Effect of Iysozyme concentration, heating at 90°C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10⁶ spores per tube. Hen egg white lysozyme (0–50 μg ml^-1) was added, and the tubes were given a heat treatment equivalent to 19·8 min at 90°C, cooled, and incubated at 8°, 12°, 16° and 25°C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6–D inactivation was therefore achieved. In tubes to which lysozyme (5–50 μg ml^-1) had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 μg ml^-1, growth was first observed after 68 d at 8°C, 31 d at 12°C, 24 d at 16°C, and 9 d at 25°C. Thus, in these circumstances, a heat treatment equivalent to 19·8 min at 90°C was not sufficient, on its own, to give a 6–D inactivation. A combination of the heat treatment, maintenance at less than 12°C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10⁶.     

Vegetable juice aids the recovery of heated spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic Clostridium botulinum at 85C for 2 min followed by plating on a standard laboratory medium reduced the count of viable spores by a factor of greater than 10⁴. A similar result was obtained when the plating medium was supplemented with juice from courgette, carrot or mung bean sprout. When plating was on media supplemented with hen egg white lysozyme or juice from turnip, swede, flat bean, cabbage or potato, heating at 85C for 10 min did not reduce the viable count by a factor of 10⁴. Thus these vegetable juices increased the measured heat resistance of spores of non-proteolytic Cl. botulinum . These findings are relevant to the safety of minimally processed (e.g. sous-vide ) foods.     

The effect of recovery medium on the estimated heat-inactivation of spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic strains of Clostridium botulinum at 85°C, followed by enumeration of survivors on a highly nutrient medium indicated a 5 decimal kill in less than 2 min. The inclusion of lysozyme or egg yolk emulsion in the recovery medium substantially increased apparent spore heat-resistance, with as little as 0.1 μg lysozyme/ml sufficient to give an increase in the number of survivors. After heating at 85°C for 2 min between 0.1% and 1% of the spores of 11 strains (5 type B, 4 type E, 2 type F) formed colonies on medium containing 10 μg lysozyme/ml. Enumeration of survivors on a medium containing lysozyme showed that heating at 85°C for 5 min resulted in an estimated 2.6 decimal kill of spores of strain 17B (type B). These findings are important in the assessment of heat-treatments required to ensure the safety with respect to non-proteolytic Clostridium botulinum of processed (pasteurized) refrigerated foods for extended storage such as sous-vide foods.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

Occurrence and Thermoresistance of Spores of Psychrophilic and Psychrotrophic Aerobic Sporeformers in Soil and Foods

Spores of psychrotrophic (able to grow at 5°C) aerobic sporeformers occurred in soil in high numbers (2 × 10³-5 × 10⁶/g), whereas psychrophilic (able to grow at 0°C) spores were present at significantly lower levels (500–10⁵/g). Psychrotrophic spores were absent in herbs and spices: in pasteurized meals prepared industrially their numbers varied from <10 to 1000/g. For spores harvested from Trypticase Soy Agar (TSA), the heat resistance of the cold-tolerant sporeformers was low with D _90°C-values from 1–11 min. The recovery of heated psychrophilic spores on this medium at 5°C was equal to their recovery at 20°C. However, the recovery of heated psychrotrophic spores was lower at 5°C than at 20°C, whereas unheated spores gave the same counts at both temperatures. The heat resistance of naturally occurring spores of cold-tolerant sporeformers washed from soil was comparable with the resistance of spores formed on TSA.     

Factors affecting growth from heat-treated spores of non-proteolytic Clostridium botulinum

Heat treatment of spores of non-proteolytic Clostridium botulinum at 85°C for 120 min followed by enumeration of survivors on a medium containing lysozyme resulted in a 4.1 and 4.8 decimal reduction in numbers of spores of strains 17B (type B) and Beluga (type E), respectively. Only a small proportion of heated spores formed colonies on medium containing lysozyme; this proportion could be increased by treatments designed to increase the permeability of heated spores. The results indicate that the germination system in spores of non-proteolytic Cl. botulinum was destroyed by heating, that lysozyme could replace this germination system, and that treatments that increased the permeability of the spore coat could increase the proportion of heated spores that germinated on medium containing lysozyme. These results are important in relation to the assessment of heat-treatments required to reduce the risk of survival and growth of non-proteolytic Clostridium botulinum in processed (pasteurized) refrigerated foods for extended storage.     

Thermal inactivation of Listeria monocytogenes during a process simulating temperatures achieved during microwave heating

Conventional heating was used to expose cells of Listeria monocytogenes , either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10⁶ upon reaching 70°C. Simulated microwave cooking of L. monocytogenes in situ , on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85°C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10⁶. To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70°C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10⁶ and 10⁸. These results show that when a temperature of 70°C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes . The survival of this organism during microwave heating when temperatures of over 70°C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.     

Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.     

A Model System for Testing the Microbiological Stability of Foods Processed in Laminated Flexible Pouches

A suitable time temperature process for packaging small potatoes in flexible pouches is described. Spores of Bacillus stearothermophilus were inoculated onto the surface of peeled potatoes in flexible pouches. These were evacuated, heat sealed and heated in a steam retort modified to allow a water cooking process with an air overpressure of ca. 68·95 kPa (10 lbf/in²). The D and z values determined in the retort were confirmed by parallel heat-resistance tests with spores in glass ampoules held in an oil bath. Heat treatment at 121·1 °C for 20 min in the retort satisfactorily killed test inocula of spores without overcooking the potatoes. Uninoculated peeled potatoes with a natural level of contamination ( ca. 4 x 10³ spores/sealed pouch) were treated in the retort for different times at 121·1 °C and then incubated at 30 °C for 6 months. No pouches heated for 17–25 min showed microbial growth. This procedure may be applied to any type of food if the numbers of naturally occurring heat-resistant contaminants can be related to the number and heat resistance of a suitable test micro-organism.     

Effect of chill and freezing temperatures on survival of Vibrio parahaemolyticus inoculated in homogenates of oyster meat

Survival of Vibrio parahaemolyticus was determined in oyster meat homogenates at various temperatures. (4°C, 0°C, -18°C and -24°C) and bacterial levels (10², 10⁴, 10⁵ and 10⁷ ml^-1). In all cases, the numbers of V. parahaemolyticus were a logarithmic function of log time. This study indicates that high numbers of V. parahaemolyticus can be inactivated at low temperatures. The time of total inactivation depends on the initial number of micro-organisms and incubation temperature. It is possible to use this information to determine the storage time necessary to reduce V. parahaemolyticus hazards in fish.     

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin^® pre-production to the milk. Each batch of cheese was inoculated with 10²–10³ cfu g⁻¹ of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l⁻¹ could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss.     

Production and Pathogenicity to Wheat of Pseudocercosporella herpotrichoides Conidia

Weekly estimates of numbers of Pseudocercosporella herpotrichoides conidia on naturally infected wheat straw, made from February to July 1982, showed there were most conidia (8.1 × 10⁶ per straw) in February and least (1.9 × 10⁴ per straw) at the end of June. The viability of these spores remained high throughout this period, with an average of 85 % germination after 24 h.
After removal of spores produced in the field, straws were incubated at 5, 10, 15, 20 or 25°C and subsequent sporulation assessed after 3 or 5 weeks. The optimum temperature for spore production was 5°C and very few spores were produced at 25°C. There was no difference in viability between spores produced at different temperatures.
Wheat seedlings placed amongst infected straw collected and retained spores on the upper and lower surfaces of all leaf blades and on outer leaf sheaths. Both naturally dispersed spores and spores sprayed on to plants were not removed by subsequent rainfall.
When wheat seedlings were inoculated between the coleoptile and outer leaf sheath with different numbers of P. herpotrichoides spores, lesion development was most rapid in seedlings inoculated with the greatest numbers of spores. However, after incubation for 12 weeks visible lesions were present on all plants inoculated with > c. 10 spores.     

Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin

We investigated the combined effects of pressure, temperature, pH, initial spore concentration and the presence of nisin on the survival of spores of Bacillus coagulans. Spores were more sensitive to pressure both at lower pH and at higher treatment temperatures. An additional 1.5-log₁₀ reduction in cfu ml^-1 was observed when pH was lowered from 7.0 to 4.0 during pressurization at 400 Mpa and 45°C. A 4-log₁₀ cfu ml^-1 reduction was observed when the temperature was increased from 25°C to 70°C during pressurization at 400 Mpa. The spores were sensitive to nisin at concentrations as low as 0.2 IU ml^-1. At least a 6-log₁₀ reduction was generally achieved with pressurization at 400 Mpa in pH 4.0 buffer at 70°C for 30 min when plated in nutrient agar containing 0.8 IU ml^-1 nisin.     

The influence of the rate of temperature change on the activation of dormant seeds of Rumex obtusifolius L.

1. One temperature shift from 20 to 30°C in darkness induces 30–40% germination in Rumex obtusifolius seeds. The same germination percentages are found with heat treatment varying between 1 and 6h duration, indicating that the total heat sum of the temperature shift is not important.
2. Germination is greatly enhanced by three consecutive heat shifts of 1h at 30°C separated by 1h periods at 20°C.
3. The seeds are activated to a small extent after a slow warming (+2°Ch^–1) from 20 to 30°C, followed by incubation for 1h at 30°C. Germination is much higher after rapid heating (+10°Ch^–1) to 30°C, followed by 1h incubation at this temperature. Repeated fast heating treatments on four consecutive days enhances germination. Moderately rapid heatings (+3·3°Ch^–1) give intermediate results.
4. The rate of cooling does not influence the germination percentage. Cooling alone cannot induce germination.
5. Heating alone from 15 to 25°C without cooling also activates germination. In this temperature range the seeds are more activated by rapid warming than by slow warming.
6. The ecological relevance of the response to different warming rate is discussed. The insensitivity of seeds to a slow warming might keep deeply buried seeds in a dormant stage.     

Delaying toxigenesis of Clostridium botulinum by sodium lactate in 'sous-vide' products

The effect of sodium lactate and storage temperature on toxigenesis by proteolytic (Pr) and nonproteolytic (Np) Clostridium botulinum spores inoculated in processed 'sous-vide'-type beef, chicken breast and salmon was explored. Three g samples of beef and salmon homogenates with 0, 2.4 and 4.8% (w/w) lactate and of chicken with 0, 1.8 and 3.6% (w/w) lactate were placed in 24-well tissue culture plates. The samples were inoculated with 10⁴ spores of pools of Pr (4A + 2B + 2F strains) or Np (4B + 4E strains), vacuum-packaged in barrier bags, and stored at 16 and 30°C for Pr and at 4, 8, 12 and 30°C for Np for up to 90 d. Lactate at 2.4% in beef and 1.8% in chicken delayed toxigenesis by Np for 40 d at 12°C and by Pr for 28 d at 16°C. Delaying toxigenesis for similar periods of time in salmon required 4.8% lactate and 12°C for Np, and 2.4% lactate and 16°C for Pr. Increasing levels of lactate and decreasing temperature significantly delayed toxigenesis of Cl. botulinum in the 'sous-vide' products.     

The effect of variation of thermal processing on the microbial spoilage of chub-packed luncheon meat

Process pasteurization values for reference temperature 70°C (P₇₀) were calculated from the temperature profiles of 250 g luncheon meat chubs cooked under experimental conditions. A simple equation relating Process P₇₀-value and the time and temperature of cooking was derived. With minimal cooking (P₇₀= 40) the surviving microflora (10³/g) was dominated by species of Lactobacillus, Brochothrix and Micrococcus. These organisms were destroyed by more intensive cooking (P₇₀= 105), leaving a flora (10²/g) composed of Bacillus and Micrococcus species. The spoilage that developed after 14 d storage at 25°C reflected the severity of the heat treatment received by each chub: with P₇₀ between 40 and 90, a Streptococcus spoilage sequence occurred; with P₇₀ between 105 and 120, a Bacillus/Streptococcus spoilage sequence occurred; with P₇₀ of 135 and above, a Bacillus spoilage sequence occurred. Cooking to a P₇₀= 75 was adequate to reduce the surviving microflora to the 10²/g level associated with current good manufacturing practice.     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

The Limulus Lysate Endotoxin Assay as a Test of Microbial Quality of Ground Beef

The Limulus lysate test (LLT) for endotoxin assay has been found to be an excellent, simple and rapid test of microbial quality of refrigerated ground beef. In fresh ground beef held at 5°C for 7–12 d, LLT titres increased from 10²–10⁵ and correlated very highly with extract-release volume (ERV) data and total viable Gram negative counts at both 5° and 30°C. The LLT was negative for fresh beef containing low numbers of bacteria and on aged beef in the absence of increasing numbers of Gram negative bacteria. Of 14 Gram negative meat isolates, all gave a positive LLT while none of eight miscellaneous Gram positive bacteria did. The use of this test provides objective information on the microbial quality of fresh refrigerated ground meats in 1 h. Based upon this study, it is suggested that a 0·1 ml inoculum from a 10³ dilution of good quality ground beef should produce a negative lysate test and thus serve as an additional rapid screening test of meat microbial quality.