CN113924920A - Bidirectional solid state fermentation process for ganoderma-American ginseng stem leaves - Google Patents
Bidirectional solid state fermentation process for ganoderma-American ginseng stem leaves Download PDFInfo
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- CN113924920A CN113924920A CN202111187211.7A CN202111187211A CN113924920A CN 113924920 A CN113924920 A CN 113924920A CN 202111187211 A CN202111187211 A CN 202111187211A CN 113924920 A CN113924920 A CN 113924920A
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- american ginseng
- ganoderma
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- fungus
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a bidirectional solid state fermentation process of ganoderma-American ginseng stems and leaves, which comprises the following steps: 1, preparing a solid culture medium, namely taking corn bran, corn yellow powder, wheat bran, American ginseng stems and leaves, monopotassium phosphate, oyster shell powder and magnesium sulfate according to a certain proportion, controlling the water content, bagging and sterilizing; inoculating, taking out the sterilized fungus bags, cooling to room temperature, inoculating, sealing the bags, fermenting in a dark room, taking out each group of fungus bag hypha after filling the bags, breaking the fungus bags, putting the fungus bags on aluminum foil paper, drying, and crushing the fungus bags and sieving by a third sieve. The process not only fully utilizes resources, reduces the generation of garbage and the consumption of raw materials, improves the industrial utilization value of the American ginseng, but also initially establishes the production process of the mycelial and the quality control index thereof, provides reference for the industrialized production of the mycelial in the Lingxi in future, provides new raw materials for the development of innovative bacteriological drugs and functional products, and provides a new idea for the biological transformation of traditional Chinese medicines.
Description
Technical Field
The invention relates to the technical field of solid state fermentation, in particular to a bidirectional solid state fermentation process for ganoderma-American ginseng stems and leaves.
Background
Ganoderma lucidum is dried fruiting body of Ganoderma lucidum Ganodermanucidum (Leys. exFr.) Karst. or Ganoderma sinense Ganodermainesezhao, XuetZhang, which is sweet in taste, neutral in nature, and enters heart, lung, liver and kidney meridians. Ganoderma lucidum mainly contains polysaccharides, triterpenes, sterols, amino acids, alkaloids, etc. Polysaccharide is one of main active ingredients of ganoderma lucidum, more than 200 kinds of polysaccharide are separated and purified at present, and related researches find that the number of transverse branches, the length of side chains and the ratio of the number of bonds of beta-glucan influence the pharmacological properties of the beta-glucan. Triterpenes are highly oxidized lanostane derivatives, have various types and complex structures, and can be divided into ganoderic acid, methyl ganoderic acid, ganoderic spore acid, ganoderic aldehyde and the like according to different linked side chains and functional groups. The content of sterol is high, and there are more than 40 compounds separated from Ganoderma lucidum, and the compounds are divided into ergosterol and cholesterol according to their skeleton. The content of amino acids accounts for more than 10% of the mass of the ganoderma lucidum, wherein the amino acids comprise lysine, leucine and other essential amino acids for human bodies. The alkaloid mainly comprises betaine, choline, gamma-trimethylaminebutyric acid, and sulfur histidine methylamine salt. According to records in compendium of materia medica, ganoderma lucidum has the effects of strengthening body resistance, consolidating constitution, nourishing, strengthening body, removing diseases and prolonging life, and modern pharmacology shows that ganoderma lucidum has the effects of reducing blood sugar, resisting tumors, resisting oxidation, regulating immunity, improving sleep and protecting liver.
American ginseng is usually used as a medicine from roots, the main bioactive substance is triterpene saponin, and researches show that the content of the triterpene saponin in American ginseng leaves is higher than that of the roots, so that the researches on stems and leaves of the American ginseng are very significant for the comprehensive utilization of American ginseng resources. In recent years, with the increase of demand, the planting area of American ginseng is larger and larger, but most of stems and leaves are thrown away as waste or directly burned in the field, which seriously causes environmental pollution and resource waste. Related researches show that the stems and leaves of American ginseng contain a large amount of ginsenoside, quantitative researches of Qu and the like show that the monomeric saponin of the American ginseng contains in different partsThe amount of ginsenoside Rg in different parts is different1、Re、F11、Rf、Rg2、Rh1、Rb1、Rc、Rb2、Rb3Rd, and Rh2The total content of the components is as follows in sequence: leaf > hairy root > rhizome > root > stem. The saponin in the stem is about 1/3-1/2 of the content of the saponin in the taproot, and the saponin in the leaf is 1.5-4 times of the content of the saponin in the taproot. Related researches find that the content of saponin in the stems and leaves of the panax ginseng is large (the content of total saponin is 2.89 times of that of the roots of the panax ginseng and is 15.99 times of that of the total saponin of the ginseng). The bioactive components of the stems and leaves of American ginseng not only contain triterpenoid saponins, but also contain flavonoids, volatile oil, lignin, steroids, saccharides, amino acids and the like. Modern pharmacological research shows that the American ginseng stem leaves have the effects of resisting aging, myocardial ischemia and arrhythmia, resisting shock, resisting hypertension, promoting hematopoiesis, enhancing immunity and the like.
In recent years, researches on ganoderma fermentation have made important progress, and researches on the chemical components of ganoderma solid fermentation mycoplasm based on burdock roots [ J ] Chinese edible fungi, 2019, 38(03):44-49 ] find that the content of polysaccharide in the burdock roots after being fermented by the ganoderma is reduced from 9.94% to 2.76%, the content of protein is increased from 13.38% to 17.28%, the content of total triterpenic acid serving as a new component is 0.14mg/g, the content of newly synthesized adenosine is reduced, and the content of total flavone is reduced. Researches on optimization of solid-state fermentation conditions of ganoderma lucidum mycelia by using rice as a substrate [ J ] in food industry science and technology, 2014, 35(02):186-191.) find that the content of polysaccharide is reduced firstly and then increased along with different fermentation days, and the content of lysine is increased. Zhangguang et al (Zhang Guo Guang, Shenlin, Huang Bingqing, etc., main active ingredients and inoxidizability of Ganoderma lucidum solid fermentation longan seed extract [ J ] food industry science and technology, 2019, 40(02):53-57.) research Ganoderma lucidum solid fermentation longan seed, 60% ethanol is used as an extraction solvent, the content of active ingredients (polyphenols and flavonoids) in the fermented extract is remarkably reduced (P <0.05), and the sugar is remarkably increased (P < 0.05). Hu Yong le and the like (Hu Yong le, Youyuang, Huang Wei, and the like, the mycoplasm active ingredient analysis before and after the ganoderma-alisma two-way fermentation [ J ]. the new village academy of academic, 2020, 37(06):10-13+204.) researches show that the contents of crude polysaccharide, flavone and total triterpene are all increased after the ganoderma-alisma two-way fermentation. Research on main functional substances and biological activity change of medical (edible) fungi fermented bean dregs [ J ] food and fermentation industry, 2020, 46(15): 100-.
In the prior art, the record about American ginseng fermentation is that American ginseng is used as a culture medium, the chemical structure of American ginseng is modified by utilizing the biological activity and biochemical reaction of microorganisms, and new bioactive components and functions are generated (Wangxhong, Liqide, Caocao's. microbial fermentation traditional Chinese medicine is new content of traditional Chinese medicine research [ J]Chinese herbal medicine, 2001(03), 77-78.) telecommunication peak et al (silver Peak, Yan Merlianthus, sun Shih, et al., research on ginsenoside in American ginseng transformed by solid state fermentation of Cordyceps militaris (L.) Link [ J ]]The research on the special byproduct of Chinese forest 2014(02) 1-3) finds that the enzyme system generated by the cordyceps militaris strain can enable the ginsenoside Rg1Is converted to Rd. Cai rains (Cai rains. microbial transformation of Panax quinquefolium and analysis of its products [ D]Haerbin university of industry, 2018.) study on fermenting radix Panacis Quinquefolii with edible and medicinal fungi, wherein in liquid fermentation, the radix Panacis Quinquefolii powder fermentation product of strains PHI30, HEE17, YC01, CH01, STHR4, 25 has higher polysaccharide content (highest 54.750 + -3.834 mg/g) than radix Panacis Quinquefolii powder, and the polysaccharide content after fermentation of other strains is lower; in the solid fermentation, the content of the fermentation products of strains HEE17, YC01 and CH01 is higher than that of the polysaccharide of American ginseng powder (the highest content is 53.880 +/-4.116 mg/g), the polysaccharide content of other strains is low after fermentation, the total saponin content of most strains is increased, the content of a small part of strains is reduced, and the variety and the content of the ginsenoside are greatly changed in the liquid fermentation and the solid fermentation.
At present, a plurality of methods for changing the drug property and improving the drug effect are utilized by bidirectional solid state fermentation, and after fungus fermentation is carried out, bioactive substances (oligosaccharides, oligopeptides, rare saponins and nucleosides) are increased. Therefore, in the invention, ganoderma lucidum and American ginseng stem and leaf are subjected to bidirectional solid state fermentation to obtain ganoderma lucidum-American ginseng stem and leaf mycoplasm (called Lingxi mycoplasm for short), in the fermentation process, enzymes generated by ganoderma lucidum can digest and decompose plant cell walls in a culture medium, and biomacromolecules in plants can be converted into micromolecular substances which can be directly absorbed by animal intestinal tracts, so that new bioactive substances are generated. Provides reference for the industrialized production of the Lingxi mycoplasm in the future, provides new raw materials for the development of innovative bacteriological drugs and functional products, and provides a new idea for the biological transformation of traditional Chinese medicines.
Disclosure of Invention
In view of the above, the invention provides a bidirectional solid state fermentation process of ganoderma lucidum-American ginseng stem leaves, the invention carries out bidirectional solid state fermentation on ganoderma lucidum and American ginseng stem leaves to obtain ganoderma lucidum-American ginseng stem leaf mycoplasm (called Lingxi mycoplasm for short), researches the Lingxi mycoplasm fermentation process through a single factor experiment and a response surface method, and detects bioactive substances before and after the fermentation of the Ganoderma lucidum-American ginseng stem leaf mycoplasm, thereby fully utilizing resources, reducing the generation of garbage and the consumption of raw materials, and improving the industrial utilization value of American ginseng.
In order to achieve the purpose, the invention adopts the following technical scheme:
a bidirectional solid fermentation process for stems and leaves of Ganoderma lucidum-American ginseng is characterized by comprising the following steps:
(1) preparing a solid culture medium of American ginseng stems and leaves;
(2) inoculating Ganoderma into solid culture medium of stem and leaf of radix Panacis Quinquefolii via Ganoderma culture solution, and fermenting to obtain fermented material;
(3) taking out the fermented material, drying and sieving to obtain a fermented product.
Preferably, the solid culture medium for preparing the stems and leaves of the American ginseng in the step (1) specifically comprises the following components: taking 60-65% of corn bran, 5-15% of corn yellow powder, 5-15% of wheat bran and 15-20% of American ginseng stem leaves according to the following mass ratio, then adding monopotassium phosphate accounting for 1% of the total mass of the four raw materials, oyster shell powder accounting for 1% of the total mass of the four raw materials and magnesium sulfate accounting for 0.5% of the total mass of the four raw materials, filling the mixture into a fungus bag, controlling the water content in the fungus bag to be 50-60%, and then sterilizing the fungus bag.
More preferably, the solid culture medium for preparing the American ginseng stems and leaves in the step (1) specifically comprises the following components: taking 65% of corn bran, 10% of corn yellow powder, 10% of wheat bran and 15-20% of American ginseng stem leaves according to the following mass ratio, then adding monopotassium phosphate accounting for 1% of the total mass of the four raw materials, oyster shell powder accounting for 1% of the total mass of the four raw materials and magnesium sulfate accounting for 0.5% of the total mass of the four raw materials, filling the mixture into a fungus bag, controlling the water content in the fungus bag to be 55%, and then sterilizing the fungus bag.
Preferably, the amount of the fungus bags filled in the step (1) is 150-300 g; the sterilization of the fungus bag comprises placing the fungus bag into a high pressure steam sterilization pot, and sterilizing at 121 deg.C for 45 min.
More preferably, the amount of the filled fungus bags in the step (1) is 200 g; the sterilization of the fungus bag comprises placing the fungus bag into a high pressure steam sterilization pot, and sterilizing at 121 deg.C for 45 min.
Preferably, the step (2) of inoculating the ganoderma lucidum into the solid culture medium of the stems and leaves of the American ginseng to ferment specifically comprises the following steps: taking out the sterilized fungus bags, standing to room temperature, inoculating Ganoderma to solid culture medium of stems and leaves of radix Panacis Quinquefolii via Ganoderma culture solution on superclean bench, sealing, and fermenting in dark room.
Preferably, the dosage of the ganoderma lucidum culture solution in the step (2) is 15-40 mL; the fermentation temperature is 25-30 ℃, and the fermentation time is 20-25 days.
More preferably, the inoculation amount in the step (2) is 30 mL; the fermentation temperature is 28 ℃, and the fermentation time is 23 days.
Preferably, the drying temperature in the step (3) is 55 ℃; the sieving is a No. three sieving.
Preferably, in the invention, the ganoderma lucidum culture solution is prepared by the following method:
(1) preparation of slant culture Medium
Peeling potato, and cutting into 1cm3Weighing 200g of the mixture, putting the mixture into a pot, adding 1000mL of water, boiling for 20-30 minutes, filtering the mixture by eight layers of gauze, and fixing the volume of the filtrate to 1000mL by using water; placing on an electromagnetic oven, heating with slow fire, adding 1g magnesium sulfate (MgSO) ground into fine powder4) 2 tablet grindingVitamin B ground into fine powder1(10 mg/tablet), 2g of monopotassium phosphate (KH)2PO4) 20g of glucose (C)6H12O6) And after the agar is completely dissolved, adding 17g of agar, continuously heating and stirring uniformly (preventing carbonization and gelatinization) until the agar is completely dissolved, and obtaining the natural pH. Adding the culture medium into a test tube while the culture medium is hot, adding the culture medium to 1/3, wiping off the excess culture medium at the mouth of the test tube, plugging the test tube with a prepared test tube tampon, filling, and sterilizing the test tube in an autoclave with sterilization parameters of 121 ℃ for 30 min. Taking out the test tube after the temperature is reduced to 45-50 deg.C (to prevent excessive water vapor from condensing on the test tube wall) after sterilization, and raising one end of the test tube tampon (with thickness of 1cm) to naturally incline the culture medium on the inner wall of the test tube to about 2/3 of the test tube, and forming a slant culture medium after solidification for use.
(2) Activation and rejuvenation of Ganoderma lucidum strains
Placing Ganoderma strain at room temperature for 2h, sterilizing the operating room with ozone, and turning on the ultraviolet lamp and exhaust fan of the superclean bench. Before inoculation, wiping a workbench, hands and an inoculation tool with alcohol, turning off an ultraviolet lamp, igniting 4 alcohol lamps, placing an inoculation shovel on the alcohol lamps for burning sterilization, holding strains with the left hand and the inoculation shovel with the right hand, cutting the strains into mung bean-sized bacterium blocks, placing the bacterium blocks in a slant of a slant culture medium, plugging a cotton plug, placing the bacterium blocks on an inoculation table, and completing inoculation. Several tubes are inoculated in parallel, so that the strain is prevented from being infected due to improper operation. After inoculation, the cells were placed in an incubator at 28 ℃ and 70% humidity for 7 days in the dark.
Repeating the above operation for 2 times after the culture is completed to obtain strong Ganoderma strain.
(3) Liquid culture of ganoderma lucidum strains
Preparation of liquid culture medium: peeling potato, and cutting into 1cm3Weighing 20% (by volume of water) and putting into a pot, adding a certain volume of water (prepared according to the required amount), boiling for 20-30 minutes, filtering with eight layers of gauze, and fixing the volume of the filtrate to a certain volume; placing on an electromagnetic oven, heating with slow fire, adding0.1% magnesium sulfate (MgSO) ground to a fine powder4) Vitamin B ground into fine powder in proper amount1(10 mg/tablet, 1 tablet/500 mL), 0.2% monopotassium phosphate (KH)2PO4) 2% glucose (C)6H12O6) And 1% peptone, continuously heating and stirring uniformly until the peptone is completely dissolved, and the pH is natural. Finally, subpackaging in conical flask, adding stopper, and sterilizing at 121 deg.C for 30 min.
Inoculation: inoculating on a clean bench, inoculating 6 pieces of Ganoderma mycelia with size of mung bean into liquid culture medium, plugging into a plug, placing on a magnetic stirrer, rotating at 200r/min for 1 day, adjusting to 600r/min, and culturing at 28 deg.C in dark place for 7 days to obtain Ganoderma culture solution.
In the invention, the culture medium added with the stems and leaves of the American ginseng and the lucid ganoderma are subjected to bidirectional solid state fermentation to obtain a fermentation product, called Lingxi mycoplasm for short.
Through the technical scheme, compared with the prior art, the invention has the following beneficial effects:
the content of biomacromolecules in an unfermented substrate is high (starch, protein, polysaccharide and polypeptide …), and the biomacromolecules are not beneficial to being directly absorbed and utilized by a human body; the American ginseng stem and leaf contains a large amount of primary glycoside, the biological activity of the primary glycoside is lower than that of secondary glycoside and rare saponin, the catabolism in the organism is slow, and the utilization degree is low; according to the invention, ganoderma lucidum and a culture medium added with American ginseng stems and leaves are subjected to bidirectional solid state fermentation to obtain medicinal mycoplasm, the medicinal mycoplasm contains higher secondary glycoside and rare saponin, the content of micromolecular bioactive substances (oligosaccharide, oligopeptide and nucleoside …) is higher, the absorption of an organism is facilitated, and the bidirectional solid state fermentation can improve the medicinal property and the medicinal effect of the American ginseng stems and leaves, reduce the toxicity of the medicament and reduce the side reaction of the medicament;
② the growth of the ganoderma lucidum needs a large amount of carbon source, the general bidirectional solid fermentation takes the corncob as the main component of the culture medium. Corn bran is a byproduct of corn deep processing, the corn bran is not subjected to a large amount of deep processing treatment in the industry at present, the utilization degree is low, the corn bran is high in content of food fiber, and contains chemical substances such as starch, protein and fat, the texture is loose, and the corn bran serving as a ganoderma lucidum solid culture medium can ensure that the ganoderma lucidum grows to have sufficient oxygen and has development and utilization values.
③ the pH of the ganoderma lucidum can be reduced in the fermentation process, the pH value of the fermentation environment is stabilized by gypsum or calcium carbonate, the invention adds 1 percent of oyster shell powder, the oyster shell powder contains a large amount of calcium carbonate with the content of more than 90 percent and also contains trace elements such as copper, iron, zinc, manganese, strontium and the like, which is more beneficial to the growth of hypha. The invention uses fermentation fungus bags, one is not easy to contaminate, and the other is used for kneading after inoculation, so that the inoculation liquid is uniform, and the invention is more beneficial to the germination of mycelium and the integral fermentation of culture medium.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a graph comparing the antioxidant capacity of different media against mycelial panaxathium;
FIG. 2 is a graph comparing the effect of different bagging amounts on the antioxidant capacity of mycelial in Lingxi;
FIG. 3 is a graph comparing the effect of different fermentation temperatures on the antioxidant capacity of mycelial in Lingxi;
FIG. 4 is a graph comparing the effect of different inoculum sizes on the antioxidant capacity of colicin;
FIG. 5 is a graph of contour lines and response surfaces of the effect of different fermentation factors on the antioxidant capacity of mycelial in Lingxi;
FIG. 6 shows ginsenoside Rg in stems and leaves of Panax Quinquefolium and Lingxi mycoplasm3Map, wherein, A, ginsenoside Rg 3; B. american ginseng stem and leaf; C. fermented lucidification;
fig. 7 is a map of ginsenoside CK in the stem and leaf of american ginseng and the mycelial of lingxi, wherein a is ginsenoside CK; B. american ginseng stem and leaf; C. fermented Lingxi mycoplasm;
FIG. 8 is a graph of nucleoside in pre-fermentation substrate and post-fermentation lucidipine, wherein, 1. cytosine; 2. uracil; 3. cytidine; 4. uridine; 5. adenine; 6. guanosine; 7. adenosine; A. a pre-fermentation substrate; B. fermented Lingxi mycoplasm; C. a cytosine; D. uracil; E. cytidine; F. uridine; G. adenine; H. guanosine; I. adenosine;
FIG. 9 is a matrix peptide profile prior to fermentation;
FIG. 10 is a mycelial peptide profile after fermentation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Bidirectional solid fermentation process for stem and leaf of Ganoderma lucidum-American ginseng
1. Preparation of solid Medium
Taking the following raw materials in percentage by mass: corn bran 65%, corn yellow powder 10%, wheat bran 10%, American ginseng stem leaf 15%, potassium dihydrogen phosphate 1%, oyster shell powder 1%, magnesium sulfate 0.5% calculated according to 100 wt% of the four raw materials, controlling the water content to be 55%, loading into fungus bags, wherein each fungus bag is 200g, after all the fungus bags are subpackaged, placing all the fungus bags in an autoclave, and sterilizing for 45min at 121 ℃.
2. Preparation of Ganoderma culture solution
(1) Preparation of slant culture Medium
Peeling potato, and cutting into 1cm3Weighing 200g of the mixture, putting the mixture into a pot, adding 1000mL of water, boiling for 25 minutes, filtering the mixture by eight layers of gauze, and fixing the volume of the filtrate to 1000mL by using water; placing on an electromagnetic oven, heating with slow fire, adding 1g magnesium sulfate (MgSO) ground into fine powder4) 2 tablets of vitamin B ground to a fine powder1(10 mg/tablet), 2g of monopotassium phosphate (KH)2PO4) 20g of glucose (C)6H12O6) And after the agar is completely dissolved, adding 17g of agar, continuously heating and stirring uniformly (preventing carbonization and gelatinization) until the agar is completely dissolved, and obtaining the natural pH. Adding the culture medium into a test tube while the culture medium is hot, adding the culture medium to 1/3, wiping off the excess culture medium at the mouth of the test tube, plugging the test tube with a prepared test tube tampon, filling, and sterilizing the test tube in an autoclave with sterilization parameters of 121 ℃ for 30 min. Taking out the test tube after the temperature is reduced to 46 ℃ (to prevent excessive water vapor from condensing on the wall of the test tube) after sterilization, and raising one end of the test tube with a cotton plug (the thickness is 1cm) to naturally incline the culture medium on the inner wall of the test tube to about 2/3 of the test tube, and forming a slant culture medium after solidification for later use.
(2) Activation and rejuvenation of Ganoderma lucidum strains
Placing Ganoderma strain at room temperature for 2h, sterilizing the operating room with ozone, and turning on the ultraviolet lamp and exhaust fan of the superclean bench. Before inoculation, wiping a workbench, hands and an inoculation tool with alcohol, turning off an ultraviolet lamp, igniting 4 alcohol lamps, placing an inoculation shovel on the alcohol lamps for burning sterilization, holding strains with the left hand and the inoculation shovel with the right hand, cutting the strains into mung bean-sized bacterium blocks, placing the bacterium blocks in a slant of a slant culture medium, plugging a cotton plug, placing the bacterium blocks on an inoculation table, and completing inoculation. Several tubes are inoculated in parallel, so that the strain is prevented from being infected due to improper operation. After inoculation, the cells were placed in an incubator at 28 ℃ and 70% humidity for 7 days in the dark.
Repeating the above operation for 2 times after the culture is completed to obtain strong Ganoderma strain.
(3) Liquid culture of ganoderma lucidum strains
Preparation of liquid culture medium: peeling potato, and cutting into 1cm3Weighing 20% (by volume of water) and putting into a pot, adding a certain volume of water (prepared according to the required amount), boiling for 25 minutes, filtering with eight layers of gauze, and taking the filtrate; placing on an electromagnetic oven, heating with slow fire, adding 0.1% magnesium sulfate (MgSO 2) ground into fine powder according to mass fraction4) Vitamin B ground into fine powder in proper amount1(10 mg/tablet, 1 tablet/500 mL), 0.2% Potassium dihydrogen phosphate(KH2PO4) 2% glucose (C)6H12O6) And 1% peptone, continuously heating and stirring uniformly until the peptone is completely dissolved, and the pH is natural. Finally, subpackaging in conical flask, adding stopper, and sterilizing at 121 deg.C for 30 min.
Inoculation: inoculating on a clean bench, inoculating 6 pieces of Ganoderma mycelia with size of mung bean into liquid culture medium, plugging into a plug, placing on a magnetic stirrer, rotating at 200r/min for 1 day, adjusting to 600r/min, and culturing at 28 deg.C in dark place for 7 days to obtain Ganoderma culture solution.
3. Inoculation of
And taking out the sterilized fungus bags, cooling to room temperature, inoculating 30mL of the fungus bags on a superclean bench, sealing the inoculated fungus bags, and fermenting in a dark room at 28 ℃. Taking out each group of fungus bags after the bags are filled with mycelia, breaking the fungus bags, putting the fungus bags on aluminum foil paper, drying the fungus bags by blowing at 55 ℃, and crushing the fungus bags through a third sieve to obtain test powder. .
Example 2
Research experiment
1.1 solid fermentation Single factor investigation
The influence of the culture medium proportion, the bagging amount, the fermentation temperature and the inoculation amount on the solid fermentation of the lucid ganoderma is investigated by taking the status of the ganoderma, the cover time, the bag filling time and the inoxidizability of the fungus quality as indexes.
Cover time is the sum of the time each fungus bag grows over the surface/total number of bags
The bag full time is the sum of the time that each fungus bag grows full of the fungus bags/the total bag number
The antioxidant determination method comprises the following steps: DPPH and ABTS methods
1.1.1 preparation of test solution and method for measuring the same
(1) The preparation method of the water extract comprises the following steps: taking 2.5g of test sample powder, placing the test sample powder in a conical flask with a plug, adding 100mL of water, performing ultrasonic extraction at room temperature for 30min, performing reflux extraction for 30min, filtering, putting the filtrate into a 100mL volumetric flask, washing filter residue with water, fixing the volume to a scale, and shaking up to obtain the test sample powder, wherein the test sample powder is diluted to a proper multiple during measurement.
(2) The preparation method of the alcohol extract comprises the following steps: taking 2.5g of test sample powder, placing in a conical flask with a plug, adding 50mL of 75% ethanol, carrying out ultrasonic extraction at 60 ℃ for 2 times, each time for 30min, filtering, combining filtrates, putting the filtrate into a 100mL volumetric flask, washing filter residue with 75% ethanol, fixing the volume to a scale, shaking up, and diluting to a proper ratio during measurement.
(3) DPPH assay: preparing 50 mu g/mL DPPH absolute ethanol solution, putting 2mL into a test tube with a plug, adding 2mL of different sample solutions, fully mixing, standing at room temperature in the dark for 30min, and measuring the absorbance at the wavelength of 517 nm.
DPPH.radical scavenging Rate ═ 1- (A)1-A2)/A0]×100％
In the formula: a. the1Absorbance of the test article
A2Background absorbance of test sample (volume of absolute ethanol instead of DPPH solution)
A0Blank control absorbance (equal volume of test solution instead of sample solution)
(4) ABTS assay: mixing 7mmol/L ABTS solution with equal volume of 4.9mmol/L potassium persulfate solution at room temperature, and standing in dark for 12 hr to obtain ABTS+A stock solution; ABTS was prepared by diluting stock solution 20-fold with phosphate buffer pH 7.4+And (4) working fluid. Adding 2mL of the solution into a test tube with a plug, adding 1mL of different sample solutions, shaking uniformly, standing at room temperature in the dark for 10min, and measuring the absorbance at 734 nm.
ABTS+Free radical scavenging rate ═ 1- (A)1-A2)/A0]×100％
In the formula: a. the1Absorbance of the test article
A2Background absorbance of test article (equal volume of distilled water instead of ABTS)+Working fluid
A0Blank control absorbance (equal volume of test solution instead of sample solution)
1.1.2 Medium mix ratio study
The following proportions of solid medium were investigated separately:
A. 70% of corn bran, 10% of corn yellow powder, 10% of wheat bran and 10% of American ginseng stem leaves;
B. 65% of corn bran, 10% of corn yellow powder, 10% of wheat bran and 15% of American ginseng stem leaves;
C. 60% of corn bran, 10% of corn yellow powder, 10% of wheat bran and 20% of American ginseng stem leaves;
D. 65% of corn bran, 5% of corn yellow powder, 15% of wheat bran and 15% of American ginseng stem leaves;
E. 65% of corn bran, 15% of corn yellow powder, 5% of wheat bran and 15% of American ginseng stem leaves.
The five groups of solid culture media are respectively added with 1 percent of monopotassium phosphate, 1 percent of oyster shell powder and 0.5 percent of magnesium sulfate, the fixed temperature is 28 ℃, the bagging amount is 200g, the inoculation amount is 30 mL/bag, and the culture is carried out in a dark room. The results are shown in Table 1:
TABLE 1 Effect of different media on Ganoderma lucidum solid fermentation
n＝5)
Referring to the table 1 and the figure 1, the experimental results show that the growth vigor of the lucid ganoderma in different culture media A > B > D > E > C, the difference between A and B is small, the growth vigor of the group C is slow, and the group C grows over the fungus bags in nearly 18 days; the mycelial oxidation resistance of different culture mediums C, B, D, E and A is less different.
According to the experimental result, the culture medium B is finally determined, namely 65% of corn husks, 10% of corn gluten meal, 10% of wheat bran and 15% of American ginseng stems and leaves.
1.1.3 bag filling amount inspection
Changing the bagging amount (150g, 200g, 250g and 300g), wherein the culture medium comprises 65% of corn husks, 10% of corn yellow powder, 10% of wheat bran, 15% of American ginseng stems and leaves, the temperature is 28 ℃, the inoculation amount is 30 mL/bag, and the results are shown in the table 2:
TABLE 2 influence of different bagging amounts on the fermentation of Ganoderma lucidum solid
n＝5)
Referring to table 2 and fig. 2, the experimental results show that the growth of ganoderma lucidum mycelia becomes weaker as the bagging amount increases; the antioxidant capacity of different bagging amounts is 200g to 250g to 150g to 300 g.
According to the experimental results, the final bagging amount was 200 g.
1.1.4 fermentation temperature investigation
The fermentation temperature was changed (25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃) and the culture medium was 65% of corn husk, 10% of corn yellow powder, 10% of wheat bran, 15% of American ginseng stem leaves, 200g of bagging amount, 30mL of inoculum size per bag, and was cultured in the dark, and the results are shown in Table 3:
TABLE 3 influence of different fermentation temperatures on the fermentation of Ganoderma lucidum (X + -SD, n ═ 5)
Referring to table 3 and fig. 3, the experimental results show that the cover-sealing time and the full-bag time of the fermentation fungus bags are substantially gradually reduced as the fermentation temperature is increased; the antioxidant capacity of the mycelial in Lingxi increases with the increase of the fermentation temperature, but gradually decreases at the temperature of 29 ℃ and 30 ℃.
According to the experimental results, the fermentation temperature was finally determined to be 28 ℃.
1.1.5 inoculum size investigation
The inoculation amount is changed for each bag (15mL, 20mL, 25mL, 30mL, 35mL, 40mL), the culture medium is 65% of corn husks, 10% of corn gluten, 10% of wheat bran and 15% of American ginseng stems and leaves, the culture temperature is 28 ℃, the bagging amount is 200g, and the cultivation is carried out in a dark place, and the results are shown in Table 4:
TABLE 4 Effect of different inoculum sizes on Ganoderma lucidum solid fermentation (X + -SD, n ═ 5)
Referring to table 4 and fig. 4, the experimental results show that the inoculum size is too small (15mL of inoculum, not full of cover in limited time), hyphae germinate slowly and grow slowly, hyphae overgrow cover time 40mL <35mL <30mL <25mL <20 mL; when the inoculation amount is 30mL, the Lingxi mycoplasm has the strongest antioxidant capacity.
According to the experimental result, the inoculation amount is finally determined to be 30 mL.
Through the above single factor experiment, the optimal fermentation conditions are obtained as follows: 65% of corn husk, 10% of corn yellow powder, 10% of wheat bran, 15% of American ginseng stem leaves and leaves in a culture medium, bagging at 28 ℃, packaging at 200g, inoculating at 30 mL/bag, and fermenting in a dark room.
1.2 solid fermentation response surface optimization
1.2.1 selection of analytical factor levels for the response surface method
Referring to the single-factor experiment result, a central group with 3 factors and 3 levels in a response surface method and a Design Box-Behnken are adopted, and Design expert.V8.0.6 software is used for carrying out response surface regression analysis. The method is characterized in that the antioxidant capacity of the ganoderma lucidum mycelia (the DPPH clearance rate of a water extract, the DPPH clearance rate of an alcohol extract, the ABTS clearance rate of a water extract and the antioxidant capacity trend of the ABTS clearance rate of the alcohol extract are consistent according to the experimental result of the two-way solid state fermentation of ganoderma lucidum-American ginseng stems and leaves, so that when the solid state fermentation condition is optimized by a response surface method, one is selected as an index, and the DPPH clearance rate of the water extract is selected as an index of the antioxidant capacity index of the experiment), the bagging amount, the fermentation temperature and the inoculation amount are optimally designed, and therefore the optimal conditions of ganoderma lucidum fermentation are determined, and the result is shown in a table 5:
TABLE 5 Box-behnken factors and levels
1.2.2 modeling regression equation and analysis of variance
Adopting Design-expert.V8.0.6 software to carry out polynomial fitting regression on the data in the following table, establishing a multiple quadratic response surface regression model, and obtaining a regression equation:
Y＝31.36+0.75A-0.56B-0.93C+0.16AB+0.26AC-0.96BC-2.61A2-2.02B2-1.89C2results of analysis of variance for each factor table 6 and table 7:
TABLE 6 response surface center combination design and results
TABLE 7 Box-Behnken Experimental design analysis of variance
According to the experimental result, F is 68.87, p in the regression model<0.0001, which indicates that the regression model is significant; in the mismatch term, p is 0.5964>0.05, the difference is not obvious, and the experiment and the model are proved to have good fitting effect; r of the model20.9888, indicating that 98.88% of the change in antioxidant capacity of the mycelial in this experiment was due to the factor selected in the experiment; correction decision coefficient Radj 20.9745, indicating that the model can account for 97.45% change in response value; the coefficient of variation CV% is 1.31%, and the smaller the coefficient of variation, the higher the reliability of the modelAnd the better the accuracy; precision of 22.546>4.0, which indicates that the signal is strong enough and the model is feasible; first order term of variance A, B, C (P) in regression equation<0.01), quadratic term A2、B2、C2(P<0.0001), interaction factor BC (P)<0.01), the effect is significant; interaction factors AB, AC (P)>0.05) not significant; the influence factor influencing the oxidation resistance of the Lingxi mycoplasm is C according to the F value>A>B, i.e. inoculum size>Amount of bagged>Fermentation temperature, BC>AC>AB, interaction of fermentation temperature and inoculum size>Interaction of bagging and inoculum size>The packaging amount and the fermentation temperature are interacted.
1.2.3 analysis of response surface interactions
The three-dimensional space diagram can intuitively reflect the influence of each variable on the response value, and in the experiment, the two-dimensional contour lines and the three-dimensional response curved surface of each factor on the oxidation resistance activity of the mycelial in Lingxi are shown in figure 5.
According to the experimental results, the interaction between the fermentation temperature and the inoculation amount is obvious and is consistent with the analysis result of variance in the table. It can be seen that the antioxidant capacity gradually increases with the increase of the fermentation temperature and the inoculation amount, and the antioxidant capacity of the lucidix is reduced when the fermentation temperature and the inoculation amount are further increased.
1.2.4 validation experiments
The optimal fermentation condition of the bidirectional solid state fermentation of the ganoderma-American ginseng stem leaves is 206.5g of bagging amount, the culture temperature is 27.92 ℃, the inoculation amount is 28.92mL, and the oxidation resistance is 31.53 percent, which is obtained by analyzing a Design-expert.V8.0.6 software model. In order to meet the experimental operation, the culture conditions are adjusted to 200g of bagging amount, the culture temperature is 28 ℃, the inoculation amount is 29mL, the culture medium comprises 65% of corn husk, 10% of corn yellow powder, 10% of wheat bran, 15% of American ginseng stem leaf, 1% of monopotassium phosphate, 1% of oyster shell powder, 0.5% of magnesium sulfate, the water content is controlled to be 55%, the bags are separated, and after high-pressure steam sterilization, light-shielding culture is carried out, and the results are shown in Table 8:
table 8 model verification test results
The experimental result shows that the average value of the antioxidant capacity of the mycelial in the Lingxi verified by the optimal fermentation condition is 30.62 +/-0.66%.
According to the experimental result, the average value of the antioxidant capacity of the mycelial in Lingxi verified by the optimal fermentation condition is 30.62 +/-0.66%, which is similar to the predicted value of 31.53%, and the mathematical model can better predict the influence of the fermentation culture condition of the mycelial in Lingxi on the DPPH clearance of the water extract of the mycelial in Lingxi, so that the culture condition is determined to be 200g of bagging amount, the culture temperature is 28 ℃, and the inoculation amount is 29 mL.
Obtaining the optimal conditions of the ganoderma lucidum solid fermentation through single factor investigation and response surface optimization: the culture medium comprises 65% of corn bran, 10% of corn yellow powder, 10% of wheat bran, 15% of American ginseng stem leaves, 1% of monopotassium phosphate, 1% of oyster shell powder and 0.5% of magnesium sulfate, the water content is controlled to be 55%, the bagging amount is 200g, the culture temperature is 28 ℃, the inoculation amount is 29mL, and the cultivation is carried out in a dark room.
2. Determination of the end-point of fermentation
According to the states of the Lingxi mycoplasm and indexes of oligosaccharide, polysaccharide, oligopeptide and ginsenoside which are detected on different fermentation days, the fermentation end point of 23 days of fermentation is obtained.
3. Detection of biologically active substances before and after fermentation
3.1 ginsenoside Rg before and after fermentation3Content change
3.1.1 chromatographic conditions
A chromatographic column: kromasil100-5-C184.6X 250 mm; mobile phase: acetonitrile (B) -water (a) (45: 55) isocratic elution; detection wavelength: 203 nm; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 1.0 mL/min.
3.1.2 preparation of test solutions
Taking a proper amount of a test sample (0.2 g of American ginseng stem leaves and 1.3g of lucidification), precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, weighing, ultrasonically extracting for 1 hour, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, washing the container and the residue by using 10mL of methanol for times, combining the filtrate and the washing liquor, evaporating to dryness, dissolving the residue in 10mL of water, extracting for 4 times by using water saturated n-butyl alcohol, combining the n-butyl alcohol solution for each time, evaporating to dryness, re-dissolving by using methanol, fixing the volume to a 10mL volumetric flask, shaking up, and passing through a 0.22 mu m microfiltration membrane to obtain the product.
3.1.3 assay, results are shown in Table 9 and FIG. 6:
TABLE 9 ginsenoside Rg in stems and leaves of Panax quinquefolium and Lingxi mycoplasm3Determination of content
The experimental result shows that the ginsenoside Rg is not detected in the stem and leaf of the American ginseng purchased in the experiment3However, the ginsenoside Rg in 6 batches of fermented lucidification3The average content of (A) was 0.1871 mg/g.
3.2 content Change of ginsenoside CK before and after fermentation
3.2.1 HPLC chromatographic conditions: a chromatographic column: kromasil 100-5-C184.6X 250 mm; mobile phase: acetonitrile (B) -water (A) (51: 49) isocratic elution; detection wavelength: 203 nm; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 1.0 mL/min.
3.2.2 preparation of test solutions
Taking a proper amount of a test sample (0.2 g of American ginseng stem leaves and 1.3g of lucidification), precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, weighing, ultrasonically extracting for 1 hour, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, washing the container and the residue by using 10mL of methanol for times, combining the filtrate and the washing liquor, evaporating to dryness, dissolving the residue in 10mL of water, extracting for 4 times by using water saturated n-butyl alcohol, combining the n-butyl alcohol solution for each time, evaporating to dryness, re-dissolving by using methanol, fixing the volume to a 10mL volumetric flask, shaking up, and passing through a 0.22 mu m microfiltration membrane to obtain the product.
3.2.3 assay, results are shown in Table 10 and FIG. 7:
TABLE 10 determination of ginsenoside CK content in stems and leaves of Panax quinquefolium and Lingxi mycoplasm
The experimental result shows that the ginsenoside CK is not detected in the stems and leaves of the panax notoginseng purchased in the experiment, but the average content of the ginsenoside CK in the fermented panaxathin is 0.3532 mg/g.
3.3 nucleoside content Change before and after fermentation
3.3.1 HPLC chromatographic conditions: a chromatographic column: COSMOSIL5C18-PAQ 4.6X 250 mm; mobile phase: methanol (C) -water (a) gradient elution, data are given in the table below; detection wavelength: 259 nm; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution;
flow rate: 1.0mL/min, gradient elution conditions are shown in Table 11:
TABLE 11 gradient elution conditions
3.3.2 preparation of test solutions: taking about 0.5g of a test sample (a substrate before fermentation and a mycelial substance after fermentation), precisely weighing, respectively placing in 10mL test tubes with plugs, precisely adding 10mL of water, uniformly mixing the plugs, ultrasonically extracting for 1 hour (500w, 40kHz), taking out the test tubes, cooling to room temperature, uniformly shaking, taking a proper amount of extracting solution, centrifuging (4000r/min, 10min), and passing the supernatant through a 0.45-micrometer microporous filter membrane to obtain the microbial inoculum.
3.3.3 nucleoside Change before and after fermentation
The change of the nucleoside in the substrate before fermentation and the mycelial prodigiosin after fermentation (the sample weighing of the test sample is the same) is researched.
Referring to fig. 8, the experimental results show that the content difference of cytosine and uracil before and after fermentation is not large and is very low; the content of cytidine, uridine, guanosine and adenosine after fermentation is increased more than before fermentation; the adenine content is reduced after fermentation.
3.4 peptide molecular weight Change before and after fermentation
3.4.1 chromatographic conditions: a chromatographic column: TSKgel G2000SWXL (30 cm. times.7.8 mm) gel column; mobile phase: 0.1mol/LPBS+0.1mol/LNa2SO4(pH 6.7); detection wavelength: 220 nm; column temperature: 30 ℃; sample introduction amount: 5 mu L of the solution;
flow rate: 0.5 mL/min.
3.4.2 preparation of test solution: taking 10g of a test sample (a substrate before fermentation and a mycelial substance after fermentation), precisely weighing, adding 30 times of water, carrying out reflux extraction for 2 hours, placing at room temperature, centrifuging, filtering, discarding a precipitate, concentrating a supernatant to about 100mL, adding ethanol, precipitating to 60% by alcohol, placing in a refrigerator at 0 ℃ for cold storage for 24 hours, centrifuging, filtering, discarding a precipitate, recovering ethanol from the supernatant, evaporating to dryness, diluting the supernatant to a proper concentration by using distilled water to a volume flask with a constant volume of 100mL, and filtering by using a 0.45-micrometer microporous filter membrane to obtain the bacillus subtilis.
3.4.3 preparation of relative molecular weight calibration curves: the method comprises the steps of establishing a standard by taking bovine serum albumin (66.4k), a trypsin inhibitor (6511), human angiotensin II (1046) and L-glyco-tripeptide (189) as relative molecular markers. The regression equation is-0.1772X +6.9993(r is 0.9978), and the linear relationship between the molecular weights is well within 189-66.4 kDa.
4.4 peptide molecular weight determination, results are shown in table 12, fig. 9 and fig. 10:
TABLE 12 results of molecular weight distribution measurement of pre-fermentation substrate and post-fermentation lucidification peptides
The experimental result shows that the molecular weight of the matrix peptide before fermentation is larger than 22.05 percent of that of 6511Da, and the mycelial substance of the Lingxi is not detected after fermentation; the molecular weight of matrix peptide before fermentation is 6511-1046 Da and accounts for 38.98%, and the content of mycelial substance after fermentation is 20.08%; the molecular weight of the matrix peptide before fermentation is less than 1046Da and accounts for 38.97%, and the mycelial mass of the fermented Lingxi accounts for 79.92%.
3.5 content changes of total ginsenosides and sapogenins before and after fermentation
3.5.1 preparation of test solution: taking about 1g of a test sample (a substrate before fermentation and a mycelial substance after fermentation), precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, heating and refluxing in a water bath for 2 hours, cooling, filtering, washing filter residues with 10mL of methanol, combining filtrates, evaporating to dryness, and dissolving residues with 10mL of water; extracting the water solution with diethyl ether for 4 times, 10mL each time, mixing diethyl ether solution (total sapogenin), volatilizing at low temperature, and metering volume of residue with methanol to 25mL volumetric flask; extracting the water solution with water saturated n-butanol under shaking for 10mL each time for 4 times, mixing n-butanol solutions (total saponins of Ginseng radix), evaporating to dryness, and adding methanol to desired volume of the residue in a 25mL volumetric flask for use.
3.5.2 creation of Standard Curve: accurately sucking 0.9916mg/mL ginsenoside Re reference substance solution 0.02mL, 0.04mL, 0.08mL, 0.12mL, 0.16mL and 0.20mL, placing in a test tube, volatilizing the solvent at low temperature, adding 1% vanillin perchloric acid test solution 0.5mL, mixing, heating in a water bath at 60 ℃ for 15 minutes, cooling in an ice water bath for 2 minutes, adding 77% sulfuric acid solution 5mL, shaking up, measuring absorbance at 540nm with the corresponding reagent as a blank, and drawing a standard curve with the mass as an abscissa (X) and the absorbance as an ordinate (Y). The regression equation is 0.0048X-0.0349(r is 0.9991), and ginsenoside Re is found to have good linear relation within the range of 19.83-198.3 mug.
3.5.3 content measurement, the results are shown in Table 13:
TABLE 13 Total ginsenoside and Total sapogenin content in Pre-and post-fermentation substrate
The experimental result shows that the content of the total ginsenoside in the substrate before fermentation is 11.37mg/g, and the content of the total sapogenin is 6.265 mg/g; the average content of total ginsenosides in 6 batches of linxiansu is 8.864mg/g, and the average content of total ginsenosides is 8.513 mg/g.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A bidirectional solid fermentation process for stems and leaves of Ganoderma lucidum-American ginseng is characterized by comprising the following steps:
(1) preparing a solid culture medium of American ginseng stems and leaves;
(2) inoculating Ganoderma into solid culture medium of stem and leaf of radix Panacis Quinquefolii via Ganoderma culture solution, and fermenting to obtain fermented material;
(3) taking out the fermented material, drying and sieving to obtain a fermented product.
2. The bidirectional solid state fermentation process of ganoderma-American ginseng stem leaves as claimed in claim 1, wherein the step (1) of preparing the American ginseng stem leaf solid medium specifically comprises: taking 60-65% of corn bran, 5-15% of corn yellow powder, 5-15% of wheat bran and 15-20% of American ginseng stem leaves according to the following mass ratio, then adding monopotassium phosphate accounting for 1% of the total mass of the four raw materials, oyster shell powder accounting for 1% of the total mass of the four raw materials and magnesium sulfate accounting for 0.5% of the total mass of the four raw materials, filling the mixture into a fungus bag, controlling the water content in the fungus bag to be 50-60%, and then sterilizing the fungus bag.
3. The bidirectional solid state fermentation process of the stem leaves of the ganoderma lucidum-American ginseng as claimed in claim 2, wherein the amount of the fungus bags filled in the step (1) is 150-300 g; the sterilization of the fungus bag comprises placing the fungus bag into a high pressure steam sterilization pot, and sterilizing at 121 deg.C for 45 min.
4. The bidirectional solid state fermentation process of ganoderma-American ginseng stem and leaf as claimed in claim 1, wherein the inoculating of ganoderma into American ginseng stem and leaf solid medium in step (2) for fermentation specifically comprises: taking out the sterilized fungus bags, standing to room temperature, inoculating Ganoderma to solid culture medium of stems and leaves of radix Panacis Quinquefolii via Ganoderma culture solution on superclean bench, sealing, and fermenting in dark room.
5. The bidirectional solid state fermentation process of the stem leaves of the ganoderma lucidum-American ginseng as claimed in claim 4, wherein the dosage of the ganoderma lucidum culture solution in the step (2) is 15-40 mL; the fermentation temperature is 25-30 ℃, and the fermentation time is 20-25 days.
6. The bidirectional solid state fermentation process of the stem leaves of the ganoderma lucidum-American ginseng as claimed in claim 1, wherein the drying temperature in the step (3) is 55 ℃; the sieving is a No. three sieving.
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