JP2015188342A - Methods for producing high value-added lipids - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing high value-added lipids such as docosahexaenoic acid, astaxanthin, squalene by microbial fermentation of mannitol derived from a brown algae (e.g., kelp) extract.SOLUTION: The method for producing high value-added lipids of the invention comprises: culturing Gluconobacter bacteria in a culture medium comprising a mannitol source such as a kelp extract and a nitrogen source; and when the conversion rate of mannitol to fructose becomes 50% or more, adding sodium chloride to the culture medium to a final concentration of 1-4%(w/v) with inoculation of a microorganism of the genus Aurantiochytrium (KH105, Accession number: FERM AP-22267).

Description

  The present invention relates to a method for producing high-value-added lipids.

  There is an increasing demand for high-value-added lipids such as docosahexaenoic acid (DHA), astaxanthin, and squalene. However, since these high-value-added lipids are difficult to obtain safely and stably from nature, methods for fermentative production of high-value-added lipids by microorganisms have been studied.

  For example, in Cited Document 1, algal bodies are stably grown by culturing marine microalgae such as Crypthecodinium cornie in a medium containing polyols such as glycerol, mannitol, sorbitol, and saccharose. It is described that the content of docosahexaenoic acid (DHA) in the lipids of algal bodies is remarkably increased.

  As microorganisms used for fermentative production of high-value-added lipids, Labyrinthula Aurantiochytrium microorganisms have attracted attention because of their excellent ability to produce high-value-added lipids.

  For example, in Reference Document 2, heterotrophic microorganisms such as Aulanthiochytrium microorganisms are efficiently cultured as heterotrophic microorganisms by culturing in stages using two types of media having different nitrogen and carbon contents. It is described that fats and oils can be produced, and that fats and oils can be produced in large quantities in a shorter period of time. In Cited Document 3, heterotrophic microorganisms such as Aulanthiochytrium microorganisms are cultivated in stages using two types of media having different sugar and glycerin contents, so that a large amount of fats and oils can be produced in a shorter period of time. It is described that it can be produced.

JP 7-87988 A (published April 4, 1995) JP2013-126404A (released on June 27, 2013) JP2013-183687A (published September 19, 2013)

  By the way, macroalgae are unused resources that exist in large quantities in the ocean, and are attracting attention as low-carbon and sustainable resources. Macroalgae do not compete with food supplies such as cereals, and can be supplied stably as resources. In terms of land use, macroalgae do not compete with food supplies such as cereals, and lead to effective use of the open ocean and coastlines. For this reason, effective utilization of large algae as marine biomass is desired. Brown algae such as kombu contains a large amount of alginate and mannitol ((a) in FIG. 1). To date, there are attempts to use alginate as marine biomass, but mannitol has not been used much.

  As described above, Auranthiochytrium microorganisms have attracted attention as microorganisms used for the fermentative production of high-value-added lipids, but Auranthiochytrium microorganisms cannot directly use mannitol as a carbon source. . If mannitol can be used as a carbon source in fermentative production of high-value-added lipids, mannitol derived from macroalgae can be used as a supply source to meet the growing demand for high-value-added lipids, and high-value-added lipids can be used. Further increase in demand can be expected due to lower prices.

  The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a method for producing high-value-added lipids in an auranthiochytrium microorganism using mannitol as a carbon source. is there.

  The present inventors can produce high-value-added lipids in auranthiochytrium microorganisms by using mannitol as a carbon source by culturing a combination of fructose-producing microorganisms and auranthiochytrium microorganisms. In the cultivation of fructose-producing microorganisms and Auranthiochytrium microorganisms, the difference in the salt requirements of these microorganisms is utilized to control the growth of fructose-producing microorganisms and For the first time, it has been found that a complex culture system capable of growing thorium microorganisms can be easily realized, and the present invention has been completed based on such new findings.

That is, in order to solve the above-described problems, the high-value-added lipid production method according to the present invention includes (a) a culture medium containing at least mannitol and a nitrogen source, and a fructose-producing microorganism and aurantiochytrium. Inoculating and culturing the genus microorganism, or (b) inoculating and culturing the fructose-producing microorganism in the culture medium, and then inoculating and culturing the auranthiochytrium microorganism.
Adding sodium chloride to the culture medium, adding sodium chloride,
Including
The fructose-producing microorganism is a microorganism that can convert mannitol into fructose and secrete the fructose out of the cell,
The sodium chloride addition step is performed when the conversion rate of the mannitol in the culture medium to fructose is 50% or more,
The sodium chloride is added to the culture medium such that the final concentration in the culture medium is a concentration at which the Aulanthiochytrium microorganism can grow and a concentration that inhibits the growth of the fructose-producing microorganism. It is characterized by that.

  In the high value-added lipid production method according to the present invention, the fructose-producing microorganism is preferably a Gluconobacter bacterium.

  In the high added value lipid production method according to the present invention, in the sodium chloride addition step, the sodium chloride is added to the culture medium so that the final concentration in the culture medium is 1 to 4% (w / v). It may be added.

The method for producing a high added-value lipid according to the present invention further includes an antibiotic addition step of adding an antibiotic that acts on the fructose-producing microorganism to the culture medium,
The antibiotic addition step may be performed when the conversion rate of the mannitol to fructose in the culture medium becomes 50% or more.

  In the method for producing a high value-added lipid according to the present invention, it is preferable to inoculate the culture medium with a fructose-producing microorganism that has been pre-cultured in a culture medium containing at least mannitol and a nitrogen source in the culture step. .

  In the method for producing a high added-value lipid according to the present invention, the culture medium preferably contains only mannitol as a carbon source.

  In the method for producing a high added-value lipid according to the present invention, the culture medium may contain a brown alga algae extract.

  In the method for producing a high value-added lipid according to the present invention, the brown alga algae may be a Compositae algae.

  In the method for producing a high value-added lipid according to the present invention, when the culture step is (b), the aurantiochytrium microorganism may be inoculated into the culture medium after the sodium chloride addition step. .

  In the method for producing a high added-value lipid according to the present invention, when the culture step is (b), the aurantiochytrium microorganism is inoculated and cultured in the culture supernatant of the fructose-producing microorganism. Good.

  In the high-value-added lipid production method according to the present invention, the Aurantiochytrium microorganism may be Aurantiochytrium sp. KH105 (reception number: FERM AP-22267).

  According to the method for producing a high-value-added lipid according to the present invention, a high-value-added lipid can be produced by an auranthiochytrium microorganism using mannitol as a carbon source. For this reason, mannitol derived from macroalgae can be used as a raw material for producing high-value-added lipids. Thereby, there exists an effect that the effective utilization of the macroalgae which was an unused resource is attained. Furthermore, there is an effect that it is possible to reduce the supply price of the high added-value lipid by reducing the price of the raw material.

  In addition, according to the method for producing a high-value-added lipid according to the present invention, it is possible to control the growth of fructose-producing microorganisms only by controlling the concentration of sodium chloride added to the medium and the timing of addition, while maintaining the genus Aurantiochytrium. A complex culture system for growing microorganisms can be easily realized. Thereby, it becomes possible to produce high-value-added lipids more easily and efficiently, and as a result, there is an effect that mass-production of high-value-added lipids becomes possible.

(A) is a figure which shows the component of saccharides and monosaccharides specific to a brown alga algae, and (b) is a graph which shows the capability to assimilate each saccharide of Aurantiochytrium sp. KH105. (A) is a graph showing the growth of Gluconobacter oxydans (NBRC 14819), saccharide consumption and saccharide production in a culture medium containing mannitol, and (b) shows G recovered at different culture times. It is a graph which shows the proliferation of Aurantiochytrium sp. KH105 in the culture medium containing the culture supernatant of oxydans (NBRC 14819). (A) is a graph showing the absorbance (OD600) at 600 nm when G. oxydans (NBRC 14819) is grown in Gluconobacter medium containing different concentrations of mannitol, (c) It is a graph which shows the change of pH of the culture medium at that time. (B) is a graph which shows the influence of the salt concentration of artificial seawater with respect to the proliferation of G. oxydans (NBRC 14819), (d) is a graph which shows the change of the pH of the culture medium at that time. (A) is a flask-scale test when Aurantiochytrium sp. KH105 was cultured using a culture solution after culturing G. oxydans (NBRC 14819) in mannitol-PY medium or kombu extract-PY medium. It is a graph which shows the fatty-acid production amount of Aurantiochytrium sp. KH105, (b) is a graph which shows the production amount of a carotenoid. (A) is a graph showing changes in absorbance, dry weight of cells, mannitol concentration and fructose concentration when performing two-stage fermentation on a jar fermenter scale using a kombu extract-PY medium, (B) is a graph showing changes in the concentration of fatty acids, and (c) is a graph showing changes in the concentration of carotenoids.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” indicating a numerical range means “A or more and B or less”. Further, “%” in this document means “% (w / v)” unless otherwise specified.

[1. (Production method of high added-value lipid)
The method for producing high-value-added lipids according to the present invention (hereinafter referred to as “the method for producing high-value-added lipids of the present invention”) includes: (a) a culture medium containing at least mannitol and a nitrogen source; Inoculating and culturing the fructose-producing microorganism and the Aurantiochytrium microorganism, or (b) inoculating the Aurantiochytrium microorganism after inoculating and culturing the fructose-producing microorganism in the culture medium. And culturing, and at least a sodium chloride addition step of adding sodium chloride to the culture medium.

  As used herein, “high-value-added lipid” means a lipid that can be produced by a microorganism belonging to the genus Aurantiochytrium, which has particularly high functionality. For example, eicosapentaenoic acid (EPA), docosa Unsaturated fatty acids such as pentaenoic acid (DPA), docosahexaenoic acid (DHA), oleic acid (OA), linoleic acid (LA), linolenic acid (ARA), dihomo-γ-linolenic acid (DGLA); astaxanthin, phenico Carotenoids such as xanthine, canthaxanthin, echinenone, β-carotene, and lycopene; hydrocarbon compounds such as squalene and sterol, and the like are listed as high value-added lipids.

(1-1. Culture process)
The culturing step includes (a) inoculating and culturing a fructose-producing microorganism and an aurantiochytrium microorganism in a culture medium containing at least mannitol and a nitrogen source, or (b) in the culture medium. Inoculating and culturing the fructose-producing microorganism, and then inoculating and culturing the Aurantiochytrium microorganism.

  Here, the “fructose-producing microorganism” is not particularly limited as long as it is a microorganism capable of converting mannitol into fructose and secreting the fructose outside the cells. The “microorganism” may be a prokaryotic organism or a eukaryotic organism.

  As a method for confirming whether or not the target microorganism is a microorganism capable of converting mannitol into fructose and secreting the fructose out of the cells (fructose-producing microorganism confirmation method), for example, a medium containing mannitol Only the target microorganism is cultured, and the amount of mannitol and fructose contained in the culture medium after completion of the culture is measured. As a result, the amount of mannitol contained in the medium after completion of the culture is smaller than the amount of mannitol contained in the medium before the start of the culture, and is contained in the medium after the end of the culture. When the amount of fructose is higher than the amount of fructose contained in the medium before the start of culture, the target microorganism can convert mannitol into fructose and secrete the fructose out of the cell It can be judged that it is a novel microorganism.

The amount of mannitol and fructose in the medium is determined using, for example, a high-performance liquid chromatography (HPLC) apparatus equipped with a Sugar-D Cosmosil column (Nacalai Tesque, Kyoto, Japan), an oven temperature of 40 ° C., and a detector temperature. It can be measured using acetonitrile: H ₂ O (75:25, v / v) set to 39 ° C. and set to a flow rate of 1.0 ml / min as an eluent.

  The medium for culturing the target microorganism in the method for confirming the fructose-producing microorganism is not particularly limited as long as it contains at least a component necessary for the target microorganism to grow in addition to mannitol. The culture conditions (temperature, time, etc.) can be appropriately set according to the type of the target microorganism, but the culture is preferably terminated before the growth curve of the target microorganism shifts from the logarithmic growth phase to the stationary phase.

  Such “fructose-producing microorganisms” are not particularly limited, and include, for example, gluconobacter bacteria, acetobacter bacteria (reference: US Pat. No. 4,734,366), and gluconoacetacter bacteria (reference: Oikawa et al). (1997) Biosci Biotechnol Biochem, 61, 1778-82). Gluconobacter bacteria are preferable because the rate of release of fructose out of the cells is high.

  The “Gluconobacter bacterium” is not particularly limited, and examples thereof include Gluconobacter oxydans. Gluconobacter oxydans is not particularly limited, and strains stored in a storage organization (NBRC, JCM, IFO, ATCC, etc.) and available to those skilled in the art can be used. For example, NBRC 14819 strain, JCM 7642 strain, IFO 12528 strain, ATCC 15163 strain, etc. can be used in the present invention.

  Further, the “aurantiochytrium microorganism” is not particularly limited as long as it is a microorganism classified into the genus Labyrinthura Aurantiochytrium. For example, Aurantiochytrium sp. KH105 (reception number: FERM AP-22267) can be mentioned. In addition, the above-mentioned “aulanthiochytrium microorganism” is also referred to as “Schizochytrium microorganism”.

  The “culture medium” used in the “culturing step” only needs to contain at least mannitol and a nitrogen source at the start of cultivation of the fructose-producing microorganism. The “culture medium” is not particularly limited, but is preferably a liquid medium for reasons such as being suitable for mass culture and easy control of culture conditions.

  The “mannitol” may be mannitol alone or a mixture of mannitol and another substance. The “mannitol” may be extracted from a natural source, may be chemically synthesized, or may be produced by a microorganism or the like.

  The concentration of mannitol added to the culture medium is preferably 5 to 200 g / L, and more preferably 30 to 200 g / L. If the mannitol concentration in the culture medium is 5 g / L or more, the Aulanthiochytrium microorganism can be grown using the culture supernatant of the fructose-producing microorganism. Further, if the mannitol concentration in the culture medium is 30 g / L or more, the aurantiochytrium microorganism can be grown using the culture supernatant of the fructose-producing microorganism to produce a high-concentration high-value-added lipid. it can. In addition, the said density | concentration of the mannitol in a culture medium is a density | concentration before the culture start of a fructose production microorganism.

  Mannitol is added to the culture medium as a carbon source, but the “culture medium” may contain other carbon sources of mannitol. The “other carbon source” is not particularly limited as long as it can be assimilated by fructose-producing microorganisms and / or auranthiochytrium microorganisms. For example, carbohydrates (monosaccharides, oligosaccharides, polysaccharides, sugar alcohols, etc.) ), Organic acids, alcohols, lipids and the like. As other carbon sources, only one type may be used, or a plurality of types may be used in combination. When another carbon source is added to the culture medium, the concentration of the other carbon source added to the culture medium is preferably 5 to 100 g / L, and more preferably 50 to 100 g / L. If the concentration of the other carbon source in the culture medium is 5 g / L or more, the fructose-producing microorganism can grow using only the carbon source. Moreover, if the density | concentration of the other carbon source in a culture medium is 50 g / L or more, a fructose production | generation microorganism can grow more highly using only the said carbon source. In addition, the said density | concentration of the other carbon source in a culture medium is a density | concentration before the culture start of a fructose production microorganism. When a plurality of types are used in combination as other carbon sources, other carbon sources may be added to the culture medium so that the total concentration of the other types of other carbon sources falls within the above range.

  When the culture medium contains a carbon source other than mannitol, the conversion rate from mannitol to fructose may be reduced when the fructose-producing microorganism uses the carbon source preferentially over mannitol. For this reason, from the viewpoint of efficiently converting mannitol into fructose by the fructose-producing microorganism, the “culture medium” preferably does not contain a carbon source that is preferentially used by the fructose-producing microorganism over mannitol. Examples of the carbon source that is preferentially used by the fructose-producing microorganism over mannitol include glucose, galactose, mannose, and the like. Therefore, the “culture medium” preferably does not contain glucose, galactose, mannose, etc., more preferably does not contain glucose, and more preferably contains only mannitol as a carbon source. Thereby, the mannitol contained in the culture medium can be efficiently converted to fructose by the fructose-producing microorganism. However, if the carbon source other than mannitol, which can be preferentially used by the fructose-producing microorganism, is cultured until it is completely depleted, and then mannitol is added, the objective can be achieved without reducing the conversion rate from mannitol to fructose. It is also possible to do.

  The “nitrogen source” is not particularly limited as long as it can be assimilated by fructose-producing microorganisms and / or auranthiochytrium microorganisms. For example, polypeptone, yeast extract, peptone, soybean flour, corn steep liquor, etc. Nitrogen-containing compounds: inorganic acids such as ammonium sulfate, ammonium nitrate, and ammonium phosphate, or ammonium salts of organic acids. As the nitrogen source, only one type may be used, or a plurality of types may be used in combination.

  The concentration of the nitrogen source added to the culture medium is preferably 1 to 50 g / L, and more preferably 5 to 20 g / L. If the nitrogen source concentration in the culture medium is 1 g / L or more, the fructose-producing microorganism can grow well. In addition, when the nitrogen source concentration in the culture medium is 5 g / L or more, the fructose-producing microorganism and the Aulanthiochytrium microorganism can grow well. In addition, the said density | concentration of the nitrogen source in a culture medium is a density | concentration before the culture start of a fructose production microorganism. When a plurality of types of nitrogen sources are used in combination, the nitrogen source may be added to the culture medium so that the total concentration of the plurality of types of nitrogen sources falls within the above range.

  The “culture medium” may contain components other than the carbon source and the nitrogen source. The “other components” are not particularly limited as long as they are generally used for culturing microorganisms, and examples thereof include inorganic salts, coenzymes such as vitamins, and pigments. The concentration of the “other components” in the culture medium can be set as appropriate.

  As the “culture medium” as described above, for example, the mannitol-PY medium (30 g / L mannitol, 10 g / L polypeptone, 5 g / L yeast extract (pH 6.0)) used in Examples described later is preferably used. be able to.

  The “culture medium” may contain a brown alga algae extract. The “brown algae algae extract” is not limited as long as it contains at least mannitol as a component derived from the brown algae algae. The concentration of mannitol in the brown algae extract can be confirmed using a high performance liquid chromatography (HPLC) apparatus.

  The method for preparing the “brown algae algae extract” is not particularly limited as long as it is a method capable of extracting mannitol from the brown algae algae. For example, it can be prepared according to a modification of the method described in Khomtimchenko, SV & Kulikova, IV 1999. Lipids of two species of brown algae of the genus Laminaria. Chem Nat Prod, 35 (1), 17-20. . Specifically, a brown algae algae extract can be prepared by adding a dry powder of brown algae algae to distilled water, stirring for 20 minutes, centrifuging, and collecting the supernatant. The dry powder of brown algae algae can be prepared, for example, by air-drying the brown algae algae and then pulverizing them. The brown alga algae may be air-dried and then fragmented by a blender, or may be used as wet algal bodies without being air-dried. As the brown algae algae extract, one prepared from only one type of brown algae algae may be used, or one prepared from a plurality of types of brown algae algae may be used in combination.

  Further, the purity of mannitol in the above-mentioned “Brown Algae Algae Extract” is not particularly limited. However, the brown algae extract may contain a component that exerts a specific action on the fructose-producing microorganism and / or the Aulanthiochytrium microorganism. For example, in the examples described below, the kombu extract contains components that negatively affect the conversion efficiency of gluconobacter bacteria from mannitol to fructose, and components that promote the growth of Aurantiochytrium microorganisms. It was suggested that it may have been. Therefore, in order to reduce the effects of components other than mannitol contained in the brown alga algae extract in the production of high value-added lipids, the brown alga algal extract is further purified to increase the purity of mannitol. May be used.

  When the above “culture medium” contains a brown alga algae extract, it may contain only a brown algae algae extract as a mannitol source, and mannitol derived from something other than a brown algae algae extract. Furthermore, you may contain.

  The concentration of the brown alga algae extract added to the culture medium is preferably 5 to 200 g / L, more preferably 30 to 200 g / L in terms of mannitol contained in the brown algae algal extract. . If the concentration of the brown algae extract in the culture medium is 5 g / L or more, the Aulanthiochytrium microorganism can be grown using the culture supernatant of the fructose-producing microorganism. In addition, if the concentration of brown algae extract in the culture medium is 30 g / L or more, auranthiochytrium microorganisms are grown using the culture supernatant of the fructose-producing microorganism, and a high concentration and high added-value lipid Can be produced. In addition, the said density | concentration of the brown algae alga extract in a culture medium is a density | concentration before the culture start of a fructose production microorganism.

  The “brown algae algae” used as a raw material for the brown algae extract is not particularly limited as long as it is an algae from which mannitol can be extracted. Can be mentioned.

  Examples of the above-mentioned “Coleoptera: Compositae Algae” include, but are not limited to, for example, algae belonging to the genus Caracombu, such as Macombu (Laminaria japonica), as well as the genus Gohei, and Nekoashikonbu.

  In the culturing step, one of the following two types of culturing methods may be performed.

(I) First embodiment (culture method (a))
In the first embodiment, a fructose-producing microorganism and an Aulanthiochytrium microorganism are inoculated and cultured in a culture medium containing at least mannitol and a nitrogen source. That is, in the first embodiment, at the start of culturing the fructose-producing microorganism, the Aulanthiochytrium microorganism is present in the culture medium.

  In the first embodiment, the fructose-producing microorganism may be inoculated with respect to the culture medium prior to the Aulanthiochytrium microorganism, or the Aulanthiochytrium microorganism before the fructose-producing microorganism. It may be inoculated or a fructose-producing microorganism and an auranthiochytrium microorganism may be inoculated at the same time.

(Ii) Second embodiment (culture method (b))
In the second embodiment, after inoculating and culturing a fructose-producing microorganism in a culture medium containing at least mannitol and a nitrogen source, an auranthiochytrium microorganism is inoculated and cultured. That is, in the second embodiment, at the start of the culture of the fructose-producing microorganism, there is no Aulanthiochytrium microorganism in the culture medium, and after a certain time has elapsed from the start of the culture of the fructose-producing microorganism, the fructose-producing microorganism. Inoculate Auranthiochytrium microorganisms into the culture medium in which

  In this case, the timing for inoculating the Aulanthiochytrium microorganism is not particularly limited, but it is preferable to inoculate the Aulanthiochytrium microorganism in the culture medium after the sodium chloride addition step (described later). Aulanthiochytrium microorganisms can be suitably grown in a medium containing 0.5-4%, preferably 1.5-3% sodium chloride. For this reason, by inoculating the Auranthiochytrium microorganism into the culture medium after the sodium chloride addition step, cultivation of the Auranthiochytrium microorganism can be started under conditions more suitable for growth.

  In the second embodiment, the aurantiochytrium microorganism may be inoculated into the culture supernatant of the fructose-producing microorganism and cultured.

  In addition, since the mannitol and nitrogen sources in the culture medium are consumed by the fructose-producing microorganism, the mannitol and nitrogen sources in the culture medium are reduced during the cultivation of the fructose-producing microorganisms unless they are additionally supplied. For this reason, in the second embodiment, mannitol and a nitrogen source may not be contained in the culture medium at the time of inoculating the Aulanthiochytrium microorganism.

  In the culturing step, a nitrogen source may be additionally supplied to the culture medium as necessary.

  In the culturing step, it is preferable to inoculate the culture medium with a fructose-producing microorganism pre-cultured in a culture medium containing at least mannitol and a nitrogen source. The culture medium used for the pre-culture of the fructose-producing microorganism is not particularly limited as long as it contains at least mannitol and a nitrogen source, but is preferably a medium having the same composition as the culture medium used in the culture step. Moreover, it is preferable that the culture medium used for preculture does not contain glucose as a carbon source. Thereby, mannitol can be more efficiently converted into fructose by the fructose-producing microorganism in the culturing step.

  In the culturing step, a pre-cultured Aulanthiochytrium microorganism may be inoculated into the culture medium. The culture medium used for the preculture of the Aurantiochytrium microorganism is not particularly limited, but is preferably a culture medium containing at least glucose and a nitrogen source. For example, 3% GPY medium (30 g / L glucose, 6 g / L polypeptone, 2 g / L yeast extract, 20 g / L sea salt, 20 g / L agar) can be used. The culture conditions for preculturing the Aulanthiochytrium microorganism are not particularly limited. For example, the culture may be performed in the 3% GPY medium at 28 ° C. with shaking for 2 days.

(1-2. Sodium chloride addition step)
The sodium chloride addition step is a step of adding sodium chloride to the culture medium. Note that mannitol in the culture medium is consumed by fructose-producing microorganisms, and the nitrogen source in the culture medium is consumed by fructose-producing microorganisms and aurantiochytrium microorganisms. In the meantime, the mannitol and nitrogen sources in the culture medium decrease. For this reason, in the sodium chloride addition step, mannitol and a nitrogen source may not be contained in the culture medium when sodium chloride is added to the culture medium.

  The sodium chloride addition step is performed when the conversion rate of mannitol in the culture medium into fructose becomes 50% or more. “Conversion rate of mannitol to fructose” means the amount of fructose produced relative to the initial concentration of mannitol at the time of cultivation of the fructose producing microorganism.

  Here, the conversion rate (hereinafter referred to as “conversion rate”) of mannitol to fructose after t hours from the start of cultivation of the fructose-producing microorganism is expressed by the following formula (1).

Conversion rate (%) = fructose production / initial concentration of mannitol × 100 (1)
(In the above formula (1), “fructose production amount” means the fructose production amount after t hours from the start of cultivation of the fructose producing microorganism, and “initial concentration of mannitol” is before the start of cultivation of the fructose producing microorganism. The concentration of mannitol in the culture medium of
The “initial concentration of mannitol” and the “fructose concentration after time t” can be confirmed by a conventionally known method using a high performance liquid chromatography (HPLC) apparatus or the like.

  In the sodium chloride addition step, sodium chloride is added to the culture medium such that the final concentration in the culture medium is a concentration at which auranthiochytrium microorganisms can grow and a concentration that inhibits the growth of fructose-producing microorganisms. Added.

  Here, the above-mentioned “concentration at which Auranthiochytrium microorganisms can grow” may be any concentration at which Auranthiochytrium microorganisms can grow in a culture medium containing sodium chloride at that concentration. The concentration may not be such that Lantiochytrium microorganisms can actively grow. For example, after culturing the Aurantiochytrium microorganism for a certain period (for example, 12 hours) in a culture medium containing a% sodium chloride, the number of Auranthiochytrium microorganisms in the culture solution is If the concentration is higher than that, it can be said that the concentration “a%” is “a concentration at which the Aulanthiochytrium microorganism can grow”.

  The above “concentration inhibiting the growth of fructose-producing microorganisms” refers to a culture containing sodium chloride at that concentration when the growth rate of fructose-producing microorganisms in a culture medium not containing sodium chloride is 100%. The concentration at which the growth rate of fructose-producing microorganisms in the medium is 80% or less, preferably 50% or less, more preferably 20% or less, even more preferably 1% or less, most preferably The concentration may be as close as possible to 0%.

  The “concentration at which auranthiochytrium microorganisms can grow and inhibit the growth of fructose-producing microorganisms” is not particularly limited, but is 1 to 4% (w / v), preferably 1.5 to 3%. (W / v), more preferably 2 to 2.5% (w / v). If the final concentration of sodium chloride added to the culture medium is within the above range, the growth of fructose-producing microorganisms can be inhibited while growing the Aurantiochytrium microorganisms. As a result, high value-added lipids can be produced efficiently.

  The “sodium chloride” may be a simple substance of sodium chloride or a mixture of sodium chloride and another substance. Examples of the “mixture of sodium chloride and other substances” include sea salt, rock salt and the like. In seawater in which Auranthiochytrium microorganisms grow naturally, it is preferable to use sea salt because minerals other than sodium are also present.

  In addition, when adding a mixture of sodium chloride and another substance as the “sodium chloride”, it may be added so that the final concentration in the culture medium falls within the above range in terms of sodium chloride.

  Only one type of sea salt or rock salt may be used, or a plurality of types may be used in combination. When a plurality of types of sea salt, rock salt, or the like are used in combination, they may be added to the culture medium so that the total concentration of sodium chloride falls within the above range.

  Fructose-producing microorganisms such as Gluconobacter bacteria cannot grow in the presence of high-concentration salts, but auranthiochytrium microorganisms rather require seawater salt concentration or about half of that amount. In the method for producing high-value-added lipids of the present invention, by utilizing this difference in salt requirement, a sodium chloride addition step is included, thereby controlling the growth of fructose-producing microorganisms such as Gluconobacter bacteria and the like. It is possible to easily realize a complex culture system for growing Lantiochytrium microorganisms.

(1-3. Antibiotic addition step)
The method for producing high-value-added lipids of the present invention may further include an antibiotic addition step in which an antibiotic that acts on fructose-producing microorganisms is added to the culture medium. By adding an antibiotic that acts on the fructose-producing microorganism to the culture medium, the growth of the fructose-producing microorganism can be inhibited. On the other hand, Aulanthiochytrium microorganisms are resistant to antibiotics. For this reason, in the method for producing high-value-added lipids of the present invention, the growth of fructose-producing microorganisms such as Gluconobacter bacteria is further achieved by further including an antibiotic addition step utilizing the difference in the activity of the antibiotics. It is possible to easily realize a complex culture system in which an aurantiochytrium microorganism is grown while controlling the above.

  Here, the “antibiotic acting on the fructose-producing microorganism” is not particularly limited as long as it acts on the fructose-producing microorganism. For example, aminoglycoside antibiotics such as streptomycin, kanamycin, gentamicin; penicillin, ampicillin And β-lactam antibiotics. As said antibiotic, only 1 type may be used and you may use in combination of multiple types. However, antibiotics that act on Aulanthiochytrium microorganisms cannot be used in excess of the working concentration.

  The concentration of the antibiotic added to the culture medium is not particularly limited as long as it is a concentration that inhibits the growth of fructose-producing microorganisms, and can be appropriately set depending on the type of antibiotic used. The above-mentioned “concentration inhibiting the growth of fructose-producing microorganisms” refers to a culture containing the antibiotic at the concentration when the growth rate of fructose-producing microorganisms in a culture medium not containing the antibiotic is 100%. The concentration at which the growth rate of fructose-producing microorganisms in the medium is 80% or less, preferably 50% or less, more preferably 20% or less, even more preferably 1% or less, most preferably The concentration may be as close as possible to 0%.

  The antibiotic addition step is performed when the conversion rate of mannitol into fructose in the culture medium becomes 50% or more. The “conversion rate of mannitol to fructose” is as described in the above section “2. Sodium chloride addition step”.

  The antibiotic addition step may be performed before the sodium chloride addition step, may be performed after the sodium chloride addition step, or may be performed simultaneously with the sodium chloride addition step.

(1-4. Other steps)
The method for producing high-value-added lipids of the present invention may further include steps other than those described above.

(I) Pre-culture step The high-value-added lipid production method of the present invention may include a pre-culture step in which a fructose-producing microorganism is pre-cultured in a culture medium containing at least mannitol and a nitrogen source. .

  The culture medium used in the pre-culture process is not particularly limited as long as it contains at least mannitol and a nitrogen source, but is preferably a medium having the same composition as the culture medium used in the culture process. Moreover, it is preferable that the said culture medium used at a pre-culture process does not contain glucose as a carbon source. As a result, it is expected that an enzyme that converts mannitol to fructose will be produced more in fructose-producing microorganisms, and mannitol can be more efficiently converted to fructose by the fructose-producing microorganisms in the culture process. .

  The culture conditions of the fructose-producing microorganism in the pre-culture step are not particularly limited. For example, in a mannitol-PY medium (30 g / L mannitol, 10 g / L polypeptone, 5 g / L yeast extract (pH 6.0)), the culture condition is 28 ° C. Incubate for 24 hours with shaking.

(Ii) High-value-added lipid recovery step The high-value-added lipid production method of the present invention may further include a high-value-added lipid recovery step for recovering the high-value-added lipid. In the high-value-added lipid recovery step, (a) high-value-added lipid may be taken out from the cells of the Aulanthiochytrium microorganism, and (b) the high-value-added lipid is taken into the cells of the Aulanthiochytrium microorganism. You may collect | recover in the accumulated state.

  In the case of (a) above, the method for recovering high-value-added lipids is not particularly limited as long as the high-value-added lipids can be taken out of the cells of the Aulanthiochytrium microorganism. For example, fatty acids can be extracted from auranthiochytrium microorganisms by using hexane or chloroform / methanol mixture (2: 1, v / v). The extracted fatty acid may then be esterified. In addition, for example, carotenoids can be extracted from an auranthiochytrium microorganism by using an acetone / methanol mixture (7: 3, v / v). In addition, for example, squalene can be extracted from an auranthiochytrium microorganism by using hexane or chloroform / methanol mixture (2: 1, v / v).

  In the case of (b) above, the recovery method is not particularly limited as long as the high-value-added lipid can be recovered in a state where it is accumulated in the cells of the Aulanthiochytrium microorganism. For example, high added-value lipid-containing cells can be recovered by centrifuging a culture solution in which an aurantiochytrium microorganism is cultured and discarding the culture supernatant.

(Iii) High-value-added lipid purification step The high-value-added lipid production method of the present invention may further include a step of purifying the high-value-added lipid. Although it does not specifically limit as a method to refine | purify a high value-added lipid, For example, a high value-added lipid can be refine | purified by methods, such as a solvent fractionation, supercritical fluid extraction, and adsorption chromatography.

[2. High value-added lipid)
The high-value-added lipid produced by the present invention includes food, cosmetics and pharmaceutical manufacturing fields that use high-value-added lipids as raw materials; industrial fields that use hydrocarbons (terpenes) as fuel; high-value-added lipid-containing cells Can be used in the fishery field and the livestock field, in which

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by an Example.

<Medium>
Gluconobacter medium: 40 g / L mannitol, 5 g / L yeast extract, 2.5 g / L magnesium sulfate heptahydrate, 1 g / L ammonium sulfate, 1 g / L monopotassium dihydrogen phosphate Mannitol medium (mannitol-PY Medium): 30 g / L mannitol, 10 g / L polypeptone, 5 g / L yeast extract (pH 6.0)
3% glucose-polypeptone-yeast extract medium (3% GPY medium): 30 g / L glucose, 6 g / L polypeptone, 2 g / L yeast extract, 20 g / L sea salt, 20 g / L agar (however, agar is a solid medium) Add only if
Kombu extract-PY medium: 10% (w / v) kombu extract, 10 g / L polypeptone, 5 g / L yeast extract, pH 6.0
Polypeptone-yeast extract medium (PY medium): 6 g / L polypeptone, 2 g / L yeast extract, 20 g / L sea salt 1% glucose-polypeptone-yeast extract medium (1% GPY medium): 10 g / L glucose, 6 g / L polypeptone, 2 g / L yeast extract, 20 g / L sea salt ・ Concentrated PY medium: 0.6 g polypeptone, 0.2 g yeast extract, 2 g sea salt, 70 ml distilled water, pH 6.0, 1 ml Antibiotic: antifungal agent Mixture The mannitol added to the above medium uses 21303-32 (product number) manufactured by Nacalai Tesque, the polypeptone uses 392-02115 (product number) manufactured by Wako Pure Chemical, and the yeast extract is Kyokuto Pharmaceutical. 551-01310-8 (product number) manufactured by Sigma Aldrich, S9883 (product number) manufactured by Sigma Aldrich, Uses Nacalai Tesque of 16805-64 (product number), antibiotics: anti-fungal agent mixture was used Nakarai Tesque 09366-44 (the number).

  If all of the sodium contained in the sea salt is present as a chloride, the sodium chloride contained in 1 g of sea salt is 0.72 g.

  Further, the kombu extract added to the above medium was described in Khomtimchenko, SV & Kulikova, IV 1999. Lipids of two species of brown algae of the genus Laminaria. Chem Nat Prod, 35 (1), 17-20. Prepared according to a method modification. Specifically, dry comb powder was added to distilled water so as to have a concentration of 10% (w / v), stirred for 20 minutes, and then centrifuged to collect the supernatant. The mannitol concentration of the extract was measured using a high performance liquid chromatography (HPLC) apparatus. Dry comb powder was prepared by pulverizing macomb after air drying.

<Microorganism>
Gluconobacter oxydans (NBRC 14819) obtained from Biological Resource Center, NITE (NBRC) was used as a bacterium belonging to the genus Gluconobacter. G. oxydans (NBRC 14819) was precultured in mannitol-PY medium at 28 ° C. for 24 hours with shaking before the test culture.

  Aurantiochytrium sp. KH105 strain (reception number: FERM AP-22267) was used as a microorganism belonging to the genus Aurantiochytrium. Aurantiochytrium sp. KH105 was pre-cultured in 3% GPY medium at 28 ° C. with shaking for 2 days before the test culture.

[Test Example 1]
(A) of FIG. 1 is a figure which shows the component of the saccharide | sugar and monosaccharide specific to a brown alga algae. Twelve kinds of saccharides derived from the brown alga algae shown in FIG. 1A were added to the PY medium so that the final concentration was 1% (w / v). The fermentability of Aurantiochytrium sp. KH105 was evaluated by measuring the absorbance at 600 nm (OD600) after shaking culture at 28 ° C. for 4 days. The test was performed in triplicate test tubes. Absorbance was measured using an absorptiometer (CO7500, manufactured by Biochrome).

  The results are shown in FIG. FIG. 1B is a graph showing the ability to assimilate each saccharide of Aurantiochytrium sp. KH105. Aurantiochytrium sp. KH105 grew well in a medium containing 4 types of monosaccharides including glucose among 12 types of saccharides derived from brown algae. In contrast, when Aurantiochytrium sp. KH105 was cultured in media containing the polysaccharides alginate, fucoidan and laminaran, respectively, when cultured in a PY medium containing no saccharide (negative) There was no significant difference in absorbance compared to the control. Further, Aurantiochytrium sp. KH105 showed lower absorbance when cultured in a medium containing glucuronate. In contrast, when Aurantiochytrium sp. KH105 was cultured in a medium containing fructose, the absorbance was significantly higher than when cultured in a medium containing glucose.

[Test Example 2]
Aurantiochytrium sp. KH105 was grown in the culture supernatant of G. oxydans (NBRC 14819) (concentrated PY medium added).

First, G. oxydans (NBRC 14819) was cultured in 175 ml of Gluconobacter medium using a 500 ml baffled flask. The growth of G. oxydans (NBRC 14819) was measured by monitoring the change in absorbance (OD600) of the culture supernatant, and saccharide consumption and saccharide production using a high performance liquid chromatography (HPLC) apparatus. Was measured. Specifically, the saccharide concentration was determined by using HPLC equipped with a Sugar-D Cosmosil column (Nacalai Tesque, Kyoto, Japan), setting the oven temperature to 40 ° C and the detector temperature to 39 ° C. Performed with acetonitrile: H ₂ O (75:25, v / v) set to a flow rate of 1.0 ml / min. Maltose was used as an internal standard for quantification.

  Collect 4.5 ml of culture supernatant at different culture times, add the collected culture supernatant to 10.5 ml of concentrated PY medium, mix them, and dispense them in equal amounts into three test tubes. did. Thereafter, Aurantiochytrium sp. KH105 was inoculated. Aurantiochytrium sp. KH105 was incubated at 28 ° C. and 140 rpm for 4 days (96 hours), and then the absorbance (OD600) was measured to measure the growth of Aurantiochytrium sp. KH105.

  The results are shown in FIG. FIG. 2A is a graph showing the growth of G. oxydans (NBRC 14819), the amount of saccharide consumed and the amount of saccharide produced in a culture medium containing mannitol. FIG. 2 (b) is a graph showing the growth of Aurantiochytrium sp. KH105 in the culture supernatant of G. oxydans (NBRC 14819) collected at different time points.

  After 18 hours of culture, mannitol (initial concentration: 32.0 g / L) was completely consumed, and the fructose concentration reached a maximum of 28.0 g / L ((a) in FIG. 2). This indicates that the conversion rate of mannitol to fructose by G. oxydans (NBRC 14819) is 87.5%.

  Aurantiochytrium sp. KH105 grew well in the culture supernatant after 9 hours of culture. The fructose concentration in the culture supernatant at this time was 20.5 g / L (concentration before adding the concentrated PY medium and diluting it twice). Aurantiochytrium sp. KH105 in culture supernatant after 9 hours culture, culture supernatant after 12 hours culture, culture supernatant after 15 hours culture, culture supernatant after 18 hours culture, and culture supernatant after 24 hours culture The final absorbance was substantially the same ((b) of FIG. 2).

[Test Example 3]
G. oxydans (NBRC 14819) is cultured in Gluconobacter medium containing mannitol at different concentrations (40 g / L, 80 g / L, 120 g / L, 160 g / L) to influence the growth rate. Examined. Using three flasks with baffles, precultured G. oxydans (NBRC 14819) was inoculated into 50 ml of the above medium, and then G. oxydans (NBRC 14819) was incubated at 28 ° C. and 140 rpm.

  The results are shown in FIG. FIG. 3 (a) is a graph showing the absorbance (OD600) when G. oxydans (NBRC 14819) is grown in Gluconobacter medium containing different concentrations of mannitol. c) is a graph showing the change in pH of the medium at that time. The error bar described in the graph of FIG. 3A represents the standard deviation of three repeated tests.

  Increasing the concentration of mannitol did not increase the growth rate of G. oxydans (NBRC 14819), and in fact, the culture time required for the growth of G. oxydans (NBRC 14819) to reach stationary phase increased ( (A) of FIG. It is speculated that mannitol was completely consumed when growth reached stationary phase.

  Regardless of the concentration of mannitol in the medium, the pH of the medium decreased to the same extent according to the culture period ((c) of FIG. 3). This suggests that the amount of acetic acid produced by G. oxydans (NBRC 14819) is probably the same regardless of the concentration of mannitol in the medium.

  Furthermore, various concentrations of sea salt were added to mannitol medium (containing 40 g / L mannitol, 10 g / L polypeptone, and 5 g / L yeast extract), and the influence on the growth of G. oxydans (NBRC 14819) was examined.

  The results are shown in FIG. FIG. 3 (b) is a graph showing the influence of sea salt concentration on the growth of G. oxydans (NBRC 14819), and FIG. 3 (d) is a graph showing the change in pH of the medium at that time. The error bar described in the graph of FIG. 3B represents the standard deviation of three repeated tests.

  Sea salt inhibited the growth of G. oxydans (NBRC 14819), and it became clear that the effect became strong when the concentration of sea salt increased (FIG. 3 (b)). The change in pH in the medium showed the same tendency, but the pH in the medium hardly changed when the concentration of sea salt was 1.8% (w / v) (FIG. 3 (d)). ). This suggests that the growth rate of G. oxydans (NBRC 14819) decreases when the concentration of sea salt is 1.8% (w / v).

[Test Example 4]
50 ml of mannitol-PY medium or kombu extract-PY medium was put in a flask with a baffle, G. oxydans (NBRC 14819) was inoculated, and cultured at 28 ° C. and 140 rpm for 12 hours. As a control, PY medium not inoculated with G. oxydans (NBRC 14819) and 1% GPY medium were incubated. The process so far is referred to as “stage 1”.

  After stage 1, 4% (w / v) sea salt solution was added to an equal volume of each culture. After thorough mixing, the pH of the mixture was adjusted to 6-7. In order to perform a test on a test tube scale, the culture supernatant of each culture solution (centrifuged medium from which G. oxydans (NBRC 14819) was removed) was transferred to a test tube. Moreover, in order to perform a flask-scale test, each culture solution (medium containing G. oxydans (NBRC 14819)) was transferred to a flask. Aurantiochytrium sp. KH105, which had been precultured and washed, was inoculated into these media and cultured for 4 days while shaking at 28 ° C. and 140 rpm. The process so far is referred to as “stage 2”.

  Absorbance at 600 nm (OD600) was measured after culture. On the other hand, the medium was sampled over the entire culture period in order to monitor changes in sugar concentration. Flask scale tests confirmed the production of fatty acids and carotenoids.

  The test tube scale test results are shown in Table 1. Table 1 shows the test in a test tube scale when Aurantiochytrium sp. KH105 was cultured using the culture supernatant after culturing G. oxydans (NBRC 14819) in mannitol-PY medium or kombu extract-PY medium. Shows the growth of Aurantiochytrium sp. KH105 and the consumption of sugars.

また、フラスコスケール試験の結果を表２に示す。表２は、マンニトール−ＰＹ培地またはコンブ抽出物−ＰＹ培地中でG. oxydans（NBRC 14819）を培養した後の培養液を用いてAurantiochytrium sp. KH105を培養した場合の、フラスコスケールの試験におけるAurantiochytrium sp. KH105の増殖および糖類の消費量を示している。誤差は、２回の反復試験の値に基づいている。

The results of the flask scale test are shown in Table 2. Table 2 shows the Aurantiochytrium in a flask scale test when Aurantiochytrium sp. KH105 was cultured using a culture solution after culturing G. oxydans (NBRC 14819) in mannitol-PY medium or kombu extract-PY medium. It shows the growth of sp. KH105 and the consumption of sugars. The error is based on the value of two replicate tests.

ステージ１では、マンニトール−ＰＹ培地を用いた場合には、G. oxydans（NBRC 14819）によるマンニトールの消費率は、試験管スケールの試験では７３．８％であり、フラスコスケール試験では７６．９％であり、同程度の消費率でマンニトールを消費した（表１および２）。一方、コンブ抽出物−ＰＹ培地を用いた場合は、G. oxydans（NBRC 14819）によるマンニトールの消費率は、試験管スケールの試験では６７．８％であったのに対して、フラスコスケールの試験では８６．４％と、わずかに高かった。また、マンニトール−ＰＹ培地を用いた場合には、G. oxydans（NBRC 14819）によってマンニトールがほぼ完全に消費されたのに対して、コンブ抽出物−ＰＹ培地を用いた場合には、消費されていないマンニトールが残存していた（表１および２）。

In stage 1, when mannitol-PY medium is used, the mannitol consumption rate by G. oxydans (NBRC 14819) is 73.8% in the test tube scale test and 76.9% in the flask scale test. Mannitol was consumed at a similar consumption rate (Tables 1 and 2). On the other hand, when the kombu extract-PY medium was used, the consumption rate of mannitol by G. oxydans (NBRC 14819) was 67.8% in the test on the test tube scale, whereas the test on the flask scale. Then it was slightly higher at 86.4%. In addition, when mannitol-PY medium is used, mannitol is almost completely consumed by G. oxydans (NBRC 14819), whereas when using kombu extract-PY medium, it is consumed. No mannitol remained (Tables 1 and 2).

  On the other hand, in stage 2 inoculated with Aurantiochytrium sp. KH105, fructose consumed by Aurantiochytrium sp. KH105 after 4 days of culturing in a test tube scale test was 76.6 when mannitol-PY medium was used. % Compared to 96.9% when using Kombu extract-PY medium, and more fructose was consumed (Tables 1 and 2).

  In flask-scale tests, this effect was not observed, and the fructose was completely removed by Aurantiochytrium sp. KH105 after 4 days incubation, both with mannitol-PY medium and with kombu extract-PY medium. Was consumed. This result shows that certain components in the kombu extract medium negatively affect the conversion efficiency of G. oxydans (NBRC 14819) from mannitol to fructose, whereas Aurantiochytrium sp. KH105 promotes growth It was considered possible.

  The final absorbance (OD600) was measured after 4 days of incubation. In the flask scale test, the absorbance was higher than in the test tube scale test. The final absorbance of Aurantiochytrium sp. KH105 was higher (flask) compared to the positive control (1% GPY medium) both when using mannitol-PY medium and when using kombu extract-PY medium. In the scale test, the final absorbance at 600 nm (OD600) was 3.63 for the positive control.)

  However, in the flask scale test, the final absorbance when using mannitol-PY medium is 4.92, and the final absorbance when using kombu extract-PY medium is 5.09, The final absorbance of Aurantiochytrium sp. KH105 was comparable. As a result of HPLC analysis, fructose in the medium was completely consumed in the flask-scale test (Table 2). In contrast, in a test on a test tube scale, fructose remained in the medium (Table 1).

[Test Example 5]
In the flask scale test, the difference in the productivity of fatty acids and carotenoids in Aurantiochytrium sp. KH105 was confirmed when different media (mannitol-PY media and kombu extract-PY media) were used.

  The culture solution of Aurantiochytrium sp. KH105 obtained in Test Example 4 was centrifuged and the supernatant was discarded. Aurantiochytrium sp. KH105 was recovered, and the cells were washed once with distilled water.

Total fatty acids (TFA) were extracted from the collected and washed cells using a chloroform / methanol mixture (2: 1, v / v), esterified with 10% hydrochloric acid diluted with methanol, and then nitrogen (N ₂ ) Dried under gas. This was dissolved in hexane and injected into a gas chromatograph (GC-17A; Shimadzu, Kyoto, Japan). Arachidic acid (20: 0) was used as an internal standard. The gas chromatograph is equipped with a split injector, a capillary column set at 190 ° C (TC-70, 0.25 mm x 30 m; GL Science, Tokyo, Japan), and a flame ionization detector set at 270 ° C. It was used.

  Carotenoids were extracted several times using an acetone / methanol mixture (7: 3, v / v), concentrated, and then dissolved in acetone. Extracts were spotted on silica gel plates (Kieselgel 60; Merck Chemicals, Darmstadt, Germany), developed with acetone: hexane (3: 7, v / v) and quantified by densitographic analysis. Β-carotene and astaxanthin were spotted as standard substances.

  The results are shown in FIG. (A) of FIG. 4 shows a flask scale when Aurantiochytrium sp. KH105 is cultured using a culture solution after culturing G. oxydans (NBRC 14819) in mannitol-PY medium or kombu extract-PY medium. FIG. 4B is a graph showing the amount of carotenoid produced. FIG. 4B is a graph showing the amount of fatty acid produced by Aurantiochytrium sp.

  The absorbance and total fatty acid amount when using the kombu extract-PY medium were lower than the absorbance and total fatty acid amount when using the mannitol-PY medium ((a) of FIG. 4). From Aurantiochytrium sp. KH105 cultured in mannitol-PY medium, 22.7 mg / L of fatty acid was extracted. On the other hand, 14.2 mg / L fatty acid was extracted from Aurantiochytrium sp. KH105 cultured in Kombu extract-PY medium. In the positive control (1% GPY medium), the total amount of lipid production was the same as that in the negative control (PY medium) ((a) of FIG. 4).

  Among polyunsaturated fatty acids, eicosapentaenoic acid (20: 5n-3) production (about 10% of TFA) is docosapentaenoic acid (22: 5n-3) production (about 20% of TFA). ). The production amount of docosahexaenoic acid (22: 6n-3) was about 40% of TFA, which was the highest. The production amounts of DHA and EPA in mannitol-PY medium were 9.4 mg / L and 2.4 mg / L, respectively. In contrast, the production amounts of DHA and EPA in the kombu extract-PY medium were 5.9 mg / L and 1.8 mg / L, respectively ((a) of FIG. 4).

  Carotenoid production was measured by thin layer chromatography. When the kombu extract-PY medium was used, the production amount of carotenoid was the highest, 15.9 mg / L. This was more than 3 times the amount of carotenoid production (4.5 mg / L) in the positive control (1% GPY medium). The amount of carotenoid produced using mannitol-PY medium was 9.1 mg / L.

  The composition analysis of carotenoids revealed that the composition was different between the positive control (1% GPY medium) and the mannitol-containing medium (mannitol-PY medium, kombu extract-PY medium). The proportion of β-carotene was similar (about 15%), but in the positive control (1% GPY medium), the proportion of astaxanthin was the highest (63%) and the proportion of canthaxanthin was the lowest (9. 7%). In contrast, when mannitol-PY medium or kombu extract-PY medium was used, the ratio of astaxanthin was small (26% and 12% of the total carotenoids, respectively). When the kombu extract-PY medium was used, canthaxanthin was 32% of the total carotenoid and astaxanthin was 12% of the total carotenoid. When mannitol-PY medium was used, the production amounts of canthaxanthin and astaxanthin were almost the same (22% and 26%, respectively).

  When mannitol-PY medium was used, production amounts of astaxanthin and β-carotene were 2.3 mg / L and 1.4 mg / L, respectively. When the kombu extract-PY medium was used, the production amounts of astaxanthin and β-carotene were 2.0 mg / L and 2.5 mg / L, respectively.

[Test Example 6]
Two-stage fermentation on a jar fermenter scale was performed using a kombu extract-PY medium. According to the same experimental system as the test of the test tube scale described in Test Example 4, a 10 L capacity jar fermenter (BE Marubishi, MDL-10L) was used to add G. oxydans (NBRC) to 3 L of kombu extract-PY medium. 14819) was then inoculated, and then supplied at a rate of 5 Sl / min at 28 ° C. and 800 rpm while controlling the pH to 6.0 and cultured for 12 hours (stage 1). Thereafter, 3 L of 4% sea salt solution was added so that the final sea salt concentration was 2%. Therefore, the sugar concentration was diluted in half. Thereafter, Aurantiochytrium sp. KH105 was inoculated, and further cultured for 7 days (168 hours) while stirring at a rate slower than 300 rpm (stage 2).

  The culture solution was sampled at different culture times, and changes in absorbance, saccharide concentration, and fatty acid and carotenoid concentrations were confirmed.

  During the jar fermenter culture, absorbance and cell dry weight (CDW) were measured to monitor the growth of both G. oxydans (NBRC 14819) and Aurantiochytrium sp. KH105. In addition, sugar consumption was monitored. The kombu extract-PY medium contained 24.2 g / L mannitol before inoculation (after 0 hour culture) of G. oxydans (NBRC 14819).

  The results are shown in FIG. FIG. 5 (a) is a graph showing changes in absorbance, dry weight of cells, mannitol concentration and fructose concentration when two-stage fermentation is performed on a jar fermenter scale using a kombu extract-PY medium. FIG. 5B is a graph showing changes in fatty acid concentration, and FIG. 5C is a graph showing changes in carotenoid concentration.

  In stage 1, the absorbance reached the maximum value (2.85) 12 hours after the start of cultivation of G. oxydans (NBRC 14819). At this time, 20.9 g / L of fructose was produced.

In stage 2, the absorbance and the dry weight of the cells reached the maximum 120 hours after starting the cultivation of G. oxydans (NBRC 14819) (108 hours after starting the cultivation of Aurantiochytrium sp. KH105) ( (9.4 and 4.3 g / L, respectively) ((a) of FIG. 5).
From this point on, the absorbance and dry weight of the cells decreased sharply until the end of the culture. This coincided with the result of disappearance (0 g / L) of mannitol and fructose 132 hours after the start of cultivation of G. oxydans (NBRC 14819).

  The production of fatty acids and carotenoids was measured. Before inoculation (after 0 hour culture) of G. oxydans (NBRC 14819), the total fatty acid contained in the medium was 41.1 mg / L. Analysis of the fatty acid composition at this point revealed that oleic acid (OA, 18: 1n-9), linoleic acid (LA, 18: 2n-6), dihomo-γ-linolenic acid (DGLA, 20: 3n-6) and Unsaturated fatty acids such as EPA (20: 5n-3) account for the majority of fatty acids, 35.8%, 8.5%, 25.8% and 6.2% of TFA, respectively. . These fatty acids were presumed to be derived from kombu.

  The total fatty acid amount (TFA) increased from the start of the culture, and reached the maximum value (176.1 mg / L) at the end of stage 1 ((b) of FIG. 5).

  In stage 2, TFA reached its maximum value (118.1 mg / L) after 132 hours (120 hours after starting cultivation of Aurantiochytrium sp. KH105) after the start of cultivation of G. oxydans (NBRC 14819). After that, it reached a minimum value (20.9 mg / L) 180 hours after culturing G. oxydans (NBRC 14819) (168 hours after culturing Aurantiochytrium sp. KH105).

  In stage 1, α-linolenic acid (ALA, 18: 3n-3) increased from 0.8 mg / L to 1.4 mg / L and DGLA increased from 10.5 mg / L to 25.9 mg / L . However, α-linolenic acid and DGLA decreased after inoculation with Aurantiochytrium sp. KH105, and then disappeared completely.

  EPA is present at 0 hours in culture (1.4 mg / L). However, it increased rapidly in stage 2, and after 60 hours (48 hours after starting cultivation of Aurantiochytrium sp. KH105) after the start of cultivation of G. oxydans (NBRC 14819), the maximum value (22.2 mg / L) ((b) of FIG. 5).

  DPA and DHA began to accumulate in stage 2. DPA and DHA reached their maximum values 60 hours after the start of cultivation of G. oxydans (NBRC 14819) (48 hours after the start of cultivation of Aurantiochytrium sp. KH105). The maximum value of DPA produced was 14.9 mg / L and the maximum value of DHA produced was 53.7 mg / L.

  The total amount of carotenoid was 10.2 mg / L at 0 hours of culture. This comes from astaxanthin and two unknown spots in TLC (Rf = 0.55 and 0.51).

  Astaxanthin was also present before inoculating G. oxydans (NBRC 14819), but was temporarily lost by inoculation of G. oxydans (NBRC 14819). Two unknown spots were also present during stage 1, but disappeared in stage 2 to which Aurantiochytrium sp. KH105 was added.

  Total carotenoid production reached a maximum value (95.4 mg / L) 156 hours after starting cultivation of G. oxydans (NBRC 14819) (144 hours after starting cultivation of Aurantiochytrium sp. KH105) did. Astaxanthin reaches its maximum concentration (18.0 mg / L) 132 hours after the start of cultivation of G. oxydans (NBRC 14819) (120 hours after the start of cultivation of Aurantiochytrium sp. KH105), and β-carotene is , 156 hours after the start of cultivation of G. oxydans (NBRC 14819) (144 hours after the start of cultivation of Aurantiochytrium sp. KH105), the maximum concentration (12.6 mg / L) was reached (FIG. 5 ( c)).

  According to the present invention, high-value-added lipids can be produced using mannitol as a carbon source. The high-value-added lipid produced by the present invention includes food, cosmetics and pharmaceutical manufacturing fields that use high-value-added lipids as raw materials; industrial fields that use hydrocarbons (terpenes) as fuel; high-value-added lipid-containing cells Can be used in the fishery field and the livestock field, in which

Claims (11)

(A) inoculating and culturing a fructose-producing microorganism and an aurantiochytrium microorganism in a culture medium containing at least mannitol and a nitrogen source, or (b) the fructose-producing microorganism in the culture medium Inoculating and culturing, and then inoculating and culturing the Aurantiochytrium microorganism,
Adding sodium chloride to the culture medium, adding sodium chloride,
Including
The fructose-producing microorganism is a microorganism that can convert mannitol into fructose and secrete the fructose out of the cell,
The sodium chloride addition step is performed when the conversion rate of the mannitol in the culture medium to fructose is 50% or more,
The sodium chloride is added to the culture medium such that the final concentration in the culture medium is a concentration at which the Aulanthiochytrium microorganism can grow and a concentration that inhibits the growth of the fructose-producing microorganism. A method for producing high-value-added lipids.   The method for producing a high added-value lipid according to claim 1, wherein the fructose-producing microorganism is a Gluconobacter bacterium.   The sodium chloride is added to the culture medium in the sodium chloride addition step so that the final concentration in the culture medium is 1 to 4% (w / v). A method for producing the described high-value-added lipid. An antibiotic addition step of adding an antibiotic that acts on the fructose-producing microorganism to the culture medium;
The said antibiotic addition process is performed when the conversion rate to the fructose of the said mannitol in the said culture medium will be 50% or more, The any one of Claim 1 to 3 characterized by the above-mentioned. High-value-added lipid production method.   5. The fructose-producing microorganism that has been pre-cultured in a culture medium containing at least mannitol and a nitrogen source is inoculated in the culture medium in the culture step. A method for producing high-value-added lipids as described in 1.   The method for producing a high added-value lipid according to any one of claims 1 to 5, wherein the culture medium contains only mannitol as a carbon source.   The method for producing a high added-value lipid according to any one of claims 1 to 6, wherein the culture medium contains a brown algae extract.   The method for producing a high value-added lipid according to claim 7, wherein the brown alga algae is a Compositae algae. In the case where the culture step is (b),
The method for producing a high added-value lipid according to claim 1, wherein the auranthiochytrium microorganism is inoculated into the culture medium after the sodium chloride addition step. In the case where the culture step is (b),
The method for producing a high added-value lipid according to any one of claims 1 to 9, wherein the auranthiochytrium microorganism is inoculated and cultured in a culture supernatant of the fructose-producing microorganism.   The high addition according to any one of claims 1 to 10, wherein the microorganism belonging to the genus Aurantiochytrium is Aurantiochytrium (Aurantiochytrium sp. KH105, receipt number: FERM AP-22267). Production method of value lipid.

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