A collagen peptide for increasing bone density and its preparation method

Abstract

This application provides a collagen peptide for increasing bone density and a method for preparing the same. The method includes the following steps: S1: dispersing bone gelatin in water to obtain a bone gelatin solution; S2: adding 0.1-1 mol / L acidic pH adjuster to the bone gelatin solution at a rate of 1-10 mL / min until the turbidity of the system increases by 10%-20% compared to the initial bone gelatin solution, then stopping the addition, allowing it to stand for 10-30 min, and filtering to obtain a bone gelatin supernatant; S3: adding an endopeptide to the bone gelatin supernatant to obtain an enzymatic hydrolysate; S4: post-processing and drying the enzymatic hydrolysate to obtain collagen peptides. This method can increase the mass ratio of low molecular weight peptides in the obtained collagen peptides, resulting in a composition of collagen peptides more suitable for bone metabolism and utilization to increase bone density.

Description

Technical Field

[0001] This application relates to the field of collagen peptide production technology, specifically to a bone collagen peptide for increasing bone density and its preparation method. Background Technology

[0002] Bone mineral density (BMD) is an important indicator of the mineral content in bone tissue and the integrity of bone structure. Its level is directly related to bone strength and resistance to fractures. With age, hormonal changes, or insufficient nutritional intake, the balance of bone metabolism is easily disrupted, leading to a decrease in bone mineral density and subsequently causing osteoporosis and other problems.

[0003] Bone tissue is mainly composed of inorganic minerals and an organic matrix, with collagen being the primary organic matrix. Collagen not only provides a structural framework for calcium salt deposition but also participates in regulating osteoblast adhesion, proliferation, and differentiation. Therefore, supplementation with collagen-related nutrients is considered an important way to improve bone metabolism and maintain or increase bone density.

[0004] Compared to intact collagen, collagen peptides have lower molecular weights and better solubility, making them more suitable for digestion and absorption to participate in metabolic processes in vivo. Existing studies have shown that collagen peptides within specific molecular weight ranges can exhibit different biological behaviors in vivo, and their effects on bone metabolism-related cells also vary. However, bone collagen peptides prepared using current technologies typically have a wide molecular weight distribution, containing both large molecular weight peptides and potentially generating numerous free amino acids or very low molecular weight peptides, making it difficult to stably obtain collagen peptide components more suitable for participating in bone matrix construction and bone metabolism regulation. For example, components with a molecular weight below 180 Da mainly exist in the form of free amino acids or very short peptides; although easily absorbed, their structural contribution to bone matrix-related metabolic processes is limited. Meanwhile, peptides with a molecular weight above 1000 Da, due to their larger molecular size, have relatively lower efficiency in digestion and absorption.

[0005] In existing preparation methods, bone gelatin or collagen raw materials typically enter the hydrolysis step directly before enzymatic hydrolysis. The molecular weight distribution, aggregation state, and conformational differences are significantly influenced by the source of the raw materials and pretreatment conditions, leading to inconsistent hydrolysis rates among different collagen components during enzymatic hydrolysis. This uneven hydrolysis behavior easily results in a low proportion of collagen peptides within the target molecular weight range and insufficient batch-to-batch stability, thus limiting the application effectiveness of bone collagen peptides in improving bone density.

[0006] Therefore, how to prepare collagen peptides with a more concentrated molecular weight distribution and better utilization by bone metabolism without relying on complex molecular weight-based separation methods remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] This application provides a collagen peptide for increasing bone density and a method for preparing the same, aiming to solve the problem that the uneven state of collagen substrate and poor controllability of enzymatic hydrolysis in the existing collagen peptide preparation process result in a small proportion of low molecular weight peptides with a molecular weight of 180~1000 Da in the obtained collagen peptide.

[0008] In a first aspect, this application provides a method for preparing collagen peptides for increasing bone density, comprising the following steps:

[0009] S1: Disperse bone gelatin in water, allowing it to swell and then dissolve to obtain a bone gelatin solution;

[0010] S2: Add 0.1-1 mol / L acidic pH adjuster to the bone gelatin solution at a rate of 1-10 mL / min until the turbidity of the system increases by 10%-20% compared to the initial turbidity of the bone gelatin solution. Stop adding the solution, let it stand for 10-30 min, and then filter to obtain the bone gelatin supernatant.

[0011] S3: Add an endopeptide to the bone gelatin supernatant to hydrolyze the collagen in the bone gelatin supernatant into bone collagen peptides, and obtain an enzymatic hydrolysate;

[0012] S4: The enzymatic hydrolysate is post-treated and dried to obtain collagen peptides.

[0013] According to this application, by regulating the state of collagen in the bone gelatin solution before the enzymatic hydrolysis reaction and then performing enzymatic hydrolysis on this basis, the uniformity and reactivity of the enzymatic hydrolysis substrate can be improved. This increases the mass ratio of low molecular weight peptides in the obtained bone collagen peptides without relying on prolonged hydrolysis time or complex post-separation processes, making the molecular weight distribution of the product more concentrated and stable, and the composition of the bone collagen peptides more suitable for bone metabolism and utilization to increase bone density.

[0014] Specifically, the inventors discovered in their research that although bone gelatin appears as a homogeneous solution macroscopically after dissolving in water, the collagen within it may still be distributed in different states within the system. These states may include collagen components with a high degree of molecular dispersion and a more open conformation, as well as collagen components with strong intermolecular interactions and a higher degree of local aggregation. The accessibility and reactivity of collagen in different states to endopeptides differ during subsequent enzymatic hydrolysis, thus affecting the overall homogeneity of the enzymatic reaction and the molecular weight distribution of the products.

[0015] In step S1, by first dispersing the bone gelatin in water and allowing it to swell, and then dissolving it, the bone gelatin molecules can fully absorb water and gradually expand. This helps to reduce the formation of local high-concentration areas and non-uniform structural states caused by direct dissolution, thereby providing a more uniform initial solution system for subsequent processing.

[0016] In step S2, an acidic pH adjuster is gradually added to the bone gelatin solution under controlled conditions, causing the system to gradually approach an unstable state from a stable state, thereby inducing flocculation of some protein components in the solution. Compared with directly adjusting the system to a fixed pH value, this implementation method does not rely on a predetermined isoelectric point parameter, but rather characterizes the degree of instability of protein components in the solution by real-time monitoring of changes in system turbidity, thereby achieving dynamic control of the flocculation process.

[0017] When turbidity begins to rise in a bone gelatin solution, it usually indicates that protein components with strong intermolecular interactions, high aggregation, or relatively dense spatial structures preferentially destabilize and form flocs; while collagen components with higher molecular dispersion and more open conformation remain in the solution at this stage. By controlling the increase in turbidity within the range of 10% to 20%, selective flocculation of the aforementioned protein components can be induced while avoiding drastic overall system instability, thereby reducing non-selective separation of enzymatically hydrolyzable collagen. Simultaneously, by controlling the addition rate of acidic pH adjusters, the instability process is made relatively gradual, which is conducive to the sequential flocculation of different protein components according to their stability differences, thus improving the controllability and repeatability of the flocculation separation process. After reaching the target turbidity change, settling and filtration can effectively separate the formed flocs from the supernatant, obtaining a bone gelatin supernatant with more homogeneous composition and state.

[0018] In step S3, the bone gelatin supernatant is used as an enzymatic hydrolysis substrate for enzymatic hydrolysis. Because the collagen in the supernatant has relatively good dispersion and accessibility, the endopeptidase can act more uniformly on the collagen molecules, thereby reducing reaction differences between different substrate components and minimizing local over-hydrolysis or under-hydrolysis. This facilitates the formation of polypeptide products with a more concentrated molecular weight distribution. Subsequently, post-processing and drying yield bone collagen peptides for increasing bone density.

[0019] In summary, by leveraging the synergistic effects of the swelling-dissolving treatment in step S1, the flocculation separation treatment in step S2, and the enzymatic hydrolysis treatment in step S3, the preparation process of collagen peptides is optimized as a whole, starting from the state of the substrate before the enzymatic hydrolysis reaction. This achieves the technical effect of increasing the proportion of low molecular weight peptides and improving the stability of molecular weight distribution.

[0020] In some embodiments, step S1 includes:

[0021] Disperse bone gelatin in water at a mass ratio of 1:8 to 20, swell at 10 to 25°C for 0.5 to 3 hours, then raise the temperature to 45 to 55°C and dissolve for 10 to 30 minutes to obtain a bone gelatin solution.

[0022] In some of the above embodiments, by controlling the mass ratio of bone gelatin to water and adopting a process of swelling followed by heating and dissolving, the bone gelatin can fully absorb water and gradually expand before dissolving. This helps to reduce the phenomenon of local high concentration and local rapid dissolution caused by direct heating and dissolution, thereby reducing the possibility of forming a heterogeneous structural state in the solution. Compared with the method of directly dissolving bone gelatin at a higher temperature, the above swelling-dissolving process can make the bone gelatin molecules more uniform in spatial distribution and hydration state, providing a more stable and controllable initial solution system for subsequent flocculation and separation steps.

[0023] By controlling the swelling process under mild conditions of 0~25℃ and then performing a medium-temperature dissolution treatment after full swelling, it is possible to ensure that the bone gelatin is fully dissolved while avoiding the adverse effects of excessively high temperatures on the collagen molecular structure, thereby helping to maintain the reactivity of collagen in subsequent steps.

[0024] In this embodiment, the bone gelatin solution obtained after step S1 is more uniform in composition and state of existence, making the flocculation separation process in the subsequent step S2 more selective, which in turn facilitates the uniform enzymatic hydrolysis reaction in step S3, thereby further increasing the proportion of low molecular weight peptides generated.

[0025] In some implementations, in step S2:

[0026] Before adding the acidic pH adjuster to the bone gelatin solution, an electrolyte salt is added to the bone gelatin solution to make the molar concentration of the electrolyte salt in the bone gelatin solution 0.05~0.2mol / L.

[0027] In some of the above embodiments, by introducing a certain amount of electrolyte salt into the bone gelatin solution before acidic pH adjustment, the electrostatic interactions between protein molecules in the solution can be moderately regulated, thereby affecting the instability behavior of different protein components during subsequent pH adjustment. Compared with the case where no electrolyte salt is added, this embodiment helps to reduce the non-uniformity of charge distribution in the system, making the response of protein molecules more consistent when approaching instability conditions.

[0028] Within the specified electrolyte salt concentration range, increased ionic strength in the solution can create a shielding effect on the surface charge of protein molecules, transforming intermolecular interactions from a simple strong electrostatic repulsion or attraction into a more moderate and tunable state. Furthermore, when an acidic pH adjuster is added, different protein components in the solution can more clearly destabilize sequentially according to their structural stability differences, thus facilitating a more controllable flocculation process.

[0029] By controlling the molar concentration of the electrolyte salt within the range of 0.05–0.2 mol / L, a synergistic regulatory effect on the flocculation process can be achieved without significantly interfering with subsequent enzymatic hydrolysis reactions. On the one hand, this ionic strength level is insufficient to cause overall salting out or non-selective sedimentation of protein components in the system; on the other hand, it can avoid sudden and uncontrollable flocculation caused by excessive local charge differences under electrolyte-free conditions.

[0030] In this implementation, the introduction of electrolyte salts and the dynamic pH adjustment process based on turbidity changes work together to make the flocculation separation in the bone gelatin solution more gentle and selective, thereby further improving the consistency of the composition and state of the bone gelatin supernatant entering the subsequent enzymatic hydrolysis step, which is conducive to the uniform function of endopeptides and ultimately further increases the mass ratio of low molecular weight peptides in bone collagen peptides.

[0031] In some implementations, in step S2:

[0032] The acidic pH adjuster includes at least one of hydrochloric acid, citric acid, malic acid, and lactic acid; the electrolyte salt includes at least one of sodium chloride and potassium chloride.

[0033] In some of the above embodiments, the acidic pH adjuster and electrolyte salt are common food-grade or industrial-grade raw materials, widely available, stable in nature, and easy to precisely control their addition amount and action process, which is conducive to the stable implementation of step S2. By selecting the above-mentioned acidic pH adjuster, the pH of the bone gelatin solution can be effectively adjusted, while avoiding the introduction of impurities that would adversely affect subsequent enzymatic hydrolysis and product quality; by selecting sodium chloride or potassium chloride as the electrolyte salt, the ionic strength of the solution can be controlled without significantly increasing the complexity of the system, and it has good compatibility in subsequent enzymatic hydrolysis and product application.

[0034] In some implementations, step S3 includes:

[0035] Based on the dry weight of bone gelatin, 0.05wt%~0.3wt% of endopeptidase was added to the supernatant of the bone gelatin, and the mixture was enzymatically hydrolyzed for 3~4 hours at pH 8.2~9.2 and temperature 55~62℃ to obtain the enzymatic hydrolysate.

[0036] In some of the above embodiments, by controlling the amount of endopeptide and the enzymatic reaction conditions within the aforementioned ranges, it is possible to ensure sufficient hydrolysis of collagen while avoiding over-hydrolysis caused by excessive enzyme dosage or harsh reaction conditions. This is beneficial for obtaining polypeptide products with a relatively concentrated molecular weight distribution. Within these conditions, the endopeptide can effectively cleave collagen in the bone gelatin supernatant, gradually transforming it from a large molecule into a polypeptide structure, while reducing the proportion of free amino acids generated.

[0037] Since the bone gelatin supernatant entering step S3 has already undergone the aforementioned treatment, its composition and state are relatively homogeneous. Under the above enzymatic hydrolysis conditions, the endopeptide can function more stably and uniformly, thereby improving the controllability and repeatability of the enzymatic hydrolysis process. Compared with the case where the substrate state is not pretreated, under the same or similar enzymatic hydrolysis conditions, it is more conducive to steadily increasing the mass proportion of low molecular weight peptides in the final bone collagen peptides.

[0038] In some embodiments, in step S3, the endopeptide includes an Alcalase enzyme preparation.

[0039] In some of the above embodiments, Alcalase enzyme preparation is selected as the endopeptide protease, which can maintain high enzyme activity and stability over a wide pH and temperature range, making it suitable for enzymatic hydrolysis of bone gelatin supernatant. By using this enzyme preparation, effective hydrolysis of collagen can be achieved without significantly increasing the enzyme dosage or prolonging the reaction time, which is beneficial for the stable progress of the enzymatic hydrolysis process. At the same time, Alcalase enzyme preparation has mature applications in the food and bioprocessing fields, stable sources, and is easy to scale up industrially, which helps to improve the operability and reproducibility of this method in actual production.

[0040] In some implementations, step S4 includes:

[0041] The enzyme hydrolysate was inactivated and then filtered through a plate and frame filter. The filtrate was collected and decolorized with activated carbon. The decolorized filtrate was then concentrated by membrane and scraper. After membrane filtration and sterilization, the concentrate was spray-dried to obtain collagen peptides.

[0042] In some of the above embodiments, by inactivating the enzyme in the hydrolysate, the enzymatic reaction can be terminated, preventing further hydrolysis in subsequent processing and thus helping to maintain the molecular weight distribution of the formed peptides. Subsequently, filtration and decolorization remove insoluble matter and impurities that affect the appearance and quality of the product, making the resulting solution clearer and more stable. The combination of membrane concentration and scraper concentration increases the solid content of the solution, which is beneficial to the smooth progress of the subsequent drying process. Membrane filtration sterilization of the concentrate before drying can improve the hygiene and safety of the product. Finally, spray drying converts the liquid material into a powder product, which is convenient for storage, transportation and application.

[0043] The above-mentioned post-processing steps are all commonly used processes in this field. When used in conjunction with the aforementioned steps, they help to stably obtain collagen peptide products without affecting the molecular weight distribution of the peptides.

[0044] In a second aspect, this application provides a collagen peptide for increasing bone density, prepared according to the method described in any embodiment of the first aspect.

[0045] According to this application, the collagen peptides are obtained by regulating the state of the bone gelatin substrate before enzymatic hydrolysis and combining it with subsequent enzymatic hydrolysis, resulting in a higher proportion of low molecular weight peptides and a more concentrated and stable molecular weight distribution in the obtained product. Compared with direct enzymatic hydrolysis or methods relying on subsequent molecular weight fractionation, these collagen peptides can be obtained without significantly increasing process complexity, exhibiting good stability and consistency. This is more conducive to the absorption and utilization of collagen peptides in vivo, making them more suitable for participating in bone matrix-related metabolic processes when used as a nutritional supplement or functional ingredient, thereby contributing to increasing bone density.

[0046] In some embodiments, the collagen peptides contain more than 70% low molecular weight peptides with a molecular weight of 180-1000 Da by mass.

[0047] In some of the above embodiments, the low molecular weight peptides in the aforementioned molecular weight range possess both good solubility and bioavailability, making them easier to absorb and utilize in vivo. Concentrating the molecular weight distribution of collagen peptides within the 180-1000 Da range, and ensuring a high proportion of peptides in this range, helps to provide peptides more suitable for participating in collagen synthesis and maintaining bone tissue structure, while maintaining absorption efficiency. This makes the resulting collagen peptides more suitable as functional raw materials for increasing bone density.

[0048] Compared with the prior art, the beneficial effects of this application are at least as follows:

[0049] 1) This application regulates the state of collagen in bone gelatin solution before enzymatic hydrolysis, making the substrate state more uniform before entering the enzymatic hydrolysis step. This is beneficial for the endopeptide to function stably and uniformly, thereby increasing the mass ratio of low molecular weight peptides in bone collagen peptides without relying on prolonged hydrolysis time or complex post-separation processes.

[0050] 2) This application adopts a processing method based on swelling-dissolution and flocculation separation to perform mild and selective pretreatment on bone gelatin solution, which helps to improve the controllability and repeatability of enzymatic hydrolysis process and makes the molecular weight distribution of the obtained bone collagen peptides more concentrated and stable.

[0051] 3) The process flow of this application is simple, and the raw materials and processing methods used are all commonly used or mature processes in the field. It is easy to scale up for industrial application and can reduce the dependence on high-cost post-processing steps such as membrane classification while ensuring stable product quality.

[0052] 4) The collagen peptides prepared by the method of this application have a higher proportion of low molecular weight peptides with a molecular weight in the range of 180~1000 Da, while the proportion of free amino acids or very low molecular weight peptides is relatively reduced. This is beneficial to provide a peptide composition that is more suitable for participating in collagen-related metabolic processes while taking into account absorption efficiency. Attached Figure Description

[0053] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0054] Figure 1 This is a molecular weight range distribution diagram of the collagen peptide in Example 1 of this application.

[0055] Figure 2 This is a molecular weight range distribution diagram of the collagen peptide in Example 2 of this application.

[0056] Figure 3 This is a molecular weight range distribution diagram of the collagen peptide in Example 3 of this application.

[0057] Figure 4 This is a molecular weight range distribution diagram of the collagen peptide in Example 4 of this application.

[0058] Figure 5 This is a molecular weight range distribution diagram of the collagen peptide in Comparative Example 1 of this application.

[0059] Figure 6 This is a molecular weight range distribution diagram of the collagen peptides in Comparative Example 2 of this application. Detailed Implementation

[0060] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0061] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0062] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0063] In this specification, unless otherwise specified, "parts" refers to "parts by weight".

[0064] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0065] Bone gelatin, food grade, type B gelatin.

[0066] Example 1

[0067] Preparation of collagen peptides for increasing bone density:

[0068] Weigh 1000g (dry basis) of bone gelatin, add 12000g of purified water (the mass ratio of bone gelatin to water is 1:12), stir and disperse at 20℃ to allow the bone gelatin to fully soak and swell, and the swelling time is 2h.

[0069] After swelling is complete, the system is heated to 50°C and dissolved for 20 minutes under continuous stirring to obtain a homogeneous bone gelatin solution.

[0070] Add sodium chloride to the above bone gelatin solution to make the molar concentration of sodium chloride in the solution reach 0.10 mol / L, and stir until completely dissolved.

[0071] Subsequently, under continuous stirring, 1.0 mol / L hydrochloric acid was added dropwise to the bone gelatin solution at a rate of 5 mL / min, and the turbidity of the solution was monitored in real time. When the turbidity of the solution increased by approximately 15% compared to the initial turbidity of the bone gelatin solution, the addition of hydrochloric acid was stopped.

[0072] After stopping the addition of acid, let the system stand for 20 minutes until the flocs are fully formed. Then, remove the flocs by filtration, collect the filtrate, and obtain the bone gelatin supernatant.

[0073] Based on the dry weight of bone gelatin, 0.15 wt% of Alcalase enzyme preparation was added to the bone gelatin supernatant. The pH of the system was adjusted to 8.8, and the temperature was controlled at 58°C. The enzymatic hydrolysis reaction was carried out under constant temperature and stirring conditions for 3.5 h.

[0074] After the enzymatic hydrolysis reaction is complete, the hydrolysate is rapidly heated to 90°C and held at this temperature for 10 minutes to completely inactivate the endopeptides in the system, thus completing the enzyme inactivation process. After enzyme inactivation, the hydrolysate is cooled to below 60°C for later use.

[0075] Subsequently, the enzyme-inactivated hydrolysate was sent to a plate and frame filter for filtration, with the filtration pressure controlled at 0.3 MPa to remove insoluble impurities, and the clear filtrate was collected.

[0076] Activated carbon was added to the filtrate at a concentration of 0.2–0.5 wt% of the filtrate mass, and the solution was stirred and decolorized at 60°C for 30–40 minutes. After decolorization, the activated carbon was removed again by plate and frame filtration to obtain the decolorized collagen peptide solution.

[0077] The decolorized solution is sent to a membrane concentration system for pre-concentration. During the concentration process, the operating temperature is controlled not to exceed 50°C, so that the solid content of the solution is increased to 25~30wt%.

[0078] Subsequently, the concentrated liquid is sent to a scraped film evaporator for further concentration. The evaporation temperature is controlled at 60°C to increase the solid content to 35-40 wt%, thus obtaining a concentrated liquid suitable for spray drying.

[0079] The above-mentioned concentrate was sterilized by membrane filtration. The sterilization membrane used had a pore size of 0.22 μm. After sterilization, the liquid directly entered the spray drying process.

[0080] The concentrate was dried by spray drying. During the spray drying process, the inlet air temperature was controlled at 160~180℃ and the outlet air temperature at 80~90℃. The dried powder was collected to obtain collagen peptides.

[0081] The molecular weight distribution of the obtained collagen peptides was tested according to GB 31645-2018 "National Food Safety Standard for Collagen Peptides". Figure 1 As shown, the mass percentage of low molecular weight peptides with molecular weights in the range of 180 to 1000 Da is 74.15%.

[0082] Example 2

[0083] Preparation of collagen peptides for increasing bone density:

[0084] Weigh 1000g (dry basis) of bone gelatin, add 12000g of purified water (the mass ratio of bone gelatin to water is 1:12), stir and disperse at 20℃ to allow the bone gelatin to fully soak and swell, and the swelling time is 2h.

[0085] After swelling is complete, the system is heated to 50°C and dissolved for 20 minutes under continuous stirring to obtain a homogeneous bone gelatin solution.

[0086] Under continuous stirring, 1.0 mol / L hydrochloric acid was added dropwise to the bone gelatin solution at a rate of 5 mL / min, and the turbidity of the solution was monitored in real time. The addition of hydrochloric acid was stopped when the turbidity of the solution increased by approximately 15% compared to the initial turbidity of the bone gelatin solution.

[0087] After stopping the addition of acid, let the system stand for 20 minutes until the flocs are fully formed. Then, remove the flocs by filtration, collect the filtrate, and obtain the bone gelatin supernatant.

[0088] Based on the dry weight of bone gelatin, 0.15 wt% of Alcalase enzyme preparation was added to the bone gelatin supernatant. The pH of the system was adjusted to 8.8, and the temperature was controlled at 58°C. The enzymatic hydrolysis reaction was carried out under constant temperature and stirring conditions for 3.5 h.

[0089] After the enzymatic hydrolysis reaction is complete, the hydrolysate is rapidly heated to 90°C and held at this temperature for 10 minutes to completely inactivate the endopeptides in the system, thus completing the enzyme inactivation process. After enzyme inactivation, the hydrolysate is cooled to below 60°C for later use.

[0090] Subsequently, the enzyme-inactivated hydrolysate was sent to a plate and frame filter for filtration, with the filtration pressure controlled at 0.3 MPa to remove insoluble impurities, and the clear filtrate was collected.

[0091] Activated carbon was added to the filtrate at a concentration of 0.2–0.5 wt% of the filtrate mass, and the solution was stirred and decolorized at 60°C for 30–40 minutes. After decolorization, the activated carbon was removed again by plate and frame filtration to obtain the decolorized collagen peptide solution.

[0092] The decolorized solution is sent to a membrane concentration system for pre-concentration. During the concentration process, the operating temperature is controlled not to exceed 50°C, so that the solid content of the solution is increased to 25~30wt%.

[0093] Subsequently, the concentrated liquid is sent to a scraped film evaporator for further concentration. The evaporation temperature is controlled at 60°C to increase the solid content to 35-40 wt%, thus obtaining a concentrated liquid suitable for spray drying.

[0094] The above-mentioned concentrate was sterilized by membrane filtration. The sterilization membrane used had a pore size of 0.22 μm. After sterilization, the liquid directly entered the spray drying process.

[0095] The concentrate was dried by spray drying. During the spray drying process, the inlet air temperature was controlled at 160~180℃ and the outlet air temperature at 80~90℃. The dried powder was collected to obtain collagen peptides.

[0096] The molecular weight distribution of the obtained collagen peptides was tested according to GB 31645-2018 "National Food Safety Standard for Collagen Peptides". Figure 2 As shown, the mass percentage of low molecular weight peptides with a molecular weight of 180~1000 Da is 71.26%.

[0097] Example 3

[0098] Preparation of collagen peptides for increasing bone density:

[0099] Weigh 1000g (dry basis) of bone gelatin, add 12000g of purified water (the mass ratio of bone gelatin to water is 1:12), stir and disperse at 20℃ to allow the bone gelatin to fully soak and swell, and the swelling time is 2h.

[0100] After swelling is complete, the system is heated to 50°C and dissolved for 20 minutes under continuous stirring to obtain a homogeneous bone gelatin solution.

[0101] Add sodium chloride to the above bone gelatin solution to make the molar concentration of sodium chloride in the solution reach 0.5 mol / L, and stir until completely dissolved.

[0102] Subsequently, under continuous stirring, 1.0 mol / L hydrochloric acid was added dropwise to the bone gelatin solution at a rate of 5 mL / min, and the turbidity of the solution was monitored in real time. When the turbidity of the solution increased by approximately 15% compared to the initial turbidity of the bone gelatin solution, the addition of hydrochloric acid was stopped.

[0103] After stopping the addition of acid, let the system stand for 20 minutes until the flocs are fully formed. Then, remove the flocs by filtration, collect the filtrate, and obtain the bone gelatin supernatant.

[0104] Based on the dry weight of bone gelatin, 0.15 wt% of Alcalase enzyme preparation was added to the bone gelatin supernatant. The pH of the system was adjusted to 8.8, and the temperature was controlled at 58°C. The enzymatic hydrolysis reaction was carried out under constant temperature and stirring conditions for 3.5 h.

[0105] After the enzymatic hydrolysis reaction is complete, the hydrolysate is rapidly heated to 90°C and held at this temperature for 10 minutes to completely inactivate the endopeptides in the system, thus completing the enzyme inactivation process. After enzyme inactivation, the hydrolysate is cooled to below 60°C for later use.

[0106] Subsequently, the enzyme-inactivated hydrolysate was sent to a plate and frame filter for filtration, with the filtration pressure controlled at 0.3 MPa to remove insoluble impurities, and the clear filtrate was collected.

[0107] Activated carbon was added to the filtrate at a concentration of 0.2–0.5 wt% of the filtrate mass, and the solution was stirred and decolorized at 60°C for 30–40 minutes. After decolorization, the activated carbon was removed again by plate and frame filtration to obtain the decolorized collagen peptide solution.

[0108] The decolorized solution is sent to a membrane concentration system for pre-concentration. During the concentration process, the operating temperature is controlled not to exceed 50°C, so that the solid content of the solution is increased to 25~30wt%.

[0109] Subsequently, the concentrated liquid is sent to a scraped film evaporator for further concentration. The evaporation temperature is controlled at 60°C to increase the solid content to 35-40 wt%, thus obtaining a concentrated liquid suitable for spray drying.

[0110] The above-mentioned concentrate was sterilized by membrane filtration. The sterilization membrane used had a pore size of 0.22 μm. After sterilization, the liquid directly entered the spray drying process.

[0111] The concentrate was dried by spray drying. During the spray drying process, the inlet air temperature was controlled at 160~180℃ and the outlet air temperature at 80~90℃. The dried powder was collected to obtain collagen peptides.

[0112] The molecular weight distribution of the obtained collagen peptides was tested according to GB 31645-2018 "National Food Safety Standard for Collagen Peptides". Figure 3 As shown, the mass percentage of low molecular weight peptides with a molecular weight of 180~1000 Da is 70.32%.

[0113] Example 4

[0114] Preparation of collagen peptides for increasing bone density:

[0115] Weigh 1000g (dry basis) of bone gelatin, add 12000g of purified water (the mass ratio of bone gelatin to water is 1:12), stir and disperse at 20℃ to allow the bone gelatin to fully soak and swell, and the swelling time is 2h.

[0116] After swelling is complete, the system is heated to 50°C and dissolved for 20 minutes under continuous stirring to obtain a homogeneous bone gelatin solution.

[0117] Under continuous stirring, 1.0 mol / L hydrochloric acid was added dropwise to the bone gelatin solution at a rate of 5 mL / min, and the turbidity of the solution was monitored in real time. The addition of hydrochloric acid was stopped when the turbidity of the solution increased by approximately 15% compared to the initial turbidity of the bone gelatin solution.

[0118] After stopping the addition of acid, sodium chloride was added to bring the molar concentration of sodium chloride in the solution to 0.10 mol / L. The system was allowed to stand for 20 minutes. After the flocs were fully formed, the flocs were removed by filtration, and the filtrate was collected to obtain the bone gelatin supernatant.

[0119] Based on the dry weight of bone gelatin, 0.15 wt% of Alcalase enzyme preparation was added to the bone gelatin supernatant. The pH of the system was adjusted to 8.8, and the temperature was controlled at 58°C. The enzymatic hydrolysis reaction was carried out under constant temperature and stirring conditions for 3.5 h.

[0120] After the enzymatic hydrolysis reaction is complete, the hydrolysate is rapidly heated to 90°C and held at this temperature for 10 minutes to completely inactivate the endopeptides in the system, thus completing the enzyme inactivation process. After enzyme inactivation, the hydrolysate is cooled to below 60°C for later use.

[0121] Subsequently, the enzyme-inactivated hydrolysate was sent to a plate and frame filter for filtration, with the filtration pressure controlled at 0.3 MPa to remove insoluble impurities, and the clear filtrate was collected.

[0122] Activated carbon was added to the filtrate at a concentration of 0.2–0.5 wt% of the filtrate mass, and the solution was stirred and decolorized at 60°C for 30–40 minutes. After decolorization, the activated carbon was removed again by plate and frame filtration to obtain the decolorized collagen peptide solution.

[0123] The decolorized solution is sent to a membrane concentration system for pre-concentration. During the concentration process, the operating temperature is controlled not to exceed 50°C, so that the solid content of the solution is increased to 25~30wt%.

[0124] Subsequently, the concentrated liquid is sent to a scraped film evaporator for further concentration. The evaporation temperature is controlled at 60°C to increase the solid content to 35-40 wt%, thus obtaining a concentrated liquid suitable for spray drying.

[0125] The above-mentioned concentrate was sterilized by membrane filtration. The sterilization membrane used had a pore size of 0.22 μm. After sterilization, the liquid directly entered the spray drying process.

[0126] The concentrate was dried by spray drying. During the spray drying process, the inlet air temperature was controlled at 160~180℃ and the outlet air temperature at 80~90℃. The dried powder was collected to obtain collagen peptides.

[0127] The molecular weight distribution of the obtained collagen peptides was tested according to GB 31645-2018 "National Food Safety Standard for Collagen Peptides". Figure 4 As shown, the mass percentage of low molecular weight peptides with a molecular weight of 180~1000 Da is 70.83%.

[0128] Comparative Example 1

[0129] Preparation of collagen peptides for increasing bone density:

[0130] Weigh 1000g (dry basis) of bone gelatin, add 12000g of purified water (the mass ratio of bone gelatin to water is 1:12), stir and disperse at 20℃ to allow the bone gelatin to fully soak and swell, and the swelling time is 2h.

[0131] After swelling is complete, the system is heated to 50°C and dissolved for 20 minutes under continuous stirring to obtain a homogeneous bone gelatin solution.

[0132] Based on the dry weight of bone gelatin, 0.15 wt% of Alcalase enzyme preparation was added to the bone gelatin solution. The pH of the system was adjusted to 8.8, and the temperature was controlled at 58℃. The enzymatic hydrolysis reaction was carried out under constant temperature and stirring conditions for 3.5 h.

[0133] After the enzymatic hydrolysis reaction is complete, the hydrolysate is rapidly heated to 90°C and held at this temperature for 10 minutes to completely inactivate the endopeptides in the system, thus completing the enzyme inactivation process. After enzyme inactivation, the hydrolysate is cooled to below 60°C for later use.

[0134] Subsequently, the enzyme-inactivated hydrolysate was sent to a plate and frame filter for filtration, with the filtration pressure controlled at 0.3 MPa to remove insoluble impurities, and the clear filtrate was collected.

[0135] Activated carbon was added to the filtrate at a concentration of 0.2–0.5 wt% of the filtrate mass, and the solution was stirred and decolorized at 60°C for 30–40 minutes. After decolorization, the activated carbon was removed again by plate and frame filtration to obtain the decolorized collagen peptide solution.

[0136] The decolorized solution is sent to a membrane concentration system for pre-concentration. During the concentration process, the operating temperature is controlled not to exceed 50°C, so that the solid content of the solution is increased to 25~30wt%.

[0137] Subsequently, the concentrated liquid is sent to a scraped film evaporator for further concentration. The evaporation temperature is controlled at 60°C to increase the solid content to 35-40 wt%, thus obtaining a concentrated liquid suitable for spray drying.

[0138] The above-mentioned concentrate was sterilized by membrane filtration. The sterilization membrane used had a pore size of 0.22 μm. After sterilization, the liquid directly entered the spray drying process.

[0139] The concentrate was dried by spray drying. During the spray drying process, the inlet air temperature was controlled at 160~180℃ and the outlet air temperature at 80~90℃. The dried powder was collected to obtain collagen peptides.

[0140] The molecular weight distribution of the obtained collagen peptides was tested according to GB 31645-2018 "National Food Safety Standard for Collagen Peptides". Figure 5 As shown, the mass percentage of low molecular weight peptides with a molecular weight of 180~1000 Da is 66.69%.

[0141] Comparative Example 2

[0142] Preparation of collagen peptides for increasing bone density:

[0143] Weigh 1000g (dry basis) of bone gelatin, add 12000g of purified water (the mass ratio of bone gelatin to water is 1:12), heat the system to 50℃, and dissolve it for 30 minutes under continuous stirring to obtain a homogeneous bone gelatin solution.

[0144] Add sodium chloride to the above bone gelatin solution to make the molar concentration of sodium chloride in the solution reach 0.10 mol / L, and stir until completely dissolved.

[0145] Subsequently, under continuous stirring, 1.0 mol / L hydrochloric acid was added dropwise to the bone gelatin solution at a rate of 5 mL / min, and the turbidity of the solution was monitored in real time. When the turbidity of the solution increased by approximately 15% compared to the initial turbidity of the bone gelatin solution, the addition of hydrochloric acid was stopped.

[0146] After stopping the addition of acid, let the system stand for 20 minutes until the flocs are fully formed. Then, remove the flocs by filtration, collect the filtrate, and obtain the bone gelatin supernatant.

[0147] Based on the dry weight of bone gelatin, 0.15 wt% of Alcalase enzyme preparation was added to the bone gelatin supernatant. The pH of the system was adjusted to 8.8, and the temperature was controlled at 58°C. The enzymatic hydrolysis reaction was carried out under constant temperature and stirring conditions for 3.5 h.

[0148] After the enzymatic hydrolysis reaction is complete, the hydrolysate is rapidly heated to 90°C and held at this temperature for 10 minutes to completely inactivate the endopeptides in the system, thus completing the enzyme inactivation process. After enzyme inactivation, the hydrolysate is cooled to below 60°C for later use.

[0149] Subsequently, the enzyme-inactivated hydrolysate was sent to a plate and frame filter for filtration, with the filtration pressure controlled at 0.3 MPa to remove insoluble impurities, and the clear filtrate was collected.

[0150] Activated carbon was added to the filtrate at a concentration of 0.2–0.5 wt% of the filtrate mass, and the solution was stirred and decolorized at 60°C for 30–40 minutes. After decolorization, the activated carbon was removed again by plate and frame filtration to obtain the decolorized collagen peptide solution.

[0151] The decolorized solution is sent to a membrane concentration system for pre-concentration. During the concentration process, the operating temperature is controlled not to exceed 50°C, so that the solid content of the solution is increased to 25~30wt%.

[0152] Subsequently, the concentrated liquid is sent to a scraped film evaporator for further concentration. The evaporation temperature is controlled at 60°C to increase the solid content to 35-40 wt%, thus obtaining a concentrated liquid suitable for spray drying.

[0153] The above-mentioned concentrate was sterilized by membrane filtration. The sterilization membrane used had a pore size of 0.22 μm. After sterilization, the liquid directly entered the spray drying process.

[0154] The concentrate was dried by spray drying. During the spray drying process, the inlet air temperature was controlled at 160~180℃ and the outlet air temperature at 80~90℃. The dried powder was collected to obtain collagen peptides.

[0155] The molecular weight distribution of the obtained collagen peptides was tested according to GB 31645-2018 "National Food Safety Standard for Collagen Peptides". Figure 6 As shown, the mass percentage of low molecular weight peptides with molecular weights in the range of 180 to 1000 Da is 68.75%.

[0156] Based on the above data (Examples 1-4 and Comparative Examples 1-2), it can be seen that each example is better than Comparative Examples 1 and 2, indicating that the preparation method provided in this application can increase the mass proportion of low molecular weight peptides (180-1000 Da) in the obtained collagen peptides without relying on prolonged enzymatic hydrolysis time or complex post-separation, and make the molecular weight distribution more concentrated and stable. The possible reason is that in Comparative Example 1, the bone gelatin solution was not pretreated with "acid-induced flocculation separation," and the substrate entering the enzymatic hydrolysis step still contained multiple states, including dispersed and aggregated states. Endopeptides react with different states... The varying accessibility of the substrate in different states leads to inconsistent hydrolysis progress within the system, easily resulting in a coexistence of "partial over-hydrolysis (increased <180Da) + partial under-hydrolysis (>1000Da residue)," thus reducing the proportion of the 180~1000Da range. In Comparative Example 2, although electrolyte salts were added and acid-induced flocculation was performed, the lack of swelling pretreatment and the longer direct heating dissolution time easily led to the formation of local high concentrations and local aggregate structures, resulting in decreased selectivity of flocculation separation and insufficient substrate homogeneity in the supernatant, thereby weakening the synchronicity of subsequent enzymatic hydrolysis and the enrichment effect of the target molecular weight range.

[0157] As shown in Examples 1 and 2, introducing a moderate ionic strength (0.10 mol / L NaCl) before acid-induced flocculation has a positive effect on increasing the proportion of peptides in the target region: Example 1 (74.15%) is higher than Example 2 (71.26%). This indicates that electrolyte salts can moderately regulate the electrostatic interactions and aggregation behavior between collagen molecules during acid addition, making flocculation more inclined to preferentially separate components with "strong interactions and aggregation / density". This results in a more consistent dispersion and accessibility of the substrate entering the supernatant, making it easier for endopeptides to achieve uniform cleavage, ultimately increasing the proportion of peptides in the 180~1000 Da range.

[0158] As shown in Examples 1 and 3, when the electrolyte concentration increased from 0.10 mol / L to 0.50 mol / L, the proportion of peptides in the target region decreased from 74.15% to 70.32%, indicating that there is a suitable range for the electrolyte concentration. The possible reason is that when the salt concentration is too high, the ionic strength of the system is too great, and flocculation separation is more likely to shift from "preferential instability of aggregated state" to a broader spectrum of desolvation / salting-out aggregation. This causes some originally dispersed, easily enzymatically hydrolyzed collagen components to be co-entrained into the precipitation, resulting in a decrease in the proportion of effective substrate in the supernatant and a more uneven state distribution. This reduces the synchronicity of subsequent enzymatic hydrolysis, leading to a weakened enrichment effect in the 180-1000 Da range.

[0159] As shown in Examples 1 and 4, adding electrolyte salts after hydrochloric acid addition (Example 4, 70.83%) resulted in a worse effect compared to adding salt first, then acid (Example 1, 74.15%). This indicates that the timing of electrolyte salt addition affects the selectivity of acid-induced flocculation. The possible reason is that during the initial acid addition, some collagen components have already undergone initial aggregation or formed unstable intermediate aggregates under low pH conditions. Subsequent addition of electrolyte salts can easily cause "secondary compression / bridging aggregation" of the already formed aggregates, making the flocculation process more non-selectively expand the aggregation range, thereby increasing false sedimentation and reducing the homogeneity of the supernatant substrate, ultimately decreasing the proportion of peptides in the target molecular weight range.

[0160] In summary, this application constructs a more uniform initial system through "swelling-dissolution" and, in conjunction with acid-induced flocculation separation using "appropriate salt concentration + reasonable feeding sequence," achieves effective screening and synchronized control of the state of the enzymatic hydrolysis substrate, thereby significantly increasing the proportion of low molecular weight peptides of 180~1000 Da, and outperforming comparative schemes that did not undergo flocculation separation or had insufficient pretreatment.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing collagen peptides for increasing bone density, characterized in that, Includes the following steps: S1: Disperse bone gelatin in water at a mass ratio of 1:8~20, swell at 10~25℃ for 0.5~3h, then raise the temperature to 45~55℃ and dissolve for 10~30min to obtain bone gelatin solution; S2: Add sodium chloride to the bone gelatin solution to make the molar concentration of sodium chloride in the bone gelatin solution 0.05~0.2mol / L; add 0.1~1mol / L hydrochloric acid to the bone gelatin solution at a rate of 1~10mL / min until the turbidity of the system increases by 10%~20% compared with the initial turbidity of the bone gelatin solution, then stop adding, let stand for 10~30min and filter to obtain bone gelatin supernatant; S3: Add Alcalase enzyme preparation to the bone gelatin supernatant to hydrolyze the collagen in the bone gelatin supernatant into bone collagen peptides to obtain enzymatic hydrolysate; S4: The enzymatic hydrolysate is post-treated and dried to obtain collagen peptides.

2. The method according to claim 1, characterized in that, Step S3 includes: Based on the dry weight of bone gelatin, 0.05wt%~0.3wt% of Alcalase enzyme preparation was added to the bone gelatin supernatant, and the mixture was enzymatically hydrolyzed for 3~4 hours at a pH of 8.2~9.2 and a temperature of 55~62℃ to obtain the enzymatic hydrolysate.

3. The method according to claim 1 or 2, characterized in that, Step S4 includes: The enzyme hydrolysate was inactivated and then filtered through a plate and frame filter. The filtrate was collected and decolorized with activated carbon. The decolorized filtrate was then concentrated by membrane and scraper. After membrane filtration and sterilization, the concentrate was spray-dried to obtain collagen peptides.

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