JPH0213388A - Preparation of lipid containing unsaturated fatty acid - Google Patents

Abstract

PURPOSE:To provide the subject lipid having an improved unsaturated fatty acid content by subjecting a lipid-producing fungus to a feed culture under a condition restricting the supply of a carbon source or to a substitution culture under a condition free of the carbon source. CONSTITUTION:An unsaturated fatty acid-producing fungus such as Mortierella.alpina is inoculated in a medium containing a saccharide such as glucose or fructose as a carbon source in an amount of the most suitable saccharide concentration or less for the growth of the lipid-producing fungus to begin the culture of the fungus. the saccharide is subsequently added to the medium in an amount of the most suitable saccharide concentration or less for the growth to multiply the fungus. The objective lipid is obtained by continuing the culture in a state free of the carbon source in the multiplication stationary period of the lipid-producing fungus or near the period after the multiplication and/or the culture of the fungus is begun.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention improves lipid productivity by culturing lipid-producing filamentous fungi in a medium with a controlled carbon source. The present invention relates to a method for producing unsaturated fatty acid-containing lipids, which is characterized by producing a high proportion of bacterial cells.

(Prior Art) It is essential to maintain homeostasis in a living body, and homeostasis is maintained by coexisting with various physiologically active substances and adjusting the actions in the living body.

These physiologically active substances include prostaglandins (
In addition to PC, there are other important physiological substances such as thromboxane (TX) and leukotriene (LT), which are considered to be members of PC in a broad sense. These PCs.

TXSLT, etc. are all produced in small amounts in living organisms as needed, and are involved in the regulation of various biological reactions in very small amounts, but many of these substance groups are derived from arachidonic acid, an unsaturated fatty acid. It is known that the arachidonic acid metabolic pathway is biosynthesized through an enzymatic reaction, and the entire arachidonic acid metabolic pathway is called the arachidonic acid cascade because it looks like a waterfall that divides into many threads and cascades down in steps. Therefore, arachidonic acid can be positioned as an extremely important unsaturated fatty acid for maintaining living organisms. Thus, it was expected that unsaturated fatty acids could be obtained industrially at low cost and in large quantities.

Because arachidonic acid has a special stereochemical structure,
It is very difficult to supply it at a low cost through chemical synthesis, so extraction and purification have been performed using naturally occurring organisms as raw materials. However, extraction from raw materials such as animal tissues and blood requires a very complicated process, and the raw materials contain only a very small amount of arachidonic acid, making industrial application difficult (M - Written by Kayla, translated by Yamakawa et al., Lipid Research Methods, page 65, 1975, Tokyo Kagaku Doujin). Therefore, as an industrial application, attempts have been made to use microorganisms to separate arachidonic acid from inexpensive and easily available raw materials through simple operations.

JP-A No. 52-64482, No. 52-64483, and No. 52-64484 include the genus Penicillium, the genus Thallatosborium, the genus Mucor, the genus Fusarium, the genus Hormopuntrum, the genus Aspergillus, or the genus Rhodotorula. A method is described in which microorganisms that have the ability to produce arachidonic acid belonging to .

However, the arachidonic acid content in the lipid obtained by this method is 7.5% or less, and the yield per dry bacterial cell is only less than 1%. Japanese Patent Publication No. 58-22199 discloses that a lipid-producing filamentous fungus belonging to the genus Mortierella, which has a high content of γ-lylunic acid, which is a precursor of arachidonic acid, is cultured in a medium using hydrocarbons as a carbon source. A method is disclosed. According to this method, its content can be up to 3
Although the lipid yield reached 4.4%, the yield of lipid was less than 100 μ/l, and the practicality was low. Further, Japanese Patent Publication No. 19154/1983 discloses a method for shortening the induction period of culture by diverting a portion of the culture to the next culture. According to this method, although the production amount of the target unsaturated fatty acid per unit time increases, there is almost no improvement in the lipid content, so it cannot be said to be a sufficiently efficient method.

(Problems to be Solved by the Invention) Therefore, an object of the present invention is to provide a method for culturing filamentous fungi that increases the amount of lipids produced by the lipid-producing filamentous fungi and improves the target content of unsaturated fatty acids. An object of the present invention is to provide a method for producing unsaturated fatty acids.

(Means for Solving the Problems) The present invention provides that when lipid-producing filamentous fungi are cultured under conditions in which the supply of carbon sources is suppressed, the accumulation of lipids within the filamentous fungi increases, and the accumulated lipids increase. This was done based on the knowledge that the degree of unsaturation of the fatty acids constituting the molecule increases, and that the above-mentioned problems can be efficiently solved by this culture method.

That is, the present invention (a) uses saccharides as a carbon source,
Optimal growth of lipid-producing filamentous fungi I! Cultivation of the filamentous fungus is started in a medium containing saccharides in an amount below the concentration, and then growth reaches optimum I!
A method of growing the filamentous fungus by adding sugars in an amount below the concentration, and/or (b) a method of culturing the lipid-producing filamentous fungus in the stationary growth phase or near the stationary growth phase in substantially no carbon source. Provided is a method for producing an unsaturated fatty acid-containing lipid, which comprises culturing a lipid-producing filamentous fungus.

The microorganism used in the present invention may be any filamentous fungus that produces lipids, preferably filamentous fungi that produce unsaturated fatty acids, particularly preferably arachidonic acid, such as Penicillium, Talatosporium, Mucor, Fusarium, Examples include bacteria belonging to genera such as Hormopuntrum, Aspergillus, Rhodotorula, Entomophthora, Delacroixia, Conideiobolus, Phytium, Phytophthora, and Mortierella. Bacteria that belong to these genera and can be suitably used in the present invention include Mortierella albina (M.
ortierella alpfna) IFO856
8, ATCC16266, ATCC32221, ATC
C42430, Mortierella bainieri (Mort
ierella bainferi) IFQ8569
, Mortierella elongata (Morterell)
aelongata) IFO8570, Mortierella exigu
a) IFO8571, Mortierella minutissima
) IFO8573, Mortierella verticillata
) IFO8575, Mortierella hygrophila I
Po 5941 Mortierella policephala (Mor
tierella polycephala) IPo
6335, etc., and all of these stones are owned by the Institute of Fermentation Foundation (IFO) or the American Type Culture Collection (A
mertcanType Culture Co11e
It is a filamentous fungus that is easily available to those skilled in the art and is listed in the bacterial strain catalog of the ``Cction, TCC''.

In the present invention, these filamentous fungi are cultivated by the above-mentioned method (a),
That is, it is characterized by culturing by a fed-batch method (hereinafter referred to as fed-batch method) in which the amount of saccharides is limited and/or the culture method (b), that is, the substitution method.

When performing the fed-batch method here, sugars are used as the carbon source, that is, single 11! Culture is usually carried out in a liquid medium using polysaccharides and polysaccharides. Here, examples of tI include glucose,
Examples include fructose, saccharose, molasses, wood saccharification liquor, and starch liquefaction products. In addition, nitrogen sources, inorganic salts, etc. that can be used in general for culturing microorganisms can be used.

For example, nitrogen sources in the medium include ammonia, ammonium salts, glutamic acid, aspartic acid, urea, etc., and these can be combined as appropriate, and if necessary, potassium, sodium, iron, magnesium, zinc, copper, manganese, etc. It can be used by adding inorganic salts such as, vitamins, etc.

Preferred media include potato, malt, peptone,
Examples include mediums prepared by yeast extract, corn steep liquor, casamino acids, bran, etc., alone or in combination, and further adding tin as necessary; particularly preferred mediums include potatoes, or a mixture of its infusion and carbohydrates, or malt extract. In general, the concentration of the nitrogen source is preferably 0.01 to 5% by weight, preferably 0.1 to 2% by weight, and the initial pH of the medium is suitably 4.0 to 7.0. In addition, when using these media, the temperature is usually 10 to 33°C.
The culture is preferably carried out at 20 to 30° C. for 2 to 30 days, and examples of the culture method include normal culture methods such as aerated agitation culture, shaking culture, and stationary culture.

When producing lipids using filamentous fungi, the sugar concentration that provides a good yield varies depending on the fungus used, so it cannot be definitively determined, but it is generally around 50 to 100 g/l. It is said to have good concentration.

Therefore, in the fed-batch culture method, I! The sugar concentration and the sugar concentration during culture are limited to the optimum concentration ~Z.

For example, when culturing Mortierella bacteria with an optimal dextrose concentration of 50 to 100 g/l, the initial dextrose concentration in the culture solution should be set to 10 to 40 g/l.
It is better to set it to j2. Further, as a method of adding saccharides in a limited manner during culture, there is a method of adding saccharides or an aqueous solution thereof to the culture solution continuously from the start of culture or in multiple portions. - In boat fishing, 1 to 10 g of sugar is consumed per day under normal culture conditions, so add sugar to the medium within a range that does not exceed the consumption amount. For example, when culturing Mortierella bacteria that consumes about 5 g/It of dextrose per day during the logarithmic growth phase, for example,
Just take 25g of dextrose on the 5th and 100th day. Although the sugar concentration may be calculated from the amount of bacteria consumed as described above, it is preferably measured over time during the culture period. An example of this method is the glucose B test (manufactured by Wako Pure Chemical Industries, Ltd.).

In the substitution method described in (b) above, after starting the culture of the fungus, the filamentous fungus is cultured in the stationary growth phase or near the stationary growth phase in a state in which a carbon source is substantially absent. That is, after a filamentous fungus takes in a carbon source into its body and multiplies, if the fungus is continued to be cultured in a state where there is substantially no carbon source, the degree of unsaturation of fatty acids constituting lipids within the fungus increases. Here, "substantially no carbon source" refers to a state in which there is no carbon source available to the bacteria in the culture medium, such as by removing the carbon source from the culture medium or stopping the supply of the carbon source.

Among these, it is best to collect the proliferated bacterial cells, wash them thoroughly with distilled water, etc., and then culture the bacteria in distilled water. still,
When performing replacement culture, it can be performed following the fed-batch method in (a) above, but replacement culture can also be performed after culturing the filamentous fungus under normal culture conditions. Here, as usual conditions, it is possible to use something other than saccharides as a carbon source, but it is preferable to use vHs and the medium described in the fed-batch method in (a) above.

To carry out replacement culture, it is usually sufficient to pass about 1/2 of the logarithmic phase of the culture, preferably in the stationary phase of the culture.
The best time is 3 to 18 days after the start of culture. Normal methods such as gauze filtration may be used to collect the bacterial cells, and then 200 ml of a carbon source-free medium per 30 g of bacterial cells, preferably 1 to 10 times with water, preferably washed with water, is added to the washing solution. Wash the bacterial cells until they become colorless and transparent, then add any amount of
It is preferable to continue shaking or stirring with 100 to 600 ml of a carbon source-free medium, preferably water, per 30 g of bacterial cells. Shaking or stirring is usually 1~
What is necessary is just to continue at 15 degreeC - 30 degreeC for 8 days, Preferably 2 to 4 days. By transferring the washed bacterial cells to a medium that does not contain a carbon source, it is possible to culture them in the absence of a carbon source, but even with this treatment process, the surface of the bacterial cells contains carbon sources. Although some medium is attached, such a state is also included in the above-mentioned replacement method (b) of the present invention.

Most of the lipids produced in the filamentous fungi cultured by the above method are contained within the fungi, so after the completion of the culture, centrifugation,
Or after obtaining bacterial cells by general separation means such as filtration,
As described in JP-A-63-44891 and JP-A-61-177990, the bacterial cells are washed with water, preferably by freeze-drying, etc. After drying, the bacterial cells are ground mechanically or physically, and then the lipids are removed using an organic solvent such as ether, hexane, methanol, chloroform, petroleum ether, a mixture thereof, or supercritical carbon dioxide. For example, the extraction method can be mentioned. Furthermore, instead of the above method, extraction may be performed using wet bacterial cells, and extraction may be performed using a water-compatible organic solvent such as methanol or ethanol. By distilling off the organic solvent from these extracts under reduced pressure, lipids containing high concentrations of unsaturated fatty acids can be obtained.

Furthermore, unsaturated fatty acids such as arachidonic acid and eicosapentaenoic acid can be isolated by decomposing the lipids by conventional methods.

(Effects of the Invention) According to the present invention, lipid-producing filamentous fungi are cultured by fed-batch culture in a state where the supply of a carbon source is limited and/or by substitution culture in a state in which there is no carbon source. It has become possible to improve the amount of lipids used and the content of unsaturated fatty acids in the lipids. Furthermore, by increasing the content, it was possible to reduce the cost of refining unsaturated fatty acids contained in lipids, and at the same time, it was possible to increase the purity of purified products.

(Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited by these Examples.

Example 1. Fed-batch method Add 5 g of dextrose (25 g/11 medium), 1 g of polypeptone (manufactured by Otama Nutritional Chemical Co., Ltd.), 150 g of CaCl, 2H to the leachate obtained by infusing 200 g of potatoes.
The culture solution (pH 5, 6) was added to 200II+1 with distilled water and put into a 500n+1 Sakalov flask, sterilized in an autoclave at 120°C for 15 minutes, and after cooling, Mortierella albina (ATCC32221
) was inoculated in a platinum loopful amount. In my home country, about 60g/! according to culture experiments conducted in advance! The dextrose concentration was optimal. Shaking culture was started at 20°C, and after 5 and 10 days, a solution of 5 g of dextrose dissolved in 10 ml of sterile water was added to increase the calculated cumulative dextrose concentration to 50 g/l and 75 g/j! Close the culture
did. Since this filamentous fungus consumed about 5 g/day of dextrose on average during culture, the dextrose concentration was always below 40 g/l throughout the culture period. After 20 days, bacteria were collected using gauze as a filter cloth, washed with distilled water, dehydrated using a centrifugal filter, dried using silica gel in a vacuum dessicator, weighed, ground in a mortar, and washed with chloroform/ Total lipids were extracted with methanol (2:1 v/V). After removing the solvent from the obtained extract, the lipids were methyl esterified by alcoholysis using sodium metal, and then the fatty acid composition in the lipids was analyzed by gas chromatography to determine the arachidonic acid content in the total lipids. In addition, at the start of culture, the dextrose concentration was adjusted to 7.
5g/j! Comparative Example 1 was conducted in which the mixture was closed and dextrose was not added in the middle. The results obtained are shown in Table 1.

Table 1 1) Analysis conditions: DEG3 15% column (190°C) Example 2. Fed-batch method Add 5 g of dextrose (25 g/per β medium), 4 g of yeast extract (manufactured by Oriental Yeast Co., Ltd.), and 150 μg of CaC1, 2 to the infusion solution obtained by infusing 80 g of potatoes.
Add H20 and make up the total volume to 200ml with distilled water to 500ml! Mortierella albina (ATCC162
66) was inoculated in a platinum loopful amount. The optimal dextrose concentration in Japan was approximately 7 Q g/1. Shaking culture was started at 20°C, and 5 days and 10 days after the start of culture in the same manner as in Example 1.
5g of dextrose every day Om! ! The culture was continued for 10 minutes by adding a solution dissolved in sterile water. After 20 days, bacteria were collected and washed, and lipid analysis was performed in the same manner as in Example 1.

Note that the dextrose concentration did not exceed 40 g/l during the culture period. Also, at the start of culture, the dextrose concentration was 15g/200n+j! (Comparative Example 2 was obtained by culturing at 75 g/j without adding dextrose. The obtained results are shown in Table 2.

Table 2 Example 3 Replacement method 20g of dextrose (100g/ji), 2g of polypeptone (manufactured by Daigo Nutrient Chemical Co., Ltd.), and 150cm of CaCl z 2 were added to the leachate obtained by leaching 200g of potatoes.
After adding H2O and making the total volume to 200 ml with distilled water into a 500 mj2 Sakalo flask and sterilizing it in the same manner as in Example 1, Mortierella albina (IFo 85
68) was inoculated in a platinum loopful amount. The optimum dextrose concentration in Japan was approximately 60 g/1. After 12 days of shaking culture at 20°C, the grown bacterial cells were thoroughly washed with sterile water using gauze as a filter cloth until the washings became colorless and transparent, and the bacterial cells were returned to the Sakalo flask and incubated at 25°C with 300I111 sterile water.
The cells were shaken and kept under starvation conditions. Four days later, bacteria were collected and dried in the same manner as in Example 1, and then lipid analysis was performed.

In addition, Comparative Example 3
And so. The results obtained are shown in Table 3.

Table 3 Example 4 Fed Batch Method + Substitution Method After steaming 10 g of potatoes for 20 minutes, they were passed through an Arashi 32 mesh to form a gruel. To this, 5 g of dextrose (25 g/l), 2 g of polypeptone (manufactured by Otama Nutritional Chemical Co., Ltd.), 150 g of CaCl z 2 HzO
5 mL of culture solution and made up to 200ml with distilled water.
After sterilizing and cooling in the same manner as in Example 1, Mortierella albina (ATCC3222
1) was inoculated with platinum.

Shaking culture was started at 20°C, and 5 g of dextrose was added at 10 mA after 5 and 9 days as in Example 1.2.
A solution dissolved in sterile water was added to continue culturing. As a result, the calculated cumulative dextrose concentration was 75 g#! It became. After 12 days, wash the grown bacteria with sterile water using gauze as a filter cloth until the washing solution becomes colorless and transparent.
Starvation was maintained by shaking for 4 days at 'C. After the above culture for a total of 16 days, bacteria were collected in the same manner as in Example 1, and after drying,
Lipid analysis was performed. Also, from the start of culture, the dextrose concentration was 15 g/200mj! (As 75g/l,
Comparative Example 4 was obtained by culturing for 16 days without dextrose feeding or replacement culture. The obtained results are shown in the fourth
Shown in the table.

Table 4 Example 5 Fed-Batch Method + Substitution Method Mortierella elongata (IFO8570') was cultured under exactly the same conditions as in Example 4 and subsequently subjected to post-treatment, and the results shown in Table 5 were obtained.

Table 5 Example 6 Fed-batch displacement method A 200ml culture solution consisting of 1% glucose, 1% polypeptone (manufactured by Daio Nutritional Chemical Co., Ltd.), and 1% yeast extract (manufactured by Oriental Yeast Co., Ltd.) was added to 500ml. Conidiobolus heterosporus (ATCC 12941) was placed in a Sakaro flask, sterilized in the same manner as in Example 1, and then left to cool. Conidiobolus heterosporus (ATCC 12941) was inoculated with platinum, and 2 and 4 days after starting culture at 28°C, Similarly, a solution of 2 g of dextrose dissolved in 5 ml of sterile water was added, and the culture was continued with calculated cumulative dextrose concentrations of 20 g/β and 30 g/β, respectively. After 6 days, the grown bacterial cells were thoroughly washed with sterile water using gauze as a filter cloth until the washing liquid became colorless and transparent, and the bacterial cells were returned to the Sakaro flask. 2 with sterile water
The mixture was shaken at 5° C. for 3 days to maintain the 11 tia condition. After culturing for a total of 9 days, bacteria were collected and dried in the same manner as in Example 1, and then lipid analysis was performed, and the same results as in Example 4 were obtained.

Claims (1)

[Claims] (b) Using sugars as a carbon source, start culturing the filamentous fungi in a medium containing sugars in an amount below the optimum sugar concentration for growth of lipid-producing filamentous fungi, and then add sugars in an amount below the optimum sugar concentration for growth of lipid-producing filamentous fungi. and/or (b) a method of culturing the lipid-producing filamentous fungus in the stationary growth phase or near the stationary growth phase in substantially the absence of a carbon source. A method for producing an unsaturated fatty acid-containing lipid, which comprises culturing.