CN117987279A - Composition and culture medium for stropharia rugoso-annulata mother culture and preparation method thereof - Google Patents
Composition and culture medium for stropharia rugoso-annulata mother culture and preparation method thereof Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of stropharia rugoso-annulata strain breeding, and in particular relates to a composition and a culture medium for stropharia rugoso-annulata mother strain culture and a preparation method thereof, wherein the composition comprises water and material components, and the material components comprise, by mass per thousandth of water: 5 to 50 per mill of agricultural and forestry waste, 2 to 3 per mill of yeast powder, 0.0025 to 0.01 per mill of zinc sulfate, 0.005 to 0.02 per mill of manganese sulfate, 0.001 to 0.004 per mill of cobalt chloride and 0.5 to 2 per mill of calcium lignosulfonate. The stropharia rugoso-annulata mother culture medium provided by the invention adopts agricultural and forestry waste as a raw material, has wide sources and low price, and the calcium lignosulfonate can obviously improve the laccase activity of stropharia rugoso-annulata. In addition, the stropharia rugoso-annulata cultivated by the culture medium has fast growth speed and vigorous growth. The formula can solve the defects that the existing stropharia rugoso-annulata mother seeds are slow in growth speed and troublesome to manufacture, and the bottle filling time of the stock seeds is too long after the stock seeds are transferred.
Description
Technical Field
The invention belongs to the field of stropharia rugoso-annulata strain breeding, and in particular relates to a composition and a culture medium for stropharia rugoso-annulata mother strain culture and a preparation method thereof.
Background
Stropharia rugoso-annulata (Stropharia rugosoannulata), also called Stropharia rugoso-annulata, has bright color and luster, thick and thick legs, is smooth, tender, crisp and rich in nutrition, and is one of the special varieties recommended to the developing countries by the International grain and agricultural organization (FAO). In recent years, stropharia rugoso-annulata has become one of the star mushrooms in the edible fungus industry in China, the cultivation amount of stropharia rugoso-annulata in China is increased year by year, and the annual yield is increased from 4.74 ten thousand tons in 2010 to 28.75 ten thousand tons in 2021. Compared with main wood rot fungi such as oyster mushrooms, mushrooms and agaric, the stropharia rugoso-annulata has stronger stress resistance and impurity resistance, is cultivated by adopting fermentation materials generally, omits the step of sterilizing cultivation bags, and is very suitable for agricultural cultivation.
The strain problem is a main factor which plagues the cultivation of stropharia rugoso-annulata. Potato dextrose agar (Potato Dextrose Agar, PDA) culture medium is the main mother culture medium of most edible fungi, but the growth speed of stropharia rugoso-annulata on the PDA culture medium is slower, 17-19 days can be used for growing into a culture dish with the diameter of 9cm, and the time required for growing oyster mushroom, mushroom and black fungus into the PDA culture dish with the diameter of 9cm is 6-7 days, 9-10 days and 14-16 days respectively (NY/T1742-2009 edible fungus strain general technical requirements).
There have been advances in related formulation studies to address the slow growth problem of the stock species, but all suffer from drawbacks, and 3 long, faster formulation specifications are now being picked. The corn flour and the compost are added to enable the stropharia rugoso-annulata mycelium to grow into a culture dish with the diameter of 9cm for 11 days, and the method is difficult to popularize, because the compost is not a standardized substance, and other people can hardly repeat the result. In the mother seed formulation using potato and trehalose as main materials, 14 days (CN 201710863770.2) are required for stropharia rugoso-annulata to grow on the culture dish. The culture dish can be fully grown after 12 days of stropharia rugoso-annulata mycelium on the culture medium of sucrose, corn flour, peptone and 6-BA. The two formulas are quick in growth speed, but the potatoes need to be boiled to obtain juice (the default practice in the field of edible fungi is that the potatoes are cut into pieces and boiled for more than 30 minutes and filtered to obtain juice), and the preparation process is time-consuming and troublesome.
Besides the slow growth rate, the low laccase activity of stropharia rugoso-annulata parent seeds of the existing formula is the greatest disadvantage. Laccase is a main enzyme for degrading lignin of edible fungi, and the activity of laccase directly determines the capability of degrading matrix and is reflected in the long speed of stock and cultivar. After the stock seeds of the stropharia rugoso-annulata are inoculated with stock seeds, the stock seeds are cultured at 23-26 ℃ and filled with bottles for about 70-80 days (Baidu encyclopedia-stropharia rugoso-annulata), and the full time of the stock seeds of oyster mushrooms, mushrooms and black fungus is respectively less than or equal to 30 days, 50 days and 45 days (NY/T1742-2009 edible fungus strain general technical requirements).
In various edible fungi, the laccase activity of the mushrooms with higher growth speed is also high; the mushroom laccase with moderate growth speed has relatively high activity; the mushroom with a slower growth rate has almost no laccase activity, and laccase in edible mushrooms plays an important role in the growth rate of hyphae. Laccase activity is positively correlated with hypha growth rate, biomass, yield.
The stropharia rugoso-annulata mother seed has low laccase activity, so that the stock seed grows very slowly, and the stropharia rugoso-annulata production process is severely restricted. The high activity of the mother strain laccase is a main screening index of the stropharia rugoso-annulata mother strain culture medium, and the growth rate of the mother strain is a secondary index. Because the mother culture medium with high laccase activity has higher mother culture growth speed; however, the laccase activity of the culture medium with high growth speed of the mother strain is not necessarily high. This is why the stock of stropharia rugoso-annulata is slowly fed after inoculation of the stock, which is faster in growth, because its laccase activity is not high.
According to statistics, the yield of agricultural and forestry waste is about 33 hundred million tons worldwide, the yield of agricultural and forestry waste per year in China is up to 8.9 hundred million tons, the stropharia rugoso-annulata can be directly converted into protein without sterilizing a culture medium, the application prospect is very wide, but the activity of laccase of the currently known stropharia rugoso-annulata mother strain is low, the growth speed is slow, the stock eating speed is slow after inoculation, the bottle filling time is too long, and the cultivation scale of the stropharia rugoso-annulata is limited.
Disclosure of Invention
Aiming at the defects and defects existing in the prior art, the invention aims to provide a composition and a culture medium for stropharia rugoso-annulata mother seed culture, which have high growth speed and high laccase activity, and a preparation method thereof; the parent strain produced by the formula can solve the defects of slow growth speed, troublesome production and long bottle filling time of the original strain after the original strain is transferred in the prior stropharia rugoso-annulata parent strain.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In a first aspect, a composition for stropharia rugoso-annulata mother culture is provided, the composition comprising water and material components, the material components comprising, in mass per thousandth of water: 5 to 50 per mill of agricultural and forestry waste, 2 to 3 per mill of yeast powder, 0.0025 to 0.01 per mill of zinc sulfate, 0.005 to 0.02 per mill of manganese sulfate, 0.001 to 0.004 per mill of cobalt chloride and 0.5 to 2 per mill of calcium lignosulfonate.
In the composition of the first aspect provided by the invention, as a preferred mode, the agricultural and forestry waste comprises straw powder and/or rape stalk powder; the mass thousandth of the agricultural and forestry waste accounting for 10 to 50 thousandth of the water.
In the composition of the first aspect provided by the invention, as a preferable mode, the agricultural and forestry waste comprises straw powder and rape stalk powder, wherein the straw powder accounts for 20% -35% of the mass of the agricultural and forestry waste; the rape straw powder accounts for 65-80% of the mass of the agricultural and forestry waste.
In the composition according to the first aspect provided by the present invention, as a preferred mode, the material components include, in terms of mass per cents of water: 15 to 25 per mill of agricultural and forestry waste, 3 per mill of yeast powder, 0.0025 to 0.005 per mill of zinc sulfate, 0.005 to 0.01 per mill of manganese sulfate, 0.001 to 0.004 per mill of cobalt chloride and 1 to 2 per mill of calcium lignosulfonate.
In the composition according to the first aspect provided by the present invention, as a preferred mode, the material components include, in terms of mass per cents of water: 20 per mill of agricultural and forestry waste, 3 per mill of yeast powder, 0.0025 per mill of zinc sulfate, 0.01 per mill of manganese sulfate, 0.004 per mill of cobalt chloride and 1.5 per mill of calcium lignosulfonate.
The invention also provides a culture medium for culturing stropharia rugoso-annulata mother strain, which is prepared by adopting the composition, and the culture medium is a liquid culture medium or a solid culture medium.
In the culture medium of the second aspect provided by the invention, as an implementation manner, the solid culture medium further comprises agar powder accounting for 20-30 per mill of the water mass.
The third aspect of the invention provides a preparation method of the culture medium, which comprises the following steps:
According to the composition in the first aspect, the agricultural and forestry waste, yeast powder, zinc sulfate, manganese sulfate, cobalt chloride and calcium lignosulfonate are added into water according to the component proportion, and are uniformly mixed to obtain a composition solution;
preparing the composition solution into a liquid culture medium; or (b)
The composition solution is prepared as a solid medium.
In the preparation method of the third aspect of the present invention, as an embodiment, the preparation of the composition solution into a liquid medium includes the steps of:
sterilizing the composition solution to obtain the liquid culture medium.
In the production method of the third aspect of the present invention, as one embodiment, the composition solution is produced as a solid medium, comprising the steps of:
mixing the composition solution with agar, and sterilizing to obtain culture medium solution;
pouring the culture medium solution into a sterile container, and solidifying to obtain the solid culture medium.
Compared with the prior art, the invention has the following advantages:
The composition for stropharia rugoso-annulata mother culture provided by the invention adopts agricultural and forestry waste as raw materials, has wide sources, is easy to obtain and low in cost, and the calcium lignosulfonate can obviously improve the laccase activity of stropharia rugoso-annulata. In the culture of stropharia rugoso-annulata by adopting the composition provided by the invention, the hypha grows fast and vigorously, the growth speed is improved by 25.6% -68.9% compared with the comparative example, and the laccase activity is improved by about 10-30 times compared with the comparative example. The parent strain produced by the formula can solve the defects of slow growth speed, troublesome production and long bottle filling time of the original strain after the original strain is transferred in the prior stropharia rugoso-annulata parent strain.
Drawings
FIG. 1 shows colony morphology of stropharia rugoso-annulata grown for 10 days on mother culture media of different straw powder and rape straw powder ratios in examples 1-7 of the present invention;
FIG. 2 shows colony morphology of stropharia rugoso-annulata grown for 10 days in mother culture media of different levels of straw powder in examples 8-13 of the present invention;
FIG. 3 shows colony morphology of stropharia rugoso-annulata grown for 10 days in mother culture media of different content of rape straw powder in examples 14-19 of the present invention.
FIG. 4 photographs of the growth of Stropharia rugoso-annulata in the examples of the present invention in the medium of comparative examples 1-2 for 10 days, wherein: (a) is comparative example 1 and (b) is comparative example 2;
FIG. 5 photographs of the growth of Stropharia rugoso-annulata in the examples of the present invention in the medium of comparative examples 3 to 4 for 10 days, wherein: (a) is comparative example 3 and (b) is comparative example 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The examples of the present invention are implemented on the premise of the technical scheme of the present invention, and detailed implementation modes and processes are given, but the protection scope of the present invention is not limited to the following examples, in which the process parameters of specific conditions are not noted, and generally according to conventional conditions.
The endpoints of the ranges and any values disclosed in the present invention are not limited to the precise range or value, and the range or value should be understood to include values close to the range or value. For numerical ranges, one or more new numerical ranges may be obtained in combination with each other between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point values, and are to be considered as specifically disclosed in the present invention.
In the present invention, unless otherwise specified and/or indicated, all numbers referring to amounts of components are by weight throughout. The process parameters for the specific conditions not noted in the examples below are generally as usual. The starting materials described in the examples below are all commercially available from the public.
The specific embodiment of the invention provides a composition for stropharia rugoso-annulata mother seed culture, which comprises water and material components, wherein the material components comprise, by mass per thousandth of water:
5 to 50 per mill of agricultural and forestry waste, 2 to 3 per mill of yeast powder, 0.0025 to 0.01 per mill of zinc sulfate (ZnSO 4), 0.005 to 0.02 per mill of manganese sulfate (MnSO 4), 0.001 to 0.004 per mill of cobalt chloride (CoCl 2) and 0.5 to 2 per mill of calcium lignosulfonate.
The inventor unexpectedly found that the soluble inducer of laccase is contained in lignin sulfonic acid calcium salt which has a phenylpropane derivative hydrophobic skeleton and also contains functional groups such as phenolic hydroxyl, alcoholic hydroxyl, carboxyl, carbonyl, sulfonic acid group and the like, and the addition of lignin sulfonic acid calcium salt in the composition for culturing stropharia rugoso-annulata mother strain can promote the secretion of laccase. Laccase can catalyze the rupture and formation of specific chemical bonds in lignin molecules, so that depolymerization and polymerization of lignin are realized, and the activity of laccase is positively related to the growth speed, biomass and yield of hyphae.
According to the embodiment of the invention, the material comprises the following components in percentage by mass:
15 to 25 per mill of agricultural and forestry waste, 3 per mill of yeast powder, 0.0025 to 0.005 per mill of zinc sulfate, 0.005 to 0.01 per mill of manganese sulfate, 0.001 to 0.004 per mill of cobalt chloride and 1 to 2 per mill of calcium lignosulfonate.
According to an embodiment of the invention, the material composition preferably comprises, in mass per cental of water: 20 per mill of agricultural and forestry waste, 3 per mill of yeast powder, 0.0025 per mill of zinc sulfate, 0.01 per mill of manganese sulfate, 0.004 per mill of cobalt chloride and 1.5 per mill of calcium lignosulfonate.
According to the embodiment of the invention, the agricultural and forestry waste can be 5%, 10%, 15%, 20%, 30%, 40%, 50% or the like of the water mass.
According to the embodiment of the invention, the yeast powder can be selected to account for 2.1 permillage, 2.3 permillage, 2.5 permillage, 2.7 permillage or 2.9 permillage of the mass of the solvent water.
According to an embodiment of the present invention, zinc sulfate may be selected to be 0.0025%, 0.005%, 0.0075%, or 0.01% of the water mass.
According to the embodiment of the invention, manganese sulfate can be selected to be 0.005 permillage, 0.01 permillage, 0.015 permillage or 0.02 permillage of water mass.
According to the embodiment of the invention, cobalt chloride can be selected to be 0.001 permillage, 0.002 permillage, 0.003 permillage or 0.004 permillage of water mass.
According to the embodiment of the invention, the calcium lignosulfonate can be selected to be 0.5 permillage, 1 permillage, 1.5 permillage or 2 permillage of water mass.
According to an embodiment of the invention, the agricultural and forestry waste comprises straw powder and/or canola straw powder.
According to the embodiment of the invention, after the straw and/or rape stalk is crushed, the agriculture and forestry waste is screened by adopting a screening device with the mesh number of 40-60 meshes, and the screened product is taken as straw powder and/or rape stalk powder used by the invention. The mesh number of the sieving device may be, for example, 40 mesh, 45 mesh, 50 mesh, 55 mesh, 60 mesh, or the like.
According to an embodiment of the present invention, preferably, the agricultural and forestry waste comprises straw powder and rape straw powder.
According to an embodiment of the invention, the straw powder accounts for 20% -35% of the mass of the agricultural and forestry waste, such as 20%, 27%, 30%, 32%, 34% or 35% and the like.
According to the embodiment of the invention, the rape straw powder accounts for 65% -80% of the mass of the agricultural and forestry waste, such as 65%, 70%, 75% or 80% and the like.
The composition for culturing stropharia rugoso-annulata mother seeds provided by the invention adopts the agricultural and forestry waste straw and the rape straw, and has the advantages of wide sources, easy acquisition and low price. In addition, in the process of culturing stropharia rugoso-annulata, the growth speed of hypha can be increased, the growth is vigorous, the growth speed is increased by 25.6-68.9% compared with the formula of the composition of the comparative example, and the laccase activity is increased by about 10-30 times. In a word, the addition of the agricultural and forestry waste straw and the rape straw selected by the invention can lead the stropharia rugoso-annulata mycelium to grow more branches, the colony edge is more neat, white and thick, the growth vigor is better, the growth speed is obviously and fast, the stropharia rugoso-annulata mycelium growth promoting effect is obvious, the laccase activity in stropharia rugoso-annulata bacterial liquid can be effectively improved, and the growth promoting effect on stropharia rugoso-annulata mycelium is more obvious when the mixture of straw powder and rape straw is used as the agricultural and forestry waste according to a specific proportion.
The specific embodiment of the invention provides a culture medium for stropharia rugoso-annulata mother culture, which is prepared by adopting the composition, and the culture medium is a liquid culture medium or a solid culture medium.
According to the embodiment of the invention, the stropharia rugoso-annulata mother strain cultivated by the mother strain culture medium of the invention is inoculated on wheat grain culture medium (18 x 36cm polypropylene bags, and about 500g of wet materials are filled), and the bags can be fully grown for 15 to 20 days.
In the above culture medium, as a preferred embodiment, agar powder of 20 to 30g per mill by mass of water, for example, 20%, 22%, 25%, 27% or 30% or the like is further included in the solid medium.
The specific embodiment of the invention provides a preparation method of the culture medium, which comprises the following steps:
S1, preparing a composition solution according to the composition component proportion;
s21, preparing a composition solution into a liquid culture medium; or (b)
S22, preparing the composition solution into a solid culture medium.
The culture medium is simple to prepare, the required components are directly weighed and uniformly mixed, and the culture medium is sterilized and does not have the similar steps of decocting potatoes for 30min and taking juice.
According to the embodiment of the invention, in S1, the preparation of the composition solution according to the composition component proportion comprises the following steps:
According to the composition component proportion, the agricultural and forestry waste, the yeast powder, the zinc sulfate, the manganese sulfate, the cobalt chloride and the calcium lignosulfonate are added into water and uniformly mixed to obtain a composition solution.
According to an embodiment of the present invention, S1 is specifically, preparing a composition solution including: weighing the components according to the weight ratio, mixing uniformly, adding into a container (such as a triangular flask), adding water according to the ratio, and shaking uniformly.
According to an embodiment of the present invention, in S21, the composition solution is prepared as a liquid medium, comprising the steps of:
sterilizing the composition solution to obtain a liquid culture medium.
According to an embodiment of the present invention, in S21, the sterilization temperature is 121 ℃ and the sterilization time is 30min.
According to an embodiment of the present invention, S21 is specifically prepared by preparing a composition solution into a liquid medium, including: covering the container with a stopper, sealing with sealing film or kraft paper, and sterilizing at 121deg.C for 30min to obtain liquid culture medium.
According to an embodiment of the present invention, in S22, the composition solution is prepared as a solid medium, comprising the steps of:
uniformly mixing the composition solution with agar, and sterilizing to obtain a culture medium solution;
the culture medium solution is poured into a sterile container (e.g., a petri dish) and solidified to obtain a solid medium.
According to an embodiment of the present invention, in S22, the sterilization temperature is 121 ℃ and the sterilization time is 30min.
According to an embodiment of the present invention, in S22, the culture medium solution is poured into a sterile culture dish, and solid culture medium is obtained after solidification, specifically including: and (3) uniformly shaking the just-sterilized culture medium solution under the sterile condition while the solution is hot before solidification (at the temperature of more than 40 ℃), pouring the solution into a sterile culture dish, and obtaining the solid culture medium after solidification.
For the medium that has solidified, it may be put in a microwave oven, heated and melted, and then the medium solution is poured into a sterile container such as a petri dish, and solidified to obtain a solid medium.
The present invention will be described in further detail with reference to specific examples.
The test materials used in the following examples and comparative examples:
The culture medium or the composition of the invention is suitable for culturing various stropharia rugoso-annulata mother strains, and the test strain 04796 is adopted for the test in the examples and the comparative examples for conveniently comparing the effects, and the culture medium or the composition is preserved in the laboratory and is a high-yield strain commonly used in production.
Pulverizing straw and rape stalk with high-speed pulverizer, sieving with 40 mesh sieve, and collecting the undersize.
Screening and exploring metal elements and calcium lignosulfonate in the composition:
(1) Influence of metallic elements on growth rate of stropharia rugoso-annulata mother seeds
Preliminary screening of metallic elements
The metal element has activating or inhibiting effect on edible fungi. Screening KH2PO4(1g/l)、MgSO4(1g/l)、CaCl2(0.1g/l)、FeCl3(0.1g/l)、ZnSO4(5mg/l)、CuSO4(10mg/l)、MnSO4(10mg/l)、Na2MoO4 H20(4mg/l)、CoCl2(2mg/l) and other 9 elements, respectively adding the elements into a culture medium formed by 20g of glucose, 3g of yeast powder, 20g of agar powder and 1000ml of water, so that the final concentration of the added metal salt in the culture medium is the bracket concentration of the corresponding salt, preparing a solid culture medium containing single metal element, and repeating the steps of the solid culture medium containing each metal salt by taking no metal element as a reference.
The method for inoculating the strain on each solid culture medium containing the single metal element is as follows:
Transferring the test strain stored in the refrigerator onto a PDA plate, and culturing in dark at 25deg.C for 10 days. Under the aseptic operation condition, preparing strain blocks with the diameter of 5mm and the thickness of 2mm by using a puncher, transferring 1 strain block to the center of the solid culture medium containing the single metal element and the center of the solid culture medium which is not contained in the single metal element and is used for comparison, carrying out light-shielding constant temperature culture at 25 ℃, recording the growth condition of the 10 th measurement hypha, measuring the colony diameter by a crisscross streaking method, and calculating the growth speed. The result shows that ZnSO 4(5mg/l)、MnSO4(10mg/l)、CoCl2 (2 mg/l) of the 3 elements can promote the growth of stropharia rugoso-annulata.
Composite screening of metallic elements
According to the results, setting the final concentration of ZnSO 4 in the culture medium to be 2.5mg/l, 5mg/l and 10 mg/l; mnSO 4 with the final concentration of 5mg/l, 10mg/l and 20mg/l is adopted in the culture medium; the final concentration of the culture medium is 1mg/l, 2mg/l and 4mg/l of CoCl 2, three metal salts are added into the culture medium formed by 20g of glucose, 3g of yeast powder, 20g of agar powder and 1000ml of water according to different final concentrations, the solid culture medium containing the composite metal element is prepared, and each solid culture medium containing the composite metal element is repeated 5 times by taking no metal element as a reference. Inoculating strain blocks with diameters of 5mm and thicknesses of 2mm into solid culture mediums containing composite metal elements according to a method for inoculating strains in preliminary screening of the metal elements, culturing at 25 ℃ in a dark constant temperature, culturing for 10 days, measuring the diameters of the strains, and calculating the growth speed.
TABLE 1 influence of combined addition of three metallic elements on the growth rate of stropharia rugoso-annulata mother seeds
The results are shown in Table 1, the growth rates of the combinations No.3, 6 and 10 are relatively fast, and especially the growth rate of the combination No. 6 (ZnSO 42.5mg/l、MnSO4 10mg/l、CoCl2 mg/l) is the fastest, which is 27.7% faster than the growth rate of CK.
(2) The calcium lignosulfonate is added into the culture medium to affect the growth speed, mycelium biomass and laccase of stropharia rugoso-annulata
Effects on growth Rate
0.5G, 1g, 1.5g and 2g (namely, the mass percent of the water is respectively 0.05%, 0.10%, 0.15% and 0.20%) of calcium lignosulfonate are respectively added into a culture medium formed by 20g of glucose, 3g of yeast powder, 20g of agar powder and 1000ml of water, and the culture medium is sterilized for 30 minutes at 121 ℃ by taking the calcium lignosulfonate which is not added as a reference, so that the solid culture medium containing the calcium lignosulfonate with different concentrations is obtained. Transferring the test strain stored in the refrigerator onto a PDA plate, and culturing in dark at 25deg.C for 10 days. Under the aseptic operation condition, preparing the flat bacterial into bacterial blocks with the diameter of 5mm and the thickness of 2mm by using a puncher, transferring 1 bacterial block into the center of a solid culture medium containing calcium lignosulfonate and a comparative solid culture medium plate without calcium lignosulfonate, and culturing at the temperature of 25 ℃ in a dark and constant temperature way, wherein the solid culture medium containing calcium lignosulfonate with each concentration is repeatedly used for 3 times. The growth of hyphae was measured at10 d and colony diameters were measured by the crisscross streak method to calculate the growth rate.
Effects on hyphal biomass
0.5G, 1g, 1.5g and 2g (namely, the mass percent of the water is respectively 0.05%, 0.10%, 0.15% and 0.20%) of calcium lignosulfonate are respectively added into liquid culture mediums formed by 20g of glucose, 3g of yeast powder and 1000ml of water to obtain liquid culture mediums containing calcium lignosulfonate with different concentrations, the culture mediums are packaged into 250ml triangular flasks, 100ml of liquid culture mediums are packaged in each flask, 5 repeats of each concentration of liquid culture medium containing calcium lignosulfonate are arranged by taking the liquid culture mediums without calcium lignosulfonate as a reference, and the culture mediums are sterilized at 121 ℃ for 30min. Inoculating 20 strain blocks (namely strain blocks obtained by transferring tested strains in primary screening of metal elements to a PDA flat plate for culturing for 10 days, wherein each strain block has the dimensions of 5mm in diameter and 2mm in thickness), homogenizing, shaking and light-shielding under the conditions of 25 ℃ and 150 r.min -1 for culturing for 10 days, filtering hypha by using gauze, drying at 60 ℃, weighing the dry weight of the hypha, and taking an average value.
Effect on laccase Activity
0.5G, 1g, 1.5g and 2g (namely, 0.05 percent, 0.10 percent, 0.15 percent and 0.20 percent of water by mass) of calcium lignosulfonate are respectively added into liquid culture mediums formed by 20g of glucose, 3g of yeast powder and 1000ml of water to obtain liquid culture mediums containing calcium lignosulfonate with different concentrations, the culture mediums are packaged into triangular flasks with 250ml, each flask is filled with 100ml of liquid culture mediums, the culture mediums without calcium lignosulfonate are used as a control, 5 repetitions of each concentration of liquid culture mediums containing calcium lignosulfonate are arranged, and the culture mediums are sterilized at 121 ℃ for 30min. Under the aseptic operation condition, the strain blocks obtained after the primary screening of metal elements and transferring the tested strain to a PDA flat plate for culturing for 10 days are added into the liquid culture mediums containing calcium lignosulfonate with different concentrations, and 20 strain blocks (the size of mycelium blocks is 5mm in diameter and 2mm in thickness) are added into each bottle. Shaking and light-shielding culturing at 25deg.C under 150 r.min -1 for 10d, and centrifuging at 4deg.C under 12000g for 15min to obtain crude enzyme solution. Laccase activity was determined using ABTS method.
The experimental results are shown in tables 2 and 3.
TABLE 2 Effect of calcium Lignosulfonate on Stropharia rugoso-annulata mother seed growth rate
TABLE 3 Effect of calcium Lignosulfonate on Stropharia rugoso-annulata mycelium biomass
| Combination serial number | Additive amount | Laccase Activity (U/mL) | Hyphal biomass (g) |
| 1 | CK | 0.87 | 0.42 |
| 2 | 0.05% Calcium lignosulfonate | 1.56 | 0.56 |
| 3 | 0.10% Calcium lignosulfonate | 2.20 | 0.56 |
| 4 | 0.15% Calcium lignosulfonate | 3.19 | 0.57 |
| 5 | 0.20% Calcium lignosulfonate | 2.72 | 0.62 |
The influence of calcium lignosulfonate on the growth speed of stropharia rugoso-annulata mother seeds is shown in table 2, and 0.05-0.02% of calcium lignosulfonate can promote the stropharia rugoso-annulata mother seeds to grow, wherein 0.05%, 0.10% and 0.15% of calcium lignosulfonate are added to promote the stropharia rugoso-annulata mother seeds more obviously, and the three dosage effects are close.
The effect of calcium lignosulfonate on mycelium biomass and laccase activity of stropharia rugoso-annulata is shown in table 3, and the addition of 0.15% of calcium lignosulfonate has the most obvious promotion, and compared with the control, the biomass is increased by 35.5%, and the laccase activity is increased by 266%. When 0.20% calcium lignosulfonate was added, the biomass increased by 46.3% and laccase activity increased by 212.8%.
At a relatively high speed, the mycelium biomass is measured more accurately than the diameter measured in a flat plate during liquid culture, so that the mycelium growth amount and laccase activity are comprehensively considered, and 0.15% of calcium lignosulfonate is selected and added into a culture medium, so that the growth of stropharia rugoso-annulata is promoted obviously.
The following are preparation examples of the medium of the present invention and preparation comparative examples of the comparative medium.
Example 1:
Preparing a liquid medium and a solid medium, comprising:
Liquid medium: 20g of rice straw with granularity of-40 meshes, 10g of rape straw, 3g of yeast powder, 2.5mg of ZnSO 4,10mg MnSO4,4mg CoCl2, 1.5g of calcium lignosulfonate, 1L of water, 100mL of each bottle of a 250mL triangular flask, and sterilizing to obtain a liquid culture medium (Z1), wherein the sterilization temperature is 121 ℃ and the sterilization time is 30min.
In the above liquid medium, 20g of agar powder was added per L of liquid medium, sterilized, and then packaged into sterilized dishes to prepare a solid medium (Z1).
Examples 2 to 7:
The procedure for preparing the liquid medium and the solid medium was the same as in example 1, except that the amounts of straw powder and canola straw powder were added in amounts of 6.67g and 13.3g (example 2) (Z2), 13.3g and 6.67g (example 3) (Z3), 5g and 15g (example 4) (Z4), 15g and 5g (example 5) (Z5), 4g and 16g (example 6) (Z6), 16g and 4g (example 7) (Z7), respectively.
Examples 8 to 13:
The procedure for preparing the liquid medium and the solid medium was the same as in example 1, except that the agronomic waste added to the medium was only straw powder, wherein the straw powder added in example 8 was 5g (D1), the straw powder added in example 9 was 10g (D2), the straw powder added in example 10 was 20g (D3), the straw powder added in example 11 was 30g (D4), the straw powder added in example 12 was 40g (D5), and the straw powder added in example 13 was 50g (D6).
Examples 14 to 19
The procedure for preparing the liquid medium and the solid medium was the same as in example 1, except that the agronomic waste added to the medium was only canola straw powder, wherein 5g (Y1) of canola straw powder was added in example 14, 10g (Y2) of canola straw powder was added in example 15, 20g (Y3) of canola straw powder was added in example 16, 30g (Y4) of canola straw powder was added in example 17, 40g (Y5) of canola straw powder was added in example 18, and 50g (Y6) of canola straw powder was added in example 19.
Comparative example 1:
Liquid medium: difco TM PDB 24g, adding water to a volume of 1L, using 250mL triangular flask, and filling 100mL of liquid culture medium per flask; comparative example 1 solid medium: difco TM PDA 39g, agar 10g, and water was added to a volume of 1L.
Comparative example 2:
comparative example 2 liquid medium: 200g of potato, 50g of trehalose, and adding water to fix the volume to 1L; this medium was supplemented with 20g of agar powder, i.e., the solid medium of comparative example 2.
Comparative example 3:
comparative example 3 liquid medium: 200g of potato, 25g of sucrose, 4g of corn flour, 1g of peptone and 6-BA0.5mg of peptone, and adding water to fix the volume to 1L; this medium was supplemented with 20g of agar powder, i.e., the solid medium of comparative example 3.
Comparative example 4:
Comparative example 4 liquid medium: 200g of potato, 20g of glucose and 1.5g of KH 2PO4 3g、MgSO4, and adding water to fix the volume to 1L; this medium was supplemented with 20g of agar powder, i.e., the solid medium of comparative example 4.
The potato treatment operation described above is as follows: cutting 200g peeled potato into small pieces of about 1cm, placing into a pot containing 1L water, heating to boil, adjusting fire, and boiling for 30min. Filtering with 8 layers of gauze to obtain juice, adding other components into the filtrate, and adding water to obtain 1L. For solid medium, 400mL of medium was added to a 500mL flask; for liquid medium, 100mL of medium was added to a 250mL Erlenmeyer flask.
Experimental example 1: long-term comparison of Stropharia rugoso-annulata cultured for 10 days in the culture medium of the example and the comparative example of the present invention
The test process comprises the following steps:
Transferring the strain stored in the refrigerator onto a PDA plate, and culturing the strain for 10 days at 25 ℃ in dark place, wherein the thickness of the plate culture medium is about 2mm. Under aseptic operation conditions, a plate strain is made into strain blocks with the diameter of 5mm and the thickness of 2mm by using a puncher, 1 strain is transferred to the center of the solid culture medium plates of the solid culture mediums of examples 1-19 and comparative examples 1-4, and the culture medium plates are cultivated at the temperature of 25 ℃ in a dark state and at the constant temperature, and 3 times of culture mediums are repeated. Growth of the 10d hyphae was recorded and colony diameter was measured by the crisscross streak method, and growth rate was calculated as an average of a plurality of replicates.
The test results are shown in table 4:
TABLE 4 Change speed of Stropharia rugoso-annulata 04796 strains on different stock formulas
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As shown in fig. 1-5 and table 4: compared with the stropharia rugoso-annulata mycelia (control group) of comparative examples 1,2, 3 and 4, the stropharia rugoso-annulata mycelia of example 1= -example 19 have more branches, more regular colony edges, white and dense colonies, better growth vigor and obviously faster growth speed, and the formula of the culture medium in the embodiment of the invention has obvious promotion effect on the growth of stropharia rugoso-annulata mycelia, especially when the total consumption of straw powder and rape straw powder is controlled to be more than 10 per mill of the mass of water in the culture medium, more preferably to be more than 20 per mill, and even more preferably, the straw powder and the rape straw powder are mixed for use. As can be seen from Table 4, in example 6, the speed is the fastest when 4g of straw powder and 16 g of rape straw powder are added, and the speed is increased by 29.6 to 68.9 percent compared with the comparative example; secondly, in the embodiment 4, 5g of straw powder and 15 g of rape straw powder are added, and the amount is increased by 26.3 to 61.8 percent compared with the comparative example; again, in example 2, 6.67 g of straw powder and 13.33 g of rape straw powder are added, which is increased by 25.6-60.8% compared with the comparative example.
Thus, it can be seen that the addition of straw powder: 6.67 g of rape straw, 13.3 g (Z2), 5g of 15g (Z4) and 4g of 16g (Z6), the colony diameter is larger, preferably the 3 proportions, namely 4-7 g of the rice straw is added per liter of culture medium, 13-16 g of rape is added, and the stropharia rugoso-annulata grows very vigorously.
Experimental example 2: laccase Activity comparison of Stropharia rugoso-annulata cultured for 10 days on the inventive example medium and the comparative example medium
Test procedure
Under aseptic conditions, the seed blocks of experiment 1 with a diameter of 5mm and a thickness of 2mm, namely the seed blocks cultured for 10d on the PDA plate of experiment 1, were added to the liquid culture media of example 16, example 10, example 6, example 4, example 2 and comparative examples 1-4, and 20 seed blocks were added per bottle, and 3 replicates were set. Shaking and light-shielding culturing at 25deg.C under 150 r.min -1 for 10d, and centrifuging at 4deg.C under 12000g for 15min to obtain crude enzyme solution. Laccase activity was determined using ABTS method.
The test results are shown in Table 5:
TABLE 5 laccase Activity comparison of Stropharia rugoso-annulata 04796 Strain on different formulations
As can be seen from Table 5, the laccase activities of the stropharia rugoso-annulata liquid (experimental group) of the examples and the stropharia rugoso-annulata liquid laccase activities of the comparative examples 1, 2,3 and 4 (control group) have significant differences, and the laccase activities of the examples reach 219-310.247U/mL; and the laccase activity of the comparative example is 0.549-16.272U/mL. Wherein the laccase in example 10 has the highest laccase activity of 310.247U/mL, which is 19 times more than that in comparative examples 2 and 3 with higher activity, and 565 times that in comparative example 4 with the lowest activity.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A composition for stropharia rugoso-annulata mother culture, which is characterized by comprising water and material components, wherein the material components comprise, in terms of mass per thousandth of water:
5 to 50 per mill of agricultural and forestry waste, 2 to 3 per mill of yeast powder, 0.0025 to 0.01 per mill of zinc sulfate, 0.005 to 0.02 per mill of manganese sulfate, 0.001 to 0.004 per mill of cobalt chloride and 0.5 to 2 per mill of calcium lignosulfonate;
The agricultural and forestry waste comprises straw powder and/or rape straw powder.
2. The composition according to claim 1, wherein the agricultural and forestry waste is present in an amount of 10 to 50% by mass per mill of water.
3. A composition according to claim 2, wherein,
The agricultural and forestry waste comprises straw powder and rape straw powder, wherein the straw powder accounts for 20% -35% of the mass of the agricultural and forestry waste; the rape straw powder accounts for 65-80% of the mass of the agricultural and forestry waste.
4. A composition according to any one of claims 1 to 3, wherein the material components comprise, in mass per thousandth of water:
15 to 25 per mill of agricultural and forestry waste, 3 per mill of yeast powder, 0.0025 to 0.005 per mill of zinc sulfate, 0.005 to 0.01 per mill of manganese sulfate, 0.001 to 0.004 per mill of cobalt chloride and 1 to 2 per mill of calcium lignosulfonate.
5. The composition of claim 4, wherein the material components comprise, in mass per thousandth of water: 20 per mill of agricultural and forestry waste, 3 per mill of yeast powder, 0.0025 per mill of zinc sulfate, 0.01 per mill of manganese sulfate, 0.004 per mill of cobalt chloride and 1.5 per mill of calcium lignosulfonate.
6. A culture medium for stropharia rugoso-annulata mother culture prepared by using the composition of any one of claims 1-5, wherein the culture medium is a liquid culture medium or a solid culture medium.
7. The culture medium according to claim 6, wherein the solid culture medium further comprises agar powder accounting for 20-30 per mill of the water mass.
8. A method of preparing the culture medium of claim 6 or 7, comprising the steps of:
Adding agricultural and forestry waste, yeast powder, zinc sulfate, manganese sulfate, cobalt chloride and calcium lignosulfonate into water according to the component proportion of the composition according to any one of claims 1-5, and uniformly mixing to obtain a composition solution;
preparing the composition solution into a liquid culture medium; or (b)
The composition solution is prepared as a solid medium.
9. The method according to claim 8, wherein,
Preparing the composition solution into a liquid culture medium, comprising the following steps:
sterilizing the composition solution to obtain the liquid culture medium.
10. The method according to claim 8, wherein,
Preparing a composition solution into a solid medium, comprising the following steps:
mixing the composition solution with agar, and sterilizing to obtain culture medium solution;
pouring the culture medium solution into a sterile container, and solidifying to obtain the solid culture medium.
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