Method for producing dipeptide or tripeptide sized collagen peptides from collagen sources and collagenase used therefor

Abstract

The invention relates to a method for obtaining collagens in tripeptide or dipeptide sizes from collagen sources in order to enable faster penetration into cells, high-specificity binding to enzymes, increased inhibitor activity, provision of anti-cancer effects and antioxidant properties, to the collagens obtained through the application of this method, and to a collagenase enzyme enabling the obtainment of these collagens.

Description

[0001] METHOD FOR PRODUCING DIPEPTIDE OR TRIPEPTIDE SIZED COLLAGEN PEPTIDES FROM COLLAGEN SOURCES AND COLLAGENASE USED THEREFOR

[0002] TECHNICAL FIELD

[0003] The invention relates to a method for obtaining collagens in tripeptide or dipeptide sizes from collagen sources in order to enable faster penetration into cells, high-specificity binding to enzymes, increased inhibitor activity, provision of anti-cancer effects and antioxidant properties, to the collagens obtained through the application of this method, and to a collagenase enzyme enabling the obtainment of these collagens.

[0004] PRIOR ART

[0005] Collagens are fibrous proteins that constitute the main structural component of connective tissues and ensure the structural integrity of various tissues in the body such as skin, bones, cartilage, tendons, and muscles. These proteins, which have a triple helix structure, strengthen the extracellular matrix and provide durability and elasticity to the tissues. Collagens are generally resistant to proteases; however, they are degraded into small peptides by specific enzymes (collagenases) and are widely used in the biomedical, cosmetic, and food industries.

[0006] Collagens have a wide range of applications in biomedical, food, cosmetic, and industrial fields. In the biomedical field, they are used in tissue engineering, wound healing treatments, orthopedic implants, and supplements supporting joint health. In the food sector, they are preferred for gelatin production, meat processing, and as a functional food component for the purpose of meat tenderization and texture improvement. In the cosmetic field, they are used in anti-aging creams, skin-renewing serums, and hair care products to increase skin elasticity and maintain moisture balance. In industrial applications, they are involved in the development of various products such as bioplastics production, adhesives, and pharmaceutical capsules.

[0007] In the relevant technical field, enzymatic collagenases are enzymes belonging to the metalloproteinase class, which cleave collagen proteins at specific sites and disrupt the triple helix structure. They can generally be obtained from bacterial (for example, Clostridium histolyticum, Vibrio spp.) or fungal sources and can be produced bybiotechnological methods. These enzymes hydrolyze collagen into smaller peptides and amino acids, thereby making it suitable for use in the biomedical, cosmetic, and food industries. Enzymatic methods are widely preferred in collagen production since they offer a more specific, controlled, and environmentally friendly process compared to chemical and physical methods.

[0008] When these small peptides are obtained through the controlled hydrolysis of collagen, they can be more easily absorbed by the body and effectively perform their biological functions. However, when the peptide sizes are not sufficiently small, various technical problems arise. Primarily, since long peptide chains are more difficult to absorb in the intestine, bioavailability is significantly reduced. Small peptides, especially dipeptides and tripeptides, are directly transported into cells and show faster effects in skin, joint, and muscle tissues. Larger peptides, on the other hand, require more processing in the digestive system for breakdown, thus becoming biologically less effective. This is an undesirable situation particularly in dietary supplements and therapeutic applications. In addition, long-chain peptides have lower solubility in water. This situation may lead to stability issues in biomedical, food, and cosmetic products. Especially in liquid-based products such as beverages, serums, and gel formulations, low solubility prevents the formation of a homogeneous structure and may reduce product quality.

[0009] Obtaining short peptides through controlled hydrolysis ensures that such formulations become more effective. In terms of functionality, long peptides may also cause undesirable effects. In particular, bitter taste and texture irregularities constitute a major problem in the food and beverage sector. While small peptides have certain biological activities (such as antioxidant, anti-inflammatory, etc.), long-chain peptides cannot fully exhibit these activities. As a result, the production of collagen hydrolysates at the tripeptide or dipeptide level through controlled hydrolysis using enzymatic collagenase is a critical process that increases both bioavailability and functionality.

[0010] In the relevant technical field, obtaining collagen in dipeptide or tripeptide sizes involves certain technical, economic, and functional challenges. Primarily, it is quite difficult to obtain specifically only dipeptides or tripeptides in the enzymatic hydrolysis process. Since collagen naturally has a large and complex structure, if the enzymatic degradation is not stopped at a certain point, the peptides may be further degraded down to single amino acids. This situation may lead to the loss of desired biological properties and a decrease in the effectiveness of the product. Furthermore, since the enzymes need to be precisely controlled, efficient management of the process becomes more difficult.From an economic perspective, additional purification and refinement processes are required to obtain dipeptides or tripeptides, which significantly increases production costs. While the production of standard collagen hydrolysates already requires certain optimizations, producing peptides of only specific sizes is a more complex and expensive process. Therefore, collagen peptides used in the food, pharmaceutical, and cosmetic sectors are generally maintained within a wide molecular size range and are optimized for general functionality rather than for a specific target.

[0011] As a result, in the relevant technical field, it is necessary to research and develop innovative methods and enzymes in order to obtain collagens in tripeptide or dipeptide sizes with high efficiency and low cost.

[0012] BRIEF DESCRIPTION OF THE INVENTION

[0013] The present invention relates to a method for obtaining collagens in tripeptide or dipeptide sizes from collagen sources, in order to eliminate the above-mentioned disadvantages and to bring new advantages to the relevant technical field.

[0014] One objective of the invention is to provide a method that enables the obtainment of collagen having tripeptide or dipeptide chain length, thereby improving bioavailability properties.

[0015] One objective of the invention is to provide a method that enables the obtainment of collagens in tripeptide or dipeptide sizes at high purities.

[0016] Another objective of the invention is to provide a method that enables the obtainment of collagens in tripeptide or dipeptide sizes with low production costs.

[0017] Another objective of the invention is to provide a collagen having tripeptide or dipeptide chain length and high protein purity.

[0018] Another objective of the invention is to provide a collagenase enzyme that enables the obtainment of collagen having tripeptide or dipeptide chain length, at high protein purities and in a homogeneous manner.

[0019] DETAILED DESCRIPTION OF THE INVENTIONIn this detailed description, the subject of the invention relates to a method for obtaining collagens in dipeptide or tripeptide sizes from collagen sources, and it is explained with examples that have no limiting effect and are provided solely for a better understanding of the subject.

[0020] The collagen referred to in the invention is a structural protein found in the body of individuals. As is known, there are 28 types of collagen reported in the literature. In the present invention, no specific information is provided regarding the obtainment of a particular type of collagen. The method provided in the present invention can also be used for the obtainment of a specifically desired type of collagen. In this respect, the present invention is not limited to the obtainment of any particular type of collagen.

[0021] In the present invention, animal sources are preferably used as the collagen source. In the present invention, species that abundantly contain collagen in their bodies can be used as the animal source. Preferably, fish, chickens, cattle, small ruminants, pigs, or eggs can be used as the animal source. However, the method subject to the present invention is not limited to these and can be applied to all animal sources for the obtainment of collagen.

[0022] The animal sources referred to as a term in the invention may include animals suitable for use in any type of collagen production, whether rich or not rich in collagen. In the invention, the term “animal source” refers to the initial raw material. The expression “collagen source” refers to pre-processed animal sources.

[0023] Another innovative aspect of this invention relates to a collagenase enzyme that enables the obtainment of collagens in dipeptide or tripeptide lengths as the final product from the collagen sources, which are the initial raw materials as mentioned. The collagenase enzyme is a metalloproteinase enzyme that can specifically cleave the collagen molecule in the desired manner. It cleaves the triple helix structure of collagen and separates it into smaller dipeptide or tripeptide collagens.

[0024] The collagenase enzyme is a protease of the metalloproteinase class that targets type I, II, III, and IV collagens and can cleave at specific amino acid sequences. The collagenase enzyme functions in a zinc- or calcium-dependent manner to enable the degradation of the triple helix structure. After binding to the collagen molecule, stabilization is achieved through specific binding regions (exosites), and the triple helix structure is relaxed by mechanical stress.Collagen has a triple helix structure characterized by Gly-X-Y repeats, and the stabilization of this structure is provided by hydrogen bonds and the content of proline and hydroxyproline. Collagenase targets this structure and performs cleavage particularly at regions containing Gly-Pro-X or Gly-X-Hyp motifs. This cleavage occurs through the activation of a water molecule by the Zn2+ion in the active site of the enzyme, and the peptide bonds are hydrolyzed. The cleavage points are generally located between glycine (Gly) and proline (Pro) or hydroxyproline (Hyp). The enzyme's ability to cleave at these regions enables the formation of smaller and biologically active peptides. Among the components resulting from the cleavage are oligopeptides, dipeptides, tripeptides, and free amino acids. In particular, peptides with Gly-Pro-Hyp and Pro-Hyp sequences are critical components that enhance the effectiveness of collagen supplements.

[0025] As a result of this specific cleavage mechanism, the resulting tripeptides are generally expected to have the Gly-Pro-Hyp sequence, while the dipeptides have the Pro-Hyp sequence. Since the bioavailability of small peptide structures is high, they are absorbed more rapidly in the body and become more effective in antioxidant, anti-inflammatory, and cellular regeneration processes. In addition, additional components such as oligopeptides, free amino acids, and bioactive peptides are formed as a result of the cleavage process. These peptides are important functional components particularly in terms of dietary supplements, pharmaceutical, and cosmetic applications. Thus, with this invention, a more controlled and optimized collagen degradation process is provided, offering high-efficiency and functional use in the biomedical, food, and cosmetic sectors.

[0026] The collagenase enzyme referred to in the invention has the SEQ 1 amino acid sequence as shown in Table 1.

[0027] MLSEPVSQVTEQHAHSAHTHGVEFNRVEYQPTATLPIQPSKATRVQSLESLDESSTACDL EALVTESSNQLISEILSQGATCVNQLFSAESRIQESVFSSDHMYNIAKHTTTLAKGYTGGG SDELETLFLYLRAGYYAEFYNDNISFIEWVTPAVKESVDAFVNTASFYENSDRHGKVLSEVI ITMDSAGLQHAYLPQVTQWLTRWNDQYAQHWYMRNAVNGVFTILFGGQWNEQFVQIIG NQTDLAKALGDFALRASSIGAEDEFMAANAGRELGRLTKYTGNASSVVKSQLSRIFEQYE MYGRGDAVWLAAADTASYYADCSEFGICNFETELKGLVLSQTYTCSPTIRILSQNMTQEQ HAAACSKMGYEEGYFHQSLETGEQPVKDDHNTQLQVNIFDSSTDYGKYAGPIFDISTDNG GMYLEGDPSQPGNIPNFIAYEASYANADHFVWNLEHEYVHYLDGRFDLYGGFSHPTEKIV WWSEGIAEYVAQENDNQAALETILDGSTYTLSEIFETTYDGFDVDRIYRWGYLAVRFMFE NHKDDVNQMLVETRQGNWINYKATITQWANLYQSEFEQWQQTLVSNGAPNAVITANSKG

[0028]

[0029] KVGESITFSSENSTDPNGKIVSVLWDFGDGSTSTQTKPTHQYGSEGEYSVSLSVTDSEGL TATATHTVVISALGGNDTLPQDCAVQSKVSGGRLTAGEPVCLANQQTIWLSVPAVNESSN LAITTGNGTGNLKLEYSNSGWPDDTNLHGWSDNIGNGECITLSNQSNYWGYVKVSGDFE NAAIVVDFDAQKCRQ

[0030]

[0031] Table 1. Sequence list of the collagenase enzyme subject to the invention.

[0032] The collagenase enzyme referred to in the invention is obtained by microbial production techniques. After the production cassette enabling the realization of these processes is prepared, the target product collagenase enzyme can be obtained in microorganisms. The collagen production cassettes contain the genetic instructions required for the efficient and accurate synthesis of the target collagenase enzyme. The collagen production cassette referred to in the invention is transferred into target organisms via at least one vector in order to enable the obtainment of the collagenase enzyme having the SEQ 2 sequence list. As the target organism referred to in this invention, at least one from the group consisting of Aspergillus oryzae (accession number: ATCC1011), Aspergillus niger (accession number: ATCC 9029), Pichia pastoris (accession number: ATCC 28485), Trichoderma reesei (accession number: ATCC 13631), Vibrio alginolyticus (accession number: ATCC 17749), Vibrio parahaemolyticus (accession number: ATCC 17802), Vibrio splendidus (accession number: ATCC 33125), Vibrio cholerae (accession number: ATCC 14035), Vibrio fischeri (accession number: ATCC 7744), Vibrio vulnificus (accession number: ATCC 27562), Vibrio harveyi (accession number: ATCC 14126) is preferably used.

[0033] In the most preferred embodiment, Aspergillus oryzae is used as the microorganism. This microorganism is a type of fungus. The reason for using this type of fungus is that it has a long history of safe use in the food industry and is recognized as "Generally Recognized As Safe" (GRAS) by the United States Food and Drug Administration (FDA). This safety profile makes it an ideal option especially in biotechnological productions. In addition, Aspergillus oryzae is highly efficient in terms of enzyme production capacity; it can produce a high amount of enzymes and easily secrete these enzymes into the culture medium. This feature increases purity in the fermentation medium and facilitates the extraction process. It is also easy to genetically modify and to insert target genes, thereby allowing optimization for the production of specific enzymes such as collagenase. Furthermore, Aspergillus oryzae can be fed with low-cost nutrient sources and is resistant to various environmental conditions, which reduces production costs and increases economic efficiency. Thanks to these features, Aspergillus oryzae stands out as a reliable, efficient, and sustainable microorganism in biotechnological enzyme production.In this invention, DNA molecules such as bacterial plasmids or viral DNA are generally used as vectors to transfer the collagenase gene into microorganisms such as Aspergillus oryzae. Plasmids are small, circular DNA molecules obtained from bacteria, which can be easily manipulated and are preferred due to their high gene carrying capacity. Viral DNA, on the other hand, is effective particularly in difficult gene transfers due to its ability to transfer genetic material into the cell through natural mechanisms. In addition, fungal plasmids specifically developed for fungal cells or artificial chromosome vectors such as Yeast Artificial Chromosomes (YACs) have the capability to carry large gene fragments and can be used to transfer genetic material into microorganisms such as Aspergillus oryzae.

[0034] The collagenase enzyme referred to in the invention has a collagenase activity of 400 CDU / mg, a gelatinase activity of 600 LGU / mg, and a caseinase activity of 4.0 caseinase / mg. While it shows a specific affinity for the collagen substrate, the effect of nonspecific proteases is at a minimal level. The optimum working pH range of the enzyme is between 6 and 10, and it exhibits maximum activity particularly at pH 8.

[0035] As stated, the scope of protection of the invention is not limited to microbial production techniques. The collagenase enzymes referred to in the invention and having the amino acid sequence list as mentioned can also be obtained by other methods known in the art. What makes the invention critical is that the collagenase enzyme having the SEQ 1 amino acid sequence enables the obtainment of collagens in dipeptide or tripeptide lengths through the production method process steps subject to this invention and the parameters characterized in these process steps.

[0036] According to what is mentioned herein, the essential novelty of the invention is the determination of suitable parameters for the application of the collagenase enzymes to collagen sources and the implementation of the processes with these parameters. Accordingly, the mentioned application process steps are characterized in the following lines.

[0037] - Preparation of the raw material to be used as the collagen source

[0038] In a preferred embodiment, animal-based raw materials are used as the collagen source. In the most preferred embodiment, powdered gelatin, liquid gelatin, collagen hydrolysate,or animal skin subjected to a liming process, all derived from animal-based raw materials, are used as the collagen source.

[0039] Gelatin is generally in the form of powdered gelatin or liquid gelatin, and provides an advantage due to its high collagen purity and suitability for controlled hydrolysis under specific processing conditions. However, prior to enzymatic hydrolysis, gelatin must be dissolved with specific temperature and pH adjustments, which leads to additional processing time and energy consumption. Collagen hydrolysate is in a partially hydrolyzed form, has a lower molecular weight, and accelerates enzymatic processes due to its rapid solubility, thereby shortening the production time. However, the use of collagen hydrolysate may limit enzymatic control and its cost is generally higher compared to gelatin. While gelatin offers low cost and flexible production as a pure collagen source, collagen hydrolysate increases process efficiency and provides a more stable and faster production advantage. The selection between these raw materials is made based on criteria such as production cost, process duration, bioavailability, and purity of the final product.

[0040] - Use of powdered gelatin as the raw material for the collagen source

[0041] If preferred, powdered gelatins having bloom values between 100 and 300 are used as gelatin. The powdered gelatins are preferably dissolved in water at a concentration ranging from 10gr / 100ml and 100gr / 100mL

[0042] Water is introduced into the reactor, and powdered gelatin is added to this reactor, allowing it to absorb the water and swell. Subsequently, a mixing process is carried out, and this process is performed at a temperature in the range of 40 to 60 °C. The pH value is adjusted to a range of 7 to 10 by means of at least one basic solution. After these processes are carried out, the temperature is adjusted and stabilized at a value in the range of 35 to 45 °C. The collagenase enzyme is then added to the raw material adjusted in this manner, thereby enabling the obtainment of tripeptide or dipeptide collagen.

[0043] - Use of liquid gelatin as the raw material for the collagen source

[0044] If preferred, liquid gelatin can be used as gelatin, and an aqueous solution having a concentration in the range of 10gr / 100ml and 100gr / 100mL The pH value is adjusted to a range of 7 to 10 by means of at least one basic solution. After these processes are carried out, the temperature is adjusted and stabilized at a value in the range of 35 to 45 °C. Thecollagenase enzyme referred to in the invention is then added to the raw material adjusted in this manner, thereby enabling the obtainment of tripeptide or dipeptide collagen.

[0045] - Use of animal skins subjected to a liming-splitting process as the raw material for the collagen source

[0046] Gelatin manufacturers, while maintaining their production capacities, can produce collagen tripeptides or dipeptides directly by using animal skins subjected to a liming-splitting process, particularly bovine hides. These processes are carried out using two different approaches: alkaline pre-treatments and acidic pre-treatments.

[0047] In the alkaline pre-treatment, the skin pieces are purified from non-collagen proteins by raising the pH to a level of 13-14 with at least one basic solution. Then, by lowering the pH to a level of 2 with at least one acidic solution, the collagen fibers are opened, and a washing process is carried out with water to remove impurities. In the final stage, the pH of the skin pieces is adjusted to a level of 4-5 through repeated washings, and they are dissolved in extraction tanks at a temperature in the range of 40 to 60 °C. The hydrolysis process is carried out with the collagenase referred to in the invention, thereby enabling the obtainment of tripeptide or dipeptide collagen.

[0048] In the acidic pre-treatment, the skin pieces are directly treated with at least one acidic solution to lower the pH to a level of 2, thereby opening the collagen fibers, and similar washing processes are applied to prepare them for the extraction process. In both methods, the molecular weight of the solution obtained in the final stage is similar to the typical gelatin molecular weight (150-250 kDa).

[0049] - Use of collagen hydrolysate as the raw material for the collagen source

[0050] If preferred, collagen hydrolysate is used as the collagen source. In a preferred embodiment, collagen hydrolysate having a molecular weight of 10,000 Da or less on average is used. The collagen hydrolysate is dissolved in water at a concentration ranging from 10gr / 100ml and 100gr / 100mlby weight. During the production process, the required amount of water is introduced into the reactor, and the predetermined amount of collagen hydrolysate is added, and dissolution is ensured by continuous mixing. Then, the pH value of the solution is adjusted to a range of 7-10 using a basic solution, and the temperature is stabilized in the range of 35-45 °C. After all temperature and pH adjustments arecompleted, while mixing continues, the collagenase enzyme referred to in the invention is added, and the production process of tripeptide or dipeptide collagen is initiated.

[0051] - Addition of the collagenase enzyme referred to in the invention to the obtained raw material and obtainment of tripeptide or dipeptide collagens through hydrolysis processes

[0052] In the previous process step, the collagen sources usable as raw materials were prepared for hydrolysis processes through pre-treatments. The collagenase enzyme having the SEQ 1 sequence list referred to in the invention is applied to the raw materials to be used as the obtained collagen sources.

[0053] Accordingly, first, a gelatin-water solution is prepared as the raw material, and the concentration value of this solution is in the range of 10gr / 100ml and 100gr / 100ml. To this solution, the collagenase enzyme is added at a concentration value in the range of 0.01% to 2% by weight. The amount of collagenase referred to in the invention is an optimized value considering the amount of substrate (collagen source) present in the solution. Low enzyme concentrations prolong the hydrolysis time, while high enzyme concentrations provide faster hydrolysis and may cause undesired degradation of the peptides.

[0054] As is known, collagenase is an enzyme belonging to the metalloproteinase class, dependent on zinc or calcium ions, and exhibits higher activity under alkaline conditions. For the hydrolysis processes to be carried out with the collagenase enzyme referred to in the invention, the pH value is in the range of 7 to 10. At lower pH values, collagenase enzyme activity decreases, while at higher pH levels, undesired side reactions occur in the peptide chains.

[0055] The temperature of the hydrolysis processes referred to in the invention is in the range of 37 to 42 °C. At lower temperatures, enzyme activity slows down, the hydrolysis time is prolonged, and the process becomes inefficient. At higher temperatures, the structure of the enzyme may be degraded (denaturation), or the targeted peptides may be excessively degraded, leading to the formation of undesired products. Therefore, the temperature is maintained in the range of 37-42 °C, and the temperature is controlled throughout the process to ensure that the enzyme operates at an optimum level.

[0056] In this process step, according to the parameters mentioned, the hydrolysis process is carried out for a period in the range of 1 to 5 hours.Inactivation of the collagenase enzyme used after the hydrolysis processes

[0057] After the hydrolysis process is completed, an inactivation process is carried out at a temperature value in the range of 45 to 55 °C in order to stop the activity of the collagenase enzyme and to prevent the hydrolysis process from continuing in an uncontrolled manner.

[0058] After the enzyme inactivation process is completed, in order to prevent the hydrolysis process from continuing in an uncontrolled manner and to increase the stability of the final product, separation and purification processes can additionally be applied in this invention. These processes increase the degree of purity of the product and ensure that the dipeptide and tripeptide content is at the targeted level.

[0059] One of these process steps, the filtration process, is applied to remove large molecular components, undissolved particles, and undesired impurities in the solution resulting from the hydrolysis processes. As the filtration process, either microfiltration or ultrafiltration can be applied.

[0060] One of these process steps, the concentration process, is carried out in order to ensure that the obtained product reaches the desired density, and the water content is reduced by vacuum evaporation or membrane technologies.

[0061] One of these process steps, the purification process, enables the obtainment of the targeted collagen peptides by separation according to molecular weight.

[0062] One of these process steps, the drying process, is applied in order to extend the shelf life of the obtained product and to maintain its stability. As the drying process, at least one of spray drying, lyophilization, or vacuum drying can be applied.

[0063] In the final stage, the final product is packaged with suitable packaging methods so as to be protected from external factors and is stored. In order to prevent external factors such as moisture, temperature, and light from affecting the stability of the product, vacuum or gas-barrier packaging methods are particularly preferred for peptide powders. As a result of these processes, the obtained collagen peptides are turned into a bioavailable, functional, and stable product, prepared for use in dietary supplements, pharmaceutical products, or cosmetic formulations.TESTS

[0064] Experiment 1

[0065] 100 grams of bovine gelatin was prepared by dissolving in 1 liter of water (10% concentration by weight). During the dissolution process, a water bath heated to 60 °C was used, and after the gelatin was completely dissolved, the pH was adjusted to 8. Then, the solution was cooled down to 37 °C. At the same time, 50 g / l collagenase was dissolved in water and stirred for 20 minutes using a magnetic stirrer. From the prepared enzyme solution, 10 ml of liquid (0.5 g collagenase - 0.5% by weight of the solid product) was added to the gelatin solution. The enzyme + substrate mixture was placed in a shaking incubator adjusted to 37 °C and shaken at a speed of 150 rpm. The moment of enzyme addition was taken as the starting point, and samples were taken at the 1st, 3rd, and 5th hours and after overnight incubation was completed. The samples were incubated at 55 °C for 15 minutes to stop enzyme activity, and HP-SEC (High-Performance Size Exclusion Chromatography) and SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) analyses were performed to determine molecular size.

[0066] Figure 1 shows the molecular weight distribution of SDS-PAGE (5-hour and overnight incubation products).

[0067] Figure 2 presents the molecular weight distribution results of HP-SEC (5-hour incubation).

[0068] Figure 3 provides the molecular weight distribution of HP-SEC as a graph (overnight incubation).

[0069] Figure 4 shows the graphical representation of the molecular weight distribution of HP-SEC.

[0070] Experiment 2

[0071] 500 grams of bovine collagen hydrolysate was prepared by dissolving in 1 liter of water (50% concentration by weight). After the collagen hydrolysate was completely dissolved, the pH was adjusted to 8 and the temperature was lowered to 37 °C. Meanwhile, 100 g / l collagenase was dissolved in water and stirred for 20 minutes using a magnetic stirrer. From the prepared enzyme solution, 25 ml of liquid (2.5 g collagenase - 0.5% by weight ofthe solid product) was added to the gelatin solution. The enzyme + substrate mixture was placed in a shaking incubator adjusted to 37 °C and shaken at a speed of 150 rpm. Samples were taken at the 1st, 3rd, and 5th hours and after overnight incubation was completed, incubated at 55 °C for 15 minutes to stop enzyme activity, and analyzed by HP-SEC and SDS-PAGE.

[0072] Figure 5 shows the molecular weight distribution of SDS-PAGE (5-hour and overnight incubation products).

[0073] Figure 6 presents the molecular weight distribution results of HP-SEC (collagen hydrolysate - control).

[0074] Figure 7 shows the graphical representation of the molecular weight distribution of HP-SEC (collagen hydrolysate - control).

[0075] Figure 8 shows the graphical representation of the molecular weight distribution of HP-SEC (1-hour incubation).

[0076] Figure 9 shows the graphical representation of the molecular weight distribution of HP-SEC (1-hour incubation).

[0077] Figure 10 shows the graphical representation of the molecular weight distribution of HP-SEC (3-hour incubation).

[0078] Figure 11 shows the graphical representation of the molecular weight distribution of HP-SEC (3-hour incubation).

[0079] Figure 12 shows the graphical representation of the molecular weight distribution of HP-SEC (overnight incubation).

[0080] Figure 13 shows the graphical representation of the molecular weight distribution of HP-SEC (overnight incubation).

[0081] Experiment 3

[0082] 350 grams of bovine gelatin was prepared by dissolving in 1 liter of water (35% concentration by weight). After the gelatin was completely dissolved, the pH was adjustedto 8 and the temperature was lowered to 37 °C. Meanwhile, 50 g / l collagenase was dissolved in water and stirred for 20 minutes using a magnetic stirrer. From the prepared enzyme solution, 35 ml of liquid (1.75 g collagenase - 0.5% by weight of the solid product) was added to the gelatin solution. The HP-SEC molecular weight distribution analysis results of the products obtained as a result of Experiment 3 were analyzed for the 1st, 3rd, and 5th hour samples.

[0083] Figure 14 shows the graphical representation of the molecular weight distribution of HP-SEC (1-hour incubation).

[0084] Figure 15 shows the graphical representation of the molecular weight distribution of HP-SEC (1-hour incubation).

[0085] Figure 16 shows the graphical representation of the molecular weight distribution of HP-SEC (3-hour incubation).

[0086] Figure 17 shows the graphical representation of the molecular weight distribution of HP-SEC (3-hour incubation).

[0087] Figure 18 shows the graphical representation of the molecular weight distribution of HP-SEC (5-hour incubation).

[0088] Figure 19 shows the graphical representation of the molecular weight distribution of HP-SEC (5-hour incubation).

[0089] Experiment 4

[0090] 350 grams of bovine gelatin was prepared by dissolving in 1 liter of water (35% concentration by weight). After the gelatin was completely dissolved, the pH was adjusted to 8 and the temperature was lowered to 37 °C. Meanwhile, 100 g / l collagenase was dissolved in water and stirred for 20 minutes using a magnetic stirrer. From the prepared enzyme solution, 35 ml of liquid (3.5 g collagenase - 1% by weight of the solid product) was added to the gelatin solution. The HP-SEC molecular weight distribution analysis results of the products obtained as a result of Experiment 4 were analyzed for the 1st, 3rd, and 5th hour samples.Figure 20 shows the graphical representation of the molecular weight distribution of HP-SEC (1-hour incubation).

[0091] Figure 21 shows the graphical representation of the molecular weight distribution of HP-SEC (1-hour incubation).

[0092] Figure 22 shows the graphical representation of the molecular weight distribution of HP-SEC (3-hour incubation).

[0093] Figure 23 shows the graphical representation of the molecular weight distribution of HP-SEC (3-hour incubation).

[0094] Figure 24 shows the graphical representation of the molecular weight distribution of HP-SEC (5-hour incubation).

[0095] Figure 25 shows the graphical representation of the molecular weight distribution of HP-SEC (5-hour incubation).

[0096] The scope of protection of the invention is specified in the attached claims and cannot be limited to those explained for sampling purposes in this detailed description. It is evident that a person skilled in the art may exhibit similar embodiments in light of the above-mentioned facts without drifting apart from the main theme of the invention.

Claims

CLAIMS1. A method for obtaining collagen in dipeptide or tripeptide sizes from a collagen source, characterized in that it comprises the following process steps:- obtaining the collagen source from animal-based raw materials,- obtaining the collagen source-water solution by dissolving the obtained collagen source in water,wherein the pH value of the collagen source-water solution is in the range of 7 to 10,- addition of the collagenase enzyme having the SEQ 1 sequence to the obtained collagen source-water solution and carrying out the hydrolysis processes, - raising the temperature of the solution to a temperature in the range of 45 to 55 °C for the inactivation of the collagenase having the SEQ 1 sequence.

2. The method according to claim 1, characterized in that the said collagen source is at least one of powdered gelatin, liquid gelatin, collagen hydrolysate, or animal skin subjected to a liming-splitting process, obtained from animal-based raw materials.

3. The method according to any one of the preceding claims, characterized in that the collagen source-water solution is at a temperature in the range of 40 to 60 °C.

4. The method according to any one of the preceding claims, characterized in that the collagenase enzyme is added to the collagen source-water solution at a temperature in the range of 35 to 45 °C.

5. The method according to any one of the preceding claims, characterized in that the collagen source is present in the collagen source-water solution at a concentration in the range of 10gr / 100ml and 100gr / 100mL6. The method according to any one of the preceding claims, characterized in that the collagenase enzyme having the SEQ 1 sequence is added to the obtained collagen source-water solution at a concentration in the range of 0.01% to 2% by weight.

7. The method according to any one of the preceding claims, characterized in that the temperature of the said hydrolysis process is in the range of 37 to 42 °C.

8. The method according to any one of the preceding claims, characterized in that at least one of the process steps of filtration, concentration, purification, or drying is applied to the product obtained after the hydrolysis processes.

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