JPH089981A - Production of highly unsaturated higher fatty acid - Google Patents

Abstract

PURPOSE:To efficiently obtain the subject higher fatty acid for health food, etc., by separating a highly unsaturated higher fatty acid such as octadecapentaenoic acid and octadecatetraenoic acid from Peridinium bipes of fresh water phytoplankton. CONSTITUTION:Red tide water containing Peridinium bipes of fresh water phytoplankton as the main component is collected and subjected to concentration by a continuous filtration concentration machine of rotary-dram type using ultrafine fiber as filter material. The concentrate is treated with a continuous dehydrator to obtain solid cakes. The obtained solid cakes are subjected to crushing e.g. by an ultrasonic crushing machine. The crushed material is added with a mixed solvent (chloroform:methanol = 2:1 in terms of volume ratio) and the mixture is treated in a constant temperature bath at 30 deg.C for 30min. After the solvent is removed from the extract by distillation, the residue is applied on a separation column and each of the components is separated to obtain the objective highly unsaturated higher fatty acid useful as an additive to health foods, milk, a fishery paste products, etc., with high efficiency.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for obtaining highly unsaturated higher fatty acids such as octadecapentaenoic acid and octadecatetraenoic acid from freshwater phytoplankton.

[0002]

Highly unsaturated higher fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).
Is expected to have various effects such as memory-improving effect, eye growth-promoting effect, cholesterol-lowering effect, anti-allergic effect, anti-cancer effect, etc., and is therefore attracting attention as an additive to health foods, milk, fish paste products, etc. .

EPA and DHA are sardines, tuna, yellowtail,
It is contained in mackerel, horse mackerel, saury, eel, bonito, etc. (especially in orbital cells of tuna and bonito), but a method for obtaining these from microorganisms (including algae) has also been proposed. .

For example, JP-A-59-157276, JP-A-59-204128 and JP-A-60.
JP-A-54675 discloses a method for obtaining EPA from algal bodies of diatom.

Japanese Unexamined Patent Publication (Kokai) No. 59-179042 discloses that unsaturated fatty acids having 14 to 18 carbon atoms are obtained from seaweeds such as green algae and red algae and used for feed.

[0006] In JP-A-60-87798, a single cell alga belonging to the genus Monodus, for example, Monodus subterraneus, is cultured under light irradiation and aeration of carbon dioxide gas, and nitrogen deficiency is caused during the culture to make EPA in the algal cells. A method for producing EPA in which a large amount of EPA is contained is shown.

Japanese Unexamined Patent Publication No. 62-239981 discloses a method for culturing algae of the genus Nannochloropsis to obtain EPA.

Japanese Unexamined Patent Publication (Kokai) No. 63-185390 discloses a method for obtaining EPA from an alga belonging to the genus Coralina, for example, Coralina pirulifera or Coralina cesiris.

Japanese Unexamined Patent Publication (Kokai) No. 2-11698 discloses that fish oil and algae contain EPA and DHA.

Japanese Patent Laid-Open No. 5-43456 discloses DHA.
Has been shown to be extracted and separated from animals, plants, algae, microorganisms and the like in the natural world.

[0011]

Although octadecapentaenoic acid and octadecatetraenoic acid are said to be effective for hypertension, there is a limitation that they cannot be obtained by synthesis. Of these, octadecapentaenoic acid is known to be contained in the body of white whales and sardines, for example, but white whales are currently uncapable and sardines contain very little. Absent. If a method can be found in which a large amount of these highly unsaturated higher fatty acids can be obtained from an underutilized one such as phytoplankton, its significance will be extremely great.

[0012] The publications cited above include EPA and DH.
Although the acquisition source of A is described, octadecapentaenoic acid and octadecatetraenoic acid are not specifically disclosed.

Journal of Plankton Research, Vol. 14,
No. 8, pp. 1055-1065 (1992), the results of analysis of fatty acid composition of phytoplankton photosynthetic products at each season and each depth of Lake Biwa water are reported. It is described in this document that a large number of fatty acids are detected in a lake water sample, and that some of the fatty acids have 18 carbon atoms and 4 or 5 unsaturated groups. This report also shows that in January, April, and May, unsaturated fatty acids having 18 carbon atoms and 3 and 4 unsaturated groups make a high contribution, and Cryptophycean algae is dominant in these months. It is also described as being observed as algae.
However, this document does not show a method for obtaining octadecapentaenoic acid and octadecatetraenoic acid on an industrial scale.

Under the above circumstances, the present invention provides useful highly unsaturated higher fatty acids, octadecapentaenoic acid and octadecatetraenoic acid (and the accompanying EPA and DHA) from phytoplankton. It is intended to provide a method of efficiently obtaining the information.

[0015]

The method for producing a polyunsaturated higher fatty acid of the present invention is a method for separating a polyunsaturated higher fatty acid containing at least octadecapentaenoic acid or / and octadecatetraenoic acid from peridinium vipes. It is characterized by doing.

The present invention will be described in detail below.

Peridinium bipe
(s) is the photosynthetic dinoflagellate genus Peridinium.
It is a freshwater phytoplankton belonging to and is also a component of the red tide. The cells are egg-shaped, pear-shaped or spherical, and a large number of armor plates separated by horizontal grooves and vertical grooves are arranged on the surface.
The sizes of the upper and lower shells are almost equal. The size is 2 in length
The width is about 8 to 40 μm and the width is about 25 to 37 μm.

There are many other species of photosynthetic dinoflagellates such as Peridinium penardii and Peridinium cunningtonii, but for the purpose of the present invention, Peridinium vipes is the source. Is most suitable as

Since piperidinium vipes is difficult to culture, it will be collected from lakes, ponds, dam lakes and the like. In Japan, it can be collected from lake water south of Nagano Prefecture.

The collected red tide water mainly composed of piperidinium bipes is generally not easy to concentrate and dehydrate, but as shown in the explanatory view of the concentrating and dehydrating apparatus in FIG. Industrial concentration and dehydration can be achieved by adopting a method in which the drum-type continuous filtration concentrator is used for concentration treatment, and then the concentrated water is supplied to a continuous dehydrator for dehydration treatment.

Since the dehydrated product obtained by using the above dehydrator becomes a solid cake, it is pelletized if necessary. Since the dehydration rate of this pellet is about 70% at best, storage in this state may cause putrefaction due to the presence of heterotrophic bacteria in the pellet. Therefore, when storing in pellet form, it is desirable to take a drying means such as freeze-drying.

After the dehydration treatment (may be further dried), it is subjected to a crushing treatment. As a crushing method (or a grinding method), an ultrasonic crushing method is preferably adopted, but another mechanical crushing method may be adopted.

After the crushing treatment, it is subjected to an extraction step. Examples of the extraction solvent include halogen-containing solvents (chloroform, etc.), ether solvents, alcohol solvents, ketone solvents, aliphatic hydrocarbon solvents, aromatic hydrocarbon solvents, ester solvents and mixed solvents thereof. Used.

As a result, the target octadecapentaenoic acid, octadecatetraenoic acid, highly unsaturated higher fatty acid such as EPA, DHA and the like can be obtained. Therefore, if necessary, a column method or other suitable method can be used. Each component is isolated by various isolation means.

The polyunsaturated higher fatty acid thus obtained can be suitably used for food additives, feed additives, cosmetic additives, medicines, foods and the like.

[0026]

According to the present invention, from the freshwater phytoplankton, which is a red tide component which has not been conventionally used, useful highly unsaturated higher fatty acids, octadecapentaenoic acid and octadecatetraenoic acid (further, EPA). And DHA)
Can be efficiently obtained.

[0027]

EXAMPLES The present invention will be further described with reference to examples.

Example <Constituent Elements> The red tide waters of Tonoyama Dam in Wakayama Prefecture and Lake Biwa in Shiga Prefecture were collected, filtered with 2.0 μm and 0.2 μm nucleopore filters, dried, and then analyzed by a Shimadzu X-ray fluorescence spectrometer to determine the constituent elements. The percentage was analyzed. According to the morphological observation with a microscope, the red tide water at the two fixed points (Yuya, Shogun) of Tonoyama was mostly occupied by peridinium bipes (more than 95%) in the red tide water, whereas in Lake Biwa (North Lake). , South Lake), the proportion of peridinium in the fraction of 2 μm or more was less than 1%. The concentration per unit volume of sample water in the field is shown in Table 1, and the concentration per dry weight is shown in Table 2.

[0029]

[Table 1]  Constituent elements (concentration per volume μg / l) Al Si PSK Ca Ti Mn Fe Tonoyama Yuya> 2.0 71.1 189 245 197 92 98.1 4.8 8.6 57.3 2.0-0.2 67.6 167 8 5 8.8 20 2.7 2.7 39.9 General Tonoyama> 2.0 88.5 208 170 135 65.7 63 4.1 7.1 55.5 2.0-0.2 68.2 165 7.5 4.8 8.7 20 2.6 2.6 38.7 Lake Biwa North Lake> 2.0 6.5 40.2 5.7 7.9 6.7 42 0 3.1 9.8 2.0-0.2 2.9 6.9 7.3 3 0.8 32.8 0.5 1.9 3 Lake Biwa South Lake> 2.0 80.8 249 9.9 11.9 11.1 67.7 2.1 25 56.8 2.0-0.2 26.2 96.2 10.9 3 2.2 12.5 2.3 9.4 30.8 Note: "> 2.0" is the amount that does not pass through the 2.0 μm filter. "2.0-0.2" means that the filter passed through the 2.0 μm filter and did not pass through the 0.2 μm filter.

Based on Table 1, FIG. 1 shows the concentration of each constituent element per volume in each water area. Looking at each water area, the water of Tonoyama has a composition pattern closer to the eutrophication of Lake Biwa Nanko than to Lake Biwa Kita. That is, the concentrations of Al, Fe, Si, and Ca show relatively close values. However, P, S, and K have overwhelmingly large fractions of 2 μm or more at Tonoyama. In each of the two fixed-point red tide waters of Tonoyama, the fraction of 2 μm or more is mostly occupied by peridinium vipes, so it can be considered that the high concentrations of P, S, and K are due to peridinium bipes.

[0031]

[Table 2]  Constituent elements (concentration per dry weight mg / g) Al Si PSK Ca Ti Mn Fe Tonoyama Yuya> 2.0 4.18 11.12 14.41 11.59 5.41 5.77 0.28 0.51 3.37 2.0-0.2 6.76 16.70 0.80 0.50 0.88 2.00 0.27 0.27 3.99 General Tonoyama> 2.0 4.66 10.95 8.95 7.11 3.46 3.32 0.22 0.37 2.92 2.0-0.2 6.82 16.50 0.75 0.48 0.87 2.00 0.26 0.26 3.87 Lake Biwa North Lake> 2.0 5.75 35.58 5.04 6.99 5.93 37.17 0.00 2.74 8.67 2.0-0.2 8.53 20.29 21.47 8.82 2.35 96.47 1.47 5.59 8.82 Lake Biwa South Lake> 2.0 5.78 17.91 0.71 0.86 0.80 4.87 0.15 1.80 4.09 2.0-0.2 8.45 31.03 3.52 0.97 0.71 4.03 0.74 3.03 9.94 Note: "> 2.0" is the amount that does not pass through the 2.0 μm filter. "2.0-0.2" means that the filter passed through the 2.0 μm filter and did not pass through the 0.2 μm filter.

Based on Table 2, the concentration of each constituent element per dry weight in each water area is shown in FIG. Looking at each element, Al does not show much difference depending on the water area. In Si, Lake Biwa is slightly higher than Tonoyama. For P, S, and K, Tonoyama and Biwakokitako are high. Ca is overwhelmingly high in the north lake of Lake Biwa. For Ti, Mn, and Fe, the north and south lakes of Lake Biwa are higher than Tonoyama. In terms of the components per dry weight, except for Ca, Tonoyama shows a composition pattern similar to that of the non-eutrophic Lake Biwa Kita Lake.

FIG. 3 shows that Al is not so different depending on the water area.
It shows the composition ratio of each element to. Si, Ti,
For Mn and Fe, the north and south lakes of Lake Biwa are higher than Tonoyama. Moreover, Ca is overwhelmingly large in Lake Biwa North Lake. However, P, S,
K has a large fraction of 2 μm or more in Tonoyama (that is, the fraction of Peridinium vipes). This may be considered as an element concentration effect of Peridinium vipes.

As described above, the pellets of Peridinium vipes of Tonoyama are characterized in that they have particularly large amounts of P, S and K components as compared with other water bodies.

<Amino acid analysis> Using ultrafine fibers as a filter medium, the red drum water of Tonoyama Dam was collected and concentrated by a rotary drum type continuous filtration concentrator and a continuous dehydrator (both manufactured by Toray Industries, Inc.) shown in FIG. The solid cake obtained was dehydrated and pelletized. As mentioned earlier, the plankton of Tonoyama Dam is mostly occupied by Peridinium vipes. (Because most of the red tide water of the Aikawa Dam in Wakayama Prefecture is also occupied by peridinium bipes, the same results as when using the red tide water of the Tonoyama Dam can be obtained.)

Samples were prepared from the pellets of Peridinium vipes by the following method. As a method of breaking the cell membrane for the preparation of the extract for measuring free amino acids,
The method generally used in yeast was adopted. A Hitachi 835 type amino acid automatic analyzer was used for the analysis. The specific procedure is as follows.

(1) Glass beads are packed in an exclusive threaded tube for grinding until the 8th minute. (2) Suspend 1 g of thawed plankton with 2-3 ml of water and fill in a tube. Allow the liquid to penetrate between the glass beads. (3) Tighten the screw and set it in the dedicated grinding device, and then 30 seconds 4
Mill twice for a total of 2 minutes. (4) Transfer the upper solution to a centrifuge tube, wash the glass beads with a small amount of water, and add this too to the centrifuge tube. (5) Centrifuge at 12000 rpm for 30 minutes and collect the supernatant (about 5 ml). (6) After deproteinizing with TCA (trichloroacetic acid) 5%, ether extraction is repeated 3 times. (7) Dilute 5 times with buffer of pH 2.2 and use for amino acid analysis.

The known amino acid standards are shown in FIG.
The analysis chart of Peridinium vipes is shown in FIG.
Comparing the two charts, it can be seen that, although the peaks of the Peridinium vipes sample are very large, very few match the amino acid positions of each standard. This unidentified peak may be a peptide of some kind, but details are unknown.

Table 3 shows the concentrations of the amino acids identified in FIG. 5 and the data obtained so far for carp and sardines.

[0040]

[Table 3] Amino Acid Concentration (mg / 100g) Amino Acid Peridinium Sardine Val 0 2.6 9.2 Leu 0 3.7 9.8 Ile 0 2.0 9.8 Pro 0 22.0 17.5 Phe 0 0.3 2.7 Tyr 0 13.1 3.5 Met 0 2.2 3.6 Arg 0 17.3 2.9 His 0 127 481 Glu 0 4.5 15.6 Tau 0 0 106 Cre 0 0 527 Cru 0 0 0 Asp 33.87 1.7 0.4 Thr 47.40 13 7.2 Ser 59.0 4 7.6 Gly 70.5 62.9 7.9 Ala 100.1 21.5 30.6 Lys 85.8 75.7 25

From Table 3, it can be seen that Peridinium vipes has a large amino acid concentration, but is significantly different from the amino acid pattern of fish and the like. It can also be seen that there is no taurine or any other sulfur containing amino acid.

<Lipid> Peridinium collected above
Lipids were analyzed from the Vipes pellets according to the following extraction method. (1) To 1 g of the pellet, 30 ml of a mixed solvent of chloroform and methanol at a volume ratio of 2: 1 is added. Leave it in a constant temperature bath at 30 ° C for 30 minutes and shake it occasionally. (2) Centrifugation (1500 rpm, 10 to 15 minutes), and the precipitation is repeated twice with 30 ml of the above mixed solvent. (3) Completely evaporate the solvent from the extract (about 90 ml) at 30 ° C or lower. (4) Dissolve in 5 ml of hexane. (5) Separation of lipids by TLC (thin layer chromatography). 1. Approximately 300 µl (10 µl x 30 times) of the remainder is applied to a silica plate in a band shape. 2. As the developing solution, a mixed solution of hexane: ether: acetic acid in a volume ratio of 50: 50: 1 is used. 3. Enclose the inside of the developing tank with filter paper so that the developing solution is saturated. 4. It develops in about 30 minutes, so cut out both ends of the plate with a diamond cutter and color with iodine to know the position. 5. Shave off the neutral lipid fraction and phospholipid fraction and put them in a small sample bottle. 6. Methylation (methylation without transesterification)
Perform the reaction. That is, 1 ml of a 15% concentration boron trifluoride methanol solution as a methylating agent was added to a sample bottle containing scraped silica and left at 100 ° C. for 40 minutes. 7. After the reaction, transfer the cooled sample to a ground stopper centrifuge tube (Spitz) for GLC analysis. Then, hexane extraction is repeated 3 times, and the sample is subjected to GLC analysis. (6) Gas Chromatograph Analysis Shimadzu Gas Chromatograph GC-9APTF was used for analysis. (7) Gas mass spectrum analysis It was measured using Hitachi M-2000A.

FIG. 6 shows a gas chromatogram of lipids in the peridinium pellet. 7 to 16 show mass spectra of each fatty acid. The fatty acids detected were all 11 kinds having an even carbon number. It is generally said that primitive organisms have fatty acids with odd carbon numbers,
Peridinium vipes does not seem to have such a fatty acid. Table 4 shows the concentration of each fatty acid (pellet 1
The number of μg in g) is shown. In the "Structure" column, m in Cm-n is the number of carbon atoms in the fatty acid, and n is the number of unsaturated groups.

[0044]

[Table 4] Structure Fatty acid concentration C12-1 Lauric acid 0.38 C14-0 Myristic acid 0.63 C16-0 Palmitic acid 1.34 C18-0 Stearic acid 0.29 C18-1 Oleic acid 1.69 C18-2 Linoleic acid 0.21 C18-3 Linolene Acid 0.28 C18-4 octadecatetraenoic acid 2.56 C18-5 octadecapentaenoic acid 3.27 C20-5 eicosapentaenoic acid 3.11 C22-6 docosahexaenoic acid 3.17

From Table 4, it can be seen that peridinium bipes has a high ratio of specific fatty acids, especially octadecatetraenoic acid and octadecapentaenoic acid, which have been difficult to obtain in large amounts in the past. It can be seen that EPA and DHA, which are currently in the spotlight, can be acquired at the same time.

[0046]

INDUSTRIAL APPLICABILITY According to the present invention, octadecapentaenoic acid and octadecatetranoic acid, which are useful highly unsaturated higher fatty acids, are obtained from the freshwater phytoplankton peridinium vipes, which is a red tide component that has not been conventionally used. Enoic acid (further, EPA or DHA) can be efficiently obtained.

[Brief description of drawings]

FIG. 1 is a graph showing each constituent element concentration per volume in each water area based on Table 1.

FIG. 2 is a graph showing each constituent element concentration per dry weight in each water area based on Table 2.

FIG. 3 is a graph showing the composition ratio of each element with respect to Al in which the difference due to the water area is hardly seen.

FIG. 4 is a chart showing known amino acid standards.

FIG. 5 is an analysis chart of Peridinium vipes.

FIG. 6 is a gas chromatogram of lipids in peridinium pellets.

FIG. 7 is a mass spectrum of methyl laurate.

FIG. 8 is a mass spectrum of methyl myristate.

FIG. 9 is a mass spectrum of methyl palmitate.

FIG. 10 is a mass spectrum of methyl stearate.

FIG. 11 is a mass spectrum of methyl oleate.

FIG. 12 is a mass spectrum of methyl linoleate and methyl linolenate.

FIG. 13 is a mass spectrum of methyl octadecatetraenoate.

FIG. 14 is a mass spectrum of methyl octadecapentanoate.

FIG. 15 is a mass spectrum of methyl eicosapentaenoate.

FIG. 16 is a mass spectrum of methyl docosahexaenoate.

FIG. 17 is an explanatory diagram of a concentration / dehydration treatment device.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yuji Watanabe 2-3-3 Nakazaki Nishi, Kita-ku, Osaka City, Osaka Prefecture Kansai General Environmental Center Co., Ltd.

Claims (4)

[Claims] 1. A Peridinium bipe
A process for producing a polyunsaturated higher fatty acid, which comprises isolating a polyunsaturated higher fatty acid containing at least octadecapentaenoic acid or / and octadecatetraenoic acid from s). 2. The process according to claim 1, wherein highly unsaturated higher fatty acids including octadecapentaenoic acid, octadecatetraenoic acid, eicosapentaenoic acid and docosahexaenoic acid are separated from Peridinium bipes. . 3. The method according to claim 1, wherein the red tide water mainly composed of Peridinium bipes is collected, concentrated, dehydrated and crushed, and then extracted. 4. The method according to claim 3, wherein the rotary cake type continuous filtration concentrator using ultrafine fibers as a filter medium is subjected to a concentration treatment, and then a continuous dehydrator is subjected to a dehydration treatment to obtain a solid cake. Law.