JPH0365948B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a method for producing wax esters that quantitatively converts the storage polysaccharide paramylon accumulated in the cells of the protozoan Euglena into wax esters simply by maintaining the cells under anaerobic conditions, and a method for producing wax esters. This invention relates to a method for producing higher fatty alcohols and higher fatty acids by saponification. Chemically, wax esters are esters of higher fatty alcohols and fatty acids, and are used to protect the epidermis of animals and plants.
It has the effect of maintaining moisture and preventing heat loss, and is industrially manufactured as a polishing agent, sizing agent, medicine, and raw material for cosmetics, but it is also manufactured using whale oil wax, wool wax, beeswax, cotton wax, carnauba wax, etc. It relies on the isolation and purification of naturally produced wax. All of these raw materials are difficult to obtain in large quantities, resulting in expensive products. In addition, higher fatty alcohols are surfactants,
It is used for medicines, cosmetics, etc., and in principle it can be produced by a chemical reduction reaction of higher fatty acids that can be easily obtained from neutral oils and fats. In reality, a considerable amount of wax is produced by saponifying naturally produced waxy substances. In view of this situation, if a method for obtaining wax esters easily and inexpensively was developed, it would make a significant contribution to the above-mentioned industrial fields. The present inventor discovered that the protozoan Euglena quantitatively converts the storage polysaccharide paramylon accumulated within its cells into wax ester by a type of fermentation phenomenon simply by placing the cells under anaerobic conditions. By technologically developing the phenomenon, we have devised a new method for producing wax esters, and by saponifying the wax esters obtained here using known methods, we can produce large quantities of higher fatty alcohols and by-products at low cost. He invented a method for producing higher fatty acids. Euglena is a single-celled protozoan, and although it is slightly larger than bacteria and yeast, it can be cultured and grown in the same way as these microorganisms, and can withstand mass production. Although Euglena can be grown under a variety of culture conditions, it always contains β-1,3 as a storage polysaccharide within its cells.
- Accumulates paramylon, a glucan, and the amount accumulated varies depending on culture conditions. According to research by the present inventors, when Euglena cells cultured aerobically are maintained under nitrogen pressure, that is, under anaerobic conditions, paramylon is rapidly decomposed and wax esters are quantitatively produced. By skillfully utilizing this principle, it is possible to efficiently produce large amounts of wax ester at low cost, and since the technology uses microorganisms, it is beneficial in terms of energy and environmental hygiene. Euglena used in this invention refers to species, varieties, and mutant strains of protozoa belonging to the genus Euglena in the zoological classification, and a representative example is Euglena gracilis Z strain (Euglena gracilis).
gracilis Z), Euglena gracilis var. bacillaris,
Examples include Euglena viridis and Astasia longa. Euglena naturally inhabits natural water systems such as ponds and swamps, so it is also possible to collect and use these. Furthermore, chloroplast-deficient mutant strains of Euglena obtained by known methods such as ultraviolet treatment, heat treatment, and antibiotic treatment can also be used. The medium used for culturing Euglena is Koren-Huttner.
and Hutner; J. Protozool. 14 , Supple. 17
(1967)), Hutner; J. Protozool. 6 ,
23 (1959)), Cramer and Meyer (1959)
The carbon source may be glucose, starch hydrolyzate, molasses hydrolyzate, glutamic acid, acetic acid, ethanol, etc., and the nitrogen source may be ammonia or ammonium. Any medium containing an appropriate combination of salts, glutamic acid, aspartic acid, etc., to which inorganic salts such as calcium, magnesium, manganese, iron, and trace amounts of vitamins B ₁ and B ₁₂ may be used. A quantitative ratio (C/N) of carbon source to nitrogen source is used in the range of 4 to 30, with relatively high values being advantageous. Culture temperature is 20℃ to 33℃, especially 27℃ to 29℃
is appropriate, and the initial pH of culture is 3.0 to 7.0, especially
3.3 to 4.5 is appropriate, and light irradiation is 400 to 10,000
Lux can be used, but it can also be done in the dark, or in twilight with indoor light without any particular irradiation. For culturing, it is desirable to perform shaking 50 to 250 times per minute or moderate agitation. The appropriate aeration rate is 0.4 to 2 liters per minute. By culturing under such aerobic conditions, Euglena reaches the stationary growth phase in about 4 to 7 days, the cell yield is 10 to 20 g as a dry weight per liter of culture solution, and the amount of paramylon contained in the cells is Although it varies greatly depending on the medium and culture conditions, it is 100 mg to 800 mg per gram of dry cell weight. In order to increase the yield of wax ester, it is desirable that the amount of paramylon contained in Euglena cells be as large as possible. For this reason, it was more effective to use a chloroplast-deficient cell line rather than a wild-type cell line containing chloroplasts and to culture it in the dark in a medium containing a relatively large amount of glucose. To maintain Euglena cells containing paramylon under anaerobic conditions, cells are isolated from the culture medium by centrifugation or other operations, suspended in an appropriate buffer or water, and nitrogen is passed through the mixture. This can be achieved by At this time, the pH may be any value as long as it does not become extremely acidic or basic, and the presence or absence of light irradiation does not affect the conversion to wax ester. The holding temperature is 27°C to 33°C, with approximately 30°C being appropriate. Usually, the conversion of paramylon to wax ester is completed in 1 to 3 days, and at this time the amount of paramylon contained in Euglena is drastically reduced to 10 mg to 25 mg (dry weight) per 1 g of cell dry weight, and the wax ester content is 150 mg. or 400 mg. Anaerobic treatment of Euglena cells can also be carried out by isolating the cells as described above and then subjecting the culture solution as it is to anaerobic conditions by nitrogen aeration without resuspending the cells. In either case, it is desirable to stir gently to prevent cells from settling and secreting mucus. To isolate wax esters from Euglena cells containing sufficient wax esters, collect the cells from suspension or culture by centrifugation, and collect the lipid fraction by a known method using a chloroform-methanol mixture. The wax ester is extracted from this fraction by silica gel column chromatography using benzene-hexane (6:4). Removal of the solvent yields the wax ester as a solid. Further, wax esters are recovered in the form of a film on the surface layer by disrupting Euglena cells in suspension or in a culture solution using physical force such as ultrasound and centrifuging. This can also be purified by fractional precipitation using various solvents. An example of the composition of the wax ester obtained here is shown in Table 1, and the main component is a wax ester with 28 carbon atoms, which is composed of myristyl myristate, that is, a fatty acid with 14 carbon atoms, and a fatty acid with 14 carbon atoms. It is an ester of 14 fatty alcohols. Other wax esters with carbon numbers around this number are included, and odd numbers indicate that either the fatty acid or the fatty alcohol has an odd number of carbon atoms. Furthermore, one of the characteristics of the wax ester obtained by this method is that it is mostly saturated wax ester, and there is extremely little unsaturated wax ester. If necessary, unsaturated wax esters can be separated by adding silver nitrate to silica gel.

[Table] Wax esters isolated from Euglena cells are saponified by a known chemical method using ethanolic potassium hydroxide or an enzymatic method using a suitable esterase in the presence of a suitable surfactant, and then equimolar of higher fatty alcohols and higher fatty acids can be obtained. Both are extracted from the saponified mixture with a suitable solvent such as hexane, and eluted with a suitable solvent such as petroleum ether-ether-acetic acid (80:20:3) by silica gel column chromatography. By doing so, they can be separated from each other. Separation between the two can also be achieved by various known reverse phase chromatography and other column chromatography. Wax esters and higher fatty alcohols isolated from Euglena cells maintained under anaerobic conditions can be used for their own specific purposes. Higher fatty acids are also used accordingly, but by re-adding them to the Euglena medium they can also serve as raw materials for new wax ester synthesis. The present invention will be further explained below with reference to Examples. Example 1 A wild strain (green strain) of a mutant Euglena gracilis bacillus was irradiated with light at 2500 lux and treated with 1% glucose, 0.1% ammonium phosphate, and 0.01% of a metal salt mixture (see Koren-Hutner reference above).
%, Vitamin B ₁ 0.0001%, Vitamin B ₁₂ 0.00001%
Inoculated into 150 ml of a medium containing
0.5 liter) and culture on a shaker (120 strokes per minute). After 6 days, cells were collected by centrifugation and added to 25mM phosphate buffer (PH6.8).
Nitrogen gas is passed through this at a rate of 20 ml per minute so that the aeration also has the effect of stirring the suspension. Nitrogen gas ventilation is performed at 30°C under indoor light. After 48 hours, cells were disrupted by sonication, lipids were extracted using a chloroform-methanol (1:2) mixture, the extract was concentrated, loaded onto a silica gel column, and benzene-hexane (6:2) was used.
4) Elute the mixture, separate the wax ester fraction, and then remove the solvent to obtain the pure wax ester mixture. The yield was approximately 0.4 g per 150 ml of culture solution. When using a Euglena wild strain, a small amount of unsaturated wax ester is present, which can be separated by adding silver nitrate to a silica gel column if necessary. Depending on the purpose of use of the wax ester, it is not necessary to specifically separate and remove the unsaturated wax ester. Example 2 Cells of a chloroplast-deficient Euglena gracilis Z strain were treated with molasses 2%, ammonium sulfate 0.1%, metal salt mixture 0.01%, vitamin B ₁ 0.0001%, vitamin
Inoculate 200 ml of medium containing 0.00001% _B12 and incubate in the dark.
Incubate at 27° C. with aeration (200 ml per minute) and agitation. After 5 days, the cell concentration was 2×10 ⁷ cells per ml (dry weight 1.5 g), and the intracellular paramylon content was 700 mg per 1 g of cell dry weight.
without separating cells from this culture medium in the dark.
Bubble with nitrogen gas at 29°C and continue stirring gently for 50 hours. This operation reduced the paramylon content to 50 mg/g of cell dry weight, while the wax ester content decreased to 1 g of cell dry weight before nitrogen gas aeration.
The dose increases from 1mg to 400mg per serving. Cells were collected by centrifugation of the culture solution, lipids were extracted with a chloroform-methanol (1:2) mixture as described in Example 1, and about 0.6 g of wax ester was extracted by silica gel column chromatography. was separated.
As shown in Table 1, the wax ester thus obtained had a main component having 28 carbon atoms.
It contained almost no unsaturated wax esters. Example 3 1 g of wax ester obtained by anaerobically treating Euglena cells was mixed with 1 g of 6% potassium hydroxide.
was added to 10 ml of 95% ethanol in which 6% potassium hydroxide had been dissolved, and the mixture was left at room temperature for 12 hours for saponification. The reaction solution was adjusted to pH 1 with 2N hydrochloric acid and extracted twice with 200 ml of n-hexane. Concentrate the extract,
After drying, the residue was dissolved in chloroform, subjected to silica gel thin layer chromatography, and fatty alcohol and fatty acid were mutually separated using a mixed solvent of petroleum ether-ethyl ether-acetic acid (80:20:3). After separation, each separated fraction was extracted with chloroform, the silica gel was removed by centrifugation, and the mixture was concentrated to dryness. The yield of higher fatty alcohol and higher fatty acid was about 420 mg each, and the recovery rate was about 85%. Example 4 After culturing Euglena according to the method of Example 1 or 2, anaerobic treatment was performed, and cells were collected by centrifugation. Cells with a wet weight of 10dl were subjected to ultrasound (10KC,
The mixture was centrifuged (15,000 rpm, 30 minutes) to separate the surface fat layer, which was extracted using 100 ml of trichlene.
After removing the solvent, the residue was recrystallized from ethanol to obtain a crude wax ester in a yield of about 2 g. This was dissolved in 25 ml of petroleum benzene at a warm temperature, cooled, and the precipitated portion was subjected to alumina chromatography and eluted with carbon tetrachloride to obtain about 1.4 g of purified wax ester. This was saponified using ethanolic potassium hydroxide according to Example 3, the fatty acid mixture was extracted as a calcium salt with acetone, the free acid was obtained using 1N hydrochloric acid, and the methyl After conversion to ester, it is fractionated into fatty acid esters of each composition by vacuum distillation. This is subjected to acid hydrolysis to separate and collect higher fatty acids of each chain length. The total yield of purified higher fatty acids was 0.6 g. On the other hand, the higher fatty alcohol portion of the saponified mixture was purified by alumina chromatography again, and then converted into an acetylated product using acetic anhydride, which was then fractionated into each constituent alcohol by vacuum distillation.
A total yield of 0.5 g of purified higher alcohol was obtained by post-saponification and fractionation.

Claims (1)

[Claims] 1. By culturing the protozoan Euglena aerobically and then maintaining the cells under anaerobic conditions, the storage polysaccharide paramylon is quantitatively converted into wax ester, which is then purified by cell disruption or solvent extraction. A method for producing a wax ester, which comprises isolating the wax ester. 2. When culturing the protozoan Euglena aerobically, a chloroplast-deficient strain is used, and a large amount of glucose or a carbohydrate that provides glucose and/or fructose is used as a carbon source in the medium, resulting in cells that accumulate a large amount of paramylon. The culture method according to claim 1, which uses 3. Wax esters produced by culturing the protozoan Euglena aerobically and then keeping them under anaerobic conditions are isolated, or they can be treated chemically or enzymatically while still contained within the cells or in a cell-disrupted mixture. Saponify the wax ester using a method that
A method for producing higher fatty alcohols and higher fatty acids, which comprises then extraction and isolation.