Coproduct method for extracting bone extract, bone oil and bone collagen peptide from bovine bone

Abstract

The application belongs to the technical field of biological products and health products processing, and provides a method for co-production of bovine bone essence, bovine bone oil and collagen peptide by using bovine bone. The method of the application can realize high-efficiency co-production of bovine bone essence, bovine bone oil and collagen peptide by using only alkaline protease in the enzymolysis process through horizontal stirring extraction, thereby saving energy and resources, simplifying the process flow, and improving the utilization rate of bovine bone and the product yield.

Description

Technical Field

[0001] This invention belongs to the field of biological products and health product processing technology, and in particular relates to a method for the co-production of bovine bone extract, bovine bone oil and collagen peptides using bovine bones. Background Technology

[0002] Beef bones are highly nutritious, containing 11-15% protein, 10-15% fat, and approximately 50-56% water. They are also rich in phospholipids, calcium, phosphorus, vitamins, and other nutrients. About 90% of the protein in beef bones is collagen.

[0003] Beef bone oil, also known as beef marrow oil, is an oil extracted and processed from beef bones. Besides retaining the unique aroma of animal fats, it is rich in unsaturated fatty acids and has wide applications in the catering and meat processing industries. Beef bone extract is a defatted beef bone extract. Its main component is beef bone collagen. During the hot-pressing extraction process, some proteins are broken down into smaller molecular fragments, low-molecular-weight flavor peptides, and amino acids. By combining bone extract, bone oil, and other raw materials, various compound seasonings can be produced, which are already popular in Asia and developed Western countries. Currently, many meat product manufacturers and catering companies in my country have improved the quality and taste of their products by using bone extract seasonings.

[0004] Collagen is a special type of protein with a triple helix structure, rich in special amino acids such as proline and hydroxyproline. Human collagen mainly originates from animal skin and bones; its absorption requires prior breakdown into small peptide molecules, and the effectiveness of direct supplementation is affected by the digestive process. Collagen peptides are easily absorbed by the human body and possess various physiological functions, including preventing stomach ulcers, promoting calcium absorption, preventing osteoporosis, promoting skin collagen metabolism, beautifying the skin, inhibiting high blood pressure, and lowering cholesterol. It is an important additive in the food and pharmaceutical industries, with continuously increasing market demand and a promising future.

[0005] Because my country's development of bovine bone extraction started relatively late, coupled with a lack of technology, capital, and understanding of meat flavor products and peptide products, production processes are relatively crude and fail to fully realize the utilization value of bovine bones. Although some enterprises utilize bovine bones to extract bovine bone oil and bone extracts, the processes, technologies, and equipment are still relatively backward, resulting in high energy consumption, heavy environmental pollution, low extraction rates, low product added value, and a failure to update the technology in a timely manner. For a long time, cage extraction has been the conventional process for extracting bone extracts and bone oil from bovine bones. To facilitate the raising and lowering of the cage, vertical tanks perpendicular to the ground are often used. The disadvantages of this method are: firstly, the gravity effect is more concentrated in a vertical tank than in a horizontal one, making it difficult to disperse bone fragments; secondly, because the cage must be placed inside the tank, stirring equipment cannot be installed, and heat transfer can only be facilitated by water circulation, resulting in a weak effect; and thirdly, the above reasons lead to a low yield of bone oil and bone extracts.

[0006] Currently, the commonly used processes for producing bovine bone collagen peptides include acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. The first two methods have many technical drawbacks and are almost obsolete. Enzymatic hydrolysis produces peptides under milder conditions, does not damage amino acids, and has higher bioactivity, making it the most commonly used method at present. However, enzymatic hydrolysis technology has only recently begun to achieve a certain scale and technological accumulation. Most existing bovine bone collagen peptide production processes employ "multiple enzymes used in combination, segmented enzyme addition, and segmented enzymatic hydrolysis," resulting in a complex, time-consuming process with low extraction rates and low purity. Many companies add a large amount of salt (approximately 10%) after extracting bone minerals to extend shelf life and enhance flavor. This necessitates desalting when reusing such bone minerals to extract collagen peptides, making the process cumbersome and wasteful of resources. For example, ZL201210166833.6 discloses a method for co-producing bovine bone oil, bovine bone extract, and bovine bone flavoring seasoning from bovine bone extract. This method uses a cage extraction process, focusing on bovine bone flavoring seasoning rather than collagen peptides, and employs multiple enzymes in the production process, making it cumbersome. ZL 202010205161.X discloses a method for co-producing bovine bone flavoring and active collagen peptides from bovine bones. However, this method discards bone oil during production, failing to fully utilize the bovine bones. Furthermore, its enzymatic hydrolysis substrate is a mixture of substances (bone protein and bone residue, etc.) with only bone oil removed, resulting in an impure substrate. Additionally, its enzymatic hydrolysis uses a traditional multi-enzyme combination, segmented enzyme addition, and segmented hydrolysis process, employing a large number of enzymes (7 types), making the process cumbersome, wasteful of resources, and severely impacting substrate purity and concentration, thus affecting the hydrolysis effect.

[0007] Therefore, there is an urgent need in this field for an effective method to co-produce bovine bone extract, bone oil, and bovine bone collagen peptides from bovine bones, in order to save energy and resources, simplify the process, and improve the utilization rate of bovine bones and the product yield. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a method for the co-production of bovine bone extract, bone oil and bovine bone collagen peptides by addressing the problems and defects of existing methods for the comprehensive utilization of bovine bones.

[0009] The method for the co-production of bovine bone extract, bovine bone oil, and collagen peptides according to the present invention includes the following steps: 1) Crush the bovine bones to a length of 3.0-4.5 cm at 0-4℃, preferably 3.5 cm; 2) Wash the broken beef bones with water by stirring. The stirring speed is 10-20 r / min, preferably 15 r / min; the washing temperature is 70-90℃, preferably 80℃; and the washing time is 20-40 min, preferably 30 min. 3) Extraction is carried out by horizontal stirring, and the beef bones are extracted by hot pressure cooking with water to obtain the extract and bone residue; 4) The extracted liquid is allowed to stand and separate to obtain beef bone clear broth (bone extract) and beef bone white broth (bone oil); 5) The osteocalcin was hydrolyzed using alkaline protease to obtain the hydrolysate; 6) Add activated carbon to the enzymatic hydrolysate and stir to decolorize and remove odor from the hydrolysate; 7) The enzymatic hydrolysate that has been decolorized and deodorized was filtered through a nanofiltration membrane with a molecular weight cutoff of 2000 Da to obtain a bovine bone collagen peptide solution. 8) The bovine bone collagen peptide solution was evaporated and concentrated to obtain a concentrated bovine bone collagen peptide solution; and 9) Dry and sterilize the bovine bone collagen peptide concentrate obtained in step 8) to obtain collagen peptide powder.

[0010] In a second aspect, the present invention provides bovine osteocalcin, bone oil, and collagen peptides produced by the method of the present invention.

[0011] This invention utilizes a method for simultaneously extracting bovine bone extract, bovine bone oil, and collagen peptides from bovine bones. Through a horizontal stirring extraction process, bovine bones are subjected to hot-pressing and steaming extraction. During enzymatic hydrolysis, only alkaline protease is used to efficiently co-produce bovine bone extract, bovine bone oil, and collagen peptides. This method technically and process-wise solves the problems and shortcomings analyzed in the background technology, effectively saving production costs, reducing energy consumption and environmental pollution, and improving the purity and extraction efficiency of bovine bone collagen peptides. It realizes a highly efficient and comprehensive production model for the combined use of bovine bones, including bone extract, bone oil, and bovine bone collagen peptides, on the same equipment and production line, through different process flow switching. This model has good economic and ecological benefits.

[0012] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0013] Figure 1 This is a flowchart illustrating the process of extracting osteocalcin, bone oil, and collagen peptides from bovine bones for the co-production of these products, as described in this invention. Detailed Implementation

[0014] As mentioned above, existing technologies for extracting bovine bone extract, bovine bone oil, and collagen peptides from bovine bones generally employ a vertical cage extraction process. This process, due to the concentrated effect of gravity, is not conducive to the dispersion of bone fragments. Furthermore, the presence of the cage means that heat transfer in this process relies solely on water circulation, without the ability to enhance heat transfer through stirring. Compared to the "stirring + water circulation" heat transfer effect of the horizontal stirring extraction process, the effect is significantly weaker, resulting in low yields of bone oil and bone extract. The inventors have discovered that using a horizontal stirring extraction process can improve production efficiency. In addition, they have found that by using a combination of basic protein, preferential basic protein, and neutral protease for a single enzymatic hydrolysis, efficient co-production of bovine bone extract, bovine bone oil, and collagen peptides can be achieved. This simplifies the complex process of using multiple enzymes, segmented enzyme addition, and segmented enzymatic hydrolysis in existing technologies, reducing the number of hydrolysis cycles and time, while simultaneously increasing the yield of bone collagen peptides.

[0015] This invention provides a method for the co-production of bovine bone extract, bovine bone oil, and collagen peptides from bovine bones, comprising the following steps: 1) Crush the bovine bones to a length of 3.0-4.5 cm at 0-4℃, preferably 3.5 cm; 2) Wash the broken beef bones with water by stirring. The stirring speed is 10-20 r / min, preferably 15 r / min; the washing temperature is 70-90℃, preferably 80℃; and the washing time is 20-40 min, preferably 30 min. 3) Extraction is carried out by horizontal stirring, and the beef bones are extracted by hot pressure cooking with water to obtain the extract and bone residue; 4) The extracted liquid is allowed to stand and separate to obtain beef bone clear broth (bone extract) and beef bone white broth (bone oil); 5) The osteocalcin was hydrolyzed using alkaline protease to obtain the hydrolysate; 6) Add activated carbon to the enzymatic hydrolysate and stir to decolorize and remove odor from the hydrolysate; 7) The enzymatic hydrolysate that has been decolorized and deodorized was filtered through a nanofiltration membrane with a molecular weight cutoff of 2000 Da to obtain a bovine bone collagen peptide solution. 8) The bovine bone collagen peptide solution was evaporated and concentrated to obtain a concentrated bovine bone collagen peptide solution; and 9) Dry and sterilize the bovine bone collagen peptide concentrate obtained in step 8) to obtain collagen peptide powder.

[0016] In an embodiment of the present invention, the beef bones are fresh or frozen beef bones.

[0017] In an embodiment of the present invention, in step 1), the bovine bones are typically crushed to a length of 3.0-4.5 cm, for example, 3.0 cm, 3.1 cm, 3.2 cm, 3.3 cm, 3.4 cm, 3.5 cm, 3.6 cm, 3.7 cm, 3.8 cm, 3.9 cm, 4.0 cm, 4.1 cm, 4.2 cm, 4.3 cm, 4.4 cm, or 4.5 cm, preferably 3.5 cm. The crushing temperature is controlled at 0-4.0℃, for example, 0℃, 0.5℃, 1.0℃, 1.5℃, 2.0℃, 2.5℃, 3.0℃, 3.5℃, or 4.0℃. The crushed bovine bones should be promptly transferred to the extraction tank, avoiding prolonged exposure to air to prevent oxidation, blackening, and off-flavoring of the bone surface, which would affect the bone extraction efficiency and product quality.

[0018] In an embodiment of the present invention, in step 2), the water used is preferably purified water, that is, water with a calcium carbonate content of less than 150 mg / L.

[0019] In an embodiment of the present invention, in step 2), the stirring speed is 10-20 r / min, for example, 10 r / min, 11 r / min, 12 r / min, 13 r / min, 14 r / min, 15 r / min, 16 r / min, 17 r / min, 18 r / min, 19 r / min, 20 r / min, preferably 15 r / min. In an embodiment of the present invention, in step 2), the cleaning temperature is 70-90℃, for example, 70℃, 71℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, preferably 80℃. In an embodiment of the present invention, in step 2), the cleaning time is 20-40 min, for example, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 38 min, 39 min, 40 min, preferably 30 min.

[0020] In the embodiments of the present invention, step 3) is typically carried out in an extraction tank. Horizontal stirring extraction in step 3) is one of the innovations of this invention. Horizontal stirring extraction means that the extraction tank lies flat during extraction, with the tank body and its internal central axis parallel to the ground. In addition to heat transfer through water circulation, heat transfer is further facilitated by the stirring action of paddles on the central axis. During the extraction process, a hanging cage is unnecessary; the crushed cattle bones are directly transferred into the horizontal tank. From the perspective of gravity, this facilitates the dispersion of the crushed cattle bones, accelerates uniform heating, and significantly improves production efficiency.

[0021] In an embodiment of the present invention, in step 3), the water used is preferably purified water, as described above.

[0022] In an embodiment of the present invention, in step 3), the mass ratio of crushed bovine bone to water can be 1:1-4, for example 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.5, 1:3.0, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, preferably 1:1.4.

[0023] In an embodiment of the invention, step 3) is typically performed in an extraction tank. In an embodiment of the present invention, in step 3), the extraction temperature is 130-145℃, for example 130℃, 131℃, 132℃, 133℃, 134℃, 135℃, 136℃, 137℃, 138℃, 139℃, 140℃, 141℃, 142℃, 143℃, 1440℃, 145℃, preferably 135℃; the extraction pressure is 0.2-0.4 MPa, for example 0.2 MPa, 0.21 MPa, 0.22 MPa, 0.23 MPa, 0.24 MPa, 0.25 MPa, 0.26 MPa, 0.27 MPa, 0.28 MPa, 0.29 MPa, 0.31 MPa, 0.31 MPa, 0.32 MPa, 0.33 MPa, 0.34 MPa, 0.35 MPa, 0.36 MPa, 0.37 MPa. MPa, 0.38 MPa, 0.39 MPa, 0.4 MPa, preferably 0.25 MPa; extraction time is 1.5-3.0 h, for example 1.5 h, 1.6 h, 1.7 h, 1.9 h, 2.0 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h, 3.0 h, preferably 2.0 h.

[0024] In an embodiment of the present invention, in step 4), during the static separation, the extracted liquid is maintained at a temperature of 80-90°C, for example, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, or 90°C, preferably 85°C. After separation, the upper layer is beef bone oil, also known as beef bone broth, and the lower layer is beef bone extract, also known as beef bone clear broth.

[0025] After the above steps, beef bone oil and beef bone extract can be mixed with different raw materials in different proportions to produce different beef-flavored seasonings, depending on the needs.

[0026] In an embodiment of the present invention, in step 5), the alkaline protease can be, for example, Novozyme 37071.

[0027] In an embodiment of the present invention, in step 5), the amount of alkaline protease added, based on bovine bone extract dry matter, can be 0.3-0.5%, for example 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, or 0.5%.

[0028] In an embodiment of the present invention, in step 5), the enzymatic hydrolysis temperature can be 50-60℃, for example 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, preferably 55℃; the pH can be 7.0-8.0, for example pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, preferably 7.5; and the enzymatic hydrolysis time can be 2-3h, for example 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, preferably 2.5h.

[0029] In an embodiment of the present invention, in step 5), a neutral protease can also be used simultaneously for enzymatic hydrolysis to remove strong umami and other off-flavor substances. In a specific embodiment, the neutral protease can be Neutrase 0.8L. In an embodiment of the present invention, the amount of neutral protease, based on bovine bone extract dry matter, can be 0.6-0.8%, for example 0.6%, 0.65%, 0.7%, 0.75%, or 0.8%.

[0030] After enzymatic hydrolysis, the enzyme can be inactivated by heating. For example, the hydrolysate can be heated to 80-100°C, preferably 95°C, and held for 10-20 min, preferably 15 min, to inactivate the enzyme.

[0031] In an embodiment of the present invention, in step 6), the amount of activated carbon used is 0.5-1.0% by weight of the enzymatic hydrolysate, preferably 0.8%. In a specific embodiment, the mixture is typically stirred for 30-60 minutes, preferably 45 minutes, until the color and odor on the surface of the enzymatic hydrolysate disappear.

[0032] In an embodiment of the present invention, in step 7), the decolorized and deisolated enzymatic hydrolysate from step 6) is filtered using a nanofiltration ceramic membrane with a molecular weight cutoff of 2000 Da, and the permeate after filtration is a collagen peptide solution.

[0033] In an embodiment of the present invention, in step 8), the collagen peptide solution is heated to 120-140°C, for example, 120°C, 121°C, 122°C, 123°C, 124°C, 125°C, 126°C, 127°C, 128°C, 129°C, 130°C, 131°C, 132°C, 133°C, 134°C, 135°C, 136°C, 137°C, 138°C, 139°C, or 140°C, preferably 130°C. The pressure is controlled at 0.6-1.3 MPa, for example, 0.6 MPa, 0.7 MPa, 0.8 MPa, 0.9 MPa, 1.0 MPa, 1.1 MPa, 1.2 MPa, or 1.3 MPa, preferably 0.8 MPa. The high-temperature and high-pressure treatment is carried out for 10-20 minutes, for example, preferably 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, or 16 minutes. The collagen peptide concentrate was concentrated under reduced pressure for 15 min, 17 min, 18 min, 19 min, and 20 min, with 15 min being the preferred time. During the concentration process, the pressure was controlled at 0.2 MPa to obtain a high-concentration collagen peptide concentrate.

[0034] In an embodiment of the invention, in step 9), drying can be performed, for example, by spraying.

[0035] This invention uses qualified bovine bones (fresh or frozen) as raw material. The process involves crushing, washing, horizontal stirring extraction, and static separation to obtain clear and white bone broths. The clear bone broth, through sterilization, homogenization, vacuum cooling, and concentration, yields bovine bone extracts in different forms. The white bone broth, after tubular centrifugation, vacuum cooling, and evaporation concentration, yields bovine bone oil with a rich meaty flavor. During the hot-press extraction process, some proteins in the bovine bones decompose into low-molecular-weight peptides and amino acids. Using bovine bone extracts and bovine bone oil as base materials, various meat-flavored seasonings can be produced by combining them with different raw materials in various proportions. Bovine bone extracts, through enzymatic hydrolysis, decolorization and impurity removal, nanofiltration, concentration, and drying, can produce bovine bone collagen peptides with multiple active functions. This invention enables the co-production of bovine bone extracts, bone oil, and bovine bone collagen peptides on a single production line, greatly improving production efficiency and economic benefits.

[0036] Specifically, in step 3) of the method of this invention, the horizontal stirring extraction method has many advantages over cage extraction. Firstly, the horizontal stirring extraction process significantly improves the yield of bovine bone extract, bone oil, and collagen peptides. Secondly, the horizontal stirring extraction equipment has lower requirements for plant height; vertical cage-type designs require tracks for lifting and transferring the cage above the extraction tank, while horizontal stirring extraction does not require such facilities, thus reducing safety hazards. The method of this invention reduces the types of enzymes, the number of enzymatic hydrolysis cycles, and the hydrolysis time, while greatly improving the yield of collagen peptides.

[0037] The technical principles and process flow of the present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The embodiments given below are only some embodiments of the present invention and are only used to explain the present invention, not to limit the scope of the present invention.

[0038] The bovine bones used in the following examples were all purchased from Keerqin Cattle Industry Co., Ltd.

[0039] Example 1 (1) Raw bone crushing: 30 kg of qualified cattle bones are crushed using a bone crusher (Zhengzhou Jukai Machinery Equipment Co., Ltd., model PG-300). The crushing length is 1.5-2.0 cm. The temperature of the crushing chamber is controlled at 0-4℃. The crushed cattle bones are then transferred into a hanging cage.

[0040] (2) Cleaning: Place the cage into the autoclave. Before placing it in, add purified water that meets the soft water standard. Turn on the water circulation pump to circulate the water. Raise the temperature to 80℃ and clean for 30 minutes. After the bovine bone blood has been fully dissolved, the blood and other impurities are discharged through the pipe at the bottom of the extraction tank.

[0041] (3) Cage extraction: Add purified water that meets the soft water standard at a ratio of 1:1.4 of beef bone to water, and close the water inlet valve after the water is added. During the water addition process, open the steam valve, and start the circulation pump to circulate the water when the temperature reaches 85℃. Extract at 135℃ and 0.25 MPa for about 2 hours to obtain the extract and bone residue.

[0042] (4) Separation of soup and residue: After extraction, the extracted soup is passed through a 60-mesh sieve, cooled and filtered through a pipeline, and then transferred to a settling tank. The bone residue is discharged through a 20-mesh sieve by vibration.

[0043] (5) Static separation: Cool the extracted liquid to 85℃ for natural separation, and let it stand for 2 hours. The upper layer of the settling tank is beef bone broth (beef bone oil), and the lower layer is beef bone clear broth (beef bone extract). Open the valve connecting the settling tank and the concentration buffer tank to pump the lower layer of beef bone clear broth into the concentration buffer tank. Observe the state of the broth through the sight glass. When obvious oil appears, close the valve at the filter and close the valve at the bottom of the settling tank. Wait for the broth to drain completely from the pipeline. The beef bone oil is transported to the finished oil storage tank by tubular centrifuge for storage and later use.

[0044] At this stage, 2.71 kg of bovine bone oil and 9.00 kg of bovine bone extract were obtained from 30 kg of bovine bones. The dry matter content of the bovine bone oil and bovine bone extract was found to be 50.00% and 45.00%, respectively.

[0045] After the above steps, beef-flavored seasonings can be produced by using beef bone oil and beef bone extract as base materials and mixing them with different raw materials as needed.

[0046] Bovine bone collagen peptides were extracted from bovine bone extract as a base material, and the following steps were continued: (6) bone extract enzymatic hydrolysis and the following steps: (6) Bone protein enzymatic hydrolysis: A two-stage low-temperature enzymatic hydrolysis and a one-stage high-temperature enzyme inactivation were employed. For the first enzymatic hydrolysis, the beef bone broth obtained in step (5) was homogenized and transferred to an enzymatic hydrolysis tank, with the temperature set at 50°C. Based on the dry matter of the bone broth, 0.8% alkaline protease and 0.8% neutral protease were added, and enzymatic hydrolysis was performed under natural pH conditions (pH 7.0-7.3, no adjustment required) for 3.5 hours to obtain the first enzymatic hydrolysate. For the second enzymatic hydrolysis, the aforementioned first enzymatic hydrolysate was set at 57°C, and based on its dry matter, 0.7% flavor protease and 0.7% papain were added. Enzymatic hydrolysis was performed under the same pH conditions as the first enzymatic hydrolysis for 1.5 hours to obtain the second enzymatic hydrolysate. The hydrolysate was then heated to 95°C and maintained for 15 minutes for enzyme inactivation. After enzyme inactivation and centrifugation, collagen peptide enzymatic hydrolysate was obtained. All proteases were purchased from Guangxi Pangbo Biotechnology Co., Ltd. (7) Decolorization and deodorization: Add 0.8% activated carbon to the enzymatic hydrolysate by weight, mix and stir for 45 minutes until the color and odor on the surface of the enzymatic hydrolysate disappear; (8) Filtration and purification: The enzymatic hydrolysate is filtered using a nanofiltration ceramic membrane with a molecular weight cutoff of 2000 Da. The permeate after filtration is the collagen peptide solution. (9) Evaporation and concentration: The collagen peptide solution is heated to 130°C and pressure is 0.8 MPa. After processing for 15 min, it is concentrated under reduced pressure. During the reduced pressure concentration, the pressure is controlled at 0.2 MPa to obtain a collagen peptide concentrate with a higher concentration. (10) Drying and sterilization: Spray dry and sterilize the concentrated solution obtained in step (9) to obtain bovine bone collagen peptide powder. After passing quality inspection, package and store it.

[0047] At this stage, the dry matter weight of collagen peptides extracted from 9.00 kg of bovine bone extract was 2.93 kg. The dry matter weight of 9.00 kg of bovine bone extract was 4.05 kg.

[0048] Example 1 describes a process for extracting bovine bone extract, bovine bone oil, and collagen peptides using a commonly used vertical cage extraction method, with data shown in Table 1. Table 1 shows that in Example 1, the yield of bovine bone oil was 9.03%, with a dry matter content of 50.00%, crude fat content of 33.00%, and crude protein content of 10.00%; the yield of bovine bone extract was 30.00%, with a dry matter content of 45.00%, crude protein content of 35.00%, and crude fat content of 1.21%; and the yield of bovine bone collagen peptides was 72.30%.

[0049] Example 2 (1) Raw bone crushing: 30kg of qualified cattle bones are crushed using a bone crusher (Zhengzhou Jukai Machinery Equipment Co., Ltd., model PG-300). The crushing length is 1.5-2.0 cm. The temperature of the crushing chamber is controlled at 0-4℃. The crushed cattle bones are then transferred into a hanging cage.

[0050] (2) Cleaning: Place the cage into the autoclave. Before placing it in, add purified water that meets the soft water standard. Turn on the water circulation pump to circulate the water. Raise the temperature to 80℃ and clean for 30 minutes. After the bovine bone blood has been fully dissolved, the blood and other impurities are discharged through the pipe at the bottom of the extraction tank.

[0051] (3) Cage extraction: Add purified water that meets the soft water standard at a ratio of 1:1.4 of beef bone to water, and close the water inlet valve after the water is added. During the water addition process, open the steam valve, and start the circulation pump to circulate the water when the temperature reaches 85℃. Extract at 135℃ and 0.25 MPa for about 2 hours to obtain the extract and bone residue.

[0052] (4) Separation of soup and residue: After extraction, the extracted soup is passed through a 60-mesh sieve, cooled and filtered through a pipeline, and then transferred to a settling tank. The bone residue is discharged through a 20-mesh sieve by vibration.

[0053] (5) Static separation: Cool the extracted liquid to 85℃ for natural separation, and let it stand for 2 hours. The upper layer of the settling tank is beef bone broth (beef bone oil), and the lower layer is beef bone clear broth (beef bone extract). Open the valve connecting the settling tank and the concentration buffer tank to pump the lower layer of beef bone clear broth into the concentration buffer tank. Observe the state of the broth through the sight glass. When obvious oil appears, close the valve at the filter and close the valve at the bottom of the settling tank. Wait for the broth to drain completely from the pipeline. The beef bone oil is transported to the finished oil storage tank by tubular centrifuge for storage and later use.

[0054] At this stage, 2.70 kg of bone oil and 9.01 kg of bone extract were obtained from 30 kg of bovine bones. The dry matter content of the bone oil and bone extract was 49.98% and 45.00%, respectively.

[0055] After the above steps, beef-flavored seasonings can be produced by using beef bone oil and beef bone extract as base materials and mixing them with different raw materials as needed.

[0056] Bovine bone collagen peptides were extracted from bovine bone extract as a base material, and the following steps were continued: (6) bone extract enzymatic hydrolysis and the following steps: (6) Bone protein enzymatic hydrolysis: The "two enzymes combined, one-time enzymatic hydrolysis" process was adopted, which was divided into low-temperature enzymatic hydrolysis and high-temperature inactivation. For low-temperature enzymatic hydrolysis, the beef bone broth obtained in step (5) was homogenized and transferred to an enzymatic hydrolysis tank, and the temperature was adjusted to 55℃ and pH 7.5. Based on the dry matter of the beef bone broth, 0.5% of Novozyme 37071 and 0.8% of Neutrase 0.8L were added for enzymatic hydrolysis, and the enzymatic hydrolysis reaction was carried out for 2.5h. Then the enzymatic hydrolysate was heated to 95℃ and held for 15 min for enzyme inactivation. After enzyme inactivation and centrifugation, the enzymatic hydrolysate was obtained. Both proteases were purchased from Beijing Novozymes Biotechnology Co., Ltd. (7) Decolorization and deodorization: Add 0.8% activated carbon to the enzymatic hydrolysate by weight, mix and stir for 45 minutes until the color and odor on the surface of the enzymatic hydrolysate disappear; (8) Filtration and purification: The enzymatic hydrolysate is filtered using a nanofiltration ceramic membrane with a molecular weight cutoff of 2000 Da. The permeate after filtration is the collagen peptide solution. (9) Evaporation and concentration: The collagen peptide solution is heated to 130°C and pressure is 0.8 MPa. After processing for 15 min, it is concentrated under reduced pressure. During the reduced pressure concentration, the pressure is controlled at 0.2 MPa to obtain a collagen peptide concentrate with a higher concentration. (10) Drying and sterilization: Spray dry and sterilize the concentrated solution obtained in step (9) to obtain bovine bone collagen peptide powder. After passing quality inspection, package and store it.

[0057] At this stage, the dry matter weight of the collagen peptides extracted from 9.01 kg of bovine bone extract was 3.07 kg. The dry matter weight of 9.01 kg of bovine bone extract was 4.05 kg.

[0058] Example 2 describes a co-production process that uses existing technology, namely a vertical cage for extracting bovine bone extract and bovine bone oil, and then employs the innovative "two-enzyme combined, one-time enzymatic hydrolysis" extraction method of this invention to extract collagen peptides. The bovine bones used in this example were all from the same breed, age, and part of the same animal from the same company. Data is shown in Table 1. Table 1 shows that in Example 2, the yield of bovine bone oil was 9.00%, with a dry matter content of 49.98%, a crude fat content of 33.02%, and a crude protein content of 10.00%; the yield of bovine bone extract was 30.03%, with a dry matter content of 45.00%, a crude protein content of 35.03%, and a crude fat content of 1.20%; and the yield of bovine bone collagen peptides was 75.80%.

[0059] Example 3 (1) Raw bone crushing: 30 kg of qualified cattle bones are crushed using a bone crusher (Zhengzhou Jukai Machinery Equipment Co., Ltd., model PG-300). The crushing length is 3.5-4.0 cm. The temperature of the crushing chamber is controlled at 0-4℃. The crushed cattle bones are then transferred to a horizontal extraction tank.

[0060] (2) Cleaning: Before feeding, add purified water that meets the soft water standard, turn on the mixer and circulation pump, and set the mixer speed to 15 r / min. The mixer shaft is a horizontal shaft that runs through the center of the horizontal extraction tank. Raise the temperature to 80℃ and clean for 30 min. After the bovine bone blood has fully dissolved, the blood and other impurities are discharged through the pipe at the bottom of the extraction tank.

[0061] (3) Horizontal stirring extraction: Add purified water that meets the soft water standard at a bone:water ratio of 1:1.4, and close the water inlet valve after the water is added. During the water addition process, open the steam valve and stir the mixer continuously at a uniform speed. When the temperature reaches 85℃, turn on the circulation pump to circulate the water. Extract at 135℃ and 0.25 MPa for about 2 hours.

[0062] (4) Separation of broth and dregs: After extraction, the bone broth is passed through a 60-mesh sieve, a cooler, and a pipeline filter to a settling tank. The bone dregs are discharged through a 20-mesh sieve by vibration.

[0063] (5) Settling and Separation: Cool the bone broth to 85°C for natural separation, and let it stand for 2 hours. The upper layer of the settling tank contains beef bone oil, and the lower layer contains beef bone broth. Open the valve connecting the settling tank and the concentration buffer tank to pump the lower layer of beef bone broth into the concentration buffer tank. Observe the state of the broth through the sight glass. When obvious oil appears, close the valve at the filter and close the valve at the bottom of the settling tank. Wait for the broth to drain completely from the pipe. The bone oil is then transported to the finished oil storage tank via a tubular centrifuge for storage and later use.

[0064] At this stage, 30 kg of bovine bone extract yielded 3.41 kg of bovine bone oil and 9.99 kg of bovine bone extract. The dry matter content of the bovine bone oil and bovine bone extract was found to be 50.00% and 45.03%, respectively.

[0065] Beef bone oil is a type of fat extracted and processed from beef bones. Besides retaining the unique aroma of beef fat, it is also rich in unsaturated fatty acids. Beef bone extract is rich in natural flavor compounds; during the hot-pressing extraction process, some proteins are broken down into low-molecular-weight fragments, peptides, and amino acids, resulting in a rich aroma. Following these steps, a series of beef-flavored seasonings that are popular with consumers can be produced using beef bone oil and beef bone extract as base materials, as needed.

[0066] Bovine bone collagen peptides were extracted and co-produced using bovine bone extract as a base material, followed by step 6) enzymatic hydrolysis of bone extract and the following steps: (6) Bone protein enzymatic hydrolysis: The "two-enzyme combined, one-time enzymatic hydrolysis" process was adopted, which involved one low-temperature enzymatic hydrolysis and high-temperature inactivation. For low-temperature enzymatic hydrolysis, the beef bone broth obtained in step (5) was homogenized and transferred to an enzymatic hydrolysis tank, and the temperature was adjusted to 55℃ and the pH to 7.5-8.0. Based on the dry matter of the bone broth, 0.5% of Novozyme 37071 and 0.8% of Neutrase 0.8L were added for enzymatic hydrolysis, and the enzymatic hydrolysis reaction was carried out for 2.5 hours. Then the enzymatic hydrolysate was heated to 95℃ and held for 15 minutes to inactivate the active enzymes. After enzyme inactivation and centrifugation, the enzymatic hydrolysate was obtained; both proteases were purchased from Beijing Novozymes Biotechnology Co., Ltd. (7) Decolorization and deodorization: Add 0.8% activated carbon to the enzymatic hydrolysate by weight, mix and stir for 45 minutes until the color and odor on the surface of the enzymatic hydrolysate disappear; (8) Filtration and purification: The enzymatic hydrolysate is filtered using a nanofiltration ceramic membrane with a molecular weight cutoff of 2000 Da. The permeate after filtration is the collagen peptide solution. (9) Evaporation and concentration: The collagen peptide solution is heated to 130°C and treated under a pressure of 0.8MPa for 15 minutes, and then concentrated under reduced pressure. During the reduced pressure concentration, the pressure is controlled at 0.2MPa to obtain a collagen peptide concentrate with a higher concentration. (10) Drying and sterilization: Spray dry and sterilize the concentrated solution obtained in step (9) to obtain bovine bone collagen peptide powder. After passing quality inspection, package and store it.

[0067] At this stage, the dry matter weight of collagen peptides extracted from 9.99 kg of bovine bone extract was 3.46 kg. The dry matter weight of 9.99 kg of bovine bone extract was 4.50 kg.

[0068] Example 3 describes the co-production of bovine bone extract, bovine bone oil, and collagen peptides using the innovative process of this invention. Specifically, it employs a "horizontal stirred extraction" + "two-enzyme combined, one-time enzymatic hydrolysis" process. The bovine bones used in this example were all from the same breed, age, and part of the same animal from the same company; data is shown in Table 1. Table 1 shows that in Example 3, the yield of bovine bone oil was 11.37%, with a dry matter content of 50.00%, crude fat content of 33.48%, and crude protein content of 9.66%; the yield of bovine bone extract was 33.30%, with a dry matter content of 45.03%, crude protein content of 35.62%, and crude fat content of 1.10%; and the yield of bovine bone collagen peptides was 76.89%.

[0069] Table 1. Data and Comparison of Bone Oil, Ostein, and Collagen Peptides Extracted from Bovine Bone in the Examples.

[0070] In summary, the yields of bovine bone oil and bovine bone extract in Example 3 were 11.37% and 33.30%, respectively, while those in Example 1 were 9.03% and 30.00% (see Table 1). This means that the yields of bovine bone oil and bone extract in Example 3 were 2.34 and 3.30 percentage points higher than those in Example 1, respectively. Similarly, the yields of bovine bone oil and bovine bone extract in Example 2 were 9.00% and 30.03% (see Table 1), indicating that the yields of bovine bone oil and bone extract in Example 3 were 2.37 and 3.27 percentage points higher than those in Example 2. This demonstrates that the horizontal stirring extraction process significantly improves the yields of bone extract and bone oil, showing a clear advantage. In Examples 2 and 3, the yields of bovine bone collagen peptides were 75.80% and 76.89%, respectively, while the yield of collagen peptides in Example 1 was 72.30% (see Table 1). This means that the yields of collagen peptides in Examples 2 and 3 increased by 3.50 and 4.58 percentage points, respectively, compared to Example 1. This demonstrates that the "two enzymes combined, one-time enzymatic hydrolysis" process significantly improves the yield of collagen peptides, showing a clear advantage.

[0071] Therefore, in summary, the process method of Example 3 has significant advantages over Examples 1 and 2, and is the best embodiment of the present invention. The horizontal stirring extraction process in this invention significantly improves the extract yield compared to the cage extraction process. The principle behind its effectiveness is as follows: First, the horizontal stirring extraction process has a central stirring shaft parallel to both the tank and the ground. The blades on the stirring shaft directly contact the bovine bone pieces, ensuring good and uniform dispersion and crushing of the bovine bone pieces in the water, and uniform emulsification and dissolution of solid particles in the liquid phase. Second, the stirring process induces physical turbulence, dispersion, and bonding of the bovine bone pieces within the extraction tank, as well as the formation of chemical reactions. Third, it enhances heat and mass transfer, and mass transfer between materials with different molecular structures, viscosity, and hygroscopicity. This is one of the important process innovations of this invention. The process of this invention reduces the types of enzymes, the number of enzymatic hydrolysis cycles, and the hydrolysis time, significantly improving the yield of collagen peptides. The principle behind this effect is that it mainly overcomes the following defects of existing technologies: First, the more types of enzymes used, the more likely the enzymes will hydrolyze each other, reducing enzyme activity; second, the use of multiple enzymes in combination, segmented enzymes, and segmented hydrolysis will frequently lead to interruptions in the hydrolysis process, affecting the hydrolysis effect.

[0072] The bovine bone extract and bone oil produced by the method of this invention can be used as base materials to formulate various seasonings, or sold as seasoning raw materials. The collagen peptides extracted by this invention are easily absorbed by the human body and have multiple physiological functions such as anti-aging, anti-oxidation, anti-hypertensive, immune enhancement, prevention and treatment of osteoporosis and arthritis, prevention of gastric ulcers, and beauty and skin care. They can be widely used in formulated foods and health products. The co-products of this invention expand the market and profit points for production units, which is the market value of this invention.

[0073] It should be noted that the above description is only a preferred embodiment of the present invention. Any changes, modifications, substitutions, and variations made within the scope of the processes, techniques, methods, formulas, market value, etc., described in this invention should be included within the protection scope of this invention. This invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to impose additional limitations on the content of this invention. Furthermore, combinations of the above-described technical features to form various embodiments not listed above are all considered to fall within the scope of this specification. Those skilled in the art, production personnel, and practitioners can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for the co-production of osteocalcin, bone oil, and collagen peptides from bovine bones, characterized in that, The method includes the following steps: 1) Crush the bovine bones to a length of 3.0-4.5 cm at 0-4℃, preferably 3.5 cm; 2) Wash the broken beef bones with water by stirring. The stirring speed is 10-20 r / min, preferably 15 r / min; the washing temperature is 70-90℃, preferably 80℃; and the washing time is 20-40 min, preferably 30 min. 3) Extraction is carried out by horizontal stirring, and the beef bones are extracted by hot pressure cooking with water to obtain the extract and bone residue; 4) The extracted liquid is allowed to stand and separate to obtain beef bone clear broth (bone extract) and beef bone white broth (bone oil); 5) The osteocalcin was hydrolyzed using alkaline protease to obtain the hydrolysate; 6) Add activated carbon to the enzymatic hydrolysate and stir to decolorize and remove odor from the hydrolysate; 7) The enzymatic hydrolysate that has been decolorized and deodorized was filtered through a nanofiltration membrane with a molecular weight cutoff of 2000 Da to obtain a bovine bone collagen peptide solution. 8) The bovine bone collagen peptide solution was evaporated and concentrated to obtain a high-concentration bovine bone collagen peptide concentrate; and 9) Drying and sterilization: Dry and sterilize the bovine bone collagen peptide concentrate obtained in step 8) to obtain collagen peptide powder.

2. The method according to claim 1, characterized in that, In step 3), the ratio of cow bone to water is 1:1-4, preferably 1:1.

4.

3. The method according to claim 1, characterized in that, In step 3), the extraction is carried out under the following conditions: temperature 130-145℃, preferably 135℃; pressure 0.2-0.4 MPa, preferably 0.25 MPa; extraction time 1.5-3.0 h, preferably 2.0 h.

4. The method according to claim 1, characterized in that, In step 4), during the static separation, the extracted liquid is kept at 80-90°C, preferably 85°C.

5. The method according to claim 1, characterized in that, In step 5), the amount of Novozyme 37071 used is 0.3-0.5% based on the dry matter of the bovine bone extract.

6. The method according to claim 1, characterized in that, Step 5) also includes simultaneously enzymatically hydrolyzing the osteocalcin with Neutrase 0.8L.

7. The method according to claim 5, characterized in that, In step 4), the amount of Neutrase 0.8L used is 0.6-0.8% based on the dry matter of the bovine bone extract.

8. The method according to claim 1, characterized in that, In step 5), the enzymatic hydrolysis is carried out under the following conditions: temperature 50-60°C, preferably 55°C, pH 7.0-8.0, preferably pH 7.

5.

9. The method according to claim 1, characterized in that, In step 6), the amount of activated carbon added is 0.5-1.0% by weight of the enzymatic hydrolysate, preferably 0.8%.

10. The method according to claim 1, characterized in that, Step 8) specifically includes heating the bovine bone collagen peptide solution to 120-140°C, preferably 130°C, controlling the pressure to 0.6-1.3 MPa, preferably 0.8 MPa, processing for 10-20 min, preferably 15 min, and then concentrating under reduced pressure at 0.2 MPa.

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