A green technological method for the production of collagen peptides

Abstract

The invention relates to method for obtaining collagen from animal sources such as fresh or saltwater fish, chicken, eggs or from parts of these animal sources that can be considered waste, and to a collagen hydrolysate obtained as a result of this method.

Description

[0001] DESCRIPTION

[0002] A GREEN TECHNOLOGICAL METHOD FOR THE PRODUCTION OF COLLAGEN PEPTIDES

[0003] TECHNICAL FIELD

[0004] The invention relates to a green technological method for obtaining collagen peptides from collagen-containing animal sources such as fresh or saltwater fish, chicken, eggs or from parts of these animal sources that can be considered waste, and to collagen peptides obtained as a result of this method.

[0005] PRIOR ART

[0006] Collagen acts as an inter-tissue adhesive and durability provider and constitutes 30% of the human body and 75% of the skin. Collagen is a structure in the human body that is lost with age. In collagen deficiency, serious problems such as wrinkles on the skin, signs of aging, problems in bones and joints, and bone resorption are seen. For this reason, individuals need to intake a certain amount of collagen into their bodies. This value is given as 10 grams per day in individuals over the age of 20.

[0007] In the art, collagen is mostly obtained from cattle, pig, and fish products. In Turkey, while various methods are applied for collagen production, mostly by-products of cattle are used. However, the production processes in the present art involving the use of these raw materials are quite insufficient and have process steps that are costly, labor intensive and time-consuming. The main reason for this is that obtaining collagen from by-products of animal sources requires very lengthy periods and complex process steps. In order for collagen and collagen-containing products to be available in the market at affordable prices, it is necessary to reduce labor costs, lower processing expenses, and complete the processes in shorter periods.

[0008] Since collagen is a food source that individuals aged 20 and over should take in daily amounts, it should be a more producible product in the market. For this, a sustainable and easily achievable production method will be able to provide innovation for the relevant technical field. In the art, high-cost acidic and / or enzymatic methods are generally used to obtain collagen. Traditional methods of acidic / basic / enzymatic extraction and hydrolysis processes are complex methods with very long process periods and low-efficiency that involve excessive consumption of resources such as energy.

[0009] With the increasing awareness of the environment day by day, the concept of green technology has gained more importance in recent years. In this context, the aim is to reduce the production and consumption of substances hazardous to the environment and human health, to reduce the amount of energy consumed, and to increase the use of renewable resources. Instead of traditional solvents used widely in the food, chemical, and pharmaceutical industries, it is recommended to use solvents that are not harmful to health and have high extraction efficiency. Green solvents are solvents that are nontoxic, recyclable, non-volatile, and do not require high costs for synthesis.

[0010] Some of the advantages of deep eutectic solvents are that they are easier to synthesize, more stable, biodegradable and have low toxicity values. Due to these properties, it has become frequently preferred in extraction processes. Another property of deep eutectic solvents is that their melting point is lower than the melting point of each constituent compound.

[0011] The main reason for the use of deep eutectic solvents in the extraction process is their high solubilization capabilities, that is, their ability to give / receive protons and electrons, which facilitates the formation of hydrogen bonds between molecules. In addition, tunable physicochemical properties of deep eutectic solvents such as viscosity and polarity are also of importance in the extraction process.

[0012] Likewise, carrying out the necessary drying processes during the production of collagen from animal sources or their wastes also has technical problems. It has been determined that in order to obtain collagen with high efficiency, the moisture of animal pieces must be reduced below 10% and the fat should be reduced below 5%.

[0013] Research and development activities should be implemented to carry out fat and moisture separation processes in animal sources used as raw materials for collagen production with higher efficiency, in an environmentally friendly and low cost manner. As a result, a method for obtaining environmentally friendly sustainable collagen peptides in a short period, with low costs, less energy consumption and waste has become a necessity in the relevant technical field.

[0014] SUMMARY OF THE INVENTION

[0015] The invention relates to a method for obtaining collagen peptides from animal sources and parts of these animal sources considered as waste.

[0016] The main object of the invention is to introduce a method for obtaining collagen peptides with high efficiency in short periods and low costs. In a preferred embodiment, wastes from animal sources are used as a source. In this way, lower costs can be achieved for obtaining unit collagen peptides.

[0017] Another object of the invention is to introduce a method for obtaining high-purity collagen peptides using environmentally friendly green solvents that cause less waste.

[0018] Another object of the invention is to introduce a method for obtaining unit collagen peptides in which less energy is spent. In order to achieve this, regulations should be made to reduce the use of solutions that pose a danger to the environment and human health, to reduce the energy consumed, and to utilize and reduce the amount of waste.

[0019] Another object of the invention is to introduce a method for obtaining collagen peptides with low molecular weight. Owing to this property, it can be used in the fields of cosmetics, pharmaceuticals, and medical techniques, and particularly in the field of food technology.

[0020] Likewise, carrying out the necessary drying processes during the production of collagen from animal sources or their wastes also has technical problems. It has been determined that in order to obtain collagen with high efficiency, the moisture of animal pieces must be reduced below 10% and the fat should be reduced below 5%.

[0021] DETAILED DESCRIPTION OF THE INVENTION In this detailed description, the subject of the invention relates to a method for obtaining collagen peptides from animal sources such as fresh or saltwater fish, chicken, eggs or from parts of these animal sources that can be considered waste, and to collagen peptides obtained as a result of this method, and is described only with the examples that will not create any limiting effect for a better understanding of the subject.

[0022] In the invention, collagen is a structural protein that is present in bodies of individuals. As is known, there are 28 types of collagen reported in the literature. In this invention, no information is provided regarding the obtaining of type-1 collagen specifically. The method provided in the present invention can be used particularly for the type of collagen that is desired to be obtained. In this aspect, the present invention is not limited to the obtaining of any special type of collagen.

[0023] In this invention, animal sources are preferably used as a source of collagen. In this invention, species that contain rich collagen in their bodies can be used as animal sources. Preferably, fish, chickens, cattle, small ruminants, pigs or eggs can be used as animal sources. However, the method of the present invention can be applied to, but not limited to, all animal sources for obtaining collagen.

[0024] Animal sources referred to as a term in the invention may include animals suitable for use for the production of any type of collagen, rich or non-collagen-rich. In the invention, animal source refers to the initial raw material. The expression "collagen source" refers to pre-treated animal sources.

[0025] Another aspect of the invention is that parts of animal sources that can be considered as waste can be used as collagen sources in order to provide certain technical advantages (such as reducing costs and recycling waste). For example, but not limited to, fish bones, skin, scales, and spines can be used as waste animal sources. In some cases, for convenience, it may be expressed simply as "source"; however, the expressions animal sources and / or waste animal sources used for obtaining collagen should be deduced from this source expression. Accordingly, when using the term waste animal sources, the person skilled in the art will be able to access this information.

[0026] The method subject to the invention may cause technical problems such as the use of solutions harmful to humans and the environment for obtaining collagen peptides, increased energy consumption due to prolonged process periods, and the generation of greater amounts of waste a result of the processes. With the method subject to the present invention, it is possible to consume less energy by carrying out processes in shorter periods, and to use solutions that do not pose a danger to the environment and human health, and to reduce waste generation compared to the embodiments in the present art.

[0027] The process steps in the method of the invention are of importance for obtaining the final product collagen peptides. The lack of one of these processing steps will not make it possible to obtain collagen peptides with the target efficiency and technical properties. Method for obtaining collagen peptides of the invention comprises the process steps of; i. pre-treating animal sources and / or waste animal sources, ii. carrying out extraction and hydrolysis processes on pre-treated collagen sources, iii. carrying out purification processes on collagen sources subjected to process step ii), iv. carrying out drying processes.

[0028] The inventors of the present invention introduce a sustainable and technically advantageous method for obtaining collagen peptides for the relevant technical field. For this purpose, the details of the i-iv process steps resulting from research-development activities for the relevant technical field are presented.

[0029] / . Pre-treating animal sources and / or waste animal sources

[0030] In this process step, processes are carried out in order to clean animal sources and to apply the next processing steps at high efficiency.

[0031] Cleaning of animal sources and / or waste animal sources, reduction to small sizes and application of the first drying processes

[0032] The pre-treatments carried out in the invention are the preparatory stage for the raw materials. The cleaned waste is reduced to smaller pieces. This process is carried out to increase the surface area of the pieces and to provide a more effective raw material environment for the next processing steps. As a result of this process, the cleaned sources are reduced to a value between 1 to 30 mm.

[0033] The first drying process is carried out to dry the sources that have been reduced to the target sizes. Said process is carried out at a temperature in the range of 40 to 95 °C. The first drying process is carried out to ensure that the particle size of the raw material reaches the target range during the grinding stage. Subsequently, the raw materials are pulverized.

[0034] One of the most important advantages of the method subject to the invention is obtaining collagen peptides while the sources are in dry from instead of wet form. The method subject to the invention is more advantageous than the methods in the present art in terms of high stocking times and low energy costs in high amounts of capacity stockpiling of sources. The advantage of stocking raw materials in dry form prevents the degradation of the protein-based product, while the pulverization process facilitates the use of the source in the process steps.

[0035] Processes of grinding sources

[0036] In this process step, the sources are pulverized. Preferably, the grinding processes are carried out until the sources are reduced to powder particles at a value of 150 to 350 microns. Other parts that cannot be ground and are outside these values are separated from the sources. This process can be carried out by means of any separator. With the application of this process step, large-sized impurities are separated, the pre-treatment of the raw material with increased surface area is carried out with high efficiencies, and pre-treatment periods are shortened.

[0037] Separation of non-collagen parts from sources and swelling of tissue matrices

[0038] For said process step, the pulverized sources are treated with at least one basic solution. Here, the basic solution can be at least one of the group of sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. Depending on the type of solvent used, the basic solution can be at a value in the range of 0.1 M to 1 M. The most important issue here is the ratio between the source and the amount of solvent. In a preferred embodiment, if it is in a ratio of basic solution / collagen source, this value is between 1 :2 to 1 :10 by weight. By carrying out this process step, it is possible to remove non-collagen proteins in ground sources and to swell tissue matrices. With the processes carried out in this range of values, the targeted swelling in the matrices is achieved, thus increasing the mass transfer rate in the tissue matrix, separating non-collagen proteins and fats, and facilitating collagen extraction. This process period is at a value between 30 minutes to 12 hours.

[0039] Carrying out the first washing and straining processes

[0040] The first washing and straining processes are carried out in order to neutralize the basic solution used in the previous process step. If preferred, this process can be performed more than once. In this way, it is ensured that the pH value of the source is neutralized.

[0041] Processes of fat removal with solvents

[0042] The process of washing with solvent carried out after the first washing and straining processes provides the fat removal process of the raw materials. In a preferred embodiment, if it is in a ratio of solvent / collagen source, this value is between 1 :2 to 1 :10 by weight. This process period is at a value between 10 minutes to 5 hours.

[0043] Carrying out the second washing and straining processes

[0044] The second washing and straining processes are carried out in order to remove the solvent and fattiness factors used in the previous process step. If preferred, this process can be performed more than once.

[0045] With the previous process steps, it is possible to reduce the pH value of the raw material particles with a pH value in the range of 8-12 to neutral values and to remove the impurities that may remain on the particles.

[0046] Carrying out demineralization process

[0047] Demineralization processes are carried out for the sources subjected to washing and straining processes. Preferably EDTA chemical compound is used for this process. The preferred EDTA solution is at a value in the range of 0.1 M to 1 M. In this way, it is possible to remove the minerals within the source. In a preferred embodiment, if it is in a ratio of EDTA solution / collagen source, this value is between 1 :2 to 1 :10 by weight. Said process period is at a value between 1 hour to 48 hours.

[0048] - Applying the third washing and straining processes

[0049] / / . Carrying out extraction and hydrolysis processes on pre-treated collagen sources the extraction processes are carried out by treating the deep eutectic solution with the solid resources obtained after the pre-treatments applied in the process step i).

[0050] In this invention, a deep eutectic solution is used for extraction processes. In the invention, deep eutectic solvents refer to a mixture which, in the phase diagram, during the mixing of two or more compounds, has a value lower than the melting points of the compounds forming the mixture. Deep eutectic solvent formulation generally consists of dual mixtures. One of the components is a hydrogen bond acceptor (HBA) and the other is a hydrogen bond donor (HBD). In deep eutectic solvent formation, hydrogen bonding occurs between the compounds rather than covalent bonding. The ratio between the hydrogen bond acceptor and the hydrogen bond donor forming the eutectic depends on the mutual hydrogen bonding abilities of the components.

[0051] In this invention, the deep eutectic solution for extraction processes contains at least one of the components choline chloride, choline acetate, choline bromide, citric acid, betaine, and glucose to provide HBA property.

[0052] In this invention, the deep eutectic solution for extraction processes contains at least one of the group of lactic acid, acetic acid, oxalic acid, glucose, glycerol, citric acid, and malic acid to provide HBD property.

[0053] The deep eutectic solution subject to this invention contains HBA:HBD at a value between 1 :0.33 to 1 :3 by weight.

[0054] The deep eutectic solution subject to the invention is contained in an aqueous solution at a value in the range of 30% to 95% by weight in a preferred embodiment. At least one of the components choline chloride-oxalic acid, choline chloride-lactic acid, choline chloride-acetic acid, lactic acid, acetic acid, oxalic acid, glucose, glycerol, citric acid, and malic acid is preferably used as a deep eutectic solvent in the invention. In the method subject to the invention, extraction and hydrolysis processes are carried out on solid form sources after the pre-treatments applied in the process step i). The purpose of the extraction process is to ensure that collagen hydrolysate is extracted efficiently and well. The purpose of the said hydrolysis processes is to shorten the amino acid chains of collagen peptides to reduce the molecular size and to obtain collagen hydrolysate with high bioavailability. The purpose of said hydrolysis processes is to ensure that the collagen source has molecular weights of 5000 daltons and lower.

[0055] The method subject to the invention carries out the extraction and hydrolysis processes in the same process step, preferably the step of treatment with deep eutectic solvents or treatment with deep eutectic solvents while promoting the hydrolysis process with at least one enzyme and / or ultrasound support is carried out, respectively.

[0056] The most critical process step in the method subject to the invention is the treatment of collagen source subjected to pre-treatment steps with deep eutectic solvents. In a preferred embodiment, the amount of deep eutectic solution is at a value between 1 :5 to 1 :20 by weight for collagen source / deep eutectic solution. Since the amount of solution will be low below these given values, the extraction process is not carried out at the targeted efficiency, and since the amount of solution increases at the upper values, additional costs are incurred. Said process period here is in the range of 6 to 24 hours. The temperature of the solvent used in these processes is in the range of 25 to 95 °C.

[0057] If the method subject to the invention is preferred, ultrasound support can be added to treatment process of collagen source with the deep eutectic solvent. Said ultrasound here has a frequency at a value in the range of 15 kHz to 45 kHz. Said ultrasound process is carried out for a period of 5 minutes to 3 hours. With the support of ultrasound, the extraction time can be shortened, the yield of collagen production is increased, the use of solutions is greatly reduced and costs can be reduced. In this aspect, ultrasound technology is an important environmentally friendly process step for sustainable green technology production. In the extraction processes performed here, the treatment of collagen source with deep eutectic solutions as well as the application of ultrasound processes as another extraction process are carried out. If the method subject to the invention is preferred, at least one enzyme is added to the extraction processes in addition to treatment process of the collagen source with the deep eutectic solvent. Protease enzyme is used as the said enzyme here. In the invention, as a protease enzyme, at least one of the group of pepsin, papain, alkalase or trypsin is preferred. The amount of protease enzyme is preferably at a value between 0.1% to 5% by weight in the collagen source.

[0058] In a preferred embodiment, collagen obtaining extraction process steps are carried out by treating with deep eutectic solvents, carrying out the ultrasound process, and treating with at least one enzyme, respectively.

[0059] If the method subject to the invention is preferred, the extraction processes are carried out in a mixer with a heater. The temperature of said mixer is at a value between 25 to 95 °C. This process period is at a value between 6 to 24 hours.

[0060] The method subject to the invention includes hydrolysis processes on the collagen source after extraction processes. The purpose of said hydrolysis processes is to ensure that the collagen source has molecular weights lower than 5000 daltons.

[0061] Hi. carrying out filtration and purification processes on collagen sources subjected to process step ii)

[0062] The filtration process is preferably carried out using one or more of the microfiltration, ultrafiltration, dialysis or membrane filtration methods.

[0063] Collagen hydrolysate solution with decreased molecular size due to the hydrolysis process is subjected to filtration processes, preferably by ultrafiltration membrane filtration method. For this process, processes are preferably carried out with a pressure in the range of 1 -15 bar.

[0064] Carrying out purification processes

[0065] Depending on the type, the collagen source may contain varying amounts of undesirable odors, mineral substances, and heavy metals. Ion exchange systems are used for these processes. In the purification stage, anionic and cationic charged minerals, heavy metals and raw material and / or process-induced odor problems are removed from the solution through resins by using anionic, cationic, and mixbed resin systems. Thus, the ash content of the final product to be obtained is reduced to less than 5%, preferably less than 1%. The protein purity of collagen hydrolysate preferably exceeds 90%. As a result of these processes, it is possible to obtain collagen peptides at values of 5000 daltons and less. This feature provides an advantage in the food and cosmetics industry. iv. Carrying out drying processes

[0066] The obtained solution of collagen peptides is subjected to drying processes.

[0067] Drying processes include at least one of the evaporator drying, spray drying with spray dryer, and freeze drying methods.

[0068] Drying processes can be carried out with a spray dry method if preferred. The entry temperature of said method is at a value in the range of 45 to 110 °C, and the exit temperature is at a value in the range of 120 to 190 °C.

[0069] Drying processes can be carried out in a lyophilizer if preferred. The temperature of said method is a value at temperatures of -50 °C or lower.

[0070] The production method subject to the present invention also introduces a specific process step for obtaining collagen peptides in the sources while removing non-collagen substances at the highest efficiency possible. Source products generally contain moisture at a value in the range of 40% to 60% and fat at a value in the range of 10% to 35%. Their removal requires the use of heat treatments and solvents. Furthermore, these animal sources contain fats. These fats can be used as raw materials in the food industry, cosmetic products, soap-detergent production, bio-diesel production, nutritional supplements, but are not limited to what is listed herein. While obtaining collagen, it is critical to obtain these value-added raw materials with high purity as a by-product.

[0071] The inventors of the present invention introduce a method with configured process steps to further reduce the energy to be used within the scope of this invention, to further reduce the amount of solution used, and to more effectively obtain by-product fats and non-collagen protein.

[0072] To achieve this, the inventors of the present invention have added the application of supercritical carbon dioxide extraction system processes to the previously characterized production method process steps. In this way, there is no need for drying processes in the pre-treatment steps of the present production method. In addition, there is no need to use fat separation solvents such as butanol, methanol, ethanol, etc. for fat separation processes.

[0073] In this invention, supercritical carbon dioxide extraction refers to an extraction process performed by using CO2 in the supercritical state to separate the components of a substance. Supercritical CO2 is in a state with both liquid and gaseous properties and can therefore be used effectively as a solvent.

[0074] As is known in the art, since CO2 in supercritical form is a material with high solubility properties, moisture in animal resources is effectively removed. In doing so, it does not leave any solvent residue on animal sources.

[0075] Likewise, CO2 in supercritical form dissolves fats in animal sources owing to its high solubility. It carries the fat molecules within its structure and then high purity fat can be obtained by separation-purification processes.

[0076] The supercritical CO2 extraction method is a method known for its use in many technical fields and the technical solutions and advantages it provides. By including it in the subject of the present invention, it is possible to eliminate the technical problems encountered in the prior version of the method.

[0077] The most important of these technical problems is the necessity of separating fat from collagen sources. Carrying out the operations of this process in short periods and with convenience without the need for any heat treatment and solvent use has been made possible by supercritical CO2 extraction processes.

[0078] The inventors of the present invention present a method for obtaining collagen peptides with sustainable and technical advantages for the relevant technical field. For this purpose, the details of the i-iv process steps resulting from research-development activities for the relevant technical field are presented.

[0079] / . Pre-treating animal sources and / or waste animal sources

[0080] In this process step, processes are carried out in order to clean and sort animal sources and to apply the next processing steps at high efficiency.

[0081] Cleaning, separating and reducing animal sources and / or waste animal sources to small sizes

[0082] The pre-treatments carried out in the invention are the preparatory stage for the raw materials. The cleaned waste is reduced to smaller pieces by the shredder. This process is carried out to increase the surface area of the pieces and to provide a more effective raw material environment for the next processing steps. As a result of this process, the cleaned sources are reduced to a value between 1 to 30 mm.

[0083] - Application of supercritical carbon dioxide extraction processes to pieces reduced to targeted sizes

[0084] The pieces reduced to the targeted sizes are added to the supercritical CO2 extraction system. In order for the processes to be carried out with high efficiency, it is critical that the parameters are applied within the specified value ranges.

[0085] The pieces are treated at a temperature range of 40 to 55 °C.

[0086] The pressure value of this process is at a value in the range of 100 to 300 bar.

[0087] With the application of this process step, it is ensured that the value of the moisture in the animal source decreases to 15% and lower values.

[0088] With the application of this process step, the fat in the animal source can be removed. In the method subject to the present invention, the necessity of treatment with solvents such as butanol and ethanol has been eliminated since the fat removal processes were carried out in the previous process steps. In this way, it has been possible to introduce a more environmentally friendly and economical fat separation process step with high efficiency and purity without the need for toxic solvents.

[0089] One of the most important advantages of the method subject to the invention is obtaining collagen peptides while the sources are in dry from instead of wet form. The method subject to the invention is more advantageous than the methods in the present art in terms of high stocking times and low energy costs in high amounts of capacity stockpiling of sources. The advantage of stocking raw materials in dry form prevents the degradation of the protein-based product, while the pulverization process facilitates the use of the source in the process steps.

[0090] Processes of grinding sources

[0091] Herein, said sources are pulverized. Preferably, the grinding processes are carried out until the sources are reduced to powder particles at a value of 150 to 350 microns. Other parts that cannot be ground and are outside these values are separated from the sources. This process can be carried out by means of any separator. With the application of this process step, large-sized impurities are separated, the pre-treatment of the raw material with increased surface area is carried out with high efficiencies, and pretreatment periods are shortened.

[0092] Separation of non-collagen parts from sources

[0093] In this process step, non-collagen protein parts are separated from the pulverized collagen sources obtained from the previous process step. Collagen is a protein that is insoluble in cold water. Therefore, the aim is for non-collagen proteins to pass into cold water. These are mostly proteins of animal sources. In order to achieve this, cold water washing processes are carried out on the sources. Non-collagen proteins are separated and obtained.

[0094] Said washing process is preferably carried out with water with a temperature at a value in the range of 5 to 25 °C.

[0095] In a preferred embodiment, the washing process is carried out for a period of 30 to 180 minutes. In the method subject to the present invention, the necessity of treatment with basic solution has been eliminated since the removal of non-collagen proteins with cold water was carried out in the previous process steps. In this way, the removal of non-collagen proteins has been introduced in a more environmentally friendly and economical way.

[0096] Carrying out demineralization process

[0097] Demineralization processes are carried out for the sources subjected to washing and straining processes. Preferably EDTA chemical compound is used for this process. The preferred EDTA solution is at a value in the range of 0.1 M to 1 M. In this way, it is possible to remove the minerals within the source. In a preferred embodiment, if it is in a ratio of EDTA solution / collagen source, this value is between 1 :2 to 1 :10 by weight. Said process period is at a value between 1 hour to 48 hours.

[0098] If leather is to be used as an animal source for this process step, it does not need to be applied. The information described here is the process steps applied for the bone, cartilage, and scale parts of animals as animal sources.

[0099] / / . Carrying out extraction and hydrolysis processes on pre-treated collagen sources the extraction processes are carried out by treating the deep eutectic solution with the solid resources obtained after the pre-treatments applied in the process step i).

[0100] In this invention, a deep eutectic solution is used for extraction processes. In the invention, deep eutectic solvents refer to a mixture which, in the phase diagram, during the mixing of two or more compounds, has a value lower than the melting points of the compounds forming the mixture. Deep eutectic solvent formulation generally consists of dual mixtures. One of the components is a hydrogen bond acceptor (HBA) and the other is a hydrogen bond donor (HBD). In deep eutectic solvent formation, hydrogen bonding occurs between the compounds rather than covalent bonding. The ratio between the hydrogen bond acceptor and the hydrogen bond donor forming the eutectic depends on the mutual hydrogen bonding abilities of the components. In this invention, the deep eutectic solution for extraction processes contains at least one of the components choline chloride, choline acetate, choline bromide, citric acid, betaine, and glucose to provide HBA property.

[0101] In this invention, the deep eutectic solution for extraction processes contains at least one of the group of lactic acid, acetic acid, oxalic acid, glucose, glycerol, citric acid, and malic acid to provide HBD property.

[0102] The deep eutectic solution subject to this invention contains HBA:HBD at a value between 1 :0.33 to 1 :3 by weight.

[0103] The deep eutectic solution subject to the invention is contained in an aqueous solution at a value in the range of 30% to 98% by weight in a preferred embodiment.

[0104] At least one of the components choline chloride-oxalic acid, choline chloride-lactic acid, choline chloride-acetic acid, lactic acid, acetic acid, oxalic acid, glucose, glycerol, citric acid, and malic acid is preferably used as a deep eutectic solvent in the invention. In the method subject to the invention, extraction and hydrolysis processes are carried out on solid form sources after the pre-treatments applied in the process step i). The purpose of the extraction process is to ensure that collagen hydrolysate is extracted efficiently and well. The purpose of the said hydrolysis processes is to shorten the amino acid chains of collagen peptides to reduce the molecular size and to obtain collagen hydrolysate with high bioavailability. The purpose of said hydrolysis processes is to ensure that the collagen source has molecular weights of 5000 daltons and lower.

[0105] In the method subject to the invention, the extraction process step is carried out and at least one enzyme is added to the same solution obtained.

[0106] The most critical process step in the method subject to the invention is the treatment of collagen source subjected to pre-treatment steps with deep eutectic solvents. In a preferred embodiment, the amount of deep eutectic solution is at a value between 1 :5 to 1 :20 by weight for collagen source / deep eutectic solution. Since the amount of solution will be low below these given values, the extraction process is not carried out at the targeted efficiency, and since the amount of solution increases at the upper values, additional costs are incurred. If the method subject to the invention is preferred, ultrasound support can be added to treatment process of collagen source with the deep eutectic solvent. Said ultrasound here has a frequency at a value in the range of 15 kHz to 45 kHz. Said ultrasound process is carried out for a period of 5 minutes to 3 hours. With the support of ultrasound, the extraction time can be shortened, the yield of collagen production is increased, the use of solutions is greatly reduced and costs can be reduced. In this aspect, ultrasound technology is an important environmentally friendly process step for sustainable green technology production. In the extraction processes performed here, the treatment of collagen source with deep eutectic solutions as well as the application of ultrasound processes as another extraction process are carried out.

[0107] If the method subject to the invention is preferred, the extraction processes are carried out in a mixer with a heater. The temperature of said mixer is at a value between 25 to 95 °C. This process period is at a value between 2 to 24 hours.

[0108] After the application of the extraction processes, separation processes are carried out with at least one separator device for separating the insoluble or suspended solids. At least one of the centrifugal, gravitational, magnetic, membrane, filtration or electrostatic separators is used as an device in the separator process. By carrying out these processes, the separation of animal fats and non-collagen proteins can be achieved.

[0109] Hydrolysis processes are carried out by adding at least one enzyme to the solution to which separator processes are applied. The purpose of said hydrolysis processes is to ensure that the collagen source has molecular weights lower than 5000 daltons. Also, with the hydrolysis process step, enzymes can be processed at certain temperatures and inactivation can be achieved. Protease enzyme is used as the said enzyme here. As a protease enzyme, at least one of the group of pepsin, papain, alkalase or trypsin is preferred. The amount of protease enzyme is preferably at a value between 0.1% to 5% by weight in the collagen source.

[0110] In a preferred embodiment, collagen obtaining extraction and hydrolysis process steps are carried out by treating with deep eutectic solvents, carrying out the ultrasound process, and treating the solution separated in the separator with at least one enzyme, respectively. The temperature of these hydrolysis processes is at a value in the range of 30 to 95 °C.

[0111] Hi. carrying out filtration and purification processes on collagen sources subjected to process step ii)

[0112] The filtration process is preferably carried out using one or more of the microfiltration, ultrafiltration, dialysis or membrane filtration methods.

[0113] Collagen hydrolysate solution with decreased molecular size due to the hydrolysis process is subjected to filtration processes, preferably by ultrafiltration membrane filtration method. For this process, processes are preferably carried out with a pressure in the range of 1 -15 bar.

[0114] Carrying out purification processes

[0115] Depending on the type, the collagen source may contain varying amounts of undesirable odors, mineral substances, and heavy metals. Activated carbon filters and ion exchange systems are used for these processes. In the purification stage, anionic and cationic charged minerals, heavy metals and raw material and / or process-induced odor problems are removed from the solution through activated carbon filters and resins by using anionic, cationic, and mixbed resin systems. Thus, the ash content of the final product to be obtained is reduced to less than 5%. It is purified from heavy metals. The protein purity of collagen hydrolysate preferably exceeds 90%. As a result of these processes, it is possible to obtain collagen peptides at values of 5000 daltons and less. This feature provides an advantage in the food and cosmetics industry.

[0116] Increasing Concentration

[0117] The purpose of the concentration increase is to increase the percentage of the amount of product to be obtained in the solution by reducing the moisture in the solution. For this, moisture is removed at a value in the range of 10% to 50% by weight in the solution. This process step is preferably carried out with the evaporator device. iv. Carrying out drying processes The obtained solution of collagen peptides is subjected to drying processes.

[0118] Drying processes include at least one of the evaporator drying, spray drying with spray dryer, and freeze drying methods.

[0119] Drying processes can be carried out with a spray dry method if preferred. The entry temperature of said method is at a value in the range of 45 to 110 °C, and the exit temperature is at a value in the range of 120 to 190 °C.

[0120] Drying processes can be carried out in a lyophilizer if preferred. The temperature of said method is a value at temperatures of -50 °C or lower.

[0121] As is known in the art, extraction processes for obtaining collagen are carried out with weak acids (usually acetic acid) and / or weak bases (usually sodium hydroxide). Subsequently, it is possible to obtain collagen in certain sizes by carrying out hydrolysis processes with the use of enzymes. The present invention presents improved extraction and hydrolysis process steps for the relevant technical field. A critical value here is that the pre-treatments are carried out with the Supercritical Carbon Dioxide Extraction System, which is an efficient, green technological method, and the deep eutectic solvents used in the extraction process, which are defined as green solvents, are not harmful to the environment, extend shelf life by reducing microbial activity, non-toxic, cheap and reusable green solvents. In this way, a less costly and environmentally friendly method is offered for the relevant technical field.

[0122] As characterized in the present invention, it is possible to separate fats, impurities and collagen-free parts that reduce the efficiency of obtaining collagen owing to the method processing steps. In this way, collagen is obtained with high amount and efficiency. In addition, it is possible to obtain raw material-specific fat and non-collagen proteins with high commercial value and high biological value.

[0123] In another aspect, the invention relates to a method which does not require the use of solvents used for fat removal processes in the methods of the present art. In the solvent fat separation process, the process of completely separating the fat from the solvent also requires a difficult process, and the remaining solvent residue in the fat is one of the factors that reduce the quality of the fat. As is known, the solvents used are harmful to human and environmental health and may also have toxic effects on humans as long as they cannot be removed from the products. As characterized in the method subject to the present invention, by applying the supercritical CO2 extraction process step, the necessity of using solvents in fat removal processes is eliminated. At the same time, with the increase in fat removal efficiency, it is possible to obtain value-added products as a by-product during the production of collagen from animal sources.

[0124] As characterized by the method subject to the present invention, the efficiency of the drying and grinding into small pieces has been increased by enabling the application of the supercritical CO2 extraction process step. By reducing animal sources with a moisture content at a value in the range of 40% and 60% to moisture at 10% and below, it can be ensured that the next collagen production method process steps are carried out with high efficiency. One of the innovative aspects of the present invention is obtaining collagen from animal sources in a dry state. In order to achieve this, it is necessary to carry out high-efficiency drying processes.

[0125] As is known in the art, extraction processes for obtaining collagen are carried out with weak acids (usually acetic acid) and / or weak bases (usually sodium hydroxide). Subsequently, it is possible to obtain collagen in certain sizes by carrying out hydrolysis processes with the use of enzymes. The present invention presents improved extraction and hydrolysis process steps for the relevant technical field. A critical value here is that deep eutectic solvents, which are defined as green solvents used in the extraction process, are not harmful to the environment, are non-toxic, inexpensive and reusable green solvents. In this way, a less costly and environmentally friendly method is offered for the relevant technical field.

[0126] As characterized in the present invention, it is possible to separate fats, impurities and collagen-free parts that reduce the efficiency of obtaining collagen owing to the method processing steps. In this way, collagen is obtained with high amount and efficiency.

[0127] As mentioned before, in the present invention, process steps of extraction processes, treatment with deep eutectic solvents, treatment with deep eutectic solvents, ultrasound process and treatment with enzymes are applied together to obtain collagen from collagen sources in high efficiency and quantity. The important point here is to obtain collagen peptides with high purity and efficiency in a short time with a green technological extraction method. The inventors of the present invention have determined that the method subject to the present invention provides technical advantages regarding collagen efficiency and amounts from the collagen sources obtained by applying the known methods in the art.

[0128] The protein purity of collagen peptides obtained from animal sources containing collagen such as fresh or saltwater fish, chicken, eggs, or wastes of these animal sources, which are used as raw materials in the method subject to the invention, is between 90% to 98%. In this way, it is possible to obtain collagen peptides suitable for use in related technical fields.

[0129] Accordingly, the ash content of collagen peptides obtained by the method subject to the present invention is less than 5%, preferably less than 1 %. In this way, it is possible to obtain collagen peptides with a high ratio of purity.

[0130] The pH value of collagen peptides obtained by the method subject to the invention is between 6 to 7. In this way, it can be suitable for use in many different technical fields.

[0131] The E. coli ratio of collagen peptides obtained by the method subject to the invention was found to be Negative / 10 g and the Salmonella ratio was at a value of Negative / 25 g. In this way, it is possible to obtain collagen peptides suitable for use especially in food and cosmetic technical fields.

[0132] The total bacterial count of collagen peptides obtained by the method subject to the invention has been determined to be approximately 1000 CFU / g and to be lower values in the studies conducted. In addition, it has been determined that the ratio of yeast and mold is at a value of approximately 30 CFU / g and less. In this way, it is possible to obtain collagen peptides suitable for use especially in food and cosmetic technical fields.

[0133] In the studies conducted of collagen peptides obtained by the method of the invention, it has been determined that collagen peptides with cadmium content at a value less than 1 ppm and preferably non-detectable, lead content at a value less than 1 ppm, mercury content at a value less than 0.1 ppm and preferably non-detectable are obtained. The scope of protection of the invention is specified in the appended claims and cannot be limited to what is described for illustrative purposes in this detailed description. It is clear that a person skilled in the art can produce similar embodiments in the light of what is explained above, without deviating from the main theme of the invention.

Claims

CLAIMS1. A method for obtaining collagen peptides from animal sources such as fresh or saltwater fish, chicken, eggs or from parts of these animal sources that can be considered waste, characterized in that it comprises the following process steps that enable obtaining collagen peptides from sources in dry form- pre-treating animal sources and / or waste animal sources,• cleaning animal sources and / or waste animal sources,• obtaining pieces by reducing the size of the cleaned sources,• subjecting the obtained pieces to the first drying processes,- the process of grinding the pieces that have undergone the drying process,- applying processes of treatment of ground sources with a basic solution, separation of non-collagen parts and swelling of tissue matrices, and fat removal by washing with solvent,- carrying out demineralization processes,- carrying out extraction and hydrolysis processes on pre-treated collagen sources,• adding at least one deep eutectic solvent for extraction processes of collagen sources,• separating non-collagen proteins and fats by passing the solution through the separator at least once,• adding at least one protease-containing enzyme to the obtained solution for hydrolysis processes and obtaining a solution containing collagen peptides as a result of the processes,- applying filtration and purification processes to the solution containing collagen peptides obtained by applying extraction and hydrolysis processes,- carrying out drying processes after purification processes and obtaining collagen peptides as a result of these processes.

2. A method according to claim 1 , characterized in that the first drying processes are carried out at a temperature value in the range of 40 to 95 °C.

3. A method according to one of the preceding claims, characterized in that the ground collagen sources are treated with at least one basic solution in order to carry out processes of separating said non-collagen parts and swelling the tissue matrices.

4. A method according to claim 3, characterized in that the amount of basic solution is at a value between 1 :2 to 1 :10 by weight for basic solution / collagen source.

5. A method according to claim 3 or claim 4, characterized in that said basic solution is at least one of the group of sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide.

6. A method according to one of claim 3-5, characterized in that the ground collagen sources purified after treatment with the basic solution are subjected to at least first washing and straining processes for neutralization.

7. A method according to claim 6, characterized in that the second washing and straining processes are carried out after the solvent washing processes are carried out.

8. A method for obtaining collagen peptides from animal sources of fresh or saltwater fish, chicken, eggs or from the parts of these animal sources that can be considered waste, characterized in that it comprises the following process steps that enable obtaining collagen peptides from sources having reduced moisture content below 15% and fat content below 10% due to pre-treatment in the supercritical carbon dioxide extraction system in order to increase the efficiency of the drying and fat separation process steps applied in the pre-treatment steps, reduce the use of solvents, eliminate the need for heat treatment, and reduce the process steps- pre-treating animal sources and / or waste animal sources,• sorting and cleaning animal sources and / or waste animal sources,• obtaining pieces by reducing the size of the cleaned sources,• obtaining collagen sources by removing fat and moisture by subjecting pieces of targeted sizes to supercritical carbon dioxide extraction processes,- the process of grinding collagen sources,- separating non-collagen parts and swelling tissue matrices by washing the ground collagen sources by treatment with cold water,- carrying out extraction and hydrolysis processes on pre-treated collagen sources,• adding at least one deep eutectic solvent for extraction processes of collagen sources,• separating non-collagen proteins and fats by passing the solution through the separator at least once,• adding at least one protease-containing enzyme to the obtained solution for hydrolysis processes and obtaining a solution containing collagen peptides as a result of the processes,- applying filtration and purification processes to the solution containing collagen peptides obtained by applying extraction and hydrolysis processes,- processing purified collagen peptides to increase concentration.

9. A method according to one of the preceding claims, characterized in that said collagen sources are subjected to processes of reduction into piecesat a value in the range of 1 to 30 mm.

10. A method according to claim 9 or claim 10, characterized in that the temperature of said supercritical CO2 extraction processes is at a value in the range of 40 to 55 °C.1 1. A method according to claim 10, characterized in that the pressure value of said supercritical CO2 extraction processes is at a value in the range of 100 to 300 bar.

12. A method according to one of the preceding claims, characterized in that said grinding processes are carried out until the sources have a size at a value in the range of 150 to 350 microns.

13. A method according to one of claim 9-13, characterized in that the water temperature used in the washing process of treatment with cold water after the grinding process is at a value in the range of 5 to 25 °C.

14. A method according to one of the preceding claims, characterized in that the deep eutectic solvent used in said extraction processes comprises at least one of the components choline chloride, choline acetate, choline bromide, citric acid, betaine, and glucose to provide HBA property.

15. A method according to one of the preceding claims, characterized in that the deep eutectic solvent used in extraction processes contains at least one of the group of at least one component lactic acid, acetic acid, oxalic acid, glucose, glycerol, citric acid, and malic acid to provide HBD property.

16. A method according to one of the preceding claims, characterized in that the deep eutectic solvent contains HBA:HBD in a ratio between 1 :0.33 to 1 :3 by weight.

17. A method according to one of the preceding claims, characterized in that the deep eutectic solvent is present in the aqueous solution at a value in the range of 30% to 98% by weight.

18. A method according to one of the preceding claims, characterized in that the amount of the collagen source is at a value in the range of 1 :5 to 1 :20 by weight for collagen source / deep eutectic solution.

19. A method according to one of the preceding claims, characterized in that the temperature of the mixture used in the extraction processes is at a value between 25 to 95 °C.

20. A method according to one of the preceding claims, characterized in that ultrasound process is applied to collagen sources for extraction processes.

21. A method according to claim 20, characterized in that said ultrasound support is performed at a frequency value in the range of 15 to 45 kHz.

22. A method according to claim 20 or claim 21 , characterized in that the ultrasound process is performed for a period of between 5 to 180 minutes.

23. A method according to one of the preceding claims, characterized in that in said hydrolysis processes, at least one of the group of pepsin, papain, alkalase or trypsin is used as a protease enzyme.

24. A method according to claim 23, characterized in that the amount of protease enzyme is at a value between 0.1% to 5% by weight in the solution.

25. A method according to one of the preceding claims, characterized in that the temperature of the solution subjected to hydrolysis processes is at a value in the range of 30 to 95 °C.

26. A method according to one of the preceding claims, characterized in that at least one of microfiltration, ultrafiltration, dialysis or membrane filtration methods is applied to the collagen hydrolysates obtained as said filtration process.

27. A method according to claim 26, characterized in that said filtration process is an ultrafiltration process.

28. A method according to claim 27, characterized in that the ultrafiltration process is carried out at a pressure value between 1 to 15 bar.

29. A method according to one of the preceding claims, characterized in that said purification process is carried out with at least one of activated carbon, anionic, cationic, and mixbed resins.

30. A method according to one of the preceding claims, characterized in that, after said concentration process step, drying is carried out with lyophilization processes at a temperature of at least -50 °C.31 . A method according to one of the preceding claims, characterized in that, after said concentration process step, the spray drying processes step is carried out at a value of an entry temperature in the range of 45 to 1 10 °C and at a temperature of an exit temperature between 120 to 190 °C.