CN106987528A - One plant production docosahexaenoic acid bacterium and its application - Google Patents
One plant production docosahexaenoic acid bacterium and its application Download PDFInfo
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Abstract
The invention discloses one plant of bacterium for producing docosahexaenoic acid and its application.The bacterium for the production docosahexaenoic acid that the present invention is provided is schizochytrium limacinum (Schizoochytrium limacinum) HS01, and it is CGMCC No.13746 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.It is demonstrated experimentally that fermented and cultured schizochytrium limacinum (Schizoochytrium limacinum) HS01, obtains zymotic fluid；The content that DHA accounts for grease in the zymotic fluid is 45.0%~60.0%.Therefore, the present invention has important application value.
Description
Technical field
The present invention relates to industrial microorganism, food and feed industry field, and in particular to one plant of two dodecahexaene of production
The bacterium of acid and its application.
Background technology
Docosahexaenoic acid (Docosahexaenoic acid, DHA) is how unsaturated fat necessary to a kind of human body
Fat acid.On molecular structure, DHA is the straight chain fatty acid containing 22 carbon atoms and 6 double bonds, because its 1st double bond occurs
On the 3rd carbon atom at fatty acid chain methyl end, therefore belong to ω -3 series fatty acids (OMEGA-3).DHA has important life
Function is managed, for example, promotes nervous system development, improves retinal function, improve eyesight, prevention of cardiovascular disease, treat cardiovascular
Disease, anti-inflammatory and suppression allergic reaction etc..Due to the DHA in diet, often content is not enough, therefore in food or milk
DHA is added in powder significant to the mankind especially infant and the elderly.
At present, production DHA method mainly has two kinds：One kind is traditional DHA sources, i.e., (mainly include from marine fishes
Salmon, mackerel, salmon and sardine) extract in tissue, but obtained by extracting fish oil quality can with fish species,
Season and fish for the change on ground and be affected, and fish oil quality also by increasingly serious fish resource is deficient and environment
The influence such as pollution；Another is emerging DHA production methods, i.e., carry out fermenting and producing DHA, this method tool using marine microorganism
Have that the cycle is short, do not influenceed by objective condition, without advantages such as fishlike smell, with extensive prospect.Sea for fermenting and producing DHA
Foreign microorganism is mainly schizochytrium limacinum (Schizochytrium), but currently used for fermenting and producing DHA schizochytrium limacinum, due to certainly
The technical indicators such as body total fatty acid content, DHA content, growth rate are limited, it is impossible to further improved yield, reduced cost.
The content of the invention
The technical problems to be solved by the invention are how to produce docosahexaenoic acid.
In order to solve the above technical problems, present invention firstly provides the bacterium of one plant of production docosahexaenoic acid.
The bacterium of production docosahexaenoic acid provided by the present invention is schizochytrium limacinum (Schizoochytrium
Limacinum) HS01, it is in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center
CGMCC No.13746。
In order to solve the above technical problems, present invention also offers a kind of microbial inoculum, the microbial inoculum contains the schizochytrium limacinum
(Schizoochytrium limacinum)HS01CGMCC No.13746。
Schizochytrium limacinum (Schizoochytrium limacinum) the HS01CGMCC No.13746 or, the microbial inoculum
Application in production docosahexaenoic acid falls within protection scope of the present invention.
In order to solve the above technical problems, present invention also offers a kind of method for producing docosahexaenoic acid, this method
Including schizochytrium limacinum described in fermented and cultured (Schizoochytrium limacinum) HS01CGMCC No.13746, two are obtained
The step of dodecahexaene acid.
In the above method, the fermentation medium solute and its solubility that the fermented and cultured is used can be：Glucose 20~
120g/L (such as 20~60g/L, 60~120g/L, 20g/L, 60g/L or 120g/L), 5~15g/L of glutamic acid or sodium glutamate
(such as 5~10g/L, 10~15g/L, 5g/L, 10g/L or 15g/L), 3~15g/L of Dried Corn Steep Liquor Powder (such as 3~10g/L, 10~
15g/L, 3g/L, 10g/L or 15g/L), NaSO45~24g/L (such as 5~14g/L, 14~24g/L, 5g/L, 14g/L or 24g/
L), 0.1~1.0g/L of KCl (such as 0.1~0.5g/L, 0.5~1.0g/L, 0.1g/L, 0.5g/L or 1.0g/L), MgSO4 1.0
~3.0g/L (such as 1.0~2.0g/L, 2.0~3.0g/L, 1.0g/L, 2.0g/L or 3.0g/L), K2SO40.3~1.5g/L is (such as
0.3~1.0g/L, 1.0~1.5g/L, 0.3g/L, 1.0g/L or 1.5g/L), KH2PO40.5~1.5g/L (such as 0.5~1.0g/
L, 1.0~1.5g/L, 0.5g/L, 1.0g/L or 1.5g/L), (NH4)2SO40.5~1.5g/L (such as 0.5~1.0g/L, 1.0~
1.5g/L, 0.5g/L, 1.0g/L or 1.5g/L), CaCl20.1~1.0g/L (such as 0.1~0.5g/L, 0.5~1.0g/L,
0.1g/L, 0.5g/L or 1.0g/L)；Solute can be water；PH value can be 5.0~6.5 (such as 5.0,6.0 or 6.5).The fermentation training
Foster base is concretely：By glucose 60g, glutamic acid or sodium glutamate 10g, Dried Corn Steep Liquor Powder 10g, NaSO4 14g、KCl
0.5g、MgSO4 2.0g、K2SO4 1.0g、KH2PO4 1.0g、(NH4)2SO41.0g and CaCl20.5g is dissolved in 1L distilled water, adjusts
PH value is saved to 6.0.
In the above method, initial biomass can be 1.0 × 10 in the fermented and cultured8~2.5 × 108Cfu/mL (such as 1.0
×108~1.5 × 108cfu/mL、1.5×108~2.5 × 108cfu/mL、1.0×108cfu/mL、1.5×108Cfu/mL or
2.5×108cfu/mL)。
In the above method, initial biomass can be 5.0 × 10 in the fermented and cultured8~3.0 × 109Cfu/mL (such as 5.0
×108~1.0 × 109cfu/mL、1.0×109~3.0 × 109cfu/mL、5.0×108cfu/mL、1.0×109Cfu/mL or
3.0×109cfu/mL)。
In the above method, the fermented and cultured inoculum concentration can for 3~10% (such as 3~5%, 5~10%, 3%, 5% or
10%).
In the above method, the condition of culture of the fermented and cultured can for 22~28 DEG C (such as 22~25 DEG C, 25~28 DEG C, 22
DEG C, 25 DEG C or 28 DEG C) culture 72~120h (such as 72~100h, 100~120h, 72h, 100h or 120h), oxyty be 5~
80% (such as 5~50%, 50~80%, 5%, 50% or 80%).
In the above method, " the schizochytrium limacinum described in fermented and cultured (Schizoochytrium limacinum)
HS01CGMCC No.13746 " may also include preparation primary seed solution and/or prepare secondary seed solution and/or prepare fermentation one-level
Seed liquor and/or preparation fermentation secondary seed solution；
The step of preparing primary seed solution can be as follows：Schizochytrium limacinum (Schizoochytrium described in Shaking culture
Limacinum) HS01CGMCC No.13746, obtain primary seed solution；
The step of preparing secondary seed solution can be as follows：Shaking culture primary seed solution, obtains secondary seed solution；
The step of preparing fermentation primary seed solution can be as follows：Fermented and cultured secondary seed solution, obtains primary seed solution of fermenting；
The step of preparing fermentation secondary seed solution can be as follows：Fermented and cultured fermentation primary seed solution, obtains two grades of kinds of fermenting
Sub- liquid.
In the preparation primary seed solution and the preparation secondary seed solution, the Shake flask medium that the Shaking culture is used
Solute and its solubility can be：10~90g/L of glucose (such as 10~50g/L, 50~90g/L, 10g/L, 50g/L or 90g/L), ferment
5~25g/L of female powder (such as 5~15g/L, 15~25g/L, 5g/L, 15g/L or 25g/L)；Solute can be water；PH is natural.It is described
The condition of culture of " Shaking culture " be 22~28 DEG C (such as 22~25 DEG C, 25~28 DEG C, 22 DEG C, 25 DEG C or 28 DEG C), 150~
250rpm/min (such as 150~200rpm/min, 200~250rpm/min, 150rpm/min, 200rpm/min or 250rpm/
Min) 24~48h of culture (such as 24~36h, 36~48h, 24h, 36h or 48h).The inoculum concentration of the Shaking culture is 3~10%
(such as 3~5%, 5~10%, 3%, 5% or 10%).The Shake flask medium is concretely：By glucose 50g and dusty yeast
15g is dissolved in 1L distilled water, and pH value is natural.
It is described to prepare in fermentation primary seed solution and preparation fermentation secondary seed solution, the kind that the fermented and cultured is used
Sub- culture medium solute and its solubility can be：20~100g/L of glucose (such as 20~60g/L, 60~120g/L, 20g/L, 60g/L
Or 120g/L), 5~15g/L of dusty yeast (such as 5~10g/L, 10~15g/L, 5g/L, 10g/L or 15g/L), NaSO45~
24g/L (such as 5~10g/L, 10~24g/L, 5g/L, 10g/L or 24g/L), 0.1~1.0g/L of KCl (such as 0.1~0.5g/L,
0.5~1.0g/L, 0.1g/L, 0.5g/L or 1.0g/L), MgSO41.0~3.0g/L (such as 1.0~2.0g/L, 2.0~3.0g/
L, 1.0g/L, 2.0g/L or 3.0g/L), K2SO40.3~1.5g/L (such as 0.3~1.0g/L, 1.0~1.5g/L, 0.3g/L,
1.0g/L or 1.5g/L), KH2Po40.5~1.5g/L (such as 0.5~1.0g/L, 1.0~1.5g/L, 0.5g/L, 1.0g/L or
1.5g/L)、(NH4)2SO40.5~1.5g/L (such as 0.5~1.0g/L, 1.0~1.5g/L, 0.5g/L, 1.0g/L or 1.5g/
L)、CaCl20.1~1.0g/L (such as 0.1~0.5g/L, 0.5~1.0g/L, 0.1g/L, 0.5g/L or 1.0g/L)；Solute can
For water；PH value can be 5.0~6.5 (such as 5.0,6.0 or 6.5).The condition of culture of " fermented and cultured " is 22~28 DEG C (such as 22
~25 DEG C, 25~28 DEG C, 22 DEG C, 25 DEG C or 28 DEG C) 24~48h of culture (such as 24~36h, 36~48h, 24h, 36h or 48h),
Oxyty is 10~80% (such as 10~50%, 50~80%, 10%, 50% or 80%).The inoculum concentration of the fermented and cultured
For 3~10% (such as 3~5%, 5~10%, 3%, 5% or 10%).The seed culture medium is concretely：By glucose 60g,
Dusty yeast 10g, NaSO4 10g、KCl 0.5g、MgSO4 2.0g、K2SO4 1.0g、KH2Po4 1.0g、(NH4)2SO41.0g and
CaCl20.5g is dissolved in 1L distilled water, regulation pH value to 6.0.
Fermented and cultured schizochytrium limacinum (Schizoochytrium limacinum) HS01, obtains zymotic fluid.As a result show,
The content that DHA accounts for grease in the zymotic fluid is 45.0%~60.0%.Therefore, the schizochytrium limacinum provided using the present invention
(Schizoochytrium limacinum) HS01 can produce DHA, with important application value.
Brief description of the drawings
Fig. 1 is schizochytrium limacinum HS01 colony morphology characteristic.
Fig. 2 is schizochytrium limacinum HS01 morphological features.
Preservation explanation
Strain name：Schizochytrium limacinum
Latin name：Schizoochytrium limacinum
Strain number：HS01
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On March 10th, 2017
Collection is registered on the books numbering：CGMCC No.13746
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetition experiments, as a result makes even
Average.
Culture medium used is as follows in following embodiments：
Wort agar culture medium：150g fructus hordei germinatus leaching powders are dissolved in 1L mixed liquors (by 1 parts by volume natural sea-water and 1 volume
Distilled water is mixed), pH value is natural；Then it is 15g/100mL, obtained culture medium to add agar powder to its concentration.
Screen fluid nutrient medium：By glucose 50g and dusty yeast 15g be dissolved in 1L mixed liquors (by 1 parts by volume natural sea-water and
1 volume distilled water is mixed), pH value is natural.
Screen solid medium：It is 15g/100mL that agar powder to its concentration is added into screening fluid nutrient medium, is obtained
Culture medium.
Screening flat board：About 55 DEG C of screening solid medium is poured into the solid plate obtained after culture dish, cooling.
Shake flask medium：Glucose 50g and dusty yeast 15g are dissolved in 1L distilled water, pH value is natural.
Seed culture medium：Glucose 60g, dusty yeast 10g, NaSO4 10g、KCl 0.5g、MgSO4 2.0g、K2SO4
1.0g、KH2Po4 1.0g、(NH4)2SO41.0g and CaCl20.5g is dissolved in 1L distilled water, regulation pH value to 6.0.
Fermentation medium：By glucose 60g, glutamic acid or sodium glutamate 10g, Dried Corn Steep Liquor Powder 10g, NaSO4 14g、
KCl 0.5g、MgSO4 2.0g、K2SO4 1.0g、KH2PO4 1.0g、(NH4)2SO41.0g and CaCl20.5g is dissolved in 1L distillations
Water, regulation pH value to 6.0.
Dried Corn Steep Liquor Powder is the product of Beijing Suo Laibao Science and Technology Ltd, and catalog number is FA0010.
Dusty yeast is the product of Angel Yeast Co., Ltd, and catalog number is LMO2.
Embodiment 1, schizochytrium limacinum (Schizoochytrium limacinum) HS01CGMCC No.13746 separation,
Identification and preservation
First, schizochytrium limacinum HS01 separation
1st, present inventor gathers schizochytrium limacinum from Zhangzhou City of Fujian Province Yunxiao County mangrove many places, and mixing is obtained
Mixed liquor；0.5mL mixed liquors are seeded to 5mL screening fluid nutrient mediums, then 25 DEG C, 200rpm/min culture 2d are trained
Bacteria liquid.
2nd, the culture bacterium solution that step 1 is obtained is spread evenly across in screening flat board, 25 DEG C of quiescent culture 2d, produces single bacterium
Fall.
3rd, complete after step 2, picking single bacterium colony is seeded to 5mL fermentation mediums respectively, then 25 DEG C, 200rpm/min trainings
2d is supported, obtains cultivating bacterium solution.
4th, the culture bacterium solution that step 3 is obtained is taken, 4 DEG C, 2000rpm centrifugation 5min collect thalline.
5th, it is 8.3mol/L's to take 1.0~2.0g of thalline to add 15mL concentration to having in plug graduated cylinder (specification is 100mL), first
HCl/water solution, covers lid, and being placed in 50~60min of hydrolysis in 70~80 DEG C of water-baths, (period is placed in eddy mixer per 10min
Upper vibration tool fills in graduated cylinder 1 time)；To be cooled first to add 10mL 95% (v/v) ethanol water to after room temperature, shake well is uniform
Afterwards, add 20mL absolute ethers shake well and extract 1~2min, be eventually adding 20mL petroleum ethers, shake well extraction 1~
Upper organic phase is placed in glass weighing disk (dried and claimed bare weight) by 2min, stratification, the glass weighing disk is placed in logical
Organic phase is fully evaporated on boiling water bath in wind cupboard totally (must fully volatilize clean), and liquid phase is grease.
6th, the grease for taking step 5 to extract, DHA content is detected according to GB 26400-2011 national food safety standard, according to
The composition and content of AOAC996.06 method detection aliphatic acid.
The higher bacterial strain of DHA content is selected, is purified 24 times repeatedly.It is to split by screen one plant of schizochytrium limacinum Strain Designation
Grow chytrid HS01.
Schizochytrium limacinum HS01 monoclonals are seeded to the generation of fermentation medium continuous passage 12 and DHA are detected according to above-mentioned steps
Content.As a result show, schizochytrium limacinum HS01 production DHA's has good stability.
2nd, schizochytrium limacinum HS01 identification
1st, Morphological Identification
Schizochytrium limacinum HS01 is seeded on wort agar culture medium, 25 DEG C of light cultures, the form of bacterium colony is observed after 5d
And pass through the morphological feature of high resolution TEM analysis and observation thalline.
Experimental result is shown in Fig. 1 and Fig. 2.As a result show, schizochytrium limacinum HS01 colony diameter is 2~4.3mm, white (after
Phase light orange), edge is irregular；Thalline is bred in fragmentation mode, and cell membrane is thin, spherical, colourless or light orange, transparent,
Size is 4.5~15.5 μm, and zoospore and expolasm net have no.
2nd, 18s rDNA sequence homology analysis
Schizochytrium limacinum HS01 18s rDNA partial sequence is as shown in the sequence 1 in sequence table.
Schizochytrium limacinum HS01 18s rDNA partial sequence is as shown in the sequence 2 in sequence table.
Each qualification result of summary, schizochytrium limacinum HS01 is schizochytrium limacinum (Schizoochytrium
limacinum)。
3rd, schizochytrium limacinum HS01 preservation
It is micro- that schizochytrium limacinum (Schizoochytrium limacinum) HS01 was preserved in China on 03 10th, 2017
(abbreviation CGMCC, address is biological inoculum preservation administration committee common micro-organisms center：BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3), deposit number is CGMCC No.13746.Schizochytrium limacinum HS01 full name is schizochytrium limacinum (Schizoochytrium
Limacinum) HS01CGMCC No.13746, referred to as schizochytrium limacinum HS01.
Embodiment 2, schizochytrium limacinum (Schizoochytrium limacinum) HS01 fermenting and producings DHA
First, schizochytrium limacinum (Schizoochytrium limacinum) HS01 fermenting and producings DHA
1st, schizochytrium limacinum (Schizoochytrium limacinum) HS01 monoclonals are seeded to and trained equipped with 2mL shaking flasks
In the shaking flask (specification is 10mL) for supporting base, 22~28 DEG C, 150~250rpm/min, 24~48h of culture obtain primary seed solution.
2nd, primary seed solution is taken, the shaking flask equipped with 250mL Shake flask mediums is seeded to 3~10% (v/v) inoculum concentration
In (shaking flask specification is 1L), 22~28 DEG C, 150~250rpm/min, 24~48h of culture obtain secondary seed solution.
3rd, secondary seed solution is taken, the fermentation tank equipped with 3L seed culture mediums is seeded to 3~10% (v/v) inoculum concentration
In (fermentation tank specification is 5L), 22~28 DEG C of 24~48h of culture (dissolved oxygen is 10~80%) obtain primary seed solution of fermenting.
4th, fermentation primary seed solution is taken, the hair equipped with 50L fermentation mediums is seeded to 3~10% (v/v) inoculum concentration
(fermentation tank specification is 100L to fermentation tank；Initial biomass is 1.0 × 10 after inoculation8~2.5 × 108Cfu/mL in), 22~28 DEG C of trainings
72~120h (dissolved oxygen is 5~80%) is supported, zymotic fluid is obtained.Contain DHA in zymotic fluid.
2nd, the analysis of the fatty acid composition in zymotic fluid
According in the step one of embodiment 15 method, the grease of zymotic fluid is extracted, then according to GB 26400-2011 food
Safe national standard detects DHA content, and the composition and content of aliphatic acid are detected according to AOAC996.06 method.
Experimental result is shown in Table 1.As a result show, the content that DHA accounts for grease is 45.0%~60.0%.
Table 1
3rd, DHA separation and Quality Identification in zymotic fluid
1st, the zymotic fluid that step one is obtained is taken, the breaking-wall cell of schizochytrium limacinum and the extraction of DHA algal oil crude oil are carried out successively
(method for carrying out the breaking-wall cell of schizochytrium limacinum and the extraction of DHA algal oil crude oil is recorded in Chinese invention patent document CN
101817738 B)。
2nd, (method of refining is recorded in Chinese invention patent document CN is refined to the DHA algal oil crude oil that step 1 is extracted
103865642 B)。
Quality index of the DHA algal oil crude oil after refining is shown in Table 2.
Table 2
Embodiment 3, schizochytrium limacinum (Schizoochytrium limacinum) HS01 large scale fermentations production DHA
1st, schizochytrium limacinum (Schizoochytrium limacinum) HS01 single bacterium colonies are seeded to and trained equipped with 20mL shaking flasks
In the shaking flask (shaking flask specification is 250mL) for supporting base, 22~28 DEG C, 150~250rpm/min, 24~48h of culture obtain one-level kind
Sub- liquid.
2nd, primary seed solution is taken, the shaking flask equipped with 250mL Shake flask mediums is seeded to 3~10% (v/v) inoculum concentration
In (shaking flask specification is 2L), 22~28 DEG C, 150~250rpm/min, 24~48h of culture obtain secondary seed solution.
3rd, secondary seed solution is taken, the fermentation tank equipped with 500L seed culture mediums is seeded to 3~10% (v/v) inoculum concentration
In (fermentation tank specification be 1000L), 22~28 DEG C of 24~48h of culture (dissolved oxygen is 10~80%), obtain biomass 15~
30g/L fermentation primary seed solution.
4th, fermentation primary seed solution is taken, is seeded to 5~15% (v/v) inoculum concentration equipped with 5000L seed culture mediums
In fermentation tank (fermentation tank specification is 8000~10000L), 22~28 DEG C of 24~48h of culture (dissolved oxygen is 10~80%) obtain
Fermentation secondary seed solution of the biomass in 15~30g/L.
5th, fermentation secondary seed solution is taken, is seeded to 5~15% (v/v) inoculum concentration equipped with 30000L fermentation mediums
(fermentation tank specification is 75000L to fermentation tank；Initial biomass is 5.0 × 10 after inoculation8~3.0 × 109Cfu/mL in), 22~28
DEG C 72~120h of culture (dissolved oxygen be 5~80%), obtains zymotic fluid.Contain DHA in zymotic fluid.
According to the method for step 2 in embodiment 2, the fatty acid composition in zymotic fluid is analyzed.As a result show, the zymotic fluid
The content that middle DHA accounts for grease is 35.0~60.0%.
Therefore, schizochytrium limacinum (Schizoochytrium limacinum) HS01 production DHA tools provided using the present invention
There is higher productive value.
<110>Xiamen Huisheng Biological Co., Ltd.
<120>One plant production docosahexaenoic acid bacterium and its application
<160> 2
<170> PatentIn version 3.5
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<213>Artificial sequence
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<223>
<400> 1
agccatgcat gtgtaagtat aagcgattgt actgtgagac tgcgaacggc tcattatatc 60
agtaataatt tcttcggtag tttcttttat atggatacct gcagtaattc tggaaataat 120
acatgctgta agagccctgt atggggctgc acttattaga ttgaagccga ttttattggt 180
gaatcatgat aattgagcag attgact 207
<210> 2
<211> 239
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
gagttctgcc tctgtccaaa aattaatcca aacagaaaca tcccatggtt tcatcggacc 60
gttcaatcgg taggtgcgac gggcggtgtg tacaaagggc agggacgtat tcaatgcaag 120
ctgatgactt gcgtttacta ggaattcctc gttggagatt aataattgca aaaatctagc 180
cccagcacga tgagcgttcc aaggattagc caggccttcc gaccaagcac tcaattcca 239
Claims (10)
- Schizochytrium limacinum 1. (Schizoochytrium limacinum) HS01, it is in Chinese microorganism strain preservation conservator The deposit number of meeting common micro-organisms center is CGMCC No.13746.
- 2. a kind of microbial inoculum, it is characterised in that：The microbial inoculum contains schizochytrium limacinum (Schizoochytrium described in claim 1 limacinum)HS01 CGMCC No.13746。
- 3. schizochytrium limacinum (Schizoochytrium limacinum) HS01 CGMCC No.13746 described in claim 1, Or, application of the microbial inoculum described in claim 2 in production docosahexaenoic acid.
- 4. a kind of method for producing docosahexaenoic acid, including schizochytrium limacinum described in fermented and cultured claim 1 (Schizoochytrium limacinum) HS01 CGMCC No.13746, the step of obtaining docosahexaenoic acid.
- 5. method as claimed in claim 4, it is characterised in that：Fermentation medium solute that the fermented and cultured is used and its molten Spend and be：20~120g/L of glucose, 5~15g/L of glutamic acid or sodium glutamate, 3~15g/L of Dried Corn Steep Liquor Powder, NaSO45~ 0.1~1.0g/L of 24g/L, KCl, MgSO41.0~3.0g/L, K2SO40.3~1.5g/L, KH2PO40.5~1.5g/L, (NH4)2SO40.5~1.5g/L, CaCl20.1~1.0g/L；Solute is water；PH5.0~6.5.
- 6. the method as described in claim 4 or 5, it is characterised in that：Initial biomass is 1.0 × 10 in the fermented and cultured8~ 2.5×108cfu/mL。
- 7. the method as described in claim 4 or 5, it is characterised in that：Initial biomass is 5.0 × 10 in the fermented and cultured8~ 3.0×109cfu/mL。
- 8. the method as described in claim 4 to 7 is any, it is characterised in that：The fermented and cultured inoculum concentration is 3~10%.
- 9. the method as described in claim 4 to 8 is any, it is characterised in that：The condition of culture of the fermented and cultured is 22~28 DEG C culture 72~120h, oxyty be 5~80%.
- 10. the method as described in claim 4 to 9 is any, it is characterised in that：" the schizochytrium limacinum described in fermented and cultured (Schizoochytrium limacinum) HS01 CGMCC No.13746 " also include preparing primary seed solution and/or preparation Secondary seed solution and/or preparation fermentation primary seed solution and/or preparation fermentation secondary seed solution；The step of preparing primary seed solution is as follows：Schizochytrium limacinum (Schizoochytrium described in Shaking culture claim 1 Limacinum) HS01 CGMCC No.13746, obtain primary seed solution；The step of preparing secondary seed solution is as follows：Shaking culture primary seed solution, obtains secondary seed solution；The step of preparing fermentation primary seed solution is as follows：Fermented and cultured secondary seed solution, obtains primary seed solution of fermenting；The step of preparing fermentation secondary seed solution is as follows：Fermented and cultured fermentation primary seed solution, obtains secondary seed solution of fermenting.
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| CN201710398286.7A CN106987528B (en) | 2017-05-31 | 2017-05-31 | Bacterium for producing docosahexaenoic acid and application thereof |
| EP18810206.5A EP3628679B1 (en) | 2017-05-31 | 2018-05-21 | Bacterium producing dha and epa, 6 gene fragments of genome of bacterium and use thereof |
| JP2019566735A JP7039625B2 (en) | 2017-05-31 | 2018-05-21 | Bacteria that produce DHA and EPA, 6 gene fragments of the bacterial genome and their use |
| PCT/CN2018/087613 WO2018219171A1 (en) | 2017-05-31 | 2018-05-21 | Bacterium producing dha and epa, 6 gene fragments of genome of bacterium and use thereof |
| US16/696,331 US10941185B2 (en) | 2017-05-31 | 2019-11-26 | Strain of bacteria producing DHA and EPA, six gene fragments in the bacterial genome and their applications |
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