CN109777754A - A method of improving blunt top spirulina beta carotene and zeaxanthin accumulation - Google Patents
A method of improving blunt top spirulina beta carotene and zeaxanthin accumulation Download PDFInfo
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- CN109777754A CN109777754A CN201910091035.3A CN201910091035A CN109777754A CN 109777754 A CN109777754 A CN 109777754A CN 201910091035 A CN201910091035 A CN 201910091035A CN 109777754 A CN109777754 A CN 109777754A
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Abstract
The present invention relates to a kind of methods of raising blunt top spirulina beta carotene and zeaxanthin accumulation, specifically one kind is by adding N doping carbon dots in culture medium, nitrogen source decrement, using suitable inoculum density, illumination, temperature, pH CMC model spirulina accumulate its beta carotene and zeaxanthin.Compared with whole process is using base culture base, the present invention makes zeaxanthin increase 29~92% in beta carotene and zeaxanthin accumulation stage using N doping carbon dots nitrogen source decrement basal medium, and beta carotene increases 45~97%.
Description
Technical field:
The invention belongs to spirulina fermentation technical field, it is related to a kind of raising blunt top spirulina beta carotene and maize
The method of matter accumulation, it is specifically a kind of by adding N doping carbon dots in culture medium, using suitable inoculum density, light
According to, temperature, pH and stress conditions culture spirulina, accumulate its beta carotene and zeaxanthin.
Background technique:
Blunt top spirulina (Spirulina platensis) is a kind of miniature cyanobacteria of rich in nutrition content, is rich in albumen
Matter, polysaccharide, unsaturated fatty acid, carotenoid (beta carotene and zeaxanthin) etc., especially beta carotene contain
Amount is 15 times of content beta-carotene in carrot.Spirulina photosynthetic efficiency is high, growth and breeding is fast, it is strong to environmental suitability and
Easily harvesting, is one of the main microalgae of current industrially scalable culture.Spirulina is considered as 21 century optimal health care product,
Spirulina the industries such as food, health care product, cosmetics, medicine, feed have been widely used in both at home and abroad at present.
Carotenoid is widely present in nature, and the carotenoid found in human body mainly includes α-carrot
Element, beta carotene, lutein, zeaxanthin, kryptoxanthin and lycopene.Wherein beta carotene etc. is VAPrecursor, be
The main source of vitamin A in human body.Zeaxanthin is a kind of oxygen containing natural carotenoid, it and lutein belong to same point
Isomers, widely distributed in nature, the zeaxanthin being mostly present in nature is all trans isomer.In human body
Zeaxanthin cannot be synthesized, needs to obtain by diet.A large number of studies show that: zeaxanthin has anti-oxidant, prevention Huang
Spot decline treats cataract, prevents cardiovascular disease, enhances immunity of organisms, slowing down the health efficacies such as atherosclerosis, and
Human health is closely related.
Summary of the invention:
The present invention is intended to provide a kind of method for improving beta carotene and zeaxanthin accumulation in blunt top spirulina simultaneously.
To achieve the goals above, the technical solution adopted by the present invention are as follows: the spirulina cells of culture to exponential phase of growth are transferred to and are added
Add the culture medium of N doping carbon dots, inventor is by the optimization to growth of spirulina platensis condition, using suitable inoculum density, light
According to, temperature and stress conditions culture spirulina, it is improved its beta carotene and zeaxanthin accumulation.
Further, the addition concentration of N doping carbon dots is 5~40mg/L in culture medium；
Further, nitrogen concentration is 1.25-2.5g/L in culture medium；
Specifically, operating procedure is as follows:
1) culture of algae
Blunt top spirulina cell is cultivated in basal medium, Initial seeding density OD560It is 0.2,28 DEG C of cultivation temperature,
Intensity of illumination is 3000Lux, culture to exponential phase of growth；
Basal medium composition is as follows: 13.6~16.8g/L of sodium bicarbonate, 2.4~4.03g/L of sodium carbonate, phosphoric acid hydrogen two
Potassium 0.5-1.0g/L, sodium nitrate 2.5-5.0g/L, potassium sulfate 1.0g/L, sodium chloride 1.0g/L, seven water and magnesium sulfate 0.2g/L, two
Water and calcium chloride 0.04g/L, green vitriol 0.01~0.20g/L, A5 component 1mL/L, remaining is water, pH value 8~
10,；
Wherein A5 component formula is as follows: boric acid 2.86g/L, tetrahydrate manganese chloride 1.86g/L, white vitriol 0.22g/L, and two
Water sodium molybdate 0.39g/L, cupric sulfate pentahydrate 0.08g/L, four water cobalt nitrate 0.05g/L, remaining is water；
2) accumulation of blunt top spirulina cell beta carotene and zeaxanthin
Using the spirulina cells of culture to exponential phase of growth as algae, algae seedling solution is obtained after centrifugation or filtration washing
Algal gel accesses fresh addition N doping carbon dots nitrogen source and is reduced basal medium, and adjusts Initial seeding density to OD560For 0.2~
0.4；26~30 DEG C of temperature, pH value 8~10, artificial Light To Dark Ratio 12h:12h illumination, culture solution light-receiving surface intensity of illumination 1500~
4500Lux cultivates 9~12 days to stationary phase harvest frustule；
The N doping carbon dots nitrogen source decrement basal medium is that the nitrogen of 10~40mg/L of addition in basal medium is mixed
Miscellaneous carbon dots, and nitric acid sodium content is adjusted to 1.25-2.5g/L；
In one embodiment of the invention, algae seedling solution is reduced basal medium through fresh addition N doping carbon dots nitrogen source
Directly it is diluted to Initial seeding density OD560It is 0.2~0.4；
The N doping carbon dots (hereinafter referred to as N CDs) use the synthesis of water-soluble bovine serum albumin(BSA) (BSA) hydro-thermal method,
Include the following steps:
After bovine serum albumin(BSA) is dissolved in ultrapure water by 1.0-1.5:100-200 (m:V), heated in 180-200 DEG C
6-8h is carbonized, and is down to room temperature after reaction, and high speed centrifugation removes carbide slag, by the yellowish comprising N@CDs of acquisition
Color liquid is filtered removal bulky grain by microfiltration membranes, and is dialysed with the dialysis membrane of 500Da, finally carries out being lyophilized spare；
Further, N@CDs's the preparation method is as follows: 0.1g BSA (66.5kD) is put into 20mL ultrapure water, in room
Ultrasonic dissolution 15min prepares BSA water solubility homogenizing fluid under conditions of temperature；It is then poly- by the liner that aqueous solution is transferred to 25ml
In the stainless steel autoclave of tetrafluoroethene, and 195 DEG C of heating 6h promote the carbonization of BSA in dry-heat air oven；Reaction knot
Shu Hou is cooled to room temperature；The carbide slag in final products solution is removed by the method for centrifugation (10000rpm, 15min)；
Removal bulky grain is filtered by 0.2 micron membrane filter comprising N@CDs weak yellow liquid by finally obtained, and with 500Da's
Dialysis membrane is dialysed for 24 hours, finally carries out being lyophilized spare.
3) after culture harvests frustule to stationary phase, beta carotene and zeaxanthin are extracted.Zeaxanthin yield reaches
0.28-0.43mg/g (frond dry weight), beta carotene yield reach 0.88-1.20mg/g (frond dry weight).
Compared with whole process is using base culture base, the present invention is used in beta carotene and zeaxanthin accumulation stage
N doping carbon dots nitrogen source decrement basal medium makes zeaxanthin increase 29~92%, and beta carotene increases 45~
97%.
The utility model has the advantages that
1, the present invention realizes the raising of beta carotene and zeaxanthin yield in blunt top spirulina: method culture according to this
Blunt top spirulina, beta carotene output increased 45~97%, zeaxanthin output increased 29~92%.
2, toxigenic capacity is low, and beta carotene and zeaxanthin accumulation are rapid: being trained using addition N doping carbon dots to basis
Base culture spirulina is supported, the product output of unit culture medium investment can be improved, advantageously reduce production cost；Beta carotene
It is accumulated in the short time in frustule with zeaxanthin, can achieve maximum production within general 9-12 days, it is with short production cycle.
In short, the present invention solves the contradiction that spirulina beta carotene and zeaxanthin cannot accumulate, realize quickly
The beta carotene and zeaxanthin in spirulina are efficiently produced, there is nutritive salt to consume less, production cost is low, β-carrot
The advantages that element is high with zeaxanthin is conducive to simplify Downstream processing operation, and rush can be widely applied to using spirulina as raw material
The related fieldss such as food, medicine, the energy.
Detailed description of the invention:
Fig. 1 adds the influence of N doping carbon dots (N CDs) and bovine serum albumin(BSA) to Growth of Spirulina Platensis；
Fig. 2 difference N doping carbon dots (N CDs) are added concentration and are accumulated to blunt top spirulina zeaxanthin and beta carotene
The influence of amount.
Specific embodiment:
In order to which the objects, technical solutions and advantages of this patent are more clearly understood, below in conjunction with attached drawing and specific implementation
Example, is further elaborated this patent.It should be appreciated that specific embodiment described herein is only used to explain that this is special
Benefit is not intended to limit the present invention.
Product measuring method of the present invention is as follows:
1) beta carotene and zeaxanthin extract: algae solution filtering is centrifuged to obtain algal gel, carries out vacuum freeze drying, is added
Acetone, ice-bath ultrasonic are crushed 20min, take supernatant is standby to survey.
2) quantitative verification: high performance liquid chromatography surveys beta carotene and zeaxanthin in spirulina, mobile phase
VMethanol: VAcetone=95:5.
Spirulina employed in embodiment is blunt top spirulina (Spirulina platensis) FACHB-904, purchase
It buys from Chinese Academy of Sciences's fresh water algae library.The strain selected above is interpreted as illustrative and non-specific restriction, the method for the present invention pair
It is applicable in the bacterial strain for the same kind being currently known.
Specific embodiment the present invention will be further explained explanation will be passed through below.
Embodiment 1 adds the influence of N doping carbon dots (N CDs) to Growth of Spirulina Platensis
Operating procedure is as follows:
1) blunt top spirulina cell is cultivated in basal medium, and culture medium is prepared with deionized water, Initial seeding density
OD560It is 0.2, pH value 8,28 DEG C of cultivation temperature, intensity of illumination 3000Lux, incubation time 9~10 days to logarithmic growth phase.
2) by culture in step 1) to OD560It is connect respectively for 1.0 or so spirulina medium with 10~15% inoculum concentration
Kind in: 1. add N doping carbon dots N CDs (20mg/L) basal medium；2. adding the base of bovine serum albumin(BSA) (20mg/L)
Basal culture medium；3. in basal medium, and adjusting cell density OD560It is 0.2, is inoculated in 500mL conical flask.Cultivation temperature
28 ± 2 DEG C, shaking speed 110r/min, artificial Light To Dark Ratio 12h:12h illumination, intensity of illumination 3000Lux is cultivated 12 days, and every
Its measurement algae solution density (OD560)。
By the measurement of algae density, compared to basal medium and bovine serum albumin(BSA) addition group, N doping carbon dots N CDs
Addition can promote the rapid growth of spirulina, and when cultivating 11 days, algae solution OD560Reach 1.0, compared with basal medium
Improve 72% (see Fig. 1).
Basal medium composition is as follows: sodium bicarbonate 13.61g/L, sodium carbonate 4.03g/L, dipotassium hydrogen phosphate 0.5g/L, nitre
Sour sodium 3g/L, potassium sulfate 1.0g/L, sodium chloride 1.0g/L, seven water and magnesium sulfate 0.2g/L, two water and calcium chloride 0.04g/L, seven
Ferrous sulfate hydrate 0.01g/L, A5 component 1mL/L, remaining is water, pH value 8~10,；
Wherein A5 component formula is as follows: boric acid 2.86g/L, tetrahydrate manganese chloride 1.86g/L, white vitriol 0.22g/L, and two
Water sodium molybdate 0.39g/L, cupric sulfate pentahydrate 0.08g/L, four water cobalt nitrate 0.05g/L；
N@CDs's the preparation method is as follows: 0.1g BSA (66.5kD) is put into 20mL ultrapure water, at room temperature
Ultrasonic dissolution 15min prepares BSA water solubility homogenizing fluid；It is then polytetrafluoroethylene (PTFE) by the liner that aqueous solution is transferred to 25ml
In stainless steel autoclave, and 195 DEG C of heating 6h come in dry-heat air oven.Promote the carbonization of BSA；After reaction, cool down
To room temperature；The carbide slag in final products solution is removed by the method for centrifugation (10000rpm, 15min)；It is obtained final
To removal bulky grain is filtered by 0.2 micron membrane filter comprising N@CDs weak yellow liquid, and it is saturating with the dialysis membrane of 500Da
Analysis for 24 hours, finally carries out being lyophilized spare.
2 comparative experiments of embodiment
Operating procedure is as follows:
1) blunt top spirulina cell is cultivated in basal medium, and culture medium is prepared with deionized water, Initial seeding density
OD560It is 0.2, pH value 8,28 DEG C of cultivation temperature, intensity of illumination 3000Lux, incubation time 9~10 days to logarithmic growth phase.
2) by culture in step 1) to OD560It is connect respectively for 1.0 or so spirulina medium with 10~15% inoculum concentration
Kind in: 1. add N doping carbon dots N CDs (20mg/L) basal medium；2. containing inorganic nitrogen-sourced (sodium nitrate 1.5g/L) is partly measured
Basal medium；3. in basal medium；And adjust cell density OD560It is 0.2, is inoculated in 500mL conical flask.Culture
28 ± 2 DEG C of temperature, shaking speed 110r/min, artificial Light To Dark Ratio 12h:12h illumination, intensity of illumination 3000Lux is cultivated 12 days,
And spirulina cells density in algae solution is made to reach OD value 0.9~1.2.
Basal medium composition is as follows: sodium bicarbonate 13.61g/L, sodium carbonate 4.03g/L, dipotassium hydrogen phosphate 0.5g/L, nitre
Sour sodium 3g/L, potassium sulfate 1.0g/L, sodium chloride 1.0g/L, seven water and magnesium sulfate 0.2g/L, two water and calcium chloride 0.04g/L, seven
Ferrous sulfate hydrate 0.01g/L, A5 component 1mL/L, remaining is water, pH value 8~10,；
Wherein A5 component formula is as follows: boric acid 2.86g/L, tetrahydrate manganese chloride 1.86g/L, white vitriol 0.22g/L, and two
Water sodium molybdate 0.39g/L, cupric sulfate pentahydrate 0.08g/L, four water cobalt nitrate 0.05g/L；
N@CDs's the preparation method is as follows: 0.1g BSA (66.5kD) is put into 20mL ultrapure water, at room temperature
Ultrasonic dissolution 15min prepares BSA water solubility homogenizing fluid；It is then polytetrafluoroethylene (PTFE) by the liner that aqueous solution is transferred to 25ml
In stainless steel autoclave, and 195 DEG C of heating 6h come in dry-heat air oven.Promote the carbonization of BSA；After reaction, cool down
To room temperature；The carbide slag in final products solution is removed by the method for centrifugation (10000rpm, 15min)；It is obtained final
To removal bulky grain is filtered by 0.2 micron membrane filter comprising N@CDs weak yellow liquid, and it is saturating with the dialysis membrane of 500Da
Analysis for 24 hours, finally carries out being lyophilized spare.
3) algae solution filter centrifugation is collected into algal gel, and carries out vacuum freeze drying for extracting zeaxanthin and β-carrot
Element, yield are as follows:
1. adding the basal medium of N doping carbon dots N CDs (20mg/L): zeaxanthin 0.26mg/ in spirulina
G, content beta-carotene 0.82mg/g.
2. containing the basal medium for partly measuring inorganic nitrogen-sourced (sodium nitrate 1.5g/L): zeaxanthin in spirulina
0.23mg/g, content beta-carotene 0.65mg/g.
3. basal medium: zeaxanthin 0.22mg/g in spirulina, content beta-carotene 0.61mg/g.
Illustrate that nitrogen source decrement and addition N doping carbon dots are all conducive to zeaxanthin and β-Hu in blunt top spirulina cell
The accumulation of radish element.
The different N doping carbon dots (N CDs) of embodiment 3 are added concentration and are accumulated to spirulina beta carotene and zeaxanthin
It influences
The N doping carbon dots N CDs concentration added in basal medium is respectively set are as follows: 5,10,20,40,80,
100mg/L, remaining condition carry out algae culture and product accumulation with embodiment 2, compare not add nitrogen in basic culture medium and mixing
Miscellaneous carbon dots N@CDs；
After culture, algae solution filter centrifugation is collected into algal gel, and carries out vacuum freeze drying for extracting zeaxanthin
And beta carotene, as a result see Fig. 2.When adding N doping carbon dots N CDs 5mg/L, zeaxanthin in spirulina
0.24mg/g, content beta-carotene 0.77mg/g；When adding N doping carbon dots N CDs 10mg/L, zeaxanthin in spirulina
Content 0.29mg/g, content beta-carotene 0.88mg/g；When adding N doping carbon dots N CDs 20mg/L, corn in spirulina
Yellow matter content 0.26mg/g, content beta-carotene 0.82mg/g；In control group, zeaxanthin 0.22mg/ in spirulina
G, content beta-carotene 0.61mg/g.
A kind of method for improving beta carotene and zeaxanthin accumulation in blunt top spirulina of embodiment 4
1) culture of algae
Blunt top spirulina cell is cultivated in basal medium, is inoculated with initial density OD560It is 0.2,28 DEG C of cultivation temperature,
Intensity of illumination is 3000Lux, culture to exponential phase of growth；
Basal medium composition is as follows: sodium bicarbonate 13.6g/L, sodium carbonate 2.4g/L, dipotassium hydrogen phosphate 0.5g/L, nitric acid
Sodium 3g/L, potassium sulfate 1.0g/L, sodium chloride 1.0g/L, seven water and magnesium sulfate 0.2g/L, two water and calcium chloride 0.04g/L, seven water
Ferrous sulfate 0.01g/L, A5 component 1mL/L are closed, remaining is water, pH value 10；
Wherein A5 component formula is as follows: boric acid 2.86g/L, tetrahydrate manganese chloride 1.86g/L, white vitriol 0.22g/L, and two
Water sodium molybdate 0.39g/L, cupric sulfate pentahydrate 0.08g/L, four water cobalt nitrate 0.05g/L；
2) accumulation of blunt top spirulina cell beta carotene and zeaxanthin
Using the spirulina cells of culture to exponential phase of growth as algae, former algae seedling solution is obtained into algal gel after being centrifuged, is connect
Entering fresh addition N doping carbon dots nitrogen source decrement basal medium, (N CDs 10mg/L, sodium nitrate 1.5g/L, basal medium is such as
Step 1)), and Initial seeding density is adjusted to OD560It is 0.2；26 DEG C of temperature, pH value 10, artificial Light To Dark Ratio 12h:12h illumination,
Culture solution light-receiving surface intensity of illumination 4500Lux cultivates 12 days to stationary phase harvest frustule；
N@CDs's the preparation method is as follows: 0.1g BSA (66.5kD) is put into 20mL ultrapure water, at room temperature
Ultrasonic dissolution 15min prepares BSA water solubility homogenizing fluid；It is then polytetrafluoroethylene (PTFE) by the liner that aqueous solution is transferred to 25ml
In stainless steel autoclave, and 195 DEG C of heating 6h promote the carbonization of BSA in dry-heat air oven；After reaction, it is cooled to
Room temperature；The carbide slag in final products solution is removed by the method for centrifugation (10000rpm, 15min)；It will finally obtain
Removal bulky grain is filtered by 0.2 micron membrane filter comprising N@CDs weak yellow liquid, and with the dialysis of the dialysis membrane of 500Da
For 24 hours, it finally carries out being lyophilized spare.
3) culture to stationary phase harvests frustule, after freeze-drying, extracts beta carotene and zeaxanthin.Zeaxanthin yield
Reach 0.43mg/g, beta carotene yield reaches 1.20mg/g.
A kind of method for improving beta carotene and zeaxanthin accumulation in blunt top spirulina of embodiment 5
1) culture of algae
Blunt top spirulina cell is cultivated in basal medium, Initial seeding density OD560It is 0.2,28 DEG C of cultivation temperature,
Intensity of illumination is 3000Lux, culture to exponential phase of growth；
Basal medium composition is as follows: sodium bicarbonate 16.8g/L, sodium carbonate 4.03g/L, dipotassium hydrogen phosphate 1.0g/L, nitre
Sour sodium 4.4g/L, potassium sulfate 1.0g/L, sodium chloride 1.0g/L, seven water and magnesium sulfate 0.2g/L, two water and calcium chloride 0.04g/L,
Green vitriol 0.20g/L, A5 component 1mL/L, remaining is water, pH value 8~10,；
Wherein A5 component formula is as follows: boric acid 2.86g/L, tetrahydrate manganese chloride 1.86g/L, white vitriol 0.22g/L, and two
Water sodium molybdate 0.39g/L, cupric sulfate pentahydrate 0.08g/L, four water cobalt nitrate 0.05g/L；
2) accumulation of blunt top spirulina cell beta carotene and zeaxanthin
Using the spirulina cells of culture to exponential phase of growth as algae, former algae seedling solution is obtained into algae after filtration washing
Mud accesses fresh addition N doping carbon dots nitrogen source decrement basal medium (N CDs 40mg/L, sodium nitrate 2.2g/L, basis training
Support base such as step 1)), and Initial seeding density is adjusted to OD560It is 0.4；30 DEG C of temperature, pH value 9, artificial Light To Dark Ratio 12h:12h
Illumination, culture solution light-receiving surface intensity of illumination 3000Lux cultivate 9 days to stationary phase harvest frustule；
N@CDs's the preparation method is as follows: 0.1g BSA (66.5kD) is put into 20mL ultrapure water, at room temperature
Ultrasonic dissolution 15min prepares BSA water solubility homogenizing fluid；It is then polytetrafluoroethylene (PTFE) by the liner that aqueous solution is transferred to 25ml
In stainless steel autoclave, and 195 DEG C of heating 6h promote the carbonization of BSA in dry-heat air oven；After reaction, it is cooled to
Room temperature；The carbide slag in final products solution is removed by the method for centrifugation (10000rpm, 15min)；It will finally obtain
Removal bulky grain is filtered by 0.2 micron membrane filter comprising N@CDs weak yellow liquid, and with the dialysis of the dialysis membrane of 500Da
For 24 hours, it finally carries out being lyophilized spare.
3) after culture to stationary phase harvest frustule freeze-drying, beta carotene and zeaxanthin are extracted.Zeaxanthin yield
Reach 0.28mg/g, beta carotene yield reaches 0.88mg/g.
A kind of method for improving beta carotene and zeaxanthin accumulation in blunt top spirulina of embodiment 6
1) culture of algae
Blunt top spirulina cell is cultivated in basal medium, Initial seeding density OD560It is 0.2,28 DEG C of cultivation temperature,
Intensity of illumination is 3000Lux, culture to exponential phase of growth；
Basal medium composition is as follows: sodium bicarbonate 15.4g/L, sodium carbonate 3.2g/L, dipotassium hydrogen phosphate 0.75g/L, nitre
Sour sodium 2.5g/L, potassium sulfate 1.0g/L, sodium chloride 1.0g/L, seven water and magnesium sulfate 0.2g/L, two water and calcium chloride 0.04g/L,
Green vitriol 0.1g/L, A5 component 1mL/L, remaining is water, pH value 8,；
Wherein A5 component formula is as follows: boric acid 2.86g/L, tetrahydrate manganese chloride 1.86g/L, white vitriol 0.22g/L, and two
Water sodium molybdate 0.39g/L, cupric sulfate pentahydrate 0.08g/L, four water cobalt nitrate 0.05g/L；
2) accumulation of blunt top spirulina cell beta carotene and zeaxanthin
Using the spirulina cells of culture to exponential phase of growth as algae, former algae seedling solution is obtained into algal gel after being centrifuged, is connect
Enter fresh addition N doping carbon dots nitrogen source decrement basal medium (N CDs 25mg/L, sodium nitrate 1.25g/L, basal medium
Such as step 1)), and Initial seeding density is adjusted to OD560It is 0.3；28 DEG C of temperature, pH value 8, artificial Light To Dark Ratio 12h:12h illumination,
Culture solution light-receiving surface intensity of illumination 1500Lux cultivates 10 days to stationary phase harvest frustule；
N@CDs's the preparation method is as follows: 0.1g BSA (66.5kD) is put into 20mL ultrapure water, at room temperature
Ultrasonic dissolution 15min prepares BSA water solubility homogenizing fluid；It is then polytetrafluoroethylene (PTFE) by the liner that aqueous solution is transferred to 25ml
In stainless steel autoclave, and 195 DEG C of heating 6h come in dry-heat air oven.Promote the carbonization of BSA；After reaction, cool down
To room temperature；The carbide slag in final products solution is removed by the method for centrifugation (10000rpm, 15min)；It is obtained final
To removal bulky grain is filtered by 0.2 micron membrane filter comprising N@CDs weak yellow liquid, and it is saturating with the dialysis membrane of 500Da
Analysis for 24 hours, finally carries out being lyophilized spare.
3) after culture to stationary phase harvest frustule freeze-drying, beta carotene and zeaxanthin are extracted.Zeaxanthin yield
Reach 0.31mg/g, beta carotene yield reaches 1.01mg/g.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
The limitation to the scope of the patents therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art,
Under the premise of not departing from this patent design, the respective embodiments described above can also make several deformations, combination and improve, these all belong to
In the protection scope of this patent.Therefore, the protection scope of this patent should be subject to the claims.
Claims (8)
1. a kind of method for improving blunt top spirulina beta carotene and zeaxanthin, which is characterized in that culture is raw to index
Long-term spirulina cells are transferred in the culture medium of addition N doping carbon dots, adjustment Initial seeding density to OD560For 0.2~
0.4；26~30 DEG C of temperature, pH value 8~10, culture solution light-receiving surface 1500~4500Lux of intensity of illumination, culture to stationary phase harvest
Frustule.
2. a kind of method for improving blunt top spirulina beta carotene and zeaxanthin as described in claim 1, feature exist
In the addition concentration of N doping carbon dots is 5~40mg/L in culture medium.
3. a kind of method for improving blunt top spirulina beta carotene and zeaxanthin as described in claim 1, feature exist
In the concentration of nitrogen source is 1.25-2.5g/L in culture medium.
4. a kind of side for improving blunt top spirulina beta carotene and zeaxanthin as claimed in any one of claims 1-3
Method, which is characterized in that steps are as follows:
1) culture of algae
Blunt top spirulina cell is cultivated in basal medium to exponential phase of growth；
2) accumulation of blunt top spirulina cell beta carotene and zeaxanthin
Using the spirulina cells of culture to exponential phase of growth as algae, algae seedling solution is obtained into algae after centrifugation or filtration washing
Mud accesses in the basal medium of fresh addition N doping carbon dots nitrogen source decrement, and adjusts Initial seeding density to OD560It is 0.2
~0.4；26~30 DEG C of temperature, pH value 8~10, artificial Light To Dark Ratio 12h:12h illumination, culture solution light-receiving surface intensity of illumination 1500~
4500Lux cultivates 9~12 days to stationary phase harvest frustule；
3) after culture harvests frustule to stationary phase, beta carotene and zeaxanthin are extracted；
The addition concentration of the N doping carbon dots is 5~40mg/L；
The concentration of the nitrogen source is 1.25-2.5g/L.
5. method as claimed in claim 4, which is characterized in that basal medium composition is as follows: sodium bicarbonate 13.6~
16.8g/L, 2.4~4.03g/L of sodium carbonate, dipotassium hydrogen phosphate 0.5-1.0g/L, sodium nitrate 2.5-5.0g/L, potassium sulfate 1.0g/
L, sodium chloride 1.0g/L, seven water and magnesium sulfate 0.2g/L, two water and calcium chloride 0.04g/L, green vitriol 0.01~
0.20g/L, A5 component 1mL/L, remaining is water, pH value 8~10,；
Wherein A5 component formula is as follows: boric acid 2.86g/L, tetrahydrate manganese chloride 1.86g/L, white vitriol 0.22g/L, molybdate dihydrate
Sour sodium 0.39g/L, cupric sulfate pentahydrate 0.08g/L, four water cobalt nitrate 0.05g/L, remaining is water.
6. method as claimed in claim 4, which is characterized in that the base that algae seedling solution is reduced through fresh addition N doping carbon dots nitrogen source
Basal culture medium is directly diluted to Initial seeding density OD560It is 0.2~0.4.
7. method as claimed in claim 4, which is characterized in that the N doping carbon dots are using water-soluble bovine serum albumin plain boiled water
The synthesis of thermal method, includes the following steps:
After bovine serum albumin(BSA) is dissolved in ultrapure water by 1.0-1.5:100-200, carbon is carried out in 180-200 DEG C of heating 6-8h
Change, be down to room temperature after reaction, high speed centrifugation removes carbide slag, by passing through comprising N@CDs weak yellow liquid for acquisition
Microfiltration membranes are filtered removal bulky grain, and are dialysed with the dialysis membrane of 500Da, finally carry out being lyophilized spare.
8. the method for claim 7, which is characterized in that the N doping carbon dots are using water-soluble bovine serum albumin plain boiled water
The synthesis of thermal method, includes the following steps:
N@CDs's the preparation method is as follows: 0.1g BSA is put into 20mL ultrapure water, at room temperature ultrasonic dissolution
15min prepares BSA water solubility homogenizing fluid；It is high for the stainless steel of polytetrafluoroethylene (PTFE) that aqueous solution is then transferred to the liner of 25ml
It presses in kettle, and 195 DEG C of heating 6h promote the carbonization of BSA in dry-heat air oven；After reaction, it is cooled to room temperature；It is logical
Cross 10000rpm, the carbide slag in the method removal final products solution of 15min centrifugation；By finally obtained comprising N@
CDs weak yellow liquid is filtered removal bulky grain by 0.2 micron membrane filter, and for 24 hours with the dialysis of the dialysis membrane of 500Da, finally
Be lyophilized spare.
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