KR102539751B1 - Method of culturing Haematococcus pluvialis by pH control - Google Patents

Abstract

The present invention relates to a method for culturing Haematococcus fluvialis using pH control, comprising the step of culturing Haematococcus pluvialis in the acidic medium of the present invention. The fluvialis culture method can completely prevent the death of Haematococcus fluvialis by contaminants through a simple method of adjusting the culture pH, so the biomass of Haematococcus fluvialis cells and astaxane caused by microbial contamination It is possible to solve problems such as a decrease in tin production capacity, and to maintain an inactivated state of contamination by microorganisms during cell culture for a long time, so that biomass and astaxanthin can be produced more uniformly from Haematococcus fluvialis cells. In addition, astaxanthin produced in this way can be used in various industries such as medicine, food science, and cosmetic raw materials as a powerful antioxidant.

Description

Method of culturing Haematococcus pluvialis by pH control}

The present invention relates to a method for culturing Haematococcus fluvialis using pH control, and more particularly, when culturing Haematococcus fluvialis, by adjusting the pH of the medium to an acidic condition at the beginning or during the culture period, external It relates to a method for culturing Haematococcus fluvialis capable of improving the biomass and astaxanthin production ability of Haematococcus fluvialis cells by preventing contamination by microorganisms.

Astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-dione), a commonly reddish ketocarotenoid, has the same chemical properties as β-carotene. A type of carotenoid-based pigment with a structure, it is an antioxidant functional substance that eliminates harmful active oxygen. It has unique molecular structural characteristics that have one more hydroxyl group (-OH) and one more ketone group (=O) at both ends compared to beta-carotene. Therefore, it has a much higher antioxidant activity than conventional antioxidants. Astaxanthin has 500 times higher antioxidant activity than vitamin E, a representative antioxidant, and 20 times higher than beta-carotene. Due to this high antioxidant activity, astaxanthin is widely used as pharmaceuticals, food additives, and feed additives for animals and fry, and its demand and application range are expected to rapidly expand.

Such astaxanthin is produced from yeast strains Phaffia rthodozyma and Bervibacterium, and is also widely distributed in marine and freshwater animals, but astaxanthin extracted from crustaceans such as shrimp and crayfish Tin is not applied due to its low content and difficult extraction process, and the Papia rodjima strain has a high growth rate but a low yield of astaxanthin.

Therefore, using Haematococcus pluvialis, which is the best microalgae in terms of accumulation content and yield of astaxanthin on earth, various methods are used to fix carbon dioxide by light and simultaneously increase the productivity of astaxanthin. Research is being conducted.

However, in order to apply the biological system using microalgae to the capture of carbon dioxide in the flue gas generated in the actual industry, a more economical microalgae mass culture system is required in reality, but Hematococcus fluvialis caused by pollutants such as external microorganisms The decrease in biomass and astaxanthin productivity has a problem of reducing economic feasibility, and this phenomenon worsens as mass culture for scale-up progresses, resulting in financial damage.

As mentioned above, the biggest challenge in mass cultivation of Haematococcus fluvialis is contamination by bacteria, fungi, and other microalgal species (Scenedesmus sp. or Chlorella sp.) (Pandey 2013, Gutman et al 2009, Shah 2016), especially Paraphysoderma known as a type of fungus. sedebokerenses is considered the most serious because it infects hematococcal cells very rapidly and kills all cells within 3-4 days (Gutman 2009).

Regarding, U.S. Patent Application No. 14/351,540 proposes a method of reducing the contamination rate by microorganisms using fungicide and a method of using a continuous device that lowers the culture temperature to a low temperature (10-22 ℃), but the effect is not perfect and the cost is high. There is, and the U.S. Patent No. 9,113,607 and U.S. Patent No. 9,392,793 discloses a method of reducing contamination by microorganisms by injecting hydrogen peroxide and salt, but since hydrogen peroxide evaporates quickly (approximately 2 hours), additional infection is monitored and the method is used. There is the hassle of having to do it repeatedly.

In addition, since hematococcus cells have a very slow growth rate compared to other microalgae, they are vulnerable to contaminants, and since all of the above methods are methods to prevent additional contamination after the contaminant is discovered, Paraphysoderma There is a problem in that it cannot be a fundamental solution in the case of infection by a fungus that infects hematococcal cells very rapidly, such as sedebokerenses .

The problem to be solved by the present invention is to obtain a biological solution from Haematococcus fluvialis cells due to contamination by external microorganisms caused by difficulties in pure culture due to the nature of the process during long-term operation of mass culture of Haematococcus fluvialis. It solves problems such as reduction in mass and astaxanthin productivity and consequent reduction in carbon dioxide conversion efficiency, and prevents infection from contaminants even in an environment where contaminants already exist as well as the possibility of inflow of contaminants during the cultivation period. It is to provide a new method for culturing Haematococcus fluvialis.

The present invention, in order to solve the above problems,

Provided is a method for culturing Haematococcus fluvialis, which includes culturing Haematococcus pluvialis in an acidic medium.

According to the present invention, the acidic condition may be pH 5.5 or less. In addition, the acidic conditions may be pH 4 to 4.5 at the initial stage of culture and maintained at pH 5.5 or less during the culture period.

According to the present invention, the acidic condition is adjusted by adding an acid to the medium, which acid is sulfuric acid, hydrochloric acid, nitric acid, acetic acid, citric acid, succinic acid, phosphoric acid, malic acid, butyric acid, propionic acid, oxalic acid, gluconic acid, tartaric acid, phthalic acid, carbonic acid And it may be at least one selected from the group consisting of ascorbic acid.

According to the present invention, the medium may include MES buffer.

The present invention also provides a method for producing astaxanthin using the Hematococcus fluvialis culture method.

The Haematococcus fluvialis culture method comprising the step of culturing Haematococcus pluvialis in an acidic medium of the present invention is a simple method of adjusting the culture pH to reduce hematosis caused by contaminating bacteria. Since the death of Coccus fluvialis can be completely prevented, problems such as reduction in biomass and astaxanthin production capacity of Haematococcus fluvialis cells due to microbial contamination can be solved, and contamination by microorganisms during cell culture Since it can maintain an inactive state for a long period of time, not only can biomass and astaxanthin be produced more uniformly from Haematococcus fluvialis cells, but also astaxanthin produced in this way is a powerful antioxidant and is used as a raw material for medicine, food science, and cosmetics. It can be used in various industries such as

Figure 1 shows the results of measuring the infection rate according to the initial pH of the culture medium when Paraphysoderma sedebokerenses fungal contaminants were inoculated into Haematococcus fluvialis culture medium and cultured.
Figure 2 is a graph showing infection rates on days 0, 6, 8, and 10 of culture according to the initial pH of the culture medium when Paraphysoderma sedebokerenses fungal contaminants are inoculated into Haematococcus fluvialis culture medium and cultured, and the color change of the culture medium This is the picture shown
Figure 3 is a Haematococcus fluvialis culture medium inoculated with Paraphysoderma sedebokerenses fungal contaminants and cultured when the initial pH of the culture medium is 4 and the pH is 7.5 (control) Hematococcus plate on day 10 of culture This is a microscope image of rubialis.
Figure 4 is a graph showing the results of comparing the growth properties when the initial pH of the culture medium is 3, 4, 7.5 when cultured in Haematococcus fluvialis culture medium.

Hereinafter, the present invention will be described in more detail.

In the present invention, biomass and astaxanthin from Haematococcus fluvialis cells due to contamination by external microorganisms caused by difficulties in pure culture due to the nature of the process during long-term operation of mass culture of Haematococcus fluvialis New Haematococcus that can solve problems such as reduced productivity and consequent reduction in carbon dioxide conversion efficiency, and prevent infection from contaminants even in an environment where contaminants already exist as well as the possibility of contaminants entering during the cultivation period It is intended to provide a method for culturing fluvialis.

To this end, the present invention provides a method for culturing Haematococcus fluvialis, which includes culturing Haematococcus pluvialis in an acidic medium.

At this time, the acidic condition is preferably maintained at pH 5.5 or less throughout the entire process of culture. In particular, as can be seen from the results of the following examples, in order to completely block contamination by microorganisms and grow cells without significant loss, culture Initially, it is most preferable to maintain pH 4 to 4.5 at the beginning of culture and pH 5.5 or less during the culture period.

The medium used for the culture of the present invention can be any medium as long as it can grow Haematococcus fluvialis and produce astaxanthin under predetermined conditions, preferably containing nitrogen sources, inorganic salts and, if necessary, vitamins. It may be a medium containing

The inorganic nitrogen source may be at least one selected from the group consisting of ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates such as potassium nitrate, ammonia, and urea. The amount added varies depending on the type of nitrogen source, and may be typically 0.1-20 g, preferably 0.2-10 g, per 1 liter of the medium.

As the organic nitrogen source, for example, 1 selected from the group consisting of cornstarch liquor (including filtered products), pharma media, soybean meal, soybean powder, peanut meal, distillers soluble, dried yeast, and monosodium glutamate There may be more than one species. The added concentration is different depending on the type of nitrogen source and may be usually 0-80 g/L, preferably 0-30 g/L.

The inorganic nitrogen source and the organic nitrogen source may be added to the medium before culturing, or additionally supplied sequentially or continuously during culturing.

Inorganic salts include, for example, phosphates, magnesium salts such as magnesium sulfate and magnesium chloride, iron salts such as iron sulfate and iron chloride, calcium salts such as calcium chloride and calcium carbonate, sodium salts such as sodium carbonate and sodium chloride, manganese sulfate Manganese salts such as cobalt chloride, cobalt salts such as cobalt chloride, copper salts such as copper sulfate, zinc salts such as zinc sulfate, molybdenum salts such as sodium molybdate, nickel salts such as nickel sulfate, selenium salts such as sodium selenate, boric acid and It may be one or more selected from the group consisting of potassium iodide. The amount added varies depending on the type of inorganic salt and may be 0.0001-15 g per 1 liter of medium. Inorganic salts may be added to the medium before culturing, or additionally supplied sequentially or continuously during culturing.

As the vitamins, for example, cyanocobalamin, riboflavin, pantothenic acid, pyridoxine, thiamine, ascorbic acid, folic acid, niacin, p-aminobenzoic acid, biotin, inositol, choline and the like can be used. The addition ratio is different depending on the type of vitamins and is usually 0.001-1000 mg, preferably 0.01-100 mg per 1 liter of the medium. Vitamins may be added to the medium before culture, or additionally supplied sequentially or continuously during culture.

The medium used in the present invention may further include an MES buffer to maintain the pH of acidic conditions, and citric acid buffer, sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, sodium carbonate aqueous solution, ammonia water, ammonia gas, sulfuric acid aqueous solution, or mixtures thereof It can also be used as a pH adjusting agent.

In addition, the acidic conditions are adjusted by adding acids to the medium, which acids include sulfuric acid, hydrochloric acid, nitric acid, acetic acid, citric acid, succinic acid, phosphoric acid, malic acid, butyric acid, propionic acid, oxalic acid, gluconic acid, tartaric acid, phthalic acid, carbonic acid and ascorbic acid. It may be one or more selected from the group consisting of.

In addition, the culture of Haematococcus fluvialis is carried out in an appropriate culture vessel. The culture vessel can be appropriately selected according to the culture capacity, and examples thereof include test tubes, flasks, fermenters, photobioreactors, and the like.

The incubation temperature may be 15 to 80 ° C, preferably 20-35 ° C, more preferably 23-30 ° C, and usually 1-90 days, preferably 5-60 days, more preferably 5-30 days It can be cultured daily.

The present invention also provides a method for producing astaxanthin using the Hematococcus fluvialis culture method.

Hereinafter, the present invention will be described in more detail through examples, but the following examples are only for helping understanding of the present invention, and do not limit the scope of the present invention.

ingredient

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

The types of media used in this experiment were both NIES-C medium and NIES-N medium, and their components are shown in Table 1 below.

In Table 1 above,

- NIES-C medium: autotrophic medium (autotrophic medium, purpose: growth)

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

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

- CO ₂ supply : 3~5%(v/v)

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

Example . Depending on the pH of the culture medium Paraphysoderma sedebokerenses Measurement of the infection rate by fungal contaminants and the pH of the culture medium hematococcal Measure impact on growth

Haematococcus in the logarithmic phase (exponenial phase) was OD in 30 ml of medium ranging from pH 3 to 9 (pH 3, 4, 4.5, 5, 6, 7, 7.5 (control), 8, 9) (680nm) = 0.5 concentration, and P. sedebokerenses fungal contaminants were inoculated by 20 μl each, and the infection rate (%) was confirmed while culturing. The inoculated P. sedebokerenses fungal contamination has been maintained by inoculating hematococcal cells at 7-day intervals, and the fungal contamination rate is 70 to 80 at a concentration of 2 × 10 ⁵ cell / ml based on hematococcal cells. Experiments were conducted using cells in the % range. The infection rate was calculated as infection rate (%) = number of infected cells/total number of cells x 100 using an optical microscope and a cell counter (hemocytometer).

In addition, the initial pH of the culture medium was set to 3, 4, or 7.5 without inoculation of the fungal contaminants, and the effect of the pH of the medium on the growth of Haematococcus fluvialis was measured.

1 is Paraphysoderma in Hematococcus fluvialis culture medium sedebokerenses Shows the results of measuring the infection rate according to the initial pH of the culture medium when inoculated with fungal contaminants and cultured. It is a photograph showing the color change, and Figure 3 is Paraphysoderma in Haematococcus fluvialis culture medium sedebokerenses These are microscopic images of Haematococcus fluvialis on the 10th day of culture when the initial pH of the culture medium is 4 and the pH is 7.5 (control) when inoculated with fungal contaminants.

Figure 4 is a graph showing the results of comparing the growth properties when the initial pH of the culture medium is 3, 4, 7.5 when cultured in Haematococcus fluvialis culture medium.

As a result of the experiment, as shown in FIG. 1, it was found that the contamination rate reached approximately 40-100% on the 18th day of culture except for the case of the initial pH 3, pH 4, and pH 4.5 of the medium. In addition, as shown in Figure 2, when the pH was in the neutral to weakly basic range of 7-8, the contamination rate was more than 80% in about 8 days, and on the 18th day of culture, the contamination of the cells in the pH range of 6-9 It was confirmed that all cells cultured at a rate of 100% were contaminated. These results can also be confirmed through the fact that the green cells on the 0th day of culture in the photograph of FIG. 2 are contaminated and turned brown. In addition, when the initial pH of the medium was 5, the contamination rate was about 50% on the 18th day of culture (Figs. 1 and 2), and it was confirmed that the contamination rate reached 100% within 30 days (not shown). ). On the other hand, when the initial pH of the medium was 3, 4, or 4.5, no contaminated cells were observed on the 18th day of culture (FIG. 1, 2), and it was confirmed that the contamination rate was maintained at 0% until the 30th day of culture. (drawing not shown). These results show that in the microscopic image of FIG. 3, all cells were contaminated by mold on the 10th day of culture in the medium with an initial pH of 7.5 and turned brown, whereas in the medium with an initial pH of 4, the cells remained clean. You can check.

In addition, as shown in Figure 4, when the initial pH of the culture medium is 3, it can be seen that the growth of the cells is very low, whereas at pH 4, the cells grow about 10-20% less than at pH 7.5, but a large loss You can see that it grows well without it.

In addition, it was confirmed that the pH of the culture medium increased through the culture process of Haematococcus fluvialis. Specifically, when the initial pH of the culture medium was 4, the pH increased to 5 on the 30th day of culture as the cells grew. rising, and when the initial pH was 5, it was confirmed that the pH rose beyond 5.5 on the 30th day of culture (not shown).

Considering these results comprehensively, in order to completely block contamination by fungi without significantly affecting the growth inhibition of Haematococcus fluvialis, the initial pH of the culture medium was set to 4 to 4.5, and the culture During the period, it was confirmed that it is preferable to maintain the pH of the culture medium at 5.5 or less.

Claims (6)

Including the step of culturing Haematococcus pluvialis in an acidic medium,
The acidic conditions are adjusted by adding acid to the medium;
The initial pH of the medium is 4 to 4.5, and the pH of the medium is maintained at 5.5 or less during the culturing process. delete delete According to claim 1,
The acid is at least one selected from the group consisting of sulfuric acid, hydrochloric acid, nitric acid, acetic acid, citric acid, succinic acid, phosphoric acid, malic acid, butyric acid, propionic acid, oxalic acid, gluconic acid, tartaric acid, phthalic acid, carbonic acid and ascorbic acid. A method for inhibiting the infection of external microorganisms against coccus fluvialis. According to claim 1,
The medium is a method for inhibiting the infection of external microorganisms for Haematococcus fluvialis, characterized in that it comprises an MES buffer. A method for producing astaxanthin using the method according to claim 1.

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