CN108342420A - A kind of mixotrophic cultivation method producing polyunsaturated fatty acid and fucoxanthine using Phaeodactylum tricornutum - Google Patents

A kind of mixotrophic cultivation method producing polyunsaturated fatty acid and fucoxanthine using Phaeodactylum tricornutum Download PDF

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CN108342420A
CN108342420A CN201810356560.9A CN201810356560A CN108342420A CN 108342420 A CN108342420 A CN 108342420A CN 201810356560 A CN201810356560 A CN 201810356560A CN 108342420 A CN108342420 A CN 108342420A
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tea polyphenols
mannitol
fucoxanthine
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CN108342420B (en
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范勇
李福利
胡光荣
王丽娟
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Abstract

The present invention relates to a kind of mixotrophic cultivation methods producing polyunsaturated fatty acid and fucoxanthine using Phaeodactylum tricornutum.The present invention includes application and tea polyphenols or tea polyphenols active constituent application in mixotrophic cultivation diatom production polyunsaturated fatty acid and fucoxanthine of the mannitol in mixotrophic cultivation diatom production polyunsaturated fatty acid and fucoxanthine.The present invention uses mannitol as simultaneous foster carbon source for the first time, it is found through experiments that, mannitol can effectively improve the speed of growth and yield of Phaeodactylum tricornutum compared with other carbon sources, and mixotrophic cultivation has better combined coefficient in photosynthetical system pigment synthesis level compared with Heterotrophic culture；In addition, the present invention uses tea polyphenols as bioactive substance culture diatom for the first time, tea polyphenols can be effectively promoted the absorption and utilization of carbon source, through actually detected, 108% can be improved to Phaeodactylum tricornutum biomass, fucoxanthine content improves 96%, and EPA content improves 122%.

Description

It is a kind of to be supported using Phaeodactylum tricornutum production polyunsaturated fatty acid and the simultaneous of fucoxanthine Cultural method
Technical field
The present invention relates to a kind of mixotrophic cultivation sides producing polyunsaturated fatty acid and fucoxanthine using Phaeodactylum tricornutum Method belongs to technical field of bioengineering.
Background technology
Fucoxanthine（Fucoxanthin）Also known as fucoxanthin or brown alga flavine, belong to the oxygen-containing derivative of carotenoid Object is common in brown alga and diatom.Fucoxanthine mainly exercises the effect that luminous energy is captured in photosynthetical system in alga cells.Rock Algae Huang matter and chlorophyllaAnd chlorophyllcForm fucoxanthine-Chlorophyll Protein Complexes（Fucoxanthin-Chlorophyll Protein, FCP）.In research in recent years, fucoxanthine has proved to be a kind of safely and effectively dietary supplements, it can be with It boosts metabolism and nervous system is not impacted, there is anti-obesity, anti-diabetic, anti-oxidant, it is anti-inflammatory, antitumor etc. A variety of physiological activity [1].Lot of experiments shows that the anti-obesity mechanism of fucoxanthine is since it can be activated to lipolysis The expression for having the UCP1 mitochondria uncoupling protein of facilitation, reaches the cell that reduces fat, prevents the effect of fat accumulation [2]。
Fucoxanthine has oxidation resistant activity.Antioxidation is one of important feature of carotenoid, many lifes Object effect is directed to the ability of Scavenger of ROS, this is its one of factor with disease prevention effect.Research is found recently Fucoxanthine can also be by adjusting Na+/K+The activity of ATP enzyme prevents peroxidation of cellular membranes.The experiment has also demonstrated due to rock The effect of algae Huang matter, catalase and glutathione turn can be gradually increased by feeding fucoxanthine to the mouse for lacking retinol The activity for moving enzyme, to reach oxidation resistant effect [3].
Eicosapentaenoic acid（Eicosapentaenoic acid, EPA, C20H30O2）It is the how unsaturated containing five double bonds Aliphatic acid（Polyunsaturated fatty acid, PUFA）, belong to omega-fatty acid.The fatty acid families include mainly α- Leukotrienes（α-Linolenic acid, ALA）, eicosapentaenoic acid（EPA）And docosahexaenoic acid （Docosahexaenoic acid, DHA）.EPA has very the prevention of developing infant brain and disease of cardiovascular system of being grown up Good effect, while also there is anti-tampon, prevent coagulating platelets, vasodilator from reducing the effects that cholesterol and blood pressure [4].It is mainly extracted from deep sea fish oil currently used for the natural EPA of food and health products, the ratio that EPA accounts for fish oil can reach 20%~30%.However, the yield of EPA is influenced by fishing type, the fishing season and place in fish oil；In addition, height contains in fish oil The cholesterol and peculiar smell of amount drastically influence the quality of fish oil.
Marine microalgae plays an important role in marine ecosystems, and as primary producer, they are marine organisms Important foodstuffs source.Marine microalgae contains a large amount of long-chain unsaturated fatty acid into the cell, and wherein EPA content is abundant.Microalgae PUFAs compositions in grease are simple, cholesterol is low.Therefore, producing the unsaturated fatty acids such as EPA using microalgae has wide city Field foreground.In marine microalgae, Phaeodactylum tricornutum（Phaeodactylum tricornutum）, micro- quasi- ball algae （Nannochloropsis oceanica）, Isochrysis galbana（Isochrysis galbana）And Nitzschia closterium minutissima（Nitzschia closterium）Deng contain higher EPA, 20% of fat content or more [5] can be reached.And color is also rich in marine microalgae Element especially fucoxanthine.Wherein Phaeodactylum tricornutum due to growth rate it is fast, the features such as fat content is high, is of interest by people. Growth rate can reach 0.38 g/L/d, and fat content can reach 48% or more, EPA yields and can reach 36 mg/L/ after harvest D produces source as new EPA in recent years.Meanwhile as typical diatom, FCP complexs, rock are rich in Phaeodactylum tricornutum Algae Huang matter content is between 15.42-16.51 mg/g.
There is the nutritional mode of microalgae diversity, most of microalgaes to use photoautotrophy（Phetoautotrophy）Mode, Many microalgaes, which have, to be carried out and is supported using organic carbon（Mixotrophy）And heterotrophism（Heterotrophy）The ability of growth.Autotrophy Light limiting factor seriously constrain micro algae biomass, and support and heterotrophism can partly or entirely eliminate light limitation [6], become and carry The effective way of high microalgae yield and intracellular activity content of material, therefore simultaneous support of microalgae becomes recent year with heterotrophism research The hot issue of outer algological biology concern.
And it is to utilize CO to support2And organic carbon, it is carried out at the same time photosynthesis and respiration, therefore under light restrictive condition, The simultaneous foster specific growth rate of most of microalgaes is approximately equal to the sum of autotrophy and heterotrophism specific growth rate [7], such as：Card moral algae （Tetraselmis sp.）, haematococcus pluvialis（Haematococcus pluvialis）And chlorella （Chlorellavulgaris）Deng.Simultaneous support provides an effective way for microalgae High Density Cultivation, and supports and be conducive to heterotrophism The accumulation of intracellular activity substance provides good approach for exploitation microalgae production intracellular activity substance.
But a kind of carbon source that suitable mixotroph utilizes of selection is to determine and support the speed of growth and economy higher than other The most critical factor of training method.《The autotrophy of Phaeodactylum tricornutum and the research of foster and heterotrophic nature》（Liu Xiaojuan etc., Ji'nan University, 2008.）Mixotrophic cultivation can be carried out using a variety of organic carbon sources by disclosing the economic microalgae Phaeodactylum tricornutum of Bacillariophyta, but simultaneous There are still easy to pollute during supporting, and the deficiencies of foster effect is not notable, the development of large-scale culture technology is limited.
Mannitol is the isomer of D-sorbite, on No. two carbon atoms of two kinds of alcohols materials hydroxyl towards different, Molecular formula is C6H14O6, molecular weight 182.17.It is soluble easily in water, it is the solid of white clear, there is the sweet taste of similar sucrose.Mesh Before, by mannitol be carbon source carry out microculture document it has been reported that but it is more concentrate on heterotrophism in the way of culture it is different Support microorganism.Such as Japanese documentation JP2004024121A（Application number JP2002185745）Disclose a kind of short-period birch Brown pore fungi Mycelium culture method, this method carry out the mycelial culture of Inonotus obliquus, fluid nutrient medium using fluid nutrient medium Involved in carbon source be a kind of non-reducible disaccharide, such as trehalose or sucrose or sugar alcohol（Such as mannitol, D-sorbite or maltose Alcohol）, the initial pH value of fluid nutrient medium is 3.5-6.5.But not to mannitol if appropriate for the carbon source as mixotroph And how the report for greatly improving mixotroph speed of growth purpose is reached using mannitol.
Invention content
Phaeodactylum tricornutum production polyunsaturated fatty acid and rock are utilized in view of the deficiencies of the prior art, the present invention provides a kind of The mixotrophic cultivation method of algae Huang matter.By the present invention in that being achieved the purpose that improve biomass with new simultaneous foster carbon source, add simultaneously Using resistance of the utilization and raising Phaeodactylum tricornutum of promoted and foster carbon source in growth course, reaches and carry The purpose of high Phaeodactylum tricornutum production yield.
The one of technical solution of the present invention is as follows：
Application of the mannitol in mixotrophic cultivation diatom production polyunsaturated fatty acid and/or fucoxanthine.
According to currently preferred, above-mentioned mannitol is configured to culture medium as carbon source, and mixotrophic cultivation diatom production is mostly not Saturated fatty acid and fucoxanthine；It is further preferred that the additive amount of the mannitol, which is every liter of culture medium, adds 1~20 g； Optimal, the additive amount of the mannitol is that every liter of culture medium adds 2 g；It is further preferred that the culture medium, every liter of component It is as follows：
1~20 g mannitol, 0.1~10 g NaNO3, 10~40 g seawater salt, 2~10 mg Na2EDTA, 1~9 mg FeCl3•6H2O, 2~12 mg NaH2PO4•12H2O, 10~60 mg Na2SiO3•7H2O, 0.001~0.1 mg CuSO4• 5H2O, 0.002~0.2 mg MgSO4•7H2O, 0.001~0.1 mg CoCl2•6H2O, 0.02~2 mg MnCl2•4H2O, 0.001~0.01 mg Na2MoO4•2H2O, 0.0001~0.01 mg biotins（Biotin）；More preferably, the culture medium, often It is as follows to rise component：
2 g mannitol, 1 g NaNO3, 30 g seawater salt, 4.37 mg Na2EDTA, 3.65 mg FeCl3•6H2O, 6.7 mg NaH2PO4•12H2O, 30 mg Na2SiO3•7H2O, 0.01 mg CuSO4•5H2O, 0.022 mg MgSO4•7H2O, 0.01 mg CoCl2•6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg Na2MoO4•2H2O, 0.0005 mg biotins（Biotin）.
According to currently preferred, the culture medium further includes tea polyphenols or tea polyphenols active constituent, concentration 0.05~2 g/L；It is further preferred that tea polyphenols or tea polyphenols activity component concentration are 0.2 g/L.
According to currently preferred, the diatom is selected from Cyclotella（Cyclotella）, Nitzschia （Nitzschia）, Navicula（Navicula）, barrel mast Trentepohlia（Cylindrotheca）Or brown algae category （Phaeodactylum）；It is further preferred that the brown algae category（Phaeodactylum）For Phaeodactylum tricornutum, Cyclotella （Cyclotella）For small ring algae.
According to currently preferred, above application, steps are as follows：
（1）Diatom is cultivated to exponential phase, seed liquor is made；
（2）By step（1）Seed liquor obtained is seeded to by 5~50% volume ratio in above-mentioned culture medium, in temperature 20~30 DEG C, 50~300 μm of ol photonsm−2·s−1It is cultivated under illumination condition 3~5 days, then maintains mannitol in cultivating system A concentration of 1~20 g/L, continues culture 4~8 days, and the zymotic fluid containing polyunsaturated fatty acid and fucoxanthine is made.
It is further preferred that the step（2）In, condition of culture is：80 μm of 23~25 DEG C of temperature, illumination condition ol photons·m−2·s−1；More preferably, the photoperiod of illumination condition is 16h illumination, 8h dark；It is further preferred that the step Suddenly（2）In, maintenance mannitol concentration is 2 g/L；It is further preferred that the step（2）In, maintain NaNO3A concentration of 0.1~ 10 g/L；More preferably, the step（2）In, maintain NaNO3A concentration of 1 g/L.
Another technical solution of the invention is as follows：
Tea polyphenols or tea polyphenols active constituent answering in mixotrophic cultivation diatom production polyunsaturated fatty acid and/or fucoxanthine With.
According to catechin compounds mass percentage content in currently preferred, described tea polyphenols be 65%~ 85%；Preferably, the tea polyphenols active constituent is selected from catechin（Flavanol compound）, flavones and flavonols, anthocyan, Phenolic acid and depside and/or polymerization phenolic compound；It is further preferred that the catechin compounds are selected from：Catechin （EC）, nutgall catechin（EGC）, catechin and gallate（ECG）And/or nutgall catechin gallic acid ester （EGCG）.
Tea polyphenols are ordinary commercial products, include the mixture of multiple compounds, and chemical composition is catechin（Flavane Alcohols）, flavones and flavonols, anthocyan, phenolic acid and depside, the compounds such as polymerization phenols complex.Wherein Tea chlorins compound is the bulk composition of tea polyphenols.Catechin compounds include mainly catechin（EC）, nutgall catechin （EGC）, catechin and gallate（ECG）And nutgall catechin gallic acid ester（EGCG）4 kinds of substances.The technical program packet Independent addition application containing above 4 kinds of catechin compounds.
According to currently preferred, above-mentioned tea polyphenols or tea polyphenols active constituent are added into culture as bioactive substance Base, mixotrophic cultivation Phaeodactylum tricornutum produce polyunsaturated fatty acid and fucoxanthine；It is further preferred that the tea polyphenols be containing There are catechin compounds to account for the 65%~85% of tea polyphenols gross mass；It is further preferred that the tea polyphenols or tea polyphenols activity The additive amount of ingredient is that every liter of culture medium adds 0.05~2 g；Optimal, the addition of the tea polyphenols or tea polyphenols active constituent Amount adds 0.2 g for every liter of culture medium；It is further preferred that the culture medium, every liter of component is as follows：
1~20 g carbon sources, 0.1~10 g NaNO3, 10~40 g seawater salt, 2~10 mg Na2EDTA, 1~9 mg FeCl3• 6H2O, 2~12 mg NaH2PO4•12H2O, 10~60 mg Na2SiO3•7H2O, 0.001~0.1 mg CuSO4•5H2O, 0.002~0.2 mg MgSO4•7H2O, 0.001~0.1 mg CoCl2•6H2O, 0.02~2 mg MnCl2•4H2O, 0.001~ 0.01 mg Na2MoO4•2H2O, 0.0001~0.01 mg biotins（Biotin）, tea polyphenols or tea polyphenols active constituent 0.05 ~2 g；More preferably, the culture medium, every liter of component are as follows：
2 g carbon sources, 1 g NaNO3, 30 g seawater salt, 4.37 mg Na2EDTA, 3.65 mg FeCl3•6H2O, 6.7 mg NaH2PO4•12H2O, 30 mg Na2SiO3•7H2O, 0.01 mg CuSO4•5H2O, 0.022 mg MgSO4•7H2O, 0.01 mg CoCl2•6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg Na2MoO4•2H2O, 0.0005 mg biotins（Biotin）, tea 0.2 g of polyphenol or tea polyphenols active constituent.
According to currently preferred, carbon source is selected from the culture medium：Mannitol, glycerine, glucose, fructose, sucrose, mountain Pears alcohol, glycine and/or acetic acid；It is further preferred that carbon source is selected from the culture medium：Mannitol and/or glycerine；It is optimal , carbon source is mannitol in the culture medium.
According to currently preferred, the diatom is selected from Cyclotella（Cyclotella）, Nitzschia （Nitzschia）, Navicula（Navicula）, barrel mast Trentepohlia（Cylindrotheca）Or brown algae category （Phaeodactylum）；It is further preferred that the brown algae category（Phaeodactylum）For Phaeodactylum tricornutum, Cyclotella （Cyclotella）For small ring algae.
According to currently preferred, above application, steps are as follows：
（1）Diatom is cultivated to exponential phase, seed liquor is made；
（2）By step（1）Seed liquor obtained is seeded to by 5~50% volume ratio in above-mentioned culture medium, in temperature 20~30 DEG C, 50~300 μm of ol photonsm−2·s−1It is cultivated under illumination condition 3~5 days, then maintains carbon source in cultivating system dense Degree is 1~20 g/L, continues culture 4~8 days, and the zymotic fluid containing polyunsaturated fatty acid and fucoxanthine is made.
It is further preferred that the step（2）In, condition of culture is：80 μm of 23~25 DEG C of temperature, illumination condition ol photons·m−2·s−1；More preferably, the photoperiod of illumination condition is 16 h illumination, 8 h dark；It is further preferred that described Step（2）In, maintenance carbon source concentration is 2 g/L；It is further preferred that the step（2）In, maintain NaNO3A concentration of 0.1~ 10 g/L；More preferably, the step（2）In, maintain NaNO3A concentration of 1 g/L.
After testing, using method mixotrophic cultivation Phaeodactylum tricornutum of the present invention, the phaeodactylum tricornutum for cultivating 12 days gained refers to Algae powder dry weight>2.5 g/L, fucoxanthine content>16 mg/g, content of polyunsaturated fatty acid>125 mg/g.Unit volume Fucoxanthine relative maximum yield in cultivating system>3.33 mg/L/ days；Polyunsaturated fatty acid relative maximum yield>26 mg/ L/ days.
The present invention has the advantages that：
1. the present invention use for the first time mannitol as and foster carbon source, mannitol be main carbohydrate in marine environment it One, widely distributed in ocean, diatom can adapt to the osmotic pressure of mannitol, and have potential generation as marine microalgae It thanks to approach to utilize mannitol, is found through experiments that, mannitol can effectively improve the growth of Phaeodactylum tricornutum compared with other carbon sources Speed and yield（Improve 80%）, and mixotrophic cultivation has preferably in photosynthetical system pigment synthesis level compared with Heterotrophic culture Combined coefficient；
2. the present invention uses mannitol as and foster carbon source, in amplifying incubation, since mannitol is not one extensive Therefore the organic carbon source utilized by microorganism can more effectively avoid the pollution of miscellaneous bacteria in environment using the carbon source so that In the short-term mixotrophic cultivation period, the failure of incubation will not be caused because of the pollution of bacterium；
3. the present invention uses tea polyphenols as bioactive substance culture diatom for the first time, tea polyphenols can be effectively promoted carbon source Absorption and utilization 108% can be improved to Phaeodactylum tricornutum biomass through actually detected, fucoxanthine content improves 96%, EPA content improves 122%；
4. the present invention is during using tea polyphenols as bioactive substance culture Phaeodactylum tricornutum, it was found that addition tea The cultivating system of polyphenol has the degeneration-resistant effect of environment well.
Description of the drawings
The growth curve of Phaeodactylum tricornutum under the conditions of Fig. 1 various concentration mannitol；
In figure：CT is that control group does not add mannitol, other are respectively to add the sweet of the g/L of a concentration of 0.5,1,2,4,10 and 15 Reveal alcohol；
Growth of Phaeodactylum tricornutum curve under the conditions of Fig. 2 difference tea polyphenols；
In figure：CT is control the case where not adding tea polyphenols, other be respectively add a concentration of 0.05,0.1,0.2,0.5,1.0, The tea polyphenols of 2.0 and 5.0 g/L；
Fig. 3 are the changes of contents of fucoxanthine of Phaeodactylum tricornutum under the conditions of mixotrophic cultivation, and TP is tea polyphenols（Tea Polyphenols）；
Fig. 4 are the yield block diagrams of EPA of Phaeodactylum tricornutum under the conditions of mixotrophic cultivation, and TP is tea polyphenols（Tea Polyphenols）；
Fig. 5 cultivate 2 days after mannitol utilization power block diagram, TP is tea polyphenols（Tea Polyphenols）；
Fig. 6 use the utilization power curve graph of HPLC analysis tea polyphenols main components in incubation；
In figure：1, EGC, epi-nutgall acid catechin；2, cianidanol (+)-catechin；3, EGCG, epi-nutgall catechu Plain gallate；4, EC, epicatechin；5, ECG, L-Epicatechin gallate；
Fig. 7 A. glycerine and mannitol are respectively as carbon source culture Phaeodactylum tricornutum, and phaeodactylum tricornutum refers under the adding conditional of tea polyphenols The growth curve of algae, TP are tea polyphenols（Tea Polyphenols）；
Fig. 7 B. glycerine and mannitol are respectively as carbon source culture Phaeodactylum tricornutum, the fucoxanthine under the adding conditional of tea polyphenols Accumulation, TP is tea polyphenols（Tea Polyphenols）.
Specific implementation mode
Technical scheme of the present invention is further elaborated with reference to embodiment and Figure of description, but the present invention is protected It is without being limited thereto to protect range.
Biological material source
Phaeodactylum tricornutum（Phaeodactylum tricornutum）, it is purchased from Chinese Marine University's microalge library, number MACC/B228；
Small ring algae（Cyclotella cryptica）, it is purchased from U.S. NCMA algaes library, number：CCMP333.
Other raw material sources
Tea polyphenols are purchased from the Shandong bio tech ltd Xi Tang, ordinary commercial products；
Mannitol is purchased from the Shandong bio tech ltd Xi Tang, ordinary commercial products；
Seawater salt is the blue treasure Blue Treasure sea salt of the salt Shui nationality Science and Technology Ltd. purchased from Qingdao sea, common city Sell product.
Detection method
1. the measurement of fucoxanthine：
Phaeodactylum tricornutum frustule is collected by centrifugation under 4000 × g speed, with distillation washing one time, centrifuges again and collects algae Cell.Frustule is resuspended to the extraction for carrying out pigment in ethanol（Ethyl alcohol:Algae solution volume=1:1；v/v）, 45 DEG C be incubated it is 2 small When, per half an hour, concussion is primary, finally, is detached the pigment of extraction with algae-residue by high speed centrifugation.
The measurement of fucoxanthine content uses Hitachi's high performance liquid chromatograph detecting system, selection anti-in the pigment extracted Phase Agilent C18Chromatographic column（2.7 μm, 100 × 4.6 mm）.Detection wavelength is 445 nm, and flow rate of mobile phase is 1 mL/min, into Sample amount：5 μL.Mobile phase is that acetonitrile and water carry out gradient elution.Acetonitrile ratio rises to 100% in 8 min by 80 %, maintains After this ratio elutes 3 min, acetonitrile ratio is linearly down to 80 % in 5 min, acetonitrile concentration is then risen to 100% again And it maintains 15 minutes.
2. the measurement of EPA
5-20 mL are collected by centrifugation in Phaeodactylum tricornutum frustule under 4000 × g speed, abandon supernatant.6 mL chloroform-methanols are added （4 mL and 2 mL）, 2 mL KCl are then added（0.9%）Fully shaking.5000 g centrifuge 5 min separation lipids and algae after concussion Slag.Subnatant is transferred in a new cuvette weighed in advance, algae oil is obtained after being dried up with nitrogen evaporator and claims immediately Amount record.
By the algae oil after drying, 1.5 mL n-hexanes and 4.5 mL, 2% H is added2SO4（It is dissolved in methanol）, 85 DEG C of esterifications Reaction 3 hours adds 0.9% KCl solution, 2 mL, fully shaking after cooling.Stand 5 minutes after concussion, take upper organic phase in from It is spare in heart pipe.If EPA concentration is low, revolving instrument concentration can be used.Finally take 200 μ L for gas-chromatography（GC）Analysis.
It is formed using the fatty acid methyl ester in Agilent 7890A gas chromatography analysis samples, chromatographic column HP-5（30 m ×320 µm×0.25 µm）.Gas chromatographic analysis program is as follows：Injector temperature：250 DEG C, sampling volume：1 μL；Carrier gas makes Use high-purity nitrogen；Temperature program is arranged：120 DEG C of 5 min of maintenance, then rise to 240 DEG C with 3.5 DEG C/min, keep 10 min.
3. the measurement of mannitol
Phaeodactylum tricornutum culture medium is centrifuged under 12000 × g speed, supernatant culture solution is collected, is diluted, made according to sample concentration With Agilent high performance liquid chromatograph Composition distribution detecting system, Bio-RAD Aminex HPX-87H chromatographic columns (300 are selected The mm of mm × 7.8), sample size is 10 μ L, and 55 DEG C of column oven, flow rate of mobile phase is 5 mol/L H2SO4, flow velocity is 0.5 mL/ Min, equal strength elute 30 minutes.
4. the measurement of tea polyphenols
Phaeodactylum tricornutum culture medium is centrifuged under 12000 × g speed, supernatant culture solution is collected, is diluted, made according to sample concentration With Hitachi's high performance liquid chromatograph detecting system, reverse phase Agilent C18 chromatographic columns (2.7 μm, 100 × 4.6mm) are selected.Detection Wavelength is 280 nm, and sample size is 10 μ L, and mobile phase is methanol:Water=24:76, flow rate of mobile phase 1mL/min, equal strength is washed It is 30 minutes de-.
1 mannitol concentration of embodiment is tested
The method that mixotrophic cultivation Phaeodactylum tricornutum produces polyunsaturated fatty acid and fucoxanthine, steps are as follows：
（1）By Phaeodactylum tricornutum culture to exponential phase（24 DEG C, 80 μm ol photonsm of temperature−2·s−1Illumination condition Lower culture 3 days）, seed liquor is made；
（2）By step（1）Seed liquor obtained is seeded to by 5~50% volume ratio in culture medium, in 23~25 DEG C of temperature, 100 μmol photons·m−2·s−1It is cultivated 10 days under illumination condition, the photoperiod is 16h illumination, 8h dark；
The step（2）In culture medium, prepare a concentration of 0.5,1,2,4,10 and 15 g/L that carbon source is mannitol respectively Culture medium is control to be not added with mannitol, and above-mentioned every liter of content of culture medium other compositions is as follows：
1 g NaNO3, 30 g seawater salt, 4.37 mg Na2EDTA, 3.65 mg FeCl3•6H2O, 6.7 mg NaH2PO4• 12H2O, 30 mg Na2SiO3•7H2O, 0.01 mg CuSO4•5H2O, 0.022 mg MgSO4•7H2O, 0.01 mg CoCl2• 6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg Na2MoO4•2H2O, 0.0005 mg biotins（Biotin）；
After testing, the results are shown in Figure 1：After first rising is presented in maximum biomass of the Phaeodactylum tricornutum under different mannitol concentrations Downward trend.For mannitol concentration in the range of 1~10 g/L, maximum biomass is more than control, but without aobvious between each group Write difference（p>0.05）.When mannitol concentration is 15 g/L, it is suppressed that the growth of Phaeodactylum tricornutum.According to economic and practical original Then, 2 g/L mannitol concentrations are chosen as best culture concentration.
2 tea polyphenols concentration experiment of embodiment
The method that mixotrophic cultivation Phaeodactylum tricornutum produces polyunsaturated fatty acid and fucoxanthine, steps are as follows：
（1）By Phaeodactylum tricornutum culture to exponential phase（24 DEG C, 80 μm ol photonsm of temperature−2·s−1Illumination condition Lower culture 3 days）, seed liquor is made；
（2）By step（1）Seed liquor obtained is seeded to by 5~50% volume ratio in culture medium, in 23~25 DEG C of temperature, 100 μmol·photons·m−2·s−1It is cultivated 8 days under illumination condition, the photoperiod is 16 h illumination, 8 h dark；
The step（2）In culture medium, respectively prepare contain a concentration of 0.05,0.1,0.2,0.5,1.0,2.0 and of tea polyphenols The culture medium of 5.0 g/L is control to be not added with tea polyphenols, and above-mentioned every liter of content of culture medium other compositions is as follows：
2 g mannitol, 1 g NaNO3, 30 g seawater salt, 4.37 mg Na2EDTA, 3.65 mg FeCl3•6H2O, 6.7 mg NaH2PO4•12H2O, 30 mg Na2SiO3•7H2O, 0.01 mg CuSO4•5H2O, 0.022 mg MgSO4•7H2O, 0.01 mg CoCl2•6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg Na2MoO4•2H2O, 0.0005 mg biotins（Biotin）；
After testing, the results are shown in Figure 2：For Phaeodactylum tricornutum under conditions of mannitol concentration is 2 g/L, maximum biomass is to add When adding 0.2 g/L tea polyphenols；But tea polyphenols concentration, in the range of 0.1-2 g/L, growth rate is more than control.Tea polyphenols When a concentration of 5 g/L, it is suppressed that the growth of Phaeodactylum tricornutum.
Embodiment 3
Method as described in Example 1, the difference is that, every liter of content of medium component is as follows：
2 g mannitol, 0.2 g/L tea polyphenols, 10 g NaNO3, 10g seawater salt, 10 mg Na2EDTA, 1mg FeCl3•6H2O, 12 mg NaH2PO4•12H2O, 10mg Na2SiO3•7H2O, 0.1 mg CuSO4•5H2O, 0.002mg MgSO4•7H2O, 0.1 mg CoCl2•6H2O, 0.02mg MnCl2•4H2O, 0.01 mg Na2MoO4•2H2O, 0.0001mg biotin.
Embodiment 4
Method as described in Example 1, the difference is that, every liter of content of medium component is as follows：
2 g mannitol, 0.2 g/L tea polyphenols, 0.1g NaNO3, 40 g seawater salt, 2mg Na2EDTA, 9 mg FeCl3•6H2O, 2mg NaH2PO4•12H2O, 60 mg Na2SiO3•7H2O, 0.001mg CuSO4•5H2O, 0.2 mg MgSO4•7H2O, 0.001 CoCl2 6H2O, 2 mg MnCl2 4H2O, 0.001mg Na2MoO4 2H2O, 0.01 mg biotins.
After testing, under the medium culture of embodiment 3,4, cell remains able to grow, but optimal conditions is embodiment 2 Culture medium condition.
Embodiment 5
Method as described in Example 1, the difference is that, using other Bacillariophyta microalgaes（Small ring algae, Cyclotella Cryptica is purchased from U.S. NCMA algaes library, algae number：CCMP333）, every liter of content of medium component is as follows：
2 g mannitol, 0.2 g/L tea polyphenols, 1 g NaNO3, 30 g seawater salt, 4.37 mg Na2EDTA, 3.65 mg FeCl3•6H2O, 6.7 mg NaH2PO4•12H2O, 30 mg Na2SiO3•7H2O, 0.01 mg CuSO4•5H2O, 0.022 mg MgSO4•7H2O, 0.01 mg CoCl2•6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg Na2MoO4•2H2O, 0.0005 mg Biotin（Biotin）；
After testing, nearly source Bacillariophyta microalgae can significantly improve biomass using the culture medium compared with photoautotrophy culture.
Embodiment 6
Method as described in Example 1, the difference is that, it is compared using different component：Add mannitol and tea simultaneously It is cultivated under the optimal conditions of polyphenol（Containing 2 g/L mannitol and 0.2 g/L tea polyphenols）；Only add the optimal conditions of mannitol Under cultivated（Containing 2 g/L mannitol）；It is cultivated under the conditions of photoautotrophy.The final rock algae of each experimental group is detected respectively Yellow matter content and EPA content.Pass through residual condition of the liquid chromatographic detection mannitol after cultivating 2 days；Pass through high-efficient liquid phase color Residual condition of the spectrum detection tea polyphenols after cultivating 2 days.Every liter of content of other medium components is as follows：
1 g NaNO3, 30 g seawater salt, 4.37 mg Na2EDTA, 3.65 mg FeCl3•6H2O, 6.7 mg NaH2PO4• 12H2O, 30 mg Na2SiO3•7H2O, 0.01 mg CuSO4•5H2O, 0.022 mg MgSO4•7H2O, 0.01 mg CoCl2• 6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg Na2MoO4•2H2O, 0.0005 mg biotins（Biotin）；
After testing, by adding mannitol, the fucoxanthine and EPA content of experimental group all increase, and further add In the case of active additive tea polyphenols, fucoxanthine（Fig. 3）And EPA（Fig. 4）Content can further increase, therefore, The program can be effectively promoted the simultaneous foster efficiency of Phaeodactylum tricornutum, reach and improve fucoxanthine and polyunsaturated fatty acid yield Purpose.
After testing, mannitol has different consumption levels two days later in culture, and there are the experimental groups of tea polyphenols to have more Fast mannitol service efficiency illustrates that tea polyphenols can promote the utilization of mannitol, achievees the purpose that promote growth（Fig. 5）.
After testing, tea polyphenols are being cultivated two days later, catechin compounds（It include mainly catechin（EC）, nutgall Theine（EGC）, catechin and gallate（ECG）And nutgall catechin gallic acid ester（EGCG）Equal substances）It gradually reduces And consumption（Fig. 6）, illustrate that the main active of tea polyphenols takes part in the physiological activity of cell, or exist in incubation Degradation process.According to this as a result, it is desirable to which semicontinuous supplement tea polyphenols are cultivated in incubation, most grown soon with reaching Purpose.
Embodiment 7
Method as described in Example 2, the difference is that prepare simultaneously carbon source be 2 g/L glycerine experimental group, with it is photosynthetic from It is control to support culture group.
The results show that using glycerine as carbon source, also there is preferable and foster effect in the case where adding tea polyphenols, together Photoautotrophy culture is compared, the case where having faster growth rate, but be slightly less than using mannitol as carbon source（Fig. 7 A）.Explanation In the case where adding tea polyphenols, the simultaneous foster efficiency of Phaeodactylum tricornutum, a variety of organic carbon sources can be promoted to can be used for phaeodactylum tricornutum and refer to The mixotrophic cultivation of algae.While with higher growth rate, the fucoxanthine content of mixotrophic cultivation group also significantly improves（Figure 7B）.
Embodiment 8
Method as described in Example 1, the difference is that in incubation semi-continuous addition organic carbon source, tea polyphenols and It is inorganic nitrogen-sourced to initial incubation concentration（Organic carbon source：2 g/L；Tea polyphenols：0.2 g/L；NaNO3：1 g/L）.
The results show that semicontinuous added with raising growth rate is conducive to, it is appropriate to be carried out in production in combination with production cost Semicontinuous addition, to reach best production efficiency.
Embodiment 9
Method as described in Example 2, the difference is that, tea polyphenols are replaced with into nutgall catechin gallic acid ester （EGCG）, a concentration of 0.2 g/L, for comparing effect of the main compound of tea polyphenols and tea polyphenols in incubation.
The results show that only addition EGCG can improve and support efficiency to a certain extent, it can be as the compounding of mixotrophic cultivation Active additive.But Synthetical cultivation cost and efficiency, addition tea polyphenols are optimal selection as built reactive additive.
The above is only the Optimizing Suggestions in presently preferred embodiments of the present invention and implementation process, not to this hair The bright limitation done in any form, therefore all contents without departing from technical solution of the present invention, according to the technical essence of the invention to Any simple modification, equivalent change and modification that upper embodiment is done, in the range of still falling within technical solution of the present invention.
Bibliography：
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[2] Maeda H, Hosokawa M, Sashima T, et al. Fucoxanthin from edible seaweed, Undaria pinnatifida , shows antiobesity effect through UCP1 expression in white adipose tissues [J]. Biochemical & Biophysical Research Communications, 2005, 332(2):392-7.
[3]Ravi K S, Narayan B, Vallikannan B. Fucoxanthin restrains oxidative stress induced by retinol deficiency through modulation of Na(+)K(+)-ATPase [corrected] and antioxidant enzyme activities in rats [J]. European Journal of Nutrition, 2008, 48(3):432-41.
[4] Milan C, Sakayu S. Biosynthesis and regulation of microbial polyunsaturated fatty acid production, Journal of Bioscience and Bioengineering[J], 1999, 87(1), 1-14.
[5]Puri M, Thyagarajan T, Gupta A, et al. Omega-3 Fatty Acids Produced from Microalgae[M] Hb25_Springer Handbook of Marine Biotechnology. Springer Berlin Heidelberg, 2015: 1043-1057.
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Claims (10)

1. application of the mannitol in mixotrophic cultivation diatom production polyunsaturated fatty acid and/or fucoxanthine.
2. application as described in claim 1, which is characterized in that the mannitol is configured to culture medium as carbon source, and supports training Support diatom production polyunsaturated fatty acid and fucoxanthine；It is further preferred that the additive amount of the mannitol is every liter of culture Base adds 1~20 g；Optimal, the additive amount of the mannitol is that every liter of culture medium adds 2 g.
3. application as described in claim 1, which is characterized in that the culture medium, every liter of component are as follows：
1~20 g mannitol, 0.1~10 g NaNO3, 10~40 g seawater salt, 2~10 mg Na2EDTA, 1~9 mg FeCl3•6H2O, 2~12 mg NaH2PO4•12H2O, 10~60 mg Na2SiO3•7H2O, 0.001~0.1 mg CuSO4• 5H2O, 0.002~0.2 mg MgSO4•7H2O, 0.001~0.1 mg CoCl2•6H2O, 0.02~2 mg MnCl2•4H2O, 0.001~0.01 mg Na2MoO4•2H2O, 0.0001~0.01 mg biotins；More preferably, the culture medium, every liter of component is such as Under：
2 g mannitol, 1 g NaNO3, 30 g seawater salt, 4.37 mg Na2EDTA, 3.65 mg FeCl3•6H2O, 6.7 mg NaH2PO4•12H2O, 30 mg Na2SiO3•7H2O, 0.01 mg CuSO4•5H2O, 0.022 mg MgSO4•7H2O, 0.01 mg CoCl2•6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg Na2MoO4•2H2O, 0.0005 mg biotins.
4. application as described in claim 1, which is characterized in that the culture medium further include tea polyphenols or tea polyphenols activity at Point, 0.05~2 g/L of concentration；It is further preferred that tea polyphenols or tea polyphenols activity component concentration are 0.2 g/L；
According to currently preferred, the diatom is selected from Cyclotella（Cyclotella）, Nitzschia（Nitzschia）, boat Shape Trentepohlia（Navicula）, barrel mast Trentepohlia（Cylindrotheca）Or brown algae category（Phaeodactylum）；Further preferably , the brown algae category（Phaeodactylum）For Phaeodactylum tricornutum, Cyclotella（Cyclotella）For small ring algae.
5. application as described in claim 1, which is characterized in that steps are as follows：
（1）Diatom is cultivated to exponential phase, seed liquor is made；
（2）By step（1）Seed liquor obtained is seeded to by 5~50% volume ratio in above-mentioned culture medium, in temperature 20~30 DEG C, 50~300 μm of ol photonsm−2·s−1It is cultivated under illumination condition 3~5 days, then maintains mannitol in cultivating system A concentration of 1~20g/L continues culture 4~8 days, and the zymotic fluid containing polyunsaturated fatty acid and fucoxanthine is made；
It is further preferred that the step（2）In, condition of culture is：80 μm of 23~25 DEG C of temperature, illumination condition ol photons·m−2·s−1；More preferably, the photoperiod of illumination condition is 16h illumination, 8h dark；It is further preferred that the step Suddenly（2）In, maintenance mannitol concentration is 2 g/L；It is further preferred that the step（2）In, maintain NaNO3A concentration of 0.1~ 10 g/L；More preferably, the step（2）In, maintain NaNO3A concentration of 1 g/L.
6. tea polyphenols or tea polyphenols active constituent are in mixotrophic cultivation diatom production polyunsaturated fatty acid and/or fucoxanthine Using.
7. application as claimed in claim 6, which is characterized in that catechin compounds mass percent in the tea polyphenols Content is 65%~85%；Preferably, the tea polyphenols active constituent be selected from catechin, flavones and flavonols, anthocyan, Phenolic acid and depside and/or polymerization phenolic compound；It is further preferred that the catechin compounds are selected from：Catechin, Nutgall catechin, catechin and gallate and/or nutgall catechin gallic acid ester；
Preferably, above-mentioned tea polyphenols or tea polyphenols active constituent are added into culture medium, mixotrophic cultivation three as bioactive substance Angle brown algae production polyunsaturated fatty acid and fucoxanthine；It is further preferred that the tea polyphenols are to contain catechin It closes object and accounts for the 65%~85% of tea polyphenols gross mass；It is further preferred that the additive amount of the tea polyphenols or tea polyphenols active constituent 0.05~2 g is added for every liter of culture medium；Optimal, the additive amount of the tea polyphenols or tea polyphenols active constituent is every liter of culture Base adds 0.2 g.
8. the use as claimed in claim 7, which is characterized in that the culture medium, every liter of component are as follows：
1~20 g carbon sources, 0.1~10 g NaNO3, 10~40 g seawater salt, 2~10 mg Na2EDTA, 1~9 mg FeCl3• 6H2O, 2~12 mg NaH2PO4•12H2O, 10~60 mg Na2SiO3•7H2O, 0.001~0.1 mg CuSO4•5H2O, 0.002~0.2 mg MgSO4•7H2O, 0.001~0.1 mg CoCl2•6H2O, 0.02~2 mg MnCl2•4H2O, 0.001~ 0.01 mg Na2MoO4•2H2O, 0.0001~0.01 mg biotins, 0.05~2 g of tea polyphenols or tea polyphenols active constituent；More Excellent, the culture medium, every liter of component is as follows：
2 g carbon sources, 1 g NaNO3, 30 g seawater salt, 4.37 mg Na2EDTA, 3.65 mg FeCl3•6H2O, 6.7 mg NaH2PO4•12H2O, 30 mg Na2SiO3•7H2O, 0.01 mg CuSO4•5H2O, 0.022 mg MgSO4•7H2O, 0.01 mg CoCl2•6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg Na2MoO4•2H2O, 0.0005 mg biotins, tea polyphenols or tea 0.2 g of polyphenol active constituent；
It is further preferred that carbon source is selected from the culture medium：It is mannitol, glycerine, glucose, fructose, sucrose, sorbierite, sweet Propylhomoserin and/or acetic acid；It is further preferred that carbon source is selected from the culture medium：Mannitol and/or glycerine；Optimal, the training It is mannitol to support carbon source in base.
9. application as claimed in claim 6, which is characterized in that the diatom is selected from Cyclotella（Cyclotella）, diamond shape Trentepohlia（Nitzschia）, Navicula（Navicula）, barrel mast Trentepohlia（Cylindrotheca）Or brown algae category （Phaeodactylum）；It is further preferred that the brown algae category（Phaeodactylum）For Phaeodactylum tricornutum, Cyclotella （Cyclotella）For small ring algae.
10. application as claimed in claim 6, which is characterized in that steps are as follows：
（1）Diatom is cultivated to exponential phase, seed liquor is made；
（2）By step（1）Seed liquor obtained is seeded to by 5~50% volume ratio in above-mentioned culture medium, in temperature 20~30 DEG C, 50~300 μm of ol photonsm−2·s−1It is cultivated under illumination condition 3~5 days, then maintains carbon source in cultivating system dense Degree is 1~20 g/L, continues culture 4~8 days, and the zymotic fluid containing polyunsaturated fatty acid and fucoxanthine is made；
It is further preferred that the step（2）In, condition of culture is：80 μm of 23~25 DEG C of temperature, illumination condition ol photons·m−2·s−1；More preferably, the photoperiod of illumination condition is 16 h illumination, 8 h dark；It is further preferred that described Step（2）In, maintenance carbon source concentration is 2 g/L；It is further preferred that the step（2）In, maintain NaNO3A concentration of 0.1~ 10 g/L；More preferably, the step（2）In, maintain NaNO3A concentration of 1 g/L.
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