Method for increasing content of ergosterol in tremella aurantialba mycelium by using adenosine

Abstract

The invention discloses a method for increasing the content of ergosterol in tremella aurantialba mycelia by using adenosine, and relates to the technical field of biological ferment.The method comprises the following steps that a sterilized liquid culture medium is inoculated with tremella aurantialba strains, and liquid culture is conducted to obtain a liquid culture solution; centrifuging the liquid culture solution to obtain mycelia; the content of ergosterol in the obtained mycelium is greater than 34.6 mg / L; a liquid fermentation culture medium is composed of 200 g / L of potatoes, 20 g / L of glucose, 3 g / L of monopotassium phosphate, 1.5 g / L of magnesium sulfate and 12.0 mg / L of vitamin B1, water is added to make the volume constant to 1000 mL, and the pH value is natural; adding adenosine into the liquid culture medium until the concentration of adenosine is greater than 1.0 g / L; the classification of the inoculated tremella auramtialba strain is named as Naematia auramtialba, and the classification is named as Naematia auramtialba; the preservation number of the strain is CGMCC (China General Microbiological Culture Collection According to the method, the content of ergosterol in the obtained tremella aurantialba mycelia can be increased to 45.8 mg / L and the biomass to 12.5 g / L under the condition that the biomass of the mycelia of the tremella aurantialba No. 2 ZJJE002 (the preservation number is CGMCC NO.40124) in the tremella aurantialba strain is not influenced, and the content of ergosterol is 1.55 times that of a control group.

Description

Technical Field

[0001] The present application relates to the field of biological fermentation technology, and particularly to a method for increasing the ergosterol content in Tremella aurantialba mycelium by using adenosine. Background Art

[0002] Tremella aurantialba Naemateliaaurantialba ), belonging to Basidiomycota ( Basidiomycota ) Tremellomycetes ( Tremellomycetes ) Tremellales ( Tremellales ) Helicogoniaceae ( Naemateliaceae ) Helicogonium ( Naematelia ). Tremella aurantialba is a well-known edible and medicinal mushroom. Its medicinal properties are warm in the middle and cold in nature, and its taste is sweet. Research shows that Tremella aurantialba has various efficacy and functions such as hypoglycemic, hypolipidemic, antioxidant, anti-inflammatory, antitussive, expectorant, antiasthmatic, and immune-enhancing effects.

[0003] Ergosterol, also known as ergosterol, is a compound commonly found in macrofungi and is a characteristic steroid of fungi. Ergosterol is an important component of the fungal cell membrane and plays an important biological role in ensuring the integrity of the membrane structure, membrane permeability, membrane fluidity, activity of membrane-bound enzymes, material transport, and cell viability. Ergosterol is an important raw material for medicine and chemical industry and is also a precursor of vitamin D2.

[0004] Adenosine is an endogenous purine nucleoside substance and is also a precursor for the synthesis of various medicinal nucleoside substances. It mainly participates in processes such as body metabolism and body regulation and plays an important regulatory role in physiological and biochemical processes. It has pharmacological effects such as improving blood circulation in the heart and brain, preventing arrhythmia, protecting the kidneys, and improving insomnia.

[0005] The yield of ergosterol in the Tremella aurantialba mycelium obtained by the existing cultivation methods is relatively low, and it is impossible to obtain a large amount of ergosterol by cultivating Tremella aurantialba.

[0006] There has been no research on using adenosine as an additive to increase the yield of ergosterol in Tremella aurantialba mycelium.

[0007] The information disclosed in the background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0008] The present application provides a method for increasing the ergosterol content in Tremella aurantialba mycelium by using adenosine. This method uses Zhongjun Tremella aurantialba No. 2 (ZJJE002) and adds adenosine during the fermentation process of this Tremella aurantialba strain to increase the ergosterol yield in the obtained fermented product, promote the synthesis of ergosterol, and achieve the purpose of obtaining high-yield ergosterol using Tremella aurantialba mycelium as the raw material through large-scale biomanufacturing. This method for producing ergosterol has low costs, easily available raw materials, high ergosterol production efficiency, and causes almost no harm to the environment.

[0009] The present application provides a method for increasing the ergosterol content in Tremella aurantialba mycelium by using adenosine, comprising the following steps: Inoculate a Tremella aurantialba strain into a sterilized liquid medium, and perform liquid culture to obtain a liquid culture solution; centrifuge the liquid culture solution to obtain mycelium; The content of ergosterol in the obtained mycelium is greater than 34.6 mg / L; The liquid medium is composed of 200 g / L of potato, 20 g / L of glucose, 3 g / L of potassium dihydrogen phosphate, 1.5 g / L of magnesium sulfate, 2.0 mg / L of vitamin B1, add water to make up the volume to 1000 mL, and the natural pH value; Add adenosine to the liquid medium until the concentration of adenosine is greater than 1.0 g / L; The classified name of the inoculated Tremella aurantialba strain is Naematelia aurantialba; the preservation certificate number is CGMCC NO. 40124.

[0010] Preferably, the concentration of adenosine in the liquid medium is 1.0 - 5.0 g / L.

[0011] Preferably, the content of ergosterol in the obtained mycelium is 34.6 - 45.8 mg / L; the biomass of the obtained mycelium is 11.3 - 12.0 g / L.

[0012] Preferably, the liquid culture conditions are: at 20 - 25 °C, with a shaking rate of 140 - 160 r / min, and the culture time is 5 - 10 days.

[0013] Preferably, the centrifugation includes the following operations: centrifuge the liquid culture solution to obtain mycelium, wash it 2 - 3 times with pure water, discard the supernatant after centrifugation, and obtain wet mycelium.

[0014] Preferably, it further includes: the determination of the ergosterol content in the mycelium; The method for determining the ergosterol content in the mycelium is to use a high-performance liquid chromatograph to determine the ergosterol content in the mycelium.

[0015] Preferably, the chromatographic determination conditions are as follows: the chromatographic column is Thermo Scientific Hypersil BDS C18, 250 mm × 4.6 mm, 5 µm, the mobile phase is methanol, the flow rate is 1.0 mL / min; the detection wavelength is 282 nm; the column temperature is 40 °C; the injection volume is 10 μL.

[0016] Preferably, it further includes: pretreating the mycelium before chromatographic determination; The pretreatment is as follows: vortex and mix the wet mycelium with 40 mL of methanol, ultrasonically extract for 30 min at 40 kHz to obtain a sample solution; centrifuge the sample solution to take the supernatant, and filter through a 0.45 µm organic microporous filter membrane to obtain a filtrate, and the obtained filtrate is used for chromatographic determination.

[0017] Preferably, the centrifugation conditions for the liquid culture medium are: using a high-speed refrigerated centrifuge, centrifuging at 14000 r / min for 10 min; The sterilization conditions for the liquid culture medium are 120 - 125 °C and 15 - 25 min.

[0018] Preferably, the conditions for centrifuging the sample solution are centrifuging at 5000 r / min for 5 min.

[0019] The beneficial effects that this application can produce include: 1) The method provided by this application for increasing the ergosterol content in Tremella aurantialba mycelium using adenosine. The culture medium used in this method can effectively increase the ergosterol content in the mycelium of Tremella aurantialba strain No. 2 ZJJE002 (preservation number: CGMCC NO. 40124), and does not affect the biomass of Tremella aurantialba mycelium. The highest ergosterol content can reach 45.8 mg / L, the biomass is 12.5 g / L, and the ergosterol content is 1.55 times that of the control group. The ergosterol content in the mycelium of Tremella aurantialba strain No. 2 ZJJE002 increases with the increase of the adenosine content, and decreases when the content in the liquid culture medium reaches 5.0 g / L. When the optimal addition amount is 4.0 g / L, the highest ergosterol content can be obtained.

[0020] 2) The method provided by this application for increasing the ergosterol content in Tremella aurantialba mycelium using adenosine. This method obtains mycelium raw materials with high ergosterol yield through the large-scale biological fermentation of Tremella aurantialba strain. This method has low cost for producing ergosterol, easily available raw materials, high efficiency in producing ergosterol, and almost no harm to the environment. This method uses the biological fermentation method of Tremella aurantialba to obtain the natural active ingredient ergosterol, providing an efficient and safe way for the production of ergosterol, and at the same time expanding the high-value development and utilization ways of Tremella aurantialba.

[0021] The Tremella aurantialba strain used is the new Tremella aurantialba strain ZJJE002, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 6, 2022; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Taxonomic name: Tremella aurantialba Naemateliaaurantialba ; The deposit number is CGMCC NO. 40124. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a bar graph of the ergosterol content in the Tremella aurantialba mycelium obtained in Examples 1-5 and the comparative examples provided in this application; Figure 2 It is a bar graph of the biomass in the Tremella aurantialba mycelium obtained in Examples 1-5 and the comparative examples provided in this application; Figure 3 It is a high-performance liquid chromatography result diagram of the ergosterol content detection of the Tremella aurantialba mycelium obtained in Example 1 provided in this application; Figure 4 It is a schematic diagram of a triangular flask of the liquid-cultured mycelium of the Tremella aurantialba strain No. ZJJE002 (deposit number CGMCC NO. 40124) in Examples 1-5 and the comparative examples provided in this application. The contents of each bottle are the fermentation broths of the comparative example and Examples 1-5 from left to right in sequence. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The present invention will be further described in detail below in conjunction with the drawings and embodiments, but the present invention is not limited in any way. Any transformation or improvement made based on the teachings of the present invention falls within the protection scope of the present invention.

[0024] Embodiment In the following embodiments, the materials and instruments used are obtained from commercial channels without special instructions; the detection methods used are existing methods without special instructions.

[0025] The strain used in each of the following embodiments is the Tremella aurantialba strain of Zhongjun Tremella aurantialba No. ZJJE002 (deposit number CGMCC NO. 40124) disclosed in CN202210600794.X. Other information not detailed can be found in CN202210600794.X.

[0026] Example 1 (1) Prepare a liquid medium: 200 g / L of potato (boiled with potato leachate for 30 minutes), 20 g / L of glucose, 3 g / L of potassium dihydrogen phosphate, 1.5 g / L of magnesium sulfate, 2.0 mg / L of vitamin B1, add water to make up to 1000 mL, natural pH value, dispense and sterilize, and the sterilization conditions are 121 °C for 20 min. Adenosine was added to the obtained liquid medium to a concentration of 1.0 g / L.

[0027] (2) Inoculate the activated Zhongjun Tremella aurantialba No. 2 ZJJE002 seed liquid into 100 ml of liquid medium at an inoculation amount of 10%, and culture it. The culture conditions are: temperature 25°C, shaking rate 150 r / min, and culture time 6 days to obtain a culture solution.

[0028] (3) Preparation of mycelia: After the culture is completed, centrifuge using a high-speed refrigerated centrifuge at 14,000 r / min for 10 min. Wash the centrifuged mycelia 2 - 3 times with pure water and then centrifuge again, discard the supernatant to obtain wet mycelia. Divide the wet mycelia into 2 equal parts with the same mass; one part of the wet mycelia is used for determining ergosterol, and the other part of the wet mycelia is dried at 40 - 60°C until the water content of the mycelia is 8% - 10% to obtain dry mycelia.

[0029] (4) Determination of mycelial biomass: Dry the wet mycelia in an oven with a temperature adjustment range of 40°C - 60°C until a constant weight is reached, and weigh the obtained substance to obtain the biomass of the mycelia.

[0030] (5) Determination of ergosterol content in mycelia: Add 40 mL of methanol to the obtained wet mycelia, vortex and mix evenly, and perform ultrasonic extraction at 40 kHz for 30 min to obtain a sample solution; centrifuge the sample solution, the centrifugation conditions of the sample solution are centrifugation at 5000 r / min for 5 min, take the supernatant, filter it through a 0.45 µm organic microporous filter membrane to obtain a filtrate, and determine the ergosterol content in the obtained filtrate using a high-performance liquid chromatograph.

[0031] The chromatographic conditions of the high-performance liquid chromatograph are: chromatographic column Thermo Scientific Hypersil BDS C18, 250 mm × 4.6 mm, 5 µm, mobile phase is methanol, flow rate is 1.0 mL / min; detection wavelength 282 nm; column temperature 40°C; injection volume 10 μL.

[0032] Results: When the concentration of adenosine in the liquid medium is 1.0 g / L, the ergosterol content in the Tremella aurantialba mycelia is 34.6 mg / L, and the biomass is 11.3 g / L.

[0033] Example 2 The difference from Example 1 is that the concentration of adenosine in the used liquid medium is 2.0 g / L.

[0034] Inoculate the Tremella aurantialba seed liquid into the liquid medium, at 25°C, shaking rate 150 r / min, and culture time 6 days. Determine the ergosterol content in the wet mycelia and the biomass of the dry mycelia.

[0035] Results: When the concentration of adenosine in the liquid medium is 2.0 g / L, the ergosterol content in the Tremella aurantialba mycelia is 37.5 mg / L, and the biomass is 11.6 g / L.

[0036] Example 3 It is different from Example 1 in that the concentration of adenosine in the liquid medium is 3.0 g / L.

[0037] The Tremella aurantialba seed liquid was inoculated into the liquid medium. At 25 °C, the shaking rate was 150 r / min, and the culture time was 6 days. The ergosterol content in the wet mycelium and the biomass of the dry mycelium were measured.

[0038] Results: When the concentration of adenosine in the liquid medium was 3.0 g / L, the ergosterol content in the Tremella aurantialba mycelium was 42.0 mg / L, and the biomass was 12.5 g / L.

[0039] Example 4 It is different from Example 1 in that the concentration of adenosine in the liquid medium is 4.0 g / L.

[0040] The Tremella aurantialba seed liquid was inoculated into the liquid medium. At 25 °C, the shaking rate was 150 r / min, and the culture time was 6 days. The ergosterol content in the wet mycelium and the biomass of the dry mycelium were measured.

[0041] Results: When the concentration of adenosine in the liquid medium was 4.0 g / L, the ergosterol content in the Tremella aurantialba mycelium was 45.8 mg / L, and the biomass was 11.8 g / L.

[0042] Example 5 It is different from Example 1 in that the liquid medium is: 200 g / L of potato (using the leaching juice, boiled for 30 minutes), 20 g / L of glucose, 3 g / L of potassium dihydrogen phosphate, 1.5 g / L of magnesium sulfate, 2.0 mg / L of vitamin B1, made up to 1000 mL with water, natural pH value, dispensed and sterilized, and the sterilization conditions were 121 °C, 20 min. Adenosine was added to the obtained liquid medium until the adenosine concentration was 5.0 g / L.

[0043] The Tremella aurantialba seed liquid was inoculated into the liquid medium. At 25 °C, the shaking rate was 150 r / min, and the culture time was 6 days. The ergosterol content in the wet mycelium and the biomass of the dry mycelium were measured.

[0044] Results: When the concentration of adenosine in the liquid medium was 5.0 g / L, the ergosterol content in the Tremella aurantialba mycelium was 45.6 mg / L, and the biomass was 12.0 g / L.

[0045] Control Example It is different from Example 1 in that adenosine is not added to the liquid medium. After the culture was completed, the ergosterol content in the wet mycelium and the biomass of the dry mycelium were measured.

[0046] Results: When adenosine was not added to the liquid medium, the ergosterol content in Tremella aurantialba mycelium was 29.6 mg / L, and the biomass was 11.6 g / L.

[0047] See Figures 1 - 2 It can be seen that when the adenosine content was 1.0 - 5.0 g / L, it had a good effect on improving the ergosterol content; the biomass did not decrease significantly.

[0048] From Figure 3 it can be seen that the method provided by this application can accurately detect the ergosterol content in the Tremella aurantialba ferment.

[0049] By comparison, it can be seen that in Example 1 compared with the comparative example, the ergosterol content in Tremella aurantialba mycelium increased by 16.9%, which was a relatively small increase. The ergosterol content in Tremella aurantialba mycelium in Example 3 was 1.42 times that of the comparative example. The ergosterol content in Tremella aurantialba mycelium in Example 4 was 1.55 times that of the comparative example. It shows that using this medium can significantly increase the ergosterol content in the mycelium and has no significant effect on the biomass.

[0050] This method can effectively increase the ergosterol content in Tremella aurantialba mycelium, and the ergosterol content in the Tremella aurantialba mycelium cultivated by this method is relatively high.

[0051] Example 6 The difference from Example 1 is that the sterilization conditions of the liquid medium are 120 °C and 15 min.

[0052] The liquid culture conditions are: at 20 °C, with a shaking rate of 140 r / min, and the culture time is 10 days.

[0053] Example 7 The difference from Example 1 is that the sterilization conditions of the liquid medium are 125 °C and 25 min.

[0054] The liquid culture conditions are: at 25 °C, with a shaking rate of 160 r / min, and the culture time is 5 days.

[0055] Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for increasing the ergosterol content in Tremella aurantialba mycelium by using adenosine, characterized in that It includes the following steps: Add adenosine to the sterilized liquid medium until the concentration of adenosine is greater than 1.0 g / L, inoculate the Tremella aurantialba strain, and perform liquid culture to obtain a liquid culture solution. Centrifuge the liquid culture solution to obtain mycelia; The liquid medium consists of 200 g / L of potato, 20 g / L of glucose, 3 g / L of potassium dihydrogen phosphate, 1.5 g / L of magnesium sulfate, 2.0 mg / L of vitamin B1, add water to make up to 1000 mL, and the natural pH value; The classified name of the inoculated Tremella aurantialba strain is Tremella aurantialba Naematelia aurantialba ; the preservation certificate number is CGMCC NO. 40124; the content of ergosterol in the obtained mycelium is greater than 34.6 mg / L.

2. The method according to claim 1, characterized in that, The concentration of adenosine in the liquid medium is 1.0 - 5.0 g / L.

3. The method according to claim 1, wherein The content of ergosterol in the obtained mycelia is 34.6 - 45.8 mg / L; the biomass of the obtained mycelia is 11.3 - 12.0 g / L.

4. The method according to claim 1, characterized in that, The liquid culture conditions are: at 20 - 25 °C, the shaking rate is 140 - 160 r / min, and the culture time is 5 - 10 days.

5. The method according to claim 1, characterized in that, Centrifugation includes the following operations: centrifuge the liquid culture solution to obtain mycelia, wash with pure water 2 - 3 times, discard the supernatant after centrifugation, and obtain wet mycelia.

6. The method according to claim 1, wherein It also includes: The determination of the ergosterol content in the mycelia; the method for determining the ergosterol content in the mycelia is to use a high performance liquid chromatograph to determine the ergosterol content in the mycelia.

7. The method according to claim 6, wherein The chromatographic determination conditions are: the chromatographic column is Thermo Scientific Hypersil BDS C18, 250 mm × 4.6 mm, 5 µm, the mobile phase is methanol, the flow rate is 1.0 mL / min; the detection wavelength is 282 nm; the column temperature is 40 °C; the injection volume is 10 μL.

8. The method according to claim 6, characterized in that, It also includes: Pretreat the mycelia before chromatographic determination; the pretreatment is: vortex and mix the wet mycelia with 40 mL of methanol, perform ultrasonic extraction at 40 kHz for 30 min to obtain a sample solution; centrifuge the sample solution and take the supernatant, filter through a 0.45 µm organic microporous filter membrane to obtain a filtrate, and the obtained filtrate is used for chromatographic determination.

9. The method according to claim 1, characterized in that The centrifugation conditions for the liquid culture solution are: use a high-speed refrigerated centrifuge, centrifuge at 14000 r / min for 10 min; the sterilization conditions for the liquid medium are 120 - 125 °C, 15 - 25 min.

10. The method according to claim 8, characterized in that, The centrifugation conditions for the sample solution are to centrifuge at 5000 r / min for 5 min.

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