CN101649339B - Method for preparing agarose - Google Patents
Method for preparing agarose Download PDFInfo
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- CN101649339B CN101649339B CN 200910192492 CN200910192492A CN101649339B CN 101649339 B CN101649339 B CN 101649339B CN 200910192492 CN200910192492 CN 200910192492 CN 200910192492 A CN200910192492 A CN 200910192492A CN 101649339 B CN101649339 B CN 101649339B
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- agarose
- nutrient solution
- little algae
- algae
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Abstract
The invention discloses a method for preparing agarose. The method comprises the following steps: adopting microalgae Neochlorisoleoabundans as a raw material and culturing the microalgae to secrete and generate a plurality of agarose; and collecting a secretory product to prepare the needed agarose. The agarose is produced by industrially culturing the microalgae; compared with a method for producing the agarose by large algae, such as ocean planted gracilaria, agar, and the like, the method has short production period, high production efficiency and favorable development prospect, can carry out production by fresh water inland and is beneficial for large-scale generalization.
Description
Technical field
The present invention relates to the polysaccharide technical field, be specifically related to a kind of preparation method of agarose.
Background technology
Agarose is by D-semi-lactosi and 3, the linear polysaccharide molecule that 6-inner ether-semi-lactosi alternately links, a kind of important biochemical reagents, being widely used in Protocols in Molecular Biology and other biochemical separate analytical techniques, is indispensable material in the senior biotechnologys such as present biochemical separation, gene recombination, immunoassay, medical diagnosis on disease.Agarose can be removed the agaropectin part and makes through separating by agar (claiming again agar-agar), and the agarose production in the whole world all is take kelp as raw material at present, makes agar, makes agarose through desulfurization again.South China mainly is to utilize fragrant plant mentioned in ancient texts, laver etc. to extract agar for raw material, and recycling agar extracts agarose.
The purposes of agarose aspect biological chemistry, medical science is increasingly extensive, along with the increasingly extensive application of biochemical technology, the demand of agarose is increased day by day.At present, agarose is mainly by import, agarose expensive.
Little algae be a class on land, the high autotrophic organism of widely distributed, the photosynthetic availability in ocean, cellular metabolism produces polysaccharide, protein, pigment etc., make it have good DEVELOPMENT PROSPECT in fields such as food, medicine, genetically engineered, liquid fuels, and the micro algae growth cycle is short, the fast heavy industrialization that is fit to of breeding is cultivated, and the acquisition of associated metabolic product.
Neochloris oleoabundans is a kind of little algae of Chlorophyta Chlorophyceae Chlorococcale Chlorococcum section, international monopoly WO2006/008401 quotes this algae as the part of multiple-microorganism and algae, described the method for the clarification substratum for preparing at least a photosynthesis water and/or fresh water microorganism, and these substratum have been used for cosmetic field.Chinese patent CN101120910 also provides a kind of cosmetic slimming composition that comprises the biomass extract of this algae.
Not yet have at present by industrialization and cultivate the report that little algae produces agarose, also do not utilize Neochloris oleoabundans to produce the correlative study report of agarose.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide a kind of with short production cycle, production efficiency is high, utilizes little algae to prepare the method for agarose.
Above-mentioned purpose of the present invention is achieved by following scheme:
A kind of preparation method of agarose, but the method is to utilize little algae metabolism to produce the products such as agar polysaccharide, protein and pigment, and the characteristics such as the growth cycle of little algae is short, breeding is fast, select a kind of little algae algae kind---Neochloris oleoabundans, by cultivating this little algae, allow its a large amount of secretions produce agarose, collect secretory product and can prepare required agarose.
Above-mentioned preparation method specifically comprises the steps:
(1) little algae is cultivated
Little algae algae kind (Neochloris oleoabundans) is inoculated in the minimal medium that contains 20～60g/l carbohydrate carbon source, cultivated 4～6 days under the condition of 25～32 ℃ of temperature condition and stirring, ventilation, illumination, thickness stops to cultivate to treat to become very by nutrient solution;
The carbohydrate carbon source that above-mentioned carbohydrate carbon source can select any one those skilled in the art to adopt when the preparation substratum is such as lactose, glucose, semi-lactosi etc.
Little algae is cultivated can also take following method:
Little algae algae kind (Neochloris oleoabundans) is inoculated in the inorganic medium, passes into CO
2Add richness (CO
2Concentration is not higher than 20%v/v) air illumination cultivation 1～3 day, make the microalgae cell content in the nutrient solution rise to OD
600Reach 5～10, add in the nutrient solution more through lactose or the glucose solution of sterilization, make that sugared concentration reaches 20～60g/l in the nutrient solution, continue to cultivate 3～4 days, treat nutrient solution very thickness stop to cultivate.
Minimal medium in above-mentioned two kinds of cultural methods is the basic medium that those skilled in the art commonly used, and its prescription is for known in those skilled in the art.
(2) extraction of agarose
Above-mentioned cultivation gained nutrient solution is heated to 50～100 ℃, then frustule is removed in centrifugation under the temperature more than 50 ℃, reclaim centrifuged supernatant, contained required agarose in the supernatant liquor this moment, follow-up any one separation of polysaccharides method of purification that needs employing those skilled in the art to commonly use, all can realize the present invention, obtain required agarose.
The invention provides a kind of from above-mentioned supernatant liquor the method for separation and purification agarose, its step is as follows:
Above-mentioned centrifuged supernatant is concentrated at 45～55 ℃ of lower reduction vaporizations,, place again freezing freezing below-10 ℃ with the enriched material cool to room temperature;
Scars are taken out, cut into fritter, thaw, use filter-cloth filtering, the gel that collection leaches after draining the water, obtains gel after cleaning with clear water, and this gel is agarose.
Above-mentioned gained agarose can also be further refining, removes impurity, and its operation steps is as follows:
Above-mentioned gel is added water and again dissolving of heating again, and reduction vaporization is concentrated.
Above-mentioned enriched material is placed freezing freezing below-10 ℃ again, then scars is taken out and thaw, drain water after, press dry, prepare required comparatively pure agarose after air-dry.
Among the above-mentioned preparation method, different according to practical use, different to the requirement of agarose quality product, can also carry out the methods such as soda boiling in later stage to the agarose for preparing and carry out further purifying.
Compared with prior art, the present invention has following beneficial effect:
The present invention cultivates little algae by industrialization and produces agarose, with produce agaroses by kelps such as ocean plantation fragrant plant mentioned in ancient texts, lavers and compare, preparation method of the present invention is with short production cycle, production efficiency is high, can be in the inland fresh water production, be conducive to large-scale promotion, have good development prospect.
Embodiment
Below in conjunction with specific embodiment the present invention is done further description, but specific embodiment is not done any restriction to the present invention.
Embodiment 1
The agarose of present embodiment, its preparation method comprises the steps:
(1) little algae is cultivated
In the minimal medium that 1L has sterilized, inoculation Neochloris oleoabundans makes cell count reach 1 * 10
6, pass into and contain 5 volume %CO
2Air, air flow 1vvm, 28 ℃ of illumination stir culture, intensity of illumination 300 μ mol/m
2Cultivate the mode that adopts purebred cultivation, cultivated 60 hours, the microalgae cell content in the nutrient solution rises to OD
600Be 5; Add the lactose solution of having sterilized of 100ml 50g/100ml in the nutrient solution, make that sugared concentration reaches about 5g/l in the nutrient solution, continue to cultivate, when total incubation time reaches 120 hours, stop cultivation;
(2) extraction of agarose
Above-mentioned little algae is cultivated the gained nutrient solution be heated to 85 ℃, collect supernatant liquor after centrifugal (3000rpm, 15min);
With above-mentioned supernatant liquor in Rotary Evaporators 55 ℃, reduction vaporization is concentrated to 1/5th of original volume, with gained concentrated solution cool to room temperature, after it becomes gel, is cut into the fritter of 1 * 1cm, and the gel that chops up is placed-10 ℃ of refrigerator overnight, freezing freezing;
The above-mentioned gel that freezes is taken out, thaws in room temperature, drain the water of thawing,
The distilled water and the heating that add 5 times are melted gel, and be concentrated at 55 ℃ of lower reduction vaporizations;
-10 ℃ freezing after, take out from refrigerator and to thaw, drain the water of thawing, with the cleaning of the cold distilled water below 15 ℃ gel, drain, use lyophilize, obtain the 20g agarose, the agarose productive rate is 20g/L (to substratum).
Embodiment 2
The agarose of present embodiment, its preparation method comprises the steps:
(1) little algae is cultivated
In the minimal medium that 10L has sterilized, inoculation Neochloris oleoabundans makes cell count reach 1 * 10
6, pass into and contain 10 volume %CO
2Air, air flow 1vvm, 30 ℃ of illumination stir culture, intensity of illumination 200 μ mol/m
2Cultivate the mode that adopts purebred cultivation, cultivated 60 hours, the microalgae cell content in the nutrient solution rises to OD
600Be 5; Add the glucose solution of having sterilized of 100ml 40g/100ml in the nutrient solution, make that sugared concentration reaches about 4g/l in the nutrient solution, continue to cultivate, when total incubation time reaches about 120 hours, stop cultivation;
(2) extraction of agarose
Above-mentioned little algae is cultivated the gained nutrient solution be heated to 90 ℃, collect supernatant liquor after centrifugal (3000rpm, 15min);
With above-mentioned supernatant liquor in Rotary Evaporators 55 ℃, reduction vaporization is concentrated to 1/5th of original volume, with gained concentrated solution cool to room temperature, after it becomes gel, is cut into the fritter of 1 * 1cm, and the gel that chops up is placed-10 ℃ of refrigerator overnight, freezing freezing;
The above-mentioned gel that freezes is taken out, thaws in room temperature, drain the water of thawing,
The distilled water and the heating that add 5 times are melted gel, and be concentrated at 55 ℃ of lower reduction vaporizations;
-10 ℃ freezing after, take out from refrigerator and to thaw, drain the water of thawing, with the cleaning of the cold distilled water below 15 ℃ gel, drain, use lyophilize, obtain the 200g agarose, the agarose productive rate is 20g/L (to substratum).
Claims (3)
1. the preparation method of an agarose is characterized in that the method is take little algae Neochloris oleoabundans as raw material, by this little algae of cultivation, thereby prepares required agarose;
The method comprises the steps:
(1) little algae is cultivated
Neochloris oleoabundans is inoculated in the minimal medium that contains 20～60g/L carbohydrate carbon source, under the condition of 25～32 ℃ of temperature condition and stirring, ventilation, illumination, cultivated 4～6 days, obtain micro algae culturing liquid;
Perhaps:
Neochloris oleoabundans is inoculated in the inorganic medium, passes into CO
2Add rich air illumination cultivation 1～3 day, make the microalgae cell content in the nutrient solution rise to OD
600Reach 5～10, add in the nutrient solution more through lactose or the glucose solution of sterilization, make that sugared concentration reaches 20～60g/L in the nutrient solution, continue to cultivate 3～4 days, obtain micro algae culturing liquid;
(2) extraction of agarose
Above-mentioned cultivation gained nutrient solution is heated to 50～100 ℃, then being higher than under 50 ℃ the temperature condition centrifugation or filtering and remove frustule, reclaims centrifuged supernatant or filtrate, separation and purification obtains required agarose from supernatant liquor or filtrate;
Wherein, in the step (1), the carbohydrate carbon source is lactose, glucose or semi-lactosi or contains these sugared carbon source raw materials.
2. described preparation method according to claim 1 is characterized in that in the described step (2), reclaim centrifuged supernatant after, supernatant liquor is concentrated at 45～55 ℃ of lower reduction vaporizations,, place again freezing freezing below-10 ℃ with the enriched material cool to room temperature; Scars are taken out, cut into fritter, thaw, the gel that collection leaches after filtering drains the water after cleaning with clear water, obtains required agarose.
3. described preparation method according to claim 2, it is characterized in that preparing the gained agarose can also further exquisite purifying, and its step is as follows: will prepare the gained agarose and add distilled water and again dissolving of heating, reduction vaporization concentrates; Enriched material is placed again freezing freezing below-10 ℃, then scars is taken out and thaw, drain water after, press dry, prepare required agarose after air-dry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910192492 CN101649339B (en) | 2009-09-18 | 2009-09-18 | Method for preparing agarose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910192492 CN101649339B (en) | 2009-09-18 | 2009-09-18 | Method for preparing agarose |
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| Publication Number | Publication Date |
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| CN101649339A CN101649339A (en) | 2010-02-17 |
| CN101649339B true CN101649339B (en) | 2013-03-13 |
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| CN 200910192492 Expired - Fee Related CN101649339B (en) | 2009-09-18 | 2009-09-18 | Method for preparing agarose |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109182133A (en) * | 2018-10-25 | 2019-01-11 | 攀枝花学院 | Microalgae pure medium and the method for isolating and purifying microalgae |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006008401A2 (en) * | 2004-07-16 | 2006-01-26 | Source Marine De L'ile Grande | Composition containing a photosynthetic marine and/or fresh-water microorganism culture medium and/or |
| CN101120910A (en) * | 2006-08-11 | 2008-02-13 | Lvmh研究公司 | Cosmetic slimming composition containing an biomass extract of the alga neochloris oleoabundans |
-
2009
- 2009-09-18 CN CN 200910192492 patent/CN101649339B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006008401A2 (en) * | 2004-07-16 | 2006-01-26 | Source Marine De L'ile Grande | Composition containing a photosynthetic marine and/or fresh-water microorganism culture medium and/or |
| CN101120910A (en) * | 2006-08-11 | 2008-02-13 | Lvmh研究公司 | Cosmetic slimming composition containing an biomass extract of the alga neochloris oleoabundans |
Non-Patent Citations (4)
| Title |
|---|
| Moore，B.G.et al.Extracellular polysaccharides of algae:Effects on life-support systems.《Science》.1964,第145卷(第3632期),586-587. * |
| Nan Wu et al.Optimization of Biopolymer Production from Lactose using a Green Alga Strain.《8th World Congress of Chemical Engineering》.2009,1-5. * |
| Sukhoverkhov S.V. |
| Sukhoverkhov,S.V.,et al.Production of agar and agarose from the red alga Ahnfeltia tobuchiensis.《Applied Biochemistry and Microbiology》.2000,第36卷(第2期),201-203. * |
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| CN101649339A (en) | 2010-02-17 |
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