CN103627639A - Method utilizing spirulina cultivation liquid to cultivate Dunaliella salina - Google Patents
Method utilizing spirulina cultivation liquid to cultivate Dunaliella salina Download PDFInfo
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Abstract
A method utilizing a spirulina cultivation liquid to cultivate Dunaliella salina belongs to the field of biotechnology and comprises the following steps: (1) preparing a Dunaliella salina culture solution; (2) adding the Dunaliella salina culture solution into an algae seed pool and then placing Dunaliella salina seeds for enlarging cultivation to the logarithmic growth phase; (3) inoculating the logphase growth algae liquid into a growth pool at a certain proportion of inoculation quantity; (4) separating the mature Dunaliella salina liquid in the growth pool through an algae liquid bed-cleaning centrifuge, and collecting the separated culture solution and algae paste, wherein the culture solution is used for preparing the Dunaliella salina culture solution in the step (1) and the algae paste is used for later processing. According to the invention, as the Dunaliella salina is cultivated by the tail liquid of a cultivation liquid for cultivating spirulina, accumulation of nutritional ingredients is facilitated, and lots of algae cells can be acquired within a short period; the product yield and added value are increased, the process is simple and the energy consumption is low; the Dunaliella salina cultivation time is shortened, the cultivation conditions are easy to control, the production cost is low and the product added value is high.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the cultural method of Dunaliella salina.
Background technology
Dunaliella salina (Dunaliella salina) is subordinate to Chlorophyta (Chlorophy ta), volvocales (Volvocales), crinosity algae section (Poly-blepharidaceae), Dunaliella (Dunaliella, Teodoresco), be a kind of unicell green alga of the acellular wall that can grow in hypersaline environment.
Dunaliella salina is single celled plant plankton, and its frond is oval, ellipse or pyriform, long 18-28 μ m, and wide 9.5-14 μ m, during motion, the bodily form changes, and under the environment such as different salinity, illumination, temperature, its metamorphosis is larger.This is that profile changes with environmental change because Dunaliella salina does not have cellulosic cell walls, and pyrenoids and starch small grain be the vicissitudinous cause with environment difference also.Dunaliella salina cell leading portion is generally recess, at recess, has two isometric flagellums, and flagellum is about 1/3 than cell; In frond, have a cup-shaped chromatoplast, the pigment in chromatoplast is mainly Chlorophylls and Carotenoids (take β-carotene as main).
Dunaliella salina be take vegetative propagation as main at growing period, and frond at the volley lobe is two, and each forms a new body.Divide when vigorous, under microscope, often can see 4-8 new individualities and form simultaneously.Division stage,, frond was less, was often green; After ripe, individuality is combined into zygote, and zygote is spherical in shape, larger than frond, has smooth thin-walled, and each zygote carries out reduction division, finally produces 16 zoospores.
Dunaliella salina contains abundant grease, β-carotene, protein, polysaccharide etc., containing mineral substance such as higher Ca, P, Zn, also contains 18 seed amino acids that comprise mankind's indispensable amino acid, the 40%-50% that the glycerine of accumulation is dry weight simultaneously.Under suitable condition, in body synthetic β-carotene can reach dry cell weight 10% on.
Dunaliella salina has unique economic worth in food, medicines and health protection and chemical industry and aquaculture.This algae has been realized suitability for industrialized production in countries such as Australia, the U.S. and Israel.The related aspect of its industrialization is mainly β-carotene class healthcare products, makeup, nutritious supplementary and algae powder etc.
Retrieval Chinese patent, finds that the patent of relevant Dunaliella salina is very few, existing:
One, the patent No. is 93106062.1, denomination of invention is " foundation of model of producing beta-carotene by cultivating marine algae ", the method is: according to dunaiella salina growth pattern and the required envrionment conditions of β-carotene accumulation mode, the environmental factor in change breeding process is to reach object.Comprising: intensity of illumination 30000 left and right, luxs, salinity by 150～180 ‰ being elevated to 240 ‰, temperature by 24～28 ℃ be elevated to 30 ℃ of above, N concentration by 1mmol/L drop to 0.5～1mmol/L, P concentration drops to 0.1mmol/L by 0.3mmol/L.The method is simple to operate, not high to equipment and technical requirements, conveniently regulating and controlling, but only considered the growth of Dunaliella salina and the accumulation of β-carotene, and do not consider the accumulation of iodine, the salt algae powder function of producing is few, and added value is not high.
Two, the patent No. is 201110314357.3, denomination of invention is " cultural method of Dunaliella salina accumulation organic iodine ", and the method is: by changing the cultivating condition of Dunaliella salina, to reach, promote it to grow, increase the effect of the semi-invariant of β-carotene and organic iodine.Comprising the cultivation of: first stage: salinity by 150 ‰～180 ‰ dropping to 30 ‰～40 ‰, intensity of illumination 10000 left and right, luxs, added KNO3600mg/L, KH2PO460mg/L, NaHCO3500mg/L; Subordinate phase cultivation: control that culturing pool temperature is controlled at 110 ‰～120 ‰ at 30～40 ℃, salinity, intensity of illumination 20000lx～40000lx, added NaHCO3500mg/L, KIO30.4～0.6g/L; The present invention has shortened Dunaliella salina culturing time, and cultivating condition is easy to control, and production cost is low, and added value of product is high.
The content of above-mentioned patent, just in order to improve the content accumulation of organic allusion quotation or β-carotene, increase the added value of Dunaliella salina, just according to dunaiella salina growth pattern and β-carotene and the required envrionment conditions of organic allusion quotation accumulation mode, environmental factor in change breeding process is to reach object cultural method, do not consider in all directions the equilibrium accumulation of the nutritive ingredient of Dunaliella salina, do not consider the problem of the required starting material recycle of algal culture.
Summary of the invention
The object of the invention is to overcome weak point in prior art, provide a kind of and change little in the situation that in the Essential Environment factor, utilize the method for spirulina breeding liquid cultivation Dunaliella salina, can guarantee the accumulation of the nutritive ingredient of Dunaliella salina comprehensively.
In order to realize object of the present invention, we are implemented the following technical scheme of employing:
A method of utilizing spirulina breeding liquid raffinate cultivation Dunaliella salina, is characterized in that: the method comprises the following steps:
(1) preparation Dunaliella salina nutrient solution;
(2) will in algae kind pond, add Dunaliella salina nutrient solution, then put into Dunaliella salina algae kind and carry out enlarged culturing to logarithmic phase, control 20～35 ℃ of temperature, salinity 12%～36%, illumination 10000lx～50000lx, the pH value 8～10 in algae kind pond, cultivate 4～6 days;
(3) the algae liquid of logarithmic phase growth is linked into growth pool by the inoculum size of 10wt%～20wt%, controls 20～35 ℃ of temperature, salinity 12%～36%, illumination 10000lx～50000lx, the pH value 8～10 of growth pool, cultivate 6～8 days;
(4) cultivating ripe Dunaliella salina algae liquid in growth pool, remove sand separated with whizzer through algae liquid, will isolate nutrient solution and algae and stick with paste and collect, nutrient solution is for the preparation of step (1) Dunaliella salina nutrient solution, and algae is stuck with paste the processing for the later stage.
The salinity kind that described spirulina breeding liquid raffinate is contained and concentration difference: sodium-chlor 1～3g/L, sodium carbonate 9～20g/L, sodium bicarbonate 0.1～2g/L, potassium primary phosphate 0.01～0.05g/L, SODIUMNITRATE 0.5～1g/L, ferrous sulfate 0.001～0.005g/L, calcium chloride 0.05～0.25g/L, Repone K 0.05～0.25g/L, magnesium sulfate 0.3～0.6g/L, magnesium chloride 0.8～1.8g/L, the pH value of spirulina breeding liquid raffinate is 10～14.
Dunaliella salina nutrient solution in described step (1) is that to utilize pH value is 10～14, carbonate content is 9～20g/L spirulina breeding liquid raffinate to add concentration be that hydrochloric acid 5.000～8.000g/L of 20% is formulated.
Described hydrochloric acid preferred content is 6.558g/L.
After step (1) has been prepared, the concentration of the sodium-chlor in Dunaliella salina nutrient solution is 12～36%.
After step (1) has been prepared, in described Dunaliella salina nutrient solution, kind and the concentration of salinity are as follows: sodium bicarbonate 0.1～0.5g/L, potassium primary phosphate 0.01～0.05g/L, SODIUMNITRATE 0.8～1.2g/L, ferrous sulfate 0.001～0.005g/L, calcium chloride 0.05～0.25g/L, Repone K 0.05～0.25g/L, magnesium sulfate 0.3～0.6g/L, magnesium chloride 0.8～1.8g/L.
The concentration of sodium bicarbonate, potassium primary phosphate, SODIUMNITRATE, ferrous sulfate, calcium chloride, Repone K, magnesium sulfate and magnesium chloride in described step (2) or step (3) Dunaliella salina nutrient solution used is respectively 0.3g/L, 0.03g/L, 1g/L, 0.002g/L, 0.15g/L, 0.18g/L, 0.45g/L, 1.2g/L.
Beneficial effect
The breeding way that the present invention adopts: utilize the raffinate of cultivating spirulina culturing liquid, utilize under cellar culture condition, adopt the environmental factors that is conducive to dunaiella salina growth, make it be conducive to nutritious accumulation, obtain in a short time a large amount of frustule numbers;
Method of the present invention has increased output and the added value of product, the simple energy consumption of technique is low, and the algae kind of each cultivation is produced after by algae kind pond enlarged culturing, has both guaranteed the superiority of technique, also guarantee the quality of algae kind, do not affected normally carrying out of production simultaneously;
The present invention has shortened Dunaliella salina culturing time, and cultivating condition is easy to control, and production cost is low, and added value of product is high.The material adding in whole breeding process meets national regulation, and product safety is reliable, can be applicable to food and medicine industry.
Accompanying drawing explanation
Fig. 1 is process flow sheet of the present invention.
Embodiment
By reference to the accompanying drawings, the present invention is described further:
As shown in Figure 1, a kind of method of utilizing spirulina breeding liquid cultivation Dunaliella salina of the present invention, the method comprises the following steps:
(1) preparation Dunaliella salina nutrient solution, this nutrient solution is that by spirulina breeding liquid raffinate, to add concentration be that 20% hydrochloric acid is formulated, this raffinate contains sodium-chlor, sodium carbonate, sodium bicarbonate, potassium primary phosphate, SODIUMNITRATE, ferrous sulfate, calcium chloride, Repone K, magnesium sulfate, magnesium chloride, the concentration of each component is respectively: sodium-chlor 1～3g/L, sodium carbonate 9～20g/L, sodium bicarbonate 0.1～2g/L, potassium primary phosphate 0.01～0.05g/L, SODIUMNITRATE 0.5～1g/L, ferrous sulfate 0.001～0.005g/L, calcium chloride 0.05～0.25g/L, Repone K 0.05～0.25g/L, magnesium sulfate 0.3～0.6g/L, magnesium chloride 0.8～1.8g/L, pH value in spirulina breeding liquid raffinate is 9～14, carbonate content is when 9～20g/L, add hydrochloric acid 5.000～8.000g/L, preferably add 6.558g/L, after adding hydrochloric acid, be mixed with Dunaliella salina nutrient solution, the salinity of Dunaliella salina nutrient solution is 12～36%, in described Dunaliella salina nutrient solution, kind and the concentration of salinity are as follows: sodium bicarbonate 0.1～0.5g/L, potassium primary phosphate 0.01～0.05g/L, SODIUMNITRATE 0.8～1.2g/L, ferrous sulfate 0.001～0.005g/L, calcium chloride 0.05～0.25g/L, Repone K 0.05～0.25g/L, magnesium sulfate 0.3～0.6g/L, magnesium chloride 0.8～1.8g/L,
Dunaliella salina nutrient solution contains sodium bicarbonate, potassium primary phosphate, SODIUMNITRATE, ferrous sulfate, calcium chloride, Repone K, magnesium sulfate heptahydrate and magnesium chloride, and the preferred concentration of each component is respectively: sodium bicarbonate 0.3g/L, potassium primary phosphate 0.03g/L, SODIUMNITRATE 1g/L, ferrous sulfate 0.002g/L, calcium chloride 0.15g/L, Repone K 0.18g/L, magnesium sulfate 0.45g/L, magnesium chloride 1.2g/L.
(2) will in algae kind pond, add Dunaliella salina nutrient solution, then put into Dunaliella salina algae kind and carry out enlarged culturing to logarithmic phase, control 20～35 ℃ of temperature, salinity 12%～36%, illumination 10000lx～50000lx, the pH value 8～10 in algae kind pond, cultivate 4～6 days;
(3) the algae liquid of logarithmic phase growth is linked into growth pool by the inoculum size of 10wt%～20wt%, controls 20～35 ℃ of temperature, salinity 12%～36%, illumination 10000lx～50000lx, the pH value 8～10 of growth pool, cultivate 6～8 days;
(4) cultivating ripe Dunaliella salina algae liquid in growth pool, remove sand separated with whizzer through algae liquid, will isolate nutrient solution and algae and stick with paste and collect, nutrient solution is for the preparation of step (1) Dunaliella salina nutrient solution, and algae is stuck with paste the processing for the later stage.
Claims (7)
1. a method of utilizing spirulina breeding liquid raffinate cultivation Dunaliella salina, is characterized in that: the method comprises the following steps:
(1) preparation Dunaliella salina nutrient solution;
(2) will in algae kind pond, add Dunaliella salina nutrient solution, then put into Dunaliella salina algae kind and carry out enlarged culturing to logarithmic phase, control 20～35 ℃ of temperature, salinity 12%～36%, illumination 10000lx～50000lx, the pH value 8～10 in algae kind pond, cultivate 4～6 days;
(3) the algae liquid of logarithmic phase growth is linked into growth pool by the inoculum size of 10wt%～20wt%, controls 20～35 ℃ of temperature, salinity 12%～36%, illumination 10000lx～50000lx, the pH value 8～10 of growth pool, cultivate 6～8 days;
(4) cultivating ripe Dunaliella salina algae liquid in growth pool, remove sand separated with whizzer through algae liquid, will isolate nutrient solution and algae and stick with paste and collect, nutrient solution is for the preparation of step (1) Dunaliella salina nutrient solution, and algae is stuck with paste the processing for the later stage.
2. a kind of method of utilizing spirulina breeding liquid raffinate cultivation Dunaliella salina according to claim 1, it is characterized in that: the salinity kind that described spirulina breeding liquid raffinate is contained and concentration difference: sodium-chlor 1～3g/L, sodium carbonate 9～20g/L, sodium bicarbonate 0.1～2g/L, potassium primary phosphate 0.01～0.05g/L, SODIUMNITRATE 0.5～1g/L, ferrous sulfate 0.001～0.005g/L, calcium chloride 0.05～0.25g/L, Repone K 0.05～0.25g/L, magnesium sulfate 0.3～0.6g/L, magnesium chloride 0.8～1.8g/L, the pH value of spirulina breeding liquid raffinate is 9～14.
3. a kind of method of utilizing spirulina breeding liquid cultivation Dunaliella salina according to claim 1, is characterized in that: the Dunaliella salina nutrient solution in described step (1) is that to utilize pH value is 10～14, carbonate content is 9～20g/L spirulina breeding liquid raffinate to add concentration be that hydrochloric acid 5.000～8.000g/L of 20% is formulated.
4. a kind of method of utilizing spirulina breeding liquid cultivation Dunaliella salina according to claim 3, is characterized in that: described hydrochloric acid preferred content is 6.558g/L.
5. a kind of method of utilizing spirulina breeding liquid cultivation Dunaliella salina according to claim 3, is characterized in that: after step (1) has been prepared, the concentration of the sodium-chlor in Dunaliella salina nutrient solution is 12～36%.
6. a kind of method of utilizing spirulina breeding liquid cultivation Dunaliella salina according to claim 3, it is characterized in that: after step (1) has been prepared, in described Dunaliella salina nutrient solution, kind and the concentration of salinity are as follows: sodium bicarbonate 0.1～0.5g/L, potassium primary phosphate 0.01～0.05g/L, SODIUMNITRATE 0.8～1.2g/L, ferrous sulfate 0.001～0.005g/L, calcium chloride 0.05～0.25g/L, Repone K 0.05～0.25g/L, magnesium sulfate 0.3～0.6g/L, magnesium chloride 0.8～1.8g/L.
7. a kind of method of utilizing spirulina breeding liquid cultivation Dunaliella salina according to claim 1, is characterized in that: the concentration of sodium bicarbonate, potassium primary phosphate, SODIUMNITRATE, ferrous sulfate, calcium chloride, Repone K, magnesium sulfate and magnesium chloride in described step (2) or step (3) Dunaliella salina nutrient solution used is respectively 0.3g/L, 0.03g/L, 1g/L, 0.002g/L, 0.15g/L, 0.18g/L, 0.45g/L, 1.2g/L.
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| CN104161047A (en) * | 2014-07-10 | 2014-11-26 | 浙江大学 | Preparation method of transpiration inhibitor based on pore immune close induced by spirulina |
| CN105176827A (en) * | 2015-10-23 | 2015-12-23 | 厦门大学 | Method for harvesting dunaliella salina by using waste spirulina culture liquid |
| CN106399110A (en) * | 2016-11-15 | 2017-02-15 | 内蒙古科技大学 | Harvesting method of Dunaliella salina |
| CN107435027A (en) * | 2017-01-09 | 2017-12-05 | 内蒙古超越健生物科技有限责任公司 | A kind of collecting method of Dunaliella salina |
| CN111205986A (en) * | 2020-03-31 | 2020-05-29 | 湛江国联水产开发股份有限公司 | Continuous cultivation method of Dunaliella salina |
| CN113234599A (en) * | 2021-05-27 | 2021-08-10 | 青岛琅琊台集团股份有限公司 | Dunaliella salina culture medium and preparation method and culture method thereof |
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| CN106399110B (en) * | 2016-11-15 | 2019-10-22 | 内蒙古科技大学 | A kind of collecting method of Dunaliella salina |
| CN107435027A (en) * | 2017-01-09 | 2017-12-05 | 内蒙古超越健生物科技有限责任公司 | A kind of collecting method of Dunaliella salina |
| CN111205986A (en) * | 2020-03-31 | 2020-05-29 | 湛江国联水产开发股份有限公司 | Continuous cultivation method of Dunaliella salina |
| CN113234599A (en) * | 2021-05-27 | 2021-08-10 | 青岛琅琊台集团股份有限公司 | Dunaliella salina culture medium and preparation method and culture method thereof |
| CN113234599B (en) * | 2021-05-27 | 2022-09-30 | 青岛琅琊台集团股份有限公司 | Dunaliella salina culture medium and preparation method and culture method thereof |
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Effective date of registration: 20170914 Address after: 017000 the Inner Mongolia Autonomous Region city Etuokeqi Erdos Wulan Town Industrial Park of Spirulina Patentee after: Etuokeqi de Rong Zaoye limited liability company Address before: 016100 the Inner Mongolia Autonomous Region city Etuokeqi Ordos town home district 10 building in 1 unit 402 room Patentee before: Zhang Derong |