JP7530698B2 - Method for producing odd-numbered fatty acid ester and culture composition - Google Patents

Description

The present 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 a culture composition containing odd-numbered fatty acid esters obtained by culturing these microorganisms.

In recent years, there has been active development of technologies to produce useful substances using microorganisms. Some types of microalgae accumulate large amounts of lipids within their cells, and technologies to produce functional ingredients, physiologically active substances, biofuels, etc. using microalgae have been developed. Photosynthetic microorganisms such as Chlorella, Spirulina, and Euglena have been used not only for substance production, but also for the algae themselves as food and feed. However, in recent years, efforts have been made to put heterotrophic microorganisms, which have excellent cultivation efficiency and are more advantageous for substance production, into practical use.

Labyrinthulae are chemoheterotrophic marine eukaryotic microorganisms belonging to the stramenopiles, and because of their high proliferation and lipid production capabilities, their application to substance production has been widely investigated. Labyrinthulae are broadly divided into the Labyrinthulidae and Thraustochytriidae families, and the Thraustochytriidae family includes the genera Aurantiochytrium, Schizochytrium, and Thraustochytrium.

It has been reported that Aurantiochytrium is highly capable of producing the omega-3 fatty acids 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 amounts of these lipids during the logarithmic growth phase, accumulating them as oil droplets within the cells.

Odd-numbered fatty acids produced by Aurantiochytrium include pentadecanoic acid (PDA), which has 15 carbon atoms, tridecylic acid (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 living organisms of mammals and other animals, and has been reported to exhibit various physiological activities, such as hair growth and hair restoration, blood pressure reduction, blood sugar level rise suppression, cell proliferation promotion, and Alzheimer's disease alleviation.

Demand for odd-numbered fatty acids, including pentadecanoic acid, is expected to increase as functional ingredients and biologically active substances in a variety of applications, including food and beverages, pharmaceuticals, and cosmetics, and there is a demand for efficient biological production methods that can produce them in a state close to natural. To date, technologies related to culture methods for producing odd-numbered fatty acids using Aurantiochytrium have been investigated.

Patent Document 1 describes a method for producing a medium for culturing Aurantiochytrium, which includes the steps of (1) treating a cell extract with a strong acid and heating it, (2) neutralizing the extract of step (1), and (3) preparing a cell culture medium based on the extract of step (2). The medium prepared by this method is said to increase the odd-numbered fatty acid content in the total fatty acids produced by Aurantiochytrium.

JP 2017-063633 A

Aurantiochytrium and other microalgae belonging to the Labyrinthule class synthesize useful odd-numbered fatty acids for various purposes as fatty acid esters such as fatty acid triglycerides and phospholipids. The fatty acid esters produced must be separated from the cells, concentrated and purified, or hydrolyzed into fatty acids. It is also anticipated that the algae containing the fatty acid esters may be used as they are as ingredients in food and beverages. Therefore, when producing odd-numbered fatty acids using microalgae such as Labyrinthules, it is important not only to have the absolute production amount of fatty acid esters, but also to have a high proportion of odd-numbered fatty acids in the total lipids.

However, commonly known production methods leave room for improvement in the amount of fatty acid esters produced, and the proportion of odd-numbered fatty acids in total lipids remains low. If the proportion of odd-numbered fatty acids is low, it is costly to concentrate and purify the fatty acid esters, and additional medium and culture costs are required to ensure production. Therefore, there is a demand for an efficient production method that increases not only the absolute production amount but also the proportion of odd-numbered fatty acids in total lipids.

Therefore, the present invention aims to provide a method for producing odd-numbered fatty acid esters that can enable Labyrinthules to efficiently produce odd-numbered fatty acid esters, and a culture composition containing the odd-numbered fatty acid esters obtained thereby.

In order to solve the above problems, the method for producing an odd-numbered fatty acid ester according to the present invention is a method for producing an odd-numbered fatty acid ester in which an odd-numbered fatty acid is ester-bonded, and includes a step of culturing a microorganism belonging to the genus Aurantiochytrium or Schizochytrium in an amino acid-supplemented medium, and 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.

The culture composition according to the present invention is a culture composition obtained by culturing a microorganism belonging to the genus Aurantiochytrium or Schizochytrium, and contains algal cells of the microorganism and an odd-numbered fatty acid ester produced by the microorganism in which an odd-numbered fatty acid is ester-bonded, and the content of the odd-numbered fatty acid per biomass of the microorganism is 0.04 g/g or more, and the proportion of the odd-numbered fatty acid in the total lipids is 25% or more.

According to the present invention, it is possible to provide a method for producing odd-numbered fatty acid esters that can efficiently produce odd-numbered fatty acid esters in microorganisms belonging to the Labyrinthula genus, and a culture composition containing the odd-numbered fatty acid esters obtained thereby.

FIG. 1 shows the relationship between pH and biomass in the culture of Aurantiochytrium. FIG. 1 is a diagram showing a schematic structure of an airlift reactor.

Below, we will explain in detail the method for producing odd-numbered fatty acid esters according to one embodiment of the present invention, and the culture composition containing the odd-numbered fatty acid esters obtained thereby.

The method for producing an odd-numbered fatty acid ester according to the present embodiment relates to a method for producing an odd-numbered fatty acid ester in which an odd-numbered fatty acid is ester-bonded by culturing a microorganism belonging to Labyrinthula. In this production method, by culturing a microorganism belonging to Labyrinthula that has the ability to produce an odd-numbered fatty acid ester, odd-numbered fatty acids useful for various applications are produced as odd-numbered fatty acid esters such as fatty acid triglycerides and phospholipids.

The present inventor attempted to develop a new medium for culturing Aurantiochytrium, which belongs to the Labyrinthula, in order to improve 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 common in nature. In addition, the production of odd-numbered fatty acids has been confirmed in Schizochytrium, a species closely related to Aurantiochytrium. Therefore, the present inventor selected precursors of propionyl CoA as candidates for medium components to be added to the new medium for Labyrinthula, 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 of 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 for the synthesis of acetyl CoA, reduced nicotinamide adenine dinucleotide (NADH), adenosine triphosphate (ATP), guanosine triphosphate (GTP), etc., and for gluconeogenesis. However, propionic acid is known as a preservative that exhibits antibacterial properties, and when added in large quantities to a medium, it may not be an optimal medium component to add to a new medium because it consumes CoA and ATP and causes changes in the cell membrane, inhibiting the growth of microorganisms.

In addition, precursors of propionyl CoA include pentanoic acid (valeric acid), which has 5 carbon atoms, heptanoic acid (enanthic acid), which has 7 carbon atoms, and nonanoic acid (pelargonic acid), which has 9 carbon atoms. However, these fatty acids have a strong odor and can transfer to the culture composition obtained by cultivation, reducing the usability of the culture composition. In addition, there is a risk that they may inhibit the growth of microorganisms due to their surfactant action, so they are not considered to be suitable as medium components to be added to new media.

On the other hand, valine, isoleucine, threonine, and methionine are also known as precursors of propionyl CoA. In cells, valine and isoleucine are converted to methylmalonyl CoA, which undergoes a reversible equilibrium reaction with propionyl CoA. Threonine is converted to propionyl CoA via α-ketobutyric acid. Methionine is converted to propionyl CoA via S-adenosylmethionine, homocysteine, etc. Unlike propionic acid, which exhibits antibacterial activity, these amino acids are unlikely to inhibit the growth of microorganisms, and are therefore considered to be suitable as medium components to be added to new media.

Therefore, in this embodiment, an amino acid-supplemented medium containing a specific amino acid corresponding to a precursor of propionyl CoA is used as a medium for culturing microorganisms belonging to the Labyrinthula genus, such as Aurantiochytrium and Schizochytrium.

The microorganisms to be cultured may be any species of microorganisms belonging to the genus Aurantiochytrium, such as Aurantiochytrium mangrovei, Aurantiochytrium limacinum, Aurantiochytrium acetophilum, or Schizochytrium mangrovei, Schizochytrium limacinum, Schizochytrium aggregatum, etc., that have the ability to produce odd-numbered fatty acid esters. Within the genera Aurantiochytrium and Schizochytrium, species have been found that accumulate large amounts of lipids as oil droplets within the cells. When Aurantiochytrium or Schizochytrium is cultured, not only fatty acid triglycerides and phospholipids composed only of odd-numbered fatty acids, but also DHA and EPA can be produced.

The microorganisms to be cultured may be any of wild strains collected from nature, mutant strains in which a mutation has been introduced into a gene, and genetically modified strains in which genes have been modified by genetic recombination technology. It is preferable to previously confirm the odd-numbered fatty acid ester production ability of the microorganisms to be cultured by test culture using a basal medium, and to isolate and use strains with high odd-numbered fatty acid ester production ability.

The amino acid-supplemented medium used for the culture may be any of synthetic, semi-synthetic, and natural media. The amino acid-supplemented medium can be prepared by adding a specific amino acid corresponding to a precursor of propionyl CoA to a basic medium containing a carbon source, a nitrogen source, vitamins, minerals, etc. It is preferable that the amino acid-supplemented medium is a seawater medium using natural seawater or artificial seawater.

As the amino acid 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 medium. The amino acid corresponding to the precursor of propionyl CoA is preferably added at a higher concentration than the amount generally added as a nitrogen source, etc., and is preferably added at a concentration of 10 mM or more per type.

The amino acid-supplemented medium preferably contains at least one of L-valine and DL-methionine at a concentration of 10 mM or more. These amino acids are relatively inexpensive, and for example, the racemic form of methionine is easily available as a chemically synthesized product, so the use of these amino acids can reduce medium costs.

It is more preferable that the amino acid-supplemented medium contains only L-valine at a concentration of 10 mM or more. As described below, L-valine is metabolically converted to odd-numbered fatty acids with high efficiency, so by using only L-valine, it is possible to increase the production amount of odd-numbered fatty acid esters and the proportion of odd-numbered fatty acids in total lipids while suppressing medium costs.

The amino acids corresponding to the precursors of propionyl CoA preferably have a concentration of 10 mM or more and 50 mM or less, more preferably 20 mM or more and 30 mM or less per type. If the concentration is 50 mM or less, even if the generated propionyl CoA is hydrolyzed, a large amount of propionic acid is not produced, so that growth inhibition due to propionic acid and inhibition of synthesis of odd-numbered fatty acids can be avoided. In addition, if the concentration is 20 mM or more and 30 mM or less, the production amount of odd-numbered fatty acid esters and the proportion of odd-numbered fatty acids in total lipids can be further increased.

Carbon sources that can be added to the amino acid-supplemented medium include, for example, glucose, fructose, mannose, galactose, sucrose, maltose, etc. Glucose is preferred as a carbon source.

As the nitrogen source to be added to the amino acid-supplemented medium, for example, amino acids such as glutamine, glutamic acid, sodium glutamate, peptides, proteins, urea, ammonia, ammonium salts, nitrates, etc. can be used. As the nitrogen source, glutamine, glutamic acid, or sodium glutamate is preferred.

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

Examples of sea salts that can be added to the amino acid-supplemented 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, boric acid, etc.

The amino acid-supplemented medium 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, as well as digests obtained by enzymatically treating these, casein hydrolysates such as tryptone, peptone, and casamino acid, soybean hydrolysates, gelatin hydrolysates, and protein hydrolysates obtained by hydrolyzing other extracts with enzymes, acids, heat, etc., can be fractionated and used as necessary.

The amino acid-supplemented medium can be prepared, for example, using GTY medium, a common medium for culturing Aurantiochytrium, as the basal medium. GTY medium contains approximately 20-100 g/L of glucose, 10-60 g/L of tryptone, and 5-40 g/L of yeast extract, and is prepared using natural seawater or artificial seawater to give a sea salt concentration of 10-40 g/L. An amino acid-supplemented medium can also be prepared by adding a propionyl CoA precursor to such a seawater medium to a concentration of 10 mM or more.

The amino acid-supplemented medium can also be prepared by adding whey from animals obtained during the production of cheese, yogurt, and other dairy products, or soy whey (soybean whey, etc.) obtained during the production of tofu, soy milk, and other processed soy products. Whey and soy whey are rich in proteins, amino acids, other nitrogenous compounds, vitamins, and minerals, and can be obtained inexpensively as by-products in product production, making them useful as nutritional sources. In addition, they do not have a strong odor, and are unlikely to transfer odors to the culture composition obtained by cultivation, and the odor of the culture composition may be masked, so the usability of the culture composition is unlikely to be impaired.

Whey from animals can be obtained by separating the water-soluble fraction from milk. For example, when making cheese, milk is curdled by fermenting it with lactic acid bacteria as a starter, or by adding milk-clotting enzymes such as chymosin, pepsin, or rennet, or by adding acid, or when making yogurt, milk is fermented with lactic acid bacteria to obtain curds whose main components are proteins and fats. When such curds are pressed or drained, whey can be obtained as the water-soluble fraction.

Milk used as a raw material for animal-derived whey may or may not have been skimmed. Furthermore, animal-derived whey may be acidic whey obtained by curdling milk at a pH of about 4.6, or sweet whey obtained by curdling milk with rennet or the like. Furthermore, animal-derived whey may or may not have been desalted of sodium, potassium, etc.

Specific examples of milk that can be used include cow's milk, buffalo milk, goat's milk, sheep's milk, yak's milk, horse's milk, and camel's milk. Of these, cow's milk, buffalo milk, goat's milk, and sheep's milk are preferred, with cow's milk being particularly preferred, because they are easily available and inexpensive.

Soy whey can be obtained by separating the water-soluble fraction from a vegetable protein source such as beans. For example, beans are ground and soaked in water, and treated under acidic conditions of pH 4.5-5.0 or lower, or heated to about 80°C or higher, to separate and remove solids such as fibers, to obtain an extract that mainly contains water-soluble components. In addition, when producing processed bean products such as tofu and fried tofu, soy milk is obtained by boiling down ground beans and then filtering out the solids. The insoluble proteins contained in the extract or soy milk obtained in this way can be salted out with a coagulant such as magnesium chloride to obtain soy whey as a water-soluble fraction.

The vegetable protein source such as beans used as the raw material for soy milk syrup may or may not be defatted. For example, soy milk syrup may be separated from soy milk made from defatted processed beans, or from soy milk made from undefatted raw beans.

As a vegetable protein source, soybeans, mung beans, black beans, etc. can be used. Among these, soybeans are preferred as a vegetable protein source because they are easily available and inexpensive. Soy whey may be directly separated from soy milk, green soy milk, black soy milk, etc., whose solid content concentration has been adjusted, to obtain soy whey, mung bean whey, black bean whey, etc.

Whey is preferably adjusted to a pH of 4 or more and 8 or less, more preferably 6 or more and 8 or less. The optimum pH for culture of Labyrinthulea species such as Aurantiochytrium and Schizochytrium is near neutral. In contrast, whey derived from animals may be acidified to about pH 4.6 by lactic acid fermentation or the addition of acid. Also, soy whey such as soy whey may be adjusted to about pH 4 to 5 for the purpose of solid coagulation or isoelectric precipitation. Therefore, by adjusting the pH of whey to near neutral in advance, it is possible to simplify the adjustment of the pH of the medium using whey.

The amino acid-supplemented medium preferably has a vitamin B12 concentration of 0.2 μg/L or less. For example, when using biological extracts, protein hydrolysates, etc. as medium components, it is preferable to use types with low vitamin B12 concentrations or fractions with low vitamin B12 concentrations. In addition, compared with animal-derived whey, which has a high vitamin B12 concentration, it is preferable to use soybean whey, which is derived from plants and has a vitamin B12 concentration below the detection limit.

The concentration of vitamin B12 is thought to affect the synthesis of odd-numbered fatty acids. Propionyl CoA is the substrate (starting material) for the synthesis of odd-numbered 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 if a precursor of propionyl CoA is added to the medium, it is thought that the amount of synthesis of odd-numbered fatty acids will decrease if the replenishing reaction (anaplerosis) that replenishes succinyl CoA is dominant.

In cells, succinyl-CoA is produced by isomerization of methylmalonyl-CoA, and this reaction is 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-added medium is high, the supplementation reaction that replenishes succinyl-CoA proceeds, and the synthesis amount of odd-numbered fatty acids decreases. In contrast, if the amino acid-added medium is prepared without actively adding vitamin B12 and the concentration of vitamin B12 is reduced to 0.2 μg/L or less, the supplementation reaction that replenishes succinyl-CoA becomes difficult to proceed, and the synthesis amount of odd-numbered fatty acids increases, so the production amount of odd-numbered fatty acid esters can be improved.

In addition to the propionyl CoA precursor, the amino acid-supplemented medium preferably contains glucose as a carbon source, glutamine, glutamic acid, or sodium glutamate as a nitrogen source, yeast extract, soy whey separated from beans, and sea salt. The concentration of the yeast extract is preferably 0.2% or less from the viewpoint of reducing the introduction of vitamin B12, which is contained at a maximum of about 0.1 μg/g. The concentration of the soy whey is preferably 0.1% or more in terms of solid content, and more preferably 0.2% or more. The concentration of the soy whey is preferably 20% or less in terms of solid content, and more preferably 10% or less.

By using such a medium, it is possible to grow Labyrinthules such as Aurantiochytrium and Schizochytrium at a sufficiently high growth rate while suppressing medium costs, thereby increasing the production amount of odd-numbered fatty acid esters and the proportion of odd-numbered fatty acids in total lipids. In particular, when soybean whey is used as the main nutrient source, unlike when protein hydrolysates and the like are used, the synthesis of odd-numbered fatty acids is less likely to be inhibited by medium components, and odd-numbered fatty acid esters can be produced efficiently.

The amino acid-supplemented medium may contain propionic acid or a salt thereof together with the propionyl CoA precursor. When propionic acid or a salt thereof is added to the amino acid-supplemented medium, the 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 mixed in advance as a medium component, or may be added during the culture as a pH adjuster.

In cells, propionic acid, which is C3, inhibits the biosynthesis of C2 molecules such as acetyl CoA. Therefore, when propionic acid or its salt is added to an amino acid-supplemented medium, the production of even-numbered fatty acids can be suppressed. In other words, the proportion of even-numbered fatty acids in total lipids can be reduced, and the proportion of odd-numbered fatty acids can be increased instead. This reduces the cost of concentrating and purifying the target odd-numbered fatty acid esters, and the total cost of the propionyl CoA precursor required for the production of odd-numbered fatty acids.

The amino acid-supplemented medium may be in any form of liquid medium, semi-solid medium, or solid medium. The amino acid-supplemented medium may contain various buffers such as phosphates, isotonicity agents such as sodium chloride, microorganisms such as bacteria, yeast, and diatoms for bipartite culture, and medium components such as agar. However, from the viewpoint of increasing the production amount of odd-numbered fatty acid esters, it is preferable that the amino acid-supplemented medium is a liquid medium that allows mass culture.

In the method for producing an odd-numbered fatty acid ester according to this embodiment, the odd-numbered fatty acid ester may be obtained as a culture composition containing the algae of the microorganism and the odd-numbered fatty acid ester by carrying out only the culture step. Alternatively, the odd-numbered fatty acid ester may be obtained in a state separated and extracted from the microorganism by carrying out the culture step and the separation step.

The culture step is a step of culturing a microorganism capable of producing an odd-numbered fatty acid ester in an amino acid-supplemented medium. Any of batch culture, continuous culture, and fed-batch culture may be used as the culture method. In addition, any appropriate culture method may be used, such as shaking culture, aeration culture, aeration and stirring culture, airlift culture, and stationary culture. Among these culture methods, continuous culture is preferred because it can maintain the glucose concentration and neutral pH. In addition, aeration and stirring culture or airlift culture is preferred because it allows for large-scale culture.

As the culture device, for example, a mechanical stirring type reactor, an airlift type reactor, a packed bed type reactor, a fluidized bed type reactor, etc. can be used depending on the culture method. In addition, as the culture vessel, various containers such as tanks, jar fermenters, flasks, dishes, culture bags, tubes, test tubes, etc. can be used depending on the culture volume, etc. The culture vessel may be made of an appropriate material such as inorganic materials such as stainless steel and glass, or organic materials such as polystyrene, polyethylene terephthalate copolymer, polypropylene, etc.

The amino acid-supplemented medium can be sterilized using an appropriate sterilization method before seeding with the microorganism capable of producing odd-numbered fatty acid esters. Sterilization methods that can be used include, for example, heat sterilization, ultraviolet sterilization, gamma ray sterilization, and filtration sterilization. In addition, the amino acid-supplemented medium can be seeded with seed cells that have been precultured using a medium of the same composition or a basal medium such as GTY medium.

Cultivation of Labyrinthula species such as Aurantiochytrium and Schizochytrium can be carried out 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.

Cultivation of Labyrinthulea species such as Aurantiochytrium and Schizochytrium can be performed for an appropriate culture time while subculturing at appropriate time intervals. However, for some species of Aurantiochytrium, the logarithmic growth phase ends about 2 days after the start of culture, and the death phase begins about 7 days later. The production amount of odd-numbered fatty acid ester increases with growth, reaching a near maximum from the end of the logarithmic growth phase to the beginning of the stationary phase, and then tends to gradually decrease. Therefore, when using such species, the time interval for subculturing is preferably 1 to 10 days, more preferably 2 to 7 days, and even more preferably 2 to 5 days.

In addition, 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 as the culture time for Aurantiochytrium and the like is extended, the amount of lipid produced increases, but the proportion of carotenoids and the like increases, and the proportion of odd-numbered fatty acids decreases. Therefore, with such a short culture time, the proportion of odd-numbered fatty acids in total lipids can be increased without continuously strictly controlling the glucose concentration and pH.

When the culture step is carried out, the cultured Labyrinthulea, such as Aurantiochytrium or Schizochytrium, produces odd-numbered fatty acid esters within the cells or in the extracellular matrix. As a result, a culture composition is obtained that contains the algae of a microorganism belonging to Labyrinthulea, such as Aurantiochytrium or Schizochytrium, and the odd-numbered fatty acid esters produced by the algae. The culture composition can be recovered by concentrating or drying the medium.

Methods for concentrating the medium include, for example, centrifugal concentration, which separates solids by centrifugal separation; sedimentation concentration, which allows solids to settle naturally; evaporation concentration, which involves heating the medium to evaporate; vacuum concentration, which involves reducing the pressure of the medium to evaporate; pressure concentration, which involves pressurizing the medium to filter it; membrane concentration, which involves filtering the medium through a separation membrane; and freeze concentration, which involves freezing the medium to remove it. Methods for drying the medium include, for example, hot air drying, cold air drying, vacuum drying, spray drying, freeze drying, infrared drying, natural drying, and drum drying, which involves heating and drying in a rotating drum.

When the culture composition is concentrated, it can have a moisture content of, for example, 80% or less, preferably 30% to 50%. When concentrated to such a moisture content, the culture composition is sufficiently reduced in volume and becomes a paste-like culture composition that is less likely to flow, making the culture composition easier to handle. When the culture composition is dried, it can have a moisture content of, for example, 10% or less.

The culture composition can be used for various purposes, such as food, feed, fertilizer, and industrial raw materials. Specific examples of food applications include general foods, health foods, food ingredients, and beverage ingredients. Specific examples of feed applications include livestock feed, poultry feed, aquaculture feed, and pet feed. Specific examples of industrial raw materials include biofuel raw materials, feed raw materials, fertilizer raw materials, chemical raw materials, and pharmaceutical raw materials.

The lipid content of the culture composition is preferably 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 necessarily high enough, so the lipid content in the culture composition remains at less than about 30% by mass, and the ratio of protein, ash, etc. is relatively high. In contrast, when culture is performed using an amino acid-supplemented medium to which a precursor of propionyl CoA has been added, the production amount of lipids including odd-numbered fatty acids increases, and a culture composition with a lipid content of 30% by mass or more to 45% by mass or more can be obtained.

The culture composition preferably has an odd-numbered fatty acid content of 0.04 g/g or more per microbial biomass (dry weight) in terms of fatty acid methyl ester. In addition, for the lipid fraction recovered by solvent extraction, the proportion of odd-numbered fatty acids in the total lipids 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 an amount of odd-numbered fatty acids can be achieved by adding a precursor of propionyl CoA, propionic acid, or a salt thereof.

Specifically, when cultured using an amino acid-supplemented medium, the total lipid production per biomass can be secured to be 0.1 g/g or more to 0.2 g/g or more and 0.25 g/g or less. In addition, the proportion of odd-numbered fatty acids in the total lipids can be secured to be 20% by mass or more to 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 efficiently produce odd-numbered fatty acid esters, and the production amount of odd-numbered fatty acid esters can be 1.0 g/L or more to 1.3 g/L or more per medium.

The separation process is a process in which the odd-numbered fatty acid ester produced by the microorganism is separated from the algae cells of the microorganism. The odd-numbered fatty acid ester can be separated by subjecting a concentrated suspension of algae cells or dried algae cells, after pretreating them as necessary, to solvent extraction, centrifugation, filtration separation, etc. Examples of pretreatment of the algae cells include flocculation treatment, chemical treatment, steaming treatment, heat treatment, and crushing treatment using various principles such as stirring, grinding, ultrasound, pressure change, chemicals, enzymes, and freezing and thawing.

Examples of solvents that can be used in solvent extraction include methanol, ethanol, diethyl ether, propylene glycol, propanol, isopropanol, acetone, chloroform, hexane, cyclohexane, benzene, acetic acid, water, and mixtures of these, supercritical fluids, and other solvents. Examples of mixed solvents include hexane-ethanol mixed solvent, chloroform-methanol mixed solvent, and ethanol-diethyl ether mixed solvent.

As a pretreatment flocculation treatment, a treatment in which a flocculant such as chitosan is added is preferably used. As a pretreatment chemical treatment, a treatment in which citric acid is added to adjust the pH is preferably used. When Aurantiochytrium or the like is made to produce a large amount of lipids, the algae become fragile and easily crushed, making it difficult to extract and separate the odd-numbered fatty acid esters. However, by adding a flocculant such as chitosan, the algae can be easily collected without damaging them. Furthermore, adjusting the pH hardens the algae, preventing them from being damaged.

The odd-numbered fatty acid esters separated from the algae of the microorganism can be purified as necessary to recover the target substance, such as odd-numbered fatty acid. For example, when recovering odd-numbered fatty acid, degumming, deacidification, decolorization, deodorization, etc. can be performed. Depending on the target substance, various methods such as solvent fractionation, distillation, molecular distillation, chromatography, membrane separation, hydrolysis, ester exchange, crystallization, inclusion complex formation, complex formation, etc. can be used in combination.

Examples of odd-numbered fatty acids produced by Labyrinthulea species such as Aurantiochytrium and Schizochytrium include tridecylic acid (C13), pentadecanoic acid (C15), and heptadecanoic acid (C17). Pentadecanoic acid can be used for purposes such as lowering blood pressure, suppressing blood sugar rise, and promoting cell proliferation. Specific examples of uses for promoting cell proliferation include healing damaged tissue, easing pain, autoimmune diseases, neurodegenerative diseases, immune diseases, diseases related to metabolic syndrome, and cancer-related diseases, reducing skin wrinkles, promoting skin metabolism, hair growth and hair care, reducing allergy symptoms, reducing muscle pain, and improving motor function.

The present invention will be explained in detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

<Culture test 1>
First, in order to determine the medium composition to be used for the production of odd-numbered fatty acid esters, a culture test of Aurantiochytrium was conducted using different types of major nutrient sources, and the production amounts of total lipids and odd-numbered fatty acids were compared.

Test media were prepared by adding either cheese whey or soy whey as the main nutrient source. Specifically, a medium was prepared containing 3.0% glucose, 0.5% sodium L-glutamate, 0.2% yeast extract, 1.2% sea salt, and 10 mM L-methionine, with cheese whey added to a concentration of 10% and soy whey added to a concentration of 10%. Each medium was sterilized at 121°C for 20 minutes using an autoclave before being used for cultivation.

The soy milk whey was prepared by heating commercially available soy milk to 80°C, adding magnesium chloride hexahydrate ( _MgCl2.6H2O ) to a concentration of 0.7%, and removing the coagulated protein by centrifugation. The supernatant soy milk whey was sterilized _at 115°C for 30 minutes using an autoclave, and then stored at 6°C before being added to the medium.

A typical GTY medium has a yeast extract concentration of about 0.5%. However, yeast extract contains vitamin B12, which promotes the supplementation reaction and reduces the synthesis of odd-numbered fatty acids. Therefore, in this culture test, 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 significant amount of vitamin B12 (about 0.3 μg/100 g), but soy milk whey does not contain detectable concentrations of vitamin B12.

The culture test was carried out by shaking culture of Aurantiochytrium Sp. SA-96 strain. For shaking culture, a 500 mL Sakaguchi flask and a reciprocating shaking incubator (manufactured by Thomas Scientific Instruments) were used. 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 was collected by centrifugation, freeze-dried, and stored until measurement.

The total lipids were quantified as follows. First, about 0.2 g of the freeze-dried Aurantiochytrium was weighed and placed in a test tube together with 10 mL of a chloroform-methanol mixed solvent (volume ratio: chloroform/methanol = 2/1). The test tube was placed in an ultrasonic cleaner, and the contents were stirred to extract the lipids into the solvent layer. The contents of the test tube were then 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, which was then stirred and centrifuged at 2800 rpm for 10 minutes to remove the water-soluble components in the upper layer, and the fat-soluble components in the lower layer were separated into another test tube. The test tube was then placed in a water bath at 40°C, and nitrogen gas was sprayed to evaporate the solvent, and the lipids extracted from the algae were dried. The weight of lipid recovered in the test tube was calculated as the total lipid production amount by Aurantiochytrium.

The quantitative determination of each fatty acid in the lipid was carried out using a GC-FID (Gas Chromatography-Flame Ionization Detector) according to the following procedure. After the quantitative determination of the total fatty acids, 14% BF ₃ -methanol (methanol solution containing 14% boric trifluoride) was added to the lipid extracted from the algae body, and the mixture was heated at 70°C for 30 minutes to methylate the acyl components in the lipid. The obtained fatty acid methyl ester (FAME) was then dissolved in n-hexane to prepare a measurement sample with a concentration of 1.0 mg/mL, which was then subjected to GC-FID.

A GC-2015 (Shimadzu Corporation) was used as the gas chromatograph. 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: carrier gas: helium (constant pressure), carrier gas pressure: 14 psi, injection amount: 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, make-up gas flow rate: 25 mL/min.

In the quantification of each fatty acid in lipids, the area obtained by subtracting the integrated area of the solvent peak from the integrated area of all peaks in the chromatogram detected by GC-FID was taken as the integrated area of all FAMEs. As a result of a total of seven measurements, the average integrated area of all FAMEs was 265,000 dots/μg-FAME. The peaks of each type of fatty acid were identified by comparing with the retention time of a fatty acid methyl ester standard (manufactured by GL Sciences). The proportion of each fatty acid in total lipids was calculated individually as the area ratio to the integrated area of all FAMEs. The production amount of each fatty acid was calculated using the results of the quantification of total lipids and the average integrated area of all FAMEs.

The average results of the cultivation tests in which the type of main nutrient source was changed are shown in Table 1. In the table below, biomass is the biomass (dry weight) of Aurantiochytrium obtained by freeze-drying after cultivation, and the proportion and production amount of odd-numbered fatty acids are the total values of tridecylic acid (C13), pentadecanoic acid (C15), and heptadecanoic acid (C17).

As shown in Table 1, when comparing the culture system to which cheese whey was added and the culture system to which soy whey was added, the production of odd-numbered fatty acids was higher in soy whey. Propionyl CoA produced by the metabolism of amino acids is used to produce odd-numbered fatty acids in the cytoplasm, but in the mitochondria, it is used in a supplementation reaction to replenish succinyl CoA. Which pathway predominates depends on the activity of methylmalonyl CoA mutase and the amount of vitamin B12, which is a cofactor for methylmalonyl CoA mutase. Cheese whey contains vitamin B12, whereas soy whey does not, so soy whey separated from beans is considered to be an appropriate major nutrient source to be added to the medium.

<Culture test 2>
Next, in order to determine the production strain to be used for producing odd-numbered fatty acid esters, culture tests were carried out using different types of Aurantiochytrium, and the production amounts of total lipids and odd-numbered fatty acids were compared.

The microorganisms used for cultivation were either Aurantiochytrium Sp. SA-89 strain or Aurantiochytrium Sp. SA-96 strain. The SA-89 strain is an isolated strain characterized by a fast growth rate and a large increase in biomass during cultivation. The SA-96 strain is an isolated strain characterized by a large amount of lipid production.

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

The culture test was carried out by shaking culture of Aurantiochytrium Sp. SA-89 strain or Aurantiochytrium Sp. SA-96 strain. A 500 mL Sakaguchi flask and a reciprocating shaking incubator (manufactured by Thomas Scientific Instruments) 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.

The average results of the culture tests using different types of Aurantiochytrium are shown in Table 2. The quantification of total lipids and each fatty acid in lipids was performed in the same manner as in the above culture test.

As shown in Table 2, in the SA-89 strain, D-methionine was not metabolized, and the production of odd-numbered fatty acids was similar to that in the case where the propionyl-CoA precursor was not added. On the other hand, L-methionine was metabolized, and the production of odd-numbered fatty acids and the proportion of odd-numbered fatty acids in total lipids increased. However, when L-methionine was metabolized, propionyl-CoA was produced, which was then hydrolyzed to produce propionic acid, causing the pH of the culture medium to drop to around 4.2. As a result, the biomass after cultivation decreased, becoming less than when the propionyl-CoA precursor was not added.

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

<Culture test 3>
Next, in order to determine the medium composition to be used for the production of odd-numbered fatty acid esters, a culture test of Aurantiochytrium was carried out using different types of propionyl-CoA precursors, and the production amounts of total lipids and odd-numbered fatty acids were compared.

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

The culture test was carried out by shaking culture of Aurantiochytrium Sp. SA-96 strain. A 500 mL Sakaguchi flask and a reciprocating shaking incubator (manufactured by Thomas Scientific Instruments) 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 pH of the culture medium was not controlled during culture. The cultured Aurantiochytrium was collected by centrifugation, freeze-dried, and stored until measurement.

The average results of the culture tests using different types of propionyl-CoA precursors are shown in Table 3. The quantification of total lipids and each fatty acid in lipids was performed in the same manner as in the above culture test.

As shown in Table 3, DL-methionine, L-valine, L-isoleucine, and L-threonine were metabolized, and the pH of the culture medium dropped to around 4 to 6. As a result, the biomass after cultivation decreased. On the other hand, L-leucine was not metabolized, and the metabolism of sodium L-glutamate added to the medium only caused a small drop in pH to around 7.1 to 7.4. When DL-methionine, L-valine, L-isoleucine, and L-threonine, which correspond to precursors of propionyl CoA, were added, the production amount of odd-numbered fatty acids and the proportion of odd-numbered fatty acids in total lipids were higher than in the control. In particular, when L-valine was added, the production amount of odd-numbered fatty acids was significantly higher.

<Culture test 4>
Next, in order to determine the culture conditions to be used for producing odd-numbered fatty acid esters, a culture test of Aurantiochytrium was carried out, and the change in biomass (biological mass) when the pH of the culture medium was changed was compared.

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

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

FIG. 1 is a diagram showing the relationship between pH and biomass in the culture of Aurantiochytrium.
As shown in Figure 1, when the pH of the culture solution was changed during the cultivation of Aurantiochytrium, the biomass increased as the pH increased. The biomass increased rapidly at pH 6.0 to 7.0, and the increase rate became significantly smaller at pH 7.4 or higher. The biomass was obtained at a substantially maximum amount in the pH range of 7.4 to 7.7, and it was confirmed that the pH of the culture solution is preferably between 7.4 and 7.7.

<Culture test 5>
Next, in order to confirm the production amount of odd-numbered fatty acid esters, a culture test of Aurantiochytrium was conducted by changing the type and concentration of propionyl-CoA precursor, and the production amount of total lipids and odd-numbered fatty acids were compared.

As test media, media were prepared to which any of L-valine, DL-methionine, and sodium propionate was added as a precursor of propionyl CoA. The L-valine concentration was changed to 10 mM, 20 mM, 50 mM, and 100 mM. The DL-methionine concentration was changed to 20 mM, 50 mM, and 100 mM. Specifically, a basic medium containing 3.6% glucose, 0.5% sodium L-glutamate, 0.2% yeast extract, 1.0% sea salt, and 10% tofu whey, and a medium to which L-valine, DL-methionine, and sodium propionate were added were prepared. Each medium was sterilized at 121°C for 20 minutes using an autoclave, and then mixed with separately sterilized glucose and tofu whey for use in the culture.

The tofu whey used was the supernatant produced when soy milk was salted out with a coagulant during the tofu manufacturing process. The tofu whey supernatant was sterilized by heating at 115°C for 30 minutes using an autoclave, and the precipitate produced by heating was aseptically filtered and added to the medium.

The cultivation test was carried out by airlift cultivation of Aurantiochytrium Sp. SA-96 strain. Aurantiochytrium was pre-cultured prior to airlift cultivation. Pre-culture was started 24 hours before airlift cultivation, and the pre-culture solution was added to the culture tank for airlift cultivation so that the concentration was 0.5%. Airlift cultivation was carried out using the airlift type reactor shown in Figure 2.

FIG. 2 is a diagram showing a schematic structure of an airlift type reactor.
As shown in Fig. 2, the airlift 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 tube 4 is supported within 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 airlift reactor 100, when air is supplied through the gas supply pipe 2, the sparger 5 diffuses the air into the culture solution 6 in the culture tank 1. The diffused air bubbles rise inside the inner cylinder 4 to form an upward flow, and when they escape to the upper side of the inner cylinder 4, they descend outside the inner cylinder 4 to form a downward flow (see the arrow in the figure). With airlift culture, the culture solution 6 is aerated and stirred in this way, so that the dissolved oxygen concentration and medium component concentration in the tank can be maintained uniformly without applying large shear forces to the microorganisms.

In the culture test using airlift culture, the capacity of the culture tank 1 as shown in Figure 2 was set to 5 L. The inner cylinder 4 was designed so that the internal area in cross section was 1/2 the internal area in cross section of the culture tank 1.

The culture conditions for airlift culture were: 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 at 7.4-7.7 with 1.0 M sodium hydroxide solution using a pH control device "mk-750pH" (Automatic System Research). The cultured Aurantiochytrium was harvested by centrifugation at 3,400 rpm for 60 minutes, freeze-dried and stored until measurement.

The average results of the culture tests in which the concentration of L-valine was changed are shown in Tables 4 and 5. The average results of the culture tests in which the concentration of DL-methionine was changed are shown in Table 6. The results of a comparison with the culture test in which propionic acid was added are shown in Table 7. The quantification of total lipids and each fatty acid in lipids was performed in the same manner as in the above culture tests.

In the table, the production amount [mM] of odd-numbered fatty acid is a value calculated based only on the molecular weight of pentadecanoic acid (M = 242). The conversion efficiency [%] of odd-numbered fatty acid is a value expressed by the following formula.
Conversion efficiency [%] = (odd-numbered fatty acid production [mM] - 0.66 [mM])
/ Concentration of amino acid corresponding to the precursor of propionyl CoA [mM]

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

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-numbered fatty acids increased to 18.6-19.3% (average 19.0%). Furthermore, 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-numbered fatty acids increased to 20.3-20.8% (average 20.6%).

On the other hand, the production of odd-numbered fatty acids reached its maximum at a concentration of 50 mM. Biomass was roughly the same regardless of the L-valine concentration, and when 100 mM L-valine was added, the total proportion of odd-numbered fatty acids was 24.3%, higher than at 50 mM. However, the production of total lipids was reduced to approximately 64% compared to 50 mM. When the concentration of L-valine is too high, propionyl CoA, a reaction substrate for odd-numbered fatty acids, is produced, but this propionyl CoA is hydrolyzed to produce propionic acid, which is thought to inhibit the synthesis of fatty acids, etc.

The conversion efficiency of odd-numbered fatty acids decreased as the concentration of L-valine increased. However, the production amount of odd-numbered fatty acids reached a maximum of approximately 1.040 g/L when 50 mM L-valine was added. Therefore, when considering both the conversion efficiency and production amount, it is believed that the optimal condition for achieving both cost-effectiveness and productivity exists at a high concentration of approximately 50 mM or less.

As shown in Table 6, when DL-methionine was added as a precursor of propionyl-CoA, the production of odd-numbered fatty acids reached a maximum at a concentration of 50 mM. However, the production of odd-numbered fatty acids was lower than when L-valine was added. In cells, methionine is converted to homocysteine, but from homocysteine, a pathway branches into one in which it is converted to propionyl-CoA via α-ketobutyric acid, and another in which it is converted to α-ketobutyric acid via cystathionine. Therefore, when DL-methionine is used as a precursor, the rate of propionyl-CoA production is slower than in the case of L-valine, and as a result, it is thought that the production of odd-numbered fatty acids is lower.

In cells, the synthesis reaction of odd-numbered fatty acids is centered on the condensation reaction of propionyl-CoA and malonyl-CoA, and fatty acid synthase plays an important role in the production of odd-numbered fatty acid esters. It is possible that the concentrations of propionyl-CoA, malonyl-CoA, and propionic acid produced by hydrolysis of propionyl-CoA, consumption of acetyl groups by propionic acid, and metabolic inhibition of amino acids are all intricately involved in the synthesis reaction of odd-numbered fatty acids, making L-valine favorable.

As shown in Table 7, a certain level of odd-numbered fatty acid production was achieved regardless of whether L-valine, DL-methionine, or propionic acid was added as the precursor to propionyl-CoA. The conversion efficiency of odd-numbered fatty acids using propionic acid as a precursor was intermediate compared to L-valine and DL-methionine, and it is believed that the fact that propionyl-CoA is produced in a single reaction was advantageous for propionic acid.

In general, potassium propionate is recognized to be acutely toxic to rats, with an LD50 of 4-5 g/kg. It is believed that the acute toxicity of potassium propionate is manifested by the inhibition of acetyl-CoA metabolism. Propionic acid is also used as a preservative in foods, etc., and is believed to inhibit fatty acid synthesis and amino acid metabolism. Therefore, L-valine and DL-methionine are suitable precursors to propionyl-CoA, with L-valine being particularly superior.

<Culture test 6>
Next, in order to investigate the distribution of odd-numbered fatty acid esters, a culture test of Aurantiochytrium was carried out, and the contents of odd-numbered fatty acids in solid lipids, liquid lipids, and phospholipids were quantified.

A test medium was prepared by adding L-valine as a precursor of propionyl CoA. Specifically, a medium was prepared by adding L-valine to a concentration of 50 mM to GTY medium containing 2% glucose, 1% tryptone, 0.5% yeast extract, and 1.0% sea salt (Red Sea Salt, Red Sea Coral Salt). The medium was sterilized at 121°C for 20 minutes using an autoclave 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 shaking incubator (manufactured by Thomas Scientific Instruments) were used for the shaking culture. The culture conditions for the shaking culture were medium volume: 250 mL, culture temperature: 25°C, culture time: 12 hours, and shaking speed: 100 strokes/min. The cultured Aurantiochytrium was collected by centrifugation at 2500 g for 15 minutes every 12 hours, washed twice with 1.5% sea 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 using a silica gel 60 column.

Neutral lipids were eluted with 4 bed volumes of chloroform. Polar lipids were eluted with 4 bed volumes of chloroform-methanol mixed solvent (volume ratio: chloroform/methanol = 1/4). Fatty acid triglycerides were then 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 applied to the column.

Hydrocarbons such as squalene, non-polar carotenoids, and sterol esters were eluted with a double bed volume of a mixed solvent of n-hexane and chloroform (volume ratio: n-hexane/chloroform = 1/1). Fatty acid triglycerides, polar carotenoids, and free fatty acids were eluted with three bed volumes of chloroform.

To purify the fatty acid triglycerides, the fatty acid triglyceride fraction was subjected to preparative thin-layer chromatography using a silica gel 60 plate. 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 Rf value of the fatty acid triglycerides shifted to the range of 0.6 to 0.8 depending on the developing solvent. The fatty acid triglyceride spots were visualized by spraying with water, and then eluted from the plate with a mixed solvent of chloroform and methanol (volume ratio: chloroform/methanol = 1:1) and collected.

To purify phospholipids, the polar lipid fraction was subjected to preparative thin-layer chromatography using a silica gel 60 plate. A mixed solvent of chloroform, methanol, acetic acid, and water (volume ratio: chloroform/methanol/acetic acid/water = 25/15/4/2) was used as the developing solvent. The Rf value of phospholipids shifted to the range of 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 mixed solvent of chloroform and methanol (volume ratio: chloroform/methanol = 1/5) and collected.

After removing the extraction solvent, the recovered fatty acid triglycerides were dissolved in a five-fold amount of n-hexane, and the solution was stored overnight at 0-4°C. After storage, the white precipitate that had precipitated 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 onto it, and the same precipitation process was repeated twice. The fatty acid triglycerides were recovered from the removed extraction solvent 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 converted to fatty acid methyl esters by reacting with 14% BF ₃ -methanol (a methanol solution containing 14% boric trifluoride) at 90° C. for 15 minutes. The fatty acid methyl esters were extracted with n-hexane and quantified by GC-FID.

The results of the analysis of the distribution of odd-numbered fatty acid esters are shown in Table 8. The quantification of total lipids and each fatty acid in lipids was performed in the same manner as in the above-mentioned culture test.

As shown in Table 8, saturated odd-numbered fatty acids, including pentadecanoic acid (C15), were highly concentrated in the solid lipid fraction. However, unlike the case where standard medium was used, odd-numbered fatty acids were also found in the liquid lipid fraction and phospholipid fraction when produced in amino acid-supplemented medium. Since the proportion of odd-numbered fatty acids in solid lipids was high at 31.5%, it is believed that precipitation processes can be used to concentrate odd-numbered fatty acids.

<Culture test 7>
Next, in order to increase the proportion of odd-numbered fatty acid esters relative to the total lipid production, L-valine was used as a precursor for propionyl CoA, and a culture test of Aurantiochytrium was conducted while varying the concentration of propionic acid, a metabolic inhibitor of acetyl CoA, and the changes in total lipid production, odd-numbered fatty acid production, and biomass were compared.

As test media, a medium containing 50 mM L-valine as a precursor of propionyl CoA, and a medium containing 50 mM L-valine and sodium propionate were prepared. The concentration of sodium propionate was changed to 10 mM, 25 mM, and 50 mM. Specifically, a medium containing 4.0% glucose, 0.5% sodium L-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 added to this medium. Each medium was sterilized at 121°C for 20 minutes using an autoclave before being used for cultivation.

The culture test was carried out by airlift culturing 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 at pH: 7.40-7.46. The other conditions were the same as in <Cultivation Test 5>.

The average results of the culture test using L-valine as the precursor of propionyl CoA and varying the concentration of propionic acid, a metabolic inhibitor of acetyl CoA, are shown in Table 9. The quantification of total lipids and each fatty acid in lipids was performed in the same manner as in the above culture test.

As shown in Table 9, the higher the propionic acid concentration, the lower the biomass, but the higher the proportion of odd-numbered fatty acids in the total lipids. In particular, the higher the propionic acid concentration, the higher the proportion of C15 pentadecanoic acid, and conversely, the lower the proportion of C16 palmitic acid. On the other hand, there was no significant change in the proportion of unsaturated fatty acids (C20, C22, etc.) synthesized via the polyketide synthesis system. It is believed that propionic acid inhibits the synthesis of acetyl CoA, resulting in a lower proportion of even-numbered fatty acids and a lower biomass, while conversely, the proportion of odd-numbered fatty acids increased.

The total lipid production was at its maximum when the propionic acid concentration was 25 mM. The odd-numbered fatty acid production was at its maximum of 1.325 g/L when the propionic acid concentration was 25 mM. The reason for the maximum production with propionic acid is unclear, but the high odd-numbered fatty acid production despite the 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 appropriate for fatty acid synthesis.

The above culture tests demonstrate Aurantiochytrium's ability to produce odd-numbered fatty acid esters, but the genera Aurantiochytrium and Schizochytrium are genera with similar lipid production abilities and were separated in 2007 due to differences in cell morphology and growth patterns. In particular, with regard to the production of odd-numbered fatty acid esters, they show very similar properties and it is considered appropriate to treat them similarly. Similar species that produce odd-numbered fatty acid triglycerides, etc. can be used to produce odd-numbered fatty acid esters using this amino acid-supplemented medium.

Reference Signs List 1 Culture tank 2 Gas supply pipe 3 Gas exhaust pipe 4 Inner cylinder 5 Sparger 6 Culture solution 100 Airlift type reactor

Claims (5)

A method for producing an odd-numbered fatty acid ester in which an odd-numbered fatty acid is ester-bonded, comprising the steps of:
The method includes culturing a microorganism belonging to the genus Aurantiochytrium or Schizochytrium in an amino acid-added medium,
The method for producing an odd-numbered fatty acid ester, 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 20 mM or more and 50 mM or less per amino acid. A method for producing an odd-numbered fatty acid ester in which an odd-numbered fatty acid is ester-bonded, comprising the steps of:
The method includes culturing a microorganism belonging to the genus Aurantiochytrium or Schizochytrium in an amino acid-added medium,
The method for producing an odd-numbered fatty acid ester, wherein the amino acid-added medium contains L-valine at a concentration of 10 mM or more. The method for producing odd-numbered fatty acid esters according to claim 1, wherein the amino acid-supplemented medium contains L-valine at a concentration of 20 mM or more and 30 mM or less. The method for producing an odd-numbered fatty acid ester according to any one of claims 1 to 3, wherein the amino acid-supplemented medium further contains propionic acid or a salt thereof at a concentration of 10 mM or more and 50 mM or less. The method for producing an odd-numbered fatty acid ester according to any one of claims 1 to 4, wherein the amino acid-added medium is a medium containing soy milk separated from beans.

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