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Mechanistic studies now emerging in the literature show that HPP inactivates micro-organisms by interrupting cellular functions responsible for reproduction and sur-vival (Figure 7.10). HPP can damage microbial membranes and thus affect transport

onstrating that leaks occur while cells are held under pressure [62]. Membrane damage occurs later than cell death and this suggests that dye exclusion measurements assess-ing this pressure effect can be used to characterize microbial pressure inactivation [63].

Knowledge of cell damage and repair mechanisms could lead to new HPP applica-tions [64,65]. For example, lysis of starter bacteria induced by HPP treatments could promote the release of intracellular proteases important in cheese ripening. Viability, morphology, lysis and cell wall hydrolase activity measurements suggest that high pressure can cause inactivation, physical damage, and lysis in Lactobacillus lactis [66].

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for partially denatured myofibrillar proteins (mostly myosin). Fully denatured

myofi-actions. Sorbitol inhibits the aggregation of myofibrillar proteins of raw

pressure-higher stabilizing effect on the ATPase activity of fish myosin than lactitol, sucrose

these products is a diminishing confidence that our diet satisfies our nutritional needs.

In 1994, 70% of women believed their diet met their nutritional needs, a figure down low-temperature, pressure-treated arrowtooth flounder mince [51]. Sorbitol shows a

phenomena involved in nutrient uptake and disposal of cell waste. Intracellular fluid compounds have been found in the cell suspending fluid after pressure treatment

dem-© 2009 by Taylor & Francis Group, LLC

Treatments at 300 MPa have shown by TEM intracellular and cell envelope damage of the cheesemaking strains Lactococcus lactis subsp. cremoris MG1363 and SK11. Cell whereas cells treated at >400 MPa had decreased cell wall hydrolase activity. How-samples indicating that this pressure activates cell wall hydrolase activity or increases cell wall accessibility to the enzyme.

HPP

Stabilizing agent H₂O

H O H₂O

HPP Native proteins

Pressure induced side-to-side aggregation

Native proteins

Stabilized proteins against side-to-side aggregation

H₂O

H H₂O

Protein structure showing intramolecular interactions Stabilizing agent molecule

Pressure induced crosslinking protein interactions Pressure effect on the protein system

FIGURE 7.9

against pressure induced aggregation. (Adapted from Uresti, R.M., Velazquez, G., Ramírez, J.A., Vázquez, M., and Torres, J.A., Effect of high pressure treatments on mechanical and mias). Journal of the Science of Food and Agriculture 84(13), 1741–49, 2004.)

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Mechanism of stabilization of sugars and polyols on myofibrillar proteins

suspensions treated at 200 or 300 MPa did not differ significantly from the control, ever, cells treated at 100 MPa released significantly more reducing sugar than all other functional properties of restructured products from arrowtooth flounder (Atheresthes

sto-© 2009 by Taylor & Francis Group, LLC

Increasing our knowledge of the behaviour of bacterial membrane proteins sub-jected to pressure under different conditions (e.g. pH or aw) will lead to effective membranes of untreated Salmonella typhimurium reveal three major and 12 minor protein bands but only two major bands after pressure treatments [67]. One band is more pressure resistant in acidic pH media suggesting a different protein conformation at this condition. HPP treatments, 345 MPa for 5 min at 25°C, alter the cell walls of Leuconostoc mesenteroides and make cell membranes permeable [68]. This damage reduces the potential gradient across membranes, preventing cells from synthesizing ATP, which activates the autolytic enzyme degradation of cell walls. Cells treated at 400 MPa for 10 min in pH 5.6 citrate buffer show no growth after 48 h of culture on plate count agar. Cells can be examined by SEM, membrane integrity by propidium

even though membrane potential decreases from −86 to −5 mV [69,70]. Membrane damage in S. typhimurium can also be measured by pH differential (pH_in–pH_out). Mor-phological changes increasing with pressure correlate with a progressive decrease of the pH differential, intracellular potassium, and ATP concentration [71,72].

Denaturation

Active enzyme

Inactive enzyme

Renaturation Enzymes

Nutrients

Waste

Leakage Membranes

(a)

(b)

Hydrostatic pressure effects on cellular functions. (Adapted from Torres, J.A.

and Velazquez, G. Commercial opportunities and research challenges in the high pressure processing of foods. Journal of Food Engineering 67, 95–112, 2005.)

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FIGURE 7.10

hurdle preservation technologies. For example, electrophoretic profiles of the outer

ies reveal no significant changes in cellular morphology while PI staining followed iodide (PI) staining and changes in membrane potential by flow cytometry. SEM stud-by flow cytometry shows a small population proportion with membrane integrity loss

© 2009 by Taylor & Francis Group, LLC

The outer membrane (OM) providing a protective barrier to Gram-negative bac-teria is susceptible to pressure-mediated permeabilization. The kinetics of OM and cytoplasmic membrane permeabilization induced by pressure treatments can be

deter-only slightly even after pressure treatments resulting in a >6 log decrease in viable observed prior to cell death. Reversible OM damage occurs rapidly and is in thermo-dynamic equilibrium with pressure conditions while irreversible OM damage is time dependent. Pressure (200 or 400 MPa) resistance of exponential-phase E. coli NCTC 8164 cells is highest for cells grown at 10°C and decreases with growth temperature up to 45°C [74]. By contrast, pressure resistance of stationary-phase cells is lowest in cells grown at 10°C and increases with growth temperature reaching a maximum at 30–37°C before decreasing at 45°C. This pressure effect can be correlated to the proportion of unsaturated fatty acids in the membrane lipids which decreases with growth temperature in both and stationary-phase cells. In

exponential-sure resistance has been observed.

7.2.1.2.2 Bacterial Spores

Although the application of 400–800 MPa inactivates pathogenic and spoilage bac-teria [4,75–77], the inactivation of bacbac-terial spores has been a major challenge to HPP process developers as these spores are extremely resistant to pressure. Therefore, current HPP products on the market rely on refrigeration, reduced water activity or low pH to prevent bacterial spore outgrowth.

surized at 980 MPa for 40 min at room temperature [78]; however, combining tem-peratures higher than 50°C with pressures above 400 MPa can be effective. For example, treating Bacillus subtilis spores at 404 MPa and 70°C for 15 min can spores of Clostridium sporogenes, considered a non-toxigenic equivalent to pro-teolytic C. botulinum and an important food spoilage bacteria, to 400 MPa at 60°C for 30 min at neutral pH yields only 1 DR [80]. The effect of 15 min hydrostatic pressure treatments (550 and 650 MPa) at 55 and 75°C in citric acid buffer (4.75 the gene that encodes the C. perfringens enterotoxin (cpe) on the chromosome (C-cpe), four isolates carrying the cpe gene on a plasmid (P-(C-cpe), and two strains of C. sporogenes was studied to develop an effective spore inactivation strategy [81].

Treatments at 650 MPa, 75°C and pH 6.5 were found to be moderately effective against spores of P-cpe (approximately 3.7 DR) and C. sporogenes (approximately 2.1 DR) but not for C-cpe (approximately 1.0 DR) spores. Treatments at pH 4.75 were moderately effective against spores of P-cpe (approximately 3.2 DR) and C.

sporogenes (approximately 2.5 DR) but not of C-cpe (approximately 1.2 DR) when combined with 550 MPa at 75°C. However, when pressure was raised to 650 MPa under the same conditions, high inactivation of P-cpe (approximately 5.1 DR) and

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Spores of six Bacillus species showed no significant inactivation when

pres-achieved five decimal reductions (DRs) at neutral pH [79]. However, subjecting

and 6.5 pH) on spores of five isolates of Clostridium perfringens type A carrying mined by staining pressure-treated cells with the fluorescent dyes propidium iodide (PI) and 1-N-phenylnaphtylamine (NPN), respectively [73]. PI fluorescence increases cell counts while increased NPN fluorescence, indicating OM permeabilization, is

phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, no simple relationship between membrane fluidity and

pres-© 2009 by Taylor & Francis Group, LLC

C. sporogenes (approximately 5.8 DR) spores, and moderate inactivation of C-cpe (approximately 2.8 DR) spores were observed. These studies show the important need for further advances in high-pressure treatment strategies to inactivate bacte-modeling of spore germination appears promising.

Many models have been developed to predict the growth of C. perfringens in meat products during the cooling stage [e.g. 82–84]. However, modeling of ger-mination has not been fully studied. Models for B. cereus spore gerger-mination in the presence of L-alanine based on the Weibull function have been proposed [85].

Germinants, that is, compounds that promote spore germination, were recently in the presence of free amino acids (e.g. L-asparagine) but fast in the presence of potassium chloride [81]. The Weibull function was used to model C-cpe spore germination as affected by pH, germinant concentration, and spore germination temperature [81]. An empirical predictive germination rate model for the germina-tion of any C. perfringens type A food poisoning isolate as a funcgermina-tion of spore ger-mination temperature was also constructed. These advances in spore inactivation will further enhance the opportunities to develop shelf-stable food products based on PATP technology.

7.3 PRESSURE ASSISTED THERMAL PROCESSING