Effect of media, additives, and incubation conditions on the recovery
of high pressure and heat-injured Clostridium botulinum spores
N.R. Reddy
a
,
*
, R.C. Tetzloff
b
,
1
, G.E. Skinner
a
a
National Center for Food Safety and Technology, U.S. Food and Drug Administration, 6502 S. Archer Road, Summit-Argo, IL 60501, USA
b
National Center for Food Safety and Technology, Illinois Institute of Technology, 6502 S. Archer Road, Summit-Argo, IL 60501, USA
article info
Article history:
Received 12 March 2009
Received in revised form
12 February 2010
Accepted 15 February 2010
Available online 19 February 2010
Keywords:
Clostridium botulinum
High pressure processing
Recovery of spores
abstract
The effect of additives and post-treatment incubation conditions on the recovery of high pressure and
heat-injured (i.e., processed at 620 MPa and 95 and 100
C for 5 min) spores of Clostridium botulinum
strains, 62-A (proteolytic type A) and 17-B (nonproteolytic type B) was studied. High pressure and heat-
injured spores were inoculated into TPGY (TrypticaseePeptoneeGlucoseeYeast extract) anaerobic broth
media containing additives (lysozyme,
L
-alanine,
L
-aspartic acid, dipicolonic acid, sodium bicarbonate,
and sodium lactate) at various concentrations (0e10
m
g/ml) individually or in combination. The spore
counts of high pressure and heat-injured 62-A and 17-B recovered from TPGY broth containing lysozyme
(10
m
g/ml) incubated for 4 months versus that recovered from peptoneeyeast extracteglucoseestarch
(PYGS) plating agar containing lysozyme (10
m
g/ml) incubated under anaerobic conditions for 5 days
were also compared. None of the additives either individually or in combination in TPGY broth improved
recovery of injured spore enumeration compared to processed controls without additives. Addition of
lysozyme at concentrations of 5 and 10
m
g/ml in TPGY broth improved initial recovery of injured spores of
17-B during the first 4 days of incubation but did not result in additional recovery at the end of the 4
month incubation compared to the processed control without lysozyme. Adding lysozyme at a concen-
tration of 10
m
g/ml to PYGS plating agar resulted in no effect on the recovery of high pressure and heat-
injured 62-A and 17-B spores. The recovery counts of high pressure and heat-injured spores of 62-A and
17-B were lower (i.e., <1.0 log units) with PYGS plating agar compared to the MPN method using TPGY
broth as the growth medium.
Published by Elsevier Ltd.
1. Introduction
Renewed interest in the use of high pressure processing (HPP) as
an alternative method for preservation of foods may be due to
increased consumer demand for minimally processed, fresh-
tasting, additive-free, and microbiologically safe shelf-stable foods
(De Heij et al., 2002, 2003; Leadley and Williams, 1997; Meyer et al.,
2000; Ting et al., 2002). HPP offers the potential for producing foods
with characteristics desired by consumers, such as natural sensory
and quality attributes, while extending shelf-life through control of
microorganisms and enzymes. HPP has been reported to be effective
in reducing or eliminating vegetative pathogens, human rotavirus,
hepatitis A virus and calicivirus in foods when processed at
250e700 MPa in combination with low to moderate (<50
C)
temperatures (Cheftel, 1995; Casadei, 2000; Linton and Patterson,
2000; Raso and Barbosa-Canovas, 2003; Khadre and Yousef, 2002;
Kingsley et al., 2002). Bacterial spores, however, are much more
resistant to various process treatments, including high pressure,
than their vegetative cell counterparts. The structure and thickness
of the bacterial spore coat is believed to account for this high
resistance. Bacterial spores cannot be inactivated by high pressure
alone (Maggi et al., 1996; Mills et al., 1998; Rovere et al., 1998). They
can survive pressure treatments above 1000 MPa unless pressuri-
zation is carried out at temperatures close to 100
C(Cheftel, 1995).
Several studies have been conducted on the inactivation of
spores of Clostridium botulinum proteolytic and nonproteolytic type
strains by HPP using various combinations of high pressure and
elevated temperatures (Gola and Rovere, 2005; Margosch et al.,
2004a,b; Reddy et al., 1999, 2000, 2003, 2006). These studies
reported various levels of inactivation and recovery of surviving
spores of C. botulinum either in phosphate buffer, mashed carrots or
crabmeat blend. Recoveries of surviving spores were enumerated
using plate count agar media with anaerobic incubation of plates for
48 h or a most probable number (MPN) method using TPGY (Tryp-
ticaseePeptoneeGlucoseeYeast extract) broth as an anaerobic
*
Corresponding author. Tel.: þ1 (708) 728 4135; fax: þ1 (708) 728 4177.
E-mail address: rukma.reddy@fda.hhs.gov (N.R. Reddy).
1
Current address: Hormel Foods, 2 Hormel Place, Austin, MN 55912, USA
Contents lists available at ScienceDirect
Food Microbiology
journal homepage: www.elsevier.com/locate/fm
0740-0020/$ e see front matter Published by Elsevier Ltd.
doi:10.1016/j.fm.2010.02.004
Food Microbiology 27 (2010) 613e617