Phellinus igniarius fermentation method for high-yield production of Hispidin and application of phellinus igniarius fermentation method

Abstract

The invention relates to the technical field of fermentation of edible and medicinal fungi, and discloses a phellinus igniarius fermentation method capable of producing high-yield Hispidin and an application of the phellinus igniarius fermentation method. The phellinus igniarius fermentation method comprises the following steps: inoculating a phellinus igniarius strain into a culture medium containing a copper ion compound, and fermenting. The phellinus igniarius mycelium obtained by adopting the fermentation method is rich in Hispidin, and can be widely used for preparing related medicines and health foods for protecting liver, resisting tumors, resisting viruses, resisting oxidation, reducing uric acid and the like. In addition, the invention also discloses a high performance liquid chromatography detection method for analyzing the content of Hispidin, by adopting the method, the content of Hispidin and precursor substances (such as protocatechuic acid, protocatechualdehyde, caffeic acid, osmundone and the like) in the phellinus igniarius leavening and derivative products thereof as well as phellinus igniarius mycelia can be effectively analyzed, and the accuracy of Hispidin detection is favorably improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method and application of mulberry linterus fermentation for high-yield hispidin. Background Art

[0002] Phellinus is a fungus belonging to the genus Sanghuangporus, Basidiomycota, Agaricomycetes, Hymenochaetales, and Hymenochaetaceae. It is found in Tibet, Sichuan, Yunnan, Hubei, Hunan, Jiangxi, Zhejiang, Shandong, Shanxi, Henan, Gansu, Shaanxi, Jilin, and Taiwan. It has the effects of promoting blood circulation and stopping bleeding, nourishing yin and tonifying the kidney, softening and dispersing nodules, stopping diarrhea and detoxifying. The main active ingredients of Phellinus are flavonoids, triterpenes, and polysaccharides, which have biological activities such as anti-cancer, anti-oxidation, lowering blood sugar, lowering blood lipids, protecting the liver, anti-inflammatory, antibacterial, and relieving gout.

[0003] Related studies have shown that there are more than 130 flavonoids isolated from Phellinus igniarius, including more than 30 pyrone compounds, Hispidin (C 13 H 10 O 5 , CAS No: 55-55-5) is one of the representative components of mulberry pyrone, which has been proven to have pharmacological effects such as liver protection, anti-tumor, anti-virus, antioxidant, and uric acid lowering. In related technologies, the regulation strategy for fermentation synthesis of Hispidin is divided into two aspects: solid-state fermentation and liquid fermentation. The former mainly focuses on the regulation of different fermentation conditions, metal ions and exogenous factors, while the latter revolves around the one-bacteria-multiple-compound strategy and the addition of exogenous factors. Among them, solid fermentation research found that Zn 2+ The effect is the best, but there is still a problem of low yield; and there are no reports on the use of metal ions to promote Hispidin synthesis in liquid fermentation. How to obtain high-yield Hispidin by liquid fermentation is still a technical problem that needs to be solved urgently.

[0004] Based on this, this study explored the regulatory strategy of liquid fermentation of Phellinus igniarius, discovered a high-yield Hispidin fermentation method for Phellinus igniarius, and established a corresponding Hispidin high-performance liquid chromatography analysis method for analysis. Summary of the invention

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

[0006] The invention also proposes an application of a mulberry linterus fermentation method in the preparation of Hispidin.

[0007] The invention also provides a method for detecting the content of Hispidin.

[0008] In a first aspect of the present invention, a method for submerged fermentation of Phellinus linteus with high yield of Hispidin is provided, comprising the following steps:

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

[0010] The method for fermenting Phellinus linteus according to an embodiment of the present invention has at least the following beneficial effects: the present invention is the first to discover that adding a certain amount of metal copper ions to the culture medium can significantly increase Hispidin in Phellinus linteus mycelium.

[0011] In some embodiments of the present invention, the mulberry linterus strain is selected from at least one of Sanghuangporus vaninii, Sanghuangporus sanghuang, Sanghuangporus baumii, and Sanghuangporus quercicola.

[0012] In some embodiments of the present invention, the Phellinus linteus strain is Phellinus linteus.

[0013] In some embodiments of the present invention, the deposit number of the Phellinus linteus strain is CGMCC 5.891.

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

[0015] In some embodiments of the present invention, 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 invention, 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 invention, the copper sulfate is selected from copper sulfate pentahydrate (CuSO 4 ·5H 2O), copper sulfate monohydrate (CuSO 4 ·H 2 Preferably, the copper sulfate is copper sulfate pentahydrate.

[0018] In some embodiments of the present invention, the final concentration of the metal copper ions in the basal culture medium is 1 mM to 5 mM.

[0019] In some embodiments of the present invention, the final concentration of the metal copper ions in the basal culture medium is 1.5 mM to 2.5 mM.

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

[0021] In some embodiments of the present invention, 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 cake powder is 2-10 g / L, the final concentration of glucose is 10-30 g / L, the final concentration of potassium dihydrogen phosphate is 0.5-2 g / L, and the final concentration of magnesium sulfate is 0.1-1 g / L.

[0022] In some embodiments of the present invention, the fermentation temperature is 28±5°C, preferably 28±2°C.

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

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

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

[0026] The second aspect of the present invention provides an application of the submerged fermentation method of Phellinus linteus as described in any one of the first aspects in the preparation of Hispidin.

[0027] The third aspect of the present invention provides a method for detecting the content of Hispidin, comprising the following steps:

[0028] The fermentation liquid, mycelium or culture obtained by the submerged fermentation method of Phellinus igniarius described in any one of the first aspects is detected by liquid chromatography.

[0029] The detection method according to the embodiment of the present invention has at least the following beneficial effects: the detection method of the Hispidin content of the present invention has good accuracy and can effectively separate the precursor substances of Hispidin in the deep fermentation broth or mycelium of Phellinus igniarius, such as protocatechuic acid, protocatechuic aldehyde, caffeic acid, porphyrin, etc.

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

[0031] Preparation of standard solution: Accurately weigh 2.00 mg of Hispidin standard and dissolve it in 50% methanol to make 0.2 mg mL -1 Hispidin standard solution is prepared for later use.

[0032] Linear relationship investigation: The Hispidin standard solution was diluted to 1 / 5, 1 / 20, 1 / 50, 1 / 100, and 1 / 200 times in sequence, and 20 μL was accurately drawn from each solution and injected into a high performance liquid chromatograph to measure the chromatographic peak area of ​​the Hispidin standard. The corresponding peak area (y) was regressed with each Hispidin standard concentration (x) to obtain the regression equation and linear range, which were y=81.694x-14.149 (R 2 =0.999) and 1.00 μg·mL -1 ~200.00μg·mL -1 , the linear relationship is good.

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

[0034] The mycelium is washed and dried, and then treated with methanol. After suction filtration and rotary evaporation, methanol is added to dissolve it again. Finally, after filtering, the filtrate is collected.

[0035] In some embodiments of the invention, the liquid chromatography method comprises the following conditions:

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

[0037] Mobile phase B: acetonitrile;

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

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

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

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

[0042] Mobile phase B: acetonitrile;

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

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

[0045] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0047] Figure 1 This is a photo of the solid culture medium culture of Phellinus linterus in the present invention.

[0048] Figure 2 This is a photo of the mycelium morphology of the Phellinus igniarius strain in Example 1 of the present invention observed under a microscope.

[0049] Figure 3 This is a diagram of the primary seed liquid culture of Phellinus igniarius in Example 1 of the present invention.

[0050] Figure 4 This is a diagram of the fermentation culture of Phellinus igniarius (containing metal copper ions) in Example 1 of the present invention.

[0051] Figure 5This is a diagram of the fermentation culture of Phellinus igniarius in Comparative Example 1 of the present invention (without metal copper ions).

[0052] Figure 6 This is a diagram of the fermentation culture (containing zinc ions) of Phellinus igniarius in Comparative Example 2 of the present invention.

[0053] Figure 7 This is the pH value test result of Phellinus igniarius liquid fermentation.

[0054] Figure 8 These are the results of the detection of Hispidin content in the mycelium after liquid fermentation of Phellinus igniarius.

[0055] Fig. 9 HPLC chromatograms of the comparison of the Hispidin reference substance group, Example 1 group, Comparative Example 1 group and Comparative Example 2 group of the present invention, wherein A is the Hispidin reference substance, B is the Example 1 group, C is the Comparative Example 1 group, and D is the Comparative Example 2 group.

[0056] Fig.10 The HPLC chromatograms of the Hispidin single-label group and the copper ion different concentration experimental groups are shown in FIG. 1 , wherein A is the Hispidin single-label group, B is the Example 1 group, C is the Example 2 group, and D is the Example 3 group.

[0057] Fig.11 This is the spectrum diagram of Hispidin of the present invention.

[0058] Fig.12 This is a group of mass spectrometry analysis chromatograms of Example 1 of the present invention, which, from top to bottom, are respectively a TIC diagram, a Hispidin maximum absorption wavelength channel diagram (380 nm), a Hispidin positive ion flow diagram, and a Hispidin negative ion flow diagram.

[0059] Fig.13 This is the mass spectrum of Hispidin of the present invention.

[0060] Fig.14 This is the secondary mass spectrum of Hispidin of the present invention. DETAILED DESCRIPTION

[0061] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

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

[0063] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

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

[0065] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0066] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0067] Example 1

[0068] 1. Bacteria activation:

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

[0070] The solid medium culture of Phellinus igniarius strain SH-H is as follows Figure 1 As shown, the results of microscopic observation of the hyphae morphology of Phellinus linteus strain SH-H are as follows Figure 2 As shown, on the PDA plate culture medium, the hyphae of Phellinus linteus are yellow-brown, and their color changes from light yellow to yellow as the culture time increases, and then continues to deepen to yellow-brown. The hyphae are dense and lush, growing radially outward in concentric circles. Microscopic observation shows that the hyphae are intertwined and arranged, with clear septa and "Y"-shaped branches.

[0071] 2. Primary liquid seed culture:

[0072] The activated bacterial strain was inoculated into a culture bottle containing a liquid seed culture medium, wherein each 250 mL culture bottle contained 100 mL of liquid, and the inoculation amount was 8 bacterial blocks (about 1 cm × 1 cm × 1 cm) per bottle, and cultured at 28°C and 150 r / min for 10 days to obtain a primary culture solution.

[0073] Among them, the culture conditions of the primary culture medium are as follows Figure 3 As shown, the bacterial liquid is brown-yellow in color, the bacterial bodies are small balls of uniform size, and powdery substances that are obviously not completely absorbed can be seen at the bottom of the liquid.

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

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

[0076] 3. Phellinus igniarius fermentation culture:

[0077] The primary culture solution (concentration of 20 g / L) was inoculated into fresh liquid seed medium at a ratio of 5% (V / V). At the same time, an appropriate amount of CuSO was added to the liquid seed medium before inoculation. 4 ·5H 2 O, so that the final concentration of copper ions after inoculation of the primary culture solution is 2 mM, and after inoculation, it is placed in a 28°C shaker and incubated at 150 r / min for 10 days, and then the fermentation broth obtained by the culture is separated by suction filtration, and then washed 3 times with distilled water to collect the mycelium (marked as Example 1 group).

[0078] Among them, the culture conditions after fermentation culture of Phellinus igniarius are as follows Figure 4 As shown, the bacterial liquid is brown-yellow, the bacteria are evenly distributed, plump and compact yellow-brown balls, and the overall density of the fermentation liquid is high.

[0079] Example 2

[0080] This embodiment provides a method for fermenting and culturing Phellinus igniarius, which differs from Embodiment 1 in that the concentration of metal copper ions is adjusted to 1 mM during the fermentation and culturing of Phellinus igniarius, and the remaining steps are the same, specifically including the following contents.

[0081] 1. Bacteria activation:

[0082] The slant cultured Phellinus linterus strain SH-H was inoculated onto a solid potato dextrose agar medium (solid PDA medium), and cultured in a 28° C. incubator for 10 days to obtain an activated strain.

[0083] 2. Primary liquid seed culture:

[0084] The activated bacterial strain was inoculated into a culture bottle containing a liquid seed culture medium, wherein each 250 mL culture bottle contained 100 mL of liquid, and the inoculation amount was 8 bacterial blocks (about 1 cm × 1 cm × 1 cm) per bottle, and cultured at 28°C and 150 r / min for 10 days to obtain a primary culture solution.

[0085] 3. Phellinus igniarius fermentation culture:

[0086] The primary culture solution (concentration of 20 g / L) was inoculated into fresh liquid seed medium at a ratio of 5% (V / V). Before inoculation, an appropriate amount of CuSO 4 ·5H 2 O, so that the final concentration of copper ions after inoculation of the primary culture solution is 2 mM, and after inoculation, it is placed in a 28°C shaker and incubated at 150 r / min for 10 days, and then the fermentation broth obtained by the culture is separated by suction filtration, and then washed 3 times with distilled water to collect the mycelium (marked as Example 2 group).

[0087] Example 3

[0088] This embodiment provides a method for fermenting and culturing Phellinus igniarius, which differs from Example 1 in that the concentration of metal copper ions is adjusted to 5 mM during the fermentation and culturing of Phellinus igniarius, and the remaining steps are the same, specifically including the following contents.

[0089] 1. Bacteria activation:

[0090] The slant cultured Phellinus linterus strain SH-H was inoculated onto a solid potato dextrose agar medium (solid PDA medium), and cultured in a 28° C. incubator for 10 days to obtain an activated strain.

[0091] 2. Primary liquid seed culture:

[0092] The activated bacterial strain was inoculated into a culture bottle containing a liquid seed culture medium, wherein each 250 mL culture bottle contained 100 mL of liquid, and the inoculation amount was 8 bacterial blocks (about 1 cm × 1 cm × 1 cm) per bottle, and cultured at 28°C and 150 r / min for 10 days to obtain a primary culture solution.

[0093] 3. Phellinus igniarius fermentation culture:

[0094] The primary culture solution (concentration of 20 g / L) was inoculated into fresh liquid seed medium at a ratio of 5% (V / V). Before inoculation, an appropriate amount of CuSO 4 ·5H 2 O, so that the final concentration of copper ions after inoculation of the primary culture solution is 2 mM, and after inoculation, it is placed in a 28°C shaker and incubated at 150 r / min for 10 days, and then the fermentation broth obtained by the culture is separated by suction filtration, and then washed 3 times with distilled water to collect the mycelium (marked as Example 3 group).

[0095] Comparative Example 1

[0096] This comparative example provides a method for fermenting and culturing Phellinus linteus, which differs from Example 1 in that CuSO is not added during the fermentation and culturing of Phellinus linteus. 4 ·5H 2 O, the remaining steps are the same, including the following.

[0097] 1. Bacteria activation:

[0098] The slant cultured Phellinus linterus strain SH-H was inoculated onto a solid potato dextrose agar medium (solid PDA medium), and cultured in a 28° C. incubator for 10 days to obtain an activated strain.

[0099] 2. Primary liquid seed culture:

[0100] The activated bacterial strain was inoculated into a culture bottle containing a liquid seed culture medium, wherein each 250 mL culture bottle contained 100 mL of liquid, and the inoculation amount was 8 bacterial blocks (about 1 cm × 1 cm × 1 cm) per bottle, and cultured at 28°C and 150 r / min for 10 days to obtain a primary culture solution.

[0101] 3. Phellinus igniarius fermentation culture:

[0102] The above-mentioned primary culture solution was inoculated into fresh liquid seed culture medium at an inoculation rate of 5% (V / V), and after inoculation, it was placed in a shaker at 28°C and incubated at 150 r / min for 10 days. The fermentation liquid obtained by the culture was then filtered and separated, and then washed with distilled water 3 times to collect the mycelium (marked as comparative example 1 group).

[0103] Among them, the culture conditions after fermentation culture of Phellinus igniarius are as follows Figure 5 As shown, the bacterial liquid is yellow, the bacteria are evenly distributed, plump and compact light yellow balls, and the overall density of the fermentation liquid is high.

[0104] Comparative Example 2

[0105] This comparative example provides a method for fermenting and culturing Phellinus linteus, which differs from Example 1 in that CuSO 4 ·5H 2 O replaced by ZnSO 4 7H 2 O, the remaining steps are the same, including the following.

[0106] 1. Bacteria activation:

[0107] The slant cultured Phellinus linterus strain SH-H was inoculated onto a solid potato dextrose agar medium (solid PDA medium), and cultured in a 28° C. incubator for 10 days to obtain an activated strain.

[0108] 2. Primary liquid seed culture:

[0109] The activated bacterial strain was inoculated into a culture bottle containing a liquid seed culture medium, wherein each 250 mL culture bottle contained 100 mL of liquid, and the inoculation amount was 8 bacterial blocks (about 1 cm × 1 cm × 1 cm) per bottle, and cultured at 28°C and 150 r / min for 10 days to obtain a primary culture solution.

[0110] 3. Phellinus igniarius fermentation culture:

[0111] The primary culture solution (concentration of 20 g / L) was inoculated into fresh liquid seed culture medium at a ratio of 5% (V / V). At the same time, an appropriate amount of ZnSO was added to the liquid seed culture medium before inoculation. 4 7H 2 O, so that the final concentration of copper ions after inoculation of the primary culture fluid is 2mM, and after inoculation, the culture fluid is placed in a shaker at 28°C and incubated at 150r / min for 10 days, and then the fermentation fluid (such as Figure 6 The mycelium was collected by suction filtration (as shown in the figure) and then washed 3 times with distilled water (marked as comparative example 2).

[0112] Test example 1: Biomass and pH value test

[0113] This test example detects the biomass of mycelium and the pH value of the fermentation liquid after the fermentation culture of Phellinus igniarius in Examples 1 to 3 and Comparative Examples 1 and 2. The specific detection method is as follows:

[0114] The fermentation broth after the fermentation culture (10d) of Phellinus igniarius of the above Examples 1 to 3 and Comparative Examples 1 and 2 was collected, the pH value was detected and then filtered and separated, and then washed with distilled water 3 times, the mycelium was collected, placed in a 40°C oven and dried at low temperature to constant weight, and the data was recorded, with three replicates in each group.

[0115] The pH test results are as follows Figure 7 As shown, compared with Comparative Examples 1 and 2, after adding copper ions, the pH value of the system shows a downward trend, and when the concentration of copper ions is 2M, the pH value is the lowest.

[0116] The biomass test results showed that adding CuSO 4 ·5H 2 O, the biomass of its mycelium increased significantly, while when CuSO 4 ·5H 2 O replaced by ZnSO 4 7H 2 After O, the amount of Phellinus igniarius mycelium obtained was significantly reduced.

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

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

[0119] (1) Intracellular sample acquisition:

[0120] The mycelium after fermentation and cultivation of Phellinus igniarius in the above-mentioned Examples 1 to 3 and Comparative Examples 1 and 2 was dried to constant weight, chopped into pieces and put into a 250 mL shake flask, immersed in 100 mL of methanol, ultrasonically crushed for 1 h, and filtered to obtain a mycelium extract, which was evaporated to dryness on a rotary evaporator, re-dissolved in methanol, filtered through a 0.22 μm microporous filter membrane, and the filtrate was taken to obtain an intracellular extract sample, which was placed in a 4°C refrigerator for standby use.

[0121] (2) Content determination method:

[0122] ① Preparation of mixed reference solution: Accurately weigh appropriate amount of each mulberry linterus reference substance (purchased from Chengdu Aifa Biotechnology Co., Ltd., mass fraction ≥ 98%), add methanol to dissolve to prepare a mixed reference solution containing protocatechuic acid, protocatechuic aldehyde, caffeic acid, porphyrin, hispidin, morin, naringenin, and mulberry flavonoids (in order of peak time). Store in a refrigerator at 4°C for future use.

[0123] ②HPLC gradient conditions: SHIMSEN Superb C18-(4.6×250mm,5μm) chromatographic column; mobile phase was 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; 60-6 7min, 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 coupled with Orbitrap Exploris 120 mass spectrometer system; Hypersil GOLDTM VANQUIS Column-(2.1×100mm,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 These are the results of the detection and analysis of the Hispidin content in the mycelium obtained from liquid fermentation of Phellinus igniarius. The calculation results based on the Hispidin content per unit mass of mycelium show that when the copper ion concentration is 1 M, the Hispidin content per unit mass of mycelium is the highest, reaching about 3.82 mg / g.

[0126] Fig. 9The HPLC chromatograms corresponding to the analysis of Hispidin in the mycelia obtained from the control group, Example 1 group, Comparative Example 1 group and Comparative Example 2 group are shown, wherein A is the Hispidin control chromatogram, B is the sample analysis chromatogram of Example 1 group, C is the sample analysis chromatogram of Comparative Example 1 group, and D is the sample analysis chromatogram of Comparative Example 2 group. The results show that compared with the Comparative Example 1 group, the Hispidin peak value of the Example 1 group can be significantly increased, while the Hispidin peak value of the Comparative Example 2 group can be significantly decreased; this indicates that Cu 2+ The mycelium can be enriched with Hispidin, while Zn 2+ It will inhibit its synthesis.

[0127] Fig.10 The chromatograms of Hispidin reference substance and HPLC chromatograms of Hispidin analysis in samples of different example groups, wherein A is the chromatogram of Hispidin reference substance, B is the chromatogram of sample analysis of Example 1 group, C is the chromatogram of sample analysis of Example 2 group, and D is the chromatogram of sample analysis of Example 3 group. The results show that the Hispidin content in the mycelium obtained by fermentation increases with Cu 2+ The increase in final concentration showed a trend of increasing first and then decreasing, with a more obvious promoting effect at a concentration of 2 mM, indicating that the maximum gain concentration may exist in the range of 2 mM to 5 mM.

[0128] Fig.11 The results show that the retention time and spectrum of the target chromatographic peak in each sample are consistent with those of the Hispidin reference substance.

[0129] Fig.12 The mass spectrometry analysis spectra of the sample in Example 1, from top to bottom, are respectively TIC diagram, Hispidin maximum absorption wavelength channel spectrum diagram (380nm), Hispidin positive ion flow diagram, and Hispidin negative ion flow diagram; Fig.13 and Fig.14 The mass spectra of Hispidin in the samples of Example 1 and the mass spectra of Hispidin control were shown in Table 1. The analysis results showed that the Hispidin signal could be detected in the mycelium of Example 1. The theoretical value of the Hispidin positive ion peak [M+H] + The positive ion [M+H] of Hispidin component in the mycelium of Example 1 is 247.0606. + The detected value is 247.0566; the theoretical value of the negative ion peak of Hispidin [MH] - The negative ion of Hispidin component in the mycelium of Example 1 group [MH] - The detection value 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 fermentation method of the present invention (adding metal copper ions) can significantly increase the content of Hispidin in the mycelium of the fermentation system Phellinus linteus. The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for fermenting Phellinus linteus to produce high yield of Hispidin, characterized in that: The following steps are involved: The Phellinus igniarius strain is inoculated into a basic culture medium containing a copper ion compound and fermented to obtain the product.

2. The deep fermentation method of Phellinus linteus according to claim 1, characterized in that: The mulberry linterus strain is selected from at least one of Sanghuangporus vaninii, Sanghuangporus sanghuang, Sanghuangporus baumii and Sanghuangporus quercicola; Preferably, the strain of Phellinus linteus is Sanghuangporus vaninii (CGMCC 5.891). Preferably, the inoculation amount of the Phellinus igniarius strain is 5% to 15% seed liquid (V:V).

3. The method for fermenting Phellinus igniarius according to claim 1, characterized in that: The copper ion compound is selected from at least one of copper sulfate, copper chloride, copper nitrate, copper acetate, and copper sulfide; Preferably, the metal copper ion compound is selected from at least one of copper sulfate, copper chloride and copper nitrate.

4. The method for fermenting Phellinus igniarius according to claim 3, characterized in that: The final concentration of the metal copper ions in the basic culture medium is 1 mM to 5 mM.

5. The method for fermenting Phellinus igniarius according to claim 1, characterized in that: The basic culture medium also contains corn flour / bran / soybean cake powder / glucose / potassium dihydrogen phosphate / magnesium sulfate and water.

6. The method for fermenting Phellinus igniarius according to claim 5, 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 bean cake powder is 2-10 g / L, the final concentration of the glucose is 10-30 g / L, the final concentration of potassium dihydrogen phosphate is 0.5-2 g / L, and the final concentration of magnesium sulfate is 0.1-1 g / L.

7. The method for fermenting Phellinus igniarius according to any one of claims 1 to 6, characterized in that: The fermentation temperature is 28±5°C; And / or, the fermentation culture time is 5 to 15 days; And / or, the initial pH value of the fermentation medium is 4-8.

8. Use of the Phellinus linteus fermentation method according to any one of claims 1 to 7 in the preparation of Hispidin.

9. A method for detecting the content of Hispidin, characterized in that: The following steps are involved: The fermentation liquid, mycelium or culture obtained by the mulberry linterus fermentation method according to any one of claims 1 to 7 is detected by liquid chromatography.

10. The detection method according to claim 9, characterized in that: The liquid chromatography method includes the following conditions: Mobile phase A: 0.05% to 0.3% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient elution program: 0min-12min, the volume fraction of the mobile phase B is 10%-15%; 12min-21min, the volume fraction of the mobile phase B increases from 10%-15% to 18%-22%; 21min-28min, the volume fraction of the mobile phase B is 18%-22%; 28min-50min, the volume fraction of the mobile phase B increases from 18%-22% to 38%-42%; 50min-54min, the volume fraction of the mobile phase B increases from 18%-22% to 38%-42% The integral fraction increases from 38% to 42% to 53% to 56%; from 54min to 56min, the volume fraction of the mobile phase B increases from 53% to 56% to 57% to 60%; from 56min to 60min, the volume fraction of the mobile phase B is 57% to 60%; from 60min to 67min, the volume fraction of the mobile phase B increases from 57% to 60% to 62% to 68%; from 67min to 75min, the volume fraction of the mobile phase B decreases from 62% to 68% to 10% to 15%; Variable wavelength program: 0min~10.5min, detection wavelength is 255nm~265nm; 10.5min~16min, the detection wavelength is 295nm~305nm; 16min~25min, the detection wavelength is 340nm~350nm; 25min~38min, the detection wavelength is 375nm~385nm; 38min~45min, the detection wavelength is 245nm~255nm; 45min~60min, the detection wavelength is 285nm~295nm; 60min~75min, the detection wavelength is 255nm~265nm.

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