At present food packaging is sterilised by treatment with a solution of hydrogen peroxide. Certain strains of microorganisms 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 3-25nm 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 300nm and is usually at least 200nm. In practice radiation is normally provided from a source having a peak intensity below 325nm, particularly at 254nm. The energy of the radiation emitted at the source is normally at least 300

• 2 " 2 microwatts/cm , particularly at least 500 microwatts/cm . Although the intensity of the radiation with which the solution is treated may be established by simple experiment, it is normally at least

2 75 and preferably at least 150 microwatters per cm .

OMH

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 C and may not exceed 100 . The temperature, at least when treating resistant organisms, is usually however at. least 85 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 general, 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

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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 micro¬ organisms i.e. the action of the radiation and hydrogen peroxide attitional to their purely additive effect is illustrated by the following Examples when taken with the comparison experiments. EXAMPLES 1 - 71 Organisms

The strains of Bacillus and Clostridium (Examples 1 - 40) , of non-sporing bacteria (Examples 4l - 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. NCD0 - National Collection of Dairy Organisms ATCC - American Type Culture Collection NCIB- - National Collection of Industrial Bacteria FRI •• - Agricultural Research Council Food Research Institute, Colney Lane, Norwich, UK. Table 1

Strain Source

B. subtilis 713 Bulk milk tank, Iran

(NCD02130)

B. subtilis 738

(NCDO 738)

(ATCC 9372)

B. subtilis 706 Rinse of farm bulk milk tank

(NDC021 ^* 29 V

Strain Source

B. subtilis var. niger

(NCIB 8058)

B. subtilis SA22 R.T. Toledo et al, Appl. Microbiόl. 26, 592-7 1973

B. globigii B17 (NCIB 8649) B. licheniformis 100 'In-line' milk B. licheniformis 117 B. licheniformis T of O.Cerf

& J. Hermier Le Lait 52, 1 - 20

1972

B. licheniformis 109 2A0 UHTr spoiled milk, 0. Cerf &

F. Metro J. Appl. Bacteriol. 42,

405-415, 1977

B. cereus 8l8 'In can' milk

B. cereus T G.J. Dring & G.W.Gould in Spores VI pp 488-494 Ed. Gerhardt P. Costilow R.N. & Sadoff, H. Washington DC American Society for Microbiology 1975

B. pumilus 312 B. pumilus EJ of 0.Cerf & J. Hermier Le Lait 52, 1 - 20 1972

B. stearothermophilus 202

(NCDO 1096)

G 12 Pond mud

Clostridium sporogenes

PA 3679 (NCIB 8053)

Escerichia coli K12 FRI

Streptococcus faecalis Chicken gut ss ^* liquefaciens EB/F/30/39

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_^ -_"

- 5 -

Strain Source

Serratia marcescens

(NCTC 10211)

PeniciIlium chrysogenum UHT spoiled milk

Spore preparation and maintenance of cultures

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 2A0, iv) a medium containing (g/l), Oxoid nutrient broth No. 2 (3.1), MnS0,.4H 0 (0.03), KgHPO, (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 .4H 0 (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 lM-NaOH and spores produced under H : CO (9:l) for C. sporogenes PA 3679 ₁ vi)

Bacillus spore agar as described by Franklin ___ ___ (1970) with

Oxoid Lab Lemco added at 0.1% (w/v) for all other strains. Growth is at 30 C for B. subtilis var. niger, B. subtilis 738, B. globigii B17, strain G12, B. cereus T and B. cereus 8l8, at 33°C for C. sporogenes PA 679, at 37 C for B. pumilus 312, B. subtilis

SA22, B. subtilis 713, B. subtilis 706, B. licheniformis 100, 13. licheniformis 117 and B. licheniformis 109 2A0 or at 55 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 times with sterile glass-distilled water before storage at -18 C» Preparation of cells of non-sporing bacteria and maintenance of cultures

Non-sporing bacteria are grown at 33 C in i ^J ) 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 25mM-potassium phosphate buffer pH 7-0 by centrifugation before resuspension at a density of q about 1 x 10 viable units/ml and were used within 4 hours.

Cultures are maintained on slopes of Heart Infusion Agar (Difco). Preparation of mould spores and maintenance of culture

Spores of Penicullium chrysogenum are grown on slopes of

Czapek Dox Agar containing (g/l); sucrose (30), K HP0, (1.0),

NaNO (2.0), MgSO, .7H 0 (0.5), KC1 (0.5), FeSO, -7H 0 (O.Ol), and

New Zealand Agar (20) at 20 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.

U.V. irradiation of bacterial spores, vegetative cells and mould spores

7 8 Bacterial spores or vegetative cells at 3 x 10 to 5 x 10 /ml and mould spores at 1 x 10 ml are suspended in 0.1M sodium phosphate buffer pH 7.0 and up to 2.5g/l00ml H O (Analar, B.D.H.)

Volumes of 4ml are rocked gently to and fro for 30 sec. at 20 C in a Petri dish base with a diameter of 9 cm at distances of 5a» ι 30cm or 33cm 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 radiation in the range l85-579nm

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with a peak intensity at 254nm; 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 (2ml) are removed and mixed with a filter sterilised solution (2ml) of catalase (Sigma Ltd) at 76 0 units/ml at 20 C before storing in ice for at least 5 min. , dilution and plating. Heat treatment (Examples 2-l6, 33-35, 31-4l, 6l and 71) Samples (2ml) are heated to 85 C: in 6θ sec, (bacterial spores), 65 C in 30 sec. (vegetative bacteria) or 5 in 15 sec. (mould spores) by adding to pre-heated screw-capped bottles before adding 2 ml of catalase pre-cooled to +1 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. Determination of survivors

Diluted spore suspensions are plated on (i) Bacillus spore agar and incubated for 2 days at 30 C (B. subtilis 713, B. subtilis 706, B. cereus T and B. cereus 8l8) or 37 C (B.licheniformis 109 2A0, B. licheniformis 100, B. licheniformis 117 and B. pumilus 312); (ii) plate count agar (Oxoid) and incubated for 2 days at 33°C (B. subtilis SA 22) or 2 days at 37 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 0 (B. stearothermophilis 202) (iv) a medium containing (g/l)χ; tryptone (Oxoid) (10), glucose (5-0) and New Zealand Agar (12) and incubated for 2 days at 30 C (G12) or (v) reinforced Clostridial medium agar of Hirsch & Grinsted (1954) but with 1.6% New Zealand Agar in place of 1.2% (C. sporogenes PA 3679).

Suspensions of vegetative cells are enumerated by diluting 100-fold in maintenance medium (Difco Bacto-peptone) , 1.0g/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 &__ _)__ 1973, Jarvis _et_ aιl_ 1977) and colonies counted after 3 days incubation at 33 C (S. faecalis), 2 days incubation at 30 C (E. coli) or

O , a.

33 C (S. marcescens).

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

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 12451-454.

Franklin J.G. , Underwood, H.M. , Perkin A.G. and Burton, H (1970) J. Dairy Res. 37, 219-226.

Hirsch, A, Grinstead, E. (1954) J. Dairy Res. 21, 101-110 Example 1

Rate of kill of spores of Bacillus subtilis 706 by U.V. irradiation and hydrogen peroxide

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 O (ig/lOOml) 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

OMPI IPO

6θ sec. respectively. In the presence of peroxide, irradiation produces a much more rapid logarithmic kill so that after 0 sec. only 0.01% of spores survive. Examples 2-l6 Several strains of Bacillus and Clostridium are irradiated for 0 sec. In _. the presence of 2.5g/l00ml H O as described o _r above followed by heating to 85 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.

Table 2

Example Strain Survivors (%)

U.V. alone* U.V. and U.V. heat** plus

H-,0 *** 2 2 and heat

2 B. subtilis SA 22 1.44 0.23 0.0004

3 B. licheniformis 109

2A0 0.045 0.73 0.004

4 B. globigii B17

(NCIB 8649) 0.44 0.47 0.008

5 B. stearothermophilus

202 (NCD0 1096) 0.64 0.14 0.004

6 B. pumilus 312 0.034 0.031 0.001

7 B. subtilis 738 0.0053 0.022 0.002 (NCD0738:ATCC 9372)

8 G12 0.067 0.00005 0.0009

9 B. licheniformis

117 0.19 0.019 0.006

10 B. subtilis 713

(NCD02130) 0.89 0.005 0.006

- CfRE tr

O PI /,, WIPO

Example Strain Survivors (%) U.V. alone* U.V. and U.V. plus

11 B. licheniformis

^" 100 0.67 1.1 0.0003

12 B. cereus 8l8 0.022 0.038 <.0001

13 B. cereus T 0.020 0.0061 0.0001

14 B. subtilis var. niger

(NCIB 8058) 0.14 0.61 0.002

15 B. subtilis 706

(NCDO 2129) 0.18 0.42 0.0008

16 Clostridium sporogenes

PA 3679 (NCIB 8053)0.21 0.0097 < 0.0001

* Lamp 5-5cm above spore suspension for 30 sec.

** Samples (2ml) removed and heated to 85 C during 60 sec

***Lamp 5.5cm above suspension with 2.5g peroxide/lOGml for

30 sec. followed by heating to 85 C during 6θ sec. Examples 17-24

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-

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Table 3

Example Hydrogen peroxide Survivors (%) (g/lOOml) after U-V. irradiation*

1.2 x lO'/ml 12 x lOVml

17 0 0.43 18 0 0.82

19 0.5 0.0073 20 0.5 0.39 21 1.0 0.0089 22 1.0 0.44

23 2.5 0.22 24 2.5 1.6

* Spores are suspended at 1.2 x 10 7 or 12 x 107/ml with hydrogen peroxide before irradiation with the U.V. lamp at 5.5cm above the suspension.

Examples 25-28

Four Bacillus strains are irradiated with U.V. in the presence of hydrogen peroxide and in two cases are subsequently o - heated to 85 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 O without U.V. irradiation.

Table 4

Comparison of spore destruction by H O and U.V. irradiation and ^b Y H O alone. 22

Strain Example H O Temperature Time*

(g/lOOml) (°C) (sec)

-

B. subtilis - 10 25 1800 Prior work var-. niger 25 1.0 + U.V.20 30

B. subtilis 738 - 25.8 24 66θ Prior work

26 1.0 + U.V.20 30

B. subtilis 713 - 3.0 90 600 Prior work

27 2.5 + U.V.20 + 85** 30 + 60**

B. licheniformis 15 8θ 126 Prior work

109 2A0 28 2.5 + U.V.20 + 85** 30 + 6θ**

* Time is that required to produce a kill of 99-99% **Spores irradiated at 20C for 30 sec. and then heated to 85 C over 6θ sec EXAMPLE 28

Spores of B. subtilis 706 are irradiated with U.V. for 6θ sec in the presence of H O (lg/lOOml). The U.V. lamp is located 30cm above the spore suspension. For comparison purposes the strain is also irradiated with U.V. in the absence of H O . Samples are removed diluted and plated as hereinbefore t ___ described. The results are shown in Table 5«

Table 5

Destruction of spores of B. subtilis 706 by U.V.

Example: U. .V. + H„0 2 2

Comparison: U.V. only

Irradiation Time (sec ) Example Comparison Survivors % Survivors %

15 1.0 -

30 0.009 20

45 0.002 8

60 0.001 2

EXAMPLES 30-35

Spores of B. pumilus 312 are irradiated with U.V. for 0 sec in the presence of H O both with and without subsequent heating to 8 C over 60 sec. The lamp is 5 ^c m above the suspension. For Of) comparison purposes spores are incubated: (i) at 20 C with hydrogen peroxide for 30 sec, (ii) with hydrogen peroxide for 30 sec. followed by heating to 85 C over 6θ sec Samples are removed at intervals, diluted and plated as hereinbefore described. The results are shown in Table 6.

Table 6 Conditions:

Examples 30-32: U.V. + H O (Comparison:H 0 ) Examples 33-35: U.V. + H O + Heat

" ' *__. u

(Comparison:H_0 + Heat)

Example Concentration of Survivors (%) Survivors (%)

U2-_ ^Q 2r, (Examples) (Comparisons)

30 0.25 0.04 90

30a 0-50 0.02 80

30b 0.75 0.01 100

31 1.0 0.01 100

32 1-75 0.3 80

32a 2.50 0.5 100

33 0.25 0.01 100

33a 0.50 0.002 80

33b 0.75 0.001 50

34 1.0 0.0002 14

35 1.75 0.0002 0.13

35a 2.50 0.0003 0.01

- ι4 - Examples 36-4l

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-

Table 7 Conditions: Examples 36-38: U.V. + H O

¹ Ct 2

(Comparison: H O only) __ _* 2

Examples 39-4l: U.V. + H O + Heat

(Comparison: H O + Heat)

2 2

Example Concentration ^* of Survivors (%) Survivors(%)

H„0„ (Examples) (Compar 2 2

36 0.5 0.014 100 37 1.0 0.17 100 38 2.5 2.0 100 39 0.5 0.005 40 40 1.0 0.005 .35 41 2.5 0.005 0.7

Examples 42-49

E. coli K-12 is irradiated with U.V. for 30 sec in the presence of varying concentrations of hydrogen peroxide. Tlie lamp is 30 cm above the suspension. For comparison purposes cells are also incubated for 0 sec. with Ig/lOOml H O in the

___ 2 absence of U.V. irradiation. Samples are removed, diluted and plated as hereinbefore described. The results are shown in Table 8.

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Table 8

Conditions:

Examples 42-49: U.V. + H O

¹ 2 ■&

Comparison Experiment 50: H O only

Example Concentration Survivors (%) ° ^f 2°2 (Examples)

42 0.001 o.4ι

43 0.005 0.26 44 0.01 0.14

45 0.025 0.08 46 0.05 0.044

47 0.075 0.017 48 0.01 0.0085

49 0.25 0.0026 50 1.0 74

Examples 51-59

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 30cm above the suspension. For comparison purposes cells are also incubated for 30 sec. with l.Og/lOOml H O in the absence of U.V. irradiation. Samples

___ ___ are removed, diluted and plated as hereinbefore described. The results are shown in Table 9-

Table 9 Conditions: Examples 51-59∑ U.V. + H O

(Comparison: Ig/lOOml H O only)

2 2

Example Concentration Survivors (%) Survivors (%) ° ^{f H} 2°2 (Examples) (Comparison)

51 0.001 1.0 52 0.005 0.7 53 0.01 0.6 54 0.025 0.32

Table 9 (continued)

Example Concentration Survivors (%) Survivors (%) of H O (Examples) (Comparison)

2 2

55: 0.05 0.29 *

56. 0.1 0.11 *

57 0.5 0.026 *

58 0.75 0.024 *

59 1.0 0.011 100

* Not tested

Examples 6θ-6l

Cells » of E. coli K12 are irradiated with U.V. for 30 sec. in the presence of l.Og/lOOml H O both with and without subsequent heating to 6 r5OC over 30 sec The lamp is 30cm above the suspension. For comparison purposes cells are (i) irradiated with U.V. for 30 sec. followed by incubation with H O for

2 2

30 sec (ii) heated to 65 C over 30 sec Samples are removed, diluted and plated as hereinbefore described. The results are shown in Table 10.

Table 10 Conditions:

Example 60s U.V. + H„0„ 2 2 (Comparison: U.V. followed by H 0 )

2 2 Example 6ls U.V. + H 0 + heat

2 2 (Comparisons heat)

Example Concentration Survivors(%) Survivors (%) of H„0_, ,_, , .. 2 2 (Examples) (Comparisons)

60 1.0 0.0044 0,14 61 1.0 < 0.00005 0.0013

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Examples 62-69

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 l.Og/lOO ml H O in

2 2 the absence of U.V. irradiation. Samples are removed, diluted and plated as hereinbefore described. The results are shown in Table 11.

Table 11 Conditions: Examples 62-69? U.V. + H O

(Comparisons l.Og/lOOml H O only)

2 2

Example Concentration of Survivors (%) Survivors (%)

H„0„ ((EExx,amples) (Comparison) 2 2

62 0.01 0.046 63 0.025 0.022 64 0.05 0.014 65 0.1 0.011 66 0.25 0.0014 67 0.5 0,00095 68 0.75 0.000i6 69 1.0 < 0.0001 86

* Not tested Examples 70-71 Spores of PeniciIlium chrysogenum are irradiated with U.V. for 30 sec. in the presence of 0.5g/l00ml H O both with and

I O without subsequent heating to 5 C over 15 sec The lamp is 5 m above the suspension. For comparison purposes spores are (i) irradiated with U.V. for 30 sec followed by incubation with H O for 30 sec. (ii) heated to 4 C over 15 sec.

Samples are removed, diluted and plated as hereinbefore described. The results are shown in Table 12.

Table 12 Conditions: Example 70; U.V. + H O

(Comparison: U.V. followed by H O ) Example 71. U.V. + H O + heat

"^* ™ 2 2

(Comparison: heat)

Example Concentration Survivors (%) Survivors (%) of H„0„ 2 2

70 0.5 0.95 3-3 71 0.5 0.0033 9.6

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