CN104311348A - Improved liquid fermentation culture medium of pleurotus eryngii and method for culturing liquid strain of pleurotus eryngii by utilizing improved liquid fermentation culture medium - Google Patents
Improved liquid fermentation culture medium of pleurotus eryngii and method for culturing liquid strain of pleurotus eryngii by utilizing improved liquid fermentation culture medium Download PDFInfo
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- CN104311348A CN104311348A CN201410568173.3A CN201410568173A CN104311348A CN 104311348 A CN104311348 A CN 104311348A CN 201410568173 A CN201410568173 A CN 201410568173A CN 104311348 A CN104311348 A CN 104311348A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B17/00—Other phosphatic fertilisers, e.g. soft rock phosphates, bone meal
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Abstract
The invention relates to a microorganism culture medium and a culture method and particularly relates to an improved liquid fermentation culture medium of pleurotus eryngii and a method for culturing liquid strain of the pleurotus eryngii by utilizing the improved liquid fermentation culture medium. The culture method comprises the following steps of selecting glucose, soybean meal, corn flour, monopotassium phosphate, magnesium sulfate, vitamin B1, gelatin, agar or mixture, preparing the fermentation culture medium of the liquid strain of the pleurotus eryngii by adopting sterile water, and preparing the liquid strain of the pleurotus eryngii by shaking culture. The improved liquid fermentation culture medium and the method have the advantages that the problems that since the quality of the liquid strain is not stable (the segments of mycelium pellets and mycelium of the bacterium liquid are larger and the bacterium suspension is non-uniform) in the existing industrialized production of the pleurotus eryngii, the production is easily infected with infectious microbes, and the production efficiency is low so as to cause unsuitability for industrialized application and the like are solved and the source of all components of the culture medium is easy; the liquid strain of the pleurotus eryngii prepared by the preparation method has the advantages that the diameter of the mycelium pellets becomes small obviously, the density of the bacterium liquid is increased and the suspension is uniform; after the liquid strain is inoculated on a solid culture medium, the growth activity is better than that of the liquid strain prepared by the traditional method, the growth speed is fast and hyphae are thick and strong and the like.
Description
Technical field
The present invention relates to a kind of microbiological culture media and cultural method, refer to the Pleurotus eryngii liquid fermentation medium of improvement especially and utilize it to cultivate the method for pleurotus eryngii liquid strain.
Background technology
Pleurotus eryngii is deeply by the nutrition and health care food that people like, in recent years due to the fast development of industrial cultivation technique, for shortening bacterial classification preparation cycle, improve strain quality and production efficiency, domestic and international manufacturer research and development liquid spawn is used for substituting during current Pleurotus eryngii is produced the solid spawn generally used, and in actual production, start application.
Pleurotus eryngii liquid strain produces great-hearted Pleurotus eryngii mycelium by liquid fermentation process.At present, domesticly not yet promulgate quality and quality that unified national standard carrys out specification pleurotus eryngii liquid strain, but people sum up effect and have worked out some region standards in actual application, as Liaoning Province's provincial standard-" DB21/T 1693-2008 edible fungi liquid strain production technology regulation " etc., clear stipulaties in these standards, qualified edible fungi liquid strain should " visible distinctive hypha form, spherical and plexi mycelium distributes in a large number, mycelia is sturdy, in mycelia, protoplasma is evenly distributed " " bacterium liquid slightly thickness, there is a large amount of sheet or spherical mycelium suspended, be evenly distributed ", that is excellent liquid spawn mycelium pellet density is high, diameter is little, be evenly distributed, on the other hand, in production application, pleurotus eryngii liquid strain carries out mechanical inoculation operation by production line inoculation device, because inoculation device spout is narrow, for preventing bacterium liquid blocking pipe, also need the pretreatment technology before increasing liquid-spawn inoculation (the general method of mechanical disintegration that adopts makes the mycelium pellet diameter in bacterium liquid diminish, and bacterium liquid is become evenly).
Therefore applicant thinks, the physical property such as size, density, uniformity coefficient of Pleurotus eryngii liquid fermenting mycelium pellet is the crucial Con trolling index of high-quality liquid spawn.
The domestic research report about Pleurotus eryngii liquid culture and patented technology concentrate on the optimization aspect of zymotechnique substantially, the research and patent that improve zymophyte pompon physical property are mainly relied in the method for mechanical disintegration or liquid medium within add granulated glass sphere, little spring reduces fermented hypha spherical diameter by the mode of breaing up mycelium pellet, increases density and uniformity coefficient.The method complex process of mechanical disintegration, very easily bacteria infection causes cultivating unsuccessfully; The method of adding granulated glass sphere and little spring is only applicable to the small-scale production of shake flask fermentation, is applied to the production of batch production fermentor tank and can operates hardly.
Goal of the invention
The object of the present invention is to provide a kind of pleurotus eryngii liquid strain fermention medium of improvement and utilize it to cultivate the method for pleurotus eryngii liquid strain.Mycelium pellet diameter can be effectively reduced, increase mycelium pellet density.
Overall technology design of the present invention is:
The pleurotus eryngii liquid strain fermention medium of improvement, this fermention medium adopts the component of following mass percentage to form: glucose 1%-2%; Analysis for soybean powder 1%-2%; Semen Maydis powder 2%-3%; Potassium primary phosphate 0.05%-0.2%; Magnesium sulfate 0.01%-0.05%; Vitamins B
10.02g/l-0.05g/l, a kind of in gelatin, agar or 0.1 gram, the mixture/100ml-0.3 gram/100ml of the two, surplus is sterilized water, pH nature.
Utilize above-mentioned fermention medium to cultivate the method for pleurotus eryngii liquid strain, be carry out cultivation in substratum pleurotus eryngii quel strains being inoculated in claim 1 to make pleurotus eryngii liquid strain, wherein culture condition is temperature is 25 ± 3 DEG C, and rotating speed is 180-200r/m; 6-8d is cultivated in shaking table concussion; Described pleurotus eryngii quel strains selects the one in Pleurotus eryngii (Pleurotus eryngii) CICC 14011, Pleurotus eryngii (Pleurotus eryngii) ACCC 51678, Pleurotus eryngii (Pleurotus eryngii) ACCC 51330.
In the present invention, the measuring method of all technical is as follows:
1, Pleurotus eryngii mycelium pellet density quantification method: evenly get 1ml nutrient solution, stroke-physiological saline solution is diluted to 10ml, mixing, gets 1mL diluent and is placed in plate, launches.Black background paper is served as a contrast, counting under plate.
2, Pleurotus eryngii mycelium pellet dry weight method (biomass): evenly get 100ml nutrient solution, filters through 80 eye mesh screens, with stroke-physiological saline solution washing, clarifies to washings.Mycelium pellet is placed on the qualitative filter paper of constant weight, 60 DEG C dry to constant weight after weigh.
3, mycelium pellet measuring diameter: get mycelium pellet 20 at random, be arranged in a straight line, survey its length with vernier callipers, calculates the mean diameter (cm) of 20 mycelium pellets, repeats 3 times.
For determining thickening material kind, applicant carried out following experiment:
One, the choosing and impact that thickening material grows Pleurotus eryngii mycelium pellet of thickening material
(1) xanthan gum, Natvosol and methylcellulose gum impact that Pleurotus eryngii mycelium pellet is grown
Experiment shows, adds xanthan gum in the medium, Natvosol or methylcellulose gum, inhibits the growth of Pleurotus eryngii mycelium pellet.
Inspection information: xanthan gum is a kind of unit cell polysaccharide produced by Rhodopseudomonas; Research in recent years about edible mushrooms bacterial disease shows, most of pathogenic bacterium are Rhodopseudomonas, therefore the growth of its metabolite to edible mushrooms also has restraining effect.Hydroxy combining on methylcellulose gum, Natvosol meeting and nutritive substance, affects mycelia absorbing liquid nutrient medium Middle nutrition material.
(2) starch and Walocel MT 20.000PV impact that Pleurotus eryngii mycelium pellet is grown
Experiment shows (as shown in Figure 1-2), adds starch or Walocel MT 20.000PV in the medium, contrasts with blank group, does not have a significant effect to mycelium pellet growth.
(3) sodium alginate impact that Pleurotus eryngii mycelium pellet is grown
Experiment shows (as shown in Figure 3), and after with the addition of sodium alginate in liquid medium within, the diameter of Pleurotus eryngii mycelium pellet is obviously greater than blank group, does not meet to produce to want little requirement to Pleurotus eryngii mycelium pellet diameter.
(4) agar and gelatin impact that Pleurotus eryngii mycelium pellet is grown
Investigate the agar adding different concns in the medium, the growing state of Pleurotus eryngii mycelium pellet, result as shown in Figure 4.When the content of agar in liquid nutrient medium is more than 0.4%, liquid nutrient medium solidifies, and cannot carry out shaking table cultivation.When the agar content in liquid nutrient medium is 0.3%, mycelium pellet diameter is minimum, is 2.41mm, compared with blank group, reduce about 18.9%, and now, the density of mycelium pellet also reaches maximum, is 3.14 × 10
4individual/L, compared with blank group, improves 57.9%.Now mycelium pellet dry weight is also higher, is 6.14g/100mL.
Investigate the gelatin adding different content in liquid medium within, the growing state of Pleurotus eryngii mycelium pellet, as shown in Figure 5.When gelatin concentration is 0.2% time, mycelium pellet diameter reaches minimum, is 2.54mm, compared with blank group, reduces about 14.5%, and now, mycelium pellet density, also close to peak value, is 3.03 × 10
4individual/L, contrasts with blank group, improves 52.3%.Mycelium pellet dry weight reaches maximum, 7.93g/100mL.
(5) contrast of 0.3% agar, 0.2% gelatin and blank group is added
According to the result of above-mentioned (4) article, when add thickening material be 0.3% agar or be 0.2% gelatin time, the Pleurotus eryngii mycelium pellet obtained meets the initial requirement of experiment most, two kinds of concentration acquired results and blank group are contrasted, as shown in Figure 6, mycelium pellet diameter, is both less than blank group; Mycelium pellet density, is both greater than blank group; And both difference on diameter and density are little.And in mycelium pellet dry weight, 0.2% gelatin group is better than blank group, blank group is better than 0.3% agar group.
(6) vigor measurement is carried out to 0.3% agar group and 0.2% gelatin group Pleurotus eryngii mycelium pellet
Experimentally the data obtained, makes Fig. 7, Fig. 8.Added 0.3% agar or 0.2% gelatin gained mycelium pellet mycelia day growth rate linear equation on plate in the medium: y=0.5443x+1.3643, R
2=0.9088 (adding 0.3% agar); Y=0.5411x+1.0257, R
2=0.9541 (adding 0.2% gelatin).By contrast, find that 0.3% agar group mycelium pellet vigor is slightly better than 0.2% gelatin group mycelium pellet vigor.And both mycelium pellet vigor are all better than control group.
Two, the liquid spawn hypha form that the pleurotus eryngii liquid strain utilizing present patent application method to obtain and traditional method obtain contrasts by applicant, and result is as follows.
As shown in Figure 9, wherein compared with control group (left figure), the liquid spawn mycelium pellet diameter utilizing the method for the application to obtain obviously diminishes, and the liquid-tight degree of bacterium improves, and even suspension.
Three, the mycelium of applicant to the pleurotus eryngii liquid strain utilizing the method for the application to obtain has carried out microscopic observation, and result is as follows.
Improved culture medium fermented hypha form is spherical or sheet; Observe under 400 power microscopes, mycelia is sturdy, and hyphal cell protoplasma fills, evenly (Figure 10).
Four, applicant contrasts the mycelial growth vigor after the pleurotus eryngii liquid strain utilizing the method for the application to obtain and conventional liq strain inoculation solid medium, and result is as follows.
After the pleurotus eryngii liquid strain inoculation solid culture medium using conventional medium to prepare, mycelial growth comparatively slow (white portion is mycelial growth overlay area, less); After using the medium preparing liquid-spawn inoculation solid culture medium after improvement, mycelial growth is grown fast (white portion is mycelial growth overlay area, more), illustrates that liquid spawn prepared by improved culture medium flushes, has using value.
The substantive distinguishing features that the present invention possesses and the remarkable technical progress obtained are:
The each Component Source of substratum in the present invention is easy, and after adding gelatin or agar, mycelium pellet diameter obviously diminishes, and the liquid-tight degree of bacterium improves, and even suspension.The pleurotus eryngii liquid strain using present method to prepare is healthy and strong, and after inoculation solid medium, growth vigor is better than traditional method liquid spawn, fast growth, and mycelia is sturdy, and hyphal cell protoplasma is evenly full.
Accompanying drawing explanation
Fig. 1 be different starch concentration Pleurotus eryngii mycelium pellet is grown affect schematic diagram.
As shown in Figure 1, add starch in the medium, contrast with blank group, mycelium pellet growth is not had a significant effect.
Fig. 2 be different Walocel MT 20.000PV concentration Pleurotus eryngii mycelium pellet is grown affect schematic diagram.
As shown in Figure 2, add Walocel MT 20.000PV in the medium, contrast with blank group, mycelium pellet growth is not had a significant effect.
Fig. 3 is that different sodium alginate concentration affects schematic diagram to the mycelia growing of pleurotus eryngii.
As shown in Figure 3, after with the addition of sodium alginate in liquid medium within, the diameter of Pleurotus eryngii mycelium pellet is obviously greater than blank group, does not meet to produce to want little requirement to Pleurotus eryngii mycelium pellet diameter.
Fig. 4 is that different agar concentration affects schematic diagram to the mycelia growing of pleurotus eryngii.
As shown in Figure 4, when the content of agar in liquid nutrient medium is more than 0.4%, liquid nutrient medium solidifies, and cannot carry out shaking table cultivation.) when the lightweight content in liquid nutrient medium is 0.3%, mycelium pellet diameter is minimum, is 2.41mm, compared with blank group, reduce about 18.9%, and now, the density of mycelium pellet also reaches maximum, is 3.14 × 10
4individual/l, compared with blank group, improves 57.9%.Now mycelium pellet dry weight is also higher, is 6.14g/100ml.
Fig. 5 is that different gelatin concentration affects schematic diagram to the mycelia growing of pleurotus eryngii.
As shown in Figure 5, when gelatin concentration is 0.2% time, mycelium pellet diameter reaches minimum, is 2.54mm, compared with blank group, reduces about 14.5%, and now, mycelium pellet density, also close to peak value, is 3.03 × 10
4individual/l, contrasts with blank group, improves 52.3%.Mycelium pellet dry weight reaches maximum, 7.93g/100ml.
The Pleurotus eryngii mycelia that Fig. 6 is interpolation 0.2% gelatin, 0.3% agar is cultivated and blank group contrast schematic diagram.
As shown in Figure 6, mycelium pellet diameter, is both less than blank group; Mycelium pellet density, is both greater than blank group; And both difference on diameter and density are little.And in mycelium pellet dry weight, 0.2% gelatin group is better than blank group, blank group is better than 0.3% agar group.
Fig. 7 is 0.3% agar mycelium pellet day growth rate diagram.
Fig. 8 is 0.2% gelatin mycelium pellet day growth rate diagram.
Experimentally the data obtained, makes Fig. 7, Fig. 8.Added 0.3% agar or 0.2% gelatin gained mycelium pellet mycelia day growth rate linear equation on plate in the medium: y=0.5443x+1.3643, R
2=0.9088 (adding 0.3% agar); Y=0.5411x+1.0257, R
2=0.9541 (adding 0.2% gelatin).By contrast, find that 0.3% agar group mycelium pellet vigor is slightly better than 0.2% gelatin group mycelium pellet vigor.And both mycelium pellet vigor are all better than Yuanping City's plate switching bacterial classification.
Fig. 9 is liquid spawn mycelium morphology (density and the diameter) schematic diagram that the pleurotus eryngii liquid strain that utilizes present patent application method to obtain and traditional method obtain.
As shown in Figure 9, wherein compared with control group (left figure), the liquid spawn mycelium pellet diameter utilizing the method for the application to obtain obviously diminishes, and the liquid-tight degree of bacterium improves, and even suspension.
Figure 10 is the pleurotus eryngii liquid strain mycelium microscopic morphology obtained and the mycelia microstructure schematic diagram that utilize method of the present invention.
Wherein Figure 10 left side is the pleurotus eryngii liquid strain mycelium microscopic morphology schematic diagram obtained utilizing method of the present invention; Figure 10 right side is the pleurotus eryngii liquid strain mycelia microstructure schematic diagram obtained utilizing microscopic examination to adopt method of the present invention.
As shown in Figure 10 left side, improved culture medium fermented hypha volume morphing is spherical or sheet; As shown in Figure 10 right side, observe under 400 power microscopes, mycelia is sturdy, and hyphal cell protoplasma fills, evenly.
Figure 11 is that the mycelial growth after the pleurotus eryngii liquid strain and conventional liq strain inoculation solid medium adopting the inventive method to prepare contrasts situation schematic diagram.
As Figure 11 is left, after the pleurotus eryngii liquid strain inoculation solid culture medium using conventional medium to prepare, mycelial growth comparatively slow (white portion is mycelial growth overlay area, less); As Figure 11 is right, after using the medium preparing liquid-spawn inoculation solid culture medium after improvement, mycelial growth is grown fast (white portion is mycelial growth overlay area, more), illustrate that liquid spawn prepared by improved culture medium flushes, have actual application value.
Embodiment
Below in conjunction with embodiment, the present invention is described further; but it is not as a limitation of the invention; the content that protection scope of the present invention is recorded with claim is as the criterion, and any equivalent technical elements made according to specification sheets is replaced, and does not all depart from protection scope of the present invention
Embodiment 1
The pleurotus eryngii liquid strain fermention medium of improvement, this fermention medium adopts the component of following mass percentage to form: glucose 1%; Analysis for soybean powder 1%; Semen Maydis powder 2%; Potassium primary phosphate 0.05%; Magnesium sulfate 0.01%; Vitamins B
10.02g/l, gelatin 0.1 gram/100ml, surplus is sterilized water, pH nature.
Utilize above-mentioned fermention medium to cultivate the method for pleurotus eryngii liquid strain, culture condition is temperature is 25 ± 3 DEG C, and rotating speed is 180-200r/m; 6-8d is cultivated in shaking table concussion.
Described pleurotus eryngii quel strains selects the one in Pleurotus eryngii (Pleurotus eryngii) CICC 14011, Pleurotus eryngii (Pleurotus eryngii) ACCC 51678, Pleurotus eryngii (Pleurotus eryngii) ACCC 51330.
Embodiment 2
The pleurotus eryngii liquid strain fermention medium of improvement, this fermention medium adopts the component of following mass percentage to form: glucose 2%; Analysis for soybean powder 2%; Semen Maydis powder 3%; Potassium primary phosphate 0.2%; Magnesium sulfate 0.05%; Vitamins B
10.05g/l, agar 0.3 gram/100ml, surplus is sterilized water, pH nature.
All the other contents are with embodiment 1.
Embodiment 3
The pleurotus eryngii liquid strain fermention medium of improvement, this fermention medium adopts the component of following mass percentage to form: glucose 2%; Analysis for soybean powder 1%; Semen Maydis powder 2%; Potassium primary phosphate 0.2%; Magnesium sulfate 0.05%; Vitamins B
10.02g/l, gelatin, agar mixture 0.3 gram/100ml, surplus is sterilized water, pH nature.
All the other contents are with embodiment 1.
Embodiment 4
The pleurotus eryngii liquid strain fermention medium of improvement, this fermention medium adopts the component of following mass percentage to form: glucose 1.5%; Analysis for soybean powder 1.5%; Semen Maydis powder 2.3%; Potassium primary phosphate 0.1%; Magnesium sulfate 0.04%; Vitamins B
10.03g/l, gelatin 0.2 gram/100ml, surplus is sterilized water, pH nature.
All the other contents are with embodiment 1.
Embodiment 5
The pleurotus eryngii liquid strain fermention medium of improvement, this fermention medium adopts the component of following mass percentage to form: glucose 2%; Analysis for soybean powder 1.5%; Semen Maydis powder 2.5%; Potassium primary phosphate 0.1%; Magnesium sulfate 0.04%; Vitamins B
10.04g/l, agar 0.25 gram/100ml, surplus is sterilized water, pH nature.
All the other contents are with embodiment 1.
Claims (2)
1. the pleurotus eryngii liquid strain fermention medium of improvement, is characterized in that this fermention medium adopts the component of following mass percentage to form: glucose 1%-2%; Analysis for soybean powder 1%-2%; Semen Maydis powder 2%-3%; Potassium primary phosphate 0.05%-0.2%; Magnesium sulfate 0.01%-0.05%; Vitamins B
10.02g/l-0.05g/l, a kind of in gelatin, agar or 0.1 gram, the mixture/100ml-0.3 gram/100ml of the two, surplus is sterilized water, pH nature.
2. utilize fermention medium as claimed in claim 1 to cultivate the method for pleurotus eryngii liquid strain, it is characterized in that pleurotus eryngii quel strains to be inoculated in and carry out cultivation in the substratum of claim 1 and make pleurotus eryngii liquid strain, wherein culture condition is temperature is 25 ± 3 DEG C, and rotating speed is 180-200r/m; 6-8d is cultivated in shaking table concussion; Described pleurotus eryngii quel strains selects the one in Pleurotus eryngii (Pleurotus eryngii) CICC 14011, Pleurotus eryngii (Pleurotus eryngii) ACCC 51678, Pleurotus eryngii (Pleurotus eryngii) ACCC 51330.
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| CN104844354A (en) * | 2015-04-29 | 2015-08-19 | 天津农学院 | High-density pleurotus eryngii liquid strain fermentation medium |
| CN105009931A (en) * | 2015-04-13 | 2015-11-04 | 鲁东大学 | Preparation of liquid strain for pleurotus eryngii and research method of culture technique of high-quality high-yield pleurotus eryngii through liquid strain |
| CN106386173A (en) * | 2016-09-18 | 2017-02-15 | 胡云龙 | Pleurotus eryngii high-yield cultivating method |
| CN107736184A (en) * | 2017-11-07 | 2018-02-27 | 江苏久禾生物科技发展有限公司 | A kind of Pleurotus eryngii industrial cultivation method based on high activity liquid spawn |
| CN108147875A (en) * | 2018-03-07 | 2018-06-12 | 常熟理工学院 | A kind of factory culture pleurotus eryngii cultivating material |
| CN109526565A (en) * | 2018-12-13 | 2019-03-29 | 成都中延榕珍菌业有限公司 | A kind of Pleurotus eryngii liquid fermentation medium and strain cultivation method and method for planting almond abalone mushroom |
| CN113186106A (en) * | 2021-03-31 | 2021-07-30 | 盐城工学院 | Pleurotus eryngii liquid culture medium and application thereof |
| CN113973646A (en) * | 2021-07-28 | 2022-01-28 | 盐城工学院 | Culture medium and culture method for improving yield of pleurotus eryngii |
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| CN109526565A (en) * | 2018-12-13 | 2019-03-29 | 成都中延榕珍菌业有限公司 | A kind of Pleurotus eryngii liquid fermentation medium and strain cultivation method and method for planting almond abalone mushroom |
| CN113186106A (en) * | 2021-03-31 | 2021-07-30 | 盐城工学院 | Pleurotus eryngii liquid culture medium and application thereof |
| CN113973646A (en) * | 2021-07-28 | 2022-01-28 | 盐城工学院 | Culture medium and culture method for improving yield of pleurotus eryngii |
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