JP7502740B2 - Method and system for destroying the outer shell of microalgae by shear processing - Google Patents

Description

The present invention relates to a method and system for destroying the outer coat of microalgae by shearing treatment. More specifically, the present invention relates to a method and system for destroying the outer coat of microalgae by shearing treatment that can efficiently release intracellular useful substances of microalgae having strong outer coats (algal coats such as cell walls, cell membranes, and cyst membranes) in a short time and without deteriorating their quality from within the cells into a treatment liquid.

Among microalgae, there are useful algae such as Haematococcus, which produces astaxanthin in the algae body, and Nannochloropsis , which produces EPA, and the contents of these algae are extracted from the cultured algae body. Since microalgae are a type of unicellular plant and have cell walls (except for some species such as Euglena), it is natural that the cell walls and cell membranes must be destroyed in order to extract the contents. For this reason, in many current processes (dry crushing processes), the algae body is dried by methods such as spray drying, drum drying, and ventilation drying, and then mechanically crushed using an atomizer, disk mill, ball mill, etc., and then extracted with a solvent, etc.

However, when carrying out the above-mentioned process, there are problems and points that need to be improved in order to increase efficiency, as described below.
(1) During heating and drying, the algae cells come into contact with hot air (oxygen), and powerful antioxidants such as astaxanthin produced by Haematococcus are oxidized during drying, resulting in a decrease in their purity and activity.
(2) In the drying process, the only way for moisture to escape from the algae, which are covered by the cell wall, is through minute passages such as plasmodesmata, which are passages for nutrients, etc.; therefore, drying of algae that are still covered by the cell wall is disadvantageous in terms of both the drying speed and the energy input.
(3) In order to extract useful contents such as astaxanthin and EPA from dried algae, it is necessary to break the outer coat of the dried algae by some kind of crushing method so that the solvent can contact and dissolve the cell contents. However, microalgae cells are very small, measuring only a few tens of microns, and their cell walls are made of tough polysaccharides such as cellulose, making it very difficult to break the outer coat. This is because even if mechanical force is applied to piled-up solid particles, the force for breaking is not transmitted uniformly, and the range of the breaking action is limited, making it particularly difficult to break down the cells of microalgae, which are not very fragile (for example, it is very difficult to crush sesame seeds in a mortar at high speed and uniformly). Therefore, for example, in the process of extracting astaxanthin from Haematococcus, the actual situation is that how to perform sufficient crushing is an important issue.

Incidentally, microalgae are not only useful as a raw material for extracting useful substances as mentioned above, but are also expected to be used as food ingredients, feed, and fertilizer raw materials, and the oils they produce can be used as cosmetic fats and oil fuels, and some of these applications are already underway. This is because microalgae grow faster than ordinary plants, have a large yield per unit area, have a high carbon dioxide fixation capacity, can be harvested throughout the year, and can be cultivated even in wasteland where crops cannot be produced if culture tanks are installed. However, even in these cases, the outer covering of the algae body is a cause of various reduced efficiencies.

That is, (4) when using algae as food materials or feed, the digestive and utilization efficiency can be improved by destroying the cell wall, because the contents of the algae are difficult to utilize as nutrients due to their indigestible cell wall. In the case of feed, it is expected to improve the nutritional efficiency, especially in monogastric animals that do not ruminate.
(5) When using algae as fertilizer, especially in composting, it is expected that the fermentation rate will be faster if the outer shell is destroyed.

For these reasons, if the outer covering of microalgae could be destroyed efficiently and sufficiently, it would be possible to develop more efficient, higher-yield processes than currently available in the areas of drying, extraction, feeding, and fertilizer production.

In addition, a process for extracting intracellular useful substances such as astaxanthin (wet crushing process) is also known in which a natural product containing astaxanthin is suspended in an organic solvent, extracted in the organic solvent while being crushed in a crusher, solids are removed, and the organic solvent is further removed to extract astaxanthin (see, for example, Patent Document 1).

A known method for destroying cell walls is the freeze-fracture method, in which slow freezing of the algae promotes the growth of ice crystals in the intracellular fluid, destroying the cell envelope from within. However, when the inventors of the present application conducted experiments in which encysted Haematococcus were cooled at cooling rates of 1.0, 1.5, and 2.0°C/min and the resulting treated cells were observed under a microscope, the destruction rate of the envelope was at most 50% or less. This suggests that the increase in volume due to the phase change of the intracellular fluid was within the elastic limit of the envelope.

JP 2006-70114 A

In the case of the above-mentioned dry crushing process, there is a problem that a large amount of thermal energy is consumed during heating, and the quality and yield of the target substance, astaxanthin, decreases due to oxidation. In addition, there is also the problem that the mechanical energy during crushing of tiny solids of several tens of microns is only transmitted to a part of the object to be crushed, and uniform and sufficient crushing is not achieved.

On the other hand, in the case of the above-mentioned wet crushing process, it is preferable to use a bead mill as the crusher. It is also described that 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, and the bead diameter used is 0.2 to 5 mm, preferably 0.5 to 2 mm (see, for example, Patent Document 1 [0026]).

However, bead mills crush objects by so-called point contact, such as collisions and shearing between beads, and as with the dry crushing process described above, mechanical energy is transmitted to only a portion of the object to be crushed, and it is thought that this is not very effective in crushing Haematococcus.

Therefore, the present invention has been made in consideration of the problems of the above-mentioned conventional technology, and its purpose is to provide a method and system for destroying the outer coat of microalgae using a shearing process that can highly efficiently destroy the outer coat of microalgae, which acts as a barrier during drying, extraction, digestion in the body, and composting, and can cause useful substances within the cells to leak into the treatment liquid from within the cells efficiently in a short period of time and without deteriorating their quality.

The method for destroying the outer coat of microalgae by shear treatment according to the present invention for achieving the above-mentioned object is characterized in that a treatment liquid containing cells that produce or contain useful substances is pressure-fed by a pump (2) while passing through a surface gap (47) formed by an immovable first surface (41b) and a movable second surface (42a) arranged opposite each other, and the treatment liquid is passed through the surface gap (47) while changing the flow direction of the treatment liquid, and the outer coat of the cells is destroyed in the treatment liquid by the shear force generated between the flow of the treatment liquid and the first surface (41b) or the second surface (42a).

In the above configuration, the treatment liquid pumped by the pump (2) passes through the narrow surface gap (47) formed between the first surface (41b) and the second surface (42a), and the strong shear force generated between the flow of the treatment liquid and the narrow flow path surface (41b, 42a) is then applied to the algae bodies in the treatment liquid. At the same time, in the portion where the flow direction of the treatment liquid changes, pressure fluctuations occur in the treatment liquid due to compression (pressure increase) and expansion (pressure decrease). As a result, the algae bodies in the treatment liquid are subjected to the load due to the pressure fluctuations in addition to the shear action.
In this way, the method for destroying the outer coat of microalgae according to the present invention destroys the outer coat of the algae bodies in the treatment liquid by using the strong shear force generated between the flow of the treatment liquid and the narrow flow path surface (41b, 42a), thereby causing intracellular useful substances to leak from within the cells into the treatment liquid.

The second feature of the method for destroying the outer covering of microalgae by shear treatment according to the present invention is that the treatment liquid is passed through a narrow passage (41c) with a narrowed flow path before passing through the surface gap (47).

In the above configuration, as the treatment liquid passes through the narrow passage (41c), a strong shear force generated between the flow of the treatment liquid and the narrow flow path surface (41c) acts on the algae bodies in the treatment liquid. Furthermore, as the flow of the treatment liquid is constricted by the narrow passage (41c), the treatment liquid is accelerated and collides with the second surface (42a). This shear force and collision can weaken the outer covering of the algae bodies in the treatment liquid before it passes through the surface gap (47).

The third feature of the method for destroying the outer covering of microalgae by shear treatment according to the present invention is that when the treatment liquid passes through the surface gap (47), the second surface (42a) is pressurized under predetermined pressure conditions in the direction of closing the surface gap (47).

In the above configuration, the treatment liquid passes through the surface gap (47) between the first surface (41b) and the second surface (42a) while pushing aside (pushing up) the second surface (42a) and forming a gap through which it can flow. This causes the algae bodies in the treatment liquid to be subjected to a stronger shearing action than when the surface gap (47) is fixed.

The fourth feature of the method for destroying the outer covering of microalgae by shear treatment according to the present invention is that the treatment liquid is passed through the narrow passage (41c) and the surface gap (47).

In the above configuration, the algae cells in the treatment liquid are subjected to "strong shear forces generated between the flow of the treatment liquid and the narrow flow path surface" and "loads due to pressure fluctuations."

The fifth feature of the method for destroying the outer coat of microalgae by shear treatment according to the present invention is that the treatment liquid is pumped by the reciprocating pump (2).

In the above configuration, the discharge pressure of the reciprocating pump can impart to the treatment liquid the fluid energy (head) required to pass the treatment liquid through the narrow passage (41c) and the narrow surface gap (47).

The sixth feature of the method for destroying the outer coat of microalgae by shear treatment according to the present invention is that the treatment liquid that has passed through the surface gap (47) is freeze-dried.

With the above configuration, it is possible to remove water from the treatment liquid while minimizing the deterioration due to oxidation of intracellular useful substances that have been dissolved into the treatment liquid from inside the algae cells by the shearing process.

The seventh feature of the method for destroying the outer shell of microalgae by shear treatment according to the present invention is that the frozen treatment liquid is placed in an environment reduced in pressure below atmospheric pressure and dried.

The above configuration makes it possible to evaporate water from the treatment solution while minimizing contact between intracellular useful substances and oxygen.

The system for destroying the outer coat of microalgae by shearing treatment according to the present invention for achieving the above-mentioned object is a system for destroying the outer coat of microalgae comprising a tank (1) for storing a treatment liquid containing cells that produce or contain useful substances, a pump (2) for pumping the treatment liquid, and a shearing treatment section (4) for crushing the outer coat of the cells, the shearing treatment section (4) comprising a first flow path (41a) for transporting the pressure-fed treatment liquid, a first surface (41b) that intersects with the first flow path (41a) and is immovable, and a second surface (41b) that intersects with the first flow path (41a) and is immovable. The device is provided with a movable second surface (42a) arranged opposite to the first surface (41b), and a second flow path (43a) accommodating the second surface (42a), wherein a first groove (41d) is formed on the surface of the first surface (41b), and a second groove (42d) capable of fitting into the first groove (41d) is formed on the surface of the second surface (42a), and the treatment liquid passes through a surface gap (47) formed between the first surface (41b) and the second surface (42a).

With the above configuration, it becomes possible to effectively carry out the method of destroying the outer coat of microalgae by shearing treatment having the first characteristic described above.

The second feature of the system for destroying the outer covering of microalgae by shear processing according to the present invention is that a narrow passage (41c) is formed in the first flow path (41a) by narrowing the flow path.

With the above configuration, it becomes possible to effectively carry out the method of destroying the outer shell of microalgae by shearing treatment having the second characteristic described above.

A third feature of the system for destroying the outer covering of microalgae by shear processing according to the present invention is that it is provided with an actuator unit (46) that applies pressure under predetermined pressure conditions in a direction pressing the second surface (42a) against the first surface (41b).

With the above configuration, it becomes possible to effectively carry out the method of destroying the outer coat of microalgae by shearing treatment having the third characteristic described above.

The fourth feature of the microalgae outer covering destruction system by shear treatment according to the present invention is that it is provided with a return pipe (6) connecting the shear treatment section (4) and the tank (1).

With the above configuration, it becomes possible to effectively carry out the method of destroying the outer coat of microalgae by shearing treatment having the fourth characteristic described above.

The fifth feature of the system for destroying the outer shell of microalgae by shear treatment according to the present invention is that the pump (2) is a reciprocating pump equipped with a cylinder (33), a piston (21) reciprocating within the cylinder (33), a headspace (32) formed by the cylinder (33) and the piston (21), a suction valve (23) for filling the headspace (32) with a treatment liquid, and a discharge valve (27) for pumping the treatment liquid out of the headspace (32).

With the above configuration, it becomes possible to effectively carry out the method of destroying the outer coat of microalgae by shearing treatment having the fifth characteristic described above.

The sixth feature of the system for destroying the outer shell of microalgae by shear treatment according to the present invention is that it is equipped with a low-temperature tank (10) for freezing the treatment liquid that has passed through the surface gap (47), and a freeze dryer (11) for drying the frozen treatment liquid.

With the above configuration, it becomes possible to effectively carry out the method of destroying the outer coat of microalgae by shearing treatment having the sixth characteristic described above.

The seventh feature of the system for destroying the outer shell of microalgae by shear treatment according to the present invention is that the freeze-dryer (11) dries the frozen treatment liquid in an environment reduced in pressure below atmospheric pressure.

With the above configuration, it becomes possible to effectively carry out the method of destroying the outer coat of microalgae by shearing treatment having the seventh characteristic described above.

The method and system for destroying the outer coat of microalgae using shear treatment according to the present invention makes it possible to efficiently extract useful substances from cells having a strong outer coat (cell wall, cell membrane, cyst membrane, etc.) in a short period of time and without deteriorating their quality, and to cause them to leak from within the cells into the treatment solution.

In addition, by destroying the microalgae's outer coat, the algae contents (useful substances) leak from within the cells into the treatment liquid, making it easier to separate and purify the algae contents (useful substances). Furthermore, if the sheared suspension is dried or otherwise processed into a food material without separating the leaked contents from the crushed outer coat, the useful components can be absorbed and utilized more efficiently in the body than if it was dried without shear destruction. Similarly, if the sheared suspension is converted into compost or other fertilizer without separating the leaked contents from the crushed outer coat, it is possible to improve fermentation efficiency.

In addition, by separating and removing the outer covering of the microalgae cells, which acts as a barrier to moisture movement, from the treated liquid, the drying time (drying rate) of the extracted algae cell contents (useful substances) is shortened (speeded up), thereby making it possible to reduce the energy required to dry the algae cell contents (useful substances).

FIG. 1 is an explanatory diagram showing a wet shear processing system according to a first embodiment of the present invention. FIG. 2 is a cross-sectional view illustrating a main portion of a pump used in the present invention. FIG. 4 is an explanatory diagram showing the suction process of the pump. FIG. 4 is an explanatory diagram showing a discharge process of a pump. FIG. 2 is a cross-sectional explanatory view showing a main part of a shearing processing unit according to the present invention. 1 is an explanatory diagram showing the shearing action on algae bodies in a treatment liquid caused by the passage of the treatment liquid through narrow passages and surface gaps of a shearing treatment unit according to the present invention. FIG. 1 is a micrograph showing a moss flax suspension (treatment liquid) before the shearing treatment according to the present invention. These are micrographs showing the state of a rock moss suspension when it was sheared for 0.3 seconds and 0.5 seconds under an applied pressure of 90 MPa. These are micrographs showing the state of a rock moss suspension when it was sheared for 0.7 seconds and 0.9 seconds under an applied pressure of 90 MPa. 1 is a graph showing the relationship between the shear force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant. 1 is a micrograph showing a Haematococcus cyst suspension before shearing treatment according to the present invention. 13 is a micrograph showing the state of a suspension of Haematococcus cysts when it was subjected to shear treatment for 0.1 seconds and 0.2 seconds under the condition of an applied pressure of 95 MPa. 13 is a micrograph showing the state of a suspension of Haematococcus cysts when the suspension was subjected to shear treatment for 0.1 seconds, 0.2 seconds, and 0.3 seconds under a pressure condition of 70 MPa. 13 is a micrograph showing the state of a suspension of Haematococcus cysts when the suspension was subjected to shear treatment for 0.1 seconds, 0.3 seconds, and 0.4 seconds under a pressure condition of 50 MPa. 1 is a photograph showing the appearance of a Haematococcus cyst suspension that has been left to stand for 30 minutes or more after shearing treatment according to the present invention. FIG. 1 is an explanatory diagram showing the correlation between absorbance at a wavelength of 470 nm (A470) and the application time of shear force. FIG. 4 is an explanatory diagram showing a wet shear processing system according to a second embodiment of the present invention. FIG. 2 is an explanatory diagram showing the extraction of astaxanthin based on the wet shear disruption method using the wet shear processing system of the present invention and the extraction of astaxanthin based on other disruption methods. FIG. 2 is an explanatory diagram showing the disruption behavior of Haematococcus cysts before and after shear treatment by the wet shear disruption method according to the present invention. FIG. 2 is an explanatory diagram showing the disruption behavior of Haematococcus cysts by ultrasonic treatment. FIG. 2 is an explanatory diagram showing an example of a chromatogram obtained by the wet shear fracture method according to the present invention. FIG. 19 is an explanatory diagram showing the amount of astaxanthin extracted from dried Haematococcus cysts obtained by methods 1 to 5 shown in FIG. 18. FIG. 1 is an explanatory diagram showing a sample of a cyst suspension subjected to wet shear disruption and freeze-drying, and a sample of a cyst suspension subjected to hot plate drying.

Below, an embodiment of the present invention is described in detail with reference to the attached drawings.

Figure 1 is an explanatory diagram showing a wet shear processing system 100 relating to the first embodiment of the present invention.

This wet shear processing system 100 is a system for destroying the outer coat of microalgae by shear processing, which performs a shear processing to destroy the outer coat (cell wall, cell membrane, cyst membrane, etc.) of the cells of algae bodies containing useful substances such as astaxanthin and EPA in a liquid, thereby leaking the useful substances contained in the cells into the liquid. The liquid from which the useful substances have leaked is separated into the useful substances and other substances by known separation methods (e.g., centrifugation or membrane filtration), and only the useful substances are extracted. In the following, liquids containing algae bodies will be distinguished as "untreated liquid" if they have not been subjected to shear processing by this wet shear processing system 100, and "treated liquid" if they have been subjected to shear processing. In addition, when there is no particular need to distinguish, they will simply be called "treated liquid".

This wet shear processing system 100 is configured to include a reserve tank 1 for storing untreated or treated liquid, a pump 2 for pumping the untreated or treated liquid, a shear processing section 4 for shearing the untreated or treated liquid pumped by the pump 2, a suction pipe 3 connecting the reserve tank 1 and the pump 2, a discharge pipe 5 connecting the pump 2 and the shear processing section 4, and a return pipe 6 connecting the shear processing section 4 and the reserve tank 1. Note that the pump 2 may be any type capable of pumping out the treated liquid at a high discharge pressure, and the method of pumping out the treated liquid is not particularly important. In this embodiment, the pump 2 is a reciprocating pump. Each component will be further described below.

Figure 2 is a cross-sectional view of the pump 2 used in the present invention. For ease of explanation, the suction valve 23 and discharge valve 27 are shown in a closed state. The pump 2 is a reciprocating pump in which the piston 21 reciprocates within the cylinder 33 to suck in untreated liquid through the suction valve 23, compress the untreated liquid in the head space 32, and pump the compressed untreated liquid through the discharge valve 27 to the downstream shear processing unit 4.

To achieve this, the pump 2 is configured to include a piston 21 that compresses the treatment liquid, a first spring 22 that biases the piston 21 to the right in the figure, an inlet valve 23 that allows only the flow of treatment liquid from the IN connector 26 to the head space 32, a first valve seat 24 that receives the inlet valve 23, a first sleeve 25 that guides the movement of the inlet valve 23, an IN connector 26 that takes in treatment liquid from the reserve tank 1, a discharge valve 27 that allows only the flow of treatment liquid from the head space 32 to the OUT connector 31, a second valve seat 28 that receives the discharge valve 27, a second spring 29 that biases the discharge valve 27 in the closing direction, a second sleeve 30 that guides the movement of the discharge valve 27, an OUT connector 31 that communicates with the shear processing section 4, a head space 32 for the piston 21 to compress the treatment liquid, a cylinder 33 for the piston 21 to reciprocate, and a connecting rod 34 and a crankshaft 35 that convert rotational energy into translational energy. For convenience of explanation, the pump 2 is configured by a single piston 21, however, the pump 2 may be configured by combining a plurality of the above components in series or in parallel.

The piston 21 is provided with a piston ring 21a that maintains liquid tightness in the head space 32. The piston 21 is also connected to a connecting rod 34, which is connected to a crankshaft 35. The crankshaft 35 is coaxially connected to an electric motor (shown). The electric motor is configured so that its rotation speed is controlled by an inverter (not shown). Therefore, the number of vibrations (frequency) per unit time of the piston 21 is configured so that it can be changed by the inverter. The operation of the pump 2 will be described below.

Figure 3 is an explanatory diagram showing the suction process of pump 2. When piston 21 moves to the right in the figure, piston 21 creates negative pressure in head space 32. As a result, "pressure at IN-side connection 26" > "pressure at head space 32". As a result, suction valve 23 opens, and treatment liquid is filled from IN-side connection 26 into head space 32. Meanwhile, discharge valve 27 remains closed, since "pressure at head space 32" < "pressure at OUT-side connection 31 + elastic force of second spring 29".

Figure 4 is an explanatory diagram showing the discharge process of the pump 2. When the piston 21 moves to the left in the figure, the processing liquid filled in the head space 32 is compressed by the piston 21. As a result, the "pressure of the head space 32" > the "pressure of the IN-side connecting part 26". As a result, the suction valve 23 closes. On the other hand, when the "pressure of the head space 32" exceeds the "pressure of the OUT-side connecting part 31 + the elastic force of the second spring 29", the discharge valve 27 opens. As a result, the compressed processing liquid is pressure-fed from the discharge valve 27 to the downstream shear processing part 4.

Since the suction valve 23 is closed, compressed untreated liquid or treated liquid is not pressure-fed to the reserve tank 1. In this way, the suction valve 23 is open when the "pressure of head space 32" < the "pressure of IN-side connecting part 26", and the discharge valve 27 functions as a check valve that is open when the "pressure of head space 32" > the "pressure of OUT-side connecting part 31".

5 is a cross-sectional view of the main part of the shear processing unit 4 according to the present invention. The shear processing unit 4 is a device for continuously applying a shear action (shear force) generated between the flow of the processing liquid and the flow channel surface to the algae bodies contained in the processing liquid by passing the flow of the processing liquid pumped by the pump 2 through a narrow passage 41c or a surface gap 47 (formed between the first surface 41b and the second surface 42a), thereby destroying the outer coat of cells that produce or contain useful substances such as astaxanthin and EPA in the processing liquid and leaking the useful substances into the processing liquid.

It is configured with a valve seat portion 41 for transferring the processing liquid pumped from the pump 2, a valve portion 42 for stopping/preventing the flow of processing liquid flowing out from the valve seat portion 41, a housing portion 43 for accommodating the valve seat portion 41 and the valve portion 42, an inlet portion 44 to which the discharge pipe 5 is connected and which takes in the pressurized processing liquid, etc., an outlet portion 45 to which the return pipe 6 is connected and from which the processed liquid flows out, and an actuator portion 46 for pressurizing the valve portion 42. Each component will be described below.

The valve seat portion 41 is composed of a straight first flow passage 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 while engaging with a step 43c of the housing portion 43. The tip of the first flow passage 41a forms a tapered narrow passage 41c (or orifice) with a reduced inner diameter. In addition, one or more first circumferential grooves 41d are formed concentrically along the circumferential direction on the first surface 41b.

The valve portion 42 is made up of a disk-shaped (disk head type) second surface 42a and a side surface 42b, an expanded diameter tip portion, and a rod-shaped rod portion 42c. The rod portion 42c is provided with an O-ring 42e that maintains the liquid-tight state of the second flow path 43a.

The second surface 42a has one or more second circumferential grooves 42d formed concentrically along the circumferential direction, which can be fitted into the first circumferential groove 41d formed in the first surface 41b.

The 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 configured to be variable. The second surface 42a is configured to be pressurized (pushed) in the direction of closing the surface gap 47 by the actuator portion 46. An electric cylinder, a pneumatic cylinder, a spring, or the like can be used as the actuator portion 36.

Between the side surface 42b of the valve portion 42 and the inner peripheral surface 43b of the second flow path 43a, a gap is formed through which the treatment liquid that has been subjected to the shear treatment can flow. Below, the shearing action on the algae bodies in the treatment liquid in the shear treatment portion 4 is described.

FIG. 6 is an explanatory diagram showing the shearing action on algae bodies in the treatment liquid caused by the passage of the treatment liquid through the narrow passage 41c and surface gap 47 of the shear treatment section according to the present invention.
6A, the treatment liquid that has flowed in through the inlet portion 44 and is pumped by the pump 2 passes through the first flow path 41a and flows into the narrow path 41c. However, the valve portion 42 is pressurized toward the first surface 41b by the actuator portion 46, and therefore the valve portion 42 is in a closed state. Therefore, the flow of the treatment liquid is stopped by the valve portion 42.

6(b), the pressure in the first flow path 41a increases due to the treatment liquid pumped from the pump 2. When the pressure of the treatment liquid in the first flow path 41a becomes equal to the pressure applied by the actuator section 46, the treatment liquid pushes the valve section 42 to the right in the figure and flows through the surface gap 47 formed between the first surface 41b and the second surface 42a. As a result, the treatment liquid is subjected to a strong shear force as it passes through the narrow passage 41c, which destroys the algae coat.

6(c), the processing liquid flowing through the surface gap 47 formed between the first surface 41b and the second surface 42a is forced to change its flow direction in a zigzag pattern as it crosses the first peripheral groove 41d and the second peripheral groove 42d. By changing the flow direction in a zigzag pattern, the processing liquid is subjected to a stronger shearing action. In addition, pressure fluctuations due to compression and expansion also occur in the area where the flow direction changes. Therefore, when the processing liquid passes through the surface gap 47 between the first surface 41b and the second surface 42a, it is subjected to loads due to shear forces and pressure fluctuations at the same time.

The treatment liquid that passes through the surface gap 47 formed between the first surface 41b and the second surface 42a collides with the inner surface 43b, changes its flow direction by 90°, and flows out from the outlet portion 45.

In this way, the processing liquid that flows in from the inlet 44 and is pumped by the pump 2 is initially prevented from flowing by the valve 42, but when the pressure of the processing liquid in the first flow path 41a becomes equal to the pressure applied by the actuator 46, it pushes the valve 42 to the right in the figure, forming a surface gap 47 between the first surface 41b and the second surface 42a through which it flows. The processing liquid is subjected to a strong shearing action as it passes through the narrow passage 41c.

Furthermore, when the processing liquid passes through the surface gap 47 formed between the first surface 41b and the second surface 42a, the first circumferential groove 41d and the second circumferential groove 42d cause the flow direction to change in a zigzag pattern. When the flow direction changes, pressure fluctuations also occur in the processing liquid. As a result, the processing liquid is subjected to a strong shearing action and a load due to the pressure fluctuations at the same time.

Therefore, by passing the treatment liquid through the shear processing section 4, it becomes possible to continuously and efficiently destroy the outer coat of the algae cells compared to the conventional method of mechanically destroying the outer coat of the fine-particle dried algae cells.

An example of shear processing of Desmodesmus sp. using the wet shear processing system 100 according to the present invention is described below. Desmodesmus sp. is a type of green algae that is representative of the genus Desmodesmus, and forms colonies with multiple cells joined together. Because its outer covering is very strong, it is said that if a method for efficiently destroying the outer covering of Desmodesmus sp. could be developed, the method would also be able to easily destroy the outer coverings of other microalgae.

Figure 7 is a micrograph showing a suspension of Fraxinus edulis before the shearing treatment according to the present invention. The magnification is 200x, and the boundary line is 0.5 mm x 0.5 mm. Two to four cells are observed to be joined together.

The crushing process was carried out by passing a cassia suspension (processing liquid) of the following concentration through the shear processing section 4 for a specified shear processing time under the shear processing conditions described below. The shear processing time was expressed as the integrated value of the residence time in the shear processing section 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 shear processing section 4 by the volumetric flow rate of the cassia suspension.

Destruction of the algal coat of the Fagaceae was evaluated by centrifuging the treated Fagaceae suspension (2000×g for 5 minutes) and expressing the amount of chlorophyll in the supernatant as a fluorescence intensity value.

(Fragaria edulis suspension)
Concentration: Approximately 0.4 g/ml dry weight
(Shearing conditions)
Volumetric flow rate of the kadamomo suspension (discharge flow rate of pump 2): 270 [ml / min]
Pressure applied to the raspberry suspension (pressure applied to the valve portion 42): 90 MPa, 80 MPa, 70 MPa, 60 MPa
(Evaluation of the destruction of the algal envelope of Ikazumo):
- Amount of chlorophyll in the centrifugal supernatant (fluorescence intensity value)

Figures 8 and 9 are micrographs showing the state of a rock stalk suspension when it is subjected to shear processing for a specified period of time under conditions of an applied pressure of 90 MPa. The magnification is 200x, and the boundary is 0.5 mm x 0.5 mm. It can be seen that the cells that were joined together after 0.3 seconds of shear processing have broken apart, and some have leaked their contents, forming a ruptured bag-like outer coat. As the shear processing time is increased, the number of cells that maintain the coat shape is greatly reduced, and at 0.9 seconds they have almost disappeared. If the photographs from 0.5 seconds onwards are enlarged and carefully observed, it can be seen that the number of cracked shell-like outer coats gradually increases, and these are also destroyed and broken into small pieces as the shear processing time increases.

Figure 10 is a graph showing the relationship between the shear force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant. The vertical axis represents the amount of chlorophyll (fluorescence intensity value) leaked out of the cells, and the horizontal axis represents the shear force application time. The solid square ■ indicates the shear force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant when the applied pressure to the cascade suspension is 60 MPa. The solid circle ● indicates the shear force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant when the applied pressure to the cascade suspension is 90 MPa. The solid triangle ▲ indicates the shear force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant when the applied pressure to the cascade suspension is 80 MPa. The hollow circle ○ indicates the shear force application time and the amount of chlorophyll (fluorescence intensity value) in the centrifugal supernatant when the applied pressure to the cascade suspension is 70 MPa.

From FIG. 10, in the case of the kadanumo suspension in which the cells are not broken (zero shear processing time), all the algae bodies are precipitated by centrifugation, so there is no chlorophyll in the centrifugation supernatant, and the fluorescence intensity is almost zero. With the shear processing according to the present invention, the fluorescence intensity increased with increasing shear force application time under all pressure conditions. That is, with increasing shear processing time, the destruction of the algae body coat progressed, and the cell contents including chlorophyll leaked into the processing liquid, increasing the chlorophyll fluorescence intensity in the centrifugation supernatant. The increasing tendency was almost the same for all processing pressures, reaching a maximum value in about 1 second. At 60 MPa, the maximum fluorescence intensity was about half that at 70 MPa or more. This was thought to be because at 60 MPa, the algae bodies remained with their coats not completely destroyed, and chlorophyll was centrifuged while still contained within the cells. That is, it was shown that it is better to process with an applied pressure of 70 MPa or more in the wet shear processing system 100 according to the present invention. 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.

Next, an example of shear processing of encysted Haematococcus using the wet shear processing system 100 according to the present invention will be described. Haematococcus, a green algae, is usually green and spherical, but when it is subjected to stress such as ultraviolet rays, it accumulates red astaxanthin inside the algae and encysts. This astaxanthin has high added value as a functional substance with strong antioxidant activity and as a natural pigment due to its bright red color, and is produced on a commercial scale for use as a raw material for health foods and skin care products. The cultured Haematococcus cysts are dried by methods such as ventilation drying, spray drying, and drum drying, and the dried algae are mechanically crushed, after which the internal astaxanthin is extracted with supercritical carbon dioxide or an organic solvent, in the conventional method. However, as mentioned above, the conventional method involving heating has a problem that astaxanthin is easily oxidized and deteriorated because it comes into contact with high-temperature oxygen during drying (astaxanthin is a natural antioxidant and is very easily combined with oxygen). In addition, it is difficult to mechanically destroy the dried, fine algal coat, and in particular, the membrane (mantle) of Haematococcus cysts exhibits gelatin-like physical properties, being elastic and tough, but not brittle, making it even more difficult to crush. Therefore, the development of an efficient method for crushing the mantle, which acts as a barrier to extraction, is an urgent and important issue.

The shearing process (crushing process) according to the present invention was carried out by passing a Haematococcus cyst suspension (processing solution) of the following concentration through the shearing process section 4 for a specified shearing process time under the following shearing process conditions, in the same manner as for the rock stalk suspension. The shearing process time is expressed as the integrated value of the residence time in the shearing process section 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 process section 4 by the volumetric flow rate of the Haematococcus cyst suspension.

(Haematococcus cyst suspension)
Concentration: Approximately 0.4 g/ml dry weight
(Shearing conditions)
Volumetric flow rate of Haematococcus cyst suspension (discharge flow rate of pump 2): 270 [ml/min]
Pressure applied to the Haematococcus cyst suspension (pressure applied to the valve portion 42): 95 MPa, 70 MPa, 50 MPa
(Evaluation of the destruction of the algal envelope of Haematococcus cysts):
Absorbance method: absorbance at a wavelength of 470 nm

Figure 11 is a micrograph showing a Haematococcus cyst suspension before shearing according to the present invention. Figure 12 is a micrograph showing the state of the Haematococcus cyst suspension when sheared under an applied pressure of 95 MPa. Figure 13 is a micrograph showing the state of the Haematococcus cyst suspension when sheared under an applied pressure of 70 MPa. Figure 14 is a micrograph showing the state of the Haematococcus cyst suspension when sheared under an applied pressure of 50 MPa. As with the case of the rock stalk suspension, the magnification is 200x and the boundary line dimensions are 0.5mm x 0.5mm.

As shown in Figure 12, it can be seen that the red cyst cells disappeared after just 0.1 seconds of shear disruption treatment. The shear treatment (shear disruption treatment) of the present invention destroys cells by shear force, i.e., the force of shearing, and as a result, the cells are observed to have a morphology that looks like their outer coat is cracked. In addition, because the shear force is transmitted using a fluid (water) as a pressure medium, the force is transmitted evenly to every corner, resulting in a characteristic that there is no unevenness in the destruction.

As shown in Figure 13, most of the cysts were disrupted by shearing for about 0.3 seconds when the pressure applied to the Haematococcus cyst suspension was 70 MPa. Also, as shown in Figure 14, most of the cysts were disrupted by shearing for about 0.4 seconds when the pressure applied to the Haematococcus cyst suspension was 50 MPa. This is because the encysted Haematococcus coat is more elastic than the rock scallop coat but less tough. As shown in Figure 10 above, an applied pressure of 70 MPa or more was required to destroy the rock scallop coat, and a long shearing time was required, whereas the Haematococcus coat can be efficiently destroyed by shearing for a short period of time even with an applied pressure of 50 MPa.

Figure 15 is a photograph showing the state of a Haematococcus cyst suspension that has been left to stand for 30 minutes or more after shearing according to the present invention. Figure 15(a) shows the Haematococcus cyst suspension when sheared for a predetermined time under a pressure condition where a pressure of 95 MPa is applied to the Haematococcus cyst suspension. Figure 15(b) shows the Haematococcus cyst suspension when sheared for a predetermined time under the same pressure condition of 50 MPa.

In untreated Haematococcus cyst suspensions, the cysts precipitated during standing, and the supernatant showed no coloration (the red line on the liquid surface is due to reflection of the precipitate). On the other hand, in the sheared solution, the liquid became colored after a short shearing treatment of 0.1 seconds at both 95 MPa and 50 MPa, indicating that the astaxanthin present inside the cysts leaked into the supernatant. To quantitatively express this leakage behavior, the sheared solution was centrifuged (2000 x g for 5 minutes) to precipitate the solids, and the absorbance of the centrifugal supernatant at 470 nm was measured, and the results are shown in Figure 16.

Figure 16 is an explanatory diagram showing the correlation between absorbance (A470) at a wavelength of 470 nm and the application time of shear force. The wavelength of 470 nm is the maximum absorption wavelength of astaxanthin, and it is safe to assume that the absorption intensity of the solution at this wavelength roughly represents (is proportional to) the amount of dissolved astaxanthin. The measurement results showed that astaxanthin in the cysts was dissolved (leaked) into the liquid by a short shear treatment of about 0.3 to 0.4 seconds. In other words, it was shown that the cysts were almost completely destroyed. This result is in good agreement with the observation results of the microscopic photographs shown in Figures 12 to 14. From this result, it was shown that the shear treatment method of the present invention can sufficiently destroy the outer coat (cyst) of Haematococcus in a short time under low pressure conditions, compared to algae with a strong outer coat such as Ikadama.

The astaxanthin that leaked into the solution has not come into contact with oxygen or been heated at all, so it is believed to have dissolved as high-quality astaxanthin. There are several options for subsequent separation and purification methods for astaxanthin, and solvent distribution is thought to be effective. In this case, the use of vacuum concentration or the like can further improve efficiency. Freeze-drying or solvent extraction after vacuum drying is also thought to be effective, making it possible to create a variety of processes. In particular, the shearing treatment and wet shearing treatment system 100 of the present invention has the advantage that the process can be continuous, such as cultivation → continuous centrifugation (suspension preparation) → continuous shear destruction → continuous centrifugation, since the algae cells after cultivation are crushed in a liquid (suspension) state.

As described above, according to the wet shear processing system 100 of the first embodiment of the present invention, the shear force generated between the flow of the processing liquid and the flow path surface (narrow passage 41c, first surface 41b, second surface 42a) with which the processing liquid comes into contact destroys the strong outer coating (algal outer coating such as cell wall, cell membrane, cyst membrane, etc.) of the microalgae in the processing liquid, thereby enabling useful substances within the cells of the microalgae to leak out from the cells into the processing liquid efficiently in a short time and without deteriorating their quality.

In addition, by forming a circumferential groove that changes the flow direction of the treatment liquid on the flow path surfaces (narrow passage 41c, first surface 41b, second surface 42a) with which the treatment liquid comes into contact, the shear force (shear action) generated between the flow of the treatment liquid and the narrow passage 41c or surface gap 47 can be increased, and it becomes possible to separately apply a load due to pressure fluctuations caused by the change in the flow direction of the treatment liquid to the microalgae bodies.

In addition, by passing the treatment liquid through the narrow passage 41c and the surface gap 47 while applying pressure in a direction that closes the surface gap 47, it is possible to increase the shear force (shear action) generated between the flow of the treatment liquid and the narrow passage 41c or the surface gap 47.

In addition, by adopting a reciprocating pump, which can easily increase the discharge pressure, as the pump 2 that pressurizes the treatment liquid, it is possible to increase the shear force (shear action) generated between the flow of the treatment liquid and the narrow passage 41c or the surface gap 47.

In addition, by passing the treatment liquid through the narrow passage 41c and the surface gap 47 and ensuring that the shear force acts on the microalgae for a long time, it is possible to efficiently destroy the outer coverings of the microalgae contained in the treatment liquid (cell walls, cell membranes, cyst membranes, and other algal outer coverings).

As a result, it becomes easier to separate and purify the algal contents (intracellular useful substances) from the treated liquid. Furthermore, if the sheared suspension is dried or otherwise processed into a food material without separating the leaked contents from the crushed outer coat, the useful components can be absorbed and utilized more efficiently in the body than if it was dried without shear destruction. Similarly, if the sheared suspension is converted into compost or other fertilizer without separating the leaked contents from the crushed outer coat, it becomes possible to improve fermentation efficiency.

In addition, by separating and removing the outer covering of the microalgae cells, which acts as a barrier to moisture movement, from the treated liquid, the drying time (drying rate) of the extracted algae cell contents (useful substances) is shortened (speeded up), thereby making it possible to reduce the energy required to dry the algae cell contents (useful substances).

Although the wet shear processing system 100 according to the first embodiment of the present invention has been described with reference to Figures 1 to 16, the embodiment of the present invention is not limited to the above. In other words, various modifications and changes can be made within the technical scope of the present invention. For example, a circumferential groove may be formed in a circular or spiral shape on the inner surface of the narrow passage 41c with which the processing liquid comes into contact. In addition, the shape of the first circumferential groove 41d and the second circumferential groove 42d may be a concentric circle, a closed curve such as an ellipse or a closed polygon, an eccentric shape as described above, or a radial shape.

In addition, the first flow path 41a may be configured with only a straight tube without providing a narrow path 41c. In addition, the number of times that the Haematococcus cyst suspension (processing liquid) passes through the shear processing unit 4 may be only one.

As described above, the method of destroying the outer coat of microalgae by shear processing according to the present invention is a far superior method of destruction compared to other methods. There are several possible methods of applying shear force to the outer coat of microalgae, but as shown in the above embodiment, the method of applying shear force to the outer coat of microalgae by passing the treatment liquid pumped by pump 2 through narrow passage 41c and surface gap 47 pressurized in the closing direction shows a very superior destruction effect.

It is noteworthy that the method of destroying the outer coat of microalgae using shearing treatment of the present invention made it possible to easily crush even Ikazumo, which has a strong outer coat and is said to be very difficult to crush after drying. In addition, since this outer coat destruction method destroys the outer coat without going through a heating and drying process as in conventional methods, the contents inside the algae are not subjected to a thermal history, and contents (intracellular useful substances) can be obtained with little deterioration. The target components (intracellular useful substances) that leak into the treatment solution can be separated and purified by solvent distribution, etc.

In addition, by treating the treatment liquid with centrifugation or membrane filtration, it is possible to easily separate the solid capsules that have been destroyed and are suspended in the treatment liquid from the dissolved intracellular useful substances. Even when a solution containing intracellular useful substances is dried by spray drying or freeze drying, it is possible to perform drying with high efficiency because the capsules, which act as a barrier to the movement of water, are not destroyed and are not included.

Microalgae with their outer coats crushed could, for example, be dried in their entirety and used as a food ingredient or feed. In this case, the outer coat, which acts as a digestive barrier, is destroyed, improving digestibility and absorption, and it is expected that nutritional value, digestibility, and feed efficiency will increase. It is also expected that the speed and efficiency of composting and other fertilizer production will improve.

Second Embodiment
FIG. 17 is an explanatory diagram showing a wet shear processing system 200 according to a second embodiment of the present invention.
Compared to the wet shear processing system 100 according to the first embodiment described above, this wet shear processing system 200 is configured to include a low-temperature tank 10 that freezes the processing liquid of the sheared Haematococcus cyst suspension, a freeze dryer 11 that dries the frozen processing liquid under reduced pressure, a first inlet pipe 12 that introduces the processing liquid from the shear processing unit 4 into the low-temperature tank 10, a first shut-off valve 12a that turns the flow of the first inlet pipe 12 on and off, a second inlet pipe 13 that introduces the processing liquid from the reserve tank 1 into the low-temperature tank 10, a second shut-off valve 13a that turns the flow of the second inlet pipe 13 on and off, and a third inlet pipe 14 that is connected to the first inlet pipe 12 and the second inlet pipe 13.

In other words, in order to increase the extraction rate of astaxanthin from the sheared Haematococcus cyst suspension, the wet shear processing system 200 freeze-dries the sheared Haematococcus cyst suspension using a low-temperature bath 10 and a freeze-dryer 11. This freeze-drying is described below.

Haematococcus lacustris dSgD-K1 strain, collected and isolated in Saga City, was cultured in AF6 medium at a temperature of 25°C, with a photon flux density of 80 μmol/ ^m2 /s and a light exposure time of 12h:12h light:dark.Then, the strain was exposed to light with a photon flux density of 300 μmol/ ^m2 /s continuously for 24 hours to induce encystment through light stress (the temperature was lowered to 15°C to suppress growth).
The obtained cysts were repeatedly suspended in deionized water and centrifuged to wash out the medium components, then suspended again in deionized water. These were then equally divided to give the same concentration and subjected to extraction tests of the five patterns (1 to 5) shown in Figure 18 below.

FIG. 18 is an explanatory diagram showing the extraction of astaxanthin based on the wet shear disruption method using the wet shear processing system 200 according to the present invention and the extraction of astaxanthin based on other disruption methods.
As shown in Figure 18, the pretreatment methods for extraction can be roughly divided into the upper "pattern of crushing, drying, and then extraction" (Methods 1 and 2) and the lower "pattern of drying, crushing, and then extraction" (Methods 3 to 5). As mentioned above, in this test, all five of these tests were compared using Haematococcus cyst suspensions prepared to the same concentration. They will be explained in order below.

<Method 1: Wet shear fracture>
This method 1 is a wet shear destruction method using the wet shear processing system 200 of the present invention. Note that the hot plate drying (120°C) is for comparison purposes to confirm the effect of freeze drying and is not included in the configuration of the present invention. In addition, the applied pressure during the shear processing is 90 MPa.

Figure 19 is an explanatory diagram showing the disruption behavior of Haematococcus cysts before and after shear treatment using the wet shear disruption method of the present invention. Figure 19(a) shows a suspension of Haematococcus cysts before disruption (hereinafter referred to as "cyst suspension"), (b) shows the cyst suspension after shear treatment, and (c) is an enlarged view of the cyst suspension in (b). The cyst suspension was diluted 200 times to facilitate microscopic observation.

As shown in Figure 19 (a), the Haematococcus cysts remain covered by the exoskeleton, and no dissolution or elution of astaxanthin from the exoskeleton is observed.

Figure 19(b) is a photograph of the cyst suspension after it had been passed multiple times through the shear processing section 4 of the shear processing system 200. The "shear processing time of 0.92 seconds" shown in the figure refers to the cumulative time that the cyst suspension remained in the shear processing section 4, i.e., the total time that it was subjected to shear force. As can be seen from Figure 19(b), most of the cyst cells were destroyed by the shear force in the very short time of 0.92 seconds.

Figure 19(c) is a further enlarged photograph showing the destruction of the cyst cells. As can be seen from Figure 19(c), the contents of the cyst have leaked out of the cells and become empty.

In this wet shear disruption method, the obtained sheared solution was dried using two types of drying methods, "freeze drying" and "hot plate drying," shown in Figure 18, and the amount of astaxanthin extracted from the obtained dried product was measured. Note that the hot plate drying was used for comparison purposes to confirm the effect of freeze drying.

Freeze-drying was performed by freezing the sheared liquid placed in a recovery flask while rotating it in a low-temperature bath at -40°C, and then drying the frozen sheared liquid in a freeze-dryer under a reduced pressure of approximately 4 Pa for more than two days.

For hot plate drying, the shearing solution was dropped with a pipette onto a hot plate (model number: AS ONE ND-2A) set to 120°C, and once the water had evaporated, it was scraped off with a scraper to obtain a dried product. At this time, the time required from dropping the shearing solution to scraping it off (drying time) was approximately 1 minute. The astaxanthin in these dried products was quantitatively analyzed using the HPLC method described below. The results of this quantitative analysis will be described later with reference to Figure 22.

<Method 2: Ultrasonic Disintegration>
Ultrasonic disruption is a conventional method widely used as a cell disruption method including microorganisms. Here, a probe-type ultrasonic disruption device (Sonifier (registered trademark) SFX 250) was used to disrupt 50 mL of cyst suspension in a plastic container under the conditions of 20 kHz and 250 W. The sample liquid (cyst suspension) was treated while being cooled on ice, but a sudden increase in temperature could not be suppressed, so the treatment was interrupted every few minutes and cooled on ice, with care taken not to allow the temperature to exceed 50°C.

Figure 20 is an explanatory diagram showing the destruction behavior of Haematococcus cysts by ultrasonic treatment. Figure 20(a) shows Haematococcus cysts before ultrasonic treatment, and (b) shows Haematococcus cysts after ultrasonic treatment.

As shown in Figure 20 (b), when ultrasonic treatment was performed on 50 mL of the cyst suspension for 15 minutes, many cells remained unbroken. This ultrasonic treatment liquid was also dried on a hot plate at 120°C, like the shear treatment liquid, to prepare a sample for astaxanthin extraction. The astaxanthin in this dried material was quantitatively analyzed by the HPLC method described below. The results of this quantitative analysis will be described later with reference to Figure 22.

Methods 3 to 5 (lower part of Figure 18) are conventional methods in which drying is carried out first, and then the dried algae are destroyed. In other words, these methods are carried out in the reverse order to the above-mentioned methods 1 and 2 (upper part of Figure 18), in which crushing is carried out first. For reference, the following methods will be explained in order, starting with method 3.

<Method 3: Hot plate drying>
In the hot plate drying method 3, first, "the Haematococcus cyst suspension was heated and dried on a hot plate" and then "the dried product was crushed with a glass homogenizer" to extract astaxanthin.

In commercial-scale astaxanthin production (hereinafter referred to as the "current method"), the astaxanthin is heated and dried using a drum dryer or similar device, then mechanically crushed in a grinder such as a disk mill or crusher, followed by solvent extraction. Therefore, method 3 performed here is the method closest to the current method, and because the outer coating is crushed in the extraction solvent, it is a method with a higher extraction efficiency than the current method.

Specifically, the untreated cyst suspension was dropped with a pipette onto a hot plate (model number: AS ONE ND-2A) set to 120°C in the same manner as in methods 1 and 2 above, and after drying, the suspension was scraped off with a scraper (approximately 1 minute from dropping to scraping). Next, 10 mg of the resulting dried cysts were weighed into a Dounce-type glass homogenizer, and a small amount of acetone was added to crush the cyst envelope. The liquid obtained by this operation was then adjusted to a constant volume in a measuring flask and used as a sample for measuring the amount of astaxanthin. In addition, extraction was also performed on samples that were only hot plate dried (without glass homogenization) to verify the effect of glass homogenization. The results of this quantitative analysis will be described later with reference to Figure 22.

Method 4: Freeze-drying
Among various drying methods, freeze-drying method 4 is the method that causes the least damage to the dried raw materials. It is the most preferable drying method especially for substances that are easily oxidized such as astaxanthin, which is the target substance in this study. Therefore, freeze-drying method was also used to dry the sheared solution in method 1.

Here, 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 approximately 4 Pa for at least two days. The resulting dried cysts were weighed into a Dounce-type glass homogenizer in the same manner as in Method 3, and a small amount of acetone was added to crush the cyst coat, after which the volume was adjusted to a constant value to prepare a sample for measuring the amount of astaxanthin. Again, extraction was performed on samples that were only freeze-dried (without glass homogenization) to verify the effect of glass homogenization. The results of this quantitative analysis will be described later with reference to Figure 22.

<Method 5: Hot air drying>
Method 5, hot air drying, involves exposing the material to be dried to high-temperature hot air, which poses a high risk of antioxidants such as astaxanthin being oxidized and deteriorated by high-temperature oxygen. However, this is the simplest and most inexpensive drying method, so it was chosen as one of the methods for comparison.

Here, approximately 25 mL of the cyst suspension was placed in a 90 mm petri dish and dried for 4 hours in a hot air dryer (model number: EYELA WFO-400W) at 95°C. The dried cysts were scraped off with a scraper and weighed into a Dounce-type glass homogenizer as in method 3, and a small amount of acetone was added to crush the cyst coat and the volume was adjusted to a constant value to obtain a sample for measuring the astaxanthin content. Again, extraction was performed on a sample that was only hot air dried (without glass homogenization) to verify the effect of glass homogenization. The results of this quantitative analysis will be described later with reference to Figure 22. The method for quantitative analysis of astaxanthin is explained below.

<Astaxanthin quantitative analysis method>
Quantitative analysis was carried out according to the Astaxanthin Quantitative Test Method set by the Astaxanthin Industry Association. Specifically, the method is as follows.

10 mg of the extraction test sample (dried sample) was weighed out and transferred to a 50 mL volumetric flask, and approximately 20 mL of acetone was added. However, for sample 3 according to method 3, sample 4 according to method 4, and sample 5 according to method 5, which were glass homogenized (Dounce type), the crushed sample in the glass homogenizer was washed out with a small amount of acetone to make approximately 20 mL.

For samples 1 to 5, about 20 mL of acetone was further added and ultrasonicated at 45 kHz for 5 minutes, and then the volume was adjusted to 50 mL with acetone. 3 mL of this sample solution was dispensed into a 15 mL centrifuge tube, and 2 mL of 0.05 M Tris-HCl buffer (pH 7.0) was added. After equilibration by heating in a 37°C incubator for 2 minutes, 600 μL of 3.4 units/mL cholesterol esterase solution was added, and the reaction was allowed to proceed for 45 minutes in a 37°C incubator while gently mixing by inversion every 10 minutes to prevent foaming. After the reaction was completed, about 1 g of sodium sulfate decahydrate and 4 mL of petroleum ether were added. The petroleum ether was added in a fume hood. After mixing by inversion 30 times, the mixture was stirred for 30 seconds with a vortex mixer and centrifuged at 1200×g for 3 minutes. Approximately 5 mL of the upper layer was transferred to a 300 mL eggplant flask using a Pasteur pipette, 4 mL of petroleum ether was added to the remaining lower layer, and the same mixing, centrifugation, and separation of the upper layer were repeated once again. The petroleum ether was removed using a rotary evaporator at 32°C, and 3 mL of a hexane-acetone mixture (82:18 V/V) was added and dissolved to prepare the HPLC analysis sample solution. The HPLC analysis conditions are as shown in Table 1 below.
[Table 1 HPLC analysis conditions]

Figure 21 is an explanatory diagram showing an example of a chromatogram obtained by the wet shear disruption method according to the present invention. After enzyme treatment, the ester peak disappears, and the trans isomer or the 9-cis and 13-cis isomer peaks become visible.

The amount of astaxanthin in the extract was calculated according to the formula recommended by the Astaxanthin Industry Association, shown below in Equation 1. For the calculation, a calibration curve was created using astaxanthin (010-27461) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. as the standard substance.
(Formula 1): P(total) = P(trans) + 1.2P(9-cis) + 1.6P(13-cis)
here,
P (trans): all-trans astaxanthin peak area 1.2P (9-cis): 9-cis astaxanthin peak area 1.6P (13-cis): 13-cis astaxanthin peak area

Figure 22 is an explanatory diagram showing the amount of astaxanthin extracted from dried Haematococcus cysts obtained by the above methods 1 to 5. The numerical values indicate the amount of astaxanthin extracted (mg) per gram of dried cysts. In conclusion, the "wet shear disruption method" (Method 1) using the above wet shear processing system 200 according to the present invention showed the best extraction efficiency, confirming the superiority of the wet shear disruption method according to the present invention. A detailed explanation is given below.

In the wet shear disruption method (Method 1) according to the present invention, two drying methods, freeze-drying and hot plate drying, were applied to dry the treated liquid after wet shear disruption, and the extraction efficiency was compared. First, the case of "freeze-drying the shear disruption liquid" shown on the left side of Figure 22 showed the largest amount of astaxanthin extraction compared to all other methods.

On the other hand, the result to the right of it, "shear crushing → hot plate drying," shows the case where the shear crushing liquid was dried on a hot plate (120°C). In this case, the amount of astaxanthin extracted decreased from 15.5 to 4.77 [mg/g-dried cysts]. This indicates that the decrease is due to the astaxanthin coming into contact with high-temperature air during drying. This also shows that it is desirable to avoid processing that exposes highly antioxidant substances such as astaxanthin to high-temperature oxygen.

The extraction yield for method 2, "ultrasonic treatment followed by freeze-drying," was only 6.15 [mg/g-dried cysts]. This indicates that the ultrasonic disruption method does not sufficiently disrupt the cysts, and therefore even when freeze-drying, which does not involve any temperature increase, is used, only a low amount of astaxanthin is extracted.

The following methods 3 to 5 are a comparison of methods that follow the same procedure as the current method: drying → crushing → extraction. Method 3, which used "hot plate drying," is the method that is closest to commercial-scale drying methods that use drum dryers and the like. Cysts dried on a hot plate were crushed together with the extraction solvent in a glass homogenizer. This crushing method provides a higher extraction efficiency than methods in which the cysts are crushed in a dry state using a disc mill or the like before extraction. Additionally, to compare the effect of drying alone, the amount of astaxanthin extracted from dried cysts that were only plate dried was also measured.

As a result, as shown in method 3 in Figure 22, drying alone yielded the lowest amount of astaxanthin extraction at 2.12 [mg/g-dried cysts]. This is thought to be because the dried cyst membrane acted as a barrier to the penetration of the extraction solvent into the algae, resulting in insufficient dissolution and elution of the astaxanthin inside. When the dried cysts were extracted while being crushed with a glass homogenizer, the amount of extraction increased to 9.66 [mg/g-dried cysts]. However, this did not reach the 15.5 [mg/g-dried cysts] obtained by the wet shear disruption method of method 1 according to the present invention.

In freeze-drying method 4, as with hot plate drying, the amount of extraction from unbroken dried cysts was limited to 2.81 mg/g-dried cysts because the dried cyst coat acted as an extraction barrier. Breaking the coat using glass homogenization increased the amount to 7.18 mg/g-dried cysts, but this was still only half the amount compared to the wet shear destruction method of method 1 according to the present invention.

In method 5, the amount of extract from the dried cysts after hot air drying was only 5.64 [mg/g-dried cysts], and even after glass homogenization, it was only 8.9 [mg/g-dried cysts]. Hot air drying is the easiest method for drying, but the dried cysts stuck firmly to the walls of the drying container, and peeling them off required a lot of energy and effort. For these reasons, it is considered to be an unsuitable method for drying cysts.

It should be noted that the amount of astaxanthin extracted is lower than the commonly-cited 30-40 mg per gram of dried cysts shown in Figure 22. This is because many of the Haematococcus cysts used as test samples were actually palmeloid cells (immature cysts). Although it is difficult to see in black and white, more than half of the cells were immature cysts, as can be seen in the photographs before destruction in Figure 19(a) and before processing in Figure 20(a). Therefore, the reason the absolute value of the amount of astaxanthin extracted was low was due to the state of the raw material, and not because the wet shear destruction method of the present invention was inferior.

Figure 23 is an explanatory diagram showing a sample in which a cyst suspension was subjected to wet shear disruption and freeze-dried, and a sample in which the cyst suspension was simply dried on a hot plate. Although it is difficult to tell because it is in black and white, the two samples differ greatly in terms of color vividness. The sample subjected to wet shear disruption and freeze-drying is red, while the sample dried on a hot plate is brown.

As described above, the wet shear disruption method using the wet shear processing system 200 of the present invention can efficiently destroy the outer coat of Haematococcus cysts, and by freeze-drying the processing liquid containing astaxanthin that has leaked from inside the cysts, it becomes possible to efficiently extract astaxanthin from the dried cysts while minimizing the oxidative degradation of astaxanthin.

Third Embodiment
The genus Botryococcus, especially Botryococcus braunii, forms cluster-like colonies in a persistent polymer matrix called alginane, and accumulates large amounts of hydrocarbon oils that can be easily used as fuel between the cells and the matrix. The amount of oil can reach 60% of the dry algae weight in some cases, making it a very promising method for liquid fuel production.

Current methods for extracting hydrocarbon oils produced within algae are carried out using various organic solvents, but by applying the wet shear disruption method of the present invention as a pretreatment, the strong shear force destroys the matrix and peels off the oil that is strongly attached to the surface of the algae cells, making it significantly easier to separate and recover the oil components. In particular, by leaving the sheared solution to stand or centrifuging it, it becomes possible to separate the released oil into layers, which makes it easier to carry out the subsequent purification process.

1 Reserve tank 2 Pump 21 Piston 22 First spring 23 Suction valve 24 First valve seat 25 First sleeve 26 In-side connecting portion 27 Discharge valve 28 Second valve seat 29 Second sling 30 Second sleeve 31 Out-side connecting portion 32 Head space 33 Cylinder 34 Connecting rod 35 Crankshaft 3 Suction pipe 4 Shear treatment portion 41 Valve seat portion 41a First flow path 41b First surface 41c Narrow passage 41d First circumferential groove 42 Valve portion 42a Second surface 42b Side surface 42c Rod portion 42d Second circumferential groove 43 Housing portion 44 Inlet portion 45 Outlet portion 46 Actuator portion 47 Surface gap 5 Discharge pipe 6 Return pipe 10 Low temperature bath 11 Freeze dryer 12 First inlet pipe 12a First shutoff valve 13 Second inlet pipe 13a Second shutoff valve 14 Third inlet pipe 100 Wet shear processing system (system for destroying the outer covering of microalgae by shear processing)
200 Wet shear processing system (system for destroying the outer covering of microalgae by shear processing)

Claims (11)

A treatment liquid containing cells having an envelope that produces or contains useful substances and that acts as a barrier during drying, extraction, digestion in a living body, and composting is pumped by a pump (2) and passes through a narrow passage (41c) with a narrowed flow path, whereby the treatment liquid collides with a second surface (42a) having a second groove (42d) formed on the movable surface, and then passes through a surface gap (47) formed by a first surface (41b) having a first groove (41d) that can fit into the second groove (42d) formed on an opposing, immovable surface, and the movable second surface (42a); and the treatment liquid passes through the surface gap (47) while changing the flow direction of the treatment liquid;
A method for destroying the outer coat of microalgae by shear treatment, characterized in that the outer coat of the cells is destroyed in the treatment liquid by shear force generated between the flow of the treatment liquid and the narrow passage (41c), the first surface (41b) or the second surface (42a). The method for destroying the outer coat of microalgae by shear treatment according to claim 1,
a step of pressing the second surface (42a) under a predetermined pressure condition in a direction in which the surface gap (47) closes when the treatment liquid passes through the surface gap (47). The method for destroying the outer coat of microalgae by shear treatment according to claim 1 or 2,
The method for destroying the outer coat of microalgae by shear treatment, characterized in that the treatment liquid is pumped by the reciprocating pump (2). The method for destroying the outer coat of microalgae by shear treatment according to any one of claims 1 to 3,
and freeze-drying the treatment liquid that has passed through the surface gap (47). The method for destroying the outer coat of microalgae by shear treatment according to claim 4,
A method for destroying the outer coat of microalgae by shear treatment, characterized in that the frozen treatment liquid is placed in an environment reduced in pressure below atmospheric pressure and dried. A tank (1) for storing a treatment liquid containing cells having an envelope that produces or contains useful substances and that acts as a barrier during drying, extraction, digestion in vivo, and composting;
A pump (2) for pumping the treatment liquid;
A system for destroying the outer coat of microalgae by shearing treatment, comprising a shearing treatment section (4) for crushing the outer coat of the cells,
The shear treatment section (4) includes a first flow path (41a) for transporting the pressure-fed treatment liquid;
a first surface (41b) that intersects the first flow path (41a) and is immovable;
a second surface (42a) disposed opposite the first surface (41b) and movable;
a second flow path (43a) that accommodates the second surface (42a);
A narrow passage (41c) in which the flow path is narrowed is formed in the first flow path (41a) in a form facing the movable second surface (42a), and a first groove (41d) is formed in the surface of the first surface (41b), and a second groove (42d) capable of being fitted into the first groove (41d) is formed in the surface of the second surface (42a),
The treatment liquid passes through the narrow passage (41c) and a surface gap (47) formed between the first surface (41b) and the second surface (42a) ,
The cells are subjected to a shear force generated between the flow of the processing liquid and the narrow passage (41c), the first surface (41b) or the second surface (42a), which causes the cell coat to break down and the useful substance to leak into the processing liquid.
A system for destroying the outer coat of microalgae by shear treatment. The system for destroying the outer coat of microalgae by shear treatment according to claim 6,
A system for destroying the outer coat of microalgae by shearing treatment, comprising: an actuator unit (46) that applies pressure under predetermined pressure conditions in a direction pressing the second surface (42a) against the first surface (41b). The system for destroying the outer coat of microalgae by shear treatment according to claim 6 or 7,
A system for destroying the outer shell of microalgae by shear treatment, comprising a return pipe (6) connecting the shear treatment section (4) and the tank (1). The system for destroying the outer coat of microalgae by shear treatment according to any one of claims 6 to 8,
the pump (2) is a reciprocating pump comprising a cylinder (33), a piston (21) reciprocating within the cylinder (33), a headspace (32) formed by the cylinder (33) and the piston (21), a suction valve (23) for filling the headspace (32) with a treatment liquid, and a discharge valve (27) for discharging the treatment liquid from the headspace (32). The system for destroying the outer coat of microalgae by shear treatment according to any one of claims 6 to 9,
a low-temperature bath (10) for freezing the treatment liquid that has passed through the surface gap (47);
A system for destroying the outer coat of microalgae by shear treatment, comprising: a freeze dryer (11) for drying the frozen treatment liquid. The system for destroying the outer coat of microalgae by shear treatment according to claim 10,
The freeze-drying machine (11) dries the frozen treatment liquid in an environment reduced in pressure below atmospheric pressure.

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