Culture method for labyrinthulids

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

[0001] The present invention relates to a method for culturing labyrinthulids, which are heterotrophic organisms.

Background Art

[0002] In recent years, technologies for producing useful substances using microalgae have been actively developed. Some microalgae accumulate a large amount of lipids in their cells, and thus, the production of functional components, cosmetic active substances, biofuels, etc. that utilize this ability is being promoted for practical use. In addition, the use of microalgae such as Chlorella, Spirulina, and Euglena is expanding for health foods and feeds because they are rich in nutrients.

[0003] In recent years, attention has also been focused on labyrinthulids, 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. It has been reported that some labyrinthulids accumulate a large amount of highly unsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and higher hydrocarbons such as squalene in their cells. In addition, they are characterized by a high growth rate and a high amino acid score.

[0004] Conventionally, marine fish such as sardines have been mainly used as sources of DHA and EPA. The fatty acids in fish oil collected from marine fish are subjected to purification treatments such as transesterification, distillation, and hydrophobic chromatography for the purpose of removing oxidation products generated during transportation, etc. and chemical substances accumulated in the sea, and increasing the purity of DHA and EPA in the fatty acid group. On the other hand, when labyrinthulids are used, competition with fishery resources is eliminated, stable supply becomes possible, and deterioration due to oxidation and contamination with impurities are also expected to be reduced. Therefore, it is expected that the cost of DHA and EPA will be reduced.

[0005] As a medium for growing the heterotrophic organism labyrinthulids, Patent Document 1 orPatent Document 2 As described in Patent Document 2 , generally, GYT medium is used. The GYT medium has a basic composition containing glucose at a concentration of 20 g / L, tryptone at 10 g / L, and yeast extract at 5 g / L, and is prepared using natural seawater or artificial seawater. However, in a medium containing expensive proteolytic products like the commonly used GYT medium, there is a problem that it is difficult to make a profit when performing mass culture of heterotrophic organisms such as labyrinthulids.

[0006] In addition, since labyrinthulids cannot grow when bacteria are mixed in, aseptic culture is required (Non-Patent Document 1). The fact that culture using a sterile tank or the like is essential has the drawback that it also costs money to maintain the culture environment of the tank. The fact that culture using a sterile tank or the like is essential has the drawback that it also costs money to maintain the culture environment of the tank.

[0007] Examples of methods for culturing at low cost by adding whey to the medium components (Patent Document 3) and a method of performing binary culture with diatoms in a non-aseptic environment and allowing labyrinthulids to grow by being preyed on the diatoms as food (Patent Document 4) can be mentioned, but none of them are sufficient and there is room for improvement.

[0008] By the way, in recent years, due to problems such as the soaring oil prices and global warming, the development of technologies using biomass, a renewable resource, as a culture sugar source has been vigorously promoted. In particular, the utilization of sugar solutions obtained by hydrolyzing woody plants and / or herbaceous plants that do not compete with food has been actively carried out. In addition to cellulose, hemicellulose (for example, mannans in conifers, xylans in broad-leaved trees, etc.) is contained in woody plants and / or herbaceous plants. Monosaccharides generated by the decomposition of such hemicellulose are used for culture.

[0009] However, when the pH of the sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants is adjusted to a weakly acidic solution around pH 5, there are problems such as the stability of the culture and the decrease in the survival of microorganisms because the sugar solution contains a large amount of culture inhibitors such as acetic acid, sulfurous acid, and furfural.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non - Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] An object of the present invention relates to a method for culturing Labyrinthula that is not inhibited even when cultured in a sugar solution whose pH has been adjusted to a weakly acidic solution around 5, which is obtained by hydrolyzing woody plants and / or herbaceous plants.

Means for Solving the Problems

[0012] As a result of intensive studies on the above problems, the present inventors have found Labyrinthula species that exhibit excellent acid resistance. And they have found that by culturing such Labyrinthula species, high - quality proteins and EPA can be produced in large quantities, thus completing the present invention. That is, the present invention is as follows in [1] to [8] below. [1] Labyrinthula species that can be cultured in a sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants. [2] The Rhizochytrium that can be cultured in the sugar solution according to claim [1] obtained by hydrolysis with sulfuric acid and / or sulfurous acid. [3] The Rhizochytrium that can be cultured in the sugar solution according to claim [1] or [2] with a pH of 4.2 to 5.5. [4] The Rhizochytrium according to claims [1] to [3] belonging to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Ulkenia. [5] The Rhizochytrium according to claims [1] to [3] belonging to the genus Aurantiochytrium (FERM AP-22414). [6] A method for culturing Rhizochytrium using the sugar solution according to claims [1] to [3]. [7] A method for culturing Rhizochytrium belonging to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Ulkenia using the sugar solution according to claims [1] to [3]. [8] A method for culturing Rhizochytrium belonging to the genus Aurantiochytrium (FERM AP-22414) using the sugar solution according to claims [1] to [3].

Advantages of the Invention

[0013] According to the present invention, it is possible to provide Rhizochytrium that is not affected by lignocellulosic biomass raw materials and shows stable culture results using the obtained sugar solution as a nutrient salt. By culturing using such Rhizochytrium, it becomes possible to supply stable proteins and EPA.

Best Mode for Carrying Out the Invention

[0014] Hereinafter, the method for culturing Rhizochytrium according to the embodiment of the present invention will be described in detail.

[0015] As the organism to be cultured in the present invention, labyrinthulids are preferred. Labyrinthulids are chemoautotrophic marine eukaryotes belonging to Stramenopiles and are classified as motile oomycetes that exhibit gliding motility and produce zoospores. Labyrinthulids have a relatively fast growth rate and the ability to accumulate various lipids such as fatty acid esters, hydrocarbons, phospholipids, and glycolipids produced by assimilation as oil droplets inside cells, and thus are suitably used for the production of useful substances.

[0016] Generally, labyrinthulids are roughly classified into the family Labyrinthulidae and the family Thraustochytriidae, and include genera such as Labyrinthula, Aurantiochytrium, Schizochytrium, Thraustochytrium, Aplanochytrium, Oblongichytrium, Botryochytrium, and Japonochytrium.

[0017] As the labyrinthulids to be cultured, the genus Aurantiochytrium, the genus Schizochytrium, or the genus Thraustochytrium is more preferred. These species have a relatively high ability to produce substances such as lipids, can produce highly unsaturated fatty acids such as DHA and EPA, carotenoids such as astaxanthin and β-carotene, and hydrocarbons such as squalene, and thus are suitably used for food applications and as raw materials for biofuels.

[0018] As the labyrinthulids to be cultured, Aureococcus is particularly preferable. Among the Aureococcus genus, there are those that produce highly unsaturated fatty acids (PUFAs) such as DHA and EPA, as well as odd-chain 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 for collagen. Since about 90% of the algal body is composed of fatty acids and amino acids, it is characterized by high nutritional value.

[0019] As a preferred form of the present invention, for example, Aureococcus sp. RD013560 strain can be mentioned. The RD013560 strain is deposited as follows. Accession number: FERM P-22414 Deposit date: March 15, 2021 Depositary: National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganisms Depositary Center (NPMD) (2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture, Japan)

[0020] Labyrinthulids are sugar solutions obtained by hydrolyzing woody plants and / or herbaceous plants, and can grow at a pH of 4.2 to 5.5.

[0021] Labyrinthulids have the ability to grow in a sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants with a pH of 4.2 to 5.5, preferably 4.4 to 5.0. This enables fermentation under weakly acidic to acidic conditions, so efficient culture can be achieved while taking measures against contamination by miscellaneous bacteria.

[0022] Examples of woody plants include various conifers (pine, cedar, fir, cypress, hemlock, Douglas fir, radiata pine, etc.) and broad-leaved trees (eucalyptus, beech, oak, maple, chestnut, elm, etc.). One of these can be selected and used, or two or more can be selected and used in combination.

[0023] Examples of herbaceous plants include straw, rice, wheat, etc. One of these can be selected and used, or two or more can be selected and used in combination.

[0024] In the present invention, a sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants is used. That is, a sugar solution obtained by hydrolyzing woody plants, a sugar solution obtained by hydrolyzing herbaceous plants, or a sugar solution obtained by hydrolyzing woody plants and herbaceous plants is used. Among them, a sugar solution obtained by hydrolyzing woody plants is preferable. The sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants can be obtained as drainage during pulp production (pulp drainage).

[0025] The conditions for hydrolysis are not particularly limited. For example, acid hydrolysis, alkali hydrolysis, hydrolysis by enzymes, etc. can be mentioned. Among these, acid hydrolysis is preferable, hydrolysis by either sulfuric acid or sulfurous acid, or both is more preferable, and hydrolysis by sulfurous acid is even more preferable. In the present invention, as sulfuric acid, sulfuric acid and its salts can be used, and as sulfurous acid, sulfurous acid and its salts can be used.

[0026] The 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 raw material woody plants and / or herbaceous plants. However, depending on whether sulfuric acid and sulfurous acid, sulfurous acid and its salts, concentrated sulfuric acid and dilute sulfuric acid are used, the preferable addition amount, the temperature during acid hydrolysis, and the acid hydrolysis time are different. Therefore, it is necessary to appropriately adjust these conditions.

[0027] The sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants is preferably adjusted (neutralized) to a pH of 4.2 to 5.5, preferably 4.4 to 4.8, before culturing Labyrinthulomycetes. Thereby, Labyrinthulomycetes can be efficiently propagated. In addition, when the sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants is within the above pH range without adjusting (neutralizing) the pH, the pH adjustment (neutralization) step can be omitted.

[0028] The pH adjustment of the sugar solution may be performed by adding reagents such as magnesium hydroxide, magnesium oxide, calcium oxide, sodium hydroxide, etc., and is not particularly limited.

[0029] When sulfurous acid is used for hydrolysis, the amount of sulfurous acid contained at the end of hydrolysis is more than 10 times the amount added as a preservative such as in grape wine. Empirically, the H++HSO3 form has the highest antibacterial property, and this form increases around pH 5 (weakly acidic). Therefore, by setting the pH of the sugar solution to around 5, contamination by miscellaneous bacteria can be effectively prevented. When the pH of the sugar solution exceeds 5.5, the above-mentioned Labyrinthulomycetes can grow stably, but in addition to the precipitation of magnesium hydroxide slurry, etc., contamination by miscellaneous bacteria is likely to occur.

[0030] The composition in the sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants varies depending on the types of the woody plants and herbaceous plants as the raw materials, the hydrolysis conditions, etc., and cannot be uniformly defined, but it contains sugars such as glucose, mannose, galactose as hexoses, and xylose, arabinose, etc. as pentoses.

[0031] The medium for culturing Labyrinthulomycetes may contain, in addition to the sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants, other nutrients such as general carbon sources, nitrogen sources, vitamins, minerals, etc., various buffers such as phosphates, isotonic agents such as sodium chloride, microorganisms such as bacteria, yeasts, diatoms, etc. for dual culture, and medium components such as agar. Also, it may be prepared using natural seawater or artificial seawater.

[0032] Specific examples of general culture medium components include carbon sources such as glucose, fructose, mannose, galactose, sucrose, maltose, etc., amino acids such as glutamic acid, sodium glutamate, etc., nitrogen sources such as peptides, proteins, urea, ammonia, ammonium salts, nitrates, etc., extracts such as yeast extract, vitamins such as thiamine, riboflavin, niacin, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, etc., and minerals such as sodium, potassium, calcium, magnesium, phosphorus, sulfur, iron, cobalt, copper, zinc, manganese, molybdenum, etc.

[0033] As seawater salts for preparing artificial seawater, for example, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, strontium chloride, ammonium chloride, iron chloride, manganese chloride, cobalt chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, 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, boric acid, etc. can be used.

[0034] In the present invention, the conditions for culturing with the sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants are not particularly limited as long as Labyrinthulaceae that can grow under conditions around pH 5 are used. Specifically, Labyrinthulaceae may be added to the sugar solution obtained by hydrolyzing woody plants and / or herbaceous plants, and stirred if necessary. The culture temperature is usually 30°C or lower, and preferably 25°C or lower from the viewpoint of the survival rate of Labyrinthulaceae. Also, the lower limit is preferably 15°C or higher to prevent the risk of a decrease in the fermentation rate. Most preferably, it is 20°C to 25°C.

[0035] The Labyrinthulomycetes may be pre-cultured before adding to the sugar solution, and examples of the medium in this case include molasses medium, GYT medium, etc. As the fermentation method, any method usually used for the fermentation of sugar solution can be adopted. Examples of such fermentation methods include batch method, repeated batch fermentation method, cell recycle continuous fermentation method, immobilized cell method, etc. Among them, the cell recycle continuous fermentation method is preferable because it can take advantage of the characteristics of the Labyrinthulomycetes in the present invention and enable stable culture while taking measures against contamination by miscellaneous bacteria.

[0036] Next, the culture composition obtained by culturing the Labyrinthulomycetes and the method for producing the culture composition will be described.

[0037] The method for producing the culture composition according to the present embodiment is a method for producing a culture composition using the above-described method for culturing the Labyrinthulomycetes, and includes a step of culturing the Labyrinthulomycetes using a medium containing whey, and a step of concentrating or drying the medium containing the cultured Labyrinthulomycetes to obtain a culture composition. According to this production method, a culture composition mainly composed of the Labyrinthulomycetes themselves can be obtained.

[0038] As the Labyrinthulomycetes to be cultured, those having the ability to produce useful substances are preferable. Examples of useful substances include higher unsaturated fatty acids such as DHA and EPA, their monoesters, diesters, triglycerides, phospholipids, glycolipids, higher alkadienes, higher alkatrienes, triterpenes, tetraterpenes and other higher hydrocarbons, essential amino acids, proteins containing them, polysaccharides, pigments, vitamins, physiologically active substances, etc.

[0039] (Highly unsaturated fatty acid) In the present invention, the highly unsaturated 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), eicosapentaenoic acid (EPA, 20:5n-3), docosatetraenoic acid (DTA, 22:4n-6), n-3 docosapentaenoic acid (n-3DPA, 22:5n-3), n-6 docosapentaenoic acid (n-6DPA, 22:5n-6), docosahexaenoic acid (DHA, 22:6n-3), etc. are exemplified. In this specification, arachidonic acid is also represented as ARA. The total fatty acid composition refers to the composition of fatty acids detected when microorganisms are cultured, freeze-dried, the fatty acids are methyl-esterified, and analyzed using GC, specifically, fatty acids having a carbon chain of 14 to 22.

[0040] As the labyrinthulids to be cultured, those that accumulate at least one of DHA and EPA intracellularly are more preferred. Also, the genus Aurantiochytrium, the genus Schizochytrium, or the genus Thraustochytrium is more preferred, and the genus Aurantiochytrium is particularly preferred. With such types, the produced DHA and EPA are accumulated in cells where they are difficult to be oxidized, so that the oxidation of DHA and EPA can be suppressed and they can be used for various applications.

[0041] Labyrinthulids can be used for various applications such as food, feed, fertilizer, industrial raw materials, etc. Specific examples of food applications include general food, health food, food ingredients, beverage ingredients, etc. Specific examples of feed include livestock feed, poultry feed, aquaculture feed, pet feed, etc. Specific examples of industrial raw materials include raw materials for biofuels, raw materials for feed, raw materials for fertilizers, raw materials for chemicals, raw materials for pharmaceuticals, etc.

Examples

[0042] Hereinafter, the present invention will be specifically described with reference to examples, but the technical scope of the present invention is not limited thereto.

[0043] (Example 1) 19.1 g of artificial seawater powder was added to 1 L of the sulfite cooking extract (solid content 15% w / v) generated when domestic broad-leaved tree chips mainly composed of domestic broad-leaved tree chips were subjected to sulfite cooking (150 °C, 9 hours), and the mixture was neutralized to pH 5.0 with magnesium hydroxide. Then, it was diluted with water so that the sugar concentration became 3.0%. This was used as a sugar solution medium (hexose concentration 0.9%, pentose concentration 2.1%). Separately, Labyrinthulomycetes, Aurantiochytrium sp. (RD013560 strain) were pre-cultured in 100 mL of GYT medium at 23 °C until full growth, and the growth was evaluated using the absorbance at OD660nm in the sugar solution medium of Example 1 as an index of the cell mass. Table 1 shows the cell concentration after 48 hours of culture.

[0044]

Table 1

[0045] (Example 2) 19.1 g of artificial seawater powder was added to 1 L of the sulfite cooking extract (solid content 15% w / v) generated when domestic coniferous tree chips mainly composed of domestic coniferous tree chips were subjected to sulfite cooking under the same conditions as in Example 1, and the mixture was neutralized to pH 5.0 with magnesium hydroxide. Then, it was diluted with water so that the sugar concentration became 3.0%. This was used as a sugar solution medium (hexose concentration 2.0%, pentose concentration 1.0%). Separately, Labyrinthulomycetes, Aurantiochytrium sp. (RD013560 strain), (A013857 strain), (A013857 strain), Schizochytrium sp. (S13702 strain), Oblongichytrium sp. (O010672 strain), Ulkenia sp. (RD013864) were pre-cultured in 100 mL of GYT medium at 23 °C until full growth, and the growth was evaluated using the absorbance at OD660nm in the sugar solution medium of Example 1 as an index of the cell mass. Table 2 shows the cell concentration after 48 hours of culture.

[0046]

Table 2

[0047] (Fatty acid analysis by GC-MS) Next, Aurantiochytrium (RD013560 strain) cultured, collected, washed in the medium of Example 1 and then lyophilized was methylated using a fatty acid methylation kit, and quantitative analysis was performed using GC-MS. The conditions are shown below. The column used was SHIMADU #CB20-M225-025, and it was subjected to the analysis conditions of 50 °C (held for 0.5 min) → temperature increase at 40 °C / min → 175 °C (held for 0 min) → temperature increase at 15 °C / min → 210 °C (held for 0 min) → temperature increase at 5 °C / min → 240 °C (held for 15 min), a total of 26.96 min. As a result, the EPA concentration was 1% and the DHA concentration was 15%. In addition, peaks derived from other PUFAs were detected.

[0048] (Figure: GC-MS chart) TIFF0007687170000003.tif84132 1. Derived from myristic acid 2. Derived from pentadecanoic acid 3. Derived from palmitic acid 4. Derived from margaric acid 5. Derived from stearic acid 9. Derived from ozbondic acid

[0049] (Protein concentration) The concentration (mass %) of organic nitrogen contained in Aurantiochytrium (RD013560 strain) cultured, collected, washed in the medium of Example 1 and then lyophilized was analyzed by the Kjeldahl method, and the protein concentration was calculated using the value calculated as the amount of organic nitrogen contained in the sample × 6.25 as the protein amount. It is shown in Table 3.

[0050]

Table 3

[0051] From the above results, it was confirmed that labyrinthulids produce abundant proteins and PUFAs.

Claims

**Claim 1**: Aureobasidium sp. with accession number FERM P-22414, which can be cultured in a sugar solution obtained by hydrolyzing woody and / or herbaceous plants by sulfuric acid and / or sulfite cooking. **Claim 2** Aureobasidium sp. with accession number FERM P-22414, which can be cultured in the sugar solution according to Claim 1, where the pH is 4.2 to 5.

5. **Claim 3**: A method for culturing Aureobasidium sp. with accession number FERM P-22414, characterized by using a sugar solution obtained by hydrolyzing woody and / or herbaceous plants by sulfuric acid and / or sulfite cooking and culturing with this sugar solution. **Claim 4** A method for culturing Aureobasidium sp. with accession number FERM P-22414, using the sugar solution according to Claim 3, where the pH is 4.2 to 5.5.

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