U.S. patent application number 08/028764 was filed with the patent office on 2003-06-05 for method, composition and device for removing oxygen from solutions containing alcohols and/or acids.
This patent application is currently assigned to Oxyrase, Inc.. Invention is credited to ADLER, HOWARD I., COPELAND, JAMES C., CROW, WELDON D..
Application Number | 20030104608 08/028764 |
Document ID | / |
Family ID | 23581302 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030104608 |
Kind Code |
A1 |
COPELAND, JAMES C. ; et
al. |
June 5, 2003 |
METHOD, COMPOSITION AND DEVICE FOR REMOVING OXYGEN FROM SOLUTIONS
CONTAINING ALCOHOLS AND/OR ACIDS
Abstract
The present invention is directed to a novel method, composition
and device for removing dissolved oxygen from solutions containing
alcohols and/or acids. By removing oxygen from various products,
the present invention is an effective antioxidant for beverages and
food products, as well as for industrial and commercial solutions
containing alcohols and/or acids.
Inventors: |
COPELAND, JAMES C.;
(ASHLAND, OH) ; ADLER, HOWARD I.; (OAK RIDGE,
TN) ; CROW, WELDON D.; (KNOXVILLE, TN) |
Correspondence
Address: |
RICHARD M. KLEIN
1100 SUPERIOR AVENUE, SUITE 700
CLEVELAND
OH
441142518
|
Assignee: |
Oxyrase, Inc.
Mansfield
OH
|
Family ID: |
23581302 |
Appl. No.: |
08/028764 |
Filed: |
March 9, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08028764 |
Mar 9, 1993 |
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07653687 |
Feb 11, 1991 |
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07653687 |
Feb 11, 1991 |
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07399870 |
Aug 29, 1989 |
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4996073 |
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Current U.S.
Class: |
435/262 |
Current CPC
Class: |
C12H 1/003 20130101;
Y10S 435/801 20130101; Y10S 435/82 20130101 |
Class at
Publication: |
435/262 |
International
Class: |
C07C 001/00 |
Claims
Having thus described the preferred embodiments, the invention is
now claimed to be:
1. A method for removing oxygen from a solution containing alcohol
comprising the steps of: a) providing a solution containing
alcohol; and, b) adding to the alcohol solution a sufficient amount
of oxygen scavenging membrane fragments to reduce the oxygen
present in the solution to water.
2. The method of claim 1, wherein said alcohol solution comprises
from about 1 to about 18 weight percent ethyl alcohol.
3. The method of claim 1, wherein said alcohol solution comprises
about 9.5 weight percent ethyl alcohol.
4. The method of claim 1, wherein oxygen scavenging membrane
fragments contain an electron transport system which reduces to
water in solutions containing alcohol.
5. The method of claim 1, wherein said oxygen scavenging membrane
fragments are derived from bacteria, yeast, fungi, plants and
animals selected from the group consisting of beef heart, potato
tubers, spinach, Saccharomyces, Neurospora, Aspergillus, Euglena,
Acetobacter, Chlamydomonas, Escherichia, Bacillus, Salmonella,
Gluconobacter, and Pseudomonas.
6. The method of claim 1, herein said oxygen scavenging membrane
fragments are cell membrane fragments derived from the organism
Escherichia coli.
7. The method of claim 1, wherein said alcohol solution is adjusted
to a pH of about 7.
8. The method of claim 1, wherein said alcohol solution is adjusted
to a pH of about 8.4.
9. The method of claim 1, wherein said alcohol solution is an
alcoholic beverage.
10. The method of claim 1, wherein said alcohol solution is
beer.
11. The method of claim 1, wherein said alcohol solution is
wine.
12. The method of claim 1, further comprising the step of adding an
organic substrate to the solution containing alcohol.
13. The method of claim 12, wherein said substrate is a compound
selected from the group consisting of lactic acid, succinic acid,
alpha-glycerol phosphate, formic acid, and malic acid or
corresponding salts thereof.
14. The method of claim 1, further comprising the step of adjusting
the pH of the alcohol solution to a pH of about 6 to about 9 prior
to the addition of the oxygen scavenging membrane fragments.
15. A device for removing oxygen from a contained alcohol solution
comprising a means for containing an alcohol solution having a
solution contact surface and a non-solution contact surface,
wherein said solution contact surface contains a sufficient amount
of oxygen scavenging membrane fragments to reduce the oxygen
present in the solution to water.
16. The device of claim 15, wherein said contained alcohol solution
is an ethanol solution having a pH of about 6 to about 9.
17. The device of claim 15, wherein said contained alcohol solution
is an ethanol solution having an alcohol content of about 1 to
about 18 weight percent.
18. The device of claim 15, wherein said contained alcohol solution
is an alcoholic beverage.
19. The device of claim 15, wherein said oxygen scavenging membrane
fragments are derived from bacteria, yeast, fungi, plants, and
animals selected from the group consisting of beef heart, potato
tubers, spinach, Saccharomyces, Neurospora, Aspergillus, Euglena,
Acetobacter, Chlamydomonas, Escherichia, Bacillus, Salmonella,
Gluconobacter, and Pseudomonas.
20. The device of claim 15, wherein said oxygen scavenging membrane
fragments are cell membrane fragments derived from the organism
Escherichia coli.
21. A deoxidizing agent for reducing the dissolved oxygen
concentration of a solution containing alcohol, comprised of
membrane fragments which possess an electron transport system that
reduces oxygen to water in a solution containing alcohol.
22. The deoxidizing agent of claim 21, wherein said oxygen
scavenging membrane fragments are derived from bacteria, yeast,
fungi, plants, and animals selected from the group consisting of
beef heart, potato tubers, spinach, Saccharomyces, Neurospora,
Aspergillus, Euglena, Acetobacter, Chlamydomonas, Escherichia,
Bacillus, Salmonella, Gluconobacter, and Pseudomonas.
23. A method for removing oxygen from an acidic solution comprising
the steps of: a) providing an acidic solution containing oxygen;
and, b) adding to the acidic solution a sufficient amount of oxygen
scavenging membrane fragments to reduce the oxygen present in the
solution to water.
24. The method of claim 23, wherein said oxygen scavenging membrane
fragments possess an electron transport system which reduces oxygen
to water in acidic solutions.
25. A method for removing oxygen from an acidic solution comprising
the steps of: a) providing an acidic solution containing oxygen;
and, b) adding to the acidic solution a sufficient amount of oxygen
scavenging membrane fragments from an organism of the genus
Acetobacter to reduce the oxygen present in the solution to
water.
26. The method of claim 25, wherein said acidic solution comprises
a pH between 2.5 and 7.
27. The method of claim 25, wherein said oxygen scavenging membrane
fragments comprise cell membrane fragments from the organism
Acetobacter aceti.
28. The method of claim 25, wherein said oxygen scavenging membrane
fragments comprise cell membrane fragments from the organism
Acetobacter aceti, ATCC No. 23746.
29. The method of claim 25, wherein said acidic solution is
wine.
30. The method of claim 25, wherein said acidic solution is
beer.
31. The method of claim 25, wherein said acidic solution comprises
fruit juices.
32. A deoxidizing agent for reducing the dissolved oxygen
concentration of an acidic solution comprising the cell membrane
fragments of an organism from the genus Acetobacter.
33. A deoxidizing agent for removing the dissolved oxygen from an
acidic solution comprising the cell membrane fragments of the
organism Acetobacter aceti.
34. An antioxidant for preventing the oxidation of an acidic
solution comprising the cell membrane fragments of the organism
Acetobacter aceti.
35. The deoxidizing agent of claim 32, wherein said cell membrane
fragments are from the organism Acetobacter aceti, ATCC No.
23746.
36. A device for removing oxygen from a contained acidic acid
solution comprising a means for containing an acidic acid solution
having a solution contact surface and a non-solution contact
surface, wherein said solution contact surface contains a
sufficient amount of oxygen scavenging membrane fragments to reduce
the oxygen present in the solution to water.
37. The device of claim 36, wherein said contained acidic solution
is an acidic solution having a pH of about 2.5 and 7.
38. The device of claim 36, wherein said oxygen scavenging membrane
fragments are cell membrane fragments derived from an organism of
the genus Acetobacter.
39. The device of claim 36, wherein said oxygen scavenging membrane
fragments are cell membrane fragments derived from the organism
Acetobacter aceti.
40. The device of claim 39 wherein said oxygen scavenging cell
membrane fragments are derived from the organism Acetobacter aceti,
ATCC No. 23746.
41. A method for removing oxygen from an acidic alcohol solution
comprising the steps of: a) providing an acidic alcohol solution
containing oxygen; and b) adding to the acidic alcohol solution a
sufficient amount of oxygen scavenging cell membrane fragments from
an organism of the genus Acetobacter to reduce the oxygen present
in the solution to water.
42. The method of claim 41, wherein said acidic ethanol solution
comprises an ethanol solution having a pH between 2.5 and 7.
43. The method of claim 42, wherein said acidic ethanol solution
comprises an acidic solution containing from about 1 to about 18
weight percent alcohol.
44. The method of claim 14, wherein said acidic alcohol solution
comprises an acidic solution containing about 9.5 weight percent
ethanol.
45. The method of claim 41, wherein said acidic alcohol solution
comprises an acidic solution containing about 13 weight percent
ethanol.
46. A method for preventing the enzymatic browning reaction of
fruit which has been cut comprising the steps of: a) providing
fruit which has been cut; and, b) coating the cut fruit with a
solution containing sufficient amount of oxygen scavenging membrane
fragments to prevent oxidation deterioration from occurring.
47. A method for preventing the enzymatic browning reaction of
vegetable which has been cut comprising the steps of: a) providing
vegetable which has been cut; and, b) coating the cut vegetable
with a solution containing sufficient amount of oxygen scavenging
membrane fragments to prevent oxidation deterioration from
occurring.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a method, composition and
device for removing dissolved oxygen from solutions containing
alcohols and/or acids. The dissolved oxygen is removed from these
solutions for the purposes of retarding oxidation deterioration,
rancidity, gum formation, etc.
[0002] Ethyl alcohol, or ethanol (CH.sub.3CH.sub.2OH), is the basis
for the very large and prosperous alcoholic beverage industry which
offers a wide range of products varying in alcohol content from
less than one percent to greater than sixty percent. In addition,
ethyl alcohol, is also utilized industrially as an intermediate
reagent in numerous processes for the production of chemicals etc.,
and is used extensively in solvents, antiseptics, anti-freezing
compounds, and fuels.
[0003] In this regard, specifically denatured alcohols (i.e. ethyl
alcohol containing added denaturants such as methyl alcohol,
pyridine, benzene, kerosene, mixtures of primary and secondary
aliphatic higher alcohols etc.) have many uses including use in
food extracts, toiletries, pharmaceuticals, and cleaning products.
As an industrial solvent, it is reported that ethyl alcohol is
second to only water, and is a critical raw material in the
manufacture of drugs, plastics, lacquers, polishes, plasticizers,
perfumes etc.
[0004] Moreover, ethyl alcohol, either alone or in combination with
a wide variety of petroleum products, may be burned as a fuel.
Mixtures of ethyl alcohol blended with various petroleum
distillates are frequently referred to by the term "gasohol".
[0005] Ethyl alcohol may be produced either synthetically from
ethylene (i.e., by either the direct or indirect hydration of
ethylene) or by the natural fermentation of sugars, starches or
cellulose. While natural fermentation is still the principal means
for producing the alcoholic content found in beverages and food
products, the synthetic process is the method most frequently used
in the production of ethyl alcohol for commercial use.
[0006] In the natural fermentation of ethyl alcohol, the ethanol
may be derived from any material which contains sugar. In this
regard, the sugar present in the raw material can be converted
directly to ethyl alcohol, or if the sugar is contained in the raw
material in more complex forms (such as starches or cellulose), the
complex forms must first be converted to simple sugars by
hydrolysis, etc. The sugars are then fermented by enzymes from
yeast etc. to produce the ethyl alcohol.
[0007] An example of the production of ethyl alcohol from complex
forms of sugar is the alcoholic fermentation of starchy raw
materials in beer production. More particularly, beer is generally
defined as an alcoholic beverage made by the fermentation of
starchy materials such as barley, along with other brewing
ingredients such as corn, rice, wheat or oats. The starchy
materials are broken down by enzymes (i.e. hydrolyzed) during the
malting process to produce less complex water soluble compounds
such as sugars and short chained peptides. The sugars are then
fermented to produce the alcoholic content of the beer, which
varies greatly depending upon the critical ingredients and
processes utilized.
[0008] Along this line, most beers have an ethyl alcohol content of
between 2-6 weight percent. In addition, the alcoholic fermentation
reaction also yields minor by-products such as glycol, higher
alcohols (fusel oil comprising a mixture of n-propyl, n-butyl,
isobutyl, amyl and isoamyl alcohols) and traces of acetaldehyde,
acetic acid and lactic acid. These minor by-products are generally
produced in almost all types of alcoholic fermentation
reactions.
[0009] An example of the production of ethyl alcohol from simpler
forms of sugars is the natural fermentation process which occurs in
wine production. Although the production of wine is generally
associated with the fermentation of sugar from the juices of
grapes, juices from other fruits and plant material such as rice
etc. may be utilized. The alcoholic content in wine varies greatly
from less than 5 weight percent to greater than 18 percent.
[0010] Although beer, wine and other alcoholic beverages and food
products are somewhat immune to microbial spoilage as a result of
their ethyl alcohol content and/or low pH, oxidation deterioration
still occurs. In this regard, it is well known that the presence of
oxygen in products, including products containing ethyl alcohol
and/or acids, can cause a great deal of detrimental damage. For
example, carbonated and non-carbonated beverages and food products
having low pH's and/or containing ethyl alcohol such as fruit
juices, soft drinks, beer, wine, jams, jellies, and preserves, pie
fillings, salad dressings, pickles, relishes, and other condiments,
olives, sauerkraut, soups, vegetable juices, and pastes, etc. may
be unstable over even a relatively short period of time due to
undesirable changes produced by oxidative deterioration. Among the
oxidative changes which beverages and food products incur over time
include changes in color, consistency, and flavor. Since these
changes in the beverages and food products greatly decrease the
product's marketability, it is desirable to reduce the presence of
oxygen in the overall product.
[0011] In addition, it is also quite desirous to remove oxygen from
various commercial products having low pH's and/or contain ethyl
alcohol. This is particularly true in a number of chemical
products, wherein the presence of oxygen can create undesirable
by-products. For example, in pharmaceutical products, it is often
quite beneficial to remove oxygen to avoid contamination, formation
of intermediate free radicals, etc.
[0012] Furthermore, it is also advantageous to remove oxygen from
low pH and/or ethyl alcohol containing products which are stored
for relatively long periods of time in order to maintain the
packaging of the product. For example, if oxygen is present in the
beverage and/or food product, the oxygen included in the product
can also cause deterioration of the container's plastic or metal
lining, packaging etc. Thus, in modern beverage and food product
preparation systems, it is desirable to remove the extraneous
oxygen from the fluids to greatly increase the shelf life of the
packaged product.
[0013] This is particularly important in modern brewing operations,
wherein the feed stock must be almost completely deoxygenated in
that the presence of even a small fraction of oxygen can result in
an unacceptable product. As a result, in modern beverage and food
product operations, various deoxygenating devices including vacuum
systems, oxygen-purging apparatuses, etc. are used to extract the
oxygen.
[0014] However, vacuum dereators and gas flushing apparatuses are
fairly expensive and they do not necessarily reduce the dissolve
oxygen content to an acceptable level. Furthermore, these
apparatuses have some drawbacks in that the oils and lubricants
used therein sometimes find their way into the fluids being
treated. The inclusion of even a small amount of such harmful
agents within the beverage and/or food product can produce
undesirable color and/or flavor changes in the overall product, as
well as toxic effects.
[0015] In addition, in order to remove some of the oxygen which
slips by the vacuum dearators and/or the gas-flushing apparatuses,
it is sometimes desirable to add various chemical antioxidants to
the product for the purposes of retarding oxidation and associated
deterioration. However, a number of chemical antioxidants useful in
industrial products such as plastics and polishes, are not suited
for food products because of their toxicity. Moreover, the
consuming public is becoming increasingly more concerned about the
uses of chemicals and preservatives in foods and beverages
including antioxidants. Thus, a great deal of research is currently
being undertaken to develop not only more universal, but also
safer, antioxidants.
[0016] Chemical antioxidants are inorganic or organic compounds
added to various materials for the purposes of retarding oxidation
and associated deterioration. They may be utilized alone or in
combination with deoxygenating processes such as those indicated
above. It is thought that some of the chemical antioxidants operate
by binding with specific intermediate free radicals (i.e. peroxy
radicals) produced during oxidation degradation. By binding with
the intermediate free radicals, the free radicals are incapable of
propagating the chain reaction to decompose into other harmful free
radicals. As a result, by binding with the intermediate reactant,
antioxidants effectively inhibit the oxidation degradation
reaction. A more detailed explanation concerning the operating
mechanism of antioxidants may be found in Van Nostrand Reinhold
Encyclopedia of Chemistry, Fourth Edition, 1984.
[0017] The use of antioxidants in foods, pharmaceuticals, and
animal feeds, as direct additives is closely regulated because of
their potential toxicity. Along this line, when used in foods,
chemical antioxidants are regulated to extremely low percentages by
the Food and Drug Administration (FDA). Although antioxidants have
been utilized for several decades and occur naturally in some food
substances, intensive research continues in order to develop
universal non-toxic antioxidants.
[0018] In this regard, the desirable properties of antioxidants,
particularly when used in food products, may be summarized as
indicated by Van Nostrand Reinhold, supra, by the following
characteristics: (1) effectiveness at low concentrations; (2)
compatibility with the substrate; (3) non-toxicity to consumers;
(4) stability in terms of conditions encountered in processing and
storage, including temperature, radiation, pH, etc.; (5)
non-volatility and non-extractability under the conditions of use;
(6) ease and safety in handling; (7) freedom from off-flavors,
off-odors, and off-colors that might be imparted to the food
products; and (8) cost effectiveness. As a result, antioxidants
vary greatly depending upon such factors as the composition of the
substrates, pH, temperature, processing conditions, impurities
etc.
[0019] An example of a common chemical antioxidant currently being
utilized in products containing alcohols and/or acids is the use of
sulfur dioxide gas (SO.sub.2) and its related sulfite salts (i.e.
sodium sulfite, potassium metabisulfite etc.) Sulfur dioxide gas
and its sulfite salts are widely used for preserving fruits and
fruit juices, alcoholic beverages produced from fruit juices,
vegetables and vegetable juices, syrups, concentrates, purees etc.
In addition, sulfur dioxide and its sulfite salts also extend the
storage life of raw fruit and vegetables by preventing the
enzymatic "browning" reactions associated with oxidative
degradation.
[0020] The effectiveness of sulfur dioxide gas and its sulfite
salts varies considerably depending upon the concentration and pH
conditions of the product desired to be protected. The preferred
operating pH range of sulfur dioxide and its sulfite salts for
preventing oxidation and inhibiting microbial degradation appears
to be about a pH of 2.5-3.5.
[0021] As a result of this effective pH range, sulfur dioxide and
its sulfite salts are used extensively in the production and
storage of wine. The sulfites are used not only for sanitizing
equipment etc., but also for inhibiting the growth of any natural
microbial flora present on the fruit prior to fermentation. This is
done prior to the addition of pure cultures of the appropriate wine
making yeast to prevent growth and competition of undesirable
organisms. During fermentation, the sulfites act not only as an
antioxidant but also as a clarifier and dissolving agent.
Furthermore, sulfur dioxide and its sulfite salts are often used
after fermentation and during storage to prevent oxidation
degradation and/or undesirable postfermentation alterations by
various microorganisms. The levels of sulfur dioxide and its
sulfite salts present in wine during storage varies greatly
depending upon the condition of the fruit, temperature, pH, sugar
concentrations etc. but is normally in the range from about 20 to
about 70 ppm.
[0022] Although the use of sulfur dioxide and other chemical
antioxidants has proven to be quite beneficial for controlling
oxidative degradation of various products, including those products
containing alcohols and/or acids, a number of serious undesirable
side effects can also be produced. This can be particularly
demonstrated in regard to the use of sulfur dioxide and/or sodium
sulfite as a chemical antioxidant in wine, fruit juices etc.
wherein a portion of the public is allergic and/or hypersensitive
to the sulfites utilized. Hence, it would be desirous to produce a
safe, non-toxic substance which continuously removes oxygen from
food products and chemical substances containing alcohols and/or
acids without producing any harmful side effects to the end
products or user.
[0023] Accordingly, the present invention is directed to a method,
composition and device for continuously removing oxygen from
solutions containing alcohols and/or acids in a safe and efficient
manner. The method and composition of the present invention may be
utilized as an antioxidant in industrial solutions containing acids
and/or alcohols such as plastics, polishes etc., as well as
beverages and food products, without altering the desired
properties of the products produced thereby. The method and
composition of the invention fulfill the desired properties of an
effective antioxidant as indicated above.
SUMMARY OF THE INVENTION
[0024] In one aspect, the present invention is directed to a method
for removing oxygen from a solution containing alcohol. The method
comprises the steps of providing a solution containing alcohol and
oxygen; and, adding to the solution a sufficient amount of oxygen
scavenging membrane fragments to reduce the oxygen present in the
solution to water. The oxygen scavenging membrane fragment utilized
in the invention contain an electron transport system which is
sufficiently effective (i.e. active) in a solution containing
alcohol to reduce oxygen to water.
[0025] In another aspect, the present invention relates to a device
for removing oxygen from a contained alcohol solution. The device
comprises a means for containing an alcohol solution having a
solution contact surface and non-solution contact surface, wherein
the solution contact surface contains a sufficient amount of oxygen
scavenging membrane fragments to reduce the oxygen present in the
solution to water.
[0026] In an additional aspect, the present invention is directed
to a deoxidizing agent for reducing the dissolved oxygen
concentration of a solution containing alcohols. The deoxidizing
agent consists of membrane fragments which possess an electron
transport system that reduces oxygen to water in solutions
containing alcohols.
[0027] In a further aspect, the present invention relates to a
method for removing oxygen from an acidic solution. The method
comprises the steps of providing an acidic solution containing
oxygen; and, adding to the acidic solution a sufficient amount of
oxygen scavenging membrane fragments to reduce the oxygen present
in the solution to water.
[0028] In still another aspect, the present invention is directed
to a method for removing oxygen from an acidic solution. The method
comprises the steps of providing an acidic solution containing
oxygen; and, adding to the acidic solution a sufficient amount of
oxygen scavenging membrane fragments from an organism of the genus
Acetobacter to reduce the oxygen present in the solution to
water.
[0029] In still a further aspect, the present invention relates to
a deoxidizing agent for reducing the dissolved oxygen concentration
of an acidic solution. The deoxidizing agent consists of membrane
fragments which possess an electron transport system that reduces
oxygen to water in solutions containing acids. More particularly,
the present invention is directed to a deoxidizing agent for
removing the dissolved oxygen from an acidic solution wherein the
deoxygenating agent consists of membrane fragments of an organism
from the genus Acetobacter.
[0030] In an additional aspect, the present invention is directed
to a device for removing oxygen from a contained acidic solution.
The device comprises a means for containing an acidic solution
having a solution contact surface and a non-solution contact
surface, wherein said solution contact surface contains a
sufficient amount of oxygen scavenging membrane fragments to reduce
the oxygen present in the solution to water.
[0031] In a still another aspect, the present invention relates to
a method for removing oxygen from a solution containing both acids
and alcohols. The method comprises the steps of providing an acidic
ethanol solution containing oxygen; and, adding to the acidic
ethanol solution a sufficient amount of oxygen scavenging membrane
fragments from an organism of the genus Acetobacter to reduce the
oxygen present in the solution to water.
[0032] In a still further aspect, the present invention is directed
to a method for presenting the enzymatic browning reaction of
sliced fruits and/or vegetables. The method comprises the steps of
providing fruit and/or vegetables which have been cut or sliced;
and, coating the exposed area with a solution containing a
sufficient amount of oxygen scavenging membrane fragments to
prevent oxidation deterioration from occurring.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention is directed to a novel method,
composition and device for removing dissolved oxygen from solutions
containing alcohols and/or acids. By removing oxygen from various
products, the present invention is an effective antioxidant for
beverages and food products, as well as for industrial and
commercial solutions containing alcohols and/or acids.
[0034] More particularly, one embodiment of the present invention
relates to a method and device for removing oxygen from solutions
containing alcohol through the use of oxygen scavenging membrane
fragments. The membrane fragments, which contain an electron
transport system that reduces oxygen to water, may be obtained from
various sources. For example, the membrane fragments may be
obtained from the cell membranes of various bacteria, such as the
organism Escherichia coli, and/or from the mitrochondria membranes
of non-bacterial organisms. Although it was known that cell
membrane fragments from various bacteria, such as the organism
Escherichia coli, possessed an electron transport system consisting
of a series of enzymes that worked in cooperation with one another
to reduce oxygen to water, it was thought that as a result of the
normal intolerance of enzymes to alcohols such as ethanol, the
electron transport system present in the cell and/or mitochondrial
membrane fragments would have been ineffective in solutions
containing alcohol.
[0035] However, as more particularly demonstrated below, the
experimental results collected by the present inventors clearly
indicate that when the pH of solutions containing acids and/or
alcohols, such as wine, were adjusted to a pH within normal
operating ranges of a particular preparation of oxygen scavenging
enzymes present in the membrane fragments (i.e. the electron
transport system of cell membrane fragments from the organism
Escherichia coli has a normal pH range of about 5.5 to about 9.5,
with an optimal pH of 8.4), the enzymes present in the membrane
fragments were still very effective in reducing the oxygen to water
and thus removing the oxygen from the product.
[0036] As stated above, this was greatly unexpected because it is
well known that ethyl alcohol denatures enzymes and other proteins
(Fruton and Sinmonds, General Biochemistry, Second Edition, John
Wiley & Sons, 1958).
[0037] In addition, further testing by the present inventors
indicated that even if very little of the membrane fragments were
added to the solutions containing alcohol, such as ethanol (i.e.
wine etc.), and the pH of the solutions were adjusted to a pH range
within the normal operating parameters of the membrane fragments,
the very low activities of the enzymes present within the membrane
fragments were sufficient to remove all of the dissolved oxygen
present in the solutions in a relatively short period of time. This
was a significant result, in that the lower effective level of the
membrane fragments, the lower the probability of any adverse
effects (i.e. changes in odor, taste and appearance) occurring in
the desired end product.
[0038] Furthermore, when the antioxidant properties of the membrane
fragments utilized in the present invention were compared with
known antioxidants of solutions containing alcohol, such as the
sulfates used in wine, the results indicated (see below) that the
membrane fragments utilized in the present invention were much more
effective than the prior art. More particularly, the results
indicated that when membrane fragments possesing the critical
electron transport system of the present invention were added to a
pH adjusted solution containing ethyl alcohol (i.e. wine), the
electron transport system utilized in the present system
immediately reduced the dissolved oxygen concentrations to very low
levels and maintained them there throughout the testing period
(which was for approximately eight weeks) at temperatures higher
than that normally used for storing wine. In contrast, the current
commercial method used (i.e. sulfited wines) showed a slow and
protracted reduction of dissolved oxygen concentration over the
testing period.
[0039] Moreover, when after the eight week testing period was
completed and the bottles of wine were open and reoxygenated, only
the wine containing the membrane fragments possessing the electron
transport system used in the present invention showed a reduction
in the reintroduced oxygen with time. Hence, the membrane fragments
utilized in the present invention clearly operated as a more
effective antioxidant in solutions containing ethanol than the
current commercial methods utilized.
[0040] The oxygen scavenging bacterial cell membrane fragments
utilized in the present invention, as well as the process for
isolating and purifying same, are similar to the membrane fragments
and filtration process disclosed in U.S. Pat. No. 4,476,224 for
"Material and Method for Promoting the Growth of Anaerobic
Bacteria", issued on Oct. 9, 1984 to Howard I. Adler, Oak Ridge,
Tenn., one of the co-inventors of the present invention. The '224
patent is incorporated herein by reference.
[0041] The '224 patent is directed to a method of removing
dissolved oxygen from a nutrient medium for anaerobic bacteria
through the use of sterile membrane fragments derived from bacteria
having membranes which contain an electron transport system which
reduces oxygen to water in the presence of a hydrogen donor in the
nutrient medium. It is known that a great number of bacteria have
cytoplasmic membranes which contain the electron transport system
that effectively reduces oxygen to water of a suitable hydrogen
donor is present in the medium. Some of the bacterial sources
identified in the '244 patent include Escherichia coli, Salmonella
typhimurium, Gluconobacter oxydans, and Pseudomonas aeruginosa.
These bacterial membranes have been highly effective in removing
oxygen from media and other aqueous and semi-solid
environments.
[0042] The same oxygen reducing effects produced by the cell
membrane fragments from the bacterial sources indicated above, are
also present in the membrane of mitochondrial organelles of a large
number of higher non-bacterial organisms. More particularly, a
great number of fungi, yeasts, and plants and animals have
mitochondria that reduces oxygen to water, if a suitable hydrogen
donor is present in the medium. Some of the sources of oxygen
reducing membranes from these mitochondria are: beef heart muscle,
potato tuber, spinach, Saccharomyces, Neurospora, Aspergillus,
Euglena and Chlamydomonas. The process of producing the useful
mitochondrial membrane fragments involves the following steps:
[0043] 1. Yeast, fungal cells, algae and protozoa, having
mitochondrial membranes containing an electron transfer system
which reduces oxygen to water, are grown under suitable conditions
of active aeration and a temperature which is conducive to the
growth of the cells, usually about 20.degree. C. to 45.degree. C.
in a broth media. Alternately, mitochondria may be obtained from
cells of animal or plant origin.
[0044] 2. The cells are collected by centrifugation or filtration,
and are washed with distilled water.
[0045] 3. For the preparation of crude mitochondrial membrane
fragments, a concentrated suspension of the cells is treated to
break up the cell walls and mitochondria. This is accomplished by
known means, for example, by ultrasonic treatment or by passing the
suspension several times through a French pressure cell at 20,000
psi.
[0046] 4. The cellular debris is removed by low speed
centrifugation or by microfiltration (cross-flow filtration).
[0047] 5. The supernatant or filtrate is subjected to high speed
centrifugation (175,000.times.g at 5.degree. C.) or
ultrafiltration.
[0048] 6. For the preparation of material of higher purity, the
cells of step 2 are suspended in a buffer containing 1.0M sucrose
and are treated by means which break up the cell walls or membranes
but leave the mitochondria intact. This is accomplished by known
means, for example, by ultrasonic treatment, passage through a
French pressure cell at low pressure, enzymatic digestion or high
speed blending with glass beads.
[0049] 7. The cellular debris from step 6 is removed by
differential centrifugation or filtration.
[0050] 8. the supernatant or retentate from step 7 is passed
through a French Press at 20,000 psi to break the mitochondria into
small pieces.
[0051] 9. Mitochondria debris from step 7 is removed by
centrifugation at 12,000.times.g for approximately 15 minutes or by
microfiltration.
[0052] 10. The supernatant or filtrate from step 9 is subjected to
high speed centrifugation (175,000.times.g at 5.degree. C.) or
ultrafiltration.
[0053] 11. The pellet or retentate from step 5 (crude mitochondrial
fragments) or the pellet or retentate from step 10 (purified
mitochondrial membrane fragments) are resuspended in a buffer
solution at a pH of about 6.0 to about 8.0. A preferred buffer
solution is 0.02M solution of
N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES)
[0054] 12. The membrane fragments in the buffer solution are then
passed under pressure through a filter having openings of about 0.2
microns.
[0055] 13. The suspension is then stored at about -20.degree. C.
for later use or it may be freeze dried.
[0056] This process, as well as the media produced thereby, is the
subject matter of a separately filed co-pending U.S. patent
application, i.e. Ser. No. 938,190, filed on Dec. 5, 1986 for
"Material and Method for Promoting Growth of Anaerobic Bacteria".
The cell and/or mitochondrial membrane fragments utilized in the
present invention are produced according to the processes set forth
in the '224 patent and the above identified co-pending
application.
[0057] The process of the present invention can be utilized for
removing oxygen in a wide variety of aqueous solutions or
semi-aqueous solutions containing alcohols. Examples of such
solution containing alcohols, are those solutions containing
ethanol including beverage and food products, such as beer and
wine, as well as industrial products such as chemical solvents,
antiseptics, antifreezing compounds and fuel.
[0058] Furthermore, while most natural beverage and food products
do not require the addition of a hydrogen donor in order for the
enzyme system present in the membrane fragments to reduce the
oxygen present in the product to water, when synthetic ethanol
containing solutions are utilized, the addition of a hydrogen donor
(i.e. an organic substrate) may be necessary in order for the
membrane fragments to perform their oxygen removing functions.
Suitable hydrogen donors are lactic acid, succinic acid,
alpha-glycerol phosphate, formic acid, malic acid and, where
available their corresponding salts.
[0059] Moreover, the temperature of the reactant solution may also
have to be adjusted to optimize the deoxygenation process. In this
regard, the temperature range for activity is wide, from a low of
5.degree. C. to a high of about 60.degree. C. Operating under
optimal conditions, the present invention can lower dissolved
oxygen to approximately 0.1 ppm or below. The membrane fragments
are equivalent in oxygen reducing ability to a strong, chemical
reducing agent, such as sodium hydrosulfite.
[0060] In addition, while the oxygen scavenging membrane fragments
may be added directly to the ethanol solution for the purposes of
deoxygenating the solution, the membrane fragments may also be
indirectly added to the ethanol solution by incorporating the
membrane fragments into the solution-contact surface of the means
utilized for containing the ethanol solution. In this regard, the
membrane fragments can be incorporated into a large variety of
solution-contact surfaces such as the polymeric liners of bottles
and cans, the plastic container itself etc. By incorporating the
membrane fragments into the solution-contact surfaces, the storage
conditions of the packaged material are improved without directly
effecting the solution.
[0061] An additional embodiment of the present invention is
directed to a method and composition for removing oxygen from
solutions containing acids. This embodiment is distinct from that
set forth above in that the presence of ethyl alcohol in the
solution is not necessary (although it can be present) and the pH
of the solution need not be adjusted to the operational ranges
required in the first embodiment.
[0062] The additional embodiment of the invention is a direct
result of the discovery that not only are the membrane fragments
effective antioxidants in solutions containing alcohols, the cell
membrane fragments from certain organisms, such as the organisms of
the genus Acetobacter, are also effective in removing oxygen from
acidic solutions. This is particularly important because unlike the
cell membrane fragments from the organism Escherichia coli, the
cell membrane fragments from the organism of the genus Acetobacter
are acceptable for food use. Hence, the cell membrane fragments
from the organism of the genus Acetobacter are efficient
antioxidants for beverage and food products in that the membrane
fragments reduce or remove oxygen from solutions containing
alcohols as well as acids.
[0063] More particularly, it was discovered that although a number
of organisms can exist in acidic environments, not all of these
organisms possess the properties necessary to be a safe and
effective antioxidant suitable for use in food products and
beverages. Along this line, the present inventors have discovered
that cell membrane fragments from organisms of the genus
Acetobacter remove oxygen not only from solutions containing
alcohols, but also from those containing acids. These cell membrane
fragments also fulfill the desired properties of an effective
antioxidant set forth above.
[0064] Specifically, the present inventors have discovered that the
electron transport system found in the cell membrane fragments of
the organism Acetobacter aceti ATCC #23746 (NCIB 8554) remove all
of the dissolved oxygen from acidic solutions containing alcohols
in a relatively short period of time, i.e., the cell membrane
fragments from the organism Acetobacter aceti ATCC #23746 removed
all of the dissolved oxygen from white wine (pH of 3.4) in 79.1
minutes at 37.degree. C. and 29.3 minutes at 32.degree. C.) See
Example 5 below. The oxygen scavenging properties of the cell
membrane fragments of Acetobacter aceti are novel in that this is,
to the inventors knowledge, the first time the properties of the
fragments have been described.
[0065] Moreover, the data produced in the above discovery indicated
that the optimum pH of the enzymes of the electron transport system
present in the cell membrane fragments of Acetobacter aceti is
about 5.2 and the operational range is from about 3 to 8.0. This is
in sharp contrast to the electron transport system for the cell
membrane fragments from Escherichia coli which has an optimum pH of
8.4 and a operational range from 6.0 to 9.0. Furthermore, the data
indicated that the cell membrane fragments from the organism
Acetobacter aceti were also quite effective in removing oxygen from
non-alcoholic acidic solutions such as tomato juice (pH=4) and cola
soft drinks (pH=3). The enzymes of the electron transport system
from the Escherichia coli cell membrane fragments failed to produce
any activity at the low pH's.
[0066] The process of the present invention directed to the removal
of oxygen from acidic solutions can be used for removing oxygen in
various acidic aqueous or semi-aqueous solutions. Examples of such
solutions include carbonated and non-carbonated beverages and food
products such as fruit juices, soft drinks, salad dressings,
pickles, relish, and other condiments, olives, sauerkraut,
vegetable juices, purees, jams, jellies and preserves, as well as
industrial and/or commercial solutions containing acids. In
addition, acidic solutions containing ethanol, such as beer and
wine, may also be utilized. The cell membrane fragments of the
organism of the genus Acetobacter can be added either (i) directly
into the acid solutions, or (ii) indirectly by incorporating the
membrane fragments into the solution-contact surface of the
packaging container.
[0067] Furthermore, while most natural beverage and food products
contain a sufficient amount of organic substrates to provide the
hydrogen needed to reduce the oxygen present in the solution to
water, additional hydrogen donors such as lactic acid, succinic
acid, alpha-glycerol phosphate, formic acid, malic acid and/or
their corresponding salts can also be added to the acidic solution,
especially those designated for commercial use.
[0068] In addition, the present invention may also be utilized for
preventing the enzymatic "browning" reaction which occurs in the
oxidation degradation of fruits and vegetables. Specifically, the
enzymes in fruits and vegetables cause apples, apricots, bananas,
potatoes, among others, to darken when they are exposed to air
after being cut, bruised or allowed to over mature. The membrane
fragments of the present invention may be utilized in order to
prevent and/or delay the enzymatic browning reaction from
occurring.
[0069] As more particularly indicated by the data set forth below,
the present inventors discovered that when the exposed portion of
freshly cut fruits or vegetables were dipped or coated with a
solution containing the membrane fragments of the present
invention, the browning reaction which occurs in oxidative
deterioration, was delayed. This was specifically true when the
fruits or vegetables used with the cell membrane fragments of the
organism Escherichia coli had a relatively neutral pH, i.e. potato
has a pH=6.5. However, when the fruits or vegetables utilized were
of low pH (i.e. less than 5), the cell membrane fragments from the
organism Escherichia coli were ineffective in preventing the
browning reaction from occurring. This is because the electron
transport system of the cell membrane fragments of the organism
Escherichia coli is inactive at such a low pH. This difficulty may
be overcome by utilitizing membrane fragments having an electron
transport system (such as cell membrane fragments from the
organisms of the genus Acetobacter) which is effective in acidic
solutions.
[0070] The following examples are set forth for the purpose of
further illustrating the preferred embodiments of the present
invention.
EXAMPLE 1
[0071] In order to determine the effectiveness of the electron
transport system of the cell membrane fragments obtained from the
organism Escherichia coli (i.e. "Oxyrase") in ethanol (ethyl
alcohol) solutions, a sufficient amount of ethanol alcohol was
added to three 0.34 units/ml of a suspension of the cell membrane
fragments to provide solutions containing 0, 9.5 and 13.0 weight
percent ethyl alcohol. In this regard, one unit of a suspension of
the cell membrane fragments is the amount of membrane fragments
that reduces 1.0% of dissolved oxygen per second per milliliter of
a solution containing 1.75 ml of a 10 mM sodium lactate solution in
20 mM phosphate buffer at a pH of 8.4 and a temperature of
37.degree. C. The membrane fragments were either isolated and
purified by the process set forth above (i.e., the process set
forth in the '224 patent and/or the '190 application) or the
membrane fragments were commercially purchased from Oxyrase, Inc.,
Ashland, Ohio. The pHs and temperatures of the three solutions were
adjusted to produce solutions having a pH of 7, and a temperature
of 37.degree. C. Two control samples, one containing 1 unit of cell
membrane fragments and the second containing phosphate buffer, were
ran at 37.degree. C. at a pH=8.4 with 0 weight percent ethanol.
Sodium lactate was added to the solutions for the purposes of
providing a substrate (i.e. hydrogen donor) for the reduction of
oxygen to water.
[0072] Upon the addition of the cell membrane fragments to the two
solutions, the amount of time (minutes) required for the electron
system present in the membrane fragments to remove 100 weight
percent oxygen from the ethanol samples was determined utilizing an
oxygen sensor (Oxygraph 5/6, Gilson International, Middleton,
Wis.). The results of are set forth in Table 1.
1TABLE 1 EFFECT OF ETHANOL ON THE ACTIVITY OF CELL MEMBRANE
FRAGMENTS FROM THE ORGANISM ESCHERICHIA COLI Time Required to
Remove Oxygen (Minutes) % Oxygen pH 8.4 pH 7.0 buffer - pH 7.0
buffer - Removed buffer pH 7.0 buffer 9.5% ethanol 13.0% ethanol 25
1.2 1.7 3.5 4.7 50 2.5 3.4 7.5 9.9 75 4.1 5.3 10.9 15.3 100 5.3 7.7
14.3 19.3
[0073] The data indicated that although affected somewhat by the pH
and ethanol concentrations, the electron transport system present
in the cell membrane fragments from the organism Esherichia coli
performed in the presence of ethanol, particularly in the amount of
ethanol normally found in wine. As indicated above, the tolerance
of the electron transport system present in the cell membrane
fragments was highly unexpected in that enzymes are often
intolerant of ethanol.
EXAMPLE 2
[0074] A similar test to that set forth above was performed on wine
samples provided by Andre Wines, Ltd., Winona, Ontario, Canada, in
order to determine whether the electron transport systems present
in the cell membrane fragments were effective in various natural
solutions containing ethanol. Since the effectiveness of the cell
membrane fragments as an antioxidant in wine was to be determined,
the wine utilized did not contain chemical antioxidants such as
potassium metabisulfite, sodium sulfite etc.
[0075] More particularly, 0.34 units/ml of a suspension of the cell
membrane fragments from the organism Escherichia coli were added to
the following wine samples provided by Andr:
[0076] White wine used: 1988 Seyval--
[0077] Residual Sugar=2.11 G/L
[0078] Sweet white used: 1988 Seyval--
[0079] Residual Sugar=24.7 G/L
[0080] Red wine used: 1988 Dechaunac--
[0081] Residual Sugar=3.8 G/L
[0082] The wine samples possessed ethanol concentrations of between
12 and 14%. Prior to the addition of the cell membrane fragments,
the pHs and temperatures of the wine samples were raised to a pH of
7 and temperature of 37.degree. C. in order to accommodate the
activity of the cell membrane fragments from Escherichia coli. As a
result of the inclusion in the various wine samples of natural
hydrogen donating substrates such as lactic acid, the addition of
an additional substrate was not required. The activities of the
electron transport systems present in the Escherichia coli cell
membrane fragments in the various wine samples were determined. The
results are set forth below in Table 2.
2TABLE 2 The Activities of Cell Membrane Fragments in Wine Time
Required to Remove Oxygen (Minutes) Percent Oxygen Removed White
Wine Red Wine Sparkling Wine 25 2.4 2.4 1.4 50 5.7 5.4 3.7 75 9.7
9.4 5.8 100 16.0 15.8 9.3
[0083] The data indicated that the electron transport system
present in the Escherichia coli cell membrane fragments removed the
dissolved oxygen in the wine samples completely in 9.3 to 16
minutes at 37.degree. C. The test results demonstrated that the
cell membrane fragments of Escherichia coli were an effective
antioxidant of pH adjusted wine and other naturally produced
ethanol containing solutions. Moreover, the results further
indicated that in order to produce an antioxidant which was
effective in natural products having low pHs, cell membrane
fragments exhibiting electron transport systems having lower pH
activity profiles were necessary.
EXAMPLE 3
[0084] In order to determine whether very low concentrations of the
electron transport systems present in the cell membrane fragments
would perform in solutions containing ethanol, the amount of time
required to remove the oxygen in previous aerated wine samples
containing 0.0075 units of the cell membrane fragments/ml at
33.degree. C. was determined. In addition, the activity of the
chemical antioxidant, potassium metabisulfite
(K.sub.2S.sub.2O.sub.2) in aerated wine, was also compared to the
activity of the low concentration of cell membrane fragments from
the organism Escherichia coli at a pH of 7. The results are set
forth in Table 3 below.
3TABLE 3 The Activities of Potassium Metabisulfite and Low
Concentrations of Cell Membrane Fragments in Wine at a pH = 7 Time
Required to Remove Oxygen (Minutes) Percent Sweet, white Sweet,
white Dry, white Oxygen wine + wine + wine + Dry, white wine +
Removed Oxyrase bisulfite Oxyrase bisulfite 25 6.3 -- 5.3 -- 50
18.0 -- 24.0 -- 75 -- -- -- -- 100 60 min. >150 min. 50 min.
>320 min. (est.) (est.) (est.) (est.) Assays run at .0075 units
oxygen/ml and 33.degree. C.
[0085] The data indicated that even as little as 0.0075 units/ml of
cell membrane fragments would remove all of the dissolved oxygen in
about 50 to 60 minutes at 33.degree. C. Furthermore, the results
indicated that potassium metabisulfite, if it is effective at all,
is much slower than the cell membrane fragments of the present
invention.
[0086] The significance of the observation that only a very small
amount of cell membrane fragments was necessary in order to remove
oxygen from ethanol containing solutions is both practical and
economic. The lower the effective level of the cell membrane
fragments, the lower the probability of adverse effects on odor,
taste and appearance. In addition, the lower the effective
concentration, the greater the opportunity for commercial
success.
EXAMPLE 4
[0087] The effectiveness of the electron transport system from the
cell membrane fragments of the organism Escherichia coli (i.e. 2.5
MG/L OXYRASE) as an antioxidant was compared to the chemical
antioxidant sulfur dioxide (50 MG/L free SO.sub.2) in pH adjusted
wines (i.e. pH=7). In this regard, the following Andr wines
utilized:
[0088] White Wine Used: 1988 Seyval--Res. Sug.=2.11 G/L
[0089] Sweet White Used: 1988 Seyval--Res. Sug.=24.7 G/L
[0090] Red Wine Used: 1988 Dechaunac--Res. Sug.=3.8 G/L
[0091] The wines were first clarified with -350 MG/L bentonite (a
colloidal clay, such as aluminum silicate, composed chiefly of
montmorillonite) and filtered in the lab via a M-70 filter. The
wines were treated and bottled. The bottles were then incubated at
elevated temperatures (i.e. at 30.degree. C..+-.1.degree. C.) to
provide accelerated stability data. Periodically, the bottles were
opened and their dissolved oxygen concentrations were determined as
indicated below in Table 4.
4TABLE 4 THE DISSOLVED OXYGEN (D.O.) CONCENTRATION IN WINE TREATED
WITH VARIOUS ANTIOXIDANTS Day Two Four Six Eight One weeks weeks
weeks weeks Com- Sample D.O. D.O. D.O. D.O. D.O. ments Dry white
8.90 2.23 1.23 0.590 0.360 Oxyrase wine - control Dry white 8.90
0.480 0.220 0.190 0.180 activity wine - sulfite Dry white 8.10
0.170 0.151 0.120 0.120 excel- wine - Oxyrase lent Sweet white 9.10
1.90 0.920 0.440 0.320 Oxyrase wine - control Sweet white 9.10
0.970 0.250 0.210 0.18 activity wine - sulfite Sweet white 8.70
0.176 0.176 0.150 0.12 excel- wine - Oxyrase lent Dry white 8.44
1.57 0.800 0.410 0.35 Oxyrase wine - control Dry white 8.44 0.470
0.350 0.190 0.15 activity wine - sulfite Dry white 8.10 0.120 0.130
0.130 0.12 excel- wine - Oxyrase lent NOTE: all units in MG/L
[0092] The data showed that the electron transport system present
in the cell membrane fragments immediately reduced the dissolved
oxygen concentrations to very low levels and maintained them there
throughout the test for eight weeks. In contrast, the sulfite
wines, the current commercial method used, showed a slow and
protracted reduction of dissolved oxygen over the test period and
did not reach the low levels produced by Oxyrase even after eight
weeks.
[0093] It was further interesting to note that the untreated wine
(control) also showed a reduction in dissolved oxygen that was
slightly behind the sulfited wines. This indicated that the
dissolved oxygen in the unprotected wines was reacting with the
wine, (i.e. the wine itself was being reduced). This is precisely
what vintners want to prevent. As a result, the data indicated that
the use of sulfites, under these conditions, as an antioxidant was
only marginal at best.
[0094] Furthermore, after eight weeks, the bottles of the sweet
white wine were opened and reoxygenated by aerating the wine to its
saturation point (.about.8.10 MG/L). After the bottles of the
reoxygenated wine were kept outside at room temperature for seven
days, the dissolved oxygen content of the reoxygenated wine was
determined as indicated in Table 5.
5 TABLE 5 Seven days Sample D.O. (MG/L) Sweet white wine--control
8.07 Sweet white wine--sulfite 8.08 Sweet white wine--Oxyrase
5.25
[0095] The results indicated that only the wine treated with the
cell membrane fragments from the organism Escherichia coli (i.e.
"Oxyrase") showed a reduction in the quantity of the reintroduced
oxygen with time. Hence, unlike sulfites, the cell membrane
fragments utilized in the present invention maintained their
activities even after eight weeks.
EXAMPLE 5
[0096] In order to obtain cell membrane fragments having low pH
optimum electron transport systems, the cell membrane fragments of
a number of acidic strains of organisms were evaluated. In this
regard, the inventors discovered that organisms from the genus
Acetobacter were particularly well suited for removing oxygen not
only from acidic solutions but also from solutions containing
ethanol. This was particularly interesting because although the
organisms of the genus Acetobacter are not the most acidic strains
of organisms available, various species of Acetobacter are
compatible with food products.
[0097] Along this line, the present inventors have discovered that
the organism Acetobacter aceti, ATCC No. 23746 (NCIB 8554) is an
excellent source for cell membrane fragments ("Aceto-Oxyrase")
having an electron transport systems which is optimum at low pHs.
In addition, not only was the electron transport system of the cell
membrane fragments from Acetobacter aceti (i.e. "Aceto-Oxyrase")
effective in acidic solutions, they were also effective in
solutions containing ethanol.
[0098] Specifically, the relative pH activities of the electron
transport system of cell membrane fragment of the organism
Acetobacter aceti in 0.02 M KH.sub.2PO.sub.4+10 mM lactate at
37.degree. C. (i.e. 40.mu. of cell membrane fragments which is
equivalent to 1.32 units of Oxyrase activity measured at optimal
pH) were determined and set forth below in Table 6.
6TABLE 6 The pH OPTIMUM OF ACETO-OXYRASE IN 0.2 M KH.sub.2PO.sub.4
+ 10 mM LACTATE AT 37.degree. C. Time for Complete pH Oxygen
Removal (sec.) % of Maximum Activity 3.5 1328 (22.1 min.) 9.90 4.0
480 (8.0 min.) 27.50 4.5 212 (3.53 min.) 62.30 5.1 132 (2.2 min.)
100.00 5.4 132 (2.2 min.) 100.00 6.1 168 (2.8 min.) 78.60 7.0 284
(4.7 min.) 46.50 8.0 1448 (24.1 min.) 9.10 8.0 156 (2.6 min.)
[0099] The data indicated that the optimum pH for the electron
transport system of the cell membrane fragments of the organism
Acetobacter aceti (Aceto-Oxyrase) is about 5.2, with an operational
range from at least about 3 to about 8.0. This is in sharp contrast
to the activity of the electron transport system present in the
cell membrane fragments of the organism Escherichia coli, which has
an optimum pH of 8.4 and an operational range from about 6.0 to
about 9.0. As a result, this novel type of electron transport
system used as an antioxidant for solutions containing alcohols and
acids has not been described before.
[0100] Furthermore, when the activity of the electron transport
system present in the cell membrane fragments of Escherichia coli
(i.e. E.C. 100 from Oxyrase, Inc., Ashland, Ohio) in white wine at
a pH=3.4 was compared with that of the cell membrane fragments of
Acetobacter aceti (i.e. "Aceto-Oxyrase") (see Table 7 below), the
data indicated that the Aceto-Oxyrase removed all of the dissolve
oxygen in white wine at 37.degree. C. in 79.1 minutes, and at
32.degree. C., in only 29.3 minutes. The electron transport system
of the cell membrane fragments of the organism Escherichia coli
showed no activity at this pH. In addition, the data showed that
the electron transport system from the cell membrane fragments of
the organism Acetobacter aceti was more effective i.e. had a higher
activity at a lower temperature than that of Escherichia coli. This
is particularly interesting since the optimal storage conditions of
many low pH and/or ethanol solutions is at lower temperatures.
7TABLE 7 ACTIVITY OF ACETO-OXYRASE AND EC 100 IN WHITE WINE Ph 3.4
Temperature Time for Complete Oxygen Removal EC 100 37.degree. C.
00 EC 100 32.degree. C. 00 Aceto-Oxyrase 37.degree. C. 4748 secs.
or 79.1 min. Aceto-Oxyrase 32.degree. C. 1760 secs. or 29.3
min.
EXAMPLE 6
[0101] The amount of time required for the electron transport
system present in the membrane fragments of the organism
Acetobacter aceti (i.e. "Aceto-Oxyrase") to remove the dissolved
oxygen completely from a number of solutions containing acids
and/or alcohols was determined according to a procedure modified
somewhat from that indicated above. The modified procedure was
necessary in order to enhance the readings produced by the Oxygraph
oxygen sensor. Preliminary experiments according to the unmodified
procedure indicated that the bubbles of certain carbonated
beverages, such as beer, as well as particles in the solutions,
such as the vegetable particles in tomato juice, interfered with
the ability of electrode in the oxygen sensor to produce smooth
tracings or readings.
[0102] In order to avoid these difficulties, the carbonated
beverages were first degassed by repeated shaking at room
temperature and/or the solutions were filtered or clarified by
centrifugation at 3,000 rpm for 10 minutes prior to analysis. The
treated solutions, i.e. beer or tomato juice, were then introduced
into the Oxygraph chamber until the chamber was completely filled
(i.e. approximately 1.75 ml). Since the solutions, i.e. beer and
tomato juice, contained natural substrates, the addition of
substrates to the solution was not necessary. The samples were
equilibrated at 25.degree. C. for five minutes. After equilibration
the oxygen saturation value was obtained and then 40 .mu.l of
"Aceto-oxyrase" (i.e. cell membrane fragments from the organism
Acetobacter aceti, approximately 28 mg/ml dry weight) was injected
into the samples. The time required to remove all measurable oxygen
was determined and set forth below in Table 8.
8TABLE 8 TIME REQUIRED FOR ACETO-OXYRASE TO REMOVE THE OXYGEN IN
VARIOUS SOLUTIONS Time (min.) Required Time (min.) Required to
remove 50% of to remove 100% of Solution pH initial Oxygen initial
Oxygen Tomato Juice 4.0 5.6 11.4 (Campbell's Soup Co. Camden, NJ)
Michelob Beer 4.7 2.8 5.3 (trademarked and sold by Anheuser- Busch,
Inc., St. Louis, MO)
[0103] The results indicated that although the electron transport
system present in the membrane fragments of the organism
Acetobaceter aceti (i.e. "Aceto-Oxyrase") was being utilized below
its optimum pH (which is approximately 5.3), the electron transport
system present in the membrane fragments successfully removed all
of the oxygen from both beer and tomato juice at 25.degree. C. in a
relatively short period of time.
[0104] In order to determine whether the electron transport system
present in membrane fragments of the organism Acetobacter aceti
(i.e. "Aceto-Oxyrase") was effective in fairly acidic solutions
which failed to contain natural hydrogen donating substrates, a
similar test to that set forth above was performed in solutions of
Classic Coke (trademarked and sold by The Coca-Cola Co., Atlanta,
Ga.) containing the added substrate, sodium lactate. In this
regard, previous experiments had indicated that no substrate was
naturally present in Classic Coke, thus 20 .mu.l of a 1 M sodium
lactate solution was also introduced into the test chamber
containing the Classic Coke. The procedure utilized was the same as
that used for the beer and tomato juice except that the analysis
was performed at 15-18.degree. C., rather than 25.degree. C. The
time required to remove all measurable oxygen was determined and
set forth below in Table 9.
9TABLE 9 TIME REQUIRED FOR ACETO-OXYRASE TO REMOVE OXYGEN IN
CLASSIC COKE Time (min.) Required Time (min.) Required to remove
50% of to remove 100% of Solution pH initial Oxygen initial Oxygen
Classic Coke 3.0 6.0 min. 12.0 min. (The Coca-Cola Co., Atlanta,
GA)
[0105] The results indicate that the electron transport system
present in the membrane fragments of the organism Acetobacter aceti
(i.e. "Aceto-Oxyrase") was effective in removing oxygen from fairly
acidic solutions which failed to contain natural substrates, if a
substrate was subsequently added to the sample.
EXAMPLE 7
[0106] In order to determine the effectiveness of the membrane
fragments for controlling the enzymatic "browning" reaction which
occurs in the oxidation degradation of fruits and vegetables,
samples of sliced fruit and vegetables were coated with a solution
of the membrane fragments. The effects the treatments had on the
"browning" of the samples were observed.
[0107] Specifically, samples of sliced or cut apples (pH=3-4),
bananas (pH=4.5-5), and potatoes (pH=6.5) were placed into separate
solutions of membrane fragments from the organism Escherichia coli
(0.3 units/ml of membrane fragments). In addition, samples of
sliced apples, bananas and potatoes were placed in a lactate
solution (0.01 M sodium lactate), a substrate for the cell membrane
fragments, to serve as controls. The effectiveness of the membrane
fragments for controlling the browning of the samples was visually
observed at both ambient (i.e. 18-20.degree. C.) and refrigerator
(i.e. 4.degree. C.) temperatures and the earliest time of
noticeable browning occurring was recorded. The results indicated
that although the browning reactions of the samples of sliced
apples and bananas were unaffected by the membrane fragments, (i.e.
the fruit turned brown over time) a strong positive result (i.e. no
observable color change) was obtained from the sample of sliced
potatoes in the Escherichia coli membrane fragments at ambient
temperatures when compared to the controls. In this regard, the
treatment of the potato slices in the solution of the cell membrane
fragments from the organism Escherichia coli delayed the onset of
browning for period of about five times (5.times.) as long as that
observed in the control.
[0108] Furthermore, by maintaining the sample of the sliced
potatoes in the solution of the membrane fragments at refrigeration
temperature (i.e. 4.degree. C.), the browning reaction was delayed
about seven times (7.times.) as long as that observed in the
lactate control at ambient temperature (i.e. 18-20.degree. C.).
However, when compared to lactate controls at refrigerated
temperatures (i.e. 4.degree. C.), the browning reaction was delayed
only slightly (i.e. 20 to 60%).
[0109] The failure of the membrane fragments of the organism
Escherichia coli in controlling the browning reaction of the sliced
apples and bananas was attributed to the low pH of the respective
fruit. As indicated above, the electron transport system present in
the membrane fragments of the organism Escherichia coli has an
optimal pH=8.4 and an operating range of about 6 to about 9. Thus,
the membrane fragments of the organism Escherichia coli were only
effective in controlling the browning reaction of the potatoes
having a pH of 6.5.
[0110] The invention has been described with reference to the
preferred embodiments. Obviously, modifications and alterations
will occur to others upon reading and understanding the preceding
detailed description. It is intended that the invention be
construed as including all such alterations and modifications
insofar as they come within the scope of the appended claims and
the equivalent thereof.
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