Clostridum botulinum can grow and form toxin at pH values lower than 4.6

Nature Vol. 281
398
2M3
Thymus
Fla. 1 Competition and absorption analysis of NCPl 50 and Pl20.
Labelled cell extracts of C57BL/6
thymus and 2M3 cells (an AMuL V in vitro derived clonal
lymphoid cellline)6 were prepared
and analysed as in Fig. 1. A final
volume of 1 ml for each cell extract
of thymus (10 7 cells) or 2M3 (S x
10! cells) was used for each tube.
All tubes contained 100 j.l.g ml- 1
unlabelled Moloney MuLV virion
proteins. Lane a, normal mouse
serum (5 j.l.1). Lane b, anti-AbT
(5 j.l.1). Lanes c, d and e, anti-AbT
(5 r-l) plus unlabelled extract of 2 x
10 , 4xl0 6 or 8xl06 A-MuLV
transformed non-producer NIH/
3T3 cells. Lanes f, g, h, antiAbT (5 j.l.1) plus unlabelled extract
of 2X106 , 4X10 6 , or 8x106
Moloney sarcoma virus transformed non-producer NIH 3T3
cells respectively. Lane i, 1: 10
diluted anti-AbT (50 j.l.1) controlmock absorbed. Lane i, 1 : 10 antiAbT (50 j.l.1) pre-absorbed with
viable A-MuLV transformed nonproducer NIH 3T3 cells (2 x 108
cells per ml diluted serum-3
cycles each 45 min; O°C). Immune
precipitates were prepared and
analysed as in Fig. 1.
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results imply that we are studying different proteins remains
unclear. One possibility is that Risser et al. were detecting
NCP150 but that the synthetic rate (as monitored by immunoprecipitation) is controlled separately from expression at the cell
surface (as monitored by cytotoxicity absorption).
We thank Drs Robert Weinberg and David Housman for
Moloney sarcoma and spleen focus-forming virus infected nonproducer NIH/3T3 cells, respectively. This work was supported
by grant VC-4J from the American Cancer Society (to D.B.),
grant CA-24220 from the NCI (to N.E.R.) and grant CA-14051
from the NCI (core grant to Dr S. E. Luria). O.N.W. is a Helen
Hay Whitney postdoctoral fellow. N .E.R. is a Research Scholar
of the American Cancer Society, Massachusetts Division. D.B.
is a Research Professor of the American Cancer Society.
Received 16 May; accepted 1 Auauatl979.
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0028~836/ 79 /400398-02$OI.OO
4 October 1979
j
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••••
j
- P120
Clostridium botulinum can grow
and form toxin
at pH values lower than 4.6
G. J. M. Raatjes & J. P. P. M. Smelt
Unilever Research Vlaardingen. PO Box 114,3130 AC Vlaardingen.
The Netherlands
It is generally accepted1 that in Clostridium botulinum both
growth and toxin formation are completely inhibited at "H
values below 4.6. This critical" H value has been confirmed by
many investigators U51n! food as substrate2-$ or culture
media 3, - . Occasionally9- 1 growth of C. botulinum and toxin
formation at pH values lower than 4.6 have been reported. In
these cases the authors ascribed the unexpected outgrowth and
toxin formation to local pH differences in inhomogeneous
media and growth of C. botulinum before pH equiUbration, or
to ~he fact that fungi created microenvironment5 within or
adjacent to the mycelial mat, where the" H wu higher than 4.6
as was demonstrated by Odiaug and Pftug l l•l l• We show here
that the general assumption that C. botulinum does not grow
below pH 4.6 is Incorrect. We have observed that growth and
toxin formation by C. botulinum can take place in homogeneous
protein rich substrates (containinl 3% or more soya or mDk
protein) at"H values lower than 4.6.
© Macmillan Journals Ltd 1979
Nature Vol. 281
399
4 October 1979
Tablel Influence of pH, type of acid, and protein content on the behaviour of C. botulinum type A and B, if inoculated together with Bacillus spores
in substrates based on protein enriched soya extracts
Protein
content
Acid
HCl
HCI
HCI
HCl
Initial pH
(%w/w)
4.0
5.5
4.2
4.4
4.4
1
(1)
+-
(2)
(3)
(J
(1 )
(2)
(3 )
5.5
5.5
3.0
Citric acid
Lactic acid
Acetic acid
4.4
4.4
4.4
4.4
Incubation (weeks at 30 ·C)
6
4
8
++
++
++
++
-+
-+
-+
-+
I
I
++
++
++
++
++
++
(1)
-+
-+
-+
-+
II
I
5.5
(J
I
++
-+
-+
-+
-+
-+
(J
I
I
NT
I
(J
(J
-+
++
++
++
(J
(J
(J
(J
(J
(J
(J
(J
(1)
+-
+-
(2)
(3)
++
-+
++
++
++
++
-+
-+
++
++
++
++
(J
(J
I
II
II
I
II
II
-+
-+
(J
(J
(J
(J
(J
(J
I
(J
-+
-+
(1)
(2)
(3)
5.5
-+
-+
(2)
(3)
(1)
(2)
(3)
5.5
(J
14
12
NT
II
(1)
0.6
I
+-
(1)
(2)
(3)
I
I
II
II
I
-+
-+
(J
(J
10
-+
-+
(J
(J
(2)
(3)
HC)
2
(J
(J
(J
(J
(J
(J
(J
I
(1) Initial inoculum 100 bacilli per ml substrate. At least hundred-fold growth of Bacillus in one sample (+) or two samples (++). Less than
hundred-fold growth of Bacillus in one sample (-) or two samples (--).
(2) Initial inoculum 1,000 C. botulinum per ml substrate. At least 10-fold growth of C. botulinum in one sample (+) or two samples (++). Less than
10-fold growth of C. botulinum in one sample (-) or two samples (--).
(3) (J, Two samples tested for toxin, no sample toxic; I, 1/2 samples toxic; II, 2/2 toxic.
NT, Not tested.
Media in screwcap bottles containing different levels of soya
or milk proteins, in which the pH was adjusted with various acids
(Table 1), were inoculated with either a mixture of spores of
C. botulinum type A strain 62A, Vh and ZK3 and C. botulinum
type B strain B6 and 2345, alone or together with a mixture of
spores of Bacillus subtilis and B. licheniformis isolated from
soya concentrates. Details are given in Table 1.
The inoculated media were filled into screwcap bottles which
were subsequently heated for 5 min at 100 ·C. After cooling, the
headspace in each bottle was filled with liquid paraffin. Duplicate bottles were examined at various intervals during incubation at 30·C for growth, pH value and toxin; the presence of
toxin was tested by intraperitoneal injection of mice.
The pH value did not increase from the initial values by more
than 0.1 pH unit during incubation. Growth and toxin formation
by C. botulinum took place at a pH value as low as 4.0 in the
presence of the bacilli if the pH was adjusted with hydrochloric
acid. Growth of the bacilli removed residual oxygen and lowered
the redox potential, thus creating more favourable conditions
for the growth of C. botulinum. When we compared the
inhibitory action of hydrochloric, citric, lactic and acetic acids at
pH 4.4, growth and toxin formation decreased in that order
(Table 1).
The level of protein in the media also affected the ability to
grow at low pH values-at pH 4.4 a minimum protein level
around 3% was necessary for growth and toxin formation.
In further experiments with 5.5% soya protein J. S.
Crowther (personal communication) observed that in anaerobic
conditions spores of C. botulinum are able to outgrow and form
toxin at pH 4.2 (if hydrochloric acid was used as acidulant) in the
absence of other microorganisms. However the results at this
pH were not always repeatable.
So far we have not found C. botulinum to grow in a wide range
of actual foods with pH value lower than 4.6, but the assumption
that C. botulinum does not grow below pH 4.6 has been shown
to be incorrect, and the understanding of conditions in which
growth may occur, requires further study.
Received 23 July; accepted 10 August 1979.
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2.
3.
4.
5.
6.
7.
8.
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© 1979 Nature Publishing Group