CN114276936B - Eurotium cristatum strain and application thereof - Google Patents
Eurotium cristatum strain and application thereof Download PDFInfo
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- CN114276936B CN114276936B CN202111587983.XA CN202111587983A CN114276936B CN 114276936 B CN114276936 B CN 114276936B CN 202111587983 A CN202111587983 A CN 202111587983A CN 114276936 B CN114276936 B CN 114276936B
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- 241001205401 Aspergillus cristatus Species 0.000 title claims abstract description 75
- 238000000855 fermentation Methods 0.000 claims abstract description 72
- 230000004151 fermentation Effects 0.000 claims abstract description 72
- 239000001052 yellow pigment Substances 0.000 claims abstract description 67
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 23
- 238000011218 seed culture Methods 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 235000013305 food Nutrition 0.000 claims description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 235000013312 flour Nutrition 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 239000004254 Ammonium phosphate Substances 0.000 claims description 3
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 3
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 3
- 239000003963 antioxidant agent Substances 0.000 claims description 3
- 230000003078 antioxidant effect Effects 0.000 claims description 3
- 239000002537 cosmetic Substances 0.000 claims description 3
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 3
- 230000036541 health Effects 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 230000001133 acceleration Effects 0.000 claims description 2
- 238000005273 aeration Methods 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000010563 solid-state fermentation Methods 0.000 claims description 2
- 235000013361 beverage Nutrition 0.000 claims 1
- 238000012262 fermentative production Methods 0.000 claims 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 description 21
- 239000000049 pigment Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 9
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- 238000002835 absorbance Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 6
- 235000010633 broth Nutrition 0.000 description 6
- 230000007760 free radical scavenging Effects 0.000 description 5
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- 235000013616 tea Nutrition 0.000 description 4
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000228347 Monascus <ascomycete fungus> Species 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000001103 potassium chloride Substances 0.000 description 3
- 235000011164 potassium chloride Nutrition 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 238000011160 research Methods 0.000 description 3
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- 239000008223 sterile water Substances 0.000 description 3
- 241000228212 Aspergillus Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
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- 239000013641 positive control Substances 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 229940123457 Free radical scavenger Drugs 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
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- 239000002775 capsule Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
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- 230000018109 developmental process Effects 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
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- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 235000019225 fermented tea Nutrition 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000037323 metabolic rate Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 239000006012 monoammonium phosphate Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
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- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000003711 photoprotective effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- -1 potassium ferricyanide Chemical compound 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 229960003351 prussian blue Drugs 0.000 description 1
- 239000013225 prussian blue Substances 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
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- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a Eurotium cristatum strain and application thereof. The invention provides a Eurotium cristatum EC-35 strain (Eurotium cristatum EC-35), wherein the Eurotium cristatum EC-35 strain (Eurotium cristatum EC-35) is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M2020230. The yellow pigment produced by fermentation of the Eurotium cristatum EC-35 strain provided by the invention has high yield.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a Eurotium cristatum strain and application thereof.
Background
Natural pigments are pigments obtained from natural sources, mainly pigments extracted from animal and plant tissues and microorganisms (culture), wherein edible pigments are widely used in food production and research as an important food additive after being developed and extracted.
The yellow pigment is an important natural pigment with nutrition and health care effects, can be used as a free radical scavenger and a photo-protecting agent in food, is more and more concerned by people, has the characteristics of good solubility, small influence on color tone by acid, alkali, salt, sugar and the like, and is heat-resistant and light-resistant, and is an ideal natural edible pigment. The yellow pigment accounts for 60% of the total pigment demand, and has wide development prospect. The traditional pigment synthesized by adopting an extraction method and oxidation has the defects of complex process, high cost and unfavorable mass industrialized production. Because of the characteristics of diversity of microorganism types, high efficiency of metabolic rate, rapidness of transformation, controllability of culture and the like, the microorganism can be used for producing pigment through large-scale culture and fermentation, compared with pigment in animals and plants, the pigment is not limited by resources, environment and existing conditions, and the method is a novel way for obtaining the available pigment with high efficiency, low cost, short production period and wide raw material source. In addition, along with the attention of people on the safety of using additives in life, the strain which naturally produces nontoxic and harmless food pigment becomes a research hot spot of many scholars, and considering the safety of the strain, only the yellow pigment produced by monascus strain can be applied in the fields of food, health care products and cosmetics at present.
The Eurotium cristatum (Eurotium cristatum) is a kind of natural probiotics produced in the process of Fuzhuan tea flowering, belonging to the genus Aspergillus, and is commonly called "Jinhua bacteria" because it produces golden-yellow closed capsule shell in the process of growth and propagation. Toxicology studies carried out in various animals show that the golden flower fungus genus has no toxic microorganism, and the fermentation product has good activities of antioxidation, bacteriostasis, intestinal flora balance regulation, immunity improvement, aging resistance, tumor resistance and the like. During the growth of Eurotium cristatum, a large amount of pigment substances, mainly yellow pigment, are usually produced by metabolism.
In the prior art, the strain with high yield of the yellow pigment is obtained by a genetic engineering means, so that hidden danger of food safety exists, and the application field is limited. The research report of producing the yellow pigment by fermenting monascus is more, and the yield of the yellow pigment is basically improved by optimizing the culture medium and the culture conditions; the excellent strain breeding of the high-yield monascus yellow pigment has food safety risk by a genetic engineering means, and the strain effect improved by an ultraviolet mutagenesis means is not obvious.
Disclosure of Invention
The invention provides a Eurotium cristatum capable of producing yellow pigment by fermentation, which belongs to aspergillus of traditional fermented tea and has no risk of food safety; the provided Eurotium cristatum has high yield in the fermentation production of the yellow pigment, and can solve the problems of complex process and high cost of the traditional yellow pigment extraction method.
In a first aspect, the invention provides a Eurotium cristatum EC-35 strain (Eurotium cristatum EC-35), wherein the Eurotium cristatum EC-35 strain (Eurotium cristatum EC-35) is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M2020230.
Preferably, the ITS gene sequence of the Eurotium cristatum EC-35 strain (Eurotium cristatum EC-35) is shown in SEQ ID NO. 1.
Preferably, the yellow pigment content in the fermentation broth obtained by fermenting the Eurotium cristatum EC-35 strain (Eurotium cristatum EC-35) is 200mg/L-450mg/L, or the yellow pigment content in the obtained solid fermentation product is 300mg/kg-400mg/kg.
In a second aspect, the invention provides an application of the Eurotium cristatum EC-35 strain in the fermentation production of yellow pigment.
In a third aspect, the present invention provides a method for fermentatively producing a yellow pigment, the method comprising the steps of: culturing the Eurotium cristatum EC-35 strain.
Preferably, the method comprises the steps of:
(1) Seed culture is carried out on the Eurotium cristatum EC-35 strain to obtain Eurotium cristatum EC-35 seed culture solution;
(2) And adding the Eurotium cristatum EC-35 seed culture solution into a fermentation medium, fermenting for 40-60h, and obtaining the yellow pigment.
Preferably, the temperature of the seed culture in step (1) is 27-29 ℃;
preferably, the seed culture is shaking culture,
preferably, the oscillation frequency is 150-200rpm;
preferably, the seed culture time is 50-70 hours.
Preferably, the fermentation in step (2) is a solid fermentation or a liquid fermentation;
preferably, the fermentation temperature in the step (2) is 30-35 ℃.
Preferably, the liquid fermentation speed is 150-220rpm;
preferably, the liquid fermentation aeration is 600L/h to 3000L/h.
Preferably, the liquid fermentation comprises a feed fermentation process.
Preferably, the feed fermentation medium comprises 15-30wt% sucrose, preferably the feed fermentation medium comprises 20wt% sucrose.
Preferably, the feed stream acceleration is in the range of 50ml/h to 750ml/h.
Preferably, the fermentation medium in step (2) is a solid medium or a liquid medium.
Preferably, the solid culture medium contains, by mass, 45% -55% of water, 0.5% -2% of glucose, 1% -4% of ammonium sulfate, 0.2% -0.75% of monopotassium phosphate, 0-0.25% of magnesium sulfate and the balance of bran and corn flour, wherein the mass ratio of the bran to the corn flour is 2-5:1.
Preferably, the liquid culture medium contains, per 15-20L of water, 150-600 g of yeast extract powder, 15-50g of ammonium phosphate and ZnSO 4 0.3-10g，FeSO 4 ·7H 2 O 0.3-10g，KCl 0-100g，MgSO 4 ·7H 2 O10-100g。
Preferably, the inoculation amount in the step (2) is 5-15% by weight.
In a fourth aspect, the invention provides a ferment produced by fermenting the Eurotium cristatum EC-35 strain.
Preferably, the yellow pigment content in the ferment is 200mg/L-450mg/L or 300mg/kg-400mg/kg.
The Eurotium cristatum EC-35 strain provided by the invention can be fermented by liquid fermentation or solid fermentation to obtain a fermented product, and in a specific embodiment provided by the invention, the Eurotium cristatum EC-35 strain is cultured by liquid fermentation, and the yellow pigment content in the obtained fermented product is 200mg/L-450mg/L. In a specific embodiment provided by the invention, the Eurotium cristatum EC-35 strain is cultured by solid fermentation, and the obtained fermentation product has the content of the yellow pigment of 300mg/kg-400mg/kg.
In a fifth aspect, the invention provides application of the Eurotium cristatum EC-35 strain or the yellow pigment prepared by the preparation method or the ferment in preparing antioxidant foods, drinks, health-care products and cosmetics.
The Eurotium cristatum EC-35 strain provided by the invention has the advantages of high growth speed, easiness in culture, high yield of yellow pigment, capability of shortening the production time and improving the production efficiency. The yellow pigment produced by fermenting the Eurotium cristatum EC-35 strain provided by the invention has strong reducing capability and the effect of scavenging free radicals.
Information on preservation of strains
The Eurotium cristatum EC-35 strain (Eurotium cristatum EC-35) is preserved in China Center for Type Culture Collection (CCTCC) at 22 days of 6 months in 2020, and the preservation number is CCTCC NO: m2020230, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: 027-68754052.
Drawings
FIG. 1 shows the reducing power of the yellow pigment solutions obtained in examples 2 to 4;
FIG. 2 shows the radical scavenging ability of the yellow pigment solutions obtained in examples 2-4.
Detailed Description
The present invention will be further described in detail with reference to specific examples for a better understanding of the present invention by those skilled in the art. It should be understood by those skilled in the art that this should not be construed as limiting the scope of the claims. It should also be noted that the reagents and apparatus of the invention are commercially available without any particular explanation.
The method for measuring the content of the yellow pigment in the invention comprises the following steps:
0.025g of yellow pigment refined product with constant weight is taken, dissolved by 95% ethanol and fixed to 50mL, and 500mg/L yellow pigment mother solution is prepared. The mother liquor is diluted to a proper concentration, scanned at 300-600nm, and the optimal detection wavelength of the yellow pigment is determined to be 405nm. The yellow pigment mother liquor was diluted to 25, 50, 75, 100, 125, 250 and 500mg/L gradient, and absorbance was measured at 405nm using 95% ethanol as a blank. And (3) taking the concentration of the yellow pigment as an abscissa (X, mg/L) and the light absorption value (A) as an ordinate to make a straight line, and calculating a standard curve regression equation of the yellow pigment. Substituting the absorbance A of the sample to be detected at 405nm into a regression equation to obtain the content of the yellow pigment.
Specific sources of reagents used in the present invention are listed in Table 1 below.
TABLE 1 manufacturer of reagents used in the present invention
| Reagents/apparatus | Purity/model | Manufacturer(s) |
| Yeast extract powder | 99％ | Angel Yeast Co.,Ltd. |
| Glucose | 99％ | Yishui Daida pharmaceutical Co Ltd |
| Corn flour | The solid content is more than or equal to 42 | Yichanghua city biological fermentation raw materials Co., ltd |
| Ammonium sulfate | 99％ | JIANGSU KOLOD FOOD INGREDIENTS Co.,Ltd. |
| Magnesium sulfate | 99％ | LAIZHOU CITY LAIYU CHEMICAL Co.,Ltd. |
| Monopotassium phosphate | 99％ | JIANGSU ZIDONG FOOD Co.,Ltd. |
| Bran | The crude protein is more than or equal to 11 percent | Henan Feitian agricultural Co Ltd |
| Monoammonium phosphate | 99％ | Hebei Runfu technology Co.Ltd |
| Zinc sulfate | 99％ | Hebei Runfu technology Co.Ltd |
| Ferrous sulfate | 99％ | Sichuan Kagbai Biotech Co.Ltd |
| Potassium chloride | 99％ | JIANGSU KOLOD FOOD INGREDIENTS Co.,Ltd. |
| Ultraviolet spectrophotometer | SP-754 | SHANGHAI SPECTRUM INSTRUMENTS Co.,Ltd. |
| Cradle | ZHWY-211D | Shanghai Zhi Cheng technology Co Ltd |
| High-speed centrifuge | Minispin | eppendorf |
| PCR instrument | C1000Touch | Bio-rad |
| 50L full-automatic fermentation tank | PUS-50L(A)-50L | SHANGHAI GUOQIANG BIOENGINEERING EQUIPMENT Co.,Ltd. |
| Biochemical incubator | SPX250BSH-II | Shanghai CIMO Medical Instrument Co.,Ltd. |
EXAMPLE 1 obtaining of Eurotium cristatum EC-35 Strain
Xiang Yifu brick tea was purchased from Tianmao APP Xiang Yi tea flagship (https:// xingyicy. Tmall. Com/.
(1) Weighing 25g of Fuzhuan tea sample, soaking in 225ml of 0.85% sterile physiological saline, adding a proper amount of sterilized glass beads, and placing on a constant temperature oscillator for oscillating for 20min at a speed of 120 r/min.
(2) Diluting with multiple ratio dilution method to obtain diluted solution 10 and diluted solution 10 respectively 2 、10 3 、10 4 Multiple times.
(3) 1mL of 10 was pipetted 4 The diluted solution is coated on a PDA flat plate, and is placed in a constant temperature incubator for culturing for 3d at 28 ℃, colonies grow, the diameter of each colony is 11-16mm, the colony is round and compact in structure, the newly generated hyphae at the edge of each colony grow vigorously, the center of each colony is golden yellow to dark brown, a small amount of brown exudates exist, and the back of each colony is dark brown. Meets the colony characteristics of the Eurotium cristatum.
(4) And (3) in a sterile state, scraping golden yellow colonies with dominant quantity and better growth vigor by an inoculating needle, inoculating on a PDA culture medium plate, culturing for 3d at 28 ℃, and repeating the transferring for 3 times, wherein the purified Eurotium cristatum is obtained.
(5) Inoculating the obtained Eurotium cristatum into PDB for culturing at 28 ℃ and 150rpm for 2d to obtain mycelium pellets;
(6) Extracting total DNA of the strain by adopting a liquid nitrogen grinding method, carrying out PCR amplification on the conserved sequence by using an ITS universal primer (ITS 1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCCGCTTATTGATATGC), and sequencing the obtained product, wherein a sequencing company is October Dingsheng biotechnology (Wuhan) Co. The ITS gene sequence is shown as SEQ ID NO. 1.
SEQ ID NO.1 sequence is as follows:
gctacggagtgcgggctctgggtcacctcccatccgtgtctatctgtaccctgttgcttcggcgtggccacggcccgccggagactaacatttgaacgctgtctgaagtttgcagtctgagtttttagttaaacaatcgttaaaactttcaacaacggatctcttggttccggcatcgatgaagaacgcagcgaaatgcgataattaatgtgaattgcagaattcagtgaatcatcgagtctttgaacgcacattgcgccccctggtattccggggggcatgcctgtccgagcgtcattgctgccctcaagcacggcttgtgtgttgggcttccgtccctggcaacggggacgggcccaaaaggcagtggcggcaccatgtctggtcctcgagcgtatggggctttgtcacccgctcccgtaggtccagctggcagctagcctcgcaaccaatctttttaaccaggttgacctcggatcaggtagggatacccgctgaacttaagcatatcaaaggcgggggaggaaa
the sequencing results were aligned in NCBI to determine their species. Finally, the strain is confirmed to be the Eurotium cristatum, namely the Eurotium cristatum EC-35 strain. The Eurotium cristatum EC-35 strain (Eurotium cristatum EC-35) is preserved in China Center for Type Culture Collection (CCTCC) at 22 days of 6 months in 2020, and the preservation number is CCTCC NO: m2020230.
Example 2 Eurotium cristatum EC-35 shake flask fermentation to produce yellow pigment
(1) Activating strains: the Eurotium cristatum EC-35 spore liquid is marked on the PDA inclined plane, and cultured for 80 hours at 29 ℃.
(2) Spore suspension preparation: adding sterile water into the activated eurotium cristatum inclined plane, scraping spores by using a scraper, and preparing spore suspension.
(3) Inoculating: the spore suspension was inoculated into a 250ml Erlenmeyer flask at 1% inoculum level, with a 100ml liquid PDB medium loading.
(4) Shaking and fermenting: culturing at 29 deg.C at 180rpm for 55 hr until the fermentation liquid turns bright yellow.
(5) And (3) measuring the yellow pigment content of the fermentation liquor: the yellow pigment content in the fermentation broth obtained by shaking flask fermentation is 220mg/L.
Example 3 Eurotium cristatum EC-35 deep liquid fermentation high yield yellow pigment
(1) Activating strains: the sporangium coronarium cryopreserved spore liquid is scratched on the PDA inclined plane, and is cultured for 80 hours at 28 ℃.
(2) Spore suspension preparation: adding sterile water into the activated eurotium cristatum inclined plane, scraping spores by using a scraper, and preparing spore suspension.
(3) Seed culture: the spore suspension was inoculated into 1L of the treated seed culture solution and shake-cultured at 28℃and 160rpm for 55 hours.
(4) And (3) fermenting a bottom material: 18L of water, 300g of yeast extract powder, 30g of ammonium phosphate and ZnSO 4 3g，FeSO 4 ·7H 2 O1g，KCl 30g，MgSO 4 ·7H 2 O 30g。
(5) Fermentation: the seed culture solution is added into a 50L fermentation tank according to 5 percent, 8L to 12L total of 20 percent sucrose is fed into the fermentation tank according to 50ml/h to 750ml/h, the rotating speed is 150rpm to 220rpm, 600L/h to 3000L/h is ventilated, and fermentation is carried out for 2 days at 30 ℃ to obtain the Eurotium cristatum fermentation solution with high content of yellow pigment.
(6) And (3) measuring the yellow pigment content of the fermentation liquor: the content of the yellow pigment in the fermentation liquor obtained by submerged liquid fermentation is 450mg/L.
EXAMPLE 4 preparation of crude yellow pigment extract by solid State fermentation of Eurotium cristatum EC-35
(1) Activating strains: the sporangium coronarium cryopreserved spore liquid is scratched on PDA inclined plane and cultured for 3d at 28 ℃.
(2) Spore suspension preparation: adding sterile water into the activated eurotium cristatum inclined plane, scraping spores by using a scraper, and preparing spore suspension.
(3) Seed culture: the spore suspension was inoculated into 0.5L of the treated seed culture solution and shake-cultured at 28℃and 160rpm for 2d.
(4) Fermentation: the solid culture medium comprises bran: 700g of corn flour mixture with the mass ratio of 3:1 is added with 700g of water, 10.5g of glucose, 21g of ammonium sulfate, 3.5g of monopotassium phosphate, 1.75g of magnesium sulfate, and fermentation is carried out for 2d at the temperature of 30-35 ℃.
(5) Preparing a crude extract of the yellow pigment: grinding the solid culture, extracting with 15 times of petroleum ether Soxhlet for 1 hr to obtain yellow pigment extractive solution, filtering, concentrating, and naturally volatilizing and drying the concentrate in ventilated place to obtain yellow pigment powder. The content of the yellow pigment in the solid fermentation product is detected to be 400mg/kg.
Example 5 determination of the reducing force of crude extract of Eurotium cristatum EC-35 yellow pigment
The reducing power of the crude extract of yellow pigment was measured by Prussian blue method.
(1) The yellow pigment-rich fermentation broths obtained in examples 2 and 3 were diluted respectively, and the yellow pigment crude extract obtained in example 4 was dissolved to prepare yellow pigment solutions of 10mg/L, 20mg/L, 40mg/L, 80mg/L, 120mg/L and 160mg/L for use.
(2) 1ml of yellow pigment solution with different concentrations is respectively taken, and 2.5ml of phosphate buffer with pH of 6.6 and 1% of potassium ferricyanide (K) by mass fraction are added 3 Fe(CN) 6 ) 2.5ml of solution, mixing, standing at 50 ℃ for 20min, adding 2.5ml of trichloroacetic acid solution with the mass fraction of 10%, mixing, taking 2.5ml of mixed solution, adding 2.5ml of distilled water and 2.5ml of ferric chloride with the mass fraction of 0.1%, mixing uniformly, standing for 10min, and measuring absorbance at 700 nm. The higher the absorbance, the better the oxidation resistance and the stronger the reducing power. The reducing power of yellow pigment solutions with different concentrations was analyzed by taking VE as a positive control.
(3) The test results show that: the reducing power of the yellow pigment solution with the concentration of 10mg/L is equivalent to VE, the reducing power is obviously enhanced along with the increase of the concentration of the yellow pigment, and the reducing power of the yellow pigment solution is obviously stronger than that of VE with the same concentration.
Example 6 determination of free radical scavenging Capacity of Eurotium cristatum EC-35 fermentation broth
Adopts DPPH free radical scavenging method
(1) The yellow pigment-rich fermentation broths obtained in examples 2 and 3 were treated respectively, and the crude yellow pigment extract obtained in example 4 was dissolved to prepare 25mg/L, 50mg/L, 75mg/L, 100mg/L, 150mg/L, 200mg/L yellow pigment solutions for use.
(2) Respectively taking 1ml of fermentation liquor with different dilutions, adding 3ml of DPPH standard solution with the concentration of 50mg/L, uniformly mixing, standing at room temperature for 30min, taking 95% ethanol as a blank control, and measuring the absorbance value A1 at 517 nm; ethanol is used for replacing sample liquid to carry out reaction, and the absorbance at 517nm is marked as A0; ethanol was used instead of DPPH standard solution to conduct the reaction, and the absorbance at 517nm was designated as A2. And (3) taking VE as a positive control, and analyzing the DPPH free radical scavenging capacity of fermentation solutions with different concentrations.
The DPPH clearance of the fermentation broth is calculated according to the following formula:
dpph. clearance (%) = [1- (A1-A2)/A0 ]. Times.100
(3) The test results show that: when the concentration of the yellow pigment is within 100mg/L, the DPPH free radical scavenging capacity of the fermentation liquor is rapidly enhanced along with the increase of the concentration, and when the concentration of the yellow pigment is higher than 100mg/L, the DPPH free radical scavenging rate of the fermentation liquor is stabilized at about 94 percent. And the clearance rate of the Eurotium cristatum fermentation liquor to DPPH free radicals is obviously higher than VE.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Hubei Angel biological group Co., ltd
<120> a Eurotium cristatum strain and application thereof
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 528
<212> DNA
<213> Eurotium cristatum (Eurotium cristatum)
<400> 1
gctacggagt gcgggctctg ggtcacctcc catccgtgtc tatctgtacc ctgttgcttc 60
ggcgtggcca cggcccgccg gagactaaca tttgaacgct gtctgaagtt tgcagtctga 120
gtttttagtt aaacaatcgt taaaactttc aacaacggat ctcttggttc cggcatcgat 180
gaagaacgca gcgaaatgcg ataattaatg tgaattgcag aattcagtga atcatcgagt 240
ctttgaacgc acattgcgcc ccctggtatt ccggggggca tgcctgtccg agcgtcattg 300
ctgccctcaa gcacggcttg tgtgttgggc ttccgtccct ggcaacgggg acgggcccaa 360
aaggcagtgg cggcaccatg tctggtcctc gagcgtatgg ggctttgtca cccgctcccg 420
taggtccagc tggcagctag cctcgcaacc aatcttttta accaggttga cctcggatca 480
ggtagggata cccgctgaac ttaagcatat caaaggcggg ggaggaaa 528
Claims (26)
1. Eurotium cristatum strainEurotium cristatum) The EC-35 strain is characterized in that the Eurotium cristatum is ]Eurotium cristatum) The EC-35 strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M2020230.
2. The Eurotium cristatum of claim 1Eurotium cristatum) The EC-35 strain is characterized in that the Eurotium cristatum EC-35 strainEurotium cristatum The ITS gene sequence of EC-35) is shown in SEQ ID NO. 1.
3. The Eurotium cristatum of claim 1 or 2Eurotium cristatum) The EC-35 strain is characterized in that the Eurotium cristatum is ]Eurotium cristatum) The yellow pigment content in the fermentation liquid obtained by fermenting the EC-35 strain is 200mg/L-450mg/L, or the yellow pigment content in the obtained solid state fermentation product is 300mg/kg-400mg/kg.
4. Use of a strain of eurotium cristatum EC-35 according to any one of claims 1 to 3 for the fermentative production of a yellow pigment.
5. A method for producing a yellow pigment by fermentation, comprising the steps of: culturing the Eurotium cristatum EC-35 strain according to any one of claims 1-3.
6. The method according to claim 5, comprising the steps of:
(1) Seed culturing the Eurotium cristatum EC-35 strain according to any one of claims 1-3 to obtain Eurotium cristatum EC-35 seed culture solution;
(2) And adding the Eurotium cristatum EC-35 seed culture solution into a fermentation medium, fermenting for 40-60h, and obtaining the yellow pigment.
7. The method according to claim 6, wherein the temperature of the seed culture in step (1) is 27-29 ℃.
8. The method of claim 6, wherein the seed culture in step (1) is shaking culture.
9. The method according to claim 8, wherein the frequency of the shaking culture in the step (1) is 150 to 200rpm.
10. The method of claim 6, wherein the seed culture time in step (1) is 50-70 hours.
11. The method according to claim 6, wherein the fermentation in step (2) is a solid fermentation or a liquid fermentation.
12. The method according to claim 6, wherein the fermentation temperature in step (2) is 30-35 ℃.
13. The method according to claim 11, wherein the liquid fermentation speed is 150-220rpm.
14. The method of claim 11, wherein the liquid fermentation aeration is 600L/h to 3000L/h.
15. The method of claim 11, wherein the liquid fermentation comprises a fed-batch fermentation process.
16. The method of claim 15, wherein the feed fermentation medium comprises 15-30wt% sucrose during the feed fermentation.
17. The method of claim 15, wherein the feed fermentation medium comprises 20wt% sucrose during the feed fermentation.
18. The method of claim 15, wherein the feed stream acceleration during the feed fermentation is 50ml/h to 750ml/h.
19. The method of claim 6, wherein the fermentation medium in step (2) is a solid medium or a liquid medium.
20. The method of claim 19, wherein the solid medium comprises, by mass, 45% -55% water, 0.5% -2% glucose, 1% -4% ammonium sulfate, 0.2% -0.75% potassium dihydrogen phosphate, 0-0.25% magnesium sulfate, and the balance bran and corn flour, wherein the mass ratio of bran to corn flour is 2-5:1.
21. The method of claim 19, wherein the liquid medium comprises, per 15-20L of water, 150-600 g yeast extract, 15-50g ammonium phosphate, znSO 4 0.3-10g，FeSO 4 ·7H 2 O 0.3-10g，KCl 0-100g，MgSO 4 ·7H 2 O 10-100g。
22. The method according to any one of claims 6 to 21, wherein the inoculum size of eurotium cristatum EC-35 in step (2) is 5% -15% by weight.
23. A ferment produced by fermentation of the eurotium cristatum EC-35 strain of any one of claims 1-3.
24. A ferment according to claim 23, wherein the ferment has a yellow pigment content of 200mg/L-450mg/L or 300mg/kg-400mg/kg.
25. Use of a strain of eurotium cristatum EC-35 according to any one of claims 1 to 3 or a yellow pigment prepared by a method of preparation according to any one of claims 6 to 22 or a ferment according to claim 23 or 24 for the preparation of antioxidant foods, health care products and cosmetics.
26. Use of a xanthogen prepared by a eurotium cristatum EC-35 strain according to any one of claims 1 to 3 or a preparation method according to any one of claims 6 to 22 or a ferment according to claim 23 or 24 for preparing an antioxidant beverage.
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