CN105779311B - A kind of rapid screening method of high proteinase yield fungal strain - Google Patents
A kind of rapid screening method of high proteinase yield fungal strain Download PDFInfo
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- CN105779311B CN105779311B CN201610328760.4A CN201610328760A CN105779311B CN 105779311 B CN105779311 B CN 105779311B CN 201610328760 A CN201610328760 A CN 201610328760A CN 105779311 B CN105779311 B CN 105779311B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a kind of rapid screening methods of high proteinase yield fungal strain, comprising the following steps: the fungal strain screened is inoculated into respectively on the raw Spore cultivation base inclined-plane of mould, then constant temperature incubation；Lower layer's control medium is first poured into sterile dry plate, then pours into upper layer screening and culturing medium, then is inserted into multiple sterile cuvettes into the double-layer plate prepared, high protein enzyme mould directed screening plate is prepared；Spore suspension is made with cultured mycotic spore to be measured, and vibrates activation culture in spore activated liquid culture medium；It is injected separately into each sterile cuvette above-mentioned again, constant temperature incubation；After the completion of culture, each transparent loop diameter on high protein enzyme mould directed screening plate is directly measured；The size of each fungal strain prolease activity is determined according to diameter, transparent circle is bigger, and fungal strain prolease activity is bigger.Method of the invention has many advantages, such as to facilitate operation, accuracy rate height, workload small and at low cost.
Description
Technical field
The present invention can be used for the screening of excellent fermenting microbe in fermented food (soy sauce, fermented soya bean, fermented bean curd etc.), belong to food hair
Ferment technical field, in particular to a kind of screening technique that high proteinase yield fungal strain is fast and convenient.
Background technique
Aspergillus oryzae, Mucor in mould etc. are widely used in fermented food, and protease abundant can will be in raw material
Protein degradation improves the digestive utilization ratio of protein at polypeptide and amino acid；Can especially generate some delicious amino acids and
Small-molecular peptides assign the good flavor of fermented food.It can be said that the moulds such as aspergillus oryzae and Mucor are the weights in fermented food production
Want strain.
The strains such as aspergillus oryzae, Mucor used in the production of the fermented foods such as soy sauce, fermented soya bean, fermented bean curd answer albumen with higher
Enzyme enzyme activity, to directly affect the utilization rate of raw material, amino nitrogen content etc. important for the proteinase activity of strain in fermented food production
Index, therefore, the fungal strain that screening obtains high protein enzyme activity are particularly important for the fermentation bean food production such as soy sauce.
Currently, common prolease activity screening technique is casein flat band method, egg is secreted on plate by measuring bacterial strain
The ratio (K value) of transparent circle and colony diameter that white enzyme generates screens high protein enzyme enzyme activity bacterial strain (referring to Fig. 1), from Fig. 1
As can be seen that this method, since colonial morphology is irregular during the growth process, blur margin is clear for mould, transparent loop diameter is more difficult
Measurement, causes the measurement result accuracy of K value lower；And the K value very little between each bacterium, it is difficult to react enzymatic productivity between each bacterium
Gap (referring to Fig. 3), as can be seen from Figure 3 the size correlation of actually measured enzyme activity size and K value is very poor, this explanation
Traditional agar-casein double-layer agar technique is unreliable to the selection result of aspergillus.Accurately to be screened, it has to be trained in koji-making
Forint- phenol law is taken to carry out the measurement of proteinase activity after supporting.Forint- phenol law measurement proteinase activity is needed using triangular flask system
Standard curve is drawn in bent, culture, although as a result accurate, equipment and reagent chemicals dosage are big, and required time is long, method
It is cumbersome, it takes time and effort.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the shortcomings of to mention in background above technology and defect, provide one
Kind facilitate that operation, accuracy rate is high, workload is small and the rapid screening method of high proteinase yield fungal strain at low cost.
In order to solve the above technical problems, technical solution proposed by the present invention is that a kind of high proteinase yield fungal strain is (special
It is preferred that aspergillus oryzae strain or Mucor bacterial strain) rapid screening method, comprising the following steps:
(1) a variety of or more plants of fungal strains screened actication of culture: are inoculated in the raw spore training of mould respectively
Base inclined-plane is supported, then constant temperature incubation；
(2) it prepares high protein enzyme mould directed screening plate: first pouring into lower layer's control training in the plate after sterilizing-drying
Base is supported, pours into upper layer screening and culturing medium again after its cooled and solidified；
(3) it places and fixes screening plant: placing the nothing of multiple same sizes during screening and culturing medium is to be coagulated on upper layer
Bacterium cuvette, sterile cuvette is fixed after culture medium solidification；
(4) preparation of mycotic spore suspension and activation to be measured: sterile saline washes cultured mould spore in lower step (1)
Son, access spore activated liquid culture medium activation after sterile lens wiping paper filtration sterilization silk, then adjust spore concentration；
(5) culture before mould measurement to be measured: each mould of equivalent aspiration step (4) preparation activates spore suspension, infuses respectively
Enter in each sterile cuvette of high protein enzyme mould directed screening plate made from above-mentioned steps (2) (in one of sterile cuvette
The sterile water that equivalent can be injected compares), inoculation is completed to be placed in constant incubator to cultivate；
(6) after the completion of the culture of above-mentioned steps (5), it is mould mold protease enzyme activity comparison screening to be measured: to measure high protein enzyme
The transparent loop diameter formed around each sterile cuvette on bacterium directed screening plate；Each mould bacterium is determined according to transparent circle diameter
Strain prolease activity size, transparent circle is bigger, and the fungal strain prolease activity being inoculated in sterile cuvette is bigger.
The present invention by by sterile cuvette and improvement casein culture medium be used cooperatively, using different size culture dish simultaneously
The screening of a variety of or more plant height protease fungal strains is carried out, method is easy, and it is time saving and energy saving, it is mould to greatly improve high protein enzyme
The accuracy and efficiency of bacterium primary dcreening operation are of great significance in the utilization of breeding screening high proteinase yield fungal strain.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is preferred that in the step (1), the mould of selection
Raw Spore cultivation base is mainly by 100~110g/L corn flour, 150~160g/L potato, 20~25g/L sucrose, 15~20g/L
Agar is formulated, and pH is natural；Potato needs peeling, stripping and slicing, cooked processing, is cooked the time and is not less than 30min, then with gauze mistake
Filter prepares culture medium using filtrate；121 DEG C of sterilizing 20min.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is preferred that in the step (2), the lower layer
Control medium is pure agar medium；The upper layer screening and culturing medium is improvement casein culture medium.The albumen that mould generates
Enzyme can hydrolyzed casein to improvement casein culture medium formed transparent circle, but because casein culture medium color be white and culture
The reasons such as ware bottom out-of-flatness are unfavorable for observing and measuring the size of transparent circle, so it is flat for lower layer to add pure agar medium
Plate is conducive to the observation and measurement of transparent circle, improves the accuracy of result.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is furthermore preferred that adding in the improvement casein culture medium
Deoxysodium cholate is added to spread inhibitor as fungal hyphae.It is furthermore preferred that the specific formula concentration of the improvement casein culture medium
It include: 3~6g/L of casein, 1.0~2.0g/L of deoxysodium cholate, potassium dihydrogen phosphate 0.36g/L, manganese sulfate 0.5g/L, chlorination
Zinc 0.014g/L, disodium hydrogen phosphate 1.07g/L, sodium chloride 0.16g/L, ferrous sulfate 0.002g/L, calcium chloride 0.002g/L, fine jade
15~20g/L of cosmetics；PH 6.5~7.0；121 DEG C of sterilizing 20min.
In above-mentioned improved plan, deoxysodium cholate is preferably added in the screening and culturing medium of upper layer, to inhibit fungal hyphae
Sprawling, improves the accuracy of measurement result.The present invention has further defined the most suitable additive amount of deoxysodium cholate, ours grinds
Study carefully show deoxysodium cholate additive amount it is excessive will lead to mould can not normal growth, transparent circle is unobvious；And deoxysodium cholate adds
Dosage is too small, inhibits mycelia effect unobvious, and mycelia spreads sterile cuvette out, influences the measurement of transparent circle.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is preferred that in the step (1), trained in insulating box
Feeding temperature is 28 ± 0.1 DEG C, incubation time 48h.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is preferred that in the step (3), screening plant without
Bacterium cuvette is using Oxford cup or the glass tubule of diameter 1cm, high 1cm, and sterile cuvette is in Φ 60mm, Φ 90mm, Φ 120mm, Φ
Placement quantity in the culture dish of 150mm is respectively 3~4,4~5,11~12,18~19；What sterile cuvette was placed
Position is at 1~2mm of ware bottom.The sterile cuvette that the present invention uses is preferred with Oxford cup；In the art, Wo Mencong
It is not found in and uses it in fungal strain prolease activity screening field, this is apparently not that those skilled in the art are readily apparent that
's.
The rapid screening method of above-mentioned high proteinase yield fungal strain, it is preferred that in the step (4), the spore is living
Change fluid nutrient medium mainly to be prepared by 10~12g/L of concentration germinating barley, 3.0~5.0g/L corn pulp, 15~20g/L glucose
It forms, pH is natural；121 DEG C of sterilizing 15min.
Cardinal principle of the invention are as follows: by the way that sterile cuvette and improvement casein culture medium to be used cooperatively, limit mycelia
It grows to outside cup, so that the transparent circle regular shape of its formation, edge clear, are convenient for measuring；Reinjected in cuvette it is isometric, etc.
The spore suspension of concentration will test quantification, significantly improve the order of accuarcy of detection the selection result；In addition, side of the invention
Method can replace the screening technique of forint- phenol law survey enzyme activity after koji-making after induction mutation of bacterium, and the sieve of mass mutation strain is rapidly completed
Choosing, efficiently reduces the screening operation amount and screening cost of mutagenic strain.
Compared with the prior art, the advantages of the present invention are as follows:
1, the deoxysodium cholate preferably added in the sterile cuvette in the method for the present invention and upper layer screening and culturing medium can all limit
Mycostatic sprawling prevents mycelia from growing to outside cup, so that the transparent circle regular shape of its formation, edge clear；Meanwhile by adding
Adding pure agar medium is that lower layer's plate is convenient for measuring so that transparent circle is high-visible；
2, isometric, isoconcentration spore suspension is injected in the sterile cuvette of the method for the present invention design, will be tested quantitative
Change, improves the order of accuarcy of the selection result；Figure 4, it is seen that using the actually measured enzyme activity size of forint- phenol law with
The transparent circle size good relationship that high protein enzyme mould directed screening flat band method of the present invention measures illustrates that high protein enzyme mould is fixed
The transparent circle size measured to screening flat board method can replace forint- phenol law to a certain extent and screen to mould, and sieve
Select result precision height.
3, method of the invention can replace the screening technique of forint- phenol law survey enzyme activity after koji-making after induction mutation of bacterium, subtract
The screening operation amount and screening cost of few mutagenic strain.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is that existing agar-casein double-layer agar technique screens high protein enzyme enzyme activity aspergillus oryzae figure.
Fig. 2 is that high protein enzyme mould directed screening flat band method of the present invention screens high protein enzyme enzyme activity aspergillus oryzae figure.From figure
It can be seen that transparent circle diameter clear, regular edges, therefore the selection result is significantly better than conventional agar-casein double-layer agar technique.
Fig. 3 is that existing agar-casein double-layer agar technique and forint- phenol law screen high protein enzyme enzyme activity aspergillus oryzae comparison diagram.
Fig. 4 is that high protein enzyme mould directed screening flat band method of the present invention and forint- phenol law screen high protein enzyme enzyme activity rice song
Mould comparison diagram.
Fig. 5 is that high protein enzyme mould directed screening flat band method screens high proteinase yield aspergillus oryzae in the embodiment of the present invention 1
As a result.
Fig. 6 is to screen high proteinase yield aspergillus oryzae using conventional agar-casein double-layer agar technique in comparative example 1 of the present invention
Result.
Fig. 7 is the result for screening high proteinase yield aspergillus oryzae in comparative example 2 of the present invention using conventional forint- phenol law.
Fig. 8 is the knot that high protein enzyme mould directed screening flat band method screens high proteinase yield Mucor in the embodiment of the present invention 2
Fruit.
Fig. 9 is to screen high proteinase yield Mucor using conventional agar-casein double-layer agar technique in comparative example 3 of the present invention
As a result.
Figure 10 is the result for screening high proteinase yield Mucor in comparative example 4 of the present invention using conventional forint- phenol law.
Specific embodiment
To facilitate the understanding of the present invention, the present invention is made below in conjunction with Figure of description and preferred embodiment more complete
Face meticulously describes, but the protection scope of the present invention is not limited to the following specific embodiments.
Unless otherwise defined, all technical terms used hereinafter and the normally understood meaning of those skilled in the art
It is identical.Technical term used herein is intended merely to the purpose of description specific embodiment, is not intended to the limitation present invention
Protection scope.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through city
Field is commercially available or can be prepared by existing method.
Embodiment 1:
A kind of rapid screening method of high proteinase yield fungal strain uses high protein enzyme mould directed screening flat band method
High protein enzyme enzyme activity aspergillus oryzae is screened, specifically includes the following steps:
(1) the 100 Aspergillus oryzae bacterial strains screened actication of culture: are transferred to the raw Spore cultivation base of mould respectively
Inclined-plane on, be placed in 28 ± 0.1 DEG C of culture 48h in insulating box.
The raw Spore cultivation based formulas of the mould of selection is corn flour 100g/L, potato 150g/L, sucrose 25g/L, agar
20g/L, pH are natural；Potato removed the peel, stripping and slicing, cooked processing, is cooked the time and is not less than 30min, then with gauze mistake
Filter prepares culture medium using filtrate；121 DEG C of sterilizing 20min.
(2) it prepares high protein enzyme mould directed screening plate: preparing the culture dish that 6 sets of specifications are Φ 150mm, sterilizing is dry
It is dry, lower layer's control medium is first poured into plate, thickness is about 2mm, pours into upper layer screening and culturing again after its cooled and solidified
Base, thickness are about 3mm.
Lower layer's control medium of selection is pure agar medium.The upper layer screening and culturing medium of selection is improvement casein culture
Base, specific formula are casein 4g/L, deoxysodium cholate 1.5g/L, potassium dihydrogen phosphate 0.36g/L, manganese sulfate 0.5g/L, chlorine
Change zinc 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, pH 6.5；121 DEG C of sterilizing 20min.
(3) it places and fixes screening plant: placing the nothing of 18 same sizes during screening and culturing medium is to be coagulated on upper layer
Bacterium Oxford cup, the position of placement are at the 2mm of ware bottom, and sterile Oxford cup is fixed after culture medium solidification.
(4) preparation of mycotic spore suspension and activation to be measured: sterile saline washes cultured aspergillus oryzae in lower step (1)
Spore, accesses spore activated liquid culture medium after sterile lens wiping paper filtration sterilization silk, 160r/min, 28 DEG C of oscillations activation 6h, then
Spore concentration is adjusted to 106A/mL.
The spore activated liquid culture medium of selection is matched by 10g/L germinating barley, 3.0g/L corn pulp, 20g/L glucose
It makes, pH is natural；121 DEG C of sterilizing 15min.
(5) culture before mould measurement to be measured: the rice aspergillus for drawing the preparation of 10 μ L steps (4) activates spore suspension, infuses respectively
Enter in each Oxford cup of high protein enzyme mould directed screening plate made from above-mentioned steps (2), is injected in one of Oxford cup
The sterile water of equivalent compares, and inoculation is completed to be placed in 28 DEG C of constant incubator to cultivate 2d.
(6) mold protease enzyme activity comparison screening to be measured: after the completion of the culture of above-mentioned steps (5), high protein enzyme rice is measured
The transparent loop diameter (referring to fig. 2) formed around each Oxford cup on aspergillus directed screening plate, selects high protein enzyme aspergillus oryzae
9 plants of best bacterium of directed screening flat band method result, as a result as shown in table 1, Fig. 5；Each mould is determined according to transparent circle diameter
Strain protein enzyme activity size, transparent circle is bigger, i.e., corresponding aspergillus oryzae strain prolease activity is bigger.
Table 1: high protein enzyme mould directed screening flat band method screens aspergillus oryzae result
Overall merit: the screening step of the present embodiment is simple, easy to operate, and the required time is about 2.5 days, the selection result
Accuracy can reach 90% or more, screens 100 plants of bacterium and only needs to prepare the culture dish that 6 sets of specifications are Φ 150mm, suitable for height
Produce the quick screening of protease mould.
Comparative example 1:
High protein enzyme enzyme activity aspergillus oryzae is screened using conventional agar-casein double-layer agar technique, concrete operations are as follows:
(1) aspergillus oryzae strain to be measured actication of culture: is inoculated in PDA culture medium inclined-plane, 28 DEG C of constant temperature incubation 48h.
(2) it prepares agar-casein double-layer plate: preparing the culture dish that 100 sets of specifications are Φ 90mm, sterilizing-drying, flat
Pure agar medium is first poured into ware, thickness is about 2mm；Casein culture medium is poured into again after its cooling, and thickness is about 3mm, system
Standby to obtain agar-casein double-layer plate: the preparation method of the preparation method of the pure agar medium of selection is the same as above-mentioned the present embodiment 1
The step of (2)；The preparation method of the casein culture medium of selection is do not have with the difference in (2) the step of above-mentioned the present embodiment 1
Add deoxysodium cholate.
(3) it prepares spore suspension: washing in lower step (1) cultured mycotic spore with sterile saline, it is sterile
Lens wiping paper filtration sterilization silk.
(4) it is inoculated with: 1mL spore suspension being taken to be coated on agar-casein double-layer plate.
(5) it cultivates: with (5) the step of above-mentioned the present embodiment 1.
(6) screening comparison: after the completion of above-mentioned steps (5) culture, the 9 plants of bacterium chosen in above-mentioned the present embodiment 1 are surveyed
It is fixed, each transparent loop diameter and colony diameter on agar-casein double-layer plate are measured, calculating K value, (transparent loop diameter and bacterium colony are straight
Diameter) result is as shown in table 2, Fig. 6.
Table 2: existing agar-casein double-layer agar technique K value screens 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 of this method is shorter (about 2.5 days), step is complicated, heavy workload, and ties
Fruit accuracy is low, screens 100 plants of bacterium and at least needs to prepare the culture dish that 100 sets of specifications are Φ 90mm, is not suitable for quickly sieving
Select high proteinase yield mould.
Comparative example 2:
Aspergillus oryzae protease enzyme activity is surveyed using conventional forint- phenol law, concrete operations are as follows:
It is measured according to National Standard of the People's Republic of China SB/T 10317-1999.
(1) koji-making: preparing 100 parts of koji-making culture mediums and be placed in triangular flask, connects in cooling and material loosening culture medium
Kind spore suspension, each triangular flask miospore number are 7.7 × 106It is a, in 32 DEG C of constant temperature incubation 36h, periodically turn over song.
The preparation method of the koji-making culture medium of selection is the following steps are included: by dregs of beans, wheat, wheat bran with the ratio of 7:2:1
Bottling (50g siccative/triangular flask) is mixed, is then soaked with water (mass ratio of amount of water and raw material is 90%), 0.1~
1~1.5h of 0.12MPa sterilizing.
(2) remaining step is carried out according to National Standard of the People's Republic of China SB/T 10317-1999.
(3) measurement result: the 9 plants of 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 activity result
Overall merit: although the selection result accuracy is high, step is complicated, and workload is huge and raw material expends greatly, required
Time it is long (about 5 days), screen 100 plants of bacterium and at least need to prepare 100 bottles of above-mentioned materials and measure, be not suitable for quickly screening
High proteinase yield mould.
It can be seen that from the Comparative result of above embodiments 1 and comparative example 1,2 using conventional agar-casein double-layer plate
The more difficult measurement of transparent loop diameter of method, causes the measurement result accuracy of K value lower, it is difficult to react producing enzyme between each aspergillus oryzae
The size correlation of the gap of ability, actually measured enzyme activity size and K value is very poor.And use forint- phenol law actually measured
The transparent circle size good relationship that enzyme activity size and high protein enzyme mould directed screening flat band method of the present invention measure, illustrates this hair
The transparent circle size that bright improvement high protein enzyme mould directed screening flat band method measures can replace Folin-Phenol to a certain extent
Method screens aspergillus oryzae strain, and the selection result accuracy is high.In addition, compared to conventional agar-casein double-layer agar technique
And forint- phenol law, high protein enzyme mould directed screening flat band method is in workload, consumptive material, time-consuming all with obvious excellent
Gesture；Therefore high protein enzyme mould directed screening flat band method is a kind of method quickly screened that can be suitable for aspergillus oryzae strain.
Embodiment 2:
A kind of rapid screening method of high proteinase yield fungal strain is that is, flat using improvement high protein enzyme mould directed screening
Plate method screens high protein enzyme enzyme activity Mucor, and the concrete operation step of the present embodiment is substantially the same manner as Example 1, pair only screened
As replacing with Mucor by aspergillus oryzae, screening bacterium number is 30 plants.Use 10 specifications for the culture dish of Φ 90mm, each culture dish is put
Set 4 sterile Oxford cups (sterile water that equivalent can be injected in one of Oxford cup compares).Select high protein enzyme mould
9 plants of best bacterium of directed screening flat band method result, as a result the selection result is as shown in the following table 4, Fig. 8.
Table 4: high protein enzyme mould directed screening flat band method screens Mucor result
Comparative example 3:
High protein enzyme enzyme activity Mucor, specific operation process and comparison are screened using conventional agar-casein double-layer agar technique
Example 1 is essentially identical, is measured to the 9 plants of bacterium chosen in above-mentioned the present embodiment 2, as a result as shown in the following table 5, Fig. 9.
Table 5: existing agar-casein double-layer agar technique K value screens 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:
Aspergillus oryzae protease enzyme activity is surveyed using conventional forint- phenol law, concrete operations and comparative example 2 are essentially identical, to upper
It states the 9 plants of bacterium chosen in the present embodiment 2 to be measured, as a result as shown in the following table 6, Figure 10.
Table 6: forint- phenol law surveys mucor protease enzyme activity result
It can be seen that from the Comparative result of above embodiments 2 and comparative example 3,4 using conventional agar-casein double-layer plate
The more difficult measurement of transparent loop diameter of method, causes the measurement result accuracy of K value lower, it is difficult to react producing enzyme between each Mucor bacterial strain
The size correlation of the gap of ability, actually measured enzyme activity size and K value is very poor.And use forint- phenol law actually measured
The transparent circle size good relationship that enzyme activity size and high protein enzyme mould directed screening flat band method of the present invention measure, illustrates this hair
The transparent circle size that bright high protein enzyme mould directed screening flat band method measures can replace forint- phenol law pair to a certain extent
Mucor bacterial strain is screened, and the selection result accuracy is high.In addition, compared to conventional agar-casein double-layer agar technique and forint-
Phenol method, high protein enzyme mould directed screening flat band method all have obvious advantage in workload, consumptive material, time-consuming；Therefore
High protein enzyme mould directed screening flat band method is a kind of method quickly screened that can be suitable for hair Aspergillus strain.
Claims (8)
1. a kind of rapid screening method of high proteinase yield fungal strain, comprising the following steps:
(1) a variety of or more plants of fungal strains screened actication of culture: are inoculated in the raw Spore cultivation base of mould respectively
Inclined-plane, then constant temperature incubation；
(2) it prepares high protein enzyme mould directed screening plate: first pouring into lower layer's control medium in the plate after sterilizing-drying,
Pour into upper layer screening and culturing medium again after its cooled and solidified；Lower layer's control medium is pure agar medium；Described
Upper layer screening and culturing medium is improvement casein culture medium；Deoxysodium cholate is added in the improvement casein culture medium as fungal hyphae
Spread inhibitor；
(3) it places and fixes screening plant: placing the sterile small of multiple same sizes during screening and culturing medium is to be coagulated on upper layer
Cup, sterile cuvette is fixed after culture medium solidification；
(4) preparation of mycotic spore suspension and activation to be measured: sterile saline washes cultured mycotic spore in lower step (1),
The activation of spore activated liquid culture medium is accessed after sterile lens wiping paper filtration sterilization silk, then adjusts spore concentration；
(5) culture before mould measurement to be measured: each mould of equivalent aspiration step (4) preparation activates spore suspension, is injected separately into
It states in each sterile cuvette of high protein enzyme mould directed screening plate made from step (2), inoculation is completed to be placed on constant temperature incubation
It is cultivated in case；
(6) after the completion of the culture of above-mentioned steps (5), it is fixed mold protease enzyme activity comparison screening to be measured: to measure high protein enzyme mould
The transparent loop diameter formed around each sterile cuvette in screening flat board；Each fungal strain egg is determined according to transparent circle diameter
White enzyme activity size, transparent circle is bigger, and the fungal strain prolease activity being inoculated in sterile cuvette is bigger.
2. rapid screening method according to claim 1, which is characterized in that in the step (1), the raw spore of the mould of selection
Sub- culture medium is mainly by 100~110g/L corn flour, 150~160g/L potato, 20~25g/L sucrose, 15~20g/L agar
It is formulated, pH is natural；Potato needs peeling, stripping and slicing, cooked processing, is cooked the time and is not less than 30min, then with filtered through gauze,
Culture medium is prepared using filtrate；121 DEG C of sterilizing 20min.
3. rapid screening method according to claim 1, which is characterized in that the specific formula of the improvement casein culture medium
Concentration include: 3~6g/L of casein, 1.0~2.0g/L of deoxysodium cholate, 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, 15~20g/L of agar powder；PH 6.5~7.0；121 DEG C of sterilizing 20min.
4. rapid screening method described in any one of claim 1 to 3, which is characterized in that in the step (3), sieve
Screening device sterile cuvette is using Oxford cup or the glass tubule of diameter 1cm, high 1cm, and sterile cuvette is in Φ 60mm, Φ 90mm, Φ
120mm, Φ 150mm culture dish in placement quantity be respectively 3~4,4~5,11~12,18~19；It is sterile small
The position that cup is placed is at 1~2mm of ware bottom.
5. rapid screening method described in any one of claim 1 to 3, which is characterized in that in the step (4), institute
Spore activated liquid culture medium is stated mainly by 10~12g/L of concentration germinating barley, 3.0~5.0g/L corn pulp, the Portugal 15~20g/L
Grape sugar is formulated, and pH is natural；121 DEG C of sterilizing 15min.
6. rapid screening method described in any one of claim 1 to 3, which is characterized in that in the step (4), institute
State spore activation condition are as follows: shaking table temperature is 28 DEG C, revolving speed 160r/min；Spore activation time is 5~6h.
7. rapid screening method described in any one of claim 1 to 3, which is characterized in that, will in the step (4)
The spore concentration for the mycotic spore suspension being prepared is adjusted to 106A/mL.
8. rapid screening method described in any one of claim 1 to 3, which is characterized in that permanent in the step (1)
The temperature cultivated in incubator is 28 ± 0.1 DEG C, incubation time 48h；In the step (5), the temperature cultivated in constant incubator
Degree is 28 ± 0.1 DEG C, and incubation time is 1~2d.
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