Production of squalene and/or sterol from cell suspensions of fermented yeast

Abstract

The present invention relates to a process for the production of squalene and/or sterol in high amounts using an alkaline solution and an organic lysis solvent at high temperature and high pressure for effectively lysing yeast cells and extracting squalene and/or sterol into an organic extraction solvent, thus obtaining squalene and/or sterolin high amount and of high purity.

Claims

1. A process for producing squalene comprising a) preparing squalene containing yeast cells by fermentation in a culture medium under batch conditions or under continuous conditions; b) lysing the squalene containing yeast cells in a lysis medium without mechanical lysis or disruption of cells and without separation of yeast cells from culture medium, wherein the lysis medium comprises the culture medium, potassium hydroxide (KOH) and a polar protic organic lysis solvent, and wherein the lysis is performed in a closed system under elevated pressure of 2 to 50 bar (2 to 5010.sup.5 Pa) and at a temperature of 140-250 C.; c) adding an non-polar organic extraction solvent to the lysis medium and extracting squalene from the lysis medium; and d) isolating squalene from the organic phase by cooling of the mixture and phase separation of organic extraction solvent phase comprising the squalene product from the lysis medium; wherein the lysis in b), extraction in c) and isolation in d) are performed under continuous conditions.

2. The process according to claim 1, wherein the organic lysis solvent is ethanol, methanol, n-butanol, isopropanol or n-propanol.

3. The process according to claim 1, wherein lysis is effected at a pressure at 6 to 40 bar (6 to 4010.sup.5 Pa).

4. The process according to claim 1, wherein the temperature is 170 to 195 C.

5. The process according to claim 1, wherein the lysis is effected within a time period of 1 to 140 minutes.

6. The process according to claim 1, wherein the potassium hydroxide is solid potassium hydroxide or potassium hydroxide solution, the organic lysis solvent is ethanol, and the lysis is performed under continuous conditions at a temperature of 140 to 250 C.

7. The process according to claim 1, wherein the organic extraction solvent is a halogenated or non-halogenated (i) alkane, (ii) cycloalkane, (iii) aromat, (iv) ketone, or (v) ether.

8. The process according to claim 7, wherein the organic extraction solvent is a non-halogenated alkane.

9. The process according to claim 1, wherein extraction is performed at a temperature of 40 to 140 C.

10. The process according to claim 9, wherein the extraction is performed at a temperature of 50 to 70 C.

11. The process according to claim 1, wherein the isolation of the squalene further comprises distillation of the organic extraction solvent phase in order to evaporate the organic extraction solvent and/or distillation of the squalene.

12. The process according to claim 11, wherein the distillation of the organic extraction solvent phase in order to evaporate the organic extraction solvent and/or distillation of the squalene is performed under continuous conditions with the lysis in b), extraction in c) and isolation in d).

13. The process according to claim 1, wherein the lysis is effected within a time period of 1 to 20 minutes.

14. The process according to claim 1, wherein the lysis medium comprises 0.1 to 5.0 gram KOH per gram dry mass of yeast cells.

Description

EXAMPLES

(1) The following is an analysis of processes of the prior art for obtaining squalene from fermented yeast cells using potassium hydroxide and ethanol as lysis agents and hexane, heptane or petrol ether (petroleum) as organic extraction solvent. The processes of the prior art are compared with the process of the present invention.

Example 1

Comparative Example: Lyophilization

(2) Comparative example 1 refers to the document of Mantzouridou et al. (2009) disclosing the production of squalene by yeast cells. Therein, yields of 5 g/l squalene are obtained in the culture medium. After fermentation, squalene is obtained by the following process steps: 1) Yeast cells are isolated by centrifugation, 2) yeast cells are washed twice with water, 3) yeast cells are lyophilized, 4) portion of dried biomass of 96 mg is added to 5 ml 60% (w/v) KOH solution in water, 7.5 ml methanol and 7.5 ml of a methanolic pyrogallol solution (0.5% w/v), 5) incubation in shaking apparatus at 45 C. overnight, 6) lipidic components are extracted three times with 10 ml hexane for each extraction, whereby phase separations are performed by centrifugation at 4000 g during 10 min, 7) optionally formed emulsions are eliminated by addition of 0.5 ml methanol, 8) the hexane fractions are dried over sodium sulfate, and 9) the solvent is then removed by 40 C. under vacuum.

(3) The following characteristics for the process can be determined in view of the indications of the amounts above. In the following, the used amounts of crude material relative to the yeast dry mass (DM) are indicated

(4) TABLE-US-00002 TABLE 2 Material Factor (g/g DM) KOH 50 EtOH 119.8 Hexane 212.5

Example 2

Comparative Example: Homogenization with Glass Beads

(5) Comparative example 2 refers to the document of Paltauf et al. (1982), disclosing a laboratory process for cell lysis. The following aspects are notable: 1) Yeast cells are separated after cultivation, suspended in water and disintegrated by shaking in a homogenizer in the presence of glass beads, 2) 2 ml ethanol and 2 ml (30% (w/v)) potassium hydroxide are added to an aliquot of total lipids (about 5 mg) and the mixture is stirred by 80 C. for 60 min, 3) non-saponifiable lipids are extracted twice with 4 ml petroleum each, and 4) the obtained extract is washed twice with 2 ml water each.

(6) The following characteristics for the process can be determined in view of the indications of the amounts above. In the following, the used amounts of crude material relative to the yeast dry mass are indicated

(7) TABLE-US-00003 TABLE 3 Material Factor (g/g DM) KOH 154.7 EtOH 316 Heptane 1088

Example 3

Comparative Example

(8) Comparative example 3 refers to the document of Singhal et al. (1990) describing a laboratory process for cell lysis. The following aspects are notable: 1) Soxhlet extraction by petrol ether (60-80 C. boiling point) as solvent 2) the resulting lipid is saponified: for saponification 60% (w/v) KOH and 20 ml ethanol are added to 1.0 g lipid and the reaction is heated under reflux for 30 min 3) non-saponifiable lipids are extracted by petrol ether 4) purification by chromatographic means.

(9) The following characteristics for the process can be determined in view of the indications of the amounts above. In the following, the used amounts of crude material relative to the yeast dry mass are indicated

(10) TABLE-US-00004 TABLE 4 Material Factor (g/g DM) KOH Not specified EtOH 15.8 Petrol ether Not specified

Example 4

General Procedure

(11) The following is an analysis of the processes of the present invention for obtaining squalene from fermented yeast cells using potassium hydroxide and ethanol as lysis agents and heptane as organic extraction solvent in the amounts and under the conditions as referred to above. The data allow a comparison with prior art data.

(12) Lysis of Yeast Cells

(13) For working with the continuously working reactor the following flows, 1) Yeast suspension, 2) Potassium hydroxide aqueous solution (50% (w/v)), and 3) Ethanol (95% (v/v)),
can be separately used or dosed to the reactor after mixing. The mixture is dosed to the continuously working reactor in a mode that the residence time is kept at 3-5 min. The working temperature is between 170 and 195 C. and thus at a pressure of about 12 to 20 bar (12 to 2010.sup.5 Pa).
Extraction with Heptane (n-Heptane or a Mixture of Heptane Isomers)

(14) The processing of the lysis fractions occurs stepwise or continuously. After cooling of the reaction mixture to a temperature range of 50-66 C., 50 ml heptane per 125 g dry cell mass yeast suspension are dosed parallel to the reaction mixture, whereby squalene is extracted into the organic phase within about 5 minutes. After a coalescence time of 1-5 min the phases are separated.

(15) The extraction can also be performed in a two step/several step extraction procedure. There are no changes of the extraction and phase separation times.

(16) Alternatively, the extraction medium can also be added directly to the lysis process.

(17) The resulting yield of squalene in the organic phase is in the range of 88-95% (w/w).

Example 5

(18) The individual material flows were pre-mixed, so that the mass proportions as indicated in table 5 could be obtained. A medium residence time of 3 min was set up. The subsequent extraction was performed according to the indications in the general part of the examples.

(19) TABLE-US-00005 TABLE 5 Extraction KOH (g/g DM) EtOH (g/g DM) Heptane (g/g DM) degree (%) 0.35 5.54 3.6 88

(20) The term extraction degree means that the given percentage of the total amount of squalene is identified in the extraction solvent; the remaining amount remains in the aqueous phase.

Example 6

(21) The individual material flows were pre-mixed, so that the mass proportions as indicated in table 6 could be obtained. A medium residence time of 5 min was set up. The subsequent extraction was performed according to the indications in the chapter General Example.

(22) TABLE-US-00006 TABLE 6 Extraction KOH (g/g DM) EtOH (g/g DM) Heptane (g/g DM) degree (%) 0.33 5.22 3.6 91

Example 7

(23) The individual material flows were pre-mixed, so that the mass proportions as indicated in table 7 could be obtained. A medium residence time of 3 min was set up. The subsequent extraction was performed according to the indications in the introductory part of the examples.

(24) TABLE-US-00007 TABLE 7 Extraction KOH (g/g DM) EtOH (g/g DM) Heptane (g/g DM) degree (%) 1.02 5.39 3.6 95

Example 8

(25) The individual material flows were pre-mixed, so that the mass proportions as indicated in table 8 could be obtained. A medium residence time of 5 min was set up. The subsequent extraction was performed according to the indications in the introductory part of the examples.

(26) TABLE-US-00008 TABLE 8 Extraction KOH (g/g DM) EtOH (g/g DM) Heptane (g/g DM) degree (%) 1.19 6.32 3.6 95

Example 9

(27) The individual material flows were pre-mixed, so that the mass proportions as indicated in table 9 could be obtained. A medium residence time of 3 min was set up. The subsequent extraction was performed according to the indications in the introductory part of the examples.

(28) TABLE-US-00009 TABLE 9 Extraction KOH (g/g DM) EtOH (g/g DM) Heptane (g/g DM) degree (%) 2.02 5.27 3.6 92

Example 10

(29) The individual material flows were pre-mixed, so that the mass proportions as indicated in table 10 could be obtained. A medium residence time of 5 min was set up. The subsequent extraction was performed according to the indications in the introductory part of the examples.

(30) TABLE-US-00010 TABLE 10 Extraction KOH (g/g DM) EtOH (g/g DM) Heptane (g/g DM) degree (%) 2.03 5.3 3.6 94

(31) As can be seen from the above, the processes of the prior art are disadvantageous due to the high consumption of solvent, basic solution and organic extraction compound. Moreover, the processes of the prior art are afflicted with the disadvantages that emulsions are formed if the amounts of alcohol is decreased. Consequently, subsequent separating of organic and aqueous phase is possible after several hours of standing or by using centrifugation. Moreover, the methods of the prior art are not suitable for a continuous processing of a reactor. However, continuous processing is necessary in order to process amounts of a culture medium as high as 60,000-200,000 liter, according to the size of the reactor, e.g. in case of a pandemia. In order to economically run a continuously working reactor, the residence times of the single steps of the processes have to be in the range of 1-30 minutes.

CITED DOCUMENTS

(32) EP 2 268 823 U.S. Pat. No. 5,460,823 FR-A-2 975 705 Bhattacharjee P. and Singhal R. S., World Journal of Microbiology & Biotechnology 19: 605-608 (2003) Beltran G. et al., International Journal of Food Microbiology 121: 169-177 (2008) Germann et al., J. Biol. Chem. 280:35904-35913 (2005) Kelly G. S., Alternative Medicine Review 4: 29-36 (1999) Kockmann N., Chemie Ingenieur Technik 84: 646-659 (2012) Kohno Y. et al. Biochimica et Biophysica Acta 1256: 52-56 (1995) Kuchta et al., FEMS Microbiol. Lett. 150: 43-47 (1997) Leber et al. European Journal of Biochemistry 286: 914-924 (2001) Mantzourido F. et al., J. Agr. Food. Chem. 57: 6189-6198 (2009) Mantzourido F. and Tsimidou M. Z., FEMS Yeast Res. 10: 699-707 (2010) Mountfort K. A. et al., Anal. Chem. 79: 2650-2657 (2007) Paltauf F. et al., Biochimica et Biophysica Acta, Lipids and Metabolism 712 (2): 268-273 (1982) Pasrija et al., J. Antimicrob. Chemother. 55:905-913 (2005) Polakowski et al., Appl. Microbiol. Biotechnol. 49:66-71 (1998) Ryder et al., Biochem. J. 230: 765-770 (1985) Ryder et al., Antimicrob. Agents Chemother. 20: 858-860 (1986) Sambrook J, Fritsch E. F. and Maniatis T., Molecular cloning: a laboratory manual. Cold Spring Harbor Press. Cold Spring Harbor (1989) Singhal R. S. et al., Journal of Food Quality 13, 375-381 (1990) Sanati et al., Antimicrob. Agents Chemother. 41: 2492-2496 (1997) Shang F. et al., Journal of Bioscience and Bioengineering 101: 38-41 (2006)