CN104988065A - Dunaliella salina culture medium and culture method - Google Patents
Dunaliella salina culture medium and culture method Download PDFInfo
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- CN104988065A CN104988065A CN201510349245.XA CN201510349245A CN104988065A CN 104988065 A CN104988065 A CN 104988065A CN 201510349245 A CN201510349245 A CN 201510349245A CN 104988065 A CN104988065 A CN 104988065A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The invention relates to a dunaliella salina culture medium. The culture medium per litter comprises 20-60 mg of NaNO3, 20-40 mg of Na3PO4, 15-20 mg of K2HPO4, 10-15 ml of iron salt stock solution, 6-8 mg of 7H2O-ZnSO4, 4-6 mg of 4H2O-MnCl2, 45-54 ug of 5H2O-CuSO4, 200-250 g of sea salt, 0.03-0.2 g of borax, 0.05-0.1 mg of CaCl2 and the balance of water. By adopting the culture medium for culturing dunaliella salina, cell proliferation of the dunaliella salina can be promoted, the content of beta-carotenes in single dunaliella salina cells can reach the maximum value, and meanwhile the synthesis quantity of dunaliella salina protein can be obviously increased.
Description
Technical field
The invention belongs to technical field of food science, be specifically related to a kind of Dunaliella salina substratum and cultural method.
Background technology
Salt algae has stronger adaptability to salinity, and can be grown on close to fresh water under the various Variation of Salinity Conditions of saturated brine, in salt lake and ocean etc., high salinity Area distribution is extensive.Salt algae is unique a kind of kind not having cell walls, and it belongs to a kind of lower eukaryotes.Salt algae is rich in the multiple nutritional components such as polysaccharide compound, trace element, protein, highly unsaturated fatty acids.At present, it has been used to multiple field, as it can be used as the bait of the young such as fish, shrimp, also can be used as important anti-oxidant and moisturizing ingredient and is applied to skin protection cosmetics field.
The molecular formula of β-carotene is C 40 H 56, and relative molecular weight is 536. 88.It is joined end to end by four isoprene double bonds and forms, and respectively there is a β-purple trailing plants ketone ring at molecule two ends, mainly contain alltrans, 9-cis, 13-cis and 15-cis four kinds of forms.Can vitamin A be changed under the effect of β-carotene enzyme in vivo, to the normal g and D of guarantee and
Anti-infective have important effect; β-carotene molecule has the special construction of multiple conjugated polyene double bond, make it can with oxygen radical generation irreversible reaction, can scavenging activated oxygen, anti-oxidant; Can strengthening immunity, improve human immune system and resist carcinogenic ability; Oral β-carotene can prevent the formation of sensitivity of light erythema person, and can reduce skin to purple
The susceptibility of outer light; To cardiovascular disorder, Myocytes Anoxia, there is provide protection simultaneously; In addition, β-carotene or occurring in nature ubiquity, the most stable natural pigment.Therefore, it is widely used in the fields such as medicine, food and makeup.Although β-carotene distribution is wide, content is atomic, so that be difficult to extract from natural resources and produce this product.And in Dunaliella salina cell, have a cup-shaped chromatoplast, pigment in chromatoplast mainly Chlorophylls and Carotenoids, report display, its carotenoid comprises 21 kinds such as xenthophylls, alpha-carotene and β-carotene, and wherein in crystal trans beta-carotene relative content be 74.26%, under extreme conditions, the content beta-carotene accumulated in salt algae can up to 14% of frond dry weight.Therefore, salt algae is made to become one of main source of the natural beta-carotin of generally acknowledging both at home and abroad.But at typical condition, in salt algae, content beta-carotene is unsatisfactory, and when changing its pathways metabolism, salt algae can be synthesized and be accumulated a large amount of β-carotenes in born of the same parents.Therefore, the scientific culture research carrying out salt algae has important significance of scientific research, and the content especially improving β-carotene in salt algae is particularly important.At present, existing correlative study shows, the suitableeest phosphorus source of dunaiella salina growth is phosphoric acid salt, the suitableeest nitrogenous source is nitrate, and about the research of other elements required for the optimum concn of these two kinds of inorganic salt and dunaiella salina growth very few, for this reason, the invention provides a kind of substratum, salt algae output can be improved and especially can improve its content beta-carotene.
Summary of the invention
The object of the invention is to solve the above-mentioned problems in the prior art, provide a kind of extraction efficiency high, β-carotene loss less and the ultrasonic extraction of the wide β-carotene of suitability.
Technical scheme of the present invention is as follows:
A kind of Dunaliella salina substratum, the prescription of described substratum and proportioning are:
NaNO3 20-60mg/L，
Na3PO4 20-40mg/L，
K2HPO4 15-20mg/L，
Mother liquid of iron salt 10-15ml/L,
7 H2O-ZnSO4 6-8mg/L，
4 H2O-MnCl2 4-6mg/L，
5 H2O- CuSO4 45-54ug/L，
Sea salt 200-250 g/L,
Borax 0.03-0.2g/L,
CaCl2 0.05-0.1 mg/L，
Surplus is water;
Preferably, the prescription of described mother liquid of iron salt and proportioning are: Na2-EDTA1.0-1.5mg/L and FeCL3.6H2O 0.25-2mg/L.
Adopt Dunaliella salina substratum as above to carry out salt algae cultural method, comprise the following steps:
(1) first prepare salt algae culture medium, described substratum prescription and proportioning are:
NaNO3 20-60mg/L，
Na3PO4 20-40mg/L，
K2HPO4 15-20mg/L，
Mother liquid of iron salt 10-15ml/L,
7 H2O-ZnSO4 6-8mg/L，
4 H2O-MnCl2 4-6mg/L，
5 H2O- CuSO4 45-54ug/L，
Sea salt 200-250 g/L,
Borax 0.03-0.2g/L,
CaCl2 0.05-0.1 mg/L，
Surplus is water.
(2) the salt algae algae kind of logarithmic growth will be in the centrifugal 5-10min of 2000-5000r/min, abandon original fluid, be placed in the substratum that step (1) prepares, insert intelligent optical after sealing and cultivate according in incubator, measure the density of Dunaliella salina cell and the content of β-carotene.
Preferably, the density method of Dunaliella salina cell is measured in step (2) for starting day to do a cell counting every other day from cultivating.
Preferably, the Cytometric concrete grammar described in step (2): get salt algae algae liquid 1-5ml, adds the anesthesia of 0.1-0.5ml dehydrated alcohol, on instillation blood counting chamber, and microscopy counting under the microscope.
Preferably, the content of the mensuration β-carotene described in step (2) is sampling and measuring when cultivating 5-7d from salt algae.
Preferably, content beta-carotene measuring method described in step (2) is: be placed in getting liquid rifle absorption 5-10ml algae liquid the centrifuge tube being added with NaOH, with the centrifugal 5-10min of 2000-5000r/min, abandon supernatant liquor, add acetone soln, ultrasonic mixing, extract pigment, with 2000-5000r/min recentrifuge 5-10min, suct clear liquid in 25ml volumetric flask, ultrasonic mixing, extract pigment, repeat to extract until frond is white, last united extraction liquid adding distil water is settled to 50ml, absorbancy under employing grating spectrophotometer survey 450nm.
Preferably, described acetone soln concentration is 70-80%, and the add-on of acetone soln is 5-10ml.
Preferably, adopt the method supplemented the nutrients according to the growth velocity of salt algae, be specially: Na3PO4 magnitude of recruitment is 0.2-0.35mg/10 6, K2HPO4 magnitude of recruitment is 0.8-1.2mg/10 6,7 H2O-ZnSO4 magnitude of recruitments be 0.06-0.1mg/L 4 H2O-MnCl2 magnitude of recruitment are 0.02-0.06m g/L.
Preferably, the culture condition described in step (2) is: daytime, culture temperature was 20-25 DEG C, and night is 15-20 DEG C, and intensity of illumination is 3000-5000Lux, and light application time is 8-12h/d.
The salt algae algae kind being in logarithmic growth described in step (1) is provided by Chinese Marine University.
Compared with prior art, beneficial effect is embodied in the present invention:
1. prior art and about in report only to report that hydrochloric acid grows best phosphorus source be phosphoric acid salt, best nitrogenous source is nitrate, and research that salt algae cultivates is affected seldom or imperfection to nutritive ingredients such as trace elements, it is required that zinc, manganese element are dunaiella salina growth institute, substratum in the present invention will with the addition of these two kinds of elements certain density, be conducive to the propagation of Dunaliella salina cell, and the strict concentration controlling these two kinds of elements, make the content beta-carotene in single Dunaliella salina cell reach the highest.Research display, manganese deficiency can affect the formation of salt algae protein, and the present invention also can significantly improve the resultant quantity of salt algae protein.
2. salt algae in the training period, except needs nitrate, phosphoric acid salt and zinc, manganese nutrition composition, also higher to demands such as Zn, Fe, Zn is the enzyme of archaeal dna polymerase, P is that DNA and RNA synthesizes required element, and Fe is the important component of respiratory chain in cell, the present invention, other compositions required for above-mentioned trace element and salt algae being cultivated press finite concentration proportion optimizing, are more conducive to the cultivation of salt algae and the synthesis of β-carotene
3. in prior art, salt algae culture medium is placed on Indoor sill and cultivates, intensity of illumination is 3000 ~ 90000Lux, when cultivating with this understanding, due in room temperature and windowsill every day intensity of illumination and light application time difference excessive, salt algae propagation and the output of β-carotene all unstable.Substratum is placed in intelligent illumination box and cultivates by the present invention, and culture condition is stablized, and is more conducive to the propagation of salt algae and the synthesis of β-carotene.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail:
The salt algae algae kind being in logarithmic growth described in step (1) is provided by Chinese Marine University.
Embodiment 1
A kind of Dunaliella salina substratum, the prescription of described substratum and proportioning are:
NaNO3 20mg/L，
Na3PO4 20mg/L，
K2HPO4 15mg/L，
Mother liquid of iron salt 10ml/L,
7 H2O-ZnSO4 6mg/L，
4 H2O-MnCl2 4mg/L，
5 H2O- CuSO4 45ug/L，
Sea salt 200 g/L,
Borax 0.03g/L,
CaCl2 0.05 mg/L，
Surplus is water.
The prescription of described mother liquid of iron salt and proportioning are: Na2-EDTA1.0mg/L and FeCL3.6H2O 0.25mg/L.
Adopt Dunaliella salina substratum as above to carry out salt algae cultural method, comprise the following steps:
(1) first prepare salt algae culture medium, described substratum prescription and proportioning are:
NaNO3 20mg/L，
Na3PO4 20mg/L，
K2HPO4 15mg/L，
Mother liquid of iron salt 10ml/L,
7 H2O-ZnSO4 6mg/L，
4 H2O-MnCl2 4mg/L，
5 H2O- CuSO4 45ug/L，
Sea salt 200 g/L,
Borax 0.03g/L,
CaCl2 0.05 mg/L，
Surplus is water;
(2) the salt algae algae kind of logarithmic growth will be in the centrifugal 5min of 2000r/min, abandon original fluid, be placed in the substratum that step (1) prepares, insert intelligent optical after sealing to cultivate according in incubator, daytime, culture temperature was 20 DEG C, and night is 15 DEG C, intensity of illumination is 3000Lux, light application time is 8h/d, does the density of a cell counting measuring Dunaliella salina cell every other day, the content of sampling and measuring β-carotene when cultivating 5d from salt algae.
Cytometric concrete grammar described in step (2): get salt algae algae liquid 1ml, adds the anesthesia of 0.1ml dehydrated alcohol, on instillation blood counting chamber, and microscopy counting under the microscope.
Content beta-carotene measuring method described in step (2) is: be placed in getting liquid rifle absorption 5ml algae liquid the centrifuge tube being added with NaOH, with the centrifugal 5min of 2000r/min, abandon supernatant liquor, add the acetone soln that 5ml concentration is 70%, ultrasonic mixing, extract pigment, with 2000r/min recentrifuge 5min, suct clear liquid in 25ml volumetric flask, ultrasonic mixing, extracts pigment, repeats to extract until frond is white, last united extraction liquid adding distil water is settled to 50ml, absorbancy under employing grating spectrophotometer survey 450nm.
Cultivation described in step (2) is adopt according to the growth velocity of salt algae the method supplemented the nutrients, and is specially: Na3PO4 magnitude of recruitment is 0.25mg/10 6, K2HPO4 magnitude of recruitment is 0.8mg/10 6,7 H2O-ZnSO4 magnitude of recruitments be 0.06mg/L 4 H2O-MnCl2 magnitude of recruitment are 0.02m g/L.
Embodiment 2
A kind of Dunaliella salina substratum, the prescription of described substratum and proportioning are:
NaNO3 60mg/L，
Na3PO4 40mg/L，
K2HPO4 20mg/L，
Mother liquid of iron salt 15ml/L,
7 H2O-ZnSO4 8mg/L，
4 H2O-MnCl2 6mg/L，
5 H2O- CuSO4 54ug/L，
Sea salt 250 g/L,
Borax 0.2g/L,
CaCl2 0.1 mg/L，
Surplus is water.
Preferably, the prescription of described mother liquid of iron salt and proportioning are: Na2-EDTA 1.5mg/L and FeCL3.6H2O 2mg/L.
Adopt Dunaliella salina substratum as above to carry out salt algae cultural method, comprise the following steps:
(1) first prepare salt algae culture medium, described substratum prescription and proportioning are:
NaNO3 60mg/L，
Na3PO4 40mg/L，
K2HPO4 20mg/L，
Mother liquid of iron salt 15ml/L,
7 H2O-ZnSO4 8mg/L，
4 H2O-MnCl2 6mg/L，
5 H2O- CuSO4 54ug/L，
Sea salt 250 g/L,
Borax 0.2g/L,
Surplus is water;
(2) the salt algae algae kind of logarithmic growth will be in the centrifugal 10min of 5000r/min, abandon original fluid, be placed in the substratum that step (1) prepares, insert intelligent optical after sealing to cultivate according in incubator, daytime, culture temperature was 25 DEG C, and night is 20 DEG C, intensity of illumination is 5000Lux, light application time is 12h/d, does the density of a cell counting measuring Dunaliella salina cell every other day, the content of sampling and measuring β-carotene when cultivating 7d from salt algae.
Cytometric concrete grammar described in step (2): get salt algae algae liquid 5ml, adds the anesthesia of 0.5ml dehydrated alcohol, on instillation blood counting chamber, and microscopy counting under the microscope.
Content beta-carotene measuring method described in step (2) is: be placed in getting liquid rifle absorption 10ml algae liquid the centrifuge tube being added with NaOH, with the centrifugal 10min of 5000r/min, abandon supernatant liquor, add the acetone soln that 10ml concentration is 80%, ultrasonic mixing, extract pigment, with 5000r/min recentrifuge 10min, suct clear liquid in 25ml volumetric flask, ultrasonic mixing, extracts pigment, repeats to extract until frond is white, last united extraction liquid adding distil water is settled to 50ml, absorbancy under employing grating spectrophotometer survey 450nm.
Cultivation described in step (2) is adopt according to the growth velocity of salt algae the method supplemented the nutrients, and is specially: Na3PO4 magnitude of recruitment is 0.35mg/10 6, K2HPO4 magnitude of recruitment is 1.2mg/10 6,7 H2O-ZnSO4 magnitude of recruitments be 0.1mg/L 4 H2O-MnCl2 magnitude of recruitment are 0.06mg/L.
Embodiment 3
A kind of Dunaliella salina substratum, the prescription of described substratum and proportioning are:
NaNO3 40mg/L，
Na3PO4 30mg/L，
K2HPO4 18mg/L，
Mother liquid of iron salt 13ml/L,
7 H2O-ZnSO4 7mg/L，
4 H2O-MnCl2 5mg/L，
5 H2O- CuSO4 50ug/L，
Sea salt 230g/L,
Borax 0.1g/L,
CaCl2 0.08mg/L，
Surplus is water.
Preferably, the prescription of described mother liquid of iron salt and proportioning are: Na2-EDTA1.3mg/L and FeCL3.6H2O 1 mg/L.
Adopt Dunaliella salina substratum as above to carry out salt algae cultural method, comprise the following steps:
(1) first prepare salt algae culture medium, described substratum prescription and proportioning are:
NaNO3 40mg/L，
Na3PO4 30mg/L，
K2HPO4 18mg/L，
Mother liquid of iron salt 13ml/L,
7 H2O-ZnSO4 7mg/L，
4 H2O-MnCl2 5mg/L，
5 H2O- CuSO4 50ug/L，
Sea salt 230g/L,
Borax 0.1g/L,
CaCl2 0.08mg/L，
Surplus is water;
(2) the salt algae algae kind of logarithmic growth will be in the centrifugal 8min of 3500r/min, abandon original fluid, be placed in the substratum that step (1) prepares, insert intelligent optical after sealing to cultivate according in incubator, daytime, culture temperature was 22 DEG C, and night is 18 DEG C, intensity of illumination is 4000Lux, light application time is 10h/d, does the density of a cell counting measuring Dunaliella salina cell every other day, the content of sampling and measuring β-carotene when cultivating 6d from salt algae.
Cytometric concrete grammar described in step (2): get salt algae algae liquid 3ml, adds the anesthesia of 0.3ml dehydrated alcohol, on instillation blood counting chamber, and microscopy counting under the microscope.
Content beta-carotene measuring method described in step (2) is: be placed in getting liquid rifle absorption 8ml algae liquid the centrifuge tube being added with NaOH, with the centrifugal 8min of 3500r/min, abandon supernatant liquor, add the acetone soln that 8ml concentration is 75%, ultrasonic mixing, extract pigment, with 3500r/min recentrifuge 8min, suct clear liquid in 25ml volumetric flask, ultrasonic mixing, extracts pigment, repeats to extract until frond is white, last united extraction liquid adding distil water is settled to 50ml, absorbancy under employing grating spectrophotometer survey 450nm.
Cultivation described in step (2) is adopt according to the growth velocity of salt algae the method supplemented the nutrients, and is specially: Na3PO4 magnitude of recruitment is 0.3mg/10 6, K2HPO4 magnitude of recruitment is 1.0mg/10 6,7 H2O-ZnSO4 magnitude of recruitments be 0.08mg/L 4 H2O-MnCl2 magnitude of recruitment are 0.04m g/L.
Above-described embodiment is only the preferred embodiment of the present invention, and should not be construed as limitation of the invention, and those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (10)
1. a Dunaliella salina substratum, is characterized in that, the prescription of described substratum and proportioning are:
2. a kind of Dunaliella salina substratum according to claim 1, is characterized in that, the prescription of described mother liquid of iron salt and proportioning are: Na2-EDTA1.0-1.5mg/L and FeCL3.6H2O 0.25-2mg/L.
3. adopt as arbitrary in claim 1 ~ 2 as described in Dunaliella salina substratum carry out salt algae cultural method, it is characterized in that, comprise the following steps:
(1) first prepare salt algae culture medium, described substratum prescription and proportioning are:
(2) the salt algae algae kind of logarithmic growth will be in the centrifugal 5-10min of 2000-5000r/min, abandon original fluid, be placed in the substratum that step (1) prepares, insert intelligent optical after sealing to cultivate according in incubator, measure the density of Dunaliella salina cell and the content of β-carotene.
4. salt algae cultural method according to claim 3, is characterized in that, measures the density method of Dunaliella salina cell for starting day to do a cell counting every other day from cultivating in step (2).
5. salt algae cultural method according to claim 4, it is characterized in that, the Cytometric concrete grammar described in step (2): get salt algae algae liquid 1-5ml, add the anesthesia of 0.1-0.5ml dehydrated alcohol, on instillation blood counting chamber, microscopy counting under the microscope.
6. salt algae cultural method according to claim 3, is characterized in that, the content of the mensuration β-carotene described in step (2) is sampling and measuring when cultivating 5-7d from salt algae.
7. salt algae cultural method according to claim 3, it is characterized in that, content beta-carotene measuring method described in step (2) is: be placed in getting liquid rifle absorption 5-10ml algae liquid the centrifuge tube being added with NaOH, with the centrifugal 5-10min of 2000-5000r/min, abandon supernatant liquor, add acetone soln, ultrasonic mixing, extract pigment, with 2000-5000r/min recentrifuge 5-10min, suct clear liquid in 25ml volumetric flask, ultrasonic mixing, extract pigment, repeat to extract until frond is white, last united extraction liquid adding distil water is settled to 50ml, absorbancy under employing grating spectrophotometer survey 450nm.
8. salt algae cultural method according to claim 7, is characterized in that, described acetone soln concentration is 70-80%, and the add-on of acetone soln is 5-10ml.
9. the method for salt algae cultivation according to claim 3, it is characterized in that, described cultivation is adopt according to the growth velocity of salt algae the method supplemented the nutrients, and is specially: Na3P04 magnitude of recruitment is 0.2-0.35mg/10 6, K2HPO4 magnitude of recruitment is 0.8-1.2mg/10 6,7 H2O-ZnSO4 magnitude of recruitments be 0.06-0.1mg/L 4 H2O-MnCl2 magnitude of recruitment are 0.02-0.06m g/L.
10. the method for salt algae cultivation according to claim 3, it is characterized in that, the culture condition described in step (2) is: daytime, culture temperature was 20-25 DEG C, and night is 15-20 DEG C, intensity of illumination is 3000-5000Lux, and light application time is 8-12h/d.
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| CN106517555A (en) * | 2016-11-21 | 2017-03-22 | 天津科技大学 | High-added-value treating method for waste culture liquid of Dunaliella salina |
| WO2019238914A1 (en) | 2018-06-15 | 2019-12-19 | Isp Investments Llc | Method for obtaining an aqueous extract of dunaliella salina and cosmetic uses of same |
| 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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| WO2019238914A1 (en) | 2018-06-15 | 2019-12-19 | Isp Investments Llc | Method for obtaining an aqueous extract of dunaliella salina and cosmetic uses of same |
| 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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