KR20190088746A - A novel Scenedesmus obliquus ABC-009 strain having high biomass and lutein productivity and uses thereof - Google Patents

Abstract

The present invention relates to a Scenedesmus obliquus ABC-009 strain with high biomass and lutein productivity and uses thereof. The Scenedesmus obliquus ABC-009 strain of the present invention has a high production rate of lutein, which is important for human health, particularly for eyes. In addition, since lutein is widely used as a raw material for food additives or cosmetics, the industrial utilization value thereof is high, so the present invention is very useful in related industries.

Description

[0001] The present invention relates to a strain of Senesmus obblus ABC-009 having high biomass and lutein productivity and a use thereof.

The present invention relates to a strain of Senesmus obovirus ABC-009 having high biomass and lutein productivity and its use.

Lutein is a type of xanthophyll family that is produced only in plants and microalgae. In addition, zeaxanthin (zeaxanthin) and only one double bond site is another isomer, and generally has poor solubility in water. Lutein is one of the main constituents of the macula of the eye, although it can not be synthesized in the body of animals, but it protects the eye by absorbing ultraviolet light, especially blue light, that stimulates the eye, . Therefore, a sufficient amount of lutein should be ingested through diet and consumed directly from plants / microalgae such as spinach, kale, and chlorella, or indirectly from predators that feed on these plant / microalgae. However, because of problems such as personal preference and time longevity, it is difficult to obtain a sufficient amount of lutein, so supplements and health supplement products are gaining popularity. Especially in the modern society, as computers and mobile phones have been used for a long time, the importance of lutein that can protect the eyes from blue light, which is a type of ultraviolet rays, is more emphasized.

The global lutein market is estimated at about $ 260 million in 2017 and is expected to grow to more than $ 350 million by 2022 (Lutein Market by Form (Powder & Crystalline, Oil Suspension, Beadlet, Emulsion ), Source (Natural, Synthetic), Application (Food, Beverages, Dietary Supplements, Animal Feed), Production Process, and Region - Global Forecast to 2022). Currently, the industrial production of lutein is mainly dependent on anchovy and other fish, and some plants such as Hong Hwangcho. Uneven catches and overly required land area are becoming a problem. However, it has been reported that the micro-algae produced lutein is about 3 to 6 times more economical than that produced by plants, and that it can be cultured with less land and industrial water.

In the meantime, Korean Patent Publication No. 2012-0007655 discloses a new strain of Semenesmus obliquus YSW15, Korean Laid-open Patent Application No. 2017-0096218 discloses a new microalgae food composition, As in the present invention, there is no known anything about the Sennesmus obblicus ABC-009 strain having high biomass and lutein productivity and its use.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and it is an object of the present invention to provide a novel Scenedesmus Obliquus ABC-009 strain was isolated and identified, and the strain was found to be much more useful as a strain for producing lutein than the other strains of Sennheis mussus which have different biomass and lutein productivity, thus completing the present invention .

In order to accomplish the object of the present invention, the present invention relates to a method for producing a biodegradable biodegradable polymer, Obliquus ) ABC-009 strain (Accession No. KCTC 18649P).

The present invention also provides a microorganism preparation for producing lutein comprising the strain, a culture solution thereof, a concentrate of the culture solution or a dried product of the culture solution as an effective ingredient.

The present invention also provides a method for producing lutein comprising culturing the strain, and recovering lutein from the cells and the culture medium.

Further, the present invention provides a food containing the strain, a culture solution thereof, a concentrate of the culture solution, or a dried product of the culture solution as an active ingredient.

The present invention also provides a feed additive comprising the strain, a culture thereof, a concentrate of the culture solution, or a dried product of the culture solution as an effective ingredient.

The inventive Scenedesmus < RTI ID = 0.0 > Obliquus ) ABC-009 strains have a high production rate of human lutein, especially in eyeballs. In addition, since lutein is widely used as a raw material for food additives and cosmetics, the lutein is highly useful in industry, and therefore, the present invention is very useful for related industries.

Fig. 1 shows the result of observing the microalgae ABC-009 selected in the present invention with an optical microscope (DM2500, Leica, Germany).
Fig. 2 shows the results of analysis of the phylogenetic tree by the 18S rRNS nucleotide sequence obtained from the microalgae ABC-009 selected in the present invention.
FIG. 3 shows the results of analyzing the biomass productivity of the microalgae ABC-009 selected in the present invention.
FIG. 4 shows the results of HPLC analysis of photosynthetic pigments in the extracts extracted from the strains after culturing the microalgae ABC-009 selected in the present invention.

In order to accomplish the object of the present invention, the present invention relates to a method for producing a biodegradable biodegradable polymer, Obliquus ) ABC-009 strain (Accession No. KCTC 18649P).

The above-mentioned Scenedesmus Obliquus ) ABC-009 strain (accession number KCTC 18649P) was collected from the Sumjin River, and single colonies were selected and identified through 18S rRNA gene sequencing analysis, and it was confirmed that the cell line was highly biomass and lutein productivity. Scribes obliquus ) ABC-009 strain was deposited on Jan. 01, 2018 by the Korea Research Institute of Bioscience and Biotechnology (KCTC) (Accession No .: KCTC 18649P).

The novel strain of Senethesmus obculus ABC-009 of the present invention exhibits high growth (biomass productivity) at room temperature, and can over-accumulate photosynthesis pigment in heterotrophic and photoindependent.

In the strain according to an embodiment of the present invention, the biomass productivity is 210 to 250 mg / L / d, the lutein productivity is 0.75 to 0.85 mg / L / d, preferably the biomass productivity is 225 to 235 mg / L / d, and the lutein productivity may be 0.80 to 0.83 mg / L / d, but is not limited thereto.

In the strain according to one embodiment of the present invention, the content of lutein per dry cell weight of the strain may be 3.0 mg / g or more, preferably 3.0 to 4.0 mg / g, more preferably 3.3 to 3.7 mg / g, and most preferably from 3.4 to 3.6 mg / g, but is not limited thereto.

The present invention also provides a microorganism preparation for producing lutein comprising the strain, a culture solution thereof, a concentrate of the culture solution or a dried product of the culture solution as an effective ingredient. The microbial agent may be selected from the group consisting of Scenedesmus obliquus ABC-009 strain (Accession No. KCTC 18649P) as an active ingredient and can be effectively used for mass production of lutein.

The present invention also provides a method for producing lutein comprising culturing the strain, and recovering lutein from the cells and the culture medium. The method of separating lutein from the culture medium of the strain may be performed by high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC), and any method known in the art may be used.

Further, the present invention provides a food containing the strain, a culture solution thereof, a concentrate of the culture solution, or a dried product of the culture solution as an active ingredient. Since the lutein has excellent eye health improving effect, the strain producing the lutein, the culture solution thereof, the concentrate of the culture solution or the food containing the dried product of the culture solution is highly nutritious. The food may be used in the form of a food, a processed food, or the like, and may be suitably used according to a conventional method. The food, the culture, the concentrate of the culture, or the dried product of the culture may be used as it is, . There is no particular limitation on the kind of the processed food. Examples of the food to which the strain, the culture liquid thereof, the concentrated liquid of the culture liquid or the dried material of the culture liquid can be added include meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, Dairy products, including soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include processed foods in the conventional sense.

The present invention also provides a feed additive comprising the strain, a culture thereof, a concentrate of the culture solution, or a dried product of the culture solution as an effective ingredient. In one embodiment of the invention, the feed additive may additionally contain a carrier which is acceptable for the feed. In the present invention, the feed additive may be added as it is, or a known carrier, stabilizer, etc. may be added. If necessary, various nutrients such as vitamins, amino acids and minerals, antioxidants, antibiotics, antibacterial agents and other additives And the shape thereof may be a suitable state such as powder, granule, pellet, suspension and the like.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1. Collection and selection of microalgae

Microalgae samples were collected in 1L bottles from the Sumjin River basin in Namwon, Jeolla province and concentrated in a laboratory using a centrifuge. In order to screen microalgae of a single species, they were separated using a microscope and a Pasteur pipette, and a dilute passage was repeated on a TAP agarose culture plate containing 100 μg / mL of ampicillin to obtain a sterile single strain ABC-009 . The composition of the TAP culture used in the experiment is as follows; L Tris, 0.375 g / L NH ₄ Cl, 0.1 g / L MgSO ₄ .7H ₂ O, 0.05 g / L CaCl ₂ .2H ₂ O, 0.0108 g / LK ₂ HPO ₄ , 0.0054 g / L KH ₂ L PO ₄ , 1 mL / L glacial acetic acid, and 1 mL / L Hutner's trace elements (50 g / L Na ₂ EDTA.H ₂ O, 22 g / L ZnSO ₄ .7H ₂ O, 11.4 g / LH ₃ BO ₃ , 5.06 g / L MnCl _{2 · 4H 2 O, 1.61g /} L CoCl 2 · 6H 2 O, 1.57g / L CuSO 4 · 5H 2 O, 1.10g / L (NH 4) 6Mo 7 O 24 · 7H 2 O and 4.99g / L FeSO ₄ · 7H ₂ O)

Example  2. Identification of isolate ABC-009

As a result of observing the selected microalgae ABC-009 with an optical microscope (DM2500, Leica, Germany), it was found that spherical single cells were morphologically composed of Chlorella sp. Or Scenedesmus sp. (Fig. 1). DNA was extracted with DNeasy Plant Mini kit (Qiagen, Hilden, Germany) in order to confirm the exact identity and novelty thereof, and PCR was performed using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 below. The two primers used in this experiment were the method of Kim et al. (2016 Biomass Bioenerg. 85: 300-305). 18SF: 5'-CCT GGT TGA TCC TGC CAG-3 '(SEQ ID NO: 1), 18SR: 5'-TTG ATC CTT CTG CAG GTT CA-

The result obtained through the PCR was cloned and the sequence was read as SEQ ID NO: 3, confirming that it was a novel species not found to date. As a result of the analysis of the phylogenetic tree using UPGMA and Maximum likity method, the new species was found to be related to Scenedesmus sp. , Especially Scenedesmus obliquus , (Fig. 2). The isolated strain was designated as Scenedesmus obliquus ) ABC-009, deposited with KCTC on Jan. 01, 2018 under the deposit number KCTC 18649P.

Example  3. Senethesmus Oblickus  Measurement of growth rate of ABC-009

To determine the growth rate of new Senesdes mousseblicus ABC-009, the cells were cultured in TAP medium for 9 days. The incubation was carried out using a light incubator with a light amount of 120 μmol photons / m ² / s. The temperature was maintained at 25 ° C., the concentration of carbon dioxide at 2% was added at 0.5 vvm, and the mixture was stirred at 150 rpm. Each experiment was carried out twice independently, and the data were analyzed by the average value. Initial concentrations were begun at 1 × 10 ⁶ cells / mL and samples were taken at 3-day intervals and used for weight analysis and photosynthetic dye content. The weight analysis was performed by measuring the weight difference between before and after drying with a 0.45 μm GF / C filter (Whatman USA). As shown in FIG. 3, it was confirmed that it grew to about 2.1 g / L in 9 days.

Example  4. Photosynthetic pigment extraction and component analysis

The 3, 6, and 9 days of Sennedy's Mousobilius ABC-009 strain were harvested by centrifugation at 3500 rpm and 25 ° C for 10 minutes in 10 mL increments. The harvested cells were stored in a 2 mL homogenizer tube and zirconia / silica beads (0.1 mm and 0.5 mm in diameter) were placed in a bead mill (Precellys 24, Bertin Technologies, USA) for 30 seconds at 6000 rpm 10 times. For each crushing, it was cooled in ice for 1 minute to prevent decomposition of the extracted photosynthetic pigment. After the extraction, the supernatant was separated by centrifugation under the same conditions as described above and stored in a screw cap vial (Agilent, USA) in an amount of 1 mL using an RC-membrane syringe filter (Sartorius Stedim Biotech, Germany). The photosynthetic dye was analyzed by HPLC (Dionex Ultimate 3000, Thermo Scientific, USA) equipped with a UV detector. The column was Acclaim 120 C18 5 μm column (Thermo Scientific, USA). For the analysis, (A) methanol-ammonium acetate solution (80:20, v / v) and (B) methanol-acetone solution (70:30, v / v) ) Was flowed at 0.8 mL / min over 1 hour until it became 100%. Purchase of Sigma Aldrich's lutein, chlorophyll a, chlorophyll b, neo xanthine, viola xanthine and beta-carotene (analysis standard,> 90%) were purchased as a control to identify substances at each peak, including lutein, among the various peaks The results of the analysis based on the results are shown in FIG.

Like most photosynthetic organisms, chlorophyll a was the most abundant, followed by lutein and chlorophyll b. In particular, the total amount of lutein production along with the growth rate was calculated, and it was verified that Lutein can be produced much higher than other Senethesmus strains (Table 1). In the following Table 1, the lutein content unit mg / g represents the lutein content per dry cell weight. At this time, the four best-known data of several Sene desmus strains reported in Minhas et al., 2016 Bioresour Technol, 211: 556-565 were compared. Based on this, it proved the superiority of the novel Sennethus mousobilius ABC-009, and this strain will be used industrially through mass cultivation in the future. This is not limited to the production of high value-added products such as lutein, but it can contribute greatly to various fields such as carbon dioxide reduction, development of eco-friendly fuel, nutritional feed of plants and plants.

Korea Biotechnology Research Institute KCTC18649P 20180104

<110> ADVANCED BIOMASS R & D CENTER <120> A novel Scenedesmus obliquus ABC-009 strain having high biomass          and lutein productivity and uses thereof <130> PN17575 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cctggttgat cctgccag 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ttgatccttc tgcaggttca 20 <210> 3 <211> 1791 <212> DNA <213> Scenedesmus obliquus ABC-009 <400> 3 cctggttgat cctgccagta gtcatatgct tgtctcaaag attaagccat gcatgtctaa 60 gtataaactg cttatactgt gaaactgcga atggctcatt aaatcagtta tagtttattt 120 ggtggtacct tactactcgg ataaccgtag taattctaga gctaatacgt gcgtaaatcc 180 cgacttctgg aagggacgta tatattagat aaaaggccga ccgagctttg ctcgacccgc 240 ggtgaatcat gatatcttca cgaagcgcat ggccttgtgc cggcgctgtt ccattcaaat 300 ttctgcccta tcaactttcg atggtaggat agaggcctac catggtggta acgggtgacg 360 gaggattagg gttcgattcc ggagagggag cctgagaaac ggctaccaca tccaaggaag 420 gcagcaggcg cgcaaattac ccaatcctga tacggggagg tagtgacaat aaataacaat 480 accgggcatt tcatgtctgg taattggaat gagtacaatc taaatccctt aacgaggatc 540 cattgaaggg caagtctggt gccagcagcc gcggtaattc cagctccaat agcgtatatt 600 taagttgttg cagttaaaaa gctcgtagtt ggatttcggg tgggttctag cggtccgcct 660 atggtgagta ctgctatggc cttcctttct gtcggggacg ggcttctggg cttcactgtc 720 cgggactcgg agtcgacgtg gttactttga gtaaattaga gtgttcaaag caggcttacg 780 ccagaatact ttagcatgga ataacacgat aggactctgg cctatcttgt tggtctgtag 840 gaccggagta atgattaaga gggacagtcg ggggcattcg tatttcattg tcagaggtga 900 aattcttgga tttatgaaag acgaactact gcgaaagcat ttgccaagga tgttttcatt 960 aatcaagaac gaaagttggg ggctcgaaga cgattagata ccgtcgtagt ctcaaccata 1020 aacgatgccg actagggatt ggcgaatgtt tttttaatga cttcgccagc accttatgag 1080 aaatcaaagt ttttgggttc cggggggagt atggtcgcaa ggctgaaact taaaggaatt 1140 gacggaaggg caccaccagg cgtggagcct gcggcttaat ttgactcaac acgggaaaac 1200 ttaccaggtc cagacatagt gaggattgac agattgagag ctctttcttg attctatggg 1260 tggtggtgca tggccgttct tagttggtgg gttgccttgt caggttgatt ccggtaacga 1320 acgagacctc agcctgctaa atagtctcag ttgctttttg cagctggctg acttcttaga 1380 gggactattg gcgtttagtc aatggaagta tgaggcaata acaggtctgt gatgccctta 1440 gatgttctgg gccgcacgcg cgctacactg atgcattcaa caagcctatc cttgaccgaa 1500 gggtctgggt aatctttgaa actgcatcgt gatggggata gattattgca attattagtc 1560 ttcaacgagg aatgcctagt aagcgcaagt catcagcttg cgttgattac gtccctgccc 1620 tttgtacaca ccgcccgtcg ctcctaccga ttgggtgtgc tggtgaagtg ttcggattgg 1680 cagcttaggg tggcaacacc tcaggtctgc cgagaagttc attaaaccct cccacctaga 1740 ggaaggagaa gtcgtaacaa ggtttccgta ggtgaacctg cagaaggatc a 1791

Claims (6)

Scenedesmus obliquus ABC-009 (Accession No. KCTC 18649P) with high biomass and lutein productivity. The strain according to claim 1, wherein the biomass productivity is 210 to 250 mg / L / d and the lutein productivity is 0.75 to 0.85 mg / L / d. A microorganism preparation for producing lutein comprising the strain of claim 1 or 2, a culture thereof, a concentrate of the culture solution or a dried product of the culture solution as an active ingredient. A method for producing lutein comprising culturing the strain of claim 1 or 2, and recovering lutein from the cells and the culture. A food comprising the strain of claim 1 or 2, a culture thereof, a concentrate of the culture solution, or a dried product of the culture solution as an active ingredient. A feed additive comprising the strain of claim 1 or 2, a culture thereof, a concentrate of the culture solution, or a dried product of the culture solution as an active ingredient.

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