Composition containing ginseng extract, preparation method and application thereof

Abstract

The present invention proposes a composition containing ginseng extract, a preparation method and application thereof, and belongs to the technical field of fermentation and extraction. The enzyme liquid is obtained by fermenting white ginseng fungus, ginseng and astragalus powder are added, enzymolysis is performed, and heating extraction is performed. The obtained product is mixed with a fermentation-promoting composition, glucose, and water, and fermented with rhamnosus lactobacillus and selenium-enriched yeast. The obtained fermentation product is added to a metal ion solution, stirred to obtain a Zn-Fe-fermentation complex, mixed with resveratrol and cordycepin, added to molten PEG6000, evenly dispersed, freeze-cured, dried, crushed, sieved, and a composition containing ginseng extract is obtained, which has good antioxidant, anti-aging, and anti-tumor effects. At the same time, it is easy for human body to absorb and contains almost no pesticide residues or heavy metals, has the characteristics of fast dissolution rate, fast drug effect, etc., and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation extraction, and in particular to a composition containing ginseng extract, a preparation method and application thereof. Background Art

[0002] Cells are the basic units of structure and function of organisms, and are also the basic units of aging. Cell aging refers to the process in which cells gradually decline in their proliferation and differentiation abilities and physiological functions over time as they perform their life activities. The life course of a cell goes through several stages: undifferentiation, differentiation, growth, maturity, aging, and death.

[0003] Although aging and death are inevitable natural laws, delaying aging, especially trying to avoid pathological aging, is possible. It is reported that by 2050, the number of people over 60 years old in the world will reach 2 billion, accounting for about 20-30% of the total population. Faced with the trend of accelerated population aging and generally increased life expectancy, ensuring that the elderly enjoy good health and a higher quality of life has become a major issue of common concern to social sciences and life sciences. In-depth research on more effective anti-aging products will certainly be conducive to the realization of my country's goal of "healthy aging". Therefore, conducting research on delaying aging and developing products that delay aging have important scientific significance and social value.

[0004] Ginseng (Panax ginseng CA Meyer) is a plant belonging to the genus Panax of the Araliaceae family. It has been used as a herbal medicine for about 2,000 years in Korea, China, Japan, etc., and is used to prevent diseases and prolong life based on experience. Ginsenoside, the representative physiologically active ingredient of ginseng, is evenly distributed in the above-ground and underground parts of ginseng. When using ginseng, its root is mainly used, but ginsenoside is also contained in ginseng leaves and ginseng seeds, and has the same effect as ginseng roots. So far, ginseng is known to have the following effects: positive effects on the central nervous system, anti-carcinogenic effects, anti-cancer activity, immune function control, anti-diabetic effects, liver function improvement effects, cardiovascular disorder improvement, anti-atherosclerosis effects, blood pressure control, menopause improvement, osteoporosis condition improvement, anti-stress and anti-fatigue effects, antioxidant effects, anti-aging effects, etc.

[0005] The current ginseng extraction methods do not produce high levels of active ingredients, and the human body has a low absorption rate. In addition, ginseng extracts prepared by different preparation methods have different activities. In addition, there are many types of pesticides remaining in ginseng and its extracts, which are not easy to degrade and generally have the risk of mutagenicity, carcinogenicity, and teratogenicity.

[0006] Chinese patent CN102771790B discloses an anti-aging nutritional health product, which is composed of yam extract, ginseng, Millettia reticulata, licorice, Polygonum multiflorum, coix seed, Poria cocos, cassia twig and oyster shell powder. The health product can increase calcium intake to slow down osteoporosis caused by aging, soften blood vessels to slow down the occurrence of cardiovascular and cerebrovascular diseases and stroke, and can alleviate the typical symptoms of aging. However, it can only improve the symptoms of aging, and has no obvious effect on delaying aging and enhancing immunity, and cannot meet the needs of modern people.

[0007] Therefore, it is of great practical significance to develop a method to obtain a ginseng extract with a high content of active ingredients, good anti-tumor, anti-aging, and antioxidant activities, easy for human body absorption, and almost no pesticide residues. Summary of the invention

[0008] The purpose of the present invention is to provide a composition containing ginseng extract and its preparation method and application, which has good antioxidant, anti-aging and anti-tumor effects, is easily absorbed by the human body and contains almost no pesticide residues or heavy metals, has the characteristics of fast dissolution rate and fast drug effect, and has broad application prospects.

[0009] The technical solution of the present invention is achieved in this way:

[0010] The invention provides a preparation method of a composition containing ginseng extract. The enzyme liquid is obtained by fermenting white ginseng fungi, ginseng and astragalus powder are added, enzymolysis is performed, and the mixture is heated to boiling water for extraction. The enzymolysis water extraction mixture is mixed with a fermentation-promoting composition, glucose and water, and fermented with rhamnosus lactobacillus and selenium-enriched yeast. The obtained fermentation product is added to a metal ion solution, stirred to obtain a Zn-Fe-fermentation complex, mixed with resveratrol and cordycepin, added to molten PEG6000, evenly dispersed, freeze-cured, dried, crushed, and sieved to obtain the composition containing ginseng extract.

[0011] As a further improvement of the present invention, the following steps are included:

[0012] S1. Preparation of enzyme fermentation medium: adding a carbon source, a nitrogen source, vitamins, and minerals to water, sterilizing, and preparing an enzyme fermentation medium;

[0013] S2. Fermentation and enzyme production: adding sterile water to the slant culture medium of the white ginseng fungus to wash out the colonies on the slant, preparing a bacterial seed solution with a buffer solution, inoculating it into the enzyme production fermentation medium in step S1, fermenting and culturing, centrifuging and sterilizing, obtaining a supernatant, adding ammonium sulfate, standing and precipitating, centrifuging, dissolving the precipitate in a buffer solution, dialyzing, centrifuging the dialyzate, collecting the supernatant, and obtaining an enzyme solution;

[0014] S3. Enzymatic water extraction: Wash ginseng and astragalus separately, dry and crush to obtain a mixed powder, add the enzyme solution prepared in step S2, heat enzymolysis, continue to heat to boiling extraction, filter, retain the solid, the filtrate is purified by macroporous resin, the eluent is mixed with the solid to obtain an enzymatic water extract mixture;

[0015] S4. Fermentation: adding the enzymatic water extract mixture obtained in step S3, the fermentation-promoting composition, and glucose into water, sterilizing, inoculating activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid, fermenting, centrifuging to remove solids, collecting the supernatant, and freeze-drying to obtain a fermentation product;

[0016] S5. Complexation of metal ions: The fermentation product obtained in step S4 is added to water, a soluble zinc salt and a soluble iron salt are added, the mixture is stirred and mixed, dried, dialyzed, and the dialyzate is dried to obtain a Zn-Fe-fermentation complex;

[0017] S6. Preparation of additives: mixing resveratrol and cordycepin to obtain an additive;

[0018] S7. Preparation of a composition containing ginseng extract: heat PEG6000 until it is melted, add the Zn-Fe-fermentation complex prepared in step S5 and the additive prepared in step S6, stir and mix evenly, freeze-solidify, dry, crush, and sieve to obtain a composition containing ginseng extract.

[0019] As a further improvement of the present invention, the mass ratio of the carbon source, nitrogen source, vitamins, minerals and water in step S1 is 70-100:7-12:1-2:0.5-1:300-400, the carbon source is sugarcane juice with a sugar content of 5-7, the nitrogen source is selected from at least one of amino acids, sodium chloride, ammonium nitrate, sodium nitrate, potassium nitrate, urea, fish meal, and peptone, the amino acids are selected from glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine, and phenylalanine, the vitamins are selected from at least one of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin E, vitamin D1, vitamin D2, and vitamin D3, and the minerals are selected from at least one of potassium chloride, sodium chloride, potassium sulfate, sodium sulfate, magnesium chloride, ferric chloride, ferrous chloride, cupric chloride, manganese chloride, zinc chloride, calcium chloride, magnesium sulfate, cupric sulfate, ferric sulfate, manganese sulfate, and calcium sulfate.

[0020] As a further improvement of the present invention, the bacterial content of the bacterial seed liquid in step S2 is 10 7 -10 8cfu / mL, the inoculation amount is 2-3v / v%, the fermentation culture conditions are 23-26°C, 50-70r / min, the fermentation is 36-48h, the amount of ammonium sulfate added is 80-85wt% of ammonium sulfate added to the system, the static sedimentation time is 2-4h, the dialysis bag pore size is 1000-1500D, and the dialysis time is 3-5h.

[0021] As a further improvement of the present invention, the mass ratio of ginseng and astragalus in step S3 is 10-15:3-5, the mass ratio of the mixed powder and the enzyme solution is 10-15:100, the temperature of the heating enzymatic hydrolysis is 35-40°C, the time is 2-4h, the time of heating to boiling extraction is 1-3h, the macroporous resin is D101 macroporous resin, HPD100 macroporous resin or SYD8 macroporous resin, and the purification method is to pass the sample through the macroporous resin at a rate of 0.1-0.2g / mL, then elute with 1.5-2.5BV water, and then elute with 3-5BV 70wt% ethanol, collect the eluate, and recover the ethanol.

[0022] As a further improvement of the present invention, the mass ratio of the enzymatic water extraction mixture, the fermentation-promoting composition, glucose, and water in step S4 is 15-20:2-3:5-7:400-500, the fermentation-promoting composition includes inulin, vitamin B6, and calcium chloride in a mass ratio of 10-12:0.5-1:0.2-0.5, the inoculation amounts of the activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid are 2-3 v / v% and 1-2 v / v%, respectively, and the bacterial content of the seed liquid is 10 8 -10 9 cfu / mL, and the fermentation culture conditions are 37-40°C, 50-70r / min, and the fermentation culture is 48-72h.

[0023] As a further improvement of the present invention, the soluble zinc salt in step S5 is selected from at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc citrate, and zinc gluconate, and the soluble iron salt is selected from at least one of ferric chloride, ferric sulfate, and ferric nitrate; the mass ratio of the fermentation product, the soluble zinc salt, and the soluble iron salt is 100:2-3:1-2, the dialysis bag pore size of the dialysis is 300-500D, and the dialysis time is 1-2h.

[0024] As a further improvement of the present invention, the mass ratio of resveratrol and cordycepin in step S6 is 3-5:7-10; the mass ratio of PEG6000, Zn-Fe-fermentation complex and additive in step S7 is 100:10-12:3-5, the freezing temperature is -25 to -20°C, the curing time is 2-4h, and the mesh size of the sieve is 80-100 mesh.

[0025] The present invention further protects a composition containing ginseng extract prepared by the above preparation method.

[0026] The present invention further protects the use of the above-mentioned composition containing ginseng extract in the preparation of anti-aging, anti-oxidation and anti-tumor medicines.

[0027] The present invention has the following beneficial effects:

[0028] The present invention firstly ferments and cultures white ginseng fungus in an enzyme-producing fermentation medium, during which a variety of enzymes can be produced, including cellulase, pectinase, hemicellulase, protease, etc., which can play a good biocatalytic role and also contain some enzymes that can promote the bioconversion of high-content saponins in ginseng into rare saponins.

[0029] The ginseng and astragalus added in the present invention have a wide range of pharmacological activities, among which the active ingredient ginsenoside of ginseng has anti-apoptosis, anti-oxidation, anti-tumor and the like effects. The active ingredient astragalus polysaccharide of astragalus has a pharmacological effect of anti-oxidative damage. The combination of the two can significantly improve the activity of superoxide dismutase (SOD), increase the activity of antioxidant enzymes, inhibit the formation of lipid peroxides, reduce the content of malondialdehyde (MDA) in various tissues, and significantly improve the body's anti-aging, anti-oxidation, anti-tumor and the like abilities.

[0030] The present invention adds ginseng and astragalus to the prepared enzyme solution, and under the action of the composite enzyme in the enzyme solution, the plant cell walls of ginseng and astragalus are broken, the content is dissolved, and the extraction rate of active substances such as ginsenosides, ginseng polysaccharides, astragalus polysaccharides, proteins, flavonoids, triterpenes, etc. is greatly improved. At the same time, cellulose in the matrix can also be catalyzed to produce chlorogenic acid, polyphenols, etc., and most of the active substances can be extracted under the action of heated water extraction.

[0031] Subsequently, the extract was filtered. Since ginseng contains relatively high levels of pesticide residues and heavy metals, the filtrate was purified by filtering with a macroporous resin. The macroporous resin utilizes the screening properties of the van der Waals force and porous mesh structure. Under the dual separation effects of adsorption capacity and mesh screening capacity, the pesticide residues and heavy metal ions are separated from other active components in the extract according to their structural characteristics and the analytical capacity of the eluent. This can significantly remove heavy metal impurities and pesticide residues. It has many advantages, such as large adsorption capacity, good selectivity, easy desorption, and convenient pretreatment. It can also further increase the content and purity of active substances in the extract.

[0032] The enzymatic water extraction mixture obtained by mixing the residual liquid after purification and recovery of ethanol with the filtered solid is inoculated with rhamnosus lactobacillus and selenium-enriched yeast. Under the synergistic action of the composite bacteria, the common saponins Rb1, Rb2, and Rc in the ginseng extract can be promoted to be transformed into rare saponins Rh1, Rg2, Rd, Rh4, Rh2, Rg1, Rg5, Rg3, etc., so that the content of rare saponins is increased. The higher the content of rare saponins, the stronger the free radical scavenging ability, the stronger the antioxidant ability, the anti-aging ability, and the anti-tumor ability. The solid residue after ginseng and astragalus extraction is promoted to ferment complex glycoproteins and other structures into small molecular active substances under the action of fermentation bacteria, and these substrates are further fermented and utilized by the fermentation bacteria to produce a large amount of short-chain fatty acids, lactic acid, oligosaccharides, polypeptides, amino acids, etc., and the enzymes secreted by microorganisms can be used to decompose and consume impurities such as sugars and proteins to the greatest extent, thereby improving the purity of ginsenosides, obtaining more types of conversion products, and significantly improving the conversion rate of rare saponins. The synergistic effect of fermentation bacteria can convert complex macromolecular phenolic substances into small molecular phenolic substances, increase the total phenol content, and destroy the plant cell wall, increasing the total flavonoid content. Selenium-enriched yeast can also promote the production of a large number of organic selenium compounds with excellent antioxidant, anti-aging and anti-tumor activities, increasing the selenium content in the product.

[0033] The fermentation-promoting composition added during the fermentation process includes inulin, vitamin B6 and calcium chloride. Inulin is a good prebiotic that can selectively promote the proliferation of probiotics including Lactobacillus rhamnosus and selenium-enriched yeast, greatly improving the bacterial content of the fermentation bacteria. Vitamin B6 can prolong the stable period of the fermentation bacteria, thereby greatly promoting the production efficiency of active substances. Calcium chloride can promote the resistance of the fermentation bacteria to harsh environments and increase its ability to resist the external environment, thereby also improving the fermentation yield.

[0034] Cordycepin is the main active ingredient in Cordyceps sinensis or Cordyceps militaris. It can effectively regulate the homeostasis of intracellular glutathione (GSH), thereby inhibiting the accumulation of reactive oxygen free radicals (ROS). By activating the PI3K / Akt signaling pathway, it upregulates the intracellular antioxidant defense level, thereby preventing cellular oxidative damage and achieving anti-aging. Resveratrol is a non-flavonoid polyphenol compound with excellent antioxidant, anti-aging and anti-cancer effects. It can activate the silent information factor SIRT1, simulate the anti-aging response of calorie restriction, participate in the regulation of the average life span of organic organisms, delay aging and prolong life, and has a significant inhibitory effect on a variety of tumor cells. The two can exert better anti-tumor, anti-aging and antioxidant effects through synergistic effects.

[0035] The present invention mixes the prepared fermentation product with zinc ions and iron ions to form a complex, which can improve the activity of multiple enzymes in the body and enhance the body's ability to scavenge free radicals, thereby effectively protecting the structure and function of the biofilm, protecting the cell membrane from damage by oxygen free radicals, extending the life of the cell, improving the body's immunity, and improving the anti-tumor ability, and the two have a synergistic effect.

[0036] The present invention adds the prepared Zn-Fe-fermentation complex and additives into a PEG6000 melt, and forms a solid dispersion of the poorly soluble Zn-Fe-fermentation complex and a suitable carrier PEG6000. Due to the crystal inhibition effect of the carrier material PEG6000, the drug exists in an amorphous dispersed state and the wettability of the drug is increased, so that after contacting with gastrointestinal fluid, the dissolution rate of the drug is accelerated, the absorption of the drug is promoted, the in vitro dissolution characteristics of the Zn-Fe-fermentation complex are improved, the dissolution rate of the drug is accelerated, and the effect of the drug is promoted.

[0037] The composition containing ginseng extract prepared by the present invention has good anti-oxidation, anti-aging and anti-tumor effects, is easily absorbed by the human body and contains almost no pesticide residues or heavy metals, has the characteristics of fast dissolution rate and fast drug effect, and has broad application prospects. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0039] White ginseng bacteria were purchased from Gulutong (Yunnan) Agricultural Development Co., Ltd.; Lactobacillus rhamnosus, 10 billion cfu / g, was purchased from Xi'an Yatu Biotechnology Co., Ltd.; selenium-enriched yeast was purchased from Angel Yeast Co., Ltd.

[0040] D101 macroporous resin was purchased from Dezhou Runxin Experimental Instrument Co., Ltd.; HPD100 macroporous resin was purchased from Langfang Miaoyang Chemical Co., Ltd.

[0041] The preparation method of activated Lactobacillus rhamnosus and selenium-enriched yeast seed solution is as follows: Lactobacillus rhamnosus and selenium-enriched yeast are inoculated into Gao's medium respectively, and fermented and cultured at 38°C and 70r / min for 24h to obtain a culture medium with a bacterial content of 10 8 cfu / mL of bacterial seed solution.

[0042] Example 1

[0043] This embodiment provides a method for preparing a composition containing ginseng extract, which specifically comprises the following steps:

[0044] S1. Preparation of enzyme fermentation medium: 70 parts by weight of sugar cane juice with a sugar content of 7, 7 parts by weight of urea, 0.3 parts by weight of vitamin A, 0.5 parts by weight of vitamin C, 0.2 parts by weight of vitamin E, 0.2 parts by weight of sodium sulfate, 0.05 parts by weight of magnesium chloride, 0.1 parts by weight of ferric chloride, 0.05 parts by weight of ferrous chloride, 0.1 parts by weight of cupric chloride were added to 300 parts by weight of water and sterilized to prepare an enzyme fermentation medium;

[0045] S2. Fermentation and enzyme production: Sterile water was added to the slant culture medium of white ginseng fungus to wash out the colonies on the slant, and a bacterial seed solution containing 10 7 cfu / mL, inoculated into the enzyme-producing fermentation medium in step S1, with an inoculation amount of 2v / v%, 23°C, 50r / min, fermented for 36h, centrifuged to sterilize, obtained supernatant, added ammonium sulfate until the content of ammonium sulfate in the system was 80wt%, allowed to stand for 4h, centrifuged, the precipitate was dissolved in PBS buffer with a pH of 7, dialyzed for 3h with a dialysis bag with a pore size of 1000D, the dialyzate was centrifuged, and the supernatant was collected to obtain an enzyme solution;

[0046] S3. Enzymatic water extraction: 10 parts by weight of ginseng and 3 parts by weight of astragalus were washed, dried, and crushed to obtain a mixed powder, 10 parts by weight of the mixed powder was added to 100 parts by weight of the enzyme solution prepared in step S2, heated to 35 ° C, enzymatic hydrolysis for 2h, continued to heat to boiling extraction for 1h, filtered, the solid was retained, the filtrate was passed through a D101 macroporous resin at a rate of 0.1g / mL, and then eluted with 1.5BV of water, and then eluted with 3BV of 70wt% ethanol, the eluate was collected, the ethanol was recovered, and the remaining liquid was mixed with the above solid to obtain an enzymatic water extraction mixture;

[0047] S4. Fermentation: 15 parts by weight of the enzymatic water extract mixture obtained in step S3, 2 parts by weight of the fermentation-promoting composition, and 5 parts by weight of glucose are added to 400 parts by weight of water, sterilized, and inoculated with activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid, wherein the inoculation amounts of the activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid are 2 v / v% and 1 v / v%, respectively, at 37°C, 50 r / min, and the fermentation is cultured for 48 hours, the solid is removed by centrifugation, the supernatant is collected, and freeze-dried to obtain a fermentation product;

[0048] The fermentation-promoting composition comprises inulin, vitamin B6 and calcium chloride in a mass ratio of 10:0.5:0.2;

[0049] S5. Complexation of metal ions: 100 parts by weight of the fermentation product obtained in step S4 was added to 500 parts by weight of water, 2 parts by weight of zinc nitrate and 1 part by weight of ferric chloride were added, stirred and mixed for 20 min, dried, dialyzed with a dialysis bag having a pore size of 300D for 1 h, and the dialysate was dried to obtain a Zn-Fe-fermentation complex;

[0050] S6. Preparation of additives: 3 parts by weight of resveratrol and 7 parts by weight of cordycepin were stirred and mixed for 15 min to obtain an additive;

[0051] S7. Preparation of a composition containing ginseng extract: Heat 100 parts by weight of PEG6000 until melted, add 10 parts by weight of the Zn-Fe-fermentation complex prepared in step S5 and 3 parts by weight of the additive prepared in step S6, stir and mix for 30 minutes, freeze and solidify at -25°C for 2 hours, dry, crush, and pass through an 80-mesh sieve to obtain a composition containing ginseng extract.

[0052] Example 2

[0053] This embodiment provides a method for preparing a composition containing ginseng extract, which specifically comprises the following steps:

[0054] S1. Preparation of enzyme fermentation medium: 100 parts by weight of sugar cane juice having a sugar content of 5, 12 parts by weight of fish meal, 0.3 parts by weight of vitamin B12, 0.5 parts by weight of folic acid, 1 part by weight of vitamin C, 0.2 parts by weight of vitamin E, 0.4 parts by weight of sodium chloride, 0.2 parts by weight of magnesium sulfate, 0.1 parts by weight of copper sulfate, 0.1 parts by weight of iron sulfate, 0.1 parts by weight of manganese sulfate, and 0.1 parts by weight of calcium sulfate were added to 400 parts by weight of water and sterilized to obtain an enzyme fermentation medium;

[0055] S2. Fermentation and enzyme production: Sterile water was added to the slant culture medium of white ginseng fungus to wash out the colonies on the slant, and a bacterial seed solution containing 10 8 cfu / mL, inoculated into the enzyme-producing fermentation medium in step S1, with an inoculation amount of 3v / v%, 26°C, 70r / min, fermented and cultured for 48h, centrifuged to sterilize, obtained supernatant, added ammonium sulfate until the content of ammonium sulfate in the system was 85wt%, allowed to stand for 2h, centrifuged, the precipitate was dissolved in PBS buffer with a pH of 7, dialyzed with a dialysis bag with a pore size of 1500D for 5h, the dialyzate was centrifuged, and the supernatant was collected to obtain an enzyme solution;

[0056] S3. Enzymatic water extraction: 15 parts by weight of ginseng and 5 parts by weight of astragalus were washed, dried, and crushed to obtain a mixed powder, and 15 parts by weight of the mixed powder were added to 100 parts by weight of the enzyme solution prepared in step S2, heated to 40°C, enzymatically hydrolyzed for 4 hours, and then heated to boiling for extraction for 3 hours, filtered, and the solid was retained. The filtrate was passed through a HPD100 macroporous resin at a rate of 0.2 g / mL, and then eluted with 2.5 BV of water, and then eluted with 5 BV of 70 wt% ethanol, the eluate was collected, the ethanol was recovered, and the remaining liquid was mixed with the above solid to obtain an enzymatic water extraction mixture;

[0057] S4. Fermentation: adding 20 parts by weight of the enzymatic water extract mixture obtained in step S3, 3 parts by weight of the fermentation-promoting composition, and 7 parts by weight of glucose to 500 parts by weight of water, sterilizing, inoculating with activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid, wherein the inoculation amounts of the activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid are 3 v / v% and 2 v / v%, respectively, at 40°C and 70 r / min, fermenting and culturing for 72 hours, removing solids by centrifugation, collecting the supernatant, and freeze-drying to obtain a fermentation product;

[0058] The fermentation-promoting composition comprises inulin, vitamin B6 and calcium chloride in a mass ratio of 12:1:0.5;

[0059] S5. Complexation of metal ions: 100 parts by weight of the fermentation product obtained in step S4 was added to 500 parts by weight of water, 3 parts by weight of zinc chloride and 2 parts by weight of ferric sulfate were added, stirred and mixed for 20 min, dried, dialyzed with a dialysis bag having a pore size of 500D for 2 h, and the dialysate was dried to obtain a Zn-Fe- fermentation complex;

[0060] S6. Preparation of additives: 5 parts by weight of resveratrol and 10 parts by weight of cordycepin were stirred and mixed for 15 min to obtain an additive;

[0061] S7. Preparation of a composition containing ginseng extract: Heat 100 parts by weight of PEG6000 until melted, add 12 parts by weight of the Zn-Fe-fermentation complex prepared in step S5 and 5 parts by weight of the additive prepared in step S6, stir and mix for 30 minutes, freeze and solidify at -20°C for 4 hours, dry, crush, and pass through a 100-mesh sieve to obtain a composition containing ginseng extract.

[0062] Example 3

[0063] This embodiment provides a method for preparing a composition containing ginseng extract, which specifically comprises the following steps:

[0064] S1. Preparation of enzyme fermentation medium: 85 parts by weight of sugar cane juice with a sugar content of 6, 10 parts by weight of peptone, 0.4 parts by weight of vitamin B1, 0.3 parts by weight of folic acid, 0.6 parts by weight of vitamin C, 0.2 parts by weight of vitamin E, 0.4 parts by weight of sodium chloride, 0.1 parts by weight of magnesium sulfate, 0.05 parts by weight of copper sulfate, 0.05 parts by weight of ferric chloride, 0.05 parts by weight of manganese chloride, 0.05 parts by weight of calcium sulfate were added to 350 parts by weight of water and sterilized to prepare an enzyme fermentation medium;

[0065] S2. Fermentation and enzyme production: Sterile water was added to the slant culture medium of white ginseng fungus to wash out the colonies on the slant, and a bacterial seed solution containing 10 8 cfu / mL, inoculated into the enzyme-producing fermentation medium in step S1, with an inoculation amount of 2.5 v / v%, 25°C, 60 r / min, fermented and cultured for 42 h, centrifuged to sterilize, obtained a supernatant, added ammonium sulfate until the content of ammonium sulfate in the system was 82 wt%, allowed to stand for precipitation for 3 h, centrifuged, the precipitate was dissolved in a PBS buffer solution with a pH of 7, dialyzed for 4 h with a dialysis bag with a pore size of 1200D, the dialyzate was centrifuged, and the supernatant was collected to obtain an enzyme solution;

[0066] S3. Enzymatic water extraction: 12 parts by weight of ginseng and 4 parts by weight of astragalus were washed, dried, and crushed to obtain a mixed powder, and 12 parts by weight of the mixed powder were added to 100 parts by weight of the enzyme solution prepared in step S2, heated to 37°C, enzymatically hydrolyzed for 3h, continued to heat to boiling and extracted for 2h, filtered, and the solid was retained. The filtrate was passed through a D101 macroporous resin at a rate of 0.15g / mL, and then eluted with 2BV of water, and then eluted with 4BV of 70wt% ethanol, the eluate was collected, the ethanol was recovered, and the remaining liquid was mixed with the above solid to obtain an enzymatic water extraction mixture;

[0067] S4. Fermentation: 17 parts by weight of the enzymatic water extract mixture obtained in step S3, 2.5 parts by weight of the fermentation-promoting composition, and 6 parts by weight of glucose are added to 450 parts by weight of water, sterilized, and inoculated with activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid, wherein the inoculation amounts of the activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid are 2.5 v / v% and 1.5 v / v%, respectively, at 38°C, 60 r / min, and the fermentation culture is carried out for 56 hours, the solid is removed by centrifugation, the supernatant is collected, and freeze-dried to obtain a fermentation product;

[0068] The fermentation-promoting composition comprises inulin, vitamin B6 and calcium chloride in a mass ratio of 11:0.7:0.35;

[0069] S5. Complexation of metal ions: 100 parts by weight of the fermentation product obtained in step S4 was added to 500 parts by weight of water, 2.5 parts by weight of zinc gluconate and 1.5 parts by weight of ferric nitrate were added, stirred and mixed for 20 min, dried, dialyzed with a dialysis bag having a pore size of 400D for 1.5 h, and the dialysate was dried to obtain a Zn-Fe- fermentation complex;

[0070] S6. Preparation of additives: 4 parts by weight of resveratrol and 8.5 parts by weight of cordycepin were stirred and mixed for 15 min to obtain an additive;

[0071] S7. Preparation of a composition containing ginseng extract: Heat 100 parts by weight of PEG6000 until melted, add 11 parts by weight of the Zn-Fe-fermentation complex prepared in step S5 and 4 parts by weight of the additive prepared in step S6, stir and mix for 30 minutes, freeze and solidify at -22°C for 3 hours, dry, crush, and pass through a 90-mesh sieve to obtain a composition containing ginseng extract.

[0072] Example 4

[0073] Compared with Example 3, the difference is that zinc gluconate is not added in step S5.

[0074] Example 5

[0075] Compared with Example 3, the difference is that no ferric nitrate is added in step S5.

[0076] Example 6

[0077] Compared with Example 3, the difference is that no inulin is added to the fermentation promoting composition, and the mass ratio of vitamin B6 and calcium chloride is 0.7:0.35.

[0078] Example 7

[0079] Compared with Example 3, the difference is that vitamin B6 is not added to the fermentation promoting composition, which includes inulin and calcium chloride in a mass ratio of 11:0.35.

[0080] Example 8

[0081] Compared with Example 3, the difference is that no calcium chloride is added to the fermentation promoting composition, and the composition includes inulin and vitamin B6 in a mass ratio of 11:0.7.

[0082] Comparative Example 1

[0083] Compared with Example 3, the difference is that no astragalus is added in step S3.

[0084] The details are as follows:

[0085] S3. Enzymatic hydrolysis and water extraction: 16 parts by weight of ginseng were washed, dried, and crushed to obtain a powder, and 12 parts by weight of the powder were added to 100 parts by weight of the enzyme solution prepared in step S2, heated to 37°C, and hydrolyzed for 3 hours, and then heated to boiling for extraction for 2 hours, filtered, and the solid was retained. The filtrate was passed through a D101 macroporous resin at a rate of 0.15 g / mL, and then eluted with 2BV of water, and then eluted with 4BV of 70wt% ethanol, the eluate was collected, the ethanol was recovered, and the remaining liquid was mixed with the above solid to obtain an enzymatic hydrolysis water extraction mixture.

[0086] Comparative Example 2

[0087] Compared with Example 3, the difference is that steps S1 and S2 are not performed, and the enzyme solution in step S3 is replaced by a mixed enzyme solution containing 100 U / mL cellulase and 50 U / mL pectinase.

[0088] The details are as follows:

[0089] S3. Enzymatic hydrolysis and water extraction: 12 parts by weight of ginseng and 4 parts by weight of astragalus were washed, dried and crushed to obtain a mixed powder. The 12 parts by weight of the mixed powder were added to 100 parts by weight of a mixed enzyme solution containing 100 U / mL cellulase and 50 U / mL pectinase, heated to 37°C, and enzymolyzed for 3 hours. The mixture was further heated to boiling and extracted for 2 hours. The mixture was filtered and the solid was retained. The filtrate was passed through a D101 macroporous resin at a rate of 0.15 g / mL, and then eluted with 2 BV of water and then with 4 BV of 70 wt% ethanol. The eluate was collected and the ethanol was recovered. The remaining liquid was mixed with the above solid to obtain an enzymatic hydrolysis and water extraction mixture.

[0090] Comparative Example 3

[0091] Compared with Example 3, the difference is that steps S1 and S2 are not performed, and enzymatic hydrolysis is not performed in step S3, and heating to boiling extraction is directly performed.

[0092] The details are as follows:

[0093] S3. Enzymatic water extraction: 12 parts by weight of ginseng and 4 parts by weight of astragalus were washed, dried and crushed to obtain a mixed powder. 12 parts by weight of the mixed powder were added to 100 parts by weight of water, heated to boiling and extracted for 2 hours, filtered, and the solid was retained. The filtrate passed through a D101 macroporous resin at a rate of 0.15 g / mL, and then eluted with 2BV of water, and then with 4BV of 70wt% ethanol. The eluate was collected, the ethanol was recovered, and the remaining liquid was mixed with the above-mentioned solid to obtain an enzymatic water extraction mixture.

[0094] Comparative Example 4

[0095] Compared with Example 3, the difference is that the Lactobacillus rhamnosus seed liquid is not inoculated in step S4.

[0096] The details are as follows:

[0097] S4. Fermentation: 17 parts by weight of the enzymatic water extract mixture obtained in step S3, 2.5 parts by weight of the fermentation-promoting composition, and 6 parts by weight of glucose are added to 450 parts by weight of water, sterilized, and inoculated with an activated selenium-enriched yeast seed solution, wherein the inoculation amount of the activated selenium-enriched yeast seed solution is 4 v / v%, 38°C, 60 r / min, and the fermentation culture is carried out for 56 hours, the solid is removed by centrifugation, the supernatant is collected, and freeze-dried to obtain a fermentation product;

[0098] The fermentation promoting composition comprises inulin, vitamin B6 and calcium chloride in a mass ratio of 11:0.7:0.35.

[0099] Comparative Example 5

[0100] Compared with Example 3, the difference is that no selenium-enriched yeast seed liquid is inoculated in step S4.

[0101] The details are as follows:

[0102] S4. Fermentation: 17 parts by weight of the enzymatic water extract mixture obtained in step S3, 2.5 parts by weight of the fermentation-promoting composition, and 6 parts by weight of glucose are added to 450 parts by weight of water, sterilized, and inoculated with activated Lactobacillus rhamnosus seed solution, wherein the inoculation amount of the activated Lactobacillus rhamnosus seed solution is 4 v / v%, 38°C, 60 r / min, fermented and cultured for 56 hours, centrifuged to remove solids, collected the supernatant, and freeze-dried to obtain a fermentation product;

[0103] The fermentation promoting composition comprises inulin, vitamin B6 and calcium chloride in a mass ratio of 11:0.7:0.35.

[0104] Comparative Example 6

[0105] Compared with Example 3, the difference is that no fermentation promoting composition is added in step S4.

[0106] The details are as follows:

[0107] S4. Fermentation: 17 parts by weight of the enzymatic water extract mixture obtained in step S3 and 6 parts by weight of glucose are added to 450 parts by weight of water, sterilized, and inoculated with activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid, wherein the inoculation amounts of the activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid are 2.5 v / v% and 1.5 v / v%, respectively. The fermentation is cultured at 38°C, 60 r / min, for 56 hours, the solids are removed by centrifugation, the supernatant is collected, and freeze-dried to obtain a fermentation product.

[0108] Comparative Example 7

[0109] Compared with Example 3, the difference is that step S4 is not performed.

[0110] The details are as follows:

[0111] S1. Preparation of enzyme fermentation medium: 85 parts by weight of sugar cane juice with a sugar content of 6, 10 parts by weight of peptone, 0.4 parts by weight of vitamin B1, 0.3 parts by weight of folic acid, 0.6 parts by weight of vitamin C, 0.2 parts by weight of vitamin E, 0.4 parts by weight of sodium chloride, 0.1 parts by weight of magnesium sulfate, 0.05 parts by weight of copper sulfate, 0.05 parts by weight of ferric chloride, 0.05 parts by weight of manganese chloride, 0.05 parts by weight of calcium sulfate were added to 350 parts by weight of water and sterilized to prepare an enzyme fermentation medium;

[0112] S2. Fermentation and enzyme production: Sterile water was added to the slant culture medium of white ginseng fungus to wash out the colonies on the slant, and a bacterial seed solution containing 10 8 cfu / mL, inoculated into the enzyme-producing fermentation medium in step S1, with an inoculation amount of 2.5 v / v%, 25°C, 60 r / min, fermented and cultured for 42 h, centrifuged to sterilize, obtained a supernatant, added ammonium sulfate until the content of ammonium sulfate in the system was 82 wt%, allowed to stand for precipitation for 3 h, centrifuged, the precipitate was dissolved in a PBS buffer solution with a pH of 7, dialyzed for 4 h with a dialysis bag with a pore size of 1200D, the dialyzate was centrifuged, and the supernatant was collected to obtain an enzyme solution;

[0113] S3. Enzymatic water extraction: 12 parts by weight of ginseng and 4 parts by weight of astragalus were washed, dried, and crushed to obtain a mixed powder, and 12 parts by weight of the mixed powder were added to 100 parts by weight of the enzyme solution prepared in step S2, heated to 37°C, enzymatically hydrolyzed for 3h, continued to heat to boiling and extracted for 2h, filtered, and the solid was retained. The filtrate was passed through a D101 macroporous resin at a rate of 0.15g / mL, and then eluted with 2BV of water, and then eluted with 4BV of 70wt% ethanol, the eluate was collected, the ethanol was recovered, and the remaining liquid was mixed with the above solid to obtain an enzymatic water extraction mixture;

[0114] S4. Complexation of metal ions: 100 parts by weight of the enzymatic water extract mixture obtained in step S3 was added to 500 parts by weight of water, 2.5 parts by weight of zinc gluconate and 1.5 parts by weight of ferric nitrate were added, stirred and mixed for 20 min, dried, dialyzed with a dialysis bag having a pore size of 400D for 1.5 h, and the dialysate was dried to obtain a Zn-Fe-fermentation complex;

[0115] S5. Preparation of additives: 4 parts by weight of resveratrol and 8.5 parts by weight of cordycepin were stirred and mixed for 15 min to obtain an additive;

[0116] S6. Preparation of a composition containing ginseng extract: 100 parts by weight of PEG6000 are heated until melted, 11 parts by weight of the Zn-Fe-fermentation complex prepared in step S4 and 4 parts by weight of the additive prepared in step S5 are added, the mixture is stirred for 30 minutes, frozen and solidified at -22°C for 3 hours, dried, crushed, and passed through a 90-mesh sieve to obtain a composition containing ginseng extract.

[0117] Comparative Example 8

[0118] Compared with Embodiment 3, the difference is that step S5 is not performed.

[0119] The details are as follows:

[0120] S1. Preparation of enzyme fermentation medium: 85 parts by weight of sugar cane juice with a sugar content of 6, 10 parts by weight of peptone, 0.4 parts by weight of vitamin B1, 0.3 parts by weight of folic acid, 0.6 parts by weight of vitamin C, 0.2 parts by weight of vitamin E, 0.4 parts by weight of sodium chloride, 0.1 parts by weight of magnesium sulfate, 0.05 parts by weight of copper sulfate, 0.05 parts by weight of ferric chloride, 0.05 parts by weight of manganese chloride, 0.05 parts by weight of calcium sulfate were added to 350 parts by weight of water and sterilized to prepare an enzyme fermentation medium;

[0121] S2. Fermentation and enzyme production: Sterile water was added to the slant culture medium of white ginseng fungus to wash out the colonies on the slant, and a bacterial seed solution containing 10 8 cfu / mL, inoculated into the enzyme-producing fermentation medium in step S1, with an inoculation amount of 2.5 v / v%, 25°C, 60 r / min, fermented and cultured for 42 h, centrifuged to sterilize, obtained a supernatant, added ammonium sulfate until the content of ammonium sulfate in the system was 82 wt%, allowed to stand for precipitation for 3 h, centrifuged, the precipitate was dissolved in a PBS buffer solution with a pH of 7, dialyzed for 4 h with a dialysis bag with a pore size of 1200D, the dialyzate was centrifuged, and the supernatant was collected to obtain an enzyme solution;

[0122] S3. Enzymatic water extraction: 12 parts by weight of ginseng and 4 parts by weight of astragalus were washed, dried, and crushed to obtain a mixed powder, and 12 parts by weight of the mixed powder were added to 100 parts by weight of the enzyme solution prepared in step S2, heated to 37°C, enzymatically hydrolyzed for 3h, continued to heat to boiling and extracted for 2h, filtered, and the solid was retained. The filtrate was passed through a D101 macroporous resin at a rate of 0.15g / mL, and then eluted with 2BV of water, and then eluted with 4BV of 70wt% ethanol, the eluate was collected, the ethanol was recovered, and the remaining liquid was mixed with the above solid to obtain an enzymatic water extraction mixture;

[0123] S4. Fermentation: 17 parts by weight of the enzymatic water extract mixture obtained in step S3, 2.5 parts by weight of the fermentation-promoting composition, and 6 parts by weight of glucose are added to 450 parts by weight of water, sterilized, and inoculated with activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid, wherein the inoculation amounts of the activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid are 2.5 v / v% and 1.5 v / v%, respectively, at 38°C, 60 r / min, and the fermentation culture is carried out for 56 hours, the solid is removed by centrifugation, the supernatant is collected, and freeze-dried to obtain a fermentation product;

[0124] The fermentation-promoting composition comprises inulin, vitamin B6 and calcium chloride in a mass ratio of 11:0.7:0.35;

[0125] S5. Preparation of additives: 4 parts by weight of resveratrol and 8.5 parts by weight of cordycepin were stirred and mixed for 15 min to obtain an additive;

[0126] S6. Preparation of a composition containing ginseng extract: 100 parts by weight of PEG6000 was heated until melted, 11 parts by weight of the fermentation product obtained in step S4 and 4 parts by weight of the additive obtained in step S5 were added, the mixture was stirred for 30 minutes, freeze-cured at -22°C for 3 hours, dried, crushed, and passed through a 90-mesh sieve to obtain a composition containing ginseng extract.

[0127] Comparative Example 9

[0128] Compared with Example 3, the difference is that resveratrol is not added in step S6.

[0129] The details are as follows:

[0130] S6. Preparation of additives: Cordycepin is an additive.

[0131] Comparative Example 10

[0132] Compared with Example 3, the difference is that cordycepin is not added in step S6.

[0133] The details are as follows:

[0134] S6. Preparation of additives: Resveratrol is an additive.

[0135] Comparative Example 11

[0136] Compared with Example 3, the difference is that no additive is added in step S7.

[0137] The details are as follows:

[0138] S7. Preparation of a composition containing ginseng extract: 100 parts by weight of PEG6000 was heated until melted, 15 parts by weight of the Zn-Fe-fermentation complex prepared in step S5 was added, stirred and mixed for 30 minutes, frozen and solidified at -22°C for 3 hours, dried, crushed, and passed through a 90-mesh sieve to obtain a composition containing ginseng extract.

[0139] Comparative Example 12

[0140] Compared with Example 3, the difference is that step S7 is a simple mixing of the Zn—Fe-fermentation complex and the additive.

[0141] The details are as follows:

[0142] S7. Preparation of a composition containing ginseng extract: 11 parts by weight of the Zn-Fe-fermentation complex prepared in step S5 and 4 parts by weight of the additive prepared in step S6 were stirred and mixed for 30 minutes to prepare a composition containing ginseng extract.

[0143] Test Example 1 Antioxidant Test

[0144] 1. Determination of hydroxyl free radical scavenging ability

[0145] Add 0.2 mL of 10 mmol / L FeSO 4 -EDTA mixed solution, 0.2mL 20mmol / L 2-D-deoxyribose solution, 0.2mL 1mg / mL composition containing ginseng extract prepared in Example 1-8 or Comparative Example 1-12, and supplemented to 1.8mL with PBS buffer at pH=7, and then added 0.2mL 10mmol / L H 2 O 2 Solution, heated in a 37℃ water bath for 1h, added 1mL 10% trichloroacetic acid to terminate the reaction, then added 1mL 1% thiobarbituric acid, mixed and heated in a boiling water bath for 10min, cooled and centrifuged to obtain the supernatant, and measured the absorbance at 532nm. Vitamin C (1mg / mL) was used as the positive control group.

[0146] The scavenging rate of hydroxyl radicals / % = (A 对照 -A 样品 ) / A 对照 ×100

[0147] 2. Effects on lipid peroxidation induced by hydrogen peroxide

[0148] The liver tissue of the mouse was washed and homogenized in an ice bath to prepare a 1 wt% suspension. 1 mL of the suspension was taken, 0.1 mL of the 1 mg / mL composition solution containing ginseng extract prepared in Example 1-8 or Comparative Example 1-12, 0.1 mL of 6 mmol / LFeSO4 Solution, 0.1mL 60mmol / LH 2 O 2 Solution, the control group added 0.1mL of pH=7 PBS buffer. After incubation at 37℃ for 1h, add 1mL of 15% trichloroacetic acid to terminate the reaction, centrifuge to take the supernatant, add 1mL of 0.67% thiobarbituric acid, boil in water bath for 15min, cool with running water, and measure the absorbance at wavelength 532nm. Vitamin C (1mg / mL) was used as the positive control group.

[0149] Lipid peroxidation inhibition rate / % = (A 对照 -A 样本 ) / A 对照 ×100

[0150] The results are shown in Table 1.

[0151] Table 1

[0152]

[0153]

[0154] It can be seen from the above table that the compositions containing ginseng extract prepared in Examples 1-3 of the present invention have good antioxidant capacity.

[0155] Test Example 2 Antioxidant Damage Capacity Test

[0156] 1. Effect on SH-SY5Y cell proliferation

[0157] SH-SY5Y cells (concentration 5.5×10 4 After inoculation with 500 μL of 50 μL DMEM medium (0.5 μg / mL), the cells were randomly divided into a zeroing group, a control group, and an experimental group (divided into Example 1-8 groups and Comparative Example 1-12 groups). After incubation for 24 hours, 200 μL of the corresponding drug-containing DMEM medium was added to the experimental group to make the final concentration of the drug 5 mg / mL, an equal amount of DMEM medium was added to the control group, and only 200 μL of medium was added to the zeroing group without drugs and cells. After 24 hours, the cell survival rate was measured. The results are shown in Table 2.

[0158] Cell survival rate = (A 实验 -A 调零 ) / (A 对照 -A 调零 )×100%, where A is the absorbance.

[0159] Table 2

[0160]

[0161]

[0162] It can be seen from the above table that the compositions containing ginseng extracts prepared in Examples 1-3 of the present invention can increase the survival rate of SH-SY5Y cells.

[0163] 2. Effects on SH-SY5Y cell damage

[0164] SH-SY5Y cells (concentration 5.5×10 4 After inoculation with 50 μg / mL of DMEM medium, the cells were randomly divided into a zeroing group, a control group, an experimental group (divided into Example 1-8 groups and Comparative Example 1-12 groups), and an injury model group. After incubation for 24 hours, 200 μL of the corresponding drug-containing DMEM medium was added to the experimental group to make the final concentration of the drug 5 mg / mL, the control group and the injury model group were added with an equal amount of DMEM medium, and only 200 μL of medium was added to the zeroing group. After pre-culture for 24 hours, 150 μmol / L of H 2 O 2 After incubation for 1.5 h, the cell viability was measured.

[0165] Cell survival rate = (A 实验 -A 调零 ) / (A 对照 -A 调零 )×100%, where A is the absorbance.

[0166] The results are shown in Table 3.

[0167] Table 3

[0168]

[0169]

[0170] As can be seen from the above table, the compositions containing ginseng extract prepared in Examples 1-3 of the present invention can significantly improve the 2 O 2 The survival rate of SH-SY5Y cells after oxidative damage was improved.

[0171] Test Example 2 Anti-aging ability test

[0172] SPF grade KM mice, male, 18-22g, were adaptively raised for 7 days and subjected to water maze experiment. The water maze was trained once a day, the first 6 trainings were carried out, and the 7th training was carried out. The qualified mice that could find the platform within 60s were selected and randomly divided into 23 groups, namely normal group, model group, positive control group, Example 1-8 group, and Comparative Example 1-12 group, 6 mice in each group; the normal group was injected subcutaneously with normal saline combined with intraperitoneal injection every day, and gavage with normal saline; the model group, positive control group, Example 1-8 group, and Comparative Example 1-12 group were injected subcutaneously with 150mg / kg D-galactose combined with intraperitoneal injection of 50mg / kg sodium nitrite to construct mouse aging model, the positive control group was gavaged with 1g / kg vitamin E after modeling, and the Example 1-8 group and Comparative Example 1-12 group were gavaged with 1g / kg of the corresponding prepared drugs after modeling. The water maze experiment was carried out at the 7th week, and all samples were dissolved in normal saline, and the experiment was carried out continuously for 8 weeks.

[0173] 1. Latency after water maze test

[0174] In the 7th week of modeling and drug administration, mice in each group were subjected to a water maze test, in which the rats were placed in the quadrant of the pool without an escape platform facing the pool wall, and the time (latency) for the rats to exit the water was recorded. The results are shown in Table 4.

[0175] Table 4

[0176]

[0177]

[0178] Note: * compared with the normal group, P < 0.05; # compared with the model group, P < 0.05.

[0179] As can be seen from the above table, the compositions containing ginseng extract prepared in Examples 1-3 of the present invention can significantly shorten the latency period in the mouse water maze experiment and have a good inhibitory effect on the reduction of learning and memory ability of mice caused by D-galactose.

[0180] 2. Detection of SOD, GSH-Px activity and MDA level in serum and various organ tissues

[0181] After the mice were given the drug for 8 consecutive weeks, the eyeballs were removed to collect blood, which was centrifuged to obtain serum; then 10% of the brain tissue was homogenized to obtain a homogenate, which was centrifuged and the supernatant was collected; the ELISA method was used to detect the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) levels in serum and various organ tissues according to the instructions of the kit. The results are shown in Table 5.

[0182] Table 5

[0183]

[0184]

[0185] Note: * compared with the normal group, P < 0.05; # compared with the model group, P < 0.05.

[0186] As can be seen from the above table, the compositions containing ginseng extracts prepared in Examples 1-3 of the present invention can significantly reduce the levels of MDA in serum and brain tissue, increase the activities of SOD and GSH-Px, and improve the body's antioxidant capacity.

[0187] Compared with Example 3, Examples 4 and 5 do not add zinc gluconate or ferric nitrate in step S5. Compared with Example 3, Comparative Example 8 does not perform step S5. Antioxidant ability, antioxidative damage and anti-aging ability are reduced. The present invention mixes the obtained fermentation product with zinc ions and iron ions to form a complex, which can improve the activity of various enzymes in the body and enhance the body's ability to scavenge free radicals, thereby effectively protecting the structure and function of the biofilm, protecting the cell membrane from damage by oxygen free radicals, prolonging cell life, improving human immunity, and improving anti-tumor ability, and the two have a synergistic effect.

[0188] Compared with Example 3, Examples 6, 7, and 8 do not include inulin, vitamin B6, or calcium chloride added to the fermentation-promoting composition. Compared with Example 3, no fermentation-promoting composition is added in step S4 in Comparative Example 6. Antioxidant capacity, antioxidant damage resistance, and anti-aging capacity are reduced. The fermentation-promoting composition added during the fermentation process includes inulin, vitamin B6, and calcium chloride. Inulin is a good prebiotic that can selectively promote the proliferation of probiotics including Lactobacillus rhamnosus and selenium-enriched yeast, greatly increasing the bacterial content of the fermentation bacteria. Vitamin B6 can prolong the stable period of the fermentation bacteria, thereby greatly promoting the production efficiency of active substances. Calcium chloride can promote the resistance of the fermentation bacteria to harsh environments, increase its ability to resist the external environment, and thus also increase the fermentation yield.

[0189] Comparative Example 1 Compared with Example 3, no astragalus was added in step S3. The ability to resist oxidative damage and anti-aging decreased. The ginseng and astragalus added in the present invention have a wide range of pharmacological activities, among which ginsenosides, the active ingredient of ginseng, have anti-apoptosis, anti-oxidation, anti-tumor and the like effects. Astragalus polysaccharide, the active ingredient of astragalus, has a pharmacological effect of resisting oxidative damage. The combination of the two can significantly increase the activity of superoxide dismutase (SOD), increase the activity of antioxidant enzymes, inhibit the formation of lipid peroxides, and reduce the content of malondialdehyde (MDA) in various tissues, which can significantly improve the body's ability to resist aging, oxidation, and tumors.

[0190] Compared with Example 3, Comparative Example 2 did not perform steps S1 and S2, and the enzyme solution in step S3 was replaced by a mixed enzyme solution containing 100U / mL cellulase and 50U / mL pectinase. Compared with Example 3, Comparative Example 3 did not perform steps S1 and S2, and no enzymolysis was performed in step S3, and the extraction was directly heated to boiling. Antioxidant capacity, antioxidant damage and anti-aging ability decreased. The present invention first ferments and cultures white ginseng fungus in an enzyme-producing fermentation medium, and can produce a variety of enzymes during the fermentation process, including cellulase, pectinase, hemicellulase, protease, etc., which can play a good biocatalytic role, and also contains some that can promote the bioconversion of high-content saponins in ginseng to rare saponins. The present invention adds ginseng and astragalus to the prepared enzyme solution, and under the action of the composite enzyme in the enzyme solution, the plant cell walls of ginseng and astragalus are broken, the content is dissolved, and the extraction rate of active substances such as ginsenosides, ginseng polysaccharides, astragalus polysaccharides, proteins, flavonoids, triterpenes, etc. is greatly improved. At the same time, cellulose in the matrix can also be catalyzed to produce chlorogenic acid, polyphenols, etc., and most of the active substances can be extracted under the action of heated water extraction.

[0191] Compared with Example 3, in Comparative Examples 4 and 5, Lactobacillus rhamnosus seed solution or selenium-enriched yeast seed solution was not inoculated in step S4. Compared with Example 3, step S4 was not performed in Comparative Example 7. Antioxidant capacity, antioxidative damage and anti-aging capacity decreased. The enzymatic water extraction mixture obtained by mixing the residual liquid after purification and recovery of ethanol with the filtered solid is inoculated with rhamnosus lactobacillus and selenium-enriched yeast. Under the synergistic action of the composite bacteria, the common saponins Rb1, Rb2, and Rc in the ginseng extract can be promoted to be transformed into rare saponins Rh1, Rg2, Rd, Rh4, Rh2, Rg1, Rg5, Rg3, etc., so that the content of rare saponins is increased. The higher the content of rare saponins, the stronger the free radical scavenging ability, the stronger the antioxidant ability, the anti-aging ability, and the anti-tumor ability. The solid residue after ginseng and astragalus extraction is promoted to ferment complex glycoproteins and other structures into small molecular active substances under the action of fermentation bacteria, and these substrates are further fermented and utilized by the fermentation bacteria to produce a large amount of short-chain fatty acids, lactic acid, oligosaccharides, polypeptides, amino acids, etc., and the enzymes secreted by microorganisms can be used to decompose and consume impurities such as sugars and proteins to the greatest extent, thereby improving the purity of ginsenosides, obtaining more types of conversion products, and significantly improving the conversion rate of rare saponins. The synergistic effect of fermentation bacteria can convert complex macromolecular phenolic substances into small molecular phenolic substances, increase the total phenol content, and destroy the plant cell wall, increasing the total flavonoid content. Selenium-enriched yeast can also promote the production of a large number of organic selenium compounds with excellent antioxidant, anti-aging and anti-tumor activities, increasing the selenium content in the product.

[0192] Comparative Examples 9 and 10 Compared with Example 3, resveratrol or cordycepin was not added in step S6. Antioxidant capacity, antioxidative damage and anti-aging ability decreased. Comparative Example 11 Compared with Example 3, no additive was added in step S7. Cordycepin is the main active ingredient in Cordyceps sinensis or Cordyceps militaris, which can effectively regulate the homeostasis of intracellular glutathione (GSH), thereby inhibiting the accumulation of a large number of reactive oxygen free radicals (ROS), by activating the PI3K / Akt signaling pathway, upregulating the intracellular antioxidant defense level, and then preventing cell oxidative damage, and achieving anti-aging. Resveratrol is a non-flavonoid polyphenol compound with excellent antioxidant, anti-aging and anti-cancer effects, which can simulate the anti-aging reaction of caloric restriction by activating the silent information factor SIRT1, participate in the regulation of the average life span of organic organisms, delay aging and prolong life, and have a significant inhibitory effect on a variety of tumor cells. Both can play a better anti-tumor, anti-aging and antioxidant effect through the synergistic effect.

[0193] Comparative Example 12 Compared with Example 3, step S7 is a simple mixing of Zn-Fe-fermentation complex and additives. Antioxidant ability, antioxidative damage and anti-aging ability are reduced. The present invention adds the prepared Zn-Fe-fermentation complex and additives to the PEG6000 melt, and forms a solid dispersion of the poorly soluble Zn-Fe-fermentation complex and the suitable carrier PEG6000. Due to the crystal inhibition effect of the carrier material PEG6000, the drug exists in an amorphous dispersed state and increases the wettability of the drug, thereby accelerating the dissolution rate of the drug after contact with the gastrointestinal fluid, promoting the absorption of the drug, improving the in vitro dissolution characteristics of the Zn-Fe-fermentation complex, accelerating the dissolution rate of the drug, and promoting the effect of the drug.

[0194] 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 a composition containing ginseng extract, It is characterized in that The enzyme solution is obtained by fermenting white ginseng fungus, ginseng and astragalus powder are added, enzymolysis is performed, and the mixture is heated to boiling water for extraction, and the enzymolysis water extraction mixture is mixed with the fermentation-promoting composition, glucose and water, and fermented with Lactobacillus rhamnosus and selenium-enriched yeast, and the obtained fermentation product is added to the metal ion solution, stirred to obtain the Zn-Fe-fermentation complex, mixed with resveratrol and cordycepin, added to molten PEG6000, dispersed evenly, freeze-cured, dried, crushed, sieved, and the composition containing ginseng extract is obtained; comprising the following steps: S1. Preparation of enzyme fermentation medium: adding a carbon source, a nitrogen source, vitamins, and minerals to water, sterilizing, and preparing an enzyme fermentation medium; S2. Fermentation and enzyme production: Add sterile water to the slant culture medium of white ginseng fungus to wash out the colonies on the slant, prepare a bacterial seed solution with a buffer solution, inoculate it into the enzyme production fermentation medium in step S1, ferment and culture, centrifuge and sterilize, obtain a supernatant, add ammonium sulfate, let stand and precipitate, centrifuge, dissolve the precipitate in the buffer solution, dialyze, centrifuge the dialyzate, collect the supernatant, and obtain an enzyme solution; the bacterial content of the bacterial seed solution is 10 7 -10 8 cfu / mL, inoculum size 2-3v / v%; S3. Enzymatic water extraction: Wash ginseng and astragalus separately, dry and crush to obtain a mixed powder, add it to the enzyme solution prepared in step S2, heat it for enzymolysis, continue to heat it to boiling for extraction, filter it, retain the solid, purify the filtrate with a macroporous resin, mix the eluent with the solid, and obtain an enzymatic water extraction mixture; the mass ratio of ginseng to astragalus is 10-15:3-5; the mass ratio of the mixed powder to the enzyme solution is 10-15:100; S4. Fermentation: adding the enzymatic water extract mixture obtained in step S3, the fermentation-promoting composition, and glucose into water, sterilizing, inoculating activated Lactobacillus rhamnosus and selenium-enriched yeast seed liquid, fermenting and culturing, removing solids by centrifugation, collecting the supernatant, and freeze-drying to obtain a fermentation product; the fermentation-promoting composition comprises inulin, vitamin B6, and calcium chloride in a mass ratio of 10-12:0.5-1:0.2-0.5; S5. Complexation of metal ions: adding the fermentation product obtained in step S4 to water, adding a soluble zinc salt and a soluble iron salt, stirring and mixing, drying, dialyzing, and drying the dialyzate to obtain a Zn-Fe-fermentation complex; the soluble zinc salt is selected from at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc citrate, and zinc gluconate, and the soluble iron salt is selected from at least one of ferric chloride, ferric sulfate, and ferric nitrate; S6. Preparation of additives: mixing resveratrol and cordycepin to obtain an additive; S7. Preparation of a composition containing ginseng extract: heat PEG6000 until it is melted, add the Zn-Fe-fermentation complex prepared in step S5 and the additive prepared in step S6, stir and mix evenly, freeze-solidify, dry, crush, and sieve to obtain a composition containing ginseng extract.

2. The preparation method according to claim 1, It is characterized in that The mass ratio of the carbon source, nitrogen source, vitamins, minerals and water in step S1 is 70-100:7-12:1-2:0.5-1:300-400, the carbon source is sugarcane juice with a sugar content of 5-7, the nitrogen source is selected from at least one of amino acids, sodium chloride, ammonium nitrate, sodium nitrate, potassium nitrate, urea, fish meal and peptone, the amino acids are selected from glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine and phenylalanine, the vitamins are selected from at least one of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin E, vitamin D1, vitamin D2 and vitamin D3, and the minerals are selected from at least one of potassium chloride, sodium chloride, potassium sulfate, sodium sulfate, magnesium chloride, ferric chloride, ferrous chloride, cupric chloride, manganese chloride, zinc chloride, calcium chloride, magnesium sulfate, cupric sulfate, ferric sulfate, manganese sulfate and calcium sulfate.

3. The preparation method according to claim 1, It is characterized in that The fermentation culture conditions in step S2 are 23-26° C., 50-70 r / min, and fermentation for 36-48 h. The amount of ammonium sulfate added is 80-85 wt % of ammonium sulfate added to the system. The static precipitation time is 2-4 h. The dialysis bag pore size is 1000-1500 D, and the dialysis time is 3-5 h.

4. The preparation method according to claim 1, It is characterized in that The temperature of the heating enzymolysis in step S3 is 35-40°C, the time is 2-4h, the time of the heating to boiling extraction is 1-3h, the macroporous resin is D101 macroporous resin, HPD100 macroporous resin or SYD8 macroporous resin, and the purification method is to pass the sample through the macroporous resin at a rate of 0.1-0.2g / mL, then elute with 1.5-2.5BV water, then elute with 3-5BV 70wt% ethanol, collect the eluate, and recover the ethanol.

5. The preparation method according to claim 1, It is characterized in that The mass ratio of the enzymatic water extraction mixture, the fermentation-promoting composition, the glucose and the water in step S4 is 15-20:2-3:5-7:400-500, the inoculation amounts of the activated Lactobacillus rhamnosus and the selenium-enriched yeast seed solution are 2-3 v / v% and 1-2 v / v%, respectively, and the bacterial content of the seed solution is 10 8 -10 9 cfu / mL, and the fermentation culture conditions are 37-40°C, 50-70r / min, and the fermentation culture is 48-72h.

6. The preparation method according to claim 1, It is characterized in that In step S5, the mass ratio of the fermentation product, the soluble zinc salt and the soluble iron salt is 100:2-3:1-2, the dialysis bag pore size of the dialysis is 300-500D, and the dialysis time is 1-2h.

7. The preparation method according to claim 1, It is characterized in that The mass ratio of resveratrol and cordycepin in step S6 is 3-5:7-10; the mass ratio of PEG6000, Zn-Fe-fermentation complex and additives in step S7 is 100:10-12:3-5, the freezing temperature is -25 to -20°C, the curing time is 2-4h, and the mesh size of the sieve is 80-100 mesh.

8. A composition containing ginseng extract obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the composition containing ginseng extract as claimed in claim 8 in the preparation of anti-aging and antioxidant medicines.

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