WO1990004336A1

WO1990004336A1 - Glucose oxidase food treatment and storage method

Info

Publication number: WO1990004336A1
Application number: PCT/FI1989/000198
Authority: WO
Inventors: Pirkko Kaarina Aaltonen; Paavo Olavi Lehtonen; Ulla Karilainen
Original assignee: Stabra Ag
Priority date: 1988-10-28
Filing date: 1989-10-26
Publication date: 1990-05-03
Also published as: US4996062A; AU4421689A; EP0396688A1; AU637585B2; CA2001752A1

Abstract

The invention relates to an enzymatic composition and a method of extending the shelf life of foodstuffs. The composition contains a glucose oxidase/catalase enzyme preparation. The method can be used both in normal and modified atmosphere packages.

Description

GLUCOSE OXIDASE FOOD TREATMENT AND STORAGE METHOD

The present invention relates to the extension of the shelf life of packaged, prepared foods, such as meat and broiler patties, smoked fish, mayonnaise based veget¬ able salads, as well as sausages, bread and egg-butter by reducing the oxygen content with an enzymatic method.

The invention also relates to a method for extend¬ ing the shelf-life of foods, whereby an enzymatic reaction creates an environment within the package that is microbi- cidal or at least microbistatic.

The invention further relates to the use of an en¬ zymatic method to reduce the population of pathogenic and food-spoilage microorganisms within a food product. The invention also relates to the use of this en¬ zymatic method alone or together with a modified atmo¬ sphere packaging technology.

The shelf life of a food product is most often- dependent on the rate of microbiological spoilage. Both the characteristics of the food product itself, such as water activity, pH, redox potential, antimicrobial agents, chemical and biological composition of the product, as well as the environment in which it is stored (tempera¬ ture, humidity and the gas composition of the package) effect the rate of spoilage.

In some cases preservatives can be added to pro¬ hibit both the growth of microorganisms and the production of toxins harmful to humans. The most widely used preser¬ vatives are benzoic acid and sodium benzoate, ethyl-, methyl- and propylparabens, sorbic acid and calcium, sodium and potassium sorbates, as well as propionic acid and calcium, sodium and potassium propionates.

Benzoic acid and sodium benzoate can be used in products with low pH values, the greatest activity being at pH values below 4.5. At neutral pH values these com- pounds are essentially ineffective. This restricts the use of benzoic acid and sodium benzoate to the clearly acidic products such as salad dressings, soft drinks and ketchups. The problem with soft drinks is that the ben- zoates may impart a disagreeable taste even at very low levels.

Ethyl-, methyl- and propylparabens are less sensi¬ tive to pH than benzoic acid and benzoates. The use of these preservatives in most foods is, however, strictly prohibited by law in many countries.

Sorbic acid and sorbates are the most widely used preservatives. Their antimicrobial activity is also de¬ pendent on the pH value but unlike benzoic acid and ben¬ zoates they still work at pH values as high as 6.0 - 6.5. However, the use of sorbates as food preservatives is limited by law to particular food products. In many countries, it is not allowed in prepared foods such as those described in the present invention.

Propionic acid and propionates are mainly used in bread and other grain products to prevent molding. In general, most countries limit the use of propionates to these types of products.

Although many of the above mentioned chemical pre¬ servatives are effective in preventing microbial growth and therefore extending the shelf-life of food products in which they are used, increased awareness of the health risks associated with such preservatives is currently leading to greater restrictions on their use. Commonly used preservatives such as sulphites have been found to cause allergic reactions in unsuspecting consumers. Even in cases where a preservative has been found to be safe, "health conscious" consumers and consequently food producers have sought to find more "natural" methods of storing foods. One such method is by the removal of oxygen. Because many of the microorganisms that are responsible for the spoilage of food require oxygen to grow, their growth in food products can be restricted or, in some cases, completely prevented by the elimination of oxygen from the environment in which they are stored.

Oxygen removal has been suggested for the purpose of minimizing detrimental oxidative processes in food. For example, processed foods have been packaged either in a vacuum or in a modified atmosphere. It is also known to remove oxygen by placing bags containing iron powder, sulfites or ascorbic acid within the packages (Prepared Foods 3: 91-98, 1988, and Food Technology 40:94-97, 1988). However, the bag is harmful to humans when consumed by accident. It has also been sug- gested to use plastic bags containing glucose, glucose oxidase, catalase and water to extend the shelf life of fat-containing products (Scott, D., Enzymes in Food Processing, 2nd ed., 1975, p. 526 and 527). Nevertheless previous attempts to develop well-functioning and effec- tive oxygen removal agents have not been successful.

With the method of the present invention, it is possible to replace the use of preservatives, anti- oxidants and oxygen removal bags harmful to humans when consumed as well as vacuum packages deforming the food products and to make the preservation of food products by protective gas more efficient.

The level of injurious free oxygen is reduced in vacuum-packaging technology, thus decreasing the rates of microbiological spoilage and oxidative reactions. The problems with vacuum-packing are that prepared foods with round shape tend to flatten, slices stick together and liquid frequently is drawn out of the product.

In modified atmosphere packaging, the shelf life of a product can be extended over that in air. The suit- able gas composition depends on the product. The most common gas compositions are 20% C0₂ + 80% 0₂, 20% C0₂ + 80% N«, 100% N₂, and different combinations of the said gases 0₂, C0₂ and N₂. The problem with modified packaging technology is that great amounts of carbon dioxide cause discolouration and packages shrink when carbon dioxide is absorbed by the product. In some cases, the carbon dioxide can cause off-odors when the package is opened. The amount of residual oxygen is at times sufficient to cause oxidative reactions. In such cases, it can be re- moved with oxygen absorbers such as iron powder, sulfites, ascorbic acid or glucose oxidase.

The use of oxygen absorbers such as iron powder, ascorbic acid or sulfites are also problematic. The addi¬ tion of ascorbic acid is regulated by law and it is not always possible to add enough ascorbic acid to prevent all oxidative reactions. Sulfites react with oxygen producing many harmful by-products and sulfites alone can cause off- odors and allergic reactions. When using iron powder care must be taken that the product is not commingled with the food.

Enzyme systems have been suggested for the removal of oxygen from packaged foods. Such systems have, how¬ ever, been wrought with difficulty as the necessary com¬ binations of enzymes, enzyme levels necessary to ensure the creation of the desired conditions, necessary reac- tants, and other factors necessary to make such enzyme systems workable have not been determined. As a result, these methods have not, to date, been successful for either preserving food products or presenting a consum- able product.

Recently, significant production breakthroughs have enabled the production of high yields of purified, highly active glucose oxidase that contains little or no contamination with the enzyme catalase. This has made it possible to develop complex enzyme compositions that solve the foregoing difficulties.

It is therefore, the object of this invention to present an enzymatic method to preserve foodstuffs whereby carefully controlled levels of glucose oxidase and catalase are added to increase the shelf life of food products.

It is also the object of this invention to provide a method whereby glucose oxidase is added to the food product to reduce the oxygen level below 1%, thereby inhibiting the growth of aerobic spoilage-causing microor¬ ganisms.

A further object of the invention is to introduce an enzymatic method whereby catalase is introduced to a food product in combination with glucose oxidase to eli- minate residual peroxide created by the reaction of glucose oxidase with glucose and oxygen.

The object of the invention is further to provide a method whereby glucose oxidase and glucose are added to a food product along with low levels of catalase, such that the microbicidal effect of the hydrogen peroxide on both food spoilage as well as pathogenic microorganisms can be accomplished prior to its elimination by the catal¬ ase.

A further object of the invention is to provide packaging technology wherein residual oxygen is removed from a food package using a glucose oxidase enzyme prep¬ aration.

According to the invention, when the ob ect is the inhibition of growth of aerobic spoilage organisms, a com- position containing a mixture of the enzymes glucose oxidase and catalase is added to a foodstuff, which is then enveloped in an air tight package in the presence of air or a substantially non-reactive gas. Preferably, the package is completely air tight but controlled leakage of air into the package can be accommodated by this method. The composition causes removal of oxygen from the air in the package. Preferably, the reaction occurs in a perfor¬ ated or highly oxygen-permeable plastic bag, a paper bag laminated with plastic, a bag of non-woven fabric, or in a separate compartment attached to the package. In this way the oxygen-binding agent does not come into contact with the food product. The agent contains an aqueous solu¬ tion of glucose oxidase absorbed in an inert carrier prov¬ iding a large surface area, such as cellulose powder, microcrystalline cellulose powder, talcum, or diatomaceous earth, as well as glucose or a glucose producing agent, water and a neutralizer safe to humans, such as calcium carbonate or sodium carbonate, which binds the gluconic acid formed in the reaction. The amount of glucose oxidase added is based on the air volume of the foodstuff package. Typically, about 200 to about 1500 units of enzyme are added per 1 liter of air in the package. It is especially preferred to use between about 300 and about 800 units of enzyme per liter of pack- age air to ensure adequate removal of oxygen, without leaving excess glucose oxidase when the package is opened. (Excess enzyme can cause rapid production of acid thus causing off flavors in the food).

Because the relative amounts of glucose oxidase and catalase are an important feature of the invention, it is most preferred to use a highly purified glucose oxidase preparation that is essentially free from contamination by catalase.

When the desired result is removal of oxygen, the ratio of glucose oxidase to catalase used is about 1:1 to 100:1, most preferably between about 3:1 and about 6:1. Preferably, glucose is also combined with the en¬ zyme mixture to form the composition to be added to the foodstuff. To ensure complete removal of oxygen from the product headspace, glucose is added to achieve a concen- tration in the food of at least 3.0 grams of glucose per liter of package oxygen with the preferred level being about 6.0 grams of glucose per liter of package oxygen.

When the object according to the invention is direct reduction of the microbial load of the food, the amount of glucose oxidase added is determined based on the volume of the food. To ensure adequate reaction of the glucose oxidase within the microenvironment of the food product, the enzyme is added at a level of 10 to 1000 units per 1 kilogram of food. Although the optimal quan¬ tities depend on the consistency of the food, as well as other conditions such as pH, temperature, etc., the most preferred level of enzyme for most applications is about 15-50 units per kilogram. When a reduction of the microbial load of the food and removal of oxygen from the product and its package headspace are desired, the glucose oxidase to catalase ratio can be adapted to maximize the exposure of suscep¬ tible microorqanisms to hydrogen peroxide, a product of the glucose oxidation process, prior to its breakdown by catalase. A preferred ratio of at least 5:1 glucose oxidase to catalase is used to accomplish this objective. The most preferred ratio is about 7:1 to about 100:1 glucose oxidase to catalase. Under this embodiment of the invention for directly reducing the microbial load, it is preferable to add glucose to the product at a level of from about 0.2 to about 1.0 gram per 1 liter of food.

It is preferred to combine the glucose oxidase, catalase and glucose into a single composition to be added to the food product. Most preferably the composition con¬ taining enzymes and glucose is combined with the product when it is prepared. Alternatively, the composition can be sprayed or spread onto the surface of the food product. Where the main objective is to maximize reduction of the microbial population of the food product prior to packaging, without reducing substantially the oxygen con¬ tent of package air, the product can be treated with glucose oxidase, then subsequently treated with catalase and packaged.

Yet another preferred embodiment of the method is to incorporate a glucose oxidase/glucose mixture into a product package to ensure removal of oxygen from the product headspace. Preferably the level of glucose oxidase incorporated is about 200 to about 1500 units per liter of package headspace. According to this embodiment, the glucose oxidase can be incorporated into a discrete compartment of the package. Glucose is also incorporated into the package such that it can be allowed to react with the enzyme and the package oxygen.

The present invention extends the shelf life of prepared foods, such as fish-, meat- and broiler patties, different kinds of meat rolls, as well as smoked fish, sausages, bread and egg-butter by removing oxygen from the package air space surrounding the prepared food, and/or by creating a microbicidal environment within the food product. The microbicidal environment is effective in reducing both the level of food spoilage and the level of pathogenic microorganisms in the foodstuff. The inven¬ tion employs an enzyme composition that can be combined with the prepared food or the package material. The com¬ position is harmless to humans and mammals and can be con¬ sumed. Although not intended to be a limitation of the invention, the invention is based on the recognition that the reaction of glucose and oxygen as catalyzed by the enzyme glucose oxidase is effective in several ways in preserving the shelf-life of food stuffs. The invention is based on the known reaction in which glucose oxidase catalyzes the reaction between glucose and oxygen. In the reaction D-gluconic acid and hydrogen peroxide are produced according to equation (1). The reaction continues until either glucose or oxygen is consumed. Hydrogen peroxide is decomposed by catalase into water and oxygen (2) .

D-glucose + 0₂ glucose oxidase D-gluconic acid + H₂0₂ (1)

H₂0₂ catalase H₂0 + 1/2 0₂ (2)

The present invention employs this reaction to increase the shelf life of foods both by creating an en¬ vironment that is not conducive to microbial growth and by producing products that have a microbicidal effect thus lowering the bacterial load. Such microbes include but are not limited to Camphylobacter jejuni, Yersinia entero- colitica, Listeria monocytogenes, Staphylococcus aureus, Shigella dysenteriae, Bacillus cereus, Salmonella typhi and paratyphi, Vibrio parahemol ticus, Escherichia coli, Pseudomonas putida and Aspergillus flavus.

The removal of oxygen from the package headspace of the food product as well as from within the product itself creates an environment that prevents the growth of aerobic spoilage organisms. In addition, gluconic acid, the oxidation product of glucose, reduces the pH of the food product, thus creating less favorable conditions for many common spoilage bacteria. This antimicrobial effect is further strengthened by hydrogen peroxide, a by-product of the glucose oxidase reaction which can effectively eliminate pathogenic organisms as well as organisms that cause the product to spoil during storage. In addition, the glucose oxidase removes glucose from the food product, thus causing selective pressure aqainst glucose dependent microorqanisms in the food.

According to the invention, the amount of glucose oxidase to be added to the product to remove oxygen is dependent upon the oxygen or air present in the product headspace. The amount is calculated to deliver units of glucose oxidase, relative to the original air volume in the package, of no less than 200 U/liter air, preferably 300-800 U/liter air. The amount of glucose oxidase pres¬ ent is suitable when it reduces the amount of oxygen to under 1% within 5 days, preferably within 0.2 - 2 days.

The enzyme composition can be added either as liguid or powder. The liquid form is prepared by combin- ing the mixture of the enzymes glucose oxidase and catal¬ ase with water, buffering agents or stabilizers and other enzymes or glucose if needed. The powder form can be prepared by combining the mixture of enzymes with a car¬ rier, such as starch, talc, cellulose or other inert solid material. With both forms the portions of mixture and liquid or solid ingredients can be adjusted to provide the appropriate, desired delivery of the enzyme mixture to the foodstuff. Preferably, proportions can be calculated to deliver a minimum amount of carrier or inert ingredient and a maximum amount of enzyme mixture. If needed, gluc¬ ose is also added in order to achieve a minimum glucose content of 3.5 g/1 air, preferably 5-6 g/1 air in the package to ensure use of all headspace oxygen.

According to the invention, if the desired result is to create a microbicidal environment within the food product, the amount of glucose oxidase and glucose that must be used are calculated to deliver a concentration of about 10 to about 1000 units per kilogram of food, with the preferred level being about 15-50 units per kilogram. Glucose is also added based on the quantity of the food product, preferably it is added to achieve a concentration of between 0.2 and 1.0 grams per liter. It should be ap¬ preciated that the levels of glucose oxidase and glucose necessary may vary depending on the amount of headspace in the product package. Thus ideally a sufficient amount of enzyme and glucose is used to ensure use of headspace and product oxygen as well as adequate production of glucose oxidative products in the foodstuff.

According to the invention, it has also been deter- mined that the ratios of glucose oxidase to catalase can be varied depending on whether the desired effect is elimination of oxygen or creation of a microbicidal en¬ vironment, or both.

According to the invention, enzyme compositions containing both glucose oxidase and catalase have been developed to take advantage of the above-described activ¬ ities depending on the particular type of food to be preserved and the packaging conditions. For example, if the desired result is to prevent the growth of aerobic organisms by providing an environment within a food product and its package headspace that contains minimal oxygen, substantially equal levels of glucose oxidase and catalase can be used. Such levels will cause rapid reaction of the environmental oxygen with glucose, and will immediately convert the resultant hydrogen peroxide to water and oxygen. The removal of hydrogen peroxide is not only important for the safety of the foods, but also to prevent inactivation of the enzyme composition. Rela¬ tive to the air volume of the foodstuff packaging, the amount of enzyme mixture added provides activity of glucose oxidase within the range of about 200 to 1500 units per liter of air in the package.

According to the invention, the glucose oxidase/ catalase composition is most easily mixed with the food product during its preparation. When prevention of sur- face growth of organisms such as molds is desired, the enzyme composition can alternatively be applied to the surface of the product, e.g. by spraying. In another em¬ bodiment, the glucose oxidase composition can be incor- porated into the package prior to addition of the food product. Such a system can be used to eliminate oxygen from the package, without the necessity for catalase.

In other cases, the desired result is a reduction of the microbial load of the product. A reduction in the population of food spoilage microorganisms increases the shelf-life of the food product. The method described herein provides a safe and effective means whereby food products can be treated with hydrogen peroxide produced as a byproduct of the glucose oxidase reaction. Such a method removes pathogenic microorganisms and is especially useful when the product cannot be treated by traditional means such as heat. In such cases, it is preferred -to allow the hydrogen peroxide to exhibit its antimicrobial effect prior to its elimination by catalase. This is ac¬ complished by adding catalase, in conjunction with glucose oxidase, at levels that are sufficient to eliminate the hydrogen peroxide prior to consumption of the product, but low enough to maximize exposure of the organisms in the food to the peroxide. Ratios of glucose oxidase to catal¬ ase of at least 5:1 have been found to best accomplish this objective, with 7:1 to 100:1 being the most preferred relative amount.

Alternatively, the food product can be treated first with glucose oxidase, followed by treatment with catalase and packaging. In this way, the exposure of the product to the glucose oxidation products is maximized, and levels of catalase can be used to ensure removal of residual peroxide. Because the activity of the enzyme is highly depen- dent upon the environmental conditions both within the product, such as pH and moisture and the external condi¬ tions, such as the temperature at which the product is stored, it should be appreciated that the levels of enzyme used and the relative amounts of glucose oxidase to catal¬ ase can vary from a ratio of about 1:1 to about 100:1.

As an alternative to combining the enzyme composi¬ tion with the food product, the composition described herein can be incorporated into a food product package to remove oxygen from the product headspace. The composition can be incorporated into a discrete compartment within the package, such as in the lid, prior to sealing. Preferably, a perforated or highly oxygen-permeable bag is used, or a separate compartment attached to the package. The bag or space containing oxygen removal agent does not contain any ingredients harmful to. humans, so that if the bag should be consumed accidentally, it is not injurious.

The method according to the invention is suitable for maintaining both the microbiological quality and sen¬ sory appreciation properties of most dry or moist solid food products, such as prepared foods, sausages, bakery products and snacks, and for extending their shelf life. The amount of glucose oxidase to be used in the method according to the invention is determined on the basis of the amount of oxygen contained in the package. For example, removal of oxygen from a 1,000 ml air space requires 300-800 U glucose oxidase. When protective gas is used, the removal of 2% residual oxygen from a 1,000 ml space correspondingly requires less, that is, 35 to 70 U glucose oxidase. A unit of glucose oxidase unit (1 U) is equivalent to an amount of enzyme required to convert 10 microliters of oxygen per minute with a substrate con¬ taining 3.3% glucose in phosphate buffer (pH 5.9) at 35°C in the presence of excess oxygen. The amount of glucose or glucose producing agent is also dependent on the amount of oxygen contained in the package and in addition to that, on the amount of glucose oxidase. For example, removal of oxygen from a 1,000 ml air space requires 3.5-7.0 g glucose. With protective gas, 0.3-0.7 g glucose is required for the removal of 2% residual oxygen from a 1,000 ml gas space.

The amount of the neutralizer depends on its neutralizing ability and the amount of oxygen. For example, the neutralization of gluconic acid formed in a 1,000 ml air space requires 1.8-2.0 g calcium carbonate.

The amount of the carrier depends on the kind of the carrier used and the amount of the other ingredients. The amount of carrier also depends on the desired humid- ity. For example, a humidity of 25% requires at least 23% of cellulose powder, calculated on the total weight of composition.

The weight of an oxygen removal bag used in the method according to the invention may thus vary from 2 to 25 g, depending on the amount of oxygen to be removed. For example, removal of oxygen from a 1,000 ml air space re¬ quires a bag of 10-20 g.

At room temperature, an oxygen removal bag placed in an air-tight food product package packed without protective gas removes oxygen from the package in 0.5-5 days, usually in 0.5-2 days, and when using a protective gas in 2-24 hours.

Although it is most preferred that the packaqe be completely air tight, the enzyme composition can accomodate some degree of air leakaqe during storage of the package. Consequently, the packaging material in all cases can be air permeable to some extent. That extent is preferably known so that the amount of composition present can be adjusted to account for the air leakage into the package. As used in this application, the term "sealed package" is intended to cover all contemplated variations of air permeability and impermeability.

The levels and ratios of glucose oxidase to catal¬ ase are an important feature of the invention claimed herein. Although it is contemplated that a wide variety of commercially available enzymes can be used to accom¬ plish the objectives of the present invention, the amount of enzymes used will vary depending on the activity of the enzyme preparation. Unit activities can be measured and reported in several ways, such as International Units or units as designated by commercial enzyme producers. A unit of glucose oxidase, as described herein, is defined as the amount of enzyme required to consume 10 microliters of oxygen per minute in a medium containing 3.3% glucose in phosphate buffer (pH 5.9) at 35°C and in the presence of excess oxygen. A unit of catalase activity is defined as the amount of enzyme which degrades about 60 micromol-es of hydrogen peroxide in one minute under assay condi- tions.

The composition and the method of the invention are further illustrated by the following examples. These examples are not meant to be limitations of the invention which is fully disclosed above. Example 1

390 U glucose oxidase which was diluted in 58% glucose syrup was added in 400 g of broiler patties before packing them in normal atmosphere. When the broiler pat¬ ties were packed in modified atmosphere (100% C0₂) the dosage was 100 U of the same enzyme preparation. The glucose oxidase catalase ratio in the preparation was 70:1. The enzyme was sterile filtrated and spread on the surface of the patties.

The broiler patties were packed with Dyno-packing machine, headspace being 750 ml and stored at +4 °C. The oxygen content, pH and total microbial count were measured after 1, 5, 11, 15, 19 and 25 days. The results were compared to control sample which was not treated with glucose oxidase and catalase and represented the nor- mal spoilage pattern. The results are shown in table 1 and 2.

In a typical method for application, the solid or liquid form of the enzyme mixture is applied onto the foodstuff surfaces as a thin film. The coated foodstuff is then packaged in an inert air package. Stability tests have shown that this method improves foodstuff shelf life several fold.

Example 2

400 g of broiler sausages were treated with glucose oxidase the same way as broiler patties in the example 1. Sausages were also packed both in normal and modified (100% C0₂) atmosphere. The results are presented in tables 3 and 4. Example 3 400 g of smoked rainbow trout was treated with glucose oxidase the same way as broiler patties and sausages in examples 1 and 2. The fish was packed both in normal and modified (100% C0₂) atmosphere. The results are shown in tables 5 and 6. Example 4

700 U glucose oxidase enzyme preparation and 10 g glucose was added in 970 g of egg-butter consisting 75% of hard-boiled eggs and 25% butter. The glucose oxidase- catalase-ratio in the preparation was 70:1. Glucose was dissolved in 20 ml of water and the enzyme preparation sterile filtrated before adding to egg-butter.

The enzyme and aqueous glucose was carefully mixed in egg-butter and the mixture was packed in 125 ml glass bottles, 50 g in each bottle. The bottles were closed with rubber caps and stored at + 10°C. The growth of lac- tic acid bacteria and total microbial count was followed during 5 weeks. The results are shown in tables 7 and 8. Example 5

Survival of bacteria in the presence of glucose oxidase and the effect of added catalase

Varying amounts of catalase (CAT) are found frequently in partially purified GO preparations. GO was commercial high purity Aspergillus niger glucose oxidase preparation made by Cultor Ltd and CAT was A. niger catal- ase by Cultor Ltd or purchased from Sigma Chemical Company. A beef liver catalase can also be used. CAT decomposes hydrogen peroxide which is considered to be the main cytotoxic substance produced by GO. The aim of this study was to find out the amount of CAT that renders GO ineffective against bacteria.

Bacteria tested were Listeria monocytogenes RHD 374, Pseudomonas aeruginosa PAOl, and Escherichia coli IH 3080. GO was a commercial glucose oxidase preparation made by Cultor Ltd. CAT was Aspergillus niger catalase purchased from Sigma Chemical Company or Cultor Ltd. The experiments were performed as checkerboard tests on micro- titer plates. The plates were incubated at 37°C for 20 hours and viable counts were determined after appropriate dilutions using a semi-quantitative spot test. The media used were Tryptic Soy Broth (TSB) and this diluted tenfold (TSB 1/10).

The results are shown as tables for each strain (Tables 9-14).

Example 6 In addition to hydrogen peroxide, GO also produces gluconic acid from glucose. To check the effect of this to the pH of the media, a series of GO dilutions was pre¬ pared without the addition of CAT. After 18 h incubation at 37^CC, the pH was measured. The results are shown in Table 15. The low pH at high GO activities may have an in¬ hibiting effect against GO during longer incubations.

Example 7

Treated Patties

225 U/kg glucose oxidase was added on the surface of hamburger patties. The glucose oxidase - catalase ratio in the preparation was 3:1. The patties were packed in normal atmosphere. The oxygen content, pH and total microbial count were measured after 1, 7, 10, 14, 11 and 28 days after packaging. The results were compared to control sample, which was not treated with glucose oxidase and represented the normal spoilage pattern.

The results are shown in Table 16.

Example 8

Treated Patties 30 U/kg glucose oxidase was added on the surface of hamburger patties. The glucose oxidase - catalase ratio was 3:1. The patties were packed in modified atmosphere

(20% C0₂ + 80% N₂). The results are shown in Table 17.

Example 9 Treated bread

400 U glucose oxidase and 5,82 g glucose was added in 400 g of rye bread. The glucose oxidase-catalase-ratio was 3:1. Breads were packed hermetically and stored at room temperature. Breads treated with glucose oxidase developed no mold for 28 days, whereas the control was molded in 3 days.

Example 10

Oxygen removal bag

Composition of oxygen removal powder: Glucose oxidase 350 U Glucose 3.0 g

Calcium carbonate 0.9 g

Cellulose powder 2.0 g

Microcrystalline cellulose powder 0.4 g Water 1.8 g (=22%)

For the laboratory tests, the powder was packed in a 6x6 cm paper bag coated with polyethene. The bag was perforated by pricking with a needle. At room temperature, the bag removed oxygen from a 500 ml air space as follows: Time (h) Oxygen content (%)

2 4.2

4 1.8

6 0.2

24 0.0 Example 11

Oxygen removal bag

Composition of oxygen removal powder: Glucose oxidase 9 U

Glucose 0.1 g Calcium carbonate 0.03 g

Cellulose powder 1.2 g

Microcrystalline cellulose powder 0.2 g Water 0.5 g (=24.6 %)

For the laboratory tests, the powder was packed in a 3x4 cm paper bag coated with polyethene. The bag was perforated by pricking with a needle. The bag removed oxygen from a 500 ml protective gas space as follows: Time (h) Oxygen content (%)

0 2.0 2 0.7

4 0.4

6 0.3

24 0.0

Example 12 Removal of oxygen from a broiler patty package Composition of oxygen removal powder: Glucose oxidase 525 U

Glucose 4.8 g

Calcium carbonate 1.44 g Cellulose powder 2.88 g

Water 2.88 g

The powder was packed in a 6x8 cm paper bag coated with polyethene. The bag was hot sealed and perforated by pricking with a needle. The oxygen removal bag was added at the packing stage to a 1150 ml plastic box containing broiler patties; the remaining empty space was 750 ml.

Removal of oxygen from the package at 5°C is shown in Table 18. Example 13

Removal of oxygen from a broiler patty package Composition of oxygen removal powder: Glucose oxidase 52.5 U

Glucose 0.48 g Calcium carbonate 0.14 g

Cellulose powder 0.29 g

Water 0.29 g

The powder was packed in a 6x8 cm paper bag coated with polyethene. The bag was hot sealed and perforated by pricking with a needle.

At the packing stage, the oxygen removal bag was placed in a 1,150 ml broiler patty plastic bag in a prot¬ ective gas atmosphere; the remaining empty space was 750 ml. Removal of oxygen from the box is shown in Table

18.

Example 14

Removal of oxygen from a broiler sausage package Example 12 was repeated except that broiler sausages were used in place of broiler patties. The re- moval of oxygen from the broiler sausage package is shown in Table 19.

Example 15

Removal of oxygen from a broiler sausage package Example 13 was repeated except that broiler sausages were used in place of broiler patties. The removal of oxygen from the broiler sausage package is shown in Table 19.

Examples 16-19 Extending the shelf life of broiler patties and broiler sausages

Examples 12-15 were repeated, and it was found that the removal of oxygen was completed in one day. Preserv- ability at +5°C was observed by measuring the total and lactic acid bacterial counts of the product as a function of time. It was found that, as compared with products packed without an oxygen removal bag, products packed in packages from which oxygen had been removed by oxygen re¬ moval bags had lower total bacterial counts throughout the preservation test. The lactic acid bacterial counts showed the same pattern as the total bacterial counts. The results of the preservation tests are shown in Tables 20 and 21.

Table 1 pH, oxygen content and total microbial count of broiler patties packed in air

pH, oxygen content and total microbial count of broiler patties packed in 100% C0₂

23

Table 3 pH, oxygen content and total microbial count of broiler sausage packed in air

Time/ Control Sample treated with days glucose oxidase

pH, oxygen and total microbial count of broiler sausage packed in 100% C0„

Table 5 pH, oxygen content and total microbial count of smoked rainbow trout packed in air

pH, oxygen content and total microbial count of smoked rainbow trout packed in CO„

Table 9

Viable counts of L.monocytogenes RHD 374 in TSB

GO, U/ml

0 0.006 0.016 0.049 0.16 0.46 1.39

CAT, U/ml

GO, U/ml

0 0.006 0.016 0.049 0.16 0.46 1.39 4.1

CAT, U/ml

VG; visible growth, not plated

VG; visible growth, not plated Table 1-3

Viable counts of E.coli IH 3080 in TSB

Viable counts of E.coli IH 3080 in TSB 1/10

VG; visible growth, not plated Table 15

Effect of gluconic acid on pH

pH, oxygen content and total microbial count of hamburger patties packed in air

pH, oxygen content and total microbial count of hamburger patties packed in modified atmosphere

SUBSTITUTE SHEET Table 18

Removal of oxygen from a broiler patty package

Removal of oxygen from a broiler sausage bag

SUBSTITUTE SHEET 31

Table 20

Bacterial count of a broiler patty

Bacterial count of a broiler sausage

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Claims

Claims :

1. A method for extending the shelf life of a food product, c h a r a c t e r i z e d in that the product is treated with a solid or liquid enzyme composition of glucose oxidase and catalase.

2. The method according to claim 1, c h a r a c¬ t e r i z e d in that the surfaces of the product are sprayed or coated with the solid or liquid enzyme composi- tion, and the treated product is packaged in an air tight package.

3. The method according to claims 1-2, c h a r ¬ a c t e r i z e d in that said glucose oxidase and said catalase are present at a ratio of between about 5:1 and 1:1.

4. The method according to claims 1-2, c h a r¬ a c t e r i z e d in that said glucose oxidase is pres¬ ent in an amount of at least about 200 units/liter of air in the food product package.

5. The method according to claims 1-2, c h a r¬ a c t e r i z e d in that glucose is combined with the enzyme composition such that the content of added glucose in the product is at least about 3.0 g/liter package air.

6. A method for reducing the microbial popula- tion of a foodstuff, c h a r a c t e r i z e d in that the product is treated with a solid or liquid composition of glucose oxidase and catalase wherein the glucose oxid¬ ase to catalase ratio is at least about 5:1.

7. The method according to claim 6, c h a r a c- t e r i z e d in that said glucose oxidase is present in an amount of at least about 0.1 U/kg product.

8. The method of claim 6, c h a r a c t e r ¬ i z e d in that glucose is combined with the enzyme com¬ position such that the content of added glucose in the product is at least 0.1 g/kg product.

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9. The method according to claims 6, 7, or 8, c h a r a c t e r i z e d in that the product is packaged in an air tight package.

10. A method for treating a foodstuff to remove pathogenic microorganisms, c h a r a c t e r i z e d in that the product is treated with a solid or liquid com¬ position of glucose oxidase and catalase wherein the glucose oxidase to catalase ratio is at least about 5:1, and that said glucose oxidase is present in an amount of at least 10 U/kg product.

11. A method for extending the shelf life of a food product, c h a r a c t e r i z e d in that the product is first treated with glucose oxidase in solid or liquid form, the glucose oxidase being added at a level of at least about 10 U/kg product, and that the product is treated with catalase.

12. A method for extending the shelf life of a food product, c h a r a c t e r i z e d in that the product is packaged in sealable packaging material and an oxygen removal agent is packed in a perforated or highly oxygen permeable plastic bag or a plastic laminated paper bag to be placed in the food product package.

13. A method according to claim 12, c h a r ¬ a c t e r i z e d in that the oxygen removal agent con- tains glucose oxidase, glucose or glucose producing agent, catalase, water, enzyme carrier and neutralizer.

14. A method according to the claims 12 and 13, c h a r a c t e r i z e d in that the oxygen removal bag contains glucose oxidase 0.2-2 U/ml air, preferably 0.35- 0.7 U/ml air.

15. A method according to the claims 12-14, c h a r a c t e r i z e d in that the oxygen removal agent contains glucose or a glucose producing agent in an amount sufficient to ensure a glucose content sufficient for the action of the enxyme, preferably 3.5-7.0 g/liter

SUBSTITUTE SHEET air, or correspondingly 0.3-0.7 g/liter protective gas.

16. A method according to the claims 12-15, c h a r a c t e r i z e d in that the oxygen removal agent contains calcium carbonate as a neutralizer, prefer- ably 1.8 g/liter air, or correspondingly 0.06 g/1 protec¬ tive gas.

17. A method according to the claims 12-16, c h a r a c t e r i z e d in that the oxygen removal agent contains cellulose powder as enzyme carrier, prefer- ably 4.0 g, and microcrystalline cellulose powder, prefer¬ ably 0.8 g, per 1 liter air, or correspondingly, cellulose powder 2.4 g and microcrystalline cellulose powder 0.4 g, per 1 liter protective gas.

18. A method according to the claims 12-15, c h a r a c t e r i z e d in that the oxygen removal bag contains humidity sufficiently to effect the enzyme reac¬ tion, preferably 15-25% by weight on the amount of oxygen removal agent.

19. The method according to claim 1, 6, 9, 10 or 12, c h a r a c t e r i z e d in that the packaging step envelops air within the package.

20. The method according to claim 1, 6, 9, 10 or 12, c h a r a c t e r i z e d in that the packaging step envelops a substantially nonoxygen atmosphere within the package.

21. An oxygen removal agent useful for removing oxygen from the headspace of a product packaged therein, c h a r a c t e r i z e d in that the package is seal- able and has disposed therein a composition of glucose oxidase.

22. The oxygen removal agent according to claim 21 c h a r a c t e r i z e d in that the composition includes glucose in contact with said glucose oxidase.

23. The oxygen removal agent according to claims 21 and 22 c h a r a c t e r i z e d in that the composi-

SUBSTΓΓUTE SHEET tion is contained within a discrete compartment joined to the packaging material.

24. A composition for extending the shelf life of food products c h a r a c t e r i z e d in that it comprises a liquid or solid mixture of glucose oxidase, glucose and a carrier.

25. The composition according to claim 24 c h a r a c t e r i z e d in that it comprises catalase.

26. The composition according to claim 25 c h a r a c t e r i z e d in that the glucose oxidase/ catalase ratio is between about 1 to 1 and 100 to 1.

27. A composition for use in an enzymatic method for extending the shelf life of food products, c h a r¬ a c t e r i z e d in that it mainly comprises glucose oxidase, a food-grade carrier providing a large surface, water and a neutralizer.

28. A composition according to claim 27, c h a r¬ a c t e r i z e d in that it contains glucose oxidase 25-40% by weight, cellulose powder 20-50% by weight of which microcrystalline cellulose powder 5-10% by weight, calcium carbonate 7-15% by weight, and water 15-25% by weight.

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