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 of the reactions. 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.
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 of the reactants 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.
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
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.
Time-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 'natural' antimicrobial agents such as benzoic or propionic
acids to help preserve the product contents.
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
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 most bacteria, yeasts and molds. 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
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 enzyme 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
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 of the enzyme 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.
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 of this patent 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.
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
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 enzymes, 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.
Head
space
Glucose
oxidase
enzyme Gluconate
Glucose
Packaged
liquid
Outside
of
container
Catalase
enzyme
Container wall
Inside of container
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 [0.21-0.01] = 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, with 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 1 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.
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 78-87% 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. Hopkins et a/. (1991) describe
a package in which alcohol or oxidase or cellular extracts of Pichia 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 whole 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
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 et al. 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.
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
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 of this enzymatic 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, flatulence 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).
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
enzyme Glucose
Galactose
Lactose
Outside
of
container
Container
wall
Inside of container
Figure 7.2 Lactose removal from liquid products.
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.
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Cholesterol
reductase
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Coprosterol
Cholesterol
Outside
of
Container
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wail
Inside of container
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