JPH02138964A - Method for enriching nutrient in alga of genus euglena - Google Patents

Abstract

PURPOSE:To obtain algae of the genus Euglena containing much various nutrients, especially readily oxidizable nutrients by adding vitamins, fatty acids, etc., to a culture solution prepared from the algae of the genus Euglena. CONSTITUTION:Algae of the genus Euglena are cultured by a conventional method. In the process, one or two or more of vitamins, fatty acids or derivatives thereof are added to a culture solution. If the additives are soluble in fats, an emulsifying agent, as desired, is added thereto.

Description

[Detailed Description of the Invention] [Industrial Field of Application] The present invention is a method for obtaining Euglena which contains large amounts of various nutrients, particularly various vitamins that are easily oxidized, and nutritionally preferable fatty acids. In other words, it relates to a method for nutritionally enriching Euglena.

[Prior art and its problems Coeuglena has the ability to biosynthesize vitamins C, E, etc., and can also biosynthesize highly unsaturated fatty acids such as eicosapencitric acid. For this reason, the conventional nutritional enrichment method is to irradiate light during Euglena culture (Agricultural and Biological Chemistry, Vol. 43, p. 2053, 1979, Vol. 50, p. 1063).
Page 1986) and methods of adding biosynthetic precursor-related substances (Japanese Patent Application Laid-open No. 57-54585, Japanese Patent Application Laid-open No. 59-20597).
6) has been proposed. However, with this method, the content of vitamins, etc. can only be increased by several times to several times that of untreated products, and the cost is high as it requires the use of expensive precursors and a light source. It was something.

[Object of the Invention] An object of the present invention is to provide Euglena which has been nutritionally enriched by a simpler and more efficient method than the conventional enrichment method described above at a low cost.

[Structure of the Invention] The present invention was completed based on the discovery that Euglena efficiently takes in water-soluble or fat-soluble vitamins, fatty acids, or derivatives thereof from the culture medium into its cells and accumulates them without decomposition. The gist is ``Nutritional enrichment of Euglena, which is characterized by adding one or more of vitamins, nutritionally preferable fatty acids, or derivatives thereof to Euglena culture solution as necessary. method”.

Euglena as used in the present invention is a protozoan that belongs to the genus Euglena (genus Euglena) according to the zoological classification, and includes all species, varieties, and variants belonging thereto. A typical example is Euglena gracilis.
ilis), Euglena gracilis var.

bacil 1aris), Euglena viridis, Astasia longa, etc.

The medium used for culturing such Euglena is Koren.
Halatner Medium (Journal of Protozoology)
(Journal of Protozoology
) Volume 14 (1967, Expanded Page 17) and Halatner Medium (Journal of Protozoology Vol. 6 (19
Known media such as 1995, p. 23) can be used. In addition, glucose, starch melt, molasses, glutamic acid, acetic acid, ethanol, etc. are used as carbon sources, and inorganic nitrogen sources such as ammonium nitrate, diammonium phosphate, ammonium sulfate, etc., glutamic acid,
Amino acids such as aspartic acid, or organic nitrogen sources such as peptone, casamino acids, yeast extract, and corn staple liquor are appropriately combined, and inorganic salts such as calcium, magnesium, manganese, and iron, and vitamins B and B1□ are combined as appropriate. It is also possible to use a medium to which a small amount has been added. Alternatively, photosynthesis of Euglena can be used without using an organic carbon source.

The appropriate culture temperature for Euglena is 20 to 35°C, and the appropriate initial pH is 3.0 to 7.5. Cultivation may be performed either under light irradiation or in the dark.

In addition, during culture, shake 50 to 250 times per minute,
It is desirable to carry out appropriate aeration and stirring.

When enriching water-soluble vitamins with the method for enriching Euglena according to the present invention, additives include vitamins (ascorbic acid, etc.) and their salts (sodium ascorbate, etc.),
Alternatively, derivatives (such as ascorbic acid stearate) can be used. When using a fat-soluble derivative (ascorbic acid stearate, etc.), it can be used alone or dissolved in a fat-soluble substance such as fatty acid, fatty acid methyl ester, oil or fat. An emulsifier can also be used if necessary. In that case, it may be emulsified before addition and then added, or it may be emulsified by stirring after addition. In this case, the amount of the additive to be added is usually 0.01 to 2%, preferably 0.01 to 1%. A larger amount may be added as long as it does not adversely affect Euglena.

In addition, when fortifying fat-soluble vitamins, vitamins (tocopherols, etc.) and their derivatives (tocopherol acetate, etc.) can be used as additives, and these should be mixed and dissolved in fat-soluble substances such as fatty acids, fatty acid methyl esters, and fats and oils. You can also do it. Of course, an emulsifier can be used if necessary. In that case, it may be emulsified before addition, or it may be emulsified by stirring during culturing. In this case, the amount of the additive to be added is usually 0.001 to 3%, preferably 0.005 to 0.5%.

A larger amount may be added as long as it does not adversely affect Euglena.

When fortifying fatty acids, not only fatty acids and their esters, but also fats and oils containing a large amount of nutritionally preferable fatty acids, salts of fatty acids, etc. can be used as additives. Since the fatty acid composition of the additive is reflected in the fatty acid composition of the enriched Euglena, any fatty acid can be enriched by selecting the additive. Emulsifiers can also be used if necessary. In that case, it may be emulsified before addition, or it may be emulsified by stirring during culturing. In this case, the amount of the additive to be added is usually 0.01 to 3%, preferably 0.05 to 1%. A larger amount may be added as long as it does not adversely affect Euglena.

Regarding the timing of addition, each can be added at the start of culture, or can be added during culture. Depending on the additive, it can also be added at different times.

The nutritional substances fortified by the present invention, namely water-soluble and fat-soluble vitamins, nutritionally preferable fatty acids (particularly those with a high degree of unsaturation), are all substances that are highly susceptible to air oxidation. The oxidative stability is improved by the incorporation into Euglena, making it possible to supply Euglena cells with high nutritional value. Its benefits include feed for Artemia, feed for rotifers, which is essential for the production of fish seedlings,
Alternatively, it can be used as feed for larvae of fish whose caliber is small, such as yellowtail grouper and white-tailed squirrel. It can also be used as various mixed feed raw materials, feed additives, health foods, etc.

Hereinafter, the present invention will be described in detail based on Examples, but the present invention is not limited thereto.

(Example 1) As an example of enrichment with water-soluble vitamins, Euglena was cultured by the following method. Five experimental areas were established, and each area was fortified with the following vitamin C.

District 1, District 2 without treatment, District 3 with 0.1% ascorbic acid, District 4 with addition of 0.1% sodium ascorbate.
0.1% ascorbic acid stearate addition 5 sections 0
Addition of 1% ascorbic acid stearate (calculated amount of additive was dissolved in 3 g of oleic acid and mixed with 0.01 g of emulsifier (Tween 80) and added.) 24 g of glucose, 8.4 g of ammonium sulfate, 7 g of magnesium sulfate. Hydrate 0.6g, potassium dihydrogen phosphate 0.6g, calcium carbonate 0.24g% EDTA disodium salt 60mg, Mohr's salt 60mg, manganese sulfate tetra-pentahydrate 21.6mg, zinc sulfate heptahydrate object 30m
g, cornstarch liquor 6.0g, vitamin 13
+ 6 mg, B1212 LLg in tap water 1.2℃
The mixture was dissolved in water, poured into a 2L jar fermenter, and sterilized. This was inoculated with 60 mI2 of a culture of Euglena gracilis that had been precultured in the same medium, and separately sterilized additives were added to adjust the pH.
Aerated culture was performed at 4, 5, and 28°C for 48 hours.

The Euglena used here is Euglena gracilis NIES 48, which was provided by the National Institute of Pollution and Microbiology (116-2 Ono, Tsukuba City, Ibaraki Prefecture), and the same can be obtained upon request.

After the cultivation was completed, Euglena was collected by centrifugation, and the total ascorbic acid content was determined by the hydrazine method. The results are shown in Table 1.

Table 1 Experimental area Vitamin C content (mg/100g dry matter equivalent,
(Same hereafter) 1st ward 2nd ward 7 3rd ward 6 4th ward 2 5th ward 6 19, 1 09, 3 57, 7 46, 7 34, 4 As a result, vitamin C and its derivatives in the culture solution enter the Euglena cells. It can be seen that a significantly large amount is taken up. In addition, in the conventional technology, vitamin C is high at 50 mg.
Only about 100g/100g of the product was obtained.

(Example 2) As an example of fortifying fat-soluble vitamins, vitamin E was fortified. The following five experimental plots were established and cultured in the same manner as in Example 1. After completion of the culture, Euglena was collected by centrifugation, and the vitamin E content in the unsaponifiable lipids was measured by high performance liquid chromatography. The results are shown in Table 2.

District 1, District 2 without treatment, District 3 with 0.01% tocopherol added 0.0
Addition of 1% tocopherol (emulsifier used) 4th section 0. 5 sections with 4% tocopherol acetate added 0.
Addition of old % tocopherol acetate (emulsifier used) Table 2 Experimental group Vitamin E content (mg/100g) 1 group
8.5 Section 2 85.3 Section 3 238.6 Section 4 156.4 Section 5 381.5 The results show that a significantly large amount of tocopherol and its derivatives in the medium are taken into Euglena cells. In addition, the vitamin E content of the conventional technology is about 80 mg/100 g at most.

(Example 3) As an example of fatty acid enrichment, ω-3, which is an essential fatty acid of marine fish,
Enriched with highly unsaturated fatty acids (eicosapentaenoic acid, docosahexaenoic acid). In the culture experiment of Example 1, the following seven experimental sections were established and Euglena was cultured. After culturing, Euglena was collected by centrifugation, lipids were extracted and methylated, and eicosapentaenoic acid (EPA) and docosahexaenoic acid (D) were extracted by gas chromatography.
The proportion of HA) in total fatty acids was measured.

District 1, District 2 without treatment, District 3 with 0.5% squid oil added, District 4 with addition of 0.5% squid oil (emulsifier used), District 4 0
．． 5% squid oil fatty acid addition 5th section 0.5% squid oil fatty acid addition (emulsifier used) 6th section 0.5% fatty acid methyl ester addition (Nister 85
, manufactured by Oriental Yeast) Section 7 0.5% fatty acid methyl ester addition (Nister 85, manufactured by Oriental Yeast) (
Emulsifier used) The proportion of EPA+DHA in the total fatty acids in the additive squid oil is 25.3%, and the proportion in Nister 85 is 76.5.
%Met.

The results are shown in Table 3.

Table 3 Experimental area Percentage of EPA+DHA in total fatty acids (%) Area 1 4.8 Area 2 8.9 Area 3 18.3 Area 4 15.2 Area 5 23.5 Area 6 22.4 Area 7 43, 3 In this way, triglycerides, fatty acids, and fatty acid methyl esters in the medium were efficiently taken into cells, making it possible to enrich Euglena with fatty acids. Furthermore, by using an emulsifier at that time, the additives were efficiently taken into the cells.

(Example 4) Nutritional enrichment of Artemia and feeding test to young red sea bream using Euglena obtained by fortifying vitamins B, C, E, pantothenic acid, and ω-3 polyunsaturated fatty acids according to the present invention I did it. The test was conducted for 3 weeks, and at the end of the test, the larvae were scooped out with reins to test total length, survival rate, and vitality.
Survival rates 24 hours after 1 minute air exposure were compared. The results are shown in Table 4.

The fortified Euglena used here contains V, B
e 20mg/100g, V, C708mg/1
00g, V, E321mg/100g, pantothenic acid 50mg/100g and ω-3 polyunsaturated fatty acid 1
It contained 0.2g/l OOg.

Section 1 = Red sea bream larva fed with Artemia immediately after conversion to M Section 2: Artemia immediately after conversion to F# fed Euglena for 24 hours Red sea bream larva fed secondary cultured Artemia Section 3: Artemia immediately after conversion to F# Survival rate (%) of red sea bream larval test area fed secondary cultured Artemia fed Euglena enriched according to the present invention for 24 hours Table 4 Total length (mm) Vitality test (%) 15.3 24.1 13 .0 46.6 25.3 44.0 78.0 29.7 96.0 By nutritionally fortifying Euglena in this way, its effectiveness as feed for Artemia has been greatly improved, demonstrating the usefulness of nutritionally fortifying. Became.

Claims (1)

[Scope of Claims] 1) A method for nutritionally enriching Euglena, which comprises adding one or more of vitamins, fatty acids, or derivatives thereof to a Euglena culture solution as necessary. 2) The method for nutritionally enriching Euglena according to item 1 above, which comprises using an emulsifier when the additive is fat-soluble.