CN113767812A - Ganoderma strain and culture method of ganoderma lucidum fruiting body - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention provides a ganoderma lucidum strain named as ganoderma lucidum ganodermashanisense, the preservation number is CGMCC NO:23077, the preservation unit is China general microbiological culture Collection center, and the preservation date is 2021, 8 and 11 days. Adding glucose into a carbon source basal culture medium to ensure that the concentration of the glucose is 30g/L, adjusting the pH value of the culture medium to 4.0, adding yeast powder into a nitrogen source basal culture medium to ensure that the concentration of the yeast powder is 2g/L, and adjusting the pH value of the culture medium to 4.0 to obtain a carbon source and nitrogen source culture medium suitable for the growth of ganoderma lucidum mycelia; then inoculating the Ganoderma strain into culture medium for Ganoderma lucidum growth, and dark culturing at 30 deg.C to obtain Ganoderma lucidum fruiting body. The ganoderma lucidum has strong natural selenium enrichment capability.
Description
Technical Field
The invention belongs to the technical field of ganoderma lucidum strains, and particularly relates to a ganoderma lucidum strain and a culture method of ganoderma lucidum fruiting bodies.
Background
Ganoderma lucidum (Ganoderma lingzhi) is one of the most famous medicinal fungi in China, and has a long medicinal history in China. Modern scientific research proves that the ganoderma lucidum has wide pharmacological activity, including immunity improvement, tumor resistance, diabetes resistance, HIV-1 virus resistance, blood sugar reduction, liver protection and the like.
The existing ganoderma lucidum varieties mostly belong to medium-high temperature varieties, the cultivation mode mainly comprises clinker cultivation and basswood cultivation, the ganoderma lucidum strains are easy to generate generation-by-generation degradation in the cultivation process, the content of active ingredients such as ganoderma lucidum polysaccharide and organic selenium is reduced generation by generation, the health care and medicinal effects are less and less obvious, and the high-efficiency medicinal development is relatively lagged.
Ganoderma lucidum has less natural selenium-rich capability, and the vigorous development of the ganoderma lucidum variety with natural selenium-rich capability is particularly important. The Chinese Nutrition society ranks selenium as one of 15 nutrients essential to human body, and a large number of clinical experiments at home and abroad show that the selenium deficiency of human body can cause dysfunction of some important organs to cause a plurality of serious diseases, more than 40 countries in the world are in selenium deficiency areas, a plurality of hundred million of people in 22 provinces in China are in selenium deficiency or low selenium zones, and the incidence of tumors, liver diseases, cardiovascular diseases and the like of population in the areas is high. Research shows that the people with low selenium or selenium deficiency can prevent tumor, liver disease, etc. by supplementing selenium with proper amount, improve immunity, maintain normal functions of heart, liver, lung, stomach and other important organs, and prevent senile cardiovascular and cerebrovascular diseases.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a Ganoderma lucidum strain and a method for culturing Ganoderma lucidum fruiting bodies thereof aiming at the defects of the prior art, wherein the Ganoderma lucidum (Ganoderma shanxi) has stronger natural selenium-rich capability.
In order to solve the technical problems, the invention adopts the technical scheme that: a Ganoderma lucidum strain is named as Ganoderma lucidum Ganoderma shanxi ense, the preservation number is CGMCC NO:23077, the preservation unit is China general microbiological culture Collection center, and the preservation address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of 2021, 8 months and 11 days.
Preferably, the ITS nucleotide sequence of the ganoderma lucidum strain is shown as SEQ ID NO. 1.
The invention also provides a method for culturing ganoderma lucidum fruiting bodies by using the ganoderma lucidum strains, which comprises the following steps:
adding glucose into a carbon source basal culture medium to ensure that the concentration of the glucose is 30g/L, and adjusting the pH value of the culture medium to 4.0 to obtain a proper culture medium for the growth of ganoderma lucidum mycelia;
or adding yeast powder into nitrogen source basal medium to make the concentration of the yeast powder be 2g/L, and adjusting the pH value of the culture medium to 4.0 to obtain a proper culture medium for the growth of ganoderma lucidum mycelia;
then inoculating the Ganoderma strain into a suitable culture medium for Ganoderma lucidum mycelium growth, and dark culturing at 30 deg.C to obtain Ganoderma lucidum fruiting body.
Preferably, the average value of the selenium content of the ganoderma lucidum fruiting body is 2.75 mg/kg-2.83 mg/kg.
Preferably, the carbon source basal medium consists of the following raw materials in mass concentration: peptone 3.0g/L, KH2PO41.0 g/L、MgSO40.45g/L agar and 12.0g/L agar; the nitrogen source basal medium consists of the following raw materials in mass concentration: glucose 20.0g/L, KH2PO42.0 g/L、MgSO40.45g/L and agar 15.0 g/L.
Preferably, the time for catalyzing the ganoderma lucidum is 1.5 d-3.0 d.
Compared with the prior art, the invention has the following advantages:
the Ganoderma lucidum (Ganoderma shanxi ense) has stronger natural selenium-rich capability, and the average value of the selenium content of the fruit body is 2.75 mg/kg-2.83 mg/kg.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a colony morphology of Ganoderma lucidum Ganoderma shannxiense of example 1 of the present invention.
FIG. 2 is a phylogenetic tree of Ganoderma lucidum ganodermatum shannxiense according to example 1 of the present invention.
FIG. 3 is a graph showing the effect of different temperatures on the growth rate of Ganoderma lucidum Ganoderma shanxi ense hyphae in example 5 of the present invention.
FIG. 4 is a graph showing the effect of different pH values on the growth rate of Ganoderma lucidum Ganoderma shanxi ense hyphae in example 5 of the present invention.
FIG. 5 is a fruit body map of Ganoderma lucidum Ganoderma shannxiense according to example 5 of the present invention.
Detailed Description
Example 1
A Ganoderma lucidum strain is named as Ganoderma lucidum Ganoderma shanxi ense, the preservation number is CGMCC NO:23077, the preservation unit is China general microbiological culture Collection center, and the preservation date is 2021 year, 8 month and 11 days.
Collecting wild Ganoderma strain in Sichuan town of Xixian province in 2018, 9/12, and obtaining pure strain (F1 generation) by tissue isolation at edible fungi institute of agricultural academy of sciences in Shanxi province, wherein the pure strain is a new species named as Shanxi Ganoderma (Ganoderma shanxi L. Fan & H. Liu sp. nov.) through morphological and its analysis.
In 2018, 9-10 months, the wild strain is purified and rejuvenated by an edible fungus research institute of academy of agricultural sciences of Shanxi province, and the specificity of the strain is preliminarily verified by a hypha antagonism method.
In 2018, the domestication of the glossy ganoderma is carried out by adopting a biological incubator in a laboratory of edible fungus institute of academy of agricultural sciences in Shanxi province from 11 months to 12 months, and the domestication is successful; the edible fungi research of Shanxi province agricultural science institute in 1 month in 2019, so that the new fruiting body is a new strain cultured by a tissue isolation method (F2 generation), and the new strain is determined to be a new strain which can be cultured by an ITS sequencing ratio in 1 month in 2019.
Performing demonstration and stable cultivation in 2019 in three different ecological areas, namely, Shanxi province Yanquan city Pingting county green and ocean agriculture development Limited company, Qin water Baiyun edible fungus planting professional cooperative society, and Shancheng county Green tripod source afforestation cooperative society, and the like, obtaining pileus-shaped glossy Ganoderma, wherein the pileus-shaped glossy Ganoderma is a russet fruiting body, separating the pileus-shaped glossy Ganoderma to form a new strain (F3), detecting the purity, the activity and the like to form a new Ganoderma strain, and naming the new Ganoderma strain as Ganoderma lucidum (Ganoderma shanxi ense); the ITS nucleotide sequence of the ganoderma lucidum strain is shown as SEQ ID NO. 1.
The Ganoderma lucidum (Ganoderma sanxinense) of this example has white and filamentous hyphae, and the bacterial colony is concentric ring striated, and is not easy to form a pellicle (LH 676 in FIG. 1). Is significantly different from the control Ganoderma strain (Ganoderma Lucidum) (GL 003 in FIG. 1). The genetic phylogenetic tree of Ganoderma lucidum (Ganoderma shanxi ense) in this example is shown in fig. 2, and the sequences of Ganoderma lucidum (Ganoderma shanxi) are gathered on separate branches and highly supported.
Example 2
This example provides the method of culturing Ganoderma lucidum fruiting body with the Ganoderma lucidum strain of example 1, which comprises:
adding glucose into a carbon source basal culture medium to ensure that the concentration of the glucose is 30g/L, and adjusting the pH value of the culture medium to 4.0 to obtain a carbon source culture medium suitable for the growth of ganoderma lucidum mycelia;
then inoculating a ganoderma lucidum strain into the culture medium for the growth of the ganoderma lucidum, and carrying out dark culture at the temperature of 30 ℃ for 1.5d to obtain ganoderma lucidum sporocarp;
the carbon source basal medium consists of the following raw materials in mass concentration: peptone 3.0g/L, KH2PO41.0 g/L、MgSO40.45g/L and agar 12.0 g/L.
Example 3
This example provides the method of culturing Ganoderma lucidum fruiting body with the Ganoderma lucidum strain of example 1, which comprises:
adding yeast powder into a nitrogen source basal culture medium to ensure that the concentration of the yeast powder is 2g/L, and adjusting the pH value of the culture medium to 4.0 to obtain a nitrogen source culture medium suitable for the growth of ganoderma lucidum mycelia;
then inoculating a ganoderma lucidum strain into the culture medium for the growth of the ganoderma lucidum, and carrying out dark culture at the temperature of 30 ℃ for 3.0d to obtain ganoderma lucidum sporocarp;
the nitrogen source basal medium consists of the following raw materials in mass concentration: glucose 20.0g/L, KH2PO42.0 g/L、MgSO40.45g/L and agar 15.0 g/L.
Example 4
The fruiting bodies of the Ganoderma Lucidum cultured in examples 2-3 and the control Ganoderma strain (Ganoderma Lucidum) were dried, crushed and sieved (60 mesh). Each group of Ganoderma encarpium is measured by taking 3 fruiting bodies. The method comprises the following steps of measuring various trace elements by adopting an atomic absorption method, wherein the zinc content is measured according to GB5009.14-2017, the iron content is measured according to GB5009.90-2016, the calcium content is measured according to GB5009.92-2016, the magnesium content is measured according to GB5009.241-2017, the germanium content is measured according to GB 5009.151-2003, the chromium content is measured according to GB 5009.123-2014, and the selenium content is measured according to GB 5009.93-2017, and the results are shown in Table 1, wherein the ganoderma lucidum sporocarp cultured in the example 2-3 has strong natural selenium-rich capacity which is 13-18 times that of the control ganoderma lucidum:
TABLE 1 content of elements (mg/kg) of fruiting bodies of Ganoderma lucidum karst and Ganoderma lucidum karst of the present invention
| Item | Zinc | Magnesium alloy | Chromium (III) | Selenium |
| Example 2 | 45.63±3.6a | 1050.1±41.8a | 0.21±0.08a | 2.75±0.68a |
| Example 3 | 46.75±2.8a | 1003.1±36.9a | 0.19±0.06a | 2.83±0.55a |
| Control group | 21.08±2.1b | 676.6±36.7b | 0.11±0.12a | 0.24±0.02b |
Note: the alphabetical identity differences were not significant (P < 0.05).
Example 5
This example is a condition screening of Ganoderma lucidum fruiting body cultured by the Ganoderma lucidum strain of example 1:
screening of culture medium: ganoderma lucidum (Ganoderma sanxinense) has a suitable carbon source of 30g/L glucose and a suitable nitrogen source of 2.0g/L yeast powder.
1. On a carbon source basal medium, 4 different carbon sources of glucose (P), sucrose (Z), lactose (R) and mannitol (G) are respectively added, and the concentration gradient of each carbon source is divided into 3 carbon sources of 10G/L, 20G/L and 30G/L, namely 12 carbon source formulas for testing are formed. Inoculating blocks with consistent growth are obtained from a strain culture dish of the ganoderma lucidum by using a completely sterilized hole puncher (phi is 6mm) to the center of a carbon source culture medium culture dish for testing, the inoculating blocks are placed at 25 ℃ for dark culture for 6d, the diameter of a bacterial colony is measured by using a vernier caliper according to a cross method, the growth speed of hyphae is calculated, and the growth vigor of the hyphae is observed. Each treatment was set to 5 replicates; the carbon source baseThe basic culture medium consists of the following raw materials in mass concentration: peptone 3.0g/L, KH2PO4 1.0g/L、MgSO40.45g/L and agar 12.0 g/L. As can be seen from Table 1, the growth rate of Ganoderma lucidum mycelia was the greatest under the condition of 30g/L glucose.
TABLE 1 Effect of different carbon sources on the growth of Ganoderma lucidum mycelia
| Different carbon sources and addition amounts | Color of hypha | Growth of hypha | Growth rate/mm. d-1 |
| 10g/L glucose | Milk white | 2+ | 2.99±0.03e |
| 20g/L glucose | Milk white | 3+ | 3.55±0.12b |
| 30g/L glucose | Milk white | 2+ | 3.87±0.05a |
| 10g/L sucrose | Milk white | 2+ | 2.71±0.07f |
| 20g/L sucrose | Milk white | 2+ | 2.94±0.07e |
| 30g/L sucrose | Milk white | 2+ | 3.51±0.01b |
| 10g/L lactose | Snow white | 3+ | 2.30±0.02h |
| 20g/L lactose | Snow white | 3+ | 2.48±0.06g |
| 30g/L lactose | Snow white | 3+ | 3.22±0.10cd |
| 10g/L mannitol | White colour (Bai) | 2+ | 2.80±0.07f |
| 20g/L mannitol | White colour (Bai) | 2+ | 3.30±0.02c |
| 30g/L mannitol | White colour (Bai) | 2+ | 3.14±0.03d |
Description of the drawings: +, 2+, 3+ indicate a gradual increase in hyphal growth. Differences were not significant when the letters were identical (P < 0.05).
2. Adding urea (the addition amount is 1.0g/L, 2.0g/L and 3.0g/L), (NH) on nitrogen source basic culture medium4)2SO4(addition amounts of 1.0g/L, 2.0g/L and 3.0g/L), peptone (addition amounts of 1.0g/L, 3.0g/L and 5.0g/L) and yeast extract powder (addition amounts of 2.0g/L, 4.0g/L and 6.0g/L)4 different nitrogen sources, to form 12 test recipes. Inoculating Ganoderma lucidum strain with consistent size to the center of the culture medium, dark culturing at 25 deg.C for 5d, observing hypha growth, and measuring hypha growth rate. The rest is the same as the carbon source test; the nitrogen source basal medium consists of the following raw materials in mass concentration: glucose 20.0g/L, KH2PO42.0g/L、 MgSO40.45g/L and agar 15.0 g/L. As can be seen from Table 2, the growth rate of Ganoderma lucidum mycelia was the greatest under the condition of 2.0g/L yeast powder.
TABLE 2 influence of different nitrogen sources on the growth of Ganoderma lucidum mycelium
| Different nitrogen sources and addition amounts | Color of hypha | Growth of hypha | Growth rate |
| Urea 1.0g/L | White colour (Bai) | 2+ | 2.11±0.02g |
| Urea 2.0g/L | White colour (Bai) | 3+ | 1.40±0.02h |
| Urea 3.0g/L | White colour (Bai) | - | - |
| (NH4)2SO41.0g/L | White in white | 2+ | 3.36±0.03b |
| (NH4)2SO42.0g/L | White in white | + | 2.78±0.06e |
| (NH4)2SO43.0g/L | White in white | + | 2.63±0.14f |
| Peptone 1.0g/L | White colour (Bai) | 2+ | 3.41±0.05b |
| Peptone 3.0g/L | White colour (Bai) | 2+ | 2.92±0.04d |
| Peptone 5.0g/L | White colour (Bai) | 3+ | 3.24±0.02c |
| Yeast powder 2.0g/L | White colour (Bai) | 3+ | 3.67±0.01a |
| Yeast powder 4.0g/L | White colour (Bai) | 3+ | 3.42±0.07b |
| Yeast powder 6.0g/L | White colour (Bai) | 3+ | 3.40±0.04b |
Description of the drawings: +, 2+, 3+ indicate a gradual increase in hyphal growth, -no statistical data. Differences were not significant when the letters were identical (P < 0.05).
Secondly, the growth of hypha of Ganoderma lucidum (Ganoderma shanxi ense) is under proper conditions: ganoderma lucidum (Ganoderma sanxinense) mycelium is cultured at 30 deg.C, and can grow at pH of 3.0-8.0, preferably pH of 4.0.
1. Temperature characteristic test: the test strains were inoculated on PDA medium and cultured in dark at 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C and 35 deg.C, respectively, with the same procedure as for carbon source test. As shown in FIG. 3, dark culture of Ganoderma at 30 deg.C has the highest growth rate of 4.69 mm. d-1。
2. And (3) matrix acid-base test: taking PDA as a basic formula, preparing 7 test formulas with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 by using 1.0M NaOH solution and 1.0M HCl solution in an ultraclean workbench before pouring a plate, inoculating test strains to the test formulas, placing the test formulas at 25 ℃ for dark culture for 5 days, observing the growth vigor of hyphae and determining the growth speed, wherein other requirements are the same as those of a carbon source test, as shown in figure 4, when the pH value is 4.0, the growth speed of the hyphae is fastest and can reach 4.96 mm d.d.d.-1。
And (III) in the Ganoderma lucidum accelerating tests at different temperatures, the Ganoderma lucidum accelerating management is carried out at the temperature of 26 ℃, the pileus of the Ganoderma lucidum is not differentiated, the Ganoderma lucidum is easy to form, and both pileus and stipe can be normally differentiated at the temperature of 30 ℃.
As shown in FIG. 5, the pileus surface and pileus of Ganoderma lucidum (Ganoderma shanxi) are reddish brown, with smooth pileus and obvious lacquer-like luster (LH 676 in FIG. 5). Compared with the control Ganoderma Lucidum (Ganoderma Lucidum) (GL 003 in FIG. 5), the difference was significant.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
SEQUENCE LISTING
<110> Shanxi functional food research institute of Shanxi agricultural university
<120> a method for culturing Ganoderma lucidum strain and Ganoderma lucidum fruiting body
<130> 2021.3.10
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 643
<212> DNA
<213> Artificial Synthesis
<400> 1
gcatacaggt tttcgtaggt gacctgcgga ggtcattatc gagttttgac tgggttgtag 60
ctggccttcc gaggcatgtg cacgccctgc tcatccactc tacacctgtg cacttactgt 120
gggtttcagg tcgcgaagcg ggctctgtac gggcttgtga agcgcatctg tgcctgcgtt 180
tatcacaaac tccataaagt attagaatgt gtattgcgat gtaacgcatc tttatacaac 240
tttcagcaac ggatctcttg gctctcgcat cgatgaagaa cgcagcgaaa tgcgataagt 300
aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcaccttg cgctccttgg 360
tattccgagg agcatgcctg tttgagtgtc atgaaatctt caacctacaa gctttgtagg 420
cttggacttg gaggcttgtt ggccgttatt ggtcagctcc tcttaaacaa attagcttga 480
ttccttgtgg atcggctctc ggtgtgataa tgtctacgcc gcgaccgtga agcgtttggc 540
gagcttctaa ccgtcccatt cggagacaac tttatgacct ctgacctcaa atcaggtagg 600
actacccgct gaacttaagc atatcaatag gccggaggaa aga 643
Claims (6)
1. A Ganoderma lucidum strain is characterized in that the Ganoderma lucidum strain is named as Ganoderma lucidum Ganoderma shanxi ense, the preservation number is CGMCC NO:23077, the preservation unit is China general microbiological culture Collection center, and the preservation date is 2021 year, 8 month and 11 days.
2. The ganoderma lucidum strain according to claim 1, wherein the ITS nucleotide sequence of the ganoderma lucidum strain is shown as SEQ ID NO. 1.
3. A method for culturing the fruiting body of Ganoderma rubrum with the Ganoderma lucidum strain of claim 1 or 2, which comprises:
adding glucose into a carbon source basal culture medium to ensure that the concentration of the glucose is 30g/L, and adjusting the pH value of the culture medium to 4.0 to obtain a proper culture medium for the growth of ganoderma lucidum mycelia;
or adding yeast powder into nitrogen source basal medium to make the concentration of the yeast powder be 2g/L, and adjusting the pH value of the culture medium to 4.0 to obtain a proper culture medium for the growth of ganoderma lucidum mycelia;
then inoculating the Ganoderma strain into a suitable culture medium for Ganoderma lucidum mycelium growth, and dark culturing at 30 deg.C to obtain Ganoderma lucidum fruiting body.
4. The method according to claim 3, wherein the Ganoderma lucidum fruiting body has an average selenium content of 2.75mg/kg to 2.83 mg/kg.
5. The method of claim 3, wherein the carbon source basal medium consists of the following raw materials in mass concentration: peptone 3.0g/L, KH2PO41.0 g/L、MgSO40.45g/L agar and 12.0g/L agar; the nitrogen source basal medium consists of the following raw materials in mass concentration: glucose 20.0g/L, KH2PO42.0 g/L、MgSO40.45g/L and agar 15.0 g/L.
6. The method as claimed in claim 3, wherein the incubation time is 1.5 d-3.0 d.
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| CN113767812B (en) | 2023-04-25 |
| ZA202209318B (en) | 2022-11-30 |
| NL2033084B1 (en) | 2023-06-30 |
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