Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk

Aims:  The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin.
Methods and Results:  Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40°C with or without 500 IU ml⁻¹ of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2·5 logs by cycled HP, while the addition of nisin (500 IU ml⁻¹) prior to HP treatment resulted in log reductions of 5·7 and 5·9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40°C in the presence of 500 IU ml⁻¹ nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed.
Conclusions:  Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments.
Significance and Impact of the Study:  Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Psychrotrophic strains of Bacillus cereus producing enterotoxin

In investigations on three outbreaks of Bacillus cereus food poisoning in Spain and The Netherlands, the causative strains grew within a temperature range of 4·37†C, but not at 43†C. Such psychrotrophic types were found to occur in various dairy products (including ca 25% of 35 samples of pasteurized milk) and some mousses and cook/chill meals. Growth of and enterotoxin production by psychrotrophic B . cereus could be prevented by temperatures below 4†C and pH-values not exceeding 5·0.     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °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.     

The use of Bacillus diarrhoeal enterotoxin (BDE) detection using an ELISA technique in the confirmation of the aetiology of Bacillus-mediated diarrhoea

A commercially available ELISA kit was used for the detection of Bacillus diarrhoeal enterotoxin (BDE) in a variety of foods and faeces. The ability of isolates of Bacillus spp., including Bacillus cereus , to produce BDE in Brain Heart Infusion broth containing 0·1% glucose was also checked by use of the kit. Results show that 29 out of 31 B. cereus isolates were enterotoxigenic. Foods positive for preformed BDE were always contaminated with >10⁵ B. cereus cfu g⁻¹, but not all foods contaminated with large numbers of B. cereus were positive for BDE. Bacillus spp., other than one isolate which closely resembled B. subtilis , were negative for BDE production. Criteria for the confirmation of Bacillus -mediated diarrhoea should now include reports of symptoms and incubation periods consistent with the diarrhoeal form of food-poisoning by Bacillus spp., together with the results of tests for enterotoxigenicity of the Bacillus isolate, and detection of BDE in either the food and/or faeces.     

Radiation inactivation of some food-borne pathogens in fish as influenced by fat levels

The influence of low (0·39–1·1%), medium (4·25%) and high (7·1–32·5%) fat levels in fish on radiation inactivation of four food-borne pathogens was investigated. Cells of Listeria monocytogenes 036, Yersinia enterocolitica F5692, Bacillus cereus and Salmonella typhimurium at logarithmic phase were inoculated in 10% fish homogenates and subjected to gamma irradiation at ice temperature (0–1 °C) with doses ranging from 0·05 to 0·8 kGy. The radiation survival curves of L. monocytogenes and B. cereus were characterized by shoulders, while a tailing effect was depicted by cells of Y. enterocolitica and B. cereus . The D₁₀ values in kGy calculated on the exponential part of the curve ranged from 0·2 to 0·3, 0·15 to 0·25, 0·1 to 0·15 and 0·09 to 0·1 for L. monocytogenes 036, B. cereus, Salm. typhimurium and Y. enterocolitica F5692, respectively. This order (D₁₀) of radiation resistance of each organism was not affected by the fat content of the fish. Inoculated pack studies carried out separately with each pathogen in fatty (Indian sardine, 7·1%) and lean (Golden anchovy, 0·39%) fish showed no difference in their survival after exposure to 1 kGy and 3 kGy doses, which corroborated the above observation. The practical significance of these results in the application of the technology is discussed.     

Heat-resistant Bacteria in Pasteurized Whole Egg

Samples of egg melange taken from an egg packing station contained an average of 7·3 x 10⁴ organisms/ml which survived laboratory pasteurization at 65°C for 3 min. Many of the organisms surviving pasteurization were found to be coryneform bacteria related to Microbacterium lacticum which could be differentiated into several groups. The remainder were a miscellaneous collection of unidentified cocci and coccobacilli and some Bacillus spp. The coryneform bacteria were shown to be the most heat-resistant isolates with negligible loss of viability after 60 min at 65°C, At least two of the representative strains were very heat-resistant, 0·01% surviving 20 and 38 min at 80°C in phosphate buffer at pH 7·1. Growth tests showed that none of the isolates grew at 5°C after 10 d incubation but those capable of growing most rapidly at 10° and 15°C were also the most heat-resistant. Such strains had a doubling time at 15°C of between 6 and 8 h in whole egg. Freezing the coryneform bacteria in liquid whole egg at –18°C had negligible effect on viability or heat-resistance at 65°C.     

Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus

Carvacrol, a natural plant constituent occurring in oregano and thyme, was investigated for its bactericidal effect towards the food-borne pathogen Bacillus cereus . Carvacrol showed a dose-related growth inhibition of B. cereus . At concentrations of 0·75 mmol l⁻¹ and above, total inhibition of the growth was observed. Below this concentration, carvacrol extended the lag-phase, reduced the specific growth rate and reduced the maximum population density. Incubation for 40 min in the presence of 0·75–3 mmol l⁻¹ carvacrol decreased the number of viable cells of B. cereus exponentially. Spores were found to be approximately 2·3-fold less sensitive to carvacrol than vegetative cells. Bacillus cereus cells showed reduced susceptibility towards carvacrol at pH 7·0 compared with different values between pH 4·5 and 8·5. The culture and exposure temperatures had a significant influence on the survival of vegetative cells. The highest death rate of cells was observed at an exposure temperature of 30 °C. Membrane fluidity was found to be an important factor influencing the bactericidal activity of carvacrol.     

Early development and growth of the laboratory reared north-east Atlantic mackerel Scomber scombrus L.

The early development, growth and morphological changes of mackerel Scomber scombrus were investigated at different incubation temperatures (8, 10, 13, 15 and 18° C). Details on the early life history are illustrated with special reference to morphological transformations. Culture techniques to rear larval mackerel stages are described using laboratory cultured foods. Artificially fertilized eggs were hatched after 80·6 h at 18·4° C and 256·8 h at 8·7° C. The standard length ( L _S) of the individuals at first feeding was 4·71 ± 0·18 mm. Four mortality critical periods and cannibalistic behaviour were identified. A maximum average larval size of 37·5 ± 4·41 mm L _S was attained 30 days post-hatch (dph) at 18·4° C. Development and growth were affected significantly by temperature during both endogenous and exogenous feeding periods. Larvae grew more rapidly at high, than at low temperatures. Daily specific growth rate (in mass) ranged from 2·4% at 10·6° C to 16·9% at 18·4° C. Likewise, average growth rate (in length) ranged from 0·05 mm day⁻¹ at 8·4° C to 0·37 mm day⁻¹ at 18·4° C. The allometric relationship of L _S, with several body measurements was not affected by temperature. Comparison with larvae collected in the Bay of Biscay did not show any significant difference in the dry mass and L _S relationship; conversely, the growth rate in length differed significantly between both laboratory and field conditions. The trends observed in the laboratory are described in relation to some aspects of the year-class strength regulation.     

Growth and physiological changes during metamorphosis of Senegal sole reared in the laboratory

The metamorphosis of Solea senegalensis was studied in larvae reared at 20° C and fed four different feeding regimes. A, Artemia (4 nauplii ml⁻¹); B, Artemia (2 nauplii ml⁻¹); C, mixed diet (2 nauplii ml⁻¹ and 3 mg ml⁻¹ microencapsulated diet); and D, microencapsulated diet (3·7 mg ml⁻¹). Rotifers were also supplied in all cases during the first days of feeding. These feeding regimes supported different growth rates during the pre-metamorphosis period (regime A, G=0·376 day⁻¹; regime B, G=0·253 day⁻¹; regime C, G=0·254 day⁻¹; regime D, G=0·162 day⁻¹). Larvae started metamorphosis 9 days after hatching (DAH) when fed the regime A, 13 DAH with regime B, 11 DAH with regime C and 15 DAH with regime D. A minimum 5·6–5·9 mm L_T was required under all feeding regimes to initiate the metamorphosis. Eye translocation was completed when the larvae reached 8·6–8·7 mm L_T (regimes A, B and C), but only 7·3 mm L_T with regime D. 4·4–6·2 days were required to complete eye migration under the regimes A, B and C, and 18·3 days under the regime D. This transformation is concomitant with changes in body reserves, and with the pattern of some digestive enzymes.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

The effect of temperature and fish size on growth and feed efficiency ratio of juvenile spotted wolffish Anarhichas minor

The growth properties of juvenile spotted wolffish Anarhichas minor reared at 4, 6, 8 and 12° C, and a group reared under 'temperature steps', (T‐step) i.e . with temperature reduced successively from 12 to 9 and 6° C were investigated. Growth rate and feed efficiency ration was significantly influenced by temperature and fish size. Overall growth rate was highest at 6° C (0·68% day⁻¹) and lowest at 12° C (0·48% day⁻¹), while the 4 and 8° C, and the T‐step groups had similar overall growth rates, i.e . 0·59, 0·62 and 0·51% day⁻¹ respectively. Optimal temperature for growth ( T _{opt G} ) and feed efficiency ratio (T_{opt FCE}) decreased as fish size increased, indicating an ontogenetic reduction in T _{opt G} and T _{opt FCE}. The results suggest a T _{opt G} of juvenile spotted wolffish in the size range 135–380 g, dropping from 7·9° C for 130–135 g to 6·6° C for 360–380 g juveniles. The T _{opt FCE} dropped from 7·4° C for 120–150 g to 6·5° C for 300–380 g juveniles. A wider parabolic regression curve between growth, feed efficiency ratio and temperature as fish size increased, may indicate increased temperature tolerance with size. Individual growth rates varied greatly at all time periods within the experimental temperatures, but at the same time significant size rank correlations were maintained and this may indicate stable size hierarchies in juvenile spotted wolffish.     

Effect of heat treatment on Cronobacter spp. in reconstituted, dried infant formula: preparation guidelines for manufacturers

Aim:  To explore safe guidelines for manufacturers and consumers to prepare, handle and store dry infant formula (DIF) to protect infants against Cronobacter spp.
Methods and Results:  Selected strains (2.45, FSM 293, ATCC-12868, FSM-271) screened from 68 strains of Cronobacter spp . were used to study growth and survival in commercial DIF. Prototype growth patterns in Enterobacteriaceae enrichment broth (EEB) containing a cocktail comprised of ATCC 12868, ATCC 29004, ATCC 29544 and ATCC 51329 showed a rapid increase in cell count (2·0 log₁₀ to 6·2 log₁₀ CFU ml⁻¹). Infant formula provided a better protective environment for the cells of Cronobacter strains than did buffered peptone water . Experiments on survival in inoculated (10⁴–10⁶ CFU ml⁻¹) reconstituted infant formula (RIF), preparation temperature, the effect of preparation volume (one-serving or two-serving) and effect of storage at room temperature for up to 10 h provided information to develop consumer guidelines for DIF preparation and handling.
Conclusions:  Reconstituted DIF in water at >70°C in larger volumes, minimizing storage time before feeding and storing unused reconstituted formulate at <4°C, may reduce the risk of Cronobacter infection in infants.
Significance and Impact of the Study:  Meningitis, necrotizing enterocolitis and bacteremia in premature babies has been linked to contaminated milk powder and DIF; better handling practices may improve the safety of these foods for neonates.     

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.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

Thermal tolerance of a northern population of striped bass Morone saxatilis

Thermal tolerance of age 0+ year Shubenacadie River (Nova Scotia, Canada) striped bass Morone saxatilis juveniles (mean ± s . e . fork length, L _F, 19·2 ± 0·2 cm) acclimated in fresh water to six temperatures from 5 to 30° C was measured by both the incipient lethal technique (72 h assay), and the critical thermal method ( C _m). The lower incipient lethal temperature ranged from 2·4 to 11·3° C, and the upper incipient lethal temperature ( I _U) from 24·4 to 33·9° C. The area of thermal tolerance was 618° C². In a separate experiment, the I _U of large age 2+ year fish (34·4 ± 0·5 cm L _F) was 1·2 and 0·6° C lower ( P < 0·01) than smaller age 1+ year fish (21·8 ± 0·5 cm L _F) at acclimation temperatures of 16 and 23° C. Using the C _m, loss of equilibrium occurred at 27·4–37·7° C, loss of righting response at 28·1–38·4° C and onset of spasms at 28·5–38·8° C, depending on acclimation temperature. The linear regression slopes for these three responses were statistically similar (0·41; P > 0·05), but the intercepts differed (25·3, 26·0 and 26·5° C; P < 0·01). The thermal tolerance of this northern population appears to be broader than southern populations.