WO2021015089A1 - Procédé et système pour rompre la coque externe d'une micro-algue par cisaillement - Google Patents

Procédé et système pour rompre la coque externe d'une micro-algue par cisaillement Download PDF

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WO2021015089A1
WO2021015089A1 PCT/JP2020/027664 JP2020027664W WO2021015089A1 WO 2021015089 A1 WO2021015089 A1 WO 2021015089A1 JP 2020027664 W JP2020027664 W JP 2020027664W WO 2021015089 A1 WO2021015089 A1 WO 2021015089A1
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shearing
microalgae
outer cover
treatment liquid
destroying
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PCT/JP2020/027664
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English (en)
Japanese (ja)
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林　信行
土井　研一
裕子 陣内
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国立大学法人佐賀大学
株式会社ミゾタ
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Priority to JP2020560292A priority Critical patent/JPWO2021015089A1/ja
Publication of WO2021015089A1 publication Critical patent/WO2021015089A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/33—Disintegrators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

  • the present invention relates to a method for destroying the outer cover of microalgae by shearing and a system for destroying the outer cover. More specifically, a treatment solution for intracellular useful substances of microalgae having a strong coat (algal skin such as cell wall, cell membrane, cyst membrane) can be treated efficiently in a short time without deteriorating the quality thereof. It relates to a method for destroying the outer cover of microalgae by shearing and a system for destroying the outer cover, which can be leaked into the cell.
  • microalgae there are useful algae such as hematococcus that produces astaxanthin in the algae and nannochlorocyps that produces EPA, and the contents of these algae are extracted from the cultured algae.
  • Microalgae are a kind of unicellular plants and have cell walls (except for some species such as Euglena), so naturally, in order to take out the contents, the outer cover such as cell walls and cell membranes must be destroyed. It doesn't become. Therefore, in many current processes (dry crushing process), the algae are dried by spray drying, drum drying, ventilation drying, etc., and then mechanically crushed using an atomizer, disc mill, ball mill, etc. The actual situation is that it is extracted with a solvent or the like.
  • the outer cover of the dried algae is crushed by some crushing method so that the solvent can be in contact with the cell contents and dissolved. Need to be crushed.
  • it is very difficult to destroy the outer cover of microalgae because the cell size is very small, several tens of microns, and the cell wall is composed of a tough polysaccharide such as cellulose.
  • microalgae are strongly expected to be used not only as raw materials for extracting useful substances as described above, but also as raw materials for food materials, feeds and fertilizers, and as oils produced for cosmetics and fuel oils.
  • it is being put to practical use. This is because microalgae grow faster than ordinary plants, the yield per unit area is large, carbon dioxide fixation is high, harvesting is possible throughout the year, and crops cannot be produced if a culture tank can be installed. This is because it can be cultivated even in wasteland. However, even in these cases, the algae coat is a cause of reducing various efficiencies.
  • a natural product containing astaxanthin is suspended in an organic solvent, extracted in an organic solvent while being crushed by a crusher, and then the solid substance is removed. Further, a step of extracting astaxanthin by removing an organic solvent (wet crushing step) is also known (see, for example, Patent Document 1).
  • a freeze crushing method in which the slow freezing of the algae promotes the growth of ice crystals in the intracellular fluid and destroys the outer cover from the inside of the cell.
  • the inventor of the present application conducted an experiment in which the cystized hematococcus was cooled at a temperature lowering rate of 1.0, 1.5, 2.0 ° C./min, and the obtained treated cells were observed under a microscope, the outside was found.
  • the destruction rate of the cover was at most 50% or less. This suggests that the volume increase due to the phase change of the intracellular fluid was within the elastic limit of the jacket.
  • the residence time is 1 to 30 minutes, preferably 2 to 15 minutes
  • the peripheral speed is 2 to 30 m / s, preferably 8 to 12 m / s
  • the bead diameter used is 0.2 to 5 mm.
  • It is preferably 0.5 to 2 mm (see, for example, Patent Document 1 [0026]).
  • the bead mill crushes the object by so-called point contact such as collision or shearing between beads, and like the above dry crushing step, mechanical energy is transmitted only to a part of the crushed object, and hematococcus. It is considered that it is not very effective against the crushing of.
  • an object of the present invention is to increase the coating of microalgae, which is a barrier during drying and extraction, in vivo digestion and composting.
  • a treatment liquid containing cells producing or containing a useful substance is pumped by a pump (2) and arranged opposite to each other.
  • the surface gap (47) formed by the immovable first surface (41b) and the movable second surface (42a) is passed, and the surface gap (47) is changed while changing the direction of the flow of the treatment liquid. 47) is passed through, and the outer cover of the cells is destroyed in the treatment liquid by the shearing force generated between the flow of the treatment liquid and the first surface (41b) or the second surface (42a).
  • the treatment liquid pumped by the pump (2) passes through a narrow surface gap (47) formed between the first surface (41b) and the second surface (42a), thereby passing the treatment liquid.
  • the strong shearing force generated between the flow and the narrow flow path surfaces (41b, 42a) will continue to act on the algae algae in the treatment solution.
  • the pressure of the treatment liquid fluctuates due to compression (pressure increase) and expansion (decompression) in the portion where the flow direction of the treatment liquid changes.
  • the algae algae in the treatment liquid are subjected to a load due to pressure fluctuation in addition to the above shearing action.
  • the outer cover of the algae algae in the treatment liquid is caused by the strong shearing force generated between the flow of the treatment liquid and the narrow flow path surfaces (41b, 42a). Will be destroyed and useful substances in the cells will leak from the cells into the treatment solution.
  • the second feature of the method for destroying the outer cover of microalgae by shearing according to the present invention is a narrow path in which the flow path of the treatment liquid is narrowed before the treatment liquid passes through the surface gap (47). It is to pass 41c).
  • the third feature of the method for destroying the outer cover of microalgae by shearing according to the present invention is that when the treatment liquid passes through the surface gap (47), the second surface is closed in the direction of closing the surface gap (47). (42a) is pressurized under a predetermined pressure condition.
  • the treatment liquid pushes away (pushes up) the second surface (42a) to form a gap through which it flows, while forming a surface gap (47) between the first surface (41b) and the second surface (42a). ) Will be passed.
  • the algae algae in the treatment liquid receive a stronger shearing action than when the surface gap (47) is fixed.
  • the fourth feature of the method for destroying the outer cover of microalgae by shearing according to the present invention is that the treatment liquid passes through the narrow path (41c) and the surface gap (47).
  • the algae algae in the treatment liquid are subject to "strong shearing force generated between the flow of the treatment liquid and the narrow flow path surface” and “load due to pressure fluctuation”.
  • the fifth feature of the method for destroying the outer cover of microalgae by shearing according to the present invention is that the treatment liquid is pumped by the reciprocating pump (2).
  • the discharge pressure of the reciprocating pump can apply the fluid energy (head) required for passing the processing liquid through the narrow path (41c) and the narrow surface gap (47) to the processing liquid.
  • the sixth feature of the method for destroying the outer cover of microalgae by shearing according to the present invention is to freeze-dry the treated liquid that has passed through the surface gap (47).
  • the seventh feature of the method for destroying the outer cover of microalgae by shearing according to the present invention is that the frozen treatment liquid is placed in an environment depressurized from atmospheric pressure to be dried.
  • the external body destruction system of microalgae by shearing includes a tank (1) for storing a treatment liquid containing cells producing or containing a useful substance, and a pump for pumping the treatment liquid.
  • a microalgae hull destruction system including (2) and a shearing treatment unit (4) for crushing the jacket of the cells, wherein the shearing treatment unit (4) transfers a pumped treatment liquid.
  • a second groove (42d) that can be fitted into the first groove (41d) is formed on the surface of the second surface (42a), and the treatment liquid is formed on the first surface (41b) and the first surface (41b). It is characterized by passing through a surface gap (47) formed between the two surfaces (42a).
  • the second feature of the external cover destruction system for microalgae by shearing according to the present invention is that a narrow path (41c) in which the flow path is narrowed is formed in the first flow path (41a).
  • a third feature of the external coating destruction system for microalgae by shearing according to the present invention is an actuator portion (which pressurizes the second surface (42a) against the first surface (41b) under a predetermined pressure condition. 46) is provided.
  • the fourth feature of the external cover destruction system for microalgae by shearing according to the present invention is to include a return pipe (6) connecting the shearing portion (4) and the tank (1).
  • the fifth feature of the external coating destruction system for microalgae by shearing according to the present invention is that the pump (2) has a cylinder (33), a piston (21) reciprocating in the cylinder (33), and the cylinder (2).
  • the sixth feature of the external coating destruction system for microalgae by shearing according to the present invention is a low temperature tank (10) for freezing the treatment liquid that has passed through the surface gap (47), and the frozen treatment liquid. It is provided with a freeze-dryer (11) for drying.
  • the seventh feature of the external coating destruction system for microalgae by shearing according to the present invention is that the freeze-dryer (11) dries the frozen treatment liquid in an environment depressurized from atmospheric pressure.
  • useful substances can be efficiently and quickly obtained from cells having a strong coat (cell wall, cell membrane, cyst membrane, etc.). It is possible to leak from the cells into the treatment solution without deteriorating the quality.
  • the algae contents leak from the cells into the treatment solution due to the destruction of the outer cover of the microalgae
  • the algae contents (useful substances) can be easily separated and purified.
  • useful components are contained in the living body as compared with the case where the suspension is dried without shear fracture. Can be efficiently absorbed and used.
  • the sheared suspension is fertilized into compost or the like without separating the leaked contents and the crushed jacket, it is possible to improve the fermentation efficiency.
  • drying time (drying rate) of the extracted algae contents (useful substances) is shortened by separating and removing the outer cover of the microalgae, which is a barrier to the movement of water, from the treated liquid. (Speeding up), which makes it possible to reduce the drying energy for the algae contents (useful substances).
  • 6 is a photomicrograph showing a state when shearing treatment was performed for 0.1 second, 0.2 second, and 0.3 second under a pressure condition where the applied pressure to the hematococcus cyst suspension was 70 MPa.
  • 6 is a photomicrograph showing a state when shearing treatment was performed for 0.1 second, 0.3 second, and 0.4 second under a pressure condition where the applied pressure to the hematococcus cyst suspension was 50 MPa.
  • FIG. 1 is an explanatory view showing a wet shearing treatment system 100 according to the first embodiment of the present invention.
  • This wet shearing treatment system 100 is subjected to a shearing treatment that destroys the outer cover (cell wall, cell membrane, cyst membrane, etc.) of algae cells containing useful substances such as astaxanthin and EPA in the liquid, and is contained in the cells. It is a system for destroying the outer cover of microalgae by shearing to leak useful substances into the liquid.
  • the liquid in which the useful substance has leaked is separated into a useful substance and another substance by a known separation method (for example, centrifugation or membrane filtration), and only the useful substance is extracted.
  • the liquid containing algae will be distinguished as "untreated liquid” if it has not been sheared by the wet shearing system 100 and "treated liquid” if it has been sheared. I will decide. Moreover, when it is not necessary to distinguish in particular, it is simply referred to as "treatment liquid".
  • the wet shearing system 100 includes a reserve tank 1 for storing the untreated liquid or the treated liquid, a pump 2 for pumping the untreated liquid or the treated liquid, and the untreated liquid or the untreated liquid pumped by the pump 2.
  • a shearing treatment unit 4 that performs shearing treatment on the treated liquid, a suction pipe 3 that connects the reserve tank 1 and the pump 2, a discharge pipe 5 that connects the pump 2 and the shearing treatment unit 4, a shearing treatment unit 4 and a reserve. It is configured to include a return pipe 6 for connecting the tank 1.
  • the pump 2 may be any pump as long as it can deliver the processing liquid at a high discharge pressure, and the method of delivering the processing liquid is not particularly limited. In the present embodiment, the pump 2 employs a reciprocating pump.
  • each configuration will be further described.
  • FIG. 2 is an explanatory cross-sectional view of a main part showing the pump 2 used in the present invention.
  • the suction valve 23 and the discharge valve 27 are shown in a closed state.
  • the pump 2 sucks the untreated liquid through the suction valve 23 by the piston 21 reciprocating in the cylinder 33, compresses the untreated liquid in the head space 32, and discharges the compressed untreated liquid from the discharge valve 27.
  • This is a reciprocating pump that pumps to the shearing unit 4 in the subsequent stage.
  • the pump 2 includes a 33, a connecting rod 34 for converting rotational energy into translational energy, and a crankshaft 35.
  • the pump 2 is composed of a single piston 21 for convenience of explanation, the pump 2 may be configured by combining a plurality of the above configurations in series or in parallel.
  • the piston 21 is provided with a piston ring 21a that maintains the liquidtightness in the head space 32. Further, the piston 21 is connected to the connecting rod 34, and the connecting rod 34 is connected to the crankshaft 35.
  • the crankshaft 35 is coaxially connected to an electric motor (not shown). Further, the electric motor is configured so that the rotation speed is controlled by an inverter (not shown). Therefore, the frequency (frequency) per unit time of the piston 21 is configured to be changed by the inverter.
  • the operation of the pump 2 will be described below.
  • FIG. 3 is an explanatory diagram showing a suction process of the pump 2.
  • the head space 32 becomes negative pressure due to the piston 21.
  • the suction valve 23 opens, and the processing liquid is filled into the head space 32 from the in-side connecting portion 26.
  • the discharge valve 27 remains closed.
  • FIG. 4 is an explanatory diagram showing a discharge process of the pump 2.
  • the processing liquid filled in the head space 32 is compressed by the piston 21.
  • the suction valve 23 is closed.
  • the discharge valve 27 opens.
  • the compressed processing liquid is pumped from the discharge valve 27 to the shearing processing unit 4 in the subsequent stage.
  • the suction valve 23 Since the suction valve 23 is in the closed state, the compressed untreated liquid or treated liquid is not pumped to the reserve tank 1. As described above, the suction valve 23 is opened when the "pressure of the head space 32" ⁇ "the pressure of the in-side connecting portion 26", and the discharge valve 27 is in the "pressure of the head space 32"> "out-side connecting portion”. Each function as a check valve that opens when the pressure is 31.
  • FIG. 5 is a cross-sectional explanatory view of a main part showing the shearing processing unit 4 according to the present invention.
  • the shearing unit 4 allows the flow of the processing liquid pumped by the pump 2 to pass through the narrow path 41c or the surface gap 47 (formed between the first surface 41b and the second surface 42a). Shearing action (shearing force) generated between the flow of the treatment liquid and the flow path surface is continuously applied to the algae algae contained in the treatment liquid to produce or contain useful substances such as astaxanthin and EPA. It is a device for breaking the jacket in the treatment liquid and leaking its useful substances into the treatment liquid.
  • the configuration includes a valve seat portion 41 for transferring the processing liquid pumped from the pump 2, a valve portion 42 that stops / prevents the flow of the processing liquid flowing out from the valve seat portion 41, and a valve seat portion 41 and a valve portion.
  • the housing portion 43 accommodating the 42, the inlet portion 44 to which the discharge pipe 5 is connected to take in the pressurized processing liquid and the like, the outlet portion 45 to which the return pipe 6 is connected and the processed liquid flows out, and the valve portion 42 are connected. It is configured to include an actuator unit 46 for pressurizing.
  • the valve seat portion 41 is composed of a straight first flow path 41a and a flange-shaped first surface 41b.
  • the valve seat portion 41 is fixed to the housing portion 43 by the inlet portion 44 in a state of being engaged with the step 43c of the housing portion 43.
  • the tip of the first flow path 41a forms a tapered narrow path 41c (or orifice) with a reduced inner diameter.
  • one or a plurality of first peripheral grooves 41d are formed concentrically along the circumferential direction.
  • the valve portion 42 is composed of a disc-shaped (disc head type) second surface 42a and a side surface 42b, an enlarged diameter tip portion, and a rod-shaped rod portion 42c.
  • the rod portion 42c is provided with an O-ring 42e that holds the liquidtight state of the second flow path 43a.
  • one or a plurality of second peripheral grooves 42d that can be fitted to the first peripheral groove 41d formed on the first surface 41b are formed concentrically along the circumferential direction.
  • the rod-shaped rod portion 42c is connected to the actuator portion 46 and is configured to be displaceable and pressurized in the axial direction (longitudinal direction). Therefore, the clearance of the surface gap 47 formed between the first surface 41b and the second surface 42a is variably configured. Further, the second surface 42a is configured to be pressurized (pressed) by the actuator portion 46 in the direction in which the surface gap 47 is closed.
  • the actuator unit 36 an electric cylinder, a pneumatic cylinder, a spring, or the like can be used as the actuator unit 36.
  • a gap is formed between the side surface 42b of the valve portion 42 and the inner peripheral surface 43b of the second flow path 43a so that the processing liquid subjected to the shearing treatment can flow.
  • the shearing action on the algae algae in the treatment liquid in the shearing treatment unit 4 will be described.
  • FIG. 6 is an explanatory view showing the shearing action on algae and algae in the treatment liquid by passing the treatment liquid in the narrow path 41c and the surface gap 47 of the shearing treatment portion according to the present invention.
  • the processing liquid pumped by the pump 2 flowing from the inlet 44 flows into the narrow passage 41c through the first flow path 41a.
  • the valve portion 42 is pressurized to the first surface 41b side by the actuator portion 46, the valve portion 42 is in a closed state. Therefore, the flow of the processing liquid is stopped by the valve portion 42.
  • the pressure in the first flow path 41a increases due to the processing liquid pumped from the pump 2.
  • the pressure of the processing liquid in the first flow path 41a becomes equal to the pressing by the actuator portion 46
  • the processing liquid pushes the valve portion 42 to the right side in the drawing, and between the first surface 41b and the second surface 42a. It flows through the formed surface gap 47.
  • the treatment liquid passes through the narrow road 41c, it receives a strong shearing force, which destroys the algae jacket.
  • the treatment liquid flowing through the surface gap 47 formed between the first surface 41b and the second surface 42a passes through the first peripheral groove 41d and the second peripheral groove 42d.
  • the direction of the flow can be changed in a zigzag shape when crossing.
  • the treatment liquid is subjected to a stronger shearing action by changing the direction of flow in a zigzag manner.
  • pressure fluctuations due to compression and expansion also occur in the portion where the flow direction changes. Therefore, when the treatment liquid passes through the surface gap 47 between the first surface 41b and the second surface 42a, it is simultaneously subjected to a load due to shearing force and pressure fluctuation.
  • the processing liquid pumped by the pump 2 flowing in from the inlet portion 44 is initially stopped from flowing by the valve portion 42, but the pressure of the processing liquid in the first flow path 41a is pressed by the actuator portion 46.
  • the valve portion 42 is pushed away to the right side in the drawing to form a surface gap 47 formed between the first surface 41b and the second surface 42a through which the valve portion 42 flows.
  • the treatment liquid passes through the narrow road 41c, it is subjected to a strong shearing action.
  • the flow direction is changed in a zigzag shape by the first peripheral groove 41d and the second peripheral groove 42d. Be made to.
  • the pressure of the treatment liquid also fluctuates. As a result, the treatment liquid is subjected to a strong shearing action and a load due to pressure fluctuation at the same time.
  • the algae jacket can be continuously and efficiently destroyed as compared with the conventional method of mechanically destroying the outer cover of the dry algae in the form of fine particles. Will be.
  • Scenedesmus is a green alga represented by the genus Desmodesmus, and forms a colony in which multiple cells are joined. Since the jacket is very strong, it is said that if a method for efficiently destroying the jacket of Scenedesmus is developed, the jacket of other microalgae can be easily destroyed by that method.
  • FIG. 7 is a photomicrograph showing the Scenedesmus suspension before the shearing treatment according to the present invention.
  • the magnification is 200 times, and the boundary line is 0.5 mm ⁇ 0.5 mm. A state in which 2 to 4 cells are joined is observed.
  • the crushing treatment was carried out by passing a scenedesmus suspension (treatment liquid) having the following concentration through the shearing treatment unit 4 for a predetermined shearing treatment time under the following shearing treatment conditions.
  • the shearing treatment time was represented by an integrated value of the residence time in the shearing treatment section 4.
  • the integrated value was calculated by multiplying the residence time per pass by the number of passes.
  • the residence time per passage was calculated by dividing the internal volume of the shearing unit 4 by the volumetric flow rate of the Scenedesmus suspension.
  • the amount of chlorophyll in the centrifugal supernatant prepared by centrifuging the treated Scenedesmus suspension was indicated by the fluorescence intensity value.
  • FIGS. 8 and 9 are micrographs showing the state of shearing treatment for a predetermined time under a pressure condition where the applied pressure to the scenedesmus suspension is 90 MPa.
  • the magnification is 200 times, and the boundary line is 0.5 mm ⁇ 0.5 mm. It can be seen that the cells that had been joined by the shearing treatment for 0.3 seconds were separated, and that some of the cells had leaked out and became a torn bag-shaped jacket. As the shearing time increased, the number of cells that maintained the morphology of the coat decreased significantly, and almost disappeared in 0.9 seconds. When the photograph after 0.5 seconds is magnified and carefully observed, it is observed that the cracked shell-like outer cover gradually increases, and they are also destroyed by the increase in the shearing time and become small pieces.
  • FIG. 10 is a graph showing the relationship between the shearing force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant.
  • the vertical axis represents the amount of chlorophyll leaked to the outside of the cell (fluorescence intensity value), and the horizontal axis represents the shearing force application time.
  • the solid square marks (1) indicate the shearing force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant when the applied pressure to the Scenedesmus suspension is 60 MPa.
  • the solid circles ⁇ indicate the shearing force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant when the applied pressure to the applied pressure on the Scenedesmus suspension is 90 MPa.
  • the solid triangle mark ⁇ indicates the shearing force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant when the applied pressure to the Scenedesmus suspension is 80 MPa.
  • Hollow circles indicate the shearing force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant when the applied pressure to the Scenedesmus suspension is 70 MPa.
  • the maximum value of fluorescence intensity was about half that of 70 MPa or more. This was considered to be due to the fact that at 60 MPa, algae bodies in which the coat was not completely destroyed remained, and the chlorophyll was centrifuged while being contained in the cells. That is, it was shown that in the wet shearing treatment system 100 according to the present invention, it is preferable to carry out the treatment with an applied pressure of 70 MPa or more. However, it is naturally expected that the required applied pressure will differ depending on the device used, the shape of the valve portion 42, and the like.
  • Hematococcus which is a green alga, usually has a green spherical shape, but when it receives stress such as ultraviolet rays, it accumulates red astaxanthin inside the algae and turns into cysts.
  • This astaxanthin has high added value as a functional substance having strong antioxidant activity and as a natural pigment due to its bright red color, and is produced on a commercial scale as a raw material for health foods and skin care products.
  • the cultured hematococcus cysts are dried by ventilation drying, spray drying, drum drying, etc., and after mechanically crushing the dried algae, the astaxanthin inside is extracted with supercritical carbon dioxide or an organic solvent.
  • This is the conventional method.
  • the conventional method involving heating has a problem that it easily leads to oxidative deterioration of astaxanthin because it comes into contact with high-temperature oxygen during drying (astaxanthin is a natural antioxidant and is very easily combined with oxygen). ..
  • a Hematococcus cyst suspension (treatment liquid) having the following concentration is subjected to a predetermined shearing treatment unit 4 under the following shearing treatment conditions, similarly to the scenedesmus suspension. This was done by passing only the processing time.
  • the shearing treatment time is represented by an integrated value of the residence time in the shearing treatment unit 4. The integrated value was calculated by multiplying the residence time per pass by the number of passes. The residence time per pass was calculated by dividing the internal volume of the shearing unit 4 by the volumetric flow rate of the hematococcus cyst suspension.
  • Hematococcus cyst suspension -Concentration: Approximately 0.4 g / ml by dry weight (Shearing conditions) -Volume flow rate of Hematococcus cyst suspension (discharge flow rate of pump 2): 270 [ml / min] -Pressure applied to the hematococcus cyst suspension (pressing against the valve portion 42): 95 MPa, 70 MPa, 50 MPa (Evaluation of the destruction of the algae coat of Hematococcus cysts): Absorbance method: Absorbance at a wavelength of 470 nm
  • FIG. 11 is a photomicrograph showing the hematococcus cyst suspension before the shearing treatment according to the present invention.
  • FIG. 12 is a photomicrograph showing a state when shearing treatment is performed under a pressure condition where the applied pressure to the hematococcus cyst suspension is 95 MPa.
  • FIG. 13 is a photomicrograph showing a state when shearing treatment is performed under a pressure condition where the applied pressure to the hematococcus cyst suspension is 70 MPa.
  • FIG. 14 is a photomicrograph showing a state when shearing treatment is performed under a pressure condition where the applied pressure to the hematococcus cyst suspension is 50 MPa.
  • the magnification is 200 times and the boundary line size is 0.5 mm ⁇ 0.5 mm.
  • the shearing treatment (shearing fracture treatment) according to the present invention destroys cells by a shearing force, that is, a shearing force, so that the outer cover is in a cracked form.
  • a shearing force that is, a shearing force
  • the shearing force is transmitted using fluid (water) as a pressure medium, the force is evenly transmitted to every corner and has the characteristic of even destruction.
  • FIG. 15 is a photograph showing the state of a hematococcus cyst suspension that has been allowed to stand for 30 minutes or more after the shearing treatment according to the present invention.
  • FIG. 15A shows a hematococcus cyst suspension when shearing treatment is performed for a predetermined time under a pressure condition where the applied pressure to the hematococcus cyst suspension is 95 MPa.
  • FIG. 15B shows a hematococcus cyst suspension when sheared for a predetermined time under the pressure condition of 50 MPa.
  • FIG. 16 is an explanatory diagram showing the correlation between the absorbance (A470) at a wavelength of 470 nm and the shearing force application time.
  • the wavelength of 470 nm is the maximum absorption wavelength of astaxanthin, and it can be considered that the absorption intensity of the solution at this wavelength approximately represents (is proportional to) the amount of dissolved astaxanthin.
  • As a result of the measurement it was shown that astaxanthin in the cyst was eluted (leaked) into the liquid by a short shearing treatment of about 0.3 to 0.4 seconds. That is, it was shown that the cysts were almost completely destroyed. This result is in good agreement with the observation results of the micrographs shown in FIGS. 12 to 14. From this result, in the shearing treatment method according to the present invention, the cyst of Hematococcus can be sufficiently destroyed in a short time under low pressure conditions as compared with algae having a strong jacket such as Scenedesmus. It has been shown.
  • Astaxanthin leaked into the solution was not in contact with oxygen at all and was not heated, so it is considered that it is dissolved as high quality astaxanthin.
  • Several options can be considered as the subsequent separation and purification method for astaxanthin, and solvent partitioning and the like are considered to be effective. At this time, the efficiency is further improved by incorporating decompression concentration or the like.
  • Solvent extraction after freeze-drying or vacuum-drying is also considered to be effective, and it is considered possible to construct various processes.
  • the shearing treatment and wet shearing treatment system 100 according to the present invention since the algae are crushed in a liquid (suspension) state after culturing, culture ⁇ continuous centrifugation (suspension preparation) ⁇ continuous shearing. There is a merit that the process can be made continuous, such as destruction ⁇ continuous centrifugation.
  • the flow of the treatment liquid and the flow path surface (narrow path 41c, first surface 41b, second surface 42a) in contact with the treatment liquid.
  • the shearing force generated between them destroys the strong coat of microalgae (algae coat of cell wall, cell membrane, cyst membrane, etc.) in the treatment solution, thereby efficiently producing the intracellular useful substance of microalgae in a short time.
  • the shearing force (shearing action) generated between the surface gap 47 and the surface gap 47 can be further increased, and the load due to the pressure fluctuation caused by the change in the flow direction of the treatment liquid can be separately applied to the microalgae.
  • a reciprocating pump that can easily increase the discharge pressure as the pump 2 that pumps the processing liquid, a shearing force (shearing action) generated between the flow of the processing liquid and the narrow path 41c or the surface gap 47 is generated. ) Can be increased.
  • the outer cover (cell wall, cell membrane, cyst membrane, etc.) of the microalgae contained in the treatment liquid, etc. It is possible to efficiently destroy the algae body coat).
  • the algae contents intracellular useful substances
  • the sheared suspension is dried without separating the leaked contents and the crushed outer cover to make a food material
  • useful components are contained in the living body more than when the suspension is dried without shear fracture. It can be efficiently absorbed and used.
  • the sheared suspension is fertilized into compost or the like without separating the leaked contents and the crushed jacket, it is possible to improve the fermentation efficiency.
  • drying time (drying rate) of the extracted algae contents (useful substances) is shortened by separating and removing the outer cover of the microalgae, which is a barrier to the movement of water, from the treated liquid. (Speeding up), which makes it possible to reduce the drying energy for the algae contents (useful substances).
  • a peripheral groove may be formed in a circular shape or a spiral shape on the inner surface of the narrow path 41c in which the treatment liquid comes into contact.
  • the shapes of the first peripheral groove 41d and the second peripheral groove 42d may be concentric circles, closed curves or closed polygons such as ellipses, eccentric left shapes, or radial shapes.
  • the first flow path 41a may be configured only with a straight pipe without providing a narrow path 41c.
  • the number of times the hematococcus cyst suspension (treatment liquid) is passed through the shearing treatment unit 4 may be only once.
  • the method for destroying the outer cover of microalgae by the shearing treatment of the present invention is a significantly superior destruction method as compared with other methods.
  • the treatment liquid pumped by the pump 2 is provided with a narrow path 41c and a surface gap 47 pressurized in the closing direction.
  • the destructive method that gives shearing force to the microalgae jacket by passing it shows a very excellent destructive effect.
  • FIG. 17 is an explanatory view showing a wet shearing treatment system 200 according to a second embodiment of the present invention.
  • the wet shearing treatment system 200 has a low temperature tank 10 for freezing the treatment liquid of the sheared hematococcus cyst suspension and a reduced pressure of the frozen treatment liquid with respect to the wet shearing treatment system 100 according to the first embodiment.
  • a freeze-dryer 11 for drying underneath, a first introduction pipe 12 for introducing the treatment liquid from the shearing treatment unit 4 into the low temperature tank 10, a first shutoff valve 12a for turning on / off the flow of the first introduction pipe 12.
  • the second introduction pipe 13 for introducing the treatment liquid from the reserve tank 1 to the low temperature tank 10, the second shutoff valve 13a for turning on / off the flow of the second introduction pipe 13, the first introduction pipe 12 and the second introduction pipe 13 It is configured to include a third introduction pipe 14 communicating with the above.
  • the sheared hematococcus cyst suspension in order to increase the extraction rate of astaxanthin from the sheared hematococcus cyst suspension, the sheared hematococcus cyst suspension is subjected to the low temperature tank 10 and the freeze dryer 11. Freeze-dried by. This freeze-drying will be described below.
  • Haematococcus lacustris dSgD-K1 strain collected and isolated in Saga City was cultured in AF6 medium at a temperature of 25 ° C., a photon bundle density of 80 ⁇ mol / m 2 / s, and a light irradiation time.
  • Light: Dark 12h: 12h
  • Light with a photon flux density increased to 300 ⁇ mol / m 2 / s was continuously irradiated for 24 hours to form cysts due to photostress (at this time, the temperature was lowered to 15 ° C. to suppress proliferation).
  • the obtained cyst was repeatedly suspended in deionized water and centrifuged to wash away the medium components, suspended in deionized water again, and evenly divided so as to have the same concentration, as shown in FIG. 18 below. It was used in the extraction test of 5 patterns (1 to 5).
  • FIG. 18 is an explanatory diagram showing the extraction of astaxanthin based on the wet shear fracture method by the wet shear treatment system 200 according to the present invention and the extraction of astaxanthin based on other fracture methods.
  • the pretreatment methods for extraction are roughly classified into the upper "pattern of crushing and then drying and then extracting" (methods 1 to 2) and the lower row of "pattern of crushing and extracting after drying”. (Method 3 to 5).
  • Methods 1 to 2 the upper “pattern of crushing and then drying and then extracting”
  • Methods 3 to 5 Methods 3 to 5
  • This method 1 is a wet shear fracture method using the wet shear treatment system 200 of the present invention.
  • the hot plate drying 120 ° C.
  • the applied pressure during the shearing process is 90 MPa.
  • FIG. 19 is an explanatory diagram showing the fracture behavior before and after the shearing treatment of Hematococcus cyst by the wet shear fracture method according to the present invention.
  • FIG. 19 (a) shows a suspension of Hematococcus cyst before destruction (hereinafter referred to as “cyst suspension”)
  • FIG. 19 (b) shows a cyst suspension after shearing treatment, which is (c). Is an enlarged view of the cyst suspension of the same (b).
  • the cyst suspension is diluted 200-fold so that it can be easily observed under a microscope.
  • the hematococcus cyst remains covered with the jacket, and no dissolution or elution of astaxanthin is confirmed from the jacket.
  • FIG. 19B is a photograph of the cyst suspension observed after passing through the shearing unit 4 of the shearing system 200 a plurality of times.
  • the “shearing time 0.92 seconds” shown in the figure is the cumulative time that the cyst suspension stayed in the shearing unit 4, that is, the total time that the cyst suspension was subjected to the shearing force.
  • FIG. 19 (b) it can be seen that most of the cyst cells were destroyed by a very short shearing force of 0.92 seconds.
  • FIG. 19 (c) is a further enlarged photograph so that the state of destruction of cyst cells can be seen. As can be seen from FIG. 19 (c), it can be seen that the cyst contents have leaked out of the cells and are emptied.
  • the obtained shearing liquid is dried by two types of drying methods, “freeze drying” and “hot plate drying” shown in FIG. 18, and the amount of astaxanthin extracted from the obtained dried product. Was measured.
  • the hot plate drying is for comparative study to confirm the effect of freeze drying.
  • the shearing solution in the eggplant flask is frozen while rotating in a low temperature bath at -40 ° C, and the frozen shearing solution is dried in a freeze-dryer under a reduced pressure of about 4 Pa for 2 days or more. I went there.
  • the ultrasonic fracturing method is a conventional method widely used as a cell crushing method including microorganisms.
  • a probe-type ultrasonic crusher (Soniffier (registered trademark) SFX 250) was used to crush a 50 mL cyst suspension in a plastic container under the conditions of 20 kHz and 250 W.
  • the sample solution (cyst suspension) was treated while being ice-cooled, but the rapid temperature rise could not be suppressed, and the treatment was interrupted every few minutes to ice-cool the temperature to 50 ° C. I was careful not to exceed it.
  • FIG. 20 is an explanatory diagram showing the destruction behavior of Hematococcus cysts by ultrasonic treatment.
  • FIG. 20 (a) shows the hematococcus cyst before the ultrasonic treatment
  • FIG. 20 (b) shows the hematococcus cyst after the ultrasonic treatment.
  • Methods 3 to 5 are conventional methods in which drying is performed first, and then the dried algae are destroyed. That is, the above-mentioned methods 1 to 2 (upper part of FIG. 18) performed the crushing in the reverse order of the crushing. Hereinafter, the method 3 will be described in order for reference.
  • Method 3 Hot plate drying> In the hot plate drying of Method 3, first, "the hematococcus cyst suspension was heat-dried on a hot plate” and “the dried product was crushed with a glass homogenizer” to extract astaxanthin.
  • the method 3 performed here is the method closest to the current method, and since the jacket is crushed in the extraction solvent, the extraction efficiency is higher than that of the current method.
  • the untreated cyst suspension was dropped on a hot plate (model number: AS ONE ND-2A) set at 120 ° C. in the same manner as in Methods 1 and 2 above with Pipetman, dried, and then scraped off with a scraper. (It takes about 1 minute from dropping to scraping).
  • 10 mg of the obtained dry cyst was weighed in a downs-type glass homogenizer, and a small amount of acetone was added to crush the cyst jacket.
  • the liquid obtained by this operation was used as a sample for measuring the amount of astaxanthin after the volume was adjusted in a volumetric flask. Samples that were only hot plate dried (without glass homogenization) were also extracted to verify the effect of glass homogenization. The results of this quantitative analysis will be described later with reference to FIG.
  • Method 4 Freeze-drying>
  • the freeze-drying method of Method 4 is a drying method that causes the least damage to the drying raw material among various drying methods. In particular, it is the most preferable drying method for substances that are susceptible to oxidation, such as astaxanthin, which is the target substance of this time. Therefore, this freeze-drying method was also used in Method 1 for drying the shearing liquid.
  • the cyst suspension was placed in an eggplant flask in the same manner as in Method 1, frozen while rotating in a low temperature bath at -40 ° C, and then dried in a freeze-dryer under a reduced pressure of about 4 Pa for 2 days or more.
  • the obtained dried cyst was weighed in a Downs-type glass homogenizer in the same manner as in Method 3, and a small amount of acetone was added to crush the cyst jacket and then the volume was adjusted to prepare a sample for measuring the amount of astaxanthin.
  • samples that were freeze-dried only (without glass homogenization) were also extracted to verify the effect of glass homogenization. The results of this quantitative analysis will be described later with reference to FIG.
  • Method 5 Warm air drying>
  • the warm air drying method of Method 5 is a method in which an antioxidant such as astaxanthin has a high risk of oxidative deterioration due to high temperature oxygen because the object to be dried is exposed to high temperature hot air.
  • an antioxidant such as astaxanthin
  • FIG. 21 is an explanatory diagram showing an example of a chromatogram obtained by the wet shear fracture method according to the present invention.
  • the ester peak disappears and the trans form or 9-cis and 13-cis form peaks are visible.
  • FIG. 22 is an explanatory diagram showing the amount of each astaxanthin extracted from the Hematococcus dried cysts obtained by the above methods 1 to 5.
  • the numerical value is the amount of astaxanthin extracted (mg) per 1 g of dry cyst.
  • shear crushing-> hot plate drying on the right side shows the case where the shear crushing liquid is dried on a hot plate (120 ° C).
  • the amount of astaxanthin extracted decreased from 15.5 to 4.77 [mg / g-dry cyst]. This indicates a decrease in astaxanthin due to contact with hot air during drying. From this, it can be seen that it is desirable to avoid the treatment of highly antioxidant substances such as astaxanthin in contact with high-temperature oxygen.
  • Method 3 in which "hot plate drying” is performed is the closest drying method to the commercial scale drying method performed using a drum dryer or the like.
  • the cysts dried on a hot plate were crushed in a glass homogenizer together with the extraction solvent.
  • This crushing method can obtain higher extraction efficiency than the method of crushing with a disc mill or the like in a dry state and then extracting.
  • the amount of astaxanthin extracted from the dried cysts that had been subjected to plate drying only was also measured.
  • the lowest astaxanthin extraction amount was 2.12 [mg / g-dried cyst] only by drying. It is considered that this is because the dried cyst membrane became a barrier to the invasion of the extraction solvent into the algae, and the dissolution and elution of astaxanthin inside were insufficient.
  • the extraction amount was improved to 9.66 [mg / g-dry cyst]. However, it was not as good as 15.5 [mg / g-dry cyst] of the wet shear fracture method of Method 1 according to the present invention.
  • the amount extracted from the dried cysts after warm air drying remained at 5.64 [mg / g-dry cysts], and even after glass homogenization, it remained at 8.9 [mg / g-dry cysts].
  • the warm air drying method is the easiest method for drying, but the dried cysts firmly stick to the vessel wall of the drying container, and the work of peeling them off requires a great deal of energy and labor. From this, it is considered that the method is not suitable as a method for drying cysts.
  • the astaxanthin extraction amount should be noted that the astaxanthin extraction amount per 1 g of dry cyst shown in FIG. 22 is lower than the generally said 30-40 mg. This is because most of the cells of the hematococcus cyst used as the test sample were actually palmeroid cells (immature cysts). Although it is difficult to understand in monochrome, more than half of the cells were immature cysts as seen in the photographs before the destruction in FIG. 19 (a) and before the treatment in FIG. 20 (a). Therefore, the absolute value of the astaxanthin extraction amount was low because of the state of the raw material, not because the wet shear fracture method according to the present invention was inferior.
  • FIG. 23 is an explanatory diagram showing a sample in which the cyst suspension is wet-shear fractured and freeze-dried, and a sample in which the cyst suspension is simply hot-plate-dried. It is difficult to understand in monochrome, but the vividness of the colors is very different between the two.
  • the sample dried by wet shear fracture and freeze-dried has a red color, whereas the sample dried by a hot plate has a brown color.
  • the outer cover of the hematococcus cyst can be efficiently destroyed, and the treatment liquid containing astaxanthin leaked from the inside of the cyst is frozen.
  • astaxanthin can be efficiently extracted from the dried cyst while minimizing the oxidative deterioration of astaxanthin.
  • the genus Botryococcus especially Botryococcus braunii, forms tufted colonies in a persistent polymer matrix called alginan and accumulates a large amount of hydrocarbon-based oil that is easily used as fuel between cells and the matrix. To do. Its content is as high as 60% of the weight of dried algae, which is very promising as a liquid fuel production method.

Abstract

L'invention concerne un procédé pour rompre la coque externe d'une micro-algue par cisaillement et un système associé, une substance utile, produite par la micro-algue, ladite micro-algue présentant une paroi cellulaire rigide, pouvant s'échapper des cellules de microalgues dans un liquide de traitement à une efficacité élevée en une courte période de temps tout en empêchant la détérioration des qualités de la substance utile. À cet effet, on utilise une première face non mobile 41b sous forme de bride, qui est perpendiculaire à un premier canal d'écoulement 41a en forme de tube droit. En outre, une deuxième face 42a, qui fait face à la première face 41b, est disposée de manière mobile. Dans la première face 41b, une ou plusieurs premières rainures circonférentielles concentriques 41d sont formées. Dans la deuxième face 42a, des deuxièmes rainures circonférentielles 42d, qui sont adaptables aux premières rainures circonférentielles 41d, sont formées. Dans la partie d'extrémité de pointe du premier canal d'écoulement 41a, le diamètre est réduit pour former un canal étroit 41c. De plus, un actionneur 46 est utilisé de manière à mettre sous pression la deuxième face 42a dans la direction de fermeture d'un interstice 47 formé entre la deuxième face 42a et la première face 41b.
PCT/JP2020/027664 2019-07-23 2020-07-16 Procédé et système pour rompre la coque externe d'une micro-algue par cisaillement WO2021015089A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
JPS6382429U (fr) * 1986-11-19 1988-05-30
JP2002320446A (ja) * 2001-04-26 2002-11-05 Asahi Denka Kogyo Kk 水中油型乳化脂の製造方法
JP2009131219A (ja) * 2007-11-30 2009-06-18 Biogenic Co Ltd アスタキサンチンの製造方法
JP2010162499A (ja) * 2009-01-16 2010-07-29 Fujifilm Corp 微生物の破砕方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6382429U (fr) * 1986-11-19 1988-05-30
JP2002320446A (ja) * 2001-04-26 2002-11-05 Asahi Denka Kogyo Kk 水中油型乳化脂の製造方法
JP2009131219A (ja) * 2007-11-30 2009-06-18 Biogenic Co Ltd アスタキサンチンの製造方法
JP2010162499A (ja) * 2009-01-16 2010-07-29 Fujifilm Corp 微生物の破砕方法

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Title
NIRO SOAVI NORTH AMERICA GEA: "High-Pressure Pumps and Homogenizers", NIRO SOAVI NORTH AMERICA GEA, 2008, Retrieved from the Internet <URL:http://www.martagan.com/cats/niro.pdf> [retrieved on 20200923] *

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