Food Protection with Chemicals, and by Biocontrol 311
Mode of Action
It appears that nitrite inhibits C. botulinum by interfering with iron–sulfur enzymes such as ferredoxin and thus preventing the synthesis of adenosine triphosphate (ATP) from pyruvate. The first
direct finding in this regard was that of Woods et al.,
212 who showed that the phosphoroclastic system
of C. sporogenes is inhibited by nitric oxide and later that the same occurs in C. botulinum, resulting
in the accumulation of pyruvic acid in the medium.
211
The phosphoroclastic reaction involves the breakdown of pyruvate with inorganic phosphate and
coenzyme A to yield acetyl phosphate. In the presence of adenosine diphosphate (ADP), ATP is
synthesized from acetyl phosphate with acetate as the other product. In the breakdown of pyruvate,
electrons are transferred first to ferredoxin and from ferredoxin to H
+ to form H 2 in a reaction catalyzed
by hydrogenase. Ferredoxin and hydrogenase are iron–sulfur (nonheme) proteins or enzymes.
Following the work of Woods and Wood,
211 the next most significant finding was that of Reddy
et al.,
155 who subjected extracts of nitrite–ascorbate-treated C. botulinum to electron spin resonance
and found that nitric oxide reacted with iron–sulfur complexes to form iron–nitrosyl complexes. The
presence of the latter results in the destruction of iron–sulfur enzymes such as ferredoxin.
The resistance of the lactic acid bacteria to nitrite inhibition is well known, but the basis is just
now clear: these organisms lack ferredoxin. The clostridia contain both ferredoxin and hydrogenase,
which function in electron transport in the anaerobic breakdown of pyruvate to yield ATP, H 2 , and
CO 2 . The ferredoxin in clostridia has a molecular weight of 6,000 and contains eight Fe atoms/mole
and eight-labile sulfide atoms/mole.
Although the first definitive experimental finding was reported in 1981, earlier work pointed to
iron–sulfur enzymes as the probable nitrite targets. Among the first were O’Leary and Solberg,
143
who showed that a 91% decrease occurred in the concentration of free–SH groups of soluble cellular
compounds of C. perfringens inhibited by nitrite. Two years later, Tompkin et al.
196 offered the
hypothesis that nitric oxide reacted with iron in the vegetative cells of C. botulinum, perhaps the
iron in ferredoxin. The inhibition by nitrite of active transport and electron transport was noted by
several investigators, and these effects are consistent with nitrite inhibition of nonheme enzymes such
as ferredoxin and hydrogenase.
159,217 The enhancement of inhibition in the presence of sequestering
agents may be due to the reaction of sequestrants to substrate iron: more nitrite becomes available for
nitric oxide production and reaction with microorganisms.
Summary of Nitrite Effects
When added to processed meats such as wieners, bacon, smoked fish, and canned cured meats
followed by substerilizing heat treatments, nitrite has definite antibotulinal effects. It also forms
desirable product color and enhances flavor in cured meat products. The antibotulinal effect consists
of inhibition of vegetative cell growth and the prevention of germination and growth of spores that
survive heat processing or smoking during postprocessing storage. Clostridia other than C. botulinum
are affected in a similar manner. Whereas low initial levels of nitrite are adequate for color and flavor
development, considerably higher levels are necessary for the antimicrobial effects.
When nitrite is heated in certain laboratory media, an antibotulinal factor or inhibitor is formed,
the exact identity of which is not yet known. The inhibitory factor is the Perigo effect/factor or Perigo
inhibitor. It does not form in filter-sterilized media. It develops in canned meats only when nitrite
is present during heating. The initial level of nitrite is more important to antibotulinal activity than
the residual level. Once formed, the Perigo factor is not affected greatly by pH changes. Measurable
Mode of Action
It appears that nitrite inhibits C. botulinum by interfering with iron–sulfur enzymes such as ferredoxin and thus preventing the synthesis of adenosine triphosphate (ATP) from pyruvate. The first
direct finding in this regard was that of Woods et al.,
212 who showed that the phosphoroclastic system
of C. sporogenes is inhibited by nitric oxide and later that the same occurs in C. botulinum, resulting
in the accumulation of pyruvic acid in the medium.
211
The phosphoroclastic reaction involves the breakdown of pyruvate with inorganic phosphate and
coenzyme A to yield acetyl phosphate. In the presence of adenosine diphosphate (ADP), ATP is
synthesized from acetyl phosphate with acetate as the other product. In the breakdown of pyruvate,
electrons are transferred first to ferredoxin and from ferredoxin to H
+ to form H 2 in a reaction catalyzed
by hydrogenase. Ferredoxin and hydrogenase are iron–sulfur (nonheme) proteins or enzymes.
Following the work of Woods and Wood,
211 the next most significant finding was that of Reddy
et al.,
155 who subjected extracts of nitrite–ascorbate-treated C. botulinum to electron spin resonance
and found that nitric oxide reacted with iron–sulfur complexes to form iron–nitrosyl complexes. The
presence of the latter results in the destruction of iron–sulfur enzymes such as ferredoxin.
The resistance of the lactic acid bacteria to nitrite inhibition is well known, but the basis is just
now clear: these organisms lack ferredoxin. The clostridia contain both ferredoxin and hydrogenase,
which function in electron transport in the anaerobic breakdown of pyruvate to yield ATP, H 2 , and
CO 2 . The ferredoxin in clostridia has a molecular weight of 6,000 and contains eight Fe atoms/mole
and eight-labile sulfide atoms/mole.
Although the first definitive experimental finding was reported in 1981, earlier work pointed to
iron–sulfur enzymes as the probable nitrite targets. Among the first were O’Leary and Solberg,
143
who showed that a 91% decrease occurred in the concentration of free–SH groups of soluble cellular
compounds of C. perfringens inhibited by nitrite. Two years later, Tompkin et al.
196 offered the
hypothesis that nitric oxide reacted with iron in the vegetative cells of C. botulinum, perhaps the
iron in ferredoxin. The inhibition by nitrite of active transport and electron transport was noted by
several investigators, and these effects are consistent with nitrite inhibition of nonheme enzymes such
as ferredoxin and hydrogenase.
159,217 The enhancement of inhibition in the presence of sequestering
agents may be due to the reaction of sequestrants to substrate iron: more nitrite becomes available for
nitric oxide production and reaction with microorganisms.
Summary of Nitrite Effects
When added to processed meats such as wieners, bacon, smoked fish, and canned cured meats
followed by substerilizing heat treatments, nitrite has definite antibotulinal effects. It also forms
desirable product color and enhances flavor in cured meat products. The antibotulinal effect consists
of inhibition of vegetative cell growth and the prevention of germination and growth of spores that
survive heat processing or smoking during postprocessing storage. Clostridia other than C. botulinum
are affected in a similar manner. Whereas low initial levels of nitrite are adequate for color and flavor
development, considerably higher levels are necessary for the antimicrobial effects.
When nitrite is heated in certain laboratory media, an antibotulinal factor or inhibitor is formed,
the exact identity of which is not yet known. The inhibitory factor is the Perigo effect/factor or Perigo
inhibitor. It does not form in filter-sterilized media. It develops in canned meats only when nitrite
is present during heating. The initial level of nitrite is more important to antibotulinal activity than
the residual level. Once formed, the Perigo factor is not affected greatly by pH changes. Measurable