Production process and application of bovine bone collagen peptide for preventing osteoporosis and delaying cell senescence

Abstract

The invention relates to the technical field of biological processing, in particular to a production process and application of bovine bone collagen peptide for preventing osteoporosis and delaying cell senescence, and the production process comprises the following steps: placing yak bone into a bone placing cylinder, and plugging a rubber plug; a first motor and a second motor are started to drive a shifting block and a pushing block to quickly move from one end of a bone placing cylinder to the other end of the bone placing cylinder in a reciprocating manner, so that a plurality of bone striking groups inserted outside the bone placing cylinder are triggered to be compressed, rebounded and released, yak bones in the bone placing cylinder are struck, and concave holes and cracks are formed in the bone surface; and then the servo motor is started to drive the bone unloading belt to unload the yak bones, and the yak bones fall into the bone crushing machine body to be crushed. The spring steel conical head is driven by the cambered surface and planar force storage group to pre-split yak bones, directional impact on the surfaces of the yak bones is completed, continuous compression and release are achieved by linkage of the two-way lead screw and the shifting block and the pushing block, full-circumferential dead-corner-free impact on the yak bones is completed, the effect of increasing the crack coverage rate is achieved, the extrusion force of a subsequent bone crusher is reduced, and the production efficiency is improved. The service life of the double-tooth roller is prolonged.

Description

Technical Field

[0001] This invention relates to the field of bioprocessing technology, specifically to a production process and application of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging. Background Technology

[0002] Currently, with the development of biotechnology, animal bones have become an important raw material for production, from which bone oil, collagen peptides, bone meal, and other products are extracted. Collagen is the most abundant protein in the human body and plays an indispensable role.

[0003] The invention with application number CN201210538195.6 discloses a production method for obtaining bone collagen oligopeptide powder, bone oil, and bone meal, including the following steps: crushing and cleaning bone materials, cooking the bone materials once in a cooking tank, separating the broth from the broth using a broth-oil separator to obtain defatted broth and bone oil, refining and packaging the bone oil; cooking the bone materials a second time after the first cooking, and storing the resulting broth in a storage tank for use in the next cooking. After secondary steaming and cooking, the bone material is dried and pulverized to obtain bone meal. The defatted broth is hydrolyzed and decolorized, filtered, concentrated, decolorized, sterilized, and dried to obtain powdered or granular bone collagen oligopeptide powder. The bone collagen oligopeptides extracted by this invention have small molecular weight, high activity, and are easily absorbed by the human body. The bone oil has high purity and reaches food grade. The process of this invention simultaneously extracts bone collagen oligopeptide powder, bone oil, and bone meal, with a short production cycle, no waste generated during production, full utilization of raw materials, and reduced production costs.

[0004] While existing collagen peptide production processes can largely achieve the reuse of agricultural byproducts, the following shortcomings have been identified due to ongoing inspections and increasingly stringent market demands: 1. Due to the significant differences in bone density between different types of animals, or even between different breeds of the same type of animal, the bone density of yak bone is 1.8 times that of ordinary cattle bone, and the general bone crushing machine involved in the original process cannot meet its requirements.

[0005] 2. If too much enzyme is added during the hydrolysis of defatted soup stock, too many hydrophobic amino acids will be exposed, resulting in too many bitter peptides that affect the taste of the product. If too little enzyme is added, the hydrolysis of large molecular proteins will be insufficient, affecting the product yield.

[0006] 3. Although the use of hydrogen peroxide in the process can completely solve the color problem of the product, it will affect the product's antioxidant properties and reduce its functionality. Summary of the Invention

[0007] In order to overcome the defects in the prior art, the purpose of this invention is to provide a production process and application of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging. The yak bone is pre-cracked by a spring steel cone driven by an arc / plane energy storage group, and continuous compression and release are achieved by a bidirectional screw linkage pusher / pusher, completing the full circumference impact of the bone body without dead angles, thus solving the problem of yak bone breakage in the above-mentioned background technology.

[0008] By using a combined extraction and hydrolysis method of ultrasound and enzyme preparations for defatted degreasing liquid, the amount of enzyme preparations used can be reduced while increasing the product yield.

[0009] Through experiments, a combination of activated carbon adsorption and membrane filtration was used to solve the product decolorization problem by reducing the amount of activated carbon used in the hydrolysate.

[0010] To achieve the above objectives, on the one hand, the present invention provides a production process for bovine bone collagen peptides that prevent osteoporosis and delay cell aging, comprising the following steps: S1. Using fresh yak bones as raw material, after removing impurities, the yak bones are first struck with a bone-breaking device to create pits and cracks, and then fall into the bone crusher body and are crushed into 1-2cm size. S2. Place the crushed aggregate into a bone washing machine and soak it in cold water. Stir, drain the cold water, and then wash it with clean water. S3. Next, the bones and water are cooked in a cooking tank with a volume ratio of 1:4. The cooking is carried out at a constant temperature and pressure of 0.5~1.5MPa and 110℃~120℃ for 2~3 hours. Then, the cooked soup is separated by a soup-oil separation device to obtain defatted soup. S4. The defatted broth is subjected to hydrolysis, decolorization, filtration, concentration, sterilization and drying processes in sequence to obtain bovine bone collagen peptide powder. Step S1 further includes the following steps: S11. Place the yak bones into the bone-inserting tube and plug it with a rubber stopper; S12. Start the first motor and the second motor to drive the paddle and push block to move quickly back and forth from one end of the bone placement tube to the other end, thereby triggering several bone-striking groups inserted outside the bone placement tube to compress and rebound and release, thus impacting the yak bone inside the bone placement tube, thereby forming pits and cracks on the bone surface. S13. The servo motor is restarted to drive the unloading belt to move a certain distance, causing the yak bones to fall into the bone crusher and be collected.

[0011] As a further improvement to this technical solution, a bone-impacting device, an arc-shaped energy storage group, and a planar energy storage group are used to strike the yak bone in a coordinated manner. The bone-impacting device includes a bone-placement tube placed horizontally with an open bottom surface, a protective cover covering the outside of the bone-placement tube, and several bone-impacting groups distributed at equal intervals around the outside of the bone-placement tube. A bone-unloading belt and a servo motor for driving the bone-unloading belt to rotate are provided below the bottom opening of the bone-placement tube. Several bone-unloading ports are opened at equal intervals on the surface of the bone-unloading belt. The arc-shaped power storage group includes a coarse reciprocating lead screw that runs through the front and rear ends of the top of the protective cover, a pair of levers threadedly connected to it, and a first motor. The levers are in the shape of isosceles triangular blocks and their hypotenuses slide in contact with a pair of impact bone groups located within the arc range. The planar power storage assembly includes a pair of thin reciprocating lead screws running through the front and rear ends of the bottom of the protective cover, a push block threadedly connected to them, and a second motor. The push block is in the shape of an isosceles triangular block and its hypotenuse slides in contact with a pair of impact bone assemblies located in the vertical plane.

[0012] The above setup, after experiments to determine whether bovine bone peptides have anti-cellular aging effects, demonstrated their ability to prevent osteoporosis and delay cellular aging. Given the high density of yak bone and its higher collagen content compared to ordinary bone materials, yak bone is subjected to bone crushing. Considering the inapplicability of existing bone crushers, a combination of impact devices, arc-shaped and planar force-saving groups is used to synergistically impact the yak bone before crushing. The arc and planar force-saving groups drive a spring steel cone to pre-crack the bone, achieving directional impact on the yak bone surface. Continuous compression and release are achieved through bidirectional screw linkage with push blocks and levers, completing a full-circumferential impact on the bone body without blind spots, achieving full crack coverage and improving the yield rate of the crushed bone.

[0013] As a further improvement to this technical solution, a bone crusher body is used to crush bones by compression. The bone crusher body includes a bone crushing box, a double toothed roller installed therein, and a main motor that drives the double toothed roller to rotate in opposite directions.

[0014] As a further improvement to this technical solution, a bone-impacting assembly is used to strike the yak bone. The bone-impacting assembly includes a coaxially sleeved striking post, a striking head, and a guide post. The striking head is a conical head made of spring steel. A spring is sleeved on the outside of the guide post. Several positioning holes are opened on the outer wall of the protective cover. The outer end of the guide post is fixedly connected to the positioning holes. Several insertion holes are opened on the outer wall of the bone placement tube. The striking post is slidably inserted into the insertion holes.

[0015] The above setup discloses the specific structure of the impactor assembly. The triangular block is driven at a fixed frequency by coarse and fine reciprocating screws, causing several sets of impactor cones to alternately impact various areas of the aggregate along the axial direction, forming a complete coverage of cracks and pits, which is equivalent to the pre-splitting process before crushing the aggregate.

[0016] As a further improvement to this technical solution, a sliding sleeve is fixedly connected between a pair of levers. The sliding sleeve is threaded onto a coarse reciprocating lead screw. A hanging rod is inserted through the top side of the sliding sleeve to restrict the axial movement of the levers. Support blocks extend from both ends of the bottom surface of the levers. The support blocks slide in contact with the large-diameter end of the striking post, guiding them to slide on the inclined surface of the levers until they slide to the top of the lever's arc. At this point, the striking post's pushing spring is compressed to its shortest length, ready to release the elastic force.

[0017] As a further improvement to this technical solution, a through hole is provided in the middle of the inclined surface of the push block, and the through hole is threaded with a fine reciprocating screw. Insert blocks are provided at both ends of the bottom surface of the push block, and the insert blocks slide in contact with the large diameter end of the striking column. A convex ring is provided at the arc end of the push block and a positioning rod is provided to restrict the axial movement of the push block.

[0018] The above-mentioned setup specifically discloses how the triangular block works in conjunction with the impact column to create an impact effect. The triangular block is driven to shuttle back and forth at the large diameter end of the impact column by a reciprocating screw, which guides the impact column to slide along its inclined plane to change its stroke, compressing the spring, and releasing the elastic force to drive the impact column to impact the aggregate.

[0019] As a further improvement to this technical solution, step S2 also includes the following steps: S21. Then add water twice the volume of the aggregate, and add sodium hydroxide at 0.8-1.5‰ of the aggregate weight, and stir for 30 minutes; after draining, add water twice the volume of the aggregate and acetic acid at 5‰, stir for 30 minutes, and then drain.

[0020] As a further improvement to this technical solution, step S4 includes the following steps: S41. Add the defatted broth obtained in step S3 to a hydrolysis tank, cool it to 50-60℃, adjust the pH value to 7-8.2, measure the concentration of the defatted broth with an instrument, add a compound biological enzyme preparation to the broth and hydrolyze for 1-1.5 hours. The weight of the enzyme preparation accounts for 0.1%-0.2% of the weight of collagen protein contained in the broth. The enzyme preparation involved in this step is a customized compound enzyme preparation, which contains acidic protease, alkaline protease and flavor enzyme in a ratio of 3:2:1, and its enzyme activity is 350,000-400,000 U / g. S42. After preliminary filtration through a 10kDa ultrafiltration membrane, add activated charcoal and heat to 100℃ while stirring, and maintain for 5 minutes. The weight of activated charcoal accounts for 2%-4% of the weight of collagen in the soup. Then cool down to 70-80℃ and let stand for 1-1.5 hours to decolorize and deodorize. S43. The hydrolysate is coarsely filtered using a candle filter with diatomaceous earth and diatomaceous earth sheets, and then finely filtered using an ultrafiltration membrane with a 5 kDa filter. S44. Use a vacuum concentrator to concentrate the filtered defatted soup stock at low temperature. S45. Using a pressure-type spray dryer, the treated concentrated defatted broth is dried into solid powder or granules to obtain bovine bone collagen peptides.

[0021] On the other hand, the present invention provides an application of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging, which is the application of the above-mentioned production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging in the preparation of bone collagen peptides with the function of preventing osteoporosis and delaying cell aging.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The production process and application of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging involves using an arc-shaped and planar force-storing group to drive a spring steel cone head to pre-crack yak bones, thereby completing the directional impact on the surface of the yak bones. The spring steel cone head, combined with a high-resilience spring, forms concave holes and mesh cracks on the bone surface, which reduces the extrusion pressure of the subsequent bone crusher and improves the life of the double-toothed roller.

[0023] 2. The production process and application of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging utilizes a bidirectional screw linkage to achieve continuous compression and release, completing the full circumferential impact of the bone without dead angles. By using coarse and fine reciprocating screws to drive triangular blocks at a fixed frequency, several sets of impact cones alternately impact the axial regions of the bone material, achieving the effect of improving crack coverage and helping the bone crusher to extrude bone fragments and improve the pass rate.

[0024] 3. The production process and application of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging achieve efficient and safe production through precise material control of the unloading belt and the closed design of the protective cover. The servo motor drives the unloading belt to rotate at a fixed angle in one go, ensuring that the unloading port rotates to the bottom of the bone placement tube for unloading. The semi-circular protective cover prevents bone fragments from splashing, thereby reducing the risk of injury to personnel and extending the equipment cleaning cycle. Attached Figure Description

[0025] The accompanying drawings described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Furthermore, the shapes and proportions of the components in the drawings are merely illustrative to aid in understanding the invention and do not specifically limit the shapes and proportions of the components. Those skilled in the art, guided by the teachings of this invention, will select various possible shapes and proportions to implement the invention according to specific circumstances.

[0026] Figure 1 This is a flowchart of the process flow of the present invention; Figure 2 This is a schematic diagram of the assembly structure of the bone crusher body and the bone-dissolving device of the present invention; Figure 3 This is a schematic diagram of the assembly structure of the bone-striking device and the arc-shaped and planar energy storage groups of the present invention; Figure 4 For the present invention Figure 3 Side view; Figure 5 This is a schematic diagram of the main body structure of the bone crusher of the present invention; Figure 6 This is an exploded view of the impactor assembly of the present invention; Figure 7 This is one of the schematic diagrams of the arc-shaped and planar energy storage assembly structure of the present invention; Figure 8 This is the second schematic diagram of the arc-shaped and planar energy storage assembly structure of the present invention; Figure 9 This is an exploded view of the impactor assembly of the present invention; Figure 10 This is a schematic diagram of the arc-shaped energy storage assembly structure of the present invention; Figure 11 This is a schematic diagram of the planar energy storage assembly structure of the present invention; Figure 12 This is a control diagram showing the effect of the bovine bone peptide of the present invention on the proliferation of MRC5 cells; Figure 13 A control diagram showing the effect of bovine bone peptides of the present invention on reducing the proliferation of MRC5 cells by hydrogen peroxide. Figure 14 This is a control diagram showing the level of intracellular ROS induced by hydrogen peroxide induced by bovine bone peptides in this invention. Figure 15 This is a control diagram showing the inhibition of hydrogen peroxide-induced cell senescence by the bovine bone peptide of the present invention. Figure 16 This is a control diagram showing how bovine bone peptides of the present invention reduce the expression of proteins related to cell senescence.

[0027] The meanings of the labels in the diagram are as follows: 100. Bone crusher body; 110. Bone crushing box; 120. Double toothed roller; 130. Main motor; 200. Bone-discharging device; 210. Bone placement tube; 211. Insertion hole; 212. Horn cover; 220. Protective cover; 221. Positioning hole; 230. Bone-discharging assembly; 231. Discharging post; 232. Discharging head; 233. Guide post; 234. Spring; 240. Bone-discharging belt; 241. Bone-discharging port; 242. Servo motor; 300. Arc-shaped power storage assembly; 310. Coarse reciprocating lead screw; 311. First motor; 320. Pulley block; 321. Support block; 322. Sliding sleeve; 330. Hanging rod; 400. Planar power storage unit; 410. Fine reciprocating lead screw; 411. Second motor; 420. Push block; 421. Insert block; 430. Positioning rod. Detailed Implementation

[0028] The details of the present invention can be more clearly understood by referring to the accompanying drawings and the description of specific embodiments. However, the specific embodiments of the present invention described herein are for illustrative purposes only and should not be construed as limiting the invention in any way. Under the teachings of this invention, those skilled in the art will conceive of any possible variations of the invention, all of which should be considered within the scope of the invention. The terms "installation" and "connection" should be interpreted broadly, referring to direct connection as well as indirect connection through an intermediate medium.

[0029] The terms "central axis," "vertical," "horizontal," "front," "rear," "upper," "lower," "left," "right," "top," "bottom," "inner," and "outer" used herein to indicate orientation or positional relationships are based on the orientation or positional relationships shown in the accompanying drawings and are used only for the convenience of describing the invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, in the description of the invention, "a number" means two or more, unless otherwise explicitly specified.

[0030] Please see Figures 1-7 As shown, this invention provides a production process for bovine bone collagen peptides that prevent osteoporosis and delay cell aging, comprising the following steps: S1. Using fresh yak bones as raw material, after removing impurities, the bone-beating device 200 first strikes the yak bones to form concave holes and cracks, and then they fall into the bone crusher body 100 and are crushed into 1-2cm size. S11. Place the yak bones into the bone holder 210 and plug it with a rubber stopper; or use a round cap with a screw thread to seal it. S12. The first motor 311 and the second motor 411 are started to drive the paddle block 320 and the push block 420 to move rapidly back and forth from one end of the bone placement tube 210 to the other end, thereby triggering several bone-breaking groups 230 inserted into the outside of the bone placement tube 210 to compress and rebound, thus impacting the yak bone inside the bone placement tube 210, thereby forming concave holes and cracks on the bone surface; so that the subsequent bone crusher can squeeze the brittle bone, reduce its shock force, and improve its service life. S13. The servo motor 242 is restarted to drive the unloading belt 240 to move a certain distance, causing the yak bones to fall into the bone crusher body 100 and be crushed and collected. S2. Place the crushed aggregate into a bone washing machine and soak it in cold water. Stir, drain the cold water, and then wash it with clean water. S21. Then add water with a volume twice that of the aggregate, and add sodium hydroxide at a weight of 0.8-1.5‰ of the aggregate, and stir for 30 minutes; after draining, add water with a volume twice that of the aggregate and acetic acid at a weight of 5‰, stir for 30 minutes, and then drain. S3. Next, the bones and water are cooked in a cooking tank with a volume ratio of 1:4. The cooking is carried out at a constant temperature and pressure of 0.5~1.5MPa and 110℃~120℃ for 2~3 hours. Then, the cooked soup is separated by a soup-oil separation device to obtain defatted soup. S4. The defatted broth is subjected to hydrolysis, decolorization, filtration, concentration, sterilization and drying processes in sequence to obtain bovine bone collagen peptide powder. S41. Add the defatted broth obtained in step S3 to a hydrolysis tank, cool it to 50-55℃, adjust the pH value to 7-8, measure the concentration of the defatted broth with an instrument, add a compound biological enzyme preparation to the broth and hydrolyze for 1-1.5 hours. The weight of the enzyme preparation accounts for 0.1%-0.2% of the weight of collagen protein contained in the broth. The enzyme preparation involved in this step is a customized compound enzyme preparation, which contains acidic protease, alkaline protease and flavor enzyme in a ratio of 3:2:1, and its enzyme activity is 350,000-400,000 U / g. S42. After preliminary filtration through a 10kDa ultrafiltration membrane, add activated charcoal and heat to 100℃ while stirring, and maintain for 5 minutes. The weight of activated charcoal accounts for 2%-4% of the weight of collagen in the soup. Then cool down to 70-80℃ and let stand for 1-1.5 hours to decolorize and deodorize. S43. Use a candle filter to coarsely filter the hydrolyzed defatted soup stock, and then use a plate and frame filter and a filter membrane for fine filtration. S44. Use a vacuum concentrator to concentrate the filtered defatted soup stock at low temperature. S45. Using a pressure-type spray dryer, the treated concentrated defatted broth is dried into solid powder or granules to obtain bovine bone collagen peptides.

[0031] Specifically, such as Figures 8-11 As shown, the production process of this invention employs a bone-impacting device 200, an arc-shaped energy storage group 300, and a planar energy storage group 400 to collaboratively impact yak bones. This causes the yak bones to be continuously impacted, forming surface cracks and pits, so that the subsequent bone crusher can reduce the reaction force and crush the bones. The bone-impacting device 200 includes a horizontally placed bone-holding cylinder 210 with an open bottom, a protective cover 220 covering the outside of the bone-holding cylinder 210, and a plurality of bone-impacting groups 23 evenly distributed around the outside of the bone-holding cylinder 210. Below the bottom opening of the bone placement tube 210, there is a bone unloading belt 240 and a servo motor 242 that drives the bone unloading belt 240 to rotate. The surface of the bone unloading belt 240 has several bone unloading ports 241 at equal intervals. The yak bone is placed in the bone placement tube 210 by the closed surface of the bone unloading belt 240 and is impacted by the bone crushing group 230. The servo motor 242 is activated to drive the bone unloading belt 240 to rotate once, so that the bone unloading port 241 is placed at the bottom opening of the bone placement tube 210 to unload the yak bone, which falls into the bone crusher body 100.

[0032] Furthermore, the present invention uses a bone crusher body 100 to crush bones. The bone crusher body 100 includes a bone crushing box 110, a double toothed roller 120 installed therein, and a main motor 130 that drives the double toothed roller 120 to rotate in the opposite direction. The above is the prior art and will not be described in detail here. One end of the bone placement tube 210 is closed and sleeved with the side end of the bone crushing box 110. The other end of the bone placement tube 210 is open and fixedly connected with a horn cover 212 for guiding yak bones into the bone placement tube 210. The protective cover 220 is a semi-cylindrical cover to fit the bone tube 210. The bone striking group 230 is arranged around the central axis of the bone placement tube 210 so as to simultaneously impact the bone surface.

[0033] Furthermore, this invention employs a striking assembly 230 to impact yak bone. The striking assembly 230 includes a coaxially sleeved striking post 231, a striking head 232, and a guide post 233. The striking head 232 is a conical head made of spring steel, such as 5160, 9260, or SUP9, known for its extremely high toughness and impact resistance, serving as the head of a chisel, wedge, or heavy-duty bone hammer capable of withstanding enormous impact forces. A spring 234, weighing 10-15 pounds, is fitted around the outside of the guide post 233 to provide the ability to damage the bone. The outer wall of the protective cover 220 has several positioning holes 221, and the outer end of the guide post 233 is fixedly connected to the positioning holes 221. The outer wall of the bone holder 210 has several insertion holes 211, and the striking post 231 is slidably inserted into the insertion holes 211 so that the rebounding striking post 231 passes through the insertion holes 211 to impact the bone.

[0034] Specifically, the arc-shaped power storage assembly 300 includes a coarse reciprocating lead screw 310 that runs through the front and rear ends of the top of the protective cover 220, a pair of levers 320 that are threadedly connected to it, and a first motor 311. The first motor 311 is fixedly connected to the side end face of the protective cover 220 by bolts. The levers 320 are in the shape of isosceles triangular blocks and their hypotenuses slide in contact with a pair of impact bone assemblies 230 located within the arc range.

[0035] A sliding sleeve 322 is fixedly connected between a pair of levers 320, and the three are made of cast iron as a single structure. The sliding sleeve 322 is threaded onto the coarse reciprocating screw 310, so that the sliding sleeve 322 reciprocates along the central axis of the bone placement tube 210, driving the levers 320 to push back several impact pins 231 and compress the springs 234, ready to release and form an impact force. A hanging rod 330 is inserted through the top side of the sliding sleeve 322, and the two ends of the hanging rod 330 pass through the two sides of the protective cover 220 to limit the axial movement of the levers 320. Support blocks 321 extend from both ends of the bottom surface of the levers 320. The support blocks 321 slide in contact with the large diameter end of the impact pins 231, guiding them to slide on the inclined surface of the levers 320 until they slide to the top of the arc of the levers 320, at which point the impact pins 231 push the springs 234 to their shortest compression, ready to release the elastic force.

[0036] Specifically, the planar power storage assembly 400 includes a pair of thin reciprocating lead screws 410 that run through the front and rear ends of the bottom of the protective cover 220, a push block 420 that is threadedly connected to them, and a second motor 411. The second motor 411 is fixedly connected to the side end face of the protective cover 220 by bolts. The push block 420 is in the shape of an isosceles triangular block and its hypotenuse slides in contact with the large diameter end of the striking column 231 of a pair of striking bone assemblies 230 located in the vertical plane, guiding it to slide on the inclined surface of the push block 420 until it slides to the top of the arc of the push block 420, at which point the striking column 231 pushes the spring 234 to its shortest compression, ready to release the elastic force.

[0037] A through hole is provided in the middle of the inclined surface of the push block 420. The through hole is threaded with the fine reciprocating screw 410, so that the push block 420 reciprocates along the central axis of the bone placement tube 210, pushing several impact pins 231 to retract and compress the spring 234, so as to release and form an impact force. Insert blocks 421 are provided at both ends of the bottom surface of the push block 420. The insert blocks 421 slide in contact with the large diameter end of the impact pin 231. The arc end of the push block 420 is provided with a convex ring and a positioning rod 430 is sleeved on it. The two ends of the positioning rod 430 pass through the two sides of the protective cover 220 to limit the axial movement of the push block 420.

[0038] This invention also provides an application of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging, which is the application of the above-mentioned production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging in the preparation of bone collagen peptides with the function of preventing osteoporosis and delaying cell aging.

[0039] In addition, an experiment was conducted to evaluate whether bovine bone peptides have anti-cellular aging effects. The following is an evaluation report of this experiment.

[0040] 1. Sample to be tested The red bovine bone peptide (HNG) and yak bone peptide (MNG) powders used in the experiment were provided by Gansu Zhongtai Biotechnology Co., Ltd. 0.88g of red bovine bone peptide was weighed and dissolved in 4.4ml of double-distilled water to a final concentration of 200mg / ml. 0.85g of yak bone peptide was weighed and dissolved in 4.25ml of double-distilled water to a final concentration of 200mg / ml.

[0041] 2. Evaluation Results 2.1 Effects of bovine bone peptides on the proliferation of MRC5 cells like Figure 12 As shown in Figures a and c, this refers to the MTT assay used to detect the proliferation rate of HNG or MNG-treated cells and control cells. MRC5 cells were seeded in 96-well plates and cultured in basal medium or medium containing a certain concentration of bovine bone peptide. After 96 hours, the cell proliferation rate was detected by the MTT assay. The proliferation rate of HNG or MNG-treated cells was higher than that of control cells, and the difference was significant.

[0042] Referring to Figures b and d, this refers to the changes in cell proliferation rate monitored by a live-cell imaging system for HNG or MNG-treated cells (*P<0.05). Similar to this result, cell proliferation was monitored using a HoloMonitor M4 live-cell imaging system. Images were taken every 12 hours in the same field of view, and the number of cells within each field was counted. Five fields of view were used per group, and cell proliferation curves were plotted after five consecutive days of data collection. The results showed that cells cultured with HNG or MNG-treated cells proliferated faster than cells cultured in basal medium, but the difference was not statistically significant.

[0043] 2.2 Bovine bone peptides alleviate the effect of hydrogen peroxide on the proliferation of MRC5 cells like Figure 13 As shown in Figures a and c, this refers to the effect of the combined use of HNG or MNG and hydrogen peroxide on the proliferation of MRC5 cells as detected by the MTT assay. MRC5 cells were seeded in 96-well plates and cultured in basal medium or medium containing a certain concentration of HNG or MNG, respectively. After cell attachment, the cells were treated with a gradient of tert-butyl peroxide. After 2 hours, the tert-butyl peroxide was removed, and the cells were cultured in the corresponding medium for another 72 hours. Cell viability was then assessed by the MTT assay. The results showed that compared with cells treated with tert-butyl peroxide alone, the cell viability was increased after treatment with bovine bone peptides, with a significant difference observed in the 200 μg / ml HNG group.

[0044] Referring to Figures b and d, these refer to the statistical analysis results of the inhibitory effect of HNG or MNG treatment on hydrogen peroxide-induced cell killing. *P<0.05,**P<0.01,***P<0.001; 100 μg / ml and 200 μg / ml HNG significantly increased the cell survival rate of cells treated with 200 μM tert-butyl peroxide from 7.3% to 28.3% and 31.3%, respectively; 200 μg / ml MNG significantly increased the cell survival rate of cells treated with 100 μM tert-butyl peroxide from 21.67% to 48.3% and the cell survival rate of cells treated with 200 μM tert-butyl peroxide from 4.3% to 29.0%. These results indicate that appropriate concentrations of bovine bone peptides can protect cells from hydrogen peroxide-induced damage.

[0045] 2.3 Bovine bone peptides significantly inhibited intracellular ROS levels induced by hydrogen peroxide. like Figure 14As shown in Figures a and c, flow cytometry analysis was used to detect the effect of HNG or MNG on the increase in cellular ROS levels induced by tert-butyl peroxide (TBHP) at a concentration of 100 μM and HNG or MNG concentrations of 100 μg / ml (+) and 200 μg / ml (++). Figures b and d show the statistical results of ROS levels, with **P < 0.01 and ***P < 0.001. Cells were seeded in 6-well plates and treated with medium containing HNG or MNG. After cell adhesion, cells were treated with 100 μM tert-butyl peroxide for 1 h and incubated with a DCF probe for half an hour. Cell fluorescence intensity was detected by flow cytometry. The intracellular ROS level in the tert-butyl peroxide-only treatment group was significantly increased, reaching approximately 2.5 times that of the control group. However, after using medium containing bovine bone peptides, the intracellular ROS level decreased significantly to 1–1.5 times, showing a significant difference.

[0046] 2.4 Bovine bone peptides inhibit hydrogen peroxide-induced cellular senescence Method 1.7: β-galactosidase staining detection Using X-Gal as a substrate, a deep blue substance is generated under the catalysis of the aging-specific protein β-galactosidase. Cells that show positive staining under an optical microscope are senescent cells. Cells treated with the above-mentioned senescence-inducing method were processed according to the kit instructions: the cell culture medium was removed, the cells were washed once with PBS, 0.5 ml of senescence fixative was added, and the cells were fixed at room temperature for 15 min. The cells were then washed twice with PBS, and 0.5 ml of staining working solution (containing 5 μl of staining solution A, 5 μl of staining solution B, 465 μl of staining solution C, and 25 μl of X-gal, freshly prepared) was added. The cells were stained overnight at 37°C. The next day, the cells were observed and photographed under an optical microscope. Three fields of view were randomly selected from each cell group, and the number of cells and positive cells in each field of view was counted using Image-Proplus. The positive cell rate (%) was calculated as: (Number of positive cells / Number of cells) x 100%.

[0047] like Figure 15 As shown in Figure a, the effect of β-galactosidase staining on HNG or MNG on tert-butyl hydroperoxide-induced cell senescence is detected, bar=100μM, the concentration of tert-butyl hydroperoxide is 100μM; the concentrations of HNG or MNG are 100μg / ml (+) and 200μg / ml (#); Figures b and c show the results of counting the number of positive cells in five fields of view in Figure a, ***P<0.001; Cells were processed according to the procedure in Method 1.7, and β-galactosidase staining was performed according to the kit instructions. In the cell group treated with tert-butyl hydrogen peroxide alone, cell volume increased, cell bodies spread out, and flattened; over 90% of the cells were stained blue-green. In contrast, cells cultured with different concentrations of HNG or MNG showed a significant reduction in senescent cells, ranging from 20% to 40%. These results indicate that bovine bone peptides can effectively protect cells and prevent hydrogen peroxide-induced cell senescence.

[0048] 2.5 Bovine bone peptides reduce the expression of proteins related to cellular senescence. like Figure 16 As shown in Figure a, the effect of HNG / MNG combined with tert-butyl hydrogen peroxide on the expression of aging-related proteins was detected by protein immunoblotting; Figure b shows the statistical results of grayscale scans of three parallel experiments, *P<0.05, **P<0.01. After processing and collecting cells according to the procedure in Method 1.7, protein immunoblotting was performed to detect the expression of aging-related proteins p16, p21, p27, and p53. Treatment of cells with tert-butyl hydrogen peroxide alone significantly increased the expression of aging-related proteins. However, after culturing cells with different concentrations of HNG and MNG, the expression of these indicator proteins significantly decreased in a dose-dependent manner.

[0049] 3. Evaluation Conclusion (1) Bovine bone peptides have good anti-hydrogen peroxide-induced cell senescence effects; (2) Bovine bone peptides have good antioxidant effects and can protect MRC5 cells from oxidative damage; (3) Bovine bone peptides have a certain effect on promoting the proliferation of MRC5 cells.

[0050] It should be noted that the fixed connections and fixing methods of the present invention are achieved using conventional fixing means such as bolt connections or welding. The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A production process for bovine bone collagen peptides that prevent osteoporosis and delay cell aging, comprising the following steps: S1. Using fresh yak bones as raw material, after removing impurities, the yak bones are first struck with a bone-breaking device to create pits and cracks, and then fall into the bone crusher body and are crushed into 1-2cm size. S2. Place the crushed aggregate into a bone washing machine and soak it in cold water. Stir, drain the cold water, and then wash it with clean water. S3. Next, the bones and water are cooked in a cooking tank with a volume ratio of 1:

4. The cooking is carried out at a constant temperature and pressure of 0.5~1.5MPa and 110℃~120℃ for 2~3 hours. Then, the cooked soup is separated by a soup-oil separation device to obtain defatted soup. S4. The defatted broth is subjected to hydrolysis, decolorization, filtration, concentration, sterilization and drying processes in sequence to obtain bovine bone collagen peptide powder. The characteristic feature is that step S1 includes the following steps: S11. Place the yak bones into the bone-inserting tube and plug it with a rubber stopper; S12. Start the first motor and the second motor to drive the paddle and push block to move quickly back and forth from one end of the bone placement tube to the other end, thereby triggering several bone-striking groups inserted outside the bone placement tube to compress and rebound and release, thus impacting the yak bone inside the bone placement tube, thereby forming pits and cracks on the bone surface. S13. The servo motor is restarted to drive the unloading belt to move a certain distance, causing the yak bones to fall into the bone crusher and be collected.

2. The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging according to claim 1, characterized in that: The bone-impacting device, the arc-shaped energy storage group, and the plane energy storage group work together to impact the yak bone. The bone-impacting device includes a bone-placement tube placed horizontally with an open bottom surface, a protective cover covering the outside of the bone-placement tube, and several bone-impacting groups distributed at equal intervals around the outside of the bone-placement tube. Below the bottom opening of the bone-placement tube, there is a bone-unloading belt and a servo motor that drives the bone-unloading belt to rotate. Several bone-unloading ports are opened at equal intervals on the surface of the bone-unloading belt. The arc-shaped power storage group includes a coarse reciprocating lead screw that runs through the front and rear ends of the top of the protective cover, a pair of levers threadedly connected to it, and a first motor. The levers are in the shape of isosceles triangular blocks and their hypotenuses slide in contact with a pair of impact bone groups located within the arc range. The planar power storage assembly includes a pair of thin reciprocating lead screws running through the front and rear ends of the bottom of the protective cover, a push block threadedly connected to them, and a second motor. The push block is in the shape of an isosceles triangular block and its hypotenuse slides in contact with a pair of impact bone assemblies located in the vertical plane.

3. The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging according to claim 2, characterized in that: The bone crusher body is used to crush bone. The bone crusher body includes a crushing box, a double toothed roller installed therein, and a main motor that drives the double toothed roller to rotate in opposite directions.

4. The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging according to claim 3, characterized in that: The yak bone is struck by a set of impacting devices. The set of impacting devices includes a striking post, a striking head, and a guide post that are coaxially connected. The striking head is a conical head made of spring steel. A spring is fitted on the outside of the guide post. The outer wall of the protective cover has several positioning holes. The outer end of the guide post is fixedly connected to the positioning holes. The outer wall of the bone placement tube has several insertion holes. The striking post is slidably inserted into the insertion holes.

5. The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging according to claim 4, characterized in that: A sliding sleeve is fixedly connected between a pair of levers. The sliding sleeve is threaded onto a coarse reciprocating lead screw. A hanging rod is inserted through the top side of the sliding sleeve to restrict the axial movement of the levers. Support blocks extend from both ends of the bottom surface of the levers. The support blocks slide in contact with the large-diameter end of the striking post, guiding them to slide on the inclined surface of the levers until they slide to the top of the lever's arc. At this point, the striking post's pushing spring is compressed to its shortest length, ready to release the elastic force.

6. The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging according to claim 5, characterized in that: The push block has a through hole in the middle of its inclined surface, which is threaded onto a fine reciprocating lead screw. The bottom surfaces of the push block have inserts extending from both ends, which slide in contact with the large-diameter end of the striking post. The arc end of the push block has a protruding ring and a positioning rod, which is used to restrict the axial movement of the push block.

7. The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging according to claim 6, characterized in that, Step S2 further includes the following steps: S21. Then add water twice the volume of the aggregate, and add sodium hydroxide at 0.8-1.5‰ of the aggregate weight, and stir for 30 minutes; after draining, add water twice the volume of the aggregate and acetic acid at 5‰, stir for 30 minutes, and then drain.

8. The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging according to claim 7, characterized in that, Step S4 specifically includes the following steps: S41. Add the defatted broth obtained in step S3 to a hydrolysis tank, cool it to 50-60℃, adjust the pH value to 7-8.2, measure the concentration of the defatted broth with an instrument, add a compound biological enzyme preparation to the broth and hydrolyze for 1-1.5 hours. The weight of the enzyme preparation accounts for 0.1%-0.2% of the weight of collagen protein contained in the broth. The enzyme preparation involved in this step is a customized compound enzyme preparation, which contains acidic protease, alkaline protease and flavor enzyme in a ratio of 3:2:1, and its enzyme activity is 350,000-400,000 U / g. S42. After initial filtration through a 10kDa ultrafiltration membrane, add activated charcoal and heat to 100℃ while stirring, maintaining the temperature for 5 minutes. The weight of the activated charcoal should be 2%-4% of the weight of the collagen in the soup. Then cool down to 70-80℃ and let stand for 1-1.5 hours to decolorize and deodorize.

9. The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging according to claim 8, characterized in that, Step S4 further includes the following steps: S43. The hydrolysate is coarsely filtered using a candle filter with diatomaceous earth and diatomaceous earth sheets, and then finely filtered using an ultrafiltration membrane with a 5 kDa filter. S44. Use a vacuum concentrator to concentrate the filtered defatted soup stock at low temperature. S45. Using a pressure-type spray dryer, the treated concentrated defatted broth is dried into solid powder or granules to obtain bovine bone collagen peptides.

10. The application of a bovine bone collagen peptide for preventing osteoporosis and delaying cell aging, characterized in that: The production process of bovine bone collagen peptides for preventing osteoporosis and delaying cell aging as described in claim 9 is applied in the preparation of bone collagen peptides that prevent osteoporosis and delay cell aging.

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