CN109553696A - A kind of preparation method of mussel class cooking liquor polysaccharide - Google Patents
A kind of preparation method of mussel class cooking liquor polysaccharide Download PDFInfo
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- CN109553696A CN109553696A CN201811576522.0A CN201811576522A CN109553696A CN 109553696 A CN109553696 A CN 109553696A CN 201811576522 A CN201811576522 A CN 201811576522A CN 109553696 A CN109553696 A CN 109553696A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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Abstract
The invention belongs to aquatic products field of deep, are related to a kind of preparation method of mussel class cooking liquor polysaccharide.Method of the invention is using mussel class machining cooking liquid as raw material, through raw material concentration, centrifugation slagging-off, ultrafiltration, ethanol precipitation, enzymatic hydrolysis, secondary alcohol precipitation, freeze-drying and etc. obtain cream powder mussel class machining cooking liquid polysaccharide finished product, polysaccharide recovery reaches 70%, and purity of polysaccharide is 80% or more.Method and process provided by the invention is simple, environmentally protective, and polysaccharide yield is high, with high purity, is suitable for industrialization amplification.Step is simple and convenient to operate, is practical.
Description
Technical field
The invention belongs to aquatic products field of deep more particularly to a kind of industrialization of mussel class machining cooking liquid polysaccharide to mention
Take method
Background technique
Both in recent years, the cultivation scale of China's Ruditapes philippinarum and scallop constantly expands, yield sharp increase, at present
Process can all be related to boiling link, can generate a large amount of cooking liquor, wherein containing many water-soluble polysaccharide components.Phase
Research is closed it has been shown that the water-soluble polysaccharide ingredient in mussel class has antitumor, anti-oxidant, raising immunity and liver protecting
Etc. different physiological roles.At this stage, for this part, cooking liquor polysaccharide is seldom recycled, mainly due to unsuitable
Recovery method, existing method is complicated for operation, polysaccharide yield is low, gained purity of polysaccharide is not high, active bad, not good application
Direction.Therefore, there is an urgent need to improve to prior art, the mussel class cooking liquor extraction method of polysaccharides of green high-efficient is developed.
The present invention provides a kind of industrialized process for preparing of mussel class cooking liquor active polysaccharide, can efficient acquisition it is more
Sugar product.The present invention helps to make full use of mussel class processing byproduct, reduces cooking liquor and discharges pollution on the environment；It can
To improve mussel class processing added value, increase economic efficiency.Gained has immunocompetent mussel class cooking liquor polysaccharide can be into one
Step exploitation is functional food.
Summary of the invention
The object of the present invention is to provide a kind of using mussel class cooking liquor as the polysaccharide industrialized process for preparing of raw material.Described
The preparation method of mussel class cooking liquor polysaccharide the following steps are included:
1. mussel class cooking liquor is carried out being concentrated into solid quality content being 8~15%, supernatant is collected in centrifugation removal of impurities
I；
2. obtaining permeate to step 1. resulting supernatant I progress ultrafiltration using the ultrafiltration membrane of ten thousand molecular weight of 1-3；
3. the ethanol water that volumetric concentration is 95% is added into permeate, stand, centrifugation, takes precipitating I；
4. it is water-dispersible to precipitate I, papain is added and is digested, is centrifuged, collects supernatant II；
5. the ethanol water that volumetric concentration is 95% is added in upward clear liquid I I, stand, centrifugation, obtains precipitating II；
After 6. precipitating II is dissolved with water, freeze-drying.
Mussel class cooking liquor polysaccharide is prepared by the above method, for gained purity of polysaccharide up to 80%, polysaccharide recovery is reachable
70% or more.And method of the invention is raw materials used from a wealth of sources, cheap, and processing method is simple, small investment, added value
Height, can be widely used in the large-scale recovery of active polysaccharide, and application prospect is very wide.The mussel class cooking liquor polysaccharide tool
It is improved the bioactivity of immunity.Therefore, the purpose of another aspect of the present invention lie also in provide mussel class cooking liquor polysaccharide and
It is preparing the application in immunostimulant.
Specific embodiment
The technical scheme is that using mussel class cooking liquor as raw material including the secondary alcohol precipitation of the heavy-enzymatic hydrolysis-of ultrafiltration -ol
Etc. processes technical solution:
Wherein, the step 1. in preferably by mussel class cooking liquor be concentrated into solid content be 12~15%.
Wherein, the step 2. in preferably 2~30,000 molecular weight of ultrafiltration membrane ultrafiltration membrane.
Wherein, the step 3. in, the preferred 1:3 of the volume ratio of permeate and ethanol water.
Wherein, the step 4. in, precipitating I it is water-dispersible after become solid quality content 10~12% system.
Wherein, the step 4. in, the preferred parameter of enzymolysis step includes: the enzyme concentration 1~3% of papain,
Digest pH be 6.0~7.5, enzymolysis time 2~6 hours, 45~65 DEG C of hydrolysis temperature.
In highly preferred technical solution, method of the present invention includes the following steps:
1. mussel class cooking liquor is carried out being concentrated into solid quality content being 12~15%, supernatant is collected in centrifugation removal of impurities
Liquid I；
2. obtaining permeate to step 1. resulting supernatant I progress ultrafiltration using the ultrafiltration membrane of ten thousand molecular weight of 1-3；
3. the ethanol water that volumetric concentration is 95% is added into permeate, make the volume of permeate and ethanol water
Than standing for 1:3, centrifugation takes precipitating I；
4. it is water-dispersible to solid quality content 10~12% to precipitate I, addition papain is digested, from
The heart collects supernatant II；Wherein, enzyme concentration 1~3%, enzymatic hydrolysis pH be 6.0~7.5, enzymolysis time 2~6 hours, hydrolysis temperature
45~65 DEG C；
5. the ethanol water that volumetric concentration is 95% is added in upward clear liquid I I, make supernatant II and ethanol water
Volume ratio is 1:3, is stood, centrifugation, obtains precipitating II；
After 6. precipitating II is dissolved with water, freeze-drying.
The contents of the present invention are further described in a manner of specific embodiment below, so that those skilled in the art are more complete
Understand to face the present invention, but do not limit the invention in any way.
Unless otherwise specified, heretofore described mussel class cooking liquor is that commodity are bought.
Embodiment 1
The production technology of Ruditapes philippinarum cooking liquor polysaccharide
(1) centrifugation removal of impurities
Ruditapes philippinarum cooking liquor (solid content 15%) is subjected to centrifugation removal of impurities with revolving speed 4000rpm, the time 20 minutes, is received
Collect supernatant.
(2) ultrafiltration
The ultrafiltration membrane that supernatant after centrifugation removal of impurities is crossed to 30,000 molecular weight, obtains permeate.
(3) alcohol precipitations
At room temperature by above-mentioned permeate, 24 hours are stood according to material liquid volume ratio 1:3 ratio using 95% ethyl alcohol.
(4) it digests
4000rpm is centrifuged 20 minutes and takes precipitating after alcohol precipitation, volatilizes ethyl alcohol, adjustment solid content is 11.6%, using wood
Melon protease hydrolyzed, pH 7.0, enzyme concentration 2%, enzymolysis time 4 hours, 65 DEG C of hydrolysis temperature.
(5) secondary alcohol precipitation
Enzymolysis liquid centrifuging and taking supernatant after enzymatic hydrolysis, carries out secondary alcohol precipitation (condition is same as above).
(6) it is freeze-dried
Alcohol precipitation system centrifuging and taking is precipitated, with a small amount of distilled water immersion, ethyl alcohol is volatilized, is freeze-dried, obtains purity
For 81.27% Ruditapes philippinarum cooking liquor polysaccharide.
Embodiment 2
The production technology of scallop cooking liquor polysaccharide
(1) centrifugation removal of impurities
Scallop cooking liquor (solid content 8.3%) is subjected to centrifugation removal of impurities with revolving speed 4000rpm, the time 20 minutes, in collection
Clear liquid.
(2) ultrafiltration
The ultrafiltration membrane that supernatant after centrifugation removal of impurities is crossed to 10,000 molecular weight, obtains permeate.
(3) alcohol precipitations
At room temperature by above-mentioned permeate, 24 hours are stood according to material liquid volume ratio 1:3 ratio using 95% ethyl alcohol.
(4) it digests
4000rpm is centrifuged 20 minutes and takes precipitating after alcohol precipitation, volatilizes ethyl alcohol, adjustment solid content is 12%, using pawpaw
Protease hydrolyzed, pH 7.0, enzyme concentration 1%, enzymolysis time 4 hours, 45 DEG C of hydrolysis temperature.
(5) secondary alcohol precipitation
Enzymolysis liquid centrifuging and taking supernatant after enzymatic hydrolysis, carries out secondary alcohol precipitation (condition is same as above).
(6) it is freeze-dried
Alcohol precipitation system centrifuging and taking is precipitated, with a small amount of distilled water immersion, ethyl alcohol is volatilized, is freeze-dried, obtains purity
For 80.25% scallop cooking liquor polysaccharide.
Embodiment 3
The test experience that Ruditapes philippinarum cooking liquor polysaccharide acts on macrophage
(1) mtt assay detection Ruditapes philippinarum cooking liquor polysaccharide promotes the value-added experiment of macrophage
1) cell culture
The cell cryopreservation tube of mononuclear macrophage (RAW264.7) is removed from liquid nitrogen and is placed in 37 DEG C of water-baths, wait freeze
It deposits after ice-nucleus substantially melts in pipe, takes out cryopreservation tube, move it in superclean bench, wipe nozzle with cotton ball soaked in alcohol.It draws
Cell suspension moves in the centrifuge tube of the 15cm of 10cm culture medium, and 1000r/min is centrifuged 5min, and Aspirate supernatant flicks centrifugation
Bottom of the tube keeps cell mass concussion evenly dispersed.Addition 1mL culture medium makes cell suspend, and then draws cell suspension instillation and is equipped with
It is shaken in the culture bottle of 10mL culture medium, keeps cell evenly dispersed, be put into CO2It is cultivated in incubator.In RPMI1640 culture medium
Middle culture macrophage RAW264.7, in culture medium containing 10% fetal calf serum (through 56 DEG C of inactivation 30min), dual anti-each
100U/mL.(37 DEG C of temperature, contain 5%CO to incubated cell in incubator2Gas), an every two days biography generation.
2) Ruditapes philippinarum cooking liquor polysaccharide improves the detection of macrophage vigor
It is careful after culture for 24 hours in the way of 100 holes μ L/ by the cell inoculation in logarithmic growth phase in 96 orifice plates
It inhales and abandons supernatant, with the culture containing corresponding final concentration sugared (200 μ g/mL, 150 μ g/mL, 100 μ g/mL, 50 μ g/mL, 25 μ g/mL)
Base processing for 24 hours, inhales and abandons supernatant, and with PBS, twice, MTT (1mg/mL) solution is then added in board-washing repeatedly, continues to cultivate 4h, again
Supernatant is abandoned, 100 μ L DMSO are added in every hole, and each hole absorbance value is detected at 570nm, and reference wavelength is set as 630nm.
The result shows that: 150 μ g/mL and the Ruditapes philippinarum cooking liquor polysaccharide of 100 μ g/mL concentration are to mononuclear macrophage
(RAW264.7) cell viability promotion effect is obvious, and opposite appreciation rate illustrates Ruditapes philippinarum cooking liquor all 50% or more
Polysaccharide can effectively enhance mononuclear macrophage vigor, improve the immunity of the human body.
(2) measurement of the Ruditapes philippinarum cooking liquor polysaccharide to macrophages phagocytic capacity
Concentration of cell suspension is adjusted to 1 × 105Mg/mL will be in logarithmic growth phase in the way of 100 holes μ L/
For cell inoculation in 96 orifice plates, supernatant is abandoned in culture afterwards for 24 hours, is cleaned 2 times through PBS, a certain amount of drug of each hole addition (200 μ g/mL,
150 μ g/mL, 100 μ g/mL, 50 μ g/mL, 25 μ g/mL) it is cultivated, each concentration repeats 6 holes.After culture for 24 hours, according to 100 μ
0.1% dimethyl diaminophenazine chloride normal saline solution is added in the principle in the hole L/, continues to inhale abandoning supernatant after cultivating 4h, is cleaned 2 times, added using PBS
Enter 100 hole μ L/ of cytolysate (glacial acetic acid: ethyl alcohol=1:1, V/V), measures each hole at 540nm after completely dissolution to cell
Absorbance value, the measurement result absorbance value the big, shows that macrophage phagocytosis dimethyl diaminophenazine chloride ability is stronger.
Measurement result is shown: Ruditapes philippinarum cooking liquor polysaccharide can be proliferated with inducing macrophage, be mentioned in 25 μ g/ml
Its high ability for swallowing dimethyl diaminophenazine chloride.
Claims (9)
1. a kind of preparation method of mussel class cooking liquor polysaccharide, comprising the following steps:
1. mussel class cooking liquor is carried out being concentrated into solid quality content being 8~15%, supernatant I is collected in centrifugation removal of impurities；
2. obtaining permeate to step 1. resulting supernatant I progress ultrafiltration using the ultrafiltration membrane of ten thousand molecular weight of 1-3；
3. the ethanol water that volumetric concentration is 95% is added into permeate, stand, centrifugation, takes precipitating I；
4. it is water-dispersible to precipitate I, papain is added and is digested, is centrifuged, collects supernatant II；
5. the ethanol water that volumetric concentration is 95% is added in upward clear liquid I I, stand, centrifugation, obtains precipitating II；
After 6. precipitating II is dissolved with water, freeze-drying.
2. the method according to claim 1, wherein the step 1. in mussel class cooking liquor is concentrated into it is solid
Shape object content is 12~15%.
3. the method according to claim 1, wherein the step 2. in ultrafiltration membrane be 2~30,000 molecular weight
Ultrafiltration membrane.
4. the method according to claim 1, wherein the step 3. in, permeate and ethanol water
Volume ratio is 1:3.
5. the method according to claim 1, wherein the step 4. in, precipitating I it is water-dispersible after solid content
Mass content 10~12%.
6. the method according to claim 1, wherein the step 4. in, the enzyme concentration 1 of papain~
3%, enzymatic hydrolysis pH be 6.0~7.5, enzymolysis time 2~6 hours, 45~65 DEG C of hydrolysis temperature.
7. the method according to claim 1, wherein including the following steps:
1. mussel class cooking liquor is carried out being concentrated into solid quality content being 12~15%, supernatant I is collected in centrifugation removal of impurities；
2. obtaining permeate to step 1. resulting supernatant I progress ultrafiltration using the ultrafiltration membrane of ten thousand molecular weight of 1-3；
3. the ethanol water that volumetric concentration is 95% is added into permeate, make the volume ratio of permeate and ethanol water
1:3 is stood, centrifugation, takes precipitating I；
4. it is water-dispersible to solid quality content 10~12% to precipitate I, papain is added and is digested, is centrifuged, receives
Collect supernatant II；Wherein, enzyme concentration 1~3%, enzymatic hydrolysis pH be 6.0~7.5, enzymolysis time 2~6 hours, hydrolysis temperature 45~65
℃；
5. the ethanol water that volumetric concentration is 95% is added in upward clear liquid I I, make the volume of supernatant II and ethanol water
Than standing for 1:3, centrifugation obtains precipitating II；
After 6. precipitating II is dissolved with water, freeze-drying.
8. the mussel class cooking liquor polysaccharide of preparation according to the method for claim 1.
9. right want 8 described in mussel class cooking liquor polysaccharide preparing the application in immunostimulant.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1583803A (en) * | 2004-06-07 | 2005-02-23 | 中国人民解放军第二军医大学 | Polysaccharose MF4 of mussel with enhancing immunity and anti-tumour activity |
| CN103191044A (en) * | 2013-04-16 | 2013-07-10 | 镇江湘兰生物科技有限公司 | Application of Ruditapes philippinarum polysaccharide in preparation of cosmetics |
| CN105852132A (en) * | 2016-03-31 | 2016-08-17 | 浙江海洋学院 | Preparation method of thick-shell mytilus edulis polysaccharide oral liquid |
| CN106749718A (en) * | 2016-11-29 | 2017-05-31 | 山东福瑞达医药集团公司 | A kind of industrial extraction method of mussel polysaccharide |
-
2018
- 2018-12-22 CN CN201811576522.0A patent/CN109553696A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1583803A (en) * | 2004-06-07 | 2005-02-23 | 中国人民解放军第二军医大学 | Polysaccharose MF4 of mussel with enhancing immunity and anti-tumour activity |
| CN103191044A (en) * | 2013-04-16 | 2013-07-10 | 镇江湘兰生物科技有限公司 | Application of Ruditapes philippinarum polysaccharide in preparation of cosmetics |
| CN105852132A (en) * | 2016-03-31 | 2016-08-17 | 浙江海洋学院 | Preparation method of thick-shell mytilus edulis polysaccharide oral liquid |
| CN106749718A (en) * | 2016-11-29 | 2017-05-31 | 山东福瑞达医药集团公司 | A kind of industrial extraction method of mussel polysaccharide |
Non-Patent Citations (3)
| Title |
|---|
| 吕学姣 等: "菲律宾蛤仔蒸煮液多糖回收工艺及微胶囊化", 《食品安全质量检测学报》 * |
| 易杨华: "《海洋药物导论》", 30 September 2004, 上海科学技术出版社 * |
| 韩青: "菲律宾蛤仔蒸煮液多糖的提取及生物活性研究", 《万方数据知识服务平台》 * |
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