JP2566377B2 - Method for producing fats and oils high in docosahexaenoic acid - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing fats and oils having a high content of docosahexaenoic acid, which are useful as foods or pharmaceuticals. Regarding the method.

[0002]

Docosahexaenoic acid (hereinafter abbreviated as DHA) is a kind of long-chain highly unsaturated fatty acid (hereinafter abbreviated as PUFA), and is known to have many physiologically active functions. In recent years, research on concentration and advanced purification technology has been actively promoted.

As a conventional method for concentrating PUFA or PUFA-containing fats and oils, a method is known in which fats and oils extracted from fish are separated and concentrated by a low temperature fractionation crystallization method, a urea addition method, a chromatography method, a lipase treatment method and the like.

[0004]

However, the above-mentioned conventional method for concentrating PUFA or PUFA-containing fats and oils involves complicated operations, and there is a problem of high cost due to the complexity of operations. When PUFA is obtained in the form of glycerin ester, there is also a problem that another step must be added after concentration.

As a method for producing a PUFA-containing fat or oil using a microorganism, a method for producing a PUFA glyceride using yeast belonging to the genus Torulopsis (Japanese Patent Laid-Open No. 2-1698).
8) is known, but it is difficult to say that this yeast lacks the assimilation ability specifically only for DHA, and thus it cannot be said that it is a sufficiently improved method in terms of DHA concentration efficiency.

As a method other than extraction and purification from fish, there is a method of extracting fats and oils containing DHA produced by microorganisms, but the yield of DHA per unit volume of culture is still low, and it is practically used. Has a cost problem.

[0007] Therefore, an object of the present invention is to solve the above-mentioned problems and to efficiently produce a fat or oil containing DHA in a high concentration in the form of glycerin ester by a simple method.

[0008]

Means for Solving the Problems As a result of extensive studies on a method for efficiently obtaining a concentrated DHA-containing fat or oil, the inventor of the present application has shown that in a medium containing a DHA-containing fat or oil as a carbon source, a specific microorganism, That is, by culturing Candida famata US-238,
We found that the content can be increased dramatically and efficiently,
The present invention has been completed.

In other words, in order to solve the above-mentioned problems, according to the present invention, Candida Famata US-2 is used.
No. 38 (FERM P-13974) was cultured in a medium containing a docosahexaenoic acid-containing oil or fat as a carbon source, and the concentration of docosahexaenoic acid glycerin ester in this medium was increased to recover the oil or fat.

Further, in the above-mentioned means,
As a medium for culturing Famata US-238 (FERMP-13974), a medium containing an oil or fat containing 5% by weight or more of docosahexaenoic acid as a carbon source can be adopted.

Alternatively, in the above-mentioned method for producing fats and oils having a high content of docosahexaenoic acid, 0.1 to 1 of one or more additives selected from the group consisting of calcium carbonate, calcium hydroxide, potassium carbonate and magnesium carbonate is added to the medium.
It is also possible to adopt a means of adding the volume (w / V)%.

The details will be described below. The% used in the present invention is the capacity (v /
W)%. Candida famata U-238 (Candida famata U used for this invention)
S-238) was adopted by the inventor of the present application as the one that most efficiently achieves the object of the present invention as a result of separating microorganisms from soils collected in various parts of Japan and examining their assimilation ability for fats and oils. This strain was isolated from the soil of Innoshima Island, Hiroshima Prefecture, and was isolated by the following screening method.

[Candida Famata US-238
Screening method] After adding a separated sample of soil to a medium A having the following composition and culturing it at 30 ° C. for 2 to 3 days, for those in which the growth of microorganisms is observed, perform enrichment culturing in the same medium, Bacteria were accumulated. And
The amount of fats and oils in the upper layer was confirmed by centrifugation of the culture solution, and a culture solution containing bacteria having the strongest ability to assimilate fats and oils was applied on a plate medium having the same composition, and after culturing, colonies were removed. Was isolated and fractionated to obtain a target strain.

Composition of medium A: (pH 8.0) Fish oil 1 ml Glucose 3.5 mg Ammonium nitrate 35 mg Dipotassium hydrogen phosphate 14 mg Magnesium sulfate 7 mg Yeast extract 7 mg Tap water 7 ml Bacteria of the strain obtained above The scientific properties are as follows.

(1) Morphological properties Morphology Oval or globular cells are one or several in series. Size (1-6) × (2-6) μ Budding multipolar budding Pseudohyphae Not developed in cornmeal medium Spontaneous mycelial formation is observed No ascospore formation is observed (2) Cultural properties (YM liquid medium) Precipitation and film formation are observed (3) Giant colony observation (YM agar medium) Good growth Upstanding trapezoid Surface condition Smooth tone Milky white (4) Physiological properties Growth pH 3-9 Optimum pH 5-7 Growth temperature Up to about 42 ° C Optimal temperature 20-35 Nitrate assimilation-Growth in vitamin-deficient medium-Diazonium blue B Color reaction- [fermentability] glucose-galactose-sucrose-maltose-lactose-raffinose-inositol- [assimilation] glucose + galactose + sucrose + Maltose + cellobiose + trehalose + lactose-raffinose + melitithose + starch + D-xylose + L-arabinose-D-ribose + L-rhamnose + erythritol-ribitol + D-mannitol + succinate + citrate- + citrate. From the results, the isolated strain was identified as Candida famata, and this strain was designated as Candida famata US-238.
(Candida famata US-238).

This strain has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology as "Microbial Accession No. Life Research Institute No. 13974 (FERM P-13974)".

The DHA used in the present invention has 6 double bonds with 22 carbon atoms, and has 3 double bonds counted from the methyl group side.
It is a fatty acid in which the double bond begins at the th carbon atom. Examples of such oils and fats containing DHA include oils (fish oils) of fish such as bonito, tuna and sardines, and oils and fats of crustaceans, marine animals, algae and the like.

In the culture according to the present invention, the above yeast is inoculated into a medium containing a fat and oil containing DHA as a carbon source,
It is carried out by aerobically culturing, and as the carbon source used in that case, saccharides such as glucose, starch, molasses and the like can be used in addition to the DHA-containing oil and fat.

Ammonium salts and other nitrogen-containing substances can be used as the nitrogen source, and magnesium salts, phosphates, potassium salts, calcium salts, iron salts, copper salts, etc. can be used as the inorganic salts. You can In addition to yeast extract, it is also preferable to add a growth promoting substance as a vitamin source.

The carbon source of the medium used in this invention is DHA.
It is preferable to use fats and oils containing 5% by weight or more.
This is because, in the present invention, the components other than DHA are assimilated by the specific yeast to concentrate the DHA in the fats and oils, and thus the fats and oils having a DHA content of less than 5% by weight deteriorate the production efficiency.

The initial concentration of DHA-containing fats and oils in the medium is 500 g / 1 or less, and a more preferable concentration range is 10 to 300 g / 1. The cultivation time becomes longer depending on the initial amount.

The culture temperature is 20 to 40 ° C., preferably 25.
The pH is 3 to 9, preferably 5 to 7 at ˜35 ° C., and usually 3 to 10 days.

Inorganic salts are often used in the range of 0.01 to 0.5% as a micronutrient for microorganisms, but in the present invention, during the culture, for example, culture 2 is used.
After 5 days, the DHA content can be further increased by adding 0.1 to 1% of one selected from calcium carbonate, calcium hydroxide, potassium carbonate, magnesium carbonate, or a mixture thereof. That is,
The microorganism used in the present invention is particularly excellent in assimilation ability of unsaturated fatty acids such as oleic acid and linoleic acid during ordinary culture.

However, the addition of the above-mentioned inorganic salt increases the assimilation ability of saturated fatty acids such as palmitic acid and stearic acid, resulting in a further increase in the DHA content.
Although the reason for the change in the assimilation ability of the microorganism due to the addition of the inorganic salt is not clear, it is considered that it causes some change in the lipid metabolism mechanism. However, the addition amount is 1.0
If it exceeds%, the concentration tends to be inhibited.

As a method for recovering the DHA-containing oil / fat, after the culture is completed, the cells are removed by centrifugation or the like, and the DHA-containing oil / fat is extracted and recovered from the obtained culture supernatant using an oil / fat extraction solvent such as hexane. Well, concentrated DHA-containing fats and oils can be obtained.

D in the obtained DHA-rich oil and fat
HA is obtained as a mixture of various glycerin fatty acid esters, particularly a mixture of triglyceride and diglyceride. If necessary, this glyceride mixture is subjected to an ester synthesis reaction with a suitable catalyst such as lipase to obtain a triglyceride having a high DHA content.

[0027]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples. [Example 1] 0.25 M containing ammonium sulfate 0.5%, magnesium sulfate 0.1%, yeast extract 0.1%
100 ml of liquid medium adjusted to pH 6 with phosphate buffer
Was added to a Sakaguchi flask and 5.0 g of fish oil was added as a DHA-containing oil and fat. Candida Famata US-
After inoculating 238 with shaking and culturing at 30 ° C. for 3 days with shaking, the culture solution was centrifuged to remove the cells, and the supernatant was extracted with hexane to recover an oil component. The fatty acid composition of the obtained fats and oils is shown in Table 1.

In the fatty acid composition analysis, fats and oils were methyl esterified, measured by GC, and essential parts were extracted from all compositions.

[0029]

[Table 1]

As is clear from the results shown in Table 1, the DHA content of the raw material fat was 19.5%, whereas the DH after the Candida famata US-238 was subjected to the culture treatment.
The A content was 35.2%. Therefore, DHA is concentrated about 1.8 times. The oil and fat recovery rate was 45%.

[Example 2] [0031] Extraction was carried out in the same manner as in Example 1 except that 0.5% of calcium carbonate was added at the start of the culture, and then extraction was carried out to obtain the fatty acid composition of fats and oils and DHA. The content was investigated and the results are also shown in Table 1.

As a result, the addition of calcium carbonate caused a change in the residual fatty acid due to the assimilation of the bacterium used in the present invention. That is, the assimilation ability of saturated fatty acids such as palmitic acid and stearic acid is increased, resulting in DHA.
The content was increasing.

[Examples 3 to 7] Ammonium sulfate 0.5
%, Magnesium sulfate 0.1%, yeast extract 0.1%, 7 ml of a liquid medium adjusted to pH 6 with a 0.25 M phosphate buffer was placed in a test tube, and fish oil (DHA 19. (5%) 0.7 g, and inoculated with Candida famata US-238, 30 ° C
The cells were shaken for 2 days.

After that, the culture was continued without adding anything (Example 3), calcium carbonate (CaC
O ₃₎ have been added 0.5% (Example 4), calcium hydroxide (Ca (OH) ₂₎ which was added 0.5% (Example 5), potassium carbonate (K ₂ CO ₃₎ 0.5 % (Example 6) and 0.5% magnesium carbonate (Example 7) were separately prepared and continuously cultured for 3 days. After culturing, the fats and oils were collected in exactly the same manner as in Example 1, and the fatty acid composition of the obtained fats and oils was examined. The results are shown in Table 2.

[0035]

[Table 2]

As is clear from the results shown in Table 2, the addition of calcium carbonate, calcium hydroxide, potassium carbonate or magnesium carbonate during the cultivation further increased the DHA content as compared with Examples 1 and 2, and It can be seen that a fat or oil in which the contained components were further concentrated was obtained.

[Examples 8 to 11] Ammonium sulfate 0.
5%, magnesium sulfate 0.1%, yeast extract 0.1%
Into a test tube, 7 ml of a liquid medium containing, and adjusted to pH 6 with a 0.25 M phosphate buffer was added, and 0.7 g of fish oil (DHA 26.8%) as a DHA-containing oil was added.
Inoculated with Candida famata US-238, 30
The cells were shaken at 3 ° C for 3 days. After that, calcium carbonate (CaCO ₃ 0.1%, 0.2% 0.5%,
1.0% was added (Examples 8, 9, 10, 1 respectively)
1), after further continuous culturing for 4 days, the oil and fat was recovered in exactly the same manner as in Example 1, and the DHA content and the oil and fat recovery rate of the obtained oil and fat were examined. The results are shown in Table 3.

[0038]

[Table 3]

As is clear from the results shown in Table 3, addition of calcium carbonate in an amount of 0.1 to 1.0% resulted in DHA.
Was further concentrated (Examples 8 to 11), and it was found that fats and oils having a high DHA content were recovered.

[Comparative Examples 1 and 2] CaC in Example 8
O ₃ was not added (Comparative Example 1), or C was added after 3 days of culture.
The same procedure as in Example 8 was carried out except that 2.0% of aCO ₃ was added (Comparative Example 2), and the DHA content and the oil / fat recovery rate of the recovered oil / fat were examined, and the results are also shown in Table 3. As a result, at 2.0%, the DHA content was slightly lower than that of the non-added one.

Example 12 Ammonium sulfate 0.5
%, Magnesium sulfate 0.1%, yeast extract 0.1%, 100 ml of a liquid medium adjusted to pH 6 with a 0.25 M potassium phosphate buffer was placed in a Sakaguchi flask, and DHA-containing fish oil (DHA 26.8% ) Is added,
Inoculated with Candida famata US-238, 30
The culture was carried out with shaking at 3 ° C. for 3 days, and the oil and fat extraction operation was performed in the same manner as in Example 1 to examine the DHA content and the oil and fat recovery rate. The results are shown in Table 4.

[0042]

[Table 4]

Comparative Examples 3 and 4 As strains other than the Candida famata US-238 strain in Example 12,
Candida famata (IFO 0664) was used (Comparative Example 3), or Torulop as a bacterium belonging to the genus Torulopsis known as an oil- and fat-assimilating bacterium.
Except that sis bombicola (IFO 10243) was used (Comparative Example 4), the culture and the oil / fat recovery were performed in exactly the same manner, and the results are also shown in Table 4.

As is clear from the results shown in Table 4, the DHA content in the recovered oil was 31% or less even when the strains other than the predetermined strain were cultured.

[Example 13] Ammonium sulfate 0.5
%, Magnesium sulfate 0.1%, yeast extract 0.1%, 100 ml of liquid medium adjusted to pH 6 with 0.25M phosphate buffer was added to a Sakaguchi flask, and further DHA
5 g of fish oil was added as a contained oil and fat, and Candida famata US-238 was inoculated, followed by shaking culture at 30 ° C. for 6 days. After that, 0.5% of CaO3 was added, the culture was continued again for 3 days, and the culture solution was centrifuged to remove the bacterial cells, and the supernatant was extracted with hexane to recover an oil component.

As a result, the DHA content of the raw material oil is 1
9.5%, whereas DHA after contact treatment with yeast
The content was 69.1%. Therefore, DHA is concentrated about 3.5 times. The oil and fat recovery rate was 12.0%.

Example 14 Next, large-scale culture was carried out using a jar famenter. The composition of the medium and the culture conditions are as shown below.

[Medium] Fish oil (DHA 19.5%) 600 g (NH ₄ ) ₂ SO ₄ 30 g KH ₂ PO ₄ 12 g MgSO ₄ .7H ₂ O 6 g Yeast extract 6 g Tap water 6000 ml [Cultivation conditions] pH 6.0 ( Adjusted with 1N NaOH) Temperature 30 ° C. Aeration rate 3 1 / min Stirring speed 300 rpm Culture time 115 hours Addition of CaCO ₃ 30 g 67 hours after the start of culture After the culture, the oil and fat were recovered in exactly the same manner as in Example 1, and recovered. The amount of oil and fat is 115 g, and the DHA content is 37.1.
It was concentrated to%.

[0049]

[Effect] As described above, the present invention efficiently concentrates and recovers DHA glycerin ester from DHA-containing fats and oils by a simple method such as culturing a predetermined strain of Candida famata in a medium containing DHA-containing fats and oils. There is an advantage that can be done.

Further, in the invention which adopts a means for adding a specific inorganic substance at the time of culturing, the DHA content can be further increased. The DHA obtained by this invention is
Since it is present in the form of glycerin ester and the free fatty acid is present in a small amount, it can be said that the oil and fat after the culture are easily purified, which is a highly practical method.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:72) (C12P 7/64 C12R 1:72)

Claims (3)

(57) [Claims] 1. Candida Famata US-238
(FERM P-13974) is cultured in a medium containing a docosahexaenoic acid-containing fat and oil as a carbon source, and the concentration of docosahexaenoic acid glycerin ester in the medium is increased to recover the fat and oil. . 2. Candida Famata US-238
The method for producing an oil and fat rich in docosahexaenoic acid according to claim 1, wherein the medium for culturing (FERM P-13974) is an oil and fat containing 5% by weight or more of docosahexaenoic acid as a carbon source. 3. The method for producing a fat or oil rich in docosahexaenoic acid according to claim 1 or 2, wherein one or more additives selected from the group consisting of calcium carbonate, calcium hydroxide, potassium carbonate and magnesium carbonate are added to the medium. 0.1 to 1 volume (w / V)% is added, The manufacturing method of the fats and oils high in docosahexaenoic acid characterized by the above-mentioned.