US 2971850 A
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Feb. 14, 1961 R. R. BARTON SCAVENGER PACKET Filed NOV. 25, 1958 643- PERMEABLE MEMBRANE "WA 7ER-IMPERMEABLQ IN V EN TOR.
RICHARD [2. BAR TON A TTORNEV United States Patent Ofii 2,971,850 Patented eh. 14, 1
SCAVENGER PACKET Richard R. Barton, Waldwick, N.J., assignor to Miles Laboratories, Inc., Elkhart, Ind., a corporation of Indiana Filed Nov. 25, 1958, Ser. No. 776,388 11 Claims. (Cl. 99-171) This invention relates to a deoxygenating process and to a product useful therein. Particularly, the invention relates to a deoxygenating body and the method of its use. More particularly, the invention relates to a deoxygenating body, or scavenger packet, which comprises an enzyme system having glucose oxidase activity, a substrate for said enzyme system and a liquid, the components of said body being confined in a water-impermeable, gas-permeable membrane in such fashion that one of the components is separated from the other components by a rupturable seal.
Those familiar with the art of enzyme chemistry are aware of enzymatic processes for the removal of oxygen or glucose from food or other products through the use of an enzyme system having glucose oxidase activity. The reaction upon which these processes are based proceeds in accordance with the following schematic diagram:
glucose glucose-l-Ori-H O gluconie acid-l-HzOz oxidase .catalase 21120: 2 20+ 9 One such process is described and claimed in US. Reissue Patent No. 23,523, issued to Dwight L. Baker, July 22, 1952. The principle of basic patent is referred to in US. Patent No. 2,758,932, issued August 14, 1956, to Don Scott.
The Scott patent teaches a method of deoxygenating a hermetically sealed container by means of a deoxygenating body comprising a dispersion of water, glucose, and an enzyme system having glucose oxidase activity. In this patent the components of the deoxygenating body are separated from the contents of the container by an oxygen permeable, moisture-proof barrier such as cellophane, wax paper, and the like.
The instant invention is closely related to the packet of the Scott patent, but, as will be pointed out below, represents an improvement thereon.
It has been found that even under the most carefully controlled conditions a deoxygenating body prepared in accordance with the teachings of the Scot patent, that is, when the components of the deoxygenating body are in contact with each other within the membrane envelope, becomes substantially inactive and loses it ability to take up oxygen after the lapse of short periods of time if exposed to the atmosphere. Preparation of these prior art packets in the absence of atmospheric oxygen, and maintaining them under a nitrogen blanket until use ha proven impractical. Attempts have been made to package the dry components and inject the requisite liquid immediately prior to use by some means such as a hypodermic needle, but this expedient also proved to be unsatisfactory on a commercial scale.
It has now been found, however, and forms the object of this invention, that the above disadvantages may be overcome if a deoxygenating body comprising a liquid, an enzyme system having glucose oxidase activity, and a substrate for said enzyme is enclosed in a packet of two ccmpartments separated by a rupturable seal, one com-. partment containing one of the three components of the body and the other compartment containing the other two. In such a device no reaction can take place since all of the components must be in intimate contact before the enzyme will oxidize the substrate. The two compartments are separated by a rupturable seal which breaks under pressure allowing the three components to mix and thus activating the deoxygenating body. Such rupture is accomplished immediately prior to use of the packet and thus the packet has all its activity preserved to the time of use.
Fig. 1 represents a schematic drawing of one embodiment of the scavenger packet of this invention. Fig. 2 represents an end view of the packet of Fig. 1. Fig. 3 represents a second embodiment of the inventive concept, and Fig. 4 represents an end view of the packet of Pi 3.
lurning now to the drawing, the packet or envelope of the figures are constructed of a gas-permeable, water-impermeable membrane such as polyethylene, laminated polyethylene, cast tefion, pliofilm, styrene, cellophane, and the like. The material must be gas-permeable to permit the passage of oxygen or oxygen containing gas such as air, and water-impermeable to prevent the passage of liquid. It will be understood that in certain embodiments of the invention, such as that shown in Fig. 3, that one compartment of the packet may be composed of a gas and liquid impermeable film. It is essential, however, that a gas-permeable liquid-impermeable film separate the total mixture from the surrounding area in order that oxygen containing gas may pass through the film and react in accordance with the equation set out above.
The packet of Figures 1 and 2 is constructed so that there is present two compartments 2 separated by a rupturable seal 4. The other edges of the compartments 6 are heat-sealed by methods known to the art.
As was stated above, the deoxygenating body enclosed within the packet comprises an enzyme having glucose oxidase activity, a substrate for that enzyme and a liquid. In the packet of Figures 1 and 2, two of these three components are confined within one of the compartments 2 and the other component is confined in the other compartment. For example, the enzyme may be confined in one compartment and the substrate and the liquid in the other; the enzyme and the liquid may be mixed and placed in one compartment and the substrate in the other; or the enzyme and substrate confined in one compartment and the liquid in the other. It is essential that the components of the deoxygenating body e kept separated prior to use to prevent deactivation of the deoxygenating body. The packet is activated immediately prior to use by breaking the rupturable seal 4 by passing the packet through low clearance rollers, or other obvious means, thus allowing the three components to become admixed. Oxygen or an oxygen-containing gas passes through the membrane and is utilized in the reaction, thus completely deoxygenating the air-space around the packet.
For use in vacuum packaging, it may be desirable that a pinhole be placed in each compartment of the packet to permit rapid passage of air and the concomitant equalization of pressure at the time of vacuum app1ica tion. This prevents rupture of the packet due to the sudden pressure differential caused by the vacuum application during vacuum packing.
The utility of the packet of the invention is obvious; one or more, as desired, may be activated and placed in a container of material subject to oxidative deterioration and the'container hermetically sealed. The oxygen in the container is consumed by the system and the contents of the container remain free of oxidative deterioration as long as the seal remains unbroken.
As has been previously stated, the components of the 'deoxygcn'ating body comprise a liquid, an enzyme system having glucose oxidase activity, and a substrate for said enzyme system. The liquid component is preferably water, although other liquids which do not impair the activity of the'enzyme system may be used. Other substances may be included, such as enzyme activators, humecta'nts, extenders, and the like. Such substances include aqueous acetate or citrate buffer, aqueous sodihm'gluconate, aqueous glycerine, dilute ethanol and the like.
Enzyme systems having glucose 'oxidase activity have as their primary and principal constituent the enzyme "glucose oxidase. One such enzyme system which also contains catalase and which is commercially available contains 1500 glucose oxidase units per gram, a unit being "defined as that amount which will take up 10 cubic millimeters of oxygen per minute under the following conditions: 3.3% cerelose, 0.4% sodium salt of dehydroactetic acid, M/ 10 phosphate butler pH 5.1 and temperature 30 C. This enzyme system also contains up to about 1100 units of catalase per gram, a unit being defined as that amount of catalase which will decompose 300 mg. of H under standard conditions. As is well known, the 'catalase breaks down hydrogen peroxide to water and molecular oxygen.
The substrate for the enzyme system will, of course, contain glucose. Preferably the substrate contains a major amount of glucose such as is present in cerelose, dextrose, corn syrup, etc.
Since the enzyme system operates most efficiently at a pH of'between 4.5 and 8.0, and since gluconic acid is one :product of the reaction, it is preferred to have present a bufiering agent in an amount sufficient to maintain the pH of the activated packet within the optimum pH range. Suitable buffers include alkali metal phosphates, calcium carbonate, sodium acetate, sodium citrates and the like.
It is preferred that the substrate for the enzyme system be thoroughly admixed with a filler material to increase surface area to speed the oxygenation reaction and :to assist in preventing the liquid from diffusing through theliquid impermeable film. The filler, preferably selected from the group of ground balsa wood pulp, cereal products, fine sawdust, carboxymethyl cellulose, agar, gelat n, gum, silica gel, cliatomaceous earth, etc., prevents the liquid from gasifying and escaping through the gaspermeable membrane.
It 1 s desired that there be present in the formulation a stabilizer material in order to insure against loss of enzyrne potency. This stabilizer material may comprise s lica gel, glycerine or similar materials. It will normally be present in amounts varying between about 0.10 to 0.33 g. per ml. of solution, preferably from about 0.15 to about 0.25 gm./ml.
It has been found advantageous to include in the formulation a preservative material in order to assist the stabilizer in insuring against loss of enzyme potency. Thispreservative may be selected from sodium gluconate solution, a solution of other salts of food acid and the like. Although it has been found that from 0.10 to 0.50 ml. of a sodium gluconate having a molarity between 0.38 and 1.5 is operable, it is preferred to use from 0.25 to 0.35 ml. of a sodium gluconate solution having molarity of 0.75 per packet.
It will be understood, of course, that difie'rent amounts 5 above that moisture content of the packet had a deleteof the components of the deoxygenating body will be used depending upon the amount of oxygen to be consumed.
Ranges of operable and preferred proportions are set outin tabular form below:
Range of components Operable Preferred Enzyme system (units) 100-550 200-400 Substrate:
Glucose (gms) 1130-2. 00 0. -1. 50 Stabilizer (mg./ml.) 0. 10-0. 33 0.15-0. 2a Preservative (g. 0.10-0.50 '0. 25-035 Filler (gum) 0. 10-1. 00 0. 30-0. 60 Buffer (gun) 0. 01-0. 30 0018-01200 Water (mL) 1. 00-3. 00 1.80-2.20
The invention described in general terms above Will be more specifically explained by reference to the following; illustrative examples:
EXAMPLE I Scavenger packets in accordance with the inventive concept were prepared usingv thefollowing formulation as the dry component:
Glucose oxida'se 2.0,lbs. Cerelose -1. 400 lbs. Filter aid ('diatomaceous'earth) 7 lbs. 9 ozs. Calcium carbonate 1.0 lbs. Sodium acetate 1 lb. 14 ozs. Citric acid .2 lbs."'8 ozs.
TABLE I Efiect of temperature on oxygen pickup Mm. O2 consumed 48 hours. 108.h0u1s EXAMPLE II.
Efiect of inois i ui'e On oxygl P Percent Mm. 02124 I120. hours absorbed EXAMPLE HI Since it was apparent from the data in Example 11 rious effect upon the rate of oxygen uptake, the packets of the following formulation were prepared:
TABLE III Grn. Mm. Or pickup Balsa Wood EXAMPLE IV Glucose oxidase was dissolved in a sodium gulconate solution of varying molarity and diluted with 30% glycerine to a potency of 150 glucose oxidase units per m1. Two (2) ml. of this liquid was placed in one compartment of a 1 mil polyethylene packet and in the other compartment was placed 1.5 g. or the following formulations:
Grams Cerelose 145.8 Silica gel 10.0 Calcium carbonate 4.0 Sodium acetate 6.9 Citric acid 9.3 Balsa wood .5 Sodium gluconate Variable The efiect of varying the amount of the sodium gluconate in the formulation on oxygen pickup is set out in Table IV below.
TABLE IV Molarity Mm. O:
Na gluconsumed conate 24 hours EXAMPLE V The liquid component of the packet of Example IV containing varying amounts of sodium gluconate wasv tested for stability over a 30 day period. Results of this stability test in glucose oxidase units per ml. are set out in Table V below.
TABLE V Potency (u./ml.) Molarity Na gluconate Original After 1 6 EXAMPLE VI The scavenger packets of Example IV were activated and immediately sealed in No. 2 cans and the oxygen uptake at the end of 24 hours was measured. The data obtained are set out in Table VI below.
TABLE VI Molarity Mm. 02
sodium consumed gluconate EXAMPLE VII In one compartment of a 1 mil polyethylene packet prepared in accordance with the concept of this invention there was placed 1.3 g. of cerelose and 0.2 g. of citric acid. In a similar packet there was placed 1.3 g. of cerelose, 0.2 g. of citric acid and 0.5 g. of ground balsa wood. In the other compartment of each packet there was placed 2 ml. of a glucose oxidase solution prepared as described in connection with Example IV above. The packets were sealed, the rupturable seal broken immediately prior to testing and the packets placed in No. 2 cans which were hermetically sealed. At the end of 24 hours the packet without the balsa Wood consumed 10,810 mm. 0 whereas the packet containing the balsa wood consumed 26,200 mm. O
EXAMPLE VIII TABLE VII Citric Acid Mm. Oz
(mgm) consumed EXAMPLE IX In one compartment of a 1 mil polyethylene packet there was placed 1.8 g. of a dry mix having the following formula:
Parts Cerelose Citric acid 2 Balsa wood 50 In the other compartment there was placed 2.0 ml. of a liquid glucose oxidase composition prepared as described in Example IV above. The packets were sealed and the rupturable seal broken immediately before placing in No. 2 cans filled with a standard volume of a white cake mix. Similar packets were placed in a No. 2 can containing walnut meats. After the expiration of one week the percentage of oxygen remaining in the head space was measured.
In the case of white cake mix, those cans containing scavenger packets were found to contain 9.5% oxygen. Control cans without scavenger packets contained 20.3% oxygen. In the case of the walnut meats those cans containing the scavenger packets contained 10.5% oxygen;
the liquid component calculated.
Parts Cerelose 130 Citric acid 2 Balsa wood 50 In the other compartment there was placed 2.0 m1. of
a liquid glucose oxidase composition prepared as described in Example IV above.
The packets were weighed and held at room temperature and room humidity for 72,hours. At the end of that period they were reweighed and the moisture loss of It was found that at the end of 72 hours the moisture loss amounted to 19.7 mg. (0.07%).
At the end of 5 /2 hours the oxygen uptake was measured to be 12,500 cu. mm. At the end of 72 hours the oxygen uptake was 19,500 cu. mm.
EXAMPLE XI Using a polyethylene having a thickness of 0.50 mil, packets were prepared as described in connection with Example X above. These packets were placed in a desiccator over calcium chloride for one week and lost 2.57% of the moisture of the liquid component during that time.
Packets prepared from this polyethylene were measured for oxygen uptake at the end of 23 hours. The oxygen uptake was found to be 56,400 cu. mm.
EXAMPLE. XII Using the components as described in connection with the packets of Example X above, but using cast Teflon films of various thicknesses, packets were prepared and the oxygen uptake measured at the end of 24 hours.
The data obtained are set out in Table IX below:
TABLE IX Teflon, Mm. 0
mil. consumed (24 hrs.)
Operable Preferred Enzyme system (units) 100-550 200-400 Substrate:
Glucose (gms.) 0. 33-2. 00 0. 75-1. 50 Stabilizer (gIlL/Hll.) 0.10-0.33 0.15-0.25
Preservative (g) 0. -0; 50 0. -0. 35
Filler (gnL) 0. 10-1. 00 0. 30-0. 60 Buffer (gm.) '0. 01-0. 30 0. 018-0. 200 Water (m1.) 1. 00-3. 00 1. 80-2. 20
Particularly preferred and contemplatedfin preferred embodiment of this invention is a eompositionotthe following formulation:
Membrane mil polyethylene 0.50 Glucose oxidase units 300 Water ml 1.4 Cerelose gm.' 1.3 Citric acid gm .02 Glycerine ml. .60 Sodium gluconate grri. 0.38 Balsa wood (6-9 1b./ cu. .ft.-coarse ground) In this preferred embodiment of this invention a solution of the sodium gluconate in the water is mixed with the glycerine and sufficient glucose oxidase to give a potency of 300 units (150 u./ ml.). This liquid is placed in one compartment of the polyethylene packet and 1.80 g. of a mix of the cerelose, balsa wood and citric acid is placed in the other compartment.
What is claimed is:
1. A deoxygenating body comprising a liquid enzyme system having glucose oxidase activity and a substrate for said enzyme system, said deoxygenating body being enclosed in a water-impermeable, gas-permeable membrane,
said liquid enzyme system being separated from said substrate by separating means including a seal rupturable upon application of pressure to said membrane.
2. A deoxygenating body according to claim 1 wherein said liquid enzyme system contains a stabilizing material.
3. A deoxygenating body according to claim 1 wherein said liquid enzyme system contains sodium gluconate.
4. A deoxygenating body according to claim 1 containing in addition to said enzyme system and said substrate a buffer material and a filler.
5. A deoxygenating body according to claim 1 wherein said substrate comprises cerelose, said body contains citric acid and ground balsa wood.
6. An article of manufacture comprising an envelope of a water-impermeable, gas-permeable membrane having two compartments separated from. each other by a seal rupturable upon application of pressure to said envelope, one of said compartments containing a liquid enzyme system having glucose oxidase activity and the other compartment containing asubstrate for said enzyme systern.
7. An article of manufacture according to claim 6 wherein said enzyme system comprises a solution of glucose oxidase in a solution of a stabilizing material.
8. An article of manufacture according to claim 6 wherein said enzyme system comprises a mixture of glucose oxidase, sodium gluconate and glycerine in a Water solution.
9. An article of manufacture according to claim 6 wherein said substrate compartment contains a mixture of a glucose source, a buffering agent and a filler.
10. An article of manufacture according to claim 6 wherein said substrate compartment contains a mixture of cerelose, citric acid, and ground balsa wood.
11. An article of manufacture according to claim 6 wherein the liquid enzyme system has a potency offrom to 550 glucose oxidase units, contains from .15 to .25 grams of sodium gluconate and from .25 to .35 ml. of glycerine per ml. of solution, and wherein said substrate compartment contains from .75 to 1.5 guns. of cerelose, from .18 to .22 gms. of citric acid, and from 0.3 to 0.6 gms. of ground balsa wood.
References Cited in the file of this patent UNITED STATES PATENTS Re. 23,523 Baker July 22, 1952 2,245,738 Taylor June 17, 1941 2,758,932 Scott Aug. 14, 1956 2,758,934 Scott Aug. 14, 1956 2,765,233 Sarrett et a1. Oct. 2, 1956 2,825,651 Loo et a1. Mar. 4, 1958