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Title:
CASEINATE-WHEY CROSSLINKED COVERING AGENT
Document Type and Number:
WIPO Patent Application WO/2000/049899
Kind Code:
A1
Abstract:
This invention provides water-insoluble caseinate-whey covering agents. Methods are provided for forming a range of coatings and films, wherein the proportion of caseinate-to-whey is chosen to optimize the desired characteristics of the covering agent, and optionally, the addition of other compounds. The covering agents of the invention, which can be edible, will find utility in the food industry, as a means of improving the shelf life of various fruits and vegetables, eliminating bacterial contamination in meats, and in reducing the amount of packaging materials required.

Inventors:
Lacroix, Monique (340 rue Green Saint-Lambert, Québec J4P 1T4, Québec J4P 1T4, CA)
Mateescu, Mircea-alexandru (377 Sherbrooke West Apt. 505 Montréal, Québec H3A 1B5, CA)
Delmas-patterson, Geneviève (551 Argyle Westmount, Québec H3Y 3B8, CA)
Application Number:
PCT/CA2000/000161
Publication Date:
August 31, 2000
Filing Date:
February 22, 2000
Export Citation:
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Assignee:
INSTITUT NATIONAL DE LA RECHERCHE SCIENTIFIQUE (2600, boulevard Laurier bureau 640 Case postale 7500 Sainte-Foy, Québec G1V 4C7, CA)
Université, Québec Montréal DU. À. (1430, rue St-Denis Case postale 8888 Centre-Ville Montréal, Québec H3C 3P8, CA)
Lacroix, Monique (340 rue Green Saint-Lambert, Québec J4P 1T4, Québec J4P 1T4, CA)
Mateescu, Mircea-alexandru (377 Sherbrooke West Apt. 505 Montréal, Québec H3A 1B5, CA)
Delmas-patterson, Geneviève (551 Argyle Westmount, Québec H3Y 3B8, CA)
International Classes:
A23B4/10; A23B4/20; A23B5/06; A23B7/154; A23B7/16; A23B9/14; A23J3/08; A23L1/00; A23L3/3418; A23P1/08; C08H1/00; C08L89/00; C09D189/00; (IPC1-7): A23P1/08; C08L89/00; A23J3/08; A23B7/16; B65D65/46
Domestic Patent References:
WO1998049237A11998-11-05
Foreign References:
US4239784A1980-12-16
EP0477832A11992-04-01
US5368871A1994-11-29
EP0782825A11997-07-09
Other References:
L.C.WU, R.P.BATES: "Influence of Ingredients upon Edible Protein-Lipid Film Characteristics", JOURNAL OF FOOD SCIENCE., vol. 38, 1973, CHICAGO US, pages 783 - 787, XP000921030
MCHUGH T H ET AL: "MILK-PROTEIN-BASED EDIBLE FILMS AND COATINGS", FOOD TECHNOLOGY,US,INSTITUTE OF FOOD TECHNOLOGISTS. CHICAGO, vol. 48, no. 1, 1 January 1994 (1994-01-01), pages 97 - 103, XP000423512, ISSN: 0015-6639
Attorney, Agent or Firm:
MBM & CO. (P.O. Box 809 Station B Ottawa, Ontario K1P 5P9, US)
Download PDF:
Claims:
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. 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 crosslinked to form a covering agent.
2. The composition of claim 1, wherein said covering agent is a film.
3. The composition of claim 2, wherein said film is edible.
4. The composition of claim 1, wherein said covering agent is a solution for dipping.
5. The composition of claim 1, wherein said covering agent is a solution for spraying.
6. The composition of claim 1 wherein said caseinate salt is calcium caseinate.
7. The composition of claim 1 wherein said whey protein is commercial whey protein.
8. The composition of claim 1 wherein said whey protein is whey protein isolate.
9. The composition of claim 1 wherein said whey protein is concentrated whey protein powder.
10. The composition of claim 1, wherein said plastisizing agent is selected from the group consisting of glycerol, propylene glycol, triethylene glycol and sorbitol.
11. The composition of claim 1, wherein said polysaccharide is selected from the group consisting of carboxymethyl cellulose, chitosam, agar and pectin.
12. The composition of claim 1, additionally comprising CaC12.
13. A process for the preparation of a covering agent, comprising the steps of : (i) preparing an aqueous solution comprising a caseinate salt, whey protein and, optionally, a plastisizing agent 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 thermal step and/or an irradiation step, wherein said heating and irradiation step causes crosslinking of caseinate and whey to produce said covering agent.
14. The process according to claim 13, wherein the thermal step is raising the temperature of the formulation to a temperature in the range 50 to 95 °C.
15. The process according to claim 13, wherein said irradiation step is a treatment with y rays of at a dose in the range of approximately 20 to 40 kGy.
16. The process according to claim 13, wherein the thermal step is raising the temperature of the formulation to a temperature of approximately 60 °C, and said yirradiative step is a treatment with yrays at a dose of approximately 30 kGy.
17. The process according to claim 13, wherein said degassed solution is produced by stirring said aqueous solution under vacuum.
18. The process according to claim 13, wherein said aqueous solution additionally comprises CaC12.
19. The use of the covering agent produced by the process of claim 13 to provide a physical barrier.
20. The use according to claim 19, wherein said physical barrier is provided to the external surface of a food product. The use according to claim 19, wherein said physical barrier is provided to the external surface or internal surface of a food packaging product. The use of the covering agent produced by the process of claim 18 in combination with an active compound to provide slow release of said active compound. The use of the covering agent according to claim 22, wherein said active compound is selected from the group comprising, an antimicrobial agent, an antifungal agent, a volatile flavoring, and a pharmaceutical.
Description:
CASEINATE-WHEY CROSSLINKED COVERING AGENT 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.

BACKGROUND OF THE INVENTION The environmental movement has promoted increased concern about reducing amounts of disposable packaging and increasing recyclability 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 produced 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 and coatings based on water-soluble proteins are typically 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) require 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 food 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, etal., J. AgandFoodChem., 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 vulnerable 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 ornamentals 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 oxygen into the product.

Fruit and vegetable decay resulting from mold growth is another concern in the industry. Rot caused by Rhizopus sp. and Aspergillus 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.

However, health concerns have limited their application (Sapers, 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 (Sapers, Food Technology, 47,75-84,1993). Another approach is the use of antibrowning agents based with citric or ascorbic acid. Although 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 (Kacem 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 Sapers 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 coatings 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. Kester and 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); coatings 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. 3,451,826 to Mulder), algin and a gelling mixture (U. S. Pat. No. 4,504,502 to Earle and McKee), protein (U. S. Pat. No. 4,344,971 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 Ukai et al.), and emulsions or suspensions of a water-soluble protein material and hydrophobic material, adjusting the pH of the protein material to its isoelectric point (U. S. Pat.

No. 5,019,403 to Krochta).

Dried foods, low moisture baked products, intermediate moisture foods and high moisture foods all exhibit potential for improvement through the use 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 the atmosphere. Low moisture baked foods are also susceptible to moisture uptake from moist fillings and toppings. Such changes can result in loss of sensory acceptability of the food product, as well as a reduced shelf-life. Many 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 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 soggy crust. 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 zein, wheat 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 when 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 nature 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- 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 formulated 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 film 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 zein, 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. Biodegradation 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 two 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/ml. p-casein is more susceptible to degradation than a for the majority of species. This phenomenon is only observed when the bacterial population is higher than 106-107 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 et al., 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 covering 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) preparing an aqueous solution comprising a caseinate salt, whey protein and, optionally, a plastisizing agent 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 Figure 1 shows elution curves for calcium 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 at 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°C 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 1.25g WPI protein and 3.75g calcium caseinate protein per 100g protein solution.

Fiugre 6 shows the puncture strength 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 ure).

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 Am).

Figure 10 presents cross sections of WPI-calcium caceinate films: a) heated at 90°C for 30 min, b) heated at 90°C for 30 min and irradiated at 32 kGy (9 mm bar = 3, um).

Figure 11 shows mold contamination (%) 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. (contrôle = control; caseinate = caseinate; lactosérum = 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 (contrôle = control; caseinate = caseinate; lactosérum = 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. (contrôle= control; caseinate = caseinate; lactosérum= 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 (contrôle = control; caseinate = caseinate; lactosérum = 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 viscoélasticité) 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 viscoélasticité) on various caseinate-whey films, ranging from 1: 99 to 99: 1 caseinate: whey and crosslinked using heat 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 (80/20); and C) 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 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 cellulose; 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 proteins, 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 enzymic 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, palmitic acid and hexadecanol.

As used herein, the term"food grade plasticizer"refers to compounds which increase the 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 undergoing 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. On 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 and 2, ß~ and K-caseins. All in all, a casein molecule consists of a primarily hydrophobic core of a and of ß- casein and surrounded by K-casein on the surface (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 the 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 Technology, 48 (1), 97-103,1994).

Caseinates are obtained either by the acidification with mineral acid (HCI 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 ammonium (Schmidt and Morris, 1984; Vuillemard et al., 1989; McHugh and Krochta, 1994). The solubilized caseinates are dehydrated thereafter. Salts of caseins thus obtained are soluble above pH 5.5.

Whey (mainly a-lactalbumine and P-lactoglobuline) or small-milk proteins, form a thermoirreversible gel, which is pH-dependent and heat sensitive (Schmidt and Morris, 1984; Vuillemard and Al, 1989). As 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 employed 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, whose 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 irridation. A 99: 1 caseinate-whey 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 is 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 material as well as improve the water resistance of such protein films (Brault et al., J. Agric. Food Chem., 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.

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 go.

Molecular weight determination 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 (PEG). 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 0.1M NaCl. 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. 45 pLm 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 calculated 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 then converted into units of force (N). Puncture tests can be carried out using a Stevens LFRA Texture Analyzer Model TA/1000 (NY, USA), as described previously by Gontard et al. (J. Food Sci., 57 (1), 190-195,1992). In this example, films are equilibrated for 48 hours in a dessicator containing a saturated NaBr solution ensuring 56% relative humidity. A cylindrical probe (0. 2 cm diameter) is moved perpendicularly at the film surface at a constant speed (1 mm/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-deformation curves are recorded.

The viscoelasticity of a film can 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 curves. 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) = (F9-F')/F0 where F° and F'were forces record initially and after 1 min of relaxation, <BR> <BR> <BR> respectively [Peleg, M., J. Food Sci. 1979,44 (1), 277]. A low relaxation coefficient (Y-> 0)<BR> <BR> <BR> <BR> <BR> indicates high film elasticity whereas 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 SetaramTM C80. 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 Teflona joints, to prevent water evaporation. The ampule with the cell is placed into the calorimetre and when the thermic equilibrium is obtained, the ampule 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 SPURRTM resin. Polymerization of the resin proceeds at 60 °C for 24 hours. Sections are made with an ultramicrotome (LKB 2128 UltrotomeTM) 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 HitachiTM 7100 transmission electron microscope operated at an accelerating voltage of 75 keV.

Water vapor permeability can be determined in a manner similar to U. S. Patent 5543164.

Briefly, test cups were made out 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 cm2, 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 (RH) using calcium sulfate Drierite desiccant (Fisher Scientific, Inc., Fair Lawn, N. J., 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 had 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. The 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 determined 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.

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, etc. 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 requirements 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 L 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) 00- [ (OD, aple/OD, 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 ColormetTM sperctrocolorimeter (Instrumar Engineering Ltd., St.

John's, NF., Canada) using the standard (1976) CIELABTM 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 to a HUE angle of 0°.

Color measurements can be taken once on each slice of fruit or vegetable, for example, for between 8 and 12 readings per data point.

Additives, including chelating agents, such as ascorbic acid and calcium disodium EDTA, antibacterial agents, flavorings, vitamins and mineras, 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 fatty acids which are useful in the 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 alcohol 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 about 2 to 15% by weight in solution.

When composite mixtures are formed 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 denaturation, the protein will typically be solubilized in an aqueous solution in a concentration range of from? to? % by weight, preferably about 5% by weight.

Methods of Crosslinking the Proteins The crosslinking of a proteinic solution can be achieved by a variety of 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.

Upon radiolysis of an aqueous 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 produce 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 bityrosine is one mechanism for causing protein aggregation, although other crosslinks can be 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 and 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 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 examples 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 the 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 whey proteins. However, heating of the whey protein solution induced cross-linking. For both cross-linked proteins, the molecular weight distribution was > 2 x106 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.

Electronmicroscope showed that the mechanical characteristics of these films are closely related to their microstructures.

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 Agriculture and Agri-food Canada and the commercial whey protein concentrate (Sapro-75,76.27% w/w protein) was purchased from Saputo cheeses Ltd (Montreal, Quebec, Canada). Whey protein isolate was produced from permeate obtained by tangential membrane microfiltration. 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 UF 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. Carboxymethyl cellulose sodium salt (CMC, low viscosity) was obtained from Sigma Chemicals (St. Louis, MO, USA). Glycerol (99.5%, reagent grade) was purchased from American Chemicals ltd (Montreal, Quebec, 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% 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 (%) fat (%) lactose (%) calcium caseinate 91.8 3.8 3.6 0.7 0.1 (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 according to Brault et al. (1997). Solutions were irradiated at a total dose of 32 kGy in a 60CO 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 cm internal diameter Petri dishes sitting on a leveled surface. Solutions were spread evenly and allowed to dry overnight at <BR> <BR> <BR> <BR> room temperature (20 2°C) in a climatic chamber (45-50% 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 , m.

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 cross-linked proteins. The total molecular weight exclusion limit was 25 x 106 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 0.02M tris buffer (pH = 8.0) and 0.1M NaCl. 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 protein solutions (0.5 % w/v) were filtered on 0.45 KLm 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 was 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 previously described. 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 (1 mm/sec) until it passed through the film. Strength and deformation values at the puncture point were used to determine hardness and deformation 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: <BR> <BR> <BR> (2) Y (1 min) = (F9-F')/F9<BR> <BR> <BR> <BR> <BR> <BR> where F° and F'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 24 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.

Analysis of variance and Duncan multiple-range tests with p 5 0.05 were used to analyze all results statistically. For puncture strength and deformation to puncture measurements, three replicates of seven films were tested. For viscoelasticity measurements, three replicate of three films were tested. The Student t-test was used and paired-comparison with p < 05 ( Snedecor and Cochran, 1978).

Figure 1 shows the elution curves obtained for native, heated or irradiated calcium caseinate.

Heating calcium caseinate at 90°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 2 x 106 daltons, an increase greater than 60-fold (Figure 1, c). Previous studies demonstrated that gamma-irradiation induced the formation of bityrosine (Davies, J. A., I. 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.,. J. Agric. Food Chem. 1998,46 (4), 1618-1623). The conditions leading to the formation of cross-links in peptides have been widely investigated (Priitz et al., Int. J.

Radiat. Biol. 1983,44 (2), 183-196). Although bityrosine is expected 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, accounting 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 at 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, a) or after heating (Figure 2, b), 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 kGy) 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 kGy on the molecular weight of whey proteins. It should be emphasized that tests were run on irradiated 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. As 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 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. Food 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 106 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, a) 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 (2 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, 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 development 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 obtained 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 indicate 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 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 the development of edible films.

The present research shows that gamma-irradiation, which induces the cross-linking of tyrosine residues in a manner similar to peroxidase (Matheis and Whitaker, J. Food Biochem. 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 strength variations of films cast from solutions containing different whey protein isolate-calcium caseinate 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/m for the films based on WPI 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 dose 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, suggests other favorable interactions than intermolecular bonding_between whey protein isolate and calcium caseinate. The puncture strength obtained for films made from mixtures of calcium caseinate 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 commercial whey proteins (CWP, Sapro-75) (Figure 6), the puncture strength of the films significantly decreased (p < 05) with increasing increasing protein concentration. These results are not surprising considering that the CWP contains substantial amounts of impurities such as lactose and 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 viscoelasticity 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 viscoelasticity 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: 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. (J. 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 (Figure 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 macroscopic 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 of 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 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 correlation 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.

EXAMPLE II : Demonstration of Effectiveness 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: caseinate 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 between the two treatments. Moreover, this example also demonstrates that the addition of calcium chloride or polysaccharides to the protein formulations increases their effectiveness by 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 Journal 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 (INFOS-Centre technique interprofessionnel des fruits et légumes, 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 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 formulations 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-Hyacinthe, Quebec, Canada). CaCl2 (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 the 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 zt 1 °C. Weight loss and mold growth (%) was noted until 100% 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 often 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 discussed by Kester and Fennema (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 Food 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 delayed mold apparition by another 3 days.

Similarly, CaClz or polysaccharides (agar and pectin) were added to the mixed-proteins formulation. It can be seen that the addition of salt or polyssacharides 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 CaClz 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 salt or polysaccharides.

The results presented in this Example demonstrate that the both caseinate: whey coating or y- 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 ffl: 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 and 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). Glycerol (99,5%, reagent grade) was purchased from American Chemicals ltd (Montreal, Quebec, Canada), carboxymethyl cellulose sodium salt (CMC, low viscosity), and calcium chloride (CaC12, laboratory reagent) was obtained from BDH Chemicals (Montreal, Quebec, Canada). All 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) CaC12 were diluted in water and mixed to obtain homogeneous solutions.

McIntosh apples (Quebec, Canada) and washed potatoes Canada #1 (product from Prince Edward Island, prepared by Emballages D. L. Inc, Laval, Qc, Canada) were purchased from a local grocery. Five slices (about 1/2 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.

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 progressively 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 enzymatic 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 apple 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 for 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 small color fluctuations are not associated with darkening (lower L*).

Nisperos-Carriedo et al. (Food Technology, 47,75-84,1991) previously reported color measurements done on sliced mushrooms coated with a formulation containing vegetable oils, cellulose gums, emulsifiers, surfactants 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 (McHugh 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 sulflhydryl-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 and 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% 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 microfiltered 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 UF 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 60CO underwater calibrator unit (UC-15b; 17.33 kGy/hour) (MDS Nordion, Kanata, Ontario, Canada) at the Canadian Irradiation Center. Films are cast by pipetting 5 mL of the solution onto smooth rimmed 8.5 cm internal diameter Petri dishes sitting on a leveled surface. <BR> <P>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 1: 1: 1 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 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 from thyme and 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) colorimetric method described by Dumoulin et al., 1996. Small rectangular pieces of films (1 x 3 cm) were introduced in electrolysis cells containing 3 ml ofKrebs- Henseleit (KH) buffer and electrolyzed at 10 mA DC for lmn using a model 1000/500 power supply (Biorad, Richmond, CA, USA) to generated oxygen free radicals (OFR). Aliquots of 200 (1 of the electrolyzed solution were 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 Figures 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% protein on whey basis) and whey protein 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, I. D.) and allowed to dry overnight at 20 t1°C in a climatic chamber (45-50%, R Humidity). The irradiated films were obtained 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, Quebec) 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 (WVP) of the 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 Chemicals, 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 pressure) 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 pentoxide. The cups 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).

= (g) (mm)/(m2). (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 (m2), p2-pl is the water vapor pressure differential across the film (32.23 and 9.82 for 100% and 56% RH, respectively).

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.