**4. The inactivation of food related bacteria**

The application of high pressure ranging from 100 to 1000 MPa, is one of the most promising methods for the food treatment and preservation at room temperature (Debs-Louka et al., 1999). High pressure inactivation of *E. coli* pressure resistant had been investigated in fruit juices and in low pH buffers. The results show that both parents and mutant strains become more pressure-sensitive in decreased pH and presence of organic acids. The high pressure treatment for 5–10 min under 300–600 MPa at 20–50°C allows the reduction of vegetative microbial cells by 4–5 log cycles. However, some enzymes, especially polyphenoloxidase in fruit juices, are more pressure-resistant and their inactivation needs additional approaches (Molin, 1983). The degradation of color and slight changes of flavor due to the higher content of dissolved oxygen in products are mentioned as an example of negative pressure effects (Knorr, 1995). Pasteurization of milk and the heat resistance of *Mycobacterium avium* subsp *paratuberculosis* (Lund et al., 2002) also vegetative and the latent form of other microorganisms have been reviewed (Sojka and Ludwig et al., 1997; Paidhungat et al., 2002; Raso et al., 1998). It seems evident that it was not possible to kill spores at room temperature with an extremely high operating pressure, up to 170 MPa. It was observed that there is an optimum range of temperature and pressure for stimulating

Supercritical Fluid Application in Food and Bioprocess Technology 559

phosphohexose isomerase, gama-glutamyltransferase, and alkaline phosphatase occurs as a result of SC treatment (Sojka and Ludwig, 1997; Ishikawa et al., 1997). Blickstad et al. (1981) reported the effect of CO2 on pork microflora and found that increasing the partial pressure

Factors, such as temperature, pressure, and moisture, contribute to a more effective treatment by increasing the diffusivity of CO2 (Isenschmid et al., 1995; Kamarei and Arlington, 1988; Schreck and Ludwig, 1997; Smelt, 1998). Within certain limits, a longer duration of exposure to CO2 permits better sterilization; exposure time can be decreased by increasing the temperature (Lund et al., 2002). Microbial resistance to CO2 also depends on the type of microorganism, the phase of growth, moisture, (Lin and Chen, 1994; Kamihira et al., 1987) and the suspension medium, the last of which can inhibit the bactericidal effect of compressed CO2, especially in some food systems rich in proteins (Ishikawa et al., 1995). Lin et al. suggested that swollen cell walls, due to the presence of water, become more CO2 permeable (Lin et al., 1994). Finally, though the use of the CO2 sterilization offers cost and environmental advantages, there is no guarantee that a CO2 sterilizer will receive FDA approval. The analysis of the CO2 sterilization shows a lower cost per cubic foot (\$6) than EtO (\$19) because of the shorter cycle time, lower cost per load, and lack of regulatory constraints

Products SCF T/ °C P/ MPa

Progestrone, Testostrone, Chlosterol CO2 with or without N2O 35-60 8-25 Glycerides of fatty acid short chain CO2 40 31 Cholesterol CO2 40-60 8-12

acyl chain length of 12-36 carbons CO2 40 13-30 Lipids from egg yolk CO2 & (methanol or ethanol) -- --

derived from cod river oil CO2 50 15

10, 16, 19 DHA from menhaden oil CO2 -- --

*saprolengia parasitica* CO2 60 35

Table 1. Some examples of supercritical fluid extraction of valuable constituents from

The CO2 technology showed some disadvantages including high capital cost; space needed to store the CO2 cylinders; design and build a prototype that satisfy the temperature, pressure, humidity, and agitation requirements for the CO2 sterilization (Schreck and Ludwig, 1997). High pressure has the advantage of retaining taste, color, and texture much better than heat treatments and also affects food constituents; proteins, lipids, and starches may undergo conformational changes. In Japan, high pressure pasteurization of acidic foods, such as fruit juices and jam, is being practiced on an industrial scale. Such "cold" processing might also be utilized in a useful way in industrial cheese-making processes of

deodorized propane -- 8

of CO2 added to the packaging atmosphere and prolonged the shelf life of the meat.

without negative environmental and health effects.

Pure saturated triglycerides with

cis-5, 8,11,14,17 EPA and Cis-4, 7,

Fatty acid-ethyl esters

PUFA from fungus

Lecithin and Soya oil Near critical CO2 +

natural materials in food industry (Nguyen et al., 1994)

the germination of spores (Nakayama et al., 1996; Roberts and Hoover, 1996). Therefore, the coupled action of hydrostatic pressure and of specific temperature was investigated in order to activate spores and consequently to inactivate their vegetative forms in a second step with higher operating pressure (Hong et al., 1999). Ludwig et al. carefully studied the behavior of spores under different operative conditions of the high temperature and pressure and introduced a cycle-type treatment (Ludwig et al., 1994; 1997); this appeared to be more efficient than the double level pressure treatment. It was concluded that at higher temperature, faster germination is obtained, as well as a wider range of pressure is suitable to this scope. Salts, glucose, and amino acids were found to enhance the rate of germination. However, until now a complete inactivation of spores has not been achieved yet. The regression analysis of inactivation rates showed that pressurization at sub-zero temperatures (−20 and −10°C) enhanced the effects of pressure as pressurization at higher temperatures (i.e., pressurization at 190 MPa and −20°C gave the same effect as pressurization at 320 MPa and room temperature). The results imply that high pressure treatment at lower temperatures has a greater effect on food sterilization without destroying the original taste and flavor. Additional effects of sugars and salts on the inactivation of yeast are also described (Hashizume et al., 1995).

Many recent studies demonstrate that SC-CO2 as a non-toxic and inexpensive gas can also be used for the inactivation of viruses (Fages et al., 1998) and pest control. It is a promising alternative method for the pasteurization and sterilization of foodstuff (particularly in the liquid phase), sterilization of thermosensitive substances, as well as thermally and hydrolytically sensitive polymeric materials in biomedical applications. Furthermore, application of SC-CO2 seems to be attractive for its economical feasibility, as it needs very low pressure (lower than 20 MPa) compared to the so–called ultra high pressure treatment (200–700 MPa) (Bertucco and Vetter, 2001). A number of papers have been addressed to the inactivation of a wide range of microorganisms, bacteria, spores, and yeasts in physiological solutions by SCF (Watanabe et al., 2004; Erkmen, 2001; Spilimbergo et al., 2002; Clery-Barraud et al., 2004).

In the field of industrial applications, it is worthy to quote some recent publications that have dealt with the inactivation in complex substrates and solid food (Haas et al. 1989; Gould, 2003; Arreola et al., 1991; Erkmen, 2000; 2001). All these authors tested the efficiency of the SC-CO2 mainly in natural foods (e.g. milk, fruit juice, and eggs) in a batch system. Spilimbergo has also checked with high pressure CO2 on red orange juice of Sicily (Spilimbergo et al., 2002). Spores of *B. coagulans, B. subtilis, B. cereus, B. licheniformis, and Geobacillus stearothermophilus* were subjected to CO2 treatment at 30–200 MPa and 35–65°C. All of the bacterial spores except the *G. stearothermophilus* spores were easily inactivated by the heat treatment. The treatment with CO2 and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation. The activation energy required for the CO2 treatment of *G. stearothermophilus* spores was lower than the activation energy for heating or pressure treatment (Matsuda et al., 2004). Lund et al. have studied heat resistance of *Mycobacterium avium* subsp *paratuberculosis* and related problems in milk pasteurization (Lund et al., 2002). Kamihira et al. found a sterilizing effect of SC-CO2 on various microorganisms at 20.3 MPa and 35°C (water content of 70 to 90) (Kamihira et al., 1987). Dried cells were not sterilized when treated under the same conditions. Bruna evaluated the composition changes of strawberry puree during high pressure pasteurization (Bruna et al., 1994). The inactivation effect of the native microorganisms in raw milk and raw cream is nearly the same. Fat does not influence the inactivation. The inactivation of milk enzymes

the germination of spores (Nakayama et al., 1996; Roberts and Hoover, 1996). Therefore, the coupled action of hydrostatic pressure and of specific temperature was investigated in order to activate spores and consequently to inactivate their vegetative forms in a second step with higher operating pressure (Hong et al., 1999). Ludwig et al. carefully studied the behavior of spores under different operative conditions of the high temperature and pressure and introduced a cycle-type treatment (Ludwig et al., 1994; 1997); this appeared to be more efficient than the double level pressure treatment. It was concluded that at higher temperature, faster germination is obtained, as well as a wider range of pressure is suitable to this scope. Salts, glucose, and amino acids were found to enhance the rate of germination. However, until now a complete inactivation of spores has not been achieved yet. The regression analysis of inactivation rates showed that pressurization at sub-zero temperatures (−20 and −10°C) enhanced the effects of pressure as pressurization at higher temperatures (i.e., pressurization at 190 MPa and −20°C gave the same effect as pressurization at 320 MPa and room temperature). The results imply that high pressure treatment at lower temperatures has a greater effect on food sterilization without destroying the original taste and flavor. Additional effects of sugars and salts on the inactivation of yeast are also described

Many recent studies demonstrate that SC-CO2 as a non-toxic and inexpensive gas can also be used for the inactivation of viruses (Fages et al., 1998) and pest control. It is a promising alternative method for the pasteurization and sterilization of foodstuff (particularly in the liquid phase), sterilization of thermosensitive substances, as well as thermally and hydrolytically sensitive polymeric materials in biomedical applications. Furthermore, application of SC-CO2 seems to be attractive for its economical feasibility, as it needs very low pressure (lower than 20 MPa) compared to the so–called ultra high pressure treatment (200–700 MPa) (Bertucco and Vetter, 2001). A number of papers have been addressed to the inactivation of a wide range of microorganisms, bacteria, spores, and yeasts in physiological solutions by SCF (Watanabe et al., 2004; Erkmen, 2001; Spilimbergo et al., 2002; Clery-

In the field of industrial applications, it is worthy to quote some recent publications that have dealt with the inactivation in complex substrates and solid food (Haas et al. 1989; Gould, 2003; Arreola et al., 1991; Erkmen, 2000; 2001). All these authors tested the efficiency of the SC-CO2 mainly in natural foods (e.g. milk, fruit juice, and eggs) in a batch system. Spilimbergo has also checked with high pressure CO2 on red orange juice of Sicily (Spilimbergo et al., 2002). Spores of *B. coagulans, B. subtilis, B. cereus, B. licheniformis, and Geobacillus stearothermophilus* were subjected to CO2 treatment at 30–200 MPa and 35–65°C. All of the bacterial spores except the *G. stearothermophilus* spores were easily inactivated by the heat treatment. The treatment with CO2 and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation. The activation energy required for the CO2 treatment of *G. stearothermophilus* spores was lower than the activation energy for heating or pressure treatment (Matsuda et al., 2004). Lund et al. have studied heat resistance of *Mycobacterium avium* subsp *paratuberculosis* and related problems in milk pasteurization (Lund et al., 2002). Kamihira et al. found a sterilizing effect of SC-CO2 on various microorganisms at 20.3 MPa and 35°C (water content of 70 to 90) (Kamihira et al., 1987). Dried cells were not sterilized when treated under the same conditions. Bruna evaluated the composition changes of strawberry puree during high pressure pasteurization (Bruna et al., 1994). The inactivation effect of the native microorganisms in raw milk and raw cream is nearly the same. Fat does not influence the inactivation. The inactivation of milk enzymes

(Hashizume et al., 1995).

Barraud et al., 2004).

phosphohexose isomerase, gama-glutamyltransferase, and alkaline phosphatase occurs as a result of SC treatment (Sojka and Ludwig, 1997; Ishikawa et al., 1997). Blickstad et al. (1981) reported the effect of CO2 on pork microflora and found that increasing the partial pressure of CO2 added to the packaging atmosphere and prolonged the shelf life of the meat.

Factors, such as temperature, pressure, and moisture, contribute to a more effective treatment by increasing the diffusivity of CO2 (Isenschmid et al., 1995; Kamarei and Arlington, 1988; Schreck and Ludwig, 1997; Smelt, 1998). Within certain limits, a longer duration of exposure to CO2 permits better sterilization; exposure time can be decreased by increasing the temperature (Lund et al., 2002). Microbial resistance to CO2 also depends on the type of microorganism, the phase of growth, moisture, (Lin and Chen, 1994; Kamihira et al., 1987) and the suspension medium, the last of which can inhibit the bactericidal effect of compressed CO2, especially in some food systems rich in proteins (Ishikawa et al., 1995). Lin et al. suggested that swollen cell walls, due to the presence of water, become more CO2 permeable (Lin et al., 1994). Finally, though the use of the CO2 sterilization offers cost and environmental advantages, there is no guarantee that a CO2 sterilizer will receive FDA approval. The analysis of the CO2 sterilization shows a lower cost per cubic foot (\$6) than EtO (\$19) because of the shorter cycle time, lower cost per load, and lack of regulatory constraints without negative environmental and health effects.


Table 1. Some examples of supercritical fluid extraction of valuable constituents from natural materials in food industry (Nguyen et al., 1994)

The CO2 technology showed some disadvantages including high capital cost; space needed to store the CO2 cylinders; design and build a prototype that satisfy the temperature, pressure, humidity, and agitation requirements for the CO2 sterilization (Schreck and Ludwig, 1997). High pressure has the advantage of retaining taste, color, and texture much better than heat treatments and also affects food constituents; proteins, lipids, and starches may undergo conformational changes. In Japan, high pressure pasteurization of acidic foods, such as fruit juices and jam, is being practiced on an industrial scale. Such "cold" processing might also be utilized in a useful way in industrial cheese-making processes of

Supercritical Fluid Application in Food and Bioprocess Technology 561

and separations. Enzymes such as alpha amylase, glucose oxidase, lipase, and catalase retained their activities in the solution of high pressure CO2 in water. All thermal and nonthermal methods to stabilize could have their own disadvantages. So, the researchers have focused on the applicability of SC in this process (Nakamura, 1990). Soybean lipoxygenase dissolved in Tris HCl buffer (0.01M; pH 9) was irreversibly inactivated by combined pressure (up to 650 MPa) and low temperature (−15 up to 35°C) treatment. The enzyme inactivation followed a first order reaction and the phase transition of water did not change

Reports have described the use of SCFs for the treatment of lignocellulosic materials, which are the major group of wastes of food industries e.g. straw and bran of corn and cereal, leaf and pomace of sugar cane, fruitwaste, etc. SCF treatment allows further utilization of lignocellulosic materials as a resource for chemicals, pulp, and energy (Puri, 1983). Pretreatment methods have been sought to remove lignin and to permit further utilization of carbohydrates contained in lignocellulosic materials. Several SCFs (e.g. SC-methanol, SCacetone, and SC-ammonia) an alternative to chemical pretreatments, which use strong acids or bases. Ammonia-treated lignocellulosic materials were neutralized and buffered to a pH value of 4.8 before being incubated at 50°C with a fractionated and partially purified commercial crude cellulose preparation from *Trichoderma reesei*. Aliquots taken at various times were filtered before being analyzed for sugars by HPLC. Two long-term experiments were made with diets consisting of preparations of spent hops and a sample of apple pomace, incubated in the Rumen Simulation Technique (RuSiTech). A significant increase in the total volatile fatty acids and methane production was observed when the preparations of spent hops were incubated in separate bags rather than in mixtures with other components (Cansell et al., 1997). Lignin, cellulose, and their mixture were gasified with a nickel catalyst in SC-water at 673 K and 25 MPa. The gasification efficiency was low, but increased with the amount of the catalyst when softwood lignin was included in the feedstock. One possible mechanism is the catalyst being deactivated by tarry products from the reaction between cellulose and softwood lignin. Sawdust and rice straw were gasified under the same

The use of the SCF as a dispersion for biocatalysis was described in 1985 and there is now a growing trend in using the SCF as a reaction media for enzymes. The advantage of using enzymes in the SCF include; synthesis reactions in which water is a product can be driven to completion, the increased solubilities of hydrophobic materials, greater thermostability of biomolecules in SCF, readily solvent recycling, integrated biochemical reactions and separations. CO2 is the most widely used SCF, however, there is a growing interest in using other SCFs (e.g., ethylene, fluoroform, ethane, sulfur hexafluoride and near critical

Enzymes such as alpha amylase, glucose oxidase, lipase and catalase retained their activities in the solution of high pressure CO2 in water. Among the enzymatic reactions in SCF, the use of lipase shows most commercial promise. A SC-CO2/H2O mixture may be used as a reaction medium for either hydrolytic or synthetic reactions catalyzed by lipase and other

the kinetic inactivation behavior.

condition (Yoshida et al., 2004).

propane).

**9. The SCF application as dispersion for biocatalysis** 

**8. The treatment of wastes of food industries** 

typical hard cheeses from "raw milk". With this process it could be possible to reduce the overall microbial load adverse to making cheese without modifying the subtle chemicophysical balance of milk. As pressure-treated milk shows modified properties during further processing, such as changed rennet or acid coagulation characteristics, coagulation time, and gel firmness. Further studies should be carried out to understand whether the structural changes of milk compounds could worsen cheese-making processes (Cuoghi, 1993). Also, the effect of high pressure on microorganisms and enzymes of ripening cheeses were studied. A significant decrease of total microbial count was obtained at the pressure above 400MPa. It was found that the inactivation of microorganisms was affected more by their initial number than by the type of cheese and its maturity. *E. coli* was completely inactivated in 400 MPa pressurized cheeses irrespective of their initial count. *Enterococci*  were inactivated at 400 MPa, while the pressure of 600 MPa was needed to achieve this effect in a 2-week-old cheese. Yeasts and moulds were inactivated with 200 MPa. Aminopeptidases and endopeptidases of both cheese and its extract lost the catalytic abilities at 600 MPa irrespective of the type and ripening time of cheeses (Reps et al., 1994). On the whole, the SCF sterilization has been reported as a successful approach in the sterilization of several kinds of food including; fruits, juices, vegetables, jam, meat, milk, wine, liquid whole egg, natural pigments, yoghurt, and even chocolate (Hammam, 1992).
