CN105779311A - Method for rapidly screening mould strains producing high-yield protease - Google Patents
Method for rapidly screening mould strains producing high-yield protease Download PDFInfo
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Abstract
The invention discloses a method for rapidly screening mould strains producing high-yield protease. The method comprises the following steps: respectively inoculating mould strains needing to be screened onto a slant of a mould spore culture medium and then carrying out constant temperature culture; firstly pouring a lower control culture medium into a sterile dried plate, then pouring an upper screening culture medium and inserting a plurality of small sterile cups in a prepared double-layer plate, thus preparing a high-protease mould directional screening plate; preparing spore suspension by using cultured mould spores to be tested and carrying out shaking activated culture in a spore activation liquid culture medium; then injecting the spore suspension into the small sterile cups respectively and carrying out constant temperature culture; after culture is completed, directly measuring the diameter of each transparent circle on the high-protease mould directional screening plate; and determining the protease activity of each mould strain according to the diameter, wherein the bigger the transparent circles are, the higher the protease activities of the mould strains are. The method has the advantages of convenience in operation, high accuracy, small workload, low cost, and the like.
Description
Technical field
The present invention can be used for the screening of excellent fermented bacterium in fermented food (soy sauce, Semen Sojae Preparatum, fermented bean curd etc.), belongs to food fermentation technical field, particularly to the screening technique that a kind of high proteinase yield fungal strain is fast and convenient.
Background technology
Aspergillus oryzae in mycete, Mucor etc. are widely used in fermented food, and the protein degradation in raw material can be become polypeptide and aminoacid by its abundant protease, improves the digestive utilization ratio of protein；Particularly to produce some delicious amino acid and small-molecular peptides, give the local flavor that fermented food is good.It can be said that the mycete such as aspergillus oryzae and Mucor is the important strain in fermented food production.
The strains such as the fermented food such as soy sauce, Semen Sojae Preparatum, fermented bean curd aspergillus oryzae used in producing, Mucor should have higher proteinase activity, in fermented food production, the proteinase activity of strain directly affects the important indicators such as the utilization rate of raw material, amino nitrogen content, therefore, the fungal strain of screening acquisition high protein enzyme activity produces particularly important for fermentation bean foods such as soy sauce.
At present, conventional prolease activity screening technique is casein flat band method, high protein enzyme enzyme live strain (referring to Fig. 1) is screened by measuring the ratio (K value) of the bacterial strain transparent circle that extracellular proteinase produces on flat board and colony diameter, as can be seen from Figure 1, this method is owing to mycete colonial morphology in growth course is irregular, blur margin is clear, the more difficult mensuration of transparent circle diameter, causes that the measurement result accuracy of K value is relatively low；And K value between each bacterium is only small, it is difficult to react the gap (referring to Fig. 3) of enzymatic productivity between each bacterium, as can be seen from Figure 3 actually measured enzyme size alive is very poor with the size dependency of K value, and this illustrates that traditional agar-casein double-layer agar technique is unreliable to the selection result of aspergillosis.For accurately screening, it has to take forint-phenol law to carry out the mensuration of proteinase activity after yeast production is cultivated.Forint-phenol law measures proteinase activity to be needed to adopt triangular flask yeast production, cultivation, drawing standard curve, although result is accurate, but equipment and reagent chemicals consumption are big, and required time is long, and method is loaded down with trivial details, takes time and effort.
Summary of the invention
The technical problem to be solved is, overcomes the deficiency and defect mentioned in background above technology, it is provided that the rapid screening method of the high proteinase yield fungal strain that a kind of convenience operates, accuracy rate is high, workload is little and cost is low.
For solving above-mentioned technical problem, the technical scheme that the present invention proposes is the rapid screening method of a kind of high proteinase yield fungal strain (particularly preferably aspergillus oryzae strain or Mucor bacterial strain), comprises the following steps:
(1) actication of culture: be inoculated in the raw Spore cultivation base inclined-plane of mycete, then constant temperature culture respectively by needing the multiple or many strains fungal strain carrying out screening；
(2) high protein enzyme mycete directed screening flat board is prepared: the plate after sterilizing-drying is first poured into lower floor's control medium, after its cooled and solidified, pours upper strata screening culture medium again into；
(3) also gravity flow screen screening device is placed: treat to place in solidifying process the sterile cuvette of multiple equal size in upper strata screening culture medium, after culture medium solidifying, sterile cuvette is fixed；
(4) mycotic spore suspension to be measured preparation and activation: physiological saline solution washes cultured mycotic spore in lower step (1), accesses the activation of spore activated liquid culture medium, then adjusts spore concentration after aseptic lens paper filtration sterilization silk；
(5) mycete to be measured is cultivated before measuring: each mycete activation spore suspension prepared by equivalent aspiration step (4), it is injected separately in each sterile cuvette of the high protein enzyme mycete directed screening flat board that above-mentioned steps (2) prepares (sterilized water that can inject equivalent in one of them sterile cuvette compares), has inoculated and be placed in constant incubator and cultivate；
(6) mold protease enzyme to be measured is lived and is contrasted screening: after the cultivation of above-mentioned steps (5) completes, and measures the transparent circle diameter formed around each sterile cuvette on high protein enzyme mycete directed screening flat board；Determining each fungal strain prolease activity size according to transparent circle diameter, transparent circle is more big, and in sterile cuvette, the fungal strain prolease activity of inoculation is more big.
The present invention by by sterile cuvette with improvement casein culture medium with the use of, the culture dish utilizing different size carries out multiple or many plant heights protease fungal strain screening simultaneously, method is easy, time saving and energy saving, drastically increase accuracy and the efficiency of high protein enzyme mycete primary dcreening operation, screen in the utilization of high proteinase yield fungal strain significant in selection-breeding.
The rapid screening method of above-mentioned high proteinase yield fungal strain, preferably, in described step (1), the raw Spore cultivation base of the mycete of selection is mainly formulated by 100～110g/L Semen Maydis powder, 150～160g/L Rhizoma Solani tuber osi, 20～25g/L sucrose, 15～20g/L agar, and pH is natural；Rhizoma Solani tuber osi needs peeling, stripping and slicing, boils process, and the time of boiling is not less than 30min, then by filtered through gauze, utilizes filtrate preparation culture medium；121 DEG C of sterilizing 20min.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is preferred that in described step (2), described lower floor's control medium is pure agar culture medium；Described upper strata screening culture medium is improvement casein culture medium.Mycete produce protease can hydrolyzed casein thus improvement casein culture medium formed transparent circle, but because casein culture medium color is the reason such as out-of-flatness bottom white and culture dish, it is unfavorable for observing and measuring the size of transparent circle, so adding pure agar culture medium is lower floor's flat board, be conducive to observation and the measurement of transparent circle, improve the accuracy of result.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is furthermore preferred that add sodium deoxycholate to spread inhibitor as fungal hyphae in described improvement casein culture medium.It is furthermore preferred that the concrete formula concentration of described improvement casein culture medium includes: casein 3～6g/L, sodium deoxycholate 1.0～2.0g/L, potassium dihydrogen phosphate 0.36g/L, manganese sulfate 0.5g/L, zinc chloride 0.014g/L, disodium hydrogen phosphate 1.07g/L, sodium chloride 0.16g/L, ferrous sulfate 0.002g/L, calcium chloride 0.002g/L, agar powder 15～20g/L；PH6.5～7.0；121 DEG C of sterilizing 20min.
In above-mentioned improved plan, upper strata screening culture medium is preferably added sodium deoxycholate, to suppress spreading of fungal hyphae, improve the accuracy of measurement result.The present invention has further defined the suitableeest addition of sodium deoxycholate, our research indicate that, sodium deoxycholate addition cross conference cause mycete cannot normal growth, transparent circle is inconspicuous；Sodium deoxycholate addition is too small then suppresses mycelia DeGrain, and mycelia spreads sterile cuvette, affects the measurement of transparent circle.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is preferred that in described step (1), the temperature cultivated in calorstat is 28 ± 0.1 DEG C, and incubation time is 48h.
The rapid screening method of above-mentioned high proteinase yield fungal strain, preferably, in described step (3), screening plant sterile cuvette adopts the glass tubule of Oxford cup or diameter 1cm, high 1cm, sterile cuvette Φ 60mm, Φ 90mm, Φ 120mm, Φ 150mm culture dish in placement quantity respectively 3～4,4～5,11～12,18～19；The position that sterile cuvette is placed is 1～2mm place at the bottom of distance ware.The sterile cuvette that the present invention adopts is preferred with Oxford cup；In the art, it is used from being not found in by we fungal strain prolease activity screening field, and this is apparently not what those skilled in the art were readily apparent that.
The rapid screening method of above-mentioned high proteinase yield fungal strain, preferably, in described step (4), described spore activated liquid culture medium is mainly formulated by concentration 10～12g/L germinating barley, 3.0～5.0g/L Semen Maydis pulp, 15～20g/L glucose, and pH is natural；121 DEG C of sterilizing 15min.
The cardinal principle of the present invention is: by by sterile cuvette with improvement casein culture medium with the use of, limit mycelia and grow to outside cup so that the transparent circle regular shape of its formation, edge clear, be convenient for measuring；Cuvette reinjects equal-volume, isocyatic spore suspension, quantification will be tested, significantly improve the order of accuarcy of detection the selection result；Additionally, forint-phenol law surveys the screening technique that enzyme is lived after the method for the present invention can replace yeast production after induction mutation of bacterium, it is rapidly completed the screening of mass mutation strain, efficiently reduces screening operation amount and the screening cost of mutagenic strain.
Compared with prior art, it is an advantage of the current invention that:
1, the sterile cuvette in the inventive method and the sodium deoxycholate that preferably adds in the screening culture medium of upper strata all can limit mycostatic spreading, and stop mycelia to grow to outside cup so that the transparent circle regular shape of its formation, edge clear；Meanwhile, it is lower floor's flat board by adding pure agar culture medium so that transparent circle is high-visible, is convenient for measuring；
2, in the sterile cuvette of the inventive method design, inject equal-volume, isocyatic spore suspension, quantification will be tested, improve the order of accuarcy of the selection result；As can be seen from Figure 4, the transparent circle size good relationship that the enzyme size alive adopting forint-phenol law actually measured records with high protein enzyme mycete directed screening flat band method of the present invention, illustrate that the transparent circle size that high protein enzyme mycete directed screening flat band method records can substitute for forint-phenol law to a certain extent and mycete is screened, and the selection result accuracy is high.
3, after the method for the present invention can replace yeast production after induction mutation of bacterium, forint-phenol law surveys the screening technique that enzyme is lived, and reduces screening operation amount and the screening cost of mutagenic strain.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below, apparently, accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, under the premise not paying creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is existing agar-casein double-layer agar technique screening high protein enzyme enzyme aspergillus oryzae figure alive.
Fig. 2 is high protein enzyme mycete directed screening flat band method of the present invention screening high protein enzyme enzyme aspergillus oryzae figure alive.As can be seen from the figure transparent circle diameter clear, regular edges, therefore the selection result is significantly better than conventional agar-casein double-layer agar technique.
Fig. 3 is existing agar-casein double-layer agar technique and forint-phenol law screening high protein enzyme enzyme aspergillus oryzae comparison diagram alive.
Fig. 4 is high protein enzyme mycete directed screening flat band method of the present invention and forint-phenol law screening high protein enzyme enzyme aspergillus oryzae comparison diagram alive.
Fig. 5 is the result of high protein enzyme mycete directed screening flat band method screening high proteinase yield aspergillus oryzae in the embodiment of the present invention 1.
Fig. 6 is the result of the agar-casein double-layer agar technique screening high proteinase yield aspergillus oryzae adopting routine in comparative example 1 of the present invention.
Fig. 7 is the result adopting conventional forint-phenol law screening high proteinase yield aspergillus oryzae in comparative example 2 of the present invention.
Fig. 8 is the result of high protein enzyme mycete directed screening flat band method screening high proteinase yield Mucor in the embodiment of the present invention 2.
Fig. 9 is the result of the agar-casein double-layer agar technique screening high proteinase yield Mucor adopting routine in comparative example 3 of the present invention.
Figure 10 is the result adopting conventional forint-phenol law screening high proteinase yield Mucor in comparative example 4 of the present invention.
Detailed description of the invention
For the ease of understanding the present invention, below in conjunction with Figure of description and preferred embodiment, the present invention is made more comprehensively, describes meticulously, but protection scope of the present invention is not limited to embodiment in detail below.
Unless otherwise defined, the implication that all technical term used hereinafter is generally understood that with those skilled in the art is identical.Technical term used herein is intended merely to the purpose describing specific embodiment, is not intended to limit the scope of the invention.
Unless otherwise specified, the various raw materials used in the present invention, reagent, instrument and equipment etc. all can be commercially available by market or can be prepared by existing method.
Embodiment 1:
The rapid screening method of a kind of high proteinase yield fungal strain, namely adopts high protein enzyme mycete directed screening flat band method screening high protein enzyme enzyme aspergillus oryzae alive, specifically includes following steps:
(1) actication of culture: the 100 Aspergillus oryzae bacterial strains carrying out screening will be needed to be transferred to respectively on the inclined-plane of the raw Spore cultivation base of mycete, be positioned in calorstat 28 ± 0.1 DEG C cultivate 48h.
The raw Spore cultivation based formulas of the mycete selected is Semen Maydis powder 100g/L, Rhizoma Solani tuber osi 150g/L, sucrose 25g/L, agar 20g/L, pH nature；Rhizoma Solani tuber osi needs to carry out removing the peel, stripping and slicing, boil process, and the time of boiling is not less than 30min, then by filtered through gauze, utilizes filtrate to prepare culture medium；121 DEG C of sterilizing 20min.
(2) high protein enzyme mycete directed screening flat board is prepared: prepare the culture dish that 6 set specifications are Φ 150mm, sterilizing-drying, first pours lower floor's control medium in plate, and thickness is about 2mm, pouring upper strata screening culture medium after its cooled and solidified again into, thickness is about 3mm.
The lower floor's control medium selected is pure agar culture medium.The upper strata screening culture medium selected is improvement casein culture medium, concrete formula is casein 4g/L, sodium deoxycholate 1.5g/L, potassium dihydrogen phosphate 0.36g/L, manganese sulfate 0.5g/L, zinc chloride 0.014g/L, disodium hydrogen phosphate 1.07g/L, sodium chloride 0.16g/L, ferrous sulfate 0.002g/L, calcium chloride 0.002g/L, agar powder 20g/L, pH6.5；121 DEG C of sterilizing 20min.
(3) placing also gravity flow screen screening device: treat to place in solidifying process the aseptic Oxford cup of 18 equal sizes in upper strata screening culture medium, the position of placement is 2mm place at the bottom of distance ware, and after culture medium solidifying, aseptic Oxford cup is fixed.
(4) mycotic spore suspension to be measured preparation and activation: physiological saline solution washes cultured aspergillus oryzae spore in lower step (1), spore activated liquid culture medium is accessed after aseptic lens paper filtration sterilization silk, 160r/min, 28 DEG C of vibration activation 6h, then spore concentration is adjusted to 106Individual/mL.
The spore activated liquid culture medium selected is formulated by 10g/L germinating barley, 3.0g/L Semen Maydis pulp, 20g/L glucose, and pH is natural；121 DEG C of sterilizing 15min.
(5) mycete to be measured is cultivated before measuring: draw 10 μ L step (4) the rice-koji bacterium prepared activation spore suspension, it is injected separately in each Oxford cup of the high protein enzyme mycete directed screening flat board that above-mentioned steps (2) prepares, the sterilized water injecting equivalent in one of them Oxford cup compares, and has inoculated cultivation 2d in the constant incubator being placed on 28 DEG C.
(6) mold protease enzyme to be measured is lived and is contrasted screening: after the cultivation of above-mentioned steps (5) completes, measure the transparent circle diameter (referring to Fig. 2) formed around the cup of each Oxford on high protein enzyme aspergillus oryzae directed screening flat board, selecting the 9 strain bacterium that high protein enzyme aspergillus oryzae directed screening flat band method result is best, result is such as shown in table 1, Fig. 5；Determining each fungal strain prolease activity size according to transparent circle diameter, transparent circle is more big, and namely corresponding aspergillus oryzae strain prolease activity is more big.
Table 1: high protein enzyme mycete directed screening flat band method screening aspergillus oryzae result
Overall merit: the screening step of the present embodiment is simple, easy to operate, the required time is about 2.5 days, and the selection result accuracy can reach more than 90%, screen 100 strain bacterium only to need to prepare the culture dish that 6 set specifications are Φ 150mm, be suitable for the rapid screening of high proteinase yield mycete.
Comparative example 1:
Adopting conventional agar-casein double-layer agar technique screening high protein enzyme enzyme aspergillus oryzae alive, concrete operations are as follows:
(1) actication of culture: aspergillus oryzae strain to be measured is inoculated in PDA medium slant, 28 DEG C of constant temperature culture 48h.
(2) agar-casein double-layer plate is prepared: preparing the culture dish that 100 set specifications are Φ 90mm, sterilizing-drying, first pour pure agar culture medium in plate into, thickness is about 2mm；Pouring casein culture medium again into after its cooling, thickness is about 3mm, prepares agar-casein double-layer plate: the preparation method of the preparation method of the pure agar culture medium of selection is with the step (2) of above-mentioned the present embodiment 1；The preparation method of casein culture medium selected and the step (2) of above-mentioned the present embodiment 1 are distinctive in that and do not add sodium deoxycholate.
(3) spore suspension is prepared: wash in lower step (1) cultured mycotic spore, aseptic lens paper filtration sterilization silk with physiological saline solution.
(4) inoculation: take 1mL spore suspension and coat agar-casein double-layer plate.
(5) cultivate: with the step (5) of above-mentioned the present embodiment 1.
(6) screening contrast: after above-mentioned steps (5) has been cultivated, the 9 strain bacterium chosen in above-mentioned the present embodiment 1 are measured, measure each transparent circle diameter and colony diameter on agar-casein double-layer plate, calculate K value (transparent circle diameter and colony diameter) result such as shown in table 2, Fig. 6.
Table 2: existing agar-casein double-layer agar technique K value screening aspergillus oryzae result
| Bacterium numbering | 1-1 | 1-2 | 1-3 | 1-4 | 1-5 | 1-6 | 1-7 | 1-8 | 1-9 |
| K value | 1.67 | 1.71 | 1.33 | 2.18 | 1.64 | 1.54 | 1.80 | 1.67 | 1.80 |
Overall merit: although the required time shorter (being about 2.5 days) of this method, but step is complicated, workload is big, and result precision is low, screen 100 strain bacterium at least to need to prepare the culture dish that 100 set specifications are Φ 90mm, be not suitable for rapid screening high proteinase yield mycete.
Comparative example 2:
Adopting conventional forint-phenol law to survey aspergillus oryzae protease enzyme to live, concrete operations are as follows:
It is measured according to National Standard of the People's Republic of China SB/T10317-1999.
(1) yeast production: prepare 100 parts of yeast production culture medium and be placed in triangular flask, at the inoculation of medium spore suspension of cooling and material loosening, each triangular flask miospore number is 7.7 × 106Individual, in 32 DEG C of constant temperature culture 36h, song is turned in timing.
The preparation method of the yeast production culture medium selected comprises the following steps: with the ratio of 7:2:1, bean cake, Semen Tritici aestivi, wheat bran are mixed bottling (50g siccative/triangular flask), then with water-wet (mass ratio of amount of water and raw material is 90%), 0.1～0.12MPa sterilizing 1～1.5h.
(2) remaining step carries out according to National Standard of the People's Republic of China SB/T10317-1999.
(3) measurement result: the 9 strain bacterium chosen in above-mentioned the present embodiment 1 are measured, as shown in table 3, Fig. 7.
Table 3: forint-phenol law surveys aspergillus oryzae protease enzyme slip-knot fruit
Overall merit: although the selection result accuracy is high, but step is complicated, workload is huge and raw material consuming is big, required time length (about 5 days), screen 100 strain bacterium at least to need prepare 100 bottles of above-mentioned bent material and measure, be not suitable for rapid screening high proteinase yield mycete.
Can be seen that from the Comparative result of above example 1 and comparative example 1,2, adopt the more difficult mensuration of transparent circle diameter of conventional agar-casein double-layer agar technique, cause that the measurement result accuracy of K value is relatively low, being difficult to react the gap of enzymatic productivity between each aspergillus oryzae, actually measured enzyme size alive is very poor with the size dependency of K value.And the transparent circle size good relationship that the enzyme size alive adopting forint-phenol law actually measured records with high protein enzyme mycete directed screening flat band method of the present invention, illustrate that the transparent circle size that the improvement high protein enzyme mycete directed screening flat band method of the present invention records can substitute for forint-phenol law to a certain extent and aspergillus oryzae strain is screened, and the selection result accuracy is high.Additionally, compared to conventional agar-casein double-layer agar technique and forint-phenol law, high protein enzyme mycete directed screening flat band method workload, consumptive material, consuming time on all there is obvious advantage；Therefore high protein enzyme mycete directed screening flat band method is a kind of method of rapid screening that can be applicable to aspergillus oryzae strain.
Embodiment 2:
A kind of rapid screening method of high proteinase yield fungal strain, namely improvement high protein enzyme mycete directed screening flat band method screening high protein enzyme enzyme Mucor alive is adopted, the concrete operation step of the present embodiment is substantially the same manner as Example 1, simply the object of screening is replaced with Mucor by aspergillus oryzae, and screening bacterium number is 30 strains.The culture dish adopting 10 specifications to be Φ 90mm, 4 aseptic Oxford cups (sterilized water that can inject equivalent in one of them Oxford cup compares) placed by each culture dish.Selecting the 9 strain bacterium that high protein enzyme mycete directed screening flat band method result is best, result the selection result is such as shown in table 4 below, Fig. 8.
Table 4: high protein enzyme mycete directed screening flat band method screening Mucor result
Comparative example 3:
Adopting conventional agar-casein double-layer agar technique screening high protein enzyme enzyme Mucor alive, specific operation process is essentially identical with comparative example 1, and the 9 strain bacterium chosen in above-mentioned the present embodiment 2 are measured, and result is such as shown in table 5 below, Fig. 9.
Table 5: existing agar-casein double-layer agar technique K value screening Mucor result
| Bacterium numbering | 2-1 | 2-2 | 2-3 | 2-4 | 2-5 | 2-6 | 2-7 | 2-8 | 2-9 |
| K value | 1.12 | 1.85 | 1.57 | 0.93 | 0.76 | 0.90 | 0.85 | 0.64 | 0.68 |
Comparative example 4:
Adopting conventional forint-phenol law to survey aspergillus oryzae protease enzyme to live, concrete operations are essentially identical with comparative example 2, and the 9 strain bacterium chosen in above-mentioned the present embodiment 2 are measured, and result is such as shown in table 6 below, Figure 10.
Table 6: forint-phenol law surveys mucor protease enzyme slip-knot fruit
Can be seen that from the Comparative result of above example 2 and comparative example 3,4, adopt the more difficult mensuration of transparent circle diameter of conventional agar-casein double-layer agar technique, cause that the measurement result accuracy of K value is relatively low, being difficult to react the gap of enzymatic productivity between each Mucor bacterial strain, actually measured enzyme size alive is very poor with the size dependency of K value.And the transparent circle size good relationship that the enzyme size alive adopting forint-phenol law actually measured records with high protein enzyme mycete directed screening flat band method of the present invention, illustrate that the transparent circle size that the high protein enzyme mycete directed screening flat band method of the present invention records can substitute for forint-phenol law to a certain extent and Mucor bacterial strain is screened, and the selection result accuracy is high.Additionally, compared to conventional agar-casein double-layer agar technique and forint-phenol law, high protein enzyme mycete directed screening flat band method workload, consumptive material, consuming time on all there is obvious advantage；Therefore high protein enzyme mycete directed screening flat band method is a kind of method of rapid screening that can be applicable to hair Aspergillus strain.
Claims (10)
1. a rapid screening method for high proteinase yield fungal strain, comprises the following steps:
(1) actication of culture: be inoculated in the raw Spore cultivation base inclined-plane of mycete, then constant temperature culture respectively by needing the multiple or many strains fungal strain carrying out screening；
(2) high protein enzyme mycete directed screening flat board is prepared: the plate after sterilizing-drying is first poured into lower floor's control medium, after its cooled and solidified, pours upper strata screening culture medium again into；
(3) also gravity flow screen screening device is placed: treat to place in solidifying process the sterile cuvette of multiple equal size in upper strata screening culture medium, after culture medium solidifying, sterile cuvette is fixed；
(4) mycotic spore suspension to be measured preparation and activation: physiological saline solution washes cultured mycotic spore in lower step (1), accesses the activation of spore activated liquid culture medium, then adjusts spore concentration after aseptic lens paper filtration sterilization silk；
(5) mycete to be measured is cultivated before measuring: each mycete activation spore suspension prepared by equivalent aspiration step (4), it is injected separately in each sterile cuvette of the high protein enzyme mycete directed screening flat board that above-mentioned steps (2) prepares, has inoculated and be placed in constant incubator and cultivate；
(6) mold protease enzyme to be measured is lived and is contrasted screening: after the cultivation of above-mentioned steps (5) completes, and measures the transparent circle diameter formed around each sterile cuvette on high protein enzyme mycete directed screening flat board；Determining each fungal strain prolease activity size according to transparent circle diameter, transparent circle is more big, and in sterile cuvette, the fungal strain prolease activity of inoculation is more big.
2. rapid screening method according to claim 1, it is characterized in that, in described step (1), the raw Spore cultivation base of the mycete of selection is mainly formulated by 100～110g/L Semen Maydis powder, 150～160g/L Rhizoma Solani tuber osi, 20～25g/L sucrose, 15～20g/L agar, and pH is natural；Rhizoma Solani tuber osi needs peeling, stripping and slicing, boils process, and the time of boiling is not less than 30min, then by filtered through gauze, utilizes filtrate preparation culture medium；121 DEG C of sterilizing 20min.
3. rapid screening method according to claim 1, it is characterised in that in described step (2), described lower floor's control medium is pure agar culture medium；Described upper strata screening culture medium is improvement casein culture medium.
4. rapid screening method according to claim 3, it is characterised in that add sodium deoxycholate in described improvement casein culture medium and spread inhibitor as fungal hyphae.
5. rapid screening method according to claim 4, it is characterized in that, the concrete formula concentration of described improvement casein culture medium includes: casein 3～6g/L, sodium deoxycholate 1.0～2.0g/L, potassium dihydrogen phosphate 0.36g/L, manganese sulfate 0.5g/L, zinc chloride 0.014g/L, disodium hydrogen phosphate 1.07g/L, sodium chloride 0.16g/L, ferrous sulfate 0.002g/L, calcium chloride 0.002g/L, agar powder 15～20g/L；PH6.5～7.0；121 DEG C of sterilizing 20min.
6. the rapid screening method according to any one of Claims 1 to 5, it is characterized in that, in described step (3), screening plant sterile cuvette adopts the glass tubule of Oxford cup or diameter 1cm, high 1cm, sterile cuvette Φ 60mm, Φ 90mm, Φ 120mm, Φ 150mm culture dish in placement quantity respectively 3～4,4～5,11～12,18～19；The position that sterile cuvette is placed is 1～2mm place at the bottom of distance ware.
7. the rapid screening method according to any one of Claims 1 to 5, it is characterized in that, in described step (4), described spore activated liquid culture medium is mainly formulated by concentration 10～12g/L germinating barley, 3.0～5.0g/L Semen Maydis pulp, 15～20g/L glucose, and pH is natural；121 DEG C of sterilizing 15min.
8. the rapid screening method according to any one of Claims 1 to 5, it is characterised in that in described step (4), described spore activation condition is: shaking table temperature is 28 DEG C, and rotating speed is 160r/min；Spore soak time is 5～6h.
9. the rapid screening method according to any one of Claims 1 to 5, it is characterised in that in described step (4), is adjusted to 10 by the spore concentration of the mycotic spore suspension prepared6Individual/mL.
10. the rapid screening method according to any one of Claims 1 to 5, it is characterised in that in described step (1), the temperature cultivated in calorstat is 28 ± 0.1 DEG C, and incubation time is 48h；In described step (5), the temperature cultivated in constant incubator is 28 ± 0.1 DEG C, and incubation time is 1～2d.
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