Inhibition of Clostridium botulinum Toxin Formation by C. sporogenes in Culture Media and in a Meat System.

Vegetative cells and spores of types A and B Clostridium botulinum and C. sporogenes were coinoculated into culture media and into mechanically deboned chicken meat. Botulinal toxin formation was inhibited; the degree of inhibition depended on the relative concentrations of the two microorganisms. Inhibition of toxin formation by C. sporogenes also occurred in the meat system, but not all strains of C. botulinum were affected.

Gamma Radiation Resistance of Clostridium botulinum 62A and Bacillus subtilis Spores in Honey.

Irradiation D values for the natural bacterial flora of two samples of raw (bulk) honey were 7.50 and 1.91 kGy; for two samples of retail honey the D values were 5.66 and 3.49 kGy. Irradiation D values of Clostridium botulinum 62A spores inoculated into three honey samples and into water were respectively, 8.11, 9.38, 12.77, and 2.07 kGy. Similar D values for Bacillus subtilis spores were 3.42, 3.35, 4.00, and 1.43 kGy. The radiation resistance of C. botulinum and B. subtilis spores in honey and in sugar syrups was a function of water content.

Gamma Radiation Inactivation of Enterococci.

Radiation survival curves were determined for 7 strains of Enterococcus faecium , 10 strains of E. faecalis , and 8 strains of the proteolytic variety of E. faecalis . The D values (i.e. the doses giving 90% reduction of viable counts) ranged from 5.0-47 kGy for the E. faecium strains, 3.5-21 kGy for the E. faecalis strains, and 3.0-4.5 kGy for the proteolytic variants of E. faecalis . The survival curves were linear for most strains but some exhibited significant non-linear trends.

Gamma Radiation Sensitivity of Listeria monocytogenes.

Seven strains of Listeria monocytogenes were irradiated in culture media or in mechanically deboned chicken meat. The survivor plots were quadratic curves when cultures were in the log phase of growth or when they were irradiated in chicken meat; cultures in the senescent phase of growth showed linear responses to irradiation. Cultures from cells surviving an irradiation dose of 1.5 kGy were no more radiation resistant that those which had had no previous exposure to irradiation. Cultures centrifuged and resuspended in water were more sensitive to radiation than those resuspended in solutions containing organic materials. These studies indicated that a dose of 2 kGy was sufficient to destroy 1 × 104 cells of L. monocytogenes .

A Procedure for the Direct Microscopic Count of Bacteria in Non-fat Dry Milk.

Bacteria in non-fat dried milk (NDM) were enumerated by a method involving preliminary solubilization of the milk proteins in 0.015 N NaOH followed by centrifuging, washing in the NaOH, and microscopically examining stained smears. The method was used to enumerate bacteria in samples of NDM obtained from government surplus stocks or from local retail sources. Bacterial counts from surplus NDM ranged from 4.64 × 105 to 2.83 × 106/g (the mean and median were, respectively, 6.23 and 2.84 × 106/g). Counts from retail samples ranged from 4.48 × 105 to 2.42 × 107/g (mean and median were 5.57 and 2.85 × 106/g). The predominant bacteria in some samples were paired streptococci; other samples contained rod-shaped bacteria, some with identifiable spores. Comparison of this method with the Levowitz-Weber method indicated that it produced fewer artifacts, was applicable to NDM samples containing a wider range of bacteria, and did not require the use of the potentially carcinogenic tetrachloroethane.

Effect of Sugar and Low-Dose Irradiation on Toxin Production by Clostridium botulinum in Comminuted Bacon.

Comminuted bacon, processed to contain target levels of 40 µg NaNO2/g and 0, 0.25 or 0.75% sucrose or 0.75% glucose, was inoculated with a mixture of spores of 20 strains of Clostridium botulinum (400 spores per g) and was canned under vacuum. Portions were irradiated using 137Cs at doses of 0, 0.19, 0.38, 0.75, 1.12 and 1.5 Mrad. Cans were incubated for 1, 2, 4 or 8 wk at 30°C. Some cans of nonirradiated bacon without or with 0.25% sucrose became toxic in 2 wk; with 0.75% sucrose, toxin production was delayed to 8 wk. Bacon irradiated at 0.75 Mrad, made with or without sucrose, became toxic in 2 to 4 wk, whereas most cans of bacon irradiated at 1.5 Mrad remained toxin-free for the 8-wk incubation period. A comparison of bacon made with 0.75% sucrose or glucose showed no difference between the sugars in the rates of toxin production by C. botulinum in irradiated cans of bacon. Irradiation at 0.19 Mrad increased the rate of toxin formation over nonirradiated bacon in sugar-containing (0.75%) bacon, but had no effect in sugar-free bacon. The pH of nonirradiated bacon containing 0.75% glucose or sucrose decreased from pH 6.12 and 6.11, respectively, to pH 5.63 and 5.67 after 8 wk of incubation at 30°C. The titratable acidity showed a concurrent increase. The pH and titratable acidity of bacon irradiated at 0.19 Mrad or higher showed no changes.

Acceptability of Bacon Stored Six Weeks at 4 or -23°C.

Bacon was obtained from production lines of two local processing plants, H and M. The slices from 8 bellies from each plant were sequentially rearranged to form composite portions representative of each belly; these were analyzed for moisture-phase NaCl and for acceptability by a panel of judges using a 9-point hedonic scale. Samples were tasted shortly after processing and again after 6 weeks of storage at 4 or -23°C. The moisture-phase NaCl content ranged from 4.62 to 7.80% (c.v. = 18.2%) for bacon from plant H; the range for bacon from plant M was 3.25 to 10.05% (c.v. = 37.7%). The belly from plant M with the highest moisture-phase NaCl content also gave the lowest hedonic score of the bacon samples tasted before storage. The average hedonic scores for bacon from the two plants were different (p<0.05). There were no significant differences due to storage condition for bacon from plant M, but bacon stored at 4°C from plant H had lower (p<0.05) average scores than the bacon sampled shortly after processing or that kept 6 weeks at -23°. Storage at 4°C caused a significant (p<0.01) reduction in hedonic score in one belly from each plant.

Inhibition of Clostridium botulinum in Comminuted Bacon by Short-Chain Alkynoic and Alkenoic Acids and Esters.

Several short-chain alkynoic and aikenoic acids and esters were screened for inhibition of gas production by Clostridium botulinum in cans of comminuted, nitrite-free, temperature-abused bacon. The most active compounds were propiolic (2-propynoic) acid, methyl and ethyl propiolate, 2-propenoic acid, methyl and ethyl propenoate, and mono- and dimethyl and ethyl fumarate. Maleic acid, methylmaleic acid, ethyl maleate, 2-methyl propenoic acid, trans-2-methyl crotonic acid, 3-methyl-2-butenoic acid, 3-methylallyl alcohol, vinyl crotonate, methylmalonic acid, dimethyl glutarate, 2,4-hexadien-1-ol, cis-3-hexenoic acid, 4-pentenoic acid, trans-2-pentenoic acid and ethylidene acetic acid were less active. A more comprehensive study on the inhibition of toxin formation by C. botulinum in comminuted nitrite-free bacon was done by comparing equimolar quantities of some of these compounds with NaNO2 at 120 µg/g and sorbic acid at 9 and 18 mM (0.10 and 0.20%). These studies showed that propiolic acid was more effective than either nitrite or sorbic acid. Compounds in this study less active than nitrite or sorbic acid were 3-methylallyl alcohol, 2,4-hexadien-1-ol, dimethyl glutarate and methylmaleic acid.

Acid Enhancement of Clostridium botulinum Inhibition in Ham and Bacon Prepared with Potassium Sorbate and Sorbic Acid.

The effectiveness of combinations of sorbic acid and other acids (hydrochloric, phosphoric, acetic, citric, lactic and succinic) on Clostridium botulinum inhibition in comminuted ham and nitrite-free bacon was studied. These acids, when added to ham to give similar pH's, did not significantly inhibit the organism, but when acetic or citric acid was added with sorbic acid, inhibition was greater than with sorbic acid alone. The acids were less effective in inhibiting C. botulinum when added to sorbic acid-containing bacon. A study of the effectiveness of three levels of potassium sorbate (0.10, 0.26 or 0.52%) or sorbic acid (0.08, 0.20 or 0.40%) and two levels of phosphoric acid (0.04 or 0.08%) in comminuted ham showed that the highest levels of sorbate or sorbic acid were sufficient to inhibit toxin production when incubated at 30°C for 180 d. The same degree of C. botulinum inhibition was afforded by 0.26% sorbate with 0.08% H3PO4 or by 0.20% sorbic acid with 0.04% H3PO4. These differences were probably due to the higher pH obtained with sorbate.

Inhibition of bacteria by lactulose preparations.

Lactulose syrups were similar to sucrose syrups in water activity-lowering effects but were more inhibitory toward test microorganisms. Heat-treated commercial lactulose syrups were most inhibitory, whereas non-heat-treated pure lactulose was only slightly more inhibitory than sucrose.

Inhibition of Clostridium botulinum by Spice Extracts and Aliphatic Alcohols.

Alcoholic extracts of spices were prepared and tested for inhibition of Clostridium botulinum in culture media. Mace (the outer covering of the seed of Myristica fragrans ) and achiote (annato, Bixa orellana ) were the most inhibitory of 33 spices studied. Also quite active were bay leaf ( Laurus nobilis ), white and black pepper ( Piper nigrum ) and nutmeg (the seed of M. fragrans ). Less active were rosemary ( Rosmarinus officinalis ), clove ( Eugenia caryophyllata ), oregano ( Oreganum vulgare ), turmeric ( Circuma longa ), thyme ( Thymus vulgaris ), and paprika ( Capsicum annuum ). Of the series C1 to C18, aliphatic straight chain alcohols of C14 or C16 chain-lengths were the most inhibitory against C. botulinum with a minimum inhibitory concentration (MIC) of 0.6 ppm. A plot of alcohol chain length versus MIC showed a highly significant (P < 0.01) cubic function.

Inhibition of Clostridium botulinum by p-hydroxybenzoic acid n-alkyl esters.

Twelve straight-chain esters, C(5) to C(14), C(16), and C(18), of p-hydroxybenzoic acid were prepared, and their melting points, solubilities in water at 25 degrees C, infrared spectra, dissociation constants (pK(a)), and activities against Clostridium botulinum were determined. These studies also included four commercial straight-chain esters, C(1) to C(4). The most potent activity was exhibited by undecyl and dodecyl esters, which are about 300 times as active as sodium nitrite. Quadratic and cubic equations were developed correlating the activity with pK(a) values and chain length of the esters, respectively.

Disappearance of patulin during alcoholic fermentation of apple juice.

Eight yeast strains were used in three typical American processes to ferment apple juice containing 15 mg of added patulin per liter. Patulin was reduced to less than the minimum detectable level of 50 microgram/liter in all but two cases; in all cases, the level of patulin was reduced by over 99% during alcoholic fermentation. In unfermented samples of apple juice, the concentration of added patulin declined by only 10% when the juice was held for 2 weeks, a period equivalent to the time required for fermentation.

Effect of Acids on Selenite Inhibition of Salmonella typhimurium and Salmonella dublin.

The minimum pH for growth (MPG) in selenite-cystine enrichment medium was determined for a number of acids. The MPG for lactic, citric, hydrochloric, aspartic, malic, succinic, pyruvic, and tartaric acids was 5.80; acetic acid was considerably more inhibitory, giving a MPG of 6.30-6.40; the MPG's for fumaric and pyruvic acids were 6.00 and 5.70, respectively. The least inhibitory acid was α-ketoglutaric acid which gave a MPG lower than or equal to 5.1. Inoculum size had little or no effect on the MPG.

Inhibition of Clostridium botulinum by 5-nitrothiazoles.

A number of 5-nitrothiazoles with various substituents in the 2-position were tested for inhibition of Clostridium botulinum in a culture medium. Thiazole itself or 2-bromo- or 2-methylthiazole at 30 mug/ml did not inhibit the organism. An amino group in the 2-position of thiazole inhibited at 10 mug/ml. Substitution of a nitro group in the 5-position of 2-aminothiazole increased the inhibitory level to 0.12 mug/ml; acetyl-, propionyl-, or butyroyl-2-amino-5-nitrothiazole inhibited at 0.04 mug/ml. Benzoyl-2-amino-5-nitrothiazole inhibited at 0.16 mug/ml; this increased to 0.01 mug/ml when the benzoyl group carried a nitro group in the m- or p-position; a nitro group in the o-position, on the other hand, inhibited at 0.04 mug/ml. Unsaturated aliphatic acyls decreased inhibition. The greatest activity was exhibited by 2-nonanoyl- and 2-lauroylamides, with minimum inhibitory concentrations of 0.005 and 0.0025 mug/ml, respectively.

Evaluation of a Rapid Test for Antibiotic Residue in Milk Using Spores of Bacillus stearothermophilus var. calidolactis 1.

Eleven analysts tested contaminated reconstituted (1:10) dry milk powders for penicillin residues using spores of Bacillus stearothermophilus var. calidolacris (Delvotest P method). Three types of responses were noted: positive, negative, and questionable. Prediction equations indicated that 95% of the time, analysts unfamiliar with the technique could detect positive results if penicillin concentrations in samples were 0.010 unit/ml or higher and positive and questionable results if the penicillin concentrations were 0.008 unit/ml. Increasing the reconstitution ratio from 1/11 to 1/4 increased the chances of detecting penicillin in milk powder. Penicillinase added to reconstituted penicillin-contaminated milks in all instances produced negative responses.

Destruction of Salmonella and Staphylococcus During Processing of a Nonfermented Snack Sausage.

Survival of Salmonella dublin , Salmonella senftenberg 775W, Staphylococcus aureus 196E, and S. aureus 184 was studied during processing of an inoculated beef, nonfermented snack sausage. No viable staphylococci or salmonellae were detected in sausages that had been heated at an internal temperature of 53.9-55.0 C or 57.8-58.9 C for 3.5 h followed by drying at 21 C and 50-55% relative humidity for 4 days. Heating at an internal temperature of 51.1-52.2 C for 3.5 h followed by drying did not produce a salmonellae- or staphylococci-free sausage.

Growth and toxin production by Clostridium botulinum in moldy tomato juice.

Tomato juice inoculated with Cladosporium sp. or Penicillium sp. developed pH gradients with the upper portions near the mold mats having pH values near neutrality and the lower portions remaining more acid. Clostridium botulinum spores in these moldy tomato juices germinated, grew out, and produced toxin.