Note: Descriptions are shown in the official language in which they were submitted.
CA 02298109 2000-02-24
FIELD OF THE INVENTION
This invention relates to water-insoluble protein-based covering agents,
including coatings and
films, methods of preparation and their use in the food industry.
B~~CKGROUND OF THE INVENTION
The environmental movement has promoted increased concern about reducing
amounts of
disposable packaging and increasing r~ecyclability of packaging, contributing
to the recent surge
of research in biodegradable and edible coatings, which function by direct
adherence to food
products and films, which act as stand-alone sheets of material used as
wrappings.
Biodegradable packaging pro~3uced from food protein offer the greatest
opportunities since their
biodegradability and environmental compatibility are assured (Krochta, J.M.,
et al., Food
Technology, 51 (2):61-74, 1997). Consumer demands for both higher quality and
longer
shelf-life foods have also stimulated research in the area of edible coating
and film research
(Chen, H., J. Dairy Sci., 78(11):2563-2583, 1995).
Edible films and coatings are capable of offering solutions to these concerns
by regulating the
mass transfer of water, oxygen, carbon dioxide, lipid, flavor, and aroma
movement in food
systems (McHugh, T. H.& Krochta, J. M., Food Technology 1994, 48(1):97-103;
and Chen,
supra1995). Edible films andf coatings based on water-soluble proteins are
typically
CA 02298109 2000-02-24
water-soluble themselves and exhibit excellent oxygen, lipid and flavor
barrier properties;
however, they are poor moisture barriers.
Individual food products within the broad food categories (dried foods,
intermediate moisture
foods, and high moisture foods) requir~° different barrier properties
in order to optimize product
quality and shelf-life. Edible films and coatings are capable of solving the
barrier problems of
these and a variety of other fo~~d systems. See: Kester, et al. , Food
Technol., 1986, 40:47-59;
Mezgheni, et al., J. Ag and Food Chem., 1998 46:318-324; and Ressouany, et
al., J. Ag and
Food Chem., 1998, 46:1618-1623.
Edible agricultural products such as fresh, frozen, whole or cut, fruits and
vegetables, meat, fish,
eggs, grains, nuts, and inedible agricultural products such as living plants,
plant products, and
ornamentals are subject to loss of quality over time from moisture loss,
enhanced respiration and
senescence, and browning and oxidative degradation. Other deterimental effects
to agricultural
products can result from microbial attack and moisture penetration.
The time these products are available in a fresh and attractive form can be
extended, if
respiration can be slowed down by limiting availability of oxygen or if the
carbon dioxide level
can be maintained at an optimum level.. Many edible products and plant
materials have
components which are vulnera-ble to oxidation, with resultant loss in quality,
as oxygen diffuses
into the tissue of the food or plant material. For example, fresh and frozen
fish, frozen fruits and
vegetables, nuts, and ornamenoals have a limited shelf-life which is due to
such oxidation. The
time these products are available in a quality form can be extended, if
oxidation can be slowed
down by limiting diffusion of ~~xygen unto the product.
Fruit and vegetable decay resulting from mold growth is another concern in the
industry. Rot
caused by Rhizopus sp. and As,r~ergillus sp. are mainly accountable for fruit
loss. In order to
control fruit and vegetable decay and losses, many studies have been done in
order to develop
new preservations methods. Yet, a need remains for methods of extending the
postharvest shelf
life.
Browning of many food products is a major problem for the food industry. Until
recently, both
enzymatic and nonenzymatic browning in foods could be inhibited by application
of sulfites.
CA 02298109 2000-02-24
However, health concerns have limited their application (Supers, Food
Technology, 47, 75-84,
1993). Other techniques, including modified atmosphere packaging (MAP) and
vacuum
packaging have been considered. While this approach can delay browning,
excessive reduction
of oxygen will damage the product by inducing anaerobic metabolism, leading to
breakdown and
off-flavor formation. Furthermore, the removal of oxygen also entails a risk
that conditions in the
product might become favorable for the growth of Clostridium botulinum
(Supers, Food
Technology, 47, 75-84, 1993). Another approach is the use of antibrowning
agents based with
citric or ascorbic acid. Althou;~h ascorbic acid can reduce enzymatic
browning, it can increase
nonenzymatic browning due to the its own oxidation into dehydroascorbic acid
(DHAA) which
then reacts with amino acids to yield brown colors by the Maillard reaction or
other
nonenzymatic means (Kaeem et al., Journal of Food Science, 52, 1668-1672,
1987).
Furthermore, high concentrations of acid or other chemical agents could
significantly alter food
flavor and odor. Also, in a recent work by Supers et al. (Journal of Food
Science, 62, 797-803,
1997), it was shown that the use of harsh chemical treatments (heated acid
solutions) can induce
severe textural damage in pre-peeled potatoes resulting in surface firming
(case hardening) and
separation of the superficial tissues that affect texture after mashing and
slicing following
cooking. Such defects would greatly limit the utilization of pre-peeled
potatoes, which have
received the anti-browning treatment.
The use of protein-based coatings, which are flavorless, odorless and
nutritious, could prove very
beneficial for controlling enzymatic browning of cut fruits and vegetables
without inducing
tissue damage. Edible coating; have already been used to effectively delay
ripening in some
climacteric fruits like mangoes;, papayas and bananas. Furthermore,
application of edible
coatings on sliced mushrooms significantly reduced enzymatic browning.
(Nisperos-Carriedo et
al., Proc. Fla. State Hort. Soc. , 104, 122-125, 1991).
Edible films have been proposed for use on foods to control respiration,
reduce oxidation, or
limit moisture loss. (See: J. J. fester arid O. R. Fennema, Food Technology
40: 47-59,1986; and
S. Guilbert, In: Food Packaging and Preservation Theory and Practice, Ed. M.
Mathlouthi,
Elsevier Applied Science Publishing Co., London, England 1986, pages 371-394).
Coatings for
edible products include wax emulsions (U.S. Pat. No. 2,560,820 to Recker and
U.S. Pat. No.
2,703,760 to Cunning); coatinl;s of natural materials including milk solids
(U.S. Pat. No.
2,282,801 to Musher), lecithin (U.S. Pat. No. 2,470,281 to Allingham and U.S.
Pat. No.
CA 02298109 2000-11-09
3,451,826 to Minder), algin and a gelling mixture (U.S. I'~tt. No. 4,504,502
to Earle and McKee),
protein (U.S. Pat. No. 4,314,971 to Garbutt), dispersions of a hydrophilic
Film former and an
edible fat (U.S. Pat. No. 3,323,922 to Durst), dispersions of hydrophobic
materials in aqueous
solutions of water-soluble high polymers (U.S. Pat. No. 3,997,674 to LJkai et
al.), and emulsions
or suspensions of a water-soluble protein material and hydrophobic m<rterial,
adjusting the pH of
the protein material to its isoelectric point (U.S. Pat. No. 5,019,4t)3 to
Krochta).
Dried foods, low moisture baked products, intermediate moisture foods and high
moisture foods
al( exhibit potential for improvement through the rise of edible coatings and
films. Dried foods
(e.g., dried vegetables and dried meats) and low moisture baked products
(e.g., crackers, cookies
and cereals) are particularly susceptible to moisture uptake from Che
atmosphere. Low moisture
baked foods are also susceptible to moisture uptal<:c from moist tillings and
toppings. Such
changes can result in loss of sensory acceptability of thc; food product, as
well as a reduced
shelf-life. Nlany dried and baked products are also susceptible to oxidation,
Lipid migration and
volatile flavor loss.
Intermediate moisture foods, such as raisins and dates, often become
unacceptable due to
moisture loss over time. Moisture loss is particularly problematic when the
moisture transfers
into lower moisture components of a food system. For example, raisins can lose
moisture to the
bran in raisin bran Nut meats, another intermediate moisture food, are
susceptible to lipid
oxidation resulting in the development of off flavors.
Many substrates such as edible products and plant materials have a high
moisture content and are
vulnerable to quality loss as they lose their moisture to the air. In
particular, fresh fruits and
vegetables, eggs, fish, living or cut trees, plants, and ornamentals, for
example, have a limited
shelf-life which is due in part to loss of moisture to the atmosphere.
Products which have peels,
skins. or shells tend to have retarded moisture loss; but over a period of
time enough moisture
can be lost to lower the product quality to the point of product rejection,
Substrates which are high in moisture content and have high moisture at the
surface are
particularly vulnerable to loss of quality due to moisture loss. Examples are
fruits and vegetables
and other foods, and plant products which have exposed tissue surfaces created
by peeling,
cutting, etc. such as peeled and/or sliced apples, sliced tomatoes, peeled
eggs, fish filets, and
CA 02298109 2000-02-24
cut-stem flowers. Because their natural skins, peels, and shells, which
normally act to retard
moisture loss have been removed, these products lose their quality quickly
High moisture food components also typically lose moisture to lower moisture
components. One
classical example of this phenomenon occurs when pizza sauce moisture migrates
into the crust
during storage, resulting in a ~~oggy crr.ist. Oxidation and flavor loss are
also problematic to high
moisture food systems. The respiration rates of whole fruits and vegetables
often dictate their
shelf lives. Minimally processed fruits and vegetables are often subject to
unacceptable levels of
oxidative browning.
Many food proteins like corn zero, whE:at gluten, soy protein isolate, whey
protein isolate and
caseins have been formulated into edible films or coatings. Proteinic films
offer better
mechanical properties but their permeability to gases and moisture are
variable. Caseins have
been widely used since this protein is abundant, cheap and readily available.
Moreover, it has
good foaming properties where mixed with fatty acids (Avena-Bustillos, R.J. &
Krochta, J.M., J.
of Food Science, 58:904-907, 1993) and can be easily polymerized into films
having good barrier
properties against gas and water vapor. An acid treatment (towards the
isoelectric point)
improves resistance to moisture transport since this treatment decreases the
mobility of the
polymer chains (Kester and Fennema, 1986; Peyron, Viandes Prod. Carnes, 12, 41-
46, 1991).
Unfortunately, the highly hydrophilic mature of these proteins limits their
ability to provide
desired edible film functions.
Water-insoluble edible films and coatings offer numerous advantages over water-
soluble edible
films and coatings for many food product applications. Increasing levels of
covalent crosslinking
in water-insoluble edible films and coatings result in better barriers to
water, oxygen, carbon
dioxide, lipids, flavors and aromas in food systems. Film mechanical
properties are also
improved. Many foods, such as fruits and vegetables, are exposed to water
during shipping and
handling. In these cases, water-insoluble films and coatings remain intact;
whereas,
water-soluble films and coatings dissolve and lose their barrier and
mechanical properties. Edible
films in the form of wraps, such as sandwich bags, also require water-
insolubility.
Edible coatings based on waxes, polysaccharides and proteins have been
developed in order to
preserve food quality and freshness. Proteins act as a cohesive, structural
matrix in multi-
CA 02298109 2000-02-24
component systems to provide: films and coatings having good mechanical
properties. Plasticizer
addition improves film mechanical properties. Such edible films could help to
reduce food
dehydration.
Edible films can be formulate~~ as composite films of heterogeneous nature
i.e. formed starting
from a mixture of polysaccharides, proteins and/or lipids. This approach
allows for the
beneficial use of the functional characteristics of each film component. The
preparation of
composite films imposes an emulsification of the lipidic material in an
aqueous phase. The
preparation technique of hydrophobic films influences its barrier properties.
A film formed
starting from a dispersed distribution of the hydrophobic material offers weak
barrier properties
to steam, compared to films with a continuous layer (Martin-Polo et al.,
1992). A dispersed
distribution is due to the difference in polarity between the support
(example: methyl cellulose)
and the hydrophobic material (technique of emulsion).
For an example of a single-protein films that is a composite of different
additives, see U.S. Patent
5543164, wherein a protein selected from the group considting of milk
proteins, whey proteins,
casein, wheat proteins, soy proteins, ovalbumin, corn zero, peanut protein and
keratin is
combined with a food grade plasticizer (sorbitol, glycerol or polyethylene
glycol) and a lipid
(fatty acids, fatty alcohols, waxes, triglycerides, monoglycerides and
mixtures thereof).
Given the environmental concerns, the biodegradability of the covering agent
is a factor for
consideration. Biodegradatior~ is a process by which bacteria, moulds, yeasts
and their enzymes
consume a substance as a source of food so that the original form of this
substance disappears
(Klemchuk, P. 1990, Pol. Degra. Stab., 27, 183-202). Under appropriate
conditions, a
biodegradation process from t'vo to three years is a reasonable period for the
assimilation and
the complete disappearance of the product (Klemchuk, supra). Pseudomonas is
recognized as
being a type of bacteria which can synthesize a very diverse number of
enzymes. Being
psychrotrophic, it is responsible for the putrefaction of refrigerated foods.
It can however
decompose certain chemicals like pesticides and is resistant to certain
disinfectants (compounds
of quaternary ammonium) and antibiotics (Tortora et al., 1989). It is found in
a majority of
natural sites (water-ground-air), foodstuffs (milk - dairy products - egg -
meats) and in some
animals (Palleroni, 1984). The majority of the Pseudomonas species degrade K-
casein before the
population reaches 104 UFC/mU. (3-casein is more susceptible to degradation
than a for the
CA 02298109 2000-02-24
majority of species. This phenomenon is only observed when the bacterial
population is higher
than 106-10' UFC/ml (Adams et al., 1976). Hence the use of Pseudomonas for the
degradation
of various components was used, given its resistance to various stress
conditions (for example:
temperature, carbon source) and its capacity to synthesize an significant
amount of enzymes
(Tortora etal., 1989).
Thus, there is a need for a water insoluble protein-based covering agent which
can be designed to
meet the individual characteristics of a wide-range of agricultural and
foodstuffs.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a caseinate-whey crosslinked
covering agent. The ratio
of caseinate-to-whey ranges from 1:99 to 99:1, and is adjusted to meet the
covering
characteristics of the substrate. Additives can be included in the film in
order to further tailor the
covering agent to its substrate. This cowering agent can be applied to
agricultural products, and
foodstuffs.
In one embodiment, this invention provides a composition for use as a covering
agent,
comprising: (i) a caseinate salt; (ii) whey protein; and optionally (iii) a
plastisizing agent,
and/or (iv) a polysaccharide, wherein said caseinate and whey molecules are
cross-linked to
form a covering agent.
In another embodiment, this invention provides a process for the preparation
of a covering agent,
comprising the steps of:: (i) poeparing an aqueous solution comprising a
caseinate salt, whey
protein and, optionally, a plastisizing a;~ent and/or a polysaccharide; (ii)
producing a
substantially degassed solution by treating said aqueous solution to remove
dissolved air; and
(iii) subjecting said degassed aqueous solution to a chemical, or thermal step
and/or an
irradiation step, wherein said chemical, or heating and/or irradiation step
causes crosslinking of
caseinate and whey to produce said covering agent.
BRIEF DESCRIPTION OF THE DRAWINGS
7
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Figure 1 shows elution curves for calciium caseinate (alanate 380): a),
native; b), heated at 90°C
for 30 minutes; or c), irradiated at 32 kGy.
Figure 2 shows elution curves for commercial whey proteins (CWP): a), native;
b), heated at
90°C for 30 minutes; or c), irradiated a.t 32 kGy.
Figure 3 presents elution curves for whey protein isolate (WPI) and calcium
caseinate with ratio
of 50- 50: a) control; b), heated at 90°(. for 30 minutes; c),
irradiated at 32 kGy; or d), combined
heat and irradiation treatment.
Figure 4 shows fraction of insoluble matter in function of the irradiation
dose. Results are
expressed as the percentage in solid yield after soaking the films 24 hours in
water.
Figure 5 demonstrates the puncture strength of unirradiated and irradiated (32
kGy) whey protein
isolate (WPI)- calcium caseinate films. Ratios express the proportion in WPI
or calcium
caseinate for a formulation based on 5% w/w total protein solution. For
instance, the formulation
25-75 represents 1.258 WPI protein and 3.75g calcium caseinate protein per
100g protein
solution.
Fiugre 6 shows the puncture sl:rength of unirradiated and irradiated (32 kGy)
commercial whey
protein-calcium caseinate films. Ratios express the proportion in CWP or
calcium caseinate for a
formulation based on 5% w/w total protein.
Figure 7 shows the viscoelasticity coefficient for unirradiated and irradiated
(32 kGy) CWP-
calcium caseinate films.
Figure 8 presents cross sections of a) unirradiated or b) irradiated (32 kGy)
calcium caseinate
films. (9 mm bar = 3 pm).
Figure 9 presents cross sections of irradiated (32 kGy) CWP-calcium caseinate
films with the
ratio : a) 50:50; b), 75:25; c) 100:0 (9 mm bar = 3 l.~m).
Figure 10 presents cross sections of WPI-calcium caceinate films : a) heated
at 90°C for 30 min,
8
CA 02298109 2000-02-24
b) heated at 90°C for 30 min and irradiiated at 32 kGy (9 mm bar = 3
pm).
Figure 11 shows mold contarr~ination (%) of coated/non-coated strawberries.
Coating based on
5% w/w mixed proteins (whey and calcium caseinate) and 2.5% w/w glycerol.
Figure 12 presents the variation of the lightness parameter (L*) as a function
of time for coated
and uncoated potato slices. (controle = control; caseinate = caseinate;
lactoserum = whey; L+C =
caseinate-whey). Measurements for each experimental condition being conducted
on the same
potato slice.
Figure 13 presents HUE angle variation for uncoated and coated potato slices
as a function of
time (controle = control; caseinate = caseinate; lactoserum = whey; L+C =
caseinate-whey).
Measurements for each experimental condition being conducted on the same
potato slice.
Figure 14 presents the variation of the lightness parameter (L*) as a function
of time for coated
and uncoated apple slices. (cocltrole = control; caseinate = caseinate;
lactoserum = whey; L+C =
caseinate-whey). Measurements for each experimental condition being conducted
on the same
apple slice.
Figure 15 presents HUE angle variation for uncoated and coated apple slices as
a function of
time (controle = control; caseinate = caseinate; lactoserum = whey; L+C =
caseinate-whey).
Measurements for each experimental condition being conducted on the same apple
slice.
Figure 16 shows the results of viscoelasticity (coefficient de
viscoelasticite) on various caseinate-
whey films, ranging from 1:99 to 99:1 caseinate:whey and crosslinked using
either heat or
irradiation.
Figure 17 shows the results of puncture strength studies (force de rupture) on
various caseinate-
whey films, ranging from 1:99 to 99:1 caseinate:whey and crosslinked using
either heat or
irradiation.
Figure 18 shows the results of results of viscoelasticity (coefficient de
viscoelasticite) on various
caseinate-whey films, ranging from 1:99 to 99:1 caseinate:whey and crosslinked
using heat
CA 02298109 2000-02-24
Figure 19 shows the results of studies (;deformation a la rupture (mm) ) on
various caseinate-
whey films, ranging from 1:99 to 99:1 caseinate:whey and crosslinked using
heat.
Figure 20 the results of puncture strength studies (force de rupture) on
various caseinate-whey
films, ranging from 1:99 to 99:1 caseinate:whey and crosslinked using heat.
Figure 21 shows the results of a comparative study of various properties of
films based on milk
proteins.
Figure 22 presents results of a study demonstrating influence of storage time
on the antioxidative
properties of calcium caseinate films containing essential oils from rosemary
and thyme.
Figure 23 presents results of a study demonstrating effects of incorporation
of lecithin on the
antioxidative properties of calcium caseinate films containing essential oils
from rosemary and
thyme.
Figure 24 presents results of a study demonstrating antioxidative activities
of calcium caseinate
films made with water and water/ethanol extracts of dry spices from rosemary
and thyme. A)
water; B) water/ethanol (80/2Ci); and C;I water/ethanol (20/80).
Figure 25 presents results of a study demonstrating influence of physical
cross-linking on the
water vapor permeability of films made with calcium caseinate and whey protein
isolate.
Figure 26 presents results of a study demonstrating the influence of physical
cross-linking on the
water vapor permeability of films made with calcium caseinate and whey protein
concentrate.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a caseinate-whey covering agent, wherein the ratio of
caseinate:whey
can be varied from 1:99 to 99:1 in order to optimize the characteristics of
the covering agent and
the final product to the requirements of the product to be covered. The total
concentration of
l0
CA 02298109 2000-02-24
protein in solution can be varied in order to meet the requirements of the
substrate to be covered.
Other additives can be included to bestow further properties to the covering
agent.
The following abbreviations are used herein: WPI, whey protein isolate; WPC,
whey protein
concentrate; WVP, water vapor permeability; PG, propylene glycol; TEG,
triethylene glycol;
CMC, carboxy methyl cellulo;~e; BSA, bovine serum albumin;
As used herein, the term "coating" refers to a thin film which surrounds the
coated object.
Coatings will not typically have the mechanical strength to exist as stand-
alone films and are
formed by applying a diluted component mixture to an object and evaporating
excess solvent.
As used herein, the term "film" refers to a stand-alone thin layer of material
which is flexible and
which can be used as a wrapping. Films of the present invention are preferably
formed from an
emulsified mixture of two proreins, optionally in combination with a lipid
and/or a plasticizer.
As used herein, the term "dissolved gases" refers to any gases, including
oxygen, nitrogen, and
air which become entrapped in the emulsified fluid mixture prior to
crosslinking.
As used herein, the term "disulfide formation" refers to the formation of new -
-S--S-- bonds
which can occur either intermolecularly or intramolecularly. These bonds can
be formed in the
proteins used in preparation of the films and coatings of the present
invention by several routes.
Disulfide formation can take place via thiol oxidation reactions wherein the
free sulfhydryl
groups of cysteine residues become oxidized and form disulfide bonds.
Additionally,
thiol-disulfide exchange reactions can take place wherein existing
intramolecular disulfide bonds
are broken by heat, chemical or enzymiic means and allowed to form new
disulfide bonds which
are a mixture of the intermolecular and intramolecular variety.
As used herein, the term "lipid component" refers to all oils, waxes, fatty
acids, fatty alcohols,
monoglycerides and triglycerides having long carbon chains of from 10 to 20 or
more carbon
atoms, which are either saturated or unsaturated. Examples of "lipid
components" are beeswax,
paraffin, carnuba wax, stearic ;acid, pahnitic acid and hexadecanol.
As used herein, the term "food grade plasticizer" refers to compounds which
increase the
11
CA 02298109 2000-02-24
flexibility of films and which have been approved for use in foods. Preferred
plasticizers are
polyalcohols such as glycerol, sorbitol and polyethylene glycol.
As used herein, the term "protein" refers to isolated proteins having either
cysteine and/or cystine
residues which are capable of undergoiing thiol-disulfide interchange
reactions and/or thiol
oxidation reactions, or proteins having tyrosine residues which are capable of
undergoing
covalent crosslinkage to form bityrosine moieties. Preferred proteins are
those which are
isolated from milk, the most preferred proteins being casein and whey.
Bovine casein is an abundant, economic and easily accessible protein. Casein
alone roughly
accounts for 80% of the total proteins in cow's milk (Schmidt and Morris,
1984). It can be
isolated from skimmed milk either by acidification with mineral acid, or by
acidification with
mixed bacterial cultures (Vuillemard and Al, 1989). It is a phosphoprotein
with amphiphilic
characteristics which binds strongly to the Ca2+ and Zn2+ ions (Schmidt and
Morris, 1984;
Vuillemard et al., 1989). Due to their absorbent character, casein films do
not produce an
effective barrier to moisture. l~n the other hand, it can act as an
emulsifying agent and create a
stable casein-lipid emulsion (Avena-Bustillos and Krochta, 1993). The gas and
moisture barrier
properties of casein-based films can be improved by the polymerization of the
protein with
calcium (Ca+2) but also by adjusting the pH of the medium at the isoelectric
point of casein. The
adjustment at the isoelectric point optimizes the protein-protein
interactions, modifies the
molecular configuration and would influence the mass transfer properties
(Krochta, 1991 and
Avena-Bustillos and Krochta, 1993).
Bovine casein is composed of four major proteinic complexes, named asl a~d a,
(3- and K-caseins.
All in all, a casein molecule consists of a primarily hydrophobic core of a
and of (3- casein and
surrounded by K-casein on the surface I;Schmidt and Morris, 1984). The
stability of micelles is
ensured by the K-caseins and the calcium colloidal phosphates found on the
periphery (Schmidt
and Morris, 1984). Casein contains many uniformly distributed proline
residues. That gives it an
open structure thus limiting thc: formation of alpha helixes and beta layers
(Modler, 1985). This
open conformation shows a certain resistance to the thermal denaturation and
offers an easy
access to the enzymatic attacks (Schmidt and Morris, 1984; Vuillemard et al. ,
1989, McHugh
and Krochta, Food Technolog~~, 48(1), 97-103, 1994).
12
CA 02298109 2000-02-24
Caseinates are obtained either by the acidification with mineral acid (HCl or
H2S04), or by the
acidification by mixed cultures made up of Streptococcus subspecies lactis
and/or cremoris, at
the isoelectric point of casein (pH of 4,6). The neutralization of the
insoluble precipitates of
casein or lactic acids by alkalis allows for the dissolution in salts of
sodium of calcium,
potassium, magnesium, or aramonium~ (Schmidt and Morris, 1984; Vuillemard et
al. , 1989;
McHugh and Krochta, 1994). The solubilized caseinates are dehydrated
thereafter. Salts of
caseins thus obtained are soluhle above pH 5.5.
Whey (mainly a,-lactalbumine and (3-lactoglobuline) or small-milk proteins,
form a
thermoirreversible gel, which is pH-dependent and heat sensitive (Schmidt and
Morris, 1984;
Vuillemard and Al, 1989). A~~ an example, heating of whey proteins at
temperatures between 70
and 85° C and to a concentration higher than 5%, forms a
thermoirreversible gel. This gel
develops by the formation of new intermolecular disulphide bonds (Vuillemard
and Al, 1989).
The gelling process of whey proteins is strongly influenced by the pH of the
medium during
heating since a pH >_ 6.5 decreases the intermolecular interactions (Schmidt
and Morris, 1984;
Xiong, 1992). High ionic forces seem to increase proteinic stability probably
through an
increase of the proteins' capacity of hydration (solubility) (Xiong, 1992).
Two different types of whey protein were used in the production of the
covering agents of the
instant invention; whey protein isolate (WPI) and whey protein concentrate
(WPC).
Ultrafiltration techniques are e:mployedl to isolate undenatured WPC's, and
high performance
hydrophilic exchange is used to purify WPI's. WPC's range from 25% to 80% whey
proteins,
whereas WPI's have protein contents greater than 80%.
Designing the Ratio of Caseinate to Whey
This covering agent differs from those known in the art, primarily because it
is comprised of two
proteins, caseinate and whey, ~Nhose ratio is determined to generate a
covering agent with
characteristics that are optimal for the product to protect.
The ratio of caseinate-to-whey is choosen to optimise the mechanical
characteristics of the
covering agent in accordance with the requirements for the food product it is
intended to protect.
The greater the proportion of caseinate, the more dependent the solution on
the dependence or
13
CA 02298109 2000-02-24
irridation. A 99:1 caseinate-mhey covering agent can be either a coating or a
film, depending
upon how the protein is crosslinked.
The greater the proportion of whey, the more the covering agent becomes a
coating that has low
physical strength, but is usable to protect fruit. The source of the whey has
a large impact on the
characteristics of the covering agent: commercially obtained whey tends to be
more denatured,
versus whey produced in a laboratory by microfiltration which tends to form
coverings with
greater puncture strength.
Example IV presents studies demonstrating the effect of varying the ratio of
caseinate to whey.
In certain embodiments of the invention, a food grade plasticizer is added to
the denatured
protein solution. The food grade plasticizer serves to increase both the
mechanical strength of the
film and its flexibility. The plasticizer its preferably a polyalcohol, for
example, sorbitol, glycerol,
triethylene glycol or polyethylene glycol. The amount of food grade
plasticizer which is added
will typically be about 1 to 15 % by weight in solution, preferably about 2 to
10 % by weight in
solution.
In other embodiments, it may be desirable to include agents such as
emulsifiers, lubricants,
binders, or de-foaming agents to influence the spreading characteristics of
the coating agent.
Determining the Mechanical and Structural Characteristics of the Covering
Agent
There are a battery of tests, well known to one skilled in the art, that can
be performed on the
coating agent to test the characteristics of the final product. The
viscoelasticity and puncture
strength of films and coatings can be measured to determine the mechanical
properties, which
can also be correlated with transmission electron microscopy observations. The
mechanical
strength of protein solutions can be increased by the formation of cross-links
which confer
elastomeric properties to the rr~aterial as well as improve the water
resistance of such protein
films (Brault et al., J. Agric. Food Chern., 24(8), 2964-2969, 1997). Size-
exclusion
chromatography can be performed on cross-linked solutions to determine the
molecular weight
distribution of the cross-linked proteins..
14
CA 02298109 2000-02-24
Film thickness can be measured using commercially available instrumentation
such as a
Mitutoyo Digimatic Indicator (Tokyo, Japan) by measuring random positions
around a film. For
example, measuring six random positions around a sample film, should provide a
film with a
thickness in the range of 45 - 60 Vim.
Molecular weight determinati~~n of the cross-linked proteins can be determined
using size-
exclusion chromatography. In one example of this method, size-exclusion
chromatography is
performed on a soluble protein fraction using a Varian Vista 5500 HPLC coupled
with a Varian
Auto Sampler model 9090, with detection of the protein solution performed
using a standard UV
detector set at 280 nm. In this example, a Supelco Progel TSK GMPW column
followed by two
Waters Hydrogel columns (2000 and 500) is used for the molecular weight
determination of the
cross-linked proteins, wherein the total molecular weight exclusion limit is
25 x 106 daltons
based on linear polyethylene glycol (P1~,G). The eluant (80% v/v aqueous and
20% v/v
acetonitrile) is flushed through the columns at a flow rate of 0.8 mL per
minute. The aqueous
portion of the eluant is 0.02M tris buffer (pH = 8) and 0.1 M NaCI. The
molecular weight
calibration curve is established using a series of protein molecular weight
markers (Sigma, MW-
GF-1000, USA) ranging from 2 x 106 daltons to 29 000 daltons. All soluble
protein solutions
0.5 % w/v ) are filtered on 0.4:5 pm prior to injection.
Insolubility measurements can be performed as in the following example,
wherein the average
dry weight of the films is determined on seven films by drying them in an oven
at 45 °C until
constant weight was achieved (6 or 7 days). Seven more films are dropped in
100 mL of boiling
water for 30 minutes. The flasks are removed from the heat and the films
remain in the water for
another 24 hours. After 24 hours, the solid films are removed and dried in the
oven as previously
described. Results are calculat~°d using the following formula: [ Dry
Weight (solid residues)/ Dry
Weight (untreated film)] x 100
The puncture strength of a film can be determined by measuring the 'breaking
load' and 'strain
at failure' which are calculated simultaneously for the samples, by recording
the application of
pressure to a film, which is them converted into units of force (N). Puncture
tests can be carried
out using a Stevens LFRA Te~saure Analyzer Model TA/1000 (NY, USA), as
described
previously by Gontard et al. ( ~T Food Sci., 57 (1), 190-195, 1992). In this
example, films are
equilibrated for 48 hours in a clessicator containing a saturated NaBr
solution ensuring 56%
CA 02298109 2000-02-24
relative humidity. A cylindrical probe ( 0.2 cm diameter ) is moved
perpendicularly at the film
surface at a constant speed (lrnm/sec) until it passes through the film.
Strength and deformation
values at the puncture point are used to determine hardness and deformation
capacity of the film.
In order to avoid any thickness variation, the puncture strength values are
divided by the
thickness of the film. The force-deforn~ation curves are recorded.
The viscoelasticity of a film c,an be measured by the relaxation curve
obtained following the
application of a force to the film. An important characteristic sought in film
products is
elasticity, hence a film having a low relaxation coefficient is preferable.
Viscoelastic properties
can be evaluated using relaxation curvca. The same procedure as the used for
the puncture test
can be used, but the probe is stopped and maintained at 3 mm deformation. The
parameter Y(1
min) _ (F'°-FI~IF° where F'~ and F' were forces record initially
and after 1 min of relaxation,
respectively [Peleg, M., J. Food Sci. 1979, 44 (1), 277]. A low relaxation
coefficient (Y --~ 0)
indicates high film elasticity v~~hereas a high coefficient (Y--~ 1) indicates
high film viscosity.
Heats of Wetting can be determined by obtaining isothermic measures using
disposable glass
ampules in a calorimetre SetaramT"' C~>0. In one exemplary method, a known
amount of the
sample, which is dessicated for a minimum of 24 hours, is placed into a vacuum
sealed ampule
and then placed into a water filled cell .equipped with TeflonT"' joints, to
prevent water
evaporation. The ampule with the cell is placed into the calorimetre and when
the thermic
equilibrium is obtained, the anlpule is broken. Water from the cell enters the
ampule due to the
negative pressure and reacts with the sample. The registered measurement is
then converted into
joules per gram giving the heat of wetting of the samples measured.
Transmission electron microscopy (TEM) can be used to examine the microscopic
structure of
the films to provide microstructure information that relates to the mechanical
characteristics of
the films. In one example, dry films are first immersed in a solution of 2.5%
glutaraldehyde in
cadodylate buffer, washed and postfixed in 1.3% osmium tetroxide in collidine
buffer. Samples
are then dehydrated in acetone (25, 50, 75, 95 and 100%) before embedding in a
SPURRT"'' resin.
Polymerization of the resin proceeds at 60 °C for 24 hours. Sections
are made with an
ultramicrotome (LKB 2128 Ultrotome'~'M) using a diamond knife and transferred
on Formvar-
carbon coated grids. Sections are stained 20 minutes with uranyl acetate (5%
in 50% ethanol)
and 5 minutes with lead citrate. Grids are observed with an HitachiT"'' 7100
transmission electron
microscope operated at an accelerating voltage of 75 keV.
16
CA 02298109 2000-02-24
Water vapor permeability can be determined in a manner similar to U.S. Patent
5543164.
Briefly, test cups were made trot of Plexiglas such that the bottom of the cup
had an outside
diameter of 8.2 cm., the area of the cup mouth was 78.5 cmz~, and the well
inside the cup had a
depth of 1.2 cm. Silicon sealant (High Vacuum Grease, Dow Corning, Midland,
Mich., U.S.A.)
and four screws, symmetrically located around the cup circumference, were used
to seal films
into test cups. Desiccator cabinets were purchased from Fisher Scientific,
Inc. (Fair Lawn, N.J.,
U.S.A.) and variable speed motors with attached fans were installed. These
cabinets were placed
in a 24°C controlled temperature room. Air speeds were measured using a
Solomat anemometer
(Stamford, Conn., U.S.A.). Fan speeds were set to achieve air speeds of 500
ft/min in the
cabinets. Each cabinet contained an Airguide hygrometer (Chicago, Ill.,
U.S.A.) to monitor the
relative humidity conditions within the cabinets. Prior to each experiment,
cabinets were
equilibrated to 0% relative humidity (R;H) using calcium sulfate Drierite
desiccant (Fisher
Scientific, Inc., Fair Lawn, N..1., U.S.A.).
Six milliliters of distilled water or equivalent amounts of saturated salt
solutions were placed in
the bottoms of the test cups to expose the film to a high percentage relative
humidity inside the
test cups. Next, films were mounted in the cups. The distance between the
solution and the film
was determined both before and after each experiment using a micrometer. After
assembly, the
test cups with films were inserted into the pre-equilibrated 0% RH desiccator
cabinets. After
about two hours, steady state load been achieved and five weights were taken
for each cup at
greater than two hour intervals. Four samples of each film were tested.
Finally, the WVP
correction method was employed to calculate the water vapor permeability
properties of the film
as described by McHugh et al. J. Food. Sci. 58:899-903 (1993). The WVP
correction method
accounts for the water vapor partial pressure gradient in the stagnant air
layer of the test cup
when testing hydrophilic edible films. ~Che conventional ASTM method for WVP
determination
does not account for this partial pressure gradient and can result in an error
of up to 35%. Use of
the WVP correction method enables accurate determination of relative humidity
conditions
during testing.
Oxygen permeability can be dtaermined for caseinate-whey coverings on a
commercial unit such
as a MOCON OXTRAN 2-20 (Minneapolis, Minn., U.S.A.). This system provides the
flexibility
of testing films under a variety of relative humidity and temperature
conditions.
17
CA 02298109 2000-02-24
Chemical Properties of the Covering Agent
There are a number of chemical properties that should be designed into the
covering agent, such
as its antioxidant properties, antibacterial properties, biodegradability,
ete. Accordingly, there
are a number of tests well known to one skilled in the art to make these
determinations.
Tests to determine the antioxidant properties of the covering agent can be
performed to optimize
this criteria depending on the oequirements of the foodstuff. Evaluation of
the antioxidative
properties of a film or coating can be measured using a model allowing the
release of oxidative
species by electrolysis of saline buffer. In this method, measurements can be
performed
following a modified procedure of the DPD (N,N-diethyl p-phenylenediamine)
colorimetric
method reported by Dumoulin et al., (Arzneim-Forsch/Drug Res., 46, 855-861,
1996). Films can
be cut in pieces of equal thickness all measuring 0.8 x 2.5 cm. They are
placed in a well
containing 3 mL of Krebs-Henseleit buffer and submitted to electrolysis for
one minute (400
Volts; 10 mA). 200 pL of the solution is sampled and added to 2 mLof DPD
solution (25
mg/mL). The oxidation species react instantly with DPD producing a red
coloration that can be
measured at 515 nm using a standard spectrophotometer. The antioxidative power
measurements describe the film's capacity to inhibit the formation of
oxidation species (red
coloration). The reaction is calibrated using the non-electrolyzed KH buffer
solution (100%
inhibition) and the electrolysed KH buffer solution (0% inhibition). The
inhibition percentage is
calculated following the equation:
[inhibition (5) -= 100 - [(ODs~,mpie/OD~o"troi) x 100]
where OD represents the relative oxidation degree intensity measured by the
spectrophotometer
at 515 nm.
Tests to determine whether the coating delays enzymatic browning can be
performed to optimize
this criteria for certain foodsuffs. Color measurements can be taken to
demonstrate whether a
coating efficiently delays enzymatic browning by acting as oxygen barrier. In
one example, can
be taken every thirty minutes for a total experimental period of five hours.
The color can be read
using a ColormetT"' sperctrocolorimeter (Instrumar Engineering Ltd., St.
John's, NF., Canada)
using the standard (1976) CIEI'-,ABT"' color system. Lightness is reported as
L~ and the HUE
angle value is given by tari ~ (li~~/a~). A.s the HUE angle decreases, red
pigmentation increases.
18
CA 02298109 2000-11-09
The a' axis {red) corresponds to a HI1E angle of 0°. Color measurements
can be taken once on
each slice of Emit or ~,~egetable, for example, for between 8 and I2 readings
per data point.
Additives, including chelating agents, such as ascorbic acid and calcium
disodium EDTA,
antibacterial agents, flavorings, vitamins and minerals, etc can be included
in the coating agent to
optimize the characteristics of the covering.
In certain embodiments of the invention, a lipid or edible oil component can
be incorporated into
the covering agent A variety of lipid components of varying chain lengths can
be used to form
effective films. The lipid component can be a fatty acid, a fatty alcohol, a
wax, a triglyceride, a
monoglyceride or any combination thereof. Examples of tatty acids which are
useful in t:he
present invention are stearic acid, palmitic acid, myristic acid and lauric
acid. Examples of fatty
alcohols which can be used in the present invention are stearyl 4~lcohol and
hexadecanol. Waxes
which are useful in the present invention include beeswax, carnuba wax,
microcrystalline wax
and paraffin wax. The lipid component will typically be present in an amount
of from 1 to 30%
by weight in solution, preferably aboc.it 2 to 15% by v~eight in solution.
When composite mixtures are fom~ed containing proteins in combination with
lipids or food
grade plasticizers or both, it is preferred to remove dissolved gases from the
mixture. As noted
above, the method of removal will typically involve subjecting the solution to
reduced pressures
by means of a vacuum pump or water aspirator.
In the present inventive method to apply the coating, the first step is the
formation of an aqueous
denatured protein solution. Prior to dcnaturation, the protein will typically
be solubilized in an
aqueous solution in a concentration range of about 5~/o by
weight.
I~efhc~ds of Crosslinking the Proteins
The crosslinking of a proteinic solution can be achieved by a variety o-f
methods including
irradiation, heat, chemical and enzymic. The preferred crosslinking treatments
of the present
invention being irradiation and heating, resulting in inter- and intra-
molecular linkages including
bityrosine residues and thiol-disulfide bridges.
19
CA 02298109 2000-02-24
Upon radiolysis of an aqueou;~ protein solution, hydroxyl radicals are
generated. Aromatic
amino acids react readily with these hydroxyl radicals. For example, tyrosine
amino acids react
with hydroxyl radicals to proaluce tyrosyl radicals. These may then react with
other tyrosyl
radicals or with tyrosine molecules to form stable biphenolic compounds, in
which the phenolic
moieties are linked through a covalent bond. The 2',2-biphenol bityrosine
moiety exhibits a
characteristic fluorescence, which provides a means of monitoring the
formation of such
crosslinks. The formation of hityrosine is one mechanism for causing protein
aggregation,
although other crosslinks can lbe formed. The gamma irradiation treatment
presents a number of
conveniences, including the production of sterile goods.
Alternatively, when heat treatment is used, the aqueous protein solution is
heated to a
temperature above the denaturation temperature of the particular protein for a
period of time
sufficient to initiate crosslinkage reactions, which are predominantly
disulfide bridges. These
thiol-disulfide interchange anti thiol oxidation reactions can be either
intramolecular or
intermolecular. The precise temperature and length of time for a given protein
can be determined
empirically, but will typically involve temperatures of from about 70 to
95°c., preferably from
about 75 to 85°c and a length of time of up to 3 hours, preferably from
about 15 to 45 minutes.
The result of this reaction is a solution of a denatured protein having a
mixture of intermolecular
and intramolecular disulfide crosslinks.
Methods of Coating the Product to be Protected
In the present inventive method, the denatured protein solution may applied to
a food item and
water is evaporated to form a coating for the food item. The method of
application is not critical
and will depend upon the particular food item. Suitable application methods
include dipping,
brushing and spraying. Similarly, the method of evaporation is not critical.
Water can be
removed by standing in air at ambient temperature. Alternatively, water can
also be removed by
gently warming the coated food item and exposing it to a stream of air or
other suitable gas such
as nitrogen.
In preferred embodiments, dissolved gases are removed from the aqueous protein
solution prior
to denaturing the protein. The removal of dissolved gases prevents formation
of air bubbles in
CA 02298109 2000-02-24
the films and increases both the mechanical strength of the film and the
ability of the film to
control mass transfer in foods. The method selected for removal of dissolved
gases is not
critical, however, a preferred method involves subjecting the solution to
reduced pressures by
means of a vacuum pump or water aspirator.
The present invention also provides foodstuffs and packagings coated with the
coating agents of
the instant invention. The following e:Kamples are provided by way of
illustration and not by
way of limitation.
The following examples describe the effect of combined physical treatments
(heat and
irradiation) on the mechanical and structural properties of milk protein-based
covering agents.
The effects of gamma-irradiation and thermal treatment of caseinate and whey
proteins solutions
has been studied using size-exclusion chromatography. Furthermore, the
puncture strength and
the viscoelastic properties of film formulations containing different protein
ratios has been
correlated with transmission electron microscopy observations.
EXAMPLES
EXAMPLE I: Mechanical and Structural Properties of Exemplary Caseinate-Whey
Coatings and Films
This example demonstrates thf: mechanical properties of cross-linked edible
films based on
calcium caseinate and two type of whey proteins (commercial and isolate). The
present study
focuses on the effect of combined physical treatments (heat and irradiation)
on the mechanical
and structural properties of milk protein-based edible films). Cross-linking
of the proteins was
carried out using thermal and radiative treatments. The effects of gamma-
irradiation and thermal
treatment of calcium caseinate and whey protein solutions was studied using
size-exclusion
chromatography. The puncture strength and the viscoelastic properties of film
formulations
containing different protein ratios was correlated with transmission electron
microscopy
observations.
Size-exclusion chromatography performed on the cross-linked proteins showed
that gamma-
irradiation increased the molecular weight of calcium caseinate while it
changed little for the
21
CA 02298109 2000-11-09
whey proteins. hlowever, heating of the whey protein solution induced cross-
linking. For both
cross-linked proteins, the molecular weight distribution was > 2 a 10~'
daltons. Combined thermal
and radiative treatments were applied to protein formulations with various
ratios of calcium
caseinate and whey proteins. Whey protein isolate could replace up to 50% of
calcium caseinate
without decreasing the puncture strength of the films. Films based on
commercial whey protein
and calcium caseinate were weaker than those containing whey protein isolate.
IJlectronmicroscope showed that the mechanical characteristics of these films
are closely related
to their microstructures.
TM
Calcium caseinate (Alanate 380, 91.8% w/w protein) was provided by New Zealand
Milk
Product Inc. (Santa Rosa, CA, USA). Whey protein isolate (WPI, 90.57% w/w
protein) was
obtained from the Food Research Center of Ag riculture and Agri-food Canada
and the
commercial whey protein concentrate (Sapro-~'S, 76.27°yo w/w protein)
was purchased from
Saputo cheeses Ltd (Montreal, Quebec, Canada). Whey protein isolate was
produced from
permeate obtained by tangential membrane rnicrofiltration. Fresh skim milk was
microfiltered
three-fold at 50 °C using an MF pilot cross-flow unit as described
previously by St-Gelais et. al.
(1995). The proteins contained in the permeate were concentrated twenty-five-
fold at 50 °C by
ultrafiltration using a OF pilot unit eduipped with a Romicon membrane (P1~I
10, total surface
area 1.3 m~). The concentrate was diafiltered five-fold by constant addition
of water and freeze-
dried before use in order to obtain WPI. Carboxymethyl cellulose sodium salt
(CN1C, low
viscosity) was obtained from Sigma Chemicals (St.Louis, MO, USA). Glycerol
(99.5%, reagent
grade) was purchased from American Chemicals ltd (Montreal, Ouebec, Canada).
Acetronitrile
(99.95%) was obtained from Anachemia Chemicals (Montreal, Quebec, Canada). AlL
products
were used as received without further purification.
All formulations were based on 5% w/w total protein, 2.5% glycerol and 0.25%o
CMC. Different
protein sources were used for the film formulations. The content in protein,
fat, lactose and ashes
are summarized in Table 1.
Table. 1. Protein, ash, fat and Lactose content of calcium caseinate (alanate
380), commercial
whey protein concentrate (CWP, Sapro-75) and whey protein isolate (WPI).
protein (%) ash (%) H20 (%) i at (%) lactose (%)
calcium caseinate 91.8 3.8 3.6 0.7 0.1.
22
CA 02298109 2000-02-24
(alante 380)
Commercial whey 76.27 3.72 0 5.79 14.22
protein concentrate
(CWP)
whey protein isolate 90.57 2.32 3.61 none 3.5
(WPI)
The components were solubilized in distilled water, under stirring, and the
solutions were heated
at 90 °C for 30 minutes. They were then degassed under vacuum to remove
dissolved air and
flushed under nitrogen accordiing to Brault et al. (1997). Solutions were
irradiated at a total dose
of 32 kGy in a 6°Co underwater calibrator unit (UC-15; 17.33 kGy/h)
(MDS Nordion, Kanata,
Ontario, Canada) at the Canadian Irradiation Center. Films were then cast by
pipetting 5 mL of
the solution onto smooth rimmed 8.5 cam internal diameter Petri dishes sitting
on a leveled
surface. Solutions were spread evenly and allowed to dry overnight at room
temperature (20 ~
2°C) in a climatic chamber (4-'i-50% R.H.). Dried films were peeled
intact from the casting
surface.
Film thickness was measured using a Mitutoyo Digimatic Indicator (Tokyo,
Japan) at six random
positions around the film. Depending on the formulation and irradiation dose,
the average film
thickness was in the range of (45-60) ~ 2 pm.
Size-exclusion chromatography was performed on the soluble protein fraction
using a Varian
Vista 5500 HPLC coupled with a Varian Auto Sampler model 9090. Proteins were
determined
using a standard UV detector set at 280 nm. Two Supelco Progel TSK PWH and
GMPW
columns followed by two Waters Hydrogel columns (2000 and 500) were used for
the molecular
weight determination of the cress-linked proteins. The total molecular weight
exclusion limit
was 25 x 10~ daltons based on linear polyethylene glycol (PEG). The eluant
(80% v/v aqueous
and 20% v/v acetonitrile) was flushed through the columns at a flow rate of
0.8 mL per minute.
The aqueous portion of the eluant was 1).02M tris buffer (pH = 8.0) and O.1M
NaCI. The
molecular weight calibration curve was established using a set of protein
molecular weight
markers MW-GF-1000 (Sigma) ranging from 2 x 106 daltons to 29 000 daltons. All
soluble
23
CA 02298109 2000-02-24
protein solutions (0.5 % w/v ) were filtered on 0.45 pm nylon membrane filters
(VWR, Nalge,
Mississauga, Ontario, Canada) prior to injection.
The average dry weight of the films was determined on seven films by drying
them in an oven at
45 °C until constant weight w;~s achieved (6 or 7 days). Seven more
films were dropped in 100
mL of boiling water for 30 minutes. The flasks were removed from the heat and
the films
remained in the water for another 24 hours. After 24 hours, the solid films
were removed and
dried in the oven as previousl~~ describ~°d. Results are calculated
using the following equation:
(1) Insoluble matter = [ Dry Weight (solid residues)/ Dry Weight (untreated
film)] x 100
Puncture tests were carried out using a Stevens LFRA Texture Analyzer Model
TA/1000 (NY,
USA), as described previously by Gontard et al. (1992). Films were
equilibrated for 48 hours in a
dessicator containing a saturated NaBr solution ensuring 56% relative
humidity. A cylindrical
probe ( 0.2 cm diameter) was moved perpendicularly to the film surface at a
constant speed
(lmm/sec) until it passed through the film. Strength and deformation values at
the puncture point
were used to determine hardness and d.°formation capacity of the film.
In order to avoid any
thickness variation, the puncture strength values were divided by the
thickness of the film. The
force-deformation curves were recorded. Viscoelastic properties were evaluated
using relaxation
curves. The same procedure was used, but the probe was stopped and maintained
at 3 mm
deformation. The parameter Y was calculated using the equation:
(2) Y(1 min) _ (F'°-,F'~lF°
where F° and FI were forces recorded initially and after 1 min of
relaxation, respectively (Peleg,
1979). A low relaxation coefficient (Y--~ 0) indicates high film elasticity
whereas a high
coefficient (Y-~ 1) indicates high film viscosity.
Dry films were first immersed in a solution of 2.5% glutaraldehyde in
cacodylate buffer, washed
and postfixed in 1.3% osmium tetroxide in collidine buffer. Samples were then
dehydrated in
acetone (25, 50, 75, 95 and 100%) before embedding in a SPURR resin.
Polymerization of the
resin proceeded at 60 °C for 2~1 hours. Sections were made with an
ultramicrotome (LKB 2128
Ultratome) using a diamond knife and transferred on Formvar-carbon coated
grids. Sections
were stained 20 minutes with uranyl acetate (5% in 50% ethanol) and 5 minutes
with lead citrate.
Grids were observed with an Hitachi 7100 transmission electron microscope
operated at an
accelerating voltage of 75 keV.
24
CA 02298109 2000-02-24
Analysis of variance and Duncan multiple-range tests with p <_ 0.05 were used
to analyze all
results statistically. For puncture strenl;th and deformation to puncture
measurements, three
replicates of seven films were tested. F'or viscoelasticity measurements,
three replicate of three
films were tested. The Studemt t-test w,as used and paired-comparison with p
_< 0.05 ( Snedecor
and Cochran, 1978).
Figure 1 shows the elution curves obtained for native, heated or irradiated
calcium caseinate.
Heating calcium caseinate at S~0°C for 30 minutes increased the
molecular weight 3 to 4-fold
(Figure 1, b). However, when the protein was submitted to gamma-irradiation at
a dose of 32
kGy, cross-linking occurred and the molecular weight distribution peak shifted
to higher
molecular weights. Based on the protein calibration curve, the molecular
weight distribution of
the cross-linked soluble calcium caseinate fraction was >_ 2 x 106 daltons, an
increase greater than
60-fold (Figure 1, c). Previous studies demonstrated that gamma-irradiation
induced the
formation of bityrosine (Davic;s, J. A., L General aspects. J. Biol. Chem.
1987, 262 (20), 9895-
9901; Brault, 1997; Mezgheni et al., J. Agric. Food Chem. 1998, 46 (1), 318-
324; Ressouany et
al., . ,l. Agric. Food Chem. 1998, 46 (4), 1618-1623). The conditions leading
to the formation of
cross-links in peptides have been widely investigated (Prutz et al., Int. J.
Radiat. Biol. 1983, 44
(2), 183-196). Although bityrosine is e:Kpected to be the major component
formed during
gamma-irradiation due to the strong characteristic fluorescence, other
mechanisms for protein
cross-linking should also be considered) (Davies et. al., . J. Biol. Chem.,
1987, 262 (20), 9902-
9907). Bityrosine is more likely to form between two protein chains
(intermolecular bonding)
than within a single protein, ac:countin~; for the increase in molecular
weight (Figure 1, c).
However, intramolecular bonding should not be totally excluded. In figure 1, a
and b, a very
small residual peak is present <~t 25 ml elution volume. This small protein
peak could be
attributed to low mass uncross-linked or intramolecularly cross-linked
proteins. In Figure 1, c,
when the irradiation was carried out at 32 kGy, this small residual peak
disappeared , an
indication that the cross-linking of caseinate by irradiation was more
efficient than by heating.
Ressouany et al. (1998) demonstrated that the maximum cross-linking density
was obtained at an
irradiation dose of 64 kGy for similar calcium caseinate solutions. A new
small residual peak at
20 ml elution volume was probably-incompletely cross-linked caseinate.
Figure 2 shows the elution curves obtained for the commercial whey proteins,
before (Figure 2,
CA 02298109 2000-02-24
a) or after heating (Figure 2, ~~), or irradiated (Figure 2, c). Gamma-
irradiation induced very little
molecular weight changes in the commercial whey. Only a broadening of the
elution peak can be
observed in Figure 2c. This feature is not surprising, considering that whey
proteins contain less
tyrosine residues than caseins (Wong et al. . Crit. Rev. Food Sci. Nutr. 1996,
36 (8), 807-844).
Our results support the report by Davies (1987), who determined bityrosine
content by
fluorescence, that in the case of a,-casein, the bityrosine concentration
quadrupled following a
low dose of irradiation (0.25 k:Gy) while it increased ten-fold in the case of
BSA. Although whey
proteins contain BSA in small amount, we expected a much more potent effect of
gamma-
irradiation at high dose of 32 hGy on the molecular weight of whey proteins.
It should be
emphasized that tests were run on irradliated WPI, yielding similar results
(not shown in Figure
2). The globular whey proteins are more prone to intramolecular cross-linking,
leading to little
change in molecular weight. ~,s expected, when the whey protein solution was
heated for 30
minutes at 90°C, it readily underwent cross-linking via the formation
of disulfide bonds. The
solution contained two distinct molecular weight fractions. The molecular
weight of the
predominant fraction was >_ 2 x 106 daltons while the smallest fraction can be
attributed to
uncross-linked protein or intramolecularly cross-linked protein. Similar
results were obtained
with heated or irradiated WPI ( not shown in Figure 2 ). These results are
consistent with those
reported by Hoffmann et al. (J: Agric. hood Chem. 1997, 45 (8), 2949-2957) on
the molecular
mass distributions of heat-induced beta-lactoglobulin; these authors were able
to separate
aggregates having a molecular mass of up to 4 x 1 O6 daltons.
Figure 3 shows the molecular mass changes in the case of a 50%-50% mixture of
whey protein
isolate and caseinate before (Figure 3, <~) or after heating (Figure 3, b), or
irradiated (Figure 3, c),
or heating at first then treated with irradiation (Figure 3, d). About 40% of
the protein was cross-
linked ( >_ 10 x 106 daltons) in the combined heating and irradiation
treatment (Figure 3, d).
The size-exclusion chromatography experiments clearly show the conditions
leading to an
increase in molecular weight in calcium caseinate and whey proteins. Mezgheni
et al. (1998)
reported that the cross-links generated by gamma-irradiation significantly
improved the
mechanical strength of calcium caseinate-based edible films. Similarly, Rayas
et al. (J. Food Sci.
1997, 62 (1), 160-162) improved the tensile strength of wheat protein films
using cystein as a
cross-linking agent. Cross-links confer elastomeric properties due to the
formation of branched
chains that increase the rigidity of a material. When the cross-linking
density is sufficiently high,
26
CA 02298109 2000-02-24
it increases the water resistance of the film (Gontard et al., 1994). Li et
al. (Proceedings of the
Institute of Food Technology meeting, July 1999 Boston, USA) demonstrated that
UV radiation
reduced the water solubility and increased the tensile strength of whey
protein-based films. Such
a feature is beneficial for the clevelopm~ent of biodegradable films and
coatings. In order to
evaluate the water solubility of the cross-linked materials, swelling
experiments were performed;
the results are shown below.
Figure 4 shows the results obt~~ined for calcium caseinate films irradiated at
different doses. The
proportion of the insoluble fraction increases with the irradiation dose up to
32 kGy, when 70%
of the film remained insoluble after 24 hours. These results are supported by
the size exclusion
chromatography results (Figure 1, 2 and 3) which suggest that a maximum cross-
linking density
was obtained at about 32 kGy. The size-exclusion chromatography results
combined with the
solubility measurements indic,~te that the irradiation of calcium caseinate
led to the formation of
an insoluble fraction of high molecular weight which accounts for 70% of the
dry matter and a
soluble protein fraction of molecular weight >_ 2 x 106. Ressouany et al.
(1998) suggested that a
maximum cross-linking density was obtained at a dose of 64 kGy. However, these
results were
obtained with caseinate films irradiated) at a mean dose rate of 1.5 kGy/hour.
In the present study,
films were irradiated at a much higher dose rate (38.1 kGy/h), which_increased
the efficiency of
the cross-linking process. Visual observation of the films that were stored in
the water for 24
hours showed that the aqueous. phase of the films irradiated at 4 kGy was
highly turbid while no
turbidity was noticed in the case of the films irradiated at a dose >_ 32 kGy.
Therefore, the
reduced weight of the films in the water might be mainly due to the uncross-
linked, soluble small
molecular mass proteins.
Enzymatic cross-linking by horseradish peroxidase has been used to cross-link
soy protein edible
films (Stuchell and Krochta, J: Food Sci. 1994, 59 (6), 1332-1337). Cross-
linking did not
improve further the water vapor permeability of these films as compared to
heat-treated films.
The films treated with the enzyme had higher soluble matter levels, which
suggests an increase
in low molecular weight material. These authors concluded that horseradish
peroxidase was not
specific enough for use in edible films, and that more specific enzymes such
as transglutaminase
should be used. However, transglutaminase is far more expensive than
horseraddish peroxidase
which greatly limits its use in l:he development of edible films. The present
research shows that
gamma-irradiation, which induces the cross-linking of tyrosine residues in a
manner similar to
27
CA 02298109 2000-02-24
peroxidase (Matheis and Whitaker, J. Food Biochem. 1987, 11, 309-327), is a
method specific
enough for the development of edible films, and particularly cost-efficient
when used on a large-
scale basis. Moreover, protein cross-linking by y-irradiation increased water-
resistance, and it
has been demonstrated that tyrosine-tyrosine cross-links improved the
mechanical resistance of
these films (Mezgheni et al., 1998; Ressouany et al., 1998). In light of these
results, a dose of 32
kGy was chosen in order to evaluate the effect of y-irradiation on the
mechanical properties of
edible films based on calcium caseinate and whey proteins.
Figure 5 shows the puncture sl:rength variations of films cast from solutions
containing different
whey protein isolate-calcium c;aseinate ratios (5% w/w total protein
solution). For instance, a
protein ratio of 50-50 corresponds to 2.5% WPI protein and 2.5% calcium
caseinate protein.
Addition of WPI in the formulations did not significantly affect the puncture
strength of the films
up to a WPI-calcium caseinate ratio of 50-50. At higher WPI concentrations,
the puncture
strength of the films was significantly reduced (p <_ 0.05) and reached a
minimal value of 0.04
N/Irm for the films based on VJPI only. Gamma-irradiation significantly
increased (p <_ 0.05) the
mechanical properties of the films by inducing cross-links between protein
chains. For instance,
for films based only on calcium caseinate (0-100), y-irradiation increased the
puncture strength
by more than 35%. This result is superior to the one reported by Ressouany et
al. (1998). These
authors used a dose rate of 2.18 kGy/h while the present experiments were
carried out at a dose
rate of 17.33 kGy/h. A higher ~~ose rate apparently increased the efficiency
of the cross-linking
mechanism. For the films containing an equal WPI-caseinate ratio (50-50),
cross-linking
increased by 20%. However, at WPI ratios higher than 50%, y-irradiation did
not affect the
puncture strength probably because the inter-molecular cross-links were only
generated between
caseinate proteins. Statistical analysis confirmed that the films cast from
solutions containing a
WPI-caseinate ratio of 0-100, 25-75 and 50-50 did not significantly differ
from one another,
whether irradiated or not. The high puncture strength of films containing 50%
WPI, comparable
to pure calcium caseinate, sugf;ests other favorable interactions than
intermolecular bonding
between whey protein isolate and calcium caseinate. The puncture strength
obtained for films
made from mixtures of calcium caseina~te and WPI might be indicative of their
phase behavior. A
greater cohesiveness between 'WPI and calcium caseinate would be expected at
WPI-caseinate
ratios of 25-75 and 50-50.
For the films containing comrr~ercial whey proteins (CWP, Sapro-75) (Figure
6), the puncture
28
CA 02298109 2000-02-24
strength of the films significantly decreased (p <_ 0.05) with increasing whey
protein
concentration. These results are not surprising considering that the CWP
contains substantial
amounts of impurities such as lactose awd fats which could act as internal
plasticizers in the
films. Results depicted in Figure 5 and 6 also shows that y-irradiation had a
more potent effect on
films richer in calcium caseinate. No statistical differences (p > 0.05) were
noted between
irradiated and control films at CWP- caseinate ratios of 75-25 and 100-0. As
established in
Figures 1 and 2, the radiative treatment: was more effective on calcium
caseinate than on whey
proteins in terms of molecular weight increase.
Figure 7 shows the viscoelasti~city coefficient of films irradiated or
unirradiated. A low
viscoelasticity coefficient means that the material is highly elastic while a
high coefficient
indicates that the material is more viscous and easily distorted. As discussed
by Mezgheni
(1997), y-irradiation decreases the visc:oelasticity coefficient of caseinate
films resulting in a
more elastic material. An addition of whey proteins (CWP) by 25% of total
total protein did not
change the viscoelasticity coefficient p 5 0.05). No statistical differences
(p > 0.05) were found
between films unirradiated or irradiated. However, the decrease from the 0-100
to the 50-50
formulations was found to be statistically significant (p <_ 0.05).
Cross-sections of the films were observed using transmission electronic
microscopy (TEM).
Figure 8 shows the micrographs that were obtained for cross-sections of films
made from
calcium caseinate. The micrographs show that the structure of these films is
highly porous.
Similar observations were made by Frinault et al. (.l. Food Sci. 1997, 62 (4),
744-747) on casein
films prepared by a modified wet spinning process. However, the microstructure
of the films that
were cast from irradiated solutions (Fig;ure 8, b) is clearly more dense than
the films cast from
unirradiated solutions (Figure 8, a). Cross-links, which are present in the
irradiated films,
increase the molecular proximity of the protein chains. This increased
molecular proximity as
well as the additional molecular bonds, proved by size exclusion
chromatography (Figures 1-3)
directly influence the macrosc~~pic characteristic of the films in terms of
mechanical strength and
water-resistance showed by physical measurements (Figures 4-7). Cross-sections
of films
containing variable amounts oaf CWP and calcium caseinate were also evaluated.
The films were
cross-linked both by heat and irradiation (32 kGy). Figure 9 a, b and c shows
the micrographs of
films containing CWP-calcium caseinate ratios of 50-50, 75-25 and 100-0. The
pore size is
highly variable depending on the proportion of commercial whey protein. For
instance, the films
29
CA 02298109 2000-02-24
made of CWP only (100-0) have a granular structure and contain numerous dense
masses that
may be attributed to impurities such as fat, lactose and mineral salts.
Addition of calcium
caseinate to the formulations rendered their microstructure smoother and
slicker. However,
major differences are seen between the micrographs of films 50-50 (Figure 9,
a) and 75-25
(Figure 9, b) in terms of pore size. The pores are obviously much larger in
the case of the films
cast from a solution containing; a protein ratio of 75-25. The variations in
pore size distribution of
these films might be correlated in part, with the variations in puncture
strength. As previously
hypothesized, the internal structure might be indicative of the protein phase
behavior. A great
difference between the microstructure of films 75-25 and 100-0 (Figure 9, c)
can also be
observed. The topography of the films varies from a porous structure to a more
granular one
(Figure 9, c). Similar correlati~~n between microstructure and mechanical
strength were seen in
films based on WPI (Figure 10). However, the structure of films containing WPI
were generally
more dense and homogeneous. The cross section of WPI-caseinate films (50-50)
heated at 90°C
for 30 minutes shows larger average pore sizes (Figure 10, a) than the same
films both heated
and irradiated at 32 kGy (Figure 10, b). After combined heat and irradiation
treatment, the
microstructure of the films was more dense, which may be caused by the higher
average
molecular mass, shown in Figure 3, c.
This example shows that y-irradiation was efficient for inducing cross-links
in calcium caseinate
edible films. Unlike enzyme treatments, y-irradiation would be particularly
cost-efficient when
used on a large-scale basis. The solubility measurements demonstrate that the
treatment is
selective enough to produce films containing a high ratio of insoluble matter.
Combination of
radiative and thermal treatments of the films based on calcium caseinate and
whey proteins
resulted in an increase in the puncture strength of the films. The mechanical
properties of the
films were influenced by the type of whey protein used. WPI could be added in
equal amount to
calcium caseinate without decreasing the puncture strength of the films. In
contrast, the addition
of CWP rapidly decreased the puncture. strength of these films, probably due
to the presence of
impurities, contained in the commercial product, which may disrupt protein-
protein interactions.
The observation of the microstructure of films by transmission electron
microscopy revealed that
all films were characterized by a highly porous structure. However, pore size
distribution varied
depending on the protein ratio and correlated in part with the mechanical
behavior of these films.
CA 02298109 2000-02-24
EXAMPLE II: Demonstration of Efi:ectiveness of Casinate:Whey Coatings to
Reduce
Water Loss and Mold Growth in Fruit
This example compares the effectiveness of edible whey:caseinate coverings
(based on 5 % w/w
protein and 2.5% w/w glycerol) to y-irradiation treatment to reduce water loss
and mold growth
in fruit The results demonstrate that both treatments are effective in
reducing water loss and
mold growth, and that whey:c,~seinate coatings are more effective than those
based on calcium
caseinate alone. Furthermore, y-irradiation was used in combination with
edible coatings for
possible synergistic effects beoween the: two treatments. Moreover, this
example also
demonstrates that the addition of calcium chloride or polysaccharides to the
protein formulations
increases their effectiveness b:y further delaying mold growth.
Strawberries were choosen as an exemplary fruit because strawberry decay
resulting from mold
growth is a common problem during fruit storage. Rot caused by Rhizopus sp.
and Aspergillus
sp. are mainly accountable for fruit loss. Because strawberries are especially
sensitive to mold
growth, its shelf life is of 2 days when stored at 15 °C. In order to
control fruit decay and losses,
many studies have been done 'in order to develop new preservation methods.
Among those tested,
gamma-irradiation has proven effective: in reducing bacterial and mold
contamination as well as
delaying the ripening of climacteric fruits (Kader, A.A., Food Technology, 6,
117-121).
Gamma-irradiation treatments have proven effective in reducing microorganisms
in fresh
strawberries (O'Connor and Mitchell, International ,lournal of Food
Microbiology, 12, 247-255.
1991) and have been used in combination with hot-water dip treatments for
inactivating yeasts
resulting in an increased stability of strawberry yoghurt (Kiss, Acta
Alimentaria, 4, 95-112.
1975). Baccaunaud and Chapon (INFG~S-Centre technigue interprofessionnel des
fruits et
legumes, 9, 43-54, 1985) have shown that modified atmosphere packaging (MAP)
followed by y-
irradiation at 2 kGy extended 'the shelf life of strawberries to over a month
when stored at 4 °C,
as compared to 14 days for heat-treated fruits (40 °C for 10 minutes)
combined with irradiation
(2 kGy).
In this example, twelve small cases of 'Kent' strawberries (300 grams each)
were used for the
31
CA 02298109 2000-02-24
analysis. Six cases were randomly chosen and irradiated at 1.5 kGy while the
other 6 remained
unirradiated. Furthermore, for each type of strawberries (irradiated and
unirradiated), 2 cases
were coated with an unirradiated calcium caseinate solution and 2 more were
coated with a
solution that was previoulsy irradiated at 32 kGy. Gamma-irradiation was
carried out in a 6°Co
irradiator (Gammacell-200, MDS Nordion, Kanata, Ontario, Canada) at the
Canadian Irradiation
Center (Laval, Quebec, Canada) at a mean dose of 1.5 kGy per hour. The fruit
cases were
irradiated for a total dose of 1.5 kGy while the protein solutions were
irradiated for a total dose
of 32 kGy.
Coating for~rnulations were based on 5 % w/w protein and 2.5% w/w glycerol.
Calcium caseinate
(New Zealand Milk Products, Santa Rosa, CA, USA) was used alone or in
combination with
whey proteins (Saputo Cheese Ltd, St-Hyacinths, Quebec, Canada). CaCIZ (0.125%
w/w) (BDH
Chemicals Ltd., Montreal, Quebec, Canada) or a mixture of polysaccharides
(0.1% agar and
0.1% pure pectin) were also added to tine coating formulations. The agar was
purchased from
Sigma Chemicals (St. Louis, MO, USA) and commercial liquid pectin (Certo
brand) was
obtained from Kraft Canada inc. (Cobourg, Ontario, Canada). Each formulation
was tested on
three cases (300 grams each) of fresh strawberries (chosen at random). After
irradiation or
coating treatment, the strawberries were stored in a large refrigerator at 4 ~
1 °C. Weight loss
and mold growth (%) was noted until 7.00% contamination was obtained.
Weight loss determination was calculated on each strawberry case. Each case
was weighed and
the ratio of the final weight on the initial weight was determined. The number
of fruits rejected
due to mold growth was determined each day of analysis.
After 17 days of storage, the control strawberries had lost about 32% (data
not shown) of their
original weight. Such a feature is of great importance for the minimally
processed or "ready-to-
eat" food market since it is of~:en limited by a series of problems related to
cell disruptions such
as leakage of nutrients, enzymatic reactions, mold growth, loss of texture and
appearance defects
(Carlin et al., Journal of Food' Science., 55, 1033-1038, 1990). In order to
minimize such defects,
Avena-Bustillos et al. (Postharvest Biology and Technology, 4, 319-329, 1994)
have
demonstrated that a casein-lipid edible coating on processed carrots can
inhibit the development
of white blush, a major cosmetic disadvantage resulting from surface
dehydration. Our results
show that fruit coating can prevent fruit dehydration during storage (Data not
shown). As
32
CA 02298109 2000-02-24
discussed by Kester and Fennc:ma (Food Technology, 12, 47-59, 1986), films
based on caseins
and fatty acids can control fruit dehydration since the fatty acids in the
formulation modify the
barrier properties of the coating.
It should be noted that previous works showed that strawberries may tolerate a
maximum
irradiation dose of 2 kGy for reducing fungal infection without quality
changes (Maxie and
Abdel-Kader, Advances in Fo~~d Research, 15, 105-138, 1966). Doses in excess
of 2 kGy often
result in softer texture due to changes in cell wall components such as
cellulose, hemicellulose
and pectic enzymes (D'Amour et al., Journal of Food Science, 58, 182-185,
1993). Likewise,
electron beam irradiation was found to have a similar effect (Yu et al.,
Journal of Food Science,
61, 844-846, 1996).
Figure 11 shows the results obtained for irradiated coating formulations based
on a mixture of
calcium caseinate and whey proteins. It can be seen that 90% of fruit
contamination was obtained
on day 20 for the mixed-proteins coated fruits while a similar number was
reached on day 17 for
the pure calcium caseinate formulation (control + film 32 kGy) (Figure 2). The
addition of whey
proteins in the formulation dellayed mold apparition by another 3 days.
Similarly, CaCl2 or
polysaccharides (agar and pectin) were; added to the mixed-proteins
formulation. It can be seen
that the addition of salt or pol~,~ssacharides improved the coating
formulations' efficiency by
further reducing mold growth on strawberries. The apparition of molds on these
samples was
observed on day 13 as compared with day 8 for samples coated with the basic
formulation. For
the uncoated fruits, a 45% fruit contamination was obtained on day 8 while a
similar number was
reached on day 15 for the strawberries coated with the basic mixed-proteins
formulation. When
CaCl2 or agar and pectin were added, mold growth was delayed another ten days,
as a 45%
contamination level was reached only on day 25 for both types of coatings. It
should be
emphasized that for these coatings, an important increase in mold growth was
not noticed until
the thirtheenth day of experimentation while a rapid increase was noted after
three days only for
the uncoated fruits and after 8 days for the fruits coated with the basic
formulation. Similarly,
total contamination (100%) was reached on day 20 for the control fruits, on
day 25 for the fruits
coated with the irradiated mixed-proteins formulation and on day 35 for the
fruits coated with
irradiated formulations with added salC or polysaccharides.
The results presented in this Example demonstrate that the both caseinate:whey
coating or y-
33
CA 02298109 2000-02-24
irradiation were effective for reducing fungal infections and extending the
shelf life of fresh
strawberries. However, no synergistic effect was observed when irradiation was
combined with
coating treatments. The use of an edible coating based on mixed proteins (whey
and caseinate)
was more effective than the formulation based only with calcium caseinate.
Moreover, the
addition of salt or polysaccharides to the formulations further increased
their effectiveness.
EXAMPLE III: The Coatings Prevent Enzymatic Browning of Fruit and Vegetables
These experiments were perfromed to demonstrate the ability of the coating
formulation to act as
an efficient oxygen barrier anti thereby delay enzymatic browning of fruit and
vegetables. Color
measurements were performed on apple and potato slices coated with calcium
casinate:whey
protein solutions in a 50:50 ratio. Results showed that the coating
efficiently delayed enzymatic
browning by acting as efficient oxygen barriers. Although slight color
variations were noted for
the entire experimental period, they were not emphasized by a darkening of the
slices.
Calcium caseinate (alanate 380) was provided by New Zealand Milk Products
(Santa Rosa, CA,
USA). Concentrated whey protein powder was obtained from Les Fromages Saputo
Ltee. (St-
Hyacinthe, Quebec, Canada). tslycerol (99,5%, reagent grade) was purchased
from American
Chemicals ltd (Montreal, Quebec, Canada), carboxymethyl cellulose sodium salt
(CMC, low
viscosity), and calcium chloridle (CaCl2,, laboratory reagent) was obtained
from BDH Chemicals
(Montreal, Quebec, Canada). Ell products were used as received without further
purification.
A dipping solution formation was prepared to generate 5 % (w/w) protein
(calcium caseinate or
whey protein powder in a 50:50 ratio), 2,5% (w/w) glycerol, 0,25% (w/w) CMC
and 0,125%
(w/w) CaCl2 were diluted in water and mixed to obtain homogeneous solutions.
McIntosh apples (Quebec, Canada) andl washed potatoes Canada #1 (product from
Prince
Edward Island, prepared by Ernballages D.L. Inc, Laval, Qc, Canada) were
purchased from a
local grocery. Five slices (about 1/a inch thick) were cut from three potatoes
and apples, dipped
one minute in the protein solutions and laid in petri dishes. Control potatoes
and apples were cut
and laid without dipping in the. dishes an exposed to atmospheric air. The
experiment was
repeated three times.
34
CA 02298109 2000-02-24
Color measurements were taken every five minutes for a total experimental
period of 130
minutes. The color was read using a Colormet spectrocolorimeter (Instrumar
Engineering Ltd,
St. John's, NF, Canada) using the standard (1976) CIELAB color system.
Lightness is reported
as L* and the HUE angle value is given by tan-~ (b*/a*). As the HUE angle
decreases, red
pigmentation increases. The a'~ axis (red) corresponds the a HUE angle of
0°. Color
measurements were taken once on each slice (potato or apple) for a total of 15
readings per data.
Figure 12 shows the variation of the lightness parameter (L*) in function of
time for coated
potato slices. For the uncoated control slices, an increase in lightness is
noted for the first fifteen
minutes. This feature is probably due to the exudation of natural juices that
contribute to increase
the surface's luminosity. Then, as enzymatic browning occurs, the brightness
of the uncoated
potato slices starts to progressiively decrease with time for the remaining
experimental period.
The lightness value for perfect white is 100 while L* = 0 corresponds to
black. The loss of
whiteness associated with enz:~matic browning can be estimated for the entire
experimental
period. Contrary to the uncoated potato slices, the coated potato slices did
not show any
evidence of darkening. A slight increase in lightness was even noticed for
both types of coated
potato slices.
Figure 13 shows the HUE angle variation for uncoated and coated potato slices.
As the HUE
angle decreases, red pigmentation becomes more pronounced. It can be seen that
the control
(uncoated) slices undergo rapid enzymatic browning as seen by the sharp
decrease of the HUE.
The sharpest decrease was noted within the first 45 minutes. Following that
decrease, the HUE
stabilized for the remainder of the experimental period. For the coated
slices, only a slight
variation of the HUE was noted for the entire experimental time period
although those small
color changes are not coupled with a darkening of the potato slices (Figure
7).
Figures 14 and 15 show the lightness (L*) and HUE angle results obtained for
apple slices.
Similarly to what was observed for potato slices, L* rapidly decreased with
time for the uncoated
apple slices.. For the coated af~ple slices, the lightness parameter remained
rather constant
showing that the protein coatings effectively protected the fruit from oxygen.
As for the HUE
(Fgure 15), results show that f~~r all types of apple slices, the angle
decreased slightly with time.
That effect seems to be somewhat less noticeable in the case of the whey
coating. As the HUE
decreases, red pigmentation develops. However, previous results (Figure 14)
show that those
CA 02298109 2000-02-24
small color fluctuations are not associated with darkening (lower L*)
Nisperos-Carriedo et al. (Fooa! Techno~!ogy, 47, 75-84, 1991) previously
reported color
measurements done on sliced mushrooms coated with a formulation containing
vegetable oils,
cellulose gums, emulsifiers, surfactant~c and fatty acids. Their work showed
that the coating
reduced enzymatic browning. After two hours, the coated mushrooms were lighter
than the
uncoated ones. Still, the coating did not completely inhibit darkening as the
coated mushrooms
were slightly darker after two hours than the fresh cut controls. Our results
showed that our
formulations based with milk proteins were more effective in controlling
enzymatic browning
since no darkening (lower L*) was noted after two hours for sliced potatoes
and apples.
Protein coatings delay browning probably by effectively reducing oxygen. It
should be
emphasized, however, that previous works have demonstrated that these coatings
are not
completely impervious to oxygen (McH ugh and Krochta, In Edible coatings and
films to
improve food quality, eds. J.M. Krochta, E.A. Baldwin and M. Nisperos-
Carriedo, pp. 139-188,
Technomic Publishing Company, Lancaster, Pennsylvania 1994). This feature
would
consequently lower the risks of creating; undesirable anaerobic conditions.
Other mechanisms
could also inhibit enzymatic browning. For instance, cysteine, a sulfhydryl-
containing amino
acid was used as a polyphenol oxidase inhibitor by acting as a coupling agent
with quinones
forming stable, colorless compounds (Dudley and Hotchkiss, Journal of Food
Biochemistry, 13,
65-75, 1989).
EXAMPLE IV: Mechanical and Structural Properties of the Extreme Ranges of
Caseinate-Whey Coatings anal Films
This Example presents demonstration of the mechanical properties of the
extreme ends of the
range of caseinate:whey (1:99 and 99:1). The viscoelestaticity (Figure 16) and
the puncture
strength data (Figure 17) are presented for these coverings, produced using
two different types of
whey protein: whey protein isolate (WPI) and commercial whey protein (WPC).
The crosslinks
are formed either by heating at 90 °C or irradiation at 32 Kgy.
Calcium caseinate (Alanate 380, 91.8%~ w/w protein) was provided by New
Zealand Milk
Product Inc. (Santa Rosa, CA, USA). Commercial whey protein concentrate (Sapro-
75, 76.27%
36
CA 02298109 2000-02-24
w/w protein) was purchased from Saputo Cheeses Ltd (Montreal, Quebec, Canada).
Whey
protein isolate (WPI, 92.52% w/w protein) was obtained from the Food Research
Center of
Agriculture Canada, wherein it was produced from permeate obtained by
tangential membrane
microfiltration. Fresh skim-milk was rr~icrofiltered three-fold at 50
°C using a MF pilot cross-
flow unit as described previously by St-Gelais et al., (Milchwissenschaft
1995, 50 (11), 614-619).
The proteins contained in the permeate were concentrated twenty-five-fold at
50 °C by
ultrafiltration using an OF pilot unit equipped with a Romicon membrane (PM
10, total surface
area 1.3 m2). The concentrate was diafiltered five-fold by constant addition
of water and freeze-
dried before use in order to obtain WPI.
The components of the films are solubilized in distilled water, under
stirring, and the solutions
are heated at 90°C for 30 minutes. They are then degassed under vacuum
to remove dissolved air
and flushed under gas according to Brault et al. (J. Agric. Food Chem., 45
(8), 2964-2969,1997).
Irradiation of the solutions at a~ total dose of 32 kGy is performed in a
6°Co underwater calibrator
unit (UC-15b; 17.33 kGy/hour) (MDS Nordion, Kanata, Ontario, Canada) at the
Canadian
Irradiation Center. Films are c,~st by pipetting 5 mL of the solution onto
smooth rimmed 8.5 cm
internal diameter Petri dishes sitting on a leveled surface. Solutions are
spread evenly and
allowed to dry overnight at room temperature (20 ~ 2°C) in a climatic
chamber (45-50% RH).
Dried films are peeled intact from the casting surface.
The viscoelasticity and puncture strength tests are performed as described
above. The results are
presented in Figures 16 - 21.
EXAMPLE V: Antioxidant Properties of Milk Protein Films
The tests for antioxidative properties were performed only with unirradiated
calcium caseinate
films in two separate experiments. In one set, dried and ground leaves from
rosemary, sage and
thyme were blended together in the ratio l:l:l and extracted in distilled
water or distilled
water/ethanol mixtures (20/80 or 80/20) following the procedures described by
Lessard et al.,
Briefly, 10 g of spice mixture were added to 30 ml boiled distilled water and
stirred at room
temperature for 1 h. Residual leaves were extracted two more times to give a
total water extract
of 100 ml. The water/ethanol extractions (20/80 or 80/20) were done in the
same manner, but at
37
CA 02298109 2000-02-24
room temperature. Calcium caseinate, carboxymethyl cellulose, and glycerol
were dissolved in
the water or water/ethanol extracts to obtain films forming solutions. In the
second set,
commercial essential oils frorr~ thyme amd rosemary were purchased from Le
Naturiste (Laval,
Quebec, Canada) and incorporated to calcium caseinate based film forming
solutions to final
concentrations of 1 % (v/v) in presence of various concentrations (1-10 %,
v/v) of lecithin
(Sigma Chemicals, St-Louis, MO, USA). All the film forming solutions were cast
as previously
described in the permeability tests experiments.
Antioxidative properties measurements
The antioxidant capacities of the films were determined by the DPD (N, N-
diethyl-p-
phenylenediamine) eolorimetric method described by Dumoulin et al., 1996.
Small rectangular
pieces of films (1 x 3 cm) were: introduced in electrolysis cells containing 3
ml of Krebs-
Henseleit (KH) buffer and electrolyzed at 10 mA DC for lmn using a model
1000/500 power
supply (Biorad, Richmond, C~,, USA) to generated oxygen free radicals (OFR).
Aliquots of 200
(1 of the electrolyzed solution ~,vere taken and mixed with 2 ml of a 2.5%
(w/v) solution of N, N-
diethyl-1,4-phenylenediamine (Aldrich chemical company, Inc., Milwaukee, WI,
USA) and
optical densities were measured at 515 nm (Varian DMS 200 spectrophotometer,
Georgetown,
Ontario, Canada). Electrolyzed and unelectrolyzed KH buffer without films
samples were treated
in the same manner and served as positive and negative controls, respectively.
The OFR react
instantly with DPD to produce a red colour, and the antioxidant capacities of
the films were
evaluated through their ability to reduce the intensity of the red colour. The
results were
expressed as OFR scavenging capacity in percentage and calculated as follows:
Scavenging capacity (%) = 100-[ (ODsample / ODcontrol).100], where ODsample
and
ODcontrol are optical densities of electrolyzed samples and positive control,
respectively.
The results are presented in Fi;;ures 22 - 24.
EXAMPLE VI: Cross-linking effect on water vapor permeability of whey protein
isolate,
whey protein concentrate, and calcium caseinate films.
Film preparation
Calcium caseinate (Alanate 380; 91.8%> protein on weight basis) was obtained
from New
Zealand Milk Product Inc. (Santa Rosa, CA, USA). Whey protein concentrate
(Sapro-75, 76.27%
38
CA 02298109 2000-02-24
protein on whey basis) and whey proteiin isolate (90.57% protein on weight
basis) were obtained
from Saputo Cheeses Ltd (Montreal, Quebec, Canada) and the Food Research and
Development
Centre (St-Hyacinthe, Quebec, Canada), respectively.
Calcium caseinate was solubilized in distilled water in presence of low
viscosity carboxymethyl
cellulose (2.5% w/v; Sigma Chemicals., St-Louis, MO, USA) and reagent grade
glycerol (2.5%
w/v; American Chemicals Ltd, Montreal, Quebec). Whey protein concentrate and
whey protein
isolate added to the solution to obtain various casein/whey protein ratios
(100/0, 75/25, 50/50,
25/75, and 0/100) with a total ;protein concentration of 5% (w/v) in the film
forming solutions.
To obtain unirradiated films, the film forming solutions were cast directly
onto smooth petri
plates (8.5 cm, LD.) and allowed to dry overnight at 20~1°C in a
climatic chamber (45-50%, R
Humidity). The irradiated films were olbtained in the same manner, but the
film forming
solutions were first irradiated at a total dose of 32 kGy in a 60Co underwater
calibrator unit (UC-
15b) (MSD, Nordion, Laval, ~~uebec) with a mean rate dose of 17.33 kGy/h
before casting. Both
the unirradiated and the irradiated film were used in the permeability
measurements.
Film thickness
The film thickness was determined using a Digimatic indicator micrometer
(Mitutoyo, Tokyo,
Japan). Measurement were taken at five locations and the means values were
used for
permeability calculations. The thichness off the films averaged 66-95 ~ 3.6 (m
depending on the
formulation.
Permeability measurements
Water Vapor Permeability (W'VP) of th.e films was determined gravimetrically
at 23°C using a
modified ASTM (1983) procedure. The test films were sealed to glass cups
contained
phosphorus pentoxide cristals (Sigma C'.hemicals, St-Louis, MO, USA) with
exposed film area of
13.40 cm2. The cups were placed in dessicators with were maintenant at
23°C under 100% RH
(21.59 mmHg water vapor pre;~sure) with distilled water of 56% RH (9.82 mmHg
water vapor
pressure) with saturated sodium bromide solution (Sigma Chemicals, St-Louis,
MO). The water
vapor transferred through the film and absorbed by the desiccant was
determined by the weight
gain of the phosporus pentoxicle. The caps were weighted initially and at 6
and 30 h, and the
permeability of the films was calculated as follows (Gontard et al. 1996).
WVP= (w.x)/A.T.(pl-p2).
39
CA 02298109 2000-02-24
_ (g).(mm)/(mz).(24h).(mmHg)
where w is the weight gain of the cups over 24 h (T), x is the film thickness
(mm), A is the area
of exposed film (mz), p2-pl is the water vapor pressure differential across
the film (32.23 and
9.82 for 100% and 56% RH, rtapective,ly).
The results are presented in Figures 25 and 26.
The invention being thus described, it will be obvious that the same may be
varied in many
ways. Such variations are not to be regarded as a departure from the spirit
and scope of the
invention, and all such modifications as would be obvious to one skilled in
the art are intended to
be included within the scope of the following claims.
