JP2015124239A - Method of extracting lipid from algae - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to break the cell wall of microalgae and efficiently recover lipid from algae.SOLUTION: A method of extracting lipid from algae comprises: a process of dissolving carbon dioxide into dispersion liquid containing microalgae; and a process of breaking microalgae by adding shear force to microalgae by a high-pressure dispersion apparatus after the process of dissolving carbon dioxide. The process of dissolving carbon dioxide is performed at the temperature of 0°C or higher and 45°C or lower and the pressure of 0.08 MPa or more and 0.5 MPa or less in absolute pressure.

Description

  The present disclosure relates to a method for extracting lipids from algae.

  In recent years, awareness and interest in environmental issues such as global warming has increased. For this reason, various investigations have been conducted on reducing carbon dioxide emissions and reducing the concentration of carbon dioxide in the atmosphere by fixing carbon dioxide. For example, attempts to use biomass, which is a carbon neutral resource instead of fossil fuel, as an energy source have been actively made.

  Algae are known to produce lipids. If lipids produced by algae can be extracted efficiently, it can be an energy source to replace fossil fuels. However, cell walls are a major obstacle to extracting lipids from algae. In general, algae cell walls are hard and flexible and cannot be easily crushed.

  As a method for crushing the cell wall of algae, for example, in Patent Document 1, hematococcus algae is suspended in a 5% to 85% ethanol aqueous solution, and the adjusted slurry is pressurized by a pump and passed through a gap to thereby remove hematococcus algae. A method of crushing is described.

  Patent Document 2 describes a method for pulverizing bacterial cells, characterized in that the bacterial cell culture solution is mixed with a dissolving solvent in a supercritical state or a pseudocritical state, and then the pressure is instantaneously reduced.

JP 2009-131219 A JP 62-285777 A

  However, Patent Document 1 has a problem that a sufficient cell crushing rate cannot be obtained, and it is difficult to efficiently recover intracellular components. Moreover, in patent document 2, although filamentous fungi are mentioned as an example of the microbial cell which can be the object of this method, it is not clear whether it can be applied to microalgae having a hard cell wall.

  An object of the present disclosure is to disrupt cell walls of microalgae so that lipids can be efficiently recovered from algae.

  The method for extracting lipids from algae according to the present disclosure applies a shearing force to a microalgae using a high-pressure dispersion device after the steps of dissolving carbon dioxide in a dispersion containing microalgae and dissolving the carbon dioxide. Thus, the step of crushing microalgae and dissolving carbon dioxide is performed at a temperature of 0 ° C. or higher and 45 ° C. or lower and a pressure of 0.08 MPa or higher and 0.5 MPa or lower in absolute pressure.

  According to the method for extracting lipid from algae of the present disclosure, the cell wall of microalgae can be crushed and lipids can be efficiently recovered from algae.

<Microalgae>
In the present disclosure, the microalgae refers to those having a cell size of 1 μm to 100 μm in diameter, except for moss plants, ferns, and seed plants from organisms performing photosynthesis that generate oxygen. The cell size is the major axis diameter of a cell measured with an optical microscope at an observation magnification of 400 times. Examples of microalgae include green algae plant gate, red plant gate, unequal hair plant gate, hapto plant gate, dinoflagellate plant gate, and Euglena plant gate (Nobu Watanabe, “Algae Handbook”, Inc. NTS, p.493).

  Specific examples of the microalgae of the green algal plant include Chlorophyceae class, Trebaux phyceae class, Cyanophyceae class, and Prasinophyceae class. Specific examples of the microalgae of the Chlorophyceae class include the Antrotrodesmus genus, the Chlorococcum genus, the Dunaliella genus, the Haematococcus genus, the Scenedesmus genus, the Desmodesmus genus, and the like. Specific examples of the microalgae of the Trebouxiophyceae class include the genus Botryococcus, the genus Chlorella, and the genus Oocystis. Specific examples of microalgae of the Cyanophyceae class include the Spirulina genus. Specific examples of the microalgae of the Prasinophyceae class include the genus Tetraselmis. Specific examples of the microalgae of the trichomes are Bacillarophyceae class, Eustigmatophyceae class, and the like. Specific examples of the microalgae of the class Bacillarophyceae include the genus Chaetoceros, the genus Nitzschia, the genus Skeletonema, and the like. Specific examples of the microalgae of the Eustigmatophyceae class include the genus Nanonochloropsis. Specific examples of the microalgae of the haptophyta include the Pavlovhyceae class, the Pavlova genus, the Prymnesiophyceae class Isochrysis genus, and the like. Specific examples of the microalgae of the dinoflagellate plant include the genus Crypthecodinium and the genus Symbiodinium. Among these, from the viewpoint of lipid productivity and lipid recoverability, Chlorella, Nannochloropsis, and Symbiodinium are preferable, and Nannochloropsis is more preferable. The microalgae may be collected from swamps, ponds, etc., cultured, or commercially obtained.

<Dispersion>
In the present disclosure, the liquid for dispersing the microalgae is not particularly limited, but water is preferable from the viewpoint of dispersibility and economical efficiency. The water may be purified or may contain impurities. Moreover, seawater may be sufficient. The dispersion is an additive comprising any one or more of a salt such as sodium chloride, a compound containing nitrogen or phosphorus, a trace metal, an inorganic flocculant, an organic flocculant, a chelating agent, and a buffer. May be included.

  When microalgae are cultured with a culture solution containing water as a main component, the culture solution of microalgae can be treated as it is. In this case, the dispersion may contain various components contained in a normal culture solution. Further, the treatment may be performed after adding other components to the culture broth, or the culture broth may be diluted with water or an aqueous solution containing a predetermined additive and then treated. You may process after replacing with the aqueous solution containing a thing.

  The concentration of the microalgae in the dispersion depends on the type of microalgae used, but is preferably 1 g / L or more, more preferably 10 g / L or more, from the viewpoint of improving the processing speed and lipid recovery. / L or more is more preferable. Moreover, from the viewpoint of lipid recovery and dispersion fluidity, it is preferably 220 g / L or less, more preferably 180 g / L or less, still more preferably 120 g / L or less, and even more preferably 80 g / L or less. The method for adjusting the density is not particularly limited. Examples of the concentration method include filtration, centrifugation, gravity sedimentation, coagulation sedimentation, and drying. Examples of the dilution method include a method of adding a liquid such as water. In addition, the density | concentration of the micro algae in a dispersion liquid is the value measured by the method described in the Example.

<Lipid>
In the present disclosure, lipids include simple lipids, complex lipids and derived lipids. Simple lipids include esters of fatty acids such as fats and oils or fatty acid esters and various alcohols. The complex lipid includes a phospholipid containing a fatty acid, alcohol and phosphoric acid, and a glycolipid containing a fatty acid, alcohol and sugar. Derived lipids are hydrolysis products of simple lipids or complex lipids, and include water-insoluble fatty acids, higher alcohols, sterols, terpenes, and fat-soluble vitamins. From the viewpoint of lipid recoverability, simple lipids or complex lipids are preferred, simple lipids are more preferred, and fats and oils are even more preferred.

-Oil-
Oils and fats mean esters of fatty acids and glycerin, specifically, neutral lipids such as monoglycerides, diglycerides, and triglycerides. The fatty acid which comprises fats and oils may not be single.

-Fatty acid-
The fatty acid may be any of short chain fatty acids having 2 to 4 carbon atoms, medium chain fatty acids having 5 to 12 carbon atoms, and long chain fatty acids having 12 or more carbon atoms. Moreover, saturated fatty acid or unsaturated fatty acid may be sufficient. Specific examples of the saturated fatty acid include decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, and icosanoic acid. Specific examples of the monovalent unsaturated fatty acid include 9-hexadecenoic acid and 9-octadecenoic acid. Specific examples of the polyunsaturated fatty acid include 9,12-octadecadienoic acid, 6,9,12-octadecatrienoic acid, 5,8,11,14-icosatetraenoic acid, 9,12,15-octa Decatrienoic acid, 5,8,11,14,17-icosapentaenoic acid, and 4,7,10,13,16,19-docosahexaenoic acid.

-Fatty acid ester-
Fatty acid esters are esters of fatty acids and alcohols other than fats and oils, waxes that are esters of long-chain fatty acids and mono- or divalent higher alcohols, and esters of medium-chain fatty acids and lower or higher alcohols. Includes medium chain fatty acid esters and the like.

<Extraction of lipids from microalgae>
The method for extracting lipids from algae of the present disclosure includes applying a shearing force to the microalgae using a high-pressure dispersion apparatus after the steps of dissolving carbon dioxide in the dispersion containing the microalgae and dissolving the carbon dioxide. And crushing the cell wall of microalgae. The step of dissolving carbon dioxide is performed at a temperature of 0 ° C. or higher and 45 ° C. or lower and a pressure of 0.08 MPa or higher and 0.5 MPa or lower in absolute pressure.

  In the method for extracting lipids from algae according to the present disclosure, carbon dioxide is dissolved in a dispersion containing microalgae, and then a shearing force is applied to the microalgae using a high-pressure dispersion device. When a shearing force is applied by a high-pressure dispersing device, it is considered that a force due to carbon dioxide foaming is also applied to the microalgae. For this reason, it is thought that the crushing efficiency of microalgae can be improved.

  When the cell wall is crushed, the lipids in the microalgae are eluted in the dispersion. The lipid eluted in the dispersion can be easily recovered.

-Dissolution of carbon dioxide-
The gas dissolution treatment for dissolving carbon dioxide in the dispersion may be performed, for example, by mixing carbon dioxide and the dispersion. By mixing carbon dioxide and the dispersion, carbon dioxide can be dissolved in the dispersion and the concentration of carbon dioxide in the dispersion can be increased. Mixing of carbon dioxide and the dispersion may be performed by putting the dispersion in an open container and venting carbon dioxide, or by putting the dispersion in a sealed container and venting carbon dioxide. Carbon dioxide and the dispersion liquid may be mixed in a sealed container by applying pressure. In the carbon dioxide dissolution treatment, stirring or the like may be performed so that the contact area between the dispersion and carbon dioxide increases. Further, instead of directly dissolving carbon dioxide in the dispersion, gas dissolution treatment may be performed by mixing another liquid in which carbon dioxide is dissolved at a high concentration with the dispersion.

  The temperature at which the carbon dioxide and the dispersion are mixed is 45 ° C. or less, preferably 30 ° C. or less, more preferably 15 ° C. or less, still more preferably 10 ° C. or less, and still more preferably, from the viewpoint of lipid recoverability. It is 8 ° C. or lower, and from the viewpoint of workability and economy, it can be 0 ° C. or higher, preferably 2 ° C. or higher, more preferably 3 ° C. or higher.

  The mixing of the carbon dioxide and the dispersion is, from the viewpoint of lipid recovery and workability, the absolute pressure of the dispersion, 0.08 MPa or more, preferably 0.09 MPa or more, more preferably 0.095 MPa or more, More preferably, it can be 0.098 MPa or more. Further, from the viewpoint of economy such as suppressing the equipment cost and pressurization cost, the absolute pressure is 0.5 MPa or less, preferably 0.3 MPa or less, more preferably 0.2 MPa or less, and further preferably 0.11 MPa or less. be able to. The pressure when mixing the carbon dioxide with the dispersion is an absolute pressure of 0.08 MPa to 0.5 MP, preferably 0.09 to 0.3 MPa, more preferably 0.095 to 0.2 MPa, and still more preferably 0.8. The pressure can be 098 to 0.11 MPa, particularly preferably atmospheric pressure (0.1 MPa). In the method of this embodiment, carbon dioxide is dissolved at a low pressure. For this reason, since the equipment cost and pressurization cost accompanying a large-sized pressurization pump, a pressure vessel, etc. used for the technique which dissolves carbon dioxide at high pressure are unnecessary, the method of this embodiment is industrially easy and inexpensive. It is also excellent in that it can be implemented on a scale.

  The mixing time for mixing the carbon dioxide and the dispersion can be 1 minute or longer, preferably 10 minutes or longer, more preferably 20 minutes or longer, from the viewpoint of lipid recovery. Moreover, from an economical viewpoint, it can be 120 minutes or less, Preferably it is 60 minutes or less, More preferably, it can be 40 minutes or less.

  When mixing carbon dioxide and dispersion liquid by aeration of carbon dioxide through the dispersion liquid, use general equipment such as single-hole nozzle, multi-nozzle, perforated pipe, sparger, air stone, or tube. Can be performed by bubbling. In addition, when carbon dioxide and the dispersion liquid are stirred in a closed container, they can be mixed using a stirring blade or the like, or mixed by rotating or shaking the closed container. Among these, from the viewpoint of the solubility of carbon dioxide, it is preferable to conduct carbon dioxide through the dispersion, and it is more preferred to conduct carbon dioxide through the dispersion while stirring.

  In the case where carbon dioxide is passed through the dispersion, from the viewpoint of lipid recoverability, it can be 50 mL / min or more, preferably 100 mL / min or more, more preferably 150 mL / min or more per liter of the dispersion. Moreover, from an economical viewpoint, it can be 1000 mL / min or less, Preferably it is 500 mL / min or less, More preferably, it can be 250 mL / min or less.

  Carbon dioxide may be pure carbon dioxide, may contain impurities, or may be a mixed gas with other gases.

−Fracture of microalgae−
The crushing process for crushing microalgae can be performed by using a high-pressure dispersion apparatus. The high-pressure disperser basically passes through a narrow channel in a pressurized state with a raw material liquid containing dispersoids such as solid particles and droplet particles, and then rapidly decompresses the solid particles, droplet particles, etc. Is a device for further dispersing or pulverizing the dispersoid. A high pressure pump can be used to pressurize the raw material liquid. The gap of the flow path through which the raw material liquid passes varies depending on the pressure applied, etc., but it cannot be generally stated, but the diameter of the straight pipe may be 1 μm to 2000 μm, and the orifice having a hole of 1 μm to 2000 μm is straight pipe Alternatively, a slit having a gap of 1 μm to 2000 μm may be disposed and formed in the middle of the straight channel. Further, the gap may be set to 1 μm to 2000 μm by forming a flow path in the gap between the tip of the valve and the valve receiver and adjusting the opening of the valve. Furthermore, the flow paths in the gap of 1 μm to 2000 μm may be arranged to face each other, and the raw material liquids may collide with each other. For example, the direction of the flow path may be suddenly changed, for example, by bending it at right angles, and the raw material liquid may collide with the wall surface of the flow path.

  As the high-pressure disperser, pressure homogenizer (SMT Co., Ltd.), high-pressure homogenizer (Izumi Food Machinery Co., Ltd.), minilab 8.3H type (Rannie), microfluidizer (Microfluidics), Nanovaita (S-G Engineering Co., Ltd.) Company, Yoshida Machine Industry Co., Ltd.), Starburst (Sugino Machine Co., Ltd.), Optimizer (Sugino Machine Co., Ltd.), Genus PY (Hakusui Chemical Co., Ltd.), DeBEE2000 (Japan BEE Co., Ltd.), and the like. Among these, a pressure type homogenizer is preferable from the viewpoint of mass processing in industrialization.

  Specifically, in the crushing process, after applying a high pressure to the dispersion, it is passed through a narrow channel that becomes a gap and rapidly reduced to near atmospheric pressure, and a high shear force is applied to the microalgae in the dispersion. This can be done by adding. Further, the microalgae that have passed through the gap may collide with the wall surface or the like. The pressure applied to the dispersion (inlet pressure) is 10 MPa or more, preferably 30 MPa or more, more preferably 50 MPa or more, and still more preferably 80 MPa or more in terms of gauge pressure from the viewpoint of algal crushability and lipid recovery. From the viewpoint of economy, it is preferably 200 MPa or less, more preferably 150 MPa or less, and still more preferably 120 MPa or less. From the viewpoint of algal crushability, lipid recoverability, and economic efficiency, the pressure (exit pressure) after depressurization can be set to atmospheric pressure (0.1 MPa in absolute pressure). Depending on the structure of the flow path and the like, the pressure may not be completely reduced to atmospheric pressure, and the outlet pressure may be 0.3 MPa or less, 0.2 MPa or less, 0.15 MPa or less in absolute pressure. 0.11 MPa or less.

  Although the temperature difference between the temperature before the crushing operation of the microalgae and the temperature after the crushing operation varies depending on the apparatus and the operating conditions thereof, it is preferably 10 ° C. or higher, for example, from the viewpoint of algal crushability and lipid recovery. Can be 15 ° C. or higher, more preferably 20 ° C. or higher. Further, from the viewpoint of cost, it is preferably 80 ° C. or lower, more preferably 60 ° C. or lower, and further preferably 40 ° C. or lower. The temperature before the crushing operation is the temperature of the dispersion immediately before being input to the crushing apparatus, and the temperature after the crushing operation is the temperature of the dispersion immediately after being discharged from the crushing apparatus.

  The microalgae crushing process may be performed once or a plurality of times. When crushing is performed multiple times, the carbon dioxide dissolution process may be performed only before the first crushing process, or the carbon dioxide dissolution process may be performed before all the crushing processes. A carbon dioxide dissolution treatment may be performed before the treatment.

  The crushing process may be a continuous process or a batch process. Moreover, you may perform a gas dissolution process and a crushing process continuously. However, there may be a step of temporarily storing the dispersion between the gas dissolution treatment and the crushing treatment. From the viewpoint of maintaining the carbon dioxide concentration in the dispersion, the storage temperature of the dispersion is preferably equal to or lower than the temperature during the gas dissolution treatment. Moreover, in order to avoid freezing of a dispersion liquid, it is preferable that it is 0 degreeC or more. Moreover, it is preferable to store in a sealed container, and carbon dioxide may be enclosed in the container.

-Recovery of lipids-
The lipid recovery method is not particularly limited as long as the lipid can be separated from the dispersion containing the crushed fine algae. For example, solvent extraction, centrifugation, stationary treatment, column chromatography and the like can be mentioned. These 1 type (s) or 2 or more types can be combined and applied. Among these, from the viewpoint of lipid recoverability, one or a combination of two or more selected from solvent extraction, centrifugation, and stationary treatment is preferable, and in particular, a combination of solvent extraction and centrifugation, or solvent extraction and stationary treatment. The combination with is preferable.

  Solvent extraction may be performed by adding an extraction solvent to the dispersion after the microalgae are crushed. Stirring may be performed after adding the solvent. Since the lipid eluted from the microalgae is dissolved in the solvent, the lipid can be recovered by separating the solvent phase from the aqueous phase and recovering the solvent phase.

  Examples of the solvent used for solvent extraction include esters such as methyl acetate and ethyl acetate; chain and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; dichloromethane, chloroform, and tetrachloride. Halogenated hydrocarbons such as carbon; hydrocarbons such as hexane, cyclohexane, and petroleum ether; aromatic hydrocarbons such as benzene and toluene; pyridines; butanol, pentanol, hexanol, and isopropyl alcohol (2- Alcohols such as propanol); polyhydric alcohols such as butylene glycol; ketones such as methyl ethyl ketone; and supercritical carbon dioxide. These may be used alone or in combination of two or more.

  Among these solvents, nonpolar solvents are preferable from the viewpoint of lipid recoverability. Specific examples of the nonpolar solvent include halogenated hydrocarbons, hydrocarbons, or aromatic hydrocarbons. Among them, hydrocarbons are preferable, and hexane is more preferable. Further, a solvent compatible with water such as methanol, ethanol, propanol, ethylene glycol, propylene glycol, and acetone can be supplementarily added to the nonpolar solvent. Further, supercritical extraction using supercritical carbon dioxide or the like can also be used. Further, dipping, decoction, leaching, reflux extraction, subcritical extraction, or the like can be used. For example, the method described in “Biochemical Experimental Method 24 Plant Lipid Metabolism Experimental Method” (edited by Yasuhiro Yamada, Society Publishing Center, p. 3-4) can be referred to.

  The temperature for solvent extraction is not particularly limited, but is preferably 10 ° C. or higher, more preferably 20 ° C. or higher, from the viewpoint of lipid recoverability. Moreover, from the viewpoints of economics such as the above viewpoint and heating of the solvent, it is preferably 60 ° C. or lower, more preferably 50 ° C. or lower, and even more preferably 40 ° C. or lower.

  Moreover, solvent extraction may be performed once and may be performed twice or more. When solvent extraction is performed twice or more, it may be performed with the same solvent or different solvents.

  Centrifugation can be performed using a general apparatus such as a separation plate type, a cylindrical type, or a decanter type. The centrifugal force in this case is preferably 500 G or more, more preferably 1000 G or more, from the viewpoint of lipid recoverability. Moreover, from an economical viewpoint, Preferably it is 10,000 G or less, More preferably, it is 5000 G or less, More preferably, it can be 2000 G or less.

  The treatment time for centrifugation is preferably 1 minute or longer, more preferably 5 minutes or longer, and even more preferably 10 minutes or longer from the viewpoint of lipid recovery. Moreover, from a viewpoint of economical efficiency, Preferably it is 80 minutes or less, More preferably, it is 40 minutes or less, More preferably, it can be 20 minutes or less.

  The temperature at the time of centrifugation is not particularly limited, but is preferably 10 ° C. or higher, more preferably 15 ° C. or higher, preferably 50 ° C. or lower, more preferably 40 ° C. or lower, from the viewpoint of lipid recovery and economy. It is.

  When combining solvent extraction and centrifugation, the solvent phase and aqueous phase can be separated quickly by centrifugation.

  In the stationary treatment, the reaction solution may be placed in a stationary state until the lipid and the aqueous phase are separated. When combined with solvent extraction, it may be kept stationary until the solvent phase and the aqueous phase are separated.

  According to the method for extracting lipids from algae according to the present disclosure, lipids can be extracted at a high recovery rate from microalgae in which lipids are accumulated in the body by such a simple operation.

  Lipids extracted from microalgae can be used as biofuel such as biodiesel fuel directly or after being subjected to purification treatment or decomposition treatment. Further, it can be used as a raw material for functional foods or pharmaceuticals, a raw material for chemical products, and a raw material for cosmetics.

  The present disclosure will be described in further detail using examples. In the following examples, Nannochloropsis in which triglycerides (TAG) are accumulated in the body will be described as an example of microalgae, but the present disclosure is not limited to TAG and Nannochloropsis.

<Measurement of concentration of microalgae in dispersion (hereinafter also referred to as algal body concentration)>
The dispersion was subjected to suction filtration with a filter (Nippon Pole Supor-450, pore size 0.45 μm) and washed with an equal amount of distilled water. The filter collecting the algal bodies was transferred to an aluminum cup and dried at 105 ° C. for 2 hours. The weight after drying was measured, the tare of the filter was subtracted, and then the algal body concentration (g / L) was obtained. Also, if the dispersion is difficult to filter by suction with a filter, dilute the dispersion to an appropriate concentration, measure the weight after drying, subtract the tare of the filter, and then multiply the dilution factor to obtain the algal body concentration ( g / L). In addition, Daigo artificial seawater SP (made by Wako Pure Chemical Industries) can be used for a dilution liquid.

<Measurement of lipid content in algal cells>
The lipid content of algal cells was analyzed according to the method for extracting lipids from biomaterials (Bligh & Dyer method) reported in 1959 by EGBligh and WJDyer (EGBligh, WJDyer, Canadian journal of biochemistry and physiology, 37 (1959 ), P.911-917).

  100 μL of 1 mg / mL 7-pentadecanone (methanol solution) was added as an internal standard to 0.5 mL of the sample before the treatment. Next, 10 μL of 2N HCl was added, and further 500 μL of chloroform and 0.9 mL of methanol were added. After stirring, the mixture was allowed to stand at 25 ° C. for 30 minutes, and 500 μL of chloroform and 500 μL of 1.5% KCl were added. After stirring, the mixture was centrifuged (centrifugal force: 1500 G, rotation speed: 3000 r / min, temperature: 25 ° C., time: 15 minutes) using a centrifuge, “himacCF7D2” (manufactured by Hitachi Koki Co., Ltd.). Next, the lower chloroform phase was collected and dried using nitrogen. Next, 0.7 mL of 0.5 N KOH-methanol solution (2.8 g of potassium hydroxide, methanol 100 mL) was added, and the mixture was kept at 80 ° C. for 30 minutes for saponification. Add 1 mL of 14% boron trifluoride solution, incubate at 80 ° C. for 10 minutes for methyl esterification, add 1 mL each of solvent and saturated saline and stir, then leave at 25 ° C. for 30 minutes The solvent phase was obtained. Hexane was used as the solvent. The obtained solvent phase was recovered, and the fatty acid ester was identified and quantified by gas chromatography (GC) under the following conditions. The identification of the fatty acid ester was judged based on whether or not the retention time was the same as that of a later-described standard substance. The amount of fatty acid ester detected by GC analysis was calculated based on the internal standard, and the total amount was determined as the amount of lipid in the dry algal cells.

  This chloroform / methanol solvent extraction method is effective for simply measuring the amount of lipid in algal cells, but from the viewpoint of the safety, recoverability, and recyclability of the solvent used, it is not suitable for industrial practical use. This is not a suitable method.

-GC analysis-
Equipment: Agilent technology 7890A
Column: DB1-MS (manufactured by J & W scientific, 20 m × 100 μm × 0.1 μm)
Oven temperature: 150 ° C. (0.5 min hold) − [40 ° C./min]−220° C. (0 min hold) − [20 ° C./min]−320° C. (2 min hold) -post run 2 min
Carrier gas: Hydrogen makeup gas: Helium sample injection amount: 5 μL
Injection method: split mode (split ratio = 75: 1)
Inlet temperature: 300 ° C
Detector: FID
Column flow rate: 0.28 mL / min Constant pressure (gauge pressure): 62.403 psi
Standard substance: The following fatty acid ester manufactured by SIGMA was used.
Methyl laurate (C12), methyl myristate (C14), methyl palmitate (C16), methyl stearate (C18), methyl palmitate (C16: 1), methyl oleate (C18: 1), methyl linoleate ( C18: 2), methyl linolenate (C18: 3), methyl eicosapentaenoate (C20: 5), methyl docosahexaenoate (C22: 6)

<Measurement of cell number>
After injecting 2 μL of the diluted dispersion into the calculation room of a bacterial counter (manufactured by Sunlead Glass), the cells were observed using an optical microscope (Nikon, ECLIPSE80i) at an observation magnification of 400 times, and the number of cells in the block was counted. A 0.05 mm square block was used in the bacterial counter calculation room. In the calculation room, there are 25 aggregates (5 vertical x 5 horizontal) composed of 16 square blocks of 0.05 mm square (vertical 4 squares x lateral 4 squares). Among these aggregates, the number of cells contained in five aggregates present on the diagonal line from the upper right to the lower left of the observation screen of the microscope was counted. That is, the number of cells in 80 squares (16 squares × 5) of a 0.05 mm square block was counted. The depth of the calculation room of the bacteria counter is 0.020 mm, and the volume per block of 0.05 mm square is 1/20000 mm ³ . Divide the total number of cells counted by the volume of the corresponding block (1/20000 mm ³ × 80 squares), and multiply this by the dilution factor of the diluent used for counting to give cells per mL of sample before dilution. I asked for a number. In addition, Daigo artificial seawater SP (made by Wako Pure Chemical Industries) was used for the dilution liquid.

<Calculation of cell disruption rate>
The number of cells in the dispersion was measured by the aforementioned method for measuring the number of cells. The number of cells after the crushing treatment was subtracted from the number of cells before the crushing treatment, divided by the number of cells before the crushing treatment, and a value multiplied by 100 was taken as the cell crushing rate (%).

<Measurement of lipid recovery rate>
Lipids were recovered by solvent extraction as shown below. The collected lipid was converted to methyl ester, and the amount of lipid was quantified by the above-mentioned GC.

-Hexane extraction-
1 mL of hexane is added to 0.5 mL of the treated sample, and the mixture is stirred for 3 minutes at 25 ° C., and then centrifuged (centrifugal force: 1500 G) using a centrifuge, “himacCF7D2” (manufactured by Hitachi Koki Co., Ltd.). , Rotation speed: 3000 r / min, temperature: 25 ° C., time: 15 minutes). Next, 400 μL of the upper hexane phase was collected, 40 μL of 1 mg / mL 7-pentadecanone (methanol solution) was added as an internal standard, and then dried using nitrogen. Next, 0.7 mL of 0.5 N KOH-methanol solution (2.8 g of potassium hydroxide, methanol 100 mL) was added, and the mixture was kept at 80 ° C. for 30 minutes for saponification. Add 1 mL of 14% boron trifluoride solution, incubate at 80 ° C. for 10 minutes for methyl esterification, add 1 mL each of solvent and saturated saline and stir, then leave at 25 ° C. for 30 minutes The solvent phase was obtained. Hexane was used as the solvent. The obtained solvent phase was recovered, and the fatty acid ester was identified and quantified by GC under the above conditions. The amount of fatty acid ester detected by GC analysis was calculated based on the internal standard, and the total amount was determined as the amount of lipid in hexane.

-Calculation of lipid recovery rate-
The lipid recovery rate with a hexane solvent was calculated by the following formula (1).
Lipid recovery rate (%) = lipid amount in hexane / lipid amount in dry alga body × 100 (1)

<Example 1>
As a dispersion liquid of microalgae, 500 g of “Yanmarine K-1” (manufactured by Chlorella Kogyo Co., Ltd., microalgae: Nannochloropsis oculata, liquid constituting the dispersion liquid: seawater, alga body concentration: 50 g / L) is charged into a glass container, and carbon dioxide Was dissolved. Dissolution of carbon dioxide is performed at a flow rate of 200 mL / min using “Ceramic Stone” (manufactured by Ibuki Co., Ltd.) as an air stone while stirring the liquid temperature of the dispersion at 5 ° C. and stirring (50 r / min). Bubbling was done. Bubbling was carried out at an absolute pressure of 0.1 MPa (atmospheric pressure) for 30 minutes. Next, a dispersion in which carbon dioxide is dissolved is charged into a pressure homogenizer (LAB 2000 manufactured by SMT), and the primary (inlet) pressure is 100 MPa in gauge pressure, and the secondary (outlet) pressure is 0.1 MPa in absolute pressure (atmospheric pressure) ) To obtain a cell disruption rate. In addition, the liquid temperature before a pressure type homogenizer process was 3 degreeC, and the liquid temperature after a process was 32 degreeC. The cell disruption rate was 46%. The lipid recovery rate after crushing was 32%.

<Example 2>
As a dispersion liquid of microalgae, 500 g of “Yanmarine K-1” (manufactured by Chlorella Kogyo Co., Ltd., microalgae: Nannochloropsis oculata, liquid constituting the dispersion liquid: seawater, alga body concentration: 50 g / L) is charged into a glass container, and carbon dioxide Was dissolved. Dissolution of carbon dioxide is performed at a flow rate of 200 mL / min using “Ceramic Stone” (manufactured by Ibuki Co., Ltd.) as an air stone while stirring (50 r / min) at a liquid temperature of 25 ° C. Bubbling was done. Bubbling was carried out at an absolute pressure of 0.1 MPa (atmospheric pressure) for 30 minutes. Next, a dispersion in which carbon dioxide is dissolved is charged into a pressure homogenizer (LAB 2000 manufactured by SMT), and the primary (inlet) pressure is 100 MPa in gauge pressure, and the secondary (outlet) pressure is 0.1 MPa in absolute pressure (atmospheric pressure) ) To obtain a cell disruption rate. In addition, the liquid temperature before a pressure type homogenizer process was 25 degreeC, and the liquid temperature after a process was 41 degreeC. The cell disruption rate was 33%.

<Comparative Example 1>
500 g of “Yanmarine K-1” (manufactured by Chlorella Kogyo Co., Ltd., microalgae: Nannochloropsis oculata, liquid constituting the dispersion: seawater, alga body concentration: 50 g / L) as a microalgae dispersion is a pressure homogenizer (LAB2000 manufactured by SMT) ), The primary (inlet) pressure was 100 MPa as the gauge pressure, and the secondary (outlet) pressure was as absolute pressure as 0.1 MPa (atmospheric pressure) in one pass to determine the cell disruption rate. In addition, the liquid temperature before a pressure type homogenizer process was 25 degreeC, and the liquid temperature after a process was 41 degreeC. The cell disruption rate was 20%. The lipid recovery rate after crushing was 16%.

<Comparative Example 2>
As a dispersion liquid of microalgae, 50 g of “Yanmarine K-1” (manufactured by Chlorella Kogyo Co., Ltd., microalgae: Nannochloropsis oculata, liquid constituting the liquid dispersion: seawater, alga body concentration: 50 g / L) is charged into a 100 mL pressure vessel. Then, carbon dioxide gas was injected to a pressure of 10 MPa with a gauge pressure, held at 45 ° C. for 30 minutes, and then the container was released to extract the carbon dioxide gas. The cell disruption rate was 0%.

  From Table 1, it can be seen that only the high-pressure dispersion treatment of Comparative Example 1 has a low microalgae crushing efficiency and a low lipid recovery rate. Moreover, it turns out that a micro algae cannot be crushed well also in the process which melt | dissolves the carbon dioxide of the supercritical state of the comparative example 2, and reduces a pressure instantaneously. On the other hand, in the method of the present disclosure in which carbon dioxide is not dissolved at a high pressure as in the supercritical state but carbon dioxide is dissolved at a low pressure in the embodiment, and then the high-pressure dispersion treatment is performed, the cell wall of the microalgae is effectively removed. It can be seen that lipids can be efficiently crushed and recovered from algae.

Claims (5)

Dissolving carbon dioxide in a dispersion containing microalgae;
A step of crushing the microalgae by applying a shearing force to the microalgae with a high-pressure dispersing device after the step of dissolving the carbon dioxide,
The step of dissolving carbon dioxide is a method for extracting lipid from algae, wherein the temperature is 0 ° C. or higher and 45 ° C. or lower, and the pressure is 0.08 MPa or higher and 0.5 MPa or lower in absolute pressure.   The method for extracting lipids from algae according to claim 1, wherein in the step of dissolving carbon dioxide, the temperature is 2 ° C. or higher and 15 ° C. or lower.   The difference between the temperature of the dispersion immediately after performing the step of crushing the microalgae and the temperature of the dispersion immediately before performing the step of crushing the microalgae is 20 ° C. or more and 80 ° C. or less. A method for extracting lipids from the algae according to claim 1 or 2.   In the step of crushing the microalgae, lipid is extracted from the algae according to any one of claims 1 to 3, wherein a pressure of 10 MPa or more is applied at a gauge pressure, and then the pressure is reduced to 0.3 MPa or less with a gauge pressure. how to.   The method for extracting lipids from algae according to any one of claims 1 to 4, further comprising a step of recovering lipids from the dispersion after the step of crushing the microalgae.