CN109793885B - Preparation method of composite polypeptide for preventing or relieving anemia - Google Patents
Preparation method of composite polypeptide for preventing or relieving anemia Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of a composite polypeptide for preventing or relieving anemia, which comprises the following steps of (1) carrying out ultrasonic crushing on a plurality of medicinal and edible plants with the function of activating blood to form plant crushing liquid, (2) adding cellulase into the plant crushing liquid for enzymolysis to obtain plant enzymolysis liquid, (3) adding plant complex enzyme into the plant enzymolysis liquid for enzymolysis to obtain micromolecule polysaccharide plant liquid, (4) adding hemoglobin powder into the micromolecule polysaccharide plant liquid, carrying out enzymolysis on the plant enzymolysis liquid to obtain micromolecule polypeptide primary liquid, (5) filtering the micromolecule polypeptide primary liquid by using a plate and frame filter to obtain polypeptide fine liquid, (6) adding auxiliary materials into the polypeptide fine liquid to obtain the composite polypeptide oral liquid or the composite polypeptide agent. The method changes the preparation method of simply adding finished hemepeptide after production and medicinal raw materials in the prior art, the composite polypeptide prepared by the method can effectively improve red blood cells, has short onset time for relieving anemia, and plays a role in preventing or relieving anemia.
Description
Technical Field
The invention relates to the field of health care products, in particular to a preparation method of a composite polypeptide for preventing or relieving anemia.
Background
Anemia is a clinical syndrome of lower than normal peripheral red blood cell volume due to various causes. The amount of hemoglobin in a volume of circulating blood by red blood cell count and the hematocrit are both below normal standards. Among them, hemoglobin is most important. According to the statistics of the world health organization: about 30 million people worldwide have different degrees of anemia. Mild anemia in adults has little effect on human bodies, but severe anemia can cause long-term blood supply insufficiency of tissues and organs, affect the functions of the tissues and the organs and possibly cause serious consequences. The number of deaths caused by anemia and various diseases is tens of millions each year. Of which about 50% of anemic patients are due to iron deficiency. Iron Deficiency Anemia (IDA), which is a kind of microcytic hypopigmentia caused by insufficient iron storage in vivo and affecting hemoglobin synthesis, is ubiquitous throughout the world, and WHO's data indicate that 30% of the global population, especially children (especially infants) and pregnant women, are affected by 50% and 40% of the total. The fourth nutrition survey in China shows that the prevalence rate of iron deficiency anemia in Chinese residents is 20.1%, wherein the prevalence rates of iron deficiency anemia in infants younger than 2 years old, elderly older than 60 years old and women of 15-50 years old are 31.1%, 29.1% and 19.9%, respectively, and the iron deficiency anemia becomes one of the most common nutritional deficiency diseases in China. Therefore, the method has important significance for the research of the prevention and treatment of the iron-deficiency anemia.
According to the research, the blood peptide can improve the red blood cell number and the hemoglobin content and has the effects of preventing and treating iron deficiency anemia. Blood peptides are mainly obtained by degrading the macromolecules of hemoglobin. Hemoglobin is a protein responsible for oxygen transport in higher organisms (Hb/HGB). Hemoglobin (Hb) is formed by the binding of globin and heme. The red color of blood is due to the presence of heme in the blood. Hemoglobin is rich in source, and the existing technology is mainly used for researching how to extract polypeptide from livestock blood, such as:
patent CN201210516085 discloses a process method for producing hemepeptide element by using pig blood, which removes impurities in the pig blood through deep filtration, and improves the purity of the hemepeptide element which is a final product.
Patent CN2012103571509 discloses a method for extracting active peptide with specific molecular weight from duck blood, which is implemented by intercepting specific active peptide through reverse osmosis ultrafiltration fractional purification, and mainly uses single-stage enzyme degradation.
Patent CN2011102863613 discloses a method for improving the yield of red blood cells of enzymolysis poultry and livestock, and special enzyme 1122 is used for single-stage degradation.
Patent CN2012103573557 discloses a process for producing small molecule hemepeptide heme iron and plasma protein by using duck blood as raw material, which mainly aims at extracting small molecule hemepeptide, heme and plasma protein through duck blood, firstly degrading by alkaline protease in the process of enzyme degradation, and then using acetic acid, ethanol and clean water for washing and purifying in the treatment process of degradation by neutral protease and flavourzyme.
The effect of the polypeptide extracted by the method is not obvious when being used alone, because the blood-enriching agent is not only required to be absorbed by a human body, but also has important effect on improving anemia. The prior art also discloses a product prepared by mixing the hemepeptide with medicinal raw materials, for example, the patent CN99125047.8 discloses a process for extracting the hemepeptide from livestock, the processing steps comprise blood collection, DC separation, primary conversion, secondary conversion, hemepeptide raw powder, preparation and product process, the cellular state is biodegraded into substances such as peptide, amino acid and the like by endopeptidase and exopeptidase to obtain the hemepeptide raw powder, and the hemepeptide raw powder is reddish brown, has slight bitter taste although no peculiar smell, and influences the taste and sense of the product. The product disclosed in this patent is a degraded hemepeptide and then formulated with a pharmaceutical material. But the final product may not taste good because of the preparation process. The prepared hemepeptide and medicinal raw materials are simply prepared, so that the active ingredients of the hemepeptide and the medicinal raw materials cannot exert more effective effects.
Disclosure of Invention
In order to achieve the above purpose, the present invention provides a method for preparing a composite polypeptide for preventing or relieving anemia, which changes the prior art of simply adding finished hemepeptide after production and medicinal raw materials, the composite polypeptide prepared by the method can effectively improve red blood cells, and the composite polypeptide has a short onset time for relieving anemia, and has the effect of preventing or relieving anemia.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a composite polypeptide for preventing or relieving anemia comprises the following preparation steps:
(1) mixing 1 part by weight of various medicinal and edible plants with the blood activating function, adding 5-15 parts by weight of water, carrying out ultrasonic disruption extraction for 2-10min under the condition of 1600-2500HZ, then cooling to 35-45 ℃, carrying out ultrasonic disruption again to achieve cell disruption and cytoplasm dissolution to form plant disruption solution;
(2) adding water into the plant crushing liquid until the solid-liquid ratio is 1:10-20, keeping the temperature to 55 ℃, adding cellulase for enzymolysis for 1-4h, and carrying out enzymolysis on plant cell walls to obtain plant enzymolysis liquid;
(3) adding plant complex enzyme into the plant enzymolysis liquid for enzymolysis for 4-10h, keeping constant temperature, boiling the solution for 5min, and inactivating enzyme to obtain micromolecular polysaccharide plant liquid;
(4) adding hemoglobin powder and deer placenta powder into small molecular polysaccharide plant liquid, adding water to 10-20 times of solid, mixing to obtain composite protein liquid, adding protease into the composite protein liquid, and performing enzymolysis to obtain small molecular polypeptide primary liquid;
(5) cooling the primary small molecular polypeptide solution to 45-65 ℃, filtering the primary small molecular polypeptide solution by using a plate and frame filter, and removing filter residues to obtain a fine polypeptide solution;
(6) adding adjuvants into the fine polypeptide filtrate, blending, sterilizing, bottling, sealing, and sterilizing at 121 deg.C under 115 deg.C to obtain composite polypeptide oral liquid or directly spray drying to obtain composite polypeptide agent.
The invention discloses a method for preparing composite polypeptide, which comprises the steps of degrading medicinal and edible plants into micromolecular polysaccharide plant liquid, adding hemoglobin powder and deer placenta powder prepared in advance, simultaneously degrading the hemoglobin powder and the deer placenta powder through enzymolysis to generate micromolecular active substances, filtering the micromolecular active substances together with the micromolecular polysaccharide plant liquid, controlling the molecular weight of all components, and ensuring that the active components can be combined with each other.
Compared with the prior art, the invention has the following beneficial effects:
1. the composite polypeptide consists of three parts: the deer placenta powder promotes blood circulation, increases absorption of organisms, and the micromolecular polysaccharide plant liquid supplements qi and blood, can realize multidirectional matching, prevents anemia, and can quickly relieve anemia symptoms.
2. The hemoglobin powder and the deer placenta powder are subjected to enzymatic degradation simultaneously, so that reaction steps are reduced, degradation effects are unified, molecular weight is controlled in a similar range after filtration, the hemoglobin powder and the deer placenta powder are easy to absorb by a human body, and effects of each other are enhanced.
Detailed Description
The present invention will be described in further detail below by way of specific embodiments:
a preparation method of a composite polypeptide for preventing or relieving anemia comprises the following preparation steps:
(1) mixing 1 part by weight of various medicinal and edible plants with the blood activating function, adding 5-15 parts by weight of water, carrying out ultrasonic disruption extraction for 2-10min under the conditions of 1600-2500HZ, then cooling to 35-45 ℃, carrying out ultrasonic disruption again to achieve cell disruption and cytoplasm dissolution to form plant disruption solution;
(2) adding water into the plant crushing liquid until the solid-liquid ratio is 1:10-20, keeping the temperature to 55 ℃, adding cellulase for enzymolysis for 1-4h, and carrying out enzymolysis on plant cell walls to obtain plant enzymolysis liquid;
(3) adding plant complex enzyme into the plant enzymolysis liquid for enzymolysis for 4-10h, keeping constant temperature, boiling the solution for 5min, and inactivating enzyme to obtain micromolecular polysaccharide plant liquid;
(4) adding hemoglobin powder and deer placenta powder into small molecular polysaccharide plant liquid, adding water to 10-20 times of solid, mixing to obtain composite protein liquid, adding protease into the composite protein liquid, and performing enzymolysis to obtain small molecular polypeptide primary liquid;
(5) cooling the primary small molecular polypeptide solution to 45-65 ℃, filtering the primary small molecular polypeptide solution by using a plate and frame filter, and removing filter residues to obtain a fine polypeptide solution;
(6) adding adjuvants into the fine polypeptide filtrate, blending, sterilizing, bottling, sealing, and sterilizing at 121 deg.C under 115 deg.C to obtain composite polypeptide oral liquid or directly spray drying to obtain composite polypeptide agent.
The various medicinal and edible plants with the function of promoting blood circulation in the step (1) comprise the following components in percentage by dry weight: 10 to 30 percent of angelica, 30 to 50 percent of red date, 20 to 50 percent of medlar and 10 to 30 percent of rhizoma polygonati.
The weight of the cellulase added in the step (2) is 2-7% calculated according to the dry weight of the medicinal and edible plants.
The weight of the plant complex enzyme added in the step (3) is 5-10% according to the dry weight of the medicinal and edible plants.
The weight of the added hemoglobin and the deer placenta powder in the step (4) is 1-5 times of the weight of the medicinal and edible plant according to the dry weight of the medicinal and edible plant, wherein the weight ratio of the hemoglobin powder to the deer placenta powder is 5-10: 1.
the enzymolysis in the step (4) comprises the following specific steps: adding NaOH into the composite protein solution, adjusting the pH value to 9.0, placing the solution in a constant temperature environment of 60 ℃, adding 0.1-0.5% of alkaline protease calculated by dry matters, maintaining for 2-6h, adjusting the pH value of the composite protein solution subjected to alkaline protease enzymolysis to 7.0, adding 0.03-0.1% of flavourzyme calculated by dry matters in a constant temperature environment of 50 ℃, performing enzymolysis for 0.5-1 h, boiling, maintaining for 2-5min, and inactivating enzyme to obtain the required primary micromolecule polypeptide solution.
The filtration comprises the following specific steps: and (3) filtering the primary filtrate of the composite polypeptide by an ultrafiltration membrane, wherein the interception amount of membrane molecules is 1-5 ten thousand Da, and treating by using a 1-2KDa membrane after the treatment is finished.
The auxiliary materials comprise the following components in percentage by volume of the polypeptide fine filtrate: 0.11g/L taurine, 2-10g/L high fructose corn syrup and 0.05-0.1g/L mint.
The auxiliary materials are calculated according to the volume of the polypeptide fine filtrate, and the auxiliary materials further comprise: 0.1 to 1 percent of mogroside and 0.1 to 0.8 thousandth of edible essence.
Deer placenta: the deer placenta and deer fetus have the efficacies of tonifying kidney and yang, and tonifying deficiency and producing sperm in traditional Chinese medicine, and are also commonly used as tonics in folk. Modern researches show that the placenta contains protein, gonadotropin, prolactin, lysozyme, polysaccharide, urokinase, various mineral elements, vitamins and the like, has various functions of promoting growth, promoting lactation, improving immunity and the like, has good regulating effect on female ovaries, and is an ideal immunomodulator and a nutritional health-care product. The small molecular polypeptide is formed after the enzymolysis, female hormone is regulated, ovary maintenance is facilitated, anemia can be promoted to be relieved, and the effect on female anemia is good.
Chinese angelica root: enriching the blood; promoting blood circulation; regulating menstruation and relieving pain; moistening dryness and smoothing intestine. The main symptoms are blood deficiency; menoxenia; amenorrhea; dysmenorrhea; the symptoms are accumulated; (ii) metrorrhagia and metrostaxis; abdominal pain due to deficiency-cold; flaccidity and paralysis; numbness of the skin; intestinal dryness and difficult defecation; severe dysentery with diarrhea; carbuncle, cellulitis, sore and ulcer; injury from falling.
Red dates: the red dates are sweet in taste and warm in nature, have the functions of tonifying middle-jiao and Qi, nourishing blood and tranquilization and moderating drug properties, and have the spleen and stomach channels. Modern pharmacological research finds that the red dates can increase the oxygen content in blood and nourish cells of the whole body, and are a tonic with mild medicinal effect.
Medlar: nourish liver, nourish kidney, moisten lung. Wolfberry leaf: tonify deficiency and replenish vital essence, clear heat and improve eyesight.
Rhizoma polygonati: invigorating qi, nourishing yin, invigorating spleen, moistening lung, and invigorating kidney; can be used for treating deficiency of spleen-stomach qi, asthenia, stomach yin deficiency, xerostomia, cough with little appetite, lung deficiency, cough with excessive consumption, hemoptysis, essence and blood deficiency, soreness of waist and knees, premature gray hair, and internal heat with thirst quenching effect.
Pig hemoglobin: the bioactive peptide with pig blood as protein source is produced through converting globin in pig blood into small molecular peptide. The small molecular pig blood peptide not only has good dissolubility, low viscosity and gel formation resistance, but also is quickly digested and absorbed in vivo. The oligopeptide consisting of 2-3 amino acids has better absorption performance than free amino acids, and the oligopeptide and the polypeptide in the diet can be completely absorbed through intestinal tracts and used as bioactive peptides to cause biological effects of organisms on the tissue level. In addition, the composition has low antigenicity, does not produce anaphylactic reaction, has physiological activity of fat metabolism and the like, so that pig blood is utilized to decompose hemoglobin in blood cells into globin and heme, the globin is further subjected to enzymolysis to obtain peptide substances with biological activity, and the composition is proved to have the effects of quickly treating and improving anemia through a white mouse experiment. The micromolecule polypeptide formed by the enzymolysis of the hemoglobin can be combined with free iron of a human body in time to form red blood cells and improve anemia symptoms.
The invention utilizes the principle of traditional Chinese medicine to carry out cell disruption and enzymolysis on medlar, red dates, sealwort, angelica and other substances with the functions of activating blood and enriching blood, and then carries out enzymolysis together with hemoglobin and deer placenta to obtain the compound polypeptide substance. The Chinese wolfberry, the red dates, the sealwort, the angelica and other substances have the functions of tonifying the kidney and helping nutrient substances to be absorbed, meanwhile, the active substances and the hemoglobin are subjected to enzymolysis together, and when the composite polypeptide is formed, the small molecular active ingredients are combined with the hemoglobin, so that the formation of the human body neohemoglobin is facilitated, and the anemia problem is helped to be treated and treated.
The hemoglobin powder can be prepared by the following method:
s1, dissolving: pouring the pig hemoglobin into an enzymolysis tank, adding purified water with the weight 5-15 times that of the pig hemoglobin, and fully stirring and dissolving.
S2, enzymolysis: adding alkaline protease accounting for 2-6% of the dry weight of the porcine hemoglobin, performing enzymolysis for 2-8 hours, and simultaneously controlling the temperature in an enzymolysis reaction tank to be 40-60 ℃ and the pH value to be 6.5-8.5;
s3, filtering: heating materials in an enzymolysis reaction tank to boil, then slightly boiling for 5-15 minutes to inactivate protease, cooling the enzymolysis reaction tank to 45-65 ℃, filtering the materials by adopting a plate and frame filter, and removing filter residues to obtain a blood peptide solution;
s4, decoloring step: adding activated carbon with the weight being 4-10% of the dry weight of the porcine hemoglobin into the filtered filtrate, fully stirring, controlling the temperature in an enzymolysis reaction tank to be 60-90 ℃, controlling the pH value to be 3-6, decoloring for 1-3 hours, then adjusting the pH value, and filtering;
s5, concentration step: introducing the filtered filtrate into a scraper evaporator according to the flow of 800kg/h, and carrying out vacuum concentration treatment at the temperature of 60-75 ℃ to obtain decolorized blood peptide feed liquid with the concentration of 40-50%;
s6, spray drying: and (4) carrying out spray drying on the decolorized blood peptide concentrated solution to obtain decolorized hemoglobin powder.
The preparation method of the composite polypeptide for preventing or relieving anemia disclosed by the invention has the following advantages:
the active peptide obtained by protein enzymolysis has good effect of relieving anemia. In addition, the source of the pig blood is rich, and the production of the bioactive peptide by using the pig blood is necessary. The bioactive peptide with pig blood as protein source is produced through converting globin in pig blood into small molecular peptide. The small molecular pig blood peptide not only has good dissolubility, low viscosity and gel formation resistance, but also is fast to digest and absorb in vivo, and has low antigenicity, no anaphylactic reaction, fat metabolism and other physiological activities. The blood peptide is supplemented with medicinal and edible materials such as angelica, red date, medlar and the like which have the functions of promoting blood circulation and removing blood stasis, and the composite polypeptide oral liquid and the composite polypeptide preparation are processed. The composite polypeptide agent and the composite polypeptide oral liquid for preventing or relieving anemia are simple to process, stable in taste, obvious in effect and free of adverse effects, and are good functional foods.
The blood peptide in the composite polypeptide disclosed by the invention is firstly subjected to enzymolysis by the haemoglobin to form macromolecular polypeptide, and then the deer placenta powder and the medicine-food homologous plant for improving anemia are added for joint enzymolysis, so that the composite polypeptide agent has better effects of preventing and relieving anemia. The small molecular active ingredients of medlar, red dates, sealwort, angelica and the like are utilized to enhance the kidney absorption effect and the blood circulation promoting effect; the deer placenta polypeptide and the blood peptide are utilized to enhance the free iron complexing ability of hemoglobin in vivo and increase the number and activity of hemoglobin in vivo, and the anemia improving effect is obvious.
Example 1 preparation of hemoglobin:
s1, dissolving: 2kg of pig hemoglobin is poured into an enzymolysis tank, 18kg of purified water is added, and the mixture is fully stirred and dissolved.
S2, enzymolysis: adding 80g of alkaline protease, carrying out enzymolysis for 3 hours, and simultaneously controlling the temperature in an enzymolysis reaction tank to be 60 ℃ and the pH value to be 8.0;
s3, filtering: heating materials in an enzymolysis reaction tank to boil, then boiling for 10 minutes slightly to inactivate protease, cooling the enzymolysis reaction tank to 50 ℃, then filtering the materials by adopting a plate-and-frame filter, and removing filter residues to obtain a blood peptide solution;
s4, decoloring step: adding 160g of active carbon into the filtered filtrate, fully stirring, controlling the temperature in an enzymolysis reaction tank to be 80 ℃ and the pH value to be 4.8, decoloring for 1 hour, then adjusting the pH value, and filtering;
s5, concentration step: introducing the filtered filtrate into a scraper evaporator according to the flow of 800kg/h, and performing vacuum concentration treatment at 60 ℃ to obtain decolorized blood peptide feed liquid with the concentration of 40-50%;
s6, spray drying: spray drying the decolorized blood peptide concentrated solution to obtain decolorized hemoglobin powder.
Example 2 composite polypeptide preparation:
1. adding 500g of angelica, 1500g of red date, 2500g of medlar and 1000g of rhizoma polygonati according to dry matter, and adding 22.5kg of purified water. And (5) carrying out ultrasonic treatment for 10min under the ultrasonic condition of 2000 HZ. Cooling to 40 deg.C, performing ultrasonic treatment for 1 time under the same conditions, crushing material cells, and dissolving out cytoplasm to obtain plant crushed solution.
2. Adding pure water into the plant crushing liquid until the solid-liquid ratio is 1:10, keeping the temperature to 55 ℃, adding 225g of cellulase, and carrying out enzymolysis for 3h to obtain plant enzymolysis liquid; adding 360g of plant complex enzyme into the plant enzymolysis liquid, carrying out enzymolysis for 7h, and keeping the constant temperature. Boiling the solution for 5min, inactivating enzyme, and preparing into small molecular polysaccharide plant liquid.
3. After the temperature of the micromolecular polysaccharide plant liquid is reduced, 3760g of hemoglobin powder and 740g of deer placenta powder are added, and 67.5kg of purified water is added to prepare the compound protein liquid.
4. Adding NaOH into the composite protein solution, adjusting the pH value to 9.0, placing the solution in a constant temperature environment of 60 ℃, adding 27g of alkaline protease, and maintaining for 3 hours.
5. Adjusting the pH value of the composite protein liquid subjected to alkaline enzymolysis to 7.0, adding 45g of flavourzyme in a constant temperature environment at 50 ℃, carrying out enzymolysis for 0.5h, boiling and maintaining for 5min to inactivate enzyme, thus obtaining the required primary micromolecule polypeptide liquid.
6. And (3) cooling the primary small molecular polypeptide solution to 45 ℃, then filtering the material by adopting a plate and frame filter, and removing filter residues to obtain the primary filter solution of the composite polypeptide.
7. And (3) passing the primary filtrate of the composite polypeptide through an ultrafiltration membrane, wherein the membrane molecular interception amount is 1WDa, treating by using a 1KDa membrane after the treatment, concentrating and filtering to prepare the composite polypeptide liquid which is high in concentration, small in molecular weight and capable of preventing and relieving anemia to a certain extent. The membrane filtration can effectively remove macromolecules, micromolecule bitter amino acids and decoloration.
8. Adding 9.9g taurine, 450g high fructose corn syrup, 5.4g mint, 960g mogroside and 48g edible essence auxiliary materials into the composite polypeptide liquid, filling and sealing, and sterilizing at 115 ℃ and 20Min to prepare the composite polypeptide for preventing and relieving anemia.
Comparative example 1:
the polypeptide preparation is prepared by only using the hemoglobin powder without adding the plant composite enzymolysis liquid and the deer placenta powder, and comprises the following specific steps:
1. adding purified water into hemoglobin powder until the dry matter is 10 times, and preparing protein solution.
2. Adding NaOH into the protein solution, adjusting pH to 9.0, placing the solution in a constant temperature environment of 60 deg.C, adding alkaline protease with dry matter content of 0.3%, and maintaining for 3 hr.
3, adjusting the pH value of the protein solution subjected to alkaline enzymolysis to 7.0, adding 0.05% flavourzyme of dry matter (based on the initial mass of the compound) into a constant temperature environment of 50 ℃, carrying out enzymolysis for 0.5h, boiling and maintaining for 5min to inactivate enzyme, thus obtaining the required primary micromolecule polypeptide solution.
4. And (3) cooling the primary small molecular polypeptide solution to 45 ℃, then filtering the material by adopting a plate and frame filter, and removing filter residues to obtain the primary polypeptide filtrate.
5. And (3) passing the primary filtrate of the polypeptide through an ultrafiltration membrane, treating the primary filtrate by using a 1KDa membrane after the primary filtrate of the polypeptide is treated by using a membrane with the membrane molecular interception amount of 1WDa, and concentrating and filtering.
6. Adding 0.11g/L taurine, 5g/L fructose-glucose syrup, 0.06g/L mint, 1% mogroside and 0.5 ‰ edible essence as adjuvants into the polypeptide liquid, bottling, sealing, sterilizing at 115 deg.C and 20Min, and making into polypeptide preparation.
Comparative example 2:
directly using hemoglobin powder and deer placenta powder for enzymolysis to prepare the compound polypeptide agent, and adding no plant compound enzymolysis liquid. The method comprises the following specific steps:
1. adding purified water into hemoglobin powder and deer placenta powder (at a dry weight ratio of 5: 1), and making into compound protein solution by 10 times of dry matter.
2. Adding NaOH into the composite protein solution, adjusting pH to 9.0, placing the solution in a constant temperature environment of 60 ℃, adding alkaline protease with dry matter content of 0.3%, and maintaining for 3 h.
3, adjusting the pH value of the compound protein solution subjected to alkaline enzymolysis to 7.0, adding 0.05% flavourzyme of dry matter (according to the initial mass of the compound) into a constant temperature environment of 50 ℃, carrying out enzymolysis for 0.5h, boiling and maintaining for 5min to inactivate enzyme, thus obtaining the required primary micromolecular polypeptide solution.
4. And (3) cooling the primary small molecular polypeptide solution to 45 ℃, then filtering the material by adopting a plate and frame filter, and removing filter residues to obtain the primary polypeptide filtrate.
5. And (3) passing the primary filtrate of the polypeptide through an ultrafiltration membrane, treating the primary filtrate by using a 1KDa membrane after the primary filtrate of the polypeptide is treated by using a membrane with the membrane molecular interception amount of 1WDa, and concentrating and filtering.
6. Adding 0.11g/L taurine, 5g/L high fructose syrup, 0.06g/L mint, 1% mogroside and 0.5 ‰ edible essence adjuvant into polypeptide liquid, bottling, sealing, sterilizing at 115 deg.C for 20Min, and making into pre-polypeptide preparation.
The effect of the comparative test is shown as follows:
after three days of acclimatization of SD rats with basal diet and normal drinking water, model-making rats (A, B, C, D, F) were changed to low-iron diet supplemented with deionized water and normal control rats (E) were kept unchanged. The drug administration of the model rats is started after the hemoglobin content is generally lower than 100g/L (namely, the model is anemia). Once daily dosing schedule and doses for each group are shown in table 1. Erythropoietin (EPO) feed was used as a drug positive control for the treatment of anemia. And collecting blood samples in a tail shearing mode every week, and performing anticoagulation by adopting a 2.5% EDTA-2Na solution in a volume ratio of 1:9 to finish routine blood detection. The effect of the complex polypeptide agent of the present invention on amelioration and treatment of anemia was determined according to the 1 month feeding period.
Wherein the low-iron feed (the iron content is 8.4mg/kg, purchased from Beijing Borai Macroda biotechnology limited); basal feed (Silekang rat breeder, available from Shanghai Proteus Biotech Co., Ltd.).
The number of red blood cells in normal rat is 5.6X 1012The normal range of the hemoglobin content is 120 g/L; the anemia reference line is the number of red blood cells 4.7X 1012The range of the hemoglobin content anemia is lower than 120 g/L.
Table 1: compound polypeptide feeding condition of mice
Comparative effect result 1:
comparing the number of red blood cells in the routine blood feeding of rats for 1 month with that in the following table 2, the red blood cells in the normal group have a normal reference value range of not less than 5.6 × 1012L, after feeding normal feed, the number of red blood cells does not greatly fluctuate at normal value within one month; anemia group (number of rat red blood cells is less than or equal to 4.7 multiplied by 10)12L), feeding low-iron feed and feeding with deionized water, and administering EPO for treating anemia to group F, wherein the red blood cells of the rat are obviously increased in 1 week and recovered to normal value in 2 weeks, and then feeding is continued, and the red blood cell number is obviously increased; when the group D is fed with normal saline and low-iron feed, the number of red blood cells of a rat is always reduced, and the anemia condition is more and more serious; when the group A is fed with the compound polypeptide agent and the low-iron feed, the normal level is basically recovered at the 2 nd week of feeding, the anemia state is improved, which is slightly lower than that of drug treatment and is obviously higher than that of the B, C two groups. When the group B is fed with the polypeptide agent and the low-iron feed of the comparative example 1, the normal level is basically recovered at week 3, the anemia state is improved, and the effect is lower than that of the group A; group C was fed with the polypeptide agent of comparative example 2 plus low iron feed, and at week 3, the level was substantially restored to normal, and the anemia state was improved, with less effect than group A.
TABLE 2 comparison of the erythrocyte content for the results of the tests
The comparative experiment shows that the content of the active ingredients,
1. the compound polypeptide agent disclosed by the invention has the effects of treating and relieving anemia similar to those of medicines;
2. according to the compound polypeptide preparation disclosed by the invention, the added medicinal and edible plants are subjected to enzymolysis together, so that the anemia relieving effect of the compound polypeptide is enhanced; mainly shows that the effect of relieving the anemia is stably improved, and is more approximate to the therapeutic effect of the medicine compared with the polypeptide without medicine-food homology.
3. The composite polypeptide agent disclosed by the invention has the advantages that the added deer placenta is subjected to enzymolysis together, so that the effect of relieving anemia of the composite polypeptide agent is enhanced, the effect is fast, and the relieving level is stable.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (6)
1. A preparation method of a composite polypeptide for relieving anemia is characterized in that: the preparation method comprises the following steps:
(1) mixing 1 part by weight of various medicinal and edible plants with the function of activating blood, adding 5-15 parts by weight of water, carrying out ultrasonic disruption extraction for 2-10min under the conditions of 1600-2500HZ, then cooling to 35-45 ℃, carrying out ultrasonic disruption again to achieve cell disruption and cytoplasm dissolution to form plant disruption solution;
(2) adding water into the plant crushing liquid until the solid-to-liquid ratio is 1:10-20, keeping the temperature to 55 ℃, adding cellulase for enzymolysis for 1-4h, and carrying out enzymolysis on plant cell walls to obtain plant enzymolysis liquid;
(3) adding plant complex enzyme into the plant enzymolysis liquid for enzymolysis for 4-10h, keeping constant temperature, boiling the solution for 5min, and inactivating enzyme to obtain micromolecular polysaccharide plant liquid;
(4) adding hemoglobin powder and deer placenta powder into small molecular polysaccharide plant liquid, adding water to 10-20 times of solid, mixing to obtain composite protein liquid, adding protease into the composite protein liquid, and performing enzymolysis to obtain small molecular polypeptide primary liquid;
(5) cooling the primary small molecular polypeptide solution to 45-65 ℃, filtering the primary small molecular polypeptide solution by using a plate and frame filter, and removing filter residues to obtain a fine polypeptide solution;
(6) adding auxiliary materials into the polypeptide fine filtrate, blending, sterilizing, filling and sealing, and then sterilizing at the temperature of 115 ℃ and 121 ℃ to obtain a composite polypeptide oral liquid or directly spray-drying to obtain a composite polypeptide agent;
the various medicinal and edible plants with the blood activating function in the step (1) comprise the following medicinal and edible plants in terms of dry weight: 10 to 30 percent of angelica, 30 to 50 percent of red date, 20 to 50 percent of medlar, 10 to 30 percent of rhizoma polygonati,
the weight of the added hemoglobin and the deer placenta powder in the step (4) is 1-5 times of the weight of the medicinal and edible plant according to the dry weight of the medicinal and edible plant, wherein the weight ratio of the hemoglobin powder to the deer placenta powder is 5-10: 1;
the enzymolysis in the step (4) comprises the following specific steps: adding NaOH into the composite protein solution, adjusting the pH value to 9.0, placing the solution in a constant temperature environment of 60 ℃, adding 0.1-0.5% of alkaline protease calculated by dry matters, maintaining for 2-6h, adjusting the pH value of the composite protein solution subjected to alkaline protease enzymolysis to 7.0, adding 0.03-0.1% of flavourzyme calculated by dry matters in a constant temperature environment of 50 ℃, performing enzymolysis for 0.5-1 h, boiling, maintaining for 2-5min, and inactivating enzyme to obtain the required primary micromolecule polypeptide solution.
2. The method for preparing a complex polypeptide for alleviating anemia according to claim 1, wherein: the weight of the cellulase added in the step (2) is 2-7% calculated according to the dry weight of the medicinal and edible plants.
3. The method for preparing a complex polypeptide for alleviating anemia according to claim 1, wherein: the weight of the plant complex enzyme added in the step (3) is 5-10% according to the dry weight of the medicinal and edible plants.
4. The method for preparing the composite polypeptide for relieving anemia according to claim 1, wherein the method comprises the following steps: the filtration comprises the following specific steps: and (3) passing the primary filtrate of the composite polypeptide through an ultrafiltration membrane, wherein the membrane molecular interception amount is 1-5 ten thousand Da, and treating by using a 1-2KDa membrane after the treatment is finished.
5. The method for preparing the composite polypeptide for relieving anemia according to claim 1, wherein the method comprises the following steps: the auxiliary materials comprise the following components in percentage by volume of the polypeptide fine filtrate: 0.11g/L taurine, 2-10g/L high fructose corn syrup and 0.05-0.1g/L mint.
6. The method for preparing the composite polypeptide for relieving anemia according to claim 5, wherein: the auxiliary materials are calculated according to the volume of the polypeptide fine filtrate, and the auxiliary materials further comprise: 0.1 to 1 percent of mogroside and 0.1 to 0.8 thousandth of edible essence.
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