CN105949345A - Extraction method of microcoleus vaginatus intracellular polysaccharide - Google Patents
Extraction method of microcoleus vaginatus intracellular polysaccharide Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
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Abstract
The invention discloses an extraction method of microcoleus vaginatus intracellular polysaccharide. The steps include: (1) subjecting microcoleus vaginatus to large-scale culture, then collecting the algal liquid, filtering the algal liquid to obtain an algal piece, and performing air-drying; (2) crushing the algal sheet to obtain algal powder; (3) mixing the algal powder with sterile water to obtain an extracted solution; (4) concentrating the polysaccharide extracted solution to obtain a polysaccharide concentrated solution; (5) using a trichloroacetic acid solution to precipitate protein, performing centrifugation and taking the supernatant; (6) using ethanol to precipitate polysaccharide, and staying the product overnight, conducting centrifugation, retaining the precipitate, dissolving ethanol precipitated crude polysaccharide in distilled water, carrying out running water dialysis, collecting polysaccharide (high molecular weight); (7) after dialysis, adding the dialysate into ethanol; (8) evaporating the ethanol in the precipitated phase to obtain polysaccharide, and conducting freeze drying. The method has the advantages of simple operation, low cost, simple equipment, high extraction rate, good product purity, and safety and reliability, reaches a multi-stage separation and extraction effect, and is suitable for industrial large-scale production.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of extraction purification intracellular polysaccharide from tool sheath micro-sheath algae powder
Method.
Background technology
Since the eighties in 20th century, in world wide, Microalgae biotechnology and microalgae metabolism physiology are rapidly sent out
Exhibition, finds and has paid attention to basis and the applied research of microalgae.Raw based on the microalgae that microdisk electrode and product thereof develop
Thing technology is that people solve one of effective ways of problem such as grain, the energy, environmental protection and medicine.Along with microalgae industry rise with
Stable development, the effect such as the antitumor of microalgal polysaccharide, antiviral, radioprotective, blood sugar lowering and immunomodulating is the most gradually by people
Recognized and accepted, developing as medicine, pharmaceutical intermediate, functional food or cosmetics for this kind of product and provide the foundation.
China's microdisk electrode and product development have become as emerging biotechnology industry, as spirulina, chlorella, Haematococcus Pluvialis, salt alga,
Nostocs etc. have realized large-scale cultivation.In recent ten years, research and application about desert microalgae cause extensive concern and weight
Depending on.In microalgae resource, desert algae is considered as a kind of challenging, promising living resources, and its exploitation application is ground
Study carefully in urgent need of strengthening.
Tool sheath micro-sheath algae (Microcoleus Vaginatus) it is that desert soil is distributed most commonly used desert algae thing
One of kind, its Biomass is sometime up to more than the 95% of soil microorganism total amount.Soil and biological breadcrust in China Desert Area
In, tool sheath micro-sheath algae is also main sociales.In recent years, tool sheath micro-sheath algae demonstrates good fixing the sand in desertification treatment
Effect and application potential, be one of the important method of the important algae kind of tackling quicksand and sand consolidation with biologic.As special in published China
Profit 102199542A describe a kind of method of isolated and purified tool sheath micro-sheath algae from biological breadcrust, it has been disclosed that Chinese patent
102268376A describes a kind of method using tool sheath micro-sheath algae to build indoor biological skinning.These reports indicate about tool
The research of sheath micro-sheath algae and application have received increasing attention.Research finds, tool sheath micro-sheath algae is except having excellent consolidating
Outside husky skinning function, possibly together with abundant polysaccharide component in its frustule, it is the microalgae resource of a kind of excellent high yield polysaccharides.
According to research reports, the physiological ecology function of tool sheath micro-sheath polysaccharides is relatively broad, and has immunomodulating, antitumor, disease-resistant
The multiple pharmacological effect such as poison, radioprotective, and almost without toxic and side effects.Additionally have sheath micro-sheath polysaccharides in diet nutritional, health care
The effect of the aspects such as conditioning, pre-anti-aging, beauty treatment is the most gradually recognized by people.Micro-sheath algae is a kind of single-row filamentous algae, algae
Filament is flourishing, and without heterocyst, not branch, sheath is firm.Owing to this frustule has a protection of firm glue sheath, thus to grinding or
The resistivity of ultrasound wave is relatively strong, badly influences the extraction of the crushing effect to this frustule and cell inclusion.Therefore, as
What high efficiency extraction micro-sheath algae intracellular polysaccharide, will become and develop micro-sheath polysaccharides resource and problem demanding prompt solution.Tool sheath is micro-
Sheath algae is widely distributed and inexhaustible microalgae living resources as one, and rich in natural polysaecharides material.Therefore, tool sheath
The development and utilization of micro-sheath polysaccharides resource will be likely to become the another emerging field of biotechnology industry, show tempting before
Scape.But the extracting method about micro-sheath algae intracellular polysaccharide has no report the most up to now, it develops not enough not yet.Mesh
Before report more about the extractive technique technique of vegetable polysaccharides and Microbial exopolysaccharides, also frequently seen plants polyose on market
With some microalgal polysaccharide product (such as spirulina polysaccharide etc.), but have no extraction process and the product of desert algae intracellular polysaccharide, more do not have
There is the report being specifically designed for having the extracting method of sheath micro-sheath algae intracellular polysaccharide.
Summary of the invention
The present invention, with one typical desert filamentous cyanobacteria tool sheath micro-sheath algae as Algal material, first carries out people to it
Work pilot scale culture, it is thus achieved that micro-algae powder；Then with algae powder as raw material, establish a set of to tool sheath micro-sheath algae intracellular polysaccharide carry out
Extract and the technical method of multiple stage separation.The present invention provides Polyose extraction purification process in a kind of tool sheath micro-sheath gonidium, and it is taked
The extracting method of the water extract-alcohol precipitation of algae powder, low cost, easy to operate, this technology can keep polysaccharide structures to destroy less, purity
High, reach the effect of multiple stage separation, energy consumption can be reduced again.The inventive method is simple and easy to do, equipment is simple, extraction ratio is high, product
Quality is good, safe and reliable, is applicable to industrial-scale production.
In order to realize above-mentioned purpose, the present invention adopts the following technical scheme that
A kind of extracting method having sheath micro-sheath algae intracellular polysaccharide, comprises the steps:
(1) first pilot scale culture is carried out by having sheath micro-sheath algae.In the present invention, use BG-11 fluid medium as culture medium
Matter, its component is as follows: sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, ethylenediaminetetraacetic acid
Disodium salt (EDTA), sodium carbonate and trace element A5 solution, be configured to culture medium by above components in certain proportion.To have the micro-sheath of sheath
Algae is respectively by I grade of algal species cultivation (being inoculated into little triangular flask by test tube original seed), II grade of algal species cultivation (proceeding to 2L-5L triangular flask)
Spreading cultivation step by step of algae kind is carried out with III grade of algae kind amplification culture (proceeding to 10L-18L bottle).Spread cultivation III grade of algae kind is transferred to light
Bioreactor (80-150L) or small-sized racetrack circulatory pool (1m3) carry out pilot scale culture.Cultivations at different levels in above algae kind
In, condition of culture is: temperature 26-30 DEG C, light intensity 3000Lux-4000Lux, continuous aerobic culture 15-30 d.After testing, training
In nutrient solution, frond Biomass reaches Chlorophyll-a Content when being 8-10 μ g/milliliter, collects algae culturing liquid, standby.
Described BG-11 fluid medium, its component is as follows: sodium nitrate (1.5 g/L), three water dipotassium hydrogen phosphates (0.04
G/L), Magnesium sulfate heptahydrate (0.07 g/L), calcium chloride dihydrate (0.036 g/L), citric acid (0.006 g/L), ferric ammonium citrate
(0.006 g/L), disodium EDTA (0.001 g/L), sodium carbonate (0.02 g/L) and trace element A5 solution
(1 mL).Wherein, A5 solution need to individually be prepared, and in every 1 liter of A5 solution, contained component is as follows: boric acid 2.86 g, molybdate dihydrate acid
Sodium 0.039 g, zinc sulphate heptahydrate 0.222 g, copper sulphate pentahydrate 0.074 g, one water manganese chloride 1.81 g, cobalt nitrate hexahydrate
0.049 g.Preparation 1 liter of BG-11 culture medium time, need to by mentioned reagent according to listed sequencing add to successively distilled water or
Subsoil water after filtered through gauze, is settled to 1 liter, and stirring and dissolving produces to without precipitation.When carrying out I grade of algal species cultivation and II grade of algae
When planting cultivation, BG-11 fluid medium need to be carried out high pressure steam sterilization (121 DEG C, 20 min), carry out III grade of algae kind and expand training
When supporting with pilot scale culture, sterilization treatment do not made by BG-11 fluid medium.In the present invention, I grade of described algal species cultivation refer to by
Algae kind test tube original seed is seeded to little triangular flask and carries out the process of activation culture；Algae kind after II grade of algal species cultivation refers to activation connects
Kind to larger capacity triangular flask carries out the process cultivated；III grade of algal species cultivation refers to that II grade of algae kind is proceeded to larger container expands
The big process cultivated.
(2) the tool sheath micro-sheath algae culturing liquid 200-500 mesh silk collected is filtered algae solution, obtain having sheath micro-sheath algae algae
Mud, air-dries algae mud the most in its natural state, obtains having sheath micro-sheath algae algae sheet.Dry tool sheath micro-sheath algae algae sheet is pulverized,
Sieve, obtain algae sheet tool sheath micro-sheath algae powder (water content ＜ 10%).Algae powder fineness requires to be advisable for 80-100 mesh.
(3) take tool sheath micro-sheath algae powder, algae powder mixed with 1:8-1:10 ratio with sterilized water, heating in water bath 2-4 hour,
Water bath heating temperature controls at 90-98 DEG C, and layer 2-3 filtered through gauze takes supernatant；Residue extracts at least 2-3 the most again
Secondary, each 1.5-2.5 hour, the supernatant extracted is merged, obtains polysaccharide extraction liquid, 4 DEG C of preservations；At above-mentioned heating in water bath
Before reason, algae powder need to mix placement 12-24 hour with sterilized water.
(4) use Rotary Evaporators (RE-5210A, Asia, Shanghai is flourish), at 30-45 DEG C, polysaccharide extraction liquid is concentrated into substance
Long-pending 1/4-1/6, obtains polysaccharide concentrated solution, 4 DEG C of preservations；
(5) with 4 DEG C of solution of trichloroacetic acid precipitating proteins, make trichloroacetic acid content reach the 4-5% of cumulative volume, place 3-4 for 4 DEG C
Hour, 4 DEG C are centrifugal 20-25 minute, take supernatant, and under the same terms (4 DEG C of solution of trichloroacetic acid precipitating proteins 3-4 hour, 4
DEG C centrifugal 20-25 minute) repeat 3-5 time, detect several measured without protein to Coomassie brilliant blue；Described centrifugal extraction
Speed is 3500-3700 rev/min.
(6) using ethanol precipitate polysaccharides, 4 DEG C stand overnight (10-14 hour), and 4 DEG C centrifugal 20-25 minute, stays precipitation, by alcohol
Heavy crude polysaccharides is substantially soluble in distilled water, utilizes bag filter (MWC is 3500 dalton) will have Polyose extraction in sheath micro-sheath gonidium
Liquid is dialysed, and collects the component of more than molecular weight 3500 dalton；
(7) dialysis solution after dialysis adds the ethanol of 3 times of volumes, and 4 DEG C stand overnight (10-14 hour), 4 DEG C of centrifugal 20-25
Minute, take precipitated phase；
(8) by dry for ethanol volatilization in precipitated phase under room temperature (20-25 DEG C, the most identical), by polysaccharide lyophilization, obtain having sheath micro-
Sheath algae intracellular polysaccharide.Tool sheath micro-sheath algae intracellular polysaccharide obtained by being extracted by this method is that a class has raising immunity, resists
The natural active product of the functions such as aging, antitumor and promotion cell growth.This intracellular polysaccharide color and luster is creamy white or light yellow,
Lyophilization is utilized to obtain its powder, easy moisture absorption, soluble in water.Polysaccharide yield computing formula is as follows:
Polysaccharide yield=polysaccharide quality (dry weight)/algae opaque amount (dry weight) × 100%.
Described tool sheath micro-sheath algae intracellular polysaccharide is a kind of typical case's filamentous cyanobacteria tool to separate from desert soil
Sheath micro-sheath algae is for extracting material, and micro-sheath algae is carried out indoor spreads cultivation and pilot scale culture step by step, and condition of culture is: BG-11 liquid
Culture medium, temperature 26-30 DEG C, light intensity 3000Lux-4000Lux, continuous aerobic culture 15-30 days, use 200-500 mesh by algae solution
Silk filters, and collects micro-sheath algae algae mud, is air-dried in its natural state by algae mud, it is thus achieved that micro-sheath algae algae sheet, by micro-sheath algae algae sheet powder
Broken, sieve after obtain algae powder, then algae powder is carried out multistage extract separate, obtain intracellular polysaccharide.
Purity of polysaccharide uses chromatography determination.Determination of polysaccharide uses Anthrone-sulfuricacid method or phend-sulphuric acid to measure.
The Anthrone-sulfuricacid method detection of polyoses content: accurately weigh and have sheath micro-sheath algae intracellular polysaccharide 20mg in 100mL beaker, the most molten
Solve, draw polysaccharide solution 1mL, add anthrone-concentrated sulphuric acid reagent 5.0mL, boil 1min, after taking-up, naturally cool to room temperature, with
Distilled water is blank, colorimetric determination under 620nm wavelength.
Relevant content in described technical scheme is explained as follows:
1, further improvement of the present invention is, first passes through and expand tool sheath micro-sheath algae step by step in step (1) and step (2)
Training and pilot scale culture obtain a large amount of frond cultures and algae sheet, and are prepared as the algae powder with certain specification requirement.
2, further improvement of the present invention is, before step (3) heating in water bath algae powder mix with sterilized water placement 12-24 little
Time.
3, further improvement of the present invention is, step (3) algae powder and sterilized water water bath heating temperature are 90-98 DEG C.
4, further improvement of the present invention is, the speed of the described centrifugal extraction of step (5) is 3500-3700 rev/min.
5, further improvement of the present invention is, the speed of step (6) and the described centrifugal extraction of step (7) is 3000-
3200 revs/min.
6, further improvement of the present invention is, step (6) and step (7) described ethanol be concentration be percent by volume
The ethanol of 90-98%.Preferably, step (6) and step (7) described ethanol be concentration be the ethanol of percent by volume 95%.
7, further improvement of the present invention is, employing trichloroacetic acid (TCA) method described in step (5) replaces Sevag method
Removing isolating protein, make polysaccharide loss rate in extraction process of the present invention reduce, protein removal rate increases.
The present invention compared with prior art, has the following advantages and benefit:
(1) extracting method product recovery rate of the present invention is high, purity is good, and in tool sheath micro-sheath gonidium, polysaccharide yield is more than 2.0%, polysaccharide
Purity more than 90.0%.
(2) present invention uses trichloroacetic acid (TCA) method to replace Sevag method to remove isolating protein, makes the extraction process after improvement
Middle polysaccharide yield increases, and protein removal is effective.
(3) problems such as the extraction process that polysaccharides is traditional also exists long flow path, and operation is complicated.Present invention process is the simplest
Single, flow process shortens further.Agents useful for same is easy to get, and organic solvent consumes less, and production cost is low, and the feature of environmental protection is good, it is easy to accomplish
Industrialization.
Accompanying drawing explanation
Accompanying drawing 1 is a kind of to have the block diagram of extraction method of polysaccharides in sheath micro-sheath gonidium.
Detailed description of the invention
In order to deepen the understanding of the present invention, the invention will be further described with embodiment below in conjunction with the accompanying drawings, this embodiment
It is only used for explaining the present invention, embodiments of the present invention and protection domain is not constituted and limit.
Embodiment 1:
Polyose extraction purification process in a kind of tool sheath micro-sheath gonidium, the steps include:
A, the preparation of tool sheath micro-sheath algae powder.Algae powder derives from the large-scale artificial of tool sheath micro-sheath algae and cultivates.Use BG-11 liquid
Culture medium is as culture matrix, and its component is following (every liter): sodium nitrate 1.5 g, three water dipotassium hydrogen phosphate 0.04 g, seven water sulphuric acid
Magnesium 0.07 g, calcium chloride dihydrate 0.036 g, citric acid 0.006 g, ferric ammonium citrate 0.006 g, disodium EDTA
0.001 g, sodium carbonate 0.02 g and 1 mL trace element A5 solution.Wherein, A5 solution (every liter) component is as follows: boric acid
2.86 g, Sodium Molybdate Dihydrate 0.039 g, zinc sulphate heptahydrate 0.222 g, copper sulphate pentahydrate 0.074 g, a water manganese chloride
1.81 g, cobalt nitrate hexahydrate 0.049 g.When preparing every 1 liter of BG-11 culture medium, need to by mentioned reagent according to sequencing successively
Add the subsoil water after distilled water or filtered through gauze to, be settled to 1 liter, and stirring and dissolving produces to without precipitation.When carrying out I grade of algae
Kind cultivate and during II grade of algal species cultivation, BG-11 fluid medium need to be carried out high pressure steam sterilization, carry out III grade of algae kind spread cultivation and
During pilot scale culture, sterilization treatment do not made by BG-11 fluid medium.
To have sheath micro-sheath algae to be trained by I grade of algal species cultivation (being inoculated into little triangular flask by test tube original seed), II grade of algae kind respectively
Support (proceeding to 2L-5L triangular flask) and III grade of algae kind amplification culture (proceeding to 10L-18L bottle) carries out spreading cultivation step by step of algae kind.To spread cultivation
III grade of algae kind be transferred to bioreactor (80-150L) or small-sized racetrack circulatory pool (1m3) carry out pilot scale culture.?
In the cultivations at different levels of above algae kind, condition of culture is: temperature 26 or 27 or 28 or 29 or 30 DEG C, light intensity 3000Lux-4000Lux,
Continuously aerobic culture 15 or after 18 or 20 or 23 or 26 or 28 or 30 days.Algae is filtered with 200 or 300 or 400 or 500 mesh silks
Liquid, collects tool sheath micro-sheath algae algae mud, is air-dried by algae mud the most in its natural state, obtains having sheath micro-sheath algae algae sheet.After testing, training
In nutrient solution, frond Biomass reaches Chlorophyll-a Content when being 8-10 μ g/milliliter, collects algae culturing liquid, standby.
B, carrying out pulverizing, sieving by dry tool sheath micro-sheath algae algae sheet, (fineness is 80 or 85 to obtain having sheath micro-sheath algae powder
Or 90 or 95 or 100 mesh).Algae powder water content ＜ 10%.Before heating in water bath, algae powder mixes with sterilized water, room temperature place 12 or 13 or
15 or 17 or 19 or 24 hours.
C, take tool sheath micro-sheath algae powder, algae powder is mixed with 1:10 ratio with sterilized water, heating in water bath 2 or 3 or 4 hours,
Described algae powder and sterilized water water bath heating temperature control, at 90 or 92 or 94 or 96 or 98 DEG C, to obtain frustule hot water extraction
Thing.By extract by 2 or 3 layers of filtered through gauze, take supernatant；Residue is brought up again 2 or 3 times as stated above, each 1.5 or 2 or 2.5
Hour, the supernatant obtained is merged, obtains polysaccharide extraction liquid, 4 DEG C of preservations.
D, use Rotary Evaporators (RE-5210A, Asia, Shanghai flourish) by Polyose extraction at 30 or 35 or 38 or 42 or 45 DEG C
Liquid is concentrated into the 1/4-1/6 of original volume, obtains polysaccharide concentrated solution, 4 DEG C of preservations.
E, with 4 DEG C of solution of trichloroacetic acid precipitating proteins, make trichloroacetic acid content reach cumulative volume 4 or 5%, 4 DEG C place 3
Or 4 hours, 4 DEG C are centrifuged 20 or 22 or 24 or 25 minutes, take supernatant, (4 DEG C of solution of trichloroacetic acid protein precipitations under the same terms
Matter 3 hours) repeat 3 or 4 or 5 times, several measured without protein to Coomassie brilliant blue detection, obtain deproteinization polysaccharide liquid.Described from
The speed that the heart extracts is 3500 or 3600 or 3700 revs/min.
F, with 3 times of volume ethanol precipitate polysaccharides, 4 DEG C stand overnight, 4 DEG C centrifugal 20 minutes, obtain polysaccharide precipitation.Precipitate with ethanol is thick
Polysaccharide is substantially soluble in distilled water, and in utilizing bag filter (MWC >=3500Da) will to have sheath micro-sheath gonidium, polysaccharide solution carries out flowing water
Dialysis, collects the polysaccharide component of more than molecular weight 3500 dalton；The speed of described centrifugal extraction is 3000 or 3100 or 3200
Rev/min；Described ethanol be concentration be percent by volume 90%, preferred concentration of alcohol is percent by volume 95%.
G, will dialysis after polysaccharide liquid add 3 times of volumes ethanol, 4 DEG C stand overnight, and 4 DEG C are centrifugal 20-25 minute, and it is heavy to take
Shallow lake phase；The speed of described centrifugal extraction is 3000 or 3100 or 3200 revs/min, described ethanol be concentration be percent by volume
90%, preferred concentration of alcohol is percent by volume 95%.
By dry for ethanol volatilization in precipitated phase at H, room temperature 20 DEG C, by polysaccharide vacuum lyophilization, obtain the tool that purity is higher
Sheath micro-sheath algae intracellular polysaccharide.
Using the technology of the present invention, the yield of tool sheath micro-sheath algae intracellular polysaccharide to may be up to 3.46%, purity of polysaccharide is 92.3%, and
Sugared content in intracellular polysaccharide is not less than 70.0%(in terms of glucose content).The technology of the present invention technique is relatively simple, it is easy to behaviour
Making, Polyose extraction reagent used is conventional chemical reagent, is easy to get, with low cost.Therefore, the present invention establishes a kind of easy
The extractive technique technique of tool sheath micro-sheath algae intracellular polysaccharide that easy and extraction effect is ideal, this technical matters is to existing
One of the biological polyoses extracting method in modern microalgae source is well supplemented.
Embodiment 2:
Polyose extraction purification process in a kind of tool sheath micro-sheath gonidium, the steps include:
(1) carry out pulverizing (fineness is 90 mesh) by dry tool sheath micro-sheath algae algae sheet, obtain having sheath micro-sheath algae powder；
(2) taking tool sheath micro-sheath algae powder, mixed with 1:8 ratio with sterilized water by algae powder, room temperature is placed 12 hours；Heating in water bath
(94 DEG C) 2 hours, filtered through gauze, take supernatant；Residue extracts at least 3 times the most again, each 2.5 hours, supernatant
Merge, obtain polysaccharide extraction liquid, 4 DEG C of preservations；
(3) use Rotary Evaporators that polysaccharide extraction liquid is concentrated at 30 DEG C the 1/5 of original volume, obtain polysaccharide concentrated solution, 4 DEG C of guarantors
Deposit；
(4) with 4 DEG C of solution of trichloroacetic acid precipitating proteins, making trichloroacetic acid content reach the 4.5% of cumulative volume, 4 DEG C of placements 4 are little
Time, 4 DEG C are centrifuged 25 minutes, take and be repeated 4 times under supernatant, the same terms, several measured without protein to Coomassie brilliant blue detection；
(5) using ethanol precipitate polysaccharides, 4 DEG C stand overnight, and 4 DEG C are centrifuged 25 minutes, stay precipitation, precipitate with ethanol crude polysaccharides is substantially soluble in steaming
In distilled water, in utilizing bag filter will to have sheath micro-sheath gonidium, polysaccharide extraction liquid is dialysed, and collects more than molecular weight 3500 dalton
Component；Described ethanol be concentration be percent by volume 92%, preferred concentration of alcohol is percent by volume 95%.
(6) dialysis solution after dialysis adds the ethanol of 3 times of volumes, and 4 DEG C stand overnight, and 4 DEG C are centrifuged 25 minutes, take precipitation
Phase；Described concentration of alcohol is percent by volume 92%, and preferred concentration of alcohol is percent by volume 95%.
(7) under room temperature, (22 DEG C), by dry for ethanol volatilization in precipitated phase, by polysaccharide lyophilization, obtain in tool sheath micro-sheath gonidium
Polysaccharide.Polysaccharide yield is 3.46%, and purity of polysaccharide is 90.8%.
Other implementation condition is same as in Example 1.
Embodiment 3:
Polyose extraction purification process in a kind of tool sheath micro-sheath gonidium, the steps include:
(1) carry out pulverizing (fineness is 100 mesh) by dry tool sheath micro-sheath algae algae sheet, obtain having sheath micro-sheath algae powder；
(2) taking tool sheath micro-sheath algae powder, mixed with 1:9 ratio with sterilized water by algae powder, room temperature is placed 18 hours；Heating in water bath
(96 DEG C) 2.5 hours, filtered through gauze, take supernatant；Residue extracts at least 2 times the most again, each 2 hours, supernatant
Merge, obtain polysaccharide extraction liquid, 4 DEG C of preservations；
(3) use Rotary Evaporators that polysaccharide extraction liquid is concentrated at 40 DEG C the 1/5 of original volume, obtain polysaccharide concentrated solution, 4 DEG C of guarantors
Deposit；
(4) with 4 DEG C of solution of trichloroacetic acid precipitating proteins, make trichloroacetic acid content reach the 4.5% of cumulative volume, place 3.5 for 4 DEG C
Hour, 4 DEG C are centrifuged 20 minutes, take and be repeated 5 times under supernatant, the same terms, several tested without protein to Coomassie brilliant blue detection
Go out；
(5) using ethanol precipitate polysaccharides, 4 DEG C stand overnight, and 4 DEG C are centrifuged 20 minutes, stay precipitation, precipitate with ethanol crude polysaccharides is substantially soluble in steaming
In distilled water, in utilizing bag filter will to have sheath micro-sheath gonidium, polysaccharide extraction liquid is dialysed, and collects more than molecular weight 3500 dalton
Component；Described ethanol be concentration be percent by volume 96%, preferred concentration of alcohol is percent by volume 95%.
(6) dialysis solution after dialysis adds the ethanol of 3 times of volumes, and 4 DEG C stand overnight, and 4 DEG C are centrifuged 20 minutes, take precipitation
Phase；Described ethanol be concentration be percent by volume 96%, preferred concentration of alcohol is percent by volume 95%.
(7) under room temperature, (25 DEG C), by dry for ethanol volatilization in precipitated phase, by polysaccharide lyophilization, obtain in tool sheath micro-sheath gonidium
Polysaccharide.Polysaccharide yield is 2.86%, and purity of polysaccharide is 91.4%.
Other implementation condition is same as in Example 1.
Embodiment 4:
Polyose extraction purification process in a kind of tool sheath micro-sheath gonidium, the steps include:
(1) carry out pulverizing (fineness is 100 mesh) by dry tool sheath micro-sheath algae algae sheet, obtain having sheath micro-sheath algae powder；
(2) taking tool sheath micro-sheath algae powder, mixed with 1:10 ratio with sterilized water by algae powder, room temperature is placed 24 hours；Heating in water bath
(98 DEG C) 4 hours, filtered through gauze, take supernatant；Residue extracts at least 2 times the most again, each 1.5 hours, supernatant
Merge, obtain polysaccharide extraction liquid, 4 DEG C of preservations；
(3) use Rotary Evaporators that polysaccharide extraction liquid is concentrated at 45 DEG C the 1/6 of original volume, obtain polysaccharide concentrated solution, 4 DEG C of guarantors
Deposit；
(4) with 4 DEG C of solution of trichloroacetic acid precipitating proteins, make trichloroacetic acid content reach the 5% of cumulative volume, place 3 hours for 4 DEG C,
4 DEG C are centrifuged 20 minutes, take and be repeated 4 times under supernatant, the same terms, several measured without protein to Coomassie brilliant blue detection；
(5) using ethanol precipitate polysaccharides, 4 DEG C stand overnight, and 4 DEG C are centrifuged 20 minutes, stay precipitation, precipitate with ethanol crude polysaccharides is substantially soluble in steaming
In distilled water, in utilizing bag filter will to have sheath micro-sheath gonidium, polysaccharide extraction liquid is dialysed, and collects more than molecular weight 3500 dalton
Component；Described ethanol be concentration be percent by volume 98%, preferred concentration of alcohol is percent by volume 95%.
(6) dialysis solution after dialysis adds the ethanol of 3 times of volumes, and 4 DEG C stand overnight, and 4 DEG C are centrifuged 20 minutes, take precipitation
Phase；Described ethanol be concentration be percent by volume 98%, preferred concentration of alcohol is percent by volume 95%.
(7) under room temperature, (25 DEG C), by dry for ethanol volatilization in precipitated phase, by polysaccharide lyophilization, obtain in tool sheath micro-sheath gonidium
Polysaccharide.Polysaccharide yield is 2.34%, and purity of polysaccharide is 92%.
What embodiments of the invention were announced is preferred embodiment, but is not limited thereto, those of ordinary skill in the art, pole
Easily according to above-described embodiment, understand the spirit of the present invention, and make different amplifications and change, but as long as without departing from the present invention's
Spirit, the most within the scope of the present invention.
Other implementation condition is same as in Example 1.
Claims (3)
1. have an extracting method for sheath micro-sheath algae intracellular polysaccharide, the steps include:
(1) mass propgation having sheath micro-sheath algae is first carried out: using BG-11 fluid medium as culture matrix, its component is such as
Under: sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, disodium EDTA, carbonic acid
Sodium and trace element A5 solution, be configured to culture medium by above components in certain proportion, will have sheath micro-sheath algae respectively by I grade of algae kind
Cultivation, II grade of algal species cultivation and III grade of algae kind amplification culture carry out spreading cultivation step by step of algae kind, spread cultivation III grade of algae kind are transferred to
Bioreactor or racetrack circulatory pool carry out pilot scale culture, and in the cultivations at different levels of above algae kind, condition of culture is: temperature
Spend 26-30 DEG C, light intensity 3000Lux-4000Lux, continuous aerobic culture 15-30d, after testing after, frond Biomass in culture fluid
Reaching Chlorophyll-a Content is 8-10 μ g/milliliter, collects algae culturing liquid, standby；
(2) the tool sheath micro-sheath algae culturing liquid 200-500 mesh silk collected is filtered algae solution, obtain having sheath micro-sheath algae algae mud,
Under naturalness, algae mud is air-dried, obtain having sheath micro-sheath algae algae sheet, dry tool sheath micro-sheath algae algae sheet is pulverized, sieved, obtains
Tool sheath micro-sheath algae powder, algae powder fineness is 80-100 mesh；
(3) take tool sheath micro-sheath algae powder, algae powder is mixed with 1:8-1:10 ratio with sterilized water, heating in water bath 2-4 hour, water-bath
Heating and temperature control at 90-98 DEG C, layer 2-3 filtered through gauze, take supernatant；Residue extracts at least 2-3 time the most again, often
Secondary 1.5-2.5 hour, the supernatant extracted is merged, obtains polysaccharide extraction liquid, 4 DEG C of preservations, before above-mentioned heating in water bath processes,
Algae powder need to mix placement 12-24 hour with sterilized water；
(4) use Rotary Evaporators that polysaccharide extraction liquid is concentrated at 30-45 DEG C the 1/4-1/6 of original volume, obtain polysaccharide dense
Contracting liquid, 4 DEG C of preservations；
(5) with 4 DEG C of solution of trichloroacetic acid precipitating proteins, make trichloroacetic acid content reach the 4-5% of cumulative volume, place 3-4 for 4 DEG C
Hour, 4 DEG C are centrifugal 20-25 minute, take supernatant, repeat 3-5 time under the same terms, detect without protein quilt to Coomassie brilliant blue
Measure；The speed of described centrifugal extraction is 3500-3700 rev/min；
(6) using ethanol precipitate polysaccharides, 4 DEG C stand overnight, and 4 DEG C centrifugal 20-25 minute, stays precipitation, by the most molten for precipitate with ethanol crude polysaccharides
In distilled water, in utilizing bag filter will to have sheath micro-sheath gonidium, polysaccharide extraction liquid carries out flowing water dialysis, collects molecular weight 3500 road
The polysaccharide component of more than Er Dun；
(7) dialysis solution after dialysis adds the ethanol of 3 times of volumes, and 4 DEG C stand overnight, and 4 DEG C centrifugal 20-25 minute, takes precipitation
Phase；
(8) by dry for ethanol volatilization in precipitated phase under room temperature, by polysaccharide lyophilization, obtain having sheath micro-sheath algae intracellular polysaccharide.
A kind of extracting method having sheath micro-sheath algae intracellular polysaccharide the most according to claim 1, it is characterised in that: described tool
Sheath micro-sheath algae intracellular polysaccharide is to have sheath micro-sheath algae with a kind of filamentous cyanobacteria rich in polysaccharide separated from desert soil to be
Extracting material, micro-sheath algae is carried out indoor and spreads cultivation step by step and pilot scale culture, condition of culture is: BG-11 fluid medium, temperature
26-30 DEG C, light intensity 3000Lux-4000Lux, continuous aerobic culture 15-30 days, algae solution 200-500 mesh silk is filtered, receives
Collect micro-sheath algae algae mud, algae mud is air-dried in its natural state, it is thus achieved that micro-sheath algae algae sheet, after micro-sheath algae algae sheet is pulverized, sieved
To algae powder, then algae powder is carried out multistage extraction and separates, obtain intracellular polysaccharide.
A kind of extracting method having sheath micro-sheath algae intracellular polysaccharide the most according to claim 1, it is characterised in that: described
BG-11 fluid medium, its component is as follows: sodium nitrate 1.5 g/L, three water dipotassium hydrogen phosphate 0.04 g/L, Magnesium sulfate heptahydrate
0.07 g/L, calcium chloride dihydrate 0.036 g/L, citric acid 0.006 g/L, ferric ammonium citrate 0.006 g/L, ethylenediaminetetraacetic acid
Wherein, A5 solution is individually prepared for disodium salt 0.001 g/L, sodium carbonate 0.02 g/L and trace element A5 solution 1 mL., and every 1
Rise contained component in A5 solution as follows: boric acid 2.86 g, Sodium Molybdate Dihydrate 0.039 g, zinc sulphate heptahydrate 0.222 g, five water
Copper sulfate 0.074 g, water manganese chloride 1.81 g, cobalt nitrate hexahydrate 0.049 g, during 1 liter of BG-11 culture medium of preparation, will
Mentioned reagent adds the subsoil water after distilled water or filtered through gauze to successively according to listed sequencing, is settled to 1 liter, and stirs
It is dissolved to produce without precipitation, carries out I grade of algal species cultivation and II grade of algal species cultivation, BG-11 fluid medium is carried out high steam
Sterilizing 121 DEG C, 20 min, carry out III grade of algae kind amplification culture and pilot scale culture, and BG-11 fluid medium is not made at sterilizing
Reason.
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