JP2001309779A - Method for producing highly unsaturated fatty acid and highly unsaturated phospholipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce highly unsaturated fatty acid-containing microorganism by culturing the microorganism in a medium prepared from a fisheries waste. SOLUTION: This highly unsaturated fatty acid-containing microorganism is efficiently produced in a short time by culturing the microorganism in a medium prepared from a fisheries waste, especially guts of fishes and shellfishes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The production of microorganisms containing polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and microorganisms containing phospholipids containing said polyunsaturated fatty acids using marine waste as a medium component The present invention relates to a method for producing polyunsaturated fatty acids or phospholipids containing the polyunsaturated fatty acids from such microorganisms. Such microorganisms or polyunsaturated fatty acids include DHA or EPA as polyunsaturated fatty acids, and are useful in the fields of foods, cosmetics, marine products, and chemical products.

[0002]

2. Description of the Related Art The amount of marine waste is increasing year by year.
Among them, inedible parts such as scallop, squid and octopus have been disposed of by incineration and landfill. However, landfills have the problem of generating bad smells and pests, and the number of landfill sites is decreasing. On the other hand, incineration requires a large amount of fuel due to the high water content of marine waste, which involves a problem that construction costs and treatment costs are increased. Therefore, there is an urgent need to develop methods for effectively using marine wastes.

On the other hand, DHA and EPA, which are highly unsaturated fatty acids of the ω-3 series, are fatty acids peculiar to marine products, and are present in large amounts in inedible parts such as squid and scallops, which are marine wastes. . For this reason, attempts have been made to obtain highly unsaturated fatty acid-containing lipids from marine wastes, and attempts to extract EPA from scallop midgut glands are already known (JP-A-11-13721).
No. 9, JP-A-11-116983), however, extraction of lipids from these tissues is not easy, requires a great deal of labor, and it can be said that the utilization of marine waste as an unused resource is sufficient. Absent. Most of the lipids obtained by this method are triacylglycerols, like the lipids obtained from fish oil.

In addition, attempts have been made to produce polyunsaturated fatty acids such as DHA and EPA by microorganisms in place of conventional fish oil. Until now, the production of polyunsaturated fatty acids in microorganisms has begun by searching for strains that highly accumulate polyunsaturated fatty acids. On the other hand, a method (JP-A-4-341180, JP-A-7-23774) for changing lactic acid bacteria that do not produce highly unsaturated fatty acids to lactic acid bacteria containing DHA is already known. In such a microorganism culture method, supplementation of nutrients such as inorganic salts, vitamins, and proteins is indispensable to maintain the activity of the bacteria and promote the growth of the bacteria. It is used. However, since the medium thus prepared is expensive, development of an inexpensive medium suitable for large-scale cultivation is urgently required. However, utilization of marine waste as a medium (Japanese Patent Laid-Open No. 11-56345) ) Has been attempted.

[0005]

An object of the present invention is to provide a method for producing a microorganism containing polyunsaturated fatty acids using marine waste as a medium component, and a method for producing a microorganism containing phospholipids containing the polyunsaturated fatty acid. And a method for producing a polyunsaturated fatty acid or a phospholipid containing the polyunsaturated fatty acid from a culture of these microorganisms.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies for the above-mentioned purpose, and as a result, have found that fish and shellfish such as squid and scallop, which accumulate DHA and EPA at a high concentration, are obtained. Regarding lipids of microorganisms cultured in the adjusted nutrient medium, 1) washing of microbial cells containing polyunsaturated fatty acids does not remove polyunsaturated fatty acids in the microbial cells; The polyunsaturated fatty acid binds to lipids in the microbial cells. 3) The growth rate of microorganisms cultured in a medium prepared from marine waste used in the present invention is high, and a large amount of microbial cells can be obtained in a short time. 4) It has been found that a bacterium containing a highly unsaturated phospholipid including DHA and EPA can be obtained by using a bacterium as a microorganism, and the present invention has been accomplished.

That is, the present invention provides a method for producing a highly unsaturated fatty acid-containing microorganism, which comprises culturing the microorganism in a medium prepared from marine waste. Further, the present invention provides a method for producing a polyunsaturated fatty acid, which comprises culturing a microorganism in a medium prepared from marine waste to produce polyunsaturated fatty acids, and collecting polyunsaturated fatty acids from the culture. is there. Furthermore, the present invention is a method for producing a phospholipid-containing microorganism containing a highly unsaturated fatty acid, which comprises culturing a microorganism having a phospholipid-producing ability in a medium prepared from marine waste. Furthermore, the present invention provides a method for culturing a microorganism having a phospholipid-producing ability in a medium prepared from marine waste to produce a polyunsaturated fatty acid-containing phospholipid, and collecting the polyunsaturated fatty acid-containing phospholipid from the culture. It is characterized by a method for producing a phospholipid containing a polyunsaturated fatty acid.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Examples of the marine waste include offal of fish and shellfish,
Specific examples include squid and scallops containing DHA and EPA at a high concentration. Here, the polyunsaturated fatty acid means an unsaturated fatty acid having a degree of unsaturation of 2 or more and a carbon number of 18 or more. Mixed fats and oils of polyunsaturated fatty acids, for example, sardines, mackerel, horse mackerel, tuna-derived fats and oils in which the proportion of polyunsaturated fatty acids in total fatty acids is 10% or more, and linole as polyunsaturated fatty acids Acid, γ-linoleic acid, arachidonic acid, α-linoleic acid, eicosapentaenoic acid, docosahexaenoic acid and salts, esters, triacylglycerol, diacylglycerol, monoacylglycerol, glycerophospholipid, glyceroglycolipid, sphingolipid A method of simultaneously accumulating various polyunsaturated fatty acids in lactic acid bacteria using many kinds of fats and oils such as glycosphingolipids, or lactic acid bacteria in a medium containing autolysates or extracts obtained from algal bodies of marine microalgae. The method of culturing is already known (JP
In these methods, various fats and oils are used as a highly unsaturated fatty acid supply source in a nutrient medium such as peptone, calf brain extract, and bovine heart extract. Is added separately from the culture medium, which requires a great deal of effort in the preparation of these fats and oils.Moreover, peptone, calf brain extract, bovine heart extract, etc. However, there is a problem that a large amount of cost is required.

Attempts have also been made to directly extract lipids from marine wastes containing high concentrations of DHA and EPA. Attempts to extract EPA from the scallop midgut gland have already been made (JP-A-11-137219, JP-A-11-116983).
However, extraction of lipids from these marine animal tissues is not easy and requires a great deal of labor. On the other hand, the feature of the present invention is to utilize marine waste as a polyunsaturated fatty acid source and a medium component, and to incorporate the polyunsaturated fatty acid derived from the marine waste into microorganisms, thereby to make the polyunsaturated fatty acid Is to provide. Microorganisms can be cultured in an inexpensive medium prepared from marine waste, and DHA and EPA can be selectively accumulated in the microorganisms, which makes it possible to extract and produce lipids economically and efficiently. Become.

Further, by using bacteria such as Vibrio lumoensis as microorganisms, DHA and EPA, which are usually present as triacylglycerol-bound forms in the visceral organs of fish and shellfish, have recently been revealed to have new physiological functions. It can be converted to phospholipid-bound DHA or EPA which is being used, and a phospholipid containing highly unsaturated fatty acids such as DHA and EPA can be obtained.

The microorganism used in the present invention may be any microorganism that can take up polyunsaturated fatty acids in marine waste, or any microorganism that has phospholipid-producing ability. It is not limited. Specific examples thereof include Escheric (Escheric)
hia coli), Bacillus subtilis, and Moritella marina strain MP-
1) and the like. These microorganisms can be easily obtained from public microorganism preservation institutions and the like. For example, Escherichia coli (Esche
richia coli) (IFO P-15044), Bacillus subtilis (B
acillus subtilis) (IAM 1026), Moritera Marina MP
-1 strain (Moritella marina strain MP-1) [ATCC 1538
1), Vibrio rumoiensis (F
ERM P-14531).

The method for producing a microorganism containing polyunsaturated fatty acids of the present invention is carried out by culturing the microorganism in a medium prepared from marine waste. The culture conditions are 4-25 ° C,
The pH is 6.5 to 7.5, and the culture period is 1 to 5 days. The production of the polyunsaturated fatty acid or the phospholipid containing the polyunsaturated fatty acid is performed by collecting fatty acids from the microorganism obtained by the above culture or a culture containing the microorganism by a conventional method such as solvent extraction.

The medium used in the present invention can be prepared as follows using, for example, a squid internal substance (Ikagoro). Sprinkle the cuttlefish on a juicer and add 4
After overnight at ℃, boil for 90 minutes. After allowing to cool, the residue is removed by squeezing with a towel, and the obtained squid extract is vacuum dried at 60 ° C. for 72 hours. The squid powder obtained at this time was 2%, sodium chloride was 3% for marine bacteria, and 0.5% for non-marine bacteria.
%, Each can be dissolved in tap water, sterilized, and then used as a cuttlefish medium.

After washing the cells of microorganisms cultured in such a medium, lipids are extracted with a solvent by a conventional method to obtain polyunsaturated fatty acids or phospholipids containing polyunsaturated fatty acids. When the extracted lipid is methyl-esterified with 10% methanolic acetyl chloride, the fatty acid composition of any fatty acid derivative ester-bound in the cells can be analyzed by gas chromatography. In addition, wet or dry cells are extracted using an organic solvent or the like,
After fractionation of the lipid by silica gel thin layer chromatography, the constituent fatty acids of the lipid can be analyzed in the same manner. The polyunsaturated fatty acids in the microbial cells of the present invention analyzed by the above method were mainly DHA and EPA. No increase in the content of other fatty acids was observed.

[0015]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples.

Example 1 Preparation of Ikagoro Medium To 150 g of Ikagoro wet weight, 450 mL of tap water was added, and the mixture was put on a juicer. After lysing the cuttlefish roe at 4 ° C. overnight, it was boiled for 90 minutes. After standing to cool, the residue was removed by squeezing with a towel, and
Vacuum dried at 72 ° C. for 72 hours. At this time, 12 g of Ikagoro powder was obtained. 2% of this squid powder
3% for marine bacteria, 0.5% for non-marine bacteria
Sodium chloride was dissolved in tap water, sterilized, and used as an Ikagoro medium.

Example 2 A DHA-producing bacterium, Morritella marina MP-1 strain (ATCC 15381), was used for comparison with the above Ikagolo medium and an LB + NaCl medium (1.
The cells were cultured at a culture temperature of 10 ° C. using 0% tryptone, 0.5% yeast extract, and 3.0% sodium chloride). Cells were collected from the culture by centrifugation and washed three times with a 0.5 M aqueous sodium chloride solution to obtain bacterial cells. Ordinary law (Bli
gh, EG, Dyer, WJ. Can. J. Microbial. 37; 911-917 (1
959)), lipid extraction was performed by chloroform-methanol extraction with an organic solvent, each sample was suspended in 10% methanolic acetyl chloride, reacted at 90 ° C. for 3 hours, and the resulting fatty acid methyl ester was analyzed by GLC. Table 1 shows the fatty acid composition of the total lipids of the Moritera marina MP-1 strain when cultured in the Ikagolo medium and the LB + NaCl medium, respectively.
The ratio of EPA, which is usually only about 1%, was increased to 11% by culturing in Ikagolo medium.

On the Ikagoro medium, Moritera Marina MP-
The yield of EPA obtained when one strain was cultured was 920
μg / L. This corresponded to about 10% of the total amount of EPA estimated to be contained in the medium. Under the same conditions, the yield of DHA was 1000 μg / L. These values were higher than the yields of EPA and DHA using the LB + NaCl medium, 9 μg / L and 183 μg / L.

Table 1

The whole lipid sample was subjected to two-dimensional thin-layer chromatography, and chloroform: methanol: water = 65: 25: 4 (V / V / V) in the first dimension and chloroform: methanol: 28 in the second dimension as the effluent solvent. % Aqueous ammonia = 65: 35: 5 (V /
(V / V), fractionated into each lipid class, scraped each lipid spot on the thin layer plate, suspended each sample in 10% methanolic acetyl chloride, reacted at 90 ° C. for 3 hours, and generated Fatty acid methyl esters were analyzed by GLC.

Table 2 shows the fatty acid composition of each lipid class of Moritella marina MP-1 strain when cultured in Ikagolo medium and LB + NaCl medium. When cultured on Ikagoro medium,
EPA is found to be present in phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) fractionated by thin-layer chromatography, and the fatty acids in the medium are taken up by bacterial cells and become components of membrane lipids. I know it exists. At this time, all the obtained PE
And the yield of total PG were 7683 μg / L and 235, respectively.
It was 9 μg / L. Therefore, DHA or EPA existing as triacylglycerol-bound type in squid
By utilizing the natural growth of bacteria, phospholipid-bound DHA
And it can be converted to the same EPA (highly unsaturated phospholipid).

Table 2

Example 3 A bacterium that does not produce polyunsaturated fatty acids, Vibrio rumoiensi
s) The S-1 strain (FERM P-14531) was used for comparison with the above Ikagolo medium and a PYS medium (1.0% polypeptone, 0.1%).
5% yeast extract, 1.0% sodium chloride) at a culture temperature of 27 ° C. The cells were collected from the culture by centrifugation and washed three times with a 0.1 M aqueous sodium chloride solution to obtain the cells. Lipids were extracted with chloroform-methanol-water according to a conventional method (Bligh-Dyer method), and the resulting lipids were suspended in 10% methanolic acetyl chloride and subjected to an esterification reaction at 90 ° C. for 3 hours. And the produced fatty acid methyl ester was analyzed by GLC.

Table 3 shows the fatty acid composition of all lipids of Vibrio lumoiensis S-1 strain when cultured in Ikagoro medium and the medium. EPA and DHA, which do not normally exist, are cultivated in the Ikagoro medium, respectively.
Had increased. At this time, the yields of EPA and DHA were 4122 μg / L and 4580 μg / L. Thus, it was found that the use of the Ikagoro medium enables production of polyunsaturated fatty acids in bacteria that do not produce polyunsaturated fatty acids. Vibrio Lumoiensis
Since most lipids of the S-1 strain are phospholipids, DHA and EPA existing as triacylglycerol-binding forms in Ikagoro were converted to phospholipid-binding DHA and It was found that it can be converted to EPA (highly unsaturated phospholipid).

Table 3

[0026]

According to the present invention, highly unsaturated fatty acids derived from the offal of fish and shellfish are specifically recovered by incorporation into the microorganism by culturing the microorganism in a medium prepared from marine waste, particularly the offal of fish and shellfish. As a result, microorganisms containing polyunsaturated fatty acids such as mainly DHA and EPA were obtained, and it was possible to efficiently produce microorganisms containing polyunsaturated fatty acids in a short time. In addition, by using bacteria as microorganisms, it is possible to reduce the amount of D which is present as a triacylglycerol-bound form in the offal of fish and shellfish.
It is possible to convert HA and EPA into phospholipid-bound DHA or EPA whose new physiological functions are being revealed recently, and to obtain highly unsaturated phospholipids containing DHA and EPA. By using a medium prepared from marine waste, particularly fish and shellfish offal, it is possible to contribute to the improvement of economic efficiency and also to the treatment and effective use of waste.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:63) C12R 1:63) (C12P 7/64 (C12P 7/64 C12R 1:01) C12R 1 : 72) Inventor Shun Yumoto F-term (reference) in Hokkaido Institute of Industrial Technology 2-1, 17-2, Tsukikanto, Toyohira-ku, Sapporo, Hokkaido 4B064 AD88 AE63 CA02 CD23 DA01 DA10 4B065 AA01X AA55X AC14 BB23 CA13 CA14 CA41 CA44 4H059 BA26 BA83 BB05 BB06 BB07 BC06 BC48 CA12 CA31 EA21

Claims (8)

[Claims] 1. A method for producing a highly unsaturated fatty acid-containing microorganism, wherein the microorganism is cultured in a medium prepared from marine waste. 2. The method for producing a highly unsaturated fatty acid-containing microorganism according to claim 1, wherein the marine waste is an offal of fish and shellfish. 3. A method for producing a polyunsaturated fatty acid, comprising culturing a microorganism in a medium prepared from marine waste to produce polyunsaturated fatty acids, and collecting the polyunsaturated fatty acids from the culture. 4. The method for producing a polyunsaturated fatty acid according to claim 3, wherein the marine waste is an offal of fish and shellfish. 5. A method for producing a phospholipid-containing microorganism containing a polyunsaturated fatty acid, comprising culturing a microorganism capable of producing phospholipid in a medium prepared from marine waste. 6. The method for producing a highly unsaturated fatty acid-containing microorganism according to claim 5, wherein the marine waste is an offal of fish and shellfish. 7. A method of culturing a microorganism having a phospholipid-producing ability in a medium prepared from marine waste to produce a polyunsaturated fatty acid-containing phospholipid, and collecting the polyunsaturated fatty acid-containing phospholipid from the culture. A method for producing a polyunsaturated fatty acid-containing phospholipid. 8. The method according to claim 7, wherein the marine waste is an offal of fish and shellfish.