CN110734954A - Rapid identification culture medium and application thereof in cellulase qualitative detection - Google Patents
Rapid identification culture medium and application thereof in cellulase qualitative detection Download PDFInfo
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 77
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- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 239000001768 carboxy methyl cellulose Substances 0.000 claims abstract description 13
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims abstract description 10
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 57
- 230000000694 effects Effects 0.000 claims description 41
- 102000004190 Enzymes Human genes 0.000 claims description 40
- 108090000790 Enzymes Proteins 0.000 claims description 40
- 229940088598 enzyme Drugs 0.000 claims description 40
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 35
- 239000011780 sodium chloride Substances 0.000 claims description 18
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 claims description 17
- 238000004043 dyeing Methods 0.000 claims description 17
- 239000008055 phosphate buffer solution Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 10
- 108010084185 Cellulases Proteins 0.000 claims description 10
- 102000005575 Cellulases Human genes 0.000 claims description 10
- 239000008272 agar Substances 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 7
- 238000007711 solidification Methods 0.000 claims description 7
- 230000008023 solidification Effects 0.000 claims description 7
- 230000002255 enzymatic effect Effects 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 238000012216 screening Methods 0.000 description 7
- 239000000306 component Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 235000010980 cellulose Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 101710111935 Endo-beta-1,4-glucanase Proteins 0.000 description 2
- 108010032083 Glucan 1,4-beta-Glucosidase Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
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- 239000012138 yeast extract Substances 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 description 1
- 241000346653 Holotrichia parallela Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 108010091371 endoglucanase 1 Proteins 0.000 description 1
- 108010091384 endoglucanase 2 Proteins 0.000 description 1
- 108010092450 endoglucanase Z Proteins 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 238000003958 fumigation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/942—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-1, 4-glucosidic bonds, e.g. cellulase
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- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the detection of cellulase, in particular to a rapid identification culture medium and application thereof in the qualitative detection of cellulase, and discloses rapid identification culture media taking sodium carboxymethylcellulose as a main component and application thereof in the qualitative detection of cellulase.
Description
Technical Field
The invention belongs to detection of cellulase, and particularly relates to a culture medium for rapid identification and application thereof in qualitative detection of cellulase.
Background
Cellulases (celluloases) are enzymes that degrade cellulose under the synergistic action of various enzyme components to convert it into reducing sugars, including Exo β -1, 4-glucanase (Exo β -1, 4-gluca Nase, EC 3.2.1.91), Endo β -1, 4-glucanase (Endo β -1, 4-glucanase, EC 3.2.1.4), β -glucosidase (β -D-gluconase, EC 3.2.1.21), Cellulases from different sources have different ratios of the above three components.
The national light industry Standard cellulase preparation (QB 2583-.
When the enzyme preparation sample is quantitatively detected, the enzyme activity of the sample to be detected has range requirements, for example, the difference value between the absorbance of the sample and the blank liquid is required to be within the range of 0.3-0.4 by the method for detecting the enzyme activity of the filter paper in QB2583-2003, and the difference value between the absorbance of the sample and the blank liquid is required to be within the range of 0.33-0.35 by the method for detecting the activity of the hydroxymethyl cellulase, whether the cellulase is contained in samples with unknown enzyme activity or not needs to be firstly evaluated during actual production application, and then the specific indexes of the cellulase activity in the samples are detected.
Currently, an unknown enzyme preparation sample is usually diluted by different times during enzyme activity determination, and then specific activity indexes of the sample with different dilution times are detected according to QB2583-2003, wherein each sample with the dilution times needs to have three times. The method needs to prepare a large amount of reagents and perform an enzyme catalysis reaction and a colorimetric process, and the quality guarantee period of some reagents is short; meanwhile, for a sample with darker color and low enzyme activity, the colorimetric method cannot be used for detecting whether the cellulase activity exists. Therefore, the QB2583-2003 method is time-consuming and labor-consuming for qualitatively evaluating whether the sample contains the cellulase, and if the method is adopted, the method can be used for rapidly identifying whether the sample contains the cellulase, so that assistance is provided for accurate quantitative measurement, and the efficiency of actual production is improved.
, which is a commonly used means in microorganism screening work, is mentioned in 2000 edition of microbiology, edited by Shenping, and there are methods reported at present for screening cellulase-producing strains by using a plate transparent ring method, such as Huang Sheng Wei in research on molecular polymorphism of intestinal microorganisms and diversity of cellulose-degrading bacteria in Holotrichia parallela larvae, for screening cellulase-producing capability of strains by using a primary screening medium, and selecting the pure cellulase-producing strainsThe application of color biological celluloses in the rapid detection of the activity of cellulase producing bacteria is disclosed in the invention patent application with the application number of 201310004456.0, the color biological cellulose gels are used as substrates to react with fermentation culture solution of the cellulase producing bacteria, the color of the solution is directly observed after the reaction is finished, a DNS reagent is not used for color reaction, culture media and methods for simultaneously screening protease and cellulase are disclosed in the invention patent application with the application number of 201810032356.1, and the culture media are prepared in a mode of simultaneously adding protease substrate gelatin protein and cellulase substrate carboxymethyl cellulose in a mode of simultaneously adding 1-3g of NaNO substrate gelatin protein and cellulase substrate carboxymethyl cellulose3；0.8-1.2g K2HPO4；0.4-0.6g MgSO4(ii) a 0.4-0.6g KCl; 1-3g carboxymethyl cellulose or microcrystalline cellulose; 8-12g gelatin; 1-3g of yeast extract powder; 16-18g agar or agarose; adding water to a constant volume of 1000 ml. A quick method for simultaneously detecting and screening the cellulase and protease active strains is established by combining the method of firstly carrying out iodine fumigation and then carrying out Coomassie brilliant blue dyeing. However, the above three methods originally aim at identifying the conditions of the enzyme-producing microorganism or the cellulase in the fermentation broth, require long-time microorganism culture or enzyme catalytic reaction, and simultaneously identify the complex components of the culture medium and have color requirements on the cellulase.
Disclosure of Invention
The invention aims to provide culture media for rapid identification and application thereof in cellulase qualitative detection, which can rapidly identify whether cellulase exists, can compare the cellulase activity of different samples and provide reference for accurate measurement of cellulase.
The overall technical concept of the invention is as follows:
the rapid identification culture medium consists of the following raw materials:
1g/L-10g/L of sodium carboxymethyl cellulose; 5g/L to 15g/L of agar; the balance of water; the pH value is 6.0-7.2.
The application of the rapid identification culture medium in the cellulase qualitative detection comprises the following process steps:
A. preparation of cellulase detection plate
Weighing 20-30mL of sterilized and unsolidified rapid identification culture medium, and placing the culture medium into a culture dish for solidification;
B. spotting of cellulases
Preparing cellulase solutions with different enzyme activities by using a phosphate buffer solution, adding 1 mu L-10 mu L of the cellulase solutions with different enzyme activities into each culture dish for rapidly identifying the culture medium under an aseptic condition, and culturing for 0.5-1 hour at the temperature of 30-50 ℃;
C. dyeing and cleaning
And D, taking out the culture dish in the step B, pouring Congo red solution into the culture dish, dyeing for 10-30 minutes, discarding the Congo red solution, adding NaCl solution, rinsing for 1-15 minutes, discarding the NaCl solution, and repeatedly rinsing for 2-5 times until a transparent ring with an obvious boundary appears.
The specific technical concept of the invention is as follows:
in order to measure the diameter of the transparent hydrolysis ring and verify the enzyme activity of the corresponding cellulase, the preferable technical implementation means is that the method further comprises step D, and the process conditions of the step D are as follows:
D. the transparent circles where distinct boundaries appear in step C are measured.
Preparing cellulase solutions with different enzyme activities by using phosphate buffer solution in the step B, wherein the cellulase solution comprises the following steps: 0U; 1: 20U; 2: 40U; 3: 100U; 4: 200U.
To verify the technical effect of the present invention, the applicant conducted the following tests:
, comparison with the Huangvicao method
1. Test materials
No. 1-screening medium for cellulase-producing strains: k2HPO41.9g/L；KH2PO40.94g/L；KCl 1.6g/L；NaCl 1.43g/L；NH4Cl 0.15g/L；MgSO4·7H2O 0.037g/L；CaCl2·2H2O is 0.017 g/L; yeastextract 0.1 g/L; 10g/L of sodium carboxymethylcellulose; 15g/L of agar; pH 7.2. (refer to the Huangvic method)
No. 2-cellulase in the present invention rapidly identifies the culture medium: 1-10g/L of sodium carboxymethyl cellulose; 5-15g/L of agar; the pH value is 6.0-7.2.
2. Test device
The method comprises the following steps of (1) sterile culture dish, measuring cylinder, ruler, sterilization pot, super clean bench, cellulase with the enzyme activity of 10 ten thousand U/g, Congo red dye solution with the concentration of 1mg/mL, NaCl solution with the concentration of 1mol/L, and phosphate buffer solution with the concentration of 0.1 mol/L, pH-6.0.
3. Test method
(1) Preparation of cellulase detection plate
Respectively weighing 20-30mL of sterilized and unsolidified No. 1 and No. 2 culture medium dosing cylinders, and pouring the weighed media into a culture dish for later use after coagulation.
(2) Spotting of cellulases of different activities
Preparing cellulase solutions with different enzyme activities by using phosphate buffer solution 0: 0U; 1: 20U; 2: 40U; 3: 100U; 4: 200U, adding 1-10 mu L of cellulase solution with different enzyme activities into each culture dish under the aseptic condition, and culturing for 0.5-1 hour at the temperature of 30-50 ℃.
(3) Dyeing and cleaning
And taking out the culture dish after the culture is finished, pouring Congo red solution into the culture dish, dyeing for 10-30 minutes, discarding the Congo red solution, adding NaCl solution, rinsing for 1-15 minutes, discarding the NaCl solution, and repeatedly rinsing for 2-5 times until a transparent ring with an obvious boundary appears.
(4) The hydrolytic circles of different enzyme activities were measured with a ruler.
4. Test results
As shown in FIG. 1, the samples with different enzyme activities all showed transparent circles except the control point 0, which indicates that the culture medium No. 2 with simple components and the complex culture medium No. 1 disclosed in the Huang Sheng Wei method can qualitatively detect the existence of the cellulase.
TABLE 1 comparison of transparent circles for different enzyme activities
It can be seen from table 1 that samples with different enzyme activities show transparent circles with different diameters, and the larger the enzyme activity is, the larger the diameter of the transparent circle is. The comparison of the enzyme activity of different cellulase samples by using the rapid identification culture medium is shown. In addition, different cellulase samples with enzyme activity difference of 20U can be compared.
Secondly, compared with the prior method, the method of the invention
TABLE 2 comparison of the method for qualitative detection of cellulase in the present invention with other methods
Compared with the existing method, the method for detecting the cellulase has the advantages of short detection time, simple culture medium components and no requirement on the color of a sample. The cellulase-containing sample in liquid or solid form can be qualitatively detected.
The invention achieves the substantive characteristics and obvious technical progress that:
compared with the reported method for detecting the cellulase-producing strain or the fermentation liquid thereof, the method for detecting the cellulase has the advantages of short detection time, simple culture medium components and no requirement on the color of a sample. The cellulase-containing sample in liquid or solid form can be qualitatively detected.
Drawings
FIG. 1 is a schematic diagram showing a comparison of transparent circles for different enzyme activities in the present invention.
Detailed Description
The invention is further described with reference to the following examples, which are not intended to limit the invention, the scope of the invention is indicated by the claims, and all technical equivalents which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Example 1
The rapid identification culture medium consists of the following raw materials:
10g/L of sodium carboxymethylcellulose; 15g/L of agar; the balance of water; pH 7.2.
The application of the rapid identification culture medium in the cellulase qualitative detection comprises the following process steps:
A. preparation of cellulase detection plate
Weighing 30mL of sterilized and unsolidified rapid identification culture medium, and placing the culture medium in a culture dish for solidification;
B. spotting of cellulases
Preparing cellulase solutions with different enzyme activities by using a phosphate buffer solution, adding 10 mu L of cellulase solution with different enzyme activities into each culture dish for rapidly identifying the culture medium under an aseptic condition, and culturing for 1 hour at 50 ℃;
C. dyeing and cleaning
Taking out the culture dish in the step B, pouring Congo red solution into the culture dish, dyeing for 30 minutes, discarding the Congo red solution, adding NaCl solution, rinsing for 15 minutes, discarding the NaCl solution, and repeatedly rinsing for 5 times until a transparent ring with an obvious boundary appears;
D. the transparent circles where distinct boundaries appear in step C are measured.
Preparing cellulase solutions with different enzyme activities by using phosphate buffer solution in the step B, wherein the cellulase solution comprises the following steps: 0U; 1: 20U; 2: 40U; 3: 100U; 4: 200U.
Example 2
This example differs from example 1 in that:
the rapid identification culture medium consists of the following raw materials:
1g/L of sodium carboxymethyl cellulose; 5g/L of agar; the balance of water; pH 6.0.
The application of the rapid identification culture medium in the cellulase qualitative detection comprises the following process steps:
A. preparation of cellulase detection plate
Weighing 20mL of sterilized and unset rapid identification culture medium, and placing the culture medium in a culture dish for solidification;
B. spotting of cellulases
Preparing cellulase solutions with different enzyme activities by using a phosphate buffer solution, adding 1 mu L of cellulase solution with different enzyme activities into each culture dish for rapidly identifying the culture medium under an aseptic condition, and culturing for 0.5 hour at the temperature of 30 ℃;
C. dyeing and cleaning
And D, taking out the culture dish in the step B, pouring Congo red solution into the culture dish, dyeing for 10 minutes, discarding the Congo red solution, adding NaCl solution, rinsing for 1 minute, discarding the NaCl solution, and repeatedly rinsing for 2 times until a transparent ring with an obvious boundary appears.
The rest is the same as in example 1.
Example 3
This example differs from example 1 in that:
the rapid identification culture medium consists of the following raw materials:
5g/L of sodium carboxymethyl cellulose; agar 10 g/L; the balance of water; pH 6.5.
The application of the rapid identification culture medium in the cellulase qualitative detection comprises the following process steps:
A. preparation of cellulase detection plate
Weighing 25mL of sterilized and unset rapid identification culture medium, and placing the culture medium in a culture dish for solidification;
B. spotting of cellulases
Preparing cellulase solutions with different enzyme activities by using a phosphate buffer solution, adding 5 mu L of cellulase solution with different enzyme activities into each culture dish for rapidly identifying the culture medium under an aseptic condition, and culturing for 0.8 hour at 40 ℃;
C. dyeing and cleaning
And D, taking out the culture dish in the step B, pouring Congo red solution into the culture dish, dyeing for 20 minutes, discarding the Congo red solution, adding NaCl solution, rinsing for 8 minutes, discarding the NaCl solution, and repeatedly rinsing for 3 times until a transparent ring with an obvious boundary appears.
The rest is the same as in example 1.
Example 4
This example differs from example 1 in that:
the rapid identification culture medium consists of the following raw materials:
3g/L of sodium carboxymethyl cellulose; 6g/L of agar; the balance of water; the pH value is 6.0-7.2.
The application of the rapid identification culture medium in the cellulase qualitative detection comprises the following process steps:
A. preparation of cellulase detection plate
Weighing 22mL of sterilized and unset rapid identification culture medium, and placing the culture medium in a culture dish for solidification;
B. spotting of cellulases
Preparing cellulase solutions with different enzyme activities by using a phosphate buffer solution, adding 3 mu L of cellulase solution with different enzyme activities into each culture dish for rapidly identifying the culture medium under an aseptic condition, and culturing for 0.6 hour at 35 ℃;
C. dyeing and cleaning
And D, taking out the culture dish in the step B, pouring the culture dish into the Congo red solution, dyeing for 15 minutes, removing the Congo red solution, adding a NaCl solution, rinsing for 5 minutes, removing the NaCl solution, and repeatedly rinsing for 3 times until a transparent ring with an obvious boundary appears.
The rest is the same as in example 1.
Example 5
This example differs from example 1 in that:
the rapid identification culture medium consists of the following raw materials:
9g/L of sodium carboxymethyl cellulose; agar 12 g/L; the balance of water; the pH value is 6.0-7.2.
The application of the rapid identification culture medium in the cellulase qualitative detection comprises the following process steps:
A. preparation of cellulase detection plate
Weighing 28mL of sterilized and unset rapid identification culture medium, and placing in a culture dish for solidification;
B. spotting of cellulases
Preparing cellulase solutions with different enzyme activities by using a phosphate buffer solution, adding 9 mu L of cellulase solution with different enzyme activities into each culture dish for rapidly identifying the culture medium under an aseptic condition, and culturing for 0.9 hour at the temperature of 45 ℃;
C. dyeing and cleaning
And D, taking out the culture dish in the step B, pouring Congo red solution into the culture dish, dyeing for 25 minutes, discarding the Congo red solution, adding NaCl solution, rinsing for 12 minutes, discarding the NaCl solution, and repeatedly rinsing for 4 times until a transparent ring with an obvious boundary appears.
The rest is the same as in example 1.
Claims (4)
1. The rapid identification culture medium is characterized by comprising the following raw materials:
1g/L-10g/L of sodium carboxymethyl cellulose; 5g/L to 15g/L of agar; the balance of water; the pH value is 6.0-7.2.
2. The application of the rapid identification culture medium in the cellulase qualitative detection according to claim 1 is characterized by comprising the following process steps:
A. preparation of cellulase detection plate
Weighing 20-30mL of sterilized and unsolidified rapid identification culture medium, and placing the culture medium into a culture dish for solidification;
B. spotting of cellulases
Preparing cellulase solutions with different enzyme activities by using a phosphate buffer solution, adding 1 mu L-10 mu L of the cellulase solutions with different enzyme activities into each culture dish for rapidly identifying the culture medium under an aseptic condition, and culturing for 0.5-1 hour at the temperature of 30-50 ℃;
C. dyeing and cleaning
And D, taking out the culture dish in the step B, pouring Congo red solution into the culture dish, dyeing for 10-30 minutes, discarding the Congo red solution, adding NaCl solution, rinsing for 1-15 minutes, discarding the NaCl solution, and repeatedly rinsing for 2-5 times until a transparent ring with an obvious boundary appears.
3. The use of claim 1, further comprising step D, wherein the process conditions of the step D are as follows:
D. the transparent circles where distinct boundaries appear in step C are measured.
4. Use according to claim 1 or 2, characterized in that in step B cellulase solutions of different enzymatic activities are prepared with phosphate buffer 0: 0U; 1: 20U; 2: 40U; 3: 100U; 4: 200U.
Priority Applications (1)
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| CN103060425A (en) * | 2013-01-07 | 2013-04-24 | 海南椰国食品有限公司 | Application of colored bio-cellulose in cellulase-producing bacterium activity rapid detection |
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| CN103060425A (en) * | 2013-01-07 | 2013-04-24 | 海南椰国食品有限公司 | Application of colored bio-cellulose in cellulase-producing bacterium activity rapid detection |
| CN105018567A (en) * | 2015-08-25 | 2015-11-04 | 山东东方海洋科技股份有限公司 | Congo red staining method |
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