CN113234605A - Simple preparation method of phlebopus portentosus solid and liquid strains - Google Patents
Simple preparation method of phlebopus portentosus solid and liquid strains Download PDFInfo
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Abstract
The invention belongs to the technical field of strain preparation, and discloses a simple preparation method of solid and liquid strains of phlebopus portentosus. The preparation method comprises the following steps: inoculating the tissue blocks of the Phlebopus portentosus fruiting body into a solid culture medium for culture to obtain solid Phlebopus portentosus strains, beating the solid strains into strain blocks, and inoculating the strain blocks into a liquid culture medium for culture to obtain the liquid strains of the Phlebopus portentosus. The culture medium of the invention is prepared from glucose, soluble starch, yeast powder and KH2PO4，MgSO4And agar. The culture medium has simple components, and easily-obtained carbon source, nitrogen source and some inorganic salts are used. The obtained solid strain has dense myceliumThe edges are neat, and extremely strong hypha activity is displayed; the obtained liquid strain has high growth speed and uniform and fine mycelium pellet, and is suitable for inoculation of modern edible fungus automatic inoculators.
Description
Technical Field
The invention belongs to the technical field of strain preparation, and particularly relates to a simple preparation method of solid and liquid strains of phlebopus portentosus.
Background
Phlebopuportentosus (Phlebopuportentosus) belongs to the family Boletaceae, the order Boletales, and the genus Boletus. Originally discovered in srilanka, and subsequently reported in southeast asian countries such as vietnam and thailand, brazil and mexico in america, and australia in the oceans, new zealand, etc., domestic distribution is mainly in the areas of guangxi, yunnan, etc. The Phlebopus portentosus fruiting body is fat, tastes delicious, belongs to high-protein and low-fat rare edible fungi which are popular with the masses. Although Phlebopus portentosus can be artificially cultured, the aspects of large-scale liquid strain preparation, optimization and the like need to be strengthened.
At present, the bolete portentosus solid and liquid strain culture media are prepared by boiling potatoes in boiling water and filtering to obtain potato juice as a basic culture medium component. Such as: zucui et al screening a culture medium of a mother species of Phlebopus portentosus (Zucui, He Ming Xia, Ji Cai Ping, et. screening of a culture medium of a mother species of Phlebopus portentosus [ J ]. southwestern agrotics 2009.22(6): 1699-; HASSAN SidikMohamed et al improved the PDA medium of Phlebopus portentosus, but potato juice was still one of the major components of the medium (HASSAN SidikMohamed, Guo Xuan Xin, Wu Zhengyan, Pan morning, Xuntang, Liu Yong. isolation and medium optimization of Phlebopus portentosus [ J ].2021.62(1): 83-86). In addition, patent literature (CN101381684A) reports the use of potato, sucrose, etc. to produce solid and liquid seed cultures; patent document (CN106929432A) mentions that potato juice obtained by boiling peeled potatoes in boiling water is used as a main component of a culture medium for the production of phlebopus portentosus liquid spawn. The potato juice can be obtained only by boiling and filtering, so the method is only suitable for small-scale production of solid and liquid strains, and the method has the obvious defects of complex operation, high cost and the like for liquid strains for large-scale industrial production. In addition, the liquid strains of the phlebopus portentosus prepared at present have the problems of overlarge fungus balls, low hypha density and the like. The nozzle opening of the modern edible fungus liquid automatic inoculation machine is easy to block due to the overlarge fungus balls; the low hypha density easily causes the pollution of fungus bags after inoculation. Therefore, it is urgently needed to provide a simple and efficient method for preparing solid and liquid strains of phlebopus portentosus, which can be used for industrial cultivation.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention aims to provide a simple preparation method of solid and liquid strains of phlebopus portentosus.
The purpose of the invention is realized by the following technical scheme:
a simple preparation method of solid and liquid strains of Phlebopus portentosus comprises the following preparation steps:
(1) inoculating the Phlebopus portentosus fruiting body tissue blocks into a solid culture medium for culturing to obtain Phlebopus portentosus solid strains;
(2) beating the solid strains obtained in the step (1) into bacterium blocks, and then inoculating the bacterium blocks into a liquid culture medium for culture to obtain phlebopus portentosus liquid strains;
the solid culture medium in the step (1) comprises the following components: 2-6% of glucose (w/v), 0.1-2% of soluble starch (w/v), 1-4% of yeast powder (w/v) and KH2PO40.05～1％，MgSO40.05-1%, agar 1-3%, and the balance of water; the pH value of the solid culture medium is 4.0-6.0;
the liquid culture medium in the step (2) comprises the following components: 2-6% of glucose (w/v), 0.1-2% of soluble starch (w/v), 1-4% of yeast powder (w/v) and KH2PO40.05～1％，MgSO40.05-1% of water, and the balance of water; the pH of the liquid culture medium is 4.0-6.0.
Further, the solid medium in the step (1) is prepared by the following method: mixing glucose, soluble starch, yeast powder, KH2Adding PO4 and MgSO4 into water, stirring and mixing uniformly, adjusting the pH value to 4.0-6.0, and then adding agarHeating until agar melts completely, packaging, sterilizing, cooling, and solidifying to obtain solid culture medium.
Further, the step of culturing in the solid culture medium in the step (1) refers to culturing in an incubator at 20-32 ℃ in the dark for 15-35 days.
Further, the beating into a mushroom block in the step (2) is to beat into a circular mushroom block with the diameter of 1 cm.
Further, the liquid medium in step (2) is prepared by the following method: mixing glucose, soluble starch, yeast powder, KH2Adding PO4 and MgSO4 into water, stirring and mixing uniformly, adjusting the pH value to 4.0-6.0, subpackaging, sterilizing and cooling to obtain the liquid culture medium.
Further preferably, the sterilization in the processes of preparing the solid culture medium and preparing the liquid culture medium means that the solid culture medium and the liquid culture medium are sterilized in a sterilization pot at 121 ℃ for 30 min.
Further, the step (2) of culturing in the liquid culture medium means that the liquid culture medium is placed in an incubator at 22-32 ℃ and is subjected to shaking culture at 90-250 rpm for 5-12 days.
Compared with the prior art, the invention has the beneficial effects that:
compared with the methods mentioned in the patent documents (CN101381684A) and (CN106929432A), the method has the following advantages:
(a) the potato juice is left as the main component of the culture medium of the solid strains and the liquid strains, so that the cost and the time for preparing the culture medium are effectively reduced;
(b) the culture medium has simple components, and easily-accessible carbon source (glucose), nitrogen source (yeast powder) and inorganic salts (KH)2PO4、MgSO4)；
(c) The solid strain prepared by the solid culture medium has dense hyphae and regular edges, and shows extremely strong hyphae activity;
(d) the liquid spawn prepared by the liquid culture medium has high growth speed and uniform and fine mycelium pellet, and is suitable for inoculation of modern edible fungus automatic inoculators.
Drawings
FIG. 1 is a schematic diagram of a solid bacterial strain obtained in example 1 of the present invention;
FIG. 2 is a schematic diagram of the liquid seed culture obtained in example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The solid and liquid strains of phlebopus portentosus of the embodiment are prepared by the following method:
1. the solid strain culture medium is used for preparing the solid strain, and the formula and the method of the solid strain culture medium are as follows:
(1) the formula and components of the culture medium are as follows: 2.5% (w/v) glucose, 0.5% (w/v) soluble starch, 1% (w/v) yeast powder, 0.1% (w/v) KH2PO40.2% (w/v) MgSO 242% (w/v) agar.
(2) Adding glucose, soluble starch, yeast powder and KH into the culture medium components in the step (1)2PO4、MgSO4Adding water to a constant volume of 1000mL, adjusting pH to 5.5, adding agar, heating until the agar is completely melted, subpackaging in conical flasks, sterilizing in a sterilizing pot at 121 ℃ for 30min, cooling to about 55 ℃, pouring 20mL of culture medium into a 90mm culture dish in an ultra-clean bench, and cooling to solidify for later use.
(3) Separating the tissue blocks of the fruiting body of Phlebopus portentosus with sterile scalpel in 0.2cm each in a clean bench3And placed in the above-mentioned culture dish, and placed in an incubator at 25 deg.C under the condition of keeping out of the sun for 20 days.
(4) In a clean bench, the cultured solid strains are beaten into round strains by a sterile hole puncher with the diameter of 1cm, and the round strains are transferred to a new culture dish for propagation, and are placed in an incubator for 20 days at 25 ℃ in a dark place.
2. The liquid culture medium is used for preparing the phlebopus portentosus liquid strain, and the formula and the method of the liquid strain are as follows:
(1) the formula and components of the culture medium are as follows: 2% (w/v) glucose, 0.8% (w/v) soluble starch, 1% (w/v) yeast powder, 0.1% (w/v) KH2PO40.2% (w/v) MgSO 24。
(2) Adding glucose, soluble starch, yeast powder and KH into the culture medium components in the step (1)2PO4、MgSO4Adding water to a constant volume of 1000mL, adjusting pH to 5.5, subpackaging in 500mL conical flasks with 200mL culture medium per flask, sterilizing in a sterilizer at 121 deg.C for 30min, and cooling for use.
(3) And (3) beating the solid strains prepared in the step (1) into bacterium blocks with the diameter of 1cm by using a sterile hole puncher with the diameter of 1cm in a super-clean workbench, inoculating the bacterium blocks into the liquid culture medium, and placing the liquid culture medium in an incubator at 26 ℃ and 120rpm for shaking culture for 7 days to obtain the phlebopus portentosus liquid strains.
The schematic diagram of the solid bacterial strain obtained in this example is shown in FIG. 1. The boletus fuscous solid strain is shown to have dense hyphae and neat edges, and shows extremely strong hyphae activity.
The schematic diagram of the liquid bacterial strain obtained in this example is shown in FIG. 2. Wherein (a) liquid seed culture in Erlenmeyer flasks; (b) liquid seed culture diluted 1, 10 and 20 times for (c) and (d), respectively. The method can be used for obtaining the phlebopus portentosus liquid strain with high growth speed and uniform and fine mycelium pellet, and is suitable for automatic inoculation and inoculation of modern edible fungi.
Example 2
The solid and liquid strains of phlebopus portentosus of the embodiment are prepared by the following method:
1. the solid strain culture medium is used for preparing the solid strain, and the formula and the method of the solid strain culture medium are as follows:
(1) the formula and components of the culture medium are as follows: 2.5% (w/v) glucose, 0.5% (w/v) soluble starch, 1% (w/v) yeast powder, 0.15% (w/v) KH2PO40.2% (w/v) MgSO 242% (w/v) agar.
(2) Adding glucose, soluble starch, yeast powder and KH into the culture medium components in the step (1)2PO4、MgSO4Adding water to 1000mL, adjusting pH to 5.5, adding agar, and heating until the agar is completely meltedChanging into chemical, subpackaging in conical flask, sterilizing at 121 deg.C for 30min in a sterilizing pot, cooling to about 55 deg.C, pouring 20mL each culture medium into 90mm culture dish in a clean bench, cooling and solidifying for use.
(3) Separating the tissue blocks of the fruiting body of Phlebopus portentosus with sterile scalpel in 0.2cm each in a clean bench3And placed in the above-mentioned culture dish, and placed in an incubator at 25 deg.C under the condition of keeping out of the sun for 20 days.
(4) In a clean bench, the cultured solid strains are beaten into round strains by a sterile hole puncher with the diameter of 1cm, and the round strains are transferred to a new culture dish for propagation, and are placed in an incubator for 20 days at 25 ℃ in a dark place.
2. The liquid culture medium is used for preparing the phlebopus portentosus liquid strain, and the formula and the method of the liquid strain are as follows:
(1) the formula and components of the culture medium are as follows: 2% (w/v) glucose, 1% (w/v) soluble starch, 1% (w/v) yeast powder, 0.1% (w/v) KH2PO40.2% (w/v) MgSO 242% (w/v) agar.
(2) Adding glucose, soluble starch, yeast powder and KH into the culture medium components in the step (1)2PO4、MgSO4Adding water to a constant volume of 1000mL, adjusting pH to 5.5, subpackaging in 500mL conical flasks with 200mL culture medium per flask, sterilizing in a sterilizer at 121 deg.C for 30min, and cooling for use.
(3) In a clean bench, the prepared solid strains are beaten into strain blocks with the diameter of 1cm by using a sterile puncher with the diameter of 1cm, the strain blocks are inoculated into the liquid culture medium, and the liquid strains are placed in an incubator at the temperature of 27 ℃ and the speed of 150rpm for shaking culture for 7 days to obtain the phlebopus portentosus liquid strains.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A simple preparation method of solid and liquid strains of Phlebopus portentosus is characterized by comprising the following preparation steps:
(1) inoculating the Phlebopus portentosus fruiting body tissue blocks into a solid culture medium for culturing to obtain Phlebopus portentosus solid strains;
(2) beating the solid strains obtained in the step (1) into bacterium blocks, and then inoculating the bacterium blocks into a liquid culture medium for culture to obtain phlebopus portentosus liquid strains;
the solid culture medium in the step (1) comprises the following components: 2-6% of glucose, 0.1-2% of soluble starch, 1-4% of yeast powder and KH2PO40.05～1％，MgSO40.05-1%, agar 1-3%, and the balance of water; the pH value of the solid culture medium is 4.0-6.0;
the liquid culture medium in the step (2) comprises the following components: 2-6% of glucose, 0.1-2% of soluble starch, 1-4% of yeast powder and KH2PO40.05～1％，MgSO40.05-1% of water, and the balance of water; the pH of the liquid culture medium is 4.0-6.0.
2. The simple preparation method of phlebopus portentosus solid and liquid strains according to claim 1, wherein the solid culture medium in the step (1) is prepared by the following method: mixing glucose, soluble starch, yeast powder, KH2Adding PO4 and MgSO4 into water, stirring and mixing uniformly, adjusting the pH value to 4.0-6.0, adding agar, heating until the agar is completely melted, subpackaging, sterilizing, cooling and solidifying to obtain the solid culture medium.
3. The method for preparing the simple solid and liquid strains of phlebopus portentosus according to claim 1 or 2, wherein the method comprises the following steps: the step (1) of culturing in the solid culture medium refers to culturing in an incubator at 20-32 ℃ in a dark place for 15-35 days.
4. The method for preparing the simplified solid and liquid strains of Phlebopus portentosus according to claim 1 or 2, wherein the step (2) of beating into a mushroom block is to beat into a round mushroom block with a diameter of 1 cm.
5. The simple preparation method of phlebopus portentosus solid and liquid strains according to claim 1, wherein the liquid culture medium in the step (2) is prepared by the following method: mixing glucose, soluble starch, yeast powder, KH2Adding PO4 and MgSO4 into water, stirring and mixing uniformly, adjusting the pH value to 4.0-6.0, subpackaging, sterilizing and cooling to obtain the liquid culture medium.
6. The method for preparing the simple solid and liquid strains of phlebopus portentosus according to claim 2 or 5, wherein the method comprises the following steps: the sterilization in the processes of preparing the solid culture medium and preparing the liquid culture medium refers to placing the culture medium in a sterilization pot for sterilization for 30min at the temperature of 121 ℃.
7. The method for preparing the simple solid and liquid strains of phlebopus portentosus according to claim 1 or 5, wherein the method comprises the following steps: the step (2) of culturing in the liquid culture medium is to put the culture medium in an incubator for 22-32 ℃ and shaking culture at 90-250 rpm for 5-12 days.
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