KR102162832B1 - Heterochlorella luteoviridis MM0014 and use thereof - Google Patents

Abstract

The present application relates to a novel microalgae, heterochlorella luteobiridis MM0014 strain and use thereof. The microalgae according to the present application is a novel strain having the ability to produce lipids including high concentrations of polyunsaturated fatty acids and saturated fatty acids, and biomass production capabilities including high concentrations of monosaccharides and proteins. These strains according to the present application can be effectively used in the development of biofuels, functional foods, natural pigments, medicinal substances, animal feed, and aquaculture feed.

Description

Heterochlorella luteoviridis MM0014 strain and use thereof {Heterochlorella luteoviridis MM0014 and use thereof}

The present application relates to a novel microalgae, heterochlorella luteobiridis MM0014 strain and use thereof.

Microalgae are photosynthetic organisms that use carbon dioxide, water, and solar energy to synthesize organic matter, which is the main energy factor, and produce oxygen. These microalgae can fix carbon dioxide in the atmosphere and have high protein and lipid content, so they can be used as biomass for food, feed, or fuel production. Specifically, lipids, sugars or proteins, which are the primary metabolites of microalgae, can be used as a source of various biofuels such as biodiesel, bioethanol or biogas, and as a raw material for polyunsaturated fatty acids (PUFAs). In addition, high value-added useful substances such as vitamins, carotenoids, polysaccharides, and lutein, which are secondary metabolites of microalgae, can be used as functional foods, natural pigments, medicinal substances, animal feeds, and aquaculture feed.

Microalgae have excellent carbon dioxide reduction effects, do not compete with farmland for the production of food resources, and have high production yields, and are recognized as an optimal material for bioenergy unlike land plants, but research on this is still insignificant.

Republic of Korea Patent No. 1525319 relates to'new microalatinium innermum NLP-F014 strain and uses thereof' and discloses a new microalgae capable of producing biofuels.

Republic of Korea Patent No. 1639467 relates to'a new microalgae Odontella and uses thereof', and a new microalgae having a high lipid content is disclosed.

Because microalgae can be photosynthesized, it has an excellent effect of reducing carbon dioxide, does not cause political and social problems related to food resources, and has a high production yield, and has the advantage of being an optimal eco-friendly material for bioenergy unlike land plants. Therefore, there is a need to develop novel microalgae with high production efficiency of materials that can be used as various useful resources.

The present application is to provide a novel microalgae with high production efficiency of materials that can be utilized as various useful resources.

In one embodiment, the present application is a base sequence of 18S rRNA (Ribosomal Ribo Nucleic Acids) represented by SEQ ID NO: 1, ITS (Internal Transcribed Spacer) represented by SEQ ID NO: 2, and rbc L (ribulose-bisphosphate carboxylase) represented by SEQ ID NO: 3 Having, it provides a microalgal heterochlorella luteobiridis MM0014 strain.

The sequences of SEQ ID NOs: 1, 2, and 3 are novel sequences that can be distinguished from other strains, which are characteristic of the microalgae according to the present application.

In one embodiment, the strain according to the present application is a heterochlorella luteobiridis MM0014 strain deposited with the accession number KCTC 13395BP.

The strain according to the present application is capable of producing high concentrations of lutein and beta-carotene, a high concentration of polyunsaturated fatty acids and a bio-oil containing saturated fatty acid, a high concentration of monosaccharide production, and a high concentration of organic matter and protein containing biomass production capacity. Have.

In one embodiment, the production capacity of lutein and beta-carotene of the strain according to the present application is 0.8±0.1 mg g ^-1 and 0.3±0.1 mg g ^-1 , respectively, based on the dry weight of the strain.

In one embodiment the strain according to the present invention is a polyunsaturated fatty acid, in particular omega-3 and omega-6, in particular linoleic acid (C18:2) and alpha linolenic acid (C18:3), respectively, about 35% and 16% of the total lipid weight. Can be produced with

In another embodiment, the strain according to the present invention can be produced as saturated fatty acid, in particular palmitic acid, in particular in a content of about 20% of the total lipid weight.

In another embodiment, the strain according to the present invention comprises a monosaccharide, particularly arabinose, which is a pentose, and the arabinose content may be produced at 44.9 mg g ^-1 based on the dry weight of the strain.

In another embodiment, the total calorific value of the biomass of the strain according to the present application is 19.7 MJ kg ^-1 and the protein is 51.5% by weight.

In another embodiment, the microalgae according to the present application has a culture temperature of 5-30° C. and a salt-free as well as NaCl resistance at a concentration of 0.5 M.

In another aspect, the present application also provides at least one heterochlorella luteo selected from the group consisting of 18S rRNA represented by SEQ ID NO: 1, which is a new sequence identified herein, ITS represented by SEQ ID NO: 2, and rbc L represented by SEQ ID NO: 3. Provides a nucleic acid for detection of Viridis MM0014 strain.

In addition, the present application includes all or partial sequences of one or more nucleic acids selected from the group consisting of 18S rRNA represented by SEQ ID NO: 1, ITS represented by SEQ ID NO: 2, and rbc L represented by SEQ ID NO: 3, and all of the one or more nucleic acids. Or it provides a nucleic acid for detection of a heterochlorella luteoviridis MM0014 strain having a sequence complementary to a partial sequence.

The sequences of SEQ ID NOs: 1, 2 and 3 are characteristic sequences that can distinguish microalgae according to the present application from other strains, for detection, isolation or identification of the strain according to the present application, for example, probes or primers therefrom. After design, synthesis, PCR (polymerase chain reaction) or hybridization (hybridization) by using a known method in the art, the strain according to the present invention can be used for detection, isolation or identification.

In another aspect, the present application provides a microbial preparation comprising the strain disclosed herein or a culture solution thereof, wherein the microbial preparation is beta-carotene, lutein, alpha linoleic acid, linolenic acid, palmitic acid, arabinose, for biomass production or each of the above It can be usefully used in the manufacture or production of health functional foods, biofuels, animal feeds, etc. using substances.

In another aspect, the present disclosure provides the steps of culturing the strain disclosed herein in suitable conditions suitable for lipid production; And it provides a lipid or bio-oil production method comprising the step of separating the lipid from the strain, or a polyunsaturated fatty acid or saturated fatty acid production / production method for separating linoleic acid, linoleic acid or palmitic acid from the lipid.

In another aspect, the present application provides a method for producing lutein or beta-carotene, comprising culturing the strain disclosed herein under appropriate conditions and separating lutein or beta-carotene from the culture medium.

Microalgae according to the present application has the ability to produce lipids including high concentrations of saturated and unsaturated fatty acids, high concentrations of lutein and beta-carotene, and is a novel strain having the ability to produce biomass containing high concentrations of monosaccharides and proteins. In addition, due to the characteristics of photothermal and euryhaline in cultivation, it has the advantage of being less affected by season and water quality when mass cultivating in outdoor cultivation facilities. These strains according to the present application can be effectively used in the development of biofuels, functional foods, natural pigments, medicinal substances, animal feed, and aquaculture feed.

1 is an optical microscope image of a heterochlorella luteobiridis MM0014 according to the present application.
2 is a scanning electron microscope photograph of a heterochlorella luteobiridis MM0014 according to the present application.
3 is a systematic relationship between the MM0014 strain and related species analyzed from the 18S rRNA sequence data according to the present application, and the phylogenetic tree was made through 1,000 bootstrap repetitions using the maximum likelihood method. Scale bars represent 1% difference in base sequence.
4 is a systematic relationship between the MM0014 strain and related species analyzed from the rbcL sequence data according to the present application, and the phylogenetic tree was made through 1,000 bootstrap repetitions using the maximum likelihood method. Scale bars represent 1% difference in base sequence.
5 is a result of analyzing the useful pigment component of the strain M0014 according to the present application by high-speed liquid chromatography. This is a natural carotenoid because the isolates of the present invention have high concentrations of antioxidants, lutein and beta-carotene. Lutein and beta, which are receiving more attention due to their ability to protect human eyes, as well as foods, medicines and cosmetics. It shows that it can be used effectively in the production of carotene.

The present application is based on the discovery of a novel single-celled green algae strain isolated from seawater, Heterochlorella luteoviridis MM0014 strain.

Green algae microalgae (green algae plant species) are the primary producers of marine ecosystems and play an important role in the global carbon, nitrogen and phosphorus cycle. Since microalgae can be converted into various high value-added organic substances by fixing carbon dioxide through photosynthesis, microalgae are attracting considerable attention in recent years, and accordingly, many studies to verify their potential as biotechnology resources have been conducted.

The microalgae heterochlorella luteoviridis disclosed herein is a green algae belonging to Heterochlorella luteoviridis , a domestic unrecorded species, aseptically isolated from the Jeongja Port in Ulsan. As a result of analyzing the morphological, molecular, and biochemical properties of microalgae disclosed herein, it was known as Chlorella luteoviridis in the past, but belongs to the heterochlorella luteoviridis re-established in 2009 as a new genus in the present application. It was identified as an unrecorded species with no official record in Korea, and was named heterochlorella luteoviridis MM0014 strain, and it was deposited with the Korean Collection for Type Cultures on July 13, 2017. No. KCTC13395BP). H. luteoviridis is currently the only taxonomically recognized species within the genus Heterochlorella, and was previously known only as a terrestrial species, but the first reported seawater species isolated from seawater.

The microalgae according to the present application exhibits very similar morphological characteristics to heterochlorella luteoviridis, such as the form of cup-shaped chloroplasts and spherical cells, as a result of optical and electron microscopy (Fig. 1, Fig. 2). In addition, the molecular biological analysis of the microalgae according to the present application was also found to belong to heterochlorella luteoviridis.

Accordingly, in one aspect, the present application relates to a novel microalgal heterochlorella luteobiridis MM0014 strain deposited under the accession number KCTC13395BP.

Microalgal heterochlorella luteoviridis MM0014 according to the present application has an 18S rRNA of SEQ ID NO: 1, ITS of SEQ ID NO: 2, and rbc L gene sequence of SEQ ID NO: 3.

Therefore, the sequence is a specific sequence present only in the strain according to the present application, and can be effectively used for the detection of the strain according to the present application, in this respect, the present application is a heterochlorella luteobiri of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 It relates to a nucleic acid or a composition for detection of diss MM0014 nucleic acid, or heterochlorella luteobiridis MM0014.

The microalgae according to the present application can survive in a wide temperature range (5-30° C.) and have NaCl resistance up to 0.5 M concentration. Due to the characteristics of light temperature and light salt properties, there is an advantage of being less affected by season and water quality when mass cultured in outdoor culture facilities, so it can be effectively used for the production of useful substances.

The microalgae according to the present application has a high content of beta-carotene and lutein production ability, a high content of polyunsaturated fatty acid and a bio-oil production ability including saturated fatty acid, a high concentration of organic matter and a biomass production ability including protein.

As used herein, “productive capacity” refers to an increase in the total amount of fatty acids per unit population or unit dry or wet weight of microalgae, an increase in the amount of saturated or polyunsaturated fatty acids, an increase in the amount of omega-3 or omega-6, or of alpha linoleic acid and linolenic acid. An increase in the amount, or an increase in the relative proportion of saturated or polyunsaturated fatty acids in the fatty acids produced, an increase in the relative proportions of omega-3 and omega-6 in the total fatty acids or polyunsaturated fatty acids produced, or an increase in the relative proportions of alpha linoleic and linolenic acids; Or an increase in the total biomass amount per unit population or unit dry or wet weight of microalgae, an increase in the amount of organic matter and protein, or an increase in the amount of arabinose, a pentose sugar, or an increase in the relative proportion of organic matter and protein in the produced biomass, or arabbi Increase in relative proportion of north; Or an increase in the total carotenoid or total pigment amount per unit population or unit dry or wet weight of microalgae, an increase in the amount of beta-carotene, an increase in the amount of lutein, or an increase in the relative proportion of beta-carotene in the produced carotenoids and lutein in the pigment. do. Any of the above may lead to an increase in the production of the desired useful substance. For example, when the relative ratio increases, the concentration of the desired useful substance increases as well as the increase, so that separation may be facilitated.

The “biomass” of the present application collectively refers to an organic material originating from microalgae that can be used to manufacture biofuels and useful materials. Such biomass may contain cellular and/or intracellular material as well as extracellular material. Extracellular substances include, but are not limited to, compounds secreted by cells.

As used herein, “biofuel” is a fuel obtained by using biomass as a resource and converting it into energy, and includes solid biofuel, liquid biofuel (bioil, bioethanol/butanol, biodiesel) and gaseous biofuel. .

Microalgae according to the present application has the ability to produce lipids or bio-oils including polyunsaturated fatty acids and saturated fatty acids at high concentration.

As used herein, "polyunsaturated fatty acid (PFUA)" refers to an unsaturated fatty acid containing two or more double bonds (π) having 18 or more carbon atoms. Polyunsaturated fatty acids such as omega-3 or omega-6 unsaturated fatty acids perform very important functions in the human body, but they are not synthesized naturally in the human body and must be consumed primarily through food.

In one embodiment, the microalgae according to the present application contain polyunsaturated fatty acids such as linoleic acid (C _18:2 ω6; Omega-6) and α-linolenic acid (linolenic acid, C _18:3 ω3; Omega-3). Contains in high content. In one embodiment, the omega 6 fatty acid linoleic acid is included in about 35.6%, the omega 3 fatty acid alpha linolenic acid is included in about 16.2%. Therefore, the microalgae according to the present application can be used for the production of omega-3 and omega-6 unsaturated fatty acids, replacing conventional vegetable oils, marine animal oils, fish oils and oilseeds. can do.

Polyunsaturated fatty acids, which have high production capacity of microalgae according to the present application, play an important role in human metabolism as a major component of cell membrane phospholipids and lipids for energy storage in cells. In addition, research results have been reported that transport substances that control inflammation, heart function and tumor growth are biosynthesized by polyunsaturated fatty acids. These properties suggest the possibility that polyunsaturated fatty acids can be used for nutritional and pharmaceutical purposes, and various commercial products containing these polyunsaturated fatty acids are widely used worldwide. Omega-3 unsaturated fatty acids are commonly derived from fish oil, and omega-6 unsaturated fatty acids are mostly obtained from plant sources such as sunflower, corn and soybean oil. Therefore, the microalgal strain of the present application can be used as a substitute for fish-derived resources, and thus has the potential as a raw material for a product for vegetarians.

In addition, microalgae according to the present application produces about 20.7% of palmitic acid, which is a saturated fatty acid. 16-carbon saturated palmitic acid, which is one of the major fatty acid components produced by the microalgae of the present application at high concentration, can be usefully used as a raw material for biofuel production.

Furthermore, as a result of analyzing the photosynthetic pigment composition of microalgae according to the present application, it was found that antioxidant carotenoid pigments, especially beta-carotene and lutein, were contained in high concentrations, and nutritionally important monosaccharide components were also biosynthesized.

Currently, major antioxidant carotenoid substances such as beta-carotene and lutein are mainly produced by chemical synthesis, but the demand for natural carotenoids in high value-added medicines, health functional foods, and cosmetics has increased, making it the main platform for producing these natural carotenoids. Can be used. In recent years, it can be effectively used for the production of lutein and beta-carotene, which are receiving more attention due to their protective function of human eyes. Accordingly, the microalgae according to the present application may be used as a raw material for functional foods, natural colors, and pharmaceutical substances.

In one embodiment, the lutein content of the strain MM0014 of the present application was about 0.8±0.1 mg g ^-1 based on the dry weight of microalgal biomass, and the beta carotene content was about 0.3±0.1 mg g ^-1 based on the dry weight of microalgal biomass. The isolate according to the present application indicates that the content of lutein is about 2 to 7 times and beta carotene is about 3 to 5 times more than spinach and kale (see Table 7), which are vegetables known to be rich in lutein and beta carotene.

In addition, the microalgae according to the present application biosynthesizes nutritionally important monosaccharide components. These monosaccharides include glucose, galactose, arabinoso and mannitol. In one embodiment, in particular, the strain according to the present application has a high production capacity of arabinose.

In one embodiment, the arabinose content (44.9 mg g ^-1 ) of the isolate according to the present application is the arabinose content (about 11.0-17.0 mg g ^-1 , Templeton) of the bagasse of sugarcane, which is the main raw material for the production of arabinose. DW et al. 2012. J. Chromatogr. A. 1270, 225-234). Therefore, the strain according to the present application is widely used in foods and pharmaceuticals as a natural low-calorie sweetener, and can be effectively used in the production of arabinose, a pentose monosaccharide, which is receiving high industrial attention due to the functions of foods and prebiotics for preventing hyperglycemia-related diseases in recent years. .

In addition, the microalgae according to the present application has a high protein content.

In one embodiment, as a result of elemental analysis, the total calorific value is 19.7 MJ kg ^-1 , which has a high protein content (51.5%), which indicates the possibility of excellent animal feed. In the animal feed, feed additives added to feed are blended in order to supply nutrients that are not synthesized in the body as essential amino acids for animal feed additives. The biomass of microalgae according to the present application can be used as a feed additive. H. luteoviridis, along with C. vulgaris and C. pyrenoidosa, were consumed as food in the European Union prior to 1997, and therefore Novel Food Regulation (EC) No. It does not fall under 258/97 and does not require a pre-commercial licensing process, making future commercial use useful in EU countries.

In addition, the total calorific value of microalgae herein indicates the possibility of microalgal biomass as a raw material for biofuels. The biomass of microalgae here is within the range of total calorific value of energy crops (17.0~20.0 MJ kg ^-1 ), which indicates that microalgal biomass production has a higher advantage than land plants when considering the high growth rate of microalgae. Show.

In another aspect, the present application also relates to a microbial preparation comprising the strain and/or a culture thereof disclosed herein, wherein the microbial preparation is the production of polyunsaturated fatty acids comprising omega-3 and/or omega-6, palmitic acid It can be effectively used for the production of saturated fatty acids, including biofuels, biomass production, beta-carotene production, lutein production, and animal feed production.

The culture medium of the strain according to the present application is a medium cultivated under appropriate conditions for the purpose of the microalgae according to the present application, the medium is a liquid composition containing nutrients necessary for culturing the microalgae and/or a substance for a special purpose, and the culture medium Silver includes all microalgae that have been removed after being cultured, or that include microalgae.

The strain according to the present application can be cultured in various media used for cultivation of microalgae. The medium used in the present invention is not limited thereto, but is preferably carried out in a medium containing a carbon source and a nitrogen source, seawater and sodium chloride, and the carbon source is glucose, fructose, galactose, glucose, glycerol, or a mixture thereof. It may be selected from the group consisting of, and the nitrogen source may be selected from the group consisting of sodium glutamate, peptone, tryptone, yeast extract, corn steep liquor, sodium nitrate, ammonium sulfate, ammonium citrate, or a mixture thereof. In one embodiment of the present application, a relatively inexpensive poor nutritional medium, R2A (Reasoner's 2A), is used for microalgal culture of the present application. The medium used for culturing the microalgae according to the present application is not limited thereto, but does not contain sodium chloride or may be cultured at a sodium chloride concentration of up to 0.5 M. The microalgae of the present application are not limited to fresh water because of their resistance to sodium chloride due to the nature of the seawater source, and abundant seawater resources can be used as a culture solution.

The microalgae according to the present application can survive in a wide temperature range (5-30° C.) and have NaCl resistance up to 0.5 M concentration. Due to the characteristics of photothermal and euryhaline, it has the advantage of being less affected by season and water quality when mass cultured in outdoor culture facilities, so it can be effectively used for the production of useful substances. have. In one embodiment, the microalgae according to the present application is 16 hours: 8 hours (day: night) cycle 18 while stirring at 160 rpm at 20 °C with a light intensity of about 40 μmole m ^-2 s ^-1 using a white fluorescent lamp in R2A medium. Incubated daily.

In another aspect, the present application also includes the steps of culturing the strain according to the present application at an appropriate temperature and medium conditions; And it relates to a method for producing polyunsaturated fatty acid comprising the step of separating the lipid from the strain and extracting the polyunsaturated fatty acid from the lipid. Culture method including the culture medium and temperature may refer to those disclosed herein, and separation or extraction may be performed using a method known in the art.

In another aspect, the present application also provides the steps of culturing the strain according to the present application under suitable temperature and medium conditions; And it relates to a beta-carotene or lutein production method using microalgae comprising the step of separating and extracting beta-carotene or lutein from the strain. Culture method including the culture medium and temperature may refer to those disclosed herein, and separation or extraction may be performed using a method known in the art.

In another aspect, the present application also relates to a method for producing biofuel using biomass derived from the strain according to the present application. As mentioned above, the biomass of the strain according to the present application can be a raw material for solid biofuels and liquid biofuels, and the method of producing biomaterials from them is described above and a method known in the art. It can be carried out, and the culture method may refer to that disclosed herein.

In addition, the present application provides a functional food composition for antioxidants comprising the microalgae or a culture medium thereof according to the present application as an active ingredient. When the present application uses microalgae as a food additive, the strain may be added as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the strain of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and there is no problem in terms of safety, so the active ingredient may be used in an amount above the above range. . In addition, the present application may include health functional foods. In addition to the active ingredients, various auxiliary ingredients such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamins It may contain vitamins such as C, vitamin D3, and vitamin E, and minerals such as copper, calcium, iron, magnesium, potassium, and zinc, or lactic acid bacteria. The amount that can be included as an active ingredient of the health functional food according to the present application may be appropriately selected according to age, sex, weight, condition, and symptoms of disease depending on the purpose, and, for example, includes about 0.01g to 10.0g per day for an adult. It is good to be, and you can obtain an antioxidant effect by ingesting a health functional food having such a content.

In addition, the present application relates to a feed additive for animals, especially fish, comprising the strain or a culture solution thereof as an active ingredient. Feed additives are added in a certain proportion to the basic feed. The basic feed may be composed of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix, mineral premix, etc. as main components. The vitamin premix may be composed of vitamin A, vitamin D, vitamin E, riboflavin and niacin, and the mineral premix may be composed of manganese, iron, zinc, calcium, copper, cobalt and selenium.

Hereinafter, examples are presented to aid in understanding the present invention. However, the following examples are provided for easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental materials and methods

Sample collection and microalgae separation

Seawater samples were collected in Jeongja Port, Jeongja-dong, Buk-gu, Ulsan (35°36'54.8"N,129°27'00.0"E) in October 2016. The water temperature of seawater was 22.7℃ and the salinity was 27.3 PSU. The sample was transferred to the laboratory and inoculated into 100 ml of BG-11 microalgal selective medium in a sterile 250 ml flask. In order to suppress the growth of bacteria, imipenem, a range antibiotic, was added to the medium at a concentration of 100 μg ml ^-1 . Flask is subjected to stationary culture under conditions of 16 hours: 8 hours (day: night) until the microalgae growth is evident under the conditions of about 40 μmole m ^-2 s ^-1 using a fluorescent lamp at 20°C. I did. Microalgal biomass was obtained by centrifuging 1.5 mL of microalgal culture solution at 3,000×g for 3 minutes. The centrifuged microalgal pellets were fractionated and inoculated on R2A agar medium containing imipenem at a concentration of 20 μg ml ^-1, and cultured in the same environment as the above-described culture conditions. Single colonies were aseptically fractionated on fresh R2A agar medium and this process was repeated until a sterile culture was obtained.

Formal sympathy

The purely isolated microalgal culture was cultured in R2A medium for 2 weeks. Live cells were harvested by centrifugation at 3,000×g for 3 minutes, washed three times with sterile distilled water, and observed at 1,000× magnification using a Zeiss Axio Imager.A2 optical microscope. For observation with a scanning electron microscope, 10 ml of culture solution was prepared at a concentration of about 1,000 cells per ml, and fixed with a final concentration of 2% (v/v) osmium tetraoxide for 10 minutes. The fixed cells were collected on a polycarbonate membrane filter having a pore size of 3 μm and washed three times with distilled water to remove the components of the medium. The membrane filter was dehydrated by treating ethanol in stages (10, 30, 50, 70, 100% and 100% twice) for 2 minutes each, and the sample dried with a supercritical dryer was used on an aluminum stub with conductive copper tape. Then, gold coating was performed using an ion sputter, and the morphological characteristics of the cell surface were observed with a scanning electron microscope.

Molecular biological identification

For molecular biological analysis, genomic DNA was extracted using the DokDo-Prep Genomic DNA kit, and further purification was performed with the Wizard Genomic DNA Clean-Up System. The NS1/NS8 and ITS1/ITS4 universal primers reported by White et al. were used to amplify 18S rRNA and ITS fragments, respectively. Because of the high conservancy of the rRNA sequence, the RuBisCO rbcL chloroplast gene was also amplified using the rbcL 7F and rbcL 1391R primers suggested by Verbruggen et al. Phylogenetic analysis using 18S rRNA and rbcL genes was performed using MEGA ver. This was done using the 6.0 software package. The nucleotide sequence of MM0014 related species was downloaded from the National Center for Biological Information (NCBI) database and sorted using ClustalW. Based on the Bayesian information standard, the best-fit nucleotide substitution model, Kimura 2-parameter + Gamma distributed, was selected for 18S rRNA, General Time Reversible + Gamma distributed, and rbcL for phylogenetic analysis. Using this model, the most likelihood tree was created through 1,000 bootstrap iterations. Auxenochlorella protothecoides MM0014 nucleotide sequences (MF040300 and MF043910) were used as subgroups in both phylogenies. The phylogenetic tree using the ITS sequence could not be prepared because sufficient data on Heterochlorella luteoviridis species did not exist in the public database. All gene analyzes were performed in triplicate, and the nucleotide sequence information obtained through this study 18s rRNA, ITS, and rbcL are each disclosed herein as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and until November 11, 2019. It was privately registered as MG491519, MG491520 and MG495094 in the U.S. National Center for Biological Information database (Table 1).

[Table 1]

Temperature and sodium chloride tolerance test

Pure cultures of heterochlorella luteobiridis were maintained through periodic subculture on R2A agar slope medium. A single colony of the MM0014 strain was split on R2A agar medium in triplicate and cultured for 21 days. As in Jang et al.'s method, the optimal growth temperature was determined by examining the survival and growth of MM0014 cells in the range of 5-35 degrees (5 degrees interval). The sodium chloride tolerance test was carried out using R2A agar medium at 20 degrees, and the experiment was conducted by adding sodium chloride at concentrations of 0.0 M, 0.5 M, 1.0 M, 1.5 M and 2.0 M.

Gas chromatograph mass spectrometry

The isolated strains were cultured heterotrophically for 18 days at 160 rpm using a shaking incubator at 20 degrees in R2A medium. For lipid analysis, biomass was harvested by centrifugation at 2,063 g. Samples were homogenized after lyophilization to increase lipid extraction efficiency. Lipid extraction was performed using a method developed by Breuer et al. Fatty acid composition was analyzed using a 7890A gas chromatograph (Agilent, Santa Clara, CA, USA) equipped with a 5975C mass selective detector. For lipid analysis, a DB-FFAP column (30 m, 250 μm ID, 0.25 μm film thickness; Agilent, Santa Clara, CA, USA) was used. The initial GV oven temperature was started at 50° C. and held for 1 minute. The temperature was increased by 10 degrees per minute to 200 degrees for 30 minutes, and then increased by 10 degrees per minute to 240 degrees and maintained for 20 minutes. The sample was injected with 1 μl at a split ratio of 20:1. Helium was used as the moving gas at a flow rate of 1 ml per minute. The mass spectrometry parameters are as follows: injector temperature: 250 degrees / source temperature: 230 degrees, electron impact mode, an acceleration voltage of 70 eV was used for ionization of the sample in the acquisition range 50-550 mz ^-1 . The identification of fatty acids was determined to be valid only with the result of a match value of 90% or more compared with the mass spectra of Wiley/NBS libraries.

Biomass characterization

The freeze-dried biomass sample is pulverized using a mortar and then ASTM No. It was homogenized using a 230 mesh (body size = 63 μm) precision standard mesh sieve. Elemental analysis was performed in duplicate using a Flash 2000 elemental analyzer to determine the carbon, hydrogen, nitrogen and sulfur content. The oxygen content was calculated by subtracting the content of carbon, hydrogen, nitrogen, sulfur and ash from 100%. The total caloric value was estimated by the formula proposed by Friedl: [total amount of heat generated ^{= 3.55C 2 - 232C - 2,230H +} 51.2C × H + 131N + 20,600 (MJ kg -1)

Example 1. Identification of heterochlorella luteoviridis MM0014 strain

The cells of this isolate were independent, non-motile, and spherical. The size of the cells was about 5 μm (young cells) to 10 μm (mature cells), and there were remarkable cup-shaped chloroplasts (FIGS. 1 and 2). Overall, the MM0014 strain exhibited typical morphological features of the heterochlorella luteobiridis species. Molecular biological identification results analyzed from 18S rRNA, ITS and rbcL sequences belonged to the heterochlorella luteobiridis group, and all molecular biological analysis results were consistent (Table 1, FIGS. 3 and 4). Therefore, this marine microalgal strain was identified as heterochlorella luteobiridis MM0014. This isolate was patented as KCTC13395BP to the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center.

Example 2. Establishment of optimal growth conditions of Heterochlorella Luteoviridis MM0014 strain

Heterochlorella luteoviridis MM0014 strain was able to grow in a temperature range of 5 to 30 degrees, and optimal growth was observed at room temperature (Table 2).

[Table 2]

It did not grow at a water temperature of 35 degrees or higher, and growth was inhibited at 5 degrees. In addition, the isolated strain was able to grow even at a concentration of 0.5 M sodium chloride, but did not survive at a concentration of 1.0 M or higher.

Example 3. Analysis of fatty acid composition of heterochlorella luteoviridis MM0014 strain

The fatty acid composition of Heterochlorella Luteoviridis MM0014 is shown in Table 3 based on the mean ± standard deviation of three repeated measurements.

[Table 3]

The main fatty acids in the isolated strain were C16:0 (20.7% ± 0.3%), C18:2 (35.6% ± 1.1%) and C18:3 (16.2% ± 0.7). In addition, unsaturated fatty acids such as C16:2 (2.5% ± 0.1%), C16:3 (2.8% ± 0.1%) and C18:1 (7.1% ± 6.0%) were also detected in the photosynthetic microorganism.

Example 4. Analysis of Biomass Components of Heterochlorella Luteoviridis MM0014 Strain

The elemental analysis results are shown in Table 4. The total calorific value and protein content based on the elemental analysis results were 19.7 MJ kg ^-1 and 51.5%, respectively.

[Table 4]

Example 5. Analysis of photosynthetic pigments of heterochlorella luteoviridis MM0014 strain

The useful pigment component of the strain according to the present application was analyzed by high-speed liquid chromatography. For this, pigment extraction was performed using a method developed by Zapata et al. (Zapata, M. et al. 2000. Mar. Ecol. Prog. Ser. 195, 29-45). Specifically, the photosynthetic pigment was extracted from 1 mg of the lyophilized biomass of this strain using 100% HPLC grade ethanol, and then filtered before injection with a Whatman PTFE syringe filter having a pore size of 0.2 μm. Samples were analyzed at 33 degrees using an Agilent 1260 Infinity HPLC system equipped with a Zorbax SB-C18 column (4.6×250 mm, 5-Micron). The mobile phase gradient is maintained at a flow rate of 1 ml min ^-1 using the method reported by Sanz et al. and methanol: 225 mM ammonium acetate (82:18, v:v) is used as solvent A and ethanol is used as solvent B. A sample of was injected and analyzed (Table 5 below). HPLC grade methanol and ethanol were used by Daejunghwa Geum Co., Ltd., and HPLC grade ammonium acetate was purchased and used by Fluka. Immediately before injection, 40 μl of HPLC grade water (Fisher) was added to 200 μl of the sample to prevent distortion of the initial peak. Standard colorants (chlorophyll a , chlorophyll b , lutein and beta-carotene) were purchased from Sigma-Aldrich. Lutein and beta-carotene content and composition were quantified by calculating the peak area from the calibration curve. Table 5 below shows the concentration gradient profile and mobile phase composition of solvents A and B.

[Table 5]

The pigment profile of heterochlorella luteobiridis MM0014 is shown in Table 6 below. As a result, the main pigments of the isolate according to the present application are chlorophyll a (Chl a , 43.6%±0.4%), chlorophyll b (Chl b , 11.3%±0.3%), lutein (27.0%±0.3%) and beta-carotene (5.2%). ±0.7%). Other fine peaks were not included in the analysis results. The lutein content of strain MM0014 was 0.8±0.1 mg g ^-1 based on the dry weight of microalgal biomass, and the beta-carotene content was 0.3±0.1 mg g ^-1 based on the dry weight of microalgal biomass. In Table 6,% indicates the content% of each pigment in the total pigment. The isolate according to the present application indicates that the content of lutein is about 2 to 7 times and beta carotene is about 3 to 5 times more than spinach and kale, which are vegetables known to be rich in lutein and beta-carotene [Table 7].

[Table 6]

[Table 7]

Carotenoid content in vegetables (unit: mg g ^-1 , US Department of Agriculture, 1998)

This indicates that the isolated strain of the present application has the ability to produce antioxidants lutein and beta-carotene at high concentration. Therefore, the strain according to the present application is a natural carotenoid, which is widely used in foods, pharmaceuticals, and cosmetics, and can be effectively used for the production of lutein and beta-carotene, which are receiving more attention due to the function of protecting human eyes in recent years.

Example 6. Analysis of Monosaccharide Components of Heterochlorella Luteoviridis MM0014 Strain

The useful monosaccharide components of the strain according to the present application were analyzed by liquid chromatography. 50 mg of freeze-resistant biomass was placed in a glass test tube, 2.5 ml of 2 N sulfuric acid was added, and then heated in a 94°C constant temperature water bath for 3 hours, and then sufficiently cooled at room temperature. The sample was transferred to a 50 ml tube and neutralized with an aqueous calcium carbonate solution (40 g 100 ml ^-1 ). The volume of the sample was adjusted to 5 ml, and 1 ml of the sample was filtered using a 0.2 μm syringe filter, and then transferred to a 1.5 ml tube. Samples were 0.01 M Ca-EDTA (50 mg l ^-1 ) at 90 degrees using the Alliance HPLC System equipped with Sugar-pak I (6.5×300 mm, Waters Co.) as a mobile phase at a flow rate of 0.2 ml min ^-1 While maintaining, 20 μl of the sample was injected and analyzed. Standard monosaccharides (sucrose, maltose, lactose, glucose, xylose, rhamnose, galactose, mannose, fructose, fucose, arabinose, mannitol, sorbitol and ribose) were purchased from Sigma-Aldrich. The content and composition of each monosaccharide was quantified by calculating the peak area from the calibration curve (Table 8 below).

[Table 8]

This indicates that the isolated strain of the present application has the ability to produce arabinose, and the content of arabinose of the isolated strain according to the present application (44.9 mg g ^-1 ) is arabinose of the bagasse of sugarcane, the main raw material for the production of arabinose. The content (about 11.0 to 17.0 mg g ^-1 , Templeton DW et al. 2012. J. Chromatogr. A. 1270, 225-234). Therefore, the strain according to the present application is widely used in foods and pharmaceuticals as a natural low-calorie sweetener, and can be effectively used in the production of arabinose, a pentose monosaccharide, which is receiving high industrial attention due to the functions of foods and prebiotics for preventing hyperglycemia-related diseases in recent years. .

Korea Research Institute of Bioscience and Biotechnology KCTC13395BP 20171117

<110> National Marine Biodiversity Institute of Korea <120> Heterochlorella luteoviridis MM0014 and use thereof <130> DP201812002P <150> KR 10-2018-0159818 <151> 2018-12-12 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1773 <212> DNA <213> Unknown <220> <223> Heterochlorella luteoviridis MM0014 <220> <221> rRNA <222> (1)..(1773) <400> 1 gtagtcatat gcttgtctca aagattaagc catgcatgtc taagtataaa ctgcttatac 60 tgtgaaactg cgaatggctc attaaatcag ttatagttta tttgatggta ctttcttact 120 cggataaccg taggaatatc tagagctaat acgtgcgtaa atcccgactc ctggaaggga 180 cgtatttatt agataaaaga ccgaccgggc ttgcccgatc cgcggtgaat catgataact 240 tcacgaatcg cacggcctcg agccggcgat gtttcattca aatttctgcc ctatcaactt 300 tcgatggtag gatagaggcc taccatggtt ttgacgggtg acggaggatt agggttcgat 360 tccggagagg gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat 420 tacccaatcc tgacacaggg aggtagtgac aagaaataac aataccgggc tttttcaagt 480 ctggtaattg gaatgagtac aatttaaaat ccttaacgag gatcaattgg agggcaagtc 540 tggtgccagc agccgcggta attccagctc caatagcgta tatttaagtt gttgcagtta 600 aaaagctcgt agttggattt cggatggggc tgatcggtcc acctatcggt gcgcactgat 660 gttgacccat ctcgctgtcg gggacgggct gctggacttc attgttcggc acccggagtc 720 gacgacgtca ctttgagtaa attagagtgt tcaaagcagg cctacgctct gaatacgtta 780 gcatggaatg acacgatagg actctggctt atcctgttgg tctgtgagac cggagtaatg 840 attaagaggg acagtcgggg gcattcgtat ttcattgtca gaggtgaaat tcttggattt 900 atgaaagacg aacttctgcg aaagcatttg tcaaggatgt tttcattaat caagaacgaa 960 agttgggggc tcgaagacga ttagataccg tcctagtctc aaccataaac gatgccgact 1020 agggatcggc aggtgtccaa tcaatgaccc tgccggcacc ttacgagaaa tcaaagtttt 1080 tgggttccgg ggggagtatg gtcgcaaggc tgaaacttaa aggaattgac ggaagaacac 1140 caccaggggt ggagcctgcg gcttaatttg actcaacacg ggaaaactta ccaggtccag 1200 acatagtgag gattgacaga ttgaaagctc tttcttgatt ctatgggtgg tggtgcatgg 1260 ccgttcttag ttggtgggtt gccttgtcag gttgattccg gtaacgaacg agacctcagc 1320 ctgctaaata gtcacgatcg gctcttgccg gtcgccaggc ttcttagagg gactatcggc 1380 gactagccga tggaagtgtg aggcaataac aggtctgtga tgcccttaga tgttctgggc 1440 cgcacgcgcg ctacactgat gcaatcaacg agcccagcct tggccgagag gcccgggtaa 1500 tcttgtaaac tgcatcgtga tggggctaga ctcttgcaat tattagtctt caacgaggaa 1560 tccctagtaa gcgcaagtca tcagcttgcg ttgattacgt ccctgttctt tgtacacacc 1620 gcccgtcgct cctaccgatt ggatgtgctg gtgaagcgtt cggattgatt gcaatcggtg 1680 gttctccgcc gttcgtgatc gagaagttcg ttaaaccctc ccatctagag gaaggagaag 1740 tcgtaacaag gtctccgtag gtgaacctgc gga 1773 <210> 2 <211> 706 <212> DNA <213> Unknown <220> <223> Heterochlorella luteoviridis MM0014 <220> <221> misc_feature <222> (1)..(706) <223> ITS <400> 2 tccgtaggtg aacctgcgga gggatcattg aatctattgg tctatatcgt ctggtgatta 60 gctgttcagt gccgccctcg gccttgacag ggcctctggc tggtgggaag gccagtggtc 120 tgtcgagggt agggtgctcc agcctctccg aggtctggtc tccctggtca ttcacgtgac 180 tggccccaaa ccccttttac tgacctagac ttgaatagct gaagcctggc tggtccagca 240 atgggcagcc aaaacaaccg agtgtacaac tctcaacaac ggatatcttg gctctcgcaa 300 cgatgaagaa cgcagcgaaa tgcgatactt aatgtgaatt gcagaattcc gtgaatcatc 360 gagtctttga acgcatattg cgcccgaggc ctcggccaag ggcacgcctg cctcagcgtc 420 ggttgacccc tcttgcctcc cttcatcggg tggcagatct ggcagtctct ctgtagccag 480 ggagtctgct gcagcacaga ggacttgaag gctctttcct ggtgtattcc aggaggggag 540 cggtttggta agtgacttca cagtcgcatg cctgccattc cctcgagcat ctacaagctc 600 cccacacgga ggcagcggct caggctgctg cccccgaccc ctcaaagttc gacctgagtt 660 cagacgagac cacccgctca acttaagcat atcaataagc ggagga 706 <210> 3 <211> 1385 <212> DNA <213> Unknown <220> <223> Heterochlorella luteoviridis MM0014 <220> <221> misc_feature <222> (1)..(1385) <223> rbcL <400> 3 ccacaaactg aaactaaagc gggtgcaggc tttaaagctg gtgttaaaga ttaccgttta 60 acgtacttta ctcctgacta tcaagtgaaa gaaactgata ttctggctgc ttttcgtatg 120 actcctcaac agggtgtacc gcctgaagaa gcaggtgctg cagttgctgc agaatcatct 180 acaggaacat ggactacagt ttggactgac ggtctgacaa gtctagatcg ttacaaagga 240 cgttgttacg atatcgaacc agtaccagga gaagatggtc aatatattgc atatgttgct 300 taccctctag atctatttga agaaggttcg gtaacgaacc tatttacttc aattgtagga 360 aacgtatttg gtttcaaagc actgcgtgca ctacgattag aagatttacg tattccgcct 420 gcgtatgtaa aaacattcca aggtgctcca catggtattc aggtagaacg tgataaacta 480 aacaaatacg gtcgttctct tttaggctgt acaattaaac caaaactagg tctatctgcg 540 aaaaactatg gtcgtgccgt ttacgaatgt ttacgtggtg gactggactt tacgaaagat 600 gacgaaaacg tgaattcaca accatttatg cgttggagag atcgtttcct atttgtagct 660 gaagctattt acaaatcgca agcagaaaca ggtgaggtaa aaggccacta cctaaacgca 720 acagcaggta cttgcgaaga gatgatgaaa agagcagaga ttgcaaaaga actgggtgtt 780 cccattatta tgcatgacta cctaacaggt ggatttactg caaacacttc gttatctcat 840 tactgccgcg ataacggact tcttttacac attcaccgtg caatgcacgc tgtaattgac 900 cgtcaaagaa accacggtat gcacttccgt gtactagcaa aagcattacg tttatcaggg 960 ggtgaccact tacactctgg aaccgttgta ggtaaactag aaggggaacg tgaagtaacg 1020 ttaggtttcg ttgaccttat gagagacgat tacattgaaa aagatagaag ccgtggtgtt 1080 tactttacac aagactgggt ttcacttcca ggtgttatgc cggttgcttc tggtggtatt 1140 cacgtttggc acatgccagc tctggttgaa atctttggag atgatgcttg tcttcaattt 1200 ggtggtggta ctctaggaca cccttggggt aacgcgccgg gtgcagcagc taaccgtgta 1260 gctcttgaag cttgtacaca agcgcgtaac gaaggtcgtg accttgcacg tgaaggtggc 1320 gatgttattc gcagtgcatg taaatggagc cctgaactgg cagccgcttg tgaagtttgg 1380 aaaga 1385

Claims (14)

Accession number having a base sequence of 18S rRNA (Ribosomal Ribo Nucleic Acids) represented by SEQ ID NO: 1, ITS (Internal Transcribed Spacer) represented by SEQ ID NO: 2, and rbc L (ribulose-bisphosphate carboxylase) represented by SEQ ID NO: 3 Heterochlorella Luteoviridis MM0014 strain deposited as KCTC 13395BP.
The method of claim 1,
The strain has the ability to produce high concentrations of lutein and beta-carotene, the ability to produce bio-oils containing high concentrations of polyunsaturated fatty acids and saturated fatty acids, the ability to produce high concentrations of monosaccharides, and the ability to produce biomass containing high concentrations of organic matter and protein. Phosphorus, heterochlorella luteobiridis MM0014 strain.
The method of claim 2,
Lutein and beta-carotene production capacity of the strain is 0.8 ± 0.1 mg g ^-1 and 0.3 ± 0.1 mg g ^-1 , respectively, based on the dry weight of the strain, heterochlorella luteobiridis MM0014 strain.
The method of claim 2,
Polyunsaturated fatty acids of the strain include linoleic acid (C18:2) and alpha linolenic acid (C18:3), and the linoleic acid (C18:2) and alpha linolenic acid (C18:3) are 35% and 16 of the total lipid weight, respectively. That is included in %, heterochlorella luteobiridis MM0014 strain.
The method of claim 2,
The saturated fatty acid of the strain is palmitic acid, and the palmitic acid is contained in 20.7% of the total lipid weight, heterochlorella luteoviridis MM0014 strain.
The method of claim 2,
The monosaccharide of the strain includes arabinose, which is a pentose, and the arabinose content is 44.9 mg g ^-1 based on the dry weight of the strain, heterochlorella luteobiridis MM0014 strain.
The method of claim 2,
The total calorific value of the biomass is 19.7 MJ kg ^-1 and the protein content is 51.5% wt%, heterochlorella luteobiridis MM0014 strain.
The method of claim 2,
The strain is a culture temperature of 5-30 ℃, which has a culture condition of NaCl concentration of 0 to 0.5 M, heterochlorella luteobiridis MM0014 strain.
A microbial preparation comprising the strain according to any one of claims 1 to 8 or a culture solution thereof,
The microbial preparation is beta-carotene, lutein, alpha linoleic acid, linolenic acid, palmitic acid, arabinose, or for the production of biomass, microbial preparation.
An antioxidant health functional food containing the strain extract according to any one of claims 1 to 8 as an active ingredient.
Animal feed containing the strain extract according to any one of claims 1 to 8 as an active ingredient.
Culturing the strain according to any one of claims 1 to 8, and extracting a pigment from the culture of the strain; And separating lutein or beta-carotene from the pigment, lutein or beta-carotene.
Culturing the strain according to any one of claims 1 to 8, and extracting bio-oil from the culture of the strain; And separating linoleic acid (C18:2) or alpha linolenic acid (C18:3) from the biooil.
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