Sterilization and virus inactivation by supercritical fluids (a review)

J. of Supercritical Fluids 66 (2012) 359–371

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Review

Sterilization and virus inactivation by supercritical fluids (a review) Michel Perrut ∗ SEPAREX, F-54250 Champigneulles, France

a r t i c l e

i n f o

Article history: Received 30 March 2011 Received in revised form 5 July 2011 Accepted 6 July 2011 Keywords: Supercritical fluid Sterilization Pasteurization Virus inactivation Carbon dioxide Hydrogen peroxide

a b s t r a c t While supercritical processes are developing both for “classical” applications in food industry and in new domains related to Health Sciences, the interactions of supercritical fluids (SCFs) with living microorganisms are of growing importance. It is known for long that supercritical fluid extraction processes do protect the processed materials from oxidation and contamination with organic solvents and prevent bio-burden increase. Moreover, SCFs were also shown to have the ability to kill most microorganisms and to “inactivate viruses”, including human pathogenic strains. This paper intends to summarize the present state-of-the-art in order to underline the promising future of SCF sterilization/pasteurization and virus inactivation as an alternative “green” method to classical processes that cannot be used in a growing number of cases: thermolabile products degrading by heat sterilization, or compounds reacting with sterilizing chemicals (hydrogen peroxide, ethylene oxide, peracetic acid, etc.), or radiolysis of biomolecules during irradiation. Process implementation and commercial development are then discussed in light of future challenges in terms of regulatory, economical and environment requirements. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological effects of supercritical fluids on microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Early work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Vegetative microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Latent forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Virus inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sterilization processes and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Sterile filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Carbon dioxide sterilization and pasteurization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Virus inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Pest control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Inactivation for immunogenic preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Elimination of endotoxins and pyrogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction For millenaries, food preservation has been fundamental for human kind as it has conditioned its survival and expansion under all climates. During the recent decades, pasteurization and ster-

∗ Tel.: +33 383 31 24 24; fax: +33 383 31 24 83. E-mail address: [email protected] 0896-8446/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2011.07.007

359 360 360 360 360 361 365 365 365 366 368 368 368 368 369 369 369 369

ilization have been a fast growing activity, especially for food preservation, medical devices and pharmaceuticals. Meanwhile the classical processes using heat cannot be used for heat-sensitive products, most operators have been more and more reluctant to move to low temperature processes based on irradiation and chemicals (like H2 O2 or ethylene oxide) for many reasons including cost, safety and environmental concerns. Living organisms are sensitive to their environment in which they can maintain metabolic activity within narrow limits of

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temperature, pH, hydrostatic pressure, and chemical composition, although a large variety of single cell organisms are capable to grow under extreme environmental conditions (i.e. deep-sea thermophilic bacteria). For long, high pressure treatment is used for pest control and sterilization in food industries – especially in Japan where irradiation was never accepted – as alternative to heat treatment that generally degrades product quality (aspect, taste, vitamin content, etc.). However, the required hydrostatic pressure for efficient sterilization is extremely high (4000–8000 bar) and exposure times are also considerable, which leads to costs incompatible with most markets. As mentioned in early publications and patents [1–13], it is known for 20 years or more that supercritical fluid exposure can be considered as a less expensive variant through various processes working at much lower pressures, where the product is contacted with carbon dioxide, possibly added with water or ethanol or other additives like acetic acid or hydrogen peroxide. More recently, much attention has been paid to supercritical fluid pasteurization of food products and sterilization of various products and items, with a special attention dedicated to inactivation of spores that are known to be highly resistant to heat, radiation, and chemical agents. In addition, few investigators worked on virus inactivation in plasma fractions and implants. As many studies were published during the last decades, it seems now timely to gather all these data – that are sometimes contradictory – and to evaluate what are the main challenges to solve to reach commercial development and acceptance of this “green” technology in regards to the regulatory, economical and environment requirements. This paper clearly intends to widespread this knowledge towards the scientists belonging to the supercritical fluid “community” that may not be aware of SCFs potential for sterilization.

2. Definitions For clarity, it seems necessary to list some basic definitions and common practices according to international standards: • Sterility: Sterility is the absence of viable microorganisms. As sterility cannot be guaranteed by testing; it has to be assured by the application of a suitably validated production process according to protocols defined by control authorities. • Sterilization: The act of rendering something free from living cells, either by removing, killing or inactivating all microorganisms, including vegetative forms and spores. • Validation: In order to validate a sterilization process, standardized preparations of selected microorganisms (called biological indicators) are used. They usually consist of a population of bacterial spores. The recommended species by the European and the US Pharmacopeia are Geobacillus stearothermophilus for steam or gas sterilization, Bacillus subtilis for dry-heat or gas sterilization, Bacillus pumilus for irradiation and Pseudomonas diminuta for sterile filtration. • Survival ratio, reduction factor and sterilization efficacy: The sterilization efficacy is often defined from the survival ratio of the number of viable microorganisms after the sterilization (N) to the number before processing (N0 ), and expressed in form of the reduction factor or degree of inactivation (DI): DI = −log10

N N0

The higher is this number, the higher is the process efficacy.

(1)

• Sterilization kinetics: For a given process operated in given conditions, changes in microbial populations versus time is commonly described by the survivor curve equation: log10

−t N = N0 D

(2)

where D is the decimal reduction time, or time required for a 1 − log reduction in the microbial population, by analogy with the first-order kinetic model for chemical reactions. Alternative models are being developed to explain microbial inactivation kinetics when the linearity of the data is questionable. • Sterility assurance level: SAL is the probability of a non-sterile item in a population. The SAL of a process for a given product is established by appropriate validation studies. A SAL value of 10−6 is generally regarded as acceptable. • Pasteurization: This word refers to a moderate heat treatment, invented by Pasteur, leading to microorganisms inactivation without significant product degradation, essentially used on food products. By extension, pasteurization is also used to designate other processes applicable to food products (such as CO2 treatment). The difference between sterilization and pasteurization is that the latter does not kill spores. 3. Biological effects of supercritical fluids on microorganisms 3.1. Early work Early work showed that gaseous CO2 and N2 O, even at low pressure (below critical pressure), inhibit the growth [1,2] and boost the inactivation rate of microorganisms including spores during irradiation [3] or thermal treatment. Heat treatment at 50–55 ◦ C in the presence of CO2 at 6 bar has the same lethal effect on several bacteria, fungi and yeasts as heat treatment at 60–65 ◦ C in presence of air, or, in other words, operating with this gas pressure could reduce by 50% the time of pasteurization at a given temperature [14]. 3.2. Vegetative microorganisms From several early sources [5,6,8,13,15], comparison of the survival curves of microorganisms in contact with a pressurised gas like nitrogen, ethane or propane, and with a sub-/super-critical fluid (carbon dioxide, ethane, propane), clearly demonstrates that the bactericidal effect of these fluids cannot be attributed to hydrostatic pressure in the range of tens or hundreds of bars, but to specific interactions depending on fluid chemical nature, nitrogen being almost inactive while CO2 , N2 O and propane are very efficient in cell inactivation. On the other hand, for long, it has been recognized that gaseous CO2 can inhibit microbial growth [1,2,4], leading to its use in the preservation of packed foods, although its inactivation effect seems reversible. Even at pressure as low as 6 bar, this gas exhibits a significant bactericide or bacteriostatic effect [14]. Moreover, this specific effect is definitely supported by the comparison of cell number decay of various microorganisms when submitted to a very high hydrostatic pressure with and without carbon dioxide [17]. For example, the decay of Escherichia coli in CO2 at 150 bar and 35 ◦ C during 15 min was similar to the one observed at 3000 bar at ambient temperature during the same period of time [17]. So, there is no doubt that this bactericidal effect is caused by specific interactions between the living cell and the fluid that readily dissolves inside the cell. As clearly shown by several authors [18–20], cell decay is considerably increased when CO2 pressure is raised beyond the critical pressure, boosting both fluid dissolution inside cell and membrane lipids interaction. A similar conclusion was raised with propane [16]. As discussed in depth by

M. Perrut / J. of Supercritical Fluids 66 (2012) 359–371

Spilimbergo et al. [21–26], many authors nisms related to alteration of cell membrane metabolism [25], although quantification of remains unknown. However, some of these alluded to when N2 O and propane are used:

proposed mechaand of its internal each contribution effects cannot be

• Cell wall rupture/perforation due to a strong interaction of the fluid with the lipids (mainly phospho-lipids), especially in case of rapid depressurization; Spilimbergo et al. [24,25] recently presented in situ monitoring of microorganisms viability by cell fluorescent staining, this method permitting to discriminate viable cells and dead cells stained due to permeabilized membrane, supporting this inactivation mechanism. • Inactivation of some key-enzymes resulting from pH decrease inside the cell [23,25], and specific inhibition of decarboxylases by excess of CO2 breaking the metabolic chain. This is to be related to other work dealing with enzymatic reactions and enzyme stability in supercritical CO2 [27–29]. • Inactivation of certain bio-reactions caused by lipid extraction [25]. • Intracellular electrolyte balance perturbation with precipitation of carbonates (Ca, Mg, etc.) from bicarbonates inside the cell when CO2 pressure is released [25]. • It is to be noticed that CO2 sorption inside Saccharomyces cerevisiae was quantified and sterilization rate described by a first order reaction, the rate constant increasing with water content and CO2 pressure [58]. As supercritical carbon dioxide rapid pressurization/depressurization cycles are known to cause membrane disruption and cell lysis [1,4,30,31], it is not surprising that pressure cycling is a very positive factor in the sterilization efficacy, and may considerably reduce the treatment time for obtaining a given degree of inactivation [15,21,31–33]. Another support for this mechanism comes from the synergetic effect of a very brief pulsed electric field pre-treatment combined with a classical high-pressure CO2 treatment that leads to a very significant improvement of the sterilization efficacy [34], as the pre-treatment may render the cell membrane more fragile (or damage it). In E. coli and Staphylococcus aureus, the inactivation rate jumps from 2.5 to 8.5 and from 3.5 to 7.8, respectively, upon processing with CO2 only or combining CO2 processing with such pre-treatment at the same conditions. Pressure, temperature and treatment duration are the basic parameters controlling microorganism survival rate during highpressure CO2 sterilization (see Table 1 and reviews [22,25]): ◦ Temperature has certainly the most important effect, and treatment efficacy rapidly decreases when the temperature is below 40 ◦ C. ◦ Pressure must be beyond the critical pressure, but as using a very high pressure (>200 bar) does not lead to significant improvement, a medium-pressure range (80–150 bar) seems adequate. Moreover, a fast pressure cycling increases the inactivation rate. ◦ Survival rate versus exposure time can be approximated by a firstorder law according to Eq. (2) after a certain period of latency [25,75]; the order of magnitude of contact time to reach the final efficacy ranges between few minutes to few hours depending on strains, matrixes and, obviously, on temperature and pressure. ◦ Moreover, there is no doubt that presence of water drastically increases the bactericidal effect of CO2 [9,33,35], probably in relation with pH effect, as it was shown during extended investigations on enzymatic reactions in supercritical media demonstrating the strong and irreversible effect of moisture excess on most enzymes [27–29]. This is also supported by the

361

fact that the medium pH has a significant influence on the survival rate as an acidic pH seems to favour inactivation [15,36]. ◦ In fact, beyond pH influence, the matrix plays an important role, as exemplified by the surprisingly different results obtained by Wei et al. [13] on different food ingredients spiked with bacteria, especially the completely different effect of the treatment on the whole egg and the egg yolk only. Note: In general, microorganisms are much more resistant to pressurised gases or supercritical fluids than mammalian cells that, as reported by Ginty et al. [77], are irreversibly inactivated in CO2 (35 ◦ C – 74 bar) in few minutes. 3.3. Latent forms As far as spores are concerned, it is not surprising to see that resistance to inactivation by contact with a sub-/super-critical fluid is much higher than for vegetative cells. Results already disclosed show a dependence on the strain, and can be summarized as follows: • Most spores are unaffected by contact with supercritical carbon dioxide at mild temperatures (<60 ◦ C) [8,9,21,22,25,39,64,65], even, in certain cases, at very high pressure (2500 bar [8]). However, fast pressure cycling improves the inactivation rate [21]. On the other hand, it seems that contacting the treated material with supercritical CO2 improves the sterilization effect of irradiation [3]. • As shown by various authors and particularly [21,22,39], temperature seems to be the key-parameter. Spore inactivation is drastically improved at temperature beyond 70 ◦ C but the required temperature for total inactivation depends on the strain [39]. Most spores including the most resistant ones (such as the heat-resistant G. stearothermophilus) can be inactivated by combination of “mild” heating (<100 ◦ C) and CO2 treatment while a sole heat treatment would require at least 120 ◦ C [39]. • Medium pH is also very important as acidity favours spore inactivation for pH lower than 4 [12]. Moreover, it was reported that CO2 concentration dissolved in the medium is an important parameter, possibly due to pH effect, as shown by injecting the fluid through a porous filter in form of so-called “micro-bubbles”, leading to a drastic increase of spore inactivation [43,45]: such an inactivation rate increase (about 3 log), caused by a fluid dispersion system is rather surprising and raises doubt on result reliability. • Addition of a strong oxidant to CO2 such as Hydrogen peroxide [65–70,76], tert-tributyl hydroperoxide (t-TBHP) or mixture of both [76], or peracetic acid or trifluoroacetic acid [71–75] even at very low concentration, permits to reach a high efficacy at mild temperature. Many other compounds (among which methanol, ethanol, formic acid, acetic acid, succinic acid, phosphoric acid) were also investigated as additives in CO2 [72,73,75,76] with limited inactivation in most cases. • As for bacteria, combination of a pulse electric field pre-treatment followed by high-pressure CO2 treatment at 40 ◦ C is very efficient on spores although each of these processes has no significant effect when operated alone [34]. The spore inactivation mechanism is not yet understood, although it seems to be caused by disruption/perforation of the spore outer layers, especially when the process is run in the presence of a strong oxidant additive in CO2 . The fibre-like exosporium – a glycoprotein layer – is significantly damaged by different treatments, while spores treated with pure CO2 are not killed. It seems that spores processed with CO2 + H2 O2 show a less distinctive

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M. Perrut / J. of Supercritical Fluids 66 (2012) 359–371

Table 1 List of microorganisms processed by supercritical fluids (n.a.: non-available). Microorganisms

Materials

Fluid Alicyclobacillus acidoterrestris (spores)

Conditions

Reduction factor −log10 N/N0

Ref.

Matrix

Pressure (bar)

Temp. (◦ C)

Time (min)

CO2 -saturated orange juice

75

45

n.a.

Total St.

[37]

Aspergillus niger

Pure CO2 +2% ethanol +0.5% acetic acid

200

35

120

Wet: 4.9 >5.6 >5.1

[9]

Bacillus anthracis (spores)

CO2 + H2 O2 (200 ppm)

275

40

240

5.74–6.14

[69]

Bacillus atrophaeus (spores)

CO2 + H2 O2 (200 ppm)

275

40

240

>6.25

[67,68]

Bacillus cereus

Pressure cycling

205

34–60

36–240

1–8

[32,33]

Bacillus cereus (spores)

Pulse electric field pre-treatment

200

40

900

1.7

[34]

300

40

1440

4

Bacillus cereus (spores)

300

35

30–120

0.7–1.5

[39]

Bacillus coagulans (spores)

300

35

30–120

0.7–1.5

[39]

Bacillus licheniformis (spores)

300

35

30–120

0.7–1.5

[39]

CO2 + water CO2 + ethanol (70%) CO2 + IPA (70%) CO2 + H2 O2 (70–200 ppm)

275 275 275 275

50–80 40 40 40–60

240 240 240 240

0.6–3.0 0.3 0.2 4.0–6.3

[66]

Bacillus pumilus (spores)

CO2 + H2 O2

275

60

120–240

4.45–6.28

[62]

Bacillus pumilus (spores)

CO2 + H2 O2 and/or t-TBHP

100

50

45

4

[76]

500–2500

n.a.

20

6

[8]

Bacillus pumilus (spores)

Bacillus subtilis Bacillus subtilis

55.1

25

60

4.4

[15]

Bacillus subtilis Bacillus subtilis

58–74 75–150

38 35–45

2.5–30 10–75

>7 5.5–>7

[21] [40]

Bacillus subtilis

Grape must

85–110

40

5–60

1–4

[41]

Bacillus subtilis

Extra- and intra-cellular pH measurement

55–80

25–30

5

3.1–5.3

[23]

200

35

120

0.32

[10]

Static

74–200

40–54

30–90

0.9–1.1

[21]

Static Cycling semi-continuous

70 150

75 3–54

1,440 1–20

>7 0.8–3.5

Bacillus subtilis (endospores)

70–150

60

360

Total St.

[42]

Bacillus subtilis (endospores)

300

35

30–120

0.5

[39]

207

60

240

Total

[71,72]

207

50

3 ˇı 40

6.0–6.9

300

40

30

Bacillus subtilis (endospores) Bacillus subtilis (endospores)

Bacillus subtilis (endospores)

CO2 + acetic or peracetic acid CO2 + acetic Cortical bone

Spores of Bacillus cereus/subtilis/megaterium/

Water

1 CO2 + water fluid injection without filter

[43,45]

M. Perrut / J. of Supercritical Fluids 66 (2012) 359–371

363

Table 1 (Continued) Microorganisms

Materials

Fluid polymyxa/coagulans/circulans/ licheniformis/macerons

Bacillus stearothermophilus (endospores) Bacillus stearothermophilus (endospores)

Conditions

Reduction factor −log10 N/N0

Matrix

Pressure (bar)

Temp. (◦ C)

Time (min)

CO2 + water fluid injection through a filter (leading to “Micro-bubbles”)

300

40

30

4

300

60

30–60

4–6

75–207

60

120

1.0

200

35

120

CO2 CO2 + water + TFA

Ref.

[71,72,75]

6.4

Pure CO2 CO2 +2% ethanol CO2 +0.5% acetic acid

0

[9]

0.2 0.4

Geobacillus stearothermophilus (spores)

300

35–95

20–120

0.5–5

[39]

Candida utilisis

60–110

10–38

5

Almost total St.

[20]

Candida albicans

500–2500

20

8

[6]

Candida albicans

CO2 and N2 O

Venous catheters

100

40

10

5.5

[61]

Clostridium sporogenes (spores)

CO2

Acidic medium 2.5 < pH < 4.0

54

70

120

0.8–7.8

[12]

Clostridium thermocellum

N2 , Ethane, propane

70

60

–

Inactivation

[16]

18–70 18–70

60 60

– –

Kinetics

Beef

1–8

38

1080

Total

[3]

Buffer pH 6.4

75

40

30

> 6.4

[36]

500–2500

n.a.

20

6–8

[8]

40–200

20–35

0–120

3.9–5.1

[9]

40–70

2–40

15–120

0–5

[15]

55.1

25

60

2.85

150

35

60

7–8

[17]

Liquid food

250

35

30

Total St.

[43,45]

Buffer solution

310

42.5

15

7

[51]

Water

140–205

25–42

30–60

8

[32,33]

Escherichia coli

Buffer pH 6.4

75

40

30

> 6.0

[36]

Escherichia coli

CO2 -saturated orange juice

75–150

25

2–9

1.7–8.6

[37]

80–200

34

10

>8

[34]

70

20

15

Total St.

50–100

20–65

30–120

Total St.

[49]

Clostridia 3679 spores

Irradiation

Enterococcus faecium Escherichia coli Escherichia coli Escherichia coli

Pressure cycling or not

Escherichia coli Escherichia coli

Micro-bubble reactor

Escherichia coli Escherichia coli

Pressure cycling

Escherichia coli

Pulse electric field pre-treatment

Escherichia coli

CO2 + water

Cotton

CO2 + propanol CO2 + triclosan

Fabric

Escherichia coli

CO2 + water

[46–48]

Cotton 50% + polyester 50% Escherichia coli

Rice

Escherichia coli

CO2 and N2 O

1–100

40–60

30

0.5–8

[50]

Venous catheters

100

40

20

4.5–5.5

[61]

Staphylococcus aureus

Acrylic hydrogel

276

40

240

8

[62]

Kloeckera apiculata

Grape juice

75

40

30

Total

[36]

Kluyveromyces fragilis

Saline water

75–100

33

5

Almost total

[20]

Lactobacillus casei

Buffer pH 6.4

75

40

30

6.3

[36]

Lactobacillus spp.

Milk serum

75

40

15

2.7

[36]

364

M. Perrut / J. of Supercritical Fluids 66 (2012) 359–371

Table 1 (Continued) Microorganisms

Materials

Conditions

Reduction factor −log10 N/N0

Ref.

Matrix

Pressure (bar)

Temp. (◦ C)

Time (min)

Lactobacillus sp.

From fermented Kimchi

71

30

200

5

[59]

Lactobacillus plantarum

From fermented Kimchi

71

30

120

8

[60]

150

5–40

60

0–7

[17]

Fluid

Lactobacillus plantarum

150

35

n.a.

Total St.

[37]

POROCRIT

CO2 -saturated orange juice Orange juice

75

40

1

>8

[38]

Liquid

Green sake

250

35

30

Total St.

[44,45]

210

35

15

Total St.

[18]

150

25

n.a.

Total St.

[37]

205

40

90

4

[32,33]

61.8 137

35 35

120 120

7 0–8

[13]

210

35

14

Total St.

[19]

Lactobacillus plantarum ®

Lactobacillus brevis Leuconostoc dextranicum Leuconostoc mesenteroides Legionella dunifii

CO2 -saturated orange j. Pressure cycling

Listeria monocytogenes

Water Various food products

Listeria monocytogenes Listeria innocua

Pressure cycling

205

34

36

3–9

[32,33]

Micrococcus luteus

CO2 + water

50–100

20–65

30–120

Total St.

[49]

Rice

1–100

40–60

30

0.5–>5

[50]

Grape must

85–110

32–40

5–60

1–4

[41]

205

34

36

8

[32,33]

500–2500

n.a.

20

8

[8]

205

34–40

36–240

6–8

[32,33]

100

40

20–50

4.5–5

[61]

Pseudomonas fluorescens

55.1

25

60

3.5

[14]

Saccharomyces cerevisiae

70–250

25–35

5–30

2–6

[44]

Saccharomyces cerevisiae

200

35

120

6.3

[9]

Saccharomyces cerevisiae

75–100

33

5

Almost total

[20]

75

40

30

> 4.6

[36]

Cotton 50% + polyester 50% Penicillium sp. Pichia awry 1272 Proteus vulgaris

Pressure cycling

Pseudomonas aeruginosa Pseudomonas aeruginosa

Pressure cycling

Pseudomonas aeruginosa

CO2 and N2 O

Saccharomyces cerevisiae

Venous catheters

Buffer pH 6.4 Grape juice

Total St.

Saccharomyces cerevisiae

CO2 -saturated orange j.

150

25

n.a.

Total St.

[37]

Saccharomyces cerevisiae (ascospores)

CO2 -saturated orange j.

150

45

n.a.

Total St.

[37]

Saccharomyces cerevisiae

Peptonated sterile water

100

36

30

3

[24]

Saccharomyces cerevisiae

CO2 Orange juice

100–200

36

50

4.7

[55–57]

N2 O

100–200

36

30

4.7

Saccharomyces cerevisiae

CO2

40–150

40

–

Inactivation kinetics

[58]

Salmonella

Irradiation

Soybean meal

6

38

n.a.

n.a.

[3]

Various food products

137

35

120

0–8

[13]

Salmonella typhimurium (food)

M. Perrut / J. of Supercritical Fluids 66 (2012) 359–371

365

Table 1 (Continued) Microorganisms

Materials

Fluid Salmonella typhimurium

CO2 + peracetic acid

Salmonella salford

Pressure cycling

Conditions

Matrix

Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus

Buffer solution

Reduction factor −log10 N/N0

Ref.

Pressure (bar)

Temp. (◦ C)

Time (min)

100

35

60

>6

[75]

205

34–40

36–240

3–9

[32,33]

200

35

120

4.8

[9]

55.1

25

60

1.71

[14]

310

42.5

15

7

[51]

Staphylococcus aureus

Pressure cycling

205

34–40

36–240

3–9

[32,33]

Staphylococcus aureus

Pulse electric field pre-treatment

80–200

34

10

>8

[34]

Staphylococcus aureus

Liquid whole egg

100

40

30

>6.3

[52]

Staphylococcus aureus

CO2 + H2 O2

Ti-surfaces

276

20–40

n.a.

Almost total

[53]

Staphylococcus aureus

CO2 and N2 O

Venous catheters

100

40

20–50

4–5

[61]

Staphylococcus aureus

Acrylic hydrogel

276

40

60

8

[62]

Streptococcus spp.

Milk serum

75

40

15

3.9

[36]

Yeast (naturally present)

Orange juice

80–90

38

30–120

4.2–6.7

[54]

envelop, both for external and internal layers, which correlates with the high killing rate [65–70].

3.4. Virus inactivation Virus contamination is a main concern for all bio-products originated from human sources: blood/plasma fractions, organ/tissue implants, and even on r-DNA products prepared by biotechnology. As these products are very fragile to temperature, irradiation and chemical agents, virus inactivation at low temperature by supercritical fluid is of key interest. The first investigation was published by Stahl et al. [6,8] who found a weak inactivation (∼2 log10 ) of Coliphage virus by very high pressure CO2 (2500 bar). The same virus type (E. coli T1 to T5 phages) was claimed to be completely inactivated by CO2 at 300 bar and 50 ◦ C during a 30-min contact between the aqueous medium and the fluid dispersed in “micro-bubbles” [43]. In 1990s, two groups disclosed attractive results on plasma fractions processing. The first one [78–80] submitted all samples to supercritical N2 O treatment at a pressure of 250 bar and temperature between 37 and 50 ◦ C for 2 h. N2 O was preferred to CO2 as it does not lead to pH decrease and irreversible denaturation of the clotting factors – that are pH-sensitive proteins – caused by CO2 in aqueous medium. Viruses exhibiting various degrees of resistance to physicochemical treatments were chosen in different classes (Table 2). Supercritical N2 O was shown to be efficient against most viruses, but inactivation is higher for enveloped viruses, probably due to interaction with the lipids of this envelop, than for non-enveloped viruses. The second group [81–84] disclosed extended results on virus inactivation by using several near-critical/supercritical fluids and compressed nitrogen. Many results (Table 2) were obtained on murine-C retrovirus (MnLV) and on several viruses including several strains of HIV. From these data released to support patent claims, it appears that supercritical N2 O and fluorocarbons (R22 and R23) are good candidates as inactivation fluids, and N2 O added with CO2 (1000 ppm to 5% CO2 , v/v) appears as the “best fluid” for HIV-1 strains inactivation [84]. However, some viruses seem particularly

resistant and, as said before, inactivation appears more important for enveloped viruses than for non-enveloped ones. According to these authors, this may be related to a partial phospholipid solubilisation/liberation and envelop disruption. But, surprisingly, ethanol-added N2 O and other fluorinated fluids look less efficient although they are known to be good lipid solvents. Another group [85–89] developed a commercial process [89] for preparation of bone allografts. On pilot plant experiments (2litre autoclave), four viruses (HIV-1, Sindbis, Polio Sabin type I and Pseudorabies) were loaded on bone samples that were submitted to supercritical CO2 processing for lipid extraction at 250 bar and 50 ◦ C for 10 min per g of bone. The virus load was heavily reduced and remained below detection level for any virus type (reduction factor over 4 log) [85–88]. Obviously, in this case, no care was taken to preservation of bio-molecules as only the mineral matrix is further used. More recently, disinfection of various viruses (TGEV, PRV, JEV, PRRSV) spiked into a representative biomaterial (different cell lines) was completed by contact with CO2 @ 40–50 ◦ C, 160 bar for 45 min, and demonstrated that supercritical CO2 can effectively inactivate viruses in a heat-sensitive biomaterial without significantly decreasing bioactivity of the biomaterial [90]. Finally, fluid interactions with virus envelop seems to be the predominant cause of inactivation. This is supported by the example of the lipid-coated Sindbis virus that is extremely resistant to high hydrostatic treatment (up to 7000 bar) [91], while it is rapidly inactivated in presence of a supercritical fluid (pure CO2 or pure N2 O).

4. Sterilization processes and equipment 4.1. Sterile filtration In an early patent [92], solution of the material (API) in a supercritical fluid (CO2 , N2 O or R13) is submitted to a sterile filtration and the solid is recovered by rapid depressurization (RESS process). Similarly, in another patent [93], solution of a steroid compound in a liquid solvent is submitted to a sterile filtration and the solid is precipitated by addition of a supercritical anti-solvent. In fact, no

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Table 2 Viral inactivation during supercritical fluid processing. Virus

Envelop

Genome

Size (nm)

Fluid and conditions

Duration (min)

Reduction factor (log10 )

Ref.

Sindbis BVDV PRV Polio 1 Reo 3

+ + + – –

RNA RNA DNA RNA RNA

55–60 55–60 120–200 25–30 60–80

N2 O 250 bar, 37 ◦ C N2 O 250 bar, 37 ◦ C N2 O 250 bar, 37 ◦ C N2 O 250 bar, 37 ◦ C N2 O 250 bar, 37 ◦ C

90 <120 90 120 120

2.4a >6.3a >4.3a 3.1 2.0

[80]

Murine-C MnlV

+

RNA

n.a.

R22 41 ◦ C, 210 bar N2 O 22–60 ◦ C, 14–340 bar N2 O + EtOH 41 ◦ C, 210 bar N2 41 ◦ C, 21–224 bar Propane 41 ◦ C, 210 bar

5–60 1–120 30 30–60 30

3.2– > 3.6 1.0– > 5.5 1.1 0.2–0.7 1.4

[82,83]

Adeno Polio HAV Reo VSV VSV Sindbis TGE BVD EMC EMC EMC EMC

– – – – + + + + + – – – –

DNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA

70–90 18–26 24–30 65–75 60–180 60–180 60–70 80–130 60–70 20–30 20–30 20–30 20–30

R22, 50 ◦ C, 210 bar R22, 50 ◦ C, 210 bar R22, 50 ◦ C, 210 bar R22, 50 ◦ C, 210 bar R22, 50 ◦ C, 210 bar N2 O, 40 ◦ C, 345 bar R22, 50 ◦ C, 210 bar R22, 50 ◦ C, 210 bar R22, 50 ◦ C, 210 bar R22, 50 ◦ C, 210 bar R134a, 50 ◦ C, 210 bar R124, 50 ◦ C, 210 bar R23, 26–58 ◦ C, 7–345 bar

>5.1 4.1–4.2 1.0–1.3 0.9–1.0 >6.5 2.5–>5.5 6.5 >2.5 2.3 4.2–5.9 0.1–1.3 0.4–0.5 0–4.6

[82]

HIV

+

RNA

80–120

N2 O/CO2 various compositions 1000 ppm to 5% CO2 , v/v 22 ◦ C, 210 bar

2–3

[84]

a

1–30

Below detection limit.

specific sterilizing efficacy of the supercritical fluid was claimed in both patents. 4.2. Carbon dioxide sterilization and pasteurization Most recent work refers to the specific properties of carbon dioxide, possibly added with another agent (such as ethanol or oxidants as H2 O2 or peracetic acid) to kill microorganisms. CO2 is also preferred since it is safe, non-toxic, inexpensive, and abundant. N2 O which seems to have a similar lethal effect on most microorganisms shall be reserved to applications where CO2 acidity is deleterious to the processed material. According to the author’s own experience and confirmed by some other undisclosed reports, it must be emphasized that bacteria inactivation results are often disappointing when a “naturally contaminated” product is treated, and are always very different from those obtained when processing the same material spiked with a given strain, as realized in most university laboratories according to scientific practice. This may be due to several reasons: matrix effect causing difficult access of the fluid to microorganisms, and/or matrix compounds interacting with the inactivation process, and/or, more probably, the presence of spores that are much more difficult to inactivate than vegetative cells, as discussed here before. This fact shall lead to prudence when extrapolating laboratory results to “real” samples processing. When the feed to be processed is a solid powder (food products [50,94] or more often pharmaceutical products) or 3D-item (i.e. natural [71,85–89,95] or synthetic [61,62,74,96] implants), the contactor is a vessel preferably equipped with a quick closure system, and having a container (basket) to facilitate charging and discharging the product, similar to currently used SCF equipment. The time duration of the operation is controlled by diffusion into the material, depending on its porosity. In most cases, it seems that performing a continuous flow of fluid is much more efficient than only using a static contact, supporting the fact that mass transfer is a significant parameter [26]. One of the most acute difficulties is related to harvesting the processed material in sterile conditions especially

when it is a fine powder. The container holding such power (called “basket”) requires a specific design to avoid both powder emission and biological contamination. As presented in Fig. 1, the process flow-sheet is very similar to the one classically implemented for supercritical fluid extraction of solid materials (Fig. 1): Liquid CO2 stored in a reservoir is subcooled and pumped to required pressure and later heated to chosen temperature, with a possible injection of a sterilant additive; the fluid percolates through the material packed in a “basket” inserted in a cylindrical vessel closed by two filters (sintered disks); the fluid is then depressurised, reheated and sent to separators to collect liquid that can be carried in the fluid. After percolation through an adsorbent filter, the gas is vented to atmosphere or recycled to the reservoir after condensation. Moreover, and different from the extraction process, it is recommended to use a high flow rate circulation pump in order to improve the contact between fluid and processed material. In fact, sterilization (or decontamination) is often an added advantage during supercritical fluid processing for other purposes: Extraction from natural products (aromas, spices, nutraceuticals and phytopharmaceuticals), particle design and formulation of active pharmaceutical ingredients (APIs), bone delipidation for implants [85–89], hydrogel drying [62,64], polymer forming, etc. Liquid pasteurization/sterilization can be carried out in continuous mode with higher performance than those obtained in static contactors, according to many authors. Counter-current columns or agitated vessels (stirred or mixed by fluid recirculation) are claimed to be effective contactors, especially when fluid dispersion into the liquid is optimised – i.e. by a micro-bubble system [43–45,102] – which demonstrates the influence of mass transfer of the fluid inside the liquid. Moreover, much attention is to be paid to membrane contactors (Fig. 2 [37,38]) that are very attractive systems for juice pasteurization in continuous mode with high throughputs, although no commercial development is yet undertaken. Table 3 shows some applications on food products, some of which being described in detail with complete evaluation of the processed material characteristics in comparison with unprocessed

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367

Fig. 1. Supercritical fluid process flow sheet.

Fig. 2. Liquid sterilization on a membrane module.

Table 3 Supercritical fluid sterilization of food products. Material

Process

Agent

Grape juice Grape must Milk serum Milk Orange juice Orange juice Apple juice Kiwi juice Peach juice Fresh Kimchi (Chinese cabbage) Tomato sauce

Cont. flow Batch Cont. flow Cont. flow Cont. flow Cont. flow membrane Semi-continuous Semi-continuous Semi-continuous Batch and semi-continuous

CO2 CO2 CO2 CO2 CO2 CO2 CO2 and N2 O CO2 and N2 O CO2 and N2 O CO2 CO2

Tomato purée Liquid whole egg Plant and animal mater. Paprika powder Rice

Batch Batch Batch Batch Batch

CO2 and N2 O CO2 CO2 + N2 O CO2 + water CO2

Conditions

Ref.

Pressure (Bar)

Temp. (◦ C)

Time (min)

75 80–110 75 150 80–90 75 100–200 100 100 71 75–150 110 100 100 1–30 60 1–100

40 32–40 40 35–38 38 40 36 35 35 10 35–45 40 33–55 40 50–70 20–95 40–60

30 5–60 15 15 30–120 1 0–50 15 15 1440 10–175 10–75 120 30 2–6 10–120 30

[36] [41] [36] [97,98] [54] [37,38] [55,56] [57] [57] [59,60,100,101] [40,41] [99] [52] [35] [94] [50]

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Table 4 Supercritical fluid sterilization of biomedical and various products (n.a.: non available). Material

Process

Solids (pharmaceut.) Powdery substance Articles (medical, linen) Porcine plasma Powder

RESS Batch Batch Batch

Predsinolone acetate PLGA and PLA particles Textile (cotton fabric)

Anti-solvent Press. cycling Batch

Textile (50% cotton–50% polyester fabric) Ti-surfaces Bone allografts

Batch

Venous catheters Acrylic hydrogel Tissue engineering scaffolds Ligament Prostheses UHW Polyethylene

Batch Batch pressure cycling

Agent

Conditions

CO2 , N2 O, R13 CO2 CO2 + addit., NH3 , CFCs CO2 + ethanol/acetic acid CO2 CO2 CO2 + water Water/propanol/triclosan CO2 + water

Batch Batch Batch

CO2 + H2 O2 CO2 + additives (acetic or peracetic or trifluoroacetic acid) CO2 CO2 CO2 + H2 O2

Batch Batch

CO2 CO2 + Ethanol/H2 O2

one, including meat [104], tomato sauce and purée [41,99], orange juice [54,103], apple juice [56], peach and kiwi juices [57]. Table 4 presents some applications on various items, with a specific interest on health materials: Drugs and biomedical systems (allografts, implants, etc.), and textiles. As sterilization equipment using a supercritical fluid process is specific to each application, there is no “generic” machinery on the market. Conversely, the main supercritical fluid equipment suppliers probably have capability to design and build pasteurization or sterilization equipment in cooperation with users who have experience in aseptic processing (clean rooms, laminar hoods, etc.) and handling/cleaning procedures [105]. The equipment design parameters can be listed as follows:

Ref.

Pressure (Bar)

Temp. (◦ C)

Time (min)

100–900 100–300 30–330 200

30–50 30–40 8–380 35

5–30 30–120 20

[92] [7] [11] [10]

205 70

25 20

60 15

[93] [32,33] [46–48]

20–65

30–120

[49]

276 207

20–40 50

n.a. 135

[53] [71]

100 276 276

40 40 40

50 60 240

[61] [62,64] [63]

170

37

140

[95] [96]

50–100

an “acceptable” loss of bio-activity although, in these conditions, the reduction factors are not high enough for all types of viruses. This means that significant work for process optimization (fluid nature, operating conditions, stabilizing agents composition) must be completed prior to considering this inactivation process as reliable for clinical products. As N2 O is recommended for such processing, possibly added with CO2 [84] or other additives, it is noticed that this fluid is hazardous and shall only be used with non-flammable products as it may behave as a strong comburant. 5. Other applications 5.1. Pest control

• Maximum operating temperature: 80 ◦ C. • Maximum pressure: 150–200 bar; with pressure cycling with a P of 100 bar. • Contact optimization: fast fluid recirculation on solids, fluid dispersion or membrane contactor for liquids in order to reduce exposure time. • Water and co-agent addition system. For the long term, it might be valuable to consider combination with a pulse electric field pre-treatment that renders cell membranes more fragile and facilitate inactivation by CO2 .

It was proved that CO2 pressure of 10–50 bar is sufficient to kill insect eggs, larva or beetles after exposure for 10–20 min; this strong effect may be connected with gas action as a respiratory analeptic [8]. A recent study [50] confirmed that the most common insects and their eggs present in rice (Sitophilus oryzae L. and Oryzaephilus surinamensis L.) can be completely eradicated by CO2 at relatively low pressure conditions (25 bar). This process is used at very large scale to treat with low pressure CO2 huge amounts of overseas supply of food products (like rice) and medicinal plants often contaminated with insects.

4.3. Virus inactivation

5.2. Inactivation for immunogenic preparations

From the presently available results, it clearly appears that virus inactivation can be operated using subcritical, near-critical or supercritical fluids:

As supercritical fluid inactivation of microorganisms is known to cause limited damages to vegetative cells and viruses, and in certain “mild” conditions to proteins present in the membrane or envelop, this technique can be envisaged for obtaining immunogenic preparation that might be used for vaccine manufacture. A recent patent [72] claims the use of near-critical or supercritical carbon dioxide for inactivating whole microorganisms, as exemplified by inactivation of Salmonella typhimurium, the causative agent of typhoid fever in human, by supercritical CO2 (103 bar, 35 ◦ C, 15 min): a positive immunologic response is expected by the inventors although not yet evidenced. A second patent [84] describes the preparation of vaccines based on virus inactivated by contact with a supercritical fluid, especially N2 O

- When there is no risk of protein degradation, it is easy to “strongly strike” and to meet the required inactivation factors; CO2 is probably the best choice. This is presently operated for bone implants during lipid extraction: A commercial plant was set to process 2kg lots of femoral head pieces by supercritical CO2 at 250 bar and 50 ◦ C during 24 h [89]; initially, bovine bones were processed now replaced by human ones to comply with regulations forbidding animal-sourced implants. - In the case of very fragile bio-molecules that are often pHsensitive, it is possible to find operating conditions leading to

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loaded with 10–1000 ppm of CO2 that is claimed to inactivate HIV viruses of different strains: when injected in mice, these treated viruses lead to an immunogenic effect similar to the one observed by heat-inactivated viruses. However, it seems that, beyond these claims, a long effort is still required to prepare safe and reliable vaccines by this method, probably in combination with existing routes. 5.3. Elimination of endotoxins and pyrogens As far as prostheses implantation and parenteral drug administration are concerned, not only the material must be sterile, but also it should be free of endotoxins/pyrogens that induce undesired side-effects like fever and inflammation. These molecules are hydrophilic lipo-polysaccharides mainly originated from cell wall of Gram negative bacteria, and their elimination is very difficult, generally operated by contact with chemicals – generally in aqueous media – that often damage the treated substrate. Treatment by supercritical fluid is inefficient as confirmed by a recent work on elimination of endotoxin from E. coli from Ti surfaces as model of biomaterial. Pure CO2 (276 bar; 25 ◦ C; 120 min) does not permit endotoxin removal but, in similar conditions, a mixture of CO2 , surfactant and water, probably forming a water-in-CO2 micro-emulsion that “washes” the endotoxins, leads to a complete elimination [62]. 6. Conclusion While pasteurization, sterilization and virus inactivation are of growing importance in food, pharmaceutical and bio-medical industries, in relation with quality and safety improvement, supercritical fluid treatments look attractive because they permit to avoid heat processing and irradiation that cannot be used in an increasing number of cases. As reviewed in this paper, much fundamental work has been being done during the two last decades to describe interactions between supercritical fluids and microorganisms both in vegetative and latent forms in order to understand the sterilization effect and try to monitor efficient non-thermal sterilization processes. Knowing that bacteria inactivation of “naturally contaminated” material is sometimes very different from what could be expected from published results of lab experiments, it is difficult to raise firm conclusions on the efficacy of the proposed methods from these available results that are often conflicting. However, main lines can be drawn: • Pest (insects) elimination from food and medicinal plants by contacting with low-pressure carbon dioxide is a commercial process used at very large scale. • Liquid food products “pasteurization”, – like fruit juices – shall be soon operative for commercial scale applications, even at very large scale. • Solid food products “pasteurization” could be envisaged caseby-case, possibly in complement of other supercritical fluid processing (extraction, deodorisation, pollutants elimination, etc.). • Due to bacteria spore resistance to supercritical CO2 , strong oxidant additives (hydrogen peroxide or peracetic acid) seem required to reach a complete “sterilization”; although this induces limitations due to possible interactions with the processed material or product, many applications are foreseen in the near future on Health Science products: supercritical fluid sterilization of bio-medical items (like implants, prostheses or medical instruments) in order to prevent nosocomial infections is effective for advantageously replacing classical methods based

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