CROSSLINKED PROTEIN AND POLYSACCHARIDE BIOFILMS FIELD OF THE INVENTION This invention relates to protein and polysaccharide-based covering materials, and is particularly concerned with coatings and films, methods of preparation and their use in the food industry and medical industry.

BACKGROUND Environmental concern about disposable packaging has contributed 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 best solutions since their biodegradability and environmental compatibility are assured (Krochta, J. M., et al., Food Technology, 51: 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: 2563-2583,1995).

Edible films have been proposed for use on foods to control respiration, reduce oxidation, or limit moisture loss. (J. J. Kester and O. R. Fennema, Food Technology 40: 47-59,1986; and S.

Gilbert 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 and U. S. Pat. No. 2,703,760); coatings of natural materials include milk solids (U. S. Pat. No. 2,282,801), lecithin (U. S. Pat.

No. 2,470,281 and U. S. Pat. No. 3,451,826), the application of an aqueous dispersion containing a water soluble algin to the surface of a food substrate, followed by a dry gelling mixture to the algin-coated food substrate (U. S. Pat. No. 4,504,502), protein such as corn steep liquor, soy whey, cheese whey, corn gluten filtrate, wheat steep liquor, brewer's wort and the like (U. S. Pat.

No. 4,344,971), dispersions of a hydrophilic film former and an edible fat (U. S. Pat. No.

3,323,922), dispersions of hydrophobic materials in aqueous solutions of water-soluble high polymers (U. S. Pat. No. 3,997,674), and emulsions or suspensions of a water-soluble protein material and hydrophobic material, with in size adjustment of the pH of the protein material to its isoelectric point (U. S. Pat. No. 5,019,403).

Edible films may be made water-soluble or water-insoluble. Water-insoluble edible films and coatings are better than water-soluble edible films and coatings for many food 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. Use of water-insoluble films and coatings allow many foods, such as fruits and vegetables, to be protected from water during shipping and handling.

Edible films in the form of wraps, such as sandwich bags, also require water-insolubility.

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 effectively to 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).

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 good 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.

Food Sci., 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, Edible film, s and coatings : A review, December, 47-59, 1986; Peyron, Viandes Prod. Carnés, 12, 41-46,1991). Unfortunately, the highly hydrophilic nature of these proteins limits their ability to provide desired edible film functions.

As raw material for the manufacture of edible coatings and films, whey proteins present an attractive and more economical alternative to the above proteins (notably, the cost of whey is half that of caseinates). Commercial whey proteins are a byproduct, mostly wasted, of cheese manufacture. Heating of whey proteins at concentrations greater than 5% at temperatures between 70 °C and 85 °C and acidic pH denature the whey proteins, resulting in a thermoirrreversible gel of polymerized whey proteins linked by disulfide bonds (McHugh, T. H.

& Krochta, J. M., Food Technology, 48: 97-103.1994). Whey protein-isolate films with improved water vapor barrier properties could be produced by heating 8-12% whey protein solutions

between 75 °C and 100 °C to denature and crosslink the proteins (McHugh, T. H., Aujard, J. F., and Krochta, J. M., J. Food Sci. 59: 416,423,1994). The films are made flexible by the inclusion of plasticizers, and in contrast to caseinate films, are water-insoluble. The use of microfluidization and ultrasound treatments during film formation improved the mechanical strengths and barrier properties of whey protein films (Chen, H., Banerjee, R. Wu, J. R. ASAE Proceedings ; ASAE: St. Joseph, MI, Paper 93-6528,1994). Lipids have also been used with heat-denatured whey to form whey-protein-lipid-emulsion edible films with improved water vapor permeability (McHugh, T. H. & Krochta, J. M., J Am. Oil Chem. Soc., 71: 307,1994).

Whey protein, with and without lipid such as acetylated monoglyceride, was also used as a coating on various foods as an antioxidant carrier, or to protect them against mechanical disintegration or oxygen penetration (see Krochta et al., Food Technology, 51: 61,1997).

Edible coatings and films based solely on lipids or solely on polysaccharides have also been developed in order to preserve food quality and freshness. Lipid components of films include waxes, oils, acetoglycerides and oleic acid. Lipid-derived coatings are notable for their ability to retard water loss and dehydration. As film formers, fatty acids and alcohols lack structural integrity and durability. As a barrier to water, the property of lipid films varies with temperature, relative humidity, the relative humidity gradient across the film, as well as the ratio of hydrophilic to hydrophobic materials in the film formulation (reviewed in Baldwin, E. A. et al.,, Food Technology, 51: 56,1997).

Polysaccharides used for edible coatings and films include cellulose derivatives (cellulose ether films made from methyl cellulose (MC), hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC) and carboxymethyl cellulose (CMC)), starch-based polysaccharides (such as amylose), alginates, carrageenan, pectins, and extracellular microbial polysaccharides such as pullulan, levan, and elsinan (Krochta et al., Food Technology, 51: 61-74, 1997). Films cast from cellulose derivatives tend to have moderate strength, and are flexible and transparent. They are good barriers against oils and fats; however, due to their hydrophilic nature, are not good moisture barriers. Edible coatings which include MC, HPMC, HPC, and CMC, have been applied to foods to provide a barrier against moisture, oxygen, or oil, and to improve better adhesion. MC and HPMC are also used in pharmaceutical tablet coating and to make edible sachets for dry food ingredients.

Among other polysaccharide coverings, starch-based films and coatings form moderate oxygen barriers, but are poor moisture barriers. The stability of starch films is affected by moisture and limits their usefulness. Starch composites can be used as biodegradable, though non-edible,

packaging. However, it is notable that for some composites, such as starch-polyethylene (PE), only the starch portion can be broken down by certain bacteria. The PE component does not degrade. Alginate coatings make good oxygen barriers while alginate films, formed by evaporation of an aqueous alginate solution followed by ionic crosslinking with a calcium salt, are impervious to oils and fats but are poor moisture barriers. Coatings that include carrageenan as a major or sole component have been applied to a variety of foods to carry antimicrobials and to reduce moisture loss, oxidation, or disintegration. A pectin derivative, low-methoxyl pectin, can be used to develop edible films whose moisture barrier properties are improved with a wax coating. Pullulan films, and coatings of pullulan, levan, and elsinan, have been used as oxygen barriers for food and pharmaceuticals.

Edible films can also 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. An emulsion of protein and lipid has potential to be superior to either protein or lipid film (McHugh, T. H. & Krochta, J. M., Food Technology, 48: 97-103,1994). Within multi-component systems, proteins act as a cohesive, structural matrix to provide films and coatings with good mechanical properties. As an example of a single-protein film that is a composite of different additives (see U. S. Patent 5,543,164), where a protein such as whey proteins, casein, wheat proteins, soy proteins, ovalbumin, corn zein, peanut protein or keratin is combined with a food grade plasticizer (sorbitol, glycerol or polyethylene glycol) and a lipid (fatty acids, fatty alcohols, waxes, triglycerides, monoglycerides).

Composite edible films made of a blend of pectin or alginate and milk proteins, including whey proteins, are described in Parris, N., Coffin, D. R., Joubran, R. F., and Pessen, H., J. Agric. Food Chem. 43: 1432-1435,1995. According to Parris et al., an aqueous solution of pectin or alginate was made, followed by the addition of plasticizer (glycerine, sodium lactate, or sorbitol), then protein, to yield a 1% (w/w) solution. The solution was then de-gassed, then cast into films and dried. The authors noted significant changes in film tensile properties with changes in plasticizer concentration. The presence of milk proteins improved the water vapor barrier properties of the films. However, film strength decreased dramatically at milk protein concentrations greater than 10%. Notably, the films described in that publication are all simple blends of various components. No attempt was made to crosslink the components.

Thus, there is a need for water-insoluble covering agents based on a combination of protein and polysaccharide, which have good mechanical and barrier properties to meet the requirements of the food and medical industries.

This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION In accordance with an aspect of the present invention there is provided a biofilm that is comprised of one or more proteins entrapped in a crosslinked polysaccharide matrix. The composition used to form the biofilm, which can range from a wrapping to a coating, comprises: one or more proteins, one or more polysaccharide and optionally plasticizing agents, functionalization agents, coupling agent and/or stabilizing agents, wherein said protein molecules are crosslinked and then entrapped within a homopolymeric or heteropolymetic polysaccharide matrix to form a covering material. The ratio of protein to polysaccharide is determined to produce a covering material with characteristics that are optimal for the particular product to be protected.

In accordance with another aspect of the present invention there is provided a process for the preparation of a wrapping biofilm, comprising the steps of: (i) preparing an aqueous solution of one or more proteins; (ii) cross-linking the protein molecules; (iii) adding one or more polysaccharides to the solution, and optionally, plasticizing agents, functionalization agents, coupling agents, and/or stabalizing agents; (iv) treating films in an alcohol/acid solution; (v) treating the film in a glycerol/acid solution; and (vi) reconditioning the biofilms.

In accordance with yet another aspect of the present invention there is provided a process for the preparation of a coating biofilm, comprising the steps of: (i) preparing an aqueous solution of one or more proteins; (ii) cross-linking the protein molecules; (iii) adding one or more polysaccharides to the solution, and optionally, plasticizing agents, functionalization agents, coupling agents and/or stabilizing agents; (iv) homogenize the solution; and (v) treat the item with the coating by either spraying, dipping or brushing methods.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates a method for the fabrication of biofilm wrapping.

Figure 2 illustrates a method for the fabrication of biofilm coatings.

Figure 3 illustrates, schematically, a possible mechanism of incorporating of whey protein into a cellulose matrix.

Figure 4 illustrates the process of preparing cellulose xanthate/ (xanthogenate).

Figure 5 describes effects of carboxymethycelulose on the protein crosslinking.

Figure 6 presents results of tests of the force required to rupture (FR) films prepared from whey protein and cellulose.

Figure 7 presents results of tests of the distance to rupture (DR) of films prepared from whey protein and cellulose.

Figure 8 presents viscoelasticity (VE) coefficients of films prepared from whey protein and cellulose.

Figure 9 presents results a time course study of the lightness parameter (L*) for uncoated (control) and coated (alanate or whey proteins) potato slices.

Figure 10 presents results of a time course study of the hue for uncoated (control) and coated (alanate or whey proteins) potato slices.

Figure 11 presents results of a time course study of the lightness parameter (L*) for uncoated (control) and coated (alanate or whey proteins) apple slices.

Figure 12 presents results of a time course study of the hue for uncoated (control) and coated (alanate or whey proteins) apple slices.

Figure 13 presents results of tests of the scavenging of oxidative species (%) for film formulations based on alanate or whey proteins with or without CMC.

Figure 14 presents elution curves in molecular exclusion chromatography for commercial whey proteins: a) native; b) heated at 90°C for 30 minutes; or c) irradiated at 32 kGy.

Figure 15 presents the results of a study demonstrating puncture strength of control, heated and y-irradiated WPC and WPI films.

Figure 16 presents results demonstrating viscoelasticity of control, heated and-irradiated WPC and WPI films.

Figure 17 presents an effect of heat and y-irradiation on water vapor permeability (WVP) of whey protein films.

Figure 18 presents an effect of heating (a) and y-irradiation (b) on the susceptibility at trypsin degradation of WPC films.

Figure 19 presents an effect of heating (a) and y-irradiation (b) on the susceptibility at trypsin degradation of WPI films.

Figure 20 presents an effect of heat and-irradiation on the susceptibility to pancreatin degradation of WPC (a) and WPI (b) films.

Figure 21 presents an effect of heat and y-irradiation on the interaction between the water and whey protein based films.

Figure 22 presents gel electrophoretic patterns for control (c), heated (H) and y-irradiated (I) WPC and WPI.

Figure 23 presents size exclusion chromatography of control (a), heated (b) and y-irradiated (c) of WPC film.

Figure 24 presents a size exclusion chromatography of control (a), heated (b) and irradiated (c) ofWPI film.

Figure 25 presents FT-IR spectra of whey protein film. Upper curve: control film; middle curve: heated film; lower curve: irradiated film.

Figure 26 presents FT-IR spectra of whey protein film. Upper curve: irradiated film; middle curve: heated film; lower curve: control film. (Data indicated in the table was obtained from 17 proteins: carbonic anhydrase, carboxypeptidase, casein, a-chymotrypsin, chymotrypsinogen, concanavalin A, elastase, immunoglobulin G, u-lactalbumin, p-lactoglobulin A, lysozyme, papain, ribonuclease A, ribonuclease S, trypsin, trypsinogen, trypsin inhibitor; Byler and Suzi, 1988).

Figure 27 presents the results of second derivative FT-IR spectra of whey protein films: (a) control film; (b) heated film; and (c) irradiated film.

Figure 28 presents the results of X-ray diffraction studies of whey protein films: (a) crystallinity degree; (b) (A/26 where A is the band intensity and 8 is the half width at half height of the band) in function of different methods and irradiation doses.

Figure 29 presents the results of X-ray diffraction of whey protein films after abstracted the amorphe: (a) crystallinity degree; (b) (A/28 where A is the band intensity and 8 is the half width at half height of the band) in function of different methods and irradiation doses.

Figure 30 presents the results of studies demonstrating effects of CMC on commercial whey protein cross-linking: (a) when CMC was added to the control protein solution, the protein molecular mass increased more than 5-fold; (b) after heating, the protein molecular mass increased an additional 5-fold as compared to heating in the absence of CMC; (c) after irradiation in the presence of CMC, the increase reached more than 100-fold, in comparison with the non- irradiated controls in the absence of CMC. Moreover, CMC may prevent protein precipitation for more than three months.

Figure 31 presents results of studies demonstrating effects of CMC on caseinate cross-linking: (a) when CMC was added to the control protein solution, the protein molecular mass increased more than 5-fold; (b) the heating treatment had little influence in the absence of CMC, while in its presence the protein molecular mass increased 5-fold; (c) after irradiation the increase was more than 5-fold, as compared with irradiation in the absence of CMC.

Figure 32 presents results of studies demonstrating effects of CMC on soy protein-whey protein isolate (50: 50%) cross-linking: (a) control without CMC; (b) after heating (90 °C for 30 minutes and irradiating at 32 kGy).

Figure 33 shows elution curves in size exclusion chromatography for calcium caseinate (alanate 380): a), native; b), heated at 90°C for 30 minutes; or c), irradiated at 32 kGy.

Figure 34 shows elution curves for commercial whey proteins (WPC): a), native; b), heated at 90°C for 30 minutes; or c), irradiated at 32 kGy.

Figure 35 shows elution curves in size exclusion chromatography 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 36 presents 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 37 presents 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.

Figure 38 shows puncture strength of unirradiated and irradiated (32 kGy) commercial whey protein-calcium caseinate films. Ratios express the proportion in WPC or calcium caseinate for a formulation based on 5% w/w total protein.

Figure 39 shows viscoelasticity coefficient for unirradiated and irradiated (32 kGy) WPC- calcium caseinate films.

Figure 40 shows elution profiles of SPI (a) heated and (b) heated in combination with gamma irradiation at 32 kGy.

Figure 41presents elution profiles of a 1: 1 mixture of SPI and WPI (a) heated and (b) heated in combination with gamma-irradiation at 32 kGy.

Figure 42 shows relaxation coefficient of films based on SPI and a 1: 1 mixture of SPI and WPI.

Figure 43 shows water vapour permeability of films based on SPI and a 1: 1 mixture of SPI and WPI.

Figure44 presents changes in total bacterial counts (APCs) on unirradiated shrimp during storage at 4 °C.

Figure 45 shows changes in total bacterial counts (APCs) on irradiated shrimp during storage at 4 °C.

Figure 46 shows helf life extension of pre-cooked shrimp as affected by gamma irradiation and antimicrobial coating during storage at 4°C 1, 2.

Figure 47 demonstrates effects of gamma irradiation and antimicrobial coating on the growth of pseudomonas putida during storage at 4°c. detailed description of the invention.

Figure 48 illustrates the FTIR spectrums and the probable structures of chitosane obtained.

Figure 49 presents FTIR spectrums for modified and reticulated (fatty acids and dialdehyde) alginate. A, non-modified alginate; B, modified and reticulated alginate; C, Probable molecular structure of the modified and reticulated alginate.

Figure 50 shows that most bioactive agents are sensitive to gastric acid and to intestinal proteolytic degradation (A), which necessitate a particular sphere structure (B).

Figure 51 shows modified chitosane-based and modified and reticulated chitosane-based tablets release profile.

Figure 52 shows modified alginate-based and modified and reticulated alginate-based tablets release profile.

THE DETAILED DESCRIPTION OF THE INVENTION It is an object of the invention to provide protein-polysaccharide biofilms that are based on crosslinked proteins versus entrapped proteins.

The ratio of protein to polysaccharide can be varied to produce a covering material with characteristics that are optimal for the particular product to be covered. Compositions used to produce the covering material of the invention, comprise whey protein and polysaccharide, with optional plasticizer, functionalization agent, and stabiliser. Other additives can be included to bestow further properties to the covering agent.

Methods for producing protein-polysaccharide biofilms that range from wrapping to coatings are also provided. In one embodiment, a biofilm with the consistency of a wrapping is prepared from the composition by: (i) preparing an aqueous solution of one or more proteins; (ii) cross-linking the protein molecules; (iii) adding one or more polysaccharides to the solution, and optionally, plasticizing agents, functionalization agents and/or stabilising agents; (iv) treating films in an alcohol/acid solution; (v) treating the film in a glycerol/acid solution; and (vi) reconditioning the biofilms.

Another embodiment provides means for preparing a protein-polysaccharide biofilm from the composition, wherein the biofilm has the consistency of a coating by: (i) preparing an aqueous solution of one or more proteins; (ii) cross-linking the protein molecules; (iii) adding one or more polysaccharides to the solution, and optionally, plasticising agents, functionalisation agents and/or stabilising agents; (iv) homogenise the solution; and (v) treat the item with the coating by either spraying, dipping or brushing methods.

Further additives can be included in the film in order to tailor the covering material to the specific application. This covering material can be applied to agricultural products, foodstuffs, and packing material used in the food industry. A composition of this covering agent may also be used as a delayed release agent to coat drugs, as a coating for pharmaceutical tablets, or as a biodegradable bandage to cover wounds.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The following abbreviations are used herein: WPI, whey protein isolate; WPC, whey protein concentrate; WVP, water vapor permeability; PG, propylene glycol; TEG, triethylene glycol; PEG, polyethylene glycol; EG, ethylene glycol; CMC, carboxymethyl cellulose; MC, methyl cellulose; HPMC, hydroxypropylmethyl cellulose; HPC, hydroxypropyl 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, also termed biofilms, are preferably formed from a crosslinked mixture of protein and polysaccharide, 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.

Ingredients of the Composition The elements to include in biofilms of this invention are: one or more proteins, one or more polysaccharides, one or more plastifying agents and one or more stabilizing agents. A functional agent is optional.

Whey protein has been used to demonstrate the biofilm and method of this invention. The use of this protein is not in any way intended to exclude the use of other such proteins or mixtures of

proteins from the invention. The novelty herein, lies in the factor that cross-linked protein molecules are entrapped within a polysaccharide matrix.

Many proteins can be used with to produce biofilms of this invention. Exemplary proteins are whey protein, soy protein, caseinate protein, etc. The proteins used in production of the covering agents of the present invention are those isolated proteins having amino acid residues with amino groups capable of functioning as nucleophiles, or 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.

Exemplary polysaccharides used in the production of the biofilms of the present invention include agar and agarose, starch and its derivatives such as amylose, cellulose and its derivatives such as CMC, alginates, which are the salts of linear co-polymers of D-mannuronic and L- glucuronic acid monomers, pectins, carrageenan, which is a complex mixture of galactose polymers, chitosan, which is de-acetylated chitin from shellfish waste, and extracellular microbial polysaccharides such as pullulan. Preferred polysaccharides are cellulose and its derivatives.

Insoluble in almost all solvents, cellulose can be solubilized by blocking the hydroxyl groups in an addition reaction with carbon disulfide. The functional group which results is called xanthate or xanthogenate. When the xanthate is treated in an acid, the insoluble polymer is regenerated.

The alginate can be used alone as the matrix or associated with cellulose (i. e. co-inclusion) for a means of protein inclusion. [see Figures 3 and 4] The ratio of whey protein to polysaccharide varies, depending on the specific requirements of the product the covering agent is meant to protect. The greater the proportion of polysaccharide, the greater the mechanical strength of the film. A typical film or coating contains between 1-10% protein, and 0.1-5.0 polysaccharide. The protein-polysaccharide covering agent can be either a coating or a film, depending on the degree of crosslinking.

Plasticizers In certain embodiments of the invention, a plasticizer is included as a component of the film. As used herein, the term"plastifying agent","plasticizer"or"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, propylene glycol, triethylene glycol, polyethylene glycol and ethylene glycol. The plasticizer serves to improve the stability of the protein within the film and its flexibility. The plasticizer may be selected from the group comprising polyalcohols, glycerol, triethylene glycol or polyethylene glycol. The amount of food grade plasticizer which is added will typically be about 1 to 10% by weight in solution, preferably about 50% by weight of the protein.

Functional Agents In certain embodiments of the invention, a functional agent is included to increase the hydrophobicity of the composition of the film, or improve its moisture barrier properties. As used herein, the term"functional agent"can be defined as a substance that it is covalently cross- linked on proteins, with or without the help of a coupling agent. It includes, but is not limited to, glyceraldehyde, PEG-epoxide, fatty acids and anhydrides. Covalent modification of the protein or polysaccharide component of the film by mechanisms such as acetylation, carboxymethylation, or fatty acid linkage to amino groups on the protein, is effected by the addition of various reactive chemicals, preferably a glyceraldehyde, a polyethyleneglycol- epoxide, a fatty acid, or an anhydride. Modification of the protein or polysaccharide component of the film may be effected by the addition of various compounds, preferably gelatin, collagen, vegetable oil, PEG-1000, polyvinyl alcohol, or triethylene glycol. The amount of functional agent used is typically from 0.1% to 10% by weight in solution.

Stabilizing Agents As used herein, the term"stabilizing agents"includes, but is not limited to gelatin, collagen prolamine, etc. There are numerous stabilizing agents known in the art, but the preferred ones are of a proteinaceous nature as these can be cross-linked or co-enclosed with raw material in the matrix. Stabilizing agents are included in the biofilms in order to: increase biofilm stability to pH limits as well as water resistance at elevated temperatures and to avoid film shrinkage at low or intermediate rates or relative humidity; etc.

Coupling Agents In certain embodiments coupling agents may be required for the covalent coupling of certain chemical compounds (acetylation, carboxymethylation, fixation of fatty acids on amino residues) or ionotropic gelation (alginate by calcium mediation). One skilled in the art will appreciate that

coupling agents are required in the biofilm solution when it is necessary to improve the hydrophobicity and water vapor barrier of the biofilm. For example, the coupling of the functional agent, caproic acid, on proteins with the help of the coupling agent, 1-ethyl-3 (3- dimethylaminopropyl) carbodiimide (coupling agent) allows a decrease in permeability of water vapor by one-half, while maintaining the appearance and mechanical properties of the biofilm.

Lipid Components In certain embodiments of the invention, a lipid or edible oil component can be incorporated into the biofilm As used herein, the term"lipid component"refers to all oils, waxes, fatty acids, fatty alcohols, a wax, monoglycerides and triglycerides having long carbon chains of from 10 to 20 or more carbon atoms, which are either saturated or unsaturated. Some examples of"lipid components"are beeswax, paraffin, microcrystalline wax, carnuba wax, stearic acid, and palmitic acid. A variety of lipid components of varying chain lengths can be used to form effective films..

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. 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.

Although some components, such as PVA, PEG and TEG might be considered to be toxic, their utilization in the formulation of films are allowed by the FDA. As well, when used in minute concentrations for films with superficial contact with food, these substances present no risk.

However, there are certain limits that need to be respected. (see: Title 21, Volume 3, Part 172- 177 for Indirect Food Additives).

In other embodiments, it may be desirable to include agents known to those skilled in the art, such as emulsifiers, lubricants, binders, or de-foaming agents to influence the spreading characteristics of the coating agent. Additives, including chelating agents, such as calcium disodium EDTA, ascorbic acid, antibacterial agents, flavorings, vitamins and minerals, etc. can be included in the coating agent to optimize the characteristics of the covering.

Methods of Making Biofilms There are different methods of crosslinking the proteins comprising the biofilms of this invention. These methods use physical (e. g., heat or irradiation), chemical (eg., epoxide resin, glutaraldehyde, glyceraldehyde), enzymatic (eg., transglutaminase).

In certain embodiments of the invention, a chemical crosslinking agent is used to effect crosslinking between the protein molecules. The chemical crosslinking agent contains several chemically reactive groups within a single chemical entity. Each reactive group is capable of forming a covalent bond with a reactive group present on the protein. The crosslinking agent is preferably a reactive resin, such as epoxide-agarose, wherein agarose is the resin which acts as a template allowing protein conjugation, and epoxide is the reactive group which forms a covalent bond with an amino group of the protein.

The epoxide groups are the reactive component of the epoxide resin. Epoxides are three- membered cyclic ethers, which react with amino groups present on the whey proteins. Since there are a number of epoxide groups on each resin particle, and since each reactive group is capable of forming a covalent bond with a reactive group present on the protein, covalent linkages are formed between a single resin bead and several protein molecules, resulting in crosslinking of the proteins.

One example of the method of using a cross-linking agent entails the use of epoxide-agarose, at a concentration from 0. 25% to 1% (w/v), which is stirred with a protein solution, at a concentration from 2% to 10% w/v (preferably 5-7% w/v) for 1 hour to 10 hours at a pH of 8.0 to 9.5. The remaining reactive sites on the epoxide-agarose are blocked by addition of a simple amine, such as ethanolamine. The proteins, crosslinked via epoxide-agarose, are washed with buffer.

Yet another method employs y-irradiation of solutions containing proteins and polysaccharides, at doses ranging from 10 to 180 kGy, to effect crosslinking of the proteins. Typically, the concentration of proteins is 1% to 10%, and the concentration of polysaccharide is 0.1% to 5%.

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 a 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.

One example of a method of making a wrapping biofilm is presented in Figure 1. The steps involved inlcude the steps of: (i) preparing an aqueous solution of the protein (eg. whey); (ii) cross-linking the protein molecules; (iii) adding one or more polysaccharides to the solution, and optionally, plasticizing agents, functionalization agents and/or stabalizing agents; (iv) treating films in an alcohol/acid solution; (v) treating the film in a glycerol/acid solution; and (vi) reconditioning the biofilms. The final step of entrapment involves use of acid to entrap protein in the polysaccharide matrix which renders the film insoluble to water. Typically, a film containing protein and polysaccharide is treated with a solution of acetic acid in ethanol, then rinsed with water to remove the excess acid. The films are then incubated at ambient temperature and 56 % relative humidity. The composition for a wrapping biofilm contains the insoluble matrix such as cellulose. Cellulose requires specific treatments (immersion for 10 min in ethanol: acetic acid (5: 1 v/v) or ethanol : H2SO4 (5: 1 v/v) to make the biofilm (wrapping) insoluble.

One example of a method of making a coating biofilm is presented in Figure 2. The steps involved include the steps of: (i) preparing an aqueous solution of protein (eg. whey); (ii) cross- linking the protein (whey) molecules; (iii) adding one or more polysaccharides to the solution, and optionally, plasticizing agents, functionalization agents and/or stabilizing agents; (iv) homogenize the solution; and (v) treat the item with the coating by either spraying, dipping or brushing methods. In choosing the ingredients for the coating biofilms, the choice of matrix varies according to requirements. In general, it consists of a soluble matrix in water such as carboxymethyl cellulose (CMC), agar or agarose, alginate, chitosane, etc.

In preferred embodiments, dissolved gases are removed from the composition of protein and polysaccharide prior to casting. 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.

In preferred embodiments, the composition of protein and polysaccharide is cast onto a smooth, level surface and allowed to dry at ambient temperature 16-24 hr at 40%-56% R. H. Dried films are peeled intact from the casting surface.

In the present inventive method, the composition of protein and polysaccharide may be 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.

Methods of Testing Biofilms 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. A number of these tests are outlined below and described in greater detail in the Examples sections. These tests will facilitate one skilled in the art to determine the additives according to the requirements for the consistency of the final biofilm.

Film thickness can be measured using a commercially available instrument i. e. Mitutoyo Digimatic Indicator (Tokyo, Japan) by measuring random positions around a film. The molecular weight of the cross-linked proteins can be determined using size-exclusion chromatography. Insolubility measurements can be performed as in the Examples section. The 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, and then converting 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., 57,190,1992). 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. For evaluation of viscoelastic properties from relaxation curves, the same procedure as used for the puncture test can be used, but the probe is stopped and maintained at 3 mm deformation. Heats of solution can be determined by obtaining isothermic measures using disposable glass ampules in a calorimetre SetaramTM C80. Transmission electron microscopy (TEM) can be used to provide microstructure information relating to the mechanical

characteristics of the films. Water vapor permeability (WVP) can be determined in a manner similar to U. S. Patent 5543164. Oxygen permeability can be determined for covering agents 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.

There are a number of chemical properties designed into the covering agent, such as antioxidant properties, antibacterial properties, biodegradability, etc. Accordingly, there are a number of well known tests that can be performed by one skilled in the art. Evaluation of the antioxidative properties of a film or coating can be measured using procedures described in Example IV.

Additionally, color measurements can be taken to demonstrate whether a coating efficiently delays enzymatic browning by acting as an oxygen barrier as described in Example IV.

Use of the Biofî1ms of the Present Invention These biofilms can be applied to food, drugs, and packaging. Dried foods, low moisture baked products, intermediate moisture foods and high moisture foods all exhibited improvement through the use of the covering material. As described below, the covering material of this invention is useful for application to many different types of products.

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, and would therefore benefit from the covering material of this invention. Low moisture baked foods are also susceptible to moisture uptake from moist fillings and toppings.

Such foods suffer a loss in quality as well as a reduced shelf-life. Many dried and baked products are also susceptible to oxidation, lipid migration and volatile flavor loss.

Many 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 dry out more slowly; but over time moisture loss can still be a problem. Intermediate moisture foods, such as raisins and dates, often become unacceptable due to moisture loss over time.

Moisture loss is also a problem when the moisture transfers from a higher to lower moisture

component of a food mix. For example, raisins can lose moisture to the bran in raisin bran, or moisture from pizza sauce migrates into the crust during storage, resulting in a soggy crust.

Products with a high moisture content, especially those with an exposed surface, are particularly vulnerable to loss of quality due to moisture loss. Examples are foods and plant products which have tissue surfaces exposed by peeling, cutting, etc. such as peeled and/or sliced apples, sliced tomatoes, peeled eggs, fish filets, and cut-stem flowers. These products lose their quality quickly because their natural coverings, normally present to retard moisture loss, have been removed.

The covering material of this invention also has medical applications. A composition of the present invention may be used as a slow/delayed release agent to coat drugs, or as a coating for pharmaceutical tablets. Films may also produced under sterile conditions and be used as a bandage to cover wounds. The film is sterile, pliable, and biodegradable, which eliminates the distressing step of removing and replacing the-bandage. It is within the scope of the present invention that medication, such as antibiotics and/or bromolin, may also be added as a component of the film. Immobilization of enzymes in the films allowing the realization of rapid measures, for example, glucose oxydase for the determination of glycemia; immobilization of medicines in films for dermal treatments or for the controlled release of drugs, etc.

The composition can be applied to cardboard boxes that hold dried food, such as cereal, crackers, grains, etc. It can also be applied to cardboard cartons that hold liquids, such as milk, juice, ice cream, etc.

The present invention also provides foodstuffs and packagings coated with the coating agents of the instant invention. The invention can be applied to products as diverse as sausage skin, medicinal coating, bacterial encapsulation, animal feed coating, silage coating, fish feed coating, etc. In another embodiment, the composition can be applied to chocolate, pretzels, cookies and probiotic bacteria. The following examples are provided by way of illustration and not by way of limitation.

EXAMPLES General Materials Cellulose, a-cellulose fibers, carboxymethyl cellulose sodium salt (CMC, low viscosity), N, N- diethyl-p-phenylenediamine (DPD), cellulose xanthogenate, glyceraldehyde, gelatin, and acetic acid were obtained from Sigma Chemicals (St-Louis, MO, USA). Acetonitrile (99.9%) was from Anachemia Chemicals (Montreal, PQ, Canada). Calcium chloride (reagent grade) was obtained from BDH Chemicals (Montreal, PQ, Canada). Glycerol (99.5% reagent grade) was purchased from American Chemicals Ltd. (Montreal, PQ, Canada). All products were used as received without further purification. Calcium caseinate (Alanate 380TM) was provided by New Zealand Milk Products (Santa Rosa, CA, USA).

Whey protein concentrate (WPC, 76.27% w/w protein) powder was supplied from Les Fromages Saputo Ltee. (St-Hyacinthe, Que, Canada).

Whey protein isolate (WPI, 90.57% w/w protein) was obtained from the Food Research Centre of Agriculture and Agri-Food Canada. WPI 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. (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 free-dried before use in order to obtain WPI.

EXAMPLE 1: Preparation of Various Biofilms This example describes the use of whey protein for the production of biofilms There are two preferred sources of whey protein for the production of the covering agents of the present invention; these are whey protein isolate (WPI) and whey protein concentrate (WPC).

Ultrafiltration techniques are employed to isolate undenatured WPCs and high performance hydrophilic exchange is used to purify WPIs. WPCs range from 25% to 80% whey proteins, whereas WPIs have greater than 80% whey protein content.

The nature of the whey protein is one of the first important considerations, when preparing the covering agents, since it directly affects the film's rheological properties. Concentrated whey contains a significant quantity of lactose (approximately 15%). Therefore, the addition of potassium sorbate, to inhibit the crystallization phenomenon, is often desirable. Alternatively, the lactose can be hydrolyzed and subsequently eliminated by ultrafiltration. It is then necessary to determine the appropriate concentration of proteins required in the formation of biofilms.

Thereafter, different physical (including thermal treatment or gamma-irradiation) and chemical (including glyceraldehyde, ethylchloroformate, etc.) cross-linking processes can be used to increase protein stability. Preferred whey proteins are those which are isolated from milk. For further discussion of whey's properties, see PCT WO 00/49899.

BiofilmFormula WPC or WPI (cross-linked) 5.00 % cellulose xanthate 0.25 % gelatin 1.00 % glycerol 2.50 % Cellulose xanthate preparation : 4% (w/w) cellulose was dissolved with approximately 18% aqueous NaOH at 20 °C. The alkali cellulose was converted to cellulose xanthate by slow addition of 1.3-1.6% (w/w) carbon disulphide. After 2-3 hr stirring, excess carbon disulphide was evacuated and the solution incubated for 3-5 days for"ripening." Cross-Linking Protein : Protein was cross-linked just prior to entrapment, using either heating,- irradiation, or chemical treatment.

Cross-linking by Heating : Aqueous solutions of 5% (w/w) whey protein (WPI or WPC) and 2.5% (w/w) glycerol were heated at 80°C for 30 minutes. Thereafter, the solution was cooled to room temperature and 0.25% w/w cellulose xanthate and 1% (w/w) gelatin were added, under constant stirring. Films were cast by pipetting 5 mL of the solution into petri dishes (Fisher Scientific, Montreal, PQ, Canada). Solutions were spread evenly and allowed to dry overnight.

Cross-linking by y-Irradiation : An aqueous solution of 5% (w/w) whey protein (WPI or WPC) and 2.5% (w/w) glycerol was degassed under vacuum to remove dissolved air and flushed under

inert atmosphere following the procedure described in Brault, D., D'Aprano, G. and Lacroix, M.

J. Agr. Food Chem., 45 (8), 2964-2969,1997. It'was transferred in to an amber glass bottle and sealed with parafilm. The solution was irradiated using a 60Co source irradiator (y-Cell 220, MDS Nordion, Canada) at the Canadian Irradiation Center for 32 kGy. Before casting onto Petri dishes, 0.25% cellulose xanthate and 1% gelatin were added.

Cross-linking by Glyceraldehyde : 5% (w/w) whey protein WPI or WPC) and 50 mg glyceraldehyde were rehydrated in Tris buffer pH 8. 2 and incubated at 37 °C. After 8 hr of the reaction, 2.5% glycerol, 0.25% cellulose xanthate and 1% gelatin were added to the mixture, always under stirring. Films were cast by pipetting 5 mL of the solution into Petri dishes (Fisher Scientific, Montreal, PQ, Canada). Solutions were spread evenly and allowed to dry overnight Film formulation by Entrapment Method : Peeled, dried films were treated in baths of an 95% ethanol/acetic acid (5: 1) for fifteen minutes. This process allows one to obtain insoluble films.

The ethanol is required to fix proteins and the acetic acid, regenerates the cellulose and includes proteins at the same time (entrapment). To remove the excess acid, some rinsing in baths of the ethanol-95/water (5: 5) and another in the 95% ethanol/glycerol/water (4: 1: 5). Before undertaking tests, films were reconditioned in a dessicator containing a saturated NaBr solution ensuring 56% relative humidity (RH) at room temperature for at least 48 hr. Permeability was approximately 0.3-0.5 gemm/m2/24h mmHg) at 56% relative humidity. Permeability was approximately 1.5 gomm/m'/24 hrmmHg at 100% relative humidity.

EXAMPLE II : Demonstration of Process Using Heat Treatment This method for the production of biofilms consists of mixing cross-linked whey protein (using heat) with the alkali-cellulose solution (cellulose xanthate). The films obtained after drying by spreading out are subsequently treated in an acid solution. The primary purpose of the last step is ensure that inclusion takes place.

Cellulose xanthate was prepared by dissolving 4-5 g of a-cellulose in 100 ml of 18% NaOH over one hour at 14-16 °C. Next, carbon disulfide (CS) was introduced, at a concentration of 2.5-3.0 g/mL, dropwise over a period of approximately two hours. The solution was stirred

continuously over 3 to 4 hours until an orange brown medium (honey) was obtained. It was stored at 4 °C (Figure 3).

To produce the biofilm, the protein concentration employed was about 5 % (w/w) either WPI, whey protein isolate, or WPC, whey protein concentrate. After stirring well, the film forming solution was heated to 75 °C for 2 hours, or 80 °C for 20 minutes, in the presence of glycerol 2.5% (w/w) and 1% gelatin (w/w). After the solution was completely cooled, cellulose xanthate 0.25% (prepared as above) was added slowly and mixed well. Five millilitres of solution was distributed in petri dishes and then left to dry for 24 hours.

The processing step allows the production of insoluble biofilms. The films, after having dried, were treated for 10-15 minutes in the acetic 95% ethanol/acetic acid (5: 1) bath. This process consists of regenerating insoluble cellulose to retain proteins in its matrix at the same time (Figure 3). Many washings in the 95% ethanol/acetic acid (5: 1) solution were necessary to remove excess thiolic derivatives. Before carrying out tests, the biofioms were immersed in the ethanol/glycerol/water solution (3.5: 1.5: 5) for 15-20 minutes, then reconditioned to 56 % relative humidity at ambient temperature.

Testing Biofilm Properties The solubility test was performed to determine the dry weight of the initial biofilms (PSI) and the dry weight of two series of treated biofilms (PST): one sample was incubated in boiling water for 30 minutes then left at room temperature (23 °C) for 24 hours and the other sample was incubated in water at 37 °C for the same 24 hour period. The solubility test of the biofilms is shown by the rate of recovery, which is calculated using the formula : Rate of recovery = (PST/PSI) x 100 The rate of recovery of the biofilms containing WPI or WPC by the various processes described above, reveals that there were no significant differences. The WPC-containing biofilms varied between 99.16 and 100 %. The results are presented below.

Treatment WPI% WPC% 100 °C/30 min and 23 °C 99. 86 0. 14 99. 82 0. 12 'C/24hr 24 hr/37 °C 99.89 0.09 99.90 0.10 The test of mechanical properties consisted of determining the breaking load, the strain at failure and the coefficient of viscoelasticity using a texturometer LFRA Stevens (Model MT 1000) according to Gontard, N., Guilbert, S. and CuQ, J.-L. (1992). J. Food Sci., 57 (1), 190-199. The mechanical properties of the biofilms are presented in Figures 6-8.

EXAMPLE III: Mechanical and Structural Properties of Exemplary Whey-Cellulose Films, Entrapment by Acid Activation The following examples describe the effect of physical and chemical treatments (irradiation and crosslinking) on the mechanical and structural properties of protein-and-polysaccharide-based covering agents. The effects of crosslinking of milk proteins and polysaccharides have 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.

As an example of a whey-cellulose film with entrapment by acid activation, films based on 5% whey, 1.1% gelatin, 2.5% glycerol and 0.25% cellulose xanthogenate are characterised in terms of their mechanical and structural properties: puncture strength; viscoelasticity; insolubility in water; and antioxidant capacities.

A solution of 5% whey protein (CWP or WPI), 0.5-1.0% gelatin, and 0.25% cellulose xanthogenate was stirred for 10 minutes at a basic pH (pH 10.0 to 11.5). The solution was cast on Petri dishes by pipetting 5 mL solution onto smooth-rimmed Petri dishes (8.5 cm internal diameter) sitting on a level surface. Solutions were spread evenly and allowed to dry overnight in a controlled atmosphere of 20 °C and 45% relative humidity. Dried films were peeled intact from the casting surface, then treated with a solution of methanolic acetic acid (0.5 M). To improve ease of handling, the film was then treated with a 30-35% solution of glycerol for 5- 10 minutes.

Statistical analysis was performed by employing analysis of variance and Duncan# multiple- range tests with p < 0.05. Using the exemplary procedures described above, puncture strength and deformation to puncture measurements, three replicates of seven films were tested. For viscoelasticity measurements, three replicate of three films are be tested. The Student t-test was utilised at the time of the analysis of variance and paired-comparison with pi 0.05 (Snedecor and Cochran,. In Statistical methods ; The Iowa State University Press: Iowa State, 1978).

'- ; Films were 100% insoluble after immersion in water at 100 °C for 30 minutes, followed by continued incubation in water for 25 h at 24 °C ; (compare with 84% for irradiated caseinate films). Anti-oxidant capacity: 76%. Puncture strength: 0. 096 N/, um (comparable to irradiated caseinate films).

Table 1 : Comparison of various structural properties of milk protein films if h is o : S a: 1 . t A e a'. r, Puncture p. pSl 0.096 0.073 0.1 TO 0.040 0.040 Strength Insolubility 100 17.6 84 84 (%) Anti-72 76 79 59 80 oxidant (%) EXAMPLE IV : Exemplary Chemically Crosslinked Whey-Cellulose Biofilm As an example of a whey-cellulose film crosslinked by epoxide-agarose, films based on 5% whey, 0. 5% gelatin, and 0.25% CMC were characterised in terms of their mechanical and structural properties: Puncture strength, viscoelasticity, insolubility in water, and anti-oxidative capacity.

Epoxide-agarose, at a concentration from 1.5% to 4% (w/v), is stirred with a 5% whey (WPC or WPI) solution for lh to 3h at a pH of 8.0 to 9.5 at room temperature. The remaining reactive sites on the epoxide-agarose were blocked by addition of a simple amine (10 mM), for example ethanolamine, for approximately 5 minutes. The proteins, crosslinked via epoxide- agarose, were washed with phosphate buffer.

EXAMPLE V: Coatings Delay Enzymatic Browning of Fruit and Vegetables Colour measurements were performed on sliced apples and potatoes coated with milk protein biofilm formulations, in order to determine their effectiveness in postponing enzymatic browning. Furthermore, antioxidant properties of films cast from whey and calcium caseinate solutions were tested using the N, N-diethyl-p- phenylenediamine (DPD) colorimetric method.

Coating Solution and Film Formation Method.

Coating solutions were prepared with 5 % (w/w) protein (calcium caseinate or whey protein powder), 2.5% glycerol, 0.25% CMC and 0.125% CaCl2. The components were mixed in distilled water to obtain homogeneous solutions.

McIntosh apples (Quebec, Canada) and Russet potatoes Canada #1 (product from Prince Edward Island, Canada) were used. Five slices (about 1 cm thick) were cut from three potatoes and three apples, dipped one minute in the protein solutions and laid on a flat surface. Control potatoes and apples were cut and laid, without dipping, in the dishes an exposed to atmospheric air. The experiment was repeated three times.

Films used for measuring antioxidant properties were based on protein, glycerol and CMC only.

The films were formed by casting the solution (5 mL) onto a 8. 5 cm internal diameter Petri dish and allowing the solution to dry overnight. The films were peeled intact from the casting surface.

Colorimetric Measurements Colour measurements were taken every five minutes for a total experimental period of 130 minutes. The colour was evaluated using a Colormet spectrocolorimeter (Instrumar Engineering Ltd, St. John's, NF, Canada) using the standard (1976) CIELAB colour system. Lightness is reported as L* and the hue angle value is given by tan~l (b*/a*). The lightness value for perfect white is 100 while L* = 0 corresponds to black. As the hue decreases, red pigmentation increases. The a* axis (red) corresponds the a hue angle of 0 °. Colour measurements were taken once on each slice (potato or apple) for a total of 15 readings per measurement.

Evaluation of Antioxidant Properties Antioxidant property measurements were performed following a modified procedure of the DPD (N, N-diethyl-p-phenylenediamine) colourimetric method (APHA, AWWA, WPCF. (1985). DPD colorimetric method. Standard method for the examination of water and wastewater. 16'edition,

New York, p. 306), as reported by Dumoulin, M.-J., et al. (1996). Arzneim.-Forsch./Drug Res., 46, 855-861.

Films were cut in pieces of equal thickness all measuring 0.8 x 2.5 cm. They were then put in a cell containing 3 mL of Krebs-Henseleit buffer and submitted to electrolysis for one minute (400 Volts; 10 mA) using a generator (Bio-Rad, Model 1000/500). Then, a volume of 200 il was sampled and added to 2 mL of DPD solution (25 mg/mL). The oxidative species react instantly with DPD producing a red coloration that can be measured at 515 nm. The colourimetric reaction was calibrated with potassium permanganate (KMnO4) solution. The oxidative capacity of 1.00 mg/L of free chlorine solution corresponds to that of 5.63 umol/L KMnO solution.

The antioxidant measurements illustrate the biofilm's capacity to inhibit the formation of oxidative species (red coloration). The reaction was calibrated using the non-electrolysed KH buffer solution (no oxidative species ascribed to 100% scavenging) and the electrolysed KH buffer solution (0% scavenging, in the absence of any anti-oxidants). The scavenging percentage was calculated using the following equation: Scavenging (%) = 100- [ (ODsample/ODcontrol) x 100] (1) where OD represents the optical density at 515 nm. The OD is directly related to the degree of oxidation of DPD reagent by the oxidative species. Thus, a film able to completely reduce the level of reactive oxidative species will have a 100% scavenging capacity.

Enzymcztic Browning The variation of the lightness parameter (L*) as function of time for coated potato slices is presented in Figure 9. For the uncoated control slices, an increase in lightness was noted for the first fifteen minutes. As enzymatic browning occurs, L* of the uncoated potato slices started to progressively decrease with time for the remaining experimental period. After 130 minutes, the L* of the control slices varied from 70.64 (at t = 0) to 66.15. The loss of lightness associated with enzymatic browning can be estimated at 4.5% for the entire experimental period (130 minutes). Under the same conditions, the coated potato slices did not show any evidence of darkening. A slight increase in lightness was even noticed for all types of coated potato slices.

After 130 minutes, the lightness of the alanate-coated slices was L* = 71.39 and a similar value (L* = 71. 72) was noted for the whey-coated slices.

Figure 10 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 underwent rapid enzymatic browning as demonstrated by the sharp decrease of the hue over the first 45 minutes. Then, the hue was stable for the remainder of the experimental period.

For the coated slices (alanate and whey proteins), only a slight variation of the hue was observed for the entire period of 130 minutes; these minor colour changes were not coupled with a darkening of the potato slices (Figure 9).

Figures 11 and 12 show the lightness (L*) and hue results obtained for apple slices. Similar to what was observed for potato slices, L* rapidly decreased with time for the uncoated apple slices.

After 130 minutes, the average L* of the uncoated apples was of 66.11 compared to 74.77 at t = 0. This represented an overall lightness loss of more than 8% for the entire period. For the coated apple slices, L* remained constant, which indicated that the protein coatings effectively protected the fruit from oxygen. As for the hue (Figure 12), results show that for all apple slices, the angle decreased slightly with time. The effect was less noticeable in the case of the whey coating.

Although the hue decreased, the results in Figure 11 show that moderate colour fluctuations were not associated with darkening. Overall results clearly suggest that protein-based edible coatings were successful in delaying enzymatic browning in sliced apples and potatoes.

In order to further evaluate the antioxidant capacities of these films, measurements were carried out using a model allowing the release of reactive oxidative species by electrolysis of saline buffer.

Antioxidant Properties of Protein Films.

The antioxidant power of protein films are presented in Figure 13. Films containing CMC had better antioxidant capacities than those based only on protein (caseinate or whey) and glycerol.

The antioxidant capacities of protein-glycerol films were of 37.63% for the alanate and of 60.21% for whey protein films. When CMC was added to the film formulations, that percentage increased to 66.14% for alanate and to 75.17% for whey. It can be seen that for both types of formulations, whey proteins exhibited a higher antioxidant power that calcium caseinate (alanate). Furthermore, the addition of CMC increased the antioxidative power of these films by 75% for the alanate formulations and by 25% for the whey protein formulations.

This example demonstrated the effect of milk protein-based edible coatings on the enzymatic browning of sliced apples and potatoes. Results confirm that the formulations were effective in

delaying browning reactions by acting as oxygen barriers or scavengers. Furthermore, an electrolysis model generating oxidative species was used to assess the antioxidant potential of these films. Whey was shown to be a better antioxidant than calcium caseinate. Such differences can be explained by the variations in amino acid composition in the two types of protein.

Furthermore, lactose which is present in the commercial whey can also account for the increased antioxidant activity, simple sugars being known for their free radical quenching effect. Similarly, the addition of a polysaccharide, like CMC, can further increase the antioxidant potential of biofilm formulations of the present invention.

EXAMPLE VI : Demonstration That Whey-Cellulose-Modifier Films Have Improved Water Barrier Properties As an example of a whey-cellulose film crosslinked with an optional functionalisation (modifying) agent, films containing various modifiers were characterised with respect to their water vapour permeability. Different modifying agents (TEG, PVA, or PEG) were added at an arbitrary concentration of 0.5% to a base formulation of 5% whey protein, 2.5% glycerol, 1% and 0.25% cellulose. The materials were obtained as described in the General Materials section of the Examples.

The whey protein was dissolved in solution at 72 °C for 2 h. After cooling to room temperature, cellulose, glycerol, gelatin, and the modifying agent were added and the mixture was stirred to dissolve the added components (note that PVA was previously dissolved in solution by stepwise addition in water at 70-80 °C ; the solution of PVA was cooled before being added to the film composition). A volume of 7 mL of the film solution was used for casting per petri dish and allowed to dry for 24h.

Table 2 demonstrates the role of the modifying agent to improve the water barrier property of the resulting film. The measurements of water vapour permeability (WVP) performed according to methods described above.

Table 2: Water vapor permeability of whey-cellulose films containing various modifying agents Modifying Agent WVP Control 1. 56 +/-0.18 TEG 1.16 +/-0.11 PVA 0.94 +/-0.09 PEG 0.86 +/-0.11

EXAMPLE VII: Mechanical and Structural Properties of Cross-Linked Whey Proteins The mechanical properties of cross-linked edible films based on two type of whey proteins (commercial and isolate) were investigated. Cross-linking of the proteins was carried out using thermal and radiative treatments. Size-exclusion chromatography performed on the cross-linked proteins showed that gamma-irradiation increased the molecular weight slightly for the whey proteins. However, heating of the whey protein solution induced a more significant amount of cross-linking, wherein the molecular weight distribution was > 2 x 106 daltons.

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. (Milchwissenschaft 1995, 50 (11), 614-619).

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.

Table 3: Protein, ash, fat and lactose content of commercial whey protein concentrate (CWP, Sapro-75) and whey protein isolate (WPI)

Protein (%) Ash {%) HzO (%) Fat (%) Lactose (%) Commerical whey protein 72.7 3.5 4.8 5.4 13.6 concentrate2 (CWP) whey protein isolate3 (WPI) 90.6 2.3 3.6 ND 3.5 2Technical bulletin of the Saputo cheeses Ltd. ; Agriculture and Agri-Food Canada, FRDC, Saint-Hyacinthe, Quebec.

Method for film preparation: 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 3. 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. (J Agric. Food Chem. 1997,45 (8), 2964-2969). 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 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 um.

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 um nylon membrane filters (VWR, Nalge, Mississauga, Ontario, Canada) prior to injection.

Determination of insoluble matter: 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. (J. Food Sci. 1992,57 (1), 190-195). 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: (2) Y (1 min) = (F0-F1)/F0 where FO and Fl were forces recorded initially and after 1 min of relaxation, respectively (Peleg, 1979). A low relaxation coefficient (Y 9 0) indicates high film elasticity whereas a high coefficient (Y- 1) indicates high film viscosity.

Transmission electron microscopy (TEM): 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.

Statistical analysis: analysis of variance and Duncan multiple-range tests with p : 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 zu 0.05 (Snedecor and Cochran, In Statistical methods ; The Iowa State University Press: Iowa State, 1978).

Figure 14 shows the elution curves obtained for the commercial whey proteins, before (Figure 14a) or after heating (Figure 14b), or irradiated (Figure 14c). Gamma-irradiation induced very little molecular weight changes in the commercial whey. Only a broadening of the elution peak can be observed in Figure 14c. This feature is not surprising, considering that whey proteins contain less tyrosine residues than caseins (Wong et al. Crit. Rev. Food Sci. Nutr, 36 (8), 807- 844,1996). Our results support the report by Davies J : Biol. Chem., 262 (20), 9895-9901, 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 14). 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 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 14). These results are consistent with those reported by Hoffmann et al. (J. Agric. Food Chem, 45 (8), 2949-2957,1997) 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.

EXAMPLE VIII: Rheological Analysis on the Effect of Heating vs-Irradiation It was an aim of this study to develop and to characterize whey protein films made by chemical and physical techniques. The methodology is essentially based on the poly-condensation followed by the inclusion of proteins in a cellulosic matrix. The microstructure of these films was examined by FTIR and X-ray diffraction analysis.

Cellulose xanthate preparation 4 % w/w cellulose were dissolved in 18 % aqueous NaOH at 20°C. The alkali cellulose is then converted to cellulose xanthate by addition slowly 1.3-1.6 % w/w carbon disulfide. After 2-3 hr stirring, the excess carbon disulfide is evacuated and the solution is incubated for a few days at 15°C for"ripening".

Preparation of the film making solution by heating Aqueous solution of 5 % w/w whey protein (WPI or WPC) containing 2.5 % w/w glycerol were heated at 80°C for 30 minutes. Then, the solution is cooled at the room temperature and 0.25 % w/w cellulose xanthate and 1 % w/w gelatin are added, always under stirring. Films were cast by applying 5 mL of the solution onto Petri dishes (Fisher Scientific, Montreal, Quebec, Canada).

Solutions were spread evenly and allowed to dry overnight.

Preparation of thefilm makingsolation by y-irradiafion The film making solution contains the same components as for heating procedure. Solution of 5 % whey protein containing 2. 5 % glycerol was degassed under vacuum to remove dissolved air and flushed under inert atmosphere in conditions described by Brault et al. (J. Agr. Food Chem., 45 (8), 2964-2969,1997). Solution was transfered in amber glass bottle and sealed with parafilm and irradiated using a 60CO source irradiator (y-Cell 220, MDS Nordion, Canada) at the Canadian Irradiation Center for 32 kGy. Before casting onto Petri dishes, 0.25 % cellulose xanthate and 1 % gelatin were added.

Film formation by entrapment method After peeled, dried films were treated in baths of ethanol-95/sulfuric acid (10: 1) solution for 15 minutes. This process is important, because it allows to obtain insoluble films. The ethanol is necessary to fix proteins and the sulfuric acid, regenerated the cellulose and include proteins at the same time (entrapment). To remove the excess of the acid, some rinsing in baths of the

ethanol-95/water (1: 1) and an other in the ethanol-95/glycerol/water (4: 1: 5). Before to undertake tests, films were reconditioned in a dessicator containing a saturated NaBr solution ensuring 56 % relative humidity (RH) at room temperature, for at least 48 hr (Gontard et al.,. J Food Sci., 57 (1), 190-1991992).

Solubility test The test consists in determine the differences between the initial dry weight (IDW) of the biofilms and the dry weight after treatment (DWT) of two series of biofilms in water. One is in the water boiling during 30 minutes and left at the room temperature during 24 hr with occasional mild agitation and the other, at 37°C during 24 hr. Therefore, the solubility of biofims translated into the yield of recovery (YR) was calculated with the formula: YR (%) = (DWT/IDW) x 100 Film thickness measurements Film thickness was measured using a Mitutoyo Digimatic Indicator (Tokyo, Japan) at five random positions around the film. Depending on the formulation, the average film thickness was in the range of 50-60 J, m.

Mechanical properties Puncture tests were done using a Stevens LFRA Texture Analyzer Model TA/1000 (NY, USA), as described by Gontard et al., supra (1992) in Example VII above.

Water vapor permeability tests Water vapor permeability tests were conducted using a modified ASTM procedure (Gontard et al., supra 1992). The film was sealed on a glass permeation cup containing 5.0 g phosphore pentoxide (0 % RH). For each film, the cup was stored at 23°C in dessicator with saturted NaBr solution (56 % RH). After steady state conditions were reached, the cups were weighed at 24 hr and water vapor permeability (WVP) of the film was calculated as follows: WVP (g. mm/m2. 24 hr. mmHg) = W. x/A. T. (P2-Pl) (1) Where W is the weight gain of the cup (g); x is the film thickness (mm) ;

A is area of exposed film (m) ; T is the time of gain (hr); P2-Pl is the difference of vapor pressure across the film (mmHg).

For each experiment, three repetitions per experiment were done.

Biodegradation by trypsin This test consist to incubate the biofilms in the trypsin solution then to quantify the proteins liberated in the medium in function of times (1 day of the interval). The enzymatic solution contained 0.05 % (w/v) of trypsine from porcine pancreas (Type II-S, Sigma Chemical CO, St Louis, MO, USA) in 20 mM Tris (pH 7.5 0.1). Each film (approximately 25 mg protein) was incubated in 5 mL enzymatic solution at room temperature (23 °C). The ratio between enzyme and substrate was approximately 1: 10. The Bradford method was used to determine protein concentration and the yield of recovery was calculated as follows: YR (%) = (QPF-QPL)/IPF x 100 Where QPF: the quantity of protein in film; QPL: the quantity of protein liberated in the medium.

Biodegradation by pancreatin This test consists to incubate biofilms in the pancreatin solution and then to determine the weight of remaining films in function of time (15 minutes of the interval). The enzymatic solution contained 1 % (w/v) of pancreatin N. F. (Fisher Scientific Company, New Jersey, USA) in 50 mM buffer of monobasic potassium phosphate, pH 7.5 0. 1. Each film (approximately 500 mg protein) was incubated in 50 mL enzymatic solution at room temperature (23 °C). The ratio between enzyme and substrate was approximately, 1 : 1.

Styzcctural analysis For electrophresis and size-exclusion chromatography analysis, studied samples were in filmogene solution containing only proteins (control and treated). For isothermal calorimetry, FT-IR and X-Ray diffraction analysis, samples were biofilms composing 5.0 % proteins, 2.5 % glycerol and 0.25 % cellulose.

Electrophoresis of the film making solution Molecular characteristics of control, heated (30 min at 80°C) and y-irradiated (32 kGy) WPC and WPI were compared from their electrophoretic patterns. Sodium Dodecyl Sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970) using a Mini-PROTEAN 11 Cell (Bio-Rad Laboratories, Hercules, CA, USA). Acrylamide stacking gel (4 %) and separating gel (12 %) were used. Gels were stained with 0.1 % Coomassie brilliant blue R-250 for 30 min in 10/40/50 acetic acid/methanol/water (v/v/v) and de-stained in the same solvent system without dye.

Size-exclusion chromatography off"zlmogen solution Size-exclusion chromatography was performed on the soluble protein fraction using Varian Vista 5500 HPLC coupled with a Varian Auto Sampler model 9090. Detection of the protein solution was done using a standard UV detector set at 280 nm. Supelco Progel TSK PWH (7.5 mm x 7.5 cm) and TSK GMPW (7.5 mm x 30 cm) guard columns (Supelco, Sigma Aldrich Canada Ltd.) followed by Ultra Hydrogel 2000 and 500 (7.5mm x 30 cm) analytical columns (Water Ltd., Mississauga, Ontario, Canada) were used for the molecular weight determination of the cross- linked proteins. The total molecular weight exclusion limit was 25 x l 06 Daltons based on linear polyethylene glycol (PEG) mass. The eluant (80 % v/v aqueous and 20 % v/v acetonitrile) was flushed through the columns at a flow rate of 0.8 mL/min. The aqueous component of the eluant was 0.02 M Tris buffer (pH 8.0) and 0.1 M NaCl. The molecular weight calibration curve was established using a serie of protein molecular weight markers (Sigma, MW-GF-1000, USA) ranging from 2 x 106 Daltons to 29 x 103 Daltons. All soluble protein solutions (0.5 % w/v) were filtered throught a Nylon membrane 0.45 pm (Nalge company, Rochester, New York, USA) prior to injection.

Isothermal calorimetry The measurements were obtained with a Setaram calorimeter in an isothermal mode (heats and swelling) allowing the study of interactions between the different components of the biofilm and the water. A known weight of dried sample ( 30 mg) was introduced in a home-made thin glass bulb and sealed under vacuum. The bulb was placed with the water within a cell equipped with Teflon joints to prevent water evaporation and then the cell was placed in the calorimeter. After thermal equilibrium, the bulb was broken by pushing gently from the top of the calorimeter a stem passing through the stopper of the cell. Due to the vacuum in the bulb, water filled the entireglass bulb once it broke and interacted with the sample. The experiment obtained after integration of the heat flow change were the sum of three contributions:

A"experimenta)"AH interaction + AH glass-breaking + AH vaporisation (2) Hglass-breaking obtained was measured by using empty bulbs and the values were-150 to-200 mJ.

For LIHyaPorisatiom a dependance on the temperature corresponding to the endothermic heat of vaporization of water in the volum of the bulb which was not satured by water vapor was formed. i\HVaporisation could be calculated from the volume of the bulb and the equilibrium water pressure at a given temperature. The heat of a blank experiment could be used to measure the second and third term of the equation (2). These heats, subtracted to #Hexperimental and in this way AH ; nteraction was obtained with values between-150 mJ at 25°C and +500 mJ at 80°C.

FTIR Spectroscopic analysis FTIR spectra were recorded using a BOMEM Hartman & Braun (Bomem, Inc., Quebec, Canada) equiped with DTGS detector (Deuterated triglycine sulfate). Spectra were analyzed using the BOMEM GRAMS software (Ver. 1. 51). The biofilms were placed in the BOMEM cell for scan spectral region from 4000-500 cm-1 and 50 scans were recorded with a 1 cm-1 resolution. The second derivative of a spectrum narrows the broad Amide I band which are related to the different protein chain conformations was equally envisaged (Dong, 1990; Byler, J. Industrial Microbiology, 3, 73-88,1988).

X-Ray difftaction The diffraction pattern of whey protein films was recorded by Siemens D-5000 diffractometer with cobalt cathod operating in reflectance mode at wavelenght 1.79019 A (30000 Volts and 16 mA). The radiation is monochromatized by a graphite crystal mounted just ahead of the scintillation counter which measures the X-ray intensity.

Solubilitytest For all types of protein based-films, the solubility in the water is one of the first important properties to study, because more than they are resistant to the water, more the potential of application of these biofilms is large. The entrapment method of cross-linked WPC and WPI allowed to obtain insoluble biofilms, especially WPI films. The Table 4 shows that the better yield of recovery is for y-irradiated WPI films (approximately 98 % for treatment at 100 °C/30 min then 23 °C/24 hr and 99 % for treatment at 37 °C/24 hr). The weight loss could be due to

the other, components of films formulation (for example, glycerol in the film could migrate in the water during the solubility test). Effect of the heating and y-irradiation on water solubility was studied, but all maner, the cross-linked proteins by heating and y-irradiation allows the increase of the insolubility films.

Table 4: Insoluble fraction of WPC and WPI films evaluated as yield of recovery, following different treatments loan YICI O CO'S IQ j r Control 67. 92 1. 74 94. 53 2. 31 (without treatment) 30 min boiling water Heated 79 11 i 2 02 97.76 il 2.00 then 24 hr/23 °C (80 °C/30 min) Irradiated 79. 36 i 2. 09 98.63 i 1 35 (32 kGy) Control 92. 86 1. 46 97.82 0. 95 (without treatment) T-Jpatpr) 24 hr/37.'C Heated 97. 81 1. 11 99. oo i 0 81 (80 MC/30 min) Irradiated 96.66 1. 57 98. 79 0. 92 (32 kGy) Afeel7atzicalproperties tests Figure 15 shows that the puncture strength of WPC and WPI films formed by heating and y- irradiation increased statistically significant as compared to that not treatment (control). These values vary between 70 and 81 N/mm compared to 59 and 65 N/mm respectively. On the contrary, films cross-linked are less extensible and elastics and there is no difference between the heating and the y-irradiation (Figure 16). Indeed, formation of cross-links by heating (training of intermolecular bridges type disulfide) or by y-irradiation (bityrosine) increases the stability of proteins, therefore, the rigidity of the biofilms.

Water vapor permeability Entrapment of cross-links proteins significantly decreased water vapor permeability of both WPC and WPI films (Figure 17), suggesting decrease of rate of moisture and increase of hydrophobicity. It is important to note that WPI films are more hydrophobic than these WPC

films despite the presence of an important quantity of fat matters in WPC. These results revealed that the method of entrapment is more efficient to form a good barrier to the water vapor that the method of fatty acid incorporation by emulsion or coupling.

Biodegradability It was found that films are definitely more stable at proteolysis than free protein preparations (Figures 18 and 19). This was found with both heated and irradiated films. Since with these data is difficult to evaluate modification produced by treatments, different films were treated in extremely high proteolytic medium based on pancreatin. The results showed that the biofilms composed with cross-links proteins are generally more stable to the filmolyse, especially the biofilms based on WPC (Figure 20). The essential factor implied in the increased stability of WPC films could be the presence in WPC of others components, i. e. fatty acids which can be constituted a congestion reducing thus the maximal catalytic activity of the enzymes. For WPI films, alone irradiated films are more resistant in enzymatic medium while films obtained by heating are degraded in the same period that the control. These results suggest that the method of cross-linking by y-irradiation affords better stability at proteolysis for short-medium intervals still monitoring the biodegradability at long term.

Electrophoresis SDS-PAGE was carried out to compare the changes in protein molecular characteristics of control and treated whey protein films. The electrophoregram (Figure 22) was showed an important accumulation of immobile protein at the gel origin (stacking gel). This accumulation was evident for both heated and irradiated WPC and WPI suggesting further development of cross-links.

Size-exclusionchromatography Chromatograms of control and treated film-making solution WPC and WPI were allowed clearly to observe the cross-linked proteins and to determine approximately their molecular weight. The cross-linking of WPC induced by heating and by y-irradiation was allowed to obtain the molecular weight approximately 3800 kDa and 2000 kDa, respectively (Figure 23). Similarly for WPI, the molecular weight of polymer by heating vary 1000-2000 kDa and by y-irradiation, 600-1000 kDa (Figure 24).

By comparing results obtained for WPC and WPI, the cross-linking by heating is more efficient for WPC and by y-irradiation for WPI.

Isothermal calorzmetry Figure 21 shows that the dispersion WPC and WPI films (control, heated and irradiated) in water results in an exothermic reaction as seen by the negative values. More than these values are small, more than the interaction of films with the water is great. In general, heating and y- irradiation possess more raised values (respectively-18 and-20 J/g for WPC;-15 and-10 J/g for WPI). These results put not only in obviousness the hydrophobicity of films, but also reinforce results obtained by the test of solubility.

The comparison between the two methods of the cross-linking was not able to be correlated. The best results for WPC films were induced by the heating and for WPI films, by y-irradiation.

However, the common ground of these two biofilms is that the molecular weight of polymers is important, resulting an efficiency of the cross-linking.

As for the comparison between WPC and WPI films, the results show that WPI is better. This is evident because WPC contains an important quantity of lactose having many hydroxyl groups that are readily for hydrogen bond with the water.

FT-IR analysis The analysis of FT-IR spectra obtained from the three series of films (control, heating and y- irradiation) were performed in two interesting spectral regions: 3600-3000 cm'and 1700-1600 cm-'(amide I).

For the spectral region 3600-3000 cm~l, a strong band was observed at 3293 cm-' (figure 25).

This last is due majority to NH stretch (protein). Although there are few studies on proteins concerning this spectral region, Bandekar (1992) has noted that the band related to NH stretch mode is generally to 3254 cm'\ There is therefore a displacement of the band that could be due to the presence of others components in the biofilm formulation, especially the cellulose and the glycerol. No difference was observed for all films (control, heating and y-irradiation). To the exception for the control film, an enlargement important of the band (3300-3600 cm-1) was observed. Generally, according to Kondo (1998), the OH groups of the cellulose film were exhibited characteristic band approximately at 3420 cm-'. Increasing the number of OH groups shows the diversity of OH frequencies in IR spectra and then the absorption band became broader. These results suggested that the y-irradiation can cross-linked proteins and, at the same

time, led an alteration of the conformation proteins while the heating was not induced the same modification.

X-Ray diffraction The diffractogram patterns were similar for control, heated and irradiated films (Figures 28 and 29). However, by increasing the y-irradiation dose from 48 to 64 kGy, the X-ray diffraction profiles an evident change was observed. This reveals that the degree of crystallisation of films increases as a function of the degree of the cross-linking. The y-irradiation, followed the chains cross-link, implies a new structure, may be more orderly and more stable. This hypothesis can equally explain the disappearance of the a-helical conformation observed by the FT-IR analysis.

The same results were obtained for different degrees of the relative humidity (0,56 and 100 % RH). These results suggest the degree of crystallisation of these films is independent of the moisture.

EXAMPLE IX: Studies Demonstrating the Effects of CMC on Protein Cross-Linking Studies were performed to demonstrate the effect of CMC on protein cross-linking. The results are presented in Figures 30-32.

EXAMPLE X: Studies Demonstrating Mechanical and Structural Properties of Films 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. 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.

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.

(Milchwissenschaft., 50 (11), 614-619,1995). 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 rn). 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.

Method for film preparation: All formulations were as described in Example VII. The content in protein, fat, lactose and ashes are summarized in Table 5. The components were prepared as in Example VII.

Table 5: Protein, ash, fat and lactose content of calcium caseinate (alanate 380), commercial whey protein concentrate (CWP, Sapro-75) and whey protein isolate (WPI). ,) protém (ta) ! ash (H2O (o) ; l : at (los f Calciumcaseinate (alante 380) (alante 380) Commercial whey protein concentrate (CWP) 76.27 3.72 0 5.79 14.22 Whey protein isolate (WPI) 90.57 2.32 3.61 none 3.5 Film thickness measurements Film thickness was measured, size-exclusion chromatograhy was performed, insoluble matter was determined, puncture tests were performed, viscoelastic properties were evaluated, transmission electron microscopy (TEM), and statistical analysis was performed as described in Example VII, above.

Figure 33 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 33, 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 33, c). Previous studies demonstrated that gamma-irradiation induced the formation of bityrosine (Davies, supra 1987; Brault, supra 1997; Mezgheni et al supra, 1998; Ressouany et al., supra, 1998). The conditions leading to the formation of cross-links in peptides have been widely investigated (putz et al., Int. J. Radiat. Biol., 1983,44,183-196,1983).

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 and Delsignore, J. Biol. Chem. 1987,262,9902-9907,1987).

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 33, C). However, intramolecular bonding should not be totally excluded. In Figure 33, 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 33, 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., supra, (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 34 shows the elution curves obtained for the commercial whey proteins, before (Figure 34A) or after heating (Figure 34 B), or irradiated (Figure 34C). Gamma-irradiation induced very little molecular weight changes in the commercial whey. Only a broadening of the elution peak can be observed in Figure 34C. This feature is not surprising, considering that whey proteins contain less tyrosine residues than caseins (Wong et al, supra, 1996). Our results support the report by Davies (supra, 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 34). 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 x 106 daltons while the smallest fraction can be attributed to uncross-linked protein or intramolecularly cross-linked protein..

Figure 35 shows the molecular mass changes in the case of a 50%-50% mixture of whey protein isolate and caseinate before (Figure 35, a) or after heating (Figure 35, b), or irradiated (Figure 35, c), or heating at first then treated with irradiation (Figure 35, d). About 40% of the protein was cross-linked ( ! 10 x 106 daltons) in the combined heating and irradiation treatment (Figure 35, 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. (1997) 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., supra, 1994). Li et al. supra (1999) 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.

Insolubility of irradiated films : Figure 36 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 33,34, and 35) 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 x 106. Ressouany et al. (supra, 199$) 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 2 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, supra, 1994). 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 invention shows that gamma-irradiation, which induces the cross-linking of tyrosine residues in a manner similar to peroxidase (Matheis and Whitaker, supra, 1987), 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., supra 1998; Ressouany et al., supra, 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.

Mechanical properties of calcium caseinate-whey protein films: Figure 37 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/pm 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. (supra, 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 38), the puncture strength of the films significantly decreased (p : 0. 05) with increasing whey protein concentration. These results are not surprising considering that the CWP contains substantial amounts of impurities such as lactose and fats which could act as internal plasticizers in the films. Results depicted in Figure 37 and 38 also shows that-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 33 and 34, the radiative treatment was more effective on calcium caseinate than on whey proteins in terms of molecular weight increase.

Figure 39 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 zu 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).

Microstructure observations: Cross-sections of the films were observed using transmission electronic microscopy (TEM). 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. (1997) on casein films prepared by a modified wet spinning process. However, the microstructure of the films that were cast from

irradiated solutions is clearly more dense than the films cast from unirradiated solutions. 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 33-35) directly influence the macroscopic characteristic of the films in terms of mechanical strength and water-resistance showed by physical measurements (Figures 36-39). 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).

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 and 75-25 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 A great difference between the microstructure of films 75-25 and 100-0 can also be observed. The topography of the films varies from a porous structure to a more granular one. Similar correlation between microstructure and mechanical strength were seen in films based on WPI. 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 than the same films both heated and irradiated at 32 kGy. 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 35, c.

The present invention demonstrates 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 behaviour of these films.

EXAMPLE XI: Mechanical and Barrier Properties of Cross-Linked Soy and Whey Protein Based Films Materials. SUPRO 500E soy protein isolate (SPI) was provided by Dupont Campbell Protein Technologies (St-Louis, MO, USA). Whey protein isolate (WPI) was lyophilised and dried for 3 h in a vacuum oven at 80°C (Model 19 Laboratory oven, Precision Scientific Inc., Chicago, IL) from the solution purified at the Food Research and Development Centre (St-Hyacinthe, Quebec, Canada). Low viscosity Carboxy methyl cellulose (CMC) sodium salt was purchased from Sigma Chemicals Co. (St-Louis, MO, USA), and lyophilised polyvinyl alcohol (PVA), 98% hydrolysis was purchased from Aldrich Chemicals Co. (St-Louis, MO, USA). Sodium carbonate monohydrate reagent and glycerol (99.5%) were obtained from American Chemicals Ltd.

(Montreal, Quebec, Canada). Phosphorous pentoxide was obtained from NDH Inc. (Toronto, Ontario, Canada).

Film formation: soy protein isolate and whey protein isolate were solubilised in distilled water, under stirring, at 90°C, to obtain a SPI/WPI ratio of (1/1), with a total protein concentration of 5% (w/v) in the film forming solution. The pH was adjusted at 8.5 with 1 M Na2CO3, and when necessary, 0.25% (w/v) CMC or 0.5% (w/v) of PVA were added. After complete solubilisation, 2.5% (w/v) of glycerol was added and the solution was then degassed under vacuum to remove dissolved air. The different protein solutions were poured in separate 240 mL amber bottles (Anachemia Sciences, Montreal, Quebec, Canada) and irradiated together at the Canadian Irradiation Centre (CIC) at a dose of 32 kGy and a mean dose rate of 31.24 kGy/h, using a 60Co source UC-15A (MDS-Nordion International Inc., Kanata, Ontario, Canada). Films were then cast by pipetting 5 mL of the solution onto smooth rimmed 8. 5 cm (i. d.) polymethacrylate (Plexiglas) plates, sitting on a levelled surface. Solutions were spread evenly and allowed to dry overnight at room temperature (20 2°C) in a climatic chamber (45-50% RH). Dried films could

be peeled intact from the casting surface. The overall experiment was performed in two separate replications.

Film thickness was measured using a Digimatic Indicator (Mitutoyo, Tokyo, Japan) at five random positions around the film, by slowly reducing the micrometer gap until the first indication of contact. Depending on the formulation the average film thickness was in the range of 45-65 2 (mi.

Puncture tests were carried out using a Stevens LFRA Texture Analyser Model TA/1000 (Stevens, NY, USA), as described in Example VII.

Water vapour permeability (WVP) of films was determined gravimetrically using a modified ASTM (1983) procedure. The films were sealed with silicone sealant High Vacuum Grease Dow Coming (Midland, MI, USA) in a glass permeation cell containing phosphorous pentoxide (0% RH, 0 mmHg water vapour pressure). All cast films were shiny on the side facing the casting plate surface and dull on the side facing the frying air during the measurements. The glass permeation cells were 3.8 cm (i. d.), 8.3 cm, (o. d.) and 13.0 cm tall, with an exposed area of 12.56 cm2. The cup was placed in a dessicator maintained at 100% RH (17.54 mmHg water vapour pressure, at 20°C) with distilled water. The water vapour transferred through the film and absorbed by the dessicant was determined from the weight gain of the cell. The assemblies were weighed initially and at 1,2,3,4,5,6,24 and 48-hours intervals. Linear regression analysis gave very significant correlation (r = 0.995). Steady state conditions were assumed to be reached when the change in weight became constant overtime (&num 6h) and the weights were recorded over 24h for all samples. The water vapour permeability was determined as follow (Heiss, 1958; Karel et al., 1959; Labuza and Contreras-Medellin, 1981): WVP = (w. x)/A. T. (pz-pl) (g. mm/m2. day. mmHg), where w is the weight gain of the cup over the time (T = 24h), x is the thickness (mm), A is the area of exposed film (m2), P2-Pl is the vapour pressure across the film (mmHg).

Size-exclusion chromatography was performed using a Varian Vista 5500 HPLC coupled with a Varian Auto Sampler model 9090 as described in Example VII..

Analysis of variance was employed to analyse statistically all results, using the program SPSS (SPSS Inc., version 6.1) Specific differences between types of films were determined by least significance difference (LSD). The Student t-test was utilised to test the difference between

irradiated and non-irradiated samples. All comparisons were made at a 5% level of significance.

For each measurements, three replicates of seven films type were tested.

Denatured SPI showed two main molecular weight peaks, in the range of ca. 60 kDa and ca.

2000 kDa (Figure 40a). When gamma-irradiation was combined to heating, further cross-links were generated in both systems investigated, S system and SW system, as suggested by the size exclusion chromatography elution patterns (Figures 40 and 41).

The irradiation of the S system at 32 kGy induced a decrease of the 60 kDa peak and an increase of a peak at ca. 200-2000 kDa (Figure 40b). The shift of the low molecular weight moiety to higher values is interpreted by an enhancement of protein aggregation of the S system due to the formation of bityrosine (i. e. cross-links). It is clear from Figure 40 that the combination of gamma-irradiation to thermal treatment generates a the much more important protein aggregation, since further cross-links are formed: bityrosine in addition to disulfide bonds. Based on the protein calibration curve, the effect of protein aggregation enhanced the molecular weight by more than 15-fold (Figure 40). The integration of the peak area indicated that this increase of the molecular weight affected only 15% of the total soy proteins When SPI was mixed to WPI in a 1: 1 ratio (SW system), the elution pattern exhibited a main peak at ca. 60 kDa, with a shoulder at ca. 2000 kDa (Figure 2a). The irradiation of SW system at 32 kGy increased protein aggregation, as confirmed by the shift of molecular weight from ca. 60 kDa to ca. 200 kDa, and the increase of the intensity of the high molecular weight moiety at 2000 kDa (Figure 41b). The formation of bityrosine, induced by gamma-irradiation is responsible for this shift of molecular weight toward higher values (Figure 41b). However, the protein aggregation in the SW system is not as important as in the S system, the shift being less than 5-fold vs. more than 15-fold, respectively. With respect to other complex systems investigated so far, the molecular weight shift was reported to be more important in caseinate/WPI system (Vachon et al., J. Agric. Food Chem., 39,3202-3209,2000). The higher amount of tyrosine in caseinate (4.0%) might account for this behaviour. Nevertheless, the use of gamma-irradiation has proven to be efficient since it resulted in an enhancement of cross-links in both systems investigated.

Cross-links confer to any material elastomeric properties, if the crosslink density does not exceed a critical value. Indeed, the higher this value is, viz. the higher branched chains are, the more rigid is the material. The effect of the combination of gamma-irradiation to the heating treatment

m the S system and SW system on the mechanical properties of films were also investigated, namely the puncture strength and puncture deformation.

Tables 6 and 7 illustrate a significant increase of the puncture strength as well as a significant increase of the puncture deformation following the irradiation of the different formulations at 32 kGy. These results clearly emphasise that the combination of gamma-irradiation to thermal treatment enhances the formation of cross-links enabling, thus, the formation of a film with improved mechanical properties. The use of gamma-irradiation can increase the mechanical properties of films by the formation of bityrosine between two protein chains. Heating treatment (30 min, 90°C), could also underwent cross-linked via the formation of disulfide and hydrophobic bonds. The cysteine groups present in proteins can undergo polymerization via sulfia'ryl-disulfide interchange reactions during heating to form a continuous covalent network upon cooling.

Table 6: Puncture strength of protein-based edible films

l m m T F'?'nu . stirn ti tm 'im o: r s o:: ao N. n.. rdxned xt rrtdxa . 2. I. S SPI: Gly (2: 1) 31.53 i 2.34 b 43.30 i 2.75b* si SPI: Gly : CMC (20: 10: 1) 48.63 2.67 d 52. 20 2.52d* S2 SPI: GIy : CMC: PVA (20 : 10 : 1: 2) 52.4g i 2.25 e 59.00 i 3. 10d* SW SPI: WPI : Gly (1 : 1 : 1) 28.60 A 2. 40 a 40.32 i 2. a* SW1 SPI: WPI: Gly : CMC (10: 10: 10: 1) 32.79 i 2.83 b 41.27 3. 52ab* SW2 SPI: WPI: Gly : CMC: PVA (10 : 10: 10 : 1: 2) 37.48 i 1. 64 8 46. 07 A 3.56C* S = films based on SPI; SW = films based on mixture of SPI and WPI ; SPI = soy protein isolate; WPI = whey protein isolate; Gly = glycerol; CMC = carboxymethylcellulose ; PVA = polyvinyl alcohol. Means followed by different letters in each column are significantly different (p < 0.05). Means followed by asterisk in each row are significantly different (p < 0.05).

Table 7 : Puncture deformation of protein-based edible films Xz ,,.. : P. 1IICtY'5'. ti II,. 'x m: s S SPI : Gly (2: l) 6. 20 0. 30' 6.31 zt 0. 25C SI SPI: Gly : CMC (20: 10: 1) 4.80 0. 340 6.21 0. 35'* S2 SPI: Gly : CMC: PVA (20: 10: 1 : 2) 5.07 i 0. 35d 5 85 i 0. 26bt SW SPI: WPI: Gly (1: 1: 1) 3.71 0. 3 osa 4.78 Ct 0. 45a* SW1 SPI: WPI: Gly : CMC (10: 10: 10: 1) 4.12 : L 0. 49 b 4.76 0. 44'* SW2 SPI: WPI: Gly : CMC: PVA (10: 10: 10: 1: 2) 4.38 A 0. 21b 4. 83 i 0. 26a* S = films based on SPI; SW = films based on mixture of SPI and WPI; SPI = soy protein isolate; WPI = whey protein isolate; Gly = glycerol; CMC = carboxymethylcellulose ; PVA = polyvinyl alcohol. Means followed by different letters in each column are significantly different (p zu 0.05). Means followed by asterisk in each row are significantly different (p <0. 05).

The enhancement of the mechanical behaviour of the films was found to be strongly related to the formulations. In both systems investigated, the effect of the irradiation on the puncture strength was weaker in presence of glycerol only (S and SW). In the S system a significant improvement (p < 0.05) of ca. 37% was observed, while in the more complex system, SW system, gamma- irradiation increased significantly the puncture strength of ca. 41% (Table 6). The addition of CMC increased significantly the puncture strength of both systems (S1 and SW1) : from 31.5 to 48.6 N/mm in the S system treated thermally, and from 28.6 to 32.8 N/mm in the SW system, following a similar treatment (Table 6). Gamma-irradiation resulted in a significant increase of the puncture strength of ca. 7% and ca. 26% in the S system and SW system respectively (Table 6).

The incorporation of PVA to both systems (S2 and SW2) enhances even more the puncture strength, going from 48.6 to 52.5 N/mm in the heated S system, and from 37. 5 to 46.1 N/mm in the non-irradiated SW system (Table 6). As for the previous formulations, once gamma- irradiation was applied to both systems that contained CMC and PVA (S2 and SW2), a significant improvement of the puncture strength values occurred: ca. 12% in S system vs. ca. 23% in SW system (Table 6).

Between the formulations investigated in both systems, the measured puncture strength values were highest in irradiated S2 and SW2 formulations, viz. in presence of CMC and PVA (Table 6).

The puncture strength values can be related to the amount of cross-links produced, that can be bityrosine and disulphide bridges, during the irradiation process. Consequently, cross-links were significantly more important in the formulations that contained the most excipients, especially PVA, viz. S2 and SW2 (Table 6). As shown in Scheme I, the OH abstract preferably hydrogen

atoms from the a-position to the PVA hydroxyl group, originating 70% of a-bonding and 30% of p-bonding fractions.

Comparing only irradiated formulations in S systems, the presence of CMC (S1) in addition to gamma-irradiation had a more pronounced effect on the puncture strength value with 21% increase of the puncture strength (Table 6). Addition of PVA to this mixture resulted in an additional 13% increase of puncture strength. In the SW system, a more pronounced effect was obtained in the presence of both CMC and PVA (SW2), in addition to gamma-irradiation representing an increase of 12% of puncture strength as compared to 2% in presence of CMC only. Hence, more cross-links were generated in S1 and in SW2 formulations. These results showed that gamma-irradiation is less efficient to increase the puncture strength in formulations containing whey protein isolate than in S formulations. According to GPC results, gamma- irradiation induced less molecular weight changes in presence of whey protein. Heating produces more disulfide linkages while gamma-irradiation produces more bityrosine linkages.

Finally, the puncture strengths of the biofilms were significantly more important in the non- irradiated and irradiated S system, than the SW system (Table 6). Consequently, the generation of cross-links were more important in the S system.

Except for the S formulation, the puncture deformation increased significantly upon gamma- irradiation at 32 kGy (Table 7), indicating that the irradiation treatment generated elastic, i. e. flexible films. In formulations that contained only glycerol, S and SW, gamma-irradiation had a significant impact only on the SW system. Indeed, following the irradiation treatment, a significant improvement of ca. 29% of the puncture deformation was observed in SW (Table 7).

The improvement of the elasticity is further confirmed by the significant decrease of ca. 27% of the relaxation coefficient (Figure 42). The addition of CMC decreased significantly the puncture deformation in the S system, from 6.20 to 4.80 mm in S1 formulation treated thermally (Table 7). In the SW system, the opposite behaviour occurred, viz. a significant increase of the puncture deformation from 3.71 to 4.12 mm upon addition of CMC (Table 7). The addition of CMC did not change significantly the relaxation coefficient in the S system S and S1). However, incorporation of CMC in SW system resulted in significant (p < 0.05) reduction of relaxation coefficient, with lower values in SW1 formulation. This is indicative of more elasticity in SW, as previously reported by Peleg (J. Food Sci., 44 (2), 277,1979). Gamma-irradiation resulted in a significant increase of the puncture deformation of ca. 29% and ca. 16% in the S system and SW system respectively (Table 7). However, gamma-irradiation did not affect significantly the

relaxation coefficient (Figure 7). The incorporation of PVA to both systems (S2 and SW2) enhances the puncture deformation from 4.80 to 5.07 mm in the non-irradiated S2 formulation, and from 4.12 to 4.38 mm in the non-irradiated SW2 formulation (Table 3). A significant decrease of ca. 11% of the relaxation coefficient resulted in S2, while in the SW system the relaxation coefficient did not vary significantly (Figure 42). On the other hand, the puncture deformation in S2 was significantly lower than the puncture deformation in S, 5.07 vs. 6.20 mm (Table 7). The puncture deformation value in SW2 formulation was significantly higher with respect to SW formulation (Table 7).

As noticed for the puncture strength, puncture deformation values were strongly dependent on the formulation of films. The highest measured puncture deformation value in both systems (S and SW) was obtained with the S formulation and the SW2 formulation (Tables 7). The relaxation coefficient of the films generated from the latter formulation are almost comparable: 0.59 vs. 0.55 (Figure 42). Therefore, the presence of excipients in the S system did not influence the elastic behaviour of the biofilms, whereas the addition of CMC and PVA to SW improved the elasticity of the films, as confirmed by the decrease of the relaxation coefficient in SW1 and SW2 with respect to SW (Figure 42).

The contribution of gamma-irradiation was more important in S1 and in SW formulations. In both cases, a significant enhancement of ca. 29% of the puncture deformation was obtained, while lowest contribution of the gamma-irradiation on the puncture deformation values were found for the formulations corresponding to S, S2, SW1 and SW2. These results can be interpreted in terms of bityrosine content, i. e. cross-links or branched chains. It seems that too many cross-links are produced in S, S2, SW1 and SW2, leading to a stiff film, whereas the amount of branched chains produced at in SI and in SW formulations seemed to be just enough to confer viscoelastic properties to the films. The puncture deformation of the biofilms were significantly more important in the non-irradiated and irradiated S system, than the SW system (Table 7). Therefore, the addition of WPI in the formulation tend to provide stiffer films with respect to SPI, as confirmed by the higher value of the relaxation coefficient found in SW vs. S formulations (Figure 42).

The results discussed so far demonstrate that the mechanical behaviour of the biofilms and the effect of the irradiation treatment are strongly sensitive to the formulation. The highest effect of gamma-irradiation on the formation of bityrosine, i. e. cross-links, in the S system occurred in the S formulation, leading to an increase of 37% of the puncture strength value. The gamma- irradiation did not show significant variations on the puncture deformation. It seems that too

many cross-links were produced, leading to a stiff three-dimensional network. The most important effect of the irradiation on the deformation was observed when CMC was added to the formulation S: an increase of 29% was measured after irradiation. These findings can be explained by the fact that lower amount of cross-links were produced in S1 with respect to S, as confirmed by the low effect of gamma-irradiation on the puncture strength values. As a consequence, films behave more similarly as elastomers.

The highest improvement of the mechanical properties following gamma-irradiation in the SW system was observed in the SW formulation: an increase of 41% was noticed between the non- irradiated and irradiated samples. It seems that the cross-links generated with this formulation were not too many, but rather sufficient to confer good elastomeric properties to the film: an increase of 29% of the puncture deformation was measured after the irradiation treatment.

Although the addition of CMC to the formulation, SW1, lowered the amount of cross-links, as inferred from the lower increase of the puncture strength value, viz. 26%, an important increase of 16% of the deformation occurred after gamma-irradiation was applied, which is not insignificant.

The largest increase of puncture deformation (29%) by the irradiation treatment was observed with S1 and SW formulations, suggesting that cross-links produced are near-optimal in these formulations, irradiated at 32 kGy. The contribution of CMC to improve the mechanical properties of the films, especially in the S system, confirm the synergy between CMC and gamma-irradiation.

Barrier properties Figure 43 presents the water vapour permeability (WVP) for the formulations investigated, expressed in g. mm/m2. 24h. mmHg. There is no significant impact of the formulation on the WVP in the S system (Figure 43). Values of WVP range between 2.90-3.16 in the non-irradiated S system. The contribution of the irradiation treatment was found to be significant only in S1 formulation, viz. in presence of CMC. Indeed, upon irradiation the WVP of S1 went from 3.16 to 2.03, representing a decrease of ca. 36% (Figure 43). This behaviour could be explain by the increase of protein-protein interactions resulting from the formation of bityrosine, viz. cross- links, which results in a decrease of the diffusivity of the permeant (Krochta et al., 1994). As for the SW system, the sole significant impact coming from the formulation was observed in SW1 formulation. The presence of CMC gave films with a lower WVP with respect to SW and SW2 formulations: 2.68 vs. 3.23-3.28 respectively. Unlike the S system, the irradiation treatment did

not influence the WVP of films obtained in the SW systems. Moreover, the addition of WPI did not bring any value to the barrier properties.

The addition of CMC was effective in both systems investigated. In the S system, its effect was combined with gamma-irradiation, whereas in the SW system the effect was observed in the non-irradiated formulation. Although more cross-links were generated in presence of PVA, this excipient did not bring further down the permeability of the corresponding films with respect to CMC.

The WVP values obtained with our systems are comparable to those reported earlier by Stuchell and Krochta (J. Food. Sci. 1994,59,1332-1337,1994) and by Jo et al. (Food and Biotechnology 1996,5,243-248,1996) for soy protein films. Non-irradiated films showed WVP values in the range of 2.9-3.2 for the S system and 2.7-3.3 for the SW system, whereas the WVP for irradiated films were in the range of 2.0-2.75 for the S system and 2.65-2.95 for the SW system. The addition of WPI did not have a significant impact on the barrier properties of the biofilms.

Nonetheless, it is worth to underline that the barrier properties for the SW system, viz. a 1: 1 mixture SPI/WPI, seem to be more efficient than those reported for SPI/PEO system : 2.65-3.3 vs. 34.6-46.1 respectively (Ghorpade etal., Cereal Chemist, 1995,72,559-563,1995).

Gamma irradiation treatment generated cross-links, increasing the molecular weight of soy protein. This investigation has clearly demonstrated the usefulness of-irradiation to improve mechanical properties of films based on SPI (S system) and on mixture of SPI with WPI (SW system). Both mechanical properties, punctures strength and puncture deformation, increased with the irradiation treatment. Films based on SPI (S system) presented the highest puncture strength and puncture deformation values than SW system. It must be emphasized that either for films based on SPI (S system) as based on SPI with WPI (SW system), there was increasing trend of puncture strength values as CMC and PVA was added. CMC increased significantly the puncture strength in both systems (S and SW). Puncture strength had its higher value with PVA in the S system. PVA was the more efficient compound to increase the puncture strength in S and SW systems while in relation to the viscoelasticity, it reduced significantly this property, contributing to a low relaxation coefficient in both systems (S and SW). The contribution of CMC in viscoelasticity was favorable only in the SW system, when it decreased significantly the relaxation coefficient. The water vapor permeability values for irradiated films were smaller than unirradiated ones, except for one case of SW system. The addition of PVA showed no contribution on permeability while CMC presented some improvements but not a trend. In S

system, CMC reduced significantly the WVP value when combined with 7-irradiation and in SW system, CMC brought a significant decrease for unirradiated films.

EXAMPLE XII: Combined Effect of Antimicrobial Coating and Gamma Irradiation on Shelf Life Extension of Pre-Cooked Shrimp (Penaeus Spp.) Soy protein isolate (SPI) containing 90 % protein (moisture free basis) was purchased from Dupont Campbell Protein Technologies (St-Louis, MO, USA). Whey protein isolate (WPI) containing 87 % (wt/wt) was produced by ultrafiltration and diafiltration at the Food Research and Development Centre (St-Hyacinthe, Qc, Canada) and transported to the Canadian Irradiation Center (Laval, Qc, Canada) under refrigerated conditions (4 2 °C). The WPI solution was lyophilized (Model 12 Research freeze dryer, The Virtis Company Gardiner, New-York, USA) and dried at 100 °C for 3 h in a model 019 vacuum oven (Precision Scientific Inc., Chicago, IL, USA) prior to incorporation in the film-forming solution. The total protein concentration in the lyophilized WPI and SPI powder were determined using a Leco FP-428 combustion oven apparatus (Leco Corporation, St-Joseph, MI, USA). SPI and WPI were mixed in a ratio of 1/1 (wt/wt) in distilled water containing 0.5 % (wt/wt) of polyvinyl alcohol (PVA) (Sigma Chemical, St-Louis, MO, USA). The total protein concentration in the solution was 5 % (wt/wt, dry weight basis). The pH of the mixture was adjusted to 8.5 with 1M Na2CO3. Glycerol and low viscosity carboxymethyl cellulose were added at the concentration of 2.5 % and 0.25 % (wt/wt), respectively and the solutions were sterilized by autoclaving (120 °C for 15 min). Antimicrobial coating solutions were obtained by incorporating trans-cinnamaldehyde (Sigma Chemicals, St- Louis, MO, USA) or thyme oil from thymus saturoides (Robert & Fils, Montreal, Qc, Canada).

Three formulations of coating solution were prepared: i) Base solution containing SPI, WPI, PVA, and Glycerol, ii) EO-0.9 containing de Base solution plus L-alpha-Phosphatidylcholine (20 %, wt/wt, Sigma Chemicals, St-Louis, MO, USA) (0.5%), thyme oil (0.75 %), and trans- cinnamaldehyde (0.15%), iii) EO-1.8 containing the base solution plus L-alpha- Phosphatidylcholine (0.5%), thyme oil (1.50 %), and trans-cinnamaldehyde (0.30%).

Shrimp samples Pre-cooked frozen peeled shrimp (Penaeus spp.) samples were purchased at a local grocery store (IGA, Laval, Qc, Canada) and transported to the Canadian Irradiation Center in a thermal container. Upon arrival (within 20 min of purchase), samples were defrosted overnight at 4 1 °C prior to application of the coating solutions.

Treatznent of shrmp Shrimp samples were randomly assigned into four treatment lots consisting of 1 control lot (uncoated) and 3 lots treated with the following coating solutions: base coating and base coating + essential oils, final concentration of 0.9 % (vol/wt) (EO09) or 1.8 % (vol/wt) (EO18). For each coated lot, approximately 200 shrimp (140 5g) were immersed for 5 min in 500 ml of the coating solution with gentle swirling using a sterile glass rod to ensure complete contact of the shrimp with the coating solution. Shrimp were removed and allowed to drain for 5 min on a pre- sterilized metal net under a biological containment hood. After draining of the excess coating solution, samples were placed into sterile Petri plates (8.1 cm i. d.) (approximately 15 shrimp/plate). Plates containing either uncoated and coated shrimp were divided into 2 groups.

One group was irradiated at the Canadian Irradiation Center (Laval, Qc, Canada) at a total dose of 3 kGy and at a dose rate of 31.24 kGy/h, using a 60Co source UC-15A (MDS-Nordion International Inc., Kanata, ON, Canada). Amber perspex 3042s (Atomic Energy Research Establishment, Harwell, OXF, UK) to validate the dose distribution throughout the samples. The irradiator was also certified by the National Institute of Standards and Technology and the (Gaithersburg, MD, USA) and the dose rate was established using a correction for decay of source. The second group served as an unirradiated control. All the plates were stored at 4 °C and duplicate samples were taken at 1,3,6,9,14, and 21 days for aerobic plate count (APC) determination. Day 1 corresponded to the day of irradiation.

In a separate experiment, the effect of gamma-irradiation and coating was evaluated on shrimp artificially contaminated with Pseudomonas putida isolated from refrigerated beef at the Food Research and Development Center (St-Hyacinthe, Qc, Canada). Samples were prepared following the procedure described above, but shrimp were first dipped in BHI broth containing approximately 105 colony forming units (CFU)/ml of P. putida. The mean level of contamination obtained at day 1 was approximately 2 loglo bacterial cells/g of shrimp before irradiation.

Microbial analysis Each shrimp sample was weighed (ca. 10 2 g) and homogenized for 2 min in 90 ml of sterile peptone water (0.1 %) using a Lab-blender 400 stomacher (Laboratory Equipment, London, UK).

From this mixture, serial dilutions were prepared and appropriate ones were spread-plated on sterile petri plates containing Plate Count Agar (Difco Laboratories, Detroit, MI, USA) and incubated at 35 1 °C for 24 h for the enumeration of total APCs. The enumeration of P. putida was done on brain infusion agar (BHA, Difco Laboratories, Detroit, MI, USA) following the

same procedure. Experiments were done in duplicate and 3 samples were analyzed at each sampling time. The limit of acceptability was calculated based on the onset of shrimp spoilage which was considered to be 10'to 10'bacteria/g (Ayres, J. Appl. Bacteriol. 23,471-486,1960).

Sensorial evaluation The sensorial evaluation was performed only on uninoculated samples. In order to minimize variations of the organoleptic properties due to difference in microbial growth, all the treatments were evaluated after 3 days of storage. The sensory testing was done at the Canadian Irradiation Center (CIC). The sensory lab was equipped with individual partitioned booths and sensorial analysis were performed by 11 trained panelist (students and employees of INRS-Institut Armand-Frappier, Laval, Qc, Canada), using a nine-point hedonic scale ranging from 1 (most disliked) to 9 (most liked) (Larmond, Laboratory methods for sensory evaluation of foods.

Research Branch of Agriculture Canada publ. 1637. Ottawa, Ontario, 1977). Odor and taste were evaluated under a red light to mask any difference of color. A second nine-point hedonic scale test was carried out under a normal light to evaluate de degree of acceptability based on appearance. Samples were heated at 50°C in a water bath, and presented with unsalted Premium crackers and drinking water on a polystyrene tray. Four different samples were simultaneously presented in bowls coded with a three digit random number. The samples were presented in a randomized complete block design. Consumers were asked to eat a bite of cracker and rinse palate with water between samples to minimize any residual effect, and to evaluate the samples from the left to the right.

Statistical analysis Data were subjected to an analysis of main effects and interaction effects of type of coating and irradiation using the ANOVA procedure of SPSS (SPSS Inc. Chicago, IL, USA). The least square significant difference (LSD) test was used at each sampling time for point-by-point determination of the influence of coating. Difference between unirradiated and irradiated samples was determined using the Student t-test. Differences between means were considered significant when p : 0. 05.

Aerobic plate counts Counts of bacterial population in unirradiated samples are shown in Figure 44. In both control (uncoated) samples and samples coated with various solutions, APCs increased significantly (p <

0.05) during the 21 days of storage. No significant difference (p > 0.05) was found between uncoated samples and samples coated with the base solution. In contrast, when essential oils were incorporated in the base solution, bacterial count decreased significantly (p < 0. 05) compared to uncoated controls. The antibacterial effectiveness of the coating solution containing 0.9 % essential oils (EO09) was significantly higher than uncoated samples until day 9, but the difference tended to disappear after 9 days of storage. At day 14 and 21, no significant difference (p > 0.05) was observed between E009 solution and uncoated samples. Bacterial counts in samples coated with E018 solution remained significantly (p < 0.05) lower than bacterial counts in uncoated samples at day 21. The patterns of bacterial growth in irradiated samples were quite different from those observed in unirradiated samples (Figure 45). The irradiation process resulted in a significant (p < 0.05) increase of lag periods before initiation of bacterial growth.

For both uncoated and coated samples no viable colony forming unit was detected during the first 7 days of storage.

In general, combination of gamma irradiation with coating resulted in a more inhibitory effect against bacterial growth. In irradiated samples, regardless of the type of coating, total APCs in coated samples were significantly (p < 0.05) lower than uncoated control samples. Based on the onset of shrimp spoilage established at 107 bacteria per g, the shelf-life periods of unirradiated and irradiated shrimp were estimated (Figure 46). Data indicated that without irradiation, limit of acceptability was reached after 7 days for uncoated, 8 days for samples coated with the base solution, and 12 days for samples coated with E009 and E018. With gamma irradiation the shelf life was 12 days for uncoated samples, 17 days for samples coated with the base coating solution, 20 days for samples coated with E009, and more than 21 days for samples coated with EO18.

Growth of Pseudomonasputida Data related to the growth of P. putida in unirradiated and irradiated shrimp are illustrated in Figure 47. Bacterial growth in unirradiated shrimp increased significantly to reach maximum values of 10.76 to 12.24 CFU/g after 21 days. Although total counts of P. putida were lower in E009 and E018 treatment, no significant (p > 0.05) effect of coating was found during the first 7 days of storage. At the end of the experimental period (21 days) only the EO18 coating solution showed a significant (p < 0.05) reduction of the growth of P. putida. When shrimp were subjected to gamma irradiation, complete inhibition of P. putida occured during the first 3 days for all the samples. The initiation of bacterial growth was observed after 3 days for control, base and E009, and after 7 days for EO18. Total APCs for both control and coated samples remained significantly lower (p c 0.05) in irradiated samples compared to unirradiated ones during the

entire storage period (21 days). Bacterial counts in samples coated with E018 solutions were significantly lower (p < 0.05) than all the other samples during all the experimental period (21 days). No significant (p > 0.05) antibacterial effect was observed for the base and E009 solutions.

Sensorial evaluation Table 8 shows the results of variance analysis relative to sensorial evaluation of shrimp. None of the sensorial parameters (appearance, odor and taste) was significantly affected by gamma irradiation (p > 0.05). Coating did not affect the appearance of shrimps, but reduced significantly (p zu 0.05) acceptability for odor and taste. There was no significant combined effect of gamma irradiation and coating on appearance, odor, or taste.

Table 8: Summarized results of variance analysis showing main effects and interaction effect of gamma irradiation and coating on the sensorial characteristics of shrimp after 3 days of storage. p (Fcritical > Fcalculated D ppearance or aste Irradiation 1 0. 851 0. 099 0.8 Coating 3 0.975 0.001 0.001 Irradiation x Coating 3 0.972 0.416 0.865 'DF : Degree of freedom Level of significance of the F test. Probability that the critical F value is greater than or equal to the calculated value of F.

Results of comparison of means for significant differences between types of coatings for unirradiated and irradiated samples are summarized in Table 9. Appearance of shrimp was not significantly (p > 0.05) affected by coating. The mean values on the hedonic scale ranged from 6.40 to 6.70 for unirradiated samples to 6.45 to 6.73 for irradiated ones. For odor and taste, no significant difference (p > 0.05) was observed between uncoated control samples and samples coated with the base solution, or with E009 solution (0.9 % essential oils). When essential oils were added to the base solution at a level of 1.8 % (EO18), odor and taste acceptability of shrimp decreased significantly (p < 0.05). In unirradiated samples, acceptability values for odor decreased from 6.89 for the base solution to 6.25 for E009 and 4.86 for E018. For taste, values were 6.78 for the base solution, 4.56 for E009 solution, and 4.17 for E018 solution. In both

cases (odor and taste), the acceptability values were significantly lower only for the coating solution containing 1.8 % (v/w) essential oils. A similar significant decrease of acceptability values was also observed in irradiated samples. <BR> <BR> <BR> <BR> <BR> <BR> <P> Table 9: Effect of coating and gamma-irradiation on the organoleptic properties of shrimp after 3<BR> <BR> <BR> days of storage' SENSORIAL PARAMETERS Appearance ~ Odor ~ Taste Unirradiated Unirradiated Unirradiate Irradiated Irradiated Irradiated Control 6.56~2.30a 6.45~1.37a 7.20~1.93a 7.22~97a 7.30~1.34a 7.70~1.49a Base 6.40~2.07a 6.55~1.57a 6.89~1.45a 6.55~1.75ab 6.78~1.20ab 6.82~1.99a EO09 6.70~2.16a 6.73~1.49a 6.25~1.49ab 4.50~1.66ab 4.56~1.46ab 5.00~2.24ab E018 6.402. 22a 6.641.57a 4. 86~1.86b 4.141.57b 4.171. 67b 4.381.92 'Means within a column bearing the same letter are not significantly different (p > 0.05) as determine by the Least Significant Difference test.

2No significant difference (p > 0.05) was found between irradiated and unirradiated samples as determined by the Student t-test.

This invention demonstrates that edible active food packaging films or coatings can be developed by incorporating natural compounds with antimicrobial properties against spoilage bacteria.

Protein-based coating containing trans-cinnamaldehyde and thyme oil were found to reduce bacterial growth on pre-cooked shrimp. The resulting films demonstrated significant antibacterial effect against Lactobacillus plantarum. It also appears that without irradiation, the inhibitory effect of the coating applied on pre-cooked peeled shrimp was closely related to the concentration of essential oils added to the solutions. With irradiation the inhibitory effect was greatly improved due to an additive interaction effect.

The present invention demonstrates a significant additive interaction effect of gamma irradiation and antimicrobial coating in reducing the growth of bacterial in pre-cooked peeled shrimp. This

effect was characterized by longer lag period, lower growth rates, and therefore significant shelf life extension in irradiated samples.

A significant additive interaction effect of gamma irradiation and coating was also observed with the base solution (without essential oils). A combination of 3 kGy irradiation of shrimp and coating with the base solution, resulted in a 5 day extension of shelf life as compared to irradiation alone, and an additional 9-day extension of shelf life as compared to coating alone. Coating acts as an additional parameter by increasing microbial susceptibility through modifications of some environmental factors such as oxygen availability at the surface of products (Cuq et al. In. Roony M. L. (Ed), Active Food Packaging. Blackie Academic & Professional. Bishopbriggs. Glasgow. pp. 111-142,1995).

From the present study, changes in appearance, odor and taste as affected by gamma irradiation was not detectable by the sensorial evaluation panelist. These results agreed with the reports of GirouxandLacroix, FoodRes. Int. 31,337-350,1998; and Kanatt et al.,. J Food Sci. 63,198- 200,1998, who found that low dose irradiation can be use to extend the shelf life of food products, without any detrimental effects on biochemical and nutritional characteristics. The irradiation dose used in the present study (3 kGy) was not high enough to induce production of unacceptable odors or flavors from lipid and protein components of shrimp. The lower scores obtained in sensorial evaluation tests for odor and taste can be related to the intrinsic sensorial characteristics of thyme oil and trans-cinnamaldehyde.

The present invention dealt with the control of bacterial growth of pre-cooked shrimp using gamma irradiation combined with edible antimicrobial coatings. This technology showed significant potential for inhibiting total aerobic counts and Pseudomonas putida, and, as a result, the microbial shelf life was extended by 5 days with gamma irradiation, and more than 11 days with gamma irradiation combined with a protein-based coating containing thyme oil and trans- cinnamaldehyde. A synergistic effect was also observed between irradiation and coating with the base solution (without essential oils). The appearance of shrimp was not affected by the treatment as well as odor and taste for essential oil concentrations of up to 0.9 %. However, incorporation of 1.8 % essentials oils in the coating solutions significantly (p zu 0.05) decreased the acceptability of the products.

EXAMPLE XIII: Biocompatible Polymers as Supports to Immobilize Bioactive Agents with Biomedical and Biotherapeutic Applications This example comprises two main parts: the first one consists in elaborating a natural origin polymer-based biocompatible matrix having the necessary characteristics to protect bioactive agents for biomedical and bioalimentary applications. The second part deals with the characterization of the newly created matrixes.

Chitosane and alginate were used as a matrix to protect the bioactive agents from denaturing factors of the external environment, while the milk proteins are used as support to immobilize or stabilize them in the matrix. These polymers generally have filmogenic characteristics, however they are not resistant to or sensitive to water, therefore they need modifications to acquire some desired characteristics (hydrophobic, acid-proof and satisfactory mechanical characteristics).

The modifications are essentially based on the coupling with a functionalisation agent (using acylation agents) or reticulation agents (bifunctional reticulating agents).

Chitosane is a polymer of animal origin obtained after partial deacetylation of chitin. The basic unit chitosane are essentially of N-glucosamine. It is at the carbon 2 level, where the amine group (NH2) is present that the coupling with fatty acids occurs (Oyrton and Claudio, Int. J Biol Macromol, 26,119-128,1999). The reticulation is also possible using a bifunctional agent such as dialdehydes allowing the formation of intermolecular bridges between the two chitosane chains. Following the Fourier transformed infrared (FTIR) analyses, the probable mechanism shows that an acylation first occurs (coupling with fatty acids). An increase of the band in the 1700 cm''spectral region appears after modification for the elongation vibration of the C=O groups. The same phenomenon is observed for the band at 2980 cm'', which might be due to the presence of C-H groups (presence of acyl chains from the fatty acids). Secondly, the formation of the imine bond (C=N) of the amine groups from chitosane and of carbonyl from dialdehyde is typical in the 1700 cm~ spectral region. The FTIR spectrums and the probable structures of chitosane obtained are illustrated in Figure 48.

Alginate is a polysaccharide produce by the Phaeophyceae algae. It is formed from the association of 2 two-acid based chains: ß-D-manuronic and a-L-guluronic (Haug, Rept. N° 30, Norwegian Institute Seaweed Research, Trondheim, Norway, 1964). Two ways of modifying alginate are presented:

Acylation is done directly after the deprotonation of the hydroxyl groups from alginate with a strong base. The spectrum interpretation of the polymer shows the same characteristic of the chitin, namely a typical band in the vicinity of 1700 cm'\ The acylation and/or the reticulation are done after coupling with ethyl amine. The process of modification and reticulation are the same as with chitosane. The spectrums (Figure 49) show the presence of new functional groups (C=N) and (C=O) in the modified alginate structure.

The mechanical properties of chitosane-based films are good. The rupture force (FR) is approximately 550N/Ilm, but no elasticity was noticed. The addition of fatty acids (functionalisation agents) allows not only to improve the hydrophobicity but also the elasticity of the films. Due to their long hydrophobic hydrocarbonized chains, the fatty acids can be inserted between the two chitosane macromolecular chains by diminishing the intermolecular hydrogen interactions and by bringing more flexibility. The viscoelasticity coefficient is approximately 68%. Although the FR is largely diminished during the acylation (from 550 to 150 N/m), this biomembrane is rigid enough to be used as wrapper.

Similarly for the alginate-based films with an initial FR of 450 N/, um and an after-acylation of N/um. This decrease is not as noticeable as the one for chitosane films. No increase of elasticity was noticed. It must be due, besides the hydrogen bonds that are largely broken by fatty acids, to ionic interactions of the carboxyl groups from the alginate (at the C6 level). The later also explains why there is no significant difference in regards of the viscoelasticity coefficient which is 44%.

Passive forms of chitosane and alginate have relatively important antioxydant properties (Dumoulin M.-J., et al., Arzneim.-Forsch. lDrug Res., 46, 855-861, 1996). Their capacity to trap free radicals is between 55 and 65%. After a polymer modification, their antioxidant power is slighty diminished (5-10%). However, by the incorporation of milk proteins in the formula, an increase was noticed and the values were raised to 70 to 80%.

In their native state, the chitosane and alginate based films are highly sensitive to water and the recovery rate (TR) is 0% (Gontard et al., J. Food Sci., 57 (1), 190-199,1992). During the coupling with fatty acids and/or the reticulation by dialdehydes, the polymers are more water resistant and the TR vary between 71 and 80%.

Based on the physical and chemical properties of the polymers, the spheres structures, according to our concept, is a combination of several components, in order to better protect bioactive agents (Figure 50A). The modified alginate is anticipated on the exterior of the sphere to act as the envelope because of its resistance in an acid environment. Chitosane, also modified and/or reticulated, is in the middle with the milk proteins and the bioactive agents. That polymer precipitates and easily turn into a neutral pH gel, including as such the proteins and bioactive agents in the matrix. Its role is essentially to support and slow down the bioactive agents degradation (enzymes for example) by intestinal proteases. The addition of milk proteins reveals several advantages, particularly the f act they are an excellent nutritional source for the growth of milk bacteria (case of probiotics immobilization). Also, they are rich in calcium which is the coupling site of the alginate envelope by the ionotropic interactions (Figure 50B). It is important to note that it is impossible to get those modified alginate spheres, alone or linked to milk proteins.

To evaluate the matrix's efficiency in the gastro-intestinal system, the study focussed on the immobilization of Lactobacillus plantarum given its sensitivity in pH< 3,0. The preliminary results showed a growth of the bacteria after 30 minutes in the gastric phase (pH=1, 5 in the presence of pepsin) and after 24 hours in the intestinal phase (pH=7,0 in the presence of pancreatic). A quantitative (bacteria count) is presently going on.

Catalase (EC 1. 11. 1. 6) is made of 4 sub-units with a mass of 25 kDa. Each sub-unit has an Fie'li ion located in an heminic nucleus identical to the one found in haemoglobin (protoporphyrin IX).

The trial consists in immobilizing this enzyme in sphere form and to evaluate the efficiency of the matrix via the catalasic activity. The latter is done by recording the disappearance of hydrogen peroxide using its absorbency at 240nm: The results showed that the immobilized catalase activity diminishes by about 50% in comparison with the free enzyme. The loss must be due to a transfer phenomenon, by the diffusion of the substrate from the external environment to the enzyme, then from the enzyme product to the external environment. Although the enzyme is protected in the modified or reticulated matrix from the gastric and intestinal degradations, the stearic encumberment problems are added. Consequently, the matrix's efficiency implies a polymer porosity large enough for the diffusion phenomenon of the substrate and the product through the semi- permeable membrane to take place. On the other hand, the results showed that the immobilized catalasic activity in the modified matrixes is greater than in the non-modified ones (40%). In the

case of the non-modified matrix, the loss of activity must be due to the catalase degradation in the gastric or intestinal phase therefore either by the acidity or by the proteases.

The use of modified polymers as matrixes for controlled release offers several interesting aspects. In effect, the 500 mg modified chitosane-based tablets (coupling with fatty acids) or modified and reticulated chitosane-based (coupling with fatty acids and reticulation by a dialdehyde) containing 100 mg of acetaminophen, were tested with a dissolution apparatus (Distek). The results show a very slow release of the medication for a period of 160 hours (approximately 7 days, Figure 51) and no differences were noticed between the two matrixes.

Consequently, this formulation does not show advantages for oral applications. However, its use could be very interesting in the case of implants or transdermic"timbres". It is important to notice that, for applicable oral formulations, the addition of one or more hydrophilic substances such as agar, carrageenan, carboxymethyl cellulose, etc. is possible. In that case, the release time depends on the concentration of the added compounds.

For the alginate-based tablets, only the modified then reticulated polymer shows interesting results (see Figure 52). The release of the medication is complete after 18 hours comparing with the release of native (1 hour) or modified (8 hours) alginate.

The use of natural polymers as support to immobilise bioactive agents reveals several interesting aspects. These polymers can be offered in different forms (spheres, films or tablets) depending on the application in the medical or alimentary field. In general, in their native state, they do not answer specific needs. Consequently, some modifications of these polymers are necessary for them to acquire the desired characteristics, in particular the resistance to water, acid or proteolytic degradation.

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