JP5943361B2 - Method for producing triacylglycerol - Google Patents

Description

  The present invention relates to a method for producing triacylglycerol (hereinafter abbreviated as TG). More specifically, the present invention relates to a method for producing TG by highly efficient accumulation of TG in microalgae for the purpose of providing edible fats and oils.

  In recent years, rice, wheat, corn, legumes have increased due to an increase in food demand due to global population growth, a decrease in crop yields due to abnormal weather, conversion of cereals and edible vegetable oils into biofuels, and inflows of investment funds. Price increases of major grains such as edible vegetable oils and fats are a problem.

  Photosynthetic microorganisms inhabit freshwater, seawater, and humid places, and also in extreme areas such as hot springs, extremely cold lakes, hot seawater, and soda lakes. Among these, microalgae are known to accumulate oils and fats and aliphatic hydrocarbons, and diversion to edible oils and fats and edible livestock feed has been proposed. However, there is no report on a method for preferentially accumulating TG, which is the main component of animal fats and vegetable fats, using microalgae with a fast growth rate due to the culture stress.

  It is known that the composition and amount of an aliphatic compound accumulated by microalgae change due to adverse environmental conditions and various stresses. In particular, it has been reported that the accumulation amount and composition of aliphatic compounds per dry weight of cells changes by adjusting nitrogen deficiency, nutrient deficiency stress, and culture conditions, making TG production easier and more efficient. A method is being considered. (For example, see Non-Patent Documents 1 and 2)

Siaut, et al. BMC Biotechnology, 11: 7-21 (2011) Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves. Chen CY, et al. Bioresour Technol, 102 (1): 71-81 (2011) Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review.

  In considering the diversion of aliphatic compounds accumulated in microalgae to edible oils and fats and edible livestock feeds, the use of edible microalgae and their wild strains with direct eating experience is advantageous from the viewpoint of security. In addition, when considering industrial use, it is important to use microalgae that are resistant to changes in environmental conditions such as temperature, humidity, and sunshine, disadvantageous growth, and various stresses.

  Although microalgae are known to accumulate aliphatic compounds, cultures that preferentially accumulate TG, which is the main component of animal and vegetable oils and fats, by using microalgae with a high growth rate and their culture stress. There is no report of the method.

  The present invention focuses on changes in the accumulation efficiency of TG derived from differences in various stress loads in culture, and optimizes the culture conditions of microalgae using wild strains of edible microalgae that are resistant to drying and high temperature conditions And applying stress load in the culturing process, especially drought stress load, and further sulfur deficiency stress load, accumulating TG in microalgae with high efficiency, and collecting TG from the culture to produce TG Objective.

  According to the present invention, a method for producing TG, comprising culturing microalgae and collecting and producing TG from the culture, comprising culturing microalgae and containing at least one of oils and fats and aliphatic hydrocarbons In the culturing step for accumulating a family compound, a method for producing TG is provided, including a culturing step for preferential accumulation of TG by culturing microalgae under drought stress load conditions.

  Microalgae are cultured after dehydration, cultured on solids in the gas phase, or cell liquid of high concentration microalgae is gradually dried, and microalgae are cultured under dry stress load conditions Is preferred.

  Incubation can also be performed under sulfur-deficient stress loading conditions.

  Cultivation can be performed under sulfur-deficient stress loading conditions using a sulfur-deficient culture solution

  The culture is preferably performed while being aerated, and is performed in a photoautotrophic manner using natural light or artificial light.

  As a light source for photoautotrophic culture, it is preferable that the culture is carried out by applying sunlight or light irradiation.

Cultivation is preferably carried out using organic substances in the cells of microalgae as the carbon source, or using CO _{2 in} the air or extracellular organic substances.

  It is preferable to culture microalgae whose concentration has been increased by pre-culture.

  The microalgae is preferably selected from the group consisting of broad green algae including treboxya algae, diatoms, true-eye algae, red algae, cyanobacterium and euglena algae.

  The microalgae is preferably an edible microalgae selected from the genus Chlorella, Donariella, Spirulina, and Euglena.

  The microalgae is preferably selected from the genus Chlorella or Parachlorella.

FIG. 1 shows the results of analysis of lipid accumulation in microalgae subjected to main culture under the dry stress load conditions of Example 1 by staining of intracellular neutral fat with Nile red staining. FIG. 2 shows the results of analysis of lipid accumulation in microalgae that were subjected to main culture under the dry stress load conditions of Example 1 by thin layer chromatography. FIG. 3 shows changes over time in the amount of lipid accumulation in microalgae that were cultured under the dry stress load conditions of Example 1. FIG. 4 shows the change over time in the degree of unsaturation of fatty acids in microalgae that were cultured under the dry stress load conditions of Example 1. FIG. 5 shows changes over time in the amount of chlorophyll and the dry weight of cells in microalgae that were cultured under the dry stress load conditions of Example 1. FIG. 6 shows the change over time in the amount of water remaining in the filter paper when the main culture is performed under the dry stress load condition of Example 1. FIG. 7 shows a comparison of the percentage of lipid accumulation in microalgae that were cultured under the dry stress load condition of Example 1 and the sulfur deficiency stress load condition of Reference Example 1. FIG. 8 shows a comparison of the degree of unsaturation of fatty acids in microalgae subjected to main culture under the dry stress load condition of Example 1 and the sulfur deficiency stress load condition of Reference Example 1. FIG. 9 shows a comparison of lipid accumulation amounts in microalgae subjected to main culture under the conditions of drought stress load in Example 1 and Gb, D.w, Wash, Triple, Dark, LL, and VLL conditions in Example 2. FIG. 10 shows a comparison of the amount of chlorophyll in microalgae subjected to main culture under the dry stress load conditions of Example 1 and the Gb, Dw, Wash, Triple, Dark, LL, and VLL conditions of Example 2. . FIG. 11 shows a comparison of the amount of water remaining in the filter paper when the main culture is performed under the dry stress load conditions of Example 1 and the Wash, Triple, LL, and VLL conditions of Example 2. FIG. 12 shows a comparison of the degree of unsaturation of fatty acids in microalgae cultured in the dry stress load conditions of Example 1 and the Gb, Dw, Wash, Triple, Dark, LL, and VLL conditions of Example 2. .

  Hereinafter, the present invention will be described in detail.

The method for producing TG of the present invention comprises culturing microalgae under a dry stress load condition in a culture step of culturing microalgae and accumulating an aliphatic compound containing at least one of oil and fat and aliphatic hydrocarbons in microalgae. Thus, a culture step for preferentially accumulating TG is included. The culture process of the present invention can also be performed under a sulfur-deficient stress load condition. Moreover, in this invention, it is preferable to culture | cultivate the micro algae which precultured and raised the density | concentration, Therefore, it is preferable to include a preculture process. TG can be obtained by the collection step of collecting TG from the microalgae culture according to the present invention.
Details of each step will be described below.

  The microalgae used in the present invention is preferably selected from the group consisting of green algae including treboxya algae, diatoms, true-eye algae, red algae, cyanobacteria and euglena algae. For example, it is desirable to be an edible aquatic organism that carries out oxygen-generating photosynthesis with chlorophyll, and can accumulate and secrete aliphatic compounds containing at least one of oil and fat and aliphatic hydrocarbons in and out of algae by photosynthesis during culture Any microalga can be used as long as it is a species of microalgae. In the present invention, edible microalgae are preferable, and at least one microalgae selected from the group consisting of Chlorella, Donariella, Spirulina, and Euglena is particularly preferable. Examples of the genus Chlorella include, for example, Chlorella kessleri 11h, Chlorella vulgaris, Chlorella pyrenoidosa, Chlorella saccharophila, Chlorella ella regularis, Chlorella relaris・ Solokiniana (Chlorella sorokiniana) and the like. Examples of the genus Donaliella include Donaliella bardawil and Dunaliella salina. Examples of the genus Spirulina include Spirulina platensis, Spirulina maxima, Spirulina geitleri, Spirulina siamese and the like. Examples of the genus Euglena include Euglena gracilis and Euglena viridis. These can be easily obtained by those skilled in the art.

  Aliphatic compounds produced by microalgae include fats and oils and aliphatic hydrocarbons. Examples of the fats and oils include aliphatic esters composed of aliphatic carboxylic acids and monovalent or trivalent aliphatic alcohols, as long as they are produced by microalgae, in addition to these, methyl laurate, myristyl myristate Further, methyl palmitate may be used. The aliphatic hydrocarbon is not particularly limited as long as it is produced by microalgae, and examples thereof include aliphatic hydrocarbons that are solid or liquid at room temperature, saturated or unsaturated in the range of 15 to 40 carbon atoms. And the following linear aliphatic hydrocarbons. TG, which is an object of the production method of the present invention, is one in which three molecules of fatty acid such as lauric acid, myristic acid, and palmitic acid are ester-bonded to one molecule of glycerol.

In the present invention, in the pre-culture step, it is preferable to cultivate microalgae by a usual method to increase the concentration of microalgae. As a medium for culturing, a general inorganic medium such as CHU medium, JM medium, MDM medium or the like can be used as long as it is a medium capable of cultivating microalgae by a usual method and increasing the concentration of microalgae. Can be used. For example, as the medium, Gamborg B5 medium, BG11 medium, HSM medium, or diluted solutions of these various media are preferable. The inorganic medium contains Ca (NO ₃ ) ₂ · 4H ₂ O, KNO ₃ and NH ₄ Cl as nitrogen sources, and KH ₃ PO ₄ and MgSO ₄ · 7H ₂ O, FeSO ₄ · 7H as other main nutrients ₂ O etc. are included. Further, an antibiotic or the like that does not affect the growth of microalgae may be added to the medium. The pH of the medium is preferably 3-10. Depending on the concentration of the microalgae to be inoculated, it is desirable that the culture period until the transition to the culture under drought stress load, and further under the sulfur-deficient stress load condition, is cultivated to about OD ₇₃₀ ≈0.5.
In the pre-culture step, the culture under the dry stress load condition or the sulfur-deficient stress load condition of the present invention may be performed, or the culture may be sequentially or simultaneously performed under these conditions.

In the TG production method of the present invention, preferably in the culturing step of accumulating an aliphatic compound containing at least one of oils and fats and aliphatic hydrocarbons in the microalgae following the pre-culture step, the microalgae are subjected to a dry stress load condition. A culturing step for preferential accumulation of TG is performed. Cultivation under a drought stress load condition is performed, for example, by transferring a standard liquid phase growth environment of microalgae called a culture solution into a gas phase growth environment. In this dry stress loading condition, microalgae grown by pre-culture in a normal liquid medium are cultured after dehydration, on solid in a gas phase, or with a high concentration of microalgae cell solution. This can be done by gradually drying. Examples of the dehydration method include physical methods such as centrifugation and filtration, and evaporation methods. After the dehydration treatment, the culture can be continued by irradiating with light by a usual method. Examples of the culture on solid include culture on an agar medium, glass fiber filter, filter paper, cloth and the like. A solid medium having the same composition as described above can be used as the medium in the culture on the solid in the gas phase. When the cell solution of high-concentration microalgae is gradually dried, it can be performed by gradually drying and culturing in a culture vessel such as a culture reactor. Cultivation under a drought stress load condition is preferably performed under conditions where water is efficiently removed and the aliphatic compounds inside and outside the microalgae are not subject to oxidative degradation. For example, it is desirable to cultivate microalgae for about 30 to 100 hours under conditions of a temperature of 30 ° C. and a humidity of 90% by using a multistage culture reactor or the like installed in the gas phase.
Thus, by culturing microalgae under dry stress loading conditions, TG can be preferentially accumulated in the body of microalgae.

In the method for producing TG of the present invention, in addition to the above-mentioned dry stress load condition, the culture can also be performed under a sulfur deficiency stress load condition. Furthermore, in order to culture | cultivate on sulfur deficiency stress load conditions, it can also culture | cultivate on sulfur deficiency stress load conditions before or after culture | cultivation on the above-mentioned dry stress load conditions.
Cultivation under a sulfur-deficient stress load condition is performed under the same conditions as the above-mentioned preculture conditions by replacing a general inorganic medium such as CHU medium, JM medium, and MDM medium used in the preculture with a sulfur deficient culture solution. It can be performed by culturing.
Or culture | cultivation on dry stress load conditions and culture | cultivation on sulfur deficiency stress load conditions can also be performed simultaneously. In order to carry out simultaneously, for example, the culture solution of the microalgae after the pre-culture is dropped on a glass fiber filter, filter paper, cloth, etc., washed with distilled water, and the culture solution containing sulfur is poured, suction filtered and dried. By a method of culturing later, or a method of culturing by dropping a culture solution of microalgae after pre-culture on a laminate of glass fiber filter, filter paper, cloth, etc. soaked with distilled water not containing sulfur Can be implemented.

When culturing under the above-mentioned dry stress load conditions, or in addition, under a sulfur-deficient stress load condition, the culture is performed while being aerated and is performed in a photoautotrophic manner using natural light or artificial light. Are preferred. As the photoautotrophic culture, it is preferable that the culture is performed with sunlight or light irradiation. In addition, as a carbon source, culturing is performed by using organic substances such as starch accumulated in the cells of microalgae or by supplying organic substances such as CO _{2 in} the air or extracellular acetate. Is preferred.

  In the collecting step of collecting TG from the culture, first, the microalgae cultured in the culturing step are separated from the medium, agar medium, glass fiber filter, filter paper, cloth or the like. Separation from the medium is preferably performed by separation of microalgae from the medium using solid-liquid separation means such as filtration or centrifugation, for example, separation with a glass micro fiber filter and an aspirator. As a means for obtaining TG from the obtained microalgae, an extraction method using an organic solvent or a pressing method can be used. As the organic solvent, ethanol, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, acetone or the like can be used. Also, for example, a supercritical extraction method can be used as an effective means.

  EXAMPLES Hereinafter, although the content of this invention is demonstrated more concretely using an Example and a reference example, this invention is not limited to a following example at all.

  In this example, we investigated the accumulation of aliphatic compounds and the efficiency of TG accumulation in drought stress loading conditions and sulfur deficiency stress loading conditions in the cultivation of Chlorella kessleri 11h strain, which is resistant to culturing under high temperature conditions. It was.

1. Pre-culture conditions Chlorella kessleri 11h strain was aerated through a foam tube (Kinoshita Ball Filter Kinoshita Rika Kogyo Co., Ltd.) with 1/4 Gambog B5 medium at a temperature of 30 ° C and 2% -CO ₂ added. Using fluorescent light fish lux 20W, it was grown up to OD ₇₃₀ ≈0.5 in a photoautotrophic state at a light intensity of 15 W / m ² .

2. Main culture conditions: Cultivation under drought stress load conditions In order to culture under drought stress load conditions, 20 mL of the pre-cultured cells are dropped into a glass micro fiber filter (Whatman GF / C) and aspirator sucked for 7 seconds. The culture solution used for preculture was removed by filtration. It is 26.5cm wide x 35.5cm wide x 5.5cm high cell culture container (Entech Hi-PACK, made of polypropylene), temperature 30 ° C, humidity 90% or higher, light intensity 3.8W / m ² , ventilation 2 % -CO placed _second condition was continued photoautotrophic cultured. In the main culture, samples were collected and analyzed at 0 hours and 96 hours.
Reference example 1

1. Pre-culture conditions Chlorella kessleri 11h strain was blown into a 3 / 10HSM medium using a culture test tube (made of glass with an internal volume of about 50 mL) and air with 2% CO ₂ added at a temperature of 30 ° C. (Kinoshita Ball Filter Kinoshita Rika Kogyo Co., Ltd.) was used for aeration and growth was performed using a Toshiba fluorescent light fish lux 20 W at a light intensity of 15 W / m ² until the photoautotrophic OD ₇₃₀ ≈0.5.

2. Main culture conditions: Cultivation under sulfur deficiency stress loading conditions Cultivation under conditions of sulfur deficiency stress loading, pre-cultured cells were centrifuged at 3000rpm, 4 ℃, 10min, supernatant was removed, then 3 / 10HSM liquid 50 mL of the medium (-S) was added and stirred, and the sulfur was washed away by centrifugation under the above conditions. This process was performed twice, and the sulfur deficiency stress load conditions were set. The cells after removal of the supernatant were suspended in 50 mL of the above-described sulfur deficient medium and placed under conditions of a temperature of 30 ° C., a light intensity of 15 W / m ² , and aeration of 2% -CO ₂ , and the culture was continued photoautotrophically. After 96 hours of culturing, 10 mL of the total volume of the cell culture solution was suction filtered with a glass micro fiber filter and dried to measure the dry weight. Further, 30 mL of the cell culture solution was centrifuged at 3000 rpm, 4 ° C., 10 minutes, and lipid extraction and analysis in the cells were performed.

As the culture conditions, unless otherwise specified, the application method to the filter paper, the culture temperature, the CO ₂ concentration, the humidity, the light source, and the light intensity are the same as those in the culture under the dry stress load condition of Example 1, and The main culture was performed under the Gb, Dw, Wash, Triple, Dark, LL, and VLL conditions described below.

1. Pre-culture conditions Chlorella kessleri 11h strain was aerated through a foam tube (Kinoshita Ball Filter Kinoshita Rika Kogyo Co., Ltd.) with 1/4 Gambog B5 medium at a temperature of 30 ° C and 2% -CO ₂ added. Using fluorescent light fish lux 20W, it was grown up to OD ₇₃₀ ≈0.5 with a Beckman DU700 spectrophotometer at a light intensity of 15 W / m ² and light autotrophic.

2. Main culture conditions: Gb, Dw, Wash, Triple, Dark, LL, and VLL conditions, dry stress load conditions, dry stress load conditions and sulfur-deficient stress load conditions Dw, Wash, Triple, Dark, The cells were cultured under various conditions of LL and VLL. Specifically, as the Gb condition, main culture was carried out by laminating Kim towel (2 sheets) impregnated with 200 mL of culture solution (1/4 Gambog B5 liquid medium), net, and filter paper in this order. As for Dw conditions, main culture was carried out by laminating Kim Towel (2 sheets) soaked with 200 mL of distilled water, nets, and filter paper in this order. Washing was carried out by washing the cells on the filter paper with 50 mL of distilled water, followed by suction filtration and carrying out main culture. In Triple conditions, the main culture was carried out with the amount of cells on the filter paper set to 3 times 60 mL. In the dark condition, the main culture was performed under the light condition as the dark condition. As for LL (Low light: 1 W / m ² ) and VLL (Very low light: 0.4 W / m ² ) conditions, the main culture was performed under low light conditions.

Test example 1
The cultures in Examples 1 and 2 and Reference Example 1 were evaluated by performing various tests.

Test method
1. In sampling sampling, one sample for chlorophyll determination, one sample for wet weight and dry weight measurement, and two samples for lipid quantification were sampled, and the reproducibility was examined by repeating this four times in total.

2. After measuring the cell weight after chlorophyll quantitative culture, 1 mL of 100% methanol was added and suspended using a vortex. After standing for 5 minutes, the mixture was centrifuged at 10000 rpm for 10 minutes at room temperature. Absorbance at 665 nm and 650 nm was measured for the supernatant. The amount of chlorophyll was quantified by the calculation formula “Chla + b = 4.0 × A ₆₆₅ + 25.5 × A ₆₅₀ ”.

3. Staining neutral lipid with Nile Red 1mL of acetone is added to 50mL of a solution of 0.5g Nile Red (9-diethylamino-5H-benzo [[a] phenoxa-] phenoxazine-5-one) in 1mL of acetone. Samples were prepared. The sample was placed on a slide glass and observed with an optical microscope while being excited with UV light (filter: WIG, excitation light 520 to 550 nm, transmitted light 580 nm).

4). Wet Weight / Dry Weight Measurement Method The mass of filter paper used in advance was measured. Thereafter, the cells after the main culture were collected. The collected cells were weighed as they were and then placed in a dryer at 55 ° C. for 5 hours or longer, completely dried and measured for dry weight. The wet weight was obtained by subtracting the mass of the filter paper and the dry weight of the cells, and obtaining only the mass of water contained in the filter paper. The dry weight was obtained by subtracting the mass of the filter paper to obtain the dry weight of the cells. In addition, since the mass of the inorganic salts remaining on the filter paper is about 0.4 mg at the maximum, it is not necessary to consider.

5. Extraction of fat-soluble component 10 mL of methanol and chloroform were added to the cells on the filter paper after the main culture and suspended using vortex to completely separate the fat-soluble component from the cells. Distilled water (5 mL) was added, and the mixture was centrifuged at 3000 rpm, 4 ° C., 15 min to separate into two phases, a methanol / water layer and a chloroform layer. Thereafter, the chloroform layer was recovered, concentrated by distilling off the solvent with an evaporator, and dissolved in a chloroform: methanol = 2: 1 (v / v) solution to obtain a fat-soluble component.

6). Separation of each lipid by thin layer chromatography Thin layer chromatography plates (5721 Silica gel 60, MERCK, Darmsadt, Germany) were treated at 120 ° C. for 2 hours to completely remove moisture. The extracted fat-soluble component was spotted on a thin layer chromatography plate, and the solvent was dried with a dryer. Developed for about 45 minutes using hexane: diethyl ether: acetic acid = 70: 30: 1 as a developing solvent. After drying the solvent of the thin-layer chromatography plate, spray the primulin solution (preparing 0.01% primulin (Tokyo Kasei Kogyo) solution with acetone: distilled water = 4: 1 (v / v) as solvent) and exciting light at 365 nm Thus, each lipid was confirmed.

7). Data obtained by a data processor (C-R7 pulas, SHIMADZU, Kyoto, Japan) was used for data analysis and quantification. The weight ratio was calculated from the peak area of each component including arachidic acid, and the number of moles of each component was calculated from the number of moles of arachidic acid.

8). Calculation of Unsaturation The number of double bonds was determined relatively. The ratio of each fatty acid was multiplied by the number of double bonds and was calculated by the following formula. {[(18: 1) + (16: 1) × 1] + [(18: 2) + (16: 2) × 2] + [(18: 3) + (16: 3) × 3]} / Total fatty acid = Unsaturation

9. Quantitative determination of fat-soluble components Spots separated by thin layer chromatography were scraped with a spatula, and a 5% (w / v) hydrochloric acid methanol solution was added. By performing heat treatment at 90 ° C. for 3 hours, acyl groups and free fatty acids in lipids are methyl esterified. After cooling, 2 mL of hexane was added and allowed to stand to separate two layers, and the upper hexane layer was recovered. To the lower layer, 2 mL of hexane was added again, and the same recovery was performed three times to improve the recovery rate. The recovered hexane solution was concentrated by distilling off the solvent with an evaporator, and quantified using a gas chromatograph. In addition, arachidic acid (Sigma, St. Louis, USA) was added in advance to a screw-cap glass test tube and evaporated to dryness with nitrogen gas, which was used as an index.

Test results
1. Neutral lipid accumulation during culture under drought stress
Using Nile Red, intracellular neutral lipids were stained and excited with light having a wavelength of 520 to 550 nm for microscopic observation. In Example 1, as a result of culturing for 96 hours under a drought stress load condition, it was shown that the neutral lipids indicated by yellow fluorescence were significantly increased (FIG. 1).

2. By thin-layer chromatography by culturing under drought stress
Separation of fat-soluble components Neutral lipids in the extracted fat-soluble components were separated by thin layer chromatography. As a result, in Example 1, it was confirmed that neutral lipids containing TG were increased by culturing for 96 hours under a dry stress load condition (FIG. 2).

3. Accumulation of TG in culture under dry stress load conditions FIG. 3 and Table 1 show changes in TG concentration with the passage of culture time under dry stress load conditions in the culture of Example 1. The dry weight of microalgae on the filter only increased by about 2.5 times from 23.6 mg to 60.0 mg, but the total fatty acid content showed a significant increase of 8.6 times from 1.5 mg to 12.9 mg. The amount of TG increased from 0.07 mg to 8.5 mg, and most of the increase in total fatty acid was attributed to TG. This result shows that the fatty acid composing TG was 4.7% of the total fatty acid content before stress loading, but increased to 65.9% of the total fatty acid content after stress loading. Indicates that 14.2% of the weight has been reached.

4). Changes over time in fatty acid composition and degree of unsaturation in culture under drought stress conditions FIG. 4 and Table 2 show changes over time in the degree of unsaturation of fatty acid composition in total lipids in the culture of Example 1. The degree of unsaturation in the proportion of fatty acid composition in the total amount of fatty acids tended to decrease slightly with the passage of culture time. On the other hand, there was no significant change in the ratio of unsaturated fatty acids in TG, and the amount of TG in all fatty acids shown in FIG. 3 tended to increase after 24 hours. The sum is expected to increase.

5. Measurement of Chlorophyll Amount and Dry Weight in Culture under Dry Stress Load Conditions FIG. 5 and Table 3 show changes over time in the chlorophyll amount and cell dry weight in the culture of Example 1. The chlorophyll content tended to decrease over time from the start of the main culture. Under these conditions, the cell growth leveled off after 48 hours, and the amount of chlorophyll per dry weight decreased from 5% to about 2%. In particular, the decrease until 24-48 hours was remarkable.

6). Change in residual amount of water in filter paper under dry stress load condition FIG. 6 shows the change over time in the residual amount of water in the filter paper under dry stress load condition in the culture of Example 1. The remaining amount of water in the filter paper gradually decreased from about 0.5 g to 0.2 g. This is thought to be because the humidity in the cell culture container is maintained at 90% or higher. When it is dried indoors (humidity: 30-50%), it dries within a few hours, and Nile No increase in lipid was observed in the staining results with Red. For this reason, it was suggested that it is important to apply moderate drought stress to lipid accumulation.

7). TG accumulation in culture under drought stress load conditions and sulfur deficiency stress load conditions Wet and dry weight measurement of fat-soluble components in the dry stress load conditions of Example 1 and the sulfur deficiency stress load conditions of Reference Example 1 Extraction, thin layer chromatography, quantification of fat-soluble components, and data analysis were performed in the same manner as described above. As a result of culturing under the sulfur deficient stress loading condition, the total lipid accumulation per dry weight was similar to that under the drought stress loading condition and the sulfur deficient stress loading condition, but the TG amount per total lipid accumulation was 52. The value was as low as% (FIG. 7 and Table 4).

8). Comparison of desaturation of fatty acids under drought stress loading conditions and sulfur deficiency stress loading conditions Fatty acid unsaturation of all fatty acids and fat in lipid deficiency stress loading conditions of Example 1 and Reference Example 1 The comparison is shown in FIG. The drought stress load condition and the sulfur deficiency stress load condition showed no significant change in fatty acid composition, but 18: 3 resulted in slightly more drought stress load conditions.

9. Comparison of drought stress load conditions and lipid accumulation amount under Gb, Dw, Wash, Triple, Dark, LL, VLL conditions Comparison of drought stress load conditions in Example 1 and TG accumulation under each condition described in Example 2 FIG. 9 and Table 6 show. In comparison between the Gb and Dw conditions, the increase in lipid accumulation was small under the Gb condition, while the increase in dry weight of cells was large. Under the Dw condition, the increase in lipid accumulation was large and the increase in dry weight of cells was small. Since the amount of lipid accumulation under the Dw condition is small compared to the drought stress load condition, it can be assumed that the amount of lipid accumulation under the drought stress load condition is not simply a nutritional deficiency stress, and is a dehydration stress in the cell body. It was suggested. Under the Wash condition, the increase in dry weight and the amount of lipid accumulation are similar to the conditions under the dry stress load in Example 1, and the presence or absence of nutrient medium remaining in the filter paper under the dry stress load condition depends on the lipid accumulation amount after 96 hours. It seems to be unrelated. Under Triple conditions, the amount of lipid accumulation per cell was about half of the dry stress load condition of Example 1. This is probably because the amount of light energy that can be used per cell decreases as a result of the tripled amount of cells on the filter paper and an increase in cell density. Under the LL and VLL conditions, the accumulation of TG and the dry weight of the cells are reduced, and it is considered that the light conditions are the same as the Triple conditions.

10. Comparison of drought stress load conditions and chlorophyll amounts under Gb, Dw, Wash, Triple, Dark, LL and VLL conditions Comparison of drought stress load conditions in Example 1 and chlorophyll amounts in each main culture condition described in Example 2 FIG. 10 and Table 7 show. The amount of chlorophyll per dry weight of the cells was small under the drought stress load condition and the Dw, Wash, and Dark conditions. The dry stress load condition of Example 1 and the Dw and Wash conditions are considered to be the result of damage to the cells due to changes in osmotic pressure due to drying and solvent replacement into distilled water, and changes to a nutrient-deficient state. In addition, it is considered that the chlorophyll is reduced under the dark condition because it is a dark condition. Under the LL and VLL conditions where the increase in TG accumulation was small, the chlorophyll content tended to be large.

11. Comparison of drought stress load conditions and residual water amounts under Wash, Triple, LL, and VLL conditions Comparison of dry stress load conditions in Example 1 with the main stress conditions described in Example 2 and FIG. Table 8 shows. Under the LL and VLL conditions where the increase in TG accumulation was small, there was a tendency for the amount of residual water to be large, similar to the amount of chlorophyll. It has been experimentally confirmed that lipid accumulation decreases when drying is insufficient under dry stress loading conditions, and the reason why the TG accumulation amount is small under the LL and VLL conditions is that the residual moisture amount is large. This is considered as one of the causes.

12 Comparison of Drought Stress Load Condition and Fatty Acid Unsaturation Conditions under Gb, Dw, Wash, Triple, Dark, LL, and VLL Conditions Dry Fat Stress Conditions in Example 1 and Fatty Acids in Each Main Culture Condition described in Example 2 A comparison of the degree of unsaturation is shown in FIG. Although there was no significant change in fatty acid composition under each condition, the degree of unsaturation of TG under LL, VLL, and Dark conditions was low. When making a double bond of fatty acids constituting TG, more energy is required compared with a single bond, and therefore the degree of unsaturation may be reduced under low light conditions.

  From the above results, it became clear that TG can be preferentially accumulated in fine cells by culturing microalgae under drought stress loading conditions. It was also clarified that TG can be preferentially accumulated in fine cells by culturing the pre-cultured microalgae under sulfur-deficient stress loading conditions. Furthermore, it became clear that TG can be preferentially accumulated in fine cells by culturing microalgae under drought stress loading conditions and sulfur deficiency stress loading conditions.

  The method for producing TG of the present invention is to produce TG by preferentially accumulating TGs in fine cells by culturing microalgae under dry stress load conditions. In culture, since it is not necessary to supply a medium or the like, TG can be easily produced at low cost. Since the accumulation ratio of TG is high, TG can be efficiently collected from microalgae cells, and TG can be produced efficiently. In addition to culturing microalgae under dry stress load conditions, TG can be preferentially accumulated in microcells by further culturing under sulfur-deficient stress load conditions, thereby producing TGs efficiently. It is also possible.

Claims (13)

A method for producing TG, comprising culturing microalgae and collecting and producing triacylglycerol (TG) from the culture,
In a culturing step of culturing a species of microalgae capable of accumulating an aliphatic compound containing at least one of oil and fat and aliphatic hydrocarbon by photosynthesis, and accumulating an aliphatic compound containing at least one of oil and fat and aliphatic hydrocarbon, A method for producing TG, comprising a culture step of preferentially accumulating TG by culturing microalgae under dry stress load conditions.   Microalgae are cultured after dehydration treatment, cultured on a solid in a gas phase, or cell liquid of high-concentration microalgae is gradually dried, and the microalgae are cultured under dry stress load conditions. The manufacturing method of TG of Claim 1.   The manufacturing method of TG of Claim 1 or 2 with which culture | cultivation is further performed on sulfur deficiency stress loading conditions.   The manufacturing method of TG of Claim 3 with which culture | cultivation is performed by a sulfur deficiency stress load condition with the culture solution of sulfur deficiency.   The method for producing TG according to any one of claims 1 to 4, wherein the culture is performed while being aerated, and is performed in a photoautotrophic manner using natural light or artificial light.   The method for producing TG according to claim 5, wherein the culture is performed by applying sunlight or light irradiation as a light source for photoautotrophic culture. The TG according to any one of claims 1 to 6, wherein the TG is cultured using, as a carbon source, organic substances in cells of microalgae, or CO _{2 in} the air or extracellular organic substances. Manufacturing method.   The manufacturing method of TG in any one of Claim 1 to 7 which culture | cultivates the micro algae which precultured and raised the density | concentration. The method for producing TG according to any one of claims 1 to 7, wherein the microalgae are edible microalgae resistant to drying conditions. The TG of any one of claims 1 to 9 , wherein the microalgae is selected from the group consisting of broadly defined green algae including treboxya algae, diatoms, true-point algae, red algae, cyanobacterium, and Euglena algae. Production method. The method for producing TG according to claim 10, wherein the microalgae is a broad green algae containing treboxya algae. The method for producing TG according to claim 10 , wherein the microalgae is an edible microalgae selected from the genus Chlorella, Donariella, Spirulina, and Euglena. The method for producing TG according to claim 12 , wherein the microalga is a genus Chlorella.