A method for extracting hydroxyapatite from animal waste bones

Abstract

The present invention provides a method for extracting hydroxyapatite from animal waste bones, belonging to the field of hydroxyapatite preparation. The present invention adopts bone powder, low eutectic solvent, surfactant and then ultrasonically disperses, adds surfactant, improves the dispersibility of cattle bone powder in the low eutectic solvent, realizes the full contact between the low eutectic solvent and the cattle bone powder, improves the extraction rate, and at the same time, the present technology does not need to use a large amount of acid and alkali, and only needs to use a small amount of alkaline solution when adjusting pH in the early stage when necessary, and does not involve the treatment of alkaline solution in the later stage, and the hydroxyapatite extraction efficiency and purity are high, the comprehensive cost is low, and it is suitable for industrial production. In addition, the reagents used in the present invention can be recycled, the recycling method is simple, further saves costs, and has environmental significance.

Description

Technical Field

[0001] The invention belongs to the field of hydroxyapatite preparation, and in particular relates to a method for extracting hydroxyapatite from discarded animal bones. Background Art

[0002] Hydroxyapatite is a functional calcium-containing inorganic salt. Due to its good biological and chemical activity, it can be used in many fields, such as food, biomedicine, electrochemistry and cosmetics. For example, it can be used as an adsorption material for heavy metals and dyes, as a drug carrier, and as a bone repair replacement material.

[0003] The preparation methods of hydroxyapatite include chemical synthesis and natural hydroxyapatite preparation. Chemical synthesis of hydroxyapatite has problems such as high reaction temperature, long reaction time, low product activity, wide size distribution range, unevenness, environmental pollution, and poor safety. Therefore, in recent years, researchers have begun to prepare natural hydroxyapatite from various biological waste materials, such as eggshells, corals, pig bones, cattle bones, fish scales, etc. Compared with chemical synthesis, the latter has the advantages of high biosafety and low cost, but there are also many problems in the preparation of natural hydroxyapatite.

[0004] The Chinese patent with application number 202210209388.0, entitled "Method for extracting hydroxyapatite from fish bones and the hydroxyapatite obtained", boils and rinses the fish bones to remove the residue, crushes them to obtain fish bone powder, and then places the fish bone powder in an alkaline solution (NaOH, KOH, LiOH) for multiple freeze-thaw cycles, takes out the fish bone powder for heat treatment, and then dialyzes the resulting product to neutrality with deionized water, and obtains hydroxyapatite by freeze-drying. However, this method uses a large amount of alkaline solution, which is costly, corrosive to the equipment, and difficult to recycle, which has a greater impact on the environment; moreover, the freeze-thaw cycle process is cumbersome, the heating extraction time is long, and large-scale production is difficult.

[0005] The Chinese patent with application number 201810899721.9 is named "Method for extracting fish scale collagen peptide powder and hydroxyapatite". The fish scales are washed, impurity-removed, and steam-exploded, and then put into hot water for leaching, followed by enzymatic hydrolysis. After enzymatic hydrolysis, the filtrate and filter residue are obtained. The filtrate is vacuum concentrated and spray-dried to obtain fish scale collagen, and the filter residue is dried, crushed, and calcined to obtain hydroxyapatite. Although this method uses enzymatic hydrolysis to obtain hydroxyapatite to avoid the use of acid and alkali solutions, the enzymatic hydrolysis method is either costly and time-consuming, or the preliminary treatment is complicated, and the final hydroxyapatite yield and purity are not enough.

[0006] Therefore, there is an urgent need for a method for extracting hydroxyapatite efficiently with low cost and low acid and alkali solution usage. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a method for extracting hydroxyapatite from discarded animal bones. The method uses a small amount of alkaline solution and is only used to adjust the pH. It has almost no corrosive effect on the equipment, and the drugs used can be recovered and reused. The preparation time is short, the cost is low, the yield and purity are high, and it is suitable for industrial production.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] A method for extracting hydroxyapatite from animal waste bones comprises the following steps:

[0010] 1) drying, crushing and sieving discarded animal bones to obtain bone powder;

[0011] 2) mixing the bone powder, the deep eutectic solvent and the surfactant and performing ultrasonic dispersion to obtain a bone powder dispersion;

[0012] 3) subjecting the bone powder dispersion to heating extraction and centrifugal separation, and taking the supernatant after the centrifugation;

[0013] 4) mixing the supernatant with methanol, allowing to stand, and filtering to obtain a filtrate and a solid precipitate;

[0014] 5) The solid precipitate is sequentially washed with water, washed with alcohol, dried, and calcined to obtain hydroxyapatite.

[0015] The present invention provides a method for extracting hydroxyapatite from discarded animal bones. The method comprises the following steps: bone powder, a low eutectic solvent and a surfactant are mixed and then ultrasonically dispersed. The surfactant is added to improve the dispersibility of the bovine bone powder in the low eutectic solvent, so as to achieve full contact between the low eutectic solvent and the bovine bone powder and improve the extraction rate. Ultrasonic technology is used for pre-dispersion to further improve the dispersion degree of the bovine bone powder. Meanwhile, ultrasound itself can also extract hydroxyapatite from the bovine bone powder, thereby improving the yield of hydroxyapatite. Meanwhile, the present technology does not require the use of a large amount of acid or alkali. Only a small amount of alkaline solution is required for adjusting the pH in the early stage, and no treatment of the alkaline solution is involved in the later stage.

[0016] In the method of the present invention, the animal waste bones in step 1) include cattle bones, sheep bones, pig bones, chicken bones or fish bones.

[0017] In the method of the present invention, the mesh size of the bone powder in step 1) is 50-200 meshes, preferably 100 meshes.

[0018] In the method of the present invention, the preparation method of the low eutectic solvent in step 2) is: melting the hydrogen bond acceptor and the hydrogen bond donor at a mass ratio of 1 to 3:2 to 4 at 80° C. into a uniform transparent liquid; wherein the mass ratio of the hydrogen bond acceptor and the hydrogen bond donor is preferably 1:2.

[0019] In the method of the present invention, the hydrogen bond acceptor includes but is not limited to choline chloride or betaine, preferably choline chloride; the hydrogen bond donor includes but is not limited to alcohols or carboxylic acids, wherein alcohols include but are not limited to ethylene glycol, propylene glycol or glycerol, and carboxylic acids include but are not limited to oxalic acid or citric acid; the hydrogen bond donor is preferably glycerol.

[0020] Furthermore, in the preparation method of the low eutectic solvent, when the hydrogen bond donor is an alcohol, it is necessary to use an alkaline solution to adjust the pH of the molten liquid to 8-14; when the hydrogen bond donor is a carboxylic acid, the pH does not need to be adjusted; the alkaline solution includes but is not limited to KOH aqueous solution, NaOH aqueous solution or ammonia water.

[0021] The low eutectic solvent prepared by the above definition of the present invention is simple to prepare, low in price, and can be reused; and the dissolution mechanism of the low eutectic solvent prepared by the present invention for extracting hydroxyapatite (HAP) conforms to the EDA theory and the hydrogen bond theory. The ionic liquid affects the bovine collagen fiber, the spatial structure of collagen, the triple helix structure, the hydrogen bonding between peptide chains, the hydrogen bonding within the peptide chain, and the peptide bond, so that the collagen is directly dissolved, the collagen undergoes irreversible denaturation, the molecular weight is reduced, and the hydrogen bonding between HAP is weakened. Under the action of external force, the HAP crystals are dispersed in the ionic liquid, that is, the purpose of separating and extracting HAP is achieved.

[0022] In the method of the present invention, the surfactant in step 2) includes but is not limited to any one of sodium dodecylbenzenesulfonate, hexadecyltrimethylammonium bromide, sodium dodecylsulfonate, sodium deoxycholate or Tween 80, preferably sodium dodecylbenzenesulfonate.

[0023] In the method of the present invention, the mass ratio of bone powder, low eutectic solvent and surfactant in step 2) is 1:10-20:0.22-3.15, preferably 1:10:1.1.

[0024] In the method of the present invention, the ultrasonic power of the ultrasonic dispersion in step 2) is 200W to 700W, preferably 300W; the ultrasonic time is 30 to 120 minutes, preferably 60 minutes.

[0025] In the method of the present invention, the extraction temperature of the heating extraction in step 3) is 80-140° C., preferably 120° C.; the extraction time is 6-24 hours, preferably 9 hours.

[0026] In the method of the present invention, in step 4), the volume ratio of the supernatant to methanol is 1:1-5, preferably 1:1; and the standing time is 10-24h, preferably 24h.

[0027] Those skilled in the art can set the parameters of ultrasonic extraction according to the type of bone powder and common knowledge in the art.

[0028] In the method of the present invention, the filtrate recovery treatment method in step 4) is: add chloroform to the filtrate, stir evenly, pour into a separatory funnel, take out the mixed solution of chloroform and protein from the lower layer after stratification, recover chloroform by vacuum distillation, and the remaining solid is protein; the remaining liquid in the separatory funnel can be separated from methanol and the low eutectic solvent by rotary evaporation. Since the filtrate obtained by suction filtration contains methanol, protein, and low eutectic solvent, the addition of chloroform can separate the protein, and the remaining liquid in the separatory funnel includes methanol and low eutectic solvent. The liquid can be separated from methanol and the low eutectic solvent by rotary evaporation, which can be used for the next extraction.

[0029] Compared with the prior art, the beneficial effects of the present invention are at least:

[0030] The method for extracting hydroxyapatite from discarded animal bones of the present invention does not require the use of a large amount of acid or alkali solution and is almost non-corrosive to equipment; and the extraction efficiency and purity of hydroxyapatite are improved through the combined action of a low eutectic solvent, a surfactant and ultrasound, with low overall cost and being suitable for industrial production.

[0031] In addition, the reagents used in the present invention can be recycled, and the recycling method is simple, which further saves costs and has environmental protection significance. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below by specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples, unless otherwise specified, are conventional methods. Unless otherwise specified, the reagents and materials used can all be obtained through commercial channels.

[0033] Example 1

[0034] This embodiment provides a method for extracting hydroxyapatite from animal waste bones, comprising the following steps:

[0035] 1) Dry, crush and sieve the cattle bones to obtain 100-mesh cattle bone powder for later use;

[0036] 2) mixing the bovine bone powder, the deep eutectic solvent, and the surfactant in a mass ratio of 1:10:1.1, and ultrasonicating at a power of 300 W for 60 min to obtain a bone powder dispersion;

[0037] The low eutectic solvent is prepared by melting choline chloride and glycerol at a mass ratio of 1:2 at 80°C to form a uniform transparent liquid, and adjusting the pH of the molten liquid to 10 using a KOH aqueous solution;

[0038] Wherein, the surfactant is sodium dodecylbenzene sulfonate.

[0039] 3) Place the evenly dispersed bone powder dispersion in a beaker and move it into an oil bath. Control the temperature at 120°C and extract for 9 hours. Centrifuge the extract after the extraction to obtain the supernatant. The solid residue is the undissolved bovine bone powder, which can be used as a raw material again in the next extraction.

[0040] 4) The supernatant was mixed with methanol in a mass ratio of 1:1 for precipitation, left to stand for 24 hours, and filtered to obtain a filtrate and a solid precipitate.

[0041] 5) The solid precipitate is washed with water, washed with alcohol, dried, and calcined to finally obtain hydroxyapatite.

[0042] 6) Add chloroform to the filtrate in step 4), stir evenly, pour into a separatory funnel, take out the mixed solution of chloroform and protein from the lower layer after stratification, recover chloroform by vacuum distillation, and the remaining solid is protein; the remaining liquid in the separatory funnel can be separated into methanol and the low eutectic solvent by rotary evaporation.

[0043] Example 2

[0044] This embodiment provides a method for extracting hydroxyapatite from animal waste bones, comprising the following steps:

[0045] 1) Dry, crush and sieve the cattle bones to obtain 50 mesh cattle bone powder for later use;

[0046] 2) mixing the bovine bone powder, the deep eutectic solvent and the surfactant in a mass ratio of 1:15:0.32 and ultrasonicating at a power of 200 W for 30 min to obtain a bone powder dispersion;

[0047] The low eutectic solvent is prepared by melting betaine and ethylene glycol at a mass ratio of 3:4 at 80°C into a uniform transparent liquid, and adjusting the pH of the molten liquid to 8 using an aqueous NaOH solution;

[0048] Wherein, the surfactant is hexadecyltrimethylammonium bromide.

[0049] 3) Place the evenly dispersed bone powder dispersion in a beaker and move it into an oil bath. Control the temperature at 110°C and extract for 6 hours. Centrifuge the extract after the extraction to obtain the supernatant. The solid residue is the undissolved bovine bone powder, which can be used as a raw material again in the next extraction.

[0050] 4) The supernatant was mixed with methanol in a mass ratio of 1:3 for precipitation, left to stand for 10 hours, and filtered to obtain a filtrate and a solid precipitate.

[0051] 5) The solid precipitate is washed with water, washed with alcohol, dried, and calcined to finally obtain hydroxyapatite.

[0052] 6) Add chloroform to the filtrate in step 4), stir evenly, pour into a separatory funnel, take out the mixed solution of chloroform and protein from the lower layer after stratification, recover chloroform by vacuum distillation, and the remaining solid is protein; the remaining liquid in the separatory funnel can be separated into methanol and the low eutectic solvent by rotary evaporation.

[0053] Example 3

[0054] This embodiment provides a method for extracting hydroxyapatite from animal waste bones, comprising the following steps:

[0055] 1) Dry, crush and sieve the cattle bones to obtain 200-mesh cattle bone powder for later use;

[0056] 2) mixing the bovine bone powder, the deep eutectic solvent and the surfactant in a mass ratio of 1:20:3 and ultrasonicating at a power of 700 W for 120 min to obtain a bone powder dispersion;

[0057] The low eutectic solvent is prepared by melting choline chloride and propylene glycol at a mass ratio of 3:2 at 80°C into a uniform transparent liquid, and adjusting the pH of the molten liquid to 14 using a KOH aqueous solution;

[0058] Wherein, the surfactant is sodium dodecyl sulfate.

[0059] 3) Place the evenly dispersed bone powder dispersion in a beaker and move it into an oil bath. Control the temperature at 140°C and extract for 24 hours. Centrifuge the extract after the extraction to obtain the supernatant. The solid residue is the undissolved bovine bone powder, which can be used as a raw material again in the next extraction.

[0060] 4) The supernatant was mixed with methanol in a mass ratio of 1:5 for precipitation, left to stand for 18 hours, and filtered to obtain a filtrate and a solid precipitate.

[0061] 5) The solid precipitate is washed with water, washed with alcohol, dried, and calcined to finally obtain hydroxyapatite.

[0062] 6) Add chloroform to the filtrate in step 4), stir evenly, pour into a separatory funnel, take out the mixed solution of chloroform and protein from the lower layer after stratification, recover chloroform by vacuum distillation, and the remaining solid is protein; the remaining liquid in the separatory funnel can be separated into methanol and the low eutectic solvent by rotary evaporation.

[0063] Example 4

[0064] This embodiment provides a method for extracting hydroxyapatite from animal waste bones, comprising the following steps:

[0065] 1) Dry, crush and sieve the cattle bones to obtain 100-mesh cattle bone powder for later use;

[0066] 2) mixing the bovine bone powder, the deep eutectic solvent, and the surfactant in a mass ratio of 1:10:1.1, and ultrasonicating at a power of 300 W for 60 min to obtain a bone powder dispersion;

[0067] The low eutectic solvent is obtained by melting choline chloride and oxalic acid at a mass ratio of 1:2 at 80°C to form a uniform transparent liquid;

[0068] Wherein, the surfactant is sodium dodecylbenzene sulfonate.

[0069] 3) Place the evenly dispersed bone powder dispersion in a beaker and move it into an oil bath. Control the temperature at 120°C and extract for 9 hours. Centrifuge the extract after the extraction to obtain the supernatant. The solid residue is the undissolved bovine bone powder, which can be used as a raw material again in the next extraction.

[0070] 4) The supernatant was mixed with methanol in a mass ratio of 1:1 for precipitation, left to stand for 24 hours, and filtered to obtain a filtrate and a solid precipitate.

[0071] 5) The solid precipitate is washed with water, washed with alcohol, dried, and calcined to finally obtain hydroxyapatite.

[0072] 6) Add chloroform to the filtrate in step 4), stir evenly, pour into a separatory funnel, take out the mixed solution of chloroform and protein from the lower layer after stratification, recover chloroform by vacuum distillation, and the remaining solid is protein; the remaining liquid in the separatory funnel can be separated into methanol and the low eutectic solvent by rotary evaporation.

[0073] Example 5

[0074] This embodiment provides a method for extracting hydroxyapatite from animal waste bones. The preparation method is basically the same as that in Example 1, with the only difference being that sheep bones are used instead of cow bones as raw materials.

[0075] Example 6

[0076] This embodiment provides a method for extracting hydroxyapatite from animal waste bones. The preparation method is basically the same as that in Example 1, with the only difference being that pig bones are used instead of cow bones as the raw materials.

[0077] Comparative Example 1

[0078] This comparative example provides a method for extracting hydroxyapatite from discarded animal bones. The specific preparation method is basically the same as that in Example 1, except that no surfactant is added in step 2).

[0079] Comparative Example 2

[0080] This comparative example provides a method for extracting hydroxyapatite from discarded animal bones. The specific preparation method is basically the same as that in Example 1, except that ultrasonic dispersion is not performed in step 2).

[0081] Experimental Example 1

[0082] The purity and yield of the hydroxyapatite in Examples 1-3 and Comparative Examples 1-2 were tested, and the test results are shown in Table 1.

[0083] Table 1: Purity and yield test table of hydroxyapatite

[0084] purity% Yield % Example 1 99 87.25 Example 2 99 84.36 Example 3 99 86.23 Example 4 99 85.37 Example 5 99 83.24 Example 6 99 84.77 Comparative Example 1 97 48.69 Comparative Example 2 98.5 60.56

[0085] Experimental Example 2

[0086] The recovery rate of the drugs recovered in Examples 1-3 was tested, and the test results are shown in Table 2.

[0087] Table 2: Drug recovery rate test table

[0088]

[0089] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for extracting hydroxyapatite from animal waste bones, characterized in that: The following steps are involved: 1) Drying, crushing and sieving discarded animal bones to obtain bone powder; 2) mixing the bone powder, the deep eutectic solvent and the surfactant and performing ultrasonic dispersion to obtain a bone powder dispersion; 3) subjecting the bone powder dispersion to heating extraction and centrifugal separation, and taking the supernatant after the centrifugation; 4) mixing the supernatant with methanol, allowing to stand, and filtering to obtain a filtrate and a solid precipitate; 5) washing the solid precipitate with water, washing with alcohol, drying and calcining in sequence to obtain hydroxyapatite; The preparation method of the low eutectic solvent in step 2) is: melting the hydrogen bond acceptor and the hydrogen bond donor at a mass ratio of 1 to 3:2 to 4 at 80° C. into a uniform transparent liquid; The hydrogen bond acceptor includes but is not limited to choline chloride or betaine; the hydrogen bond donor includes but is not limited to alcohols or carboxylic acids, wherein alcohols include but are not limited to ethylene glycol, propylene glycol or glycerol, and carboxylic acids include but are not limited to oxalic acid or citric acid; In the preparation method of the low eutectic solvent, when the hydrogen bond donor is an alcohol, it is necessary to use an alkaline solution to adjust the pH of the molten liquid to 8-14; when the hydrogen bond donor is a carboxylic acid, it is not necessary to adjust the pH; The surfactant in step 2) includes but is not limited to any one of sodium dodecylbenzene sulfonate, hexadecyltrimethylammonium bromide, sodium dodecyl sulfonate, sodium deoxycholate or Tween 80; The ultrasonic power of the ultrasonic dispersion in step 2) is 200W to 700W; and the ultrasonic time is 30 to 120 minutes.

2. The method for extracting hydroxyapatite from animal waste bones according to claim 1, characterized in that: The animal waste bones in step 1) include cattle bones, sheep bones, pig bones, chicken bones or fish bones.

3. The method for extracting hydroxyapatite from animal waste bones according to claim 1, characterized in that: In the step 2), the mass ratio of bone powder, low eutectic solvent and surfactant is 1:10-20:0.22-3.

15.

4. The method for extracting hydroxyapatite from animal waste bones according to claim 1, characterized in that: The extraction temperature of the heating extraction in step 3) is 80-140° C. and the extraction time is 6-24 hours.

5. The method for extracting hydroxyapatite from animal waste bones according to claim 1, characterized in that: In the step 4), the volume ratio of the supernatant to methanol is 1:1-5; and the standing time is 10-24 hours.

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