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Development of a heat-pasteurization process is described for picked meat of Dungeness crabs (Cancer magister) contained in oxygen-impermeable flexible pouches, For each time-temperature treatment, 30 samples, each inoculated with an equal mixture of three strains of C. botulinum nonproteolytic type B, for a total of 107 spores, provided the basis for calculation of the thermal resistance (a 7D process). Following heat processing, the crabmeat was removed from the pouches and transferred to enrichment medium where it was incubated anaerobically for 150 days. Endpoints at which spores survived were determined by the presence of toxin in the enrichment medium. Process times ranged from 90 min at 88.9°C to 20.3 min at 94.4°C. D values (the time at each temperature required to reduce the inoculum by 1 log) ranged from 12.9 for the 88.9°C process to 2.9 for the 94.4°C process. The relative sterilization value, F0 was .054 and the pasteurization value, , was 240. This pasteurization process safely extends refri...

L. monocytogenes was inoculated onto the surface of brined salmon steaks and heat processed in a commercial smokehouse to simulate a hot process for preparing smoked fish. The minimum temperature required for inactivation of L. monocytogenes was 153°F (67.2°C) when generated smoke was applied throughout the entire process. When generated smoke was added only during the last half of the process, L. monocytogenes was recovered from steaks heated to temperatures as high as 176°F (80.0°C). When smoke was not applied during the process, L. monocytogenes survived on steaks heated to internal temperatures between 131°F and 181°F (55.0 to 82.8°C) but was not isolated from steaks heated above 181°F (82.8°C). When liquid smoke CharSol C-10 was applied as a full-strength (100%) dip before processing, L. monocytogenes was inactivated in samples processed at temperatures as low as 138°F (58.9°C). When liquid smoke 10-Poly or CharSol C- 10 was applied at a concentration of 50%, the lethal temperature was increased to the range of 145 to 150°F (62.8 to 65.6°C). With further dilution of C-10 to 25% and 10% the inactivation temperatures increased to 156°F (68.9°C) and 163°F (72.8°C). A full-strength dip of CharOil, the oil-soluble fraction of CharSol C-10. was less effective, and L. monocytogenes survived in salmon steaks processed to an internal temperature of 166°F (74.4°C), the highest temperature tested with this liquid smoke. This study provides evidence that heat alone is not reliable for inactivation of L. monocytogenes during the hot-smoking process. The proper stage and duration of smoke application or proper composition and concentration of liquid smoke in combination with heat are critical for inactivation of the organism.

Comminuted raw salmon containing various concentrations and combinations of sodium lactate, sodium chloride, and sodium nitrite was inoculated with 10 Listeria monocytogenes cells per g (150 cells/15-g sample), vacuum-packaged in oxygen-impermeable film and stored at 5 or 10°C. Samples were examined for growth of L. monocytogenes and total aerobic microorganisms at specific intervals for up to 50 d. Sodium lactate exhibited a concentration-dependent antilisterial effect that was enhanced by nitrite and/or increased concentrations of NaCl. At 5°C, total inhibition of L monocytogenes was achieved for up to 50 d by 2% sodium lactate in combination with 3% water-phase NaCl. At 10°C, total inhibition was achieved for up to 35 d by 3% sodium lactate in combination with 3% water-phase NaCl, or by 2% sodium lactate in combination with 125 ppm sodium nitrite and 3% water-phase NaCl. Sodium lactate and the other additives also inhibited growth of the aerobic microflora but to a lesser degree than L. monocytogenes .

Three selective media were evaluated for direct plating recovery and enumeration of Listeria monocytogenes in the presence of high levels of a variety of microorganisms occurring on cold-process (smoked) salmon products. Sliced salmon was brined to contain either no added salt, 3, or 5% water-phase NaCl, or 3 or 5% NaCl plus 140 ppm NaNO2. The slices were packaged in oxygen-permeable film or sealed under vacuum in oxygen-impermeable film, and stored at 10°C or 5°C until total microbial loads reached 106 to 109 CFU/g. Oxford formulation of Listeria selective agar and Lee's modification of Listeria selective agar achieved quantitative recovery of 102 cells per ml of L. monocytogenes strain Scott A in the presence of diluted slurries of these fish containing 104 to 108 CFU/ml of background organisms. A modification of lithium chloride-phenylethanol-moxalactam agar containing an iron-esculin indicator system sometimes failed because of interfering growth by the background microflora.

This study demonstrates the feasibility of a heat-pasteurization process for certain vacuum-packaged hot-smoked fishery products for inactivation of the spores of the nonproteolytic Group II Clostridium botulinum types B, E, and F. This process permits the use of lower concentrations of salt and other inhibitors without jeopardizing safety and quality of the products during prolonged refrigerated storage. The pasteurization treatment was developed based upon the inactivation of nonproteolytic types B or E in hot-process (smoked) salmon. Smoke was not applied to the samples inoculated with types B and E because of its possible inhibitory effects. After processing in the smokehouse, each sample was cooled to 34°F (1.1°C), injected with 106 spores, vacuum-packaged, and then heat-pasteurized in a water bath held at a constant temperature. A total of 85, 65, and 55 min in the 185°F (85°C), 192°F (88.9°C), and 198°F (92.2°C) baths, respectively, prevented toxin production by type E during 21 d of incubation at 25°C. Longer times, 175, 85, and 65 min, respectively, were required to prevent toxin production by nonproteolytic type B. Toxin production by type E during 120 d of storage at 10°C was prevented by a 45-minute treatment in the 198°F (92.2°C) bath. When heat-pasteurized samples were transferred into TPGY broth and incubated anaerobically for 150 d at 25°C, outgrowth and toxin production by type E was prevented by a 55-minute process at 198°F (92.2°C) and type B was prevented by a 65-minute process. This process does not, however, inactivate the more heat-resistant proteolytic strains of C. botulinum Group I or other spore-formers. The packages and master cartons of these pasteurized products therefore should follow the existing recommendations for smoked fishery products and be labeled "Keep refrigerated - Store below 38°F (3.3°C)."

Whitefish steaks were brined in NaCl, KCl, or equimolar NaCl:KCl to contain similar chloride ion concentration and inoculated intramuscularly with 10 or 100 spores of Clostridium botulinum type E per gram of fish. Steaks were then heated in a simulated (i.e., without smoke) hot-smoke process to internal temperatures of 62.8° to 76.7°C (145°-170°F) for the final 30 min of a 2- to 3-h process, packaged under vacuum in oxygen-impermeable film, and stored at abuse temperature of 25°C. During 7 d of storage, toxin production was inhibited in steaks containing more than 0.66 ionic strength NaCl, 0.64 KCl, or 0.71 equimolar NaCl:KCl. The results indicate that it is feasible to substitute KCl for NaCl in hot-process smoked fish for inhibition of outgrowth and toxin production by Clostridium botulinum type E.

Mixtures of proteolytic and nonproteolytic strains of toxigenic Clostridium botulinum types A, B, and F; nonproteolytic types B, E, and F; Clostridium sporogenes; and nontoxic E-like organisms resembling nonproteolytic C. botulinum were tested against each other for the purpose of selecting a mixture of compatible C. botulinum strains for inoculated pack studies on the basis of their sensitivity to bacteriophages and bacteriocin-like agents. All of the proteolytic strains produced bacteriocin-like agents that were inhibitory to three or more of the other proteolytic types and C. sporogenes. When selected strains of proteolytic types A and B were grown together, type A cultures produced neurotoxin, but type B toxin production was inhibited. Nonproteolytic strains of C. botulinum also produced bacteriocin-like agents against each other. Of these, type E strain EF4 produced bacteriocin-like agents against both proteolytic and nonproteolytic types of C. botulinum and C. sporogenes. EF4, however, was not inhibitory to the nontoxigenic E-like strains. When EF4 was grown with type A strain 62A, it had an inhibitory effect on type A toxin production. Strain 62A inactivated the type E toxin of EF4 after 7 to 21 days at 30 degrees C. On the basis of the production of these bacteriocin-like agents by different strains of C. botulinum and their potential effect on neurotoxin production, it is very important that compatible strains are used in mixtures for inoculated pack studies to determine the safety of a food process or product.

Microbiological and chemical changes were determined during the smoking and drying of salmon strips processed at 29 to 318C for 4 days at a facility in Alaska in 1993. During the process, Staphylococcus aureus populations increased to more than 10 5 CFU/g after 2 to 3 days of processing. Subsequent laboratory studies showed that a pellicle (dried skinlike surface) formed rapidly on the strips when there was rapid air circulation in the smokehouse and that bacteria embedded in or under the pellicle were able to grow even when heavy smoke deposition occurred. Under these conditions, an inoculum of 26 CFU/ g of S. aureus increased to 10 5 CFU/g after 3 days of processing. Elimination of preprocess drying and reduction in air e ow during smoking resulted in smoke deposition before pellicle formation and enabled the product to reach levels of water-phase salt and water activity that inhibit the growth of S. aureus and Listeria monocytogenes. In 1994, these modie cations were then applied during processing at an Alaskan facility, and S. aureus could not be detected in the e nished product. L. monocytogenes was detected in the raw product area, on the processing tables, and on the raw salmon strips, but it was not detected in the e nished product when the smoke was applied before pellicle formation. Smoking and drying are among the oldest methods of food preservation. Even though we have had considerable experience with these processes, food poisoning outbreaks occasionally occur. Improperly processed products and the lack of understanding that some products may become hazardous if they are stored at temperatures that permit the growth of microbial pathogens have caused these food poisoning outbreaks. In Alaska, native smoked and dried salmon products are processed during the summer months when the e sh are returning to the rivers during the spawning season. Since processing is done under ambient temperature and humidity, the processing time can vary from 4 days to 1 or 2 weeks, depending on weather conditions. This long drying time raises concern, because the drying temperatures are ideal for the growth of bacterial pathogens. During processing, there is a race between the product reaching inhibitory levels of water-phase salt (WPS), moisture, and water activity (aw) and the growth of pathogenic and spoilagetype bacteria. If the product is processed at temperatures of 608C or higher, the nonspore-forming bacteria are usually inactivated. In 1993, we worked with an Alaskan native processor along the Yukon River to learn more about the processes used and the microbiological and chemical composition of smoked and dried salmon. The processor had developed a fairly modern processing facility with stainless steel tabletops, a chlorinated water supply, a refrigeration room, and walls and e oors that could be cleaned and sanitized.

Water phase sodium chloride (WPS) levels of 1.8 to 3.0% in combination with heat pasteurization for 15 min at temperatures of 75, 80, 85, and 90 degrees C were evaluated as methods for the inactivation or inhibition of nonproteolytic, psychrotrophic Clostridium botulinum types B and E in crab analogs (imitation crab legs) subsequently stored at 10 and 25 degrees C. Samples inoculated with 10(2) type B or E spores per g prior to pasteurization remained nontoxic for 120 days at 10 degrees C and for 15 days at 25 degrees C. With 10(4) type E spores per g and 80 degrees C pasteurization, > or = 2.4 and 2.7% WPS was required for inhibition at 10 and 25 degrees C storage, respectively. Pasteurization at 85 degrees C decreased the inhibitory level of WPS to 2.1% at 10 degrees C and to 2.4% at 25 degrees C. When the inoculum was 10(4) type B spores per g, samples with 2.7% WPS were toxic after 80 days of storage at 10 degrees C. Samples inoculated with 10(3) type B spores per g and processed at 85 degrees C remained nontoxic for 15 days at 25 degrees C with a WPS of > or = 2.4%. When pasteurization was carried out before inoculation and packaging, 1.8% WPS prevented toxin production by 10(2) and 10(4) type E spores per g for 30 days at 10 degrees C, and this time period increased as the WPS concentrations increased. Three percent WPS prevented toxin production by 10(4) type E spores per g in vacuum-packaged analogs stored 110 days at 10 degrees C. Pasteurization processes used in these experiments, however, do not inactivate the heat-resistant proteolytic types of Clostridium botulinum. Therefore, the most important factor controlling the growth of this bacterium is continuous refrigeration below 3.0 degrees C or frozen storage of the finished product.

Cold-smoked salmon processing plants were surveyed to determine the occurrence and sources of L. monocytogenes contamination. Sanitation and cleanup procedures adequately eliminated L. monocytogenes from the processing line and equipment, but recontamination occurred soon after resumption of processing. The primary source of contamination proved to be the surface areas of frozen or fresh raw fish coming into the plant. Listeria species were not isolated from the flesh except when they were introduced during the filleting operation, by the injection of recirculated brine, or in localized areas where there were pre-existing bruises. Penetration of L. monocytogenes into intact flesh via the vascular system did not occur when fresh fish were immobilized with carbon dioxide and bled, nor when frozen, headed, and eviscerated fish were thawed for 20 h in water inoculated with 44 L. monocytogenes organisms per ml. Populations of L. monocytogenes inoculated onto the surface of brined salmon portions changed very little during a cold-smoke process at 72 to 87°F (22.2 to 30.6°C) for 20 h, with or without applied smoke; but when the processing temperature was lowered to 63 to 70°F (17.2 to 21.1°C), populations decreased 10- to 25-fold when smoke was applied. L. monocytogenes injected into the interior of these portions increased 2- to 6-fold at 63 to 70°F (17.2 to 21.1°C) and 100-fold at 72 to 87°F (22.2 to 30.6°C), regardless of the presence of smoke. L. monocytogenes was enumerated in 48 contaminated finished products collected from six different processing plants. L. monocytogenes populations ranged from 0.3 to 34.3 cells per g, with a mean of 6.2 per g and a median of 3.2 per g.

The behavior of Listeria monocytogenes in relation to sodium nitrite (NaNO2) in combination with sodium chloride (NaCl) was evaluated in cold-process (smoked) salmon during storage at 5 or 10°C in either oxygen-permeable film or vacuum-sealed impermeable film. Salmon slices containing either 3 or 5% water-phase NaCl, with or without 190–200 ppm of NaNO2, were inoculated with 10 or 327 CFU/g (150 or 4.9 × 103 CFU/15-g sample) of strain Scott A. The inhibitory contribution of NaNO2 was relative to inoculum size, storage time and temperature, packaging method, and concentration of NaCl. There was less growth of L. monocytogenes in vacuum-packaged samples as compared to those packaged in oxygen-permeable film. The most inhibition was achieved in vacuum-packaged products stored at 5°C, where NaNO2 in combination with 5% water-phase NaCl prevented any increase in a 10 CFU/g-inoculum during 34 d storage. At 10°C, inhibition was initially enhanced by NaNO2, but by 32 d L. monocytogenes populations had increased f...

The behavior of Listeria monocytogenes (10 Scott A cells per g) in cold-process (smoked) salmon containing 3, 5, or 6% water-phase NaCl was evaluated during 30 to 40 d storage at 5 or 10°C in either oxygen-permeable film or vacuum-sealed impermeable film. At 10°C, L. monocytogenes grew to 106 to 108 CFU/g by the second week, with no differences attributed to NaCl concentration except for an initial lag in the 6% NaCl samples. Vacuum packaging suppressed growth of L. monocytogenes by 10- to 100-fold in samples with 3 or 5% NaCl. Inhibition related to NaCl concentration was most apparent at 5°C and L. monocytogenes populations were held below 102 CFU/g by 6% NaCl. Growth of a 327 CFU/g inoculum was about 10-fold greater than a 10 CFU/g inoculum at 10°C and 100-fold greater at 5°C. Growth of two strains isolated from naturally contaminated, commercially prepared, cold-smoked fish did not differ from Scott A. The use of sugar in the product did not influence growth of L. monocytogenes . Maximum populations of aerobic microorganisms reached at 5 and 10°C were similar, although the rate of growth was somewhat delayed at 5°C, and some inhibition was shown by 5 and 6% NaCl and by vacuum packaging.

Hemolytic ceftazidime lithium chloride agar (HCLA) has been developed as a selective and differential plating medium for the isolation of Listeria monocytogenes from fishery products. Selectivity is based upon lithium chloride, colistin methane sulfonate, and ceftazidime. When horse blood was incorporated in the agar overlay, L. monocytogenes colonies were easily distinguished by their characteristic blue-gray color surrounded by narrow zones of ß-hemolysis. Listeria innocua , a species commonly present in foods, does not produce hemolysis on the medium. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua . When used in combination with Food and Drug Administration and U.S. Department of Agriculture enrichment methodology, HCLA was effective in inhibiting competitive organisms, differentiating colonies of L. monocytogenes by their ß-hemolysis, and shortening the incubation time at 35°C for presumptive identification to 17-24 h.

Liquid smoke in combination with NaCl was an effective inhibitor of outgrowth and toxin production by Clostridium botulinum types A and E spores in hot-processed whitefish, chub and carp stored at an abuse temperature of 25°C for 7 or 14 d. Surface-inoculated type E produced toxin in control samples containing 3.7% water-phase NaCl, but not in liquid smoke-treated samples having less than 2.0% water-phase NaCl. Liquid smoke was less effective when type E spores were injected intramuscularly. Liquid smoke lowered the concentration of NaCl required to inhibit toxin production by surface-inoculated type A from 4.6 to 2.8% in samples stored 7 d. Liquid smoke enhanced the ability of NaCl to prevent toxin production, but should not be considered a substitute for NaCl or refrigerated storage (below 3.3°C).

When Clostridium botulinum type C is cured of its prophage it simultaneously ceases to produce toxin. This nontoxigenic culture can then be converted to another toxigenic bacterial species, Clostridium novyi type A or to toxigenic Clostridium botulinum types C or D, by specific bacteriophages. The toxigenicity and type of toxin produced by these cultures depends upon the continued presence of these bacteriophages.