CN104086649B - A kind of method of grading extraction nutrient substance from spirulina - Google Patents
A kind of method of grading extraction nutrient substance from spirulina Download PDFInfo
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Abstract
The present invention provides a kind of method of grading extraction nutrient substance from spirulina, with fresh spirulina algae mud as a raw material, high concentration ethanol is used spirulina algae mud to carry out dehydration and extracts chlorophyll and carotenoid, the algae-residue after alcohol extraction extract with phosphate buffer phycobniliprotein and water soluble polysaccharide simultaneously.The method has that energy consumption is low, the feature of low cost, and it passes through grading extraction, can obtain the multiple nutrients material in spirulina, and its extraction conditions is gentle, extract the nutrient substance obtained and be difficult to be destroyed.
Description
[technical field]
The invention belongs to bioengineering field, relate to a kind of method of grading extraction nutrient substance from spirulina.
[background technology]
Spirulina (Spirulina), has another name called arthrospira, is a kind of cyanophyceae or cyanobacteria, provides nutrition by photosynthesis for self,
Mainly live in salt water and brackish water.It is the high-protein plant food of a kind of comprehensive nutrition, equilibrium, its protein content
Up to 55%-70% (wherein phycobniliprotein accounts for dry weight 14%-20%), water soluble polysaccharide accounts for the 7%～8% of spirulina dry weight, and
There is abundant pigment (chlorophyll such as 1% and 0.5% carotenoid).Additionally, spirulina is without cholesterol, fat content
Only account for the 5% of dry weight, containing abundant unsaturated fatty acid such as gamma-Linolenic acid, be especially suitable for obese people and eat.Because it has rich
Nutritional labeling rich, comprehensive, has in fields such as food service industry, cosmetic industry, pharmaceutical sector, feed industry and environmental protection industries at present
Extensively application, has the most been furtherd investigate and widely used has been had phycobniliprotein, chlorophyll, carotenoid, water soluble polysaccharide
And various trace element.
At present, the common method extracting spirulina Determination of Chlorophyll and carotenoid has: organic solvent extraction, ultrasonic extraction
And supercritical CO2Extraction.During conventional organic solvent extraction operating cost, solvent-oil ratio is big, toxicity is big, cost is high and
Easily make Chlorophylls and Carotenoids decompose under high temperature thus reduce extraction ratio and biological activity.The extracting method master of phycobniliprotein
Multigelation method to be had, supercritical ultrasonics technology and CaCl2Swelling method.Owing to the polypeptide moiety of phycobniliprotein is quick to illumination, temperature, pH
Sense, the most existing method separating phycobniliprotein from spirulina there is limitations, such as degeneration can inactivate and destroy algae gallbladder
Albumen, longer extraction time and high cost.Additionally, phycobniliprotein is higher to the requirement of extraction environment, needs at low temperature, keep away
Light, the environment such as aseptic are carried out, and phycobniliprotein is the most rotten, cause anticorrosion, preserve difficulty and increase, and this makes phycobniliprotein
Extraction be difficult to put into actual production.The extracting method of water soluble polysaccharide is broadly divided into three kinds, it may be assumed that hot water extraction method, alkali
Water extraction method and microwave-assisted extraction.Ordinary test frequently with hot water extraction method, but need high temperature (more than 85 DEG C) and long time
Between (more than 8h) extract, so method exists time-consuming, power consumption and polysaccharide yield and purity is not the highest shortcoming.Except this it
Outward, traditional extraction technique many employings spirulina powder is raw material, the high energy consumption of dry run, and can destroy nutritional labeling.
Carry out in the extraction process of spirulina utilization at present, often only take into account and obtain a kind of effective ingredient by a kind of extraction process
Or nutrient substance, such as use higher temperature to extract to obtain merely the water soluble polysaccharide of relatively high yield pulp1, but now can make
Become the inactivation of phycobniliprotein.So cause the great wasting of resources, therefore provide one by grading extraction with from spirulina
The method obtaining multiple nutrients material is highly desirable to.
[summary of the invention]
It is an object of the invention to provide a kind of method of grading extraction nutrient substance from spirulina, the method have energy consumption low,
The feature of low cost, it passes through grading extraction, can obtain the multiple nutrients material in spirulina, and its extraction conditions is gentle, carries
The nutrient substance taking acquisition is difficult to be destroyed.
The technical scheme that the present invention takes is as follows:
The fractional extraction method of a kind of nutritious spirulina material, comprises the following steps:
(1) fresh spirulina algae mud is put in centrifuge tube, be added thereto to the ethanol solution that volume fraction is more than 80% and carry out
Concussion is extracted, and is then centrifuged separating, and gained supernatant is Chlorophylls and Carotenoids extracting solution；Obtain after being centrifuged
Algae-residue is placed in the environment that temperature is-20 DEG C and stands overnight, then by algae-residue lyophilizing, standby,
(2) algae-residue after the lyophilizing in step (1) is placed in centrifuge tube, is added thereto to phosphate buffer, then enters
Row concussion is extracted, and is centrifuged extracting the extracting solution obtained, gained supernatant, is phycobniliprotein and water soluble polysaccharide extracts
Liquid.
Described nutritious spirulina material includes phycobniliprotein, chlorophyll, carotenoid and water soluble polysaccharide.
Further, in step (1), fresh spirulina algae mud is 1:4-1:8 with the mass ratio of ethanol solution, step (1)
The temperature carrying out concussion extraction is 25-40 DEG C, and the time is 15-60min.
Preferably, in described step (1), fresh spirulina algae mud is 1:6 with the mass ratio of ethanol solution, and step (1) is carried out
The temperature that concussion is extracted is 30 DEG C, and the time is 30min.
Further, in described step (2), the algae-residue after described lyophilizing is 1:12-1:16 with the mass ratio of phosphate buffer,
Step (2) carries out the temperature of concussion extraction and is 40-50 DEG C, and extraction time is 3-5h.
Preferably, in described step (2), the algae-residue after described lyophilizing is 1:14 with the mass ratio of phosphate buffer, extracts
Temperature is 45 DEG C, and extraction time is 3h.
Preferably, the concentration of the phosphate buffer in described step (2) be 0.1mol/L, pH be 6.8-7.2.
Described volume fraction be more than 80% ethanol solution be preferably dehydrated alcohol.
In concrete operations, the method for grading extraction nutrient substance from spirulina of the present invention specifically can be carried out as follows:
1) fresh spirulina algae mud is placed in centrifuge tube, adds the body that solid-liquid ratio (algae mud: ethanol, g/g) is 1:4-1:8
Fraction is the ethanol solution (Duplicate Samples is three) of more than 80%, wraps tinfoil after shaking up.Centrifuge tube is put into 25-40 DEG C,
Vibrate in the constant-temperature table of 150-250r/min 15-60min, extracts and is centrifuged 8min in desk centrifuge after terminating, centrifuge
Rotating speed is 5000-14000r/min.Gained supernatant is Chlorophylls and Carotenoids extracting solution, supernatant is poured out and in
Preserving in-20 DEG C of refrigerators, algae-residue puts into freezer dryer lyophilizing after the refrigerator internal memory of-20 DEG C lets slip night, standby.
2) the spirulina algae-residue of above-mentioned lyophilizing is placed in centrifuge tube, according to solid-liquid ratio (algae-residue: phosphate buffer, g/ml)
Adding 0.1mol/L, pH6.8-7.2 phosphate buffer for 1:12-1:16, wrap tinfoil after shaking up, putting into design temperature is
40-50 DEG C, the constant-temperature table of 200r/min extracts 3-5h.Sample after extracting puts into 20 DEG C, centrifugal in desk centrifuge
5min, the rotating speed of centrifuge is 7000-12000r/min.Gained supernatant is phycobniliprotein and water soluble polysaccharide extracting solution.
Measure the yield of phycobniliprotein in this extracting solution as follows: supernatant is carried out in screw socket pipe constant volume, with 0.1mol/L,
The phosphate buffer of pH7.0 makees blank, measures sample at 620nm, 650nm and 490nm with ultraviolet-uisible spectrophotometer
Absorbance, calculates the yield of phycobniliprotein.Water soluble polysaccharide yield in this extracting solution utilizes sulfuric acid-phynol method to measure.
Advantages of the present invention and beneficial effect:
(1) traditional extraction technique generally uses spirulina powder, and the present invention is directly using fresh spirulina mud as raw material, greatly
Reduce high energy consumption and the high cost that drying process is brought, can ensure that again nutrient substance is not destroyed simultaneously, it is achieved thereby that
Doulbe-sides' victory in economic benefit and environmental benefit.
(2), when the present invention uses high concentration ethanol to process spirulina algae mud, it not only has the effect of dehydration, Er Qietong
Shi Zuowei extracts the solvent of chlorophyll and carotenoid, and when can improve conventional organic solvent extraction operating cost, solvent disappears
Consumption is big, toxicity is big, cost is high and easily make under high temperature Chlorophylls and Carotenoids decompose thus reduce the shortcomings such as extraction ratio.
(3) present invention uses extract with phosphate buffer phycobniliprotein and water soluble polysaccharide, and Extracting temperature is relatively low, water soluble polysaccharide
It is not destroyed with protein active, greatly improves in traditional extracting method and albumen can be made to become for acquisition high-purity water soluble polysaccharide
Property inactivation problem.
(4) ethanol dehydration of the present invention, two key technologies of wet underwater welding alcohol soluble substance, greatly reduce total cost of production more than 30%,
Can be by two kinds of extract classifications such as ethanol extract (Chlorophylls and Carotenoids) and water extracts (phycobniliprotein and water soluble polysaccharide)
Prepare so that the nutritional labeling in spirulina is extracted fully, and total raw material utilization rate reaches more than 80%, and residue can conduct
Feedstuff.
[detailed description of the invention]
Below in conjunction with specific embodiment, the invention will be further described, but the comprised scope of the present invention is not limited to
This.
Embodiment 1
(1) dehydration: obtain fresh spirulina algae mud from SPIRULINA CULTIVATION pond, accurately weigh the spirulina of 2g with electronic balance
Algae mud, in 50mL centrifuge tube, adds the dehydrated alcohol (Duplicate Samples is three) that solid-liquid ratio (algae mud: ethanol, g/g) is 1:4,
Tinfoil is wrapped after shaking up.Centrifuge tube is put into 40 DEG C, mechanical shaking extraction 15min in the constant-temperature table of 150r/min, extracts after terminating
Being centrifuged 8min in desk centrifuge, the rotating speed of centrifuge is 5000r/min, and gained supernatant is that Chlorophylls and Carotenoids carries
Take liquid, and be stored in the refrigerator of-20 DEG C.Algae-residue is put into freezer dryer after the refrigerator internal memory of-20 DEG C lets slip night and is frozen
Dry, standby.
The extraction ratio of the Chlorophylls and Carotenoids of determination step (1): by the algae-residue after step (1) lyophilization with 90%
Acetone soln extract pigment utilize ultraviolet-uisible spectrophotometer to measure its suction at 480nm, 510nm, 647nm, 664nm
Luminosity, and substitute into following empirical equation and calculate the extraction ratio of its Chlorophylls and Carotenoids.Calculate after testing, the present embodiment
The extraction ratio of the Chlorophylls and Carotenoids in 1 step 1 is respectively 90.64%, and 90.87%.
Chlorophyll concentration (μ g/ml) a=-1.7858 × A647+11.8668×A664
Total carotinoid concentration (μ g/ml)=7.6 × (A480-1.49×A510)
Content mg × 100% of the chlorophyllous quality mg/ raw material Determination of Chlorophyll that chlorophyll extraction ratio=extraction obtains.
The content of carotenoid in the quality mg/ raw material of the carotenoid that Extraction of carotenoid pigment rate=extraction obtains
Mg × 100%.
The mensuration of raw material Determination of Chlorophyll/carotenoid content is by weighing quantitative fresh algae mud, directly carrying out lyophilization,
Then extraction and determination gained is carried out with 90% acetone soln.
(2) hydrotrope is extracted: accurately weigh in 1g step (1) the spirulina algae-residue after lyophilizing with electronic balance, be placed on
In centrifuge tube, it is that 1:14 adds 0.1mol/L, pH7.0 phosphate according to solid-liquid ratio (algae-residue: phosphate buffer, g/ml)
Buffer, wraps tinfoil after shaking up, putting into design temperature is 45 DEG C, mechanical shaking extraction 3h in the constant-temperature table of 200r/min.Will
Sample after extraction puts into 20 DEG C, and centrifugal 5min in desk centrifuge, the rotating speed of centrifuge is 8000r/min's.Gained supernatant
Liquid is the extracting solution of phycobniliprotein and water soluble polysaccharide.
Determination step (2) phycobniliprotein and the extraction ratio of water soluble polysaccharide: supernatant is carried out in screw socket pipe constant volume, with
The phosphate buffer of 0.1mol/L, pH7.0 makees blank, measures sample at 620nm, 650nm with ultraviolet-uisible spectrophotometer
With the absorbance of 490nm, the yield calculating phycobniliprotein is 5.53%, utilizes sulfuric acid-phynol method to record obtaining of water soluble polysaccharide
Rate is 1.13%, wherein:
Weight mg/ raw material algae grain weight amount mg × 100% of phycobniliprotein yield=extraction gained phycobniliprotein；
Weight mg/ raw material algae grain weight amount mg × 100% of water soluble polysaccharide yield=extraction gained water soluble polysaccharide.
Embodiment 2
1) dehydration-extract alcohol soluble substance: obtain fresh spirulina algae mud from SPIRULINA CULTIVATION pond, accurately weigh 2g with electronic balance
Spirulina algae mud in 50mL centrifuge tube, add solid-liquid ratio (algae mud: ethanol, g/g) be the volume fraction of 1:6 be 80%
Ethanol solution (Duplicate Samples is three), wraps tinfoil after shaking up.Centrifuge tube is put into 30 DEG C, in the constant-temperature table of 200r/min
Mechanical shaking extraction 30min, extracts and is centrifuged 8min in desk centrifuge after terminating, and the rotating speed of centrifuge is 8000r/min, on gained
Clear liquid is Chlorophylls and Carotenoids extracting solution, and is stored in the refrigerator of-20 DEG C.Algae-residue is in the refrigerator internal memory of-20 DEG C
Freezer dryer lyophilizing is put into after letting slip night, standby.
Algae-residue after step (1) lyophilization is extracted pigment with the acetone soln of 90% and utilizes ultraviolet-uisible spectrophotometer to survey
Determining its absorbance at 480nm, 510nm, 647nm, 664nm, the extraction ratio calculating Chlorophylls and Carotenoids is respectively
93.18%, 93.33%.
(2) extract the hydrotrope: accurately weigh the spiral algae-residue after 1g alcohol extraction in centrifuge tube with electronic balance, add solid-liquid ratio
(algae-residue: phosphate buffer, g/ml) is 0.1mol/L, pH6.8 phosphate buffer of 1:16, wraps tinfoil after shaking up,
Putting into design temperature is 40 DEG C, mechanical shaking extraction 4h in the constant-temperature table of 200r/min.Sample after extracting puts into 20 DEG C, platform
Centrifugal 5min in formula centrifuge, the rotating speed of centrifuge is 12000r/min, and gained supernatant is phycobniliprotein and water soluble polysaccharide
Extracting solution.
Supernatant is carried out in screw socket pipe constant volume, makees blank with the phosphate buffer of 0.1mol/L, pH7.0, can by ultraviolet
Seeing the spectrophotometric determination sample absorbance at 620nm, 650nm and 490nm, the yield calculating phycobniliprotein is 4.30%,
The yield utilizing sulfuric acid-phynol method to record water soluble polysaccharide is 1.16%.
Embodiment 3
(1) dehydration-extract alcohol soluble substance: obtain fresh spirulina algae mud from SPIRULINA CULTIVATION pond, accurately weigh with electronic balance
The spirulina algae mud of 2g is in 50mL centrifuge tube, and adding the volume fraction that solid-liquid ratio (algae mud: ethanol, g/g) is 1:8 is 90%
Ethanol solution (Duplicate Samples is three), wrap tinfoil after shaking up.Centrifuge tube is put into 25 DEG C, the constant-temperature table of 250r/min
Middle vibration 60min, extracts and is centrifuged 8min in desk centrifuge after terminating, and the rotating speed of centrifuge is 14000r/min.Gained supernatant
Liquid is Chlorophylls and Carotenoids extracting solution, and is stored in the refrigerator of-20 DEG C.Algae-residue is deposited in the refrigerator of-20 DEG C
Put into freezer dryer lyophilizing the most afterwards.Algae-residue after lyophilization extracts pigment with the acetone soln of 90% and utilizes the ultraviolet can
See spectrophotometric determination its at the absorbance of 480nm, 510nm, 647nm, 664nm, and calculate chlorophyll and carotenoids
The extraction ratio of element is respectively 93.10%, and 92.52%.
(2) extract the hydrotrope: accurately weigh the spirulina algae-residue after 1g step (1) lyophilizing with electronic balance, be placed on from
In heart pipe, it is that 1:12 adds 0.1mol/L, pH7.2 phosphate-buffered according to solid-liquid ratio (algae-residue: phosphate buffer, g/ml)
Liquid, wraps tinfoil after shaking up, putting into design temperature is 50 DEG C, mechanical shaking extraction 5h in the constant-temperature table of 200r/min.After extracting
Sample put into 20 DEG C, centrifugal 5min in desk centrifuge, the rotating speed of centrifuge is 7000r/min, and gained supernatant is algae
Biliprotein and the extracting solution of water soluble polysaccharide.
Supernatant is carried out in screw socket pipe constant volume, makees blank with the phosphate buffer of 0.1mol/L, pH7.0, can by ultraviolet
Seeing the spectrophotometric determination sample absorbance at 620nm, 650nm and 490nm, the yield calculating phycobniliprotein is 5.12%,
The yield utilizing sulfuric acid-phynol method to record water soluble polysaccharide is 1.23%.
The above, be only presently preferred embodiments of the present invention, and the present invention not does any pro forma restriction, thus all not
Depart from technical solution of the present invention content, according to the present invention technical spirit to any simple modification made for any of the above embodiments, etc.
With change and modification, all still fall within the range of technical solution of the present invention.
Claims (7)
1. the method for grading extraction nutrient substance from spirulina, it is characterised in that said method comprising the steps of:
(1) putting in centrifuge tube by fresh spirulina algae mud, be added thereto to the ethanol solution that volume fraction is more than 80%, carry out mechanical shaking extraction under dark condition, be then centrifuged separating, gained supernatant is Chlorophylls and Carotenoids extracting solution；The algae-residue obtained after being centrifuged is placed in the environment that temperature is-20 DEG C and stands overnight, then by algae-residue lyophilizing, standby；
(2) being placed in centrifuge tube by the algae-residue after the lyophilizing in step (1), be added thereto to phosphate buffer, then carry out mechanical shaking extraction, be centrifuged by the extracting solution of acquisition, gained supernatant is phycobniliprotein and water soluble polysaccharide extracting solution,
Described nutrient substance includes chlorophyll, carotenoid, phycobniliprotein and water soluble polysaccharide.
The method of grading extraction nutrient substance from spirulina the most according to claim 1, it is characterized in that, in described step (1), fresh spirulina algae mud is 1:4-1:8 with the mass ratio of ethanol solution, step (1) carries out the temperature of mechanical shaking extraction and is 25-40 DEG C, and the time is 15-60min.
The method of grading extraction nutrient substance from spirulina the most according to claim 2, it is characterized in that, in described step (1), fresh spirulina algae mud is 1:6 with the mass ratio of ethanol solution, and it is 30 DEG C that step (1) carries out the temperature of mechanical shaking extraction, and the time is 30min.
The method of grading extraction nutrient substance from spirulina the most according to claim 1, it is characterized in that, in described step (2), algae-residue after described lyophilizing is 1:12-1:16 with the mass ratio of phosphate buffer, step (2) carries out the temperature of mechanical shaking extraction and is 40-50 DEG C, and extraction time is 3-5h.
The method of grading extraction nutrient substance from spirulina the most according to claim 4, it is characterized in that, in described step (2), the algae-residue after described lyophilizing is 1:14 with the mass ratio of phosphate buffer, Extracting temperature is 45 DEG C, and extraction time is 3h.
6. according to the method for grading extraction nutrient substance from spirulina according to any one of claim 1-5, it is characterised in that the concentration of the phosphate buffer in described step (2) be 0.1mol/L, pH be 6.8-7.2.
7. according to described in any one of claim 1-5 from spirulina the method for grading extraction nutrient substance, it is characterised in that described volume fraction be the ethanol solution of more than 80% be dehydrated alcohol.
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| CN104974268A (en) * | 2015-06-18 | 2015-10-14 | 中山鼎晟生物科技有限公司 | Hot water extraction method of dunaliella salina polysaccharide |
| CN106923337A (en) * | 2017-03-15 | 2017-07-07 | 福建农林大学 | A kind of preparation method and applications of spirulina full agonist |
| CN108047344B (en) * | 2017-12-08 | 2019-12-31 | 江西中烟工业有限责任公司 | Preparation method of soybean spirulina compound extract and application of soybean spirulina compound extract in tipping paper |
| IT202000032636A1 (en) | 2020-12-29 | 2022-06-29 | Consiglio Nazionale Ricerche | METHOD FOR THE EXTRACTION OF HIGH PURITY PHYCOBILIPROTEINS FROM CYANOBACTERIA AND/OR ALGAE BIOMASS |
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| CN101575359A (en) * | 2008-05-05 | 2009-11-11 | 青岛海之骄生物科技有限公司 | Extraction method of water-soluble spirulina phycobiliprotein |
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