WO1998048856A1

WO1998048856A1 - Pressure-enhanced oxidative sterilization process

Info

Publication number: WO1998048856A1
Application number: PCT/US1998/008069
Authority: WO
Inventors: Robert E. Marquis
Original assignee: University Of Rochester
Priority date: 1997-04-29
Filing date: 1998-04-28
Publication date: 1998-11-05
Also published as: AU7253498A

Abstract

A process for sterilizing an item comprises subjecting the item concurrently to treatment with a composition comprising an inorganic or organic hydroperoxide and to an applied hydrostatic pressure of about 25 MPa to 250 MPa. Substantially all bacterial spores infecting the item are thereby killed. Preferably, the process is carried out at a temperature of about 20 °C to 45 °C, the pH of the hydroperoxide-containing composition is about 7, and the applied pressure is about 50 MPa to 200 MPa. The invention provides an effective and inexpensive method of sterilizing heat- and/or acid-sensitive items.

Description

PRESSURE-ENHANCED OXIDATIVE STERILIZATION PROCESS

Field of the Invention

This invention relates to oxidative sterilization of microorganisms and, more particularly, to a process for an oxidative sterilization process enhanced by hydrostatic pressure .

Background of the Invention

Hydrostatic pressure is being used more and more as a biocide, especially in the food industry to reduce bacterial numbers in a variety of foods and thereby extend shelf-life-. A major advantage of pressure preservation compared with, say, heat preservation, is that pressure-treated foods tend to retain natural flavors and quality. There is also growing interest in the use of hydrostatic pressure for general sterilization and disinfection. One example of this use of pressure is described in Jurkiewicz et al . , " Inactivation of simian immunodeficiency virus by hydrostatic pressure" in

Proceedings Of the National Academy of Science USA, 1995, vol. 92, pp 6935-6937.

The major barrier to widespread use of hydrostatic pressure for sterilization is the high pressure resistance of some bacterial spores. Currently, in the food industry pressure can be used only for disinfection, that is, reducing the concentration of microorganisms rather than substantially eliminating them. Even for the killing of vegetative microorganisms, which are typically more pressure-sensitive than spores, high pressures are required, for example, 4 kilobar (400 MPa) applied for up to an hour. As described in Gould and Sale, "Role of pressure in the stabilization and destabilization of bacterial spores" in Sleigh and MacDonald, editors, The Effects of Pressure on Organisms, Academic Press, New York, 1972, pp 147-157, the disclosure of which is incorporated herein by reference, lower pressures can induce germination of spores, and the germinated forms are then more sensitive than dormant forms to pressure killing. Also, Bender and Marquis, "Sensitivity of various salt forms of Bacillus megaterium spores to the germinating action of hydrostatic pressure" in Canadian Journal of Microbiology, 1982, vol.28, no. 6, pp 643-649, the disclosure of which is incorporated herein by reference, reports variations in the germinating effect of hydrostatic pressure, depending on the mineral content of the spores.

However, many spores are retractile to pressure germination, and attempts to kill spores in mixed populations by pressurization have met with only limited success. Roberts and Hoover, "Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity, and nisin" in Journal of Applied Bacteriology, 1996, vol. 81, pp 363-368, the disclosure of which is incorporated herein by reference, describes the combination of high pressure, heat, acidity, and other agents to destroy bacterial spores. Marquis, "Effects of hydrostatic pressure on bacterial acid sensitivity and adaptation" in Medsubhyp Int . , 1995, vol.5, pp 113-117, the disclosure of which is incorporated herein by reference, describes the acid- promoted killing of the vegitative microorganism Enterococcus hirae ATTC9790 at pressurization to 40 to 60 MPa.

Brief Description of the Figure

Fig. 1 graphically depicts the effect of hydrostatic pressure on the killing of B . subtilis subsp . niger spores at 25°C.

Summary of the Invention

In accordance with the present invention, a process for sterilizing an item comprises subjecting the item concurrently to a composition comprising an inorganic or organic hydroperoxide and to an applied hydrostatic pressure of about 25 MPa to 250 MPa. Substantially all bacterial spores infecting the item are thereby killed. The process is preferably carried out at a temperature of about 20°C to 45°C, with a pH of the hydroperoxide-containing composition of about 3 to 8 , and an applied pressure of about 50 MPa to 200 MPa. The present invention provides an effective and inexpensive method of sterilizing heat- and/or acid-sensitive items.

Detailed Description of the Invention

The sterilization process of the present invention comprises subjecting an item concurrently to treatment with a composition comprising an inorganic or organic hydroperoxide and to an applied hydrostatic pressure of about 25 MPa to 250 MPa, preferably about 50 MPa to 200 MPa. In a more preferred embodiment, the applied pressure is about 100 MPa to 150 MPa. The composition comprises a hydroperoxide selected from the group consisting of hydrogen peroxide and tertiary butyl hydroperoxide, preferably at a concentration of about 3 mM to 3 M, more preferably, at a concentration of about 30 mM to 1 M. In especially preferred embodiments, the composition has a pH of about 3 to 8 and comprises hydrogen peroxide at a concentration of about 30 mM to 1 M or tertiary butyl hydroperoxide at a concentration of about 70 mM to 1 M. Most preferably, the pH of the composition is about 7. In a particularly preferred embodiment of the process of the invention, the composition has a pH of about 7 and comprises hydrogen peroxide at a concentration of about 30 mM to 1 M; the applied hydrostatic pressure is about 50 MPa to 100 MPa. The process is preferably carried out at a temperature of about 25°C to 30°C for a time period of about 1 hour to 4 hours . The following examples further illustrate the invention :

Example 1 -- Spore preparation and sporicidal assays Spores of Bacillus subtilis subsp . niger and

B . mega terium ATCC19213 were prepared as described previously in Bender and Marquis, "Spore heat resistance and specific mineralization" in Applied and Environmental Microbiology, 1985, vol. 50, pp 1414-1421, the disclosure of which is incorporated herein by reference. The spores were washed free of vegetative debris by means of differential centrifugation, and the cleaned, pelleted spores were stored under 95% ethanol until needed.

For killing experiments, a spore suspension in water was prepared so as to provide an initial count of between 10⁷ and 10⁸ colony-forming units per ml. Following addition of the test agents to the spore suspension, part of the resulting suspension was incubated at 0.1 MPa, and part was pressurized using procedures and pressure chambers described previously in Marquis, "High-pressure microbial physiology" in Advances in Microbial Physiology, Academic Press, New York, 1976, Vol. 14, pp 163-167, the disclosure of which is incorporated herein by reference. At intervals, the pressurized suspension was decompressed and sampled. The unpressurized suspension was sampled at the same time. The samples were immediately diluted 1:10 in 1 percent Difco-peptone broth (available from Difco Laboratories, Detroit MI) to stop hydroperoxide action. A series of 1:10 dilutions in peptone broth was then prepared, and 0.1-ml samples were spread plated on trypticase-soy agar plates.

The plates were incubated at 37°C until colony formation was complete, and numbers of colonies were counted directly.

H₂0₂ was obtained from Sigma Chemical Co. (St. Louis, MO) as a stabilized 30 weight percent solution and appropriately diluted with water at the time of use.

Peracetic acid was obtained as a 32 weight percent solution from Aldrich Chemical Co. (Milwaukee, I) and was diluted and neutralized with KOH solution at the time of use. Tertiary butyl hydroperoxide (t-BOOH) was obtained from Arco Specialty Chemicals (Philadelphia, PA) as a 70 weight percent solution in water. All other chemicals were from Sigma Chemical Co.

Example 2 -- Pressure enhancement of the sporicidal action of H₂0₂

As shown by the data presented in Fig. 1., a 3 weight percent (ca. 978 mM) solution of H₂0₂ only slightly reduced numbers of spores of B . subtilis subsp. niger at 0.1 MPa but nearly completely sterilized suspensions at 100 MPa after 2 to 3 hours of exposure. Samples taken after 3 hours contained no viable cells. At 100 MPa, the spores were not killed in the absence of H₂0₂, nor were there signs of germination when the spores were examined by phase microscopy. For these tests, the spores were suspended in water at pH 7. However, if they were suspended in 1 percent Difco peptone broth at pH 7, the same enhancement of killing was observed. Estimation of the volume of activation (ΔV#) for pressure enhancement of spore killing by 100 MPa pressure over the interval from 1 hour to 2.3 hours indicated a value of some -45 ml/mol. Pressurization to 50 MPa also accelerated the rate of killing, although the increase was not as great as at 100 MPa. The calculated activation volume was -61 ml/mol.

Example 3 -- Pressure effects on sporicidal actions of various oxidative agents

Table 1 presents summary results from a series of tests using a variety of oxidative agents with spores of B . megaterium ATCC19213 and B . subtilis subsp. niger . The latter microorganism is reported to be more resistant than the former to killing by t-BOOH (cf. Marquis et al . ,

"Molecular mechanisms of resistance to heat and oxidative damage" in Journal of Applied Bacteriology Symposium Supplement , 1994, vol. 74, pp 40S-48S) . Large negative values of Δ V^# indicate pressure enhancement of spore destruction, while Δ V^# values near zero indicate that applied pressure has little effect on the spore killing rate. Pressure enhancement of sporicidal potency was observed for solutions containing H₂0₂ as well as for those containing the organic hydroperoxide t-BOOH. However, there was essentially no effect of pressure on spore killing by peracetic acid or by sodium hypochlorite , which is considered to act oxidatively (cf. Bloomfield and Arthur, "Mechanisms of inactivation and resistance of spores to chemical biocides" in Journal of Applied Bacteriology Symposium Supplement, 1994, vol. 76, pp 91S-104S; Dukan and Touati, "Hypochlorous acid stress in Escherichia col i : Resistance, DNA damage, and comparison with hydrogen peroxide stress" in Journal of Bacteriology, 1996, vol. 178, pp 6145-6150) . The data in Table 1 show also that pressure enhancement of spore killing occurs at a higher temperature of 45°C as well as at 25°C.

The data presented in Table 1 demonstrate that hydrostatic pressure can be used in conjunction with H₂0₂ az a pH of 7 to achieve sterilization of spore suspensions, including suspensions of spores of B . subtilis subsp. niger, which have relatively high resistance to killing by H₂0₂, as reported in Shin et al . , "Microscopic and thermal characterization of hydrogen peroxide killing and lysis of spores and protection by transition metal ions, chelators, and antioxidants" in Applied and Environmental Microbiology, 1994, vol. 60, pp 3192-3197, the disclosure of which is incorporated herein by reference. Pressurization alone to 100 MPa was not lethal, and the enhancing effect of pressure was observed with H₂0₂ and t-BOOH but not with peracetic acid or sodium hypochlorite. Significant levels of germination in the spore populations would be expected to result in increased sensitivity to all of the agents and not just H₂0₂ and t-BOOH. In fact, if pressure acted to reduce barriers to entry of the agents, such as those posed by the coats-outer-membrane complex, then enhancement of peracetic acid or hypochlorite action should have been observed, since decoating of spores enhances sensitivity to these agents (cf. Marquis et al . , "Sporicidal action of peracetic acid and protective effects of transition metal ions" in Journal of Industrial Microbiology, 1995, vol. 15, pp 486-492) . Thus, increased hydrostatic pressure, like increased temperature, enhances hydroperoxide-promoted spore killing. The sporicidal activity of H₂0₂ and t-BOOH increases substantially with increasing temperature, while the activity of peracetic acid and sodium hypochlorite is only slightly increased at higher temperatures (cf. Marquis et al . 1995) . At the higher temperature of 45°C, at which both H₂0₂ and t-BOOH are more effective sporicides than at 25°C, pressure still acted to enhance killing, and complete sterilization could be achieved in only a few hours with 0.1 weight percent (ca. 33 mM) H₂0₂ or 72 mM t-BOOH. Although the applicant is not to be bound by the following proposed interpretation of the observed results, enzymes with high degrees of sensitivity to oxidative damage may be the major targets for sporicidal action of H₂0₂, as reported in Palop et al . , "Hydroperoxide inactivation of enzymes within spores of Bacillus mega terium ATCC19213" in FEMS Microbiology Letters, 1996, vol. 142, pp 283-287, the disclosure of which is incorporated herein by reference. Pressure may act to enhance sensitivity either by increasing exposure of sensitive sites through molecular rearrangements of the enzymes or by facilitating localized radical production, possibly by weakening electrostatic binding of transition metal cations to specific anionic sites. The values for activation volumes for pressure enhancement of killing are of intermediate magnitude, being too large to be accounted for by breakage of single bonds but not so large as to indicate highly cooperative molecular phenomena. Whatever the precise mode of action, relatively low pressurization, in accordance with the present invention, substantially enhances the sporicidal action of hydroperoxides , particularly H₂0₂, provides at ambient or slightly elevated temperatures an inexpensive, highly effective, environmental benign process for the sterilization of heat- and/or acid- sensitive materials.

Table 1 Effect of hydrostatic pressure on sporicidal activities of oxidative chemicals

"Apparent activation volumes were calculated by use of the equation ΔV^#=2.3 (RT/P) log (D_p/D_{01 MPa}) , where ΔV^# is the apparent activation volume, R is the gas constant, T is the Kelvin temperature, P is the pressure, D_p is the D value for killing 90 percent of the population under the applied pressure, and D_{0 MPa} is the corresponding D value at 0.1 MPa. All tests were carried out at pH 7 and at a temperature of 25°C, except where otherwise indicated. ^bValues are each averages of two experiments carried out at separate times but with the same spore suspension . °The temperature of the experiment was 45°C instead of 25°C.

The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims

What Is Claimed Is:

1. A process for sterilizing an item, said process comprising: subjecting the item concurrently to treatment with a composition comprising an inorganic or organic hydroperoxide and to applied hydrostatic pressure of about 25 MPa to 250 MPa, thereby killing substantially all bacterial spores infecting said item.

2. The process of claim 1 carried out at a temperature of about 20┬░C to 45┬░C.

3. The process of claim 2 wherein said temperature is about 25┬░C to 30┬░C.

4. The process of claim 1 wherein said composition has a pH of about 3 to 8.

5. The process of claim 4 wherein said pH is about 7.

6. The process of claim 1 wherein said applied hydrostatic pressure is about 50 MPa to 200 MPa.

7. The process of claim 6 wherein said applied hydrostatic pressure is about 100 MPa to 150 MPa.

8. The process of claim 1 wherein said inorganic or organic hydroperoxide is selected from the group consisting of hydrogen peroxide and tertiary butyl hydroperoxide .

9. The process of claim 8 wherein said inorganic or organic hydroperoxide is present in said composition at a concentration of about 3 mM to 3 M.

10. The process of claim 9 wherein said concentration of inorganic or organic hydroperoxide is aboui 30 mM to 1 M.

11. The process of claim 10 wherein said composition comprises hydrogen peroxide at a concentration of about 30 mM to 1 M.

12. The process of claim 10 wherein said composition comprises tertiary butyl hydroperoxide at a concentration of about 70 mM to 1 M.

13. The process of claim 1 wherein said composition comprises hydrogen peroxide at a concentration of about 30 mM to 1 M and has a pH of about 7, and said applied hydrostatic pressure is about 50 MPa to 100 MPa.

14. The process of claim 13 carried out at a temperature of about 25┬░C to 30┬░C.

15. The process of claim 14 carried out for a time period of about 1 hour to 4 hours.