Search

CN-122010673-A - Method for efficiently extracting intracellular products

CN122010673ACN 122010673 ACN122010673 ACN 122010673ACN-122010673-A

Abstract

The invention relates to the field of fermentation product extraction methods, in particular to a method for efficiently extracting intracellular products. The invention applies staged extraction and ultrasonic combination to intracellular product extraction, and can obviously improve the extraction rate. After the thalli in the fermentation liquor are crushed, the first extraction adopts nonpolar solvent extraction, ultrasonic short-time low-temperature treatment is adopted in the extraction process, the extraction efficiency of a target product can be obviously improved, the second extraction adopts the ultrasonic short-time low-temperature treatment again by adding polar solvent into the extraction phase of the first extraction, the extraction amount of intracellular products is improved, the process does not need complex equipment, the operation time is short, the extraction efficiency is high, and the method is suitable for large-scale industrial production.

Inventors

  • JIANG XIJUAN
  • ZHOU YUN
  • ZHANG YAPING
  • WU JIHUA
  • LI MINGJUN

Assignees

  • 万华化学集团股份有限公司

Dates

Publication Date
20260512
Application Date
20260106

Claims (10)

  1. 1. A method for efficiently extracting an intracellular product, comprising the steps of: S1, taking a microbial fermentation liquid, and crushing thalli of the microbial fermentation liquid, wherein the microbial fermentation liquid contains a target intracellular product; S2, adding a nonpolar extractant into the microbial fermentation broth after thallus breakage, performing first extraction under an ultrasonic condition, centrifuging, and collecting an extraction phase; s3, adding a polar extractant into the extraction phase in the step S2, performing second extraction under the ultrasonic condition, centrifuging, and collecting a nonpolar extraction phase.
  2. 2. The method of claim 1, wherein the target intracellular product in the microbial fermentation broth comprises farnesene, lycopene, beta-carotene, squalene or other yeast-producible intracellular products.
  3. 3. The method for efficiently extracting an intracellular product according to claim 1 or 2, wherein the method for disrupting a cell comprises a bead mill method, a high pressure homogenization method, a freeze-milling method, an enzymolysis method, a chemical reagent method or a liquid nitrogen disruption method.
  4. 4. A method of efficiently extracting an intracellular product in accordance with any one of claims 1-3 wherein said nonpolar extractant comprises at least one of n-hexane, cyclohexane, heptane, carbon tetrachloride, n-dodecane, petroleum ether, methyl tertiary butyl ether.
  5. 5. The method for efficiently extracting intracellular products according to any one of claims 1 to 4, wherein the addition amount of the nonpolar extractant is 5 to 100 times the volume of the microbial fermentation broth; Optionally, the addition amount of the nonpolar extractant is 10-50 times of the volume of the microbial fermentation broth.
  6. 6. The method of claim 1-5, wherein the polar extractant comprises at least one of methanol, ethyl acetate, isopropanol, ethanol, methylene chloride, dimethyl sulfoxide, and acetone.
  7. 7. The method for efficiently extracting intracellular products according to any one of claims 1 to 6, wherein the addition amount of the polar extractant is 5 to 30 times the volume of the microbial fermentation broth; optionally, the addition amount of the polar extractant is 5-20 times of the volume of the microbial fermentation broth.
  8. 8. The method for efficiently extracting intracellular products according to any one of claims 1 to 7, wherein in the step S2 or the step S3, the ultrasonic condition is that the ultrasonic power is 50 to 500w, the time is 2 to 30min, and the temperature is not more than 30 ℃; Optionally, the ultrasonic time is 5-20 min, preferably 5-10 min; optionally, the temperature of the ultrasound is 20-28 ℃.
  9. 9. The method for efficiently extracting intracellular products according to any one of claims 1 to 8, wherein the centrifugation conditions in the step S2 or the step S3 are 3000 to 8000rpm, and the centrifugation is performed for 5 to 30 minutes.
  10. 10. The method for efficient extraction of intracellular products of any one of claims 1 to 9, further comprising the step of removing the organic phase after said collecting of the non-polar extract phase; Optionally, removing the organic phase by rotary steaming at 30-80deg.C and vacuum degree of 20-90kPa; optionally, removing the organic phase by adopting a rotary steaming method, wherein the rotary steaming temperature is 40-60 ℃ and the vacuum degree is 30-50kPa.

Description

Method for efficiently extracting intracellular products Technical Field The invention relates to the field of fermentation product extraction methods, in particular to a method for efficiently extracting intracellular products. Background With the continuous progress of synthetic biology technology, more and more petrochemical products and fine chemicals are being turned to fermentation production. The method has the remarkable advantages of high production efficiency, high product purity, unrestricted raw material sources and the like, and is widely focused by scientific research institutions and enterprises. Fermentation products are largely classified into extracellular products and intracellular products. Extracellular products (e.g., alkaline protease, saccharifying enzyme) are secreted directly outside the cell, and are present in the supernatant for extraction without disruption of the cell. In contrast, intracellular products are compounds or biological macromolecules produced by cells during metabolism or biosynthesis, which are mainly present inside the cells and which are not normally actively secreted outside the cells and must be extracted by a cell disruption step. Taking saccharomycetes fermentation as an example, common intracellular products comprise squalene, beta carotene, farnesene, arbutin, erythritol and the like, and the products can be obtained by extracting after the thallus is crushed. Squalene, also known as thirty-carbon hexaene, is a colorless oily acyclic triterpene, is unstable and easily oxidized, and generates odor in the air. The hydrogenated stable squalane is commonly used in the fields of cosmetics and personal care as an emollient and an antioxidant, penetrates deep into skin and reduces water loss and fine wrinkles. The squalene is unstable and easy to cause acne, and is mainly used as a raw material of vaccine adjuvants and health products. In the field of vaccine adjuvants, squalene can increase antigen immunogenicity, is used for vaccine development and disease prevention, and can enhance vaccine efficacy by maintaining immune response and controlling response type, thereby providing virus protection. Farnesene, also known as farnesene, is an acyclic sesquiterpene. It is a colorless or pale yellow oily liquid, possessing various isomers, of which alpha-farnesene and beta-farnesene are most common. Alpha-farnesene has the faint scent of fresh petals, and beta-farnesene emits a glue-like scent. The biosynthesis is mainly completed through microbial fermentation, and the method has the outstanding advantages of environmental protection and sustainability. The application field is very wide, and the water-based oil-based emulsion is capable of covering a plurality of industries such as energy chemical industry, medicine, pesticide, cosmetics, food and the like, and has great potential in the fields of biofuel, lubricant, surfactant, tire manufacturing and the like. Beta-carotene is a natural orange pigment with a chemical formula of C 40H56 and a molecular weight of 536.87. It belongs to fat-soluble compound, is insoluble in water, but is easily dissolved in oil and fat and organic solvents such as ethyl alcohol and diethyl ether. Can be converted into vitamin A in vivo, thus having important nutrition and health care value. As a powerful antioxidant, it can neutralize free radical, reduce oxidation stress, prevent cell damage and chronic diseases, and as a precursor of vitamin A, it is converted into retinaldehyde to assist in maintaining normal vision and preventing night blindness and macular degeneration. As a natural pigment with multiple benefits, the beta-carotene is widely applied to the food and cosmetic industries and has positive influence on human health. At present, the steps of extracting the intracellular products such as squalene, beta carotene, farnesene and the like mainly comprise thallus crushing, product extraction, solvent removal, decolorization, chromatography, concentration and the like, wherein the thallus crushing method mainly comprises a mechanical crushing method such as a bead grinding method, a high-pressure homogenizing method, a freeze grinding method and the like, in addition, the method also comprises an enzymolysis method, a chemical reagent method, a liquid nitrogen crushing method and the like, the general mechanical crushing method is simple to operate and low in cost, is suitable for large-scale production, the product extraction generally adopts an organic solvent, but the organic solvent extraction has the problems of low extraction efficiency, large solvent consumption and incomplete extraction, the problems of the mainly influenced factors such as the distribution coefficient of solutes, the nature and the consumption of the solvent and the like, the extraction effect is generally improved by adopting a plurality of extraction modes, improving the pressure, adding a dispersing agent, adjusting the pH value and the like, but