Effect of Carbon Dioxide and Pressure Processing on Microbial and Enzyme Inactivation in Food and Beverages

Tatiana Koutchma, Illinois Institute of Technology National Center for Food Safety and Technology Summit Agro, Illinois, USA Edgar Murakami, U.S. Food and Drug Administration National Center for Food Safety and Technology Summit Agro, Illinois, USA

INTRODUCTION

High pressure processing (HPP) is a minimal treatment employing mild processing conditions to produce ‘fresh-like’ foods. HPP had been found effective in inactivating various vegetative bacterial cells^24,28. However, to be effective against microbial spores HPP has to be performed at high temperatures^3,24. High temperature negatively affects food quality and complicates equipment design and operation. Carbon dioxide (CO₂) has the potential for enhancing the effectiveness of HPP for inactivating microorganisms and enzymes. The addition of CO₂ may allow for lower process parameters and consequently encourage new applications for the technology²¹. This article will review recently published reports on HPP with CO₂. It will cover information on target microorganisms, process parameters, carbonation procedures and suggested mechanisms for microbial and enzymatic inactivation.

CO₂ PROPERTIES AND CARBONATION PROCEDURES

CO₂ is a non-toxic gas with antimicrobial properties and it is an accepted ingredient in foods and beverages⁸. CO₂ can be easily added and removed by manipulating the pressure and temperature of a solution. The solubility of CO₂ in water is expressed as the volume of dissolved CO₂ per unit volume of water. The solubility increases with pressure and decreases with temperature. With changing pressure (P) and temperature (T) the physical state of CO₂ is affected. Figure 1 shows a pressure-temperature (P-T) phase diagram for CO₂. Antimicrobial effectiveness of CO₂ depends on the pressure and temperature, but is also affected by the state of CO₂, such as gaseous, liquid or supercritical fluid (SC), and the concentration of dissolved CO₂ (Table 1). When used in modified atmosphere packaging, CO₂ also has inhibiting properties⁸. For CO₂ the SC region is above the critical temperature of 31°C (Tc) and critical pressure of 7.4 MPa (Pc) in the (P-T) diagram³². SC-CO₂ has properties between liquid and gas. Its relatively high density gives it excellent dissolving and penetrating powers, facilitating entry through cell walls and into spores where CO₂ could lower the pH or even extract the contents of the bacteria.

	Density, g/cc	Viscosity, cP	Diffusion Coefficient, cm2/s
Gas	0.002	0.014	0.01
Super Critical	0.5	0.02-0.12	0.0001
Liquid	1.0	1.0	0.00001
Table 1. Typical physical properties associated with different phase states of CO232.
Figure 1. The phase diagram for CO2 indicating the solid, gas, liquid and supercritical fluid (SCF) regions32

Carbonation is the process of adding CO₂ to the sample solution. Some carbonation techniques are:

1. bubbling CO₂ in a vessel at low temperature and at slightly elevated pressures and then packing in flexible containers⁷;
2. adding COs to the head space of a compressible container²¹;
3. using CO₂ as the pressuring medium by injecting into a vessel at high pressure^3,4,10. A variation of this technique is bubbling CO₂ through a microfilter producing microbubbles that stay suspended in the solution³³.

EFFECT OF PRESSURISED CO₂ ON BACTERIAL SPORES

Bacterial spores are resistant to high pressures (up to 827 MPa at temperatures below 35°C) and they are inactivated only at high pressures combined with elevated temperatures²⁴. Published reports of effects of pressure and CO₂ on bacterial spores are summarised in Table 2. The carbonation levels were estimated based on temperature and pressure during the carbonation process¹⁹.

Table 2. Published studies on high pressure with CO2 on bacterial spores

Watanabe et al³³ evaluated the effectiveness of heat alone (95°C), high pressure CO₂ with heat (65°C, 200MPa) and moderately pressurised CO₂ (35°C, 30 MPa). In general, the most effective treatment was HPP with CO₂ and the least effective treatment was moderately pressurised CO₂ . However, all the treatments were relatively ineffective against Geobacillus stearothermophilus. When heat was added to the moderately pressurised CO₂ , the D-value for G. stearothermophilus was 30 minutes at 95°C. Heat alone at 95°C was ineffective; HPP with CO₂ with heat resulted in the D-value of 75 minutes. By this means, in the moderately pressurised CO₂, accelerated inactivation occurs at a temperature level close to 95°C. Against the spores of Bacillus subtilis, the heat resulted in a D-value of 19 min and HPP with CO₂ with heat produced a D-value of 9 min. B. subtilis was the most resistant spore by one order of magnitude to moderately pressurised CO₂ even compared with G. stearothermophilus. It can be concluded that the target bacteria must be determined for each particular process technology.

Table 3. Published studies on high pressure with CO2 on yeasts and moulds (vegetative cells and spores)



Table 4. Published studies on high pressure with CO2 on vegetative bacterial cells

Ballestra and Cuq³ reported that even at relatively low pressure, the addition of CO₂ during HPP accelerated the inactivation of bacterial and fungal spores. They evaluated the effect of HPP with CO₂ in a Ringer solution as a function of process temperature (80 and 90°C) and water activity (aw). The addition of CO₂ during HPP at 5 MPa enhanced inactivation of B. subtilis spores, Bacillus fulva ascopores and Aspergillus niger conidia (Tables 2 and 4). The most pronounced effects were at low temperatures when HPP alone could not inactivate any of the spores evaluated. The inactivation of A. niger conidia by heat alone increased at aw levels from 0.90 to 0.99. This effect was enhanced with the addition of pressurised CO₂ at 5MPa.

Kamihira et al¹⁶ evaluated the effect of the moisture content of microbial cells and spores and the use of entrainer (ethanol and acetic acid) on the inactivation of various species. Regardless of the moisture content, spores of B. subtilis and G. stearothermophilus could not be destroyed at P= 20.3 MPa, T=35°C and process time of 2 hours. A. niger (conidia) could only be completely inactivated at those conditions when the cells were wet. Dry cells of A. niger were only destroyed with the addition of either entrainer. At the above conditions, HPP and CO₂ were not effective against G. stearothermophilus, regardless of moisture content and entrainer added.

Generally, published reports have shown that the addition of CO₂ during HPP enhanced the inactivation of bacterial spores, even those that are resistant to heat alone and HPP at elevated temperatures. Current information on treatments of bacterial spores using high pressure and CO₂ is limited in the range of process parameters such as temperature, pressure and carbonation level. Published studies on the effects of entrainers are encouraging and need to be continued.

EFFECT OF PRESSURISED CO₂ ON VEGETATIVE BACTERIA, YEASTS AND MOULDS

In recent years, the effects of high-pressure CO₂ on the vegetative cells of various species of bacteria were demonstrated^{29,11,12,13,14}. Published studies on HPP with CO₂ for inactivating vegetative cells of bacteria yeasts and moulds, and spores of certain moulds are outlined in Tables 3 and 4. Lin¹² demonstrated that pressurised CO₂ was effective for the inactivation of Listeria monocytogenes and yeast cells. A minimum D-value of L. monocytogenes was obtained under 6.1 MPa CO₂ pressure at 45°C11. Hong¹³ reported a reduction of more than 6 logs of Lactobacillus plantarum after 30 minutes under a CO₂ pressure of 3.8MPa at 30°C.

Murakami et al²¹ processed inoculated water samples in syringes at 35°C and pressures of up to 400 MPa for 300s. CO₂ enhanced the inactivation of L. innocua (Figure 2). While HPP was effective in inactivating Escherichia coli K12, the addition of either air or CO₂ did not provide additional benefits. The same result was reported by Corwin and Shellhammer⁷.

Figure 2. Effect of adding CO2 during HPP on inactivation of L. innocua21

Rasanayagam et al²³ processed orange juice saturated with CO₂ using membrane technology at near critical condition of 7.9 MPA and 40°C in continuous mode (Figure 3). More than a 6-log reduction of the pathogenic and non-pathogenic bacteria (E. coli K12, yeasts, lactic acid bacteria, E. coli O157:H7, Salmonella spp., or L. monocytogenes) was achieved after a 2-minute treatment.

Figure 3. Experimental set-up of continuous flow high pressure and supercritical CO2 processing of orange juice (Courtesy of Rasanayagam, American Air Liquide, Chicago, IL)

Kamihihra et al¹⁶ investigated the effect of the state of CO₂ on the inactivation of individual cells (not suspended) of E. coli at 4.1 MPa (40 atm) for a processing time of 2 hours. There was no difference in inactivation between gaseous CO₂ (T=35°C) and liquid CO₂ (T=20°C), despite the difference in temperature. However, when the process condition was elevated to the supercritical condition, the inactivation of E. coli increased. It was difficult to determine if the addition of CO₂ or the higher-pressure level caused the greater inactivation. During investigation of the effect of moisture content of E. coli and baker’s yeast it was found that wet cells were easier to inactivate than dry ones. No studies were done in the samples without CO₂ to separate pressure effects.

The effect of the CO₂ on the destruction of E. coli and Saccharomyces cerevisiae at 6 MPa and 35°C was studied by Shimoda et al^26,27. Shimoda²⁵ also observed enhanced antimicrobial effects of dissolved CO₂ in an aqueous medium by pressurising with microbubbles of CO₂ up to 19 MPa on the death kinetics of Aspergillus niger spores. He found that the antimicrobial activity of CO₂ was dependent on the concentration of dissolved CO₂ but not on the process pressure. A solvent macroscopic effect of compressed CO₂, temperature and dissolved CO₂ concentration was reported by Isenschmidt¹⁴ in yeasts cells over a range of pressure and temperature.

HPP is effective for E. coli K12 destruction. However, reports on the effectiveness of the addition of CO₂ during HPP against E. coli are contradictive. This is partly due to studies that did not include comparison of treatments using HPP with CO₂ versus HPP at the identical process conditions. Most published studies were geared toward effectiveness evaluation that was a reasonable goal for a new application. However, this approach does not clearly separate the effects of various parameters such as pressure, carbonation, level and other critical parameters.

EFFECT OF PRESSURISED CO₂ ON ENZYMES

The disadvantage of HPP technology is the poor inactivation of enzymes. This lack of inactivation can cause deterioration in quality of some juices, fruits and vegetables¹⁸. The reported data of effects of pressure and CO₂ on enzymes are summarised in Table 5. Arreola et al¹, Balaban et al² and Boff et al⁶ applied pressured SC-CO₂ and CO₂ -assisted HPP to inactivate pectinesterase (PE) in orange juice. PE was inactivated with SC-CO₂ treatment below temperatures necessary for thermal treatment such as 31 MPa and 60°C. The treatment did not significantly change pH and oBrix values; however, it stabilised and enhanced cloud, improved colour and slightly decreased total acidity. Sensory evaluations showed colour and cloudness of SC-CO₂ treated juice also were better than in untreated samples. Tedjo et al³¹ reported the adequacy of HPP and SC-CO₂ on the inactivation of lipoxygenase (LOX) and peroxidase (POD) in a batch mode. LOX was more pressure sensitive and temperature sensitive under SC-CO₂ treatment. A protective effect of sucrose and buffer at pH 7.0 on the enzymes treated by various SC-CO₂ was demonstrated. Yoshimira et al³³ applied continuous treatment by SC-CO₂ with microbubbles to inactivate (-amylase and acid protease and found that the inactivation efficiency was affected by initial pH and the buffer action of samples. Ishikawa et al¹⁵ reported lower residual activities of glucoamylase and acid protease with increasing density of SC-CO₂.

Table 5. Published studies on high pressure with CO2 on enzymes

Published studies on HPP with CO₂ have demonstrated the effectiveness of this treatment for enzyme inactivation. More studies need to be conducted to relate enzyme and microbial destruction¹⁸.

MECHANISMS OF CO₂ AND PRESSURE INACTIVATION

In general, studies have shown that microbial inactivation by HPP with CO₂ increases with increasing temperature, pressure, time, dissolved CO₂ and decreasing aw^25,21,4. Several mechanisms have been proposed in the literature such as deprivation, extraction and acidification theories. One of the earliest hypotheses was the deprivation theory in which the presence of CO₂ deprived cells of oxygen. However, this was disproved when it was shown that CO₂ also affected anaerobic bacteria⁸.

According to the cell extraction theory, inactivation by CO₂ occurs due to penetration through the cell membrane, dissolving in cell fluid and then extracting the cellular fluid when pressure is released. Park et al²² measured quantity of extracted cell materials with increase in pressure and found crevices on membranes of inactivated cells. Enomoto et al¹⁰, comparing slow and explosive releases of CO₂, found more proteins were released from cells after an explosive CO₂ release; however, the microbial reduction was not influenced by the rate of release. They suggested that inactivation was achieved during the pressurisation step.

The acidification theory suggests that CO₂ reacts with water to form carbonic acid and lowers the pH of microbial cells. Acid formation occurs either outside or inside the cells⁴. This theory was supported by studies of wet and dry cells^17,16. However, there are studies that found that CO₂ affected the non-acid sensitive bacteria such as Listeria, not those that are acid sensitive such as E. coli^7,21. Some published reports have indicated that it is not just the low pH that is effective in killing bacteria but the type of acid^5,8. Bang⁵ adjusted the pH of buffer and apple juice by adding either CO₂ or citric acid and found that the acid is more effective than CO₂ . Daniels et al⁸ suggested that inactivation might not be due to acidification alone. Other authors^25,8 concluded that a combination of different mechanisms inactivates the cells. More work remains to define the mechanisms of CO₂ and pressure microbial inactivation.

CONCLUSIONS

In summary, intensive research of CO₂ assisted pressure and SC-CO₂ treatments demonstrate their effectiveness to control microorganisms and enzymes in foods. Food processing applications of HPP with CO₂ are becoming more popular as economically viable alternatives to heat treatments. Potential applications for HPP with CO₂ are in products that are sensitive to heat and pressure such as fruit juice and beverages^2,34, fresh fish⁹ and smoked fish¹⁸. However, despite much effort spent on the study of this technology, the fundamental mechanisms of microbial inactivation by pressurised CO₂ are not fully understood. For instance, if microbial inactivation is similar to supercritical solvent extraction, then it is necessary to have a supersaturated CO₂ at process conditions. It appears to be that a combination of several mechanisms inactivates the cells. However, studies need to be conducted to determine what mechanism dominates under various process conditions.

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BIOGRAPHIES

Dr. Koutchma obtained her Ph.D. from Moscow State University of Food Technology, Russia, in 1989. She was a Research Associate at McGill University, Department of Food Science and Agricultural Chemistry, Montreal (1997-2000) and joined the National Centre for Food Safety and Technology at the Illinois Institute of Technology in June 2000 as a Research Assistant Professor in Food Process Engineering. Her research fields are novel methods of thermal and non-thermal processing such as electron beam and ultraviolet irradiation; radio frequency, microwave, and ohmic heating; combined processing to enhance food safety. Her current research is in the determination and development of process indicators to control irradiation of foods. Dr Koutchma also teaches Food Process Engineering to graduates and is also involved in promoting food safety.

Dr. Edgar G. Murakami is a Food Engineer with the US Food and Drug Administration, and is located at the National Center for Food Safety and Technology in Chicago, USA. He had been a member of several research teams studying the inactivation of bacteria in foods using novel processing techniques such as ohmic heating, UV light and high pressure processing. His current research interest is the addition of gases during high pressure processing. Dr. Murakami is also involved in the regulatory evaluation of aseptic processing and packaging systems used within the United States.

CONTACT

Address: Tatiana Koutchma, Illinois Institute of Technology, National Center for Food Safety and Technology, 6502 South Archer Road, Summit Agro, IL, 60501, USA

Edgar Murakami, U.S. Food and Drug Administration, National Center for Food Safety and Technology, 6502 South Archer Road, Summit Agro, IL, 60501, USA