KR20130121560A - Novel chlorella vulgaris and culturing method thereof - Google Patents

Abstract

The present invention relates to a novel microalgal strain which enables superior heterotrophic cultures, Chlorella vulgaris KCTC 12163BP. More particularly, the novel microalgal strain has superior heterotrophic culture ability and high fat content and enables high density culture, easy harvesting, and a minute manipulation of a culture environment. Accordingly, a method for culturing a large amount of Chlorella vulgaris KCTC 12163BP enables mass production of functional products using chlorella with various physical activities and draws high economic values.

Description

Novel Chlorella vulgaris strain and its culturing method

The present invention relates to a novel Chlorella vulgaris strain (KCTC 12163BP) capable of nutrient culture and its culture method.

Chlorella is a spherical or oval green algae plant with a diameter of less than 10 micrometers in diameter of single cells, naturally growing in fresh and sea water. These chlorella contain functional materials such as chlorophyl, CGF (Chlorella Growth Factor) and beta glucan, as well as high-quality proteins, carbohydrates, various vitamins and minerals, and have recently been spotlighted as health foods. have. In addition, the rotifer (rotifer), which is used as a feed for the fry, is used as a food organism, and there is a lot of demand.

To isolate new chlorella species from natural seawater and freshwater, pure strains are obtained by diluting the water in which chlorella grows, diluting it to a certain concentration, incubating it, and incubating it again in agar medium to isolate single colony. I'm using a method of obtaining it. In addition, if you look at the existing chlorella mass cultivation method, first cultivated chlorella seedlings and inoculated the seedlings in the culture medium at indoor or outdoor breeding ground at a concentration of about 3 × 10⁴ cells / ml, maintaining the equator temperature 25 ℃, and supplying oxygen In order to incubate slowly for about 7-10 days to grow to a concentration of 30 million cells / ml, it is common to use this product through the processing process.

Unlike general bacteria, chlorella has a long incubation period and low cell concentration, which is costly to commercialize. In order to solve this problem, various methods of culturing chlorella at high concentration have been attempted by adjusting the culture conditions. In other words, incubation at a high growth rate at the initial high temperature, gradually cooling the temperature and adding pure oxygen at low temperature to cultivate relatively high yield of chlorella (Korean Patent 2001-0019217) and culturing high concentration chlorella under cancer culture conditions. There is a method (Korean Patent Publication No. 1997-0043017), but they still have the disadvantage of slow growth.

On the other hand, as a method of culturing by changing the composition of chlorella culture medium, using pure organic materials such as persimmon leaf, seaweed extract and aquatic product extract to commercialize chlorella without separation process when using a chemical medium (Korea Patent Publication 1998-025344) and It is known to add natural minerals such as ginseng extract and elvan to the culture to increase the functionality (Korean Patent Publication 1984-0000010).

Currently, 3,000 tonnes of chlorella are cultured annually worldwide. Chlorella is an antioxidant and vegetable nutrient that is nutrients such as vegetable protein, essential amino acid, essential fatty acid, glycoprotein, vitamins, minerals, dietary fiber, chlorophyll, nucleic acid (DNA, RHA), beta carotenoid, flavonoid, and leucine. (Phytochemical, phytosterol), sterols, gamma linolenic acid and other nutrients, including a wide range of uses for health foods, food additives, feed, pharmaceutical ingredients, etc., the demand is increasing every year.

In recent years, global competition is being made to secure new biological resources, which requires the development of technologies for the isolation and production of new chlorella species. With the conventional techniques mentioned above, it is not possible to remove many microorganisms contained in the sample collected at the time of separation of the new species, and the pure separation and cultivation of the new species is very difficult. In addition, in order to meet the increasing demand for phytoplankton such as chlorella, various methods such as expansion of production facilities have been proposed, but there are limitations.

Therefore, there is a need for a new culture method for mass culture of chlorella.

The present inventors have found that Chlorella vulgaris (Chlorella, heterotrophic growth conditions in the active novel strain (KMMCC174) while trying to proceed with the studies for the mass culture of Chlorella vulgaris ) Was isolated and identified for the first time, and the present invention was completed by establishing optimum culture conditions of the strain.

Therefore, an object of the present invention is a high density cultivation, easy to harvest, large cell size and cell growth rate Chlorella vulgaris ( Chlorella vulgaris ) to provide KCTC 12163BP.

Another object of the present invention is the Chlorella vulgaris ( Chlorella vulgaris ) to provide a method for mass culturing KCTC 12163BP microalgae.

In order to achieve the above object, the present invention is a new strain chlorella vulgaris excellent Chlorella nutrition ( Chlorella vulgaris ) to provide KCTC 12163BP microalgae.

In one embodiment of the present invention, the microalgae may be using glucose as a carbon source.

In one embodiment of the present invention, the microalgae may be cultured by additionally adding 1% glucose at intervals of 3 to 4 days after incubation at a concentration of 3% or less at the initial inoculation of glucose.

In one embodiment of the present invention, the microalgae may be grown at a high temperature of 30 ℃ ~ 32 ℃.

In one embodiment of the present invention, the microalgae may be cultured by adding Ca (NO ₃ ) ₂ and NaNO ₃ twice as nitrogen sources as compared to JM medium which is a conventionally used medium.

In addition, the present invention, using glucose as a carbon source, incubated at a concentration of 3% or less at the initial glucose inoculation, and then cultured with an additional 1% glucose at intervals of 3-4 days, the culture temperature is 26 comprising the step of culturing in a ~ 32 ℃, paragraph 1 of chlorella vulgaris (chlorella vulgaris ) to provide a method of mass culturing KCTC 12163BP as a taga nutrient.

In one embodiment of the present invention, the concentration of nitrogen and phosphorus in the culture medium may be included in a weight ratio of 2: 1 nitrogen: phosphorus based on the JM medium concentration.

The novel Chlorella vulgaris according to the present invention (Chlorella vulgaris ) KCTC 12163BP microalgae have high lipid content and excellent nutrient cultivation ability, enabling high density cultivation, easy harvesting and fine manipulation of culture environment.

Therefore, the present invention Chlorella vulgaris (Chlorella vulgaris ) KCTC 12163BP microalgae cultivated in the mass culture method of the present invention is capable of mass production of chlorella products having a variety of physiological activity is considered to have high economic value.

1 is a result of measuring the daily growth rate after culturing by adding 1%, 2% of glucose, acetate, glycerol as a carbon source to three chlorella according to an embodiment of the present invention. (C4-3: KMMCC-882, C4-4: New microalgae of the present invention KCTC 12163BP, C4-8: KMMCC-143)
Figure 2 is a graph showing the daily growth rate value according to the glucose concentration by temperature, three species of chlorella according to an embodiment of the present invention.
Figure 3 is a graph showing the results of measuring the growth density by temperature of the three chlorella cultured in 1% glucose treatment according to an embodiment of the present invention.
Figure 4 is a graph showing the results of measuring the daily growth rate of the N: P ratio of three chlorella grown in JM medium at 26 ℃ according to an embodiment of the present invention.
Figure 5 is a graph showing the results of measuring the growth density for each N: P ratio of three chlorella grown in JM medium at 26 ℃ according to an embodiment of the present invention.
Figure 6 is a graph showing the results of measuring the growth density of the three chlorella autotrophic medium and the growth value of the other nutrient medium at 26 ℃ by treating twice the nitrogen or phosphorus in the medium according to an embodiment of the present invention to be.
FIG. 7 shows the daily growth rate of autotrophic cultivation at different concentrations of N: P and the daily growth rate of tagase nutrients at 26 ° C. by treating twice with nitrogen or phosphorus in a medium according to one embodiment of the present invention. A graph showing one result.
Figure 8 is a graph showing the results of measuring the cell size of chlorella grown in autotrophic medium and the cell size of chlorella grown in tagatrophic medium when treated with twice the nitrogen or phosphorus in the medium according to an embodiment of the present invention to be.
Figure 9 shows the dry weight of chlorella grown in autotrophic medium by N: P concentration of the three chlorella grown in JM medium at 26 ℃ according to one embodiment of the present invention and the dry weight of chlorella grown in the nutrient medium It is a graph showing the result of a measurement.
10 is a graph showing the results of measuring the dry weight of 25ml harvested in autotrophic medium according to the N: P concentration of three chlorella according to an embodiment of the present invention and the dry weight of 25ml harvested in a nutrient medium .
FIG. 11 shows growth at the time of 3% glucose and 3% of glucose at 3 days intervals at an initial temperature of 26 ° C. in a JM medium containing 2x nitrogen. It is a graph showing the result of measuring density.
12 is a graph showing the results of measuring the cell density during mass culturing of nutrient culture and autotrophic culture according to an embodiment of the present invention.
Figure 13 is a graph showing the results of measuring the cell size at the time of mass culturing of nutrient culture and autotrophic culture in accordance with an embodiment of the present invention.
Figure 14 is a graph showing the results of measuring the dry weight per cell in the mass culture of the taga nutrient culture and autotrophic culture in accordance with an embodiment of the present invention.

The present invention is a novel chlorella vulgaris ( Chlorella) easy to mass cultivation excellent in nutrient culture vulgaris ) KCTC 12163BP microalgae and a method of culturing the same. Chlorella (Chlorella) is observable in most known as microalgae Like cheongtae (靑苔) in diameter with a microscope in a round about 8㎛ belonging to Chlorophyta size.

Chlorella is recognized as a nutritious food containing a large amount of protein, dietary fiber, vitamins and minerals. Unlike other food ingredients, Chlorella is expected to be a future food because of its rapid growth rate. in particular CGF (chlorella growth Factor) component contained in the chlorella (chlorella) has been reported to be a major and effective for growth and development, disease recovery, in addition, the high value-added products by experiments, such as research and antioxidants, whitening as geriatric treatment with anticancer agents (Hasegawa et al., 1994; Justo et al., 2001; Kim et al., 2008).

In addition, Chlorella (Chlorella) has got different lipid composition has attracted attention as a raw material of recent bioenergy biodiesel one (Miao and Wu, 2004a, b ;. Xiong et al, 2008).

Therefore, in the method of mass culturing of this useful chlorella, research is being actively carried out to mass cultivation with taga nutrients rather than autotrophs.

In general, "taga" refers to a nutritional form that depends on organics made by other organisms, or "dependent nutrition".

In addition, "self-nourishment" means that nutrients do not require organic compounds such as proteins and sugars, and assimilate carbon dioxide gas in the presence of inorganic essential nutrients to synthesize and synthesize all necessary organic substances.

When cultivating microalgae in a nutrient culture method, unlike autotrophic culture, there is no restriction on cell culture due to the inhibition of light transmission, and microalgae can be systematically controlled, high density cultivation, and low cost in harvesting. There is this. However, in spite of these advantages, it is necessary to secure microalgae that can withstand such an environment because it is cultured in the dark state of fermenter.

In the present invention, the result selecting a heterotrophic culture capable of excellent Chlorella (Chlorella) for industrial use with Chlorella (Chlorella) as heterotrophic culture method, by analyzing the base sequence of 18S rRNA review of phylogenetic classification, the same base Since no microalgae with sequence existed, the present inventors deposited the new microalgae selected in the present invention at the Korea Institute of Biotechnology and Biotechnology Center on March 27, 2012 and received the accession number KCTC 12163BP, which was “Chlorella vulgaris ( Chlorella vulgaris). vulgaris ) KCTC 12163BP microalgae ”.

Chlorella vulgaris ( Chlorella isolated and identified in the present invention) vulgaris ) KCTC 12163BP microalgae is characterized by excellent nutrient cultivation and high fat content.

According to one embodiment of the present invention, to find out whether the nutrient culture of the microalgae of the present invention is significantly superior to other microalgae and its optimal culture conditions Pukyong National University Korea Marine Microalgae Bank (Korea Marine) KMMCC-882: Chlorella, sold from the Microalgae Culture Center (KMMCC) sp . (Below, KMMCC-882 is designated as "C4-3") , KMMCC-143: Chlorella vulgaris (Below, KMMCC-143 is designated as "C4-8") and the organics used as carbon sources (glycerol, acetate, glucose) for the microalgae of the present invention and the new microalgae (KCTC 12163BP, "C4-4") of the present invention As a result, the growth was not observed in all of the experimental groups cultured with 1% and 2% of glycerol and acetate, respectively, but growth was observed in the experimental groups containing 1% and 2% of glucose, indicating that the carbon source suitable for the present invention was glucose. (See FIG. 1).

In addition, as a result of measuring the daily growth rate according to the glucose concentration by temperature in order to determine the optimum culture temperature and glucose concentration of the new microalgae of the present invention, C4-3 species was cultured by adding 1% and 2% glucose at 28 ℃ One experimental group showed the highest daily growth rate, C4-8 species showed the highest daily growth rate in the experimental group added with 1% and 2% glucose at 26 ° C, and the novel microalgae of the present invention (KCTC 12163BP, "C4-4") showed the highest daily growth rate in the experiment cultured with 1% glucose at 26 ° C, indicating that the preferred temperature and glucose concentration were different (see FIGS. 2 and 3). .

In addition, the preferred temperature and glucose concentration of each species is slightly different as a result, but after initiating the culture using the concentration of 3% or less at the initial inoculation, 1% glucose is added at an interval of 3 to 4 days and then continued. It was found that it was best to induce growth.

In the case of the experimental culture incubated at 30 ℃ two species except the novel microalgae (KCTC 12163BP, "C4-4") of the present invention was confirmed that the growth rate is rapidly reduced, but the new microalgae of the present invention is gentle even at 32 ℃ It is a photothermal species that shows high growth and can be grown at high temperature.

According to one embodiment of the present invention, Ca (NO ₃ ) ₂ and NaNO _{3 in} the JM medium to determine the optimum ratio of nitrogen and phosphorus in the JM medium The total amount of nitrogen added in the form of 0.0166 g / L and the total amount of phosphorus added in the form of KH ₂ PO ₄ and Na ₂ HPO ₄ were 0.0107 g / L as a control having a nitrogen to phosphorus ratio of 1: 1. The growth rate of chlorella was increased by doubling and doubling the amount of chlorine, and when C4-3 and C4-4 were added only twice as much nitrogen, C4-8 was added twice as much nitrogen and phosphorus. It was found that the highest (see Figs. 4, 5).

Furthermore, the present inventors confirmed the cell growth rate, cell size, and dry weight of the three chlorellas in order to confirm that the growth of taga nutrient culture is actually higher than that of autotrophic culture. Significantly higher values indicate that the nutrient culture actually showed higher growth than the autotrophic culture (see FIGS. 6 to 10).

The present invention also provides a novel Chlorella vulgaris ( Chlorella) of the present invention. vulgaris ) KCTC 12163BP can be provided in a large amount culture method, preferably using glucose as a carbon source, incubated at a concentration of 3% or less at the initial inoculation of glucose, and then 1% glucose every 3-4 days In addition to the culture, the culture temperature may include the step of culturing at 26 ~ 32 ℃.

In addition, when the nitrogen: phosphorus in the culture medium in a weight ratio of 2: 1, it is possible to mass culture in accordance with the growth rate.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example >

Statistical analysis

One-way ANOVA test was used for all test results, and Duncan's multiple range test was used for significance (P <0.05). All statistical analyzes were performed using SPSS (SPSS Inc., 2008;

< Example  1>

A new strain  Chlorella Bulgarian ( Chlorella vulgaris ) KCTC  12163 BP Separation of

<1-1> Isolation of strain

The present inventors collected chlorella in Jinhae to isolate novel chlorella micro strains with excellent nutrient culture and selected morphological and bacteriological characteristics by selecting strains showing excellent growth rate in nutrient culture.

<1-2> Identification of selected strains

a. Morphological identification

The morphological shape of the strain was observed through the microscope of the strain selected in Example <1-1>.

As a result, the cells of the identified strains were observed to have a spherical form of 2-5 μm (result not shown).

b. Molecular biology identification

The base sequence of 18S rRNA used in the art was analyzed for molecular biological identification of the strain selected in Example <1-1>.

As a result, the strains identified in the present invention were found to belong to the genus Chlorella, and the classification positions were found to belong to Chlorophycea - Chlorococum - Chlorella , and C. vulgaris and C. ellipsoidea are known as the same species. have.

Furthermore, the present inventors analyzed the nucleotide sequence of the identified strain, the nucleotide sequence is shown in SEQ ID NO: 1, the sequencing analysis confirmed that it is a new strain having a new nucleotide sequence that does not exist conventionally, March 2012 On 27th, it was deposited with the Korea Institute of Bioscience and Biotechnology, and received the accession number KCTC 12163BP. Hereinafter, in the present invention, the identified new strain was named " Chlorella vulgaris KCTC 12163BP".

< Example  2>

Strain Growth Analysis in Taga Nutrition

The present inventors have a number of advantages, such as the nutrient cultivation is possible without the light inhibition phenomenon, it is possible to harvest a high density, and easy to harvest, but due to the characteristics of the culture vessel called fermentation tank can be grown quickly in the dark and the cells should be robust, etc. In consideration of the fact that many conditions are required, the optimal value of the nutrient value of the mycobacteria according to the present invention was analyzed.

<2-1> As a carbon source  Organic matter screening

In order to select the optimal carbon source of the new strain according to the present invention was tested on glucose, glycerol, acetate, 100% JM medium dispensed in a 250 mL flask (Thompson et al., 1988) and three carbon sources 1% and It was added at a concentration of 2%. The experiments were conducted in the dark at 28 ° C., and the growth rate of chlorella was compared according to the type of carbon source and the concentration of carbon source.

As a result, the growth of the strain was not observed in all of the experimental groups (1% and 2% concentration test) cultured by adding glycerol and acetate as a carbon source, whereas the experimental group to which 1% and 2% glucose was added was present. It was shown that the new strain of growth well, the optimal carbon source of the new strain of the present invention was found to be glucose (see Fig. 1).

<2-2> Temperature and Glucose  Growth rate measurement by concentration

In order to determine the growth of the new strains of the present invention according to the temperature and glucose concentration, 100 mL JM medium was dispensed into a 250 mL flask, and then 1% to 5% glucose was added to each of 24 ° C, 26 ° C, 28 ° C, and 30 ° C. Stationary culture was carried out at temperature conditions. In addition, it was confirmed whether the growth at a temperature of 32 ℃ for the strain showed a growth at 30 ℃.

 As a result, in the experimental group incubated at 24 ° C, all three species showed significantly higher growth rate in the experimental group to which 1% glucose was added. In the experimental group incubated at 26 ° C, C4-3 and C4-4 Species showed the highest daily growth rate in the experimental group to which 1% glucose was added. In addition, the C4-8 species showed the highest daily growth rate in the experimental group to which the 1% glucose concentration was added and the experimental group to which the 2% glucose was added. C4-3 and C4-8 showed the highest daily growth rate, but C4-4 did not show any significant trend.

In addition, the experimental strain cultured at 30 ℃ the new strain Chlorella vulgaris of the present invention ( Chlorella vulgaris ) Two species except C4-4, KCTC 12163BP, showed a sharp decrease in growth rate, but the new strain of the present invention did not rapidly decrease in the daily growth rate even at 30 ° C., especially at 32 ° C. (See FIGS. 2-3). Therefore, the strain according to the present invention was found to grow actively even at a high temperature of 30 ℃ ~ 32 ℃.

<2-3> Nitrogen and phosphorus ratio in the medium

Novel strain of the present invention Chlorella vulgaris (Chlorella vulgaris ) In order to investigate the optimal culture conditions of KCTC 12163BP, the optimum nitrogen and phosphorus content of the culture medium were analyzed. For this purpose, conventionally used JM medium was used, and Ca (NO ₃ ) ₂ and NaNO ₃ were added to the JM medium. The total amount of nitrogen added in the form of 0.0166 g / L and the amount of phosphorus added in the form of KH ₂ PO ₄ and Na ₂ HPO ₄ were 0.0107 g / L with a nitrogen to phosphorus ratio of 1: 1. Phosphorus was added twice and three times to examine the growth rate of the strains for the groups of nitrogen: phosphorus ratios of 2: 1, 2: 2, 2: 3, 3: 1, 3: 2, 3: 3.

As a result, C4-3 and the new strain Chlorella vulgaris of the present invention ( Chlorella) vulgaris ) C4-4, KCTC 12163BP, was significantly higher in N: P 2: 1 medium inoculated with Ca (NO ₃ ) ₂ and NaNO _3, which are nitrogen sources in the existing JM medium, respectively, twice as much as the conventional medium. The daily growth rate was shown, and in case of C4-8, nitrogen source and KH ₂ PO ₄ The concentration of Na ₂ HPO ₄ also showed the highest daily growth rate in the double-inoculated N: P 2: 2 medium. Especially, in case of C4-8 species, there was no significant difference between the daily growth rate value of the experimental group added with 2 times of nitrogen and the value of the experimental group added with 1 times of phosphorus. It seems to have a wider adaptation than the species. In addition, all three species showed the lowest growth in N: P 3: 3 medium containing triple nitrogen and phosphorus, and N: P 3: 2 medium containing triple nitrogen and phosphorus doubled phosphorus. The addition was found to negatively affect growth (see FIGS. 4-5).

< Example  3>

Measurement of Taga Nutrition and Autotrophic Culture

<3-1> Cell growth rate measurement

In Example <2-1> by confirming that the growth rate of the strain of the present invention is higher than other ratios of nitrogen and phosphorus when nitrogen is added to the existing medium twice, 1 to the JM medium in which nitrogen or phosphorus is added twice. Incubated in control JM medium containing 1x nitrogen and 1 fold of nitrogen in the same conditions as in the other cultures in the presence of 2% glucose and incubated at 26 ° C in the dark, continuous 60 μmol photons m ^-2 s ^-1 And cultured in each of the experimental groups cultured in the control JM medium added with nitrogen 1-fold under the same conditions as the autotrophic test cells cultured in JM medium added with nitrogen or phosphorus 2 times at 26 ℃. Each cell was initially inoculated at a density of 10 ⁶ cells / mL and incubated for 3 days with 3 repetitions to measure the growth rate of the cells.

As a result, the experimental values cultivated by increasing the concentration of nitrogen and phosphorus at a ratio of 2: 2 for C4-8 and 2: 1 for C4-3 and C4-4, and the daily cultivation of the experimental zone incubated with the existing JM medium As a result of evaluating the growth rate, all three species showed significantly higher growth rate than the self-nourishment, and in the case of other nutrient cultures, the experimental group that doubled the nitrogen value was higher than the conventional JM medium. In the case of autotrophic cultivation, the experimental group cultured in the medium containing twice the nitrogen tended to show lower daily growth rate. Cell densities in autotrophic cultures and in other nutrients were similar at the first day after inoculation, but from day 2 after the inoculation, the nutrient-incubated cells were more than twice as high as in the autotrophic cultures. (See Figures 6-7).

<3-2> Measuring cell size

The average cell size was obtained by measuring the size of the long side and the short side (Moticam Pro 205A, Motic) by 50 individuals daily.

As a result, the cell size differed by about 0.5 μm even in the same experimental group because the time of division and growth was different for each cell, indicating that the margin of error was larger. In all species, the size of the cells in the nutrient culture was greater than the size of the cells in the autotrophic culture, which was significantly larger than 1 μm (see FIG. 8).

<3-3> Dry weight measurement

After measuring the size of the cells and dried 1.2 μm glass microfiber filter (GF / C, Whatman) at 60 ℃ for 2 hours on the last day of the culture and weighed, 25 mL of the culture medium was filtered and dried again for 2 hours The dry weight was measured by measuring the weight.

As a result, the dry weight value per cell of all three species showed significantly higher (P <0.05) 2-3 times higher value in the nutrient culture group than the autotrophic culture group. ). In case of C4-8, the control group without added doubling nitrogen was the highest at 0.0316 ng. In case of C4-8 and C4-3, nitrogen was 2 The medium added in multiples showed significantly higher values. The dry weight per cell was significantly higher (P <0.05) for C4-3 cultured with 2x nitrogen and incubated with nutrients (0.068 ng).

The dry weight value of 25 mL after 6-day cultivation of three species was significantly higher (P <0.05) and 0.02 g (0.8 g / kg). L) Next, C4-4 species cultured in double nutrients with doubled nitrogen were 0.017 g (0.68 g / L), and C4-3 species using JM medium as control were 0.015 g (0.6 g / L) in order. See FIG. 10).

<3-4> Fatty Acid Analysis

After culturing three chlorella through the Example <3-1> and harvested with a GF / C filter and stored at -80C it was analyzed three times the fatty acid. Fatty acid was methylated with BF3-methanol according to the method of Metcalfe et al. (1966), followed by three times gas chromatography (6890 plus, Agilent, USA).

As a result, heterotrophic Chlorella by nutrient culture showed a lower content of fat than the result by the self-nutrient culture Chlorella, self nutrition in the case of the present invention novel strain Chlorella vulgaris (Chlorella vulgaris) a C4-4 KCTC12163BP species The medium containing 2 times of nitrogen showed 64.3% fat content, showing the highest tendency compared to other species. However, the change in fat content according to the difference between nitrogen 1 times and 2 times was different according to species. In the case of taga nutrition, C4-4 species, the new strain of the present invention, showed a significantly higher fat content as compared to other species with nitrogen at 38.0% (see Tables 1 to 2).

Therefore, from these results, it was confirmed that the new strain of the present invention is excellent in fat content as compared to other species.

Analysis of Fatty Acids in Chlorella Cultured with Autotrophs (%) Fatty acids C4-3 C4-4 C4-8 2N JM JM 2N JM JM 2N JM JM C10: 0 2.7 ± 3.77 2.6 ± 3.71 7.6 ± 0.07 3.1 ± 4.44 6.2 ± 0.14 2.7 ± 3.8 C11: 0 3.1 ± 0.25 3.2 ±± 0.24 3.9 ± 0.38 3.7 ± 0.82 3.2 ± 0.18 3.3 ± 0.1 C12: 0 2.0 ± 2.76 4.2 ±± 0.54 5.7 ± 0.09 5.5 ± 1.23 4.8 ± 0.16 4.6 ± 0.68 C13: 0 3.7 ± 0.16 3.9 ±± 0.28 3.6 ± 0.36 3.7 ± 0.83 3.4 ± 0.14 3.3 ± 0.04 C14: 0 4.0 ± 0.73 3.9 ±± 0.33 5.1 ± 0.10 4.9 ± 1.02 4.4 ± 0.30 4.1 ± 0.44 C14: 1 2.7 ± 0.39 2.3 ±± 0.24 2.8 ± 0.02 2.8 ± 0.66 2.5 ± 0.16 2.3 ± 0.37 C15: 0 0.9 ± 1.22 2 ±± 0.16 2.4 ± 0.12 2.3 ± 0.4 2.0 ± 0.09 2.1 ± 0.22 C15: 1 2.2 ± 0.31 2 ±± 0.71 2.6 ± 0.07 2.6 ± 0.47 2.3 ± 0.04 2.3 ± 0.11 C16: 0 19.0 ± 1.09 18.5 ±± 0.71 14.9 ± 1.21 15.5 ± 2.11 14.7 ± 0.08 15.3 ± 0.47 C16: 1 2.5 ± 0.48 2.3 ±± 0.1 2.9 ± 0.08 2.9 ± 0.51 2.6 ± 0.08 2.5 ± 0.2 C17: 0 1.8 ± 0.38 1.7 ±± 0.14 1.1 ± 1.58 0.9 ± 1.3 1.9 ± 0.05 1.8 ± 0.25 C17: 1 - 0 ±± 0 2.5 ± 3.55 5.4 ± 0.67 6.2 ± 0.05 7.0 ± 0.55 C18: 0 2.5 ± 0.50 2.7 ±± 0.09 2.9 ± 0.04 3.5 ± 0.47 2.7 ± 0.09 2.3 ± 0.34 C18: 1n9 6.8 ± 1.7 6.93 ±± 0.4 8.0 ± 0.01 8.7 ± 1.34 7.2 ± 0.09 6.5 ± 0.87 C18: 2n6 19.7 ± 1.02 18.5 ±± 0.83 15.5 ± 0.60 16 ± 0.65 12.6 ± 0.12 14.9 ± 0.25 C18: 3n6 - - - - - - C18: 3n3 19.0 ± 0.86 17.7 ±± 0.81 12.1 ± 0.57 11.5 ± 0.27 15.7 ± 0.23 18.7 ± 0.05 C20: 0 4.3 ± 0.74 4 ±± 0.35 5.3 ± 0.02 2.2 ± 3.12 4.7 ± 0.20 4.4 ± 0.61 C20: 1n9 - 0.9 ±± 1.24 - - - - C20: 2 1.9 ± 0.44 1.8 ±± 0.13 1.1 ± 1.62 2.5 ± 0.42 2.0 ± 0.08 1.8 ± 0.29 C20: 3n6 - - - - - - C20: 3n3 - - - 1 ± 1.4 - - C20: 4 n6 (AA) 1.1 ± 1.61 0.8 ±± 1.14 - - - - C20: 5n3 (EPA) - - - 1.1 ± 1.59 1.1 ± 1.58 - C22: 1n9 - - - - - - C22: 2 - - - - - - C23: 0 - - - - - - C24: 0 - - - - - - C24: 1 - - - - - - C22: 6n3 (DHA) - - - - - - Saturated 44.0 ± 5.0 b 46.7 ± 4.82 ab 52.6 ± 4.0 a 45.3 ± 7.08 ab 47.9 ± 3.9 ab 43.9 ± 3.96 ab Mono-
saturated 7.4 ± 7.7 b 6.6 ± 5.28 b 10.8 ± 5.9 ab 13.7 ± 9.46 a 13.6 ± 3.4 a 14.1 ± 5.91 a Poly-
saturated 48.6 ± 15.9 a 46.6 ± 10.16 ab 36.6 ± 11.1 c 40.8 ± 14.45 bc 38.6 ± 6.7 c 41.9 ± 9.69 bc Total lipid (%) 36.7 ± 8.15 b 52.7 ± 5.28 a 64.3 ± 0.24 a 58.3 ± 12.27 a 48.4 ± 1.73 ab 35.4 ± 5.07 b

 Analysis of Fatty Acids in Chlorella Cultured with Taga Nutrition (%) Fatty acids C4-3 C4-4 C4-8 2N JM JM 2N JM JM 2N JM JM C10: 0 0.5 ± 0.78 0.6 ± 0.87 0.6 ± 0.78 0.7 ± 0.02 1.5 + 0.04 0.8 ± 1.15 C11: 0 0.6 ± 0.01 0.3 ± 0.4 0.6 ± 0.16 0.4 ± 0 0.7 ± 0.07 0.8 ± 0.07 C12: 0 0.8 ± 0.00 0.4 ± 0.62 0.4 ± 0.59 0.6 ± 0.12 1.2 ± 0.02 1.5 + 0.04 C13: 0 1.3 ± 0.02 1.2 ± 0.1 1.4 ± 0.08 1.1 ± 0.12 1.5 ± 0.26 1.4 ± 0.01 C14: 0 1.1 ± 0.01 0.5 ± 0.73 1.2 ± 0.05 0.6 ± 0.06 1.3 ± 0.05 1.4 ± 0.06 C14: 1 0.8 ± 0.00 1 ± 0.13 0.5 ± 0.07 0.5 ± 0.01 0.7 ± 0.01 1.6 ± 0.01 C15: 0 0.5 ± 0.05 0.6 ± 0.14 0.6 ± 0.10 0.3 ± 0.01 0.6 ± 0.12 0.6 ± 0.09 C15: 1 0.4 ± 0.01 0.9 ± 0.06 1.1 ± 0.12 0.3 ± 0.14 1.0 ± 0.05 0.7 ± 0.06 C16: 0 25.3 ± 0.08 22.9 ± 2.42 25.5 ± 1.26 25.6 ± 0.64 21.5 ± 3.03 27.3 ± 2.11 C16: 1 2.3 ± 0.04 1.3 ± 0.14 1.5 ± 0.10 1.3 ± 0.05 4.6 ± 0.07 3.5 ± 0.39 C17: 0 0.6 ± 0.01 0.7 ± 0.11 0.7 ± 0.05 0.4 ± 0.02 0.8 ± 0.05 0.8 ± 0.13 C17: 1 0.3 ± 0.02 4.9 ± 0.66 2.2 ± 2.54 0.3 ± 0 2.7 ± 3.18 7.4 ± 0.39 C18: 0 2.8 ± 0.03 2.3 ± 0.4 3.0 ± 0.28 2.3 ± 0.01 2.4 ± 0.04 2.6 ± 0.21 C18: 1n9 4.8 ± 0.04 4.6 ± 0.77 5.6 ± 0.48 6.1 ± 0.02 23.6 ± 0.28 3.2 ± 0.29 C18: 2n6 37.5 ± 0.16 40.1 ± 4.18 39.7 ± 2.82 47.1 ± 0.48 20.3 ± 0.74 30.7 ± 1.23 C18: 3n6 - - - 0.3 ± 0.04 - 0.5 ± 0.07 C18: 3n3 14.8 ± 0.03 13.1 ± 1.47 10.5 ± 0.90 10 ± 0.03 10.6 ± 0.44 12.4 ± 0.48 C20: 0 1.0 ± 0.06 1.3 ± 0.44 1.4 ± 0.14 0.4 ± 0.6 1.5 ± 0.02 1.6 ± 0.31 C20: 1n9 0.4 ± 0.02 0.2 ± 0.32 0.5 ± 0.06 0.1 ± 0.21 0.5 ± 0.03 - C20: 2 0.5 ± 0.03 0.6 ± 0.13 0.5 ± 0.03 0.4 ± 0.06 0.5 ± 0.02 0.6 ± 0.15 C20: 3n6 - - - - - 0.7 ± 0.94 C20: 3n3 - - 0.2 ± 0.28 - - - C20: 4 n6 (AA) 0.2 ± 0.26 0.5 ± 0.16 0.2 ± 0.26 0.1 ± 0.15 - - C20: 5n3 (EPA) 0.4 ± 0.02 0.6 ± 0.17 0.5 ± 0.04 0.2 ± 0.25 0.6 ± 0.02 - C22: 1n9 - - - - - - C22: 2 - - - - - - C23: 0 - - - - - - C24: 0 2.8 ± 0.17 1.3 ± 1.9 1.5 ± 2.19 0.8 ± 1.15 1.7 ± 2.38 - C24: 1 - - - - - - C22: 6n3 (DHA) - - - - - - Saturated 37.5 ± 7.0 a 32.1 ± 6.40 a 36.9 ± 7.1 a 33.1 ± 7.20 a 34.8 ± 5.9 a 38.8 ± 7.61 a Mono-
saturated 3.9 ± 1.3 d 8.1 ± 7.72 bc 5.3 ± 7.3 cd 2.4 ± 2.37 d 9.1 ± 8.9 b 13.2 ± 4.29 a Poly-
saturated 58.6 ± 11.1 a 59.7 ± 20.24 a 57.7 ± 16.4 a 64.3 ± 14.56 a 56.2 ± 12.7 ab 48.1 ± 12.84 b Total lipid (%) 12.8 ± 0.33 b 14.9 ± 5.54 b 13.5 ± 1.84 b 38.0 ± 1.74 a 16.8 ± 0.61 b 14.8 ± 2.27 b

< Example  4>

Initial in badge Glucose  Chlorella growth rate by concentration

To determine whether it is beneficial to grow by adding 1% glucose in the medium every three days or 3% glucose at the beginning of the culture, in a double-nitrogen-added JM medium on the day of inoculation 3 The experimental group cultured with the addition of% glucose and the experimental group cultured by adding 1% glucose on the inoculation day, 3 days after inoculation, and 6 days after inoculation were compared. The culture was carried out in a 250mL flask on a 100mL medium scale and incubated in the dark at 26 ° C.

As a result, in the case of C4-3, the experimental group to which 3% glucose was added was significantly higher than the experimental group to which 1% glucose was added three times. It was confirmed that the experimental group to which% glucose was added was better in growth (see FIG. 11). However, in case of C4-4, the experimental group added with 3% glucose and the experimental group added with 1% glucose did not show any significant difference. In the case of C4-8, 1% glucose was added to the experimental group added significantly more than 3% glucose on the 3rd and 9th day after incubation (P <0.05) 1% glucose added 3 times It was confirmed that the growth of one experimental section is better.

< Example  5>

Comparison of Growth Rate between Autotrophic and Tagatrophic Cultures in Mass Culture

The inventors of the present invention, Chlorella vulgaris ( Chlorella) of the present invention to determine whether there is a difference in the growth rate of autotrophic and other nutrients in the mass culture vulgaris ) Chlorella incubated in 250 mL flasks for C4-4, KCTC 12163BP, was gradually scaled up to 2 L flasks and 10 L round plastic tanks, and finally, 20 L round plastic tanks. Incubated in the. Autotrophic culture was irradiated with a continuous light of 60 μmol photons m ^-2 s ^-1 , and the nutrient culture was incubated with 3% glucose in the dark. Inoculate 3 L of chlorella culture in log phase into 15 L JM medium containing 2x nitrogen and adjust the initial inoculation density to 700 ± 50 (x10 ⁴ cells / mL), and then ventilate at 26 ℃ for 7 days. It was. Cell density and dry weight per cell were measured daily and 50 cells were randomly measured on the last day of culture. Cell density was counted by hemocytometer and daily growth rate was determined based on the highest cell density days. The dry weight per cell was measured by drying the GF / C filter at 60 ° C. for 2 hours, diluting 10 mL of the culture solution in 100 mL of distilled water, filtering, and drying again in the same manner. All experiments were repeated three times.

As a result, the cell density during the mass culture showed a rapid growth of 2.25 from the first day after the cultivation of the nutrient culture, whereas the growth of the cell density was 0.3. After 3 days of incubation, the TGA cultivation showed a higher daily growth rate of 0.95, which was about 6 times higher than that of the 0.17 cultivation of the autotrophic cultivation. The size of the cells measured on the last day of the mass culture was significantly larger in the nutrient culture compared to the autotrophic culture (P <0.05) (see FIG. 13).

The dry weight of one cell measured during the incubation period was significantly higher than that of autotrophic culture in all the experimental sections except for the first day of inoculation (P <0.05) (see FIG. 14). In autotrophic and other nutrient cultures, the dry weight of cells tended to increase significantly with increasing incubation time (P <0.05).

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Korea Biotechnology Research Institute KCTC12163BP 20120327

<110> Pukyong National University Industry-University Cooperation Foundation <120> Novel Chlorella vulgaris and culturing method <130> PN1202-018 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1671 <212> DNA <213> Chlorella vulgaris <400> 1 ggctcattaa atcagttata gtttatttga tggtacctac tactcggata cccgtagtaa 60 atctagagct aatacgtgcg taaatcccga cttctggaag ggacgtattt attagataaa 120 aggccgaccg ggctctgccc gactcgcggt gaatcatgat aacttcacga atcgcatggc 180 cttgcgccgg cgatgtttca ttcaaatttc tgccctatca actttcgatg gtaggataga 240 ggcctaccat ggtggtaacg ggtgacggag gattagggtt cgattccgga gagggagcct 300 gagaaacggc taccacatcc aaggaaggca gcaggcgcgc aaattaccca atcctgacac 360 agggaggtag tgacaataaa taacaatact gggccttttc aggtctggta attggaatga 420 gtacaatcta aaccccttaa cgaggatcaa ttggagggca agtctggtgc cagcagccgc 480 ggtaattcca gctccaatag cgtatattta agttgctgca gttaaaaagc tcgtagttgg 540 atttcgggtg gggcctgccg gtccgccgtt tcggtgtgca ctggcagggc ccaccttgtt 600 gccggggacg ggctcctggg cttcactgcc cgggactcgg agtcggcgct gttactttga 660 gtaaattaga gtgttcaaag caggcctacg ctctgaatac attagcatgg aataacacga 720 taggactctg gcctatcctg ttggtctgta ggaccggagt aatgattaag agggacagtc 780 gggggcattc gtatttcatt gtcagaggtg aaattcttgg atttatgaaa gacgaactac 840 tgcgaaagca tttgccaagg atgttttcat taatcaagaa cgaaagttgg gggctcgaag 900 acgattagat accgtcctag tctcaaccat aaacgatgcc gactagggat cggcggatgt 960 ttcttcgatg actccgccgg caccttatga gaaatcaaag tttttgggtt ccggggggag 1020 tatggtcgca aggctgaaac ttaaaggaat tgacggaagg gcaccaccag gcgtggagcc 1080 tgcggcttaa tttgactcaa cacgggaaaa cttaccaggt ccagacatag tgaggattga 1140 cagattgaga gctctttctt gattctatgg gtggtggtgc atggccgttc ttagttggtg 1200 ggttgccttg tcaggttgat tccggtaacg aacgagacct cagcctgcta aatagtcacg 1260 gttggctcgc cagccggcgg acttcttaga gggactattg gcgactagcc aatggaagca 1320 tgaggcaata acaggtctgt gatgccctta gatgttctgg gccgcacgcg cgctacactg 1380 atgcattcaa cgagcctagc cttggccgag aggcccgggt aatctttgaa actgcatcgt 1440 gatggggata gattattgca attattaatc ttcaacgagg aattcctagt aagcgcaagt 1500 catcagcttg cgttgattac gtccctgccc tttgtacaca ccgcccgtcg ctcctaccga 1560 ttgggtgtgc tggtgaagtg ttcggattgg cgaccggggg cggtctccgc tctcggccgc 1620 cgagaagttc attaaaccct cccacctaga ggaaggagaa acgtaacaag g 1671

Claims (7)

Heterotrophic cultured Chlorella vulgaris is available (Chlorella vulgaris ) KCTC 12163BP microalgae. The method of claim 1,
The microalgae are Chlorella vulgaris ( Chlorella), characterized in that using glucose as a carbon source vulgaris ) KCTC 12163BP microalgae. The method of claim 1,
The microalgae are cultured at a concentration of 3% or less at the initial inoculation of glucose, and then cultured by adding 1% of glucose at intervals of 3 to 4 days ( Chlorella). vulgaris ) KCTC 12163BP microalgae. The method of claim 1,
Chlorella vulgaris ( Chlorella) characterized in that the microalgae can be grown at high temperatures of 30 ℃ ~ 32 ℃ vulgaris ) KCTC 12163BP microalgae. The method of claim 1,
Wherein the microalgae is a conventional medium used causes nitrogen than JM medium is Ca (NO _{3) 2} and NaNO ₃ 2 Chlorella further comprising a step of adding to the culture times vulgaris (Chlorella vulgaris) KCTC 12163BP microalgae. Glucose is used as the carbon source, and incubated at the concentration of 3% or less at the initial inoculation of glucose, followed by incubation with an additional 1% glucose at intervals of 3 to 4 days, and the culture temperature is incubated at 26 to 32 ° C. a step, of claim 1 chlorella vulgaris (chlorella vulgaris ) A method of mass culturing KCTC 12163BP as a taga nutrient. The method according to claim 6,
Culture medium nitrogen: phosphorus is 2: 1 of claim 1 Chlorella vulgaris (Chlorella such a manner that a weight ratio of vulgaris ) A method of mass culturing KCTC 12163BP as a taga nutrient.

Images