CN110423794A - A kind of method of Xianggu mushroom strain fruiting ability quick predict - Google Patents
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Abstract
The present invention relates to a kind of methods of Xianggu mushroom strain fruiting ability quick predict, to contain the low melting-point agarose of guaiacol as chromogenic substrate, the mushroom mycelium that preculture obtains is inoculated in chromogenic substrate, after enzymatic reaction, the OD value of chromogenic substrate is read with spectrophotometry, the concentration of low melting-point agarose and guaiacol is respectively 1% and 0.02% in the chromogenic substrate, and the fusing point of the low melting-point agarose is 65 DEG C.A kind of method of Xianggu mushroom strain fruiting ability quick predict of the invention, pass through qualitative method and the spectrophotometry combination of developing the color, the continuity variation of the enzyme activity of high-volume bacterial strain can not only quickly be detected, fruiting situation can also be predicted according to enzyme activity result obtained, it is no longer than 13d from mycelia preculture to detection enzyme activity, predictablity rate is up to 85% or more.
Description
Technical field
The invention belongs to fungal enzyme technical fields, and in particular to a kind of method of Xianggu mushroom strain fruiting ability quick predict.
Background technique
The method of measurement fungi enzyme activity is varied at present, none unified standard, these methods, which can arrange, to be divided into
Two classes, method for qualitative analysis and quantitative analysis method are in accordance with what the compound that ectoenzyme and substrate generate was analyzed.Example
Such as, the substrate of laccase is usually guaiacol, ABTS, adjacent ditolyl etc.；3 kinds of hydrolases of cellulase are usually to be with filter paper
Substrate；In addition to enzyme solution, buffer in polyphenol oxidase reaction system, it is also necessary to add catechol as substrate.Enzyme Qualitative analysis
Method is usually plate colour developing circle method, and sooner or later, shade simply judges enzyme activity to the appearance according to discoloration circle.This kind of side
Method is easy to operate, quick, suitable for the primary dcreening operation of a large amount of samples, but can not confirm the definite situation of producing enzyme；Quantitative analysis method has
O2, microcalorimetric method, pulsed laser light sound analysis method, high performance liquid chromatography and polarography, these types of method can be direct or indirect
Measure specific enzyme activity, accuracy is high, but operates relative complex, and cost is also higher, therefore nowadays nor very much.It is divided light
For the relatively these types of method of degree method, at low cost, operation is simpler, can be according to the relationship meter that light absorption value (OD) is changed over time
Enzyme activity is calculated, is current most common method both at home and abroad.But the method even has some limitations problem in application.Its
One, when a large amount of samples need to detect, liquid fermentation and culture is cumbersome and time consuming；Second, not being suitable for detecting some period
Interior continuous enzyme activity can not recognize the variation of the producing enzyme situation of the early stage of ectoenzyme.
In addition, the research to catalyzed by laccase from dried mushrooms has been reported that there is laccase high yield strain breeding thereof more, there is induction laccase yield to increase
, there are also the transcriptional expressions of the laccase family to growth period each during mushroom growth to study, and in laccase family
The discussion of isodynamic enzyme function is studied.But by the enzyme activity changing rule in the mycelia stage of bacterial strain early stage and the connection of fruiting ability one
The experimental study risen but has not been reported.
Therefore, what the enzyme activity changing rule and fruiting ability for studying a kind of mycelia stage by bacterial strain early stage linked together
The great realistic meaning of method of Xianggu mushroom strain fruiting ability quick predict.
Summary of the invention
The purpose of the present invention is overcome above-mentioned lack in the prior art by the enzyme activity variation rule in the mycelia stage of bacterial strain early stage
The defect for the experimental study that rule and fruiting ability link together, provides a kind of side of Xianggu mushroom strain fruiting ability quick predict
Method quickly detects the continuity variation of the enzyme activity of high-volume bacterial strain, root by qualitative method and the spectrophotometry combination of developing the color
It is predicted according to fruiting situation of the enzyme activity result obtained to Xianggu mushroom strain.
In order to achieve the above objectives, the technical solution adopted by the present invention are as follows:
A kind of method of Xianggu mushroom strain fruiting ability quick predict, to contain the low melting-point agarose of guaiacol as aobvious
Color base matter, the chromogenic substrate have good transparency and reactive adaptation, the mushroom mycelium that preculture obtains are inoculated in aobvious
After enzymatic reaction, the OD value of chromogenic substrate is read with spectrophotometry for color base matter.
Its preferred technical solution are as follows:
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, low melting point agar in the chromogenic substrate
The concentration of sugar and guaiacol is respectively 1% and 0.02%.
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, the fusing point of the low melting-point agarose are
65℃。
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, the specific steps are as follows:
(1) mushroom mycelium is seeded in activation culture on PDA plate, mycelia growth conditions are consistent and quantity is more to obtain
Inoculation block, punched in activated bacterial strain mycelia edge, cell age about 10 days at this time, be considered as activation and complete, punching aperture is
PDA plate aperture, separately take culture medium thickness be 3~4mm PDA plate and in the flat plate the heart be inoculated with, repetition connect muti-piece put down
Plate cultivates the PDA plate after inoculation in constant incubator, is inoculated with visible obvious newborn mycelia around block and colony edge is clear
Chu Hou was drawn lines using the methods of right-angled intersection every 24 hours, measured bacterial strain diameter, recorded strain growth speed, repeated above-mentioned behaviour
Make, until bacterial strain diameter reaches culture dish edge, stops setting-out, the time of activation culture is no more than 13 days on PDA plate；
(2) guaiacol solution is added into the low melting-point agarose solution for be cooled to 60 DEG C, guaiacol exists after addition
Concentration in low melting-point agarose solution is 0.02%, shakes up, obtains color-developing substrate solution；Using quantitative liquid-distributor, in cell
Appropriate color-developing substrate solution is injected separately into hole on culture plate, tissue culture plate may be selected to be the more of 12 holes, 24 holes or 48 holes
Kind cell culture plate, the technical program are preferably 24 porocyte culture plates, and the color-developing substrate solution injected at this time is 2mL,
Using needing to adjust filling fast eolor base quality when other tissue culture plates, to guarantee that stromal thickness is being suitble in range, solidify standby
With, obtain agarose colour developing plate；
(3) to guarantee that each inoculation block growth time is consistent, mycelia is punched according to the position of setting-out on PDA plate, and punching is straight
Diameter is culture plate aperture, and into the hole of agarose colour developing plate, center is put into bacterial spawn block, mycelia upward, a Kong Fangyi bacterial spawn
Agarose colour developing plate is placed in constant incubator enzymatic reaction 8h after putting well by block；Agarose colour developing plate is placed in constant temperature incubation
The time of case enzymatic reaction is 8h, which is the optimization time by groping, and is inoculated with block, that is, mycelia at that time
Enzyme activity can be necessary and sufficiently reacts, and when time of enzymatic reacting is less than 8h, the chromogenic reaction result of enzymatic is insufficient, and is greater than 8h
When, the chromogenic reaction result of enzymatic is excessive；
(4) the agarose colour developing plate after taking out enzymatic reaction, measures its OD value using spectrophotometry.
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, the culture of PDA culture medium in step (1)
Condition are as follows: temperature is 23 DEG C.
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, in step (2), the low melting point agar
The concentration of sugar juice is 1.0g/100mL, preparation method are as follows: water-bath adds after 1.0g agarose is dissolved in the distilled water of 100mL
Heat, wherein the time of heating water bath is 40min, and temperature is 90 DEG C.
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, in step (2), the guaiacol is molten
The volume fraction of liquid is 4%, preparation method are as follows: 4mL guaiacol is dissolved in the ethyl alcohol of 96mL.
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, in step (3), bacterial spawn block is in 24 hole fine jades
The modes of emplacement that lipolysaccharide develops the color in plate well are as follows: the mycelia of bacterial spawn block faces upward, and bacterial spawn block is placed on the center position in hole, protects
It demonstrate,proves in chromogenic reaction chromogenic substrate and is occurred with center to uniform around.
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, the survey of spectrophotometry in step (4)
Amount condition are as follows: microplate reader is that the other methods of such as spectrophotometer are generally not suitable for solid herein using the reason of microplate reader
Chromogenic substrate, and microplate reader can OD value to solid matrix carry out high-throughput detection, model: Tecan infinite
M200PRO；Wavelength: 465nm.
A kind of method of Xianggu mushroom strain fruiting ability quick predict as described above, the middle measurement OD value of step (4) before will
Bacterium colony block takes out, and prevents the presence of inoculation block from generating interference to light absorption value.
Beneficial effect
(1) method of a kind of Xianggu mushroom strain fruiting ability quick predict of the invention, by will develop the color qualitative method and point
Light photometry combines, and can not only quickly detect the continuity variation of the enzyme activity of high-volume bacterial strain, additionally it is possible to according to obtained
Enzyme activity result predicts fruiting situation；
(2) method of a kind of Xianggu mushroom strain fruiting ability quick predict of the invention, from mycelia preculture to detection enzyme activity
13d is only needed, the preculture in the prior art-shake flask fermentation culture-traditional method of extraction crude enzyme liquid then needs preculture
10d, fermented and cultured 10d, the entire time for detecting enzyme activity at least reduce 35%；
(3) method of a kind of Xianggu mushroom strain fruiting ability quick predict of the invention, cannot smooth fruiting according to 0D≤1.5
Test strain is predicted, accuracy rate is up to 85% or more, and this not only reduces because being unable to fruiting bring economic loss
And the loss of time, while reducing a large amount of workload；
(4) method of a kind of Xianggu mushroom strain fruiting ability quick predict of the invention eliminates Liquid Culture and extracts enzyme
The step of liquid, can not only observe the enzyme activity situation of early stage mycelia, additionally it is possible to obtain the daily amount of having a net increase of, this method can be same
When make multiple groups experiment and be repeated several times, both having saved the time also reduces economic cost.
Detailed description of the invention
Fig. 1 is PDA punching in a kind of bacterial strain preculture of the method for Xianggu mushroom strain fruiting ability quick predict of the invention
Explanatory diagram.
Specific embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, art technology
Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Embodiment 1
A kind of method of Xianggu mushroom strain fruiting ability quick predict, the specific steps of which are as follows:
(1) mushroom mycelium preculture.36 kinds of common mushroom myceliums are seeded in activation culture 10d on PDA plate, In respectively
Activated bacterial strain mycelia edge punching (punching aperture d=5mm), separately taking culture medium thickness is that the PDA plate of 3~4mm exists
And the heart is inoculated in the flat plate, is repeated aforesaid operations 5 times, the PDA plate after inoculation is cultivated in the incubator, cultivation temperature is
23 DEG C, incubation time 13d, until visible obvious newborn mycelia and colony edge understands around inoculation block, using right-angled intersection
It draws lines within method every 24 hours, measures bacterial strain diameter, record strain growth speed, repeat aforesaid operations, bacterial strain diameter reaches within the tenth day
Culture dish edge stops setting-out；
(2) agarose colour developing flat panel production.The specific steps are that:
A) 4mL guaiacol is dissolved in the ethyl alcohol of 96mL, 4% guaiacol is prepared；
B) 1.0g agarose being dissolved in heating water bath after the distilled water of 100mL, wherein the time of heating water bath is 40min,
Temperature is 90 DEG C, and the low melting-point agarose solution of 1.0g/100mL is prepared；
C) after the agarose solution after dissolving is cooled to 60 DEG C, 4% guaiacol is added with quantitative liquid shifter, after addition
Final concentration of the 0.02% of guaiacol, shakes up, obtains color-developing substrate solution；
D) it is injected separately into 2ml color-developing substrate solution in each hole in 24 porocyte culture plates, solidifies spare；
The enzymatic reaction of (3) 24 porocyte culture plates.As Fig. 1 distribution shown in, on PDA plate mycelia according to setting-out position
Punching, punching emptying aperture diameter d are 5mm, are put into bacterial spawn block into the hole of agarose colour developing plate, bacterial spawn block develops the color in 24 hole agaroses
Modes of emplacement in plate well are as follows: the mycelia of bacterial spawn block faces upward, and bacterial spawn block is placed on the center position in hole, a Kong Fangyi bacterium
Agarose colour developing plate is placed in 28 DEG C of constant incubator enzymatic reaction 8h after putting well by column block；
(4) enzyme activity is detected.Agarose colour developing plate after taking out enzymatic reaction, chooses inoculation block, with OD value for enzyme activity list
Position, using the OD value at microplate reader measurement agarose colour developing plate 465nm；
(5) mushroom strain different to above-mentioned 36 plants of enzyme activity carries out subsequent mushroom producing culture.
Using the enzyme activity of method as described above detection bacterial strain early stage continuous mycelia, in step (1), around inoculation block
It can be seen that it is obvious new life mycelia and colony edge understand, the time that PDA plate is cultivated in the incubator at this time be 13d；In step (3)
It can be observed that rufous is presented in bacterial spawn block first, over time, the color of bacterial spawn block and agarose plate gradually adds
It is deep；Testing result are as follows: the OD value of most strains is increased as growth number of days increases, and rest part bacterial strain OD value increases
Slowly, or horizontality is kept, wherein OD value there are 15 plants lower than 1.5；According to testing result prediction OD value is smaller, enzyme activity compared with
Low bacterial strain cannot smooth fruiting；During subsequent mushroom producing culture, in 15 plant bacterial strains of the OD value lower than 1.5, there are 2 plants smoothly to go out
Mushroom, 13 plants cannot smooth fruiting, prediction Xianggu mushroom strain fruiting ability accuracy rate be 87%；
Repeat the enzyme activity using method repeated detection bacterial strain early stage continuous mycelia as described above, wherein most bacterial strain
OD value be all with growth number of days increase and increase, rest part bacterial strain OD value increase slowly, or holding horizontality, portion
Divide in experiment, OD value there are 15 plants, 16 plants, 14 plants lower than 1.5；Still prediction OD value is smaller according to testing result, enzyme activity is lower
Bacterial strain cannot smooth fruiting；During subsequent mushroom producing culture, in bacterial strain of the OD value lower than 1.5, practical fruiting result and pre-
The accuracy rate for surveying Xianggu mushroom strain fruiting ability see the table below.
Bacterial strain Enzyme activity assay result and the practical non-fruiting number table of comparisons
Table in analysis it is found that a kind of Xianggu mushroom strain fruiting ability quick predict of the invention method, not according to 0D≤1.5
Smoothly it can predict that test strain, accuracy rate is up to 85% or more by fruiting.
Comparative example 1
A kind of method of Xianggu mushroom strain fruiting ability quick predict, it is substantially the same manner as Example 1, the difference is that, step
Suddenly in (2) agarose colour developing flat panel production, the color-developing substrate solution injected in each hole in 24 porocyte culture plates is free of 4%
Guaiacol.
Using the enzyme activity of method as described above detection bacterial strain early stage continuous mycelia, testing result are as follows: most strains
OD value be all with growth number of days increase and increase, rest part bacterial strain OD value increase slowly, or holding horizontality,
Middle OD value has 14 plants lower than 1.5；According to testing result prediction OD value is smaller, the lower bacterial strain of enzyme activity cannot smooth fruiting；Afterwards
During continuous mushroom producing culture, in 14 plant bacterial strains of the OD value lower than 1.5, there is 4 plants of smooth fruitings, 10 plants cannot smooth fruiting, in advance
The accuracy rate for surveying Xianggu mushroom strain fruiting ability is 71%.
Comparative example 2
A kind of method of Xianggu mushroom strain fruiting ability quick predict, it is substantially the same manner as Example 1, the difference is that, it will
The time that agarose colour developing plate is placed in constant incubator enzymatic reaction is 7h.
Using the enzyme activity of method as described above detection bacterial strain early stage continuous mycelia, testing result are as follows: most strains
OD value be all with growth number of days increase and increase, rest part bacterial strain OD value increase slowly, or holding horizontality,
Middle OD value has 15 plants lower than 1.5；According to testing result prediction OD value is smaller, the lower bacterial strain of enzyme activity cannot smooth fruiting；Afterwards
During continuous mushroom producing culture, in 15 plant bacterial strains of the OD value lower than 1.5, there is 3 plants of smooth fruitings, 13 plants cannot smooth fruiting, in advance
The accuracy rate for surveying Xianggu mushroom strain fruiting ability is 80%.
Comparative example 3
A kind of method of Xianggu mushroom strain fruiting ability quick predict, it is substantially the same manner as Example 1, the difference is that, it will
The time that agarose colour developing plate is placed in constant incubator enzymatic reaction is 9h.
Using the enzyme activity of method as described above detection bacterial strain early stage continuous mycelia, testing result are as follows: most strains
OD value be all with growth number of days increase and increase, rest part bacterial strain OD value increase slowly, or holding horizontality,
Middle OD value has 16 plants lower than 1.5；According to testing result prediction OD value is smaller, the lower bacterial strain of enzyme activity cannot smooth fruiting；Afterwards
During continuous mushroom producing culture, in 16 plant bacterial strains of the OD value lower than 1.5, there is 4 plants of smooth fruitings, 12 plants cannot smooth fruiting, in advance
The accuracy rate for surveying Xianggu mushroom strain fruiting ability is 75%.
Claims (10)
1. a kind of method of Xianggu mushroom strain fruiting ability quick predict, characterized in that with the low melting point agar containing guaiacol
Sugar is used as chromogenic substrate, and the mushroom mycelium that preculture obtains is inoculated in chromogenic substrate, after enzymatic reaction, is read with spectrophotometry
Take the OD value of chromogenic substrate.
2. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 1, which is characterized in that described aobvious
The concentration of low melting-point agarose and guaiacol is respectively 1% and 0.02% in color base matter.
3. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 1, which is characterized in that described low
The fusing point of melt agarose is 65 DEG C.
4. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 1, which is characterized in that specific step
It is rapid as follows:
(1) mushroom mycelium is seeded in activation culture on PDA plate, is punched in activated bacterial strain mycelia edge, separately takes training
Support base with a thickness of 3~4mm PDA plate and in the flat plate the heart be inoculated with, repeatedly connect muti-piece plate, the PDA after inoculation is put down
Plate is cultivated in constant incubator, after visible obvious newborn mycelia and colony edge understand around inoculation block, is drawn lines within every 24 hours,
Bacterial strain diameter is measured, strain growth speed is recorded, repeats aforesaid operations, until bacterial strain diameter reaches culture dish edge, stops picture
Line；
(2) guaiacol solution is added into the low melting-point agarose solution for be cooled to 60 DEG C, shakes up, it is molten to obtain chromogenic substrate
Liquid.It is injected separately into appropriate color-developing substrate solution in hole on tissue culture plate, solidifies, obtains agarose colour developing plate；
(3) mycelia is punched according to the position of setting-out on PDA plate, is put into bacterial spawn block, mycelia into the hole of agarose colour developing plate
Upward, agarose colour developing plate is placed in constant incubator enzymatic reaction 8h after putting well by a Kong Fangyi bacterial spawn block；
(4) the agarose colour developing plate after taking out enzymatic reaction, measures its OD value using spectrophotometry.
5. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 4, which is characterized in that step
(1) condition of culture of PDA culture medium in are as follows: temperature is 23 DEG C.
6. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 4, which is characterized in that described low
The concentration of melt agarose sugar juice is 1.0g/100mL, preparation method are as follows: after 1.0g agarose is dissolved in the distilled water of 100mL
Heating water bath, wherein the time of heating water bath is 40min, and temperature is 90 DEG C.
7. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 4, which is characterized in that step
(2) in, the volume fraction of the guaiacol solution is 4%, preparation method are as follows: 4mL guaiacol is dissolved in the second of 96mL
Alcohol.
8. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 4, which is characterized in that step
(3) in, modes of emplacement of the bacterial spawn block in 24 hole agaroses colour developing plate well are as follows: the mycelia of bacterial spawn block faces upward, and bacterial spawn block is put
Set the center position in hole.
9. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 4, which is characterized in that step
(4) measuring condition of spectrophotometry in are as follows: microplate reader, model: Tecan infinite M200PRO；Wavelength: 465nm.
10. a kind of method of Xianggu mushroom strain fruiting ability quick predict according to claim 4, which is characterized in that step
(4) bacterium colony block is taken out before measurement OD value in.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112113921A (en) * | 2020-08-12 | 2020-12-22 | 华南农业大学 | High-throughput detection method for ergothioneine content between samples to be detected and application thereof |
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2019
- 2019-09-10 CN CN201910853786.4A patent/CN110423794B/en active Active
Non-Patent Citations (3)
| Title |
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| S OHGA: "Transcriptional regulation of laccase and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust", 《FEMS MICROBIOL LETT.》 * |
| 任鹏飞: "香菇胞外酶活性变化及其与农艺性状的相关性分析", 《山东农业科学》 * |
| 王祎宁: "漆酶及其引用的研究进展", 《生物技术通报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112113921A (en) * | 2020-08-12 | 2020-12-22 | 华南农业大学 | High-throughput detection method for ergothioneine content between samples to be detected and application thereof |
| CN112113921B (en) * | 2020-08-12 | 2021-11-26 | 华南农业大学 | High-throughput detection method for ergothioneine content between samples to be detected and application thereof |
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