Deer myocardial peptide capable of replenishing qi and enriching blood as well as preparation method and application of deer myocardial peptide

Abstract

The invention discloses deer myocardial peptide capable of replenishing qi and enriching blood as well as a preparation method and application of the deer myocardial peptide, and relates to the technical field of functional foods. The preparation method comprises the following steps: uniformly mixing fresh deer heart and a fermentation composition, and fermenting to obtain fermentation liquor; homogenizing the fermentation liquor to obtain homogenized liquor; mixing the homogenized liquid with compound enzyme, and performing enzymolysis to obtain enzymatic hydrolysate; and carrying out ultrafiltration concentration on the enzymatic hydrolysate, and drying to obtain the deer myocardial peptide. The micromolecular deer myocardial peptide prepared by the invention is high in purity and high in activity, and shows a good improvement effect in the aspects of replenishing qi and enriching blood, so that the application of the deer myocardial peptide is expanded.

Description

Technical Field

[0001] The invention relates to the technical field of functional foods, and in particular to a deer myocardial peptide for replenishing qi and blood, and a preparation method and application thereof. Background Art

[0002] Anemia refers to a syndrome caused by a decrease in the volume of red blood cells in the human body's peripheral blood, which is below the lower limit of the normal range and cannot transport enough oxygen to the tissues. Causes of anemia include anemia caused by reduced erythropoiesis, hemolytic anemia, and hemorrhagic anemia. Insufficient or impaired utilization of hematopoietic raw materials is one of the common causes of anemia. Hematopoietic raw materials refer to substances necessary for the proliferation, differentiation, and metabolism of hematopoietic cells, such as proteins, lipids, vitamins (folic acid, vitamin B12, etc.), trace elements (iron, copper, zinc, etc.). Insufficient or impaired utilization of any hematopoietic raw material may lead to reduced erythropoiesis. For example: (1) Anemia caused by deficiency or impaired utilization of folic acid or vitamin B12: Megaloblastic anemia caused by absolute or relative deficiency or impaired utilization of folic acid or vitamin B12 in the body due to various physiological or pathological factors. (2) Anemia caused by iron deficiency and impaired utilization: This is the most common anemia in clinical practice. Iron deficiency and impaired utilization of iron affect heme synthesis, and this type of anemia is called anemia caused by abnormal heme synthesis. In this type of anemia, the red blood cells become smaller and the central pale-stained area expands, which is a type of microcytic hypochromic anemia.

[0003] Deer myocardial peptide is a natural nutritional supplement that has been used in the fields of health supplementation and physical recovery, and has shown certain potential in the treatment of cardiovascular and cerebrovascular diseases, anti-tumor, neurodegenerative diseases, and immune system diseases.

[0004] Chinese patent CN118987162A discloses a composition for improving myocardial vitality, myocardial cell metabolism and heart failure and its application, the composition includes the following components: trisodium salt of fructose 1,6-diphosphate, myocardial peptide, mangosteen powder, elderberry powder, black fruit glandular rib fruit concentrated powder, noni pulp powder, taurine, oligosaccharides, D-ribose, ginseng powder, daidai pollen, vitamins, minerals. The composition has high safety, rigorous and scientific compatibility, and implements the core concept of "from cause to effect, syndrome differentiation and treatment". It improves antioxidant capacity and hypoxia tolerance to achieve the purpose of improving myocardial vitality, promoting myocardial cell energy metabolism, and reducing myocardial infarction area. It has a good effect on improving and conditioning cardiovascular and cerebrovascular diseases such as heart failure, myocardial infarction, myocardial ischemic injury, angina pectoris and arrhythmia.

[0005] At present, research on the extraction of deer myocardial peptide is relatively scarce, and a method for extracting deer myocardial peptide with high activity and purity is still needed. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a deer myocardial peptide for invigorating qi and nourishing blood, and a preparation method and application thereof. The small molecule peptide composed of 2-10 amino acids is prepared by using freshly farmed deer heart as raw material and enzymatic hydrolysis using fermentation technology and biological enzymatic cutting technology. The peptide has high purity and activity, and shows good improvement effects in invigorating qi and nourishing blood, thereby expanding the application of deer myocardial peptide.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a method for preparing a deer myocardial peptide for replenishing qi and blood, comprising the steps of:

[0009] (1) Fermentation: mixing fresh deer heart and a fermentation composition, and fermenting to obtain a fermentation liquid; the fermentation composition is a combination of Lactobacillus reuteri, Aspergillus niger and Bacillus;

[0010] (2) homogenizing: homogenizing the fermentation liquid obtained in step (1) to obtain a homogenized liquid;

[0011] (3) enzymatic hydrolysis: mixing the homogenate obtained in step (2) with a composite enzyme, and performing enzymatic hydrolysis to obtain an enzymatic hydrolyzate; the composite enzyme is a combination of α-amylase, neutral protease and papain;

[0012] (4) ultrafiltration and concentration of the enzymatic hydrolysate obtained in step (3), and drying to obtain the deer myocardial peptide.

[0013] Preferably, in step (1), the mass of the fermentation composition is 1-10% of the mass of deer myocardin, and more preferably 5%.

[0014] Preferably, in step (1), the fermentation conditions are: constant temperature fermentation at 25-50°C for 12-48 hours, with stirring every 6-14 hours; more preferably: constant temperature fermentation at 35°C for 24 hours, with stirring every 12 hours.

[0015] Preferably, in step (1), the mass ratio of Lactobacillus reuteri, Aspergillus niger and Bacillus is 0.5-2:0.5-2:1-5, more preferably 1:1:2.

[0016] Preferably, in step (2), the homogenization is performed 1 to 5 times, more preferably 2 times.

[0017] Further preferably, the homogenization conditions are: homogenization pressure 50-100 MPa, rotation speed 3000-10000 rpm, 5-15 min each time; further preferably: homogenization pressure 80 MPa, rotation speed 5000 rpm, 8 min each time.

[0018] Preferably, in step (3), the amount of the complex enzyme added is 1-5% by mass of the homogenous liquid, and more preferably 3%.

[0019] Preferably, in step (3), the mass ratio of the complex enzyme being α-amylase, neutral protease and papain is 0.1-2:2-10:1-10, more preferably 1:3:3.

[0020] Preferably, in step (3), the enzymatic hydrolysis conditions are: 45-55°C for 0.5-1.5h; more preferably: 50°C for 1h.

[0021] Preferably, in step (4), the ultrafiltration concentration is specifically: ultrafiltration concentration using an ultrafiltration membrane with a molecular weight of 10,000.

[0022] As a specific example of the present invention, the preparation method comprises the steps of:

[0023] (1) Fermentation: sterilize fresh deer heart, place in a fermentation tank, add a fermentation composition with a mass percentage of 5% of deer myocardial peptide, mix thoroughly, and ferment at 35° C. for 24 hours, stirring once every 12 hours. After fermentation is complete, filter with gauze to obtain a fermentation liquid; the fermentation composition is a combination of Lactobacillus reuteri, Aspergillus niger and Bacillus in a mass ratio of 1:1:2;

[0024] (2) Homogenization: Pour the fermentation liquid obtained in step (1) into an ultra-high pressure microfluidizer and homogenize twice at a homogenization pressure of 80 MPa and a rotation speed of 5000 rpm for 8 min each time to obtain a homogenized liquid;

[0025] (3) Enzymolysis: the homogenate obtained in step (2) and the composite enzyme are fully mixed, the amount of the composite enzyme added is 3% of the mass fraction of the homogenate, and the enzymolysis is carried out at 50° C. for 1 hour. After the enzymolysis is completed, the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution; the composite enzyme is a combination of α-amylase, neutral protease and papain in a mass ratio of 1:3:3;

[0026] (4) The enzymatic hydrolysate is concentrated by ultrafiltration (ultrafiltration membrane molecular weight 10000), sterilized, and spray-dried to obtain the deer myocardial peptide.

[0027] On the other hand, the present invention provides deer myocardial peptide prepared by the above preparation method.

[0028] Finally, the present invention provides the use of the deer myocardial peptide prepared by the above preparation method in the preparation of qi-invigorating and blood-tonifying products.

[0029] The beneficial effects of the present invention are:

[0030] (1) The present invention optimizes the extraction process of deer myocardin, performs fermentation, homogenization, and enzymatic hydrolysis in sequence, optimizes the compounding of fermentation bacteria and enzymatic hydrolysis enzymes, and adjusts the compounding ratio, so as to efficiently obtain high-quality deer myocardin products.

[0031] (2) The deer myocardial peptide prepared by the present invention shows a good improvement effect in invigorating qi and nourishing blood, thus expanding the application of deer myocardial peptide. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0033] In the following embodiments, all the raw materials used can be obtained through conventional commercial channels. Specifically, Lactobacillus reuteri has an activity of 10 billion cfu / g, Aspergillus niger has an activity of 2 billion cfu / g, Bacillus licheniformis has an activity of 20 billion cfu / g, α-amylase has an activity of 100,000 U / g, neutral protease has an activity of 200,000 U / g, papain has an activity of 100,000 U / g, and the deer heart is the heart of an artificially raised sika deer.

[0034] Example 1

[0035] (1) Fermentation: sterilize fresh deer heart, place in a fermentation tank, add a fermentation composition of 5% by weight of deer myocardial peptide, mix thoroughly, and ferment at 35° C. for 24 hours, stirring once every 12 hours. After fermentation, filter with gauze to obtain a fermentation liquid; the fermentation composition is a combination of Lactobacillus reuteri, Aspergillus niger and Bacillus licheniformis in a mass ratio of 1:1:2;

[0036] (2) Homogenization: Pour the fermentation liquid obtained in step (1) into an ultra-high pressure microfluidizer and homogenize twice at a homogenization pressure of 80 MPa and a rotation speed of 5000 rpm for 8 min each time to obtain a homogenized liquid;

[0037] (3) Enzymolysis: the homogenate obtained in step (2) and the composite enzyme are fully mixed, the amount of the composite enzyme added is 3% of the mass fraction of the homogenate, and the enzymolysis is carried out at 50° C. for 1 hour. After the enzymolysis is completed, the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution; the composite enzyme is a combination of α-amylase, neutral protease and papain in a mass ratio of 1:3:3;

[0038] (4) The enzymatic hydrolyzate obtained in step (3) is concentrated by ultrafiltration (ultrafiltration membrane molecular weight 10000), sterilized, and spray-dried to obtain the deer myocardial peptide.

[0039] Example 2

[0040] (1) Fermentation: sterilize fresh deer heart, place in a fermentation tank, add a fermentation composition of 1% by weight of deer myocardial peptide, mix thoroughly, and ferment at 25° C. for 48 hours, stirring once every 14 hours. After fermentation, filter with gauze to obtain a fermentation liquid; the fermentation composition is a combination of Lactobacillus reuteri, Aspergillus niger and Bacillus licheniformis in a mass ratio of 0.5:2:5;

[0041] (2) Homogenization: Pour the fermentation liquid obtained in step (1) into an ultra-high pressure microfluidizer and homogenize 5 times at a homogenization pressure of 50 MPa and a rotation speed of 10,000 rpm for 5 min each time to obtain a homogenized liquid;

[0042] (3) Enzymolysis: the homogenate obtained in step (2) and the composite enzyme are fully mixed, the amount of the composite enzyme added is 1% of the mass fraction of the homogenate, and the enzymolysis is carried out at 45° C. for 1.5 h. After the enzymolysis is completed, the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution; the composite enzyme is a combination of α-amylase, neutral protease and papain in a mass ratio of 0.1:10:10;

[0043] (4) The enzymatic hydrolyzate obtained in step (3) is concentrated by ultrafiltration (ultrafiltration membrane molecular weight 10000), sterilized, and spray-dried to obtain the deer myocardial peptide.

[0044] Example 3

[0045] (1) Fermentation: sterilize fresh deer heart, place in a fermentation tank, add a fermentation composition of 10% by weight of deer myocardial peptide, mix thoroughly, and ferment at 50° C. for 12 hours, stirring once every 6 hours. After fermentation, filter with gauze to obtain a fermentation liquid; the fermentation composition is a combination of Lactobacillus reuteri, Aspergillus niger and Bacillus licheniformis in a mass ratio of 2:0.5:1;

[0046] (2) Homogenization: Pour the fermentation liquid obtained in step (1) into an ultra-high pressure microfluidizer and homogenize once at a homogenization pressure of 100 MPa, a rotation speed of 3000 rpm, and a time of 15 min to obtain a homogenized liquid;

[0047] (3) Enzymolysis: the homogenate obtained in step (2) and the composite enzyme are fully mixed, the amount of the composite enzyme added is 5% of the mass fraction of the homogenate, and the enzymolysis is carried out at 55° C. for 0.5 h. After the enzymolysis is completed, the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution; the composite enzyme is a combination of α-amylase, neutral protease and papain in a mass ratio of 2:2:1;

[0048] (4) The enzymatic hydrolyzate obtained in step (3) is concentrated by ultrafiltration (ultrafiltration membrane molecular weight 10000), sterilized, and spray-dried to obtain the deer myocardial peptide.

[0049] Comparative Example 1

[0050] (1) Homogenization: Sterilize fresh deer heart and homogenize twice in an ultra-high pressure microfluidizer at a homogenization pressure of 80 MPa and a rotation speed of 5000 rpm for 8 min each time to obtain a homogenized liquid;

[0051] (2) Enzymolysis: the homogenate obtained in step (1) and the composite enzyme are fully mixed, the amount of the composite enzyme added is 3% of the mass fraction of the homogenate, and the enzymolysis is carried out at 50° C. for 1 hour. After the enzymolysis is completed, the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution; the composite enzyme is a combination of α-amylase, neutral protease and papain in a mass ratio of 1:3:3;

[0052] (3) The enzymatic hydrolyzate obtained in step (3) is concentrated by ultrafiltration (ultrafiltration membrane molecular weight 10000), sterilized, and spray-dried to obtain the deer myocardial peptide.

[0053] Comparative Example 2

[0054] (1) Homogenization: Sterilize fresh deer heart and homogenize twice in an ultra-high pressure microfluidizer at a homogenization pressure of 80 MPa and a rotation speed of 5000 rpm for 8 min each time to obtain a homogenized liquid;

[0055] (2) Enzymolysis: the homogenate obtained in step (2) and the composite enzyme are fully mixed, the amount of the composite enzyme added is 3% of the mass fraction of the homogenate, and the enzymolysis is carried out at 50° C. for 1 hour. After the enzymolysis is completed, the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution; the composite enzyme is a combination of α-amylase, neutral protease and papain in a mass ratio of 1:3:3;

[0056] (3) Fermentation: placing the enzymatic hydrolyzate in a fermentation tank, adding a fermentation composition with a mass percentage of 5% of deer myocardial peptide, mixing thoroughly, and fermenting at a constant temperature of 35° C. for 24 hours, stirring once every 12 hours, and filtering with gauze after the fermentation is completed to obtain a fermentation liquid; the fermentation composition is a combination of Lactobacillus reuteri, Aspergillus niger and Bacillus licheniformis in a mass ratio of 1:1:2;

[0057] (4) The fermentation broth obtained in step (3) is concentrated by ultrafiltration (ultrafiltration membrane molecular weight 10000), sterilized, and spray-dried to obtain the deer myocardial peptide.

[0058] Comparative Example 3

[0059] The difference from Example 1 is that in step (1), the fermentation composition is a combination of Lactobacillus reuteri and Aspergillus niger in a mass ratio of 1:1. The other parameters are the same.

[0060] Comparative Example 4

[0061] The difference from Example 1 is that in step (3), the compound enzyme is a combination of bromelain, neutral protease and papain in a mass ratio of 1:1:1. The other parameters are the same.

[0062] Comparative Example 5

[0063] The difference from Example 1 is that in step (3), the compound enzyme is a combination of bromelain, neutral protease and lipase in a mass ratio of 3:3:1. The other parameters are the same.

[0064] Comparative Example 6

[0065] The difference from Example 1 is that in step (1), the mass ratio of Lactobacillus reuteri, Aspergillus niger and Bacillus licheniformis is 5:1:1. The other parameters are the same.

[0066] Comparative Example 7

[0067] The difference from Example 1 is that in step (3), the mass ratio of α-amylase, neutral protease and papain is 5:1:1. The other parameters are the same.

[0068] Results

[0069] 1. Molecular weight distribution detection

[0070] According to the AGPC / UV test in Appendix GB 31645-2018, the proportion of peptides with a relative molecular weight not exceeding 10,000 and not exceeding 2,000 was counted. The calculation method is illustrated by the measurement results of the product in Example 1. The measurement results of the product in Example 1 are:

[0071]

[0072]

[0073] According to calculation, peptides with a relative molecular weight not exceeding 10,000 account for 99.9%, and peptides with a relative molecular weight not exceeding 2,000 account for 90.8%.

[0074] The calculation results of the embodiments and comparative examples are as follows:

[0075]

[0076] 2. Protein and peptide content detection

[0077] The protein content was determined according to the first method of GB5009.5-2016; the peptide content was determined according to Appendix BB of GB22492-2008.

[0078] The results are as follows:

[0079]

[0080]

[0081] 3. Biological activity detection

[0082] The activity was tested using the T lymphocyte-E rosette test (reference: Cai Hongjie, "Improvement of calf thymosin production process and analysis of preparations", No. 1672-0369 (2011) 19-2414-04), and the results are as follows:

[0083]

[0084] The above results show that the deer myocardial peptide powder prepared in Examples 1-3 of the present invention has an increased content of short peptide chains and a high peptide content. In addition, compared with commercially available products and the control group, the activity is significantly improved. Compared with Examples 1-3, the short peptide chain content, peptide content and active bacteria in Comparative Examples 1 and 2 decreased, proving that the production process of first fermentation, then homogenization and then enzymolysis of the present invention can improve the shearing degree and improve the peptide content and activity of the product. Compared with Example 1, Comparative Examples 3 and 6 show that the specific fermentation bacteria combination screened by the present invention can obtain higher quality deer myocardial peptide. The results of Comparative Examples 4, 5 and 7 show that the specific enzyme combination screened for the preparation of deer myocardial peptide by the present invention also significantly improves the quality of deer myocardial peptide.

[0085] 4. Blood-enriching effect experiment

[0086] (1) Experimental drugs

[0087] Deer myocardial peptide prepared in Example 1;

[0088] Ferrous sulfate (National Medicine Approval No. H37020689).

[0089] (2) Modeling

[0090] Male Kunming mice (18-22g) were selected and adaptively raised for one week. The temperature of the raising environment was 20±2℃, the relative humidity was 45%-65%, and they were raised with low-iron feed and deionized water. They were given free drinking water and diet. Non-iron cage covers and plastic cages were used during the raising process to avoid iron contamination. The blank group was fed with normal feed and normal drinking water.

[0091] After 2 weeks of feeding, tail blood was collected to measure hemoglobin (Hb), red blood cell count (RBC), and hematocrit (HCT). Compared with the blank group, there were significant differences, with Hb lower than 100g / L and RBC ≤7.5×10 12 / L, hematocrit (HCT) ≤ 35%, indicating successful modeling.

[0092] (3) Grouping and dosing

[0093] The mice with successful modeling were randomly divided into groups, with 10 mice in each group, and were administered with 100 mg / kg (L-LXJT) of the low dose of Example 1, 200 mg / kg (H-LXJT) of Example 1, and 5 mg / kg of ferrous sulfate (LST), respectively, once a day by gavage (10 mL / kg). The control group was gavaged with an equal volume of normal saline and continued to be fed with low-iron feed and deionized water. After continuous administration for 1 week, blood counts were measured. The results are as follows:

[0094]

[0095]

[0096] In the table, compared with the blank group, *P<0.05, **P<0.01; compared with the drug group, #P<0.05, ##P<0.01.

[0097] The results show that the deer myocardial peptide powder prepared by the present invention has a significant blood-enriching effect and can be used for the preparation of blood-enriching health foods.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing deer myocardial peptide for replenishing qi and blood, characterized in that: Includes steps: (1) Fermentation: mixing fresh deer heart and a fermentation composition, and fermenting to obtain a fermentation liquid; the fermentation composition is a combination of Lactobacillus reuteri, Aspergillus niger and Bacillus; (2) homogenizing: homogenizing the fermentation liquid obtained in step (1) to obtain a homogenized liquid; (3) enzymatic hydrolysis: mixing the homogenate obtained in step (2) with a composite enzyme, and performing enzymatic hydrolysis to obtain an enzymatic hydrolyzate; the composite enzyme is a combination of α-amylase, neutral protease and papain; (4) ultrafiltration and concentration of the enzymatic hydrolysate obtained in step (3), and drying to obtain the deer myocardial peptide.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass of the fermentation composition is 1-10% of the mass of deer myocardin.

3. The preparation method according to claim 1, characterized in that: In step (1), the fermentation conditions are: constant temperature fermentation at 25-50°C for 12-48 hours, and stirring every 6-14 hours.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of Lactobacillus reuteri, Aspergillus niger and Bacillus is 0.5-2:0.5-2:1-5.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the Lactobacillus reuteri, Aspergillus niger and Bacillus is 1:1:

2.

6. The preparation method according to claim 1, characterized in that: In step (2), the homogenization is performed 1-5 times, and the homogenization conditions are: homogenization pressure 50-100 MPa, rotation speed 3000-10000 rpm, and each time for 5-15 min.

7. The preparation method according to claim 1, characterized in that: In step (3), the amount of the complex enzyme added accounts for 1-5% of the mass fraction of the homogenous liquid.

8. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of the complex enzyme, α-amylase, neutral protease and papain, is 0.1-2:2-10:1-10, preferably 1:3:

3.

9. Deer myocardial peptide prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the deer myocardial peptide according to claim 9 in the preparation of qi-invigorating and blood-tonifying products.

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