The Behaviour of a Food Poisoning Strain of Clostridium welchii in Beef

S ummary : An inoculum of 10⁵ spores of Clostridium welchii F2985/50 in meat survived steaming at 100° for 5 h, the number being reduced sevenfold for every hour of steaming. They also survived for at least 6 months in frozen meat stored at -5° and -20°, whereas vegetative cells died more rapidly at -5° than at -20°. In beef stored for 13 days at 1°, 5°, 10° and 15° there was no multiplication but a slow destruction of vegetative cells, but there was little change in the spore count. Slow multiplication occurred at 20° but at 25° and 37° growth was rapid. Only about 3% of the spores germinated without prior heat shock, so the majority failed to germinate in raw meat stored at any temperature, but did so once the meat had been heated. In meat which had been heated and allowed to cool almost all of the spores had lost their heat resistance.
It was found that the minimal growth temperature was related to pH and medium, so that meat with a pH higher than that used in these experiments (pH 5°7–5°8) would probably have a lower minimal growth temperature for these organisms and would thus be more susceptible to spoilage.     

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, α-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 °C for 15, 30 and 60 min, or 121 °C for 15 min. Lactococcin R remained active after storage at −20 and −70 °C for 3 months and after exposure to a pH of 2–9. The molecular weight of lactococcin R was about 2·5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml⁻¹) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0·5% glucose, at 6·5–7·0 initial pH values, 30 °C temperature and 18–24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6–7 (95%). Crude lactococcin R at 1280 AU ml⁻¹ was bactericidal, reducing colony counts of Listeria monocytogenes by 99·98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2·5 kDa vs 3·4 kDa, and in being sensitive to pepsin and α-chymotrypsin to which nisin is resistant.     

Conditions affecting growth and enterotoxin production by Staphylococcus aureus on temperature-abused chicken meat

Growth of Staphylococcus aureus at 15°C, with and without addition of representative spoilage bacteria, was studied in cooked, whole chicken meat and chicken broth. In the absence of competitors, the organism grew better in broth culture than on whole meat, but multiplied more slowly in broth when other organisms were present, even from twice the previous level of inoculum. The presence of competitors had no marked effect on the growth of Staph. aureus on whole meat. Enterotoxin A was not produced at 15°C on either whole meat or in broth, and occurred at 20°C only in pure culture. At 30° and 37°C, toxin was produced whether or not competitors were present. Toxin production by Staph. aureus appeared to be influenced more by growth temperature than by bacterial competition.     

Survival of Campylobacter jejuni in raw, pasteurized and ultra-heat-treated goat's milk stored at different temperatures

The survival of a human strain of Campylobacter jejuni in raw, pasteurized and ultra-heat-treated goat's milk stored at 5°, 10°, 15° and 20°C was studied. No viable units were detected in raw milk after 24 h at 20°C and 48 h at 15°C. None were detected in pasteurized milk after 48 h at 20°C. In all other samples, there was a decline in viable units in the first 24 h but very little decline in the next 24 h period. The organism survived best at 5° and 10° C.     

Incidence and Spoilage Potential of Isolates from Vacuum-packaged Meat of High pH Value

Meat of high pH value (6·6) showing dark-cutting characteristics was vacuum-packaged and stored for up to 8 weeks at 0–2°C. 'Off'-odours were detected on opening the packages after 6 weeks of storage. Total counts at this stage were ca. 10⁷/cm² of which lactobacilli were the major component, with ca. 10⁶/cm² Gram negative organisms. Psychrotrophic Enterobacteriaceae represented a major proportion of the microflora only after the full 8 weeks of storage and were not detected previously. Aerobic storage of steaks cut from the vacuum packaged meat stored for 8 weeks resulted in a predominantly Gram negative spoilage flora.
Inoculation studies on meat of normal pH value (5·4) and appearance using representative isolates from the vacuum-packaged meat microflora indicated that most of the test organisms were capable of causing spoilage under aerobic conditions but few under vacuum-packaging when incubated at 4°C. On meat of higher pH value (6·15) many of the Gram negative isolates did not grow as well, whereas the Gram positive isolates grew better than on meat of normal pH value when held under aerobic conditions. Under vacuum-packaging all but one isolate grew as well or better on meat of high pH value than on normal meat at 4°C and objectionable odours were more marked.     

Growth and Sporulation of Clostridium welchii in Breast and Leg Muscle of Poultry

Growth of a heat resistant, food poisoning strain of Clostridium welchii was followed in raw, minced breast and leg muscle of the chicken. Within the range 22–50° growth was slightly more rapid in the leg (pH 6·5–6·7) than in the breast (pH 5·6–5·7) and was fastest in leg muscle at 50°. No growth occurred at 15 or 52°.
In a comparison between chicken and turkey, inoculated breast and leg muscle were cooked for 1 h at 85° and held at 37°. Multiplication of surviving organisms was initiated much more rapidly in chicken than in turkey meat, though the growth rates were comparable in each case.
Sporulation of several strains of CI. welchii , including other heat resistant, food poisoning types, was generally 10–100 times greater in leg than in breast muscle of the chicken. Differences in sporulation could be attributed both to differences in pH and type of meat.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni . The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO₂+ 80% N₂, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O₂+ 10% CO₂+ 85% N₂. The packaging material in the first two treatments was PA 80/PE 100–PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37°C, 20°C and 4°C for 48 h, 4 days and 25 days, respectively. At 37°C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20°C and at 4°C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37°C its numbers increased only in the optimal gas atmosphere; at 20°C the strain was not detectable after 24 to 48 h storage and at 4°C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

Factors affecting the production of hydrogen sulphide by Lactobacillus sake L13 growing on vacuum-packaged beef

Lactobacillus sake L13 produced hydrogen sulphide during growth at 0°C on vacuum-packaged beef of normal pH (5·6–5·8) when the packaging films used had oxygen permeabilities as high as 200 ml/m²/24 h/atm (measured at 25°C and 98% relative humidity. No hydrogen sulphide was detected when the film permeability was 300 ml/m²/24 h/atm. Sulphmyoglobin was formed whenever hydrogen sulphide was present except when the film permeability was very low (1 ml of oxygen/m²/24 h/atm). Lactobacillus sake L13 also produced hydrogen sulphide when grown on beef under anaerobic conditions at 5°C. When meat pH was high (6·4–6·6) hydrogen sulphide was first detected after incubation for 9 d. When 250 μg of glucose was added to each g of high pH meat, or when meat pH was normal (5·6–5·8), hydrogen sulphide was first detected after incubation for 18 d. The spoilage of beef by hydrogen sulphide-producing lactobacilli is more rapid when the pH of the meat is high because high-pH meat contains less glucose. Sulphmyoglobin formation and greening can be prevented by the use of packaging films of very low oxygen permeability.     

Changes in Escherichia coli O157:H7 numbers during holding on excised lean, fascia and fat beef surfaces at different temperatures

Aim:  To investigate changes in Escherichia coli O157:H7 numbers on excised beef carcass surfaces over 72 h at different temperatures.
Methods and Results:  Excised lean meat, fascia and fat were inoculated with E. coli O157:H7 and held in an environmental chamber for 72 h, at air speed 0·5 m s⁻¹, relative humidity (RH) 90%, and temperatures 4, 8 and 12°C. On lean, pathogen counts increased significantly at 12°C. On fascia, significant reductions in counts occurred at 4 and 8°C. Pathogen numbers were significantly reduced on fat at 4, 8 and 12°C (64 h). Counts on fat were significantly less at all temperatures, compared to lean or fascia and surface water activity, a_w, decreased significantly over time on fat at 4°C. Significant decreases in surface pH values were recorded on all meat substrates.
Conclusions:  The survival of E. coli O157:H7 varied in relation to the meat substrate and the holding temperature. Reductions in counts on fat surfaces appeared to be related to low surface a_w values. No relationship between pathogen survival and surface pH was established.
Significance and Impact of the Study:  The use of excised meat pieces in an environmental cabinet offers a more flexible approach to determining the use of different chilling regimes in the production of safe meat.     

Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkalinesoda lake

An alkaline xylanase-producing alkaliphilic Bacillus sp. AR-009 was isolated from analkaline soda lake in Ethiopia. The enzyme was optimally active at pH 9 and was stable over abroad pH range. The optimum temperature for xylanase activity, assayed at pH 9, was60°–65°C. Measured at pH 8 and 9, the enzyme had good stability at 55° and60°C. At both pH values, over 80% of its original activity was retained after heating for2·5 h at 55°C. At 60°C, the enzyme maintained 63% of its original activity after2·5 h incubation while at pH 9 it retained 54% of its original activity after 1 h heating. Theseproperties qualify the enzyme to be novel and potentially important for application in someindustrial processes.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5·8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15°C in (1) aerobic conditions; (2) 30% CO₂+ air; (3) 30% CO₂+ N₂; and (4) 100% CO₂. When samples were held at 1°C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6°C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1°C rather than 37°C. In CO₂ atmospheres growth of L. monocytogenes was inhibited on meat held at 6°C, especially under 100% CO₂. By contrast, storage at 15°C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

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°.     

Temperature-induced Changes in the Synthesis of Unsaturated Fatty Acids by Acanthamoeba castellanii

ABSTRACT. Major fatty acid components of Acanthamoeba castellanii lipids extracted after growth at 30°C include myristate, palmitate, stearate and the polyunsaturates linoleate, eicosadienoate, eicosatrienoate and arachidonate, with oleate as the sole major monounsaturated fatty acid. By comparison, growth at 15°C gave increased linoleate, eicosatrienoate and arachidonate, but decreased oleate and palmitate. When the growth temperature was shifted downwards from 30°C to 15°C, increased lipid unsaturation occurred over a period of 24 h; thus decreases of oleate and eicosadienoate were accompanied by increases in linoleate, eicosatrienoate, arachidonate and eicosapentaenoate. An upwards shift from 15°C to 30°C gave negligible alterations in fatty acid composition over a similar period. At 15°C organisms rapidly use [1-¹⁴C] acetate for de novo fatty acid synthesis; stearate is converted via oleate to further desaturation and chain elongation products. Similar short term experiments at 30°C indicate only de novo synthesis and Δ9-desaturation; synthesis of polyunsaturates was a much slower process. Rapid incorporation of [1-¹⁴C] oleate at 30°C was not accompanied by metabolic conversion over two hours, whereas at 15°C n-6 desaturation to linoleate was observed. Temperature shift of organisms from 15°C to 30°C in the presence of [1-¹⁴C] acetate revealed that over half of the fatty acids in newly-synthesised lipids were saturated, but the proportions of unsaturated fatty acids increased with time until the total polyenoate components reached 17% after 22 h. A shift of temperature in the reverse direction gave a corresponding figure of 60% for polyunsaturated fatty acids. These results emphasize the importance of n-6 desaturation in the low temperature adaptation of Acanthamoeba castellanii .     

Influence of photoperiod on low temperature acclimation for cold-hardiness in Drosophila auraria

ABSTRACT. Imagines of Drosophila auraria Peng, a reproductive diapause species, developed cold-hardiness at low temperatures to a greater extent when exposed to a diapause-inducing photoperiod (LD10:14 h) than when exposed to a diapause-preventing photoperiod (LD 16:8h). Imagines kept at 18°C, which was the temperature at which they were reared to eclosion, did not survive a test exposure to -5°C for 8 days regardless of age or photoperiod. When transferred to 10 or 5°C, either from eclosion or from 8 days after eclosion, the survival rate, on testing, rose with time since transfer and rose faster and higher with a photoperiod of LD 10:14h than with LD16:8h. Flies transferred to 15°C only showed improved ability to survive the test if they were kept in LD 10:14h. When cultured at 18°C to the age of 8 days after eclosion, diapause was terminated in about 30% of females even at LD 10:14h. In these post-diapause females the ability to develop cold-hardiness at lower temperatures was somewhat less than in the diapausing females, but apparently greater than in the non-diapause females. These results suggest that the physiological mechanism which promotes cold-hardiness under a diapause-inducing photoperiod is not directly linked to the process causing reproductive diapause.
In Sapporo, flies from a natural population became tolerant to cold in October when they entered diapause and daily mean temperature fell below 15°C and the light/dark cycle fell below LD 12:12h.     

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.     

Changes in the Microbiology of Vacuum-packaged Beef

The development of the microbial flora on meat stored in vacuum-bags at 0–2° for up to 9 weeks was studied. Although the proportion of lactic acid bacteria increased relative to the aerobic spoilage organisms, the numbers of the latter continued to increase throughout storage. The initial contamination of the meat before vacuum-packaging was important; meat with a very low initial number had lower numbers of bacteria throughout storage for up to 9 weeks and steaks cut from such meat which had been stored always had 1–2 days' additional aerobic shelf life at 4°. Spoilage of these steaks was due either to slime formation and off-odour associated with high counts of presumptive Pseudomonas spp., or by discoloration and souring (lactic acid bacteria). Extract release volume and pH measurements performed on the vacuum-packaged primal joints were only of value in determining the onset of aerobic spoilage when large numbers of Gram negative organisms were present, whereas the titrimetric method of spoilage evaluation of the vacuum-packaged meat showed a correlation with spoilage due to lactic organisms.     

Effect of temperature in the regulation of DNA synthesis in synchronous cultures of Chlorella

The influence of temperature on DNA replication of Chlorella pyrenoidosa grown at 30 °C was studied in synchronous cultures. Final DNA content and cell counts/ml were 30 to 40% less than controls after a 4 h exposure to 15 °C, but reached normal levels after being kept temporarily at 10 °C. The rates of DNA synthesis at these two temperatures were, respectively, one-sixth and one-tenth of the rate at 30 °C, but during the 15 °C treatment the cells lost the ability to enter a further replication cycle more rapidly than at 10 °C. This loss of capacity to initiate further replication cycles was prevented by inhibiting protein synthesis with cycloheximide.     

The effect of cold, anoxia and ethylene on the flowering ability of buds of Cichorium intybus

The apical bud and the axillary buds of Witloof chicory ( Cichorium intybus L. cv. Tardive d'Anvers) remain in the vegetative state if they are left on the root and maintained at 18°C. Flowering occurs in long days of 16 h after a pretreatment of either 8 weeks at 3°C, 3 days in complete anoxia at 15°C, or 4 days in the presence of ethylene (1000 ppm) at 15°C. In contrast, the adventitious buds which spread out on the root after ablation of the collar flower in a photoperiod of 16 h without particular pretreatment. The grafting of apical buds onto roots after different treatments shows that cold and ethylene act on the root, whereas anoxia acts directly at the level of the bud. It seems that the inhibition of the flowering of preformed buds (apical and axillary) stems from the collar. A hypothesis is proposed to explain this inhibition and why it is broken by cold, anoxia and ethylene.     

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.     

Interactions of temperature and photoperiod in the control of flowering of latitudinal and altitudinal populations of wild strawberry (Fragaria vesca)

Floral induction and development requirements of a range of latitudinal and altitudinal Norwegian populations of the wild strawberry Fragaria vesca L. have been studied in controlled environments. Rooted runner plants were exposed to a range of photoperiods and temperatures for 5 weeks for floral induction and then transferred to long day (LD) at 20°C for flower development. A pronounced interaction of temperature and photoperiod was shown in the control of flowering. At 9°C, flowers were initiated in both short day (SD) and LD conditions, at 15 and 18°C in SD only, whereas no initiation took place at 21°C regardless of daylength conditions. The critical photoperiod for SD floral induction was about 16 h and 14 h at 15 and 18°C, respectively, the induction being incomplete at 18°C. The optimal condition for floral induction was SD at 15°C. A minimum of 4 weeks of exposure to such optimal conditions was required. Although the populations varied significantly in their flowering performance, no clinal relationship was present between latitude of origin and critical photoperiod. Flower development of SD-induced plants was only marginally advanced by LD conditions, while inflorescence elongation and runnering were strongly enhanced by LD at this stage. The main shift in these responses took place at photoperiods between 16 and 17 h. Unlike all other populations studied, a high-latitude population from 70°N ('Alta') had an obligatory vernalization requirement. Although flowering and fruiting in its native Subarctic environment and after overwintering in the field in south Norway, this population did not flower in the laboratory in the absence of vernalization, even with 10 or 15 weeks of exposure to SD at 9°C. Flowering performance in the field likewise indicated a vernalization requirement of this high-latitude population.