Medium for heterotrophic microorganisms or cultured cells, method for culturing heterotrophic microorganisms or cultured cells, method for producing oil or fat, and additive for medium

Abstract

To provide a novel medium in which heterotrophic microorganisms, especially those capable of producing oil or fat, can be grown, and a method for culturing heterotrophic microorganisms using the medium.SOLUTION: A medium for heterotrophic microorganisms or cultured cells is prepared that contains a microalgal extract obtained by protease and lipase treatment of microalgae, and a carbon source required for growth. Further provided is a method of producing oil or fat that includes: a step of culturing, in the medium, a heterotrophic microorganism having oil-producing capability; and a step of extracting oil or fat from the heterotrophic microorganism cultured therein.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to a medium for heterotrophic microorganisms or cultured cells, a method for culturing heterotrophic microorganisms or cultured cells, a method for producing fats and oils, and an additive for a medium for heterotrophic microorganisms or cultured cells. [Background technology]

[0002] Biofuels are a type of renewable energy produced from biological resources and are attracting attention as an alternative energy source to fossil fuels. The development of practical biofuel production technologies is one of the important efforts toward reducing greenhouse gas emissions and ensuring a sustainable energy supply. In particular, the development of sustainable aviation fuel (SAF) is expected to reduce the environmental impact of the aviation industry. Recently, biological processes using microalgae and oleaginous yeast (oleaginous yeast) have been investigated as a method for producing SAF. In this method, the oils produced by these microorganisms are converted into hydrocarbons that can be used as SAF through extraction and purification processes.

[0003] In addition to microorganisms, terrestrial plants, primarily palm trees, can also be used as raw materials for biofuels. However, palm oil plantation agriculture places a heavy burden on the environment, such as ecosystem destruction and soil degradation, and is fraught with issues related to working conditions and human rights. On the other hand, the advantages of biofuel production using microalgae and oleaginous yeast include (1) the ability to utilize abandoned farmland and thus not competing with food production, and (2) a short growth cycle and high biomass production capacity per unit area. Microalgae, in particular, are capable of carbon dioxide fixation through photosynthesis, raising hopes for carbon-neutral oil production. However, the amount of oil produced by microalgae is currently limited, and the high cost of oil production prevents practical application. Research is also being conducted on genetically modified microalgae with enhanced oil production (see Patent Document 1), but given legal restrictions on the use of genetically modified organisms, outdoor mass cultivation of genetically modified microalgae is extremely difficult. Furthermore, research has been conducted into producing triacylglycerol by culturing algae under a specific light irradiation environment (see Patent Document 2), but this requires irradiation with light of a specific wavelength.

[0004] In this context, oleaginous yeast has a higher oil-producing capacity than microalgae and a fatty acid composition similar to that of vegetable oils, and is therefore expected to be a substitute for edible oils. Technologies for improving oil-producing capacity have also been developed; for example, a method for improving oil-producing capacity by culturing oleaginous yeast at a predetermined sugar concentration (see Patent Document 3) has been disclosed. However, because oleaginous yeast is a heterotrophic organism, it must be supplied with the necessary nutrients. This requires the preparation of a nutrient-rich medium containing sugars such as glucose, protein digests such as peptone and tryptone, yeast extract, etc., which has led to a bottleneck in that the culture costs are high.

[0005] Regarding microalgae, techniques have been developed in which a target substance is produced in the microalgae and then the microalgae are treated to extract the target substance. For example, a method for producing a target peptide by culturing microalgae and then treating them with a protease to extract peptides into an aqueous solution has been disclosed (see Patent Document 4), and a method for producing a marine microalgae extract for use in food production by extracting marine microalgae with alcohols, sugars, sugar alcohols, etc. has been disclosed (see Patent Document 5).

[0006] Furthermore, a method has been disclosed in which microalgae are treated with a protease to extract peptides into an aqueous solution, and then insoluble matter in the aqueous solution is removed to obtain peptides consisting of an L-amino acid sequence (see Patent Document 6).

[0007] In addition, a method has been disclosed in which algae are hydrolyzed with acids such as sulfuric acid and hydrochloric acid to extract algae components, which are then used as an additive to a microbial culture medium such as yeast extract (see Patent Document 7). However, sulfuric acid is added for hydrolysis, and then high-temperature heat treatment at 121°C is performed, which requires energy. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-135906 [Patent Document 2] Japanese Patent Application Publication No. 2016-029901 [Patent Document 3] Japanese Patent Application Laid-Open No. 2010-158219 [Patent Document 4] Japanese Patent Application Laid-Open No. 2007-295842 [Patent Document 5] Japanese Patent Application Publication No. 8-266243 [Patent Document 6] Japanese Patent Application Laid-Open No. 2007-297326 [Patent Document 7] Japanese Patent Application Laid-Open No. 2011-229439 Summary of the Invention [Problem to be solved by the invention]

[0009] In light of the above background, an object of the present invention is to provide a new culture medium capable of growing heterotrophic microorganisms and cultured cells, particularly heterotrophic microorganisms capable of producing fats and oils, without using high-temperature treatment or ultrasonic devices, and to provide a method for culturing heterotrophic microorganisms in such a culture medium. [Means for solving the problem]

[0010] In the course of intensive research to solve the above problems, the present inventors used a microalgae extract obtained by enzymatically treating Spirulina (Arthrospira), which is cultivated industrially as a model of microalgae, as a medium to culture Lipomyces starkeyi JCM 5995 as a model of oleaginous yeast. Sufficient cell growth was confirmed, and the present invention was completed.

[0011] That is, the present invention is as follows. [1] A culture medium for heterotrophic microorganisms or cultured cells, comprising a microalgae extract obtained by treating microalgae with protease and lipase, and a carbon source necessary for growth. [2] The medium described in [1] above, characterized in that the microalgae extract is a microalgae extract obtained by treating microalgae with a mixed enzyme containing protease and lipase. [3] The medium according to [2] above, wherein the mixed enzymes are enzymes derived from mammalian pancreatic juice. [4] The medium according to [1] or [2] above, wherein the heterotrophic microorganism is a heterotrophic microalga, yeast, or heterotrophic bacterium. [5] The medium according to [1] or [2] above, characterized in that the microalgae is spirulina. [6] The medium described in [1] or [2] above, wherein the carbon source is glucose. [7] A method for culturing heterotrophic microorganisms or cultured cells in the medium according to any one of [1] to [6] above. [8] A step of culturing a heterotrophic microorganism capable of producing fats and oils in the medium according to any one of [1] to [6] above; extracting fats and oils from the cultivated heterotrophic microorganisms capable of producing fats and oils; A method for producing fats and oils, comprising: [9] An additive for a culture medium for heterotrophic microorganisms or cultured cells, comprising a microalgae extract obtained by treating microalgae with a protease and a lipase. [Effects of the Invention]

[0012] The medium for culturing heterotrophic microorganisms of the present invention can be used for culturing various heterotrophic microorganisms, particularly for culturing yeast or heterotrophic microalgae. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of measuring total nitrogen (TN) and total organic carbon (TOC) in spirulina extract when pancreatin was added to final concentrations of 0, 0.25, 0.5, 1, 2, and 4 mg / mL in Example 1. [Figure 2] FIG. 2 shows the results of measuring TN and TOC in Example 2 when the reaction time was set to 0, 0.5, 1, 2, 4, 8, and 24 hours. [Figure 3] FIG. 3 shows the results of measuring TN and TOC in Example 3 when the reaction temperatures were 30, 40, 45, 50, 55, and 65°C. [Figure 4] FIG. 4 shows the results of Example 4, comparing TN and TOC in the control Spirulina extract (without), Spirulina extract SE (without), and YM medium (YM). [Figure 5] FIG. 5 shows the results of Example 5, in which glucose was added to Spirulina extract SE to final concentrations of 5, 10, 20, 40, and 50 mg / mL, and the growth of Lipomyces starchii was evaluated. [Figure 6]FIG. 6 shows the results of evaluating the growth of Lipomyces starkeyi in Example 5 using a medium in which glucose was added to Spirulina extract SE to a final concentration of 20 mg / mL (SE+Glu20) or YM medium. [Figure 7] Figure 7 shows the results of analyzing the fatty acid composition of lipids obtained by culturing Lipomyces starchyi in Spirulina Extract SG or YM medium in Example 6, extracting fatty acid methyl esters (FAMEs) from the culture medium on the 7th day. [Figure 8] Figure 8 shows the results of measuring TN and TOC in Example 7, where microalgae extracts were prepared from cultures rather than dried algae, using Anabaena or Synechocystis instead of Spirulina, with and without the addition of pancreatin. [Figure 9] FIG. 9 shows the results of measuring TN and TOC for pepsin only, lipase only, pepsin and lipase (pepsin + lipase), and pancreatin in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0014] The medium for heterotrophic organisms of the present invention is not particularly limited as long as it is a medium for heterotrophic microorganisms or cultured cells, containing a microalgae extract obtained by treating microalgae with protease and lipase, and an assimilable carbon source; hereinafter, this medium is also referred to as "the medium of the present invention." Furthermore, the method for culturing heterotrophic microorganisms or cultured cells of the present invention is not particularly limited as long as it is a method for culturing heterotrophic microorganisms or cultured cells in the medium of the present invention; hereinafter, this method is also referred to as "the culture method of the present invention." Furthermore, the method for producing oils and fats of the present invention is not particularly limited as long as it comprises the steps of culturing heterotrophic microorganisms capable of producing oils and fats in the medium of the present invention and extracting oils and fats from the cultured heterotrophic microorganisms capable of producing oils and fats; hereinafter, this method is also referred to as "the method for producing oils and fats." Furthermore, the additive for the medium for heterotrophic microorganisms or cultured cells of the present invention is not particularly limited as long as it is an additive for the medium for heterotrophic microorganisms or cultured cells, containing a microalgae extract obtained by treating microalgae with protease and lipase; hereinafter, this method is also referred to as "the additive of the present invention." The medium may be a liquid medium, or a gel medium containing agar, agarose, gelatin, or the like.

[0015] ■Microalgae extract (microalgae) As used herein, "microalgae" refers to algae with a major axis of 1 μm to 1 mm per individual. Furthermore, the microalgae used to prepare the microalgae extract herein are preferably algae that do not produce substances toxic to the growth of heterotrophic microorganisms or cultured cells. Examples of such microalgae include blue-green algae (cyanobacteria), as well as algae belonging to the genera Chlorella, Scenedesmus, Microcystis, Anchistrodesmus, Gorenkinia, Selenastrum, Protosiphon, Chlorococcus, Gramidmonas, Haematococcus, Euglena, Botryococcus, Nannochloropsis, and Synechococcus. Examples of blue-green algae include Spirulina, Anabaena, and Synechocystis. Examples of substances toxic to growth include microcystin, nodularin, anatoxin, and saxitocine.

[0016] The microalgae used to prepare the microalgae extract can be cultured by known microalgae culture methods or purchased commercially. Commercially available microalgae may be dried or in the form of dried microalgae tablets. Furthermore, live or dried microalgae may be crushed in a mortar, ball mill, or the like before being treated with protease and lipase.

[0017] (Lipase and protease treatment) Microalgae extracts can be obtained by enzymatically treating microalgae with protease and lipase, and may also be further treated with amylase. These enzymatic treatments may be carried out using a mixed enzyme solution in which the enzymes are mixed at once, or may be carried out stepwise with each enzyme, but treatment with a mixed enzyme solution in which the enzymes are mixed at once is preferred.

[0018] The protease may be an endopeptidase or an exopeptidase, and the exopeptidase may be an aminopeptidase or a carboxypeptidase. Examples of the protease include serine proteases such as trypsin, chymotrypsin, and proteinase K (ProK), cysteine ​​proteases such as papain, metalloproteases such as thermolysin, aspartyl proteases such as pepsin, matrix metalloproteases (MMPs), and pronase, and mixtures of proteases may also be used in combination.

[0019] The lipase may be any enzyme that hydrolyzes the ester bond of lipids, and examples thereof include triacylglyceride lipase, phospholipase, lysophospholipase, lipoprotein lipase, monoglyceride lipase, and elastase, and these may also be used in combination.

[0020] Furthermore, when treating microalgae with protease and lipase, digestive enzymes including protease and lipase may be used. Examples of the digestive enzymes include digestive enzymes derived from the pancreas of edible animals such as pigs, cows, sheep, and goats, more preferably pancreatin and pancrelipase.

[0021] Pancreatin contains at least trypsin, chymotrypsin, elastase, hydroxylipase, and amylase.

[0022] When treating with protease and lipase, the microalgae may be treated with distilled water, a buffer solution, or the like containing protease and lipase.

[0023] The treatment temperature with the protease and lipase can be adjusted appropriately depending on the protease and lipase or other enzymes used, but can be 20 to 60°C, preferably 25 to 58°C, and the treatment time can be 3 to 48 hours, preferably 12 to 36 hours.

[0024] When pancreatin is used as the protease and lipase, the concentration of pancreatin in the enzyme-treated solution can be adjusted to 0.1 to 10 mg / mL, preferably 0.5 to 2 mg / mL.

[0025] The microalgae extract may be an extract obtained by treating microalgae with the protease and lipase. However, it is preferable to use the supernatant obtained by removing the microalgae bodies after the protease and lipase treatment by centrifugation, filtration, or the like. Centrifugation can be performed at 10,000 to 30,000 g for 5 to 20 minutes. Furthermore, the microalgae extract is preferably sterilized by autoclaving or the like before use as a culture medium. Since enzymes such as proteases and lipases are inactivated by autoclaving, the proteases and lipases do not act on the products of the heterotrophic microorganisms or cultured cells being cultured. Furthermore, the pH may be adjusted to 5 to 9, preferably 6 to 8, by adding an acid or alkali, as needed.

[0026] ■ Heterotrophic microorganisms or cultured cells As used herein, heterotrophic microorganisms include yeast, heterotrophic bacteria, and heterotrophic microalgae.

[0027] Examples of the yeast include oleaginous yeasts such as those of the genus Lipomyces, such as Lipomyces starchii, Cryptococcus, Rhodosporidium, Rhizopus, Yarrowia, Trichosporon, Candida, Rhodotorula, and Apiotrichum, as well as yeasts of the genus Saccharomyces, Pichia, and Kluyveromyces, and mutant strains thereof are also included.

[0028] Examples of the heterotrophic microorganisms include oil-producing bacteria such as Fusarium, Mucor, Aspergillus, Aspergillus, Humicola, and the like, and heterotrophic bacteria such as Bacillus, Lactobacillus, Paracoccus, Pseudomonas, Rhodopseudomonas, Bacteroides, Bifidobacterium, Lactococcus, and Streptococcus, including mutant strains thereof.

[0029] Examples of the heterotrophic microalgae include the genus Aurantiochytrium. (cultured cells) The cultured cells used herein are not particularly limited, but include fibroblasts, vascular endothelial cells, corneal epithelial cells, conjunctival epithelial cells, basal cells, myoblasts, osteoblasts, chondrocytes, glial cells, epidermal keratinocytes, oral mucosal cells, myeloma cells, nerve cells, cardiac myocytes, hepatocytes, adipocytes, mesenchymal stem cells, somatic stem cells, pluripotent stem cells, and blood cells, including mutant strains thereof. The origin of the above cells may be human, dog, cat, rabbit, rat, or pig.

[0030] The method for obtaining the heterotrophic microorganisms or cultured cells is not particularly limited and can be selected appropriately depending on the purpose. Examples include collecting them from nature or living organisms, using commercially available products, and obtaining them from preservation institutions or depository institutions.

[0031] ■ Carbon source and medium contents necessary for growth (carbon source) The carbon source contained in the medium of the present invention may be any carbon source necessary for the growth of the heterotrophic microorganisms or cultured cells being cultured, in other words, any carbon source necessary for proliferation, and examples of such carbon sources include sugars, organic acids, starch, and glycerin. Examples of sugars include monosaccharides, disaccharides, trisaccharides, polysaccharides, and sugar alcohols.

[0032] Examples of monosaccharides include glucose, fructose, galactose, mannose, xylose, ribose, and arabinose, and they may be in either the D- or L-form, but are preferably in the D-form. Examples of disaccharides include sucrose, maltose, trehalose, and lactose. Examples of trisaccharides include raffinose. Examples of polysaccharides include oligosaccharides, starch, and glycogen. Examples of sugar alcohols include sorbitol, mannitol, galactitol, and maltitol.

[0033] Examples of organic acids include acetic acid, citric acid, fumaric acid, glycolic acid, lactic acid, malic acid, propionic acid, pyruvic acid, succinic acid, glucuronic acid, galacturonic acid, uronic acid, chlorogenic acid, and lignocellulosic acid.

[0034] The amount of carbon source contained in the medium of the present invention can be determined appropriately within a range that does not impair the effects of the medium and / or taking into consideration costs. When sugars are used as carbon sources when added to the medium, the final concentration at the start of culture (initial concentration) can be, for example, 5 to 200 mg / mL.

[0035] (Contents of the medium) The present medium may contain only the microalgae extract and a carbon source necessary for growth, but may also contain other nutritional components.

[0036] The medium may contain a nitrogen source, and examples of the nitrogen source include amino acids, nucleic acids, ammonia, and urea.

[0037] The amino acids are not particularly limited as long as they are amino acids or derivatives thereof, or salts thereof that do not inhibit the growth of heterotrophic microorganisms or cultured cells, and include 20 types of amino acids or derivatives thereof, or salts thereof, namely aspartic acid (Asp), arginine (Arg), lysine (Lys), proline (Pro), glutamic acid (Glu), alanine (Ala), methionine (Met), serine (Ser), threonine (Thr), glycine (Gly), asparagine (Asn), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), tryptophan (Trp), tyrosine (Tyr), glutamine (Gln), histidine (His), and cysteine ​​(Cys), or their derivatives or salts thereof, or combinations thereof. Furthermore, the L-form of each of these amino acids is preferred.

[0038] Examples of nucleic acids include nucleic acids such as uracil, adenine, guanine, cytosine, and thymine, or derivatives thereof, or salts thereof, and these may be used in combination.

[0039] The medium may contain vitamins such as biotin, pantothenic acid, niacin, pyridoxine, thiamine, etc., metal ions such as zinc ions and iron ions, inorganic ions such as potassium ions and magnesium ions, etc. The medium may also contain yeast extract and polypeptone, but from the viewpoint of cost reduction, the content of yeast extract or polypeptone can be 0.3% or less, preferably 0.1% or less, and more preferably 0.01% or less.

[0040] The medium of the present invention can be adjusted appropriately depending on the heterotrophic microorganism or cultured cells to be cultured, but preferably contains 1 to 100 mg of microalgae in terms of dry weight per mL of medium.

[0041] ■ Oil and fat production methods In the present method for producing fats and oils, the heterotrophic microorganisms capable of producing fats and oils may be the fat-producing yeasts, fat-producing heterotrophic bacteria, or fat-producing microalgae described in the above section "Heterotrophic microorganisms or cultured cells."

[0042] In a method for culturing heterotrophic microorganisms capable of producing fats and oils, the heterotrophic microorganisms capable of producing fats and oils may be cultured in the medium of the present invention. The culture temperature and time can be adjusted appropriately depending on the type of heterotrophic microorganism to be cultured and the composition of the medium, but examples include a culture temperature of 15 to 55°C, preferably 20 to 40°C, and a culture time of 12 to 168 hours, preferably 24 to 72 hours. Furthermore, CO2 or O2 may be added to the medium as needed, and a predetermined amount of light may be irradiated.

[0043] Methods for extracting fats and oils include methods in which cultured heterotrophic microorganisms are removed from the medium by centrifugation, filtration, or the like, followed by extraction using heat treatment, organic solvent treatment, enzyme treatment, ultrasonic treatment, acid treatment, alkali treatment, bead crushing, or the like. Specific examples include a method in which heterotrophic microorganisms recovered by centrifugation are heat-treated in methanolic hydrochloric acid to methylate the lipids and convert them into fatty acid methyl esters (FAMEs), and the Bigh & Dyer method (A rapid method of total lipid extraction and purification Can J Biochem Physiol. 1959 Aug;37(8):911-7. doi: 10.1139 / o59-099.), in which microorganisms are suspended in a mixed solvent of methanol / chloroform / water, extracted, centrifuged, and the recovered chloroform layer is evaporated to dryness by spraying with nitrogen.

[0044] ■Additives The additive of the present invention is not particularly limited as long as it contains the microalgae extract, but may also contain nutritional components necessary for the growth of heterotrophic microorganisms or cultured cells, as necessary. Examples of media to which the additive can be added include known media for yeast, media for heterotrophic bacteria, and media for heterotrophic microalgae. The addition of the additive of the present invention improves the growth of heterotrophic microorganisms or cultured cells, making it possible to reduce the content of nutrient-rich medium components such as yeast extract and polypeptone.

[0045] Examples of known yeast media to be added include synthetic minimal (SD) medium, yeast nitrogen base (YNB) medium, drop out base (DOB) medium, YM medium, YPD medium, and PD medium. Examples of known heterotrophic bacterial media to be added include minimal salts (M9) medium, terrific broth (TB), and lysogeny broth (LB). Examples of known heterotrophic microalgae media to be added include HUT medium and GTY medium. Examples of known cell culture media include Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), and Roswell Park Memorial Institute (RPMI) 1640 medium.

[0046] The ratio of the present additive to the medium is not particularly limited, but examples include the present additive:medium=5:95 to 95:5, 10:90 to 90:10, 20:80 to 80:20, 30:70 to 70:30, 40:60 to 60:40, and 50:50. [Example]

[0047] The present invention will be described in more detail below with reference to examples. The examples are not limiting.

[0048] Materials and Methods First, the TOC measurement method, TN measurement method, glucose quantity quantification method, amino acid analysis method, and fatty acid analysis method in the following examples will be explained.

[0049] 1.TOC measurement method The total organic carbon (TOC) content of Spirulina extract was measured using a total organic carbon analyzer (TOC-V-VCPN, Shimadzu Corporation) equipped with an infrared detector, measuring the carbon dioxide produced by catalytic oxidation at 680°C. A 2.125 g / L aqueous solution of potassium hydrogen phthalate was used as a 1000 mg C / L standard solution.

[0050] 2.TN measurement method The total nitrogen (TN) content in the spirulina extract was measured using ultraviolet absorptiometry. 0.2 mL of a mixed solution of 0.04 g / L sodium hydroxide and 0.03 g / L potassium peroxodisulfate was added to 1 mL of sample, and the mixture was heated at 121°C for 30 minutes using an autoclave. After cooling, 0.24 mL of hydrochloric acid (1:16) was added. This process converted the nitrogen compounds into nitrate ions, and the absorbance at a wavelength of 220 nm was measured using a spectrophotometer (UH5300, Hitachi High-Tech Corporation). A nitrate nitrogen standard solution of 1000 mg N / L was used.

[0051] 3. Glucose quantification method The amount of glucose in the spirulina extract was measured using a glucose quantification kit (Lab Assay Glucose: Fujifilm Wako Pure Chemical Industries, Ltd.).

[0052] 4. Amino acid analysis method The amino acids in the Spirulina extract were analyzed using a fully automated amino acid analyzer (JLC-500 / V2, JEOL Ltd.). The sample was mixed with equal amounts of a buffer solution (lithium citrate buffer solution (P-21), pH 2.98, JEOL Ltd.), filtered through a 0.22 μm pore size filter, and then subjected to analysis.

[0053] 5.Fatty acid analysis The cells collected by centrifugation were suspended in 300 μL of methanol and transferred to a glass test tube. After thorough drying using a centrifugal concentrator (VR-36R: Itec), the residue was suspended in 0.1 M methanolic hydrochloric acid (Wako Pure Chemical Industries, Ltd.). The tube was sealed and incubated in boiling water to methylate the acyl groups in the lipids and convert them to fatty acid methyl esters (FAMEs). The resulting FAMEs were recovered with n-hexane. The recovered hexane phase was evaporated, and the residue containing FAMEs was dissolved in 200 μL of n-hexane. Qualitative analysis of palmitic acid (16:0), palmitoleic acid (16:1Δ9), stearic acid (18:0), 18:1Δ9, linoleic acid (18:2Δ9,12), γ-linolenic acid (18:3Δ6,9,12), α-linolenic acid (18:3Δ9,12,15), and stearidonic acid (18:4Δ6,9,12,15) was performed using a gas chromatograph (GC-2014, Shimadzu Corporation) equipped with a mass spectrometer. Helium was used as the carrier gas, and the analysis was performed in splitless mode at a constant flow rate of 1.25 mL / min. An SH-FAME column (Shimadzu Corporation) was used. To quantify FAMEs, a gas chromatograph equipped with a flame ionization detector (Shimadzu Corporation) was used. The operating conditions were the same as those for the qualitative analysis described above.

[0054] [Example 1] (Spirulina Extract Extraction Conditions - Enzyme Concentration) 1. Preparation of Spirulina Extract Dried Spirulina tablets purchased from Japan Algae were crushed in a mortar to prepare a dried powder of Spirulina cells, which was used as the source of Spirulina extract. Spirulina cells were suspended in distilled water to a final concentration of 20 mg / mL. Pancreatin (Japan Algae) was added to this suspension as an enzyme source at final concentrations of 0, 0.25, 0.5, 1, 2, and 4 mg / mL. 20 mL of this Spirulina and pancreatin mixture was sealed in a 50 mL tube and incubated at 45°C in a thermostatic water bath for 24 hours. After heating, the pH was adjusted to 6.2 with 1 M sodium hydroxide solution. This solution was centrifuged at 20,000 × G for 10 minutes, and the supernatant was used as Spirulina extract for subsequent procedures.

[0055] The TN and TOC of the above Spirulina extract were measured using the above TOC and TN measurement methods. The results are shown in Figure 1.

[0056] As shown in Figure 1, both TN and TOC were increased by the pancreatin-treated Spirulina extract. TN was maximized at 0.5 mg / mL, and TOC was maximized at 1 mg / mL, so in subsequent experiments, a pancreatin concentration of 1 mg / mL was used.

[0057] [Example 2] (Spirulina Extract Extraction Conditions - Reaction Time) Spirulina extracts were prepared using the method described in Example 1, except that the pancreatin content was 1 mg / mL and the incubation time was 0, 0.5, 1, 2, 4, 8, or 24 hours at 45°C, and TN and TOC were measured. The results are shown in Figure 2.

[0058] As can be seen from Figure 2, both TN and TOC increased with reaction time. In subsequent experiments, the reaction time was set at 24 hours. [Example 3] (Spirulina Extract Extraction Conditions - Reaction Time) Spirulina extract was prepared and its TN and TOC were measured in the same manner as in Example 1, except that the pancreatin content was 1 mg / mL, the reaction time was 24 hours, and the reaction temperatures were 30, 40, 45, 50, 55, and 65° C. The results are shown in Figure 3.

[0059] As shown in Figure 3, TN increased with increasing temperature from 30°C, but decreased at 65°C. TOC was almost the same between 30 and 55°C, but decreased at 65°C. In subsequent experiments, the reaction time was set at 55°C.

[0060] [Example 4] (Compared to YM medium) Based on the results of Examples 1 to 3 above, Spirulina extract was prepared by the method described in Example 1, except that the pancreatin concentration was 1 mg / mL, the reaction time was 24 hours, and the reaction temperature was 55°C. Hereinafter, the Spirulina extract obtained under these conditions will also be referred to as "Spirulina extract SE." Three solutions were prepared using the same method as in Example 1 except that pancreatin was not added: control Spirulina extract, Spirulina extract SE, and YM medium (yeast extract-malt extract medium: yeast extract 0.3%, malt extract 0.3%, peptone 0.5%, glucose 1%), which is used as a nutrient-rich medium for yeast. The TN and TOC of each solution were measured and the results are shown in Figure 4. In Figure 4, "without" represents the control Spirulina extract, "with" represents Spirulina extract SE, and "YM" represents YM medium.

[0061] As shown in Figure 4, compared to the control Spirulina extract, the pancreatin-treated Spirulina extract SE showed a 1.3-fold increase in TN and a 2-fold increase in TOC. This confirms that pancreatin treatment efficiently digests yeast. Furthermore, compared to YM medium, pancreatin treatment increased TN by approximately 10% compared to the nutrient-rich YM medium, and maintained TOC at approximately 70% of that of YM medium. This also confirms that pancreatin treatment is effective in digesting yeast. Furthermore, pancreatin treatment consumes less energy than ultrasonication, high-temperature treatment, acid treatment, etc., confirming that the medium can be prepared with less energy.

[0062] The glucose content, the main organic carbon source utilized by L. starkeyi, and the amino acids, the main organic nitrogen source, in Spirulina Extract SE medium and YM medium were measured using the glucose quantification method and amino acid analysis method described above, respectively. Measurement of glucose content revealed that YM medium contained 11.8 g / L of glucose, while Spirulina Extract SE contained approximately 1 g / L of glucose. Furthermore, measurement of amino acid content revealed that the total amounts in SE and YM medium were similar, at 1.35 mg / mL and 1.40 mg / mL, respectively.

[0063] [Example 5] (Growth evaluation of L. starkeyi) A culture test of L. starkeyi was conducted using Spirulina extract SE. First, Spirulina extract SE was autoclaved, and the oleaginous yeast Lipomyces starkeyi (L. starkeyi) JCM 5995 strain was cultured in a medium consisting solely of the sterilized Spirulina extract SE. Growth of L. starkeyi was minimal. The TOC value of Spirulina extract SE was approximately 60% of that of YM medium, and the glucose content was approximately one-tenth of that of YM medium, as described in Example 4. Therefore, we suspected that the carbon source contained in Spirulina extract SE was insufficient. Therefore, Spirulina extract SE was autoclaved, and glucose was added to the sterilized Spirulina extract SE to final concentrations of 5, 10, 20, 40, and 50 mg / mL, and the growth of L. starkeyi JCM 5995 strain was evaluated.

[0064] Growth evaluation was performed as follows. First, the oil-producing yeast L. starkeyi was pre-cultured at 25°C for 7 days in YM medium (Yeast Extract-Malt Extract medium: Yeast extract 0.3%, Malt extract 0.3%, Peptone 0.5%, Glucose 1%) supplemented with ampicillin. For main culture, 20 mL of the above Spirulina Extract SE or Spirulina Extract SE medium supplemented with glucose was placed in a 50 mL Erlenmeyer flask, and the culture was carried out at 25°C and 250 rpm, followed by the addition of 1 / 1000th of the above L. starkeyi pre-culture solution. Cell growth was monitored using a spectrophotometer (UH5300, Hitachi High-Technologies Corporation) to measure OD 600 The OD was measured on the 3rd, 4th, 5th, 6th, 7th, 10th, 12th, and 14th days of culture. 600 The measurement results are shown in Figure 5. Note that the enzymes contained in Spirulina Extract SE are inactivated by autoclaving.

[0065] The results in Figure 5 show that the addition of glucose significantly increased the growth of L. starkeyi. The amount of glucose contributed to improved growth up to a concentration of 20 mg / mL, and at concentrations of 40 and 50 mg / mL, growth was further improved, although growth was slightly delayed.

[0066] In addition, on a different day from the above, the OD values ​​on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, and 9th days after cultivation were measured when L. starkeyi was cultivated in YM medium using the same method as above, and when it was cultivated in Spirulina extract SE (SE+Glu20) or YM medium supplemented with 20 mg / mL of glucose. 600 The measurement results are shown in Figure 6. It was revealed that Spirulina Extract SE is a medium with high growth potential comparable to that of YM.

[0067] [Example 6] (Evaluation of lipid productivity of L. starkeyi) L. starkeyi was cultured in Spirulina Extract SG or YM medium, and fatty acid methyl esters (FAMEs) were extracted from the culture medium on day 7, and the fatty acid composition of the lipids was analyzed. The results are shown in Figure 7.

[0068] When Spirulina Extract SG was used, the fatty acid composition was comparable to that when YM medium was used. Therefore, no significant changes in growth or fatty acid productivity were observed when Spirulina Extract SG was used. Furthermore, when the amount of FAME per mL of culture medium was measured on the 7th day, it was 1.97 mg / mL for Spirulina Extract SG and 2.14 mg / mL for YM medium. Therefore, when growing oil-producing yeast, Spirulina Extract SG can be said to be a medium that has growth ability comparable to nutrient-rich commercially available media and maintains fatty acid production within the yeast.

[0069] [Example 7] (Study of microalgae) In the above examples, microalgae extracts were prepared from dried Spirulina. However, other microalgae were cultured and enzymatically treated without drying the cultures, rather than using dried forms. The microalgae used were Anabaena and Synechocystis, both of which belong to the cyanobacteria. Anabaena and Synechocystis were cultured in BG-11 medium, and OD 730A culture solution equivalent to 50 ml of TN was prepared. TN and TOC were measured in the same manner as in Example 1, except that 1 mg / ml of pancreatin was added to this culture solution and the reaction temperature was set at 55°C. The results are shown in Figure 8. In the figure, Pancreatin- indicates the case where no pancreatin was added, and Pancreatin+ indicates the case where pancreatin was added.

[0070] The TN and TOC values ​​of both Anabaena and Synechocystis increased with pancreatin treatment. Specifically, in the case of Anabaena, TN increased by 1.4 times and TOC increased by about 1.4 times with pancreatin treatment, while in the case of Synechocystis, TN increased by 1.3 times and TOC increased by about 1.2 times with pancreatin treatment. Therefore, it was confirmed that the TN and TOC values ​​can be increased even when microalgae other than Spirulina are used to prepare microalgae extracts, and even when live cells are used without drying.

[0071] [Example 8] (Spirulina Extract Extraction Conditions - Types of Enzymes) In the above example, pancreatin was used as the enzyme, but comparisons were also made with the digestive fluids pepsin alone, lipase alone, and pepsin plus lipase. TN and TOC were measured in the same manner as in Example 1, except that 1 mg / mL pancreatin, 1 mg / mL pepsin (162-18721: Wako Pure Chemical Industries, Ltd.), 0.5 mg / mL lipase (Nacalai Tesque, Inc.), or 0.5 mg / mL pepsin and 0.5 mg / mL lipase were added and the reaction temperature was set to 55°C. A control without enzymes was also performed in the same manner. The results are shown in Figure 9.

[0072] When treated with pepsin alone, both TN and TOC increased by only about 10% compared to the control without enzyme addition. On the other hand, when treated with pancreatin, both TN and TOC increased by about 1.3 times and 2 times compared to the control without enzyme addition. Furthermore, TN and TOC values ​​were equivalent to those of pancreatin when treated with pepsin and lipase. Therefore, it was thought that pepsin alone was not sufficient for yeast digestion, and that pepsin and lipase were necessary.

[0073] The results of the above examples demonstrate that oil-producing yeast can be cultured using Spirulina, a photosynthetic organism. Conventionally, heterotrophic organisms such as yeast extract have been used as the main amino acid source for culturing oil-producing yeast, but this example demonstrates that Spirulina extract can replace this yeast extract. In the future, if a culture system for oil-producing yeast can be established using inedible microalgae that grow on abandoned farmland or in the aquatic environment as an extract source, it is expected that this will contribute to the industrial application of carbon-neutral technology.

[0074] Furthermore, previous technologies required expensive medium substrates containing large amounts of organic nitrogen, such as amino acids and peptides, to cultivate yeast, which has poor inorganic nitrogen assimilation ability. By using the medium and additives of the present invention to replace such expensive medium substrates with low-cost extracts of microalgae that grow autotrophically, the cost of culturing yeast, including oil-producing yeast, can be reduced. Furthermore, the carbon dioxide emitted when burning the oils produced by this method can be refixed by microalgae, allowing for the creation of a carbon-neutral material production system.

Claims

1. A culture medium for heterotrophic microorganisms or cultured cells, comprising a microalgae extract obtained by treating microalgae with protease and lipase, and a carbon source necessary for growth.

2. 2. The culture medium according to claim 1, wherein the microalgae extract is obtained by treating microalgae with a mixed enzyme containing protease and lipase.

3. 3. The medium according to claim 2, wherein the mixed enzymes are enzymes derived from mammalian pancreatic juice.

4. 3. The culture medium according to claim 1 or 2, characterized in that the heterotrophic microorganism is a heterotrophic microalga, yeast, or heterotrophic bacterium.

5. 3. The culture medium according to claim 1, wherein the microalgae is spirulina.

6. 3. The medium according to claim 1, wherein the carbon source is glucose.

7. A method for culturing heterotrophic microorganisms or cultured cells in the medium according to any one of claims 1 to 6.

8. A step of culturing heterotrophic microorganisms capable of producing fats and oils in the medium according to any one of claims 1 to 6; extracting fats and oils from the cultured heterotrophic microorganisms capable of producing fats and oils; A method for producing fats and oils, comprising:

9. An additive for a culture medium for heterotrophic microorganisms or cultured cells, comprising a microalgae extract obtained by treating microalgae with a protease and a lipase.

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