Microculture model studies on the effect of various gas atmospheres on microbial growth at different temperatures

A microculture technique, employing 96-well tissue culture plates in plastic bags, was used to test the effect of different gas atmospheres (vacuum, air, nitrogen, and carbon dioxide) on the growth of Escherichia coli, Bacillus macerans, Salmonella typhimurium. Candida albicans, Lactobacillus plantarum, Pseudomonas/Acinetobacter/moraxella-group, Brochothrix thermosphacta and Yersinia enterocolitica at 2, 6, and 20 degrees C. In general, carbon dioxide was the most effective inhibitor. The inhibition increased with decreasing temperature. Only the combination of carbon dioxide and 2 degrees C provided complete inhibition of Broch. thermosphacta and Y. enterocolitica.     

Storage of poultry meat under modified atmospheres or vacuum packs: possible role of microbial metabolites as indicator of spoilage

The effect of carbon dioxide (100%), nitrogen (100%), carbon dioxide/oxygen (20% : 80%) or vacuum pack at 3 and 10°C was studied on the microbial flora, in skinless poultry breast fillets or thigh meat. Lactic acid bacteria and Brochothrix thermosphacta were the predominant organisms in samples stored in vacuum packs, carbon dioxide and nitrogen. Pseudomonads grew only in oxygen/carbon dioxide packaging systems. The concentration of lactate diminished in both thigh and breast meat during storage at 3 and 10°C. This decrease was more pronounced in thigh meat stored under 20% : 80% carbon dioxide/oxygen. Acetate increased to varying degrees in all samples regardless of the storage conditions.     

The effect of carbon dioxide on the growth of Yersinia enterocolitica in a simulated milk medium

Carbon dioxide (30 mmol/l) was shown to inhibit the growth of four strains of Yersinia enterocolitica grown separately in a simulated milk medium at 7°C and 60 rev/min for 4 d. This indicates that addition of CO₂ to refrigerated raw milk supplies is likely to be a safe process with respect to that organism.     

Effect of formulation pH and storage temperatures on the preservative efficacy of some gases used as propellants in cosmetic aerosols

The effects of changes in formulation pH and storage temperature on the preservative activities of some aerosol propellants—butane, carbon dioxide, dimethylether and their combinations were investigated. A preservative challenge test method was used to determine the survival rates of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus niger at formulation pH levels 5·80, 7·28 and 8·10 and storage temperatures of 20°, 30° and 40°C.
A significant decrease in the pH of formulations was observed with no corresponding changes in the antimicrobial effectiveness when carbon dioxide was incorporated. Alterations in the antimicrobial profiles of these propellants due to changes in formulation pH were dependent on the propellant and the species of the micro-organism, especially when single propellants were used. Results also showed that the propellants exert antimicrobial activities against the various organisms at the three storage temperatures but there were significantly greater inhibitory activities at 40°C. With a combination of 10% butane/dimethylether (1:2) and 10 bar carbon dioxide there were no differences in the degree of microbial inhibition at the various formulation pH levels and storage temperatures. In most cases, the organisms were completely inactivated within 24 h. These findings showed that the combination of butane/dimethylether with carbon dioxide could be used to protect against microbial contamination and spoilage of formulations of different pH levels as well as those meant for storage at different temperatures.     

Cell location and partial characterization of Brochothrix thermosphacta and Lactobacillus curvatus lipases

The lipases of Brochothrix thermosphacta and Lactobacillus curvatus were in the soluble fraction of the cell and not membrane- or wall-bound. Their optimal activities were around pH 6mD5 and at 37°C. Enzymes were stable between 4 and 30°C and at freezing temperatures (— 20°C and —60°C). The lipase of Lact. curvatus was stable in the pH range of 4mD0 to 11mD0, whereas that of B. thermosphacta was denatured at pH lower than 5mD0 and greater than 9mD0. Among triglycerides tested, maximal activity was found with tributyrin and hydrolysis of other triglycerides decreased as the length of fatty acid increased.     

Factors affecting growth and lipase production by meat lactobacilli strains and Brochothrix thermosphacta

Lipolytic activity of lactobacilli strains and Brochothrix thermosphacta was cellrelated; no significant activity was found in the supernatant fluids. Most lipase was produced during the logarithmic phase of growth and was greatly affected by growth conditions. The optimal temperatures for growth and lipase production were respectively 24°C for B. thermosphacta and 30°C for lactobacilli. For all strains, an initial pH of around 7-0 for the medium and low glucose concentration stimulated lipase production. Tributyrin inhibited both growth and lipase production at a concentration of 0-1% for B. thermosphacta or 1% for lactobacilli. Butyric acid (0-1%) and anaerobic culture inhibited lipase production by B. thermosphacta while these two factors had no effect on enzyme production by lactobacilli.     

Incorporation of nisin into a meat binding system to inhibit bacteria on beef surfaces

In two separate experiments, the bacteriocin, nisin, was incorporated into a commercially available meat binding system (Fibrimex^®) and applied to meat surfaces as a way of inhibiting the meat spoilage organism, Brochothrix thermosphacta during extended refrigerated storage. In experiment 1, pre-rigor lean beef carcass tissue (BCT) was inoculated with B. thermosphacta , left untreated (U), treated with 10 μg ml⁻¹ nisin (N), Fibrimex^® (F) or Fibrimex^® containing 10 μg ml⁻¹ nisin (FN), held aerobically at 4 °C for up to 7 d, and populations of B. thermosphacta and nisin activity determined. Experiment 2 determined the effects of the same treatments but on post-rigor, frozen and thawed lean BCT that was inoculated, vacuum-packaged, and stored at 4 °C for up to 14 d. In both experiments, N- and FN-treated tissues exhibited significantly lower populations of B. thermosphacta compared to U- and F-treated tissues, for the duration of refrigerated storage. Nisin activity was detected up to 7 d in N- and FN-treated samples from experiment 1. However, activity was detected only to days 0 and 2 in FN- and N-treated samples, respectively, from experiment 2. These studies indicate that the addition of a bacteriocin to a meat binding system and application to meat surfaces may be useful in reducing undesirable bacteria in restructured meat products.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25°C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22°C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22°C and plating on CIN agar (48 h at 25°C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

A note on the microbiology of retail packs of prepared salad vegetables

Retail packs of mixed, prepared salad vegetables from two different manufacturers were stored at 7°C until the end of storage-life (sell-by date plus 1 d), when the microbial flora was examined. The quality of the salads was acceptable at the end of storage-life. The oxygen concentrations in packs were lower, and the carbon dioxide concentrations were higher, than those in air. High numbers of bacteria were present, with Pseudomonas spp. and Enterobacter agglomerans predominating in packs of both salads, together with lactic acid bacteria in one of the salads. Significant numbers of pectolytic bacteria including Pseudomonas spp. and Erwinia carotovora were detected. Despite the presence of high numbers of coliform bacteria, Escherichia coli was not detected in batches of one salad, and was detected in relatively low numbers in batches of the other. Yersinia spp., predominantly environmental strains of Yersinia enterocolitica , were isolated by enrichment from all samples tested; Staphylococcus aureus and enterococci were not detected.     

Effect of modified atmosphere composition on the metabolism of glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO(2) percentages, glucose metabolism remained anaerobic under greater oxygen contents.     

A comparison of carbon dioxide production and oxygen uptake in sediment cores from four south Swedish lakes

Carbon dioxide production and oxygen uptake were measured in undisturbed sediment cores taken during winter from four lakes of different trophic state. Respiration was measured at 5, 10, 15 and 20°C at high oxygen saturation (75–100%). The respiratory quotient, calculated from the mean values of carbon dioxide production and oxygen uptake at each temperature for each lake, was 0.83–0.96 with a mean value for the four lakes of 0.90. At very low oxygen saturations (<10%) carbon dioxide production was 21–42% of the production at 20°C and high oxygen saturations. The results indicate that under aerobic conditions, oxygen uptake and carbon dioxide production are closely-coupled processes in these lake sediments.     

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y . enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp . Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp , including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y . enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.     

Fate of Aeromonas and Yersinia on modified-atmosphere-packaged (MAP) cod and trout

A.R. DAVIES AND A. SLADE. 1995. The growth/survival of Aeromonas spp. and Yersinia enterocolitica on cod and trout stored in two modified atmospheres at 0, 5 and 12°C was compared with that on aerobically stored fish. Both organisms grew on aerobically stored fish at all three temperatures. Growth on the modified-atmosphere-stored fish was never greater and was generally markedly less than on aerobically stored fish. This reduction in growth was greater in the higher carbon dioxide-containing atmosphere and at the lower temperature(s).     

The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogenes in beef cattle slurry is temperature-dependent; they decline more rapidly at 17°C than at 4°C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T₉₀ values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T₉₀ value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.     

Effect of Packaging under Carbon Dioxide, Nitrogen or Air on the Microbial Flora of Pork Stored at 4°C

Changes in the microbial flora of pork packed in laminated plastic bags and stored at 4 °C were studied in an initial atmosphere of carbon dioxide, nitrogen or air. The time needed for the total aerobic count at 28 °C to reach 5 × 10⁶ organisms/cm² was about 7 times longer in carbon dioxide than in air, whilst in nitrogen it was about twice as long.
The predominant organisms on fresh pork taken directly from the processing line were: Acinetobacter calcoaceticus , non-fluorescent Pseudomonas spp. and Flavobacterium spp. After storage in air for 7 d, more than 90% of the flora consisted of non-fluorescent Pseudomonas spp. After storage in nitrogen for 10 d, 70% of the flora consisted of non-fluorescent Pseudomonas spp. with lower levels of fluorescent Pseudomonas spp., Kurthia zopfii, Aeromonas hydrophila and Lactobacillus plantarum. The non-fluorescent Pseudomonas spp. could be divided into three different groups, on proteolytic and lipolytic ability; the distribution of the groups was markedly different between pork loins stored in air and nitrogen.
On pork stored in carbon dioxide for 21 d the flora consisted of L. plantarum together with lower levels of heterofermentative lactic acid bacteria. When the storage time in carbon doxide was prolonged to 35 d, the proportion of heterofermentative lactic acid bacteria increased to about 50% of the flora.     

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l^-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32°C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37°C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the vir F gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37°C on Yersinia selective agar.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22°C and 37°C; production was optimum at 37°C in the stationary phase (14–16 h). A decrease in phospholipase C activity at various storage temperatures (—5°C, 4°C, 37°C) was also observed, although the enzyme was active over a wide range of temperature (5–65°C) and pH (3mD5–7mD5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.