Improvements in Methods of Sterilization

This invention relates to a method of sterilisation in which a microorganism is rendered non-viable.

At present food packaging is sterilised by treatment with a solution of hydrogen peroxide. Certain strains of microoganisms are however resistant to such treatment and a small, though significant, number of spores survive with consequent risk of spoilage to the contents.

A method of sterilisation has now been found which reduces the number of surviving spores of resistant organisms more efficiently.

According to the present invention a method of sterilisation comprises treating a microorganism with an ultraviolet irradiated solution of hydrogen peroxide the wavelength of the radiation being wholly or predominantly below 325 nm and the concentration of the hydrogen peroxide solution being no greater than 10% by weight and such that the microorganism is rendered non-viable by synergism between the radiation and hydrogen peroxide.

The concentration of the solution of hydrogen peroxide, which is usually aqueous, generally does not exceed 6% by weight and preferably does not exceed 3.0% by weight. The concentration is normally at least 0.01% by weight, a concentration at least 0.25% by weight being preferred especially when the microorganism is present in the form of spores, and a concentration at least 0.5% especially so.

In general the wavelength of the ultraviolet radiation is wholly or predominantly below 300 nm and is usually at least 200 nm. In practice radiation is normally provided from a source having a peak intensity below 325 nm, particularly at 254 nm. The energy of the radiation emitted at the source is normally at least 300 microwatts/cm.sup.2, particularly at least 500 microwatts/cm.sup.2. Although the intensity of the radiation with which the solution is treated may be established by simple experiment, it is normally at least 75 and preferably at least 150 microwatts per cm.sup.2.

Although spores of many microorganisms are destroyed by treatment as hereinbefore described at ambient temperatures, it may be desirable, particularly when treating especially resistant microorganisms, to maintain the solution at an elevated temperature either during irradiation or subsequent thereto. In general, such temperatures do not exceed 120.degree. C. and may not exceed 100.degree.. The temperature, at least when treating resistant organisms, is usually however at least 85.degree. C.

The method of the present invention is applicable to a wide variety of microorganisms, including moulds, yeasts, bacteria, viruses and protozoa and finds particular application in the destruction of spore-forming bacteria, especially those which are dairy contaminants.

Although microorganisms in vegetative form may be treated, the present invention is of particular interest for the destruction of spores, especially those of resistant Bacillus and Clostridium strains, e.g. strains of B. subtilis and B. stearothermophilus such as B. subtilis (ATCC 9372) and B. stearothermophilus (NCDO 1096)

In genral, the irradiation period increases with the resistance of the organism, at least 10 sec. generally being required. Heating of the solution, which may, as hereinbefore indicated, be conducted synchronously with irradiation or subsequent thereto is normally carried out for at least 10 sec. and periods of at least 30 sec., e.g. 60 sec. or more may be desirable.

Although the present method may be applied to sterilisation of liquids, e.g. waste water and cannery cooling water, it is of particular interest for the sterilisation of surfaces, for example surfaces of walls and furniture in hospitals and the surfaces of food containers. The latter surfaces may be treated with the peroxide solution, for example by passing the container or material from which the container is fabricated through a tank containing the solution or by spraying the walls of the container or the material with solution. Irradiation may be carried out by a lamp so disposed that containers or packaging materials which have emerged from the tank or spray are subjected to the method of the present invention.

The synergistic action of the present method on the microorganisms i.e. the action of the radiation and hydrogen peroxide additional to their purely additive effect is illustrated by the following Examples when taken with the comparison experiments.

The strains of Bacillus and Clostridium (Examples 1-40), of non-sporing bacteria (Examples 41-69) and of moulds (Examples 70-71) and where appropriate the origins thereof are listed in Table 1.

The following abbreviations apply:

NIRD--National Institute of Research in Dairying, Shinfield, Reading, UK.

NCDO--National Collection of Dairy Organisms

ATCC--American Type Culture Collection

NCIB--National Collection of Industrial Bacteria

FRI--Argicultural Research Council Food Reserach Institute, Colney Lane, Norwich, UK.

C. sporogenes is maintained in Robertson's cooked meat medium; the other strains used are maintained on slopes of Oxoid nutrient agar. Spores are produced by growth on one of the following: (i) the potato agar of Gould, Stubbs & King (1970) for B. subtilis var. niger (ii) Oxoid nutrient agar for B. stearothermophilus 202, (iii) the agar medium of Wang, Scharer & Humphrey (1964) for B. licheniformis 109 2AO (iv) a medium containing (g/l), Oxoid nutrient broth No. 2 (3.1), MnSO.sub.4.4H.sub.2 O (0.03), K.sub.2 HPO.sub.4 (0.25) and New Zealand Agar (15) for strain G12, (v) a medium containing (g/l); Oxoid skim milk powder (50), Difco yeast extract (3.0), Oxoid peptone (5.0), BBL trypticase (5.0), MnCl.sub.2.4H.sub.2 O (0.072) BDH cysteine hydrochloride (0.5), Davis agar (15); the medium is adjusted to a final pH of 7.0 to 7.2 with 1 M-NaOH and spores produced under H.sub.2 :CO.sub.2 (9:1) for C. sporogenes PA 3679, (vi) Bacillus spore agar as described by Franklin et al (1970) with Oxoid Lab Lemco added at 0.1% (w/v) for all other strains. Growth is at 30.degree. C. for B. subtilis var. niger, B. subtilis 738, B. globigii B17, strain G12, B. cereus T and B. cereus 818, at 33.degree. C. for C. sporogenes PA 3679, at 37.degree. C. for B. pumilus 312, B. subtilis SA22, B. subtilis 713, B. subtilis 706, B. licheniformis 100, B. licheniforms 117 and B. licheniformis 109 2AO or at 55.degree. C. for B. stearothermophilus 202.

Spores of B. stearothermophilus 202 are produced on 30 ml slopes in 100 ml screw-capped bottles; those of other strains on agar in Petri dishes. Sporulation is detected by the appearance of bright spores by phase contrast microscopy and incubation continued until the highest percentage of free spores is observed (after one to nine days, depending on strain). The cultures are harvested and washed 5 times with sterile glass-distilled water before storage at -18.degree. C.

Non-sporing bacteria are grown at 33.degree. C. in (i) heart infusion broth for E. coli and S. faecalis (ii) glycerol-salts medium as described by Dimmick (1965) for S. marcescens on a gyratory shaker (Gallenkamp, 150 rev/min) for 18 hours. The cells are harvested and washed with 50 ml sterile 25 mM-potassium phosphate buffer pH 7.0 by centrifugation before resuspension at a density of about 1.times.10.sup.9 viable units/ml and were used within 4 hours. Cultures are maintained on slopes of Heart Infusion Agar (Difco).

Spores of Penicillium chrysogenum are grown on slopes of Czapek Dox Agar containing (g/l); sucrose (30), K.sub.2 HPO.sub.4 (1.0), NaNO.sub.3 (2.0), MgSO.sub.4.7H.sub.2 O (0.5), KCl (0.5) FeSO.sub.4.7H.sub.2 O (0.01), and New Zealand Agar (20) at 20.degree. C. for at least 7 days before harvesting in sterile glass distilled water containing 0.1% Tween 80 and washing by centrifuging with sterile glass distilled water. Cultures were also maintained on Czapek Dox Agar.

Bacterial spores or vegetative cells at 3.times.10.sup.7 to 5.times.10.sup.8 /ml and mould spores at 1.times.10.sup.6 ml are suspended in 0.1 M sodium phosphate buffer pH 7.0 and up to 2.5 g/100 ml H.sub.2 O.sub.2 (Analar, B.D.H.). Volumes of 4 ml are rocked gently to and fro for 30 sec. at 20.degree. C. in a Petri dish base with a diameter of 9 cm at distances of 5.5, 30 cm or 33 cm from an Hanovia Chromatolite low-pressure Hg lamp (Hanovia Ltd., Slough, England) from which the filter is removed as described by W. M. Waites & B. A. Fry J. Gen. Microbiol 34, 413-426 (1964). The lamp emits radiation in the range 185-579 nm with a peak intensity at 254 nm; the latter radiation is the only radiation of significant practical value produced by the lamp, the other spectral lines and groups being of much lower intensity. The energy emitted is 300-500 microwatts/sq. cm. at source. Samples (2 ml) are removed and mixed with a filter sterilised solution (2 ml) of catalase (Sigma Ltd) at 7650 units/ml at 20.degree. C. before storing in ice for at least 5 min., dilution and plating.

Samples (2 ml) are heated to 85.degree. C. in 60 sec., (bacterial spores), 65.degree. C. in 30 sec. (vegetative bacteria) or 54.degree. in 15 sec. (mould spores) by adding to pre-heated screw-capped bottles before adding 2 ml of catalase pre-cooled to +1.degree. C. and containing 7650 units/ml and plunging into ice. The suspensions are stored in ice for at least 5 min. before diluting and plating.

Diluted spore suspensions are plated on (i) Bacillus spore agar and incubated for 2 days at 30.degree. C. (B. subtilis 713, B. subtilis 706, B. cereus T and B. cereus 818) or 37.degree. C. (B. licheniformis 109 2AO, B. licheniformis 100, B. licheniformis 117 and B. pumilus 312); (ii) plate count agar (Oxoid) and incubated for 2 days at 33.degree. C. (B. subtilis SA 22) or 2 days at 37.degree. C. (B. globigii B17, B. subtilis 738 and B. subtilis var. niger); (iii) a medium containing (g/l); tryptone (Oxoid) (5): yeast extract (Oxoid) (2.5), glucose (10) and New Zealand Agar (28) and incubated for 3 days at 55.degree. C. (B. stearothermophilis 202) (iv) a medium containing (g/l): tryptone (Oxoid) (10), glucose (5.0) and New Zealand Agar (12) and incubated for 2 days at 30.degree. C. (G12) or (v) the reinforced Clostridial medium agar of Hirsch & Grinsted (1954) but with 1.6% New Zealand Agar in place of 1.2% (C. sporogenses PA 3679).

Suspensions of vegetative cells are enumerated by diluting 100-fold in maintenance medium (Difco Bacto-peptone), 1.0 g/l; NaCl, 5.0 g/l; adjusted to pH 7.0-7.1) and plating on Heart Infusion Agar (Difco) (E. coli and S. faecalis) or Trypticase Soy agar (BBL) (S. marcescens) using a Spiral Plate Maker (Spiral Systems, Cincinnati, Ohio, USA) (Gilchrist et al 1973, Jarvis et al 1977) and colonies counted after 3 days incubation at 33.degree. C. (S. faecalis), 2 days incubation at 30.degree. C. (E. coli) or 33.degree. C. (S. marcescens).

Suspension of mould spores are enumerated by diluting in sterile glass distilled water, plating on Czapek Dox Agar and colonies counted after 9 days incubation at 20.degree. C.

Full references to the publications hereinbefore cited are as follows:

Dimmick R. C. (1965) J. Bacteriology 89, 791-798. Gilchrist, J. E., Campbell, J. E. Donnelly, C. B., Peeler, J. T. & Delaney, J. M. (1973) Applied Microbiol. 25, 244-252

Jarvis, B. Lach, V. H. & Wood J. (1977) J. Applied Bacteriol 43/, 149-157

Gould G. W., Stubbs, J. M. & King W. L. (1970) J. General Microbiology 60, 347-355.

Wang, D. I. C. Scharer, J. G. & Humphrey A. E. (1964) Applied Microbiol 12 451-454.

Franklin J. G., Underwood, H. M., Perkin A. G. and Burton, H. (1970) J. Dairy Res. 37, 219-226. Hirsch, A. Grinsted, E. (1954) J. Dairy Res. 21, 101-110

Spores of Bacillus subtilis 706 are irradiated as described above but in the presence or, for purposes of comparison, in the absence of a concentration of H.sub.2 O.sub.2 (1 g/100 ml) which was previously found not to kill spores at comparable temperatures. In the absence of peroxide, irradiation produces a logarithmic rate of kill which results in 19 and 2% survivors after 30 and 60 sec. respectively. In the presence of peroxide, irradiation produces a much more rapid logarithmic kill so that after 30 sec. only 0.01% of spores survive.

Several strains of Bacillus and Clostridium are irradiated for 30 sec. In the presence of 2.5 g/100 ml H.sub.2 O.sub.2 as described above followed by heating to 85.degree. C. during 60 sec. The results are shown in Table 2 which also shows the results obtained from irradiation with U.V. alone and with U.V. followed by heat treatment.

B. subtilis 713 is irradiated with U.V. in the presence of varying concentrations of hydrogen peroxide at two different spore concentrations. The conditions and results are shown in Table 3.

Four Bacillus strains are irradiated with U.V. in the presence of hydrogen peroxide and in two cases are subsequently heated to 85.degree. C. over 60 sec. The conditions and results are shown in Table 4 which also shows for comparison purposes the results obtained from prior work using H.sub.2 O.sub.2 without U.V. irradiation.

Spores of B. subtilis 706 are irradiated with U.V. for 60 sec. in the presence of H.sub.2 O.sub.2 (1 g/100 ml). The U.V. lamp is located 30 cm above the spore suspension. For comparison purposes the strain is also irradiated with U.V. in the absence of H.sub.2 O.sub.2. Samples are removed diluted and plated as hereinbefore described. The results are shown in Table 5.

Spores of B. pumilus 312 are irradiated with U.V. for 30 sec. in the presence of H.sub.2 O.sub.2 both with and without subsequent heating to 85.degree. C. over 60 sec. The lamp is 5 cm above the suspension. For comparison purposes spores are incubated: (i) at 20.degree. C. with hydrogen peroxide for 30 sec., (ii) with hydrogen peroxide for 30 sec. followed by heating to 85.degree. C. over 60 sec. Samples are removed at intervals, diluted and plated as hereinbefore described. The results are shown in Table 6.

The procedure of Examples 30-35 is repeated using B. subtilus 713 in place of B. pumilus 312. The results are shown in Table 7.

E. coli K-12 is irradiated with U.V. for 30 sec. in the presence of varying concentrations of hydrogen peroxide. The lamp is 30 cm above the suspension. For comparison purposes cells are also incubated for 30 sec. with 1 g/100 ml H.sub.2 O.sub.2 in the absence of U.V. irradiation. Samples are removed, diluted and plated as hereinbefore described. The results are shown in Table 8.

S. faecalis ss liquefaciens EB/F/30/39 is irradiated with U.V. for 30 sec. in the presence of varying concentrations of hydrogen peroxide. The lamp is 30 cm above the suspension. For comparison purposes cells are also incubated for 30 sec with 1.0 g/100 ml H.sub.2 O.sub.2 in the absence of U.V. irradiation. Samples are removed diluted and plated as hereinbefore described. The results are shown in Table 9.

Cells of E. coli K12 are irradiated with U.V. for 30 sec. in the presence of 1.0 g/100 ml H.sub.2 O.sub.2 both with and without subsequent heating to 65.degree. C. over 30 sec. The lamp is 30 cm above the suspension. For comparison purposes cells are (i) irradiated with U.V. for 30 sec. followed by incubation with H.sub.2 O.sub.2 for 30 sec. (ii) heated to 65.degree. C. over 30 sec. Samples are removed, diluted and plated as hereinbefore described. The results are shown in Table 10.

Cells of S. marcescens are irradiated with U.V. for 30 sec. in the presence of varying concentrations of hydrogen peroxide. The lamp is 33 cm above the suspension. For comparison purposes cells are also incubated for 30 sec. with 1.0 g/100 ml H.sub.2 O.sub.2 in the absence of U.V. irradiation. Samples are removed, diluted and plated as hereinbefore described. The results are shown in Table 11.

Spores of Penicillium chrysogenum are irradiated with U.V. for 30 sec. in the presence of 0.5 g/100 ml H.sub.2 O.sub.2 both with and without subsequent heating to 54.degree. C. over 15 sec. The lamp is 5 cm above the suspension. For comparison purposes spores are (i) irradiated with U.V. for 30 sec. followed by incubation with H.sub.2 O.sub.2 for 30 sec. (ii) heated to 54.degree. C. over 15 sec. Samples are removed diluted and plated as hereinbefore described. The results are shown in Table 12.