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ABSTRACT: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage.
Fresh ground beef was inoculated with a five-strain cocktail of E. coli O157:H7 vacuum-packaged, pressure-treated at 400 MPa for 10 min at -5 or 20 degrees C and stored at -20 or 4 degrees C for 5-30 days. A 3-log CFU g(-1) reduction of E. coli O157:H7 in the initial inoculum of 1 x 10(6) CFU g(-1) was observed immediately after pressure treatment at 20 degrees C. During frozen storage, levels of E. coli O157:H7 declined to <1 x 10(2) CFU g(-1) after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55-65 degrees C.
High-pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g(-1) and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage.
HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.
Journal of Applied Microbiology 08/2009; 108(4):1352-60. · 2.34 Impact Factor
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ABSTRACT: 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.
Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40 degrees 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 degrees 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.
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.
Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.
Journal of Applied Microbiology 07/2008; 105(1):78-87. · 2.34 Impact Factor
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ABSTRACT: 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.
Journal of Applied Microbiology 06/2008; 105(1):78 - 87. · 2.34 Impact Factor
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ABSTRACT: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination.
Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment.
VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear.
There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.
Journal of Applied Microbiology 02/2007; 102(1):65-76. · 2.34 Impact Factor
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ABSTRACT: Prior genetic analysis suggests that there may exist an interaction between the products of the vaccinia virus genes A18R, a putative negative transcription elongation factor, and G2R, a putative positive transcription elongation factor. In addition, affinity purification of polyhistidine-tagged G2R protein overexpressed in vaccinia virus-infected cells, reported here, results in copurification of the vaccinia H5R protein, previously characterized as a late viral transcription factor. We have therefore used several methods to screen further for interactions among the G2R, A18R, and H5R proteins. Methods include copurification or co-immunoprecipitation of proteins overexpressed during vaccinia virus infection, activation of the gal 4 promoter by gal 4 fusions in the yeast two-hybrid system, and co-immunoprecipitation of proteins synthesized in vitro in a rabbit reticulocyte lysate. The results reveal interactions which include all possible pairwise combinations of the three proteins G2R, A18R, and H5R; however, not all possible permutations of the interactions are observed and the interactions are not observed in all environments tested. The results suggest that the vaccinia virus proteins G2R, A18R, and H5R interact as part of a higher order transcription complex.
Virology 07/1998; 245(2):313-22. · 3.35 Impact Factor
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ABSTRACT: The phenotypic defects of two mutants of vaccinia virus, the lesions of which map to gene G2R, were characterized in vivo, and the results suggest a role for the G2R protein in viral transcription elongation. Both a temperature-sensitive mutant, Cts56, and an isatin-beta-thiosemicarbazone-dependent deletion mutant, G2A, in gene G2R have a characteristic and unique defect in late viral gene expression. The G2R mutants synthesize early viral RNA, early viral proteins, and viral DNA normally under nonpermissive conditions. In G2R mutants, late viral protein synthesis begins at the normal time, low-molecular-weight viral proteins are synthesized in normal quantities, but synthesis of high-molecular-weight viral proteins is reduced in amount. Intermediate and late promoter utilization is normal in G2R mutants, but intermediate and late RNAs are reduced in size. The reduction in length of the intermediate and late mRNAs represents a truncation of mRNA 3' ends. Thus, intermediate and late RNAs are too short to encode large proteins but long enough to encode small proteins, therefore accounting for the protein synthesis phenotype. These results suggest that the G2R protein acts to regulate the elongation potential of the viral RNA polymerase late during a vaccinia virus infection.
Journal of Virology 01/1996; 70(1):47-54. · 5.40 Impact Factor
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ABSTRACT: Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.
Journal of Virology 10/1995; 69(9):5516-27. · 5.40 Impact Factor
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ABSTRACT: In this study the potential of using selected bacteriophages as pressure surrogates for hepatitis A virus (HAV) and Aichi virus (AiV) was investigated. The coliphages included, T4, MS2, Qβ, λ imm 434, λ cI 857 and λ cI 857A. T4 displayed similar pressure responses as HAV and was chosen for further study. The most pressure-resistant phage, MS2, was selected as a possible surrogate to estimate AiV inactivation by high pressure processing (HPP). HAV, AiV and their selected bacteriophage surrogates were treated at a range of pressures and times in three different media. All four were treated in phosphate-buffered saline (PBS), artificial seawater (ASW) or oyster slurry (OS) at 250, 400 or 500 MPa for 1, 5 or 10 min at 20 °C. While T4 had similar pressure resistance to HAV under conditions of high (500 MPa) and lower pressure (250 MPa), inactivation trends were very different following treatment at 400 MPa and when the viruses were suspended in OS. MS2 showed similar resistance as AiV but at ambient treatment temperatures only. The highest levels of inactivation of MS2 were achieved at 60 °C and 500 MPa. AiV was eliminated at 60 °C for 5 min at ambient pressure, but > 3 log survived exposure to 60 °C at 500 MPa. This degree of protection by pressure may be important in determining the mechanisms of pressure and heat resistances in other viruses.Industrial relevanceGreater knowledge of the responses of viruses and their surrogates to high pressure will aid in the validation of new high pressure-processed food that may be at risk to contamination from HAV or other enteric viruses.
Innovative Food Science & Emerging Technologies.
