KR20200072383A - Heterochlorella luteoviridis MM0014 and use thereof - Google Patents

Abstract

The present application relates to a novel microalga, Heterochlorella luteoviridis MM0014 strain and a use thereof. The microalga 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 including high concentrations of monosaccharides and proteins. The strain according to the present application can be effectively used in the development of biofuels, functional foods, natural pigments, medicinal substances, animal feed, aquaculture feed, and the like.

Description

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

The present application relates to a novel microalgae, heterochlorella luteobirides MM0014 strain and uses thereof.

Microalgae are photosynthetic organisms that synthesize carbon dioxide, water, and solar energy to produce organic energy, which is a major energy factor, and produce oxygen. These microalgae can fix carbon dioxide in the atmosphere and can be utilized as biomass for food, feed or fuel production because of high protein and lipid content. Specifically, lipids, sugars or proteins, which are primary metabolites of microalgae, can be used as a source of various biofuels such as biodiesel, bioethanol or biogas, and raw materials of 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 feed for fish farming.

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

Korean Registered Patent No. 1525319 relates to a'new micro-latinium inner NLP-F014 strain and its use' and a new microalgae capable of producing biofuel is disclosed.

Korean Registered Patent No. 1639467 relates to'a new microalgae odontella and its use' and a new microalgae with high lipid content is disclosed.

Microalgae have the advantage of being photosynthetic, so they have excellent carbon dioxide reduction effects, no political and social problems related to food resources, and high production yields. Therefore, it is necessary to develop new microalgae with high production efficiency of materials that can be utilized as various useful resources.

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

In one aspect, the present application provides 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 To provide a microalgae heterochlorella luteobiridis MM0014 strain.

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

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

The strain according to the present application is capable of producing high concentrations of lutein and beta-carotene, producing high concentrations of polyunsaturated fatty acids and saturated fatty acids, producing high concentrations of monosaccharides, and producing high concentrations of organic substances and proteins. Have

In one embodiment, the lutein of the strain according to the present application and the production capacity of the beta-carotene are 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 strains according to the present application are polyunsaturated fatty acids, 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, respectively. Can be produced.

In another embodiment, the strain according to the present application can be produced as saturated fatty acids, especially palmitic acid, especially in an amount of about 20% of the total lipid weight.

In another embodiment, the strain according to the present application comprises monosaccharides, particularly arabinose which is a pentasaccharide, and the arabinose content can 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 salt-free, NaCl resistance at a concentration up to 0.5 M.

In another aspect the present application is also one or more heterochlorella luteos selected from the group consisting of a new sequence identified herein, the 18S rRNA represented by SEQ ID NO: 1, the ITS represented by SEQ ID NO: 2 and the rbc L represented by SEQ ID NO: 3 Provided is a nucleic acid for detecting a Viridis MM0014 strain.

In addition, the present application is all or a partial sequence 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, all of the one or more nucleic acids Alternatively, a nucleic acid for detecting heterochlorella luteobiridis MM0014 strain having a complementary sequence to a partial sequence is provided.

The sequence of SEQ ID NO: 1, 2 and 3 is a characteristic sequence that can distinguish the microalgae according to the present application from other strains, for detection, separation or identification of the strain according to the present application, for example, probes or primers therefrom After designing, synthesizing, PCR (polymerase chain reaction) or hybridization (hybridization) can be used in the detection, isolation or identification of strains according to the present application using methods known in the art.

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

In another aspect, the present application comprises culturing the strain disclosed herein under suitable conditions suitable for lipid production; And it provides a method for producing a lipid or bio-oil comprising 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, wherein the strain disclosed herein is cultured under appropriate conditions, and lutein or beta-carotene is separated from the culture medium.

The microalgae according to the present application is a novel strain having a lipid production capacity including high concentrations of saturated and unsaturated fatty acids, a high concentration of lutein and beta-carotene, and a biomass production capacity comprising high concentrations of monosaccharides and proteins. In addition, due to the characteristics of photothermal (eurythermal) and photo-inflammatory (euryhaline) in culture, it has the advantage of being less affected by the season and water quality when mass cultured in outdoor culture facilities. These strains according to the present application can be effectively used in the development of biofuels, functional foods, natural pigments, medicinal materials, animal feeds, and fish feed for aquaculture.

1 is an optical microscope photograph of a heterochlorella luteobirides MM0014 according to the present application.
Figure 2 is a scanning electron microscope photograph of a heterochlorella luteobiridis MM0014 according to the present application.
Figure 3 is a phylogenetic relationship between the MM0014 strain and related species analyzed from the 18S rRNA sequence data according to the present application, the phylogenetic tree was made through 1,000 bootstrap iterations using the maximum likelihood method. The scale bar represents a 1% difference in sequence.
Figure 4 is a phylogenetic relationship between MM0014 strain and related species analyzed from rbcL sequence data according to the present application, the phylogenetic tree was made through 1,000 bootstrap iterations using the maximum likelihood method. The scale bar represents a 1% difference in sequence.
5 is a result of analyzing the useful pigment component of the M0014 strain according to the present application by high-speed liquid chromatography. This isolate is a natural carotenoid that has high concentration production capacity of lutein and beta-carotene, which are antioxidants, and lutein and beta, which are receiving more attention due to their ability to protect human eyes as well as food, medicine, and cosmetics. It can be used effectively for the production of carotene.

The present application is based on the discovery of a novel unicellular green algae strain, Heterochlorella luteoviridis MM0014 strain, isolated and identified from seawater.

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

The microalgae heterochlorella luteobiridis disclosed herein is a green algae belonging to the heterologella luteoviridis , a domestic unrecorded species aseptically isolated from the sperm port of Ulsan. As a result of analyzing the morphological, molecular, and biochemical properties of the microalgae disclosed herein, it was previously known as Chlorella luteoviridis, but belongs to the heterochlorella luteobiridis, which was redefined as a new genus in 2009. It was revealed that there is no official record in Korea, and it was named a heterochlorella luteobiridis MM0014 strain, and it was deposited with the Korea Collection for Type Cultures on July 13, 2017. Number KCTC13395BP). H. luteoviridis is currently the only taxonomically recognized species in the genus Heterochlorella, and the first known species of seawater isolated from seawater, although previously known only as terrestrial species.

The microalgae according to the present application show morphological characteristics very similar to heterochlorella luteobiridis, such as cup-shaped chloroplasts and spherical cell morphology, as a result of optical and electron microscopic examination (Fig. 1, Fig. 2). In addition, the microalgae according to the present application was also found to belong to heterochlorella luteobiridis as a result of molecular biological analysis.

In this aspect, the present application relates to a novel microalgae heterochlorella luteobiridis MM0014 strain deposited with accession number KCTC13395BP.

Microalgae heterochlorella luteobiridis MM0014 according to the present application has the 18S rRNA of SEQ ID NO: 1, the ITS of SEQ ID NO: 2, and the 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 aspect, the present application is the heterochlorella luteobiri of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. DIS MM0014 nucleic acid, or heterochlorella luteobidis MM0014 relates to a nucleic acid or composition for detection.

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 these characteristics of photo-temperature and photo-salt properties, it has the advantage of being less affected by the season and water quality when mass-cultured in outdoor culture facilities, and can be effectively used for producing useful materials.

The microalgae according to the present application has a high content of beta-carotene and lutein production, a high content of bio-oil production including polyunsaturated fatty acids and saturated fatty acids, and a biomass production ability including high concentrations of organic substances and proteins.

“Production capacity” as used herein refers to an increase in the total amount of fatty acids per unit of microalgae or per unit dry or wet weight, 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 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 acid and linolenic acid; Or increase in the total amount of biomass per microalgal unit population or unit dry or wet weight, increase in the amount of organic matter and protein, or increase in the amount of arabinose which is a pentose sugar, or increase in the relative proportion of organic and protein in the produced biomass, or Increased relative proportions of North; Or microalgal unit population or increase in total carotenoid or total pigment amount per unit dry or wet weight, increase in the amount of beta-carotene, increase in the amount of lutein, or increase in the relative proportion of beta-carotene in the produced carotenoid, lutein in the pigment do. Any one of the above may lead to an increase in the production of the desired useful substance, for example, when the relative ratio increases, the desired useful substance increases, as well as the concentration increases, and separation can be facilitated.

The term “biomass” as used herein refers to an organic material derived from microalgae that can be used in the production of biofuels and useful materials. Such biomass may contain extracellular and/or intracellular material. Extracellular materials include, but are not limited to, compounds secreted by cells.

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

Microalgae according to the present application has the ability to produce lipids or bio-oils containing high concentrations of polyunsaturated fatty acids and saturated fatty acids.

As used herein, “polyunsaturated fatty acid (PFUA)” means 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, play a very important function in the human body, but are not synthesized naturally in the human body and must be consumed mainly through food.

In one embodiment, the microalgae according to the present application are polyunsaturated fatty acids such as linoleic acid (C _18:2 ω6; Omega-6) and α-linolenic acid (C _18:3 ω3; Omega-3). High content. In one embodiment, the omega 6 fatty acid linoleic acid comprises about 35.6%, and the omega 3 fatty acid alpha linolenic acid comprises 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.

The polyunsaturated fatty acid having high production capacity of microalgae according to the present application plays an important role in the metabolic process of the human body as a major component of cell membrane phospholipids and lipids for energy storage in cells. In addition, research results have been reported that transporters 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 often extracted from fish oil, and omega-6 unsaturated fatty acids are mostly obtained from plant sources such as sunflower, corn and soybean oil. Therefore, the microalgae strain of the present application may be used as a substitute for fish-derived resources, and thus has potential as a raw material for vegetarian products.

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

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

Currently, major antioxidant carotenoids such as beta-carotene and lutein are mainly produced by chemical synthesis, but the demand for natural carotenoids in pharmaceuticals, health functional foods or cosmetics with high added value has increased, and as a main platform for producing these natural carotenoids. Can be used. Recently, it can be effectively used for the production of lutein and beta-carotene, which are getting more attention due to the function of protecting the human eye. Accordingly, the microalgae according to the present application may be used as raw materials for functional foods, natural pigments, and medicinal substances.

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

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

In one embodiment, the content of arabinose (44.9 mg g ^-1 ) of the isolates according to the present application is the arabinose content (about 11.0 to 17.0 mg g ^-1 , Templeton) of the bagasse of sugar cane, 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 food and medicine as a natural low-calorie sweetener, and in recent years, it can be effectively used for production of pentasaccharide monosaccharide arabinose, which is receiving high industrial attention due to functions such as hyperglycemia-related diseases prevention food and prebiotics. .

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 potential as an excellent animal feed. In the animal feed, a feed additive added to the feed is added to supply nutrients that are not synthesized in the body as essential amino acids for animal feed addition. Microalgae biomass according to the present application may 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, so Novel Food Regulation (EC) No. It is not applicable to 258/97 and there is no need for a pre-commercialization licensing process, so future commercial applications are useful in European Union countries.

In addition, the total heat generation amount of the microalgae of the present application represents the possibility of bioalgae as a biofuel raw material. The biomass of microalgae of the present application is within the total calorific value range of energy crops (17.0~20.0 MJ kg ^-1 ), which indicates that microalgal biomass production has a higher advantage than terrestrial plants when considering the high growth rate of microalgae. Shows.

In another aspect the present application also relates to a microbial agent, comprising the strains and/or cultures thereof disclosed herein, the microbial agent producing polyunsaturated fatty acids comprising omega-3 and/or omega-6, palmitic acid It can be effectively used for the production of saturated fatty acids containing, biomass production as a raw material for biofuel, beta-carotene production, lutein production, and animal feed production.

The culture medium of the strain according to the present application is a medium cultured under appropriate conditions for the microalgae according to the present application, and the medium is a liquid composition containing a nutritional substance and/or a substance for a specific purpose required for the culture of the microalgae, and the culture medium Silver microalgae include those that have been removed after culture or contain microalgae.

Strains according to the present application can be cultured in various media used for the culture of microalgae. The medium used in the present invention is not limited to this, but is preferably carried out in a medium containing a carbon source and a nitrogen source and seawater and sodium chloride, and the carbon source is glucose, fructose, galactose, glucose, glycerol, or a mixture thereof. 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 mixtures thereof. In one embodiment of the present application, R2A (Reasoner's 2A), which is a relatively low-cost nutrient medium for microalgae culture of the present application, is used. 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 up to 0.5 M. The microalgae of the present application are resistant to sodium chloride due to the nature of the seawater origin, and thus are not limited to fresh water, and abundant seawater resources can be used as a culture medium.

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 (eurythermal) and photo-inflammatory (euryhaline), it has the advantage of being less affected by the season and water quality when mass cultured in outdoor culture facilities, so it can be effectively used for the production of useful materials. have. In one embodiment, the microalgae according to the present application are stirred in a R2A medium using a white fluorescent lamp at a light intensity of about 40 μmole m ⁻² s ⁻¹ at 20° C. at 160 rpm for 16 hours: 8 hours (day: night), 18 Incubated daily.

In another aspect, the present application also comprises culturing the strain according to the present application at an appropriate temperature and medium condition; And separating lipids from the strain and extracting polyunsaturated fatty acids from the lipids. The culture method including the culture medium and temperature may refer to those disclosed herein, and separation or extraction may be performed using methods known in the art.

In another aspect, the present application also comprises culturing the strain according to the present application at an appropriate temperature and medium condition; And it relates to a method for producing beta-carotene or lutein using microalgae comprising the step of separating and extracting beta-carotene or lutein from the strain. The culture method including the culture medium and temperature may refer to those disclosed herein, and separation or extraction may be performed using methods known in the art.

In another aspect, the present invention also relates to a method for producing biofuel using biomass derived from a strain according to the present application. As mentioned above, the biomass of the strain according to the present application may be a raw material for solid biofuels and liquid biofuels, and methods for producing biomaterials therefrom using the aforementioned matters and methods known in the art It may be performed, and the culture method may refer to those disclosed herein.

In addition, the present application provides a functional food composition for antioxidant comprising a microalgae or a culture medium 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 used with other foods or food ingredients, and may be suitably used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to its purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of 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 health and hygiene purposes or for health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. . In addition, the present application may include a health functional food, in addition to the above-mentioned active ingredients, various auxiliary ingredients, for example, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamins Vitamins such as C, vitamin D3, and vitamin E, and minerals or lactic acid bacteria such as copper, calcium, iron, magnesium, potassium, and zinc. The amount that can be included as an active ingredient of the health functional food according to the present application can be appropriately selected according to the age, gender, weight, condition, and symptoms of the disease according to the purpose, for example, about 0.01g to 10.0g per adult basis It is good to be able to obtain an antioxidant effect by ingesting a health functional food having such a content.

In addition, the present application relates to feed additives for animals, particularly fish, which include the strain or a culture medium thereof as an active ingredient. The feed additive is added to the basic feed at a certain rate. The basic feed may consist of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix and mineral premix. Vitamin premix may be composed of vitamin A, vitamin D, vitamin E, riboflavin and niacin, and mineral premix may be composed of manganese, iron, zinc, calcium, copper, cobalt and selenium.

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

Example

Experimental materials and methods

Sampling and separation of microalgae

Seawater samples were collected in October 2016 at Jeongja-Jajeong Port (35°36'54.8"N,129°27'00.0"E) in Buk-gu, Ulsan. Seawater had a water temperature of 22.7°C and a salinity of 27.3 PSU. The sample was transferred to a laboratory and inoculated into 100 ml of BG-11 microalgae selection medium in a sterilized 250 ml flask. To inhibit the growth of bacteria, imipenem, a range antibiotic, was added to the medium at a concentration of 100 μg ml ⁻¹ . The flask was subjected to static culture until the growth of microalgae was pronounced under conditions of 16 hours: 8 hours (day: night) by using a fluorescent lamp at 20°C and giving a light amount of about 40 μmole m ^-2 s ^-1 . Did. The microalgal biomass was obtained by centrifuging the 1.5 mL microalgae culture at 3,000 x g for 3 minutes. The centrifuged microalgae pellets were inoculated by stroke-fractionation on an 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. A single colony was aseptically fractionated onto fresh R2A agar medium and the process was repeated until an aseptic culture was obtained.

Morphological sympathy

Purely isolated microalgae cultures were cultured in R2A medium for 2 weeks. The living cells were harvested by centrifugation at 3,000 x g for 3 minutes, washed three times with sterile distilled water, and observed at a magnification of 1,000 x using a Zeiss Axio Imager.A2 optical microscope. For observation under a scanning electron microscope, 10 ml of the culture solution was prepared at a concentration of about 1,000 cells per ml and fixed at 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 treated with ethanol stepwise (10, 30, 50, 70, 100%, and 100% twice) for 2 minutes each to perform dehydration, and the supercritical dryer used a conductive copper tape on an aluminum stub to dry samples. Then, gold coating was performed using an ion sputter to observe the morphological characteristics of the cell surface using a scanning electron microscope.

Molecular biology identification

For molecular biological analysis, genomic DNA was extracted using the DokDo-Prep Genomic DNA kit and additional purification was performed with the Wizard Genomic DNA Clean-Up System. NS1/NS8 and ITS1/ITS4 universal primers reported by White et al. were used to amplify 18S rRNA and ITS fragments, respectively. Due to the high conservation of the rRNA sequence, the RuBisCO rbcL chloroplast gene was amplified using the rbcL 7F and rbcL 1391R primers proposed by Verbruggen et al. Systematic analysis using 18S rRNA and rbcL genes was performed using MEGA ver. 6.0 software package. The base sequences of MM0014 related species were downloaded from the National Biological Information Center (NCBI) database and sorted using ClustalW. Based on Bayesian information criteria, the best-fit nucleotide substitution model, Kimura 2-parameter + Gamma distributed, was selected for 18S rRNA, General Time Reversible + Gamma distributed, and rbcL, respectively, for systematic analysis. Using this model, the highest-order phylogenetic tree was created through 1,000 bootstrap iterations. Auxenochlorella protothecoides MM0014 nucleotide sequences (MF040300 and MF043910) were used as an outgroup in two phylogenetic trees. Phylogenetic tree using ITS sequence was not prepared because there is not enough data for Heterochlorella luteoviridis species in public database. All gene analyzes were conducted in 3 repetitions, and the sequencing information obtained through this study, 18s rRNA, ITS, and rbcL were disclosed herein as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, until November 11, 2019 Privately registered in the U.S. National Biological Information Center database as MG491519, MG491520 and MG495094 (Table 1).

[Table 1]

Temperature and sodium chloride resistance test

The pure culture of heterochlorella luteobiridis was maintained through periodic passages on R2A agar slope media. A single colony of strain MM0014 was streaked in 3 replicates on R2A agar medium and cultured for 21 days. As in the method of Jang et al., the optimal growth temperature was determined by examining the survival and growth of MM0014 cells in the range of 5-35 degrees (5 degree intervals). The sodium chloride resistance test was conducted using an R2A agar medium at 20 degrees, and experiments were conducted by adding 0.0 M, 0.5 M, 1.0 M, 1.5 M, and 2.0 M sodium chloride.

Gas chromatograph mass spectrometry

The isolated strain was cultured for 18 days in a heterotrophic manner at 160 rpm using a shake incubator at 20 degrees in R2A medium. For lipid analysis, biomass was harvested by centrifugation at 2,063 g. The sample was lyophilized and then homogenized to increase the lipid extraction efficiency. For the lipid extraction, a method developed by Breuer et al. was used. Fatty acid composition was analyzed using a 7890A gas chromatograph (Agilent, Santa Clara, CA, USA) equipped with a 5975C mass selective detector. For the 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 started at 50° C. and was maintained for 1 minute. The temperature was increased by 10 degrees per minute for 30 minutes to 200 degrees, and then raised by 10 degrees per minute to 240 degrees for 20 minutes. The sample was injected with 1 μl at a split ratio of 20:1. Helium was used as the mobile gas at a flow rate of 1 ml per minute. The mass spectrometry factors are as follows: injector temperature: 250 degrees / source temperature: 230 degrees, electron impact mode was used for ionization of samples in the acquisition range 50-550 mz ^-1 with an acceleration voltage of 70 eV. The fatty acid was identified as valid only when the match value was over 90% compared to the mass spectra of the Wiley/NBS libraries.

Biomass Characterization

The lyophilized biomass sample was ground using a pestle and then ASTM No. Homogenized using a 230 mesh (body size = 63 μm) precision standard mesh. Elemental analysis was carried out in two iterations using the Flash 2000 Elemental Analyzer to determine the carbon, hydrogen, nitrogen and sulfur contents. The oxygen content was calculated by subtracting the contents 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 luteobidis MM0014 strain

Cells of this isolate were independent, non-motile, and spherical. Cells ranged in size from about 5 μm (young cells) to 10 μm (mature cells) with significant cup-shaped chloroplasts (FIGS. 1 and 2 ). Overall, the MM0014 strain exhibited typical morphological characteristics of the heterochlorella luteobiris species. Molecular biological identification results analyzed from 18S rRNA, ITS and rbcL base sequences The isolated strains belong to the Heterochlorella luteobiridis group and all molecular biological analysis results were consistent (Table 1, Figures 3 and 4). Therefore, this marine microalgae strain was identified as Heterochlorella luteobidis MM0014. This isolate was deposited as a patent for KCTC13395BP to the Korea Research Institute of Bioscience and Biotechnology's Biological Resource Center.

Example 2. Establishment of optimal growth conditions for the heterochlorella luteobidis MM0014 strain

Heterochlorella luteoviridis MM0014 strains were 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 temperature of 35 degrees or higher, and growth was inhibited at 5 degrees. In addition, the isolates were able to grow up to a concentration of 0.5 M sodium chloride, but did not survive at a concentration above 1.0 M.

Example 3. Fatty acid composition analysis of heterochlorella luteoviridis MM0014 strain

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

[Table 3]

The main fatty acids of the isolated strains 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 luteobidis MM0014 strain

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

[Table 4]

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

The useful pigment component of the strain according to the present application was analyzed by high-speed liquid chromatography. To this end, 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 using 100% HPLC grade ethanol from the biomass of the 1 mg lyophilized strain and filtered before injection with a Whatman PTFE syringe filter with 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 60 μl using methanol: 225 mM ammonium acetate (82:18, v:v) as solvent A and ethanol as solvent B. The sample was injected and analyzed (Table 5 below). HPLC grade methanol and ethanol were used by Daejung Chemical 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 pigments (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 the mobile phase composition of solvents A and B.

[Table 5]

The pigment profile of heterochlorella luteoviridis MM0014 is shown in Table 6 below. As a result, the main pigments of the isolates 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 found to be 0.8±0.1 mg g ^-1 based on the dry weight of the microalgae biomass, and the beta-carotene content was found to be 0.3±0.1 mg g ^-1 based on the dry weight of the microalgae biomass. In Table 6,% represents the content of each pigment in the total pigment. The isolates according to the present application show that lutein has a content of about 2 to 7 times, and beta-carotene has a content of about 3 to 5 times more than spinach and kale, which are known to be rich in lutein and beta-carotene [Table 7].

[Table 6]

[Table 7]

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

This indicates that the isolates of the present application have a high concentration of antioxidant lutein and beta-carotene. Therefore, the strain according to the present application is a natural carotenoid, and is widely used in food, medicine, and cosmetics, and can be effectively used in the production of lutein and beta-carotene, which are recently receiving more attention due to a function of protecting the human eye.

Example 6. Analysis of monosaccharide components of heterochlorella luteobidis MM0014 strain

The useful monosaccharide component of the strain according to the present application was analyzed by liquid chromatography. 50 mg of freeze-tightened biomass was placed in a glass test tube, and 2.5 ml of 2N sulfuric acid was added, followed by heating in a 94-degree constant temperature water bath for 3 hours and sufficiently cooling at room temperature. The sample was transferred to a 50 ml tube, and then 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. For the sample, a flow rate of 0.2 ml min ^-1 with 0.01 M Ca-EDTA (50 mg l ^-1 ) as a mobile phase at 90 degrees using an Alliance HPLC System equipped with Sugar-pak I (6.5×300 mm, Waters Co.) And analyzed by injecting 20 μl of the sample. 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 isolate strain of the present application has the production capacity of arabinose, and the content of arabinose (44.9 mg g ^-1 ) of the isolate strain according to the present application is arabinose of bagasse of sugar cane, which is the main raw material for the production of arabinose. 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 food and medicine as a natural low-calorie sweetener, and in recent years, it can be effectively used for production of pentasaccharide monosaccharide arabinose, which is receiving high industrial attention due to functions such as hyperglycemia-related diseases prevention food and prebiotics. .

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)

Deposition number having the base sequence of 18S rRNA (Ribosomal Ribo Nucleic Acids) represented by SEQ ID NO: 1, Internal Transcribed Spacer (ITS) represented by SEQ ID NO: 2, and rbc L (ribulose-bisphosphate carboxylase) represented by SEQ ID NO: 3 Heterochlorella luteobiris MM0014 strain deposited as KCTC 13395BP.
According to claim 1,
The strain having a high concentration of lutein and beta-carotene production capacity, a high concentration of polyunsaturated fatty acids and saturated fatty acids, bio-oil production capacity, high concentration of monosaccharides production capacity, high concentration of organic matter and biomass production capacity containing proteins Phosphorus, heterochlorella luteobidis MM0014 strain.
According to claim 2,
The production capacity of the lutein and the beta-carotene 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, the heterochlorella luteobiridis MM0014 strain.
According to claim 2,
The 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, respectively, of the total lipid weight. What is included in %, heterochlorella luteoviridis MM0014 strain.
According to claim 2,
The saturated fatty acid of the strain is palmitic acid, wherein the palmitic acid is contained in 20.7% of the total lipid weight, heterochlorella luteobidis MM0014 strain.
According to claim 2,
The monosaccharide of the strain includes arabinose which is a pentasaccharide, and the content of arabinose is 44.9 mg g ^-1 based on the dry weight of the strain, heterochlorella luteobiridis MM0014 strain.
According to claim 2,
The total heat generation amount of the biomass is 19.7 MJ kg ^-1 and the protein content is 51.5% by weight, heterochlorella luteobiris MM0014 strain.
According to claim 2,
The microalgae is a culture temperature of 5-30 ℃, having a culture condition of NaCl concentration of 0 to 0.5 M, heterochlorella luteobiridis MM0014 strain.
A microorganism preparation comprising a strain according to any one of claims 1 to 8 or a culture medium thereof,
The microbial agent is beta-carotene, lutein, alpha linoleic acid, linolenic acid, palmitic acid, arabinose, or biomass production, microbial agent.
A health functional food for antioxidant 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.
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 bio-oil.
18S rRNA represented by SEQ ID NO: 1, ITS represented by SEQ ID NO: 2 and rbc L represented by SEQ ID NO: 3 All or a partial sequence of one or more nucleic acids selected from the group consisting of, all or a partial sequence of the one or more nucleic acids Nucleic acid for detecting heterochlorella luteobiridis MM0014 strain having a sequence complementary to it.

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