JOURNAL OF FOOD SCIENCE
MICROBIOLOGY
Prevention of Clostridium botulinum Type A,
Proteolytic B and E Toxin Formation in
Refrigerated Pea Soup by Lactobacillus
plantarum ATCC 8014
G.E. Skinner, H.M. Solomon and G.A. Fingerhut
ABSTRACT
The ability of Lactobacillus plantarum ATCC 8014 to inhibit
Clostridium botulinum toxin production in pea soup was in-
vestigated. Soup containing C. botulinum spores (103/g) with
and without L. plantarum (106/g) were evaluated. Soup con-
taining only type A spores was toxic on days 1 and 2 when
incubated at 358C and 258C, respectively. Soup containing
only proteolytic type B spores was toxic on days 2 and 5 at
358C and 258C, respectively. Soup containing only type E
spores was toxic at 258C, 158C, and 58C in 7, 7, and 63 days
respectively. No toxin was found in soup containing C. botuli-
num spores plus L. plantarum at any temperature studied.
Key Words: biocontrol, Clostridium botulinum, storage sta-
bility, refrigerated food, Lactobacillus plantarum, pea soup
INTRODUCTION
FOODS OFTEN RECEIVE SOME TYPE OF HEAT TREATMENT TO IN-
crease their shelf life and rely on refrigeration to ensure their safety.
Such foods have been designated as minimally processed refrigerated
foods (Kvenberg, 1990), new generation refrigerated foods (Rhode-
hamel, 1992), chilled foods (Richardson, 1994), extended shelf life
refrigerated foods (Conner et al., 1989), “sous-vide” (Mossel and
Struijk, 1991; Rhodehamel, 1992), or refrigerated pasteurized foods
of extended durability (REPFEDs) (Notermans et al., 1990; Lund and
Notermans, 1993). The thermal treatment applied to such products,
often referred to as pasteurization, is insufficient to inactivate all spores
of Clostridium botulinum. Many such foods require refrigeration to
avoid microbial spoilage or toxicity. Additional barriers could help
increase the safety of such foods in the event they are exposed to
elevated temperatures. Traditional hazard analyses of refrigerated foods
identify nonproteolytic types of C. botulinum as a primary concern
because of their ability to form toxin at refrigerated temperatures.
Discussions on the risks associated with extended shelf life refriger-
ated foods have traditionally focused on the potential for toxin pro-
MICROBIOLOGY
duction by nonproteolytic types of C. botulinum (Conner et al., 1989;
Notermans et al., 1990; Doyle, 1991; Rhodehamel, 1992; Lund and
Notermans, 1993; Peck, 1997). This is a result of the reduction of the
resident, competitive microflora during pasteurization, the ability of
nonproteolytic C. botulinum to grow under refrigeration temperatures
and extended storage times (which give C. botulinum spores more
time to germinate and produce toxin). Also, such foods are often
packaged under vacuum or some modified atmosphere inhibiting
growth of traditional aerobic spoilage microbes. Nevertheless, out-
Author Skinner is with the U.S. Food & Drug Administration, Division of Food
Processing & Packaging, Food Process Hazard Analysis Branch / National
Center for Food Safety & Technology, Summit-Argo, IL, 60501. Author Solomon
is with the U.S. Food & Drug Administration, 200 C St., SW, Washington, DC
20204. Author Fingerhut is with the National Center for Food Safety & Technol-
ogy / IIT, Summit- Argo, IL 60501. Address inquiries to Dr. Guy E. Skinner.
724 JOURNAL OF FOOD SCIENCE—Volume 64, No. 4, 1999
breaks of botulism have been associated with refrigerated commercial
clam chowder and bean dip that were attributed to toxin production by
proteolytic types of C. botulinum in which improper storage tempera-
ture was implicated (Trevejo, 1995). These indicate that processors
need to expand the hazard analysis for extended shelf life refrigerated
foods to encompass risks associated with these types of spores as
well.
Lactic acid bacteria (LAB) may have the potential to serve as an
additional protective barrier in extended shelf life refrigerated foods
(Gombas, 1989; Crandall and Montville, 1993). Lactic acid bacteria
may serve as antagonists against various bacteria through the produc-
tion of compounds such as bacteriocins and/or diacetyl, or by altering
the environment by the production of acids (Ray and Daeschel, 1992).
Low refrigeration temperatures inhibit growth of the LAB in the food
and prevent toxin formation by C. botulinum, resulting in minimal
flavor changes in properly stored products. However, if the food is
exposed to elevated temperatures, the LAB inoculum strain will grow
rapidly and out-compete microbial pathogens that may be present.
LAB produce lactic acid, reducing the pH in the food and creating an
environment less conducive to growth and/or toxin production by
pathogenic bacteria such as C. botulinum. Such biocontrol represents
a “natural” form of food preservation, and there is increased interest in
its potential as a secondary barrier in refrigerated foods.
Biocontrol was approved by the USDA in 1986 for use in bacon. It
has been evaluated for effectiveness in delaying spoilage of beef steaks,
poultry meat, mechanically deboned poultry meat and other commodi-
ties (Gombas, 1989). These have included a model gravy system (Cran-
dall and Montville, 1993), a model “sous-vide” beef product (Crandall
et al., 1994), and “sous-vide” salmon and rice (Simpson et al., 1993).
Lee et al. (1974) reported that radiation-killed Pediococcus cerevisiae
along with 1% glucose inhibited C. botulinum toxin production in ham
and vacuum packed smoked turkey. Biocontrol inhibited C. botulinum
toxin formation in frozen chicken a la king (Saleh and Ordal, 1955a, b),
chicken salad (Hutton et al., 1991), and model gravy (Crandall and
Montville, 1993) that had been exposed to temperature abuse. Before
biocontrol could be utilized as an additional barrier, it must be evaluated
for its effectiveness in preventing growth and toxin formation by patho-
gens such as C. botulinum in extended shelf life refrigerated foods
under temperature abuse. Our objective was to evaluate the ability of L.
plantarum to inhibit toxin production by C. botulinum in pea soup at
refrigeration and abuse storage temperatures.
MATERIALS & METHODS
Pea soup prepration
Refrigerated pea soup was prepared by aseptically combining equal
portions of a commercial, canned condensed pea soup with sterile
distilled water as per can instructions. The soup was mixed and heated
for approximately 30 min in a large beaker covered with aluminum
foil using a Model PC-351 hot plate stirrer (Corning Inc., Corning,
NY) on the high setting, then cooled to room temperature in approxi-
mately 40 min in a Model CH/P circulating water bath (Forma Scien-
tific, a division of Mallinckrodt, Inc., Marietta, OH) set at 28C. After
© 1999 Institute of Food Technologists
cooling the pea soup, filter sterilized glucose was added to a final
concentration of 0.5% and the soup was distributed into sterile tubes
at 40 mL per tube. For inoculation studies, four treatments of pea soup
were prepared: (1) soup with no inoculum; (2) soup with Lactobacil-
lus plantarum (106/g); (3) soup with a C. botulinum spores (103/g);
(4) soup with L. plantarum (106/g) plus C. botulinum spores (103/g).
Spores and/or cells were gently stirred into the soup to minimize the
incorporation of oxygen, which might delay or inhibit C. botulinum
germination or toxin formation. After mixing, soup was aseptically
dispensed into sterile 50 mL test tubes (40 mL/tube) and incubated at
predesignated temperatures. The initial pH of the pea soup was 6.2,
and the water activity, aw, measured with an Aqualab CX-2 water
activity meter (Decagon Devices, Pullman, WA) was 0.99. We recog-
nize that type E may not be considered a notable inherent risk from
nonseafood products such as pea soup. However, nonproteolytic types
are of great concern with respect to refrigerated foods, particularly
those which have some seafood ingredient. Pea soup served as a
model to evaluate the potential for biological control.
Lactic acid bacteria inoculum
Frozen preparations of Lactobacillus plantarum ATCC 8014 (bac-
teriocin negative) were made by growing the cultures overnight at
358C, followed by centrifugation using a Sorvall Instruments Centri-
fuge Model RC5C (DuPont Co., Newtown, CT) in the cold (48C) at
5000 rpm for 5 min, and washing the pellet with sterile 0.1% peptone.
This procedure was repeated 3 times. Pellets of the LAB were then
resuspended in sterile MRS broth with 20% sterile glycerol, after
which they were transferred to 2 mL cryovials and stored at 2808C.
Final concentrations of cells in the cryovials were 109/mL. For inocu-
lation studies, a 12 h L. plantarum ATCC 8014 culture was obtained
by thawing a frozen vial at room temperature, inoculating 0.5 mL of
the thawed culture into 100 mL MRS broth and incubating at 358C.
After 12 h of incubation, the culture was mixed and inoculated into the
room temperature pea soup at levels of 1 3 106 cells/mL.
Clostridium botulinum type spore preparations
The C. botulinum cultures were obtained from the U.S. Food &
Drug Administration (FDA), Washington, DC. Spore suspensions of
individual strains were obtained by growing in trypticase-peptone-
glucose-yeast extract (TPGY) medium. Proteolytic spores were pro-
duced by incubating the TPGY broth for 7 days at 358C (Solomon and
Kautter, 1988) while type E spores were produced by incubating for
5–7 days at 268C (Solomon et al., 1977). Spores of each strain were
harvested by centrifugation, washed 3 times with sterile distilled wa-
ter, and resuspended in sterile distilled water. Spore numbers of each
C. botulinum strain suspension were estimated using the 3-tube most
probable number (MPN) method using TPGY broth as the culture
medium, and pour plating with trypticase soy agar (TSA) followed by
anaerobic incubation at 358C for 5 to 7 days (Hutton et al., 1991).
Individual type E cultures were frozen at 2158C for 6 days, then
heated to 708C for 10 min to produce toxin free spore suspensions.
Equal numbers of spores from each strain were mixed then diluted
using sterile distilled water. The proteolytic spore cocktails used in
each inoculation study were heat shocked at 808C for 10 min prior to
inoculation, whereas type E spores were not heat shocked. Spores
were inoculated into the room temperature pea soup at 103/g. The C.
botulinum type A inoculum included equal numbers of 62A, 73A and
OS3A strains, the proteolytic type B inoculum included equal num-
bers of Mush2 B, Mush3 B and TJ B strains, and the type E cocktail
contained equal numbers of Beluga E and 070E strains.
Challenge study and analysis for C. botulinum toxin
Separate inoculation studies were performed with C. botulinum
type A and proteolytic type B spores at 358C, 258C, and 158C. Inocu-
lation studies with type E spores were performed at 258C, 158C and
58C. In each study, LAB growth was monitored by removing tubes of
the LAB coinoculated soup with C. botulinum spores (BL), pour
plating in MRS agar (with bromocresol purple added as an acid indi-
cator) and incubating at 358C for 48 h. The pH of the BL soup (con-
taining LAB and C. botulinum spores) was monitored during the
study. At each sampling interval, two test tubes of soup containing
both spores and LAB (BL) and/or two tubes of soup with only spores
(B) were removed from each storage temperature at determined inter-
vals along with negative controls (1 test tube each of soup and soup
containing LAB) and assayed for the presence of C. botulinum toxin
and pH. These same soup treatments were assayed for toxin presence
when pH of the pea soup containing C. botulinum spores and L.
plantarum (BL) decreased below 4.6. Each tube of pea soup coinoc-
ulated with LAB and spores of C. botulinum was analyzed for pH and
LAB CFU/ mL. Dilutions were made in 0.1% peptone, pour plated in
MRS agar (containing bromocresol purple indicator) and incubated at
358C for 48h. LAB counts were expressed as CFU/ mL soup. Soup
pH was monitored using a Corning pH/ion analyzer Model 350 (Corn-
ing Incorporated, Corning, NY). Extracts were prepared for toxin
analysis by pouring the soup contents from the test tube into sterile
centrifuge tubes, centrifuging tube contents using a Sorvall Instru-
ments Model RC5C Centrifuge (DuPont Co., Newtown, CT) at 48C
for 25 min at 10,000 rpm, then pouring the supernatant into scintilla-
tion vials and refrigerating. Refrigerated test extracts were analyzed
for presence of toxin using the mouse bioassay procedure (FDA,
1995) with a minor modification to reduce the trypsin solution con-
centration (5% trypsin solution used instead of 10%) for the type E
extracts. Trypsinization was performed on the type E C. botulinum
extracts by treating them with trypsin (1:250 from Difco Laboratories,
Detroit, MI) for 1 h at 358C (1.8 mL clear extract filtrate, 0.2 mL 5%
trypsin solution) to potentiate type E toxin. Test extracts were injected
intraperitoneally into two mice (0.5 mL/mouse) and the mice were
observed for 48 h for typical botulism symptoms. The inoculation
study was continued for approximately 25 days for both types of
proteolytic spores and for approximately 90 days with type E.
RESULTS & DISCUSSION
GROWTH STUDIES SHOWED THAT L. PLANTARUM ATCC 8014 PRO-
liferated most rapidly at 358C, with growth rate decreasing as incuba-
tion temperature decreased to 58C (Fig. 1). Growth of L. plantarum in
the type A, proteolytic type B and type E inoculation studies was
consistent between experiments performed at duplicated temperatures,
with counts increasing from 106 to approximately 5 3 108/ mL at
358C, 258C, and 158C. Typical growth at 358C, 258C, 158C, and 58C at
short times (Fig. 1), was compared with LAB growth up to 91 days
(2184 h) (Fig. 3). At 58C L. plantarum cell numbers remained at 106/
MICROBIOLOGY
Fig. 1—Representative growth of Lactobacillus plantarum ATCC 8014
in pea soup coinoculated with spores of Clostridium botulinum.
Volume 64, No. 4, 1999—JOURNAL OF FOOD SCIENCE 725
 C. botulinum Toxin Inhibition in Pea Soup . . .

mL through day 42 then increased to levels of 107/mL by day 49, Table 1—Toxigenesis of pea soup Inoculated with C. botulinum type
A, proteolytic type B or type E (103/mL) with and without L. plantarum
staying constant through the end of the study (day 91). ATCC 8014 (106/mL)
In inoculation experiments with LAB and C. botulinum types A,
Storage C. botulinum L. plantarumb Toxin formation
proteolytic B and E, the time necessary for L. plantarum ATCC 8014 temp (°C) typea (106/ mL) (days
to lower the pea soup pH varied with storage temperature. The pea
soup pH was reduced by L. plantarum to less than 4.6 at all tempera- 35 A — >0, <1
A + >24c
tures studied (Fig. 2, 4). All pea soup contained 0.5% glucose asepti- pB — >1, <2
cally added during formulation because preliminary studies had shown pB + >22c
that glucose enhanced pH reduction (preliminary data not shown) and 25 A — >1, <2
A + >24c
Crandall and Montville (1993) had reported that the addition of more pB — >4, <5
than 0.5% glucose had no additional effect on pH reduction. Various pB + >22c
researchers have reported that addition of carbohydrate to a system E — >1.3, <7
E + >21c
may decrease the time for the LAB to reduce the system pH (Riemann 15 A — >42c
et al., 1972; Tanaka et al, 1985a, b; Crandall and Montville, 1993). At A + >24c
358C, soup pH decreased below 4.6 in 18 or 16 h in the type A and pB — >22c
pB + >22c
proteolytic type B inoculation experiments, respectively (Fig. 2). At E — >4.2, <7
258C, 28 and 27 h were necessary for the pH to drop below 4.6 in the E + >35c
type A and proteolytic type B inoculation experiments, respectively 5 E — >49, <63
E + >91c
(Fig. 2). At 158C, the L. plantarum required 89 and 93 h to reduce the
apB–C. botulinum proteolytic type B inoculum, (103/mL).
pH to less than 4.6 in the type A and proteolytic type B inoculation b+ = L. plantarum (106/mL)inoculated into soup containing C. botulinum spores (103/mL).
experiments, respectively (Fig. 2). In the type E inoculation study c—Last day the pea soup was tested for C. botulinum toxin, at which point no toxin was
detected.
performed at 58C, L. plantarum required 63 days to reduce the pea
soup pH below 4.6 (Fig. 4). Reduction of pH occurred as L. pla-
narum cells produced lactic acid as a by-product of carbohydrate
fermentation. As pH decreases from its initial value of 6.1 (pea soup) toxic until day 5. Periodic sampling for toxin showed pea soup con-
to 4.6 the food matrix becomes less ideal for growth, and spores of C. taining only type A and proteolytic type B spores stored at 158C to be
botulinum subsequently would require longer times to germinate and negative on days 42 and 22, respectively, the last sampling day at that
produce toxin. It is generally accepted that under normal conditions, temperature for both studies.
C. botulinum will not produce toxin below pH 4.6. An important C. botulinum toxin was found in pea soup inoculated with the
finding was that the pH remained low and did not increase at extended mixture of type E spores without LAB at 258C, 158C, and 58C. Type
periods of time. E C. botulinum toxin was found in pea soup containing only spores
Incubation time necessary for C. botulinum toxin formation in pea that was incubated at 258C on day 7, but not after 1.3 days (sampling
soup containing only spores varied with incubation temperature (Ta- period when the pH of soup containing spores and LAB fell below
ble 1). Soup containing C. botulinum spores and no LAB (Treatment 4.6). At 158C, toxin was also found in pea soup inoculated with spores
(B)) was the only treatment to support toxin formation in the pro- and no LAB on day 7 but not on day 4.2 (sampling period when the
teolytic studies. No toxin was found in any negative controls nor any pH of soup containing spores and LAB fell below 4.6). Pea soups
soups tested on day 0. Type A C. botulinum produced toxin in pea stored at 58C were negative for toxin on day 49 but toxin was found in
soup incubated at 358C in 1 day, the first sampling period, while one of two day 63 soup tubes and both tubes of soup from day 91. As
proteolytic type B toxin was not found on day 1 but was present on was found in the studies with proteolytic strains of C. botulinum, no
day 2. At 258C storage, pea soup inoculated with type A was found toxin was found in any pea soup containing both type E spores of C.
positive for toxin on day 2, while proteolytic type B soup was not botulinum and L. plantarum ATCC 8014.
MICROBIOLOGY

Fig. 2—Representative pH reduction of pea soup coinoculated with Fig. 3—Representative long term growth of Lactobacillus plantarum
Lactobacillus plantarum ATCC 8014 and spores of Clostridium botu- ATCC 8014 in pea soup coinoculated with spores of Clostridium
linum. botulinum.

726 JOURNAL OF FOOD SCIENCE—Volume 64, No. 4, 1999


Fig. 4—Representative long term pH reduction of pea soup
coinoculated with Lactobacillus plantarum ATCC 8014 and spores of
Clostridium botulinum.
Pea soup inoculated with both L. plantarum and spores of C.
botulinum and stored at 358C, 258C, 158C, and 58C were tested for the
presence of C. botulinum toxin within 6 h after the pH decreased to
less than 4.6 and at other intervals. No toxin was found in any pea
soup containing both spores of C. botulinum and cells of L. plan-
tarum ATCC 8014. The pH of the pea soup containing both organ-
isms did not increase over time. Thus, we assumed that if toxin was
not found in the sampling period just after the pH of the pea soup
containing both L. plantarum and spores of C. botulinum decreased
below 4.6, then the coinoculated soups would remain free of toxin
through the end of their shelf-life.
At inoculum levels of 106/g, L. plantarum ATCC 8014 successful-
ly inhibited toxin formation by spores of type A, proteolytic B, and
type E C. botulinum (inoculated at levels of 103/g) in pea soup at the
severe and moderate abuse temperatures tested (358C, 258C, and 158C)
as well as at extended periods at refrigeration temperatures (58C). In
the pea soup food system, L. plantarum ATCC 8014, a LAB which
does not produce a bacteriocin, adequately reduced the pH of the
system before C. botulinum toxin could form. This demonstrates that
biological control, if applied properly, may serve as an additional
barrier, along with refrigeration, to help prevent C. botulinum toxin
formation when a refrigerated food is exposed to temperature abuse.
The efficacy of biological control depends on several factors, includ-
ing product pH, inoculum level, inoculum type, type and level of
carbohydrate, buffering capacity and presence of inhibitory compounds
(Gombas, 1989). Therefore, application of biological control into real
food systems would require challenge studies on a product-to-product
basis (Conner et al., 1989).
In refrigerated pea soup, L. plantarum inhibited toxin formation
by C. botulinum type A, proteolytic B and E strains under a range of
Reprinted from J. Food Sci. 64(4): 724–727
©1999 Institute of Food Technologists
temperatures. If it is to be effectively used as an additional barrier to
help maintain the safety of a food, this technology needs to be proven
effective for each specific product application. The incorporation of
technologies such as competitive microflora as additional barriers for
food safety should never be considered as a replacement for Good
Manufacturing Practices (Gombas, 1989).
REFERENCES
Conner, D.E., Scott, V.N. and Bernard, D.T. and Kautter, D.A. 1989. Potential Clostrid-
ium botulinum hazards associated with extended shelf-life refrigerated foods: A
review. J. Food Safety. 10: 131-153.
Crandall, A.D., Winkowski, K. and Montville, T.J. 1994. Inability of Pediococcus pen-
tosaceus to inhibit Clostridium botulinum in sous vide beef with gravy at 4 and
10°C. J. Food Prot. 57(2): 104-107.
Crandall, A.D. and Montville, T.J. 1993. Inhibition of Clostridium botulinum growth
and toxigenesis in a model gravy system by coinoculation with bacteriocin-produc-
ing lactic acid bacteria. J. Food Prot. 56(6): 485-488.
Doyle, M.P. 1991. Evaluating the potential risk from extended shelf-life refrigerated
foods by Clostridium botulinum inoculation studies. Food Technol. 45(4): 154-
156.
FDA. 1995. Bacteriological Analytical Manual, 8th Ed., Association of Official Analyt-
ical Chemists, Arlington, VA.
Gombas, D.E. 1989. Biological competition as a preserving mechanism. J. Food Safety.
10: 107-117.
Hutton, M.T., Chehak, P.A. and Hanlin, J.H. 1991. Inhibition of botulinum toxin pro-
duction by Pediococcus acidilactici in temperature abused refrigerated foods. J.
Food Safety. 11: 255-267.
Kvenberg, J.E. 1990. Microbiological criteria and regulatory aspects of minimally
processed refrigerated foods. J. Food Prot. 53: 910.
Lee, W.H., Riemann, H.P. and Al-Mashat, A.J. 1974. Controlled fermentation and pre-
vention of undesirable bacterial grown in food (U.S. Patent #3,794,739)
Lund, B.M. and Notermans, S.H.W. 1993. Potential hazards associated with REPFEDS.
In Clostridium botulinum, Ecology and Control in Foods. 279-303. A.W. Hauschild
and K.L. Dodds (Ed.). Marcel Dekker, Inc., New York, NY.
Mossel, D.A.A. and Struijk, C.B. 1991. Public health implication of refrigerated pas-
teurized (sous-vide) foods. Int. J. Food Microbiol. 13: 187-206.
Notermans, S., Dufrenne, J. and Lund, B.M. 1990. Botulism risk of refrigerated, pro-
cessed foods of extended durability. J. Food Prot. 53(12): 1020-1024.
Peck, M.W. 1997. Clostridium botulinum and the safety of refrigerated processed foods
of extended durability. Trends Food Sci. Technol. 8(6): 186-192.
Ray, B. and Daeschel, M. 1992. Food Biopreservatives of Microbial Origin, CRC Press,
Boca Raton, FL.
Rhodehamel, E.J. 1992. FDA’s concerns with Sous vide Processing. Food Technol.
46(12): 73-76.
Richardson, K.C. 1994. Microbiological safety of chilled foods. Food Australia. 46(6):
277-278.
Riemann, H, Lee, W.H. and Genigeorgis, C. 1972. Control of Clostridium botulinum
and Staphylococcus aureus in semi-preserved meat products. J. Milk Food Technol.
35: 514-523.
Saleh, M.A. and Ordal, Z.L. 1955a. Studies on growth and toxin production of Clostrid-
ium botulinum in a precooked frozen food. I. Some factors affecting growth and
toxin production. Food Res. 20: 332-339.
Saleh, M.A. and Ordal, Z.L. 1955b. Studies on growth and toxin production of Clostrid-
ium botulinum in a precooked frozen food. II. Inhibition by lactic acid bacteria. Food
Res. 20: 340-350.
Simpson, M.V., Smith, J.P., Simpson, H.S., Ramaswamy, H.S. and Dodds, K. 1993. Chal-
lenge studies with Clostridium botulinum in minimally processed “sous vide” prod-
ucts. Proceedings of Food Processing 2000, October 19, 1993. Natick, MA.
Solomon, H.M. and Kautter, D.A. 1988. Outgrowth and toxin production by Clostrid-
ium botulinum in chopped garlic and oil. J. Food Protection. 51(11): 862-865.
Solomon, H.M., Lynt, R.K., Lilly Jr., T. and Kautter, D.A. 1977. Effect of low tempera-
tures on growth of Clostridium botulinum spores in meat of the blue crab. J. Food
Protection 40(1): 5-7.
Tanaka, N., Meske, L., Doyle, M.P., Traisman, E., Thayer, D.W. and Johnston, R.W. 1985a.
Plant Trials of bacon made with lactic acid bacteria, sucrose and lowered sodium
nitrite. J. Food Prot. 48(8): 679-686.
Tanaka, N., Meske, L., Doyle, M.P., Traisman, E., Thayer, D.W. and Johnston, R.W. 1985b.
Sensory characteristics of reduced nitrite bacon manufactured by the Wisconsin
process. J. Food Prot. 48(8): 687-692.
Trevejo, R.T. 1995. Foodborne outbreaks in California. Dairy, Food Environ. Sanit. 15:
611-615.
Ms received 7/1/98; revised 2/17/99; accepted 3/6/99. MICROBIOLOGY
We thank Mr. Bob Pekelnicky of World’s Finest Chocolates, Chicago, Illinois, for assistance in
performance of many of the lactic acid bacteria (LAB) growth curves and preparation of the frozen
LAB preparations. We also thank Dr. D.E. Gombas for technical support and background informa-
tion regarding biological control and Dr. T.J. Montville, Rutgers University, for supplying some LAB
cultures for the preliminary portions of this study. This publication was partially supported by Coop-
erative Agreement No. FD-000431 from the U.S. Food & Drug Administration and the National
Center for Food Safety & Technology. Its contents are solely the opinions of the authors and do not
necessarily represent official views of the U.S Food & Drug Administration.
Volume 64, No. 4, 1999—JOURNAL OF FOOD SCIENCE 727