CN104531602B - A kind of method that Phaeodactylum tricornutum fucoxanthin content is improved with Cericammoniumsulfate - Google Patents
A kind of method that Phaeodactylum tricornutum fucoxanthin content is improved with Cericammoniumsulfate Download PDFInfo
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- CN104531602B CN104531602B CN201410743644.XA CN201410743644A CN104531602B CN 104531602 B CN104531602 B CN 104531602B CN 201410743644 A CN201410743644 A CN 201410743644A CN 104531602 B CN104531602 B CN 104531602B
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Abstract
The invention discloses a kind of method that Phaeodactylum tricornutum fucoxanthin content is improved with Cericammoniumsulfate, take sterilizing seawater to be mixed with MAD mother liquors and obtain nutrient solution, Phaeodactylum tricornutum mother liquor culture is added in nutrient solution, after culture 2 days, Cericammoniumsulfate is added, algae solution is collected after continuing culture 6 days, is centrifuged, abandon supernatant, frond is collected, frond freeze-drying process 2 days, grinding is standby, grinding gained frond is dissolved with absolute ethyl alcohol, stand 1h in 4 DEG C of environment, 4 DEG C, 13000rpm, 10min is centrifuged, obtains supernatant.The invention is simple to operate, and expense is low, with short production cycle, suitable for batch processing and factorial praluction.
Description
Technical field
The invention belongs to biomedicine technical field, and Phaeodactylum tricornutum rock algae is improved with Cericammoniumsulfate more particularly, to one kind
The method of flavine content.
Background technology
Fucoxanthin (Fucoxanthin) is the carotenoid of a kind of crocus, and it is primarily present in edible brown alga
In.Its chemical constitution is special polyenoid chain (conjugated double bond, epoxide group and the conjugation carbonyl with inoxidizability).Exactly
Because it has this structure, therefore fucoxanthin is also considered as a kind of lutein.Treatment of the fucoxanthin in human diseases
In there is extremely important effect, in the diseases such as anti-obesity, anticancer, the research to its mechanism of action there has also been certain base
Plinth.
Phaeodactylum tricornutum (phaeodactylum tricornutum) can synthesize the grease of high level, institute due to it
To be constantly subjected to pay close attention to and study.Research finds fucoxanthin in Phaeodactylum tricornutum be present.Sang Min Kim et al et al.
Demonstrated by the method for mass spectrum and nuclear magnetic resonance and fucoxanthin really in Phaeodactylum tricornutum be present and by trans efficient liquid
Mutually quantified.Elicitor can be to purpose secondary metabolite regulation and control and biosynthesis play the role of it is important, in certain journey
The metabolite content of culture can be effectively improved on degree.Cericammoniumsulfate just belongs to the category of elicitor.At present, utilization is various
Elicitor is not reported also to improve the pertinent literature of fucoxanthin content.Handled herein by using the Cericammoniumsulfate of various concentrations
Phaeodactylum tricornutum, to study the upgrowth situation of Phaeodactylum tricornutum, separation and fucoxanthin and profit in extraction Phaeodactylum tricornutum
Measure with HPLC to fucoxanthin content after the Cericammoniumsulfate processing Phaeodactylum tricornutum of various concentrations.And then draw most suitable sulfuric acid
The concentration of cerium ammonium.Strong experimental basis and means are provided to improve fucoxanthin content.
The content of the invention
Present invention aims at a kind of extracting method of Phaeodactylum tricornutum fucoxanthin is provided, this method is to improving triangle
Fucoxanthin content significant effect in brown algae, simple to operate, expense is low, with short production cycle, suitable for batch processing and
Factorial praluction.
The present invention realizes especially by following technical scheme：
A kind of method that Phaeodactylum tricornutum fucoxanthin content is improved with Cericammoniumsulfate, specifically includes following steps：
1) take sterilizing seawater to be mixed with MAD mother liquors and obtain nutrient solution；
2) Phaeodactylum tricornutum mother liquor culture is added in nutrient solution；
3) after cultivating 2 days, Cericammoniumsulfate is added；
4) algae solution is collected after continuing culture 6 days, is centrifuged, abandons supernatant, collects frond；
5) it is frond freeze-drying process 2 days, grinding is standby.
Often 100ml is the formula of described MAD mother liquors of the present invention：KNO3：10g, KH2PO4：1g, FeSO4·
7H2O：0.25g, MnSO4：0.025g, EDTANa2：1g, VB1：0.0006g, deionized water：100ml.
Condition of culture of the present invention is：5000 luxs, 25 DEG C.
The volume ratio of described sterilizing seawater and MAD mother liquors is 1000: 1.
The volume ratio of described nutrient solution and Phaeodactylum tricornutum mother liquor is 10: 1.
The concentration of Cericammoniumsulfate is 0mg/L~0.2mg/L after nutrient solution addition Cericammoniumsulfate in step (3).
Drying process condition described in step (5) is：- 70 DEG C of temperature, pressure 999Pa.
The advantage of the invention is that：Invention makes public for the first time extracts fucoxanthin from Phaeodactylum tricornutum, to improve rock algae
Flavine content also using the Cericammoniumsulfate processing Phaeodactylum tricornutum of various concentrations, finds most suitable Cericammoniumsulfate concentration for the treatment of,
Handled by induction of the concentration to Phaeodactylum tricornutum, the content increase of its fucoxanthin is notable.The invention is simple to operate,
Expense is low, with short production cycle, suitable for batch processing and factorial praluction.Because fucoxanthin is to anticancer, hyperglycaemia, cardiovascular disease
Disease, diabetes have significantly effect, improve the needs that the content of fucoxanthin will meet people and cure the disease, and can improve warp
Ji benefit.
Brief description of the drawings
Fig. 1 is growth curve after various concentrations of embodiment of the present invention Cericammoniumsulfate processing Phaeodactylum tricornutum；
Fig. 2 is growth of Phaeodactylum tricornutum curve of the embodiment of the present invention；
Fig. 3 is fucoxanthin changes of contents after various concentrations of embodiment of the present invention Cericammoniumsulfate processing Phaeodactylum tricornutum.
Embodiment
With reference to embodiment, the present invention is described further, as described below, is only the preferable implementation to the present invention
Example, is not limited the present invention, any person skilled in the art is possibly also with the disclosure above
Technology contents be changed to the equivalent embodiment changed on an equal basis.It is every of the invention without departing from the present invention program content, foundation
Technical spirit any simple modification that following examples are made or equivalent variations, all fall within protection scope of the present invention.
First, experimental method
1st, the culture of Phaeodactylum tricornutum
Phaeodactylum tricornutum stoste is provided by bioengineering key lab of oceanography institute of University Of Ningbo teacher Xie Zhihao.Took
Filter and the seawater of high-temperature sterilization, add 1: 1000 sterilizing MAD mother liquors.Often 100ml is the formula of MAD mother liquors：KNO3：10g,
KH2PO4：1g, FeSO4·7H2O：0.25g, MnSO4：0.025g, EDTANa2：1g, VB1：0.0006g, deionized water：100ml.
Algae solution is accessed with 1: 10 ratio, is positioned over 5000lx, temperature is 25 DEG C, in the incubator of 12h/12h dark/illumination.Daily
Divide three periods (8：00,12：00,20：00) frond is uniformly shaken.
2nd, the measure of growth of Phaeodactylum tricornutum curve
300ml 3 groups of sterilizing MAD nutrient solutions are taken respectively.The algae solution that 30ml is poured into parallel to every group, is put into illumination cultivation
Cultivated in case.Shake algae daily to be cultivated three times, and (frond is surveyed every time using the OD values of spectrophotometric machine measure frond
Algae solution should be shaken up before OD values).
Frond number is measured with OD680 by the method and counting method of blood cell that dilute in equal volume.Take 1ml algae
Liquid, it is diluted respectively, is diluted to 10000 times always, and to each group of diluent determining OD680 value.
3rd, Phaeodactylum tricornutum is handled using Cericammoniumsulfate
MAD nutrient solutions are added into sterilizing seawater, Phaeodactylum tricornutum stoste are added with 1: 10 ratio, by triangle algae solution
It is put in illumination box and cultivates (condition of culture is same as above) 2 days, allows Phaeodactylum tricornutum to adapt to new nutrient environment.Toward often
The Cericammoniumsulfate of various concentrations is added in group experimental group.Since that day of Phaeodactylum tricornutum access culture medium, profit is needed daily
Determine the OD values of every group of algae solution with spectrophotometric machine, then METHOD FOR CONTINUOUS DETERMINATION 6 days.
This experiment fucoxanthin standard items used are derived from bioengineering key lab of oceanography institute of University Of Ningbo Zhang Jin
Flourish teacher (standard items are prepared for oneself, purity 80%).
The measure of the standard curve of standard items：0.1mg fucoxanthin standard items are weighed in 400 μ l absolute ethyl alcohol,
Solution is resuspended up and down using liquid-transfering gun so that fucoxanthin is completely dissolved in chromatogram methanol.Standard items is dilute in this experiment
Multiple is released to be 5 times, 10 times, 20 times, 50 times, 100 times and be dissolved in 400 μ l fucoxanthin standard items.
This experiment 250 × 4.6mml.D.S-5 μm of pillar model used, 12nmAA12S05-2546WT
No.0425057734.HPLC types used are Waters 2695Separations Module.
This experiment condition is：90% methanol 30min, 100% methanol 10min, the lasting 15min of 90% methanol.200-
700nm wavelength.It is monitored at 445nm.
2nd, Application Example
Influence of the Cericammoniumsulfate of embodiment 1 to fucoxanthin in Phaeodactylum tricornutum
300ml 18 groups of sterilizing MAD nutrient solutions, the ratio access Phaeodactylum tricornutum frond of access 1: 10 are taken respectively.Use
Concentration is respectively 0.2mg/L, 0.4mg/L, 0.8mg/L, and 1.6mg/L, 2mg/L Cericammoniumsulfate handle Phaeodactylum tricornutum and do not done
Parallel 3 groups of any processing.Since that day of Phaeodactylum tricornutum access culture medium, need to survey using spectrophotometer daily
The concentration of fixed every group of algae solution, then determine 6 days.
As a result as shown in figure 1, the extinction value changes of each group are not very bright after Cericammoniumsulfate (CAS) of various concentrations is added
Aobvious, comparatively light absorption value of several groups than other will be big for the light absorption value in the control group logarithm end of term, at 0.4mg/L Cericammoniumsulfate
After managing frond, OD values are minimum.Wherein the OD values of control group are that the OD values of 0.4322,0.4mg/L experimental group are 0.3371, control
Group is its 1.3 times.Illustrate after being handled by Cericammoniumsulfate, the biomass of Phaeodactylum tricornutum declines.It is from this figure it can be seen that sharp
After handling Phaeodactylum tricornutum with Cericammoniumsulfate, all in decline phase since the 5th day, its light absorption value is all in downward trend.
Embodiment 2
400ml 7 groups of sterilizing seawater is taken, is separately added into 400 μ l MAD mother liquors.Add 40ml Phaeodactylum tricornutum mother liquor
Cultivated in nutrient solution in illumination box.After culture 2 days, the sulphur of various concentrations is added in 6 groups of algae culturing liquids wherein
Sour cerium ammonium, its concentration are respectively 0mg/L, 0.2mg/L, 0.4mg/L, 0.8mg/L, 1.6mg/L, 2.0mg/L.
As shown in Fig. 2 the measure of the growth curve from Phaeodactylum tricornutum, cultivate to understanding that phaeodactylum tricornutum refers to after the 6th day
Algae has reached the logarithm end of term, can obtain the biomass of maximum.Therefore just need to collect when to culture by the 6th day
Algae solution.
Every group of Phaeodactylum tricornutum is taken into 80ml in centrifuge tube respectively, 4 DEG C, 5000rpm, 10min is centrifuged, abandons supernatant, take
1ml MAD sea water mediums, centrifugation frond is resuspended, and algae solution is moved into 1.5mlEP pipes, 4 DEG C, 5000rpm, centrifugation
10min, abandon supernatant and weigh its fresh weight.The mouth of pipe of EP pipes is sealed with sealed membrane, pricked sealed membrane with transfer needle several small
Hole, it is put in freeze drier and dries 2 days, takes out EP pipes, sealed membrane is torn off, because Phaeodactylum tricornutum belongs to Diatomeae, because
This its cell membrane is very thick, it is impossible to takes out its content.Using the Phaeodactylum tricornutum frond of grinding rod mill-drying, make its cell
Wall fully ruptures, and takes 1ml absolute ethyl alcohol in EP pipes, and is resuspended up and down with liquid-transfering gun so that fucoxanthin fully dissolves
In absolute ethyl alcohol.1h is stood in 4 DEG C of environment, 4 DEG C, 13000rpm, centrifuges 10min.Aspirate supernatant is in new 1.5ml EP
Guan Zhong.
Using HPLC (high performance liquid chromatography) fucoxanthin is monitored under 445nm absorbing wavelength.The sulfuric acid of various concentrations
It is 0mg/L~0.2mg/L in concentration range as a result as shown in Figure 3 and Table 1 by calculating after cerium ammonium processing Phaeodactylum tricornutum
When, incremental trend, the rock during 0.2mg/L-2mg/L is presented in the fucoxanthin content obtained by every gram of Phaeodactylum tricornutum fresh weight
The trend successively decreased is presented in phycoxanthine content.As can be seen from the figure the institute after 0.2mg/L Cericammoniumsulfate processing Phaeodactylum tricornutum
The fucoxanthin content obtained is most, is 0.398mg/g fresh weights, Cericammoniumsulfate concentration is 2mg/L, and the fucoxanthin content of gained is most
It is low, it is 0.158mg/g fresh weights.The content of untreated its fucoxanthin of Phaeodactylum tricornutum is 0.249mg/g fresh weights.
Influence of the various concentrations Cericammoniumsulfate of table 1 processing to Phaeodactylum tricornutum fucoxanthin content
| Cericammoniumsulfate concentration (mg/L) | 0 | 0.2 | 0.4 | 0.8 | 1.6 | 2.0 |
| Fucoxanthin content (mg/g fresh weights) | 0.249 | 0.398 | 0.325 | 0.186 | 0.199 | 0.158 |
Claims (1)
- A kind of 1. method that Phaeodactylum tricornutum fucoxanthin content is improved with Cericammoniumsulfate, it is characterised in that comprise the following steps：1) take sterilizing seawater to be mixed with MAD mother liquors and obtain nutrient solution；Often 100ml is the formula of the MAD mother liquors：KNO3：10g, KH2PO4：1g, FeSO4·7H2O：0.25g, MnSO4：0.025g, EDTANa2：1g, VB1：0.0006g, deionized water：100ml； And the volume ratio of the sterilizing seawater and the MAD mother liquors is 1000: 1；2) Phaeodactylum tricornutum mother liquor culture is added in nutrient solution；Condition of culture is：5000 luxs, 25 DEG C；And the battalion Nutrient solution and the volume ratio of the Phaeodactylum tricornutum mother liquor are 10: 1；3) after cultivating 2 days, Cericammoniumsulfate is added；And the concentration of Cericammoniumsulfate is 0.2mg/L；4) algae solution is collected after continuing culture 6 days, is centrifuged, abandons supernatant, collects frond；5) it is frond freeze-drying process 2 days, grinding is standby；The drying process condition is：- 70 DEG C of temperature, pressure 999Pa.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010233517A (en) * | 2009-03-31 | 2010-10-21 | Teijin Ltd | Cultured microalgae containing fucoxanthin and method for producing the same |
| CN103833692A (en) * | 2014-02-21 | 2014-06-04 | 宁波大学 | Method for extracting high-purity fucoxanthin from seaweeds |
| CN103965143A (en) * | 2013-01-30 | 2014-08-06 | 中国科学院海洋研究所 | Method for extracting and separating fucoxanthin from marine unicellular diatom |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010233517A (en) * | 2009-03-31 | 2010-10-21 | Teijin Ltd | Cultured microalgae containing fucoxanthin and method for producing the same |
| CN103965143A (en) * | 2013-01-30 | 2014-08-06 | 中国科学院海洋研究所 | Method for extracting and separating fucoxanthin from marine unicellular diatom |
| CN103833692A (en) * | 2014-02-21 | 2014-06-04 | 宁波大学 | Method for extracting high-purity fucoxanthin from seaweeds |
Non-Patent Citations (2)
| Title |
|---|
| 几种释放因素对紫杉醇合成与释放的影响;余斐，等;《华中科技大学学报》;20010630;第29卷(第6期);第100-102页 * |
| 诱导子对雨生红球藻虾青素含量的影响及其分子机理的初步研究;王丽丽;《中国优秀硕士学位论文全文数据库 基础科学辑》;20120315;摘要，第13页第2段第1.3节至第18页第1段，第35页第3.3.3节，第45页讨论部分最后1段，图 3.11，表 3.6 * |
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