Review High pressure carbon dioxide pasteurization of solid foods: Current knowledge and future...

Trends in Food Science & Technology 22 (2011) 427e441

Review

* Corresponding author.

0924-2244/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2011.04.009

High pressure carbon

dioxide

pasteurization of

solid foods: Current

knowledge and

future outlooks

Giovanna Ferrentino* and
Sara Spilimbergo
Department of Materials Engineering and Industrial

Technologies, University of Trento, via Mesiano 77,

Trento 38050, Italy (Tel.: D39 0461 282 485;

e-mail: [email protected])

High pressure carbon dioxide (HPCD) technology applied to

foods has gained a particular scientific interest considering

the number of publications and patents published in the last

decades. Although the antimicrobial effect of HPCD has

been demonstrated mainly for liquid foodstuffs, very few

research papers investigated the possibility to exploit the treat-

ment on solid foods. In this concern, the main objective of the

present review is to give a general survey of the published

knowledge concerning the HPCD applied to solid foods.

Remarks and future outlooks will be highlighted with the

aim to suggest a research line to follow for future studies.

IntroductionManufacturers of food products are currently under in-

creasingly stringent demands concerning the productionprocesses. This is essentially caused by the growing interestof consumers for high-quality and “minimal processing”products, as well as for energy saving and safer productionprocesses. Minimally processed foods are obtained withmethods of food safety and preservation that are designed

to retain the natural and fresh properties of foods(Manvell, 1997).

Most of the food products contain high levels of nutri-ents or a high water activity; therefore they are particularlysusceptible of microbial spoilage which results in a deterio-ration of their organoleptic characteristics, and may evenrisk the health of immune-compromised individuals(Tournas, Heeres, & Burgess, 2006).

Preservation of food products reduces the number of un-desirable microorganisms below a specific critical valueduring the shelf storage period of the product. It is usuallyperformed through different methods as thermal pasteuriza-tion up to 80 �C and sterilization up to 120 �C (e. g. fruits,food powders and meat), drying (e. g. vegetables, andherbs), freezing (e. g. meats, fishes, and vegetables), addi-tion of preservatives (e. g. meats, and vegetables), autoclav-ing (e. g. herbs), gamma irradiation (e. g. vegetables), orethylene oxide and methyl bromide exposure (e. g. vegeta-bles; spices). These treatments are successful to eliminatethe degenerative effects of enzymes and microorganisms,but on the other hand they may also reduce the food qualityby causing alterations in the taste and in the organolepticfeatures of food products. Several authors observed a reduc-tion in vitamin C retention, color degradation, and changesin other quality indicators due to the thermal treatmentsapplied to fruits (Awuah, Ramaswamy, & Economides,2007; Vikram, Ramesh, & Paprulla, 2005). Others reportedthat the application of both methyl bromide and ethyleneoxide is extremely toxic. Methyl bromide is potentiallycapable of depleting the atmospheric ozone layer. Ethyleneoxide has been banned in Europe because of safety and en-vironmental concerns, and its use for the treatment ofground spices has been revoked in the United States(Thayer, Josephson, Brynjolfsson, & Giddings, 1996).Autoclaving, freezing, drying or exposure to steam havebeen described as methods which cause degradation of bio-active compounds and the destruction of the delicate tissueof the foods (Howard, 2008).

Alternatives to traditional treatments as the use ofozone, radical species, and high hydrostatic pressurehave been proposed in these years (San Martin,BarbosaeCanovas, & Swanson, 2004). The latter hasbeen already used for few years at commercial scale forthe decontamination of solid foods such as meat products(Shigehisa, Ohmori, Saito, Taji, & Hayashi, 1991) orready to eat meals (Cheftel, 1995). Pressures ranging

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428 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441

from 300 to 700 MPa are usually applied. The process re-sulted effective on different microbial species but the highequipment cost, the difficulty of controlling and managingan operating pressure in such extreme range of values, thesafety concerns represent the potential drawbacks to beovercome.

Among the other innovative preservation methods, highpressure carbon dioxide process (HPCD) has gained greatinterest in the scientific field. Since the 1980s it has beenincreasingly investigated as a promising technique to in-duce a pasteurizing/sterilizing effect when applied both tosolid and liquid matrixes. The HPCD preservation methodprovides several advantages. Carbon dioxide (CO2) usedin this process is not only a powerful solvent for a widerange of compounds of interest in food processing, but isrelatively inert, inexpensive, nontoxic, nonflammable, recy-clable and readily available in high purity leaving no resi-due when removed after the process (Clifford &Williams, 2000). Furthermore, it is considered to bea GRAS (Generally Recognized as Safe) solvent, whichmeans it can be used in food products.

So far most of the research has been focused on suspen-sions of pure cultures of different microorganisms inocu-lated or naturally occurring in liquid food products (e.g.fruit juices, beer, wine and milk) which affect the foodspoilage and give main concern to health (Arreola,Balaban, Wei, Peplow, & Marshall, 1991; Del Pozo-Insfran, Balaban, & Talcott, 2006). The method seems tobe very promising because HPCD (or dense phase CO2)is able of killing bacteria, yeast and fungi at moderate pres-sure and low temperature preserving more quality attributesof the products than traditional treatments (Ferrentino,Plaza, Ramirez-Rodrigues, Ferrari, & Balaban, 2009;Spilimbergo & Ciola, 2010).

Three recent reviews compile the relevant currentknowledge about the potential of HPCD treatment andsummarize the most significant state of the art, includingthe most important applications and data for the treatmentapplied to a wide range of microorganisms mainly in liquidsubstrates (Damar & Balaban, 2006; Garcia-Gonzalezet al., 2007; Spilimbergo & Bertucco, 2003).

Compared to liquids, the HPCD process applied to solidfoods is less studied due to the complexity of the matrix,which can make the CO2 bactericidal action more difficult,and to the lack of information about the inactivation mech-anism which is almost obscure and scarcely studied.

The aim of the present review is to provide the status ofthe art of HPCD process applied to solid foodstuffs with theexamination of almost all the literature published on thissubject including the most relevant patents.

The review has been constructed as follows: a descriptionof the application of the process for each category of solidfoods tested so far (meats, fruits and vegetables, food pow-ders, sprouts seeds, spices and herbs, fishes) has beenaddressed. For each category of solid foods a discussionof the main effects of the HPCD treatment on microbial

inactivation and quality attributes of the foods has been re-ported considering all significant articles published in thefield.

A brief section of the review is dedicated to the descrip-tion of the main results obtained by different authors whenthe HPCD process is carried out in combination with somepretreatments or additives.

Finally a reflection on the important aspects of the pro-cess, its limits and potentials, the lack of scientific informa-tion to fill up, and a direction to orientate the research willbe evidenced.

Application of HPCD treatment to solid foodsThe effects of the HPCD treatment on the microbial

inactivation and quality attributes of food matrixes arereported in the following sections. The process has been ap-plied to meats (chicken, pork, and beef), vegetables (celery,and spinach), seeds and food powders (alfalfa seeds, cocoapowder, and ginseng), fruits (cut pieces of pears, straw-berries, honeydew melon, and cucumber), spices and herbs(chives, thyme, oregano, parsley, and mint), and fish(shrimp, and oyster). Considering the different characteris-tics and behaviors of each substrate to the treatment, the lit-erature review has been reported separately for eachcategory of solid matrix.

Table 1 is a compilation of the experimental results thatcan be found in the literature with indication of the type offood, microorganism, the present industry conventionaltreatment, and the conditions of HPCD treatment with thecorresponding achieved microbial inactivation. Table 2 re-ports the desired level of microbial inactivation requiredby law (Regulation (EC) No. 2073/2005) for each type ofmicroorganism and foodstuffs mentioned. Table 3 summa-rizes the description and the main observations of the ef-fects of the treatment on the appearance, and somequality attributes of the foods subjected to the process.

MeatsMicrobial inactivation

The HPCD technology in particular in supercritical state(31 �C and 7.4 MPa) has been widely investigated and ap-plied to meat considering the ability of CO2 to extract andfractionate fats from ground beef into lower melting tem-perature components, as well as for the removal of choles-terol (Chao, Mulvaney, Bailey, & Fernando, 1991). Apartfrom this application, the treatment has been also appliedto test the efficacy on the inactivation of bacteria strainsto induce pasteurization. Sirisee, Hsieh, and Huff (1998)tested the tolerance of Escherichia coli and Staphylococcusaureus in ground beef to HPCD treatment carried out at42.5 �C and 31.03 MPa. Ground beef samples were mixedwith the suspended microbial cultures and then placed inthe reactor to be processed. The experimental results high-lighted the longer treatment time needed to inactivate bothtarget microbes compared to the same treatment carried outon the same microorganisms in a liquid phosphate buffer

Table 1. Application of HPCD treatment on microbial forms detected on solid substrates. Type of food, microorganism, conventional treatment, and HPCD process conditions with thecorresponding microbial inactivation and references are reported.

Food Target microorganism Process conditions Microbial inactivation Reference Conventional treatment

Flour Mold 6.2 MPa, 23 �C, 2 h 99.8% Steam, microwaves, Joule effect, thermal treatmentsBacteria 99.6%

Strawberries 6.2 MPa, �22 �C, 2 h 99% Chemical preservation (acidulants, antioxidants, chlorine orantimicrobials); gas and controlled modified atmosphere;refrigeration; moisture reduction

Mozzarella cheese Bacteria 6.2 MPa, 23 �C, 16 h 87% (Haas et al., 1989) Antimicrobial packaging, gas and controlled modifiedatmosphere, refrigerationParmesan cheese 1.4 MPa, 23 �C, 168 h 50%

Romano cheese 1.4 MPa, 23 �C, 168 h 99%Onions 5.5 MPa, 23 �C, 2 h 90% Chemical preservation (acidulants, antioxidants, chlorine or

antimicrobials); gas and controlled modified atmosphere;refrigeration; moisture reduction

Dry peppers(30% moisture added)

5.5 MPa, 23 �C, 2 h 90% Sterilization with ethylene oxide; ionizing radiation; steamtreatment

Chives 5.5 MPa, 45 �C, 2 h Totalinactivation

Chemical preservation (acidulants, antioxidants, chlorine orantimicrobials); gas and controlled modified atmosphere;refrigeration; moisture reduction

ThymeOreganoParsleyMintFresh celery leavesand leafstalks

Natural microorganisms 6.9, 31.4 and62.8 MPa,40 or 60 �C, 30 or60 min

4 Log (cfu/g) (Kuhne & Knorr,1990)


Chicken meat Salmonella typhimurium 13.7 MPa, 35 �C, 2 h 94e98% (Wei et al., 1991) Addition of organic acids (acetic and lactic acids); ionizingradiation; steam pasteurizationListeria monocytogenes

ATCC1531379e84%

Shrimp L. monocytogenesATCC15313

99% Quick freezing, heat e cool pasteurization

Ground beefsystems

Escherichia coli 31.03 MPa, 42.5 �C,180 min

1 Log (cfu/g) (Sirisee et al.,1998)

Addition of organic acids (acetic and lactic acids); ionizingradiation; steam pasteurization

Staphylococcus aureus 31.03 MPa, 42.5 �C,120 min

3 Log (cfu/g)

Kimchi vegetables Lactic acid bacteria 6.9 MPa, 10 �C,24 h

4 Log (cfu/ml) (Hong & Park,1999)


Skinned beef meat Brochothrix thermosphacta 6.1 MPa, 45 �C,150 min

5 Log (Erkmen, 2000) Addition of organic acids (acetic and lactic acids); ionizingradiation; steam pasteurizationMinced beef meat 1 Log

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Table 1 (continued )

Food Target microorganism Process conditions Microbial inactivation Reference Conventional treatment

Alfaalfa seeds Escherichia coli K12 27.6 MPa, 50 �C,60 min

92.8% (Mazzoni et al.,2001)

Chemical preservation (chlorine compounds; acidified sodiumchlorite, hydrogen peroxide, trisodium phosphate, peraceticacid, ethanol and commercial cleaning solutions)

Total aerobic bacteria 85.6%

Beef trimmings Total plate count 10.3 MPa, 36 �C,15 min

0.83 Log (Meurehg, 2006) Addition of organic acids (acetic and lactic acids); ionizingradiation; steam pasteurizationE. coli O157:H7 0.93 Log

E. coli 1.00 LogSalmonella spp. 1.06 Log

Ground beef Total plate count 10.3 MPa, 36 �C,15 min

0.78 LogE. coli O157:H7 0.94 LogE. coli 0.94 LogSalmonella spp. 1.23 Log

Cocoa powder Aerobic mesophilicspores

30.0 MPa, 65 �C,40 min

Totalinactivation

(Calvo et al., 2007) Steam, microwaves, Joule effect, thermal treatments

Aerobic thermophilicsporesMesophilic thermoresistant sporesThermophilicthermo resistant sporesTotal plate count

Fresh SpinachLeaves

E. coli K12 10 MPa, 40 �C,10 min

5 Log (cfuper leaf)

(Zhong et al., 2008) Chemical preservation (acidulants, antioxidants, chlorineor antimicrobials); gas and controlled modified atmosphere;refrigeration; moisture reduction

Ginseng Powder Total aerobicmicrobial count

10 MPa, 60 �C,15 h

2.67 Log (cfu/g) (Dehghani et al., 2008) Sterilization with ethylene oxide; ionizing radiation; steamtreatment

Alfaalfa seeds E. coli O157:H7 15 MPa, 35 �C,10 min

3.51 Log (cfu/g) (Jung et al., 2009) Chemical preservation (chlorine compounds; acidified sodiumchlorite, hydrogen peroxide, trisodium phosphate, peraceticacid, ethanol and commercial cleaning solutions)Listeria Monocytogenes 10 MPa, 45 �C,

5 min2.65 Log (cfu/g)

S. typhimurium 2.48 Log (cfu/g)Boneless pork loins Escherichia coli 12 MPa, 35 �C,

30 min1.5 Log (cfu/cm2) (Choi et al., 2009b) Addition of organic acids (acetic and lactic acids); ionizing

radiation; steam pasteurizationL. monocytogenes 1.4 Log (cfu/cm2)S. typhimurium 1.56 Log (cfu/cm2)E. coli O157:H7 1.0 Log (cfu/cm2)

Soy sauce pastemarinated porkloins

Escherichia coli 14 MPa, 45 �C,40 min

33.81% (Choi et al., 2009a)L. monocytogenes 37.96%S. typhimurium 34.48%E. coli O157:H7 36.84%

Hot e pepperpaste marinatedpork loins

Escherichia coli 26.42%L. monocytogenes 27.59%S. typhimurium 32.74%E. coli O157:H7 28.28%

Pears S. cerevisiae 10 MPa, 50 �C,10 min

4 Log (cfu/g) (Valverde et al., 2010) Chemical preservation (acidulants, antioxidants, chlorineor antimicrobials); gas and controlled modified atmosphere;refrigeration; moisture reduction

Oyster Aerobic Plate Count 17.2 MPa, 60 �C,60 min

3 Log (cfu/g) (Meujo et al., 2010) Quick freezing, heat e cool pasteurization

Paprika powder Mesophilic aerobicmicroorganisms

30.0 MPa, 90 �C,45 min

5.5 Log (cfu/g) (Calvo & Torres, 2010) Sterilization with ethylene oxide; ionizing radiation;steam treatment

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Table 2. Desired level of microbial inactivation based on the type of food and microorganism.

Inactivation target degree (cfu/g)

Food Total platecount

Aerobic thermo-resistantspores

Yeasts/Moulds Enterobacteriacee E. coli Salmonella Listeria

Cocoa product <103 <10 <10 <10 Total absence Total absence Total absenceGinseng powder <104 ND <102 ND Total absence Total absence Total absenceFruits and vegetables <104 ND ND <102 <20 Total absence Total absenceMeat products <105 ND ND <102 <50 Total absence Total absenceDairy products <105 ND ND <102 102 < 103 Total absence Total absenceFishery products <106 ND ND <102 <20 Total absence Total absenceSprouted seeds <103 ND ND <102 <20 Total absence Total absenceSpices and herbs <103 ND ND <102 <20 Total absence Total absence

ND ¼ not defined.

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solution (Sirisee et al., 1998). One log cycle reduction of E.coli in ground beef took 178 min, but only 1.7 min wasneeded to achieve the same inactivation level in liquidphosphate buffer solutions. Same experimental evidenceswere observed for S. aureus inoculated in ground beefsystem and in liquid phosphate buffer solutions. The expla-nation was related to the presence of fats and proteins inground beef which could play an important role in protect-ing microorganisms from high pressure CO2 bactericidalaction and to the lower moisture content (72%) whichreduced the amount of CO2 able to dissolve in the foodmatrix.

The protective effect of carbohydrate and other or-ganic compounds in foods was also reported byErkmen (2000) who inoculated Brocothrix thermos-phacta microbial cells on minced and skinned beefmeat and carried out the HPCD treatment (6.1 MPa,45 �C for 150 min) in a batch device. Also in this study,the results demonstrated that the treatment was not as ef-fective as for a liquid substrate. B thermosphacta sus-pended in brain heart infusion broth was completelyinactivated under 6.1 MPa after 80, 50 and 30 min at25, 35 and 45 �C respectively. The sterilization effectat 6.1 MPa and 45 �C on B thermosphacta was observedafter 150 min in skinned meat. The increase of microbialresistance to the treatment was attributed to the nature ofthe matrix and compounds (carbohydrates and fats) aspreviously observed by Sirisee et al. (1998). Howevera reduction equal to 5 and 1 log cycles was observedfor B. thermosphacta inoculated on skinned and mincedmeat, respectively.

In a further study, the treatment was applied to chickenmeat strips (breast meat with no skin) to inactivate Salmo-nella and Listeria cultures in which the chicken sampleswere dipped (Wei, Balaban, Fernando, & Peplow, 1991).The samples treated at 13.7 MPa and 35 �C for 2 h andspiked with Salmonella were shown to reduce the bacterialnumbers by 94e98% while those spiked with Listeria wereonly reduced by 79e84%.

Choi, Bae, Kim, Kim, and Rhee (2009a) conducteda study to evaluate the effects of supercritical carbon

dioxide treatment in marinated pork products (bonelesspork loins) in soy sauce and hot e pepper paste for the in-hibition of generic E. coli, Listeria monocytogenes, Salmo-nella typhimurium and E. coli O157:H7. Publishedinactivation kinetics showed that two stages were observedin the microbial survival curves. The early stage was char-acterized by a slow rate of microbial reduction which thensharply decreased during the later stage. The results dem-onstrated that the inactivation rate increased with increas-ing pressure, temperature, and exposure time but that itwas also depended on the initial number of cells and thesuspending medium. Carrying out a treatment at 14 MPa,45 �C for 40 min, the observed reduction levels were equalto 37.96% for L. monocytogenes, 34.48% for S. typhimu-rium, 33.81% for generic E. coli, and 36.84% for E. coliO157:H7 in soy sauce marinated pork. Conversely, whenthe hot e pepper paste marinated pork was subjected tothe HPCD treatment, the reduction level was 26.42% forgeneric E. coli; 27.59% for L. monocytogenes; 32.74% forS. typhimurium and 28.28% for E. coli O157:H7. Howeverthe inactivation results were too low if compared with stan-dard pasteurization methods. As shown in Table 2 total ab-sence of Listeria was needed to consider the food safe forhuman consumption.

Effect on quality attributesOnly visual observations of the ground beef sample

treated by HPCD were reported by Sirisee et al. (1998). Ac-cording to these authors, the color of ground beef changedafter the treatment and looked like cooked ground beef.This observation was also supported by the experimentalresults reported by Brown and Mebine (1969) who statedthat high concentrations of CO2 could cause darkening intissues by combining with myoglobin to form metmyoglo-bin. This effect could be limited overcome if this techniquehas to be applied to beef meats which contains high levelsof myoglobin (myoglobin content: 0.05e0.20% for whitechicken meat, 0.1e0.3% for pork and veal, and0.4e2.0% for beef).

Qualitative analyses were performed on the samples ofchicken treated by HPCD (Wei et al., 1991). Also in this

Table 3. Description of the effect of HPCD treatment on some quality attributes of treated foods.

Food Observations Reference

Strawberries, honeydew melon, cucumber Tissue destruction (Haas et al., 1989)

Chives, oregano Enhanced aroma

Parsley Similar taste of the untreated sampleSlight off aroma

Thyme Worst taste compared to the untreated sample

Mint Better taste compared to the untreated sampleEnhanced aroma

Chicken meat and shrimp Color change to whitishCooked appearanceLoss of liquid

(Wei et al., 1991)

Ground beef Color change to darkCooked appearance

(Sirisee et al., 1998)

Kimchi Higher pHLower titratableBetter sensory propertiesNo significant color, flavor and texture changes

(Hong & Park, 1999)

Alfaalfa seeds No detrimental effect on viability of the seedsNo detrimental effect on germination rate of the seeds

(Mazzoni et al., 2001)

Ground beef Higher tendernessNo significant changes in juiciness, flavor intensityand off flavor intensity

(Meurehg, 2006)

Cocoa powder Decreased water contentNo effect on physical aspect

(Calvo et al., 2007)

Meat of porcine longissimus dorsi muscle No effect on muscle pHNo effect on tendernessNo effect on water e holding capacityIncreased lightnessSarcoplasmic protein denaturation

(Choi et al., 2008)

Spinach leaves DiscolorationDecreased leaf firmness

(Zhong et al., 2008)

Cabbage, lettuce, mizuna Soften structure (Matsufuji et al., 2009)

Soy sauce and hot pepper marinatedpaste marinated pork

No differences in the surface color intensityOverall acceptability

(Choi et al., 2009a)

Alfalfa seeds No effect on germination rate (Jung et al., 2009)

Paprika powder Slight reduction of color intensityDecrease of water content

(Calvo & Torres, 2010)

Pears Consistency lossSofter aspectLoss of liquidTransition to brown coloring

(Valverde et al., 2010)

Oyster Retention of the overall acceptability (Meujo et al., 2010)


case the authors reported a color change of the sampleswhichturned whitish and seemed to be cooked or soaked in acid. Inaddition, the treated samples showed a liquid loss from thetissue which was completely absent in the untreated sample.

A more complete analysis was recently performed byChoi et al. (2008) who investigated the effect of supercrit-ical carbon dioxide treatment for sterilization purpose onmeat quality and protein denaturation of the porcine


longissimus dorsi muscle. During the treatment, CO2 waseasily adsorbed into the meat lowering the meat pH withthe formation of carbonic acid and its subsequent dissocia-tion in bicarbonate and hydrogen ions (Jacobsen &Bertelsen, 2002). It was observed that the pressure and tem-perature of the HPCD treatment could affect molecular in-teractions and proteins conformation, leading to proteindenaturation and aggregation in the meat (Messens, VanCamp, & Huyghebaert, 1997). In the same study, theHPCD treatment (7.4 MPa at 31.1 �C for 10 min) was re-ported not to affect the muscle pH, total weight, tendernessand water holding capacity of the treated meat. Howeverthe treated samples had a higher lightness and a lower red-ness value. The change in color was correlated to a denatur-ation process of proteins occurred during the treatment. Theauthors also verified that, while the HPCD treatment had noeffect on myofibrillar protein solubility including myosinprotein, the SDS e PAGE analysis revealed a denaturationof sarcoplasmic protein (phosphorylase b, creatine kinase,triosephophate isomerase and one unknown protein) whichmasked the red color of the sarcoplasm, causing the muscleto become pale.

Despite these findings, sensory evaluations of soy sauceand hot-pepper paste marinated pork samples revealed nodifferences in the surface color intensity and an overall ac-ceptability of controls and samples treated with supercriti-cal CO2 at 15.2 MPa and 31.1 �C for 10 min (Choi et al.,2009a).

Several studies demonstrated the efficiency of the su-percritical carbon dioxide process which increased thequality of meats inducing the extraction of cholesteroland other lipids. This potential is becoming more andmore attractive due to consumer concern over dietary in-take of fat and cholesterol. It has been reported that CO2

under supercritical conditions solubilizes a portion of thelipid components and removes them from the food ma-trix. Chao et al. (1991) found that up to 36.9% of thecholesterol and 71.2% of total lipids in the meat couldbe removed during the process. However, such processhad limited effects on fat extraction of meat productscontaining high levels of fats (Clarke, 1991). A negativeeffect of the extraction with CO2 was associated to thedehydratation of the muscle tissue which resulted in a de-creased solubility of some muscle proteins and otherchanges in functional properties (Hardardottir &Kinsella, 1988).

Fruits and vegetablesMicrobial inactivation

Very few researches have been addressed applying theHPCD treatment to fruits. The first study was carried outin 1989 by Haas, Prescott, Dudley, Dik, Hintlian, & Keaneon fresh strawberries, honeydew melon and cucumber inorder to delay surface molding. The treatment had a positiveeffect on molding especially for strawberries showing a pre-servative action.

Apart from this study, only recently theHPCD technologyhas been applied and tested more in deep on freshe cut con-ference pears to test the inactivation of Saccharomyces cere-visiae isolated from the natural flora of the pear puree(Valverde, Mar�ın-Iniesta, & Calvo, 2010). The results dem-onstrated the key role of the temperature in the inactivationrate and how the nature of the substrate influenced the choiceof the process conditions. The yeast survival ratio did not lin-early depend on temperature. The survival curves presentedtwo stages: a first stage up to 35 �C, where inactivation wasminimal, and a second one from this temperature to 55 �C,where the inactivation rate rapidly increased. The reasonfor a two stage dependence on temperature could be relatedto the growth mechanism of mesophile microorganisms,since their optimal temperature for multiplication occursbetween 25 and 40 �C. Above that temperature, proteinsdenature, cytoplasmic membranes collapse, and cells lyseare deactivated. In addition the results demonstrated that anincrease in pressure from 6 to 30 MPa did not significantlyincrease the inactivation rate and the same happened forthe treatment time. Completemicrobial inactivation occurredat 10 MPa, 55 �C for 10 min. The lesser water activity of thesolid pear could justify the lower inactivation rate of S. cere-visiae in comparison to liquid media, aside from their differ-ent compositions and physical states (Lin, Yang, & Chen,1992).

The HPCD treatment has been also applied to some veg-etables. Kuhne and Knorr (1990) carried out experimentson fresh celery and leafstalks showing a substantial inacti-vation effect on total count. Their data indicated that a treat-ment carried out for 30 min at 40 �C and 62.8 MPa induceda reduction in the total plate count of approximately104 cfu g�1 (from 1.7$107 cfu g�1 in the untreated sampleto 9.8 103 cfu g�1 after the treatment). They also reportedthe inefficiency to inactivate spores on the same substratesand under the same experimental conditions.

The HPCD treatment (7.0 MPa for 24 h) performed onthe fermented Baechu kimchi, a Chinese cabbage froma group of traditional vegetable foods in Korea at aboutpH 4.45, brought insufficient reduction of lactic acid bacte-ria (Hong & Park, 1999).The results were significantlydifferent from previous studies for pure cultures of lacticacid bacteria. As a matter of fact, the same research groupisolated Lactobacillus sp. from the late stages of kimchi fer-mentation and showed that the HPCD treatment inducedinactivation suspending the microbial cells in the culturebroth (Hong & Park, 1997). The different result was attrib-uted to the lowered diffusivity and the limited mass transferof CO2 due to the compact structure of the salted Chinesecabbage and to the lack of kimchi juice in the solid samplewhich could act as an easy penetration medium.

The last application of the HPCD treatment to vegetableswas reported by Zhong, Black, Davidson, and Golden (2008)who evaluated the potential of the treatment on the inactiva-tion of inoculated E. coli Ke12 and background microfloraon fresh spinach leaves. The study demonstrated that the


microbial reduction obtained in CO2 supercritical conditions(7.5 and 10MPaat 40 �Cand 40min)was significantly higherthan that in subcritical state (5 MPa at 40 �C and 40 min). At5 MPa and 10 min, the microbial reduction of E. coli andbackground microflora was about 2 and 1 log cycles respec-tively while in supercritical conditions a reduction to unde-tectable level (w 5 Log cycles) was obtained. However noinactivation evidences were reported to highlight theefficacy of the treatment against pathogens such as E. coliO157:H7 and Salmonella, very common microorganismson this substrate.

Effect on quality attributesResults on the evaluation of the physical attributes of

fruits treated by HPCD showed negative effects of the treat-ment on the products. Although just a visual observationwas addressed, the treatment resulted in gross tissue de-struction of strawberries, honeydew melon and cucumber(Haas et al., 1989).

Quantitative analyses were performed on the HPCDpears treated by Valverde et al. (2010) in terms of pH,sugar content and color. The results showed that the pro-cess did not reduce the pH and did not alter the sugar con-tent of the pears although a loss of consistency wasobserved and manifested as a softer aspect and a loss ofliquid. A change in the color parameters (decrease of theluminosity L, increase of the a value to the red and reduc-tion of the b value to the blue) of the fruits was also de-tected mainly due to the reduced vitamin Cconcentrations and to the partial and ineffective inactiva-tion of the peroxidase enzyme activity.

From these early findings, it is evident that the primaryproblem of applying this technology to the preservation offruits is the adverse effect this process produces on the re-duction of fruit firmness and texture due to the use of thehigh pressure. It is possible to conclude that the HPCD pro-cess is more adequate for products where a firm texture isnot essential, such as fruit cocktails, creams, juices, or othertypes of fruit preserves.

Few analyses have been also performed to evaluate theimpact of the process on the quality attributes of vegetables.Results of sensory analyses performed by Hong and Park(1999) on kimchi samples during storage at 10 �C showedthat the treated vegetables had a higher pH, lower titratableacidity and better sensory properties. No differences weredetected for color, off e flavor and hardness comparingthe samples with the untreated.

The study carried out by Zhong et al. (2008) showed theimportance of the choice of the process conditions. Digitalphotographs of HPCD spinach leaves demonstrated that thetreatment performed at longer processing time (40 min) andhigher pressure (10 MPa) resulted in a greater leaf discolor-ation and decrease of leaf firmness. Discoloration waslikely caused by the CO2 dissolution into leaf tissues, whichacidified the leaves and degraded the chlorophylls(Mingotaud, Chauvet, & Patterson, 1996).

Food powdersMicrobial inactivation

In the field of food powders, the HPCD treatment has beenonly applied for the inactivation of naturalmicroflora of a nonfermented high polyphenol cocoa powder with a butter con-tent of 10.5% obtained from the grinding of cocoa seeds(Calvo, Muguerza, & Cienfuegos-Jovellanos, 2007). The ef-ficacy of the HPCD treatment was tested for the inactivationof mesophilic and thermophilic aerobic microorganisms. Inaddition considering that the natural contamination of the ca-caowas due to bacteria spores, the efficiency of the treatmentwas also tested on Aspergillus niger (CECT 2574, ATCC16404) and Aspergillus ochraceus spores spiked on cocoasamples. Results demonstrated that an increase in pressurefrom 13 to 30MPa at 65 �C for 40min did not show any effecton the microbial inactivation of mesophilic and thermophilicaerobic microorganisms. No microbial inactivation was alsoobserved operating the treatment at 30 MPa although thetemperature was increased to 40, 65 and 80 �C. Same resultswere also obtained applying twelve cycles of compression/decompression up to 30 MPa to the cocoa sample heated at80 �C. The inactivation target degree was only achievedwith the combination of higher temperatures (80 or 65 �C)and some extra humidity (5 or 10%) at a pressure of30 MPa concluding that the initial water content was one ofthe key factors affecting the inactivation mechanism. Resultson cocoa samples contaminated with spores demonstratedthat the inactivation of both types of fungi spores was fullyachieved at 30 MPa, 80 �C for 30 min after the addition of5% water.

Effect on quality attributesAnalysis on the quality attributes of the cocoa powder

were performed in particular to detect the effect of theHPCD treatment on polyphenolic compounds as (þ) - cat-echin, (�) - epicatechin, proanthocyanidins and anthocya-nins. These compounds could be extracted if wet CO2 isused as solvent. On the other hand, the temperatures ap-plied to achieve sterilization could provoke the flavanolspolymerization. Contrary to what it could be expected,the polyphenolic compounds of the samples treated at30 MPa, 80 �C with 10% water addition were not extracted.The chromatographic profiles of the extracts of the treatedsamples did not show the presence of phenolic acids or fla-vonoids. Moreover, the water content of the sample afterthe treatment decreased to about the initial level. This as-pect is of great importance for the storage, shelf life, andphysical aspect of the cocoa powder, as well for its furtherapplications, since the humidity content of the final productshould not exceed 9%, as specified by law for this type ofcompounds (Calvo et al., 2007).

Sprouts seedsMicrobial inactivation

Few papers have been published reporting the effect ofHPCD on microorganisms occurred in sprouts seeds, in


particular alfalfa seeds considered a valuable dietary foodand commonly consumed raw or slightly cooked in saladsor sandwiches (Jung, Choi, & Rhee, 2009; Mazzoni,Sharma, Demerci, & Ziegler, 2001). Mazzoni et al.(2001) demonstrated the efficacy of the treatment on thenatural indigenous aerobic microorganisms and ona non e pathogenic strain of E. coli K12 inoculated onthe seeds. For the aerobic plate count, at a fixed temperatureof 50 �C, the treatment carried out at 13.8 MPa for 15 minresulted in less than 10% inactivation, while 85.6% inacti-vation was achieved increasing the treatment time to60 min and the pressure to 27.6 MPa. Results demonstratedthat the treatment was also effective on E. coli K12 with re-ductions equal to 26.6, 68.1, and 81.3% at 13.8, 20.7 and27.6 MPa for 15 min and 50 �C, respectively.

The high level of microbial inactivation obtained in thestudy of Mazzoni et al. (2001) showed that the supercriticalCO2 was not serving as only bactericidal agent but it mightbe reaching microorganisms in hidden in crevices or be-tween the cotyledon and the head of the sprouts due tothe high pressure applied.

An extensive inactivation study was also carried out byJung et al. (2009) who contaminated alfalfa sprouts withE. coli O157:H7, L. monocytogenes and S. typhimurium mi-crobial strains. The results demonstrated the efficiency ofthe treatment, although a limited step of the process wasthe low CO2 solubility due to the solid nature of the matrix(Choi et al., 2009a; Wei et al., 1991). An HPCD treatmentperformed at 20 MPa, 45 �C for 15 min determined morethan 7 log reductions for the three tested pathogens.

Effect on quality attributesThe rate of germination is the quality attribute moni-

tored during the treatment of alfalfa seeds indicating thenutritional value of seeds: fats and carbohydrates are bro-ken down and the starch digestibility is increased duringthe germination process (Vidal-Valverde et al., 2002).

The study carried out by Mazzoni et al. (2001) showedthat the HPCD treatment did not have any detrimentaleffect on the viability of alfalfa seeds. The percent ofgermination for all treatment conditions was over 90%and no significant differences in the germination ratewere detected between the treated and untreated seeds,indicating that the HPCD process could be effective forthe treatment of alfalfa seeds at commercial level withoutcompromising the germination quality.

Jung et al. (2009) demonstrated that treating seeds withHPCD at high treatment conditions (20 MPa at 40 and45 �C) impaired the germination capability of alfalfa seeds.To not influence the germination capability of alfalfa seedsafter three days of germination it was necessary to applyprocess conditions which did not guarantee the microbialsafety (>5 log reductions of foodborne pathogens). Thecontrasting conclusions obtained by the only two paperspublished on this matter, suggest that further studies are

needed to gain more insights on the effects of the treatmentboth on germination rate and sprouting conditions.

Spices and herbsMicrobial inactivation

One of the first applications of the HPCD treatment tospices and herbs has been published by Dehghani,Annabi, Titus, Valtchev, and Tumilar (2008) for the sterili-zation of ginseng powder. Considering that the cultivationof ginseng usually takes up to 6 years and the species aresubmerged in wild soil for the entire period, over than78% of the extracted ginseng products are highly contami-nated with fungi and bacteria. The common sterilizationmethods used for this product include autoclaving, ethyleneoxide exposure, and g-irradiation. These methods havebeen proven to achieve complete removal of many microor-ganisms including several bacteria classes such as E. coli,S. aureus, and Bacillus cereus (Byun et al., 1998). The gin-seng powder used in the study of Dehghani et al. (2008)could not be released into the market due to the high levelof total bacteria (5$107 cfu g�1) and also to the presence offungi. The acceptable levels for oral use by the TherapeuticGoods Administration in Australia (TGA) are contamina-tion less than 104 cfu g�1 and 102 cfu g�1 for total aerobicmicrobial count and fungi, respectively. HPCD treatmentwas applied to evaluate the possibility to achieve completeinactivation of fungi and bacteria. However the results ofthe study confirmed that the only bactericidal action ofCO2 was not efficient to completely inactivate microorgan-isms in ginseng samples.

There was not significant effect of changing the process-ing time from 15 min to 2 h at 15.0 MPa and 60 �C on totalaerobic microbial count reduction. At 15.0 MPa and pro-cessing time of 2 h, increasing the temperature from25 to 60 �C slightly decreased the CO2 efficiency for totalaerobic count reduction. Increasing the processing time to15 h at 60 �C only decreased the total aerobic count to2.67 log cycle that is still beyond the limit for oral admin-istration by TGA (e.g., total aerobic microbial countdecreased from 5$107 cfu g�1 to 1.10$105 cfu g�1). Inaddition such long processing times are not desirable andpractical for commercial purposes.

More recently Calvo and Torres (2010) applied the HPCDtreatment for the inactivation of natural microflora of paprikapowder, the dried and milled product of selected varieties ofred pepper from the genusCapsicum. The effects of the treat-ment were tested on the initial microbial load (aerobic mes-ophilic count, Enterobacteriaceae, E. coli, S. aureus, mouldsand yeasts) and on spores isolated from paprika powder bycultivation. Results demonstrated that themicrobial inactiva-tion was a strong function of the water activity of paprikasamples reaching a value of 1.5 log cycle at 6.0 MPa and80 �C for 30 min with a water percentage of 35%.

Although the beneficial effects of the initial water mois-ture were evident, higher amounts of water activity werenot investigated because the powder became a paste and


was difficult to handle also with the addition of anti e cak-ing agents in the vessel. Information about the behavior ofthe inactivation rate with the change of the process param-eters were also reported in the study. Slight variations of theinactivation rate occurred increasing the pressure from 6.0to 30.0 MPa obtaining less than a 1 log cycle reductiondifference.

The higher resistance of the natural microflora of pa-prika powder to the HPCD treatment could be linked tothe protective effect due to the interaction with the food in-gredients or to the insufficient contact between the CO2 andthe microbial cells. To investigate the possible protectiveeffect of the matrix, the authors inoculated the natural mi-croflora isolated from paprika powder on different sterilizedmaterials (ground coffee, cocoa powder and flour) whoseappearance and texture was similar to that of paprika.The results indicated that no important differences wereobtained independently of the chosen inoculated product.

Furthermore to improve the contact between the CO2 andthe microorganisms and get better results in the microbialinactivation some options were taken into account. The firstattemptwas the use of an antie caking agent (CaCO3)widelyapplied in the food industry in proportions less than 2% toavoid the formation of lumps. The intercalation of inert buthighly porous particles such as perforated beads, bentoniteand activated carbon was also taken into account to increasethe porosity of the bed in order to prevent the possible forma-tion of preferential channels that could lead to “dry or dead”zones. The last attempt was the increase of the flow rate bytripling the total mass of CO2 in contact with the paprika.The experimental results demonstrated no remarkableimprovements for any of these attempts.

The authors concluded that the only factor influencing thesterilization process was the resistance of the natural micro-flora which was higher in a low water activity environment.Optimizing the initial moisture between 25 and 30%, temper-ature between 85 and 90 �C, pressure between 6.0 and10.0 MPa and treatment times between 30 and 45 min, itwas possible to achieve the disinfection and the total countreduction required by law (as shown in Table 2).

The moisture content was found to be an important pa-rameter to be controlled also for the application of HPCDtreatment to wheat flour. Haas et al. (1989) reported thatat higher moisture level equal to 28%, the inactivation ofbacterial and molds populations in wheat flour was en-hanced operating in a temperature range of 25 and 50 �Cat 6.2 MPa for two hours. Good microbial reduction wasalso observed when flour with the addition of 25% of glyc-erin (to increase the water activity to 0.91) was inoculatedwith S. senftenberg, S. aureus and E. coli. A further in-crease of glycerin to 75% corresponding to water activityequal to 0.61 greatly reduced the killing power. In thesame study experiments carried out on dry peppers andremoistened pepper (30%) showed similar results. Testingdry materials with high microbial counts such as rosemary,onion powder and bell pepper demonstrated that without

rehydration no killing effect due to the CO2 treatmentwas evident while there was complete kill in fresh spicesof thyme, mint, chives, and oregano.

Effect on quality attributesFew studies have been performed to characterize the

quality of food powders after the HPCD treatment. Theonly results were the one published on paprika powdersand expressed in ASTA units which take into account thecolor intensity of the powder (Calvo & Torres, 2010).The authors reported that only a slight color reduction, anaverage of 6 ASTA units, was detected in the treated sam-ples independently from the temperature and the final watercontent of the powder. However, some browning was ob-served when treatment times were prolonged over 60 minat temperatures over 90 �C. Considerable color loss oc-curred at the highest pressures over 25 MPa due to the ex-traction of oleoresin and pigments. In conclusion the studyreported that factors influencing paprika quality were: thewater content and the process temperature. In the studyof Haas et al. (1989) quality analyses were performed onfresh herbs exposed to 5.5 MPa, 45 �C for 2 h. The resultsdemonstrated that thyme, mint, chives, and oregano had en-hanced aromas after the treatment. However some herbsshowed a different taste after the treatment. Parsley tastedsimilar to the untreated but developed a slight off aroma af-ter treatment while untreated thyme and mint tasted betterthan the treated sample. The results seemed to be dependenton the variety, and maturity of the herbs which on the otherhand affected the taste, aroma and the palatabilityevaluations.

FishesMicrobial inactivation

The first attempt to apply the HPCD treatment for thepreservation of fish was carried out by Wei et al. in 1991.In this study, shrimp samples spiked with Listeria weretreated by HPCD at 5.85 MPa, 35 �C for 2 h. The resultsdemonstrated that the treatment reduced the bacterialcounts by only 35e45% but increasing the pressure to13.7 MPa a microbial reduction equal to 99% was obtained.More recently an innovative approach to post harvestprocessing of oyster has been introduced focusing on theeffects of supercritical carbon dioxide on bacterial contam-inants trapped in the digestive system of oysters (Meujoet al., 2010). To observe the effects of CO2 as warm pas-teurization technique, experiments were performedin vitro on bacterial culture of a non e pathogenic strainof Vibrio used as a model for Vibrio spp. and several bac-terial isolates from an oyster homogenate. The authors re-ported that the level of total bacterial inactivationachieved with the HPCD treatment (100 bar for 30 min at37 �C or 172 bar for 60 min at 60 �C), was comparableto that achieved with several FDA approved post harvestprocessing for oysters, namely, high hydrostatic pressureand quick frozen (Prapaiwong, Wallace, & Arias, 2009).

Table 4. Application of HPCD treatment coupled with additives or pretreatments on microbial forms in food system. Type of food, microorganism, process conditions, and additives orpretreatments employed are reported with the corresponding microbial inactivation and references.

Additives/Pretreatments Target microorganism Food system Process conditions Microbial inactivation Reference

100% CO2 at atmospheric pressure Anaerobe spores Fresh celeryleaves andleafstalks

HPCD (30 min at 40 �C and 62.8 MPa)CO2 flushed on the sample for 60 minat 27 �C, 0.1 MPa

50% (Kuhne & Knorr, 1990)

H2O2 Thermo e resistant spores Cocoapowder

HPCD (2 h at 40 �C and 30.0 MPa)(CO2 saturated in H2O2 as solvent)

No inactivation (Calvo et al., 2007)

Ethanol HPCD (2 h at 40 �C and 30.0 MPa) CO2

saturated in ethanol

Acetic acidLactic acid

Escherichia coli Bonelesspork loins

HPCD (30 min at 35 �C and 12 MPa) 3%solution of acetic acid (samples immersedfor 1 min at 4 �C) 3% solution of lacticacid (samples immersed for 1 min at 4 �C)

2.58 Log (cfu/cm2) (Choi et al., 2009b)Listeria monocytogenes 2.60 Log (cfu/cm2)Salmonella typhimurium 2.33 Log (cfu/cm2)E. coli O157:H7 2.10 Log cfu/cm2)

NaOCl Natural microflora Red paprika HPCD (10 min at 35 �C and 6.0 MPa)100 ppm NaOCl solution (samplesimmersed for 10 min at room temperature)

4 Log (cfu/g) (Matsufuji et al., 2009)Green pepper 3.8 Log (cfu/g)Cucumber 4.2 Log (cfu/g)Cabbage 3 Log (cfu/g)Lettuce 3 Log (cfu/g)Mizuna (Brassica) 4 Log (cfu/g)Celery 3.5 Log (cfu/g)Onion 2.5 Log (cfu/g)Carrot 2 Log (cfu/g)Bean sprout 7 Log (cfu/g)

Water Total aerobicmicrobial count

Ginseng powder HPCD (1 h at 60 �C and 10.0 MPa) 1 mLof water in 70 mL of CO2

0.1 Log (cfu/g) (Dehghani et al., 2008)

Ethanol HPCD (1 h at 60 �C and 10.0 MPa) 1 mLof ethanol in 70 mL of CO2

0.2 Log (cfu/g)

H2O2 HPCD (1 h at 60 �C and 10.0 MPa) 1 mLof H2O2 in 70 mL of CO2

0.4 Log (cfu/g)

Water þ ethanol þ H2O2 HPCD (1 h at 60 �C and 10.0 MPa) 1 mLof water þ1 mL of ethanol þ 1 mL ofH2O2 in 70 mL of CO2

1.7 Log (cfu/g)

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Effect on quality attributesQuality observations of shrimp samples treated by Wei

et al. (1991) showed some color change after the HPCDtreatment carried out at 13.7 MPa, 35 �C for 2 h. As ob-served for the chicken samples, the authors reported thatthe outer layer of the shrimps turned whitish and gave theappearance of being cooked rapidly at low temperature orsoaked in acid. Also loss of small amount of liquid fromthe tissue was observed giving the idea of some tissue dam-aging caused by the treatment.

The study on oysters treated by supercritical CO2 re-ported a sensory analysis in terms of physical appearance,smell and texture assessed by a panel of 13 people(Meujo et al., 2010). The treated oysters were comparedwith two standards: a freshly shucked, untreated oysterand an oyster left at room temperature for about two daysto develop a mild odor associated with spoiled oysters.The sensory analysis revealed that oysters remained accept-able considering their physical appearance, texture andsmell after the exposure to CO2 process (10 and 20 MPafor 20 and 50 min at 37 �C).

Additives and treatments combined with HPCDtreatment

Few research papers have been published demonstratingan increase of the microbial inactivation rate by addingsome co e solvents or coupling the effects of HPCD treat-ment with a pretreatment. Table 4 is a compilation of the ex-perimental results found in literature concerning the effect ofcoupling additives and pretreatments to the HPCD treatment.Indications of the types of food,microorganism, process con-ditions, and employed additives or pretreatments are reportedwith the corresponding microbial inactivation.

The addition of co e solvents or additives to pressurizedCO2 has been proposed in a consistent number of publica-tions mainly applied to liquid substrates. The synergistic orantagonist effect of the process has been explained basedon the nature of the co e solvent able to increase theamount of CO2 in contact with the substrate and in turnavailable to act as antimicrobial agent (Ferrentino,Balaban, Ferrari, & Poletto, 2010; Has & Herman, 1977;Haas et al., 1989).

Choi, Kim, Kim, Kim, and Rhee (2009b) reported theantimicrobial effects of coupling organic acids (acetic orlactic acid solutions for 1 min) and HPCD treatments(12 MPa and 35 �C for 30 min) under various conditionson the reduction of a nonpathogenic E. coli and three

Table 5. Relevant patents accomplished for the application of HPCD trea

Assignee Number Observations

Swift & Company, Chicago, Ill. US 3,483,005 Method of sterilizsusceptible to the

Air Liquid, Houston, TX US 2008/01711 A1 Method to pasteuor near room temcarbon dioxide

pathogenic bacteria in fresh pork. Experimental resultsshowed that the combined treatments had no synergistic ef-fect and no significant differences were observed betweenthe samples treated coupling acetic acid and HPCD or lac-tic acid and HPCD.

Combination treatments applied to solid foods is a com-pletely undiscovered field. Only two papers investigated theefficacy of the HPCD treatment combined with pretreat-ments (Calvo et al., 2007; Kuhne & Knorr, 1990) for theinactivation of spores as shown in Table 4.

Since spores could not be inactivated exposing fresh cel-ery leaves and leafstalks to only HPCD treatment at62.8 MPa for 30 min at 40 �C, Kuhne and Knorr (1990)proposed a pretreatment with the exposure of the samplesto 100% CO2 at atmospheric pressure and 27 �C for60 min. At the end of the combined treatments, 50% reduc-tion of anaerobe spores was observed.

The use of combined treatments was also suggested byCalvo et al. (2007) to inactivate thermo e resistant sporesin cocoa powder coupling the effect of ethanol and hydro-gen peroxide (H2O2) with HPCD (30.0 MPa and 40 �C for2 h). The results showed that the application of a combinedtreatment was useless with the presence of both ethanol andH2O2. Hemmer, Drews, LaBerge and Matthews (2006) pub-lished experimental data demonstrating that adding H2O2

(<100 ppm), 6 log reductions on populations of Geobacil-lus stearothermophilus and Bacillus atrophaeus sporesstrips (impregnated on cellulose supports) were completelydeactivated using supercritical CO2 in 1 h at 40 �C and20.0 MPa. In the same year, Zhang et al. (2006) showedwith transmission electron microscopy images (TEM) thatB. atrophaeus spore envelope (growth on strips and glassvials) was damaged by supercritical CO2 allowing the pen-etration of H2O2 and the oxidation of some vital internalstructures causing spore death.

PatentsPatents related to the use of HPCD pasteurization/steril-

ization of solid foods are scarce. Two are the most relevantshown in Table 5 where is reported the number of the pat-ent, the assignee and the description of the method. Thefirst patent described the coupled effect of pressurizedCO2 with another non e thermal technique (Urbain,Shank, & Kauffman, 1969). The food, mainly meat, wasexposed to an atmosphere of pressurized carbon dioxideand subsequently to relatively low energy ionizing radia-tion. The patent reported that the combined process resulted

tment to solid foods.

Reference

ing food products which aredevelopment of flavor degradation

(Urbain et al., 1969)

rize pre-packaged food at,perature, using supercritical

(Rasanayagam & Yuan, 2008)


in a truly sterile food product free from spoilage, with anextended storage life, without discoloration and with un-changed flavor.

Rasanayagam and Yuan (2008) granted a patent to pas-teurize pre e packed foods at, or near room temperature,using supercritical CO2. The invention described a methodto use supercritical CO2 to inactivate pathogenic microor-ganisms in foods, and at the same time, to carbonate thefood in a pre e packed pouch or container with a pre de-fined polymer structure that allowed CO2 to permeate whilediscouraging the permeation of other substances. Themethod was able to induce a substantial pasteurization (3or 4 log cycles), and to inhibit enzymes in the foodstuff.Additionally the polymer pouch was able to retain CO2 toa predetermined level (between 0 and 5 volumes of CO2)in a way to establish a modified atmosphere suitable forthe storage of the pasteurized food.

Potentials of HPCD technology and future outlooksThe review clearly shows that the treatment has poten-

tials in reducing microbial loads improving the safety ofthe food. However a great challenge is the control of pro-cess parameters (pressure, temperature and treatmenttime) considering that they can affect molecular interac-tions and protein conformation causing color and structuralchanges in food products. This aspect seems to be a seriousconcern to extensively apply the technology. It is evidentthat the treatment seems suitable not for all solid foodstuffsdue to the physical consistency of the products and theirability to positively react to the process in terms of micro-bial inactivation and retention of quality attributes. The ef-fects of the treatment on the quality attributes reported sofar clearly evidence that the process cannot be applied tosoft foods or leafy vegetables. It seems suitable for foodswhich retaine their structure or maintaine an appeal forthe consumers although in soft form (i.e. fruits in syrup).

A further fundamental aspect to take into account is theweakest knowledge of mechanistic studies and mathemati-cal modeling of the inactivation kinetics which are essentialto understand the fundamentals of the HPCD treatment, tooptimize the process parameters and propose a safe, cheapand time consuming technique. In addition it is evident thata great challenge for the application of the technology is theneed to investigate and understand the mechanisms bywhich HPCD inactivates pathogens and microbes andhow the food matrix interferes with the inactivation.

The overview of the scientific works published so far re-veals that most of the results on the effects of the HPCDtreatment on solid products are just qualitative. Very fewsystematic or quantitative analyses have been performedto investigate the chemical and physical effects of the treat-ment. To confirm the potentials of the treatment froma quantitative point of view more scientific evidences areneeded.

In this concern, the definition of the foods suitable forthe treatment is necessary carrying out screening tests

with a wide range of product types to enable the selectionof suitable candidate products. The achievement of kineticsof microbial and enzymatic inactivation at different processconditions are necessary at lab scale testing both typicalpathogens but also natural microbes occurring in foods.

In addition, extensive studies need to be performed toevaluate the shelf life and long term safety of the best can-didate products to the treatment. Once all these missing in-formation will be filled, the subsequent step will be thescaling of such novel technology into production processes,including design of appropriate equipment and screening ofprocessing parameters after scale e up suitable for foodmatrixes.

Only with scientific results, convincing data, and provi-sion of clear, objective and unbiased information, includingthe potentially negative aspects of the technology and theirlimitations, the HPCD treatment could become one of themost available emergent technologies applied to solidfoods.

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Review High pressure carbon dioxide pasteurization of solid foods: Current knowledge and future...

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