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.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats III. The effect of potassium sorbate

The growth of Clostridium botulinum types A and B spores, at 10¹ or 10³ per container, was studied in a pork slurry system containing nitrite (40 μg/g), sodium chloride (2.5, 3.5, 4.5% w/v) sodium isoascorbate (550 μg/g) at varying pH levels, with or without potassium sorbate (0.26% w/v), without heating and after two heat treatments (80°C for 7 min, and 80°C for 7 min + 70°C for 1 hr) followed by storage at 15, 17.5, 20 or 35°C for up to 6 months. At a given spore inoculum, potassium sorbate significantly decreased toxin production, as did increasing NaCl, decreasing pH or decreasing storage temperature. Heat treatment did not significantly affect spoilage or toxin production overall, but interacted significantly with some factors. The effect of sorbate was greater at 3.5% NaCl than at 2.5%, at pH values below 6.0, and at low storage temperature.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized cured meats VI. Nitrite monitoring during storage of pasteurized pork slurries

Residual nitrite levels were monitored during storage for up to 6 months, in a model pork slurry system used to study the relative effects of curing ingredients and additives used in pasteurized cured meats to control the growth of Clostridium botulinum.
In 'low' pH slurries the rate of loss of nitrite fell with reducing storage temperature. Less residual nitrite remained after HIGH heat treatment but the rate of loss of that residual nitrite was slower during storage than nitrite remainly after LOW heat treatment. Inclusion of nitrate resulted in higher residual nitrite levels, particularly after HIGH heat and if stored below 20°C. If isoascorbate was added nitrite became undetectable within circa 30 days, even when nitrate had been added. The rate of loss of nitrite was slower in 'high' pH slurries (pH 6.3–6.8).
Monitoring levels of nitrite in the product soon after production would detect its accidental overuse but monitoring nitrite in the product during distribution or at retail, without knowledge of the composition and prior history of the product, gives little indication of the amount used at manufacture. The level of residual nitrite was not directly related to the ability of the curing mixture to control the growth of CI. botulinum types A and B. Some slurries in which C1. botulinum grew least during 6 months' storage contained no residual nitrite because isoascorbate was also present.     

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.     

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.     

Effects of Sodium Chloride Reduction and Polyphosphate Addition on Clostridium Botulinum Toxin Production in Turkey Frankfurters

Mechanically deboned turkey meat emulsions were made with 1.0, 1.5, 2.0, 2.5, or 3.0% salt (NaCl), or with combinations of 1.5% or 2.0% salt with 0.4% sodium tripolyphosphate (TPP), sodium hexametaphosphate (HMP), or sodium acid pyrophosphate (SAPP). Sodium nitrite levels were constant at 150 ppm. Emulsions were inoculated with a mixture of 10 strains of C. botulinum (10³/g) and incubated at 27°C. Increasing NaCl content from 1.0% to 3.0% delayed toxin production by 3 days on the average. Toxin production was detected earlier when TPP was added, HMP had no effect, and SAPP delayed toxin production.     

The effect of pH,sodium chloride,sodium nitrite and storage temperature on the growth of Clostridium perfringens and faecal streptococci in laboratory media

The effects of salt concentration (1–12% w/v) in combination with unheated sodium nitrite (0–400 μg/ml) on growth of mixed strains of Clostridium perfringens and of faecal streptococci at three pH values (5.6, 6.2, 6.8) and storage temperatures ranging from 10°C to 35°C is reported. At pH 6.2, following storage at 15°C, 1% salt and 50 μg/ml nitrite inhibited growth of C. perfringens. At 20°C and pH 6.2, 200 μg/ml nitrite plus 3% salt, or 50 μg/ml plus 4% salt were required to inhibit growth. Growth of C. perfringens was prevented by levels of curing salts used commercially providing the pH was 6.2 or below. At pH 6.8 or above at least 4% salt and 50 μg/ml nitrite was required to prevent growth at 20°C. The faecal streptococci grew in medium containing 6% salt and 400 μg/ml nitrite irrespective of pH or storage temperature. In 8% salt growth was prevented by storing at or below 17.5°C or, if pH was 6.2 or lower, by adding 200 μg/ml nitrite irrespective of storage temperature. Growth of faecal streptococci was not controlled by concentrations of curing salts which would be acceptable in meat products.     

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.     

Chilled storage life of hot-boned, pre-rigor, salted minced beef

Pre-rigor beef mince with 2% added salt was stored under CO₂ at −1.5 °C (A). The same mince with 100 ppm sodium nitrite was stored under CO₂ at 5 °C (B) and −1.5 °C (C), and under vacuum at −1.5 °C (D). Microbiological and sensory analyses were carried out for up to 21 weeks. Indicative storage life was taken as the time for microbial numbers to reach 10⁷ colony forming units per g. Mince stored under regimes B or D attained these numbers by 6 and 14 weeks, respectively; mince stored under regimes A and C had not attained these numbers by the end of the storage trial. Mince stored at 5 °C developed storage flavours of sufficient intensity to be detectable by consumers by 9 weeks storage. In general, the other minces did not develop unacceptable levels of storage or off flavours. Over 90% of the added sodium nitrite had disappeared after 10 weeks of storage, partly through conversion to sodium nitrate. Mince pH was not affected by the storage conditions and remained at about 6.0. The water holding capacity of the pre-rigor mince deteriorated during prolonged storage.     

Microbiological aspects and technological need: technological needs for nitrates and nitrites.

Nitrate and nitrite are used in meat and fish curing, and in the manufacture of certain cheeses. Nitrate itself has little antimicrobial effect and in most applications could be replaced by lower concentrations of nitrite. Further, improved hygiene diminishes the need for nitrite. The antimicrobial activity and technological needs for nitrate and nitrite are reviewed. It is concluded that the technological needs for nitrite in meat products stored at < 10 degrees C could be met by added nitrite concentrations of 50 mg/kg. The overall effect of nitrate in salted fish appears to be marginal. In such products, Vibrio parahaemolyticus does not grow at salt concentrations of 10% and food poisoning by this organism is not related to the absence of nitrate or nitrite; growth of Clostridium botulinum Type E (the predominant cause of botulism from fish products) is arrested by salt concentrations of 3-4%. Listeria monocytogenes in seafood cannot be controlled by nitrite. The use of nitrate in cheese production could be avoided, or at least reduced to a low level by avoiding silage with a high count of gas-producing Clostridia and hygienic milk collection.     

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.     

Effect of the Parabens With and Without Nitrite on Clostridium botulinum and Toxin Production in Canned Pork Slurry

Antimiciobials were evaluated in thioglycollate broth at pH 6.5 for the ability to inhibit growth and toxin production by C. botulinum 12885A and ATCC 7949 (Type B). Methyl, ethyl, propyl, and butyl parabens (0.1%) and sorbic acid (0.2%) were effective in inhibiting growth of C. botulinum 12885A and ATCC 7949 in broth. Ethyl, propyl, and butyl parabens (0.1%) and sorbic acid (0.2%) inhibited toxin production by both strains in culture medium. Ethyl, propyl, butyl parabens (0.1%) and sorbic acid (0.2%) were individually added to a comminuted pork slurry having salt and sugar, with or without 40 ppm sodium nitrite. Cans were inoculated with a mixture of C. botulinum 12885A and ATCC 7949 spores. The canned product was abused by holding at 27°C and was observed over a 3-month period for swollen cans. Swollen cans were examined for botulinal toxin by the mouse bioassay. Propyl and butyl paraben did not inhibit or delay toxin production. Ethyl paraben with or without nitrite delayed toxin production for 4 wk. Sorbic acid inhibited toxin for 3 wk; when 40 ppm nitrite was added to the sorbic acid treatment, toxin production was delayed for 4 wk.     

SPECIFICITY OF THE FERRIC-H2SO4 REAGENT FOR ALGINATES

SUMMARY— Alginate in foods was isolated from papain digests of the samples as the insoluble calcium salt. dispersed by sodium hexametaphosphate and a 1- to 5-ml aliquot of the dispersion 50–250 μg of sodium alginate) dried at 100°C. Heating of the anhydrous sample in the presence of the ferric-H₂SO₄ reagent for 12 min at 60°C produced a pink color with maximum absorption at 490–515 mμ and maximum density after aging for 90 min at 29±1°C. Under these conditions, proteins, amino acids (except tryptophane), other carbohydrates, vitamins. food additives and incipients did not interfere. The presence of water in the medium rendered the reaction nonspecific and increasing the temperature above 60°C or heating for longer periods at lower temperatures resulted in color formation by other polyuronides, especially pectin and pectic acid. Recoveries of alginate from several products were good: Milk, 94–98%; ice cream, 93.5–98%; pasteurized process cheese spread. 92.6-95%; chocolate milk, 92.4-95%; dietetic foods, 90-97.2%.     

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.     

The effect of pH,water activity,sodium nitrite and storage temperature on the growth of enteropathogenic Escherichia coli and salmonellae in a laboratory medium

The effect of salt concentration (1–10% w/v) in combination with sodium nitrite (0–400 μg/ml) on growth of mixed strains of Salmonella and enteropathogenic E. coli at three pH values (5.6, 6.2, 6.8) and storage temperatures ranging from 10°C to 35°C is reported. E. coli tended to be more tolerant of salt and nitrite than Salmonella.     

Cooked and brined shrimps packed in a modified atmosphere have a shelf-life of >7 months at 0 °C, but spoil in 4–6 days at 25 °C

Summary Spoilage and safety of cooked, brined and modified atmosphere packed shrimps were studied at 0, 5, 8, 15 and 25 °C. Shrimps from two sources, cold and warm waters, were brined in a sodium–chloride brine containing benzoic, citric and sorbic acids. Shelf-life was above 7 months at 0 °C but only 4–6 days at 25 °C. Apparent activation energy for the effect of temperature on shelf-life was > 100 kJ mol^-1. This pronounced effect of temperature was explained by changes in spoilage microflora at different storage temperatures. Simple and empirical mathematical models for rates of spoilage were developed for the prediction of shelf-life at different temperatures. To evaluate safety, products were challenged with Listeria monocytogenes and spores of Clostridium botulinum . Above 5 °C growth responses of L. monocytogenes followed the square root model with a T_min-value of +0.2 °C. Cl. botulinum produced toxin at the time of spoilage at 25 °C but only in shrimps with < 3% water-phase salt.     

Effect of polyphosphates on the survival of pre-stressed Salmonella typhimurium cells in frozen chicken meat

Six different commercial polyphosphate preparations were evaluated for their lethal efficacies against inoculated pre-stressed stationary phase cells of Salmonella typhimurium LT₂ in frozen comminuted poultry meat subsequently stored at ?18°C for 28 days. At the 0·3% wt/wt concentration used, Puron 6040 was found most potent, achieving approximately 2 log cycle viability reduction, closely followed by Fibrisol N7 and sodium tripolyphosphate with 94·7% and 84·5% viability reductions, respectively. Curaphos 700 instant and tetrasodium pyrophosphate No. 7 both exhibited similar potency magnitudes of approximately 70% viability reduction each, while acid sodium pyrophosphate was the least potent. The polyphosphates slightly altered sample pH values and aided fluid retention without any detectable influence on the a_w value. The findings demonstrate the potential antimicrobial benefits derivable from polyphosphates incorporation coupled with the conferrable textural advantages. It is considered that the observed potencies are influenced by chemical factors including chain length, pH and the subsequent relative stabilities of the polyphosphates in the menstruum system.     

Nitrates, nitrites and N-nitrosocompounds: a review of the occurrence in food and diet and the toxicological implications

Data on occurrence of nitrate, nitrite and N-nitrosocompounds in food and drinking water, and on total dietary intakes are reviewed. Metabolic, toxicological and epidemiological studies are surveyed and the implications with respect to safety evaluation are addressed. It is concluded that, on the basis of recent long-term animal studies and of clinical experience in man, the current Acceptable Daily Intake (ADI) allocated to nitrate by the Joint FAO/WHO Expert Committee on Food Additives of 0-5 mg/kg body weight/day (expressed as sodium nitrate) might be increased to 0-25 mg/kg body weight/day. Based on similar criteria, the ADI for nitrite would be 0-0.1 mg/kg body weight/day (expressed as sodium nitrite). In view of the known carcinogenicity of N-nitrosocompounds, exposure to these compounds in food should be minimized by appropriate technological means, such as lowering the nitrite concentration in preserved foods to the minimum required to ensure microbiological safety and use of inhibitors of nitrosation like alpha-tocopherol or ascorbic acid. Further work is needed to define the minimal levels of nitrite in foods needed to inhibit outgrowth of Clostridium botulinum and toxin production.     

Effect of Meat Binding Formulations on Thermal Inactivation of Escherichia coli O157:H7 Internalized in Beef

ABSTRACT:  This study evaluated the effects of meat binding or restructuring formulations, including salt/phosphate, algin/calcium, Activa™RM, and Fibrimex®, with or without 0.27% (wt/wt) lactic acid, on thermal inactivation of internalized Escherichia coli O157:H7 in ground beef, serving as a model system for restructured products. Ground beef batches (700 g; approximately 5% fat) were mechanically mixed with a 5-strain composite of E. coli O157:H7 (7 log CFU/g) and then with the restructuring formulations. Product portions (30 g) were extruded into plastic test tubes (2.5 × 10 cm) and stored at 4 °C (18 h), before heating to 60 or 65 °C in a circulating water bath to simulate rare or medium-rare doneness of beef, respectively. Cooking to 60 or 65 °C reduced ( P < 0.05) bacterial counts of control samples by 1.8 and 3.2 log CFU/g, respectively. Thermal destruction at 60 °C was not different ( P > 0.05) among all treatments and the control. At 65 °C, greater ( P < 0.05) thermal inactivation of E. coli O157:H7, as compared to the control, was obtained in samples treated with lactic acid alone (reductions of 4.9 log CFU/g), whereas for all other treatments, microbial destruction (reductions of 2.2 to 4.5 log CFU/g) was comparable ( P > 0.05) to that of the control. Cooking weight losses were lower ( P < 0.05) in salt/phosphate samples (<1%) compared to other formulations and the control (7.4% to 15.9%). Findings indicated that, under the conditions examined, restructuring of beef with salt/phosphate, algin/calcium, Activa™RM, or Fibrimex® did not affect inactivation of internalized E . coli O157:H7 in undercooked (60 or 65 °C) samples, whereas inclusion of lactic acid (0.27%) in nonintact beef products enhanced pathogen destruction at 65 °C.     

INSTABILITY OF SODIUM NITRITE IN A CHEMICALLY DEFINED MICROBIOLOGICAL MEDIUM

Mixtures of sodium nitrite, amino acids and ascorbic acid at pH 6.3 or 7.2 were filter sterilized or heated for various times at 121°C. Samples were analyzed for nitrite concentration immediately after treatment and after storage at 37 or 43°C. Heating the complete medium for 15 min at 121°C reduced the nitrite concentration by 30–50% independent of the initial nitrite concentration. Storage of complete filter sterilized medium at 43°C for 18–24 hr resulted in 50–65% loss of nitrite while in heat sterilized medium the loss was as great as 90%. None of the 19 amino acids stored individually with sodium nitrite at 37°C for up to 4 hr affected the nitrite concentration, whereas ascorbic acid resulted in total disappearance of nitrite. During heating at 121°C of the 19 amino acids only cystine resulted in any significant nitrite loss after 15 min of treatment while ascorbic acid effected total nitrite disappearance after 5 min. The defined medium containing nitrite demonstrated inhibitory activity against seven strains of Clostridium perfringens.