JPS61254193A - Production of unssaturated wax ester - Google Patents

Abstract

PURPOSE:To obtain unsaturated wax ester suitable as a substitute raw material for a sperm oil in high yield of production, by cultivating aerobically Euglena of a protozoan and changing a medium to an anaerobic state. CONSTITUTION:A microorganism of the genus Euglena (e.g., Englena gracillis Z strain) is inoculated into a medium consisting of a carbon source (e.g., glucose), a nitrogen source (e.g., NH4NO3 or peptone), ca, vitamin B1, etc. At the early stage of culture or after the mold is grown at a certain degree, 0.1-5wt% based on the medium of an unsaturated fatty acid (e.g., linoleic acid) is added to the medium, and the mold is cultivated at 20-33 deg.C at 3.0-7.0pH under an aerobic condition such as aeration or stirring for 3-6 days. Then when the growth of the mold in the medium is approximately in a stationary phase, nitrogen is introduced to the medium which is changed to an anaerobic state, the medium is kept in the state for 1-5 days, unsaturated wax ester is accumulated in the mold cell and it is extracted and separated from the mold.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to the protozoan Euglena,
The present invention relates to a method for producing unsaturated wax esters in high yield by culturing Euglena (euglena).

Chemically speaking, wax esters are esters of higher aliphatic alcohols and higher fatty acids, and have 11 functions such as protecting the epidermis, maintaining moisture, and preventing heat loss.
Industrially, it is used in large quantities as a lubricant, polishing agent, 1σ water solution, raw material for cosmetics, etc.

Conventionally, unsaturated wax esters have mostly been produced from natural products derived from animals and plants (1), and the supply of (1) has not always been stable.

In particular, wax esters suitable as high-quality lubricants are obtained from whale oil, but in recent years there has been a need for alternative raw materials due to supply concerns stemming from the ban on whaling.

Matukola whale oil is a linear Nisdelic acid consisting of a long-chain fatty acid and a long-chain alcohol with carbon numbers of 14 to 18, respectively.
It is unsaturated with one double bond.

This Matukora whale oil substitutes 4 raw materials for deep-sea fish A-range roughfish (1-1oplostethus atl).
anticus) oil, plant jojoba (3 immonds)
ia chinensiS) oil is attracting attention. Orange roughy oil has 16 to 22 carbon atoms each, and is mainly composed of saturated or unsaturated esters of long-chain fatty acids and long-chain alcohols having one double bond. ) jojoba oil has carbon numbers of 20 and 22, respectively.
The main components are unsaturated long-chain fatty acids and unsaturated long-chain alkylene esters, each of which has a double bond. These (matsu kola whale oil) have a similar effect to oil, and are used as raw materials and oil, but the former has restrictions on blood crimes, In addition, the latter requires a long period of time to harvest from the 1IIIi seeds, and there is no sufficiently stable source of supply. 41-yield η-
There are known methods to do this. This b) method is excellent because it allows for rapid growth of micro-products and easy adjustment of productivity.

According to the conventional technology, as a method of protecting wax esters from a physical standpoint, M
erificans to produce valmisan Fl'2 from AIJJ゛kan (U.S. Pat. No. 3/109,506), protozoa to 1-Gnorl, J-Li, carbon number 26... Method for producing 30 saturated II 1-chain wax Nisdel (Japanese Unexamined Patent Publication No. 118090/1989)
No.), Parmidin PI9 Sebru from Rhizocane using Nocardia h'Al1il bacterium ', 1
i'i''zuru person 〈 △ dv △ p
pl M 1crohio1. 1/I
, 5 (1971 I') p. 93), △ci in
Using bacteria of the genus C.I.A.C.T.E.R., saturated hydrocarbons are converted to pine-]whale oil, 1゛)4, and a chemical similar to small butterfly oil. Method (U.S. 121st Edition 7'I/I O/I 2
B3'';5) and the like are known.

131.1 of 131.1, which is solved by the invention.
All were saturated 1 sox-esters. Pine - one j! ：J oil must satisfy 1) as a lubricant (as a lubricant, as a lubricant, 114 catty properties, fire resistance 1), and therefore constitutes 1m + 1, wax ester. It can be omitted that the number of carbon atoms in fatty acids and alcohols is 17'IJx 10, and that it is necessary to have an unsaturated 211 bond in at least one of the fatty acids and alcohols. ing. Follow (, front) small J, eel saturated wax 1-stell, pine-] one?・:t 1
Tll's %j jBij%'! (as a person) is not unreasonable.

(Previously) Bo's traditional method patent for use of rice/I40/l
If the method of No. 283 is used, unsaturated wax nitrogen is produced, but the productivity is low and although it is a preferred method, it is not suitable.

The present invention has been made in consideration of such circumstances, and by culturing animal Euglena, it is possible to obtain lubricating oil, etc. to produce suitable unsaturated wax esters in high yields.
The purpose of this is to provide a method for

As a means of solving the problem, the present invention provides a method of cultivating protozoa - Grena in a medium aerobically, adding a fatty acid to the medium, and then shifting the medium to an anaerobic state. It is a characteristic.

The coater used in the present invention is I-LSI, which is a protozoan that belongs to the genus Euglena (genus Euglena) according to the zoological classification, and includes Masu, Hengeshi, and Mutant strains belonging to this genus. A representative example is Coglena gracilis 7 strains (F 1ljJlenil QI'aC!
l :S7), Fuglena (1raciliS bac
illiaris),] - Gubreno hiridis (FF u(11ena viritlis),
Examples include Astasia longa (△StaSialonga).

]-Gubure naturally inhabits natural water systems such as ponds and swamps, and it is also possible to collect and use them1.They can also be treated with ultraviolet light, heat, antibiotics, Various mutant strains treated with known methods such as 21-herosoguanidine treatment can be used.

The medium used for the cultivation of Euglena is -1-Len Hachner medium (Journal
our own of Protozoology)
1 / l 3 < 196 F [) (enlarged p. 17)), Hatsu I-ner medium (S 17-) -l Δbup [11 to Zo Δ Roshi vol. 16 (1959) p. 23], etc. Known media can be used.

In addition, the nitrogen source is nitrate-ammonium, nitrate-ammonium, etc., using glucose, starch hydrolyzate, molasses hydrolyzate, glutamic acid, acid resistance, ethanol, etc.
,/7 soil - ammonium oxide, etc. 2 ~ s, -'' - nsboob liquor, etc. (if how many nitrogen 1 element, j Nishi fil contains 11, oh, calcium, nucnesium, 7 nkan, 8th grade, l!!!, 1. ] oil and vitamin Strebium B124 (respectfully 1111). , 1) Tesaru.

Ding 7 - Kurena's Cultivation 1:,! The temperature is 20 to 33 degrees Celsius, iV+, Netoko fil 1-1 fit, 3.0
□-7,0 is iia 3. It is p.

j and 1 anzu are light 04'-F:! ;4 can be done with 11j'i black bottom 1f, yt, taIf'; 3
:'! For II-in, use a shaker at a rate of 50°/2F]O times per minute, or perform moderate aeration stirring! It's adorable.

By culturing under 'l'l' airways A'1, the stationary phase of growth was reached at about 30 to 30 ml, and '1 Ill fl follicles were grown. 11'11'
・If'<j'¥<12 dryness per 1Q of liquid! ``・It's about 10 to 20 grams at first.

In the present invention, the vesicles added to the culture medium 41!
Gal'lein 0 (2, bon 1 ~ inic acid, se 1 ~ lein 6
1. Erucic acid (tq) is mentioned (J), but it is also possible to use [111% of wax varnish, 4j% of wax varnish, various fatty acids depending on silk formation, 8 types of amine salts thereof, and metals]. Dezaru.

Addition of unsaturated fatty acids j) to the medium is from 0.1 to 5
% 1α, and 0.5 to 2% culm degree is appropriate for 1'1. 1% or less, more preferably 0.2 to 0.5%
It is better to spell it out.

The addition rtl and period of these unsaturated fatty acids to 18 regions are as follows:
After the start of culture, it may be 3 times. Added to the medium from the beginning L/-U i15 i (4:>
J, I (1) 'r Alh', Mf y! '7')
If high-temperature fat 117 + acid is present at the beginning of 1v1, it will delay the growth of the dormant, so it is preferable to add it after the bacteria has grown to a certain extent.

In addition, when transferring the medium to an anaerobic state in the culm of the culm, it is desirable that the added 1) and 1) fatty acids have been taken into the cells by the anaerobic state. It is most preferable to add it 1 to 2 times before the transition to the state.

Of medium! The transition to +1F air condition is carried out by passing nitrogen into the medium. The timing of the transition is during the stationary phase of Coglena growth (preferably around 1.5 pm; the medium is shifted to an anaerobic state).
After ζ, unsaturated wax Nisdel is accumulated in the cells by maintaining the state between 1 and 5B.

In order to accumulate 1 NOx ester in the cells in this way, remove the culture solution/infected bacterial cells by a method such as Raba or centrifugal tube, and extract the lipids using a silk paste. Subsequently, the wax stellate is separated from the lipid.

For extraction of fat V, wet bacterial cells are chloroform/methanol d
This can be done by using a liquid extraction process blade.
Also, from this lipid, wax esters are extracted using a column using silica gel [1Madograph-r-, etc.]. ).

Action = 9 - Hugue 1 is a protozoan with 111 cells, which is larger than bacteria -) yeast, etc., or it can have a 111-cell structure similar to these microscopic organisms, Can be produced in large quantities. [Also]-Cultivate Coufre aerobically 1. :+'A! It is stated in the above-mentioned Japanese Patent Application Laid-Open No. 11n 59 that wax-■-stellate is formed when the wax is kept under atmospheric conditions.
-118090 is known as d3, and its 41-product has a dry bacterial weight of up to 50% of 1, and has an extremely high δl)C productivity.

, by Corklena! The wax ester generator B+f IJl in 111i air conditioner A'1 is believed to be as follows. 1 - That is, when Euglena cultured under aerobic conditions is placed in anaerobic conditions ■, baramin 11, which is a multi-W1 stored under aerobic conditions (! It is decomposed into ri and becomes pyruvic acid through glycolysis 1. Production (Iζ Pyruhin + J: , Alejylcoene IJ"Ino, △l) + r et al fat /
Only one is synthesized. The fat R91J synthesized through this process is a saturated straight chain fatty acid with 11 to 17 carbon atoms, with the center being 14. The Nisdell reaction occurs between both components of A and wax ester is produced. Therefore, in culture in a normal medium, both fatty acids and alcohols are saturated at °C. 1q of wax esters are the only saturated ones.

During the wax ester production process, the vesicle +1/I acid taken in from the culture medium is present in the C1 cells, and some of it is converted into glucose. Between the 4-saturated long-chain al T'-ru derived from -
J-sprue bonds are formed to produce esters of vesicle Japanese 117im and saturated alcohols, and the other part is reduced to unsaturated long-chain alcohols, followed by saturated fatty acids derived from glucose and unsaturated alcohols taken from the medium. Between saturated fatty acids
[It forms a Sual bond.]

Effects of the Invention According to the present invention, in this way Coglena's = 1
1 = In addition to the saturated wax-esters produced during cultivation under normal anaerobic conditions, the unsaturated wax-esters derived from the vesicles added to the medium were added to the culture medium to efficiently (1-) This is possible.

Therefore, according to the present invention, 1.
CJ-Tari, which has a high proportion of unsaturated double bonds in its components,
It has a chemical structure similar to that of Matsukora whale fill, and can be used as an alternative raw material for a wide range of applications such as lubricants.

However, 21-grena is a protozoan that is extremely easy to obtain, and by culturing it, it is possible to stably supply unsaturated wax esters in large quantities.

In addition, in the intracellular accumulated paramylon, the supply amount of the enzyme Δ source such as glucose from the medium, and the JF
4 if! ! By adjusting the amount of unsaturated fatty acid added to the rod and its feed tank, it is possible to change the composition of the wax ester produced.

Example = 12 = Examples of the present invention will be described below.

Example-1 Glucose 2g, Voripeb 1-0.5g, ammonium sulfate 25mQ, ammonium bicarbonate 25mQ, diammonium phosphate 25m01 magnesium carbonate 60mq
, calcium carbonate 12mg, Mohr's salt 5m9, manganese sulfate hydrate 1.8mg, zinc sulfate heptahydrate 2.5mg
, vitamin B+ 0.25mg and vitamin BI20.
0005…q was dissolved in 100 mQ of deionized water, and the pH
was adjusted to 4.5. This was placed in a 500 mQ volume shaker flask to serve as a medium, and Euglena gracilis 7 strains were inoculated therein, shaken 120 times per minute, and cultured in the dark at 28°C for 3 days.

After that, Olein M! manufactured by Nippon Oil & Fats Co., Ltd. 2 (7 Ruin A
1293.5%, linoleic acid 1.7%, stearic acid 2
．． 4%) was added thereto, and the culture was continued for 1 day.

Next, transfer the entire contents of flask T to a 200 ml H-necked round bottom flask (), adjust the pH to 6.5, and add 2O'C.
Cultivation was continued for an additional 5 days while gently stirring and aerating nitrogen gas under room light.

Next, the bacterial cells were collected by centrifugation, washed twice with 0.85% saline, and then immersed in a mixture of chlorol 11/methanol (1:1). Homogenization was performed using waves to disrupt the bacterial cells and extract lipids. The extracted lipids were loaded onto a silica gel column and eluted with hexane/ethyl acetate (10:0.5) ifP solution to obtain the pure wax ester fraction.

The yield of wax ester is 100mf of culture solution! Approximately 0.85 hit! In II.)1.

The separated wax ester was saponified with an alkali according to a conventional method to separate the fatty acid component and the alcohol component ('J). The results are shown in Table 1.

= 14 = Execution 1]-2 Seven wild strains of E. noglena gracilis were grown in Koren-Hara 1-ner medium with 1% glucose as the main carbon source in a 500-capacity long-neck shake flask. Inoculated to 100 resistant,
After culturing for 14 days, the cells were cultured at 28°C under nR black while shaking 120 times per minute at First Light 11)-13,5.
Collect cells by centrifugal force l (19 A rain?!+
2 (same as Example-1) and resuspended in 100ynQ]-Len Hatsu]-ner medium <pl-17,0),
In the same manner as in Example 1, the cells were cultured for 5 days at room temperature (10-15° C.) under nitrogen gas.

Thereafter, the cells were collected in the same manner as in Example 1), the lipids were extracted, and the wax ester was separated. The yield of wax ester is approximately 0.0% per 100 ml of culture medium. '1
．． It was OQ. The composition of the constituent components of the wax Nissal was as shown in Table 2.

Example 3 The unsaturated fatty acids added to the culture medium were linoleic acid 88.7%, A-leic acid 9.8%, α-lylunic acid O 95%, manufactured by Wako Pure Chemical Industries, Ltd. Palmitin Mt
The procedure was the same as in Example 2 except that 1q of -C wax ester was used, except that 19% of 0.6%) was used. 11 fβ (approximately 0.80 per 100 mf! of one medium!), and the composition of its constituent components is as shown in Table 3.

Example-4 As an unsaturated fatty acid added to 18 bases, α-lylunic acid (α-lyllun general 7
2.7%, linoleic acid 19.7%, A-leic acid 7.1%
) A wax ester was obtained in the same manner as in Example 2, except that 19 was used. The yield of wax ester is medium 10.
It was about 0.91q per 0mQ, and the composition of its constituent components was as shown in Table 4.

From the above examples, the unsaturated fatty acids taken into the cells of Euglena from the medium and the unsaturated long-chain alcohols that were reduced by the long-chain fatty acid (long-chain fatty acids) that Euglena has, respectively. It can be seen that the unsaturated wax esters, which have different constituent components, are produced efficiently.

In addition, as in Example 1, if unsaturated fatty acids are added to the medium and the medium is not immediately converted to an anaerobic state but subjected to aerobic culture, the vesicles 11 flit taken into the bacterial cells.
1I7i n: 2/'l Unsaturated fatty acids with reduced chain length, which is thought to be caused by receiving oxidation, and reduced chain length, which is thought to be caused by reduction of tere. Some unsaturated alcohols are produced.

Claims (1)

[Claims] 1. A method for producing an unsaturated wax ester, which comprises aerobically cultivating the protozoan Euglena in a medium, adding an unsaturated fatty acid to the medium, and then shifting the medium to an anaerobic state.