CN103012615A - Method for efficiently extracting sepia acidic polysaccharose - Google Patents
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- CN103012615A CN103012615A CN2013100050805A CN201310005080A CN103012615A CN 103012615 A CN103012615 A CN 103012615A CN 2013100050805 A CN2013100050805 A CN 2013100050805A CN 201310005080 A CN201310005080 A CN 201310005080A CN 103012615 A CN103012615 A CN 103012615A
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Abstract
The invention relates to a method for efficiently extracting sepia acidic polysaccharose, which comprises the following steps: defreezing cryopreserved sepia, evenly mixing with ethyl acetate, carrying out ultrasonic treatment, soaking, carrying out vacuum filtration under reduced pressure to remove the ethyl acetate, and carrying out freeze-drying to obtain sepia powder; and carrying out enzymolysis on the powder with papain under optimum parameter conditions, inactivating the papain, removing proteins, precipitating with anhydrous ethanol, and carrying out freeze-drying to obtain the sepia acidic polysaccharose. The invention utilizes the neural network technique to optimize the extraction parameters, and implements the efficient preparation of sepia acidic polysaccharose in combination with the papain enzymolysis technique, so that the relative extraction effect is up to 91.4%. The method is simple and convenient to operate, is high in extraction rate, provides theoretical references for development and research of sepia acidic polysaccharose in future, better promotes the development of sepia acidic polysaccharose in the aspects of medicine, functional health food and the like, and has wide market prospects.
Description
Technical field
The present invention relates to a kind of extracting method of polysaccharide, be specifically related to a kind of method of high efficiency extraction squid ink acidic polysaccharose, belong to functional health-care food or biomedicine field.
Background technology
Polysaccharide is to be present in the occurring in nature resource than the carbohydrate of horn of plenty, extensively is present in the organisms such as plant, animal and microorganism.Polysaccharide is as a kind of natural polymer seed extract of almost non-toxic side effect, aspect the prevention and treatment of numerous disease, particularly some stubborn diseases, difficult disease such as aspects such as tumour, hypoimmunity, hyperglycemia, aging, virus diseases, have the effect of himself uniqueness.Along with the continuous progress of science and technology, and the research and development of molecular biology and novel drugs resource, the research of polysaccharide is paid attention to more and more widely, and the research report of polysaccharide is also in cumulative year after year.Educational circles generally believes that as protein, nucleic acid epoch, 21 century is the epoch of " polysaccharide life science " both at home and abroad.
Because polysaccharide has diversified bioactive functions and at functional food be widely used clinically, the explo iting and researching of polysaccharide Biological resources is become increasingly active, become the study hotspot of natural drug, biological chemistry, life science, up to the present, existing more than 300 kind of polysaccharide compound is separated from natural product.Yet, with the day by day scarcity of landing field Chinese herbal medicine resource, the human abundant oceanic resources of the length and breadth of land and reserves that the research visual angle of active polysaccharide stretched to.
Cuttlefish is one of China's four large marine products, and only the annual production of Sepiella maindroni just reaches 70,000 tons in recent years.The cuttlefish ink sac accounts for its overall proportion 10%, and every annual amount can reach 7 kilotons.In China, the cuttlefish ink sac causes the wasting of resources and environmental pollution always as waste.China's medicine is long to understanding and the applicating history of squid ink, sees the earliest the Compendium of Material Medica of the Ming Dynasty, is mainly used in treating stenocardia.Modern study confirms that squid ink has widely biological action, such as effects such as hemostasis and treatment gynecopathy, antitumor, anti-oxidant, radioprotective, anti-retroviral, antibiotic, reducing blood-fat and liter are white.In recent years, we find that squid ink has the toxic side effect of stronger alleviation chemotherapeutics, and the reduction chemotherapeutics is to the toxic damages of body healthy tissues organ.Our sufficient early-stage Study result shows that with a small amount of other research reports polysaccharide is activeconstituents main in the squid ink, and we have confirmed that polysaccharide has obvious chemotherapeutic protection effect.Now prove, why polysaccharide has widely biological effectiveness, and with wherein contained acidic polysaccharose is directly related, acidic polysaccharose is the main component of its performance function, even having report to think, the function that how much has determined this polysaccharide of contained sulfate group is strong and weak in the polysaccharide.
Squid ink is the abundant but marine active substances that discarded by food-processing industry of a kind of reserves, although existing a small amount of research relates to its activity at present, even to the activity research of polysaccharide component wherein, but there is not yet report for extraction preparation and the biological function thereof of its main active ingredient acidic polysaccharose.In Global land resource day by day precious today, from terrestrial plant exploitation natural radioactivity polysaccharide since the geography of cultivation require tight, yield poorly, the factor such as cost of material height, so that the application of various natural radioactivity polysaccharide is subject to great restriction, comparatively speaking, marine algae resource can not take the agricultural land, abundant in natural resources, and aquaculture cost is lower, therefore, the development and use of squid ink resource will fully develop talents.Therefore, the exploitation squid ink acquisition economic benefit that not only can turn waste into wealth can also be improved environment and obtain good social benefit, the more important thing is that the medicine source is abundant, excavate its potential pharmaceutical use for taking full advantage of oceanic resources, enlarge the medicine source and have far reaching significance and wide prospect.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of efficient, stable, easy squid ink acidic polysaccharose.
For achieving the above object, the present invention is by the following technical solutions:
A kind of method of high efficiency extraction squid ink acidic polysaccharose comprises the steps: that described squid ink acidic polysaccharose is take squid ink as raw material, and the frozen squid ink that thaws is used the ethyl acetate mixing, ultrasonication; Suction filtration goes ethyl acetate, and lyophilize obtains black powder; Artificial neural network technology is optimized the papain enzymolysis parameter, and the ethanol precipitation namely obtains acidic polysaccharose behind the zymolyte Deproteinization after the lyophilize, and concrete steps are as follows:
(1) squid ink that under 3-5 ℃ of condition, thaws, with isopyknic ethyl acetate mixing, described volume unit is milliliter;
(2) the mixture ultrasonication that step (1) is obtained, the treatment time is 4-6s, interval 8-12s, number of processes is 15-25 time;
(3) step (2) gains are stirred 6-10h under 3-5 ℃ of condition, remove ethyl acetate with the decompress filter method, black squid ink powder is processed to get in lyophilize;
(4) with papain enzymolysis squid ink powder, boiling water bath inactivated proteases 10min, get supernatant liquor after centrifugal, add chloroform/propyl carbinol mixed solution in the supernatant liquor, magnetic stirrer 20min, stirring velocity is 1000 rpm, the centrifugal supernatant liquor that gets, the dehydrated alcohol that adds 4 times of volumes in the supernatant liquor, the precipitation lyophilize namely gets the squid ink acidic polysaccharose; The mass ratio of described papoid and squid ink powder is papoid: squid ink powder=5:95; The volume that adds chloroform/propyl carbinol (V/V=4/1) mixed solution in the described supernatant liquor is 1/4 of supernatant liquor cumulative volume; Add in the described supernatant liquor that both ratios are chloroform/propyl carbinol=4/1 in chloroform/propyl carbinol mixed solution; Described processing parameter with papain enzymolysis squid ink powder adopts artificial neural network technology optimization, and its optimum processing parameter is that pH 7.0, enzymolysis time are 7h, and hydrolysis temperature is 52 ℃, and solid-liquid ratio is 1 g/35ml;
A kind of squid ink acidic polysaccharose that adopts the aforesaid method preparation;
Described employing artificial neural network technology is optimized every factor that affects extraction efficiency according to the Box-behnken experimental design.According to neural network platform and the testing data of JMP software, select the method match response target of " K is folding " cross validation, it is 5 that the cross validation group is counted K, and over-fitting penalizes 0.001, and course is several 20, greatest iteration number 50, convergence criterion 0.00001.Coefficient of determination R by model
2And cross validation R
2Value is determined the concealed nodes number.By 2-7 concealed nodes carried out model training, repeat 5 times at every turn, show R
2Value increases along with the increase of concealed nodes number, and levels off to 1, cross validation R
2Increase afterwards first and descend, it is excessive that explanation can match occur at concealed nodes above 3, and cause match not enough less than 3, therefore selecting the concealed nodes number is 3, adopts three layers of nerve net of 3 * 3 * 1 structure, i.e. 3 input neurons, represent respectively pH(X1), enzymolysis time (X2), solid-liquid ratio (X3), 3 concealed nodes, 1 output neuron represents the extraction yield Y of polysaccharide.
Description of drawings
Fig. 1 is the extraction flow process of squid ink acidic polysaccharose;
Fig. 2 is the R of different concealed nodes models
2And cross validation R
2
Embodiment
Come the present invention is described in further details below in conjunction with drawings and Examples and testing data, these embodiment only are used for illustrating the present invention, do not limit the scope of the invention.
Embodiment 1
The artificial neural network technology optimal preparation technology
(1) get under 4 ℃ of the freezing squid ink and thaw, add the equal-volume ethyl acetate, ultrasonication (treatment time 5s, interval 10s, number of times 20 times) is soaked 8h;
(2) adopt decompress filter to remove the organic solvent ethyl acetate of soaking, black powder is processed to get in the squid ink lyophilize;
(3) according to the Box-behnken test design, such as table 1,2;
(4) take by weighing the squid ink powder of 1g, directly add 5% papoid pulvis (the relatively weight of squid ink powder), under 52 ℃ of conditions, add solid-liquid ratio by above-mentioned design, regulate pH, the controlled enzymatic hydrolysis time;
(5) enzymolysis time is to the enzyme 10min that goes out under boiling water bath afterwards, and centrifugal (5000rpm, 10min) removes precipitation 2-3 time;
(6) adopt the Sevag method remove albumen (chloroform/propyl carbinol that namely adds 1/4 volume, volume ratio 4:1 shakes up 20min at magnetic stirring apparatus, the centrifugal 10min of 5000rpm gets supernatant liquor and obtains jonquilleous polysaccharide liquid;
(7) the Sepia polysaccharide constant volume that adopts each different volumes that the colorimetric cylinder of 50ml will obtain adopts the By Anthrone Sulphuric acid method to measure the content of Sepia polysaccharide in the solution to 50ml, and the calculating yield;
(8) coefficient of determination R by model
2And cross validation R
2Value is determined concealed nodes number such as Fig. 2; R
2Arrived more than 0.99, illustrated that actual value and predictor have good dependency.
Table 1 is Box-Behnken test design level of factor table:
Table 2 is Box-behnken test design scheme and result:
The high-efficient extraction technology step of squid ink acidic polysaccharose
(1) get under 4 ℃ of the freezing squid ink and thaw, add the equal-volume ethyl acetate, ultrasonication (treatment time 5s, interval 10s, number of times 20 times) is soaked 8h;
(2) adopt decompression to filter and remove the organic solvent ethyl acetate of soaking, black powder is processed to get in the squid ink lyophilize;
(3) get squid ink powder and papoid mixture (W/W=95/5) and dissolve with distilled water (pH 7.0), solid-liquid ratio (W/V) is 1g/35ml, 52 ℃ of enzymolysis 7h;
(4) the boiling water bath enzyme 10min that goes out, the centrifugal 10min of 5000g goes precipitation, repeats 2 times;
(5) Sevag method Deproteinization: add the chloroform/propyl carbinol (V/V=4/1) of 1/4 volume, stir 20min at magnetic stirring apparatus, stirring velocity 1000 rpm, behind the 20min clock, the centrifugal 10min of 5000g gets supernatant liquor;
(6) 4 times of volume dehydrated alcohol precipitations, lyophilize namely gets the squid ink acidic polysaccharose.
(1) get under 3 ℃ of the freezing squid ink and thaw, add the equal-volume ethyl acetate, ultrasonication (treatment time 4s, interval 8s, number of times 15 times) is soaked 6h;
(2) adopt decompression to filter and remove the organic solvent ethyl acetate of soaking, black powder is processed to get in the squid ink lyophilize;
(3) get squid ink powder and papoid mixture (W/W=95/5) and dissolve with distilled water (pH 7.0), solid-liquid ratio (W/V) is 1g/35ml, 52 ℃ of enzymolysis 7h;
(4) the boiling water bath enzyme 10min that goes out, the centrifugal 10min of 5000g goes precipitation, repeats 2 times;
(5) Sevag method Deproteinization: add the chloroform/propyl carbinol (V/V=4/1) of 1/4 volume, stir 20min at magnetic stirring apparatus, stirring velocity 1000 rpm, behind the 20min clock, the centrifugal 10min of 5000g gets supernatant liquor;
Embodiment 4
(1) get under 5 ℃ of the freezing squid ink and thaw, add the equal-volume ethyl acetate, ultrasonication (treatment time 6s, interval 12s, number of times 25 times) is soaked 10h;
(2) adopt decompression to filter and remove the organic solvent ethyl acetate of soaking, black powder is processed to get in the squid ink lyophilize;
(3) get squid ink powder and papoid mixture (W/W=95/5) and dissolve with distilled water (pH 7.0), solid-liquid ratio (W/V) is 1g/35ml, 52 ℃ of enzymolysis 7h;
(4) the boiling water bath enzyme 10min that goes out, the centrifugal 10min of 5000g goes precipitation, repeats 2 times;
(5) Sevag method Deproteinization: add the chloroform/propyl carbinol (V/V=4/1) of 1/4 volume, stir 20min at magnetic stirring apparatus, stirring velocity 1000 rpm, behind the 20min clock, the centrifugal 10min of 5000g gets supernatant liquor.
Claims (6)
1. the method for a high efficiency extraction squid ink acidic polysaccharose, it is characterized in that: described squid ink acidic polysaccharose is take squid ink as raw material, the frozen squid ink that thaws is used the ethyl acetate mixing, ultrasonication; Suction filtration goes ethyl acetate, and lyophilize obtains black powder; Artificial neural network technology is optimized the papain enzymolysis parameter, and the ethanol precipitation namely obtains acidic polysaccharose behind the zymolyte Deproteinization after the lyophilize, and concrete steps are as follows:
(1) squid ink that thaws under 3-5 ℃ of condition is with isopyknic ethyl acetate mixing;
(2) the mixture ultrasonication that step (1) is obtained, the treatment time is 4-6s, interval 8-12s, number of processes is 15-25 time;
(3) step (2) gains are stirred 6-10h under 3-5 ℃ of condition, remove ethyl acetate with the decompress filter method, black squid ink powder is processed to get in lyophilize;
(4) with papain enzymolysis squid ink powder, boiling water bath inactivated proteases 10min, get supernatant liquor after centrifugal, repeat 2 times, add chloroform/propyl carbinol mixed solution in the supernatant liquor, magnetic stirrer 20min, stirring velocity is 1000 rpm, the centrifugal supernatant liquor that gets, the dehydrated alcohol of 4 times of volumes of adding in the supernatant liquor, the precipitation lyophilize namely gets the squid ink acidic polysaccharose.
2. the method for a kind of high efficiency extraction squid ink acidic polysaccharose according to claim 1, it is characterized in that: the mass ratio of the described papoid of step (4) and squid ink powder is papoid: squid ink powder=5:95.
3. the method for a kind of high efficiency extraction squid ink acidic polysaccharose according to claim 1, it is characterized in that: the volume that adds chloroform/propyl carbinol (V/V=4/1) mixed solution in the described supernatant liquor of step (4) is 1/4 of supernatant liquor cumulative volume.
4. it is characterized in that according to claim 1 or the method for 3 described a kind of high efficiency extraction squid ink acidic polysaccharoses: add in the described supernatant liquor of step (4) that both ratios are chloroform/propyl carbinol=4/1 in chloroform/propyl carbinol mixed solution.
5. according to claim 1 or the method for 3 described a kind of high efficiency extraction squid ink acidic polysaccharoses, it is characterized in that: the described processing parameter with papain enzymolysis squid ink powder of step (4) adopts artificial neural network technology optimization, its optimum processing parameter is that pH 7.0, enzymolysis time are 7h, hydrolysis temperature is 52 ℃, and solid-liquid ratio is 1 g/35ml.
6. squid ink acidic polysaccharose that adopts method preparation claimed in claim 1.
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| CN104004109A (en) * | 2014-06-09 | 2014-08-27 | 浙江海洋学院 | Ocean sulfated glycosaminoglycan SE-3 and preparation method thereof |
| 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 |
| CN106636275A (en) * | 2016-12-12 | 2017-05-10 | 钦州学院 | Method for extracting proteins in sipunculus nudus |
| CN106832033A (en) * | 2017-01-23 | 2017-06-13 | 石狮市华宝海洋生物医药有限公司 | The technique that segmentation complex enzyme hydrolysis extract Cuttlefish Ink small molecule mucoitin sulfate |
| CN108084289A (en) * | 2017-12-29 | 2018-05-29 | 舟山富晟食品科技有限公司 | The extracting method of squid ink polysaccharide |
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| CN110092845A (en) * | 2019-05-07 | 2019-08-06 | 山东大学 | A kind of low molecular weight Sepia polysaccharide and preparation method and application |
| CN115919890A (en) * | 2022-07-08 | 2023-04-07 | 南方医科大学顺德医院(佛山市顺德区第一人民医院) | Application of sepia polysaccharide in preparation of medicine for treating skin photoaging |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004109A (en) * | 2014-06-09 | 2014-08-27 | 浙江海洋学院 | Ocean sulfated glycosaminoglycan SE-3 and preparation method thereof |
| CN104004109B (en) * | 2014-06-09 | 2016-08-31 | 浙江海洋学院 | Ocean Sulfation glycosaminoglycans SE-3 and preparation method thereof |
| 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 |
| CN106636275A (en) * | 2016-12-12 | 2017-05-10 | 钦州学院 | Method for extracting proteins in sipunculus nudus |
| CN106832033A (en) * | 2017-01-23 | 2017-06-13 | 石狮市华宝海洋生物医药有限公司 | The technique that segmentation complex enzyme hydrolysis extract Cuttlefish Ink small molecule mucoitin sulfate |
| CN106832033B (en) * | 2017-01-23 | 2019-03-26 | 石狮市华宝海洋生物医药有限公司 | It is segmented the technique that complex enzyme hydrolysis extracts Cuttlefish Ink small molecule mucoitin sulfate |
| CN108084289A (en) * | 2017-12-29 | 2018-05-29 | 舟山富晟食品科技有限公司 | The extracting method of squid ink polysaccharide |
| CN108250314A (en) * | 2017-12-29 | 2018-07-06 | 舟山富晟食品科技有限公司 | A kind of squid ink active polysaccharide |
| CN110092845A (en) * | 2019-05-07 | 2019-08-06 | 山东大学 | A kind of low molecular weight Sepia polysaccharide and preparation method and application |
| CN115919890A (en) * | 2022-07-08 | 2023-04-07 | 南方医科大学顺德医院(佛山市顺德区第一人民医院) | Application of sepia polysaccharide in preparation of medicine for treating skin photoaging |
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