CN102199541B - Schizochytrium sp.TIO1101 strain with high-yield DHA (docosahexaenoic acid) and fermentation method thereof - Google Patents

Abstract

The invention discloses a Schizochytrium sp.TIO1101 strain with high-yield DHA (docosahexaenoic acid) and a fermentation method thereof. The Schizochytrium sp.TIO1101 strain provided by the invention has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No.4603. After the Schizochytrium sp.TIO1101 is subjected to high-density heterotrophic fermentation for 72h, the biomass liveweight, the lipid content and the DHA are respectively 120g/L, 54.3% and 51.4% of the content of total lipid, therefore the Schizochytrium sp.TIO1101 has development and application values extremely. The Schizochytrium sp.TIO1101 disclosed by the invention has the advantages that: the Schizochytrium sp.TIO1101is obtained through separation; the high-density heterotrophic fermentation of the Schizochytrium sp.TIO1101 is realized; and the lipid extracted from the Schizochytrium sp.TIO1101 cells obtained under the fermentation condition has high DHA content and purer components.

Description

High yield DHA's splits the algae strain of kettle algae and fermentation process thereof

Technical field

What the present invention relates to a kind of high yield DHA splits the algae strain of kettle algae and fermentation process thereof.

Background technology

Docosahexenoic acid (DHA) mainly is present in human brain grey matter and the retina, it is the indispensable fatty acid composition of keeping the human normal physiological metabolism, in infant's brain and visual acuity, has very important physiological action, but and Cardiovarscular, improve body immunity etc.DHA causes the great interest of people because of its distinctive physiological regulation function and is widely used.

The tradition source of DHA is fish oil, but the polyunsaturated fatty acid that extracts from fish oil (PUFAs) exists the output shakiness, yield is low, cost is high and contain other problems such as ω-6PUFAs, and along with the growing tension of fishing resources, oneself can't satisfy the growing market requirement traditional DHA resource.Therefore, the exploitation DHA newborn resource oneself become new study hotspot.Microbial fermentation is produced DHA becomes one of focus of domestic and international research with its unique advantage, wherein utilizes little algae to cultivate production DHA and has larger advantage and produce potential.

Little algae has that growth cycle is short, reproduction speed is fast, plasticity-is strong, nutritive factor is required the characteristics such as simple, and part can reach by the control of biological engineering method and culture condition high density fermentation and cultivates.Found at present to be rich in polyunsaturated fatty acid (PUFAs) in multiple little algae, as split kettle algae, Crypthecodinium cohnii etc.Little algae is the initial production person of polyunsaturated fatty acid in the marine food chain, many saturated fatty acids DHA in some frustule, EPA content are higher, its relative content is up to the 5%-6% of dry cell weight, and contained polyunsaturated fatty acid kind is more simple, the separating-purifying that carries out single component is relatively easy, thereby to utilize marine microalgae to produce polyunsaturated fatty acid be a very promising commercial field.Little algae oil has been widely used in the fields such as medicine, food, makeup, feed because being rich in polyunsaturated fatty acid.

Split DHA rich content in the kettle frustule, and other unsaturated fatty acid content is very low.This algae is used safety, and it has carried out a series of security detection to white mouse, rabbit the humans such as Hammond, does not find any toxic side effect.The security of splitting the kettle algae has obtained the approval of U.S. food and medicine section (Food and Drug Administration).The research that kettle algae fermentation culture is produced DHA is split at present existing more utilization both at home and abroad, but mostly has the not high problem of output.T.Yokocki etc. adopt several kinds of carbon source and nitrogenous source counterincision kettle algae Schizochytriumlimacinum SR21 to be optimized cultivation in shaking flask, and the DHA production peak that obtains is 4g/L.The people such as Kw Fan are split kettle algae (Schizochytrium mangroves) take glucose and yeast extract as the carbon and nitrogen sources cultivation, and the output of DHA only is 2.79g/L.The output of DHA is not very high in more than cultivating, and this also has direct relation except outside the Pass having with selected algae strain with culture process.

Summary of the invention

What the purpose of this invention is to provide a kind of high yield DHA splits the algae strain of kettle algae and fermentation process thereof.

The invention provides and split kettle algae Schizochytrium sp.TIO1101, its deposit number is CGMCCNo.4603.

Be used for the described fermention medium that splits kettle algae TIO1101, the preparation method is as follows: glucose 100-130g, yeast extract 15-30g, peptone 10-20g, potassium primary phosphate 2-4g, sal epsom 1-3g, micro-complex liquid 8-11ml and VITAMIN complex liquid 4-8ml are settled to 1L with 0.5 * seawater; Described 0.5 * seawater is mixed to get 1 parts by volume seawater and 1 parts by volume water.

In the described fermention medium: described glucose is that 120g, described yeast extract are that 20g, described peptone are that 15g, described potassium primary phosphate are that 3g, described sal epsom are that 2g, described micro-complex liquid are that 10ml, described VITAMIN complex liquid are 5ml.

The preparation method of described micro-complex liquid is specific as follows: Na ₂EDTA 6.0g, FeCl ₃6H ₂O 0.29g, H ₃BO ₃6.84g, MnCl ₂4H ₂O 0.86g, ZnCl ₂0.06g, CoCl ₂6H2O 0.026g, NiSO ₄6H ₂O 0.052g, CuSO ₄5H ₂O 0.002g, NaMoO ₄2H ₂O 0.005g, water is settled to 1L.

The preparation method of described VITAMIN complex liquid is specific as follows: VITMAIN B1 (thiamin) 100mg, and vitamin H (biotin) 0.5mg, vitamin B12 (cyanocobalamin) 0.5mg, water is settled to 1L.

The present invention also protects a kind of described method of splitting kettle algae TIO1101 of cultivating, and is at 25-30 ℃, pH 5.5-7.5, condition under cultivate the described kettle algae TIO1101 that splits.In the described cultural method, temperature can be 25 ℃, and pH can be 6.0.Described cultivation is preferably carried out in described fermention medium.The described inoculum density of kettle algae TIO1101 in described fermention medium that split can be 1.0 * 10 ⁶Individual/mL.

The described kettle algae TIO1101 that splits can be used for preparing grease and/or docosahexenoic acid.

The present invention also protects a kind of method for preparing grease and/or docosahexenoic acid, is the described kettle algae TIO1101 that splits of fermentation, obtains grease and/or docosahexenoic acid.The parameter of described fermentation can be: 25-30 ℃, and pH 5.5-7.5.The parameter of described fermentation is preferably 25 ℃, and pH 6.0, dissolved oxygen 10%.Can adopt the rotating speed of 200-400rpm in the described fermentation, preferably adopt the rotating speed of 300rpm.The time of described fermentation specifically can be 96 hours.Described fermentation is preferably carried out in described fermention medium.The described inoculum density of kettle algae TIO1101 in described fermention medium that split can be 1.0 * 10 ⁶Individual/mL.In the process of described fermentation, can carry out batch feeding.For every 6L fermention medium, described batch feeding specifically can be: begin stream from moment of fermentation 12h and add glucose solution and (be comprised of glucose and water, the concentration of glucose is 50g/L) and yeast extract paste solution (formed by yeast extract paste and water, the concentration of yeast extract paste is 10g/L) until the moment of fermentation 50h, the flow velocity of glucose solution is that the flow velocity of 50mL/h, yeast extract paste solution is 20mL/h.

Behind the fermentation 72h, the biomass, fat content and the DHA that split kettle algae TIO1101 account for total lipid content and are respectively 120g/L, 54.3% and 51.4%, and the dried frond of every 100g can obtain 54.3g grease, 27.9gDHA, has development and application values.

Beneficial effect of the present invention: (1) separation has obtained to split kettle algae TIO1101; (2) realized splitting the high-density heterotrophic fermentation of kettle algae TIO1101; (3) kettle algae TIO1101 cell is carried in the grease DHA content height and composition is comparatively simple for splitting of obtaining under this fermentation condition.

Description of drawings

Fig. 1 is for splitting kettle algae TIO1101 18S rRNA gene PCR electrophorogram.

Fig. 2 is for splitting kettle algae TIO1101 18S rRNA gene evolution tree.

Fig. 3 splits kettle algae TIO1101 dry cell weight with the variation diagram of fermentation time.

Fig. 4 is the color atlas that splits after the esterification of kettle algae TIO1101 oil sample.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.

Seawater: take from Xiamen sea area.

The preparation method of trace element complex liquid: Na ₂EDTA 6.0g, FeCl ₃6H ₂O 0.29g, H ₃BO ₃6.84g, MnCl ₂4H ₂O 0.86g, ZnCl ₂0.06g, CoCl ₂6H2O 0.026g, NiSO ₄6H ₂O 0.052g, CuSO ₄5H ₂O0.002g, NaMoO ₄2H ₂O 0.005g, distilled water is supplied 1L.

The preparation method of VITAMIN complex liquid: VITMAIN B1 (thiamin) 100mg, vitamin H (biotin) 0.5mg, vitamin B12 (cyanocobalamin) 0.5mg, distilled water is supplied 1L.

The preparation method of fermention medium: glucose 120g, yeast extract 20g, peptone 15g, potassium primary phosphate 3g, sal epsom 2g, micro-complex liquid 10ml, VITAMIN complex liquid 5ml are settled to 1L with 0.5 * seawater (with the seawater of 1 times of fresh water dilution).

Embodiment 1, split separation and the evaluation of kettle algae TIO1101

One, splits the separation (Pollen Pini fish method) of kettle algae TIO1101

Get the corruption fallen leaves in the mangrove forest of Sha Men Yuandang Ya lake, be cut into the sequin that diameter is about 1.5cm, clean with the aseptic seawater that contains Streptomycin sulphate and each 1g/L of penicillin, be inoculated on the solid separating plate, each plate 3-5 sheet, add the aseptic seawater of about 3mL and a small amount of aseptic Pollen Pini on flat board, at 28 ℃ of lower 4-5d that cultivate, the picking Pollen Pini, observation adheres to the culture of growth at Pollen Pini, the Pollen Pini that adheres to culture is transferred to the separation of ruling on the new flat board again, until obtain pure growth.With a strain algae called after TIO1101 who obtains.

Two, split the evaluation of kettle algae TIO1101

1, algae falls and the cellular form observation

Culture is observed fall form and observe its cellular form by film-making under powerful microscope of the algae that grows after the separation and purification flat board is cultivated 5d.Split that kettle algae algae falls to being faint yellow, opaque, the edge is irregular, its vegetative cell is elliposoidal, diameter 7-13 μ m, the growth of row binary fission.

2, Molecular Identification

(1) extracting genome DNA

Adopt the cracking process of improvement to extract total DNA.

Get fermented liquid 5mL in centrifuge tube, the centrifugal 5min of 8000r/min, centrifugal collection frond under the similarity condition after sterile distilled water washs a time.Place aseptic mortar to add an amount of liquid nitrogen grinding fresh frond.Frond after the grinding is transferred in the centrifuge tube, adds 2.4ml lysate (0.25mol/L Tris-Cl pH 8.2,0.1mol/L EDTA pH 8.0,2%SDS, 0.1mol/L NaCl), cracking 40min behind the mixing.Add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extraction, isopropanol precipitating, washing with alcohol.The DNA that carries adds the TE solution dissolving of 50 μ l10mmol/L ,-20 ℃ of Refrigerator stores.

(2) 18S rRNA amplification, order-checking

Amplimer is: forward Pf:5 '-CCAACCTGGTTGATCCTGCCAGTA-3 ';

Reverse Pr:5 '-CCTTGTTACGACTTCACCTTCCTCT-3.

50 μ L PCR reaction compositions: 10 * PCR buffer (5 μ L), dNTP (4 μ L), forward primer (1 μ L), reverse primer (1 μ L), TaKaRa Taq (0.2 μ L, 1.25U), template DNA (2 μ L, 0.6～1.5 μ g), distilled water complements to 50 μ L.

The pcr amplification condition: 95 ℃ of denaturation 5min, 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, last 72 ℃ are extended 10min.The sepharose of employing 1% is seen Fig. 1 to PCR product electrophoresis detection, produces the 18S rRNA gene that size is about 1700bp.，

Pcr amplification product is connected with the T carrier, checks order in Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Sequence total length 1707bp (seeing the sequence table 1 of sequence table).

(3) Phylogenetic Analysis

The 18S rRNA gene order that order-checking is obtained compares by Blast program and GenBank amplifying nucleic acid data, utilizes ClustalX 1.8 and Mega2 software building phylogenetic tree, carries out Phylogenetic Analysis.The result shows that this algae strain 18S rRNA gene and Schizochytrium mangrovei have the homology more than 99%, utilize the systematic evolution tree of ClustalX 1.8 and Mega2 software building to see Fig. 2, systematic evolution tree shows that it is one that this algae strain and Schizochytrium mangrovei gather, and sibship is nearest.

Fall and the result of cellular form and Molecular Identification according to algae, TIO1101 belongs to and splits kettle algae (Schizochytriumsp.).Split kettle algae Schizochytrium sp.TIO1101 and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (abbreviation CGMCC on 02 23rd, 2011, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4603.Split kettle algae Schizochytrium sp.TIO1101CGMCC No.4603 abbreviation and split kettle algae TIO1101.

Embodiment 2, split the high-density heterotrophic fermentation of kettle algae TIO1101

One, fermentation

In the 10L fermentor tank, add the 6L fermention medium, then add 0.6L and split kettle algae TIO1101 bacterium liquid, make and split the concentration of kettle algae TIO1101 in substratum and be about 1.0 * 10 ⁶Individual/mL; Fermentation culture conditions is: 25 ℃ of temperature, and pH value 6.0, rotating speed 300rpm (200-400rpm all can), dissolved oxygen 10% is cultivated 96h; Carrying out 10L fermentor tank batch feeding cultivates: begin stream from moment of fermentation 12h and add glucose solution and (be comprised of glucose and water, the concentration of glucose is 50g/L) and yeast extract paste solution (formed by yeast extract paste and water, the concentration of yeast extract paste is 10g/L) until the moment of fermentation 50h, the flow velocity of glucose solution is that the flow velocity of 50mL/h, yeast extract paste solution is 20mL/h.

Two, biomass, grease and DHA content detection

1, biomass

Get the fermented liquid detection of biological amount (in the frond dry cell weight of collecting) of different fermentations time in the step 1, method is as follows: get the 100mL fermented liquid and pack in the centrifuge tube of weighing in advance, the centrifugal 10min collecting precipitation of 8000r/min, after sterile distilled water cleaning 3 times, vacuum-drying is weighed to constant weight, is dried frond.Split kettle algae dry weight (i.e. the weight of dried frond) with variation such as Fig. 3 of fermentation time.Behind the fermentation culture 72h, splitting the kettle algae, to obtain high-biomass be 140g/L.

2, grease

Pulverize rear employing Soxhlet extraction process (GB5009.6-85) extracting grease and weighing of the dried frond that step 1 is obtained.Fat content in the dried frond is higher, reaches 54.3%.

3、DHA

Get the grease obtained sample of 0.1g extracting and place 10ml centrifuge tube with cover, add the 1mL ether: (ether: normal hexane is 2: 1 to the normal hexane mixed solution; Volume ratio), shake up the KOH/CH that adds 0.8mol/L behind the 15min ₃OH solution 2mL shakes up rear 50 ℃ of reaction 15min, adds water to 10mL scale place along wall, and standing demix is got supernatant liquor and carried out the capillary gas chromatography analysis.

The parameter that capillary gas chromatography is analyzed: Varian GC3800 gas chromatograph, fid detector, the CP-sil5CB capillary column (0.32mm * 0.25 μ m * 30m); Carrier gas is nitrogen, and flow velocity is 30.0mL/min; Injector temperature is 250 ℃, and detector temperature is 280 ℃; Column temperature employing program is given birth to temperature: 160 ℃ of initial temperatures (keeping 3min), be raised to 230 ℃ with 6 ℃/min speed, and keep 8min.Adopting methyl margarate is that interior mark carries out the inner mark method ration analysis.

The typical curve function is Y=1.4648X-0.0087, and correlation coefficient r is 0.9999.Wherein Y-axis represents the ratio of analyte peak size and interior mark peak size.X-axis represent advance the amount of analyte and the ratio of the interior scalar of advance.Interior mark methyl margarate and split kettle algae oil sample color atlas and see Fig. 4.

The result shows that split the content that kettle algae DHA accounts for total fat is 51.4%.

The dried frond of every 100g can obtain the 54.3g grease, obtains 27.9gDHA.

Claims (6)

1. split the kettle algae ( SchizochytriumSp.) TIO1101, its deposit number are CGMCC No.4603.

2. cultivating the described method of splitting kettle algae TIO1101 of claim 1 for one kind, is to cultivate the described kettle algae TIO1101 that splits in fermention medium;

The condition of described cultivation is: 25 ℃, pH 6.0, dissolved oxygen 10%, rotating speed are 300rpm, 96 hours;

The preparation method of described fermention medium is as follows: glucose 120g, yeast extract 20g, peptone 15g, potassium primary phosphate 3g, sal epsom 2g, micro-complex liquid 10ml and VITAMIN complex liquid 5ml are settled to 1L with 0.5 * seawater; Described 0.5 * seawater is mixed to get 1 parts by volume seawater and 1 parts by volume water;

The preparation method of described micro-complex liquid is as follows: Na ₂EDTA 6.0g, FeCl ₃6H ₂O 0.29g, H ₃BO ₃6.84g, MnCl ₂4H ₂O 0.86g, ZnCl ₂0.06g, CoCl ₂6H ₂O 0.026g, NiSO ₄6H ₂O 0.052g, CuSO ₄5H ₂O 0.002g, NaMoO ₄2H ₂O 0.005g, water is settled to 1L;

The preparation method of described VITAMIN complex liquid is as follows: VITMAIN B1 100mg, and vitamin H 0.5mg, vitamin B12 0.5mg, water is settled to 1L.

3. the described kettle algae TIO1101 that splits of claim 1 is in the application of preparation in the grease.

4. application as claimed in claim 3 is characterized in that: described grease is docosahexenoic acid.

5. a method for preparing grease is the described kettle algae TIO1101 that splits of fermentation claim 1 in fermention medium, obtains grease;

The condition of described fermentation is: 25 ℃, pH 6.0, dissolved oxygen 10%, rotating speed are 300rpm, 96 hours;

The preparation method of described fermention medium is as follows: glucose 120g, yeast extract 20g, peptone 15g, potassium primary phosphate 3g, sal epsom 2g, micro-complex liquid 10ml and VITAMIN complex liquid 5ml are settled to 1L with 0.5 * seawater; Described 0.5 * seawater is mixed to get 1 parts by volume seawater and 1 parts by volume water;

The preparation method of described micro-complex liquid is as follows: Na ₂EDTA 6.0g, FeCl ₃6H ₂O 0.29g, H ₃BO ₃6.84g, MnCl ₂4H ₂O 0.86g, ZnCl ₂0.06g, CoCl ₂6H ₂O 0.026g, NiSO ₄6H ₂O 0.052g, CuSO ₄5H ₂O 0.002g, NaMoO ₄2H ₂O 0.005g, water is settled to 1L;

The preparation method of described VITAMIN complex liquid is as follows: VITMAIN B1 100mg, and vitamin H 0.5mg, vitamin B12 0.5mg, water is settled to 1L.

6. method as claimed in claim 5, it is characterized in that: described grease is docosahexenoic acid.

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