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.

Contribution of Composition, Physicochemical Characteristics and Polyphosphates to the Microbial Safety of Pasteurized Cheese Spreads

1994 ◽  
Vol 57 (4) ◽  
pp. 295-300 ◽  
Author(s):  
KARL F. ECKNER ◽  
WENDY A. DUSTMAN ◽  
ANNA A. RYŚ-RODRIGUEZ
Keyword(s):  
Clostridium Botulinum ◽  
Toxin Production ◽  
Physicochemical Characteristics ◽  
The Other ◽  
Microbial Safety ◽  
Processed Cheese ◽  
Moisture Contents ◽  
Glass Containers ◽  
Different Types ◽  
Process Cheese

Pasteurized process cheese spread was manufactured with moisture contents of 52, 54, 56 and 60%. Three different types of phosphate emulsifier were used, disodium ortho-phosphate and two commercially-available polyphosphates, S9 and S9H. Pasteurized, processed cheese spreads were inoculated with approximately 1 × 104 Clostridium botulinum spores/gram cheese in the cook kettle, held 3 min at 80°C, hot-filled into glass containers, and incubated at 30°C. Samples were analyzed over 30 weeks for growth of C. botulinum and toxigenesis. Toxin was first detected in 60% moisture cheese with disodium ortho-phosphate as the emulsifier at 8 weeks and in 60% moisture cheese with the test polyphosphates as the emulsifier when tested at 20 weeks. None of the other cheese formulations were toxic at 20 weeks. Toxin production correlated statistically to time, moisture, pH and phosphate type.

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.

Antibotulinal Activity of Process Cheese Ingredients

2004 ◽  
Vol 67 (8) ◽  
pp. 1765-1769 ◽  
Author(s):  
KATHLEEN A. GLASS ◽  
ERIC A. JOHNSON
Keyword(s):  
Whey Protein ◽  
Clostridium Botulinum ◽  
Antimicrobial Effect ◽  
Toxin Production ◽  
Whey Protein Concentrate ◽  
Protein Concentrate ◽  
Fat Replacer ◽  
Blue Cheese ◽  
Sweet Whey ◽  
Process Cheese

Ingredients used in the manufacture of reduced-fat process cheese products were screened for their ability to inhibit growth of Clostridium botulinum serotypes A and B in media. Reinforced clostridial medium (RCM) supplemented with 0,0.5, 1, 2, 3, 5, or 10% (wt/vol) of various ingredients, including a carbohydrate-based fat replacer, an enzyme-modified cheese (EMC) derived from a Blue cheese, sweet whey, modified whey protein, or whey protein concentrate, did not inhibit botulinal growth and toxin production when stored at 30°C for 1 week. In contrast, RCM supplemented with 10% soy-based flavor enhancer, 10% Parmesan EMC, or 5 or 10% Cheddar EMC inhibited botulinal toxin production in media for at least 6 weeks of storage at 30°C. Subsequent trials revealed that the antibotulinal effect varied significantly among 13 lots of EMC and that the antimicrobial effect was not correlated with the pH or water activity of the EMC.

Antibotulinal Effectiveness of Nisin in Pasteurized Process Cheese Spreads

1987 ◽  
Vol 50 (10) ◽  
pp. 842-848 ◽  
Author(s):  
EILEEN B. SOMERS ◽  
STEVE L. TAYLOR
Keyword(s):  
Sodium Chloride ◽  
Clostridium Botulinum ◽  
Toxin Production ◽  
Disodium Phosphate ◽  
Process Cheese

Pasteurized process cheese spreads were prepared at moisture levels ranging from 52 to 57% with added sodium chloride at levels from 0 to 2.0%, with disodium phosphate levels ranging from 1.4 to 2.5%, and with nisin levels of 0 to 250 ppm. Clostridium botulinum spores were added at a level of approximately 1000 spores per gram of cheese spread except for control batches and one experiment where the spore levels were varied (10–1000 spores/g). The cheese spreads were incubated at 30°C for up to 48 weeks. Nisin is an effective antibotulinal agent in pasteurized process cheese spreads. Addition of nisin allows formulation of pasteurized process cheese spreads with reduced sodium levels (addition of 1.4% disodium phosphate and no added sodium chloride) or slightly higher moisture levels (55–57%) by comparison to typical commercial pasteurized process cheese spreads. Higher levels of nisin (100 and 250 ppm) were required to prevent outgrowth of botulinal spores in cheese spreads with highest moisture levels or most greatly reduced sodium levels. However, in a cheese spread of 52% moisture prepared with 2.5% disodium phosphate but no added sodium chloride, a nisin level of 12.5 ppm was able to prevent completely outgrowth and toxin production by C. botulinum.

Growth of Proteolytic Clostridium botulinum in Process Cheese Products: I. Data Acquisition for Modeling the Influence of pH, Sodium Chloride, Emulsifying Salts, Fat Dry Basis, and Temperature

1995 ◽  
Vol 58 (10) ◽  
pp. 1091-1099 ◽  
Author(s):  
PIETER F. TER STEEG ◽  
HENK G. A. M. CUPPERS ◽  
JOHAN C. HELLEMONS ◽  
GUUS RIJKE
Keyword(s):  
Lactic Acid ◽  
Sodium Chloride ◽  
Clostridium Botulinum ◽  
Raw Materials ◽  
Challenge Test ◽  
Type A ◽  
Cheese Products ◽  
Process Cheese ◽  
Emulsifying Salts ◽  
Toxin Formation

Outgrowth of proteolytic Clostridium botulinum type A and B spores in pasteurized process cheese products was assessed to acquire data for improved models of botulinum stability. High-moisture (58.5%) products were made with different levels of pH (5.45 to 5.9), sodium chloride (1.1 to 2.8%, wt/wt) and citrates or phosphates as emulsifying salts (1.5 to 2%, wt/wt), and held at 15 to 30°C. Supplemental experiments were carried out to address the effect of lactic acid concentration originating from the nonfat and 50% fat dry basis (FDB) cheese raw materials, of moisture (50 to 69%), and of total fat (0.1 to 41%, wt/wt). Colony counts were recorded as substitutes for the traditional times to toxin formation. In the last experimental series a polyclonal ELISA against type A and B toxins was carried out as an alternative to the mouse challenge test. Very low spore levels could lead to detectable toxin formation. Temperature strongly influenced outgrowth. At 18°C outgrowth only occurred in 3 months at favorable aw (0.966) and pH (5.9). At 25°C, outgrowth occurred within one week under favorable conditions. No growth occurred within 3 months when aw and pH were 0.95 and 5.55 respectively. Polyphosphate appeared to be more inhibitory than citrate. Moisture is a frequently used indicator of botulinum stability, but when the FDB deviates from 50%, moisture is actually a poor indicator. Components such as NaCl, emulsifying salts, and lactic acid determine stability. Fat does not contribute to stability. Increased fat levels can reduce moisture without a concomitant increase in stability.

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.

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.

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.

Evaluation of Factors Involved in Antibotulinal Properties of Pasteurized Process Cheese Spreads

1986 ◽  
Vol 49 (7) ◽  
pp. 526-531 ◽  
Author(s):  
N. TANAKA ◽  
E. TRAISMAN ◽  
P. PLANTINGA ◽  
L. FINN ◽  
W. FLOM ◽  
...  
Keyword(s):  
Sodium Chloride ◽  
Water Activity ◽  
Clostridium Botulinum ◽  
Toxin Production ◽  
Inhibitory Effect ◽  
Low Ph ◽  
Disodium Phosphate ◽  
Minimum Number ◽  
Process Cheese ◽  
Design Experiments

Pasteurized process cheese spreads with various levels of sodium chloride, disodium phosphate, moisture and pH were challenged with spores of Clostridium botulinum types A and B. Response surface methodology was used to design experiments that would yield maximum results with the minimum number of trials. Supplemental experiments were added to further clarify the response and to examine combinations of special interest. A total of 304 treatment combinations (batches) was incubated at 30°C, and five samples from each batch were taken at predetermined intervals up to 42 wk of incubation and tested for botulinal toxin. Sodium chloride and disodium phosphate inhibited botulinal toxin production with similar effectiveness. The inhibitory effect of low pH (<5.7) and low moisture (<54%) levels on botulinal toxin production was as expected, i.e., as either pH or moisture went up, it was necessary to increase sodium chloride and/or phosphate concentrations to compensate. Differences in water activity between cheese spreads with different compositions were observed but they were too small to use for controlling the properties of the products, e.g., a range of 9% in moisture level (51 to 60%) produced only 0.022 variation in water activity. Combinations of the above factors were developed for safe pasteurized process cheese spreads containing up to 60% moisture.

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.

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