KR102647563B1 - Lutein and oleic acid-producing novel microalgae of Desmodesmus sp. DSHM22 and its culturing method - Google Patents

Abstract

The present invention relates to the novel Desmodesmus genus DSHM22 microalgae and its culture method. It provides microalgae of the Desmodesmus genus that produce useful xanthophylls and fatty acids such as lutein and oleic acid, and determines the most optimized culture conditions. By providing a method to efficiently produce tophylls and fatty acids, it can provide potential as raw materials for health functional foods, pharmaceuticals, biofuels, feed, cosmetics, and pharmaceuticals.

Description

DSHM22 microalgae of the novel Desmodesmus genus producing lutein and oleic acid and culture method thereof {Lutein and oleic acid-producing novel microalgae of Desmodesmus sp. DSHM22 and its culturing method}

The present invention provides a novel Desmodesmus genus DSHM22 microalgae and a method for cultivating the same.

Carotenoids are organic pigments with high added value ranging from yellow, orange, and red in color and are bioactive substances with high antioxidant, anti-inflammatory, and anti-obesity effects. Carotenoids are used as additives in food, feed, health functional foods, and cosmetics industries. Carotenoids can be produced through chemical synthesis, but as restrictions on the use of artificial additives become more stringent, the importance of natural pigments is gradually increasing. Among these, lutein is a carotenoid of the xanthophyll series that protects eyes, acts as an antioxidant, and has anti-inflammatory properties. For this reason, it is considered a notable useful ingredient in the medical field. Currently, the only commercial source of lutein is marigold flower petals. However, land plants require a large amount of farmland and have various limitations such as low yields and seasonal changes, so it is important to develop alternative, highly efficient natural resources. It is necessary.

Therefore, microalgae biomass, which has higher productivity per unit area than marigold petals and can be produced year-round, is emerging as a promising material for economical lutein production.

Oleic acid (C18:1) is a mono-unsaturated fatty acid containing one double bond, also called omega-9 fatty acid, and is known to have various health effects. Oleic acid, also known as the main component of olive oil, not only has a strong antioxidant effect, preventing cell damage and improving immunity, but is also known to protect the human heart health by showing strong effects such as lowering blood pressure and improving blood circulation. In addition, oleic acid has the effect of lowering bad cholesterol (LDL) and increasing good cholesterol (HDL), which can help prevent arteriosclerosis and maintain heart health. In addition, it has quite a variety of effects, including strengthening immunity, preventing infection, strengthening memory, preventing arthritis, alleviating menopausal symptoms, improving digestive function, improving skin, and anti-obesity. In addition, oleic acid (C18:1) is a main ingredient that can produce high-quality biodiesel and can affect the oxidation stability and cetane number of biodiesel.

Microalgae are defined as small organisms that perform photosynthesis at a microscopic size. They contain a large amount of high value-added nutrients and bioactive substances such as carotenoids and fatty acids that are beneficial to humans and animals, and serve as primary producers and a major source of nutrients in the Earth's ecosystem. is in charge of These microalgae have a very high photosynthetic efficiency compared to land plants, so they grow quickly and are less limited by space, so they have the advantage of being highly sustainable even when grown in large quantities. However, low biomass productivity resulting from the photoautotrophic cultivation system for industrial use of microalgae is considered a major obstacle to industrial application of microalgae. Therefore, research on increasing biomass productivity through heterotrophic cultivation using organic carbon sources such as glucose, fructose, and sucrose has been actively conducted. These heterotrophic cultivation methods include photoheterotrophic cultivation and chemical heterotrophic cultivation. Both of these culture methods are known to exhibit biomass productivity that is several to ten times higher than photoautotrophic culture. However, heterotrophic microalgae that grow by absorbing organic carbon sources are quite limited, so there is a need to continuously discover these microalgae to increase their industrial utilization.

Therefore, the present invention aims to confirm the presence or absence of heterotrophic culture of DSHM22 of the genus Desmodesmus, a novel microalgae, and to increase the productivity of biomass and useful substances such as lutein and oleic acid under the confirmed heterotrophic culture conditions. Completed.

1. Japanese Patent Publication No. 29000001 (published on January 5, 2017)

The purpose of the present invention is to provide microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP.

Another object of the present invention is (1) inoculating microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP into a culture medium; and (2) adding an organic carbon source to the DSHM22 microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22 ) deposited as KCTC15412BP inoculated into the culture medium and irradiating the light source. sp. DSHM22) to provide a culture method.

Another object of the present invention is to provide a composition for enhancing carotenoid or fatty acid production containing a culture of Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP.

In order to achieve the above object, the present invention provides microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP.

In addition, the present invention includes the steps of (1) inoculating microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP into a culture medium; and (2) adding an organic carbon source to the DSHM22 microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22 ) deposited as KCTC15412BP inoculated into the culture medium and irradiating the light source. sp. DSHM22) to provide a culture method.

In addition, the present invention provides a composition for enhancing carotenoid or fatty acid production containing a culture of Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP.

The present invention relates to a novel Desmodesmus genus DSHM22 microalgae and a culture method thereof, which provides microalgae of the genus Desmodesmus that produce lutein and oleic acid and efficiently produces lutein and oleic acid using the most optimized culture conditions. It provides a method and can suggest potential as an additive in health functional food compositions, feed, and cosmetics.

Figure 1 shows the vertex coordinates (A) of the area where DSHM22 of the genus Desmodesmus was collected, the results of observation using an optical microscope (B), and the results of analyzing biomass productivity under light culture conditions (C).
Figure 2 shows the results of confirming the form of DSHM22 in Desmodesmus using a scanning electron microscope.
Figure 3 shows the results of analyzing the genetic phylogenetic tree of Desmodesmus genus DSHM22 based on the base sequence of the ITS-2 gene region.
Figure 4 shows the results of screening Desmodesmus genus DSHM22 by culturing it in various agricultural irrigation fertilizers (A) and the daily growth change (B) according to the concentration of the irrigation fertilizer selected through screening.
Figure 5 shows the photoheterotrophic (PH) and chemoheterotrophic (HT) cultures of Desmodesmus genus DSHM22 using photoautotrophic (PA) and 1 g/L glucose. This is the daily growth result when cultured under each condition for 10 days.

Hereinafter, the present invention will be described in more detail.

The present invention provides microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP.

The microalgae may produce one or more selected from the group consisting of neoxanthin, lutein, α-carotene, β-carotene, and zeaxanthin, It is not limited to this.

The microalgae may produce one or more selected from the group consisting of palmitic acid, oleic acid, linoleic acid, and alpha-linoleic acid, but are not limited thereto. no.

In addition, the present invention includes the steps of (1) inoculating microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP into a culture medium; and (2) adding an organic carbon source to the DSHM22 microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22 ) deposited as KCTC15412BP inoculated into the culture medium and irradiating the light source. sp. DSHM22) culture method is provided.

The culture medium in step (1) may contain agricultural fertilizer.

The agricultural fertilizer may include one or more selected from the group consisting of nitrogen (N), phosphorus (P), and potassium (K), but is not limited thereto.

The organic carbon source in step (2) may be glucose.

The culture method is to extract neoxanthin, lutein, α-carotene, and β-carotene from the Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP. and zeaxanthin, but is not limited thereto.

The culture method is performed by extracting palmitic acid, oleic acid, linoleic acid and alpha-linoleic acid from the Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP. ) can increase the production of one or more selected from the group consisting of, but is not limited to this.

In addition, the present invention provides a composition for enhancing carotenoid or fatty acid production containing a culture of Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP.

The composition can be used as an active ingredient in health functional foods, pharmaceutical compositions, feed compositions, feed additive compositions, or cosmetic compositions, but is not limited thereto.

Hereinafter, to aid understanding of the present invention, it will be described in detail through examples. However, the following examples only illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

<Example 1> Collection of microalgae

First, a total of 6 weeks of marine microalgae were secured from the East Sea waters of Korea, and among the secured microalgae, microalgae with the highest biomass productivity were selected. As shown in Figure 1, 6 weeks of microalgae (#1 to #6) were secured from Gangwon-do, the East Sea region, and the secured microalgae were smeared several times on BG-11 agar smear medium to make them sterile. Afterwards, the biomass productivity was analyzed after culturing in BG-11 medium for 10 days at 28°C under continuous light of 60 μmol/m ² /s under shaking culture conditions at 150 rpm in a 25 mL T-Flask (lat. : 38°23' 2.95”, longitude: 128°29' 38.47”) It was found that microalgae showed the highest biomass productivity.

<Example 2> Identification of microalgae

Morphological identification

#3 microalgae, which showed the highest biomass productivity, was analyzed by scanning electron microscopy. For scanning electron microscopy analysis, 10 mL of microalgae culture was first subjected to lipid fixation with 1% osmium tetroxide (OsO ₄ ). It was filtered using a membrane filter with a mesh size of 3 μm. Afterwards, it was washed twice with distilled water to remove residual salts, and the membrane filter with the filtered cells attached was sequentially washed with ethanol at concentrations of 10, 20, 30, 40, 50, 70, 80, 90, and 100%. The cells were dehydrated and dried using an automatic critical point dryer (EM CPD300, Leica, Wetzlar, Germany), and the dried filter was mounted on an aluminum stub (Electron Microscopy Sciences, Hatfield, PA, USA) using copper conductive double-sided tape. , was coated with gold using ion sputter (MC1000, Hitachi). Afterwards, it was observed using a FE-SEM field emission scanning electron microscope (Sigma 500/VP, Carl Zeiss, Oberkochen, Germany).

As a result, according to Figure 2, four elongated-shaped cells forming a coenobium were observed in many cases, and often one or two cells instead of four were observed. It had spines extending from the cell wall surrounded by granules. Therefore, through scanning electron microscopy, it was confirmed that it was a morphologically typical microalgae belonging to the genus Desmodesmus.

2. Genetic identification

#3 1 mL of microalgae was collected during the exponential growth phase and the cells were concentrated using a centrifuge. Afterwards, the supernatant was removed from the tube to obtain a pellet, and #3 microalgae DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Primer sets ITS-F and ITS-R were used for internal transcribed spacer 2 (ITS-2) sequence analysis. Primers A and SSU-inR were used for 18S rRNA analysis.

In the case of ITS-2, the same result was obtained as the base sequence in Sequence Listing 1, and in the case of 18S rRNA, the result was the same as the base sequence in Sequence Listing 2.

Based on the analyzed base sequences, the phylogenetic tree of the ITS-2 region with related species was analyzed. For phylogenetic tree analysis, sequence alignment was performed using the GeneiousPrime v.2022.2.2 (BiomattersLtd., Auckland, New Zealand) program, and Bayesian analysis was performed using MrBayesv. 3.2.7 was conducted using the default GTR + G + I model, ML (Maximum-likelihood) analysis was performed with RAxMLv.8.2.10, and the bootstrap value was performed with 1,000 replicates. As a result of the phylogenetic tree analysis, according to Figure 3, the obtained Desmodesmus genus showed genetically similar characteristics to various previously reported Desmodesmus genera, and was #3 microalgae based on morphological characteristics and genetic phylogenetic analysis results. was named Desmodesmos genus DSHM22 microalgae.

<Example 3> Confirmation of optimal culture conditions for DSHM22 in Desmodesmos

1. Drench fertilizer

BG-11 medium, a microalgae culture medium, is quite expensive and is not easy to use for mass culture. Therefore, considering future mass culture of microalgae, microalgae can be grown in four types of water-soluble irrigation fertilizers sold on the market. was compared with BG-11 medium.

As a result, according to Figure 4, it was confirmed that Nutrivit (Nutrivival Products Nederland, Netherland) and high-N S-feed (FarmHannong, South Korea) corresponding to irrigation fertilizers B and C showed the highest growth, and microalgae It showed twice as high photoculture growth as the culture medium BG-11. In addition, it was confirmed that Nutrivit generates sediment when dissolved at more than 1 g/L. As a result of measuring the growth of microalgae in a gradient concentration range of 0 to 1 g/L, it was confirmed that it grew best at 1 g/L. .

2. Culture method

Cultivation of microalgae can be divided into photoautotrophic cultivation (PA), photoheterotrophic cultivation (PH), dark culture, or heterotrophic cultivation (HT). In particular, some microalgae contain organic carbon. It is possible to grow under dark culture (HT) and photoheterotrophic culture (PH) conditions, which show higher growth than light culture (PA) under the included conditions, and DSHM22 of the Desmodesmos genus is capable of dark culture and light heterotrophic culture. To confirm whether it was microalgae, glucose was added at a concentration of 1 g/L and culture was performed under light and dark conditions.

As a result, according to Figure 5, growth was not observed under dark culture conditions, but the highest growth was observed under photoheterotrophic culture (PH) conditions, and the biomass (g/L) was approximately twice as high after 10 days. It was confirmed that .

<Example 4> Confirmation of photosynthetic pigment production by DSHM22 in Desmodesmos

DSHM22 of the Desmodesmos genus was cultured in two types of light culture (Light) and photoheterotrophic culture (light culture + glucose), and after 10 days, microalgae cells were harvested and photosynthetic pigments were analyzed. Photosynthetic pigments were analyzed using HPLC-DAD (Agilent 1260, Germany) and analyzed under gradient conditions using both solvent A and solvent B. First, for solvent A, a solution of 0.1M Tris, acetonitrile, and methanol was prepared in a ratio of 14:82:2, and for solvent B, methanol and ethyl acetate ( Ethyl acetate) was mixed in a ratio of 68:32. Afterwards, the solvent was sonicated for 1 hour to remove dissolved bubbles. The column used was a Waters Spherisorb® 5.0 umODS2 (4.6 mm Each analysis was performed. The flow rate was set at 1.2 mL/min, 10 μL was injected, and analysis was performed for 25 minutes. Gradient conditions were as follows. 0 min: 100% solvent A, 15 min: 100% solvent B, 18 min: 100% solvent B, 19 min: 100% solvent A, 25 min: 100% solvent A. To quantify photosynthetic pigments, a calibration curve was created using a carotenoid standard solution (DHI, USA) available in liquid form from DHI, and the pigments were quantified by comparing the area values of the analyzed peaks.

As a result, according to Table 1 below, it was confirmed that DSHM22 of the Desmodesmos genus is a microalgae capable of producing various carotenoid pigments. In particular, lutein productivity was the highest among carotenoid pigments, and when comparing photoculture conditions and photoheterotrophic culture conditions, lutein content and It was confirmed that productivity increased significantly, and the productivity of neoxanthin, zeaxanthin, alpha-carotene, and beta-carotene also increased significantly. This was found to be due to a significant increase in biomass productivity under photoheterotrophic culture conditions.

pigment RT (min) peak area Amount (mg/g) productivity
(mg/L/d) Photoculture neoxanthin 7.588 171.53 0.3751 23.4378 Violaxanthin 8.944 107.46206 0.0104 0.6499 lutein 11.730 463.80 0.6672 41.6917 Zeaxanthin 12.024 44.15 0.0975 6.09303 chlorophyll b 14.398 46.39 0.2468 15.4244 chlorophyll a 15.730 975.66 7.0464 440.3322 alpha-carotene 18.719 87.17 0.1142 7.13330 beta-carotene 18.909 18.06 0.0241 1.50576 Photoculture
+ glucose neoxanthin 7.537 343.04 0.3807 54.2146 Violaxanthin 8.895 213.49461 0.0207 2.9446 lutein 11.676 1037.99 0.7575 107.8803 Zeaxanthin 11.984 76.09 0.0854 12.1674 chlorophyll b 14.376 715.33 1.9207 273.5560 chlorophyll a 15.718 1561.60 5.7147 813.8997 alpha-carotene 18.743 197.82 0.1317 18.7500 beta-carotene 18.934 32.77 0.0235 3.3416

<Example 5> Confirmation of fatty acid production of DSHM22 in Desmodesmos

Fatty acid composition of microalgae was analyzed using a 7890A gas chromatograph (Agilent, Santa Clara, CA, USA) equipped with a 5975C mass selective detector. Crude lipids were extracted, esterified with hexane, and analyzed using a DB-FFAP column (30 m, 250 μm ID, 0.25 μm film thickness; Agilent, Santa Clara, CA, USA). The GV oven temperature started at 50°C and was maintained for 1 minute. The temperature was raised to 200°C at a rate of 10°C per minute for 30 minutes, then raised to 240°C at a rate of 10°C per minute and maintained for 20 minutes. 1 μl of the sample was injected at a split ratio of 20:1, and helium was flowed as a carrier gas at a flow rate of 1 ml/min. Mass spectrometry was analyzed under the following conditions. Sample temperature: 250 ℃, source temperature: 230 ℃, electron impact mode, an acceleration voltage of 70 eV was used for ionization of the sample in an acquisition range of 50-550 m·z ^-1 . For the identification of fatty acids, only results with a match value of 90% or more were judged valid compared to mass spectrometry of Wiley/NBS libraries.

As a result of fatty acid composition analysis, as shown in Table 2 below, in the case of Desmodesmos genus DSHM22, palmitic acid, oleic acid, linoleic acid, and alpha-linolenic acid are included in the fatty acid composition. It was confirmed that it accounts for most of the , and the fatty acid composition accumulated under photoculture and photoheterotrophic culture conditions was similar.

fatty acid type Photoculture Photoculture + glucose palmitic acid C16:0 - 24.63 ± 0.08 25.82 ± 1.76 palmitoleic acid C16:1 - 0.56 ± 0.01 0.43 ± 0.17 oleic acid C18:1 omega-9 30.97 ± 0.06 30.41 ± 0.86 linoleic acid C18:2 omega-6 21.94 ± 0.00 21.29 ± 0.93 Gamma-linolenic acid (GLA) C18:3 omega-6 3.61 ± 0.00 3.25 ± 0.51 Alpha-linolenic acid (ALA) C18:3 omega-3 15.32 ± 0.02 16.04 ± 1.03 Eicosenoic acid C20:0 - 0.34 ± 0.00 0.39 ± 0.08 Behen Mountain C22:0 - 1.02 ± 0.02 1.00 ± 0.06 Lignoceric acid C24:0 - 1.60 ± 0.03 1.38 ± 0.35

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15412 20230414

Claims (10)

delete delete delete (1) inoculating the microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP into the culture medium; and
(2) Adding an organic carbon source to the DSHM22 microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP inoculated into the culture medium and irradiating the light source. .DSHM22) Culture method. In claim 4,
The culture medium in step (1) is a method of cultivating microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP, characterized in that it contains agricultural fertilizer. In claim 5,
The agricultural fertilizer is Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP, characterized in that it contains at least one selected from the group consisting of nitrogen (N), phosphorus (P), and potassium (K). Culture method. In claim 4,
A method of cultivating microalgae of the genus Desmodesmus ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP, wherein the organic carbon source in step (2) is glucose. In claim 4,
The culture method is to extract neoxanthin, lutein, α-carotene, and β-carotene from the Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP. And a method of cultivating Desmodesmus sp. DSHM22 microalgae ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP, characterized in increasing the production of one or more selected from the group consisting of zeaxanthin. In claim 4,
The culture method is performed by extracting palmitic acid, oleic acid, linoleic acid and alpha-linoleic acid from the Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP. ) Cultivation method of Desmodesmus genus DSHM22 microalgae ( Desmodesmus sp. DSHM22) deposited as KCTC15412BP, characterized in increasing the production of one or more selected from the group consisting of. A composition for enhancing carotenoid or fatty acid production comprising a culture of Desmodesmus sp. DSHM22 microalgae deposited as KCTC15412BP.