JP7836849B2 - Culture composition containing odd fatty acid esters - Google Patents

Description

This invention relates to a method for producing odd-numbered fatty acid esters using microorganisms belonging to the Labyrinthula genus, such as Aurantiochytrium and Schizochytrium, and to a culture composition containing odd-numbered fatty acid esters obtained by culturing these microorganisms.

In recent years, technologies for producing useful substances using microorganisms have been actively developed. Certain microalgae accumulate large amounts of lipids within their cells, leading to the development of technologies for producing functional components, physiologically active substances, and biofuels using these microalgae. Photosynthetic microalgae such as Chlorella, Spirulina, and Euglena have been utilized not only for substance production but also for their algal bodies themselves as food and animal feed. However, in recent years, the practical application of heterotrophic microorganisms, which offer superior cultivation efficiency and are more advantageous for substance production, is also progressing.

Labyrinthulidae are chemosynthetic heterotrophic marine eukaryotic microorganisms belonging to the stramenopile family. Due to their high growth and lipid production capabilities, their applications in substance production are being widely explored. Labyrinthulidae are broadly classified into the Labyrinthulidae family and the Thraustochytriidae family. The Thraustochytriidae family includes genera such as Aurantiochytrium, Schizochytrium, and Thraustochytrium.

Aurantiochytrium has been reported to have a high capacity to produce omega-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), as well as hydrocarbons such as squalene and odd-numbered fatty acids. Aurantiochytrium is known to produce large quantities of these lipids during the logarithmic growth phase, accumulating them as oil droplets within the cell.

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 present in small amounts in the bodies of mammals and other organisms, and has been reported to exhibit various physiological activities, including hair growth promotion, blood pressure lowering, blood glucose level suppression, cell proliferation promotion, and Alzheimer's disease alleviation.

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

Patent Document 1 describes a method for producing a culture medium for culturing Aurantiochytrium, comprising: (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 culture medium prepared by this method is said to increase the content of odd fatty acids in the total fatty acids produced by Aurantiochytrium.

Japanese Patent Publication No. 2017-063633

Aurantiochytrium and other microalgae belonging to the Labyrinthula class synthesize odd-numbered fatty acids useful for various applications as fatty acid esters such as fatty acid triglycerides and phospholipids. The produced fatty acid esters need to be concentrated and purified, or hydrolyzed back into fatty acids, after being isolated from the cells. Furthermore, the algal bodies 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.

However, generally known production methods have 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, and also incurs extra costs for culture media and cultivation to ensure sufficient production. Therefore, there is a need for efficient production methods that not only increase absolute production volume but also increase the proportion of odd-numbered fatty acids in total lipids.

Therefore, the present invention aims to provide a method for efficiently producing odd fatty acid esters in Labyrinthula species, and a culture composition containing the odd fatty acid esters obtained thereby.

To solve the aforementioned problems, the present invention provides a method for producing odd-numbered fatty acid esters, comprising the step of culturing microorganisms belonging to the genera 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.

Furthermore, the culture composition according to the present invention is a culture composition obtained by culturing microorganisms belonging to the genus Aurantiochytrium or Schizochytrium, comprising the algal bodies of the microorganisms and odd fatty acid esters formed by esterification of odd fatty acids produced by the microorganisms, wherein the content of odd fatty acids per biomass of the microorganisms is 0.04 g/g or more, and the proportion of odd fatty acids in the total lipids is 25% or more.

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 genus, and a culture composition containing the odd fatty acid esters obtained thereby can be provided.

This figure shows the relationship between pH and biomass in the culture of Aurantiochytrium. This is a schematic diagram illustrating the structure of an air-lift type reactor.

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

This embodiment relates to a method for producing odd-numbered fatty acid esters by culturing microorganisms belonging to the Labyrinthula genus to produce odd-numbered fatty acid esters in which odd-numbered fatty acids are ester-bonded. This method involves culturing microorganisms belonging to the Labyrinthula genus that have the ability to produce odd-numbered fatty acid esters, thereby producing odd-numbered fatty acids useful for various applications as odd-numbered fatty acid esters such as fatty acid triglycerides and phospholipids.

The inventors attempted to develop a novel culture medium for Aurantiochytrium, a member of the Labyrinthula genus, with the aim of improving the production of odd-numbered fatty acid esters. The substrate (starting material) for the synthesis reaction of odd-numbered fatty acids by Aurantiochytrium is propionyl-CoA, which has three carbon atoms, unlike the even-numbered fatty acids commonly found in nature. 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.

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), and gluconeogenesis. However, propionic acid is known as a preservative exhibiting antibacterial properties, and when added in large quantities to culture media, it can consume CoA and ATP, alter cell membranes, and inhibit microbial growth. Therefore, it may not be an optimal component to add to new culture media.

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 its usability. Additionally, their surfactant properties may inhibit microbial growth. Therefore, they are considered unsuitable as culture medium components to be added to new culture media.

On the other hand, valine, isoleucine, threonine, and methionine are also known precursors of propionyl-CoA. In cells, valine and isoleucine are converted to methylmalonyl-CoA through a reversible equilibrium reaction with propionyl-CoA. Threonine is converted to propionyl-CoA via α-ketobutyrate. Methionine is converted to propionyl-CoA via S-adenosylmethionine, homocysteine, etc. Unlike propionic acid, which exhibits antibacterial activity, these amino acids do not easily inhibit microbial growth, and are therefore considered suitable as culture medium components to be added to new culture media.

Therefore, in this embodiment, an amino acid supplement medium containing a predetermined amino acid equivalent to a precursor of propionyl-CoA is used as the culture medium for culturing microorganisms belonging to the Labyrinthula group, such as Aurantiochytrium and Schizochytrium.

The microorganisms used for culture include any species belonging to the genus Aurantiochytrium, such as Aurantiochytrium mangrovei, Aurantiochytrium limacinum, and Aurantiochytrium acetophilum, or to the genus Schizochytrium, such as Schizochytrium mangrovei, Schizochytrium limacinum, and Schizochytrium aggregatum, that have the ability to produce odd fatty acid esters. Species of the genera Aurantiochytrium and Schizochytrium have been found that accumulate large amounts of lipids as oil droplets within their cells. When Aurantiochytrium and Schizochytrium are cultured, they can produce not only fatty acid triglycerides and phospholipids composed solely of odd-numbered fatty acids, but also DHA and EPA.

The microorganisms used for culture may include wild-type strains collected from nature, mutant strains with introduced gene mutations, or genetically modified strains whose genes have been altered using genetic engineering technology. It is preferable to pre-confirm the ability of the microorganisms to produce odd fatty acid esters by performing test cultures using a basic culture medium, and then isolate and use strains with high odd fatty acid ester production ability.

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, equivalent to a precursor of propionyl-CoA, to a basic medium containing a carbon source, nitrogen source, vitamins, minerals, etc. It is preferable to use seawater medium, either natural or artificial seawater, for the amino acid supplemented medium.

As amino acids equivalent 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 equivalent to the propionyl-CoA precursor at a higher concentration than that generally added as a nitrogen source, and preferably at a concentration of 10 mM or more per amino acid.

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; for example, racemic methionine is readily available as a chemically synthesized product. Therefore, using these amino acids can reduce the cost of the medium.

Furthermore, it is more preferable that the amino acid supplemented medium contains only L-valine at a concentration of 10 mM or higher. As described later, L-valine has a high conversion efficiency to odd-numbered fatty acids; therefore, using only L-valine allows for increased production of odd-numbered fatty acid esters and a higher proportion of odd-numbered fatty acids in the total lipids, while suppressing medium costs.

The amino acids corresponding to the precursors of propionyl-CoA are preferably concentrated at a concentration of 10 mM to 50 mM, 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 production of odd fatty acid esters and the proportion of odd fatty acids in the total lipids can be further increased.

As carbon sources to be added to amino acid-supplemented culture media, for example, glucose, fructose, mannose, galactose, sucrose, maltose, etc., can be used. Glucose is preferred as the carbon source.

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.

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 such as sodium, potassium, calcium, magnesium, phosphorus, sulfur, iron, cobalt, copper, zinc, manganese, and molybdenum can also be used.

Examples of sea salts that can be added to amino acid-supplemented culture media 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.

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 their enzymatic digests, as well as protein hydrolysates obtained by decomposing casein hydrolysates such as tryptone, peptone, and casamino acids, soy hydrolysates, gelatin hydrolysates, and other extracts using enzymes, acids, heat, etc., can be fractionated and used as needed.

Amino acid supplemented media can be prepared, for example, using GTY medium, a common culture medium for Aurantiochytrium, as a base medium. GTY medium is prepared using natural or artificial seawater, containing approximately 20-100 g/L of glucose, 10-60 g/L of tryptone, and 5-40 g/L of yeast extract, with a sea salt 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.

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 nutritional sources because they can be obtained inexpensively as by-products of product manufacturing. In addition, they do not have a strong odor, making odor transfer to the culture composition obtained through cultivation unlikely, and the odor of the culture composition may be masked, thus minimizing the usability of the culture composition.

Whey derived from animals can be obtained by separating the water-soluble fraction from milk. For example, during 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, during yogurt production, lactic acid fermentation of milk yields curd, which is mainly composed of protein and fat. By pressing or draining this curd, whey can be obtained as the water-soluble fraction.

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, etc. Additionally, the animal-derived whey may or may not have its sodium, potassium, etc., removed.

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 due to their easy availability and low cost, with cow's milk being particularly preferred.

Soy milk can be obtained by separating the water-soluble fraction from plant-based protein sources such as beans. For example, grinding beans, immersing them in water, and treating them under acidic conditions (pH 4.5-5.0 or lower) or heating conditions (80°C or higher) to separate and remove solid components such as fiber yields an extract primarily containing water-soluble components. Alternatively, during the production of soy products such as tofu and fried tofu, grinding the beans and then straining out the solids yields soy milk. 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.

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, undefatted beans.

As a plant-based protein source, soybeans, mung beans, black beans, etc., can be used. Among these, soybeans are preferred due to their easy availability and low cost. Soy milk may also be obtained by directly separating it from soy milk, mung bean milk, black bean milk, etc., with adjusted solid content, as soybean whey, mung bean whey, black bean whey, etc.

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, animal-derived whey may be acidified to around pH 4.6 due to lactic acid fermentation or acid addition. Furthermore, soy whey may be adjusted to a pH of around 4-5 due to solid coagulation or isoelectric point precipitation treatments. Therefore, pre-adjusting the pH of the whey to near neutral simplifies the pH adjustment of the culture medium using whey.

The amino acid supplemented culture medium preferably has a vitamin B12 concentration of 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 low vitamin B12 concentrations. Furthermore, compared to animal-derived whey with high vitamin B12 concentrations, it is preferable to use plant-derived soy milk with a vitamin B12 concentration below the detection limit.

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 in the synthesis of acetyl CoA, NADH, ATP, GTP, etc., and in gluconeogenesis. Therefore, even when 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.

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, reducing the synthesis of odd fatty acids. Conversely, by preparing the amino acid-supplemented medium without actively adding vitamin B12, and lowering the vitamin B12 concentration to 0.2 μg/L or less, the supplementation reaction to replenish succinyl CoA becomes less likely to proceed, increasing the synthesis of odd fatty acids and thus improving the production of odd fatty acid esters.

The amino acid supplemented medium preferably contains, in addition to a propionyl-CoA precursor, 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 approximately 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.

Using such a culture medium allows for the growth of Labyrinthula 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, unlike when using protein hydrolysates, inhibits the synthesis of odd fatty acids less by the medium components, thus enabling efficient production of odd fatty acid esters.

The amino acid supplement 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 supplement 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 medium component, or it may be added during cultivation as a pH adjuster.

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.

The amino acid supplemented medium may be in any form: liquid medium, semi-solid medium, or solid medium. The amino acid supplemented medium may also 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.

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

The culture process involves culturing microorganisms capable of producing odd-numbered fatty acid esters in an amino acid-supplemented medium. Any culture method may be used, including batch culture, continuous culture, and 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.

Depending on the culture method, various types of culture apparatus can be used, such as mechanically agitated reactors, air-lift reactors, packed-bed reactors, and fluidized-bed reactors. Furthermore, 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. Culture vessels may be made of suitable materials, including inorganic materials such as stainless steel and glass, or organic materials such as polystyrene, polyethylene terephthalate copolymer, and polypropylene.

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

Labyrinthula 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.

The cultivation of Labyrinthullae species such as Aurantiochytrium and Schizochytrium can be carried out with appropriate subculturing intervals and culture durations. However, some Aurantiochytrium species complete their logarithmic growth phase approximately two days after the start of cultivation and enter the death phase approximately seven 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 subculturing interval is preferably between one and ten days, more preferably between two and seven days, and even more preferably between two and five days.

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 longer culture times for Aurantiochytrium and similar species increase lipid production, the proportion of carotenoids 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 total lipids without strictly controlling glucose concentration or pH.

During the culture process, cultured Labyrinthula species such as Aurantiochytrium and Schizochytrium produce odd-numbered fatty acid esters both intracellularly and in the extracellular matrix. As a result, a culture composition is obtained containing the algal bodies of microorganisms belonging to the Labyrinthula species (Aurantiochytrium, Schizochytrium, etc.) 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.

Various methods can be used to concentrate the culture medium, such as centrifugal concentration (centrifugation of solids), sedimentation concentration (natural sedimentation of solids), evaporation concentration (heating and evaporating the medium), vacuum concentration (evaporating the medium under reduced pressure), pressure concentration (pressure filtration), membrane concentration (filtration through a separation membrane), and freeze concentration (freezing and removing the medium). Furthermore, various methods can 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 (heat drying in a rotating drum).

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

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.

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, resulting in a lipid content of less than approximately 30% by mass in the culture composition, while the proportions of protein, ash, etc., are relatively high. In contrast, when culturing is performed using an amino acid-supplemented medium to which a propionyl-CoA precursor is added, the production of lipids, including odd fatty acids, increases, making it possible to obtain a culture composition with a lipid content of 30% by mass or more to 45% by mass or more.

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 odd fatty acid content can be achieved by adding propionyl-CoA precursors, propionic acid, or their salts.

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 an odd-numbered fatty acid ratio of 20% to 40% or more, and 50% or less, while suppressing unsaturated fatty acids to 50% or less. Compared to conventional production methods, it is possible to efficiently produce odd-numbered fatty acid esters, and the production volume of odd-numbered fatty acid esters can be increased from 1.0 g/L or more to 1.3 g/L or more per culture medium.

The separation process involves separating odd-numbered fatty acid esters produced by microorganisms from the microbial algal bodies. The odd-numbered fatty acid esters can be separated by solvent extraction, centrifugation, filtration, etc., after pre-treatment of a concentrated algal suspension or dried algal body as needed. Examples of algal pre-treatment include flocculation, chemical treatment, steaming, heat treatment, stirring, crushing, ultrasonic treatment, pressure changes, chemicals, enzymes, and freeze-thaw cycles.

For solvent extraction, various solvents can be used, 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.

For pretreatment, a flocculant such as chitosan is preferably used. Furthermore, for pretreatment, a treatment to adjust the pH by adding citric acid is preferably used. When Aurantiochytrium and other algal cells produce large amounts of lipids, the algal body becomes fragile and easily breaks, making the extraction and separation of odd-numbered fatty acid esters difficult. However, by adding a flocculant such as chitosan, the algal body can be easily collected without damaging it. Additionally, pH adjustment hardens the algal body, preventing damage.

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. Furthermore, 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.

Odd fatty acids produced by Labyrinthula species 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.

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

<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.

For the test culture medium, we prepared media containing either cheese whey or soy milk whey as the main nutrient source. Specifically, we prepared two media: one 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 another containing soy milk whey at a concentration of 10%. Each medium was sterilized in an autoclave at 121°C for 20 minutes before being used for cultivation.

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.

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), while soy milk whey does not contain vitamin B12 at a detectable concentration.

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 were: medium volume: 200 mL, culture temperature: 25°C, culture time: 72 hours, shaking speed: 115 strokes/min. The cultured Aurantiochytrium was collected by centrifugation, freeze-dried, and stored until measurement.

The total lipid content was determined using the following procedure. First, approximately 0.2 g of freeze-dried 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 collected in another test tube. 1.0 mL of physiological saline was added to the collected 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 collected in another test tube. This test tube was then placed in a 40°C water bath, and the solvent was removed by blowing nitrogen gas, drying 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.

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.

A GC-2015 gas chromatograph (Shimadzu Corporation) was used. An Agilent J&W GC column DB-23 (Agilent Technologies, length: 30 m, inner diameter: 0.25 mm, film thickness: 0.25 μm) was used as the column. The measurement conditions were as follows: Carrier gas: Helium (constant pressure), Carrier gas pressure: 14 psi, Injection volume: 1 μL, Injection conditions: Split/Splitless, Injection port temperature: 250°C, Split ratio: 50:1, Heating conditions: 50°C (1 min) → 25°C/min to 175°C → 4°C/min to 230°C → 230°C (5 min), FID temperature: 280°C, Hydrogen flow rate: 40 mL/min, Air flow rate: 400 mL/min, Makeup gas flow rate: 25 mL/min.

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.

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

As shown in Table 1, when comparing culture systems supplemented with cheese whey and those supplemented with soy milk whey, the soy milk whey system produced higher levels 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 specifically on the amount of vitamin B12, a cofactor of methylmalonyl CoA mutase. Since cheese whey contains vitamin B12, while soy milk whey does not, soy milk serum isolated from legumes is considered appropriate as the main nutrient source to add to the culture medium.

<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.

For culturing, either Aurantiochytrium sp. SA-89 strain or Aurantiochytrium sp. SA-96 strain was used. Strain SA-89 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.

As test media, two types were prepared: a medium without the addition of a propionyl-CoA precursor, and a medium 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.

The culture test was performed by shaking culture of Aurantiochytrium sp. SA-89 strain or 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 were: medium volume: 200 mL, culture temperature: 25°C, culture time: 72 hours, shaking speed: 115 strokes/min. The cultured Aurantiochytrium was collected by centrifugation, freeze-dried, and stored until measurement.

Table 2 shows the average results of culture tests using different Aurantiochytrium species. The quantification of total lipids and individual fatty acids within the lipids was performed in the same manner as in the previous culture tests.

As shown in Table 2, in strain SA-89, D-methionine was not metabolized, and the production of odd fatty acids was similar to that of the case without the addition of propionyl-CoA precursor. 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 that of the case without the addition of propionyl-CoA precursor.

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 in strain SA-89, and the proportion of pentadecanoic acid (C15) was also sufficiently high.

<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.

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. A control (negative control) medium containing L-leucine was also prepared. 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. Finally, a medium was modified by adding leucine to the basic medium to a concentration of 10 mM each. Each medium was sterilized in an autoclave at 121°C for 20 minutes before use for cultivation.

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 were: medium volume: 200 mL, culture temperature: 25°C, culture time: 72 hours, shaking speed: 115 strokes/min. The pH of the culture medium was not controlled during cultivation. The cultured Aurantiochytrium was collected by centrifugation, freeze-dried, and stored until measurement.

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 previous culture tests.

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.

<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.

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 using an autoclave at 121°C for 20 minutes before use.

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 were: culture temperature: 25°C, culture time: 72 hours, shaking speed: 115 strokes/min. The pH of the culture medium was varied within the range of pH 4–8 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.

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 of 7.4 to 7.7 is preferable.

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

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. This basic medium was then modified by adding L-valine, DL-methionine, and sodium propionate, 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 use for cultivation.

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 autoclaved at 115°C for 30 minutes, and the precipitate formed by heating was aseptically filtered and added to the culture medium.

The culture test was performed using Aurantiochytrium sp. SA-96 strain by airlift culture. The Aurantiochytrium was pre-cultured prior to 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.

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.

In the air-lift type 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 bubbles rise inside the inner cylinder 4, forming an upward flow. After exiting the upper side of the inner cylinder 4, they descend outside the inner cylinder 4, forming a downward flow (see arrows in the diagram). With air-lift culture, the culture medium 6 is aerated and stirred in this manner, thus maintaining uniformity of dissolved oxygen concentration and culture medium component concentration within the tank without applying significant shear force to the microorganisms.

In the culture test using air-lift culture, a culture tank 1 with a capacity of 5 L was used, as shown in Figure 2. The inner cylinder 4 was designed so that its internal area in a cross-sectional view was half the internal area of the culture tank 1 in a cross-sectional view.

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.

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, the results of comparisons with culture tests with added propionic acid are shown in Table 7. The quantification of total lipids and individual fatty acids within lipids was performed in the same manner as in the aforementioned culture tests.

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

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 propionyl-CoA precursor was added, the proportion of pentadecanoic acid (C15) was 3.4%. However, 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%).

Furthermore, the addition of 20 mM L-valine increased the proportion of C15 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, the addition of 50 mM L-valine increased the proportion of C15 to 16.7-18.4% (average 17.7%), and the total proportion of odd fatty acids became 20.3-20.8% (average 20.6%).

On the other hand, the production of odd-numbered fatty acids reached its maximum value at a concentration of 50 mM. Biomass remained approximately the same regardless of the L-valine concentration. When 100 mM L-valine was added, the total proportion of odd-numbered 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-numbered fatty acids, is generated. This propionyl CoA then hydrolyzes to produce propionic acid, which inhibits the synthesis of fatty acids and other substances.

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.

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, the pathway branches into two: one via α-ketobutyrate to propionyl-CoA, and the other via cystathionine to α-ketobutyrate. Therefore, it is thought that when DL-methionine was used as a precursor, the rate of propionyl-CoA production was slower than with L-valine, resulting in a decrease in the production of odd fatty acids.

Within 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.

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. For propionic acid, the advantage of producing propionyl-CoA in a single reaction is considered to be a key factor.

Generally, potassium propionate has been shown to exhibit 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.

<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.

As the test medium, a culture medium containing L-valine as a precursor of propionyl-CoA was prepared. Specifically, 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.) was prepared, to which L-valine was added to a concentration of 50 mM. The medium was sterilized in an autoclave at 121°C for 20 minutes before being used for cultivation.

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 were: medium volume: 250 mL, culture temperature: 25°C, culture time: 12 hours, shaking speed: 100 strokes/min. The cultured Aurantiochytrium was collected every 12 hours by centrifugation at 2500 g for 15 minutes, washed twice with a 1.5% seawater salt solution, freeze-dried, and stored until measurement.

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

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.

Hydrocarbons such as squalene, nonpolar carotenoids, and sterol esters were eluted using a mixed solvent of n-hexane/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 using chloroform at three times the bed volume.

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 an Rf value range of 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).

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 recovered by elution from the plate with a chloroform-methanol mixed solvent (volume ratio: chloroform/methanol = 1/5).

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 in the n-hexane solution was washed with a small amount of cold n-hexane solution to recover the 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.

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.

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

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 phospholipid fraction when produced in amino acid-supplemented media. Since the proportion of odd-numbered fatty acids in the solid lipid fraction is high at 31.5%, precipitation treatment or similar methods can be used to concentrate odd-numbered fatty acids.

<Culture Test 7>
Next, in order to increase the proportion of odd fatty acid esters to 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.

As test media, two 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 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, and a medium containing 10 mM, 25 mM, or 50 mM sodium propionate was prepared by adding these to the medium. Each medium was sterilized in an autoclave at 121°C for 20 minutes before being used for cultivation.

The culture test was performed using Aurantiochytrium sp. SA-96 strain by airlift culture. 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 7.40–7.46. Other conditions were the same as in <Culture Test 5>.

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 aforementioned culture tests.

As shown in Table 9, biomass decreased as the concentration of propionic acid increased, 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.

The total lipid production showed a maximum at a propionic acid concentration of 25 mM. Furthermore, the production of odd fatty acids reached a maximum value of 1.325 g/L at a propionic acid concentration of 25 mM. While the reason for the maximum at propionic acid is unclear, the high production of odd fatty acids, despite a low proportion of lipid fractions containing carotenoids, sterols, etc., 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.

The above culture tests demonstrate the ability of Aurantiochytrium to produce odd-numbered 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, their properties regarding the production of odd-numbered fatty acid esters are extremely similar, and it is considered appropriate to treat them similarly. Similar species that produce odd-numbered fatty acid triglycerides, etc., can be used in the production of odd-numbered fatty acid esters using this amino acid-supplemented medium.

1. Culture tank 2. Gas supply pipe 3. Gas exhaust pipe 4. Inner cylinder 5. Sparger 6. Culture medium 100. Air-lift type reactor

Claims (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.