High pressure inactivation kinetics of<i>bacillus subtilis</i>cells by a three‐state‐model considering distributed resistance mechanisms

128

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30°C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10°C and by 6-log-unit cycles at temperatures of 20 and 30°C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20°C and >400 MPa at 30°C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20°C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.

Section: Discussionsupporting

confidence: 65%

Use of Hydrostatic Pressure for Inactivation of Microbial Contaminants in Cheese

O’Reilly

O’Connor

Kelly

et al. 2000

128

“…Using two strains of Clostridium botulinum type E, Reddy et al (31) also observed differences in pressure resistance within one species. Remarkably, the strain B. licheniformis TMW 2.492, used in this study, exhibited a intermediate pressure resistance compared to other food isolates of B. subtilis and B. amyloliquefaciens but a higher resistance than other strains of B. subtilis for which literature data are available (10,11,16,18,23,41). This finding highlights the need for studies with food isolates to establish pressure processes in food preservation.…”

Section: Vol 70 2004 Pressure Inactivation Of Bacillus Endospores 7325mentioning

confidence: 70%

“…Therefore, those target organisms used to determine suitable process conditions for the thermal treatments of foods are not suitable target organisms for pressure processes. On the basis of published data, spores of B. amyloliquefaciens Fad 11/2, Fad 77, Fad 82, Fad 99, and Fad 108 are more pressure resistant than spores of Bacillus, Geobacillus, Alicyclobacillus, or Clostridium species, including strains of C. botulinum type A and type E (1,5,9,10,11,16,18,20,23,29,30,31,32,41,45). Therefore, we currently consider them relevant target organisms for the pressure sterilization of foods.…”

Section: Vol 70 2004 Pressure Inactivation Of Bacillus Endospores 7325mentioning

confidence: 99%

Pressure Inactivation of Bacillus Endospores

Margosch

Gänzle

Ehrmann

et al. 2004

139

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80°C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70°C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60°C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.High-hydrostatic-pressure treatment of foods is a process for food preservation at moderate temperatures. Pressure in the range of 200 to 800 MPa is effective at eliminating vegetative bacteria in food (37,39). Pressure treatment at ambient temperatures initiates germination of bacterial endospores (13), but they are not inactivated at 25°C and a pressure over 1,000 MPa (36, 37). Inactivation of bacterial endospores requires a combination of pressure and moderate heat (21), and the efficacy of pressure treatment is enhanced by the presence of nisin (33, 41), by low pH (41, 43), and by oscillatory compression procedures (10,14). Currently available data indicate that endospores of Bacillus and Clostridium species are inactivated by treatments with pressures ranging from 500 to 800 MPa at temperatures ranging from 60 to 80°C.Studies on the pressure effects on vegetative cells of bacteria have demonstrated that the resistance to pressure strongly varies within strains of one species (4, 12). Likewise, the heat resistance of endospores of various strains of one species may exhibit strong variations (38). The majority of studies on the pressure resistance of bacterial endo...

“…However, for Bacillus species (16,28) and for E. coli (15), it was shown that pressure inactivation curves may exhibit a pronounced shoulder and tailing. The shoulder of pressure inactivation curves was explained by phenotypic heterogeneity in the population, and a mathematical model that used a Weibulldistributed kinetics for the transition from a stable to a metastable state during pressure treatments fitted experimental death-time data well (16). If pressure treatment of bacteria indeed is a two-stage inactivation process leading to metastable or sublethally injured cells, it should be possible to identify physiological changes occurring during the shoulder of pressure death-time curves.…”

Section: Discussionmentioning

confidence: 99%

Effects of High Pressure on Survival and Metabolic Activity of Lactobacillus plantarum TMW1.460

Ulmer

Gänzle

Vogel

2000

124

The application of high pressure (HP) for food preservation requires insight into mechanisms of HPmediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10 6 cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.