WO2020251072A1 - Procédé de culture en intérieur de cellules individuelles de prasiola japonica - Google Patents

Procédé de culture en intérieur de cellules individuelles de prasiola japonica Download PDF

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WO2020251072A1
WO2020251072A1 PCT/KR2019/007042 KR2019007042W WO2020251072A1 WO 2020251072 A1 WO2020251072 A1 WO 2020251072A1 KR 2019007042 W KR2019007042 W KR 2019007042W WO 2020251072 A1 WO2020251072 A1 WO 2020251072A1
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weight
parts
culture
freshwater
medium
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PCT/KR2019/007042
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주성수
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강릉원주대학교산학협력단
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Priority to PCT/KR2019/007042 priority Critical patent/WO2020251072A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

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  • the present invention relates to a freshwater laver single cell indoor culture method, and more particularly, to a freshwater laver single cell indoor culture method capable of mass cultivation into single cells by separating from freshwater laver collected in nature.
  • Laver which lives in the sea, is red algae ( Phylum rhodophyta ), a seaweed food that is mainly eaten at home by farming.
  • Prasiola japonica a green algae (Phylum chlorophyta) that grows in rivers other than the sea, grows in small rivers, not in the sea. In Korea, most of them disappear or are no longer collected, or freshwater laver grows in some areas of Sohancheon in Samcheok, Gangwon-do, and is used by residents as food.
  • Such domestic freshwater laver is 100% identical in gene sequence to Kawanori, which grows near Mt. Fuji and in Kyushu, Japan, so the species name of freshwater laver is Prasiola japonica .
  • Korean Patent Registration No. 1223232 discloses a method for cultivating freshwater laver including a culture solution manufacturing step of preparing a culture solution by mixing purified water and freshwater laver fronds.
  • An object of the present invention is to provide a freshwater laver single cell indoor culture method capable of securing a large amount of physiologically active substances similar to freshwater laver fronds by grafting indoor culture technology through single cell culture of freshwater laver differentiated from spore culture of conventional freshwater laver. do.
  • these problems are exemplary, and the scope of the present invention is not limited thereby.
  • a solid medium manufacturing step of preparing a solid medium by mixing agar with sterilized fresh water A solid culture step of crushing and smearing the fronds of freshwater laver ( P. japonia ) collected on the solid medium and culturing them to form filaments; And a liquid culture step of inoculating the filamentous body in a liquid medium prepared by mixing sterilized fresh water and nutrient medium and supplying air to maintain a single cell state and culturing it.
  • limited biomass produced from rare areas through mass culture of freshwater laver single cells in a standardized indoor space using a single cell indoor culture method of freshwater laver ( Prasiola japonica ) It is possible to realize the effect of overcoming and securing a large amount of natural resources with high pharmacological activity.
  • the scope of the present invention is not limited by these effects.
  • FIG. 1 is a microscopic photograph showing the result of culturing a solid medium of freshwater laver fronds of the present invention (100X). * A, B) culture group with nutrient medium added, C, D) culture group without nutrient medium, A, C) culture day 2, B, D) culture day 8. Arrows: filaments grown from fronds.
  • FIG. 2 is a diagram showing the result of sequencing of the filamentous bodies of the freshwater laver solid medium culture group of the present invention. * P. japonica NCBI gene accession number (gene accession number) showed 100% homology to KR261683.1.
  • Figure 3 is a micrograph showing the independent subculture results of the solid medium cultured filaments grown from freshwater laver fronds of the present invention (100X).
  • FIG. 4 is a diagram showing the results of secondary sequencing after subculture of solid medium cultured filamentous bodies grown from freshwater laver fronds of the present invention. * P. japonica NCBI gene accession number (gene accession number) showed homology of KR261683.1 and 100.
  • Figure 5 is a microscopic photograph of the appearance of single cells shown as a result of smearing the solid medium cultured filamentous body of the present invention. *A) Single cells observed on day 1 of culture (100X), B) Conjugates observed on day 14 of culture (400X), arrows: circumferential single cells.
  • Figure 6 is a photograph showing the results of the freshwater laver liquid culture of the present invention. Single-celled freshwater laver culture is possible only when air injection and nutrient medium are supplied.
  • FIG. 7 is a photograph of observing the change in culture of freshwater laver according to the amount of nutrient medium added. * Observation of nutrient medium A)1 mL/L, B)5 mL/L, C)10 mL/L, D) nutrient medium 10 mL/L culture group.
  • FIG. 8 is a photograph of the freshwater laver culture of the present invention and the result of nucleotide sequence analysis thereof. Suspended cultures were identified as single cells with 100% nucleotide sequence homology to freshwater laver, whereas cultures grown by sedimentation and attachment to the wall have nucleotide sequences of other species (93% homology to Ankistrodemus).
  • Figure 9 is a photograph of observing the changes that appear when cultured by treating with 5 mL/L of nutrient medium in sterilized filtration fresh water of the present invention and adjusting the pH. A)pH 5.5, B) 7.5, C) 11.5.
  • Prasiola japonica used in this document is a kind of green algae, which grows wildly in valleys, not in the sea, and grows asexually from August to October by attaching to rocks with a fast flow rate and sunlight.
  • Freshwater laver is currently known to inhabit only Samcheok in Korea and some regions of Japan (Kyushu). It only lives in cold and clear freshwater. It is known that the content of calcium, phosphorus, zinc and unsaturated fatty acids is up to four times higher than that of sea laver.
  • thallus used in this document is a plant in which the organs of stem, leaf, and root are not differentiated, and is a simple structure without vascular bundles, and the whole acts as a leaf, absorbing water and nutrients, and performing photosynthesis. It consists of cells in the shape of a thread or plate, and varies in size. Algae such as seaweed and seaweed, fungi, and other lower organisms belong to this.
  • a solid medium manufacturing step of preparing a solid medium by mixing agar with sterilized fresh water A solid culture step of crushing and smearing the fronds of freshwater laver ( P. japonia ) collected on the solid medium and culturing them to form filaments; And a liquid culture step of inoculating the filamentous body in a liquid medium prepared by mixing sterilized fresh water and nutrient medium and supplying air to maintain a single cell state and culturing it.
  • the nutrient medium is NaNO 3 90 to 100 parts by weight, Na 2 EDTA 30 to 50 parts by weight, H 3 BO 3 25 to 45 parts by weight, NaH 2 PO 4 ⁇ 2H 2 O 15 to 35 parts by weight, FeCl 3 ⁇ 6H 2 O 0.5 to 2.5 parts by weight, MnCl 2 ⁇ 4H 2 O 0.3 to 0.5 parts by weight, ZnCl 2 0.02 to 0.04 parts by weight, CoCl 2 ⁇ 6H 2 O 0.02 to 0.04 parts by weight, (NH 4 ) 6 M o7 O 24 ⁇ 4H 2 O 0.02 to 0.04 parts by weight, CuSO 4 ⁇ 5H 2 O 0.04 to 0.06 parts by weight, vitamin B 1 (Thiamine) 0.04 to 0.06 parts by weight and vitamin B 12 (Cobalamin) 0.005 It may contain to 0.02 parts by weight.
  • the mixing ratio of the sterilized fresh water and the nutrient medium may be 1:300 to 1:500.
  • the liquid culture step may be cultured at a temperature of 15 to 19°C and an illuminance of 1000 to 3000 lux, and the liquid culture may be an air-lift culture using an air pump.
  • freshwater laver P. japonica
  • P. japonica requires a limited and unique habitat environment (low water temperature, fast flow rate, and abundant minerals) for growth, and is the only green algae that grows naturally in Samcheok Sohancheon in South Korea. It has been reported that spore cultivation is possible due to the feature that hermaphrodites appear in the fronds bearing the stalk and disappear while forming monospores, but the P. japonica non-spore single cell culture presented in the present invention has not yet been reported in the world, so the Convention on the Conservation of Biodiversity ( Convention on biological diversity) is not only a technology irrelevant to the limitations of resources, but also the linkage of the patent of the'Pharmaceutical Composition for Cancer Treatment' by P.
  • the present inventors have collected freshwater laver fronds and river water (freshwater) used for indoor cultivation from Sohancheon, Samcheok-si, Gangwon-do, and then used ampicillin and ampicillin to remove the collected fronds.
  • the mixture was rotated for 24 hours in a mixture treated with 100 ⁇ g/mL of kanamycin, and fresh water was autoclaved at 121° C. for 15 minutes, then filtered through 0.22 ⁇ m filter paper and used for final indoor culture.
  • the liquid medium used for indoor culture was prepared by dissolving Conwy medium, trace metal solution, and vitamin mix in a total volume of 1 L of distilled water as shown in Table 1 below.
  • Composition table of nutrient medium for indoor culture of freshwater laver (component content per unit volume)
  • Ingredient name Content (g/L) NaNO 3 100 Na 2 EDTA 40 H 3 BO 3 35 NaH 2 PO 4 2H 2 O 25 FeCl 3 6H 2 O 1.5 MnCl 2 4H 2 O 0.4 ZnCl 2 0.03 CoCl 2 6H 2 O 0.03 (NH 4 ) 6 M o7 O 24 4H 2 O 0.03 CuSO 4 5H 2 O 0.05
  • the solid medium used to induce the growth of filamentous bodies from the fronds was prepared by mixing 0.8% bacto agar per volume of sterilized fresh water and 1 mL/L of the nutrient medium of Table 1, and the prepared solid medium A frond of 1x1 cm (width x length) was sliced and spread on a medium, followed by incubation at 18°C and 2000 lux temperature and illuminance. Thereafter, the filaments grown from the fronds were isolated for secondary proliferation and cultured for 4 to 5 days in the same medium under the same conditions.
  • Example 3 Liquid culture (single cell separation)
  • the freshwater laver filaments produced in the solid culture were inoculated into a glass bottle of 500 mL of sterilized fresh water in which 2.5 mL/L of the nutrient medium of Table 1 was mixed, and air-floating culture was performed using an air pump. , The temperature and illuminance of 2000 lux were maintained. Thereafter, the cultured freshwater laver single cells were set to have a turbidity of 1 or more (600 nm) and a chromaticity close to green as a logarithmic phase, and passage culture was performed at a ratio of 1:10.To prevent contamination of microorganisms such as bacteria, 150 ⁇ g/ Ampicillin was added at a concentration of mL.
  • the present inventors extracted total cell DNA (total cell DNA) from the single cell pellet after completion of culture in order to confirm the nucleotide sequence of single cells isolated from the obtained freshwater laver fronds.
  • total cell DNA total cell DNA
  • lyticase, lysozyme, and Intron's I-genomic plant DNA extraction kit were used.
  • a primer of ribulose-1,5-bisphosphate carboxylase/oxigenase large subunit (rbcL, NIBRCL0000100964-8) of freshwater laver ( P. japonica ) was prepared, and gradient polymerase chain reaction (gradient PCR). ) To amplify the gene.
  • the gene amplified through the PCR was loaded on a 1.2% agarose gel, and a band of about 500 bp was identified, and then DNA was extracted and cloning was performed using a T-easy vector system. That is, 1 ⁇ L of pGEM ® T-easy vector, 1 ⁇ L of T4 DNA ligase, 5 ⁇ L of 2X rapid ligation buffer (Promega, USA), and 150 ng of the obtained gene were prepared as a reaction solution, and then reacted at room temperature for 1 hour.
  • coli cell colony from which the transformation was successfully induced was cultured for 16 hours in a liquid medium containing ampicillin (100 ⁇ g/mL) to obtain sufficient cells. It was obtained using a mini kit (RBC, Taiwan), and the insertion gene was confirmed by treatment with an Eco RI restriction enzyme to identify a gene of about 500 bp.
  • the nucleotide sequence analysis was requested by SolGent (Korea) and confirmed through gene matching analysis.
  • the nucleic acid sequences of the primers used in the PCR reaction are summarized in Table 2 below, and the PCR reaction conditions are summarized in Table 3 below.
  • Primer sequence used for PCR primer Nucleic acid sequence (5 ⁇ 3 ⁇ ) location Sequence number rbcL_F AGTTCCGGCTGAAGAATGT 111 ⁇ 130 One rbcL_R CGCATAAATGGTTGTGAG 591 ⁇ 608 2
  • the filament obtained through additional culture was spread thinly on a new solid medium, and as a result of culturing, a number of curved single cells (A) were identified, and as a result of culturing them for about two weeks, the characteristics of single cells conjugating to each other (B) As a result, it was analyzed that spores could be generated by returning to the life cycle of freshwater laver that reproduces asexually and sexually (Fig. 5).
  • the nutrient medium of Table 1 was added to fresh water sterilized according to an embodiment of the present invention at a concentration of 1, 5, and 10 mL/L to perform air-lift culture.
  • Optimal green single cell growth was confirmed in the added experimental group (FIG. 6), and the group added with a high concentration of 10 mL/L nutrient medium grew in the form of filamentous bodies growing from the fronds and did not meet the single cell growth conditions (FIG. 7).
  • the optimum conditions for the single cell indoor culture method of the present invention are 1) sterile filtration fresh water, 2) nutrient medium 5 mL/L, 3) air-lift culture, 4) water temperature 15 ⁇ 18°C and 5) irradiance 2000 lux.
  • the result of sequencing by amplifying the ribulose-1,5-bisphosphate carboxylase/oxigenase large subunit (rbcL) gene of freshwater kim ( P. japonica ) as a template using total cell DNA extracted from floating single cells .
  • the sequence was 100% identical to the freshwater laver gene, confirming that single-cell isolation and culture were successful, and the precipitated or adherent cells cultured at the same time showed 93% homology with Ankistrodemus, a kind of green algae, and thus symbiotic with wild freshwater laver. It was confirmed as a species (Fig. 8).
  • the culture was cultured in an acidic to alkaline order (pH 5.5, 7.5, 11.5), and as a result, the acidic condition showed growth inhibition and the alkaline condition showed active growth activity, so the optimum pH of liquid culture was 9.5. It was understood that it was before and after.
  • the method of culturing freshwater laver ( P. japonica ) single-cell indoor of the present invention can secure a large amount of physiologically active substances similar to freshwater laver fronds in a standardized indoor space that differentiates freshwater laver single cell culture from the conventional freshwater laver spore cultivation. Therefore, it can be used to overcome limited production from rare areas and secure large quantities of natural resources with high pharmacological activity.

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Abstract

La présente invention concerne un procédé de culture en intérieur de cellules individuelles de Prasiola japonica. Le procédé de culture en intérieur de cellules individuelles de Prasiola japonica, qui est différent de la technique classique de culture de spores de Prasiola japonica, peut être utilisé pour la culture en masse, ce qui permet de résoudre un problème associé à une biomasse limitée produite à partir de zones rares et d'obtenir une grande quantité de ressource naturelle pour une activité pharmacologique.
PCT/KR2019/007042 2019-06-12 2019-06-12 Procédé de culture en intérieur de cellules individuelles de prasiola japonica WO2020251072A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3042673B2 (ja) * 1995-10-04 2000-05-15 富山県 新規な淡水性微細藻類
JP2008125422A (ja) * 2006-11-20 2008-06-05 Mie Prefecture 海苔の単細胞化方法及び養殖方法
KR101223232B1 (ko) * 2012-04-18 2013-01-17 임봉규 민물 김의 양식방법
KR20140019288A (ko) * 2010-09-15 2014-02-14 페르망탈그 섬광 형태로 빛의 불연속적인 공급하의 혼합영양 단세포 조류의 배양 방법
KR102018118B1 (ko) * 2018-03-12 2019-09-04 강릉원주대학교 산학협력단 민물 김(Prasioloa japonica) 단세포 실내 배양방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3042673B2 (ja) * 1995-10-04 2000-05-15 富山県 新規な淡水性微細藻類
JP2008125422A (ja) * 2006-11-20 2008-06-05 Mie Prefecture 海苔の単細胞化方法及び養殖方法
KR20140019288A (ko) * 2010-09-15 2014-02-14 페르망탈그 섬광 형태로 빛의 불연속적인 공급하의 혼합영양 단세포 조류의 배양 방법
KR101223232B1 (ko) * 2012-04-18 2013-01-17 임봉규 민물 김의 양식방법
KR102018118B1 (ko) * 2018-03-12 2019-09-04 강릉원주대학교 산학협력단 민물 김(Prasioloa japonica) 단세포 실내 배양방법

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