The need for a food preservation method is to make safe, inexpensive foods with preservation of heat-sensitive compounds.

CO2 is used because of its safety, low cost, and high purity.

Dense-phase carbon dioxide (DPCD) treatment is a non-thermal treatment of liquid foods or liquid model solutions which inactivates micro-organisms without the loss of nutrients or quality changes that may occur due to thermal effects.

It is also called as “cold pasteurization”.

In the DPCD process, food is contacted with pressurized sub- or supercritical CO2 for a period of time in a batch, semi-batch or continuous manner.

The CO2 pressures can range from 7.0 to 40.0 MPa. Process temperatures can range from 20 to 60C.

The treatment times can range from about 3 to 9 min for continuous, or from 120 to 140 min for semi-continuous or batch DPCD processes.

Source:- Novel Thermal and Non-Thermal Technologies for Fluid Foods.

3 types of treatment systems are used:

• Batch type:- Both CO2 and treatment solution are stationary in a container during treatment

• Semi-continuous type:- continuous flow of CO2 through the chamber while liquid food is stationary

• Continuous type:- Flow of both CO2 and the liquid food

Batch type DPCD system (Hong and Pyun 1999)

A typical batch system mainly has a CO2 gas cylinder, a pressure regulator, a pressure vessel, a water bath or heater, and a CO2 release valve.

The sample is placed into the pressure vessel and temperature is set to the desired value.

Then, CO2 is introduced into the vessel until the sample is saturated at the desired pressure and temperature.

The sample is left in the vessel for a period of time and then CO2 outlet valve is opened to release the gas.

Some systems contain an agitator to decrease the time to saturate the sample with CO2.

Ishikawa et al.,1995 developed a semi-continuous system using a cylindrical filter to micro-bubble CO2 entering into the pressure vessel.

The inactivation of enzymes using a micropore filter was 3 times more than without using it.

CO2 is increased from 0.4 to 0.92 mol/L in the sample at 25 MPa and 35 °C.

Continuous type DPCD system (Shimoda et al., 2001)

A continuous micro-bubble system, very effective in inactivating microorganisms.In this system, liquid CO2 and a saline solution were pumped through a vessel.Liquid CO2 was changed to gas using an evaporator and then dispersed into the saline solution from a stainless steel mesh filter with 10-m pore size.The micro-bubbles of CO2 moved upward while dissolving into the solution.Then, the solution saturated with CO2 was passed through a heater to reach the desired temperature. Another coil with a heater was used to adjust the residence time.

Different researchers proposed different mechanism of inactivation of micro-organisms in DPCD process.Some of them are given below:

pH lowering effect (Meyssami et al.,1992)

Inhibitory effect of molecular CO2 and bicarbonate ion (Ishikawa et al.,1995)Physical disruption of cells (Fraser,1951)Modification of cell membrane and extraction of cellular components (Kamihira et al.,1987)

CO2 can lower pH when dissolved in the aqueous part of a food by forming carbonic acid, which further dissociates to give bicarbonate, carbonate and H+ ions lowering extracellular pH.

CO2 + H2O ↔ H2CO3

H2CO3 ↔ H+ + HCO3

HCO3 ↔ H+ + CO3

The internal pH of microbial cells has the largest effect on their destruction.

Sufficient CO2 in the environment penetrates through the cell membrane and lowers internal pH by exceeding the cell’s buffering capacity.

Cells maintain a pH gradient between the internal and external environments by pumping H+ ions out of the cell.

This may inactivate microorganisms by inhibiting essential metabolic systems including enzymes.

Bacterial enzymes may be inhibited by CO2.

At low pH, protein-bound arginine may interact with CO2 to form a bicarbonate complex, inactivating the enzyme (Weder et al.,1992).

Complete inactivation of alkaline protease at 35°C, 15 MPa was done by pH lowering by dissolved CO2 and lipase was done by sorption of CO2 into the enzyme molecules.

Another proposed mechanism is precipitation of intracellular carbonate Ca+2, Mg+2 from bicarbonate (Lin et al.,1993) which causes a lethal change to the biological system.

Inactivation of E. coli cells was done at 50.7 MPa in less than 5 min by bursting due to the rapid pressure release and the expansion of CO2 within the cell.

Indication of cell rupture can be observed by measuring the total protein concentration in the supernatant of DPCD-treated samples (Spilimbergo et al.,2003).

Morphological changes caused by DPCD may differ based on treatment conditions, gas release rate, or the type of microorganism.

Untrated Saccharomyces cerevisiae cells

DPCD treated Saccharomyces cerevisiae cells

Source:-Folkes, 2004

This concept is based on hydrophilicity and solvent characteristics of CO2.

Kamihira et al.(1987) observed that the extraction of intracellular substances such as phospholipids is a possible mechanism of microbial inactivation.

Isenchmid et al.(1995) proposed that diffused and accumulated CO2 increases fluidity of the membrane due to the order loss of the lipid chains, also called the “anesthesia effect,” and this causes an increase in permeability which causes disruption.

Untreated and DPCD treated Lactobacillus

plantarum cellsSource:-Hong and Pyun,1999

Spores are highly resistant forms of bacteria to the physical treatments such as heat, drying, radiation, and chemical agents (Watanabe et al.,2003).Processing temperature had a significant role in inactivation of spores by DPCD and high temperature is required to kill bacterial or fungal spores (Enomoto et al.,1997).Inactivation is done by 2 steps (Ballestra and Cuq,1998):– penetration of CO2 into the cells with heat activation of the

dormant spores– increase in sensitivity of spores to the antimicrobial effects of

CO2 by heat activationKamihira et al. (1987) did not observe any killing effect of DPCD on Bacillus stearothermophilus spores and observed only 53% inactivation of Bacillus subtilis spores by DPCD treatment at a relatively low temperature (35 °C) where survival decreased dramatically by increasing temperature from 50°C to 60 °C.

Another technique to achieve significant amount of spore inactivation at relatively low temperatures is by using continuous DPCD treatment systems that are more efficient than batch systems.

Ishikawa et al. (1997) achieved 6 log reduction in Bacillus polymyxa, B. cereus, and B. subtilis spores at 45 °C, 50 °C, and 55 °C, respectively, by using a continuous micro-bubble system.

DPCD had more killing effect than HHP treatment or heat treatment alone, showing that CO2 had a unique role in inactivation (Watanabe et al.,2003).

DPCD can inactivate certain enzymes at temperatures where thermal inactivation is not effective (Balaban et al.,1991).

It can be done mainly due to 3 causes:• lowering of pH• conformational changes of the enzyme• inhibitory effect of molecular CO2 on enzyme activity

Pectinesterase (PE) inactivation in orange juice can be done by lowering the pH to 2.4 (Balaban et al.,1991).

The extent of enzyme inactivation by DPCD is affected by the type and source of the enzyme, DPCD treatment conditions such as pressure, temperature, and time, and treatment medium properties.

DPCD has been applied mostly to liquid foods, particularly fruit juices. Some of are mentioned below:

Sl. No.

Name of Fruit

Reference Findings

1. Orange juice Arreola et al.,1991 Improvement of physical and nutritional quality attributes like color, and ascorbic acid retention and stability

2. Carrot juice Park et al.,2002 Cloud retention

3. Beer Folkes, 2004 Aroma and flavor retention in pasteurized beer

4. Mandarin juice Yagiz et al.,2005 Improvement of cloud formation, color, titrable acidity

5. Coconut water based beverages

Balaban, 2005 Improvemeent of shelf life for 9 weeks under refrigerated storage

6. Milk Tomasula 1997; Hofland 1999; Tisi

2004

Increase in lipolytic activity during storage and casein production, due to lower pH

Food Target micro-organism

Microbial inactivation

Reference

Flour Mold 99.8% Hass et al.,1989

Strawberries Bacteria 99.6% Hass et al.,1989

Onion Bacteria 99% Hass et al.,1989

Chicken meat Salmonella typhimurium

94-98% Wei et al.,1991

Beef Escherichia coli 1 log (cfu/g) Sirisee et al.,1991

Kimchi vegetables Lactic acid bacteria 4 log (cfu/ml) Hong and Park,1999

Leafstalks Natural micro-organisms

4 log (cfu/g) Kuhne and Khorr,1990

Retention of antioxidants, phytochemicals, organoleptic attributes such as taste, color, appearance (Kincal et al., 2006).

Relatively low process temperature so beneficial for heat sensitive compounds.

Lack of oxygen and lower pH prevents microbial growth. Retention of vitamin-C (Arreola et al.,1991).

Challenge to accept a new technology. Lack of the first commercially successful DPCD operation. Operational cost is higher. Greenhouse effect of CO2 gas.

DPCD is a non-thermal technology that can inactivate certain microorganisms and enzymes at temperatures low enough to avoid the thermal effects of traditional pasteurization.

DPCD treatment does not only improve food quality, but also promote shelf life and (long-term) safety by inactivating spoilage and pathogenic microorganisms.

An emerging technology among all other technologies of future generation.

More research is essential to demonstrate and explain the effect of DPCD preservation on the shelf life and safety of food products.

Effect of sensory and nutritional quality of both liquid and solid foods should be more thoroughly investigated.

Economics of the process must be assessed.

Commercialization of DPCD must be required.

Arreola AG, Balaban MO, Marshall MR, Peplow AJ, Wei CI, Cornell JA. 1991a. Supercritical CO2 effects on some quality attributes of single strength orange juice. J Food Sci 56(4):1030–3.

Chen JS, Balaban MO, Wei CI, Gleeson RA, Marshall MR. 1993. Effect of CO2 on the inactivation of Florida spiny lobster polyphenol oxidase. J Sci Food Agric 61:253–9.

Folkes G. 2004. Pasteurization of beer by a continuous dense-phase CO2 Gainesville,Univ. of Florida, Aug 10, 2005.

Fraser D. 1951. Bursting bacteria by release of gas pressure. Nature 167:33–4.

Hong SI, Park WS, PyunYR. 1999. Non-thermal inactivation of Lactobacillus plantarum as influenced by pressure and temperature of pressurized carbon dioxide. Int J Food Sci Technol 34:125–30.

Ishikawa H, Shimoda et al.,1995a. Inactivation of enzymes in an aqueous solution by micro-bubbles of supercritical CO2. Biosci Biotechnol Biochem 59(4):628–31.

Park SJ, Lee JI, Park J. 2002. Effects of combined process of high pressure CO2 and high hydrostatic pressure on the quality of carrot juice. JFS: Food Eng Phys Prop 67(5):1827–33.

Damar S., Balaban MO., Review of Dense Phase CO2 Technology: Microbial and Enzyme Inactivation, and Effects on Food Quality, Journal of Food Science —Vol. 71, Nr. 1, 2006.