AU2020103076A4 - Method of preparing Lepista sordida culture spawn with mushroom residue - Google Patents
Method of preparing Lepista sordida culture spawn with mushroom residue Download PDFInfo
- Publication number
- AU2020103076A4 AU2020103076A4 AU2020103076A AU2020103076A AU2020103076A4 AU 2020103076 A4 AU2020103076 A4 AU 2020103076A4 AU 2020103076 A AU2020103076 A AU 2020103076A AU 2020103076 A AU2020103076 A AU 2020103076A AU 2020103076 A4 AU2020103076 A4 AU 2020103076A4
- Authority
- AU
- Australia
- Prior art keywords
- culture
- lepista sordida
- spawn
- residue
- formula
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- 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
- A01G18/00—Cultivation of mushrooms
-
- 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
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Mushroom Cultivation (AREA)
Abstract
The present invention discloses a culture material for Lepista sordida culture spawn.
The culture material for Lepista sordida culture spawn consists of the following
components in percentage by weight: 38% of chicken manure, 35% of Flammulina
velutipes residue, 25% of wheat straw, 1% of gypsum and 1% of calcium carbonate.
Meanwhile, the present invention discloses a method for cultivating Lepista sordida
culture spawn by the Lepista sordida culture material. The Flammulina velutipes
residue are added into the culture material of the Lepista sordida culture spawn, so
that the nutrition of a culture medium is increased, the vitality of the strain is
enhanced, a hypha has a strong germination force and grows rapidly, and a mycelium
grows densely and stoutly. Compared with the traditional culture spawn, the culture
material provided by the invention has rich and stable raw material sources and low
cost, the culture spawn prepared from the culture material is rich in extracellular
enzyme system, rapid in germination, high in feeding ability and easy to promote and
popularize, and the problem about efficient recycling of the Flammulina velutipes
residue is solved well.
Description
METHOD OF PREPARING Lepista SORDIDA CULTURE SPAWN WITH
The present invention belongs to the field of biotechnology, and in particular,
relates to a method of preparing Lepista sordida culture spawn with mushroom
residue.
In recent years, with the improvement of people's living standard and the popular support of the healthy diet concept, the market demand of the edible mushroom has been rising continuously. High-grade rare edible mushroom varieties are favored by people, with price being several times, even dozens of times that of the traditional numerous edible mushroom and with considerable profits. Therefore, people have gradually realized that it is of great significance in conducting scientific research on edible mushroom cultivation, especially on high-grade rare edible mushroom cultivation. Lepista sordida, as a rare edible mushroom variety, has great development potential. The Lepista sordida taxonomically belongs to Basidiomycota, Agaricomycetes, Agaricales, TricholomataceaeLepista. In the fruiting body of the fresh Lepista sordida, the pileus is flat hemispherical and is purple or pinkish purple, and the stipe is cylindrical and is light purple or dusty pink. The Lepista sordida has delicious taste and attractive color and luster, and has very high medicinal value and nutritional value, but the wild Lepista sordida strains are rare, difficult to cultivate and very precious. As early as 2000, there were a few reports about the Lepista sordida in Fujian, Yunnan and the like in China, but most of them was still in the primary stage of research; and only a small scale of successful cultivation reports occurred, but still showed the main problems of low biotransformation rate, even no fruiting. Therefore, the research on artificial cultivation of the Lepista sordida strain is an urgent problem to be solved at present, and the selection of the formula of the Lepista sordida culture spawnawn is the key to solve the problem. The Lepista
sordida belongs to a straw rotting fungus, and macromolecular substances such as cellulose, hemicellulose, lignin, starch and the like mainly serve as the carbon source in the growth process; therefore, whether the Lepista sordida culture spawn can secrete rich extracellular enzyme systems capable of degrading the macromolecular substances as well as the level of the enzyme activity directly affect the germination and feeding ability of the hypha in the later period, which is crucial to the cultivation of the Lepista sordida. Flammulina velutipes has become an indispensable part of people's daily diet due to its advantages of high protein, low fat, low energy, rich mineral elements, vitamin and the like. After entering the 1980s, China has developed mushroom rapidly and become a superpower of mushroom. Flammulina velutipes is the mushroom species with the highest industrialized production degree in China. A huge number of Flammulina velutipes residue with stable source and controllable quality are produced continuously every day. With the development of Flammulina velutipes, the Flammulina velutipes residue, namely the waste after cultivation of the Flammulina velutipes, is also increasing, and there are millions of tons of Flammulina velutipes residue produced in China every year. Since most of the cultivators don't know much about the nutritional value of the mushroom residue, the mushroom residue of the Flammulina velutipes are often discarded or burned everywhere, or concentrated in the fields of feed and fertilizer, and diversified utilization of the mushroom residue cannot be realized. The mushroom residue still contain a large number of mycelia and rich nutritional substances, and mycetes and pests are easily reproduced and proliferated in the mushroom residue, so on one hand, resources are greatly wasted. On the other hand, the growth of the mycetes and pests will inevitably increase the number of the mycotic spores and pests in the air, resulting in air pollution. Therefore, we have innovatively researched the formula of the Flammulina velutipes residue-containing culture material for the Lepista sordida culture spawn, so as to open up a new path for the artificial cultivation of the high-grade rare mushroom and the efficient utilization of the Flammulinavelutipes residue.
To solve the above problems, the present invention discloses a culture material for Lepista sordida culture spawn. The culture material for Lepista sordida culture spawn consists of the following components in percentage by weight: 38% of chicken manure, 35% of Flammulina velutipes residue, 25% of wheat straw, 1% of gypsum and 1% of calcium carbonate. Meanwhile, the present invention discloses a method for cultivating Lepista sordida by the culture material. A certain theoretical basis is provided for further exploring the formula of high-yield cultivation, reasonable cultivation management and efficient utilization of the Flammulinavelutipes residue. The present invention is implemented by the following technical solutions: a Lepista sordida culture material consists of the following components in percentage by weight: 15-45% of chicken manure, 10-50% of Flammulina velutipes residue, 10- 3 0 % of wheat straw, 0.5- 3 .5 % of gypsum and 0.5- 3 % of calcium carbonate. Preferably, the Lepista sordida culture material consists of the following components in percentage by weight: 38% of chicken manure, 35% of Flammulina velutipes residue, 2 5 % of wheat straw, 1% of gypsum and 1% of calcium carbonate. A method for cultivating Lepista sordida culture spawn by the Lepista sordida culture material includes the following steps: (1) prewetting: weighing the chicken manure, the Flammulinavelutipes residues and the wheat straws according to a formula, adding water according to a material-to-water ratio of 1:1.2 to 1:1.4, and soaking and prewetting with limewater with a mass fraction of 1-2% for 12-36 h in advance, wherein the Flammulina velutipes residue are crushed into granules with a grain diameter of 0.2-0.7 m after being dried in the sun, and the wheat straws are cut into small sections with a length of 1-4 cm; (2) bagging: accurately weighing the calcium carbonate and the gypsum according to the formula, dissolving the calcium carbonate and the gypsum with water and stirring uniformly, adding into other prewetted culture materials, adjusting
the water content of a culture medium to 5 5 -6 5 %, adjusting the pH value of the culture medium to pH7.5-pH9, and charging with a polypropylene bag with a specification of 17cm*33cm, wherein 1000 g of wet materials are charged in each bag; (3) sterilization and cooling: maintaining an outer wall of the fungal bag filled with the culture material clean, fastening a plastic cover with a ventilation filter membrane and sealing, conducting steam sterilization under high pressure at 121°C for 2 h, cooling to below 25°C and then conducting inoculation; (4) inoculation: transferring the Lepista sordida strain to an improved PDA culture plate and conducting standing culture in an incubator at 25°C for 6-10 d under a dark condition to obtain an activated plate strain; selecting 8 to 10 activated Lepista sordida strain blocks (each block is 0.5cm2 ), inoculating into a liquid culture medium, conducting shake culture in a shaking table at a temperature of 25°C to 26°C and at a rotating speed of 120 r/min for 2 d, adjusting the temperature of the shaking table to 24°C to 25°C and the rotating speed to 130-150 r/min, continuously cultivating for 4-5 d to obtain a liquid strain; conducting sterile operation, inoculating 20 mL of Lepista sordida strain into each bag, and adding into the Lepista sordida culture material containing Flammulina velutipes residue formula which is sterilized and cooled in the step (3); and (5) cultivation: adjusting the constant-temperature and constant-humidity incubator, and conducting cultivation for 50-60 d by controlling the temperature to 24°C to 25°C and the humidity to 5 5 -6 5 % to obtain the Lepista sordida culture spawn. Preferably, a formula of the improved PDA culture plate is: 20% of potato, 1% of glucose, 1% of sucrose, 0.2% of yeast powder and 2% of agar. Preferably, a formula of the liquid culture medium is: 1% of corn residue, 1% of glucose, 1% of maltose, 0.2% of peptone, 0.3% of yeast powder, 0.1% of monopotassium phosphate and 0.05% of magnesium sulfate heptahydrate. The Lepista sordida culture spawn prepared by the method may be transferred to a culture bag to conduct Lepista sordida fruiting cultivation.
The Lepista sordida culture spawn prepared by the method may be directly subjected to Lepista sordida fruiting cultivation after being covered with soil. Beneficial Effects: The Flammulina velutipes residue is added into the culture material of the Lepista sordida, so that the nutrition of a culture medium is increased, the vitality of the strain is enhanced, a hypha has a strong germination force and grows rapidly, and a mycelium grows densely and stoutly. Compared with the traditional culture spawn, the culture material provided by the invention has rich and stable raw material sources and low cost, the culture spawn prepared from the culture material is rich in extracellular enzyme system, rapid in germination, high in feeding ability and easy to promote and popularize, and the problem about efficient recycling of the Flammulina velutipes residue is solved well. The residue of mushroom medium is the culture medium remained after fresh mushroom is harvested when the mushroom is cultivated by the leftovers of subsidiary agricultural products such as straws, wood dust and the like, and is a compound of mushroom hypha residue and crude fiber which is subjected to enzymatic hydrolysis by the mushroom and qualitative change in structure. The mushroom residue is rich in protein, polysaccharide and other nutritional ingredients, which are very suitable for the growth of the hyphae. When the formula into which % of Flammulinavelutipes residue is added serves as a culture medium, the culture medium is particularly suitable for the cultivation of the Lepista sordida and can obviously promote the growth of the Lepista sordida. Through measurement of enzymatic activity, the enzymatic activity of four enzymes in the Lepista sordida strain is the highest compared with those of the conventional culture material formula and other mushroom dreg culture material formulas. The enzymatic activity of carboxymethycellulose (CMC) of the Lepista sordida strain is up to 1.00 U, the hemicellulase activity of the Lepista sordida is 1.78 U, the enzymatic activity of the amylase is up to 1.58 U, and the enzymatic activity of the laccase is up to 0.90 U. Therefore, the formula (38% of chicken manure, 35% of Flammulina velutipes residue, 25% of wheat straw, 1% of gypsum and 1% of calcium carbonate) may be
proved to be the optimal culture medium from three aspects such as the growth condition of the hypha, the growth rate of the hypha and the activity of the extracellular enzyme. It is of great significance in solving the defects of low yield, high cost, long cycle and the like in the artificial domesticated cultivation of the Lepista sordida in the future. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a glucose determination standard curve;
FIG. 2 shows a xylose determination standard curve;
FIG. 3 shows comparison of the growth rate of hypha of Lepista sordida in
different cultivation time;
FIG. 4 shows comparison of the growth rate of Lepista sordida with different
culture spawn formulas;
FIG. 5 shows comparison of the enzymatic activity of CMC of Lepista sordida
with different culture spawn formulas;
FIG. 6 shows comparison of hemicellulase enzymatic activity of Lepista sordida
culture spawn with different culture spawn formulas;
FIG. 7 shows comparison of amylase activity of Lepista sordida culture spawn
with different culture spawn formulas; and
FIG. 8 shows comparison of laccase activity of Lepista sordida culture spawn
with different culture spawn formulas.
Embodiments of the present invention are described below in detail. The
embodiments are implemented on the premise of the technical solution of the present
invention, and the detailed implementation manner and the specific operation process
are given, but the protection scope of the present invention is not limited to the
following embodiments.
Embodiment 1
1 Materials and Methods
1.1 Materials
1.1.1 Tested strains The Lepista sordida (LS) strain was separated and preserved by Shandong Provincial Key Laboratory of Applied Mycology, Qingdao Agricultural University.
1.1.2 Drugs and reagents Table 1 Test Drugs Drug Name Production Place Gypsum Available in the market Fermented chicken manure Available in the market Wheat straw Available in the market 2-hydroxypropane-1,2,3-tricarboxylic acid Tianjin Biochemical Co., Ltd. (China) Potassium sodium tartrate Tianjin Biochemical Co., Ltd. (China) Glucose Tianjin Biochemical Co., Ltd. (China) Absolute ethyl alcohol Qingdao Ruijinte Technology Limited Liability Company Glacial acetic acid Qingdao Ruijinte Technology Limited Liability Company Anhydrous sodium acetate Chongqing Xinke Reagent Co., Ltd. 3.5-dinitrosalicylic acid Shandong Laiyang Kangde Chemical Co., Ltd. (USA) Phenol Tianjin Standard Science and Technology Limited Liability Company (China) Xylan Shanghai Sangon Biotechnology Co., Ltd. Anhydrous sodium sulfite Tianjin Bodi Chemical Co., Ltd. (China) ABTS Shanghai Fumin Chemical Reagent Factory Co., Ltd. (China) Calcium Carbonate Shandong Damao Chemical Reagent Factory Flammulinavelutipes residue Provided by Gaomi Huide Agricultural Product Co., Ltd. Pleurotuseryngii residue Provided by Shandong Baoyi Biotechnology Limited Liability Company Lentinus edodes residue Provided by Shandong Qihe Biotechnology Limited Liability Company
1.1.3 Formula of tested culture material Table 2 Culture spawn Formula Design Serial Number Formula A chicken manure 63% wheatstraw35% gypsum11% calcium carbonate 1%
DESCRIPTION B chicken manure 31% cornstraw67% gypsum% calcium carbonate 1% C chicken manure 38% wheatstraw25% gypsum% calcium carbonate 1% Flammulina velutipes residue 35% D chicken manure 38% wheatstraw25% gypsum% calcium carbonate 1% Lentinus edodes residue 35% E chicken manure 38% wheatstraw25% gypsum% calcium carbonate 1% Pleurotus eryngii residue 35%
1.2 Reagent preparation
1.2.1 Preparation of DNS reagent 1 L of distilled water was added into a flask, a water bath kettle was turned on at the same time, the temperature was adjusted to below 45C, and the flask was put into the water bath kettle. 12.6 g of dinitrosalicylic acid and 370 g of potassium sodium tartrate was accurately weighed by a balance and was sequentially added into the flask, 524 ml of NaOH with the concentration of 2 mol/L was measured and added into the flask, and 10 g of phenol and 10 g of sodium sulfite were added into the flask. Drugs were completely dissolved by a magnetic stirrer. Finally, distilled water was supplemented to 2 L after the temperature decreased.
1.2.2 Preparation of ABTS reagent Preparation of an ABTS solution of 0.5 mmol/L: 0.13 g of drug ABTS was accurately weighed by an analytical balance. The drug ABTS was added into a 2-hydroxypropane-1,2,3-tricarboxylic buffer solution with a pH of 4.0 which was prepared in advance, stirring was conducted while adding, and finally a volume was determined to 1 L after the drug was completely dissolved.
1.2.3 Preparation of buffer solution
1.2.3.1 Preparation of citric acid buffer solution with a pH of 4.8 84.4 g of 2-hydroxypropane-1,2,3-tricarboxylic acid and 56.8 g of NaH 2PO 4 were accurately weighed by an analytic balance and were poured into a flask, and a certain amount of distilled water was added, wherein it is necessary to continuously stir drugs in this process. Finally, the drug was completed dissolved, a volume was determined to 2 L, and liquid A and liquid B were respectively filled in two reagent bottle cups.986 mL of liquid A and 1014 mL of liquid B were measured accurately
DESCRIPTION and were mixed uniformly to obtain the buffer solution.
1.2.3.2 Preparation of citric acid buffer solution with a pH of 4.0
21.lg of citric acid and 28.4 g of NaH 2PO 4 were accurately weighed by an
analytic balance and were poured into a flask, wherein it is necessary to continuously
stir drugs in this process so as to prepare a uniform solution-drug complete solution.
Finally, distilled water was added to a constant volume of 1 L, and liquid A and liquid
B were respectively filled in two reagent bottle cups. 385.5 mL of liquid A and 614.5
mL of liquid B were measured accurately and were completely mixed uniformly.
1.2.3.3 Preparation of acetic acid buffer solution with a pH of 4.6 4.1 g of anhydrous sodium acetate was accurately weighed by an analytic balance, an acetic acid solution with a concentration of 0.2 mol/L was measured to dissolve drugs, and the acetic acid solution of 0.2 mol/L was added into a volumetric flask until a volume is accurately determined to 500 mL.
1.2.3.4 Preparation of xylan solution of 10 mg/mL 5 g of xylan was accurately weighed by an analytic balance, and an acetic acid solution was added while stirring, wherein it is necessary to continuously stir drugs in this process so as to prepare a uniform completely dissolved solution drug. Finally, a volume was accurately determined to 500 mL. 1.2.3.5 Preparation of sodium carboxymethylcellulose (CMC-Na) solution of 1% 5 g of sodium carboxymethylcellulose was accurately weighed by an analytic balance for future use, the acetic acid buffer solution which was prepared in advance in 1.2.3.3 was measured, the acetic acid buffer solution was added while stirring to completely dissolve the sodium carboxymethylcellulose, and finally, the acetic acid buffer solution was added into a volumetric flask until a volume was determined to 500 mL.
1.3 Method for observing character of hypha and measuring growth rate of hypha 1.3.1 Preparation of culture spawn
Prewetting: 7500 g of chicken manure, 1500 g of Flammulinavelutipes residue, 1500 g of Lentinus edodes residue, 1500 g of Pleurotus eryngii residue, 4700 g of wheat straw and 3000 g of corn straw were accurately weighed according to a formula. The wheat straws and the corn straws were cut into sections with a length of 1-4 cm, and the Flammulina velutipes residue, the Lentinus edodes residue and the Pleurotus eryngii residue were dried in the sun and crushed. The materials were soaked and prewetted according to a material-to-water ratio of 1:1.2 to 1:1.3 for 24 h in advance. Bagging: the water absorbing condition of a culture material was recorded, the culture material was weighed and a water content was calculated, bagging was conducted after the culture material absorbed water completely, calcium carbonate and gypsum which were weighed and dissolved in advance were stirred uniformly and added into the culture material, and a water content of a culture medium was adjusted to be about 60%. The pH was adjusted and controlled to 8.5. The materials were charged by a polypropylene bag with a specification of 17cm*33cm and weighing was conducted after the mushroom dregs are charged, so that 1000 g of wet materials were charged in each bag. Sterilization and cooling: an outer wall of the fungal bag filled with the culture material was maintained clean, a plastic cover with a ventilation filter membrane was fastened and sealed, sterilization was conducted under high pressure at 121°C for 2 h,
cooling was conducted to below 25°C and then inoculation was conducted.
Inoculation: a tested strain was transferred to an improved PDA culture plate, and standing culture was conducted in an incubator at 25°C under a dark condition to
obtain an activated plate strain. 8 to 10 activated Lepista sordida strain blocks (each block is 0.5cm 2) were selected and inoculated into a liquid culture medium, and shake culture was conducted in a shaking table at a temperature of about 25°C and at a
rotating speed of about 120 r/min for 6-8 d to obtain a liquid strain. Sterile operation was conducted, and 20 mL of Lepista sordida strain was inoculated into each bag. Cultivation: a temperature of a constant-temperature and constant-humidity
incubator was controlled to about 25°C, and a humidity was controlled to about 60%.
The inoculated Lepista sordida culture spawn were put into the incubator and were subjected to light-tight cultivation, and germination and growth conditions were observed and recorded regularly every day. 1.3.1.4 Measurement of growth condition of hypha The growth vigour and the color of the hypha were recorded regularly every day, and finally, the number of overgrown days of the hypha was recorded. The growth vigour of the hypha is divided into three grades: dense, relatively dense and relatively sparse, and the color of the hypha is divided into three grades: pinkish purple, light purple and relatively white. After the hyphae were full of the material surface, the growth condition of the hypha was measured every other 10 d, and the growth rate of the hypha was calculated. The growth rate of the hypha (cm/d)= the growth depth of the hypha (cm)/cultivation time (d).
1.4Specific steps of measuring enzymatic activity of four extracellular enzymes of
Lepista sordida
1.4.1 Measurement of activity of laccase
A crude enzyme solution was diluted with the buffer solution prepared in
1.2.3.2, 3 mL of diluted crude enzyme solution was added into a test tube, 2 mL of
ABTS of 0.5 mmol/L was added to determine a volume to 10 mL, a water bath kettle
was turned on at 30°C, and reagents used in the experiment were put in the water bath
kettle at 30°C. The ABTS solution and the diluted crude enzyme solution were mixed
uniformly, enzymatic hydrolysis reaction was started, a light absorption value was
measured under 420 nm, and the change condition of an OD value of reaction liquid
was recorded every 3 min, wherein it is necessary to record 6 data in the enzymatic
hydrolysis process.
A calculation formula of the enzymatic activity of the laccase:
U/L=A*V*10^6/(sLtv)
A: a difference value between two consecutive light absorption values;
V: a total volume of a reaction system;
P=3.6*10^4(mol/L)-1cm-1;
L: a diameter of a cuvette;
t: reaction time;
v: sample volume;
1.4.2 Measurement of enzymatic activity of carboxymethycellulose
A carboxymethycellulose solution of 1% was added into a test tube to dilute a
crude enzyme solution, 1 mL of diluted crude enzyme solution was put into the test
tube, a water bath kettle was turned on, a temperature was adjusted to 40°C, water
bathing was conducted for 30 min, 1.5 mL of 3.5-dinitrosalicylic acid reagent was
added into the test tube, the temperature of the water bath kettle was adjusted to
100°C, water bathing was conducted for 5 min, distilled water was added to
determine a volume to 12.5 mL after cooling was conducted to a room temperature,
and a light absorption value was measured under 540 nm, wherein an inactivated
diluted crude enzyme solution serves as blank control. The glucose content obtained
by degrading sodium carboxymethylcellulose with the carboxymethycellulose was
calculated according to a glucose standard curve measured by DNS, wherein the
enzymatic activity is defined as the amount of enzyme required to produce Ipmol of
glucose in one minute, U=1pmol/(min.mL enzyme)*dilution ratio.
1.4.3 Measurement of enzymatic activity of hemicellulase
A crude enzyme solution was diluted by an appropriate multiple, 0.25 mL of
crude enzyme solution was added into a test tube, 0.75 mL of xylan (10 mg/mL) was
put into the test tube, a water bath kettle was turned on, a temperature was adjusted to
°C, enzymatic hydrolysis was conducted in water bath for 30 min, 1.5 mL of
3.5-dinitrosalicylic acid reagent was added, the temperature of the water bath kettle
was adjusted to 100°C, boiling water bathing was conducted for 5-10 min, cooling
was conducted to a room temperature, a volume was determined to 12.5 mL, and an
value A was measured under 540 nm, wherein an inactivated crude enzyme solution
serves as control. The enzymatic activity is defined as the amount of enzyme required
to produce 1pmol of xylose in one minute, U=pmol/(min.mL enzyme)*dilution
ratio. An enzymatic activity formula is: U=S*D*1000/150/30/L. S: the measured amount of xylose corresponding to light absorption on a standard curve, mg; D: dilution ratio of enzyme solution;
1000: a conversion coefficient between mg and pg;
150: a molecular weight of xylose, g/mol;
30: enzymatic hydrolysis time;
L: absorbed enzyme solution volume. 1.4.4 Measurement of activity of amylase 950 pL of soluble starch of 1% and 50pL of crude enzyme solution were taken
by a pipette and were added into a test tube, the temperature of a water bath kettle
was adjusted to 50°C, the test tube was put into the water bath kettle, 1.5 mL of
3.5-dinitrosalicylic acid reagent was added at once after water bath heat preservation
was conducted for 30 min, the temperature of the water bath kettle was adjusted to
100°C, boiling water bathing was conducted for 5 min, the test tube was taken out
and cooled to room temperature, dilution was conducted with distilled water to
determine a volume to 12.5 mL, and a light absorption value was measured under 540
nm. A reducing sugar standard curve was measured by 3.5-dinitrosalicylic acid, and
the content of glucose obtained by decomposing the soluble starch by the amylase
was calculated. 1 enzymatic activity unit is defined as the amount of enzyme required
to produce Ipmol of xylose in each minute. Three repeated samples were prepared
for each sample, and a crude enzyme solution inactivated by boiling water bath
serves as control. The enzymatic activity is defined as the amount of enzyme required
to produce 1pmol of glucose in one minute, U=1pmol/(min.mL enzyme)*dilution
ratio. An enzymatic activity calculation formula: U=S*D*1000/180/30/L (S is the
content of glucose on the standard curve, D is dilution ratio, 180 is the molecular
weight of the glucose, 30 is the enzymatic hydrolysis time, and L is sample volume).
1.5 Preparation of reducing sugar standard curved measured by DNS
1.5.1 Glucose standard curve
250 mL of standard glucose solution (1 mg/L) was prepared. Glucose standard
solution with different gradients were accurately taken by a pipette and were added
into a test tube, a volume was determined with distilled water to 1 mL, a water bath
kettle was turned on, the temperature was adjusted to 100°C, 1.5 mL of DNS reagent
which was prepared one week in advance was added and was subjected to water
bathing for 10 min, the test tube was taken out and cooled, distilled water was added
to accurately determine a volume of the solution in the test tube to 12.5 mL after the
temperature is reduced to room temperature, mixing was conducted uniformly, a
wavelength of a spectrophotometer was adjusted to 540 nm, the spectrophotometer
was turned on in advance and preheated for 30 min, a light absorption value of each
gradient was measured by taking the distilled water as blank control, the glucose
standard curve was drawn by taking a glucose concentration (mg/mL) as an
x-coordinate and a light absorption value as a y-coordinate, and a linear formula was
obtained (FIG. 1).
1.5.2 Xylose standard curve
250 mL of standard xylose solution (10mg/L) was prepared. Glucose standard
solutions with different gradients were added into a 4-mL centrifugal tube, a volume
was determined by distilled water to 2.5 mL, mixing was conducted uniformly, 0.25
mL of xylose solution was added into each test tube, 0.75 mL of prepared disodium
hydrogen phosphate-citric acid with a pH of 4.8 was added into the test tube, 1.5 mL
of DNS reagent was added into the test tube, a water bath kettle was turned on in
advance, the temperature was adjusted to 100°C, water bathing was conducted for 5
min, the test tube was taken out and cooled, distilled water was added to accurately
determine a volume of the solution in the test tube to 12.5 mL after the temperature
was reduced to room temperature, mixing was conducted uniformly, a wavelength of
a spectrophotometer was adjusted to 540 nm, the spectrophotometer was turned on in
advance and preheated for 30 min, the distilled water served as blank control, the
xylose standard curve was drawn by taking a xylose concentration (10 mg/mL) as an
x-coordinate and a light absorption value as a y-coordinate, and a linear formula was
obtained (FIG. 2).
2. Results and Analysis
2.1 Comparison of the growth condition of hypha of Lepista sordida with different
culture spawn formulas In the experiment, the growth condition of the hyphae in different culture mediums are observed by inoculating the Lepista sordida liquid strains into five culture mediums, placing the culture mediums at 22°C to 25°C and controlling the air
humidity to about 60%, as shown in table 5. It can be concluded from table 4 that in five different culture mediums, the growth condition of the Lepista sordida strain in the culture medium C is the best, and the hyphae are dense and pinkish purple and can grow full of the fungal bag from the beginning of inoculation to the 53rd day. The second best is in the culture mediums D and E, the hyphae are dense and light purple, and can grow full of the fungal bag from the beginning of inoculation to the 56th day. The third best is in the culture medium A, although the hyphae grow full of the fungal bag on the 54th d after inoculation, the hyphae are relatively dense and white. The worst is in the culture medium B, the growth of the hyphae is the weakest, the hyphae are relatively sparse and white, and it is necessary for the hyphae to spend 60 d after inoculation to grow full of the fungal bag.
Table 3 Comparison of the Growth Character of Hypha of Lepista sordida with Different Culture spawn Formulas
Formula Hypha Growth Color Number of Vigour Overgrown Days A Relatively dense Relatively white 54 B Relatively sparse Relatively white 60 C Dense Pinkish purple 53 D Dense Light purple 56 E Dense Light purple 56
2.2 Comparison of the growth rate of hyphae of Lepista sordida with different culture spawn formulas Table 4 Analysis on the Growth Rate Variance of Hyphae of Lepista sordida with Different Culture Spawn Formulas
Difference Sum of Degree of Sum of F Significance Source Deviation Freedom Mean Squares Squares Inter-group 0.354 4 0.089 0.1160.000 Intra-group 15.972 21 0.761 Total 16.326 25
The result in FIG. 3 shows that the growth rates of the Lepista sordida culture
spawn in different culture time are different. In the first 10 days of cultivation, there
is no significant difference in the growth rate of the Lepista sordida culture spawn
with five formulas, in the 10 to 40 days of continuous cultivation, the growth rate of
the Lepista sordida culture spawn cultivated by the formula C is the highest and is
obviously more excellent than those of the Lepista sordida cultivated by four other
formulas, and the difference is significant. In the 50 to 60 days of cultivation, there is
no significant difference in the growth rate of the Lepista sordida culture spawn with
the five formulas.
On the 10th day of cultivation, the growth rate of the culture spawn in the five
formulas is about 0.27 cm/d. In the 10th day to the 20th day of continuous
cultivation, the lowest growth rate of the Lepista sordida culture spawn in the
formula B is 0.23 cm/d, and the highest growth rate of the Lepista sordida culture
spawn in the formula C is 0.40 cm/d. On the 30th day of cultivation, the lowest
growth rate of the Lepista sordida culture spawn in the formula B is 0.20 cm/d, and
the highest growth rate of the Lepista sordida culture spawn in the formula C is 0.30
cm/d. On the 40th day of cultivation, the lowest growth rate of the Lepista sordida
culture spawn in the formula B is 0.12 cm/d, and the highest growth rate of the
Lepista sordida culture spawn in the formula C is 0.20 cm/d. On the 50th day of
cultivation, the growth rate is about 0.12 cm/d; and on the 60th day of cultivation, the
growth rate is about 0.11 cm/d.
The result in FIG. 4 shows that in the cultivation formulas A, B, C, D and E,
there is significant different in the growth rate of the Lepista sordida culture spawn in
different time, wherein when the formulas A and B are selected, the growth rate of the
Lepista sordida culture spawn decreases with the change of time through
measurement. In the formula A, the growth rate decreases to 0.12 cm/d on the 50th
day of cultivation and the 60th day of cultivation from 0.28 cm/d in the first 10 days
of the primary cultivation. In the formula B, the growth rate decreases to 0.10cm/d on
the 50th day of cultivation and the 60th day of cultivation from 0.25cm/d in the first
days of the primary cultivation. When the formulas C, D and E are selected, the
growth rate of the Lepista sordida culture spawn increases first and then decreases
with the change of time. In the formula C, the growth rate increases to the maximum
0.40 cm/d in the 10th day to 20th day of cultivation from 0.30 cm/d in the first 10
days of the primary cultivation, and then gradually decreases to 0.12 cm/d in the 50th
day to the 60th day of cultivation. In the formula D, the growth rate increases to the
maximum 0.26 cm/d in the 10th day to 20th day of cultivation from 0.25 cm/d in the
first 10 days of the primary cultivation, and then gradually decreases to 0.11 cm/d in
the 50th day to the 60th day of cultivation. In the formula E, the growth rate increases
modestly in the 10th day to 20thday of cultivation from 0.27 cm/d in the first 10 days
of the primary cultivation and then significantly decreases to 0.09 cm/d in the 50th
day to the 60th day of cultivation.
2.3 Measurement of the enzymatic activity of extracellular enzyme of Lepista sordida
culture spawn with different formulas
2.3.1 Comparison of the enzymatic activity of CMC of Lepista sordida culture spawn
with different formulas
Table 5 Analysis on the Enzymatic Activity Variance of CMC of Lepista sordida Culture spawn
with Different Formulas Difference Sum of Degree of Mean F Significanc Source Squares Freedom Square e Inter-group 0.455918 4 0.113979 32.00425 0.000 Intra-group 0.035614 10 0.003561 Total 0.491531 14
It can be seen from analysis in FIG. 5 that there is significant difference in the
enzymatic activity of CMC of the Lepista sordida culture spawn produced by five
formulas of culture materials. When the formula C is selected, the enzymatic activity
of CMC of the Lepista sordida culture spawn is the highest, reaching to 1.00 U,
which is significantly different from that of the other four formulas. The enzymatic
activity of CMC is the second highest, reaching about 0.69 U when the formulas A, D
and E are selected for treatment; and the enzymatic activity of CMC of the Lepista
sordida culture spawn is the lowest, only about 0.47 U when the formula B is
selected.
2.3.2 Comparison of the enzymatic activity of hemicellulase of Lepista sordida
culture spawn with different formulas
Table 6 Analysis on the Enzymatic Activity Variance of Hemicellulase of Lepista sordida Culture
Spawn with Different Formulas
Difference Sum of Degree of Mean Significan
Source Squares Freedom Square F ce
Inter-group 2.417893 4 0.604473 22.74162 0.000
Intra-group 0.2658 10 0.02658
Total 2.683694 14
It can be seen from analysis in FIG. 6 that there is significant difference in the
enzymatic activity of the hemicellulase of the Lepista sordida culture spawn
produced by five formulas of culture materials. The enzymatic activity of the
hemicellulase of the Lepista sordida culture spawn using the formula C is the highest,
reaching 1.78 U; the enzymatic activity in the formula E is the second highest,
reaching 1.70 U; the enzymatic activity in the formula D and the formula A is the
third highest; and the enzymatic activity of the hemicellulase of the Lepista sordida
culture spawn using the formula B is the lowest, only 0.17 U.
2.3.3 Comparison of the enzymatic activity of amylase of Lepista sordida culture
spawn with different formulas
Table 7 Analysis on the Enzymatic Activity Variance of Amylase of Lepista sordida Culture spawn
with Different Formulas Difference Sum of Degree of Mean F Significan Source Squares Freedom Square ce Inter-group 4.730063 4 1.182516 1067.596 0.000 Intra-group 0.011076 10 0.001108 Total 4.741139 14
It can be seen from analysis in FIG. 7 that there is significant difference in the
enzymatic activity of the amylase of the Lepista sordida culture spawn produced by
the five tested formulas. The enzymatic activity of the amylase of the Lepista sordida
culture spawn using the formula C is the highest, reaching 1.58 U; the enzymatic
activity of the amylase in the formulas D and E is the second highest, about 0.98 U;
and the enzymatic activity of the amylase in the formula A and the formula B is the
lowest, only 0.46 U and 0.51 U.
2.3.4 Comparison of the enzymatic activity of laccase of Lepista sordida culture
spawn with different formulas
Table 8 Analysis on the Enzymatic Activity Variance of Laccase of Lepista sordida Culture spawn
with Different Formulas Difference Sum of Degree of Significan Source Squares Freedom MS F ce Inter-group 1.109649 4 0.277412 588.8937 0.000 Intra-group 0.004711 10 0.000471 Total 1.11436 14
It can be seen from analysis in FIG. 8 that there is significant difference in the
enzymatic activity of the amylase of the Lepista sordida culture spawn produced by
the five tested formulas. There is no significant difference in the enzymatic activity of
the laccase of the Lepista sordida culture spawn using the formulas A, C, D and E,
and the enzymatic activity is about 0.83 U, wherein the enzymatic activity in the
formula C is up to 0.90 U. The enzymatic activity of the laccase in the formula B is
the lowest, only 0.19 U.
Researches on the biological characteristics, genetic breeding and high-yield
cultivation of the Lepista sordida, as to-be-developed wild edible mushroom, are
rarely reported in China. According to the experiment, the optimal formula suitable
for the growth of the Lepista sordida culture spawn is initially explored from three
perspectives of the growth character of the Lepista sordida culture spawn, the growth
rate of the culture spawn and the activity of the extracellular enzyme of the culture
spawn. The growth character of the Lepista sordida culture spawn and the growth
rate of the culture spawn may directly reflect the optimal culture medium suitable for
the growth of the Lepista sordida culture spawn. The activity of the extracellular
enzyme of the Lepista sordida culture spawn in different culture medium formulas
may directly reflect the utilization of nutrients by the hyphae, and the activity is
closely related to the growth condition. The Lepista sordida culture spawn cultivated
by the culture medium with the formula C (38% of chicken manure, 35% of
Flammulina velutipes residue, 25% of wheat straw, 1 % of gypsum and 1% of
calcium carbonate) has the best growth condition, and the overall growth rate of the
culture spawn is the highest and tends to increase first and then decrease; and
furthermore, the growth rate of the culture spawn in the 10th day to the 20th day is
the highest, reaching 0.40 cm/d. When the formula C added with 35% of Flammulina
velutipes residue is selected, the enzymatic activity of the measured four extracellular
enzymes is the highest in the formulas of the tested Lepista sordida culture spawn
through enzymatic activity measurement. The enzymatic activity of the CMC is up to
1.00 U, the enzymatic activity of the hemicellulase reaches 1.78 U, the enzymatic
activity of the amylase reaches 1.58 U, and the enzymatic activity of the laccase
reaches 0.90 U. Therefore, the formula C (38% of chicken manure, 35% of
Flammulina velutipes residue, 25% of wheat straw, 1 % of gypsum and 1% of
calcium carbonate) may be proved to be the optimal culture medium from three
aspects such as the growth condition of the hypha, the growth rate of the hypha and
the activity of the extracellular enzyme. It is of great significance in overcoming the
defects of low yield, high cost and long cycle of the Lepista sordida in the future.
It will be apparent to those skilled in the art that various modifications and variations of the present invention can be made without departing from the spirit or scope of the invention. If these various modifications and variations of the present invention belong to the scope of the claim and equivalent technical scope, the
invention is intended to comprise these modifications and variations.
Claims (7)
- What is claimed is: 1. A culture material for Lepista sordida culture spawn, consisting of the following components in percentage by weight: 15-45% of chicken manure, 10- 3 0% of wheat straw, 0.5-3.5% of gypsum, 0.5-3% of calcium carbonate and 10-50% of Flammulinavelutipes residue.
- 2. The culture material for Lepista sordida culture spawn according to claim 1, consisting of the following components in percentage by weight: 38% of chicken manure, 35% of Flammulina velutipes residue, 25% of wheat straw, 1% of gypsum and 1% of calcium carbonate.
- 3. A method for cultivating Lepista sordida culture spawn by the Lepista sordida culture material according to claim 1 or 2, comprising the following steps: (1) prewetting: weighing the chicken manure, the Flammulina velutipes residue and the wheat straws according to a formula, adding water according to a material-to-water ratio of 1:1.2 to 1:1.4, and soaking and prewetting with limewater with a mass fraction of 1-2% for 12-36 h in advance, wherein the Flammulina velutipes residue are crushed into granules with a grain diameter of 0.2-0.7 m after being dried in the sun, and the wheat straws are cut into small sections with a length of 1-4 cm; (2) bagging: accurately weighing the calcium carbonate and the gypsum according to the formula, dissolving the calcium carbonate and the gypsum with water and stirring uniformly, adding into other prewetted culture materials, adjusting the water content of a culture medium to 55-65%, adjusting the pH value of the culture medium to pH7.5-pH9, and charging with a polypropylene bag with a specification of 17cm*33cm, wherein 1000 g of wet materials are charged in each bag; (3) sterilization and cooling: maintaining an outer wall of the fungal bag filled with the culture material clean, fastening a plastic cover with a ventilation filter membrane and sealing, conducting steam sterilization under high pressure at 121°C for 2 h, cooling to below 25°C and then conducting inoculation; (4) inoculation: transferring the Lepista sordida strain to an improved PDACLA I MSculture plate and conducting standing culture in an incubator at 25°C for 6-10 d under a dark condition to obtain an activated plate strain; selecting 8 to 10 activated Lepista sordida strain blocks (each block is 0.5cm2 ), inoculating into a liquid culture medium, conducting shake culture in a shaking table at a temperature of 25°C to 26°C and at a rotating speed of 120 r/min for 2 d, adjusting the temperature of the shaking table to 24°C to 25°C and the rotating speed to 130-150 r/min, continuously cultivating for 4-5 d to obtain a liquid strain; conducting sterile operation, inoculating 20 mL of Lepista sordida strain into each bag, and adding into the Lepista sordida culture material containing the Flammulina velutipes residue formula which is sterilized and cooled in the step (3); and (5) cultivation: adjusting the constant-temperature and constant-humidity incubator, and conducting cultivation for 50-60 d by controlling the temperature to 24°C to 25°C and the humidity to 55-65% to obtain the Lepista sordida culture spawn.
- 4. The method according to claim 3, wherein a formula of the improved PDA culture plate is: 20% of potato, 1% of glucose, 1% of sucrose, 0. 2 % of yeast powder and 2% of agar.
- 5. The method according to claim 3, wherein a formula of the liquid culture medium is: 1% of corn residue, 1% of glucose, 1% of maltose, 0. 2 % of peptone, 0.3% of yeast powder, 0.1% of monopotassium phosphate and 0.05% of magnesium sulfate heptahydrate.
- 6. Lepista sordida culture spawn prepared by the method according to claim 3 may be transferred to a culture bag to conduct Lepista sordida fruiting cultivation.
- 7. Lepista sordida culture spawn prepared by the method according to claim 3 may be directly subjected to Lepista sordida fruiting cultivation after being covered with soil.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010039580.0A CN111066574B (en) | 2020-01-15 | 2020-01-15 | Method for preparing Lepista sordida cultivars by using mushroom dregs |
| CN202010039580.0 | 2020-01-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020103076A4 true AU2020103076A4 (en) | 2020-12-24 |
Family
ID=70323164
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020103076A Ceased AU2020103076A4 (en) | 2020-01-15 | 2020-10-28 | Method of preparing Lepista sordida culture spawn with mushroom residue |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN111066574B (en) |
| AU (1) | AU2020103076A4 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115474652A (en) * | 2022-09-29 | 2022-12-16 | 黑龙江绿和宝农业科技有限公司 | Preparation method of corn straw microbial fermentation feed |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113348966A (en) * | 2021-06-07 | 2021-09-07 | 云南省热带作物科学研究所 | Culture medium and culture method for Boletus zhonghuasheng liquid strain |
| CN114946531B (en) * | 2022-06-27 | 2024-03-26 | 黑龙江省农业科学院牡丹江分院 | Lepista sordida cultivation method |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007135565A (en) * | 2005-11-22 | 2007-06-07 | Saitama Prefecture | New strain belonging to lepista sordida and artificial culture method |
| CN103947454A (en) * | 2014-05-08 | 2014-07-30 | 鲁东大学 | Artificial culturing method of lepista sordida mycelium and culturing medium thereof |
| US20170079214A1 (en) * | 2014-05-27 | 2017-03-23 | Novozymes A/S | Methods for Mushroom Cultivation |
| CN105779313A (en) * | 2016-05-23 | 2016-07-20 | 沈阳德宝嘉泓农业科技有限公司 | Method for preparing needle mushroom liquid strain by taking mushroom bran extracting solution as raw material |
| CN106278460A (en) * | 2016-08-09 | 2017-01-04 | 上海市农业科学院 | The method of needle mushroom dreg factory culture Agaricus bisporus and culture material formula thereof |
| CN107082698A (en) * | 2017-05-15 | 2017-08-22 | 达州市农业科学研究院 | Corncob Lepista sordida compost and the method for cultivating Lepista sordida |
| CN108157744B (en) * | 2018-01-02 | 2021-04-23 | 青岛农业大学 | Fresh-keeping agent |
| CN108243832A (en) * | 2018-02-02 | 2018-07-06 | 金珠满江农业有限公司 | The artificial method for planting of paint face mushroom |
| CN108293599B (en) * | 2018-02-28 | 2020-05-19 | 中华全国供销合作总社昆明食用菌研究所 | Lepista sordida, and separation propagation method and soil-covering cultivation method thereof |
| CN108925364A (en) * | 2018-08-24 | 2018-12-04 | 杨杰 | A kind of implantation methods of Lepista sordida |
-
2020
- 2020-01-15 CN CN202010039580.0A patent/CN111066574B/en active Active
- 2020-10-28 AU AU2020103076A patent/AU2020103076A4/en not_active Ceased
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115474652A (en) * | 2022-09-29 | 2022-12-16 | 黑龙江绿和宝农业科技有限公司 | Preparation method of corn straw microbial fermentation feed |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111066574A (en) | 2020-04-28 |
| CN111066574B (en) | 2021-09-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020103076A4 (en) | Method of preparing Lepista sordida culture spawn with mushroom residue | |
| CN102550293B (en) | Method for liquid fermentation cultivation of Agaricus bisporus strain | |
| CN105009931A (en) | Preparation of liquid strain for pleurotus eryngii and research method of culture technique of high-quality high-yield pleurotus eryngii through liquid strain | |
| CN106146179B (en) | A method of utilizing agaricus bisporus windrow water extract culture agaricus bisporus strain | |
| CN101897270A (en) | Production technology of Cordyceps sinensis mycelium | |
| CN110249912A (en) | A kind of method that Phellinus industrial bottle is planted | |
| CN101880699B (en) | Method for producing chitooligosaccharides by using microbial fermentation | |
| CN106922392A (en) | A kind of compost of selenium-rich pleurotus cornucopiae and the cultural method of selenium-rich pleurotus cornucopiae | |
| CN101451107B (en) | Method for large scale preparing Gliocladium chlamydospore | |
| CN106588277A (en) | Cultivation method for increasing yield of straw mushrooms | |
| CN1986827B (en) | Truffle polyose preparing process | |
| CN110713956B (en) | Lysine bacillus S12 and application thereof | |
| CN106434603B (en) | A method of cellulase is produced using neutral ammonium sulfite process waste liquid fed-batch fermentation | |
| TWI385248B (en) | A formula of culturing medium for cordyceps spp. | |
| CN102550294A (en) | Method for liquid fermentation cultivation of Pleurotus cornucopiae strain | |
| CN1579197A (en) | Bacterisugar mono-cell protein feed producing method | |
| CN100564510C (en) | A kind of substratum of liquid deep fermentation for producing truffle polysaccharide | |
| CN103497941B (en) | Method for preparing cellulase through trichoderma viride high-efficiency fermentation | |
| CN107099465B (en) | Growth-promoting bacterial strain and its microorganism seedling medium for promoting cuttage tea shoot to take root | |
| CN108849241A (en) | A method of utilizing agricultural crop straw culturing edible fungus | |
| CN112243798B (en) | Energy-saving and efficient tremella strain breeding and planting method | |
| CN107337488A (en) | A kind of preparation method of zinc-rich germ bran biological fertilizer | |
| CN1685796A (en) | Method of raising cordyceps production stability | |
| CN104641930B (en) | A kind of agrocybe industrial planting method | |
| CN109673386A (en) | A kind of Lepista sordida bacterium culture medium and preparation method and cultural method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) | ||
| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |