JPS58165791A - Production of squalene - Google Patents

Abstract

PURPOSE:A filamentous fungus in Mortierella is cultured in a medium containing carbon hydrate or hydrocarbon as a carbon source to produce squalene from the culture mixture in high yield. CONSTITUTION:Carbon hydrates such as glucose, starch or the like and hydrocarbons such as kerosine, n-alkane or the like are used as carbon sources. Further, nitrogen sources such as ammonium nitrate or urea, inorganic salts such as potassium dihydrogen phosphate, socium chloride or the like are used for the culture medium. A filamentous fungus in Mortierella is cultured in a liquid medium through standing, vibration or aeration and agitation process at a pH of 4-6 and 10-33 deg.C for 5-30 days. The squalene is collected by separating cell bodies, extracting them with a solvent to collect lipids and subjecting the extract to a liquid chromatography using silicic acid as a filler to separate squalene from the lipids.

Description

[Detailed Description of the Invention] Squalene is contained in a large amount in the liver oil of a special deep-sea shark, and squalane obtained by hydrogenating it is known to be used as a base oil for cosmetics.

Squalene, a precursor of sterols, has also been found as a lipid component in animals, plants, and microorganisms. The existence of squalene as a lipid component within microorganisms is known, but the amount of squalene present in filamentous fungi is at most 0.01% of the dry bacterial weight, and usually from 0.01% to less. [Suzuki, Yokochi, Oil Chemistry, current, 52 (19
82)) Although an industrial synthesis method for squalane has already been established,
(1978)) When seeking a thick phase other than shark liver oil for the production of natural squalene, production by accumulation in microbial cells has the highest practical significance. As a result of finding microorganisms with high squalene content, increasing the amount of accumulated squalene by changing culture conditions, and producing fats and oils using microorganisms, Morteierella. Isabelina (Mortie-rella.
isabellina) TPO7884, 78
The present invention was completed based on the discovery that filamentous fungi such as No. 24 can produce squalene at an unprecedentedly high level in a medium using carbohydrates or hydrocarbons as a carbon source.

That is, the present invention is a method for culturing a filamentous fungus of the genus Morteierella in a patent characterized by carbohydrates or hydrocarbons, and producing squalene from the culture.

Specific examples of the bacteria used by the present inventors include the above-mentioned filamentous fungi, but any Mortierella genus bacteria can be used as the bacteria of the present invention.

Incidentally, the above-mentioned bacteria are all produced by the Fermentation Research Foundation; decane, undecane, hexadecane, pentadecane, etc. are used. In addition, as a nitrogen source, for example, NH4NO3, (
Inorganic nitrogen sources such as Nl-I4), , 804, etc., or urea, peptone, corn extract, corn, stave.

Organic nitrogen sources such as liquors are used.

Examples of inorganic salts include KH2PO4, K2T (PO4
, NaC1! .

Pe5o4.7H20, MgSO4,7I-120,z
nso4.7H20& is used. Add trace elements and other nutritional sources as necessary.

The above-mentioned filamentous fungi are usually cultured in a liquid medium by static culture, shaking culture, aerated agitation culture, or the like.

Culture medium) The pH is preferably 40 to 60, and the culture is usually carried out at 10 to 33°C for about 5 to 30 days. Thus, squalene is produced in the culture. When collecting squalene from a culture, it is easily separated from the culture because squalene is contained in the lipids in the cells of filamentous fungi.

Next, embodiments of the invention will be shown, but the present invention is not limited thereto.

Example 1 n-decane 12.5d, Kl-T2PO42ji
, Mg504- , +7H20Q, :3 g
, N a C110.1g, F e S04.7 H
2010mg.

CaCl! 2-2 O20], Om9, Cu
S 04-5 O200, 2”FA.

ZnSO4,7I(201,0m9.MnC12,4H
20LOm9゜'I"hi, amins -T(Cl
2mg, I)-Biotjn Ooo 2mg and NI (4No30.91g, Arui (NH
40.94 g of 4112So (C/N ratio (weight of carbon atoms in n-decane/weight of nitrogen atoms in nitrogen source) are both 24.1) was mixed with 10,100 O of deionized water, and I) H
was adjusted to 46.

Put 400ml of this synthetic medium into 11 Erlenmeyer flasks,
Each strain was inoculated and incubated at a specified temperature for a specified period of 150 hrs.
Shaking culture was performed at rpm. After culturing, chloroform-methanol (2: IV/V
) was added and homogenized in the presence of glass peas to simultaneously crush the bacterial cells and extract the lipids. In order to perform the extraction completely, this was repeated five times and all the extracts were collected. After the above-mentioned extract was purified by the pl and och distribution washing method, the solvent was distilled off under reduced pressure, and the total lipid amount was measured by gravimetric method.

5- The lipids extracted from the bacterial cells and precisely weighed are made using silicic acid as a filler.
The lipids were separated into neutral lipids and polar lipids by column chromatography using chloroform and methanol as developing solvents. Since squalene is contained in neutral lipids, the squalene content was determined by gas chromatography analysis of the separated neutral lipids. [Squalene analysis method, precious wood, Yokochi, oil chemistry, ±1, 52 (]
982)) From the results of Table 1 for Mortierella filamentous fungi determined by this method, the strain Mortierella
ellaieabellina IFO7824 uses n-decane as a carbon source, regardless of the culture conditions, the squalene content is 0.4% or more, and the amount of squalene produced in the medium is 10. A value of 0 m9/1 or more was observed, and if the culture conditions were selected, a production amount of 50 m2/1 or more was found.

6- -7- JP-A-58-IG5791 (3) Example 2 Glucose 30,9. Kii, PO43, @ , (
Nl14)2SO43,1,9, MgSO4,7H20
0.5g, NaCe 01! j.

Fe, SO4, 782010m9. ca(472,2H
,,010m9.

CuSO4,5I-f□00.2m9. ZnSO4,
7H201, O+ng.

MnC13,4H2O], Qm9. Thiamine
-HCl 2mg, l) -Biotin Q, 0
Mix 2 mg in 1000 ml deionized water and adjust the pH to 46.
Synthetic medium adjusted to (C/N ratio at this time is 1), 4
) 400m1f ] 11 Place in an Erlenmeyer flask and inoculate with each bacterial strain, and heat at the specified temperature and 150 rpm for the specified period.
1.2 mg per medium even when lucose is used as a carbon source]-
/ 7 also found to produce more than one squalene.

8- 9-

Claims (1)

[Claims] (1) A method for producing squalene, which comprises culturing Morteierella bacteria in a medium containing carbohydrates or hydrocarbons as a carbon source, and collecting squalene from the culture.