CN110066349A - A kind of low molecular weight blackberry, blueberry polysaccharide and preparation method thereof - Google Patents

A kind of low molecular weight blackberry, blueberry polysaccharide and preparation method thereof Download PDF

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CN110066349A
CN110066349A CN201910268825.4A CN201910268825A CN110066349A CN 110066349 A CN110066349 A CN 110066349A CN 201910268825 A CN201910268825 A CN 201910268825A CN 110066349 A CN110066349 A CN 110066349A
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blackberry
polysaccharide
molecular weight
blueberry
low molecular
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CN110066349B (en
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扶雄
窦祖满
陈春
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Zhuhai Sino-American Play Health Technology Co Ltd
Zhuhai Institute of Modern Industrial Innovation of South China University of Technology
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Zhuhai Institute of Modern Industrial Innovation of South China University of Technology
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof

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Abstract

The invention discloses a kind of low molecular weight blackberry, blueberry polysaccharide and preparation method thereof.The weight average molecular weight of low molecular weight blackberry, blueberry polysaccharide is ten thousand Da of 18-59；Grain diameter in the solution is 400~1000nm, and dispersion index is 0.18~0.29；It is made of arabinose, galactolipin, glucose, galacturonic acid, glucuronic acid, and its weight percent accounting is respectively as follows: 9.8~11.2%, 3.1~3.6%, 2.7~3.3%, 81.9~83.5% and 0.40~0.42%；To ABTS free radical scavenging activity IC50Value is 1.47~1.62mg/mL, to alpha-glucosaccharase enzyme inhibition activity IC50Value is 1.11~1.59mg/mL.The present invention obtains low molecular weight blackberry, blueberry polysaccharide using water extraction and alcohol precipitation method combining ultrasonic edman degradation Edman, which has both excellent anti-oxidant and hypoglycemic activity.

Description

A kind of low molecular weight blackberry, blueberry polysaccharide and preparation method thereof
Technical field
The present invention relates to a kind of polysaccharide, and in particular to a kind of low molecular weight blackberry, blueberry polysaccharide and preparation method thereof；Belong to blackberry, blueberry Extract technical field.
Background technique
Diabetes can influence a variety of organ lesions as a kind of chronic metabolic diseases, such as: kidney, eyes and nerve. According to World Health Organization, lethality caused by diabetes is up to the 7th, the world in the year two thousand thirty.Participate in the enzyme of glycometabolism It is often used as the therapy target of new antidiabetic medicine exploitation.Most effective one method is by inhibiting alpha-glucosaccharase Enzymatic activity slows down the formation of glucose, to reduce the absorption to glucose, achievees the purpose that control postprandial blood sugar.
Currently, the hypoglycemic drugs such as the acarbose clinically used, Miglitol are by inhibiting α-in the short time Glucosidase activity controls blood glucose, but takes for a long time and can bring certain side effect, such as: hypoglycemia, flatulence, diarrhea Deng.Meanwhile numerous studies prove that vivo oxidation stress be the major reason that diabetes are formed.Therefore, anti-oxidant answer is had both Swash and the natural active products of alpha-glucosidase is inhibited to verify the adjuvant treatment for controlling postprandial blood sugar and diabetes with great Facilitation.
In recent years, people have the structure for finding polysaccharide compared with in-depth study to its biology to the functional activity of plant polyose Activity has important influence, as being unfavorable for its bioactivity due to structure and physicochemical property obstacle there are polysaccharide in nature It plays or some polysaccharide molecular weights isolated and purified from natural plants is big, active weaker.
Summary of the invention
The problem to be solved in the present invention is to overcome the defect of prior art preparation blackberry, blueberry polysaccharide, provide it is a kind of do not destroy it is black The basic structure of certain kind of berries polysaccharide, low molecular weight, while it is more to have both excellent anti-oxidant and alpha-glucosaccharase enzyme inhibition activity blackberry, blueberry Sugar and preparation method thereof.
Blackberry, blueberry, also referred to as raspberry, the dew certain kind of berries, are the fruits of the perennial liana of rose family rubus.Blackberry, blueberry is as a kind of Integration of drinking and medicinal herbs fruit, it is increasing to the research of its bioactivity.Inventor find blackberry, blueberry polysaccharide have it is significant anti-oxidant and Hypoglycemic activity, and the molecular weight of blackberry, blueberry polysaccharide is than more uniform, the cellular construction of preparation method blackberry, blueberry polysaccharide through the invention Do not destroy.Therefore, it prepares a kind of with anti-oxidant and hypoglycemic activity blackberry, blueberry polysaccharide, is more advantageous to the whole benefit of blackberry, blueberry fruit With the exploitation of value.
The purpose of the present invention is achieved through the following technical solutions:
A kind of low molecular weight blackberry, blueberry polysaccharide, weight average molecular weight are ten thousand Da of 18-59；
The grain diameter of the low molecular weight blackberry, blueberry polysaccharide in the solution be 400~1000nm, dispersion index be 0.18~ 0.29；
The low molecular weight blackberry, blueberry polysaccharide is by arabinose, galactolipin, glucose, galacturonic acid, glucuronic acid group At, and its weight percent accounting be respectively as follows: 9.8~11.2%, 3.1~3.6%, 2.7~3.3%, 81.9~83.5% and 0.40~0.42%；
The low molecular weight blackberry, blueberry polysaccharide has both antioxidant activity and hypoglycemic activity, wherein living to ABTS radicals scavenging Property IC50Value is 1.47~1.62mg/mL, to alpha-glucosaccharase enzyme inhibition activity IC50Value is 1.11~1.59mg/mL.
A kind of preparation method of the low molecular weight blackberry, blueberry polysaccharide, comprising the following steps:
(1) pre-treatment: blackberry, blueberry fresh fruit is cleaned, and drying pulverizes after cooling, crosses mesh, obtains blackberry powder, spare；
(2) degreasing: being added ethyl alcohol into step (1) treated blackberry powder, and 2~4h, centrifugation are heated at 70~90 DEG C After filter, collect filter residue by its 45~60 DEG C dry, it is spare；
(3) hot water extracts: be added into the blackberry, blueberry filter residue after step (2) degreasing 30~40 times of blackberry, blueberry filter residue quality go from Sub- water heats and stirs 2~4h in 85~95 DEG C of water-baths, filters after centrifugation, collects filtrate；
(4) it takes off albumen: the supernatant in step (3) is concentrated, add Sevag reagent, be centrifuged after oscillation, collect supernatant Liquid, then albumen is taken off through Sevag method, repeat this step 9~12 time；
(5) it decolourizes: the de- protein extract rotary evaporation in step (4) being removed into extra Sevag reagent, and thereto The AB-8 macroreticular resin of extracting solution quality, vibrates filtering and collecting filter liquid after 16~18 times of de- albumen of addition；
(6) ethanol precipitation: dehydrated alcohol is added into the extracting solution after decoloration in step (5), keeps its mass concentration final Be 80%~90%, at 0~4 DEG C stand 12~for 24 hours；Precipitating is collected after centrifugation；
(7) be ultrasonically treated: by step (6) sediment dissolution in deionized water, ultrasonic power be 250~ 300w, ultrasonic treatment 8 at 70~90 DEG C of ultrasonic temperature~for 24 hours；
(8) it dialyses: by the 24~48h that dialyses under the polysaccharide solution bag filter after ultrasonic treatment in step (7)；
(9) polysaccharide solution after dialysis in step (8) is freeze-dried at -55 DEG C~-80 DEG C, it is more obtains blackberry, blueberry Sugar.
To further realize the object of the invention, it is preferred that drying temperature described in step (1) is 50~55 DEG C；It is dry 12~for 24 hours；Crossing grit number is 60~100 mesh.
Preferably, amount of alcohol added described in step (2) is 3~5 times of blackberry powder quality；Drying temperature is 45~60 ℃。
Preferably, the quality additional amount of deionized water described in step (3) is 35~40 times of blackberry powder filter residue amount；It stirs Mix 2.5~3.5h；Centrifugation described in step (3) is that 4500g is centrifuged 10min.
Preferably, oscillation is 20~30min of oscillation at 200~300r/min in step (4).
Preferably, the speed vibrated in step (5) is 200~300r/min, and the time of oscillation is 8~12h.
Preferably, the mass concentration of ethyl alcohol is 80%~85% in step (6)；Centrifugation described in step (6) is 4000 ~4500g is centrifuged 5~8min.
It preferably, is 10~15h in sonication treatment time described in step (7)；The ultrasonic treatment temperature is 75~85 ℃。
Preferably, the size of bag filter described in step (8) is 3000~3500Da.
Compared with the existing technology, the present invention has the advantages that
(1) blackberry, blueberry polysaccharide weight average molecular weight prepared by the present invention is between 180,000 Da to 590,000 Da；The low molecular weight is black The grain diameter of certain kind of berries polysaccharide in the solution is between 400~1000nm, and dispersion index is between 0.18~0.29；The low molecule Amount blackberry, blueberry polysaccharide is made of arabinose, galactolipin, glucose, galacturonic acid, glucuronic acid, and its weight percent score Not are as follows: between 9.8~11.2%, 3.1~3.6%, 2.7~3.3%, 81.9~83.5% and 0.40~0.42%；Simultaneously Have both antioxidant activity and hypoglycemic activity；
(2) preparation method agents useful for same of the present invention is environmentally protective, equipment is simple, low in cost, and operation is easy, to middle-size and small-size Enterprise's industrialized production is extremely beneficial.
(3) compared to enzyme process and chemical method, the method for the present invention expends low and environmentally protective efficient preparation method of the present invention；Phase And operation more simpler than the appliance arrangement needed for other physical methods such as microwave method and radiation method, the present invention is easy, and is degraded simultaneously The cellular construction for not destroying polysaccharide in the process, retains the bioactivity of polysaccharide.
(4) the blackberry, blueberry active polysaccharide of low molecular weight prepared by the present invention, ABTS free radical scavenging activity IC501.47~ Between 1.62mg/mL, to alpha-glucosaccharase enzyme inhibition activity IC50Between 1.11~1.59mg/mL.
Detailed description of the invention
Fig. 1 is the blackberry, blueberry polysaccharide under the preparation condition of the embodiment of the present invention 1,2,3 in different NaOH concentration solution Helix-coil transition analysis.
Fig. 2 is that blackberry, blueberry polysaccharide presses down ABTS free radical scavenging activity half under the preparation condition of the embodiment of the present invention 1,2,3 Concentration IC processed50Value.
Fig. 3 is blackberry, blueberry polysaccharide under the preparation condition of the embodiment of the present invention 1,2,3 to alpha-glucosaccharase enzyme inhibition activity half Inhibition concentration IC50Value.
Specific embodiment
In order to better understand the present invention, the present invention, but protection model of the invention are further illustrated below with reference to embodiment Enclose the range for being not limited merely to implementation column statement.
Detection method
(1) measurement of the polysaccharide content
Using sugared content in phend-sulphuric acid measurement sample: 105 DEG C of the accurate weighing glucose 10mg that drying to constant weight, Be dissolved in deionized water, with volumetric flask constant volume to 100mL, be made into the glucose standard of 100ug/mL, pipette 0 respectively, 0.2, 0.4,0.6,0.8,1mL glucose standard and with deionized water moisturizing to 1mL, is then added 5% phenol 0.5mL and the concentrated sulfuric acid 2.5mL after mixing well, is protected from light and stands 20min, measure its light absorption value A at 490nm with microplate reader490.With concentration of glucose For abscissa, with A490For ordinate, standard curve is drawn.Appropriate polysaccharide sample accurately is weighed, it is molten to be made into 100ug/mL polysaccharide Liquid, it is accurate to draw 1mL polysaccharide solution, light absorption value is measured by aforesaid operations step, calculates the sugar in polysaccharide sample with standard curve Content.
(2) content of reducing sugar measures
Using content of reducing sugar in DNS method measurement polysaccharide sample: 105 DEG C of glucose that drying to constant weight of accurate weighing 10mg, is made into the glucose standard of 1mg/mL, pipette 0 respectively, 0.1,0.2,0.3,0.4,0.5mL glucose standard is used in combination Then deionized water moisturizing is added 1mL DNS reagent (mention the last week configure), after mixing well, boils 5min to 0.5mL, After being cooled to room temperature plus deionized water is to 5ml, measures its light absorption value A at 540nm with microplate reader540.It is cross with concentration of glucose Coordinate, with A540For ordinate, standard curve is drawn.Appropriate polysaccharide sample accurately is weighed, is made into 1mg/mL polysaccharide solution, accurately 0.5mL polysaccharide solution is drawn, measures light absorption value by aforesaid operations step, calculates the sugared content in polysaccharide sample with standard curve.
(3) protein content determination
Using protein content in Coomassie Brilliant Blue measurement polysaccharide sample.Accurate weighing bovine serum albumin 5mg, is made into 50ug/mL standard protein solution pipettes 0,0.1,0.2,0.3,0.4,0.5mL protein standard liquid and with deionized water moisturizing respectively To 0.5mL, 2.5mL Coomassie Brillant Blue solution is then added, after mixing well, is protected from light 10min, measures it at 595nm Light absorption value A595.Using protein concentration as abscissa, with A595For ordinate, standard curve is drawn.Accurately weigh appropriate polysaccharide sample Product are made into 2mg/mL polysaccharide solution, accurate to draw 0.5mL polysaccharide solution, measure light absorption value by aforesaid operations step, with standard song Protein content in line computation polysaccharide sample.
(4) molecular weight distribution
Using the molecular weight distribution and homogeneity of high performance liquid chromatograph measurement sample.2mg polysaccharide sample is weighed to be dissolved in In the potassium phosphate buffer (0.02M) of 2mL, 0.22 μm of moisture film is crossed, is analyzed.Standard curve uses Propiram standard Product (molecular weight is respectively 642000,337000,194000,21100,9600,6100Da).Liquid phase operating condition: chromatographic column is TSK-gel G5000PWXL (7.8 × 300mm I.D., 10 μm) and TSK-gel G3000PWXL (7.8 × 300mm I.D., 7 μ M) gel column is used in series；Detector: 1260 Composition distribution of Agilent；Mobile phase: 0.02M potassium phosphate buffer (pH6.0)；Column temperature: 35 ± 0.1 DEG C；Detector temperature: 45 ± 0.1 DEG C；Flow velocity: 0.6mL/min；Sample volume: 20 μ L.
(5) monosaccharide composition and glucuronic acid content measurement
Using the monosaccharide and uronic acid composition in ion chromatography (IC) detection polysaccharide sample.Hydrolyzation sample: 5mL 2mol/ is used Trifluoroacetic acid (TFA) reagent of L sufficiently dissolves 10mg sample, is then transferred into 10mL ampoul tube, will be pacified with alcohol blast burner After the bottle sealing of small jar bottle, in reacting 6h in 105-110 DEG C of drying box.Ampoule nozzle is broken into pieces after the completion of hydrolysis, and sample is fallen Enter in revolving bottle, excessive TFA is evaporated off in 50 DEG C of backspins, and 4mL Chromatographic Pure Methanol is added into revolving bottle, after dissolution, depressurizes dense It is reduced to and is completely dried, repetitive operation five times.Ultrapure water dissolved residue is finally used, and is settled to 100mL.Take 1mL sample solution saturating 0.22 μm of moisture film is crossed, is injected in liquid phase bottle, IC detection is carried out, the group of monosaccharide and uronic acid in sample is calculated according to standard curve At.Standard curve draws (fucose, arabinose, galactolipin, glucose, xylose, fruit using monosaccharide and uronic acid standard items Sugar, mannose, galacturonic acid, glucuronic acid).Ion chromatography operating condition are as follows: chromatographic column: CarboPacTMPA20 analysis Column, CarboPacTMPA1 guard column；Detector: pulsed amperometry (PAD)；Column temperature: 30 DEG C；Input sample: 10 μ L；Flowing Phase: A liquid is ultrapure water, and B liquid is 200mM NaOH solution, and C liquid is 500mM CH3COONa solution, D liquid are that 20mM NaOH is molten Liquid；Flow velocity: 0.5mL/min；Elution program: B liquid (0-2min), 90% A liquid and 10% D liquid (3-20min), B liquid (20- 30min)。
(6) partial size and monodispersity index measurement
The average grain diameter of particle and monodispersity index are measured using dynamic optical diffraction (DLS) technology in polysaccharide solution.It will be more Sugar-like product are made into the solution of 0.5mg/mL, and Malvern Nano ZS instrument is then used to analyze: He-Ne (633nm) is as laser Device, angle of scattering are 173 °, test temperature is 25 DEG C.
(7) infrared spectrum analysis
The polysaccharide sample of 3mg is weighed by KBr tabletting, using Fourier infrared spectrograph in 400-4000cm-1Wave number model It is scanned to obtain scanning spectra in enclosing.
(8) triple helix structure determination
It is identified using triple helix structure of the Congo red reagent method to blackberry, blueberry polysaccharide.Polysaccharide is configured to distilled water The solution of 1mg/ml, while Congo red reagent (100 μM) are prepared, polysaccharide solution and Congo red reagent are mixed well in equal volume, are used NaOH solution (4M) adjusts the NaOH concentration of mixed liquor in 0-0.5M range.In 200-600nm wave-length coverage, carries out spectrum and sweep It retouches, measures different NaOH concentration sample maximum absorption wavelengths, and make with the curdlan (curdlan) with triple helix structure For control.
(9) ABTS free radical scavenging activity measures
ABTS solution allocation: 7mM ABTS solution and 2.45mM K2S2O8By the proportional arrangement of 1:1, after being protected from light 12h It generates.Use ABTS+Working solution configuration: it is 0.70 ± 0.02 that ABTS solution, which is diluted with water the absorbance under 732nm wavelength,.96 30 μ L polysaccharide sample solution and 225 μ LABTS are added in hole elisa Plates+Working solution, oscillation mix, are protected from light 30min, use enzyme Mark instrument measures absorbance A under 732nm wavelength732.Deionized water makees blank control, and vitamin C (Vc) makees positive control.ABTS Free radical scavenging activity calculation formula is as follows:
ABTS free radical scavenging activity (%)=[1- (A0-A1)/A2] × 100
Wherein, A0 indicates 30 μ L sample solution and 225 μ L ABTS+Light absorption value after working solution hybrid reaction；A1 indicates 30 The light absorption value that μ L sample solution is mixed with 225 μ L deionized waters；A2 indicates 30 μ L deionized waters and 225 μ L ABTS+Working solution is mixed The light absorption value of conjunction.
(10) alpha-glucosaccharase enzyme inhibition activity
Add polysaccharide solution, the 50 μ L alpha-glucosidase (0.25U/ of 50 μ L various concentrations respectively in 96 hole elisa Plates ML), oscillation mixes, and 37 DEG C of incubation 6min continuously add 100 μ L 4- nitrophenols-a-D glucofuranose glycosides (pNPG, 5mM), incubate Educate 10min.Finally, the Na of the 0.2M of 1mL is added2CO3Solution terminates reaction.With suction of the microplate reader detection reaction solution at 405nm Light value A405.Deionized water is as blank control, and acarbose is as positive control.Calculation formula is as follows:
Inhibiting rate (%)=[1- (Asample-Ablank)/Acontrol] × 100
In formula: Acontrol is no polysaccharide sample, only adds the absorbance of alpha-glucosidase；Asample expression adds Add the absorbance of sugar juice and alpha-glucosidase；Ablank indicates polysaccharide solution, not the extinction of alpha-glucosidase Degree.
Embodiment 1
A kind of preparation method of low molecular weight blackberry, blueberry polysaccharide, comprising the following steps: blackberry, blueberry fresh fruit is chosen, after cleaning up, It is dried at 45 DEG C for 24 hours, smashes it through 60 meshes, obtain blackberry powder.
50g blackberry powder accurately is weighed, 95% (percent by volume) ethyl alcohol of 3 times of blackberry powder quality is added, in 75 DEG C of conditions Under, 4h is heated and stirred, is filtered after centrifugation, filter residue is dried for 24 hours under the conditions of 45 DEG C.
The deionized water of 30 times of blackberry, blueberry filter residue quality is added into the blackberry, blueberry filter residue of drying, is added and stirs under the conditions of 85 DEG C 4h is mixed, filtrate is collected after 5000g centrifugation 10min, repetitive operation 2 times, merges filtrate twice and is concentrated under reduced pressure into 200ml, be added The Sevag reagent (n-butanol: chloroform=1:4, v/v) of 1/4 volume of the concentrated liquid vibrates 20min, 5000g centrifugation under 200r/min Supernatant is collected after 10min, then takes off albumen through Sevag method, is repeated 12 times.
Extracting solution rotary evaporation after de- albumen is removed to extra Sevag reagent, and 16 times of de- albumen are added thereto The AB-8 macroreticular resin of extracting solution quality afterwards, vibrates 8h, filtering and collecting filter liquid at 200r/min.
Dehydrated alcohol, which is added, makes the mass concentration 80% that ethyl alcohol is final in solution, is stood for 24 hours at 4 DEG C, is centrifuged and receives Collection precipitating.
With deionized water dissolving sediment, dialyse 48h under the bag filter of 3000Da, and dialyzate is freezed at -55 DEG C It is dry, blackberry, blueberry polysaccharide is obtained, BBP is recorded as.
Embodiment 2
A kind of preparation method of low molecular weight blackberry, blueberry polysaccharide, comprising the following steps: blackberry, blueberry fresh fruit is chosen, after cleaning up, 12h is dried at 45 DEG C, 60 meshes is smashed it through, obtains blackberry powder.50g blackberry powder accurately is weighed, 3 times of blackberry powder quality are added 95% (white point of volume ratio) ethyl alcohol heat and stir 4h under the conditions of 75 DEG C, filtered after centrifugation, by filter residue in 45 DEG C of conditions Lower drying 12h.The deionized water of 30 times of blackberry, blueberry filter residue quality is added into the blackberry, blueberry filter residue of drying, is added simultaneously under the conditions of 85 DEG C 2h is stirred, filtrate is collected after 5000g centrifugation 10min, repetitive operation 2 times, merges filtrate twice and is concentrated under reduced pressure into 200ml, add Enter the Sevag reagent (n-butanol: chloroform=1:4, v/v) of 1/4 volume of the concentrated liquid, vibrate 20min under 200r/min, is centrifuged Supernatant is collected after 10min, then takes off albumen through Sevag method, is repeated 12 times.Extracting solution rotary evaporation after de- albumen is removed more Remaining Sevag reagent, and the AB-8 macroreticular resin of extracting solution quality after 17 times of de- albumen is added thereto, it shakes at 200r/min Swing 8h, filtering and collecting filter liquid.Dehydrated alcohol, which is added, makes the mass concentration 80% that ethyl alcohol is final in solution, stands at 4 DEG C For 24 hours, 4000g collects precipitating after being centrifuged 5min.It is 150w in ultrasonic power, ultrasonic temperature is with deionized water dissolving sediment It is ultrasonically treated 12h under the conditions of 70 DEG C, then dialyses under the bag filter through 3000Da for 24 hours, dialyzate is freezed at -55 DEG C dry It is dry, blackberry, blueberry polysaccharide is obtained, BBP-12 is recorded as
Embodiment 3
A kind of preparation method of low molecular weight blackberry, blueberry polysaccharide, comprising the following steps: blackberry, blueberry fresh fruit is chosen, after cleaning up, It is dried at 60 DEG C for 24 hours, smashes it through 100 meshes, obtain blackberry powder.50g blackberry powder accurately is weighed, 4 times of blackberry, blueberry silty are added 95% ethyl alcohol of amount heats and stirs 4h under the conditions of 75 DEG C, filter after centrifugation, and filter residue is dried for 24 hours under the conditions of 60 DEG C. The deionized water of 40 times of blackberry, blueberry filter residue quality is added into the blackberry, blueberry filter residue of drying, is added and stirs 4h under the conditions of 85 DEG C, from Filtrate is collected after the heart, repetitive operation 2 times, is merged filtrate twice and is concentrated under reduced pressure into 200ml, 1/4 volume of the concentrated liquid is added Sevag reagent (n-butanol: chloroform=1:4, v/v) vibrates 20min under 200r/min, collects supernatant after 5000g centrifugation 10min Liquid, then albumen is taken off through Sevag method, it is repeated 12 times.Extracting solution rotary evaporation after de- albumen is removed to extra Sevag reagent, And the AB-8 macroreticular resin of extracting solution quality after 18 times of de- albumen is added thereto, 12h is vibrated at 300r/min, is received after filtering Collect filtrate.Dehydrated alcohol, which is added, makes the mass concentration 85% that ethyl alcohol is final in solution, stands at 4 DEG C for 24 hours, 5000g centrifugation Precipitating is collected after 10min.It is 300w in ultrasonic power, ultrasonic temperature surpasses under the conditions of being 90 DEG C with deionized water dissolving sediment For 24 hours, then dialyse under the bag filter through 3500Da 48h for sonication, and dialyzate is freeze-dried at -80 DEG C, it is more to obtain blackberry, blueberry Sugar is recorded as BBP-24.
Blackberry, blueberry Polysaccharide B BP, BBP-12, BBP-24 under the middle different preparation methods described to embodiment 1,2,3 is according to this tool The test method that body embodiment defines carries out polysaccharide yield measurement, total sugar content measures, content of reducing sugar measures, protein contains Measure fixed, molecular weight distribution determination, partial size and dispersibility measurement, antioxidant activity and the analysis of alpha-glucosaccharase enzyme inhibition activity.
By detection, blackberry, blueberry Polysaccharide B BP, BBP-12 and BBP-24 total sugar content under different preparation conditions is respectively 56 ± 0.56%, 55 ± 0.31% and 56.48 ± 0.88；Content of reducing sugar be respectively 2.97 ± 0.32%, 3.54 ± 0.41% and 4.12 ± 0.57%；Protein content is respectively 1.77 ± 0.04%, 1.76 ± 0.05% and 1.86 ± 0.04%；Wherein implement Total sugar content and protein content are without significant difference between example 1, implementation column 2 and implementation column 3；But embodiment 1 to embodiment 3 is made In standby polysaccharide, content of reducing sugar is in rising trend.
To blackberry, blueberry Polysaccharide B BP, BBP-12, BBP-24's under embodiment 1, embodiment 2, the different preparation conditions of embodiment 3 It is as follows that molecular weight analyzes result: it is big that the appearance time of Polysaccharide B BP calculates its molecular weight at 21.249min, according to standard curve Small is 5.91 × 105Da；It is big that the appearance time of Polysaccharide B BP-12 calculates its molecular weight at 22.272min, according to standard curve Small is 3.26 × 105Da；At 23.447min, calculate its molecular size range according to standard curve is the appearance time of BBP-24 1.77×105Da, polysaccharide prepared by embodiment 1 to embodiment 3, molecular weight successively decline.
The monosaccharide of blackberry, blueberry Polysaccharide B BP, BBP-12, BBP-24 under the middle different preparation conditions described to embodiment 1,2,3 and Uronic acid composition is as shown in table 1.
Table 1
The monosaccharide of blackberry, blueberry Polysaccharide B BP, BBP-24 and alditol acid analysis in table 1 the result shows that, in embodiment 1, embodiment 2 not With blackberry, blueberry Polysaccharide B BP, BBP-24 under preparation condition by arabinose, galactolipin, glucose, galacturonic acid, glucose Aldehydic acid composition.Wherein, the ratio in BBP is respectively 9.79%, 3.04%, 3.22%, 83.53%, 0.42%；In BBP-12 Ratio is respectively 11.23%, 3.46%, 3.01%, 81.86%, 0.41%；Ratio in BBP-24 is respectively 10.67%, 3.68%, 2.71%, 82.55%, 0.39%.
Blackberry, blueberry Polysaccharide B BP, BBP-24 under the different preparation conditions of embodiment 1, embodiment 2, embodiment 3 particle in the solution Particle size distribution it is as shown in table 2.
Table 2
Blackberry, blueberry Polysaccharide B BP, BBP-12, BBP-24 particle size of particle and monodispersity index analysis in the solution in table 2 The result shows that blackberry, blueberry Polysaccharide B BP, BBP-12 and BBP-24 under the middle different preparation conditions of embodiment 1,2,3 are in the solution Granular size be respectively 1070.5 ± 19nm, 598.6 ± 6.6nm, 395.4 ± 5.56nm；BBP, BBP-12 and BBP-24 exist Monodispersity index in solution is respectively 0.293 ± 0.022,0.214 ± 0.016,0.172 ± 0.015, show embodiment 1 to Polysaccharide partial size prepared by embodiment 3 gradually reduces, and dispersibility in the solution steps up.
Blackberry, blueberry Polysaccharide B BP, BBP-12 and BBP-24's under the different preparation conditions of embodiment 1, embodiment 2, embodiment 3 is red External spectrum analysis is as shown in table 3.
Table 3
The results of IR of blackberry, blueberry Polysaccharide B BP, BBP-12 and BBP-24 show under different preparation conditions in table 3 Blackberry, blueberry Polysaccharide B BP, BBP-12 and BBP-24 in 4000~400cm-1Occurs apparent polysaccharide molecule absorption peak in range. BBP, BBP-12 and BBP-24 are in 3361,3340,3294cm-1O-H stretching vibration peak is nearby presented；2937,2938 and 2935cm-1C-H stretching vibration peak is presented in place；In 1745,1745,1746cm-1Place is presented C=O and shrinks vibration peak；1631, 1618 and 1614cm-1There is strong absworption peak in place, shows that 3 kinds of polysaccharide have carboxylic group；In 1442,1441 and 1443cm-1It is left Right place shows C-H angle vibration peak；In 1235,1239 and 1235cm-1C-O-H stretching vibration peak is presented in place；3 kinds of polysaccharide exist 1100cm-1C-O-H stretching vibration peak nearby is showed, meanwhile, it also indicates that this 3 kinds of polysaccharide, is pyranose；919,913 and 921cm-1Place is presented compared with strong absworption peak, further demonstrates the presence of glucopyranosyl in 3 kinds of polysaccharide, three kinds of polysaccharide it is infrared Spectroscopic analysis results show that ultrasonotomography does not destroy the basic structure of blackberry, blueberry polysaccharide.
The triple helix knot of blackberry, blueberry Polysaccharide B BP, BBP-24 under the different preparation conditions of embodiment 1, embodiment 2, embodiment 3 Structure analysis is as shown in Figure 1.
The result shows that with the increase of NaOH concentration, tri- kinds of polysaccharide of BBP, BBP-12, BBP-24 and Congo red mixed solution Maximum absorption wavelength gradually increases, and shows identical trend with positive control curdlan, shows three kinds of polysaccharide in alkaline ring There is triple helix structure under border, triple helix results of structural analysis shows that ultrasonotomography does not destroy three strands of blackberry, blueberry polysaccharide Helical structure.
The ABTS radicals scavenging of blackberry, blueberry Polysaccharide B BP, BBP-12, BBP-24 under the different preparation conditions of embodiment 1,2,3 It is active as shown in Figure 2.
The result shows that its ABTS free radical scavenging activity of Polysaccharide B BP IC in embodiment 150For 1.62mg/mL, embodiment 2 In Polysaccharide B BP-12 to ABTS free radical scavenging activity IC50Value is 1.56mg/mL, and BP-24 pairs of the Polysaccharide B in embodiment 3 ABTS free radical scavenging activity IC50Value is 1.47mg/mL, and blackberry, blueberry polysaccharide of the embodiment 1 into embodiment 3 is clear to ABTS free radical Except active IC50It is worth in a slight decrease.
Embodiment 1, embodiment 2, blackberry, blueberry Polysaccharide B BP, BBP-12, BBP-24 couple in embodiment 3 under different preparation conditions Alpha-glucosaccharase enzyme inhibition activity is as shown in Figure 3.
The result shows that the Polysaccharide B BP in embodiment 1 is to alpha-glucosaccharase enzyme inhibition activity IC50Value is 1.59mg/mL, real The Polysaccharide B BP-12 in example 2 is applied to alpha-glucosaccharase enzyme inhibition activity IC50Value is 1.32mg/mL, the Polysaccharide B BP- in embodiment 3 24 couples of alpha-glucosaccharase enzyme inhibition activity IC50Value is 1.11mg/mL, and clinical medicine acarbose is in same alpha-glucosidase To alpha-glucosaccharase enzyme inhibition activity IC under vigor50Value 3.35mg/mL, blackberry, blueberry prepared by embodiment 1, embodiment 2 and embodiment 3 Polysaccharide a- glucosidase inhibitory active is 2.11 times, 2.54 times and 3.02 times of acarbose, embodiment 1 to embodiment respectively Blackberry, blueberry polysaccharide in 3 is in rising trend to alpha-glucosaccharase enzyme inhibition activity.
Blackberry, blueberry has anti-aging, raising immunity, promotion brain metabolism, blood pressure lowering, drop as a kind of integration of drinking and medicinal herbs fruit Blood lipid and anti-arrhythmia and other effects.Wherein, polysaccharide plays physiological function in blackberry, blueberry as a kind of important bioactive substance In play an important role, and molecular size range plays an important role to the performance of polysaccharide bioactivity.
Compare current research, Yu Yang et al. (Xu, Y.；Guo,Y.；Duan,S.；Wei,H.；Liu,Y.；Wang,L.； Huo,X.；Yang,Y.,Effects of ultrasound irradiation on the characterization and bioactivities of the polysaccharide from blackcurrant fruits.Ultrason Sonochem 2018,49,206-214.) use 40% ethyl alcohol (m/v) to carry out alcohol precipitation to blackcurrant fruit extracting solution, and study Influence of the ultrasonotomography to blackcurrant fruit polysaccharide to alpha-glucosaccharase enzyme inhibition activity, the research is only with 40% ethyl alcohol (m/v) carrying out alcohol precipitation is not settled out polysaccharide completely, causes the waste of fruit raw material resources, does not meet economical and eco-friendly development Requirement, and the polysaccharide and degradation after polysaccharide clinical medicine acarbose is weaker than to alpha-glucosaccharase enzyme inhibition activity, make it Using being restricted.
And the present invention carries out alcohol precipitation to blackberry, blueberry extracting solution using 80% ethyl alcohol (m/v), keeps the polysaccharide in extracting solution complete It is settled out entirely, and (molecular weight, partial size, infrared spectroscopy and triple helix structure on the basis of not destroying polysaccharide basic structure Data prove), blackberry, blueberry polysaccharide is ultrasonically treated, to obtain the blackberry, blueberry polysaccharide of low molecular weight.Implementation column 1,2 and of embodiment Polysaccharide prepared by embodiment 3 is to ABTS free radical scavenging activity IC50Value respectively reaches 1.62,1.56 and 1.47mg/mL, thus says Bright, the blackberry, blueberry polysaccharide of low molecular weight is more advantageous to the promotion of antioxygenic property.
Low molecular weight blackberry, blueberry polysaccharide prepared by implementation column 1, embodiment 2 and embodiment 3 is to alpha-glucosaccharase enzyme inhibition activity IC50Value respectively reaches 1.59mg/mL, 1.32mg/mL and 1.11mg/mL, and clinical medicine acarbose is in same phlorose To alpha-glucosaccharase enzyme inhibition activity IC under glycosides enzyme activity50It is prepared by value 3.35mg/mL, embodiment 1, embodiment 2, embodiment 3 Blackberry, blueberry polysaccharide a- glucosidase inhibitory active is 2.11 times, 2.54 times and 3.02 times of acarbose respectively, it is seen that the present invention Low molecular weight blackberry, blueberry polysaccharide obtained shows more strong inhibitory activity to a- glucuroide, considerably beyond clinical application Drug acarbose shows very excellent alpha-glucosaccharase enzyme inhibition activity.
In the Chinese patent of 104987431 A of publication number CN, traditional water extraction preparation is combined using freeze thawing wall breaking technology Mulberries polysaccharide, alpha-glucosaccharase enzyme inhibition activity just correspond to the 70% of acarbose；102775512 A's of publication number CN In Chinese invention patent, marginal tuber polysaccharide (IC50Value is 0.36mg/mL) it is acarbose to alpha-glucosaccharase enzyme inhibition activity (IC50Being worth is 1.28 times of 0.46mg/mL), and low molecular weight blackberry, blueberry polysaccharide alpha-glucosaccharase enzyme inhibition activity prepared by the present invention It is 2.11~3.02 times of acarbose, the alpha-glucosidase for being apparently higher than mulberries polysaccharide and marginal tuber inhibits to live Property.Therefore, the low molecular weight blackberry, blueberry polysaccharide of the technology of the present invention preparation can be used as control postprandial blood sugar and diabetes auxiliary is controlled The good selection treated；The especially entire techniqueflow of the present invention meets environment protection requirement, is particularly suitable for industrialized production.
Embodiments of the present invention are simultaneously not restricted by the embodiments, other any real without departing from spirit of the invention Made changes, modifications, substitutions, combinations, simplifications under matter and principle, should be equivalent substitute mode, are included in the present invention Protection scope within.

Claims (10)

1. a kind of low molecular weight blackberry, blueberry polysaccharide, which is characterized in that the weight average molecular weight of the low molecular weight blackberry, blueberry polysaccharide is 18-59 Ten thousand Da；
The grain diameter of the low molecular weight blackberry, blueberry polysaccharide in the solution is 400~1000nm, and dispersion index is 0.18~0.29；
The low molecular weight blackberry, blueberry polysaccharide is made of arabinose, galactolipin, glucose, galacturonic acid, glucuronic acid, and Its weight percent accounting is respectively as follows: 9.8~11.2%, 3.1~3.6%, 2.7~3.3%, 81.9~83.5% and 0.40~ 0.42%；
The low molecular weight blackberry, blueberry polysaccharide has both antioxidant activity and hypoglycemic activity, wherein to ABTS free radical scavenging activity IC50Value is 1.47~1.62mg/mL, to alpha-glucosaccharase enzyme inhibition activity IC50Value is 1.11~1.59mg/mL.
2. a kind of preparation method of low molecular weight blackberry, blueberry polysaccharide as described in claim 1, it is characterised in that the following steps are included:
(1) pre-treatment: blackberry, blueberry fresh fruit is cleaned, and drying pulverizes after cooling, crosses mesh, obtains blackberry powder, spare；
(2) degreasing: being added ethyl alcohol into step (1) treated blackberry powder, and 2~4h, mistake after centrifugation are heated at 70~90 DEG C Filter collects filter residue and dries it at 45~60 DEG C, spare；
(3) hot water extracts: the deionization of 30~40 times of blackberry, blueberry filter residue quality being added into the blackberry, blueberry filter residue after step (2) degreasing Water heats and stirs 2~4h in 85~95 DEG C of water-baths, filters after centrifugation, collects filtrate；
(4) it takes off albumen: the supernatant in step (3) is concentrated, add Sevag reagent, be centrifuged after oscillation, collect supernatant, then Albumen is taken off through Sevag method, repeats this step 9~12 time；
(5) it decolourizes: the de- protein extract rotary evaporation in step (4) being removed into extra Sevag reagent, and is added thereto The AB-8 macroreticular resin of extracting solution quality after 16~18 times of de- albumen vibrates filtering and collecting filter liquid；
(6) ethanol precipitation: being added dehydrated alcohol into the extracting solution after decoloration in step (5), makes its mass concentration finally be 80%~90%, at 0~4 DEG C stand 12~for 24 hours；Precipitating is collected after centrifugation；
(7) it is ultrasonically treated: in deionized water by the sediment dissolution in step (6), being 250~300w in ultrasonic power, surpass At 70~90 DEG C of sound temperature ultrasonic treatment 8~for 24 hours；
(8) it dialyses: by the 24~48h that dialyses under the polysaccharide solution bag filter after ultrasonic treatment in step (7)；
(9) polysaccharide solution after dialysis in step (8) is freeze-dried at -55 DEG C~-80 DEG C, obtains blackberry, blueberry polysaccharide.
3. the preparation method of low molecular weight blackberry, blueberry polysaccharide according to claim 2, which is characterized in that described in step (1) Drying temperature be 45~60 DEG C；Dry 12~for 24 hours；Crossing grit number is 60~100 mesh.
4. the preparation method of low molecular weight blackberry, blueberry polysaccharide according to claim 2, which is characterized in that described in step (2) Amount of alcohol added be 3~5 times of blackberry powder quality；The temperature of drying is 50~55 DEG C.
5. the preparation method of low molecular weight blackberry, blueberry polysaccharide according to claim 2, which is characterized in that described in step (3) The quality additional amount of deionized water is 35~40 times of blackberry powder filter residue amount；Stir 2.5~3.5h；Described in step (3) from The heart is that 4500g is centrifuged 10min.
6. the preparation method of low molecular weight blackberry, blueberry polysaccharide according to claim 2, which is characterized in that oscillation in step (4) It is 20~30min of oscillation at 200~300r/min.
7. the preparation method of low molecular weight blackberry, blueberry polysaccharide according to claim 2, which is characterized in that oscillation in step (5) Speed be 200~300r/min, time of oscillation is 8~12h.
8. the preparation method of low molecular weight blackberry, blueberry polysaccharide according to claim 2, which is characterized in that ethyl alcohol in step (6) Mass concentration be 80%~85%；Centrifugation described in step (6) is that 4000~4500g is centrifuged 5~8min.
9. the preparation method of low molecular weight blackberry, blueberry polysaccharide according to claim 2, which is characterized in that described in step (7) It is 10~15h in sonication treatment time；The ultrasonic treatment temperature is 75~85 DEG C.
10. the preparation method of low molecular weight blackberry, blueberry polysaccharide according to claim 2, which is characterized in that described in step (8) The size of bag filter is 3000~3500Da.
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