CN105969666B - Double eyebrow algaes and its application in generation diesel oil, EPA, fucoxanthin - Google Patents

Double eyebrow algaes and its application in generation diesel oil, EPA, fucoxanthin Download PDF

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CN105969666B
CN105969666B CN201610414424.1A CN201610414424A CN105969666B CN 105969666 B CN105969666 B CN 105969666B CN 201610414424 A CN201610414424 A CN 201610414424A CN 105969666 B CN105969666 B CN 105969666B
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刘志媛
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Abstract

The invention discloses one plant of double eyebrow algae (Amphora sp.) and its applications in generation diesel oil, EPA, fucoxanthin.The deposit number of double eyebrow algaes of the invention is CGMCC No.11816.Double eyebrow algaes of the invention are the excellent algae strains of one plant of Comprehensive Traits.Algae strain growth is fast, Yi Caishou, and biomass production cost is lower than most microalgaes.While preparing biodiesel, the high value added products such as EPA, carotenoid and fucoxanthin can be obtained using biorefining technology, exocellular polysaccharide and silica silicon shell are also commercially valuable.

Description

Double eyebrow algaes and its application in generation diesel oil, EPA, fucoxanthin
Technical field
The present invention relates to one plant of oil-rich microalgae and its applications in generation diesel oil, EPA, fucoxanthin, in particular to One plant of double eyebrow algae and its application in generation diesel oil, EPA, fucoxanthin.
Background technique
Bring problem of environmental pollution when fossil shortage of fuel oil and its burning, makes reproducible substitute fuel --- biological bavin Oil is concerned.Microalgae can large-scale culture on the Degraded Lands such as beach, salt-soda soil, clayed ground and in shallow sea and lake, Ground is not striven with grain, does not strive grain with people, and can fresh-water-saving resource, the carbon dioxide treatment and emission reduction that can also discard with factory mutually tie It closes.Therefore, microalgae is considered as the biodiesel raw material of great exploitation potential.In recent years, researchers obtained many and had The oil-rich microalgae of biodiesel potentiality to be exploited.However, causing microalgae biological due to the high cost of microdisk electrode, harvesting and processing The production cost of diesel oil still cannot limit commercially producing for microalgae biodiesel compared with fossil fuel oil.
Currently, researchers are it is believed that by microalgae production biodiesel algae and microalgae biorefining (Biorefining) It will solve the high-cost effective way of microalgae biodiesel that technology, which combines,.Microalgae is rich in polyunsaturated fatty acid, high-quality egg White, polysaccharide, pigment and a variety of special cometabolism substances, while producing biodiesel, progress polyunsaturated fatty acid, The cost of microalgae bio-fuel-oil will be effectively reduced in the production of the high value added products such as albumen or pigment.
Summary of the invention
The present invention provides one plant of double eyebrow algae (Amphora sp.) and its generation diesel oil, EPA and fucoxanthin rich oil are micro- Application in algae.
Double eyebrow algaes (Amphora sp.) of the invention are this laboratories by screening for many years, and it is excellent to obtain one plant of Comprehensive Traits Good diatom strain, is named as BQWSM, is preserved in China General Microbiological culture presevation administrative center on January 8th, 2016 (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica), deposit number CGMCC No.11816.The form of double eyebrow algae algae strain BQWSM is shell surface ellipse, 10~12.5 microns long, 6~7.5 microns wide.Two ends Shape, 2.8~3.5 microns wide, back edge protrusion, ventral margin indent, shell seam is straight, bilateral symmetry.
For the present invention by culture detection discovery, double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816 environment are suitable Ying Xingqiang is easily cultivated, and Yi Caishou is described in detail below:
(1) thermal adaptability is strong: belonging to high temperature resistant algae, grows optimum temperature: 30-38 DEG C, being resistant to 4 DEG C of low temperature and 45 DEG C of height Temperature.Hainan can long-term outside scenery, be not necessarily to temperature control device.
(2) salinity scanning is strong: belonging to eurysalinity algae, experiment shows in the seawater that fresh water to salinity is 80 ‰ at present Can grow, optimal salinity is 30 ‰ -- 60 ‰, highest salinity is not yet tested.
(3) attachment culture can be carried out and the culture that suspends.
(4) be easy harvesting: the algae is in group's cellular morphology of larger particles shape or sheet under different condition of culture, can It is harvested with the screen to filtrate, reduces harvesting cost.
Double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816 fat contents are up to dry cell weight 30-60%.? Under the conditions of 35 DEG C, F/2 sea water medium culture 3 days, total lipid content can reach 20% or more；Culture 7 days, when reaching exponential growth When the middle and later periods, frustule total lipid content is up to 30% or more；When reaching Later growth or environmental stimulus (such as low temperature or item with high salt Part culture), frustule total lipid content can reach 60% or more.
Double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816 algae oil main ingredient are:
Palmitinic acid (C16:0): 30% or so；
Palmitoleic acid (C16:1): 25% or so；
EPA (eicosapentaenoic acid, C20:5): 3% -25% or so
Palmitinic acid (C16:0) and palmitoleic acid (C16:1) ratio are high, account for 50% or more, and two kinds of fatty acid carbon chains are shorter, satisfy It is higher with spending, it is suitble to do biodiesel.
Double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816 are free of DHA containing only EPA, thus are easier to obtain EPA sterling.In addition, double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816 algae strain easily harvestings and culture, biomass are raw Cost is relatively low for production, and fat content is high, and EPA accounts for fatty acid amount most up to 25%, is higher than most microalgaes, thus is comparatively ideal preparation The raw material of EPA.
Application of double eyebrow algae (Amphora sp.) CGMCC No.11816 in production EPA also belongs to protection of the invention Range.
In the application, producing in EPA includes cultivating double eyebrow algae (Amphora sp.) CGMCC No.11816 for extracting EPA, double eyebrow algae (Amphora sp.) CGMCC No.11816 are according to the method culture included the following steps:
1) double eyebrow algaes (Amphora sp.) CGMCC No.11816 culture medium for being seeded to silicate-containing is stood or is ventilated Algae is cultivated, cultivation temperature is 30~38 DEG C, obtain algae culture solution；
2) culture medium of the algae culture solution of step 1) culture and silicate-containing is placed in training according to volume ratio 1:20-120 It supports in container and carries out mass propgation, by the solid lamina affixad adhered to for diatom or attachment net (such as PC plate or the attachment of other solids Material) it is fixed in culture vessel (perpendicular to container bottom) vertically, it is formed and is used for the attached plate that double eyebrow algaes adhere to, adjacent two 3-6 centimetres of interval, air is passed through into culture vessel, makes 30-38 DEG C of temperature of sea water medium, to diatom attached plate irradiation light Light, intensity of illumination 10-80mol.m-2.s-1, light application time is 12-15 hours daily, after culture 3 to 10 days, harvesting algae strain cell.
It further include low temperature stimulation after double eyebrow algaes (Amphora sp.) CGMCC No.11816 culture, the low temperature thorn Sharp step are as follows: be placed in 10-25 DEG C of low temperature environment and give low temperature stimulation 0.5-2 days.
The preferred 15-25 DEG C of low temperature environment of low temperature stimulation, the method for low temperature stimulation can be to be placed in low-temperature circulating water Bath moderate stimulation.
Application of double eyebrow algae (Amphora sp.) CGMCC No.11816 in production biodiesel also belongs to of the invention Protection scope.
Application of double eyebrow algae (Amphora sp.) CGMCC No.11816 in production carotenoid also belongs to the present invention Protection scope.
In the carotenoid, higher content is fucoxanthin, therefore, double eyebrow algae (Amphora of the invention Sp.) CGMCC No.11816 can be used for high efficiency extraction fucoxanthin.
In conclusion double eyebrow algaes (Amphora sp.) BQWSM CGMCC No.11816 of the invention is one plant comprehensive The excellent algae strain of shape.Algae strain growth is fast, Yi Caishou, and biomass production cost is lower than most microalgaes.In the same of preparation biodiesel When, the high value added products such as EPA, carotenoid and fucoxanthin, exocellular polysaccharide can be obtained using biorefining technology It is also commercially valuable with silica silicon shell.
Detailed description of the invention
Fig. 1 is photo under double eyebrow algae (Amphora sp.) BQWSM microscopes.
Specific embodiment
Screening, separation and the identification of 1. pairs of eyebrow algaes of embodiment (Amphora sp.) BQWSM
1, the screening of double eyebrow algae (Amphora sp.) BQWSM
We obtain the excellent diatom strain of one plant of Comprehensive Traits: double eyebrow algaes (Amphora sp.) by screening BQWSM, the specific method is as follows:
Haikou coastal seawater is taken, nutritive salt enrichment is added, bottom of bottle grows brown algae and falls behind, and brown algae is taken to fall painting plate (plate is formulated with+1.5% agar of F/2 culture medium) obtains multiple double eyebrow algae monoclonals, the multiple monoclonal F/2 of picking Fluid nutrient medium culture isolates and purifies, isolating and purifying through high temperature, screening adaptability with high salt through ten several generations using plate is applied The fast algae strain of Johnson & Johnson head, room temperature culture detect EPA content, the highest algae strain of picking EPA content, then carry out plate purifying, train It supports.One plant of double eyebrow algae is obtained, BQWSM is named as.
2, the identification of double eyebrow algae (Amphora sp.) BQWSM
2.1 Morphological Identification
Double eyebrow algae (Amphora sp.) BQWSM (Fig. 1) are observed under 100 times of oil mirrors and laser confocal microscope, it is seen that Frustule shell surface ellipse, it is 10~12.5 microns long, it is 6~7.5 microns wide.Both ends capitiform, 2.8~3.5 microns wide, back edge is convex Out, ventral margin indent, shell seam is straight, bilateral symmetry.
2.2,18s rDNA and ITS sequence method for identifying molecules: have been measured
Logarithmic phase microalgae cell is collected by centrifugation, CTAB method extracts genomic DNA.Respectively with Eukaryotic Algae 18srDNA and ITS The 18srDNA sequence and ITS sequence of universal primer amplification frustule.
18srDNA primer sequence are as follows: 18SF:5 ' GGA TCA GAA TTC TAT CTG GTT GAT CCT GCC AG 3'；
18SR:5’-CTC AGT AAG CTT GAT CCT TCC GCA GGT TCA CC-3’。
ITS primer sequence are as follows: ITSF:5 '-GGAAGTAAAA GTCGTAACAAGG-3 '；
ITSR:5’-TCCTCCGCTTATTGATATGC-3’
PCR reaction condition is as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 1min, 55 DEG C of annealing 30s, 72 DEG C extend 1.5min, 30 circulations；, last 72 DEG C of extensions 8min.Glue PCR product after the recovery and pMD 18-T carrier are (precious purchased from Dalian Biology) it is ligated and transformed into competence E.coliJM109 (Beijing institute of microbiology).It selects 5 transformed bacterias and drops into row PCR inspection It surveys, then will be enlarged by the recombination bacterium colony after culture and send to the raw work in Shanghai and the completion sequencing of Shanghai JaRa company.
Sequencing result show 18srDNA sequence as shown in sequence 1 in sequence table, ITS sequence such as 2 institute of sequence in sequence table Show.Resulting 18S rDNA will be sequenced using Blast and ITS sequence carries out sequence analysis, the results showed that, algae strain The similarity 98%. of 18srDNA sequence and Amphora cofeaefomis 18S rDNA sequence；ITS sequence and Amphora The similarity 95% of cofeaefomis sequence, the similarity 97% with Amphora salina sequence.
According to morphology and molecular biological variety identification method, identify that the algae is double eyebrow algae spp, name are as follows: double eyebrow algaes (Amphora sp.) BQWSM, double eyebrow algae (Amphora sp.) BQWSM have been preserved on January 8th, 2016 Chinese common micro- Biological inoculum preservation administrative center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microbe research Institute), deposit number is CGMCC No.11816.
The Performance Testing of embodiment 2. pairs of eyebrow algaes (Amphora sp.) BQWSM CGMCC No.11816
Applicant passes through to double eyebrow algaes various overall merits of (Amphora sp.) BQWSM CGMCC No.11816 point Analysis finds that algae strain is one plant of production EPA and fucoxanthin oil-rich microalgae, and the potentiality with business development are described in detail below:
One, the environmental suitability detection of double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816:
By culture detection discovery, double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816 environmental suitabilities are strong, Easily culture, Yi Caishou are described in detail below:
(1) thermal adaptability is strong: belonging to high temperature resistant algae, grows optimum temperature: 30-38 DEG C, can outdoor training throughout the year in Hainan It supports, is not necessarily to temperature control device.
Temperature experiment:
Algae culture: double eyebrow algaes are with f/2 culture medium list algae static gas wave refrigerator.Wait cultivate to after exponential phase of growth, algae solution is taken It is inoculated in 3L Erlenmeyer flask in 1:8 ratio.It is sub-packed in after shaking up in 500ml triangular flask, every bottle of 400ml algae solution.Experiment sets 4 A processing: 20 DEG C, 30 DEG C, 35 DEG C, 38 DEG C, 3 repetitions of each processing.Photoperiod is 14h:8h, intensity of illumination 4000Lux. Seawater is derived from Haikou City white sand door, salinity 30 ‰, pH8.1.High pressure sterilization after filtering.
Growth measurement: centrifugation (5000g) is collected after double eyebrow algaes are cultivated 6 days, and deionized water cleaning is primary, and centrifugation obtains again Frustule precipitating.With analytical precision balances, (MA200, upper current chart level instruments and meters have after frustule freeze-drying (Labconco) Limit company) weighing.The relative growth rate (K) and cell doubling time of double eyebrow algaes are calculated according to following formula:
K=(lnN2-lnN1)/(t2-t1)
T=0.6931/K (2)
In formula, K is relative growth rate；T1, t2 are corresponding incubation time (3 days)；N1 and N2 is respectively t1, t2 period Dry cell weight；T is the mean doubling time of cell.
Experimental result: the specific growth rate of cell and doubling time such as table 1 under the conditions of static gas wave refrigerator
Table 1
Remarks: abc indicates variance analysis difference degree, and being indicated between the processing of same letter in same column does not have significance difference It is different, P > 0.05；Not no significant difference between the processing of same letter in same column, Pp < 0.05.
(2) eurysalinity: belonging to eurysalinity algae, and experiment at present shows give birth in the seawater that fresh water to salinity is 80 ‰ Long, salinity is 10 ‰ -- growth rate is not yet tested without significant difference (P < 0.05) (table 2), highest salinity under conditions of 60 ‰.
Salinity experiment:
Algae culture: double eyebrow algaes are with F/2 culture medium list algae static gas wave refrigerator.Wait cultivate to after exponential phase of growth, algae solution is taken It is inoculated in 5L Erlenmeyer flask in 20mg/L ratio.It is sub-packed in after shaking up in 500ml triangular flask, every bottle of 400ml algae solution.Experiment The culture solution of 6 salinity gradients (10 ‰ -60 ‰) is set, stationary culture in incubator is placed in, condition of culture is the photoperiod to be 14h:8h, intensity of illumination 4000Lux, 30 DEG C of temperature.Each gradient sets 3 in parallel.
F/2 culture medium is to add following element: NaNO3 75mg, NaH2PO42H2O 5.65mg in every liter of seawater, Na2EDTA 4.16mg,FeCl3·6H2O 3.15mg,Na2SiO3·9H2O 20mg, CuSO4·5H2O 0.01mg,ZnSO4· 7H2O 0.022mg,CoCl2·6H2O 0.01mg, MnCl2·4H2O 0.18mg,Na2MoO4·2H2O 0.006mg, Vitamin B12 0.0005mg,Vitamin B1 0.1mg,Biotin 0.0005mg。
Growth measurement: the growth measurement method in synthermal experiment.
Experimental result is as shown in table 2:
Table 2
Remarks: abc indicates variance analysis difference degree, and being indicated between the processing of same letter in same column does not have significance difference It is different, P > 0.05；Not no significant difference between the processing of same letter in same column, Pp < 0.05.
(3) attachment culture can be carried out and the culture that suspends.
(4) be easy harvesting: the algae, in larger particles shape or the colony morphology of sheet, can use sieve under different condition of culture Net filtration harvesting reduces harvesting cost.
The bis- eyebrow algaes of two, (Amphora sp.) BQWSM CGMCC No.11816 cell grease assay
Double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816 fat contents are up to dry cell weight 30-60%.? Under the conditions of 35 DEG C, F/2 sea water medium culture 3 days, total lipid content can reach 20% or more；Culture 7 days, reaches in exponential growth In the later period, total lipid content is up to 30% or more；Frustule total lipid content under Later growth or low temperature or high salt conditions reaches as high as To 60% or more.
3. yield of biomass is high:
(1) stationary culture: every liter of inoculum concentration 20mg frustule (weight in wet base), 38 DEG C, intensity of illumination 4000Lux, 400ml F/2 sea water medium, 500ml conical flask stationary culture 7 days, biomass be about 4.5g ㎡, 0.2g dry weight/L, be higher than two plants of seas Yield of biomass under water chlorella Chlorela ssp. same culture conditions.
(2) blowing air culture: inoculum concentration 20mg frustule (weight in wet base)/L is passed through air (air stream using 50L incubator Amount: 15L/min) culture, 30 DEG C, intensity of illumination 4000Lux, photoperiod 14h:8h, yield of biomass is about 3g/m2/ d, About 0.2g/L/d.
3, fatty acid compositional analysis shows that this plant of algae can be used as production EPA and biodiesel raw material
Condition of culture are as follows: 500ml conical flask stationary culture, intensity of illumination 4000Lux, photoperiod 14h:8h, temperature 30℃.If 2 processing: (1) 30 DEG C after CMC model 7 days, harvesting, freeze-drying；(2) 30 DEG C after CMC model 7 days, are transferred to 20 It is further cultured for 2 days, harvests under the conditions of DEG C, freeze-drying.
The algae powder 0.3 of freeze-drying is ground, the total rouge of chloroform-methanol method extraction (Bligh, E.G., Dyer, W.J., 1959.A rapid method of total lipid extraction and purification.Can.J.Biochem .Physiol.37,911-917.), then esterification, esterification reaction of organic acid is as follows:
Esterification process: 60 DEG C of water-bath 15min of 3ml 0.5mol/LKOH/ methanol solution are first added, 2ml is added after cooling 14% BF3 solution vibrates 2min in 60 DEG C of water-baths, puts to room temperature, and 2ml n-hexane is added, and 1ml saturation is added in shaken well Sodium chloride solution and a small amount of anhydrous sodium sulfite stand, take 1ml in 2ml teat glass, appropriate carclazyte is added and removes depigmentation, takes Supernatant carries out GC analysis.
GC chromatographic condition: during gas chromatographic analysis, sample volume is 1 μ L, split ratio 30:1.Chromatographic condition: FFAP Column is 30m*0.25mm, 0.50 μm of film thickness；Injector temperature is 250 DEG C, and detector temperature is 280 DEG C；Temperature programming, at 50 DEG C Retain 1min., is raised to 200 DEG C with 25 DEG C/min, then be increased to 230 DEG C with 3 DEG C/min, retains 10min. total run time 27min.Carrier gas is high pure nitrogen.Pass through sample retention time and 37 kinds of fatty acid methyl ester standard items (Supelco 37Component FAME mix, St.Louis, MO, USA) chromatogram retention time comparison, determine fatty acid methyl in sample The ingredient of ester, (it is then same Fatty Acid Methyl Esters that i.e. retention time is identical).
As a result as follows:
Double eyebrow algae (Amphora sp.) BQWSM CGMCC No.11816 algae oil main ingredient are:
Palmitinic acid (C16:0): 30%；
Palmitoleic acid (C16:1): 25%；
EPA (eicosapentaenoic acid, C20:5): 4%~10% (30 DEG C of cultures) 20% -25% (30 DEG C are cultivated 7 days, then It is transferred to 20 DEG C to cultivate 2 days)
Palmitinic acid (C16:0) and palmitoleic acid (C16:1) ratio are high, account for 50% or more, and two kinds of fatty acid carbon chains are shorter, satisfy It is higher with spending, it is suitble to do biodiesel.
EPA belongs to the serial polyunsaturated fatty acid of Ω -3, is the main component of fish oil, has multiple pharmacological effect.EPA tool It is helpful to reduce cholesterol and content of triglyceride, the effect for promoting internal saturated fat acid metabolic, to reduce blood viscousness Degree promotes blood circulation, prevents fat in the deposition of vascular wall, the formation and development of prevention of arterial atherosis, prevention brain blood The cardiovascular diseases such as bolt, cerebral hemorrhage, hypertension.
Currently commercially EPA main source is deep sea fish oil, since the uncertainty of abyssal fishes growth and the mankind are frequent Fishing, in addition various pollutions cause the stock of fish is more next to reduce, quality also accordingly declines.Therefore, other alternate resources are found As new research hotspot.The polyunsaturated fatty acid in microalgae source is easy to get with raw material, and supply is stablized, and does not destroy ecology The advantages that balance.Many microalgaes such as triangle brown fat algae (Phaeodactylum tricornutum) and Nannochloropsis oculata (Nannochloropsis oculata) can synthesize EPA, but often contain a certain amount of DHA simultaneously, and the two is not readily separated, point From to be mixture that product is often EPA and DHA.Our algae strain is free of DHA containing only EPA, thus is easier to obtain EPA Sterling.In addition, Amphora sp.BQWSM algae strain easily harvesting and culture, biomass lower production costs, fat content is high, EPA Fatty acid amount is accounted for most up to 25%, is higher than most microalgaes, thus is the comparatively ideal raw material for preparing EPA.
4, carotenoid and fucoxanthin (fucoxanthin) content are high
Carotenoid is a kind of antioxidant, there is the medication performance for alleviating cardiovascular disease, its world market pin in 2018 It sells total value and is up to 3.09 hundred million dollars.Currently, industrially the primary raw material of production carotenoid is rhodotorula and Dunaliella salina, Its highest content is respectively the 3.5% and 10% of dry matter weight.
According to radish cellulose content measuring method (bibliography: Lichtenthaler HK.Chlorophylls and Carotenoids:pigments of photosynthetic biomembranes.Methods in Enzymology, 1987,148:350-382.) Amphora sp.BQWSM algae strain carrotene is measured:
90% acetone method extracts pigment, calculates in extracting solution according to the following formula
The content of carotenoid:
Carotenoid (mg/L)=(1000 × D470-3.27 × Chla-104 × Chlb)/229
We the experimental results showed that, the carotenoid content of Amphora sp.BQWSM algae strain is up to 10% or more.
Fucoxanthin is a kind of fat-soluble natural carotenoid, accounts for 10% or more carotenoid total amount, has antioxygen Change activity, anti-inflammatory, it is antitumor and anti-fat the effects of, can be used in food processing and animal feed, be pre- anti-cancer and subtract The active drug of fertilizer.Fucoxanthin is largely present in brown alga, diatom and sea mollusk in nature, at present mostly from kelp Middle extraction preparation.Research shows that (Yan little Jun, Fan Xiao, Lou Qingxiang wait the extraction of carotenoid and [J] containing measurement in seaweed Marine Sciences collected papers, 2001,6 (43): 108-114), kelp fucoxanthin content is about 0.5mg/g of dry matter weight or so, mouse Fucoxanthin content is 0.724mg/g in tail algae, and fucoxanthin content is 0.680mg/g in sargassum kjellmanianum Yendo, and rock algae is yellow in thallus laminariae Cellulose content is 1.141mg/g.
(Liu Liang hooks bright Yue etc., the optimization Dalian work of kelp fucoxanthin extraction process with measuring method for fucoxanthin extraction Industry college journal, 2010,29 (8): 406-408):
A certain amount of diatom dry powder (30mg) is weighed, 20 μ LDMSO and 0.5mL85% methanol of addition are protected from light in mortar to be ground Grind 10min.85% methanol is added, is placed in 40 DEG C of water-baths and is protected from light extraction 60min, stand, take supernatant, extract repeatedly, merge supernatant Liquid measures light absorption value at 445nm.
In formula, A is light absorption value of the sample at 445nm；A1cm is the 1g/L fucoxanthin in the cuvette of 1cm optical length Theoretical absorption value, i.e., 1 600；N is extension rate；V is extracting solution total volume, mL；M is sample quality, g.
We, which test, shows under above-mentioned condition of culture (30 DEG C, salinity 30, illumination 4000lux) Amphora sp.BQWSM Algae strain fucoxanthin content is up to 10mg/g or more.Therefore, it can use double eyebrow algaes (Amphora sp.) BQWSM of the invention CGMCC No.11816 extracts production fucoxanthin.Extracting method can be existing fucoxanthin extracting method.
In conclusion Amphora sp.BQWSM algae strain is the excellent algae strain of one plant of Comprehensive Traits.Algae strain growth is fast, Yi Cai It receives, biomass production cost is lower than most microalgaes.While preparing biodiesel, it can be obtained using biorefining technology The high value added products such as EPA, carotenoid and fucoxanthin, exocellular polysaccharide and silica silicon shell also have business valence Value.
The application of embodiment 3, double eyebrow algaes (Amphora sp.) BQWSM CGMCC No.11816 in production EPA
As described in example 2 above, EPA in double eyebrow algaes (Amphora sp.) BQWSM CGMCC No.11816 of the invention Content is very high, and is free of DHA, is easier to extract and purify, and the present inventor provides a kind of double eyebrow algae productions of culture The method of EPA, specific as follows:
1, algae incubation step:
F/2 sea water medium stationary culture algae；
Cultivation temperature is 30-38 DEG C, and incubation time is 7 days, until algae concentration 0.1g/L or so.
The formula of F/2 sea water medium is to add following element: NaNO in every liter of seawater375mg,NaH2PO4·2H2O 5.65mg,Na2-EDTA 4.16mg,FeCl3·6H2O 3.15mg,Na2SiO3·9H2O 20mg, CuSO4·5H2O 0.01mg, ZnSO4·7H2O 0.022mg,CoCl2·6H2O 0.01mg,MnCl2·4H2O 0.18mg,Na2MoO4·2H2O 0.006mg, Vitamin B120.0005mg,Vitamin B10.1mg,Biotin 0.0005mg。
2, mass propgation step:
40 liters of F/2 sea water mediums are added into 50 liters of culture vessels, sea water medium salinity is 35 ‰ to 55 ‰, will The algae of upper step culture is placed in culture vessel by stationary culture algae volume and sea water medium volume ratio 1:100, by PC Plate is fixed in culture vessel (perpendicular to bottom of culture vessel) vertically, forms multiple attached plates for double eyebrow algae attachments, adjacent Two 3 centimetres of attached plate intervals blow air (10L-25L/min) to diatom attached plate, make the temperature 30-38 of sea water medium DEG C, light, intensity of illumination 10-80mol.m are irradiated to diatom attached plate-2.s-1, make daytime: night ratio 14 hours: 10 hours；
It harvests step: after being cultivated 3 to 5 days in culture vessel, taking out diatom attached plate, be placed in the solution for having added film In (added the F/2 culture medium or seawater of film, rise film addition concentration be 70g/L), after 1 to 2 day, be attached to diatom attached plate On frustule can be suspended in water body in membranaceous or bulk sheet, harvest frustule with screen filtration；Film is played under The group for stating weight fraction ratio is grouped as NH3SO4:NaNO3=5:1.
Low temperature stimulation step: the frustule after harvesting is put together, and is placed in recirculated water bath and is given 15,18,20 Or 25 DEG C low temperature stimulation 1 day, then frustule is freeze-dried.
3, EPA detection and extraction (survey fatty acid method see the GC of embodiment 2, comprising: extract total rouge, esterification reaction of organic acid, Gas chromatographic analysis EPA and various fatty acid percentage compositions), extracting method can be extracted according to the prior art.
Diatom cultural method of the invention includes low temperature stimulation step, the frustule grease through analyzing, after each low temperature stimulation For content up to 50%, it is 3 times without low-temperature treatment frustule that EPA content, which can account for 20% or more of fatty acid composition,.
Described above to be merely exemplary for the purpose of the present invention, and not restrictive, those of ordinary skill in the art understand, In the case where not departing from spirit and scope as defined in the appended claims, many modifications, variation or equivalent can be made, but all It will fall within the scope of protection of the present invention.

Claims (7)

1. one plant of double eyebrow algae Amphora sp., which is characterized in that entitled double eyebrow algaes of double eyebrow algae Amphora sp. Amphora sp.BQWSM, deposit number are CGMCC No.11816.
2. application of couple eyebrow algae Amphora sp.BQWSM CGMCC No.11816 in production EPA.
3. application according to claim 2, which is characterized in that include cultivating double eyebrow algae Amphora in production EPA Sp.BQWSM CGMCC No.11816, double eyebrow algae Amphora sp.CGMCC No.11816 are according to including the following steps Method culture:
1) double eyebrow algae Amphora sp.BQWSM CGMCC No.11816 are seeded to the culture medium culture algae of silicate-containing, Cultivation temperature is 25 DEG C~38 DEG C, obtains algae culture solution；
2) the algae culture solution of step 1) culture and the culture medium of silicate-containing culture is placed according to volume ratio 1:20-120 to hold Mass propgation is carried out in device, and the solid lamina affixad adhered to for diatom or attachment net are fixed in culture vessel vertically, formed For the attached plate of double eyebrow algaes attachment, two adjacent 3-6 centimetres of attached plate intervals are passed through air into culture vessel, train seawater The temperature for supporting base is 30-38 DEG C, irradiates light, intensity of illumination 10-80mol.m to diatom attached plate-2.s-1, light application time is daily After 12-15 hours, culture 3 to 10 days, harvesting algae strain cell.
4. application according to claim 3, which is characterized in that double eyebrow algae Amphora sp.BQWSM CGMCC It further include low temperature stimulation after No.11816 culture, the step of the low temperature stimulation are as follows: give 10-25 DEG C of low temperature stimulation 0.5-2 days.
5. application of couple eyebrow algae Amphora sp.BQWSM CGMCC No.11816 in production biodiesel.
6. application of couple eyebrow algae Amphora sp.BQWSM CGMCC No.11816 in production carotenoid.
7. application according to claim 6, which is characterized in that the carotenoid is fucoxanthin.
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Non-Patent Citations (3)

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Title
2株微藻对养殖废水中氮磷去除率的影响;许云等;《热带生物学报》;20140930;第5卷(第3期);228-232 *
5株新分离海洋硅藻总脂和脂肪酸组成的比较研究;高秀芝等;《生物学杂志》;20140228;第31卷(第1期);60-63、81 *
许云等.2株微藻对养殖废水中氮磷去除率的影响.《热带生物学报》.2014,第5卷(第3期),228-232. *

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