KR101554536B1 - Development of contamination control technology for pure culture of Haematococcus pluvialis using surfactant - Google Patents

Abstract

The present invention relates to a development of a contamination control technology for pure culture of Haematococcus pluvialis using an ionic surfactant. According to various embodiments of the present invention, a method for culturing cells of Haematococcus pluvialis comprising a step for removing contaminated microorganisms by adding a surfactant to a cell culture medium can solve a problem of a decrease in productivity of biomass and astaxanthin caused by inhibition of growth of the cells of Haematococcus pluvialis and synthesis of astaxanthin due to contamination by the microorganisms, such as bacteria, microalga, etc, in the cell culture medium when mass photo-culturing the cells of Haematococcus pluvialis, a kind of microalgae, under an autotrophic condition. More specifically, using a surfactant when culturing the cells, a condition of inactivation of bacterial contamination can be kept for a long time, thereby being useful for producing more uniform biomass and astaxanthin from the cells of Haematococcus pluvialis, and further, the astaxanthin produced by the method can be widely used in various industrial fields such as medicine, sitology, raw materials for cosmetics, etc as a powerful antioxidant.

Description

[0001] The present invention relates to the development of contamination control technology for the pure culture of horseradish peroxidized fluvialis using a surfactant,

The present invention relates to the development of contamination control technology for the pure culture of hippocampus flucytialis cells using a surfactant, and it is an object of the present invention to effectively remove external microorganisms when culturing hippocampal flora, The present invention relates to an improvement of process technology capable of enhancing the biomass and astaxanthin production capacity of Tokocus pluvialis cells.

Astaxanthin (3,3'-dihydroxy-β, β'-carotene-4,4'-dione), a ketocarotenoid with a red color in general, is a chemical compound such as β-carotene Carotenoid-based coloring matter, which has a unique molecular structural property that has one hydroxyl group (-OH) and one ketone group (= O) at both terminal ends, as compared with beta-carotene, as an antioxidative functional substance that eliminates harmful free radicals Therefore, it has much higher antioxidant activity than conventional antioxidants. Astaxanthin has antioxidant activity 500 times higher than vitamin E and 20 times higher than beta-carotene. Because of its high antioxidant activity, astaxanthin is widely used as a feed additive for pharmaceuticals, food additives and animal and poultry, and its demand and application range is expected to increase rapidly.

These astaxanthin are produced by yeast strains Phaffia rthodozyma and Bervibacterium , and are distributed widely in marine and freshwater animals. However, astaxanthin is produced from yeast strains, such as Phaffia rthodozyma and Bervibacterium , Tin has a low content, is difficult to be extracted and is not applied, and has a problem in that the yield of the astaxanthin is low although the growth rate of the Papiadoderm strains is high.

Conventionally, KR2005-0005341 A discloses a method for producing a high content of astaxanthin by culturing a strain capable of producing astaxanthin, thereby increasing the amount of astaxanthin produced by the microorganism to produce astaxanthin KR2010-0105193 A discloses a method for producing astaxanthin by irradiation with radiation, and the method for producing astaxanthin is disclosed. In this method, the best microalgae Various studies have been carried out to enhance the productivity of astaxanthin at the same time as immobilization of carbon dioxide by light using Haematococcus pluvialis .

However, in order to actually apply the biological system using microalgae to the capture of carbon dioxide in the flue gas generated from industry, it is necessary to realize a microalga mass culture system which is more economical in reality. However, in order to apply the microalgae- The decrease in biomass and astaxanthin productivity has a problem of reducing the economical efficiency. As the phenomenon progresses to mass culture for scale-up, the situation becomes worse and monetary hits become worse.

In particular, hippocampus Tocococcus pluvialis has its own reactive mechanism sensitive to oxidative stress and is very sensitive to the biotic stresses caused by contamination of bacteria and other microalgae (chlorella), which leads to inhibition of growth and production of astaxanthin Which is directly related to the decrease in productivity of biomass and astaxanthin due to degradation (see FIG. 1).

Therefore, in order to produce biomass and astaxanthin more uniformly than in the case of large-scale cultivation of horseradish peroxidase, it is urgently necessary to develop a pollution control technology capable of controlling the biomass and astaxanthin production.

Various techniques have been known as methods for preventing microbial contamination of conventional cell culture media. KR10-0988017 discloses a method for culturing mycoplasma contaminated cells and a method for removing mycoplasma contamination of cells. amphidinols). In KR10-1131867, polygamma glutamic acid is disclosed as a biomaterial for preventing cell damage during cell culture. KR2005-0093632 discloses a method of using δ-levulinic acid The present invention relates to a method for producing a chlorella culture medium and a culture medium containing the chlorella culture medium and a culture medium for preventing microbial contamination of the microorganism, In WO 2005/056741, the above nonionic surfactant is used as a packaging material, A technique for preventing contamination by bacteria and fungi by depositing or coating on the surface of media, building materials, construction auxiliary materials, fabrics, fur, paper, leather or leather has been disclosed, and in WO 2012/168003, ionic surfactants Discloses a method for selectively dissolving eukaryotic cells in a microorganism sample. However, a technique for preparing a solution having a pH of 9 or higher by adding an ionic surfactant and then performing a process of dissolving eukaryotic cells, i.e., blood cells, There is no mention of the increase in productivity of biomass and astaxanthin in the hippocampus Tokubus pluvialis.

The problem to be solved by the present invention is to provide a method for the production of biomass from the hippocampus Tococulus pluvialis cells due to contamination by external microorganisms caused by the difficulty of pure culture due to the nature of the process during the long- A method for culturing new horseradish peroxidase pluvialis cells in order to solve problems such as reduced productivity of astaxanthin and reduction of conversion efficiency of carbon dioxide.

According to a representative aspect of the present invention, a method for culturing a hippocampus pluvialis cell comprising the step of adding a surfactant to a cell culture medium to remove contaminating microorganisms is disclosed.

According to various embodiments of the present invention, a method for culturing a hippocampus flocalys cell comprising the step of adding a surfactant to the cell culture medium of the present invention to remove contaminating microorganisms is a method of culturing microbial cells, In the case of massive optical culture of Cucumber pluvialis cells, the biomass and astaxanthin caused by the inhibition of astaxanthin synthesis and the growth of hippocampal Tococulus pluvialis cells due to contamination by microorganisms such as bacteria and microalgae in cell culture medium It is possible to maintain the state of inactivation of the bacterial contamination for a long period of time when the cell culture is carried out. Therefore, it is possible to obtain a more uniform biomass and astaxanthin from hippocampus Tococulus pluvialis cells, In addition to being useful for tin production, Xanthine is a potent antioxidant and can be used in a variety of industries including medicine, food science, and cosmetic raw materials.

FIG. 1 is a graph showing a decrease in productivity of biomass and astaxanthin due to cell death due to bacterial contamination during astaxanthin synthesis in a large-scale photo-cultivation field of outdoor horseradish peroxidase according to an embodiment of the present invention Fig.
FIG. 2 is a graph showing changes in cell wall characteristics and the merits of green dormant cells of each cell when transformed into a green capsule state through green dormant cells in the state of green monocotyledonous birds of horseradish peroxidase Fluvialys according to an embodiment of the present invention It is a chart.
FIG. 3 is a graph showing changes in the productivity of biomass and astaxanthin after cell death due to bacterial contamination during astaxanthin synthesis in a large-scale photo-cultivation field of outdoor horseradish peroxidase according to an embodiment of the present invention FIG.
FIG. 4 is a graph showing the growth of horseradish peroxidase according to the presence or absence of SDS pretreatment in various bacterial contamination conditions according to an embodiment of the present invention.
FIG. 5 is a graph showing the results of SDS pretreatment of external contaminants through continuous passaging of inoculated Seed after removal of bacteria present in the contaminated cell culture solution by SDS pretreatment technique in a large- It is a graph that verifies the effect of technology.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

According to an aspect of the present invention, there is provided a method for producing a hippocampus fluvialis cell, comprising the steps of: (a)

(b) primary centrifuging the culture solution containing the hippocampal Tococles pluvialis cells in the dormant state obtained in the step (a);

(c) adding a surfactant to the culture medium containing hippocampus pluvialis cells obtained in the dormant state after performing the step (b), and then performing shaking motion;

(d) performing a second centrifugal separation of the culture solution after the step (c), separating the killed microorganisms other than the hippocampal Tococulus pluvialis cells in a dormant state in the culture liquid and washing the surfactant; And

(e) reacting the obtained hippocampal state hippocampal pluck cell with the obtained growth medium after the step (d), and culturing the hippocampus pluvialis cells.

Unlike other microalgae, the hippocampus Tococulus pluvialis cells are unique in that their shape, size and weight, including cell wall components, are changed by external environmental stress. As shown in Fig. 2, Ruby Alice cells are transformed from rusted flagellar algae cells into immature red-cyst cells through immature green algae dormant cells, while the cell wall properties change from hydrophilic to hydrophobic as the transformation proceeds The cell walls are more robust, making it difficult to penetrate foreign materials.

Thus, when horseradish peroxidase cells are cultured in a culture medium contaminated with contaminating microorganisms including bacteria or general microalgae, the hippocampal flocalys cells in the cell culture medium can be classified into two types depending on the degree of contamination of the bacteria: green flagellate state to a green resting state. If the pollution is not severe, a large number of hippocampal flounder cells in the form of a rusty flagella algae are present. When a surfactant is added to a culture solution, The process of inhibiting the penetration of the surfactant component into the hippocampal Tococles pluvialis cells due to the problem that the ionic surfactant component kills not only the bacteria and the general microalgae cells to be removed but also the hippocampal Tococulus pluvialis cells , Wherein the process comprises the steps of: (a) A step of switching the hippocampus Flags Tokoro kusu ruby Alice cells with green algae hyumyeongi state.

In the step (a), when the horseradish peroxidase activity is converted into the dormant state in the first culture medium, if nitrogen deficiency is induced for 5-6 days after the growth inhibition medium is additionally added, To convert the hippocampus Tocococcus pluvialis cells to a state of alveolar dormancy.

(B) centrifuging the first culture solution containing the hippocampal Tococulus pluvialis cells obtained in the step (a) in the dormant state to remove contaminating microorganisms such as bacteria or light microalgae contained in the first culture solution by 1 Wherein the first culture medium containing hippocampal Tococulus pluvialis cells in a dormant state is centrifuged at a low speed of 1000-1500 rpm to remove bacteria or light microalgae contained in the first culture medium .

At this time, it is preferable that centrifugation is carried out for 1-3 minutes repeatedly 3-5 times, and the removal efficiency of contaminating microorganisms such as bacteria or light microalgae is increased more than centrifugation for a long time Can be achieved.

According to an embodiment of the present invention, the step (c) comprises the step of allowing the surfactant component to penetrate into the cells of contaminating microorganisms such as bacteria or microalgae cells other than hippocampal flora, The surfactant is added at a working concentration of 10-30 g / L and shaken at a rate of 100-120 rpm for 20-40 minutes. In this process, most cells including the chlorophyll of bacteria and microalgae The components escape from the cell wall and most of the bacteria and microalgae cells die.

When the surfactant is added at a concentration of less than 10 g / L, bacterial or microalgae cells are not completely killed and the ability to accumulate biomass and astaxanthin is lowered. When the surfactant is added in an amount exceeding 30 g / L, There is a problem that the growth of green cyst cells may be delayed after regeneration of the surfactant.

In another embodiment of the present invention, the surfactant of step (c) is selected from the group consisting of sodium dodecylsulfonate (SDS), sodium laurylsulfate (SLS), sodium lauryl ether sulfate (SLES), linear alkylbenzenesulfonate LAS), monoalkyl phosphate (MAP), fatty acid metal salt, and the like.

On the other hand, when performing the shaking motion, it is possible to use a hand shaker or a shaker, but the present invention is not limited thereto.

According to an embodiment of the present invention, in the step (d), the culture medium containing the contaminating microorganisms killed by the addition of the surfactant obtained in the step (c) and the hippocampus hippocampal pluck bacillus cells is applied to the second Separating the killed contaminating microorganisms except for the hippocampal Tococulus pluvialis cells in a dormant state by centrifugation and washing the surfactant, wherein the secondary centrifugation is carried out at a speed of 1000-1500 rpm for 1-3 minutes It is preferred to perform 5-15 repetitions, and centrifugation at a speed exceeding 1500 rpm, or centrifugation at a rate of 1000-1500 rpm for more than 3 minutes, It is impossible to achieve the effect of removing all the microorganisms.

According to an embodiment of the present invention, the step (e) is a step of reactivating the hippocampal state hippocampal pluck baculovirus cells in a growth medium for use as a hippocampus flocalys cell culture seed, , Wherein the available growth medium composition is CaCl ₂ .2H ₂ O, KCl, Na ₂ Glycerophosphate · 5H ₂ O, MgSO ₄ · 7H ₂ O, Tris-aminomethane, Thiamine, Biotin, Vitamin B12, PIV metal solution, Na ₂ EDTA, FeCl ₃ · 6H ₂ O, MnCl ₂ · 4H ₂ O, ZnSO ₄ But are not limited to, 7H ₂ O, CoCl ₂ .6H ₂ O, and Na ₂ MoO ₄ .2H ₂ O.

As described above, the method of culturing the hippocampal T. fucillus alliance cells of the present invention is a method for culturing microbial cells, hippocampus pluvialis cells under autotrophic conditions, in a large scale optical culture, in which microorganisms such as bacteria and microalgae It is possible to solve the problems such as the growth of hippocampal T. fucillus pluvialis cells and the decrease of biomass and astaxanthin production capacity caused by the inhibition of astaxanthin synthesis. In particular, when a surfactant is used, It is possible to maintain the state of inactivation of bacterial contamination for a long time during culturing and thus can be usefully used for production of more uniform biomass and astaxanthin from hippocampus Tococulus pluvialis cells.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.

material

The strain used in the present invention was purchased from Haematococcus pluvialis NIES-144 from National Institute for Environmental Studies, Tsukuba, Japan.

All of the media used in this experiment were NIES-C medium and NIES-N medium, and their constituents are shown in Table 1 below.

NIES-C NIES-N ingredient Content (/ L) ingredient Content (/ L) Ca (NO _{3) 2} 0.15 g CaCl ₂ .2H ₂ O 0.13 g KNO ₃ 0.10 g KCl 0.07 g Na ₂ Glycerophosphate · 5H ₂ O 0.05 g Na ₂ Glycerophosphate · 5H ₂ O 0.05 g MgSO ₄ .7H ₂ O 0.04 g MgSO ₄ .7H ₂ O 0.04 g Tris-aminomethane 0.05 g Tris-aminomethane 0.05 g Thiamine 0.01 mg Thiamine 0.01 mg Biotin 0.10 [mu] g Biotin 0.10 [mu] g Vitamin B12 0.01 [mu] g Vitamin B12 0.01 [mu] g PIV metal solution
(per liter)
Na ₂ EDTA 1 g
FeCl ₃ .6H ₂ O 0.196 g
MnCl ₂ .4H ₂ O 0.036 g
ZnSO ₄ .7H ₂ O 0.022 g
CoCl ₂ .6H ₂ O 4 mg
Na ₂ MoO ₄ .2H ₂ O 2.5 mg 3 ml PIV metal solution
(per liter)
Na ₂ EDTA 1 g
FeCl ₃ .6H ₂ O 0.196 g
MnCl ₂ .4H ₂ O 0.036 g
ZnSO ₄ .7H ₂ O 0.022 g
CoCl ₂ .6H ₂ O 4 mg
Na ₂ MoO ₄ .2H ₂ O 2.5 mg 3 ml

In Table 1,

- NIES-C medium: autotrophic medium (objective: growth)

- NIES-N medium: autotrophic medium (purpose: growth inhibition, light induction, astaxanthin production)

- Medium volume: 150 mL (flask), 20 L (outdoor light incubator)

- CO ₂ supply: 3% (v / v)

- Light condition: 20 μE / m ² / s during 'green' stage (vegetative growth); 150 μE / m ² / s during 'red' stage (inductive growth)

- SDS (Sodium Dodecyl Sulfate): 20 g / L, gentle shaking for 30 min

- Bacteria: Bacillus subtilis family (separated from contaminated optical incubator in outdoor light culture)

Example 1

After preparing a culture medium of horseradish peroxidase, which is contaminated with bacteria or external microalgae cells, NIES-N medium is injected, and nitrogen deficiency is induced for 5-6 days, Pluvialis cells were transformed from green flagellate to green resting state.

Then, the contaminated cell culture fluid containing the hippocampus pluvialis cells in the greenhouse dormant state was washed four times for 2 minutes at a low rate of 1,000-1,500 rpm to primarily remove bacteria and light microscopic algae cells Sodium dodecyl sulfate (SDS) was injected at a working concentration of 20 g / L into the cell culture medium from which the primary contaminated microorganisms were removed, and the cells were slowly shaken for 30 minutes. Min for 8-10 min to isolate the killed microbial cells, except for the hippocampus Tococulus pluvialis cells in the dormant state of the greenhouse. The sodium dodecyl sulphonate was washed away and the bacteria and mild microalgae cells were washed 2 Was removed by car.

The green algae dormant hippocampal T. flebis alice cells in which the bacteria and microalgae were removed were reactivated in NIES-C medium for one day and used as an outdoor culture seed (Seed).

Experimental Example 1: Contamination of external microorganisms (bacteria, microalgae) in a culture medium during large-scale cultivation of microalgae cells using an optical incubator under outdoor self-nutrient conditions. Growth and development of astaxanthin in the hippocampus Tokusux pluvialis cells Influence on astaxanthin accumulation

In order to investigate the effect of contaminating microorganisms (bacteria, microalgae) in cell culture medium on the accumulation of astaxanthin in the hippocampal flora, in a large scale culture of microalgae cells using an optical incubator under outdoor self-nutrient conditions Experiments were performed.

(20 μE / m ² / s) and only carbon dioxide in the NIES-C medium lacking the organic carbon source and bacterial contamination in the cell culture medium (OD ₆₈₀ = approx. 0.8) coli, Bacillus subtilis, and Agrabacterium strains) were further screened, and NIES-N medium in the photoreactor was further injected to inhibit vegetative growth (cell growth) in the 'green' stage Increase in biomass of horseradish peroxidase during the 30 days of incubation (astaxanthin synthesis) in 'red' stage with strong light (150 μE / m ² / s) Increase) and astaxanthin accumulation, and the results are shown in Fig.

As shown in Fig. 3, the biomass production and astaxanthin accumulation capacity of hippocampus Tocococcus pluvialis due to bacterial contamination under the condition of self-nutrition decreased by 31% and 49%, respectively, on the 18th day.

The production of astaxanthin in the hippocampal flora ally cells increases in proportion to the amount of light received per unit cell and depends on the photoreaction. As a result, the biomass decreased because of the biotic stress due to bacterial contamination, green resting state, and the cell growth rate was significantly reduced. The reason for the decrease in astaxanthin accumulation capacity is that the amount of light received per unit hippocampus Tokubus pluvialis cells was reduced due to the shading effect due to bacterial contamination .

Experimental Example 2: Culturing of hippocampal Tocococcus pluvialis cells under autotrophic conditions Whether the application of the SDS pretreatment technique in the various contamination conditions of the bacteria in the initial medium occurred during the vegetative (green) stage of the hippocampal Tococulus pluvialis cells growth

The following experiments were conducted to investigate the effect of vegetative growth of horseradish tobacco pluvialis cells under autotrophic conditions during the 'green' stage depending on the degree of contamination of external microorganisms (bacteria, microalgae) in the cell culture medium .

In the NIES-C medium lacking the organic carbon source, only carbon dioxide was used as the sole carbon source and cultured for 12 days in the 'green' stage with light of low light intensity (20 μE / m ² / s) Alice 's biomass vegetative growth was analyzed. The results are shown in Fig.

As shown in Fig. 4, as the absorbance value of the bacteria in the initial culture medium of hippocampal T. flebiiis cells increased from 0.01 to 0.1, the increase in the biomass of hippocampal T. flebis alice cells decreased sharply. This is probably due to the fact that the cell shape was changed to green resting cells, which are relatively slow cell proliferation in the green flagellate state, where cell proliferation is active due to bacterial contamination.

On the other hand, cell cultures contaminated with bacteria at different concentrations were similar in growth pattern to Control cells in SDS pretreated cells.

Accordingly, the SDS pretreatment method according to the present invention can effectively inactivate bacteria regardless of the degree of contamination in the cell culture fluid to be used as seed inoculation, and consequently maintains high biomass productivity of the horseradish peroxidase pluvialis cells .

Experimental Example 3: Cell culture medium in which external microbial contaminants (bacteria, microalgae) were removed through SDS pretreatment was used to determine the persistence of biomass growth in a subculture culture of hippocampal flora, effect

In order to confirm the sustainability of the technical effect developed in the present invention in the case of mass culture of hippocampus Tokubus pluvialis cells using an optical incubator under outdoor self-nutritional conditions, external microbial sources (bacteria, microalgae) in the cell culture medium were subjected to SDS pretreatment (20 μE / m ² / s), followed by continuous subculture in NIES-C medium lacking organic carbon source, and the pattern of vegetative growth (cell number increase) was analyzed The results are shown in Fig.

As shown in Fig. 5, it was confirmed that the non-growth rate of hippocampal T. fucifloris cells was steadily increased as the subculture continued. This phenomenon occurs when the cells maintain the exponential phase continuously during the incubation period. Therefore, the maintenance of the cell cycle can be said to disprove the utility of the SDS pretreatment technique.

Claims (6)

(a) converting hippocampal Tococles pluvialis cells into a dormant state in a culture medium;
(b) primary centrifuging the culture solution containing the hippocampal Tococles pluvialis cells in the dormant state obtained in the step (a);
(c) adding a surfactant to the culture medium containing hippocampus pluvialis cells obtained in the dormant state after performing the step (b), and then performing shaking motion;
(d) performing a second centrifugal separation of the culture solution after the step (c), separating the killed microorganisms other than the hippocampal Tococulus pluvialis cells in a dormant state in the culture liquid and washing the surfactant; And
(e) reacting the obtained hippocampal state hippocampal pluck cell with the obtained growth medium, after the step (d), and culturing the hippocampal pluck cell.
The method according to claim 1, wherein the primary centrifugation in step (b) is performed at a speed of 1000-1500 rpm for 1-3 minutes.
The method according to claim 1, wherein the surfactant of step (c) is added at a concentration of 10-30 g / L.
The method of claim 1, wherein the surfactant in step (c) is selected from the group consisting of sodium dodecylsulfonate (SDS), sodium laurylsulfate (SLS), sodium lauryl ether sulfate (SLES), linear alkylbenzenesulfonate (LAS) Wherein the at least one ionic surfactant is at least one selected from the group consisting of monoalkyl phosphates (MAP) and fatty acid metal salts.
The method according to claim 1, wherein the shaking motion of step (c) is performed at a speed of 100 to 120 rpm for 20 to 40 minutes.
The method according to claim 1, wherein the secondary centrifugation in step (d) is performed at a speed of 1000-1500 rpm for 1-3 minutes.

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