Sheep bone marrow peptide composition capable of activating osteoblasts to be rich in bone repair factors

Abstract

The invention relates to the technical field of biological medicine and food processing, and discloses a sheep bone marrow peptide composition for activating osteoblasts to be rich in bone repair factors, the sheep bone marrow peptide composition has an ordered assembly structure mediated by electrostatic adsorption, and the sheep bone marrow peptide composition is prepared from the following raw materials in parts by weight: 1000-1500 parts of sheep bone marrow peptide powder; 250 to 350 parts of sweet orange fruit powder; 450 to 700 parts of a non-denatured type II collagen protein; 800 to 1200 parts of hydrolyzed type II collagen protein; 1-10 parts of ginseng peptide powder; 1-10 parts of Chinese wolfberry fruit peptide powder; and 5 to 15 parts of stevioside. According to the invention, an interface proton buffer layer is constructed by utilizing the zwitterionic characteristics of the hydrolyzed type II collagen, and a microenvironment with high buffer capacity is formed on the surface of the non-denatured type II collagen, so that the permeation of gastric acid hydrogen ions is effectively blocked, and the local pH value of core protein is kept stable.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and food processing technology, specifically to a sheep bone marrow peptide composition that activates osteoblasts and is rich in bone repair factors. Background Technology

[0002] Bone loss and articular cartilage degeneration are common problems affecting quality of life as people age. Activating osteoblast activity to promote bone repair while inhibiting articular cartilage wear and inflammation is key to maintaining skeletal health. Sheep bone marrow peptides are rich in various osteogenic factors that can effectively induce osteoblast proliferation; non-denatured type II collagen helps regulate joint immune responses through a specific oral immune tolerance mechanism. Combining these two active ingredients can exert a synergistic repair effect on both bones and joints, representing an important intervention in the field of bone health.

[0003] Current technologies primarily employ physical mixing processes, directly combining sheep bone marrow peptide powder, type II collagen, and conventional excipients to prepare dosage forms such as solid beverages, capsules, or compressed candies. This preparation method features a short process flow and highly versatile equipment, enabling large-scale industrial production at low cost. Furthermore, the resulting products provide consumers with multi-source protein and peptide nutritional supplements, offering excellent convenience in storage, portability, and consumption as a basic dietary supplement.

[0004] However, compositions prepared using conventional processes face significant challenges in bioavailability during practical applications. First, the immune tolerance efficacy of undenatured type II collagen relies heavily on its intact triple-helix structure, which is extremely sensitive to gastric acid. In existing products, the active particles are directly exposed, lacking a microscopic interface buffer. Upon ingestion, hydrogen ions rapidly disrupt the hydrogen bonds maintaining the helix, causing acid-induced denaturation before reaching the intestinal target site. Second, highly active pepsin in gastric juice has a non-specific cleavage effect on protein chains. Traditional formulations, with homogeneous distribution of components, lack a competitive consumption mechanism against enzymatic hydrolysis, resulting in the core components being directly exposed to the enzymatic environment and rapidly truncated and inactivated. Third, the surface charge of naturally occurring sheep bone marrow peptides is electrically repulsive to that of collagen particles. Simple mixing cannot overcome the microscopic repulsion between components, leading to a highly dispersed distribution during digestive tract transport, making it difficult for both to reach the target site synchronously and achieving the desired synergistic effect. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts. This addresses the problem that existing sheep bone marrow peptide and non-denatured type II collagen compositions lack microstructural protection and orderly binding mechanisms in the digestive tract environment, leading to acid-induced denaturation, enzymatic inactivation, and component separation of active ingredients, thereby reducing the bioavailability of bone repair factors and the activation efficiency of osteoblasts.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts, using the following technical solution: A sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts has an ordered assembly structure mediated by electrostatic adsorption. The composition is made from raw materials comprising the following parts by weight: 1000 to 1500 parts sheep bone marrow peptide powder, 250 to 350 parts sweet orange fruit powder, 450 to 700 parts non-denatured type II collagen, 800 to 1200 parts hydrolyzed type II collagen, 1 to 10 parts ginseng peptide powder, 1 to 10 parts wolfberry peptide powder, and 5 to 15 parts steviol glycosides. The microstructure of the composition comprises: a core of non-denatured type II collagen, the surface of which is coated with an interfacial proton buffer layer composed of partially hydrolyzed type II collagen; an acid-activated mixture of sheep bone marrow peptide powder and sweet orange fruit powder is adsorbed on the outside of the interfacial proton buffer layer to form a peptide-protein ordered assembly complex; the remaining hydrolyzed type II collagen is distributed around the ordered assembly complex to form a competitive enzymatic sacrificial matrix.

[0007] By employing the above technical solution, this invention utilizes mechanochemical modification and electrostatic self-assembly technology to construct a multi-level microstructure with a "core-buffer layer-adsorption layer-sacrificial layer". This structure enhances the acid resistance and bioactivity retention of the composition through the following physicochemical mechanisms: First, an interfacial proton buffer system was constructed. This invention utilizes the zwitterionic properties of hydrolyzed type II collagen, pre-coating it onto the surface of the undenatured type II collagen core. Because hydrolyzed type II collagen contains abundant amino and carboxyl side chains, it can bind hydrogen ions in an acidic environment, thus forming a high-buffering-capacity barrier at the micro-interface of the undenatured type II collagen particles. This barrier effectively blocks the penetration of high concentrations of hydrogen ions from gastric juice into the core, maintaining a relatively stable pH value in the microenvironment of the undenatured type II collagen and preventing it from losing its triple-helix structure due to acid-induced denaturation.

[0008] Secondly, it achieves charge-mediated ordered assembly. Under normal conditions, both sheep bone marrow peptides and type II collagen are negatively charged and difficult to bind. This invention introduces sweet orange fruit powder as a proton donor, using high shear mechanical force to protonate the amino groups on the surface of the sheep bone marrow peptides, changing their zeta potential from negative to positive. This positively charged modified peptide can be directionally adsorbed onto the surface of negatively charged undenatured type II collagen and its buffer layer through strong electrostatic attraction. This ordered assembly not only achieves physical encapsulation of the active ingredients but also prevents stratification and segregation of the components during storage and transportation.

[0009] Third, a competitive enzymatic sacrificial mechanism was established. This invention places partially hydrolyzed type II collagen in the outermost layer as a sacrificial matrix. Upon entering the gastric juice environment, due to its looser structure and exposed cleavage sites, the hydrolyzed type II collagen, as a high-affinity substrate, preferentially binds to and is hydrolyzed by pepsin. This process depletes the enzyme activity in the local environment, competitively reducing the attack of pepsin on the core undenatured type II collagen, thereby providing a time window for the active ingredients to reach the intestines through the stomach.

[0010] Preferably, the raw materials are in the following proportions by weight: 1100 to 1200 parts sheep bone marrow peptide powder, 280 to 320 parts sweet orange fruit powder, 500 to 650 parts non-denatured type II collagen, 900 to 1100 parts hydrolyzed type II collagen, 4 to 6 parts ginseng peptide powder, 4 to 6 parts wolfberry peptide powder, and 8 to 12 parts steviol glycosides.

[0011] By adopting the above technical solution, the ratio of each component is optimized to ensure that a buffer layer and an adsorption layer with complete coverage can be formed at the microscopic level, while ensuring sufficient thickness of the external sacrificial matrix, so that the structural integrity and functionality of the composition can achieve a good synergistic effect.

[0012] Preferably, the raw materials have the following characteristics: the average molecular weight (Mw) of the sheep bone marrow peptide powder is distributed between 500 Da and 1000 Da, and the proportion of components with a molecular weight less than 1000 Da is greater than or equal to 90%; the undenatured type II collagen retains the complete triple helix active structure of collagen; the average molecular weight (Mw) of the hydrolyzed type II collagen is distributed between 1500 Da and 3000 Da, and its 10% aqueous solution has a pH value of 5.5 to 6.5, exhibiting zwitterionic properties.

[0013] By adopting the above technical solution, selecting sheep bone marrow peptides with a specific molecular weight can ensure that they have sufficient specific surface area to accept protonation modification, while small molecule peptides are more likely to fill tightly in the adsorption layer; limiting the molecular weight and isoelectric point characteristics of hydrolyzed type II collagen is to ensure that it has suitable film-forming properties and buffering capacity. Too large a molecular weight is not conducive to interface spreading, while too small a molecular weight is difficult to form an effective physical barrier.

[0014] Preferably, the mixture of acid-activated sheep bone marrow peptide powder and sweet orange fruit powder is an acid-high potential activated sheep bone marrow peptide mixture, which is obtained by the following preparation method: in an environment with a relative humidity of 30% to 40%, sheep bone marrow peptide powder and sweet orange fruit powder are mixed and subjected to high shear treatment at a temperature of 25 to 35°C. The amino groups on the surface of the sheep bone marrow peptide powder are protonated by the mechanochemical action and the organic acids in the sweet orange fruit powder, causing its Zeta potential to shift from a negative value to a positive value.

[0015] By employing the above-mentioned technical solution and strictly controlling the temperature, humidity, and shear conditions during the activation process, mechanical energy is used to induce chemical adsorption and proton transfer reactions between solid organic acid molecules and amino groups on the peptide chain surface. This solid-phase mechanochemical modification avoids the energy consumption and activity loss associated with later drying in liquid-phase reactions, successfully achieving charge reversal on the peptide powder surface, and providing the necessary electrochemical basis for subsequent electrostatic assembly.

[0016] Preferably, the structure with the core surface coated with the interfacial proton buffer layer is formed by: utilizing the zwitterionic properties and high specific surface area of ​​hydrolyzed type II collagen, it is gently mixed with undenatured type II collagen under low shear force, so that the hydrolyzed type II collagen forms an isolation buffer layer with controllable thickness on the surface of the undenatured type II collagen through physical adsorption; wherein, the weight parts of the hydrolyzed type II collagen used to construct the interfacial proton buffer layer are 80 to 150 parts.

[0017] By employing the above technical solution and a low-shear gentle mixing process, the natural structure of non-denatured type II collagen is avoided from being damaged by strong mechanical forces. Limiting the proportion of the buffer layer ensures that the core particle surface is completely covered without excessive accumulation, allowing subsequent positively charged peptide layers to still sense the negative potential field of the core, thereby guaranteeing the tightness of the multilayer structure.

[0018] Preferably, the competitive enzymatic sacrificial matrix is ​​composed of 850 to 950 parts by weight of hydrolyzed type II collagen, ginseng peptide powder, wolfberry peptide powder and steviol glycoside. The sacrificial matrix is ​​loosely distributed in the gaps and outer surface of the peptide-protein ordered assembly complex, and is used to preferentially bind to digestive enzymes in the digestive tract environment.

[0019] By adopting the above technical solution, the loose outer layer structure constructed by multiple components has a large specific surface area, which can quickly adsorb and consume digestive enzymes that invade the particle interior, and protect the orderly assembled complex with intact internal structure through substrate competition mechanism.

[0020] Secondly, the present invention provides a method for preparing a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts, using the following technical solution: A method for preparing a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts includes the following steps: S1. Preparation of activated phase: Sheep bone marrow peptide powder and sweet orange fruit powder are mixed under high shear, and an acidic high potential activated sheep bone marrow peptide mixture is prepared by mechanochemical action. S2. Buffer core construction: Undenatured type II collagen and partially hydrolyzed type II collagen are gently mixed, and an interfacial proton buffer layer is formed on the surface of undenatured type II collagen by physical adsorption, thus obtaining undenatured type II collagen particles with an interfacial proton buffer layer. S3, Ordered Assembly: The particles obtained in step S2 are used as the base material. The mixture obtained in step S1 is added in a mixed state, and a polar initiator is sprayed in. Electrostatic adsorption assembly is carried out by charge reversal and liquid bridge force to obtain peptide-protein ordered assembly complex. S4. Total Mixing and Post-processing: The remaining hydrolyzed type II collagen, ginseng peptide powder, wolfberry peptide powder and steviol glycosides are premixed and then added to the complex in step S3 for mixing to construct a competitive sacrificial matrix. The residual polar initiator is then removed to obtain the sheep bone marrow peptide composition.

[0021] By adopting the above technical solution, this method controls the distribution of components in the microscopic space through a step-by-step assembly strategy. The technical principle is as follows: First, the activated phase preparation step utilized a high shear field to achieve surface modification of sheep bone marrow peptides under solid-state conditions. Mechanical energy broke up powder agglomeration, increased the reaction contact area, promoted the uniform distribution of organic acid molecules on the peptide chain surface, and induced protonation, thereby endowing the originally negatively charged peptide powder with a positive surface potential, establishing the electrochemical basis for subsequent electrostatic adsorption.

[0022] Secondly, the buffer core construction step employs a gentle dry coating technique, utilizing the van der Waals forces and electrostatic interactions between undenatured type II collagen and hydrolyzed type II collagen to pre-form a dense buffer medium on the core surface. This independent step prevents the activated acidic peptides from directly contacting the unprotected core protein, avoiding the risk of protein denaturation due to excessively high local acid concentrations.

[0023] Furthermore, the ordered assembly step incorporates polar initiator-assisted electrostatic adsorption. The polar solvent micromist not only wets the particle surface, inducing interfacial dissolution and recrystallization to form solid-liquid bridges, but also acts as a dielectric to enhance the interaction between positively and negatively charged groups. This process firmly anchors the modified peptide to the buffer core surface, forming a stable core-shell structure.

[0024] Finally, the final mixing step constructs a sacrificial layer on the exterior of the formed complex. This step, by controlling the mixing intensity, ensures that the remaining excipients, such as hydrolyzed collagen, adhere in a looser state, guaranteeing their rapid dispersion and competitive substrate role upon entering the gastric fluid.

[0025] Preferably, in step S1, the spindle speed of the high-shear mixer is 100 to 300 r / min, the high-speed flying knife speed is 1500 to 2500 r / min, the mixing time is 10 to 15 minutes, and the material temperature is controlled at 25 to 35°C during the mixing process.

[0026] By employing the above technical solution, the high-speed flying knife provides sufficient shear energy to break up the powder and induce mechanochemical reactions, while the lower spindle speed ensures the uniformity of macroscopic mixing. Strict temperature control prevents the peptides from undergoing thermal denaturation or Maillard reactions under prolonged shear heating, thus preserving their biological activity.

[0027] Preferably, in step S3, the polar initiator is food-grade ethanol, and its injection amount is 0.5% to 1.0% of the total weight of the raw materials, and it is uniformly sprayed through a high-pressure atomizing nozzle at a rate of 5 to 10 mL / min; the spray pressure is controlled to be 0.2 to 0.4 MPa.

[0028] By employing the above technical solution, trace amounts of ethanol are sufficient to induce a liquid bridging effect at the particle contact point, but not enough to dissolve or damage the protein structure or cause material agglomeration. High-pressure atomization ensures that the droplet size is small and uniformly distributed, avoiding localized over-wetting. The high volatility of ethanol also facilitates its removal in subsequent processes, ensuring product safety.

[0029] Preferably, in step S4, the residual polar initiator is removed by fluidized bed purging with clean, dry air for 10 to 20 minutes; the mass ratio of the remaining hydrolyzed type II collagen to the hydrolyzed type II collagen used in step S2 is 5.6 to 11.5:1.

[0030] By employing the above technical solution, fluidized bed purging technology can efficiently remove ethanol residue at low temperatures, avoiding damage to heat-sensitive components caused by heating and drying. By limiting the mass ratio of the inner and outer hydrolyzed collagen layers, the density of the interfacial buffer layer and the capacity of the outer sacrificial layer are balanced, thus balancing the functional allocation between internal protection and external consumption.

[0031] This invention provides a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts. It possesses the following beneficial effects: 1. This invention utilizes the zwitterionic properties of hydrolyzed type II collagen to construct an interfacial proton buffer layer, forming a microenvironment with high buffering capacity on the surface of undenatured type II collagen. This effectively blocks the penetration of gastric hydrogen ions and maintains the local pH stability of the core protein. Compared with conventional physical mixing techniques where the core components are directly exposed to a strong acid environment, this solution effectively solves the problem of undenatured type II collagen rapidly losing its triple-helix active structure due to acid-induced denaturation.

[0032] 2. This invention uses mechanochemical action to protonate and flip the charge of sheep bone marrow peptides. Combined with the induction of a polar initiator, it achieves the directional and dense assembly of positively charged peptide layers on the surface of a negatively charged core, which enhances the binding force between particles and prevents the components from separating and stratifying during storage and transportation. Compared with the disordered and discrete distribution of components in the prior art, this solution overcomes the defect of premature disintegration and separation of active ingredients in the front of the digestive tract due to loose structure.

[0033] 3. This solution adopts an inner and outer layered raw material distribution strategy, constructing a loose hydrolyzed collagen sacrificial matrix on the periphery of the ordered assembly. This outer layer, as a high-affinity substrate, preferentially binds to pepsin, consuming local enzyme activity through a substrate competition mechanism. Compared with the traditional method of uniformly mixing excipients and active ingredients, this solution solves the technical problem of pepsin directly attacking the core non-denatured type II collagen, which leads to a significant reduction in its bioavailability. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0036] Sheep bone marrow peptide powder is a commercially available food-grade raw material with an average molecular weight (Mw) ranging from 500 Da to 1000 Da. Among them, the proportion of components with a molecular weight less than 1000 Da is ≥90%, and the isoelectric point (pI) is 6.5 to 7.5. Non-denatured type II collagen, retaining the complete triple helix active structure of collagen, with a total type II collagen content of ≥25%, Hubei Hege Composite Bone Collagen Biotechnology Co., Ltd. The average molecular weight (Mw) of hydrolyzed type II collagen ranges from 1500 Da to 3000 Da, and the pH of a 10% aqueous solution is 5.5 to 6.5. (Hubei Hege Composite Bone Collagen Biotechnology Co., Ltd.) The total acid content of sweet orange fruit powder, calculated as citric acid, is ≥2.0%; Both ginseng peptide powder and wolfberry peptide powder are made from commercially available food-grade raw materials, with a total peptide content of ≥60%. Steviosides CAS No. 57817-89-7, total glycoside content ≥95%; Pepsin CAS No. 9001-75-6, enzyme activity ≥3000U / mg; Hydrochloric acid CAS number 7647-01-0.

[0037] Preparation Example 1: This preparation example provides an acidic, high-potential activated sheep bone marrow peptide mixture, comprising the following steps: 1180g of sheep bone marrow peptide powder and 300g of sweet orange fruit powder were put into a high-shear mixer with a temperature-controlled jacket. The relative humidity in the mixing chamber was controlled at 35%, and the jacket cooling water circulation was turned on to control the material temperature at 30℃. The spindle speed was set to 200r / min and the high-speed fly knife speed was set to 2000r / min, and shearing and mixing were performed for 12 minutes. The surface of the sheep bone marrow peptide powder was protonated and the Zeta potential was shifted to the positive direction by the mechanochemical action and organic acids in the fruit powder. The mixture was then discharged to obtain an acidic high-potential activated sheep bone marrow peptide mixture.

[0038] Preparation Example 2: This preparation example provides an acidic, high-potential activated sheep bone marrow peptide mixture, comprising the following steps: 1180g of sheep bone marrow peptide powder and 300g of sweet orange fruit powder were put into a high-shear mixer with a temperature-controlled jacket. The relative humidity in the mixing chamber was controlled at 30%, and the jacket cooling water circulation was turned on to control the material temperature at 25℃. The spindle speed was set to 100r / min and the high-speed fly knife speed was set to 1500r / min. Shearing and mixing were carried out for 10 minutes. The surface of the sheep bone marrow peptide powder was protonated and the Zeta potential was shifted to the positive direction by the mechanochemical action and organic acid in the fruit powder. The mixture was discharged to obtain an acidic high-potential activated sheep bone marrow peptide mixture.

[0039] Preparation Example 3: This preparation example provides an acidic, high-potential activated sheep bone marrow peptide mixture, comprising the following steps: 1180g of sheep bone marrow peptide powder and 300g of sweet orange fruit powder were put into a high-shear mixer with a temperature-controlled jacket. The relative humidity in the mixing chamber was controlled at 40%, and the jacket cooling water circulation was turned on to control the material temperature at 35℃. The spindle speed was set to 300r / min and the high-speed fly knife speed was set to 2500r / min for shearing and mixing for 15 minutes. The surface of the sheep bone marrow peptide powder was protonated and the zeta potential was shifted to the positive direction by the mechanochemical action and organic acid in the fruit powder. The mixture was then discharged to obtain an acidic high-potential activated sheep bone marrow peptide mixture.

[0040] Preparation Example 4: This preparation example provides a non-denatured type II collagen particle with an interfacial proton buffer layer, comprising the following steps: Weigh 500g of undenatured type II collagen and 100g of hydrolyzed type II collagen and put them into a zero-gravity mixer; set the mixing speed to 30r / min and gently mix for 6 minutes; utilize the adsorption and zwitterionic properties of hydrolyzed type II collagen to form a physical isolation and buffer layer on the surface of undenatured type II collagen, discharge the material, and obtain undenatured type II collagen particles with an interfacial proton buffer layer.

[0041] Preparation Example 5: This preparation example provides a non-denatured type II collagen particle with an interfacial proton buffer layer, comprising the following steps: Weigh 500g of undenatured type II collagen and 80g of hydrolyzed type II collagen and put them into a zero-gravity mixer; set the mixing speed to 20r / min and mix gently for 5 minutes; utilize the adsorption and zwitterionic properties of hydrolyzed type II collagen to form a physical isolation and buffer layer on the surface of undenatured type II collagen, discharge the material, and obtain undenatured type II collagen particles with an interfacial proton buffer layer.

[0042] Preparation Example 6: This preparation example provides a non-denatured type II collagen particle with an interfacial proton buffer layer, comprising the following steps: Weigh 500g of undenatured type II collagen and 150g of hydrolyzed type II collagen and put them into a zero-gravity mixer; set the mixing speed to 40r / min and mix gently for 8 minutes; utilize the adsorption and zwitterionic properties of hydrolyzed type II collagen to form a physical isolation and buffer layer on the surface of undenatured type II collagen, discharge the material, and obtain undenatured type II collagen particles with an interfacial proton buffer layer.

[0043] Example 1: This embodiment provides an ordered assembly composition of sheep bone marrow peptides with an interfacial proton buffering effect, the total feed amount of which is 3000g, and includes the following steps: (1) Charge-mediated electrostatic adsorption assembly: 600g of the non-denatured type II collagen particles with an interfacial proton buffer layer prepared in Preparation Example 4 were put into a gravity-free mixer and the mixer speed was kept at 30r / min. 1480g of the acidic high potential activated sheep bone marrow peptide mixture prepared in Preparation Example 1 was added to the mixer in three portions, with an interval of 1.5 minutes between each addition. During the addition process, 21g of food-grade ethanol was uniformly sprayed into the mixing chamber at a rate of 8mL / min through a high-pressure atomizing nozzle and the spray pressure was set to 0.3MPa. After the spraying was completed, the mixture was mixed for another 10 minutes to allow the acidic high potential activated sheep bone marrow peptide mixture to be tightly adsorbed on the surface of the non-denatured type II collagen particles with an interfacial proton buffer layer, forming an ordered peptide-protein assembly complex.

[0044] (2) Construction of competitive sacrificial matrix and total mixture: Weigh 5g ginseng peptide powder, 5g wolfberry peptide powder, 10g steviol glycoside, and 900g of remaining hydrolyzed type II collagen in a small three-dimensional mixer for 8 minutes to obtain sacrificial matrix premix; add the sacrificial matrix premix to a gravity-free mixer containing peptide-protein ordered assembly complex, set the mixing speed to 40r / min, and mix at room temperature for 12 minutes; then pass clean and dry air through for fluidized purging for 15 minutes to remove residual ethanol, discharge the material, and obtain sheep bone marrow peptide ordered assembly composition with interfacial proton buffering effect.

[0045] Example 2: This embodiment provides an ordered assembly composition of sheep bone marrow peptides with an interfacial proton buffering effect, the total feed amount of which is 3000g, and includes the following steps: (1) Charge-mediated electrostatic adsorption assembly: 580g of the non-denatured type II collagen particles with an interfacial proton buffer layer prepared in Preparation Example 5 were put into a gravity-free mixer and the mixer speed was kept at 20r / min. 1480g of the acidic high potential activated sheep bone marrow peptide mixture prepared in Preparation Example 2 was added to the mixer in two portions, with an interval of 1 minute between each addition. During the addition process, 15g of food-grade ethanol was uniformly sprayed into the mixing chamber at a rate of 5mL / min through a high-pressure atomizing nozzle and the spray pressure was set to 0.2MPa. After the spraying was completed, the mixture was mixed for another 8 minutes to allow the acidic high potential activated sheep bone marrow peptide mixture to adsorb onto the surface of the non-denatured type II collagen particles with an interfacial proton buffer layer, forming a peptide-protein ordered assembly complex.

[0046] (2) Construction of competitive sacrificial matrix and total mixture: Weigh 5g ginseng peptide powder, 5g wolfberry peptide powder, 10g steviol glycoside, and 920g of remaining hydrolyzed type II collagen in a small three-dimensional mixer for 5 minutes to obtain sacrificial matrix premix; add the sacrificial matrix premix to a gravity-free mixer containing peptide-protein ordered assembly complex, set the mixing speed to 30r / min, and mix at room temperature for 10 minutes; then pass clean and dry air through for fluidized purging for 10 minutes to remove residual ethanol, discharge the material, and obtain sheep bone marrow peptide ordered assembly composition with interfacial proton buffering effect.

[0047] Example 3: This embodiment provides an ordered assembly composition of sheep bone marrow peptides with an interfacial proton buffering effect, the total feed amount of which is 3000g, and includes the following steps: (1) Charge-mediated electrostatic adsorption assembly: 650g of the non-denatured type II collagen particles with an interfacial proton buffer layer prepared in Preparation Example 6 were put into a gravity-free mixer and the mixer speed was kept at 40r / min. 1480g of the acidic high potential activated sheep bone marrow peptide mixture prepared in Preparation Example 3 was added to the mixer in 3 portions, with an interval of 2 minutes between each addition. During the addition process, 30g of food-grade ethanol was uniformly sprayed into the mixing chamber at a rate of 10mL / min through a high-pressure atomizing nozzle and the spray pressure was set to 0.4MPa. After the spraying was completed, the mixture was mixed for 12 minutes to allow the acidic high potential activated sheep bone marrow peptide mixture to be tightly adsorbed on the surface of the non-denatured type II collagen particles with an interfacial proton buffer layer, forming a peptide-protein ordered assembly complex.

[0048] (2) Construction of competitive sacrificial matrix and total mixture: Weigh 5g ginseng peptide powder, 5g wolfberry peptide powder, 10g steviol glycoside, and 850g of remaining hydrolyzed type II collagen in a small three-dimensional mixer for 10 minutes to obtain sacrificial matrix premix; add the sacrificial matrix premix to a gravity-free mixer containing peptide-protein ordered assembly complex, set the mixing speed to 50r / min, and mix at room temperature for 15 minutes; then pass clean and dry air through for fluidized purging for 20 minutes to remove residual ethanol, discharge the material, and obtain sheep bone marrow peptide ordered assembly composition with interfacial proton buffering effect.

[0049] Example 4: This embodiment provides an ordered assembly composition of sheep bone marrow peptides with an interfacial proton buffering effect, the total feed amount of which is 3000g, and includes the following steps: (1) Charge-mediated electrostatic adsorption assembly: 600g of the non-denatured type II collagen particles with an interfacial proton buffer layer prepared in Preparation Example 4 were put into a gravity-free mixer and the mixer speed was kept at 35r / min. 1480g of the acidic high potential activated sheep bone marrow peptide mixture prepared in Preparation Example 1 was added to the mixer in three portions, with an interval of 1.5 minutes between each addition. During the addition process, 18g of food-grade ethanol was uniformly sprayed into the mixing chamber at a rate of 6mL / min through a high-pressure atomizing nozzle and the spray pressure was set to 0.25MPa. After the spraying was completed, the mixture was mixed for another 9 minutes to allow the acidic high potential activated sheep bone marrow peptide mixture to adsorb onto the surface of the non-denatured type II collagen particles with an interfacial proton buffer layer, forming an ordered peptide-protein assembly complex.

[0050] (2) Construction of competitive sacrificial matrix and total mixture: Weigh 5g ginseng peptide powder, 5g wolfberry peptide powder, 10g steviol glycoside, and 900g of remaining hydrolyzed type II collagen in a small three-dimensional mixer for 6 minutes to obtain sacrificial matrix premix; add the sacrificial matrix premix to a gravity-free mixer containing peptide-protein ordered assembly complex, set the mixing speed to 35r / min, and mix at room temperature for 11 minutes; then pass clean and dry air through for fluidized purging for 12 minutes to remove residual ethanol, discharge the material, and obtain sheep bone marrow peptide ordered assembly composition with interfacial proton buffering effect.

[0051] Comparative Example 1: This comparative example provides a sheep bone marrow peptide composition. The difference from Example 1 is that it does not employ a step-by-step assembly process, but rather a one-time physical mixing method. Specifically, sheep bone marrow peptide powder, sweet orange fruit powder, non-denatured type II collagen, hydrolyzed type II collagen, and other trace components are directly added to a mixer according to the proportions in Example 1, and mixed at room temperature for 20 minutes until homogeneous. No ethanol is injected, and no pretreatment is performed. The specifications of the remaining raw materials are the same as in Example 1.

[0052] Comparative Example 2: Compared with Example 1, the difference lies in omitting the step of constructing the interface buffer layer with undenatured type II collagen. Specifically, in step (1), the untreated undenatured type II collagen was directly added to a zero-gravity mixer and spray-assembled with an acidic high-potential activated sheep bone marrow peptide mixture; the 100g of hydrolyzed type II collagen originally planned to be used to construct the buffer layer was incorporated into step (2) and added together as a sacrificial matrix. The remaining process parameters were the same as in Example 1.

[0053] Comparative Example 3: Compared with Example 1, the difference is that the acid-induced activation step of sheep bone marrow peptides was omitted. Specifically, in step (1), the raw sheep bone marrow peptide powder without high shear treatment was directly mixed and assembled with non-denatured type II collagen particles with an interfacial proton buffer layer; the 300g of sweet orange fruit powder originally intended for activation was moved to the total mixing stage in step (2). The remaining process parameters were the same as in Example 1.

[0054] Comparative Example 4: Compared to Example 1, the difference lies in the altered order of addition of hydrolyzed type II collagen, which disrupts the external sacrificial layer structure. Specifically, all 1000g of hydrolyzed type II collagen in the formulation is mixed with non-denatured type II collagen before step (1), and then peptide powder assembly is performed. The remaining process parameters are the same as in Example 1.

[0055] Comparative Example 5: Compared to Example 1, the difference lies in the change of the liquid medium in the assembly process. Specifically, in the feeding assembly process of step (1), an equal amount of purified water is used instead of food-grade ethanol for spray initiation. The remaining process parameters are the same as in Example 1.

[0056] Test Example 1: Test objective: This test case verifies the surface charge regulation effect of sheep bone marrow peptides and the formation of ordered assembly structures between particles by measuring the zeta potential and particle size distribution of raw materials and products at each processing stage.

[0057] Experimental methods: Zeta potential determination: Raw sheep bone marrow peptide powder, the acidic high-potential activated sheep bone marrow peptide mixtures obtained from Preparation Examples 1 to 3, and the unactivated mixture from Comparative Example 3 were dispersed in deionized water at pH 7.0 to prepare dispersions with a concentration of 0.1 mg / mL. The zeta potential of each sample was measured using a laser Doppler electrophoresis light scattering instrument at 25°C.

[0058] Particle size distribution determination: Undenatured type II collagen (UC-II), peptide-protein ordered assembly complexes prepared in Examples 1 to 3 (i.e., intermediate products before the addition of external sacrificial matrix), and a simple physical mixture obtained in Comparative Example 1 were used. A dry laser particle size analyzer was employed. The dispersion pressure was set to 2.0 bar, and the volume average particle size (D50 and D90) of each sample were measured.

[0059] The experimental data are shown in Table 1.

[0060] Table 1: Summary of surface electrochemical properties and particle size distribution test data for each component

[0061] Note: — indicates that this indicator is not the focus of the evaluation for this sample or is not applicable.

[0062] Results and conclusions: Table 1 shows the changes in electrochemical properties and assembly effects of the raw materials before and after mechanochemical modification.

[0063] The raw material, sheep bone marrow peptide powder, has a Zeta potential of -2.43 mV in a near-neutral aqueous environment, exhibiting weak negative charge. This results in electrostatic repulsion between the product and the similarly negatively charged undenatured type II collagen (-5.12 mV), making spontaneous adsorption difficult. After processing as described in Preparation Example 1, the Zeta potential of the product changed to +18.74 mV. This change indicates that under mechanochemical action, organic acid molecules effectively contacted the peptide chain, protonating the amino groups on the peptide chain surface and establishing a positive potential surface, providing the necessary conditions for electrostatic adsorption. In Preparation Example 2, due to the decrease in shear speed, the Zeta potential shift decreased to +12.31 mV, indicating that shear strength affects the dispersion uniformity of acid molecules and proton transfer efficiency.

[0064] Particle size data reflects the coating condition of the particles. The D50 of the raw material, undenatured type II collagen, is 42.15 μm. The D50 of the product in Example 1 increased to 64.82 μm, with a particle size increase of approximately 22 μm. This increase corresponds to the thickness of the modified sheep bone marrow peptides and buffer layer adsorbed on the surface of the core particles. In Example 3, by increasing the proportion of buffer layer material and the amount of ethanol, the D50 further increased to 75.05 μm, indicating that the coating layer thickness can be controlled by process parameters.

[0065] In contrast, the D50 of the simple physical mixture in Comparative Example 1 was 43.08 μm, which was not significantly different from the particle size of the raw materials (the difference was within the measurement error range). This indicates that in the absence of acid-induced activation and polar initiators, the components were discretely distributed and did not form an effective coating structure. The experimental results confirm that the process of the present invention successfully constructed an assembled structure with undenatured type II collagen as the core and modified peptides and buffer proteins as the shell.

[0066] Test Example 2: Test objective: This test case examines the retention of active ingredients in the composition under simulated gastric juice conditions and the physicochemical stability under high temperature and high humidity conditions, in order to verify the actual effect of the interfacial buffering effect and the competitive enzymatic hydrolysis mechanism.

[0067] Experimental methods: Simulated gastric juice (SGF) tolerance test: Simulated gastric juice containing pepsin (3000 U / mg) was prepared, and the pH was adjusted to 2.0. 1.0 g of each sample from the examples and comparative examples was weighed and placed in simulated gastric juice at 37°C with shaking digestion for 2 hours. After the reaction, the solution was neutralized, centrifuged, and the supernatant was collected. The antigen activity retention rate of non-denatured type II collagen was detected by enzyme-linked immunosorbent assay (ELISA).

[0068] pH buffering capacity determination: Weigh 0.5g of the sample and dissolve it in deionized water. Use an automatic potentiometric titrator to titrate with 0.1mol / L hydrochloric acid solution, and record the volume of hydrochloric acid consumed when the pH of the solution drops to 2.0. The larger the volume consumed, the stronger the buffering capacity of the sample to an acidic environment.

[0069] Accelerated stability test: The sample was sealed and placed in a constant temperature and humidity chamber at 40℃ and 75% relative humidity for 3 months. After removal, it was reconstituted, and the activity retention rate of the non-denatured type II collagen was measured.

[0070] The experimental data are shown in Table 2.

[0071] Table 2: Summary of Data from Simulated Gastric Fluid Tolerance, Buffer Capacity, and Accelerated Stability Tests

[0072] Results and conclusions: The data in Table 2 show that the structure constructed in this invention significantly improves the acid resistance and enzymatic hydrolysis resistance of the active ingredients.

[0073] In terms of simulating gastric juice tolerance, the activity retention rates of Examples 1 to 4 were all above 79%, with Example 3 achieving a retention rate of 88.13% due to its thicker adsorption layer and higher buffering capacity. Comparative Example 1, using simple physical mixing, had an activity retention rate of only 32.15%, demonstrating that unprotected, non-denatured type II collagen is rapidly inactivated under the action of gastric acid and pepsin.

[0074] The buffer capacity test results were positively correlated with the digestion experiment results. Example 1 consumed 12.35 mL of hydrochloric acid, significantly higher than the 8.09 mL consumed in Comparative Example 2. Although Comparative Example 2 contained the same amount of hydrolyzed collagen, its lack of a pre-constructed interfacial buffer layer on the core particle surface resulted in insufficient local buffering capacity, allowing acidic ions to more easily penetrate the core and damage the protein structure. This confirms the necessity of enriching hydrolyzed collagen at the interface.

[0075] Regarding the competitive sacrificial mechanism, Comparative Example 4 placed all hydrolyzed collagen internally without an external sacrificial matrix, resulting in a decrease in its SGF retention rate to 38.76%. This data indicates that without an external high-affinity substrate to preferentially consume pepsin, simple physical encapsulation is insufficient to resist enzymatic degradation. The examples demonstrated that by distributing hydrolyzed collagen in an internal and external layer, the external sacrificial layer delayed the enzyme's contact time with the core component.

[0076] Regarding stability, the activity retention rates of the example groups were all above 92% after accelerated aging. Comparative Example 5, using water as the granulation medium, had a retention rate of only 62.19% after aging. This is because the water medium induced localized collagen hydrolysis and structural collapse during preparation, and the residual moisture accelerated the degradation reaction. The examples used trace amounts of ethanol as an initiator, which achieved liquid bridging between particles while avoiding the introduction of moisture, thus ensuring the physicochemical stability of the product under long-term storage conditions.

[0077] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts, characterized in that, The sheep bone marrow peptide composition has an ordered assembly structure mediated by electrostatic adsorption and is made from raw materials comprising the following parts by weight: Sheep bone marrow peptide powder: 1000-1500 parts; Sweet orange powder: 250-350 servings; Undenatured type II collagen: 450-700 parts; Hydrolyzed type II collagen: 800-1200 parts; Ginseng peptide powder: 1-10 parts; Goji berry peptide powder: 1-10 parts; Steviosides: 5-15 parts; The composition comprises: a core of non-denatured type II collagen, the surface of which is coated with an interfacial proton buffer layer composed of partially hydrolyzed type II collagen; a mixture of acid-activated sheep bone marrow peptide powder and sweet orange fruit powder is adsorbed on the outside of the interfacial proton buffer layer to form a peptide-protein ordered assembly complex; and the remaining hydrolyzed type II collagen is distributed around the ordered assembly complex to form a competitive enzymatic sacrificial matrix.

2. The sheep bone marrow peptide composition rich in bone repair factors for activating osteoblasts according to claim 1, characterized in that, The weight parts of the raw materials are: Sheep bone marrow peptide powder: 1100-1200 parts; Sweet orange powder: 280-320 servings; Undenatured type II collagen: 500-650 parts; Hydrolyzed type II collagen: 900-1100 parts; Ginseng peptide powder: 4-6 parts; Goji berry peptide powder: 4-6 parts; Steviosides: 8-12 parts.

3. The sheep bone marrow peptide composition rich in bone repair factors for activating osteoblasts according to claim 1, characterized in that, The raw material has the following characteristics: The average molecular weight (Mw) of the sheep bone marrow peptide powder is distributed between 500 Da and 1000 Da, and the proportion of components with a molecular weight less than 1000 Da is ≥90%. The undenatured type II collagen retains the complete triple helix active structure of collagen; The average molecular weight (Mw) of the hydrolyzed type II collagen is distributed between 1500 Da and 3000 Da, and its 10% aqueous solution has a pH value of 5.5 to 6.5, exhibiting zwitterionic properties.

4. The sheep bone marrow peptide composition rich in bone repair factors for activating osteoblasts according to claim 1, characterized in that, The mixture of acid-activated sheep bone marrow peptide powder and sweet orange fruit powder is an acid-high potential activated sheep bone marrow peptide mixture, which is obtained by the following preparation method: In an environment with a relative humidity of 30%-40%, sheep bone marrow peptide powder is mixed with sweet orange fruit powder and subjected to high shear treatment at a temperature of 25-35℃. The mechanochemical action and the organic acids in the sweet orange fruit powder cause the amino groups on the surface of the sheep bone marrow peptide powder to be protonated, causing its Zeta potential to shift from negative to positive.

5. The sheep bone marrow peptide composition for activating osteoblasts and rich in bone repair factors according to claim 1, characterized in that, The structure in which the core surface is covered with an interface proton buffer layer is formed in the following manner: Utilizing the zwitterionic properties and high specific surface area of ​​hydrolyzed type II collagen, it is gently mixed with undenatured type II collagen under low shear force, allowing the hydrolyzed type II collagen to form a controllable thickness isolation buffer layer on the surface of the undenatured type II collagen through physical adsorption; wherein, the weight parts of hydrolyzed type II collagen used to construct the interfacial proton buffer layer are 80-150 parts.

6. The sheep bone marrow peptide composition rich in bone repair factors for activating osteoblasts according to claim 1, characterized in that, The competitive enzymatic sacrificial matrix is ​​composed of 850-950 parts by weight of hydrolyzed type II collagen, ginseng peptide powder, wolfberry peptide powder and steviol glycosides. It is loosely distributed in the gaps and outer surface of the ordered peptide-protein assembly complex, and is used to preferentially bind to digestive enzymes in the digestive tract environment.

7. A method for preparing a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Preparation of activated phase: Sheep bone marrow peptide powder and sweet orange fruit powder are mixed under high shear, and an acidic high potential activated sheep bone marrow peptide mixture is prepared by mechanochemical action. S2. Buffer core construction: Undenatured type II collagen and partially hydrolyzed type II collagen are gently mixed, and an interfacial proton buffer layer is formed on the surface of undenatured type II collagen by physical adsorption, thus obtaining undenatured type II collagen particles with an interfacial proton buffer layer. S3, Ordered Assembly: The particles obtained in step S2 are used as the base material. The mixture obtained in step S1 is added in a mixed state, and a polar initiator is sprayed in. Electrostatic adsorption assembly is carried out by charge reversal and liquid bridge force to obtain peptide-protein ordered assembly complex. S4. Total Mixing and Post-processing: The remaining hydrolyzed type II collagen, ginseng peptide powder, wolfberry peptide powder and steviol glycosides are premixed and then added to the complex in step S3 for mixing to construct a competitive sacrificial matrix. The residual polar initiator is then removed to obtain the sheep bone marrow peptide composition.

8. The method for preparing a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts according to claim 7, characterized in that, In step S1, the spindle speed of the high-shear mixer is 100-300 r / min, the high-speed flying knife speed is 1500-2500 r / min, the mixing time is 10-15 minutes, and the material temperature is controlled at 25-35℃ during the mixing process.

9. The method for preparing a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts according to claim 7, characterized in that, In step S3, the polar initiator is food-grade ethanol, and its injection amount is 0.5%-1.0% of the total weight of the raw materials. It is uniformly sprayed through a high-pressure atomizing nozzle at a rate of 5-10 mL / min; the spray pressure is controlled at 0.2-0.4 MPa.

10. The method for preparing a sheep bone marrow peptide composition rich in bone repair factors that activates osteoblasts according to claim 7, characterized in that, In step S4, the residual polar initiator is removed by fluidized purging with clean, dry air for 10-20 minutes; the mass ratio of the remaining hydrolyzed type II collagen to the hydrolyzed type II collagen used in step S2 is 5.6-11.5:1.

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