FI118506B - Protein solution for use in forming protein film for e.g. food products, contains modified protein, which is modified by cleaving disulfide bond(s) originally present in the protein by sulfitolysis to obtain free sulfhydryl groups - Google Patents

Abstract

A protein solution for use in protein film formation, has a pH of = 7 and contains modified protein, which is modified by cleaving at least one disulfide bond originally present in the protein by sulfitolysis to obtain free sulfhydryl groups. These free sulfhydryl groups are able to cause an interchange reaction to form disulfide bonds between the proteins. Independent claims are also included for: (1) a protein-based film comprising a protein network formed by disulfide bonds between the proteins, where the protein network is formed by treating protein with modified protein in a solution having a pH of = 7; (2) a food product being coated with or containing substances coated with the above film; (3) baby's milk formula containing the above film as emulsion; (4) pharmaceutical product containing at least one therapeutically active agent and being coated with the above film; (5) a container coated with the above film; and (6) method for preparing a protein-based film comprising providing the inventive protein solution, and forming the solution into the protein-based film.

Description

1 8506

A method of making film coatings and film coating

The invention relates to a process for the production of proteinaceous coating films, microcapsules and the like, and to encapsulation of solids.

The invention also relates to proteinaceous coating films.

BACKGROUND OF THE INVENTION

Use of whey proteins as film - forming agents

In recent decades, there has been increasing interest in the use of protein-rich membranes for the protection of food and nutritional products. These membranes are intended to be edible and are digested in the human digestive tract and are biodegradable in nature. Such films can significantly reduce the use of synthetic non-biodegradable packaging materials.

The first edible protein films were made from plant-derived proteins. The purpose of these membranes was to increase the shelf life of the product by reducing water evaporation (drying), preventing oxygen from entering the product, and preventing microbial contamination. Wheat isolated gluten and maize isolated zein were the most commonly used proteins for this purpose. The membranes were prepared with solvent: V: Rack proteins in ethanol and glycerol as the plasticizer. The mixture was heated to 75-77 ° C and immediately before casting the films, the mixture was allowed to cool. Something- • · · • .1. the films were then allowed to dry at 35 ° C for at least 15 hours, after which they were removed from the molds. Films made from wheat gluten and maize zein effectively inhibited oxygen and carbon dioxide permeability but were sensitive to moisture permeability, a property that was dependent on the relative humidity of the environment (Aydt et al. 1991, Gennadois et al.) . 1993).

* · · * ··· 30 The high water permeability was reduced by adding various lipids or fat soluble compounds to the membranes. The best results were obtained with diacetyl tartaric acid esters of monoglycerides, since the use of these compounds increased the mechanical strength of the films and the transparency of the films. (Gontard et al. 1994).

According to U.S. Patent No. 4,720,390, whey protein gels in the form of 4% to 12% (w / w) solution in food, and from 2.5% to 40% fat can be added to this solution: (volume / volume). The amount of protein required to form a gel is reduced to a certain extent by increasing the amount of fat / oil. Warming the protein 90 118506 to 2 ° C for at least 30 minutes is a prerequisite for successful gel formation in a neutral solution. Sugars such as dextrose, lactose and sucrose as well as spices, salt and preservatives may be added.

Gelation and the uniform composition of the gel are highly dependent on the concentration of whey proteins and the heat treatment (e.g. temperature and time). The SH / SS exchange reaction results in the formation of disulfide bonds (SS). These covalent bonds are the most important binding forces that influence gel strength (Shimada and Cheftel 1988).

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According to another US patent 5,543,164, protein membranes can be prepared from a 10% heparin solution by heating the solution to 90 ° C for 30 minutes. After heating, the solution is cooled to room temperature and the air bubbles removed in vacuo. The same treatment can also be done before heating. After cooling, a plasticizer such as glycerol, sorbitol or polyethylene glycol (2-10% by weight of the solution) may be added. In addition, fats / oils or fat-soluble compounds can be added in an amount of 2-15% (w / w) by heating the fat until it is liquid and homogenizing it into an emulsion. The primary function of fats is to prevent the passage of water, oxygen, carbon dioxide, fats, and odors and flavors. Ho-20 mogenation also increases the mechanical strength of the films.

: Y: The protein solution can be poured or (molded) into molds and a film of a certain strength can be obtained by: *** drying the solution by an appropriate method. Drying time • · ·: usually about 18 hours at room temperature. Upon drying, the liquid forms a film that is insoluble in water and any free SH groups are oxidized to SS groups / bonds. Oxidation can be promoted by the use of oxygen or an oxidizing compound.

• · • · ·

By appropriate methods, the protein solution can be applied to the surface of the food and, after drying, a uniform film is formed after drying as described in WO9319615. Film formation can be promoted as described above. 1 · The main limitation to the production of edible membranes from native plant proteins is their almost total insolubility in water. However, whey proteins are also highly soluble in water, but the main limitation in the use of whey proteins is the preparation of a film-forming solution. It is known in the art that to obtain good quality films it is essential to heat the solution to 90 ° C for 30 minutes.

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Upon heating, the solution forms disulfide bonds, which are considered important film-binding forces, and the addition of sulfhydryl (SH-) groups accelerates film formation. The use of chemical substances such as mercaptoethanol, cysteine, dithiothole-5-ritol or sulphite is not possible in food, or is restricted to a certain amount, or their methods and processes are unknown.

Conversion of whey proteins by heating results in the formation of lysinoalanine in a neutral or basic environment. In addition, the nutritional value of the protein 10 decreases and lysinoalanine can cause adverse side effects. Heating proteins with sugars (e.g., aldehyde-containing glucose or lactose) produces chemical compounds formed early in the Maillard reaction. These compounds include the Amador compound, which may cause a reduction in the nutritional value of the protein and the compound formed may be allergenic (Friedman 15 1994). The process described above involves one difficult step of removing the dissolved gases from the solution under vacuum to avoid gas bubbles that can increase membrane moisture or oxygen permeability.

Use of whey proteins in emulsions and microencapsulation

A first description of whey proteins as an emulsifier for fats and fat soluble materials is disclosed in U.S. Patent 4,790,998. The patented process was capable of producing oils which also contained aromatic compounds or were self-contained. weather aromatic (e.g., lemon oil), microcapsules with an average halogen content of 25 pm at 1 pm. These microcapsules were used as artificial turbidity ♦ in acidic drinks.

• · * 9 • 99 9 · * ··· Emulsions were made from the original whey protein concentrate (55% protein content). The solution had a whey protein content of 7.6% by weight, a soybean oil content of 4.5% by weight and the pH was adjusted to 2.2. The solution was heated to 75 ° C for 5 minutes and then homogenized in two steps (4500 psi and 500 psi). After homogenization, the solution was cooled to 20 ° C. The emulsion was used in sour drinks ..., · to obtain a cloudy result. In the manufacture of microcapsules, the emulsion also • ·. spray dried or freeze dried and the resulting solid microcapsules • · ·: 35 were used as dispersible in beverage powders.

U.S. Patent No. 5,601,760 describes the use of a mixture of native whey proteins, whey protein concentrate and isolate, β-lactoglobulin and a mixture of β-lactoglobulin and α-lactalbumin 118506 4 as an emulsifier for fats, oils and other. microencapsulation of fat soluble compounds.

The amount of whey protein and lactose or other carbohydrate (e.g. emulsifier or micro-encapsulating agent) in the solution will generally vary between 10% and 30% by weight. The amount of the microencapsulated substance or mixture may vary between 5% and 95% by weight and the amount of milk fat between 25% and 75% by weight based on the weight of the emulsifier.

Alternatively, it is preferred that the amount of emulsifier is about 10% by weight based on the weight of the solution. The solution may be heated, for example, to 80 ° C for 30 minutes and then emulsified by homogenization.

Depending on the properties of the grease portion, the temperature of the mixture is raised to 60 ° C and the air 15 is removed by vacuum. The emulsion can then be prepared in two steps. In the first step, the fat is dispersed in the solution with a blender and then the mixture is homogenized at 25-80 MPa several times so that the final droplet size is> 1 µm. The emulsion can be spray-dried using a blowing temperature of 160 ° C and an outlet temperature of 80 ° C.

20 Film coating of nuts and seeds would be an interesting way of improving, for example, the appearance, taste, aroma and shelf life of the final product. This type of • 9. * ··. There are very few published studies on these applications in Kiqality. The main reasons for this may be difficulties in the coating process (Mate et al.

25 1996).

• * • · 9 * · ♦ Film Coating for Medicinal Products φ · • «99 9 In drug manufacturing, film coating is a method that can effectively protect the product from chemical and physical stresses in the environment,

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This may result in an unpleasant taste and / or smell of the drug or achieve controlled release of the drug from the preparation, either in time or space. The base of the coating ..... is a film former, which is generally a high molecular weight polymer and. which is soluble or dispersed in the coating medium. · ···: 35 can also be used to modify the properties of the end product and!. **: to improve the coating process, eg. plasticizers, dyes, pigments and anti-caking agents. When the polymer-containing coating fluid is sprayed onto the cores, a film is formed and adheres to the cores immediately upon simultaneous drying.

Over the past 30 years, aqueous coating fluids have displaced coating fluids containing or-5 organic solvents in the pharmaceutical industry due to increasing demands from society for product safety and environmental friendliness. because of the economics of the method. A wide variety of commercially available synthetic cellulose derivatives for water-based film coating of tablets is available today. Hydroxypropylmethylcellulose (HPMC), Hydroxy-10 Cipropylcellulose (HPC) and Sodium Carboxymethylcellulose (NaCMC) are currently quite often used as soluble film polymers to mask the unpleasant taste and / or odor of the active ingredient. Other cellulose derivatives which are insoluble in an acidic medium but which are soluble at pH 5-6 may be used as so-called. enteric coatings (i.e., the release of the active ingredient is targeted to the gut). As an example of enteric polymers having cellulose structure, e.g. cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), and hydroxypropyl methylcellulose acetate succinate (HPMCAS). Ethyl cellulose (EC) is used to control (prolong) the release of the active ingredient from the drug in the gastrointestinal tract by means of a film coating. Due to their chemical nature, the compounds which can be used in aqueous coatings are e.g. acrylates, vinyls and glycols. All of the above coating materials have their own advantages and limitations as well as the • ·. · · \ The process and the characteristics of the end product.

In the future, the number of different peptide and protein structured drugs ... is expected to grow rapidly as they pass through the preclinical phase I research phase • · * and attention has been paid to their compatibility with existing pharmaceuticals. Whey proteins are common byproducts of the dairy and cheese manufacturing industry and have a similar chemical structure to new peptide drugs. Whey proteins are also produced in large quantities worldwide. Lg), α-lactalbumin (α-La), bovine serum albumin (BSA) and certain immunoglobulins (Dybing and Smith 1991), of which β-lactoglobulin is the major component of whey proteins (about 50-60% of the protein). It is a globular molecule (spherical ··· V: 35) with a known secondary structure well (15% α-thread, 50% β-level and 15-20% inverse structure). In the pH ranges of physiological fluids, it occurs as dimers. Each monomer contains 162 amino acids and two intracellular disulfide 118506 6 disions as well as one free cysteine (Wong et al. 1996). Unlike milk gelatine, for example, milk-derived proteins have not been found to have a BSE risk.

The use of native and modified whey proteins as pharmaceutical film coatings and coating processes has so far not been disclosed in the art. The use and properties of native whey proteins as edible membranes in foods and various nutritional products are known (Gennadios et al. 1993, McHugh and Krochta 1994ab, Kim and Morr 1996, Anker et al. 2002). Generally, in these applications, whey proteins are first heated to denature the proteins and to have internal sulfhydryl groups, whereby disulfide bonds can be formed between the molecules, affecting the membrane structure. The aforementioned disulfide bonds as well as molecular interactions between protein chains (hydrogen bonds, hydrophobic interactions and electrostatic forces) lead to the formation of a brittle film.

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Conventional native whey proteins are considered to be good film formers when it is desired to prevent the passage of air through the oxygen and the product at relatively low relative humidity. In addition, the mechanical durability of whey protein membranes is good. However, the water vapor permeability of membranes is questionable due to the hydrophilicity of the aforementioned proteins (Anker et al. 2002). Gennadios and coworkers (1993) investigated the effect of temperature on the oxygen permeability of edible whey protein membranes. McHugh and Krochta (1994ab) investigated the use of glycerol and sorbitol for soft. permeability of the edible protein membranes to oxygen in the air and their mechanical resistance. Oxidation properties and mechanical resistance of the aforementioned films were good and even better than those found with reference films made of synthetic materials. Later, Kim and Morr (1996) reported on the suitability of various food proteins for microencapsulation of substances. the physical and chemical properties of microcapsules made from proteins.

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Free films have proven to be excellent research tools for studying the film forming and coating capabilities and coating properties of new polymers. opportunities. Free films may be prepared by casting or spraying.

. * The latter is generally considered to be more realistic in representing the film in its ultimate operating mode. However, the final film properties should always be investigated when incorporated into drug manufacturing, either in drum coating and / or air suspension coating processes.

118506 7

Literature sources (referred to)

Anker, M .; Bemtsen, J.; Hermanson, A.-M. and Stading, M., Improved water vapor barrier of whey protein Films by addition of an acetylated monoglyceride. Innova-5 five Food Sci. & Emerging Technologies 3, 81-92 (2002)

Aydt, T.P., Weller, C.L. and Testin, R.F. Mechanical and barrier properties of edible com and wheat protein Films. Am. Soc. Agric. Eng. 34,207-211 (1991) 10 Dybing, S. T., and Smith, D. E., Relation of Chemistry and Processing Procedures to Whey Protein Functionality: A Review. Cult. Dairy Prod. J. 57, 377-391 (1991).

Friedman, M. Improvement in Safety of Foods by SH-Containing Amino Acids and Peptides. A review. J. Agric. Food Chem., 42.3-20. (1994) 15

Gennadios A., Weller C.L. and Testin R.F., Temperature Effect on Oxygen Permeability of Edible Protein-Based Films. J. Food Sci., 1993, 58,212-214,219.

Gontard, N., Duchez, C., Cuq, J-L. and Guilbert, S. Edible Composite Films of Wheat 20 Gluten and Lipids: Water Vapor Permeability and Other Physical Properties. Int. J. Food Sci and Technol. 29, 39-50 (1994) • · * · · • ♦ φ, · », Kim Y.D. and Morr C.V., Microencapsulation properties of gum arabic and several · food Proteins: Spray-dried orange oil emulsion particles. J. Agric. Food Chem., 44,: * 5 25 25 1314-1320 (1996) · »» · · **: Mate, J.; Frankel, E.N .; Krochta J.M., Whey protein isolate edible coatings: Effect 4 9 · on the randomisation process of dry roasted peanuts. J. Agric. Food Chem., 44, 1736-1740 (1996) *: · 30 ··· *

McHugh T.H. and Krochta J.M., Milk Protein-Based Edible Films and Coatings. Food Technology, 48, 97-103 (1994).

McHugh T.H. and Krochta J.M., Sorbitol vs. glycerol-plasticized whey protein edi-35 ble Films: Integrated oxygen permeability and tensile property evaluation. J. Agric. Food Chem., 42, 841-845 (1994).

• · 118506 8

Shimada, K. and Cheftel, J.C., Texture characteristics, protein solubility, and sulfhydryl group / disulfide bond contents of heat-induced gels of whey protein isolate. J. Agric. Food Chem. 36: 1018-1025 (1988) 5 Stevenson E.M.; Law, J.R.; Leaver, J. Heat-induced aggregation of whey proteins is enhanced by the addition of thiolated β-casein. J. Agric. Food Chem., 44: 2825-2828 (1995).

Wong, D.W.S., Camirand, W.M. and Pavlath, A.E., Structures and Functionalities of 10 Milk Proteins Crit. Rev. Food Sci Nutr. 36, 807-844 (1996).

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a novel process for the preparation of aqueous protein films without long term heat treatment at high temperatures. These films can be used to coat many types of different materials. It is a further object of the present invention to provide films which can be effectively modified by various treatments or by the addition of adjuvants to modify the properties of the films for certain applications. One additional objective is also to provide functional and health-promoting end products. The proteins used in the process of the present invention are proteins which naturally contain ···. at least one disulfide bond, preferably whey proteins.

It is a further object of the present invention to provide novel films and coatings, capsules, microcapsules and the like, and emulsions for various uses in food technology, in different areas of pharmaceutical manufacturing and agriculture.

* -: Preferably, these membranes and coatings contain activated soluble whey protein (ASWP). In the field of pharmaceutical manufacture, the formation of a water-based ASWP coating and a process with good film * coating capability and producing film coatings with water (WVT) and oxygen overlay.) Are described. headforms are low and have satisfactory mechanical strength properties.

• ·. It is a further object of the present invention to provide aqueous film coatings that can be successfully applied to solid dosage forms: (e.g., granules, pellets and tablets) and food in established industrial coating processes, and that these films are stable during storage. . It is a further object of the present invention to develop novel capsules, primarily ASWP-based, which could replace gelatin capsules for encapsulation of various solid and semi-solid materials.

The present invention is based on the surprising finding that when a protein-containing solution is treated with a modified protein that has been modified by opening at least one of its originally disulfide bond to obtain free sulfhydryl groups, and free sulfhydryl groups induce a disulfide bond formation between the disulfide bonds. According to one embodiment of the present invention, this modified protein is an activated soluble whey protein (ASWP) fraction obtained from a protein isolation process such as that described in FI107116.

In the conversion reaction, disulfide bonds (SS) between the amino acid chains of the proteins are opened and free sulfhydryl groups (SH) are formed. Such a protein is referred to herein as a 'modified protein' or 'activated protein', and both terms may be used alternatively. The conversion reaction can be carried out in many different ways, but most are not applicable to food or pharmaceutical products, i.e., edible products. For example, one such method for increasing the amount of free SH groups is described in Stevenson et al. (J. Agric. 20 Food Chem. 1995, 44: 2825-2828), which produces a synthetic protein containing free SH groups and multiple SS bonds. Thus, according to the present invention, it is practical to use only proteins which initially contain at least one disulfide linkage, i.e., before conversion.

In a preferred embodiment, the protein is converted by treating it with a sulfite ion-forming compound to sulfonate the protein. The primary sulfite ion-forming compounds are soluble food grade sulfites, such as alkali metal or alkaline earth metal sulfides, hydrogen sulfites, or metabisulfites, or combinations thereof. Preferred sulfite is sodium sulfite. Preferably, a separate oxidant or cat-30 lytic is not added. This method of protein sulfonation is described in FI 101514 and FI 107116, in which the conversion reaction is carried out to isolate whey proteins *. by changing their structure. These documents do not describe any specific additional applications or application methods for the modified proteins. In the isolation process, some of the modified protein is precipitated at low pH and some remains soluble. These fractions can be further used in the process of the present invention.

• t * · ♦ • * · • ·

An important factor affecting the degree of protein conversion is the amount of sulfite per amount of protein used. According to current practice, the amount of sulfite in sodium metabisulphite is about 0.01-0.06% (w / v), while the amount of protein in the solution is 10-11% (w / v) at 50-60 ° C and pH 6. -7. Surprisingly, the amount of sulfite required was found to be significantly lower than that described by FI101514 and FI107116.

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The reaction time at which the sulfonation reaction / sulfitolysis occurred was 30 minutes. Subsequently, the pH was adjusted to 2-3 to liberate SC 5 from the sulfonate derivatives of the protein and the residual sulfite. SO2 was blown air out of the reactor and reused as sulfite. Subsequently, the pH was adjusted to 4-6 and the modified protein-10 concentrate was washed with water and ultrafiltered to a desired concentration, e.g., 10-20% protein content.

For the fractionation, the modified whey protein concentrate was microfiltered to separate the fractions, the precipitate fraction and the soluble fraction. Both fractions were washed and concentrated to 10-50% (w / v) required for use by ultrafiltration.

The proteins useful in the method of the present invention are all natural proteins which contain at least one disulfide bond which is opened in the conversion step.

Preferred proteins are whey proteins such as the ASWP described herein.

Whey proteins and their fractions are used in this and subsequent examples as examples to illustrate the present invention. Other types of proteins may also be used provided they can be modified as described herein. One useful type of protein is soy protein, which is widely used in the food industry and contains SS bonds in its natural form.

ASWP can be proposed and proposed as a starting material for film and drug film coating and for encapsulation of solids and semi-solids. This ASWP contains remarkably pure β-lactoglobulin which is activated differently. as before (McHugh and Krochta 1994) and wherein the number of SH groups] has been added without heat treatment. It is evident that this new activated soluble whey protein has many advantages over the formation of protein membranes and the properties of the finished: T: 35 membrane compared to conventional native whey proteins used as an edible membrane material for coating food and nutrients. This protein invention also enables the use of spray technology in the manufacture of films and allows the well-known limitations associated with the use of gelatin as a raw material for encapsulation to be avoided. In addition, the spray-dried ASWP powder can be readily transported to a film coating plant and subsequently dissolved in an aqueous coating solution just prior to film coating. This has major advantages for the pharmaceutical and food industries, for example, in terms of transportation, storage, raw material shelf life and final applicability.

In the present invention, it has been found that aqueous protein membranes can be prepared from modified protein, primarily activated soluble whey protein fraction, by the addition of a plasticizer, e.g., glycerol, sorbitol or polyethylene glycol (PEG) (or mixtures thereof). After preparation, the solution can be applied to a mold and left to dry, for example, overnight in a ventilated room (25 ° C / 40-50% RH). The dried film is then ready to be removed.

Further, it is found that AS WP as a film former can be combined, e.g., with native whey proteins or with a concentrate (75% or more) or other isolate, preferably close protein, in an exchange reaction (Figure 1), thereby modifying the physico-chemical and pharmaceutical properties of the films. As a result of the exchange reaction, the proteins form a three-dimensional network that plays an essential role in the formation of gel-20 and well structures. SH groups prevent the initiation of harmful side reactions and the formation of by-products, such as lysinoalanine and compounds formed * at the beginning of the Maillard reaction (e.g., Amador's compound) (Figure 2).

The number of SH groups does not decrease in the exchange reaction. The number of SH groups can be reduced by oxidizing them with oxygen in the air to form disulfide groups, i.e., 2 x SH + · · · · · · · · ·, · · · · · · · · · · · · S + H 2 O, which strengthens the gel or film structure. Depending on the intended use, it is useful to leave a suitable number of SH groups, because SH-. the groups act as antioxidants, neutralize toxic compounds of plant or microbial origin ··· and inactivate e.g. acrylamide.

• · «« · «» *: ··; In addition, the beneficial effects of SH groups are due to the chelation of metals, where sulfur ligands isolate oxidizing Cu2 + and Fe2 + and possibly toxic As3 + '

^ I ^ I ^ 1 _ ^ I ^ I

·, Cd, Co, Hg, Pb and Se in both inorganic and organic compounds.

• · · 35 • · * · *: SH groups can prevent 1) Amador compound formation, which occurs

At the beginning of the Maillard reaction and 2) the formation of lysinoalanine, which in turn occurs when the protein is treated in an alkaline environment, particularly when heated (Friedman 1994).

The addition of certain excipients can modify the physico-chemical and far-5 mass properties of gels and films. Fat-soluble compounds such as soybean oil and other oils, and emulsifying these compounds into protein structures, can reduce membrane moisture and water vapor permeability and strengthen the protein structure. Addition of carbohydrates such as maltodextrin can slow down the action of proteolytic enzymes and increase the mechanical strength of the protein structure.

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Other types of additives may also be included, for example, to improve film retention. Such additives include anti-adhesive agents such as T1O2, antimicrobial agents such as natamycin (E 235), labeled with E code, and preservatives such as sorbic acid (E 200) and its salts, benzoic acid (210) and its salts, parabens (E 214- 219), lactic acid (E 270) and its salts, propionic acid (E 280) and its salts and similar substances.

The film coatings of the present invention have many applications in the fields of food technology, medicine and agriculture. The properties of the films of this invention can be modified and used (1) as food coatings to protect them from mechanical stress, dehydration, oxidation or harmful external agents, (2) tablets, granules and the like: ** in the form of coatings for solid solid dosage forms, (3) capsule shells for phasic and similar purposes, and (4) as the basic raw material for the manufacture of microcapsules, nano-capsules, emulsions and the like.

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ILLUSTRATIONS

Figure 1. Exchange reaction and exchange conversion.

5 Figure 2. Formation of the Amador compound.

Figure 3. Scanning electron microscope (SEM) image of micro-encapsulated rapeseed oil.

10 Figure 4A-B. Scanning electron microscope (SEM) images of the surface of aged (free) films. The membranes are made from an aqueous solution of activated soluble whey proteins (ASWP) (film: ASWP 3%, glycerol 1%, heat treatment at 70 ° C for 10 min, storage: 1 month, 25 ° C / 60% R.H.). Magnifications A) x500 and B) x10000.

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Figure 5A-D. Scanning electron microscope (SEM) images of the surface of aged (free) films. The membranes are prepared from an aqueous solution of activated soluble whey proteins (ASWP) (membrane: ASWP 4%, glycerol 2%, heat treatment at 70 ° C for 10 min, storage: 1 month, 25 ° C / 60% R.H.). Magnifications at A) x 100, 20 B) x 500, C) x 800 and D) x 1000.

· * * · • · ·

Figure 6A-B. Scanning electron microscope (SEM) images of the surface of aged (free) films. The membranes are made of activated soluble | aqueous solution of whey proteins (ASWP) (membrane: ASWP 3%, glycerol 2%, heat treatment at 70 ° C for 20 min, storage: 1 month, 25 ° C / 60% R.H.). Magnifications A) x500: V and B) x5000.

· · · · · · · ·

Figure 7A-C. Scanning electron microscope (SEM) images of the surface of aged (free) films. The membranes are made from an aqueous solution of activated soluble: "* · 30 Whey Protein (ASWP) (film: ASWP 4%, glycerol 1%, heat treatment at 70 ° C for 20 min, storage: 1 month, 25 ° C / 60% RH Magnifications A) x 100, [B) x 500 and C) x 100.

Figure 8A-C. Scanning electron microscope (SEM) images of the surface of aged (free) films. The membranes are prepared from an aqueous solution of activated soluble whey proteins (ASWP) (membrane: ASWP 3%, glycerol 2%, heat treatment at 80 ° C for 10 min, storage: 1 month, 25 ° C / 60% R.H.). Magnifications A) x 100, B) x 500 and C) x 100.

Figure 9A-B. Scanning electron microscope (SEM) images of the surface of aged (free) films. The membranes are prepared from an aqueous solution of activated soluble whey proteins (ASWP) (film: ASWP 4%, glycerol 1%, heat treatment at 80 ° C for 10 min, storage: 1 month, 25 ° C / 60% R.H.). Magnifications A) x500 and B) x1000.

10 Figure 10A-C. Scanning electron microscope (SEM) images of the surface of aged (free) films. The membranes are prepared from an aqueous solution of activated soluble whey proteins (ASWP) (film: ASWP 3%, glycerol 1%, heat treatment at 80 ° C for 20 min, storage: 1 month, 25 ° C / 60% R.H.). Magnifications A) xlOO, B) x500and C) xlOOO.

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Figure 11A-C. Scanning electron microscope (SEM) images of the surface of aged (free) films. The membranes are prepared from an aqueous solution of activated soluble whey proteins (ASWP) (film: ASWP 4%, glycerol 2%, heat treatment at 80 ° C for 20 min, storage: 1 month, 25 ° C / 60% R.H.). Magnifications A) x100, 20 B) x500andC) x100.

: V: Figure 12. Nuclear power microscope (AFM) images available: ***; On the surface of ASWP films. The average treatment seems to produce smaller droplets,:. as shown in Figure B.

.... ί 25 a · ... Figure 13. Scanning electron microscope (SEM) images of the surface of non-pigmented ASWP films (composition 1 in Table 21). Magnifications A) x500, B) x10000 and C) x675.

30 Figure 14. Scanning electron microscope (SEM) images of the surface of pigmented ASWP films (composition 3 in Table 21). Magnifications A) x500, B) x100O and C) x550.

• *. Figure 15. Scanning electron microscope (SEM) images of maltose: 35 dextrin-containing ASWP membranes (ASWP / P67 7.5%, maltodextrin DE9 5%, glycerol 4%, sorbitol 1%, heat treatment at 70 ° C for 1 h). Magnifications A) x500, B) x100O and C) x5500.

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Figure 16A-D. X-ray diffraction (XRD) images of fresh and aged ASWP membranes without pigment (Compositions 1 and 2 in Table 21). Storage: 0 to 6 months in controlled room conditions (25 ° C / 60% R.H) and in stressed conditions (50 ° C). Film composition 1 stored A) 25 ° C / 60% R.H and B) 50 ° C 5 (top two images); Film composition 2 stored at C) 25 ° C / 60% R.H and D) 50 ° C (bottom two images). The figure shows the intensity on the y-axis and 2-theta (in degrees) on the x-axis.

Figure 17A-D. X-ray diffraction (XRD) images of fresh and aged ASWP membranes containing pigment (Compositions 3 and 4 in Table 21). Storage: 0 to 6 months in controlled room conditions (25 ° C / 60% R.H) and in stressed conditions (50 ° C). Film composition 3 stored A) 25 ° C / 60% R.H and B) 50 ° C (top two images); Film composition 4 stored at C) 25 ° C / 60% R.H and D) 50 ° C (bottom two images). The figure shows the intensity on the y-axis and 2-theta (in degrees) on the x-axis.

• • • • 1 »• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 1 · M 1 • • • • • • • • • DETAILED DESCRIPTION OF THE INVENTION Transparencies and Coatings 16 1 1 8506 DETAILED DESCRIPTION OF THE INVENTION Films and Coatings According to one embodiment of the present invention, the soluble whey protein fraction obtained from the whey protein isolation process (based on FI 107116) is used as a water-soluble film-forming agent for edible films. This protein contains activated pure β-lactoglobulin (greater than 95% by weight on a dry basis) with the addition of SH groups (up to 40 pmol / g) without heat treatment. Protein-10 liner films are prepared from ASWP at concentrations of 3-10% (w / v). Glycerol, sorbitol, polyethylene glycol (PEG) or mixtures thereof 1-6% (w / v) of the total solution may be used as the Peh meter. The pH of the membrane solutions may range from 4.5 to 7.0. The films are prepared without heat treatment but heating (e.g. 70-80 ° C, 10-20 min) improves e.g. mechanical strength of membranes; and pH resistance. ASWP transparencies are clear and almost transparent.

In another embodiment of the present invention, the soluble whey protein as a film-former can be replaced by an activated exchanged protein containing 15-30% soluble fraction and the remainder of the protein containing microfiltrated whey protein 20 concentrate or isolate. The exchange reaction generally requires heating at 70-80 ° C for 10-20 min. The resultant films are almost clear and transparent: V: transparent.

In a further embodiment of the present invention, mechanical strength and e.g.

The stability of pepsin hydrolysis can be enhanced by adding carbohydrates, such as maltodextrins, to the composition of these types of membranes. This addition generally requires · · heating at 70-80 ° C for 10-20 min. The resulting films are • • * * * * * almost clear and transparent.

φ 30 The physico-chemical properties of the protein membranes can be changed by adding excipients. In one embodiment of the present invention, the addition of fat-soluble compounds (e.g., addition of stearates at about 1-2% followed by homogenization at 80 ° C) improves the moisture resistance of the films. Still together with this invention, • ·. In an embodiment of the invention, the addition of a pigment dye, such as titanium oxide, for example, v: 35 0.5-1.5% provides effective protection against UV light and corresponding radiation.

• * • · · · · ·

When the protein solution is made, the temperature and pH of the solution are adjusted to the appropriate level for the intended application. The protein solution may be used either in liquid form or the solution may also be dried to a powder form by spray drying (or the like). These proteins in the form of solid powders offer significant advantages because the powder can be easily stored for later use and reconstituted to a suitable concentration just prior to use for topping or similar processes. Solutions with a protein concentration of 5 to 14% by weight are preferably used to form the films and this solution can also be used for film coating of foodstuffs and pharmaceuticals (e.g. tablets, capsules, granules, pellets and microcapsules). In order to prepare capsule shells, the protein solution must have a higher viscosity and the protein concentration may be 30-10% by weight.

To prepare the membranes, a predetermined amount of the protein solution is gently applied to the mold and allowed to dry at room temperature (21-23 ° C / 40-50% RH) for 18-20 hours. Homogeneous films of a given thickness are obtained.

15

When making edible films on foods, the protein solution can be applied gently by brushing, spreading, spraying or dipping. Film formation can be promoted by simultaneously blowing warm air to dry the film surface. As the free SH groups oxidize to the SS groups, the resulting m.p. ·. ·. 20 is a very solid and mechanically strong film.

For the coating of drugs containing active therapeutic agents (e.g., tablets, capsules, granules, pellets and microcapsules), the protein solution is sprayed. onto the substance (core) using a suitable spraying method and: the V 25 liquid is evaporated simultaneously by heating the coating chamber. Any known pot, drum or air suspension coating technique, or variants thereof, may be used. These techniques are well known in the art. The resultant film coating is homogeneous, solid and mechanically strong.

In one embodiment of the present invention, protein-based capsule shells (which are an alternative to gelatin capsules) are prepared by dipping. rod for protein solution. The protein-coated rod can then be dried in t 35 warm air. Both the top and bottom of the capsule can be made by this method. After filling the capsule, the top and bottom of the capsule are connected and locked. This technique is known in the art as a process for making gelatin-based capsules and can be readily applied to the process of the present invention.

118506 18

Emulsions and Microcapsules A surprising finding of the present invention is that modified proteins such as ASWP based on FI 107116, both modified whey protein and precipitate fraction, can be used in the preparation of emulsions and that, for example, an emulsion prepared from a soluble fraction can be further microencapsulated.

Still another embodiment of the present invention provides a method for emulsifying fats / oils, fat-soluble compounds, and particles with proteins such as ASWP or soluble whey protein fraction. As a result of this method, proteins have free SH groups. AS WP and the whey protein fraction alone or in combination with the original whey proteins or other suitable native proteins form an emulsifying layer of protein around the fat drop. The protein layer of Prote-15 is formed as a result of a three-dimensional network. This network is provided by SH groups which open disulfide (SS) bonds and form new bonds with SH groups released upon heating (e.g., pasteurization). The emulsifying protein layer is generally formed at pH 2-8. This emulsion may be microencapsulated, e.g., by freeze-drying or spray-drying.

20

By emulsifying a suitable emulsifier such as ASWP, it can significantly: V: increase the shelf life of fats, oils, and fat soluble compounds (e.g., aromatic compounds and spices) in foods and aqueous products. Example- · ·:. ·. Therefore, the release of fatty substances and volatile compounds in spices 25 can be controlled.

t · ·· ♦ ♦ · · • ·

In yet another embodiment, microcapsules are prepared by spray drying the emulsions described in this invention. Microcapsules as solids last longer than, for example, emulsions and provide better protection of encapsulated materials against external physicochemical stress. The shielding is dependent on the structure and thickness of the · · # # protein film covering the microcapsules. Microencapsulation is used to protect materials such as oxygen, UV radiation and harmful compounds.

On the other hand, microencapsulation is a useful technique for controlling the rate or site of release of (active) substances.

0 ": 35 • ·:. * ·· Another important embodiment of the present invention is the preparation of a breast milk substitute from the precipitate fraction which acts as an ingredient and emulsifier. The precipitate fraction contains virtually all whey protein α-lactalbumin. It is important because α-118506 19 lactalbumin is the only The precipitate fraction also acts as an emulsifier for oil, such as rapeseed oil, and no other emulsifiers are needed.

5 EXAMPLE 1

Method for Preparation of Activated Soluble Whey Protein (ASWP) Films 10 AS WP (or activated soluble whey protein) membranes were prepared from a fraction obtained from a protein isolation process such as described in patent FI 107116. This AS WP contains activated pure β-lactoglobulin wherein of groups (35-45 pmol / g protein) was obtained without heat treatment.

An aqueous solution was prepared from ASWP containing 4% protein and 2% glycerol by weight. The pH of the solution was adjusted to pH 7.0 using 1 M NaOH solution. The solution was stirred well and carefully poured (20 ml) into petri dishes (85 mm diameter > 20 mm polystyrene) to prepare free films. Free · · · L1 films were allowed to dry horizontally at 22 ° C / RH 45% for at least 22 hours.

After drying, the films were carefully removed. They were transparent and elastic.

* · · • · ··· · · ·

Example 2! ···. 25 • · · »·

The effect of heating on the formation and properties of ASWP films.

Aqueous solutions of ASWP containing 3% and 4% of protein and 1% and 2% glycerol as a plasticizer were prepared. Eight different combinations of these were made, containing four different heat treatments: 70 ° C / 10 min; 70 ° C / 20 .... · 30 min; 80 ° C / 10 min; 80 ° C / 20 min (Table 1).

• · * * ·· * · · • 1 · · • 1 · • ·· • · 20 1 1 8 5 0 6

Table 1. ASWP solution compositions used in heating experiments.

Ingredient / Composition (%) Sample No. 12345678 ASWP 34343434

Glycerol 12 2 12 112

Heating 70 ° C / 10min 70 ° C / 20min 80 ° C / 10min 80 ° C / 20min ASWP solutions were mixed and samples (compositions 1-8) were heated in a water bath. After a predetermined heating time (10 min or 20 min), the samples were cooled to approximately room temperature (20-22 ° C) and carefully pipetted into Teflon molds (6.6 mL each). After drying, transparent and elastic films were obtained. The film adhesion was lower if the heating temperature was kept high and the heating time was longer. The film forming properties are shown in Example 15.

10 EXAMPLE 3 · · · · · ·, · 1 ·. Exchanged Free Membranes' 1 ··· 1: From the original pre-filtered whey protein concentrate and soluble fraction ·····, a 9% aqueous solution was prepared with a mixing ratio of the original whey concentrate to the soluble fraction of 70:30. The solution used glycerol as a plasticizer at 3% and sorghum. bitol 1% and the pH of the solution was adjusted to pH 7.0 (1 M NaOH). The solution was heated for 30 min at 80 ° C, cooled to room temperature (20-22 ° C), and poured into Tef. lon-molds. The films were dried at room temperature at 21 ° C and 45% relative humidity overnight. The resulting films were transparent and stretch-resistant.

• · • · ··· * · 1 «· 1 f • · ·

• M

• · EXAMPLE 4 21 118506

Aqueous ASWP coating solutions Aqueous 5% and 6% protein and a mixture of glycerol 5 (1-3%) and sorbitol (1-3%) were used as a plasticizer in the composition of the aqueous ASWP solutions. The pH of the solution was adjusted to pH 7.0 (1 M NaOH). The assay consisted of a total of 14 different combinations of film former and softener (Tables 2 and 3).

Table 2: Film Coating Tests (Part 1).

10 _

Koe__Koostumus (%) ___ __ASWP__Glyseroli__Sorbitoli Päällystysliuos_ 1. (C) 5__0__0__Esilämmitys_ 2 (J) 5__1__0__Esilämmitys_ 3 (D) 5__0__1__Esilämmitys_ 4 (A) 5__1__1__Esilämmitys_ 5 (E) 5__2__0__Esilämmitys_ 6 (F) 5__0__2__Esilämmitys_ 7 (B) 5__2__2__Esilämmitys_ 8 . (I) 5__3__0__Preheat_year; 9. (K) 5__0__3__Preheating_ 1Q. (G) 5__3__3__Preheating_ • · • ♦ · • «· * ·· · • · · *; Table 3: Coating Tests (Part 2). -.

Experiment Composition (%) ___ __AS WP__Glycerol__Sorbitol Coating Solution_ _L__5__1__1__No Preheat_ "* 2 .__ 5__1__1__Preheat_ _3 .__ § .__ 1,2,1

• · «• * EXAMPLE 5 22 118506

Adding maltodextrin to the ASWP film composition

From the fraction of activated soluble whey proteins (ASWP), a 7.5% ve-5 silica solution was prepared which also contained maltodextrin (degree of hydrolysis 9%) 5% as well as glycerol 4% and sorbitol 1% as plasticizers. The pH of the solution was adjusted to pH 7.0 (1 M NaOH). The solutions were heated in an oven at 70 ° C (A) and 80 ° C (B) for 1 hour and then allowed to cool to room temperature (20-22 ° C), after which the solutions were poured into Teflon molds (free film casting) ). The membranes 10 were dried horizontally at 21 ° C / 45% RH for a total of 48 hours (A) or 24 hours (B). After drying, the films were carefully removed from the mold and were translucent and elastic. The films of treatment A were readily adhered to the mold, which was not observed with the films of treatment B.

EXAMPLE 6 Acid Resistance of ASWP Films The dissolution of ASWP films was studied at pH 2.0 and 6.8. The original pre-filtered whey protein concentrate and ASWP fraction were used in a ratio of 70:30 when a 9% aqueous solution was prepared. The solution also contained 5% maltodextrin (DE9) and 3%

·· .1. glycerol and 1% sorbitol as plasticizers. The pH of the solution was adjusted to pH 7.0 (1 M

NaOH). The solution was heated in an oven for 30 minutes at 85 ° C and allowed to cool to room temperature (20-22 ° C), after which the solution was poured into 1 Teflon. molds (casting of free films) The films were dried horizontally • · · 25 at 21 ° C / 45% RH for a total of 24 hours. After drying, the films were removed! ··: Carefully molded and examined. In 1 M HCl (pH 2) at 37 ° C for 6 to 7 hours, after which they were dissolved, and the membranes also remained unopened initially in 0.1 M HCl (pH 2) at 37 ° C for 4 hours and followed by 0.1 M phosphate citrate buffer solution (pH 6.8) at 37 ° C for 4 hours.

* ·: ··: 30 • 118506 23 EXAMPLE 7 Enzyme Treatment of Membranes

The original pre-filtered whey protein concentrate and ASWP fraction were used in 5: 70:30 ratio when preparing a 9% aqueous solution. The solution also contained 5% maltodextrin (DE9), 3% glycerol and 1% sorbitol as plasticizers. The pH of the solution was adjusted to pH 7.0 (1 M NaOH). The solution was heated in an oven for 30 minutes at 85 ° C and then allowed to cool to room temperature (20-22 ° C), after which the solution was poured into Teflon molds (free film casting). The free films were dried horizontally at 21 ° C / 45% RH for a total of 24 hours. After drying, the films were carefully removed from the mold and examined. The membranes were incubated in pepsin (0.1%) containing medium (0.1 M HCl; pH 2) at 37 ° C until dissolved in 30-45 minutes.

15 EXAMPLE 8

Emulsion and microencapsulation

The original pre-filtered whey protein concentrate and ASWP fraction were used in a ratio of 70:30 to prepare an aqueous 5% solution. Rape oil was added 13

20% (based on the weight of the solution) and the mixture was adjusted to pH 6.5 (1M

: ***: NaOH). The mixture was heated in a water bath to 60 ° C, then homogenized with Ultra Turrax to obtain an emulsion and finally passed through an FT-9 homogenizer three times. The emulsion was pasteurized at 75-78 ° C for 5 minutes and cooled to room temperature (20-22 ° C). The final emulsion was stored at • 25 ° C, 8 ° C. For the manufacture of microcapsules, the emulsion was warmed to “2 room temperature (20-22 ° C) and spray dried on a laboratory size spray dryer (Buechi Mini Spray Dryer B-191, Switzerland). Inlet and outlet temperatures ··· were 170 ° C and 90 ° C, respectively. The injection pressure was maintained at 5 bar. By this method, rapeseed oil was microencapsulated with good success and the resulting product (i.e., m · ...: 30 microcapsules) was a white, free-flowing powder having a particle size of 1 to 2 µm (Figure 3).

• · · · · · · · · · · ··· 2 • 1 EXAMPLE 9 24 118506

Peanut Coating - Composition and Preparation of Film Coating Solution 5 The concentration of activated soluble whey protein (ASWP) in the coating solution was 5%. As a plasticizer, a mixture of 1% glycerol and 1% sorbitol (based on the total weight of the solution) was used and added to the solution with stirring. The pH of the solution was adjusted to pH 7.0 (1 M NaOH). The solution was heated in an oven for 60 minutes at 70 ° C and then allowed to cool to room temperature (20-22 ° C). The ready-to-use solution was stored at 8 ° C for 5 months before coating. The results of film coating are shown in Example 14.

EXAMPLE 10 15 Free Films; test series The ASWP fraction was used to make free films. The aqueous solutions contained 7.5% or 10% ASWP, 3% glycerol and 1% sorbitol as plasticizers (based on the total weight of the solution). Titanium dioxide (1%) was added to some solutions to prevent the film from sticking. The solutions were heated in an oven 60 for 20 minutes at 70 ° C (except for one solution prepared without. ·· *. Heat treatment) and then allowed to cool to room temperature (20-22 ° C) and then poured into Teflon molds ( free film casting).

The films were dried horizontally at 21 ° C / 45% RH for a total of 24 hours (excluding, except for films cast from a solution prepared without heat treatment; these films had a drying time of 48 hours). All films were translucent • · '···' and elastic.

· »·

Table 4: Composition of Free ASWP Films.

φ * • · φ · · "*: Experiment__Composition (%) ___ ·: **: __ ASWP__Glycerol__Sorbitol Titanium Dioxide 1. 7.5 *] 3 1 *: * *. 2. 10, O * 1 3__1 __: _ ** _3. ____3__1__1_ 4. 7.5 * 2 1 3 11 11 25 1 1 8506 * l Heat treatment at 70 ° C for 1 h; * 2 No heat treatment

Free films were used for the shelf-life assay and the results are shown in Example 19 5 EXAMPLE 11 Preparation of Coating Solutions The ASWP fraction was used to prepare four experimental coating solutions. The aqueous solutions contained 5.0% ASWP as well as 1% glycerol and 1% sorbitol as soft 10 (based on the total weight of the solution). The pH of the solutions was adjusted to pH 7.0 (1 M NaOH). They were then heated in an oven for 60 minutes at 70 ° C and then allowed to cool to room temperature (20-22 ° C). The coating solutions were stored cool at 6-8 ° C. The membrane solid excipients (magnesium stearate, titanium dioxide) were added to the solutions just prior to coating, after which the coating solutions were homogenized to form a milky suspension (= ready-to-use coating solution). Magnesium stearate and titanium dioxide were used in three coating solutions at 0.5 to 2% (based on the total weight of the solution) to prevent the film from adhering (see Table 5).

20 Table 5: ASWP Film Compositions.

• * * · · • · • · .11:. ·. Experiment__Composition (%) ____! ". · ASWP Glycerol Sorbitol Magn. Titanium-Quinoline, • · ... Stear. Oxide yellow • * * • · • · ··· _ J _L__5__1__1 __-__-__-_ 2._ 5_ 1 1 1 _ 1 • '' I - I - .il - --- - ^. »: * _3 .__ 5__1__1__0 * 5__0 * 5__0 * 1_ C: 4. 5 1 1 2 2 0.1 • • ·

The results of the corresponding coating tests with these coating compositions are shown in Example 17.

• ·· • · · • · ·:. 25 EXAMPLE 12 Manufacture of Capsule Shells

The capsule shell solutions contained 9% protein (30% soluble fraction (ASWP) 5 and 70% original whey protein concentrate), glycerol 4% and sorbitol 1%. The pH was adjusted to 5.0 (1 M NaOH). The solutions were heated in a heating cabinet at 70 ° C for 1 h and cooled to room temperature (20-22 ° C).

For preparation of capsule shells, the solution was dried on a laboratory size spray dryer (Buechi Mini Spray Dryer B-191, Switzerland). The feed temperature was 170 10 ° C, the exit temperature 90 ° C and the spray pressure 5 bar.

The capsule shell preparation solution was made from spray-dried powder (protein content 53.1%). The solution contained 40% protein (15 g of powder was dissolved in 20 ml of purified water and the pH was adjusted to 6.5 with 5 M NaOH). Reconstitution occurred in a half-gram portions while stirring continuously (was a little air bubbles). The capsule shell was prepared by dipping the rod once into the solution and drying with warm air for about 5 minutes so that the solution no longer drained. Finally, the protein-coated rod was left to dry at room temperature (20-22 ° C) for 4-5 hours, after which the capsule shell was ready to be removed from the rod.

· * · * · 20 i * i EXAMPLE 13 • · ··· • ♦ * · «« t The basic formula for infant milk • The basic infant formula was made from skimmed milk (Valio), whey «* * ·· ** Protein Precipitation Fraction (P 13), Rapeseed Oil (Raisio Group Ltd) and Lactose (JuustoKaira Oy) »The basic model of infant formula contained 30 • · Itii! Protein 1.5% • ·. Whey Protein 1.0% ·. · · Casein 0.5%

Fat 3.5% Rapeseed oil 35 Carbohydrates 7.3% Lactose 118506 27

The whey protein precipitate fraction served as an emulsifier; no other emulsifier was used

Skimmed milk content: 5 Protein 3.3%

Casein 2.5%

Whey Protein 0.6%

Other nitrogen tips. substances 0.2%

Carbohydrate: 10 Lactose 4.9%

Fat 0%

The precipitation fraction (P13) contained: 15 Protein 7.93% 79.3, g / L

Solids 8.92% 89.2 g / L

Carbohydrate etc. 0.85%

Ash (salts) 0.14% 20

Compilation of the basic model: • 20 liters of the basic model are required: • 25% casein 0.5%.

• · ... 3.70 liters of skimmed milk are obtained from casein.

Whey Protein 1.0% 3.70 liters of skimmed milk contains 22 g of whey protein. In 20 liters, 1.0% whey protein-30 contains 200 g protein, so the protein requirement was 178 g. It needed a whey protein -:> 4 *: inine fraction (P 13) 2.25 liters • · t Lactose 7.3% • ·. The amount of lactose in skim milk was 3.70 liters at 181 g. 20 liters of standard model V: 35 required 1460 g of lactose, so the lactose supplement needed was 1.28 kg.

• ♦ • · ♦ • ♦♦ • ·

Fat 3.5%

The fat was added as rapeseed oil. It needed 35 g / l, so the total need was 700 g.

... 28 Manufacture of basic breast milk substitute

The basic model was made in 40 liter heated and stirred containers. Until step 5, 12 L of microfiltrated water was taken and heated to 45 ° C. First, 1.28 kg of lactose was dissolved in warm water. Next, 2.25 liters of precipitate fraction was added. It was mixed into a uniform suspension. The pH was adjusted to 6.5 with 4.0 N NaOH. Skimmed milk, 3.70 liters, was then added. Lastly, rapeseed oil 700 g or 800 ml was added.

10

The suspension was stirred vigorously so that the oil was homogeneously mixed with the basic suspension. The temperature of the suspension was raised to 63 ° C and stirring was continued.

Λ

The heated suspension was first homogenized at 70 kg / cm. During homogenization, the suspension turned white and resembled whole fat milk. The second generation of homo-15 occurred at 120 kg / cm. The temperature was then maintained at 50 ° C. Immediately after homogenization, pasteurization was performed at 78 ° C and the residence time was about 35 seconds. After pasteurization, the pH of the suspension was 6.58. It was sampled for analysis and cooled to 8 ° C where it was stored.

The dry matter, protein content, sulphate ash, and stability and protein hydrolysis were determined from the suspension / substitute. Stability was determined at room temperature (22 ° C) and 8 ° C. Three 100 ml volumetric flasks were filled with the prepared · · · ···. suspension and held for 24 hours at room temperature (22 ° C) and three replicate lanes at 8 ° C for 2 weeks, after which the homogeneity of the substitute / suspension or separation of the layers was checked.

At room temperature, the substitute remained homogeneous after 24 hours and was not distinguishable from * ··· *. At 8 ° C, the surrogate remained homogeneous for two weeks and was still homogeneous after four weeks and no separation was observed.

*: * 30

M »· -'V

:] *]: The base model "0" of the infant formula produced was subjected to a hydrolysis test with pepsin at pH 2 for 3 hours and then with trypsin at pH 8 for 2 hours. mimicking the conditions of the digestive tract. The degree of hydrolysis was determined by the OPA method. For comparison two commercial infant formulas "P" (powder) V: 35 and "T" (ready for use) were used.

• · • · · • »I« · 118506 29

Table 6: Degree of hydrolysis of infant formulas Replacement Products (O, P, T) __ Hydrolysis -% ___

Time (h) __ O__P__T__Handling_ 1 7.49__7.51__4.40 Pepsin pH 2_ 2__10.43__7.91__7.00 Pepsin pH 2_ _3__10.55 8.87__6.33__Pepsin pH 2_ 3.5__17.95__15.05 9.69 Trypsin βH8_ 55__16.05__11.09 Trypsin pH 8-5 EXAMPLE 14

Method for Filming Peanuts with ASWP Solution Using Side-Ventilated Drum Coating Device 10 Non-Pigmented Coating Solution

Performing Film Coating: A: The materials and method for preparing the coating solution are described in Example 9.

: ***: 15 The aqueous solutions contained 5% ASWP plus 1% glycerol and 1% sorbitol as plasticisers (based on the total weight of the solution). The kernels used were tn · .... peanuts in shell or in shell.

·· · • · · · ·] ..! The peanuts were coated with a laboratory-scale instrumented tablet drum * ·· * '20 (Thai coater, model 15, Pharmaceuticals and Medical Supply

Ltd. Partnership, Thailand). The batch size was 900 grams of peanuts. Prior to coating, the peanuts were preheated for 5 minutes until the drum temperature had risen to 40 ° C. Other process parameters during coating were as follows: a ·: ··: pump feed speed 2.2 rpm, spray pressure 300 kPa, drum rotation speed 5 rpm, drum vacuum 5 Spout air outflow from drum 20 1 / s . 221 g of coating solution was sprayed per batch. Immediately after stopping the coating solution feed V *, the peanuts were dried in a rotating drum at 40 ° C for an additional 5: 5 minutes. The coated peanuts were then brought to room temperature (25 ° C / RH 60%) for at least 24 hours before being examined.

118506 30

On the basis of the organoleptic assessment, the appearance of the ASWP-coated peanuts was satisfactory and did not adhere to one another during coating and drying. The coating of peanuts with ASWP aqueous solutions proceeded without any technical problems or difficulties.

EXAMPLE 15 10 Method for Making ASWP Films and Filming Properties

Manufacture and examination of free films

Free ASWP films using glycerol as a plasticizer were made with an oath-bat. The preparation of aqueous ASWP solutions was previously described in Example 2 and their compositions are shown in Table 7 below.

Table 7: Composition of aqueous ASWP solutions.

20 __

Substance Composition (%) _____ j * (a) 2 * (a) 3 * (V) 4 * (b) 5 * (°) 6 * (c) 7 * (d) g * (d) AS WP_ 3% 4% 3% 4% 3% 4% 3% 4%

Glycerol__1% 2% 2% 1% 2% 1% 1% 2%

Purified water q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s.

^ * Heat treatments: (a) 70 ° C / 10 min; (b) 70 ° C / 20 min; (c) 80 ° C / 10 min; (d) 80 • '.. f ° C / 20 min • »• · ie«

The membranes were stored for 1 week at controlled room conditions (25 ° C / 60% 25 RH) prior to their solid state structural examination (XRD, • · · atomic power microscope AFM). After approximately 1 month of storage (25 ° C / 60% RH), the above membranes were examined for appearance and structure by scanning electron microscopy (SEM). XRD analysis of membrane samples was performed under symmetric reflectance ♦ ·. mode in CuK ^ radiation (1.54 Angstroms). The angular space was between 2 ° and 60 ° (20): 30 at 0.02 ° intervals and the measurement time was 20 s / phase for all measurements. The Atomic Power Microscope (AFM) was a Park Scientific Instruments Autoprobe CP (Thermomicroscopes, USA) equipped with a special probe. Measurements were made in IC-AFM (intermittent 118506 31 contact AFM) mode. For phase imaging, the atomic power microscope was equipped with an M.A.P. © module, which enables measurement of force as well as measurement of phase difference signals. A scanning electron microscope, SEM (Jeol JSM-840A, Jeol, Japan) was used to examine changes in membrane appearance and morphology during storage (1 month at 25 ° C / 60% RH).

Morphology and physical properties of membranes

From an organoleptic point of view, the ASWP films were translucent, clear, and easy to handle as they did not adhere to much. Short term storage (1 month at 25 ° C / 60% RH) did not change the appearance of the film except for slight darkening. However, films containing 4% ASWP and 1% glycerol as a plasticizer (tests 4 and 6) were clearly more brittle than the other films. The brittleness of the membranes may be due to insufficient amount of plasticizer (glycerol) or partial removal of plasticizer from the well structure (dripping) during storage.

Scanning electron microscope (SEM) images show that membrane morphology and structure are not very dependent on coating solution preparation (temperature and time) and short term storage (Figures 4A-H). The membranes softened with higher concentrations of glycerol (2%) showed less surface defects than the other membranes (Figures A, C, E, G). The films softened with a small amount of glycerol (1%) (1%) showed relatively large irregularities: ***; feminine patches and splinters (Figures A, D, F, G). According to SEM images, films ··· • prepared by a longer heat treatment of the coating solution (20 min) were ··· ♦ · 25 essentially homogeneous in structure. If the amount of plasticizer was low (1%), they too; ·. ♦. however, there was a tendency for the films to fragment.

The XRD results indicated that no crystallization occurred in the ASWP membranes during the short term storage test (1 week at 25 ° C / 60% RH). No crystalline peaks ··· ♦ · 30 were detected on the diffractionograms. Accordingly, the present invention. the films are predominantly amorphous in structure and the film former is in non-linear form in the film matrix. The atomic force microscope (AFM) images allow three distinct phases to be distinguished in each of the films studied. The separated phase droplets appear to be the largest in lots 1 and 2 and the smallest in lots 3 to 7. Films made through the middle treatment * · *: 35 have the smallest '' droplets '' (Figure 5). No correlation was observed between membrane size (* heat treatment) and large patches / apertures (when examined at 60 x 60 pm), small patches (examined from small images), or apertures.

118506 32 EXAMPLE 16

Method of Film Coating Tablets with ASWP Solution Using a Page-5 Air-conditioned Coating Machine

Non-pigmented coating liquid

Performing a film overlay 10

The substances and the method for preparing the coating liquid and / or dispersion are described in Example 4. The aqueous solutions contained 5% and 6% ASWP (Tables 2 and 3). Glycerol and sorbitol and a mixture thereof (1: 1) were added and blended into the solution as a Peh dimension. The coating solution vessel was allowed to remain in a water bath at 75 ° C for 15 minutes prior to use (heat treatment, see Tables 2 and 3).

The tablet cores (substrates) contained: theophylline (Ph.Eur.) 5%, lactose monohydrate 30%, microcrystalline cellulose 56%, talc 8% and magnesium stearate 1%.

The tablets were coated with a laboratory-scale instrumented drum coating device (Thai coater, model 15, Pharmaceuticals and Medical Supply Ltd. Partner, Thailand). The batch size was 1000 g of tablet cores. Before coating, tablets · ·. ***. and preheated for 10 minutes until the drum temperature had risen to 40 ° C. 325 g of coating liquid was sprayed per batch. Immediately after stopping the feeding of the coating solution 25, the tablets were dried in a rotating drum at 40 ° C. for another 5 minutes. Other coating process parameters are shown in Table 1. * * 8. The coated tablets were then brought to room temperature (25 ° C / RH 60 • · * · · * *%) for at least 24 hours before being examined.

The coated tablets were examined for appearance (organoleptic and stereomicroscopic • · ·), tablet weight and weight variation (n = 20), radial fracture strength (Schleuniger; n = 10), dissolution with the European Pharmacopoeia lapam method (n = 6), and dimensions before and after coating (Sony- ♦. micrometer; n = 10).

··· * ♦ «~.

·. ♦ * 35 • ♦:. * ·· Coating experiments were performed according to the experimental design scheme shown in Example 4 (Tables 2 and 3) and individual experiments were performed in random order (see Example 4).

Table 8: Coating Parameters.

33 118506

Process Parameter__Part 1__Part 2_ Coating Fluid Feed Rate 3.5 5.0 (g / min) __ (= 2.2 rpm) (= 3.0 rpm)

Injection pressure (kPa) __ 300__300_

Drum temperature (° C) __ 40__50_

Drum Speed (RPM) 7__5_

Drum Vacuum (Pa) __ ^ 5 __- 5_

Exhaust Air Flow Rate (1 / s) _Q8_ | jjS_ 5 Availability of ASWP Solutions in the Coating Process

The coating of the tablets with aqueous solutions of ASWP proceeded without any technical problems or difficulties. In the lot 1 coating batch, the adherence of the tablets to the drum walls was hardly observed during the coating. However, it should be noted that slight mechanical erosion and abrasion of the surface of the tablet-10 cores was observed, which affected the appearance of the coated tablets. A summary of the success (usability) of the Part 2 coatings is given in Table 9.

• • • 1 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • ••••••••••••••••••••••••••• · · · · · · · · · · · · · · · · · · · · · ··· ······················· ········· Member next Members of the Board ·

Table 9: Availability of ASWP Solutions in the Tablet Coating Process (Part 2).

Experiment__Composition (%) _ Description of Coating Process ASWP Gly Sor (drum rotation speed 5 rpm; 50 'C; _____ fluid feed rate 3.0 rpm) 1. * __ 5__1__1__No coating sticking problems. noticeable on the drum and blades (especially _____ at the end of the coating) ._ 3. * 6 1,2 1,2 Clear adhesion of tablets to drum walls and blades (multiple tablets). The composite is not suitable for coating. A_ | _6_ | 1.2 1 1.2 Clear adhesion of tablets._ * Coating fluid not heat treated.

5 Examination of Coated Tablets The appearance of the coated tablets varied greatly, indicating that not all 10 experimental coating compositions were suitable for their intended use and their use; that the coating compositions were also sensitive to the coating conditions. The best end result was obtained with a coating composition 4 containing 5% ASWP and 1% softener (glycerol and sorbitol 1: 1).

Table 10: Appearance of coated tablets as sensory. (scoring 0-10).

···. Experiment / Part 1 Composition (%) _ _ Appearance * ** "__ ASWP__Glycerol__Sorbitol __ (Score 0-10) V L__5__0__0__4_ *: · *: 2 .__ 5__1__0__4_ ·: ·: _3 .__ 5__0__1__6_ a_j__i__i_i_ V! · -— - - - - ** * 5 6 .__ 5__0__2__4_ 7. 15 \ 2 \ 2 11 118506 35 8. Is 13 10 "1_ 9. __5__0__3__6_ 10. 5 I 3 I 3 10 ♦ Mechanical erosion and abrasion of the tablet core were observed , which affected the appearance of the coated tablets.

The tablet weight gain and weight variation after coating was good in all coating batches, which contributed to the usefulness of the studied coating formulations in drum coating (Table 11).

Table 11: Tablet weight and weight variation after coating (n = 20).

10__

Experiment / Part 1 Composition (%) ___ Average Weight and Weight Variation __ASWP Glycerol Sorbitol Ka (mg) S.D. RSD%

Tablet core __-__-__ 498.7__3J5__0 ^ 8_ J .__ 5__0__0__509.6 9.8 1.9 2, __ 5__1__0__507.5 3.2 0.6 _3 .__ 5__0__1__507.8 4.8 1.0 A .__ 5__1__1__509.6 4.0 0.8 _5 .__ 5_2__0__508.6 12.8 2.5: X: 6, __ 5_0_2__511.7 4.3 0.8 O _7 .__ 5_ 2_ 2__512.9 3.0 0.6 _8 .__ 5_ 3__0__515.7 4, 5 0.9 9 .__ 5__0__3__510.1 5.7 1.1 f \: 1 10. I 5 13 13 518.4 6.0 1.2 · · · · · · · · · · · · · · · · · · · · large but slightly "··. unexpectedly, the fracture strength had not increased with respect to the fracture strength of the tablet cores • · 15. With the exception of two coating batches, the tablet fracture variation *: ** · the coating batches were low, demonstrating the availability of ASWP coating solutions .

· 1 · * 1 1 • · · · «· i • · · • ·

Table 12: Mechanical strength of tablets after coating (n = 10).

36 118506

Experiment / Part 1 Composition (%) ___ Mechanical Durability_ ASWP Glycerol Sorbitol Ka (N) S.D .__ RSD%

Tablet core ___-__ 99.6__43__43_ _L__5__0__0__86.1 193 22.5 2 .__ 5__1__0__81.6 3.4 4.1 _3 .__ 5__0__1__77.5 5.3 6.9 A .__ 5 1__1__81.9 5.3 6.4 _5 .__ 5__2__0__77 , 6 6.0 7.8 _6 .__ 5__0__2__74.9 5.6 7.5 _7 .__ 5__2__2__76.4 4.6 6.0 _8 .__ 5__3__0__78.9 6.7 8.5 9. __5__0__3_ 87.8 11.0 12.5 10. 1 5 1 3 | 78.9 5.4 6.8 ASWP-coated tablets can be classified as rapidly disintegrating formulations, since 5 drugs (theophylline) were released very rapidly (t50% values in less than 10 min) in each of the coating batches examined (Table 13). The dissolution of ASWP coating and drug release appeared to be independent of the pH of the dissolution medium between pH 1.2 and 6.8.

Table 13: Dissolution of Coated Tablets in Vitro (n = 6) ._ · · · Experiment / Part 1 Composition (%) __ T50% (min) __ *: ··: ASWP Glycerol Sorbitol 0.1NHC1 0.1NHC1 I pH 6.8 ______ + pepsin__

Tabl.ydin - ___-__ 33 __ * __ 33_ _L__5 0__0__43 __ * __: _ 2 .__ 5 1__0__73 __ * _ j43_. "I. _3 .__ 5 0__1__53 __ * __: _ A .__ 5 1 1__33 __ * __: _ _5 .__ 5_2__o__33. .__ 5 0__2_j43 __ * __ 53_ _7 .__ 5 2__2 __: __ * __: _: 8._ 5 3 0 7,5 * • ·· - ————........—---—— - - 9. __5__0__3__33 __ * __ 33_ 10. 1 5 1 3 I 3 | 3,4 I * 1 - 118506 37 EXAMPLE 17

Method for Film Coating Tablets with ASWP Layer Using a Page-5 Air-conditioned Drum Coating Device

Pigmented coating solution

Performing Film Coating 10

The materials and method for preparing the coating solution (dispersion), the composition of the tablet cores (substrates) and the film coating process and equipment are described in Example 16. The compositions of the pigmented coating solutions are shown in Table 14. The aqueous dispersions contained 5% (w / w) ASWP. A mixture of 15 glycerol and sorbitol (1: 1) was used as a plasticizer and added to the protein-containing solution. Solid film auxiliaries (magnesium stearate, titanium dioxide) were added to the coating solution and finally the mixture was homogenized to form a milky dispersion. The coating solution (dispersion) was used for drum coatings at 600 g / coat.

20

Table 14: Composition of Pigmented Coating Solutions (Dispersions).

• · · • · · _ _. _______ • · | - --— · 1 "· Experiment Composition (%) _____ * · 1:. ·. ASWP Glycerol Sorbitol Mg.stear.

1. 5 1 1 _ - * · · 1 1 ^ __ 2. __5__i__l__l_j__ _3 .__ 5__1__1__0,5__OjS__0,1 4. 5 1 1 2 2 0.1 *

»•• I

· «» • • • • • • M «• · * 1 · · · 1 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Table 15: Coating Parameters.

38 118506

Process Parameter__Sample 1 and 3__Sample 2 and 4_ Coating Solution Feed Rate 3.5 3.5 (g / min) __ (= 2.2 rpm) (= 2.2 rpm)

Injection pressure (kPa) __ 300__300__

Drum temperature (° C) __ 40__50_

Drum speed (rpm) 8 * __ 8 ^ _

Drum Vacuum (Pa) __ ^ 5 __ ^ _

Exhaust Air Flow Rate (1 / s) _20 _ [_ 20_ * Preheat the tablet cores at 3 rpm and start coating for 10-15 minutes at 5 rpm.

5 ** Preheat the tablet cores at 3 rpm and start coating for 5 minutes at 5 rpm.

The coated tablets were examined for appearance (organoleptically and by stereomicroscope 10), tablet weight and weight variation (n = 20), radial fracture strength (Schleuniger; n = 10), disintegration time with European Pharmacopoeia equipment (n = 6), and tablet dimensions before and after coating (Sony micrometer; n = 10).

: 7: 15 I Applicability of Pigmented ASWP Dispersions in the Coating Process

Generally, no significant technical problems or difficulties were encountered in coating the tablets with pigmented aqueous ASWP dispersions. In all of the investigated casings. However, some sticking of tablets was observed in 20 tyser • · · • ** | during such coating. This phenomenon was particularly evident when coating dispersion: ···: sprayed on tablet cores over 300 g (i.e., more than half of the target amount) and / or ·; ··· for 90 minutes from the start of coating. However, the adhesion of the tablets was significantly lower than that of the corresponding coating formulations without magnesium stearate. The addition of magnesium stearate to the coating composition reduces the adhesion of the tablets to the drum walls and blades, which improves the adhesion of the tablets.

usability of the coating form for drum coating. However, it should be noted that slight mechanical erosion and abrasion of the surface of the tablet cores was observed, which affected the appearance of the coated tablets.

Examination of Coated Tablets Coated tablets were evaluated organoleptically and the results are shown in Table 16 (score 0 to 10).

10

Table 16: Appearance of coated tablets, organoleptically assessed (score 0 to 10).

Experiment Composition (%) _____ Appearance AS WP Gly Sorb Mg. Titanium Quinoline (Score 0-10) _____stear. yellow yellow .__ 1. 5 1 1 -__: _ 2? _ 2. 5 111__1__ 3. 5 11 0.5 0.5__0J__6_ 4. 1 ~ 5 1 1 I 1 12 12 10.1 141 * Clear adhesion of tablets to drum wall and shovel finishing at 15 o'clock • 1 · · · · · · · · · · · · · · · · · · · · · · · · · · The coating progress and ASWP film formation were examined by sampling (20 tablets / sample) at regular intervals during the coating process. (coatings according to Experiment 4: 0, 20, 40, 60, 80, 100, 120, 140 and 160 min). The results are presented in Table 17.

· ♦ · ♦ ··· ♦ · 1 · • ♦ • · · · · · · · · · · · · · · · · · · · · · ··· ·

Table 17: Appearance and coating quality of tablets taken during the coating process, as sensed (score 0 to 10).

Test 4 Sampling ___ Appearance_ Coating- Coating Performance- Coating Theoretical (Score 0 to 10) Working Time Tea Consumption Quantity (min) (g) (ASWP) _% mg / cm2 _ a. 20 65.0_ 0.3 0.6__9_ b. 40__135J__0 , 7 1.1__8_ c. 60 208.3__1.0 1.8__7_ d. 80 285.1__1.4 2.4__7_ e 100 360.7__1.8 3.1__6_ f 120 435.6_ 2.2 3.7 6_ g. 140 570.0_ 2.8 4.8__5_ h. __160__a 600____5J__4_ 5 Table 18: Tablet weight and weight variation after coating (n = 10).

Experiment Composition (%) Mean-standard deviation - ^^^^ -. Fr- «a, -, - ASWP Gly Sorb Mg. Titanium-Ka S.D. RSD% • · [··· '_____ stear. dioxide (mg) ___: Tabl.ydin __-__-__-__ 498.7 3.8 0.8 * "** J _.__ 5 1 1_ -__ 517.5 3.7 0.7 fV 2 .__ 5 111 ___ 521.0 4.4 0.8 O _3 .__ 5 1 1_ 0.5 0.5 518.4 4.0 0.8 4. 15 I 1 I 1 12 2 522.4 2.7 0.5 • · · • # * · • · · • · • ·

Ml • • • • • • • • • • · · · · · · · ·

• I I

• ·· • · 118506 41

Table 19: Mechanical strength of tablets after coating (n = 10). Experiment Composition (%) Mean Deviation __ (n = 10) _

ASWP Gly Sorb Mg. Titanium-Ka S.D. RSD

_____stear. dioxide (N) ___%

Tabl.ydin __-__-__-__ 99.6 4.3 4.3 __5 11 -___ 93.7 5.9 6.3 2 .__ 5 111 ___ 85.6 3.8 4.4 _3 .__ 5 1 1_ 0.5 0 , 5 79.3 7.1 8.9 4. 15 I 1 I 1 12 12 105.1 5.1 4.8 5 Table 20: In vitro disintegration of coated tablets (n = 3-6).

Experiment Composition (%) Tablet disintegration time _______in vitro (n = 3-6) _ ASWP Gly Sorb Mg. Titanium _____stear. dioxide__

Tabl.ydin __-__-__-__ <0.5 min_ 1. 5 11 - - <1 min • 1 - - "" - 'V .: 2 .__ 5__1 1__1 __-__ <1.5 min_ O _3 .__ 5 1 1 0.5 0.5 <1.5 min_ 4. [5 1 1 1 1 2 2 [<1.5 min • · EXAMPLE 18 ♦ · • · 1 φ • · · 1 10 ASWP transparencies containing as auxiliary maltodextrin, was prepared by casting softened solutions into molds and drying. The dried films were then carefully removed from the molds. Glycerol and sorbitol were used as plasticizers. Although small pores were formed in the • • • • • • • • • • • • • the membranes were significantly better in mechanical strength than the corresponding membranes in the absence of maltodextrin.

•••••••••••••••••• 118506 42 EXAMPLE 19

Shelf Life of Free Films and Coated Tablets 5 Structural Studies of Free ASWP Films

Free ASWP films plasticized with glycerol and sorbitol were prepared by the casting process. The compositions of the ASWP film solutions are shown in Table 21.

10

Table 21: Compositions of ASWP-aqueous solutions used to make free films.

Substance Composition (%) ___ l * (a) 2 * (a) 3 * (a) 4 * 00 ASWP ____ 10__7 ^ 5__7J5_

Glyseroli__3__3__3__3_

Sorbitoli__1__1__1__1_

Titanium dioxide __-__-__ 1__1_

Purified water_q.s._q.s._q.s._q.s._ * Heat treatment of aqueous ASWP solution: (a) 70 ° C / 1 h; (b) no heat treatment: X: 15 Free membranes were stored under controlled room conditions (25 ° C / 60% RH) and ·; ··· under stressed conditions (50 ° C) for 6 months. . The membranes were examined for structural properties (XRD and NIR assays) at 1, 3, and 6 months. Starting 20 exams. The analytical methods are described in the previous sections.

• · ···. Scanning Electron Microscope (SEM) Images of Fresh ASWP Films · ;;; (reference films) indicate that the films are homogeneous and of good quality (Figures 6 and 7). The results of the shelf life assay are shown in Figures 9 and 10. XRD- *: · *: 25 showed no crystalline * ···· reasons for non-pigmented ASWP during storage (Experiments 1 and 2). On the other hand, the crystallinity of the pigmented ASWP films has not increased during the storage period because of the additional f | no peaks were formed compared to the corresponding fresh reference film • (· in addition to the titanium dioxide peak) (Experiments 3 and 4). ASWP films appear to be very physically stable and thus useful for coating applications. under conditions (50 ° C), the appearance of the ASWP films clearly changed and the films became tacky (including film darkening).

The invention has been described herein with reference to certain preferred embodiments and applications. However, it will be apparent to one skilled in the art that modifications to the preferred embodiments may be made and practiced, and the invention may be practiced within the scope of the appended claims, except as specifically provided herein.

• 1 • · · · · 1 • 1 • · «• · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ···· ECE ♦ · · • «« · ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦

Claims (26)

118506 A process for preparing a protein-based membrane comprising a protein network formed by inter-protein disulfide bonds, characterized in that it comprises the steps of: - forming a protein-containing solution having a pH of 7 or less, modified by sulfonation by opening at least one disulfide bond originally present therein to obtain free sulfhydryl groups, said free sulfhydryl groups being capable of effecting an exchange reaction reaction, wherein said disulfide bonds are formed between proteins to form said membrane, and - said protein base is formed from said solution. A method according to claim 1, characterized in that said modified protein comprises a whey protein, such as a soluble fraction or precipitate fraction of a modified whey protein, or a combination thereof. Process according to claim 1 or 2, characterized in that said protein is sulfonated by treatment with a sulfite ion-forming reagent. 20 A process according to claim 3, characterized in that said sulfite ion-forming reagent comprises an alkali metal or alkaline earth metal sulphate. * · ·. phytis, hydrogen sulfite or metabisulfite, or a combination thereof. * · · · · · · · · · · · The process according to any one of the preceding claims, characterized in that the amount of free sulfhydryl groups in the total protein of the solution before the exchange reaction is from 0.5 to 60 pmol / g of protein. • · • * · »· Method according to any one of the preceding claims, characterized in that the protein solution is further heated to promote conversion. • · · • · · · ·; ·) ·. A method according to any one of the preceding claims, characterized in that the film is formed on the material to coat it. • · • · · "*" 35 A method according to any one of the preceding claims, characterized in that the film is formed on the foodstuff. 118506 Method according to one of the preceding claims, characterized in that the film is formed on a substance containing a therapeutically active substance, such as a tablet, granule, pellet or the like. Method according to one of the preceding claims, characterized in that the film is formed into a capsule shell. Method according to one of the preceding claims, characterized in that the film is formed around a lipid, an oil, a lipophilic compound or a combination thereof to form an emulsion or a microcapsule. Process according to any one of the preceding claims, characterized in that a plasticizer or lipophilic compound such as stearate, oily butter fat or real oil or a combination thereof is further added. 15 Process according to any one of the preceding claims, characterized in that a strength enhancing compound such as a carbohydrate such as maltodextrin or other starch hydrolyzate is further added. A method according to any one of the preceding claims, characterized in that a pigment dye such as titanium oxide, an antifouling agent, is further added. *. an antimicrobial agent or preservative. 15. A protein-based membrane having a protein network formed by inter-protein disulfide bonds, characterized by a protein network formed in solution at pH is 7 or less and contains a modified protein which has been converted by opening * at least one of the originally disulfide bonds in it to obtain the sulfhydryl groups on the ends, whereby said free sulfhydryl groups have occurred exchange reaction in which said disulfide bonds formed between proteins. • * · • · • * # ·· ··] ·. A protein-based membrane according to claim 15, characterized in that said modified protein comprises a whey protein, such as a soluble fraction or precipitate fraction of a modified whey protein - **, or a combination thereof. ·: * ·: 35 A protein-based membrane according to claim 15 or 16, characterized in that said protein is sulfonated by treatment with a sulfite ion-forming reagent. 118506 Protein-based membrane according to claim 15, characterized in that said sulfite ion-forming reagent comprises an alkali metal or alkaline earth metal sulfite, hydrogen sulfite or metabisulfite or a combination thereof. Protein-based membrane according to any one of claims 15 to 18, characterized in that the amount of free sulfhydryl groups in the total protein of the solution prior to the conversion reaction was between 0.5 and 60 pmol / g of protein. A protein-based membrane according to any one of claims 15 to 19, characterized in that the protein solution is heated to promote conversion. Protein-based membrane according to one of Claims 15 to 20, characterized in that a plasticizer or lipophilic compound such as stearate, oily butter fat or real oil or a combination thereof is added thereto. 15 Protein-based film according to one of Claims 15 to 21, characterized in that a strength enhancing compound such as a carbohydrate such as maltodextrin or other starch hydrolyzate is further added thereto. Protein-based membrane according to any one of claims 15 to 22, characterized in that a pigment dye such as titanium oxide, an antifouling agent, antimicrobial agent or preservative is further added thereto. • »··» • « 24. Food product characterized in that it is surrounded by or contains ingredients which are inherited from the film according to any one of claims 15 to 23. • · • · 2 ^: t Infant formula, characterized in that it contains as an emulsion a lipid, an oil, a lipophilic compound or a combination thereof, surrounded by a film according to any one of claims 15 to 20 in the form of an emulsion or microcapsule. 30: 3: A pharmaceutical product containing at least one therapeutically active substance, characterized in that it is surrounded by a film according to any one of claims 15 to 23. φ · • · M »· 2« · · • • • 3 • «♦ 118506