CN107446826B - Screening culture medium for filamentous fungi and preparation method and application thereof - Google Patents
Screening culture medium for filamentous fungi and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a screening culture medium of filamentous fungi, a preparation method and application thereof, belonging to the technical field of microorganisms. The screening culture medium of the filamentous fungi comprises a chromogenic culture medium, an enzymolysis culture medium and a nutrient culture medium which are arranged from bottom to top in sequence; the chromogenic medium contains agarose and a calcium ion indicator, and the enzymolysis medium contains agarose and calcium phytate. The screening culture medium has a unique three-layer structure, and by adopting the screening culture medium, the screening target is wide, the screening efficiency is high, and the filamentous fungi with better enzyme activity can be screened out.
Description
Technical Field
The invention relates to a screening culture medium of filamentous fungi, a preparation method and application thereof, belonging to the technical field of microorganisms.
Background
Filamentous fungi are widely used in the food processing industry, and many fermented foods and food materials such as fermented bean curd, fermented soybeans, sauce, soy sauce, citric acid, etc. are produced and processed with the participation of the filamentous fungi. Most filamentous fungi can convert carbohydrates such as starch and sugar, nitrogen-containing compounds such as protein, and other compounds in the raw materials to make various foods, seasonings, and food additives. In industrial production, higher benefits are achieved under the condition of not increasing the investment of manpower, financial resources and the like, and one of the most direct and effective measures is to perform strain transformation. Through strain breeding, the purposes of improving the utilization rate of raw materials, shortening the fermentation period, enhancing the flavor of fermented food and the like can be achieved, and the production requirements of enterprises are met.
There are currently two major directions for strain engineering of filamentous fungi: one is a method of genetic engineering; one is the traditional breeding method, including chemical mutagenesis, physical mutagenesis, protoplast fusion and other techniques. At present, because the application of genetic engineering breeding in the fermented food industry is controversial, the traditional breeding is very important for strain modification in the food industry. For traditional breeding, the mutagenesis means is various, but the screening method is very limited. The strain screening process is to rapidly and accurately screen out the required target strain by adopting different screening strategies according to production requirements and strain characteristics, and the most common effective method is an agar plate observation method, including a transparent ring method and a chromogenic ring method.
Filamentous fungi are rich in exocrine enzyme systems such as protease, amylase, glucoamylase, cellulase, pectinase and the like during long-term evolution, and detailed transparent circle and color circle plate screening methods (casein for protease, starch for amylase, iodine solution and the like) have been established for mutagenesis and screening of strains corresponding to these enzyme systems. Application No.: 201510130064.8 the patent of the method for preparing taste peptide and taste amino acid by Aspergillus oryzae fermented peanut meal and its application is to screen out mutant strain with improved protease by casein culture medium.
The existing screening technology aiming at the filamentous fungus enzyme systems generally has the following defects: the screening enzyme activity target is single, the composition of the screening culture medium is basically a single substrate, and the obtained strains cannot be subjected to comprehensive enzyme system investigation; moreover, long-term use of these induced substrate culture media leads to higher resistance saturation and unbalanced distribution of enzyme systems of the strains, so that production strains meeting production requirements cannot be quickly and effectively screened out.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a screening culture medium of filamentous fungi and a preparation method thereof, and the screening culture medium can be used for quickly and efficiently screening filamentous fungi strains with better performance.
In addition, the invention also provides application of the screening culture medium in screening filamentous fungi.
In order to achieve the purpose, the invention adopts the technical scheme that: a screening culture medium for filamentous fungi comprises a chromogenic culture medium, an enzymolysis culture medium and a nutrient culture medium which are arranged from bottom to top in sequence; the chromogenic medium contains agarose and a calcium ion indicator, and the enzymolysis medium contains agarose and calcium phytate.
The screening culture medium has a three-layer structure which is uniquely associated with each other and is respectively a chromogenic culture medium, an enzymolysis culture medium arranged on the chromogenic culture medium and a nutrient culture medium arranged on the enzymolysis culture medium. The nutrient medium is used for culturing, domesticating and screening the filamentous fungi, and phytase produced by the filamentous fungi permeates into the enzymolysis culture medium, can decompose calcium phytate and liberate calcium ions; calcium ions permeate into the chromogenic culture medium to be developed with the calcium ion indicator, and then the strain can be selected through the transparent color circle.
By adopting the screening culture medium, the filamentous fungi with better performance can be screened, and the screening culture medium can shorten the time for observing and selecting the fungi to 48 hours in a multilayer form; the method is also more approximate to the real enzyme production condition in the strain propagation stage, and the screening efficiency can be obviously improved.
In a preferred embodiment of the screening medium for filamentous fungi of the present invention, the chromogenic medium is prepared from a starting material comprising:
(a) the calcium ion indicator solution consists of a calcium ion indicator and a buffer solution, wherein the ratio of the mass of the calcium ion indicator to the volume of the buffer solution is (0.1-2 g): 100 mL; and
(b) the agarose solution consists of agarose and a buffer solution, wherein the ratio of the mass of the agarose to the volume of the buffer solution is 0.2-1.0 g: 100 mL;
the volume ratio of (a) to (b) is 1-10: 100.
as a more preferred embodiment of the screening medium for filamentous fungi of the present invention, the calcium ion indicator is Chromol T. The chrome black T is a metal indicator which can be complexed with calcium ions to form a complex, and is blue in pH range of 7-11 and red in complex with calcium ions.
In a preferred embodiment of the screening medium for filamentous fungi of the present invention, the enzymolysis medium is prepared from:
(c) calcium phytate suspension, wherein the content of calcium phytate in the calcium phytate suspension is 0.02-0.2 g/mL; and
(b) the agarose solution consists of agarose and a buffer solution, wherein the ratio of the mass of the agarose to the volume of the buffer solution is 0.2-1.0 g: 100 mL;
the volume ratio of (c) to (b) is 1: 10.
as a preferred embodiment of the screening medium for filamentous fungi of the present invention, the buffer solution is Tris-HCl buffer solution, and the pH value of the buffer solution is 7.1-8.9. Within this pH range, the growth of the filamentous fungi is best and the coloration reaction is optimal.
As a preferred embodiment of the screening medium for filamentous fungi according to the invention, the nutrient medium comprises inorganic salts and the following components in amounts:
in the screening culture medium, the carbon sources of the nutrient culture medium adopt 2-deoxyglucose and xylose, wherein the 2-deoxyglucose is a structural analogue of glucose, and the xylose is pentose, so that the inhibition of sugar source metabolites such as hexose and the like is relieved, the utilization of the xylose by filamentous fungi is improved, and the generation of important enzymes (protease, amylase and glutaminase) in the fermentation process of the screening strain is improved.
Calcium phytate is added into the nutrient medium, and has the main functions of regulating and controlling the nutrient composition of the medium, changing the state of the medium, facilitating the shortening of the screening culture period, and simply, intuitively and accurately selecting the target mutant strain within 48 hours. Furthermore, the nutrient medium contains lactic acid, which contributes to the improvement of the water solubility of calcium phytate.
As a more preferred embodiment of the screening medium for filamentous fungi of the present invention, the inorganic salts include potassium nitrate, ammonium sulfate, sodium chloride and magnesium chloride; in the nutrient medium, the concentration of the 2-deoxyglucose is 5-30mmol/L, and the content of the calcium phytate is 0.3-0.6% (w/v).
As a preferred embodiment of the screening medium for filamentous fungi of the present invention, the thickness of the chromogenic medium is 1/6-1/3; the thickness of the enzymolysis culture medium is 1/4-1/3 of the thickness of the screening culture medium; the thickness of the nutrient medium is 1/3-7/12 of the thickness of the screening medium.
In addition, the invention also provides a preparation method of the screening culture medium of the filamentous fungi, which comprises the following steps:
preparation of a chromogenic medium: dissolving a calcium ion indicator into a buffer solution according to the proportion of adding 0.1-2 g of the calcium ion indicator into every 100mL of the buffer solution, and filtering and sterilizing to obtain a calcium ion indicator solution; dissolving agarose into a buffer solution according to the proportion of adding 0.2-1.0 g of agarose into every 100mL of the buffer solution, sterilizing, cooling, adding the prepared calcium ion indicator solution, shaking uniformly, and pouring the plate to obtain a chromogenic medium;
preparing an enzymolysis culture medium: adding calcium phytate into water, soaking overnight, sterilizing, cooling to room temperature, centrifuging, discarding supernatant to obtain calcium phytate precipitate, and adding water dispersed calcium phytate precipitate to obtain calcium phytate suspension; dissolving agarose into a buffer solution according to the proportion of adding 0.2-1.0 g of agarose into every 100mL of the buffer solution, sterilizing, cooling, adding the prepared calcium phytate suspension, shaking uniformly, and pouring the mixture to obtain an enzymolysis culture medium;
preparation of a nutrient medium: dissolving calcium phytate in water, and adding lactic acid while stirring to obtain a mixed solution A; dissolving 2-deoxyglucose in water, filtering and sterilizing to obtain 2-deoxyglucose aqueous solution; dissolving potassium nitrate and magnesium chloride in water, and filtering and sterilizing to obtain a mixed solution B; adding xylose, sodium chloride and ammonium sulfate into the mixed solution A, quantifying, uniformly stirring, adjusting the pH value, adding agarose, sterilizing and cooling to obtain a mixed solution C; adding the 2-deoxyglucose aqueous solution and the mixed solution B into the mixed solution C, shaking uniformly, and then pouring the plate to obtain a nutrient medium;
adding the enzymolysis culture medium to the cooled chromogenic culture medium to obtain a pre-screening culture medium;
and after the enzymolysis culture medium is cooled, adding the nutrient culture medium to the enzymolysis culture medium in the pre-screening culture medium to obtain the screening culture medium of the filamentous fungi.
In addition, the invention also provides application of the screening culture medium of the filamentous fungi in screening the filamentous fungi. Preferably, the filamentous fungus is Aspergillus oryzae, Aspergillus niger or Trichoderma reesei.
Compared with the prior art, the invention has the beneficial effects that:
(1) the screening medium has the advantages of wide screening targets: the main components of the nutrient medium comprise deoxyglucose, xylose, calcium phytate and the like, so that the comprehensive capability of various enzyme activities of glutaminase, aminopeptidase, protease, amylase, cellulase, pectinase and the like of the screened mutant strain can be ensured to be better, and the balance among enzyme systems can be further ensured.
(2) By adopting the screening culture medium, the screening efficiency is high: the screening culture medium can shorten the time for observing and selecting bacteria to 48 hours in a form of a multilayer chromogenic culture medium; the method is also more approximate to the real enzyme production condition in the strain propagation stage, and the screening efficiency can be obviously improved.
(3) The screening culture medium of the invention has uniform dissolution and good mixing property: lactic acid is added into the culture medium, which is also beneficial to improving the water solubility of calcium phytate.
(4) The preparation method of the screening culture medium can crush the calcium phytate particles with poor water solubility to smaller particle size.
Drawings
FIG. 1 is a radar chart of sensory evaluation in effect example 3 of the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically mentioned, are commercially available.
In the following examples, the percentages are by mass unless otherwise specified.
Example 1
In an embodiment of the screening medium for filamentous fungi of the present invention, the screening medium for filamentous fungi of this embodiment includes a chromogenic medium, an enzymatic medium, and a nutrient medium, which are sequentially disposed from bottom to top;
the raw materials for preparing the chromogenic medium contain a calcium ion indicator solution and an agarose solution, and the volume ratio of the calcium ion indicator solution to the agarose solution is 2: 100, respectively; the calcium ion indicator solution consists of a calcium ion indicator and a Tris-HCl buffer solution with the pH value of 8.0, wherein the ratio of the mass of the calcium ion indicator to the volume of the buffer solution in the calcium ion indicator solution is 0.5 g: 100 mL; the agarose solution consists of agarose and a Tris-HCl buffer solution with the pH value of 8.0, and the ratio of the mass of the agarose to the volume of the buffer solution in the agarose solution is 0.3 g: 100 mL;
the preparation raw materials of the enzymolysis culture medium contain calcium phytate suspension and agarose solution, and the volume ratio of the calcium phytate suspension to the agarose solution is 1: 10; wherein the content of calcium phytate in the calcium phytate suspension is 0.05 g/mL; the agarose solution consists of agarose and a Tris-HCl buffer solution with the pH value of 7.5, and the ratio of the mass of the agarose to the volume of the buffer solution in the agarose solution is 0.2 g: 100 mL;
the nutrient medium contains the following components in percentage by weight: 5mmol/L of 2-deoxyglucose, 0.1% (w/v) of calcium phytate, 20mmol/L of lactic acid, 1% (w/v) of xylose, 1.5% (w/v) of potassium nitrate, 0.5% (w/v) of ammonium sulfate, 0.5% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride and 1% (w/v) of agarose with a pH value of 6.5;
wherein the calcium ion indicator is chrome black T.
Further, in the screening medium for filamentous fungi of the present example, the thickness of the chromogenic medium is 1/4 times the thickness of the screening medium; the thickness of the enzymolysis culture medium is 1/4 of the thickness of the screening culture medium; the thickness of the nutrient medium is 1/2 the thickness of the screening medium.
The preparation method of the screening medium for filamentous fungi of this example was:
(1) preparation of a chromogenic medium: weighing 0.5g of chrome black T, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 8.0, and filtering and sterilizing to obtain a calcium ion indicator solution; weighing 0.3g of agarose, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 8.0, sterilizing at 121 ℃ for 15min, cooling to about 45 ℃, adding 2mL of sterile calcium ion indicator solution, shaking uniformly, and pouring the plate to obtain a chromogenic medium; wherein, Tris and HCl reagents in Tris-HCl buffer solution are more than analytical purity, and the purity of water used simultaneously is deionized water and the grade above;
(2) after the chromogenic culture medium in the step (1) is cooled, adding an enzymolysis culture medium, wherein the preparation process of the enzymolysis culture medium is as follows: weighing calcium phytate according to the proportion of adding 1g of calcium phytate into 10mL of water, soaking overnight, and sterilizing at 121 ℃ for 15 min; after the sterilized calcium phytate solution is cooled to room temperature, centrifuging by using a centrifugal method, discarding supernatant, washing calcium phytate precipitate by using sterile water for 3 times, and adding a proper amount of sterile water to disperse the calcium phytate precipitate again to prepare sterile calcium phytate suspension; weighing 0.2g of agarose, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 7.5, sterilizing at 121 ℃ for 15min, cooling to about 45 ℃, adding 10mL of calcium phytate suspension containing 0.5g of calcium phytate, shaking uniformly, and pouring to obtain an enzymolysis culture; the Tris and HCL reagents in the Tris-HCl buffer solution are more than analytical purity, and the water purity used at the same time is deionized water and the grade above the deionized water;
(3) after the enzymolysis culture medium in the step (2) is cooled, adding a nutrient culture medium to obtain a screening culture medium of the filamentous fungi of the embodiment; wherein the preparation process of the nutrient medium is as follows: weighing calcium phytate, dissolving the calcium phytate in deionized water, putting the calcium phytate into a rotor with 3-5 particles, magnetically stirring the calcium phytate for more than 5 hours by using a magnetic stirrer, slowly adding lactic acid in the stirring and dissolving process, and continuously stirring to obtain a mixed solution A; weighing 2-deoxyglucose, dissolving the 2-deoxyglucose in deionized water, and sterilizing the dissolved 2-deoxyglucose solution through a 0.22um filter membrane to obtain a sterile 2-deoxyglucose aqueous solution; weighing potassium nitrate and magnesium chloride in proportion, dissolving in deionized water, filtering and sterilizing to obtain a mixed solution B; adding xylose, sodium chloride and ammonium sulfate into the mixed solution A in sequence, stirring uniformly after quantification, adjusting the pH to 6.5, adding agarose, and sterilizing at 121 ℃ for 15min to obtain a mixed solution C; adding sterile 2-deoxyglucose aqueous solution and the mixed solution B into the mixed solution C, shaking uniformly, and then pouring the plate to obtain the nutrient medium.
Example 2
In an embodiment of the screening medium for filamentous fungi of the present invention, the screening medium for filamentous fungi of this embodiment includes a chromogenic medium, an enzymatic medium, and a nutrient medium, which are sequentially disposed from bottom to top;
the raw materials for preparing the chromogenic medium contain a calcium ion indicator solution and an agarose solution, and the volume ratio of the calcium ion indicator solution to the agarose solution is 10: 100, respectively; the calcium ion indicator solution consists of a calcium ion indicator and a Tris-HCl buffer solution with the pH value of 7.1, wherein the ratio of the mass of the calcium ion indicator to the volume of the buffer solution in the calcium ion indicator solution is 0.1 g: 100 mL; the agarose solution consists of agarose and a Tris-HCl buffer solution with the pH value of 7.1, and the ratio of the mass of the agarose to the volume of the buffer solution in the agarose solution is 1.0 g: 100 mL;
the preparation raw materials of the enzymolysis culture medium contain calcium phytate suspension and agarose solution, and the volume ratio of the calcium phytate suspension to the agarose solution is 1: 10; wherein the content of calcium phytate in the calcium phytate suspension is 0.2 g/mL; the agarose solution consists of agarose and a Tris-HCl buffer solution with the pH value of 7.1, and the ratio of the mass of the agarose to the volume of the buffer solution in the agarose solution is 1.0 g: 100 mL;
the nutrient medium contains the following components in percentage by weight: 18mmol/L of 2-deoxyglucose, 0.3% (w/v) of calcium phytate, 5mmol/L of lactic acid, 3% (w/v) of xylose, 1.5% (w/v) of potassium nitrate, 0.5% (w/v) of ammonium sulfate, 0.5% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride and 2% (w/v) of agarose with a pH value of 6.5;
wherein the calcium ion indicator is chrome black T.
Further, in the screening medium for filamentous fungi of the present example, the thickness of the chromogenic medium is 1/3 times the thickness of the screening medium; the thickness of the enzymolysis culture medium is 1/3 of the thickness of the screening culture medium; the thickness of the nutrient medium is 1/3 the thickness of the screening medium.
The method for preparing the screening medium for filamentous fungi of this example includes the same steps as those of step (3) of example 1 in step (3), and includes the following steps:
(1) preparation of a chromogenic medium: weighing 0.1g of chrome black T, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 7.1, and filtering and sterilizing to obtain a calcium ion indicator solution; weighing 1.0g of agarose, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 7.1, sterilizing at 121 ℃ for 15min, cooling to about 45 ℃, adding 10mL of sterile calcium ion indicator solution, shaking uniformly, and pouring to obtain a chromogenic medium; wherein, Tris and HCl reagents in Tris-HCl buffer solution are more than analytical purity, and the purity of water used simultaneously is deionized water and the grade above;
(2) after the chromogenic culture medium in the step (1) is cooled, adding an enzymolysis culture medium, wherein the preparation process of the enzymolysis culture medium is as follows: weighing calcium phytate according to the proportion of adding 1g of calcium phytate into 10mL of water, soaking overnight, and sterilizing at 121 ℃ for 15 min; after the sterilized calcium phytate solution is cooled to room temperature, centrifuging by using a centrifugal method, discarding supernatant, washing calcium phytate precipitate by using sterile water for 3 times, and adding a proper amount of sterile water to disperse the calcium phytate precipitate again to prepare sterile calcium phytate suspension; weighing 1.0g of agarose, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 7.1, sterilizing at 121 ℃ for 15min, cooling to about 45 ℃, adding 10mL of calcium phytate suspension containing 2g of calcium phytate, shaking uniformly and turning over to obtain enzymolysis culture; the Tris and HCL reagents in the Tris-HCl buffer solution are more than analytical purity, and the water purity used at the same time is deionized water and the grade above.
Example 3
In an embodiment of the screening medium for filamentous fungi of the present invention, the screening medium for filamentous fungi of this embodiment includes a chromogenic medium, an enzymatic medium, and a nutrient medium, which are sequentially disposed from bottom to top;
the raw materials for preparing the chromogenic medium contain a calcium ion indicator solution and an agarose solution, and the volume ratio of the calcium ion indicator solution to the agarose solution is 1: 100, respectively; the calcium ion indicator solution consists of a calcium ion indicator and a Tris-HCl buffer solution with the pH value of 8.9, wherein the ratio of the mass of the calcium ion indicator to the volume of the buffer solution in the calcium ion indicator solution is 2 g: 100 mL; the agarose solution consists of agarose and a Tris-HCl buffer solution with the pH value of 8.9, and the ratio of the mass of the agarose to the volume of the buffer solution in the agarose solution is 0.2 g: 100 mL;
the preparation raw materials of the enzymolysis culture medium contain calcium phytate suspension and agarose solution, and the volume ratio of the calcium phytate suspension to the agarose solution is 1: 10; wherein the content of calcium phytate in the calcium phytate suspension is 0.02 g/mL; the agarose solution consists of agarose and a Tris-HCl buffer solution with the pH value of 8.9, and the ratio of the mass of the agarose to the volume of the buffer solution in the agarose solution is 0.2 g: 100 mL;
the nutrient medium contains the following components in percentage by weight: 30mmol/L of 2-deoxyglucose, 0.6% (w/v) of calcium phytate, 30mmol/L of lactic acid, 0.5% (w/v) of xylose, 1.5% (w/v) of potassium nitrate, 0.5% (w/v) of ammonium sulfate, 0.5% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride and 0.5% (w/v) of agarose with a pH value of 6.5;
wherein the calcium ion indicator is chrome black T.
Further, in the screening medium for filamentous fungi of the present example, the thickness of the chromogenic medium is 1/6 times the thickness of the screening medium; the thickness of the enzymolysis culture medium is 1/4 of the thickness of the screening culture medium; the thickness of the nutrient medium is 7/12 the thickness of the screening medium.
The method for preparing the screening medium for filamentous fungi of this example includes the same steps as those of step (3) of example 1 in step (3), and includes the following steps:
(1) preparation of a chromogenic medium: weighing 2g of chrome black T, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 8.9, and filtering and sterilizing to obtain a calcium ion indicator solution; weighing 0.2g of agarose, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 8.9, sterilizing at 121 ℃ for 15min, cooling to about 45 ℃, adding 1mL of sterile calcium ion indicator solution, shaking uniformly, and pouring the plate to obtain a chromogenic medium; wherein, Tris and HCl reagents in Tris-HCl buffer solution are more than analytical purity, and the purity of water used simultaneously is deionized water and the grade above;
(2) after the chromogenic culture medium in the step (1) is cooled, adding an enzymolysis culture medium, wherein the preparation process of the enzymolysis culture medium is as follows: weighing calcium phytate according to the proportion of adding 1g of calcium phytate into 10mL of water, soaking overnight, and sterilizing at 121 ℃ for 15 min; after the sterilized calcium phytate solution is cooled to room temperature, centrifuging by using a centrifugal method, discarding supernatant, washing calcium phytate precipitate by using sterile water for 3 times, and adding a proper amount of sterile water to disperse the calcium phytate precipitate again to prepare sterile calcium phytate suspension; weighing 0.2g of agarose, dissolving in 100mL of Tris-HCl buffer solution with the pH value of 8.9, sterilizing at 121 ℃ for 15min, cooling to about 45 ℃, adding 10mL of calcium phytate suspension containing 0.2g of calcium phytate, shaking uniformly, and pouring to obtain an enzymolysis culture; the Tris and HCL reagents in the Tris-HCl buffer solution are more than analytical purity, and the water purity used at the same time is deionized water and the grade above.
Example 4
An example of a screening medium for filamentous fungi of the present invention that differs from the screening medium for filamentous fungi of example 1 only in that: in this example, the nutrient medium contained the following components: 5mmol/L of 2-deoxyglucose, 0.45% (w/v) of calcium phytate, 20mmol/L of lactic acid, 1% (w/v) of xylose, 1.5% (w/v) of potassium nitrate, 0.5% (w/v) of ammonium sulfate, 0.5% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride and 1% (w/v) of agarose with a pH value of 6.5.
The screening medium for filamentous fungi of this example was prepared in the same manner as in example 1.
Example 5
An example of a screening medium for filamentous fungi of the present invention that differs from the screening medium for filamentous fungi of example 1 only in that: in this example, the nutrient medium contained the following components: 1% (w/v) of calcium phytate, 20mmol/L of lactic acid, 1% (w/v) of xylose, 1.5% (w/v) of potassium nitrate, 0.5% (w/v) of ammonium sulfate, 0.5% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride and 1% (w/v) of agarose with a pH value of 6.5.
The screening medium for filamentous fungi of this example was prepared in the same manner as in example 1.
Example 6
An example of a screening medium for filamentous fungi of the present invention that differs from the screening medium for filamentous fungi of example 1 only in that: in this example, the nutrient medium contained the following components: 50mmol/L of 2-deoxyglucose, 0.6% (w/v) of calcium phytate, 20mmol/L of lactic acid, 1% (w/v) of xylose, 1.5% (w/v) of potassium nitrate, 0.5% (w/v) of ammonium sulfate, 0.5% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride and 1% (w/v) of agarose with a pH value of 6.5.
The screening medium for filamentous fungi of this example was prepared in the same manner as in example 1.
Comparative example 1
A culture medium of this comparative example comprising the following components in amounts: 1.5% (w/v) of potassium nitrate, 0.5% (w/v) of ammonium sulfate, 0.05% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride, 1% (w/v) of agarose with a pH of 6.5, 5mmol/L of 2-deoxyglucose, and 0.6% (w/v) of xylose.
The preparation method of the culture medium of the comparative example comprises the following steps: potassium nitrate, ammonium sulfate, sodium chloride, magnesium chloride, and an agarose solution having a pH of 6.5 were mixed, sterilized at 121 ℃ for 15min, cooled to about 65 ℃, and added sequentially with a 50X 2-deoxyglucose mother liquor and a 50X xylose mother liquor sterilized with a 0.22um sterile filter to obtain the culture medium of the present comparative example.
Comparative example 2
A culture medium of this comparative example comprising the following components in amounts: 1.5% (w/v) of potassium nitrate, 0.4% (w/v) of calcium phytate, 0.5% (w/v) of ammonium sulfate, 0.05% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride, 1% (w/v) of agarose with a pH value of 6.5, and 0.6% (w/v) of glucose.
The preparation method of the culture medium of the comparative example comprises the following steps: potassium nitrate, calcium phytate, ammonium sulfate, sodium chloride, magnesium chloride, and agarose solution with pH of 6.5 were mixed, sterilized at 121 ℃ for 15min, cooled to about 65 ℃, and added with 50 × glucose mother liquor sterilized with 0.22um sterile filter to obtain the culture medium of this comparative example.
Comparative example 3
A culture medium of this comparative example comprising the following components in amounts: 1.5% (w/v) of potassium nitrate, 20mmol/L of lactic acid, 0.6% (w/v) of xylose, 5mmol/L of 2-deoxyglucose, 0.4% (w/v) of calcium phytate, 0.5% (w/v) of ammonium sulfate, 0.05% (w/v) of sodium chloride, 0.05% (w/v) of magnesium chloride and 1% (w/v) of agarose.
The preparation method of the culture medium of the comparative example comprises the following steps:
(1) dissolving calcium phytate: dissolving calcium phytate in deionized water, placing into 3-5-particle rotor, magnetically stirring for more than 5h by magnetic stirrer, and slowly adding lactic acid during dissolving process.
(2) Preparing a sterile 2-deoxyglucose solution: dissolving 2-deoxyglucose in deionized water, sterilizing with 0.22um filter membrane, and refrigerating at 4 deg.C.
(3) Preparing a sterile inorganic salt mixed solution: weighing sodium chloride and magnesium chloride in proportion, dissolving in deionized water, filtering for sterilization, and storing at room temperature.
(4) Adding xylose, potassium nitrate and ammonium sulfate into the dissolved calcium phytate solution in sequence, stirring uniformly after quantifying, and adjusting the pH value to 6.5.
(5) And (3) high-pressure sterilization: weighing agarose according to a certain proportion, adding into the above mixed culture medium, and sterilizing at 121 deg.C for 15 min.
(6) Cooling to about 65 ℃, sequentially adding the prepared sterile 2-deoxyglucose solution and the sterile inorganic salt mixed solution according to the proportion, shaking uniformly, and then pouring the plate to obtain the culture medium of the comparative example.
Effect example 1 mutagenesis method and Process
Acquisition of mutant colonies-Aspergillus oryzae strains mutagenized by ARTP
1. Lethality analysis of the starting strain:
selecting a mature inclined plane of a normally growing and rich-sporulation aspergillus oryzae strain, hooking spores of about 3 rings, transferring the spores into a 100mL triangular flask, adding sterilized 0.85% physiological saline about 20mL in advance into the flask, and adding a proper amount of normal saline
The glass strain of (1); the vortex was shaken for a period of time in order to disperse the spores into a spore suspension. Microscopic examination is carried out, and the concentration of spore suspension is adjusted to 5 × 107About one/mL.
And opening an ARTP mutagenesis equipment power supply and a gas cylinder valve, and uniformly coating 10uL of spore suspension on the surface of the sterilized slide glass. From the groove of the turntable No. 1, the sample slide to be processed is sequentially placed in the slide fixing circular groove above the automatic turntable clockwise by using an elbow tweezers. The processing power of each slide was set to 120W, the air flow rate was set to 10SLM, the processing distance was set to 2mm, and the processing time was set to 0s, 2s, 10s, 20s, 30s, 60s, and 80s in this order, and the sample processing was started after the setting. After all samples were processed, the slides were placed individually into EP tubes containing 1mL sterile water using sterile forceps. Placing the EP tube on an oscillator, oscillating for 1min, and eluting the spores attached to the slide glass into sterile water to form new bacterial suspension; gradually diluting the spore suspension after mutagenesis to 104. And the obtained spores are respectively coated on the culture media of comparative example 1, comparative example 3 and example 1 to obtain lethality curves of different culture media.
2. Screening of mutant strains
Selecting the condition that the spore lethality rate is more than 90%, and carrying out mutation breeding. And the spores after mutagenesis were spread and diluted on the culture media of comparative example 1, comparative example 3 and example 1, respectively, and culture screening was performed.
The bacterial strain which is grown on the culture medium of the comparative example 1 and has the colony size which is improved by more than 50 percent compared with the wild type is transferred to the culture medium of the comparative example 2 for further screening the decomposition effect of calcium phytate. And continuously passaging for 3 passages on the culture media of the comparative example 1 and the comparative example 2.
Strains which are grown on the culture medium of the comparative example 3 and have the colony size which is improved by more than 50 percent compared with the wild type and have obvious decomposition rings are screened, and the strains are continuously propagated on the culture medium of the comparative example 3 for 3 generations.
Strains in which the size of growing colonies on the medium of example 1 was increased by 50% or more compared to the wild type and the coloration circles were evident were selected and passaged continuously for 3 generations on the medium of example 1.
The number of positive bacteria, the positive rate and the time consumption were examined for different media, and the results are shown in Table 1 below.
TABLE 1
Note: the positive bacteria refer to the strains which grow on a specific screening culture medium and have the colony size which is improved by more than 50 percent compared with the wild type (the decomposition ring is obvious simultaneously, and the ring diameter ratio of the color development ring is more than 1.2 times of that of the wild type).
Effect example 2 screening of strains and enzyme Activity measurement
Selecting 15 mutant bacteria with good effect obtained by three different screening methods in the effect example 1 respectively, performing starter propagation fermentation analysis for 40 hours, performing 3 times of repeated tests on each mutant bacteria, and determining the activity of glutaminase, aminopeptidase, protease, amylase, cellulase, pectinase and phytase on the obtained starter, wherein the detection method and the enzyme activity definition refer to national standards, and the determination results are shown in table 2.
TABLE 2
The experimental result shows that 3 strains of the 15 strains screened from the culture media of the comparative example 1 and the comparative example 2 have certain advantages and stable properties compared with the comprehensive enzyme system of the original strain; 5 strains of the 15 strains screened from the culture medium of the comparative example 3 have certain advantages and stable properties compared with the comprehensive enzyme system of the original strain; of the 15 strains selected from the medium in example 1, 12 strains had some advantages over the original strain of the integrated enzyme system and had stable properties. The comparison shows that the mutant strains screened by the screening culture medium have good enzyme activity stability and basically do not fluctuate.
Effect example 3 Soy sauce fermentation parameters of the selected strains
Two of the novel strains selected from the medium of example 1, which had high enzyme activity and excellent stability, were selected as 417 and 438, and soy sauce fermentation was carried out (three parallel experiments were carried out for each strain). After the transfer slant is activated in sequence, the culture is enlarged in a triangular flask, and then the production simulation starter propagation small test and fermentation are continuously carried out. (the slant culture medium is soybean juice culture medium; the triangular flask culture medium is bran and flour; the starter propagation culture medium is prepared by raw materials such as soybean and wheat.) after the fermentation is finished, the indexes of amino nitrogen, total nitrogen, reducing sugar, glutamic acid, precipitate, turbidity and the like of the crude oil are detected, and the results are shown in Table 3; and heating the crude oil for sensory evaluation, and the radar chart of the sensory evaluation is shown in FIG. 1.
Wherein the bean juice culture medium comprises the following components in percentage by weight: bean juice (4-5 ° Be'), soluble starch 2% (w/v), potassium dihydrogen phosphate 0.1% (w/v), magnesium sulfate 0.025% (w/v), ammonium sulfate 0.05% (w/v), agar powder 1.5% (w/v), and pH 6.5.
TABLE 3
As can be seen from Table 3, the amino nitrogen of the new strains 417 and 438 is higher than that of the original strain by more than 5 percent, the total nitrogen is higher than that of the original strain by more than 2 percent, and the glutamic acid is higher than that of the original strain by more than 10 percent; the turbidity and the heat-treated precipitate were significantly lower than the starting strain. As can be seen from FIG. 1, the fresh and sweet taste of the crude oil of the 417 strain is obviously improved; 438 the crude oil of the strain is red and strong in color and luster, clear in body state, fresh and cool in taste, fresh and sweet and delicious.
The inventors also examined the effects of the culture media of examples 2 to 5 by referring to the methods of effect examples 1 to 3, and found that the culture media of examples 2 to 5 all had the same screening effect as the culture media of examples, which is not repeated herein.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A screening medium for filamentous fungi, comprising: comprises a chromogenic culture medium, an enzymolysis culture medium and a nutrient culture medium which are arranged from bottom to top in sequence; the chromogenic medium contains agarose and a calcium ion indicator, and the enzymolysis medium contains agarose and calcium phytate.
2. The screening medium for filamentous fungi of claim 1, wherein: the raw materials for preparing the chromogenic medium comprise:
(a) the calcium ion indicator solution consists of a calcium ion indicator and a buffer solution, wherein the ratio of the mass of the calcium ion indicator to the volume of the buffer solution is (0.1-2 g): 100 mL; and
(b) the agarose solution consists of agarose and a buffer solution, wherein the ratio of the mass of the agarose to the volume of the buffer solution is 0.2-1.0 g: 100 mL;
the volume ratio of (a) to (b) is 1-10: 100.
3. the screening medium for filamentous fungi of claim 2, wherein: the calcium ion indicator is chrome black T.
4. The screening medium for filamentous fungi of claim 1, wherein: the preparation raw materials of the enzymolysis culture medium comprise:
(c) calcium phytate suspension, wherein the content of calcium phytate in the calcium phytate suspension is 0.02-0.2 g/mL; and
(b) the agarose solution consists of agarose and a buffer solution, wherein the ratio of the mass of the agarose to the volume of the buffer solution is 0.2-1.0 g: 100 mL;
the volume ratio of (c) to (b) is 1: 10.
5. the screening medium for filamentous fungi according to any one of claims 2 to 4, wherein: the buffer solution is Tris-HCl buffer solution, and the pH value of the buffer solution is 7.1-8.9.
6. The screening medium for filamentous fungi of claim 1, wherein: the nutrient medium contains inorganic salt and the following components in percentage by weight:
7. the screening medium for filamentous fungi of claim 6, wherein: the inorganic salt comprises potassium nitrate, ammonium sulfate, sodium chloride and magnesium chloride; in the nutrient medium, the concentration of the 2-deoxyglucose is 5-30mmol/L, and the content of the calcium phytate is 0.3-0.6% w/v.
8. The screening medium for filamentous fungi of claim 1, wherein: the thickness of the chromogenic medium is 1/6-1/3 of the thickness of the screening medium; the thickness of the enzymolysis culture medium is 1/4-1/3 of the thickness of the screening culture medium; the thickness of the nutrient medium is 1/3-7/12 of the thickness of the screening medium.
9. A method for preparing a screening medium for filamentous fungi according to any one of claims 1 to 8, comprising: the method comprises the following steps:
preparation of a chromogenic medium: dissolving a calcium ion indicator into a buffer solution according to the proportion of adding 0.1-2 g of the calcium ion indicator into every 100mL of the buffer solution, and filtering and sterilizing to obtain a calcium ion indicator solution; dissolving agarose into a buffer solution according to the proportion of adding 0.2-1.0 g of agarose into every 100mL of the buffer solution, sterilizing, cooling, adding the prepared calcium ion indicator solution, shaking uniformly, and pouring the plate to obtain a chromogenic medium;
preparing an enzymolysis culture medium: adding calcium phytate into water, soaking overnight, sterilizing, cooling to room temperature, centrifuging, discarding supernatant to obtain calcium phytate precipitate, and adding water dispersed calcium phytate precipitate to obtain calcium phytate suspension; dissolving agarose into a buffer solution according to the proportion of adding 0.2-1.0 g of agarose into every 100mL of the buffer solution, sterilizing, cooling, adding the prepared calcium phytate suspension, shaking uniformly, and pouring the mixture to obtain an enzymolysis culture medium;
preparation of a nutrient medium: dissolving calcium phytate in water, and adding lactic acid while stirring to obtain a mixed solution A; dissolving 2-deoxyglucose in water, filtering and sterilizing to obtain 2-deoxyglucose aqueous solution; dissolving potassium nitrate and magnesium chloride in water, and filtering and sterilizing to obtain a mixed solution B; adding xylose, sodium chloride and ammonium sulfate into the mixed solution A, quantifying, uniformly stirring, adjusting the pH value, adding agarose, sterilizing and cooling to obtain a mixed solution C; adding the 2-deoxyglucose aqueous solution and the mixed solution B into the mixed solution C, shaking uniformly, and then pouring the plate to obtain a nutrient medium;
adding the enzymolysis culture medium to the cooled chromogenic culture medium to obtain a pre-screening culture medium;
and after the enzymolysis culture medium is cooled, adding the nutrient culture medium to the enzymolysis culture medium in the pre-screening culture medium to obtain the screening culture medium of the filamentous fungi.
10. Use of a screening medium for filamentous fungi according to any one of claims 1 to 8 for screening filamentous fungi.
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