Materials and Methods for Producing Cardiolipin-Like Phospholipids
The subject invention provides methods for producing phospholipids using yeasts not previously known to produce high concentrations of such phospholipids. In particular, Wickerhamomyces anomalus is cultivated in a specially-tailored nutrient medium and under cultivation conditions such that the yeast unnaturally produces high concentrations of phospholipids that resemble human cardiolipin and/or precursors thereof in structure and/or function. Yeast culture compositions are also provided, comprising yeast cells, growth medium, and high concentrations of phospholipids.
1 . A method for producing a phospholipid, the method comprising:
inoculating a nutrient medium with an inoculum of a Wickerhamomyces anomalus yeast to produce a yeast culture comprising yeast cells and medium; and
cultivating the yeast culture for 3 to 9 days under conditions favorable for the production of the phospholipid, said phospholipid having a structure according to:
where Z is H, a serine group, choline group, ethanolamine group, inositol group, or glycerol group, and
where R1-R4 are the same or different fatty acid side chains having between 14 and 22 carbon atoms and between 0 and 6 double-bonded carbon atoms.
2 . (canceled)
3 . The method of claim 1 , wherein the phospholipid is a phosphatidylglycerol having a structure according to General Formula 1, and wherein Z is a glycerol group.
4 . The method of claim 1 , wherein the phospholipid is a cardiolipin having a structure according to General Formula 1.
5 . The method of claim 4 , wherein R1, R2, R3 and R4 are linoleoyl groups.
6 . (canceled)
7 . The method of claim 1 , wherein the favorable conditions include dissolved oxygen measurement of about 20% to about 50% of saturation.
8 . The method of claim 1 , wherein the favorable conditions include cultivation pH of about 3.0 to about 7.0.
9 . The method of claim 1 , wherein the nutrient medium comprises one or more inorganic salts; one or more sources of nitrogen; one or more sources of vitamins, proteins, growth factors, and/or trace nutrients; one or more sources of fatty acids; and one or more sources of carbon.
10 . The method of claim 9 , wherein the medium comprises one or more inorganic salts selected from ammonium sulfate, magnesium sulfate, di-potassium phosphate, monosodium phosphate, and potassium phosphate.
11 . The method of claim 9 , wherein the medium comprises one or more sources of nitrogen selected from potassium nitrate, ammonium nitrate ammonium sulfate, ammonium phosphate, ammonia, urea, and ammonium chloride.
12 . The method of claim 9 , wherein the medium comprises yeast extract.
13 . The method of claim 9 , wherein the medium comprises one or more sources of fatty acids selected from canola oil, sunflower oil, and soybean oil.
14 - 15 . (canceled)
16 . The method of claim 9 , wherein the nutrient medium comprises ammonium sulfate, magnesium sulfate, di-potassium phosphate, monosodium phosphate, potassium phosphate, biotin, acetyl L-carnitine, alpha-lipoic acid, inositol, urea, yeast extract, glucose, soybean and/or sunflower oil, and trace elements.
17 . The method of claim 9 , wherein the nutrient medium comprises 5 g/L to 20 g/L inositol.
18 - 19 . (canceled)
20 . The method of claim 1 , further comprising purifying the phospholipid from the yeast culture.
21 . The method of claim 20 , wherein purification comprises mixing the yeast culture with ethyl acetate at a ratio of 1:1 by volume to form a mixture;
mixing the mixture for about 40 to 100 hours, followed by centrifuging the mixture for 20 to 30 minutes to produce a cell pellet layer, a water layer, a middle solid layer and an ethyl acetate layer;
collecting the water layer; and
evaporating the water layer to produce a viscous brown mass comprising purified phospholipids.
22 - 24 . (canceled)
25 . A yeast culture comprising cells of a phospholipid-producing yeast, a nutrient medium, and a high concentration of a phospholipid, wherein the yeast is Wickerhamomyces anomalus , and wherein the phospholipid is retained in the cells of the yeast and/or is present as an extracellular secretion in the nutrient medium; wherein the high concentration of phospholipid is 0.1 g/L to 55.0 g/L of the medium.
26 . (canceled)
27 . The yeast culture of claim 25 , wherein the phospholipid has a structure according to
where Z is H, a serine group, choline group, ethanolamine group, inositol group, or glycerol group, and
where R1-R4 are the same or different fatty acid side chains having between 14 and 22 carbon atoms and between 0 and 6 double-bonded carbon atoms.
28 - 29 . (canceled)
30 . An anti-aging composition for reversing and/or slowing a sign of aging, the composition comprising a phospholipid molecule having a structure according to:
where Z is H, a serine group, choline group, ethanolamine group, inositol group, or glycerol group, and
where R1-R4 are the same or different fatty acid side chains having between 14 and 22 carbon atoms and between 0 and 6 double-bonded carbon atoms, and
wherein the phospholipid molecule was produced by cultivating a Wickerhamomyces anomalus yeast.
31 . (canceled)
32 . The anti-aging composition according to claim 30 , formulated as a cosmetic composition, wherein the composition further comprises a dermatologically-acceptable carrier and, optionally, one or more additional skin-active agents.