Method for preparing micromolecular active substance by taking aronia and medlar as raw materials

Abstract

The embodiment of the invention discloses a method for preparing a small-molecule active substance by taking aronia and medlar as raw materials. The method comprises the following steps: cleaning and crushing fresh aronia fruits and fresh Chinese wolfberry fruits to obtain slurry; protease, pectinase and cellulase are added into the pulp for an enzymolysis reaction, and an enzymolysis product is obtained; and heating the enzymolysis product to terminate enzymatic activity, purifying, and drying to obtain the micromolecular active substance. Through reasonable process design and parameter control, the purity and activity of the micromolecular active substance are effectively improved, and the prepared micromolecular active substance is more easily absorbed by a human body, has more prominent functional activity and is suitable for development of functional food and health care products.

Description

Technical Field

[0001] The embodiments of the present invention relate to the fields of functional food and biotechnology, and specifically to a method for preparing small molecule active substances using aronia berry and wolfberry as raw materials. Background Art

[0002] Aronia, also known as blackberry, has been shown in clinical studies to have a variety of health benefits, mainly including: antioxidant effect, aronia is rich in antioxidants such as anthocyanins and polyphenols, which can neutralize free radicals, reduce oxidative stress, and have a protective effect on cells; cardiovascular health, clinical trials have shown that aronia can lower blood pressure and blood lipid levels, improve arterial elasticity, and reduce the risk of cardiovascular disease; anti-inflammatory effect, its active ingredients have anti-inflammatory properties, can reduce inflammatory responses, and help relieve inflammation-related diseases; blood sugar regulation, some studies have found that aronia helps lower blood sugar levels and improve insulin sensitivity, which may be beneficial for diabetic patients; immune system support, the polyphenols in aronia can enhance immune function and increase the body's resistance to pathogens; liver protection: antioxidant components help protect liver cells and promote liver detoxification function; antibacterial and antiviral, experimental studies have shown that aronia extract has an inhibitory effect on certain bacteria and viruses. At the same time, clinical application studies have confirmed that after taking wild aronia, the fasting blood sugar of patients with type 2 diabetes is significantly reduced, and the levels of glycosylated hemoglobin (HbAle), total cholesterol and lipids in the patients' plasma are also reduced.

[0003] In addition, wild aronia is rich in various vitamins, including: vitamin C, which helps to strengthen the immune system, promotes collagen synthesis, and has antioxidant effects; vitamin K, which plays an important role in blood coagulation and bone health; vitamin E, a powerful antioxidant that protects cells from damage by free radicals; B vitamins, among which vitamin B1 (thiamine), helps convert food into energy and supports nervous system function; vitamin B2 (riboflavin), participates in energy metabolism and maintains skin and eye health; vitamin B6, participates in protein and amino acid metabolism, supports brain health; folic acid, is essential for cell division and DNA synthesis.

[0004] Lycium barbarum is rich in a variety of vitamins and minerals, including vitamins such as vitamin A, which helps maintain healthy vision and skin; vitamin C, which enhances immune system function and has antioxidant effects; B vitamins: vitamin B1 (thiamine), vitamin B2 (riboflavin) and vitamin B6, which help energy metabolism and nervous system function, and minerals such as iron, which promotes the formation of red blood cells and prevents anemia; zinc, which supports immune function and wound healing; calcium, which contributes to the health of bones and teeth; potassium, which maintains normal heart function and blood pressure; and selenium, which has antioxidant effects and protects cells from free radical damage. In addition, wolfberries are rich in polysaccharides and carotenoids, which help improve immunity and protect eye health.

[0005] Both aronia and wolfberry are known as superfoods. Aronia is rich in vitamins, minerals, antioxidants and some other important polyphenols, which have the effect of fighting cancer cells. It is rich in various bioactive ingredients, such as polyphenols, flavonoids and polysaccharides. However, most of these ingredients exist in the form of macromolecules with low bioavailability, which limits their functional activity.

[0006] In view of this, the present invention application is specially proposed. Summary of the invention

[0007] To this end, an embodiment of the present invention provides a method for preparing a small molecule active substance using aronia berry and wolfberry as raw materials. The present invention combines aronia berry and wolfberry and uses modern biotechnology to prepare a small molecule active substance to improve its efficiency of absorption and utilization by the human body and enhance its functional activity. The small molecule active substance prepared by the present invention has good application potential in the fields of food, health products and medicine.

[0008] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0009] According to a first aspect of an embodiment of the present invention, the present invention provides a method for preparing a small molecule active substance using aronia berry and wolfberry as raw materials, the method comprising the following steps:

[0010] (1) Wash fresh aronia berries and fresh wolfberries, crush them, and sieve them to obtain a slurry;

[0011] (2) adding protease, pectinase and cellulase to the slurry to carry out enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0012] (3) The enzymatic hydrolysis product is heated to terminate the enzyme activity, purified, and dried to obtain the small molecule active substance.

[0013] Furthermore, in step (1), the mass ratio of the fresh aronia berry to the fresh wolfberry is 5:1;

[0014] The particle size of the mesh is 100 μm-200 μm.

[0015] Furthermore, in step (2), based on the mass of the slurry, the mass percentages of protease, pectinase and cellulase are 0.5-1.5%, 0.2-1.2% and 0.1-1.1% respectively;

[0016] The protease is selected from one or more of alkaline protease, neutral protease and trypsin.

[0017] Furthermore, in step (2), the conditions of the enzymatic hydrolysis reaction are: temperature 40-50°C, pH value 6.0-7.0, and reaction time 2-3h.

[0018] Furthermore, in step (3), the heating conditions are: temperature 90° C., time 10 min.

[0019] Furthermore, in step (3), purification is performed using a nano-ultrafiltration membrane with a molecular weight cutoff of 1,000-5000Da.

[0020] Furthermore, in step (3), the drying process includes spray drying or freeze drying.

[0021] According to a second aspect of the embodiments of the present invention, the present invention provides a small molecule active substance, which is prepared by any of the methods described above.

[0022] According to a third aspect of the embodiments of the present invention, the present invention provides the use of the small molecule active substance as described above in functional foods and health products.

[0023] The present invention provides a biotechnology method for preparing small molecule active substances using aronia and wolfberry, which comprises mixing and crushing the two raw materials, adding a specific enzyme preparation for enzymolysis, purifying the small molecule substances using ultrafiltration technology, and using a nanoparticle ultrafiltration membrane with a molecular weight cutoff of 3,000Da for purification, which is a key step in the deep processing of aronia wolfberry peptides. The present invention effectively improves the purity and activity of small molecule active substances through reasonable process design and parameter control, and the prepared small molecule active substances are more easily absorbed by the human body, have more prominent functional activity, and are suitable for the development of functional foods and health products.

[0024] The embodiments of the present invention have the following advantages:

[0025] Improve absorption rate: Through enzymatic hydrolysis and ultrafiltration technology, macromolecules are converted into small molecules, thereby improving the bioavailability of active substances.

[0026] Enhanced functional activity: Small molecule active substances can more easily penetrate the cell membrane and exert their antioxidant, immune regulation, cardiovascular protection, and blood sugar lowering functions.

[0027] Comprehensive utilization of resources: The nutrients of aronia and wolfberry are utilized simultaneously to exert synergistic effects and enhance the comprehensive functions of the product. DETAILED DESCRIPTION

[0028] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] This embodiment provides a method for preparing a small molecule active substance using aronia berry and wolfberry as raw materials, comprising the following steps:

[0031] (1) Raw material preparation: prepare 100 kg of fresh aronia berries and 20 kg of fresh wolfberries, wash them and remove impurities.

[0032] (2) Crushing treatment: The cleaned raw materials are crushed and passed through a 100 μm mesh sieve to make a fine slurry.

[0033] (3) Enzymatic hydrolysis: 1.8 kg of neutral protease (1.5% by mass), 1.44 kg of pectinase (1.2% by mass) and 1.32 kg of cellulase (1.1% by mass) were added to the slurry obtained in step (2), and the mixture was reacted at 40° C. and a pH value of 6.0 for 3 hours.

[0034] (4) Enzyme inactivation treatment: After the enzymatic hydrolysis is completed, the reaction product of step (3) is heated to 90° C. and maintained for 10 minutes to terminate the enzyme activity.

[0035] (5) Ultrafiltration purification: Centrifuge at 8000 rpm for 10 minutes to remove insoluble matter and collect the supernatant. Use an ultrafiltration membrane with a molecular weight cutoff of 3,000 Daltons (Da) for molecular retention and collect the permeate.

[0036] (6) Drying and molding: The permeate is spray-dried to obtain a small molecule active substance powder.

[0037] (7) Product testing: The powder was analyzed for components to determine the content of small molecule peptides, polyphenols and polysaccharides. The peptides were separated from other components using the HPLC method, and the experimental measurement showed that the main peak corresponding to the wild aronia wolfberry peptide accounted for 85% of the total peak area. That is, the estimated purity of the wild aronia wolfberry peptide was 85%.

[0038] The experimental results show that the produced peptide is a pentapeptide, which is composed of 5 amino acids, and the chemical formula of amino acids is C5H9NO3.

[0039] Example 2

[0040] This embodiment provides a method for preparing a small molecule active substance using aronia berry and wolfberry as raw materials, comprising the following steps:

[0041] (1) Raw material preparation: prepare 100 kg of fresh aronia berries and 20 kg of fresh wolfberries, wash them and remove impurities.

[0042] (2) Crushing treatment: The cleaned raw materials are crushed and passed through a 200 μm mesh sieve to form a fine slurry.

[0043] (3) Enzymatic hydrolysis: 0.6 kg of neutral protease (mass percentage of 0.5%), 0.24 kg of pectinase (mass percentage of 0.2%) and 0.12 kg of cellulase (mass percentage of 0.1%) were added to the slurry obtained in step (2), and the reaction was carried out at a temperature of 50° C. and a system pH of 7.0 for 2 hours.

[0044] (4) Enzyme inactivation treatment: After the enzymatic hydrolysis is completed, the reaction product of step (3) is heated to 90° C. and maintained for 10 minutes to terminate the enzyme activity.

[0045] (5) Ultrafiltration purification: Use nanoparticle ultrafiltration membrane for molecular interception, and use ultrafiltration membrane with a molecular weight cutoff of 3,000 Daltons (Da) to separate small molecule peptides, purify the target peptide segment, and collect the permeate. Centrifuge at 8000 rpm for 10 minutes to remove insoluble substances and collect the supernatant.

[0046] (6) Drying and molding: The permeate is spray-dried to obtain a small molecule active substance powder.

[0047] (7) Product testing: HPLC-MS reverse phase C18 column analysis:

[0048] Mobile phase: Gradient elution of water (containing 0.1% trifluoroacetic acid) and acetonitrile (containing 0.1% trifluoroacetic acid) is commonly used.

[0049] The absorbance of peptide bonds was detected at 220 nm or 280 nm.

[0050] Peptide formula:

[0051] The molecular weight of the peptide was determined by mass spectrometry, and the amino acid composition and molecular formula of the peptide were analyzed by mass spectrometry. The results are as follows:

[0052] Pentapeptide: C 15 H 35 N7O 10

[0053] Decapeptide: C 50 H 70 N 14 O 20

[0054] Pentapeptide: C 75 H 105 N 21 O 30

[0055] 19-peptide: C 95 H 133 N 27 O 38

[0056] According to the peptide purity calculation, the total peptide purity of the small molecule active substance prepared in this example is 82%, wherein the content of 5-peptide to 19-peptide accounts for 68% of the total peptide.

[0057] Peptide content determination

[0058] The experimental results showed that the total peptide content was 7.5%, of which the 5-10 peptide content was 3%, the 11-15 peptide content was 2.5%, and the 16-19 peptide content was 2%.

[0059] In the experiment of the present invention, by adjusting the type and amount of enzymes, the ratio of alkaline protease, neutral protease and trypsin, pectinase and cellulase, the invention has formed the optimal process parameters to produce a mixture of multiple peptides rather than a single peptide molecule. The present invention uses experimental ratios to develop products with different functional properties.

[0060] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for preparing small molecule active substances using aronia berry and wolfberry as raw materials, characterized in that: The method comprises the following steps: (1) Wash fresh aronia berries and fresh wolfberries, crush them, and sieve them to obtain a slurry; (2) adding protease, pectinase and cellulase to the slurry to carry out enzymatic hydrolysis to obtain an enzymatic hydrolysis product; (3) The enzymatic hydrolysis product is heated to terminate the enzyme activity, purified, and dried to obtain the small molecule active substance.

2. The method for preparing small molecule active substances using aronia berry and wolfberry as raw materials according to claim 1, characterized in that: In step (1), The mass ratio of fresh aronia berry to fresh wolfberry is 5:1; The particle size of the mesh is 100 μm-200 μm.

3. The method for preparing small molecule active substances using aronia berry and wolfberry as raw materials according to claim 1, characterized in that: In step (2), Based on the mass of the slurry, the mass percentages of protease, pectinase and cellulase are 0.5-1.5%, 0.2-1.2% and 0.1-1.1% respectively; The protease is selected from one or more of alkaline protease, neutral protease and trypsin.

4. The method for preparing small molecule active substances using aronia berry and wolfberry as raw materials according to claim 1, characterized in that: In step (2), The conditions of the enzymatic hydrolysis reaction are: temperature 40-50° C., pH value 6.0-7.0, and reaction time 2-3 h.

5. The method for preparing small molecule active substances using aronia berry and wolfberry as raw materials according to claim 1, characterized in that: In step (3), The heating conditions are: temperature 90° C., time 10 min.

6. The method for preparing small molecule active substances using aronia berry and wolfberry as raw materials according to claim 1, characterized in that: In step (3), Purification was performed using a nanofiltration membrane with a molecular weight cut-off of 1,000-5,000 Da.

7. The method for preparing small molecule active substances using aronia berry and wolfberry as raw materials according to claim 1, characterized in that: In step (3), the drying process includes spray drying or freeze drying.

8. A small molecule active substance, characterized in that: It is prepared by the method according to any one of claims 1 to 7.

9. Use of the small molecule active substance according to claim 8 in functional foods and health products.