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 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.     

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.     

Packaging of Scallops with Sorbate: An Assessment of the Hazard from Clostridium botulinum

Toxin production in shucked scallops by a mixture of 10 strains of Clostridium botulinum (types A, B, E, F) was studied using mouse bioassay. Scallops, with or without 0.1% potassium sorbate, were packaged in air-permeable film or vacuum packaged in impermeable film. Two trials were done in which packages were inoculated with different spore levels and held at 4°, 10° and 27°C. Bacterial counts were made on various selective media. Only type A toxin was observed and this only in evacuated packages held at 27°C. Toxin developed more rapidly in the presence of sorbate. All packages in which toxin was recorded had offensive odors and were visibly spoiled. The potential hazard from botulism is discussed.     

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

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.     

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.     

Effects of Pulsed Electric Fields on Microorganisms in Orange Juice Using Electric field Strengths of 30 and 50 kV/cm

ABSTRACT: Inactivation of 4 microorganisms in orange juice was investigated in a 100 L/h flowing pulsed electric field (PEF) system. Electric field levels of 30 kV/cm and 50 kV/cm were applied, and Leuconostoc mesenteroides, E. coli , and Listeria innocua were inactivated by as much as 5 log cycles at 30 kV/cm and 50 °C. Saccharomyces cerevisiae ascospores were the least susceptible to PEF at all treatment levels, and a maximum of 2.5 log reduction was achieved at 50 kV/cm and 50 °C. Both electric field levels were effective in inactivating microorganisms at temperatures below standard thermal treatment, however, the number of pulses applied was particularly important in inactivation.     

Aflatoxin Inactivation Using Aqueous Extract of Ajowan (Trachyspermum ammi) Seeds

ABSTRACT: Aqueous extract of ajowan seeds was found to contain an aflatoxin inactivation factor (IF). Thin layer chromatography analysis of the toxins after treatment with IF showed relative reduction of aflatoxin G₁ > G₂ > B₁ > B₂. Quantification of toxin using a fluorotoxin meter as well as the Enzyme Linked Immuno s orb ent Assay (ELISA) confirmed these findings. An approximate 80% reduction in total aflatoxin content over the controls was observed. This observed phenomenon of reduction in total toxin was referred to as toxin inactivation. Temperature was found to influence the rate of toxin inactivation. At 45 °C, it was found to be rapid during the initial 5 h and slowed later. The IF was found to retain considerable activity even after boiling and autoclaving, indicating partial heat stability. The activity was lost below pH 4.0. Above pH 4.0, it increased gradually, reaching the maximum at pH 10.0. IF was found to be stable to gamma irradiation. Toxin decontamination in spiked corn samples could be achieved using IF. This study emphasizes the potential of ajowan IF in aflatoxin removal from contaminated food commodities. However, the biological toxicity, if any, of the IF inactivated aflatoxins needs to be confirmed, and the work in this direction is in progress.     

CHARACTERISTICS OF PROGENY OF ETHYLENE OXIDE TREATED Clostridium botulinum TYPE 62A SPORES

SUMMARY– Spores of Clostridium botulinum Type 62A were exposed to a mixture of 12% ethylene oxide (ETO) and 88% dichlorodifluoromethane to determine if the ETO resistance, toxin producing ability and spore producing ability would be retained in progeny of successive generations exposed to ETO. D values for the spores exposed to 700 mg per liter of ETO, at 40°C and 47% relative humidity, showed that there was no significant difference in ETO resistance through four successive generations of survivors. Toxin producing ability of the various generations was shown to be qualitatively similar by injecting mice intraperitoneally with culture supernatants. Microscopic examination of the different generations revealed no difference in rate or extent of spore production. Therefore, according to these criteria, there appeared to be no adaptations or mutations caused by four ETO exposures.     

Synergistic Effect of Electrolyzed Water and Citric Acid Against Bacillus Cereus Cells and Spores on Cereal Grains

ABSTRACT:  The effects of acidic electrolyzed water (AcEW), alkaline electrolyzed water (AlEW), 100 ppm sodium hypochlorite (NaClO), and 1% citric acid (CA) alone, and combinations of AcEW with 1% CA (AcEW + CA) and AlEW with 1% CA (AlEW + CA) against Bacillus cereus vegetative cells and spores was evaluated as a function of temperature (25, 30, 40, 50, or 60 °C) and dipping time (3 or 6 h). A 3-strain cocktail of Bacillus cereus cells or spores of approximately 10⁷ CFU/g was inoculated in various cereal grains (brown rice, Job's tear rice, glutinous rice, and barley rice). B . cereus vegetative cells and spores were more rapidly inactivated at 40 °C than at 25 °C. Regardless of the dipping time, all treatments reduced the numbers of B . cereus vegetative cells and spore by more than 1 log CFU/g, except the deionized water (DIW), which showed approximately 0.7 log reduction. The reductions of B . cereus cells increased with increasing dipping temperature (25 to 60 °C). B . cereus vegetative cells were much more sensitive to the combined treatments than spores. The effectiveness of the combined electrolyzed water (EW) and 1% CA was considerable in inhibiting B . cereus on cereal grains. The application of combined EW and CA for controlling B . cereus cells and spores on cereal grains has not been previously reported. Therefore, the synergistic effect of EW and CA may provide a valuable insight on reducing foodborne pathogens on fruits, vegetables, and cereal grains.     

Quality changes in fresh-cut Fuji apple as affected by ripeness stage, antibrowning agents, and storage atmosphere

ABSTRACT:  The effect of ripening state, modified atmosphere, and the use of antibrowning agents was investigated in an attempt to determine optimum ripeness and processing conditions for extending the shelf-life of fresh-cut Fuji apple. Apples were classified in 3 groups: mature-green, partially ripe, and ripe; after peeling and slicing, fruits were treated with 1% (w/v) N -acetylcysteine, or 1% (w/v) ascorbic acid (control), and then packed into polypropylene trays with air or a gas mixture (2.5% O₂+ 7% CO₂+ 90.5% N₂) and sealed. Trays containing the apple slices were stored in darkness at 4 °C ± 1 °C and analyzed periodically during 43 d. Changes in atmosphere composition, color, and firmness were examined. Partially ripe apples, based on their lower ethanol production and maintenance of their original color and firmness, were the most suitable to prepare the fresh-cut commodities. A postcutting dip in 1% (w/v) N -acetylcysteine was the most effective treatment to prevent cut surface browning and preserve the initial appearance of Fuji apple slices during more than 1 mo at 4 °C. Low O₂ and elevated CO₂ (2.5% O₂+ 7% CO₂) atmosphere extended the shelf life of apple slices because of a significant inhibition of ethylene production.     

EFFECTS OF CULTURAL CONDITIONS ON THE SYNTHESIS OF SALMONELLA TOXIN

The influence of culture age, pH, aeration, and temperature on Salmonella toxin production was investigated using fermentor cultures containing casamino acids-yeast extract as a growth medium. About 75% or more of total Salmonella toxin was synthesized and released when the cultures were harvested during stationary phase of growth. Increased production and release of the toxin was also observed when the cultures were regulated at pH 6.0, 7. 0, or under alkaline control. The optimal temperature for the synthesis of Salmonella toxin was at 37°C; increased aeration of the cultures (500 rpm) appeared to enhance Samonella toxin production.     

Influence of temperature shifts on survival, growth, and toxin production by psychrotrophic and mesophilic strains of Bacillus cereus in potatoes and chicken gravy.

A study was done to determine the influence of temperature on growth and toxin production characteristics of psychrotrophic and mesophilic strains of Bacillus cereus when inoculated into mashed potatoes and chicken gravy containing various concentrations of sodium chloride and held at temperatures different from those at which cells had been cultured. Logarithmic growth phase cells (10 h, 30 degrees C) of psychrotrophic (F3802A/84) and mesophilic (B4ac-1) strains of Bacillus cereus were inoculated into rehydrated commercially processed instant mashed potatoes and chicken gravy supplemented with 0, 2, or 4% sodium chloride. Growth, survival, and diarrheal toxin production in potatoes and gravy held at 30, 37, and 10 degrees C (strain F3802A/84) or 30, 40, and 10 degrees C (strain B4ac-1) were monitored. Both strains grew in both foods containing no added sodium chloride or 2% sodium chloride when held at 30, 37, or 40 degrees C for 2 days. Strain B4ac-1 grew better than strain F3802A/84 in foods containing 4% sodium chloride. Maximum amounts of enterotoxin (1024 ng/g) were produced by strain B4ac-1 in chicken gravy held at 30 and 40 degrees C. Strain F3802A/84 grew to populations of 7 log10 CFU/g in foods containing no added sodium chloride or 2% sodium chloride at 10 degrees C. Strain F3802A/84 produced the highest amount of enterotoxin (1024 ng/g) at 30 degrees C in chicken gravy containing 0.7 or 2% sodium chloride; however, little or low amounts of toxin (4-16 ng/g) were produced in chicken gravy at 10 degrees C. Compared to strain B4ac-1, cells of strain F3802A/84 subjected to a downward shift in incubation temperature (10 degrees C) grew more rapidly in chicken gravy. Strain B4ac-1 produced the highest amount of toxin (1024 ng/g) at 30 degrees C in gravy containing 4% sodium chloride and at 40 degrees C in gravy containing 0.7% sodium chloride. Toxin was not detected in inoculated mashed potatoes. Results of this study indicate that shifts in incubation temperature influence growth and toxin production by psychrotrophic and mesophilic strains of B. cereux differently. It is important to store pasteurized, ready-to-eat foods at a temperature low enough to prevent the growth of B. cereus.     

Effectiveness of chemical sanitizers against Campylobacter jejuni-containing biofilms

Survival of Campylobacter jejuni in mixed-culture biofilms was determined after treatment with chemical sanitizers including chlorine, quaternary ammonia, peracetic acid (PAA), and a PAA/peroctanoic acid mixture (PAA/POA). Biofilm-producing bacteria (gram-positive rods, Y1 and W1) were isolated from chicken house nipple drinkers. A meat plant isolate (Pseudomonas sp.) was also included as a biofilm producer. Two-day-old biofilms grown on polyvinyl chloride (PVC) plastic coupons in R2A broth at 12 degrees C were incubated with 10(6) CFU/ml C jejuni for 6 h to allow attachment. The coupons were then rinsed and incubated in fresh media for an additional 24 h. C. jejuni-containing biofilms were detached by vortexing with glass beads in modified brucella broth, which was then enumerated for C. jejuni on selective/differential media. The presence of biofilm enhanced (P < 0.01) the attachment and survival of C. jejuni After the 24-h incubation, only 20 CFU/cm2 of C. jejuni were recovered from the control without biofilms compared to 2,500 to 5,000 CFU/cm2 in samples with preexisting biofilms. The presence of biofilm microflora decreased (P < 0.01) the effectiveness of sanitizers against C. jejuni. Chlorine was the most effective sanitizer since it completely inactivated C. jejuni in the biofilms after treatment at 50 ppm for 45 s. C. jejuni in biofilms was susceptible to all sanitizers tested but was not completely inactivated by treatment with quaternary ammonia, PAA, or PAA/POA mixture at 50 and 200 ppm for 45 s.     

Responses of dairy cows to dietary aflatoxin: feed intake and yield, toxin content, and quality of milk of cows treated with pure and impure aflatoxin

Ten fistulated Holstein cows in midlactation were given daily doses of 13 mg of aflatoxin B1 for 7 days. Six received pure aflatoxin B1; three received an impure preparation that contained aflatoxin B1 plus other aflatoxins and metabolites produced by Aspergillus parasiticus in culture. Toxin was administered to each animal twice daily, one-half of the total dose each time, via the rumen orifice. Morning and evening milks were collected and analyzed for aflatoxin M1. Milk production and feed intake were monitored for 5 days before, every day during, and for 8 days after treatment with aflatoxin B1. Milk contained from 1.05 ppb to 10.58 ppb of aflatoxin M1. None was in milk 4 days after administration of toxin had stopped. Somatic cell counts and standard plate counts from milks of two cows were not affected appreciably by administration of toxin. Fluctuations in feed intake and milk production occurred in all animals during the treatment period with a significant decrease in milk production of those cows receiving 13 mg of impure aflatoxin B1 per day. Differences in results when cows received equal amounts of aflatoxin B1 may be attributable to the type of toxin administered (pure versus impure).     

复合菌系降解黄曲霉毒素B_1的效果及组成多样性研究

采用"外淘汰法"筛选了一组对黄曲霉毒素B_1(aflatoxin B_1,AFB_1)具有高效降解能力的复合菌系FBAD-2,该复合菌系在120 h内能将质量浓度为2 000μg/L的AFB_1完全降解,对质量浓度为5 000μg/L的AFB_1能降解90%。FBAD-2在30~70℃的范围内均能保持对AFB_1的高效降解能力,其最适温度为60℃。毒素降解实验分析表明,FBAD-2对AFB_1的降解主要是胞外酶的作用,其最适产酶时间为24 h,此时的胞外粗酶液在48 h内能将5 000μg/L的AFB_1完全降解。16S rDNA基因组测序分析结果表明,FBAD-2的微生物组成主要包括土芽孢杆菌(Geobacillus)、嗜热小杆菌(Symbiobacterium thermopilum)、梭菌(Clostridium)和热厌氧杆菌(Tepidanaerobacter)等。     

Bacillus cereus emetic toxin production in cooked rice

Three mesophilic strains of Bacillus cereus known to produce emetic toxin were used to model germination, growth and emetic toxin production in boiled rice cultures at incubation temperatures ranging from 8°C to 30°C. Minimum temperatures for germination and growth in boiled rice were found to be 15°C for all strains. Toxin production at 15°C was found to be significantly greater (P<0·01; reciprocal toxin titre of 373±124) than at 20°C and 30°C (reciprocal toxin titres 112±37 and 123±41, respectively). Toxin production became detectable after 48 h incubation at 15°C, with a maximum titre reached by 96 h. At 20°C and 30°C, toxin production was detected at 24 h incubation, with a maximum titre reached by 72 h. Toxin production at 15°C was detectable at lower bacterial counts (6·2 log₁₀ cfu g⁻¹), than with incubation at 20°C and 30°C (>7·0 log₁₀ cfu g⁻¹). In this study, the lower temperature limit for germination and growth on solid laboratory medium was found to be 12°C for all strains, i.e. 3°C lower than that observed in boiled rice.     

Improved multiplex PCR assay for simultaneous detection of Bacillus cereus emetic and enterotoxic strains

Toxin producing Bacillus cereus can cause enterotoxic and/or emetic food poisoning. In the present study, a multiplex PCR assay was developed to detect all toxin genes known to be involved in food poisoning of B. cereus in a single reaction. Specific primers for the detection of enterotoxic (entFM, hblC, nheA, and cytK) genes and emetic toxin production (2 primer pairs: ces, CER) were designed based on the GeneBank sequences. The developed multiplex PCR assay was evaluated in pure culture and artificially inoculated milk, using 43 B. cereus strains and non-target strains. In brief, sensitivity in pure culture was 10-fold or more higher than artificially inoculated milk in multiplex PCR detection limit assay. The presented PCR assay is a developed molecular tool for the rapid simultaneous detection of emetic and enterotoxin producing B. cereus 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.