CN109369800B - Method for extracting high-purity collagen from bovine bone - Google Patents
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The invention relates to the technical field of biological collagen preparation, in particular to a method for extracting high-purity collagen from bovine bone, which comprises the following steps: s1, cleaning fresh ox bones; s2, putting the cleaned ox bone into a reactor, adding an ethanol solution, ventilating, sealing and reacting to prepare ox bone slurry; s3, processing the bovine bone slurry by using a high-pressure homogenizer to form uniform bovine bone slurry; s4, adding an acid solution into the ox bone slurry, uniformly mixing, performing ultrahigh pressure impinging stream treatment and closed reaction kettle treatment, performing high-voltage pulse electric field treatment, performing closed reaction kettle treatment, and performing solid-liquid separation by using a filter press to obtain an extracting solution; s4, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen; the purity of the collagen of the invention is more than 98 percent, the yield of the collagen is more than 90 percent, the bioactivity of the collagen is improved, the absorption and utilization rate of a human body is improved to more than 98.5 percent, and the extraction time is short.
Description
Technical Field
The invention relates to the technical field of biological collagen preparation, in particular to a method for extracting high-purity collagen from bovine bone.
Background
Collagen is a natural biological macromolecule synthesized by animal cells, widely exists in skins, bones, cartilages, teeth, tendons, ligaments and blood vessels of animals, is an extremely main structural protein of connective tissues, and plays roles in supporting structural organs and protecting organisms. Collagen has stable property, is difficult to decompose at common processing temperature and short-time heating, is difficult to be absorbed by human body, and has low edible utilization rate.
At present, the common extraction methods are chemical method and enzyme degradation method. The chemical method is a method of promoting the peptide chain of protein molecules to be broken into small molecular substances at a certain temperature by using chemical reagents such as acid, alkali and the like. The prior art, such as Chinese patent with patent number CN201710823158.2, discloses a method for extracting bovine bone collagen peptide, comprising the following steps of firstly, breaking bone; secondly, feeding and cleaning; thirdly, feeding, cooking and discharging soup; fourthly, hydrolysis and filtration; fifthly, concentration; sixthly, drying and spraying powder; the ox bone is fully utilized, and some nutrient substances which are not easy to be applied in the ox bone are fully released through deep processing, so that the ox bone can be utilized by the human body. Also, for example, chinese patent No. CN201710904024.3 discloses a method for extracting type I collagen from animal hard bone by acid-enzyme combination, comprising the following steps: step one, pretreating bone raw materials, and then decalcifying with hydrochloric acid, wherein the concentration of hydrochloric acid is 0.01-0.02 mol/L, and the soaking time is 10-12 h; cleaning the mixture to a pH value of 3-5 after decalcification treatment; step two, heating and stewing, wherein the stewing temperature is 90-95 ℃ and 2-3 h; cooling, adding protease for enzymolysis, wherein the protease is pepsin, the addition amount of the protease is 3-5 per mill of the bone raw material, the enzymolysis temperature is 50-55 ℃, the enzymolysis time is 5-7 hours, and the pH is kept at 5-7; separating to obtain enzymatic hydrolysate; and step five, performing post-treatment to obtain the bone I type collagen powder. The invention extracts the type I collagen, thereby improving the utilization value of the edible bone; the preparation method is simple and has high production efficiency; the product obtained by adopting the decoloring and desalting process is white in color and high in purity.
In the prior art, the acid method is not suitable for industrial application because of severe strong acid reaction and severe equipment corrosion and the alkali method is subjected to racemization reaction. The enzymatic extraction is characterized in that certain temperature and pH are controlled, protease is used for enzymolysis of raw materials, the collagen is extracted through impurity removal and fine filtration, the period is short, the process is finished within 1-24h, the protein yield is high, the damage of amino acid is less, the method is environment-friendly, and the defects of wide molecular weight distribution, high cost and more impure proteins are overcome. Therefore, the research on the extraction method of the high-purity collagen in the bovine bone is of great significance.
Disclosure of Invention
The invention provides a method for extracting high-purity collagen from bovine bone to solve the technical problems.
The method is realized according to the following technical scheme:
a method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning fresh ox bones with clear water, removing meat residues on the surfaces of the ox bones, and then cleaning the ox bones with citric acid solution;
s2, putting cleaned ox bones into a reactor, adding an ethanol solution, ventilating to the temperature of 110-130 ℃, stopping ventilating, adjusting the pressure to 80-95MPa, carrying out closed reaction for 1-2h, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an acid solution into the ox bone slurry, uniformly mixing, treating for 10-15min by ultrahigh pressure impinging stream, treating for 20-30min in a closed reaction kettle, treating the mixed solution for 60-90S by a high-voltage pulsed electric field, treating for 20-30min in the closed reaction kettle, and performing solid-liquid separation by a filter press to obtain an extracting solution;
s5 ultrafiltering the extractive solution, collecting ultrafiltrate, and spray drying to obtain collagen.
The mass concentration of the citric acid solution is 7-10%.
The adding amount of the ethanol solution is 4-7 times of the mass of the cleaned cattle bone.
The mass concentration of the ethanol solution is 45-50%.
The aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is (3-5) to 1.
The acid solution is an organic acid solution with the mass concentration of 55-65%.
The organic acid comprises the following substances in parts by weight: 8-11 parts of lactic acid, 4-7 parts of tartaric acid, 1-2 parts of gallic acid and 2-5 parts of acetic acid.
The adding amount of the acid solution is 5-6 times of the mass of the cleaned cattle bone.
The pressure in the ultrahigh pressure impinging stream treatment is 180-230MPa, and the impinging distance is 2-4 mm.
The electric field intensity in the high-voltage pulse electric field treatment is 10-14kV/cm, and the pulse number is 10-15.
The molecular weight of the ultrafiltration membrane is 0.1-0.7 ten thousand daltons.
The invention has the beneficial effects that:
the purity of the collagen is more than 98 percent, the yield of the collagen is more than 90 percent, the biological activity of the collagen is improved, the absorption and utilization rate of a human body is improved to more than 98.5 percent, and the extraction time is short.
Firstly, the invention uses citric acid to treat the ox bone, and removes grease and calcium components on the surface of the ox bone; secondly, in the pulping process, ethanol and aeration treatment are utilized to transfer impurity components such as calcium element and the like from a water phase to an organic phase, so that the yield and the purity of the collagen are improved; and finally, extracting the collagen by using an acidic solution, combining ultrahigh pressure impinging stream, effectively destroying the stable structure of the collagen, combining a high-voltage pulse electric field, generating an electromagnetic effect, shortening the extraction time, preventing collagen molecular chains from being broken, preventing collagen molecules from being degraded, and improving the bioactivity of the collagen.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
A method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning fresh ox bones with clear water, removing meat residues on the surfaces of the ox bones, and then cleaning the ox bones with a citric acid solution with the mass concentration of 10%;
s2, putting the cleaned ox bone into a reactor, adding an ethanol solution with the mass concentration of 50% and the mass of 7 times that of the cleaned ox bone, ventilating to 130 ℃, stopping ventilating, adjusting the pressure to 95MPa, sealing for reaction for 2 hours, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an organic acid solution with the mass concentration of 65% into the bovine bone slurry, uniformly mixing, performing ultrahigh pressure impinging stream treatment for 15min, performing closed reaction kettle treatment for 30min, performing high-voltage pulsed electric field treatment on the mixed solution for 90S, performing closed reaction kettle treatment for 30min, and performing solid-liquid separation by using a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is 5: 1;
the organic acid is composed of the following substances: 11kg lactic acid, 7kg tartaric acid, 2kg gallic acid, 5kg acetic acid;
the adding amount of the organic acid solution is 6 times of the mass of the cleaned beef bones;
the pressure in the ultrahigh pressure impinging stream treatment is 230MPa, and the impinging distance is 4 mm;
the electric field intensity in the high-voltage pulse electric field treatment is 14kV/cm, and the pulse number is 15;
the molecular weight of the ultrafiltration membrane is 0.7 ten thousand daltons.
Example 2
A method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning fresh ox bones with clear water, removing meat residues on the surfaces of the ox bones, and then cleaning the ox bones with a citric acid solution with the mass concentration of 7%;
s2, putting the cleaned ox bone into a reactor, adding an ethanol solution with the mass concentration of 45% and the mass of 4 times that of the cleaned ox bone, ventilating to 110 ℃, stopping ventilating, adjusting the pressure to 80MPa, sealing for reaction for 1h, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an organic acid solution with the mass concentration of 55% into the bovine bone slurry, uniformly mixing, performing ultrahigh pressure impinging stream treatment for 10min, performing closed reaction kettle treatment for 20min, performing high-voltage pulsed electric field treatment on the mixed solution for 60S, performing closed reaction kettle treatment for 20min, and performing solid-liquid separation by using a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is 3: 1;
the organic acid is composed of the following substances: 8kg lactic acid, 4kg tartaric acid, 1kg gallic acid, 2kg acetic acid;
the adding amount of the organic acid solution is 5 times of the mass of the cleaned beef bones;
the pressure in the ultrahigh pressure impinging stream treatment is 180MPa, and the impinging distance is 2 mm;
the electric field intensity in the high-voltage pulse electric field treatment is 10kV/cm, and the pulse number is 10;
the molecular weight of the ultrafiltration membrane is 0.1 ten thousand daltons.
Example 3
A method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning fresh ox bones with clear water, removing meat residues on the surfaces of the ox bones, and then cleaning the ox bones with a citric acid solution with the mass concentration of 8%;
s2, putting the cleaned ox bone into a reactor, adding an ethanol solution with the mass concentration of 48 percent and the mass of 5 times that of the cleaned ox bone, ventilating to 120 ℃, stopping ventilating, regulating the pressure to 85MPa, carrying out closed reaction for 1.5h, then stirring at high speed to form slurry, and sieving by a 60-mesh sieve to obtain the ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an organic acid solution with the mass concentration of 60% into the bovine bone slurry, uniformly mixing, carrying out ultrahigh pressure impinging stream treatment for 12min, carrying out closed reaction kettle treatment for 25min, carrying out high-voltage pulsed electric field treatment on the mixed solution for 75S, carrying out closed reaction kettle treatment for 25min, and carrying out solid-liquid separation by using a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is 4: 1;
the organic acid is composed of the following substances: 9.5kg lactic acid, 5kg tartaric acid, 1.5kg gallic acid, 3kg acetic acid;
the adding amount of the organic acid solution is 5.6 times of the mass of the cleaned beef bones;
the pressure in the ultrahigh pressure impinging stream treatment is 200MPa, and the impinging distance is 3 mm;
the electric field intensity in the high-voltage pulse electric field treatment is 12kV/cm, and the pulse number is 12;
the molecular weight of the ultrafiltration membrane is 0.5 ten thousand daltons.
Example 4
A method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning 50kg of fresh beef bones with clear water, removing meat residues on the surfaces of the fresh beef bones, and then cleaning the beef bones with a citric acid solution with the mass concentration of 9%;
s2, putting the cleaned ox bone into a reactor, adding an ethanol solution with the mass concentration of 50% 6 times that of the cleaned ox bone, ventilating to 120 ℃, stopping ventilating, adjusting the pressure to 90MPa, carrying out closed reaction for 1h, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an organic acid solution with the mass concentration of 60% into the bovine bone slurry, uniformly mixing, performing ultrahigh pressure impinging stream treatment for 10min, performing closed reaction kettle treatment for 25min, performing high-voltage pulsed electric field treatment on the mixed solution for 60S, performing closed reaction kettle treatment for 20min, and performing solid-liquid separation by using a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is 3.5: 1;
the organic acid is composed of the following substances: 10kg lactic acid, 6kg tartaric acid, 2kg gallic acid, 4kg acetic acid;
the adding amount of the organic acid solution is 5.5 times of the mass of the cleaned beef bones;
the pressure in the ultrahigh pressure impinging stream treatment is 205MPa, and the impinging distance is 3 mm;
the electric field intensity in the high-voltage pulse electric field treatment is 13kV/cm, and the pulse number is 10;
the molecular weight of the ultrafiltration membrane is 0.4 ten thousand daltons.
Comparative example 1
A method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning 50kg of fresh beef bones with clear water, removing meat residues on the surfaces of the fresh beef bones, and then cleaning the beef bones with a citric acid solution with the mass concentration of 9%;
s2, putting the cleaned ox bone into a reactor, adding an ethanol solution with the mass concentration of 50% 6 times that of the cleaned ox bone, ventilating to 120 ℃, stopping ventilating, adjusting the pressure to 90MPa, carrying out closed reaction for 1h, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an organic acid solution with the mass concentration of 60% into the bovine bone slurry, uniformly mixing, treating for 25min in a closed reaction kettle, treating the mixed solution for 60S in a high-voltage pulsed electric field, treating for 20min in the closed reaction kettle, and performing solid-liquid separation by using a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is 3.5: 1;
the organic acid is composed of the following substances: 10kg lactic acid, 6kg tartaric acid, 2kg gallic acid, 4kg acetic acid;
the adding amount of the organic acid solution is 5.5 times of the mass of the cleaned beef bones;
the electric field intensity in the high-voltage pulse electric field treatment is 13kV/cm, and the pulse number is 10;
the molecular weight of the ultrafiltration membrane is 0.4 ten thousand daltons.
Comparative example 2
A method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning 50kg of fresh beef bones with clear water, removing meat residues on the surfaces of the fresh beef bones, and then cleaning the beef bones with a citric acid solution with the mass concentration of 9%;
s2, putting the cleaned ox bone into a reactor, adding an ethanol solution with the mass concentration of 50% 6 times that of the cleaned ox bone, ventilating to 120 ℃, stopping ventilating, adjusting the pressure to 90MPa, carrying out closed reaction for 1h, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an organic acid solution with the mass concentration of 60% into the bovine bone slurry, uniformly mixing, performing ultrahigh pressure impinging stream treatment for 10min, sealing the reaction kettle for 45min, and performing solid-liquid separation by using a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is 3.5: 1;
the organic acid is composed of the following substances: 10kg lactic acid, 6kg tartaric acid, 2kg gallic acid, 4kg acetic acid;
the adding amount of the organic acid solution is 5.5 times of the mass of the cleaned beef bones;
the pressure in the ultrahigh pressure impinging stream treatment is 205MPa, and the impinging distance is 3 mm;
the molecular weight of the ultrafiltration membrane is 0.4 ten thousand daltons.
Comparative example 3
A method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning 50kg of fresh beef bones with clear water, removing meat residues on the surfaces of the fresh beef bones, and then cleaning the beef bones with a citric acid solution with the mass concentration of 9%;
s2, putting the cleaned ox bone into a reactor, adding an ethanol solution with the mass concentration of 50% 6 times that of the cleaned ox bone, ventilating to 120 ℃, stopping ventilating, adjusting the pressure to 90MPa, carrying out closed reaction for 1h, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding 60% hydrochloric acid solution into the ox bone slurry, mixing uniformly, performing ultrahigh pressure impinging stream treatment for 10min, performing closed reaction kettle treatment for 25min, performing high-voltage pulsed electric field treatment on the mixed solution for 60S, performing closed reaction kettle treatment for 20min, and performing solid-liquid separation by using a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is 3.5: 1;
the addition amount of the hydrochloric acid solution is 5.5 times of the mass of the cleaned beef bones;
the pressure in the ultrahigh pressure impinging stream treatment is 205MPa, and the impinging distance is 3 mm;
the electric field intensity in the high-voltage pulse electric field treatment is 13kV/cm, and the pulse number is 10;
the molecular weight of the ultrafiltration membrane is 0.4 ten thousand daltons.
Comparative example 4
A method for extracting high-purity collagen from bovine bone comprises the following steps:
s1, cleaning 50kg of fresh beef bones with clear water, removing meat residues on the surfaces of the fresh beef bones, and then cleaning the beef bones with a citric acid solution with the mass concentration of 9%;
s2, putting the cleaned ox bone into a reactor, adding an ethanol solution with the mass concentration of 50% 6 times that of the cleaned ox bone, ventilating to 120 ℃, stopping ventilating, adjusting the pressure to 90MPa, carrying out closed reaction for 1h, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an animal protease solution with the mass concentration of 60% into the bovine bone slurry, uniformly mixing, performing ultrahigh pressure impinging stream treatment for 10min, performing closed reaction kettle treatment for 25min, performing high-voltage pulsed electric field treatment on the mixed solution for 60S, performing closed reaction kettle treatment for 20min, and performing solid-liquid separation by using a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is 3.5: 1;
the adding amount of the animal protease solution is 5.5 times of the mass of the cleaned cattle bone;
the pressure in the ultrahigh pressure impinging stream treatment is 205MPa, and the impinging distance is 3 mm;
the electric field intensity in the high-voltage pulse electric field treatment is 13kV/cm, and the pulse number is 10;
the molecular weight of the ultrafiltration membrane is 0.4 ten thousand daltons.
Test examples
Accurately weighing 5.0mg of hydroxyproline, dissolving in a small amount of water, diluting to 100mL of constant volume, and taking 0.05mg/mL of standard solution, then taking 0.05, 0.125, 0.25, 0.375 and 0.5mL of standard solution, respectively adding 0.1mol/L of sodium acetate to 1mL, measuring absorbance by using a high performance liquid chromatography, and drawing a standard curve;
then, 5.0mg of the sample of example 4 and comparative examples 1 to 4 was taken, and then a sample solution of 0.05mg/mL was prepared, and the collagen content was obtained by measuring the absorbance by high performance liquid chromatography in combination with a standard curve, and the collagen extraction rate and purity were calculated, wherein the collagen extraction rate is (collagen weight in sample × sample weight)/(bovine bone collagen weight × bovine bone weight); collagen purity-weight of collagen in sample/weight of sample; the results are shown in table 1:
TABLE 1
| Example 4 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | |
| Extraction ratio (%) | 93.2 | 86.5 | 80.3 | 95.4 | 90.5 |
| Purity (%) | 98.6 | 94.1 | 95.7 | 89.4 | 88.3 |
Test example 2
The determination of the heavy metal content was carried out for examples 1 to 4: inorganic arsenic and total arsenic were detected according to GB/T5009.11 "determination of Total arsenic and inorganic arsenic in food", lead (in terms of Pb) was detected according to GB5009.12 "determination of lead in food", cadmium (in terms of Cd) was detected according to GB/T5009.15 "determination of cadmium in food", mercury (in terms of methyl mercury) was detected according to GB/T5009.17 "determination of Total Mercury and organic Mercury in food", and the results are shown in Table 2:
TABLE 2
| Example 1 | Example 2 | Example 3 | Example 4 | |
| Inorganic arsenic (mg/kg) | 0.12 | 0.08 | 0.10 | 0.07 |
| Total arsenic (mg/kg) | 0.23 | 0.18 | 0.20 | 0.15 |
| Cadmium (mg/kg) | <0.01 | <0.01 | <0.01 | <0.01 |
| Lead (mg/kg) | 0.03 | 0.05 | 0.05 | 0.04 |
| Mercury (mg/kg) | <0.01 | <0.01 | <0.01 | <0.01 |
Claims (3)
1. The method for extracting the high-purity collagen from the bovine bone is characterized by comprising the following steps of:
s1, cleaning fresh ox bones with clear water, removing meat residues on the surfaces of the ox bones, and then cleaning the ox bones with citric acid solution;
s2, putting cleaned ox bones into a reactor, adding an ethanol solution, ventilating to the temperature of 110-130 ℃, stopping ventilating, adjusting the pressure to 80-95MPa, carrying out closed reaction for 1-2h, then stirring at high speed to form slurry, and sieving with a 60-mesh sieve to obtain ox bone slurry;
s3, processing the ox bone slurry by a high-pressure homogenizer, ejecting the material from the flow-limiting gap, cutting by turbulent flow, and colliding to enable the material to be ultra-fine to form extremely small particles to form uniform ox bone slurry;
s4, adding an acid solution into the ox bone slurry, uniformly mixing, treating for 10-15min by ultrahigh pressure impinging stream, treating for 20-30min in a closed reaction kettle, treating the mixed solution for 60-90S by a high-voltage pulsed electric field, treating for 20-30min in the closed reaction kettle, and performing solid-liquid separation by a filter press to obtain an extracting solution;
s5, performing ultrafiltration treatment on the extracting solution, collecting ultrafiltrate, and performing spray drying to obtain collagen;
the adding amount of the ethanol solution is 4-7 times of the mass of the cleaned beef bones;
the mass concentration of the ethanol solution is 45-50%;
the aeration refers to mixed gas of nitrogen and carbon dioxide, wherein the dosage ratio of the nitrogen to the carbon dioxide is (3-5) to 1;
the acid solution is an organic acid solution with the mass concentration of 55-65%;
the organic acid comprises the following substances in parts by weight: 8-11 parts of lactic acid, 4-7 parts of tartaric acid, 1-2 parts of gallic acid and 2-5 parts of acetic acid;
the pressure in the ultrahigh pressure impinging stream treatment is 180-230MPa, and the impinging distance is 2-4 mm;
the electric field intensity in the high-voltage pulse electric field treatment is 10-14kV/cm, and the pulse number is 10-15.
2. The method for extracting highly pure collagen from bovine bone according to claim 1, wherein the citric acid solution has a mass concentration of 7-10%.
3. The method for extracting highly pure collagen from bovine bone according to claim 1, wherein the ultrafiltration membrane used in the ultrafiltration treatment has a molecular weight of 0.1 to 0.7 ten thousand daltons.
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| US5138030A (en) * | 1989-10-10 | 1992-08-11 | Pachence James M | Process for extracting type I collagen form an avian source, and applications therefor |
| CN101792382A (en) * | 2010-03-10 | 2010-08-04 | 宋艳 | Method for preparing disinfected-decolorized fish-squamosum organic acid calcium and non-denatured collagen |
| CN106939325A (en) * | 2016-01-05 | 2017-07-11 | 许昌学院 | A kind of preparation method of ox bone collagen albumen |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5138030A (en) * | 1989-10-10 | 1992-08-11 | Pachence James M | Process for extracting type I collagen form an avian source, and applications therefor |
| CN101792382A (en) * | 2010-03-10 | 2010-08-04 | 宋艳 | Method for preparing disinfected-decolorized fish-squamosum organic acid calcium and non-denatured collagen |
| CN106939325A (en) * | 2016-01-05 | 2017-07-11 | 许昌学院 | A kind of preparation method of ox bone collagen albumen |
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| CN109369800A (en) | 2019-02-22 |
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