CN108587913B

CN108587913B - Scenedesmus with high alpha-linolenic acid content, and culture method and application thereof

Info

Publication number: CN108587913B
Application number: CN201810337916.4A
Authority: CN
Inventors: 徐旭东; 高宏; 孟霞
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2017-06-08
Filing date: 2018-04-16
Publication date: 2021-09-03
Anticipated expiration: 2038-04-16
Also published as: CN107254410A; CN108587913A

Abstract

The invention relates to scenedesmus with high alpha-linolenic acid content, a culture method and application thereof. The fatty acid of the Scenedesmus total lipid mainly comprises hexadecane, oleic acid, linoleic acid and alpha-linolenic acid, and the content of the alpha-linolenic acid in the total lipid is stabilized at 35-45% under the optimized culture condition. For example, in a 3L fermentation experiment, after 120 h, the dry cell weight of the culture can reach 19.48 g L^‑1The total lipid content was 12.1%, the content of alpha-linolenic acid in the total lipid was 42.42%, and the yield of alpha-linolenic acid was 0.2 g L^‑1d^‑1. The strain can be used as an additive for producing animal feed to meet the requirements of animal growth on nutrients such as alpha-linolenic acid and the like, and can also be used as a raw material for industrially preparing the alpha-linolenic acid.

Description

Scenedesmus with high alpha-linolenic acid content, and culture method and application thereof

Technical Field

The present invention relates to microalgae and a culture technique thereof, and more particularly, to scenedesmus having a high ALA content, a culture method thereof, and applications thereof.

Background

Polyunsaturated Fatty acids (PUFAs) are straight-chain Fatty acids having 18 to 22 carbon atoms in length and containing two or more double bonds, and mainly include Linoleic Acid (LA), gamma-linolenic Acid (GLA), alpha-linolenic Acid (ALA), Arachidonic Acid (AA), eicosapentaenoic Acid (EPA), and docosahexaenoic Acid (DHA). In PUFAs, the double bond furthest from the carboxyl end is referred to as the omega-3 (n-3) series at the 3 rd carbon atom, and the omega-6 (n-6) series at the 6 th carbon atom.

Alpha-linolenic acid (ALA) is all-

cis

9,12, 15-octadecatrienoic acid, belongs to omega-3 series polyunsaturated fatty acid, and can finally form eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) through elongation and further desaturation of a carbon chain. EPA is the precursor of triene prostaglandin (such as PGI3, TXA 3) in animal body, and DHA is the main component of nervous system phospholipid such as brain, retina, etc. The important roles of ALA and its derivatives EPA and DHA in the aspects of brain tissue development, signal conduction of nervous system, development of cardiovascular system and cellular antioxidation of animals are known and accepted by the public. Since ALA is not synthesized by humans and other mammals themselves, ALA required in the body must be taken from food; furthermore, too low a ratio of ingested omega-3/omega-6 fatty acids may also result in an inefficient conversion of ALA to DHA and EPA, leading to psychological and cardiovascular diseases. Therefore, timely supplementation with alpha-linolenic acid is essential for human and animal health. The european union food safety agency (EFSA) also proposed in 2011 in nutritional science announcements that ALA is closely related to brain and nervous tissue development, and that ALA is added to the daily diet.

Human omega-3 polyunsaturated fatty acids are derived from the oil and fat content of foods such as plants and fish. The omega-3 series of polyunsaturated fatty acids are also required for growth and development of fish, and in nature are mainly derived from ingested algae and other plants. Researches show that ALA is added into the feed to meet the requirements of farmed fish on the fatty acid and promote the growth and health of the farmed fish.

Microalgae are a group of lower plants that are unicellular or multicellular. Many types of microalgae are rich in nutritional ingredients such as protein, oil and fat, polysaccharide and the like, and other types of microalgae are rich in physiological active ingredients such as unsaturated fatty acid, carotenoid, vitamin and the like. The dry powder of algae such as spirulina, chlorella, etc. as feed additive can significantly promote the growth, metabolism and disease resistance of aquatic animals. However, the existing microalgae industry mainly relies on autotrophic growth for open pond culture, has low total yield and high price, can only meet higher-end consumption, is difficult to be used as an additive for the aquatic animal feed industry, and has low ALA content in spirulina and other algae species. Therefore, the method needs to screen the algae species which are rich in ALA and can grow heterotrophically, develop the fermentation technology of the microalgae and greatly improve the yield of the microalgae per unit area. The invention provides scenedesmus (Scenedesmus obliquus) which is rich in ALA and can grow completely heterotrophicallyScenedesmussp.) and optimizing the fermentation culture conditions.

Requirement of priority

The present application claims priority of the patent application entitled "scenedesmus having a high GLA content, a method for culturing the same and use thereof" filed by the applicant at 8.6.2017 under application No. 2017104292799.

Disclosure of Invention

The inventor discovers in the research process that Scenedesmus obliquus can perform photoautotrophic growth, photoautotrophic growth (autotrophic/heterotrophic mixed growth) and pure heterotrophic growth under different conditions, high-content oil and fat can be accumulated in the growth process, ALA accounts for more than 20% of the total fat content, and the content of ALA in the total fat can reach 45% when the culture medium components are adjusted, so that the Scenedesmus obliquus can be used for producing ALA or feed additives or food additives with high ALA content.

Based on the findings and researches, the invention provides scenedesmus, which is preserved in the China center for type culture Collection in 2017, 5 months and 24 days with the preservation number: CCTCC NO: m2017282.

The fatty acid of the total lipid of scenedesmus mainly consists of sixteen carbonsOleic acid, linoleic acid and ALA, and the content of ALA in the total lipid is stabilized at 35-45% under the optimized culture condition. For example, in a 3L fermentation experiment, after 120 h, the dry cell weight of the culture can reach 19.48 g L^-1The total lipid content was 12.1%, the ALA content was 42.42% and the ALA yield was 0.2 g L^-1d^-1. Therefore, the strain has the potential of being used as a raw material for industrial production of ALA.

The invention also provides a culture method of the scenedesmus, and the scenedesmus is cultured by illumination or heterotrophic culture.

In one embodiment, the scenedesmus is heterotrophically cultured, comprising the step of culturing the scenedesmus under dark conditions using a basal medium supplemented with a carbon source and a nitrogen source. Under dark conditions, the algal strain can be completely heterotrophic, and the growth speed is faster than that under light conditions.

In a preferred embodiment, the basal medium is an aqueous solution comprising: KH (Perkin Elmer)₂PO₄，0.7 g L^-1；K₂HPO₄，0.3 g L^-1；MgSO₄ 7H₂O，0.3 g L^-1；FeSO₄ 7H₂O，3 mg L^-1(ii) a Glycine, 0.1 g L^-1；vitamin B₁，0.01 mg L^-1；A5，1 ml L^-1. Wherein the component A5 is boric acid (H)₃BO₃) 2.86 g/L, manganese chloride tetrahydrate (MnCl)₂·4H₂O) 1.81 g/L, zinc sulfate heptahydrate (ZnSO)₄·7H₂O) 0.22 g/L, sodium molybdate dihydrate (Na)₂MoO₄·2H₂O) 0.39 g/L, copper sulfate pentahydrate (CuSO)₄·5H₂O) 0.08 g/L, cobalt chloride hexahydrate (CoCl)₂·6H₂O）0.01 g/L。

The basic medium components are used only to test the kind and content of the nitrogen source and carbon source suitable for scenedesmus of the present invention, and do not limit the present invention. One skilled in the art can select any required basal medium to culture scenedesmus of the present invention, for example, BG11 medium can also be selected as the basal medium.

In a preferred embodiment, the carbon source is 10 to 40 g L^-1The glucose of (4). Although the amount of glucose added did not greatly affect the initial growth rate of the algal strain, 20-40 g L was added after 96 hours of culture^-1In the glucose culture medium, the algae are still in an exponential growth stage, so that the algae can finally reach higher concentration, and the yield of fatty acid in a unit volume of culture solution is improved.

In a preferred embodiment, the nitrogen source is urea, potassium nitrate and/or yeast extract. Any one or a combination of urea, potassium nitrate and yeast extract. An appropriate nitrogen source is beneficial to the growth of the algal strains.

In a preferred embodiment, the nitrogen source is from 1 to 10 g L^-1Urea. Urea is cheaper than other nitrogen sources. The addition amount of urea is 1-10 g L^-1Has little influence on the total lipid content. However, when the urea content is 4-7 g L^-1When the content is within the range of (4), the ALA content is high.

In a preferred embodiment, the scenedesmus is cultured at a temperature of 25-35 ℃. The algae can grow well at 25-35 ℃, especially at 30-35 ℃, the growth speed is faster, the state is better, and the growth speed of the algae is obviously reduced when the temperature is lower than 20 ℃. In the range of 25-30 deg.C, ALA content is high.

The invention also provides application of Scenedesmus in preparation of ALA.

The invention also provides the application of Scenedesmus obliquus in preparing feed additive or food additive with high ALA content

The present invention also provides a method for producing fatty acids, in particular ALA, using the scenedesmus described above.

In a preferred embodiment, the scenedesmus used for the production of fatty acids is cultured by the above-mentioned culture method.

Preservation of microorganisms

The scenedesmus related to the invention is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 5 and 24 months, and the preservation number is as follows: CCTCC NO: m2017282. The survival of the scenedesmus is detected by the China center for type culture Collection in 2017, 6 and 8 months, and the result is survival.

Drawings

FIG. 1 is an optical micrograph of Scenedesmus HSJ 296;

FIG. 2 is a graph of Scenedesmus HSJ296 growth under different nitrogen sources;

FIG. 3 is a graph of Scenedesmus HSJ296 growth at different urea concentrations;

FIG. 4 is the total lipid content of Scenedesmus HSJ296 at different urea concentrations;

FIG. 5 is a graph of Scenedesmus HSJ296 growth at different temperatures;

FIG. 6 shows the total lipid content of Scenedesmus HSJ296 at different temperatures;

FIG. 7 is a graph of Scenedesmus HSJ296 growth at different glucose concentrations;

FIG. 8 is the total lipid content of Scenedesmus HSJ296 at different glucose concentrations;

FIG. 9 is a graph of Scenedesmus HSJ296 growth in fermentors.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

1. Screening and preservation of algal strains

Researchers separate 232 strains of green algae from a water sample collected in the field by a separation and purification method, and the green algae can be used for screening green algae with high ALA content and capable of completely heterotrophic growth.

The inventors inoculated 232 sterilized and purified green algae into a container containing 5 ml of glucose with a concentration of 30 g L^-1The BG11 test tube was cultured by a temperature-controlled shaking table at 30 ℃ and 150 rpm for 5 days. Through observation, 30 completely heterotrophic green algae were selected. 30 heterotrophically culturable green algae were inoculated into 500 ml Erlenmeyer flasks containing 120 ml BG11, and cultured under the same conditions as described above. After culturing for 5 days, the algae liquid is collected by centrifugation, freeze-dried to constant weight, and 0.1 g of algae powder is weighed and extracted to obtain the oil. Methyl esterifying the oil, and measuring the oil by gas chromatographyThe fatty acid composition and the relative content of each component.

The algal strain with the highest ALA content in the total lipid is screened by the method, and the ALA content in the total lipid is 25.78% when the culture conditions are not optimized. The morphology of the algal cells was observed using an optical microscope and was shown to have scenedesmus characteristics (fig. 1). Extracting algae cell DNA as template, amplifying with 18S rDNA universal PCR primer to obtain 18S rDNA, and sequencing to obtain the sequence shown in SEQ ID NO. 1. The sequence of the product is aligned to confirm that the product belongs to Scenedesmus (Scenedesmus) It is named Scenedesmus HSJ296 (Scenedesmussp. HSJ296）

The inventor sends the strain to a culture collection center (CCTCC) in Wuhan university No. eight 299 of Wuhan district in Wuchan city, Hubei province for microbial preservation in 2017, 24 months and 5 months, wherein the preservation numbers are as follows: CCTCC NO: m2017282.

2. Optimization of Scenedesmus HSJ296 culture conditions

Basic culture medium: KH (Perkin Elmer)₂PO₄，0.7 g L^-1；K₂HPO₄，0.3 g L^-1；MgSO₄ 7H₂O，0.3 g L^-1；FeSO₄7H₂O，3 mg L^-1(ii) a Glycine, 0.1 g L^-1；vitamin B₁，0.01 mg L^-1；A5，1 ml L^-1. The basic medium components are only used for providing a plurality of necessary nutrient elements for Scenedesmus obliquus HSJ296, and are used for testing the optimal carbon source and nitrogen source required for culturing Scenedesmus obliquus HSJ296, and the concentration thereof, and the invention is not limited. The person skilled in the art can use other combinations of components of the basal medium depending on his conditions and needs.

2.1 optimization of Nitrogen sources

When scenedesmus HSJ296 was shake-cultured in a basal medium under a heterotrophic dark culture condition at 30 ℃ in a shake flask, the effects of 5 nitrogen sources, i.e., urea, potassium nitrate, ammonium nitrate, glycine and yeast extract, on the growth of algal strains were compared. The glucose concentration in the culture medium was 6 g L^-1The concentrations of the 5 nitrogen sources were all 1 g L^-1And carrying out shaking culture for 120 h.

The results are shown in FIG. 2, where urea is most suitable for the growth of Scenedesmus HSJ296 compared to other nitrogen sources. Secondly, Scenedesmus HSJ296 grew well when yeast extract or potassium nitrate was used as a nitrogen source. However, scenedesmus HSJ296 grew very slowly when ammonium nitrate or glycine was used as the nitrogen source.

To further investigate the effect of urea on Scenedesmus HSJ296 growth, we set 1-10 g L^-1Four urea concentration gradients within the range were used for algal strain cultivation. The growth curve of Scenedesmus HSJ296 at different urea concentrations is shown in FIG. 3, 1-10 g L^-1The difference of the growth rates of the algae plants is not large in the urea concentration range of (4) 4 g L^-1It is slightly better. The total lipid content is shown in FIG. 4, with urea concentrations of 1, 4, 7 and 10 g L^-1Then, the total lipid content was 9.83%, 10.93%, 10.33% and 10.78%, respectively. The urea concentration is 4 g L^-1Its total lipid content is slightly higher.

Thus, the urea concentration was 4 g L^-1In this case, Scenedesmus HSJ296 culture is most suitable.

The fatty acid composition of scenedesmus HSJ296 total lipid at different urea concentrations is shown in table 1.

Table 1 fatty acid composition (mol%) of scenedesmus HSJ296 total lipid at different urea concentrations

2.2 optimization of culture temperature

Under the condition of heterotrophic culture in the dark, when scenedesmus HSJ296 is cultured by using basal medium in a shake flask, the urea concentration in the basal medium is 4 g L^-1Glucose concentration of 20 g L^-1The effect of temperature on the growth of algal strains was investigated. We set up four temperature gradients and shake culture for 120 h.

The growth rates of scenedesmus HSJ296 at different temperatures are shown in FIG. 5, and the growth rate of scenedesmus HSJ296 is fastest when the culture temperature is 30 ℃; the growth rate of Scenedesmus HSJ296 was slower at both 20 ℃ and 25 ℃; the growth state was good at 35 ℃. The experimental results prove that the temperature has a great influence on the growth rate of Scenedesmus HSJ 296. The total lipid content is shown in fig. 6, and the total lipid content of scenedesmus HSJ296 is 11.43% at 30 ℃; the total lipid content was 10.63%, 10.87% and 10.93% at 20 ℃, 25 ℃ and 35 ℃, respectively. The temperature is 30 deg.C, and the oil content of the algae is highest. The experimental results prove that the temperature has little influence on the grease accumulation of Scenedesmus HSJ 296.

Therefore, 30 ℃ is the optimum culture temperature for Scenedesmus HSJ296 from the viewpoint of both growth rate and oil content.

The fatty acid composition of scenedesmus HSJ296 total lipids at different temperatures is shown in table 2.

TABLE 2 fatty acid composition (mol%) of Scenedesmus HSJ296 total lipid at different temperatures

2.3 sugar concentration optimization

Glucose is the most common carbon source for maintaining heterotrophic growth of algal strains. In exploring the effect of initial glucose concentration on Scenedesmus HSJ296 biomass accumulation, we set the glucose concentration gradients at 10, 20, 30, and 40 g L^-1Under the condition of dark heterotrophic culture, the scenedesmus HSJ296 is cultured by shaking the shake flask. The urea concentration in the basal medium was 4 g L^-1The culture temperature was 30 ℃. Of course, glucose is only an example of a carbon source used for algal cell growth and is not intended to limit the present invention. Other carbon sources can be used by those skilled in the art depending on their conditions and needs.

The growth curves of Scenedesmus HSJ296 at different glucose concentrations are shown in FIG. 7 at 10-40 g L^-1The initial glucose concentration range, within 96 h, had little effect on the growth of scenedesmus HSJ 296. But the initial glucose was 10 g L^-1When the culture is carried out for 96 hours, the algae basically do not grow; and initial sugar concentrations of 20, 30 and 40 g L^-1In the meantime, the algal strains still grow exponentially after 96 hours of culture.

From the above data, it can be seen that the initial glucose concentration in the basal medium was 10 g L^-1When the culture is continued for 96 hours, glucose in the medium is substantially consumed, the carbon source in the medium is deficient, and the algal strain stopsAnd (5) growing. After 120 h of culture, the yield of the algae strains is 4.97, 9.34, 10.4 and 11.02 g L respectively^-1(not shown in the figure). The above data demonstrate that glucose has a greater effect on microalgae growth under heterotrophic culture conditions.

For total lipid accumulation, as shown in FIG. 8, when the glucose concentration was 10-40 g L^-1In between, the total lipid content was 10.43%, 10.22%, 10.42% and 10.06%, respectively, with slight fluctuations. The initial glucose concentration had little effect on the accumulation of this algal oil.

The total lipid fatty acid composition of scenedesmus HSJ296 at different glucose concentrations is shown in table 3.

TABLE 3 fatty acid composition (mol%) of Scenedesmus HSJ296 total lipids at different glucose concentrations

According to the experimental results, in the subsequent fermentation culture experiment, the fermentation requirement can be 20-40 g L^-1The fermentation is carried out by selecting an appropriate sugar concentration within the glucose concentration range of (1).

3. Cultivation in fermenter

Heterotrophic culture of Scenedesmus HSJ 296120 h with basal medium at 30 deg.C and initial glucose concentration of 20 g L^-1Urea concentration of 4 g L^-1The rotation speed was 200 rpm, and pH was not controlled.

As shown in FIG. 9, the culture time was between 52-88 h, the cells began to grow exponentially, and after 90 h, the algae entered plateau. At 52 h, glucose also began to be consumed rapidly, and at 76 h, the glucose concentration dropped to 4 g L^-1(data not shown). Culturing for 120 h, stopping the growth of Scenedesmus HSJ296, and making the dry weight of cells reach 19.48 g L^-1The total lipid content was 12.1% and the ALA content was 42.42% of the total lipid. The yield of ALA can reach 1 g L^-1I.e. an ALA yield of 0.2 g L^-1 d^-1。

The total lipid fatty acid composition of Scenedesmus HSJ296 cultured in the fermentor is shown in Table 4.

TABLE 4 Total fatty acid composition (mol%) of Scenedesmus HSJ296 cultured in fermenter

The growth rate and the ALA yield of the microalgae are improved by improving the culture conditions and the culture modes of the microalgae, and the result shows that the optimal culture temperature of Scenedesmus HSJ296 is 30 ℃, and the optimal nitrogen source is 4 g L^-1The initial concentration of urea and glucose is 20-40 g L^-1All within the scope of the growth of the algae. Under optimized culture conditions, the fatty acids of Scenedesmus HSJ296 total lipid are mainly composed of hexadecane (C16: 0), oleic acid (C18: 1), linoleic acid (C18: 2) and ALA, and the ALA content in the total lipid is stabilized between 35-45%.

3L fermentation experiment is carried out, after 120 h, the dry cell weight of Scenedesmus HSJ296 can reach 19.48 g L^-1The total lipid content was 12.1%, the ALA content was 42.42% and the ALA yield was 0.2 g L^-1d^-1. In the future, with the further optimization of fermentation conditions, the ALA content and the yield are expected to be remarkably improved. Scenedesmus HSJ296 has potential of producing animal feed microalgae additive to supplement ALA and other nutrients, and can also be used as raw material for extracting and preparing ALA.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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<120> Scenedesmus with high ALA content, its culture method and application

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aaagattacc cggacctttc ggtcaaggat aggcttgttg aatgcatcag tgtagcgcgc 360

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acggccatgc accaccaccc atagaatcaa gaaagagctc tcaatctgtc aatcctcact 600

atgtctggac ctggtaagtt ttcccgtgtt gagtcaaatt aagccgcagg ctccacgcct 660

ggtggtgccc ttccgtcaat tcctttaagt ttcagccttg cgaccatact ccccccggaa 720

cccaaaaact ttgatttctc ataaggtgct ggcgaagtca ttaaaaaaac attcgccaat 780

ccctagtcgg catcgtttat ggttgagact acgacggtat ctaatcgtct tcgagccccc 840

aactttcgtt cttgattaat gaaaacatcc ttggcaaatg ctttcgcagt agttcgtctt 900

tcataaatcc aagaatttca cctctgacat gaaatacgaa tgcccccgac tgtccctctt 960

atcattactc cggtcctaca gaccaacaag ataggccaga gtcctatcgt gttattccat 1020

gctaaagtat tctggcgtaa gcctgctttg aacactctaa tttactcaaa gtaaccacgt 1080

cgactccgag tcccggacag tgaagcccag aagcccgtcc ccgacagaaa ggaaggccat 1140

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ataagcagtt tatacttaga catgcatggc ttaatctttg agacaagcat atgactactg 1800

gcaggatcaa ccaaatctct agaggatccc cgggtaccga gctcgaatc 1849

Claims

1. Scenedesmus, which is preserved in China center for type culture Collection in 2017, 5 months and 24 days with the preservation number: CCTCC NO: m2017282.

2. The method for culturing Scenedesmus as claimed in claim 1, wherein light culture or heterotrophic culture is carried out.

3. The culture method according to claim 2, wherein the scenedesmus is heterotrophically cultured, comprising the step of culturing the scenedesmus under dark conditions using a basal medium supplemented with a carbon source and a nitrogen source.

4. According to claimThe culture method of 3, wherein the carbon source is 20 to 40 g.L^-1The glucose of (4).

5. The culture method according to claim 3, wherein the nitrogen source is any one or a combination of urea, potassium nitrate and yeast extract.

6. The culture method according to claim 5, wherein the nitrogen source is 1 to 10 g.L^-1Urea.

7. The culture method according to any one of claims 3 to 6, wherein the culture temperature of Scenedesmus is 25 to 35 ℃.

8. Use of scenedesmus as claimed in claim 1 for the preparation of alpha-linolenic acid.

9. Use of scenedesmus as claimed in claim 1 for the preparation of a feed additive or food additive with a high alpha-linolenic acid content.

10. A method of producing α -linolenic acid, comprising extracting α -linolenic acid from scenedesmus as claimed in claim 1.