0.30 1.66 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.76 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.58) (0.0) (0.0) (0.0) (0.0) (0.90) (0.0) (0.0) (0.0) (0.0) (0.40) (0.0) (0.0) (0.0) (0.0) 

[5/6] [6/6] [6/6] [6/6] [6/6] [5/5] [5/5] [6/6] [6/6] [4/5] [6/6] [6/6] 

14 days 1.88 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.64 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.09 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.31) (0.0) (0.0) (0.0) (0.0) (0.75) (0.0) (0.0) (0.0) (0.0) (1.1) (0.0) (0.0) (0.0) (0.0) 

[5/6] [5/6] [6/6] [6/6] [6/6] [5/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] 

28 days 1.09 ≤0.30 ≤0.30 ≤0.30 ≤0.30 2.45 ≤0.30 ≤0.30 ≤0.30 ≤0.30 1.07 ≤0.30 ≤0.30 ≤0.30 ≤0.30 

(0.53) (0.0) (0.0) (0.0) (0.0) (0.50) (0.0) (0.0) (0.0) (0.0) (0.31) (0.0) (0.0) (0.0) (0.0) 

[6/6] [6/6] [6/6] [6/6] [5/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] [6/6] 

a Log CFU/g average of 6 samples (±standard deviation) (N=2 trials, n=3 replicates; detection limit by direct plating=≤0.3 log CFU/g).b Values in brackets are numbers of samples negative by enrichment from among the total number of samples that were enriched.
A 

B 

72 A.C.S. Porto-Fett et al. / International Journal of Food Microbiology 140 (2010) 61–75

larvae/g of batter) of T. spiralis to below detection (Fig. 1). The loss of

viability and/or infectivity of the larvae within Genoa salami was

confirmed by both microscopy and mouse bioassays. Likewise,

regardless of the time or level of pressure treatment, HPP was

sufficient for reducing the initial load (about 3.4 log larvae/g) of T.

spiralis larvae in pig masseter muscle to below detection. The loss of

viability and/or infectivity of the larvae in masseter following HPP was

also confirmed by both microscopy and mouse bioassays.

4. Discussion

Genoa salami has been prepared using traditional processes and

has been enjoyed and consumed worldwide largely without incident

for centuries. Since Genoa is typically (primarily) made from pork,

processors are required to practice heating, freezing (sometimes

referred to as certification), and/or curing to control T. spiralis in pork.

Among these, certification, the process of storing pork for prescribed

times at relatively cold temperatures, is perhaps the most practiced,

but probably not the most cost effective approach. Likewise, heating is

not an option for some meat products, including Genoa salami, due to

likely untoward effects on quality. Therefore, curing, the process of

holding and drying salami for prescribed times and temperatures

based on casing size and on salt content is a viable, but not necessarily

preferred or effective, approach for trichinae control. Given that the

existing make procedures also impact both the safety and quality of

Genoa, one objective of the present study was to evaluate the

equivalency of HPP as an alternative to freezing or curing for ensuring

Fig. 1. Panel A. Live larvae (coiled) of T. spiralis recovered before fermentation/drying of

from Genoa salami. Panel B. Dead larvae (uncoiled) T. spiralis recovered after

fermentation/drying of Genoa salami.

that pork and pork products are trichinae-free. A second objective was

to assess the lethality contributed by fermentation/drying and a postprocess treatment via HPP on viability of L. monocytogenes, Salmonella

spp., and/or E. coli O157:H7. For these reasons, it was essential that we

obtained pork that was harvested from swine that were experimentally inoculated with Trichinella larvae and assemble a multidisciplinary team to address these objectives, so that the results

could be directly used by the industry, so that the process would pass

muster with regulators, and so that the finished product would satisfy

consumers. As described herein, these objectives were met in full.

With respect to the microbial pathogens tested in the present

study, our results established total reductions of about 1.1 to 1.3,

about 1.1 to 2.2, and about 4.2 to 4.8 log CFU/g of L. monocytogenes, E.

coli O157:H7, and Salmonella, respectively, following fermentation

and drying of Genoa salami. These results are in general agreement

with other reports wherein cells of L. monocytogenes were more

resistant than cells of E. coli O157:H7 and S. typhimurium to

fermentation and drying of fermented sausages (Nightingale et al.,

2006; Nissen and Holck, 1998; Porto-Fett et al., 2008). In general, for

all treatments, an additional reduction of about 1.6 to ≥5.0, ≥4.7 to

≥5.8, and ≥1.9 to ≥2.4 log CFU/g in L. monocytogenes, E. coli O157:H7,

and S. typhimurium numbers, respectively, was achieved after Genoa

salami was pressurized at 600 MPa for up to 5 min or at 483 MPa for

up to 12 min and then stored refrigerated for up 28 d. Previous studies

reported that Gram-positive bacteria are generally more pressure

resistant than Gram-negative bacteria, presumably due to intrinsic

differences in their morphology, cell membrane, and cell wall, as well

as to differences in how they respond to biochemical and genetic

stresses (Mackey et al., 1994; Simpson and Gilmour, 1997; Ritz et al.,

2000; Tholozan et al., 2000). However, in this study, differences in

pressure resistance between L. monocytogenes and Salmonella could

not be established since comparatively too few cells of the latter were

available following fermentation and drying to squarely quantify the

subsequent lethality of HPP towards these two pathogens. A direct

comparison was feasible between L. monocytogenes and E. coli O157:

H7, because both of these pathogens survived at similar levels after

drying. In this regard, our data are in complete agreement with data

published by others in that L. monocytogenes (Table 5) was more

resistant to pressurization than E. coli O157:H7 (Table 6). That being

said, our findings showed that L. monocytogenes was inactivated to a

greater extent after pressurization in treatments B, C, and D when

compared to treatment A. These results may be attributed to the lower

aw value for chubs inoculated with L. monocytogenes that were

processed by treatment A (about aw value of 0.88) when compared

with aw values of chubs processed by the other treatments (about aw

values of ≥0.92). In general, lower aw values have a baro-protective

effect for pathogens when they are subjected to high pressure

treatments (Rubio et al., 2007).

Our findings validate that processing Genoa salami under the

conditions tested herein does not provide a favorable environment for

survival of L. monocytogenes, E. coli O157:H7, or Salmonella. Our

findings also validate that HPP is an effective post-processing

intervention for reducing relatively low levels of L. monocytogenes,

E. coli O157:H7, or Salmonella in Genoa salami. Collectively, the

conditions used in this study for fermentation, drying, HPP, and

storage of Genoa salami are sufficient to meet the current performance standards of a 5.0-log reduction for E. coli O157:H7 and a 6.5-

log reduction for Salmonella (Nickelson et al., 1996; Reed, 1995,

United States Department of Agriculture/Food Safety and Inspection

Service, USDA-FSIS, 2001b). It also satisfies compliance guidelines for

L. monocytogenes that requires processors of RTE meat products to

deliver a post-process lethality treatment, in this case HPP, and/or

allow for no more than a 1.5- to 2.0-log increase over shelf life (United

States Department of Agriculture/Food Safety and Inspection Service,

USDA-FSIS, 2001b; Anonymous, 2003). For treatments A, B, and D we

observed both lethality and inhibition. For treatment C we observed
A.C.S. Porto-Fett et al. / International Journal of Food Microbiology 140 (2010) 61–75 73

lethality, but levels of listeriae increased by N2.0 log CFU/g during

refrigerated storage. Thus, relative to L. monocytogenes, these data

make it possible for a processor to self affirm Alternative 1 status by

following the make protocol described and validated herein for

treatments A, B, and D, and to self affirm Alternative 2 status for

treatment C. These data should also be applicable to related products

without the need to conduct a bona fide process validation for each

pathogen and each process and/or at each establishment. More

specifically, it is neither possible nor practicable for a given plant to

obtain trichinae-infected pork and/or to obtain pork that may be

naturally contaminated with L. monocytogenes, Shiga toxin-producing

E. coli, and/or Salmonella spp. Likewise, it would not be prudent or

allowable to intentionally contaminate product with these pathogens

in a plant that processes food for human consumption. Furthermore,

there is a paucity of information on acceptable surrogates for the

above mentioned pathogens and considerable ongoing debate as to

the selection and comparative utility of using surrogates rather than

pathogens for challenge studies. As such, it would be more beneficial

from a public health standpoint to conduct and subsequently publish

a scientifically-sound validation study, such as the one detailed

herein, that was performed by an accredited/competent facility and to

then subsequently verify that the validated processing conditions and

product type/formulation were reduced to routine practice in the

processing plant. It is probable that such conditions would be equally

effective for like processes and for processes that are less stringent

from the perspective of pathogen viability. It is also probable that the

results validated herein would be directly applicable to like products

and to products with formulations that are less favorable to the

survival/growth of target pathogens. The precedent for this strategy

can be found in the NCBA Blue Ribbon Task Force (see Nickelson et al.,

1996; Reed, 1995).

With respect to the nematode tested, as an alternative to curing

and/or certification, one objective of the present study was to evaluate

the effect of HPP as a post-process intervention following fermentation/drying to inactivate T. spiralis larvae in both infected pig muscle

and in Genoa salami. Regardless of the processing treatment,

fermentation and drying of Genoa salami was sufficient to inactivate

T. spiralis larvae by ≥2.3 log larvae/g. These results are in general

agreement with other studies reporting that curing and fermentation/

drying reduced levels of T. spiralis larvae in Genoa salami. As an

example, Childers et al. (1982) reported that when Genoa salami

(stuffed into a 98-mm diameter casing) was formulated with 1.67 or

3.3% of sodium chloride, fermented at 35 °C or 46.1 °C, and then dried

at 7.8 °C for up 35 d, no viable larvae were recovered by artificial

digestion after 10 and 5 d of drying time, respectively. Similar results

were reported by Smith et al. (1989) who evaluated the effect of salt

level on the viability of T. spiralis larvae in Genoa salami (stuffed into a

80-mm diameter) that were dried for up to 90 d at 7 °C and 80% RH.

They reported loss of viability and/or infectivity of larvae from Genoa

salami formulated with 2.0% (aw= 0.955), 2.75% (aw= 0.950), or

3.3% (aw= 0.930) salt after 13 d of drying.

In the present study, pressures of either 483 or 600 MPa for 1.0 or

0.5 min, respectively, inactivated T. spiralis larvae in masseter muscle

(about 3.4 log larvae/g) harvested from infected pigs. Thus, if

fermentation and drying were not effective, then HPP would

effectively inactivate the about 2.3 log larvae/g of T. spiralis larvae

that were present in the Genoa salami prepared in this study. Our

findings are also in general agreement with those of Ohnishi et al.

(1992) who demonstrated that if muscle tissue from mice infected by

oral gavage with 200 larvae was subsequently treated with ≥200 MPa

(29,000 psi), then the T. spiralis larvae were inactivated. In a related

study, Ohnishi et al. (1994) demonstrated that pressures ≥200 MPa

for 10 min induced histological and morphological changes in T.

spiralis that may result in the loss of viability and/or infectivity of the

larvae. In contrast, Gamble et al. (1998) reported that hydrodynamic

treatments of 55 to 60 MPa when applied to infected pork tenderloin

were not sufficient to completely inactivate T. spiralis larvae.

Collectively, these data confirm that pressures of ≥200 MPa are

required to insure for inactivation of trichinae in muscle tissue/meat.

For the Genoa salami evaluated in the present study, the standard/

traditional make protocol was established based on specified holding

and drying times and temperatures determined by its salt content and

casing size as prescribed for trichinae control (United States

Department of Agriculture/Food Safety and Inspection Service,

2001a). On occasion, consumer preferences may prompt processors

to alter certain parameters, such as the salinity, acidity, and/or

moisture content, to generate a product that is more or less tart, salty,

and/or firm. However, in practice, this is not so easily done, because as

mentioned above, the basic parameters for fermentation and drying

are essentially fixed based on salt content and casing diameter. As

such, to comply with existing policies, it would be necessary to

include some other “intervention” for trichinae control before key

processing parameters could be modified. The technology evaluated

in the present study as an alternate method for trichinae control was

HPP. However, since holding and drying alone were effective at

reducing the population of viable larvae from an initial level of about

2.3 larvae/g to below detection as determined using a mouse bioassay,

it was not possible to quantify the efficacy of HPP towards T. spiralis

directly in Genoa. Nonetheless, it was possible to quantify the effect of

HPP on trichinae larvae in pig masseter muscle, a raw, whole-muscle

piece of tissue harvested directly from trichinae-infected pigs. As an

aside, meat from the same pigs from which masseter tissue was

harvested was also used to prepare the Genoa salami in this study. Our

data validated that a 3.2-log reduction of trichinae larvae was

achieved in as little as 1.0 min using 483 MPa and in as little as

0.5 min using 600 MPa. Based on these data, HPP would be an

effective alternative to heating, freezing, or curing for control of

trichinae in Genoa-type salami and related whole-muscle products

such as prosciutto, capocollo, and Parma ham. Moreover, when

applied after fermentation and drying, HPP was also quite effective at

reducing levels of L. monocytogenes, E. coli O157:H7, or Salmonella.

These data are significant in the context of HPP being used as a postprocess intervention to provide lethality towards L. monocytogenes

and for limiting its subsequent outgrowth during shelf life. These data

also validate that treatments A, B, C, and D along with HPP satisfy the

requisite performance standards for red meat and poultry products

for E. coli O157:H7 (N6.2 log CFU/g reduction) and Salmonella

(≥6.6 log CFU/g reduction) (Nickelson et al., 1996; Reed, 1995;

United States Department of Agriculture/Food Safety and Inspection

Service, USDA-FSIS, 2001b).

Collectively, our findings confirmed that Genoa salami prepared

using the make protocols described in this study generates appreciable reductions in levels of L. monocytogenes, E. coli O157:H7,

Salmonella, and T. spiralis, and meets all existing regulatory requirements for product wholesomeness. Regarding the potential impact

and practical application of the findings of this study, our validation of

HPP as an alternative technology for trichinae control would allow

processors to adjust the prescribed times and temperatures for

holding and/or drying to produce a product that was more desirable

to some consumers from a taste/texture perspective and still be in

compliance with requisite regulatory policies. As an added benefit,

HPP would also make it possible for a processor to satiate the

performance standards for E. coli O157:H7 and Salmonella as well as

the current guidelines for L. monocytogenes.

Acknowledgments

We extend our appreciation to the following individuals who in

large measure contributed to the successful completion of this study

by sharing their time, talents, resources, and/or opinions: Rosemary

Martinjuk, Nelly Osoria, Evan Griffin, Jennifer Levi, Nicole Posey, Peggy

Williamson, John Phillips (all of the USDA/ARS/ERRC in Wyndmoor,
74 A.C.S. Porto-Fett et al. / International Journal of Food Microbiology 140 (2010) 61–75

PA), Giorgio Muraro (of Santa Maria Foods ULC in Brampton, Ontario,

Canada), and Mitch Fournet (of the USDA/ARS/BARC in Beltsville,

MD). This study was funded, in part, by Santa Maria Foods through a

Trust Fund Cooperative Agreement with JBL. Lastly, we respectfully

dedicate this manuscript to our dear friend and long time colleague/

collaborator, Jean L. Smith, who unexpectedly passed away as this

project/paper was wrapping up. Her many and valued contributions

and her kindness and inspiring spirit are not forgotten. “Peace out cub

scouts! — Jean”.

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