JP2023047453A - Method for culturing labyrinthulomycetes - Google Patents

Abstract

To provide methods for culturing Labyrinthulomycetes that do not inhibit cultivation even in a sugar solution obtained by hydrolyzing woody plants and/or herbs and adjusting the pH of the sugar solution to a weakly acidic solution of around 5, and to provide Labyrinthulomycetes showing stable culture results by using the obtained sugar solution as a nutrient salt without being affected by woody biomass raw materials.SOLUTION: The inventors of the present invention have found Labyrinthulomycetes showing excellent acid resistance as a result of intensive studies on the above problems. And the inventors found that it is possible to produce a large amount of high-quality protein and EPA by culturing such Labyrinthulomycetes and completed the invention.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a method for culturing Labyrinthulids, which are heterotrophic organisms.

In recent years, techniques for producing useful substances using microalgae have been actively developed. Since certain kinds of microalgae accumulate a large amount of lipids in their cells, practical use of the production of functional ingredients, purifying active substances, biofuels, etc. using this ability is underway. Microalgae such as chlorella, spirulina, and euglena contain abundant nutrients, and their use in health foods and feeds is expanding.

Also, in recent years, attention has been focused on Labyrinthula, which are protists closely related to microalgae. Labyrinthulids are heterotrophic marine eukaryotic microorganisms that do not perform photosynthesis, and are widely distributed mainly in subtropical and tropical regions. Labyrinthula contains polyunsaturated fatty acids (PUFA), including docosahexahydrochloride (DHA) and eicosapentaenoic acid (EPA), polyunsaturated fatty acids (PUFA), polyunsaturated fatty acids (PUFA), and higher hydrocarbons such as squalene. In addition to being reported to accumulate in large amounts within the body, it is characterized by a fast growth rate and a high amino acid score.

Conventionally, marine fish such as blue fish have been mainly used as sources of supply of DHA and EPA. Fatty acids in fish oil collected from marine fish are transesterified, distilled, and processed for the purpose of removing oxidants generated during transportation and chemical substances accumulated in the sea, and increasing the purity of DHA and EPA in the fatty acid group. It has undergone purification treatment such as hydrophobic chromatography. In contrast, the use of Labyrinthulas eliminates competition with marine resources, enables stable supply, and is expected to reduce deterioration due to oxidation and contamination with impurities, resulting in lower costs for DHA and EPA. is expected to progress.

As a medium for growing heterotrophic organism Labyrinthulea, GYT medium is generally used as described in Patent Document 1 or Patent Document 2 . GYT medium has a basic composition containing 20 g/L glucose, 10 g/L tryptone, and 5 g/L yeast extract, and is prepared using natural seawater or artificial seawater. A medium containing such an expensive protein hydrolyzate has the problem that it is difficult to make a profit when mass-cultivating heterotrophic organisms such as Labyrinthulids.

In addition, since Labyrinthulids cannot proliferate if they are contaminated with bacteria, sterile culture was required (Non-Patent Document 1). The necessity of culturing in an aseptic tank or the like has the disadvantage that maintenance of the culturing environment of the tank is costly. The fact that culturing in aseptic tanks, etc. is essential has the disadvantage that maintenance of the culturing environment in the tank is costly.

A method of adding whey to the medium component for low-cost culture (Patent Document 3), and a two-membered culture with diatoms as a non-sterile environment, preying on diatoms as food to grow Labyrinthula. There is a method (Patent Document 4) that can be used, but none of them can be said to be sufficient and there is room for improvement.

In recent years, due to problems such as soaring oil prices and global warming, technological development using biomass, which is a renewable resource, as a source of cultured sugar has been vigorously pursued. In particular, sugar solutions obtained by hydrolyzing woody plants and/or herbaceous plants, which do not compete with food, are being actively used. Woody plants and/or herbaceous plants contain hemicelluloses (for example, mannans in softwoods and xylans in hardwoods) in addition to cellulose. Monosaccharides produced by degrading such hemicellulose are used for culture.

However, when the pH of the sugar solution obtained by hydrolyzing woody plants and/or herbs is adjusted to a weakly acidic solution around 5, the sugar solution contains a large amount of culture inhibitors such as acetic acid, sulfurous acid, and furfural. Due to its inclusion, there were problems such as stability of culture and reduced survival of microorganisms.

JP 2006-304685 A International Publication No. 2012-077799 Patent No. 6429162 JP 2017-051187 A Porter, (1990) Phylum Labyrinthulomcota. In: Margulis L, Corliss JO, Melkonian M, Chapman DJ (eds) Handbook of Protoctista. Jones and Bartlett, Boston, pp 8388

An object of the present invention is to provide a method for culturing Labyrinthula in which the cultivation is not inhibited even in a sugar solution obtained by hydrolyzing woody plants and/or herbs and adjusting the pH of the sugar solution to a weakly acidic solution of around 5.

The inventors of the present invention have found Labyrinthula showing excellent acid resistance as a result of intensive studies on the above problems. Then, the inventors found that by culturing such labyrinthulids, it is possible to produce a large amount of high-quality protein and EPA, thereby completing the present invention.
That is, the present invention is the following [1] to [8].
[1] Labyrinthula that can be cultured in a sugar solution obtained by hydrolyzing woody plants and/or herbs.
[2] Labintula species culturable in the sugar solution according to [1], which are obtained by hydrolysis with sulfuric acid and/or sulfurous acid.
[3] Labyrinthula that can be cultured in the sugar solution according to [1] or [2], which has a pH of 4.2 to 5.5.
[4] Labyrinthula according to [1] to [3], which belong to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Ulkenia.
[5] Labyrinthula according to [1] to [3], which belong to the genus Aurantiochytrium (FERM AP-22414).
[6] A method for culturing Labyrinthulea using the sugar solution according to any one of claims [1] to [3].
[7] A method for culturing Labyrinthula belonging to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Ulkenia, using the sugar solution according to any one of [1] to [3].
[8] A method for culturing Labyrinthulea belonging to the genus Aurantiochytrium (FERM AP-22414) using the sugar solution according to any one of claims [1] to [3].

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide Labyrinthula that show stable culture results by using the obtained sugar solution as a nutrient without being affected by woody biomass raw materials. By culturing using such labyrinthulids, it is possible to stably supply protein and EPA.

Hereinafter, a method for culturing Labyrinthula according to an embodiment of the present invention will be described in detail.

Labyrinthuleas are preferred as the organisms to be cultured in the present invention. Labyrinthulids are chemoheterotrophic marine eukaryotes belonging to the stramenopile family, and are classified as oomycetes with motility, such as producing gliding movements and migratory cells. Labyrinthulas grow relatively quickly and have the ability to accumulate various lipids such as fatty acid esters, hydrocarbons, phospholipids, and glycolipids produced by assimilation in the form of oil droplets in their cells, making them useful for the production of useful substances. It is preferably used.

In general, Labyrinthula is roughly divided into Labyrinthulidae and Thraustochytriidae, and Labyrinthula, Aurantiochytrium, Schizochytrium ), the genus Thraustochytrium, the genus Aplanochytrium, the genus Oblongichytrium, the genus Botryochytrium, the genus Japonochytrium, and the like.

Labyrinthula to be cultured are more preferably those of the genus Aurantiochytrium, Schizochytrium, or Thraustochytrium. These types have a relatively high ability to produce lipids and the like, and can produce highly unsaturated fatty acids such as DHA and EPA, carotenoids such as astaxanthin and β-carotene, and hydrocarbons such as squalene. It can be suitably used for applications, raw materials for biofuels, and the like.

As Labyrinthula to be cultured, the genus Aurantiochytrium is particularly preferred. The genus Aurantiochytrium includes polyunsaturated fatty acids (PUFA) such as DHA and EPA, as well as odd-numbered fatty acids that are said to be effective in improving Alzheimer's disease and type 2 diabetes. In addition, it is rich in essential amino acids such as lysine and proline, which is a raw material of collagen, and about 90% of the algal body is composed of fatty acids and amino acids, so it is characterized by high nutritional value.

A preferred embodiment of the present invention includes, for example, the Aurantiochytrium genus RD013560 strain. Strain RD013560 has been deposited as follows.
Accession number: FERM P-22414
Acceptance date: March 15, 2021 Depositary: National Institute of Technology and Evaluation Biotechnology Center Patent Microorganism Depositary Center (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan)

Labyrinthulas are sugar solutions obtained by hydrolyzing woody plants and/or herbaceous plants, and can grow at pH 4.2 to 5.5.

Labyrinthulas have the ability to proliferate in a sugar solution obtained by hydrolyzing woody plants and/or herbs at a pH of 4.2 to 5.5, preferably 4.4 to 5.0. As a result, fermentation can be carried out under weakly acidic to acidic conditions, so efficient cultivation can be achieved while taking measures against contamination with various bacteria.

Examples of woody plants include various conifers (pine, cedar, Japanese cypress, cypress, hemlock, Douglas fir, radial pine, etc.) and broad-leaved trees (eucalyptus, alder, beech, birch, oak, shiitake, etc.). One of these may be selected and used, or two or more may be selected and used in combination.

Examples of herbaceous plants include straw, rice, and wheat. One of these may be selected and used, or two or more may be selected and used in combination.

In the present invention, a sugar solution obtained by hydrolyzing woody plants and/or herbs is used. That is, sugar solutions obtained by hydrolyzing woody plants, sugar solutions obtained by hydrolyzing herbs, or sugar solutions obtained by hydrolyzing woody plants and herbs are used. Among them, a sugar solution obtained by hydrolyzing woody plants is preferable. A sugar solution obtained by hydrolyzing woody plants and/or herbs can be obtained as waste fluid (pulp waste fluid) during pulp production.

Conditions for hydrolysis are not particularly limited. Examples thereof include acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Of these, acid hydrolysis is preferred, hydrolysis with either or both of sulfuric acid and sulfurous acid is more preferred, and hydrolysis with sulfurous acid is even more preferred. In the present invention, sulfuric acid and its salts can be used as sulfuric acid, and sulfurous acid and its salts can be used as sulfurous acid.

Conditions for hydrolyzing woody plants and/or herbaceous plants with sulfuric acid and/or sulfurous acid are not particularly limited. Sulfuric acid, sulfurous acid, or both sulfuric acid and sulfurous acid can be added to the woody and/or herbaceous raw materials. Depending on which one is used, the preferred addition amount, temperature during acid hydrolysis, and acid hydrolysis time differ, so it is necessary to adjust these conditions as appropriate.

The sugar solution obtained by hydrolyzing woody plants and/or herbs is adjusted (neutralized) to pH 4.2 to 5.5, preferably pH 4.4 to 4.8, before culturing Labyrinthula. ) is preferred. As a result, Labyrinthulids can be propagated efficiently. In addition, if the sugar solution obtained by hydrolyzing woody plants and/or herbs is within the above pH range without adjusting (neutralizing) the pH, adjusting (neutralizing) the pH A process can be omitted.

The pH of the sugar solution may be adjusted by adding reagents such as magnesium hydroxide, magnesium oxide, calcium oxide and sodium hydroxide, and is not particularly limited.

When sulfurous acid is used for hydrolysis, the amount of sulfurous acid contained at the end of hydrolysis is 10 times or more the amount added as a preservative to wine or the like. Empirically, the H++HSO3 form of sulfurous acid has the highest antibacterial activity, but this form increases around pH 5 (weakly acidic). Therefore, by setting the pH of the sugar solution to around 5, contamination with various bacteria can be effectively prevented. If the pH of the sugar solution exceeds 5.5, the Labyrinthula described above can grow stably, but precipitation of magnesium hydroxide slurry and the like are likely to occur, and contamination with various germs is likely to occur.

The composition of the sugar solution obtained by hydrolyzing woody plants and/or herbs varies depending on the woody plants and/or herbaceous plants as raw materials, the hydrolysis conditions, etc., and cannot be defined uniformly. Although not possible, hexoses contain sugars such as glucose, mannose, and galactose, and pentoses contain sugars such as xylose and arabinose.

The medium for culturing Labyrinthula includes sugar solutions obtained by hydrolyzing woody plants and/or herbs, as well as other nutrients such as common carbon sources, nitrogen sources, vitamins and minerals. , various buffers such as phosphate, tonicity agents such as sodium chloride, microorganisms such as bacteria, yeast and diatoms for two-membered culture, and medium components such as agar. Moreover, it may be prepared using natural seawater, or may be prepared using artificial seawater.

Specific examples of common medium components include carbon sources such as glucose, fructose, mannose, galactose, sucrose, and maltose, amino acids such as glutamic acid and sodium glutamate, peptides, proteins, urea, ammonia, ammonium salts, nitrates, and the like. and extracts such as yeast extract, vitamins such as thiamine, riboflavin, niacin, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, sodium, potassium, calcium, magnesium, phosphorus, sulfur, iron, Minerals such as cobalt, copper, zinc, manganese, and molybdenum are included.

Seawater salts for preparing artificial seawater include, for example, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, strontium chloride, ammonium chloride, iron chloride, manganese chloride, cobalt chloride, sodium dihydrogen phosphate, hydrogen phosphate Disodium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium nitrate, sodium carbonate, sodium silicate, sodium fluoride, magnesium sulfate, cobalt sulfate, copper sulfate, zinc sulfate, sodium molybdate, potassium bromide, boron An acid or the like can be used.

In the present invention, the conditions for culturing in a sugar solution obtained by hydrolyzing woody plants and/or herbaceous plants are not particularly limited as long as Labyrinthula species that can grow at around pH 5 are used. Specifically, Labyrinthulas may be added to a sugar solution obtained by hydrolyzing woody plants and/or herbs, and the mixture may be stirred as necessary. The culture temperature is usually 30° C. or lower, preferably 25° C. or lower from the viewpoint of the survival rate of Labyrinthulids. Moreover, it is preferable that the lower limit is 15° C. or more from the viewpoint of preventing a decrease in the fermentation rate. Most preferably, it is between 20°C and 25°C.

Labyrinthula may be pre-cultured before being added to the sugar solution, and examples of the medium in that case include molasses medium and GYT medium. As the fermentation method, any method that is usually used for fermentation of sugar solution can be employed. Examples of such fermentation methods include a batch method, a repeated batch fermentation method, a bacterial body recycling continuous fermentation method, an immobilized bacterial cell method, and the like. Among them, the continuous fermentation method with bacterial cell recycling is preferable because it enables stable cultivation while taking measures against contamination by bacteria by making use of the characteristics of Labyrinthula in the present invention.

Next, a culture composition obtained by culturing Labyrinthula and a method for producing the culture composition will be described.

The method for producing a culture composition according to the present embodiment is a method for producing a culture composition using the method for culturing Labyrinthulea described above, and is a step of culturing Labyrinthulea using a medium containing whey. and a step of concentrating or drying the medium containing the cultured Labyrinthulea to obtain a culture composition. According to this production method, a culture composition containing Labyrinthula itself as a main component is obtained.

Labyrinthula to be cultured are preferably those having the ability to produce useful substances. Useful substances include, for example, higher unsaturated fatty acids such as DHA and EPA, their monoesters, diesters, and triglycerides, phospholipids, glycolipids, higher hydrocarbons such as higher alkadienes, higher alkatrienes, triterpenes, and tetraterpenes. , essential amino acids, proteins containing them, polysaccharides, pigments, vitamins, physiologically active substances, and the like.

(Highly unsaturated fatty acid)
In the present invention, the polyunsaturated fatty acid (PUFA) is a fatty acid having 18 or more carbon atoms and 3 or more double bonds, more preferably a fatty acid having 20 or more carbon atoms and 3 or more double bonds. Specifically, linoleic acid (LA, 18:2n-6), α-linolenic acid (ALA, 18:3n-3), γ-linolenic acid (GLA, 18:3n-6), stearidonic acid (STA, 18 :4n-3), dihomo-γ-linolenic acid (DGLA, 20:3n-6), eicosatetraenoic acid (ETA, 20:4n-3), arachidonic acid (ARA, 20:4n-6), ethyl icosapentaenoic acid (EPA, 20:5n-3), docosatetraenoic acid (DTA, 22:4n-6), n-3 docosapentaenoic acid (n-3DPA, 22:5n-3), n-6 docosapentaenoic acid Examples include enoic acid (n-6DPA, 22:5n-6) and docosahexaenoic acid (DHA, 22:6n-3). Arachidonsan is also referred to herein as ARA. The total fatty acid composition refers to the composition of fatty acids, specifically fatty acids with 14 to 22 carbon chains, detected when microorganisms are cultured, freeze-dried, fatty acids are methyl-esterified, and analyzed using GC. .

Labyrinthula to be cultured are more preferably those that accumulate at least one of DHA and EPA in cells. Moreover, the genus Aurantiochytrium, Schizochytrium, or Thraustochytrium is more preferable, and the genus Aurantiochytrium is particularly preferable. With such a type, the produced DHA and EPA are accumulated in cells that are difficult to be oxidized, so that the oxidation of DHA and EPA can be suppressed and used for various purposes.

Labyrinthula can be used for various purposes such as food, feed, fertilizer, and industrial raw materials. Specific examples of edible uses include general foods, health foods, food materials, and beverage materials. Further, specific examples of feeds include livestock feeds, poultry feeds, aquaculture feeds, pet feeds, and the like. Specific examples of industrial raw materials include biofuel raw materials, feed raw materials, fertilizer raw materials, chemical raw materials, pharmaceutical raw materials, and the like.

EXAMPLES The present invention will be specifically described below by showing examples, but the technical scope of the present invention is not limited to these.

(Example 1)
19.1 g of artificial seawater powder was added to 1 L of cooking extract (solid content 15% w/v) generated when sulfite cooking (150 ° C, 9 hours) of chips composed mainly of domestic broadleaf miscellaneous trees was performed, and hydroxylation was performed. Neutralized to pH 5.0 with magnesium. Then, it was diluted with water so that the sugar concentration was 3.0%. This was used as a sugar solution medium (0.9% hexose concentration, 2.1% pentose concentration).
Separately, Labyrinthula and the genus Aurantiochytrium (RD013560 strain) were precultured in 100 mL of GYT medium at 23 ° C. to full growth, and the absorbance at OD 660 nm in the sugar solution medium of Example 1 was used as a cell mass index. was evaluated. Table 1 shows the cell concentration after 48 hours of culture.

(Example 2)
19.1 g of artificial seawater powder was added to 1 L of cooking extract (solid content: 15% w/v) generated when chips composed mainly of domestic coniferous miscellaneous trees were digested with sulfite under the same conditions as in Example 1, and water was added. Neutralized to pH 5.0 with magnesium oxide. Then, it was diluted with water so that the sugar concentration was 3.0%. This was used as a sugar solution medium (hexose concentration: 2.0%, pentose concentration: 1.0%).
Separately, Labyrinthula, Aurantiochytrium (RD013560 strain), (A013857 strain), (A013857 strain), Schizochytrium (S13702 strain), Obrongichytrium (O010672 strain), and Ulkenia (RD013864) were added to the GYT medium. Preculture was carried out at 100 mL at 23° C. to full growth, and proliferation was evaluated in the sugar solution medium of Example 1 using the absorbance at OD 660 nm as an indicator of the amount of cells. Table 2 shows the cell concentration after 48 hours of culture.

(Fatty acid analysis by GC-MS)
Next, Aurantiochytrium (RD013560 strain), which was cultured, harvested, washed, and then freeze-dried in the medium of Example 1, was methylated using a fatty acid methylation kit and quantitatively analyzed using GC-MS. did Conditions are shown below. SHIMADU #CB20-M225-025 was used as a column, and the temperature was increased from 50°C (held for 0.5 min) to 40°C/min to 175°C (held for 0 min) to 15°C/min to 210°C (held for 0 min). →Temperature rising at 5°C/min→240°C (retained for 15 minutes) Under the analysis conditions of a total of 26.96 minutes, the EPA concentration was 1% and the DHA concentration was 15%. Other peaks derived from PUFA were also detected

１．ミリスチン酸由来
２．ペンタデシル酸由来
３．パルミチン酸由来
４．マルガリン酸由来
５．ステアリン酸由来
９．オズボンド酸由来 (Figure: GC-MS chart)

1. 2. derived from myristic acid; Derived from pentadecylic acid3. Derived from palmitic acid4. Derived from margaric acid5. Derived from stearic acid9. Derived from ospondic acid

(protein concentration)
The concentration (% by mass) of organic nitrogen contained in the Aurantiochytrium genus (RD013560 strain) that was cultured, harvested, washed, and then freeze-dried in the medium of Example 1 was analyzed by the Kjeldahl method and contained in the sample. The protein concentration was calculated using the value calculated by multiplying the amount of organic nitrogen by 6.25 as the amount of protein. Table 3 shows.

From the above results, it was confirmed that labyrinthuleas produce abundant proteins and PUFAs.

Claims (8)

Labyrinthula that can be cultured in a sugar solution obtained by hydrolyzing woody plants and/or herbs. Labyrinthula culturable in the sugar solution according to claim 1, obtained by hydrolysis with sulfuric acid and/or sulfurous acid. Labyrinthula that can be cultured in the sugar solution according to [1] or [2], which has a pH of 4.2 to 5.5. The Labyrinthula according to any one of claims 1 to 3, which belongs to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Ulkenia. Labyrinthula according to claims [1] to [3], which belong to the genus Aurantiochytrium (FERM AP-22414). A method for culturing Labyrinthulea using the sugar solution according to any one of claims [1] to [3]. A method for culturing Labyrinthula belonging to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Ulkenia using the sugar solution according to any one of claims [1] to [3]. A method for culturing Labyrinthulea belonging to the genus Aurantiochytrium (FERM AP-22414) using the sugar solution according to any one of claims [1] to [3].