Nostoc sp. strain of cyanobacteria producing 2-methylisoborneol, and method for culturing same

Abstract

The present invention relates to: a Nostoc sp. strain of cyanobacteria that produces 2-methylisoborneol; and a method for culturing same. The Nostoc sp. strain according to the present invention is the first ever Nostoc sp. strain that produces 2-methylisoborneol, and considering the positive effect that 2-methylisoborneol has on the human body and the fact that 2-methylisoborneol is also used as a raw material for perfume, the HNIBRCY4 strain, which is an edible Nostoc sp. strain capable of being mass-produced, can be effectively used to produce 2-methylisoborneol.

Description

Nostoc strain, a soil cyanobacteria producing 2-methylisoborneol, and its cultivation method

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0094202, filed on July 17, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a Nostoc sp. strain, a soil blue-green algae producing 2-methylisoborneol, and a method for culturing the same, and more particularly, to a Nostoc sp. HNIBRCY4 strain having a deposit number of KCTC15834BP, a composition for producing 2-methylisoborneol containing the same as an active ingredient, and a method for producing 2-methylisoborneol using the same.

[0003]

[0004] Geosmin is an irregular sesquiterpene, C 12 H 22 It is a bicyclic alcohol with the chemical formula O, and 2-MIB is an irregular monoterpene with C 11 H 20 These are substances with the chemical formula O. These substances have a very low threshold (< 10 ng / L) so that many people can easily recognize them, and it is known that sensitive people can recognize them even at less than 5 ng / L. These odor-inducing substances, geosmin and 2-MIB, do not pose a health hazard to humans or aquatic animals, but people perceive them as having an earthy or moldy smell, and when water smells like this, they judge that the water is unsafe to drink, and it is regulated only for aesthetic reasons.

[0005] However, recent research results have revealed that geosmin and 2-MIB have positive effects on brain activity, such as increasing blood serotonin levels and improving concentration, and are also used as raw materials for perfumes overseas, increasing the possibility of industrial use. Meanwhile, various studies are being conducted to identify the causal species that produce geosmin and / or 2-MIB in aquatic systems through genetic analysis, but there are few cases in which the possibility of industrial use has been confirmed by isolating pure blue-green algae that produce geosmin and / or 2-MIB in the environment. In particular, 2-MIB compounds are known to be mainly produced by actinobacteria, cyanobacteria, myxobacteria, and proteobacteria, and among the cyanobacteria, the genera Oscillatoria, Phormidium, and Planktothrix are included, but there is no known strain belonging to the genus Nostoc that is edible and capable of mass production. Therefore, there is a growing need to discover new strains that can produce these odor-inducing substances and also be utilized industrially.

[0006]

[0007] Accordingly, the inventors of the present invention have made great efforts to discover a novel strain producing 2-methylisoborneol (2-MIB), and as a result, have confirmed that the strain producing 2-MIB belongs to the genus Nostoc, and have also confirmed the optimal conditions for the strain to produce 2-MIB, thereby completing the present invention.

[0008]

[0009] Accordingly, the purpose of the present invention is to provide a Nostocsp. HNIBRCY4 strain having the deposit number KCTC15834BP.

[0010]

[0011] Another object of the present invention is to provide a composition for producing 2-methylisoborneol (2-MIB) comprising the strain as an active ingredient.

[0012]

[0013] Another object of the present invention is to provide a method for producing 2-methylisoborneol, comprising the steps of (a) culturing the strain; and (b) isolating 2-methylisoborneol from the culture obtained in step (a).

[0014]

[0015] Another object of the present invention is to provide a composition for producing 2-methylisoborneol comprising the strain.

[0016]

[0017] Another object of the present invention is to provide a composition for producing 2-methylisoborneol, which essentially consists of the above strain.

[0018]

[0019] To achieve the above purpose, the present invention provides a Nostocsp. HNIBRCY4 strain having the deposit number KCTC15834BP.

[0020]

[0021] In order to achieve another object of the present invention, the present invention provides a composition for producing 2-methylisoborneol (2-MIB) comprising the strain as an active ingredient.

[0022]

[0023] In order to achieve another object of the present invention, the present invention provides a method for producing 2-methylisoborneol, comprising: (a) a step of culturing the strain; and (b) a step of separating 2-methylisoborneol from the culture obtained in step (a).

[0024]

[0025] In order to achieve another object of the present invention, the present invention provides a composition for producing 2-methylisoborneol comprising the strain.

[0026]

[0027] In order to achieve another object of the present invention, the present invention provides a composition for producing 2-methylisoborneol, which essentially consists of the strain.

[0028]

[0029]

[0030] The present invention is described in detail below.

[0031]

[0032] The present invention provides a Nostocsp. HNIBRCY4 strain having the deposit number KCTC15834BP.

[0033]

[0034] In this specification, the term “comprising” is used with the same meaning as “including” or “characterized by,” and does not exclude additional components or method steps, etc. that are not specifically mentioned in the composition or method according to the present invention. In addition, the term “consisting of” means excluding additional elements, steps, or components, etc. that are not separately described. The term “essentially consisting of” means that, in the scope of the composition or method, it may include materials or steps, etc. that do not substantially affect the basic characteristics thereof, in addition to the materials or steps described.

[0035] The HNIBRCY4 strain according to the present invention is a novel strain of terrestrial cyanobacteria isolated and identified from a soil sample collected from Gohado, Mokpo-si, Jeollanam-do. Meanwhile, the term 'cyanobacteria' above is a traditional term referring to cyanobacteria, and in this specification, 'cyanobacteria' is used with the same meaning as 'cyanobacteria' or 'cyanobacteria'.

[0036] In one embodiment of the present invention, the strain isolated by the inventor was analyzed using a cyanobacteria-specific identification primer (CYA primer) for microbial identification and classification, and as a result, it was confirmed that the strain belongs to the genus Nostoc sp. The inventor deposited the Nostoc sp. HNIBRCY4 strain with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on February 26, 2024 in accordance with the Budapest Treaty (Accession No. KCTC15834BP).

[0037] In addition, it was confirmed that the strain according to the present invention has the characteristic of producing 2-methylisoborneol (2-MIB), which means that the inventors have confirmed for the first time that the strain belongs to the genus Nostoc, which has not been known so far as a strain producing 2-MIB.

[0038] In the present invention, the 2-methylisoborneol (2-MIB) is C 11 H 20 It refers to a compound having an irregular monoterpene having the chemical formula O, synthesized by terpene synthase, and having a chemical structure represented by the following chemical formula 1.

[0039] <Chemical Formula 1>

[0040]

[0041] Accordingly, the present invention provides a composition for producing 2-methylisoborneol comprising the HNIBRCY4 strain as an effective ingredient.

[0042]

[0043] The HNIBRCY4 strain included in the composition according to the present invention may preferably mean a live cell of the HNIBRCY4 strain, but is not limited thereto, and may mean a fragmented cell wall fraction, dead cells, dried cells, culture, or culture supernatant of the HNIBRCY4 strain, as needed.

[0044] The term "culture" in the present invention refers to the result of inoculating the HNIBRCY4 strain of the present invention into a medium and culturing it. Specifically, the culture may refer to the HNIBRCY4 strain of the present invention, the medium, or the culture itself, which is obtained by culturing the strain of the present invention in a medium, including a metabolic product of the strain.

[0045] The composition according to the present invention may additionally include various nutrient media, buffer solutions, etc. as needed in addition to the HNIBRCY4 strain, and may also be a composition further including at least one of other strains known to produce 2-MIB, for example, strains belonging to actinobacteria, cyanobacteria, myxobacteria, and proteobacteria.

[0046] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. In addition, the composition according to the present invention may further include additives such as a carrier, excipient, or diluent in addition to the active ingredient. The composition for producing 2-methylisoborneol of the present invention may include 0.1 to 99.9 wt% of the strain of the present invention, and 99.9% to 0.1 wt% of the carrier.

[0047] In addition, the present invention,

[0048] (a) a step of culturing the HNIBRCY4 strain according to the present invention; and

[0049] (b) a step of separating 2-methylisoborneol from the culture obtained in step (a);

[0050] A method for producing 2-methylisoborneol including

[0051]

[0052] Below, the present invention is described in detail according to each step of the above method.

[0053]

[0054] (a) a step of culturing the HNIBRCY4 strain according to the present invention;

[0055]

[0056] In the present invention, the term "cultivation" refers to growing the HNIBRCY4 strain according to the present invention under appropriately controlled environmental conditions. The culturing process can be performed according to suitable media and culture conditions known in the art. This culturing process can be easily adjusted and used by a person skilled in the art depending on the selected microorganism, and specifically, it can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0057] According to one embodiment of the present invention, the HNIBRCY4 strain according to the present invention exhibited high 2-MIB production under autotrophic or diazotrophic conditions without organic matter. In addition, considering that the biomass production of the strain was the highest under mixotrophic conditions, the strain was first cultured under autotrophic conditions and then converted to mixotrophic conditions, and as a result, the highest daily 2-MIB production (3.37 μg / g / d) was achieved. Therefore, in the present invention, the 'cultivation' is preferably performed under photoautotrophic conditions or diazotrophic conditions, and most preferably, it is performed under complex growth conditions in which culture is started under autotrophic conditions and then converted to mixotrophic conditions, but is not limited thereto. In addition, in the case of starting cultivation under independent nutrient conditions and then switching to mixed nutrient conditions, the initial culturing period under independent nutrient conditions may be 4 to 12 days, preferably 6 to 10 days, and most preferably 7 to 9 days, and then it is preferable to switch to mixed nutrient conditions, but this is not limited thereto, and a person skilled in the art can appropriately adjust the above period by considering various culturing conditions of the strain according to the present invention.

[0058]

[0059] The above 'medium' refers to a material containing nutrients as main components necessary for culturing the HNIBRCY4 strain according to the present invention, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium is preferably BG11, which is mainly used for culturing algae, but is not limited thereto, and any medium used for culturing general microorganisms including Nostoc, containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, etc. as needed, and well known to those skilled in the art, may be used without any particular limitation.

[0060] The carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0061] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0062] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in a batch or continuous manner, but are not limited thereto.

[0063] Additionally, during the above culturing, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. Additionally, during the culturing, foaming may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Additionally, to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or to maintain anaerobic and microaerobic states, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0064] In culturing the strain according to the present invention, the culture temperature may be maintained at 15°C to 40°C, preferably 20°C to 30°C, but is not limited thereto.

[0065]

[0066] (b) a step of separating 2-methylisoborneol from the culture obtained in step (a);

[0067]

[0068] In the step of separating 2-methylisoborneol from the medium (medium in which the culture was performed) or culture according to the above culture, the 'separation' may be collecting the target 2-MIB using a suitable method known in the art according to the culture method of the microorganism, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and a person skilled in the art can separate the target 2-MIB from the medium or culture using a suitable method known in the art.

[0069] Additionally, the production method according to the present invention may additionally include a purification step. This purification may be performed using any suitable method known in the art. In one example, if the production method includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of order, or simultaneously or integrated into a single step, but this is not limited thereto.

[0070]

[0071] The Nostoc sp. HNIBRCY4 strain, accession number KCTC15834BP, according to the present invention is the first Nostoc sp. strain to produce 2-methylisoborneol. Considering the positive effects of 2-methylisoborneol on the human body and its use as a perfume ingredient, the HNIBRCY4 strain according to the present invention, a Nostoc sp. strain suitable for both edible use and mass production, can be usefully utilized for producing 2-methylisoborneol.

[0072]

[0073] Figure 1 shows a phylogenetic tree that identifies the HNIBRCY4 strain.

[0074] Figure 2 shows a photograph of the HNIBRCY4 strain observed under a microscope.

[0075] Figure 3 shows the results of confirming the growth rate of the HNIBRCY4 strain under diazotrophic, autotrophic, mixotrophic, and heterotrophic conditions.

[0076] Figure 4 shows the content of 2-MIB in the HNIBRCY4 strain under diazotrophic, autotrophic, and mixotrophic conditions.

[0077] Figure 5 shows the daily production of 2-MIB of the HNIBRCY4 strain according to changes in nutritional conditions.

[0078]

[0079] The present invention is described in detail below.

[0080] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0081]

[0082] Example 1. Pure isolation and identification of a strain of the soil cyanobacterium Nostoc producing the odorous substance 2-methylisoborneol (2-MIB).

[0083] A specific blue-green algae was purified from a soil sample collected from Gohado, Mokpo-si, Jeollanam-do, and while culturing it, it was confirmed that it produced an 'earthy smell'. The substance responsible for the smell was analyzed using SPME-GC (solid phase microextraction-gas chromatography), and it was confirmed that the substance was 2-methylisoborneol.

[0084] The isolated cyanobacteria were analyzed using cyanobacteria-specific identification primers (CYA primers), and as shown in Figure 1, it was confirmed to be a Nostoc sp. strain. Subsequently, it was observed under a microscope (see Figure 2), and finally identified as Nostoc sp. and named HNIBRCY4.

[0085] Afterwards, the strain was deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on February 26, 2024 in accordance with the Budapest Treaty and was issued with the accession number (KCTC15834BP).

[0086]

[0087] Example 2. 2-MIB production characteristics according to culture conditions of HNIBRCY4 strain in Nostoc.

[0088] In order to confirm the growth rate and 2-MIB production characteristics of the HNIBRCY4 strain identified in Example 1, culture was performed under diazotrophic, autotrophic, mixotrophic, and heterotrophic conditions, respectively, and the growth rate, biomass content, and 2-MIB content of the strain were confirmed.

[0089] The HNIBRCY4 strain was cultured by adding 250 ml of BG11 medium to a 300-mL photobioreactor. At this time, 1 g / L of glucose was added to the medium under mixed nutrient conditions, and BG110 medium with NaNO3, the nitrogen source in BG11, removed was supplied under nitrogen autotrophic conditions. The medium was aerated with 5% carbon dioxide under medium temperature (25°C), and 100 μmol / m2 of plant growth LED was used. 2 / s and cultured for 12 days. At this time, light was not irradiated under heterotrophic conditions.

[0090] As a result, as can be confirmed in Fig. 3, the highest biomass production (1.81 g / L) was achieved under mixed nutrient conditions supplied with 1 g / L of organic glucose, and no significant growth was observed after 6 days under heterotrophic conditions supplied with the same organic matter, confirming that light conditions are essential for the cultivation of the HNIBRCY4 strain.

[0091]

[0092] In addition, the 2-methylisoborneol content of the cultured HNIBRCY4 strain was analyzed using SPME-GC (Varian US / CP3800, CA, USA), and as can be seen in Fig. 4, it was confirmed that the content was high under organic-free photoautotrophic conditions (62.1 μg / g) and nitrogen-autotrophic conditions (55.9 μg / g).

[0093]

[0094] Based on these results, the HNIBRCY4 strain was first cultured under complex growth conditions in which the culture conditions were changed from autotrophic, mixotrophic, and single growth conditions to autotrophic-mixotrophic and mixotrophic-autotrophic conditions on the 8th day of cultivation, thereby securing a large amount of biomass and exploring the conditions for maximum production of 2-methylisoborneol. As a result, as can be confirmed in Fig. 5, the maximum daily 2-MIB production of 3.37 μg / g / d was achieved under autotrophic-mixotrophic conditions.

[0095]

[0096] The Nostoc sp. HNIBRCY4 strain, accession number KCTC15834BP, according to the present invention is the first Nostoc sp. strain to produce 2-methylisoborneol. Considering the positive effects of 2-methylisoborneol on the human body and its use as a perfume ingredient, the HNIBRCY4 strain according to the present invention, which is a Nostoc sp. strain suitable for both edible use and mass production, can be usefully used to produce 2-methylisoborneol, and thus has high potential for industrial application.

[0097]

[0098] [Accession number]

[0099] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0100] Accession number: KCTC15834BP

[0101] Date of acceptance: 20240226

[0102] [Correction pursuant to Rule 91, November 13, 2024]

Claims

1. Nostocsp. HNIBRCY4 strain with accession number KCTC15834BP.

2. In the first paragraph, the strain is characterized by having the ability to produce 2-methylisoborneol (2-MIB).

3. A composition for producing 2-methylisoborneol, comprising the strain of claim 1 or 2 as an effective ingredient. 4.(a) A step of culturing a strain according to paragraph 1 or 2; and (b) a step of separating 2-methylisoborneol from the culture obtained in step (a); A method for producing 2-methylisoborneol comprising:

5. A method for producing 2-methylisoborneol, characterized in that in the fourth paragraph, step (a) is cultured under organic-free photoautotrophic conditions or nitrogen-autotrophic conditions.

6. A method for producing 2-methylisoborneol, characterized in that in the fourth paragraph, step (a) is performed by converting the cultivation to mixed nutrient conditions after culturing under independent nutrient conditions.

7. A method for producing 2-methylisoborneol, characterized in that the step (a) in paragraph 4 is performed at 15°C to 40°C.

8. A method for producing 2-methylisoborneol, characterized in that the step (a) in paragraph 4 is performed at 20°C to 30°C.

9. A method for producing 2-methylisoborneol, characterized in that the separation in step (b) is performed by one or a combination of two or more methods selected from the group consisting of centrifugation, filtration, treatment with a crystallization protein precipitant, extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, affinity chromatography, and HPLC.

10. A method for producing 2-methylisoborneol, characterized in that the method for producing 2-methylisoborneol in claim 4 additionally includes a purification step.

11. A method for producing 2-methylisoborneol, characterized in that the method for producing 2-methylisoborneol in the fourth paragraph additionally includes a recovery step.

12. A method for producing 2-methylisoborneol, characterized in that in paragraph 5, the independent nutritional conditions are converted to mixed nutritional conditions after culturing for 4 to 12 days.

13. A method for producing 2-methylisoborneol, characterized in that in paragraph 5, the independent nutritional conditions are converted to mixed nutritional conditions after culturing for 6 to 10 days.

14. A method for producing 2-methylisoborneol, characterized in that in paragraph 5, the independent nutritional conditions are converted to mixed nutritional conditions after culturing for 7 to 9 days.

15. A method for producing 2-methylisoborneol, characterized in that in paragraph 9, the purification step and the recovery step are performed continuously or discontinuously, regardless of the order, or are performed simultaneously or integrated into one step.

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