CN101375691B - Lepista sordida extract as well as preparation method and use thereof - Google Patents
Lepista sordida extract as well as preparation method and use thereof Download PDFInfo
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Abstract
The invention provides an extract of a Lepista sordida, a preparation method and an application thereof. The method comprises the following steps: (1) mixing the Lepista sordida entity powder with a phosphate buffer, the PH value of which is 6-9; leaching at the temperature ranging from 0 to 4 DEG C; centrifuging to extract a supernatant fluid to acquire a crude extract from the Lepista sordida; (2) adding ammonium sulphate in the supernatant fluid to achieve 50 percent of saturation and centrifuging the supernatant fluid after the supernatant fluid stands for 8-16 hours at the temperature ranging from 0 to 4 DEG C; (3) adding ammonium sulphate in the supernatant fluid to achieve 70 percent of saturation, and centrifuging the supernatant fluid after the supernatant fluid stands for 8-16 hours at the temperature ranging from 0 to 4 DEG C; (4) dissolving with distilled water and precipitating, so as to acquire the protein of the Lepista sordida which resists TMV. The extract of the Lepista sordida belongs to the natural extract, is not harmful to the human beings, and is environment-friendly. Furthermore, the purifying method of the extract of the Lepista sordida is very simple, detailed in steps, and easy to operate.
Description
Technical field
The present invention relates to a kind of paint face mushroom extract and its preparation method and application, specially refer to a kind of paint face mushroom Antiphytoviral protein extract and its preparation method and its application in preventing and treating the viroses of plant.
Background technology
The viroses of plant are class diseases that are difficult to control in producing, and have the title of " plant cancer ".According to estimates, the loss that whole world every year causes because of viral diseases of plants is above 15,000,000,000 dollars, wherein the harm of tobacco mosaic virus disease (Tobacco mosaicvirus) cause every year with a toll of more than 100,000,000 dollars (Wu Yunfeng. world agriculture, 1995, (5): 35～36).(Tobacco mosaic virus is a kind of very important plant virus TMV) to tobacco mosaic virus (TMV), and host range is extensive, can endanger various plants.At present, also there is not the very effective method of preventing and treating for the tobacco mosaic virus disease evil.In recent years, find that from natural extract some albumen can have certain prevention effect to this virus, the antiviral protein that especially extracts from higher fungi has caused widely to be paid close attention to.And this natural extract is minimum to the hazardness of human and environment, belongs to environmentally friendly.
Edible mushrooms is extensively understood by people institute as a kind of food, and its contained kinds of protein is various and content abundant, is the better source material of antiviral protein screening.But the research aspect the preventing and treating of the viroses of plant of the activeconstituents in the edible mushrooms up to the present is also fewer, several special case report both domestic and external operation is all complicated unusually, be unfavorable for production application, and the bacterial classification source mainly is the artificial culture kind, lacks from the popularity of wild edible bacterial classification natural product screening.Paint face mushroom (Lepista sordida) belongs to Basidiomycotina (Basidiomycotina) Lepista lentinus (Lepista).The general median size of its sporophore, rich in protein, higher in the output of summer and autumn, main wild in the Inner Mongol, ground such as Heilungkiang, northeast and Fujian, at present as yet not relevant for any report of active substance Antiphytoviral in the paint face mushroom.
Summary of the invention
The purpose of this invention is to provide a kind of paint face mushroom extract and other preparation method and application.
Paint face mushroom preparation method of extract provided by the present invention is that paint face mushroom fruit body powder is mixed with the phosphoric acid buffer of pH value for 6-9, and 0-4 ℃ of lixiviate, its supernatant liquor of centrifuging and taking promptly obtains paint face mushroom crude extract solution.
In the described method, the blending ratio of paint face mushroom fruit body powder dry weight and phosphoric acid buffer is a 1g paint face mushroom fruit body powder dry weight: the 10-20ml phosphoric acid buffer.
In the described method, the pH value of phosphoric acid buffer is preferably 8.0; The time of described lixiviate is preferably 2-6 hour.
In the described method, the particle diameter of paint face mushroom fruit body powder is below 60 orders.Described paint face mushroom fruit body powder be with paint face mushroom sporophore 20-40 ℃ of oven dry, pulverize, cross the paint face mushroom sporophore powder that 60 mesh sieves obtain.
In the described method, the ratio that the described centrifugal precipitation that obtains can also 1g:10-20ml is mixed with the phosphoric acid buffer of pH value for 6-9,0-4 ℃ of lixiviate, obtains paint face mushroom crude extract solution.
In the described method, paint face mushroom extract solution also extracts through following step:
1) be 50% with adding ammonium sulfate to saturation ratio in the described paint face mushroom crude extract solution, after 8-16 hour, centrifugal 0-4 ℃ of placement, collect supernatant liquor;
2) adding ammonium sulfate to saturation ratio in the supernatant liquor that step 1) is obtained is 70%, and after 8-16 hour, centrifugal, collecting precipitation obtains paint face mushroom protein extract 0-4 ℃ of placement.
The paint face mushroom protein extract that paint face mushroom extract provided by the present invention is a method for preparing.
Paint face mushroom protein extract of the present invention can be used for preventing and treating tobacco mosaic virus (TMV).
The present invention is a material with the paint face mushroom first, its sporophore has been carried out the active ingredient analysis, and the active substance of its anti-TMV has been carried out purifying and property research, for the exploitation of the biogenic pesticide of anti-TMV provides another brand-brand-new way.Paint face mushroom protein extract of the present invention has the good restraining effect to TMV, transfer to after protein concentration is 60 μ g/ml at extract, adopt the mode of cauline leaf spraying to handle tobacco plant, can evoking tobacco to the resistance of TMV, infecting inhibiting rate and can reach more than 77% tobacco mosaic virus (TMV).Paint face mushroom extract of the present invention belongs to natural extract, and is all harmless to the mankind and environment.The simple operation easily of the preparation method of paint face mushroom protein extract of the present invention.
Description of drawings
Fig. 1 is the schematic flow sheet of paint face mushroom protein extract preparation method of the present invention
Fig. 2 is the anti-TMV experiment of a paint face mushroom protein extract of the present invention photo
Embodiment
Method described in the following embodiment if no special instructions, is ordinary method.
Percentage composition shown in the following embodiment if no special instructions, is the quality percentage composition.
The acquisition of embodiment 1, a kind of paint face mushroom extract and effect experiment thereof
One, the acquisition of paint face mushroom crude extract
1, the paint face mushroom sporophore of above-mentioned acquisition is dried in 30 ℃ of baking ovens, pulverize, cross 60 mesh sieves and obtain paint face mushroom sporophore powder.
2, paint face mushroom sporophore powder that the 10g step 1) is obtained and the phosphoric acid buffer of 0.025mol/L pH8.0 are according to the mixed of 1g/10ml; At 4 ℃, lixiviate is after 4 hours, centrifugal 20 minutes of 10000rpm/min, collect supernatant liquor, with precipitation again in the phosphoric acid buffer of 0.025mol/L pH8.0 mixed, centrifugal collection supernatant liquor according to 1g/10ml, merge the supernatant liquor that obtains for twice, obtain 200ml paint face mushroom crude extract.Detect the protein concentration of this paint face mushroom crude extract with BCA protein concentration test kit (U.S. Pierce company), measuring method carries out according to the specification sheets of test kit.
Two, the anti-TMV active substance of paint face mushroom extract separates
1, the paint face mushroom crude extract that step 1 is obtained, carry out following fractionation precipitation:
1) 0-30% ammonium sulfate precipitation: paint face mushroom buffer extraction liquid adding ammonium sulfate to the saturation ratio that step 1 is obtained is 30%, places 4 ℃ of precipitation 12h down, and 10000rpm/min is centrifugal, and the 20min precipitation is the 0-30% ammonium sulfate precipitated protein.Collect supernatant liquor simultaneously and wait until subsequent operations.
2) the centrifugal supernatant liquor that obtains of step 1) being continued to add ammonium sulfate to saturation ratio is 40%, places 4 ℃ of precipitation 12h down, and 10000rpm/min is centrifugal, and the 20min precipitation is 30%～40% ammonium sulfate precipitated protein.Collect supernatant liquor simultaneously and wait until subsequent operations.
3) with step 2) the centrifugal supernatant liquor that obtains continues to add ammonium sulfate to saturation ratio is 50%, places 4 ℃ of precipitation 12h down, and the centrifugal 20min of 10000rpm/min precipitates and is 40%～50% ammonium sulfate precipitated protein.Collect supernatant liquor simultaneously and wait until subsequent operations.
4) the centrifugal supernatant liquor that obtains of step 3) being continued to add ammonium sulfate to saturation ratio is 60%, places 4 ℃ of precipitation 12h down, and 10000rpm/min is centrifugal, and the 20min precipitation is 50%～60% ammonium sulfate precipitated protein.Collect supernatant liquor simultaneously and wait until subsequent operations.
5) the centrifugal supernatant liquor that obtains of step 1) being continued to add ammonium sulfate to saturation ratio is 70%, places 4 ℃ of precipitation 12h down, and 10000rpm/min is centrifugal, and the 20min precipitation is 60%～70% ammonium sulfate precipitated protein.Collect supernatant liquor simultaneously and wait until subsequent operations.
6) the centrifugal supernatant liquor that obtains of step 1) being continued to add ammonium sulfate to saturation ratio is 85%, places 4 ℃ of precipitation 12h down, and 10000rpm/min is centrifugal, and the 20min precipitation is 70%～85% ammonium sulfate precipitated protein.
2, the component that the paint face mushroom crude extract that respectively step 1 is obtained and above-mentioned each fractionation precipitation obtain is used 0.025mol/L, and the phosphoric acid buffer of PH=8.0 is diluted to total protein concentration and is 60 μ g/ml.Handle tobacco according to following method, and detect their inhibiting rates TMV.
1) plant is handled: with the above-mentioned extract that obtains and each fractionation precipitation solution (total protein concentration is 60 μ g/ml), respectively the withered spot three lives cigarette (Nico tiana tabacum SamsunNN) of seedling age unanimity (3-4 leaf) is put in order the strain spraying, three repetitions are done in each processing, each repeats three strains, three leaves of every strain.Contrast (CK) (contrast is only with the clear water processing of spraying) is set simultaneously.
2) preparation of virus inoculation liquid: virus is that (with common cigarette (Ncotiana tabacumHawana38) is the breeding host in the common strain of TMV system, this research department's live body is preserved), its inoculation liquid acquisition methods is: the tobacco leaf that will infect the common cigarette of TMV is removed master pulse, mix with 1:20 (g/ml) with the phosphoric acid buffer of 0.01mol/L pH=8.0, every 20ml phosphoric acid buffer adds 0.015g silicon carbide, blade is ground, obtain inoculation liquid.
3) tobacco that method spraying inoculation: will be set by step 1) obtained after 72 hours is with step 2) inoculation liquid that obtains carries out frictional inoculation (planting the disease research method third edition, Fang Zhongda, Chinese agriculture press), uses flushing with clean water after the inoculation immediately, in hot-house culture.Culture condition is 28 ℃ of illumination 12 hours, and 18 ℃ of unglazed photographs 12 hours hocket.
(4) inhibiting rate calculates: inoculate 72 hours " Invest, Then Investigate " incidences, investigate every blade the withered spot number amount, be calculated as follows out withered spot inhibiting rate.
X=(CK-Y)/CK * 100 (formula I)
Among the formula I, X is withered spot inhibiting rate, unit: %; CK is the average withered spot number of clear water contrast blade, unit: individual; Y is for inducing the average withered spot number of handling rear blade, unit: individual through paint face mushroom sporophore crude extract.
The result is as shown in table 1, and the result shows that 50-60% ammonium sulfate precipitation component and 60-70% ammonium sulfate precipitation component are the highest to the TMV inhibiting rate.
The TMV of table 1. paint face mushroom extract suppresses experimental result
3, paint face mushroom extract further extracts
According to the conclusion that above-mentioned steps 1 and step 2 obtain, the paint face mushroom extract that step 1 is obtained is further extracted according to following method:
1) adding ammonium sulfate to saturation ratio in the paint face mushroom crude extract that step 1 obtains is 50%, and after 12 hours, centrifugal 20 minutes of 10000rpm/min collects supernatant liquor 4 ℃ of placements.
2) in the supernatant liquor that step 1) obtains, continue to add ammonium sulfate to saturation ratio 70%, 4 ℃ place 12 hours after, centrifugal 20 minutes of 10000rpm/min, collecting precipitation promptly obtains the anti-TMV protein extract of paint face mushroom.
The paint face mushroom protein extract that this step is obtained according to the method for step 2 carries out TMV to be suppressed to detect, and the result is shown in table 2 and Fig. 2 institute, and the result shows that the ammonium sulfate precipitation of this saturation ratio is better to the inhibition effect of TMV, can reach 77.1% in concentration during for 60ug/ml.A is that the paint face mushroom crude extract resisting tobacco mosaic virus that step 1 obtains suppresses the experiment photo among Fig. 2, wherein left side figure be contrast (CK) the plant leaf photo of frictional inoculation after clear water was sprayed 72 hours, and right side figure is the blade photo that paint face mushroom crude extract that step 1 obtains sprays the plant of frictional inoculation after 72 hours; B is that the TMV of the paint face mushroom protein extract that obtains of above-mentioned steps 3 suppresses the experiment photo among Fig. 2, wherein left side figure be the adjoining tree blade photo of frictional inoculation after clear water was sprayed 72 hours, and right side figure is the plant leaf photo that paint face mushroom protein extract that above-mentioned steps 3 obtains sprays frictional inoculation after 72 hours.
The TMV of table 2. paint face mushroom extract suppresses experimental result
The extracting method of embodiment 2, a kind of paint face mushroom extract and TMV thereof suppress experiment
The extraction schema of paint face mushroom extract of the present invention as shown in Figure 1, concrete grammar is as described below:
1) the paint face mushroom sporophore of above-mentioned acquisition is dried in 20 ℃ of baking ovens, pulverize, cross 60 mesh sieves and obtain paint face mushroom sporophore powder.
2) (0.02M is pH7.0) according to the mixed of 1g/20ml for paint face mushroom sporophore powder that the 10g step 1) is obtained and phosphoric acid buffer; At 4 ℃, lixiviate is after 2 hours, centrifugal 20 minutes of 10000rpm/min, collect supernatant liquor, will precipitate again that (0.02M is pH7.0) according to the mixed of 1g/20ml with phosphoric acid buffer, centrifugal collection supernatant liquor merges the supernatant liquor that obtains for twice, obtains 400ml paint face mushroom crude extract.Detect the protein concentration of this paint face mushroom crude extract with BCA protein concentration test kit (U.S. Pierce company), measuring method carries out according to the specification sheets of test kit.
3) in step 2) to add ammonium sulfate to saturation ratio in the paint face mushroom crude extract crude extract that obtains be 50%, 4 ℃ place 8 hours after, centrifugal 20 minutes of 10000rpm/min collects supernatant liquor.
4) in the supernatant liquor that step 1) obtains, continue to add ammonium sulfate to saturation ratio 70%, 4 ℃ place 8 hours after, centrifugal 20 minutes of 10000rpm/min, collecting precipitation promptly obtains the anti-TMV protein extract of paint face mushroom.
According to the method for the step 3 of the step 2 of embodiment 1, respectively to step 2), the paint face mushroom crude extract that obtains of step 4) carries out anti-TMV inhibiting rate test experience, the result is as shown in table 3.
The TMV of the paint face mushroom extract of this example of table 3. suppresses experimental result
The extracting method of embodiment 3, a kind of paint face mushroom extract and TMV thereof suppress experiment
The extraction schema of paint face mushroom extract of the present invention as shown in Figure 1, concrete grammar is as described below:
1) the paint face mushroom sporophore of above-mentioned acquisition is dried in 35 ℃ of baking ovens, pulverize, cross 60 mesh sieves and obtain paint face mushroom sporophore powder.
2) paint face mushroom sporophore powder that the 10g step 1) is obtained and phosphoric acid buffer (0.03M pH=6.0) are according to the mixed of 1g/10ml; At 4 ℃, lixiviate is after 6 hours, centrifugal 20 minutes of 10000rpm/min, collect supernatant liquor, will precipitate again and the mixed of phosphoric acid buffer (0.03M PH=6.0) according to 1g/10ml, centrifugal collection supernatant liquor, merge the supernatant liquor that obtains for twice, obtain 200ml paint face mushroom crude extract.Detect the protein concentration of this paint face mushroom crude extract with BCA protein concentration test kit (U.S. Pierce company), measuring method carries out according to the specification sheets of test kit.
3) in step 2) to add ammonium sulfate to saturation ratio in the paint face mushroom crude extract that obtains be 50%, 4 ℃ place 16 hours after, centrifugal 20 minutes of 10000rpm/min collects supernatant liquor.
4) in the supernatant liquor that step 1) obtains, continue adding ammonium sulfate to saturation ratio is 70%, 4 ℃ place 16 hours after, centrifugal 20 minutes of 10000rpm/min, collecting precipitation promptly obtains the anti-TMV protein crude extract of paint face mushroom.
According to the method for the step 3 of the step 2 of embodiment 1, respectively to step 2), the paint face mushroom extract that obtains of step 4) and step 6) carries out tobacco mosaic virus (TMV) inhibiting rate test experience, the result is as shown in table 4.
The tobacco mosaic virus (TMV) inhibiting rate test experience result of table 4. paint face mushroom of the present invention extract
The acquisition of embodiment 4, a kind of paint face mushroom extract and TMV thereof suppress experiment
The extraction schema of paint face mushroom extract of the present invention as shown in Figure 1, concrete grammar is as described below:
1) the paint face mushroom sporophore of above-mentioned acquisition is dried in 40 ℃ of baking ovens, pulverize, cross 60 mesh sieves and obtain paint face mushroom sporophore powder.
2) (0.025mol/L is PH=9.0) according to the mixed of 1g/10ml for paint face mushroom sporophore powder that the 10g step 1) is obtained and phosphoric acid buffer; At 4 ℃, lixiviate is after 3 hours, centrifugal 20 minutes of 10000rpm/min, collect supernatant liquor, will precipitate again that (0.025mol/L is pH=9.0) according to the mixed of 1g/10ml with phosphoric acid buffer, centrifugal collection supernatant liquor merges the supernatant liquor that obtains for twice, obtains 200ml paint face mushroom crude extract.Detect the protein concentration of this paint face mushroom crude extract with BCA protein concentration test kit (U.S. Pierce company), measuring method carries out according to the specification sheets of test kit.
3) in step 2) to add ammonium sulfate to saturation ratio in the paint face mushroom crude extract that obtains be 50%, 4 ℃ place 12 hours after, centrifugal 20 minutes of 10000rpm/min collects supernatant liquor.
4) in the supernatant liquor that step 1) obtains, continue to add ammonium sulfate to saturation ratio 70%, 4 ℃ place 8 hours after, centrifugal 20 minutes of 10000rpm/min, collecting precipitation promptly obtains paint face mushroom resisting tobacco mosaic virus protein extract.
According to the method for the step 3 of the step 2 of embodiment 1, respectively to step 2), the paint face mushroom extract that obtains of step 4) and step 6) carries out tobacco mosaic virus (TMV) inhibiting rate test experience, the result is as shown in table 5.
The tobacco mosaic virus (TMV) inhibiting rate test experience result of table 5. paint face mushroom of the present invention extract
The acquisition of embodiment 5, paint face mushroom mushroom extract of the present invention and effect experiment thereof
The extraction schema of paint face mushroom extract of the present invention as shown in Figure 1, concrete grammar is as described below:
1) the paint face mushroom sporophore of above-mentioned acquisition is dried in 30 ℃ of baking ovens, pulverize, cross 60 mesh sieves and obtain paint face mushroom sporophore powder.
2) (0.02M is pH8.0) according to the mixed of 1g/10ml for paint face mushroom sporophore powder that the 10g step 1) is obtained and phosphoric acid buffer; At 4 ℃, lixiviate is after 4 hours, centrifugal 20 minutes of 10000rpm/min, collect supernatant liquor, will precipitate again that (0.02mol/L is pH=8.0) according to the mixed of 1g/10ml with phosphoric acid buffer, centrifugal collection supernatant liquor merges the supernatant liquor that obtains for twice, obtains 200ml paint face mushroom crude extract.Detect the protein concentration of this paint face mushroom crude extract with BCA protein concentration test kit (U.S. Pierce company), measuring method carries out according to the specification sheets of test kit.
3) in step 2) to add ammonium sulfate to saturation ratio in the paint face mushroom extract crude extract that obtains be 50%, 4 ℃ place 12 hours after, centrifugal 20 minutes of 10000rpm/min collects supernatant liquor.
4) in the supernatant liquor that step 1) obtains, continue to add ammonium sulfate to saturation ratio 70%, 4 ℃ place 12 hours after, centrifugal 20 minutes of 10000rpm/min, collecting precipitation promptly obtains paint face mushroom resisting tobacco mosaic virus protein extract.
According to the method for the step 3 of the step 2 of embodiment 1, respectively to step 2), the paint face mushroom extract that obtains of step 4) carries out resisting tobacco mosaic virus inhibiting rate test experience, the result is as shown in table 6.
The TMV of table 6. paint face mushroom of the present invention mushroom extract suppresses experimental result
Claims (8)
1. the preparation method of a paint face mushroom protein extract is that paint face mushroom fruit body powder is mixed with the phosphoric acid buffer of pH value for 6-9, and 0-4 ℃ of lixiviate, the centrifuging and taking supernatant liquor obtains paint face mushroom extract solution; Described paint face mushroom extract solution also passes through the extraction of following step:
1) be 50% with adding ammonium sulfate to saturation ratio in the described paint face mushroom extract solution, after 8-16 hour, centrifugal 0-4 ℃ of placement, collect supernatant liquor;
2) adding ammonium sulfate to saturation ratio in the supernatant liquor that step 1) is obtained is 70%, and after 8-16 hour, centrifugal, collecting precipitation obtains paint face mushroom protein extract 0-4 ℃ of placement.
2. method according to claim 1 is characterized in that: the blending ratio of described paint face mushroom fruit body powder and described phosphoric acid buffer is a 1g paint face mushroom fruit body powder dry weight: the 10-20ml phosphoric acid buffer.
3. method according to claim 1 is characterized in that: the pH value of described phosphoric acid buffer is 8, and the time of described lixiviate is 2-6 hour.
4. method according to claim 1 is characterized in that: the particle diameter of described paint face mushroom fruit body powder is below 60 orders.
5. method according to claim 4 is characterized in that: described paint face mushroom fruit body powder for paint face mushroom sporophore 20-40 ℃ of oven dry, pulverize, cross the paint face mushroom sporophore powder that 60 mesh sieves obtain.
6. method according to claim 5, it is characterized in that: described centrifuging and taking supernatant liquor obtains the ratio that precipitation that paint face mushroom extract solution obtains simultaneously can also 1g: 10-20ml to be mixed with the phosphoric acid buffer of pH value for 6-9,0-4 ℃ of lixiviate, obtain paint face mushroom extract solution.
7. the paint face mushroom protein extract that any described method obtains among the claim 1-6.
8. the application of the described paint face mushroom of claim 7 protein extract in preventing and treating tobacco mosaic virus (TMV).
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| CN101671386B (en) * | 2009-09-25 | 2012-05-02 | 北京市农林科学院 | Lepista sordida protein LsAPI, preparation method and application thereof |
| CN101665531B (en) * | 2009-09-25 | 2012-05-23 | 北京市农林科学院 | Lepista sordida protein LsAPII as well as preparation method and application thereof |
| CN104472563B (en) * | 2014-12-31 | 2017-07-28 | 四川省自然资源科学研究院 | Common waaterrthacinth growth inhibitor and preparation method thereof |
| CN115058347B (en) * | 2022-06-07 | 2023-05-12 | 青岛农业大学 | Preparation and application of lepista sordida and non-saccharide extract thereof |
Citations (1)
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| CN1136393A (en) * | 1996-01-19 | 1996-11-27 | 济南科贝尔生物工程有限公司 | Agricultural chemical for preventing and controlling plant virus prepared by liquid fermentation and its producing technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1136393A (en) * | 1996-01-19 | 1996-11-27 | 济南科贝尔生物工程有限公司 | Agricultural chemical for preventing and controlling plant virus prepared by liquid fermentation and its producing technology |
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| 刘艳如,等.12种食用菌凝集素的筛选.《福建师范大学学报(自然科学版)》.2003,第19卷(第2期),65-68. * |
| 孙慧,等.杨树菇(Agrocybe aegerita)中一种抑制TMV侵染的蛋白质纯化及部分特性.《生物化学与生物物理学报》.2001,第33卷(第3期),351-354. * |
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