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.     

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.     

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.     

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.     

Effects of mastic resin and its essential oil on the growth of proteolytic Clostridium botulinum

Studies were done to determine the effect of mastic resin and its essential oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of Clostridium botulinum in media, and on neurotoxin production in challenge studies with English-style crumpets. Preliminary studies, using a spot-on-the-lawn method, indicated that high levels of mastic resin in ethanol ( approximately 8% w/w) were required for complete inhibition of all strains of C. botulinum tested, but mastic resin in ethanol had a greater anti-botulinal effect than ethanol alone. However, only low levels of mastic oil ( approximately 0.3% v/v) were required for inhibition of proteolytic strains of C. botulinum. Both studies showed a strain specific inhibition, with C. botulinum type A strains being more sensitive to mastic resin and its essential oil than type B strains. However, mastic resin in ethanol proved to be more effective when used as a vapor phase inhibitor applied to cotton pads and placed inside inoculated plates than when added directly to media. While both mastic resin and its essential oil inhibited the growth of proteolytic strains of C. botulinum in vitro, they failed to inhibit neurotoxin production in challenge studies with C. botulinum in English-style crumpets.     

CHALLENGE STUDIES WITH CLOSTRIDIUM BOTULINUM TYPE E IN A VALUE-ADDED SURIMI PRODUCT STORED UNDER A MODIFIED ATMOSPHERE

Challenge studies were carried out on raw, cooked, and sterilized surimi nuggets, inoculated with 10⁴ spores/g of C. botulinum type E spores. All products were packaged in air and air with an Ageless SS oxygen absorbent and stored at 4, 12 and 25C. Toxin was not detected in any raw product throughout storage (28 days). The absence of toxigenesis was attributed to the low pH (4.1–4.3) due mainly to the growth of lactic acid bacteria (10⁷CFU/g). Toxin was also not detected in any cooked product after 28 days. Product pH did not decrease as previously (due to the absence of LAB), but counts of C. botulinum still decreased throughout storage.
In sterile nuggets , C. botulinum counts increased to 10⁶ cfu/g at both 12 and 25C, respectively, by 28 days. Lactic acid bacteria and Bacillus spp. were not detected throughout the 28 days storage period. Toxin was detected by days 28 and 14 at 12 and 25C, respectively, and toxigenesis preceded spoilage. The absence of toxin in cooked nuggets was attributed to the anti-botulinal role by Bacillus species, the predominant spoilage bacteria in cooked nuggets.     

Growth and toxin production of proteolytic Clostridium botulinum in aseptically steamed rice products at pH 4.6 to 6.8, packed under modified atmosphere, using a deoxidant pack

Demand for aseptically steamed rice products has been increasing rapidly in Japan over the past 10 years. In our previous study, we showed that proteolytic Clostridium botulinum produce toxins in steamed rice products packaged under a modified atmosphere of < or =0.3% oxygen. In the present study, we examined the effect of pH to control botulism risk in steamed rice products packaged under modified atmosphere (5% CO2 and 95% N2 as the balance) with the inclusion of a deoxidant pack to produce an oxygen concentration of < or =0.3%. A mixture of 10 strains of proteolytic C. botulinum (5 type A strains and 5 type B strains) was inoculated into steamed rice products at pH values between 4.6 and 6.8 prior to packaging. All samples were stored at 30 degrees C for 24 weeks. Samples at higher pH showed earlier starts of neurotoxin production. Neurotoxin was detected after 2 weeks of incubation in samples at pH 5.4 or above, whereas it took 4 weeks for the toxin to be detected in samples at pH 5.2 to 5.3 and 12 weeks in samples at pH 5.0 to 5.1. In samples at pH 4.9 or below, no toxin was detected during the experimental period. Apparent sample spoilage did not occur before C. botulinum produced neurotoxin in most of the samples. Based on these results, we conclude that aseptically steamed rice products must be packaged at pH 4.9 or below under modified atmosphere containing < or =0.3% oxygen, with the inclusion of a deoxidant pack.     

Clostridium botulinum Growth and Toxigenesis in Shelf-Stable Noodles

Unacidified and acidified noodles were inoculated with a composite of C. botulinum types A and B spores. Noodles were cooked, packaged in oxygen-permeable polypropylene bags, steamed, cooled, then stored aerobically at 7 or 23°C, or anaerobically at 30°C. Toxin did not develop in any aerobically stored samples at pH ±4.5. Toxin developed in samples with pH >5.0, stored aerobically and anaerobically. In addition, toxin was detected in one anaerobically incubated acidified unit where pH had been increased by microbial growth. Proper acidification of product prevented toxic growth of C. botulinum.     

Clostridium botulinum spores and toxin in mascarpone cheese and other milk products.

A total of 1,017 mascarpone cheese samples, collected at retail, were analyzed for Clostridium botulinum spores and toxin, aerobic mesophilic spore counts, as well as pH, a(w) (water activity), and Eh (oxidation-reduction potential). In addition 260 samples from other dairy products were also analyzed for spores and botulinum toxin. Experiments were carried out on naturally and artificially contaminated mascarpone to investigate the influence of different temperature conditions on toxin production by C. botulinum. Three hundred and thirty-one samples (32.5%) of mascarpone were positive for botulinal spores, and 7 (0.8%) of the 878 samples produced at the plant involved in an outbreak of foodborne botulism also contained toxin type A. The chemical-physical parameters (pH, a(w), Eh) of all samples were compatible with C. botulinum growth and toxinogenesis. Of the other milk products, 2.7% were positive for C. botulinum spores. Growth and toxin formation occurred in naturally and experimentally contaminated mascarpone samples after 3 and 4 days of incubation at 28 degrees C, respectively.     

Botulism challenge studies of a modified atmosphere package for fresh mussels: inoculated pack studies

A series of botulism challenge studies were performed to determine the possibility of production of botulinum toxin in mussels (Mytilus edulis) held under a commercial high-oxygen (60 to 65% O(2)), modified atmosphere packaging (MAP) condition. Spore mixtures of six strains of nonproteolytic Clostridium botulinum were introduced into mussel MAP packages receiving different packaging buffers with or without the addition of lactic acid bacteria. Dye studies and package flipping trials were conducted to ensure internalization of spores by packed mussels. Inoculated mussel packages were stored at normal (4°C) and abusive (12°C) temperatures for 21 and 13 days, respectively, which were beyond the packaged mussels' intended shelf life. Microbiological and chemical analyses were conducted at predetermined intervals (a total of five sampling times at each temperature), including total aerobic plate counts, C. botulinum counts, lactic acid bacterial counts, package headspace gas composition, pH of packaging buffer and mussel meat, and botulinum toxin assays of packaging buffer and mussel meat. Results revealed that C. botulinum inoculated in fresh mussels packed under MAP packaging did not produce toxin, even at an abusive storage temperature and when held beyond their shelf life. No evidence was found that packaging buffers or gas composition influenced the lack of botulinum toxin production in packed mussels.     

The effect of 100% CO2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures

The growth of a cocktail of spores from six nonproteolytic Clostridium botulinum type B and E isolates at 5 and 10 degrees C was used to assess the combined effect of NaCl (0.5-4.5% w/v), pH (5.5-6.5) and atmosphere (10% H2:90% N2, 5% CO2:10% H2:85% N2, or 100% CO2) in buffered peptone, yeast, glucose, starch broth with an Eh of approximately -350 mV. Under all atmospheres growth tended to be slower as the concentration of NaCl increased and with NaCl combined with pH levels below 6.0. Of the atmospheres tested, growth occurred at a slower rate and over a narrower range of conditions when C. botulinum was exposed to 100% CO2. This effect was enhanced when the incubation temperature was 5 degrees C. The results indicate that while CO2 decreased C. botulinum growth at chill temperatures, prevention of growth also depended on the NaCl concentration and the pH of the medium.     

Effects of modified atmosphere packaging on toxin production by Clostridium botulinum in raw aquacultured summer flounder fillets (Paralichthys dentatus)

Packaging fishery products under vacuum atmosphere packaging (VAC) and modified atmosphere packaging (MAP) conditions can significantly extend the shelf life of raw, refrigerated fish products. There is considerable commercial interest in marketing VAC and MAP refrigerated (never frozen) raw fish fillets. The objective of this study was to determine if Clostridium botulinum toxin development precedes microbiological spoilage in raw, refrigerated flounder fillets. Aquacultured flounder (Paralichthys dentatus) individual fish fillets either were packed with a film having an oxygen transmission rate (OTR) of 3000 cm3 m(-2) 24 h(-1) at 22.8 degrees C or were vacuum packaged or packaged under 100% CO2 with a film having an OTR of 7.8 cm3 m(-2) 24 h(-1) at 21.1 degrees C and were stored at 4 and 10 degrees C. Samples were analyzed by aerobic plate count (APC) for spoilage and qualitatively for botulinum toxin with a mouse bioassay. The results demonstrate that flounder fillets (4 degrees C) packaged with a film having an OTR of 3,000 were microbiologically spoiled (APC, > 10(7) CFU/g) on day 15, but there was no toxin formation, even after 35 days of storage. However, at 10 degrees C, toxin production occurred (day 8), but it was after microbial spoilage and absolute sensory rejection (day 5). Vacuum-packaged fillets and 100% CO2 fillets (4 degrees C) packaged with a film having an OTR of 7.8 were toxic on days 20 and 25, respectively, with microbial spoilage (APC, >10(7) CFU/g) not occurring during the tested storage period (i.e., >35 days). At 10 degrees C, in vacuum-packaged flounder, toxin formation coincided with microbiological spoilage (days 8 to 9). In the 100% CO2-packaged fillets, toxin formation occurred on day 9, with microbial spoilage occurring on day 15. This study indicates that films with an OTR of 3,000 can be used for refrigerated fish fillets and still maintain the safety of the product.     

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.     

Toxin Development by Clostridium botulinum in Modified Atmosphere-Packaged Fresh Tilapia Fillets During Storage

Potential for toxin development by Clostridium botulinum type E was investigated in retail-type packages of fresh tilapia fillets packaged in high barrier film under selected atmospheres [100% air, a modified atmosphere (MA) containing 75%CO₂:25%N₂, and vacuum] and stored under refrigeration (4°C) and abuse temperatures (8 and 16°C). Toxin development coincided with sensory spoilage at 16°C storage for fillets packaged in either MA, or vacuum. At 8°C, toxin development in fillets packaged in either of the atmospheres occurred 7-23 days after sensory spoilage. At 4°C, none of MA-packaged fillets became toxic until 10 days after onset of sensory spoilage. Toxin development did not precede sensory spoilage in any treatments or temperatures studied.     

Microbiological quality and production of botulinal toxin in film-packaged broccoli, carrots, and green beans.

The production of toxin by a 10-strain mixture of proteolytic Clostridium botulinum in fresh produce packaged in polyethylene films with different oxygen permeability was determined. Broccoli florets, shredded carrots, and green beans inoculated with approximately 10(2) C. botulinum spores per g were placed in bags (1.4 kg per bag) composed of four films with different oxygen transmission rates (OTRs). Broccoli was packaged in bags with OTRs of 3 (7,000 cm3/m2/24 h) and 4 (16,000 cm3/m2/24 h), and green beans were packaged in bags with OTRs of 2 (6,000 cm3/m2/24 h) and 4. Broccoli and green beans in bags were compressed and heat-sealed. Shredded carrots were packaged in bags with OTRs of 1 (3,000 cm3/m2/24 h) and 3 and vacuum-sealed. Produce was stored at 4, 13, and 21 degrees C for up to 27 (broccoli) or 28 (carrots and green bean) days and analyzed periodically. At each sampling time, gas composition within the bags, pH of the produce microbial population (total aerobic and anaerobic microorganisms, lactic acid bacteria, psychrotrophic bacteria, yeasts, and molds), and the presence or absence of botulinal toxin were determined. Packaging material affected the quality of vegetables, especially broccoli stored at 4 and 13 degrees C. For example, broccoli was scored as "good" after 22 days at 4 degrees C when it was packaged in film with higher gas permeability (OTR of 4), whereas broccoli appeared to be in "poor" condition when packaged in film with lower gas permeability (OTR of 3). With the exception of lactic acid bacteria, packaging material did not noticeably influence the growth of microorganisms. Lactic acid bacteria grew better in broccoli packaged in bags with an OTR of 3 than in those with an OTR of 4 at all temperatures. Botulinal toxin was detected in broccoli packaged in bags with an OTR of 3 and stored at 13 degrees C for 21 days and in those with an OTR of 4 and 3 and stored at 21 degrees C for 10 days. All toxic samples were visibly spoiled. Toxin was not detected in produce packaged under any other test conditions.     

COMPARISON OF MARGIN OF SAFETY BETWEEN SENSORY SPOILAGE AND ONSET OF CLOSTRIDIUM BOTULINUM TOXIN DEVELOPMENT DURING STORAGE OF MODIFIED ATMOSPHERE (MA)-PACKAGED FRESH MARINE COD FILLETS WITH MA-PACKAGED AQUACULTURED FISH FILLETS

The margin of safety between shelf-life (onset of sensory spoilage) and potential time to toxin production by Clostridium botulinum type E in retail type packages of fresh marine cod fillets packaged in high barrier film under selected atmospheres [100% air, a modified atmosphere (MA) containing 75% CO₂:25% N₂, and vacuum] and stored under refrigeration (4C) and temperature-abuse conditions (8 and 16C) was investigated. Margin of safety data of MA-packaged marine cod fillets was also compared with MA-packaged aquacultured tilapia, catfish, and salmon fillets. Sensory spoilage preceded onset of toxin presence for the marine cod fillets packaged in any of the atmospheres and storage temperatures tested. At 4C, none of the marine cod fillets packaged in either atmosphere developed toxin, even 20 days after spoilage, as determined by sensory characteristics. During storage at refrigeration and mild (8C) temperature-abuse conditions, MA-packaged marine cod showed a greater margin of safety compared to aquacultured tilapia, catfish, and salmon packaged under the same atmospheres and conditions. Fat content appeared to potentially influence the margin of safety in MA-packaged aquacultured fresh fish fillets during storage.     

Evaluation of botulinal toxin production in packaged fresh-cut cantaloupe and honeydew melons.

The ability of Clostridium botulinum to produce toxin on cubed, packaged melons was investigated relative to microbial spoilage at various incubation temperatures and in different packaging systems. Freshly cut cubes (approximately 2.5 cm3) of cantaloupe and honeydew melons were surface inoculated with a 10 strain mixture of proteolytic and nonproteolytic spores of C. botulinum (10 to 15 cubes per package; approximately 100 total spores per package). To initially evaluate toxin production and spoilage in a passively modified atmosphere, melon cubes were loosely packaged in air in polyethylene pouches, sealed, and incubated at 7 or 15 degrees C for up to 21 days. At various sampling intervals, samples were tested for headspace oxygen and carbon dioxide levels, pH, presence of botulinal toxin, aerobic and anaerobic plate counts, and counts of yeasts and molds. During incubation, headspace oxygen levels decreased, headspace carbon dioxide levels increased, aerobic and anaerobic plate counts increased, and the pH remained constant or decreased slightly. Botulinal toxin was not detected in any cantaloupe samples or in honeydew samples incubated at 7 degrees C. Botulinal toxin was detected in some honeydew samples at 15 degrees C after 9 days of incubation, but the toxic honeydews were severely spoiled and considered organoleptically unacceptable. A similar second experiment was performed in which half of the melon cubes were treated with UV light to inactivate vegetative organisms before packaging, and these were incubated at 7, 15, or 27 degrees C. In this second experiment, toxin production occurred in the UV-treated samples at 15 degrees C with gross spoilage and at 27 degrees C with only marginal spoilage. These data indicate that inhibition of spoilage organisms with UV light could result in botulinal toxin formation in packaged melons before overt spoilage.     

Risk of Clostridium botulinum type E toxin production in blue crab meat packaged in four commercial-type containers.

The aim of this investigation was to determine if a risk of Clostridium botulinum growth and toxin production existed in four different packaged crabmeat products. Freshly picked blue crab meat was inoculated with 10(3) to 10(4) spores per g of a mixed pool of four strains of C. botulinum type E (Beluga, Minnesota, G21-5, and 070). The lump crabmeat was packaged in four different packaging containers: (i) 12-oz copolymer polyethylene cups currently used by most crab processors; (ii) 12-oz copolymer polyethylene cups with heat-shrink, tamper-evident low-density polypropylene seals; (iii) 8-oz copolymer polyethylene cups with easy-open aluminum ends: and (iv) 8-oz copolymer polypropylene cups with integral tamper-evident pull-tabs. The packages were stored at either 4 degrees C for 21 days or 10 degrees C for 15 days. Storage at 10 degrees C was used to simulate temperature abuse. The mouse bioassay was used to detect the presence of C. botulinum toxin. Psychotrophic and anaerobic populations were enumerated and were found to increase with time regardless of packaging type. No botulinum toxin was detected in any of the four packaging types stored at 4 degrees C or 10 degrees C throughout the entire storage period.     

Shelf Life and Clostridium botulinum Toxin Development during Storage of Modified Atmosphere-packaged Fresh Catfish Fillets

Shelf life (onset of sensory spoilage) and potential for toxin production by Clostridium botulinum type E in retail type packages of fresh catfish fillets in high barrier film were investigated under selected atmospheres when stored under refrigeration and temperature-abuse conditions. Shelf life of fillets in all atmospheres decreased with increase of storage temperature from 4°C to 16°C. Trimethylamine content associated with onset of spoilage was different for each storage temperature and atmosphere. Surface pH and K-values were not good indicators of onset of sensory spoilage. Toxin development coincided with sensory spoilage at 16°C storage for fillets packaged in either atmosphere. At 4°C, none of the MA-packaged fillets became toxic, even after 37 days of sensory spoilage.     

Clostridium botulinum type E outgrowth and toxin production in vacuum-skin packaged shrimp

White and brown shrimp (Penaeus spp.), harvested from the Georgia coast, were inoculated with log 3-4 spores of a mixed pool of four strains of Clostridium botulinum type E (Beluga, Minnesota, G21-5, and 070). Oxygen barrier film was used to vacuum-skin package the shrimp. Packaged shrimp was stored at either 4°C or 10°C. Storage at 10°C was used to simulate temperature abuse. Anaerobes were enumerated by Most Probable Number method using TPYGT broth incubated in anaerobic jars. Clostridium botulinum toxin production was evaluated using the mouse bioassay. After six days at 10°C, botulinum toxin was detected in the packaged shrimp, but the shrimp was noticeably spoiled at this point. No botulinum toxin was detected in the packaged shrimp stored at 4°C for 21 days, although spoilage was noted after 6 days.