KR20130048941A - Novel chlorella vulgaris capable low temperature growth and use thereof - Google Patents

Abstract

PURPOSE: A novel strain, Chlorella vulgaris, capable of growing at low temperatures is provided to ensure excellent growth rate at 4-15 deg. C and salt tolerance, and to be used as feed for fish and shellfish by containing amino acids and essential fatty acids. CONSTITUTION: Chlorella vulgaris KCTC 11964BP microalgae for culturing rotifer is able to grow at low temperatures, 4-15 deg. C. The rotifer is Brachionus plicatilis. The microalgae enhance the content of amino acids and unsaturated fatty acids needed for growing rotifer. The microalgae have salt tolerance at a salinity of 15-35psu. A composition for feed for rotifer contains the microalgae or a culture medium thereof. A formulation for aquaculture feed contains the composition as an active ingredient. A method for culturing the large amount of rotifer comprises a step of culturing the rotifer using the microalgae from November to February.

Description

Novel Chlorella vulgaris capable low temperature growth and use

The present invention is a new strain Chlorella vulgaris ( Chlorella) that can be grown at low temperatures vulgaris) and relates to the use thereof, is easy for mass cultivation at a low temperature and a novel strain which can be used as food for rotifers Chlorella vulgaris (Chlorella vulgaris ) KCTC 11964BP microalgae, a composition for rotifer feed comprising the same, a method for culturing rotifers at low temperature and a preparation for aquatic feed.

The living animals and plants that are aquaculture for aquatic organisms are called food organisms. Marine microalgae are important food organisms for the production of seedlings of aquaculture animals. In addition, marine microalgae are the primary producers of marine ecosystems and play a role in determining the biological productivity of the ocean.

Rotifers (Rotifer) is the snapper Pagrus first developed in the 1960s It is one of the major animal feeders of major larvae. It is small in size and weak in motility and has high density culture and is one of the most widely used animal feed organisms in the production of artificial seedlings of marine fish (Fukusho K. 1989. Biology and mass production of the rotifer, Brachionus plicatilis. Int J Aqu Fish Technol 1, 232-240).

Microalgae and yeast are currently used as feed organisms for mass cultivation of rotifers. For microalgae , Nannochloropsis and Chlorella And Nannochloris And yeast Saccharomyces cerevisiae and marine yeast Candida utilis is representative.

However, although yeast is easier to use than microalgae in that it is more economical, there is a problem of lack of unsaturated fatty acids necessary for growth of rotifers (Watanabe T, et. al . 1980.Relationship between dietary value of brine shrimp Artemia salina and their content of ω-yeast highly unsaturated fatty acids. Bull jap Soc Sci Fish 46, 35-41). Therefore, to solve this problem, ω-yeast was emulsified with squid oil in baker's yeast (Hirayama K and Funamoto H. 1983. Supplement effect of several nutrient on nutritive deficiency of baker's yeast for population growth of the rotifer Brachionus plicatilis . Nippon suisan Gakkaishi 49, 505-510), which is also less nutritious compared to microalgae, there is a problem that causes water pollution.

Therefore, most hatcheries still prefer living microalgae, despite the high cost of rotifer mass cultivation.

The microalgae used for the rotifer culture, especially Chlorella, Nannochloris, Nannochloropsis Eustigmatophyceae of mass culture is easy and suited to the culture of rotifers high content of protein and polyunsaturated fatty acids.

On the other hand, even microalgae used for the cultivation of rotifers are often suddenly killed in high water temperature of 30 ° C. or higher, and growth is very low in low temperature water of 10 ° C. or lower, especially in mass culture of rotifers in summer and high temperature of winter. Many difficulties are involved (Fukusho K, et. al . 1985.Food value of a rotifer, Brachionus plicatilis cultured with Tetraselmis tetrathele for larvae of a flounder Paralichthys olivaceus . Bull Natl Res Aquacult 7, 29-36; Hur SB 1991.The selection of optimum phytoplankton species for rotifer culture during cold and warm seasons their nutritional value for marine finfish larvae. In: Rotifer and Microalgae Culture Systems, Proceedings of a US-Asia Workshop, Fulks W and Main K, eds. The Oceanic Institute . 163-173).

Therefore, it is necessary to develop a novel microalgae suitable for the cultivation of rotifers due to easy survival and mass culture at high and low water temperature.

Therefore, the present inventors have discovered a novel microalgae Chlorella vulgaris strain, which is easy to mass cultivate at low water temperature and suitable as a rotifer feeding organism. The present invention was completed by separation and identification for the first time.

It is therefore an object of the present invention is 4 ℃ ~ 15 ℃ possible growth at a low temperature rotifers (Rotifer) culture Chlorella vulgaris for (a Chlorella vulgaris ) to provide KCTC 11964BP microalgae.

Another object of the present invention is to provide a composition for rotifer (Rotifer) feed comprising the Chlorella vulgaris KCTC 11964BP microalgae or a culture thereof according to the present invention.

Another object of the present invention is to provide a formulation for aquatic feed comprising the composition for the Rotifer feed according to the present invention as an active ingredient.

In addition, another object of the present invention is to provide a method for culturing a large number of rotifers (Rotifer) during November-February, which is a cold water phase using the Chlorella vulgaris KCTC 11964BP microalgae according to the present invention as food. .

In order to achieve the above object, the present invention provides a Chlorella vulgaris KCTC 11964BP microalgae for rotifer (Rotifer) culture that can be grown at a low temperature of 4 ℃ ~ 15 ℃.

In one embodiment of the invention, the rotifer is Brachionus It can be plicatilis .

In one embodiment of the present invention, the microalgae may promote the growth of rotifers by increasing the content of amino acids and unsaturated fatty acids required for the growth of rotifers.

In one embodiment of the present invention, the microalgae may have flame resistance in the condition that the salinity is 15 ~ 35psu (practical salinity unit).

In addition, the present invention is the Chlorella vulgaris ( Chlorella) according to the present invention vulgaris ) KCTC 11964BP provides a composition for rotifer (Rotifer) feed comprising a microalgae or a culture thereof.

The present invention also provides a formulation for aquatic feed comprising the composition for the Rotifer feed according to the present invention as an active ingredient.

In addition, the present invention is the Chlorella vulgaris ( Chlorella) according to the present invention vulgaris ) KCTC 11964BP microalgae as a food, provides a method of cultivating rotifers (Rotifer) during the cold season from November to February.

The novel Chlorella vulgaris according to the present invention (Chlorella vulgaris ) KCTC 11964BP microalgae has excellent growth rate compared to other microalgae under low temperature of 4 ℃ ~ 15 ℃ and flame resistance that can grow under salt condition of seawater. It contains abundant amino acids and essential fatty acids that have various physiological functions, and ultimately, it can be very useful as a food organism and feed for the culture of zooplankton such as rotifers and the aquaculture industry of fish and shellfish that feed on these zooplankton. There is.

1 is a novel strain Chlorella vulgaris ( Chlorella) according to the present invention vulgaris ) KCTC 11964BP (KMMCC 120) and other microalgae in the algae growth rate at a low temperature of 10 ℃.
Figure 2 is a novel strain Chlorella vulgaris ( Chlorella) according to the present invention vulgaris ) In order to confirm the flame resistance of KCTC 11964BP (KMMCC 120), it is a graph showing the growth rate of salt at 30 psu (black graph) and 15 psu (white graph).

The present invention provides a novel Chlorella vulgaris microalgae for rotifer (Rotifer) cultivation, which is excellent in growth even at low temperatures, and can solve the shortage of food supply for cultivation of rotifers during the cold season. have.

In general, rotifers are zooplankton of Rotifera, which are mainly found in slow-flowing freshwater such as dams and reservoirs, and there are about 2,500 species worldwide.

These rotifers are used as feed organisms for aquatic life, which means plants and zooplankton that aquatic creatures eat at an early age. In addition, rotifers have the nutrients needed for aquatic organisms, especially larvae, which make them a much better food source than commercially available formulas.

In addition, some of the invertebrates, including fish larvae, which are actually breeding and aquaculture organisms in the fisheries sector, can survive only after hatching in fertilized eggs and rotifers as food. Without the rotifers, it is impossible to breed animals in the area of aquaculture, so rotifers are recognized for their industrial value as useful food organisms for mass production of these aquatic species.

On the other hand, rotifers that can be used as food organisms in the production of marine fish seedlings are greatly influenced by the type of microalgae that feed rotifers during the cultivation process (Hirayama et.al., 1979). Due to the low temperature of the rotifers, the growth of the microalgae is very low, there is a problem that the mass culture is difficult.

Therefore, in the present invention, in order to discover a novel rotifer culture microalgae that can enable large-scale cultivation of rotifers in winter, after collecting phytoplankton in the coastal and brackish waters, new microalgae which is active at low temperature Isolation and identification, and the analysis of the nucleotide sequence of the 18S rRNA analysis of the phylogenetic classification, the microalgae having the same nucleotide sequence did not exist, it was confirmed that belongs to Chlorella vulgaris systematically.

Therefore, the present inventors deposited the new microalgae isolated and identified in the present invention to the Microbial Resource Center of Korea Research Institute of Bioscience and Biotechnology as of June 14, 2011 and received the accession number KCTC 11964BP, which was “Chlorella vulgaris ( Chlorella vulgaris ) KCTC 11964BP microalgae ”.

Chlorella vulgaris ( Chlorella isolated and identified in the present invention) vulgaris ) KCTC 11964BP microalgae is characterized by active growth or growth at low temperatures, preferably at low temperatures of 4 ℃ to 15 ℃.

According to one embodiment of the present invention, to determine whether the low temperature growth characteristics of the novel microalgae of the present invention is significantly superior to other microalgae Pukyong National University Korea Marine Microalgae Culture Center (KMMCC) KMMCC-33: Nannochloropsis sp., KMMCC-13: Nannochloropsis oceanica , KMMCC-16: Nannochloris oculata , KMMCC-119: Nannochloris sp., KMMCC-395: Nannochloris sp. As a result of comparing the growth rate of the microalgae and the novel microalgae of the present invention at a low temperature of 10 ° C, the new microalgae of the present invention, Chlorella vulgaris ( Chlorella) vulgaris ) The other microalgae except KCTC 11964BP showed low growth, whereas the microalgae of the present invention showed a growth rate of three times higher than those of the control microalgae (see FIG. 1).

In addition, Chlorella vulgaris ( Chlorella isolated and identified in the present invention) vulgaris ) KCTC 11964BP microalgae have a characteristic of flame resistance, and especially have a characteristic showing active growth even under the salinity condition of 10psu ~ 35psu.

The novel microalgae identified in the present invention can be used as a feed microalgae for cultivation of rotifers. Currently, there are about 10 kinds of rotifers inhabiting pure seawater, and some of freshwater-derived species It is known to inhabit and adapt to areas of moderate salinity, that is, brackish water in natural waters.

Therefore, in order to cultivate rotifers that live in the brackish salt region as well as seawater, it may be essential that the microalgae that are prey have flame resistance.

The present inventors have identified Chlorella vulgaris ( Chlorella) identified in the present invention. vulgaris ) KCTC 11964BP microalgae to measure the degree of flame resistance, in one embodiment of the present invention was investigated the growth of microalgae under the salinity condition of 15psu and 30psu, Chlorella vulgaris ( Chlorella of the present invention compared to other microalgae) vulgaris ) KCTC 11964BP microalgae showed superior growth rate in both salinity conditions of 15 psu and 30 psu (see FIG. 2).

The term "psu" used as the salinity unit means a practical salinity unit, which is a practical salinity unit, and represents salinity as the ratio (psu) of the g mass of the dissolved substance dissolved in 1 kg of seawater. In general, the average salinity of seawater is known to represent 35 psu of salinity, but the salinity varies considerably with coastal rainfall.

Therefore, although the new microalgae according to the present invention were collected in the intermediate region of fresh water and salt concentrations of seawater, it showed active growth even under the salinity condition of 10psu ~ 35psu, so that the culture was easy under the seawater condition. In general, organisms that are collected and separated from the lower concentration of the brackish water than the sea water is often killed and failed to adapt to salt when transferred directly to the sea water, but the new microalgae of the present invention can grow well even in the salt concentration of sea water, which will fail to adapt to salt. There is no concern.

Furthermore, the present inventors have found a novel Chlorella vulgaris ( Chlorella). vulgaris ) To determine whether KCTC 11964BP microalgae is actually useful for cultivation of rotifers in the nutritional aspect, the components and contents of amino acids and fatty acids contained in rotifers were analyzed in rotifers cultured with microalgae of the present invention.

According to the results according to an embodiment of the present invention, the rotifers cultured with the new microalgae of the present invention showed higher amino acid content than the rotifers using other microalgae as a control, and particularly the content of essential amino acid. Was higher than other controls (see Table 5).

In addition, fatty acid analysis, novel Chlorella vulgaris (Chlorella vulgaris ) The rotifers cultured with KCTC 11964BP microalgae were significantly higher in unsaturated fatty acids than rotifers cultured with other microalgae, especially EPA (eicosapentaenoic acid). ) And DHA (decosahexaenoic acid) and polyunsaturated fatty acids (PUFA) containing the same was also found to be significantly higher (see Table 6).

Among polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA) are known to be included in the back blue fish. If EPA or DHA is not supplied enough to fry, the immune function is lowered and the mortality rate is high. Ingredients that must be supplied as ingredients.

In light of this, the novel Chlorella vulgaris KCTC 11964BP microalgae isolated and identified in the present invention is rich in amino acids and unsaturated fatty acids, particularly EPA (eicosapentaenoic acid) and DHA (decosahexaenoic acid) in rotifers. The aquatic organisms such as the fry cultured with the microalgae of the present invention can be excellent in nutritional aspects.

Therefore, the present invention relates to novel Chlorella vulgaris (Chlorella according to the present invention vulgaris ) KCTC 11964BP microalgae or a rotifer feed composition comprising a culture solution thereof can be provided.

The rotifer feed composition is a novel Chlorella vulgaris ( Chlorella) as an active ingredient vulgaris ) KCTC 11964BP microalgae or the culture of the microalgae, the culture of the microalgae may be a culture containing the microalgae, or centrifuged to remove the culture medium in the culture and recover only concentrated cells Or it may include a concentrated cell obtained through the filtration process and the concentrated cell can be preserved so as not to lose its activity by frozen or lyophilized according to a conventional method. Preferably, the microalgae and the culture medium of the microalgae may use living microalgae.

In addition, the present invention can provide a formulation for fish feed containing the rotifer feed composition as an active ingredient.

The aquatic feed formulation according to the present invention may be prepared as a form in which the feed preparation of the present invention is added to a feed composition of aquaculture fish, feed additives or commercially available feed compositions according to each aquaculture seafood, and the aquaculture seafood is limited thereto. All but rotifer-fed fish may be included, for example, larvae of fish, crustaceans (shrimp, crab, crayfish, lobster, etc.) and cephalopods (octopus, squid, octopus, etc.). .

Furthermore, the present invention is a novel Chlorella vulgaris ( Chlorella) discovered in the present invention. vulgaris ) KCTC 11964BP microalgae can be provided a mass culture method of rotifers, in particular can provide a method for mass culture of rotifers during cold water phase.

New chlorella according to the present invention as previously described vulgaris (Chlorella vulgaris ) KCTC 11964BP microalgae is characterized by active growth even at low temperatures of 4 ~ 15 ℃, when using the new microalgae, it is possible to cultivate a large number of rotifers outdoors during the cold season, winter.

The mass culturing method of the rotifer is a novel chlorella vulgaris ( Chlorella) of the present invention instead of the conventional food in the rotifer culture method vulgaris ) KCTC 11964BP microalgae can be used as a food for cultivation, and rotifer culture methods include batch culture method, semi-continuous culture method and high density culture method, and can be cultured by any one of them. .

The batch culture method is a method of culturing using a plurality of relatively small culture tank as the most common culture method, the semi-continuous culture method is cultured in a relatively large tank and harvesting a certain amount of rotifer in the same tank every day for a certain period of time Afterwards, the culture water corresponding to the harvested amount is supplemented with rotifers or new culture water. In addition, the high-density culture method is a method of culturing while maintaining the individual density of rotifers 10,000 to 20,000 individuals per ml, which is a method of culturing by installing a system for controlling the concentration of oxygen deficiency and ammonia caused by the increase in the number of rotifers. .

Therefore, the rotifer mass cultivation method provided by the present invention may be cultured by any of the above-described methods, and the novel microalgae of the present invention or the rotifer feed composition comprising the same may be added to the rotifer culture water or added to the rotifer feed. It can be cultured by using.

In addition, the culturing method provided by the present invention can be cultured in a large amount during the low-temperature water, preferably from November to February.

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

A new strain  Chlorella Bulgarias KCTC  11964 BP Separation of

<1-1> Isolation of Strains

The present inventors collected tide using 20 μm phytoplankton net in tidal pool of Hwajinpo, Donghae, Gangwon-do, to discover new microalgae that can be grown and grow at low temperature and can be used for cultivation of rotifers. After culturing by spreading on agar solid medium (LDM medium, see Table 1), the strain showing growth was isolated and cultured strain using f / 2 medium (see Table 2) known as a culture medium for seawater. Morphological and bacteriological properties were also analyzed.

<1-2> Identification of Selected Strains

a. Morphological identification

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

As a result, the cells of the identified strains were observed to have a spherical morphology as small green algae, 2-5 μm in diameter, without flagella, and no motility (results not shown).

b. Molecular biology identification

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

As a result, the strain identified in the present invention was found to belong to the genus Clonella, and the classification position was found to belong to Chlorophyceae - Chlorococcales - Chlorellaceae - Chlorella , and in particular, it belonged to Chlorella vulgaris . The present inventors analyzed the nucleotide sequence of the identified strain, the nucleotide sequence is shown in SEQ ID NO: 1, by confirming that the strain having such a nucleotide sequence is a novel strain that does not exist, June 14, 2011 As of date, it was deposited with the Korea Institute of Biotechnology and Microbial Resources Center and was given accession number KCTC 11964BP. Hereinafter, in the present invention, the identified new strain “ Chlorella vulgaris KCTC 11964BP ”.

c. Mycological  Sympathy

Identified above Chlorella , the new strain The bacteriological characteristics of the vulgaris KCTC 11964BP strain were examined, and the results showed that photosynthetic growth was possible by photosynthesis, and the growth temperature was well grown even at low temperatures of 4-15 ℃. , Especially at temperatures of 10 ° C.

In addition, the salinity that can be grown is prepared by mixing the filtered seawater and distilled water into 15 psu and 30psu, and the growth was examined under these conditions, it was found that it is possible to grow in both 15 psu and 30psu (see Fig. 2), Was found to range from 60 μmol m ⁻² s ⁻¹ to 100 μmol m ⁻² s ⁻¹ .

In particular, the growth investigation under the salt condition is Chlorella which is a new strain of the present invention. vulgaris KCTC 11964BP (aka 'KMMCC-120') and KMMCC-137: Chlorella sp. and KMMCC-327: Nannochloropsis sp.

< Example  2>

Strain Growth Analysis at Low Temperature

The inventors of the present invention, in order to solve the problem of lack of winter food supply, which has been a major problem in the cultivation of rotifers, in order to check whether the new strains identified in the present invention can be easily used as food for winter rotifers, Check if possible. That is, the microalgae that grow well due to the active cell growth even under cold water conditions in winter can be easily used for mass cultivation of winter rotifers. Therefore, the growth rate of the new strain and other microalgae strains identified in the present invention was analyzed at a low temperature of 10 ° C. At this time, the microalgal strains used for comparison also KMMCC-33: Nannochloropsis sp., KMMCC-13: N as a strain used for rotifer culture. oceanica , KMMCC-16: Nannochloris oculata , KMMCC-119: Nannochloris sp., KMMCC-395: Nannochloris sp. were used from the Korea Marine Microalgae Bank of Pukyong National University. Each of these strains was inoculated at a density of 100 × 10 ⁴ cells / mL in f / 2 medium and incubated for 10 days in a continuous illumination of 100 μmol m ^-2 s ^-1 under saline conditions of 15 psu. Repeat counting four times at the same time every day, the growth rate was calculated by Guillard's method [sgr gr 3.322 × log (N ₁ / N ₀ ) / t, (t : Number of days of culture after inoculation, N ₀ , N ₁ : Cell density after inoculation and after t days)].

In addition, the statistical treatment of all experimental results in the present invention was carried out a one-way ANOVA test, the significance (P <0.05) between the treatment average was Duncan's multiple range test. All statistical analyzes were performed using the SPSS (SPSS Inc., 2008; Ver. 17) program.

As a result, as shown in Fig. 1, Chlorella which is a new strain identified in the present invention. All except vulgaris KCTC 11964BP (KMMCC-120) showed low growth of less than 1,000 × 10 ⁴ cells / mL (0.0109 ~ 0.3303 growth rate). The species with the lowest growth is N. oculata (KMMCC-16) and Nannochloris sp. (KMMCC-119), Chlorella , a new strain identified in the present invention. vulgaris KCTC 11964BP (KMMCC-120) showed a significantly higher growth rate (0.5052; peak cell density of 3,316x10 ⁴ cells / mL) at 10 ° C compared to other species ( P <0.05). It was found that the strain is very suitable for rotifer mass culture of.

< Example  3>

Nutrition Facts

Furthermore, the inventors have identified C. vulgaris Amino acids and fatty acids were analyzed to analyze the nutritional components of KCTC 11964BP strains and rotifers cultured with food. The rotifer used was Brachionus of the Culture Collection of Useful Marine Plankton (CCUMP), Pukyong National University. plicatilis (CCUMP-35, L-type) was used and the daily supply of microalgae per rotifer was 30 × 10 for the smallest Nannochloris sp. (KMMCC-119) considering the cell volume of each microalgae. ⁴ cells, Nannochloropsis sp. (KMMCC-33) is 22 × 10 ⁴ cells, Chlorella vulgaris ( KMMCC-120) was supplied in sufficient quantity at 15 × 10 ⁴ cells. Since the rotifer was inoculated at 10 ° C / mL in a 250 mL Erlenmeyer flask (100 mL volume) in 26 ℃, 15 psu, 60 μmol m ^-2 s ^-1 continuous illumination was incubated for 5 days in three replicates.

C cultivated by the same method described above for this purpose. vulgaris KCTC 11964BP strains and rotifers cultured with food were harvested at the end of the logarithmic growth period, and the harvested cells were then stored frozen at -80 ° C until analysis. In this case, samples using KMMCC-33: Nannochloropsis sp. And KMMCC-119: Nannochloris sp. Were used in addition to the strains identified in the present invention for comparative analysis. That is, each of the strains used for comparative analysis and rotifers cultured with these strains were used as a control sample.

First, 20 mg of each frozen cell harvested for free amino acid analysis was placed in a test tube, and 15 mL of 6N HCl was added thereto, followed by sealing for 24 hours at 110 ° C. The digestion solution was filtered and dried under reduced pressure to completely remove HCl, and then quantified to 25 mL with sodium dilution buffer (pH 2.2). It was analyzed by the ninhydrin method. Analytical conditions were: column size 4 mm × 150 mm, absorbance 570 nm and 440 nm, solution flow rate 0.25 mL / min, buffer flow rate 0.45 mL / min, reactor temperature 120 ° C, reactor size 15 m, analysis time 65 minutes And analyzed.

In addition, fatty acid analysis was performed by adding 2 mL of 10% BF ₃ -methanol to the same amount of sample (20 mg or more) used above in a 15 mL test tube, filling with nitrogen, and then heating at 85 ° C. for 1 hour and 30 minutes to obtain a methyl ester. (Morrison and Smith, 1964; Budge, 1999). After the sample was cooled to about 40 ° C., fatty acids were separated and extracted by adding water and hexane. The extracted fatty acids were analyzed using HP GC 6890 Plus (Agilent, USA) with HP autosampler. The GLC used for fatty acid analysis was DB-225 (20 m × 0.1 mm, id, 0.1 μm film thickness, J & W Scientific, Agilent Technologies, USA). Analysis conditions were such that the column temperature is 60 ~ 195 ℃ (25 ℃ / min), temperature rising conditions 195 ~ 205 ℃ (3 ℃ / min), 205 ~ 230 ℃ (8 ℃ / min), injection temperature 250 ℃, detection Temperature 250 degreeC and the transport gas He (60 cm / sec) were used. Fatty acid analysis was performed using standard (PUFA 1, 10 and 37 component FAME Mix, Supelco, Ontaro, Canada) analyzed under the same conditions.

As a result, amino acid analysis and fatty acid analysis results for each microalgae used as a rotifer food are shown in Tables 3 and 4, and the amino acid content and fatty acid content were significantly different between the new strain and other strains identified in the present invention. It was found to contain all amino acids and fatty acids as a whole.

LDM Badge Composition Table Bristol solution 100 mL Biotin 0.25 ㎍ Vitamin B ₁₂ 0.15 μg PIV metal solution 6 mL Tryptone 1 g Agar 12 g Filtered Sea water 900 mL Bristol solution stock NaNO ₃ 10.0 g / 400 mL dH ₂ O CaCl ₂ 1.0 g / 400 mL dH ₂ O MgSO ₄ 7H ₂ O 3.0 g / 400 mL dH ₂ O K ₂ HPO ₄ 3.0 g / 400 mL dH ₂ O KH ₂ PO ₄ 7.0 g / 400 mL dH ₂ O NaCl ₂ 1.0 g / 400 mL dH ₂ O 10 mL of each stock solution is added to 940 mL of distilled water. To this add a drop 1.0% FeCl ₃ solution. PIV metal solution Na ₂ -EDTA 750 mg FeCl ₂ 6H ₂ O 97 mg MnCl ₂ 4H ₂ O 41 mg ZnCl ₂ 5 mg CoCl ₂ 6H ₂ O 2 mg Na ₂ MoO ₄ 4 mg Distilled water 1,000 mL

f / 2 badge composition table NaNO ₃ 1 mL Primary trace metals NaH ₂ PO ₄ 9H ₂ O 1 mL CuSO ₄ 5H ₂ O 9.8 g / L dH ₂ O Na ₂ SiO ₃ 9H ₂ O 1 mL Na ₂ MoO ₄ 2H ₂ O 6.3 g / L dH ₂ O trace metal solution 1 mL ZnSO ₄ 7H ₂ O 22.0 g / L dH ₂ O vitamin solution 0.5 mL CoCl ₂ 6H ₂ O 10.0 g / L dH ₂ O filtered seawater 950 mL MnCl ₂ 4H ₂ O 180.0 g / L dH ₂ O Stock solution vitamin solution NaNO ₃ 75 g / L work stock NaH ₂ PO ₄ 9H ₂ O 5 g / L Vitamine B ₁₂  1 mL Na ₂ SiO ₃ 9H ₂ O 30 g / L Biotin  10 mL Trace metal solution ThiamineHCl  200 mg FeCl ₃ 6H ₂ O 3.15 g distilled water 1,000 mL Na ₂ EDTA2H ₂ O 4.36 g Primary stock Primary trace metals 1 mL each Vitamine B ₁₂ 1.0 g / L dH ₂ O distilled water 1,000 mL Biotin 0.1 g / L dH ₂ O

Analysis of Amino Acid Content in Microalgae (%) KMMCC
No.
Amino acid 33 13 16 119 395 120 (of the present invention
New strain) Arginine 3.16 3.33 2.14 3.14 4.29 2.59 Histidine 1.12 1.31 1.51 1.76 2.52 1.42 Isoleucine 2.15 2.72 1.59 2.48 3.13 2.18 Leucine 4.07 5.06 3.56 4.76 6.38 4.28 Lysine 3.07 3.66 2.75 3.40 4.61 2.73 Phenylalanine 2.74 2.97 3.18 3.25 4.29 2.55 Throneine 2.22 2.62 2.11 2.54 3.42 2.04 Valine 2.93 3.39 2.71 3.10 3.87 2.60 Alanine 3.08 3.61 3.80 5.21 6.20 3.94 Aspartic acid 4.39 5.02 4.81 5.59 7.49 5.02 Glutamic acid 6.96 8.12 7.95 9.36 13.05 6.77 Glycine 2.59 3.23 2.58 3.04 4.08 2.87 Proline 7.29 6.40 3.37 3.36 3.89 2.70 Serine 1.93 2.16 1.96 2.37 3.10 2.33 Tyrosine 1.84 1.98 3.01 2.05 2.68 1.70 Total 49.54 55.58 47.03 55.41 72.97 45.72 EAA 21.46 25.06 19.55 24.43 32.51 20.39 NEAA 28.08 30.52 27.48 30.98 40.49 25.33 EAA / NEAA 0.76 0.82 0.71 0.79 0.80 0.80

KMMCC-33: Nannochloropsis sp., KMMCC-13: N. oceanica , KMMCC-16: Nannochoris oculata , KMMCC-119: Nannochoris sp., KMMCC-395: Nannochoris sp., KMMCC-120: Chlorella vulgaris , EAA: essential amino acid, NEAA: non-essential amino acid

 Analysis of fatty acid content of microalgae (%) KMMCC
No.
Fatty acid 33 13 16 119 395 120 (new strain of the present invention) 8: 0 0.41 0.20 0.66 0.16 0.09 0.40 10: 0 0.26 0.20 - 0.16 - - 11: 0 0.07 - 0.45 0.04 0.19 0.21 12: 0 0.29 0.30 - 0.74 - - 13: 0 0.87 0.30 0.42 0.04 0.22 0.23 14: 0 4.28 4.20 3.61 4.80 3.50 2.25 14: 1 0.64 0.20 1.23 0.48 0.36 1.20 15: 0 0.29 - 0.68 0.59 0.27 0.37 15: 1 0.19 - 1.42 0.12 0.40 0.61 16: 0 17.49 24.30 9.24 26.83 15.40 10.96 16: 1 25.75 19.00 1.54 5.33 8.69 3.04 17: 0 0.18 - 6.97 0.28 1.18 7.55 17: 1 0.74 - 16.14 8.48 3.24 11.66 18: 0 0.19 0.60 1.10 2.08 2.99 0.99 18: 1n9 3.31 10.20 6.23 10.14 29.59 5.64 18: 2 n9 4.50 5.70 1.29 15.41 5.73 2.924 18: 3 - 0.20 - 23.63 - - 18: 3 n6 0.16 - 14.28 0.017 0.11 22.32 18: 3 n9 0.16 - - - 4.87 - 20: 0 0.06 - 32.26 0.02 8.21 24.64 20: 1 0.05 - - 0.02 4.12 0.94 20: 2 0.05 - 1.47 0.08 1.66 - 20: 3 0.16 0.90 - 0.04 0.11 - 20: 4 0.38 3.10 - 0.04 - - 21: 0 4.07 - - - 0.71 0.34 20: 5n-3 34.88 19.20 0.38 0.35 6.28 3.74 22: 0 - - - 0.03 - - 22: 1 0.02 - 0.635 0.02 - - 22: 2 0.10 - - 0.04 2.09 - 24: 0 0.03 - - 0.02 - - 22: 6n-3 0.29 - - 0.02 - - others - 11.30 - - - - TOTAL 100 100 100 100 100 100 Saturated 28.49 30.10 55.39 35.75 32.76 47.94 Monounsaturated 30.70 29.40 27.19 27.59 46.40 23.09 Polyunsaturated 40.68 29.10 17.42 39.63 20.85 29.98 EPA + DHA 35.17 19.20 0.38 0.37 6.28 3.74

KMMCC-33: Nannochloropsis sp., KMMCC-13: N. oceanica , KMMCC-16: Nannochoris oculata , KMMCC-119: Nannochoris sp., KMMCC-395: Nannochoris sp., KMMCC-120: Chlorella vulgaris , EPA: eicosapentaenoic acid, DHA: docosahexaenoic acid

In addition, among these microalgae, amino acid content and fatty acid content of the rotifers cultured with three microalgae, KMMCC 33, 119, 120 (new strain of the present invention), were shown in Tables 5 and 6, respectively. .

Analysis of amino acid content of rotifers cultured with each microalgae (%) KMMCC
No.
Amino acid 33 119 120
(The present invention)
New strain) Arginine 3.48 4.16 3.99 Histidine 1.24 1.39 1.37 Isoleucine 2.54 2.86 3.02 Leucine 4.08 4.5 4.83 Lysine 4.09 4.44 4.34 Phenylalanine 3.17 2.95 3.2 Throneine 2.24 2.21 2.59 Valine 3.10 3.58 3.67 Alanine 2.32 2.77 3.13 Aspartic acid 5.47 6.01 5.97 Glutamic acid 7.69 8.03 8.85 Glycine 2.03 2.45 2.57 Proline 3.87 3.85 4.08 Serine 2.68 2.72 3.10 Tyrosine 2.54 2.09 2.44 Total 50.54 54.01 57.15 EAA 23.94 26.09 27.01 NEAA 26.6 27.92 30.14 EAA / NEAA 0.90 0.93 0.90

KMMCC-33: Nannochloropsis sp., KMMCC-119: Nannochoris sp., KMMCC-120: Chlorella vulgaris , EAA: essential amino acid, NEAA: non-essential amino acid.

Analysis of fatty acid content of rotifers cultured with each microalgae (%) KMMCC
No.
Amino acid 33 119 120
(New invention strain) 8: 0 0.07 0.05 0.05 10: 0 0.06 0.03 - 11: 0 - - - 12: 0 0.40 0.14 0.06 13: 0 0.47 0.54 0.55 14: 0 4.49 2.94 2.27 14: 1 0.40 0.85 - 15: 0 0.56 0.56 - 15: 1 - 0.17 - 16: 0 14.32 15.94 12.63 16: 1 16.14 9.26 8.35 17: 0 0.49 0.47 0.51 17: 1 0.80 0.63 0.26 18: 0 3.06 3.29 2.98 18: 1n9 1.85 1.38 - 18: 2 n9 11.0 24.60 41.62 18: 3 n6 3.96 7.92 4.16 18: 3 n9 0.81 0.18 - 20: 0 - 7.38 0.68 20: 1 0.77 2.08 3.75 20: 2 1.36 2.08 2.43 20: 3 0.55 0.83 0.18 20: 4 - 1.26 0.12 21: 0 3.67 2.39 1.13 20: 5n-3 15.27 9.27 5.61 22: 0 - 0.37 0.30 22: 1 - 0.92 1.49 22: 2 11.12 - - 24: 0 5.11 3.14 1.51 22: 6n-3 3.29 0.80 9.39 TOTAL 100 100 100 Saturated 32.70 37.24 22.67 Monounsaturated 19.96 15.29 13.85 Polyunsaturated 47.36 46.94 63.51 EPA + DHA 8.56 10.07 15.00

KMMCC-33: Nannochloropsis sp., KMMCC-119: Nannochoris sp., KMMCC-120: Chlorella vulgaris , EPA: eicosapentaenoic acid, DHA: docosahexaenoic acid.

Among these results, in particular, the results of analyzing the amino acid content and fatty acid content in the rotifers, the total amino acid content of the rotifers cultured with each microalgae is C. The highest level of vulgaris KCTC 11964BP (KMMCC-120) was found to be 57.15% in the rotifers.

In addition, as a result of fatty acid analysis of rotifers, C18: 2n9 is C. new strain of the present invention. In the group using the vulgaris KCTC 11964BP (KMMCC-120) with 41.62% Nannochloropsis sp. (KMMCC-33) and Nannochloris sp. Compared to 11.0% and 24.60% of the (KMMCC-119) group, the rate was significantly higher. All of C16: 0 were 12.63 ~ 15.94%, which was relatively similar. Total PUFA content is also C. The highest was 63.51% in rotifers fed vulgaris KCTC 11964BP (KMMCC-120). EPA is Nannochloropsis sp. (KMMCC-33) was the highest in the group fed 15.27%, DHA was C. The highest was 9.39% in the rotifer fed vulgaris (KMMCC-120).

Based on these results, the present inventors identified C. strain C. , which was isolated and identified in the present invention. When vulgaris KCTC 11964BP is used as a microflora for rotifers, it promotes the growth of rotifers, and the amino acid and fatty acid components of rotifers are richer than those of other microalgae. From the point of view of the rotifers was found to be more suitable as a micro strain for feeding. As well as C. vulgaris KCTC 11964BP has the characteristics of active growth compared to other microorganisms even at a low temperature of 10 ℃ can be used as a food strain for the cultivation of rotifers, especially in winter.

     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 KCTC11964BP 20110614

<110> Industry Academic Cooperation Foundation of Pukyong National University <120> Novel Chlorella vulgaris capable low temperature growth and use          the <130> NP11-1219 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1672 <212> DNA <213> Chlorella vulgaris KCTC 11964BP_gene sequence <400> 1 ggctcattaa atcagttata gtttatttga tggtacttac tactcggata cccgtagtaa 60 atctagagct aatacgtgcg taaatcccga cttctggaag ggacgtattt attagataaa 120 aggccgaccg ggcttctgcc cgactcgcgg tgaatcatga taacttcacg aatcgcatgg 180 ccttgtgccg gcgatgtttc attcaaattt ctgccctatc aacttttgat ggtaggatag 240 aggcctacca tggtggtaac gggtgacgga ggattagggt tcgattccgg agagggagcc 300 tgagaaacgg ctaccacatc caaggaaggc agcaggcgcg caaattaccc aatcctgaca 360 cagggaggta gtgacaataa ataacaatac tgggcctttt caggtctggt aattggaatg 420 agtacaatct aaacccctta acgaggatca attggagggc aagtctggtg ccagcagccg 480 cggtaattcc agctccaata gcgtatattt aagttgctgc agttaaaaag ctcgtagttg 540 gatttcgggt gggacctgcc ggtccgccgt ttcggtgtgc actggcaggg ctcaccttgt 600 tgccggggac gggctcctgg gcttcactgt ccgggactcg gagtcggcgc tgttactttg 660 agtaaattag agtgttcaaa gcaggcctac gctctgaata cattagcatg gaataacacg 720 ataggactct ggcctatcct gttggtctgt aggaccggag taatgattaa gagggacagt 780 cgggggcatt cgtatttcat tgtcagaggt gaaattcttg gatttatgaa agacgaacta 840 ctgcgaaagc atttgccaag gatgttttca ttaatcaaga acgaaagttg ggggctcgaa 900 gacgattaga taccgtccta gtctcaacca taaacgatgc cgactaggga tcggtggatg 960 tttcttcgat gactccgccg gcaccttatg agaaatcaaa gtttttgggt tccgggggga 1020 gtatggtcgc aaggctgaaa cttaaaggaa ttgacggaag ggcaccacca ggcgtggagc 1080 ctgcggctta atttgactca acacgggaaa acttaccagg tccagacata gtgaggattg 1140 acagattgag agctctttct tgattctatg ggtggtggtg catggccgtt cttagttggt 1200 gggttgcctt gtcaggttga ttccggtaac gaacgagacc tcagcctgct aaatagtcac 1260 ggttggttcg ccagccggcg gacttcttag agggactatt ggcgactagc caatggaagc 1320 atgaggcaat aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact 1380 gatgcattca acgagcctag ccttggccga gaggcccggg taatcttcga aactgcatcg 1440 tgatggggat agattattgc aattattaat cttcaacgag gaatgcctag taagcgcaag 1500 tcatcagctt gcgttgatta cgtccctgcc ctttgtacac accgcccgtc gctcctaccg 1560 attgggtgtg ctggtgaagt gttcggattg gcgacctggg gcggtctccg ctctcggccg 1620 ccgagaagtt cattaaaccc tcccacctag aggaaggaga aacgtaacaa gg 1672

Claims (8)

Possible growth at a low temperature of 4 ℃ ~ 15 ℃ rotifers (Rotifer) Chlorella vulgaris for culture (Chlorella vulgaris ) KCTC 11964BP microalgae. The method of claim 1,
The rotifers are beuraki O Taunus replicon car subtilis (Brachionus plicatilis) rotifers (Rotifer) for culture Chlorella vulgaris, characterized in that (Chlorella vulgaris ) KCTC 11964BP microalgae. The method of claim 1,
The microalgae rotifers (Rotifer), comprising a step of increasing the content of amino acids and unsaturated fatty acids required for the growth of rotifers promote the growth of rotifers culture Chlorella vulgaris for (Chlorella vulgaris ) KCTC 11964BP microalgae. The method of claim 1,
The microalgae Chlorella vulgaris for rotifer (Rotifer) culture, characterized in that the salt has a salt resistance under the condition of 15 ~ 35psu (practical salinity unit) vulgaris ) KCTC 11964BP microalgae. Claim 1 Chlorella vulgaris (Chlorella vulgaris ) KCTC 11964BP microalgae or a composition for a rotifer (Rotifer) feed comprising a culture solution thereof. A formulation for fish feed comprising the composition for a Rotifer feed of claim 5 as an active ingredient. Method of culturing a large number of rotifers (Rotifer) during the November-February, which is a cold water season using the microalgae of claim 1. Chlorella vulgaris ( Clorella) characterized in that the microalgae of claim 1 are incubated at a temperature of 4 ℃ ~ 15 ℃ during November ~ February, which is a low temperature water temperature vulgaris ) mass culture method of KCTC 11964BP.

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