CN104429604A - Sparassis crispa liquid strain culture medium and culture method - Google Patents
Sparassis crispa liquid strain culture medium and culture method Download PDFInfo
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Abstract
The invention belongs to the technical field of industrial cultivation for edible funguses, and particularly relates to a sparassis crispa liquid strain culture medium and a culture method. The formula of the culture medium comprises 0-145 g/L of glucose, 0-70 g/L of glutinous rice flour, 1-6 g/L of fish meal peptone, 0-5 g/L of KH2PO4 and 0-3 g/L of MgSO4.7H2O, the original pH is 5-6, and the culture medium is cultured by preparation of sparassis crispa liquid mother strains and preparation of sparassis crispa liquid original strains. Aiming at the defects that the sparassis crispa liquid culture medium is high in cost and limited in raw material sources, the invention provides the sparassis crispa liquid strain culture medium and the culture method. The production cost is reduced, and the production efficiency is improved. The liquid culture medium has the advantages of being simple in formula and convenient in raw material sources and is suitable for large-scale production. The liquid strain culture process is simple, and the operation is convenient.
Description
Technical field
The invention belongs to edible fungus industrial cultivation technical field, be specifically related to a kind of Sparassis crispa liquid spawn culture medium and cultural method.
Background technology
Sparassis crispa (
sparasis latifolia) have another name called silk ball gill fungus, silk ball mushroom, because of its sub real limb complications, be similar to huge silk ball and gain the name, being described as the magical mushroom of illusion in Japan, is a kind of food (medicine) edible mushroom new varieties recently developed, and its famous and precious degree can the treasure such as Cordyceps sinensis, hickory chick, ferfas shoulder to shoulder.According to surveying and determination, crude protein content 12.9 % in Sparassis crispa fruit body dry product, raw fiber 13.7 %, amino acid classes is comparatively complete, and containing a large amount of mineral matters, as iron (48.1 mg/kg), zinc (38.5 mg/kg), manganese (30.6 mg/kg) equal size are all higher, vitamin content is also very abundant, as vitamin C, vitamin D, vitamin E etc., wherein content of vitamin E occupies Homonemeae food prostatitis in " composition table of foods ".Sparassis crispa not only delicious flavour, be of high nutritive value, and there is good medical treatment, health-care efficacy.The result of study in japanese food research center (Japan Food Research Laboratories) shows, in Sparassis crispa fruit body polyoses content enrich, every 100 grams containing beta glucan be 43.6 grams, higher than edible and medical fungis such as glossy ganoderma, Agricus blazei, Cordyceps militaris.Beta glucan in Sparassis crispa can improve body immunity and body hematopoiesis function, there is effect that is anticancer, anti-cancer, can prevent and improve the many diseases such as hypertension, hyperglycaemia because hematic acid, allergy and habits and customs produce, also have certain prevention and prohibition effect to some tumour, the mixed extract of Sparassis crispa and grifola frondosus can also Therapeutic cancer and acquired immune deficiency syndrome (AIDS) (JP 2003265139-A).Therefore, Sparassis crispa is health and the health food of a kind of " food and medicine consangunity ".
Because Sparassis crispa has good nutritive value and health-care efficacy, receive the very big concern of Chinese scholars and consumer in recent years gradually.Through studying for many years; Edible Fungus Research Institute, Fujian Academy of Agricultural Sciences can artificial cultivation Sparassis crispa; and achieve scale, factorial praluction (patent: a kind of Sparassis crispa industrialized cultivation medium formula and production technology; application number: 201010286462.6), but still adopt solid spawn in described production technology.The more traditional solid spawn of liquid spawn has many advantages, as: bacterial classification is with short production cycle, cost is low; The spawn activity produced is strong, purity is high; Inoculation is convenient, and after inoculation mycelium germination point many, spread rapidly, suitable edible mushroom large-scale planting etc.Therefore, liquid spawn is adopted to become the inexorable trend of edible fungus industrial development.
Pass through literature survey, current Sparassis crispa is cultivated bacterial classification used and is solid spawn, although there is the report about Sparassis crispa liquid fermentation technology, but be applied to cultivation and still have many limitation, as the optimal liquid medium that Jia Peipei etc. filters out, its hypha biomass is only 4.2g/L, and needs to add the nutriment such as honey powder, brewer's yeast in formula.By formula improvement, although Some Domestic patent improves a lot in hypha biomass, but still be more difficultly applied to large-scale planting.As patent [liquid fermentation culturing method of rare edible and medical fungi Sparassis crispa and special culture media thereof] (application number: 201110220578.4), medium needs the raw materials such as pine needle, and pine needle use before need through pre-treatment, be unfavorable for operation; And patent [a kind of wide leaf Sparassis crispa synthetic medium] (application number: the synthetic medium 201210370266.6) has into advantages such as distinguishing one from the other, steady quality, impurity are few, but be only suitable for orientation regulation and control and the metabolic flux analysis of the synthesis of Sparassis crispa metabolite, and the separation and Extraction of downstream metabolites.
In sum, about the research of Sparassis crispa liquid spawn, there are the following problems at present: 1, hypha biomass is low; 2, culture medium raw material source inconvenience, raw material supply is limited; 3, operation inconvenience, is unfavorable for large-scale production.Therefore, develop a kind of raw material sources extensively, handled easily, suitable Sparassis crispa large-scale production liquid spawn compounding method have great importance.
Summary of the invention
The present invention is directed to the shortcomings such as Sparassis crispa liquid nutrient medium cost is high, raw material sources are restricted, provide a kind of Sparassis crispa liquid spawn culture medium and cultural method, reduce production cost, enhance productivity.
Sparassis crispa liquid spawn culture medium optimization formula provided by the invention is as follows: glucose 0-145g/L, glutinous rice flour 0-70g/L, fish meal protein peptone 1-6g/L, KH
2pO
40-5g/L, MgSO
47H
2o 0-3g/L, initial pH 5-6.
More preferably, the formula of described medium is: glucose 10-145g/L, glutinous rice flour 35-60g/L, fish meal protein peptone 2-3g/L, KH
2pO
40-3g/L, MgSO
47H
2o 0-1.5g/L, initial pH 5.5-6.
It is convenient that this medium has raw material sources, simple operation and other advantages.
Sparassis crispa strain cultivation method of the present invention comprises the steps:
(1) Sparassis crispa liquid mother plants and prepares: first prepare liquid mother culture media, the formula of liquid mother culture media is: peeled potatoes 200g/L, glucose 20g/L, peptone 3g/L, initial pH 5-6; Then the test tube slant mother inoculating activation plants, and cultivation temperature 23-25 DEG C, shaking flask rotating speed 150rpm, cultivate 7-10 days, obtains liquid mother and plants;
(2) Sparassis crispa liquid original seed preparation: first prepare liquid pedigree seed culture medium, the formula of liquid pedigree seed culture medium is: glucose 0-145g/L, glutinous rice flour 0-70g/L, fish meal protein peptone 1-6g/L, KH
2pO
40-5g/L, MgSO
47H
2o 0-3g/L, initial pH 5-6; Then inoculate liquid mother to plant, cultivation temperature 23-25 DEG C, rotating speed 150rpm, cultivate and be Sparassis crispa liquid original seed after 15-20 days.
More specifically, the concrete steps of described cultural method are as follows:
1, Sparassis crispa liquid mother plants and prepares
(1) potato (peeling) 200g is taken, glucose 20g, fish meal protein peptone 2g, KH
2pO
43g, MgSO
47H
2o 1g;
(2) potato slices, add water 1L, to boil after 20min by four layers of filtered through gauze, obtain potato juice for subsequent use;
(3) glucose, fish meal protein peptone, KH is added by above-mentioned formula in potato juice
2pO
4, MgSO
47H
2o dissolves, and be settled to 1L with water, adjustment pH is 5-6, obtains liquid mother culture media;
(4) be distributed in 250ml triangular flask by aforesaid liquid mother culture media, liquid amount is 100ml, bottle plug silica gel plug, 0.1Mpa, 121 DEG C of high pressure steam sterilization 30min;
(5) under aseptic condition, in cooled liquid mother culture media, inoculate PDA inclined-plane bacterium block (0.5cm × 0.5cm) 5-8 block, be placed in shaking table and cultivate, rotating speed 150rpm, temperature 23-25 DEG C, cultivate after 7-10 days and be Sparassis crispa liquid mother kind.
2, Sparassis crispa liquid original seed preparation
After Sparassis crispa liquid Mother culture terminates, carry out the preparation of original seed, specific as follows:
(1) glucose 0-145g/L, glutinous rice flour 0-70g/L, fish meal protein peptone 1-6g/L, KH
2pO
40-5g/L, MgSO
47H
2o 0-3g/L; (2) 0.8L that adds water in glutinous rice flour fully stirs, and is heated to boiling while stirring;
(3) by glucose, KH
2pO
4and MgSO
47H
2o adds in glutinous rice flour solution, is settled to 1L after stirring, and adjustment pH is 5-6, is liquid pedigree seed culture medium;
(4) aforesaid liquid pedigree seed culture medium is distributed in 250ml triangular flask, liquid amount 100ml, bottle plug silica gel plug, 0.1Mpa, 121 DEG C of high pressure steam sterilization 30min;
(5) under aseptic condition, by 8%(v/v) inoculum concentration liquid mother is planted in access liquid pedigree seed culture medium, after inoculation, shaking table is cultivated, and rotating speed 150rpm, temperature 23-25 DEG C, cultivate and be Sparassis crispa liquid original seed after 15-20 days.
Beneficial effect of the present invention:
Method of the present invention has the following advantages:
1, liquid culture based formulas is simple, and raw material sources are convenient, suitability for scale production.
2, strain cultivation process is simple, easy to operate.
Embodiment
The present invention's Sparassis crispa bacterial classification used is ' Fujian embroiders No. 1 ', derives from Edible Fungus Research Institute, Fujian Academy of Agricultural Sciences.
Embodiment one:
1, Sparassis crispa liquid Mother culture
(1) female kind activates.Sparassis crispa is inoculated in PDA(peeled potatoes 200g, glucose 20g, agar powder 16g) in slant medium, cultivate 25 days, obtain the silk ball starter kind of activation for 23-25 DEG C;
(2) prepare liquid mother to plant.Peeled potatoes 200g cuts into slices, and the 1L that adds water boils, and adds glucose 20g after filtration, peptone 2g, KH
2pO
43g, MgSO
47H
2o 1g, is settled to 1L after stirring, and regulates pH to be 5.5;
(3) aforesaid liquid medium is distributed in 250ml triangular flask, liquid amount 100ml, high pressure steam sterilization 30min;
(4) the inclined-plane mother inoculating activation after sterilizing plants bacterium block (0.5cm × 0.5cm) 5-8 block, cultivates, rotating speed 150rpm, temperature 23-25 DEG C in shaking table, cultivates after 10 days and namely obtains liquid mother kind.
2, Sparassis crispa liquid Primary spawn
(1) liquid pedigree seed culture medium: glucose 20g, fish meal protein peptone 6g, KH
2pO
45g, MgSO
47H
2o 3g, initial pH are 5.
Control medium: potato 200g, glucose 20g, fish meal protein peptone 2g, pH value nature, is settled to 1L.
(2) cultured liquid mother culture media is inoculated in aforesaid liquid pedigree seed culture medium, inoculum concentration is 8%, cultivation temperature 23 ~ 25 DEG C, shaking flask rotating speed 150rpm, shaken cultivation is carried out 15 days under natural lighting condition, obtain Sparassis crispa liquid original seed, measure bacterium bulb diameter, Peloton density and mycelial biomass, concrete assay method is as follows:
Bacterium bulb diameter (mm): the some mycelium pellet alinements of random taking-up from medium, surveys total length, averages.
Peloton density (individual/ml): with liquid-transfering gun random sampling from medium, measures bacterium ball number.
Mycelial biomass (g/L): filtered by medium double gauze, the mycelium pellet clear water after filtration fully rinses, and puts into 40 DEG C of baking ovens and dries to constant weight, be hypha biomass with after scales/electronic balance weighing.
After measured, bacterium bulb diameter is 2.7mm, and bacterium ball is close is 5.1/ml, hypha biomass 1.3g/L.
(3) by 8% inoculum concentration, liquid mother culture media is inoculated in control medium, cultivation temperature 23 ~ 25 DEG C, shaking flask rotating speed 150rpm, shaken cultivation 15 days under natural lighting condition.After measured, bacterium bulb diameter is 2.0mm, and Peloton density is 12.5/ml, and mycelial biomass is 0.6g/L.
Embodiment two:
1, Sparassis crispa liquid Mother culture
Female kind activates with liquid Mother culture by the method in embodiment one.
2, Sparassis crispa liquid Primary spawn
(1) liquid pedigree seed culture medium: glutinous rice flour 70g, fish meal protein peptone 1g, KH
2pO
41.5g, MgSO
47H
2o 3g, initial pH are 5.
Control medium: potato 200g, glucose 20g, fish meal protein peptone 2g, pH value nature, is settled to 1L.
(2) be inoculated in aforesaid liquid pedigree seed culture medium by cultured liquid mother culture media, inoculum concentration is 8%, cultivation temperature 23 ~ 25 DEG C, and shaking flask rotating speed 150rpm carries out shaken cultivation 15 days under natural lighting condition, obtain Sparassis crispa liquid original seed.By the method in embodiment one, after measured, bacterium bulb diameter is 1.9mm, and bacterium ball is close is 49.3/ml, hypha biomass 6.7g/L.
(3) by 8% inoculum concentration, liquid mother culture media is inoculated in control medium, cultivation temperature 23 ~ 25 DEG C, shaking flask rotating speed 150rpm, shaken cultivation 15 days under natural lighting condition.After measured, bacterium bulb diameter is 2.0mm, and Peloton density is 12.5/ml, and mycelial biomass is 0.6g/L.
Embodiment three:
1, Sparassis crispa liquid Mother culture
Female kind activates with liquid Mother culture by the method in embodiment one.
2, Sparassis crispa liquid Primary spawn
(1) liquid pedigree seed culture medium: glucose 10g, glutinous rice flour 60g, fish meal protein peptone 2g, KH
2pO
43g, MgSO
47H
2o 1.5g, initial pH are 5.5.
Control medium: potato 200g, glucose 20g, fish meal protein peptone 2g, pH value nature, is settled to 1L.
(2) be inoculated in aforesaid liquid pedigree seed culture medium by cultured liquid mother culture media, inoculum concentration is 8%, cultivation temperature 23 ~ 25 DEG C, and shaking flask rotating speed 150rpm carries out shaken cultivation 15 days under natural lighting condition, obtain Sparassis crispa liquid original seed.By the method in embodiment one, after measured, bacterium bulb diameter is 1.7mm, and bacterium ball is close is 48.0/ml, hypha biomass 7.6g/L.
(3) by 8% inoculum concentration, liquid mother culture media is inoculated in control medium, cultivation temperature 23 ~ 25 DEG C, shaking flask rotating speed 150rpm, shaken cultivation 15 days under natural lighting condition.After measured, bacterium bulb diameter is 2.0mm, and Peloton density is 12.5/ml, and mycelial biomass is 0.6g/L.
Embodiment four:
1, Sparassis crispa liquid Mother culture
Female kind activates with liquid Mother culture by the method in embodiment one.
2, Sparassis crispa liquid Primary spawn
(1) liquid pedigree seed culture medium: glucose 40g, glutinous rice flour 20g, fish meal protein peptone 3g, initial pH are 6.
Control medium: potato 200g, glucose 20g, fish meal protein peptone 2g, pH value nature, is settled to 1L.
(2) be inoculated in aforesaid liquid pedigree seed culture medium by cultured liquid mother culture media, inoculum concentration is 8%, cultivation temperature 23 ~ 25 DEG C, and shaking flask rotating speed 150rpm carries out shaken cultivation 15 days under natural lighting condition, obtain Sparassis crispa liquid original seed.By the method in embodiment one, after measured, bacterium bulb diameter is 1.9mm, and bacterium ball is close is 15.3/ml, hypha biomass 5.8g/L.
(3) by 8% inoculum concentration, liquid mother culture media is inoculated in control medium, cultivation temperature 23 ~ 25 DEG C, shaking flask rotating speed 150rpm, shaken cultivation 15 days under natural lighting condition.After measured, bacterium bulb diameter is 2.0mm, and Peloton density is 12.5/ml, and mycelial biomass is 0.6g/L.
Embodiment five:
1, Sparassis crispa liquid Mother culture
Female kind activates with liquid Mother culture by the method in embodiment one.
2, Sparassis crispa liquid Primary spawn
(1) liquid pedigree seed culture medium: glucose 145g, glutinous rice flour 35g, fish meal protein peptone 3g, initial pH are 6.
Control medium: potato 200g, glucose 20g, fish meal protein peptone 2g, pH value nature, is settled to 1L.
(2) be inoculated in aforesaid liquid pedigree seed culture medium by cultured liquid mother culture media, inoculum concentration is 8%, cultivation temperature 23 ~ 25 DEG C, and shaking flask rotating speed 150rpm carries out shaken cultivation 15 days under natural lighting condition, obtain Sparassis crispa liquid original seed.By the method in embodiment one, after measured, bacterium bulb diameter is 2.1mm, and bacterium ball is close is 18.8/ml, hypha biomass 9.5g/L.
(3) by 8% inoculum concentration, liquid mother culture media is inoculated in control medium, cultivation temperature 23 ~ 25 DEG C, shaking flask rotating speed 150rpm, shaken cultivation 15 days under natural lighting condition.After measured, bacterium bulb diameter is 2.0mm, and Peloton density is 12.5/ml, and mycelial biomass is 0.6g/L.
Embodiment six
1, Sparassis crispa liquid Mother culture
Female kind activates with liquid Mother culture by the method in embodiment one.
2, Sparassis crispa liquid Primary spawn
(1) liquid pedigree seed culture medium: glucose 55g, glutinous rice flour 40g, fish meal protein peptone 3g, KH
2pO
43g, MgSO
47H
2o 1g, initial pH are 5.5.
(2) be inoculated in aforesaid liquid pedigree seed culture medium by cultured liquid mother culture media, inoculum concentration is 8%, cultivation temperature 23 ~ 25 DEG C, and shaking flask rotating speed 150rpm carries out shaken cultivation 15 days under natural lighting condition, obtain Sparassis crispa liquid original seed.By the method in embodiment one, after measured, bacterium bulb diameter is 1.5mm, and bacterium ball is close is 48.7/ml, hypha biomass 9.7g/L.
Interpretation of result:
Liquid spawn is applied to edible fungus industrial cultivation, and in bacterial classification self-characteristic, need possess following characteristic: first, bacterium bulb diameter should be the smaller the better, can avoid like this blocking inoculating gun in seeded process; Secondly, Peloton density is the bigger the better, and after inoculation, mycelium germination point is many, mycelia front cover time shorten, reduces and pollutes probability; 3rd, possessing while above 2, improving mycelial biomass in medium as far as possible.In production technology, wide material sources answered by required raw material, and operating process is simple.
As can be seen from above example, in production cost, medium component of the present invention simple (only needing 2 kinds of carbon sources, a kind of nitrogenous source), material source is convenient, suitable large-scale fermenting and producing, simultaneously, without the need to pre-treatment when described medium uses, use simple, handled easily.Carry out fermented and cultured by medium of the present invention, Sparassis crispa hypha biomass can reach 9.7g/L, and higher than the result of study (4.3g/L) of contrast (0.6g/L) and Jia Peipei etc., Peloton density is 48.7/ml, suitability for scale production.
Claims (3)
1. a Sparassis crispa liquid spawn culture medium, is characterized in that: the formula of described medium is: glucose 0-145g/L, glutinous rice flour 0-70g/L, fish meal protein peptone 1-6g/L, KH
2pO
40-5g/L, MgSO
47H
2o 0-3g/L, initial pH 5-6.
2. Sparassis crispa liquid spawn culture medium according to claim 1, is characterized in that: described Sparassis crispa bacterial classification is ' Fujian embroiders No. 1 '.
3. a Sparassis crispa strain cultivation method, is characterized in that: described cultural method comprises the steps:
(1) Sparassis crispa liquid mother plants and prepares: first prepare liquid mother culture media, the formula of liquid mother culture media is: peeled potatoes 200g/L, glucose 20g/L, peptone 3g/L, initial pH 5-6; Then the test tube slant mother inoculating activation plants, and cultivation temperature 23-25 DEG C, shaking flask rotating speed 150rpm, cultivate 7-10 days, obtains liquid mother and plants;
(2) Sparassis crispa liquid original seed preparation: first prepare liquid pedigree seed culture medium, the formula of liquid pedigree seed culture medium is: glucose 0-145g/L, glutinous rice flour 0-70g/L, fish meal protein peptone 1-6g/L, KH
2pO
40-5g/L, MgSO
47H
2o 0-3g/L, initial pH 5-6; Then inoculate liquid mother to plant, cultivation temperature 23-25 DEG C, rotating speed 150rpm, be Sparassis crispa liquid original seed after cultivating 15-20d.
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| CN106365800A (en) * | 2016-08-29 | 2017-02-01 | 福建容益菌业科技研发有限公司 | Matrix for cultivating Sparassis crispa |
| CN111837811A (en) * | 2020-07-27 | 2020-10-30 | 芜湖野树林生物科技有限公司 | Sparassis crispa liquid strain culture medium and culture method |
| CN112646730A (en) * | 2020-12-22 | 2021-04-13 | 福建容益菌业科技研发有限公司 | Sparassis crispa liquid fermentation process |
| CN113846021A (en) * | 2021-09-06 | 2021-12-28 | 融和梦(福建)生物科技有限公司 | Liquid culture method of sparassis crispa, freeze-dried powder and application of freeze-dried powder |
| CN115141864A (en) * | 2022-09-06 | 2022-10-04 | 山东久茸生物科技有限公司 | Processing method of selenium-rich sparassis crispa polysaccharide |
| CN115226571A (en) * | 2022-07-25 | 2022-10-25 | 江苏品品鲜生物科技股份有限公司 | Sparassis crispa liquid strain culture medium and culture method thereof |
| CN117487672A (en) * | 2023-12-29 | 2024-02-02 | 云南菌视界生物科技有限公司 | Yellow or pale yellow Sparassis crispa strain suitable for artificial cultivation |
| CN117487671A (en) * | 2023-12-29 | 2024-02-02 | 云南菌视界生物科技有限公司 | Sparassis crispa strain suitable for industrial cultivation and cultivation method thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106365800A (en) * | 2016-08-29 | 2017-02-01 | 福建容益菌业科技研发有限公司 | Matrix for cultivating Sparassis crispa |
| CN106365800B (en) * | 2016-08-29 | 2019-05-17 | 福建容益菌业科技研发有限公司 | A kind of matrix for silk ball bacteria cultivation |
| CN111837811A (en) * | 2020-07-27 | 2020-10-30 | 芜湖野树林生物科技有限公司 | Sparassis crispa liquid strain culture medium and culture method |
| CN112646730A (en) * | 2020-12-22 | 2021-04-13 | 福建容益菌业科技研发有限公司 | Sparassis crispa liquid fermentation process |
| CN113846021A (en) * | 2021-09-06 | 2021-12-28 | 融和梦(福建)生物科技有限公司 | Liquid culture method of sparassis crispa, freeze-dried powder and application of freeze-dried powder |
| CN115226571A (en) * | 2022-07-25 | 2022-10-25 | 江苏品品鲜生物科技股份有限公司 | Sparassis crispa liquid strain culture medium and culture method thereof |
| CN115141864A (en) * | 2022-09-06 | 2022-10-04 | 山东久茸生物科技有限公司 | Processing method of selenium-rich sparassis crispa polysaccharide |
| CN117487672A (en) * | 2023-12-29 | 2024-02-02 | 云南菌视界生物科技有限公司 | Yellow or pale yellow Sparassis crispa strain suitable for artificial cultivation |
| CN117487671A (en) * | 2023-12-29 | 2024-02-02 | 云南菌视界生物科技有限公司 | Sparassis crispa strain suitable for industrial cultivation and cultivation method thereof |
| CN117487670A (en) * | 2023-12-29 | 2024-02-02 | 云南菌视界生物科技有限公司 | Sparassis crispa high-yield strain, molecular marker, identification and industrial cultivation method |
| CN117487672B (en) * | 2023-12-29 | 2024-03-22 | 云南菌视界生物科技有限公司 | Yellow or pale yellow Sparassis crispa strain suitable for artificial cultivation |
| CN117487671B (en) * | 2023-12-29 | 2024-03-22 | 云南菌视界生物科技有限公司 | Sparassis crispa strain suitable for industrial cultivation and cultivation method thereof |
| CN117487670B (en) * | 2023-12-29 | 2024-03-22 | 云南菌视界生物科技有限公司 | Sparassis crispa strain JSJ-Spar4-2 and industrial cultivation method |
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