CN107119099A - The method for producing fucoxanthin using the smooth rhombus algae of illumination cultivation - Google Patents
The method for producing fucoxanthin using the smooth rhombus algae of illumination cultivation Download PDFInfo
- Publication number
- CN107119099A CN107119099A CN201710525234.1A CN201710525234A CN107119099A CN 107119099 A CN107119099 A CN 107119099A CN 201710525234 A CN201710525234 A CN 201710525234A CN 107119099 A CN107119099 A CN 107119099A
- Authority
- CN
- China
- Prior art keywords
- fucoxanthin
- algae
- smooth
- rhombus algae
- illumination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the culture of microorganism, the method that a kind of smooth rhombus algae of utilization illumination cultivation produces fucoxanthin is particularly related to.Extract the step such as fucoxanthin in zymotic fluid after preparation, fermented and cultured, fermentation ends including seed liquor, the present invention solves present in prior art the biomass concentration of smooth rhombus algae, fucoxanthin yield, the low problem of fucoxanthin content under phototrophic conditions.The content for drying fucoxanthin in algae powder with prepared smooth rhombus algae is high, the yield of fucoxanthin is high, the fucoxanthin in relatively large marine alga source is safer, while production efficiency, reduction production cost is improved, the advantages of greatly reducing the pollution risk in incubation.
Description
Technical field
The invention belongs to the culture of microorganism, one kind is particularly related to using the smooth rhombus algae production fucoxanthin of illumination cultivation
Method.
Background technology
Fucoxanthin (Fucoxanthin), also known as fucoxanthine, pheophytin are big essentially from big algae, diatom and chrysophyceae etc.
Type marine alga and microalgae, are a kind of natural carotenoid, participate in the reaction of Photosystem I I in photosynthesis.Because it has uniqueness
Allene structure, fucoxanthin is a kind of bioactive molecule with strong anti-oxidation.Recent study finds that fucoxanthin exists
Cell, animal and human body are all confirmed with a variety of functional activities, including anti-oxidant, anti-inflammatory, anticancer, anti-fat, anti-glycosuria
The physiologically actives such as disease, anti-angiogenesis and antimalarial, and there is protection to organs such as liver, cerebral vessels, bone, skin and eyes
Effect.In summary function, fucoxanthin is a kind of natural products with wide health food and drug development prospect, rock algae
500 tons of the market capacity of flavine, the fucoxanthin medicinal extract price of 10% content is up to 40000 yuan/kilogram, therefore with huge
Market value.
Remarkable effect in effect of fucoxanthin in terms of fat-reducing receives more and more attention it, and it can pass through suppression
The generation of fat cell processed and acceleration two approach of catabolism of fat reach the effect of fat-reducing.Fucoxanthin master in the market
If being obtained by being extracted in the tangleweeds such as undaria pinnitafida (Undariapinnatifida), sea-tangle (Laminaria japonica)
.But the above method has tangleweed in cell wall thickness, high polysaccharose substance content, purification difficult and marine pollution etc.
Fucoxanthin content is extremely low in problem, and tangleweed (being only the 0.01%-0.07% of dry weight), in summary, fucoxanthin
Product quality be difficult to ensure that to a certain extent, and downstream separation purifying difficulty increase, because it is to extraction separation and purification
There are higher technical requirements, so as to cause high-purity fucoxanthin expensive, further limit the application of fucoxanthin.
For relatively large-scale marine alga, marine microalgae is that fucoxanthin preferably substitutes source.Tangleweed in the market
The content of the concentration fucoxanthin of middle extraction is generally less than 1%, and fucoxanthin content is to be up in some marine microalgae cells
0.6%, it is nearly 100 times of tangleweed.Diatom is widely distributed marine microalgae, on the surface of ocean, fresh water and humidity
It is widely present, generally relies on luminous energy and synthesize nutrient needed for itself.Smooth rhombus algae (Nitzchia laevis) is a kind of single
Cell algae, belongs to Bacillariophyta (Bacillariophyta).
The research for producing fucoxanthin using smooth rhombus algae is less.Applicant is by retrieving discovery, Application No.
A kind of cultural method for improving fucoxanthin yield in diatom, the patent mistake are disclosed in 201310329269.X patent document
Optimal Medium, addition tomato extract promotes to have synthesized fucoxanthin to a certain extent, and the later stage is final using photo-irradiation treatment
So that small ring algae reaches 7.77mg/ (Ld) fucoxanthin yield, wherein proposing that smooth rhombus algae gives birth to available for illumination cultivation
Fucoxanthin is produced, but the document only discloses the method, step and process conditions that fucoxanthin is prepared using small ring algae.Not
The production of fucoxanthin can be carried out using other strains by announcing, while wherein also not related condition of culture, intensity of illumination, training
Support the series of key techniques information such as cultivation results and fucoxanthin content and yield such as base composition, biomass concentration.Guo and
Designer in the application is by screening, and acquisition can synthesize several plants of diatoms of fucoxanthin, find small ring algae Cyclotella
Cryptica CCMP 333 fucoxanthin content, up to 0.77%, is most suitable fucoxanthin production algae in more than ten strain microalgaes
Strain, but its biomass concentration and fucoxanthin yield are relatively low, and industrial applications are still difficult.Under condition of culture wherein used, put down
Sliding rhombus algae Nitzschia laevis UTEX 2047 produce fucoxanthin under illumination autotrophic condition, and its content (accounts for cell
Dry weight) it is up to 0.7%, but biomass concentration<0.2g/L, yield is only 0.06mg/ (Ld), and it is not entered in this paper
The optimization (Guo, et al., 2016) of row condition of culture.It is therefore desirable to pass through the skills such as Optimal Medium composition and condition of culture
Art means, improve the biomass concentration and fucoxanthin yield of smooth rhombus algae under phototrophic conditions, while further improving rock algae
Flavine content.
The content of the invention
It is an object of the invention to provide the method that a kind of smooth rhombus algae of utilization illumination cultivation produces fucoxanthin, by excellent
Changing condition of culture can realize that high yield prepares fucoxanthin.
The overall technology of the present invention is conceived：
1st, the method for producing fucoxanthin using the smooth rhombus algae of illumination cultivation, it is characterised in that comprise the following steps：
A, seed liquor preparation
The smooth rhombus algae activated is placed in Heterotrophic culture in aseptic seed culture medium and seed liquor is made within 3-9 days, makes to put down
Sliding rhombus frustule is in exponential phase；
B, fermented and cultured
Seed liquor in step A is transferred in no bacteria fermentation culture medium according to volume ratio for 3%-20% inoculum concentration,
Culture of being ventilated under illumination condition prepares zymotic fluid, and culture medium loading amount is 20%-80%, 20 DEG C -30 DEG C of cultivation temperature, culture week
4-12 days phase；
Described seed culture medium and fermentation medium include the raw material of following content range：
NaCl 10g/L-32g/L；Na2SiO3·9H2O 30mg/L-700mg/L；MgSO4·7H2O 1.09g/L-
2.18g/L；CaCl2·2H2O 0.1g/L-0.27g/L；KH2PO40.031g/L-0.062g/L；K2HPO4 0.00375g/L-
0.0075g/L；FeCl3·6H2O 0.291mg/L-0.582mg/L；MnCl2·4H2O 0.025mg/L-0.246mg/L；ZnCl2
0.031mg/L-0.311mg/L；CoCl2·6H2O 0.0114mg/L-0.0228mg/L；Na2MoO4·2H2O 0.012mg/L-
0.024mg/L；H3BO33.06g/L-30.56g/L；(NH4)6MO7O24·4H2O 0.028mg/L-0.278mg/L；Tris-
buffer 0.089g/L-0.892g/L；H2SO41.64μg/L-16.4μg/L；vitamin B12 1.5g/L-15×10-5g/
L；biotin 2.5g/L-25×10-5g/L；Nitrogen source 0.2g/L-7g/L；PH=6-9, the nitrogen source is from organic nitrogen source, inorganic
Nitrogen source or its combination, wherein organic nitrogen source includes but is not limited to：Yeast extract, peptone, yeast extract, amino acid, urea, egg
White hydrolysate or its combination, it is inorganic nitrogen-sourced to include but is not limited to from potassium nitrate, sodium nitrate, ammonium chloride, ammonium hydrogen carbonate or its group
Close；
Described intensity of illumination is no more than 200 μm olm-2·s-1；
Described smooth rhombus algae is selected from smooth rhombus algae (Nitzschia laevis) UTEX 2047 and (is purchased from U.S.'s moral
Ke Sasi universities Austin microalgae preservation storehouse, Culture Collection of Algae at The University
Of Texas at Austin, abbreviation UTEX), smooth rhombus algae (Nitzschia laevis) CCMP559 (be purchased from U.S. ocean
Microalgae and Organism Depositary, National Center for Marine Algae and Microbiota, referred to as
NCMA) or smoothly rhombus algae (Nitzschia laevis) CCMP 1092 (is purchased from U.S.'s marine microalgae and microbial preservation
The heart, National Center for Marine Algae and Microbiota, abbreviation NCMA), wherein it is preferred that smooth rhombus
Algae (Nitzschia laevis) UTEX 2047.
C, from step B fermentation terminate after zymotic fluid in extract fucoxanthin.
Belong to because separating smooth rhombus frustule from zymotic fluid and fucoxanthin being extracted from smooth rhombus frustule
Prior art, applicant will not be repeated here.
The concrete technical scheme of each step is as follows in the present invention：
For ease of the progress of industrial fermentation processes, described culture medium is placed in bioreactor, and bioreactor is selected
Triangular flask or pillar bioreactor.
Illumination light source therein can take various forms, and all without departing from the essence of the present invention, technical scheme preferably is,
Light source used in illumination includes but is not limited to sunshine, fluorescent lamp light, LED light or its combination.
The condition of culture of illumination cultivation is that concussion and cultivate in illumination shaking table, shaking speed are placed on during using triangular flask
For 100 revs/min -240 revs/min；The nothing that carbon dioxide volume content is less than 5% is passed through during using pillar bioreactor
Bacterium air jet flow, throughput is 3 liters/min.
To improve the yield and content of fucoxanthin, culture bar preferably is that the nitrogen source in fermentation medium is from as follows
The raw material of content：NaNO31g/L；Peptone 1g/L, intensity of illumination is 30 μm of olm-2·s-1。
The condition of culture being more highly preferred to is also to include following content range in described seed culture medium and fermentation medium
Raw material：Glucose 5-40g/L.
Further optimal technical scheme is, is 5-200 μm of ol in intensity of illumination from pillar bioreactor
m-2·s-1Under conditions of ventilate culture.
The detection method being related in the present invention is as follows：
1st, the measure of smooth rhombus frustule dry weight
3mL zymotic fluids were taken every 24 hours after inoculation, are centrifuged 5 minutes under conditions of rotating speed is 3000 rev/min,
ddH2Centrifuge, be repeated 2 times again after O washings；Zymotic fluid is filtered to preweighted filter paper, is put into 80 DEG C of vacuum drying chambers and dries
Do to constant weight.
2nd, the detection of fucoxanthin
Current applicant does not have found country or the company standard of fucoxanthin detection, mainly passes through UV, visible light light absorption method
(UV methods) and high performance liquid chromatograph (HPLC) are detected, because of UV method poor specificities, are easily disturbed by other pigments, therefore HPLC methods
It is the presently preferred detection means of fucoxanthin.The application reference Guo et al. research, and be improved on its basis, have
Body is as follows：
Weigh and 5mL absolute ethyl alcohols concussion extraction 10 minutes is added after the algae powder after 20mg is freezed, cryogrinding, centrifuge (bar
Part is 4 DEG C of temperature, 3000 revs/min of rotating speed, 5 minutes time) supernatant is collected, the concussion of 3mL absolute ethyl alcohols is rejoined in precipitation
Extract, until algae powder is white.Extract solution is collected, 10 points are centrifuged under conditions of 4 DEG C of temperature, rotating speed is 12000 revs/min
Clock, takes supernatant nitrogen to dry up, and adds 1mL absolute ethyl alcohols dissolving pigment, crosses high performance liquid chromatograph (HPLC) after film and analyzes, whole
Carried out under the conditions of individual process lucifuge.
3rd, HPLC analysis methods
High performance liquid chromatograph waters2695, configures PDA detectors, Detection wavelength 450nm, from C18 reversed-phase columns
(250mm×4.6mm×5mm).Mobile phase is：A phases are pure ethyl acetate, and B phases are acetonitrile：Methanol：Water=84:2:14, C phases are
Pure methanol, using gradient elution, mobile phase uses HPLC grades.
Condition of gradient elution is as follows：
| Time (min) | A (%) | B (%) | C (%) | Flow velocity (mL/min) |
| 0 | 0 | 100 | 0 | 0.8 |
| 15 | 32 | 0 | 68 | 0.8 |
| 30 | 32 | 0 | 68 | 0.8 |
| 35 | 0 | 100 | 0 | 0.8 |
The substantive distinguishing features and the notable technological progress of acquirement that the present invention possesses are：
1st, present invention firstly provides fucoxanthin is produced by scale fermentation process using smooth rhombus algae, through applicant
It is experimentally confirmed that prepared smooth rhombus algae dry fucoxanthin in algae powder content it is high (at least up to 0.7-1.0%, highest
Up to 1.38%, improved 97.1%) compared with prior art, the yield height of fucoxanthin is (at least up to 1.02mg/ (Ld), highest
Up to 9.88mg/ (Ld), 163.7 times are improved compared with prior art).
2nd, the method that the present invention is provided has the potentiality applied to industrialized production fucoxanthin.One is optimum culture condition
The fucoxanthin content of smooth rhombus algae is greatly improved afterwards；Two be fucoxanthin yield be far above all diatoms for reporting at present and
Other algae.
3rd, the smooth rhombus algae powder that the present invention is obtained, can stablize in the case where not limited by external condition and realize continuous industry
, can be from the ocean common contaminants such as Sources controlling heavy metal, the Polychlorinated biphenyls of culture medium, relatively large sea on the basis of metaplasia production
The fucoxanthin in algae source is safer.
4th, other relative microdisk electrodes produce fucoxanthin, and the production cycle greatly shortens, most short to terminate within 4 days fermentation,
While improving production efficiency, reduction production cost, the pollution risk in incubation is greatly reduced.
Brief description of the drawings
Fig. 1 is smooth rhombus algae in autotrophy (phototrophy sugar-free) and raises together with and (add 5g/L grapes under illumination condition in culture medium
Sugar) growth curve under the conditions of initial concentration.
It can be seen that the biomass concentration of wherein autotrophy is extremely low, biomass is only 0.5g/L after 6 days, and raises together with condition
Under biomass be more than 5 times of autotrophy.
Fig. 2 is the content of fucoxanthin of the smooth rhombus algae under autotrophy (phototrophy sugar-free) and 5g/L glucose initial concentrations
The comparison of (i.e. the mass ratio of fucoxanthin/freeze-dried algae powder) and yield.
It can be seen that wherein A represents phototrophy sugar-free, M represents phototrophy 5g/L glucose：Not only biomass is raised together with significantly to carry
Height, fucoxanthin content is significantly improved simultaneously, reaches as high as 1.2%, and autotrophy is only 1.0%；Fucoxanthin yield also above
Autotrophic condition (autotrophy is 1.02mg/ (Ld)), up to 4.59mg/ (Ld).
Fig. 3 is growth curve of the smooth rhombus algae in different illumination intensity.
It can be seen that low light intensity is more beneficial for smooth rhombus algae biomass under the conditions of raising together with, intensity of illumination is 5 μm of ol
m-2·s-1When biomass concentration highest, as light intensity is strengthened, biomass concentration is significantly reduced, when being 70 μ for intensity of illumination
mol·m-2·s-1When, biomass is about 0.7g/L.
Fig. 4 is influence of the different illumination intensity to fucoxanthin content and yield in smooth rhombus frustule.
It can be seen that while low light intensity is beneficial to the growth of smooth rhombus algae, also beneficial to the accumulation of fucoxanthin.Illumination is strong
Spend for 5 μm of olm-2·s-11When, the content and yield of fucoxanthin are highest, and maximum concentration and yield are respectively up to 1.38%
With 9.88mg/ (Ld), 97.1% and 163.7 times is respectively increased compared with prior art, while being also the mesh that applicant is recognized
Preceding existing microdisk electrode produces the maximum yield of fucoxanthin.
Embodiment
Embodiments of the invention are further described below in conjunction with accompanying drawing, but not as a limitation of the invention, this hair
Bright protection domain is defined by the content that claim is recorded, any to be replaced according to the equivalent technical elements that specification is made,
Protection scope of the present invention is not departed from.
Embodiment 1
Smooth rhombus algae autotrophy culture
Produce strain (big purchased from Texas ,Usa from smooth rhombus algae (Nitzschia laevis) UTEX 2047
Learn Austin microalgae preservation storehouse, Culture Collection of Algae at The University of Texas
At Austin, abbreviation UTEX).
Processing step is as follows：
A, by after activation produce strain smooth rhombus algae be seeded in heterotrophism in the aseptic seed culture medium being placed in shaking flask
Culture is made smooth rhombus frustule in seed liquor, seed liquor for 3 days and is in exponential phase；
B, using 250mL conical flasks as culture vessel, be transferred to fermentation medium culture medium 100mL and sterilize.It is by volume
Seed liquor access in step A is prepared zymotic fluid by 10% inoculum concentration without progress shaking table autotrophy culture in bacteria fermentation culture medium,
Condition of culture is as follows：Intensity of illumination is 30 μm of olm-2·s-1, temperature is 23 DEG C, and rotating speed is 150 revs/min, cultivation cycle 5
My god.
C, from step B fermentation terminate after zymotic fluid in extract fucoxanthin：
Zymotic fluid, centrifuge washing, freeze-drying are collected in culture after terminating.
Seed culture medium includes the raw material of following content：
NaCl 10g/L；Na2SiO3·9H2O 60mg/L；MgSO4·7H2O 2.18g/L；CaCl2·2H2O 0.27g/
L；KH2PO40.062g/L；K2HPO40.0075g/L；FeCl3·6H2O 0.582mg/L；MnCl2·4H2O 0.246mg/L；
ZnCl20.311mg/L；CoCl2·6H2O 0.0228mg/L；Na2MoO4·2H2O 0.024mg/L；H3BO330.56g/L；
(NH4)6MO7O24·4H2O 0.278mg/L；Tris-buffer 0.892g/L；H2SO416.4μg/L；vitamin B12 15
×10-5g/L；biotin 25×10-5g/L。
Fermentation medium also includes the raw material of following content on the basis of seed culture medium：NaNO31g/L, peptone
1g/L, pH=8.5.
Embodiment 2
Smooth rhombus algae raises together with culture
The difference of embodiment 2 and embodiment 1 is, is also included in the seed culture medium and fermentation medium in embodiment 2
There is the raw material of following content：Glucose 5g/L, remaining content is same as Example 1.
The effect analysis of embodiment 1,2：
Smooth rhombus algae is compared in autotrophy and under the conditions of raising together with, and is raised together with culture growth conditions preferably, is raised together with biomass highest
Up to 2.68g/L, autotrophy is only 0.70g/L (see Fig. 1).Such as Fig. 2, autotrophy fucoxanthin content is 1.0%, is raised together with up to
1.2%.Calculate fucoxanthin yield under the conditions of raising together with and be higher than autotrophy, optimal yield is 4.59mg/ (Ld).
Embodiment 3
Smooth rhombus algae raises together with culture
The difference of embodiment 3 and embodiment 2 is original of the seed culture medium with also including following content in fermentation medium
Material：Glucose 20g/L, production strain (is purchased from U.S. ocean from smooth rhombus algae (Nitzschia laevis) CCMP 559
Microalgae and Organism Depositary, National Center for Marine Algae and Microbiota, referred to as
NCMA), remaining content be the same as Example 2.
The effect analysis of embodiment 3：
Preferably, biomass is up to 2.88g/L to production growth state in the present embodiment.Autotrophy fucoxanthin content
For 0.9%.The production strain fermentation production fucoxanthin yield calculated in the present embodiment is higher than autotrophy, and optimal yield is
4.32mg/(L·d)。
Embodiment 4
The difference of embodiment 4 and embodiment 2 is that bioreactor selects 250mL pillar bioreactors, while
It is 5 μm of olm to be aided with intensity of illumination in step B-2·s-1Illumination, throughput is 3L/ minutes, remaining content and the phase of embodiment 2
Together.
Embodiment 5-8
The difference of embodiment 5-8 and embodiment 4 be the intensity of illumination in embodiment 5-8 step B respectively be 15,
30th, 50 and 70 μm of olm-2·s-1, cultivation cycle is 4 days in step B；Remaining content is same as Example 4.
Embodiment 4-8 effect analysis：
Referring to Fig. 3 and Fig. 4, photo-irradiation treatment auxiliary culture is carried out, intensity of illumination is 5-200 μm of olm-2·s-1.At illumination
Reason can further improve fucoxanthin content so that fucoxanthin is further improved, when intensity of illumination is 5 μm of olm-2·s-1
When, fucoxanthin content is up to 1.38%, and now the yield of fucoxanthin reaches as high as 9.88mg/ (Ld), maximum output
863.9% is improved before being relatively not optimised.
Embodiment 9
The difference of embodiment 9 and embodiment 4 is：Strain is produced in embodiment 9 and selects smooth rhombus algae (Nitzschia
Laevis) CCMP1092 (is purchased from U.S.'s marine microalgae and Organism Depositary, National Center for Marine
Algae and Microbiota, abbreviation NCMA), throughput is 1 liter/min in step B, and seed liquor is 3% according to volume ratio
Inoculum concentration transfer in no bacteria fermentation culture medium, culture of being ventilated under illumination condition prepares zymotic fluid, and culture medium loading amount is
80%, 25 DEG C of cultivation temperature, cultivation cycle 12 days；Described seed culture medium and fermentation medium include following content range
Raw material：NaCl 32g/L；Na2SiO3·9H2O 410mg/L；MgSO4·7H2O 2.18g/L；CaCl2·2H2O 0.1g/L；
KH2PO40.031g/L；K2HPO40.0055g/L；FeCl3·6H2O 0.291mg/L；MnCl2·4H2O 0.025mg/L；
ZnCl20.031mg/L；CoCl2·6H2O 0.0180mg/L；Na2MoO4·2H2O 0.012mg/L；H3BO316.1g/L；
(NH4)6MO7O24·4H2O 0.278mg/L；Tris-buffer 0.089g/L；H2SO49.4μg/L；vitamin B12 15×
10-5g/L；biotin 0.21g/L；Nitrogen source 0.2g/L；PH=6, the nitrogen source is constituted using the raw material of following mass fraction：Ferment
Female cream：Ammonium hydrogen carbonate：Peptone：Urea=1：2：1：2；Described intensity of illumination is 200 μm of olm-2·s-1, light is LED
Source.Remaining content is same as Example 4.
The effect analysis of embodiment 9：
Fucoxanthin content is 1.21%, and fucoxanthin yield is 9.1mg/ (Ld).
Embodiment 10
The difference of embodiment 10 and embodiment 4 is：Strain is produced in embodiment 9 and selects smooth rhombus algae (Nitzschia
Laevis) CCMP1092 (is purchased from U.S.'s marine microalgae and Organism Depositary, National Center for Marine
Algae and Microbiota, abbreviation NCMA), seed culture medium is 40g/L, seed with concentration of glucose in fermentation medium
Liquid is transferred according to the inoculum concentration that volume ratio is 20% in no bacteria fermentation culture medium, and ventilation culture prepares fermentation under illumination condition
Liquid, throughput 3L/min (wherein air：Carbon dioxide ratio is 95:5), culture medium loading amount is 20%, 30 DEG C of cultivation temperature, training
Support 4 days cycles；Described seed culture medium and fermentation medium include the raw material of following content range：NaCl 10g/L；
Na2SiO3·9H2O 30mg/L；MgSO4·7H2O 1.09g/L；CaCl2·2H2O 0.16g/L；KH2PO40.062g/L；
K2HPO40.00375g/L；FeCl3·6H2O 0.582mg/L；MnCl2·4H2O 0.112mg/L；ZnCl20.031mg/L；
CoCl2·6H2O 0.0228mg/L；Na2MoO4·2H2O 0.024mg/L；H3BO33.06g/L；(NH4)6MO7O24·4H2O
0.140mg/L；Tris-buffer 0.892g/L；H2SO416.4μg/L；vitamin B121.5g/L；biotin 25×10-5g/L；Nitrogen source 0.2g/L-7g/L；PH=6-9, the nitrogen source is constituted from the raw material of following mass fraction：Peptone：Yeast is carried
Take thing：Amino acid：Potassium nitrate=1：1：1：2；Described intensity of illumination is no more than 5 μm olm-2·s-1, light source is masking
Sunshine after adjustment.
The effect analysis of embodiment 10：
Fucoxanthin content is 1.11%, and fucoxanthin yield is 7.8mg/ (Ld).
Embodiment 11
The difference of embodiment 11 and embodiment 10 is：Seed culture medium is 20g/ with concentration of glucose in fermentation medium
L, seed liquor is transferred according to the inoculum concentration that volume ratio is 14% in no bacteria fermentation culture medium, the ventilation culture system under illumination condition
Preparation zymotic fluid, throughput 3L/min (wherein air：Carbon dioxide ratio is 98:2), culture medium loading amount is 40%, cultivation temperature
20 DEG C, cultivation cycle 7 days；Described seed culture medium and fermentation medium include the raw material of following content range：NaCl 22g/
L；Na2SiO3·9H2O 700mg/L；MgSO4·7H2O 1.66g/L；CaCl2·2H2O 0.27g/L；KH2PO40.045g/L；
K2HPO40.0075g/L；FeCl3·6H2O 0.431mg/L；MnCl2·4H2O 0.246mg/L；ZnCl20.151mg/L；
CoCl2·6H2O 0.0114mg/L；Na2MoO4·2H2O 0.018mg/L；H3BO330.56g/L；(NH4)6MO7O24·4H2O
0.028mg/L；Tris-buffer 0.451g/L；H2SO41.64μg/L；vitamin B120.1g/L；biotin 2.5g/
L；Nitrogen source 0.2g/L-7g/L；PH=6-9, the nitrogen source is constituted from the raw material of following mass fraction：Amino acid：Proteolysis
Thing：Sodium nitrate：Ammonium chloride=2：2：1：2；Described intensity of illumination is no more than 100 μm olm-2·s-1, light source is fluorescence
Lamp.
The effect analysis of embodiment 11：
Fucoxanthin content is 1.11%, and fucoxanthin yield is 7.8mg/ (Ld).
Claims (7)
1. the method for producing fucoxanthin using the smooth rhombus algae of illumination cultivation, it is characterised in that comprise the following steps：
A, seed liquor preparation
The smooth rhombus algae activated is placed in Heterotrophic culture in aseptic seed culture medium and seed liquor is made within 3-9 days, makes smooth water chestnut
Shape frustule is in exponential phase；
B, fermented and cultured
Seed liquor in step A is transferred in no bacteria fermentation culture medium according to volume ratio for 3%-20% inoculum concentration, in light
Culture of being ventilated according under the conditions of prepares zymotic fluid, and culture medium loading amount is 20%-80%, 20 DEG C -30 DEG C of cultivation temperature, cultivation cycle 4-
12 days；
Described seed culture medium and fermentation medium include the raw material of following content range：
NaCl 10g/L-32g/L；Na2SiO3·9H2O 30mg/L-700mg/L；MgSO4·7H2O 1.09g/L-2.18g/L；
CaCl2·2H2O 0.1g/L-0.27g/L；KH2PO40.031g/L-0.062g/L；K2HPO4 0.00375g/L-0.0075g/
L；FeCl3·6H2O 0.291mg/L-0.582mg/L；MnCl2·4H2O 0.025mg/L-0.246mg/L；ZnCl2
0.031mg/L-0.311mg/L；CoCl2·6H2O 0.0114mg/L-0.0228mg/L；Na2MoO4·2H2O 0.012mg/L-
0.024mg/L；H3BO33.06g/L-30.56g/L；(NH4)6MO7O24·4H2O 0.028mg/L-0.278mg/L；Tris-
buffer 0.089g/L-0.892g/L；H2SO41.64μg/L-16.4μg/L；vitamin B12 1.5g/L-15×10-5g/
L；biotin 2.5g/L-25×10-5g/L；Nitrogen source 0.2g/L-7g/L；PH=6-9, the nitrogen source is from organic nitrogen source, inorganic
Nitrogen source or its combination, wherein organic nitrogen source includes but is not limited to：Yeast extract, peptone, yeast extract, amino acid, urea, egg
White hydrolysate or its combination, it is inorganic nitrogen-sourced to include but is not limited to from potassium nitrate, sodium nitrate, ammonium chloride, ammonium hydrogen carbonate or its group
Close；
Described intensity of illumination is no more than 200 μm olm-2·s-1；
Described smooth rhombus algae is selected from smooth rhombus algae (Nitzschia laevis) UTEX 2047, smooth rhombus algae
(Nitzschia laevis) CCMP559 or smooth rhombus algae (Nitzschia laevis) CCMP 1092；
C, from step B fermentation terminate after zymotic fluid in extract fucoxanthin.
2. the method that the smooth rhombus algae of utilization illumination cultivation according to claim 1 produces fucoxanthin, it is characterised in that
Fermentation medium in described step B is placed in bioreactor, and bioreactor is anti-from triangular flask or pillar photo-biological
Answer device.
3. the method that the smooth rhombus algae of utilization illumination cultivation according to claim 1 produces fucoxanthin, it is characterised in that
Light source used in illumination includes but is not limited to sunshine, fluorescent lamp light, LED light or its combination.
4. the method that the smooth rhombus algae of utilization illumination cultivation according to claim 2 produces fucoxanthin, it is characterised in that
Concussion and cultivate in illumination shaking table is placed on during using triangular flask, shaking speed is 100 revs/min -240 revs/min；Using post
Filtrated air culture is passed through during formula bioreactor, throughput is 1-3 liters/min.
5. the method that the smooth rhombus algae of utilization illumination cultivation according to claim 1 produces fucoxanthin, it is characterised in that
Nitrogen source in described seed culture medium and fermentation medium selects the raw material of following content：NaNO31g/L；Peptone 1g/L,
Intensity of illumination is 30 μm of olm in step B-2·s-1。
6. the method that the smooth rhombus algae of utilization illumination cultivation according to claim 1 produces fucoxanthin, it is characterised in that
Also include the raw material of following content range in described seed culture medium and fermentation medium：Glucose 5-40g/L.
7. the method that the smooth rhombus algae of utilization illumination cultivation according to claim 6 produces fucoxanthin, it is characterised in that
Fermentation medium in step B selects pillar bioreactor, is 5-200 μm of olm in intensity of illumination-2·s-1Condition
Lower ventilation culture.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710525234.1A CN107119099B (en) | 2017-06-30 | 2017-06-30 | Method for producing fucoxanthin by culturing rhombohedral algae with light |
| PCT/CN2018/093490 WO2019001548A1 (en) | 2017-06-30 | 2018-06-29 | Method for preparing fucoxanthin by fermental cultivation of nitzschia laevis |
| US16/310,367 US11572577B2 (en) | 2017-06-30 | 2018-06-29 | Fermentation method for production of fucoxanthin by Nitzschia laevis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710525234.1A CN107119099B (en) | 2017-06-30 | 2017-06-30 | Method for producing fucoxanthin by culturing rhombohedral algae with light |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107119099A true CN107119099A (en) | 2017-09-01 |
| CN107119099B CN107119099B (en) | 2021-01-12 |
Family
ID=59731233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710525234.1A Active CN107119099B (en) | 2017-06-30 | 2017-06-30 | Method for producing fucoxanthin by culturing rhombohedral algae with light |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107119099B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624646A (en) * | 2018-06-15 | 2018-10-09 | 北京大学 | The method that fucoxanthin zymotic fluid is prepared using stream plus ventilation culture |
| CN108841887A (en) * | 2018-06-15 | 2018-11-20 | 北京大学 | The method for improving fucoxanthin content in the smooth diamond shape algae fermentation liquid of Heterotrophic culture using illumination |
| WO2019001548A1 (en) * | 2017-06-30 | 2019-01-03 | 北京大学 | Method for preparing fucoxanthin by fermental cultivation of nitzschia laevis |
| CN109644858A (en) * | 2018-12-07 | 2019-04-19 | 浙江海洋大学 | The method for improving brown alga flavine in brown alga |
| CN111961593A (en) * | 2019-12-24 | 2020-11-20 | 华南理工大学 | Method for increasing fucoxanthin yield in diatom and application thereof |
| CN112094798A (en) * | 2020-09-22 | 2020-12-18 | 深圳大学 | Culture medium for improving fucoxanthin yield in rhombohedral alga and application thereof |
| CN114214203A (en) * | 2021-12-27 | 2022-03-22 | 威海迪普森生物科技有限公司 | Method for increasing fucoxanthin yield through polyculture production of nitzschia closterium |
| CN116286379A (en) * | 2023-04-03 | 2023-06-23 | 广东海洋大学 | Method for promoting microalgae to accumulate fucoxanthin and synthesizing lipid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102007216A (en) * | 2008-04-22 | 2011-04-06 | 日本水产株式会社 | Process for production of fucoxanthin, and microalga for use in the process |
| CN103396979A (en) * | 2013-07-31 | 2013-11-20 | 华南理工大学 | Culture method for increasing yield of fucoxanthin contained in diatom |
| US20130309719A1 (en) * | 2010-10-06 | 2013-11-21 | Photonz Corporation Limited | Heterotrophic microbial production of xanthophyll pigments |
-
2017
- 2017-06-30 CN CN201710525234.1A patent/CN107119099B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102007216A (en) * | 2008-04-22 | 2011-04-06 | 日本水产株式会社 | Process for production of fucoxanthin, and microalga for use in the process |
| US20130309719A1 (en) * | 2010-10-06 | 2013-11-21 | Photonz Corporation Limited | Heterotrophic microbial production of xanthophyll pigments |
| CN103396979A (en) * | 2013-07-31 | 2013-11-20 | 华南理工大学 | Culture method for increasing yield of fucoxanthin contained in diatom |
Non-Patent Citations (1)
| Title |
|---|
| 陈明耀: "《生物饵料培养》", 31 October 1999, 中国农业出版社 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019001548A1 (en) * | 2017-06-30 | 2019-01-03 | 北京大学 | Method for preparing fucoxanthin by fermental cultivation of nitzschia laevis |
| CN108624646A (en) * | 2018-06-15 | 2018-10-09 | 北京大学 | The method that fucoxanthin zymotic fluid is prepared using stream plus ventilation culture |
| CN108841887A (en) * | 2018-06-15 | 2018-11-20 | 北京大学 | The method for improving fucoxanthin content in the smooth diamond shape algae fermentation liquid of Heterotrophic culture using illumination |
| CN108841887B (en) * | 2018-06-15 | 2022-02-08 | 北京大学 | Method for improving fucoxanthin content in heterotrophic culture rhombohedral alga fermentation liquid by utilizing illumination |
| CN109644858B (en) * | 2018-12-07 | 2021-01-26 | 浙江海洋大学 | Method for increasing fucoxanthin in brown algae |
| CN109644858A (en) * | 2018-12-07 | 2019-04-19 | 浙江海洋大学 | The method for improving brown alga flavine in brown alga |
| CN111961593A (en) * | 2019-12-24 | 2020-11-20 | 华南理工大学 | Method for increasing fucoxanthin yield in diatom and application thereof |
| CN111961593B (en) * | 2019-12-24 | 2022-08-12 | 华南理工大学 | Method for increasing fucoxanthin yield in diatom and application thereof |
| CN112094798A (en) * | 2020-09-22 | 2020-12-18 | 深圳大学 | Culture medium for improving fucoxanthin yield in rhombohedral alga and application thereof |
| CN114214203A (en) * | 2021-12-27 | 2022-03-22 | 威海迪普森生物科技有限公司 | Method for increasing fucoxanthin yield through polyculture production of nitzschia closterium |
| CN114214203B (en) * | 2021-12-27 | 2023-12-15 | 威海迪普森生物科技有限公司 | Method for improving fucoxanthin yield by mixing and preserving of nitenpyram |
| CN116286379A (en) * | 2023-04-03 | 2023-06-23 | 广东海洋大学 | Method for promoting microalgae to accumulate fucoxanthin and synthesizing lipid |
| CN116286379B (en) * | 2023-04-03 | 2024-02-23 | 广东海洋大学 | Method for promoting microalgae to accumulate fucoxanthin and synthesizing lipid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107119099B (en) | 2021-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107119099A (en) | The method for producing fucoxanthin using the smooth rhombus algae of illumination cultivation | |
| CN107099564A (en) | The method for producing fucoxanthin using the smooth rhombus algae of Heterotrophic culture | |
| CN104328064B (en) | A kind of bafillus natto and its application in fermenting and producing farnoquinone | |
| CN110250210B (en) | Optimal DSE strain for promoting corn seed soaking and rooting | |
| Tran et al. | Cultivation of Haematococcus pluvialis for astaxanthin production on angled bench-scale and large-scale biofilm-based photobioreactors | |
| CN108410737B (en) | A kind of two-steps tissue culture method of purple ball algae | |
| CN108841887B (en) | Method for improving fucoxanthin content in heterotrophic culture rhombohedral alga fermentation liquid by utilizing illumination | |
| CN105861342A (en) | Phaffia rhodozyma strain rich in astaxanthin and screening method and application of Phaffia rhodozyma strain | |
| US11572577B2 (en) | Fermentation method for production of fucoxanthin by Nitzschia laevis | |
| CN101591617B (en) | Docosahexaenoic acid-producing strain and mutation and screening method and application thereof | |
| Maneechote et al. | Chitosan-coated oleaginous microalgae-fungal pellets for improved bioremediation of non-sterile secondary effluent and application in carbon dioxide sequestration in bubble column photobioreactors | |
| Yu | Effect of mixed carbon substrate on exopolysaccharide production of cyanobacterium Nostoc flagelliforme in mixotrophic cultures | |
| CN108203729B (en) | Preparation method of kelp antioxidant peptide | |
| CN111925943A (en) | Chlorella vulgaris, and its culture method and application | |
| CN109481388B (en) | Active microalgae skin moisturizer as well as preparation method and application thereof | |
| CN109294920A (en) | A method of addition nano material induces haematococcus pluvialis efficient accumulation astaxanthin | |
| CN101186892A (en) | Breeding for oligo-acid production bacterium and method for producing oligo-acid thereof | |
| Zhu et al. | Fluid flow induced shear stress affects cell growth and total flavone production by Phellinus igniarius in stirred-tank bioreactor | |
| CN114058514A (en) | Method for accumulating starch by using marine green algae, Qingdao and Pantoea galbana | |
| CN107254413A (en) | A kind of method by being co-cultured with immobilized microorganism using starch Heterotrophic culture microalgae | |
| CN110699258B (en) | Culture method for improving chlorella cell biomass | |
| CN106148214B (en) | A kind of streptomyces ansochromogenes and the method for preparing Nikemycin | |
| CN102618592A (en) | Method for producing EPA (Eicosapentaenoic Acid) by using eustigmatoa cf. polyphem | |
| CN101280281B (en) | Paclitaxel genome rearrangement bacterial strain HDFS4-26 | |
| CN105695554B (en) | A method of it is co-cultured by bacterium algae and improves lutein yield |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |