Culture composition containing odd fatty acid esters

Abstract

To provide a culture composition containing odd-numbered fatty acid ester obtained by efficiently producing odd-numbered fatty acid ester in Labyrinthula.SOLUTION: The culture composition containing odd-numbered fatty acid ester of the present invention includes a step of culturing a microorganism belonging to the genus Aurantiochytrium or Schizochytrium in an amino acid-added medium containing one or more amino acids selected from the group consisting of L-valine, L-isoleucine, L-threonine, L-methionine, D-methionine, and DL-methionine at a concentration of 10 mM or more per type, and contains the algae of the microorganism belonging to the genus Aurantiochytrium or Schizochytrium and odd-numbered fatty acid esters produced by the microorganism in which odd-numbered fatty acids are ester-bonded. The odd-numbered fatty acid content per biomass of the microorganism is 0.04 g / g or more, and the ratio of the odd fatty acid in the total lipid is 25% or more.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing odd-chain fatty acid esters using microorganisms belonging to Labyrinthulomycetes such as Aurantiochytrium and Schizochytrium, and a culture composition containing odd-chain fatty acid esters obtained by culturing these microorganisms.

Background Art

[0002] In recent years, technologies for producing useful substances using microorganisms have been actively developed. Since certain microalgae accumulate a large amount of lipids in cells, technologies for producing functional components, bioactive substances, biofuels, etc. using microalgae have been developed. As microalgae, photosynthetic microorganisms such as Chlorella, Spirulina, and Euglena have been used not only for substance production but also for the algal bodies themselves as foods, feeds, etc. However, in recent years, the practical application of heterotrophic microorganisms with excellent culture efficiency and more advantageous for substance production has also been promoted.

[0003] Labyrinthulomycetes are chemoheterotrophic marine eukaryotic microorganisms belonging to Stramenopiles, and due to their high growth ability and lipid production ability, their application to substance production has been widely studied. Labyrinthulomycetes are roughly classified into the family Labyrinthulidae and the family Thraustochytriidae, and the family Thraustochytriidae includes genera such as Aurantiochytrium, Schizochytrium, and Thraustochytrium.

[0004] It has been reported that Aurantiochytrium has a high ability to produce hydrocarbons such as squalene and odd-chain fatty acids in addition to docosahexaenoic acid (DHA), which is an omega-3 fatty acid, and eicosapentaenoic acid (EPA). Aurantiochytrium is known to produce large amounts of these lipids during the logarithmic growth phase and accumulate them as oil droplets in cells.

[0005] Odd-numbered fatty acids produced by Aurantiochytrium include pentadecanoic acid (PDA), which has 15 carbon atoms; tridecylic acid, which has 13 carbon atoms; and heptadecanoic acid (margaric acid), which has 17 carbon atoms. Pentadecanoic acid is also found in small amounts in the bodies of mammals and other organisms, and has been reported to exhibit various physiological activities such as promoting hair growth, lowering blood pressure, suppressing blood glucose elevation, promoting cell proliferation, and alleviating Alzheimer's disease.

[0006] Odd-numbered fatty acids, including pentadecanoic acid, are expected to see increased demand in various applications such as food and beverages, pharmaceuticals, and cosmetics as functional ingredients and physiologically active substances. Therefore, there is a need for efficient production methods that allow for biological production in a near-natural state. To date, technologies related to cultivation methods for producing odd-numbered fatty acids using Aurantiochytrium have been investigated.

[0007] Patent Document 1 describes a method for producing a culture medium for culturing Aurantiochytrium, comprising the steps of (1) treating a cell extract with a strong acid and heating it; (2) neutralizing the extract from step (1); and (3) preparing a cell culture medium based on the extract from step (2). The medium prepared by this method is said to increase the content of odd fatty acids in the total fatty acids produced by Aurantiochytrium. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2017-063633 [Overview of the project] [Problems that the invention aims to solve]

[0009] Microalgae belonging to the Labyrinthula genus, such as Aurantiochytrium, synthesize odd-numbered fatty acids useful for various applications as fatty acid esters such as fatty acid triglycerides and phospholipids. After the produced fatty acid esters are separated from the cells, they need to be concentrated and purified or hydrolyzed back into fatty acids. It is also conceivable that the algae themselves containing the fatty acid esters may be used directly as ingredients in food and beverages. Therefore, when producing odd-numbered fatty acids using microalgae such as Labyrinthula, it is important not only to have a high absolute production volume of fatty acid esters, but also a high proportion of odd-numbered fatty acids in the total lipids.

[0010] However, with commonly known production methods, there is room for improvement in the amount of fatty acid esters produced, and the proportion of odd-numbered fatty acids in total lipids remains low. A low proportion of odd-numbered fatty acids increases the cost of concentrating and purifying fatty acid esters, as well as the extra costs of culture media and cultivation required to ensure sufficient production. Therefore, there is a need for efficient production methods that not only increase the absolute production volume but also increase the proportion of odd-numbered fatty acids in total lipids.

[0011] Therefore, the present invention aims to provide a method for producing odd fatty acid esters that can efficiently produce odd fatty acid esters in Labyrinthula species, and a culture composition containing the odd fatty acid esters obtained thereby. [Means for solving the problem]

[0012] To solve the aforementioned problems, the present invention provides a method for producing odd fatty acid esters in which odd fatty acids are esterified, comprising the step of culturing a microorganism belonging to the genus Aurantiochytrium or Schizochytrium in an amino acid-supplemented medium, wherein the amino acid-supplemented medium contains one or more amino acids selected from the group consisting of L-valine, L-isoleucine, L-threonine, L-methionine, D-methionine, and DL-methionine, at a concentration of 10 mM or more per amino acid.

[0013] Furthermore, the culture composition according to the present invention is a culture composition obtained by culturing a microorganism belonging to the genus Aurantiochytrium or Schizochytrium, comprising the algal body of the microorganism and an odd fatty acid ester formed by ester bonding of odd fatty acids produced by the microorganism, wherein the content of odd fatty acids per biomass of the microorganism is 0.04 g / g or more, and the proportion of odd fatty acids in the total lipids is 25% or more. [Effects of the Invention]

[0014] According to the present invention, a method for producing odd fatty acid esters that can efficiently produce odd fatty acid esters in microorganisms belonging to the Labyrinthula class, and a culture composition containing the odd fatty acid esters obtained thereby can be provided. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the relationship between pH and biomass in the culture of Aurantiochytrium. [Figure 2] This is a schematic diagram illustrating the structure of an air-lift type reactor. [Modes for carrying out the invention]

[0016] The following describes in detail a method for producing odd fatty acid esters according to one embodiment of the present invention, and a culture composition containing the odd fatty acid esters obtained thereby.

[0017] The present embodiment relates to a method for producing odd fatty acid esters by culturing microorganisms belonging to the Labyrinthula class to produce odd fatty acid esters in which odd fatty acids are ester-bonded. In this production method, by culturing microorganisms belonging to the Labyrinthula class that have the ability to produce odd fatty acid esters, odd fatty acids useful for various applications are produced as odd fatty acid esters such as fatty acid triglycerides and phospholipids.

[0018] The inventors attempted to develop a novel culture medium for Aurantiochytrium, a member of the Labyrinthula family, with the aim of improving the production of odd-numbered fatty acid esters. Unlike even-numbered fatty acids, which are common in nature, the substrate (starting material) for the synthesis reaction of odd-numbered fatty acids by Aurantiochytrium is propionyl-CoA, which has three carbon atoms. Furthermore, the production of odd-numbered fatty acids has also been confirmed in Schizochytrium, a close relative of Aurantiochytrium. Therefore, the inventors selected propionyl-CoA precursors as candidate culture medium components to be added to the novel culture medium for Labyrinthula species such as Aurantiochytrium and Schizochytrium, and confirmed and evaluated the effectiveness of various precursors through culture experiments, as described below.

[0019] Propionic acid is known as a precursor to propionyl-CoA. In cells, propionic acid is converted to succinyl-CoA via S-methylmalonyl-CoA and R-methylmalonyl-CoA. Succinyl-CoA enters the citric acid cycle and is used in the synthesis of acetyl-CoA, reduced nicotinamide adenine dinucleotide (NADH), adenosine triphosphate (ATP), guanosine triphosphate (GTP), etc., as well as in gluconeogenesis. However, propionic acid is known as a preservative that exhibits antibacterial activity, and when added in large quantities to culture media, it can consume CoA and ATP, cause cell membrane alteration, and inhibit microbial growth, so it may not be an optimal component to add to new culture media.

[0020] Furthermore, precursors of propionyl-CoA include pentanoic acid (valeric acid) with 5 carbon atoms, heptanoic acid (enanthic acid) with 7 carbon atoms, and nonanoic acid (pelargonic acid) with 9 carbon atoms. However, these fatty acids have a strong odor, which can transfer to the culture composition obtained through cultivation, impairing the usability of the culture composition. Additionally, their surfactant properties may inhibit the growth of microorganisms, making them unsuitable as culture medium components to be added to new culture media.

[0021] On the other hand, valine, isoleucine, threonine, and methionine are also known as precursors of propionyl-CoA. In cells, valine and isoleucine are converted into methylmalonyl-CoA, which undergoes a reversible equilibrium reaction with propionyl-CoA. Threonine is converted into propionyl-CoA via α-ketobutyric acid. Methionine is converted into propionyl-CoA via S-adenosylmethionine, homocysteine, etc. These amino acids are considered to be suitable as medium components to be added to a new medium because, unlike propionic acid that exhibits antibacterial action, they are difficult to inhibit the growth of microorganisms.

[0022] Therefore, in the present embodiment, an amino acid-added medium in which a predetermined amino acid corresponding to a precursor of propionyl-CoA is added is used as a medium for culturing microorganisms belonging to Labyrinthulomycetes such as Aurantiochytrium and Schizochytrium.

[0023] As the microorganisms to be cultured, any species having the ability to produce odd-chain fatty acid esters can be used, such as microorganisms belonging to the genus Aurantiochytrium, including Aurantiochytrium mangrovei, Aurantiochytrium limacinum, Aurantiochytrium acetophilum, etc., and microorganisms belonging to the genus Schizochytrium, including Schizochytrium mangrovei, Schizochytrium limacinum, Schizochytrium aggregatum, etc. In the genus Aurantiochytrium and the genus Schizochytrium, species that accumulate a large amount of lipids as oil droplets inside cells have been found. When Aurantiochytrium or Schizochytrium is cultured, not only fatty acid triglycerides and phospholipids composed only of odd-chain fatty acids, but also DHA and EPA can be produced.

[0024] Any of the following microorganisms can be used for cultivation: wild strains collected from nature, mutant strains with introduced gene mutations, or genetically modified strains whose genes have been altered using genetic engineering technology. When culturing microorganisms, it is preferable to first confirm their ability to produce odd fatty acid esters by performing test cultures using a basic culture medium, and then isolate and use strains that have a high ability to produce odd fatty acid esters.

[0025] The amino acid supplemented medium used for cultivation may be a synthetic medium, a semi-synthetic medium, or a natural medium. The amino acid supplemented medium can be prepared by adding a predetermined amino acid, which corresponds to a precursor of propionyl-CoA, to a basic medium containing a carbon source, a nitrogen source, vitamins, minerals, etc. It is preferable to use seawater medium, either natural or artificial seawater, for the amino acid supplemented medium.

[0026] As amino acids corresponding to the precursor of propionyl-CoA, one or more selected from the group consisting of L-valine, L-isoleucine, L-threonine, L-methionine, D-methionine, and DL-methionine can be added to the culture medium. It is preferable to add the amino acids corresponding to the precursor of propionyl-CoA at a higher concentration than the amount generally added as a nitrogen source, and it is preferable to add them at a concentration of 10 mM or more per amino acid.

[0027] The amino acid supplemented medium preferably contains at least one of L-valine and DL-methionine at a concentration of 10 mM or higher. These amino acids are relatively inexpensive, and for example, racemic methionine can be readily obtained as a chemically synthesized product; therefore, using these amino acids can reduce the cost of the medium.

[0028] Furthermore, it is more preferable that the amino acid supplemented medium contains only L-valine at a concentration of 10 mM or higher. As will be described later, L-valine has a high conversion efficiency when metabolized into odd fatty acids, so using only L-valine allows for a higher production of odd fatty acid esters and a higher proportion of odd fatty acids in the total lipids while suppressing the cost of the medium.

[0029] The amino acids corresponding to the precursors of propionyl-CoA are preferably concentrated at a concentration of 10 mM to 50 mM per type, and more preferably at 20 mM to 30 mM. When the concentration is 50 mM or less, even if the generated propionyl-CoA is hydrolyzed, a large amount of propionic acid is not produced, thus avoiding inhibition of growth and synthesis of odd fatty acids by propionic acid. Furthermore, when the concentration is 20 mM to 30 mM, the amount of odd fatty acid esters produced and the proportion of odd fatty acids in the total lipids can be further increased.

[0030] Examples of carbon sources that can be added to amino acid-supplemented culture media include glucose, fructose, mannose, galactose, sucrose, and maltose. Glucose is preferred as the carbon source.

[0031] As nitrogen sources to be added to amino acid-supplemented culture media, for example, amino acids such as glutamine, glutamic acid, and sodium glutamate, as well as peptides, proteins, urea, ammonia, ammonium salts, and nitrates can be used. Glutamine, glutamic acid, or sodium glutamate are preferred as nitrogen sources.

[0032] Examples of vitamins that can be added to the amino acid-supplemented culture medium include thiamine, riboflavin, niacin, pantothenic acid, vitamin B6, biotin, and folic acid. Minerals that can be added include sodium, potassium, calcium, magnesium, phosphorus, sulfur, iron, cobalt, copper, zinc, manganese, and molybdenum.

[0033] Examples of sea salts that can be added to the amino acid-supplemented culture medium include 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, and boric acid.

[0034] Amino acid supplemented culture media can also be prepared by adding biological extracts, protein hydrolysates, etc. For example, extracts such as yeast extract, meat extract, fish extract, and plant extract, or digests obtained by enzymatic treatment of these, as well as protein hydrolysates obtained by decomposing other extracts with enzymes, acids, heat, etc., such as tryptone, peptone, casamino acids, soybean hydrolysates, gelatin hydrolysates, and other extracts, can be fractionated and used as needed.

[0035] Amino acid supplemented media can be prepared, for example, using GTY medium, which is commonly used as a culture medium for Aurantiochytrium, as a base medium. GTY medium is prepared using natural or artificial seawater to contain glucose at concentrations of approximately 20-100 g / L, tryptone at 10-60 g / L, yeast extract at 5-40 g / L, and sea salt at a concentration of 10-40 g / L. Amino acid supplemented media can also be prepared by adding a propionyl-CoA precursor to such a seawater medium at a concentration of 10 mM or higher.

[0036] Furthermore, amino acid-supplemented culture media can also be prepared by adding animal-derived whey obtained during the manufacturing process of cheese, yogurt, and other dairy products, or soy whey obtained during the manufacturing process of tofu, soy milk, and other soy products. Whey and soy whey are rich in proteins, amino acids, other nitrogen compounds, vitamins, and minerals, and are useful as a nutrient source because they can be obtained inexpensively as by-products of product manufacturing. In addition, they do not have a strong odor, making it less likely for odors to transfer to the culture composition obtained by cultivation, and the odor of the culture composition may be masked, thus not impairing the usability of the culture composition.

[0037] Whey of animal origin can be obtained by separating the water-soluble fraction from milk. For example, in cheese production, milk is curdled by lactic acid fermentation as a starter, or by adding rennet enzymes such as chymosin, pepsin, and rennet, or by adding acid. Similarly, in yogurt production, milk is fermented with lactic acid bacteria to obtain curd, which is mainly composed of protein and fat. By pressing or draining such curd, whey can be obtained as a water-soluble fraction.

[0038] The milk used as a raw material for animal-derived whey may or may not have its milk fat removed. Furthermore, the animal-derived whey may be acidic whey obtained by coagulating milk at a pH of approximately 4.6, or sweet whey obtained by coagulating milk with rennet or the like. In addition, the animal-derived whey may or may not have its sodium, potassium, etc. removed.

[0039] Specifically, milks such as cow's milk, buffalo milk, goat's milk, sheep's milk, yak's milk, mare's milk, and camel's milk can be used. Among these, cow's milk, buffalo milk, goat's milk, or sheep's milk are preferred, with cow's milk being particularly preferred, due to their easy availability and low cost.

[0040] Soy milk can be obtained by separating the water-soluble fraction from plant-based protein sources such as beans. For example, by grinding beans, immersing them in water, and treating them under acidic conditions such as pH 4.5 to 5.0 or below, or under heating conditions of 80°C or above, solid components such as fiber can be separated and removed to obtain an extract mainly containing water-soluble components. Also, during the production of soy products such as tofu and fried tofu, soy milk can be obtained by boiling down ground beans and then straining out the solid components. By salting out the insoluble proteins contained in the extract or soy milk obtained in this way with a coagulant such as magnesium chloride, soy milk can be obtained as a water-soluble fraction.

[0041] The plant-based protein source, such as beans, used as the raw material for soy milk may or may not have its oil removed. For example, soy milk may be separated from either soy milk made from defatted processed beans or soy milk made from unprocessed beans that have not been defatted.

[0042] As a plant-based protein source, soybeans, mung beans, black beans, etc., can be used. Among these, soybeans are preferred as a plant-based protein source because they are readily available and inexpensive. Soy milk may also be obtained as soybean whey, mung bean whey, black bean whey, etc., by directly separating it from soy milk, mung bean milk, black bean milk, etc., whose solid content has been adjusted.

[0043] Whey is preferably adjusted to a pH of 4 to 8, and more preferably to a pH of 6 to 8. Labyrinthula species such as Aurantiochytrium and Schizochytrium have an optimal culture pH near neutral. In contrast, whey derived from animals may be acidified to around pH 4.6 due to lactic acid fermentation or the addition of acid. Also, soy whey may be adjusted to a pH of around 4-5 due to processes to coagulate solids or isoelectric point precipitation. Therefore, by adjusting the pH of the whey to near neutral beforehand, the pH adjustment of the culture medium using whey can be simplified.

[0044] In amino acid supplemented culture media, the vitamin B12 concentration is preferably 0.2 μg / L or less. For example, when using biological extracts, protein hydrolysates, etc., as culture medium components, it is preferable to use types or fractions with a low vitamin B12 concentration. Furthermore, compared to animal-derived whey with a high vitamin B12 concentration, it is preferable to use plant-derived soy milk with a vitamin B12 concentration below the detection limit.

[0045] The concentration of vitamin B12 is thought to affect the synthesis of odd fatty acids. Propionyl-CoA is a substrate (starting material) for the synthesis of odd fatty acids, but in cells it is converted to succinyl-CoA via S-methylmalonyl-CoA and R-methylmalonyl-CoA. Succinyl-CoA enters the citric acid cycle and is used for the synthesis of acetyl-CoA, NADH, ATP, GTP, etc., and for gluconeogenesis. Therefore, even if a precursor of propionyl-CoA is added to the culture medium, if the succinyl-CoA replenishment reaction (anaplerosis) is dominant, the amount of odd fatty acid synthesis is thought to decrease.

[0046] In cells, succinyl-CoA is produced by the isomerization of methylmalonyl-CoA, a reaction catalyzed by methylmalonyl-CoA mutase. Methylmalonyl-CoA mutase is an enzyme that uses vitamin B12 as a cofactor. Therefore, if the concentration of vitamin B12 in the amino acid-supplemented medium is high, the supplementation reaction to replenish succinyl-CoA proceeds, and the amount of odd fatty acid synthesis decreases. In contrast, if the amino acid-supplemented medium is prepared without actively adding vitamin B12, and the vitamin B12 concentration is lowered to 0.2 μg / L or less, the supplementation reaction to replenish succinyl-CoA becomes less likely to proceed, and the amount of odd fatty acid synthesis increases, thus improving the production of odd fatty acid esters.

[0047] The amino acid supplemented medium preferably contains, in addition to a precursor of propionyl-CoA, glucose as a carbon source, glutamine, glutamic acid, or sodium glutamate as a nitrogen source, yeast extract, soy milk separated from legumes, and sea salt. The concentration of yeast extract is preferably 0.2% or less from the viewpoint of reducing the importation of vitamin B12, which is present at a maximum of about 0.1 μg / g. The concentration of soy milk is preferably 0.1% or more on a solid basis, and more preferably 0.2% or more. Furthermore, the concentration of soy milk is preferably 20% or less on a solid basis, and more preferably 10% or less.

[0048] Using such a culture medium allows for the growth of Labyrinthullae species such as Aurantiochytrium and Schizochytrium at sufficiently high growth rates while suppressing medium costs, thereby increasing the production of odd fatty acid esters and the proportion of odd fatty acids in total lipids. In particular, using soy milk as the main nutrient source allows for the efficient production of odd fatty acid esters because, unlike when using protein hydrolysates, the synthesis of odd fatty acids is less likely to be inhibited by the culture medium components.

[0049] The amino acid supplemented medium may contain propionic acid or a salt thereof, along with a precursor of propionyl-CoA. When propionic acid or a salt thereof is added to the amino acid supplemented medium, its concentration is preferably 10 mM to 50 mM, more preferably 10 mM to 40 mM, and even more preferably 20 mM to 30 mM. As a salt of propionic acid, for example, sodium propionate can be used. Propionic acid or a salt thereof may be pre-mixed as a component of the medium, or it may be added during cultivation as a pH adjuster.

[0050] In cells, propionic acid, a C3 molecule, inhibits the biosynthesis of C2 molecules such as acetyl-CoA. Therefore, adding propionic acid, or its salt, to an amino acid-supplemented culture medium can suppress the production of even-numbered fatty acids. In other words, it can reduce the proportion of even-numbered fatty acids in total lipids and increase the proportion of odd-numbered fatty acids. Therefore, it is possible to reduce the cost of concentrating and purifying the target odd fatty acid esters, as well as the total cost of the propionyl-CoA precursors required for the production of odd fatty acids.

[0051] The amino acid supplemented medium may be in any form from liquid medium, semi-solid medium, and solid medium. The amino acid supplemented medium may contain various buffering agents such as phosphates, isotonic agents such as sodium chloride, microorganisms such as bacteria, yeasts, and diatoms for two-member culture, and medium components such as agar. However, from the viewpoint of increasing the production of odd fatty acid esters, the amino acid supplemented medium is preferably a liquid medium that allows for large-scale culture.

[0052] In the method for producing odd fatty acid esters according to this embodiment, odd fatty acid esters may be obtained as a culture composition containing microbial algae and odd fatty acid esters by performing only the culture step. Alternatively, odd fatty acid esters may be obtained as a state separated and extracted from microorganisms by performing both the culture step and the separation step.

[0053] The culture process involves culturing microorganisms capable of producing odd fatty acid esters in an amino acid-supplemented medium. Any culture method may be used, such as batch culture, continuous culture, or fed-batch culture. Furthermore, various culture methods such as shaking culture, aeration culture, aeration-stirred culture, air-lift culture, and static culture can be used. Among these culture methods, continuous culture is preferred because it allows for the maintenance of glucose concentration and a neutral pH. Aeration-stirred culture or air-lift culture is preferred because it enables large-scale culture.

[0054] Depending on the culture method, the culture apparatus can be a mechanically agitated reactor, an air-lift reactor, a packed-bed reactor, a fluidized-bed reactor, etc. Depending on the culture volume, various containers such as tanks, jar fermenters, flasks, dishes, culture bags, tubes, and test tubes can be used as culture vessels. The culture vessels may be made of suitable materials, such as inorganic materials like stainless steel and glass, or organic materials like polystyrene, polyethylene terephthalate copolymer, and polypropylene.

[0055] The amino acid-supplemented medium can be sterilized using an appropriate sterilization method before seeding microorganisms capable of producing odd fatty acid esters. Suitable sterilization methods include, for example, heat sterilization, ultraviolet sterilization, gamma ray sterilization, and filtration sterilization. Furthermore, seed cells pre-cultured in a medium of the same composition or a basal medium such as GTY medium can be seeded into the amino acid-supplemented medium.

[0056] Labyrinthurium species such as Aurantiochytrium and Schizochytrium can be cultured under appropriate culture conditions. The culture temperature is preferably 10°C to 35°C, more preferably 10°C to 30°C, and even more preferably 24.5°C to 27.5°C. The pH is preferably 4 to 9, more preferably 6 to 8, and even more preferably 7.4 to 7.7.

[0057] Labyrinthurium species such as Aurantiochytrium and Schizochytrium can be cultured for an appropriate duration while subculturing at appropriate time intervals. However, some Aurantiochytrium species complete their logarithmic growth phase approximately 2 days after the start of culture and enter the death phase approximately 7 days later. The production of odd fatty acid esters increases with growth, reaching a near-maximum from the end of the logarithmic growth phase to the beginning of the quiescent phase, and then gradually decreases. Therefore, when using such species, the time interval for subculturing is preferably between 1 and 10 days, more preferably between 2 and 7 days, and even more preferably between 2 and 5 days.

[0058] Furthermore, the culture time for labyrinthula species such as Aurantiochytrium and Schizochytrium is preferably 10 days or less, more preferably 7 days or less, and even more preferably 5 days or less. It has been confirmed that while the production of lipids increases with longer culture times for Aurantiochytrium and the like, the proportion of carotenoids and other similar substances increases, while the proportion of odd fatty acids decreases. Therefore, with such short culture times, it is possible to increase the proportion of odd fatty acids in the total lipids without strictly controlling glucose concentration and pH.

[0059] During the culture process, cultured Labyrinthula species such as Aurantiochytrium and Schizochytrium produce odd-numbered fatty acid esters in the cells and extracellular matrix. As a result, a culture composition is obtained containing the algal bodies of microorganisms belonging to the Labyrinthula species such as Aurantiochytrium and Schizochytrium, and the odd-numbered fatty acid esters produced by the algal bodies. The culture composition can be recovered by concentrating or drying the culture medium.

[0060] Various methods can be used to concentrate the culture medium, such as centrifugal concentration by centrifugation of the solids, sedimentation concentration by allowing the solids to settle naturally, evaporation concentration by heating and evaporating the medium, vacuum concentration by reducing the pressure and evaporating the medium, pressure concentration by pressurizing and filtering the medium, membrane concentration by filtering the medium through a separation membrane, and freeze concentration by freezing and removing the medium. Various methods can also be used to dry the culture medium, such as hot air drying, cold air drying, vacuum drying, spray drying, freeze drying, infrared drying, natural drying, and drum drying by heating and drying in a rotating drum.

[0061] When the culture composition is concentrated, for example, it can have a water content of 80% or less, preferably 30% to 50%. Concentrating it to such a water content significantly reduces the volume of the culture composition and makes it a paste that is less fluid, thus improving its handling properties. When the culture composition is dried, for example, it can have a water content of 10% or less.

[0062] The culture composition can be used for various purposes, such as food, animal feed, fertilizer, and industrial raw materials. Specific examples of food uses include general foods, health foods, food ingredients, and beverage ingredients. Specific examples of animal feed include livestock feed, poultry feed, aquaculture feed, and pet feed. Specific examples of industrial raw materials include raw materials for biofuels, animal feed, fertilizer, chemicals, and pharmaceuticals.

[0063] The culture composition preferably has a lipid content of 30% by mass or more, more preferably 40% by mass or more, and even more preferably 45% by mass or more, based on the mass of solids. In conventional production methods, the lipid production efficiency is not always sufficiently high, so the lipid content in the culture composition remains below about 30% by mass, and the proportions of protein, ash, etc., become relatively high. In contrast, when cultivation is performed using an amino acid supplement medium to which a propionyl-CoA precursor is added, the production of lipids, including odd fatty acids, increases, so a culture composition with a lipid content of 30% by mass or more to 45% by mass or more can be obtained.

[0064] The culture composition preferably contains 0.04 g / g or more of odd fatty acids per unit of microbial biomass (dry weight), in terms of fatty acid methyl esters. Furthermore, the proportion of odd fatty acids in the total lipid fraction recovered by solvent extraction is preferably 25% by mass or more, more preferably 30% by mass or more, even more preferably 40% by mass or more, and even more preferably 45% by mass or more. Such amounts of odd fatty acids can be achieved by adding propionyl-CoA precursors, propionic acid, or salts thereof.

[0065] Specifically, when culturing using an amino acid-supplemented medium, it is possible to ensure a total lipid production of 0.1 g / g or more, 0.2 g / g or more, and 0.25 g / g or less per biomass. Furthermore, it is possible to ensure that the proportion of odd fatty acids in the total lipids is 20% by mass or more, 40% by mass or more, and 50% by mass or less, while suppressing unsaturated fatty acids to 50% by mass or less. Compared to conventional production methods, it is possible to produce odd fatty acid esters more efficiently, and the production volume of odd fatty acid esters can be increased from 1.0 g / L or more to 1.3 g / L or more per culture medium.

[0066] The separation process involves separating odd fatty acid esters produced by microorganisms from the microbial algal bodies. The odd fatty acid esters can be separated by solvent extraction, centrifugation, filtration, etc., after pre-treating a concentrated suspension of algal bodies or dried algal bodies as necessary. Examples of pre-treatment of algal bodies include flocculation, chemical treatment, steaming, heat treatment, stirring, grinding, ultrasonic treatment, pressure changes, chemicals, enzymes, and crushing treatments using various principles such as freeze-thaw cycles.

[0067] Various solvents can be used for solvent extraction, such as methanol, ethanol, diethyl ether, propylene glycol, propanol, isopropanol, acetone, chloroform, hexane, cyclohexane, benzene, acetic acid, water, mixed solvents of these, and supercritical fluids. Examples of mixed solvents include hexane-ethanol mixed solvents, chloroform-methanol mixed solvents, and ethanol-diethyl ether mixed solvents.

[0068] For pretreatment, a flocculant such as chitosan is preferably used. For pretreatment, a chemical treatment involving the addition of citric acid to adjust the pH is also preferably used. When large amounts of lipids are produced in Aurantiochytrium and other algal bodies, the algae become fragile and easily broken, making the extraction and separation of odd-numbered fatty acid esters difficult. However, by adding a flocculant such as chitosan, the algae can be easily collected without damaging them. Furthermore, pH adjustment hardens the algae, preventing damage.

[0069] Odd-numbered fatty acid esters isolated from microbial algal bodies can be purified as needed to recover target substances such as odd-numbered fatty acids. For example, when recovering odd-numbered fatty acids, degumming, deacidification, decolorization, and deodorization treatments can be performed. Depending on the target substance, various methods such as solvent fractionation, distillation, molecular distillation, chromatography, membrane separation, hydrolysis, transesterification, crystallization, inclusion complex formation, and complex formation can be used in combination.

[0070] Odd fatty acids produced by Labyrinthullae such as Aurantiochytrium and Schizochytrium include, for example, tridecyl acid (C13), pentadecanoic acid (C15), and heptadecanoic acid (C17). Pentadecanoic acid can be used for purposes such as lowering blood pressure, suppressing blood glucose elevation, and promoting cell proliferation. Specific examples of its use in promoting cell proliferation include healing damaged tissue, alleviating pain, autoimmune diseases, neurodegenerative diseases, immune diseases, metabolic syndromes, and cancer-related diseases, reducing skin wrinkles, promoting skin metabolism, promoting hair growth, reducing allergy symptoms, reducing muscle pain, and improving athletic performance. [Examples]

[0071] The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited thereto.

[0072] <Culture Test 1> First, to determine the culture medium composition to be used for the production of odd fatty acid esters, we conducted culture tests of Aurantiochytrium with different types of major nutrients and compared the total lipid production and the production of odd fatty acids.

[0073] For the test culture medium, we prepared a medium supplemented with either cheese whey or soy milk whey as the main nutrient source. Specifically, we prepared a medium containing 3.0% glucose, 0.5% L-sodium glutamate, 0.2% yeast extract, 1.2% sea salt, and 10 mM L-methionine, with cheese whey added to a concentration of 10%, and a medium with soy milk whey added to a concentration of 10%. Each medium was sterilized in an autoclave at 121°C for 20 minutes before being used for cultivation.

[0074] For the soy milk whey, commercially available soy milk was heated to 80°C, magnesium chloride hexahydrate (MgCl2·6H2O) was added to a concentration of 0.7%, and the coagulated protein was removed by centrifugation. The supernatant soy milk whey was sterilized in an autoclave at 115°C for 30 minutes, then stored at a cold temperature of 6°C before being added to the culture medium.

[0075] Typical GTY culture medium contains yeast extract at a concentration of approximately 0.5%. However, yeast extract contains vitamin B12, which promotes the replacement reaction and reduces the synthesis of odd fatty acids. Therefore, in this culture experiment, the concentration of vitamin B12 was changed to 0.2% to limit it to the minimum amount necessary for growth. Note that cheese whey contains a considerable amount of vitamin B12 (approximately 0.3 μg / 100g), but soy milk whey does not contain vitamin B12 at a detectable concentration.

[0076] The culture test was performed by shaking culture of Aurantiochytrium Sp. SA-96 strain. A 500 mL Sakaguchi flask and a reciprocating shaker (manufactured by Thomas Scientific Instruments Co., Ltd.) were used for the shaking culture. The culture conditions for the shaking culture were: medium volume: 200 mL, culture temperature: 25 °C, culture time: 72 hours, and shaking speed: 115 strokes / min. The cultured Aurantiochytrium was collected by centrifugation, freeze-dried, and stored until measurement.

[0077] The total lipid content was determined using the following procedure. First, approximately 0.2 g of lyophilized aurantiochytrium was weighed and placed in a test tube with 10 mL of a chloroform-methanol mixed solvent (volume ratio: chloroform / methanol = 2 / 1). This test tube was placed in an ultrasonic cleaner and the contents were stirred to extract the lipids into the solvent layer. Next, the contents of the test tube were centrifuged at 2800 rpm for 10 minutes, and 5.0 mL of the supernatant was separated into another test tube. 1.0 mL of physiological saline was added to the separated solution, stirred, and then centrifuged at 2800 rpm for 10 minutes to remove the water-soluble components in the upper layer, and the lipid-soluble components in the lower layer were separated into another test tube. The test tube was then placed in a 40°C water bath, and nitrogen gas was blown in to remove the solvent and dry out the lipids extracted from the algae. The weight of the lipids recovered in the test tube was determined as the total lipid production by Aurantiochytrium.

[0078] The quantitative determination of each fatty acid in the lipids was performed using a GC-FID (Gas Chromatography - Flame Ionization Detector) in the following procedure. After the total fatty acid content was determined, 14% BF3-methanol (a methanol solution containing 14% boric acid trifluoride) was added to the lipids extracted from the algae, and the mixture was heated at 70°C for 30 minutes to methylate the acyl components in the lipids. The resulting fatty acid methyl ester (FAME) was then dissolved in n-hexane to prepare a sample with a concentration of 1.0 mg / mL, which was then subjected to GC-FID testing.

[0079] A GC-2015 gas chromatograph (manufactured by Shimadzu Corporation) was used. An Agilent J&W GC column DB-23 (manufactured by Agilent Technologies, length: 30m, inner diameter: 0.25mm, film thickness: 0.25μm) was used as the column. The measurement conditions were as follows: carrier gas: helium (constant pressure), carrier gas pressure: 14psi, injection volume: 1μL, injection conditions: split / splitless, inlet temperature: 250℃, split ratio: 50:1, heating conditions: 50℃ (1 min) → 25℃ / min to 175℃ → 4℃ / min to 230℃ → 230℃ (5 min), FID temperature: 280℃, hydrogen flow rate: 40mL / min, air flow rate: 400mL / min, makeup gas flow rate: 25mL / min.

[0080] For the quantification of each fatty acid in lipids, the integrated area of ​​the total FAME was calculated by subtracting the integrated area of ​​the solvent peak from the integrated area of ​​all peaks in the chromatogram detected by GC-FID. After a total of seven measurements, the average integrated area of ​​the total FAME was 265,000 dots / μg-FAME. The peaks for each type of fatty acid were identified by comparing them with the retention times of fatty acid methyl ester standards (GL Sciences). The proportion of each fatty acid in the total lipid was individually determined as the area ratio to the integrated area of ​​the total FAME. Furthermore, the production amount of each fatty acid was calculated using the results of the quantification of the total lipid and the average value of the integrated area of ​​the total FAME.

[0081] Table 1 shows the average results of culture tests with varying types of main nutrients. In the table below, biomass refers to the biomass (dry weight) of Aurantiochytrium obtained by freeze-drying after culture, and the percentage and production of odd fatty acids are the sum of tridecyl acid (C13), pentadecanoic acid (C15), and heptadecanoic acid (C17).

[0082] [Table 1]

[0083] As shown in Table 1, when comparing culture systems supplemented with cheese whey and those supplemented with soy milk whey, soy milk whey resulted in higher production of odd fatty acids. Propionyl-CoA, produced by amino acid metabolism, is used in the cytoplasm for the production of odd fatty acids, but in the mitochondria, it is used in a replenishment reaction to replenish succinyl-CoA. Which pathway is dominant is thought to depend on the activity of methylmalonyl-CoA mutase and the amount of vitamin B12, a cofactor of methylmalonyl-CoA mutase. Cheese whey contains vitamin B12, while soy milk whey does not; therefore, soy milk, isolated from legumes, is considered appropriate as the main nutrient source to add to the culture medium.

[0084] <Culture Test 2> Next, in order to determine the production strain to be used for odd-numbered fatty acid ester production, culture tests were conducted using different types of Aurantiochytrium, and the total lipid production and odd-numbered fatty acid production were compared.

[0085] For culturing, we used either Aurantiochytrium Sp. SA-89 or Aurantiochytrium Sp. SA-96. The SA-89 strain is an isolated strain characterized by its rapid growth rate and large increase in biomass during cultivation. The SA-96 strain is an isolated strain characterized by its high lipid production.

[0086] As test media, two types of media were prepared: one without the addition of a propionyl-CoA precursor, and another with either D-methionine or L-methionine added as a propionyl-CoA precursor. Specifically, a basic medium containing 3.0% glucose, 0.5% L-sodium glutamate, 0.2% yeast extract, 1.2% sea salt, and 10% soy milk whey was prepared; a medium to which D-methionine was added to this basic medium to a concentration of 10 mM; and a medium to which L-methionine was added to this basic medium to a concentration of 10 mM. Each medium was sterilized in an autoclave at 121°C for 20 minutes before being used for cultivation.

[0087] Culture tests were performed by shaking culture of either Aurantiochytrium Sp.SA-89 or Aurantiochytrium Sp.SA-96 strains. A 500 mL Sakaguchi flask and a reciprocating shaker (manufactured by Thomas Scientific Instruments Co., Ltd.) were used for shaking culture. The culture conditions for shaking culture were: medium volume: 200 mL, culture temperature: 25°C, culture time: 72 hours, and shaking speed: 115 strokes / min. The cultured Aurantiochytrium were collected by centrifugation, freeze-dried, and stored until measurement.

[0088] Table 2 shows the average results of culture tests using different types of Aurantiochytrium. The quantification of total lipids and individual fatty acids within the lipids was performed in the same manner as in the culture tests described above.

[0089] [Table 2]

[0090] As shown in Table 2, in the SA-89 strain, D-methionine was not metabolized, and the production of odd fatty acids was similar to that when no propionyl-CoA precursor was added. On the other hand, L-methionine was metabolized, resulting in higher production of odd fatty acids and a higher proportion of odd fatty acids in total lipids. However, when L-methionine is metabolized, propionyl-CoA is produced, and propionyl-CoA is hydrolyzed to produce propionic acid, causing the pH of the culture medium to drop to around 4.2. As a result, the biomass after culturing decreased, becoming less than when no propionyl-CoA precursor was added.

[0091] In contrast, in strain SA-96, both D-methionine and L-methionine were metabolized, resulting in higher production of odd fatty acids and a higher proportion of odd fatty acids in total lipids. When a propionyl-CoA precursor was added, the pH of the culture medium decreased to around 4.1. As a result, the biomass after culturing decreased, but the production of odd fatty acids was higher than that of strain SA-89, and the proportion of pentadecanoic acid (C15) was also sufficiently high.

[0092] <Culture Test 3> Next, in order to determine the culture medium composition to be used for the production of odd fatty acid esters, we conducted culture tests of Aurantiochytrium using different types of propionyl-CoA precursors and compared the total lipid production and the production of odd fatty acids.

[0093] As test media, culture media were prepared by adding one of the following as a precursor of propionyl-CoA: DL-methionine, L-valine, L-isoleucine, and L-threonine. In addition, a culture medium with L-leucine added was prepared as a control (negative control). Specifically, a basic medium containing 3.0% glucose, 0.5% L-sodium glutamate, 0.2% yeast extract, 1.2% sea salt, and 10% soy milk whey was prepared. This basic medium was then modified by adding DL-methionine, L-valine, L-isoleucine, and L-threonine to a concentration of 10 mM each, and by adding leucine to a concentration of 10 mM each. Each medium was sterilized in an autoclave at 121°C for 20 minutes before being used for cultivation.

[0094] The culture test was performed by shaking culture of Aurantiochytrium Sp. SA-96 strain. A 500 mL Sakaguchi flask and a reciprocating shaker (manufactured by Thomas Scientific Instruments Co., Ltd.) were used for the shaking culture. The culture conditions for the shaking culture were: culture medium volume: 200 mL, culture temperature: 25 °C, culture time: 72 hours, and shaking speed: 115 strokes / min. The pH of the culture medium was not controlled during the culture. The cultured Aurantiochytrium were collected by centrifugation, freeze-dried, and stored until measurement.

[0095] Table 3 shows the average results of culture tests using different types of propionyl-CoA precursors. The quantification of total lipids and individual fatty acids within the lipids was performed in the same manner as in the culture tests described above.

[0096] [Table 3]

[0097] As shown in Table 3, DL-methionine, L-valine, L-isoleucine, and L-threonine were metabolized, causing the pH of the culture medium to decrease to around 4-6. As a result, the biomass after culturing decreased. On the other hand, L-leucine was not metabolized, and the metabolism of L-sodium glutamate added to the culture medium resulted in only a small decrease in pH to around 7.1-7.4. When DL-methionine, L-valine, L-isoleucine, and L-threonine, which are precursors of propionyl-CoA, were added, the production of odd fatty acids and the proportion of odd fatty acids in total lipids were higher than in the control group. In particular, the production of odd fatty acids increased significantly when L-valine was added.

[0098] <Culture Test 4> Next, in order to determine the culture conditions to be used for the production of odd fatty acid esters, we conducted culture tests of Aurantiochytrium and compared the changes in biomass (biomass) when the pH of the culture medium was changed.

[0099] A culture medium was prepared containing 3.0% glucose, 0.5% L-sodium glutamate, 0.2% yeast extract, 1.2% sea salt, and 10% soy milk whey. The medium was sterilized in an autoclave at 121°C for 20 minutes before use.

[0100] The culture test was performed by shaking culture of Aurantiochytrium Sp.SA-96 strain. A 500 mL Sakaguchi flask and a reciprocating shaker (manufactured by Thomas Scientific Instruments Co., Ltd.) were used for shaking culture. The culture conditions for shaking culture were: culture temperature: 25°C, culture time: 72 hours, and shaking speed: 115 strokes / min. The pH of the culture medium was varied in the range of pH 4 to 8 using 1.0 M hydrochloric acid and 1.0 M sodium hydroxide from the end of the logarithmic growth phase to the beginning of the quiescent phase.

[0101] Figure 1 shows the relationship between pH and biomass in the culture of Aurantiochytrium. As shown in Figure 1, when the pH of the culture medium was changed during the cultivation of Aurantiochytrium, the biomass increased as the pH rose. The biomass increased rapidly between pH 6.0 and 7.0, and the increase became significantly smaller above pH 7.4. Nearly the maximum amount of biomass was obtained in the pH range of 7.4 to 7.7, confirming that a culture medium pH between 7.4 and 7.7 is preferable.

[0102] <Culture Test 5> Next, to confirm the production of odd fatty acid esters, we conducted culture tests of Aurantiochytrium with varying types and concentrations of propionyl-CoA precursors, and compared the production of total lipids and odd fatty acids.

[0103] As test media, culture media were prepared by adding one of the following as a precursor of propionyl-CoA: L-valine, DL-methionine, or sodium propionate. The concentration of L-valine was varied to 10 mM, 20 mM, 50 mM, and 100 mM. Similarly, the concentration of DL-methionine was varied to 20 mM, 50 mM, and 100 mM. Specifically, a basic medium containing 3.6% glucose, 0.5% L-sodium glutamate, 0.2% yeast extract, 1.0% sea salt, and 10% tofu whey was prepared, as well as media to which L-valine, DL-methionine, and sodium propionate were added, respectively. Each medium was sterilized using an autoclave at 121°C for 20 minutes, and then mixed with separately sterilized glucose and tofu whey before being used for cultivation.

[0104] For the tofu whey, we used the supernatant obtained when soy milk was salted out with a coagulant during the tofu manufacturing process. The tofu whey supernatant was heat-sterilized in an autoclave at 115°C for 30 minutes, and the precipitate formed by heating was aseptically filtered and added to the culture medium.

[0105] The culture test was performed by airlift culture of Aurantiochytrium Sp.SA-96 strain. Aurantiochytrium was pre-cultured before airlift culture. Pre-culture was started 24 hours before airlift culture, and the pre-culture solution was added to the airlift culture vessel to a concentration of 0.5%. Airlift culture was performed using the airlift reactor shown in Figure 2.

[0106] Figure 2 is a schematic diagram showing the structure of an air-lift type reactor. As shown in Figure 2, the air-lift type reactor 100 has a cylindrical culture tank 1. A gas supply pipe 2 and a gas exhaust pipe 3 are connected to the culture tank 1. A cylindrical inner cylinder 4 is supported inside the culture tank 1. A sparger 5 connected to the gas supply pipe 2 is provided at the bottom of the culture tank 1.

[0107] In the airlift reactor 100, when air is supplied through the gas supply pipe 2, it is diffused into the culture medium 6 in the culture tank 1 by the sparger 5. The diffused air bubbles rise inside the inner cylinder 4, forming an upward flow, and when they exit to the upper side of the inner cylinder 4, they descend outside the inner cylinder 4, forming a downward flow (see arrows in the figure). With airlift culture, the culture medium 6 is aerated and stirred in this manner, so uniformity of dissolved oxygen concentration and culture medium component concentration in the tank can be maintained without applying a large shear force to the microorganisms.

[0108] In the culture test using air-lift culture, a culture tank 1 with a capacity of 5L was used, as shown in Figure 2. The inner cylinder 4 was made to a size such that its internal area in a cross-sectional view was half the internal area of ​​the culture tank 1 in a cross-sectional view.

[0109] The culture conditions for airlift culture were as follows: medium volume: 3.0 L, culture temperature: 24.5~27.5°C, culture time: 72 hours, aeration rate (per culture tank): 1.2~1.3 v / v / min. The pH of the culture medium was continuously controlled to 7.4-7.7 using a pH control device "mk-750pH" (manufactured by Automatic System Research Co., Ltd.) with a 1.0 M sodium hydroxide solution. The cultured Aurantiochytrium were collected by centrifugation at 3400 rpm for 60 minutes, freeze-dried, and stored until measurement.

[0110] The average results of culture tests with varying L-valine concentrations are shown in Tables 4 and 5. The average results of culture tests with varying DL-methionine concentrations are shown in Table 6. Furthermore, Table 7 shows the results of comparisons with culture tests with added propionic acid. The quantification of total lipids and individual fatty acids within the lipids was performed in the same manner as in the aforementioned culture tests.

[0111] In the table, the production amount of odd fatty acids [mM] is calculated using only the molecular weight of pentadecanoic acid (M=242). Furthermore, the conversion efficiency of odd fatty acids [%] is expressed by the following formula. Conversion efficiency [%] = (Production amount of odd fatty acids [mM] - 0.66 [mM]) Concentration [mM] of the amino acid equivalent to the precursor of propionyl-CoA

[0112] [Table 4]

[0113] [Table 5]

[0114] [Table 6]

[0115] [Table 7]

[0116] As shown in Tables 4 and 5, when L-valine was added as a precursor to propionyl-CoA, the proportion of odd fatty acids in the total lipids increased as the concentration of L-valine increased, and in particular, the proportion of pentadecanoic acid (C15) increased. When no precursor to propionyl-CoA was added, the proportion of pentadecanoic acid (C15) was 3.4%, but when 10 mM L-valine was added, the proportion of C15 increased sharply to 11.8-13.4% (average 12.6%), and the total proportion of odd fatty acids became 16.2-16.4% (average 16.3%).

[0117] Furthermore, when 20 mM L-valine was added, the proportion of C15 increased to 15.6-16.2% (average 15.9%), and the total proportion of odd fatty acids became 18.6-19.3% (average 19.0%). Additionally, when 50 mM L-valine was added, the proportion of C15 increased to 16.7-18.4% (average 17.7%), and the total proportion of odd fatty acids became 20.3-20.8% (average 20.6%).

[0118] On the other hand, the production of odd fatty acids was maximized at a concentration of 50 mM. Biomass remained approximately the same regardless of the L-valine concentration, and when 100 mM L-valine was added, the total proportion of odd fatty acids increased to 24.3%, higher than at 50 mM. However, the production of total lipids decreased to approximately 64% compared to the 50 mM case. It is thought that when the L-valine concentration is too high, propionyl-CoA, a reaction substrate for odd fatty acids, is generated, and that propionyl-CoA hydrolyzes to produce propionic acid, inhibiting the synthesis of fatty acids and other substances.

[0119] The conversion efficiency of odd-numbered fatty acids decreased as the L-valine concentration increased. However, the production volume of odd-numbered fatty acids reached its maximum value of approximately 1.040 g / L when 50 mM L-valine was added. Therefore, considering both conversion efficiency and production volume, it is thought that the optimal conditions for balancing cost and productivity exist at high concentrations of approximately 50 mM or less.

[0120] As shown in Table 6, when DL-methionine was added as a precursor to propionyl-CoA, the production of odd fatty acids reached its maximum at a concentration of 50 mM. However, the production of odd fatty acids was lower compared to when L-valine was added. In cells, methionine is converted to homocysteine, but from homocysteine, there is a branching pathway: one that converts to propionyl-CoA via α-ketobutyrate, and another that converts to α-ketobutyrate via cystathionine. Therefore, it is thought that when DL-methionine was used as a precursor, the rate of propionyl-CoA production was slower than when L-valine was used, resulting in a decrease in the production of odd fatty acids.

[0121] In cells, the synthesis of odd-numbered fatty acids primarily involves the condensation reaction between propionyl-CoA and malonyl-CoA, with fatty acid synthases playing a crucial role in the production of odd-numbered fatty acid esters. It is possible that L-valine was favored due to the complex interplay of factors in the synthesis of odd-numbered fatty acids, including the concentration of propionyl-CoA, the concentration of propionic acid produced by the hydrolysis of propionyl-CoA, the consumption of acetyl groups by propionic acid, and the inhibition of amino acid metabolism.

[0122] As shown in Table 7, adding L-valine, DL-methionine, or propionic acid as a precursor to propionyl-CoA ensured a certain level of odd-numbered fatty acid production. The conversion efficiency of odd-numbered fatty acids using propionic acid as a precursor was intermediate compared to L-valine and DL-methionine, suggesting that propionic acid's advantage lay in its ability to produce propionyl-CoA in a single reaction.

[0123] In general, potassium propionate has been shown to have acute toxicity in rats, with an LD50 of 4-5 g / kg. This acute toxicity is thought to be caused by the inhibition of acetyl-CoA metabolism. Furthermore, propionic acid is used as a preservative in foods and is known to inhibit fatty acid synthesis and amino acid metabolism. Therefore, L-valine and DL-methionine are suitable precursors for propionyl-CoA, with L-valine being particularly superior.

[0124] <Culture Test 6> Next, to investigate the distribution of odd-numbered fatty acid esters, a culture test of Aurantiochytrium was performed, and the content of odd-numbered fatty acids in solid lipids, liquid lipids, and phospholipids was quantified.

[0125] As the test medium, a culture medium was prepared by adding L-valine as a precursor of propionyl-CoA. Specifically, a medium was prepared by adding L-valine to a GTY medium containing 2% glucose, 1% tryptone, 0.5% yeast extract, and 1.0% sea salt (Red Sea Coral salt, manufactured by Red Sea Salt Co., Ltd.) to a concentration of 50 mM. The medium was sterilized in an autoclave at 121°C for 20 minutes before being used for cultivation.

[0126] The culture test was performed by shaking culture of Aurantiochytrium Sp. SA-96 strain. A 500 mL Sakaguchi flask and a reciprocating shaker (manufactured by Thomas Scientific Instruments Co., Ltd.) were used for shaking culture. The culture conditions for shaking culture were: medium volume: 250 mL, culture temperature: 25 °C, culture time: 12 hours, and shaking speed: 100 strokes / min. Every 12 hours, the cultured Aurantiochytrium was collected by centrifugation at 2500 g for 15 minutes, washed twice with a 1.5% seawater salt solution, freeze-dried, and stored until measurement.

[0127] First, lipids were extracted from the freeze-dried aurantiochytrium using a chloroform-methanol mixed solvent (volume ratio: chloroform / methanol = 2 / 1). Then, the extracted lipids were fractionated using chloroform by column chromatography with a silica gel 60 column.

[0128] Neutral lipids were eluted with chloroform at four times the bed volume. Polar lipids were then eluted with a chloroform-methanol mixed solvent at four times the bed volume (volume ratio: chloroform / methanol = 1 / 4). Subsequently, fatty acid triglycerides were fractionated by column chromatography using silica gel 60. The silica gel 60 column was prepared using n-hexane. The neutral lipid fraction was dissolved in n-hexane and then subjected to column chromatography.

[0129] Hydrocarbons such as squalene, nonpolar carotenoids, and sterol esters were eluted with a mixed solvent of n-hexane and chloroform (volume ratio: n-hexane / chloroform = 1 / 1) at twice the bed volume. Then, fatty acid triglycerides, polar carotenoids, and free fatty acids were eluted with chloroform at three times the bed volume.

[0130] To purify fatty acid triglycerides, fractions of fatty acid triglycerides were subjected to preparative thin-layer chromatography using silica gel 60 plates. A mixed solvent of n-hexane, diethyl ether, and acetic acid (volume ratio: n-hexane / diethyl ether / acetic acid = 82 / 18 / 1) was used as the developing solvent. The fatty acid triglycerides moved to a range of Rf values ​​from 0.6 to 0.8 depending on the developing solvent. The fatty acid triglyceride spots were visualized by spraying water, and then recovered by elution from the plate with a mixed solvent of chloroform and methanol (volume ratio: chloroform / methanol = 1:1).

[0131] Furthermore, to purify the phospholipids, the polar lipid fraction was subjected to preparative thin-layer chromatography using a silica gel 60 plate. A chloroform-methanol-acetic acid-water mixed solvent (volume ratio: chloroform / methanol / acetic acid / water = 25 / 15 / 4 / 2) was used as the developing solvent. The phospholipids moved within a range of Rf values ​​from 0.2 to 0.8 depending on the developing solvent. The phospholipid spots were visualized by spraying Zinzadze reagent and water, and then eluted from the plate with a chloroform-methanol mixed solvent (volume ratio: chloroform / methanol = 1 / 5) and recovered.

[0132] The recovered fatty acid triglycerides were dissolved in five times the volume of n-hexane after removing the extraction solvent, and the solution was stored overnight at 0-4°C. After storage, the white precipitate that had settled in the n-hexane solution was washed with a small amount of cold n-hexane solution and recovered as a solid lipid fraction. The removed extraction solvent was concentrated at 35°C by blowing nitrogen gas, and the same precipitation treatment was repeated twice. From the removed extraction solvent, the fatty acid triglycerides were recovered as a liquid lipid fraction by thin-layer chromatography.

[0133] The fatty acid triglycerides contained in the solid lipid fraction, the fatty acid triglycerides contained in the liquid lipid fraction, and the recovered phospholipids were reacted with 14% BF3-methanol (a methanol solution containing 14% boric acid trifluoride) at 90°C for 15 minutes to convert them to fatty acid methyl esters. The fatty acid methyl esters were extracted with n-hexane and quantified by GC-FID.

[0134] Table 8 shows the results of the analysis of the distribution of odd-numbered fatty acid esters. The quantification of total lipids and each fatty acid within the lipids was performed in the same manner as in the culture test described above.

[0135] [Table 8]

[0136] As shown in Table 8, saturated odd-numbered fatty acids, including pentadecanoic acid (C15), were highly concentrated in the solid lipid fraction. However, unlike when using standard media, odd-numbered fatty acids were also found in the liquid lipid fraction and the phospholipid fraction when produced in amino acid-supplemented media. Since the proportion of odd-numbered fatty acids in the solid lipid is high at 31.5%, it is considered that precipitation treatment or other methods can be used to concentrate odd-numbered fatty acids.

[0137] <Culture Test 7> Next, in order to increase the proportion of odd fatty acid esters in the total lipid production, L-valine was used as a precursor of propionyl-CoA, and the concentration of propionic acid, a metabolic inhibitor of acetyl-CoA, was varied in Aurantiochytrium culture tests. The changes in total lipid production, odd fatty acid production, and biomass were then compared.

[0138] Two test media were prepared: one containing 50 mM L-valine as a precursor of propionyl-CoA, and another containing 50 mM L-valine and sodium propionate. The concentration of sodium propionate was varied to 10 mM, 25 mM, and 50 mM. Specifically, a culture medium containing 4.0% glucose, 0.5% L-sodium glutamate, 0.2% yeast extract, 1.2% sea salt, 10% tofu whey, and 50 mM L-valine was prepared, as well as a culture medium to which 10 mM, 25 mM, or 50 mM sodium propionate was added. Each medium was sterilized in an autoclave at 121°C for 20 minutes before being used for cultivation.

[0139] The culture test was performed by airlift culture of Aurantiochytrium Sp.SA-96 strain. The culture conditions for airlift culture were: medium volume: 3.0 L, culture temperature: 24.3~26.8°C, culture time: 72 hours, aeration rate (per culture tank): 1.0~1.2 v / v / min. The pH of the culture medium was continuously controlled to pH: 7.40~7.46. Other conditions were the same as in <Culture Test 5>.

[0140] Table 9 shows the average results of culture tests using L-valine as a precursor to propionyl-CoA, and varying the concentration of propionic acid, an acetyl-CoA metabolic inhibitor. The quantification of total lipids and individual fatty acids within the lipids was performed in the same manner as in the culture tests described above.

[0141] [Table 9]

[0142] As shown in Table 9, as the concentration of propionic acid increased, biomass decreased, but the proportion of odd-numbered fatty acids in total lipids increased. In particular, as the concentration of propionic acid increased, the proportion of pentadecanoic acid (C15) increased, while the proportion of palmitic acid (C16) decreased. On the other hand, the proportion of unsaturated fatty acids (C20, C22, etc.) synthesized via the polyketide synthesis system did not change significantly. It is thought that propionic acid inhibited the synthesis of acetyl-CoA, resulting in a decrease in the proportion of even-numbered fatty acids and a decrease in biomass, while conversely, the proportion of odd-numbered fatty acids increased.

[0143] Total lipid production reached a maximum at a propionic acid concentration of 25 mM. Similarly, odd fatty acid production reached a maximum of 1.325 g / L at a propionic acid concentration of 25 mM. While the reason for the maximum production at propionic acid is unclear, the high production of odd fatty acids, despite a low proportion of lipid fractions containing carotenoids, sterols, and other components in addition to triglycerides and phospholipids, suggests that the ratio of C2 molecules such as acetyl-CoA to C3 molecules such as propionyl-CoA may have been optimal for fatty acid synthesis.

[0144] The above culture tests demonstrate the ability of Aurantiochytrium to produce odd fatty acid esters. Aurantiochytrium and Schizochytrium are genera with similar lipid-producing capabilities and were separated in 2007 based on differences in cell morphology and proliferation patterns. In particular, they exhibit extremely similar properties regarding the production of odd fatty acid esters, and it is considered appropriate to treat them similarly. Similar species that produce odd fatty acid triglycerides, etc., can be used in the production of odd fatty acid esters using this amino acid-supplemented medium. [Explanation of Symbols]

[0145] 1 Culture tank 2 Gas supply pipe 3. Gas exhaust pipe 4 Inner cylinder 5 Super 6 Culture solution 100 Air-lift type reactor

Claims

[Claim 1] Microorganisms belonging to the genera Aurantiochytrium or Schizochytrium, A culture composition obtained by culturing in an amino acid supplement medium containing L-valine at a concentration of 20 mM to 50 mM, The material comprises the algal body of the aforementioned microorganism and an odd fatty acid ester formed by esterification of odd fatty acids produced by the aforementioned microorganism. A culture composition wherein the content of odd fatty acids per unit of biomass of the microorganism is 0.04 g / g or more.

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