CN108949575A - A kind of hidden dinoflagellate of high DHA content and its application - Google Patents
A kind of hidden dinoflagellate of high DHA content and its application Download PDFInfo
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- CN108949575A CN108949575A CN201810480850.4A CN201810480850A CN108949575A CN 108949575 A CN108949575 A CN 108949575A CN 201810480850 A CN201810480850 A CN 201810480850A CN 108949575 A CN108949575 A CN 108949575A
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- hidden dinoflagellate
- culture medium
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- dinoflagellate
- dha
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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
- C12N1/125—Unicellular algae isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
Abstract
The present invention relates to a kind of hidden dinoflagellate of high DHA content, the hidden dinoflagellate is preserved in China typical culture collection center on November 13rd, 2017, and deposit number is CCTCC NO:M2017690；Further relate to the hidden dinoflagellate is preparing the application in DHA；Further relate to the cultural method of the hidden dinoflagellate.Hidden dinoflagellate W1 growth of the invention is fast, can carry out complete heterotrophic growth.In the case where nitrogen stress induction, the hidden dinoflagellate can largely accumulate DHA.Therefore, hidden dinoflagellate of the invention and cultural method can be used for preparing DHA or food additives or feed addictive with high DHA content, with good economic efficiency.
Description
Technical field
The present invention relates to microdisk electrode and unsaturated fatty acid production field, more specifically it relates to a kind of high DHA content
Hidden dinoflagellate and its application.
Background technique
Polyunsaturated fatty acid (Polyunsaturated Fatty Acid, PUFA) refers to and contains two or more
Double bond and carbon length are the straight chain fatty acid of 18-22 carbon atom, mainly include linoleic acid (LA), gamma-Linolenic acid (GLA), α-
Linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) etc..In PUFA,
It is known as ω -3 (n-3) series on the 3rd carbon atom away from the farthest double bond of c-terminus, ω-is known as if on the 6th carbon atom
6 (n-6) series.
Docosahexaenoic acid (Docosahexaenoic acid, DHA) is presently found carbochain longest, degree of unsaturation
Highest long-chain highly unsaturated fatty acid.DHA is the important feature component of body biomembrane, in infant's neurodevelopment, view
Nethike embrane is formed, intellectual development etc. plays a significant role, and is also prostaglandin, the tool such as change Prostaglandins and Leukotrienes element
There is the precursor of itself Auto-regulator of strong physiological activity, to prevention cardiovascular disease, anti-lipid, blood pressure lowering, inhibits tumour shape
There is remarkable effect at equal.Since with above-mentioned a variety of important physiological functions, DHA, which has become, is worth high, the extensive rouge in market
Fat acid product.
Microalgae is a kind of unicellular or many cells rudimentary plant.Numerous species are rich in protein, grease, polysaccharide in microalgae
Etc. nutritional ingredients, type also is rich in physiologically active ingredients such as unsaturated fatty acid, carotenoid, vitamins.But mesh
Preceding microalgae industry relies primarily on autophyting growth and carries out open pond culture, and DHA overall productivity is low, price is high, limits DHA and is seeking
It supports and the application in scientific research.
Summary of the invention
Inventor samples isolated one plant in Wuhan City of Hubei China province waters and can survive in high salinity medium
The microalgae of growth, which exists in the form of unicellular, spherical in shape, 4-30 μm of diameter, does not move, atrichia, is under microscope
It is transparent spherical.According to cellular morphology and life habit and 18SrDNA sequencing display, belong to eucaryote domain, vesica algae circle
Chromalveolata, different whip algae (Heterokontophyta or Stramenopiles), bicyclic door (Bigyra),
Labyrinthulomycetes mesh, Thraustochytriaceae section, hidden dinoflagellate category (Schizochytrium).
Based on the above discovery, the present invention provides a kind of hidden dinoflagellate of high DHA content, the hidden dinoflagellate is in November, 2017
It is preserved within 13rd China typical culture collection center, deposit number is CCTCC NO:M2017690.
The present invention also provides above-mentioned hidden dinoflagellates to prepare the application in DHA.
The present invention also provides a kind of methods for cultivating above-mentioned hidden dinoflagellate, comprising the following steps:
S1: cultivating the hidden dinoflagellate in full culture medium, accumulates biomass；
S2: the hidden dinoflagellate frustule of S1 culture is collected；
S3: the hidden dinoflagellate frustule being forwarded in nitrogen stress culture medium and is cultivated, and obtains the hidden dinoflagellate culture.Pass through
The method of segmented culture can guarantee that first proliferation obtains a large amount of biomass to class pot algae in a short time, then can induce by nitrogen stress
The hidden a large amount of DHA of dinoflagellate cylinder accumulation.
In a preferred embodiment, the full culture medium is to contain 20g/L glucose, 5g/L yeast extract, 5g/L
The aqueous solution of peptone, 1mL/L A5 solution and the artificial sea salt of 24g/L.
In a preferred embodiment, the pH of the full culture medium is 6-8, the dissolved oxygen of the full culture medium 2% with
On.
In a preferred embodiment, cultivation temperature is 25 DEG C in S1.Through our research indicate that, the hidden dinoflagellate is 25
It is grown at DEG C most fast.
In a preferred embodiment, the other components of the nitrogen stress culture medium are identical as the full culture medium, and nitrogen contains
Amount is the 1/4-1/2 of the full culture medium.
In a preferred embodiment, the pH of the nitrogen stress culture medium is 6-7, and the dissolved oxygen of the nitrogen stress culture medium is
0.5-2%.
In a preferred embodiment, cultivation temperature is 18-22 DEG C in S2.
Hidden dinoflagellate W1 growth of the invention is fast, can carry out complete heterotrophic growth.In the case where nitrogen stress induction, the hidden first
Algae can largely accumulate DHA.Therefore, hidden dinoflagellate of the invention and cultural method can be used for preparing DHA or with high DHA contents
Food additives or feed addictive, it is with good economic efficiency.
Microbial preservation
Hidden dinoflagellate of the invention has been sent on November 13rd, 2017 to No. 299 Wuhan of Wuhan City, Hubei Province Wuchang District Bayi Road
China typical culture collection center (CCTCC) in institution of higher education carries out preservation, and deposit number is CCTCC NO:M2017690, life
Entitled hidden dinoflagellate W1, Classification system are Schizochytrium sp.W1.
Detailed description of the invention
Fig. 1 is the photo of hidden dinoflagellate W1 of the invention under an optical microscope；
Fig. 2 is the growth curve that hidden dinoflagellate W1 of the invention is cultivated.
Specific embodiment
Principles and features of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
1. the acquisition and preservation of hidden dinoflagellate W1
The water sample that hidden dinoflagellate of the invention is acquired from Wuhan City East Lake screens isolated, the microalgae by artificial seawater
It is survived in the form of single celled, spherical in shape, atrichia does not move, and 4-30 μm of cell dia, be in colorless and transparent ball under microscope
Body can carry out complete heterotrophic growth.It is sequenced through 18S rDNA and Morphological Identification, the microalgae belongs to eucaryote domain, vesica algae
Boundary Chromalveolata, different whip algae (Heterokontophyta or Stramenopiles), bicyclic door (Bigyra),
Labyrinthulomycetes mesh, Thraustochytriaceae section, hidden dinoflagellate category (Schizochytrium).
The microalgae is sent on November 13rd, 2017 to No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road in the school
China typical culture collection center (CCTCC) carries out preservation, and deposit number is CCTCC NO:M2017690, is named as hidden dinoflagellate
W1, Classification system are Schizochytrium sp.W1.
2. the culture of hidden dinoflagellate W1
It carries out preliminary experiment to grope the condition of culture of hidden dinoflagellate W1, experimental result is shown, the most adaptability of hidden dinoflagellate W1
Long temperature is 25 DEG C, and dim light is liked under the conditions of heterotrophic growth or is protected from light culture, optimal pH 6-8.
It is 25 ± 1 DEG C by culture environment condition setting based on the above research, initial pH is adjusted to 7.0 ± 0.1, and shaking flask is returned
Rotation reciprocating rate is 150rpm, and 500mL shaking flask liquid amount is 100mL, in black undercover culture.Culture medium is to contain 20g/L
Glucose, 5g/L yeast extract, 5g/L peptone, 1mL/L A5 solution (table 1) and the artificial sea salt of 24g/L (ingredient such as 2 institute of table
Show) aqueous solution.
The ingredient of 1 A5 solution of table
The ingredient of the artificial sea salt of table 2
Initial inoculation concentration is the biomass of 0.5g/L, is protected from light in incubation, and control dissolved oxygen 2% or more.Culture 5
It, biomass is surveyed in sampling daily, draws growth curve.As a result as shown in Figure 1, before hidden dinoflagellate W1 4 days growth it is faster, to the 5th
Its growth slows down.
The induction of 3.DHA generates
Hidden dinoflagellate W1 is induced to generate DHA by the change of nitrogen stress and some other condition of culture, in this step, training
The other compositions for supporting base are constant, and the concentration of yeast extract is only adjusted to 1-2g/L.And pH is adjusted to 6-7, holding cultivation temperature is
18-22 DEG C, and it is maintained at dissolved oxygen between 0.5-2%.After induction 2-3 days, DHA accounts for dry weight in obtained hidden first algae powder
11.4%, total rouge accounts for 39% of dry weight or more.
The present invention not yet deeply gropes the DHA accumulation condition of algae strain, it is therefore believed that in culture and inductive condition
Afterwards, hidden dinoflagellate W1 of the invention can accumulate higher DHA.
Claims (9)
1. a kind of hidden dinoflagellate of high DHA content, the hidden dinoflagellate is preserved in Chinese Typical Representative culture guarantor on November 13rd, 2017
Hiding center, deposit number are CCTCC NO:M2017690.
2. hidden dinoflagellate described in claim 1 is preparing the application in DHA.
3. a kind of method for cultivating hidden dinoflagellate described in claim 1, which comprises the following steps:
S1: cultivating the hidden dinoflagellate in full culture medium, accumulates biomass；
S2: the hidden dinoflagellate frustule of S1 culture is collected；
S3: the hidden dinoflagellate frustule being forwarded in nitrogen stress culture medium and is cultivated, and obtains the hidden dinoflagellate culture.
4. according to the method described in claim 3, it is characterized in that, the full culture medium is to contain 20g/L glucose, 5g/L ferment
Mother soaks the aqueous solution of powder, 5g/L peptone, 1mL/L A5 solution and the artificial sea salt of 24g/L.
5. according to the method described in claim 4, it is characterized in that, the pH of the full culture medium be 6-8, the full culture medium
Dissolved oxygen is 2% or more.
6. according to the method described in claim 3, it is characterized in that, cultivation temperature is 25 DEG C in S1.
7. method according to claim 3, which is characterized in that the other components of the nitrogen stress culture medium and the full culture medium
Identical, nitrogen content is the 1/4-1/2 of the full culture medium.
8. the method according to the description of claim 7 is characterized in that the pH of the nitrogen stress culture medium is 6-7, the nitrogen stress culture
The dissolved oxygen of base is 0.5-2%.
9. the method according to any one of claim 3-8, which is characterized in that cultivation temperature is 18-22 DEG C in S2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591617A (en) * | 2009-06-15 | 2009-12-02 | 南京工业大学 | A kind of docosahexaenoic acid-producing strain and mutagenesis screening method thereof and its application |
| CN106010970A (en) * | 2016-07-13 | 2016-10-12 | 中国科学院青岛生物能源与过程研究所 | Crypthecodinium cohnii and application thereof |
-
2018
- 2018-05-18 CN CN201810480850.4A patent/CN108949575A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591617A (en) * | 2009-06-15 | 2009-12-02 | 南京工业大学 | A kind of docosahexaenoic acid-producing strain and mutagenesis screening method thereof and its application |
| CN106010970A (en) * | 2016-07-13 | 2016-10-12 | 中国科学院青岛生物能源与过程研究所 | Crypthecodinium cohnii and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| ANA MENDES ET AL: "Crypthecodinium cohnii with emphasis on DHA production: a review", 《JOURNAL OF APPLIED PHYCOLOGY》 * |
| 常桂芳: "氧对裂壶藻利用甘油产生DHA影响机制及其高密度发酵控制策略的研究", 《中国博士学位论文全文数据库（电子期刊）》 * |
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Application publication date: 20181207 |