Fermentation method of high-yield hispidin phellinus and application thereof

Abstract

The present application relates to the technical field of edible and medicinal fungus fermentation, and discloses a fermentation method of Phellinus baumii with high Hispidin yield and application.The fermentation method of Phellinus baumii in the present application comprises inoculating Phellinus baumii strains into a culture medium containing copper ion compounds, and then fermenting to obtain the product.The mycelium of Phellinus baumii obtained by the fermentation method of the present application is rich in Hispidin, and can be widely used in the preparation of related drugs and health foods for liver protection, anti-tumor, anti-virus, oxidation resistance, uric acid reduction, etc.In addition, the present application also discloses a high-performance liquid chromatography detection method for analyzing the content of Hispidin, which can effectively analyze the content of Hispidin and its precursor substances (such as protocatechuic acid, protocatechuic aldehyde, coffee acid, and aspidin) in the fermentation product of Phellinus baumii, its derivative product, and the mycelium of Phellinus baumii, and is helpful to improve the accuracy of Hispidin detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, in particular to a fermentation method of Phellinus baumii for high yield of Hispidin and application thereof. BACKGROUND

[0002] Phellinus baumii belongs to the fungal kingdom Basidiomycota, Agaricomycetes, Hymenochaetales, and Hymenochaetaceae, and is found in Tibet Autonomous Region, Sichuan, Yunnan, Hubei, Hunan, Jiangxi, Zhejiang, Shandong, Shanxi, Henan, Gansu, Shaanxi, Jilin and Taiwan. Sanghuangporus Phellinus baumii has the effects of activating blood and stopping bleeding, nourishing yin and kidney, softening and resolving, and relieving diarrhea and detoxification. The main active components of Phellinus baumii are Phellinus baumii flavonoids, Phellinus baumii triterpenes, Phellinus baumii polysaccharides, etc., which have biological activities such as anticancer, antioxidant, hypoglycemic, hypolipidemic, liver protection, anti-inflammatory, antibacterial, and gout relief.

[0003] Related studies have shown that more than 130 flavonoids have been isolated from Phellinus baumii, including more than 30 pyrones, and Hispidin (C 13 H 10 O5, CAS No: 55-55-5) is one of the representative components of Phellinus baumii pyrones, which has been confirmed to have the pharmacological effects of liver protection, anti-tumor, anti-virus, antioxidant, and uric acid reduction. In related technologies, the regulation strategies for fermentation synthesis of Hispidin are divided into solid-state fermentation and liquid-state fermentation. The former mainly focuses on the regulation of different fermentation conditions, metal ions and exogenous factors, and the latter focuses on the one-bacterium-multiple-compounds strategy and the addition of exogenous factors. Among them, the solid-state fermentation research found that Zn 2+ has the best effect in promoting the synthesis of Hispidin, but there is still the problem of low yield; and there is no report on the use of metal ions to promote the synthesis of Hispidin in liquid fermentation. How to obtain high-yield Hispidin by liquid fermentation is still a technical problem to be solved at present.

[0004] Therefore, based on the regulation strategy of liquid-state fermentation of Phellinus baumii, a fermentation method of Phellinus baumii for high yield of Hispidin is found, and a corresponding high-performance liquid chromatography analysis method for Hispidin is established for analysis. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a fermentation method of high-yield Hispidin Phellinus and application thereof. The fermentation method of the present application can significantly improve the content of Hispidin in Phellinus mycelium.

[0006] The present application also provides application of the fermentation method of Phellinus in preparation of Hispidin.

[0007] The present application also provides a detection method of Hispidin content.

[0008] In the first aspect of the present application, a fermentation method of high-yield Hispidin Phellinus is provided, comprising the following steps:

[0009] The Phellinus strain is inoculated into the basic medium containing copper ion compound, and then fermented to obtain the product.

[0010] The fermentation method of Phellinus according to the embodiments of the present application has at least the following beneficial effects: the present application first discovers that adding a certain content of metal copper ion into the culture medium can significantly improve the content of Hispidin in Phellinus mycelium.

[0011] In some embodiments of the present application, the Phellinus strain is selected from at least one of Phellinus vaninii (P. vaninii), Phellinus baumii (P. baumii), Phellinus baumii (P. baumii), Phellinus baumii (P. baumii), and Phellinus baumii (P. baumii). Sanghuangporus vaninii In some embodiments of the present application, the Phellinus strain is selected from at least one of Phellinus vaninii (P. vaninii), Phellinus baumii (P. baumii), Phellinus baumii (P. baumii), Phellinus baumii (P. baumii), and Phellinus baumii (P. baumii). Sanghuangporus sanghuang Sanghuangporus baumii In some embodiments of the present application, the Phellinus strain is selected from at least one of Phellinus vaninii (P. vaninii), Phellinus baumii (P. baumii), Phellinus baumii (P. baumii), Phellinus baumii (P. baumii), and Phellinus baumii (P. baumii). Sanghuangporus quercicola

[0012] In some embodiments of the present application, the Phellinus strain is P. vaninii.

[0013] In some embodiments of the present application, the Phellinus strain has a preservation number of CGMCC 5.891.

[0014] In some embodiments of the present application, the inoculation amount of the Phellinus strain is 5% to 15% seed liquid (V:V).

[0015] In some embodiments of the present application, the copper ion compound is selected from at least one of copper sulfate, copper chloride, copper nitrate, copper acetate, and copper sulfide.

[0016] In some embodiments of the present application, the copper ion compound is selected from at least one of copper sulfate, copper chloride, and copper nitrate.

[0017] In some embodiments of the present application, the copper sulfate is selected from at least one of copper sulfate pentahydrate (CuSO4·5H2O) and copper sulfate monohydrate (CuSO4·H2O). Preferably, the copper sulfate is copper sulfate pentahydrate.​​

[0018] In some embodiments of the present application, the final concentration of the metal copper ion in the basic medium is 1 mM to 5 mM.

[0019] In some embodiments of the present application, the final concentration of the metal copper ion in the basic medium is 1.5 mM to 2.5 mM.

[0020] In some embodiments of the present application, the basic medium further comprises corn flour, bran, soybean meal, glucose, potassium dihydrogen phosphate, magnesium sulfate and water.

[0021] In some embodiments of the present application, the final concentration of the corn flour is 10 to 40 g / L, the final concentration of the bran is 5 to 20 g / L, the final concentration of the soybean meal is 2 to 10 g / L, the final concentration of the glucose is 10 to 30 g / L, the final concentration of the potassium dihydrogen phosphate is 0.5 to 2 g / L, and the final concentration of the magnesium sulfate is 0.1 to 1 g / L.

[0022] In some embodiments of the present application, the temperature of the fermentation is 28 ± 5℃. Preferably, it is 28 ± 2℃.

[0023] In some embodiments of the present application, the fermentation time is 5 to 15 days; preferably, it is 8 to 12 days.

[0024] In some embodiments of the present application, the pH value of the fermentation is 4 to 8.

[0025] In some embodiments of the present application, the fermentation method comprises cascade fermentation, such as primary fermentation and secondary fermentation.

[0026] In a second aspect of the present application, there is provided a use of the Phellinus igniarius submerged fermentation method according to any one of the first aspect in the preparation of Hispidin.

[0027] In a third aspect of the present application, there is provided a detection method of the content of Hispidin, comprising the following steps:

[0028] The fermentation broth or mycelium or culture obtained by the Phellinus igniarius submerged fermentation method according to any one of the first aspect is detected by liquid chromatography.

[0029] According to the detection method of the present application, the content of Hispidin can be detected accurately, and the precursor substances of Hispidin, such as protocatechuic acid, protocatechualdehyde, caffeic acid and aspidin, can be effectively separated from the Phellinus igniarius submerged fermentation broth or mycelium.

[0030] In some embodiments of the present application, the detection further comprises calculating the content of Hispidin in the fermentation broth or mycelium with Hispidin as a reference. The standard sample calculation method comprises the following conditions:

[0031] Preparation of standard sample solution: precisely weigh 2.00 mg of Hispidin standard sample, dissolve in 50% methanol to prepare 0.2 mg·mL Hispidin standard sample solution, and reserve. -1

[0032] Investigation of linear relationship: sequentially dilute the Hispidin standard sample solution to 1 / 5, 1 / 20, 1 / 50, 1 / 100, and 1 / 200, and precisely take 20 µL of each, inject into the high performance liquid chromatograph, and measure the chromatographic peak area of the Hispidin standard sample. Regress the concentration (x) of each Hispidin standard sample and the corresponding peak area (y) to obtain the regression equation and linear range, which are y = 81.694x - 14.149 (R 2 =0.999) and 1.00 µg·mL -1 ~200.00 µg·mL -1 , respectively, and the linear relationship is good.

[0033] In some embodiments of the present application, the mycelium further comprises pretreatment, specifically:

[0034] After washing and drying the mycelium, methanol is added for treatment, and after suction filtration and rotary evaporation, methanol is added for redissolution, and finally after filtration treatment, the filtrate can be collected.

[0035] In some embodiments of the present application, the liquid chromatography comprises the following conditions:

[0036] Mobile phase A: 0.05%~0.3% formic acid aqueous solution;

[0037] Mobile phase B: acetonitrile;

[0038] ​Gradient elution procedure: 0 min to 12 min, the volume fraction of the mobile phase B is 10% to 15%; 12 min to 21 min, the volume fraction of the mobile phase B is raised from 10% to 15% to 18% to 22%; 21 min to 28 min, the volume fraction of the mobile phase B is 18% to 22%; 28 min to 50 min, the volume fraction of the mobile phase B is raised from 18% to 22% to 38% to 42%; 50 min to 54 min, the volume fraction of the mobile phase B is raised from 38% to 42% to 53% to 56%; 54 min to 56 min, the volume fraction of the mobile phase B is raised from 53% to 56% to 57% to 60%; 56 min to 60 min, the volume fraction of the mobile phase B is 57% to 60%; 60 min to 67 min, the volume fraction of the mobile phase B is raised from 57% to 60% to 62% to 68%; 67 min to 75 min, the volume fraction of the mobile phase B is reduced from 62% to 68% to 10% to 15%;

[0039] Variable wavelength procedure: 0 min to 10.5 min, the detection wavelength is 255 nm to 265 nm; 10.5 min to 16 min, the detection wavelength is 295 nm to 305 nm; 16 min to 25 min, the detection wavelength is 340 nm to 350 nm; 25 min to 38 min, the detection wavelength is 375 nm to 385 nm; 38 min to 45 min, the detection wavelength is 245 nm to 255 nm; 45 min to 60 min, the detection wavelength is 285 nm to 295 nm; 60 min to 75 min, the detection wavelength is 255 nm to 265 nm.

[0040] In some embodiments of the application, the liquid chromatography comprises the following conditions:

[0041] Mobile phase A: 0.05% to 0.2% formic acid aqueous solution by mass fraction;

[0042] Mobile phase B: acetonitrile;

[0043] Gradient elution procedure: 0 min~12 min, the volume fraction of mobile phase B is 12%~13%; 12 min~21 min, the volume fraction of mobile phase B is raised from 12%~13% to 18%~20%; 21 min~28 min, the volume fraction of mobile phase B is 18%~20%; 28 min~50 min, the volume fraction of mobile phase B is raised from 18%~20% to 38%~40%; 50 min~54 min, the volume fraction of mobile phase B is raised from 38%~40% to 53%~55%; 54 min~56 min, the volume fraction of mobile phase B is raised from 53%~55% to 57%~58%; 56 min~60 min, the volume fraction of mobile phase B is 57%~58%; 60~67 min, the volume fraction of mobile phase B is raised from 57%~58% to 62%~65%; 67~75 min, the volume fraction of mobile phase B is reduced from 62%~65% to 10%~13%;

[0044] Variable wavelength procedure: 0 min ~10.5min, detection wavelength is 255 nm ~265nm; 10.5 min ~16min, detection wavelength is 295 nm ~305nm; 16 min ~25min, detection wavelength is 340 nm ~350nm; 25 min ~38min, detection wavelength is 375 nm ~385nm; 38 min ~45min, detection wavelength is 245 nm ~255nm; 45 min ~60min, detection wavelength is 285 nm ~295nm; 60 min ~75min, detection wavelength is 255 nm ~265nm.

[0045] Other features and advantages of the present application will be set forth in the accompanying description. BRIEF DESCRIPTION OF DRAWINGS

[0046] The present application will be further described with reference to the accompanying drawings and examples, in which:

[0047] Figure 1 It is a picture of the solid culture of Phellinus baumii in the present application.

[0048] Figure 2 It is a picture of the microscopic observation of the mycelium morphology of Phellinus baumii in Example 1 of the present application.

[0049] Figure 3 It is a picture of the primary seed liquid culture of Phellinus baumii in Example 1 of the present application.

[0050] Figure 4 It is a picture of the fermentation culture of Phellinus baumii in Example 1 of the present application (containing metal copper ions).

[0051] Figure 5 Figure for fermentation culture of Phellinus sp. in the present application comparative example 1 (without copper ion).

[0052] Figure 6 Figure for fermentation culture of Phellinus sp. in the present application comparative example 2 (with zinc ion).

[0053] Figure 7 Detection results of pH value of liquid fermentation of Phellinus sp.

[0054] Figure 8 Detection results of Hispidin content in mycelium after liquid fermentation of Phellinus sp.

[0055] Figure 9 HPLC chromatograms of each comparison of the present application Hispidin reference substance group, example 1 group, comparative example 1 group and comparative example 2 group, wherein A is Hispidin reference substance, B is example 1 group, C is comparative example 1 group, and D is comparative example 2 group.

[0056] Figure 10 HPLC chromatograms of the present application Hispidin single standard group and different concentrations of copper ion experimental group, wherein A is Hispidin single standard, B is example 1 group, C is example 2 group, and D is example 3 group.

[0057] Figure 11 Spectrum of the present application Hispidin.

[0058] Figure 12 Mass spectrum analysis chromatogram of the present application example 1 group, from top to bottom are TIC chart, Hispidin maximum absorption wavelength channel chart (380 nm), Hispidin positive ion flow chart, and Hispidin negative ion flow chart.

[0059] Figure 13 Mass spectrum of the present application Hispidin.

[0060] Figure 14 Secondary mass spectrum of the present application Hispidin. DETAILED DESCRIPTION

[0061] The concept and technical effects of the present application will be described below in combination with examples to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0062] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0063] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0064] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0065] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0066] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0067] Example 1

[0068] 1. Activation of microbial strains:

[0069] The *Sanghuang* strain used in this embodiment ( Sanghuangporus vaninii The strain was purchased from the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC 5.891, hereinafter referred to as SH-H. The SH-H strain, cultured on agar slant, was inoculated onto solid potato dextrose agar (PDA) medium and incubated at 28°C for 10 days to obtain the activated strain. The formulation of the solid potato dextrose agar medium was as follows: 24 g / L potato dextrose (PDB), 20 g / L agar, and water.

[0070] The culture condition of the Phellinus sp. strain SH-H solid medium is shown in Figure 1 The result of the microscopic observation of the mycelium morphology of the Phellinus sp. strain SH-H is shown in Figure 2 As shown in the figure, the mycelium of the Phellinus sp. is yellowish brown, and its color changes from light yellow to yellow and then to yellowish brown with the increase of the culture time. The mycelium is dense and luxuriant, and grows in a concentric circle outward. The microscopic observation shows that the mycelium is arranged in an interwoven manner, and has clear septa and "Y" branches.

[0071] 2. Primary liquid seed culture:

[0072] The activated strain is inoculated into a culture bottle containing the liquid seed culture medium, wherein the liquid volume of each 250 mL culture bottle is 100 mL, the inoculation amount is 8 mycelial blocks (about 1 cm x 1 cm x 1 cm) per bottle, and the culture is carried out at 28°C with 150 r / min shaking for 10 days to obtain the primary culture liquid.

[0073] As shown in the figure, the culture condition of the primary culture liquid is shown in Figure 3 The culture condition of the primary culture liquid is shown in

[0074] The formula of the liquid seed culture medium (SF2 culture medium) is as follows:

[0075] 30 g / L of corn flour, 10 g / L of bran, 7 g / L of soybean meal, 15 g / L of glucose, 1 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, and water.

[0076] 3. Phellinus sp. fermentation culture:

[0077] The primary culture liquid (concentration of 20 g / L) is inoculated into fresh liquid seed culture medium at a ratio of 5% (V / V), and an appropriate amount of CuSO4•5H2O is added to the liquid seed culture medium before inoculation, so that the final concentration of copper ions after inoculation of the primary culture liquid is 2 mM. After inoculation, the culture is incubated at 28°C on a shaking table at 150 r / min for 10 days, and then the fermentation liquid obtained by the culture is separated by suction filtration, and then washed with distilled water for 3 times, and the mycelium is collected (marked as Example 1 group).

[0078] As shown in the figure, the culture condition of the Phellinus sp. fermentation culture is shown in Figure 4 The culture condition of the Phellinus sp. fermentation culture is shown in

[0079] Example 2

[0080] The embodiment provides a fermentation culture method of Phellinus baumii, which is different from the method in Embodiment 1 in that the concentration of metal copper ions is adjusted to 1 mM in the fermentation culture process of the Phellinus baumii, and the rest steps are the same, and specifically includes the following contents.

[0081] 1. Strain activation:

[0082] The slope-cultured Phellinus baumii strain SH-H is inoculated on a solid potato dextrose agar medium (solid PDA medium), and then cultured in a 28°C incubator for 10 days, so that the activated strain is obtained.

[0083] 2. First-stage liquid seed culture:

[0084] The activated strain is inoculated into a culture bottle containing a liquid seed culture medium, wherein the liquid volume of each 250 mL culture bottle is 100 mL, the inoculation amount is 8 mycelial blocks (about 1 cm*1 cm*1 cm) per bottle, and the culture is carried out at 28°C and 150 r / min for 10 days, so that the first-stage culture liquid is obtained.

[0085] 3. Fermentation culture of Phellinus baumii:

[0086] The first-stage culture liquid (20 g / L) is inoculated into fresh liquid seed culture medium at a ratio of 5% (V / V), and an appropriate amount of CuSO4*5H2O is added to the liquid seed culture medium before inoculation, so that the final concentration of copper ions is 1 mM after inoculation of the first-stage culture liquid; after inoculation, the culture is incubated at 28°C and 150 r / min for 10 days, and then the obtained fermentation liquid is separated by suction filtration, and then washed with distilled water for three times, and the mycelium is collected (marked as the group of Embodiment 2).

[0087] Embodiment 3

[0088] The embodiment provides a fermentation culture method of Phellinus baumii, which is different from the method in Embodiment 1 in that the concentration of metal copper ions is adjusted to 5 mM in the fermentation culture process of the Phellinus baumii, and the rest steps are the same, and specifically includes the following contents.

[0089] 1. Strain activation:

[0090] The slope-cultured Phellinus baumii strain SH-H is inoculated on a solid potato dextrose agar medium (solid PDA medium), and then cultured in a 28°C incubator for 10 days, so that the activated strain is obtained.

[0091] 2. First-stage liquid seed culture:

[0092] The activated strain is inoculated into a culture bottle containing liquid seed culture medium, with a liquid volume of 100 mL per 250 mL culture bottle, and an inoculation amount of 8 mycelial blocks (about 1 cm x 1 cm x 1 cm) per bottle, and is cultured at 28°C with 150 r / min shaking for 10 days to obtain a primary culture solution.

[0093] 3. Fermentation culture of Phellinus baumii

[0094] The primary culture solution (20 g / L) is inoculated into fresh liquid seed culture medium at a ratio of 5% (V / V), and an appropriate amount of CuSO4·5H2O is added to the liquid seed culture medium before inoculation, so that the final concentration of copper ions after inoculation of the primary culture solution is 5 mM. After inoculation, the culture is incubated at 28°C on a shaker at 150 r / min for 10 days. The fermentation solution obtained by culture is then separated by suction filtration, and then washed with distilled water three times to collect the mycelium (marked as Example 3 group).

[0095] Comparative Example 1

[0096] This comparative example provides a fermentation culture method of Phellinus baumii, which is different from Example 1 in that CuSO4·5H2O is not added during the fermentation culture of Phellinus baumii, and the remaining steps are the same, specifically including the following contents.

[0097] 1. Strain activation

[0098] The Phellinus baumii strain SH-H inoculated on the slope culture is inoculated on solid potato dextrose agar medium (solid PDA medium), and is cultured in a 28°C incubator for 10 days to obtain an activated strain.

[0099] 2. Primary liquid seed culture

[0100] The activated strain is inoculated into a culture bottle containing liquid seed culture medium, with a liquid volume of 100 mL per 250 mL culture bottle, and an inoculation amount of 8 mycelial blocks (about 1 cm x 1 cm x 1 cm) per bottle, and is cultured at 28°C with 150 r / min shaking for 10 days to obtain a primary culture solution.

[0101] 3. Fermentation culture of Phellinus baumii

[0102] The primary culture solution is inoculated into fresh liquid seed culture medium at a ratio of 5% (V / V), and is incubated at 28°C on a shaker at 150 r / min for 10 days after inoculation. The fermentation solution obtained by culture is then separated by suction filtration, and then washed with distilled water three times to collect the mycelium (marked as Comparative Example 1 group).

[0103] The culture conditions after fermentation of Phellinus baumii are as follows Figure 5As shown, the bacterial solution is yellow, the bacterial body is evenly distributed and full and close light yellow small ball, the overall density of the fermentation broth is high.

[0104] Comparative Example 2

[0105] This comparative example provides a fermentation culture method of Phellinus baumii, which is different from example 1 in that CuSO4·5H2O is replaced by ZnSO4·7H2O in the fermentation culture process of Phellinus baumii, and the remaining steps are the same, which specifically includes the following contents.

[0106] 1. Strain activation:

[0107] Inoculate the slant culture of Phellinus baumii strain SH-H on the solid potato dextrose agar medium (solid PDA medium), and culture in a 28℃ incubator for 10d to obtain the activated strain.

[0108] 2. Primary liquid seed culture:

[0109] Inoculate the above activated strain into the culture bottle containing liquid seed culture medium, wherein the liquid volume of each 250 mL culture bottle is 100 mL, and the inoculation amount is 8 bacterial blocks (about 1cm×1cm×1cm) per bottle, and culture at 28℃, 150 r / min shaking for 10d to obtain the primary culture liquid.

[0110] 3. Phellinus baumii fermentation culture:

[0111] Inoculate the above primary culture liquid (concentration of 20g / L) into fresh liquid seed culture medium at an inoculation amount of 5% (V / V), and add an appropriate amount of ZnSO4·7H2O to the liquid seed culture medium before inoculation, so that the final concentration of copper ions after inoculation of the primary culture liquid is 2mM, and then incubate at 28℃ on a shaking table at 150 r / min for 10d. Then, the fermentation broth obtained by culture (as shown in Figure 6 ) is separated by suction filtration, and then washed with distilled water for 3 times, and the mycelium is collected (marked as comparative example 2 group).

[0112] Detection example 1: biomass and pH value detection

[0113] This detection example detects the biomass of mycelium and the pH value of fermentation broth after the fermentation culture of Phellinus baumii in examples 1-3 and comparative examples 1 and 2, and the specific detection method is as follows:

[0114] Collect the fermentation broth after the fermentation culture (10d) of Phellinus baumii in examples 1-3 and comparative examples 1 and 2, detect the pH value, then separate by suction filtration, then wash with distilled water for 3 times, collect the mycelium, place it in a 40℃ oven for low temperature drying until the weight is constant, record the data, and each group is repeated three times.

[0115] The pH detection analysis results are as follows:Figure 7 As shown, it is shown that the pH value of the system shows a downward trend after the addition of copper ions, and when the concentration of copper ions is 2M, the pH value is the lowest.

[0116] The biomass detection results show that after further adding CuSO4•5H2O on the basis of the liquid seed culture medium, the biomass of the mycelium is significantly increased, and when CuSO4•5H2O is replaced by ZnSO4•7H2O, the amount of the obtained Phellinus mycelium is significantly reduced.

[0117] Detection Example 2: Hispidin content detection and analysis

[0118] This detection example detects the Hispidin content in the mycelium of Phellinus after fermentation culture of Examples 1-3 and Comparative Examples 1 and 2, and the specific detection method is as follows:

[0119] (1) Intracellular sample acquisition:

[0120] The mycelium of Phellinus after fermentation culture of Examples 1-3 and Comparative Examples 1 and 2 was dried to constant weight, cut into 250 mL shake flasks, immersed in 100 mL methanol, ultrasonically broken for 1 h, filtered, and the mycelium extract was obtained. The rotary evaporator was evaporated to dryness, methanol was added for redissolution, filtered through a 0.22 µm microporous filter membrane, and the filtrate was obtained, which was placed in a 4°C refrigerator for standby.

[0121] (2) Content determination method:

[0122] ① Preparation of mixed reference solution: accurately weigh the appropriate amount of each Phellinus reference (purchased from Chengdu Efa Biological Technology Co., Ltd., mass fraction ≧98%) and dissolve in methanol to prepare a mixed reference solution containing protocatechuic acid, protocatechuic aldehyde, coffee acid, erythronol, Hispidin, morin, naringenin, and phellinus flavone (in order of peak time). Store in a refrigerator at 4°C for standby.

[0123] ② HPLC gradient conditions: SHIMSEN Superb C18-(4.6x250mm, 5μm) column; mobile phase: 0.1% formic acid aqueous solution (A)-acetonitrile (B), gradient elution program: 0~12min, 13%B; 12~21min, 13~20%B; 21~28min, 20%B; 28~50min, 20~40%B; 50~54min, 40~55%B; 54~56min, 55~58%B; 56~60min, 58%B; 60~67min, 58~65%B; 67~75min, 65~13%B; variable wavelength program detection, detection wavelength 260nm, 10.5~16min, 300nm; 16~25min, 344nm; 25~38min, 380nm; 38~45min, 250nm; 45~60min, 290nm; 60~75min, 260nm; injection volume 20μL; column temperature 25℃; volume flow rate 1.0mL•min -1 .

[0124] ③ Mass spectrometry gradient conditions: Vanquish Flex UHPLC combined with Orbitrap Exploris 120 mass spectrometer system; Hypersil GOLDTM VANQUIS Column-(2.1x100mm, 1.9μm); mobile phase: 0.1% formic acid aqueous solution (A)-acetonitrile (B); gradient elution program: 0~4min, 10%B; 4~8min, 10~13%B; 8~25min, 13~18%B; 25~30min, 18~20%B; 30~40min, 20~23%B; 40~48min, 23~30%B; 48~70min, 30~31%B; 70~75min, 31~36%B; 75~80min, 36~40%B; 80~85min, 40~65%B; 85~88min, 65%B; 88~93min, 65~95%B; detection wavelength: 260nm, 350nm, 380nm; injection volume 10μL; column temperature 25℃; volume flow rate 1.0mL•min -1 .

[0125] Figure 8 The detection and analysis results of Hispidin content in the mycelium obtained by liquid fermentation of Phellinus baumii showed that when the copper ion concentration was 2mM, the Hispidin content per unit mass of mycelium was the highest, reaching about 3.82mg / g.

[0126] Figure 9HPLC chromatograms of Hispidin analysis in mycelium of control group, Example 1 group, Comparative Example 1 group and Comparative Example 2 group, wherein A is the chromatogram of Hispidin control, B is the chromatogram of Example 1 group, C is the chromatogram of Comparative Example 1 group, and D is the chromatogram of Comparative Example 2 group. The results show that, compared with Comparative Example 1 group, the peak value of Hispidin in Example 1 group is significantly increased, while the peak value of Hispidin in Comparative Example 2 group is significantly decreased; indicating that Cu 2+ can enrich Hispidin in mycelium, while Zn 2+ inhibits the synthesis of Hispidin.

[0127] Figure 10 HPLC chromatograms of Hispidin analysis in mycelium of control group, Example 1 group, Comparative Example 1 group and Comparative Example 2 group, wherein A is the chromatogram of Hispidin control, B is the chromatogram of Example 1 group, C is the chromatogram of Comparative Example 1 group, and D is the chromatogram of Comparative Example 2 group. The results show that, compared with Comparative Example 1 group, the peak value of Hispidin in Example 1 group is significantly increased, while the peak value of Hispidin in Comparative Example 2 group is significantly decreased; indicating that Cu 2+ can enrich Hispidin in mycelium, while Zn + inhibits the synthesis of Hispidin.

[0128] Figure 11 Hispidin spectrum, the results show that the retention time and spectrum of the target chromatographic peak in each sample are consistent with those of the Hispidin control.

[0129] Figure 12 TIC spectrum, Hispidin maximum absorption wavelength channel spectrum (380 nm), Hispidin positive ion flow spectrum, Hispidin negative ion flow spectrum, from top to bottom; Figure 13 and Figure 14 Hispidin mass spectrum of Example 1 group and Hispidin control, the analysis results show that Hispidin signal can be detected in the mycelium of Example 1 group, the theoretical value of Hispidin positive ion peak [M+H] + is 247.0606, the detected value of Hispidin component in the mycelium of Example 1 group is 247.0566; the theoretical value of Hispidin negative ion peak [M-H] + is 245.0450, the detected value of Hispidin component in the mycelium of Example 1 group is 245.0425. - -

[0130] It can be seen that, compared with the blank control group (comparative example 1) and the metal Zn2+ ion group (comparative example 2), the content of Hispidin in the fermentation system of the mycelium of Piptoporus betulinus can be significantly improved by using the fermentation mode (adding metal copper ions) of the present application.

[0131] The above has made a detailed description of the embodiments of the present application, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A fermentation method of Phellinus ribis for high production of Hispidin, characterized by, The method comprises the following steps: Sanghuangporus The Trametes versicolor strain is Trametes versicolor with a preservation number of CGMCC 5.891 vaninii The copper ion compound is at least one selected from copper sulfate and copper nitrate; the final concentration of copper ions in the basic medium is 1.5 mM-2.5 mM. ) The basic medium further comprises corn flour, bran, soybean meal, glucose, potassium dihydrogen phosphate, magnesium sulfate and water.

2. The fermentation method of Phellinus baumii according to claim 1, characterized in that, The final concentration of the corn flour is 10-40 g / L, the final concentration of the bran is 5-20 g / L, the final concentration of the soybean meal is 2-10 g / L, the final concentration of the glucose is 10-30 g / L, the final concentration of the potassium dihydrogen phosphate is 0.5-2 g / L, and the final concentration of the magnesium sulfate is 0.1-1 g / L.

3. The fermentation method of Phellinus baumii according to claim 2, characterized in that, The fermentation temperature is 28±5℃; 4. The fermentation method of Phellinus baumii according to any one of claims 1 to 3, characterized in that, The fermentation culture time is 5-15 days; The initial pH value of the fermentation medium is 4-8.

5. Application of the Sanghuangporus fermentation method according to any one of claims 1-4 in the preparation of Hispidin. ​

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