Botulinum Strains Research Articles

Sodium hypophosphite inhibition of the growth of selected Gram-positive foodborne pathogenic bacteria

Sodium hypophosphite (SHP) was evaluated for inhibition of growth of seleceed Gram-positive food-borne pathogenic bacteria in Trypticase Soy Broth. In addition, the effects of pH and sodium chloride (NaCl) alone and in combination with (SHP) were also examined. All inhibition studies were performed with optimal or nearly optimal growth conditions for each bacterium. Growth was monitored by determining culture optical density at 600 nm, and a time to significant growth determined for each test media. Ratios of time to significant growth for each control over that in test variables were used to evaluate the effect of SHP and other variables on growth. SHP was effective in inhibiting growth of Clostridium perfringens and Clostridium botulinum strains 62A 52A and Lamanna B, but generally ineffective against Staphylococcus aureus and Bacillus cereus. Results from this investigation show that SHP has potential as a food ingredient for the inhibition of certain Gram-positive foodborne pathogens.

A simple method of detection of toxigenic Clostridium botulinum type B strains

39 spontaneous nontoxigenic (tox −) mutants were isolated from 20 toxigenic (tox +) strains of Clostridium botulinum types B, A and F. The growth of tox + strains different from that of tox − mutants on a medium supplemented with Tween 80. On VL-Tween agar, tox − mutants formed irregular, glisteing, translucent colonies whereas in tox + colonies the centers were covered by an opaque (‘hoarfrosting’) substance. In Tween-liquid medium, tox + cultures were distinguished from tox − cultures by the formation of a turbid and flocculent growth. Of four different Tween preparations tested (Tweens 20, 40, 60 and 80), only Tween 80 proved to be capable of detecting toxigenic strains. The results indicated a correlation between toxigenicity of C. botulinum and its ability to metabolize Tween 80. The metabolites form the characteristic ‘hoarfrosting’ substance on Tween agar. We recommend the use of Tween 80 for the detection of tox + C. botulinum strains in genetic studies.

Activator Protein Supporting the Botulinum ADP-ribosyltransferase Reaction

The ADP-ribosyl moiety of NAD was transferred to proteins with Mr values of 22,000 and 25,000 when bovine brain cytosol was incubated with a botulinum ADP-ribosyltransferase C3 (BT-C3) which was purified from the culture medium of a type C strain of Clostridium botulinum. Any protein fraction eluted from a chromatographic column to which the cytosol had been applied, however, was not significantly ADP-ribosylated by BT-C3, unless the reaction mixture was further supplemented with a small amount of the cytosol. Thus, substrate protein(s) could be partially purified based on their ability to be ADP-ribosylated by BT-C3 in the presence of the cytoplasmic activator(s). The rate of ADP-ribosylation of the substrates was extremely low by itself but was increased enormously and progressively when increasing amounts of cytosol were added, affording a reliable means for assay of the activator contained therein. The activator was separated from the substrate proteins and partially purified from the cytosol by sequential chromatography steps with an anion exchanger and a gel filtration column. The activity of the partially purified activator was heat-labile and protease-sensitive, suggesting that the activator was a protein or had a protein component necessary for activity. The action of the activator protein(s) was specific for BT-C3-catalyzed ADP-ribosylation; cholera toxin-catalyzed ADP-ribosylation of GTP-binding protein (Gs) was not supported by this activator. Thus, this is the first report to show that botulinum ADP-ribosyltransferase-catalyzed reaction can proceed significantly only in the presence of other protein factor(s), just as has been observed with an ADP-ribosylation factor required for cholera toxin-induced similar reaction.

Immuno-crossreactivity between botulinum neurotoxin type C1 or D and exoenzyme C3

Botulinum neurotoxin type D and exoenzyme C3 have been separately purified from Clostridium botulinum strain D-1873 to apparent homogeneity. Both ADP-ribosylated a rat liver cytosolic protein of 24 kDa. The N-terminal amino acid sequence of C3 was determined and showed a low degree of homology with those of the light and heavy chains of neurotoxins of various types which have been reported previously. However, a polyclonal antibody raised against C3 cross-reacted with the light chains, but not with the heavy chains, of type C1 and D neurotoxins. Furthermore, a monoclonal antibody recognizing the light chains of type C1 and D neurotoxins interacted with C3. These results suggest that the light chain of type C1 or D neurotoxin and exoenzyme C3 share at least one epitope in common with each other.

Binding of Clostridium botulinum type C neurotoxin to different neuroblastoma cell lines

Binding of type C neurotoxin (C1 toxin) from Clostridium botulinum (strain Stockholm) to neuroblastoma cell lines was studied by using biotinylated anti-toxin antibody and avidin-biotinylated peroxidase complex. The neurotoxin bound with high efficiency to mouse neuroblastoma (NS-20Y and NIE-115) cells and to hybridomas of rat glioblastoma and mouse neuroblastoma (NG108-C15) cells. The toxin bound little to human neuroblastoma, rat astrocytoma, and nonneural cell lines. Binding of the neurotoxin to NG108-C15 cells was inhibited by gangliosides (GT1b and GM1) and by monoclonal antibodies (CA-12 and C-9), although inhibition was not complete. Sequential preincubation of C1 toxin with GT1b and CA-12 caused complete inhibition. A Scatchard plot of binding of 125I-labeled C1 toxin to NG108-C15 cells showed a hyperbolic curve. Monoclonal antibody CA-12 but not C-9 neutralized the lethal activity of the toxin toward mice. Only C-9 clearly inhibited toxin binding to GT1b. These results suggest that NG108-C15 cells have at least two kinds of receptors for C1 toxin. From the results of binding tests with neuraminidase-, pronase-, and trypsin-treated NG108-C15 cells, the chemical nature of the high-affinity site was presumed to be a glycoprotein containing sialic acid. GT1b may have an important role in low-affinity sites.

Botulinum neurotoxin type A: Cleavage of the heavy chain into two halves and their partial sequences

The 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall). The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond. The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT ((1987) FEBS Lett. 226, 115–120). Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies. Two different approaches were taken to cut the H chain with trypsin and isolate the fragments. In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment. The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography. The 1–27 residues of 49-kDa N-terminal half of the H chain were AlaLeuAsnAspLeuCysIleLysValAsnAsnTrpAsp LeuPhePheSerProSerGluAspAsnPheThr AsnAspLeu. The sequence of the other half of the H chain (44 kDa) was X-IleIleAsnLeuXIleLeuAsnLeuArgTyrGluX AsnHisLeuIleAspLeuLysX-TyrAlaSer. In the second method, the H chain was first separated from the L chain, purified, and then cleaved. One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method. The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain, (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain, (iii) For structure-function studies chromatographic isolation of the following four different fragments of the NT under nondenaturing condition is feasible: L chain (53 kDa), N-terminal half of the H chain (49 kDa), C-terminal half of the H chain (44 kDa), and the NT minus the C-terminal half (i.e., L chain linked to the N-terminal half of the H chain via -S-S- bond, 94 kDa). The sequences of 48 residues of the H chain 27 at the N-terminal end and 21 at about the midpoint of the H chain are now known. Certain homologies in the amino acid sequence were found between these two regions of type A botulinum NT and the corresponding regions of tetanus toxin (produced by Clostridium tetani).

Inhibition of Growth ofClostridium botulinumby Intestinal Microflora Isolated from Healthy Infants

Infant botulism results when Clostridium botulinum spores germinate, colonise, and produce botulinum neurotoxin in the intestine. As the normal intestinal microflora is important in resistance to C. botulinum colonisation in the mouse model of infant botulism, we investigated the effect of intestinal bacteria isolated from six healthy infants (two breastfed, two formula-fed, and two both formula and breast-fed) on the growth of 14 strains of C. botulinum (three Type A, four Type B, three Type E, two Type F, and two Type G) using zone inhibition procedures. We tested 88 anaerobic and 64 aerobic and facultative isolates. Strains of bifidobacteria, lactobacilli, propionibacteria and enterococci inhibited the growth of C. botulinum , as did several strains of the Bacteroides fragilis group. The only clostridium inhibiting C. botulinum growth was C. ramosum , which inhibited both of the Type G strains and one of the two Type F strains. At least two bacterial isolates from each faecal sample inhibited most of the 14 C. botulinum strains. Co-culture experiments with intact faecal samples and C. botulinum spores or vegetative cells indicated that the normal flora of healthy infants had a bacteriostatic, rather than a bacteriocidal, effect on the growth of C. botulinum . Thus, susceptibility to infant botulism may result in part from the absence of these inhibitory organisms from the normal flora of the infant intestine. Keywords: Infant botulism; Colonisation resistance; Bacterial antagonism; Clostridium botulinum .

The heat resistance of spores of Clostridium botulinum 213B over the temperature range 120 to 140°C

SummaryThe heat resistance of spores of Clostridium botulinum strain 213B heated in phosphate buffer (pH 7.0) was determined over the temperature range 120 to 140°C using a computer controlled thermoresistometer. The predicted D121°C value of approximately 0.13 min and z value of 11.0°C (obtained by linear regression) was close to the classical D121°C of 0.2 min and z value of 10°C used in process calculations. These data indicated that, according to the heat resistance of this culture, the z value of C. botulinum can be considered constant over the whole range 120 to 140°C. This also provided support for the recommended classical process calculations, using a z value of 10.0°C, being adequate if extrapolated to 140°C.

Zinc stimulates sporulation inClostridium botulinum 113B

The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to ≥98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.

Factors affecting the toxicity of rotting carcasses containing Clostridium botulinum type E.

Mice killed shortly after receiving c. 2000 spores of a type E strain of Clostridium botulinum per os were incubated at one of five chosen temperatures together with bottles of cooked meat medium seeded with a similar inoculum. After incubation the rotting carcasses were homogenized. Sterile membrane filtrates of the homogenates (10%, w/v) and pure cultures were then titrated for toxicity. Some of the main findings were confirmed with two further type E strains. Toxicity produced at 37 degrees C was poor in both carcasses and cultures (200-20,000 mouse intraperitoneal LD/g or ml). It was good in both systems at 30 and 23 degrees C, usually reaching 20,000-200,000 LD/g or ml, and in carcasses occasionally more; at 30 degrees C maximal toxicity was reached more quickly in carcasses than in cultures. Prolonged incubation (36-118 days) at 30 or 23 degrees C resulted in complete loss of toxicity in virtually all carcasses but not in cultures. At 16 degrees C the development of toxicity in carcasses was strikingly greater than in cultures. At 9 degrees C neither system produced more than slight toxicity after prolonged incubation. Trypsinization increased the toxicity of cultures but not usually of carcasses. Unfiltered carcass homogenate (10%, w/v) with maximal intraperitoneal toxicity was harmless for mice by mouth in doses of 0.25 ml. These findings differed in important respects from those made earlier with a type C strain.

Botulinum neurotoxin type B (strain 657): partial sequence and similarity with tetanus toxin

The type B neurotoxin (NT) isolated from Clostridium botulinum (strain 657) behaved as a mixture of single (unnicked) and dichain (nicked) proteins, both of M r ∼ 150 kDa. When the dichain NT was reduced by mercaptoethanol, the two chains migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as separate polypeptides of M r ∼ 100 and 50 kDa that appeared similar to the heavy and light chains of other serotypes of botulinum NT. The N-terminal amino acid sequences of the two chains were determined. They were as follows: light chain: Pro-Val-Thr-Ile- Asn-- Asn - Phe - Asn - Tyr - Asn - Asp - Pro - Ile - Asp - Asn - Asn - Asn - Ile - Ile - Met - Met - Glu - Pro - Pro - Phe - Ala - Arg - Gly - Met - Gly - Arg - Tyr - Tyr - Lys - Ala - Phe - Lys - Ile - Thr - Asp - Arg - Ile - Trp - Ile -; and heavy chain: Ala - Pro - Gly - Ile - X - Ile - Asp - Val - Asp - Asn - Glu - Asp - Leu - Phe - Phe - Ile - Ala - Asp - Lys - Asn - Ser - Phe - Arg - Asp - Asp - Leu -. These two sequences matched exactly with those of the light and heavy chains of type B NT (strain Okra) of which only 16 and 18 residues were known ( J. Biol. Chem. (1985) 260, 10461). The above sequences were different from those of type A NT. Immunoprecipitation reactions of type B NT isolated from strains 657 and Okra were indistinguishable against polyclonal anti-type B NT serum. These two preparations did not produce precipitin reactions with polyclonal anti-type A NT serum. The tetanus NT (from C. tetani) resembles botulinum NT in relative molecular mass, dichain structure and probably in the mode of action; but the two NTs differ in their sites of action and their serological specificity. Comparison of amino acid sequences of botulinum NT, reported here, and tetanus NT ( EMBO J. (1986) 5, 2495) shows that their two heavy chains are less homologous (13 out of 26 residues matched in position) than the two light chains (31 out of 44 residues matched). Three stretches of residues: 3–7, 18–24 and 32–44, on the light chains of the two NTs are identical.

Proteins as potential bacterial growth inhibitors in foods: Suppression of the activity of water by proteins as determined by NMR relaxation methods

Food microbiologists have long known that suppression of the activity of water,aw, can retard microbial growth in food systems. Traditionally,aw, suppression has been achieved by addition of salts or humectants to foods. To limit the amount of preservatives added to food products, studies were initiated to assess the feasibility of using proteins to suppressaw to a practical value for retarding bacterial growth and to determine the optimum environmental condition for maximizing this effect for milk proteins. New expressions were developed relating observed longitudinal and transverse NMR relaxation rates, in the absence of cross-relaxation, to protein hydration\(\bar \upsilon _w \), to the protein activity coefficient, γp, and to the correlation time of the bound water, τc. From γp, the second virial coefficient of the protein,Bo, can be found. By use of\(\bar \upsilon _w \) andBo,aw could then be directly evaluated at any protein concentration. Resulting expressions were tested by2H-NMR relaxation measurements made as a function of protein concentration, for: β-lactoglobulin A (the major whey protein) under nonassociating (pH 6.0) and associating (pH 4.65) conditions; and for casein (the major milk protein) in the micellar (with added Ca2+) and submicellar (without Ca2+) forms. Values ofaw calculated from these2H-NMR data show that casein, at all the concentrations and temperatures examined, suppressesaw more than does β-lactoglobulin A because of a largerBo. In turn, micellar casein suppressesaw to a larger extent than does submicellar casein because of a larger\(\bar \upsilon _w \). Extrapolation ofaw at 4°C to a concentration ten times that in normal milk yields a value, ofaw of less than 0.95, at whichSalmonella and some strains ofClostridium botulinum no longer grow. These results are in agreement with what is known about storageability of condensed milk. Generalizations regarding the types of proteins and cosolutes to be used for suppressingaw will be discussed. Structural information on these proteins calculated from τc will also be presented.

Packaging of Scallops with Sorbate: An Assessment of the Hazard from Clostridium botulinum

ABSTRACTToxin production in shucked scallops by a mixture of 10 strains of Clostridium botulinum (types A, B, E, F) was studied using mouse bioassay. Scallops, with or without 0.1% potassium sorbate, were packaged in air‐permeable film or vacuum packaged in impermeable film. Two trials were done in which packages were inoculated with different spore levels and held at 4°, 10° and 27°C. Bacterial counts were made on various selective media. Only type A toxin was observed and this only in evacuated packages held at 27°C. Toxin developed more rapidly in the presence of sorbate. All packages in which toxin was recorded had offensive odors and were visibly spoiled. The potential hazard from botulism is discussed.

Evidence that subunits of type A botulinum toxin need not be linked by disufide

Type A neurotoxin of Clostridium botulinum strain 62A was purified by a modification of the procedure of Tse et al (1982). Electrophoresis in sodium dodecyl sulfate - polyacrylamide gels (SDS - PAGE) indicated the mol. wt of the intact dichain molecule is 140,000 and that of its L subunit is 52,000, both expected from published values. However the mol. wt of 83,000 for the H subunit was lower than the mol. wt of 97,000 in the literature. The purified toxin separated in SDS - PAGE into H and L subunits when pretreated with 2-mercaptoethanol but it unexpectedly behaved similarly without the pretreatment. Specific toxicity (∼3 × 10 8 mouse ld 50/mg protein) was not affected by the spontaneous molecular change that made dissociation into subunits possible. The subunits of dichain botulinum toxins are believed to be covalently joined by intersubunit disulfide(s) since they have been demonstrated only when samples are treated with 2-mercaptoethanol or dithiothreitol. Since it is not always needed, the pretreatment is apparently not reducing a disulfide that connects the subunits. The strong chelating activity also possessed by the pretreating agents suggest that the subunits may be joined by a metallic divalent cation.

PLASMIDS OF CLOSTRIDIUM BOTULINUM TYPE A, B, E, F, AND OTHER CLOSTRIDIAL SPECIES

ABSTRACTPlasmids from 46 strains of Clostridium botulinum types A, B, E, and F were isolated using a modification of the Minton and Morris procedure (1981). When these strains were examined, 54% contained one or more plasmids ranging in molecular weight from ∼ 69 to 3.4 megadaltons (MDa). Attempted curing of plasmids for two C. botulinum type A strains, 73A and Hall, using acridine orange and elevated growth temperatures (42 to 45°C) produced no changes in plasmid profiles or toxicity as determined by agarose gel electrophoresis and immunodiffusion tests, respectively. However, exposure of type E C. botulinum strain BL764 to 1000 Krad gamma irradiation resulted in loss of one plasmid (8.4 MDa) but toxicity was retained.

Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease.

Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes.

The effect of acid pH on the probability of growth of proteolytic strains of Clostridium botulinum

In low-acid foods, where the pH is not sufficiently acidic to prevent growth of Clostridium botulinum, sub-optimal pH may nevertheless contribute to preservative systems based on combinations of inhibitory factors. The effect of pH in the range 4.5–7.0 on the probability, P, of growth of single vegetative bacteria of proteolytic strains of C. botulinum at 30°C has been determined by comparison of the most probable number (MPN) count of the bacteria in nutrient medium adjusted to the required pH with HCl, and in the basal medium at pH 6.8–7.0. The medium was maintained in strictly anaerobic conditions at a redox potential equivalent to lower than −300 mV at pH 7. At pH values between 5.0 and 7.0 log 10 P for growth of strain ZK3 (type A) in 3 days was between 0 and −1. Between pH 4.6 and pH 4.9 growth was delayed and incubation for up to 14 days was required; log 10 P decreased sharply with decrease in pH to a value of −5.6 or lower at pH 4.60 for growth within 14 days. For an inoculum containing a mixture of 4 additional type A strains and 5 type B strains log 10 P for growth within 14 days at pH 4.66 was −5.6. A mathematical model was developed that related the effect of acid pH and incubation time to the probability of growth of strain ZK3 in the above conditions.

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid.

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)

PROBABILITY OF GROWTH OF CLOSTRIDIUM BOTULINUM AS AFFECTED BY STRAIN, CELL AND SEROLOGIC TYPE, INOCULUM SIZE AND TEMPERATURE AND TIME OF INCUBATION IN A MODEL BROTH SYSTEM

ABSTRACTUsing factorial design experiments and MPN methodology, we evaluated the probability (P) of growth initiation of 17 individual proteolytic (A,B,F) and non‐proteolytic (B,E,F) C. botulinum strains (spores and cells) in a model broth as it is affected by strain, cell and serologic type, inoculum size (100–106), temperature (4–47°C) and time of incubation (up to 28 days). Regression analyses of the P of growth of the most capable strains for all conditions tested, allowed the development of quantitative equations relating P of growth initiation by one spore or cell to the variables studied. The close agreement between observed and predicted Ps demonstrated the potential usefullness of the modeling approach in studying microbial interactions with food environments.

An Evaluation of Antibotulinal Activity in Nitrite-Free Curing Systems Containing Dinitrosyl Ferrohemochrome

The ability of the pigment dinitrosyl ferrohemochrome to mimic the cured meat color function attributed to nitrite, was evaluated in a number of nitrite-free, model meat systems. In addition, compounds with reported antibotulinal properties were compared to the antibotulinal effect of nitrite. Fifteen treatments were evaluated and compared to 50 and 150 ppm nitrite. Two processing conditions (short and extended heating) were also compared for their ability to enhance pigment color and eliminate the natural meat microbial population. Meat slurries varying in cure composition were inoculated with a composite of six different strains of Clostridium botulinum, types A and B. After processing, the packages were incubated at 10 and 27°C, and were analyzed for toxin. The treatment containing 3000 ppm sodium hypophosphite most closely resembled the 150 ppm nitrite control in its ability to prevent spore outgrowth and toxin production. The treatment containing 1250 ppm monomethyl fumerate also scored better than the other treatments including ethylene diamine tetraacetic acid (EDTA), potassium sorbate and tertiary butyl hydroquinome (TBHQ), but was slightly less inhibitory than sodium hypophosphite. The longer heat treatment eliminated all the natural meat flora (lactic acid bacteria) and enhanced the color production of the pigment.