WO1995017214A1

WO1995017214A1 - Ethene as a sterilizing agent

Info

Publication number: WO1995017214A1
Application number: PCT/NZ1994/000151
Authority: WO
Inventors: Warwick Murrow Jameson
Original assignee: The Anchor Brewing Company (Nz) Ltd
Priority date: 1993-12-23
Filing date: 1994-12-22
Publication date: 1995-06-29
Also published as: AU1328995A

Abstract

The use of ethene, when solubilised, as a sterilizing agent is provided. Ethene may be solubilised in a liquid to sterilize or inhibit the growth of microorganisms in that liquid. In this way, ethene has been found to act as an antibiotic, inhibiting or eliminating bacteria, viruses and/or fungi. In particular, the addition of ethene to beer or beer wort has been found to sterilize the beer or beer wort. Ethene may also be solubilised to provide a solution for human consumption. The solution is useful in preventing or eliminating microorganisms from the body.

Description

Ethene As A Sterilizing Agent

TECHNICAL FIELD

The invention relates to the use of ethene as a sterilizing agent.

The applicant has found that ethene may be useful in treating a wide range of substances, especially liquids to help prevent growth of micro-organisms such as bacteria, viruses and fungi. More specifically, ethene may be used to prevent contamination of beer by micro-organisms.

BACKGROUND OF INVENTION

One of the main avenues for the introduction of spoilage organisms such as bacteria into beer is via the yeast used to ferment the beer. Yeast contaminated with bacteria, if cropped from one brew for use in another, can quickly give an increase in bacteria levels that will require the yeast to be replaced with a fresh, uncontaminated one. The preparation of large amounts of fresh sterile yeast from a small culture can be a costly, time-consuming and inconvenient chore for a brewery. Production may be disrupted until a new yeast is ready to use.

Methods have been devised in an effort to kill bacteria contaminating yeast. Certain antibiotics have been tried but with the fear the bacteria may become resistant to them or may already have resistance.

A process called "acid washing" may be used whereby the yeast is washed in a sterilising solution of low pH. The disadvantage of this process is that it may not kill all bacteria and it may damage the yeast, thus ruining it for future use. Beer itself, apart from the yeast, can also become contaminated with bacteria, wild yeasts and moulds, even in the most hygienic of breweries. Therefore, most modern day beers are subject to some type of sterilising process.

One such sterilising process is called micro (or ultra) filtration in which the contaminants are sieved from the beer. Unfortunately much flavour can also be sieved from the beer and contaminants may be reintroduced during post-filtration processing.

Chemical sterilants can be used. Although these may be effective, the ideas of introducing foreign chemicals to the beer has little public support. These chemical sterilants may also cause undesirable flavour and appearance changes in the beer.

Another method of sterilising beer is by heat treatment (pasteurisation) . This method is effective but can cause undesirable flavour changes. It is also costly in terms of energy use, manpower and the machinery required. It tends also to be a slow process and product loss arises through bottle breakages.

It is therefore an object of this invention to go some way in overcoming these disadvantages or to at least provide the public with a useful choice.

For many years, brewers have used hops to flavour beers. However, brewers have also found that hops added to beer exert a preservative effect. The precise component of the hops that exerts the preservative effect has never been discovered.

Today, opinion is that hop resins contribute little to the biological stability of beer. However, when hops were added to beer for post-primary fermentation preservative effect in earlier days, beers generally contained yeast. This is not the case with most modern beers. It is known that yeast produces ethene.

DISCLOSURE OF THE INVENTION

The applicant has, surprisingly, found that ethene, also known as ethylene, can be used as a nutrient for, for example yeast. Also, the applicant believes that yeast may produce ethene from alcohol by dehydration of alcohol. The applicant has further surprisingly found that hops contained in beer can be induced into producing ethene by the addition of an amount of ethene to the beer. In a trial, an ethanol extract of one kilogram of hops contained in beer, after the addition of some ethene, produced a further 25 volumetric litres of free ethene.

The applicant has surprisingly found that ethene can be used as a sterilizing agent. Although ethene can be used to sterilize a wide range of products, it is especially useful as an antibacterial agent when added to beer. It can also prevent growth of moulds and other micro-organisms.

Ethene production by certain plants (except hops in beer) is well known. Ethene production by Penicillium digitatum is explained in The Chemistry and Biochemistry of Plant Hormones Vol 7, Academic Press, New York and London 1974. Ethene production is not just confined to plants, it is also produced by animal and human tissues (Ethylene Production by Subcellular Particles from Rat Liver, Rat Intestinal Mucosa and Penicillium Digitatum, NATURE Vol 197, Jan 26th 1963, 366-367) and in particular it was noted that rats with sarcoma or abscesses exhaled more ethene than healthy rats. It is notable however that molecular disruption of cell mitochondria increases ethene production by the mitochondria. Production of ethene by beef heart mitochondria (Production of Ethylene by Beef Heart Mitochondria, Biochimica Et Biophysica Acta 78 (1963) 528-530) was increased 10 fold when the mitochondria was sonicated. Sonication (ultrasonic vibration) will case cavitation within mitochondria and thus a loss of its molecular integrity.

The applicant believes that production of ethene by body tissue is in response to damage of cell mitochondria by invading bacteria, viruses or other micro-organisms, this response being a defence mechanism directed at killing the invading micro¬ organisms.

The applicant's trials show that consumption of beer containing ethene can rapidly clear bacterial throat infections in as little time as 12 hours. Regular drinkers of beer containing the beer's own natural antibiotic, ethene, were informally questioned over a period of 12 months and it was found that these beer drinkers, almost without exception, reported an immunity to cold viruses (rhinoviruses) .

Ethene may therefore also be effective as a virucide. Also ethene may be added to a liquid to provide a sterilising solution which is intended for human consumption and which assists in eliminating or inhibiting micro-organisms in the consumer's body upon consumption.

The applicant has surprisingly found that ethene can be added to beer wort to destroy or inhibit contaminants such as bacteria and moulds that may have been carried into the wort by the yeast, or that may already be in the wort.

The applicant has also surprisingly found that ethene can be added to finished beer prior to its packaging so as to prevent or limit the spoilage of the beer by micro-organisms. The applicant has surprisingly found that ethene can be added to beer wort to perform the dual function of providing a nutrient for yeast, thus enhancing fermentation characteristics, whilst at the same time being effective in killing unwanted contaminants such as bacteria.

The applicant has surprisingly found that hops in beer will generally not readily produce ethene until stimulated into doing so by contact with a small amount of introduced ethene. It may be that the antibacterial metabolite of yeast is ethene and that this ethene contracts hop material in beer, triggering further ethene production from the hop material so ethene produced by hop material in fermenting beer may have a sterilizing effect whilst producing ethene as a nutrient for yeast thus enhancing fermentation characteristics.

These findings are of great importance in the fermentation industry.

Of course, the use of ethene as a sterilising agent is not limited to its use in beer or beer wort. Ethene may be added to any liquid to produce a solution substantially free of contaminating micro-organisms. For example, it could be added to soft drinks such as fruit juice, cordials or water to produce a drink which is substantially free of micro-organisms.

Also, it is envisaged that a consumable solution may be produced by adding ethene to water, cordial, fruit juice etc and that the solution so produced may be used as a medicament to assist in inhibiting or eliminating micro-organisms in the consumer's body.

The invention provides the use of ethene as a preservative, preventing, inhibiting or eliminating growth of micro-organisms in a substrate. The term "substrate" can be solid, liquid or gaseous.

The invention provides a method of sterilising or micro¬ organism inhibiting the growth of micro-organisms in a liquid comprising adding ethene to the liquid.

The invention also provides a sterilising or micro-organism inhibiting solution comprising ethene combined with a suitable liquid. The sterilising or inhibiting solution may be suitable for human consumption.

The invention also provides a liquid comprising solubilised ethene, the liquid being adapted for human consumption and wherein the ethene acts as a preservative, preventing, inhibiting or eliminating growth of micro-organisms in the liquid.

Preferably the liquid is beer or beer wort.

The invention also provides a method of solubilising ethene in a liquid to provide a consumable sterilising solution which sterilises or inhibits the growth of micro-organisms in the body when consumed.

Within this specification the term "micro-organisms" is used in a broad sense and includes fungi including moulds and yeast, bacteria and viruses and includes the term "protista". The term "sterilising" when used in this specification also takes a broad meaning and includes total elimination and partial elimination of micro-organisms.

The ethene may be mixed with another gas prior to its addition to the liquid. This may enhance its solubility and/or reduce the flammability of the ethene in the liquid. Preferably the ethene is mixed with carbon dioxide. More preferably the gas mixture is 75% carbon dioxide, 25% ethene (vol/vol). However, any admixture of gas or gases may be used according to the nature of the matter requiring sterilisation. The admixture of gas or gases may be, for example nitrogen and/or oxygen.

An additive may be provided to the liquid which will enhance the sterilizing power of the ethene. The additive may be ascorbic acid.

The ethene may have an antioxidant effect.

The invention also provides a method of mixing ethene with another gas or a liquid for medical application. The invention also provides a solution containing dissolved ethene, the solution being intended for human consumption and adapted to steriliser inhibit micro-organisms in a person's body. In its natural uncombined state, ethene is non-toxic to humans.

More particularly the invention provides a method of adding ethene to beer to produce a sterilising and/or preservative effect.

Ethene may be used as a virucide, bactericide and/or a fungicide when added to a liquid.

An advantage of using ethene as an antibiotic/virucide is that it is a natural antibiotic/virucide and is small in size. It is also an unsaturated molecule and therefore may be able to form molecular structures by combining with other molecules or atoms within the body with the ability to do this giving it an antibiotic/virucide property. Ethene may be incorporated into solution for example directly into blood.

To assist effectiveness as a medicament, ethene is preferably solubilised within a liquid such as water for example. This water/ethene mixture may then be consumed to allow the ethene to diffuse into the bloodstream via the intestinal tract. Such a liquid/ethene mix may contain 20% v/v ethene to water for example but other ratios may be used. The advantage of this method is that the ethene has only one main means of escape and that is by diffusion into the surrounding tissue then into the bloodstream. An ethene/liquid mix could be introduced into a body by injection.

Ethene thus introduced into the intestinal tract may stimulate the production of further ethene by food material within the tract, or may stimulate bodily production of ethene.

Any method that allows for internal absorption is envisaged. Ethene may though be applied as an inhalant.

Ethene application may be enhanced by the use of pressure or a vacuum.

Ethene application may be enhanced by using varying temperatures.

Ethene effectiveness may be enhanced by atmospheric humidification.

Depending on the method of use, rate of use and other factors, such as the concentration of ethene used and the susceptibility of the cells to be destroyed, ethene may be lethal or at least limit the growth of a wide range of micro¬ organisms including viruses, bacteria and fungi. The ethene may also act as a nutrient for other micro-organisms.

The invention is particularly directed to using ethene as an antibacterial agent in beer or wort and as a preservative agent in bottled beer.

BEST MODE OF CARRYING OUT THE INVENTION

Embodiments of the invention will now be described, by way of example only. EXAMPLE 1

use of ethene as a combined nutrient and sterilant

This process involves solubilising ethene, into a liquid so that the ethene is able to contact micro-organisms contained in the liquid to selectively destroy unwanted micro-organisms, such as bacteria, while performing a dual function of being a nutrient for desirable micro-organisms such as yeast.

Four identical beer fermentations were set up. Each fermentation was of 1,000 litres of beer wort at an original gravity of 0G1.030. The wort sugars comprised of 75% wort extract and 25% sucrose. Each tank was innoculated with 2 kilograms of brewers yeast (£. cerevisiae ) . A recirculating pump was attached to each tank and 16 litres by volume of oxygen was metered into the wort in each tank by admitting the oxygen on the suction side of the pump, the pumps revolving impellor acting as a gas diffuser to get the oxygen into solution.

Wort samples (A) were then taken from each tank via sterilized sample cocks and a one millilitre sample of each was incubated on proprietory agar media to determine the presence of gram-negative bacteria.

Two litres by volume of ethene gas was then dissolved into each of the trial tanks, tanks 'A' and 'B' by using the same method used for the oxygen. Tanks 'C and 'D' were used as controls with no added ethene.

Samples were taken from each tank at 24 hour intervals and the specific gravity of each determined by hydrometer.

The results in Table 1 show that the degree of attenuation was greater in the trials than the controls and therefore more ethanol was produced by the trials than the controls. Table 1

Trialis Controls

'A' 'B' 'C 'D'

Original Gravity 1.030 1.030 1.030 1.030

24 hours Specific Gravity 1.016 1.016 1.018 1.018

4-Q '» << " 1.008 1.008 1.010 1.010

7 it ιι 1.004 1.004 1.006 1.006

Ethanol by Volume 3.4% 3.4% 3.13% 3.13% (attenuation degrees x .13059)

At 72 hours elapsed fermentation time, fermentation had ceased and samples (B) were again taken from each of the four tanks and incubated on proprietory agar media to determine gram- negative bacterial growth. Incubation of all samples was at 37C. Each sample was one millilitre. The results are shown in Table 2

Table 2

Growth of Gram-negative Colonies.

Trials Controls

'A' 'B' 'C 'D'

Original Gravity sample A 6 6 6 6 Finishing Gravity sample B 0 0 18 18

The presence of bacteria on the 'A' samples was confirmed by examination under a microscope. The total absence of bacteria in sample 'B' from trial tanks A and B was confirmed by examination under a microscope as was the presence of bacteria in the control samples. This result demonstrates that ethene destroys bacteria. Similar results are obtained where the micro-organisms are gram- positive bacteria or moulds and much higher levels of contamination are encountered, e.g. 400 colonies per millilitre.

In conclusion, ethene added to beer worth in controlled amounts, provided a nutrient for fermentive micro-organisms such as yeast thereby enhancing fermentation characteristics such as ethanol production. At the same time the added ethene destroyed unwanted micro-organisms such as bacteria.

Bacteria, for example Acetobacter, can impart unwanted flavours and aromas in edible substances, beer for example. Therefore ethene can limit or prevent the formation of undesirable flavours in edible substances such as beer.

EXAMPLE 2

use of ethene as an antibiotic sterilant in a consumable substance, e.g. beer

Bacteria or other micro-organisms in beer can multiply to cause spoilage of the beer. Two examples, brews A & B show the effect of added ethene on bacteria levels in beer.

Brew 'A' . This beer had a finishing gravity of SGI.010 thus nutrients were available for bacterial growth. A sample taken and incubated on proprietory agar media at 37^βC gave a total bacterial colony count of 17 per millilitre.

The beer was then passed through a carbonator to charge it with carbon dioxide. 1.57% of ethene gas vol/vol beer was solubilised in the beer by addition via the carbonator. The beer was then bottled into crown sealed glass bottles. After one months storage at ambient temperatures, a sample bottle was tested to determine bacterial levels in the beer. Six bacterial colonies developed per millilitre of beer, a reduction of 11 colonies per millilitre. Without ethene in the beer, the number of colonies would be expected to increase with the passage of time.

Brew 'B' . With a finishing specific gravity of 1.018, this beer also contained bacterial nutrients. Treatment was as per Brew 'A' and gave a total bacterial colony count of 14. However, the ethene level was increased to 2.5% ethene vol/vol beer. A bottle was tested for bacteria after one month in the same manner as previous samples. This sample gave a total bacterial count per millilitre of beer of zero.

In conclusion, these examples showed the effectiveness of ethene in destroying bacteria in substances such as beer but also showed that enough ethene must be used to obtain an effective kill rate. Generally the applicant has found that 2.5% ethene vol/vol in beer at bottling will destroy all micro¬ organisms in the beer obviating the need to pasteurise or sterile filter the beer.

EXAMPLE 3

Enhancement of the amount of free ethene in a substance, e.g. beer

Ethene, if added to beer, will reduce in amount as the ethene reacts with other molecules in the beer, e.g. phosphate. A solid substance, ascorbic acid, can be added to beer to help maintain ethene levels. Hydrogen released by ascorbic acid competes for sites that would normally take up hydrogen from ethene. Ascorbic acid is an oxygen scavenger in beer and appears to react more readily with oxygen than ethene. Like ascorbic acid, ethene also serves as an anti-oxidant in beer. During trials it was noted that ethene had the ability to de¬ oxidise oxidised plant material, notably hops. The anti-oxidant nature of ethene can exert a preservative effect on edible substances such as beer. A batch of beer containing ethene was divided into two. One half remained untreated while the other half was treated by the addition of 30 ppm w/v of ascorbic acid. Samples of each were analysed using gas chromatography for residual ethene content. Results are expressed on a mole/mole basis.

Component With ascorbic No ascorbic

Ethene 0.22%+/- 0.01% 0.056% +/-0.005%

This result shows that another substance, e.g. ascorbic acid, can be used to enhance free ethene levels in substances, notably beers and that ethene competes as an anti-oxidant with ascorbic acid in beers. Enhanced levels of ethene in a substance will result in an enhanced kill rate of unwanted micro-organisms.

EXAMPLE 4

Another method of enhancing ethene levels in a substance, e.g. beer

The applicant has found that plant material, e.g. hops or an ethanol extract of hops for example, can be mixed into beer and induced into producing ethene by mixing ethene with a liquid and contacting that ethene/liquid mix with the immersed plant material.

One kilogram of dried hops was extracted with ethanol and water and the resultant extract added to beer prior to filtration. After a period in conditioning storage and after filtration, the beer was charged with ethene and carbon dioxide using a carbonating machine. The ratio of ethene to carbon dioxide was 1.32% v/v. After one month, the gas content of the beer was analysed by gas chromatography. It was found that ethene levels had increased by 41.6% to 1.87% v/v of carbon dioxide. In a similar trial in which only the resin fraction of hops was added to beer, ethene levels decreased by 83.3%. This shows that the resin fraction of hops may not produce ethene and therefore may not contribute to the antibiotic nature of hops.

By using this method to contact immersed plant material such as hops with ethene, overall ethene levels are increased, further enhancing the destruction of unwanted micro-organisms such as bacteria.

EXAMPLE 5

Demonstration of ethene's antiviral effect

Gas; Ethene technical grade

Virus: Rhinovirus 'M' type clinical isolate

Cells; MEK (embryonic monkey kidney epithelial cell)

Method; Medium 199 was sparged with ethene for 30 minutes.

Virus was titrated in gassed medium and all dilutions incubated at 37°C for 2 hours. The control was treated similarly with the exception of non-gassed medium used. The test virus/cell mixture was incubated in a container with a blanket atmosphere of ethene and the controls in a normal atmosphere. Cultures were incubated for 5 days and read microscopically from days 3 to 5.

Results

_5 Virus endpoint in ethene medium = 1 x 10

_5 Virus endpoint in control medium = 2 x 10

Conclusions

There was an apparent twofold reduction of Rhinovirus titre in the ethene (trial) medium compared with the control medium. EXAMPLE 6

Gas; Ethene technical grade

Virus; Rhinovirus clinical isolate

Cells; Human skin fibroblast (HF)

Method; Serum-free medium 199 was sparged with ethene for 1 hour. Foetal calf serum was added to a concentration of 2%, virus was added and incubated 2 hours at 37°C.

After incubation, virus was titrated in 0.3 log steps in the gassed medium with 2% foetal calf serum and incubated into HF cells. The air space in the cell culture tubes was purged with ethene gas for 30 seconds. Cultures were incubated in a roller drum incubator at 35°C and read microscopically twice weekly.

Control

As for test method with exception of ethene gassing procedures. All control incubations and procedures were carried out simultaneously and were identical to the test sample.

Results

Virus endpoint titration in ethene medium = 10 —1 38 Virus endpoint titration in control = 10 -1^*75

Conclusions

There was a 2.4 fold reduction in Rhinovirus in the trial compared to the same virus incubated under control conditions.

It is to be understood that the scope of the invention is not limited to the described embodiments and therefore that numerous variations and modifications may be made to these embodiments without departing from the scope of the invention as set out in the claims. For example, ethene may be used to sterilize a wide range of liquids, not merely beer. Such liquids may include wine, water, or even blood for example.

INDUSTRIAL APPLICABILITY

The invention may find wide applicability in the inhibition or elimination of micro-organisms in liquids. In particular, the invention will be useful in helping eliminate unwanted micro-organisms from beer or beer wort. It will therefore be of enormous benefit in the brewing industry. However, the invention may be used to provide medicaments for human use which help inhibit or eliminate the growth of micro-organisms in the human body.

Claims

1. A sterilising or micro-organism inhibiting solution comprising ethene solubilised in a suitable liquid.

2. A sterilising solution as claimed in claim 1 in which the ethene is combined with another gas.

3. A sterilising solution as claimed in claim 2 in which the other gas enhances the solubility or reduces the flammability of the ethene in the liquid.

4. A sterilising solution as claimed in claim 2 or 3 in which the other gas is carbon dioxide.

5. A sterilising solution according to claim 4 in which the gas mixture is 75% carbon dioxide and 25% ethene (vol/vol) .

6. A sterilising solution according to any preceding claim further comprising an enhancer which enhances the sterilising power of ethene.

7. A sterilising solution according to claim 6 in which the enhancer is ascorbic acid.

8. A sterilising solution according to any preceding claim wherein the sterilising solution is adapted for animal consumption.

9. A sterilising solution according to claim 8 which is adapted for human consumption.

10. A sterilising solution according to claim 8 or claim 9 in which the solution is particularly adapted to kill bacteria.

11. A sterilising solution according to claim 8 or 9 in which the solution is particularly adapted to kill fungi.

12. A sterilising solution according to claim 8 or claim 9 in which the solution is particularly adapted to kill viruses.

13. A sterilising solution according to any one of claims 1 to 12 wherein the sterilising solution is beer or wort.

14. A sterilising solution according to any preceding claim in which the ethene also has an antioxidant effect.

15. A liquid comprising solubilised ethene, the liquid being adapted for human consumption and wherein the ethene acts as a preservative, preventing, inhibiting or eliminating growth of micro-organisms in the liquid.

16. A liquid according to claim 15 in which the ethene also acts as a nutrient for micro-organisms.

17. A liquid according to claim 16 in which the ethene prevents, inhibits or eliminates the growth of certain micro-organisms whilst acting as a nutrient for other micro-organisms.

18. A liquid according to any one of claims 15 to 17 which is beer or beer wort.

19. A liquid according to any one of claims 15 to 18 wherein another gas is added to the liquid in addition to ethene.

20. A liquid according to claim 19 in which the other gas enhances the solubility of the ethene or reduces the flammability of the ethene in the liquid.

21. A liquid according to claims 19 or 20 in which the other gas is carbon dioxide.

22. A liquid according to claim 21 in which the gas mixture is 75% carbon dioxide, 25% ethene (vol/vol) .

23. A liquid according to claim 18 in which the ethene has been added to beer prior to packaging of the beer.

24. A liquid according to any one of claims 15 to 23 further comprising an enhancer which enhances the sterilising power of ethene.

25. A liquid according to claim 24 in which the enhancer is ascorbic acid.

26. A liquid according to any one of claims 15 to 25 in which the ethene also has an antioxidant effect.

27. A method of providing a consumable sterilising solution which sterilises or inhibits the growth of micro-organisms in the body when consumed comprising solubilising ethene in a liquid.

28. A method according to claim 27 in which the ethene is combined with another gas.

29. A method according to claim 28 in which the other gas enhances the solubility or reduces the flammability of the ethene in the liquid.

30. A method according to claim 29 in which the other gas is carbon dioxide.

31. A method according to claim 30 in which the ratio of ethene to carbon dioxide is 25:75 (vol/vol).

32. A method according to any one of claims 27 to 31 further comprising the addition of an enhancer to enhance the sterilising power of the ethene.

33. A method according to claim 32 in which the enhancer is ascorbic acid.

34. A method of sterilising or inhibiting the growth of micro¬ organisms in a liquid comprising adding ethene to the liquid.

35. A method according to claim 34 in which the liquid is beer or beer wort.

36. A method according to claim 34 or claim 35 which further comprises adding a second gas to the liquid.

37. A method according to claim 36 in which the second gas enhances the solubility or reduces the flammability of ethene in the liquid.

38. A method according to claim 37 in which the second gas is carbon dioxide.

39. A method according to claim 38 in which the ratio of ethene to carbon dioxide is 25:75 (vol/vol).

40. A method according to claim 34 or 35 in which the liquid contains 20% ethene (vol/vol) .

41. A method according to claim 34 in which the ethene is added to beer prior to the packaging of the beer.

42. A method according to any one of claims 34 to 41 further comprising the addition of an enhancer to enhance the sterilising power of the ethene.

43. A method according to claim 42 in which the enhancer is ascorbic acid.

44. The use of ethene as a preservative, preventing, inhibiting or eliminating growth of micro-organisms in a substrate.