Basic information and application of dense phase carbon-dioxide

2. The need for a food preservation method is to make safe,
inexpensivefoodswith preservation of heat-sensitivecompounds.
CO2 isused becauseof itssafety, 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-organismswithout thelossof nutrientsor quality changesthat
may occur dueto thermal effects.
It isalso called as“cold pasteurization”.

3. In the DPCD process, food is contacted with
pressurized sub- or supercritical CO2 for a period of
timein abatch, semi-batch or continuousmanner.
The CO2 pressures can range from 7.0 to 40.0 MPa.
Processtemperaturescan rangefrom 20 to 60°C.
The treatment times can range from about 3 to 9 min
for continuous, or from 120 to 140 min for semi-
continuousor batch DPCD processes.
Source:- Novel Thermal and Non-Thermal Technologies for Fluid Foods.

4. 3 typesof treatment systemsareused:
• Batch type:- Both CO2 and treatment solution are
stationary in acontainer during treatment
• Semi-continuous type:- continuous flow of CO2
through thechamber whileliquid food isstationary
• Continuous type:- Flow of both CO2 and the liquid
food

5. Batch type DPCD system (Hong and Pyun 1999)

6. 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
thedesired value.
Then, CO2 is introduced into the vessel until the sample is saturated at
thedesired pressureand temperature.
The sample is left in the vessel for a period of time and then CO2 outlet
valveisopened to releasethegas.
Some systems contain an agitator to decrease the time to saturate the
samplewith CO2.

7. Ishikawa et al.,1 995 developed a semi-continuous
system using a cylindrical filter to micro-bubble CO2
entering into thepressurevessel.
The inactivation of enzymes using a micropore filter
was3 timesmorethan without using it.
CO2 is increased from 0.4 to 0.92 mol/L in the sample
at 25 MPaand 35 °C.

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

9. A continuous micro-bubble system, very effective in inactivating
microorganisms.
In this system, liquid CO2 and a saline solution were pumped
through avessel.
Liquid CO2 was changed to gas using an evaporator and then
dispersed into the salinesolution from astainlesssteel mesh filter
with 10-µm poresize.
The micro-bubbles of CO2 moved upward while dissolving into
thesolution.
Then, the solution saturated with CO2 was passed through a
heater to reach the desired temperature. Another coil with a
heater wasused to adjust theresidencetime.

10. Different researchers proposed different mechanism
of inactivation of micro-organismsin DPCD process.
Someof them aregiven below:
pH lowering effect (Meyssami et al.,1 992)
Inhibitory effect of molecular CO2 and bicarbonate ion (Ishikawa
et al.,1 995)
Physical disruption of cells(Fraser,1 951 )
Modification of cell membrane and extraction of cellular
components(Kamihira et al.,1 98 7 )

11. 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 H↔ 2CO3
H2CO3 H↔ +
+ HCO3
HCO3 H↔ +
+ CO3
The internal pH of microbial cells has the largest
effect on their destruction.

12. Sufficient CO2 in the environment penetrates through
the cell membrane and lowers internal pH by
exceeding thecell’sbuffering 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 systemsincluding enzymes.

13. Bacterial enzymesmay beinhibited by CO2.
At low pH, protein-bound arginine may interact with CO2 to form a
bicarbonatecomplex, inactivating theenzyme(Weder et al.,1 992).
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 theenzymemolecules.
Another proposed mechanism is precipitation of intracellular
carbonate Ca+2
, Mg+2
from bicarbonate (Lin et al.,1 993) which causes
alethal changeto thebiological system.

14. Inactivation of E. co li 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 thecell.
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

15. This concept is based on hydrophilicity and
solvent characteristicsof CO2.
Kamihira et al.(1 98 7 ) observed that the
extraction of intracellular substances such
as phospholipids is a possible mechanism
of microbial inactivation.
Isenchmid et al.(1 995) 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 cells
Source:-Hong and Pyun,1999

16. Sporesarehighly resistant formsof bacteriato thephysical treatmentssuch 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
(Eno mo to et al.,1 997 ).
Inactivation isdoneby 2 steps(Ballestra and Cuq,1 998 ):
– penetration of CO2into thecellswith heat activation of thedormant spores
– increasein sensitivity of sporesto theantimicrobial effectsof CO2 by heat
activation
Kamihira et al. (1 98 7 ) did not observeany killing effect of DPCD on Bacillus
stearo thermo philus 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.

17. Another technique to achieve significant amount of spore
inactivation at relatively low temperatures is by using continuous
DPCD treatment systemsthat aremoreefficient than batch systems.
Ishikawa et al. (1 997 ) achieved 6 log reduction in Bacillus
po lymyxa, B. cereus, and B. subtilis spores at 45 °C, 50 °C, and 55
°C, respectively, by using acontinuousmicro-bubblesystem.
DPCD had more killing effect than HHP treatment or heat treatment
alone, showing that CO2 had auniquerolein inactivation (Watanabe
et al.,2003).

19. DPCD can inactivate certain enzymes at temperatures where thermal
inactivation isnot effective(Balaban et al.,1 991 ).
It can bedonemainly dueto 3 causes:
• lowering of pH
• conformational changesof theenzyme
• inhibitory effect of molecular CO2 on enzymeactivity
Pectinesterase (PE) inactivation in orange juice can be done by lowering
thepH to 2.4 (Balaban et al.,1 991 ).
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.

21. DPCD has been applied mostly to liquid foods, particularly fruit juices.
Someof arementioned 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

22. 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

23.  Retention of antioxidants, phytochemicals, organoleptic attributes
such astaste, color, appearance(Kincal et al., 2006 ).
 Relatively low process temperature so beneficial for heat sensitive
compounds.
 Lack of oxygen and lower pH preventsmicrobial growth.
 Retention of vitamin-C (Arreo la et al.,1 991 ).
 Challengeto accept anew technology.
 Lack of thefirst commercially successful DPCD operation.
 Operational cost ishigher.
 Greenhouseeffect of CO2gas.

24. 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 spoilageand pathogenic microorganisms.
An emerging technology among all other technologies
of futuregeneration.

25. More research is essential to demonstrate and explain the
effect of DPCD preservation on theshelf lifeand safety of
food products.
Effect of sensory and nutritional quality of both liquid and
solid foodsshould bemorethoroughly investigated.
Economicsof theprocessmust beassessed.
Commercialization of DPCD must berequired.

26. Arreola AG, Balaban MO, Marshall MR, Peplow AJ, Wei CI,
Cornell JA. 1991a. Supercritical CO2 effects on some quality
attributesof singlestrength orangejuice. JFood 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. JSci Food Agric 61:253–9.
FolkesG. 2004. Pasteurization of beer by acontinuousdense-phase
CO2 Gainesville,Univ. of Florida, Aug 10, 2005.
Fraser D. 1951. Bursting bacteriaby releaseof gaspressure. Nature
167:33–4.

27. Hong SI, Park WS, PyunYR. 1999. Non-thermal inactivation of
Lacto bacillus plantarum as influenced by pressure and temperature of
pressurized carbon dioxide. Int JFood 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 thequality of carrot juice. JFS: Food
Eng PhysProp 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.