CN101235096B - Method for extracting polysaccharide from phormidium tenue - Google Patents
Method for extracting polysaccharide from phormidium tenue Download PDFInfo
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- CN101235096B CN101235096B CN2008100467704A CN200810046770A CN101235096B CN 101235096 B CN101235096 B CN 101235096B CN 2008100467704 A CN2008100467704 A CN 2008100467704A CN 200810046770 A CN200810046770 A CN 200810046770A CN 101235096 B CN101235096 B CN 101235096B
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- phormidium tenue
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Abstract
The invention discloses a method for extracting polysaccharide from phormidium tenue, which comprises preparing sodium nitrate, dibasic potassium phosphate, magnesium sulfate, calcium chloride, citrate, ammonium iron citrate, disodium editate, sodium carbonate and micro element solution into culture medium, arranging phormidium tenue into the culture medium to cultivate and prepare phormidium tenue powder, extracting and purifying polysaccharide from the phormidium tenue polysaccharide powder, harvesting and drying. The invention has simple process, easy operation, high reliability, yield and quality, and the equipments are simple, thus is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of method of from Phormidium tenue, extracting polysaccharide, belong to biological technical field.
Background technology
Phormidium tenue belongs to Cyanophyta, Cyanophyceae, hormogon[ium order, Oscillariaceae, Phormidium.In the natural algae skinning of arid, semiarid zone, often there is Phormidium tenue to distribute.At present, the utilization of Phormidium tenue generally only rested on form artificial algae skinning and improve the soil and administer desertification, to other application and developments of Phormidium tenue still blank out almost.It is that polysaccharide content is generally all at more than 36% of its dry weight because it is rich in polysaccharide that Phormidium tenue is used to form the major reason that artificial algae skinning carries out desert treatment.Algal polysaccharides can change the flow of solution feature, increases the viscosity of solution, forms stable gel, can be used as flocculation agent; The wetting ability of polysaccharide macro-molecular and hydrophobicity can be used as emulsifying agent or biological flocculation and precipitation agent; It can be used for removing the heavy metal ion in the waste water in conjunction with the ability of electric charge.Therefore, they can be used for foodstuffs industry, pharmaceutical industry, cosmetic industry, industrial textile and petroleum industry etc.The polysaccharide that utilizes is to extract to obtain from kelp mostly at present, and their polysaccharide content is lower than the Phormidium tenue mostly, in addition, cultivates the cost of kelp and wants high more than the cost of cultivating Phormidium tenue, thereby improved the price of polysaccharide.
Summary of the invention
The object of the present invention is to provide a kind of method of extracting polysaccharide from Phormidium tenue, this is easy to implement the method, easy to operate, safe and reliable, and cost is low, good product quality, and equipment used simply is suitable for suitability for industrialized production.
The method of extracting polysaccharide from Phormidium tenue comprises the steps:
(1) preparation of nutrient solution: add SODIUMNITRATE 0.8～2.5g by every premium on currency, dipotassium hydrogen phosphate 0.02～0.05g, sal epsom 0.06～0.09g, calcium chloride 0.02～0.05g, citric acid 0.004～0.008g, ferric ammonium citrate 0.004～0.008g, disodium EDTA 0.001～0.003g, yellow soda ash 0.01～0.04g, trace element solution 0.5～2ml, wherein trace element solution is by adding boric acid 270～300mg in every 100ml distilled water, tetrahydrate manganese chloride 170～200mg, Zinc Sulphate Heptahydrate 18～25mg, Sodium Molybdate Dihydrate 15～30mg, cupric sulfate pentahydrate 6～10mg stirs evenly after the dissolving fully and makes nutrient solution;
(2) cultivation of Phormidium tenue: at first be that one-level is cultivated, the Phormidium tenue kind is placed static cultivation of nutrient solution or aerated culture, regulate air-flow at 1.5～3.5L.min
-1, light intensity is controlled at 65～85 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, after cultivating 5～10 days, is transferred to secondary and cultivates; Next is that secondary is cultivated, and the culture of one-level being cultivated gained is linked in the new nutrient solution, and aerated culture is regulated air-flow at 2.5～5L.min
-1, aseptically process, light intensity are controlled at 75～95 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, after cultivating 5～10 days, is transferred to three grades of cultivations; The 3rd is three grades of cultivations, and the culture of secondary being cultivated gained is linked in the new nutrient solution, and aerated culture is regulated air-flow at 3～6L.min
-1, aseptically process, light intensity are controlled at 70～100 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, cultivates after 5～10 days the inoculation provenance of gained algae kind for continuing to cultivate; Continue cultivating at first is that the partial circulating cultivation pool is cultivated, and adopts the algae kind of gained after three grades of cultivations, access partial circulating cultivation pool, ratio in every liter of nutrient solution 0.2～0.5g weight in wet base inserts, controlled temperature utilizes natural light to carry out illumination at 22～35 ℃, and the control intensity of illumination is at 120～180 μ E.m
-2.s
-1, stir substratum; Next is that the systemic circulation cultivation pool is cultivated, the Phormidium tenue of cultivating in the partial circulating cultivation pool 4～7 days is transferred in the systemic circulation cultivation pool, the volume ratio of nutrient solution is 1: 20～25 in partial circulating cultivation pool and the systemic circulation cultivation pool, with controlled temperature in the systemic circulation cultivation pool at 22～35 ℃, utilize natural light to carry out illumination, the control intensity of illumination is at 120～180 μ E.m
-2.s
-1The Phormidium tenue screening of cultivating after 10～20 days is received to such an extent that the Phormidium tenue algae is starched;
When above-mentioned Phormidium tenue is inoculated, in the ratio access of every liter of nutrient solution 0.1～0.5g weight in wet base.
(3) preparation of Phormidium tenue powder: the Phormidium tenue algae is starched centrifugal acquisition Phormidium tenue algae mud, then Phormidium tenue algae slurry is obtained the Phormidium tenue powder after concentrated, dehydration, drying, pulverizing;
(4) extracting of Phormidium tenue polysaccharide: the distilled water that in the Phormidium tenue powder, adds 7～10 times of weight, stir evenly, then under 70～90 ℃, stir extracting 3～5 hours, centrifugal 10～15min under 3000～6000rpm condition, collect supernatant liquor, in supernatant liquor, add isopyknic chloroform～propyl carbinol 4～7: 1v/v solution, vibration 20～30min, the centrifugal 10～15min of 3000～6000rpm, collect upper solution, the freezing in advance dehydrated alcohol to 0～4 ℃ that adds 3～5 times of volumes shakes up, and leaves standstill 10～20min, the white flocculent precipitate of separating out is the Phormidium tenue polysaccharide, and the centrifugal 5～10min of 3000～6000rpm collects polysaccharide;
(5) purifying, results and dry: at first be purifying, above-mentioned gained polysaccharide is dissolved in 4～6 times of distilled water, with 10～15 times of 0～4 ℃ of cold absolute ethanol washings; Next is results, with the centrifugal 5～10min of above-mentioned solution 8000～10000rpm in whizzer, collects the polysaccharide precipitation thing; The 3rd is dry, with the polysaccharide precipitation thing collected-60～-110 ℃ of following lyophilizes, polysaccharide product.
The present invention has the following advantages and distinguished effect: easy to implement the method, economy is quick, easy to operate, safe and reliable, output is high, good product quality, and equipment simply is applicable to commercial scale production.
Embodiment
The present invention extracts polysaccharide by following steps from Phormidium tenue:
The preparation of A, nutrient solution
Add SODIUMNITRATE 1.5g, dipotassium hydrogen phosphate 0.04g, sal epsom 0.075g, calcium chloride 0.036g, citric acid 0.006g, ferric ammonium citrate 0.006g, disodium EDTA 0.001g, yellow soda ash 0.02g by every premium on currency, trace element solution 1ml, wherein trace element solution stirs evenly after the dissolving fully by adding boric acid 286mg, tetrahydrate manganese chloride 181mg, Zinc Sulphate Heptahydrate 22.2mg, Sodium Molybdate Dihydrate 25.2mg, cupric sulfate pentahydrate 7.9mg in every 100ml distilled water.
The cultivation of B, Phormidium tenue
(1) one-level is cultivated: the Phormidium tenue kind is inserted the triangular flask that nutrient solution is housed, and (in 150～200ml), static cultivation or aerated culture during aerated culture, are regulated the air-flow size at 2.5L.min
-1, air-flow carries out aseptically process through cotton ball, and light intensity is controlled at 70 μ E.m
-2.s
-1, temperature is controlled at 30 ℃.After cultivating 7～10 days, be transferred to secondary and cultivate.
(2) secondary is cultivated: the culture that one-level is cultivated gained is linked into the culturing bottle that nutrient solution is housed, and (in 500～1000ml), aerated culture is regulated the air-flow size at 3L.min
-1, aseptically process, light intensity are controlled at 80 μ E.m
-2.s
-1, temperature is controlled at 30 ℃.After cultivating 7～10 days, be transferred to three grades of cultivations.
(3) three grades of cultivations: the culture that secondary is cultivated gained is linked into the culturing bottle that nutrient solution is housed, and (in 5000～20000ml), aerated culture is regulated the air-flow size at 5L.min
-1, aseptically process, light intensity are controlled at 90 μ E.m
-2.s
-1, temperature is controlled at 30 ℃.After cultivating 7～10 days, just can be used as the inoculation provenance that continues cultivation.
(4) continue to cultivate
1. partial circulating cultivation pool (1～2m
3) cultivate: adopts the algae kind of gained after three grades of cultivations, access partial circulating cultivation pool.Adopt glass temperature canopy controlled temperature at 30 ℃, utilize natural light to carry out illumination, adopt shading screen control intensity of illumination at 120～180 μ E.m
-2.s
-1, impeller or submersible pump stir substratum.
2. systemic circulation cultivation pool (40～60m
3) cultivate: the Phormidium tenue of cultivating in the partial circulating cultivation pool 4～7 days is transferred in the systemic circulation cultivation pool, and the volume ratio of nutrient solution is 1: 20～25 in partial circulating cultivation pool and the systemic circulation cultivation pool.Other culture condition are identical with the culture condition of partial circulating cultivation pool.
When above-mentioned Phormidium tenue is inoculated, in the ratio access of every liter of nutrient solution 0.1～0.5g weight in wet base.
The preparation of C, Phormidium tenue powder
The Phormidium tenue of cultivating in the systemic circulation cultivation pool after 15～20 days is extracted into filtration unit with pump---on the vibration inclined-plane sieve, behind vibration inclined-plane sieve, receive Phormidium tenue algae slurry, then with the algae slurry through the centrifugal acquisition Phormidium tenue of whizzer algae mud.Phormidium tenue algae mud is obtained the Phormidium tenue powder after concentrated, dehydration, drying, pulverizing.
The extracting of D, Phormidium tenue polysaccharide
In the Phormidium tenue powder, add the distilled water of 7～10 times of weight, fully stir evenly.Under 80 ℃, utilize agitator ceaselessly to stir extracting 3～5 hours then.In whizzer, the centrifugal 10～15min of 3000～6000rpm collects supernatant liquor.Isopyknic chloroform～the propyl carbinol of adding in supernatant liquor (5: 1, v/v) solution, vibration 20～30min, the centrifugal 10～15min of 3000～6000rpm.Lower floor is chloroform, butanol solution, and the middle level is for mainly comprising proteinic suspension, and the upper strata is the Crude polysaccharides solution that mainly comprises polysaccharide.Collect Crude polysaccharides solution, the freezing in advance dehydrated alcohol to 0～4 ℃ that adds 3～5 times of volumes shakes up, and leaves standstill 10～20min, and the white flocculent precipitate of separating out is the Phormidium tenue polysaccharide.Centrifugal 5～the 10min of 3000～6000rpm collects polysaccharide.
E, purifying, results and drying
Purifying: above-mentioned gained polysaccharide is dissolved in 4～6 times of distilled water (w/v), with 10～15 times (w/v) 0 ℃ of cold absolute ethanol washing;
Results: with the centrifugal 5～10min of above-mentioned solution 8000～10000rpm in whizzer, collecting precipitation thing;
Dry: with the polysaccharide precipitation thing collected-100 ℃ of following lyophilizes, polysaccharide product.
Claims (3)
1. a method of extracting polysaccharide from Phormidium tenue is characterized in that comprising the steps:
(1) preparation of nutrient solution: add SODIUMNITRATE 0.8～2.5g by every premium on currency, dipotassium hydrogen phosphate 0.02～0.05g, sal epsom 0.06～0.09g, calcium chloride 0.02～0.05g, citric acid 0.004～0.008g, ferric ammonium citrate 0.004～0.008g, disodium EDTA 0.001～0.003g, yellow soda ash 0.01～0.04g, trace element solution 0.5～2ml is mixed with nutrient solution, wherein trace element solution adds boric acid 270～300mg for pressing in every 100ml distilled water, tetrahydrate manganese chloride 170～200mg, Zinc Sulphate Heptahydrate 18～25mg, Sodium Molybdate Dihydrate 15～30mg, cupric sulfate pentahydrate 6～10mg stirs evenly after the dissolving fully and promptly makes nutrient solution;
(2) cultivation of Phormidium tenue: at first be that one-level is cultivated, the Phormidium tenue kind is placed static cultivation of nutrient solution or aerated culture, regulate air-flow at 1.5～3.5L.min
-1, light intensity is controlled at 65～85 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, cultivates and is transferred to the secondary cultivation after 5～10 days; Next is that secondary is cultivated, and the culture of one-level being cultivated gained is linked into aerated culture in the new nutrient solution, regulates air-flow at 2.5～5L.min
-1, aseptically process, light intensity are controlled at 75～95 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, cultivates after 5～10 days, is transferred to three grades of cultivations; The 3rd is three grades of cultivations, and the culture of secondary being cultivated gained is linked in the new nutrient solution, and aerated culture is regulated air-flow at 3～6L.min
-1, aseptically process, light intensity are controlled at 70～100 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, cultivates after 5～10 days the inoculation provenance of gained algae kind for continuing to cultivate; Continue cultivating at first is that the partial circulating cultivation pool is cultivated, and adopts the algae kind of gained after three grades of cultivations, inserts the partial circulating cultivation pool, and controlled temperature utilizes natural light to carry out illumination at 22～35 ℃, controls intensity of illumination at 120～180 μ E.m
-2.s
-1, stir substratum; Next is that the systemic circulation cultivation pool is cultivated, the Phormidium tenue of cultivating in the partial circulating cultivation pool 4～7 days is transferred in the systemic circulation cultivation pool cultivates, at 22～35 ℃, utilize natural light to carry out illumination controlled temperature in the systemic circulation cultivation pool, the control intensity of illumination is at 120～180 μ E.m
-2.s
-1The Phormidium tenue screening of cultivating after 10～20 days is received to such an extent that the Phormidium tenue algae is starched;
(3) preparation of Phormidium tenue powder: the Phormidium tenue algae is starched centrifugal acquisition Phormidium tenue algae mud, and Phormidium tenue algae mud obtains the Phormidium tenue powder after concentrated, dehydration, drying, pulverizing then;
(4) extracting of Phormidium tenue polysaccharide: the distilled water that in the Phormidium tenue powder, adds 7～10 times of weight, stir evenly, then under 70～90 ℃, stir extracting 3～5 hours, centrifugal 10～15min under 3000～6000rpm condition, collect supernatant liquor, in supernatant liquor, add isopyknic chloroform butanol solution, vibration 20～30min, the centrifugal 10～15min of 3000～6000rpm, collect upper solution, the freezing in advance dehydrated alcohol to 0～4 ℃ that adds 3～5 times of volumes shakes up, and leaves standstill 10～20min, the white flocculent precipitate of separating out is the Phormidium tenue polysaccharide, and the centrifugal 5～10min of 3000～6000rpm collects polysaccharide;
(5) purifying, results and dry: at first be purifying, above-mentioned gained polysaccharide is dissolved in 4～6 times of distilled water, with 10～15 times of 0～4 ℃ of cold absolute ethanol washings; Next is results, and the solution behind the purifying with the centrifugal 5～10min of 8000～10000rpm, is collected the polysaccharide precipitation thing; The 3rd is dry, with the polysaccharide precipitation thing collected-60～-110 ℃ of following lyophilizes, polysaccharide product.
2. the method for extracting polysaccharide from Phormidium tenue according to claim 1, it is characterized in that: the volume ratio of nutrient solution is 1: 20～25 in partial circulating cultivation pool and the systemic circulation cultivation pool.
3. the method for extracting polysaccharide from Phormidium tenue according to claim 1, it is characterized in that: chloroform and propyl carbinol volume ratio are 4～7: 1 in the used chloroform butanol solution.
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| CN2008100467704A CN101235096B (en) | 2008-01-25 | 2008-01-25 | Method for extracting polysaccharide from phormidium tenue |
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| CN105218696A (en) * | 2015-10-31 | 2016-01-06 | 仇颖超 | A kind of preparation method of high purity fucoidin |
| CN114796968A (en) * | 2022-04-15 | 2022-07-29 | 阿尔格生命科学(江苏)有限公司 | Preparation method of algae active high-efficiency crude oil degradation agent |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297948A (en) * | 1999-11-30 | 2001-06-06 | 中国科学院南海海洋研究所 | Algae polysaccharide and its preparation and use |
| JP2005110620A (en) * | 2003-10-10 | 2005-04-28 | Eisaku Oikawa | Method for identifying phormidium tenue and primer used for the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297948A (en) * | 1999-11-30 | 2001-06-06 | 中国科学院南海海洋研究所 | Algae polysaccharide and its preparation and use |
| JP2005110620A (en) * | 2003-10-10 | 2005-04-28 | Eisaku Oikawa | Method for identifying phormidium tenue and primer used for the same |
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