JP2018093765A - Method for producing oils and fats containing docosahexaenoic acid produced by microalga belonging to genus aurantiochytrium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing oils and fats having a step of extracting and separating oils and fats containing a higher unsaturated fatty acid such as docosahexaenoic acid (DHA) produced by microalga belonging to the genus Aurantiochytrium.SOLUTION: A culture liquid obtained by culturing microalga belonging to the genus Aurantiochytrium in a salt-containing culture medium is mixed with an organic solvent without pretreatment such as concentration, dehydration, or desiccation, and oils and fats including DHA, etc. are extracted in the organic solvent when the cell membrane of the culture alga contained in the culture liquid is crushed. The oils and fats are separated in the extraction solvent phase, the crushed algal residue is separated in the dehydrated solid phase, and the culture liquid is separated in the drainage phase. After washing with water and removing contaminants contained in the extraction solvent phase such as salts and gum, the extraction solvent phase is treated with a solvent separation and recovery system and solvent is separated and recovered from oils and fats and recycled. The separated oils and fats is made into oil and fat products by separation and removal treatment from remaining solvent, and through purification treatment such as deoxidation and decoloration.SELECTED DRAWING: Figure 1

Description

  The present invention is a process for directly extracting and separating fats and oils containing useful components such as docosahexaenoic acid produced by microalgae from cultured algal cells belonging to the genus Aurantiochytrium cultivated in a culture medium containing salt The present invention relates to a method for producing fats and oils containing docosahexaenoic acid.

Patent Document 1 shows that the growth rate of microalgae strains belonging to the genus Aurantiochytrium is very fast. From the microalgae, it became clear that the microalgae of the genus Aurantiochytrium are capable of producing large quantities of useful substances such as squalene and docosahexaenoic acid (hereinafter referred to as “DHA”) at a high growth rate. Manufacturing methods that enable efficient and economical production of fats and oils including squalene, DHA, and the like have been studied.
For example, Patent Document 2 relates to a method for producing an oil produced by a specific strain of microalga belonging to the genus Navikura (JPCC DA0580 strain). In Patent Document 2, the cultured microalgae are concentrated and recovered by centrifugal precipitation, filter filtration, or the like, and the oil is extracted from the recovered alga bodies with an organic solvent. In Patent Document 2, it is preferable to physically destroy algal bodies with an ultrasonic homogenizer or the like in order to increase extraction efficiency with a solvent, and it is said that purification of the extracted oil may be performed by a known method. That is, Patent Document 2 discloses that algae are separated and recovered from a culture solution as a pretreatment for extraction.
According to Patent Document 3, an organic solvent dimethyl ether (DME) is permeated into cells and vaporized under reduced pressure to crush the cell membrane, and then an extraction solvent is separately added to and mixed with the crushed cell-containing oil and fat for extraction. It is shown.
In Patent Document 4, after the target microalgae are subjected to solid-liquid separation, that is, concentration and dehydration, extraction is performed with an extraction solvent containing a nonpolar organic solvent in a state where the cell membrane is not destroyed or dissolved.
Patent Document 5 describes a method for optimizing the production of hydrocarbons by microorganisms such as microalgae belonging to the genus Aurantiochytrium.
Further, in Patent Document 6, chitosan as a flocculant is added to agglomerate, oil and fat contained in the collected microalgae is extracted, and algal residue after oil and fat extraction is effectively used.

International Publication No. 2012/077779 International Publication No. 2010/116611 JP 2011-31170 A JP 2014-140308 A Japanese Patent Laid-Open No. 2015-96050 JP 2014-14284 A

The methods described in Patent Documents 2 to 6 have been made for the purpose of improving the economics of production. However, the method for extracting, separating and refining oils and fats from microalgae has only improved the partial treatment means and the general functions of each process, and further improved economics and stable operation. Means for solving the technical problems included in each of these steps in improving the above are not shown. In particular, when the methods disclosed in Patent Documents 2 to 6 are used to extract and separate oils and fats from microalgae belonging to the genus Aurantiochytrium, there is room for further improvement in economic efficiency and stable operability. There is.
That is, when separating and recovering oils and fats from microalgae cultured in a culture medium containing salt, such as microalgae belonging to the genus Aurantiochytrium, microalgae are considered from the viewpoint of their efficient, economical and stable production. The problem is how to perform dehydration / concentration, extraction with an organic solvent, and separation of the extracted oil and fat from the solvent, and how to treat the algal residue.
Dehydration and concentration of microalgae belonging to the genus Aurantiochytrium is very fine, algal body size of several μm to several tens of μm, and the algal body concentration (dry mass) is as large as 35 g / L to 150 g / L. In many cases, it is very difficult to recover algal bodies by simple dehydration and concentration operations such as filtration.
In addition, the extraction operation with an organic solvent must crush the cell membrane of the microalgae and thoroughly mix the oil and fat produced therein and the solvent to perform extraction and separation as an extraction solvent phase efficiently.
Further, when the solvent separated into the extraction solvent phase is separated and recovered from the fats and oils, in consideration of the influence of the salt content accompanying the solvent phase, the portion in contact with the salt content of the equipment for the organic solvent evaporation treatment including the distillation apparatus is removed. Expensive equipment such as titanium or seawater resistant stainless steel is required. Here, a normal metal material can be used by reducing the salinity, but an additional step for reducing the salinity is required.
Moreover, it is preferable that contamination of a solvent is made into the minimum on the quality of product fats and oils, and the impurity gum substance in fats and oils is also removed efficiently.
An object of the present invention is to efficiently and economically and stably extract and separate oils and fats containing useful components such as DHA produced by microalgae belonging to the genus Aurantiochytrium cultivated in a culture medium containing salt. It is in providing the manufacturing method of DHA containing fats and oils which can do.

As a result of repeated studies to overcome the above-mentioned problems, the present inventors have found a method that enables production of oils and fats containing useful components such as DHA efficiently, economically and stably, and complete the present invention. It came to.
In particular, the present inventors pay attention to the fact that the algae bodies of microalgae belonging to the genus Aurantiochytrium are fine, and the separation operation from the culture solution is not easy by simple separation operations such as filtration. In addition, the cultured algal bodies were crushed and the pulverized extraction method, in which the fats and oils were directly extracted into the organic solvent for extraction added to the culture solution, was intensively studied to determine whether or not it could be applied to the production of fats and oils. As a result, the cultured alga of microalgae belonging to the genus Aurantiochytrium can be directly crushed in the presence of an organic solvent for extracting fats and oils in the culture solution. We obtained new knowledge that the fats and oils encapsulated in the cell membrane can come into contact with the organic solvent and can be extracted into the organic solvent in the culture medium. The present invention has been made based on such new knowledge of the present inventors.
In Patent Document 3, after cell disruption by DME vaporization, an extraction solvent is added and mixed to extract an organic substance with the extraction solvent, and the disruption and extraction are not performed simultaneously.
That is, the method for producing DHA-containing fats and oils from the microalga belonging to the genus Aurantiochytrium according to the present invention,
An organic solvent for extraction of fats and oils in a culture solution containing cultured algal bodies containing DHA-containing fats and oils obtained by culturing with a culture solution containing a salt content of microalgae belonging to the genus Aulanthiochytrium that produces DHA-containing fats and oils Crushing and extracting step of crushing the cell membrane of the cultured alga body in the presence, extracting the DHA-containing fat with the organic solvent, and forming an extraction solvent phase containing the DHA-containing fat;
A separation and recovery step for separating and recovering the extraction solvent phase obtained in the crushing extraction step;
An oil and fat recovery step of recovering DHA-containing oil and fat from the separated and recovered extraction solvent phase;
It is characterized by having.

  According to the method for producing DHA-containing fats and oils according to the present invention, it is possible to efficiently, economically and stably produce fats and oils containing useful components such as DHA and squalene produced by microalgae belonging to the genus Aurantiochytrium. It becomes possible.

It is explanatory drawing which shows the structure of the manufacturing method of the fats and oils containing useful components, such as DHA and squalene which the microalga which belongs to Aulanthiochytrium which concerns on one Embodiment of this invention produces. It is explanatory drawing which shows the structure of the manufacturing method which concerns on other embodiment of this invention. It is a figure which shows the particle size distribution measurement result before and behind algal body crushing by a pressure type homogenizer. It is a figure which shows the particle size distribution measurement result before and behind algal body crushing by a pressure type homogenizer. It is a figure which shows the measurement result of the amount of extraction fats and oils after crushing extraction.

The culture solution of microalgae belonging to the genus Aurantiochytrium inhabiting aerial waters contains salt. The proposition is to efficiently and economically and stably separate fats and oils containing DHA and the like produced from algae (cultured algae) cultured in a culture solution containing salt. The concentration of the microalgae in the harvested culture, the location of the fats and oils, the size / shape and physical strength of the microalgae are greatly involved in determining the production method.
The microalgae belonging to the genus Aulanthiochytrium that produces DHA, etc., has an algal body concentration (dry mass) in the harvested culture solution of 35 to 150 g / L, an algal body size of several μm to several tens of μm, Exists inside the cell membrane. Separation of fats and oils from alga bodies usually involves separation of the alga bodies from the culture solution, reduction of water from the separated alga bodies or drying of the alga bodies, that is, dehydrating and concentrating the alga bodies. It is common to perform extraction with However, for the microalgae, the concentration of algal bodies in the harvested culture solution is high and the size of the algal bodies is very fine. It is difficult. The inventors of the present application pay attention to the technical problems peculiar to such algae, and intensively study a method capable of extracting fats and oils without separating the cultured algal bodies from the culture solution. New knowledge that it can be used for extraction of oils and fats. That is, according to the crushing and extracting step according to the present invention, the harvested culture solution is mixed with an organic solvent, and the algal cell membrane is crushed with a crushing device, and at the same time, the oil and fat is extracted by contact with the organic solvent and the fat and oil sufficiently mixed, The liquid after extraction can be separated into an extraction solvent phase, an extraction residue algae (solid content phase), and a culture solution drainage phase. As an organic solvent for extracting fats and oils, non-polar organic materials such as n-hexane which can extract fats and oils containing at least DHA and can be easily separated from fats and organic solvents by evaporation or distillation operations. A solvent can be used.
Furthermore, it was found that the disruption of cell membranes and the extraction of fats and oils can be carried out simultaneously with a crushing device such as a pressure homogenizer, and phase separation after extraction can be efficiently achieved with a centrifuge. In addition, crushing and / or extraction operation may be repeated twice or more in order to improve the extraction rate.
The culture solution containing cultured algal cells is mixed with a non-polar organic solvent such as n-hexane, and then the crushing and solvent extraction are carried out at the same time, so that most of the gums of impurities in fats and oils are in sufficient contact with water. It moves into the water phase, and the burden of gum removal in the downstream oil refining process is reduced.
In the extraction of fats and oils, supercritical or subcritical extraction methods using carbon dioxide from dried algae are conceivable. However, desalting and dehydration from the culture broth and evaporative removal of water require great energy and cost. Absent.

The manufacturing method of the DHA containing fats and oils based on this invention formed based on each above matter has the following processes.
(1) An organic solvent for oil and fat extraction is added to a culture solution obtained by culturing microalgae belonging to the genus Aurantiochytrium that produces DHA-containing fats and oils in a culture solution containing salt, A crushing and extracting step of crushing a cell membrane of cultured algal bodies, extracting DHA-containing fats and oils with an organic solvent, and obtaining a liquid containing an extraction solvent phase and an aqueous phase composed of an organic solvent phase containing at least the DHA-containing fats and oils.
(2) A separation and recovery step of separating and recovering the extraction solvent phase from the liquid containing the extraction solvent phase and the aqueous phase obtained by the crushing extraction step.
(3) Oil / fat recovery step of recovering DHA-containing oil / fat from the separated and recovered extraction solvent phase.
The culture solution to be treated in the crushing and extracting step contains at least a DHA-containing oil or fat, that is, a cultured algal body having a DHA-containing oil or fat in the cell membrane. By crushing the cell membrane of cultured algal cells in the culture solution, the DHA-containing fat can be extracted with the organic solvent in contact with the organic solvent for fat extraction added to the culture solution. Therefore, the culture solution containing the cultured algal bodies is supplied to the crushing and extraction step without being concentrated and dehydrated and / or dried after the completion of the culture operation.
Useful components remain in the residue (hereinafter referred to as “algae residue”) generated by crushing the algal bodies after the solvent extraction, and are useful as additives such as feed and fertilizer. Algae residue is refined | miniaturized by the crushing with the alga body crushing apparatus, and it can obtain as a microparticles | fine-particles whose particle diameter size is 10 micrometers or less 90 volume% or more.
Therefore, in addition to the crushing and extracting step, the separation and recovery step, and the oil and fat recovery step, a step of separating and recovering the solid content including the algal residue can be added as necessary.

For the separation and recovery step of the algae residue, a separation and recovery method using a flocculant can be preferably used.
The separation and recovery step using the flocculant can be performed on the phase that is the target of separation and recovery of the algal residue. That is, the step of separating and recovering algae residues using a flocculant may be applied to a two-phase liquid aqueous phase and / or extraction solvent phase including an aqueous phase and an extraction solvent phase formed by the crushing extraction step. it can.
When processing both the aqueous phase and the extraction solvent phase, the flocculant is added to the entire two-phase liquid containing the aqueous phase and the extraction solvent phase formed by the crushing extraction process, so that it is contained in the two-phase liquid. The algae residue can be separated and recovered by a separation and recovery step in which the solid content including the algae residue is agglomerated and flocked to form an aqueous phase, an extraction solvent phase, and a solid content phase, and each phase is separated and recovered. The extraction solvent phase recovered by this separation and recovery process is supplied to the oil and fat recovery process.
In addition, when algae residues are dispersed in the two-phase liquid aqueous phase formed by the crushing extraction process, the aqueous phase is separated and treated with a flocculant to agglomerate and floc the algae residues. Can be recovered.
As described above, if the flocculant is added to agglomerate and floc the algae residue, it can be dehydrated and concentrated, and the algae residue can be effectively used.
Although there is a report that agglomeration of algae in a culture solution containing seawater is possible by pH adjustment, it is effective for algal residues that are crushed algal bodies of the present invention. Rather, addition of a flocculant that can achieve agglomeration and flocification quickly and efficiently is suitable.

Conventionally, concentrated dehydration of algae residues has been performed on the extraction residual water phase separated by a two-phase separation centrifuge, etc., of the extraction solvent phase (light liquid) and the extraction residual aqueous phase (heavy liquid) after extraction of the contained fats and oils. A two-stage separation method is generally used in which a flocculant is added to agglomerate / floc the algae residue contained in the extraction residual aqueous phase and concentrate and dehydrate again by centrifugation or filtration.
In the present invention, it is possible to add a flocculant to the entire two-phase liquid after the crushing extraction process, concentrate and dehydrate the algae residue in a three-phase separation centrifuge, and separate the algae residue as a solid phase in one step. became. This one-stage separation method is particularly preferable for reducing the number of steps.
When using algae residues as feed additives, use a flocculant for food additives such as water-soluble chitosan. When using algae residues as fertilizer additives, use a general flocculant such as cation-modified polyacrylamide or polysilica iron. The alga residue can be dehydrated and concentrated to a water content of about 85% by mass.
In addition, when not using an algal residue effectively and making only fats and oils into a product, the coagulant | flocculant is not added but the extraction residual water phase containing an algal residue is made into waste_water | drain.

The oil and fat recovery step may include a water washing step of washing and removing impurities contained in the extraction solvent phase and water-soluble impurities in the oil and fat or water-soluble impurities or having an affinity for water, if necessary. it can.
The separated extraction solvent phase contains water dissolved in an organic solvent and a small amount of entrained water. Since this moisture contains salt and may corrode metal materials such as carbon steel in a temperature range above room temperature, the chloride ion concentration in the moisture is reduced to 200 mg / L (milligram / liter) or less. It is preferable to reduce the salt concentration by washing the extraction solvent phase with water having a product ion concentration of 150 mg / L or less.
In addition, although the fats and oils in the extraction solvent phase contain a small amount of gum, the water-soluble gums are dissolved and transferred to the wash water in the main water washing, and it is possible to further reduce the gum quality in the fats and oils. Become.
Mixing with water having a chloride ion concentration of 150 mg / L or less in an amount of 2 to 25% by mass of the amount of the organic solvent in the extraction solvent phase, separation of precipitation, etc., to make the chloride ion concentration 200 mg / L or less, and It has been found that further reduction of gum quality is possible.
If the amount is less than 2% by mass of the amount of the organic solvent, the chloride ion concentration in the water accompanying the solvent increases, and the risk of corroding the metal material of the equipment may increase. In addition, washing with a large amount of additional water exceeding 25% by mass of the organic solvent amount increases the capacity of the washing / separating apparatus and the wastewater treatment facility.
Moreover, an oil-and-fat collection | recovery process can have an organic-solvent removal process which removes an organic solvent from an extraction solvent phase, and obtains oil and fat. For removal of the organic solvent from the extraction solvent phase, a method of evaporating the organic solvent and condensing and recovering the evaporated organic solvent can be used, and a distillation method can be suitably used for this treatment.
The organic solvent removed from the extraction solvent phase can be recycled as an organic solvent for oil and fat extraction in the crushing extraction step.

By combining the organic solvent removal step after the water washing step, more efficient separation and recovery of the organic solvent can be performed.
The extraction solvent phase containing oils and fats with a chloride ion concentration of 200 mg / L or less and reduced gummy substance concentration strictly separates the oils and solvents in a two-stage evaporation or distillation operation in order to avoid mixing the solvent into the product oils and fats. Thus, the solvent is preferably recycled.
In the first stage of the evaporation or distillation operation, the organic solvent is evaporated or distilled and separated so as to reduce the solvent concentration in the oil separated from the extraction solvent phase to 2 to 50% by mass, preferably 9 to 25% by mass. To do. Under these conditions, for example, when n-hexane is used as the organic solvent, 99% by mass or more of the supplied amount of n-hexane can be separated and recovered at a condensation temperature of 20 to 90 ° C.
Subsequently, in the second stage of evaporation or distillation, the residual organic solvent can be strictly separated and removed from the fats and oils by an operation under a high vacuum of 0.08 to 2 kPaA, more preferably 0.1 to 1 kPaA. The second stage is an operation under a high vacuum, but the second stage distillation treatment feed rate can be 1/100 (mass basis) or less of the amount of organic solvent in the extraction solvent phase. A method has been established that can be economically and stably produced by a combination of evaporation or distillation operations in the first stage.
The separated organic solvent for extraction, such as n-hexane, can be reused again in the crushing extraction process, and if there is a deficiency due to loss or the like, a new organic solvent is replenished as necessary. .
The fats and oils from which the residual solvent has been removed can be further processed in a purification step if necessary. That is, refined fats and oils can be produced by subjecting fats and oils to degumming, deoxidation and decolorization according to conventional methods. In addition, the degumming process included in the normal purification process may be omitted because the gum concentration is greatly reduced in the previous process such as the crushing extraction process and the water washing process added as necessary.

Hereinafter, an embodiment of a manufacturing method according to the present invention will be described.
<Manufacture of fats and oils>
The fats and oils produced in the present invention are obtained by culturing microalgae belonging to the genus Aurantiochytrium. The fats and oils can be produced by extracting and separating the fats and oils contained in the cultured microalgae.
<Microalgae>
As the microalgae belonging to the genus Aurantiochytrium in the present invention, any microalga can be used without particular limitation as long as it can be used for the production of the DHA-containing fats and oils intended in the present invention. For example, a strain collected by a conventional method from the natural environment where such microalgae inhabit, or a strain that can be used for the production of the DHA-containing fat and oil of the present invention can be selected from known strains. As strains of such microalgae, the SR21 strain (Accession No. FERM BP-5034) described in Japanese Patent No. 2746572, the Aurantiochytrium / tsukuba-3 strain (Accession No. FERM described in Patent No. 5842197) BP-11442) and other microalgae strains belonging to the genus Aurantiochytrium which are heterotrophic algae.
<Culture conditions>
The growth and culture of microalgae belonging to the genus Aurantiochytrium of the present invention is carried out by seeding the microalgae strain in a culture solution appropriately adjusted with tap water and artificial seawater and culturing according to a conventional method. Any known culture solution can be used. For example, the carbon source is glucose, fructose, etc., the nitrogen source is yeast extract, corn steep liquor, sodium glutamate, ammonium sulfate, etc., and the culture solution can contain vitamins and the like as appropriate, and tap water or seawater in the culture solution Is about 50% by weight. The culture solution in which the components are adjusted is appropriately sterilized, and after cooling, the pre-cultured microalgae strain is added, and shaking culture or aeration-agitation culture is performed at an appropriate temperature of 10 to 30 ° C.
The salinity concentration of the culture solution is preferably selected from the range of 6.5 g / L to 14 g / L as the chloride ion concentration.

<Fracture extraction, separation, water washing, solvent separation and recovery, residual solvent removal, purification, algae residue and wastewater treatment>
The culture broth containing the cultured microalgae is mixed with an organic solvent, and crushing and extraction are performed simultaneously. The oil and fat is separated as an extraction solvent phase, washed with water, and the solvent is recovered and recycled. The process of separating the algae residue and the wastewater can be carried out with the configuration of the processing method of FIG. 1 or FIG.
The culture solution containing the cultured microalgae and the mixing ratio (volume ratio) of the organic solvent for oil extraction may be set so that the desired oil extraction effect can be obtained, and is not particularly limited. For example, it can be selected from the range of (organic solvent for oil extraction) / (cultured medium containing cultured microalgae) = 0.1 to 1.0.
FIG. 1 is an explanatory diagram showing, in a process flow diagram, the configuration of a method for producing fats and oils containing useful components such as DHA and squalene produced by microalgae belonging to the genus Aurantiochytrium according to one embodiment of the present invention.
In the manufacturing method having the process flow shown in FIG. 1, first, an organic solvent for oil and fat extraction is added to a culture solution containing cultured algae of microalga belonging to the genus Aurantiochytrium from the culture tank 1, and then extracted by crushing. Provided to step 2.
At the initial stage of the production process, the organic solvent is supplied from the organic solvent supply unit 8 for extracting oil and added to the culture solution. In a state where the organic solvent described later can be recycled, the organic solvent returned from the solvent separation and recovery steps 5 and 6 is added to the culture solution for oil and fat extraction. The organic solvent from the supply unit 8 and the organic solvent returned from the solvent separation and recovery steps 5 and 6 may be mixed and used.
In the crushing and extracting step 2, the cell membrane of the cultured alga body is disrupted and the DHA-containing oil and fat produced in the cultured algal body is extracted with an organic solvent.
Furthermore, in the crushing extraction step 2, a two-phase liquid of an aqueous phase and an extraction solvent phase containing the extracted DHA-containing oil and fat is formed, and a flocculant is added to the two-phase liquid. By adding the flocculant, fine solids such as cell residues contained in the two-phase liquid are agglomerated, flocked and settled, and the aqueous phase, extraction solvent phase and solid phase are separated from the two-phase liquid. Three phases are formed.
Each of the three phases is separated in the separation step 3 using a separation device such as a three-phase separation centrifuge, and the aqueous phase is discharged as wastewater and the solid phase is discharged out of the system as algae residue. . Water for washing is added to the extraction solvent phase separated from the three phases, and water washing treatment is performed in the water washing step 4. The aqueous phase obtained after the water washing treatment and the extraction solvent phase are separated, and the aqueous phase is combined with the aqueous phase separated from the three phases formed after the addition of the flocculant and treated as waste water.
The organic solvent is separated and recovered from the extraction solvent phase obtained after the water washing treatment in the separation and recovery step 5, and the recovered organic solvent is returned to the addition position to the culture solution and reused for oil and fat extraction. The organic solvent can be recycled.
Oils and fats can be obtained by the solvent separation and recovery step 5. The residual concentration of the organic solvent contained in the oil and fat is further reduced in the residual solvent removing step 6, and the oil and fat is purified by a conventional method in the subsequent purification step 7.
Preferably, the solvent separation / recovery step 5 uses the first stage operating conditions in the evaporation or distillation operation described above, and the residual solvent removal step 6 uses the second stage operating conditions in the evaporation or distillation operation described above. Can do.
In the purification step 7, a purification method for obtaining a target oil and fat component with a target purity from known purification methods can be appropriately selected and used.

FIG. 2 is a process flow diagram illustrating the structure of a method for producing fats and oils containing useful components such as DHA and squalene produced by microalgae belonging to the genus Aurantiochytrium according to another embodiment of the present invention. .
The manufacturing process shown in FIG. 2 has the same configuration as the manufacturing process shown in FIG. 1 except that the introduction position of the treatment with the flocculant is changed. In the production process of FIG. 2, the separation of the algae residue component by the flocculant is performed on the aqueous phase (extraction residual aqueous phase) obtained from the separation process 3. The flocs containing algae residue obtained by adding the flocculant are dehydrated in the algae residue dehydration step 9, and the water generated by the dehydration is joined to the drainage path from the water washing step 4 and discharged out of the system. In addition, the dehydrated algae residue is collected as algae residue.
The manufacturing process shown in FIGS. 1 and 2 can be performed by a batch method, a semi-continuous method, or a continuous method.
In addition, when the organic solvent used for various treatments such as the organic solvent for oil and fat extraction is volatile in the treatment environment (for example, treatment temperature) using the treatment, the treatment using the organic solvent is performed in a sealable system. It is preferable to carry out in a sealed state.

Hereinafter, the present invention will be described in more detail with reference to examples. In addition, each measured value in the following examples is a value obtained by a conventional method.
(Example)
An aqueous solution containing 20 g of glucose per liter, 10 g of polypeptone, and 5 g of yeast extract was sterilized at 130 ° C. for 15 minutes and then cooled to 25 ° C., and this was mixed with filter-disinfected sea water and tap water. A pre-culture solution of a microalgae strain belonging to the genus Aulanthiochytrium selected as a strain producing oils and fats containing at least DHA is put into a culture solution adjusted to a seawater ratio of about 50% by mass, and sterilized at 25 ° C. for 4 days. The glucose and sodium glutamate aqueous solution were added as appropriate, and the sterilized air was aerated and cultured with stirring.
In addition, as a strain capable of producing fats and oils containing at least DHA, heterotrophic including SR21 strain (Accession No. FERM BP-5034), Aurantiochytrium / tsukuba-3 strain (Accession No. FERM BP-11442), etc. Microalgal strains belonging to the genus Aurantiochytrium can be used as well.
The culture broth harvested after completion of the culture has an algal body concentration (dry mass) of about 60 g per liter, the size of the microalgae is 4 to 30 μm, the frequency average particle size 9.9 μm, and the volume average particle size 10.4 μm. there were. The harvest culture is directly mixed with the organic solvent n-hexane at a volume ratio of organic solvent / harvest culture = 1.0 and supplied to the pressure homogenizer. The microalga contained in the harvest culture is the presence of n-hexane. It was crushed below. By this crushing treatment, the cell membrane of the microalgae was also crushed, and the fat and oil containing at least DHA produced in the cell membrane was released and extracted with n-hexane.
As a result of observation with a laser diffraction / scattering particle size measuring device and an optical microscope, the size of the algal residue and oil produced by crushing the microalgae is 0.3 to 30 μm, and the frequency average particle It has a diameter of 1.5 to 2.2 μm, a volume average particle size of 2.5 to 4 μm, an oil and fat average particle size of 1 μm, and an algal residue (cell debris) average particle size of 5 to 8 μm. In addition, it was confirmed that efficient solvent extraction can be achieved by making mixed contact between the solvent and the oil and fat.
3 (A) to 3 (C) and FIGS. 4 (D) to (F) and Table 1 show the particle size distribution, frequency, and volume average particle size before and after cell disruption under each processing pressure of a pressure homogenizer. It is. FIG. 5 and Table 2 show the oil and fat extraction results with n-hexane after crushing extraction. Good extraction results were obtained at a processing pressure of 20 to 60 MPaG as a pressure homogenizer.

In addition, after ultrasonically crushing the harvested culture solution sample, the total amount of the fats and oils extracted with chloroform / methanol was 15.9 g / L (Table 2), which was determined by n-hexane at a processing pressure of 40 MPaG using a pressure homogenizer. The difference from the extracted oil and fat amount of 15.0 g / L (Table 2) was estimated as the gum mass.
That is, most of the gums produced in the microalgae were dissolved in the water of the culture solution due to the algal bodies (cells) being crushed, indicating that the gum content in the fats and oils was reduced.
The culture broth after crushing extraction became a two-phase liquid that separated into two phases, a light liquid phase (extraction solvent phase) and a heavy liquid phase (aqueous phase) when left standing, but in this example, after the extraction The algae residue was uniformly dispersed in both phases and did not settle. Therefore, a water-soluble chitosan flocculant for food additives (trade name: Fujiclean KT-250, Fuji Engineering Co., Ltd.) is added to the entire two-phase liquid after crushing and extraction, and the algae residue is agglomerated and flocked. It separated into the extraction solvent phase (light liquid phase), the drainage phase (heavy liquid phase), and the algae residue (dehydrated solid content phase) with a separation centrifuge. The same applies when Keithlock C-3298 (trade name, Kyowa Sangyo Co., Ltd.), a general cation-modified polyacrylamide, or PSI-025 (trade name, Suido Kiko Co., Ltd.) of polysilica iron is used as a flocculant. The algae residue could be concentrated and dehydrated.
In the case of this example, if the whole liquid of the two-phase system obtained by crushing extraction is centrifuged without adding a flocculant, solid content such as algae residue is not dispersed in the light liquid phase, Dispersed in the heavy liquid phase (extraction residual aqueous phase). Therefore, the flocculant Fujiclean KT-250 was added to the extraction residual aqueous phase separated into two phases by a centrifuge, and the algae residue in the extraction residual aqueous phase was aggregated and flocked and recovered. Algae residues could be recovered in the same manner when general-purpose Keith lock C-3298 or PSI-025 was used as the flocculant. FIG. 2 shows a configuration of a method of adding a flocculant to the extraction residual aqueous phase when the entire two-phase liquid obtained by crushing extraction is centrifuged, and concentrating and dewatering the algal residue.
The separated extraction solvent phase is washed with water having a chloride ion concentration of about 150 mg / L in order to reduce the concentration of chloride ions in the dissolved and entrained water to 200 mg / L or less and further reduce the concentration of the remaining gum. did.
Since the dissolved and entrained water in the extraction solvent phase contains a chloride ion concentration of about 8 g / L and the extracted oil contains about 3 g / L of gum, the amount of organic solvent is 23% by mass. After injecting water having a chloride ion concentration of about 150 mg / L, mixing and washing with an in-line mixer, the aqueous phase was separated and removed from the extraction solvent phase by stationary sedimentation separation. It was confirmed that the chloride ion concentration of water in the extraction solvent phase after washing was 180 mg / L or less, and the gum concentration in fats and oils was 400 mg / L or less.
The extraction solvent phase having a chloride ion concentration of 180 mg / L or less and reduced gum concentration is obtained by separating and recovering the oil and fat separated and recovered under the atmospheric pressure and the n-hexane condensation temperature of 70 ° C. in the first-stage distillation separation operation. The residual n-hexane concentration was 9% by mass, and 99.8% of the organic solvent n-hexane in the extraction solvent phase was separated and recovered. In the second stage, distillation was performed under a high vacuum of 0.12 kPaA to obtain fats and oils substantially free from the solvent n-hexane.
The oil and fat from which the solvent had been removed was further subjected to deoxidation and decolorization to produce purified oil and fat.

DESCRIPTION OF SYMBOLS 1 Culture tank 2 Crushing extraction 3 Separation 4 Water washing 5 Solvent separation collection 6 Residual solvent removal 7 Purification 8 Organic solvent supply part 9 Algae residue dehydration

Claims (12)

A method for producing docosahexaenoic acid-containing fats and oils,
Organics for extraction of fats and oils in a culture solution containing cultured algal bodies containing docosahexaenoic acid-containing fats and oils obtained by culturing with a culture solution containing salinity of microalgae belonging to the genus Aulanthiochytrium that produce docosahexaenoic acid-containing fats In the presence of a solvent, the cell membrane of the cultured alga body is crushed, the docosahexaenoic acid-containing fats and oils are extracted with the organic solvent, and a crushing extraction step for forming an extraction solvent phase containing the docosahexaenoic acid-containing fats and oils;
A separation and recovery step for separating and recovering the extraction solvent phase obtained in the crushing extraction step;
An oil and fat recovery step of recovering docosahexaenoic acid-containing oil and fat from the separated and recovered extraction solvent phase;
A process for producing a docosahexaenoic acid-containing fat or oil characterized by comprising:   By the crushing extraction step, a two-phase liquid containing the aqueous phase and the extraction solvent phase is formed, and a flocculant is added to the entire two-phase liquid, and the cultured algae contained in the two-phase liquid Claims characterized by having a separation and recovery step of agglomerating and flocking the solid content containing the body residue to form three phases of an aqueous phase, an extraction solvent phase and a solid content phase, and separating and recovering these phases from each other. Item 2. The manufacturing method according to Item 1.   The method according to claim 2, wherein the separated and recovered extraction solvent phase is supplied to the oil and fat recovery step.   The said oil and fat collection | recovery process has a water washing | cleaning process of washing and removing the impurities containing the salt content contained in the said extraction solvent phase, and the gum | gum substance in fats and oils, The water washing process of Claim 1 characterized by the above-mentioned. Production method.   In the water washing step, the extraction solvent phase is washed with water having a chloride ion concentration of 150 mg / L or less in an amount of 2 to 25% by mass of the amount of the organic solvent in the extraction solvent phase, and contained in the extraction solvent phase. 5. The production method according to claim 4, wherein impurities including greasy salt and oily fat are removed from the extraction solvent phase.   The said oil and fat collection | recovery process has an organic solvent removal process which removes the organic solvent for extraction contained in this extraction solvent phase from the said extraction solvent phase, and obtains fats and oils. The production method according to item.   In the organic solvent removal step, the concentration of the organic solvent in the oil and fat is reduced to 2 to 50% by mass in the first stage, and the oil and fat is concentrated. In the second stage, the organic solvent in the oil and fat is vacuumed at an operating pressure of 0.08 to 2 kPaA. The production method according to claim 6, further comprising a two-stage process for further reducing the residual solvent.   The manufacturing method according to claim 7, wherein the concentration of the organic solvent in the fat is reduced to 9 to 25% by mass in the first stage.   The manufacturing method according to claim 7 or 8, wherein the operation pressure of the second stage is 0.1 to 1 kPaA.   The manufacturing method according to claim 6, wherein the organic solvent removing step is performed after the water washing step.   The manufacturing method according to any one of claims 6 to 10, wherein the organic solvent obtained in the organic solvent removal step is separated and recovered and recycled for oil and fat extraction in the crushing extraction step.   The manufacturing method according to any one of claims 6 to 11, wherein the fat and oil from which the residual solvent has been removed is further processed in a purification step.

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