KR20170122889A - Efficient Recovery of Oil from Oleaginous Microorganisms - Google Patents

Abstract

The present invention relates to an efficient extraction method of oil from oleaginous microorganisms. By using the extraction method of oil of the present invention, a drying process of microorganisms and an evaporation process of an organic solvent are not required, and therefore, the process costs for oil extraction can be greatly reduced and the method can be widely used in medicine, cosmetics and food industries.

Description

Technical Field [0001] The present invention relates to a method for efficiently extracting oil from an oil-containing microorganism,

The present invention relates to a method for efficiently extracting oil from an oil-containing microorganism. More specifically, the present invention relates to a method for efficiently extracting oil from an oil-containing microorganism, which does not require a step of drying microorganisms and a step of evaporating an organic solvent.

The world uses fossil fuels as its main energy source, but the carbon dioxide produced during the process is causing global warming. Currently, various studies have been made to reduce carbon dioxide, and studies on microalgae have been actively carried out. Microalgae have carbon assimilation ability to fix carbon dioxide by using light energy, and can produce various useful substances including lipids and proteins. In addition, some microalgae have high oil content and can be used as a raw material for biodiesel or green diesel, which has the advantages of reducing carbon dioxide, producing alternative fuels and useful materials. In addition, much attention has been paid to the cultivation of microalgae containing high-value-added medicines, pigments, carbohydrates and fine chemicals as well as useful substances. As such, the demand for natural materials in various fields such as foods, pharmaceuticals, and chemicals is rapidly increasing, and interest in microalgae as a source of natural substances as well as materials useful for bacteria and fungi are also increasing. Biomass produced through mass culture of microalgae contains various useful substances as shown in Table 1 below.

On the other hand, hematococcus pluvialis ( Haematococcus pluvialis ) contains astaxanthin represented by the following formula (1) which is a strong red antioxidant, and astaxanthin is a raw material for medicines, cosmetics and health functional plants. Hematococzuks initially grew into green with a high chlorophyll content, and then accumulated astaxanthin in red in the cells over time. Health functional foods In order to obtain Hematococcus extract as a functional raw material, supercritical carbon dioxide extraction method or acetone extraction method should be used in accordance with the standard of manufacture of the food.

Korean Patent Laid-Open Publication No. 2011-0116722 discloses a method of using supercritical carbon dioxide as an extraction solvent in the method of producing astaxanthin-containing extract from green algae. However, in this case, the process is performed under an ultra-high pressure condition of 600 to 1000 atm And there is a problem of process complexity such as necessity of a pretreatment process for pulverizing the hematococzus powder or extraction twice.

In the case of extracting astaxanthin with acetone under wet conditions, astaxanthin, acetone and water are mixed in the extraction mixture. In order to prevent decomposition of astaxanthin, Acetone is relatively easy to evaporate. However, acetone extraction method is not yet practical because it requires a lot of energy cost to evaporate water.

Under these circumstances, the inventors of the present invention have found that it is possible to directly extract a microorganism in a watery state without a drying process, to reduce the energy cost by eliminating the need for evaporation of the solvent, As a result of efforts to develop a more advantageous extraction method for weak oil extraction, it has been confirmed that the oil to be separated is recovered by gravity sedimentation or centrifugation by adding an additional oil to the oil extract of the oil-containing microorganism.

Korean Patent Laid-Open Publication No. 2011-0116722

An object of the present invention is to provide an extraction method for recovering a target oil with high efficiency from an oil-containing microorganism.

Another object of the present invention is to provide an extraction method for efficiently recovering astaxanthin from Haematococcus pluvialis .

It is still another object of the present invention to provide a cosmetic or food composition containing astaxanthin.

In order to accomplish the above object, the present invention provides a method for purifying an object oil (first oil) from a microorganism by adding an appropriate amount of an additional oil (second oil) to a physically disrupted microorganism containing the object oil The present invention provides an oil extraction method for efficiently extracting oil.

Specifically, the oil extraction method of the present invention comprises the steps of: preparing a mixture in which a water-miscible organic solvent is added to an aqueous culture medium containing a microorganism containing a target oil (first oil); Obtaining a physically disrupted lump of said mixture; Separating the pulverized microorganisms from the crushed product and recovering a first mixed layer containing a first oil, a water miscible organic solvent and water; Adding a second oil, which is the same as or different from the first oil, to the first mixed layer, wherein the water-miscible organic solvent and the water-containing second mixed layer and the first oil and the second oil- Adding oil; And recovering the third mixed layer.

A second aspect of the present invention provides a method for extracting astaxanthin from hematococzial floualis using the oil extraction method of the present invention.

A third aspect of the present invention provides a method for producing a cosmetic composition or a food composition containing astaxanthin obtained by using the oil extraction method of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for separating a target first oil from a microorganism in a water culture broth by using a water miscible organic solvent and separating the first oil from an aqueous culture broth by adding the same or different second oil which is miscible with the first oil , It has surprisingly been found that the water-miscible organic solvent and the water-containing mixed layer and the first oil and the second oil-containing mixed layer are layered. The present invention is based on this. At this time, in order to separate the layers, the second oil is added in an amount capable of separating the water-miscible organic solvent and the water-containing mixed layer and the first oil and the second oil-containing mixed layer.

When the first oil is present in a small amount in a mixture of water and a water-miscible organic solvent (e.g., acetone), the first oil is uniformly dispersed in the form of fine particles in a water miscible organic solvent. It is not easy. However, if a second oil, which is the same as or compatible with the first oil, is added to the first oil dispersed in the aqueous culture medium, since the binding force between the first oil and the second oil is greater than the binding force with the other solvent, And it is conjectured that the flocculation has finally become possible.

At this time, since the density of the first oil and the second oil-containing mixed layer can be changed according to the amount of the second oil added, the density of the second oil and the second oil can be differentiated from the density of the water miscible organic solvent and the water- The addition amount can be controlled.

Therefore, in order to distinguish the density of the water-miscible organic solvent and the water-containing mixed layer from the density of the first oil and the second oil-containing mixed layer, it is preferable that the concentration of the organic solvent added to extract the first oil from the microorganism- A method in which the amount of the organic solvent is adjusted, or a part of the organic solvent is evaporated after the extraction of the first oil is completed, and then the second oil is added. In addition, centrifugation can be performed to increase the layer separation efficiency, and it is possible to accelerate agglomeration by raising the chance of contact between the oil dispersed by the centrifugal force.

The oil-containing microorganism may be a microalgae, a bacteria, a yeast or a fungus. The oil contained in the microorganism may be selected from the group consisting of? -Carotene,? -Carotene,? -Cryptoxanthin,? -Carotene, Or zeaxanthin.

Specific examples of the oil-containing microorganisms include Haematococcus pluvialis , Botryococcus brownies, braunii), cylinder throw teka (Cylindrotheca sp.), Chemnitz cyano (Nitzschia sp.), seukijo kit Solarium (Schizochytrium sp.), bakteo (Arthrobacter sp.), ahsine Sat bakteo knife core Shetty kusu (Acinetobacter calcoaceticus) in Aspergillus, Rhodococcus opacus), Bacillus know Phil sword Luce (Bacillus alcalophilus), Candida Mercure Bata (Candida curvata), Cryptococcal Syracuse Albi Douce (Cryptococcus albidus , Lipomyces starkeyi , Rhodotorula < RTI ID = 0.0 > glutinis), Aspergillus Duck Party (Aspergillus oryzae , Mortierella isabellina , Humicola < RTI ID = 0.0 > lanuginosa or Mortierella vinacea . It has been reported that the hematococzual fluvialis can accumulate astaxanthin up to 8%, and it is possible to extract astaxanthin from it Research is actively under way.

As used herein, the term " Haematococcus pluvialis "is a brown algae with flagella, having long hairs at the top and swim through it, with relatively thick cell walls (20 [mu] m) , And the space occupied by the cytoplasm is small. If the environment is not good, the cytoplasm is full, and it becomes a dormant, rounded cell with no flagella (cyst cell), which is a secondary metabolite in the body and accumulates a large amount of astaxanthin, a kind of carotenoid pigment, .

The term " astaxanthin "used in the present invention is a kind of carotenoid pigment having a structural formula such as the following formula (2) And is widely distributed in microalgae. It is a fat-soluble coloring matter with a red color and is a representative strong antioxidant.

As can be seen in Table 2 below, the oil content in the dry state of the oil-containing microorganisms which are not astaxanthin but contain other oils is found to be less than 16% by weight and more often up to 86% by weight.

Examples of microalgae in the oil-containing microorganisms of the present invention include, but are not limited to, the microalgae of Table 2, and the microalgae of Anacystis nidulans , Ankistrodesmus sp., Biddulpha < RTI ID = 0.0 > aurita , Chaetoceros sp., Chlamydomonas sp. applanata ), Chlamydomonas reinhardtii , Chlorella sp.), chlorella erlipsoidia ( Chlorella ellipsoidea), Chlorella emulsion Sony (Chlorella emersonii), Chlorella More Koh prototype Death (Chlorella protothecoides), Chlorella frame noise Dosa (Chlorella pyrenoidosa), Chlorella Thoreau Kearney Ana (Chlorella sorokiniana), Chlorella vulgaris (Chlorella vulgaris , Chlorella minutissima , Chlorococcus littorale , Cyclotella cryptica , Dunaliella bardawil , Dunaliella salina), two analytic Ella Hotel Tio rekta (Dunaliella tertiolecta), two analytic Ella Primo rekta (Dunaliella primolecta), jimno Stadium (Gymnodinum sp.), Hime eggplant grandma (Hymenomonas br TB Carterae , Isochrysis galbana , Isochrysis sp., Microcystis aeruginosa , Micromonas pusilla , Monodus suberaceae, subterraneous , Nannochloris sp., Nannochloropsis sp., Nannochloropsis atomus , Nannochloropsis salina , Navicula < RTI ID = 0.0 > pelliculosa), Chemnitz Asia Claus teddy Leeum (Nitzscia closterium), Chemnitz Asia Palais Ah (Nitzscia palea , Oocystis polymorpha , Ourococcus sp., Oscillatoria rubescens , Pavlova lutheri), paeoh duct Solarium tricot nutum (Phaeodactylum tricornutum), peak no Lactococcus Pro Solid bars (Pycnococcus provasolii), minnow furnace eggplant koreuda other (Pyramimonas cordata , Spirulina platensis , Stephanodiscus minutulus , Stichococcus sp., Synedra ulna , Scenedesmus spp. obliquus), skeletal Barinas Tram Le Grassi (Selenastrum gracile), skeletal Norma Leto Costa Rum (Skeletonoma costalum ), Tetraselmis chui ), Tetraselmis maculata), tetra-cell Miss (Tetraselmis sp.), Brassica (Tetraselmis tetra cells can miss suecica , Thalassiostra pseudomona), Ana times (Anabaena sp.), Carl load Riggs (Calothrix sp.), in the Atacama Chiffon (Chaemisiphon sp.), co-Roc CD option System (Chroococcidiopsis sp.), cyano dese (Cyanothece sp.), published Such as Cylindrospermum sp., Dermocarpella sp., Fischerella sp., Gloeocapsa sp., Myxosarcina sp., Nostoc sp. ), Oscillatoria sp., Phormidium corium, Pleurocapsa sp., Prochlorococcus sp., Pseudanabaena sp., Cenacococcus sp. There the like (s ynechococcus sp.), cine Cauchy seutiseu (Synechocystis sp.), tall report Riggs (Tolypothrix sp.) or Xeno Cocos (Xenococcus sp.), using an oil extraction process of the present invention from these five days Can be extracted.

Examples of bacteria in the oil-containing microorganisms of the present invention include, but are not limited to, the bacteria of Table 2, such as E. coli , Rhodocista centenaria , Rhodospira trueperi ), Rhodospirillum fulvum , Rhodospirillum molischianum , Rhodospirillum < RTI ID = 0.0 > photometricum , Rhodospirillum rubrum , Rhodospirillum salexigens , Rhodospirillum < RTI ID = 0.0 > salinarutn , Rhodospirillum sodomense , Rhodospirillum mediosalinum , Rhodopseudomonas spp. sp.), Pseudomonas rodop also know Fira (Rhodopseudomonas acidophila), Pseudomonas rodop Cobb Peninsula tooth (Rhodopseudomonas capsulatus), Pseudomonas rodop Pearl forest leases (Rhodopseudomonas palustris , Rhodopseudomonas spp. sphaeroides , Rhodobacter capsulatus or Rhodobacter sphaeroides . From these, the oil extraction method of the present invention can be used to extract the oil.

The water miscible organic solvent used in the oil extraction method of the present invention includes, but is not limited to, acetone, ethanol, acetic acid, N, N-dimethylformamide or N, N-dimethylacetamide, In the case of using a microorganism in a state in which moisture is not present, if the amount of oil contained in the microorganism is small, the oil, the water miscible organic solvent and the water may be separately mixed without being separated. Microalga Hematococcus Fluvialis contains astaxanthin, a useful substance, and the organic solvent that can be used for the extraction of astaxanthin as a health functional food is limited to acetone, which is a hydrophilic solvent.

Therefore, when extracting the oil containing astaxanthin from Hematococz < RTI ID = 0.0 > pluvialis < / RTI > in a dry state with water, acetone, water and oil are mixed without layer separation, The use of the oil extraction method of the present invention does not require the evaporation step of organic solvent and water.

According to an embodiment of the present invention, the concentration of the oil-containing microorganism in the aqueous culture medium for culturing the oil-containing microorganism is preferably 50 to 400 g / L. When the concentration of microorganisms is less than 50 g / L, the water content is very high compared to the amount of microorganisms. Therefore, the efficiency of the process is low due to the oil extraction rate relative to the volume. When the microorganism concentration exceeds 400 g / L, There is a disadvantage that the dehydration cost is rapidly increased.

In order to disrupt microorganisms in the mixture of the organic solvent and the water culture liquid, the present invention uses a physical disruption method. Examples of the physical disruption method include, but are not limited to, ultrasonic wave, high speed dispersion, high pressure dispersion, ball mill or french press Can be used.

Although chemical methods using chemicals for cell disruption can be considered, extraction of astaxanthin from hematococzial pluvialis is limited because of the limitations of the chemicals that can be used when the extract is used as a food Physical cell disruption without the use of chemicals is preferred.

The ultrasonic disruption method is a method in which a shock wave generated when a cavitation bubble is generated in a cell by an ultrasonic wave is broken and then a shock wave generated in the cell is broken, and a fast dispersion disintegration method is performed in a narrow space between a rotor rotating at high speed and a stator The cell is broken by shearing force. In addition, the high pressure dispersion crushing method is a method in which cells are crushed by forces such as cavitation, shearing, impact and rupture which are received while cells pass through small spaces of the valve by high pressure, and are used in the oil extraction method of the present invention The high-pressure dispersion crushing method is preferable as the microbial crushing method.

The method of recovering the remaining organic solvent layer (also referred to as a first mixed layer, which is an organic solvent layer in which water, a water-miscible organic solvent and an oil are mixed) after the microorganisms are disrupted, is not limited to this However, it can be recovered by a method such as filtration or centrifugation. Filtration through a filter may be used for the solid-liquid separation step of separating the crushed microbial particles from the first mixed layer, and separation of the crushed microbial particles and the first mixed layer may be performed by centrifugation.

The recovered first mixed layer contains a target oil (referred to as a first oil) to be extracted, for example, astaxanthin, and the like. In the first mixed layer containing the objective oil, the objective oil Or an oil containing a target oil (referred to as a second oil) is further added, so that the first oil and the second oil coagulate and precipitate downward, and the objective oil can be easily separated by a method such as centrifugation.

For example, in the case where the oil-containing microorganism is Hematococcus pluvialis, the cell is physically disrupted by high pressure dispersion crushing or the like using acetone as an extraction solvent in a water culture medium of Hematococzus pluvialis After that, a first mixed layer in which water, acetone, and astaxanthin, which is a target first oil are mixed together, is obtained. At this time, astaxanthin (second oil), which is the same as the objective oil, is added to the first mixed layer , The second mixed layer in which water and acetone are mixed is located in the upper layer and the third mixed layer containing astaxanthin oil is precipitated in the lower layer and separated. When the precipitated third mixed layer is recovered by centrifugal separation or the like, an astaxanthin-containing oil can be obtained without any additional process.

The second oil used in the present invention, i.e., the oil (second oil) to be additionally added to separate the objective oil, is not limited thereto, but is most preferably an oil extracted from the same microorganism. In addition, an oil obtained by mixing the oil extracted from the same microorganism with soybean oil may be used. As the process is repeated, the amount of extracted oil is constantly increased, and the concentration of the useful substance contained in the oil is kept constant, and the amount of the useful substance is continuously increased. The oil extracted from Hematococcus pluvialis contains about 3 to 5% by weight of astaxanthin, which is a useful substance, in terms of the weight of the oil. When the oil derived from the same Hematococcus pluvialis is added to recover the oil, The concentration of taenzin is maintained.

The oil extraction method of the present invention is advantageous in that the microorganisms in the state of moisture can be extracted directly without a drying process and the evaporation of the solvent is not required and the energy cost can be reduced, It may be more advantageous for extraction of weak oil. In particular, the process temperature proposed to prevent decomposition of astaxanthin is 50 DEG C or less, and if it is necessary to evaporate water at 50 DEG C, a large energy cost is required. If the oil extraction method of the present invention is used, No process is required.

As another aspect of the present invention, there is provided a process for producing a cosmetic composition containing astaxanthin.

Specifically, the method for preparing a cosmetic composition containing astaxanthin comprises the steps of: preparing a mixture comprising a water-miscible organic solvent in an aqueous culture medium containing astaxanthin-containing microorganisms; Obtaining a physically disrupted lump of said mixture; Separating the pulverized microorganisms from the lysate, recovering a first mixed layer containing astaxanthin, a water miscible organic solvent and water; Adding astaxanthin or an oil compatible therewith to the first mixed layer, wherein a water-miscible organic solvent and a water-containing second mixed layer and a third mixed layer containing astaxanthin and further astaxanthin or a miscible oil thereof Adding astaxanthin or an oil compatible therewith in a layer separable amount to the mixed layer; And recovering a third mixed layer containing astaxanthin, wherein the water miscible organic solvent is selected from the group consisting of acetone, ethanol, acetic acid , N, N-dimethylformamide, N, N-dimethylacetamide, and the like.

As another embodiment of the present invention, there is provided a process for producing a food composition containing astaxanthin.

 Specifically, the method for producing a cosmetic composition containing astaxanthin comprises the steps of: preparing a mixture of an aqueous culture solution containing astaxanthin-containing microorganisms and acetone; Obtaining a physically disrupted lump of said mixture; Separating the pulverized microorganisms from the lysate, recovering a first mixed layer containing astaxanthin, acetone and water; Adding a second mixed layer containing acetone and water and a third mixed layer containing astaxanthin and further astaxanthin or an oil capable of mixing therewith to the first mixed layer, Adding astaxanthin or an oil compatible therewith in a detachable amount; And recovering a third mixed layer containing astaxanthin. The present invention also provides a method for producing astaxanthin-containing food composition.

The method of extracting oil from an oil-containing microorganism of the present invention does not require a step of drying microorganisms and a step of evaporating an extraction solvent, so that it is possible to extract oils that are weak in heat at low cost and can be widely used in medicine, cosmetics and food industry.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the movement of an oil layer when an oil is additionally added externally to a water-acetone-oil mixture. FIG.
Fig. 2 is a microscopic photograph (cell concentration: 10 g / L) according to the passage number of the wet hematococzial pluvialis passing through the high-pressure dispersing device.
FIG. 3 is a graph showing the distribution of intact cells according to the number of passages and cell concentration of the high-pressure dispersing device of wet Hematococcus pluvialis.
FIG. 4 is a graph showing changes in the oil extraction rate depending on the number of passes and cell concentration of the high-pressure dispersing device of wet Hematococcus pluvialis.
FIG. 5 is a graph showing changes in the total astaxanthin content according to the passage count and cell concentration of the high-pressure dispersing apparatus of wet Hematococcus pluvialis.
6 is a graph showing the distribution of oil according to the addition amount of acetone when the oil content of the microalgae is set to 50%.
7 is a graph showing a change in the distribution of oil according to the addition amount of acetone when the oil content of the microalgae is set to 25%.
8 is a graph showing changes in oil recovery rate due to the addition of oil after evaporation of acetone.
9 is a graph showing changes in the oil recovery rate due to the addition of oil according to the amount of wet micro-algae treatment.
10 is a graph showing changes in the oil recovery rate due to the addition of oil according to microalgae species.

Hereinafter, the present invention will be described more specifically in the following examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.

Example  1: Oil extraction from wet microalgae by physical disruption using acetone

Hematococcus pluvialis cultivated in the company JENJIN INTECH was partially centrifuged to remove water and a 200 g / L wet microalgae solution was prepared. Acetone and a wet microalgae solution were mixed to prepare a wet microalgae solution containing 10, 20 and 40 g / L acetone. The cells were disrupted by passing through a high pressure disperser 1, 2, or 3 times, and the disrupted cells were removed by filtration, and the oil-containing acetone containing a part of water was recovered. Water and acetone were removed through evaporation and the oil extraction rate and astaxanthin content were measured.

It was observed that spherical cells pass through a high-pressure dispersing device and cell breakage occurs. As the number of passages increases, more lysed cells can be observed (see FIG. 2). As shown in FIG. 3, the number of intact cells retaining spherical shape was significantly decreased after one pass of the high-pressure dispersing device, and the number of intact cells was steadily decreased after passing 2 or 3 times. When the initial cell concentration was high, the number of cells maintaining the spherical shape was relatively high at one time of passage, but at the third passage, it was low regardless of the concentration, indicating that most of the cells were disrupted.

In addition, the oil extraction rate of wet Hematococcus pluvialis showed a high extraction rate of more than 40% even after one pass through the high-pressure dispersing device. As the number of passages increased, the oil extraction rate also increased. %. There is no significant difference in the performance of the high-pressure dispersing device that performs physical fracturing even when the cell concentration increases (see FIG. 4).

Fig. 5 shows the total amount of astaxanthin, which is a useful substance contained in oil. As the number of passages increased, the extraction rate of astaxanthin increased as the oil extraction rate increased. It was confirmed that oil and astaxanthin were extracted very efficiently by physical fracturing.

Example 2: Comparison of power consumption of acetone and water evaporation

Hematococcus Soluble in astaxanthin as a functional food in Pluvialis is limited to acetone. When oil is extracted with acetone under wet condition, oil, acetone, and water are mixed. To remove the oil, an evaporation process is required. The process temperature to prevent decomposition of astaxanthin is less than 50 ℃. Acetone volatilization is relatively easy at 50 ℃, but water requires a lot of energy cost to evaporate at 50 ℃.

Table 3 shows measured values of energy required for evaporation of water and acetone under reduced pressure at 50 占 폚.

As shown in Table 3, the amount of power required for evaporation of 1 L of water was 5.5 times higher than that of acetone of the same amount, and the treatment time of water was 4.8 times longer than that of acetone. Thus, evaporation of acetone is relatively easy, but evaporation of water consumes a lot of energy, and when water is mixed with acetone and microalgae oil, evaporation of water in the mixture requires more time and energy. Therefore, a method of recovering oil by adding oil as in the present invention is considered to be a preferable method of reducing energy cost.

Example  3: Oil recovery rate (microalgae oil content 50%) of wet microalgae according to the amount of acetone added

An oil recovery experiment using oil was carried out by mixing the oil extracted from Hematococcus pluvialis with soybean oil. Under the conditions of 200 g / L wet microalgae, acetone was added at 10, 20, 30, 40, and 50 times the dry weight of microalgae as shown in Table 4 below, passed through a high pressure dispersing device, and the shredded cells were removed. The experiment was carried out assuming acetone-oil solution. The microalgae oil content was set at 50% (containing 1 g of oil in 2 g of microalgae).

Depending on the amount and proportion of acetone and water, the oil is mostly recovered to the oil layer, and some oil is distributed to the upper layer containing acetone-water (see FIG. 6). As the amount of acetone increased, the recovery of oil in the oil layer decreased and the amount of oil dissolved in the solvent layer increased. When the amount of acetone added was 10 times that of the microalgae, 98% of the oil was recovered into the oil layer, and the amount of oil remaining in the upper layer was almost zero. When the amount of added acetone was increased, the distribution of the oil to the organic solvent layer was accelerated, and when the amount of added acetone was 50 times that of the microalgae, the oil recovery was reduced to 55%.

Example  4: Oil recovery rate of micro-algae according to the amount of acetone added (microalgae oil content 25%)

An oil recovery experiment using oil was carried out by mixing the oil extracted from Hematococcus pluvialis with soybean oil. Under the condition of 200 g / L wet microalgae, acetone was added at 10, 20, 30, 40, and 50 times the dry weight of microalgae as shown in Table 5 below, passed through a high pressure dispersing device, and the shredded cells were removed. The experiment was carried out assuming acetone-oil solution. The microalgae oil content was set at 25% (containing 1 g of oil in 4 g of microalgae).

Depending on the amounts and ratios of acetone and water, the oil is mostly recovered into the oil layer, and some oils are distributed in the upper layer containing acetone-water (cf. FIG. 7). As the amount of acetone increased, the recovery of oil in the oil layer decreased and the amount of oil dissolved in the solvent layer increased. When the amount of acetone added was 10 times that of the microalgae, 96% of the oil was recovered into the oil layer, and the amount of oil remaining in the upper layer was 0.5%. When the amount of added acetone is increased, the distribution of the oil to the organic solvent layer is accelerated. When the amount of added acetone is 50 times that of the microalgae, the oil recovery is 3%, the oil layer is hardly separated and the oil is dissolved in the solvent layer .

Example 5: Oil recovery rate after oil evaporation of wet microalgae with oil

An oil recovery experiment using oil was carried out by mixing the oil extracted from Hematococcus pluvialis with soybean oil. Acetone was added 50 times as much as the dry weight of the microalgae under the conditions of 200 g / L wet microalgae, and the acetone-oil solution containing some of the recovered water after passing through the high pressure dispersing device, As a result, a comparative experiment with or without the acetone evaporation process was carried out. The microalgae oil contents were set at 25% and 50%, and 80% of the acetone was evaporated in each sample.

As the amount of acetone increases, the amount of oil to be added to the solvent layer increases and the amount of recovered oil decreases. Therefore, it is desirable to reduce the amount of acetone. However, when the amount of the solvent increases, the contact between the microalgae and the solvent increases, which is more advantageous in extracting the oil. Therefore, after extracting the oil with an appropriate amount of acetone, evaporating a part of the acetone and recovering the oil by adding oil from the remaining acetone and water Method. When the oil content of the microalgae was 50%, the oil recovery increased from 55% to 95% (see FIG. 8). When the oil content of the microalgae was 25%, the oil recovery rate significantly increased from 3% to 95%. Therefore, it is possible to recover more oil efficiently by adding oil after removing part of acetone by evaporation process.

Example  6: Oil recovery rate with increasing throughput of wet microalgae

An oil recovery experiment using oil was carried out by mixing the oil extracted from Hematococcus pluvialis with soybean oil. Acetone was added 10 times as much as the dry weight of microalgae under the condition of 200 g / L wet microalgae, and the acetone-oil solution containing some of the recovered water after passing through the high pressure dispersing device, Experiments were conducted under the same sample conditions. The microalgae oil content was set at 25% and 50%.

In experiments using small amounts of microalgae, it was difficult to recover microalgae and oil fractions on the container wall or pipette, resulting in loss of performance. However, it is confirmed that the loss problem is reduced when scale up. The amount of microalgae used was increased from 2 g to 20 g (oil content 50%) and from 4 g to 40 g (oil content 25%).

As shown in Fig. 9, when the oil content of microalgae was 50%, the oil loss decreased from 2.0% to 0.9%. When the oil content of the microalgae was 25%, the oil loss decreased from 3.5% to 1.5%. Less oil loss was observed in oils with higher oil content and it was expected that the oil loss would be minimized when the process was scaled up.

Example  7: Oil recovery rate depending on species of wet microalgae Hematokokusu Fluvialis  And CSS mix)

The oil recovery by oil addition was investigated for the microalgae CSS mix produced by Hematococcus pluvialis and Enlpi produced by Wefirst Biotechnology Co.. Under the condition of 200 g / L wet microalgae (oil content 25%), acetone was added 10 times as much as the dry weight of microalgae, passed through a high-pressure dispersing apparatus, and the shredded cells were removed. (See Table 8).

The physical recovery of the microalgae and the recovery of oil by oil addition resulted in different oil recovery rates depending on the species of the samples (see Fig. 10). Hematococcus pluvialis showed more than 82% oil recovery. CSS mix sample showed 86% of oil recovery, slightly higher than Hematococcus pluvialis. The difference in oil recovery rate depending on species of microalgae It was found that there was almost no.

Through the above-mentioned embodiments, the microorganisms in the state of moisture are disrupted by the physical cell disruption method, and then the oil contained in the microorganisms is extracted with an organic solvent such as acetone or the like, and then the oil is precipitated The target oil can be easily separated and recovered. The use of the oil extraction method as in the present invention does not require the step of drying the microorganisms and the step of evaporating the organic solvent, thereby greatly reducing the process cost.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described examples are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, as well as all changes or modifications derived from the meaning and scope of the appended claims and their equivalents.

Claims (14)

i) preparing a mixture in which a water-miscible organic solvent is added to an aqueous culture medium containing a first oil-containing microorganism;
ii) obtaining a physically disrupted lump of the mixture of step i);
iii) separating the pulverized microorganism from the disruption of step ii), recovering the first oil, water miscible organic solvent and water-containing first mixed layer;
iv) adding a second oil, which is the same as or different from the first oil, to the first mixed layer in step iii), wherein the water-miscible organic solvent and the second mixed layer containing water and the first mixed oil- Adding a second oil in a detachable amount; And
v) recovering the third mixed layer in step iv)
Containing microorganisms. ≪ Desc / Clms Page number 13 > The method according to claim 1,
Wherein the first oil-containing microorganism is a microalgae, a bacteria, a yeast or a fungus. 3. The method of claim 2,
The first oil containing microorganism may be Haematococcus pluvialis , Botryococcus brownie, braunii), cylinder throw teka (Cylindrotheca sp.), Chemnitz cyano (Nitzschia sp.), seukijo kit Solarium (Schizochytrium sp.), bakteo (Arthrobacter sp.), ahsine Sat bakteo knife core Shetty kusu (Acinetobacter calcoaceticus) in Aspergillus, Rhodococcus opacus , Bacillus alcalophilus Candida curvata , Cryptococcus albidus , Lipomyces starkeyi , Rhodotorula < RTI ID = 0.0 > glutinis , Aspergillus oryzae , Mortierella < RTI ID = 0.0 > isabellina , Humicola lanuginosa , and Mortierella vinacea. < Desc / Clms Page number 19 > The method according to claim 1,
The first oil contained in the microorganism is? -Carotene,? -Carotene,? -Cryptosanthin. gamma-carotene, lutein, astaxanthin, and zeaxanthin. The method according to claim 1,
Wherein the water miscible organic solvent in step i) is one or more selected from the group consisting of acetone, ethanol, acetic acid, N, N-dimethylformamide and N, N-dimethylacetamide. The method according to claim 1,
Wherein the concentration of the oil-containing microorganism in the water culture medium of step i) is 50 to 400 g / L. The method according to claim 1,
Wherein the mixture of step ii) is pulverized by at least one method selected from the group consisting of ultrasonic waves, high-speed dispersion, high-pressure dispersion, a ball mill and a French press. The method according to claim 1,
Wherein the first mixed layer in step iii) is recovered by filtration or centrifugation. The method according to claim 1,
And the second oil in step iv) is an oil extracted from the first oil-containing microorganism. The method according to claim 1,
And the third mixed layer in the step v) is recovered by gravity sedimentation or centrifugation. 11. The method according to any one of claims 1 to 10,
Wherein the first oil-containing microorganism is Haematococcus pluvialis , and the first oil is astaxanthin-containing oil. A method for producing cosmetic compositions containing astaxanthin,
i) preparing a mixture in which a water-miscible organic solvent is added to an aqueous culture medium containing an astaxanthin-containing microorganism;
ii) obtaining a physically disrupted lump of the mixture of step i);
iii) separating the pulverized microorganism from the disruption in the step ii), recovering astaxanthin, the water miscible organic solvent and the water-containing first mixed layer;
iv) adding astaxanthin or an oil compatible therewith to the first mixed layer in step iii), wherein the water-miscible organic solvent and the water-containing second mixed layer, and the astaxanthin and the additional astaxanthin, Adding astaxanthin or an oil compatible therewith in a layer separable amount to a third mixed layer containing an oil as possible; And
v) recovering a third mixed layer containing astaxanthin in step iv)
≪ / RTI > wherein the cosmetic comprises astaxanthin. 13. The method of claim 12,
Wherein the water miscible organic solvent in step i) is at least one selected from the group consisting of acetone, ethanol, acetic acid, N, N-dimethylformamide and N, N-dimethylacetamide. A method of preparing a food composition comprising astaxanthin,
i) preparing an aqueous culture solution containing an astaxanthin-containing microorganism and acetone-added mixture;
ii) obtaining a physically disrupted lump of the mixture of step i);
iii) separating the pulverized microorganism from the disruption of step ii), recovering the first mixed layer containing astaxanthin, acetone and water;
iv) adding astaxanthin or an oil compatible therewith to the first mixed layer in step iii), wherein a second mixed layer containing acetone and water, and astaxanthin and further astaxanthin or a miscible oil thereof Adding astaxanthin or an oil compatible therewith in a layer separable amount to the third mixed layer; And
v) recovering a third mixed layer containing astaxanthin in step iv)
≪ / RTI > wherein the composition comprises astaxanthin.

Images