CN114214216B - Selenium-enriched yeast strain and application thereof - Google Patents
Selenium-enriched yeast strain and application thereof Download PDFInfo
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- CN114214216B CN114214216B CN202111183007.8A CN202111183007A CN114214216B CN 114214216 B CN114214216 B CN 114214216B CN 202111183007 A CN202111183007 A CN 202111183007A CN 114214216 B CN114214216 B CN 114214216B
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- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 136
- 239000011669 selenium Substances 0.000 title claims abstract description 136
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 135
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims description 75
- 229940091258 selenium supplement Drugs 0.000 claims description 128
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 74
- 239000001963 growth medium Substances 0.000 claims description 53
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 51
- 229960001471 sodium selenite Drugs 0.000 claims description 51
- 235000015921 sodium selenite Nutrition 0.000 claims description 51
- 239000011781 sodium selenite Substances 0.000 claims description 51
- 238000012258 culturing Methods 0.000 claims description 42
- 238000000855 fermentation Methods 0.000 claims description 25
- 230000004151 fermentation Effects 0.000 claims description 25
- 238000009630 liquid culture Methods 0.000 claims description 21
- 239000000843 powder Substances 0.000 claims description 21
- 230000001580 bacterial effect Effects 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 20
- 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 17
- 239000001888 Peptone Substances 0.000 claims description 16
- 108010080698 Peptones Proteins 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 235000019319 peptone Nutrition 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 11
- 238000001694 spray drying Methods 0.000 claims description 11
- 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 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- QDLHCMPXEPAAMD-UHFFFAOYSA-N wortmannin Natural products COCC1OC(=O)C2=COC(C3=O)=C2C1(C)C1=C3C2CCC(=O)C2(C)CC1OC(C)=O QDLHCMPXEPAAMD-UHFFFAOYSA-N 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 7
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 230000000813 microbial effect Effects 0.000 claims description 2
- 230000008021 deposition Effects 0.000 claims 1
- 241000235342 Saccharomycetes Species 0.000 abstract description 9
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 235000013305 food Nutrition 0.000 abstract description 4
- 238000004321 preservation Methods 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 3
- 239000003674 animal food additive Substances 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
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- 238000012545 processing Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 28
- 229940041514 candida albicans extract Drugs 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000012138 yeast extract Substances 0.000 description 10
- 238000002703 mutagenesis Methods 0.000 description 9
- 231100000350 mutagenesis Toxicity 0.000 description 9
- GGLZPLKKBSSKCX-YFKPBYRVSA-N L-ethionine Chemical compound CCSCC[C@H](N)C(O)=O GGLZPLKKBSSKCX-YFKPBYRVSA-N 0.000 description 6
- 238000004806 packaging method and process Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
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- 238000007865 diluting Methods 0.000 description 4
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- 235000011389 fruit/vegetable juice Nutrition 0.000 description 4
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- 239000000725 suspension Substances 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
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- 210000004027 cell Anatomy 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000006481 glucose medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000006587 Glutathione peroxidase Human genes 0.000 description 1
- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 208000019926 Keshan disease Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010039921 Selenium deficiency Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
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- 239000008121 dextrose Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
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- -1 potassium ferricyanide Chemical compound 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 125000003748 selenium group Chemical group *[Se]* 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
- A23L31/10—Yeasts or derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/14—Yeasts or derivatives thereof
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/16—Inorganic salts, minerals or trace elements
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen
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Abstract
The invention relates to a saccharomycete processing technology, and discloses a selenium-enriched saccharomycete strain and application thereof, wherein the selenium-enriched saccharomycete strain is preserved in the microorganism strain preservation center of Guangdong province, the preservation date is 2021, 9 and 9 days, the preservation number is GDMCC No.61733, the organic selenium conversion rate of the saccharomycete selenium prepared by the production of the saccharomycete strain is as high as 93 percent or more, the selenium content is greater than 7600 mug/g, and the produced saccharomycete selenium can improve the digestion utilization rate of the organic selenium when applied to food, health care products or feed additives and is beneficial to the growth of organisms.
Description
Technical Field
The invention relates to a yeast processing technology, in particular to a selenium-enriched yeast strain and application thereof.
Background
Selenium is an essential trace element for human and animals, is an important component of various enzymes, is an important component of glutathione peroxidase, and protects a biological membrane from damage through oxidation reduction, and maintains normal functions of cells. Selenium also has immunity enhancing, cardiovascular protecting, growth promoting, antitumor, toxic materials removing, and metabolism regulating effects. The local diseases caused by selenium deficiency in China include keshan disease and large bone joint disease.
The yeast has high selenium-rich capability and can convert inorganic selenium into organic selenium, so that the selenium-rich yeast is widely used as a source for supplementing trace element selenium in the industries of foods, health-care products and feeds. The selenium-enriched yeast is a microbial fermentation product obtained by adding inorganic selenium in the form of sodium selenite and the like in the process of culturing the yeast, converting the inorganic selenium into an organic selenium form and enriching the organic selenium in the selenium-enriched yeast.
In recent years, research on selenium-enriched yeasts at home and abroad is relatively more, but the selenium-enriched yeasts can be rarely produced in large scale, and the main problems are as follows: (1) The selenium content is lower, generally below 1000mg/kg, and high at 1500mg/kg. (2) The organic selenium content is low, some of the organic selenium is only simple yeast and selenium adsorption, and the methionine selenium content is lower. (3) Some are not suitable for large scale production, have poor reproducibility, and some are productive but have poor efficiency.
Disclosure of Invention
Therefore, the application of the selenium-enriched yeast strain is needed to solve the problems of low selenium content of the existing selenium-enriched yeast.
In order to achieve the above purpose, the invention provides a selenium-enriched yeast strain which is preserved in the microorganism strain collection center of Guangdong province, wherein the preservation date is 2021, 9 and 9 days, and the preservation number is GDMCC No.61733.
Further, the conversion rate of the organic selenium reaches more than 93 percent.
Selenium yeast is prepared from the selenium-enriched yeast strain, wherein the selenium content is greater than 7600 mug/g, and the organic selenium accounts for more than 93 percent:
the preparation method of the yeast selenium comprises the following steps:
(1) Inoculating the yeast strain into a YEPD culture medium for activation, and culturing until the logarithmic phase to obtain an activated bacterial liquid;
(2) Inoculating the activated bacterial liquid into a YEPD culture medium according to the proportion of 1-10%, and carrying out shaking culture;
(3) When the OD value of the bacterial liquid is 0.6, adding sodium selenite solution with the volume amount of 0.1% of the YEPD culture medium, and then adding sodium selenite solution with the volume amount of 0.2% of the YEPD culture medium every 1 hour until the total volume of the sodium selenite solution is 0.7% -1.1% of the volume of the YEPD culture medium;
(4) Continuing to culture for no more than 36 hours;
(5) Centrifuging and freeze-drying to obtain the yeast selenium.
Further, the concentration of the sodium selenite solution is 300-800mg/ml.
Further, the bacterial liquid after the activation in the step (2) is inoculated in the YEPD culture medium according to 4 percent, and the total volume of the sodium selenite solution in the step (3) is 0.9 percent of the volume of the YEPD culture medium.
The preparation method of the yeast selenium comprises the following steps:
(1) Inoculating the yeast strain of claim 1 onto selenium-enriched YEPD solid medium, and culturing at 25-37 ℃ for 24-48 h;
(2) Inoculating selenium-enriched yeast single colony of YEPD solid culture medium into shake flask liquid culture medium, culturing at 25-37deg.C for 24-48 hr, shake flask liquid culture medium comprising: malt extract powder with weight fraction of 2-20%, sodium selenite with weight fraction of 10-300ug/ml, peptone with weight fraction of 5-25mg/ml, shake flask liquid culture medium pH value of 4-7;
(3) Adding the shake flask liquid culture solution into a culture medium with a seed tank, and culturing at 25-37 ℃ for 16-48 h, wherein the seed tank culture medium comprises 5-25% of sugar and 10-300ug/ml sodium selenite by weight, and the pH value of the seed tank culture medium is 4-7, wherein the sugar is wort, glucose, sucrose or mixed sugar;
(4) Adding the seed tank culture solution into a fermentation tank for culturing, wherein the fermentation tank comprises 5-25% of sugar, 10-300ug/ml sodium selenite and 1-5% of ammonium hydrogen phosphate, and pH value is 4-7, wherein the sugar is wort, glucose, sucrose or mixed sugar, a nutrient source is fed in during fermentation, and sodium selenite solution is fed in batches;
(5) Spray drying at 100-180deg.C to obtain yeast selenium.
8. The method of preparing selenium yeast of claim 7, wherein in step (3) and step (4), the sugar is one or more of wort, glucose, or sucrose.
The application of the yeast selenium in food, health care products or feed additives.
The technical scheme has the following beneficial effects:
1. the selenium-enriched yeast obtained by the preparation method has the selenium content of more than 7000ppm, wherein the organic selenium content can reach 93-99%, and the invention provides two preparation methods of the yeast selenium, one is prepared in a laboratory, the other is prepared in a large-scale fermentation tank, and both preparation methods can effectively prepare the yeast selenium with high selenium content.
2. In the invention, the activated strain is screened and purified in the selenium-enriched culture medium with multiple concentration gradients, so that the yeast strain capable of growing in the high-selenium culture medium is selected.
3. The yeast strain provided by the invention uses a sodium selenite resistance plate and an ethionine resistance plate to screen out yeast with double resistance, and the selenium-enriched amount of the double-resistant yeast is better than that of the monoclonal antibody yeast.
Drawings
The growth curve of single colonies screened in the example of FIG. 1.
Detailed Description
In order to describe the technical content, constructional features, achieved objects and effects of the technical solution in detail, the following description is made in connection with the specific embodiments in conjunction with the accompanying drawings.
Example 1
The embodiment discloses a selenium-enriched yeast strain, and the mutagenesis screening method comprises the following steps:
(1) Commercial Strain activation
Inoculating strain Saccharomyces cerevisiae (accession number: CICC 33033) into 100ml YEPD at 28deg.C and 200rpm for about 24-48 hr, and culturing to logarithmic phase;
yeast extract peptone glucose medium (YEPD medium) component content: 20g of glucose, 20g of peptone and 10g of yeast extract powder, distilled water is added to 1L, and the mixture is autoclaved at the natural pH and 105 ℃ for 30min.
(2) Isolation and identification of strains
Diluting the activated bacterial liquid into 10 -1 、10 -2 、10 -3 、10 -4 And (3) respectively absorbing 200 mu L of concentration gradient, coating the concentration gradient on a selenium-enriched culture medium, performing 3 parallel coatings on each concentration, culturing the culture medium in a constant temperature incubator at 28 ℃ for 48 hours, streaking and purifying to obtain single colonies, performing PCR amplification identification on the single colonies, and accurately and then placing the single colonies in a refrigerator at-20 ℃ for later use.
The selenium-enriched culture medium comprises the following components: 20g of glucose, 20g of peptone, 10g of yeast extract powder, xmg of sodium selenite, distilled water to 1L, natural pH and autoclaving at 121 ℃ for 20min.
Xmg sodium selenite preparation gradient concentration selenium-rich culture medium, in this embodiment, the selenium content of the selenium-rich culture medium is 2×10 in sequence -4 molSe 4+ /L、4×10 -4 molSe 4+ /L、8× 10 -4 molSe 4+ /L、1×10 -3 molSe 4+ /L、2×10 - 3 molSe 4+ /L。
And (3) streaking and purifying the single colony on the selenium-enriched culture medium with gradient concentration, selecting a strain capable of growing in the selenium-enriched culture medium with higher concentration, and carrying out PCR amplification identification.
(3) Determination of Strain growth Curve
Inoculating the activated bacteria liquid into 100mL YEPD culture medium according to 4%, drawing a growth curve of saccharomycetes by adopting a light absorption photometry method, shake culturing at 28 ℃ at 180r/min, taking the curved line as a starting point, measuring the number of bacteria by using a blood cell counting plate, sampling every 4 hours (taking 2mL by using a pipette) in the culturing process, measuring and recording the absorbance at an OD600nm, measuring for 48 hours in total, and drawing a strain growth curve, wherein the growth curve is shown in figure 1.
(4) Preparation of mutant Strain suspension
Culturing activated strain to logarithmic phase, collecting 5ml of bacterial liquid 4800r/min, centrifuging for 5min, washing with sterile water for 2 times, discarding supernatant, adding sterile water again, shaking on vortex mixer to obtain bacterial suspension, measuring the number of bacterial strain in the bacterial suspension with blood cell counting plate, gradient diluting with sterile water, and keeping the number of bacterial strain at 10 6 About, the prepared bacterial suspension is used for mutagenesis.
(5) DES mutagenesis
Adding 0.8% DES, shaking at 25deg.C and 180r/min for 80min, adding 6% sodium thiosulfate solution, stopping reaction, centrifuging, washing with sterile water for 2 times, adding 5mLYEPD culture medium, and culturing overnight.
(6) Screening of sodium selenite resistant plates
Centrifugally collecting thallus, washing 2 times, diluting and coating 2X 10 -3 molSe 4+ L sodium selenite resistant plates.
(7) Screening of ethionine resistant plates
Colonies growing on sodium selenite resistant plates were subjected to liquid culture, DES mutagenesis was again performed as described above in step (5), cultured overnight, and after centrifugation to collect the cells, which were washed 2 times with water and plated on 3.0g/L ethionine resistant plates.
Ethionine resistant plates: the ethionine solution filter sterilized with the filter was added to the sterilized YNB medium. The selenium-enriched amount of the yeast is better than that of the yeast.
(8) UV mutagenesis and screening of sodium selenite resistant plates
The colony plate growing on the ethionine plate is streaked on the sodium selenite plate, and after the colony plate grows out, the plate is placed in an 8wUV light source for mutagenesis, the mutagenesis distance is 20cm, and the mutagenesis distance is 90s. And (3) continuously culturing the plate after mutagenesis, picking out a monoclonal with large growth in light orange yellow, and inoculating the monoclonal into a YEPD culture medium for expansion culture.
The screened selenium-enriched yeast (Saccharomyces cerevisiae) is preserved in the microorganism strain collection of Guangdong province at 2021, 9 and 9, address: building 59 of Guangzhou Mr. first, china, no. 100 university, with deposit number GDMCC No.61733.
Selenium yeast was prepared using the strain of example 1. The selenium content of the finished product yeast is more than 7600 mug/g, and the organic selenium accounts for more than 93 percent.
Example 2
A preparation method of yeast selenium comprises the following steps:
(1) Inoculating the yeast strain of the example 1 into a YEPD culture medium for activation, and culturing until the logarithmic phase to obtain an activated bacterial liquid;
(2) Inoculating the activated bacterial liquid into 100ml of YEPD culture medium according to a proportion, shake culturing,
(3) When the OD value of the bacterial liquid is 0.6, adding 100 mu L of sodium selenite solution, and then adding 200 mu L of sodium selenite solution every 2 hours;
(4) Continuously culturing for 24 hours;
(5) Centrifuging and freeze-drying to obtain the yeast selenium.
Wherein the yeast extract peptone glucose medium (yeast extract peptone dextrose medium, YEPD): 20g of glucose, 20g of peptone, 10g of yeast extract powder, 1L of distilled water, naturally p H, autoclaving at 105 ℃ for 30min,
the inoculation proportion of the bacterial liquid, the concentration of the sodium selenite solution and the total volume of the sodium selenite solution are designed into a four-level orthogonal test with 3 factors.
The selenium-enriched yeast products of each orthogonal test group are respectively measured for selenium content, and the selenium content measurement specifically comprises the following steps:
determination of selenium content in yeast: accurately weighing 0.1g of selenium-rich product, placing the selenium-rich product into a flask, adding 15mL of digestive juice, cooling, heating to a volume of about 2.5mL, adding 2.5mL of hydrochloric acid, heating to smoke, cooling, transferring the digestive juice into a 50mL volumetric flask, fixing the volume by water, diluting the digestive juice by 50-100 times, taking 20mL of final digestive juice in the 50mL volumetric flask, adding 8mL of hydrochloric acid, adding 2mL of potassium ferricyanide solution, fixing the volume by water, and measuring the selenium concentration by an atomic fluorescence spectrometry.
(2) Organic selenium content:
accurately weighing 0.1g of saccharomycete selenium-rich product, putting the saccharomycete selenium-rich product into a dialysis bag, fully dialyzing for 24 hours, removing inorganic selenium, taking substances in the dialysis bag, and measuring the selenium content according to the method;
(3) comprehensively considering the selenium enrichment and the organic selenium conversion rate to determine the optimal selenium concentration of the culture medium, wherein,
total selenium content μg/g = total selenium μg/dry weight g of product
Organic selenium conversion% = organic selenium content μg/g +.total selenium content μg/g x 100%.
The results obtained are shown in Table 1.
TABLE 1 orthogonal experiments and determination of Yeast selenium content
As shown in Table 1, the yeast prepared by the method has higher selenium content, the selenium content is more than 7000ppm, and compared with the single-resistant yeast strain, the organic selenium conversion rate of the double-resistant yeast strain is also greatly improved, and the maximum organic selenium conversion rate can be up to 99.5 percent.
Example 3
A preparation method of yeast selenium comprises the following steps: the method comprises the steps of flat bacterial strain culture, liquid seed culture, seed tank fermentation culture, fermentation tank fermentation, spray drying and packaging, and specifically comprises the following steps:
1) Culturing the flat plate strain: the selenium-enriched yeast strain is inoculated on a selenium-enriched YEPD solid culture medium, 20mg/ml glucose, 20mg/ml peptone, 10mg/ml yeast extract powder, 10ug/ml sodium selenite and 20mg/ml agar are cultured for 24-48h at 25-37 ℃.
2) Culturing liquid seeds: inoculating selenium-enriched yeast single colony in a flat plate into 100-1000ml of shake flask liquid culture medium, wherein the shake flask liquid culture medium is 10% of malt extract powder by weight, 10ug/ml of sodium selenite, 5-25mg/ml of peptone, pH value is 4-7, and culturing at 25-37 ℃ for 24-48 h.
3) Fermentation culture in a seed tank: adding the shake flask liquid culture solution into 15-40L seed tank containing 10% sugar, 10ug/ml sodium selenite, and pH 4-7, wherein sugar is wort, and culturing at 25-37deg.C for 16-48 hr.
4) Fermenting in a fermentation tank: adding the seed tank culture solution into 50-200L fermentation tank containing 10 wt% sugar, 10ug/ml sodium selenite, 3 wt% ammonium hydrogen phosphate and pH 4-7, culturing at 25-37deg.C for 16-48 hr, adding nutrition source, and adding sodium selenite solution in batches.
5) Spray drying: spray drying at 100-180deg.C to obtain powder.
6) And (3) packaging: the selenium-enriched yeast powder is packaged in various forms, and after mixing and dilution, the total selenium content is 100-1000mg/Kg, and the organic selenium content is 98% of the total selenium content by weight.
Example 4
1) Culturing the flat plate strain: the selenium-enriched yeast strain is inoculated on a selenium-enriched YEPD solid culture medium, 20mg/ml glucose, 20mg/ml peptone, 10mg/ml yeast extract powder, 100ug/ml sodium selenite and 20mg/ml agar are cultured for 24-48h at 25-37 ℃.
2) Culturing liquid seeds: inoculating selenium-enriched yeast single colony in a flat plate into 100-1000ml of shake flask liquid culture medium, wherein the shake flask liquid culture medium is 2% of malt extract powder by weight, 100ug/ml of sodium selenite, 5-25mg/ml of peptone, pH value is 4-7, and culturing at 25-37 ℃ for 24-48 h.
3) Fermentation culture in a seed tank: adding the shake flask liquid culture solution into 15-40L seed tank containing 5-25% sugar, 100ug/ml sodium selenite, pH 4-7, wherein the sugar is wort and glucose, and culturing at 25-37deg.C for 16-48 hr.
4) Fermenting in a fermentation tank: adding the seed tank culture solution into 50-200L fermentation tank containing 5 wt% sugar, 100ug/ml sodium selenite, 1-5 wt% ammonium hydrogen phosphate and pH 4-7, culturing at 25-37deg.C for 16-48 hr, adding nutrition source, and adding sodium selenite solution in batches.
5) Spray drying: spray drying at 100-180deg.C to obtain powder.
6) And (3) packaging: the selenium-enriched yeast powder is packaged in various forms, and after mixing and dilution, the total selenium content is 1000-4000mg/Kg, and the organic selenium content is 97% of the total selenium content by weight.
Example 5
1) Culturing the flat plate strain: the selenium-enriched yeast strain is inoculated on a selenium-enriched YEPD solid culture medium, 20mg/ml glucose, 20mg/ml peptone, 10mg/ml yeast extract powder, 300ug/ml sodium selenite and 20mg/ml agar are cultured for 24-48h at 25-37 ℃.
2) Culturing liquid seeds: inoculating selenium-enriched yeast single colony in a flat plate into 100-1000ml of shake flask liquid culture medium, wherein the shake flask liquid culture medium is 20% of malt extract powder by weight, 300ug/ml of sodium selenite, 5-25mg/ml of peptone, pH value is 4-7, and culturing at 25-37 ℃ for 24-48 h.
3) Fermentation culture in a seed tank: adding the shake flask liquid culture solution into 15-40L seed tank containing 25% sugar, 300ug/ml sodium selenite, and pH 4-7, wherein the sugar is wort, glucose and sucrose, and culturing at 25-37deg.C for 16-48 hr.
4) Fermenting in a fermentation tank: adding the seed tank culture solution into 50-200L fermentation tank containing 25 wt% of sugar, 300ug/ml of sodium selenite, 5 wt% of ammonium hydrogen phosphate and pH value of 4-7, wherein the sugar is wort, glucose and sucrose, culturing at 25-37 ℃ for 16-48 h, feeding nutrition source during fermentation, and adding sodium selenite solution in batches.
5) Spray drying: spray drying at 100-180deg.C to obtain powder.
6) And (3) packaging: the selenium-enriched yeast powder is packaged in various forms, and the total selenium content is 5000-9000mg/Kg after mixing and dilution or direct packaging, and the organic selenium content is 95% by weight of the total selenium content.
Example 6
1) Culturing the flat plate strain: the selenium-enriched yeast strain is inoculated on a selenium-enriched YEPD solid culture medium, 20mg/ml glucose, 20mg/ml peptone, 10mg/ml yeast extract powder, 100ug/ml sodium selenite and 20mg/ml agar are cultured for 24-48h at 25-37 ℃.
2) Culturing liquid seeds: inoculating selenium-enriched yeast single colony in a flat plate into 100-1000ml of shake flask liquid culture medium, wherein the shake flask liquid culture medium is 20% of malt extract powder by weight, 100ug/ml of sodium selenite, 5-25mg/ml of peptone, pH value is 4-7, and culturing at 25-37 ℃ for 24-48 h.
3) Fermentation culture in a seed tank: adding the shake flask liquid culture solution into 15-40L seed tank containing 5-25% sugar, 100ug/ml sodium selenite, and pH 4-7, wherein the sugar is wort, glucose, sucrose, and culturing at 25-37deg.C for 16-48 hr.
4) Fermenting in a fermentation tank: adding the seed tank culture solution into 50-200L fermentation tank containing 25 wt% sugar, 100ug/ml sodium selenite, 3 wt% ammonium hydrogen phosphate, 0.5-3 wt% ammonia water and pH 4-7, culturing at 25-37 deg.c for 16-48 hr, adding nutrient source, and adding sodium selenite solution in batches.
5) Spray drying: spray drying at 100-180deg.C to obtain powder.
6) And (3) packaging: the selenium-enriched yeast powder is packaged in various forms, and after mixing and dilution, the total selenium content is 1000-4000mg/Kg, and the organic selenium content is 95% of the total selenium content by weight.
The yeast selenium prepared by the embodiment of the invention is applied to medicines, foods and feeds, can be easily digested and absorbed, and improves the digestion and utilization rate of organic selenium.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the statement "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article or terminal device comprising the element. Further, herein, "greater than," "less than," "exceeding," and the like are understood to not include the present number; "above", "below", "within" and the like are understood to include this number.
While the embodiments have been described above, other variations and modifications will occur to those skilled in the art once the basic inventive concepts are known, and it is therefore intended that the foregoing description and drawings illustrate only embodiments of the invention and not limit the scope of the invention, and it is therefore intended that the invention not be limited to the specific embodiments described, but that the invention may be practiced with their equivalent structures or with their equivalent processes or with their use directly or indirectly in other related fields.
Claims (4)
1. A selenium-enriched yeast strain Saccharomyces cerevisiae HJC 01001, which is deposited with the cantonese collection of microbial strains, wherein the date of deposition is 2021, 9 and 9, and the accession number is GDMCC No.61733.
2. The preparation method of the yeast selenium is characterized by comprising the following steps:
(1) Inoculating the selenium-enriched yeast strain of claim 1 into a YEPD culture medium for activation, and culturing until the logarithmic phase to obtain an activated bacterial liquid;
(2) Inoculating the activated bacterial liquid into a YEPD culture medium according to the proportion of 1-10%, and carrying out shaking culture;
(3) When the OD value of the bacterial liquid is 0.6, adding sodium selenite solution with the volume amount of 0.1% of the YEPD culture medium, and then adding sodium selenite solution with the volume amount of 0.2% of the YEPD culture medium every 1 hour until the total volume of the sodium selenite solution is 0.7% -1.1% of the volume of the YEPD culture medium; the concentration of the sodium selenite solution is 300-800 mg/ml;
(4) Continuing to culture for no more than 36 hours;
(5) Centrifuging and freeze-drying to obtain the yeast selenium.
3. The method for preparing yeast selenium according to claim 2, wherein the activated bacterial liquid in the step (2) is inoculated in the YEPD medium according to 4%, and the total volume of the sodium selenite solution in the step (3) is 0.9% of the volume of the YEPD medium.
4. The preparation method of the yeast selenium is characterized by comprising the following steps:
(1) Inoculating the selenium-enriched yeast strain of claim 1 onto a selenium-enriched YEPD solid medium, and culturing at 25-37 ℃ for 24-48 h;
(2) Inoculating selenium-enriched yeast single colony of YEPD solid culture medium into shake flask liquid culture medium, culturing at 25-37deg.C for 24-48 hr, shake flask liquid culture medium comprising: malt extract powder with weight fraction of 2-20%, sodium selenite with weight fraction of 10-300ug/ml, peptone with weight fraction of 5-25mg/ml, shake flask liquid culture medium pH value of 4-7;
(3) Adding the shake flask liquid culture solution into a seed tank culture medium, and culturing at 25-37 ℃ for 16-48 h, wherein the seed tank culture medium comprises 5-25% of sugar and 10-300ug/ml sodium selenite by weight, and the pH value of the seed tank culture medium is 4-7, wherein the sugar is one or more of wort, glucose or sucrose;
(4) Adding the seed tank culture solution into a fermentation tank for culturing, and culturing at 25-37 ℃ to obtain 16-48 h fermentation tank, wherein the fermentation tank comprises 5-25% of sugar, 10-300ug/ml of sodium selenite, 1-5% of ammonium hydrogen phosphate and pH value of 4-7, the sugar is one or more of wort, glucose or sucrose, a nutrient source is fed in during fermentation, and sodium selenite solution is fed in batches;
(5) Spray drying at 100-180deg.C to obtain yeast selenium.
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