EFFECT OF FILMS OF DIFFERENT OXYGEN TRANSMISSION RATE ON TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM TYPE E IN VACUUM PACKAGED COLD AND HOT SMOKED TROUT FILLETS

Studies were done to determine the effect of film oxygen transmission rate (OTR) on the time to toxicity in vacuum packaged cold and hot smoked rainbow trout fillets challenged with C. botulinum type E (10² spores/g) and stored at refrigerated conditions (4C), and under mild (8C) and moderate (12C) temperature abuse conditions. While no samples were toxic at 4C, toxin was detected within 28 days at 8C for cold smoked trout fillets vacuum packaged in films with high OTR. Toxin was also detected for most vacuum packaged hot smoked trout fillets within 14–28 days at 8C, with the exception of trout fillets packaged in films with an OTR > 10,000 cc/m²/day. In most cases at 8C, spoilage, based on odor/color scores, preceded or occurred simultaneously with toxigenesis. At 12C all cold and hot smoked trout were toxic after 14–21 days and samples packaged in films with an OTR <5000 cc/m²/day became toxic before, or at the same time as, samples became spoiled. This study has shown that vacuum packaging of trout fillets in low gas barrier films, ranging in OTR from approximately 3,000 to approximately 10,000 cc/m²/day at 24C and 0% relative humidity (RH), did not prevent the growth and toxin production by C. botulinum in vacuum packaged cold and hot smoked trout fillets at 12C. Additional barriers, other than the OTR of the packaging film, need to be considered to ensure the safety of vacuum packaged trout fillets, particularly at mild to moderate temperature abuse storage conditions.     

CHALLENGE STUDIES WITH PROTEOLYTIC CLOSTRIDIUM BOTULINUM IN YEAST AND CHEMICALLY LEAVENED CRUMPETS PACKAGED UNDER MODIFIED ATMOSPHERES

Challenge studies were done with proteolytic Clostridium botulinum (10³ spores/g) in yeast-and chemical-leavened crumpets (50-g) packaged in air with an ethanol vapor (2-G Ethicap®) generator or in 100% CO₂ and stored at ambient temperature (25C) for 30 days. Neurotoxin was detected in all gas- (CO₂) packaged crumpets after 5 days regardless of the method of leavening. While neurotoxin was delayed for 10 days in chemical-leavened Ethicap®-packaged crumpets, it was not detected in any similarly packaged yeast-leavened crumpets throughout storage. This inhibition of growth and neurotoxin production by C. botulinum was attributed to the production of ethanol by Saccharomyces cerevisiae in the yeast leavened crumpets, in conjunction with the ethanol vapor generated by the Ethicap® sachets (2-G), to levels to inhibitory to the growth of C. botulinum (>2.8% v/v). Subsequent challenge studies in sterile crumpets inoculated with either C. botulinum (10³ spores/g) or a co-inoculum of C. botulinum (10³ spores/g) and S. cerevisiae (10⁵ CFU/g) confirmed the significant role (p<0.001) of S. cerevisiae in enhancing the antibotulinal efficacy of ethanol vapor. These studies showed that the method of crumpet leavening could have a profound effect on the growth of and neurotoxin production by C. botulinum in crumpets packaged under modified atmospheres.     

EFFECT OF HEADSPACE OXYGEN AND FILMS OF DIFFERENT OXYGEN TRANSMISSION RATE ON TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM TYPE E IN RAINBOW TROUT FILLETS STORED UNDER MODIFIED ATMOSPHERES

Studies were conducted to determine the effect of various levels of headspace oxygen (0–100%, balance CO₂) or film oxygen transmission rate (OTR) on the time to toxicity in modified atmosphere packaged (MAP) fresh trout fillets challenged with C. botulinum type E (10² spore/g) and stored under moderate temperature abuse conditions (12C). In all cases, trout were toxic within 5 days, irrespective of the initial levels of oxygen in the package headspace. However, spoilage preceded toxigenesis. Packaging of trout fillets in low gas barrier films, with OTRs ranging from 4,000 to 10,000 cc/m²/day at 24C and 0% relative humidity, also had no effect on time to toxicity in all MAP trout fillets. All fillets were toxic within 4–5 days and spoilage again preceded toxigenesis. This study has shown that the addition of headspace O₂, either directly to a package or indirectly by using a low gas barrier film, had no influence on the time to toxigenesis or spoilage. Additional barriers, other than headspace O₂ or film transmission rate, need to be considered to ensure the safety of MAP trout fillets, particularty at moderate temperature abuse conditions.     

EARLY DETECTION OF CLOSTRIDIUM PERFRINGENS ENTEROTOXIN AND ITS RELATIONSHIP TO SENSORY QUALITY OF COOKED CHICKEN

Growth, sporulation, and enterotoxin formation by Clostridium perfringens NCTC 8239 were determined in chicken thigh meat incubated at 45°C for 1.5 h and 37°C for up to 12.5 h. With an inoculum of 10⁶ vegetative cells per g, the cell counts reached mean log ₁₀ 7.32/g after 6 h of incubation and remained in that range through 14 h. Heat-resistant spores (log₁₀ 2.48/g) were first detected at 4 h, and the number increased to log₁₀ 5.19/g at 14 h. Enterotoxin (0.19 μg/g) was first detected after 2 h of incubation (1.5 h at 45°C and 0.5 h at 37°C) in the absence of detectable sporulation, and the enterotoxin concentration increased to 0.76 μg/g after 14 h. Significant differences (p < 0.01) in the odor, color, and texture scores for inoculated versus uninoculated cooked chicken following 2 h incubation correlated with the production of enterotoxin and suggested that these parameters could be used as indices of chicken spoilage by C. perfringens.     

Effect of pH and CO2 on growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres.

The effect of pH and CO2 on both growth of and toxin production by Clostridium botulinum in English-style crumpets, packaged under modified atmospheres was investigated using a 2 x 2 factorial experiment. English-style crumpets (water activity, 0.990; pH 6.5 and 8.3) were inoculated with C. botulinum spores types A and proteolytic B (500 spores/g), packaged in either 60% CO2 (balance N2) or 100% CO2, stored at ambient temperature (25 degrees C), and monitored daily for toxicity. Toxin was detected after 4 days in crumpets packaged in 60% CO2, irrespective of initial product pH. Toxin production was delayed 1.5 to 3 days in crumpets packaged under 100% CO2. Analysis of variance indicated a significant interaction effect of pH and %CO2 on time of earliest toxin detection. Delay of toxin production was greatest for high pH (8.3) crumpets. All products were organoleptically acceptable at the time of toxigenesis, and therefore, high moisture-high pH bakery products, if contaminated with spores of C. botulinum, could become hazardous if packaged in atmospheres containing CO2.     

EFFECTS OF VARIOUS FRESH MEAT STORAGE METHODS ON THE COLOR OF COOKED GROUND PORK

The effects of microbial growth in raw materials on cooked pork color were investigated. In two trials with sow meat held aerobically at 2C for 3 weeks, microbial load reached spoilage levels (10⁷ cfu/g), pH increased to 6.46, and samples cooked to 71C had red exudate, shown by absorption spectroscopy to contain myoglobin and cytochrome c. Samples cooked to 82C also received high panel ratings for red color, due to red, flocculent precipitate in the exudate, but undenatured myoglobin levels were low. In sow meat held frozen or vacuum-packaged at 2C, pH after 3 weeks was 6.03 and 6.18, and plate counts were 10⁴ and 10⁷, respectively, but exudates after cooking were much less red. In five trials with fresh pork legs, total plate counts also reached 10⁷ cfu/g by 3 weeks storage, and pH increased to 6.37, but cooked samples were not red. Higher myoglobin levels in sow meat probably accounted for the red color and level of undenatured myoglobin remaining after cooking of high pH, spoiled samples.     

INACTIVATION OF BACTERIAL SPORES BY COMBINED ACTION OF HYDROSTATIC PRESSURE AND BACTERIOCINS IN ROAST BEEF

Foodborne bacterial spores are normally resistant to high hydrostatic pressure; however, at moderate pressure, they can be induced to germinate and outgrow. At this stage, they can be killed by bacteriocin-based biopreservatives (BP-containing pediocin and nisin at 3:7 ratio; BP_X, BP + 100 μg/mL lysozyme; BP_Y, BP_X+ 500 μg/mL Na-EDTA). Based on this principle, spores of the meat spoilage organism, Clostridium laramie (1–2 × 10² spores/bag) alone or a mixture of four clostridial spores (5 × 10³ spores/bag), Clostridium sporogenes, Clostridium perfringens, Clostridium tertium, and Clostridium laramie, were inoculated in roast beef in the presence of 5000 AU/g of bacteriocin-based biopreservatives. The roast beef samples were subjected to hydrostatic pressure (HP) at 345 MPa for 5 min at 60C and stored at 4 or 12C for 84 days or at 25C for 7 days. The HP treatment of roast beef samples inoculated with a mixture of clostridial spores could be stored for 42 days at 4C. The HP in combination with either BP_X or BP_Y extended the shelf-life of roast beef up to 7 days at 25C. The combined treatment of HP and BP controlled the growth of C. laramie spores and extended the shelf-life of roast beef for 84 days when stored at 4C.     

Nonproteolytic Clostridium botulinum toxigenesis in cooked turkey stored under modified atmospheres

The ability of nonproteolytic Clostridium botulinum type B spores to grow and produce toxin in cooked, uncured turkey packaged under modified atmospheres was investigated at refrigeration and mild to moderate abuse temperatures. Cook-in-bag turkey breast was carved into small chunks, surface-inoculated with a mixture of nonproteolytic C. botulinum type B spores, packaged in O2-impermeable bags under two modified atmospheres (100% N2 and 30% CO2:70% N2), and stored at 4, 10, and 15 degrees C. Samples were analyzed for botulinal toxin and indigenous microorganisms, as well as subjected to sensory evaluation, on days 0, 7, 14, 28, 42, and 60. Given sufficient incubation time, nonproteolytic C. botulinum type B grew and produced toxin in all temperature and modified atmosphere treatment combinations. At moderate temperature abuse (15 degrees C), toxin was detected by day 7, independent of packaging atmosphere. At mild temperature abuse (10 degrees C), toxin was detected by day 14, also independent of packaging atmosphere. At refrigeration temperature (4 degrees C), toxin was detected by day 14 in product packaged under 100% N2 and by day 28 in product packaged under 30% CO2:70% N2. Reduced storage temperature significantly delayed toxin production and extended the period of sensory acceptability of cooked turkey, but even strict refrigeration did not prevent growth and toxigenesis by nonproteolytic C. botulinum. At all three storage temperatures, toxin detection preceded or coincided with development of sensory characteristics of spoilage, demonstrating the potential for consumption of toxic product when spoilage-signaling sensory cues are absent.     

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.     

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.     

EFFECT OF ETHANOL VAPOR ON GROWTH AND TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM IN A HIGH MOISTURE BAKERY PRODUCT

To determine the effect of ethanol vapor on toxin production by Clostridium botulinum, studies were done in English style crumpets (a_w 0.990, pH 6.5) challenged with 500 spores/g C. botulinum types A and proteolytic B and packaged in high gas barrier bags [ethanol transmission rate (ETR) 0.21 g/m²/day @ 25 C]. Crumpets were packaged in air with either commercially available ethanol vapor generators (Ethicap® 2, 4 or 6G) or cotton wool pads saturated with 2, 4 or 6 g of 95% food grade ethanol and stored at 25C. Toxin was detected in all inoculated control crumpets (0% ethanol) after 5 days at ambient temperature (25C). Ethicap® 2G delayed toxicity for 10 days while complete inhibition (>21 days) was observed in all crumpets packaged with 4 or 6G Ethicap® or with 2, 4 or 6 g of ethanol per pad. However, all crumpets were overtly spoiled by this time. Both headspace ethanol and absorption of ethanol by crumpets increased as a function of Ethicap® size/weight of ethanol. Based on these preliminary studies, ethanol vapor would appear to be an effective additional barrier to control the growth and toxin production by C. botulinum in high moisture bakery products and ensure the safety of these products at ambient temperature.     

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.     

Growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres.

To determine the safety of a high moisture bakery product, packaged under modified atmospheres, challenge studies were done on English-style crumpets (water activity [a(w)] 0.990, pH 6.5) inoculated postbaking with Clostridium botulinum types A and proteolytic B spores (5 X 10(2) spores/g). Products were packaged either in air, in air with an Ageless FX200 oxygen absorbent, or in a CO2/N2 (60:40) gas mixture, stored at ambient temperature (25 degrees C), and monitored for toxicity daily. All inoculated crumpets were toxic within 4 to 6 days and were organoleptically acceptable at the time of toxigenesis. Counts of C. botulinum increased to approximately 10(5) CFU/g at the time of toxicity. To determine the effect of baking on product safety, subsequent challenge studies were done on crumpets inoculated with 5 x 10(2) spores/g (baked weight basis) prior to baking. All crumpets were toxic after only 6 days, irrespective of packaging conditions, and toxigenesis again preceded spoilage. Temperature profile studies showed that the maximum internal temperature reached during baking was 97 degrees C, and the total baking process was equivalent to 0.03 min at 121 degrees C. The actual time to toxin production in both studies (4 to 6 days) correlated well with the predicted time (3.4 days) using the U.S. Department of Agriculture Pathogen Modeling Program (version 5.1) for proteolytic strains of C. botulinum. These studies confirm that high moisture bakery products, if contaminated with C. botulinum spores either pre- or postbaking, could pose a public health hazard, if packaged in air (in a high gas barrier package where O2 was depleted and CO2 was generated during storage) or under modified atmosphere packaging conditions and stored at ambient temperature.     

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.     

SENSORY AND MICROBIOLOGICAL QUALITY EVALUATION OF VACUUM-PACKED SLICED COOKED CHICKEN BREAST. SHELF-LIFE ESTIMATION

Abstract The shelf-life of vacuum packed, sliced, cooked chicken-breast based on sensory and microbial changes as a function of temperature (2.3, 6.5, 10, 13.5 and 17.7C) was determined. Sensory evaluation and a microbiological study charted the development of lactic acid and psychotropic bacteria and of Brochothrix thermosphacta. Six different sensory methods were used to estimate product shelf-life; of these, the method based on average smell and taste was deemed the most suitable, since these parameters had a greater impact on shelf-life. From a microbiological point of view, mean shelf-life times were estimated at each temperature and compared with the estimates of the tasting panel. In the samples stored at the three lowest study temperatures (2.3, 6.5 and 10C), lactic acid and psychotropic bacteria counts of 10⁷–10⁸ cfu/g were not achieved; this agrees with the absence of sensory rejection at the end of the experiment. At 13.5 and 17.7C, mean shelf-life estimated microbiologically was shorter than that estimated using sensory methods. This difference, which here amounted to at least 8 days, is due to the so-called "delayed change", reported in previous experiments with cooked meat products.     

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.     

Model of the Inactivation of Bacterial Spores by Moist Heat and High Pressure

ABSTRACT: Formulae for the prediction of inactivation and accumulated lethality of bacterial spores under moist heat and high pressures were derived on the basis of classic thermodynamic and kinetics principles. The capability of the model to describe the inactivation of bacterial spores was verified using 2 independent data sets corresponding to Clostridium botulinum processed at 60°C to 75°C and Bacillus stearothermophilus processed at 92°C to 110°C. Both sets included pressures between 5 × 10⁸ Pa and 7 × 10⁸ Pa. The equation fit explained more than 86% of the variation of the rate constant data. The developed equations establish a strong foundation on which to compare high-pressure processing treatments of different systems. This is especially useful because most systems have different transient temperature-pressure conditions.     

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

Using 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.     

SHELF-LIFE OF COLESLAW MADE FROM CABBAGE TREATED WITH GASEOUS ACETIC ACID

Coleslaw containing 25% mayonnaise was formulated with untreated cabbage (control) and cabbage fumigated with two levels of gaseous acetic acid: 0.08 mL and 0.50 mL/100 g cabbage. A device consisting of a rotating barrel and vaporizing chamber was used to fumigate the shredded cabbage. Populations of aerobic microorganisms in coleslaw made from untreated cabbage reached 10⁸ cfu/g after 15 days in storage at 5C. Growth of lactic acid bacteria was extensive and in some cases this group was chiefly responsible for spoilage. Microorganisms were not detected in coleslaw made from cabbage fumigated at higher levels of acetic acid during 22 days in storage. Mayonnaise had a lethal effect on the microflora of cabbage, and fumigation with acetic acid prior to formulation of coleslaw increased the effect. The color of coleslaw was not affected by fumigation but CO₂ production during storage was reduced.     

Incidence, source and some properties of psychrotrophic Bacillus spp found in raw and pasteurized milk

Spores of psychrotrophic Bacillus spp were isolated from 58% of farm bulk tank milks and about 69% of pasteurized milks. Counts of Bacillus spp in about 10% of raw milk samples reached 1 × 10⁵ cfu/ml and above within seven days at 6°C. Psychrotrophic spore counts in pasteurized milks ranged from <0.5 to 170 spores/litre with an average of about 17/1. There was little correlation between the total bacterial count of the raw milk and presence of psychrotrophic Bacillus spores. There was some evidence that the bulk tank itself may be a source of contamination. The spores in pasteurized milk probably were not the result of postpasteurization contamination. The optimum germination temperature for psychrotrophic Bacillus spores was lower than that for spores of mesophilic strains. About 50% of the psychrotrophic Bacillus strains isolated from milk were capable of growth at 2°C.