Enzymatic extraction method for a wild mountain ginseng-like product

Abstract

The present invention discloses a method for enzymatic hydrolysis extraction of a wild ginseng-like product, belonging to the technical field of wild ginseng. Some rare saponins in wild ginseng can promote cell growth and division and improve the body. Through specific active enzymes, the prototype saponins can be enzymatically hydrolyzed to generate different rare saponins. In the present invention, through the action of radio frequency plasma, a mixed gas of α-linolenic acid vapor and nitrogen is used to treat the ultrafiltration membrane, thereby introducing double bond groups onto the ultrafiltration membrane; an esterification reaction occurs between p-hydroxycinnamic acid and cellulase to generate a product with reactive activity; these products undergo graft polymerization with the double bonds on the surface of the ultrafiltration membrane to form a more stable and more functional modified ultrafiltration membrane; the present invention can effectively separate and extract the target substances through a combination of enzymatic hydrolysis and ultrafiltration membrane fractionation; the molecular weight distribution of the peptides released during the enzymatic hydrolysis process is within a certain range, which helps to precisely separate these substances through the ultrafiltration membrane, and the extraction rate is high.

Description

Technical Field

[0001] The present invention relates to the technical field of wild mountain ginseng, and in particular to a method for enzymatic extraction of wild mountain ginseng-like products. Background Art

[0002] Some compounds in wild mountain ginseng have the effect of promoting cell growth and division, which helps to accelerate the wound healing process. It also has the characteristics of promoting cell regeneration, regulating immune function and moisturizing effect. Research shows that ginseng can stimulate the proliferation of basal epidermal cells and promote the accelerated repair of damaged skin.

[0003] The seeds of wild mountain ginseng are spread by birds and animals. It grows in the needle-broadleaf mixed forest composed of arbors, shrubs and weeds. It is distributed between 33 degrees and 48 degrees north latitude, and is mainly produced in: the Far East region of Russia and the Changbai Mountain range near the Changbai Mountain range in China. Wild mountain ginseng has the effects of tonifying deficiency and rescuing collapse, greatly tonifying primordial qi, etc., and can strengthen the essence, build up the body and prolong life.

[0004] Chinese Patent CN106727807B: It relates to the technical field of traditional Chinese medicine, and discloses a ginseng enzymatic hydrolysate, its preparation process and application. The preparation process of the present invention first extracts ginseng to obtain a ginseng extract, concentrates it and adjusts the pH value to acidic to obtain a concentrated ginseng solution; a composite enzyme is added to the concentrated ginseng solution for enzymatic hydrolysis, and after enzymatic hydrolysis, it is dried to obtain a ginseng enzymatic hydrolysate; wherein, the composite enzyme is cellulase and one or more enzymes selected from pectinase, hemicellulase and β-glucanase. The present invention enzymatically hydrolyzes the ginseng extract through a suitable enzyme combination and enzymatic hydrolysis method to obtain a ginseng enzymatic hydrolysate with a specific saponin composition.

[0005] Chinese Patent CN114917264A: It discloses a wild mountain ginseng-like composition, its preparation method, and its application in anti-tumor, immune enhancement and anti-aging. This composition realizes the molecular reconstruction of the active ingredients of ordinary ginseng, obtains saponin components similar to or even higher in content than wild mountain ginseng, and more than a dozen active peptides similar to wild mountain ginseng-related protein fragments. It is highly similar to wild mountain ginseng in terms of the richness of active ingredients and the composition ratio of various components, thus achieving a comprehensive therapeutic effect similar to that of wild mountain ginseng.

[0006] Chinese Patent CN107375374B: A fermentation extraction method of American ginseng extract or ginseng extract with the function of assisting in improving sleep is disclosed, including the following steps: (1) Raw material treatment: Using American ginseng or ginseng as raw materials, after pulverization and puffing treatment, they are reserved for use; (2) Primary fermentation: Using cellulase to enzymatically hydrolyze the American ginseng or ginseng after puffing treatment to obtain an enzymatic hydrolysate of American ginseng or ginseng, then inactivating the enzymatic hydrolysate, and screening it for standby; (3) Secondary fermentation: Preparing a fermentation medium with the enzymatic hydrolysate, carbon source and nitrogen source, then inoculating Aspergillus oryzae liquid, and fermenting and decomposing at 35-45°C to obtain enzyme solution; (4) Concentration: Filtering the obtained enzyme solution, concentrating the obtained supernatant to obtain an extract of American ginseng or ginseng.

[0007] Currently, the extraction methods for pseudo-wild ginseng are mainly decocting, cooking, making soup, stewing, enzymatic hydrolysis, etc. These methods are costly, complex in operation, and will cause serious waste of materials and low extraction rate. Summary of the Invention

[0008] To solve the above problems, the present invention provides an enzymatic hydrolysis extraction method for pseudo-wild ginseng products, and its operation steps are as follows:

[0009] S1: Put 5-10 parts of pseudo-wild ginseng into an ultrafine pulverizer for pulverization, add 100-150 parts of distilled water, soak for 30-60 min, reflux in a water bath at 100°C for 60-90 min, repeat the extraction 2 times, and combine the extraction solutions;

[0010] S2: Add 0.1-1.5 parts of enzyme to the extraction solution, raise the temperature for enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0011] S3: After the enzymatic hydrolysis product is extracted by a modified ultrafiltration membrane, rotary evaporation and drying are carried out to obtain a pseudo-wild ginseng extract.

[0012] In an embodiment of the present invention, the frequency of the ultrafine pulverization in S1 is 20-30 Hz, and the time is 60-90 s.

[0013] In an embodiment of the present invention, the enzyme in S2 is one of cellulase, hemicellulase, acid protease, pectinase, and β-glucanase.

[0014] In an embodiment of the present invention, the enzymatic hydrolysis temperature in S2 is 40-60°C, and the time is 3-6 days.

[0015] In an embodiment of the present invention, the preparation mechanism of the modified ultrafiltration membrane is as follows:

[0016] It mainly includes introducing double bonds through plasma treatment and the esterification reaction occurring between p-hydroxycinnamic acid and cellulase; in the preparation process of the modified ultrafiltration membrane, first, through the action of radio frequency plasma, a mixture of α-linolenic acid vapor and nitrogen is used to treat the ultrafiltration membrane, thereby introducing double bond groups onto the ultrafiltration membrane; the purpose of this step is to form active sites on the membrane surface that can participate in further chemical reactions; an esterification reaction occurs between p-hydroxycinnamic acid and cellulase to generate reactive products; finally, these products are graft-polymerized with the double bonds on the ultrafiltration membrane surface to form a more stable and functional modified ultrafiltration membrane.

[0017] Furthermore, the preparation method of the modified ultrafiltration membrane is as follows:

[0018] A1: Place the ultrafiltration membrane in a radio frequency plasma generator and treat it with a mixture of α-linolenic acid vapor and nitrogen, where the ratio of the mixture is α-linolenic acid vapor: nitrogen = 1:5 - 8, and the treatment time is 30 - 60 seconds to obtain an ultrafiltration membrane with double bond groups;

[0019] A2: In a stirring kettle, mix 2 - 5 parts of p-hydroxycinnamic acid, 0.01 - 0.3 parts of cellulase (CAS No.: 9012 - 54 - 8), 0.03 - 0.5 parts of acetic acid, and 200 - 350 parts of ethanol, heat up to 60 - 70 °C, and stir at this temperature for 2 - 5 hours to obtain a mixed solution;

[0020] A3: Add 20 - 30 parts of the ultrafiltration membrane with double bond groups and 0.002 - 0.05 parts of potassium peroxide to the mixed solution obtained in step 2, and continue to stir at 60 - 70 °C for 5 - 10 hours;

[0021] A4: Take out the ultrafiltration membrane treated in step 3 from the stirring kettle, wash it with absolute ethanol, and then dry it to obtain the modified ultrafiltration membrane.

[0022] Furthermore, the power of the radio frequency plasma generator is 50 - 100 W, the gas flow rate is 10 - 20 sccm, and the working pressure is controlled at 15 - 20 Pa.

[0023] An enzymatic hydrolysis extraction method for a product similar to wild ginseng of the present invention. Compared with the prior art, the present invention has the following remarkable effects:

[0024] 1. Improvement in the efficiency of purification and impurity removal: By combining enzymatic hydrolysis and ultrafiltration membrane fractionation, the target substances can be effectively separated and extracted; the peptide molecular weights released during the enzymatic hydrolysis process are distributed within a certain range, which helps to precisely separate these substances through the ultrafiltration membrane;

[0025] 2. The fouling resistance of the membrane and the convenience of cleaning and maintenance are improved. Detailed implementation manners

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] Extraction rate:

[0028]

[0029] In the formula: m1—the mass of the extract of pseudo-wild ginseng after drying, g,

[0030] m0—the mass of pseudo-wild ginseng before extraction, g.

[0031] Example 1

[0032] A method for enzymatic extraction of pseudo-wild ginseng products, the operation steps of which are as follows:

[0033] S1: Put 5 g of pseudo-wild ginseng into an ultrafine pulverizer, pulverize it, add 100 g of distilled water, soak for 30 min, reflux in a water bath at 100 °C for 60 min, repeat the extraction 2 times, and combine the extraction solutions;

[0034] S2: Add 0.1 g of enzyme to the extraction solution, raise the temperature for enzymatic hydrolysis, and obtain an enzymatic hydrolysis product;

[0035] S3: After the enzymatic hydrolysis product is extracted by a modified ultrafiltration membrane, rotary evaporation and drying are carried out to obtain an extract of pseudo-wild ginseng.

[0036] The frequency of the ultrafine pulverization in S1 is 20 Hz and the time is 60 s.

[0037] The enzyme in S2 is cellulase.

[0038] The enzymatic hydrolysis temperature in S2 is 40 °C and the time is 3 days.

[0039] The preparation method of the modified ultrafiltration membrane is as follows:

[0040] A1: Place the ultrafiltration membrane in a radio frequency plasma generator, and use a mixed gas of α-linolenic acid vapor and nitrogen for treatment, wherein the ratio of the mixed gas is α-linolenic acid vapor: nitrogen = 1:5, and the treatment time is 30 seconds to obtain an ultrafiltration membrane with double bond groups;

[0041] A2: In a stirring kettle, 2 g of p-hydroxycinnamic acid, 0.01 g of cellulase (CAS No.: 9012-54-8), 0.03 g of acetic acid and 200 g of ethanol are mixed, heated to 60 °C, and stirred at this temperature for 2 hours to obtain a mixed solution;

[0042] A3: Add 20 g of ultrafiltration membrane with double bond groups and 0.002 g of potassium peroxide to the mixed solution obtained in step 2, and continue to stir at 60 °C for 5 hours;

[0043] A4: Take out the ultrafiltration membrane treated in step 3 from the stirring kettle, wash it with absolute ethanol, and then dry it to obtain a modified ultrafiltration membrane.

[0044] The power of the radio frequency plasma generator is 50 W, the gas flow rate is 10 sccm, and the working pressure is controlled at 15 Pa.

[0045] After analysis and calculation, the enzymatic hydrolysis extraction rate of the wild ginseng product in this example is 85.46%.

[0046] Example 2

[0047] A method for enzymatic hydrolysis extraction of a wild ginseng-like product, the operation steps of which are:

[0048] S1: Put 6 g of wild ginseng-like product into an ultrafine grinder to crush it, add 110 g of distilled water, soak for 40 min, reflux in a water bath at 100 °C for 70 min, repeat the extraction 2 times, and combine the extraction solutions;

[0049] S2: Add 0.5 g of enzyme to the extraction solution, heat it for enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0050] S3: After the enzymatic hydrolysis product is extracted by the modified ultrafiltration membrane, it is rotary evaporated and dried to obtain a wild ginseng-like product extract.

[0051] The frequency of the ultrafine grinding in S1 is 25 Hz, and the time is 70 s.

[0052] The enzyme in S2 is hemicellulase.

[0053] The enzymatic hydrolysis temperature in S2 is 45 °C, and the time is 4 days.

[0054] The preparation method of the modified ultrafiltration membrane is:

[0055] A1: Place the ultrafiltration membrane in a radio frequency plasma generator, and treat it with a mixed gas of α-linolenic acid vapor and nitrogen, where the ratio of the mixed gas is α-linolenic acid vapor: nitrogen = 1:6, and the treatment time is 40 seconds to obtain an ultrafiltration membrane with double bond groups;

[0056] A2: In a stirring kettle, 3 g of p-hydroxycinnamic acid, 0.1 g of cellulase (CAS No.: 9012-54-8), 0.2 g of acetic acid and 250 g of ethanol are mixed, heated to 65 °C, and stirred at this temperature for 3 hours to obtain a mixed solution;

[0057] A3: Add 23 g of a double-bonded ultrafiltration membrane and 0.02 g of potassium peroxide to the mixed solution obtained in step 2, and continue to stir at 65 °C for 6 hours;

[0058] A4: Take out the ultrafiltration membrane treated in step 3 from the stirring kettle, wash it with absolute ethanol, and then dry it to obtain a modified ultrafiltration membrane.

[0059] The power of the radio frequency plasma generator is 60 W, the gas flow rate is 15 sccm, and the working pressure is controlled at 16 Pa.

[0060] After analysis and calculation, the enzymatic hydrolysis extraction rate of the wild ginseng product in this example is 86.51%.

[0061] Example 3

[0062] A method for enzymatic hydrolysis extraction of a wild ginseng-like product, the operating steps of which are:

[0063] S1: Put 9 g of wild ginseng-like product into an ultrafine grinder for pulverization, add 140 g of distilled water, soak for 50 min, reflux in a water bath at 100 °C for 80 min, repeat the extraction 2 times, and combine the extraction solutions;

[0064] S2: Add 1 g of enzyme to the extraction solution, heat it for enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0065] S3: After the enzymatic hydrolysis product is extracted by the modified ultrafiltration membrane, rotary evaporation and drying are carried out to obtain a wild ginseng-like product extract.

[0066] The frequency of the ultrafine pulverization in S1 is 25 Hz and the time is 80 s.

[0067] The enzyme in S2 is acid protease.

[0068] The enzymatic hydrolysis temperature in S2 is 55 °C and the time is 5 days.

[0069] The preparation method of the modified ultrafiltration membrane is:

[0070] A1: Place the ultrafiltration membrane in a radio frequency plasma generator, and treat it with a mixed gas of α-linolenic acid vapor and nitrogen, where the ratio of the mixed gas is α-linolenic acid vapor: nitrogen = 1:7, and the treatment time is 50 seconds to obtain a double-bonded ultrafiltration membrane;

[0071] A2: In a stirring kettle, 4 g of p-hydroxycinnamic acid, 0.2 g of cellulase (CAS No.: 9012-54-8), 0.4 g of acetic acid and 300 g of ethanol are mixed, heated to 65 °C, and stirred at this temperature for 4 hours to obtain a mixed solution;

[0072] A3: Add 28 g of double-bonded ultrafiltration membrane and 0.04 g of potassium peroxide to the mixed solution obtained in step 2, and continue to stir at 65 °C for 9 hours;

[0073] A4: Take out the ultrafiltration membrane treated in step 3 from the stirring kettle, wash it with absolute ethanol, and then dry it to obtain a modified ultrafiltration membrane.

[0074] The power of the radio frequency plasma generator is 90 W, the gas flow rate is 15 sccm, and the working air pressure is controlled at 18 Pa.

[0075] Through analysis and calculation, the enzymatic hydrolysis extraction rate of the wild ginseng product in this example is 87.35%.

[0076] Example 4

[0077] A method for enzymatic hydrolysis extraction of a wild ginseng-like product, the operation steps of which are:

[0078] S1: Put 10 g of wild ginseng-like product into an ultrafine pulverizer for pulverization, add 150 g of distilled water, soak for 60 min, reflux in a water bath at 100 °C for 90 min, repeat the extraction 2 times, and combine the extraction solutions;

[0079] S2: Add 1.5 g of enzyme to the extraction solution, raise the temperature for enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0080] S3: After the enzymatic hydrolysis product is extracted by the modified ultrafiltration membrane, rotary evaporation and drying are carried out to obtain a wild ginseng-like product extract.

[0081] The frequency of the ultrafine pulverization in S1 is 30 Hz and the time is 90 s.

[0082] The enzyme in S2 is β-glucanase.

[0083] The enzymatic hydrolysis temperature in S2 is 60 °C and the time is 6 days.

[0084] The preparation method of the modified ultrafiltration membrane is:

[0085] A1: Place the ultrafiltration membrane in a radio frequency plasma generator, and treat it with a mixed gas of α-linolenic acid vapor and nitrogen, wherein the ratio of the mixed gas is α-linolenic acid vapor: nitrogen = 1:8, and the treatment time is 60 seconds to obtain a double-bonded ultrafiltration membrane;

[0086] A2: In a stirring kettle, 5 g of p-hydroxycinnamic acid, 0.3 g of cellulase (CAS No.: 9012-54-8), 0.5 g of acetic acid and 350 g of ethanol are mixed, heated to 70 °C, and stirred at this temperature for 5 hours to obtain a mixed solution;

[0087] A3: Add 30 g of ultrafiltration membrane with double bond groups and 0.05 g of potassium peroxide to the mixed solution obtained in step 2, and continue to stir at 70 °C for 10 hours;

[0088] A4: Take out the ultrafiltration membrane treated in step 3 from the stirring kettle, wash it with anhydrous ethanol, and then dry it to obtain a modified ultrafiltration membrane.

[0089] The power of the radio frequency plasma generator described is 100 W, the gas flow rate is 0 sccm, and the working pressure is controlled at 20 Pa.

[0090] Through analysis and calculation, the enzymatic hydrolysis extraction rate of the wild ginseng product in this example is 88.92%.

[0091] Comparative Example 1

[0092] Do not modify the ultrafiltration membrane, and the others are the same as in Example 1.

[0093] Through analysis and calculation, the enzymatic hydrolysis extraction rate of the wild ginseng product in this example is 58.37%.

[0094] Comparative Example 2

[0095] Do not add α-linolenic acid, and the others are the same as in Example 1.

[0096] Through analysis and calculation, the enzymatic hydrolysis extraction rate of the wild ginseng product in this example is 66.29%.

[0097] Comparative Example 3

[0098] Do not add cellulase, and the others are the same as in Example 1.

[0099] Through analysis and calculation, the enzymatic hydrolysis extraction rate of the wild ginseng product in this example is 70.31%.

[0100] Through the data analysis of the above examples and comparative examples, the modified ultrafiltration membrane prepared by the present invention can effectively improve the extraction rate of wild ginseng-like products.

[0101] The above examples of the present invention are only illustrations for clearly explaining the present invention, and are not limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for enzymatically extracting a wild ginseng-like product, the operating steps of which are as follows: S1: Put 5 - 10 parts of wild ginseng-like product into an ultrafine pulverizer for pulverization, add 100 - 150 parts of distilled water, soak for 30 - 60 min, reflux in a water bath at 100 °C for 60 - 90 min, repeat the extraction 2 times, and combine the extracts; S2: Add 0.1 - 1.5 parts of enzyme to the extract, raise the temperature for enzymatic hydrolysis to obtain an enzymolysis product; S3: After the enzymolysis product is extracted by a modified ultrafiltration membrane, rotary evaporation and drying are carried out to obtain a wild ginseng-like product extract; The preparation method of the modified ultrafiltration membrane is as follows: A1: Place the ultrafiltration membrane in a radio frequency plasma generator and treat it with a mixed gas of α-linolenic acid vapor and nitrogen, where the ratio of the mixed gas is α-linolenic acid vapor : nitrogen = 1 : 5 - 8, and the treatment time is 30 - 60 seconds to obtain an ultrafiltration membrane with double bond groups; A2: In a stirring kettle, mix 2 - 5 parts of p-coumaric acid, 0.01 - 0.3 parts of cellulase, 0.03 - 0.5 parts of acetic acid and 200 - 350 parts of ethanol, raise the temperature to 60 - 70 °C, and stir at this temperature for 2 - 5 hours to obtain a mixed solution; A3: Add 20 - 30 parts of the ultrafiltration membrane with double bond groups and 0.002 - 0.05 parts of potassium peroxide to the mixed solution obtained in step 2, and continue to stir at 60 - 70 °C for 5 - 10 hours; A4: Take out the ultrafiltration membrane treated in step 3 from the stirring kettle, wash it with absolute ethanol, and then dry it to obtain a modified ultrafiltration membrane; The enzyme in S2 is one of cellulase, hemicellulase, acid protease, pectinase, and β-glucanase.

2. The enzymatic extraction method of a wild mountain ginseng-like product according to claim 1, characterized in that: The frequency of the ultra-fine pulverization in S1 is 20 - 30 Hz, and the time is 60 - 90 s.

3. A method for enzymolysis extraction of a wild mountain ginseng-like product according to claim 1, characterized in that: The enzymatic hydrolysis temperature in S2 is 40 - 60 °C, and the time is 3 - 6 days.

4. A method for enzymatic extraction of a wild mountain ginseng-like product according to claim 1, characterized in that: The power of the radio frequency plasma generator is 50 - 100 W, the gas flow rate is 10 - 20 sccm, and the working air pressure is controlled at 15 - 20 Pa.