CN110156797A - A kind of technique for extracting chlorophyll from spirulina - Google Patents
A kind of technique for extracting chlorophyll from spirulina Download PDFInfo
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Abstract
The invention discloses a kind of from spirulina extracts the technique of chlorophyll, including pretreatment of raw material, immersion, enzymatic hydrolysis, filtering, baking step.When the application extracts spirulina Determination of Chlorophyll, chlorophyll is obtained by the way of immersion, enzymatic hydrolysis, microwave and ultrasonic treatment, avoids the use of organic solvent, improves the edible safety of chlorophyll；Enzymolysis processing is carried out to spirulina using enzymes such as protease, cellulase, pectase, ligninase, amylase, esterases, by other mass degradations in spirulina in addition to chlorophyll, and the chlorophyll in spirulina will not be destroyed, extract the chlorophyll contained in spirulina as far as possible, and product does not contain other impurities, improve the yield and purity of chlorophyll, chlorophyll extracts yield and is above 99.5%, it is higher by 8.8% or more than traditional solvent extraction method, chlorophyll purity is above 99.96%.
Description
Technical field
The invention belongs to technical field more particularly to a kind of techniques that chlorophyll is extracted from spirulina.
Background technique
Spirulina (scientific name: Spirulina) is a kind of unicellular lower eukaryote, has reduction cholesterol, adjusting blood glucose, enhancing immune
System, anti-oxidant, antifatigue, treatment anemia, antitumor, anti-cancer tumor suppressor health-care efficacy, mitigate the poison of cancer radiation, chemotherapy
Side reaction improves immune function, reduces blood lipid and other effects.The characteristics of nutritive component of spirulina is protein content height, and fat,
Content of cellulose is low, and also contains miscellaneous vitamin, it is vitamin B12 and the highest food of content beta-carotene
Product.In addition, absorbability irony content is highest in it or all foods, while it also found that it contains with anti-cancer, for cancer
The algae protein of effect and other a large amount of mineral elements and the bioactive substance for improving immunity of organisms.Spirulina is still
The natural treasure-house of chlorophyll, how of fine quality amount is, accounts for the 1.1% of frond, is 2~3 times of most of terrestrial plants, is common vegetable
10 times.The type of chlorophyll contained by spirulina is mainly chlorophyll a, and it is the mankind that the ferroheme of molecular structure and people are quite similar
The direct material for synthesizing hemoglobin, can be rated as " green blood ", and content is up to 7600mg/kg algae powder.Spirulina is as one
Kind novel dual-purpose of drug and food resource, active constituent have phycobniliprotein, chlorophyll, carotenoid, water-soluble polysaccharide and various
Microelement has epochmaking nutrition, health care and the potentialities to be exploited such as medicinal, and becomes the health that FAO (Food and Agriculture Organization of the United Nation) is recommended
Food.
Chlorophyll is a kind of safe and non-toxic natural fat-soluble pigment, be structure is nitrogen on a magnesium and four pyrrole rings
In conjunction with using porphyrin as the general name of the green pigment of skeleton.Chlorophyll is not bery stable, and light, acid, alkali, oxygen, oxidant etc. can all make it
It decomposes.Under acid condition, chlorophyll molecule is easy to lose the magnesium in porphyrin ring as de-magnesium pheophytin.Chlorophyll have hematopoiesis,
The multiple uses such as vitamin, removing toxic substances, disease-resistant are provided.The structure and ferroheme of chlorophyll are extremely similar, are easily absorbed by the body, to human body
There is good physiological function, has extensive use in medicine, foods and cosmetics industry.
The extracting method of spirulina Determination of Chlorophyll and carotenoid is roughly divided into 3 kinds, it may be assumed that organic solvent extraction surpasses
Sound extraction method, supercritical CO2Extraction.When common organic solvent extraction operating cost, solvent-oil ratio is big, and toxicity is big, breaks
Bad nutritional ingredient is easy to decompose chlorophyll under at high cost and high temperature, to reduce yield and bioactivity.Though currently, there is phase
The document report that chlorophyll is extracted from spirulina is closed, but the prior art is to the comprehensive development and utilization skill of the biomass such as spirulina
Art is also immature, and the biomass such as most of spirulina could not be fully utilized, and causes the huge waste of biomass energy；Moreover,
It is not soluble in water since the chlorophyll in spirulina can be dissolved in the organic solvents such as acetone, methanol, ethyl alcohol, ethyl acetate, so
It extracts and all spirulina algal gel is dehydrated using organic solvent when processing, extract chlorophyll, precipitated after alcohol extracting,
The operation such as filtering, and the solubility of carrotene, lutein in organic solvent is larger, contains in chlorophyll made from this method
Carrotene, lutein contain a certain amount of organic solvent in chlorophyll obtained, can cause to health after people are edible
It influences.Such as:
1, it is more to disclose phycocyanin in fresh Spirulina, chlorophyll and spirulina by patent application CN201710169066.7
The extraction process of sugar, it is related to the extraction process technology field of active material in spirulina.Fresh spirulina is weighed, then from
The heart washs 1-3 times, and algal gel of weighing adds 0.The water of 5-4 times of algal gel weight, stirs evenly, and freeze thawing is twice, cold at subzero 20 DEG C
Freeze overnight, dissolve at room temperature, after second dissolves, add the water of 2-8 times of weight of algal gel, after placing 2-8h, centrifugation is obtained
Clear liquid a and solid b, solid b obtain extracting solution and solid c after ethyl alcohol soxhlet extraction mode is extracted, and wherein supernatant a is used for algae
The extraction of azurin；The extracting solution of solid b is used for the extraction of sodium zinc chlorophyllin；Solid c is used for the extraction of spirulina polysaccharide；This
Technique is relatively simple, sufficiently effective can utilize existing spirulina resource, realize the comprehensive utilization and depth to spirulina
Exploitation has positive meaning to spirulina industry development.
2, patent application CN201510528132.6 discloses a kind of preparation method of high-purity chlorophyll a, the method
The step of it is as follows: (1) using cultivate to logarithmic growth phase latter stage blunt top spirulina as raw material, frond is collected by centrifugation；(2) frond is used
Ethyl alcohol extracts three times, and extract liquor merges；(3) appropriate n-hexane or petroleum ether are added in extract liquor, adds water to split-phase and is stripped
It takes, the solution containing pigment is washed twice with water；(4) 100~200 mesh neutral alumina column chromatography for separation purify chlorophyll a, first
With the n-hexane or petroleum ether solution rinse alumina column for containing 0.3% ethyl alcohol, ethyl alcohol-n-hexane or ethyl alcohol-stone are used after loading
Oily ether mixed solvent gradient washes；(5) solution containing chlorophyll a depressurizes rotary evaporation recycling design, can be obtained high-purity
Chlorophyll a.Chlorophyll a prepared by the present invention is free of chlorophyll b, and HPLC purity is greater than 99.5%.
3, patent application CN200910016683.9, disclose one kind from spirulina extract phycocyanin, chlorophyll and
The technique of spirulina polysaccharide, comprising the following steps: take fresh spirulina, weigh, centrifuge washing, algal gel of weighing adds into supernatant
Water stirs evenly, and freeze thawing (makes cell wall rupture) twice, after second dissolves, adds water, places 2~8h, and centrifugation obtains
Clear liquid and precipitate A, precipitate A are divided into extracting solution and precipitate B through ethyl alcohol soxhlet extraction, wherein supernatant mentioning for phycocyanin
It takes；Precipitate A is used for the extraction of sodium zinc chlorophyllin；Precipitate B is used for the extraction of spirulina polysaccharide.Extraction process collection algae of the invention
Azurin, chlorophyll, spirulina polysaccharide isoreactivity substance are extracted from one, and existing spirulina money is substantially effectively utilized
Source avoids the waste of resource.
Therefore, how to make full use of the biomass energy of spirulina, reduce the wasting of resources, improve chlorophyll recovery rate and pure
Degree, it is existing in the prior art to need to solve that healthy and safe, up-to-standard, stable pure natural chlorophyll product is produced in guarantee
Certainly the technical issues of.
Summary of the invention
The present invention is in order to solve the above technical problems, provide a kind of technique for extracting chlorophyll from spirulina.
In order to reach purpose described above, the invention adopts the following technical scheme:
A kind of technique for extracting chlorophyll from spirulina, comprising the following steps:
(1) pretreatment of raw material: fresh spirulina is cleaned, 80~90min of dark processing at 1~9 DEG C, then 1
It was crushed to 50~100 meshes at~8 DEG C, is sent into the centrifuge tube equipped with steel ball, liquid nitrogen is poured into, when liquid nitrogen is just evaporated completely
It covers centrifuge tube and is placed in vortex instrument and be vortexed, be vortexed to the spirulina powder for crossing 250~350 meshes；
(2) it impregnates: under the conditions of dark and argon gas or nitrogen protection, deionized water is added in Xiang Shangshu spirulina powder end and impregnates
Processing handles auxiliary using microwave and ultrasound and impregnates, obtains spirulina aqueous solution；
(3) digest: be added under the conditions of dark and argon gas or nitrogen protection, in Xiang Shangshu spirulina aqueous solution mixed enzyme A into
Row enzymatic hydrolysis, and microwave treatment assistance enzymolysis is used, mixed enzyme B is then added, further uses ultrasonication assistance enzymolysis, obtains
Mixture must be digested；
(4) it filters: under the conditions of dark and argon gas or nitrogen protection, above-mentioned enzymatic hydrolysis mixture being carried out using ultrafiltration membrane
Filter removes filtrated stock, obtains chlorophyll semi-finished product；
(5) it dries: under the conditions of dark and argon gas or nitrogen protection, chlorophyll semi-finished product obtained above being sent into drying machine
In dried, sealed package is kept in dark place, and obtains the chlorophyll.
Further, it is overturned repeatedly centrifuge tube 3~5 times during step (1), the vortex.
Further, the mode impregnated in step (2), the microwave and ultrasound processing auxiliary are as follows: first in microwave power
For 50~60W, microwave temperature be at 2~6 DEG C progress 45~65s of microwave treatment, then ultrasonic power be 105~120W,
Ultrasonic temperature is that 3~7min of ultrasonication is carried out at 18~23 DEG C, in the above manner alternately microwave and ultrasound
Processing 2~3 times.
Further, in step (2), the state modulator of the immersion are as follows: immersion pressure is 0.05~0.09MPa, is impregnated
Temperature is 1 DEG C~8 DEG C, and soaking time is 2.5~3h.
Further, in step (3), the microwave treatment: microwave power is 85~95W, and the microwave time is 60~120s,
Microwave temperature is 2~7 DEG C.
Further, in step (3), the ultrasonication: ultrasonic power is 105~120W, sonication times 3
~9min, ultrasonic temperature are 18~25 DEG C.
Further, in step (3), the mixed enzyme A is protease, cellulase, pectase, hemicellulase, mistake
Oxide enzyme is according to mass ratio=3:1~2:1:0.3:0.1~0.2.
Further, it in step (3), is added before mixed enzyme B, is first warming up to 40~50 DEG C and enzyme inhibitor adjustment is added
PH carries out enzyme deactivation, and mixed enzyme B is added after cooling to 1~10 DEG C；After ultrasonication assistance enzymolysis, material is warming up to
It 42~50 DEG C and enzyme inhibitor adjustment PH is added carries out enzyme deactivation, then cool to 1~8 DEG C of acquisitions and digest mixture.The enzyme inhibits
Agent is protease inhibitors, cellulase inhibitor, pectin enzyme inhibitor, hemicellulose enzyme inhibitor, peroxidase inhibition
One of agent, wooden enzyme inhibitor, amylase inhibitor, lipoxidase inhibitor or more than one.
Further, in step (3), mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase, fruit
Glue enzyme is according to mass ratio=1~2:2:1:0.5:0.5:1~1.5.
Further, in step (5), the temperature of the decompression drying is 26~30 DEG C, and relative air humidity≤65% dries
The dry time is 22~30min, is dried to chlorophyll water content lower than 20%.
Since present invention employs above technical schemes, have the advantages that
(1) it when the application extracts spirulina Determination of Chlorophyll, is obtained by the way of immersion, enzymatic hydrolysis, microwave and ultrasonic treatment
Chlorophyll is obtained, the use of organic solvent is avoided, improves the edible safety of chlorophyll, enzyme cellulase used and fruit
Glue enzyme can degrade the cellulose for forming spirulina cells wall and pectin skeleton, and then destroy the cell wall skeleton knot of spirulina
Structure, increases the dissolution of cell inner chlorophyll, and chlorophyll extracts yield and is above 99.5%, is higher by than traditional solvent extraction method
8.8% or more, chlorophyll purity is above 99.96%.
(2) the application using the enzymes such as protease, cellulase, pectase, ligninase, amylase, esterase to spirulina into
Row enzymolysis processing, by other mass degradations in spirulina in addition to chlorophyll, and the chlorophyll in spirulina will not be destroyed,
Extract the chlorophyll contained in spirulina as far as possible, and product does not contain other impurities, improves the yield of chlorophyll
And purity.
(3) since spirulina contains more protein, and chlorophyll is very tight in conjunction with protein, and the application first carries out
It impregnates, then carries out enzymatic treatment again, facilitate going on smoothly for enzymatic hydrolysis, and first use the protein of proteasome degradation spirulina, have
Help cellulase etc. to degrade to the cellulose and pectin skeleton of spirulina cells wall, is conducive to improve chlorophyll extraction
Rate.
(4) the application decomposes the protein outside spirulina chlorophyll using protease and esterase, using peroxidase
With the substrate oxidation of lipoxygenase catalytic protein enzyme and esterase protein degradation matter, reaction is promoted to carry out, accelerate reaction speed, reduced
Time that chlorophyll extracts.
(5) each step of the application is carried out under gas shield being protected from light completely, and isolation chlorophyll connects with oxygen and light
Touching, avoids spirulina Determination of Chlorophyll content from being degraded during the extraction process, shortens chlorophyll extraction time, increases mentioning for chlorophyll
Take efficiency.
(6) ultrasonic wave extraction that the application uses has good cavitation effect and mechanical effect, and microwave energy generates
Good fuel factor makes intracellular polar substances generate amount of heat, and easy to operate, shortening extraction time, reduction energy consumption,
Save the cost, moreover it is possible to effectively improve recovery rate.
(7) due to spirulina cells be easy it is swollen, organic solvent extract when will in view of whether can destroy spiral phycobniliprotein,
But the application.
(8) the application is in pretreatment of raw material, and carrying out pulverization process to spirulina is that joined liquid nitrogen, and liquid nitrogen is when being vortexed
Gasification energy instantaneous coagulation and the temperature for reducing spirulina, are conducive to be fully ground and improve grinding efficiency, and be able to maintain and grind
Honed journey carries out at low temperature, can effectively prevent the degradation of chlorophyll.
(9) when the application pre-processes spirulina, shady place reason is first carried out before crushing, reduces the aqueous of spirulina
Amount, facilitate it is subsequent chlorophyll is extracted, improve chlorophyll extract yield.
Detailed description of the invention
In order to illustrate more clearly of present example or technical solution in the prior art, to embodiment or will show below
There is in technical description required attached drawing do simply to introduce, it should be apparent that, the accompanying drawings in the following description is only the present invention
Some examples to those skilled in the art, can also be attached according to these under the premise of not paying creativeness
Figure obtains other attached drawings:
Fig. 1 is a kind of process flow chart that chlorophyll is extracted from spirulina of the application.
Specific embodiment
Specific embodiments of the present invention will be described in further detail below, but the invention is not limited to these realities
Mode is applied, it is claimed to still fall within the claims in the present invention for any improvement or replacement on the present embodiment essence spirit
Range.
Embodiment 1
A kind of technique for extracting chlorophyll from spirulina, comprising the following steps:
(1) pretreatment of raw material: fresh spirulina is cleaned, and dark processing 80min, then crushes at 8 DEG C at 1 DEG C
To 50 meshes are crossed, it is sent into the centrifuge tube equipped with steel ball, pours into liquid nitrogen, centrifuge tube is covered when liquid nitrogen is just evaporated completely and is placed on
It is vortexed, is vortexed to the spirulina powder for crossing 250 meshes in vortex instrument；It is overturned repeatedly centrifuge tube 3 times during the vortex；
(2) it impregnates: under the conditions of dark and argon gas or nitrogen protection, deionized water is added in Xiang Shangshu spirulina powder end and impregnates
Processing handles auxiliary using microwave and ultrasound and impregnates, obtains spirulina aqueous solution；
The mode that microwave and ultrasound processing auxiliary is impregnated are as follows: first microwave power is 50W, microwave temperature is 2 DEG C
Then lower progress microwave treatment 45s carries out ultrasonication in the case where ultrasonic power is 105W, ultrasonic temperature is 18 DEG C
3min, alternately microwave and ultrasound is handled 2 times in the above manner；The state modulator of the immersion are as follows: impregnating pressure is
0.05MPa, soaking temperature are 1 DEG C, soaking time 2.5h；
(3) digest: be added under the conditions of dark and argon gas or nitrogen protection, in Xiang Shangshu spirulina aqueous solution mixed enzyme A into
Row enzymatic hydrolysis, and microwave treatment assistance enzymolysis is used, mixed enzyme B is then added, further uses ultrasonication assistance enzymolysis, obtains
Mixture must be digested；
The microwave treatment: microwave power 85W, microwave time are 60s, and microwave temperature is 2 DEG C；At the ultrasonic wave
Reason: ultrasonic power 105W, sonication times 3min, ultrasonic temperature are 18 DEG C；The mixed enzyme A is protease, fibre
Plain enzyme, pectase, hemicellulase, peroxidase are tieed up according to mass ratio=3:1:1:0.3:0.1；It is added before mixed enzyme B,
It is first warming up to 40 DEG C and enzyme inhibitor adjustment PH is added and carry out enzyme deactivation, mixed enzyme B is added after cooling to 1 DEG C；Ultrasonication
After assistance enzymolysis, material is warming up to 42 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, then cools to 8 DEG C of acquisition enzymes
Solve mixture；Mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase, pectase according to mass ratio=1:
2:1:0.5:0.5:1；
(4) it filters: under the conditions of dark and argon gas or nitrogen protection, above-mentioned enzymatic hydrolysis mixture being carried out using ultrafiltration membrane
Filter removes filtrated stock, obtains chlorophyll semi-finished product；
(5) it dries: under the conditions of dark and argon gas or nitrogen protection, chlorophyll semi-finished product obtained above being sent into drying machine
In dried, sealed package is kept in dark place, and obtains the chlorophyll；The temperature of the decompression drying is 26 DEG C, and air is opposite
Humidity≤65%, drying time 22min are dried to chlorophyll water content lower than 20%.
Embodiment 2
A kind of technique for extracting chlorophyll from spirulina, comprising the following steps:
(1) pretreatment of raw material: fresh spirulina is cleaned, and dark processing 90min, then crushes at 1 DEG C at 9 DEG C
To sieving with 100 mesh sieve, it is sent into the centrifuge tube equipped with steel ball, pours into liquid nitrogen, centrifuge tube lid is covered when liquid nitrogen is just evaporated completely and is set
In being vortexed in vortex instrument, it is vortexed to the spirulina powder for crossing 350 meshes；It is overturned repeatedly centrifuge tube 5 times during the vortex；
(2) it impregnates: under the conditions of dark and argon gas or nitrogen protection, deionized water is added in Xiang Shangshu spirulina powder end and impregnates
Processing handles auxiliary using microwave and ultrasound and impregnates, obtains spirulina aqueous solution；
The mode that microwave and ultrasound processing auxiliary is impregnated are as follows: first microwave power is 60W, microwave temperature is 6 DEG C
Then lower progress microwave treatment 65s carries out ultrasonication in the case where ultrasonic power is 120W, ultrasonic temperature is 18 DEG C
7min, alternately microwave and ultrasound is handled 3 times in the above manner；The state modulator of the immersion are as follows: impregnating pressure is
0.09MPa, soaking temperature are 8 DEG C, soaking time 3h；
(3) digest: be added under the conditions of dark and argon gas or nitrogen protection, in Xiang Shangshu spirulina aqueous solution mixed enzyme A into
Row enzymatic hydrolysis, and microwave treatment assistance enzymolysis is used, mixed enzyme B is then added, further uses ultrasonication assistance enzymolysis, obtains
Mixture must be digested；
The microwave treatment: microwave power 95W, microwave time are 120s, and microwave temperature is 7 DEG C；At the ultrasonic wave
Reason: ultrasonic power 120W, sonication times 9min, ultrasonic temperature are 25 DEG C；The mixed enzyme A is protease, fibre
Plain enzyme, pectase, hemicellulase, peroxidase are tieed up according to mass ratio=3:2:1:0.3:0.2；It is added before mixed enzyme B,
It is first warming up to 50 DEG C and enzyme inhibitor adjustment PH is added and carry out enzyme deactivation, mixed enzyme B is added after cooling to 10 DEG C；Ultrasonication
After assistance enzymolysis, material is warming up to 50 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, then cools to 1 DEG C of acquisition enzyme
Solve mixture；Mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase, pectase according to mass ratio=2:
2:1:0.5:0.5:1.5；
(4) it filters: under the conditions of dark and argon gas or nitrogen protection, above-mentioned enzymatic hydrolysis mixture being carried out using ultrafiltration membrane
Filter removes filtrated stock, obtains chlorophyll semi-finished product；
(5) it dries: under the conditions of dark and argon gas or nitrogen protection, chlorophyll semi-finished product obtained above being sent into drying machine
In dried, sealed package is kept in dark place, and obtains the chlorophyll；The temperature of the decompression drying is 30 DEG C, and air is opposite
Humidity≤65%, drying time 30min are dried to chlorophyll water content lower than 20%.
Embodiment 3
A kind of technique for extracting chlorophyll from spirulina, comprising the following steps:
(1) pretreatment of raw material: fresh spirulina is cleaned, and dark processing 82min, then crushes at 7 DEG C at 2 DEG C
To 60 meshes are crossed, it is sent into the centrifuge tube equipped with steel ball, pours into liquid nitrogen, centrifuge tube is covered when liquid nitrogen is just evaporated completely and is placed on
It is vortexed, is vortexed to the spirulina powder for crossing 270 meshes in vortex instrument；It is overturned repeatedly centrifuge tube 5 times during the vortex；
(2) it impregnates: under the conditions of dark and argon gas or nitrogen protection, deionized water is added in Xiang Shangshu spirulina powder end and impregnates
Processing handles auxiliary using microwave and ultrasound and impregnates, obtains spirulina aqueous solution；
The mode that microwave and ultrasound processing auxiliary is impregnated are as follows: first microwave power is 52W, microwave temperature is 3 DEG C
Then lower progress microwave treatment 50s carries out ultrasonication in the case where ultrasonic power is 110W, ultrasonic temperature is 19 DEG C
4min, alternately microwave and ultrasound is handled 2 times in the above manner；The state modulator of the immersion are as follows: impregnating pressure is
0.06MPa, soaking temperature are 2 DEG C, soaking time 2.6h；
(3) digest: be added under the conditions of dark and argon gas or nitrogen protection, in Xiang Shangshu spirulina aqueous solution mixed enzyme A into
Row enzymatic hydrolysis, and microwave treatment assistance enzymolysis is used, mixed enzyme B is then added, further uses ultrasonication assistance enzymolysis, obtains
Mixture must be digested；
The microwave treatment: microwave power 87W, microwave time are 80s, and microwave temperature is 3 DEG C；At the ultrasonic wave
Reason: ultrasonic power 110W, sonication times 4min, ultrasonic temperature are 19 DEG C；The mixed enzyme A is protease, fibre
Plain enzyme, pectase, hemicellulase, peroxidase are tieed up according to mass ratio=3:1.2:1:0.3:0.12；Be added mixed enzyme B it
Before, it is first warming up to 42 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, mixed enzyme B is added after cooling to 2 DEG C；At ultrasonic wave
After managing assistance enzymolysis, material is warming up to 43 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, then cools to 3 DEG C of acquisitions
Digest mixture；Mixed enzyme B be ligninase, cellulase, amylase, esterase, lipoxygenase, pectase according to mass ratio=
1.2:2:1:0.5:0.5:1.2；
(4) it filters: under the conditions of dark and argon gas or nitrogen protection, above-mentioned enzymatic hydrolysis mixture being carried out using ultrafiltration membrane
Filter removes filtrated stock, obtains chlorophyll semi-finished product；
(5) it dries: under the conditions of dark and argon gas or nitrogen protection, chlorophyll semi-finished product obtained above being sent into drying machine
In dried, sealed package is kept in dark place, and obtains the chlorophyll；The temperature of the decompression drying is 27 DEG C, and air is opposite
Humidity≤65%, drying time 25min are dried to chlorophyll water content lower than 20%.
Embodiment 4
A kind of technique for extracting chlorophyll from spirulina, comprising the following steps:
(1) pretreatment of raw material: fresh spirulina is cleaned, and dark processing 88min, then crushes at 3 DEG C at 8 DEG C
To 90 meshes are crossed, it is sent into the centrifuge tube equipped with steel ball, pours into liquid nitrogen, centrifuge tube is covered when liquid nitrogen is just evaporated completely and is placed on
It is vortexed, is vortexed to the spirulina powder for crossing 330 meshes in vortex instrument；It is overturned repeatedly centrifuge tube 5 times during the vortex；
(2) it impregnates: under the conditions of dark and argon gas or nitrogen protection, deionized water is added in Xiang Shangshu spirulina powder end and impregnates
Processing handles auxiliary using microwave and ultrasound and impregnates, obtains spirulina aqueous solution；
The mode that microwave and ultrasound processing auxiliary is impregnated are as follows: first microwave power is 58W, microwave temperature is 5 DEG C
Then lower progress microwave treatment 60s carries out ultrasonication in the case where ultrasonic power is 1115W, ultrasonic temperature is 22 DEG C
6min, alternately microwave and ultrasound is handled 3 times in the above manner；The state modulator of the immersion are as follows: impregnating pressure is
0.08MPa, soaking temperature are 6 DEG C, soaking time 2.9h；
(3) digest: be added under the conditions of dark and argon gas or nitrogen protection, in Xiang Shangshu spirulina aqueous solution mixed enzyme A into
Row enzymatic hydrolysis, and microwave treatment assistance enzymolysis is used, mixed enzyme B is then added, further uses ultrasonication assistance enzymolysis, obtains
Mixture must be digested；
The microwave treatment: microwave power 93W, microwave time are 100s, and microwave temperature is 6 DEG C；At the ultrasonic wave
Reason: ultrasonic power 115W, sonication times 8min, ultrasonic temperature are 24 DEG C；The mixed enzyme A is protease, fibre
Plain enzyme, pectase, hemicellulase, peroxidase are tieed up according to mass ratio=3:1.8:1:0.3:0.18；Be added mixed enzyme B it
Before, it is first warming up to 48 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, mixed enzyme B is added after cooling to 8 DEG C；At ultrasonic wave
After managing assistance enzymolysis, material is warming up to 49 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, then cools to 7 DEG C of acquisitions
Digest mixture；Mixed enzyme B be ligninase, cellulase, amylase, esterase, lipoxygenase, pectase according to mass ratio=
1.8:2:1:0.5:0.5:1.4；
(4) it filters: under the conditions of dark and argon gas or nitrogen protection, above-mentioned enzymatic hydrolysis mixture being carried out using ultrafiltration membrane
Filter removes filtrated stock, obtains chlorophyll semi-finished product；
(5) it dries: under the conditions of dark and argon gas or nitrogen protection, chlorophyll semi-finished product obtained above being sent into drying machine
In dried, sealed package is kept in dark place, and obtains the chlorophyll；The temperature of the decompression drying is 29 DEG C, and air is opposite
Humidity≤65%, drying time 28min are dried to chlorophyll water content lower than 20%.
Embodiment 5
A kind of technique for extracting chlorophyll from spirulina, comprising the following steps:
(1) pretreatment of raw material: fresh spirulina is cleaned, and dark processing 85min, then crushes at 4 DEG C at 5 DEG C
To 80 meshes are crossed, it is sent into the centrifuge tube equipped with steel ball, pours into liquid nitrogen, centrifuge tube is covered when liquid nitrogen is just evaporated completely and is placed on
It is vortexed, is vortexed to the spirulina powder for crossing 300 meshes in vortex instrument；It is overturned repeatedly centrifuge tube 4 times during the vortex；
(2) it impregnates: under the conditions of dark and argon gas or nitrogen protection, deionized water is added in Xiang Shangshu spirulina powder end and impregnates
Processing handles auxiliary using microwave and ultrasound and impregnates, obtains spirulina aqueous solution；
The mode that microwave and ultrasound processing auxiliary is impregnated are as follows: first microwave power is 55W, microwave temperature is 4 DEG C
Then lower progress microwave treatment 55s carries out ultrasonication in the case where ultrasonic power is 112W, ultrasonic temperature is 20 DEG C
5min, alternately microwave and ultrasound is handled 3 times in the above manner；The state modulator of the immersion are as follows: impregnating pressure is
0.07MPa, soaking temperature are 5 DEG C, soaking time 2.8h；
(3) digest: be added under the conditions of dark and argon gas or nitrogen protection, in Xiang Shangshu spirulina aqueous solution mixed enzyme A into
Row enzymatic hydrolysis, and microwave treatment assistance enzymolysis is used, mixed enzyme B is then added, further uses ultrasonication assistance enzymolysis, obtains
Mixture must be digested；
The microwave treatment: microwave power 90W, microwave time are 90s, and microwave temperature is 5 DEG C；At the ultrasonic wave
Reason: ultrasonic power 113W, sonication times 6min, ultrasonic temperature are 21 DEG C；The mixed enzyme A is protease, fibre
Plain enzyme, pectase, hemicellulase, peroxidase are tieed up according to mass ratio=3:1.5:1:0.3:0.15；Be added mixed enzyme B it
Before, it is first warming up to 45 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, mixed enzyme B is added after cooling to 5 DEG C；At ultrasonic wave
After managing assistance enzymolysis, material is warming up to 46 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, then cools to 4 DEG C of acquisitions
Digest mixture；Mixed enzyme B be ligninase, cellulase, amylase, esterase, lipoxygenase, pectase according to mass ratio=
1.5:2:1:0.5:0.5:1.3；
(4) it filters: under the conditions of dark and argon gas or nitrogen protection, above-mentioned enzymatic hydrolysis mixture being carried out using ultrafiltration membrane
Filter removes filtrated stock, obtains chlorophyll semi-finished product；
(5) it dries: under the conditions of dark and argon gas or nitrogen protection, chlorophyll semi-finished product obtained above being sent into drying machine
In dried, sealed package is kept in dark place, and obtains the chlorophyll；The temperature of the decompression drying is 28 DEG C, and air is opposite
Humidity≤65%, drying time 26min are dried to chlorophyll water content lower than 20%.
Comparative example 1
Embodiment in patent application CN201710169066.7 carries out chlorophyll extraction.
Comparative example 2
Embodiment in patent application CN201510528132.6 carries out chlorophyll extraction.
Comparative example 3
Embodiment in patent application CN200910016683.9 carries out chlorophyll extraction.
Chlorophyll is extracted using by the method for the embodiment of the present application 1~5 and comparative example 1~3, chlorophyll is examined to extract
Rate, the purity and organic solvent content of obtained chlorophyll, experimental result are as shown in table 1 below.
Table 1
| Group | Extract yield | Purity | Organic solvent content |
| Embodiment 1 | 99.7% | 99.98% | 0% |
| Embodiment 2 | 99.5% | 99.96% | 0% |
| Embodiment 3 | 99.7% | 99.98% | 0% |
| Embodiment 4 | 99.6% | 99.97% | 0% |
| Embodiment 5 | 99.8% | 99.99% | 0% |
| Comparative example 1 | 90.5% | 97.03% | 2.7% |
| Comparative example 2 | 90.7% | 99.52% | 0.4% |
| Comparative example 3 | 87.6% | 96.15% | 3.5% |
As 1 experimental data of table it is found that chlorophyll made from the application method is free of organic solvent, and chlorophyll extracts yield
It is above 99.5%, is higher by 8.8% or more than extracting yield using traditional solvent chlorophyll, chlorophyll purity is above
99.96%.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, nothing
By from the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended
Claim rather than above description limit, it is intended that by the institute in the meaning and scope for the equivalent loins for falling in claim
It changes and includes within protection scope of the present invention.
Claims (10)
1. a kind of technique for extracting chlorophyll from spirulina, which comprises the following steps:
(1) pretreatment of raw material: fresh spirulina is cleaned, 80~90min of dark processing at 1~9 DEG C, then at 1~8 DEG C
Under be crushed to 50~100 meshes, be sent into the centrifuge tube equipped with steel ball, pour into liquid nitrogen, covered when liquid nitrogen is just evaporated completely
Centrifuge tube is placed in vortex instrument and is vortexed, and is vortexed to the spirulina powder for crossing 250~350 meshes；
(2) impregnate: under the conditions of dark and argon gas or nitrogen protection, Xiang Shangshu spirulina powder is added at deionized water immersion in end
Reason handles auxiliary using microwave and ultrasound and impregnates, obtains spirulina aqueous solution；
(3) it digests: mixed enzyme A is added under the conditions of dark and argon gas or nitrogen protection, in Xiang Shangshu spirulina aqueous solution and carries out enzyme
Solution, and microwave treatment assistance enzymolysis is used, mixed enzyme B is then added, further uses ultrasonication assistance enzymolysis, obtains enzyme
Solve mixture；
(4) it filters: under the conditions of dark and argon gas or nitrogen protection, above-mentioned enzymatic hydrolysis mixture being filtered using ultrafiltration membrane, is removed
Filtrated stock is removed, chlorophyll semi-finished product are obtained；
(5) dry: under the conditions of dark and argon gas or nitrogen protection, by chlorophyll semi-finished product obtained above be sent into drying machine into
Row drying, sealed package are kept in dark place, and obtain the chlorophyll.
2. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step (1),
It is overturned repeatedly centrifuge tube 3~5 times during the vortex.
3. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step (2),
The mode that the microwave and ultrasound processing auxiliary is impregnated are as follows: first in the case where microwave power is 50~60W, microwave temperature is 2~6 DEG C
45~65s of microwave treatment is carried out, is then surpassed in the case where ultrasonic power is 105~120W, ultrasonic temperature is 18~23 DEG C
3~7min of sonicated, alternately microwave and ultrasound is handled 2~3 times in the above manner.
4. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step (2),
The state modulator of the immersion are as follows: immersion pressure is 0.05~0.09MPa, and soaking temperature is 1 DEG C~8 DEG C, soaking time 2.5
~3h.
5. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step (3),
The microwave treatment: microwave power is 85~95W, and the microwave time is 60~120s, and microwave temperature is 2~7 DEG C.
6. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step (3),
The ultrasonication: ultrasonic power is 105~120W, and sonication times are 3~9min, and ultrasonic temperature is 18~25
℃。
7. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step (3),
The mixed enzyme A is protease, cellulase, pectase, hemicellulase, peroxidase according to mass ratio=3:1~2:
1:0.3:0.1~0.2.
8. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step (3),
It is added before mixed enzyme B, is first warming up to 40~50 DEG C and enzyme inhibitor adjustment PH progress enzyme deactivation is added, after cooling to 1~10 DEG C
Add mixed enzyme B；After ultrasonication assistance enzymolysis, material is warming up to 42~50 DEG C and enzyme inhibitor adjustment is added
PH carries out enzyme deactivation, then cools to 1~8 DEG C of acquisition enzymatic hydrolysis mixture.
9. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step (3),
Mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase, pectase according to mass ratio=1~2:2:1:
0.5:0.5:1~1.5.
10. a kind of technique for extracting chlorophyll from spirulina according to claim 1, it is characterised in that: in step
(5), the temperature of the decompression drying is 26~30 DEG C, relative air humidity≤65%, and drying time is 22~30min, drying
It is lower than 20% to chlorophyll water content.
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