CN112501027A - Eurotium cristatum strain and domestication and fermentation method - Google Patents
Eurotium cristatum strain and domestication and fermentation method Download PDFInfo
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Abstract
The invention discloses a Eurotium Cristatum strain which is preserved in China general microbiological culture Collection center in 2018, 4 and 2 months, and the strain is Eurotium Cristatum XD-01 with the preservation number of CGMCC No.15395, Eurotium Cristatum XD-02 with the preservation number of CGMCC No.15396 and Eurotium Cristatum XD-03 with the preservation number of CGMCC No. 15397. In addition, the invention also discloses a method for domesticating and fermenting the strain. The eurotium cristatum strain is derived from Fuzhuan tea, and is ecologically safe and harmless.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a eurotium cristatum strain and a domestication and fermentation method thereof.
Background
Selenium is a trace element essential to human body, and has effects of resisting oxidation, scavenging free radicals, protecting heart, and preventing and treating myopia, cataract, AIDS, diabetes, and cancer. The world health organization has clear regulation on the daily selenium content of human bodies, and the Chinese Nutrition society regulates that the daily selenium intake of adults is 50-250 micrograms. In China, 70% of land is deficient in selenium, the daily intake of selenium for adults is about 20-30 micrograms, so that the safe selenium supplement is widely researched, especially inorganic selenium is converted into organic selenium through probiotics, and currently reported probiotics capable of enriching selenium comprise lactobacillus rhamnosus, streptococcus thermophilus, lactobacillus acidophilus, bifidobacteria and the like.
Zinc is a necessary trace element for human body, and has important functions in maintaining normal metabolism, promoting development of body and intelligence, and enhancing immunity. Nearly one third of people in the world face the health problem caused by zinc deficiency, the zinc deficiency problem of children and old people in China is particularly serious, the zinc deficiency of the children can influence the development of the bodies and intelligence of the children, and the zinc deficiency of the old people can cause the reduction of the immunity of the organism, so that various diseases can be caused.
The iron element is an essential nutrient element for human and animal life process, is an indispensable trace element, and particularly participates in the transportation process of oxygen in blood. Iron deficiency can cause the human and animal bodies to suffer from symptoms such as "iron deficiency anemia". In daily life, people mainly take iron element in an inorganic state, but the use of the iron element is limited due to stimulation of iron ions to mucous membranes, large side effect of gastrointestinal tracts, low absorption rate and the like.
Eurotium cristatum, also known as eurotium cristatum, belongs to Aspergillus glaucus of Eurotium of Tricholomataceae of Ascomycetales. The three screened eurotium cristatum strains can convert inorganic selenium, zinc and iron into organic state, solve the potential safety hazard existing after the inorganic state is ingested, and provide reliable probiotic strains for producing tea drinks and other products.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide an eurotium cristatum strain aiming at the above-mentioned deficiencies of the prior art. The strain is derived from Fuzhuan tea, and is ecologically safe and harmless.
In order to solve the technical problems, the invention adopts the technical scheme that: a Eurotium cristatum strain is characterized by comprising a Eurotium cristatum XD-01 selenium-rich strain which is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 15395; the preservation date is as follows: year 2018, 4 month 2 day, depository: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;
or the Eurotium cristatum zinc-rich strain is Eurotium cristatum XD-02, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 15396; the preservation date is as follows: year 2018, 4 month 2 day, depository: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;
or the Eurotium cristatum iron-rich strain Eurotium cristatum XD-03 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 15397; the preservation date is as follows: year 2018, 4 month 2 day, depository: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
In addition, the invention also provides a method for screening the eurotium cristatum wild strain, which is characterized in that the method for screening the eurotium cristatum selenium-rich wild strain comprises the following steps: weighing 10g of Fuzhuan tea with brilliant yellow cyst shell from commercial Jingwei Fuzhuan tea, grinding, adding 90mL of sterile water, and oscillating and mixing uniformly; diluting the mixture after shaking and mixing with sterile water by 10 times, respectively selecting 10-3And 10-4Coating the two gradient dilutions on a PDA solid culture medium containing 10mg/L selenium element, and standing and culturing at 30 ℃ for 60 h; selecting a colony which is large and bright yellow in color, scribing on a PDA flat plate containing 10mg/L of selenium element, continuously screening for 8-10 generations, and storing at 4 ℃ in a PDA slant culture medium for later use to obtain the eurotium cristatum selenium-rich wild strain;
the screening method of the zinc-rich wild strain of eurotium cristatum comprises the following steps: weighing 10g of Fuzhuan tea with brilliant yellow cyst shell from commercial Jingwei Fuzhuan tea, grinding, adding 90mL of sterile water, and oscillating and mixing uniformly; diluting the mixture after shaking and mixing with sterile water by 10 times, respectively selecting 10-3And 10-4Coating the two gradients of diluent on a PDA solid culture medium containing 5mg/L zinc element, and standing and culturing at 30 ℃ for 60 h; selecting a colony which is large and bright yellow in color, scribing on a PDA (personal digital Assistant) plate containing 5mg/L zinc element, continuously screening for 8-10 generations, and storing at 4 ℃ in a PDA slant culture medium for later use to obtain a zinc-rich wild strain of eurotium cristatum;
the screening method of the iron-rich wild strain of eurotium cristatum comprises the following steps: weighing 10g of Fuzhuan tea with brilliant yellow cyst shell from commercial Jingwei Fuzhuan tea, grinding, adding 90mL of sterile water, and oscillating and mixing uniformly; diluting the mixture after shaking and mixing with sterile water in 10 times gradient, and respectively selectingGet 10-3And 10-4Coating the two gradients of diluent on a PDA solid culture medium containing 500mg/L iron element, and standing and culturing at 30 ℃ for 60 h; and selecting a colony which is large and bright yellow in color, streaking the colony on a PDA plate containing 500mg/L iron element, continuously screening for 8-10 generations, and storing in a PDA slant culture medium at 4 ℃ for later use to obtain the iron-rich wild strain of the eurotium cristatum.
Further, the present invention provides a method for acclimatizing the wild strain of Eurotium cristatum selected as described above, which is characterized in that,
the method for domesticating the eurotium cristatum selenium-rich wild strain comprises the following steps: resuspending the lawn of the selenium-rich wild strain of Eurotium cristatum stored in claim 2 in normal saline to a concentration of 2X 106～2×108Carrying out ultraviolet mutagenesis on the spore/mL heavy suspension, respectively inoculating the heavy suspension subjected to ultraviolet mutagenesis to PDA solid plate culture media containing 0mg/L, 5mg/L, 10mg/L, 15mg/L, 20mg/L and 25mg/L of inorganic selenium, uniformly coating, and culturing at 30 ℃ for 60 hours; selecting a colony with a larger colony and a bright color, streaking the colony on a PDA solid plate containing 10mg/L inorganic selenium, and comparing the growth conditions of different strains; selecting a colony with the largest colony and the brightest yellow color as a starting strain, separating and purifying for 8-10 generations, and storing at a 4 ℃ inclined plane to obtain the eurotium cristatum selenium-rich strain of claim 1;
the method for domesticating the zinc-rich wild strain of eurotium cristatum comprises the following steps: resuspending the lawn of the zinc-rich wild strain of Eurotium cristatum stored in claim 2 in physiological saline to a concentration of 2X 106～2×108Carrying out ultraviolet mutagenesis on the spore/mL heavy suspension, respectively inoculating the ultraviolet mutagenized heavy suspension to PDA solid plate culture media containing 0mg/L, 5mg/L, 10mg/L, 15mg/L and 20mg/L inorganic zinc, uniformly coating, and culturing at 30 ℃ for 60 h; selecting a colony which is large and bright in color, scribing the colony on a PDA solid plate containing 5mg/L inorganic zinc, and comparing the growth conditions of different strains; selecting a colony which is the largest and brightest and yellow as a starting strain, separating and purifying for 8-10 generations, and storing at a 4 ℃ inclined plane to obtain the eurotium cristatum zinc-rich strain of claim 1;
the method for domesticating the iron-rich wild strain of eurotium cristatum comprises the following steps: resuspending the lawn of the iron-rich wild strain of Eurotium cristatum stored in claim 2 in physiological saline to a concentration of 2X 106～2×108Carrying out ultraviolet mutagenesis on the spore/mL heavy suspension, respectively inoculating the ultraviolet mutagenized heavy suspension to PDA solid plate culture media containing 0mg/L, 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L inorganic iron, uniformly coating, and culturing at 30 ℃ for 60 h; selecting a colony with a larger colony and a bright color, streaking the colony on a PDA solid plate containing 500mg/L inorganic iron, and comparing the growth conditions of different strains; selecting a colony which is the largest and brightest yellow as a starting strain, separating and purifying for 8-10 generations, and storing at a 4 ℃ inclined plane to obtain the eurotium cristatum iron-rich strain of claim 1.
Further, the present invention provides a fermentation method of the eurotium cristatum strain, which is characterized by comprising: resuspending the lawn of the Eurotium cristatum strain of claim 1 in physiological saline to give a concentration of 2X 106～2×108Inoculating the heavy suspension liquid into a liquid seed culture medium, performing shaking culture at 150rpm and 30 ℃ for 72h, inoculating the heavy suspension liquid into a fermentation culture medium according to the inoculation ratio of 5%, and performing shaking culture at 150rpm and 30 ℃ for 72 h; the liquid seed culture medium comprises the following components: sucrose 15g/L, peptone 2g/L, yeast extract 10g/L and KH2PO4 2g/L。
The fermentation method described above is characterized in that,
when the eurotium cristatum strain is the eurotium cristatum selenium-rich strain, the fermentation medium comprises the following components: 10 g/L-80 g/L of cane sugar or glucose, 2 g/L-15 g/L of corn steep liquor dry powder, 2 g/L-10 g/L of peptone, 2 g/L-10 g/L of yeast extract and KH2PO4 1g/L～8g/L，MgSO4·7H2O 1.0g/L～7.5g/L，(NH4)2SO42.5-8.0 g/L of selenium element 10 mg/L;
when the eurotium cristatum strain is an eurotium cristatum zinc-rich strain, the fermentation medium comprises the following components: sucrose or glucose 10-80 g/L, jade2-15 g/L of rice milk dry powder, 2-10 g/L of peptone, 2-10 g/L of yeast extract and KH2PO4 1g/L～8g/L，MgSO4·7H2O 1.0g/L～7.5g/L，(NH4)2SO42.5-8.5 g/L of zinc element 5 mg/L;
when the eurotium cristatum strain is an iron-rich eurotium cristatum strain, the fermentation medium comprises the following components: 10 g/L-80 g/L of cane sugar or glucose, 2 g/L-15 g/L of corn steep liquor dry powder, 2 g/L-10 g/L of peptone, 2 g/L-10 g/L of yeast extract and KH2PO4 1g/L～8g/L，MgSO4·7H2O 1.0g/L～7.5g/L，(NH4)2SO42.5-7.0 g/L of iron element 500 mg/L.
The fermentation method described above is characterized in that,
when the eurotium cristatum strain is the eurotium cristatum selenium-rich strain, the fermentation medium comprises the following components: 20 g/L-30 g/L of cane sugar or glucose, 3 g/L-5 g/L of corn steep liquor dry powder, 3 g/L-6 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO44.0-5.5 g/L of selenium element 10 mg/L;
when the eurotium cristatum strain is an eurotium cristatum zinc-rich strain, the fermentation medium comprises the following components: 20 g/L-30 g/L of cane sugar or glucose, 3 g/L-6 g/L of corn steep liquor dry powder, 3 g/L-5 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO44.0-5.5 g/L of zinc element 5 mg/L;
when the eurotium cristatum strain is an iron-rich eurotium cristatum strain, the fermentation medium comprises the following components: 20 g/L-28 g/L of cane sugar or glucose, 3 g/L-6 g/L of corn steep liquor dry powder, 3 g/L-6 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO4 4.0g/L-5.5 g/L, iron element 500 mg/L.
The eurotium cristatum selenium-rich strain has the following properties:
morphological characteristics: the eurotium cristatum selenium-rich strain is inoculated on a PDA solid flat plate, and a white villiform colony can be seen after 24 hours of culture at 28 ℃, the diameter of the colony can reach 15mm after 48 hours, and hyphae at the center of the colony begin to turn yellow. The whole colony is bright yellow after 72 hours, and the diameter can reach 45-50 mm. And continuously culturing for 144h until the central part of the bacterial colony has a dark brown exudate, wherein the whole bacterial colony is brown, a brown pigment also exists on the back of the culture medium, and the diameter of the bacterial colony is 50-55 mm. The colony morphology of the cultured cells inoculated into the Czochralski culture medium is basically the same as that of a PDA (personal digital assistant) plate, but the cells grow slowly, and the diameter of the final colony is small and is 35-40 mm.
Physiological and biochemical characteristics: the strain has strong aerobic property and poor shearing resistance, and hyphae are easy to break and grow slowly when the stirring speed exceeds 300 rpm. The pH range of growth is 3.0-6.0, and the optimum pH is 5.0. The maximum growth temperature is 38 ℃, and the optimal growth temperature is 28 ℃. The strain can utilize various carbon sources, but the optimal carbon source is glucose; various nitrogen sources can be utilized, the inorganic nitrogen source is poor, and the organic nitrogen source is the best of corn steep liquor. The strain has strong inorganic selenium tolerance, strong inorganic selenium conversion capability to organic selenium, and 690mg/kg of organic selenium in hypha.
The strain is identified by China industrial microorganism strain preservation management center (FMIC-QO 01-003 fungus multiphase identification and detection method is adopted for identification, QO-03-02 microorganism bacterium molecular biology identification operating procedures), and is determined as follows: eurotium Cristatum (Eurotium Cristatum).
The eurotium cristatum zinc-rich strain has the following properties:
morphological characteristics: the zinc-rich strain of eurotium cristatum is inoculated on a PDA solid plate, and a white velvet colony can be seen after 24 hours of culture at 28 ℃, the diameter of the colony can reach 15mm after 48 hours, and hypha at the center of the colony begins to turn yellow. The whole colony is bright yellow after 72 hours, and the diameter can reach 45-50 mm. And continuously culturing for 144h until a dark brown exudate exists in the central part of the bacterial colony, wherein the whole bacterial colony is brown, the edge of the bacterial colony is yellow brown, a brown pigment also exists on the back of the culture medium, and the diameter of the bacterial colony is 50-55 mm. The colony morphology of the cultured cells inoculated into the Czochralski culture medium is basically the same as that of a PDA (personal digital assistant) plate, but the cells grow slowly, and the diameter of the final colony is small and is 35-40 mm.
Physiological and biochemical characteristics: the strain has strong aerobic property and poor shearing resistance, and hyphae are easy to break and grow slowly when the stirring speed exceeds 200 rpm. The pH range of growth is 3.8-6.0, and the optimum pH is 5.5. The maximum growth temperature is 38 ℃, and the optimal growth temperature is 30 ℃. The strain can utilize various carbon sources, but the optimal carbon source is glucose; various nitrogen sources can be utilized, the availability of inorganic nitrogen sources is poor, and corn steep liquor is the best of organic nitrogen sources. The strain has strong inorganic zinc tolerance, strong inorganic zinc conversion capability to organic zinc, and 380mg/kg of organic zinc in hypha.
The strain is identified by China industrial microorganism strain preservation management center (FMIC-QO 01-003 fungus multiphase identification and detection method is adopted for identification, QO-03-02 microorganism bacterium molecular biology identification operating procedures), and is determined as follows: eurotium Cristatum (Eurotium Cristatum).
The eurotium cristatum iron-rich strain has the following properties:
morphological characteristics: the eurotium cristatum iron-rich strain is inoculated on a PDA solid plate, a white villiform bacterial colony can be seen after 24 hours of culture at 28 ℃, the diameter of the bacterial colony can reach 15mm after 48 hours, and hyphae at the central position of the bacterial colony begin to turn yellow. The whole colony is bright yellow after 72 hours, and the diameter can reach 45-50 mm. And continuously culturing for 144h until the central part of the colony has a dark brown exudate, wherein the whole colony is brown, the back of the culture medium is also dark brown, and the diameter of the colony is 50-55 mm. The colony morphology of the cultured cells inoculated into the Czochralski culture medium is basically the same as that of a PDA (personal digital assistant) plate, but the cells grow slowly, and the diameter of the final colony is small and is 35-40 mm.
Physiological and biochemical characteristics: the strain has strong aerobic property and poor shearing resistance, and hyphae are easy to break and grow slowly when the stirring speed exceeds 300 rpm. The pH range of growth is 3.5-6.5, and the optimum pH is 5.0. The maximum growth temperature is 35 ℃, and the optimal growth temperature is 30 ℃. The strain can utilize various carbon sources, preferably glucose; various nitrogen sources can be utilized, the inorganic nitrogen source is poor, and the organic nitrogen source is the most optimal corn steep liquor. The strain has strong inorganic iron tolerance, strong inorganic iron conversion capability to organic iron, and organic iron in hypha can reach 6000 mg/kg.
The strain is identified by China industrial microorganism strain preservation management center (FMIC-QO 01-003 fungus multiphase identification and detection method is adopted for identification, QO-03-02 microorganism bacterium molecular biology identification operating procedures), and is determined as follows: eurotium Cristatum (Eurotium Cristatum).
Compared with the prior art, the invention has the following advantages:
1. the eurotium cristatum strain is derived from the Fu tea, and is ecologically safe and harmless.
2. The invention carries out ultraviolet mutagenesis and inorganic selenium domestication culture on the eurotium cristatum selenium-rich wild strain, and the screened eurotium cristatum selenium-rich strain can tolerate inorganic selenium and efficiently convert the inorganic selenium into organic selenium.
3. The invention carries out ultraviolet mutagenesis and inorganic zinc domestication culture on the zinc-rich wild strain of the eurotium cristatum, and the screened zinc-rich strain of the eurotium cristatum can tolerate inorganic zinc and efficiently convert the inorganic zinc into organic zinc.
4. The invention carries out ultraviolet mutagenesis and inorganic iron domestication culture on the eurotium cristatum iron-rich wild strain, and the screened eurotium cristatum iron-rich strain can tolerate inorganic iron and efficiently convert the inorganic iron into organic iron.
5. According to the invention, by optimizing the fermentation culture medium, the eurotium cristatum selenium-rich strain can tolerate inorganic selenium on the optimized culture medium, and can efficiently convert the inorganic selenium into organic selenium, the content of intracellular organic selenium is up to 690mg/kg, and the eurotium cristatum selenium-rich strain can be used as a production strain for efficiently converting the inorganic selenium into the organic selenium, so that the requirement of industrial production is met.
6. According to the invention, by optimizing the fermentation medium, the eurotium cristatum zinc-rich strain can tolerate inorganic zinc on the optimized culture medium and efficiently convert the inorganic zinc into organic zinc, the intracellular organic zinc content is as high as 380mg/kg, and the eurotium cristatum zinc-rich strain can be used as a production strain for efficiently converting the inorganic zinc into the organic zinc, so that the industrial production requirement is met.
7. According to the invention, by optimizing the fermentation medium, the eurotium cristatum iron-rich strain can tolerate inorganic iron on the optimized culture medium and efficiently convert the inorganic iron into organic iron, the intracellular organic iron content is up to 6000mg/kg, and the eurotium cristatum iron-rich strain can be used as a production strain for efficiently converting the inorganic iron into the organic iron, so that the requirement of industrial production is met.
The technical solution of the present invention is further described in detail by the following examples.
Drawings
FIG. 1 is an appearance diagram of lawn of the eurotium cristatum selenium-rich strain of the invention which grows for 60 hours on PDA slant culture medium.
FIG. 2 is an appearance diagram of lawn of the eurotium cristatum selenium-rich strain of the invention which grows for 72h on PDA culture medium.
FIG. 3 is a photomicrograph of cystosepiment formed by the selenium-rich strain of Eurotium cristatum on PDA medium.
FIG. 4 is a photomicrograph of the ascospores and ascospores produced by the selenium-rich strain of eurotium cristatum of the present invention.
FIG. 5 is an appearance diagram of the Eurotium cristatum selenium-rich strain cultured in Erlenmeyer flask for 72 h.
FIG. 6 is an appearance diagram of the Eurotium cristatum selenium-rich strain cultured in a 125L fermentation tank for 72 h.
FIG. 7 is an appearance diagram of lawn of zinc-rich Eurotium cristatum strain of the present invention grown on PDA slant culture medium for 60 h.
FIG. 8 is an appearance diagram of lawn of zinc-rich Eurotium cristatum strain of the present invention grown on PDA medium for 72 h.
FIG. 9 is a photomicrograph of cystosepiment formed by zinc-rich strains of Eurotium cristatum of the present invention on PDA medium.
FIG. 10 is a photomicrograph of ascospores and ascospores produced by zinc-rich strains of eurotium cristatum of the present invention.
FIG. 11 is an appearance diagram of the zinc-rich strain of Eurotium cristatum of the present invention cultured in Erlenmeyer flask for 72 h.
FIG. 12 is an appearance diagram of the zinc-rich strain of Eurotium cristatum of the present invention cultured in a 125L fermenter for 72 hours.
FIG. 13 is an appearance diagram of lawn of iron-rich Eurotium cristatum strain of the present invention grown on PDA slant culture medium for 60 h.
FIG. 14 is an appearance diagram of lawn of iron-rich Eurotium cristatum strain of the present invention grown on PDA medium for 72 h.
FIG. 15 is a photomicrograph of cystosepiment formed by iron-rich strains of Eurotium cristatum of the present invention on PDA medium.
FIG. 16 is a photomicrograph of the ascospores and ascospores produced by the iron-rich strain of eurotium cristatum of the present invention.
FIG. 17 is an appearance diagram of iron-rich strains of Eurotium cristatum cultured in Erlenmeyer flask for 72 h.
FIG. 18 is an appearance diagram of iron-rich strain of Eurotium cristatum of the present invention cultured in a 125L fermenter for 72 hours.
Detailed Description
Example 1 screening of Eurotium cristatum selenium-enriched wild strains
10g of Fuzhuan tea with brilliant yellow encysted shell is weighed from commercial Jingwei Fuzhuan tea in Xian, and is put into a mortar for rough grinding, and then is put into a conical flask with 3-5 glass beads and 90mL of sterile water. Mix by shaking at 150rpm for about 30 min. Taking 1mL spore mixed solution to perform 10-fold gradient dilution by using sterile water, and respectively selecting 10-3And 10-4Two gradients of spore suspension are sucked up by 0.1mL and spread on PDA solid culture medium containing 10mg/L selenium element, and then the solid culture medium is kept still for 60h at 30 ℃. Selecting large colonies and bright yellow colonies, streaking on PDA plate containing 10mg/L selenium element, screening for 8-10 generations, performing slant culture on PDA, and storing at 4 deg.C.
EXAMPLE 2 mutagenesis, acclimatization, isolation and purification of wild strains
The lawn selected in example 1 was eluted with sterile physiological saline and broken up with glass beads to adjust the concentration to 2X 106-2×108Taking a certain amount of spore suspension per mL of spore suspension, placing the spore suspension into a culture dish (the liquid level is not more than 3mm), irradiating for 25min by using an ultraviolet lamp tube with the distance of 20W and 30cm, taking 0.1mL of the spore suspension, inoculating into PDA solid plates containing different concentrations of (0; 5; 10; 15; 20; 25mg/L) inorganic selenium, and coating. Culturing at 30 deg.C for 60 h. Selecting several colonies with larger colonies and bright colors on different concentrations of selenium element, and streaking on a strip containing 10And comparing the size and appearance of the colony again by using the PDA solid plate of the mg/L inorganic selenium, and selecting the dominant bacterium with the largest colony and the brightest color. Separating and purifying for 8-10 generations by scribing method to obtain a eurotium cristatum strain which can tolerate inorganic selenium and efficiently convert the inorganic selenium into organic selenium, and preserving on PDA slope at 4 ℃ for later use.
The strain is identified by China industrial microorganism strain preservation management center (FMIC-QO 01-003 fungus multiphase identification and detection method is adopted for identification, QO-03-02 microorganism bacterium molecular biology identification operating procedures), and is determined as follows: eurotium Cristatum (Eurotium Cristatum).
Example 3 preservation of Eurotium cristatum selenium-enriched strains
Selecting the selenium-rich Eurotium cristatum lawn selected in example 2, culturing at 30 deg.C for 60 hr, adding 20mL sterile water, scraping with bamboo stick, and mixing to obtain spore with concentration of 2 × 106--2×108one/mL. Mixing the raw materials in a ratio of 1:1 volume ratio, adding PDA liquid culture medium containing 50% glycerol, and mixing. Pre-freezing at-20 deg.C, and storing at-80 deg.C for a long period with spore germination rate of above 90%.
Example 4 culture Medium screening of Eurotium cristatum selenium-enriched strains
Selecting four factors of carbon source glucose, organic nitrogen source corn steep liquor dry powder, inorganic nitrogen source ammonium sulfate and trace element selenium, wherein the glucose is selected from 10g/L, 25g/L and 35 g/L; selecting 2g/L, 5g/L and 8g/L of corn steep liquor dry powder; selecting 2g/L, 3.5g/L and 5g/L ammonium sulfate; the selenium element is selected from three levels of 10mg/L, 15mg/L and 20 mg/L; the results are shown in Table 1.
TABLE 1 orthogonal test factor horizon
As can be seen from the table, the main nutrients in the fermentation medium were confirmed by four-factor three-level orthogonal optimization: 25g/L of glucose; 5g/L of corn steep liquor dry powder; 5g/L of ammonium sulfate; the selenium enrichment capacity of Eurotium cristatum is strongest when the selenium element (sodium selenite) is 10 mg/L.
Determining the preferable composition of the fermentation medium of the eurotium cristatum selenium-rich strain: 20 g/L-30 g/L of cane sugar or glucose, 3 g/L-5 g/L of corn steep liquor dry powder, 3 g/L-6 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO44.0-5.5 g/L of selenium element 10 mg/L.
Example 5 fermentation culture of Eurotium cristatum selenium-enriched Strain
The lawn of the selenium-rich Eurotium cristatum strain selected in example 2 was made into bacterial suspension with normal saline to make spore concentration 2 × 106～2×108Sucking 1mL of the bacterial suspension, and inoculating into a liquid seed culture medium (100mL), wherein the liquid seed culture medium comprises the following components: sucrose 15g/L, peptone 2g/L, yeast extract 10g/L and KH2PO42g/L at 150rpm for 72h at 30 ℃ and inoculated with 200mL/500mL of the fermentation medium selected in example 4 at an inoculation ratio of 5% (v/v) for 72h at 150rpm at 30 ℃ with shaking. Filtering to collect mycelium, repeatedly washing mycelium with deionized water for 5-8 times until the filtrate is clear (more extracellular pigment is secreted during culture), filtering again to collect mycelium, and freeze drying to detect organic selenium content in Eurotium cristatum (Turcz.) Pilat up to 690 mg/kg.
Example 6 screening of Zinc-enriched wild strains of Eurotium cristatum
10g of Fuzhuan tea with brilliant yellow encysted shell is weighed from commercial Jingwei Fuzhuan tea in Xian, and is put into a mortar for rough grinding, and then is put into a conical flask with 3-5 glass beads and 90mL of sterile water. Mix by shaking at 150rpm for about 30 min. Taking 1mL spore mixed solution to perform 10-fold gradient dilution by using sterile water, and respectively selecting 10-3And 10-4Two gradients of spore suspension are sucked up by 0.1mL and spread on PDA solid medium containing 5mg/L zinc element, and then static culture is carried out for 60h at 30 ℃. Selecting large colonies and bright yellow colonies, streaking on PDA plate containing 5mg/L zinc element for further screening for 8-10 generations, performing slant culture on PDA, and storing at 4 deg.C.
Example 7 mutagenesis, domestication, isolation and purification of wild strains
The lawn selected in example 6 was eluted with sterile physiological saline and broken up with glass beads to adjust the concentration to 2X 106-2×108Sucking a certain amount of spore suspension into a culture dish (the liquid level is not more than 3mm), irradiating for 25min by using an ultraviolet lamp tube with the distance of 20W and 30cm, sucking 0.1mL of the spore suspension, inoculating into PDA solid plates containing inorganic zinc with different concentrations (0; 5; 10; 15; 20mg/L) and coating. Culturing at 30 deg.C for 60 h. Selecting a plurality of colonies with larger colonies and bright colors on zinc elements with different concentrations, streaking the colonies on a PDA solid plate containing 5mg/L inorganic zinc, comparing the size and appearance of the colonies again, and selecting the dominant bacterium with the largest colony and the brightest color. Separating and purifying for 8-10 generations by a scribing method to obtain an eurotium cristatum strain which can tolerate inorganic zinc and efficiently convert the inorganic zinc into organic zinc, and preserving on a PDA inclined plane at 4 ℃ for later use.
The strain is identified by China industrial microorganism strain preservation management center (FMIC-QO 01-003 fungus multiphase identification and detection method is adopted for identification, QO-03-02 microorganism bacterium molecular biology identification operating procedures), and is determined as follows: eurotium Cristatum (Eurotium Cristatum).
Example 8 preservation of Zinc-enriched Strain of Eurotium cristatum
Selecting zinc-rich Eurotium cristatum from example 7, culturing at 30 deg.C for 60 hr, adding 20mL sterile water, scraping with bamboo stick, and mixing to obtain spore with concentration of 2 × 106～2×108Then adding PDA liquid culture medium containing 50% glycerol in a volume ratio of 1:1, and mixing uniformly. Pre-freezing at-20 deg.C, and storing at-80 deg.C for a long period with spore germination rate of above 90%.
Example 9 Medium selection of Zinc-enriched Strain of Eurotium cristatum
Selecting four factors of carbon source glucose, organic nitrogen source corn steep liquor dry powder, inorganic nitrogen source ammonium sulfate and trace element selenium, wherein the glucose is selected from 10g/L, 25g/L and 35 g/L; selecting 3g/L, 5.5g/L and 8g/L of corn steep liquor dry powder; selecting 2g/L, 3.5g/L and 5g/L ammonium sulfate; the zinc element is selected from three levels of 5mg/L, 10mg/L and 20 mg/L; the results are shown in Table 2.
TABLE 2 orthogonal test factor horizon
As can be seen from the table, the main nutrients in the fermentation medium were confirmed by four-factor three-level orthogonal optimization: 25g/L of glucose; 5.5g/L of corn steep liquor dry powder; 5g/L of ammonium sulfate; when the zinc element (zinc sulfate) is 5mg/L, the zinc-rich capacity of eurotium cristatum is strongest.
Determining the preferable composition of the fermentation medium of the eurotium cristatum zinc-rich strain: 20 g/L-30 g/L of cane sugar or glucose, 3 g/L-6 g/L of corn steep liquor dry powder, 3 g/L-5 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO44.0-5.5 g/L of zinc element and 5mg/L of zinc element.
Example 10 fermentation culture of Zinc-enriched Strain of Eurotium cristatum
The fungal lawn of the zinc-rich Eurotium cristatum strain selected in example 7 was suspended in physiological saline to give a spore concentration of 2X 106～2×108Sucking 1mL of the bacterial suspension, and inoculating into a liquid seed culture medium (100mL), wherein the liquid seed culture medium comprises the following components: sucrose 15g/L, peptone 2g/L, yeast extract 10g/L and KH2PO42g/L at 150rpm for 72h at 30 ℃ and inoculated with 200mL/500mL of the fermentation medium selected in example 9 at an inoculation ratio of 5% (v/v) for 72h at 150rpm at 30 ℃ with shaking. Filtering to collect mycelium, repeatedly washing mycelium with deionized water for 5-8 times until the filtrate is clear (more extracellular pigment is secreted during culture), filtering again to collect mycelium, and freeze drying to detect the content of organic zinc in Eurotium cristatum cell to 380 mg/kg.
Example 11 selection of iron-rich wild strains of Eurotium cristatum
Weighing 10g Fuzhuan tea with brilliant yellow encysted shell from commercial Jingwei Fuzhuan tea in Xian, placing into a mortar for rough grinding, and then placing into 90mL sterile water with 3-5 glassGlass beads in a conical flask. Mix by shaking at 150rpm for about 30 min. Taking 1mL spore mixed solution to perform 10-fold gradient dilution by using sterile water, and respectively selecting 10-3And 10-4Two gradients of spore suspension, 0.1mL of spore suspension is sucked and spread on PDA solid medium containing 500mg/L iron element, and static culture is carried out for 60h at 30 ℃. And selecting colonies which are large in colony size and bright yellow in color, streaking the colonies on a PDA plate containing 500mg/L iron element, continuously screening for 8-10 generations, carrying out slant culture on the PDA plate, and preserving at 4 ℃.
EXAMPLE 12 mutagenesis, domestication, isolation and purification of wild strains
The lawn selected in example 11 was eluted with sterile physiological saline and broken up with glass beads to adjust the concentration to 2X 106-2×108Sucking a certain amount of spore suspension into a culture dish (the liquid level is not more than 3mm), irradiating for 25min by using an ultraviolet lamp tube with the distance of 20W and 30cm, sucking 0.1mL of the spore suspension, inoculating into PDA solid plates containing different concentrations of (0; 500; 1000; 1500; 2000mg/L) inorganic iron, and coating. Culturing at 30 deg.C for 60 h. Selecting a plurality of colonies with larger colonies and bright colors on iron elements with different concentrations, streaking the colonies on a PDA solid plate containing 500mg/L inorganic iron, comparing the size and appearance of the colonies again, and selecting the bacteria with the largest colonies and the brightest colors as dominant bacteria. Separating and purifying for 8-10 generations by a marking method to obtain an eurotium cristatum strain which can tolerate inorganic iron and efficiently convert the inorganic iron into organic iron, and preserving on a PDA inclined plane at 4 ℃ for later use.
The strain is identified by China industrial microorganism strain preservation management center (FMIC-QO 01-003 fungus multiphase identification and detection method is adopted for identification, QO-03-02 microorganism bacterium molecular biology identification operating procedures), and is determined as follows: eurotium Cristatum (Eurotium Cristatum).
Example 13 preservation of iron-rich Strain of Eurotium cristatum
Selecting the Fe-rich Eurotium cristatum from example 12, culturing at 30 deg.C for 60 hr, adding 20mL sterile water, scraping with bamboo stick, and mixing to obtain spore with concentration of 2 × 106～2×108one/mL, then adding PDA liquid culture containing 50% glycerol at a volume ratio of 1:1Nourishing the base and mixing evenly. Pre-freezing at-20 deg.C, and storing at-80 deg.C for a long period with spore germination rate of above 90%.
Example 14 media selection of iron-rich strains of Eurotium cristatum
Selecting four factors of carbon source glucose, organic nitrogen source corn steep liquor dry powder, inorganic nitrogen source ammonium sulfate and trace element selenium, wherein the glucose is selected from 10g/L, 25g/L and 35 g/L; selecting 3g/L, 5g/L and 8g/L of corn steep liquor dry powder; selecting 2g/L, 3.5g/L and 5g/L ammonium sulfate; the iron element is selected from three levels of 500mg/L, 1000mg/L and 1500 mg/L; . The specific results are shown in Table 3.
TABLE 3 orthogonal test factor table
As can be seen from the table, the main nutrients in the fermentation medium were confirmed by four-factor three-level orthogonal optimization: 25g/L of glucose; 5g/L of corn steep liquor dry powder; 5g/L of ammonium sulfate; the iron-rich capacity of eurotium cristatum is strongest when the iron element (ferrous sulfate) is 500 mg/L.
Determining the preferable composition of the fermentation medium of the eurotium cristatum iron-rich strain: 20 g/L-28 g/L of cane sugar or glucose, 3 g/L-6 g/L of corn steep liquor dry powder, 3 g/L-6 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO44.0 g/L-5.5 g/L of iron element 500 mg/L.
Example 15 fermentation culture of iron-rich Strain of Eurotium cristatum
The bacterial lawn of the Eurotium cristatum-rich strain selected in example 12 was suspended in physiological saline to give a spore concentration of 2X 106～2×108Sucking 1mL of the bacterial suspension, inoculating into a liquid seed culture medium (100mL), wherein the liquid seed culture medium comprises a packageComprises the following steps: sucrose 15g/L, peptone 2g/L, yeast extract 10g/L and KH2PO42g/L at 150rpm for 72h at 30 ℃ and inoculated with 200mL/500mL of the fermentation medium selected in example 14 at an inoculation ratio of 5% (v/v) for 72h at 150rpm at 30 ℃ with shaking. Filtering to collect mycelium, repeatedly washing mycelium with deionized water for 5-8 times until the filtrate is clear (more extracellular pigment is secreted during culture), filtering again to collect mycelium, and freeze drying to detect organic iron content of Eurotium cristatum at 6000 mg/kg.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (6)
1. A Eurotium cristatum strain is characterized by comprising a Eurotium cristatum XD-01 selenium-rich strain which is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 15395;
or the Eurotium cristatum zinc-rich strain is Eurotium cristatum XD-02, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 15396;
or the Eurotium cristatum iron-rich strain XD-03 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 15397.
2. A method for screening a eurotium cristatum wild strain is characterized in that,
the screening method of the eurotium cristatum selenium-rich wild strain comprises the following steps: weighing 10g of Fuzhuan tea with brilliant yellow cyst shell from commercial Jingwei Fuzhuan tea, grinding, adding 90mL of sterile water, and oscillating and mixing uniformly; diluting the mixture after shaking and mixing with sterile water by 10 times, respectively selecting 10-3And 10-4Coating the two gradients of diluent on PDA solid medium containing 10mg/L selenium element, and standing at 30 deg.C for culture60 h; selecting a colony which is large and bright yellow in color, scribing on a PDA flat plate containing 10mg/L of selenium element, continuously screening for 8-10 generations, and storing at 4 ℃ in a PDA slant culture medium for later use to obtain the eurotium cristatum selenium-rich wild strain;
the screening method of the zinc-rich wild strain of eurotium cristatum comprises the following steps: weighing 10g of Fuzhuan tea with brilliant yellow cyst shell from commercial Jingwei Fuzhuan tea, grinding, adding 90mL of sterile water, and oscillating and mixing uniformly; diluting the mixture after shaking and mixing with sterile water by 10 times, respectively selecting 10-3And 10-4Coating the two gradients of diluent on a PDA solid culture medium containing 5mg/L zinc element, and standing and culturing at 30 ℃ for 60 h; selecting a colony which is large and bright yellow in color, scribing on a PDA (personal digital Assistant) plate containing 5mg/L zinc element, continuously screening for 8-10 generations, and storing at 4 ℃ in a PDA slant culture medium for later use to obtain a zinc-rich wild strain of eurotium cristatum;
the screening method of the iron-rich wild strain of eurotium cristatum comprises the following steps: weighing 10g of Fuzhuan tea with brilliant yellow cyst shell from commercial Jingwei Fuzhuan tea, grinding, adding 90mL of sterile water, and oscillating and mixing uniformly; diluting the mixture after shaking and mixing with sterile water by 10 times, respectively selecting 10-3And 10-4Coating the two gradients of diluent on a PDA solid culture medium containing 500mg/L iron element, and standing and culturing at 30 ℃ for 60 h; and selecting a colony which is large and bright yellow in color, streaking the colony on a PDA plate containing 500mg/L iron element, continuously screening for 8-10 generations, and storing in a PDA slant culture medium at 4 ℃ for later use to obtain the iron-rich wild strain of the eurotium cristatum.
3. A method of acclimatizing the wild strain of Eurotium cristatum selected by the method according to claim 2,
the method for domesticating the eurotium cristatum selenium-rich wild strain comprises the following steps: resuspending the lawn of the selenium-rich wild strain of Eurotium cristatum stored in claim 2 in normal saline to a concentration of 2X 106～2×108Carrying out ultraviolet mutagenesis on the heavy suspension of spores/mL, and respectively inoculating the heavy suspension subjected to ultraviolet mutagenesis into PD (deoxyribonucleic acid) containing 0mg/L, 5mg/L, 10mg/L, 15mg/L, 20mg/L and 25mg/L of inorganic seleniumA, uniformly coating on a solid plate culture medium, and culturing for 60 hours at 30 ℃; selecting a colony with a larger colony and a bright color, streaking the colony on a PDA solid plate containing 10mg/L inorganic selenium, and comparing the growth conditions of different strains; selecting a colony with the largest colony and the brightest yellow color as a starting strain, separating and purifying for 8-10 generations, and storing at a 4 ℃ inclined plane to obtain the eurotium cristatum selenium-rich strain of claim 1;
the method for domesticating the zinc-rich wild strain of eurotium cristatum comprises the following steps: resuspending the lawn of the zinc-rich wild strain of Eurotium cristatum stored in claim 2 in physiological saline to a concentration of 2X 106～2×108Carrying out ultraviolet mutagenesis on the spore/mL heavy suspension, respectively inoculating the ultraviolet mutagenized heavy suspension to PDA solid plate culture media containing 0mg/L, 5mg/L, 10mg/L, 15mg/L and 20mg/L inorganic zinc, uniformly coating, and culturing at 30 ℃ for 60 h; selecting a colony which is large and bright in color, scribing the colony on a PDA solid plate containing 5mg/L inorganic zinc, and comparing the growth conditions of different strains; selecting a colony which is the largest and brightest and yellow as a starting strain, separating and purifying for 8-10 generations, and storing at a 4 ℃ inclined plane to obtain the eurotium cristatum zinc-rich strain of claim 1;
the method for domesticating the iron-rich wild strain of eurotium cristatum comprises the following steps: resuspending the lawn of the iron-rich wild strain of Eurotium cristatum stored in claim 2 in physiological saline to a concentration of 2X 106～2×108Carrying out ultraviolet mutagenesis on the spore/mL heavy suspension, respectively inoculating the ultraviolet mutagenized heavy suspension to PDA solid plate culture media containing 0mg/L, 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L inorganic iron, uniformly coating, and culturing at 30 ℃ for 60 h; selecting a colony with a larger colony and a bright color, streaking the colony on a PDA solid plate containing 500mg/L inorganic iron, and comparing the growth conditions of different strains; selecting a colony which is the largest and brightest yellow as a starting strain, separating and purifying for 8-10 generations, and storing at a 4 ℃ inclined plane to obtain the eurotium cristatum iron-rich strain of claim 1.
4. A process for the fermentation of the Eurotium cristatum strain of claim 1, wherein the process comprisesThe method comprises the following steps: resuspending the lawn of the Eurotium cristatum strain of claim 1 in physiological saline to give a concentration of 2X 106～2×108Inoculating the heavy suspension liquid into a liquid seed culture medium, performing shaking culture at 150rpm and 30 ℃ for 72h, inoculating the heavy suspension liquid into a fermentation culture medium according to the inoculation ratio of 5%, and performing shaking culture at 150rpm and 30 ℃ for 72 h; the liquid seed culture medium comprises the following components: sucrose 15g/L, peptone 2g/L, yeast extract 10g/L and KH2PO4 2g/L。
5. The fermentation process according to claim 4,
when the eurotium cristatum strain is the eurotium cristatum selenium-rich strain, the fermentation medium comprises the following components: 10 g/L-80 g/L of cane sugar or glucose, 2 g/L-15 g/L of corn steep liquor dry powder, 2 g/L-10 g/L of peptone, 2 g/L-10 g/L of yeast extract and KH2PO4 1g/L～8g/L，MgSO4·7H2O 1.0g/L～7.5g/L，(NH4)2SO42.5-8.0 g/L of selenium element 10 mg/L;
when the eurotium cristatum strain is an eurotium cristatum zinc-rich strain, the fermentation medium comprises the following components: 10 g/L-80 g/L of cane sugar or glucose, 2 g/L-15 g/L of corn steep liquor dry powder, 2 g/L-10 g/L of peptone, 2 g/L-10 g/L of yeast extract and KH2PO4 1g/L～8g/L，MgSO4·7H2O 1.0g/L～7.5g/L，(NH4)2SO42.5-8.5 g/L of zinc element 5 mg/L;
when the eurotium cristatum strain is an iron-rich eurotium cristatum strain, the fermentation medium comprises the following components: 10 g/L-80 g/L of cane sugar or glucose, 2 g/L-15 g/L of corn steep liquor dry powder, 2 g/L-10 g/L of peptone, 2 g/L-10 g/L of yeast extract and KH2PO4 1g/L～8g/L，MgSO4·7H2O 1.0g/L～7.5g/L，(NH4)2SO42.5-7.0 g/L of iron element 500 mg/L.
6. The fermentation process of claim 5,
when the eurotium cristatum strain is the eurotium cristatum selenium-rich strain, the fermentation medium comprises the following components: 20 g/L-30 g/L of cane sugar or glucose, 3 g/L-5 g/L of corn steep liquor dry powder, 3 g/L-6 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO44.0-5.5 g/L of selenium element 10 mg/L;
when the eurotium cristatum strain is an eurotium cristatum zinc-rich strain, the fermentation medium comprises the following components: 20 g/L-30 g/L of cane sugar or glucose, 3 g/L-6 g/L of corn steep liquor dry powder, 3 g/L-5 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO44.0-5.5 g/L of zinc element 5 mg/L;
when the eurotium cristatum strain is an iron-rich eurotium cristatum strain, the fermentation medium comprises the following components: 20 g/L-28 g/L of cane sugar or glucose, 3 g/L-6 g/L of corn steep liquor dry powder, 3 g/L-6 g/L of peptone, 3.0 g/L-5.5 g/L of yeast extract and KH2PO4 1.2g/L～2.5g/L，MgSO4·7H2O 1.5g/L～2.2g/L，(NH4)2SO44.0 g/L-5.5 g/L of iron element 500 mg/L.
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| JP2009029735A (en) * | 2007-07-26 | 2009-02-12 | Ikeda Shokken Kk | Prophylactic and therapeutic agent of hepatitis |
| CN106497806A (en) * | 2017-01-06 | 2017-03-15 | 青岛农业大学 | A kind of coronoid process dissipate capsule bacterium strain and its application |
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| CN116970665B (en) * | 2023-09-22 | 2023-12-19 | 凡可生物技术(广州)有限公司 | Extraction method and application of tremella aurantialba polysaccharide fermented by tremella aurantialba |
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