Factors controlling the growth of Clostridium botulinum types A and B in pasteurized cured meats IV. The effect of pig breed, cut and batch of pork

Pork slurries were prepared from leg or shoulder muscle from three animals from each of three breeds of pig (Pietrain, Gloucester Old Spot and Large White × Landrace cross). Slurries (pork:water, 1:1.5) contained NaNO₂ (100 μ/g), NaCl (2.5, 3.5 or 4.5% w/v on the water), were subjected to one of three heat treatments (unheated, 80°C for 7 min, 80°C for 7 min plus 70°C for 1 hr) and stored at 35, 20, 17.5 or 15°C for up to 6 months to determine the relative effects of the above factors on growth (spoilage) and toxin production by Clostridium botulinum types A and B at 10³ spores per bottle.
Increasing salt or heat treatment, or decreasing storage temperature or inoculum level all reduced spoilage and toxin production. Both 'animal' and 'cut' significantly affected spoilage and toxin production. More spoilage and toxin production occurred in meat from the shoulder cut than from the leg cut. In both cases there was considerable variation between animals within breed, but there was no systematic difference between breeds. There is no obvious explanation for the variation in meat between animals, but it should be borne in mind when planning and assessing results of large multifactor experiments. Although there was more spoilage and toxin production after 6 months' than 3 months' storage, the statistical analyses yielded essentially similar conclusions.     

Production of Enterotoxin-B in Cured Meats

SUMMARY— A variety of laboratory cured hams were inoculated with 10³−10⁶ cells of S. aureus strain S-6 and incubated at 10, 22 and 30°C anaerobically for up to 16 weeks. Enterotoxin-B was detected by gel-diffusion in hams with original pH over 5.30, up to 9.2% NaCl (brine) and 0.54 ppm undissociated nitrous acid. There was better toxin production at 30° than at 22° or 10°C. Toxin was detected at 10°C after at least 2 weeks incubation and in most samples after 8 weeks when pH was greater than 5.6. Toxic hams had more than 4 × 10⁶ cells/g. Contaminants were always less than 10⁵/g. Tween 80 inhibited toxin production at 30° but not at 10°C. Toxic hams looked normal even after 2 months incubation at 10°C.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats

The effect of combinations of sodium chloride (2.5, 3.5, 4.5% w/v on water), sodium nitrite (100, 200, 300 μg/g), sodium nitrate (0, 500 μg/g), sodium isoascorbate (0,1000 μg/g, or equimolar with nitrite level) and polyphosphate (Curaphos 700; 0, 0.3% w/v), on the growth of Clostridium botulinum types A and B was studied in an experimental pork slurry system, without heating and after two heat treatments (80°C for 7 min and 80°C for 7 min plus 70°C for 1 hr) followed by storage at: 15, 17.5, 20 or 35°C for up to 6 months.
Statistical analyses showed that increasing salt or nitrite levels, adding isoascorbate or nitrate, using the highest heat treatment or decreasing the storage temperature all significantly reduced toxin production by Cl. botulinum . The addition of 0.3% polyphosphate (Curaphos 700) significantly increased toxin production. There were many significant two-factor interactions; the effect of increasing nitrite was relatively less if isoascorbate was present, at 4.5% salt, or at low storage temperature. The presence of isoascorbate also counteracted the increase in toxin production attributed to the presence of polyphosphate.     

DEVELOPMENT OF STRESS AND SURVIVAL OF SALMONELLA TYPHIMURIUM ATCC 7136 IN A TAPIOCA SOYSTARCH PRODUCT CONTAINING POTASSIUM SORBATE

The effects of reduced water activity and 0.1% sorbate on Salmonella typhimurium ATCC 7136 in a soy-starch product were examined. The formulated product (final pH 6.7), was inoculated with cells to give a final concentration of approximately 2 × 10⁶ cells/g. Ten gram samples were placed in a series of desiccators over saturated salt solutions and stored at either 22°C or 40°C. Water activities ranged from 0.79 to 0.97. Samples were withdrawn at prescribed intervals and appropriate dilutions made. A dual plating procedure was used to monitor growth (tryptic soy agar) and development of injury (Levine's Eosin Methylene Blue Agar supplemented with 2% NaCl and 0.05% sodium desoxycholate). Cells stored at 22°C remained viable longer at an a_w of 0.79 than at a_w values of 0.96 to 0.97. As storage time increased, cells were stressed at a faster rate at higher a_w values. This effect was accelerated at 40°C and as storage time increased.     

Prediction of toxin production by Clostridium botulinum in pasteurized pork slurry

A model pork slurry system was used to study factors controlling the growth of Clostridium botulinum types A & B (Roberts, Gibson & Robinson, 1981a,b). The following factors were studied in combination: sodium chloride (2.5, 3.5, 4.5% w/v on water); sodium nitrite (100, 200, 300 μg/g); sodium nitrate (0, 500 μg/g); sodium isoascorbate (0, 1000 μg/g); polyphosphate (Curaphos 700, 0, 0.3% w/v); heat treatment (none, 8°C/7 min, 80°C/7 min + 70°C/1 hr); at two pH levels and stored at: 15, 17.5, 20 or 35°C.
Analyses of results yielded a statistical model providing two formulae (for 'low' and 'high' pH slurries) which estimate the probability of toxin production in the pork slurry system within the limits defined above.     

INHIBITION OF STAPHYLOCOCCUS AUREUS AND BACILLUS CEREUS ON A VEGETARIAN FOOD TREATED WITH NISIN COMBINED WITH EITHER POTASSIUM SORBATE OR SODIUM BENZOATE

Various amounts of nisin (0, 10³ and 5 × 10³ IU/g) in combination with either potassium sorbate (0, 2, and 3%) or sodium benzoate (0, 0.06 and 0.12%) were tested for effectiveness in inhibiting growth of Staphylococcus aureus C10 and Bacillus cereus B7 inoculated on a vegetarian food. The strains used were isolated from vegetarian foods obtained commercially in Taiwan, and the test food, spice and dried bean curd, was selected for the study based on ability to support the growth of these organisms. After treatment with a preservative combination, the surfaces of sterilized food samples were inoculated, samples were stored in vacuum or nonvacuum packages at either 4C or 30C, and at appropriate times, tested for microbial growth. Growth of both isolates was unaffected by vacuum-packaging treatment; however, a bacteriostatic effect was found at 4C. Data indicated that during the 14-day storage at 4C, vacuum-packaged samples treated with 5 × 10³ IU/g nisin and 0.12% sodium benzoate significantly (p < 0.05) decreased the counts of S. aureus C10 and B. cereus B7 by 2.61 and 3.02 log₁₀ CFU/g, respectively. In the vacuum-packaged samples treated with 5 × 10³ IU/g nisin and 3% potassium sorbate, counts for C10 and B7 were decreased by 2.35 and 2.64 log₁₀ CFU/g, respectively. Thus, the combined treatment extended the shelf-life of the vegetarian food .     

GROWTH OF STAPHYLOCOCCUS AUREUS AND ENTEROTOXIN A PRODUCTION IN FOODS CONTAINING POLYPHOSPHATES

Effects of pyro-, tripoly- and hexametaphosphates (0.5 and 1%, w/w) on growth of Staphylococcus aureus strain 196E and enterotoxin A (SEA) production were studied in cooked custard and beef at 22 and 30°C. No effect was observed in custard, where cell numbers/g increased from 10³ to 10⁸ and SEA reached 4.2 ng/g after 48 h at 22°C, irrespective of treatment. Cell numbers in cooked beef were ca. 10⁹/g after 48 h, and reduced numbers (by 1.5–2 log cycles) were found in samples containing 0.5 and 1% pyrophosphate during incubation at 22%, but not at 30°C. SEA concentrations in beef were 28 ng/g after 48 h at 22°C, and 93 and 184 ng/g after 24 and 48 h, respectively, at 30°C. SEA concentration correlated with amount of growth, and was nondetectable when cell numbers were ± 10⁶/g. Reduction of the meat pH by sodium acid pyrophosphate contributed to the observed inhibitory effect.     

INFLUENCE OF FAT CONTENT AND PRESERVATIVES ON THE BEHAVIOR OF LISTERIA MONOCYTOGENES IN BEAKER SAUSAGE

Behavior of Listeria monocytogenes Scott A in pork liver sausage containing 22–67% fat, and antilisterial activities of sodium lactate, sorbic acid, potassium sorbate and sodium propionate were studied during storage at 4C and 10C. Commercial pork liver sausage batter (22% fat), alone and with additions of lard (15, 30, and 45% by weight) were tested. Concentrations of 1.8% sodium lactate, 0.1% sorbate as the acid or the potassium salt, and 0.2% sodium propionate were tested in heat sterilized sausage inoculated with a 24 h culture of the organism (10⁴ CFU/g). Fat content alone caused small reductions in cell numbers by the end of the storage periods: from log CFU/g of 9.9 to 9.4 after 14 days at 10C, and from 7.3 to 6.5 after 50 days at 4C in the basic sausage formulation and with 45% added fat, respectively. The inhibitory activities of lactate and propionate increased with increase in fat content, and were more pronounced at 4C, where the effects were listericidal. Inhibition by sorbic acid was least influenced by the fat content, and the potassium salt was less antilisterial than the acid.     

Inactivation of Listeria monocytogenes on Beef Bologna and Cheddar Cheese Using Polyvinyl-idene Chloride Films Containing Sorbic Acid

ABSTRACT: Polyvinylidene chloride (PVDC, Saran^R F-310) films containing sorbic acid (0%, 1.5%, and 3.0% w/v) were prepared with use of a solvent-casting method and were then placed between slices of commercially produced beef bologna that were previously surface-inoculated with L. monocytogenes at 10³ or 10⁵ CFU/g. In addition, cubes of commercial Cheddar cheese were surface-inoculated to contain 10³ or 10⁵ Listeria monocytogenes colony-forming units (CFU) /g and then wrapped with the sorbic acid-containing films. Films containing 1.5% and 3.0% (w/ v) sorbic acid prevented growth of L. monocytogenes on bologna slices with populations as much as 7.1 logs lower after 28 d of storage at 4 °C compared with the sorbic acid-free controls. In contrast, numbers of Listeria remained relatively stable on Cheddar cheese with populations decreasing < 1.3 logs after 35 d of storage. With use of the sorbic acid-containing films, common spoilage organisms were also inhibited on both products. After 28 d of contact with bologna and Cheddar cheese, these films retained 7% and 60% of their original sorbic acid content, respectively, with the control film retaining 85% of its original sorbic acid content. Given these findings, sorbic acid-containing films may be useful in enhancing the safety and shelf-life of ready-to-eat delicatessen products.     

EFFECT OF NITRITE AND STORAGE TEMPERATURE ON THE ORGANOLEPTIC QUALITY AND TOXINOGENESIS BY Clostridium botulinum IN VACUUM-PACKAGED SIDE BACON

The effect of nitrite and storage temperature and toxinogenesis by Clostridium botulinum in vacuum-packed side bacon was investigated. In two series of experiments (A & B) bacon packs were prepared with levels of 0, 50, 100, 150 and 200 ppm nitrite and inoculated with C botulinum at 10² spores/g and 10⁴ spores/g. Packs A were incubated at 20 and 30° C and packs B at 30°C only. Both were held for a maximum of 32 days and analyzed for toxin at intervals of 2, 4, 8, 16 and 32 days. At 20°C none of the controls without nitrite was found to be toxic after 32 days. At 30°C inhibition of toxin formation at the higher nitrite levels was observed at 32 days. Organoleptic evaluation of the bacon packs stored at 30° C showed about one-third of the toxic samples examined were acceptable to the panel.     

Modeling the Growth Inhibition Kinetics of Baker's Yeast by Potassium Sorbate Using Statistical Approaches

Potassium sorbate is widely used as a preservative in food products. The inhibition kinetics of yeast growth by potassium sorbate was determined in order to predict the inhibition mechanism and develop an antimicrobial activity model. Linear regression resulted in uncompetitive growth inhibition using different concentrations of nutrients in liquid media containing yeast extract, malt extract (YM broth) and potassium sorbate. At 30°C, constant K_s of YM broth to yeast was 0.254%, maximum growth rate (μ_max) of yeast was 0.008 min^-1, and inhibition constant (K_i) of potassium sorbate to yeast growth was 0.324%. This statistical method could reliably determine the growth inhibition mechanism and kinetics.     

Modeling the Growth/No-Growth Interface of Zygosaccharomyces bailii in Mango Puree

Probabilistic microbial modeling using logistic regression was used to predict critical temperatures to inhibit for at least 35 d Zygosaccharomyces bailii growth in a pH 3.5 mango puree formulated with 1000 ppm of potassium sorbate (KS) or sodium benzoate (NaB) at selected a_w (0.99, 0.98, or 0.97). The probability of growth was calculated, thereby ascertaining the set conditions and critical temperatures required inhibiting yeast growth for different storage times. Using the logistic model, with a growth probability of 0.05, critical temperatures were higher for KS than for NaB. Use of KS to inhibit Z. bailii growth enabled for mango puree 30 d of storage at 6.4 °C.     

Low Temperature Growth of Type E Clostridium botulinum Spores. 1. Effects of Sodium Chloride, Sodium Nitrite and pH

SUMMARY— The effects of pH and the addition of sodium chloride (NaCl) or sodium nitrite (NaNO₂) to trypticase-peptone-glucose (TPGI and trypticase-peptone-sucrose-yeast extract (TPSY) media upon spore outgrowth were investigated using seven Type E Clostridium botulinum strains. An inoculum of 1.0 × 10⁵ spores/ml was used and the pH was adjusted with hydrochloric acid to cover the range of 5.2 to 6.6. To define salt tolerance, NaCl was added to the media at intervals of 0.5% in the range of 2.0 to 5.0% and at 0.1% intervals in the 4.5 to 5.0% range. The effect of NaNO₂ was investigated with the addition of 100 and 200 ppm NaNO₂ to the media. Samples were incubated at 30, 15.6, 10, 7.2, 5.0 and 3.4°C. Outgrowth of all strains tested was inhibited at pH 5.2 at 15.6°C. Inhibition occurred at higher pH at lower temperatures. None of the strains showed outgrowth with 4.87% NaCl in the media at any of the incubation temperatures used. Addition of 100 and 200 ppm NaNO₂ to the media inhibited the outgrowth of the Minneapolis, 517, 26080 and A6247 strains but not the Kalamazoo and Seattle Forks strains.     

Prediction of Listeria spp. growth as affected by various levels of chemicals, pH, temperature and storage time in a model broth

The effects of concentration of NaCl (0.5 to 12.5%), methyl paraben (0.0 to 0.2%), sodium propionate (0.3%), sodium benzoate (0.1%), potassium sorbate (0.3%), pH (>5.9) temperature (4 to 30°C), storage time (up to 58 d) and inoculum (>10⁵ to >10⁻² per ml) on the log₁₀ probability percentage of one cell of Listeria spp. to initiate growth in a broth system were evaluated in a factorial design study. At pH 5.96 and temperature ranging from 4 to 30°C the concentrations of sodium propionate, potassium sorbate, and sodium benzoate examined allowed growth of L. monocytogenes with lag phases at 4°C of 18, 27 and 21 days, respectively. For 0.1 and 0.2% methyl paraben growth of all Listeria spp. was initiated at 8°C and 30°C, respectively. At pH 6, concentration of 12% NaCl supported the growth of L. monocytogenes at 8 to 30°C, whereas 12.5% inhibited all Listeria species. Four regression equations were derived relating probability of growth initiation to temperature, concentrations of NaCl and preservatives storage time, and Listeria species specific effects. From these equations, the number of cells needed for growth initiation can be calculated. The impact of this type of quantitative study and its possible application on the development of microbial standards for foods is discussed.     

ENTEROTOXIN FORMATION IN FOODS BY CLOSTRIDIUM PERFRINGENS TYPE A

Growth, sporulation and enterotoxin formation in various foods inoculated with a Clostridium perfringens type A enterotoxin-producing strain were studied. Good vegetative growth, 10⁷–10⁸ cells/g, was obtained after 4 hr of anaerobic growth and remained almost the same throughout the 20–24 hr observation in most of the foods. A gradual increase in spore count to the level of 10⁴–10⁵/g was observed with an increase in the incubation time. Enterotoxin was detected in moist cooked chuck roast, ground beef and turkey as well as in moist cooked and dry roasted chicken at levels up to 0.125μg/g. The earliest time at which enterotoxin was detected was after 10 hr of anaerobic growth in moist cooked turkey at 37°C. Although growth and some sporulation occurred, enterotoxin was not detected in dry roasted beef or turkey with or without gravy, or in moist cooked pork or lamb. Poor growth and sporulation also were obtained with chicken broth, chicken gravy and beef gravy. In moist cooked turkey that had been temperature abused for 6 hr at 37°C, held cold for 15 hr and reheated to 37°C, toxin could be detected after only 5 hr of holding at 37°C. The ability of certain foods to support sporulation and enterotoxin formation indicates that such preformed enterotoxin may contribute to early onset of symptoms in some cases of C. perfringens food poisoning.     

HEAT PASTEURIZATION OF CRAB AND SHRIMP FROM THE PACIFIC COAST OF THE UNITED STATES: PUBLIC HEALTH ASPECTS

ABSTRACT— A study of the potential public health hazard presented by coagulase-positive staphy-lococci, salmonellae and Clostridium botulinum in the meat of Dungeness crab and Pacific Coast shrimp pasteurized in flexible plastic containers revealed potentially toxinogenic staphylococci on the commercial nonpasteurized product, in 15% of the shrimp and 9% of the crab samples. No salmonellae or Cl. botulinum were isolated from, respectively. 26 and 54 samples of shrimp or 74 samples of crab. Pasteurization for 1 min at 180°F destroyed large inocula (10⁷ and 10⁸ cells) of staphylococci and salmonellae introduced into packages of the products, but processing for 5 min at 180°F allowed some members of an inoculum containing 10³ spores of Cl. botulinum type E to survive. While storage at 40°F prevented the growth on crab and shrimp meat of all staphylococci and salmonellae tested, it permitted growth and toxin formation by Cl. botulinum type E after 30–40 days. No toxin could be detected in packages inoculated with type A and proteolytic B spores and held at 50°F or lower. A 0.1% dip of sodium benzoate, with or without fumaric acid, did not prevent growth and toxinogenesis by Cl. botulinum types A, proteolytic B or E. It was concluded that for complete safety a holding temperature of 36°F or lower at all times would be required, but that it could not be expected to be maintained in commercial channels.     

Postharvest Microbiology of Farm-reared, Tropical Freshwater Prawn (Macrobrachium Rosenbergii)

ABSTRACT: The microbiological quality of farm-reared, tropical freshwater prawn ( Macrobrachium rosenbergii ) stored at 2 different temperatures was studied. The prawn muscle was found to have the initial bacterial load of 10⁴ cfu/g. The lactics and vibrios were in the range of 10² cfu/g, while the E. coli , aeromonads, staphylococci, anaerobes, and molds were in the level of 10¹ cfu/g. Salmonella and Vibrio cholerae were present in the prawn muscle. The prawn muscle held at room temperature (28 ± 2 °C) was organoleptically acceptable up to 8 h, when the bacterial load was more than 10⁶ cfu/g. However, the prawn muscle stored at freezer temperatures (−10 to −15 °C) was found to be in acceptable condition even after 30 d of storage and the bacterial load was fluctuating in the range of 10³ to 10⁴ cfu/g.     

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

Combinations 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.     

Quenching Mechanism and Kinetics of Ascorbic Acid on the Photosensitizing Effects of Synthetic Food Colorant FD&C Red Nr 3

ABSTRACT:  The pH effect on the oxidative stability of ascorbic acid in the presence of food colorant FD&C Red Nr 3 during storage with or without light was investigated. The quenching mechanism and kinetics of ascorbic acid on the FD&C Red Nr 3 photosensitized oxidation in an aqueous system at 25 °C were also studied by measuring the degradation of ascorbic acid or depletion of headspace oxygen. Red Nr 3 had no influence on the oxidation of ascorbic acid under dark storage, but accelerated its oxidation rate under light storage. The oxidative stability of ascorbic acid decreased as the pH increased from 4 to 7 under light without FD&C Red Nr 3. The quenching rates of ascorbic acid on the singlet oxygen by measuring the degradation of ascorbic acid in the presence of Red Nr 3 under light storage were 1.53 ± 0.15 × 10⁸, 1.86 ± 0.25 × 10⁸, and 1.19 ± 0.12 × 10⁸ M⁻¹S⁻¹ at pH 4, 5.6, and 7, respectively.     

The effects of different incubation temperatures on the acetaldehyde content and viable bacteria counts of bio-yogurt made from ewe's milk

Yogurt and bio-yogurt were manufactured from ewe's milk using a starter culture and a probiotic culture. Incubation was carried out at 37°C and 42°C until pH 4.6 was reached and the yogurts were stored at 4 +1°C for 14 days. Analysis after 1, 7 and 14 days showed that incubation temperature and storage time significantly influenced overall properties of the samples. During the storage, whey separation and pH decreased, but titratable acidity, lactic acid and volatile fatty acid contents increased. Viable bacterial counts in all bio-yogurts were above 10⁷ cfu g⁻¹ at the end of storage.