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CN-121992054-A - Diacetylated lactone type sophorolipid, and preparation method and application thereof

CN121992054ACN 121992054 ACN121992054 ACN 121992054ACN-121992054-A

Abstract

The invention discloses a diacetylated lactone type sophorolipid, a preparation method and application thereof, comprising the steps of fermenting yeast cells for producing sophorolipid in a culture medium containing a hydrophobic carbon source to obtain a sophorolipid fermentation crude product; centrifuging the sophorolipid fermentation crude product, removing supernatant, dissolving precipitate with ethanol, centrifuging to remove thalli, distilling ethanol phase under reduced pressure to obtain crude sophorolipid, crystallizing the crude sophorolipid in PBS buffer solution for multiple steps, filtering, and oven drying to obtain lactone type crystal. The invention prepares the product rich in diacetylated lactone type sophorolipid (> 80%), and then obtains the product with purity of >99% by means of crystallization technology. The product has good antibacterial activity, and the minimum antibacterial concentration of the product on bacillus cereus and other bacteria is as low as 8-32 mg/L. The invention prepares a large amount of single type sophorolipid structures through a green and environment-friendly process, avoids the use of organic solvents, and lays a good foundation for the application of the diacetylated lactone type sophorolipid in the field of antibiosis.

Inventors

  • LONG XUWEI
  • MA QIANQIAN

Assignees

  • 南京理工大学

Dates

Publication Date
20260508
Application Date
20251105

Claims (10)

  1. 1. A method for preparing and separating diacetylated lactone-type sophorolipids, comprising: Fermenting the yeast cells for producing sophorolipid in a culture medium containing a hydrophobic carbon source to obtain a sophorolipid fermentation crude product; centrifuging the sophorolipid fermentation crude product, removing supernatant, dissolving precipitate with ethanol, centrifuging to remove thalli, and distilling ethanol phase under reduced pressure to obtain crude sophorolipid; the crude sophorolipid is crystallized in multiple steps through PBS buffer solution, and then the lactone type crystal is obtained after filtration and drying.
  2. 2. The method for preparing and separating di-acetylated lactone type sophorolipid according to claim 1, wherein the hydrophobic carbon source is oleic acid, soybean oil, n-hexadecane or palm oil.
  3. 3. A process for the preparation and isolation of bis-acetylated lactone-type sophorolipids according to claim 1 or 2, characterised in that the hydrophobic carbon source is preferably oleic acid.
  4. 4. The method for preparing and separating diacetylated lactone type sophorolipids according to claim 1, wherein the culture medium is divided into 10% w/v oleic acid, 100 g/L glucose, 5 g/L yeast extract, 0.7 g/L peptone ;1 g/L KH 2 PO 4 ;0.5 g/L MgSO 4 ·7 H 2 O;0.1 g/L CaCl 2 ·2H 2 O;0.1 g/L NaCl;pH=4.0.
  5. 5. The process for the preparation and isolation of diacetylated lactone-type sophorolipids according to claim 1, characterized in that the yeast cell producing sophorolipids is candida apis, preferably Candida bombicola (ATCC 22214).
  6. 6. The method for preparing and separating di-acetylated lactone type sophorolipids according to claim 1, wherein the multi-step crystallization of crude sophorolipids in PBS buffer comprises: Adding sufficient PBS buffer solution into the crude sophorolipid, fully stirring, centrifuging to separate supernatant and precipitate, and crystallizing; Filtering and separating crystals in the supernatant after crystallization is finished, drying to obtain a diacetyl lactone type product, washing the precipitate with PBS for many times until the precipitate is white, and directly drying after separating the precipitate to obtain the diacetyl lactone type product; collecting washing liquid, crystallizing, separating crystal and stoving to obtain diacetylated lactone product.
  7. 7. The method for preparing and separating di-acetylated lactone type sophorolipids according to claim 1, wherein the multi-step crystallization of crude sophorolipids with PBS buffer further comprises: adding sufficient PBS buffer into crude sophorolipid, stirring at 30deg.C, and Crystallization was performed at 4 ℃.
  8. 8. A diacetylated lactone-type sophorolipid produced by the preparative separation method according to any one of claims 1 to 7.
  9. 9. Use of a diacetylated lactone-type sophorolipid of claim 8 or of a diacetylated lactone-type sophorolipid produced by the preparative separation process of any of claims 1-7 in antibacterial activity.
  10. 10. The use according to claim 9, wherein the inhibition ratio of staphylococcus aureus, bacillus subtilis and bacillus cereus is more than 95% when the concentration of the diacetylating lactone type sophorolipid is 8-32 mg/L.

Description

Diacetylated lactone type sophorolipid, and preparation method and application thereof Technical Field The invention belongs to the technical field of microorganisms and biology, and particularly relates to diacetylated lactone type sophorolipid, a preparation method and application thereof. Background Biosurfactants sophorolipids (Sophorolipids, SLs) are a class of glycolipid biosurfactants. Sophorolipids produced by natural fermentation are a mixture of various structures, and are mainly classified into lactone type and acid type, wherein the lactone type and the acid type are further classified into four structures of deacetylation, monoacetylation at two different acetylation positions and diacetylation according to the degree of acetylation, and sophorolipid components produced under different fermentation conditions, such as different carbon sources, are different, so that the physicochemical and biological properties of the obtained products are greatly different. Generally speaking, acid type sophorolipids have better emulsifying and wetting effects, while lactone type sophorolipids have better antibacterial properties. It has also been found that the more hydrophobic the sophorolipid molecule, the more bactericidal activity it is. Therefore, in order to enhance the antibacterial feasibility of SLs, it is important to obtain highly hydrophobic SLs, and currently existing processes can obtain lactone-type sophorolipids by means of chromatography or crystallization, but it is difficult to obtain single type of highly hydrophobic diacetylated lactone-type sophorolipids. Disclosure of Invention In view of the above, the invention provides a diacetylated lactone-type sophorolipid, and a preparation method and application thereof. In a first aspect, embodiments of the present invention provide a method for preparing and separating a diacetylated lactone-type sophorolipid, the method comprising: Fermenting the yeast cells for producing sophorolipid in a culture medium containing a hydrophobic carbon source to obtain a sophorolipid fermentation crude product; centrifuging the sophorolipid fermentation crude product, removing supernatant, dissolving precipitate with ethanol, centrifuging to remove thalli, and distilling ethanol phase under reduced pressure to obtain crude sophorolipid; the crude sophorolipid is crystallized in multiple steps through PBS buffer solution, and then the lactone type crystal is obtained after filtration and drying. Further, the hydrophobic carbon source is oleic acid, soybean oil, n-hexadecane, palm oil. Further, the hydrophobic carbon source is preferably oleic acid. Further, the culture medium is divided into 10% w/v oleic acid, 100 g/L glucose, 5.5 g/L yeast extract powder and 0.7. 0.7 g/L peptone ;1 g/L KH2PO4;0.5 g/L MgSO4·7 H2O;0.1 g/L CaCl2·2H2O;0.1 g/L NaCl;pH=4.0. Further, the yeast cell producing sophorolipids is candida apis, preferably Candida bombicola (ATCC 22214). Further, the multi-step crystallization of crude sophorolipids by PBS buffer comprises: Adding sufficient PBS buffer solution into the crude sophorolipid, fully stirring, centrifuging to separate supernatant and precipitate, and crystallizing; Filtering and separating crystals in the supernatant after crystallization is finished, drying to obtain a diacetyl lactone type product, washing the precipitate with PBS for many times until the precipitate is white, and directly drying after separating the precipitate to obtain the diacetyl lactone type product; collecting washing liquid, crystallizing, separating crystal and stoving to obtain diacetylated lactone product. Further, the multi-step crystallization of crude sophorolipids by PBS buffer also includes: adding sufficient PBS buffer into crude sophorolipid, stirring at 30deg.C, and Crystallization at 4 ℃; In a second aspect, embodiments of the present invention provide a diacetylated lactone-type sophorolipid, prepared by the above-described preparative separation method. In a third aspect, embodiments of the present invention provide an application of diacetylated lactone-type sophorolipids in antibacterial applications. Further, the inhibition rate of staphylococcus aureus, bacillus subtilis and bacillus cereus is more than 95% when the concentration of the diacetylating lactone type sophorolipid is 8-32 mg/L. Compared with the prior art, the invention has the beneficial effects that: The invention provides a preparation method of diacetylated lactone-type sophorolipid, which selects oleic acid as a hydrophobic carbon source to carry out fermentation production of sophorolipid, wherein the content of the diacetylated lactone-type sophorolipid in the finally obtained product is more than 80 percent, and the invention optimizes the crystallization process of the lactone-type sophorolipid, and can finally obtain the lactone-type content of more than 99 percent by a multi-step crystallization method, wherein the content of the diacetylated lactone-type is m