CN111118105A - Method and application of rapid determination of strain degradation of Pleurotus eryngii based on mycelial characteristics and biochemical indexes - Google Patents
Method and application of rapid determination of strain degradation of Pleurotus eryngii based on mycelial characteristics and biochemical indexes Download PDFInfo
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Abstract
本发明提供了一种基于菌丝特征与生化指标的杏鲍菇菌种衰退的方法及其应用。本申请的判断方法，通过杏鲍菇菌种连续传代培养，分析测定不同代数杏鲍菇菌种酶活性、菌丝特征，探讨杏鲍菇连续传代过程中的菌种退化情况，以明确杏鲍菇菌种可使用种代数及菌种出现衰退的菌丝特征及其酶活表现，为杏鲍菇工厂化栽培时菌种质量提供快速鉴定方法。利用菌丝特点及其平板显色法定性测定酶活适合于杏鲍菇菌种的快速检测，耗时短、成本低、结果稳定可靠，可用作工厂化菌种质量判断。The present invention provides a method and application for the decline of Pleurotus eryngii strains based on mycelial characteristics and biochemical indexes. The judgment method of the present application, through the continuous subculture of Pleurotus eryngii, analyzes and measures the enzyme activity and mycelial characteristics of the Pleurotus eryngii strains of different generations, and discusses the degradation of the Pleurotus eryngii during the continuous passage of Pleurotus eryngii, so as to clarify the degradation of Pleurotus eryngii during the continuous subculture process. Mushroom strains can use the species algebra, the hyphal characteristics of the strains' declining hyphae and their enzymatic activity performance, and provide a rapid identification method for the quality of the strains during the industrial cultivation of Pleurotus eryngii. The use of the characteristics of mycelium and the method for qualitative determination of enzyme activity by plate chromogenic method is suitable for the rapid detection of Pleurotus eryngii strains.
Description
Technical Field
The invention belongs to the technical field of agriculture, and particularly relates to a method for rapidly judging pleurotus eryngii strain degradation based on hypha characteristics and biochemical indexes and application thereof.
Background
Pleurotus eryngii (Pleurotus eryngii)Pleurotus eryngii) Taxonomic localization belongs to the phylum Eumycota, Basidiomycotina, Hymenomycetes, Agaricales, Pleurotaceae. Also called Pleurotus Citrinopileatus Sing, contains abundant nutrients such as protein, vitamins, carbohydrate, etc., and mineral elements such as zinc, magnesium, calcium, copper, etc. The pleurotus eryngii fruiting body is rich and thick in meat quality, crisp and tender in texture and fresh and cool in taste, can enhance the human body immunity, has the effects of reducing blood fat, beautifying, moistening intestinal tracts, resisting cancer and the like, and is a rare edible fungus variety which is deeply loved by consumers and integrates the functions of food, medicine and therapy. At present, pleurotus eryngii is mainly from industrial cultivation, and the key problem influencing the industrial cultivation is strain degradation. The strain degeneration refers to the phenomenon that economic characters of edible fungus populations are deteriorated, so that the yield, quality, resistance and the like of the edible fungus populations are changed from human needs. The fungus can have strain degeneration phenomenon in the process of subculture on an artificial culture medium, and has the appearance of thin and weak hyphae, sparse aerial hyphae, slow hyphae growth and easy pollution by mixed bacteria in external form; the production is characterized by delayed fruiting period, reduced fruiting number, irregular fruiting, reduced yield and the like. Thereby causing huge losses of manpower, material resources and financial resources. Most pleurotus eryngii production factories still adopt sporocarp separation and strain subculture for preserving and using strains, but the factories do not have a set of rapid judgment indexes for whether the pleurotus eryngii strains degenerate or not, and more or more fruiting yields are used for judging, so that the method is time-consuming, and the economic benefit of the factories is greatly influenced. Therefore, it is imperative to find a set of indexes for rapidly judging strain degeneration.
Disclosure of Invention
At present, the research on the pleurotus eryngii mainly focuses on the aspects of cultivation technology, breeding technology and active substance analysis, and the relevant research on the strain degradation problem in the pleurotus eryngii factory production has no substantial progress; and the strain degradation of the factory still remains to be judged through the change of yield, which wastes time and labor. Therefore, the experiment aims to provide a method for rapidly judging the degradation of the pleurotus eryngii strains by researching hypha characteristics and physiological and biochemical indexes of the degraded strains.
In order to achieve the purpose, the invention adopts the following technical scheme:
(1) measuring the growth speed of hyphae, the diameter of the hyphae, the branch quantity of the hyphae and the colony morphology of the strain: inoculating Pleurotus eryngii strain to PDA culture medium, culturing at 25 deg.C for 7d, and activating strain; inoculating the activated pleurotus eryngii strains of different generations to a PDA culture medium for culture, and compared with the first generation of original strains, reducing the average growth speed of hyphae by more than or equal to 22 percent, reducing the diameter of the hyphae by more than or equal to 16 percent, reducing hyphae branches, stopping the growth of the hyphae, enabling colonies to rise irregularly, enabling thin edges of the hyphae to be in an extremely irregular round shape, white, cotton-like and judging the hyphae to be degenerated;
(2) determination of cellulase secretion Capacity: the colony diameter, the color-changing ring diameter and the enzyme index are respectively 24.50-32.50 mm, 26.00-42.00 mm and 1.10-1.30 EI, and the degeneration is judged;
(3) determination of amylase secretion capacity: the colony diameter, the color-changing ring diameter and the enzyme index are respectively 52.00-70.00 mm, 13.50-26.00 mm and 0.25-0.37 EI, and the degradation is judged;
(4) manganese peroxidase secretion ability: when the colony diameter, the color-changing ring diameter and the enzyme index are respectively in the range of 52.00-71.00 mm, 13.50-26.00 mm and 0.25-0.37 EI or less than the range, judging the degradation;
(5) LBL decolorizing capacity: when the decolorization rate is within the range of 48.00% -55.00% or lower than the range, judging the degradation;
(6) speed of decomposing cultivation material: the average growth rate of the hyphae of the pleurotus eryngii strains in the cultivation material is reduced by more than or equal to 7.5 percent, and the pleurotus eryngii strains are judged to be degenerated.
The method for measuring the growth speed of the hyphae comprises the following steps: measuring the growth speed of the hyphae every two days after the hyphae germinate until the hyphae grow over the flat plate; average rate of hypha growth mm/d = hypha length over plate/number of days hyphae over plate.
The observation method of the hypha diameter, the hypha branch quantity and the bacterial colony morphology of the strain comprises the following steps: inoculating the activated pleurotus eryngii strains of each generation to a PDA culture medium, culturing at 25 ℃, observing the change condition of colony morphology in the culture process until hyphae grows over a flat plate, selecting a small piece of culture medium at the same position on the activated pleurotus eryngii strain PDA culture medium of each generation, placing the small piece of culture medium on a glass slide, observing the hyphae morphology under a 40-time lens after slicing, and measuring the hyphae diameter of each generation by using Image-Pro Plus software.
The method for measuring the cellulase secretion capacity, the amylase secretion capacity and the manganese peroxidase secretion capacity comprises the following steps: respectively inoculating the activated different generations of pleurotus eryngii strains into a congo red-sodium carboxymethyl cellulose culture medium, a PDA soluble starch culture medium and a PDA-guaiacol culture medium by using a puncher with the aperture of 1cm, culturing at 25 ℃, measuring the colony diameter and the color change ring diameter at the 7 th day, and calculating an enzyme index EI; EI = discolouration circle diameter/colony diameter, results are expressed as mean ± standard deviation of 5 replicates.
The congo red-sodium carboxymethylcellulose culture medium comprises the following components in percentage by weight: sodium carboxymethylcellulose 2.0g/L, MgSO4·7H2O1.88g/L，KH2PO40.5g/L, 2.0g/L of peptone, 0.25g/L of Congo red and 15g/L of agar powder;
the PDA soluble starch culture medium comprises the following components: 200g/L of potato, 10g/L of glucose and 2g/L of soluble starch;
the PDA-guaiacol culture medium comprises the following components in parts by weight: 200g/L of potato, 10g/L of glucose and MgSO4·7H2O 1g/L，KH2PO41g/L, vitamin B10.02 g/L, agar 15g/L and guaiacol 0.1 g/L.
The LBL decolorization capacity determination method comprises the following steps: inoculating the preserved pleurotus eryngii strains of all generations to a PDA culture medium for activation, moving 5 mycelium blocks with the diameter of 1cm from the activated PDA culture medium of different generations of pleurotus eryngii strains to an LBL liquid culture medium, and carrying out liquid culture at the temperature of 25 ℃, wherein each generation has 5 biological repetitions; after liquid culture for 3d, centrifuging 10 ml of culture solution at 8000 r/min and 4 deg.C for 15 min, transferring supernatant, and measuring absorbance at 615nm, wherein the decolorization rate DR = (1- (A)615nm strain/A615nm blank))×100%。
The method for measuring the speed of decomposing the cultivation material comprises the following steps: transferring 1 mycelium block with the diameter of 1cm from activated PDA culture medium of Pleurotus eryngii strains of different generations into a large test tube filled with 40g of cultivation material, performing biological repetition for 5 generations, culturing at 25 deg.C for 30 d, measuring growth speed every 5 days, and observing the condition of mycelium decomposition cultivation material; the formula of the cultivation material comprises the following components in parts by mass: 14% of cottonseed hulls, 64% of sawdust, 20% of bran, 1% of gypsum and 1% of sugar.
Further, the method is applied to rapid judgment of pleurotus eryngii strain decline.
The invention has the advantages that: according to the invention, the enzyme activity and the hypha characteristics of pleurotus eryngii strains of different generations are analyzed and measured through continuous subculture of the pleurotus eryngii strains, the strain degeneration condition in the process of continuous subculture of the pleurotus eryngii is discussed, the hypha characteristics and the enzyme activity expression of the strain that the strain generations and the strains decline can be used by the pleurotus eryngii strains are determined, and a rapid identification method is provided for the strain quality during industrial cultivation of the pleurotus eryngii. The method for qualitatively determining the enzyme activity by utilizing the characteristics of the hypha and the plate color development method is suitable for the rapid detection of the pleurotus eryngii strains, has the advantages of short time consumption, low cost and stable and reliable results, and can be used for judging the quality of the industrial strains.
Drawings
FIG. 1 is a microscopic structure of pleurotus eryngii hyphae of different generations under a microscope view of 40 times;
FIG. 2 shows the strain morphs of Pleurotus eryngii of different generations;
FIG. 3 is a comparison of the hypha decomposition material capability of different generations of Pleurotus eryngii strains.
Detailed Description
1. Test strains: the method comprises the steps of separating pleurotus eryngii sporocarp, obtaining target strains through subculture, and storing 8 generations of strains through subculture in life science college of Fujian agriculture and forestry university, wherein the pleurotus eryngii sporocarp comes from Zhangzhou green biological science and technology limited company.
2. Culture medium
(1) PDA solid enriched medium (1L): 200g of potato, 20g of glucose, 2g of yeast powder and peptone3g, 1.5g potassium dihydrogen phosphate, magnesium sulfate, 7H2O1.5 g, vitamin B10.1g of agar and 20g of agar.
(2) Standard PDA solid medium (1L): 200g of potato, 20g of glucose and 20g of agar.
(3) LBL medium (1L): 200g of potato, 20g of lactose and NH4NO32g，MgSO4·7H2O 0.5g，KH2PO41.5g， BTB 0.06g，pH 7.0。
(4) Crabapple red-sodium carboxymethylcellulose medium (1L): sodium carboxymethylcellulose 2.0g, MgSO4·7H2O1.88g，KH2PO40.5g, peptone 2.0g, Congo red 0.25g and agar powder 15 g.
(5) PDA soluble starch medium (1L): 200g of potato, 10g of glucose and 2g of soluble starch.
(6) PDA-tannic acid medium: 200g of potato, 10g of glucose and MgSO4·7H2O 1g，KH2PO41g, vitamin B10.02 g, agar 15g and tannic acid 0.4 mmol.
(7) PDA-guaiacol medium: 200g of potato, 10g of glucose and MgSO4·7H2O 1g，KH2PO41g, vitamin B10.02 g, agar 15g and guaiacol 0.1 g.
(8) The formula of the cultivation material is as follows: 14% of cottonseed hulls, 64% of sawdust, 20% of bran, 1% of gypsum and 1% of sugar.
Sterilizing the culture medium at 121 deg.C for 30min, sterilizing the cultivation material at 121 deg.C for 4 hr, and cooling.
3. Laboratory apparatus
A constant temperature incubator, an ultra-clean workbench, an optical microscope and a spectrophotometer.
4. Experimental methods
(1) And (3) measuring the growth speed of pleurotus eryngii hyphae: inoculating the preserved Pleurotus eryngii strains of each generation to PDA culture medium, culturing at 25 deg.C for about one week, and activating the strains. Inoculating the activated pleurotus eryngii strains of different generations to a PDA culture medium, wherein the inoculation area is 5mm multiplied by 5mm, 10 times of treatment is set, and the growth speed of the mycelia is measured every two days after the mycelia germinate until the mycelia grow over the flat plate.
Average rate of hypha growth (mm/d) = hypha length (mm) over plate/number of days (d) for hypha to grow over plate
(2) Measurement of the diameter of pleurotus eryngii hyphae and observation of colony morphology: inoculating the activated pleurotus eryngii strains of each generation to a PDA culture medium, culturing at 25 ℃, observing the change condition of colony morphology in the culture process until hyphae grows over a flat plate, selecting a small piece of culture medium at the same position on the activated pleurotus eryngii strain PDA culture medium of each generation, placing the small piece of culture medium on a glass slide, observing the hyphae morphology under a 40-time lens after slicing, and measuring the hyphae diameter of each generation by using Image-Pro Plus software.
(3) Plate color change ring experiment: inoculating the activated different generations of pleurotus eryngii strains to the center of a corresponding test culture medium (a congo red-sodium carboxymethyl cellulose culture medium (a cellulase system is determined), a PDA soluble starch culture medium (an amylase is determined), and a PDA-guaiacol culture medium (a manganese peroxidase is determined)) by using a puncher (the aperture is 1 cm), culturing at 25 ℃, measuring the diameter of a colony and the diameter of a color change ring at 7d, measuring the diameter of the colony at an amylase index, removing hyphae on the surface of the colony, dyeing by using a Lugol iodine solution method, and finally measuring the diameter of an area which is not dyed into blue. The Enzyme Index (EI) EI = color change circle diameter/colony diameter was calculated and the results are expressed as the mean ± standard deviation of 5 replicates.
(4) LBL medium decolorization experiment: inoculating the preserved Pleurotus eryngii strains of each generation to PDA culture medium for activation, transferring 5 mycelium blocks with diameter of 1cm from the activated PDA culture medium of different generations of Pleurotus eryngii strains to LBL liquid culture medium, and performing liquid culture at 25 deg.C with 5 biological repetitions per generation. After liquid culture for 3d, 10 ml of culture solution is taken, centrifuged for 15 min at 8000 r/min and 4 ℃, supernatant is removed, and the absorbance value is measured at 615nm, and the decolorization rate is calculated by using a decolorization rate formula.
DR=（1－(A615nm strain/A615nm blank))×100%。
In the formula, DR represents a decolorization rate, A615 nm strain is an absorbance value after the inoculated strain is cultured, and A615 nm is an absorbance value of a blank culture solution. The higher the DR value, the higher the hyphal activity and the higher the strain quality.
(5) Pleurotus eryngii hypha decomposition cultivation material experiment: inoculating the preserved Pleurotus eryngii strains of each generation to a PDA culture medium for activation, transferring 1 mycelium block with the diameter of 1cm from the activated PDA culture medium of different generations of Pleurotus eryngii strains into a large test tube filled with 40g of cultivation material, repeating the steps for 5 generations, culturing at 25 ℃ for 30 days, measuring the growth speed every 5 days, and observing the condition of mycelium decomposition cultivation material.
5. Results and analysis
(1) Variation of growth speed of different generations of Pleurotus eryngii strains
As can be seen from Table 1, the growth rate of the hyphae of the Pleurotus eryngii strain is significantly reduced at G7, the average growth rate is 3.00 +/-0.02 mm/d, and the growth rate is reduced by 21.88% compared with that of G1. Therefore, when the average growth rate of the pleurotus eryngii hyphae is reduced by more than 22 percent, the strains are degraded to a certain degree, and are not suitable for being used as seeds for factory cultivation and production.
TABLE 1 growth rate of hyphae of Pleurotus eryngii strain of different generations
(2) Variation of hypha diameters of different algebraic strains of pleurotus eryngii
As can be seen from Table 2, the hypha diameter of G7 is 2.93 +/-0.20 mu m, the obvious reduction is realized, the reduction percentage reaches 16.76%, and the hypha diameter of G15 continuously descends along with the subculture, the hypha diameter is only 2.56 +/-0.23 mu m, and the reduction percentage reaches 27.27%. Therefore, when the reduction percentage of the pleurotus eryngii hypha diameter is more than 16 percent, the strain can be shown to be degraded to a certain degree, and is not suitable for being used as a plant cultivation and production seed any more.
TABLE 2 hypha diameters of different generations of Pleurotus eryngii strains
(3) Variation of hypha microscopic structure of different generations of pleurotus eryngii strains
As is clear from FIG. 1, as the number of generations increased, hyphal branching decreased gradually, the number of hyphae in the microscopic field from G7 decreased significantly, and hyphal branching decreased significantly. Therefore, when the hypha amount and hypha branches are greatly reduced under the visual field of a microscope of 40 times, the bacterial strain can be shown to be degraded to a certain degree, and is not suitable for being used as a plant cultivation production seed any more.
(4) Variation of colony morphology of different algebraic strains of pleurotus eryngii
As can be seen from FIG. 2, the bacterial colony from G7 appears, the hyphae can stagnate when growing on the culture medium to a certain extent but not growing on the plate, and the hyphae grown subsequently are weak, the bacterial colony structure is compact, the interior is slightly concave, the hyphae are vigorous, the edge is in a regular round shape, thick white and felt shape, and the variation degree gradually deepens along with the successive transfer; when the bacterial colony reaches G15, the bacterial colony is flat, the surface has irregular uplift, the hypha is thin, the edge is in an extremely irregular round shape, the bacterial colony is white and flocculent, and the hypha stagnation phenomenon is more obvious. Therefore, when the pleurotus eryngii hyphae are cultured on the culture medium, the situation can indicate that the strains are degraded to a certain degree and are not suitable for being used as seeds for factory cultivation and production.
(5) Results of plate discoloration circles experiment of different generations of pleurotus eryngii strains
As can be seen from tables 3, 4 and 5, after the mycelium is cultured for 7 days, the results of the three color change circle experiments show that G7 shows obvious reduction in the corresponding colony diameter, color change circle diameter and enzyme activity index, and the reduction trend is continued as the subculture is carried out. Therefore, the colony diameter, the discoloring ring diameter and the enzyme index of the pleurotus eryngii strain hyphae on the congo red-sodium carboxymethylcellulose culture medium are respectively 24.50-32.50 mm, 26.00-42.00 mm and 1.10-1.30 EI; the colony diameter, the color change circle diameter and the enzyme index on the PDA soluble starch culture medium are respectively 52.00-70.00 mm, 13.50-26.00 mm and 0.25-0.37 EI; when the colony diameter, the color change circle diameter and the enzyme index on the PDA-guaiacol culture medium are respectively in the range of 52.00-71.00 mm, 13.50-26.00 mm and 0.25-0.37 EI or less than the range, the bacterial strain can be shown to be degenerated to a certain degree.
TABLE 3 Congo Red-sodium carboxymethylcellulose test results
TABLE 4 starch-Lugol iodine test results
TABLE 5 PDA-guaiacol Experimental results
(6) LBL decolorization experimental result of different generations of pleurotus eryngii strains
As can be seen from Table 6, there was a significant change in the initial decolorization rate from G7, while the lower the decolorization rate, the weaker the activity of the strain, the decolorization rate of G7 was 54.29. + -. 0.52%. Therefore, when the LBL decolorization rate of the pleurotus eryngii strain hyphae is in the range of 48.00% -55.00% or is lower than the range, the strain activity is reduced, and the strain is degenerated.
TABLE 6 LBL decolorization test of Pleurotus eryngii strains of different generations
(7) Experimental result of decomposing cultivation material by using pleurotus eryngii strains of different generations
As can be seen from Table 7 and FIG. 3, the rate and ability of mycelia to decompose the cultivation material from G7 began to be significantly reduced, and the growth rate of mycelia in the cultivation material was 3.24. + -. 0.06mm/d, and the percentage of reduction was 7.95%. Therefore, when the average growth rate of the pleurotus eryngii strain hyphae in the cultivation material is reduced by more than 7.5 percent, the strain is characterized to be degraded to a certain extent on the production level.
TABLE 7 growth rate of hyphae in cultivation material of Pleurotus eryngii strain of different generations
6. Summary of the invention
By researching the hypha characteristics and physiological and biochemical indexes of strains of each generation, the results show that different generations of strains have certain differences in the seven aspects of hypha growth speed, hypha diameter, hypha microstructure, colony morphology, color change circle test, LBL decolorization test, hypha decomposition cultivation material speed and the like, and all show that the indexes can be used as indexes for representing strain degradation from G7. The various indexes for judging the degradation of the pleurotus eryngii strains determined by the research can quickly judge whether the strains degrade or not, and can provide important reference for factories in quickly judging the strains.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (8)
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