envases.pdf

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7 Enzymes as active packaging agents

A.L. BRODY and J.A. BUDNY

This chapter discusses the role of enzymes in active packaging, especially as

oxygen scavengers.

Almost all mechanisms in which packaging structures function in

response to a stimulus involve physical, chemical or physiochemical actions.

In a physical action, the active element of the material, which is usually

external, opens, expands, closes, contracts, etc. as one or more variables are

changed. In chemical or physiochemical reactions, a component of the total

package reacts with the package structure or component, usually in an

irreversible manner, with the result that the active component is effectively

consumed or changed as the internal package environment is changed.

However, in catalytic processes physiochemical or chemical reactions

occur in which the catalyst remains effective and intact. Enzymes, which are

biological catalysts, accelerate chemical reactions but are not consumed as a

result ofthereactions. Within limits, for as long as reactants or substrates are

present, enzymes will function to catalyze chemical, or more specifically

biochemical, reactions. When the proper enzymes are introduced under the

proper conditions, they are capable of catalyzing reactions which can either

prevent the product from being changed or extend the function of packaging

beyond its accepted or previously understood functions by actively serving

as a processing unit.

7.1 Enzymes

Enzymes are biological catalysts which are found in all living cells, whether

plant or animal. These macromolecular proteins exhibit two outstanding

characteristics in addition to the fact that they occur naturally and are found

in living systems.

The first characteristic is their catalytic power. Enzymes accelerate

chemical reactions that occur in biological systems by factors that exceed a

million over their uncatalyzed rate. In essence, enzymes allow living

systems to carry out reactions that would not ordinarily occur or occur so

slowly that the rates would not be of any practical significance. A simple

reaction of the formation of carbonic acid from carbon dioxide and water

occurs 10

7

times faster with the enzyme carbonic anhydrase than the non-

enzymatic or chemical reaction.


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The second important characteristic of enzymes is their specificity.

Enzymatic specificity takes on two distinct forms: the type of chemical

reaction; and for any type of chemical reaction, a specificity for the reactant

or substrate. Consequently, for each chemical reaction that occurs in a

biological system, there is a unique enzyme required for the optimal

production of reaction products. With so many different biological reactions,

it follows that there are many different enzymes.

As with all catalysts, enzymes do not alter equilibrium conditions. An

enzyme increases a forward reaction in the same way and to the same extent

that it increases the reverse reaction, i.e., enzymes accelerate the rate at

which a chemical equilibrium is reached but an enzyme does not distort the

ratio of the equilibrium concentrations of the products to reactants.

The catalytic potential of enzymes and the speed at which they facilitate

chemical reactions lies in their ability to reduce the Gibbs Free Energy of

Activation (E

a

). Enzymes accomplish their catalytic objectives, not by

reducing the E

a

of the uncatalyzed reaction but by creating a new and

different transition state and hence a different reaction path or mechanism.

This new or different transition state is the enzyme-substrate complex (ESC)

where the reactant becomes associated or bound to the free enzyme at the

reactive center or site, followed by the release of the product which

generates the free enzyme again. The now free enzyme is once again

available to combine with another molecule of reactant to repeat the process.

The net effect of this sequence is that reactant or substrate becomes product

and the enzyme is unchanged.

The active site is an area or region of an enzyme where the bond-breaking

and bond-forming ofthereactants and products occur. The participation, and

hence the reactivity, of an enzyme for a particular substrate-product pair is

determined by the amino acid sequence and the geometric or spatial

arrangement of the enzyme. Because enzymes are high molecular weight

polymers which are made up of amino acids, it is not surprising that the

active site represents only a small percentage of the total enzyme. It is also

not surprising that the polymeric catalysts are three dimensional, and

consequently the active site has a size (volume) shape to it. This spatial

characteristic of the active site defines the size, shape and type of substrates

or reactants which can be catalyzed by the enzyme.

The kinetics of enzyme reactions are obviously of great importance in

considering their potential commercial applications. At the outset, enzymatic

reaction rates are linear with time until all of the free enzyme is used to form

the ESC. When all of the enzyme exists as ESC, or as soon as the product

is formed, the enzyme reacts with another reactant or substrate molecule,

and the rate of conversion of reactant to product plateaus at the maximal

reaction rate or velocity. Once the initial velocity has been achieved, all of

the enzyme exists as the ESC.


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Although enzymes may be classified according to the substrates they

affect, as, for example, proteases for proteins, lipases for lipids, etc., in

reality, these are designations for broad families to break proteins of entities

that are specific to a single protein or lipid under a particular set of

circumstances. An enzyme suitable for a single 16 carbon fatty acid

oxidation reaction will not catalyze an 18 carbon fatty acid oxidation even

though the actual reactions at the sites may be identical. This characteristic

may be viewed as beneficial in that only the specific reaction and no other

is catalyzed by the enzyme. On the other hand, this attribute may be

regarded as undesirable since a specific enzyme is required for a specific

reaction, and no single enzyme can effect a series of related reactions.

Enzymes are proteins whose reactivity is quite sensitive to temperature.

At temperatures as low as 140

0

F (68

0

C), the catalytic reactivity of the

enzymes may be temporarily or permanently disrupted, thus rendering

enzymes among the most vulnerable of all biological matter. This tem-

perature sensitivity is an important consideration in the commercial

application of enzymes in processing operations.

Among the many enzymes functioning in reactions that have been and are

being used commercially are rennin (chymosin) to precipitate the casein of

milk in cheese making; proteases in laundry detergents to assist in protein

stain removal; amylase to convert starch to sugar for brewing; lactase to

break down lactose in milk; various oxidases to accelerate oxidative

reactions; and catalase to remove hydrogen peroxide that might be formed

during prior oxidative reactions. Other, more generic applications of

enzymes include stereospecific amino acid production, high fructose sugar

production, beer and wine fermentation, tenderizing meats, milling and

baking, juice and wine clarification, juice extraction from fruits and

production of flavor enhancers, to cite a few.

7.2 Potential roles of enzymes in active packaging

In many commercial situations, enzymes may be viewed as chemicals to be

added to the product to catalyze a reaction as one way to affect batch

processing. The addition can occur to the in-plant batch or individual

package. For in-package situations, the enzyme may be added directly to the

product to effect a reaction or may be incorporated into the package

structure. To function within a package material, the enzyme must be

immobilized and the substrate, reactant or a constituent circulated past the

site to initiate a reaction. Immobilization of an enzyme, or placing it in a

static position where it may function for an indefinite period, may be

accomplished by making the enzyme an integral part of the packaging

material.

Active packaging in general often involves the incorporation of a


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chemical into the package material. Active packaging employing one or

more enzymes involves the incorporation into the package material of the

specific enzymes in much the same manner as the incorporation of a more

conventional chemical to create the active package. The key differences are

that the enzyme is not changed by the reaction and can continue to function

indefinitely; the enzyme is vulnerable to variations in temperature, pH, etc.;

and the range of environmental conditions for the functioning of the enzyme

is a relatively narrow band. These key considerations which affect the ability

of the enzyme to function require special processes and techniques for

incorporating enzymes into packaging materials. Often, harsh manufacturing

processes, geometric configurations, etc. that are adequate and even

appropriate for non-enzyme packaging components render the use of

enzymes inappropriate. Consequently, new and innovative methods are

likely to be required for the incorporation of enzymes into packaging

materials.

Although a broad range of enzymatic reactions stemming from enzyme

incorporation into package materials can be conceived, only a relatively

small number have actually been attempted on a practical basis. Examples of

those that have been actively pursued include:

Oxygen removed by means of glucose oxidase plus catalase.

Removal of products of microbiological degradation by glucose oxidase/

catalase.

Incorporation of lactase to remove lactose from milk.

Incorporation of cholesterol-changing enzymes to remove cholesterol

from liquid egg or milk.

Tim e-temperature integrator indicators which are triggered enzymat-

ically.

Examples of enzymatic reactions that have not found general use but

which might have some future potential and require development include:

Conversion of sugar into alcohol and carbon dioxide in secondary

fermentations of wine to produce champagne-like products.

A United Kingdom patent application (Thomas and Harrison, 1983)

which describes an in-package secondary fermentation system using

immobilized yeast within a liquid porous container immersed in an

alcoholic beverage. In one manifestation, the container was a flexible

pouch. Further, the inventors refer to isolated enzyme complexes as

being useful as yeast substitutes. The objective here was to consume the

residual fermentable sugars, converting them into carbon dioxide and

water.

In-package production of lactic acid for pickles, sauerkraut or sour dairy

products.

Production of 'natura l' antimicrobial agents such as benzoic or propionic

acids to help preserve the product contents.


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Destruction of natural toxins in foods.

Removal of undesirable respiratory end products such as ethylene that

accelerate the respiratory processes of fresh fruits and vegetables.

Removal of undesirable end products of microbiological or endogenous

enzymatic reactions such as polypeptides, carbonyls, ketones, volatile

acids, etc.

Tenderizing of fresh meat such as beef by proteases such as papain.

7.3 History

Although many enzymes and their roles have been known for several

decades, the notion of incorporating them into package materials to achieve

a desirable result dates back only to the 1940s. Almost simultaneously with

the idea of protecting against browning of dry foods such as eggs by

removing residual oxygen, the notion of in-package glucose oxidase/catalase

reactions was born. In reality, the initial action of glucose oxidase is with

residual quantities of glucose, a reducing sugar active in the non-enzymatic,

non-oxidative Maillard browning reactions. Highly reactive hydrogen

peroxide is produced by glucose oxidase, and is removed by catalase which

breaks it into water and oxygen. This concept was put into practice by

employing porous packets of the enzyme mix in which the enzymes slowly

reacted with minute quantities of residual oxygen, an analogue of the

commercial incorporation of sachets of desiccants to reduce the in-package

relative humidity. The applications during the 1940s and 1950s appear to

have been largely confined to very long term storage of military foods.

The concern for the adverse effects of temperature abuse on frozen foods

led to numerous ventures into development of time-temperature indicators,

among which have been enzymatically actuated versions, beginning in the

1970s.

The exponential growth of modified atmosphere packaging in the 1980s

led to the notions of oxygen and carbon dioxide and moisture control using

in-package sachets of chemicals. Some enzymatic agents were included in

these chemicals.

Towards the end of the 1980s, interest increased with the formation of

PharmaCal, Ltd. whose objective was to develop the application of enzymes

in unit size situations. This company and its principal, the co-author of this

article, suggested and, in some instances, physically evaluated three areas in

which immobilized enzymes within package structures would catalyze

reactions of products contained within packages.

Lactase to remove lactose.

Cholesterol reductase to remove cholesterol.

Glucose oxidase/catalase to remove oxygen.

Whether or not the communications emanating from PharmaCal were


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directly responsible, several other enzymatically based active packaging

oxygen control devices have been proposed since that time.

7.4 Oxygen removal

As is documented elsewhere in this book, oxygen is a highly reactive gas

which can cause deterioration of almost all food products in terms of flavor,

color, nutritional value and safety. Further, the presence of oxygen is

essential to the growth and potential deteriorative effects of aerobic

microorganisms including m ost bacteria, yeasts and m olds. Thus, minimiza-

tion or removal of oxygen is an important factor in prolonging the quality

retention of many food products.

According to Baker (1949, but evidently conceived in 1944) the addition

of an oxidase to liquid-containing food products such as beer, peas, corn,

milk, apple cider or orange juice, protects them from oxidation. 'In some

instances, it is better to produce in the product a substrate for the oxidase

that is to be introduced rather than to use a substrate already present.' For

example, glucose originally present or added can be oxidized to gluconic

acid. Baker's patent indicates that if the oxidase produces an objectionable

end product such as hydrogen peroxide, then an additional enzyme might be

introduced to remove the undesirable end product.

Among the interesting aspects of this early patent is the notion that as the

oxygen in the product is removed, free oxygen in the headspace is further

dissolved by equilibrium dynamics, thus removing oxygen from the

headspace. The reaction, now very well known, is

2G + 2O

2

+ 2H

2

O -> GO + 2H

2

O

2

where G is the substrate. Since hydrogen peroxide is a very good oxidizing

agent, it is 'just as objectionable, or even more so, than is the original

molecular oxygen.' Thus, catalase is introduced to break down the hydrogen

peroxide

2H

2

O

2

+ catalase - 2H

2

O

2

+ catalase

The sum of these two reactions yields half the oxygen originally present and

therefore ultimately the free oxygen approaches zero.

Baker's invention was implemented by introducing one or more pellets of

the enzyme into the product such as beer or orange juice. The patent also

mentions the incorporation of lactase to hydrolyze lactose into glucose and

galactose which are then oxidized in the presence of oxidases. Perhaps

without realizing the significance of this assertion, the patent suggested the

use of enzymes to reduce the lactose content of milk.

The patent does not explicitly describe precisely how the enzymes are

incorporated. The inference is that the enzymes are introduced directly into


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the product. Expressed differently, this patent does not indicate that the

enzymes are either part of the package structure or in an independent packet

within the primary package. Thus, although the 1949 patent described

perhaps for the first time the employment of enzymes to eliminate in-

package oxygen, it did not indicate that the enzymes were part of the

package material or structure.

This concept of enzyme incorporation into a package material was first

overtly described in a 1956 patent (Sarett, 1956). (Sarett, incidentally, was

the assignee for the Baker patent.) In this patent, the same basic enzymatic

reactions as in the Baker patent were reiterated as a reference, but the

enzymes glucose oxidase and catalase in a solution were impregnated into or

on a moistureproof or fabric sheet. The enzym e was bound to the sheet with

a water-dispersible adhesive such as polyvinyl alcohol, starch, casein or

carboxymethyl cellulose. The enzyme-coating face must contact the moist

product to ensure that the requisite oxygen reduction reactions take place.

The enzyme system was indicated to serve as a barrier to oxygen which

would otherwise be transmitted through the sheet. Products described as

being benefited by this system of oxygen reduction include cheese, butter,

frozen foods subject to browning, etc.

Although during the period of the patent a Kraft packaging paper called

moistureproof (which, as it happens, was not actually moistureproof) was

often used to package butter and cheese, the patent does not indicate the use

of this material. Rather, the package material is described as having \ .. an

exposed surface covered with a gas-permeable packaging material and

having an inter layer between and in contact with packaging material and . . .

food . .. inter layer providing an oxygen barrier. ...' The specific package

materials identified were moistureproof cellophane, paper, rubber hydrochlo-

ride with impregnation employed for the papers and coating for the plastic

and cellulose films. Also cited as being suitable substrates were wax paper,

styrene, polyethylene and vinyls.

Experiments discussed in the body of the patent indicated results in which

oxidation of cheese surfaces was retarded by the presence of the enzyme-

containing package material.

In 1958, Scott (co-inventor on the 1956 Sarett patent) of Fermco

Laboratories, published a paper on Enzymatic Oxygen Removal from

Packaged Foods in which enzymes were incorporated into packaging

materials or introduced into packets. Fermco Laboratories was a manu-

facturer of enzymes, one category of which was labeled Fermcozyme

antioxidants, and of the packets which were named Oxyban. This paper

marked the first publication to our knowledge on the use of packets of

chemicals in packages.

The glucose oxidase/catalase systems were derived from mold mycelia

which were disrupted, filtered and further purified. To be effective in


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reducing oxygen, glucose oxidase/catalase systems must be used in gas-tight

packages. Among the applications indicated were:

Aqueous foods

direct incorporation, in mayonnaise or carbonated beverages;

surface treatment in canned dog food;

in packets in situations in which the enzyme and the product should be

kept separate.

Non-aqueous foods

direct incorporation;

in packets, as for chow mein noodles.

The mayonnaise and carbonated beverage examples involved incorpora-

tion oftheenzyme system directly into the products, with oxidative rancidity

delayed in the former class of products and color fading (e.g., grape-flavor

carbonated beverages) as well as flavor oxidations delayed in the latter. The

dog food example was also a direct addition to retard surface discoloration

on the top of the dog food in retorted cans.

As a coating, the dried enzyme system was coated on the surfaces of

package materials for processed cheese. Deposition of the enzymes was in

solution form or via incorporation into a dry starch mixture prior to 'dusting'

the package material surface. When the dry and therefore inactive enzyme

picked up moisture from the product, it was activated and was a sufficiently

good oxygen interceptor to control the formation of brown ring. Another

series of experiments focused on obviating oxidative gray coloration on the

surfaces of luncheon meats.

Fermco's Oxyban product was a dry glucose oxidase/catalase/glucose/

buffers blend to be incorporated into products to reduce headspace and

occluded and dissolved oxygen in dry foods such as coffee or soup. In

another manifestation, the Oxyban was placed in small packets in which it

reacted with oxygen in packages of roasted and ground coffee, smoked yeast

or

egg

solids. Exactly how the enzyme was activated without moisture was

not indicated, but clearly some moisture from the product was required. The

author noted that this in-package packet was analogous to the desiccant

packet.

Three years later, Scott, then with Hammer (Scott and Hammer, 1958),

elaborated on the oxygen-scavenging packet for in-package deoxygenation.

Using the same glucose oxidase/catalase packet system described earlier

from their laboratory experiments, they proceeded forward to a more

commercially viable mechanism. Among the problems they enumerated

were:

Oxygen-scavenger surface area owing to the gas phase reaction.

The need for moisture (cited above).

Necessity to neutralize gluconic acid to avoid enzyme deactivation.


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Package material structure allowing passage of oxygen but not mois-

ture.

The gluconic acid problem was obviated using phosphate buffers. As little as

15 g of Oxyban enzyme mix in a packet was capable of removing all

measurable oxygen from a sealed No. 2 size can held at ambient

temperature. Once again, the type of package material used for the packet

was not indicated.

An interesting side note was an exploration of the use of glucose oxidase

alone which, of course, led to an increase in the amount of hydrogen

peroxide which would, in turn, slow the subsequent rate of oxygen

uptake.

The products benefited by the total system were primarily dry milk, potato

granules and ice cream mix.

An international patent application (Lehtonen et ai 1991) described a

package material containing an enzyme system to remove oxygen from the

interior of the package by enzymatic reaction. By removing the oxygen, the

growth of aerobic microorganisms was significantly retarded, and so this

technology was favorable to shelf-life from both microbiological and

chemical standpoints. The enzyme, for example, glucose oxidase, was

incorporated into a package material with a gas-impermeable layer on the

exterior and a gas permeable layer on the interior, i.e., the layer containing

the oxygen-consuming enzyme was sandwiched between two plastic film

layers.

The background ofthispatent cited a 1969 German publication describing

the use of glucose oxidase in package materials for the surface protection of

meats, fish and cheese products but without elaboration. And, of course, the

classical review paper by Labuza

et al.

(1989) described a similar

technology of coating plastic film with glucose oxidase catalase, with the

enzyme system activated by moisture from the food as Scott had previously

cited.

This patent application from Cultor Ltd. of Helsinki, Finland, details a

flexible package structure containing an enzyme system in the liquid phase

trapped between films, the outer of which might be polyamide or

polyvinylidene-coated polyester. The inner film would be polyethylene

which is generally not a good gas barrier.

The enzymes of choice were oxidases of the oxidoreductase family using

oxygenases and hydroxylases which bind oxygen to oxdizable molecules.

The enzyme solution contains a buffer and a stabilizer, and may also be

mixed with a filler. The enzyme layer was applied on the film by gravure or

screen technique with the layer thickness being about 12 fim. The enzyme is

not directly in contact with the contents.

The film produced was employed either as the cover film layer or as the

thermoformable bottom layer for tray-type packages.


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The inventors noted that with increasing temperature, the gas permeability

of package materials increases and so also does the ability of the enzyme

system to reduce the oxygen content from the 20.9 of air to about 1 at

ambient temperature within 24 hours.

From technological and potential commercial perspectives, this Finnish

work is so precise as to imply a major advance in the ability to implement

the principles of enzymes as active package components.

Co-author Budny and his company PharmaCal, Ltd. have been actively

researching enzymes for active packaging since the 1980s. The contribution

of PharmaCal, Ltd. to enzymes in active packaging was to expand the

concept of packaging beyond the two long-regarded functions of packaging:

containment of the product; and protection of the contents. These require-

ments originally were embodied in wine skins that ancient goat- and sheep-

herders used for their sustenance beverages. Throughout history, while there

have been advancements in materials and approaches, there have not been

any fundamental changes or additions to the necessary requirements for

containers or packages. Whether they are animal skins, a lid or a multi-layer

stock, they should protect the contents and not leak.

PharmaCal, Ltd. added a third dimension to packaging by allowing an

individual package to become a processing unit or to perform a process step

or function that previously was limited to in-plant operations. With a

combination of patent applications and proprietary technology, PharmaCal,

Ltd. has been able to expand the concept of packaging to include processing

steps, value-addition to packaged products and increased processing effi-

ciencies.

PharmaCal, Ltd. has developed a two-enzyme system involving glucose

oxidase and catalase to intercept oxygen and has applied the technology for

enzymes in active packaging to improve the proven concept of oxygen

removal with the dual enzyme system of glucose oxidase and catalase. The

use of the enzymes to remove oxygen has been acknowledged as not new,

but their role in enzyme-based active packaging has been regarded as a more

advanced application. Figure 7.1 illustrates the mechanism in which

packaged liquid reacts enzymatically with glucose in the package wall to

form gluconate. The resulting hydrogen peroxide is enzymatically reacted

with catalase to produce oxygen and water that re-enter the contained

product liquid.

A container with an internal reactor, in reality an integral section of the

package wall through which the liquid contents may flow, permits the

enzymes to be retained for a reaction described in a 1989 patent application

(Budny, 1989).

A 1991 patent (Ernst, 1991), described a glucose/glucose oxidase enzyme

mixture in a porous precipitated silica acid carrier. Calcium carbonate,

calcium hydrogen phosphate, magnesium carbonate or disodium hydrogen


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Figure 7.1 Oxygen removal from liquid products.

carbonate may also be employed as carriers or reaction accelerators. The

oxygen scavenger may be in the interior of in-package sachets.

A 1991 patent (Copeland et al 1991) describes the incorporation of

oxygen scavenging cell membrane fragments which contain an electron

transfer system in solutions containing alcohol or acids to reduce oxygen to

water. Although neither purified nor crude enzym es, the active component of

membrane fragments in this technology must constitute the enzyme

system.

The inventors note that the major mechanism to effect the reaction is

incorporation of the membrane fragments into the product, and that these

active components may also be made part of the package structure. Sources

of the membrane fragments were cell membrane of bacteria such as

Escherichia coli and/or mitochondrial membranes.

Examples of products from which oxygen might be removed by the

system include beer, wine, fruits, juices and a variety of non-food products.

Both red and white wines were treated with materials supplied by Oxyrase,

Inc., which is also the patent assignee. Dissolved oxygen was removed

within 16 minutes at 37C. Less than 12 minutes was required to remove

100

of the oxygen from beer or tomato juice. A five-fold increase in the

time to the onset of browning of cut surfaces of bananas and apples was

observed at ambient temperature.

Developers from chewing gum producer, William Wrigley, Jr., have

described the use of porous polymeric beads containing glucose oxidase in

multilayer flexible package materials (Courtright

et al

1992). The porous

particles are made from styrene divinyl benzene, with the enzyme incorpo-

rated mechanically. The beads are then blended into a thermoplastic coating

in the multilayer film.

H e a d

space

Glucose

oxidase

enzymeluconate Glucose

Packaged

liquid

Outside

of

container

ata lase

e nz y me

Container wall

Inside of container


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Labuza and Breen (1989) have analyzed the issues involved in the

incorporation of glucose oxidase into package materials.

To counteract the quantity of oxygen passing through an aluminum foil

lamination an enzyme surface will have to react with oxygen in the

following manner:

Rate = permeability X area X oxygen pressure difference

between the outside and inside

Rate = 0.1 X 1 [0 .2 1-0 .0 1] = 0.2 ml per day per m

2

= 20 jxl/day

The calculation above assumes air outside and < 1 oxygen inside. For the

worst case and with a pinhole or cracked score, there would be the need to

scavenge 1 ml/day. A film could be made equivalent to a barrier by binding

the oxygen scavenging enzyme to the inside surface of the film to react with

the excess oxygen.

Glucose oxidase transfers two hydrogens from the -CHOH group of

glucose to oxygen with the formation of glucono-delta-lactone and hydrogen

peroxide. The lactone then spontaneously reacts with water to form gluconic

acid. One mole of glucose will consume one mole of oxygen and so a

package with 500 ml headspace is required, to reach zero oxygen, w ith only

0.0043 mole of glucose needed as a substrate. The major factors are the

speed at which the enzyme works, the amount of glucose available, and the

rate at which oxygen permeates into the package. In the presence of catalase,

a normal contaminant of commercial glucose oxidase, the hydrogen

peroxide is broken down, and so with catalase one mole of glucose will react

with only a half mole of oxygen, decreasing the overall effectiveness of the

system. Pure glucose oxidase without catalase is reportedly expensive.

If no surface exists for the peroxide for diffusion, the glucose oxidase will

be inactivated, precluding this application. Since many foods may have

minimal contact with the package surface, except on the sides and bottom,

this may not be the best approach for oxygen scavenging.

At 30-40

0

C, pure glucose oxidase has a rate of oxygen consumption of

about 150 000 (il/h/mg. Based on this, and spreading mg per m

2

on a film,

this would be equivalent to reacting with all the oxygen passing through a

film with an oxygen permeability of about 18 000 ml/day m

2

atm.

Thus at room temperature, a i m square surface with 1 mg of enzyme

spread out on it should be able to handle all the oxygen passing through any

package film. One advantage is that both polypropylene and polyethylene

are good substrates for immobilizing enzymes. One factor to take into

account is the stability of the enzyme when bound to the film. An unknown

factor is how stable the enzyme will be on the film over time. Glucose

oxidase bound to a plastic surface has been shown to undergo a 50 drop in

activity in 2-3 weeks followed by little loss over the next four weeks.


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The Japanese have worked on binding of enzymes to chitosan, which is an

insoluble polymeric carbohydrate from shellfish shells, but a 70 loss in

activity for bound glucose oxidase has been reported. Glucose oxidase

immobilized on polyethylenimine-coated glass beads retained 78-8 7 of its

activity and was more stable to heat inactivation. Since the enzyme is a

protein and can serve as a nutrient for microbes along with the glucose

substrate, a microbial inhibitor may be needed in the film.

Besides glucose oxidase mentioned previously, other enzymes have

potential. One such enzyme is ethanol oxidase which oxidizes ethanol to

acetaldehyde. The reaction is extremely rapid. Hopkinseta/. (1991) describe

a package in which alcohol or oxidase or cellular extracts of

ichia

pastoris

cells containing alcohol oxidase are the enzymes used for oxygen scaveng-

ing in dry foods. An alcohol substrate either from the product or introduced

into the package from the exterior is required to remove the oxygen from the

package headspace.

7.5 Antimicrobial effects

The use of enzymes in active packaging to control microbial growth and

subsequent packaged-product degradation can be achieved by two independ-

ent approaches. By controlling the amount of available oxygen, selective

control of aerobic bacteria can occur. However, this method of bacterial

control can, under certain circumstances, allow the overgrowth of patho-

genic anaerobic bacteria which, from a human view, may be worse than

aerobic bacterial overgrowth. A second approach that has been implemented

by several investigators is non-specific relative to oxygen requirements and

is a direct attack on the organisms present, independent of whether the

organisms are aerobic or anaerobic. This second approach can be either by

a direct attack on bacteria (both aerobic and anaerobic) or by the production

of broad-spectrum antimicrobial agents.

Neither the literature nor the memories of the authors indicates the

commercial implementation of the Fermco products. Meanwhile, the use of

immobilized enzymes in commerce has increased significantly. During the

1970s, Scott (1975), in his continuing research on the technology of glucose

oxidase, noted that catalase-free glucose oxidase might exert antimicrobial

effects due to the production of hydrogen peroxide. At the University of

Rhode Island, Rand and his co-workers conducted research and development

on catalase-free glucose oxidase as a food preservative, especially with

regard to fish (Field et aL 1986).

The enzymes (not coincidentally, supplied by Fermco) were applied to

fresh flounder fillets or w hole fish by dips, immersion in ice or by enzyme/

algin blankets. In some experiments, the enzyme system included catalase

and/or glucose. The university researchers' experiments (which had begun


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during the early 1980s) demonstrated that the enzyme treatments retarded

the onset and magnitude of adverse microbiologically triggered spoilage

odors. The researchers explained the result as due to reductions in surface

pH under the refrigerated conditions of the test. These changes influenced

the metabolism of putrefactive microorganisms. They also suggested that the

generation of hydrogen peroxide might inhibit the growth of psychrotropic

microorganisms which are reported to be sensitive to the chemicals used.

Other possible microbistatic agents include gluconic acid, reportedly a metal

complexing agent, and gluconolactone, reported to be a binding agent for

water and metal ions. Another factor reported by the group was an altered

gaseous microenvironment in which oxygen in the muscle interstices was

depleted by the enzymatic action thus retarding the growth of aerobic

psychrophiles. This last, of course, is synergistic with the oxygen removal

aspects of the enzyme system.

The authors cited a Japanese patent in which catalase-free glucose oxidase

was demonstrated to be effective in preserving other proteinaceous foods

such as ground chicken and tofu (Fukazawa, 1980).

Although the Rand

etal

work did not specifically state the incorporation

of enzymes into package materials, the implications were sufficiently clear

in the examples of the enzyme-containing ice and the enzyme-containing

algin blanket. Either of these could have been relatively easily substituted

with a skin package material which had been surface tested with the enzyme

system. The notion of hydrogen peroxide as an intentional active anti-

microbial agent is somewhat of a contradiction since this chemical is quite

reactive with many food constituents, especially lipids, and residual free

hydrogen peroxide is not readily accepted by regulatory officials. If the

hydrogen peroxide is fully reacted with microorganisms as in aseptic

packaging, however, perhaps the proposed system may warrant further

consideration. Unfortunately, work at the University of Rhode Island on this

topic has been discontinued.

A German patent assigned to Continental Group (Anon. 1977) describes

incorporation of biologically active enzymes into polymers on the interiors

of package structures to destroy microorganisms of contained products. The

enzymes were intended to destroy microorganisms by breaking cell walls

and also to consume oxygen, thus increasing shelf-life without heat. The

applicable products were beer and fruit juices.

Enzymes such as muramidase for cell wall destruction and glucose

oxidase for oxygen interception were attached to the internal polymer by

covalent bonds. 'Non-essential' functional groups such as NH

2

, COOH, OH

phenol, imidazole and sulfhydryl were cited as examples.

The polymer was described as a terpolymer of monomer alkyl acrylate

and vinyl aromatic applied to the interior of a glass container from a solvent

and dried by heat. The enzyme was subsequently applied as a coating from

an aqueous dispersion.


15/19

Tests indicated highly significant reductions in oxygen concentrations

within the glass jars due to the conversion of glucose to gluconate in an

oxidative enzymatic reaction.

This appears to be the first reference to actually incorporating an enzyme

into an interior package wall to achieve an enzymatic antimicrobial

effect.

7.6 Time-temperature integrator-indicators

For many years, efforts have been underway to develop a practical, accurate,

reliable and economic indicator of total temperature-time exposure of food

products. Among the routes has been the application of the principles of

temperature sensitivities of enzymes. Although the original objectives were

aimed at frozen food defrosting devices, more recent interest has been

focused on chilled foods. Among the issues are activation only when

actually at the beginning of shelf-life, accuracy over the entire range, how

reflective the integrator-indicator is of the actual temperature-time experi-

ence,

and another basic question, how well the measurement represents the

effect of the temperature-time integral on the food

itself

Kramer and Farquhar (1976) listed a number of the problems in their

evaluation of five commercial, time-temperature indicating and defrosting

devices. No descriptions were given the mechanisms for sensing, integrating

or measuring time-temperature.

On the other hand, Blixt and Tiru (1976) described a commercial

enzymatic time-temperature monitor, called I-point TTM. The authors, of

Kockums Chemicals of Malmo, Sweden, stated that their device met all the

requirements of reliability, accuracy, size, cost, understandable message and

ability to integrate ' . . . both length and degree of all temperature

exposures.'

The reaction was based on enzymatic degradation to colored end points.

The device was a two-part system, one containing an enzyme and pH

indicator since the system was based on pH change caused by enzymatic

activity plus a substrate. Because of the enzymatic core of the pH change,

the temperature response was exponential with increasing temperature, and

so evidently indicative of actual biochemical changes arising due to the

temperature-time experience. Although the indicators reportedly functioned

very effectively, no reference was made to the type of enzyme used. One

might speculate on the simple glucose oxidase-catalase system producing

gluconic acid as the reaction proceeded.

This product was another manifestation of the application of enzymes in

package systems to an inactive mode.

A 1989 US patent (Klibanov and Dordich, 1989) claimed a temperature-

change indicator composed of an enzyme and substrate, a colorimetric


16/19

indicator and a trigger mechanism of a solid organic solvent system that

melted when a specific temperature range was reached to permit the enzyme

system to respond to temperature stimulus over time. The enzyme and

substrate cited in the reduction process was peroxidase and peroxide with a

/7-anisdine colorimetric indicator. Another enzyme cited as being effective

was polyphenol oxidase. The organic solvents claimed were basically

paraffins. Applications were as monitors on the exterior of distribution

packages of pharmaceutical and food products.

No further reference to the use ofthisenzymatic temperature indicator has

been found in the literature.

7.7 Lactose removal

Lactose intolerance is a dietary problem affecting a minor but nevertheless

substantial fraction of the population. Individuals affected by this problem

suffer from a lack of the enzyme lactase in their intestinal wall. Lactase is

necessary to break the disaccharide lactose, or milk sugar, into its

component parts glucose and galactose. Since lactose cannot be absorbed

from the gastrointestinal tract, its presence can cause discomfort in the form

of

cramps,

bloating, flatu lence and diarrhea. Persons with lactose intolerance

either avoid milk or introduce lactase enzyme into their milk prior to

consumption.

A British patent assigned to Tetra Pak International AB (Anon., 1975)

describes incorporation of lactase into pasteurized or sterilized milk prior to

packaging to split the lactose after packaging. The lactose must be sterile

and is added aseptically. The patent notes that the milk must remain for

about a day at a temperature of at least 8

0

C for the lactase to function.

The Tetra Pak approach differs from the previously discussed examples of

active packaging because the enzyme has no relationship to the packaging

material. Rather, a solution of enzyme is added directly to the individual

package just prior to sealing. In reality, the Tetra Pak approach is batch

processing done on a miniature scale, within the individual container.

However, this approach does point out that an active enzymatic process can

be carried out in a sealed container.

PharmaCal, Ltd. extended and improved the Tetra Pak approach and made

the process a true enzymatic active packaging process. Budny, at Pharma-

Cal, Ltd. (1990) incorporated the lactase, using proprietary technology of

PharmaCal, Ltd. with the result that 30 -70 of the lactose was removed in

24-36 hours at 3-4C. PharmaCal, Ltd. has proprietary designs and

approaches for commercializing this active package (Figure 7.2).


17/19

7.8 Cholesterol removal

The widespread information on the effects of excess cholesterol in the diet

does not require discussion here. To demonstrate the awareness in the USA

of the cholesterol content of foods, all food packages in the United States

must be labeled for cholesterol content.

Co-author Budny (1990) suggests the removal of cholesterol which is

present in whole milk by incorporating the enzyme, cholesterol reductase, in

the package structure. Using much the same proprietary technology of

PharmaCal, Ltd. as he employed for enzymatic oxygen removal or lactose

splitting, the fluid milk contents are exposed to the enzyme to convert its

cholesterol to coprosterol which is not absorbed by the intestine.

This system, illustrated in Figure 7.3, reduces the extensive in-plant

processing required by supercritical fluid extraction systems to produce

cholesterol-reduced fluid milk products. Rather, active packaging and the

technology of PharmaCal, Ltd. allows untreated fluid milk to be packaged,

Milk

Lactase

e n z y m e

Glucose

Galactose

Lactose

Outside

of

container

ontainer

wal l

Inside of container

Figure 7.2 Lactose removal from liquid products.


18/19

Figure 7.3 Cholesterol removal from liquid products.

and in the time taken to transport the package to the consumer, it

conceivably could become free of cholesterol. While the commercial

implementation has not yet been completed, the component elements of the

application have been successfully demonstrated.

References

Anon. 1977)

Package d foods and drinks in containers coated internally with polymer carrying

enzyme with sterilising action.

German Patent DE2817854A.

Anon. 1990) Packaged milk containing lactose enzyme-giving milk with reduced lactose

content.

UK Patent Application.

Baker, D.L. 194 9)

Deoxygenation Process.

20 September. US Patent 2482724.

Be st, D . 19 90 ) Fermentation opportunities ripen.

Prepared Foods,

159, 5.

Blixt, K. and Tiru, M. 19 77 )

An Enzymatic Time/Temperature Device for Monitoring the

Handling of Perishable Commodities. International Symposium on Freeze-Drying Biolog-

ical Products, 36, 237.

Budny, J. 198 9) A transporting storage or dispensing container with enzym atic reactor.

International Patent Application WO89/06273.

Bu dny , J. 19 90 ) Presentation at Pack Alimen taire, San Fran cisco, California, M ay.

Milk

holesterol

reductase

enzyme

oprosterol

holesterol

Outside

of

ontainer

container

wail

Inside of co ntainer


19/19

Copeland, J. C , A dler, H.I. and Crow, W .D . 199 1) Method and composition for removing

oxygen from solutions containing alcohols an d/or acids.

X S Patent 4996073.

Copeland, R.A. 199 4) Enzym es, the catalysts of l ife. T oday s Chem ist at Work,March.

Courtland, S.B., McG rew, G.N. and Richey, L. 19 92)

Food packaging improvements,

30 June.

US Patent 5126174.

Ernst, R. 19 91 )

Oxygen absorbent and use thereof 2

July. US Patent 5028578.

Field, C , Pivarnik, L.F., Barnett, S.M. and Rand, A.G . 19 86 ) Utilization of glucose ox idase

for extending the shelf-life of fish. J. Food Science, 5 1 .

Fukazawa, R. 19 80)

Methods of preventing spoilage of foods.

Japanese Patent 23071180.

Hopk ins, T.R., Smith, V J . and Banasiak, D .S. 19 91 )

Process utilizing alcohol oxidase,

10

December. US Patent 5071660.

Klibanov, A.M . and Dordich, J.S. 198 9) Enzymatic temperature change indicator, 2 May. US

Patent 4826762.

Kramer, A . and Farquhar, J.W. 19 76 ) Tes ting of time-temp erature indicating and defrost

devices.

Food Technology,

30, 56.

Labuza, T. and Breen, W . 198 9) Active Packaging. J. Food Processing and Preservation, 13,

1.

Lehton en, P., Karilainen, U ., Jaakkola R. and Ky mo lainen, S. 19 91 ) A

packaging ma terial

which removes oxygen from a package and a method of producing the material. International

Patent Application WO 91/13556.

Sarett, B.L . and Scott, D . 19 56 ) Enzym e treated sheet product and article wrapped therewith.

US Patent 2765233.

Scott, D . 19 58) Enzymatic oxy gen removal from packaged foods.

Food Technology,

12 7),

7.

Scott, Do n and Hammer, F. 19 61) O xygen scavenging packet for in-packet deoxygenation.

Food Technology,

15 12) , 99.

Scott, D . 196 5) Oxidoreductase.

Enzymes in Food Processing,

Academic Press, NY.

Thom as, K. and Harrison, R J . 19 85) Method and apparatus for secondary fermentation of

beverages. UK Patent Application 21 435 44 A.

W iseman , A . 19 75 ) En zym e utilization in industrial proce sses, Handbook of Enzyme

Biotechnology, Ellis Horw ood, UK .

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