Growth Of Clostridium Botulinum Research Articles

Quantitative Evaluation of Clostridium botulinum Nonproteolytic Types B, E, and F Growth Risk in Fresh Salmon Tissue Homogenates Stored under Modified Atmospheres

The potential risk of C. botulinum growth in fresh fish stored under modified atmospheres (MA) remains unclear, as few qualitative studies have identified certain conditions leading to toxigenesis. This is the second paper of a series attempting to quantify the effect of selected parameters on the probability (P) of toxigenesis by one spore in fish. The factorially designed experiments included fresh salmon tissue homogenate with 3 levels of initial microbial flora (IMF) inoculated with a pool of spores of 13 nonproteolytic type B, E and F strains at 7 levels (10−2–104/3-g sample) and incubated at 1, 4, 8, 12, 16, and 30°C under 3 MA (vacuum, 100% CO2, 70% CO2 + 30% air) for up to 60 d. The earliest we observed toxicity at 30, 16, 12, 8 and 4°C irrespective of MA were 1, 2, 6, 9-12 and 15–>60 days and required 10°, 103–104, 102–103, 101–104 and 104 spores/sample, respectively. The probability of toxigenesis was affected significantly (P<0.05) by IMF, MA, storage temperature (T), storage time (ST) and the interactions T × ST, MA ×T, MA × ST, and IMF × T. Only type B toxin was detected in toxic samples. Using linear and logistic regression models, equations were derived which could estimate the length of the lag phase and the P of toxigenesis by one spore under a particular storage condition.

Water Relations of Foodborne Bacterial Pathogens - An Updated Review

The effects of aw limitation on growth and metabolic activities of foodborne bacterial pathogens continue to be actively investigated in laboratories throughout the world. Perhaps the most intensive work over the past 10 years has centered on growth of Clostridium botulinum in multicomponent systems. This emphasis undoubtedly has been the result of concern about the role played by sodium nitrate in formation of nitrosamines and the possibility of a prohibition of the addition of this preservative to foods. While investigations have continued on C. botulinum and more “traditional” foodborne pathogens, a “new” group of pathogens, some of them opportunistic, has emerged. Several of these organisms are covered in this review whereas others are not for the simple reason that the water requirements of these organisms have not as yet been investigated. Particularly surprising is the lack of aw-related information on Listeria monocytogenes, Aeromonas hydrophila and colohemmorrhagic Escherichia coli. In fact, the water requirements of gram-negative bacteria in general and the Enterobacteriacae in particular seem to have been somewhat neglected. Researchers intending to do aw-related research should consider trends in the American diet and in commercial food processors that supply much of it. For example, no one can deny that consumption of fish and seafood products has increased in the diets of many Americans yet potential pathogens indigenous to these products have received little investigative work in terms of their water requirements. Scombroid poisoning, a form of histamine poisoning, may be caused by several species of gram-negative bacteria, yet we know nothing of the effect of aw on these organisms, their heat resistance, combinations with modified atmospheres, pH, preservatives, etc. Similarly, limited application of gamma irradiation for sterilization of spices has been approved by the FDA, however, the effect of aw in these irradiated systems is largely unknown. Certainly greater thermal resistance at low aw levels has been reported; however, it is surprising that investigators have not searched for a similar effect with irradiation. Despite these shortcomings, a sizeable body of literature and knowledge of aw and its effects on microorganisms has emerged. Some of the research has only begun to exploit the hard-won knowledge of how microorganisms adapt to, and cope with, environments of low aw. Based on extrapolations of these efforts and our greater awareness of these physiological facts, one can only predict even greater advancements during the next decade.

Probability of growth and toxin production by nonproteolytic Clostridium botulinum in rockfish stored under modified atmospheres

The potential risk of C. botulinum growth in fresh fish stored under modified atmospheres remains unclear. Few studies have identified qualitatively certain conditions leading to toxigenesis. This paper is the First of a series attempting to quantify the effect of a variety of parameters on the probability ( P) of toxigenesis by one spore in fish. The factorial design experiments included red snapper tissue homogenate inoculated with a pool of nonproteolytic spores (5 type E, 4 type B and 4 type F strains) at 7 levels (10 4−10 −2 per 3 g sample) and incubated at 4, 8, 12, 17 and 30°C under 3 modified atmospheres (vacuum, 100% CO 2 and 70% CO 2+30% air) for up to 21 days. At the 10 0 spore/sample level the earliest time to detect toxin production at 4, 8, 12, 17 and 30°C under all modified atmospheres was > 21, 12, 9, 6 and 2 days, respectively. At the 10 1 spores/sample level the earliest times for the same temperatures were > 21, 9, 6, 3–6 and 1–2 days, respectively. The probability of toxigenesis was affected significantly ( P < 0.005) by temperature, storage time, atmosphere×temperature, and temperature×time but not by atmosphere ( P > 0.1). Using linear and logistic regression models, equations were derived which can predict the P of 1 spore initiating growth and toxigenesis by a particular day and at a particular temperature of storage. Studies involving other fish substrates are in progress.

Kinetics of growth and toxigenicity of Clostridium botulinum in experimental wound botulism.

An animal model of wound botulism was developed in mice using an inoculum of Clostridium botulinum type A spores. The number of C. botulinum in infected wounds was quantitated by culturing on egg yolk agar, and the level of C. botulinum toxin in infected wound tissue was measured by a bioassay in mice and by an enzyme-linked immunosorbent assay. All infected mice receiving no further treatment developed neuroparalytic symptoms consistent with botulism after an incubation period of ca. 48 h, and all of these animals died. Serotherapy with C. botulinum type A antitoxin initiated 24 h postchallenge reduced the mortality rate to 5%. Treatment with metronidazole 2 to 24 h postchallenge resulted in recovery rates of 40 to 91%.

Inhibition of Clostridium botulinurn Type E in Model Acidified Food Systems

ABSTRACTThe potential for growth and toxigenesis of a mixture of three strains of C. botulinum type E in model food systems at pH levels of 4.600 and 4.200 was investigated. Whole shrimp, shrimp puree, tomato puree, and tomato and shrimp puree were acidified to pH levels of 4.200 and 4.600 with acetic or citric acid. Inoculated and uninoculated control samples were tested during 8 wk incubation at 26°C. No significant C. botulinum growth or toxigenesis was detected in the food systems over the 8 wk test period and the pH of the various foods remained generally constant. This investigation affirms the safety of the generally accepted inhibitory pH level of 4.6 in the food systems studied.

Acid Equilibrium Development in Mushrooms, Pearl Onions and Cherry Peppers

Mushrooms, pearl onions, and cherry peppers were packed with either citric acid or acetic acid to determine the time required for the pH inside the particulates to decrease to 4.8 or less. The blanched products were packed into home canning jars, covered with acidified brine, and pasteurized in steam at 100°C. After storage at 25°C, pH measurements were taken on samples of drained brine, individual particulates, and a puree of all particulates. With acetic acid, equilibrium pH was achieved in approximately 1, 15, and 30 d for mushrooms, onions, and peppers, respectively. Longer times would be required to achieve equilibrium pH for each product with citric acid. The time required for the pH inside the particulates to decrease to 4.8 or less was a function of acid type, initial acid concentration, and the product being acidified. With sufficient acidulant to achieve an equilibrium pH of 4.6 or less, the time required for particulates of mushrooms, onions, or peppers to decrease to pH 4.8 or less was 7 d or less in all instances. Since studies (inoculated packs) of similar products have shown that a minimum of about 10 d is required for Clostridium botulinum growth at a pH of 5.0, the rate of acidification for these products was sufficient to prevent growth of C. botulinum.

Inhibitory Effects of Various Salts and/or Ionic Strengths on Growth from Clostridium botulinum 52A Spores or Vegetative Cells

Growth response from spores and vegetative cells of Clostridium botulinum strain 52A in peptone-yeast extract-glucose (PYEG) broth at two pH levels (5.55 or 5.85) containing sodium acid pyrophosphate (SAPP) (0, 0.2, 0.4%), NaCl (0, 1.25, 2.50%) and/or potassium sorbate (KS) (0, 0.13, 0.26%) was measured as the mean A630 nm of 20 tubes at 37°C. Additional treatments contained KC1 and MgCl2 (0, 1.25, 2.50%) without SAPP or KS. Growth ratios (GR = treatment/control) based on time to reach A630 = 0.35 were calculated to compare effects of additives on strain 52A. Growth from spores was affected significantly (p≤0.01) by pH level. KS and KS/pH interactions were also significant factors in growth from both spores and vegetative cells; SAPP/pH interactions were significant for cell growth, only. Combinations of SAPP (0.2, 0.4%) NaCl (0%) and KS (0.26%) were the most favorable treatments for delaying growth from spores or vegetative cells. NaCl (1.25, 2.50%) decreased antibotulinal effects produced by combinations of SAPP and KS. Elimination of NaCl enhanced antibotulinal effects. Formulations containing KC1 or MgCl2 (without SAPP and KS) at the same molarity as the NaCl in earlier treatments (0.21, 0.43) resulted in inhibition of growth from vegetative cells greater than growth from spores in the presence of MgCl2 at M = 0.43 (ionic strength = 1.29). This inhibition was more evident at pH 5.55 than pH 5.85. This study in a model system suggests ionic strength and/or chloride salt may be important considerations when manipulating formulations of additives designed to control C. botulinum growth.

Effect of Polyphosphates in Combination with Nitrite-Sorbate or Sorbate on Clostridium botulinum Growth and Toxin Production in Chicken Frankfurter Emulsions

Sodium nitrite, sorbic acid, potassium sorbate and polyphosphates (sodium acid pyrophosphate, SAPP; sodium hexametaphosphate, SHMP; and sodium tripolyphosphate, STPP) were tested at similar preadjusted (before cooking) pH levels (in the range of pH 5.78 to 6.19 after cooking) to determine effective combinations capable of controlling Clostridium botulinum growth and toxin production in mechanically deboned chicken meat frankfurter emulsions incubated at 27°C. In combination with low levels of nitrite (40 ppm), potassium sorbate (0.26%, pH 6.06) was more effective than sorbic acid (0.20%, pH 6.03) in delaying toxin production (>27 d vs. 6 d) and in controlling growth. In formulations containing combinations of nitrite (40 ppm) and sorbic acid (0.20%) or nitrite (40 ppm) and potassium sorbate (0.26%), the addition of polyphosphates (0.4%) resulted in a greater delay of toxin production (8 to 25 d for nitrite-sorbic acid-SAPP vs. 28 d for nitrite-potassium sorbate-SAPP) at similar pH levels. Under these conditions, SAPP delayed production of detectable toxin longer (25 d) than did either SHMP (6 to 11 d) or STPP (4 to 14 d). The addition of polyphosphates to nitrite-free emulsions containing sorbic acid (0.20%) or potassium sorbate (0.26%) did not delay the development of botulinal toxin when the pH was essentially equivalent in the range of 5.78 to 6.07.

Effect of Formulation on Water Activity and Sensory Attributes of Egg Sausage

The effect was investigated of sodium chloride (1.5, 3.0, 4.5%), hydrolyzed plant protein (2.0, 3.0, 4.0%), and whey (2.0, 3.0, 4.0%) on the water activity of egg sausage. Egg sausage, similar to breakfast sausage, contains approximately 44% whole egg, 24% pork fat and 18% mechanically deboned spent fowl meal. Water activity was determined using an electric hygrometer. Both sodium chloride and hydrolyzed plant protein significantly lowered water activity whereas whey did not. Mean water activity values ranged from .855 to .937. A sensory evaluation was performed in order to determine the highest level of each solute that could be used without impairing the taste and texture of the product. The combination of test variables most preferred was 1.5% sodium chloride, 3.0% hydrolyzed plant protein, and 2.0% whey and had a water activity of .925. This water activity is below that required for growth of Clostridium botulinum.

Effect of Sodium Acid Pyrophosphate in Combination with Sodium Nitrite or Sodium Nitrite/Potassium Sorbate on Clostridium botulinum Growth and Toxin Production in Beef/Pork Frankfurter Emulsions

ABSTRACTCombinations of sodium acid pyrophosphate (SAPP) with added sodium nitrite and/or potassium sorbate were tested at various pH levels to determine effectiveness in delaying Clostridium botulinum growth and toxin production in frankfurter emulsions. Formulations containing sodium nitrite (40 ppm), potassium sorbate (0.26%) and SAPP (0.4%) resulted in a greater delay of toxin production (12–18 days) than other combinations (6–12 days) having similar pH values. Treatments containing 0.4% SAPP appeared to be more inhibitory than their counterparts without SAPP, displaying less numbers of toxic samples during the 53‐day storage period at 27°C. Aerobic mesophilic colony counts and residual nitrite data showed little difference among treatments.

Nitrite, nitrite alternatives, and the control of Clostridium botulinum in cured meats.

Historically, nitrite has been a component of meat-curing additives for several centuries. In recent years the safety of nitrite as an additive in cured meats has been questioned mainly because of the possible formation of carcinogenic nitrosamines. Nitrite has many important functions in meat curing including its role in color development, flavor, antioxidant properties, and antimicrobial activity. The inhibition of Clostridium botulinum growth and toxin production is an especially important antimicrobial property of nitrite. This review discusses the effects of processing, curing ingredients (especially nitrite), and storage of cured meats in relation to the control of C. botulinum. If nitrite is eliminated from cured meats or the level of usage decreased, then alternatives for the antibotulinal function of nitrite need to be considered. Several potential alternatives including sorbates, parabens, and biological acidulants are discussed.

Inhibition of Clostridium botulinum by antioxidants, phenols, and related compounds

A total of 75 compounds, including antioxidants, preservatives, gallic acid and p-hydroxybenzoic acid esters, hydroquinones, hydroxyquinolines, phenol derivatives, and related compounds, were screened for their antibotulinal activity in prereduced Thiotone-yeast extract-glucose broth. The most effective inhibitors of Clostridium botulinum growth and toxin production were long-chain esters of p-hydroxybenzoic acid and gallic acid, antioxidants, and butylphenol derivatives. The antioxidant nordihydroguaiaretic acid at 100 microgram/ml delayed the growth and toxin production for the entire incubation period (7 days). Other antioxidants, such as butylated hydroxytoluene, butylated hydroxyanisole, and tert-butylhydroquinone were also very effective (at 200 to 400 microgram/ml) for the inhibition of C. botulinum growth and toxin production. Toxin was detected, although no detectable growth was found by daily absorbance measurements, in the prereduced medium containing 50 to 400 microgram of 8-hydroxyquinoline, pentylphenol, tert-pentylphenol, 3,5-ditert-butylphenol, 3,5-ditert-butylcatechol, (2-hydroxydiphenyl)methane, or (4-hydroxydiphenyl)methane per ml.

Effect of temperature on spore germination and vegetative cell growth of Clostridium botulinum.

Spore germination and vegetative growth of Clostridium botulinum type E strain VH at 2 to 50 degrees C were studied. At all of these temperatures, germination began immediately after the addition of the spores to the germination medium. Microscopic observations during germination revealed three types of spores: phase bright (ungerminated), phase variable (partially germinated), and phase dark (fully germinated). At all temperatures except 50 degrees C, there was a pronounced lag between the initial appearance of phase-variable spores and their eventual conversion to phase-dark spores. The number of partially germinated spores increased steadily, reaching 40 to 60% by 18 to 21 h of incubation. During this time, phase-dark, fully germinated spores developed slowly and did not exceed 28% in any of the samples. At 18 to 26 h of incubation, the rate of full germination increased abruptly four-fold. There was extensive and relatively rapid germination at 2 degrees C, the lowest temperature tested, yielding about 60% phase-variable spores by 18 h, which became phase-dark by 26 h of incubation. The optimum temperature for partial and full germination was consistently 9 degrees C. Germination at 50 degrees C was exceptionally rapid and was completed within 1 to 2 h, although 40% remained phase bright. Vegetative cells showed detectable growth at 6 to 41 degrees C, with a distinct optimum at 32.5 degrees C. No growth occurred at 50 degrees C, and only marginal growth was observed at 6 to 14 degrees C. The psychrophilic nature of the germination process coupled with the cold tolerance of vegetative growth appears to give C. botulinum type E an advantage in cold climates as well as in cold-stored foods.

Biochemical Basis for Nitrite-Inhibition of Clostridium botulinum in Cured Meat

The biochemistry of the anaerobic spore-forming clostridia and the chemistry of the nitrite reactivity during meat curing are reviewed. Direct and indirect mechanisms for nitrite inhibition of Clostridium botulinum growth and toxin formation are presented. The “Perigo effect” produced by heating nitrite in laboratory media may be produced by artifactual oxidants, and is not identical to that produced in cured meats. Inhibition of C. botulinum by nitrite in cured meats is most likely due to several interacting mechanisms: (a) reaction and oxidation of cellular biochemicals within the spores and vegetative cells; (b) restriction of use of iron (or other essential metal ions) through inhibition of solubilization, transport, or assimilation, thus interfering with metabolism and repair mechanisms and (c) cell surface membrane activity limiting substrate transport by the outgrowing cell.

PH, Acidity, and Vitamin C Content of Fresh and Canned Homegrown Washington Tomatoes

The safety of home canned tomato products in the Pacific Northwest has been questioned because of publicity surrounding low-acid tomato varieties recommended for gardens. One hundred fifty-two samples of home grown tomatoes collected from 19 counties in Washington were subjectively ranked into maturity categories by appearance. They were found to decrease in acidity and increase in pH with maturity, while the vitamin C content increased to a maximum in slightly overripe tomatoes. Ripe tomatoes grown in eastern Washington under low relative humidity and high temperatures may have greater pH values than tomatoes grown under high relative humidity and lower temperatures in western Washington. Ninety-one of the fresh tomato samples were canned with salt in a boiling water bath and stored 4 months at 4 C. The pH of the canned tomatoes decreased by 0.10 unit from the fresh values, while acidity did not change; vitamin C decreased by 30%. Overripe tomatoes had the lowest acidity and greatest pH. Thirty-one jars of tomatoes canned by home canners were collected and analyzed. The pH was sufficient in all samples to inhibit growth of Clostridium botulinum; however, two jars canned with no acidulant added had a pH> 4.6. Vacuum in jars ranged from 10.6 to 22.9 inches of mercury. All jars submitted were adequate for the home preservation of tomatoes.

INFLUENCE OF pH ON Clostridium botulinum CONTROL BY SODIUM NITRITE AND SORBIC ACID IN CHICKEN EMULSIONS

ABSTRACTThe influence of hydrogen‐ion concentration on the effectiveness of sodium nitrite and/or sorbic acid to control Clostridium botulinum growth during elevated temperature abuse (27°C) of mechanically deboned chicken meat frankfurter‐type emulsions was examined. Toxin production, spore germination (loss of heat resistance) and outgrowth, residual nitrite levels, and total microbial growth (aerobic CFU) were determined at specified times during the incubation period. The effect of sorbic acid (0.2%), alone or in combination with nitrite (40, 156 μg/g), in significantly (p &lt; 0.05) inhibiting spore germination, growth, and toxin production was pH dependent. This effect was not observed at pH values above 6.20 and it increased with decreasing pH. Inclusion of nitrite in the formulation increased the effective pH for sorbic acid inhibition of toxin production. The rapid rate of germination and outgrowth in both control and nitrite‐containing treatments was not influenced by pH in the range examined (5.93‐6.93). When sorbic acid was included in the formulation, there was a slower nitrite depletion during storage. This effect was also pH dependent and it was not observed at higher pH values (7.15). Total microbial growth was affected by decreasing pH, but not by the inclusion of nitrite and/or sorbic acid in the formulation.

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

It is generally accepted that in Clostridium botulinum both growth and toxin formation are completely inhibited at pH values below 4.6. This critical pH value has been confirmed by many investigators using food as substrate or culture media. Occasionally 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 equilibration, or to the fact that fungi created microenvironments within or adjacent to the mycelial mat, where the pH was higher than 4.6 as was demonstrated by Odlaug and Pflug. 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 (containing 3% or more soya or milk protein) at pH values lower than 4.6.

Botulism Control by Nitrite and Sorbate in Cured Meats: A Review

Nitrite plays a major role in the botulinal safety of cured meat products. When used at appropriate levels, it retards Clostridium botulinum growth and delays production of its deadly neurotoxin. Even though the incidence of botulinal spores in meat is very low, factors such as the tonnage of cured meats consumed, the potential for mishandling such products, and the nature of the disease necessitate the use of nitrite or other equally effective compound(s) for extra safety. Residual nitrite and nitrosamine levels in cured meat products have been decreasing in recent years through control and research conducted by the meat industry and related institutions. Such levels are minimal compared to total nitrate and nitrite amounts ingested or formed in the human body. Sorbate, especially in combination with nitrite at concentrations adequate only for cured meat color and flavor development, is at least as effective as currently used nitrite levels in delaying C. botulinum growth and toxin production. The mechanism(s) through which nitrite and/or sorbate perform their actions upon C. botulinum remains to be found. A review of the pertinent research completed to date should be helpful in the search for the mechanism(s).

SODIUM NITRITE AND SORBIC ACID EFFECTS ON Clostridium botulinum TOXIN FORMATION IN CHICKEN FRANKFURTER‐TYPE EMULSIONS

ABSTRACT C. botulinum growth and toxin production and residual nitrite depletion were studied in a mechanically deboned chicken meat (MDCM) frankfurter‐type product, during 27°C temperature abuse. A series of three trials were conducted in order to determine the effects of sodium nitrite and sorbic acid on the above parameters. Low nitrite concentrations (20 and 40 μg/g), did not influence C botulinum growth and toxin production. The addition of sorbic acid (0.2%) to these nitrite levels resulted in a significant extention of the time necessary for toxin to develop. The 0.1% sorbic acid level was ineffective. Nitrite concentrations of 156 μg/g doubled the time necessary, for botulinal toxin production, as did 0.2% sorbic acid alone. The magnitude of toxin production delay was increased five fold when 156 μg/g nitrite and 0.2% sorbic acid were combined. Nitrite depletion, was rapid during processing and 27°C incubation. Low residual nitrite concentrations were present for a longer time in nitrite‐sorbic acid combination treatments, indicating that the presence of sorbic acid may delay depletion of residual nitrite. Gas production and spoilage of the product followed the same rate of development as toxin.

Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis.

The ability of spores of one type A and one type B strain of Clostridium botulinum to grow and produce toxin in tomato juice was investigated. The type A strain grew at pH 4.9, but not at pH 4.8; the type B strain grew at pH 5.1, but not at pH 5.0. Aspergillus gracilis was inoculated along with C. botulinum spores into pH 4.2 tomato juice; in a nonhermetic unit, a pH gradient developed under the mycelial mat, resulting in C. botulinum growth and toxin production. In a hermetic unit, mold growth was reduced, and no pH gradient was detected; however, C. botulinum growth and low levels of toxin production (less than 10 50% lethal doses per ml) still occurred and were associated with the mycelial mat. The results of tests to find filterable or dialyzable growth factors were negative. It was demonstrated that for toxin production C. botulinum and the mold had to occupy the same environment.