Thermoplastic collagen composition containing polyester

Abstract

The present invention relates to a blended composition comprising i) collagen, ii) water, iii) a plasticizer in particular, and iv) a polyester having a melting point of 120°C or less, wherein the weight ratio of collagen to polyester is 2:1 to 1:20. The blended composition can provide a sheet having a water solubility of 40% w / w or less, and when subjected to blow film extrusion, it can provide a homogeneous film. The present invention also relates to solid articles made from the blended composition, and to the use of these materials in the food industry, pet food sector, agricultural sector, or pharmaceutical sector.

Description

[Technical Field]

[0001] This application claims priority to European Patent Application No. 23382462.2, filed on 17 May 2023.

[0002] This invention relates to the field of collagen-based materials, and more particularly to thermoplastic collagen compositions, including thermoplastic collagen and polyester. These compositions possess sufficient mechanical properties to be adapted for various solid molded articles. [Background technology]

[0003] Collagen is the most abundant extracellular matrix protein in animals, accounting for nearly 30% of the total protein content. Collagen is primarily found in connective tissues such as cartilage, bone, tendons, ligaments, and skin. Up to 28 different types of collagen have been described in the literature. The basic structural unit of collagen is a heterotrimer consisting of three helical protein chains (known as alpha chains) twisted together in a triple helix form. These molecules are arranged in parallel and alternating directions to form fibrils and fibers.

[0004] Collagen is one of the most successfully applied proteins in the industry. Among other fields, collagen is used in the food industry (e.g., as a film-forming material in packaging) or the pet food sector (e.g., chewing bones or functional foods), the agricultural sector, and for pharmaceutical or medical purposes (e.g., as a biocompatible implant or as a carrier for cell culture in in vitro studies). The biodegradability of collagen is a significant advantage in terms of waste management after use.

[0005] Collagen-containing tissues can be used as raw materials in the preparation of various industrial products. The technologies that produce these products utilize collagen at different stages of processing and degradation, ranging from intact, undenatured collagen fibers to highly degraded gelatin. In some applications, one of the drawbacks of gelatin is its high water solubility, which results from the fragmentation of protein chains and damage to crosslinks in the collagen structure due to hydrolysis during the denaturation process.

[0006] As a biopolymer, collagen, like synthetic polymers, can be processed by thermoplastic techniques such as extrusion or injection molding to obtain articles in various forms, including casts or molded pieces, sheets, films, or coatings. Scaffolds made from natural biopolymers such as collagen have the advantages of their biocompatibility and biodegradability. In such processes, intact, undenatured collagen fibers or highly degraded gelatin are generally unsuitable. To use collagen as a thermoplastic biopolymer, partially denatured collagen that maintains long protein chains but reduces their crosslinking and entanglement must be used. Such collagen may be named thermoplastic collagen (TC). Such collagen is produced from collagenous tissue, such as bovine or pigskin, and involves a denaturation process. After production, it is mixed with water. Other additives such as glycerin, dyes, or inorganic salts may be optionally added. Extrusion or injection molding of thermoplastic collagen is operated at moderate temperatures (90-100°C) and yields general-purpose products such as pellets, threads, sheets, films, or molded parts.

[0007] Thermoplastic collagen is described in the prior art. For example, European Patent No. 2727938 discloses a method for obtaining collagen from animal skin, including processing the skin in a mixing cylinder by mechanical grinding at 50-70°C. The physical properties of the collagen material thus obtained make it easily convertible by thermoplastic conversion techniques. However, in the hands of the inventors, thermoplastic collagen obtained according to this document was found to exhibit high solubility, almost comparable to that of gelatin. Furthermore, depending on the number of days elapsed since its manufacture, blow film extrusion made from thermoplastic collagen disclosed in European Patent No. 2727938 was impossible or resulted in films exhibiting heterogeneity, high thickness variation, and extremely high film tackiness. Moreover, such thermoplastic collagen was found to show a significant decrease in melt flow rate (MFR) after 7 or 23 days.

[0008] Furthermore, International Publication No. 2007104322 (Naturin) discloses modified or partially modified dried collagen powder based on fibril-forming collagen, and its use for preparing thermoplastic collagen compositions containing it with water and optionally a plasticizer. However, in our hands, blow extrusion of single-layer films made from such thermoplastic collagen compositions proved problematic, and the quality of the samples was unacceptable. The films had uneven thickness and exhibited a very rough surface with visible granules within the film. Moreover, the films became very brittle after 24 or 48 hours. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] European Patent No. 2727938 [Patent Document 2] International Publication No. 2007104322 [Overview of the project] [Problems that the invention aims to solve]

[0010] Therefore, despite what is known in the state of the art and the progress made in the field, there remains a need to provide a thermoplastic collagen composition having improved mechanical properties, which can be used as a highly customizable scaffold.

Means for Solving the Problems

[0011] The inventors have found that when adding a polyester having a melting point of 120 °C or lower to thermoplastic collagen made from a mixture of collagen, water, and especially a plasticizer, a blend composition can be obtained that exhibits mechanical properties sufficient to adapt them to various solid molded articles such as complex films, unidirectional filaments, blow-extruded bottles, 3D printed objects, or injection molded objects. In particular, the blend composition of the present invention can provide a homogeneous film by blow film extrusion without visible granules, and also exhibits a lower solubility compared to the thermoplastic collagen disclosed in the prior art without the presence of gelatin or polyester.

[0012] As demonstrated in the experimental section of the present application, the collagen-based blend composition of the present invention can provide a material exhibiting many technical advantages listed below. Lower variability of melt flow rate (melt fluidity) over time Good processability in thermoforming processes Lower dependence of mechanical properties on ambient relative humidity Lower water solubility, i.e., higher water resistance Lower oxygen transmission rate and water vapor transmission rate (OTR, WVTR) Good processability in 3D printing (fused deposition modeling, FDM) Significant increase in tensile strength of unidirectional (thread) test specimens Improved adhesion to polyester, and thus improved cell viability.

[0013] Therefore, a first aspect of the present invention is i) collagen, ii) water, iii) in particular a plasticizer, and iv) a polyester having a melting point of 120° C or lower, where the weight ratio of collagen to polyester is 2:1 to 1:20, relates to a collagen-based blend composition comprising a blend composition.

[0014] A second aspect of the present invention is a) a step of melt-blending a collagen-based blend composition as defined herein, and b) a step of extruding, casting or blowing the blend of step a) onto a solid article, relates to a solid molded article that can be obtained by a method comprising.

[0015] Furthermore, the examples also show that the collagen-containing blend composition of the present invention can provide a film obtained by a platen press having improved stability. Samples containing thermoplastic collagen in the absence of polyester cannot withstand cell culture conditions (37° C in culture medium) because the collagen dissolves in a short time, which was found not to occur when thermoplastic collagen was mixed with a polyester such as PCL. Therefore, it has been found that the degradation and resorption rates can be adjusted by changing the ratio of collagen to polyester. The higher the ratio of collagen to polyester, the shorter the degradation and resorption times, and vice versa. This imparts interesting properties to the blend composition of the present invention for use in implants and cell culture.

[0016] Therefore, a third aspect of the present invention relates to the use of a blend composition comprising collagen and polyester of the first aspect as a support for in vitro assays.

[0017] Further aspects of the present invention relate to the use of collagen-based blend compositions or solid molded articles as defined herein in the food industry, pet food industry, agricultural industry, or pharmaceutical industry. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows the cell viability (%V) for different films. As a positive control C+, cells were seeded in a standard commercially available polystyrene well plate (Corning ref 3595) treated for optimal cell adhesion. Film 1 corresponds to Viscofan's Collagen Cell Carrier® membrane (CCC), films 2 and 3 correspond to the films according to the present invention (film 2: CF30+PCL(30:70), film 3: CF30+PCL(70:30)), and film 4 corresponds to 100% PCL). [Figure 2] This is a photograph of material printed by a domoBIO 2A (Domotek, Spain) printer, having blends according to the present invention including CF30+PCL (70:30), (50:50), and (30:70). [Modes for carrying out the invention]

[0019] All terms used herein are to be understood in the ordinary sense known in the art unless otherwise specified. Other more specific terms used herein are set forth below and are intended to be applied uniformly throughout the specification and claims unless the definitions explicitly stated provide a broader definition.

[0020] For the purposes of this invention, all given ranges include both the lower and upper endpoints of the range. Given ranges such as temperature, time, and weight should be considered approximate values ​​unless otherwise specified.

[0021] As used herein, the terms “about,” “near,” or “approximately” refer to values ​​within a range of ±10% of a given value. For example, the expression “about 10” or “nearly 10” includes ±10% of 10, i.e., 9 to 11.

[0022] The collagen-based blend compositions of the present invention are thermoplastic. For the purposes of the present invention, the term “thermoplastic” refers to a plastic polymer material or composition that becomes flexible or moldable at a certain high temperature and solidifies upon cooling. Thermoplastic materials can repeatedly soften upon heating and harden upon cooling. As used herein, the term “thermoplastic collagen” always refers to partially modified collagen produced from collagen tissue, such as bovine or pigskin, which exhibits thermoplastic properties when mixed with water and especially plasticizers.

[0023] As used herein, the term “plasticizer” refers to a substance that, when added, can produce or promote plasticity.

[0024] As used herein, the term “biodegradable” refers to a composite or product that can be broken down over time in the environment of use (for example, metabolized and / or hydrolyzed into harmless degradation products).

[0025] As used herein, the term "biocompatible" refers to a material that does not cause adverse reactions in living organisms. For example, a suitable biocompatible material, when introduced into a human subject, does not itself evoke a significant immune response and is not toxic to the subject.

[0026] The terms "molecular weight," "average molecular weight," and "Mw" have the same meaning and are used interchangeably herein. Molecular weight is calculated using the following formula:

number

[0027] For the purposes of this invention, the term “melt flow rate” (MFR), also known as the melt flow index (MFI), refers to a measure of the flowability of a polymer molten material. It is defined as the mass (in grams) of polymer flowing through a capillary tube of a specific diameter and length in 10 minutes at a predetermined temperature and pressure applied through a predetermined alternative gravimetric weight. The MFR variation rate after n days is calculated by the following formula: MFR change (%) = (MFR on day n - MFR on day 0) / (MFR on day 0) × 100 Here, the MFR value is measured at g / 10 min as detailed in the examples.

[0028] The term "oxygen permeability" (OTR) refers to the percentage of oxygen that passes through the film when measured according to the method described in the examples.

[0029] As used herein, the term "water vapor transmission rate" (WVTR) refers to the percentage of water vapor that passes through a film when measured according to the method described in the Examples.

[0030] The term "tensile breaking strength" refers to the tensile stress at the moment a test sample breaks. Tensile stress is calculated by dividing the force applied to the test sample by the cross-sectional area of ​​the sample.

[0031] The expression "can be obtained by" is used herein to define a product (e.g., a blended composition or a solid molded article) by the preparation method, and refers to a product that can be obtained through the preparation method disclosed herein. For the purposes of the present invention, the expressions "can be obtained," "obtained," and similar equivalent expressions are used interchangeably, and in either case, the expression "can be obtained" encompasses the expression "obtained."

[0032] A first aspect of the present invention relates to a blended composition comprising i) collagen, ii) water, iii) a plasticizer in particular, and iv) a polyester having a melting point of 120°C or less.

[0033] In one embodiment, optionally combined with one or more features of the various embodiments described above or below throughout this description, the blend composition is meltable. For the purposes of the present invention, the term “meltable” means that the blend composition melts and / or becomes fluid when heated to its melting temperature and solidifies when cooled below its melting temperature.

[0034] In another embodiment, optionally combined with one or more features of the various embodiments described above or below throughout this description, the blend composition is solid at room temperature (20-25°C), and more specifically, solid at temperatures below 40°C. More specifically, the solid is in the form of solid pellets.

[0035] In another embodiment, optionally combined with one or more features of the various embodiments described above or below throughout this description, the blend composition is solid at a temperature of 40°C or less and is meltable.

[0036] In another embodiment, optionally combined with one or more features of the various embodiments described above or below throughout this description, the blended composition may be thermoformed by a step of melt-blending a collagen-based blended composition as defined herein, or by a step of extruding, casting, or blowing the blend of step a) into a solid article.

[0037] In another embodiment, optionally combined with one or more features of the various embodiments described above or below throughout this description, the collagen in the blend composition is partially denatured. For the purposes of this invention, the term “partially denatured” refers to collagen having a degree of denaturation of 80–95% as measured by DSC (heating index of 5 K / min) by rehydrating the collagen sample with water overnight, introducing the rehydration product into a differential scanning calorimetry (DSC) vessel, sealing it, and recording the DSC. The degree of denaturation of the collagen sample can be assessed from the relative area below the peak around 60°C (typically observed for completely natural collagen and absent for completely denatured collagen).

[0038] In other specific embodiments, optionally combined with one or more features of the various embodiments described above or below throughout this description, water is present in an amount of 5 to 40%, more specifically 5 to 35%, more specifically 7 to 30%, more specifically 7 to 25%, and more specifically 7 to 15% by weight relative to the total weight of the blend composition. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, water is present in the composition in an amount of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 35%, or about 40% by weight relative to the total weight of the blend composition.

[0039] The collagen-based blend composition of the present invention may optionally contain a plasticizer. According to one embodiment, optionally, in combination with one or more features of the various embodiments described above or below throughout this description, the collagen-based blend composition contains a plasticizer.

[0040] In a particular embodiment, optionally combined with one or more features of the various embodiments described above or below, the weight ratio of collagen to plasticizer is 100:5 to 100:60, more specifically 100:10 to 100:40, and even more specifically 100:10 to 100:30. In another embodiment, optionally combined with one or more features of the various embodiments described above or below, the plasticizer is present in the composition in such an amount that the weight ratio of plasticizer to collagen is about 100:5, about 100:10, about 100:15, about 100:20, about 100:25, about 100:30, about 100:35, about 100:40, about 100:45, about 100:50, about 100:55, or about 100:60.

[0041] In other specific embodiments, optionally, in combination with one or more features of the various embodiments described above or below throughout this description, the plasticizer may be glycerin, ethylene glycol, mono-, di-, or triesters of glycerin and carboxylic acid, for example (C3-C3 12Alkan acid, sulfated fatty acid, lecithin, xylitol, sorbitol, ethylene glycol, 1,2-propylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol (e.g., 200-10000 Da), polypropylene glycol, ethylene diglycol, propylene diglycol, ethylene triglycol, propylene triglycol, polyethylene glycol, polypropylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol The following are selected from the group consisting of tandiol, 1,6-hexanediol, 1,5-hexanediol, 1,2,6-hexanetriol, 1,3,5-hexanetriol, neopentyl glycol, trimethylolpropane, pentaerythritol, inositol, mannitol, triethanolamine, sorbitol ethoxylate, glycerin ethoxylate, pentaerythritol ethoxylate, sorbitol acetate, pentaerythritol acetate, formamide, acetamide, N,N-dimethylformamide, N,N-dimethylacetamide, and mixtures thereof.

[0042] Non-specific examples of sulfated fatty acids include sulfated castor oil, sulfated olive oil, sulfated soybean oil, and sulfated sunflower oil. Glycerin tri(C3-C) 12 Non-limiting examples of alkyl esters include glycerol triacetate, glycerol tributylate, glycerol trioleate, glycerol tripalmitate, and glycerol tristearate.

[0043] In other specific embodiments, optionally, in combination with one or more features of the various embodiments described above or below throughout this description, the collagen-based blend composition comprises glycerin, ethylene glycol, and triglycerin (C3-C3). 12The material comprises a plasticizer selected from the group consisting of alkyl esters, sulfated fatty acids, lecithin, and combinations thereof, more specifically the plasticizer being glycerin, and more specifically the amount of glycerin present such that the weight ratio of glycerin to collagen is 5:100 to 60:100, more specifically 10:100 to 40:100, and more specifically 10:100 to 30:100.

[0044] The collagen-based blend composition of the present invention further comprises a polyester having a melting point of 120°C or lower. The use of a polyester with this melting point has the advantage of lowering the processability temperature of the blend composition for conversion into other materials such as films, compared to polyesters with higher melting points, and thus avoiding the presence of water vapor. As a result, a homogeneous film with low roughness may be obtained. In contrast, with polyesters having a melting point higher than 120°C, such as polylactic acid (PLA) (see Table 11), no films could be extruded.

[0045] There are no restrictions on the types of polyester that can be used, as long as they have a melting point of 120°C or lower, especially at 101,300 Pa.

[0046] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polyester has a melting point of 115°C or lower, 110°C or lower, 100°C or lower, 90°C or lower, 80°C or lower, 70°C or lower, 60°C or lower, 55°C or lower, and particularly 101300 Pa.

[0047] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polyester has a melting point of 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 85°C or higher, and particularly 101300 Pa.

[0048] According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polyester is selected from the group consisting of polycaprolactone (PCL), polybutylene succinate co-adipate (PBSA), polyethylene adipate (PEA), polybutylene succinate (PBS), or polybutylene adipate terephthalate (PBAT), and combinations thereof. More specifically, the polyester is polycaprolactone (PCL) or polybutylene succinate co-adipate (PBSA).

[0049] According to one embodiment, optionally combined with one or more features of the various embodiments described above or below, the weight ratio of thermoplastic collagen to polyester is 1.3:1 to 1:18. According to another embodiment, optionally combined with one or more features of the various embodiments described above or below, the weight ratio of thermoplastic collagen to polyester is 1:1.2 to 1:18. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the weight ratio of thermoplastic collagen to polyester is about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 1:1, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, or about 1:20.

[0050] According to one embodiment, a collagen-based blend composition, a) Collagen, b) water; c) Plasticizers, especially glycerin, ethylene glycol, and triglycerin (C3-C3) 12 ) Selected from the group consisting of alkyl esters, sulfated fatty acids, lecithin, and combinations thereof, more specifically, the plasticizer is glycerin. b) A polyester having a melting point of 120°C or less, particularly polycaprolactone (PCL) or polybutylene succinate-co-adipate (PBSA), wherein the weight ratio of collagen to polyester is 2:1 to 1:20, more specifically 1.9:1 to 1:18, more specifically 1.3:1 to 1:18, and more specifically 1:1.2 to 1:18. More specifically, in the above embodiments, the weight ratio of collagen to plasticizer is 100:5 to 100:60, more specifically 100:10 to 100:40, and more specifically 100:10 to 100:30.

[0051] Furthermore, combinations of the collagen-based blend composition of the present invention with other proteins may provide further process improvements or modifications to mechanical properties such as tensile properties. Therefore, in one embodiment, the collagen-based blend composition may optionally further include soy protein or gluten in combination with one or more features of the various embodiments described above or below.

[0052] In another embodiment, optionally combined with one or more features of the various embodiments described above or below, the collagen-based blend composition is in the form of pellets.

[0053] It is also part of the present invention, namely a method for preparing a collagen-based blend composition as defined herein, a) A step of mixing thermoplastic collagen containing collagen, water, and especially a plasticizer with polyester, b) A step of melting and blending the mixture from step a) and extruding it, To form a method that includes

[0054] The present invention also, a) A step of mixing thermoplastic collagen containing collagen, water, and especially a plasticizer with polyester, b) A step of melting and blending the mixture from step a) and extruding it, This relates to collagen-based blend compositions as defined herein, which can be obtained by a method comprising the following.

[0055] It is also part of the present invention, namely a method for preparing a collagen-based blend composition as defined herein, a') A step of pre-mixing collagen powder, water, and especially a plasticizer, b') A step of mixing the composition of step a') with polyester, c') The process of melting and blending the mixture from step b') and extruding it, To form a method that includes

[0056] The present invention also, a') A step of pre-mixing collagen powder, water, and especially a plasticizer to provide a mixture, b') A step of mixing the mixture from step a) with polyester, c') The process of melting and blending the mixture from step b') and extruding it, This relates to collagen-based blend compositions as defined herein, which can be obtained by a method comprising the following.

[0057] In particular, in the above embodiments, the weight ratio of the composition of step a) or alternatively step a') to polyester is 75:25 to 5:95, more specifically 70:30 to 10:90. In particular, in the above embodiments, the weight ratio of collagen to polyester is 2:1 to 1:20, more specifically 1.9:1 to 1:18.

[0058] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the thermoplastic collagen of step a) is i) 40-75%, more specifically 50-70%, and even more specifically 55-65% collagen by weight. ii) 10-40% by weight, more specifically 15-35%, and even more specifically 20-30% water, and iii) Contains 5-50% by weight, more specifically 5-35%, and even more specifically 5-20% plasticizers, The weight percentage is expressed relative to the total weight of thermoplastic collagen, where the sum of all components of thermoplastic collagen is 100%.

[0059] The thermoplastic collagen in step a) is commercially available (Ekomat CF10 and CF30, Ekolber SL). Alternatively, it can be prepared by methods well known in the art, for example, as disclosed in European Patent No. 2727938.

[0060] In one embodiment, the thermoplastic collagen of step a) is optionally combined with one or more features of the various embodiments described above or below, as disclosed in European Patent No. 2727938, particularly in Example 1, but with adjustments to the amounts of water and plasticizer as needed.

[0061] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the thermoplastic collagen and polyester used in step a) are in the form of pellets.

[0062] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the mixture of step a') contains i) 40-75%, more specifically 45-55%, collagen by weight. ii) 10-40% by weight, more specifically 30-40% water, and iii) Contains 5-50% plasticizer by weight, more specifically 10-20%, The weight percentage is expressed relative to the total weight of the mixture in step a'), where the sum of all components in the mixture in step a') is 100%.

[0063] The collagen in step a') is commercially available (Kapro B95 SF, DCP). Alternatively, it can be prepared by methods well known in the art, for example, as disclosed in International Publication No. 2007104322.

[0064] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the collagen powder used in step a') is disclosed in International Publication No. 2007104322. More specifically, the collagen powder used in step a') is a dried collagen powder based on modified or partially modified fibril-forming collagen exhibiting an average molecular weight of at least 500 kDa, a solubility of 25% or more in water at 60°C, and an average particle size between 30 μm and 350 μm.

[0065] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polyester used in step b') is in the form of pellets.

[0066] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, step b') is carried out in a mixer, particularly an experimental z-blade mixer, for a suitable time and rpm conditions, for example, 100 rpm for 5 minutes.

[0067] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the melt blending and extrusion steps b) or c') are carried out in a co-rotating twin-screw extruder at an actual melting temperature of 120-130°C or less, as measured by a thermometer in either of the twin-screw extruders. For example, a 100 rpm co-rotating twin-screw extruder Leistritz GL27:D=27mm, L / D=36 may be used at a set extrusion temperature of 50°C-100°C from the feed zone to the die head at 4 kg / h to obtain two strands of 3 mm in diameter at the die outlet, which are then cooled before pelletization.

[0068] As described above, the collagen-based blend composition of the present invention can provide a sheet having a water solubility of 40% w / w or less. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the water solubility is 35% w / w or less, 30% w / w or less, 25% w / w or less, 20% w / w or less, 15% w / w or less, and 10% w / w or less.

[0069] The water solubility is a) i) collagen, ii) water, iii) a plasticizer in particular, and iv) a step of providing a 1 mm sheet at about 95 °C by a platen press from a blend composition containing a polyester having a melting point of 120 °C or lower, b) a step of cutting the obtained sheet into small pieces having a width and length of about 0.5 to 2 cm, c) a step of drying the sheet in a vacuum oven at about 160 °C until it reaches a constant weight, d) a step of collecting a sample of about 5 g from the sheet and measuring its initial weight (W i ), e) a step of immersing the sample of step c) in 100 mL of water at about 60 °C for about 1 hour with stirring, f) a step of separating and recovering the insoluble immersion sheet, g) a step of drying the immersion sheet in a vacuum oven at about 160 °C until it reaches a constant weight, h) a step of measuring the final weight (W f ) of the sheet of step g), and i) a step of applying the following formula: Water solubility (%) = (W i - W f ) / W i * 100 and can be determined by

[0070] Furthermore, the collagen-based blend composition of the present invention can provide a homogeneous film when subjected to blow film extrusion.

[0071] More specifically, the extrusion is carried out in an extruder at an actual melting temperature that is about +15 °C higher than the melting point of the polyester and less than 135 °C, measured by any of the thermometers of the extruder.

[0072] For the purposes of this invention, the term “homogeneous film” refers to the fact that no visible granules are observed. A method for evaluating the presence or absence of visible granules is to measure the surface roughness. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the blow-extruded film has low roughness. The roughness may be determined as shown in the following examples.

[0073] Further aspects of the present invention include: a) A step of melt-blending a collagen-based blend composition as defined herein, b) The process of extruding, casting, or blowing the blend from step a) into a solid article, This relates to a solid molded article that can be obtained by a method including the following.

[0074] According to one embodiment, the solid molded article may optionally be a film, an injection molded article, a blow molded article, a monofilament, a single-oriented filament, an extruded blow bottle, a 3D printed object, an injection molded object, or a machined piece, in combination with one or more features of the various embodiments described above or below.

[0075] The film according to the present invention may have a single layer (single-layer film) or two or more layers (multilayer film). Therefore, in one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the solid molded article is a film, more specifically a film selected from a single-layer film or a multilayer film.

[0076] In a more specific embodiment, optionally in combination with one or more features of the various embodiments described above or below, the film is a single-oriented film, more specifically a film oriented 2.5 to 6 times in the longitudinal direction.

[0077] The multilayer thermoplastic film according to the present invention may have 2 to 13 layers, particularly 2 to 11 layers, more preferably 2 to 9 layers, and even more preferably 2 to 7 layers. In certain embodiments, the multilayer thermoplastic film may have 3 or 5 layers, optionally in combination with one or more features of the various embodiments described above or below.

[0078] The multilayer film may include one or more layers containing caseinate, as disclosed in the patent document European Patent No. 2596051.

[0079] In a more specific embodiment, optionally in combination with one or more features of the various embodiments described above or below, the film is a single-layer film comprising a blended composition as defined herein, optionally further comprising soy protein or gluten.

[0080] In another, more specific embodiment, optionally in combination with one or more features of the various embodiments described above or below, the film is a multilayer film, where at least one layer of the multilayer film comprises a blended composition as defined herein, optionally further comprising soy protein or gluten, and the remaining layers comprise a polymer and optionally a compatibilizer.

[0081] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the film is a multilayer film comprising at least three layers having an A / / B / / C structure, wherein layer B comprises i) a collagen-based blend composition as defined herein, optionally further comprising soy protein or gluten, and ii) optionally a compatibilizer, and layers A and C comprise a polymer and optionally a compatibilizer.

[0082] In a particular embodiment, the polymer of the embodiment described above is selected from the group consisting of polyethylene (PE), polypropylene (PP), polycaprolactone (PCL), polyethylene adipate (PEA), polybutylene succinate-co-adipate (PBSA), polyhydroxyalkanoate (PHA), polyglycolide (PGA), polylactide (PLA), polylactide-co-glycolide (PLGA), polydioxanone (PDO), polybutylene succinate (PBS), polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), and polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), maleic anhydride grafted polyolefin (PE-g-MAH, PP-g-MAH), polyamide (PA), thermoplastic starch (TPS), thermoplastic cellulose, ethylene vinyl alcohol (EVOH), polyvinyl alcohol (PVOH), and combinations thereof. More specifically, the polymer is selected from the group consisting of polyethylene (PE), polycaprolactone (PCL), polybutylene succinate-co-adipate (PBSA), and combinations thereof.

[0083] In another specific embodiment, the compatibilizer of the above embodiment is selected from the group consisting of ethylene vinyl acetate (EVA), maleic anhydride grafted onto ethylene vinyl acetate copolymer (EVA-g-MAH), polyvinyl acetate (PVAc), vinyl acetate ethylene copolymer (VAE), ethylene ethyl acrylate copolymer (EEA), ethylene methyl acrylate copolymer (EMA), and combinations thereof.

[0084] In a particular embodiment, layers A and C are the same. More specifically, layers A and C comprise polymers selected from the group consisting of polyethylene (PE), polycaprolactone (PCL), polybutylene succinate-co-adipate (PBSA), and combinations thereof.

[0085] In another specific embodiment, layers A and C differ from those in the above embodiment. More specifically, each of layers A and C independently comprises polycaprolactone (PCL) or polybutylene succinate-co-adipate (PBSA).

[0086] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the film is a multilayer film comprising at least five layers having an A / / D / / B / / D / / A structure, wherein layer B comprises i) a collagen-based blend composition as defined herein, optionally further comprising soy protein or gluten, and ii) optionally a compatibilizer, layer A comprises the polymer defined above, and layer D comprises the compatibilizer defined above.

[0087] In one embodiment, optionally combined with one or more features of the various embodiments described above or below throughout this description, the film of the present invention has a thickness of 10 to 200 μm, more specifically 20 to 100 μm, when measured by a scanning electron microscope (SEM).

[0088] According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the solid molded article is selected from the group consisting of injection molded articles, blow molded articles, mechanical parts, and fused deposition modeling (FDM) filaments, and comprises a collagen-based blend composition as defined herein, optionally further comprising soy protein or gluten, and optionally a compatibilizer.

[0089] In another embodiment, optionally combined with one or more features of the various embodiments described above or below, the solid molded article is a bottle or another 3D multilayer article comprising at least three layers having an A / / B / / C structure, wherein layer B comprises a collagen-based blend composition as defined herein, optionally further comprising soy protein or gluten, and layers A and C comprise the polymers defined above.

[0090] In another embodiment, optionally combined with one or more features of the various embodiments described above or below, the solid molded article is a bottle or another 3D multilayer article comprising at least five layers having an A / D / B / D / A structure, wherein layer B comprises a collagen-based blend composition as defined herein, optionally further comprising soy protein or gluten, and layers A and C comprise the polymers defined above.

[0091] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the solid molded article is a film and has the following characteristics: a) The melt flow rate fluctuation at 90°C and a load of 9.6 kg is -50% or less after 7 days. b) The melt flow rate fluctuation at 120°C and a load of 9.6 kg is -50 or less after 23 days. c) The melt flow rate fluctuation at 90°C and a load of 9.6 kg was -30% or less after 7 days. d) The melt flow rate variation at 120°C and a load of 9.6 kg was -40% or less after 23 days. e) The oxygen permeability (OTR) at 23°C and 50% relative humidity is 600 cm³. 3 / m 2 ·days or less, f) The water vapor transmission rate (WVTR) at 23°C and 85% relative humidity is 800 g / m². 2 ·days or less, g) The tensile breaking strength in the mechanical direction is 3 to 50 mPa. h) Transverse tensile breaking strength is 3-30 mPa. i) The tensile elongation at break in the mechanical direction is 9-450%, and j) Transverse tensile elongation at break is 10-150%, It has one or more of the following.

[0092] Throughout the specification and claims, the word “including” and variations thereof are not intended to exclude other technical features, additives, components, or processes. Furthermore, the word “including” encompasses the case of “consisting of.” Further objects, advantages, and features of the present invention may become apparent to those skilled in the art upon consideration of the description or may be acquired through the practice of the present invention. The following examples and drawings are provided for illustrative purposes only and are not intended to limit the present invention. Furthermore, the present invention encompasses all possible combinations of the specific embodiments and preferred embodiments described herein. [Examples]

[0093] 1.Material The commercially available thermoplastic collagen grades Ekomat CF10 and CF30 are supplied by Ekolber SL. Ekomat collagen is prepared according to the method described in European Patent No. 2727938. Bovine collagen powder (DCP, supplied by the Netherlands, Kapro B95 SF) with a protein content of 95% or more, a fat content of 3% or less, and a 95% particle size of less than 200 μm. This corresponds to the dried collagen powder precursor disclosed in International Publication No. 2007104322 (Naturin). Food-grade bovine gelatin was supplied by Gelita in bloom grade 220 and mesh 18. 99.5% pure glycerin was provided by Tata Genaro. Polycaprolactone (PCL) pellets from Natureplast, PBI 012, Mw 50,000, melt flow rate (ISO 1133, 160℃, 2.16kg) 7g / 10min, and melting point 58℃. Natureplast poly(butylene succinate-cobutylene adipate) (PBSA) pellets PBE 001, melt flow rate (ISO 1133, 160℃, 2.16kg) = 4-5g / 10min, melting point 88℃. Natureworks polylactic acid (PLA) pellets Ingeo 4043D, melt flow rate (ASTM D1238, 210℃, 2.16kg) 6g / 10min, peak melting temperature 145-160℃. Dupont ethylene vinyl acetate EVA-g-MAH Bynel 3810 grafted with maleic anhydride, melt flow rate (ASTM D1238, 190°C, 2.16 kg) 2.6 g / 10 min, melting point 75°C.

[0094] 2. General procedure for preparing a blend containing collagen or gelatin and biodegradable polyester

[0095] Step 1. Preparation of a thermoplastic blend containing collagen and biodegradable polyester. Both commercially available Ekomat grade (composition shown in Table 1) and biodegradable polyester in pellet form were supplied in different ratios as shown in Table 3 and melt-blended in a 100 rpm co-rotating twin-screw extruder, Leistritz GL27: D=27 mm, L / D=36, at a set extrusion temperature of 50°C to 100°C from the supply zone to the die head at 4 kg / h, yielding two strands with a diameter of 3 mm at the die outlet, which were cooled before pelletization. A comparative blend CF0 was prepared similarly.

[0096] Step 2. Preparation of a thermoplastic blend containing collagen and biodegradable polyester. Thermoplastic collagen using Naturin was prepared by pre-mixing collagen powder (Kapro B95 SF), water, and glycerin in an experimental z-blade mixer at 100 rpm for 5 minutes. The resulting composition (shown in Table 2) and biodegradable polyester pellets were fed in different ratios as shown in Table 3 and melt-blended in a 50 rpm co-rotating twin-screw extruder, Leistritz GL27: D=27 mm, L / D=36, at a throughput of 4 kg / h and a set extrusion temperature of 50°C to 100°C from the feed zone to the die head, yielding two strands with a diameter of 3 mm at the die outlet, which were cooled before pelletization.

[0097] Step 3. Preparation of a comparative blend containing thermoplastic gelatin and biodegradable polyester. Thermoplastic gelatin (tGel) was prepared by pre-mixing gelatin and glycerin in an experimental z-blade mixer at 100 rpm for 5 minutes in weight ratios of 80% and 20% of the total mixed weight, respectively. The mixed material and biodegradable polyester were fed in different ratios as shown in Table 3 and melt-blended in a 50 rpm co-rotating twin-screw extruder, Leistritz GL27: D=27 mm, L / D=36, at a throughput of 4 kg / h and a set extrusion temperature of 50°C to 100°C from the feed zone to the die head, yielding two strands with a diameter of 3 mm at the die outlet, which were cooled before pelletization.

[0098] [Table 1]

[0099] [Table 2]

[0100] Table 3 shows the composition of compounds prepared by melt-blending component "a" (one of the following: thermoplastic collagen Ekomat, thermoplastic collagen Naturin, or thermoplastic gelatin (tGel)) and component "b" (biodegradable polyester), where a:b is the weight ratio of "a" to "b". The compound blend is named a+b.

[0101] [Table 3]

[0102] 3. Procurement of blow film samples Single-layer and triple-layer blow film samples were produced using one or three single-screw extruders, 20 mm diameter, L / D 30, on a Labtech LF 250 blow film extrusion line. For compound blends containing PCL, the extruder temperature profile was set to 50–100°C from the feed extruder zone to the metering extruder zone, and the die head temperature was maintained at 100°C. For compound blends containing PBSA, the extruder temperature was set to 50–110 or 120°C from the feed extruder zone to the metering extruder zone, and the die head temperature was maintained at 110–120°C. Three weeks after the production of the compound samples, the film samples were extruded.

[0103] 4. Testing and Evaluation i. Water solubility ii. Homogeneity of extruded films from compound blends iii. Heat processability - blow film extrusion iv. Meltflow rate (MFR) v. Breaking tensile stress and breaking strain of film, and water swelling vi.Oxygen transmission rate (OTR) vii. Water vapor transmission rate (WVTR) viii. Cell proliferation and cytotoxicity ix.3d printability x. Single-oriented monofilament xi. Thermoprocessability (injection molding, 3D printing blow film extrusion)

[0104] i. Water solubility Sample preparation for water solubility testing Samples were prepared by supplying materials to an internal mixer by Brabender in the ratios shown in Table 4. Mixing conditions were 90°C, 60 rpm, and 5 minutes after filling with components and stabilizing the torque. After mixing, the blended compounds were collected and a 1 mm thick sheet was produced at 95°C using an experimental platen press by Dr. Collin. The sheet was cut into pieces with a width and length of approximately 0.5–2 cm.

[0105] Evaluation Test Procedure 3-5 grams of the sample prepared above were weighed using an analytical balance and dried in a vacuum oven at 160°C until the weight was constant. Initial dry weight of the sample (W) i The weight was recorded to four decimal places. The samples were immersed in 100 mL of distilled water and stirred manually or orbitally in a thermostat-controlled bath at 60°C for 1 hour. Samples containing gelatin were completely solubilized, while samples containing collagen were only partially solubilized. In the latter case, the insoluble portion was separated with the help of a strainer, collected, and dried in a vacuum oven at 160°C until dry weight was obtained. Final dry weight (W) f The values ​​were recorded to four decimal places. To determine the percentage of dissolved protein, calculations regarding water solubility were performed considering the initial weight, initial moisture content, and dry weight, according to the following formula: Water solubility (%) = (W i -W f ) / W i *100 In the formula, W i This is the dry weight of the sample before immersion in water (dried in a vacuum oven at 160°C until it reaches a certain weight), and W f This is the dry weight of the sample after immersion in water (dried in a vacuum oven at 160°C until a certain weight is reached).

[0106] The following results were obtained.

[0107] [Table 4]

[0108] ii and iii. Homogeneity and processability of extruded blown films Pellet samples having the compositions shown in Tables 5 and 6 were prepared as described in steps 1, 2, and 3 above. The films were extruded as detailed in Section 3 above. Roughness measurements (Ra, Rz values, at least 10 measurements in the longitudinal direction) were performed using a Brukner profilometer model DektakXT in accordance with the ISO 4287 test standard.

[0109] [Table 5] ×Cannot be extruded ▲Although extrudeable, the film exhibits heterogeneity, particularly in the case of Ekomat CF30, with significant variations in thickness and very high adhesiveness. ○ Extrusion and homogeneous film with no significant problems (no visible granules) 1 Ekolber SL CF30 film extrusion 3 days ago 2 Ekolber SL CF30 film extrusion 3 weeks ago

[0110] [Table 6] The average Rz amplitude represents the average of the maximum peak-to-trough distances obtained for each of the divided fundamental lengths of the measured length. Ra (Arithmetic Mean Roughness): This is the arithmetic mean of the deviations of the roughness profile from the center line along the evaluation length lm.

[0111] The results demonstrate that the compositions according to the present invention (CF30+PCL in 70:30, 50:50, or 30:70 ratios) provide a significant decrease in water solubility compared to blended compounds containing Ekomat-grade CF30 and thermoplastic gelatin, and simultaneously provide a single-phase film free of visible granules in the film (not shown in the results).

[0112] Blow film extrusion of CF30 pellets manufactured by Ekolber three weeks prior to extrusion proved impossible because the molten material barely flowed into the extruder at the set processing temperature. Furthermore, a blend containing glycerin-free collagen, water, and PCL, CF0, was found to be unprocessable by film extrusion several days after its preparation (data not shown in table).

[0113] iv. Meltflow rate (MFR) The internal procedure based on ISO 1133 was followed. 3-5 gram pellet samples were filled into the cylinder of the MFR measuring device, the sample was manually compressed with a rod, the piston was inserted into the cylinder, and it was ensured that the sample had been at the test temperature for at least 15 minutes before filling. If the test temperature was 90°C, the sample remained in the cylinder for a 5-minute preheating time before applying the test load to the piston and starting the test. If the test temperature was 100°C or higher, the preheating time was 1 minute. The extruded material flowing from the test machine die was cut and weighed every 2 minutes for 10 minutes. Extruded samples showing bubbles were discarded, and at least three measurements were performed.

[0114] All (pellet) samples were packed in sealed polyethylene bags at 23±2°C before testing 3, 7, and 23 days after the manufacturing date.

[0115] Tables 7-10 below show the MFR values ​​measured at 0, 3, 7, and 23 days for blends of CF30+PCL, CF30+PBSA, and CF10+PBSA, as well as the MFR difference (g / 10 min and %) at 7 and 23 days relative to the initial MFR value. For comparison, the MFR values ​​of thermoplastic collagens CF30 and CF10 were also measured. Furthermore, Table 10 shows the MFR test data for CF10 pellet samples stored at -18°C and room temperature after 3, 7, and 23 days.

[0116] [Table 7]

[0117] [Table 8]

[0118] [Table 9]

[0119] [Table 10]

[0120] result The table above shows that 23 days after the supplier's manufacturing date, Ekomat CF30 and CF10 showed little to no flow, did not flow homogeneously, and exhibited a very irregular surface (data not shown in the table). Compound samples containing 10% PCL or PBSA also showed similar problems and a significant decrease in melt fluidity after 7 and 23 days. In contrast, the compound samples according to the present invention showed a less significant decrease in MFR after 23 days from the compound sample preparation.

[0121] The melt flow rate test results are consistent with the fact that Ekomat, as well as compounds containing Ekomat and 10% PCL or PBSA, could not be extruded to produce blown film samples even after being stored at room temperature for 21 days, while compound samples containing Ekomat and at least 30% PCL or PBSA could be extruded.

[0122] Table 10 shows the MFR values ​​of Ekomat CF10 pellets stored at 23°C and -18°C. Storing Ekomat under freezing conditions minimizes the decrease in melt flow observed in the same pellets stored at room temperature, demonstrating the influence of storage temperature on the time course of melt flow. This fact highlights the potential limitations and problems in the industrial management of collagen thermoplastic pellets, as Ekomat grades must be stored under freezing conditions or, if stored at room temperature, must be thermoformed within a very short time from the Ekomat manufacturing date.

[0123] In two additional MFR tests, the test temperatures were set to 125°C and 110°C to increase the melt flow of Ekomat CF10. However, this caused bubbles to form in the CF10 extruded due to water vaporization, and therefore melt flow rate data could not be obtained. In contrast, CF10 + PBSA 30:70 tested at high temperatures provided a smooth, homogeneous, and bubble-free extruded material.

[0124] conclusion The test results showed that the compound according to the present invention, containing a blend of Ekomat and at least 30% PCL or PBSA, provided higher melt-flow stability during storage at room temperature than comparative thermoplastic collagen samples without a polyester compound blend. Furthermore, when stored at room temperature, the molten Ekomat flowed very little or irregularly after 7 days, and especially after 23 days, following production. Increasing the test temperature or water content may increase the Ekomat MFR, but this resulted in water vaporization and a decrease in extruder quality.

[0125] v. Mechanical properties (tensile stress-strain) and water swelling of blown film Film samples (monolayer films having the composition shown below and prepared as described in Section 3) were tested 7 days after extrusion. Forty-eight hours prior to testing, the samples were conditioned and maintained at a controlled temperature of 23±2°C and relative humidity (RH) of 45±5% or 100%. Tests were performed to determine the tensile breaking strength and tensile elongation in the mechanical direction (MD) and transverse direction (TD) of the film samples, according to ISO 527-3. Swelling in water was determined by the weight difference of the film sample before and after immersion in water at 23±2°C for 4 hours. Swelling, expressed as a percentage, was determined as (W1-W2 / W1)*100, where W1 and W2 are the weights of the sample before and after immersion in water, respectively.

[0126] [Table 11]

[0127] The swelling of CF30 + PCL (30:70) (in water at 23°C for 4 hours) was 34%.

[0128] result Extrusion of single-layer blow-blown films using ET-G15W35 thermoplastic collagen presented problems, and the sample quality was unacceptable. The films had uneven thickness, a very rough surface, and became extremely brittle after 24 or 48 hours.

[0129] A single-layer film containing Ekomat CF10 and CF30, in which the Ekomat pellets were stored at -18°C before extrusion to avoid loss of their melt fluidity, was optically single-phase but became very brittle after 1 day and also exhibited high water solubility (Table 4, CF30 + PCL100:0).

[0130] In contrast, the thermoplastic blend film containing Ekomat and polyester according to the present invention provided better extrudeability and mechanical properties, less brittleness, and a higher elongation at break.

[0131] Furthermore, they exhibited significantly lower water solubility than single-layer films containing CF10 and CF30 (Table 4 CF30+PCL blends 70:30, 50:50, 30:70), and they provided better resistance to high %RH atmospheres (100%, data not shown in table).

[0132] The minimum processing temperature required to process the blend resulted in bubbles and foaming of the extruded material due to water vaporization, making it impossible to extrude a single-layer film of a 50:50 blend of PLA (which has a melting point of approximately 145-160°C) and CF30 (i.e., outside the claims). To melt the PLA material, the extruder temperature profile at the die was set low at 170°C.

[0133] vi. Oxygen permeability (OTR) of blown film samples The oxygen permeability (OTR) of the film samples was analyzed using a Mocon Ox-Tran® transmission analyzer at 23°C and 50% relative humidity, according to the DIN 53 380 standard test method.

[0134] [Table 12]

[0135] The films according to the present invention exhibited, as expected, lower oxygen barrier (i.e., higher OTR values) than films based on thermoplastic collagen. Furthermore, they provided a better (higher) oxygen barrier than low-density polyethylene films (data not shown).

[0136] vii. Water vapor transmission rate (WVTR) of blown film samples Water vapor transmission rates were determined using a Mocon Permatran-W® transmission analyzer at 23°C and 85% relative humidity, according to the DIN 53-122 standard test method. Prior to testing, samples were conditioned at 23°C and 50% RH for 48 hours.

[0137] [Table 13]

[0138] The film according to the present invention improved water vapor barrier properties by more than 30% compared to a collagen film that does not contain PCL.

[0139] viii. Cell proliferation and cytotoxicity The cytotoxicity of films prepared from the blend according to the present invention was evaluated and compared with other films. The following films were tested. Film 1: Viscofan Collagen Cell Carrier® membrane (CCC) Film 2: CF30 + PCL (30:70) Film 3: CF30 + PCL (70:30) Film 4: 100% PCL

[0140] Films 2 and 3 were prepared by heat-pressing pelletized collagen or collagen + PCL using an experimental heated platen press to obtain plates with a thickness of approximately 1 mm.

[0141] Human dermal fibroblasts were seeded onto films pre-sterilized with 25 kGy (gamma irradiation). As a positive control (C+), cells were seeded onto standard commercially available polystyrene well plates (Corning ref 3595) treated for optimal cell adhesion. As a negative control, untreated commercially available polystyrene well plates (Corning ref 351178) were used. To seed onto the films, cell suspensions were prepared, and 50 μl droplets were deposited onto each film, resulting in a cell density of 14,000 cells / cm². 2The final density was obtained. The samples were placed in 24-well plates and incubated at 37°C and 5% CO2 for 3 hours. They were then immersed in 1 mL of complete medium. After 24 hours of incubation, four replicates of each sample were analyzed by WST-1 and two replicates were analyzed by Live / Dead.

[0142] The WST-1 assay was performed using the commercially available "WST-1 Cell Growth Reagent" kit from Roche Applied Science. The film was incubated in 300 μl of serum-free WST-1 solution at 37°C and 5% CO2 for 4 hours. Subsequently, 100 μl of each sample was extracted and analyzed for absorbance at 440 nm using a plate spectrophotometer (Sinergy HT, Biotek).

[0143] For the Live / Dead assay, samples were washed with saline buffer and incubated in calcein solution at 37°C and 5% CO2 for 20 minutes. The solution was removed, and dead cells were stained with propidium iodide solution for 5 minutes. Samples were washed with saline buffer, and fluorescence images were acquired using a confocal microscope (Stellaris 5, Leica Geosystems, Switzerland).

[0144] As shown in Figure 1, cell viability in the films showed significant differences among different types of samples. Films 1 (Viscofan Collagen Cell Carrier® membrane (CCC)) and 2 (CF30+PCL (30:70)) showed the highest viability, indicating that these were the samples with the highest cell adhesion. A slight decrease in cell viability was observed in film 2, but it was not statistically significant. Film 3 (CF30+PCL (70:30)) showed a viability value of 50%, which was slightly lower than that of films 1 and 2. Finally, film 4 (100% PCL) and the negative control (data not shown) both showed the same values, indicating a significant decrease in cell viability. None of the films showed any signs of toxicity.

[0145] ix.3D printability The following pellet samples were used.

[0146] [Table 14]

[0147] For printing these materials, a domoBIO 2A (Domotek, Spain) printer was used, configured with a pellet extruder head (Mahor V4) and a high-performance heated bed. Additionally, a Buildtack printing surface was used to ensure proper adhesion of the materials. Due to the low melting point of the materials, the hopper fan was kept running to prevent the materials from melting before entering the extruder screw and blocking them. The parameters used for printing were as follows:

[0148] [Table 15]

[0149] Figure 2 shows printing materials having the blend according to the present invention. All materials containing the compound according to the present invention were printable under the conditions described. In contrast, Ekomat CF30 could not be printed. The extruded material was highly irregular, and there was no homogeneous feed.

[0150] x. Monofilament extrusion As described in Procedure 1, Ekomat CF30 grade and PCL were both supplied in pellet form in a 1:1 weight ratio and melt-blended in a 150 rpm co-rotating twin-screw extruder, Leistritz GL27: D=27mm, L / D=36, at a set extrusion temperature of 70°C-90°C from the supply zone to the die head at 6 kg / h, yielding two strands of 3 mm diameter at the die exit, which were cooled before being continuously wound into filament reels.

[0151] After 24 hours, the filament reel was unwound and uniaxially stretched using a Collin Teach Line MDO-AT. The stretching process consists of passing a preheated filament through a series of rollers at 45°C. The rollers rotate at a specified constant angular velocity, with each roller moving faster than the previous one. A larger difference in roller speed results in greater stretching of the monofilament. The orientation ratio is defined herein as v2 / v1, where v1 and v2 are the speeds of the first and subsequent rollers in meters per minute.

[0152] [Table 16] *Orientation ratio: Ratio of the speed (m / min) of the second (tensile) nip roll to the first nip roll.

[0153] The results obtained showed that the monofilament containing Ekomat and PLC according to the present invention exhibited a significant increase in tensile strength when stretched.

[0154] xi. Thermoprocessability (blow film extrusion, injection molding, 3D printing) As shown in the table below, compound blends of Ekomat CF10 or CF30 and PCL or PBSA in ratios of 70:30 to 10:90 are more easily heat-processed, while high-content Ekomat (90:10) resulted in extremely difficult processes or were not industrially feasible.

[0155] [Table 17]

[0156] Prior art documents European Patent No. 2727938 International Publication No. 2007104322 European Patent No. 2596051 ISO 1133-3(Third edition,1997-01-15).Plastics-Determination of the melt mass-flow rate(MFR)and the melt volume-flow rate(MVR) of thermoplastics ISO 527-3(First edition,1995-08-01).Plastics-Determination of tensile properties-Part 3:Test conditions for films and sheets ISO 4287(First edition Premiere edition 1997-04-01).Geometrical Product Specifications(GPS)-Surface texture:Profile method-Terms, definitions and surface texture parameters

[0157] For completeness, various aspects of the present invention are described in the following numbered clauses.

[0158] Clause 1. A blended composition, i) Collagen, ii) water; iii) Plasticizers in particular, and iv) Containing polyester having a melting point of 120°C or lower, A blended composition in which the weight ratio of collagen to polyester is 2:1 to 1:20.

[0159] Clause 2. A blend composition according to Clause 1, which is meltable.

[0160] A blended composition according to any one of Clauses 1 to 2, which is in a solid state at a temperature of 40°C or below, or at room temperature (20-25°C), and more specifically in the form of solid pellets.

[0161] Clause 4. A blended composition according to any one of Clauses 1 to 3, which can be thermoformed by a step of melt-blending a collagen-based blended composition as defined herein, or by a step of extruding, casting, or blowing the blend of step a) onto a solid article.

[0162] Clause 5. A blended composition according to any one of Clauses 1 to 4, which can provide a homogeneous film when subjected to blow film extrusion.

[0163] A blended composition according to any one of Clauses 1 to 5, which can provide a sheet having a water solubility of 40% w / w or less.

[0164] Clause 7. A blended composition according to any one of Clauses 1 to 6, wherein the collagen is partially denatured.

[0165] Clause 8. A blended composition according to any one of Clauses 1 to 7, wherein the polyester is selected from the group consisting of polycaprolactone (PCL), polybutylene succinate-co-adipate (PBSA), polyethylene adipate (PEA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof.

[0166] Clause 9. The blended composition according to Clause 8, wherein the polyester is polycaprolactone (PCL) or polybutylene succinate-co-adipate (PBSA).

[0167] Clause 10. A blended composition according to any one of Clauses 1 to 9, wherein water is present in an amount of 5 to 40% by weight relative to the total weight of the blended composition.

[0168] Clause 11. A blended composition according to any one of Clauses 1 to 10, wherein the weight ratio of collagen to polyester is 1.9:1 to 1:18.

[0169] Clause 12. A blended composition according to any one of Clauses 1 to 11, comprising a plasticizer.

[0170] Clause 13. Plasticizers may be glycerin, ethylene glycol, glycerin tri(C3-C) 12 ) A blended composition according to Clause 12, selected from the group consisting of alkyl esters, sulfated fatty acids, lecithin, and combinations thereof.

[0171] Clause 14. A blended composition according to any one of Clauses 12 to 13, wherein the weight ratio of collagen to plasticizer is 100:5 to 100:60.

[0172] Clause 15. A blended composition according to any one of Clauses 1 to 14, further comprising soy protein or gluten.

[0173] Clause 16.a) A step of melt-blending a blended composition as described in any one of Clauses 1 to 15, b) The process of extruding, casting, or blowing the blend from step a) into a solid article, A solid molded article that can be obtained by a method including the following.

[0174] Clause 17. A solid molded product as described in Clause 16, selected from the group consisting of films, injection molded articles, blow molded articles, monofilaments, single-oriented filaments, extruded blow bottles, 3D printed objects, injection molded objects or machined pieces, and filaments for 3D printing by fused deposition modeling (FDM).

[0175] Clause 18. A solid molded article as described in Clause 17, which is a film selected from the group consisting of single-layer films and multi-layer films.

[0176] Clause 19. The following characteristics: a) The melt flow rate fluctuation at 90°C and a load of 9.6 kg is -50% or less after 7 days. b) The melt flow rate fluctuation at 120°C and a load of 9.6 kg is -50 or less after 23 days. c) The melt flow rate fluctuation at 90°C and a load of 9.6 kg was -30% or less after 7 days. d) The melt flow rate variation at 120°C and a load of 9.6 kg was -40% or less after 23 days. e) The oxygen permeability (OTR) at 23°C and 50% relative humidity is 600 cm³. 3 / m 2 ·days or less, f) The water vapor transmission rate (WVTR) at 23°C and 85% relative humidity is 800 g / m². 2 ·days or less, g) The tensile breaking strength in the mechanical direction is 3 to 50 mPa. h) Transverse tensile breaking strength is 3-30 mPa. i) The tensile elongation at break in the mechanical direction is 9-450%, and j) Transverse tensile elongation at break is 10-150%, A solid molded article as described in Clause 18, having one or more of the following:

[0177] Clause 20. Use of any one of the blend compositions described in Clauses 1 to 15 as a support for in vitro assays.

[0178] Clause 21. Use of any blended composition described in any one of Clauses 1 to 15 or any solid molded article described in any one of Clauses 16 to 19 in the food industry, pet food industry, agricultural industry, or pharmaceutical industry.

Claims

1. A blended composition, i) Collagen, ii) water; iii) Plasticizers, and iv) Containing polyester having a melting point of 120°C or lower, A blended composition in which the weight ratio of collagen to polyester is 2:1 to 1:

20.

2. The blend composition according to claim 1, which is in a solid state at a temperature of 40°C or lower and is meltable.

3. The blended composition according to any one of claims 1 to 2, wherein the polyester is selected from the group consisting of polycaprolactone (PCL), polybutylene succinate-co-adipate (PBSA), polyethylene adipate (PEA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof.

4. The blend composition according to claim 3, wherein the polyester is polycaprolactone (PCL) or polybutylene succinate-coadipate (PBSA).

5. The blended composition according to any one of claims 1 to 4, wherein water is present in an amount of 5 to 40% by weight relative to the total weight of the blended composition.

6. The blended composition according to any one of claims 1 to 5, wherein the weight ratio of the collagen to the polyester is 1.9:1 to 1:

18.

7. The plasticizer is glycerin, ethylene glycol, or triglycerin (C ３ -C １２ A blended composition according to any one of claims 1 to 6, selected from the group consisting of alkyl esters, sulfated fatty acids, lecithin, and combinations thereof.

8. The blended composition according to any one of claims 6 to 7, wherein the weight ratio of the collagen to the plasticizer is 100:5 to 100:

60.

9. A blended composition according to any one of claims 1 to 8, further comprising soy protein or gluten.

10. a) A step of melt-blending the blended composition according to any one of claims 1 to 9, b) A step of extruding, casting, or blowing the blend from step a) into a solid article, A solid molded article that can be obtained by a method including the following.

11. A solid molded article according to claim 10, selected from the group consisting of films, injection molded articles, blow molded articles, monofilaments, single-oriented filaments, extruded blow bottles, 3D printed objects, injection molded objects or machined pieces, and 3D printing filaments by fused deposition modeling (FDM).

12. The solid molded article according to claim 11, wherein the film is selected from the group consisting of single-layer films and multi-layer films.

13. The following characteristics: a) The melt flow rate variation at 90°C and a load of 9.6 kg is -50% or less after 7 days. b) The melt flow rate fluctuation at 120°C and a load of 9.6 kg is -50 or less after 23 days. c) The melt flow rate fluctuation at 90°C and a load of 9.6 kg is -30% or less after 7 days. d) The melt flow rate variation at 120°C and a load of 9.6 kg is -40% or less after 23 days. e) The oxygen permeability (OTR) at 23°C and 50% relative humidity is 600 cm³. ３ / m ２ ・Days or less f) The water vapor transmission rate (WVTR) at 23°C and 85% relative humidity is 800 g / m². ２ ・Days or less g) The tensile breaking strength in the mechanical direction is 3 to 50 mPa. h) Transverse tensile breaking strength is 3 to 30 mPa. i) The tensile elongation at break in the mechanical direction is 9 to 450%, and j) Transverse tensile elongation at break is 10-150%, A solid molded article according to claim 12, having one or more of the following.

14. Use of the blend composition according to any one of claims 1 to 9 as a support for an in vitro assay.

15. Use of the blended composition according to any one of claims 1 to 9 or the solid molded article according to any one of claims 10 to 13 in the food industry, pet food industry, agricultural industry, or pharmaceutical industry.

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