CN111321086A - Screening method of tannase-producing candida - Google Patents
Screening method of tannase-producing candida Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/72—Candida
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Abstract
The invention belongs to the technical field of strain screening and identification, and discloses a screening method of tannase-producing candida, which comprises the following steps: obtaining a soil sample, placing the soil sample in a 250mL triangular flask, adding normal saline, and carrying out oscillation enrichment culture in a shaking table to obtain a soil suspension; the soil suspension is diluted with sterile water in a gradient way and is coated on a PDA separation culture medium for culture; randomly selecting a single colony with typical tannase-producing candida colony characteristics, and carrying out streaking separation and purification for 2-3 times; after purification, inoculating a single colony into an enrichment culture solution for culturing for 18h, performing shake-flask culture for 72h, measuring the enzyme activity of the single colony, and determining a target strain; and carrying out primary screening and secondary screening tests on the determined target strains, identifying the enzyme yield of the tannase-producing candida, and storing the tannase-producing candida in 30% glycerol. The method is simple to operate, high in efficiency and small in error, and the obtained tannase-producing candida is high in application value and has extremely high social and economic benefits.
Description
Technical Field
The invention belongs to the technical field of strain screening and identification, and particularly relates to a screening method of tannase-producing candida.
Background
At present, tannin is a polyphenol substance, the tannin content is low except algae, lichen and moss, and the tannin content of tea, sorghum, persimmon, oak, quercus liaotungensis, grape seeds, grape skin and other plants in the immature stage is higher. Tannic acid has unique astringent taste, and can form insoluble complex with protein, pectin, alkaloid, etc. Tannins can be classified into two major categories, hydrolyzed tannins and condensed tannins, according to their chemical structure and characteristics. The hydrolyzed tannic acid molecule contains ester bond, and can be hydrolyzed into gallic acid or gallic acid and glucose under the action of tannase, dilute alkali or dilute acid. Condensed tannic acid is a flavanol derivative, and aromatic rings in the molecule are connected by a C-C bond, and therefore, the condensed tannic acid does not have an ester structure and is not easily hydrolyzed by tannase.
However, the tannase produced by the prior art has low yield and high market price. Tanninyl hydrolase (e.c.3.1.1.20), commonly known as tannase, is occasionally found in an assay for the synthesis of gallic acid from aqueous tannic acid. Tannase hydrolyzes tannic acid completely into gallic acid and glucose, and the intermediate products of conversion are 2, 3, 4, 6-tetragalloylglucose and two types of mono-galloylglucose. Tannase exists in plants, animals and microorganisms, but it is produced mainly by microorganisms such as bacteria, yeast and fungi. The method has the advantages that the bacteria for producing the tannase are less in amount, the yield is low, the stability is poor, the bacteria are easy to pollute, the extraction process is complex and the like, and the tannase producing yeast has few literature reports, only KenjiaOki purifies the tannase from the culture solution of Candida in 1976, and the method is reported so far. The tannase has wide application, and research and application of the tannase are deeply carried out in the processes of food processing, feed processing and cosmetic production. However, the existing tannase-producing strains have low enzyme activity, and tannase is mixed with a substrate and a product in the whole reaction system, so that the tannase is difficult to recycle, and the factors such as the enzymology property, the optimal fermentation expression and the industrial mass production are not well understood, so that the tannase is currently deficient in the practical application.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) the tannase produced by the prior art has low yield and high market price; the tannase-producing yeast has few literature reports and is only reported to date.
(2) The prior art has the advantages of less bacteria, low yield, poor stability, easy pollution and complex extraction process.
(3) The existing tannase producing strain has low enzyme activity, the tannase is mixed with a substrate and a product in the whole reaction system and is difficult to recycle, and factors such as the enzymology property, the optimal fermentation expression, the industrial large-scale production and the like are not well understood, so the tannase is deficient in the actual application at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a screening method of tannase-producing candida.
The screening method of the tannase-producing candida is realized in the way, and comprises the following steps:
preparing an enrichment medium, a PDA (personal digital Assistant) separation medium and a YPD (YPD) medium; obtaining 5.0-7.0 g of soil sample, placing the soil sample in a 250mL triangular flask filled with 45-50 mL enrichment medium, adding 100mL of normal saline, and performing shaking enrichment culture in a shaking table to obtain a soil suspension;
step two, sterilizing the prepared PDA separation culture medium, cooling to about 40 ℃, pouring the sterilized PDA separation culture medium into a sterile culture medium, performing gradient dilution on the soil suspension obtained in the step one by using sterile water, sucking the diluted soil suspension by using a suction pipe, dropwise adding the diluted soil suspension into the PDA separation culture medium, slightly spreading the dropwise soil suspension by using a coating rod, uniformly distributing the dropwise soil suspension into the PDA separation culture medium, and performing standing culture in an incubator at 25 ℃ for 3 d;
randomly selecting a single colony with typical tannase-producing candida colony characteristics, carrying out streaking separation and purification for 2-3 times, transferring to an inclined plane of a YPD culture medium, and storing at 4 ℃ for later use;
step four, inoculating the single colony into an enrichment culture solution after purification is finished, culturing for 18 hours, and performing shake flask culture for 72 hours at the temperature of 30-35 ℃ according to the frequency of 180-200 r/min; taking fermented broth, centrifuging at 4000rpm for 10min, discarding supernatant, collecting thallus, dissolving in citric acid-sodium citrate buffer solution with pH of 5.0, recovering to original volume to obtain crude enzyme solution, and measuring enzyme activity to determine target strain;
and step five, performing primary screening and secondary screening tests on the target strains determined in the step four, identifying the enzyme yield of the tannase-producing candida, and storing the tannase-producing candida in 30% glycerol.
Further, in the first step, the method for configuring the enrichment medium, the PDA separation medium and the YPD medium comprises the following steps:
enrichment culture medium: adding glucose into a 250ml conical flask filled with 100ml of water according to the proportion of 50g/L of glucose and 1g/L of urea, and then continuously adding 1.25g of yeast extract and agar powder into the conical flask; heating the conical flask while stirring, cooling, testing the alkalinity of the culture medium, adjusting the pH value to 7.2 by using 1mol/L sodium chloride, and sterilizing at 121 ℃ for 20 min;
PDA isolation medium: sterilizing glucose, peptone, agar, yeast extract and magnesium sulfate at 115 deg.C for 20 min; sterilizing tannin and bromophenol blue at 115 deg.C for 20 min; then naturally cooling to 45 ℃, and mixing the two;
YPD medium: sterilizing peptone and yeast extract at 121 deg.C under high pressure for 20 min; adding 100ml sterilized glucose solution.
Further, in the first step, the temperature of the shaking enrichment culture is 30-35 ℃, the time is 24-28 h, and the shaking frequency is 180-200 r/min.
Further, in the third step, the method for separating and purifying the tannase-producing candida bacterial colony comprises the following steps:
(1) sucking 1.0mL of enrichment solution, diluting the enrichment solution to a proper concentration in a gradient manner by 10 times, coating the enrichment solution on a plate identification culture medium, and culturing the enrichment solution in an incubator for 3-4 days at a constant temperature of 30-35 ℃;
(2) selecting a single bacterial colony, streaking and purifying the single bacterial colony on an identification culture medium, culturing the single bacterial colony for 3-4 days at a constant temperature of 30-40 ℃, and continuously culturing for 5 generations;
(3) the content of tannase can be known from the color shade and diameter of the color-changing circle, and strains with stable enzyme activity are selected and stored for later use.
Further, in the third step, the preservation method of the separated and purified tannase-producing candida is as follows:
selecting a tannase-producing candida strain with relatively stable enzyme activity, transferring the strain to a slant culture medium, culturing the strain at 40-45 ℃ and 150-170 rmp/min for 3-4 days, and storing the strain in a refrigerator at 3-5 ℃ for later use.
Further, in step four, the method for measuring enzyme activity comprises:
1) taking 7 clean test tubes with plugs, 1 blank tube, 3 test tubes and a control tube and 3 test tubes respectively, and placing the propyl gallate solution with the concentration of 0.005moI/L and the crude enzyme solution into a water bath at 40 ℃ for heat preservation for 5-10min before the reaction;
2) adding 0.25mL of propyl gallate solution into all test tubes, adding 0.25mL of pH5.0 and 0.1mol/L of sodium citrate buffer solution into a blank tube, adding 0.25mL of crude enzyme solution into the test tubes, and placing all test tubes into a 40 ℃ water bath for heat preservation for 5 min;
3) adding 0.3mL of 0.05mol/L methanol rhodanine solution into all the test tubes respectively, and preserving the heat in a water bath at 40 ℃ for 5 min;
4) 0.25ml of crude enzyme solution in a control tube; adding 0.2mL of KOH solution with the concentration of 0.5mol/L into the control tube and the test tube; keeping the temperature in 40 ℃ water bath for 5 min;
5) adding 4mL of distilled water into all the test tubes for dilution, and preserving the heat in a water bath at 40 ℃ for 5 min;
6) measuring the light absorption value of the reaction mixture by using distilled water as a blank at the position of 520nm of an ultraviolet spectrophotometer; all treatment tubes were set to 3 replicates and the average of 3 replicates was taken.
Further, in the fifth step, the primary screening method of the target strain comprises the following steps:
selecting a ring of target strains to inoculate on an identification medium, and performing static culture in an incubator at 25-30 ℃ for 24-28 h; if the strain produces tannase, the tannic acid in the culture medium is hydrolyzed to produce gallic acid, so that a bromophenol blue indicator in the culture medium is changed from blue purple to yellow, an obvious color change ring is formed around a colony, and the strain producing the color change ring is reserved.
Further, the bromocresol green culture medium comprises the following components: PDA basal medium, 0.01% tannin indicator, 0.015% bromophenol blue indicator, pH 6.0.
Further, in the fifth step, the re-screening test of the target strain comprises:
respectively preparing a primary screened target strain and industrial commonly-used tannase-producing candida as bacterial suspensions with the same concentration, pouring a layer of identification culture medium which just covers the bottom of a culture dish into the culture dish, putting an oxford cup after the culture medium is solidified, pouring a layer of identification culture medium, taking the oxford cup out after the culture medium is solidified, inoculating 0.1mL of bacterial suspension at the oxford cup, standing and culturing for 24 hours in an incubator at 25 ℃, observing a color change ring, judging the acid production capacity according to the size of the color change ring, wherein the larger the color change ring is, the stronger the acid production capacity is, otherwise, the weaker the color change ring is, and the selected target strain is larger than the commercial tannase-producing candida.
The tannase-producing candida is round, thick and smooth in surface, moist, non-transparent and uniform in color of the edge and the central part; the culture is elliptical at the initial stage and is a single-cell individual; after 3 days of culture, the cells were larger with smaller daughter cells on the larger cells.
By combining all the technical schemes, the invention has the advantages and positive effects that: according to the screening method of the tannase-producing candida, provided by the invention, a soil suspension is obtained by a soil sample oscillation enrichment culture mode and then is subjected to separation culture, single strains with typical tannase-producing candida colony characteristics are randomly selected and then are subjected to streak separation and purification, and the obtained target strains are subjected to primary screening and secondary screening tests.
Drawings
FIG. 1 is a flow chart of a screening method of Candida tannase provided in an embodiment of the present invention.
FIG. 2 is a schematic diagram of the morphological identification of Candida tannase provided in the example of the present invention;
in the figure, A represents tannase-producing colonies morphology, B represents initial yeast culture (40 ×), C represents yeast culture 3D (40 ×), and D represents yeast stain (40 ×).
Fig. 3 is a schematic diagram of the gallic acid absorption peak provided by the examples of the present invention.
Fig. 4 is a graph of gallic acid standard curve provided by the embodiment of the present invention.
FIG. 5 is a graph showing the peak absorption of tannic acid according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a screening method of tannase-producing candida, and the invention is described in detail with reference to the attached drawings.
As shown in fig. 1, the screening method of candida tannase provided by the embodiment of the present invention includes the following steps:
s101, preparing an enrichment medium, a PDA (personal digital Assistant) separation medium and a YPD (YPD) medium; obtaining 5.0-7.0 g of soil sample, placing the soil sample in a 250mL triangular flask filled with 45-50 mL enrichment medium, adding 100mL physiological saline, and performing shaking enrichment culture in a shaking table to obtain a soil suspension.
S102, sterilizing the prepared PDA separation culture medium, cooling to about 40 ℃, pouring the sterilized PDA separation culture medium into a sterile culture medium, performing gradient dilution on the soil suspension obtained in the step S101 by using sterile water, sucking the diluted soil suspension by using a suction pipe, dropwise adding the diluted soil suspension into the PDA separation culture medium, slightly spreading the dropwise soil suspension by using a spreading rod, uniformly distributing the dropwise soil suspension into the PDA separation culture medium, and performing standing culture in an incubator at 25 ℃ for 3 d.
S103, randomly selecting a single colony with typical tannase-producing candida colony characteristics, carrying out streaking separation and purification for 2-3 times, transferring to an inclined plane of a YPD culture medium, and storing at 4 ℃ for later use.
S104, after purification, inoculating the single colony into an enrichment culture solution for culturing for 18h, and performing shake flask culture at the temperature of 30-35 ℃ for 72h according to the frequency of 180-200 r/min; taking the fermented fermentation liquor, centrifuging at 4000rpm for 10min, discarding supernatant, collecting thallus, dissolving in citric acid-sodium citrate buffer solution with pH of 5.0, recovering to original volume to obtain crude enzyme solution, and measuring enzyme activity to determine target strain.
And S105, performing primary screening and secondary screening tests on the target strain determined in the step S104, identifying the enzyme yield of the tannase-producing candida, and storing the tannase-producing candida in 30% glycerol.
In step S101, the method for configuring the enrichment medium, PDA isolation medium, and YPD medium provided in the embodiments of the present invention includes:
enrichment culture medium: adding glucose into a 250ml conical flask filled with 100ml of water according to the proportion of 50g/L of glucose and 1g/L of urea, and then continuously adding 1.25g of yeast extract and agar powder into the conical flask; heating the conical flask while stirring, cooling, testing the alkalinity of the culture medium, adjusting the pH value to 7.2 by using 1mol/L sodium chloride, and sterilizing at 121 ℃ for 20 min;
PDA isolation medium: sterilizing glucose, peptone, agar, yeast extract and magnesium sulfate at 115 deg.C for 20 min; sterilizing tannin and bromophenol blue at 115 deg.C for 20 min; then naturally cooling to 45 ℃, and mixing the two;
YPD medium: sterilizing peptone and yeast extract at 121 deg.C under high pressure for 20 min; adding 100ml sterilized glucose solution.
In step S101, the temperature of the shaking enrichment culture provided by the embodiment of the invention is 30-35 ℃, the time is 24-28 h, and the shaking frequency is 180-200 r/min.
In step S103, the method for separating and purifying the tannase-producing candida bacterial colony provided by the embodiment of the present invention comprises:
(1) and (3) sucking 1.0mL of enrichment solution, diluting the enrichment solution to a proper concentration in a gradient manner by 10 times, coating the enrichment solution on a plate identification culture medium, and culturing the enrichment solution in an incubator for 3-4 days at a constant temperature of 30-35 ℃.
(2) And (3) selecting a single colony, streaking and purifying the single colony on an identification culture medium, culturing the single colony for 3-4 days at a constant temperature of 30-40 ℃, and continuously culturing for 5 generations.
(3) The content of tannase can be known from the color shade and diameter of the color-changing circle, and strains with stable enzyme activity are selected and stored for later use.
In step S103, the method for preserving the isolated and purified tannase-producing candida albicans provided by the embodiment of the present invention comprises: selecting a tannase-producing candida strain with relatively stable enzyme activity, transferring the strain to a slant culture medium, culturing the strain at 40-45 ℃ and 150-170 rmp/min for 3-4 days, and storing the strain in a refrigerator at 3-5 ℃ for later use.
In step S104, the method for measuring an enzymatic activity provided in the embodiment of the present invention includes:
1) taking 7 clean test tubes with plugs, 1 blank tube, 3 test tubes and a control tube and a test tube respectively, and putting the propyl gallate solution with the concentration of 0.005moI/L and the crude enzyme solution into a water bath with the temperature of 40 ℃ for heat preservation for 5-10min before the reaction starts.
2) 0.25mL of propyl gallate solution was added to all tubes, 0.25mL of pH5.0, 0.1mol/L sodium citrate buffer was added to the blank tube, 0.25mL of crude enzyme solution was added to the test tube, and all tubes were incubated in a 40 ℃ water bath for 5 min.
3) 0.3mL of 0.05mol/L methanolic rhodanine solution was added to each tube and incubated in a 40 ℃ water bath for 5 min.
4) 0.25ml of crude enzyme solution in a control tube; adding 0.2mL of KOH solution with the concentration of 0.5mol/L into the control tube and the test tube; keeping the temperature in a water bath at 40 ℃ for 5 min.
5) All tubes were diluted with 4mL distilled water and incubated in a 40 ℃ water bath for 5 min.
6) Measuring the light absorption value of the reaction mixture by using distilled water as a blank at the position of 520nm of an ultraviolet spectrophotometer; all treatment tubes were set to 3 replicates and the average of 3 replicates was taken.
In step S105, the preliminary screening method for the target strain provided by the embodiment of the present invention includes: selecting a ring of target strains to inoculate on an identification medium, and performing static culture in an incubator at 25-30 ℃ for 24-28 h; if the strain produces tannase, the tannic acid in the culture medium is hydrolyzed to produce gallic acid, so that a bromophenol blue indicator in the culture medium is changed from blue purple to yellow, an obvious color change ring is formed around a colony, and the strain producing the color change ring is reserved.
The bromocresol green culture medium provided by the embodiment of the invention comprises the following components: PDA basal medium, 0.01% tannin indicator, 0.015% bromophenol blue indicator, pH 6.0.
In step S105, the rescreening test of the target strain provided in the embodiment of the present invention includes: respectively preparing a primary screened target strain and industrial commonly-used tannase-producing candida as bacterial suspensions with the same concentration, pouring a layer of identification culture medium which just covers the bottom of a culture dish into the culture dish, putting an oxford cup after the culture medium is solidified, pouring a layer of identification culture medium, taking the oxford cup out after the culture medium is solidified, inoculating 0.1mL of bacterial suspension at the oxford cup, standing and culturing for 24 hours in an incubator at 25 ℃, observing a color change ring, judging the acid production capacity according to the size of the color change ring, wherein the larger the color change ring is, the stronger the acid production capacity is, otherwise, the weaker the color change ring is, and the selected target strain is larger than the commercial tannase-producing candida.
The tannase-producing candida provided by the embodiment of the invention is round, the surface is sticky and smooth, the candida is moist and non-transparent, and the color of the edge and the color of the central part are uniform; the culture is elliptical at the initial stage and is a single-cell individual; after 3 days of culture, the cells were larger with smaller daughter cells on the larger cells.
The present invention will be further described with reference to the following examples.
Example (b): morphological characterization of strains
1. The morphological identification method of the strain provided by the embodiment of the invention comprises the following steps:
(1) the tannase-producing candida bacterial colonies are scattered in sterile water, diluted by proper times, coated on a tannin identification culture medium plate in a sterile operation mode, cultured for 3 days at the constant temperature of 30 ℃, and then the morphological characteristics of the bacterial colonies are observed.
(2) The cell morphology of the strain was observed under an optical microscope at the initial stage of culture and after 3 days of culture: under the aseptic operation condition, a small amount of fresh thalli is picked up on a clean glass slide, and dipped in a small amount of sterile water for dilution and microscopic examination.
2. Results
(1) The morphological identification scheme of the yeast provided by the embodiment of the invention is shown in figure 2, wherein A is the colony morphology of tannase producing bacteria, B is the initial culture stage of the yeast (40 ×), C is the culture stage of the yeast (3D (40 ×), and D is the staining of the yeast (40 ×).
As shown in FIG. 2A, the yeast cells were round, sticky and smooth in surface, moist, opaque, and easy to pick. The color of the edge and the central part are uniform. As shown by B, C, D in FIG. 2, at the initial stage of culture, the microscopic cells were all elliptical and were single-cell individuals. After 3 days of culture, the cells were large with small spores on the large cells. The bacterium is bluish purple after dyeing.
(2) Determination of maximum absorption peak wavelength of gallic acid
The absorption peak of gallic acid is shown in FIG. 3, and the gallic acid has maximum absorption peak at 520nm when the visible region scans the wave band of gallic acid.
(3) Drawing of gallic acid standard curve
The gallic acid standard curve graph is shown in FIG. 4, the absorbance value of gallic acid standard solution at 520nm is linearly related, the regression equation is Y ═ 3.239X-0.0613, and the correlation coefficient R20.9995. And obtaining the concentration of the gallic acid through a light absorption value.
(4) The maximum absorption peak of tannic acid at 640nm is shown in figure 5, which is different from the maximum absorption peak 760nm reported in the literature.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A screening method of tannase-producing candida is characterized by comprising the following steps:
preparing an enrichment medium, a PDA (personal digital Assistant) separation medium and a YPD (YPD) medium; obtaining 5.0-7.0 g of soil sample, placing the soil sample in a 250mL triangular flask filled with 45-50 mL enrichment medium, adding 100mL of normal saline, and performing shaking enrichment culture in a shaking table to obtain a soil suspension;
step two, sterilizing the prepared PDA separation culture medium, cooling to about 40 ℃, pouring the sterilized PDA separation culture medium into a sterile culture medium, performing gradient dilution on the soil suspension obtained in the step one by using sterile water, sucking the diluted soil suspension by using a suction pipe, dropwise adding the diluted soil suspension into the PDA separation culture medium, slightly spreading the dropwise soil suspension by using a coating rod, uniformly distributing the dropwise soil suspension into the PDA separation culture medium, and performing standing culture in an incubator at 25 ℃ for 3 d;
randomly selecting a single colony with typical tannase-producing candida colony characteristics, carrying out streaking separation and purification for 2-3 times, transferring to an inclined plane of a YPD culture medium, and storing at 4 ℃ for later use;
step four, inoculating the single colony into an enrichment culture solution after purification is finished, culturing for 18 hours, and performing shake flask culture for 72 hours at the temperature of 30-35 ℃ according to the frequency of 180-200 r/min; taking fermented broth, centrifuging at 4000rpm for 10min, discarding supernatant, collecting thallus, dissolving in citric acid-sodium citrate buffer solution with pH of 5.0, recovering to original volume to obtain crude enzyme solution, and measuring enzyme activity to determine target strain;
and step five, performing primary screening and secondary screening tests on the target strains determined in the step four, identifying the enzyme yield of the tannase-producing candida, and storing the tannase-producing candida in 30% glycerol.
2. The method of claim 1, wherein the enrichment medium, PDA isolation medium, YPD medium are configured in a manner that comprises:
enrichment culture medium: adding glucose into a 250ml conical flask filled with 100ml of water according to the proportion of 50g/L of glucose and 1g/L of urea, and then continuously adding 1.25g of yeast extract and agar powder into the conical flask; heating the conical flask while stirring, cooling, testing the alkalinity of the culture medium, adjusting the pH value to 7.2 by using 1mol/L sodium chloride, and sterilizing at 121 ℃ for 20 min;
PDA isolation medium: sterilizing glucose, peptone, agar, yeast extract and magnesium sulfate at 115 deg.C for 20 min; sterilizing tannin and bromophenol blue at 115 deg.C for 20 min; then naturally cooling to 45 ℃, and mixing the two;
YPD medium: sterilizing peptone and yeast extract at 121 deg.C under high pressure for 20 min; adding 100ml sterilized glucose solution.
3. The screening method of Candida tannase as claimed in claim 1, wherein in the first step, the temperature of shaking enrichment culture is 30-35 ℃, the time is 24-28 h, and the shaking frequency is 180-200 r/min.
4. The screening method of tannase-producing candida as claimed in claim 1, wherein in the third step, the method for separating and purifying the tannase-producing candida colonies comprises the following steps:
(1) sucking 1.0mL of enrichment solution, diluting the enrichment solution to a proper concentration in a gradient manner by 10 times, coating the enrichment solution on a plate identification culture medium, and culturing the enrichment solution in an incubator for 3-4 days at a constant temperature of 30-35 ℃;
(2) selecting a single bacterial colony, streaking and purifying the single bacterial colony on an identification culture medium, culturing the single bacterial colony for 3-4 days at a constant temperature of 30-40 ℃, and continuously culturing for 5 generations;
(3) the content of tannase can be known from the color shade and diameter of the color-changing circle, and strains with stable enzyme activity are selected and stored for later use.
5. The screening method of tannase-producing candida as claimed in claim 1, wherein in the third step, the preservation method of the isolated and purified tannase-producing candida is as follows:
selecting a tannase-producing candida strain with relatively stable enzyme activity, transferring the strain to a slant culture medium, culturing the strain at 40-45 ℃ and 150-170 rmp/min for 3-4 days, and storing the strain in a refrigerator at 3-5 ℃ for later use.
6. The method for screening Candida tannase according to claim 1, wherein in step four, the method for measuring the enzymatic activity comprises:
1) taking 7 clean test tubes with plugs, 1 blank tube, 3 test tubes and a control tube and 3 test tubes respectively, and placing the propyl gallate solution with the concentration of 0.005moI/L and the crude enzyme solution into a water bath at 40 ℃ for heat preservation for 5-10min before the reaction;
2) adding 0.25mL of propyl gallate solution into all test tubes, adding 0.25mL of 0.1mol/L sodium citrate buffer solution with pH of 5.0 into a blank tube, adding 0.25mL of crude enzyme solution into the test tubes, and placing all test tubes into a 40 ℃ water bath for heat preservation for 5 min;
3) adding 0.3mL of 0.05mol/L methanol rhodanine solution into all the test tubes respectively, and preserving the heat in a water bath at 40 ℃ for 5 min;
4) 0.25ml of crude enzyme solution in a control tube; adding 0.2mL of KOH solution with the concentration of 0.5mol/L into the control tube and the test tube; keeping the temperature in 40 ℃ water bath for 5 min;
5) adding 4mL of distilled water into all the test tubes for dilution, and preserving the heat in a water bath at 40 ℃ for 5 min;
6) measuring the light absorption value of the reaction mixture by using distilled water as a blank at the position of 520nm of an ultraviolet spectrophotometer; all treatment tubes were set to 3 replicates and the average of 3 replicates was taken.
7. The screening method of tannase-producing candida as claimed in claim 1, wherein in the fifth step, the primary screening method of the target strain comprises the following steps:
selecting a ring of target strains to inoculate on an identification medium, and performing static culture in an incubator at 25-30 ℃ for 24-28 h; if the strain produces tannase, the tannic acid in the culture medium is hydrolyzed to produce gallic acid, so that a bromophenol blue indicator in the culture medium is changed from blue purple to yellow, an obvious color change ring is formed around a colony, and the strain producing the color change ring is reserved.
8. The screening method of tannase-producing candida as claimed in claim 7, wherein said bromocresol green medium comprises the following components: PDA basal medium, 0.01% tannin indicator, 0.015% bromophenol blue indicator, pH 6.0.
9. The screening method of tannase-producing candida as claimed in claim 1, wherein in the fifth step, the rescreening test of the target strain comprises:
respectively preparing a primary screened target strain and industrial commonly-used tannase-producing candida as bacterial suspensions with the same concentration, pouring a layer of identification culture medium which just covers the bottom of a culture dish into the culture dish, putting an oxford cup after the culture medium is solidified, pouring a layer of identification culture medium, taking the oxford cup out after the culture medium is solidified, inoculating 0.1mL of bacterial suspension at the oxford cup, standing and culturing for 24 hours in an incubator at 25 ℃, observing a color change ring, judging the acid production capacity according to the size of the color change ring, wherein the larger the color change ring is, the stronger the acid production capacity is, otherwise, the weaker the color change ring is, and the selected target strain is larger than the commercial tannase-producing candida.
10. The tannase-producing candida screened by the tannase-producing candida screening method according to any one of claims 1 to 9, wherein the tannase-producing candida is round, sticky and smooth in surface, moist and opaque, and uniform in color at edges and central parts; the culture is elliptical at the initial stage and is a single-cell individual; after 3 days of culture, the cells were larger and the larger cells were accompanied by smaller daughter cells.
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| CN115232856A (en) * | 2022-07-27 | 2022-10-25 | 河南省健康元生物医药研究院有限公司 | High-throughput screening method for acremonium chrysogenum based on solid fermentation |
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