Effect of Ethanol on the Growth of Clostridium botulinum

2003 ◽  
Vol 66 (4) ◽  
pp. 610-617 ◽  
Author(s):  
DAPHNE PHILLIPS DAIFAS ◽  
JAMES P. SMITH ◽  
BURKE BLANCHFIELD ◽  
BRIGITTE CADIEUX ◽  
GREG SANDERS ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Ethanol Concentration ◽  
Toxin Production ◽  
Lag Phase ◽  
Mouse Bioassay ◽  
Bacteriostatic Effect ◽  
Maximum Probability ◽  
Ethanol Level ◽  
Inhibition Of Growth ◽  
Group I

Model broth studies were carried out to investigate the effect of ethanol on the growth of proteolytic (group I) strains of Clostridium botulinum. Ethanol extended the pathogen's lag phase, decreased its exponential growth rate, and decreased its final level of growth in the stationary phase. In all cases, botulinum neurotoxin production was associated with growth. Micrographs of C. botulinum cultures grown at 37°C in trypticase peptone glucose yeast extract (TPGY) broths containing 2 and 4% ethanol showed elongation of vegetative cells and interference with cell division. The inhibition of growth and toxin production at the ethanol level predicted (5.5%, wt/wt) was confirmed by microscopy and by the mouse bioassay. A subsequent study was carried out to determine the combined effect of ethanol (0 to 8% [wt/wt]), water activity (aw; 0.953 to 0.997), and pH (6.2 to 8.2) on the probability of the growth of and neurotoxin production by proteolytic strains of C. botulinum (103 spores per ml). Growth and neurotoxin production occurred in 1 to 3 days in TPGY broths without ethanol (0%) and in 2 to 4 days in broths containing 2% ethanol regardless of the aw or pH levels (P < 0.005). Growth and neurotoxin production were delayed by an ethanol concentration of 4% ethanol and completely inhibited by a concentration of 6%. At an ethanol concentration of 4%, the probability of growth and toxin production over 365 days (Pt) was influenced by aw and pH. After 365 days, the maximum probability of growth and toxin production (Pmax) was 1 for all but one combination. However, τ, the time it took for 50% of all eventually positive replicates for any given combination of barriers to show growth and/or turbidity, ranged from <3 to 229 days. All tubes of TPGY broths that showed no growth after 365 days were subcultured in fresh TPGY broths. In all cases, growth and toxin production occurred within 24 h at 37°C, indicating the reversible (sporostatic and/or bacteriostatic) effect of ethanol on C. botulinum.

Toxin Production by Clostridium botulinum Type E in Packaged Channel Catfish

1997 ◽  
Vol 60 (11) ◽  
pp. 1358-1363 ◽  
Author(s):  
PING CAI ◽  
MARK A. HARRISON ◽  
YAO-WEN HUANG ◽  
JUAN L. SILVA
Keyword(s):  
Channel Catfish ◽  
Clostridium Botulinum ◽  
Modified Atmosphere Packaging ◽  
Toxin Production ◽  
Storage Conditions ◽  
Mouse Bioassay ◽  
Modified Atmosphere ◽  
Oxygen Barrier ◽  
Botulinum Type ◽  
Clostridium Botulinum Type E

Channel catfish were inoculated with 3 to 4 log spores/g of a mixed pool of four strains of C. botulinum type E (Beluga, Minnesota, G21-5, and 070) and were packaged with an oxygen-permeable overwrap, in an oxygen-barrier bag with a modified atmosphere of CO2-N2 (80:20) or in a master bag with the same modified atmosphere. Packaged fish were stored at either 4°C and sampled at intervals over 30 days or at 10°C and sampled at intervals over 12 days. An additional master bag treatment in which overwrap-packaged catfish was stored first at 4°C, then removed from the master bags and stored at 10°C, was sampled at intervals over 18 days. Toxin production was evaluated using the mouse bioassay. Aerobic psychrotrophic and anaerobic populations were enumerated, and product spoilage characteristics were noted. Under abusive storage conditions of 10°C, there was no difference among the potential for toxin production in the packaged fish, with botulinum toxin detected on fish from each package type by day 6. At 4°C, toxin production was detected on day 9 in the overwrapped packages, while it was on day 18 in the modified atmosphere packaging. No toxin was found in the master bags held continually at 4°C. Toxin was detected on day 18 from samples initially held at 4°C in the master bag and subsequently held at 10°C. Spoilage preceded toxin production for samples stored at 4°C for each type of packaging. At 10°C, spoilage and toxin detection times coincided.

Risk of Growth and Toxin Production by Clostridium botulinum Nonproteolytic Types B, E, and F in Salmon Fillets Stored Under Modified Atmospheres at Low and Abused Temperatures

1987 ◽  
Vol 50 (4) ◽  
pp. 330-336 ◽  
Author(s):  
GENERO W. GARCIA ◽  
CONSTANTIN GENIGEORGIS ◽  
SEPPO LINDROTH
Keyword(s):  
Clostridium Botulinum ◽  
Storage Time ◽  
Toxin Production ◽  
Lag Phase ◽  
Type B ◽  
Inoculum Level ◽  
Modified Atmospheres ◽  
Botulinum Type ◽  
Best Fit ◽  
Design Experiments

In factorial design experiments we inoculated fresh salmon fillets with a spore pool of 13 nonproteolytic strains of Clostridium botulinum type B, E, and F at 6 levels (10−1 to 104/50 g of fillet), and incubated at 1, 4, 8, 12 and 30°C under modified atmospheres (MA) of vacuum, 100% CO2 and 70% CO2 + 30% air for up to 60 d. The earliest time we detected toxin in the fillets at 30, 12 and 8°C, irrespective of MA, was after 1, 3–9 and 6–12 d of storage and required 100–103, 101–103, 101–102 spores/fillet. The probability (P) of toxin production was significantly (P<0.05) affected by temperature (T), MA storage time (ST), MA × T, MA × ST and T × ST. Only type B toxin was detected in the toxic fillets. No toxin was detected in fillets stored at 4°C for up to 60 d. Toxin detection coincided with spoilage at 30°C, but preceded spoilage at 8 and 12°C, and followed spoilage at 4°C. Using linear and logistic regression analysis, best fit equations were derived relating the length of the lag phase and P of toxin production to T, ST, MA and spore inoculum level.

scholarly journals Phage lysin that specifically eliminates Clostridium botulinum Group I cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhen Zhang ◽  
Meeri Lahti ◽  
François P. Douillard ◽  
Hannu Korkeala ◽  
Miia Lindström
Keyword(s):  
Vegetative Growth ◽  
Clostridium Botulinum ◽  
Early Stage ◽  
Divalent Cations ◽  
Toxin Production ◽  
Lytic Activity ◽  
Group I ◽  
Agricultural Industries ◽  
I Cells ◽  
Phage Lysin

AbstractClostridium botulinum poses a serious threat to food safety and public health by producing potent neurotoxin during its vegetative growth and causing life-threatening neuroparalysis, botulism. While high temperature can be utilized to eliminate C. botulinum spores and the neurotoxin, non-thermal elimination of newly germinated C. botulinum cells before onset of toxin production could provide an alternative or additional factor controlling the risk of botulism in some applications. Here we introduce a putative phage lysin that specifically lyses vegetative C. botulinum Group I cells. This lysin, called CBO1751, efficiently kills cells of C. botulinum Group I strains at the concentration of 5 µM, but shows little or no lytic activity against C. botulinum Group II or III or other Firmicutes strains. CBO1751 is active at pH from 6.5 to 10.5. The lytic activity of CBO1751 is tolerant to NaCl (200 mM), but highly susceptible to divalent cations Ca2+ and Mg2+ (50 mM). CBO1751 readily and effectively eliminates C. botulinum during spore germination, an early stage preceding vegetative growth and neurotoxin production. This is the first report of an antimicrobial lysin against C. botulinum, presenting high potential for developing a novel antibotulinal agent for non-thermal applications in food and agricultural industries.

Inhibition of Clostridium botulinum in Model Reduced-Sodium Pasteurized Prepared Cheese Products

2017 ◽  
Vol 80 (9) ◽  
pp. 1478-1488 ◽  
Author(s):  
Kathleen A. Glass ◽  
Ming Mu ◽  
Brian LeVine ◽  
Frank Rossi
Keyword(s):  
Clostridium Botulinum ◽  
Sorbic Acid ◽  
Interactive Effect ◽  
Stable Process ◽  
Toxin Production ◽  
Mouse Bioassay ◽  
Linear Quadratic ◽  
Cheese Products ◽  
Potassium Replacement ◽  
Process Cheese

ABSTRACT The 1986 Food Research Institute–Tanaka et al. model predicts the safety of shelf-stable process cheese spread formulations using the parameters of moisture, pH, NaCl, and disodium phosphate (DSP) to inhibit toxin production by Clostridium botulinum. Although this model is very reliable for predicting safety for standard-of-identity spreads, the effects of additional factors have not been considered. The objective of this study was to create a predictive model to include the interactive effect of moisture, pH, fat, sorbic acid, and potassium-based replacements for NaCl and DSP to reflect modern reduced-sodium recipes. Eighty formulations were identified using a central composite design targeting seven factors: 50 to 60% moisture, pH 5.4 to 6.2, 0 to 0.2% sorbic acid, 10 to 30% fat, 1.7 to 2.4% NaCl, 0.8 to 1.6% DSP, and 0 to 50% potassium replacement for sodium salts. Samples were inoculated with proteolytic C. botulinum spores at 3 log spores per g, hot filled into sterile vials, and stored anaerobically at 27°C. Samples were assayed at 0, 1, 2, 3, 4, 8.5, 17.5, 26, and 40 weeks for the presence of botulinum toxin using the mouse bioassay. A parametric survival model was fit to the censored time-to-toxin data. All linear, quadratic, and pairwise effects were considered for model fit. As hypothesized, the effects of pH, sorbate, moisture, DSP, and NaCl were highly significant (P < 0.001). Fat concentration and potassium replacement effects were significant at P < 0.021 and P < 0.057, respectively. The model consistently predicted the safety failure of the toxic samples, but it also predicted failure for some samples that were not toxic. This model is an adjunct to existing models by adding the factors of potassium salts, fat, and sorbic acid to predict the botulinal safety of prepared process cheese products but is not intended to be a substitute for formulation evaluation by a competent process authority.

Amplified Immunoassay ELISA-ELCA for Measuring Clostridium botulinum Type E Neurotoxin in Fish Fillets

1994 ◽  
Vol 57 (11) ◽  
pp. 985-990 ◽  
Author(s):  
MICHAEL G. ROMAN ◽  
JOHN Y. HUMBER ◽  
PAUL A. HALL ◽  
N. RUKMA REDDY ◽  
HAIM M. SOLOMON ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Toxin Production ◽  
Coagulation Cascade ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mouse Bioassay ◽  
Sampling Sequence ◽  
Botulinum Type ◽  
Catfish Fillets ◽  
Clostridium Botulinum Type E ◽  
Coagulation Assay

The measurement of Clostridium botulinum type E toxin in fish was accomplished using an amplified immunoassay (enzyme-linked immunosorbent assay-enzyme-linked coagulation assay [ELISA-ELCA]) based on the coagulation cascade. Fresh catfish fillets inoculated with a mixture of spores from five strains of C. botulinum type E were packaged in high barrier film with air, vacuum and modified atmosphere and stored at 4, 8 or 16°C for up to 75 days. Toxin production was monitored during storage by both mouse bioassay (trypsin and non-trypsin treated) and ELISA-ELCA on the non-trypsinized samples. All 26 inoculated products that were positive by the mouse bioassay were also positive by ELISA-ELCA. Of 35 uninoculated samples which were not toxic in mouse bioassay, none were positive by ELISA-ELCA; of 73 inoculated samples which were not toxic by mouse bioassay, 14 had toxin measurable by the ELISA-ELCA. The position of these immunoassay-positives in the sampling sequence indicated that the toxin was identified by the immunoassay before it was found in the mouse bioassay. These results suggest that the ELISA-ELCA technique is a usable alternative to the mouse bioassay for monitoring C. botulinum type E toxin production in fish challenge studies.

Growth and Production of Toxin of Clostridium botulinum Type E in Rainbow Trout under Various Storage Conditions

1995 ◽  
Vol 58 (8) ◽  
pp. 863-866 ◽  
Author(s):  
DONNA M. GARREN ◽  
MARK A. HARRISON ◽  
YAO-WEN HUANG
Keyword(s):  
Rainbow Trout ◽  
Botulinum Toxin ◽  
Clostridium Botulinum ◽  
Toxin Production ◽  
Most Probable Number ◽  
Mouse Bioassay ◽  
Oxygen Barrier ◽  
Probable Number ◽  
Botulinum Type ◽  
Clostridium Botulinum Type E

Rainbow trout (Oncorhynchus mykiss) were inoculated with 3 to 4 1og10 spores per g of fish of a mixed pool of four strains of Clostridium botulinum type E (Beluga, Minnesota, G21-5, and 070). The trout were vacuum-skin packaged with either oxygen-barrier or oxygen-permeable films. Trout packaged with oxygen-permeable film were stored at 4°C for 21 days, while trout packaged with oxygen-barrier film were stored either at 4°C for 21 days or at 10°C for 15 days. Storage at 10°C was used to simulate commercial temperature abuse. Clostridium botulinum outgrowth was determined by a most probable-number (MPN) method using (tryptone peptone yeast extract glucose trypsin) anaerobic broth. Toxin production was evaluated using a mouse bioassay. Psychrotrophic and anaerobic populations increased with time regardless of packaging type. After 6 days at l0°C, botulinum toxin was detected in the packaged trout; however, the fish was noticeably spoiled before that time. No botulinum toxin was detected in trout packaged with either barrier or permeable films and stored at 4°C for 21 days, although the product was considered spoiled by day 12.

Influence of pH and Temperature on the Growth of and Toxin Production by Neurotoxigenic Strains of Clostridium butyricum Type E

2002 ◽  
Vol 65 (8) ◽  
pp. 1267-1270 ◽  
Author(s):  
FABRIZIO ANNIBALLI ◽  
LUCIA FENICIA ◽  
GIOVANNA FRANCIOSA ◽  
PAOLO AURELI
Keyword(s):  
Low Temperature ◽  
Yeast Extract ◽  
Food Industry ◽  
Room Temperature ◽  
Toxin Production ◽  
Clostridium Butyricum ◽  
Mouse Bioassay ◽  
Inhibition Of Growth ◽  
Foodborne Botulism ◽  
Influence Of Ph

Strains of Clostridium butyricum that produce botulinal toxin type E have been implicated in outbreaks of foodborne botulism in China, India, and Italy, yet the conditions that are favorable for the growth and toxinogenesis of these strains remain to be established. We attempted to determine the temperatures and pH levels that are most conducive to the growth of and toxin production by the six strains of neurotoxigenic C. butyricum that have been implicated in outbreaks of infective and foodborne botulism in Italy. The strains were cultured for 180 days on Trypticase–peptone–glucose–yeast extract broth at various pHs (4.6, 4.8, 5.0, 5.2, 5.4, 5.6, and 5.8) at 30°C and at various temperatures (10, 12, and 15°C) at pH 7.0. Growth was determined by checking for turbidity; toxin production was determined by the mouse bioassay. We also inoculated two foods: mascarpone cheese incubated at 25 and 15°C and pesto sauce incubated at 25°C. The lowest pH at which growth and toxin production occurred was 4.8 at 43 and 44 days of incubation, respectively. The lowest temperature at which growth and toxin production occurred was 12°C, with growth and toxin production first being observed after 15 days. For both foods, toxin production was observed after 5 days at 25°C. Since the strains did not show particularly psychrotrophic behavior, 4°C can be considered a sufficiently low temperature for the inhibition of growth. However, the observation of toxin production in foods at room temperature and at abused refrigeration temperatures demands that these strains be considered a new risk for the food industry.

Incidence of Clostridium botulinum in Vegetables Packaged under Vacuum or Modified Atmosphere

1996 ◽  
Vol 59 (1) ◽  
pp. 59-61 ◽  
Author(s):  
TIMOTHY LILLY ◽  
HAIM M. SOLOMON ◽  
E. JEFFERY RHODEHAMEL
Keyword(s):  
Clostridium Botulinum ◽  
Type A ◽  
Toxin Production ◽  
Mouse Bioassay ◽  
Modified Atmosphere ◽  
Type B ◽  
Green Pepper ◽  
Botulinum Type ◽  
Negative Controls ◽  
Anaerobic Environment

Because modified atmosphere-packaged (MAP) vegetables may provide an anaerobic environment conducive to Clostridium botulinum growth and toxin production, the incidence of C. botulinum spores in commercially available, precut MAP vegetables was determined. One-pound (454-g) packages of MAP vegetables were aseptically opened, added to freshly steamed and cooled sterile trypticase-peptone-glucose-yeast extract broth and incubated at 35°C for 7 days. Positive and negative controls were included with each sampling. After incubation the broth cultures were tested for toxicity by the standard mouse bioassay. Of the 1,118 MAP vegetable packages examined, one package each of shredded cabbage, chopped green pepper, and Italian salad mix contained C. botulinum type A spores. One additional salad mix (main ingredient, escarole) contained both C. botulinum type A and type B spores. Results indicated a low overall incidence rate (0.36%) of C. botulinum spores in commercially available precut MAP vegetables.

scholarly journals Inhibition of Growth of Nonproteolytic Clostridium botulinum Type B in Sous Vide Cooked Meat Products Is Achieved by Using Thermal Processing but Not Nisin

2001 ◽  
Vol 64 (6) ◽  
pp. 838-844 ◽  
Author(s):  
MIIA LINDSTRÖM ◽  
MIRJA MOKKILA ◽  
EIJA SKYTTÄ ◽  
EIJA HYYTIÄ-TREES ◽  
LIISA LÄHTEENMÄKI ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Clostridium Botulinum ◽  
Ground Beef ◽  
Toxin Production ◽  
Heat Processing ◽  
Type B ◽  
Treatment Needs ◽  
Inhibition Of Growth ◽  
Botulinum Type ◽  
Sous Vide

The safety of refrigerated processed foods of extended durability (REPFEDs) with respect to nonproteolytic Clostridium botulinum is under continuous evaluation. In the present study, mild ( values 0 to 2 min [P, pasteurization value; z-value 7.0°C; reference temperature 85.0°C]) and increased ( values 67 to 515 min) heat treatments were evaluated in relation to survival of nonproteolytic C. botulinum type B spores in sous vide processed ground beef and pork cubes. The use of two concentrations of nisin in inhibition of growth and toxin production by nonproteolytic C. botulinum in the same products was also evaluated. A total of 96 samples were heat processed and analyzed for C. botulinum by BoNT/B gene-specific polmerase chain reaction and for botulinum toxin by a mouse bioassay after storage of 14 to 28 days at 4 and 8°C. Predictably, after mild processing all samples of both products showed botulinal growth, and one ground beef sample became toxic at 8°C. The increased heat processing, equivalent to 67 min at 85°C, resulted in growth but not toxin production of C. botulinum in one ground beef sample in 21 days at 8°C; in the pork cube samples no growth was detected. The increased heating of both products resulted in higher sensory quality than the milder heat treatment. Nisin did not inhibit the growth of nonproteolytic C. botulinum in either product; growth was detected in both products at 4 and 8°C, and ground beef became toxic with all nisin levels within 21 to 28 days at 8°C. Aerobic and lactic acid bacterial counts were reduced by the addition of nisin at 4°C. The study demonstrates that the mild processing temperatures commonly employed in sous vide technology do not eliminate nonproteolytic C. botulinum type B spores. The intensity of each heat treatment needs to be carefully evaluated individually for each product to ensure product safety in relation to nonproteolytic C. botulinum.

Risk of Clostridium botulinum Type E Toxin Production in Blue Crab Meat Packaged in Four Commercial-Type Containers

1996 ◽  
Vol 59 (3) ◽  
pp. 257-260 ◽  
Author(s):  
MARK A. HARRISON ◽  
DONNA M. GARREN ◽  
YAO-WEN HUANG ◽  
KEITH W. GATES
Keyword(s):  
Botulinum Toxin ◽  
Blue Crab ◽  
Clostridium Botulinum ◽  
Storage Period ◽  
Toxin Production ◽  
Mouse Bioassay ◽  
Botulinum Type ◽  
Clostridium Botulinum Type E ◽  
Entire Storage Period ◽  
Crab Meat

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 103 to 104 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°C for 21 days or 10°C for 15 days. Storage at 10°C was used to simulate temperature abuse. The mouse bioassay was used to detect the presence of C. botulinum toxin. Psychrotrophic 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°C or 10°C throughout the entire storage period.

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