A kind of Poria cocos fermented bacteria powder and its preparation method and application

Abstract

The present application relates to the field of biological fermentation engineering, and discloses a kind of Poria cocos fermentation powder and its preparation method and application. The preparation method of the present invention domesticates Eurotium guanidinum and Poria cocos with dihydromyricetin and hesperidin respectively, and uses Eurotium guanidinum capsule as a carrier to embed Poria cocos, so that Poria cocos can tolerate high temperature environment, and can artificially control intermittent high temperature in the process of fermenting Chinese herbal medicine, which can prevent contamination by miscellaneous bacteria, ensure purity, and do not affect normal fermentation, and the cost is lower than that of using special aseptic operation equipment. The Poria cocos fermentation powder is used to ferment tangerine peel, Ligusticum chuanxiong, Cyclocarya paliurus, Eucommia ulmoides leaves and dandelions under intermittent high temperature conditions, which can increase the content of effective active ingredients of Chinese herbal medicines compared with ordinary Poria cocos fermentation, and there is no black Aspergillus contamination. The present invention provides a new idea for solving the problems faced by Chinese herbal medicine fermentation, and lays a solid foundation for technological progress and industrial upgrading in the field of Chinese herbal medicine fermentation.

Description

Technical Field

[0001] The present application relates to the field of biological fermentation engineering, and more specifically, to a Poria cocos fermentation bacteria powder and a preparation method and application thereof. Background Art

[0002] Traditional Chinese herbal medicine fermentation uses natural fermentation of microorganisms in the air. The fermentation process is difficult to control artificially, and the product quality cannot be guaranteed. Modern Chinese herbal medicine fermentation uses modern biotechnology to monitor fermentation parameters including temperature, pH, fermentation time, number of strains and status, which helps to achieve product quality standardization. Bidirectional fermentation refers to using Chinese herbal medicine as the medicinal matrix and adding fungi for fermentation. Its bidirectionality lies in that while fungi obtain the nutrients needed for growth from Chinese herbal medicine, they also affect the metabolic activity of fungi, which can improve the extraction rate of active ingredients in Chinese herbal medicines, enhance the efficacy, and produce new active ingredients. Therefore, the use of fungi to ferment Chinese herbal medicines is more superior than general physical or chemical processing methods.

[0003] Poria cocos is the dried sclerotium of the fungus Poria cocos of the Polyporaceae family. It mostly parasitizes on the roots of Pinus massoniana or Pinus densiflora. It is a traditional Chinese medicinal material in my country. In commonly used Chinese medicine prescriptions, the compatibility rate of Poria cocos is as high as 70%. Poria cocos tastes sweet and light, and has a mild medicinal property. It has the effects of diuresis, invigorating the spleen and stomach, and calming the mind. It is known as the "holy medicine for dehumidification" and the "top grade of immortal medicine". Modern medical research shows that Poria cocos also has the effects of enhancing the body's immunity, anti-tumor, and lowering blood sugar. The main components of Poria cocos are Poria cocos polysaccharides and triterpenoid compounds. Poria cocos polysaccharides have biological activities such as anti-tumor activity, enhancing immunity, promoting cell division, and complement activation. The inhibition rate of mouse sarcoma S-180 is as high as 96.88%.

[0004] Domestication of fungi generally refers to a method of adapting fungi to certain conditions through artificial measures, thereby selectively breeding fungi. For the domestication of medicinal edible fungi, a culture medium containing certain nutrients is usually used for domestication, but many Chinese herbal medicines produce active substances with antibacterial effects during fermentation, which inhibit the growth of fungi and the fermentation effect, such as dihydromyricetin, an active ingredient of berry tea, hesperidin, an active ingredient of tangerine peel, phloridzin, an active ingredient of cinnamon leaf, and chlorogenic acid, an active ingredient of honeysuckle. Therefore, the present invention selects dihydromyricetin and hesperidin, active substances with antibacterial effects, to domesticate fungi, so that fungi can adapt to the antibacterial environment during the fermentation process and improve the fermentation effect of medicinal edible fungi. Dihydromyricetin is a dihydroflavonol flavonoid compound, which is widely present in plants of the genus Ampelopsis in the family Ampelopsis, and is the main active ingredient in Ampelopsis, which has the effect of resisting pathogenic microorganisms, and has a significant inhibitory effect on bacteria such as Bacillus subtilis and Staphylococcus aureus, as well as fungi such as Aspergillus niger and Aspergillus flavus. Hesperidin is widely found in natural plants such as tangerine peel and citrus peel. It has multiple biological activities, such as antibacterial, anti-inflammatory, and antiviral effects, especially a broad spectrum antibacterial effect on food contamination.

[0005] Since most medicinal and edible fungi are not resistant to high temperatures, the fermentation temperature can only be maintained at 20-35°C during the fermentation of Chinese herbal medicines using medicinal and edible fungi, which can easily lead to contamination by foreign bacteria, especially Aspergillus niger. Aspergillus niger will not be killed until it lasts for 60 minutes in an environment of 60°C, and the cost of using special aseptic operation equipment is very high, which is not suitable for large-scale promotion. Therefore, the present invention adopts the high-temperature resistant capsule of Aspergillus cristatus as a carrier to embed Poria cocos, so that Poria cocos can tolerate high-temperature environment, so that intermittent high-temperature conditions can be artificially controlled during the fermentation of Chinese herbal medicines, the growth of foreign bacteria can be inhibited, the purity of the fermentation product can be ensured, and the normal fermentation of medicinal and edible fungi will not be affected. This method greatly reduces the production cost. Summary of the invention

[0006] In order to overcome the deficiencies in the prior art, the present invention provides a Poria cocos fermented bacteria powder and its preparation method and application. The preparation method of the present invention, by using antibacterial active substances to domesticate the cosmidia and Poria cocos and using the capsule of the cosmidia as a carrier to embed the Poria cocos mycelium, obtains Poria cocos that can adapt to the antibacterial environment and high temperature environment during the fermentation process, and then adds an interfering agent, freeze-dried and crushed, and finally makes high-quality Poria cocos fermented bacteria powder. The Poria cocos fermented bacteria powder is used to ferment tangerine peel, Ligustrum lucidum, Cyclocarya paliurus, Eucommia ulmoides leaves and dandelions, which can enhance the effect of Chinese herbal medicine fermentation, increase the content of Chinese herbal medicine active substances, and inhibit the growth of miscellaneous bacteria, especially Aspergillus niger, to ensure the purity of the Chinese medicine fermentation process. The preparation method is conducive to promoting the continuous innovation and development of the Chinese herbal medicine fermentation industry.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0008] In a first aspect, the present application discloses a method for preparing Poria cocos fermented bacteria powder, comprising the following steps:

[0009] S1, preparation of vector:

[0010] S1-1, take 25g of golden flower from black tea, put it into 225ml of sterilized saline, shake well, let it stand for 10 minutes, take 1ml of the mixture, inoculate it into PDA solid culture medium, and culture it in a 26℃ incubator for 5-7 days;

[0011] S1-2, picking a well-growing colony of Eurotium cristatum from the PDA solid medium obtained in step S1-1 and inoculating it into the PDA solid medium, and culturing it at 26° C. for 3-5 days to obtain a slant strain of Eurotium cristatum;

[0012] S1-3, picking a well-growing colony of Eurotium cristatum from the PDA solid culture medium obtained in step S1-2, inoculating it into a PDB liquid culture medium, and culturing it on a shaker at a speed of 150 r / min and a temperature of 26° C. for 10 days to obtain a bacterial solution enriched with Eurotium cristatum spores;

[0013] S1-4, taking 1 ml of the bacterial solution prepared in step S1-3, inoculating it into a PDA solid culture medium containing a taming agent, and culturing it at 26° C. for 3 days to acclimate the Eurotium cristatum;

[0014] S1-5, picking a colony with good growth status from the PDA solid culture medium obtained in step S1-4, inoculating it into a PDB liquid culture medium containing an acclimator, and culturing it on a shaker at a speed of 150 r / min and a temperature of 26° C. for 5 days to obtain an acclimated Eurotium cristatum bacterial liquid, wherein the dry weight of mycelium in the bacterial liquid is 3.0 to 3.5 g / L;

[0015] S1-6, placing the culture solution of the Eurotium cristatum expanded in step S1-5 in a culture chamber at 28°C for 1 day, then raising the temperature to 30°C for 1 day, and finally raising the temperature to 32°C for 1 day, taking the bacteria produced in the upper layer, inactivating them at high temperature, and freeze-drying them, thereby obtaining the cysts of the Eurotium cristatum;

[0016] S2, preparation of Poria cocos powder:

[0017] S2-1, inoculate the Poria cocos strains into PDA solid medium and culture at 24 °C for 3 days;

[0018] S2-2, picking a well-growing Poria cocos colony from the PDA solid culture medium obtained in step S2-1, inoculating it into a PDB liquid culture medium, and culturing it on a shaker at a speed of 170 r / min and a temperature of 24° C. for 10 days to obtain a bacterial liquid enriched with Poria cocos mycelium;

[0019] S2-3, taking 1 ml of the bacterial solution prepared in step S2-2, inoculating it into a PDA solid culture medium containing a taming agent, and culturing it at 24° C. for 3 days to taming the Poria cocos;

[0020] S2-4, picking a well-growing Poria cocos colony from the PDA solid culture medium obtained in step S2-3, inoculating it into a PDB liquid culture medium containing a taming agent, culturing it on a shaker at a speed of 170 r / min and a temperature of 24° C. for 10 days to obtain a tamed Poria cocos bacterial liquid, wherein the dry weight of mycelium in the bacterial liquid is 5.0 to 5.5 g / L, filtering to obtain wet Poria cocos cells, freeze-drying the wet cells and crushing them to obtain tamed Poria cocos powder;

[0021] S3, preparation of Poria cocos fermented bacteria powder:

[0022] S3-1, using the capsule of the Aspergillus niger obtained in step S1-6 or dextrin as a carrier, mixing it with the Poria cocos powder domesticated in step S2-4, adding distilled water at a mass ratio of 1:100, stirring at 37° C. for 3 h to fully dissolve it, embedding the Poria cocos, concentrating the solution, filtering it with suction, and freeze-drying the precipitate to obtain embedded Poria cocos;

[0023] S3-2, crushing the embedded Poria cocos in step S3-1 to make fine and uniform powder, adding an interfering agent and mixing evenly to obtain Poria cocos fermented fungus powder.

[0024] Furthermore, in step S3-1, the capsule of Eurotium cristatum obtained in step S1-6 is preferably used as a carrier.

[0025] Furthermore, the acclimation agent is selected from any one of chlorogenic acid, dihydromyricetin or hesperidin.

[0026] Furthermore, in steps S1-4 and S1-5, the acclimating agent is dihydromyricetin, and the content of the dihydromyricetin powder is 0.9 g / g.

[0027] Furthermore, in steps S2-3 and S2-4, the acclimating agent is hesperidin, and the content of the hesperidin powder is 0.9 g / g.

[0028] Furthermore, the dosage of the taming agent is 0.5-2.0%.

[0029] Furthermore, the dosage of the taming agent is 1.0%.

[0030] Furthermore, in step S3-1, the mass ratio of the domesticated Poria cocos powder and the Eurotium cristatum shell carrier is 1:(1.0-1.5).

[0031] Furthermore, in step S3-1, the mass ratio of the domesticated Poria cocos powder and the Eurotium cristatum shell carrier is 1:1.2.

[0032] Furthermore, in step S3-2, the interfering agent is dihydromyricetin, and the mass ratio of the interfering agent added is 1%.

[0033] Furthermore, the PDA solid culture medium, also known as potato dextrose agar culture medium, comprises 6.0 g / L potato flour, 20.0 g / L glucose, 20.0 g / L agar, and 0.1 g / L chloramphenicol. 46.0 g of the prepared PDA powder is added to 1 L of distilled water, heated and stirred until completely dissolved, and sterilized in a high-pressure steam sterilizer at 121° C. for 20 min for use.

[0034] Furthermore, the PDB liquid culture medium, also known as potato glucose broth culture medium, includes 5.0 g / L potato flour, 15.0 g / L glucose, 10.0 g / L peptone, and 5.0 g / L sodium chloride. 35.0 g of the prepared PDB powder is added to 1 L of distilled water, heated and stirred until completely dissolved, placed in a high-pressure steam sterilizer and sterilized at 121° C. for 20 minutes, and set aside.

[0035] In a second aspect, the present application discloses a fermented Poria cocos powder, which is prepared by any preparation method described in the first aspect.

[0036] In a third aspect, the present application discloses an application of Poria cocos fermented fungus powder prepared by any preparation method described in the first aspect in the field of Chinese herbal medicine fermentation.

[0037] The application includes application in fermenting dried tangerine peel, Ligusticum wallichii (sweet tea), Cyclocarya paliurus, Eucommia ulmoides leaves and dandelion.

[0038] The application includes application at normal temperature (24°C) and intermittent high temperature (70°C, 30 min) conditions.

[0039] In summary, compared with the prior art, the beneficial effects or advantages of the present invention are mainly reflected in the following aspects:

[0040] 1. The fermented Poria cocos powder prepared by the method of the present invention can be used for the fermentation of Chinese herbal medicines, which overcomes the problem that the fermentation effect of Poria cocos is affected by the antibacterial substances of Chinese herbal medicines and the fermentation process is prone to contamination by foreign bacteria. It provides a new type of efficient, convenient and low-cost fermentation method for the fermentation of Chinese herbal medicines, and is expected to promote the rational and full utilization of Chinese herbal medicine resources.

[0041] 2. Since many Chinese herbal medicines will produce active substances with antibacterial effects during fermentation, which inhibit the growth of fungi, the present invention uses dihydromyricetin and hesperidin with antibacterial effects as taming agents to tame the Aspergillus niger and Poria cocos, respectively, so that they can better adapt to the antibacterial environment during the fermentation process and improve the fermentation effect.

[0042] 3. The cost of using special aseptic operation equipment is high, which is not conducive to large-scale promotion. The present invention solves this problem in a targeted manner, and the capsule of Eurotium cristatum is used to embed Poria cocos hyphae, so that Poria cocos can be more tolerant to high temperature environment, so that intermittent high temperature conditions can be artificially controlled during the fermentation of Chinese herbal medicine, effectively inhibiting the growth of miscellaneous bacteria, especially Aspergillus niger, and ensuring the purity of fermentation products. In addition, interfering agents are added to the embedded Poria cocos, and the growth of miscellaneous bacteria is further inhibited. The preparation method of this kind of Poria cocos fermented bacteria powder is not seen in other documents and patents. DETAILED DESCRIPTION

[0043] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0044] Example 1: Preparation of Poria cocos fermented bacteria powder

[0045] This embodiment provides a method for preparing fermented Poria cocos powder, and the specific steps are as follows:

[0046] S1, preparation of vector:

[0047] S1-1, take 25g of golden flower from black tea, put it into 225ml of sterilized saline, shake well, let it stand for 10 minutes, take 1ml of the mixture, inoculate it into PDA solid culture medium, and culture it in a 26℃ incubator for 7 days;

[0048] S1-2, picking a well-growing colony of Eurotium cristatum from the PDA solid medium obtained in step S1-1 and inoculating it into the PDA solid medium, and culturing it at 26° C. for 5 days to obtain a slant strain of Eurotium cristatum;

[0049] S1-3, picking a well-growing colony of Eurotium cristatum from the PDA solid culture medium obtained in step S1-2, inoculating it into a PDB liquid culture medium, and culturing it on a shaker at a speed of 150 r / min and a temperature of 26° C. for 10 days to obtain a bacterial solution enriched with Eurotium cristatum spores;

[0050] S1-4, taking 1 ml of the bacterial solution prepared in step S1-3, inoculating it into a PDA solid culture medium containing 1% dihydromyricetin, and culturing it at 26° C. for 3 days to acclimate the Eurotium cristatum;

[0051] S1-5, picking a colony with good growth status from the PDA solid culture medium obtained in step S1-4, inoculating it into a PDB liquid culture medium containing 1% dihydromyricetin, and culturing it on a shaker at a speed of 150 r / min and a temperature of 26° C. for 5 days to obtain an acclimated Eurotium cristatum bacterial liquid, wherein the dry weight of mycelium in the bacterial liquid is 3.0 g / L;

[0052] S1-6, placing the culture solution of the Eurotium cristatum expanded in step S1-5 in a culture chamber at 28°C for 1 day, then raising the temperature to 30°C for 1 day, and finally raising the temperature to 32°C for 1 day, taking the bacteria produced in the upper layer, inactivating them at high temperature, and freeze-drying them, thereby obtaining the cysts of the Eurotium cristatum;

[0053] S2, preparation of Poria cocos powder:

[0054] S2-1: Inoculate the Poria cocos strains with complete certificates purchased from Fumei Strain Business Department in Jiuzhen, Xishui County into PDA solid medium and culture at 24°C for 3 days;

[0055] S2-2, picking a well-growing Poria cocos colony from the PDA solid culture medium obtained in step S2-1, inoculating it into a PDB liquid culture medium, and culturing it on a shaker at a speed of 170 r / min and a temperature of 24° C. for 10 days to obtain a bacterial liquid enriched with Poria cocos mycelium;

[0056] S2-3, taking 1 ml of the bacterial solution prepared in step S2-2, inoculating it into a PDA solid culture medium containing 1% hesperidin, and culturing it at 24° C. for 3 days to acclimate the Poria cocos;

[0057] S2-4, picking a well-growing Poria cocos colony from the PDA solid culture medium obtained in step S2-3, inoculating it into a PDB liquid culture medium containing 1% hesperidin, and culturing it on a shaker at a speed of 170 r / min and a temperature of 24° C. for 10 days to obtain an acclimated Poria cocos bacterial liquid, wherein the dry weight of mycelium in the bacterial liquid is 5.0 g / L, filtering to obtain wet Poria cocos cells, freeze-drying the wet cells and crushing them to obtain acclimated Poria cocos powder;

[0058] S3, preparation of Poria cocos fermented bacteria powder:

[0059] S3-1, using the cysts of the Aspergillus niger obtained in step S1-6 as a carrier, mixing with the Poria cocos powder domesticated in step S2-4 at a mass ratio of 1:1.2, adding distilled water at a mass ratio of 1:100, stirring at 37° C. for 3 hours to fully dissolve it, embedding the Poria cocos, concentrating the solution, filtering it with suction, and freeze-drying the precipitate to obtain Poria cocos embedded in the cysts of the Aspergillus niger;

[0060] S3-2, crushing the Poria cocos embedded in the capsule of Eurotium cristatum in step S3-1 to make fine and uniform powder, adding 1% interfering agent dihydromyricetin and mixing evenly, so as to obtain Poria cocos fermented fungus powder.

[0061] In the above embodiment, the PDA solid culture medium, also known as potato dextrose agar culture medium, includes 6.0 g / L potato flour, 20.0 g / L glucose, 20.0 g / L agar, and 0.1 g / L chloramphenicol. 46.0 g of the prepared PDA powder is added to 1 L of distilled water, heated and stirred until completely dissolved, and placed in a high-pressure steam sterilizer for sterilization at 121° C. for 20 min for standby use.

[0062] In the above embodiment, the PDB liquid culture medium, also known as potato dextrose broth culture medium, includes 5.0 g / L potato flour, 15.0 g / L glucose, 10.0 g / L peptone, and 5.0 g / L sodium chloride. 35.0 g of the prepared PDB powder is added to 1 L of distilled water, heated and stirred until completely dissolved, and placed in a high-pressure steam sterilizer for sterilization at 121° C. for 20 min for use.

[0063] Example 2: Single factor test of preparation process

[0064] (1) Investigation of Poria cocos vector types

[0065] In order to enable Poria cocos to tolerate the high temperature environment controlled during the fermentation of Chinese herbal medicine, the Poria cocos mycelium was embedded with a carrier, and the carrier type was selected from the shell of Aspergillus niger or dextrin. The Poria cocos powder and the carrier were mixed in a mass ratio of 1:1, and distilled water was added in a ratio of 1:100. The mixture was stirred at 37°C for 3 hours for embedding. The mixed solution was concentrated, filtered, and the precipitate was freeze-dried and crushed to obtain Poria cocos powder embedded with the shell of Aspergillus niger or the dextrin carrier. The embedded Poria cocos powder was dissolved in an appropriate amount of distilled water and sprayed on tangerine peel to ferment the tangerine peel. The temperature was raised to 70°C for 30 minutes every day, and then cooled to 30°C to maintain the fermentation process. The fermentation time was 30 days, and the content of hesperidin in the fermented product of tangerine peel was determined as an evaluation index. After the extracted hesperidin sample was dissolved in 50% ethanol, the absorbance was measured at 360 nm using a UV spectrophotometer using the standard curve method to calculate the hesperidin content (mg / g) in the sample to be tested.

[0066] The test results are shown in Table 1. This test investigated the content of hesperidin after the Poria cocos encapsulated with the capsule of Eurotium guanidinum or dextrin as a carrier fermented tangerine peel under high temperature conditions, and used this as an evaluation index to determine the type of Poria cocos carrier. As can be seen from Table 1, the average content of hesperidin after the domesticated Poria cocos fermented tangerine peel was 1.099 mg / g, which is higher than the average content of hesperidin after the Poria cocos fermented tangerine peel with dextrin as a carrier. Therefore, Eurotium guanidinum can be selected as the preferred Poria cocos carrier.

[0067] Table 1 Hesperidin content of tangerine peel fermented with Poria cocos embedded in different carriers

[0068]

[0069] (2) Investigation of the types of acclimation agents for Eurotium cristatum

[0070] The types of acclimation agents for the crown-stage Eurotium are chlorogenic acid, dihydromyricetin, and hesperidin, and the dosage of the acclimation agent is 0.5%. The acclimated crown-stage Eurotium is used to ferment the old leaves of berry tea, and the total flavonoids content in the berry tea fermentation product is determined as an evaluation index. The specific operation method is: the crown-stage Eurotium is inoculated into PDA solid culture medium containing 0.5% chlorogenic acid, 0.5% dihydromyricetin, and 0.5% hesperidin, and the bacteria with good growth are selected and inoculated into PDB liquid culture medium containing 0.5% different acclimation agents, and the crown-stage Eurotium is acclimated. The fermented berry tea was sprinkled on old leaves of berry tea with a water content of 60% according to a 1% inoculum, and 3 replicates were set for each experimental group. The tea was cultured at 28°C for 4 days, 30°C for 1 day, and 32°C for 1 day, and then dried. The dried fermented berry tea was crushed into a powder of 0.85 mm (20 mesh), 0.5 g was weighed, 95% ethanol was added to dissolve and the volume was adjusted to 50 ml, and the sample was filtered after standing to obtain a test sample. 1 mL of the filtrate was added to 95% ethanol and diluted 25 times as a test sample. 1 mL of the test sample was drawn into a 10 mL colorimetric tube, 3 mL of aluminum chloride solution was added, and then 95% ethanol was added to adjust the volume to 10 mL, mixed evenly, and allowed to stand at room temperature in the dark for 40 minutes. The absorbance was measured at 291 nm using an ultraviolet spectrophotometer, and the content of total flavonoids in the test sample (mg / mL) was obtained by the standard curve, and finally the content of total flavonoids in the test sample (mg / g) was obtained by multiplying the dilution multiple.

[0071] The test results are shown in Table 2. This test investigated the total flavonoid content of old berry tea leaves fermented by Eurotium coronarius domesticated with chlorogenic acid, dihydromyricetin and hesperidin as domesticators, and used this as an evaluation index to determine the domesticator type of Eurotium coronarius. As can be seen from Table 2, the average content of total flavonoids in old berry tea leaves fermented by Eurotium coronarius domesticated with dihydromyricetin is 3.829 mg / g, which is higher than the average content of total flavonoids in old berry tea leaves fermented by Eurotium coronarius domesticated with chlorogenic acid and hesperidin. Therefore, dihydromyricetin can be selected as the preferred domesticator of Eurotium coronarius.

[0072] Table 2 Total flavonoids content of old berry tea leaves fermented by Eurotium cristatum domesticated with different domestication agents

[0073]

[0074]

[0075] (3) Investigation of the types of tuckahoe fungus acclimation agents

[0076] The taming agent type of Poria cocos is chlorogenic acid, dihydromyricetin, and hesperidin, and the dosage of the taming agent is 0.5%. The tamed Poria cocos is used to ferment tangerine peel, and the content of hesperidin in the fermented tangerine peel is determined as an evaluation index. The specific operation method is: the Poria cocos is inoculated in PDA solid culture medium containing 0.5% chlorogenic acid, 0.5% dihydromyricetin, and 0.5% hesperidin, and the bacteria with good growth are selected and inoculated in PDB liquid culture medium containing 0.5% different taming agents, and the Poria cocos is tamed. According to the 1% inoculation amount, it is sprinkled on the tangerine peel after high temperature sterilization. Each experimental group is set up with 3 replicates, cultured at 24℃ for 30d, and dried; the standard curve method is also used, and the absorbance is measured at 360nm using an ultraviolet spectrophotometer to calculate the content of hesperidin (mg / g) in the sample to be tested.

[0077] The test results are shown in Table 3. This test investigated the content of hesperidin after the tuckahoe fermented tangerine peel with chlorogenic acid, dihydromyricetin, and hesperidin as taming agents, and used this as an evaluation index to determine the taming agent type of the tuckahoe. As can be seen from Table 3, the average content of hesperidin, an active substance produced by the tuckahoe fermented tangerine peel with hesperidin is 1.177 mg / g, which is higher than the average content of hesperidin after the tuckahoe fermented tangerine peel with chlorogenic acid and dihydromyricetin. Therefore, hesperidin can be selected as the preferred tuckahoe taming agent.

[0078] Table 3 Hesperidin content of tangerine peel fermented by Poria cocos tamed with different taming agents

[0079]

[0080] Example 3: Orthogonal test optimization of preparation process

[0081] Based on the above single factor test results, the carrier-capsule of Eurotium cristatum (A), the acclimation agent of Eurotium cristatum-dihydromyricetin (B), and the acclimation agent of Poria cocos-hesperidin (C) were used as three factors, and L9 (3 3 ) Orthogonal test method, taking the content of hesperidin in the fermentation product of tangerine peel fermented with Poria powder as the evaluation index, the mixing ratio of the bacteria and the ascariasis of Eurotium cristatum and the percentage of the acclimation agents dihydromyricetin and hesperidin were determined, and the ratios were all mass ratios.

[0082] The domesticated Poria mycelium was mixed with the capsule extracted from the domesticated Eurotium cristatum, and 1% interfering agent was added to mix, freeze-dried, and crushed to obtain Poria powder. The Poria powder was dissolved with an appropriate amount of water, and the powder liquid was evenly sprayed on the tangerine peel for fermentation. During the fermentation process, intermittent high temperature conditions were controlled, and the temperature was raised to 70°C for 30 minutes every day, and then cooled to 30°C to maintain the fermentation process. The fermentation time was 30 days. L9(3 3 ) The orthogonal test factors and levels are shown in Table 4. L9(3 3 ) The orthogonal experimental design and results are shown in Table 5.

[0083] The results of the range R value in Table 5 showed that the content of hesperidin in the fermentation product of tangerine peel fermented by Poria powder was affected by various factors in the following order: Poria acclimator-hesperidin>Ganoderma rubra acclimator-dihydromyricetin>carrier-Ganoderma rubra shell. The results of the intuitive analysis of the mean values ​​K1, K2, and K3 showed that the optimal preparation conditions of Poria fermented powder were: Ganoderma rubra acclimated with 1.0% dihydromyricetin, Poria acclimated with 1.0% hesperidin, and the acclimated Poria acclimated with the Ganoderma rubra shell carrier in a ratio of 1:1.2.

[0084] Table 4 L9(3 3 )Orthogonal experimental factors and levels

[0085]

[0086] Table 5 L9(3 3 )Orthogonal Experimental Design and Results

[0087]

[0088]

[0089] Example 4: Application of Poria cocos fermented bacteria powder in the field of Chinese herbal medicine fermentation

[0090] This example investigates the application of the fermented Poria cocos powder prepared in Example 1 in the field of Chinese herbal medicine fermentation, and determines the fermentation effect of the fermented Poria cocos powder by fermenting dried tangerine peel, Ligusticum chuanxiong (sweet tea), Cyclocarya paliurus, Eucommia ulmoides leaves, and Taraxacum officinale as examples.

[0091] (1) Fermented tangerine peel

[0092] Dissolve the fermented Poria cocos powder in an appropriate amount of pure water, and evenly spray the powder liquid on the tangerine peel according to the inoculation amount of 1%. Control the intermittent high temperature conditions, raise the temperature to 70℃ for 30min every day, and then cool it down to 30℃ to maintain the fermentation process. The fermentation time is 30d. At the same time, another experimental group sets the temperature at 24℃ for 30d. The untamed common Poria cocos purchased from the strain company was used as a control. After the hesperidin sample extracted from the fermentation product was dissolved in 50% ethanol, the absorbance was measured at 360nm using an ultraviolet spectrophotometer, and the hesperidin content (mg / g) in the sample to be tested was calculated according to the standard curve.

[0093] The test results are shown in Table 6. In this test, the hesperidin content of tangerine peel fermented by Poria cocos fermented powder was determined, and this was used as an evaluation index to investigate the fermentation effect of Poria cocos fermented powder on tangerine peel. As can be seen from Table 6, at a fermentation temperature of 24°C, the hesperidin content of tangerine peel fermented by Poria cocos fermented powder was significantly higher than that of the ordinary Poria cocos fermented group. The reason for this is that the ordinary Poria cocos has not been domesticated and cannot adapt to the antibacterial component hesperidin produced by tangerine peel fermentation, and the growth of Poria cocos is inhibited, while the Poria cocos strain of Poria cocos fermented powder has been domesticated with an antibacterial agent. Therefore, even at the optimal fermentation temperature of Poria cocos, the effect of ordinary Poria cocos fermenting tangerine peel is significantly lower than that of the Poria cocos fermented powder fermented group. Under controlled intermittent high temperature conditions, because ordinary Poria cocos is not heat-resistant, it is inactivated under high temperature conditions, while the Poria cocos of Poria cocos fermented powder is embedded in the capsule of Aspergillus niger and tolerates high temperature environment, so the fermentation effect of Poria cocos fermented powder is significantly higher than that of ordinary Poria cocos.

[0094] Table 6 Hesperidin content of dried orange peel fermented by Poria cocos

[0095]

[0096]

[0097] (2) Fermented Ligusticum chuanxiong

[0098] Dissolve the Poria cocos fermentation powder with an appropriate amount of pure water, and spray the powder liquid evenly on the leaves of Ligustrum lucidum with a water content of 65% according to a 1% inoculation amount, control the intermittent high temperature conditions, and ferment for 30 days. At the same time, the experimental group was set to ferment for 30 days at 24 ° C. The untamed common Poria cocos purchased from the strain company was used as a control. After the fermentation is completed, take 1mL of the phloridzin sample extracted from the fermentation product in a colorimetric tube, dilute it to 10mL with distilled water, and use an ultraviolet spectrophotometer to measure the absorbance at 284nm. The content of phloridzin in the sample to be tested (mg / g) is calculated according to the standard curve. In addition, the number of Aspergillus niger was counted at 30 days of fermentation to evaluate the contamination of the fermentation product by miscellaneous bacteria.

[0099] The test results are shown in Table 7. In this test, the phloridzin content after the fermentation of the Poria cocos powder with the leaves of the ginger was determined, and this was used as an evaluation index to investigate the fermentation effect of the Poria cocos powder on the leaves of the ginger, and the number of Aspergillus niger in the fermentation product was counted to evaluate the contamination of miscellaneous bacteria. As can be seen from Table 7, at a fermentation temperature of 24°C, due to the weak antibacterial effect of the active substances produced by the fermentation of the leaves of the ginger, the growth of the ordinary Poria cocos was not completely inhibited, so the ordinary Poria cocos was still able to ferment the leaves of the ginger to produce phloridzin, but the phloridzin content was lower than that of the Poria cocos fermentation powder group; the ordinary Poria cocos fermentation group was contaminated with Aspergillus niger, and the Poria cocos fermentation powder contained 1% interfering agent to inhibit the growth of miscellaneous bacteria, so the contamination degree of Aspergillus niger was low. Under the condition of controlling intermittent high temperature, the phloridzin content of the Poria cocos fermentation powder group was significantly higher than that of the ordinary Poria cocos group, because the ordinary Poria cocos could not grow in a high temperature environment; there was no Aspergillus niger contamination in both groups.

[0100] Table 7 Phloridzin content and Aspergillus niger count in Poria cocos fermented Zingiber officinale

[0101]

[0102]

[0103] (3) Fermented Cyclocarya paliurus

[0104] Use an appropriate amount of pure water to dissolve the Poria cocos fermentation powder, and spray the powder liquid evenly on the water content of 65% of the green willow according to the 1% inoculation amount, control the intermittent high temperature conditions, and ferment for 30 days. At the same time, the experimental group was set to ferment at 24 ° C for 30 days. The untamed common Poria cocos purchased from the strain company was used as a control. After the fermentation is completed, take 1mL of gallic acid extract, add 5mL of ultrapure water, 1mL of sodium tungstate-sodium molybdate mixed solution and 3mL of sodium carbonate solution, place it for 2h, and use an ultraviolet spectrophotometer to measure the absorbance at 765nm. The content of gallic acid in the sample to be tested (mg / g) is calculated according to the standard curve. In addition, the number of Aspergillus niger was counted at 30 days of fermentation to evaluate the contamination of the fermentation product by miscellaneous bacteria.

[0105] The test results are shown in Table 8. In this test, the gallic acid content of Cyclocarya paliurus fermented by Poria cocos fermented powder was determined, and this was used as an evaluation index to investigate the fermentation effect of Poria cocos fermented powder on Cyclocarya paliurus, and the number of Aspergillus niger in the fermented product was counted to evaluate the contamination of miscellaneous bacteria. As can be seen from Table 8, at a fermentation temperature of 24°C, due to the weak antibacterial effect of Cyclocarya paliurus fermented product, the growth of ordinary Poria cocos was not completely inhibited, so ordinary Poria cocos was still able to ferment Cyclocarya paliurus to produce gallic acid, but the gallic acid content was lower than that of the Poria cocos fermented powder group; the ordinary Poria cocos fermented group was contaminated with Aspergillus niger, and the Poria cocos fermented powder contained 1% interfering agent to inhibit the growth of miscellaneous bacteria, so the contamination degree of Aspergillus niger was low. Under the condition of controlling intermittent high temperature, the gallic acid content of the Poria cocos fermented powder group was significantly higher than that of the ordinary Poria cocos group, because ordinary Poria cocos could not grow in a high temperature environment; no Aspergillus niger contamination occurred in both groups.

[0106] Table 8 Gallic acid content and Aspergillus niger count of Cyclocarya paliurus fermented by Poria cocos

[0107]

[0108]

[0109] (4) Fermented Eucommia Leaves

[0110] Use an appropriate amount of pure water to dissolve the Poria cocos fermentation powder, and spray the powder liquid evenly on the Eucommia ulmoides leaves with a water content of 65% according to the inoculation amount of 1%, control the intermittent high temperature conditions, and ferment for 30 days. At the same time, the experimental group was set to ferment at 24°C for 30 days. The untamed common Poria cocos purchased from the strain company was used as a control. After the fermentation is completed, chlorogenic acid is extracted with 50% methanol, and the absorbance is measured at 330nm using an ultraviolet spectrophotometer after filtration. The content of chlorogenic acid in the sample to be tested (mg / g) is calculated according to the standard curve. In addition, the number of Aspergillus niger was counted at 30 days of fermentation to evaluate the contamination of the fermentation product by miscellaneous bacteria.

[0111] The test results are shown in Table 9. In this test, the content of chlorogenic acid after the fermentation of Eucommia ulmoides leaves by Poria cocos fermented powder was determined, and this was used as an evaluation index to investigate the fermentation effect of Poria cocos fermented powder on Cyclocarya paliurus, and the number of Aspergillus niger in the fermentation product was counted to evaluate the contamination of miscellaneous bacteria. As can be seen from Table 9, at a fermentation temperature of 24°C, due to the strong antibacterial effect of the Eucommia ulmoides fermented product, the growth of ordinary Poria cocos was inhibited, so the fermentation of Eucommia ulmoides leaves by ordinary Poria cocos was affected, and the chlorogenic acid content produced was significantly lower than that of the Poria cocos fermented powder group; the ordinary Poria cocos fermented group was contaminated with Aspergillus niger, and the Poria cocos fermented powder contained 1% interfering agent to inhibit the growth of miscellaneous bacteria, so the contamination degree of Aspergillus niger was low. Under the condition of controlling intermittent high temperature, the chlorogenic acid content of the Poria cocos fermented powder group was significantly higher than that of the ordinary Poria cocos group, because ordinary Poria cocos cannot grow in a high temperature environment; no Aspergillus niger contamination occurred in both groups.

[0112] Table 9 Chlorogenic acid content and Aspergillus niger count of Eucommia ulmoides leaves fermented by Poria cocos

[0113]

[0114]

[0115] (5) Fermented dandelion

[0116] Use an appropriate amount of pure water to dissolve the fermented Poria cocos powder, spray the powder liquid evenly on dandelions with a water content of 65% according to a 1% inoculation amount, control intermittent high temperature conditions, and ferment for 30 days. At the same time, the experimental group was set to ferment at 24°C for 30 days. The untamed common Poria cocos purchased from the strain company was used as a control. After the fermentation was completed, chlorogenic acid was extracted with 50% methanol, and the absorbance was measured at 330nm using an ultraviolet spectrophotometer after filtration. The content of chlorogenic acid in the sample to be tested was calculated according to the standard curve (mg / g). In addition, the number of Aspergillus niger was counted at 30 days of fermentation to evaluate the contamination of the fermentation product by miscellaneous bacteria.

[0117] The test results are shown in Table 10. In this test, the content of chlorogenic acid after the fermentation of Eucommia ulmoides leaves by Poria cocos fermented powder was determined, and this was used as an evaluation index to investigate the fermentation effect of Poria cocos fermented powder on Cyclocarya paliurus, and the number of Aspergillus niger in the fermented product was counted to evaluate the contamination of foreign bacteria. As can be seen from Table 10, at a fermentation temperature of 24°C, due to the strong antibacterial effect of dandelion fermentation products, the growth of common Poria cocos was inhibited, so the fermentation of dandelions by common Poria cocos was affected, and the chlorogenic acid content produced was significantly lower than that of the Poria cocos fermented powder group; the common Poria cocos fermentation group was contaminated with Aspergillus niger, and the Poria cocos fermented powder contained 1% interfering agent to inhibit the growth of foreign bacteria, so the contamination degree of Aspergillus niger was low. Under the condition of controlling intermittent high temperature, the chlorogenic acid content of the Poria cocos fermented powder group was significantly higher than that of the common Poria cocos group, because the common Poria cocos could not grow in a high temperature environment; there was no Aspergillus niger contamination in both groups.

[0118] Table 10 Chlorogenic acid content and Aspergillus niger count of dandelion fermented with Poria cocos

[0119]

[0120]

[0121] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing Poria cocos fermented bacteria powder, characterized in that: The following steps are involved: S1, preparation of vector: S1-1, take 25g of golden flower from black tea, put it into 225ml of sterilized saline, shake well, let it stand for 10 minutes, take 1ml of the mixture, inoculate it into PDA solid culture medium, and culture it in a 26℃ incubator for 5-7 days; S1-2, picking a well-growing colony of Eurotium cristatum from the PDA solid medium obtained in step S1-1 and inoculating it into the PDA solid medium, and culturing it at 26° C. for 3-5 days to obtain a slant strain of Eurotium cristatum; S1-3, picking a well-growing colony of Eurotium cristatum from the PDA solid culture medium obtained in step S1-2, inoculating it into a PDB liquid culture medium, and culturing it on a shaker at a speed of 150 r / min and a temperature of 26° C. for 10 days to obtain a bacterial solution enriched with Eurotium cristatum spores; S1-4, taking 1 ml of the bacterial solution prepared in step S1-3, inoculating it into a PDA solid culture medium containing a taming agent, and culturing it at 26° C. for 3 days to acclimate the Eurotium cristatum; S1-5, picking a colony with good growth status from the PDA solid culture medium obtained in step S1-4, inoculating it into a PDB liquid culture medium containing an acclimator, and culturing it on a shaker at a speed of 150 r / min and a temperature of 26° C. for 5 days to obtain an acclimated Eurotium cristatum bacterial liquid, wherein the dry weight of mycelium in the bacterial liquid is 3.0 to 3.5 g / L; S1-6, placing the culture solution of the Eurotium cristatum expanded in step S1-5 in a culture chamber at 28°C for 1 day, then raising the temperature to 30°C for 1 day, and finally raising the temperature to 32°C for 1 day, taking the bacteria produced in the upper layer, inactivating them at high temperature, and freeze-drying them, thereby obtaining the cysts of the Eurotium cristatum; S2, preparation of Poria cocos powder: S2-1, inoculate the Poria cocos strains into PDA solid medium and culture at 24 °C for 3 days; S2-2, picking a well-growing Poria cocos colony from the PDA solid culture medium obtained in step S2-1, inoculating it into a PDB liquid culture medium, and culturing it on a shaker at a speed of 170 r / min and a temperature of 24° C. for 10 days to obtain a bacterial liquid enriched with Poria cocos mycelium; S2-3, taking 1 ml of the bacterial solution prepared in step S2-2, inoculating it into a PDA solid culture medium containing a taming agent, and culturing it at 24° C. for 3 days to taming the Poria cocos; S2-4, picking a well-growing Poria cocos colony from the PDA solid culture medium obtained in step S2-3, inoculating it into a PDB liquid culture medium containing a taming agent, culturing it on a shaker at a speed of 170 r / min and a temperature of 24° C. for 10 days to obtain a tamed Poria cocos bacterial liquid, wherein the dry weight of mycelium in the bacterial liquid is 5.0 to 5.5 g / L, filtering to obtain wet Poria cocos cells, freeze-drying the wet cells and crushing them to obtain tamed Poria cocos powder; S3, preparation of Poria cocos fermented bacteria powder: S3-1, using the capsule of the Aspergillus niger obtained in step S1-6 or dextrin as a carrier, mixing it with the Poria cocos powder domesticated in step S2-4, adding distilled water at a mass ratio of 1:100, stirring at 37° C. for 3 h to fully dissolve it, embedding the Poria cocos, concentrating the solution, filtering it with suction, and freeze-drying the precipitate to obtain embedded Poria cocos; S3-2, crushing the embedded Poria cocos in step S3-1 to make fine and uniform powder, adding an interfering agent and mixing evenly to obtain Poria cocos fermented fungus powder.

2. The preparation method according to claim 1, characterized in that: The acclimation agent is selected from any one of chlorogenic acid, dihydromyricetin or hesperidin.

3. The preparation method according to claim 2, characterized in that: In steps S1-4 and S1-5, the taming agent is dihydromyricetin, and the content of the dihydromyricetin powder is 0.9 g / g; In steps S2-3 and S2-4, the acclimating agent is hesperidin, and the content of the hesperidin powder is 0.9 g / g.

4. The preparation method according to claim 1, characterized in that: The dosage of the taming agent is 0.5-2.0%.

5. The preparation method according to claim 4, characterized in that: The dosage of the taming agent is 1.0%.

6. The preparation method according to claim 1, characterized in that: In step S3-1, the mass ratio of the tamed Poria powder and the Eurotium cristatum shell carrier is 1:(1.0-1.5).

7. The preparation method according to claim 6, characterized in that: In step S3-1, the mass ratio of the domesticated Poria cocos powder and the Eurotium cristatum shell carrier is 1:1.

2.

8. The preparation method according to claim 1, characterized in that: In step S3-2, the interfering agent is dihydromyricetin, and the mass ratio of the interfering agent added is 1%.

9. A fermented Poria cocos powder prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the fermented Poria cocos powder prepared by the preparation method according to any one of claims 1 to 8 in the field of Chinese herbal medicine fermentation.

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