Methods for Isolating Single-Molecule Products
The subject invention provides materials and methods for producing, isolating, extracting and purifying single-molecule products. The subject invention provides materials and methods for extracting microbial metabolites at a high level of purity, for example, a purity of at least 80% by weight, and preferably at least 95% by weight or more. Specifically, the subject invention provides materials and methods for isolating or extracting biosurfactants and polyketides at a high level of purity. Preferably, the biosurfactant is a sophorolipid (SLP).
1 . A method for extracting one or more microbial metabolites at a high level of purity, the method comprising:
1) cultivating a metabolite-producing microorganism to produce a microorganism culture broth, said microorganism culture broth comprising liquid fermentation medium, microorganism cells and one or more microbial metabolites;
2) mixing the culture broth with a non-polar solvent;
3) centrifuging the mixture of step 2) to cause phase separation in the mixture, wherein a non-polar layer, an interphase layer, a water layer and a cell layer are separated;
4) separately collecting one or more layers selected from the non-polar layer, interphase layer, water layer and cell layer;
5) independently centrifuging one or more collected layers of step 4) and independently collecting one or more supernatants from one or more collected layers; and
6) obtaining one or more microbial metabolites by evaporation.
2 . The method of claim 1 , wherein the metabolite-producing microorganism is a yeast.
3 . The method of claim 2 , wherein the yeast is Starmerella bombicola or Meyerozyma guilliermondii.
4 . The method of claim 1 , wherein the non-polar solvent is ethyl acetate.
5 . The method of claim 1 , wherein the culture broth and the non-polar solvent is mixed at a ratio of from 1:10 to 10:1.
6 . The method of claim 5 , wherein the culture broth and the non-polar solvent is mixed at a ratio of 1:1.
7 . The method of claim 1 , wherein the high level of purity is at least 95%.
8 . The method of claim 1 , wherein the microbial metabolite is a sophorolipid (SLP).
9 . The method of claim 8 , wherein the SLP is hydrophilic SLP comprising di-acetylated linear SLP and/or mono-acetylated linear SLP.
10 . The method of claim 1 , wherein the water layer is collected in step 4).
11 . The method of claim 10 , wherein the method further comprising isolating one or more microbial metabolites from the water layer, which comprises:
i) obtaining an evaporated product of the water layer;
ii) mixing the evaporated product of the water layer with an alcohol;
iii) centrifuging the mixture of step ii) and collecting the alcohol layer; and
iv) obtaining the microbial metabolite by evaporating the alcohol.
12 . The method of claim 1 , wherein the alcohol is methanol or ethanol.
13 . The method of claim 1 , wherein the non-polar layer is collected in step 4).
14 . The method of claim 13 , wherein the method further comprising purifying one or more microbial metabolites from the non-polar layer, by:
i) obtaining an evaporated product of the non-polar layer;
ii) mixing the evaporated product of the non-polar layer with chloroform;
iii) centrifuging the mixture of step ii) and collecting the top layer; and
iv) obtaining the microbial metabolite by evaporation.
15 . The method of claim 10 , wherein the method further comprising purifying each of one or more microbial metabolites from the water layer, which comprises a plurality of purifying cycles using solvents with increased non-polarity, each cycle comprising:
i) obtaining an evaporated product of the water layer;
ii) mixing the evaporated product of the water layer with a solvent;
iii) centrifuging the mixture from step ii) and collecting the solvent layer; and
iv) obtaining a microbial metabolite by evaporating the solvent;
wherein each cycle uses a different solvent that is more non-polar than the solvent used in the previous cycle.
16 . The method of claim 15 , wherein the solvents in the plurality of purifying cycles are used in an order of: methanol, ethanol, 1-propanol, 1-butanol, 1-hexanol, 1-heptanol, 2-propanol, 2-butanol, acetone, and chloroform.
17 . The method of claim 13 , wherein the method further comprises purifying each of the one or more microbial metabolites from the non-polar layer, which comprises a plurality of purifying cycles using solvents with increased polarity, each cycle comprising:
i) obtaining an evaporated product of the non-polar layer;
ii) mixing the evaporated product of the non-polar layer with a solvent;
iii) centrifuging the mixture from step ii) and collecting the solvent layer; and
iv) obtaining a microbial metabolite by evaporating the solvent;
wherein each cycle uses a different solvent that is more polar than the solvent used in the previous cycle.
18 . The method of claim 17 , wherein the solvents in the plurality of purifying cycles are used in an order of: chloroform, acetone, 2-butanol, 2-propanol, 1-heptanol, 1-hexanol, 1-butanol, 1-propanol, ethanol, and methanol.
19 . A composition produced by the method of claim 1 , the composition comprising a purified SLP and a percentage of water, wherein the percentage of water is less than 50%, and the SLP has a purity of at least 95%.
20 . The composition of claim 19 , wherein the percentage of water is 20% to 30%.
21 - 26 . (canceled)