WO2022143113A1 - Levure riche en chrome ayant une absorption élevée de chrome, son procédé de préparation et son application - Google Patents

Levure riche en chrome ayant une absorption élevée de chrome, son procédé de préparation et son application Download PDF

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WO2022143113A1
WO2022143113A1 PCT/CN2021/137249 CN2021137249W WO2022143113A1 WO 2022143113 A1 WO2022143113 A1 WO 2022143113A1 CN 2021137249 W CN2021137249 W CN 2021137249W WO 2022143113 A1 WO2022143113 A1 WO 2022143113A1
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chromium
yeast
saccharomyces cerevisiae
enriched yeast
source
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肖明华
翟明
俞灿
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安琪酵母股份有限公司
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K10/00Animal feeding-stuffs
    • A23K10/10Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/14Fungi; Culture media therefor
    • C12N1/16Yeasts; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/14Fungi; Culture media therefor
    • C12N1/16Yeasts; Culture media therefor
    • C12N1/18Baker's yeast; Brewer's yeast
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/36Adaptation or attenuation of cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/645Fungi ; Processes using fungi
    • C12R2001/85Saccharomyces
    • C12R2001/865Saccharomyces cerevisiae
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to the technical field of microorganism application, in particular to a chromium-enriched yeast with high chromium absorption rate and a production method and application thereof.
  • Chromium yeast is one of the sources of chromium nutritional supplements designated by the State Food and Drug Administration.
  • Yeast is the earliest and most widely used pure natural nutritional microorganism, and it is also a natural nutritional treasure and an ideal biological carrier.
  • Yeast chromium is to cultivate yeast cells in a medium containing trivalent chromium, and convert inorganic chromium into organic chromium through biotransformation, so as to improve the absorption and utilization rate of chromium in the body, reduce its toxic and side effects, and better play its role. Regulates blood sugar, lipid-lowering and cholesterol-lowering effects.
  • the existing products mainly use Saccharomyces cerevisiae as a strain, add carbon, nitrogen and phosphorus sources, and use inorganic chromium as a chromium source. After fermentation and culture, the chromium-enriched yeast yeast milk is finally obtained, and then the product is obtained by drying.
  • inorganic chromium has the following defects: the absorption rate is low, generally less than 10%; it has no biological activity and needs to be converted into biologically active GTF chromium to regulate metabolism. effect.
  • the body of patients with diabetes and coronary heart disease basically does not have this transformation ability; the harmful effects of inorganic chromium have been found in animal experiments.
  • the problem of the prior art solved by the present invention is as follows: the existing products mainly use Saccharomyces cerevisiae as a strain, add carbon, nitrogen and phosphorus sources, and use inorganic chromium as a chromium source. product.
  • inorganic chromium there are more or less inorganic chromium in the existing products, the absorption rate of inorganic chromium is low, and it needs metabolic regulation to play a role, and some patients cannot even use it.
  • inorganic chromium is easy to produce and trace amount of hexavalent chromium in the process product of chromium source, and trace amount of hexavalent chromium also exists in the chromium-enriched yeast in the present technology. Hexavalent chromium is harmful to human and animal health.
  • the invention adopts organic chromium as the chromium source, eliminates the inorganic chromium source, the main raw material of the present invention (the chromium source is organic chromium) from the source, and uses the organic chromium raw material to produce the production process of chromium-enriched yeast, and there is no inorganic chromium in the finished product. , and the product has higher chromium content and chromium absorption rate.
  • the present invention proposes the following technical solutions.
  • the invention provides a chromium-enriched yeast, wherein the mass content of chromium in the chromium-enriched yeast is greater than 38,000 ppm, the organic chromium ratio in the chromium-enriched yeast is greater than 97% by mass, and the chromium-enriched yeast has a chromium absorption rate by mass. >50%, there is no hexavalent chromium in the chromium-rich yeast.
  • the mass content of chromium in the chromium-enriched yeast is greater than 40,000 ppm
  • the organic chromium ratio in the chromium-enriched yeast is greater than 95% by mass
  • the chromium-enriched yeast is more than 60% by mass in the absorption rate of chromium.
  • the present invention also provides the preparation method of the above-mentioned chromium-enriched yeast, characterized in that it comprises the following steps:
  • Step 1 Amplify Saccharomyces cerevisiae to provide Saccharomyces cerevisiae seeds required for fermentation;
  • Step 2 transfer the Saccharomyces cerevisiae seeds obtained in step 1 into a 50L fermentation tank to continue fermentation, and when the wet weight of Saccharomyces cerevisiae reaches 60-90g/L, add chromium source;
  • Step 3 growing the Saccharomyces cerevisiae obtained in Step 2, when the wet weight of the yeast reaches 120-140g/L, continue fermentation to obtain yeast milk;
  • Step 4 separate the yeast milk obtained in step 3, and then dry to obtain the chromium-enriched yeast;
  • the chromium source is organic chromium
  • the organic chromium is selected from one or more of chromium nicotinate, chromium picolinate and chromium methionine.
  • the fermentation temperature is 25-35° C., preferably, the dissolved oxygen is less than or equal to 30 ⁇ mol/L, and more preferably, the concentration of ethanol is less than or equal to 0.3%.
  • the chromium source is added in flow, preferably, the amount of the chromium source is 2-4mol, the chromium The source is all added within 2-10h.
  • the pH is controlled to be 3.9-4.5. 18h.
  • the pH is raised to 5-8 within 1-4 hours before the end of the fermentation, until the end of the fermentation, to obtain yeast milk.
  • Saccharomyces cerevisiae described in step 1 is selected from Saccharomyces cerevisiae Z1.3 (Saccharomyces cerevisiae HANSEN Z1.3), Saccharomyces cerevisiae Z2.1 (Saccharomyces cerevisiae HANSEN Z2.1) and Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae HANSEN Z2.1) One or more of FX-2).
  • the components of the medium used in the culture in steps 1-3 contain one or more of carbon sources, nitrogen sources, phosphorus sources and trace elements.
  • the carbon source is selected from molasses and/or glucose
  • the nitrogen source is selected from one or more of ammonium sulfate, peptone, yeast extract and aqueous ammonia
  • the phosphorus source is selected from dihydrogen phosphate Potassium
  • the trace element is selected from zinc sulfate and/or magnesium sulfate.
  • the concentration of the molasses is 20-40% by mass, and the concentration of the glucose is 20-40% by mass.
  • the purity of the potassium dihydrogen phosphate is greater than 96%, the purity of the ammonium sulfate is greater than 99%, preferably, the purity of the zinc sulfate is greater than 99%, and the purity of the magnesium sulfate is greater than 99% %.
  • the carbon source, the nitrogen source and the phosphorus source are added by means of feed addition.
  • the present invention also provides a chromium-enriched yeast, which is characterized in that it is produced by the above-mentioned method.
  • the present invention also provides the application of the chromium-enriched yeast in the field of animal husbandry, preferably the application in the field of feed or feed additive.
  • the invention adopts organic chromium to replace traditional inorganic chromium as the chromium source, the absorption rate of chromium by yeast is greatly improved, and the absorption rate of the shake flask is increased from 1% to 2% to more than 60%;
  • the total chromium of the chromium-enriched yeast can easily break through the upper limit of the traditional chromium-enriched yeast total chromium of 8000ppm, the minimum content is also above 38000ppm, can reach above 40000ppm, as high as 43096ppm, the mass content of organic chromium is more than 97%, and even can reach 98% The above; there is inorganic chromium that is difficult to wash in traditional chromium-enriched yeast.
  • the new chromium-enriched yeast eliminates inorganic chromium from the source, and what remains is organic chromium, which is more conducive to human absorption and animal absorption; organic chromium sources are used to produce rich Chromium yeast can greatly reduce the chromium content in production wastewater and reduce environmental pressure; there is no hexavalent chromium in the chromium-enriched yeast produced by this process.
  • the strain Saccharomyces cerevisiae Z1.3 (Saccharomyces cerevisiae Hansen Z1.3) used in the present invention has been biologically preserved in the China Center for Type Culture Collection (CCTCC, Wuhan University, zip code 430072) on October 25, 2005, The deposit number is CCTCC M 205125, and this strain has been recorded in the patent publication with the application number CN200610066365.X.
  • Saccharomyces cerevisiae FX-2 Saccharomyces cerevisiae FX-2 used in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on August 1, 2016, and the deposit number is CCTCC NO: M2016418, and the deposit address is China. Wuhan. Wuhan University, Postal Code: 430072; Tel: (027)-68754052, the strain has been recorded in the patent publication with the application number CN201611141122.8.
  • the strain Saccharomyces cerevisiae Z2.1 (Saccharomyces cerevisiae Hansen Z2.1) used in the present invention was preserved in the China Center for Type Culture Collection on October 25, 2005, the strain preservation number is CCTCC NO: M205127, and the preservation address is: China . Wuhan. Wuhan University, Postcode: 430072, Tel: (027) 68752319, the strain has been recorded in the patent publication with the application number CN201710522840.8.
  • the invention provides a chromium-enriched yeast, wherein the mass content of chromium in the chromium-enriched yeast is greater than 38,000 ppm, the organic chromium ratio in the chromium-enriched yeast is greater than 97% by mass, and the chromium-enriched yeast has a chromium absorption rate by mass. >50%, there is no hexavalent chromium in the chromium-rich yeast.
  • the mass content of chromium in the chromium-enriched yeast is greater than 40,000 ppm
  • the organic chromium ratio in the chromium-enriched yeast is greater than 95% by mass
  • the chromium-enriched yeast is more than 60% by mass in the absorption rate of chromium.
  • the present invention also provides the preparation method of the above-mentioned chromium-enriched yeast, characterized in that it comprises the following steps:
  • Step 1 Amplify Saccharomyces cerevisiae to provide Saccharomyces cerevisiae seeds required for fermentation;
  • Step 2 transfer the Saccharomyces cerevisiae seeds obtained in step 1 into a 50L fermentation tank to continue fermentation, and when the wet weight of Saccharomyces cerevisiae reaches 60-90g/L, add chromium source;
  • Step 3 growing the Saccharomyces cerevisiae obtained in step 2, when the wet weight of the yeast reaches 120-140 g/L, continue fermentation to obtain yeast milk;
  • Step 4 separate the yeast milk obtained in step 3, and then dry to obtain the chromium-enriched yeast;
  • the chromium source is organic chromium
  • the organic chromium is selected from one or more of chromium nicotinate, chromium picolinate and chromium methionine.
  • the present invention also provides a chromium-enriched yeast, which is characterized in that it is produced by the above-mentioned method.
  • the present invention also provides the application of the chromium-enriched yeast in the field of animal husbandry, preferably the application in the field of feed or feed additive.
  • the mass content of chromium in the chromium-enriched yeast is 0-40000 ppm, that is, the amount of chromium absorption is calculated by mass, and the chromium content per gram of yeast is 0-0.04 g;
  • Chromium absorption rate total chromium mass content in yeast (product) / mass content of chromium in the added chromium source;
  • organic chromium ratio intracellular organic chromium mass content/yeast (product) total chromium mass content
  • the chromium content in the chromium-enriched yeast of the present invention refers to the chromium mass content of beneficial trivalent chromium, and at the same time, hexavalent chromium cannot exist.
  • Chromium source chromium nicotinate or chromium methionine or chromium pyridinecarboxylate
  • the yeast milk is dusted by a dusting and drying tower to obtain a chromium-enriched yeast product.
  • Step 1 the Saccharomyces cerevisiae Chromium-enriched Saccharomyces cerevisiae Z1.3 (Saccharomyces cerevisiae HANSEN Z1.3) is picked a ring to carry out shake-flask culture—the step-by-step amplification culture of seed culture, to provide the Saccharomyces cerevisiae seeds required for fermentation;
  • the components of the culture medium in the shake flask are: 100 g of sucrose, 20 g of yeast extract, 1 g of magnesium sulfate, 1 g of potassium dihydrogen phosphate, pH 4.8, cultured at 31°C for 24 hours, 5L shake flask with a liquid volume of 1L, and the shaker rotation speed 180rpm/ min;
  • Step 2 transfer the Saccharomyces cerevisiae seeds of step 1 gained into 50L fermentor, control fermentation temperature to be 35 °C, dissolved oxygen 30 ⁇ mol/L, after 2 hours of fermentation, flow the carbon source (molasses concentration is 30%) of 4000g.
  • Step 3 add 8000g carbon source (molasses concentration is 30%), 200g nitrogen source (ammonium sulfate), 200g phosphorus source (potassium dihydrogen phosphate), control the ethanol concentration to be 0.07% ⁇ 0.03%, for the growth of Saccharomyces cerevisiae , when the wet weight of the yeast reaches 120g/L, continue to maintain the pH at 3.9, control the dissolved oxygen at 30 ⁇ mol/L by ventilation and stirring, continue the fermentation for 9h, start 4h before the end of the fermentation, and gradually increase the pH to 6 with ammonia water , until the end of fermentation, to obtain yeast milk;
  • Step 4 The chromium-enriched yeast product A is obtained by spray-drying the yeast milk obtained in step 3 after separating impurities.
  • the chromium content of the prepared chromium-enriched yeast product A is 40563 ppm; the organic chromium ratio is 98.39%; the chromium absorption rate is 66.45%, and the prepared Chromium-enriched yeast product A has no hexavalent chromium.
  • the steps are the same as those in Example 1, except that the chromium source fed in the step 2 is 3 mol of chromium methionine.
  • the chromium content of the prepared chromium-enriched yeast product B was 38451 ppm; the organic chromium ratio was 97.78%; the chromium absorption rate was 61.03%, and the prepared chromium-enriched yeast product B had no hexavalent chromium.
  • the steps are the same as in Example 1, except that the chromium source streamed in the step 2 is 3mol chromium picolinate.
  • the prepared chromium-enriched yeast product C has a chromium content of 43096 ppm, an organic chromium ratio of 98.78%, and a chromium absorption rate of 68.4%.
  • the prepared chromium-enriched yeast product C has no hexavalent chromium.
  • Example 2 The rest of the experimental steps are the same as in Example 1, except that when the wet weight of Saccharomyces cerevisiae reaches 90 g/L in step 2, after adjusting the pH to 3.9, the chromium source (0.01 mol/L chromium nicotinate for a total of 3 mol , the chromium source is all added within 10h, and then the chromium-enriched yeast product D is obtained.
  • the prepared chromium-enriched yeast product D has a chromium content of 41,025 ppm, an organic chromium ratio of 98.78%, and a chromium absorption rate of 65.18%. There is no hexavalent chromium in the chromium-enriched yeast product D.
  • step 3 when the wet weight of the yeast reaches 140 g/L, the pH is controlled to 3.9, the dissolved oxygen is controlled to 30 ⁇ mol/L by ventilation and stirring, and the fermentation is continued for 18 h.
  • the pH was raised to 6 within 4 hours before the end of the fermentation until the end of the fermentation to obtain yeast milk, and then the chromium-enriched yeast product E was obtained.
  • the prepared chromium-enriched yeast product E has a chromium content of 40127 ppm, an organic chromium ratio of 98.67%, and a chromium absorption rate of 63.76%.
  • the prepared chromium-enriched yeast product E has no hexavalent chromium.
  • step 2 the fermentation temperature of the Saccharomyces cerevisiae seeds obtained in step 1 is controlled to be 25°C; afterward, the chromium-enriched yeast product F is obtained.
  • the prepared chromium-enriched yeast product F has a chromium content of 41357 ppm, an organic chromium ratio of 98.88%, and a chromium absorption rate of 65.71%.
  • the prepared chromium-enriched yeast product F has no hexavalent chromium.
  • step 2 when the wet weight of Saccharomyces cerevisiae reaches 60 g/L, after adjusting the pH to 4.5, maintain the pH at 4.5 (the pH drops and then adds ammonia water to raise the pH to 4.5). ); and then the chromium-enriched yeast product G was obtained.
  • the prepared chromium-enriched yeast product G has a chromium content of 40357 ppm, an organic chromium ratio of 98.38%, and a chromium absorption rate of 64.12%.
  • the prepared chromium-enriched yeast product G has no hexavalent chromium.
  • Example 2 The rest of the experimental conditions are the same as those in Example 1, except that the chromium source fed in in step 2 is chromium chloride; the chromium-enriched yeast product D1 is then obtained.
  • the chromium content of the prepared chromium-enriched yeast product D1 was 2044.78ppm; the organic chromium ratio was 89.71%; the chromium absorption rate was 2.22%; and the hexavalent chromium was 0.933ppm.
  • step 2 chromium nicotinate is not added, and in step 3, when the wet weight reaches 120 g/L, 3 mol of chromium nicotinate is added within 1 hour; then the chromium-enriched yeast product D2 is obtained. .
  • the total chromium content in the chromium-enriched yeast product D2 was 32647ppm, the absorption rate of chromium was 51.87%, and the proportion of organic chromium was 98.52%.
  • Example 2 The rest of the experimental conditions are the same as in Example 1, except that 3 mol of chromium nicotinate was added at the beginning of the fermentation in step 3, and the total amount of chromium nicotinate added was exactly the same as in Example 1; the chromium-enriched yeast product D3 was obtained later.
  • the total chromium content in the chromium-enriched yeast product D3 was 35612ppm, the absorption rate of chromium was 56.58%, and the proportion of organic chromium was 98.41%.
  • Example 2 The rest of the experimental conditions are the same as in Example 1, the difference is only that the fermentation pH is controlled at 5.5 in Steps 2 and 3, and then the chromium-enriched yeast product D4 is obtained.
  • the total chromium content in the chromium-enriched yeast product D4 was 31985ppm, the absorption rate of chromium was 50.82%, and the proportion of organic chromium was 97.89%.
  • Example 2 The rest of the experimental conditions are the same as in Example 1, the only difference is that the fermentation temperature is controlled at 38° C. in Steps 2 and 3; then the chromium-enriched yeast product D5 is obtained.
  • the total chromium content in the chromium-enriched yeast product D5 was 26854ppm, the absorption rate of chromium was 42.67%, and the proportion of organic chromium was 98.14%.
  • step 3 the alcohol of the fermentation broth is controlled at 1.0%; then the chromium-enriched yeast product D6 is obtained.
  • the total chromium content in the chromium-enriched yeast product D6 is 19688ppm, the absorption rate of chromium is 31.28%, and the proportion of organic chromium is 98.36%.
  • step 3 the dissolved oxygen in the fermentation broth is controlled at 60 ⁇ mol/L; then the chromium-enriched yeast product D7 is obtained.
  • the total chromium content in the chromium-enriched yeast product D7 was 22457ppm, the absorption rate of chromium was 35.68%, and the proportion of organic chromium was 98.41%.
  • Example 1 40563 98.39 66.45
  • Example 2 38451 97.78 61.03
  • Example 3 43096 98.78 68.4 Example 4 41025 98.78 65.18
  • Example 5 40127 98.67 63.76
  • Example 6 41357 98.88 65.71
  • Example 7 40357 98.38 64.12 Comparative Example 1 2044.78 89.71 2.22
  • Comparative Example 2 32647 98.52 51.87
  • Comparative Example 3 35612 98.41 56.58 Comparative Example 4 31985 97.89 50.82
  • Comparative Example 6 19688 98.36 31.28 Comparative Example 7 22457 98.41 35.68
  • Comparative Example 4 shows that fermentation pH has a significant impact on the absorption rate of chromium; Comparative Example 5 is compared with Example 1, and it can be seen that fermentation temperature has a significant impact on the absorption rate of chromium; Comparative Example 6 Compared with Example 1, it can be seen that the alcohol content of the fermentation broth has a significant impact on the absorption rate of chromium; in Comparative Example 7, compared with Example 1, it can be seen that the dissolved oxygen in the fermentation broth has a significant impact on the absorption rate of chromium.

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Abstract

L'invention concerne une levure riche en chrome, son procédé de préparation et son application. La levure riche en chrome a une teneur massique en chrome supérieure à 38 000 ppm, une proportion de chrome organique supérieure à 97 % en masse et un taux d'absorption du chrome supérieur à 50 % en masse, et elle ne contient pas de chrome hexavalent. Le procédé de préparation de la levure riche en chrome utilise du chrome organique pour remplacer le chrome inorganique classique en tant que source de chrome. L'absorption du chrome par la levure est considérablement augmentée, l'absorption du chrome est élevée, et la teneur en chrome dans les eaux usées produites est fortement réduite, ce qui réduit les contraintes liées à la protection de l'environnement.
PCT/CN2021/137249 2020-12-30 2021-12-10 Levure riche en chrome ayant une absorption élevée de chrome, son procédé de préparation et son application WO2022143113A1 (fr)

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CN202011603128.9A CN114686383B (zh) 2020-12-30 2020-12-30 高铬吸收率的富铬酵母及其制备方法和应用

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