Journal nf Food Protection, Vol. 55, No. I, Pages 23-27 (January 1992)
`Copyrighte. International Association of Milk, Food and Environmenlal Sanitarians
`
`23
`
`Effect of Headspace Oxygen Concentration
`on Growth and Toxin Production by
`Proteolytic Strains of Clostridium botulinum
`
`R. C. WHITING* and K. A. NAFTULIN
`
`U.S. Department of Agriculture, ARS, Eastern Regional Research Center. 600 E. Mermaid Lane, Philadelphia, Pennsylvania 19118
`
`(Received for publica1ion September 11. I 990)
`
`ABSTRACT
`
`A series of experiments was conducted to determine growth
`and toxin formation by proteolytic strains of Closlridium botuli(cid:173)
`num in broth media that have known pH values (5-7), NaCl
`concentrations (0-4%). and controlled oxygen-nitrogen a1mo(cid:173)
`spheres. Lower pH and higher NaCl levels inhibited growth and
`toxin production by vegetative cells, but 15% oxygen in the
`headspace was insufficient for inhibition in all media. When
`spores were used as inocula and the tubes were gas flushed,
`outgrowth and toxin production generally occurred only under a
`I% or less oxygen atmosphere. Occurrence of growth and toxin
`was favored by high pH and low NaCl levels and was related to
`spore inoculum size. Spores were also inoculated into a mixed
`fennenter with controlled oxygen levels in the headspacc. Times
`to measurable turbidity increased with greater oxygen levels from
`36 h at 0.005% 0 2 to 109 h at 0.7% 0 2; however. growth rates
`
`were unaffected by headspace oxygen levels. No 10xin was ob(cid:173)
`, further demonstrating that the critkal level
`served with 0.9% 0 2
`of oxygen for germination and growth is approximately I%.
`
`Clostridiu.m botulinum is characterized as an obligate
`anaerobe. However, growth and toxin production were
`demonstrated after outbreaks and in model foods under
`ostensibly aerobic conditions. For example. sliced luncheon
`meats, air-packed smoked fish, sailed-dried fish, sauteed
`onions left on the grill, packaged fresh mushrooms, and
`oriental noodles in oxygen permeable plastic have all sup(cid:173)
`ported toxin formation (9). Nonproteolytic type E strains in
`fish-based model substrates developed toxicity in aerobic
`packaging (I .14.17,27).
`Early work by Meyer (20) and Dack et al. (3-5)
`indicated proteolytic strains could grow in broth media with
`headspace oxygen concentrations of 1.7%. Type A spores
`were reported to grow in the Eh range of -436 to -6 mV
`(22). Spores placed in trypticase soy broth with cysteine
`and sparged with N1 (-145 mV) or air (-60 mV) grew
`
`Me11tio11 of brand nr Jim, 11ames d oes not constitute an endorsement br the
`U.S. Department of Agric11/111re over others o( a .<imilar nature 1101
`mentirmed.
`
`equally well at favorable conditions, but growth was de(cid:173)
`layed with air sparging of broth containing 5-6% NaCl, pH
`5.3, or 30% sucrose (26).
`Pred icting the growth of microorganisms in foods re(cid:173)
`quires knowledge of the simultaneous influence of all
`significant factors. A model for toxin production in fish by
`nonproteolytic type E botulinum was developed with coef(cid:173)
`ficients for fish type, spore type, type of atmosphere (none
`) , temperature, spore inoculum. and aerobic
`containing 0 2
`plate count(/). Roberts and Jarvis (23) modeled the growth
`of type A spores in pasteurized pork slurries. T heir model
`included storage temperature. NaCl, n itrite, ascorbate, heat
`treatment, polyphosphate, and high (6.3-6.77) or low (5.54-
`6.36) pH. Montville (21) followed the interaction of pH
`and NaCl in broth on the culture density of type A vegeta(cid:173)
`tive cells. Dodds (6) measured the lag time for toxin
`production by type A and B spores in cooked, vacuum(cid:173)
`packed potatoes with controlled aw and pH. Regression
`equations for the probability of one spore to produce toxin
`at a specific aw -pH were calculated. None of these studies.
`however, used contro lled oxygen levels as a variable.
`There is concern that the increasing commercial use of
`modified atmosphere packaging and precooked foods is
`increasing the risk of foodbome microbial illnesses includ(cid:173)
`ing botulism (/ I). A better understanding of the oxygen
`tolerance of C. bo1ulinum as it interacts with pH. salt. and
`inoculum size is needed. This work examined the growth
`and toxin formation by proteolytic strains of C. botulinum
`in media that had known pH and NaCl concentrations and
`were maintained under a controlled oxygen-nitrogen atmo(cid:173)
`sphere headspace. Time for growth and toxin production
`from both spores and vegetative cells in static broth tubes
`and from spores in an agitated fermenter was studied.
`
`:11ATF.RJALS ANO METHODS
`
`C. botulinum
`Proteolytic type B strain spores (ATCC 7949) were grown at
`35°C for 3 weeks in cooked meat media inside an anaerobic
`chamber flushed with 5% H, : 10% CO2:&S% N2 (Coy Laboratory
`Products, Inc., Ann Arbor. Ml). The spore cullure was stored
`
`JOURNAL OF FOOD PROTECTION. VOL. 55, JANUARY 1992
`
`Nexus Ex. 1032
`Page 1 of 5
`
`

`

`24
`
`WHl'rlNG AND NATTUUN
`
`ins ide the chamber. The spore preparations were heat shocked at
`80°C for IO min and enumerated inside the anaerobic chamber by
`diluting w ith sterile 0. 1% peptone, surface plating on botulinal
`assay medium (BAM) agar (/2) with a spiral plater (Spiral
`Systems. [nc., Bethesda. MD). and incubating at 35°C. BAM
`consisted of 5.0 g yeast extract. 5.0 g tryptone, 2.65 g nutrient
`broth, 1.2 g dipo1assium phosphate. and 2.0 g dextrose per L (pl-I
`7.3). For studies with vegetative cells, spores were heat shocked
`and IO µI
`inoculared inw 20 ml of BAM brorh inside the
`anaerobic chamber. After 48 h at 35°C. 0.1 ml was transferred to
`each of four tubes containing 20 ml BAM. After 24 h the cultures
`were washed and resuspended in 20 ml peptone water (total
`volume).
`In the spore germination experiment. spores were heat shocked
`and diluted with peptone water. Diluted spores (0.10 ml) were
`then added to BAM media (5.0 ml) to provide a level of 500
`spores per ml. For the experiment in the fermenter. three indi(cid:173)
`vidual strains of type A (69, FDA: 62, FDA; 33. US Army Lab,
`Natick, MA) and three strains of type B (169. FDA; 999, FDA:
`A TCC 7949) were individually heat shocked and 0.1 ml inocu(cid:173)
`lated into BAM (100 ml). After incubating inside the anaerobic
`ch.amber at 35°C for 3 weeks, the spores were washed, resuspended
`in water, heat shocked, and enumerated as described above. T he
`individual strains were d iluted with water and combined to give
`an equal strnin mixture totaling 2.5 x 101 spores per ml. The spore
`preparation was stored at 6°C for use as inoculum for the fer(cid:173)
`menter.
`
`Growth of vegetative cells
`Culture tubes containing BAM (5.0 ml) with varying pH (pH
`5-7 by 0.1 N HCI) and added NaCl (0-4%) were autoclaved and
`gas equilibrated overnight (ca. 15 h) inside the anaerobic cham(cid:173)
`ber. The loose-capped tubes were inoculated with 0.1 ml vegeta(cid:173)
`tive cells (ATCC 7949) to give an average log CFU per ml counts
`of 3.8. The tubes were placed immediately inside jars fitted with
`plastic connectors, transferred to an incubator at 20°C, and con(cid:173)
`nected together serially. An oxygen-ni trogen gas mixture with the
`desired oxygen concentration was then passed through the jars at
`a constant flow rate of 500 cc/min. The outlet tube of the last jar
`was submerged in water 10 create a slight positive pressure in the
`jars and visually confim1 gas flow and absence of leaks. At each
`sampling time over a 14-d period, the end jar was removed and
`immediately transferred into the anaerobic chamber where the
`clostridia in the tubes were enumerated. The tubes were then
`removed from the chamber and duplicate samples (0.2 ml) placed
`in a well of a ELISA plate for turbidity measurement at 630 nm.
`The remainder was stored at 6°C for toxin assay by ELISA (/3).
`The amount of toxin from one tube from each run was quantified
`by mouse bioassay (8), and a sample was placed on each ELISA
`plate as a toxin standard for the other samples on the plate. Two
`or three replicate tubes were sampled at each time for every pH(cid:173)
`NaCl combination. The medium pH and NaCl levels were se(cid:173)
`lected 10 incorporate the anticipated range of growth using a
`response surface design. This permilled determination of the
`variable interactions without the large number of samples required
`by a full factorial design.
`The gas mixtures were fonnulated by metering and mixing
`ultrahigh purity nitrogen and custom grade I%, I 0% or 20%
`oxygen-in-nitrogen (Lindy Division, Union Carbide, Philadelphia.
`PA). Oxygen levels of the mixtures were assayed with an oxygen
`analyzer (Systech Instruments. McHenry, IL). The procedure was
`repeated seven times with oJ<ygen levels of 0.0% (three times),
`1.6 %, 3.5%. 10.0%, and 15.0%.
`To estimate the redox potentials (Eh). uninoculared beakers
`containing BAM (pH 7.0 and no added NaCl) with the same
`surface to volume ratio as the culture tubes were autoclaved and
`equilibrated inside the anaerobic chamber. The beakers were then
`
`removed, placed under a flowing nitrogen or 10% oxygen- in(cid:173)
`nitrogen, and the platin um and reference electrodes (Radiometer,
`Denmark) carefully placed I cm from the top or bot1om of the
`BAi\1 broth. The Eh was followed until the media were in
`equilibration with the headspace oxygen. The electrodes were
`standardized with potassium hydrogen phthalate-quinhydrone (18).
`
`Spore outgrowth
`The spore inoculation experiment used 5.0 ml BAM with
`varying pl-ls and NaCl additions in 15-ml culture tubes. After
`autoclaving, media were equilibrated inside the anaerobic cham(cid:173)
`ber overnight and inoculated with O. IO ml ATCC 7949 spores.
`The rubes were transferred to a plastic glove bag and the bag was
`flushed three times with the desired oxygen-nitrogen gas mixture.
`Tubes were then individually flushed for 10 s with the gas mixture
`and immediately resealed with gas impermeable butyl rubber
`stoppers (Bellco Glass, Inc., Vineland, NJ). After all tubes were
`flushed and capped, the tubes were transferred to anaerobic jars
`which were then flushed for 10 min with 2 Umin of the same gas
`mixtures and sealed. The jars were incubated at 20°C for up to 90
`d. When growth in a tube was evident. the IUbe was withdrawn
`and the jar reflushed. The number of days for visible growth was
`recorded, and the tube was stored at 6°C for confirmation of toxi n
`formation wi th the ELISA test. The jars were reflushed at 2-week
`intervals if not previously opened lO remove a tube with growth.
`
`Spore outgrowth in a fermemer
`Two liters of BAM with 0.5% sodium thioglycolate (pH 7.0
`with no added NaCl) was sterilized iriside the 6-L bowl of a
`Techne BR-06 Bioreacter (Techne, Jnc .. Princeton, NJ). Tem(cid:173)
`perature was maintained at 35.0°C and the broth mixed at 100
`rpm. The oxygen-nitrogen gas mixture flowed through a microbial
`filter into the headspace at 500 cc/min. The oxygen meter was
`attached to the gas outlet to confin11 the oxygen level. The pH
`was monitored by a submerged electrode interfaced with a micro(cid:173)
`computer (Leading Edge D2) with control software by Nomad,
`Inc. (Livennore, CA). Broth was continuously pumped through a
`LKB 2138 Uvicord S column densitom eter (LKB Instruments,
`Jnc .. Gaithersburg. MD) equipped with 408-nm filter and inter(cid:173)
`faced with the computer. Turbidity and pH values were printed
`and stored on the m icrocomputer's hard disk every 30 min. Before
`inoculation. the fermenter was operated for 24 h to permit detec(cid:173)
`tion of any contaminant. The gas mix ture was flowing for a
`minimum of 6 h before inoculation 10 establish equilibra tion
`between headspace and d issolved oxygen. Broth was inoculated
`with he.at-shocked spore mixt ure (10-ml) containing a total of 2.8
`x 106 spores. lmmediately after inoculating. a sample (4 ml) was
`withdrawn for Eh measurement. A rubber stopper with Eh elec(cid:173)
`trodes and syringe needle was pressed upon the sample to exclude
`air and the measurement taken after about 5 min when the reading
`stabilized. Another sample was transferred to the anaerobic cham(cid:173)
`ber and surface plated on BAM agar plates to confirm inoculum
`size. Other plates were incubated aerobically to detect contamina(cid:173)
`tion by facultative anaerobes. T he remaining portion of this
`sample was mixed with an equal portion of glycerol and stored at
`- I 5°C for mouse bioassay. At the end of the fellllenter run,
`another set of samples was taken and analyzed for Eh, bacterial
`population, Gram stain. catalase activity. possible contamination,
`and presence of toxin. Preliminary trials were conducted with
`water and a dissolved oxygen electrode (Associated Bio-engineers
`and Consultants, Inc., Bethlehem, PA) in the fermenter under
`identical condition as above 10 determine the rate of oxygen
`equilibration after changing from air to nitrogen.
`Turbidity data were entered onto an RS/I table (BBN Soft(cid:173)
`ware Products Corp .. Cambridge, MA) and fitted to the Gompertz
`equation using a VAX computer and a Gauss-Newwn iteration
`procedure (2). Lag times and rate of turbidity increase were
`calculated from the Gomperrz coefficienL~.
`
`JOURNAL OF FOOD PROTECTION. VOL. .55, JANUARY 1992
`
`Nexus Ex. 1032
`Page 2 of 5
`
`

`

`HEADSPACE OXYGEN ON C. BOTULINUM
`
`25
`
`RESULTS ANO DISCUSSION
`
`Vegetative cells
`The Eh near the botLom of the pH 7 (no added NaCl)
`medium increased from -117 m V (equi librated in the reduc(cid:173)
`ing atmosphere of the anaerobic chamber) to + 107 mY
`with t1owing nitrogen. Equilibration was 90% complete
`within 4 h. When 10% oxygen was purged over the me(cid:173)
`dium, the Eh attained +275 mY within 2 h.
`With no oxygen in the nitrogen atmosphere above the
`medium, growth and toxin were consistently observed in
`media having pH ~ 5.5 and NaCl $. 3.0 % (Table !).
`Growth and toxin were not observed at pH 6.0 with 4.0%
`NaCl. When the oxygen level was increased to 1.6%, no
`toxin was detected at the 3.0 and 4.0% NaCl levels and at
`pH 5.0. Increasing the oxygen levels did not prevent toxin
`formation in the other five NaCl-pH treatments until 15%
`flowing headspace oxygen. At that oxygen level, growth
`and toxin still occurred in two media with pH 6.0 and 7.0
`and no added NaCl. For a given medium. the time for
`measurable toxin formation was only slightly increased by
`increasing oxygen concentrations.
`
`TABLE I • .Interaction of oxygen levels with pH and sail content
`for production of toxin from vegetative cells of C. botulinum at
`20°c.
`
`did show an inverse relationship with the time for toxin
`formation.
`
`TABLE 2. Effect of inoculum size on the growth and toxin
`production by vegetative cells grown with a 10% oxygen atmo-
`sphere over broth media at 20°C.
`
`pH
`
`7.0
`
`6.5
`
`6.0
`
`6.0
`
`5.5
`
`5.0
`
`Added
`NaCl
`(%)
`
`0.0
`
`1.0
`
`0.0
`
`2.0
`
`1.0
`
`0.0
`
`lnoc.
`(log)
`
`Time
`toxin
`(d)
`
`Toxin
`(MLD/ml)
`
`1.7
`3.5
`5.7
`1.7
`3.5
`5.7
`1.7
`3.5
`5.7
`1.7
`3.5
`5.7
`1.7
`3.5
`5.7
`1.7
`3.5
`5.7
`
`9
`3
`
`7
`
`3
`I
`
`7
`4
`
`7
`2
`
`3
`
`9
`21
`52
`0
`22
`128
`0
`16
`58
`0
`20
`7
`0
`30
`110
`0
`0
`42
`
`% oxygen above media
`
`•-- No growth observed within I 4 d.
`
`pH
`
`Added
`NaCl(%)
`
`7.0
`7.0
`6.5
`6.5
`6.0
`6.0
`6.0
`5.5
`5.5
`5.0
`
`0.0
`4.0
`1.0
`3.0
`0.0
`2.0
`4.0
`1.0
`3.0
`0.0
`
`0.0
`
`2•
`7*
`3
`6
`4
`3
`NT
`5
`5*
`7*
`
`1.6
`
`2
`NT
`2
`NT
`2
`3
`NT
`3
`NT
`NT
`
`3.5
`
`3
`NT
`3
`NT
`3
`8
`NT
`6
`NT
`NT
`
`10.0
`
`15.0
`
`3
`
`7
`
`3
`7
`
`7
`
`NT
`
`3
`
`NT
`
`7
`NT
`
`NT
`
`NT
`
`' Time toxin first detected in days.
`* Toxin detected in 2 of 3 runs within 14 d.
`NT-No toxin or growth detected within 14 d.
`Blank space indicates sample not run.
`
`Douglas and Rigby (7) claimed the Eh fell during
`spore germination and emergence, and Siegel and Metzger
`(24,25) demonstrated that a growing culture in a fermenter
`can reduce the Eh by 150 mY. We surmise that under
`favorable pH and NaCl conditions. the vegetative cells
`were able to maintain the initial Eh in the media and
`continue to g row, even with subsequently high headspace
`oxygen concentrations.
`The inoculum size affected the time for toxin under
`10% oxygen atmosphere in every pH-NaCl medium (Table
`2). With 50 CFU per ml inoculum. growth and toxin
`occurred only at 7.0 with no added NaCl within 1he 14-d
`incubation at 20°C. With 5 x 105 CFU per ml inoculum.
`growth and toxin were observed in all media. The amount
`of toxin produced was not great with this strain (24), but it
`
`Spore inocu/a
`When spores were inoculated into media and sealed
`under varying headspacc oxygen concentrations for up to
`90 d at 20°C, the oxygen levels that allowed growth and
`toxin formation were much lower than those observed with
`vegetative cells (Table 3). At 0.5% or more oxygen, no
`growth was observed at pH 5.0. The frequency of growth
`in the other media decreased at 0.5 and 1.0% compared to
`0% oxygen. At 2.0% oxygen only one tube had growth but
`no detectable toxin. Eight tubes, includ ing the tube at 2%
`oxygen, had growth but no toxin and were confirmed to
`have gram-positive, catalase-negative rods. It was probable
`that these tubes were removed for storage at 6°C for the
`toxin assay without allowing sufficient time for formation
`of detectable amounts of toxin. Our experience with this
`ELISA showed it could detect approximately 10 mouse
`unit~ per ml of toxin.
`
`Fermenter
`The previous culture lubes were not agitated. undoubt(cid:173)
`edly a factor in allowing the continuing growth of the
`vegetative cel ls. A series of runs in the fermenter was
`intended to collaborate the observations of spore growth
`presented on Table 3 but with mixing and monitored
`headspace oxygen levels. Trials measuring dissolved oxy(cid:173)
`gen showed a 90% reduction in the first I 1/2 h after
`initiating flow of nitrogen. All factors were made optimum
`for growth including the addition of thioglycolate to the
`media. The times to turbidity increases were 36 to 60 h with
`less than 0.4% 0 2 and 110 h with 0.5 to 0.7% 0 2 (Table
`
`JOURNAL OF POOD PROTECTION, VOL. 55, JANUARY 1992
`
`Nexus Ex. 1032
`Page 3 of 5
`
`

`

`26
`
`WIIITING AND NAfTULIN
`
`TABLE 3. Time for growth and toxin production of spores of C.
`botulinum with varying headspace oxygen, pH, and salt levels at
`20°c.
`
`Oxygen
`(%)
`
`pH
`
`Added
`NaCl
`(%)
`
`Ave. time
`growth
`(d)'
`
`Growth
`
`Toxin
`
`0.0
`
`0.5
`
`1.0
`
`2.0
`
`7.0
`7.0
`6.0
`6.0
`5.0
`5.0
`7.0
`7.0
`6.0
`6.0
`5.0
`5.0
`7.0
`7.0
`6.0
`6.0
`5.0
`5.0
`7.0
`7.0
`6.0
`6.0
`5.0
`5.0
`
`0.0
`3.0
`0.0
`1.5
`0.0
`3.0
`0.0
`3.0
`0.0
`1.5
`0.0
`3.0
`0.0
`3.0
`0.0
`1.5
`0.0
`3.0
`0.0
`3.0
`0.0
`1.5
`0.0
`3.0
`
`6
`8
`8
`9
`35
`b -
`4
`74
`89
`79
`
`5
`32
`34
`26
`
`76
`
`10/10'
`10/IO
`10/10
`10/10
`5/10
`0/IO
`5/5
`4/5
`1/5
`1/5
`0/5
`0/5
`3/5
`4/5
`2/5
`1/5
`0/5
`0/5
`1/10
`0/10
`0/IO
`0/10
`0/10
`0/IO
`
`10/10
`10/10
`10/10
`10/10
`5/6d
`0/4
`4/5
`3/5
`1/5
`1/3
`0/3
`0/1
`1/5
`2/4
`1/4
`1/3
`0/2
`0/2
`0/8
`0/7
`0(7
`0(7
`0(7
`0(7
`
`• Days to visible growth.
`•- No tubes showed turbidity or contained toxin within 90 d.
`• Numerator is number of positive tubes, denominator total
`number of tubes tested.
`" Not all nongrowth tubes were assayed for t0xin.
`
`4 ). The presence of toxin was confirmed by mouse bioassay
`in all runs and plate counts showed counts reached 107 to
`L08 CFU per ml. At 0.9 and 1.2% oxygen, no turbidity
`increases indicating vegetative growth were observed after
`over 325 h incubation and neither run had detectable
`amounts of toxin. However, plate counts at the end of the
`runs of 105 CFlJ per ml indicated 2-log cycles of growth
`had occurred. The absence of toxin may be from insuffi(cid:173)
`cient number of cells to produce detectable amounts of
`toxin, inability of the organism to produce toxin under
`these conditions, or inactivation of toxin by proteases,
`denaturation or other chemical reaction (9).
`Once turbidity began to increase, growth was rapid at
`all permissive oxygen levels and not consistently affected
`by the oxygen levels. After maximum growth, the pH
`declined 0.6 to 1.5 units (average final pH was 6.24) and
`the Eh declined from an average of +287 to -38 mV (pH
`corrected). This lowering of redox potential despite con(cid:173)
`tinuously flowing oxygen and mixing showed again the
`capability of C. botulinum to create a more favorable
`environment for itself. These Eh values are higher than the
`+ 144 mV in sterilized milk and tryptone medium with
`
`TABLE 4. Time to turbidity and growth rate of C. botulinum in
`a Jermemer with varying headspace oxygen levels at 35°C.
`
`Oxygen(%)
`
`T ime turbidity (h)
`
`Growth rate (Abs/h)
`
`0.005
`0.007
`0.08
`0.15
`0.3
`0.4
`0.5
`0.6
`0.7
`0.9
`1.2
`
`36
`45
`56
`53
`60
`44
`105
`106
`109
`>325
`>380
`
`0.70
`0.51
`0.50
`0.28
`0.65
`0.56
`
`0.15
`0.35
`NT•
`NT
`
`• Lag time determined from pH decline.
`• No increase in turbidity observed.
`
`lactose that permitted growth by strain 62A (15 ,16).
`Nonproteolytic type E strains grew in media with Eh values
`between+ 100 and +250 mV (14,22). Lund and colleagues
`(18,19) showed that the number of type E spores necessary
`to initiate growth must increase exponentially with increas(cid:173)
`ing Eh. Redox potential measurements in a particular com(cid:173)
`plex medium are not specifically rela.ted to oxygen concen(cid:173)
`trations because of the many redox couples involved, some
`not rapidly reversible or able to react with oxygen (10). The
`controlling factor in growth by anaerobes is the concentra(cid:173)
`tion of oxygen and oxygen radicals in the medium and the
`organism's ability to protect itself from them (15).
`In summary, this work showed tllat the critical range of
`oxygen tolerance of spores of proteolytic strains of C.
`botulinum is approximately I%. Conditions inside the fer(cid:173)
`menter of a favorable environment, high spore numbers,
`and mixing permitted significant grnwth only at 5,0.7%
`headspace oxygen. However, the number of trials was very
`limited and the maximum time allowed for growth was 14
`d. The spores in static tubes had lower numbers of spores,
`a more realistic abuse temperature of 20°C, and were
`incubated for 90 d. They grew under 1.0% oxygen in
`media having pH values of 6.0 and 7 .0. Even though the
`absolute minimum pH is generally considered to be 4.6
`(9,2/ ), this work suggested that reducing the pH to 5.0
`greatly restricts the ability of the C. botulinum to grow.
`This work also demonstrated the interaction of pH, NaCl,
`and headspace oxygen in delaying growth and toxin forma(cid:173)
`tion. Additional modeling for spore loads realistically ex(cid:173)
`pected to be present in foods intended for refrigerated
`storage is needed to confidently explo it multiple barriers to
`growth when the food is temperature abused.
`Allowing the spores to germinate and multiply before
`placing them under oxygen atmospheres in unagitated broths
`demonstrated a great ability by the organism to maintain a
`suitable environment for continuing growth and toxin pro(cid:173)
`duction. These quiesence broths could represent a solid
`food or a liquid during storage and implied that the oxygen
`surrounding a food is an unreliable barrier to C. borulinum
`growth.
`
`JOURNAL OF FOOD PROTECTION, VOL. 55, JANUARY 1992
`
`Nexus Ex. 1032
`Page 4 of 5
`
`

`

`HEADSPACE OXYGEN ON C. BOTl/LfNUM
`
`27
`
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`JOURNAL OF FOOD PROTECTION, VOL. 55. JANUARY 1992
`
`Nexus Ex. 1032
`Page 5 of 5
`
`