CN111718825A - Sturgeon bone peptide wine with effects of tonifying kidney and benefiting yang as well as preparation method and application thereof - Google Patents
Sturgeon bone peptide wine with effects of tonifying kidney and benefiting yang as well as preparation method and application thereof Download PDFInfo
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- CN111718825A CN111718825A CN202010586231.0A CN202010586231A CN111718825A CN 111718825 A CN111718825 A CN 111718825A CN 202010586231 A CN202010586231 A CN 202010586231A CN 111718825 A CN111718825 A CN 111718825A
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- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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Abstract
The invention discloses sturgeon bone peptide wine with the effects of tonifying kidney and tonifying yang, and a preparation method and application thereof. The sturgeon bone peptide wine is prepared by dissolving sturgeon bone peptide with the mass ratio of 3-10% in white spirit; the alcohol degree of the white spirit is between 40% Vol and 60% Vol. The preparation method comprises the following steps: (1) adding the hydrolyzed sturgeon bone peptide dry powder into the white spirit according to the formula proportion, and uniformly mixing to obtain a suspension; (2) soaking the suspension obtained in the step (1) for more than 30 days to obtain a leaching mixture; (3) and (3) pouring the leaching mixture obtained in the step (2) into a jar to obtain an upper clear solution, thus obtaining the sturgeon bone peptide wine. The sturgeon bone peptide wine provided by the invention can be applied to the preparation of health care products, medicines or foods for preventing, treating or assisting in treating kidney-yang deficiency.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to sturgeon bone peptide wine with effects of tonifying kidney and tonifying yang, and a preparation method and application thereof.
Background
The Chinese sturgeon is powerful in shape, large in size and long in service life, the longest-lived fish can reach 40 years old, and the Chinese sturgeon is the fish with the largest individual and the longest service life in freshwater fish. They are not only a group between cartilaginous fishes (sharks, etc.) and teleost fishes, but also a more primitive group of teleost fishes, and the fishbone thereof has several unique component structures.
Chinese sturgeon is a high-protein and fatty fish. The fish skin can be used for preparing leather, the roe can be used for preparing sauce, the fish gallbladder can be used as medicine, the fish meat, fish intestines, fish meat, fish bones and the like are all first-class delicacies, and the air bladder and notochord can be used for preparing fish glue. The protein content in muscle is 16.42-20.41%, and the fat content is 3.05-4.32%; the protein content in liver is 10.31-16.26%, and the fat content is 16.63-27.58%; the egg granules contain 24.90-29.70% of protein and 18.06-24.00% of fat. Both muscle and egg contain 17 common amino acids that can be measured. It contains 8 kinds of amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, threonine and valine in addition to tryptophan which is destroyed by acid hydrolysis); two semi-essential amino acids (arginine and histidine, both essential for infants); and 8 optional amino acids (alanine, aspartic acid, cystine, glutamic acid, glycine, proline, serine, histidine). The amino acids in muscle are glutamic acid, aspartic acid, lysine and leucine; the low content of amino acids include histidine, methionine and cystine; the high and low arrangement of the rest amino acid content are different. The muscle of the female individual contains 48.54% of essential and semi-essential amino acids, and the egg granules contain 48.80%; male muscle was 49%. The compendium of materia Medica records: its liver is mainly used for treating scabies, its flesh can tonify deficiency and replenish qi, it can be used for treating blood stranguria, its nose flesh can tonify deficiency and make qi downward, its seeds can be used for curing small insects in abdomen. "
The comprehensive utilization of Chinese sturgeons has wide prospects, and the application technology of the Chinese sturgeon fishbone is very limited at present.
Disclosure of Invention
Aiming at the defects or improvement requirements of the prior art, the invention provides sturgeon bone peptide wine with the effects of tonifying kidney and yang, a preparation method and application thereof, and aims to verify that the sturgeon bone peptide wine provided by the invention is prepared according to a unique process, can effectively improve the anti-fatigue capability and the cold resistance of a kidney-yang deficiency syndrome mouse, and anatomical experiments show that the weight and the weight of immune organs of the kidney-yang deficiency syndrome mouse of the sturgeon bone peptide wine provided by the invention are obviously increased, so that a health care product, a medicine or a food for preventing, treating or assisting in treating the kidney-yang deficiency syndrome is provided.
In order to achieve the above object, according to one aspect of the present invention, there is provided sturgeon bone peptide wine, which is a white wine in which 3% to 10% by mass of sturgeon bone peptide is dissolved; the alcohol degree of the white spirit is between 40% Vol and 60% Vol.
Preferably, the sturgeon bone peptide wine has the mass fraction of polypeptides with the sturgeon bone peptide molecular weight less than or equal to 250Da of more than 80%.
Preferably, the sturgeon bone peptide wine has the sturgeon bone peptide selenium content of 0.7mg/kg to 0.9mg/kg
Preferably, the sturgeon bone peptide wine is a pure brewed Maotai-flavor wine.
Preferably, the sturgeon bone peptide wine is prepared by dissolving sturgeon bone peptide with a mass ratio of 6% in the white wine; the alcohol degree of the white spirit is 53 percent Vol.
According to another aspect of the present invention, there is provided a method for preparing sturgeon bone peptide wine according to the present invention, which comprises the steps of:
(1) adding the hydrolyzed sturgeon bone peptide dry powder into the white spirit according to the formula proportion, and uniformly mixing to obtain a suspension;
(2) soaking the suspension obtained in the step (1) for more than 30 days to obtain a leaching mixture;
(3) and (3) pouring the leaching mixture obtained in the step (2) into a jar to obtain an upper clear solution, thus obtaining the sturgeon bone peptide wine.
Preferably, in the step (2), the suspension container is inverted every 10-12 days of standing, and the suspension container is inverted and mixed for more than 3 times to obtain an extraction mixture.
Preferably, the preparation method of the sturgeon bone peptide wine comprises the following steps:
(A1) crushing sturgeon fishbone raw materials, and sieving the crushed sturgeon fishbone raw materials with a sieve of 160-300 meshes to obtain fishbone powder;
(A2) performing first enzymolysis on the fishbone powder obtained in the step (A1), and degrading a fishbone collagen skeleton to obtain an enzymolysis crude product;
(A3) performing secondary enzymolysis on the enzymolysis crude product obtained in the step (A2) to obtain an enzymolysis refined product, so that the selenoglycoprotein is degraded into small molecular polypeptide; the method comprises the following specific steps:
adding alkali liquor into the enzymolysis crude product obtained in the step (A2) to adjust the pH value to 7.5-8.0, adding 3-5% by mass of polyethylene glycol and 0.5-1.0% by mass of mixed protease into the fishbone powder, carrying out enzymolysis for more than 2 hours, stirring once every 0.5-2 hours to maintain the system uniform, and monitoring the pH value to maintain the pH value between 7.5-8.0;
(A4) desalting the enzymolysis refined product obtained in the step (A3) to obtain a desalted enzymolysis refined product;
(A5) and (D) carrying out solid-liquid separation on the desalted enzymolysis refined product obtained in the step (A4), and then taking a liquid phase for drying to obtain the sturgeon bone peptide.
Preferably, in the preparation method of the sturgeon bone peptide wine, the mixed protease in the step (A3) is a mixture of subtilisin, pancreatin and bromelain in a mass ratio of 1: 1-2.5: 0.8-1.2, preferably 1:2.5: 1.
According to another aspect of the invention, the sturgeon bone peptide wine is provided, and the sturgeon bone peptide wine is applied to the preparation of health products, medicines or foods for preventing, treating or assisting in treating kidney-yang deficiency.
Generally, compared with the prior art, the technical scheme provided by the invention degrades the protein in the sturgeon fishbone into the small molecular peptide with good alcohol solubility by means of repeated hydrolysis, and verifies the treatment effect of the small molecular peptide on the kidney-yang deficiency syndrome, so that the health-care product, the medicine or the food for preventing, treating or assisting in treating the kidney-yang deficiency syndrome is provided.
Drawings
FIG. 1 is a record of the normal group weight test experiment provided in example 4;
FIG. 2 is the record of the kidney-yang deficiency syndrome model group weight test provided in example 4;
FIG. 3 is the weight test record of the Feitian couchgrass group provided in example 4;
FIG. 4 is a record of the sturgeon bone peptide wine group weight test provided in example 4;
FIG. 5 is a spleen dissection experimental record of sturgeon bone peptide wine group provided in example 4;
FIG. 6 is the thymus dissection experimental record of sturgeon bone peptide wine group provided in example 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention provides sturgeon bone peptide wine, which is prepared by dissolving sturgeon bone peptide with the mass ratio of 3-10% in white spirit; the alcohol degree of the white spirit is between 40% Vol and 60% Vol. Preferably, 6% of sturgeon bone peptide is dissolved in the white spirit; the alcohol degree of the white spirit is 53 percent Vol, and the Maotai-flavor white spirit brewed by pure grains is preferred.
The mass fraction of polypeptide with the sturgeon bone peptide molecular weight less than or equal to 250Da is more than 80%, and the selenium content is between 0.7mg/kg and 0.9 mg/kg; the preparation method comprises the following steps:
(A1) crushing sturgeon fishbone raw materials, and sieving the crushed sturgeon fishbone raw materials with a sieve of 160-300 meshes to obtain fishbone powder; experiments show that the crushing fineness of the fishbone raw material influences the overall effect of the extraction process, the polypeptide activity of the finally obtained product is poor due to the excessively fine fineness, the enzymolysis is incomplete due to the excessively coarse fineness, the dissolution rate and the absorption rate of the organic selenium and the molecular weight of the peptide after enzymolysis are large, and the overall nutritive value is reduced.
(A2) Performing first enzymolysis on the fishbone powder obtained in the step (A1), degrading a fishbone collagen skeleton, and exposing the selenoglycoprotein to obtain an enzymolysis crude product;
the method comprises the following specific steps:
adding water into the fishbone powder obtained in the step (A1) according to a solid-to-liquid ratio (mass ratio) of 1: 1-1: 2 to prepare a suspension, adding collagenase and cellulase for enzymolysis, wherein the mass of the collagenase is 0.3-0.5% of the mass of the fishbone powder, the mass of the cellulase is 0.1-0.4% of the mass of the fishbone powder, and the enzymolysis is carried out for 0.5-1.5 hours while continuously stirring and maintaining the temperature at 25-30 ℃; heating to above 90 deg.C, and maintaining for 30min to inactivate enzyme.
(A3) And (D) carrying out second enzymolysis on the enzymolysis crude product obtained in the step (A2) to obtain an enzymolysis refined product, so that the selenoglycoprotein is degraded into small molecular polypeptide, wherein the mass proportion of the polypeptide with the molecular weight of less than 250Da exceeds 80%, and the small molecular polypeptide becomes the selenium-enriched polypeptide, thereby improving the absorptivity of selenium element.
The method comprises the following specific steps:
adding alkali liquor into the enzymolysis crude product obtained in the step (A2) to adjust the pH value to 7.5-8.0, adding 3-5% by mass of polyethylene glycol and 0.5-1.0% by mass of mixed protease into the fishbone powder, carrying out enzymolysis for more than 2 hours, stirring once every 0.5-2 hours to maintain the system uniform, and monitoring the pH value to maintain the pH value between 7.5-8.0;
the mixed protease is a mixture of subtilisin, pancreatin and bromelain according to a mass ratio of 1: 1-2.5: 0.8-1.2, preferably 1:2.5: 1.
The selection, the proportion and the enzymolysis environment of the mixed protease determine the site and the efficiency of enzyme digestion, so that the activity of peptide fragments is different, and the enzyme digestion results of the mixed protease with different proportions on complex proteins in the fishbone are greatly different. Meanwhile, the enzyme digestion result also determines the absorption and utilization degree of the selenium element, and the anti-aging activity difference of different selenium-containing peptide fragments is obvious. Therefore, the selection of the mixed protease and the corresponding enzymolysis conditions are unpredictable, and even if the mixed protease is the same protease, the enzyme conformation is changed due to the difference of enzymolysis environments, including the difference of the pH value of an enzymolysis system and the polarity of a solvent, so that the specific enzyme cutting site is uncertain. According to the preferable scheme, the subtilisin, the pancreatin and the bromelain are mixed according to the mass ratio of 1: 1-2.5: 0.8-1.2 for mixed enzymolysis, the polyethylene glycol is added to change the polarity of the solution and the solid-liquid contact surface, and the content of polypeptide with the enzymolysis product being less than 243 daltons is more than 80% and has excellent activity.
(A4) Desalting the enzymolysis refined product obtained in the step (A3) to obtain a desalted enzymolysis refined product, preferably performing reverse osmosis desalting.
The reverse osmosis desalination treatment parameters are as follows: the pressure is 1MP to 1.5MP, the temperature is 25 ℃, the acetate fiber membrane has the salt rejection rate of 70 percent or more.
(A5) And (D) carrying out solid-liquid separation on the desalted enzymolysis refined product obtained in the step (A4), and then taking a liquid phase for drying to obtain the sturgeon bone peptide.
The preparation method of the sturgeon bone peptide wine provided by the invention comprises the following steps:
(1) adding the hydrolyzed sturgeon bone peptide dry powder into the white spirit according to the formula proportion, and uniformly mixing to obtain a suspension;
(2) inverting the suspension obtained in the step (1) in a suspension container every 10-12 days of standing, wherein the total soaking time is more than 30 days, and inverting and mixing for more than 3 times to obtain an extraction mixture; practice shows that in the process of inverting the container, the sturgeon bone peptide deposited at the bottom of the container slowly descends and is fully fused with white spirit, compared with stirring, the sturgeon bone peptide is accelerated, the efficiency is higher, and sensory evaluation shows that the stability and the quality of a finished product are superior to those of a finished product dissolved by adding the sturgeon bone peptide by stirring.
(3) And (3) pouring the leaching mixture obtained in the step (2) into a jar to obtain an upper clear solution, thus obtaining the sturgeon bone peptide wine.
Experiments prove that the sturgeon bone peptide wine provided by the invention has the obvious effects of preventing and treating animal weight loss, aversion to cold, autonomous activity reduction and immune organ atrophy caused by kidney-yang deficiency. Compared with the liquor used as the base liquor alone, the sturgeon bone peptide liquor provided by the invention shows obviously better kidney-tonifying and yang-reinforcing effects on a kidney-yang deficiency model control group and a base liquor gavage control group in a mouse gavage experiment. In conclusion, the sturgeon bone peptide wine provided by the invention can be used for preparing health-care products, medicines or foods for preventing, treating and assisting in treating kidney-yang deficiency.
The following are examples:
example 1
Sturgeon bone peptide wine is prepared by dissolving sturgeon bone peptide with mass ratio of 3% in Chinese liquor; the alcohol degree of the white spirit is 40% Vol. Preferably, sturgeon bone peptide with a mass ratio is dissolved in the white spirit; the liquor is Maotai-flavor liquor (base liquor 1) brewed from pure grains.
The mass fraction of the polypeptide with the sturgeon bone peptide molecular weight less than or equal to 243Da is 87.27%, and the selenium content is 0.71 mg/kg; the preparation method comprises the following steps:
(A1) crushing sturgeon fishbone raw materials, and sieving the crushed sturgeon fishbone raw materials with a 160-mesh sieve to obtain fishbone powder;
(A2) performing first enzymolysis on the fishbone powder obtained in the step (A1), degrading a fishbone collagen skeleton, and exposing the selenoglycoprotein to obtain an enzymolysis crude product;
the method comprises the following specific steps:
adding water into the fishbone powder obtained in the step (A1) according to the solid-to-liquid ratio (mass ratio) of 1:1 to prepare a suspension, adding collagenase and cellulase for enzymolysis, wherein the mass of the collagenase is 0.5% of the mass of the fishbone powder, the mass of the cellulase is 0.4% of the mass of the fishbone powder, and performing enzymolysis for 1.5 hours while continuously stirring and maintaining the temperature at 25-30 ℃; heating to above 90 deg.C, and maintaining for 30min to inactivate enzyme.
(A3) And (D) carrying out second enzymolysis on the enzymolysis crude product obtained in the step (A2) to obtain an enzymolysis refined product, so that the selenoglycoprotein is degraded into small molecular polypeptide, wherein the mass proportion of the polypeptide with the molecular weight of less than 250Da exceeds 80%, and the small molecular polypeptide becomes the selenium-enriched polypeptide, thereby improving the absorptivity of selenium element.
The method comprises the following specific steps:
adding alkali liquor into the enzymolysis crude product obtained in the step (A2) to adjust the pH value to 7.5-8.0, adding 3% by mass of polyethylene glycol 4000, adding 0.5% by mass of mixed protease of fishbone powder, performing enzymolysis for 3 hours, stirring every 1 hour during the enzymolysis to maintain the system uniform, and monitoring the pH value to maintain the pH value between 7.5-8.0;
the mixed protease is subtilisin, pancreatin and bromelain according to the mass ratio of 1:2.5: 1.
(A4) And (D) performing reverse osmosis desalination treatment on the enzymolysis refined product obtained in the step (A3) to obtain a desalted enzymolysis refined product.
The reverse osmosis desalination treatment parameters are as follows: pressure 1MP, temperature 25 ℃, salt rejection rate 70%, cellulose acetate membrane.
(A5) And (D) carrying out solid-liquid separation on the desalted enzymolysis refined product obtained in the step (A4), and then taking a liquid phase for drying to obtain the sturgeon bone peptide.
The extracted product of the selenium-rich sturgeon bone peptide prepared by the embodiment is detected according to the national standard GB 5009.93-2017, and the selenium content is 0.71 mg/kg. The results of testing the polypeptide content and molecular weight distribution by the research institute of light-analysis chemical technology in Beijing central office (food laboratory) were as follows, and the polypeptide content with a molecular weight of less than 243 was 87.27%:
| detecting items | The result of the detection | Unit of |
| Total amount of peptide | 89.17 | ％ |
| Molecular weight distribution | 242 | Da |
| Molecular weight distribution (Da) | Percent (%) |
| ＞2300 | 0.0 |
| 2300-1200 | 0.42 |
| 1199-580 | 5.62 |
| 579-242 | 6.69 |
| ＜243 | 87.27 |
The preparation method of the sturgeon bone peptide wine provided by the invention comprises the following steps:
(1) adding the hydrolyzed sturgeon bone peptide dry powder into the white spirit according to the formula proportion, and uniformly mixing to obtain a suspension;
(2) inverting the suspension obtained in the step (1) in a suspension container every 10 days of standing, and inverting and mixing for 3 times to obtain a leaching mixture, wherein the total soaking time is 30 days;
(3) and (3) pouring the leaching mixture obtained in the step (2) into a jar to obtain an upper clear solution, thus obtaining the sturgeon bone peptide wine.
Example 2
Sturgeon bone peptide wine is prepared by dissolving sturgeon bone peptide 6 wt% in Chinese liquor; the alcohol degree of the white spirit is 53% Vol. Preferably, sturgeon bone peptide with a mass ratio is dissolved in the white spirit; the liquor is a Maotai-flavor liquor brewed from pure grains, and the purchased Feitian Maotai liquor is used as base liquor.
The mass fraction of the polypeptide with the sturgeon bone peptide molecular weight less than or equal to 243Da is 81.03%, and the selenium content is 0.77 mg/kg; the preparation method comprises the following steps:
(A1) crushing sturgeon fishbone raw materials, and sieving the crushed sturgeon fishbone raw materials with a 200-mesh sieve to obtain fishbone powder;
(A2) performing first enzymolysis on the fishbone powder obtained in the step (A1), degrading a fishbone collagen skeleton, and exposing the selenoglycoprotein to obtain an enzymolysis crude product;
the method comprises the following specific steps:
adding water into the fishbone powder obtained in the step (A1) according to a solid-to-liquid ratio (mass ratio) of 1:1.5 to prepare a suspension, adding collagenase and cellulase for enzymolysis, wherein the mass of the collagenase is 0.4% of the mass of the fishbone powder, the mass of the cellulase is 0.1% of the mass of the fishbone powder, and performing enzymolysis for 1 hour while continuously stirring and maintaining the temperature at 25-30 ℃; heating to above 90 deg.C, and maintaining for 30min to inactivate enzyme.
(A3) And (D) carrying out second enzymolysis on the enzymolysis crude product obtained in the step (A2) to obtain an enzymolysis refined product, so that the selenoglycoprotein is degraded into small molecular polypeptide, wherein the mass proportion of the polypeptide with the molecular weight of less than 250Da exceeds 80%, and the small molecular polypeptide becomes the selenium-enriched polypeptide, thereby improving the absorptivity of selenium element.
The method comprises the following specific steps:
adding alkali liquor into the enzymolysis crude product obtained in the step (A2) to adjust the pH value to 7.5-8.0, adding polyethylene glycol 4000 with the mass fraction of 4%, adding mixed protease with the mass of 0.8% of the fishbone powder, performing enzymolysis for 2 hours, stirring every 0.5 hour during the enzymolysis to maintain the system uniform, and monitoring the pH value to maintain the pH value between 7.5-8.0;
the mixed protease is subtilisin, pancreatin and bromelain according to the mass ratio of 1:2: 0.8.
(A4) And (D) performing reverse osmosis desalination treatment on the enzymolysis refined product obtained in the step (A3) to obtain a desalted enzymolysis refined product.
The reverse osmosis desalination treatment parameters are as follows: pressure 1MP, temperature 25 ℃, salt rejection rate 70%, cellulose acetate membrane.
(A5) And (D) carrying out solid-liquid separation on the desalted enzymolysis refined product obtained in the step (A4), and then taking a liquid phase for drying to obtain the sturgeon bone peptide.
The extracted product of the selenium-rich sturgeon bone peptide prepared by the embodiment is detected according to national standard GB 5009.93-2017, and the selenium content is 0.77 mg/kg. The results of testing the polypeptide content and molecular weight distribution by the research institute of the light-analysis chemical technology in Beijing central science (food laboratory) are as follows, and the polypeptide content with the molecular weight less than 243 is 81.03 percent:
| detecting items | The result of the detection | Unit of |
| Total amount of peptide | 90.20 | ％ |
| Molecular weight distribution | 312 | Da |
| Molecular weight distribution (Da) | Percent (%) |
| ＞2300 | 0.0 |
| 2300-1200 | 1.37 |
| 1199-580 | 7.51 |
| 579-242 | 10.09 |
| ＜243 | 81.03 |
The preparation method of the sturgeon bone peptide wine provided by the embodiment comprises the following steps:
(1) adding the hydrolyzed sturgeon bone peptide dry powder into the white spirit according to the formula proportion, and uniformly mixing to obtain a suspension;
(2) inverting the suspension obtained in the step (1) in a suspension container every 11 days of standing, and inverting and mixing for 3 times to obtain a leaching mixture, wherein the total soaking time is 40 days;
(3) and (3) pouring the leaching mixture obtained in the step (2) into a jar to obtain an upper clear solution, thus obtaining the sturgeon bone peptide wine.
Example 3
A sturgeon bone peptide wine is prepared by dissolving sturgeon bone peptide 10 wt% in Chinese liquor; the alcohol degree of the white spirit is 60% Vol. Preferably, sturgeon bone peptide with a mass ratio is dissolved in the white spirit; the liquor is Maotai-flavor liquor (base liquor 2) brewed from pure grains.
The mass fraction of the polypeptide with the sturgeon bone peptide molecular weight less than or equal to 243Da is 80.73%, and the selenium content is 0.80 mg/kg; the preparation method comprises the following steps:
(A1) crushing sturgeon fishbone raw materials, and sieving the crushed sturgeon fishbone raw materials with a 300-mesh sieve to obtain fishbone powder;
(A2) performing first enzymolysis on the fishbone powder obtained in the step (A1), degrading a fishbone collagen skeleton, and exposing the selenoglycoprotein to obtain an enzymolysis crude product;
the method comprises the following specific steps:
adding water into the fishbone powder obtained in the step (A1) according to a solid-to-liquid ratio (mass ratio) of 1:2 to prepare a suspension, adding collagenase and cellulase for enzymolysis, wherein the mass of the collagenase is 0.3% of the mass of the fishbone powder, the mass of the cellulase is 0.1% of the mass of the fishbone powder, and performing enzymolysis for 1.5 hours while continuously stirring and maintaining the temperature at 25-30 ℃; heating to above 90 deg.C, and maintaining for 30min to inactivate enzyme.
(A3) And (D) carrying out secondary enzymolysis on the enzymolysis crude product obtained in the step (A2) to obtain an enzymolysis refined product, so that the selenoglycoprotein is degraded into small molecules to become selenium-rich polypeptide, and the absorption rate of selenium element is improved.
The method comprises the following specific steps:
adding alkali liquor into the enzymolysis crude product obtained in the step (A2) to adjust the pH value to 7.5-8.0, adding polyethylene glycol 4000 with the mass fraction of 5%, adding mixed protease with the mass of 1.0% of the fishbone powder, performing enzymolysis for 2 hours, stirring every 1 hour during the enzymolysis to maintain the system uniform, and monitoring the pH value to maintain the pH value between 7.5-8.0;
the mixed protease is subtilisin, pancreatin and bromelain according to the mass ratio of 1:1: 1.2.
(A4) And (D) performing reverse osmosis desalination treatment on the enzymolysis refined product obtained in the step (A3) to obtain a desalted enzymolysis refined product.
The reverse osmosis desalination treatment parameters are as follows: pressure 1MP, temperature 25 ℃, salt rejection rate 70%, cellulose acetate membrane.
(A5) And (D) carrying out solid-liquid separation on the desalted enzymolysis refined product obtained in the step (A4), and then taking a liquid phase for drying to obtain the sturgeon bone peptide.
The extracted product of the selenium-rich sturgeon bone peptide prepared by the embodiment is detected according to the national standard GB 5009.93-2017, and the selenium content is 0.80 mg/kg. The results of testing the polypeptide content and molecular weight distribution by the research institute of light-analysis chemical technology in Beijing central office (food laboratory) were as follows, and the polypeptide content with a molecular weight of less than 243 was 80.73%:
the preparation method of the sturgeon bone peptide wine provided by the embodiment comprises the following steps:
(1) adding the hydrolyzed sturgeon bone peptide dry powder into the white spirit according to the formula proportion, and uniformly mixing to obtain a suspension;
(2) inverting the suspension obtained in the step (1) in a suspension container every 12 days of standing, and inverting and mixing for 3 times to obtain a leaching mixture, wherein the total soaking time is 40 days;
(3) and (3) pouring the leaching mixture obtained in the step (2) into a jar to obtain an upper clear solution, thus obtaining the sturgeon bone peptide wine.
Example 4
Verification of effects of sturgeon bone peptide prepared in examples 1 to 3 on kidney-tonifying and yang-reinforcing for a long time
Test objects: sturgeon bone peptide wine prepared in examples 1 to 3; reference base liquor 1, base liquor 2, commercially available Feitianfu liquor; the testing process comprises the following steps: mouse modeling experiment:
1 experimental materials:
1.1, the mice are bred by SPF-level Kunming, the breeding environment is a laboratory animal room of 5 th floor in the center of stem cells of Guangzhou city, the temperature is 21-22 ℃, the humidity is 55 +/-5%, the bright/dark cycle is carried out every 12h, and the feed and the water are not limited.
1.2 sturgeon bone peptide wine prepared in examples 1 to 3 was provided by Guizhou Gudelenin wine industry Co.
1.3 base liquor 1, base liquor 2, commercially available Feitianfu Maotai (Maotai flavor type 53 degree)
2 methods and results
2.1 influence of sturgeon bone peptide wine on mouse kidney yang deficiency caused by hydrocortisone
2.1.1 Effect on the anti-fatigue ability of Experimental Kidney Yang deficiency mice
Every 10 male mice of Kunming species are used as a group, one test object is tested, the age of the mice is 10-12 months, and the weight of the mice is 50g +/-3 g:
(1) in the normal control group, distilled water is administered by intragastric administration;
(2) in the model group of yang deficiency syndrome, 25mg/kg of hydrocortisone is injected intramuscularly, and distilled water is given by intragastric administration;
(3) the control group of the base liquor is injected with hydrocortisone with the same quantity of 25 mg/kg. Simultaneously, 100 mu L of base wine 1, base wine 2 and Feitianfu Maotai (Maotai flavor type 53 degrees) are added in the stomach;
(4) the sturgeon bone peptide wine group is injected with hydrocortisone with the same dose of 25 mg/kg. Simultaneously gavage 100 μ L of sturgeon bone peptide wine prepared in examples 1 to 3;
the test groups (1) to (4) were gavaged 1 time/day each day for 6 consecutive days, and the test groups (2) to (4) were given intramuscular injection of hydrocortisone for 5 consecutive days.
3. Results of the experiment
3.1 weight test
The weights of the animals in each group were recorded as shown in the following table:
the experimental record of the normal group is shown in fig. 1, the experimental record of the kidney-yang deficiency model group is shown in fig. 2, the experimental record of the flying couchgrass platform group is shown in fig. 3, and the experimental record of the sturgeon bone peptide wine of the example 2 is shown in fig. 4.
Experiments show that the sturgeon bone peptide wine has obvious weight increasing effect on weight loss caused by kidney-yang deficiency, and the base wine 1, the base wine 2 and the market-sold Feitian Maotai cannot obviously increase the weight of a mouse with the kidney-yang deficiency.
3.2 fatigue resistance test
Half an hour after the last gastric lavage, the mice were placed in a swimming box (40cm × 40cm × 40cm, water depth of 30cm, water temperature of 28 ± 1 ℃) for swimming, and the duration of swimming of the mice was recorded (the mice were provided with a lead patch at the tail part according to 10% of the body weight during swimming, and the mice did not float after the head part was completely submerged below the water surface from the beginning of swimming as the end point of the experiment).
The duration of swimming (' x +/-smin) of the mice of the normal control group, the yang deficiency syndrome model group, the base liquor control group and the sturgeon bone peptide liquor group is calculated as follows:
| group of | Duration of swimming |
| Normal control group | 8.14±1.32 |
| Model group of yang deficiency syndrome | 1.53±0.56 |
| |
1.48±0.33 |
| |
1.52±0.68 |
| Flying cogongrass platform | 1.87±0.75 |
| Example 1 | 3.88±0.85 |
| Example 2 | 4.86±1.13 |
| Example 3 | 3.61±1.13 |
Through statistical treatment, compared with a normal control group, the anti-fatigue capability of mice in a kidney-yang deficiency syndrome model group given hydrocortisone is remarkably reduced; the intragastric base liquor 1 and the base liquor 2 of the mice of the kidney-yang deficiency model group and the commercially available Feitian Maotai have no obvious difference compared with the mice of the kidney-yang deficiency model group; the sturgeon bone peptide wine prepared in the gastric perfusion examples 1 to 3 can effectively enhance the fatigue resistance of mice with yang deficiency syndrome, and compared with a model group with yang deficiency syndrome, the sturgeon bone peptide wine group has very obvious difference, wherein the sturgeon bone peptide wine prepared in the example 2 is best modified, and has more obvious fatigue enhancement effect compared with the sturgeon bone peptide wine prepared in the examples 1 and 3.
3.3 Cold resistance test:
after 30min of the last administration, each group of mice was placed in a refrigerator at-18 ℃, and the number of deaths of the animals was observed after 60min, and the experimental results are shown in the following table:
influence of sturgeon bone peptide wine on cold resistance of mice with syndrome of kidney yang deficiency
X2The test was △ P < 0.05 compared to the normal control group.
It can be seen from the above table that, in the model of kidney-yang deficiency caused by hydrocortisone, the cold tolerance of the mice is obviously reduced, the cold tolerance of the mice with kidney-yang deficiency cannot be improved by the intragastric base liquor (including Feitian Maotai), and the cold tolerance of the mice with kidney-yang deficiency can be improved after the sturgeon bone peptide liquor is taken. Warp X2The test shows that the sturgeon bone peptide wine group has significant difference with the kidney-yang deficiency model group.
3.4 Effect on the weight of the immune organs of mice with syndrome of Experimental Kidney Yang deficiency
40 Kunming male mice with the age of 10-12 months and the weight of 50g +/-3 g are grouped and administered as above. After giving the wine half an hour on day 5, the spleen and thymus of the mice were then picked and weighed, and thymus (mg)/body weight (10g) and spleen (mg)/body weight (10g) were calculated and t-test was performed between groups. The results are detailed in the following table:
influence of sturgeon bone peptide wine on weight of immune organs of experimental kidney-yang deficiency mice
In the sturgeon bone peptide wine gavage experiment of example 2, the spleen dissection process is shown in fig. 5, and the thymus weighing process is shown in fig. 6.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. Sturgeon bone peptide wine is characterized in that sturgeon bone peptide with the mass ratio of 3% to 10% is dissolved in white wine; the alcohol degree of the white spirit is between 40% Vol and 60% Vol.
2. The sturgeon bone peptide wine according to claim 1, wherein the mass fraction of polypeptides with molecular weight of less than or equal to 250Da is more than 80%.
3. Sturgeon bone peptide wine according to claim 1, characterised in that the sturgeon bone peptide selenium content is between 0.7mg/kg and 0.9 mg/kg.
4. The sturgeon bone peptide wine according to claim 1, characterized in that the white wine is a pure brewed Maotai-flavor white wine.
5. The sturgeon bone peptide wine according to any one of claims 1 to 4, characterized in that 6% by mass of sturgeon bone peptide is dissolved in the white wine; the alcohol degree of the white spirit is 53 percent Vol.
6. A method of preparing sturgeon bone peptide wine according to any one of claims 1 to 5, characterized by comprising the steps of:
(1) adding the hydrolyzed sturgeon bone peptide dry powder into the white spirit according to the formula proportion, and uniformly mixing to obtain a suspension;
(2) soaking the suspension obtained in the step (1) for more than 30 days to obtain a leaching mixture;
(3) and (3) pouring the leaching mixture obtained in the step (2) into a jar to obtain an upper clear solution, thus obtaining the sturgeon bone peptide wine.
7. The preparation method of the sturgeon bone peptide wine according to claim 6, wherein in the step (2), the suspension container is inverted every 10-12 days of standing, and the mixture is inverted and mixed for more than 3 times to obtain an extraction mixture.
8. The method for preparing sturgeon bone peptide wine according to claim 6, wherein the sturgeon bone peptide is prepared according to the following method:
(A1) crushing sturgeon fishbone raw materials, and sieving the crushed sturgeon fishbone raw materials with a sieve of 160-300 meshes to obtain fishbone powder;
(A2) performing first enzymolysis on the fishbone powder obtained in the step (A1), and degrading a fishbone collagen skeleton to obtain an enzymolysis crude product;
(A3) performing secondary enzymolysis on the enzymolysis crude product obtained in the step (A2) to obtain an enzymolysis refined product, so that the selenoglycoprotein is degraded into small molecular polypeptide; the method comprises the following specific steps:
adding alkali liquor into the enzymolysis crude product obtained in the step (A2) to adjust the pH value to 7.5-8.0, adding 3-5% by mass of polyethylene glycol and 0.5-1.0% by mass of mixed protease into the fishbone powder, carrying out enzymolysis for more than 2 hours, stirring once every 0.5-2 hours to maintain the system uniform, and monitoring the pH value to maintain the pH value between 7.5-8.0;
(A4) desalting the enzymolysis refined product obtained in the step (A3) to obtain a desalted enzymolysis refined product;
(A5) and (D) carrying out solid-liquid separation on the desalted enzymolysis refined product obtained in the step (A4), and then taking a liquid phase for drying to obtain the sturgeon bone peptide.
9. The method for preparing sturgeon bone peptide wine according to claim 8, wherein the mixed protease in the step (A3) is a mixture of subtilisin, pancreatin and bromelain in a mass ratio of 1: 1-2.5: 0.8-1.2, preferably 1:2.5: 1.
10. Use of sturgeon bone peptide wine according to any one of claims 1 to 5 for the preparation of a health product, a pharmaceutical product or a food product for the prevention, treatment or adjuvant treatment of kidney-yang deficiency.
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| CN113150937A (en) * | 2021-04-14 | 2021-07-23 | 嫦娥创新（武汉）生物科技有限公司 | White spirit curing process for reducing liver damage of high-alcohol white spirit |
| CN115093920A (en) * | 2022-07-13 | 2022-09-23 | 贵州古得宁酒业有限公司 | Preparation of sturgeon bone peptide Maotai-flavor liquor capable of improving human immunity and resisting aging |
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