KR20160045168A - The production of high purity phycocyanin from cycanobacterium Spirullina platensis using LEDs based two-stage cultivation - Google Patents

Abstract

The development of a two-stage cultivation method for producing high-purity phycocyanin relates to a novel cultivation method using photosynthesis cycanobacterium Spirulina platensis which can increase the contents of phycocyanin inside a cell. The method is capable of controlling the strength and the wavelength required to improve the purity of phycocyanin, and produces high-purity phycocyanina by effectively providing light source conditions of a device for cultivating Spirulina, thereby being efficiently used for producing a photosynthetic pigment effective for antimicrobial activities, anti-inflammatory activities, anticancer activities, and the like.

Description

The present invention relates to a two-stage culture method for producing high purity phycocyanin,

The present invention relates to a method for producing high purity phycocyanin using microalgae.

Microalgae are single cell organisms that produce photosyntheses by themselves as producers of marine ecosystems. There are hundreds of thousands of microalgae, and they produce various physiologically active substances depending on the growth environment. Especially microalgae growing in extreme environment such as deep sea lava and lava zone, new functional materials not found in the land are found.

Therefore, microalgae can be considered as a report of various physiologically active substances. And microalgae are easy to mass-produce. Indeed, in the industrial sector, microalgae have been cultured in large quantities for food or aquaculture. In recent years, in order to utilize microalgae as biomass in the field of bio-energy, studies for increasing the production of microalgae are actively carried out, and the industrial value of microalgae will be further increased in the future.

One of Spirulina platen sheath (Spirullina platensis) of blue-green algae (cyanobacteria) is a photosynthetic microorganism having a filament in the form of multi-cellular. It has been used for a long time as a food rich in nutrients such as protein, carbohydrate, vitamins and minerals. Due to its high nutritional value, the United Nations Food and Agriculture Organization (UNFAO) has been designated as food for the future. Currently, there are many studies on spirulina culture worldwide.

Phycocyanin is a blue pigment found in cyanobacteria, rhodophytes, and cryptophytes. Especially, the fact that cyanobacteria have their own blue color contains a large amount of phycocyanin to be. Pycocyanin dissolves in water and exhibits very strong fluorescence and antioxidant properties. In fact, pycocyanin is a key ingredient in the use of cyanobacteria in food, cosmetics, and medicine. Therefore, research on picosan is also very active, and recently, the number of papers and patents in biotechnology has also increased.

Pycocyanin is the major picofile protein of Spirulina, accounting for 29% of dry weight. As a blue photosynthetic pigment, it absorbs light energy in the photosynthesis system and transfers it to chlorophyll a. It is used as a natural coloring agent and a fluorescent marker, and antibacterial, anti-inflammatory, antioxidant and anti-cancer properties are reported. Accordingly, various methods for efficiently producing phycocyanin from strains have been proposed. Pycocyanin determines purity by absorbance A680 / A620 ratio and is divided into food grade (> 0.7) and analytical grade (≥4.0). Since the primary liquid extracted from the cells has a low purity of less than 0.7, several additional purification steps are required depending on the purpose of use. Pycocyanin is priced according to its purity.

To obtain a picocyanin having a purity of 0.7 or more, ammonium sulfate precipitation, ultrafiltration and the like are used. Gel filtration chromatography and ion-exchange chromatography ) Or the like, a picocyanin having a purity of 4.0 or more can be obtained. Such an increase in purification steps leads directly to production costs, resulting in economic and temporal losses.

Refining at the Food grade level should proceed to a minimum level. Each step requires a very high cost in the industrial field. The synthesis of phycocyanin in cells is affected by the light intensity and the culture conditions such as wavelength, salinity, temperature, nitrogen source concentration, and thus purity and content are influenced by the extraction.

Conventional culture methods use solar light, fluorescent light, light emitting diode of single wavelength, etc. as a light source, which has a problem that it is difficult to easily control irradiation conditions of a light source during a culture period. The pigment content of photosynthetic microorganisms varies with the intensity and wavelength of light. The density of photosynthetic photons reaching the cells is negatively correlated with the content of the pigments. Depending on the wavelength range, the growth of the cells and the types of pigments synthesized have a considerable effect on the photosynthetic photon flux density. When extracting useful substances from spirulina dry powder, the recovery efficiency is lower than that of unripe viable cells. This can have a significant impact on the purity of the picosanine extracted from Spirulina. When the drying method is used, there is a problem that a loss of about 50% of the phycocyanin and 10-20% of the protein occurs.

Accordingly, the present inventors have made efforts to develop an effective production method for the pigment protein picocyanin. As a result, they have newly developed a photosynthetic cyanobacteria spirulina culture method, which is a cyanobacterium that can increase the content of picosian in the cell, The present invention has been accomplished by providing a high purity picocyanin production method capable of controlling the pilling and intensity required for improving purity and effectively showing the light source conditions of the spirulina culture apparatus.

SUMMARY OF THE INVENTION

1) culturing Spirullina ;

2) primary culture by irradiating red and blue mixed light;

3) irradiating primary cultured spirulina of step 2) with blue light to secondary culture; And

4) purifying phycocyanin from the second cultivated spirulina of step 3). ≪ IMAGE >

In addition, the object of the present invention is to provide a method for producing a high-purity phycocyanin, which further comprises the step of purifying by using affinity precipitation and ammonium sulfate precipitation.

Further, the object of the present invention is

1) culturing spirulina;

2) primary culture by irradiating red and blue mixed light; And

3) irradiating primary cultured spirulina of step 2) with blue light to secondary culture, and a method for cultivating spirulina for producing high purity phycocyanin.

In order to achieve the above object,

1) culturing Spirullina ;

2) primary culture by irradiating red and blue mixed light;

3) irradiating primary cultured spirulina of step 2) with blue light to secondary culture; And

4) purifying phycocyanin from the secondary cultured spirulina of step 3) above.

The present invention also provides a method for producing a high-purity phycocyanin characterized by further comprising the step of purification using affinity precipitation and ammonium sulfate precipitation.

Further,

1) culturing spirulina;

2) primary culture by irradiating red and blue mixed light; And

3) irradiating primary cultured spirulina of step 2) with blue light to secondary culture the spirulina.

2-step culture method for the production of high purity and not when the Pico of the present invention is developed photosynthetic cyanobacteria Spirulina platen sheath (cycanobacterium Spirullina Platensis ) can be used to increase the content of phycocyanin. It is possible to control the wavelength and intensity necessary for the improvement of picosyin purity and to produce a high purity phycocyanin by effectively presenting the light source conditions of the spirulina culture apparatus Antimicrobial, anti-inflammatory, anti-cancer, and the like.

FIG. 1 is a graph showing the relationship between the content of picosene, the purity, the content of alopycosa, and the content of crude extract in white light emitting diode light when the absorbance (680 nm) Ratio, and the content of whole picofile protein.
FIG. 2 is a graph showing the correlation between the absorbance and the dry weight of each sample taken at a point when the dry weight corresponds to 1 g · L ^-1 . The content of picocyanin in the crude extract obtained from the spirulina according to the wavelength of the light source , Purity, content of alopicocaine, percentage, content of whole picofile protein, maximal biomass production rate ( μ _max ), biomass and maximum biomass concentration ( X _max ).
FIG. 3 is a graph showing the content, purity and recovery rate of phycocyanin according to each purification step.
Fig. 4 is a diagram showing a spirulina culture apparatus. Fig.
FIG. 5 is a diagram showing the phycocyanin of the crude extract showing the content and purity change according to the wavelength of the light source.
FIG. 6 is a graph showing changes in picosene content and purity of biomass and crude extract using a two-step spirulina culture method in which the wavelength and intensity of light are controlled according to the culture step.

Hereinafter, the present invention will be described in detail.

The present invention

1) culturing Spirullina ;

2) primary culture by irradiating red and blue mixed light;

3) irradiating primary cultured spirulina of step 2) with blue light to secondary culture; And

4) purifying phycocyanin from the secondary cultured spirulina of step 3) above.

The spirulina of step 1) is characterized by being Spirullina platensis .

The irradiation of step 2) is preferably irradiated with a light intensity of 20-150 μmol˙m ^-2 s ^-1 , and the culturing is preferably performed for 3 -7 days, but is not limited thereto.

The irradiation of Step 3) is preferably performed at a light intensity of 50 to 350 μmol˙m ^-2 s ^-1 , and the culturing is preferably performed for 12 to 16 days, but is not limited thereto.

It is preferable that the irradiation of the step 2) and the step 3) is carried out by using a light emitting diode.

In addition, the picocyanin purified from the spirulina of step 4) is characterized by having a food grade purity.

The present invention may further comprise purification using affinity precipitation and ammonium sulfate precipitation.

The tablets are to separate the analytical grade of phycocyanin.

The present invention

1) culturing spirulina;

2) primary culture by irradiating red and blue mixed light; And

3) irradiating primary cultured spirulina of step 2) with blue light to secondary culture the spirulina.

Spirulina is cultivated at a light intensity ranging from 20 to 200 μm ˙ ² s ^-1 using white, red and blue light emitting diodes. However, after a certain period of time, the content and purity of the picosene are determined by the intensity and wavelength The results are as follows. As a result, it was found that the intensity of the light source and the content of the phycocyanin were inversely correlated with each other in a certain range. The rate of production of biomass was fast under the red wavelength (660 nm) Under low purity and blue wavelength (450 nm), the production rate of biomass was slow, and the content and purity of phycocyanin were high. Since the amount and purity of the pigment protein that can be extracted depend on the light source condition, the pycocyanin content and purity of the extracted crude extract are classified into food grade, To the maximum. In the case of red, blue, and mixed light, the growth rate was moderate in the experimental group using red and blue, and the content of phycocyanin during the incubation period was similar to that of red single light. Then, the mixture was incubated to the middle of the growing stage using the mixed light, and the wavelength of the light source was changed with blue single light favorable for securing the content of picosylan. The culture vessel may be a flask or a rectangular reactor having a thickness of about 5 cm. In order to uniformly irradiate the light, the height of the culture medium should not exceed 5 cm in the irradiation direction of the light source, do.

In a specific example of the present invention, the difference in the content of picocyanin according to the intensity of light was investigated by incubating Spirullina platensis . As a result, it was found that in the set range of 25 and 75 μmolm ^-2 s ^-1 The content and purity of phycocyanin were high (see Fig. 1). As a result of measuring the content of phycocyanin according to the wavelength of light, it was confirmed that the blue light was advantageous for the synthesis of phycocyanin and had a high purity value of 2.78 (see FIG. 2). In order to obtain the maximum purity of ude extract by maximizing the phycocyanin synthesis, the first step was the red blue light and the second step was the blue light alone. As a result, the biomass (See Figs. 3, 5 and 6).

Accordingly, high-purity 2-step culture method Pico development for the production and not during the present invention is the photosynthetic cyanobacteria Spirulina (cycanobacterium Spirullina platensis ) and can control the wavelength and intensity required for the improvement of picosynthetic purity. It is also useful for the production of photosynthetic pigments effective for antibacterial, antiinflammatory and anticancer activities by effectively producing the light source conditions of the spirulina culture apparatus and producing high purity picocyanin Lt; / RTI >

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> Spirulina  Culture conditions and methods for measuring picocyanin content

Spirullina platensis AP- 20590 , purchased from Korea Biotechnology Institute, was inoculated in 200 ml of SOT medium at 2 g / ℓ and incubated at 30 ℃ ± 1, pH 9.0, 50 μmol˙m ^-2 s ^-1 [660 nm, 450 nm] for 24 hours under light condition. The periphery of the incubator was closed to minimize the inflow of unnecessary light other than the used light source. The growth of the strain was measured using an absorbance of 680 nm wavelength using a spectrometer. A standard curve was established using a correlation between the previously measured absorbance and the dry cell mass, and the correlation equation was shown in the following equation (1).

The content of picocyanin was determined by measuring the maximum absorbance at 620 nm and 652 nm using a spectrophotometer. Generally, the content of phycocyanin can be measured using the following formula (2) by the method proposed by Bennett and Bogorad. Absorbance 620 nm is the maximum absorbance of the phycocyanin and 280 nm is the absorbance of the whole protein of the extract.

< Example  2> Spirulina Platensis  Growth and phycocyanin (PC) content evaluation

<2-1> Measurement of phycocyanin content by light dose

The following experiment was conducted to investigate the difference in the content of phycocyanin according to the intensity of light.

Specifically, 25, 75, 125, and 200 μmolm ^-2 s ^-1 were incubated under four different light doses with the same conditions except for light intensity. At this time, when the absorbance was measured at 680 nm at 1.0, samples were collected and the content of phycocyanin was measured.

As a result, as shown in FIG. 1, the growth rate was highest when the intensity was the strongest within the intensity range of light which was set so as not to cause light inhibition, but the final content and purity of the phycocyanin were the lowest. The content and purity of the phycocyanin were high at the set range of 25, 75 μmolm ^-2 s ^-1 (FIG. 1).

<2-2> The wavelength of light light Wavelength ) To measure the content of phycocyanin

 The following experiment was carried out to investigate the difference of the growth rate and the content of the phycocyanin according to the wavelength of light after the intensity of light.

Specifically, a light emitting diode of red light (Red, 660 nm), blue light (Blue, 450 nm) and red (red and blue mixed light, RB) was used as a light source. At this time, the light intensity is 75 μmol˙m ^-2 s ^- was fixed to ¹ in all conditions other than the wavelength of the Example <2-1> The method was the same.

As a result, as shown in Fig. 2, when the dry weight was 1.0 g / l, the difference according to wavelength was sampled and the difference in the content of phycocyanin and the growth rate of the cells was measured. Under the red light, the growth rate was the fastest, the content and purity of the phycocyanin were the lowest, the growth rate was the slowest under blue light, and the content and purity of phycocyanin were the highest. Blue light was more advantageous for the synthesis of phycocyanin among the photosynthetic pigments at a ratio of alopicocaine. And a value of 2.78, which is a relatively high purity in the case of blue light, was confirmed (Fig. 2). Therefore, light intensity and wavelength for obtaining a crude extract having a high picocyanin content and purity can be selected through the above experimental results.

< Example  3> Evaluation of pycocyanin content and purity elevation using 2-step culture method

In order to obtain the maximum purity of ude extract by maximizing the picocyanin synthesis after confirming the growth rate and the content of phycocyanin with respect to light intensity and wavelength obtained from the above Example 2, Experiments were performed.

In particular, when the red and blue colors are used together, the growth rate is faster and the picocyanin purity is higher than that when the red and blue colors are used together. As a first step, the first step is to cultivate for 5 days using red-blue mixed light to increase the biomass to the midpoint (OD ₆₈₀ = 1.6) until the middle of the growing season, The content and purity of phycocyanin were greatly increased. The red-blue mixed light has a light intensity of 100 μmolm ^-2 s ^-1 (FIG. 4).

As a result, as shown in FIG. 3, FIG. 5 and FIG. 6, when the red blend light was used up to the midpoint of the first stage (OD ₆₈₀ = 1.6), the purity of the crude extract was 2.0 . In the second stage using blue light, crude extracts of up to 2.7 purity were obtained on the 14th day of culture. The total incubation period was reduced by about 7 days compared with the experiment in which the same wavelength of blue light with a purity of 2.7 was recorded. In the second culture stage, the increase of biomass was faster in the order of blue light 75, 150, and 300 μm ˙ ^-2 s ^-1 , and the maximum biomass amount was similar regardless of culture medium. In the case of 150 μmol˙m ^-2 s ^{- 1} , purity of more than 2.5 was continuously recorded from 10 days after culture (FIGS. 3, 5 and 6).

< Example  4> Pycocyanin tablets

In order to extract the water-soluble pigment picocyanin, the following experiment was conducted.

Specifically, cells were lysed using 40 mM CaCl ₂ solvent (pH 4). First, 5 ml of the culture solution was centrifuged to remove the supernatant except for the pellet. After washing twice with CaCl ₂ solvent, 40 mM CaCl ₂ After 1.25 ml of the solvent was added and the mixture was stored at 4 ° C for 12 hours, the mixture was centrifuged again to separate only the deep blue supernatant dissolved in the phycocyanin. Only the dark blue supernatant, in which the phycocyanin was dissolved, was isolated. The ratio of biomass to solvent was kept at 4: 1 so that the content of picosylane could be accurately compared. The cell debris of the crude extract obtained from Spirulina platensis was removed through glass fiber filter paper and subjected to two-step purification. First, the crude extract from which debris was removed was mixed with chitosan, and the column containing activated charcoal was passed for 5 minutes. Then, ammonium sulfate fractionation was performed by adjusting the saturation concentrations of ammonium sulfate to 30% and 60%, respectively. The phycocyanin concentrated in the form of pellet was dissolved in 20 mM potassium phosphate buffer (pH 6.9) and dialyzed for desalting. After dialysis, 20 mM potassium phosphate buffer containing 5 mM sodium azide was used as a storage buffer.

As a result, as shown in Fig. 3 CaCl ₂ The purity of analytical grade, purity 4.0 or higher, was purified through crude extract extraction from fresh biomass to a picosian purity of 2.5, and the purity after passing through the column was increased to 3.1. After the ammonium sulfate fractionation process, a final picocyanin of 4.2 was obtained and the final recovery rate was 65% relative to the crude extract (FIG. 3).

Claims (11)

1) culturing Spirullina ;
2) primary culture by irradiating red and blue mixed light;
3) irradiating primary cultured spirulina of step 2) with blue light to secondary culture; And
4) purifying phycocyanin from the secondary cultured spirulina of step 3).
The method of claim 1, wherein the spirulina of step 1) is Spirullina platensis .
2. The method of claim 1, wherein the irradiation of step 2) is performed at a light intensity of 20 - 150 μmolm ^-2 s ^-1 .
2. The method of claim 1, wherein the irradiation of step 2) and step 3) is carried out using a light emitting diode.
The method according to claim 1, wherein the culture of step 2) is cultivated for 3 to 7 days.
The method according to claim 1, wherein the step 3) is irradiated with a light intensity of 50 - 350 μmolm ^-2 s ^-1 .
The method according to claim 1, wherein the culture of step 3) is cultured for 12 to 16 days.
The method of claim 1, wherein the purified picosian from the spirulina of step 4) has a food grade purity.
2. The method of claim 1, further comprising the step of purifying using affinity precipitation and ammonium sulfate precipitation.
10. The method of claim 9, wherein the tablet is for separating an analytical grade of phycocyanin.
1) culturing spirulina;
2) primary culture by irradiating red and blue mixed light; And
3) a step of secondary culturing by irradiating the primary cultured spirulina of step 2) with blue light, and culturing the spirulina for high purity phycocyanin.

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