CN102234671A - Method for extracting polysaccharide from longan pulp - Google Patents
Method for extracting polysaccharide from longan pulp Download PDFInfo
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Abstract
The invention discloses a method for extracting polysaccharide from longan pulp. The method comprises the following steps of: (1) carrying out enzymolysis on the longan pulp by using cellulase, and recording the obtained product as an enzymolysis product; (2) removing solid residues in the enzymolysis product, and recording the residual liquid as a residue-removing product; and (3) removing protein and pigment in the residue-removing product, and recording the obtained product as a protein-removing product, thereby obtaining the polysaccharide. In the method disclosed by the invention, the longan pulp is used as a raw material, and the technologies of carrying out enzymolysis with cellulase, carrying out suction filtration and concentration, removing protein with trichloroacetic acid, removing impurities by dialysis, drying by alcohol precipitation and the like are used for preparing longan polysaccharide. In the extraction method disclosed by the invention, the cellulase is used for carrying out hydrolysis on pulp cell wall tissues to dissolve out polysaccharide substances in cells, thus polysaccharide components in pulp cells can be fully extracted, and the extraction ratio is obviously improved; the hydrolysis temperature is low, and the extraction conditions are mild, thus ensuring the biological activity of the longan polysaccharide; and the water ratio of the used process material is low, thus saving resources, greatly lowering the workload of the subsequent processes such as concentration, alcohol precipitation and the like, and obviously improving the production efficiency.
Description
Technical field
The present invention relates to a kind of method of from longan pulp, extracting polysaccharide.
Background technology
Longan (Dimocarpus longan Lour.) has another name called longan, is a kind of subtropics characteristic fruit, mainly is distributed in south east asia, particularly the southern china province.Be China's south subtropics famous and expensive special local product, the title in north, south " longan " " genseng " in history.Longan fruit is rich in nutrition, liked by people, more is considered as precious tonic, and its nourishing function is apparent.Longan is nutritious, is precious nourishing reinforcer.Fruit also can be made into can, wine, cream, sauce etc. except that eating raw, also can be processed into garden, osmanthus biltong etc.The leaf of longan, flower, root, nuclear all can be used as medicine in addition.Longan is wooden hard, and the careful grace of texture is a raw material of making noble furniture, can be engraved as various exquisite artworks again.Longan Flower is a kind of important nectariferous plant, and honey of lungan flowers is the first-class honey in the honey.
The longan polysaccharide dry product is generally the brown amorphous solid, and the moisture absorption very easily obtains white powder through after the separation and purification, be insoluble to ethanol, organic solvents such as acetone, propyl carbinol, be insoluble in cold water.
Summary of the invention
An object of the present invention is to provide a kind of method of from longan pulp, extracting polysaccharide.
The method of extracting polysaccharide from longan pulp provided by the present invention comprises the steps:
1) uses the cellulase degradation longan pulp, the note of all substances in the container behind the enzymolysis is made enzymolysis product;
2) remove solid residue in the described enzymolysis product, the fluent meterial note that obtains is gone bottoms product;
3) remove described protein and the pigment that goes in the bottoms product, the product note that obtains is made the Deproteinization product, promptly obtain polysaccharide.
In the aforesaid method, after described step 3), comprise the steps 4): remove the small molecular weight impurity in the described Deproteinization product, the product note that obtains is removed the small molecules product.
In the aforesaid method, after described step 4), comprise the steps 5): precipitate polysaccharide from described going the small molecules product with ethanol, obtain the polysaccharide of post precipitation.
In the aforesaid method, in the described step 1), described method with the cellulase degradation longan pulp is: with homogenate, cellulase and the water mixing of described longan pulp, in temperature is under 40 ℃-50 ℃ or 40 ℃ or 45 ℃ or 50 ℃, the condition of pH value for 5.5-8.0 or 5.5 or 6.5 or 8.0, reaction 2.5h-3.5h or 2.5h or 3h or 3.5h;
In the aforesaid method, described step 2) in, described method of removing the solid residue in the described enzymolysis product comprises the steps: described enzymolysis product centrifugal, gets supernatant liquor;
In the aforesaid method, in the described step 3), describedly remove the described protein in the bottoms product and the method for pigment of going and comprise the steps: to described step 2) to add concentration in the product that obtains be that trichloroacetic acid solution to the pH value of 3%-5% or 3% or 4% or 5% (quality percentage composition) is 3.5-5.5 or 3.5 or 4.0 or 5.5, under 4 ℃ condition, leave standstill 3h-6h or 3h or 4h or 6h, centrifugal again, get supernatant liquor, be described Deproteinization product;
In the aforesaid method, in the described step 4), described method of removing the small molecular weight impurity in the described Deproteinization product is dialysis.
In the aforesaid method, in the described step 1), the proportioning of the homogenate of described longan pulp, cellulase and water is: 1g: (600-800) U: (3-7) g is specially 1g: 600U: 3g or 1g: 700U: 5g or 1g: 800U: 7g;
In the aforesaid method, described step 2) in, described get supernatant liquor after, comprise the steps: described supernatant liquid filtering, collect filtrate, more described filtrate is concentrated, obtain concentrated solution.
In the aforesaid method, in the described step 1), described enzymolysis carries out under the condition of vibration, and the speed of vibration is 140r/min-180r/min, is specially 140r/min, 160r/min or 180r/min, and rotation radius is 12mm;
In the aforesaid method, described step 2) in, described centrifugal speed is 4200r/min-4800r/min, be specially 4200r/min, 4600r/min or 4800r/min, rotation radius is 12mm, and the described centrifugal time is 15min-25min, is specially 15min, 20min or 25min; Described filtering method is a vacuum filtration; Described spissated method is that rotary evaporation concentrates;
In the aforesaid method, in the described step 3), described centrifugal speed is 4200r/min-4800r/min, be specially 4200r/min, 4600r/min or 4800r/min, rotation radius is 12mm, and the described centrifugal time is 15min-25min, is specially 15min, 20min or 25min;
In the aforesaid method, in the described step 4), the method for described dialysis comprises the steps: with deionized water dialysis 24h-54h or 24h or 36h or 54h under 2 ℃-8 ℃ or 2 ℃ or 4 ℃ or 8 ℃ of conditions, changes deionized water one time every 1h-3h or 1h or 2h or 3h;
In the aforesaid method, described step 2) in, the spissated temperature of described rotary evaporation is 55 ℃-65 ℃, is specially 55 ℃, 60 ℃ or 65 ℃.
In the aforesaid method, after described step 1), described step 2) preceding, comprise the go out step of enzyme of described enzymolysis product; And/or the method for the described enzyme that goes out is for being to place 5min-20min or 5min or 10min or 20min under 100 ℃ the condition in temperature with described enzymolysis product;
In the described method, after described step 5), comprise the steps 6): with the polysaccharide of the described post precipitation of washing with alcohol, the polysaccharide after the washing is carried out drying;
In the aforesaid method, described step 2) in, described simmer down to is concentrated into described filtrate the 1/3-1/2 of original volume;
In the aforesaid method, in the described step 5), described alcoholic acid volume is 2 times-4 times of described small molecules product volume, is specially 2 times, 3 times or 4 times; The described sedimentary time is 12h-24h, is specially 12h, 18h or 24h; Described temperature of precipitation is 2 ℃-8 ℃ or 2 ℃ or 4 ℃ or 8 ℃;
In the aforesaid method, in the described step 6), described drying is lyophilize, and the described cryodesiccated time is 24h-48h or 24h or 36h or 48h; Described cryodesiccated temperature is-60 ℃; The number of times of described washing is 4 times-6 times, is specially 4 times, 5 times or 6 times;
In the aforesaid method, in the described step 1), described water is distilled water or deionized water; Described longan pulp homogenate is to obtain according to the method that comprises the steps: described longan in-20 ℃ of cold storage, with the peeling of the longan after freezing stoning, is obtained longan pulp, described longan pulp is broken into homogenate, promptly obtain longan pulp homogenate.
The polysaccharide that is obtained by above-mentioned arbitrary described method also belongs to protection scope of the present invention.
The polysaccharide that above-mentioned arbitrary described method obtains also belongs to protection scope of the present invention in the application that preparation has in the product of anti-oxidant function.
In the above-mentioned application, described anti-oxidant function is for removing free radical; Described free radical is at least a in ultra-oxygen anion free radical, hydroxy radical qiao and the DPPH free radical.
In the above-mentioned application, described product can be healthcare products, medicine or makeup etc.
Method of the present invention is a raw material with fresh longan pulp, by cellulase hydrolysis, suction filtration concentrate, trichloroacetic acid method removes technology such as albumen, dialysis removal of impurities and alcohol precipitation drying, the preparation longan polysaccharide.Extracting method of the present invention adopts cellulase hydrolysis flesh cell wall tissue, makes polysaccharide material stripping in the cell, can fully extract polysaccharide component in the flesh cell, has significantly improved yield; Hydrolysis temperature is low, and the extraction conditions gentleness can keep the biological activity of polysaccharide to greatest extent; The technology material-water ratio that uses is low, and water consumption only is 1/3 of a traditional hot water leaching, has not only economized on resources but also reduces the workload of technologies such as follow-up concentrated alcohol precipitation greatly, significantly improves production efficiency.The finished product yield 〉=1.24% of the longan polysaccharide that the inventive method obtains, purity 〉=80%, and have high anti-oxidant function.The longan polysaccharide of method preparation of the present invention can be widely used in industries such as food, medicine, daily use chemicals.
Description of drawings
Fig. 1 is the preparation flow figure of longan polysaccharide.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The fresh longan of using among the following embodiment can be bought from the food market and obtain.
Cellulase is available from Denmark Novozymes Company, and products catalogue is Celluclast1.5L.
The preparation of embodiment 1, longan polysaccharide
One, the preparation of polysaccharide
Concrete steps are as follows:
1. longan pre-treatment
Fresh longan in-20 ℃ of low temperature cold storage, with freezing longan peeling stoning, is obtained longan pulp again; Accurately take by weighing the 1000g longan pulp, the 5min that pulls an oar in hollander obtains longan pulp homogenate.
2. cellulase degradation
The fresh longan pulp homogenate that 1. step is obtained mixes with distilled water that (mass ratio is 1000g: 5000g), the vibration mixing, (proportioning of fresh longan pulp homogenate and cellulase is 1g: 700U) to wherein adding cellulase again, be 6.5 in the pH value, temperature is that 3.0h is extracted in vibration under 45 ℃ the condition, the speed of vibration is 180r/min, rotation radius is 12mm, and the product note that obtains is made enzymolysis product; Enzymolysis product is boiled the 5min enzyme that goes out under 100 ℃.
3. remove solid residue
The product that 2. step is obtained separates the juice slag with the centrifugal 20min of rotating speed (rotation radius is 12mm) of 4600r/min, gets supernatant liquor and is longan fruit juice;
Adopt the vacuum filtration method that the trace of the residue in longan fruit juice pomace is removed, collect filtrate;
Adopt the rotary evaporation method of enrichment, under 60 ℃ of conditions, filtrate is concentrated into 1/3 of filtrate original volume, obtain concentrated solution;
4. the TCA method is removed albumen
Dropwise adding concentration in the concentrated solution that 3. step obtains is the trichloroacetic acid solution of 4% (quality percentage composition), reaches 4.0 until the pH value, and this mixed solution is left standstill 4h in 4 ℃, and centrifugal 20min under the 4600r/min rotating speed collects supernatant liquor.
5. the removal of impurities of dialysing
The supernatant liquor that 4. step is obtained is transferred in the dialysis tubing, is placed in 4 ℃ of deionized waters to dialyse, and changes deionized water one time every 2h, removes residual small molecular weight impurity behind the processing 36h.
6. alcohol precipitation, washing and drying
Add 3 times of volume dehydrated alcohols in the product that 5. step obtains, behind alcohol precipitation 18h under 4 ℃ of conditions, centrifugal 20min under the 4200rmp condition, collecting precipitation promptly obtain the longan polysaccharide precipitation;
Use the 50mL absolute ethanol washing centrifugal this polysaccharide precipitation at every turn, repeat this operation 4 times;
Longan polysaccharide after the washing is deposited in lyophilize 24h under-60 ℃ of conditions, obtains longan polysaccharide.
3 repetitions are established in experiment, and the result takes the mean.
The result shows, it is even white powder that aforesaid method extracts the longan polysaccharide that obtains, the polysaccharide quality average out to 12.4g that 3 repeated experiments obtain, the product yield average out to 1.24% of 3 repeated experiments.
The calculation formula of product yield is: longan pulp homogenate quality * 100% of product yield=longan polysaccharide quality/be used to extract, i.e. product yield=12.4g/1000g * 100%=1.24%.
Two, the analysis of physical and chemical property of polysaccharide
1, measure each substances content in the longan polysaccharide according to following method:
The mensuration of total nitrogen adopts Kjeldahl determination, and (GB/T 5009,5-2003);
The mensuration of fat adopts the AOAC method; (GB/T 5009,6-2003)
The mensuration of ash content; (GB/T 5009,4-2003)
The mensuration of polysaccharide adopts the phenolsulfuric acid method;
Uronic acid is measured the sulfuric acid-carbazole method that adopts.
3 repetitions are established in experiment, and the result takes the mean.
Measurement result shows that the quality percentage composition that aforesaid method extracts crude protein in the longan polysaccharide obtain is 1.05%, and longan polysaccharide content 74.32%, fat mass percentage composition are 0.06%, and ash content quality percentage composition is 2.37%, glucuronic acid content 0.49%; Carry out dry constant weight at 80 ℃ and measure, the result shows that the weight loss on drying of the longan polysaccharide that the aforesaid method extraction obtains is 5%.
2, the detection of longan polysaccharide solubleness
Under the normal temperature (25 ℃), the longan polysaccharide stirring with 1g is dissolved in the 10mL distilled water respectively, whirlpool mixing 20min, and centrifugal 20min separation of supernatant under the 4800r/min condition is weighed 100 ℃ of dryings, measures longan polysaccharide solubleness; The result show aforesaid method extract the longan polysaccharide obtain at normal temperatures the solubleness in the water solvent be 85.42%.Under 20,30,40,50 and 60 ℃, as stated above the stirring of 1g longan polysaccharide is dissolved in the 10mL distilled water respectively, centrifugal drying is weighed, and calculates solubleness.As a result, solubleness at various temperatures is as follows: 20 ℃ 85.21%, 30 ℃ 86.73%, 40 ℃ 87.15%, 50 ℃ 87.76%, 60 ℃ 88.92%.Above-mentioned experiment shows, the longan polysaccharide that the present invention obtains in water solvent, the rising of solubility with temperature and slightly rising, average solubleness is 87.34% (w/w), promptly the 1g longan polysaccharide can dissolve 0.8734g in 10mL water.
The preparation of embodiment 2, longan polysaccharide
One, the preparation of polysaccharide
Concrete steps are as follows:
1. longan pre-treatment
Fresh longan in-20 ℃ of low temperature cold storage, with freezing longan peeling stoning, is obtained longan pulp again; Accurately take by weighing the 1000g longan pulp, the 5min that pulls an oar in hollander obtains longan pulp homogenate.
2. cellulase degradation
The longan pulp homogenate that 1. step is obtained mixes with distilled water that (mass ratio is 1000g: 7000g), the vibration mixing, (proportioning of longan pulp homogenate and cellulase is 1g: 800U) to wherein adding cellulase again, be 8.0 in the pH value, temperature is that 3.5h is extracted in vibration under 50 ℃ the condition, the speed of vibration is 160r/min, rotation radius is 12mm, and the product note that obtains is made enzymolysis product; Enzymolysis product is boiled the 20min enzyme that goes out under 100 ℃.
3. remove solid residue
The product that 2. step is obtained separates the juice slag with the centrifugal 25min of rotating speed (rotation radius is 12mm) of 4800r/min, gets supernatant liquor and is longan fruit juice;
Adopt the vacuum filtration method that the trace of the residue in longan fruit juice pomace is removed, collect filtrate;
Adopt the rotary evaporation method of enrichment, under 65 ℃ of conditions, filtrate is concentrated into 1/2 of filtrate original volume, obtain concentrated solution;
4. the TCA method is removed albumen
Dropwise add 5% (quality percentage composition) trichloroacetic acid solution in the concentrated solution that 3. step obtains, reach 5.5 until the pH value, this mixed solution is left standstill 6h in 4 ℃, centrifugal 25min under the 4800r/min rotating speed collects supernatant liquor.
5. the removal of impurities of dialysing
The supernatant liquor that 4. step is obtained is transferred in the dialysis tubing, is placed in 8 ℃ of deionized waters to dialyse, and changes deionized water one time every 3h, removes residual small molecular weight impurity behind the processing 54h.
6. alcohol precipitation, washing and drying
Add 4 times of volume dehydrated alcohols in the product that 5. step obtains, behind alcohol precipitation 24h under 8 ℃ of conditions, centrifugal 20min under the 4200rmp condition, collecting precipitation promptly obtain the longan polysaccharide precipitation;
Use the 50mL absolute ethanol washing centrifugal this polysaccharide precipitation at every turn, repeat this operation 6 times;
Longan polysaccharide after the washing is deposited in lyophilize 48h under-60 ℃ of conditions, obtains longan polysaccharide.
3 repetitions are established in experiment, and the result takes the mean.
The result shows, it is even white powder that aforesaid method extracts the longan polysaccharide that obtains, the polysaccharide quality average out to 11.5g that 3 repeated experiments obtain, the product yield average out to 1.15% of 3 repeated experiments.
The calculation formula of product yield is: longan pulp homogenate quality * 100% of product yield=longan polysaccharide quality/be used to extract, i.e. product yield=11.5g/1000g * 100%=1.15%.
Two, the analysis of physical and chemical property of polysaccharide
1, measure each substances content in the polysaccharide according to following method:
The mensuration of total nitrogen adopts Kjeldahl determination, and (GB/T 5009,5-2003);
The mensuration of fat adopts the AOAC method; (GB/T 5009,6-2003)
The mensuration of ash content; (GB/T 5009,4-2003)
The mensuration of polysaccharide adopts the phenolsulfuric acid method;
Uronic acid is measured the sulfuric acid-carbazole method that adopts.
3 repetitions are established in experiment, and the result takes the mean.
Measurement result shows that the quality percentage composition that aforesaid method extracts crude protein in the longan polysaccharide obtain is 1.05%, and longan polysaccharide content 79.31%, fat mass percentage composition are 0.06%, and ash content quality percentage composition is 2.37%, glucuronic acid content 0.54%; Carry out dry constant weight at 80 ℃ and measure, the result shows that the weight loss on drying of the longan polysaccharide that the aforesaid method extraction obtains is 4%.
2, the detection of longan polysaccharide solubleness
Under the normal temperature (25 ℃), the longan polysaccharide stirring with 1g is dissolved in the 10mL distilled water respectively, whirlpool mixing 20min, and centrifugal 20min separation of supernatant under the 4800r/min condition is weighed 100 ℃ of dryings, measures longan polysaccharide solubleness; The result show aforesaid method extract the longan polysaccharide obtain at normal temperatures the solubleness in the water solvent be 86.99%.Under 20,30,40,50 and 60 ℃, as stated above the stirring of 1g longan polysaccharide is dissolved in the 10mL distilled water respectively, centrifugal drying is weighed, and calculates solubleness.As a result, solubleness at various temperatures is as follows: 20 ℃ 86.01%, 30 ℃ 86.92%, 40 ℃ 88.75%, 50 ℃ 89.67%, 60 ℃ 90.12%.Above-mentioned experiment shows, the longan polysaccharide that the present invention obtains in water solvent, the rising of solubility with temperature and slightly rising, average solubleness is 89.32% (w/w).
The preparation of embodiment 3, longan polysaccharide
One, the preparation of polysaccharide
Concrete steps are as follows:
1. longan pre-treatment
Fresh longan in-20 ℃ of low temperature cold storage, with freezing longan peeling stoning, is obtained longan pulp again; Accurately take by weighing the 1000g longan pulp, the 5min that pulls an oar in hollander obtains longan pulp homogenate.
2. cellulase degradation
The longan pulp homogenate that 1. step is obtained mixes with distilled water that (mass ratio is 1000g: 3000g), the vibration mixing, (proportioning of longan pulp homogenate and cellulase is 1g: 600U) to wherein adding cellulase again, be 5.5 in the pH value, temperature is that 2.5h is extracted in vibration under 40 ℃ the condition, the speed of vibration is 140r/min, rotation radius is 12mm, and the product note that obtains is made enzymolysis product; Enzymolysis product is boiled the 10min enzyme that goes out under 100 ℃.
3. remove solid residue
The product that 2. step is obtained separates the juice slag with the centrifugal 15min of rotating speed (rotation radius is 12mm) of 4200r/min, gets supernatant liquor and is longan fruit juice;
Adopt the vacuum filtration method that the trace of the residue in longan fruit juice pomace is removed, collect filtrate;
Adopt the rotary evaporation method of enrichment, under 55 ℃ of conditions, filtrate is concentrated into 1/3 of filtrate original volume, obtain concentrated solution;
4. the TCA method is removed albumen
Dropwise add 3% (quality percentage composition) trichloroacetic acid solution in the concentrated solution that 3. step obtains, reach 3.5 until the pH value, this mixed solution is left standstill 3h in 4 ℃, centrifugal 15min under the 4200r/min rotating speed collects supernatant liquor.
5. the removal of impurities of dialysing
The supernatant liquor that 4. step is obtained is transferred in the dialysis tubing, is placed in 2 ℃ of deionized waters to dialyse, and changes deionized water one time every 1h, removes residual small molecular weight impurity behind the processing 24h.
6. alcohol precipitation, washing and drying
Add 2 times of volume dehydrated alcohols in the product that 5. step obtains, behind alcohol precipitation 12h under 2 ℃ of conditions, centrifugal 20min under the 4200rmp condition, collecting precipitation promptly obtain the longan polysaccharide precipitation;
Use the 50mL absolute ethanol washing centrifugal this polysaccharide precipitation at every turn, repeat this operation 5 times;
Longan polysaccharide after the washing is deposited in lyophilize 36h under-60 ℃ of conditions, obtains longan polysaccharide.
3 repetitions are established in experiment, and the result takes the mean.
The result shows, it is even white powder that aforesaid method extracts the longan polysaccharide that obtains, the polysaccharide quality average out to 11.0g that 3 repeated experiments obtain, the product yield average out to 1.10% of 3 repeated experiments.
The calculation formula of product yield is: longan pulp homogenate quality * 100% of product yield=longan polysaccharide quality/be used to extract, i.e. product yield=11.0g/1000g * 100%=1.10%.
Two, the analysis of physical and chemical property of polysaccharide
1, measure each substances content in the polysaccharide according to following method:
The mensuration of total nitrogen adopts Kjeldahl determination, and (GB/T 5009,5-2003);
The mensuration of fat adopts the AOAC method; (GB/T 5009,6-2003)
The mensuration of ash content; (GB/T 5009,4-2003)
The mensuration of polysaccharide adopts the phenolsulfuric acid method;
Uronic acid is measured the sulfuric acid-carbazole method that adopts.
3 repetitions are established in experiment, and the result takes the mean.
Measurement result shows that the quality percentage composition that aforesaid method extracts crude protein in the longan polysaccharide obtain is 1.05%, and longan polysaccharide content 73.23%, fat mass percentage composition are 0.06%, and ash content quality percentage composition is 2.37%, glucuronic acid content 0.39%; Carry out dry constant weight at 80 ℃ and measure, the result shows that the weight loss on drying of the longan polysaccharide that the aforesaid method extraction obtains is 5%.
2, the detection of longan polysaccharide solubleness
Under the normal temperature (25 ℃), the longan polysaccharide stirring with 1g is dissolved in the 10mL distilled water respectively, whirlpool mixing 20min, and centrifugal 20min separation of supernatant under the 4800r/min condition is weighed 100 ℃ of dryings, measures longan polysaccharide solubleness; The result show aforesaid method extract the longan polysaccharide obtain at normal temperatures the solubleness in the water solvent be 84.23%.Under 20,30,40,50 and 60 ℃, as stated above the stirring of 1g longan polysaccharide is dissolved in the 10mL distilled water respectively, centrifugal drying is weighed, and calculates solubleness.As a result, solubleness at various temperatures is as follows: 20 ℃ 84.24%, 30 ℃ 84.92%, 40 ℃ 85.25%, 50 ℃ 86.17%, 60 ℃ 86.89%.Above-mentioned experiment shows, the longan polysaccharide that the present invention obtains in water solvent, the rising of solubility with temperature and slightly rising, average solubleness is 85.36% (w/w).
The antioxidation in vitro functional experiment of embodiment 4, longan pulp polysaccharide
The longan polysaccharide that obtains with embodiment 1,2 and 3 carries out following each experiment respectively.
Sample solution: polysaccharide is soluble in water, obtain the sample solution of different concns; Polysaccharide concentration is shown in table 1-3 in the sample solution.
1. the removing of ultra-oxygen anion free radical
Adding ionic strength in a series of test tube is 0.05M, pH is 8.2 Tris-Hcl damping fluid 9ml, add sample solution 100 μ l more respectively, blank group adds the distilled water with volume, add 0.045M pyrogallol (with the 0.01MHCL preparation) 100 μ l again, concussion timing three minutes adds 10% xitix, 100 μ l termination reactions, surveys light absorption value immediately at the 325nm place.
SA-removes the ability of free radical, % in the formula; A
0-blank sample measured value; A
S-be the sample determination value.
2. the removing of hydroxy radical qiao
In test tube, add 9.1mM Whitfield's ointment-ethanolic soln 0.5ml successively, sample 0.5ml, 9.1mM Fe
2+Solution 0.5ml, distilled water 3.5ml adds 88mM H at last
2O
25ml after colour developing shakes up, measures under 510nm and removes activity.
SA-removes the ability of free radical, % in the formula; A
0-be blank sample measured value; A
S-be the sample determination value.
3. DPPH free radical
Add successively, the DPPH-95% aqueous ethanolic solution 1.5ml of 0.1mM, sample 1.5ml at room temperature places 30min after the concussion, and 517nm measures absorbancy.
SA-removes the ability of free radical, % in the formula; A
1-DPPH+ sample; A
2-95% aqueous ethanolic solution+sample; A
3-DPPH+ distilled water.
The functionally active detected result of gained longan polysaccharide is shown in table 1,2,3 among the embodiment 1.
Table 1, removing DPPH free radical:
| Sample concentration (mg/mL) | 5 | 10 | 15 | 20 | 25 |
| Clearance rate (%) | 27.12 | 47.43 | 62.39 | 73.83 | 81.40 |
IC
50：10.10mg/mL。
Table 2, removing hydroxy radical qiao:
| Sample concentration (mg/mL) | 5 | 10 | 15 | 20 | 25 |
| Clearance rate (%) | 29.32 | 41.42 | 54.88 | 65.21 | 77.13 |
IC
50：11.51mg/mL。
Table 3, removing ultra-oxygen anion free radical:
| Sample concentration (mg/mL) | 10 | 20 | 30 | 40 | 50 |
| Clearance rate (%) | 23.01 | 46.11 | 57.76 | 73.32 | 82.13 |
IC
50：21.82mg/mL。
The active no significant difference of anti-oxidant function of gained longan polysaccharide among the anti-oxidant function activity of the longan polysaccharide that obtains among embodiment 2 and the embodiment 3 and the embodiment 1.
Claims (10)
1. a method of extracting polysaccharide from longan pulp comprises the steps:
1) uses the cellulase degradation longan pulp, the note of all substances in the container behind the enzymolysis is made enzymolysis product;
2) remove solid residue in the described enzymolysis product, the fluent meterial note that obtains is gone bottoms product;
3) remove described protein and the pigment that goes in the bottoms product, the product note that obtains is made the Deproteinization product, promptly obtain polysaccharide.
2. method according to claim 1 is characterized in that: in the described method, after described step 3), comprise the steps 4): remove the small molecular weight impurity in the described Deproteinization product, the product note that obtains is removed the small molecules product.
3. method according to claim 2 is characterized in that: in the described method, after described step 4), comprise the steps 5): precipitate polysaccharide from described going the small molecules product with ethanol, obtain the polysaccharide of post precipitation.
4. according to arbitrary described method among the claim 1-3, it is characterized in that: in the described step 1), described method with the cellulase degradation longan pulp is: with homogenate, cellulase and the water mixing of described longan pulp, in temperature is under 40 ℃-50 ℃ or 40 ℃ or 45 ℃ or 50 ℃, the condition of pH value for 5.5-8.0 or 5.5 or 6.5 or 8.0, reaction 2.5h-3.5h or 2.5h or 3h or 3.5h;
Described step 2) in, described method of removing the solid residue in the described enzymolysis product comprises the steps: described enzymolysis product centrifugal, gets supernatant liquor;
In the described step 3), describedly remove the described protein in the bottoms product and the method for pigment of going and comprise the steps: to described step 2) to add concentration in the product that obtains be that trichloroacetic acid solution to the pH value of 3%-5% or 3% or 4% or 5% (quality percentage composition) is 3.5-5.5 or 3.5 or 4.0 or 5.5, under 4 ℃ condition, leave standstill 3h-6h or 3h or 4h or 6h, centrifugal again, get supernatant liquor, be described Deproteinization product;
In the described step 4), described method of removing the small molecular weight impurity in the described Deproteinization product is dialysis.
5. according to arbitrary described method among the claim 1-4, it is characterized in that: in the described step 1), the proportioning of the homogenate of described longan pulp, cellulase and water is: 1g: (600-800) U: (3-7) g is specially 1g: 600U: 3g or 1g: 700U: 5g or 1g: 800U: 7g;
Described step 2) in, described get supernatant liquor after, comprise the steps: described supernatant liquid filtering, collect filtrate, more described filtrate is concentrated, obtain concentrated solution.
6. according to arbitrary described method among the claim 1-5, it is characterized in that: in the described step 1), described enzymolysis carries out under the condition of vibration, and the speed of vibration is 140r/min-180r/min, be specially 140r/min, 160r/min or 180r/min, rotation radius is 12mm;
Described step 2) in, described centrifugal speed is 4200r/min-4800r/min, is specially 4200r/min, 4600r/min or 4800r/min, and rotation radius is 12mm, and the described centrifugal time is 15min-25min, is specially 15min, 20min or 25min; Described filtering method is a vacuum filtration; Described spissated method is that rotary evaporation concentrates;
In the described step 3), described centrifugal speed is 4200r/min-4800r/min, is specially 4200r/min, 4600r/min or 4800r/min, and rotation radius is 12mm, and the described centrifugal time is 15min-25min, is specially 15min, 20min or 25min;
In the described step 4), the method for described dialysis comprises the steps: with deionized water dialysis 24h-54h or 24h or 36h or 54h under 2 ℃-8 ℃ or 2 ℃ or 4 ℃ or 8 ℃ of conditions, changes deionized water one time every 1h-3h or 1h or 2h or 3h.
7. according to arbitrary described method among the claim 1-6, it is characterized in that: described step 2), the spissated temperature of described rotary evaporation is 55 ℃-65 ℃, is specially 55 ℃, 60 ℃ or 65 ℃.
8. according to arbitrary described method among the claim 1-7, it is characterized in that: in the described method, after described step 1), described step 2) preceding, comprise the go out step of enzyme of described enzymolysis product; And/or the method for the described enzyme that goes out is for being to place 5min-20min or 5min or 10min or 20min under 100 ℃ the condition in temperature with described enzymolysis product;
In the described method, after described step 5), comprise the steps 6): with the polysaccharide of the described post precipitation of washing with alcohol, the polysaccharide after the washing is carried out drying;
Described step 2) in, described simmer down to is concentrated into described filtrate the 1/3-1/2 of original volume;
In the described step 5), described alcoholic acid volume is described 2 times-4 times of removing small molecules product volume, is specially 2 times, 3 times or 4 times; The described sedimentary time is 12h-24h, is specially 12h, 18h or 24h; Described temperature of precipitation is 2 ℃-8 ℃ or 2 ℃ or 4 ℃ or 8 ℃;
In the described step 6), described drying is lyophilize, and the described cryodesiccated time is 24h-48h or 24h or 36h or 48h; Described cryodesiccated temperature is-60 ℃; The number of times of described washing is 4 times-6 times, is specially 4 times, 5 times or 6 times;
In the described step 1), described water is distilled water or deionized water; Described longan pulp homogenate is to obtain according to the method that comprises the steps: with described longan in-20 ℃ freezing, with the longan after freezing peeling stoning, obtain longan pulp, described longan pulp is broken into homogenate, promptly obtain longan pulp homogenate.
9. the polysaccharide that obtains by arbitrary described method among the claim 1-8.
10. polysaccharide described in the claim 9 has application in the anti-oxidant function product in preparation;
And/or described anti-oxidant function is for removing free radical;
And/or described free radical is at least a in ultra-oxygen anion free radical, hydroxy radical qiao and the DPPH free radical.
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| CN103837671A (en) * | 2012-11-26 | 2014-06-04 | 天士力制药集团股份有限公司 | Method for measuring free polysaccharide content in polysaccharide protein conjugate |
| CN104431851A (en) * | 2014-12-10 | 2015-03-25 | 华南农业大学 | Longan product rich in fructo-oligose and preparation method thereof |
| CN106265397A (en) * | 2015-05-19 | 2017-01-04 | 成都宝科生物科技有限公司 | A kind of anti-ageing face mask |
| CN106755181A (en) * | 2016-12-13 | 2017-05-31 | 广西壮族自治区农业科学院农产品加工研究所 | A kind of method for extracting longan seed polysaccharide |
| CN106749726A (en) * | 2016-12-13 | 2017-05-31 | 广西壮族自治区农业科学院农产品加工研究所 | A kind of method for extracting Arillus longan polysaccharide |
| CN106946997A (en) * | 2016-08-19 | 2017-07-14 | 武汉轻工大学 | The enhanced longan pulp polysaccharide of functional activity and its green modification method for preparing combined based on endogenous protein |
| CN108948220A (en) * | 2018-06-28 | 2018-12-07 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of low viscosity, the preparation method of the prebiotic active polysaccharide of highly dissoluble longan |
| CN109485706A (en) * | 2018-11-02 | 2019-03-19 | 浙江省疾病预防控制中心 | A kind of cordate houttuynia glycoprotein and its preparation and application |
| CN109880864A (en) * | 2019-03-06 | 2019-06-14 | 福州大学 | The enzyme process method for integrated extraction of function polysaccharide and procyanidine in a kind of longan peel |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103837671A (en) * | 2012-11-26 | 2014-06-04 | 天士力制药集团股份有限公司 | Method for measuring free polysaccharide content in polysaccharide protein conjugate |
| CN104431851A (en) * | 2014-12-10 | 2015-03-25 | 华南农业大学 | Longan product rich in fructo-oligose and preparation method thereof |
| CN106265397A (en) * | 2015-05-19 | 2017-01-04 | 成都宝科生物科技有限公司 | A kind of anti-ageing face mask |
| CN106946997A (en) * | 2016-08-19 | 2017-07-14 | 武汉轻工大学 | The enhanced longan pulp polysaccharide of functional activity and its green modification method for preparing combined based on endogenous protein |
| CN106755181A (en) * | 2016-12-13 | 2017-05-31 | 广西壮族自治区农业科学院农产品加工研究所 | A kind of method for extracting longan seed polysaccharide |
| CN106749726A (en) * | 2016-12-13 | 2017-05-31 | 广西壮族自治区农业科学院农产品加工研究所 | A kind of method for extracting Arillus longan polysaccharide |
| CN106749726B (en) * | 2016-12-13 | 2018-12-25 | 广西壮族自治区农业科学院农产品加工研究所 | A method of extracting Arillus longan polysaccharide |
| CN108948220A (en) * | 2018-06-28 | 2018-12-07 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of low viscosity, the preparation method of the prebiotic active polysaccharide of highly dissoluble longan |
| CN109485706A (en) * | 2018-11-02 | 2019-03-19 | 浙江省疾病预防控制中心 | A kind of cordate houttuynia glycoprotein and its preparation and application |
| CN109880864A (en) * | 2019-03-06 | 2019-06-14 | 福州大学 | The enzyme process method for integrated extraction of function polysaccharide and procyanidine in a kind of longan peel |
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