KR101883733B1 - Production method of microalgae and separation method of fucoxhantin by the production method - Google Patents

Abstract

The present invention relates to a method for mass production of microalgae and to fuchocanthin derived from microalgae produced by the method, and more particularly, to a method for mass production of microalgae using phaeodactylum tricornutum, which is excellent in the ability to produce fucoxanthin, A method for mass production of microalgae capable of mass production in a short period of time, and fuocoanthin derived from microalgae produced by the above method.
According to the present invention, microalgae having excellent fucosanthin producing ability can be mass-produced at a low cost, and fucosanthin can be isolated from the microalgae produced by the above method at a high yield.

Description

TECHNICAL FIELD The present invention relates to a method for mass production of microalgae and a method for separating fucoxanthin from microalgae produced by the above method,

The present invention relates to a method for mass production of microalgae and a method for separating fucosantine derived from microalgae produced by the above method. More particularly, the present invention relates to a method for mass production of microalgae using phaeodactylum tricornutum, The present invention relates to a method for mass production of microalgae capable of mass production in a short period of time after culturing and a method for separating fucosantine derived from microalgae produced by the above method.

Marine organisms account for over 80% of all species in the planet, with more than 500,000 species. These marine organisms are characterized by the diversity of species, the identity of the evolutionary process, the special environment of marine environment, and their specific physiological activity and efficacy due to factors such as growth and metabolism. Therefore, new types of biosignals and active ingredients It is an important resource. In addition, there are specific enzymes and cofactors involved in biodegradation of their biosynthetic enzymes, and research and interest in the development of physiologically functional new materials and the development of health functional foods, cosmetics, and new drugs using them have been concentrated worldwide. In addition, due to factors such as food shortage due to the increase in world population, decrease of land cultivation area due to rising sea level due to global warming, and marine environment of Korea, which is the sea on three sides, marine biotechnology is developing as a future- The possibility of the related technology is required. Fucoxanthin in marine biomass has been reported to be beneficial to human health. Fucoxanthin, which is sold overseas, is mainly extracted from seaweed and kelp, and its content is low. In addition, large brown algae However, since microalgae can be produced throughout the year, it can be said that it is more economical in terms of feed supply of fucosanthin.

Korean Patent Publication No. 10-2016-0114962 Korean Patent No. 10-1070846

An object of the present invention is to provide a method for culturing a plant, comprising: (a) inoculating a culture solution with Phaeodactylum tricornutum and culturing at a temperature of 18 to 22 ° C for 5 to 10 days at a light intensity of 1000 to 2000 lux; (b) adding a coagulant to the cultured medium to precipitate for 2 to 6 hours; And (c) recovering the precipitated paedoatilum tricornatum and obtaining a paedoatilium tricornatum concentrate through centrifugation; To provide a mass production method for the paedoatilium tricornitum.

Yet another object of the present invention is to provide a method for culturing a host cell comprising the steps of (a) inoculating a culture solution with Phaeodactylum tricornutum and culturing at a temperature of 18 to 22 ° C for 5 to 10 days at a light intensity of 1000 to 2000 lux; (b) adding a coagulant to the cultured medium to precipitate for 2 to 6 hours; (c) recovering the precipitated faecal tilum tricornatum and obtaining a paedoatilium tricornatum concentrate through centrifugation; The present invention also provides a method for separating fucoxanthin derived from paeodactyltricornitum, which comprises (d) adding ethyl acetate to the concentrate, ultrasonication and then extracting the extract for 20 to 48 hours.

(A) inoculating a culture solution with Phaeodactylum tricornutum and culturing at a temperature of 18 to 22 ° C for 5 to 10 days at a light intensity of 1000 to 2000 lux; (b) adding a coagulant to the cultured medium to precipitate for 2 to 6 hours; And (c) recovering the precipitated paedoatilum tricornatum to obtain a paedoatilium tricornatum concentrate through centrifugation. The present invention further provides a method for mass production of paedoatilum tricornitum comprising the steps of:

In one embodiment, the culture medium of step (a) may be 10 to 20 L of seawater to which 0.3 to 0.7% (v / v) of conway is added.

In one embodiment, the phaedoatilum tricornatum of step (a) may be inoculated at a ratio of 1: 400 to 1: 600 relative to the volume of the culture.

In one embodiment, the flocculant of step (b) may be FeCl ₂ with a concentration of 0.01 to 0.25 g / L.

(A) inoculating a culture solution with Phaeodactylum tricornutum and culturing at a temperature of 18 to 22 ° C for 5 to 10 days at a light intensity of 1000 to 2000 lux; (b) adding a coagulant to the cultured medium to precipitate for 2 to 6 hours; (c) recovering the precipitated faecal tilum tricornatum and obtaining a paedoatilium tricornatum concentrate through centrifugation; And (d) ultrasonically treating the concentrate with ethylacetate and then extracting the extract for 20 to 48 hours. The present invention also provides a method for separating fucoxanthin from paeodactyltricornitum.

In one embodiment, the sonication of step (d) may be performed three or more times for 3 to 8 minutes.

According to the present invention, microalgae having excellent fucosanthin production ability can be cultured, mass-produced at a low cost in a short period of time, and fucosanthin can be isolated at high yield from microalgae produced by the above method.

FIG. 1 is a graph showing the proliferation tendency of Phaeodactylum tricornutum according to the illuminance and microalgae inoculation ratio.
FIG. 2 is a bar graph showing the proliferation rate of the microalgae, in which phaeodactylum tricornutum was grown according to the concentration of the medium.
FIG. 3 is a graph showing the growth rate over 11 days in comparison with the initial inoculum absorbance at 10 L and 20 L for the comparison of the growth level according to the microalgae culture volume.
Fig. 4 is a comparative photograph of the phaeodactylum tricornutum growth pattern (x200, bar = 200 占 퐉).
(A) 10 L culture, (B) 20 L culture.
FIG. 5 is a graph showing the measurement of the cohesion rate according to the concentration of the coagulant.
(A) FeCl ₂ , (B) MgCl ₂ , (C) FeCl ₂ + MgCl ₂ (unit: g / L)
FIG. 6 shows the result of comparing the flocculation level of Phaeodactylum tricornutum according to the concentration at 1 hour after the flocculant treatment.
(A) Ctrl, (B) MgCl 2 1 g / L, (C) FeCl 2 0.1 g / L, (D) FeCl 2 0.25 g / L, (E) FeCl 2 0.5 g / L, (F) FeCl 2 1 g / L, (g) FeCl 2 + MgCl 2 0.1 g / L, (H) FeCl 2 + MgCl 2 0.25 g / L, (I) FeCl 2 + MgCl 2 0.5 g / L, (J) FeCl 2 + MgCl ₂ 1 g / L (× 200, scale bar = 20 μm).
FIG. 7 shows the comparison result of the residual level of the iron salt in the upper layer according to the concentration of the flocculant. The arrows indicate Phaeodactylum tricornutum, which has not been submerged. (× 400, scale bar = 20 μm).
Fig. 8 shows the result of the coagulation degree comparison of the coagulant concentration of 0.1 g / L or less.
Figure 9 shows the results of 10 L and 20 L coagulation rates when the same amount of FeCl ₂ 0.1 g / L coagulant was treated for mass culture.
FIG. 10 is a photograph of a 10 L mass cultured and treated with 0.1 g / L of coagulant and observed over time.
FIG. 11 is a separation diagram for securing fucoxanthin derived from Phaeodactylum tricornutum.
12 is a TLC analysis result of a natural pigment obtained through a silica gel column after 24 hours (A) and 48 hours (B) extraction of an ethyl acetate extraction process.
13 shows the results of the extraction solvent and extraction yield over time.
14 is a graph of UV spectra absorbance against fucosanthin.
FIG. 15 is a graph showing the results of HPLC analysis of the final product according to the separation process of the present invention. (A) Analysis of components of the fucoxanthin expected substance using LC-MS. (B) All molecular weights and fucosanthin expected molecular weights at 58.762 min.
FIG. 16 shows the results of peaks of fucoxanthin standard peaks at various concentrations using LC-MS.
(A) 250 μg / mL, (B) 125 μg / mL, (C) 62.5 μg / mL, and (D) 31.25 μg / mL.
Figure 17 is a calibration curve of the Fucoxanthin standard.
FIG. 18 shows the results of LC / MS analysis of peak and area of fucoxanthin 3 mg / mL extracted from Phaeodactylum tricornutum.
Figure 19 shows the GC-MS analysis of the Fucoxanthin complex (information on the numbers is given in Table 3).
Figure 20 shows the results of the DPPH scavenging assay of the fucoxanthin complex derived from Phaeodactylum tricornutum (*, P <0.05; **, P <0.01; ***, P <0.001).
FIG. 21 shows the result of protein degradation assay of the fucoxanthin complex derived from Phaeodactylum tricornutum.
FIG. 22 shows the results of inhibition of tyrosinase activity using fucoxanthin.
(A) Melanin forming microscope photograph (x200, bar = 20 μm),
(B) Quantitative comparison of melanin formation level
23 shows the results of the inhibition of melanin biosynthesis after the treatment of fucoxanthin complex with B16F10 melanoma cells.
24 shows the result of the cell protection effect test by the fucoxanthin complex in CCD986sk cell.

(A) inoculating a culture solution with Phaeodactylum tricornutum and culturing at a temperature of 18 to 22 ° C for 5 to 10 days at a light intensity of 1000 to 2000 lux; (b) adding a coagulant to the cultured medium to precipitate for 2 to 6 hours; And (c) recovering the precipitated paedoatilum tricornatum to obtain a paedoatilium tricornatum concentrate through centrifugation. The present invention further provides a method for mass production of paedoatilum tricornitum comprising the steps of:

In one embodiment, the culture medium of step (a) may be 10 to 20 L of seawater to which 0.3 to 0.7% (v / v) of conway is added, preferably 10 to 20 liters of sea water to which conway 0.5% . The culture solution is suitable for mass production while promoting the growth of microalgae.

In one embodiment, the phaedoatilum tricornatum of step (a) may be inoculated at a ratio of 1: 400 to 1: 600 relative to the volume of the culture, preferably at a ratio of 1: 500 to the volume of the culture It is preferred to be inoculated. When the paedoatilum tricornatum is less than 1: 400 in terms of the volume of the culture medium, there is a problem that the initial inoculation concentration is small and the growth of microalgae is delayed. When the volume of the culture medium exceeds 1: 600, And the effect on microalgae growth is not large.

In one embodiment, the flocculating agent in step (b) may be in the concentration range of 0.01 to 0.25 g / L FeCl _2, preferably, the concentration is preferably 0.1 g / L. When the concentration of the flocculant is lower than 0.01 g / L, there is a problem that the flocculation is not performed well. When the concentration is higher than 0.25 g / L, a large amount of iron salt is consumed and the economical cost is increased.

(A) inoculating a culture solution with Phaeodactylum tricornutum and culturing at a temperature of 18 to 22 ° C for 5 to 10 days at a light intensity of 1000 to 2000 lux; (b) adding a coagulant to the cultured medium to precipitate for 2 to 6 hours; (c) recovering the precipitated faecal tilum tricornatum and obtaining a paedoatilium tricornatum concentrate through centrifugation; And (d) ultrasonically treating the concentrate with ethylacetate and then extracting the extract for 20 to 48 hours. The present invention also provides a method for separating fucoxanthin from paeodactyltricornitum.

In one embodiment, in the step (d), the ultrasonic treatment may be performed three or more times for 3 to 8 minutes, and preferably, the ultrasonic treatment is performed three times for 5 minutes. When the ultrasonic treatment is performed for less than 3 minutes, the cell wall is not sufficiently broken so that the fucosanthin is difficult to be separated and the yield is low. When the ultrasonic treatment is performed for 8 minutes or longer, the structural change due to the physical environment change such as high temperature is caused there is a problem.

Hereinafter, the configuration of the present invention will be described in detail.

< Example  1> Target microalgae

Phaeodactylum tricornutum was used as a microalgae to ensure optimal culture rate and maximum biomass in indoor culture conditions. Paeo-anthylum tricornatumum was purchased from Restorabs Co., Ltd. and used in the experiment of the present invention.

&Lt; Example 2 >

The microalgae phaeodactylum tricornutum selected in Example 1 was cultured under controlled light conditions to obtain final biological resources for development of marine biomaterials. (500, 1000, 1500, and 2000 lux), respectively, when irradiated with artificial light in consideration of the variable of indoor space, 2000 lux), and the microalgae inoculation rates were 2, 4, and 6% of the total culture media, respectively. The initial incubation rates of the microalgae were set at about 0.43, 0.49, and 0.65 nm, respectively.

Phaeodactylum tricornutum showed a smooth propagation under light condition of 1500 lux or more and showed no significant difference from 2000 lux. If there is no big difference between 1500 and 2000 lux, it is not necessary to irradiate with higher lux. Therefore, when the efficiency is considered, the optimum light condition (illumination) is confirmed to be 1500 lux. Similar proliferation levels were observed over time, regardless of the inoculation rate (2%, 4%, 6%) (see Figure 1).

< Example  3> Determination of optimal culture condition of selected microalgae

3-1. Comparison of growth level according to microalgae culture medium concentration

In order to investigate the growth rate of microalgae according to the concentrations of inorganic salts and vitamins, which are known to be required for microalgae proliferation, the addition of a conway medium commonly used for culturing Phaeodactylum tricornutum The growth rate was determined according to the concentration of the medium. For each experimental group, the composition of the conway medium was added at 0%, 0.25%, 0.5%, 0.75% and 1% (v / v) ratio and filtered air was injected through a 0.22 μm filter. And cultured at 20 ± 2 ° C for 5 days.

After 5 days of incubation for each concentration, no change was observed until day 2, but a visible growth rate change was observed from day 3. In the other experimental groups except for the 3-day culture-free group (0%), similar proliferation rates were observed. On the 4th day, 0.75% and 1% were not significantly different from the other groups but tend to have high proliferation. However, after 5 days of incubation, 1% was somewhat decreased, and 0.5% and 0.75% showed favorable proliferation rate compared with other experimental groups and showed the highest proliferation rate at 0.5% when compared with the initial cultivation time. Therefore, it was confirmed that the culture condition of 0.5% (v / v) for 5 days when the culture medium of Phaeodactylum tricornutum was cultured was optimal (see FIG. 2).

3-2. Comparison of growth levels according to microalgae culture volume

In order to establish the optimum growth condition of Phaeodactylum tricornutum and to cultivate from 10 L or more, the growth rate and the amount of growth of microalgae were evaluated to establish conditions for mass culture. . Therefore, in order to derive the optimal culture scale, 10 L and 20 L of Paeodactylum tricornutum were grown under the same environmental conditions for 11 days, (Fig. 3 and Fig. 4). On the other hand, at 10 L, the cells continued to proliferate even after 6 days, while the 20 L showed a gradually decreasing trend.

Therefore, 10 L and 20 L cultures showed higher efficiency than 10 L cultures in the current culture system environment, so 10 L was found to be the most suitable culture scale.

3-3. Comparison of microalgae coagulation level

In order to establish a stable and optimized harvesting method, the present invention provides a method of harvesting paedoatilium tricornatum (pectin) in order to establish an optimized harvesting method according to the study, Phaeodactylum tricornutum) were cultured and physical and chemical aggregation methods were performed.

The chemical agglomeration method is a method of collecting and collecting agglomerated agglomerates by binding density of an organic polymer flocculant or an inorganic flocculant using the electrostatic property possessed by the cell wall of the microalgae and increasing density and volume. The most commonly used coagulants are aluminum sulfate, aluminum potassium sulfate, sodium aluminate, ferrous sulfate, ferric sulfate, ferric chloride, ferrous chloride and magnesium chloride. Considering that the object of the present invention is development of a functional bio raw material, a substance containing aluminum, which is known to be highly toxic, is excluded, and the most common substance among iron (FeCl ₂ ) and magnesium (MgCl ₂ ) It was selected as a flocculant for testing.

To confirm the efficacy of iron (FeCl ₂ ) and (MgCl ₂ ), 50.0 mL of Phaeodactylum tricornutum, which was cultured at 675 nm to an absorbance level of 0.06 to 0.07, was dispensed in a 50 mL conical tube, Was added so that the final concentrations were 0.1, 0.25, 0.5, and 1 g / L. After adding the coagulant, the samples were collected at 3 cm from the water surface to confirm the change over time up to 12 hours. Were measured. The centrifugation method was limited in nature when it was applied in the future. On the other hand, when FeCl ₂ treatment was conducted to induce the aggregation of Phaeodactylum tricornutum, the color of the culture medium became darker than that of the control group, and gradually changed to transparent state over time Respectively. The par ohdak tilrum tree cor nutum (Phaeodactylum tricornutum) for the addition of FeCl ₂ in a non-culture broth were measured by absorbance, was set as a reference value to correct the absorbance measured in the culture group, par by FeCl ₂ ohdak tilrum tree cor The formula for calculating the coagulation rate of Phaeodactylum tricornutum is as follows.

Flocculation efficiency (%) = [1- (A-B) / C] 100

A: Absorbance of FeCl ₂ added culture,

B: absorbance of seawater added with FeCl ₂ ,

C: Absorbance of the control without FeCl ₂

The coagulation rate was about 80% or more from 1 hour at all concentrations after FeCl ₂ addition, and it was found to have a coagulation rate close to 100% after 2 hours. In addition, FeCl ₂ and MgCl ₂ were added at the same concentration Showed similar tendency to FeCl ₂ addition. However, when MgCl _{2 was} added, the flocculation did not occur even at a high concentration, and the flocculation effect was confirmed to be due to FeCl ₂ (see FIG. 5). The results of similar coagulation rates at all concentrations from 1 hour after the addition of the coagulant were judged to be due to the fact that the aggregation of microalgae was effectively formed by the iron ions and the sedimentation rate was increased. However, in order to visually confirm the formation of aggregates The same amount of precipitated microalgae was sampled and observed under a microscope. Phaeodactylum tricornutum agglutinates were observed in all experimental groups treated with FeCl ₂ concentration. Relatively small aggregates were formed at a concentration of 0.1 g / L, and at concentrations higher than 0.25 g / L A similar level of Phaeodactylum tricornutum aggregate was observed irrespective of the concentration (see FIG. 6). Therefore, for the cultivation of Phaeodactylum tricornutum, it is recommended to add FeCl ₂ at a concentration of 0.1 g / L or more to the total culture volume, Respectively.

In the culture medium without microalgae, yellow color was observed after FeCl ₂ addition. In order to confirm whether the cause of color development in the supernatant of cultured group was microalgae which did not precipitate or the residue of added iron ions 200 μL was collected at 3 cm from the surface of water and placed in a 96-well plate and observed under a microscope. In order to visually confirm the total amount suspended by time, all of the iron salts in the collected samples were taken after being left for 6 hours after the sampling. In the first hour, a part of Phaeodactylum tricornutum not precipitated with iron salts was observed in the supernatant liquid (white arrow in FIG. 7), but only iron salts were found in the upper layer after 2 hours , And the iron salt density of the supernatant decreased with time (see FIG. 7).

Phaeodactylum tricornutum, which remained in the upper layer at 1 hour after the addition of FeCl ₂ , was found to be relatively small at a high concentration but not significantly different from the concentration after 2 hours, The same flocculation and sedimentation rates were observed, but there was no significant difference in concentration. Therefore, it is considered that the lowest concentration of iron salt is 0.1 g / L in order to reduce the cost of harvesting after cultivation of Phaeodactylum tricornutum. In case of low iron salt addition, So that a more efficient and economical process can be established.

On the other hand, in order to investigate the coagulation and sedimentation rate at a concentration of 0.1 g / L or less, the dilution was continuously diluted from 0.1 g / L to 0.01 g / L and observed under the same conditions. As a result, the initial sedimentation rate and the time-dependent sedimentation rate until 1 hour were lower than 0.1 g / L, and the final concentration of the iron salt as the flocculant was 0.1 g / L (see FIG. 8).

3-4. Optimization of immersion concentration for mass culture of microalgae

Phaeodactylum tricornutum cultivated from the parental species in 10 L and 20 L of seawater added with 0.5% (v / v) Conway medium to determine optimal and maximum culture capacity under the current limited environment, Were inoculated at a ratio of 1: 500 based on the volume of the culture medium. The aggregation rate of microalgae was measured spectrophotometrically at absorbance of 675 nm and the supernatant was collected 3 cm below the surface of microalgae culture before flocculation to confirm the absorbance. After the addition of the coagulant solution to the microalgae culture solution at a concentration of 0.1 g / L, the precipitate was sedimented, and then the supernatant was collected 3 cm below the water surface Respectively.

The agglomeration rate was found to be 46% at 20 L and 70% at 1 hour after 10 L treatment, and 80% or more at 10 L after 2 hours. Whereas at 20 L, it was 50%. On the other hand, in the 10 L culture group, there was almost no change in the increase rate of cohesion after 4 hours, whereas in the 20 L culture group, the coagulation rate was lower than the coagulation rate of 2 hours in the 10 L culture group And 10).

As a result of comparing the growth levels of 10 L and 20 L, not only the relative growth was observed at a capacity of 10 L but also the coagulation efficiency was also high. Thus, it is considered that the culture of 10 L in terms of time and space is appropriate . It was also confirmed that it was optimal to set the incubation period to 7 to 10 days and the settling time after the coagulant treatment to 4 hours.

< Example  4> Originated microalgae Fucozanthin  detach

4-1. Extraction and Separation Process

Phaeodactylum tricornutum was grown from 675 nm to an absorbance of 0.06 to 0.07 using a mass production method of Phaeodactylum tricornutum identified in Examples 2 and 3, followed by chemical agglutination And 0.1 g / L of FeCl ₂ was added to precipitate the precipitate. The supernatant was discarded and the precipitate was transferred to a 50-mL conical tube. After removing the supernatant by centrifugation at 4000 rpm for 15 min, ethyl acetate (ethyl acetate) was added to the microalgae in which water was completely removed by freeing the water as much as possible.

1 g of lyophilized phaeodactylum tricornutum was weighed and added to 1 L of ethyl acetate, and the mixture was sonicated three times for 5 minutes per 30% AMPL intensity After that, the content of fucoxanthin was determined by ethyl acetate extraction time, and the mixture was stirred for 24 hours and 48 hours with a stirrer and extracted at room temperature. After 24 hours and 48 hours of extraction, the solution was completely concentrated using a vacuum concentrator.

A concentrated solution of Phaeodactylum tricornutum extract was dissolved in the same solvent as the silicagel filler to a volume of less than 5 mL to prepare a loading sample. A silica gel layer of about 15 cm was filled to perform silica gel column chromatography and then slurry was applied to a solvent system having a ratio of hexane to ethyl acetate (EA) of 1: The column was filled and loaded, and the orange and red pigments, typical of fucoxanthin, were separated and purified for each fraction eluted through a flash column (see FIG. 11).

The purity of the fucoxanthin complex isolated from Phaeodactylum tricornutum was analyzed by TLC. TLC eluting solvents were hexane (Hexane) and ethyl acetate (ethyl acetate) at a ratio of 1: 1. The separated fucoxanthin was spotted on a TLC plate at 10 μL. Next, the TLC plate with the sample was poured, the developing solvent was poured, the lid was closed, the sample was gently inserted into a TLC container in which the inside was saturated with the developing solvent, and developed for about 5 minutes. After the development, the TLC plate was dried using a drier and analyzed.

Phaeodactylum tricornutum was extracted with ethyl acetate and the fucoxanthin was separated with a silica gel column. Fucoxanthin was analyzed by TLC. As a result, the Rf value was found to be 0.33 to 0.35, and both the 24 hr and 48 hr extracts were observed at the same position (see FIG. 12). These samples were compared with fucoxanthin standard material by high performance liquid chromatography (HPLC) analysis.

For the Acetone extraction process, 15 mg was isolated from 1.5 g of lyophilized Phaeodactylum tricornutum, which was added to 1% of dry weight of lyophilized Phaeodactylum tricornutum And confirmed by the corresponding yield. On the other hand, 20 mg of fucoxanthin isolated from lyophilized 1 g of Phaeodactylum tricornutum was obtained at 24 hours after extraction with ethyl acetate, yielding an average yield of 2% Respectively. Also, it was confirmed that there was no significant difference between the results obtained by extracting for 48 hours and the results obtained by extracting for 24 hours, and it was confirmed to be most efficient to extract for 24 hours using ethyl acetate (see FIG. 13) .

4-2. HPLC -MS Fucoxanthin  Qualitative and quantitative analysis

The fucoxanthin obtained from the silica gel column was sampled at a concentration of 5 mg / mL in ethanol and centrifuged at 13000 rpm for 1 min. The supernatant was filtered using a 0.22 μm syringe filter And analyzed by HPLC-MS.

Fucoxanthin complex was analyzed by LC-MS (HP 1100 series, Agilent, CA, USA) to determine the content and content of fucoxanthin complex. The analytical conditions were diluted with ethanol and water (A) and acetonitrile (HPLC grade, Thermo Fisher Scientific, Waltham, MA, USA) / min and the absorbance was analyzed at 450 nm. The column used for the analysis was a Zorbax SB-Aq C18 column (4.615 mm, 5 μm) set at a column temperature of 30.0 ° C, and 10 μL of the sample was injected. The mobile phase conditions are as follows (see Table 1).

 LC-MS analysis conditions for fucosanthin analysis Parameters Conditions
LC / MS model HP 1100 series, Agilent,
Column Zorbax SB-Aq C18 column (4.615 mm, 5 [mu] m) Column Temperature 30 ℃ Flow rate 1 mL / min Injection 10 μL A water (in 0.02% TFA) B Acetonitrile (in 0.02% TFA) Time (min) A (%) B (%) Gradient step
0 0 100 60 100 0 Detector Agilent 1100/1200 Diode Array Detector (450nm)

In order to confirm the UV spectra absorbance of fucoxanthin, fucosanthin was measured by wavelength band. As a result, fucosanthin was found to exhibit the largest absorption at 450 nm when measured in a wavelength range of 250 to 600 nm. Based on this, in this study, the wavelength for detection of fucosanthin contained in standard substances and extracted samples was set to 450 nm through LC-MS (see FIG. 14).

As a result of the analysis, a peak at a high wavelength was measured at 58 min (see FIG. 15A), and a mass spectrometry analysis confirmed that the molecular weight expected to be fucozanthine was 58.762 min (see FIG. It was confirmed that the content and purity of fucoxanthin were relatively high in the fucoxanthin complex obtained by the improved extraction method as well as showing a single peak shape and confirming the same molecular weight as the molecular weight of fucozanthin.

Based on the analytical results, it was confirmed that the area value of the peak corresponding to fucoxanthin was 22632.3 mAU * S in terms of the total area value of 24351.3 mAU * S, and the purity was 92.24%.

4-3. Verification of contents using standard materials

The standard substance for calibration curve was prepared by dissolving fucosanthin standard substance (Sigma-aldrich 16337) in ethanol to make 1 mg / mL as fucosanthin and diluting to final concentrations of 250, 125, 62.5 and 31.25 μg / mL .

3 mg of fucosanthin isolated from Phaeodactylum tricornutum was completely dissolved in ethanol and filtered with a 0.22 μm syringe filter. The prepared standard sample and test solution were sealed with foil to block light. And stored at 4 ° C in a refrigerator.

The linearity of the fucosanthin standard was evaluated in order to confirm whether a linear measurement of the amount of fucozanthin in a certain concentration range of the sample can be obtained. The standard calibration curve showed a linearity of correlation coefficient (R2) of 0.9989 at a concentration of 31.25 - 250 μg / mL (see FIG. 17). The calculation formula of the fucosanthin calibration curve obtained from the standard sample was y = 55.8740x + 520.0583 [y = fucoxanthin / the ratio of the peak area of the internal standard material, x = concentration of fucoxanthin (μg / mL)]. The concentration of fucosanthin isolated from Phaeodactylum tricornutum was determined. For the analysis, the analysis conditions were adjusted as shown in Table 2 below.

 LC-MS analysis conditions for fucosanthin analysis Parameters Conditions LC-MS model HP 1100 series, Agilent, Column Zorbax SB-Aq C18 column (4.615 mm, 5 [mu] m) Column Temperature 30 ℃ Flow rate 1 mL / min Injection 10 μL Mobile Phaeodactylum tricornutumse A water (in 0.02% TFA) B Acetonitrile (in 0.02% TFA) Time (min) A (%) B (%) Gradient step 0 100 0 3 70 30 8 40 60 12 10 90 15 0 100 18 20 80 22 100 0 Detector Agilent 1100/1200 Diode Array Detector (450nm)

As a result of the standard analysis, peaks were observed in the range of retention time 17.59 ~ 18.4 min. In the case of fucoxathin complex derived from Phaeodactylum tricornutum, peaks were formed in the range of 17.87 ~ 17.98 min The fucoxathin and fucoxathin - like pigments were the most abundant in the extracts.

The peak height was 2539.7876 mAU and the area was 22632.3 mAU * S when LC-MS was used with 3 mg / mL of fucoxathin complex extracted from Phaeodactylum tricornutum. 18). Substituting this into the formula calculated from the calibration curve of the reference material yields a value of 395.7519, indicating that 395.7519 μg / mL fucoxanthin is contained within 3 mg / mL of fucoxanthin.

Thus, when 1 g of Phaeodactylum tricornutum was extracted, an average of 20 mg of fucoxanthin complex was isolated. Of these, fucosanthin was found to be 2638.35 μg, having a yield of 2.638 mg / g, The standard substance of the fucoxanthin complex obtained according to the above process was identified as fucoxanthin.

According to previous studies on fucoxanthin content, fucoxanthin content of seaweed was 87.6 mg / 100 g in seaweed, 62.4 mg / 100 g in seaweed, and 127.7 mg / 100 g in seaweed (SJ Kim et. al., 2004), and the content of fucoxanthin extracted from Sargassum filipendula was known to be 0.1957 ± 0.0173 mg / g (Kartini Zailanie and Sukoso, et al., 2014) .

4-4. Identification of secondary metabolites in the fucoxanthin complex

After performing the silica gel column fraction, the kind of the low-molecular secondary metabolites contained in the fucoxanthin complex was confirmed, and GC-MS analysis was performed to provisionally evaluate the substance that would cause synergy in the future Respectively. GC-MS (Agilent Technologies 5975C) equipped with a CTC CombiPAL autosampler system (Palo, Alto, CA, USA) was used for GC-MS analysis. The column used for the sample analysis was fucoxanthin extracted from Phaeodactylum tricornutum in ethanol with HP-5 column (Agilent Technologies, 250 μm × 0.25 μm × 30 m) And 5 μL of injection at 10 mg / mL was analyzed. The split ratio was 2: 1 and the injector temperature was 250 ° C. After the GC oven was stopped for 5 minutes at 50 ° C, it was measured to 310 ° C at 10 ° C intervals and stopped for 5 minutes. Data were collected and analyzed after 4 minutes in consideration of the solvent peak, and Wiley7N library data was used for component analysis (see FIG. 19).

GC-MS analysis showed that 4-Dehydroxy-N- (4,5-methylenedioxy-2-nitrobenzylidene) was the most abundant substance in fucoxanthin extracted from Phaeodactylum tricornutum. tyramine, and p-cumylphenol, as well as BENZENE, 1,4-BIS (TRIMETHYLSILYL) and Diisooctyl phthalate (Table 3).

Representative low molecular weight secondary metabolites contained in fucosanthin complexes Peak RT (min) Library Area% Qual% One 32.0798 4-Dehydroxy-N- (4,5-methylenedioxy-2-nitrobenzylidene) tyramine 16.789 52 2 22 p-Cumylphenol 13.7589 72 3 31.095 BENZENE, 1,4-BIS (TRIMETHYLSILYL) - 7.9733 46 4 27.9217 Diisooctyl phthalate 4.1052 91 5 30.2003 Cyclononasiloxane, octadecamethyl- 4.095 43 6 27.3296 2-, 2-Dibenzylethanol 3.7917 25 7 13.1496 Benzene, 1,2,4,5-tetramethyl- 2.4987 97 8 28.707 4H-Dibenz [de, g] isoquinoline, 5,6,6a, 7-tetrahydro-1,2,9,10-tetramethoxy-5- methyl- (CAS) 1.7206 86 9 14.5078 2-Hydroxypyridine 1.6013 30 10 29.7884 Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl- 1.4382 80 11 25.714 (E) -2-Methyl-1- (2-methylphenyl) -1-phenyl-1-butene 1.2777 64 12 19.2001 s-Hydrindacene, 1,1,7,7-tetramethyl- 1.0044 58 13 22.4184 Bicyclo [4.3.0] nonan-1-ol, 7,9-bis (methylene) -2,2,6-trimethyl- 0.9808 86 14 23.5384 Benzene, (3-methyl-2-butenyl) - 0.9781 42 15 27.7544 Nitrazepam 0.5323 22

Example 5 Evaluation of antioxidant activity of fucosanthin derived from microalgae

5-1. DPPH scavenging assay

1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a chemically stabilized water-soluble free radical, a violet compound that exhibits characteristic light absorption at 517 nm. This radical is very stable in an organic solvent such as alcohol and when it comes into contact with a substance having an antioxidative activity, the radical (DPPH) is extinguished by giving electrons and the color changes from purple to yellow in the first solution, Is a representative antioxidant activity test method that can be easily observed. For the radicals, a methanol solution containing 0.3 mM of DPPH was prepared, mixed with 100 μg / mL of the test substance, reacted at room temperature for 10 minutes, and absorbance was measured at 517 nm.

Serial dilution of the fucoxanthin complex (100 μg) from the dose of 100 μg resulted in the DPPH reactive oxygen scavenging activity. No significant effect was observed in the ethanol extract of Phaeodactylum tricornutum ethanol, while the fucoxanthin complex In the fucoxanthin complex, the lowest effect was observed at a dose of 1.56 μg. When the concentration was more than 25 μg, the level of ascorbic acid was significantly different from that of the control (see FIG. 20).

5-2. Protein degradation assay

The protein degradation assay is a method to identify the effects of antioxidants that protect proteins and enzymes from damage by reactive oxygen species (ROS) using metal ion catalysis. A Target protein BSA (bovine serum albumin, Sigma-Aldrich, MO, USA) a was ^{used, Cu 2 + (100 μM)} , H2O2 (2.5 mM) by the addition of 10% after the mixing and reaction with the test substance SDS-polyacryamide gel electrophoresis to confirm degradation inhibition of BSA protein. As a positive control, ascorbic acid (50 μM), which has an excellent antioxidative effect, was used.

In the experimental group treated with the addition of hydrogen peroxide and Cu ^{2 +} alone, BSA was almost completely degraded, whereas the ethanol extract showed a protein protective ability equivalent to that of ascorbic acid at a dose of 25 μg or more. The fucoxanthin complex ) Was confirmed to have a high protective efficacy over 6.25 μg. These results demonstrate that the fucoxanthin complex has a higher inhibitory effect on the protein degradation by the active oxygen than the ethanol extract and that the relative concentration of the substances exhibiting high antioxidative activity in the fucoxanthin complex is high (See Fig. 21).

< Example  6> Origin of microalgae Fucoxanthin  Whitening efficacy evaluation

6-1. Non-cell Tyrosinase  Activity inhibition experiment

Disodium hydrogen phosPhaeodactylum tricornutumte 12-water and sodium dihydrogen phosPhaeodactylum tricornutumte dihydrate were dissolved in distilled water (DW) to prepare a 100 mM sodium phos- phaeodactylum tricornutum buffer (pH 6.5) Tyrosine (Sigma, St. Louis, Mo., USA) was dissolved in 100 mM sodium phosphate buffer to prepare 1.5 mM L-tyrosine solution. Mushroom tyrosinase (Sigma), which acts as a reactive enzyme, was dissolved in 100 mM sodium phos- phateodactylum tricornutum buffer to prepare 2,500 units / mL tyrosinase. The reaction mixture was mixed with 153 μL of a 100 mM sodium phos- phaeodactylum tricornutum buffer, 36 μL of a 1.5 mM L-tyrosine solution, 5 μL of a sample and 6 μL of tyrosinase, and reacted at 37 ° C. for 1 hour. , And ImageJ software (version 1.51 Java, USA).

When the fucoxanthin complex was treated with 3.12, 6.25, 12.5, 25, 50, 100 and 200 μg, respectively, the inhibitory activity of tyrosinase activity was measured by a microscope. As a result, 12.5 μg It is possible to confirm that the formation of melanin is remarkably inhibited from the above capacity (see Fig. 22A). The microscopic photographs showed statistical significance at a concentration of 25 μg or more and inhibition of tyrosinase activity. From 100 μg or more, inhibition of tyrosinase activity similar to positive control ascorbic acid (See Fig. 22B). Thus, the above results confirmed the whitening effect of the fucoxanthin complex through direct inhibition of tyrosinase activity.

6-2. Experiment to measure the inhibitory effect of melanin biosynthesis in cells

In order to measure the inhibition effect of melanin biosynthesis by using B16F10 cells, the cells were seeded in a 6-well plate at 1 × 10 ⁵ cells / well, treated with fucoxanthin complex at a concentration of 25, 50 μg / mL, For induction, IBMX (200 μM) was treated in each well except for the control group and cultured for 48 hours. After incubation, 300 μL of 1N NaOH was added and incubated for 1 hour at 60 ° C in an incubator to completely dissolve the melanin. Absorbance was measured at 490 nm. As a positive control, ascorbic acid (10 mM) was used.

The results showed that the inhibition rate of the fucoxanthin complex was significantly higher than that of the melanin accumulation in the experimental group induced by IBMX. Especially, the positive control group, ascorbic acid, And a similar level of melanin biosynthesis inhibitory activity was confirmed (see Fig. 23).

6-3. Protection effect of UV rays on skin cells

To examine the effect of UV-B on cell damage and cell protection, CCD986sk cells were seeded on a 4-well chamber slide and treated with 6.25, 12.5 μg / mL fucoxanthin complex for 6 hours. Gt; UV-B &lt; / RTI &gt; The nucleus morphology was observed under fluorescent microscope after fluorescent staining of the cell nuclei of each experimental group irradiated with UV-B.

As a result of observation, a clear nucleation condensation was observed in the UV-B treatment group, while the fucoxanthin complex retained the nucleus morphology similar to the control, thus proving the UV-blocking and cytoprotective effect of the fucosanthin complex (see FIG. 24 ).

Claims (6)

(a) inoculating Phaeodactylum tricornutum at a ratio of 1: 400 to 1: 600 with respect to the volume of the culture medium, and culturing at a temperature of 18 to 22 DEG C for 5 to 10 days at an illumination condition of 1500 to 2000 lux ;
(b) adding a coagulant to the cultured medium to precipitate for 2 to 6 hours; And
(c) recovering the precipitated faecal tilum tricornatum and obtaining a paedoatilium tricornatum concentrate through centrifugation;
In a method for mass production of paedoatilum tricornitum comprising:
Wherein the culture medium of step (a) is 10 to 20 L of seawater to which 0.5 to 0.7% (v / v) of conway is added and the flocculant of step (b) is FeCl ₂ of concentration 0.025 to 0.1 g / Mass production method. delete delete delete (a) inoculating Phaeodactylum tricornutum at a ratio of 1: 400 to 1: 600 with respect to the volume of the culture medium, and culturing at a temperature of 18 to 22 DEG C for 5 to 10 days at an illumination condition of 1500 to 2000 lux ;
(b) adding a coagulant to the cultured medium to precipitate for 2 to 6 hours;
(c) recovering the precipitated faecal tilum tricornatum and obtaining a paedoatilium tricornatum concentrate through centrifugation; And
(d) a method for separating fucoxanthin derived from paeodactylum tricornatum, which comprises adding ethylacetate to the concentrate, ultrasonication and then extracting for 20 to 48 hours,
Wherein the culture medium of step (a) is 10 to 20 L of seawater to which 0.5 to 0.7% (v / v) of conway is added and the flocculant of step (b) is FeCl ₂ of concentration 0.025 to 0.1 g / Derived fucosanthin. 6. The method of claim 5,
Wherein the ultrasonic treatment in step (d) is performed three or more times in 3 to 8 minutes.

Images