Method for extracting amino acid and compound calcium byproduct bone meal from bovine bone
The multi-enzymatic method of enzymatic decomposition of bovine bones, using complex enzymes and high-temperature treatment, solved the problems of low amino acid yield and waste of resources in the prior art, and achieved efficient utilization and environmentally friendly extraction of bovine bone components.
Patent Information
- Application Number
- CN202510553006.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-29
- Publication Date
- 2025-07-25
AI Technical Summary
The existing bovine bone extraction process has problems such as low amino acid yield, insufficient enzymatic lysis efficiency, waste of resources and environmental pollution, especially the limited efficiency of single enzyme method, insufficient pretreatment and insufficient waste utilization.
The multi-enzymatic method is used to enzymatically dissolve bovine bone, including pretreatment, enzymatic decomposition and separation steps, and the enzyme is performed using complex enzymes (papain and isopentyl transferase), combined with high-temperature treatment and ultrafiltration technology, amino acid powder and compound calcium products are prepared, and the residue is prepared into feed-grade bone meal.
It significantly improves the yield of amino acids and enzymatic lysis efficiency, realizes the full utilization of bovine bone components, and has good economic benefits and environmental protection effects.
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Figure BDA0005382620080000071
Abstract
Description
Technical Field
[0001] The present invention relates to a chemical extraction technology, especially a technology for extracting components from bovine bones. Background Art
[0002] As a by-product of meat processing, bovine bones contain abundant collagen (accounting for 20% - 30% of the bone weight) and minerals (such as calcium and phosphorus), and have extremely high resource utilization value. However, the traditional extraction processes have the following problems:
[0003] 1. Treatment with strong acids and bases: Although the acid hydrolysis method (such as hydrochloric acid or sulfuric acid) has low cost, it can cause thermal denaturation or oxidative decomposition of amino acids (such as tryptophan and cysteine), reducing the biological activity of the product. At the same time, a large amount of acid-containing waste liquid is generated, causing serious environmental pollution.
[0004] 2. Destruction of components at high temperature: When extracting calcium by the calcination method, high temperature (800 - 1000 °C) is likely to damage the collagen structure, resulting in amino acid loss, and the energy consumption is high.
[0005] Low separation efficiency: When simultaneously extracting calcium and amino acids, the mineral components and organic components interfere with each other, and the purity of the product is insufficient (for example, the purity of amino acids after acid hydrolysis is only 60% - 70%).
[0006] In recent years, the enzymatic hydrolysis method has gradually become a research hotspot. It hydrolyzes collagen through specific proteases, with mild conditions and a high retention rate of product activity. For example, some studies have shown that papain can efficiently hydrolyze the Gly-X-Y repeat sequence, releasing characteristic amino acids such as glycine and proline. However, the existing technologies still have the following deficiencies:
[0007] 1. Limited efficiency of single enzyme: A single protease (such as papain) can only hydrolyze some peptide bonds, and the yield of free amino acids is relatively low (about 12% - 15%);
[0008] 2. Insufficient pretreatment: The dense structure of bovine bones is not fully destroyed, resulting in too long enzymatic hydrolysis time (more than 48 hours);
[0009] 3. Problem of waste treatment: The residue after enzymatic hydrolysis is not effectively utilized, resulting in resource waste.
[0010] Therefore, it is urgent to develop an efficient and environmentally friendly enzymatic hydrolysis process for bovine bone amino acids to achieve the full utilization of all components. Summary of the Invention
[0011] To improve the amino acid yield and enzymatic hydrolysis efficiency and achieve the full utilization of bovine bone components, the present invention provides a method for extracting amino acids and co-producing bone meal with composite calcium from bovine bones.
[0012] The technical solution adopted by the present invention is: A method for extracting amino acids and co-producing bone meal with composite calcium from bovine bones, comprising the following steps:
[0013] S1. Remove the fascia from fresh bovine bones and crush them to a particle size of 1 - 10 mm to obtain bone residues.
[0014] S2. Cook the bone residues in a pressure vessel at 110 - 140 °C for 0.5 - 2 hours to obtain cooked bone residues.
[0015] S3. Freeze the cooked bone residues at -15 - 22 °C for at least 15 h, then take them out and let them thaw naturally to room temperature to obtain pretreated bone residues, and record the mass of the pretreated bone residues.
[0016] S4. Place the pretreated bone residues in an enzymatic hydrolysis container, add water according to the ratio of residue to water of 1:8 - 14 based on the mass of the pretreated bone residues, then add a composite enzyme for enzymatic hydrolysis treatment. The enzymatic hydrolysis temperature is 38 - 50 °C, and the enzymatic hydrolysis time is more than 1.5 h to obtain enzymatically hydrolyzed bone residues.
[0017] S5. Raise the internal temperature of the enzymatic hydrolysis container to 85 - 95 °C and maintain it for 8 - 15 min; then perform solid - liquid separation to obtain enzymatically hydrolyzed residues and enzymatically hydrolyzed liquid.
[0018] S6. After the enzymatically hydrolyzed liquid is ultrafiltered to remove macromolecular substances, it is then spray - dried to obtain amino acid powder.
[0019] The enzymatically hydrolyzed residues produced by the above - mentioned scheme can also be used to produce composite calcium products. Specifically, it can be achieved according to the following steps: The enzymatically hydrolyzed residues are leached with hydrochloric acid to obtain a filtrate and a residue. The pH of the filtrate is adjusted to 8.7 - 9.2 with a regulator, and the obtained precipitate is subjected to solid - liquid separation and drying to obtain a composite calcium product. The regulator can be sodium carbonate.
[0020] The residues produced by the above - mentioned scheme can also be used to prepare feed - grade bone meal. Specifically, it can be achieved according to the following steps: The residues are neutralized to a pH of 6.8 - 7.3, and then sequentially dried, sterilized, and pulverized to obtain feed - grade bone meal.
[0021] As a further improvement of the present invention, the composite enzyme includes papain. In the present invention, the use of papain can break down macromolecular proteins into small - molecule peptide segments or amino acids by cleaving peptide bonds in protein molecules.
[0022] More preferably, the composite enzyme is composed of papain and isopentenyl transferase. More preferably, in step S4, the E / S of the composite enzyme is 2000 - 7000 U / g, and the ratio of the activity concentration of papain to isopentenyl transferase is: papain:isopentenyl transferase = 1:0.3 - 1.
[0023] The hydrochloric acid used for leaching enzymolysis residues can be 4M hydrochloric acid. The leaching temperature can be set at 65 - 72°C, and the leaching time is preferably not less than 2h.
[0024] The present invention also discloses an amino acid product, which is prepared by the method for extracting amino acids and by - product bone meal of composite calcium from bovine bone of the present invention.
[0025] The present invention also discloses a protein powder, which is characterized in that its components include the amino acid product of the present invention.
[0026] The beneficial effects of the present invention are as follows: 1) Experiments prove that the method of the present invention can significantly improve the amino acid yield and enzymolysis efficiency; 2) The method of the present invention can realize the full utilization of bovine bone components and has good economic benefits. Detailed implementation mode
[0027] The present invention will be further described below in conjunction with embodiments.
[0028] The bone residues in the following examples and comparative examples are of the same batch, and the protein content of the bone residues measured by the Kjeldahl method is 26.7%.
[0029] Isopentenyl transferase was purchased from Hubei Yuansheng Peptide Biotechnology Co., Ltd.
[0030] Papain was purchased from Shanghai Zhongfeng Biotechnology Co., Ltd.
[0031] Example 1:
[0032] Extract amino acids and composite calcium from bovine bone according to the following steps:
[0033] (1) Remove the fascia from fresh bovine bone and crush it to a particle size of 3 - 7mm to obtain bone residues;
[0034] (2) Steam the bone residues in a pressure cooker at 120°C for 1 hour to obtain steamed bone residues;
[0035] (3) Freeze the steamed bone residues at - 18°C for 24h and then take them out to thaw naturally to room temperature to obtain pretreated bone residues, and record the mass of the pretreated bone residues;
[0036] (4) Place the pretreated bone residues in an enzymolysis container, add water according to the ratio of residue to water of 1:10 according to the mass of the pretreated bone residues, and then add a composite enzyme composed of papain and isopentenyl transferase for enzymolysis treatment according to the enzyme - substrate ratio of 5000U / g, wherein the ratio of the activity concentration of papain to isopentenyl transferase is: papain:isopentenyl transferase = 1:0.5; the enzymolysis temperature is 45°C, and the enzymolysis time is 2h to obtain enzymolyzed bone residues;
[0037] (5) Raise the internal temperature of the enzymatic hydrolysis container to 90 °C and maintain for 12 min; then perform solid-liquid separation to obtain enzymatic hydrolysis residue and enzymatic hydrolysis solution;
[0038] (6) After the enzymatic hydrolysis solution removes macromolecular substances by ultrafiltration, it is then spray-dried until the moisture content is less than 1% to obtain amino acid powder.
[0039] (7) The enzymatic hydrolysis residue is extracted with 4M hydrochloric acid at 68 °C for 3 h, followed by solid-liquid separation to obtain a filtrate and a residue. The pH of the filtrate is adjusted to 9.0 with the regulator sodium carbonate. The obtained precipitate is subjected to solid-liquid separation and dried at 80 °C until the moisture content is less than 2% to obtain a composite calcium product.
[0040] (8) The residue is neutralized to pH 7.0, then successively dried at 80 °C until the moisture content is less than 2%, sterilized at 160 °C, and pulverized to obtain feed-grade bone meal.
[0041] Example 2:
[0042] Extract amino acids and composite calcium from bovine bones according to the following steps:
[0043] (1) Remove the fascia from fresh bovine bones and crush them to a particle size of 3 - 7 mm to obtain bone residues;
[0044] (2) Cook the bone residues in a pressure cooker at 110 °C for 1.5 hours to obtain cooked bone residues;
[0045] (3) Freeze the cooked bone residues at -20 °C for 20 h, then take them out and thaw naturally to room temperature to obtain pretreated bone residues, and record the mass of the pretreated bone residues;
[0046] (4) Place the pretreated bone residues in an enzymatic hydrolysis container, add water according to the ratio of residue to water of 1:12 based on the mass of the pretreated bone residues, and then add a composite enzyme composed of papain and isopentenyl transferase according to the enzyme-substrate ratio of 4500 U / g for enzymatic hydrolysis treatment, where the ratio of the activity concentrations of papain to isopentenyl transferase is: papain:isopentenyl transferase = 1:0.3; the enzymatic hydrolysis temperature is 42 °C, and the enzymatic hydrolysis time is 2.5 h to obtain enzymatically hydrolyzed bone residues;
[0047] (5) Raise the internal temperature of the enzymatic hydrolysis container to 85 °C and maintain for 15 min; then perform solid-liquid separation to obtain enzymatic hydrolysis residue and enzymatic hydrolysis solution;
[0048] (6) After the enzymatic hydrolysis solution removes macromolecular substances by ultrafiltration, it is then spray-dried until the moisture content is less than 1% to obtain amino acid powder.
[0049] (7) The enzymolysis residue is leached with 4M hydrochloric acid at 65 °C for 3.5 h, followed by solid-liquid separation to obtain a filtrate and a residue. The pH of the filtrate is adjusted to 9.1 with the regulator sodium carbonate. The obtained precipitate is subjected to solid-liquid separation and dried at 80 °C until the moisture content is less than 2% to obtain the composite calcium product.
[0050] (8) The residue is neutralized to a pH of 6.9, and then successively dried at 80 °C until the moisture content is less than 2%, sterilized at 160 °C, and pulverized to obtain feed-grade bone meal.
[0051] Example 3:
[0052] Extract amino acids and composite calcium from bovine bones according to the following steps:
[0053] (1) Fresh bovine bones are removed of fascia and crushed to a particle size of 3 - 7 mm to obtain bone residues;
[0054] (2) The bone residues are cooked in a pressure cooker at 123 °C for 1 hour to obtain cooked bone residues;
[0055] (3) The cooked bone residues are frozen at -16 °C for 26 h and then taken out and naturally thawed to room temperature to obtain pretreated bone residues, and the mass of the pretreated bone residues is recorded;
[0056] (4) The pretreated bone residues are placed in an enzymolysis container, water is added according to a ratio of 1:8 of the residue-to-water ratio based on the mass of the pretreated bone residues, and then a composite enzyme composed of papain and isopentenyl transferase is added at an enzyme-to-substrate ratio of 5500 U / g for enzymolysis treatment, where the ratio of the activity concentration of papain to isopentenyl transferase is: papain:isopentenyl transferase = 1:0.7; the enzymolysis temperature is 48 °C, and the enzymolysis time is 1.8 h to obtain enzymolyzed bone residues;
[0057] (5) The internal temperature of the enzymolysis container is raised to 94 °C and maintained for 10 min; then solid-liquid separation is carried out to obtain enzymolysis residues and enzymolysis liquid;
[0058] (6) The enzymolysis liquid is ultrafiltered to remove macromolecular substances and then spray-dried until the moisture content is less than 1% to obtain amino acid powder.
[0059] (7) The enzymolysis residue is leached with 4M hydrochloric acid at 71 °C for 2.5 h, followed by solid-liquid separation to obtain a filtrate and a residue. The pH of the filtrate is adjusted to 8.6 with the regulator sodium carbonate. The obtained precipitate is subjected to solid-liquid separation and dried at 80 °C until the moisture content is less than 2% to obtain the composite calcium product.
[0060] (8) The residue is neutralized to a pH of 7.2, and then successively dried at 80 °C until the moisture content is less than 2%, sterilized at 160 °C, and pulverized to obtain feed-grade bone meal.
[0061] Comparative Example 1:
[0062] This comparative example is a control experiment for Example 1, which is carried out according to the same steps and conditions as Example 1, and the difference is that: papain is used to replace the composite enzyme, and the enzyme-substrate ratio remains unchanged. The specific scheme is as follows:
[0063] (1) After removing the fascia from fresh bovine bones, they are crushed to a particle size of 3 - 7 mm to obtain bone residues;
[0064] (2) The bone residues are steamed in a pressure cooker at 120 °C for 1 hour to obtain steamed bone residues;
[0065] (3) The steamed bone residues are frozen at -18 °C for 24 h and then taken out and naturally thawed to room temperature to obtain pretreated bone residues, and the mass of the pretreated bone residues is recorded;
[0066] (4) The pretreated bone residues are placed in an enzymatic hydrolysis container, water is added according to a ratio of 1:10 of the residue-water ratio according to the mass of the pretreated bone residues, and then papain is added at an enzyme-substrate ratio of 5000 U / g for enzymatic hydrolysis treatment; the enzymatic hydrolysis temperature is 45 °C, and the enzymatic hydrolysis time is 2 h to obtain enzymatically hydrolyzed bone residues;
[0067] (5) The internal temperature of the enzymatic hydrolysis container is raised to 90 °C and maintained for 12 min; then solid-liquid separation is carried out to obtain enzymatically hydrolyzed residues and enzymatically hydrolyzed liquid;
[0068] (6) The enzymatically hydrolyzed liquid is ultrafiltered to remove macromolecular substances and then spray-dried to a moisture content of less than 1% to obtain amino acid powder.
[0069] (7) The enzymatically hydrolyzed residues are extracted with 4M hydrochloric acid at 68 °C for 3 h and then solid-liquid separated to obtain a filtrate and residues. The pH of the filtrate is adjusted to 9.0 with a regulator sodium carbonate, and the obtained precipitate is solid-liquid separated and dried at 80 °C to a moisture content of less than 2% to obtain a composite calcium product.
[0070] (8) The residues are neutralized to a pH of 7.0, and then successively dried at 80 °C to a moisture content of less than 2%, sterilized at 160 °C, and pulverized to obtain feed-grade bone meal.
[0071] Comparative Example 2:
[0072] This comparative example is a control experiment for Example 1, which is carried out according to the same steps and conditions as Example 1, and the difference is that: isopentenyl transferase is used to replace the composite enzyme, and the enzyme-substrate ratio remains unchanged. The specific scheme is as follows:
[0073] (1) After removing the fascia from fresh bovine bones, they are crushed to a particle size of 3 - 7 mm to obtain bone residues;
[0074] (2) The bone residues are steamed in a pressure cooker at 120 °C for 1 hour to obtain steamed bone residues;
[0075] (3) Freeze the cooked bone residue at -18°C for 24 hours and then take it out to thaw naturally to room temperature to obtain the pretreated bone residue, and record the mass of the pretreated bone residue;
[0076] (4) Place the pretreated bone residue in an enzymatic hydrolysis container, add water according to the ratio of residue to water of 1:10 based on the mass of the pretreated bone residue, and then add isopentenyl transferase at an enzyme-substrate ratio of 5000 U / g for enzymatic hydrolysis; the enzymatic hydrolysis temperature is 45°C and the enzymatic hydrolysis time is 2 hours to obtain the enzymatically hydrolyzed bone residue;
[0077] (5) Raise the internal temperature of the enzymatic hydrolysis container to 90°C and maintain it for 12 minutes; then perform solid-liquid separation to obtain the enzymatically hydrolyzed residue and the enzymatically hydrolyzed liquid;
[0078] (6) After the enzymatically hydrolyzed liquid is ultrafiltered to remove macromolecular substances, it is then spray-dried to a moisture content of less than 1% to obtain amino acid powder.
[0079] (7) The enzymatically hydrolyzed residue is extracted with 4M hydrochloric acid at 68°C for 3 hours and then solid-liquid separated to obtain a filtrate and a residue. The pH of the filtrate is adjusted to 9.0 with the regulator sodium carbonate. The obtained precipitate is solid-liquid separated and dried at 80°C to a moisture content of less than 2% to obtain a composite calcium product.
[0080] (8) The residue is neutralized to a pH of 7.0, and then successively dried at 80°C to a moisture content of less than 2%, sterilized at 160°C, and pulverized to obtain feed-grade bone meal.
[0081] Comparative experiment:
[0082] For each example and comparative example, the calcium carbonate content of the obtained composite calcium product is detected according to GB1886.214-2016, and the yield is calculated; the calcium content of the obtained feed-grade bone meal is detected according to GB / T6436-2018, and the phosphorus content of the obtained feed-grade bone meal is detected according to GB / T6437-2018; the protein content of the bone residue (all from the same batch) used in each example and comparative example is detected by the Kjeldahl method.
[0083] The free amino acid content in the amino acid powder is detected by high performance liquid chromatography:
[0084] Respectively take 2 g of the amino acid powder of each example and comparative example, add 6 g of 0.1 mol / L HCl, centrifuge at 4°C and 8000 r / min for 20 minutes, take the supernatant, filter it through a 0.45 μm nylon filter membrane, and wait for instrument detection. Each sample is done in 3 parallels.
[0085] Determination conditions: The chromatographic column was Zorbax Eclipse-AAA (4.6 mm × 150 mm × 5 μm), mobile phase A was a 40 mmol / L NaH2PO4 solution with a pH value of 7.8; mobile phase B was V(methanol)∶V(acetonitrile)∶V(water) = 45∶45∶10, the flow rate was 1 mL / min, the injection volume was 1 μL, the detection wavelength was set at 338 nm, and the column temperature was set at 45 °C. The gradient elution program was as follows: at 0 min, 0% mobile phase B; at 1.9 min, 0% mobile phase B; at 18.1 min, 37% mobile phase B; at 23.2 min, 37% mobile phase B; at 24.0 min, 100% mobile phase B; at 26.0 min, 100% mobile phase B; at 27.0 min, 0% mobile phase B; at 30.0 min, 0% mobile phase B. The free amino acid yield was calculated based on the detection results, and the results are shown in Table 1.
[0086] Table 1 Detection results of comparative experiments
[0087]
[0088] From the detection results of Examples 1 to 3 in Table 1, it can be seen that the free amino acid yield using the method of the present invention can reach more than 30%. It can be seen that the method of the present invention can significantly improve the free amino acid yield of extracting amino acids from bovine bone.
[0089] From the comparison of Example 1, Comparative Example 1 and Comparative Example 2 in Table 1, it can be seen that when papain or isopentenyl transferase was used alone under the condition that the enzyme-substrate ratio and other conditions were exactly the same, the free amino acid yields were 10.6% and 2.7% respectively. However, the free amino acid yield of Example 1 using the composite enzyme prepared by compounding the two was as high as 26.4%. It can be seen that papain and isopentenyl transferase in the present invention have an obvious synergistic effect in improving the free amino acid yield of the process of extracting amino acids from bovine bone. The inventor believes that the reason may be due to the specificity of the pretreated bone residue substrate, and the composite enzyme releases free amino acids or promotes the dissociation of the protein structure by catalyzing the modification of specific amino acid side chains.
Claims
1. A method for extracting amino acids and by - product bone meal of composite calcium from bovine bones, comprising the following steps: S1. Remove the fascia from fresh bovine bones and crush them to a particle size of 1 - 10 mm to obtain bone residues; S2. Cook the bone residues in a pressure - bearing container at 110 - 140 °C for 0.5 - 2 hours to obtain cooked bone residues; S3. Freeze the cooked bone residues at - 15 - 22 °C for at least 15 h, then take them out and thaw naturally to room temperature to obtain pretreated bone residues, and record the mass of the pretreated bone residues; S4. Place the pretreated bone residues in an enzymatic hydrolysis container, add water according to the ratio of residue to water of 1:8 - 14 based on the mass of the pretreated bone residues, then add a composite enzyme for enzymatic hydrolysis treatment. The enzymatic hydrolysis temperature is 38 - 50 °C, and the enzymatic hydrolysis time is more than 1.5 h to obtain enzymatically hydrolyzed bone residues; S5. Raise the internal temperature of the enzymatic hydrolysis container to 85 - 95 °C and maintain it for 8 - 15 min; then perform solid - liquid separation to obtain enzymatically hydrolyzed residues and enzymatically hydrolyzed liquid; S6. After the enzymatically hydrolyzed liquid is ultra - filtered to remove macromolecular substances, it is then spray - dried to obtain amino acid powder.
2. The method for extracting amino acids, composite calcium and by - product bone meal from bovine bones according to claim 1, characterized in that: It further includes the step of preparing composite calcium using the enzymatically hydrolyzed residues, specifically: the enzymatically hydrolyzed residues are leached with hydrochloric acid to obtain a filtrate and residues, the pH of the filtrate is adjusted to 8.7 - 9.2 with a regulator, and the obtained precipitate is subjected to solid - liquid separation and drying to obtain a composite calcium product.
3. The method for extracting amino acids and by - product bone meal of composite calcium from bovine bones according to claim 2, characterized in that: It further includes the step of preparing feed - grade bone meal using the residues: the residues are neutralized to a pH of 6.8 - 7.3, and then sequentially dried, sterilized, and pulverized to obtain feed - grade bone meal.
4. The method for extracting amino acids and composite calcium by-products bone meal from cattle bones according to claim 2, characterized in that: The regulator is sodium carbonate.
5. The method for extracting amino acids and composite calcium by-product bone meal from bovine bones according to any one of claims 1 to 4, characterized in that The composite enzyme includes papain.
6. The method for extracting amino acids and composite calcium by-products of bone meal from bovine bones according to claim 5, characterized in that: The composite enzyme is composed of papain and isopentenyl transferase.
7. The method for extracting amino acids and composite calcium from bovine bones with by - product bone meal as claimed in claim 6, wherein: In step S4, the E / S of the composite enzyme is 2000 - 7000 U / g, and the ratio of the activity concentration of papain to isopentenyl transferase is: papain:isopentenyl transferase = 1:0.3 - 1.
8. The method for extracting amino acids and composite calcium from bovine bones with by - product bone meal as claimed in claim 2, characterized in that: The hydrochloric acid is 4M hydrochloric acid, the leaching temperature is 65 - 72 °C, and the leaching time is not less than 2 h.
9. An amino acid product prepared by the method for extracting amino acids and by - product bone meal of composite calcium from bovine bones according to any one of claims 1 - 8.
10. A protein powder, characterized in that: Its components include the amino acid product described in claim 9.