KR20120048729A

KR20120048729A - Method and device for producing cell and fat solubles material by culturing cell

Info

Publication number: KR20120048729A
Application number: KR1020100108998A
Authority: KR
Inventors: 김성천
Original assignee: 김성천
Priority date: 2010-11-04
Filing date: 2010-11-04
Publication date: 2012-05-16
Also published as: KR101251191B1

Abstract

The present invention relates to a method and apparatus for producing cells and liposoluble materials through cell culture. The method of the present invention comprises the steps of culturing a cell containing a fat-soluble substance; Dissolving a lipid-soluble substance in a lipid-soluble substance-extracting solvent by contacting the lipid-soluble substance-extracting solvent with a cell culture liquid and bringing the lipid-soluble substance-extracting solvent into contact with the cell culture liquid; The process of aggregating, stagnating, and separating cells from the mixture; Separating the remaining solution from which the cells have been separated into a lipophilic substance in which a lipophilic substance is dissolved in a lipophilic substance extraction solvent - a solvent and water; And a step of obtaining a separated cell and a lipid-soluble substance-solvent, respectively; . An apparatus of the present invention comprises: a culture tank (10) for culturing cells containing a lipid-soluble substance; A solvent tank (20) for storing a lipid-soluble substance extraction solvent in which the lipophilic substance is dissolved; A mixing tank 30 for mixing the culture medium of the cells from the culture tank 10 and the liposoluble material extraction solvent from the solvent tank 20; A separating tank 40 having a cell separating device 40a for separating, stagnating and separating cells from the mixed liquid from the mixing tank 30; A fractionation tank (50) for separating the solution from the separation tank (40) from which the cells are separated into a liposoluble substance in which the liposoluble material of the cell culture liquid is dissolved in a liposoluble material extraction solvent and a water; A cell-receiving device 80 for receiving or treating cells separated from the separating tank 40; And a lipophilic substance-receiving device 90 for receiving or treating the lipophilic substance-solvent fractionated from the fractionation tank 50; .

Description

TECHNICAL FIELD The present invention relates to a method and apparatus for producing cells and liposoluble materials through cell culture,

The present invention relates to a method and apparatus for producing cells and liposoluble materials through cell culture, and more particularly, to a method and apparatus for producing low-cost, high-efficiency cells and lipid-free materials from cell culture fluids.

Various kinds of biomass obtained from cultured cells have been widely used as raw materials for health functional foods and medicinal products, and their availability has been expanded due to production of raw materials for feed, alternative energy, and production of biochemical materials.

The economics of mass production of biomass are ensured by the enlargement of cell culture. For example, in the case of conventional bach fermentation, the economics depend on the size of the stirred tank or the airlift fermenters.

Continuous perfusion is a method in which cells are harvested continuously in a fermentation machine containing a product but not in a cell, and the cells are continuously cultured for several weeks or months in a state where a new medium is continuously supplied to the fermenter As a technique that can be used, it is possible to cultivate cells at a high concentration, to convert a substrate into a product, and to use the fermenter in a short period of time, so that a desired purpose can be achieved by using a small fermenter.

Continuous perfusion culture consists of a process in which the cells are separated from the medium containing the product, while the cells are transferred to the fermenter and the remainder are harvested. At this time, the cell separation should be very sophisticated because the cells are sensitive to physical impact, operate in a sterilized state, and have no process problems. In addition, the design must be simple, robust, economically scalable, and sealed to enable hygienic and dangerous organisms to grow.

Ultrasonic resonance field, gravitational settling devices, spin filter device, filtration membrane and centrifugal separator are used for congestion and separation of cells cultured in a bioreactor or a fermenter in continuous perfusion culture.

Ultrasonic resonance or gravitational sedimentation cell separation device is a device that can perform the cell separation function almost permanently while consuming very little power with a simple device and its greatest advantage is that it does not damage the cell without the addition of other mechanical devices It can be separated while keeping it.

Application of ultrasonic resonance or gravitational settling to the separation of cells can overcome the problems encountered when using conventional filtration membranes. For example, the conventional filtration membrane method is troublesome to replace the membrane due to the clogging of the membrane when it is used for a long period of time. However, the device using the ultrasonic resonance field or the gravity sedimentation can not only reduce such inconvenience, .

In addition, centrifugation, which is one of the conventional cell recovery methods, makes it difficult to apply a fermenter to an on-line system, while ultrasonic resonance or gravitational sedimentation can be applied to an on-line system, and ultrasound resonance or gravity sedimentation Separation device enables on-line clarification and perfusion culture of the fermenter. Therefore, application of ultrasound resonance device or gravitational sedimentation device to the recovery of cells or extracellular products is expected to replace the existing processes using ultrafiltration and microfiltration.

The separation of cells and microorganisms using ultrasound resonance or gravity sedimentation is known to have little effect on microbial and cell viability due to the use of very little power and is used for high concentration cultivation of plant cells, insect cells and animal cells It has been used as a device to maintain cells without death in perfusion culture and is also used for continuous perfusion culture for the production of monoclonal antibodies.

Various cells contain not only lipid, protein and carbide, which are primary metabolites, but also various kinds of physiologically active substances which are secondary metabolites. The most abundant substance that can be obtained from a cell is a protein or protein, which is a component to be considered in terms of using the cell itself. When viewed from the viewpoint of using a substance produced by a cell, lipids, carbons, pigments, vitamins, minerals And special components. In addition, since substances involved in the regulation of intermediate metabolites and metabolism in the synthesis and degradation processes of carbohydrates, proteins, nucleic acids, and lipids are essential compounds of cells, these compounds can also be obtained from cells.

There are many compounds (mainly secondary metabolites) that are not essential for survival in plants, and more than 100,000 species have been known so far, and these compounds are often distributed in only a few or a few plants . Secondary metabolites can be divided into alkaloids, phenolic compounds, terpenes, and other compounds depending on the structure and synthesis process.

Conventional extraction methods for obtaining various useful substances from cell biomass are to remove the water as much as possible through centrifugation, filtration and drying processes that inhibit cell growth, and then to separate and purify useful substances through a cell crushing process .

However, such a conventional extraction method has problems in that the cell is destroyed in the dehydration process and the extraction process, and other useful materials flow out and disappear, making it difficult to re-use or re-use the cells. Have.

In order to solve the problems of the conventional method for extracting useful substances from cells (microalgae), the present inventors have proposed a method of extracting biofuels by microalgae cultivation in Korean Patent Application No. 10-2010-0043955 (filed on May 11, 2010) Method and Apparatus for Producing High Density Microalgae and Concentrated Lipophilic Material through Microalgae Cultivation "of Korean Patent Application No. 10-2010-0059100 (filed on June 22, 2010).

An object of the present invention is to provide a method for producing a lipid-soluble substance by mixing a cell culture fluid containing a lipid-soluble substance with a lipid-soluble substance extraction solvent to dissolve the lipid-soluble substance in the cell into a lipid- Then, the remaining cell-free solution is fractionated into water and a lipophilic substance-solvent (a solution in which the lipophilic substance is dissolved in a lipid-soluble substance-extracting solvent), followed by regenerating the separated cells, The process of recontacting with the solvent of the material extraction solvent or the separated liposoluble material-solvent is repeated as necessary until the final product of the liposoluble substance-solvent , And the cells of the former are used for the production of biocomponents, medicines, health functional foods, biofuels, and protein seaweeds Liposoluble substance extracted from the latter lipophilic substance-solvent can be used for the production of medicines, health functional food, biodiesel, etc., and can be used at low cost through cell culture, And to provide a method and an apparatus for production in a yield.

According to the present invention, there is provided a method and apparatus for producing cell and liposoluble material through cell culture.

A method for producing a cell and a lipid-soluble substance through cell culture according to the present invention comprises the steps of: culturing a cell containing a lipid-soluble substance; Dissolving the liposoluble material in the liposoluble material extraction solvent by mixing the liposoluble material extraction solvent with the culture solution of the cell and bringing the liposoluble material extraction solvent into contact with the cell culture liquid; A step of aggregating, stagnating and separating cells from the mixed solution; A step of fractionating the remaining solution from which the cells have been separated into the liposoluble material-solvent and water in which the liposoluble material is dissolved in the liposoluble material extraction solvent; And a step of obtaining the separated cells and the lipid-soluble substance-solvent, respectively; .

Preferably, the method for producing a cell and a lipid-soluble material through cell culture according to the present invention comprises the steps of separating the cells from the mixture by applying an ultrasonic resonance field to the mixture, or applying gravity sedimentation to the mixture, .

Preferably, the method for producing a cell and a lipid-soluble substance through cell culture according to the present invention is a method for producing a cell and a lipid-soluble substance by cultivating the cell, separating the cultured cell culture liquid from the liposoluble material- Soluble substance in the liposoluble material extraction solvent, and further repeating the process, wherein the re-dissolution of the cell, the re-dissolution of the liposoluble material, the re-separation of the cell, And the fat-soluble substance-solvent is subjected to a plurality of times of re-fractionation to obtain the desired high density of the cells and the fat-soluble substance-solvent containing the desired concentrated fat-soluble substance.

Preferably, the method for producing a cell and a lipid-soluble substance through cell culture according to the present invention comprises the steps of vibrating and pulverizing the cell culture broth and stirring the mixture when the cell culture broth and the liposoluble material extraction solvent are mixed, One process is performed to increase the contact between the cell culture medium and the liposoluble material extraction solvent.

An apparatus for producing a cell and a lipid-soluble substance through cell culture according to the present invention comprises: a culture tank for culturing cells containing a lipid-soluble substance; A solvent tank for storing a lipid-soluble substance-extracting solvent in which the lipid-soluble substance is dissolved; A mixing tank for mixing the culture liquid of the cells from the culture tank and the liposoluble material extraction solvent from the solvent tank; A separating tank provided with a cell separating device and capable of aggregating, stagnating and separating the cells from a mixed solution from the mixing tank; A fractionation tank for separating the solution from the separation tank into which the cells have been separated (removed) into the liposoluble material extraction solvent, the liposoluble material in which the liposoluble material is dissolved, and water; A cell-receiving device for receiving or treating the cells separated from the separation tank; And a lipophilic substance-receiving device for containing or treating the lipophilic substance-solvent fractionated from the fractionation tank; .

Preferably, in an apparatus for producing a cell and a lipid-soluble substance through cell culture according to the present invention, the cell separation apparatus comprises an ultrasonic resonance field generating device for separating the cells from the mixed solution by applying an ultrasonic resonance field to the mixed solution, At least one of gravity settling apparatus for separating the cells from the mixed liquid by applying gravitational settling to the mixed liquid.

Preferably, the apparatus for producing a cell and liposoluble material through cell culture according to the present invention comprises a cell-circulation line circulating the separated cells from the separation tank to the culture tank; And a solvent-circulation line circulating the fractioned liposoluble material from the fractionation tank-solvent into the solvent combination; . Wherein said cell-containing device is adapted to receive or process said high-density cells, which have been cultivated once or more than twice in said culture tank through said cell-circulatory line, and finally fractionated from said separation tank; The fat-soluble substance-receiving apparatus accommodates or processes the concentrated fat-soluble substance-solvent, which is once or more than once fractionated in the fractionation tank through the solvent-circulation line and then finally fractionated from the fractionation tank.

Preferably, the apparatus for producing cells and liposoluble material through cell culture according to the present invention comprises a first peristaltic pump for supplying a predetermined amount of the cell culture liquid of the culture tank and the liposoluble material extraction solvent of the solvent tank to the mixing tank; A second peristaltic pump for selectively transferring the cells separated in the separation tank to the culture tank through the cell-circulation line or transferring the cells to the cell-receiving device according to the density; And a third peristaltic pump for transferring the lipid-soluble substance-solvent fractionated in the fractionation tank to the solvent tank or transferring the solvent to the lipid-soluble substance-receiving device according to the degree of concentration thereof; .

Preferably, the apparatus for producing a cell and a lipid-soluble substance through cell culture according to the present invention is characterized in that the mixing tank is provided with a vibration crushing apparatus for vibrating and crushing the cell culture liquid so as to increase contact between the cell culture liquid and the liposoluble material- And an agitating device for agitating the mixed liquid.

In the method and apparatus for producing cells and liposoluble material through cell culture according to the present invention, preferably, the liposoluble material extraction solvent is a hydrocarbon solvent.

The 'lipophilic substance' in the present invention is a substance which dissolves in oil and is a substance which dissolves phospholipids, free fatty acids, esters of fatty acids, triacylglycerols, sterols and sterol esters, carotenoids, xanthophylls (for example, oxycarotenoids), hydrocarbons, Phenolic compounds, terpenes, isoprenoid-derived compounds and other substances which dissolve in oil.

In the present invention, the cell includes an animal cell, a plant cell, a fungus, a diatomaceous earth, a mussel, a mussel, a bird mussel, a mussel, a flour, a green river, and a prokaryote.

The method and apparatus for producing cells and liposoluble material through cell culture according to the present invention are characterized in that a culture solution of a cell containing liposoluble material is mixed with a liposoluble material extraction solvent so that the liposoluble material of the cell is dissolved in the liposoluble material extraction solvent, Is applied to a cell separator to separate cells, and the cells are removed, and the remaining solution is fractionated with a lipophilic substance-solvent and water to separate the lipophilic substance-solvent containing the lipophilic substance- A method and apparatus for obtaining high density and high concentration without the need for separation pretreatment. It is capable of obtaining cells and lipid-free substances without cell damage at a low production cost with low operation cost, thereby enabling the production of biocompounds, medicines, , Cells (biomass) that can be used for the production of biofuels and proteins, and drugs, medicines, The productivity of the liposoluble material which can be used for the production of health functional food, biodiesel and the like can be maximized.

The apparatus and method according to the present invention are characterized in that the cell isolated by the cell separator is regrown, cells are separated from the regenerated cells, and the subsequent process of extracting the lipophilic substance is repeatedly performed as necessary, A lipophilic substance-solvent containing substance can be obtained at high density and high concentration.

The method and apparatus according to the present invention are capable of producing a high density cell and a concentrated lipid material by repeatedly regrowing, re-separating and re-fractionating using peristaltic pumps, unlike the prior art culture, harvest and extraction methods, Can be produced at low cost.

The method and apparatus according to the present invention can prevent the loss of other useful components in cells by extracting the lipid-soluble substance without destroying the cells, thereby improving the availability of the lipid-soluble substance-extracted cells, Thereby maximizing the productivity of the cells.

The method and apparatus according to the present invention can further improve the production yield of lipophilic substances and cells through effective contact between the cell culture medium and the liposoluble material extraction solvent.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view of an exemplary apparatus to which a method of producing a cell and liposoluble material through cell culture according to the present invention is applied;
2 is a schematic diagram of an exemplary cell separation device (gravity settling device) applied to the present invention,
FIG. 3 is a graph showing the effect of the liposoluble material extraction solvent on cell survival,
FIG. 4 is a graph showing the effect of a solvent (alkane) for extracting a lipid-soluble substance and vibration pulverization on lipid-soluble substance extraction from cells,
FIG. 5 is a graph showing the effect of a solvent (alkane) for extracting a lipid-soluble substance and ultrasound resonance on the extraction of liposoluble material from cells,
FIG. 6 is a graph showing GC-TOF-MS analysis results of biodiesel extracted from cells according to the present invention.

Hereinafter, a method and apparatus for producing a cell and a lipid-soluble substance through cell culture according to the present invention will be described in detail. The following examples are illustrative of the present invention but are not intended to limit the scope of the present invention.

First, referring to FIG. 1, an apparatus 1 for producing cells and liposoluble materials through cell culture according to the present invention will be described.

1, the apparatus 1 according to the present invention basically comprises a culture tank 10, a solvent tank 20, a mixing tank 30, a separation tank 40, a fractionation tank 50, Containing device 80 and a lipophilic substance-receiving device 90 and preferably further comprises a cell-circulating line 60 and a solvent-circulating line 70. The cell-

The culture tank 10 is a device for culturing cells and supplying the cultured cell culture liquid to the next step. The culture tank 10 can be widely used as a system suitable for culturing cells. The incubation tank 10 may be equipped with a pond, an artificial incubator, bioreactors, plastic bags, tubes, fermentors, shake flasks, pneumatic lift columns lift columns), and there is no limit to the type of cells that can be cultured. The culture can be done by independent nutrient culture or heterotrophic culture alone, or by autotrophic culture after autotrophic culture or heterotrophic culture after autotrophic culture.

The solvent tank 20 is a device for storing and supplying a liposoluble material extraction solvent for extracting liposoluble substances contained in cells of a cell culture liquid.

The mixing tank 30 uniformly mixes the cell culture liquid from the culture tank 10 and the liposoluble material extraction solvent from the solvent tank 20 so that the liposoluble material contained in the cell of the cell culture liquid is in contact with the liposoluble substance And a lipophilic substance-solvent dissolved in the substance extraction solvent. Preferably, the mixing tank 30 mixes the cell culture medium and the liposoluble material extraction solvent at a ratio of about 5: 1.

Preferably, the vibrating mill 31 is mounted on the mixing chamber 30 to vibrate the cell culture liquid. The preferred vibration shredding apparatus 31 is to treat the culture liquid with ultrasonic waves. Vibrational pulverization pulverizes molecular aggregates to separate or permeate the molecular aggregates. Vibratory pulverization improves the extraction efficiency of lipophilic substances by making the cell culture liquid into small particles so that the cells are exposed (contacted) with more lipophilic substance extraction solvent.

A stirring device 32 for stirring the mixed solution of the cell culture solution and the liposoluble material extraction solvent may be provided in place of the vibration shredding device 31 or the vibration shredding device 31. The agitation device 32 may also be provided with a cell culture liquid The extraction efficiency of the lipophilic substance is improved by increasing the contact of the lipophilic substance extraction solvent.

Preferably, the cell culture liquid in the culture tank 10 and the liposoluble material extraction solvent in the solvent tank 20 are transferred to the mixing tank 30 at a predetermined flow rate through the first peristaltic pump 2.

The line 11 from the culture tank 10 and the line 21 from the solvent tank 20 are respectively connected to the first peristaltic pump 2 and the line 21 from the first peristaltic pump 2, (33) is connected to the mixing chamber (30).

The separating tank 40 is continuously subjected to a perfusion culture in which a supersonic resonance field or gravity sedimentation is applied to the mixed liquid transferred from the mixing tank 30 through the line 34 from the mixing tank 30, And the cells of the extracted cell culture liquid are agglutinated to each other to thereby cause cells to flocculate, stagnate and separate from the mixed solution. To this end, a cell separation device 40a is installed in the separation tank 40.

As the cell separation device 40a provided in the separation tank 40, an ultrasonic resonance generation device 41 for separating cells by an ultrasonic resonance field or a gravitic sedimentation device 46 for separating cells by gravitational sedimentation can be applied .

As the ultrasonic resonance generating device 41, for example, an acoustic cell filter 41 (Nature Biotechnology 12, 281 - 284 (1994) can be applied. Ultrasonic resonance field is applied to cause the cells to flocculate and separate. Such an ultrasonic resonance field generating device 41 is disclosed in, for example, U.S. Patent No. 5711888 (registered on Feb. 27, 1998), Mutilayered plezoelectric resonator for the separation suspended particles'.

The acoustic cell filter 41 illustrated in FIG. 1 includes an acoustic chamber 42, an ultrasonic oscillator 43, an ultrasonic transducer 44, and a reflector 45, Ultrasonic resonance is applied to the cells to allow the cells to aggregate in the ultrasound resonance to form aggregates.

The ultrasonic oscillator 44 generates the first traveling wave by the action of the ultrasonic oscillator 43 and the ultrasonic oscillator 44 generates the ultrasonic wave by the ultrasonic oscillator 44, The first traveling wave applied from the ultrasonic transducer 45 to the reflection film 45 is reflected in the reverse direction in the reflection film 45 to generate the second traveling wave and the first traveling wave generated by the ultrasonic oscillator 44 is reflected by the reflection film 45, The ultrasonic vibrator 44 generates a standing wave between the ultrasonic transducer 44 and the reflection film 45. In this case, Standing waves are formed by combining two independent traveling waves coming from opposite directions.

The standing waves of the ultrasonic waves are structured by a structure of an ultrasonic transducer (e.g., a piezoelectric transducer) and a reflection film 45 which are spaced apart from each other by a predetermined distance and two independent ultrasonic transducers .

The standing wave of ultrasound has node and antinode part. The pressure amplitude of the standing wave of this ultrasonic wave is the largest at the double part and has the minimum value at the node and appears twice at one wavelength. Due to the discontinuity of particles, cells, or droplets in the ultrasound resonance field, the ultrasonic resonance field generates position-dependent acoustic potential energy. By this phenomenon, the cell moves to the lowest acoustic potential energy and is entrapped in the standing wave of the ultrasonic wave. This causes the cells to be trapped at a pressure node that is present every half of the wavelength. The collected particles aggregate within the standing wave to form aggregates.

As another ultrasonic resonance generating device 41, there are a piezoelectric transducer connected to both ends of the upper glass substrate and converting electrical input from outside into mechanical vibration and applying it to the upper glass substrate, and a piezoelectric transducer, Wherein the upper glass substrate and the lower substrate are filled with a fluid mixture of cells and the electrodes are placed in a direction perpendicular to the longitudinal direction of the piezoelectric transducer And the N electrodes are arranged at regular intervals along the longitudinal direction of the piezoelectric transducer (not shown).

According to such a cell separating apparatus, the vibration applied to the upper glass substrate by the piezoelectric transducer generates surface waves traveling on the upper glass substrate, collides with other surface waves traveling in a direction opposite to the traveling direction of the surface waves, Generates a standing wave that moves particles in the vertical direction of the piezoelectric transducer perpendicular to the longitudinal direction of the piezoelectric transducer and in a direction perpendicular to the upper glass substrate. The surface wave generates acoustic waves traveling from the upper glass substrate to the lower substrate in the fluid, and the acoustic waves are reflected by the lower substrate and reflected by the upper glass substrate to generate an ultrasonic resonance field Respectively.

The gravitational settling device 46 agglutinates and separates cells using a difference in sedimentation velocity between cells and a medium, a slope of a tube through which the solution flows in the apparatus, and an electromagnetic vibrator. As the gravitational settling apparatus 46, for example, a cell settler (Biotechnology Solutions, USA) may be used.

The gravitational set-up device 46 shown in Fig. 2 has a plurality of inclined plates 46a having a plurality of inclined planes arranged at regular intervals in a predetermined interval. In the illustrated embodiment, the inclined plate 46a has upper and lower It consists of two stages. The swash plate 46a is supported by the upper, middle and lower mounting frames 46b at regular intervals. The upper swash plate 46a and the lower swash plate 46a are staggered from each other in order to improve the sedimentation efficiency of the cells by reducing the flow rate of the downflow.

The mixed liquid of the cell culture liquid and the liposoluble material extraction solvent from the mixing tank 30 flows into the gravity settling apparatus 46 through the inlet port 46c connected to the line 34 and is subjected to the settling process. A cell collection unit 46d for collecting and discharging the sedimented cells is provided at the rear end of the swash plate 46a and the cells collected in the cell collection unit 46d are provided at the cell outlet 46e As shown in FIG. The solution from which the precipitated cells have been removed flows through the solution outflow port 46g beyond the upper outflow way 46f to the next treatment stage.

Preferably, the gravitational set-up device 46 is provided with a vibration generating portion 46h so as to flow the swash plate 46a to the right, thereby effectively separating the agglomerated and stagnant cells inside the swash plate.

1, the mixing tank 30 and the separating tank 40 are constructed separately from each other. However, as shown by a dotted line, the mixing tank 30 and the separating tank 40 ) May be integrated into one reaction tank 100 in which their functions are integrated.

The fractionation tank 50 separates the solution from which the cells transferred from the separation tank 40 have been removed through the line 51 from the separation tank 40 into a lipophilic substance-solvent (layer) A solution dissolved in a lipid-soluble substance-extracting solvent) and water. That is, in the fractionation tank 50, the solution from the separation tank 40 is fractionated into the upper layer of the lipophilic substance-solvent (layer) and the lower layer of water.

As will be described later, the cells separated in the separation tank 40 are transferred to the cell-receiving apparatus 80 and used for the subsequent intended use, and the upper lipophilic substance-solvent (fractionated in the fractionation tank 50) Layer) is transferred to the lipophilic material-receiving device 90 and is then used for the intended use.

The cell-circulation line 60, which can be selectively added, circulates the separated cells in the lower layer of the separation tank 40 to the culture tank 10 selectively. That is, in a preferred embodiment, the cells of the separation chamber 40 are delivered to the cell-receiving device 80 or re-supplied to the culture tank 10 via the cell-circulation line 60.

The solvent-circulating line 70 that can be selectively added is a line for circulating the lipid-soluble substance-solvent separated in the upper layer in the fractionation tank 50 to the solvent tank 20 again. That is, in a preferred embodiment, the lipophilic substance-solvent of the fractionation tank 50 is transferred to the lipid-soluble substance-receiving device 90 or is returned to the solvent tank 20 through the solvent-circulation line 70.

The circulated cells are regenerated in the culture tank 10 and the regenerated cells and the liposoluble material extraction solvent or the circulated liposoluble material-solvent of the solvent tank 20 are supplied to and mixed with the mixing tank 30, Soluble substance of the liposoluble material is further advanced, the mixture is further supplied to the separation tank 40 and the fractionation tank 50, so that the cells are separated and the liposoluble material-solvent fraction is repeatedly performed as described above.

Preferably, the re-fractionation / re-separation of such cells and the re-fractionation of the liposoluble material-solvent are performed once until the cells are densified to the desired degree of density and the liposoluble material dissolved in the liposoluble solvent is concentrated to the desired degree Or it can be repeated many times more than once.

The cell-receiving device 80 receives the cells once separated in the separation tank 40 or circulates the cell-circulation line 60 and circulates the cells one or more times to be densified to a desired density The high density cells are transferred from the separation tank 40 and temporarily accommodated for the purpose of the subsequent treatment or subjected to a desired treatment.

For example, in the cell-receiving device 80, high-density cells are subjected to various treatments such as ethanol fermentation, butanol fermentation, organic acid fermentation, etc., and various useful substances such as biocompounds, medicines, health functional foods, biofuels, And temporarily accepts them before sending them to a processing device for producing such useful materials.

For example, in the case of performing various fermentations such as ethanol fermentation in the cell-receiving device 80, the cell-receiving device 80 becomes a fermenter, and the cell-receiving device 80 is temporarily The cell-receiving device 80 becomes a reservoir.

The lipophilic material-receiving device 90 may be configured to receive the lipophilic substance-solvent fractionated in the fractionation tank 50 directly or to perform the fractionation at least once in the fractionation tank 50 while circulating the solvent- Solubilized substance-solvent when the liposoluble material of the solvent is concentrated to the desired degree of concentration, the liposoluble substance-solvent is transferred from the fractionation chamber 50 to temporarily accept or perform the desired treatment for the subsequent desired treatment.

For example, the lipophilic substance-receiving device 90 may be configured to extract a lipophilic substance from a lipophilic substance-solvent and to produce various useful substances such as medicines, health functional foods, biodiesel, etc. from lipophilic substances by various treatments, It is temporarily accommodated before sending it to the processing unit for production.

For example, in the case of extracting a lipophilic substance from a lipophilic substance-solvent in a lipophilic substance-receiving device 90 and producing biodiesel from the extracted lipophilic substance, the lipophilic substance-receiving device 90 may be a distillation- And the lipid-soluble substance-receiving device 90 becomes a reservoir if the lipid-soluble substance-solvent is temporarily stored in a separate distillation-biodiesel production tank before sending it to the distillation-biodiesel production tank.

Preferably, the device 1 of the present invention is adapted to transfer the separated cells in the separating tank 40 to the culture tank 10 selectively via the cell-circulation line 60 according to their density, And a second peristaltic pump (3) for transferring the second peristaltic pump (80). A second peristaltic pump 3 is provided in the cell-circulation line 60 between the separation tank 40 and the culture tank 10 and an additional line 81 from the second peristaltic pump 3 Is connected to the cell-receiving device (80).

In a preferred embodiment, when the density of the cells of the separation tank 40 does not reach the desired density by the action of the second peristaltic pump 3, the cells are repeatedly circulated to the culture tank 10 and repeatedly The amount of cultivation, the dissolution of liposoluble material in the cells, and the separation of cells are carried out, and when they reach the desired density, they are transferred to the cell-receiving device 80.

Preferably, the apparatus 1 of the present invention further comprises a third interlocking device 70 for transferring the lipophilic substance-solvent fractionated in the fractionation tank 50 to the solvent tank 20 or transferring the solvent to the lipophilic substance- And a pump 4. To this end, a third peristaltic pump 4 is installed in the solvent-circulation line 70 between the fractionation tank 50 and the solvent tank 20 and an additional line 91 is provided from the third peristaltic pump 4 And is connected to a lipophilic material-receiving device (90).

In a preferred embodiment, when the concentration of the lipophilic substance in the lipid-solvent of the fractionation tank 50 does not reach the desired concentration by the action of the third peristaltic pump 4, the lipophilic substance- And is used for re-fractionation at least once, and is conveyed to the lipid-soluble substance-receiving device 90 when the desired concentration is reached.

Hereinafter, a method for producing a cell and liposoluble material through cell culture according to the present invention will be described with reference to FIGS. 1 to 6. FIG.

1. Cell (high density) culture

Cells to be cultured in the culture tank 10 include plant cells, molds, diatoms, bivalves, algae, mulberry, red flour, green algae, prokaryotes and the like.

For example, Chlorella protothecoides may be used in the present invention. C. protothecoides can be cultured at a cell density 10 times higher than most microalgae, which is very suitable for securing biomass. C. protothecoides can harvest biomass up to 35 gfw / L in ideal conditions under heterotrophic conditions, and approximately 55% of the biomass can be stored as a lipid-soluble material.

Assuming a relatively constant rate of production of liposoluble material by the cell, it is natural that higher biomass density will increase the total amount of useful material produced per volume. Current conventional fermentation methods for growing cells produce biomass densities of about 50 to about 80 g / L or less.

The present inventors have found that by applying the method of the present invention, significantly higher biomass densities than currently known biomass concentrations can be achieved.

Preferably, the method of the present invention comprises at least about 100 g / L, more preferably at least about 130 g / L, more preferably at least about 150 g / L, even more preferably at least about 170 g / Achieving a biomass density of cells greater than 200 g / L.

Thus, at such high cell biomass densities, the rate of total useful material production per volume is significantly higher than currently known processes, although the rate of production of useful materials in cells is slightly reduced.

In a preferred embodiment, the method of the present invention is characterized in that after culturing the cells, the cells are first cultivated and the regenerated cells are re-cultured after mixing the cell culture medium with the liposoluble material extraction solvent, The process of mixing, coagulating and re-separating the liposoluble substance-extracting solvent or the lipophilic substance-solvent (the liposoluble substance of the cell culture liquid dissolved in the liposoluble substance-extracting solvent) is repeatedly carried out as necessary a plurality of times, To ensure high productivity by culturing the cells at a high density to the desired density.

C. protothecoides can grow sub-nutritionally to glucose or corn sweetener hydrolyzate (CSH). Heterotrophic growth can increase the fat-soluble material content and reduce the direct dependence on solar energy. The energy density of biodiesel produced from C. protothecoides is substantially equivalent to that of petroleum-based diesel. Chlorella is easy to engineer in molecular biology and is able to grow in large scale photobiorectors with enhanced CO ₂ .

2. Extraction of lipid-soluble substances from cells

The major cost associated with the production of biomass and useful materials using cells arises in the process of harvesting cells from large volume cultures. Harvesting the cells from the cell culture medium, and drying and then destroying the cells to extract lipophilic substances. In the conventional method for producing lipophilic materials, the cost of such a process accounts for 40 to 60% of the total cost.

In the conventional method, it is difficult to harvest the biomolecules other than the lipid-soluble material by destroying the cells in the process of extracting the lipophilic material. However, the present invention mitigates and overcomes this conventional problem by the low-cost non-destructive recycling culture.

The lipid-soluble substance-extracting solvent used in the present invention is a solvent which is highly selective for lipophilic substances and is biologically compatible and can contact cells without significant loss in cell activity. Generally, the number of octanol (log Poct, Logarithm of the partition coefficient) is 5 or more (Dodecanone is an exception to this rule). Hexane and heptane are toxic to cells and decanol and dipentyl ether are harmless to cells in solvents with 4-5 octanol.

Exemplary lipophilic substance extraction solvents that can be used in the present invention include 1,12-dodecanedioic acid diethyl ether, n-hexane, n-heptane, n-heptane, n-octane, n-dodecane, dodecyl acetate, decane, dihexyl ether, isopar ), 1-dodecanol, 1-octanol, butyoxyethoxyehteane, 3-octanone, cyclic paraffins, varsol, isoparaffin isoparaffins, branched alkanes, oleyl alcohols, dihecylether, 2-dodecane, and the like.

The liposoluble material extraction solvent used in the present invention may include one or more C4-C16 hydrocarbons and may include C10, C11, C12, C13, C14, C15 or C16 hydrocarbons.

Ultrasonic irradiation of the microorganism without cell damage by the vibration shredding apparatus 31 is dose-dependent at low frequencies. As the frequency increases, microorganisms survive even at long exposure times. Studies have been carried out to determine the strengths over a frequency range and other exposure times that are appropriately subdivided in frequency norms (20 kHz to 1 MHz) that have no effect on cell activity and can optimize the extraction of lipid substances, And exposure time influenced the extraction efficiency.

Can be used to optimize the extraction of fat-soluble materials without damaging the cells with an ideal intensity over the other frequency range (20 kHz to 60 kHz) at different exposure times. That is, frequency, intensity, and exposure time affect lipophilic substance extraction efficiency. Because the cell size, cell shape, cell wall composition, and physiological condition have a complex effect on the interaction of cells and ultrasonic waves, 20 kHz and 1 MHz, 20-100 kHz, 20-60 kHz, 30 -50 kHz, or 40 kHz, among other various frequencies. With the proper combination of lipophilic substance extraction solvent and vibratory milling, extraction efficiency of lipophilic substances (10% of total cellular fatty acids) can be achieved up to almost 100%.

Soluble material extracting solution is stirred by the agitator 32 instead of vibrating milling, the contact between the cell culture liquid and the lipid-soluble substance-extracting solvent is increased (improved), thereby improving the extraction efficiency of the lipid-soluble substance . It is, of course, also possible to carry out vibration pulverization and stirring together.

3. Coagulation, stagnation and separation of cells without cell damage

The separating tank 40 is provided with a cell separation device 40a for continuous perfusion culture, which can precisely and efficiently perform cell migration, flocculation, stagnation and separation without damaging the cells. As the cell separation device 40a, an ultrasonic resonance field generating device 41 to which the ultrasonic resonance field is applied or a gravity sedimentation device 46 to which gravity sedimentation is applied can be applied.

For example, when the ultrasonic resonance generating device 41 is applied to the cell separating device 40a, an appropriate combination of the ultrasonic frequency of the liposoluble material extraction solvent and the ultrasonic resonance resonator, the distance between the ultrasonic vibrator 44 and the reflective film 45, , The extraction efficiency of fat-soluble substances (10% of total cellular fatty acids) can be achieved up to almost 100%.

Hereinafter, a method for producing a cell and a lipid-soluble substance through cell culture according to the present invention will be described with reference to specific examples.

Example 1. Culture of cells

Chlorella protothecoides were used in the present invention while being maintained on a proteose agar slant.

KH ₂ PO ₄ (0.7 g), K ₂ HPO ₄ (0.3 g), MgSO ₄ .7H ₂ O (0.3 g) and FeSO ₄ .7H ₂ O (3 mg) were added to the basic medium. Urea (1 g), Arnon's A solution (1 ml), thiamine hvdrochloride (10 ㎍), pH 6.3. The cultures were carried out in 5% CO ₂ , 20 °, 15,000 lux fluorescent lamps.

The composition of the Arnon's A5 solution was H ₂ BOS ₃ (2.9 g), MnCl ₂ .4H ₂ O (1.8 g), ZnSO ₄ .7H ₂ O (0.22 g), CuSO ₄ .5H ₂ O ₃ (0.018 g).

The heterotrophic culture of Chlorella protothecoides was carried out by adding 0.01% urea and 4.0% glucose instead of 0.1% urea in the basal medium.

Example 2. Effect of solvent for extracting lipophilic substances on cells

C. protothecoides culture medium was treated with a 5: 1 ratio of C10 to C16 alkanes in a ratio of 5: 1, 1 ml of fractionated C. protothecoides solution was diluted 1 / 100,000 times and coated on a 1.5% agar plate The results are shown in FIG. 3. It can be seen that the solvent for extracting the lipid-soluble substance had no effect on the survival of the cells .

Example 3 Effect of Solvent and Ultrasonic Vibrating Powder for Extracting Lipid Soluble Substance from Cells

The growth rate of C. protothecoides was treated with the culture medium in the log section, hexane and decane solvent (liposoluble material extraction solvent) for 5 minutes at a ratio of 5: 1, and then shaken at 40 kHz for 2 seconds in a water bath .

The fat-soluble substance extracted with the liposoluble material extraction solvent was saponified, and the free fatty acid was measured by LC-MS analysis using C17 as a standard. The results are shown in FIG. 4, in which 10% total cellular fatty acid was extracted by mixing the liposoluble material extraction solvent for 5 minutes and the vibration pulverization for an additional 2 seconds. Short vibration milling increased the degree of lipophilic substance extraction by 75%.

Example 4. Effect of solvent and ultrasonic resonance on extracting lipid-soluble substances from cells

The growth rate of C. protothecoides was determined by treating the culture medium in the log section with hexane or decane solvent (liposoluble material extraction solvent) for 5 minutes at a ratio of 5: 1 and then separating the acoustic cell filter 41 The cells were transferred by transfer to the bath 40, and transferred to the fractionation tank 50 continuously to fractionate the lipophilic substance-solvent.

The acoustic cell filter 41 is composed of an acoustic chamber 42, a 3 MHz ultrasonic oscillator 43, an ultrasonic vibrator 44 and a reflection film 45 as shown in Fig. The acoustic chamber 42 was manufactured using an acrylic tube, and the reflective film 45 was made of glass. It was confirmed that cell aggregation by ultrasonic resonance occurs in the acoustic chamber 42.

The fat-soluble substance extracted with the liposoluble material extraction solvent was saponified, and the free fatty acid was measured by LC-MS analysis using C17 as a standard. The results are shown in Fig. 5, in which 10% of the total cellular fatty acids were extracted by 5 minutes of the lipid-soluble substance extraction solvent mixture and the treatment with the acoustic cell filter. The acoustic cell filter 41 increased the degree of lipophilic substance extraction by about 75%.

Example 5. Cell stagnation, separation and lipid-soluble material-fraction of solvent

C. protothecoides were incubated in a 5L incubator (10) at a stirring speed of 150 rpm under a light intensity of 15,000 lux or under a dark reaction until the log interval.

The cell culture medium of the culture tank 10 and the decane solvent (lipophilic substance extraction solvent) of the solvent tank 20 were transferred to the mixing tank 30 at a ratio of 5: 1 and mixed.

The resultant mixed solution was transferred to a separation tank 40 operated by CS 10 Cell settler (Biotechnology Solutions, USA) as an acoustic cell filter as the ultrasonic resonance generating device 41 or a gravity settling device 46, And precipitated down while forming cell aggregates. The remaining solution except the cells stagnated and stagnated and separated was continuously transferred to the fractionation tank 50 to be fractionated, whereby the lipophilic substance-solvent (layer) and water (layer) were fractionated into upper and lower parts.

The cells of the separation tank 40 were then transferred to the culture tank 10 through the cell-circulation line 60 and cultivated by adding a culture medium. The lipid-soluble substance-solvent (layer) Was transferred to the solvent tank (20) through the circulation line (70).

Example 6 (High Density) Cultivation of Cells

After the liposoluble material obtained from Example 5 was extracted and subjected to regeneration, the cell culture medium, which had been inhibited by the liposoluble material extraction solvent by the liposoluble material extraction, was transferred to the culture tank 10 As a result, the growth rate was 0.028 g / h.

In comparison with the growth rate of about 0.033 g / h in the general fermenter culture in the culture tank, it was found that there is no problem in cultivating the strain after the simultaneous extraction.

The cells are cultured at a density of 50 to 200 gfw / L by repeating the above culturing, mixing, separation and fractionation processes once a day or twice to 20 times per day to obtain a liposoluble substance- Respectively. As a result of repeating the separation, fractionation and culturing process, it was confirmed that the cell density and lipid - soluble material increased by 2 to 3 times at every step.

Example 7. Fractionated lipid soluble material-extraction of fatty acids in solvent

Fatty acids were extracted from a Buchi 210/215 rotary evaporator (Buchi, Switzerland) equipped with a round bottom flask corresponding to the example of the lipophilic substance-receiving device (90). The fractionated lipid soluble material-solvent was transferred to an evaporator and placed in a round bottomed flask. The cold raw water was introduced into the condenser and the oil bath of the distillation flask was set at 174 ° C.

When the distillation started, a decane passed through the apparatus and flowed into the condenser and collected into the receiving flask in liquid form. At the end of distillation, the volume of recovered decane in the receiving flask and the volume of cell-origin lipophilic material left in the distillation flask were measured and determined by LC-MS analysis with C17 as the standard. The recovered decane solvent was transferred to the solvent tank 20 for reuse.

Example 8. Biodiesel Extraction from Cellular Fatty Acids

Methanol and caustic soda were mixed to prepare methoxide, the prepared methoxide was added to a stirrer and stirred, and the extracted cell lipophilic substance was reacted with a methoxide to form a biodiesel, a glycerin and a soap solid component . These products were supplied to a centrifuge and centrifuged. The biodiesel was placed at the upper part due to the difference in specific gravity of each product, and the upper part and lower part of the lower part were separated so that heavy glycerin and soap components were positioned.

The thus-separated glycerin and soap components were discharged into separate glycerin storage tanks. Water in the upper portion of the biodiesel and twice the amount of biodiesel was introduced into a stirrer and stirred, thereby being contained in biodiesel The glycerin, the soap component and the methanol component were dissolved in water, and the thus stirred solution was separated into a centrifugal separator to separate the components such as glycerin dissolved in the water. And then discharged to a glycerin storage tank through a separate discharge line. Finally, a distillation unit, which is a heater unit, was operated to distill 1 to 2% of water remaining in the biodiesel, Respectively.

The GC-TOF-MS (GC-6890N, Agilent Technologies, USA) analysis of the harvested biodiesel is shown in FIG.

Example 9. Fractionated Lipophilic Substances - Analysis of Beta-Carotene in Solvents

The content of beta-carotene in the liposoluble material-solvent fractioned above was determined using a HPLC (Hewlett Packard Series model 1100) equipped with a Waters Spherisorb S5 ODS2 cartridge column (4.6 x 250 mm).

In order to separate the coloring matter, 90% of acetonitrile, 9.99% of distilled water, 0.01% of triethylamine, 86% of acetonitrile and 8.99% of distilled water were used for 2 to 14 minutes while flowing the solvent at a rate of 1.0 ml / %, Triethylamine 0.01% and ethyl acetate 5%, and for 15 to 21 minutes 100% ethyl acetate was used. The post-run was 9 minutes with the starting solvent. The beta-carotene pigment was detected at 445 nm when the reference (Jin et al., 2001 Biochim Biophys Acta 1506: 244-2597) was set at 550 nm, and a standard curve of beta-carotene (DHI water and environment, Denmark) As a result, the amount of beta-carotene was measured to be 8.72x10 < ^-10 >

Example 10. Ethanol fermentation of the fractionated cells

The example of the cell-receiving device 80 is a fermenter using a microbial fermenter (INNO 200603, Innobio, Korea). Clostridium phytopermans were grown in culture tubes containing GS-2 medium containing the indicated amounts of each of the fractionated cells.

The GS-2 medium contained yeast extract 6.0, urea 2.1, K ₂ HPO ₄ 2.9, KH ₂ PO ₄ 1.5, MOPS 10.0, triosodium citrate dihydrate 3.0, and cysteine hydrochloride 2.0 (each expressed in g / L) . The initial pH of the culture medium was 7.5, and the initial Clostridium phytopermementus concentration was 0.8-1.1 × 10 ⁷ cells / mL and cultured under an atmosphere of N ₂ at 30 ° C.

The ethanol concentration at the completion of fermentation of the fractionated cells was determined. Clostridium phytopermantans produced hydrogen simultaneously with ethanol fermentation. The ethanol concentration was analyzed using HPLC (Breeze HPLC system, Waters Co., USA) equipped with RI detector. The column was Aminex HPX-87H (3007.8 mm, Bio-rad).

When the fractionated cell density was 10 g / L, the concentration of ethanol was 0.23-0.26% (v / v). When the fractionated cell density was 20 g / L, the concentration of ethanol was 0.42-0.54% (v / v). When the fractionated cell density was 40 g / L, the concentration of ethanol was 0.92 to 1.20% (v / v). Ethanol distillation was carried out using a distillation column.

These results indicate that higher density fractionated cells do not inhibit the action of clostridial peatotermentans, as the concentration of ethanol increases as the density of the fractionated cells increases. Finally, it can be seen that these cellulose feedstocks are fermented with ethanol without the chemical pretreatment of the cell feedstock and the addition of cellulases or other enzymes to the clostridial piotopermants.

Example 11. Extraction of ethanol

The fermentation products produced by ethanol fermentation were transferred to a still and distilled with ethanol. At this time, the oil bath was operated to evaporate the ethanol and heated to the temperature higher than the evaporation temperature of ethanol. When the temperature is higher than the evaporation temperature of water, the water is evaporated and the concentration of ethanol may be lowered by mixing with ethanol. Therefore, the vaporization temperature is formed between 78.3 and 85 ° C and heated for a certain period of time. In an ethanol storage tank.

Example 12. Butanol fermentation of fractionated cells

The example of the cell-receiving device 80 is a fermenter using a microbial fermenter (INNO 200603, Innobio, Korea).

Clostridium Thermocellum was anaerobically cultured in DSM medium at 60 ℃, anaerobic condition, 150 rpm. The fractionated cells were transferred to a 5 L fermenter (50), inoculated with the C. thermocellum culture (5%, v / v), saccharified by incubation at 60 ° C under anaerobic conditions and 150 rpm for 3 days [ Biotechnology Letters Vol 7 No 7 509-514 (1985)]. After inoculation, nitrogen gas was injected into the culture solution to maintain anaerobic conditions.

For butanol fermentation, Clostridium acetobutylicum , a spore suspension, was heated at 80 ° C for 10 min. Then, anaerobically cultured in a medium at a temperature of 37 ° C.

The fermenter was inoculated with the C. acetobutylicum culture (5%, v / v), and anaerobic butanol fermentation was carried out at 37 ° C under anaerobic conditions with stirring at 180 rpm. During the fermentation period, samples were taken periodically to analyze microbial growth and butanol concentration.

The used DSM medium contained 1.3 g of (NH ₄ ) ₂ SO ₄ per liter, 2.6 g of MgCl ₂ .6H ₂ O, 1.43 g of KH ₂ PO ₄ , 7.2 g of K ₂ HPO ₄ .3H ₂ O, 0.13 g of CaCl ₂ ₂ · 6H ₂ O, 1.1mg of FeSO ₄ · 7H ₂ O, 6.0g of sodium β-glycerophosphate, yeast extract of 4.5g (yeast extract), a carbon source of 10g (filter paper, a lump or cell BIOS processed into cellulose) , 0.25 g of reduced glutathione and 1 mg of resazurin. The pH was adjusted from 5.0 to 8.0 with 1 M HCl or 1 M NaOH.

The C. acetobutylicum culture medium contained 0.75 g KH ₂ PO ₄ , 0.75 g K ₂ HPO ₄ , 0.4 g MgSO ₄ .H ₂ O, 0.01 g MnSO ₄ .H ₂ O, 0.01 g FeSO ₄ .7H ₂ O, 0.5 g of cysteine; 5 g of yeast extract, 2 g of asparagine.H ₂ O, 2 g of (NH ₄ ) ₂ SO ₄ .

The acetone, butanol and ethanol produced by the microorganisms after the continuous process were quantitatively analyzed using gas chromatography (Agilent technology 6890N Network GC system) equipped with a FID (Flame Ionization Detector), and the column was HP-INNOWAX (30 cm x 250 Mu m x 0.25 mu m, Agilent technology) was used. The temperature of the sample injecting portion and the detecting portion was set to 250 DEG C, and the oven was set to rise to 170 DEG C at a rate of 10 DEG C / minute at 50 DEG C. [ The fermentation broth contained 13 g / L of butanol, 8 g / L of acetone and 0.5 g / L of ethanol.

Example 13. Extraction of butanol

(1-butyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide], and BMIM-PF6 (trifluoromethylsulfonyl) Butanol was extracted with 1-butyl-3-methyl imidazolium hexafluorophosphate (1-butyl-3-methyl imidazolium hexafluorophosphate). Butanol was extracted by vortexing the fermentation broth of the fermentation broth with BMIM-TFSI (Sigma Aldrich, USA) in the same amount, and BMT-PF6 (Sigma Aldrich, USA) in the fermentation broth. In this case, it is also possible to use a solution prepared by using a general ionic liquid production method. As a result, 60 ㅁ 1% of butanol was extracted when BMIM-TFSI was used, and 64 ± 1% of butanol was extracted when BMIM-PF6 was used.

Example 14. Organic acid fermentation in fractionated cells

The example of the cell-receiving device 80 is a fermenter using a microbial fermenter (INNO 200603, Innobio, Korea). Lactobacillus brevis subsp. brevis were cultured in PYG medium (medium composition: 20.0 g of peptone, 5.0 g of glucose, 10.0 g of yeast, 0.08 g of NaCl, 0.5 g of cysteine hydrochloride, 0.008 g of calcium chloride, 0.008 g of MgSO ₄ , 0.04 g of K ₂ HPO ₄ , KH ₂ PO ₄ 0.04 g, sodium bicarbonate 0.4 g, pH 7.1-7.3), harvested by centrifugation, and washed with 0.085% saline. This was inoculated into a fermenter (50) containing the transferred cells. The initial pH of the medium was 7.2, and the initial lactic acid fermentation strain was 0.8-1.1 × 10 ⁸ cells / mL and cultured with N ₂ gas at 30 ° C. The concentrations of malate, lactate, acetate, citrate and butyrate were determined at the completion of fermentation.

Concentrations of malic acid, lactic acid, acetic acid and ascorbic acid, organic acids, were measured by HPLC (HP placard, Japan) with a Platinum EPS C18 organic acid analytical column (250 mm x 4.6 mm, 5 μm) and 0.05 M KH ₂ PO ₄ at pH 2.4. The nitric acid was analyzed by gas chromatography (HP placard, Japan) using CP 58 Wax (FFAP) (30 cm x 0.25 mm ID, 0.25 m) column.

The results showed that the concentrations of malic acid, lactic acid, acetic acid, citric acid and lactic acid were 870.30 + 13.15, 746.16 + 8.91, 4,233.23 + 76.06, 318.04 + 47.75 and 1.99 + 1.99 mM, respectively.

These results show that these cellulosic feedstocks are fermented with lactic acid without chemical pretreatment of the cell feedstock from which the lactic acid fermentor strain is fractionated and without the addition of cellulases or other enzymes.

Example 15. Extraction of lactic acid

Ca (OH) ₂ was added to the fermentation broth to adjust the pH to 10 and then heated to increase the solubility of calcium lactate (Ca-lactate), to kill lactic acid bacteria and to coagulate the protein. It was filtered at high temperature and recovered with calcium lactate and cooled to precipitate calcium lactate. After dissolving calcium lactate at high temperature, CaSO ₄ was precipitated by treatment with sulfuric acid, and lactic acid was recovered.

1: device 2 of the present invention: first peristaltic pump
3: second peristaltic pump 4: third peristaltic pump
10: culture tank 11, 21, 33, 34, 51, 81, 91: line
20: solvent tank 30: mixing tank
31: Vibrating milling device 32: stirring device
40: Separation tank 40a: Cell separation device
41: Ultrasonic Resonance Generator (Acoustic Cell Filter)
42: Acoustic chamber 43: Ultrasonic oscillator
44: ultrasonic vibrator 45:
46: gravity needle apparatus 46a: swash plate
46b: mounting frame 46c: inlet
46d: cell collection part 46e: cell outlet
46f: Outflow Way 46g: Solution Outlet
46h: vibration generating unit 50:
60: cell-circulation line 70: solvent-circulation line
80: Cell-receiving device 90: Lipophilic substance-receiving device

Claims

A process for culturing cells containing lipid-soluble substance;
Dissolving the liposoluble material in the liposoluble material extraction solvent by mixing the liposoluble material extraction solvent with the culture solution of the cell and bringing the liposoluble material extraction solvent into contact with the cell culture liquid;
A step of aggregating, stagnating and separating cells from the mixed solution;
A step of fractionating the remaining solution from which the cells have been separated into the liposoluble material-solvent and water in which the liposoluble material is dissolved in the liposoluble material extraction solvent; And
Obtaining the separated cells and the liposoluble material-solvent, respectively;
&Lt; / RTI > The method of claim 1, wherein the cell is cultured.

The method according to claim 1, wherein the cells are separated from the mixture by applying an ultrasonic resonance field to the mixed solution.

The method according to claim 1, wherein the cells are separated from the mixture by applying gravity sedimentation to the mixture.

4. The method according to any one of claims 1 to 3,
The separated cells are regrown, and the regenerated cell culture fluid is mixed with and contacted with the liposoluble material extraction solvent or fractionated liposoluble material-solvent to further dissolve the liposoluble material in the cell into the liposoluble material extraction solvent , And then repeating the process, wherein the re-fractionation of the cells, the re-dissolution of the liposoluble material, the re-separation of the cells, and the re-fractionation of the liposoluble material-solvent are repeated one or more times, Solubilizing the cell and the liposoluble substance-solvent containing the desired concentrated liposoluble substance to produce a cell and liposoluble material through cell culture.

4. The method according to any one of claims 1 to 3,
Wherein at least one of the steps of vibrating and pulverizing the cell culture liquid and stirring the mixture liquid is performed to mix the cell culture liquid and the liposoluble material extraction solvent to increase the contact between the cell culture liquid and the liposoluble material extraction solvent Wherein the cells are cultured in a cell culture medium.

A culture tank (10) for culturing cells containing a fat-soluble substance;
A solvent tank (20) storing a liposoluble material extraction solvent in which the lipophilic substance is dissolved;
A mixing tank (30) for mixing the culture medium of the cells from the culture tank (10) and the liposoluble material extraction solvent from the solvent tank (20);
A separating tank 40 having a cell separating device 40a for separating, coagulating, and separating the cells from the mixed liquid from the mixing tank 30;
A fractionation tank (50) for fractionating the solution from the separation tank (40) from which the cells are separated (removed) into the liposoluble material extraction solvent, the liposoluble material in which the liposoluble material of the cell culture liquid is dissolved, and water;
A cell-receiving device 80 for receiving or treating the cells separated from the separation tank 40; And
A lipophilic substance-receiving device 90 for receiving or treating the lipophilic substance-solvent fractionated from the fractionation tank 50;
Characterized in that the cell and the liposoluble material are produced by cell culture.

The apparatus of claim 6, wherein the cell separator (40a) comprises: an ultrasonic resonance field generating device (41) for applying an ultrasonic resonance field to the mixed solution to separate the cells from the mixed solution; And a gravity settling device (46) for separating the cells from the gravity settling device (46).

8. The method according to claim 6 or 7,
A cell-circulation line (60) circulating the separated cells from the separation tank (40) to the culture tank (10); And
A solvent-circulation line (70) circulating the fractioned lipid-soluble substance-solvent from the fractionation tank (50) to the solvent tank (20); Further comprising:
The cell-receiving device 80 is adapted to be cultivated in the culture tank 10 through the cell-circulation line 60 once or twice or more, Accepting or treating cells;
The fat-soluble substance-receiving device 90 is a device for collecting the fat-soluble substance-containing device 90 from the fractionation tank 50 after it has been subdivided once or more than twice in the fractionation tank 50 through the solvent- Characterized in that the lipophilic substance-solvent is received or treated.

9. The method of claim 8,
A first peristaltic pump (2) for supplying a predetermined amount of the cell culture liquid of the culture tank (10) and the liposoluble material extraction solvent of the solvent tank (20) to the mixing tank (30);
The cells separated from the separation tank 40 are selectively transferred to the culture tank 10 through the cell-circulation line 60 according to their density or transferred to the cell- Peristaltic pump (3); And
A third peristaltic pump 4 for transferring the lipid-soluble substance-solvent fractionated in the fractionation tank 50 to the solvent tank 20 or transferring it to the lipid-soluble substance-receiving device 90 according to the degree of enrichment;
Characterized in that the cell and the liposoluble material are produced by cell culture.

8. The method according to claim 6 or 7,
The mixing tank 30 is provided with an oscillation crushing device 31 for vibrating and pulverizing the cell culture liquid and an agitator 32 for agitating the mixture liquid so as to increase the contact between the cell culture liquid and the liposoluble material extraction solvent The device for producing cell and liposoluble material through cell culture, characterized in that it further comprises one device.