CN108239176A - Low molecular weight sea grass polysaccharide and preparation method thereof, sulphation low molecular weight sea grass polysaccharide and preparation method thereof and application - Google Patents
Low molecular weight sea grass polysaccharide and preparation method thereof, sulphation low molecular weight sea grass polysaccharide and preparation method thereof and application Download PDFInfo
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Abstract
The present invention provides a kind of low molecular weight sea grass polysaccharide, the weight average molecular weight of the low molecular weight sea grass polysaccharide is 4.48 × 104Da；The low molecular weight sea grass polysaccharide is rhamnose by molar ratio：Glucose：Galactolipin：Xylose：Arabinose=1.65：1.00：0.09：0.57: 0.17 monosaccharide composition.The present invention obtains the low molecular weight sea grass polysaccharide of specified molecular weight and monosaccharide composition using enzymatic isolation method, which has higher antioxidant activity.In addition, carrying out further sulphation modification on the basis of the low molecular weight sea grass polysaccharide, the low molecular weight sea grass polysaccharide of obtained sulphation also has good antioxidant activity.
Description
Technical field
The invention belongs to Enteromorpha extract technical fields, and in particular to low molecular weight sea grass polysaccharide and preparation method thereof and
Sulphation low molecular weight sea grass polysaccharide and preparation method thereof.
Background technology
Human body can generate free radicals in metabolic processes, and this bioactive molecule has one or several unpaired electronics
Atomic group or atom, have the characteristics that certain stability, high oxidative and chemical property are active.Under normal circumstances, freely
Base cell proliferation, differentiation, apoptosis, necrosis have adjustment effect；Numerous body tissues can just be made by relying on free radical and oxidation process
Generate energy；The oxidative and anti-oxidative of body is in dynamic balance.But various exogenous and endogenic response to oxidative stress
Biological interior free yl dynamic equilibrium can be influenced, especially excessive active oxygen can be generated when morbid state occurs in body, lead to machine
Body is in oxidative stress status.Excessive free radical and its metabolite cause cell metabolism function obstacle and damage in body,
The large biological molecules such as excessive free radical attack DNA, protein and carbohydrate, generate a variety of different consequences so that from
It is closely related by base and the generation of a variety of diseases, if cancer, angiocardiopathy, rheumatoid arthritis and atherosclerosis are outside it
The organism aging process that age increases and occurs is also closely related with this.Interior free yl can generally pass through antioxidant for clearing.At present
Common antioxidant has antrancine 12 preparation (BHA) and butylated hydroxytoluene (BHT).Although BHA and BHT has very
Good antioxidant effect, but with the enhancing of environmental consciousness, people start to suspect that the antioxidant of this kind of chemical synthesis may
It induces hepatic injury and leads to cancer.Therefore, it is an important directions from the searching novel antioxidant of natural products.
In recent years, people have compared with in-depth study the antioxidant activity effect of algal polysaccharides and oligosaccharide.But with people
Deepen continuously to the research of polysaccharide composition, property, structure, function, it is noted that problems with：In nature there are polysaccharide not
All there is bioactivity；Some polysaccharide are unfavorable for the performance of its bioactivity due to obstacles such as structure or physicochemical properties；Also have
A little polysaccharide can also generate adverse reaction or even toxic side effect simultaneously although drug effect is good；Some are detached out of natural biological body
Polysaccharide molecular weight it is high but activity is weaker, need to be further improved.Therefore, certain method is taken to degrade or tie polysaccharide
Structure modification is the fundamental way to solve the above problems.
Enteromorpha is the common green alga of Along Qingdao Coast Area, resourceful, is easily acquired.Principle active component of the polysaccharide as Enteromorpha,
It is increased to the research of its bioactivity.Prove that sea grass polysaccharide has with the previous work of the applicant according to the literature
Antioxidant activity, but this antioxidant activity is significantly insufficient.Therefore, a kind of side for improving sea grass polysaccharide antioxidant activity is found
Method is more advantageous to improving the utility value of Enteromorpha.
Invention content
In view of this, the technical problem to be solved in the present invention is to provide a kind of low molecular weight sea grass polysaccharide and its preparation side
Method and sulphation low molecular weight sea grass polysaccharide and preparation method thereof, the present invention provide the sea grass polysaccharide of low molecular weight with higher
Antioxidant activity.
The present invention provides a kind of low molecular weight sea grass polysaccharide, the weight average molecular weight of the low molecular weight sea grass polysaccharide is
4.48×104Da；The low molecular weight sea grass polysaccharide is rhamnose by molar ratio：Glucose：Galactolipin：Xylose：Arabinose
=1.65：1.00：0.09：0.57：0.17 monosaccharide composition.
Preferably, the polydispersity coefficient of the low molecular weight sea grass polysaccharide is 1.648, the low molecular weight sea grass polysaccharide
Number-average molecular weight is 2.72 × 104Da；The Z-average molecular weight of the low molecular weight sea grass polysaccharide is 2.326 × 105Da。
The present invention also provides a kind of preparation methods of above-mentioned low molecular weight sea grass polysaccharide, include the following steps：
A) sea grass polysaccharide of purifying is made with being fermented by the feed series bacillus that deposit number is CGMCC NO.12912
After the sea grass polysaccharide degrading enzyme mixing obtained carries out enzyme digestion reaction, successively by enzyme deactivation, centrifugation, supernatant liquor is obtained；
B it) will be centrifuged after the supernatant liquor alcohol precipitation, obtain low molecular weight sea grass polysaccharide solution, by the low molecular weight waterside
Tongue polysaccharide solution obtains low molecular weight sea grass polysaccharide by concentration, dialysis and freeze-drying.
Preferably, the sea grass polysaccharide degrading enzyme is prepared as follows：
Step 1：The feed series bacillus that deposit number is CGMCC NO.12912 is activated, seed liquor is made；
Step 2：The seed liquor is seeded in culture medium, fermented acquisition fermentate；
Step 3：Supernatant is taken after the fermentate is centrifuged, after degerming, concentration, desalination, dry obtained sea grass polysaccharide
Degrading enzyme.
Preferably, the mass ratio of the sea grass polysaccharide of the purifying and the sea grass polysaccharide degrading enzyme is 50:1.
The present invention also provides a kind of preparation methods of the low molecular weight sea grass polysaccharide of sulphation, include the following steps：
A) low molecular weight sea grass polysaccharide is scattered in organic solvent, adds esterifying reagent and reacted, reacted
Liquid, the low molecular weight sea grass polysaccharide are arbitrary for the low molecular weight sea grass polysaccharide described in claims 1 or 2 or claim 3~5
The low molecular weight sea grass polysaccharide that preparation method described in one is prepared；
B) lye is added in into the reaction solution and neutralized, obtain neutralizer；
C) neutralizer is centrifuged successively, dialyses, concentrates and alcohol precipitation, obtain refined solution；
D) refined solution is centrifuged successively, weight it is molten and freeze-drying obtain sulphation low molecular weight Enteromorpha it is more
Sugar.
Preferably, the esterifying reagent is prepared as follows：
It is 2 by volume ratio:After 1 anhydrous pyridine is mixed with chlorosulfonic acid, heating stirring obtains esterifying reagent.
It is described the present invention also provides a kind of low molecular weight sea grass polysaccharide for the sulphation that above-mentioned preparation method is prepared
The weight average molecular weight of the low molecular weight sea grass polysaccharide of sulphation is 5.99 × 104Da；Sulfate radical degree of substitution is 0.81, the sulfuric acid
The low molecular weight sea grass polysaccharide of change is rhamnose: glucose: galactolipin: xylose: arabinose=1.09 by molar ratio: 1.00:
0.06: 0.10: 0.11 monosaccharide composition.
Preferably, the polydispersity coefficient of the low molecular weight sea grass polysaccharide of the sulphation be 2.501, the sulphation it is low
The number-average molecular weight of molecular weight sea grass polysaccharide is 2.40 × 104Da；The equal molecules of Z of the low molecular weight sea grass polysaccharide of the sulphation
Measure is 1.574 × 105Da。
The present invention also provides a kind of antioxidants, are prepared including above-mentioned low molecular weight sea grass polysaccharide or above-mentioned preparation method
The low molecular weight sea grass polysaccharide for the sulphation that obtained low molecular weight sea grass polysaccharide or above-mentioned preparation method is prepared or on
State the low molecular weight sea grass polysaccharide of sulphation.
Compared with prior art, the present invention provides a kind of low molecular weight sea grass polysaccharide, the low molecular weight sea grass polysaccharides
Weight average molecular weight be 4.48 × 104Da；The low molecular weight sea grass polysaccharide is rhamnose by molar ratio：Glucose：Galactolipin：
Xylose：Arabinose=1.65：1.00：0.09：0.57：0.17 monosaccharide composition.The present invention obtains specific point using enzymatic isolation method
The low molecular weight sea grass polysaccharide of son amount and monosaccharide composition, the low molecular weight sea grass polysaccharide have higher antioxidant activity.In addition,
Further sulphation modification is carried out on the basis of the low molecular weight sea grass polysaccharide, the low molecular weight Enteromorpha of obtained sulphation
Polysaccharide also has good antioxidant activity.
Biological deposits explanation
EP-1, Classification And Nomenclature：Feed series bacillus Paenibacillus pabuli, on 08 29th, 2016 are preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), address is：BeiChen West Road, Chaoyang District, BeiJing City 1
Number No. 3 Institute of Microorganism, Academia Sinica of institute.Deposit number is CGMCC No.12912.
Description of the drawings
Fig. 1 is sea grass polysaccharide DEAE Sepharose Fast Flow step gradient curves；
Fig. 2 is the Sepharose 6B gel permeation chromatography figures of sea grass polysaccharide；
Fig. 3 is the infrared spectrogram of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation；
Fig. 4 is the superoxide radical removing work of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation
Property；
Fig. 5 is the hydroxyl radical free radical removing work of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation
Property；
Fig. 6 is the DPPH radicals scavengings work of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation
Property；
Fig. 7 is the reducing power of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation；
Fig. 8 is the metal chelation abilities of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation.
Specific embodiment
The present invention provides a kind of low molecular weight sea grass polysaccharide, the weight average molecular weight of the low molecular weight sea grass polysaccharide is
4.48×104Da；The low molecular weight sea grass polysaccharide is rhamnose by molar ratio：Glucose：Galactolipin：Xylose：Arabinose
=1.65：1.00：0.09：0.57：0.17 monosaccharide composition.
The polydispersity coefficient of the low molecular weight sea grass polysaccharide is 1.648, and the number of the low molecular weight sea grass polysaccharide is divided equally
Son amount is 2.72 × 104Da；The Z-average molecular weight of the low molecular weight sea grass polysaccharide is 2.326 × 105Da。
The present invention also provides a kind of preparation methods of above-mentioned low molecular weight sea grass polysaccharide, include the following steps：
A) sea grass polysaccharide of purifying is made with being fermented by the feed series bacillus that deposit number is CGMCC NO.12912
After the sea grass polysaccharide degrading enzyme mixing obtained carries out enzyme digestion reaction, successively by enzyme deactivation, centrifugation, supernatant liquor is obtained；
B it) will be centrifuged after the supernatant liquor alcohol precipitation, obtain low molecular weight sea grass polysaccharide solution, by the low molecular weight waterside
Tongue polysaccharide solution obtains low molecular weight sea grass polysaccharide by concentration, dialysis and freeze-drying.
In the present invention, the Enteromorpha as made from the feed series bacillus fermentation that deposit number is CGMCC NO.12912 is more
Sugar degrading enzyme is prepared as follows：
Step 1：The feed series bacillus that deposit number is CGMCC NO.12912 is activated, seed liquor is made；
Step 2：The seed liquor is seeded in culture medium, fermented acquisition fermentate；
Step 3：Supernatant is taken after the fermentate is centrifuged, after degerming, concentration, desalination, dry obtained sea grass polysaccharide
Degrading enzyme.
The step of pre-treatment being further included before activation described in step 1；It by deposit number is CGMCC that the pre-treatment, which is,
The feed series bacillus of NO.12912 is in scribing line culture 5 days~7 days on the solid medium containing sea grass polysaccharide.
The feed series bacillus that deposit number is CGMCC NO.12912 is activated in the embodiment of the present invention, in step 1
Culture medium includes：
The pH value of the culture medium is 6.0~8.0, preferably 7.0.
For activating in the culture medium of strain provided by the invention, the peptone is tryptone or casein peptone；Institute
Yeast extract is stated as yeast extract or yeast extract；Preferably yeast extract.
In some embodiments, the culture medium for activating strain provided by the invention includes
Culture medium is provided by the present invention used by fermenting in step 2, including：
The carbon source is selected from alpha-cyclodextrin, beta-cyclodextrin, dextrin, starch, amarogentin, ursin, cellobiose, D- fruits
Sugar, D- galactolipins, gentiobiose, alpha-D-glucose, α-D- lactose, lactose, maltose, mannose, PEARLITOL 25C, D- sweet dews
Sugar, melezitose, melibiose, Alpha-Methyl-D- galactosides, Beta-methyl-D- galactosides, 3- methyl-glucose, Alpha-Methyl-D-
Glucoside, Beta-methyl-D-Glucose glycosides, Alpha-Methyl-D-MANNOSE glycosides, isomaltoketose, D-Psicose, D- raffinoses,
Salicin, D-glucitol, stachyose, sucrose, D-Tag, D- trehaloses, turanose, acetic acid, beta-hydroxybutyric acid, pyruvic acid first
Ester, pyruvic acid, monomethyl succinate, glycerine, adenosine, 2'- desoxyadenossines, inosine, thymidine, uridine, adenosine 5'-phosphate, chest
Glycosides -5'- phosphoric acid, uridine-5'-phosphate, 6- phospho-fructoses or 6- phosphate-dextroses；
Carbon source is selected from glucose, xylose, rhamnose, fructose, maltose, sucrose, lactose, starch, Enteromorpha in the culture medium
Polysaccharide.In some specific embodiments, the carbon source of the culture medium is sea grass polysaccharide.
A concentration of 0.4g/L~4g/L of carbon source.Specially 0.4g/L, 0.6g/L, 0.8g/L, 1g/L, 2g/L, 3g/L or
4g/L.The concentration of carbon source influences the growing state of thalline and generates the vigor of enzyme, with the growth of the raising thalline of carbon source concentration
Situation is deteriorated, and enzyme activity then shows first to increase the trend reduced again with the raising of carbon source concentration.
The nitrogen source is selected from NaNO3、NH4Cl、NH4NO3, urea, yeast extract, peptone, gelatin.The present invention is implemented
In example, nitrogen source is peptone and yeast extract.In some specific embodiments, the yeast extract is yeast extract；It is described
Peptone is tryptone or casein peptone.The mass ratio of the peptone and yeast extract is (4~16)：(2~14).
In the embodiment of the present invention, a concentration of 4g/L~16g/L of peptone.Specially 4g/L, 6g/L, 8g/L, 10g/L,
12g/L, 14g/L or 16g/L.The concentration of peptone influences the growing state of thalline and generates the vigor of enzyme, as peptone is dense
The growing state presentation of the raising thalline of degree first increases the trend that reduces again, and peak appears in 10g/L, enzyme activity also with
The raising of peptone concentration shows first to increase the trend reduced again, is up to appear in 6g/L.
Similarly, a concentration of 3g/L~15g/L of yeast extract.Specially 3g/L, 5g/L, 7g/L, 9g/L, 11g/L,
13g/L or 15g/L.The concentration of yeast extract influences the growing state of thalline and generates the vigor of enzyme, with yeast extract concentration
The growing state presentation of raising thalline first increase the trend that reduces again, peak appears in 9g/L, and enzyme activity is also with ferment
The raising of mother's leaching powder concentration shows first to increase the trend reduced again, is up to appear in 7g/L.
In the embodiment of the present invention, a concentration of 5g/L~35g/L of NaCl；Specially 5g/L, 10g/L, 15g/L, 20g/L,
25g/L, 30g/L or 35g/L.Experiment shows that the concentration of NaCl influences the growing state of thalline and generates the vigor of enzyme, with
The growing state of the raising thalline of NaCl concentration is deteriorated, and the vigor of enzyme also reduces.
In the embodiment of the present invention, Na2HPO4A concentration of 1mmol/L~9mmol/L；Specially 1mmol/L, 2mmol/L,
3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L or 9mmol/L.Experiment shows Na2HPO4It is dense
Degree influences the growing state of thalline and generates the vigor of enzyme, with Na2HPO4The growing state of the raising thalline of concentration is deteriorated, with
Na2HPO4The vigor of the raising enzyme of concentration first increases to be declined afterwards, and peak appears in 6mmol/L.
Experiments indicate that the pH value of culture medium influences thalli growth situation and generates the vigor of enzyme, the present invention is implemented
In example, the pH value of the culture medium is 6.0~8.0.Specially 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5 or 9.0.Thalline
Growth and enzyme activity peak appear in pH value for 7.0 when.
FePO4Hydrate be seven water ferric phosphates；MgSO4Hydrate be epsom salt.
The culture medium of verification the verifying results includes in embodiment：
Empirical tests, the preferable culture medium of effect include：
It is furthermore preferred that culture medium includes：
The culture medium that pre-treatment uses is to add agar in the culture medium used in above-mentioned fermentation, forms it into solid culture
Base, specifically, solid medium includes：
The condition activated described in step 1 is 24 DEG C~36 DEG C, shaking table culture 12~for 24 hours；The rotating speed of the shaking table is 150
~210rpm.
The condition fermented described in step 2 is 24 DEG C~36 DEG C, 24~48h of shaking table culture；The rotating speed of the shaking table is 150
~210rpm.
Fermentation temperature, rotating speed, inoculum concentration and fermentation volume are screened in embodiment.
Wherein, fermentation temperature is 20 DEG C, 24 DEG C, 28 DEG C, 32 DEG C, 36 DEG C.The result shows that 28 DEG C are most suitable fermentation temperature.
The rotating speed of fermentation is 40rpm, 80rpm, 120rpm, 160rpm, 200rpm, 240rpm.The result shows that 200rpm is
Most suitable rotating speed.
Inoculum concentration is 1%~8%, specially 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%.Take off the result shows that, most
Suitable inoculum concentration is 3%.
Fermentation volume be 50mL~120mL, specially 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 110mL,
120mL.The result shows that most suitable fermentation volume is 70mL.
The present invention research shows that, under suitable condition, for 24 hours fermentation gained sea grass polysaccharide degrading enzyme activity and amount i.e. can reach
Higher level.
The rotating speed centrifuged described in step 3 is 6000rpm, time 20min.
Centrifugation, degerming, concentration, desalination described in step 3 are all in 4 DEG C of progress.
The degerming uses filtration sterilization, and filter diameter is 0.22 μm.
Concentration is using ultrafiltration concentration.
Desalination is using dialysis desalination.
The aperture of dialysis is 8000-14000Da Da.
The component of dialyzate includes：5mMPBS buffer solutions
Dry is vacuum freeze drying.
There is good enzymatic activity with the sea grass polysaccharide degrading enzyme that strain provided by the invention and fermentation generate, can degrade
Sea grass polysaccharide, and the sea grass polysaccharide oligosaccharides of different polymerization degree can be prepared according to different parameters.Sea grass polysaccharide provided by the invention
The activity of degrading enzyme is up to 1.03U/mL.
The sea grass polysaccharide as made from the feed series bacillus fermentation that deposit number is CGMCC NO.12912 that will be obtained
Degrading enzyme is mixed with the sea grass polysaccharide purified, obtains mixture.
The sea grass polysaccharide of the purifying is prepared as follows：
1) polysaccharide in Enteromorpha is extracted using Hot water extraction, obtains Enteromorpha Thick many candies；
2) after the Enteromorpha Thick many candies are mixed with water, Enteromorpha Thick many candies aqueous solution, the Enteromorpha Thick many candies aqueous solution are obtained
By being eluted through DEAE Sepharose Fast Flow Weak anion-exchange chromatographies column, eluent is obtained；
3) it by the eluent after concentration and dialysis, with Sepharose 6B gel chromatography column purifications, is purified
Sea grass polysaccharide aqueous solution；
4) by the sea grass polysaccharide aqueous solution of the purifying by concentration, dialysis and freeze-drying, the sea grass polysaccharide purified.
The Hot water extraction is specifically operated as follows：
Clean sea grass polysaccharide after cleaning with water is mixed and carries out heating extraction, obtains reaction product；
The reaction product is filtered, obtains filtrate；
The filtrate is decolourized and removed to concentrate after smell, obtains concentrate；
The concentrate is filtered, is cooled down after centrifugation, obtains gel；
It mixes, is dehydrated with the ethyl alcohol of volumetric concentration >=95% after the gel is freezed, obtained dewatered
Polysaccharide；
The dewatered polysaccharide is dried, obtains sea grass polysaccharide.
Wherein, the present invention mixes with water first by the clean sea grass polysaccharide after cleaning and carries out heating extraction, obtains anti-
Answer product.
The cleaning of the sea grass polysaccharide carries out as follows：
Sea grass polysaccharide is placed in water soaking and stirring, the soaking time is preferably 10~40min, and yarn is used after immersion
Cloth filters, and removes filtrate.
The number of the cleaning is preferably 3~4 times.
Clean sea grass polysaccharide after obtained cleaning is mixed with water carries out heating extraction, the Extracting temperature for 80~
100 DEG C, solid-liquid ratio 1：20~1：40, extraction pH is 6~7, and extraction time is 2~3 hours.
According to said extracted method, obtained sea grass polysaccharide yield is higher, and obtained sea grass polysaccharide solution is easier to
By dissolved colloidal state indexing gel state.
Most preferably, the extraction conditions are 90 DEG C of temperature, pH 6, extraction time 3h, solid-liquid ratio 1:40.
The reaction product is filtered, obtains filtrate, obtained filtrate was further carried out with the bolting silk of 500 mesh
Filter, removes insoluble impurity.
Then, the filtrate is carried out being concentrated to 1/5th of original volume, obtains concentrate.Be allowed to be formed gel or
Person is to add in cleaned second of extraction of Enteromorpha progress secondary to the filtrate carried, to improve the polyoses content in filtrate, is made
Filtrate forms gel as early as possible.
After obtaining concentrate, concentrate is heated to 40~50 DEG C, is then centrifuged in concentrate, the time of centrifugation is
5~15 minutes, rotating speed was 5000~10000r/min, further removed the insoluble matter in polysaccharide extraction liquid.
Under conditions of the polysaccharide solution centrifuged is placed in 4 DEG C, gel is obtained.
It mixes, is dehydrated with the ethyl alcohol of volumetric concentration >=95% after the gel is freezed, obtained dewatered
Polysaccharide.
The temperature of the freezing is -20 DEG C, time >=8 hour of the freezing.The number of the dehydration is preferably 2~3
It is secondary.
The sea grass polysaccharide obtained using above-mentioned dewatering type can protect the structure of polysaccharide, thus be more advantageous to the shape of microballoon
Into.
The dewatered polysaccharide is dried, obtains Enteromorpha Thick many candies.
The temperature of the drying is 50 DEG C, and the time of the drying is 4~5 hours.
Finally sea grass polysaccharide is crushed.
After obtaining Enteromorpha Thick many candies, after the Enteromorpha Thick many candies are mixed with water, Enteromorpha Thick many candies aqueous solution is obtained, it is described
Enteromorpha Thick many candies aqueous solution passes through to be eluted through DEAE Sepharose Fast Flow Weak anion-exchange chromatographies column, is eluted
Liquid；
Specifically, taking Enteromorpha Thick many candies, with appropriate water dissolution, insoluble matter is centrifuged off, supernatant passes through DEAE
Sepharose Fast Flow Weak anion-exchange chromatography columns isolate and purify Thick many candies, and specific isolation and purification method is：
Successively to distill 2 column volumes of water elution, 0.5mol/LNaCl elutes 2 column volumes, and 1.07mol/L NaCl are washed
3 column volumes are taken off, elution curve is drawn with sulfuric acid-phynol method.0mol/L NaCl eluents are merged according to elution curve.
It is pure with Sepharose 6B gel chromatographic columns by the eluent after concentration and dialysis after obtaining eluent
Change, the sea grass polysaccharide aqueous solution purified；
To the method for the concentration and dialysis there is no specifically limited, well known to a person skilled in the art methods to be the present invention
It can.
By the eluent it is concentrated, dialysis after, further with Sepharose 6B gel chromatography column purifications, specific method
It is as follows：
Using water as mobile phase, flow velocity 1mL/min merges eluent, the sea grass polysaccharide purified according to elution curve
Aqueous solution.
By the sea grass polysaccharide aqueous solution of the purifying by concentration, dialysis and freeze-drying, the sea grass polysaccharide purified.
The sea grass polysaccharide of purifying is made with being fermented by the feed series bacillus that deposit number is CGMCC NO.12912
Sea grass polysaccharide degrading enzyme mixing carry out enzyme digestion reaction after, successively by enzyme deactivation, centrifugation, obtain supernatant liquor；
The sea grass polysaccharide of the purifying is 25 with the mass ratio of sea grass polysaccharide degrading enzyme：1~100:1.
In the present invention, it is 60h~96h that the temperature of the enzyme digestion reaction, which is the time of 30~60 DEG C of enzyme digestion reactions,.
Product after enzymolysis is subjected to enzyme deactivation, centrifugation, obtains supernatant liquor.
The enzyme deactivation boils enzyme deactivation using boiling water, and the time boiled is 10min, is protein inactivation.
The rotating speed of the centrifugation is 8000rpm, and the time of centrifugation is 10min.
After obtaining supernatant, it will be centrifuged after the supernatant liquor alcohol precipitation, low molecular weight sea grass polysaccharide solution obtained, by institute
Low molecular weight sea grass polysaccharide solution is stated by concentration, dialysis and freeze-drying, obtains low molecular weight sea grass polysaccharide.
Specifically, the ethanol precipitation that 95% is added in into the supernatant removes unreacted polysaccharide.Supernatant is through rotation
Turn to be concentrated by evaporation, dialyse (≤2000Da), freeze-drying obtains low molecule low molecular weight sea grass polysaccharide (DUP).
The present invention also provides a kind of preparation methods of the low molecular weight sea grass polysaccharide of sulphation, include the following steps：
A) low molecular weight sea grass polysaccharide is scattered in organic solvent, adds esterifying reagent and reacted, reacted
Liquid, the low molecular weight sea grass polysaccharide are above-mentioned low molecular weight sea grass polysaccharide or the low molecule being prepared according to upper preparation method
Measure sea grass polysaccharide；
B) lye is added in into the reaction solution and neutralized, obtain neutralizer；
C) neutralizer is centrifuged successively, concentration and alcohol precipitation obtain refined solution；
D) refined solution is centrifuged successively, weight it is molten and freeze-drying obtain sulphation low molecular weight Enteromorpha it is more
Sugar.
Low molecular weight sea grass polysaccharide is scattered in organic solvent by the present invention first, is added esterifying reagent and is reacted,
Obtain reaction solution.
The esterifying reagent is prepared as follows：
It is 2 by volume ratio:After 1 anhydrous pyridine is mixed with chlorosulfonic acid, heating stirring obtains esterifying reagent.
Specifically, anhydrous pyridine ice-water bath is cooled to 0 DEG C, chlorosulfonic acid is slowly added under magnetic agitation, 95 DEG C continue
30min is stirred, faint yellow solid is made fully to dissolve, is refrigerated in refrigerator after sealing, in 1 week effectively.
The organic solvent is preferably formamide.
In the present invention, described react is specially：
Low molecular weight sea grass polysaccharide powder is suspended in organic solvent, magnetic stirring is lower to be added dropwise the esterifying reagent, 95
DEG C reaction 3h, obtain reaction solution.
After reaction, 0 DEG C will be cooled in the reaction solution ice-water bath, have been pre-cooled adding in thereto to 0 DEG C
4mol/L NaOH solutions neutralize, obtain neutralizer；
Then, by the neutralizer centrifugation, dialysis, rotary evaporation in vacuo concentration after add in 3 times of volumes 95% ethyl alcohol into
Row alcohol precipitation, obtains refined solution；
Refined solution centrifugation, heavy molten, freeze-drying are obtained into sulphation low molecular weight sea grass polysaccharide (SDUP).
In the present invention, the sulfate radical degree of substitution (DS) is calculated according to formula (I) formula：
DS=1.62 × S%/(32-1.02 × S%), formula (I)；
In formula (I), S% is the mass fraction of sulphur atom.
The present invention also provides a kind of low molecular weight sea grass polysaccharide for the sulphation being prepared using above-mentioned preparation method,
The weight average molecular weight of the low molecular weight sea grass polysaccharide of the sulphation is 5.99 × 104Da；Sulfate radical degree of substitution is 0.81, described
The low molecular weight sea grass polysaccharide of sulphation is rhamnose by molar ratio：Glucose：Galactolipin：Xylose：Arabinose=1.09:
1.00：0.06：0.10：0.11 monosaccharide composition.
The polydispersity coefficient of the low molecular weight sea grass polysaccharide of the sulphation is 2.501, the low molecular weight of the sulphation
The number-average molecular weight of sea grass polysaccharide is 2.40 × 104Da；The Z-average molecular weight of the low molecular weight sea grass polysaccharide of the sulphation is
1.574×105Da。
The present invention also provides a kind of antioxidant, the antioxidant is selected from above-mentioned low molecular weight sea grass polysaccharide or above-mentioned sulphur
The low molecular weight sea grass polysaccharide of acidification.
The present invention obtains the low molecular weight sea grass polysaccharide of specified molecular weight and monosaccharide composition, the low molecular weight using enzymatic isolation method
Sea grass polysaccharide has higher antioxidant activity.In addition, carry out further sulphur on the basis of the low molecular weight sea grass polysaccharide
Acidification modification, the low molecular weight sea grass polysaccharide of obtained sulphation also have good antioxidant activity.
For a further understanding of the present invention, with reference to embodiment to low molecular weight sea grass polysaccharide provided by the invention and its
Preparation method, sulphation low molecular weight sea grass polysaccharide and preparation method thereof and application illustrate, protection scope of the present invention
It is not limited by the following examples.
1 sea grass polysaccharide of embodiment extracts
Enteromorpha dry powder is cleaned with tap water and (removed desalination and other impurities), is dried for standby in 60 DEG C.Use Hot water extraction
Polysaccharide is carried, by Enteromorpha：Water (w/v)=1:30 add in distilled water, and 100 DEG C of heating stirrings extract 2h, with 200 mesh silk cover filterings, filter
Slag repeats to extract once, merges coarse filtration liquid twice, measures volume.Coarse filtration liquid placement is cooled to gel, cuts after -20 DEG C of refrigerators
Freezing.It takes out, break into pieces after the complete deep colling of adhesive tape, add in 95% alcohol of 1.5 times of coarse filtration liquid volumes, be placed at room temperature for, make ice cube
It thaws dehydration.With 200 mesh silk cover filterings, water and alcohol are squeezed with hand.Filter residue is further taken off with a certain amount of 95% alcohol again
Water then in 60 DEG C of drying, obtains Enteromorpha Thick many candies.
2 sea grass polysaccharide of embodiment isolates and purifies
Thick many candies are isolated and purified using DEAE Sepharose Fast Flow Weak anion-exchange chromatography columns,
Fraction is collected with fraction collector, total sugar content is detected using sulfuric acid-phynol method.According to linear elution as a result, respectively with distillation
2 column volumes of water elution, 0.5mol/L NaCl elute 2 column volumes, and 1.07mol/L NaCl elute 3 column volumes, 4mol/L
It elutes 2 column volumes, then 6 column volumes is rinsed with deionized water (purpose is to remove extra salting liquid)；Column volume：60mL；On
Sample amount：30mg；Flow velocity：3.5mL/min.Fraction collector is collected, and sulfuric acid-phynol method detection total sugar content with elution volume, is inhaled
Luminosity and salinity mapping.The result is shown in Figure 1, Fig. 1 are washed for sea grass polysaccharide DEAE Sepharose Fast Flow stage gradients
De- curve.Distilled water elution fraction is collected, dialysis desalination, concentration, freeze-drying are placed in drier and preserve.
Through the isolated polysaccharide component of DEAE Sepharose Fast Flow anion-exchange chromatographies, continue through
Sepharose 6B gel chromatographic columns (2.6 × 80cm) are further isolated and purified.Using water as eluent, flow velocity 1mL/
min.Component (10mL/ pipes) is collected using fraction collector；Total sugar content is detected with sulfuric acid-phynol method, is surveyed at 490nm wavelength
Its fixed absorption value, is mapped with absorbance-elution time.As a result it is that the Sepharose 6B gels of sea grass polysaccharide ooze to see Fig. 2, Fig. 2
Saturating chromatogram.Merge the high component of sugar content through dialysis desalting, freeze-drying, be placed in drier and preserve.
The preparation of 3 sea grass polysaccharide degrading enzyme of embodiment
1), pre-treatment：The feed series bacillus that deposit number is CGMCC NO.12912 is lined sea grass polysaccharide to consolidate
On body culture medium, 5d is cultivated at 28 DEG C；
Solid medium be sea grass polysaccharide 2g/L, peptone 6g/L, yeast extract 7g/L, NaCl 5g/L, Na2HPO4
6mmol/L,MgSO41g/L, FePO4·7H2O 0.05g/L, agar 15g/L.
2) it, activates：Solid medium bacterial strain is connect into 1~3 ring in the test tube of seed culture fluid, with 200rpm at 28 DEG C
Cultivate 12h.
Culture solution be sea grass polysaccharide 2g/L, peptone 6g/L, yeast extract 7g/L, NaCl 5g/L.
3) it, ferments：Seed culture medium is accessed in fermentation medium, with 28 DEG C of cultures of shaking table for 24 hours, obtains fermentate.
Fermentation medium is sea grass polysaccharide 2g/L, peptone 6g/L, yeast extract 7g/L, NaCl5g/L, Na2HPO4
6mmol/L,MgSO41g/L, FePO4·7H2O 0.05g/L.After fermentation, bacterium solution OD is measured600It is 1.223 to be worth, gained bacterium
Liquid 6000rpm centrifugations 20min obtains fermented supernatant fluid；Under the conditions of 4 DEG C, gained fermented supernatant fluid is crossed into 0.22 μm of filter membrane degerming, is surpassed
Filter concentration, dialysis desalination, vacuum freeze drying, obtain sea grass polysaccharide degrading enzyme.
Measure enzyme activity：The detection method of enzyme activity is DNS methods, and enzyme activity defines：1mL enzyme solutions 1min generates 1 μ g reduced sugars
As a unit of activity, result 1.03U/ml.
It is prepared by 4 low molecular weight sea grass polysaccharide of embodiment
The preparation of low molecular weight sea grass polysaccharide passes through enzymatic hydrolysis process.It is dropped using the sea grass polysaccharide that embodiment 3 is prepared
The sea grass polysaccharide that solution enzyme and embodiment 2 are prepared is 50 according to mass ratio:1 reacts 72h in 45 DEG C, and boiling water boils 10min, makes
Protein inactivation, 8000rpm centrifugation 10min collect the unreacted polysaccharide of ethanol precipitation removal that supernatant adds in 95%.Supernatant
Concentrated through rotary evaporation, dialyse (≤2000Da), freeze-drying obtains low molecular weight sea grass polysaccharide (DUP).By the low molecular weight
Sea grass polysaccharide carries out examination of infrared spectrum, and it is the low molecular weight of low molecular weight sea grass polysaccharide and sulphation as a result to see Fig. 3, Fig. 3
The infrared spectrogram of sea grass polysaccharide.
The low molecular weight sea grass polysaccharide of 5 sulphation of embodiment
Sulphation modification uses chlorosulfonic acid-pyridine method.Chlorosulfonic acid, which meets water, can generate fierce reaction or even explosion, therefore
Operating process will avoid touching water.
1) preparation of esterifying reagent:Adding 200mL anhydrous pyridines, ice-water bath is cooled to 0 DEG C, magnetic in 500mL round-bottomed flasks
100mL chlorosulfonic acids are slowly added under power stirring, 95 DEG C are persistently stirred 30min, are that faint yellow solid fully dissolves, ice after sealing
It is refrigerated in case, in 1 week effectively.
2) synthesis of sulphation low molecular weight sea grass polysaccharide:Low molecular weight sea grass polysaccharide powder prepared by 1g embodiments 4 hangs
Float in 40ml formamides, magnetic stirring is lower to be added dropwise 7.5ml esterifying reagents, 95 DEG C of reaction 3h.After, 0 is cooled in ice-water bath
DEG C, addition has been pre-cooled into 0 DEG C of 4mol/L NaOH solutions and solution acid alkalinity, centrifugation, dialysis, rotary evaporation in vacuo
95% ethyl alcohol of 3 times of volumes is added in after concentration, centrifugation, heavy molten, freeze-drying obtain sulphation low molecular weight sea grass polysaccharide
(SDUP).The low molecular weight sea grass polysaccharide of the sulphation is subjected to examination of infrared spectrum, it is low molecular weight as a result to see Fig. 3, Fig. 3
The infrared spectrogram of sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation.
Sulfate radical degree of substitution (DS) calculates：DS=1.62 × S%/(32-1.02 × S%)
S%：The mass fraction of sulphur atom.
6 study on the biochemical characters of embodiment
(1) total sugar content is measured using phend-sulphuric acid.The drafting of standard curve：The accurate sandlwood for preparing 0.1mg/mL
Standard for Sugars solution.0.0,0.1,0.15,0.2,0.25,0.3,0.35,0.4mL standard solution is drawn respectively, and moisturizing makes final volume
For 0.5mL.6% phenol of 0.3mL, the 1.5mL concentrated sulfuric acids are added in, oscillation shakes up, and 15min is heated in 100 DEG C of water-baths, after cooling
490nm measures absorbance A.With rhamnose concentration (mg/mL) for abscissa, A is ordinate, draws standard curve.
The low molecular weight waterside of sulphation that low molecular weight sea grass polysaccharide prepared by embodiment 4 is prepared with embodiment 5 respectively
Tongue polysaccharide sample is configured to 5mg/mL solution, and the used time is diluted to 0.1mg/mL.0.5mL is taken, remaining repeats the operation of above-mentioned standard product,
Total sugar content is calculated with standard curve.
(2) glucuronic acid content is measured using sulfate-carbazole.Carbazole reagent：0.1g carbazoles are dissolved in 95% second of 90mL
Alcohol, constant volume to 100ml.Sulfuric acid-borax soln：0.9534g boraxs are dissolved in 10mL water, and stirring and dissolving adds in ice-water bath
The 90mL concentrated sulfuric acids are stood overnight after mixing spare.The drafting of standard curve：Accurate compound concentration is 0.25mg/mL grape alditols
Sour standard solution.0,10,20,30,40,50,65,70,80 μ L of standard solution are drawn respectively, are added water to final volume as 100 μ L, are added
Enter 0.6mL sulfuric acid-borax soln and 20 μ L carbazoles, shake up, 100 DEG C of heating 10min cool down, absorbance A is measured at 530nm.With
Glucuronic acid concentration (mg/mL) is abscissa, and light absorption value is ordinate, draws standard curve.The measure of sample：Sample is suitable
Under concentration, 100 μ L is taken to be operated by above-mentioned steps.Glucuronic acid content in sample is calculated with standard curve.
(3) sulfate radical content is measured using barium chloride-gelatin method.Sulfate radical standard solution:Accurately weigh in 105 DEG C of conditions
Lower drying is dissolved, compound concentration is 0.6mg/mL sulfate radical standard solution to the potassium sulfate of constant weight with 1mol/L hydrochloric acid solutions.
0.5% gelatin solution:2.5g gelatin is weighed, is dissolved in 500mL, 70 DEG C or so of steaming feedback water, stands overnight, 4 DEG C save backup.
Barium chloride-gelatin solution：0.5g barium chlorides are weighed, are dissolved in 100mL oneself 0.5% gelatin solutions that stand overnight, 4 DEG C of preservations are standby
With.The drafting of sulfate radical standard curve：It is accurate to draw 0.00,0.04,0.08,0.12,0.16 and of sulfate radical standard solution
0.20mL is mended with the HC1 solution of 1mol/L to 0.20mL in test tube, and 3.8mL, 8% trichloroacetic acid are separately added into each pipe
With 1.0mL barium chlorides-gelatin solution, oscillation is stood after shaking up, and reacts 15min at room temperature, and extinction is measured at wavelength 360nm
Value A1；Barium chloride-gelatin solution is replaced with the gelatin solution of same volume 0.5%, light absorption value A is measured at wavelength 360nm2。
Using sulfate radical content as abscissa, (A1-A2) light absorption value for ordinate draw standard curve.It respectively prepared by embodiment 4 low
The low molecular weight sea grass polysaccharide sample of sulphation prepared by molecular weight sea grass polysaccharide and embodiment 5 adds in 1mol/L HCl, 100 DEG C
6h is hydrolyzed, adds in 3.8mL, 8% trichloroacetic acid and 1.0mL barium chlorides-gelatin solution, oscillation is stood after shaking up, at room temperature instead
15min is answered, light absorption value A is measured at wavelength 360nm1；Replace barium chloride-gelatin molten with the gelatin solution of same volume 0.5%
Liquid.Light absorption value A is measured at 360nm2, it is that reference substance makees standard curve with sodium sulphate, calculates to obtain sulfate radical content.
(4) low molecular weight sea grass polysaccharide and the sulphur of the preparation of embodiment 5 prepared by embodiment 4 is measured using Coomassie Brilliant Blue
Protein content in the low molecular weight sea grass polysaccharide sample of acidification.
Obtain following table 1 result：
The physicochemical property of 1 low molecular weight sea grass polysaccharide of table and its sulphation polysaccharide derivative
In table 1, Nd is expressed as not detecting.Yield is that the low molecular weight Enteromorpha of sulphation is prepared by low molecular weight sea grass polysaccharide
The yield of polysaccharide.
7 monosaccharide component of embodiment measures
Monosaccharide component is measured using PMP column front derivations efficient liquid phase chromatographic analysis (PMP-HPLC).With methanol compound concentration
PMP solution for 0.5mol/L；Mixing monosaccharide (L- rhamnoses, D- xyloses, D-Glucose, the D- half that compound concentration is 10mg/mL
Lactose, D-arabinose) standard solution；Low molecular weight sea grass polysaccharide prepared by embodiment 4 is prepared with embodiment 5 respectively
The low molecular weight sea grass polysaccharide sample trifluoroacetic acid solution complete hydrolysis of sulphation, after 40 DEG C of rotations of hydrolysate are evaporated, then
Repeatedly plus CH3OH is evaporated, until removing extra trifluoroacetic acid.It rear sample will be evaporated will be dissolved in 100 μ L water and 10mg/mL samples are made
Solution.Sample and each 100 μ L of standard monosaccharide solutions are taken, the NaOH for adding in 0.5mol/mL PMP solution 120 μ L, 0.3mol/L is molten
Liquid 100 μ L, 70 DEG C of water-bath 1h, the hydrochloric acid solution that 100 a concentration of 0.3mol/L of μ L are added in after being cooled to room temperature neutralize, dichloro
Methane extracts 3 times, and supernatant liquor is taken to cross 0.22 μm of miillpore filter to get derivative sample and standard items.Chromatographic condition is：Chromatography
Column：Agilent TC-C18 chromatographic columns；Column temperature：30℃；Mobile phase：0.02mol/ml phosphate buffers (pH6.8)/CH3CN
=83:17(V/V).Gradient elution is (0min, 17%B；5min, 18%B；10min, 19%B；30min, 20%B).In elution
Sample amount：20μL；Flow velocity：1.0mL/min；Detector：UV(250nm).According to the appearance time and peak area ratio in sample
Monosaccharide forms and molar ratio.Obtain following table 2 result：
The monosaccharide component of 2 low molecular weight sea grass polysaccharide of table and its sulphation polysaccharide derivative
8 molecular weight determination of embodiment
The measure of molecular weight is using High Performance Gel Permeation liquid chromatography (HPGPC).Using High Performance Gel Permeation color
Spectrometry (HPGPC) measures the molecular weight of polysaccharide.HPGPC conditions:Analytical column is SB-806HQ and SB-804HQ columnss in series；Mobile phase
For 0.1mol/L NaNO3；Flow velocity is 1mL/min；Column temperature is 25 DEG C；Sample size is 20 μ L；Composition distribution detects.Respectively into
20 μ L, GPC softwares of sample are different according to the appearance time of 6 different molecular weight standard specimens, draw standard curve.The measure of sample:Point
The inaccurate low molecular weight sea grass polysaccharide for weighing the preparation of embodiment 4 and the low molecular weight Enteromorpha of sulphation prepared by embodiment 5 are more
Sugar-like product add and flow the solution that phased soln is made 5%, centrifuging and taking supernatant, and 20 μ L, GPC softwares of sample introduction are right according to appearance time
Answer standard curve.The peak molecular weight of sample is calculated, molecular weight distribution spread factor (D) is calculated according to peak width.Obtain following table 3
As a result：
The molecular weight and its polydispersity coefficient of 3 low molecular weight sea grass polysaccharide of table and its sulphation polysaccharide derivative
9 superoxide radical scavenging capacity of embodiment is tested
The sulphation low molecular weight sea grass polysaccharide that low molecular weight sea grass polysaccharide prepared by embodiment 4 is prepared with embodiment 5
The experiment of superoxide radical scavenging capacity is using NADH-PMS-NBT methods.
Concrete operations are：Various concentration sample (0.01-0.5mg/mL) is prepared with Tris-HCl buffer solutions (pH8.0).Often
A sample takes 0.1ml, adds in 557 μM of NADH of 1ml (being dissolved in 16mM Tris-HCl, pH 8.0), and 45 μM of PMS of 1ml (are dissolved in
16mM Tris-HCl, pH 8.0), 108 μM of NBT of 1ml (being dissolved in 16mM Tris-HCl, pH 8.0).5min is reacted at room temperature, in
It is A that 560nm, which surveys light absorption value,1；A0Include all reagents but no sample；A2By sample and reagent but replace NBT with Tris-HCl.
Superoxide radical clearance rate calculation formula：
Superoxide radical clearance rate (%)=[1- (A1-A2)/A0] × 100%
As a result see the super oxygen freedom that Fig. 4, Fig. 4 are low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation
Base scavenging capacity.As shown in Figure 4, sample concentration is in 2mg/mL, the low molecular weight of low molecular weight sea grass polysaccharide and sulphation
The superoxide radical scavenging capacity of sea grass polysaccharide is respectively 72.48%, 93.06%, and the clearance rate with the Vc of concentration is
82.95%.Illustrate sea grass polysaccharide after sulphation modification, superoxide radical clearance rate is remarkably reinforced, and higher than with concentration
Vc。
10 hydroxyl radical free radical scavenging capacity of embodiment is tested
The sulphation low molecular weight sea grass polysaccharide that low molecular weight sea grass polysaccharide prepared by embodiment 4 is prepared with embodiment 5
Hydroxyl radical free radical scavenging capacity is tested
Prepare the polysaccharide sample (0.25-10.0mg/mL) of various concentration, 2 ml of each sample adds in the FeSO of 5mmol/L4
(2mL), the salicylic acid ethanol solution (2mL) of 5mmol/L；Then 2ml 5mmol/LH are added in2O2.37 DEG C of water-bath 30min, cooling
To room temperature, it is A that light absorption value is surveyed at 510nm1, positive control is done with ascorbic acid.A0For reagent (no sample)；A2For no hydroxyl
Free radical.
Hydroxyl radical free radical clearance rate calculation formula：
Hydroxyl radical free radical clearance rate=[1- (A1-A2)/A0] × 100%
As a result it is the hydroxyl free of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation to see Fig. 5, Fig. 5
Base scavenging capacity.As shown in Figure 5, when sample concentration is less than 4mg/mL, the low molecule of low molecular weight sea grass polysaccharide and sulphation
The hydroxyl radical free radical clearance rate of amount sea grass polysaccharide is significantly higher than Vc；And sample concentration be higher than 4mg/mL when, low molecular weight Enteromorpha is more
The hydroxyl radical free radical clearance rate of sugar and the low molecular weight sea grass polysaccharide of sulphation is less than Vc；In a concentration of 10mg/mL, low molecule
It is respectively 54.29% to measure sea grass polysaccharide, the low molecular weight sea grass polysaccharide of sulphation and the hydroxyl radical free radical clearance rate of Vc,
73.21%, 100%.Illustrate at low concentrations, the hydroxyl of the low molecular weight sea grass polysaccharide of low molecular weight sea grass polysaccharide and sulphation
Base free radical scavenging activity is higher than Vc；And at high concentrations, the hydroxyl radical free radical clearance rate of sea grass polysaccharide is low after sulphation modification
In the Vc of same concentration, but it is higher than the hydroxyl radical free radical clearance rate of unmodified sea grass polysaccharide.
11 DPPH radicals scavengings of embodiment are tested
The DPPH ethanol solutions of 0.2mmol/L are prepared, it is now with the current.Prepare the polysaccharide concentration (0.25- of various concentration
10mg/mL).1ml samples is taken to add in 95% ethyl alcohol of DPPH and 2ml of 2ml.Reaction solution is vigorously mixed, and is put in darkroom reaction
30min, it is A that light absorption value is surveyed at 517nm1。A02ml DPPH and 3ml ethyl alcohol is included for reaction solution.A2For 1ml and 4ml 95%
Ethyl alcohol.The DPPH free radical scavenging activity calculation formula of polysaccharide：
DPPH free radical scavenging activities (%)=[1- (A1-A2)/A0] × 100%
As a result see the DPPH freedom that Fig. 6, Fig. 6 are low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation
Base scavenging capacity.It will be appreciated from fig. 6 that the DPPH free radicals of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation
Clearance rate is less than Vc；But in sample concentration 10mg/mL, the DPPH free radical scavenging activities of the low molecular weight sea grass polysaccharide of sulphation
Close to Vc；In a concentration of 10mg/mL, low molecular weight sea grass polysaccharide, the low molecular weight sea grass polysaccharide of sulphation and the DPPH of Vc
Free radical scavenging activity is respectively 43.67%, 93.26%, 98.05%.Illustrate sea grass polysaccharide after sulphation modification, DPPH is free
Base clearance rate significantly increases.
12 reducing power of embodiment measures
With 0.2mmol/mL phosphate buffers (pH6.6) configuration various concentration sample (0.25-10.0mg/mL).Specific side
Method adds in 1mL (1%, w/v) potassium ferricyanide, 50 DEG C of warm bath 20min to take sample 1.0mL.Then 2.0ml trichloroacetic acids are added in
(10%, w/v) terminates reaction.1.2ml iron chloride (0.1%, w/v) is added in into reaction solution again, light absorption value is surveyed at 700nm.
Light absorption value is bigger, and reducing power is stronger.As a result it is low molecular weight sea grass polysaccharide and the low molecular weight Enteromorpha of sulphation to see Fig. 7, Fig. 7
The reducing power of polysaccharide.As shown in Figure 7, in a concentration of 10mg/mL, low molecular weight sea grass polysaccharide, sulphation low molecular weight waterside
The reducing power of tongue polysaccharide and Vc are respectively 0.9713,2.0449,3.8013；With the low molecular weight Enteromorpha of sulphation under concentration
Though the reducing power of polysaccharide is apparently higher than non-sulphation modification low molecular weight sea grass polysaccharide not as good as Vc.Illustrate sulphation modification
Its reducing power can be remarkably reinforced in low molecular weight sea grass polysaccharide.
13 metal chelation abilities of embodiment measure
The metal chelation abilities of polysaccharide measure bibliography and slightly change.Prepare the sample (0.2-10mg/ of various concentration
ML), 2mM FeCl are added in2(0.1mL) and 5mM phenanthrene alloxazine (0.4mL) is placed at room temperature for 10min after mixing.EDTA is as positive right
According to the survey light absorption value at 562nm.Metal ion chelating capacity calculation formula：
Metal chelation abilities (%)=100% × (A0-A)/A0
A0For the light absorption value of no sample, A is sample mixed liquor light absorption value.
As a result it is the metal-chelating of low molecular weight sea grass polysaccharide and the low molecular weight sea grass polysaccharide of sulphation to see Fig. 8, Fig. 8
Ability.As shown in Figure 8, in a concentration of 10mg/mL, low molecular weight sea grass polysaccharide, sulphation low molecular weight sea grass polysaccharide and
The metal ion chelating capacity of EDTA is respectively 97.69%, 66.78%, 99.70%；With low molecular weight sea grass polysaccharide under concentration
Metal ion chelating capacity be apparently higher than sulphation modification low molecular weight sea grass polysaccharide, and close to the metal ion chela of EDTA
Conjunction ability.Illustrate that the metal ion chelating capacity of the low molecular weight sea grass polysaccharide through sulphation modification is declined.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of low molecular weight sea grass polysaccharide, which is characterized in that the weight average molecular weight of the low molecular weight sea grass polysaccharide is 4.48
×104Da；The low molecular weight sea grass polysaccharide is rhamnose by molar ratio：Glucose：Galactolipin：Xylose：Arabinose=
1.65：1.00：0.09：0.57：0.17 monosaccharide composition.
2. low molecular weight sea grass polysaccharide according to claim 1, which is characterized in that the low molecular weight sea grass polysaccharide it is more
The coefficient of dispersion is 1.648, and the number-average molecular weight of the low molecular weight sea grass polysaccharide is 2.72 × 104Da；The low molecular weight waterside
The Z-average molecular weight of tongue polysaccharide is 2.326 × 105Da。
3. a kind of preparation method of low molecular weight sea grass polysaccharide as claimed in claim 1 or 2, which is characterized in that including following
Step：
A) by the sea grass polysaccharide of purifying and as made from the feed series bacillus fermentation that deposit number is CGMCC NO.12912
After the mixing of sea grass polysaccharide degrading enzyme carries out enzyme digestion reaction, successively by enzyme deactivation, centrifugation, supernatant liquor is obtained；
B it) will be centrifuged after the supernatant liquor alcohol precipitation, obtain low molecular weight sea grass polysaccharide solution, the low molecular weight Enteromorpha is more
Sugar juice obtains low molecular weight sea grass polysaccharide by concentration, dialysis and freeze-drying.
4. preparation method according to claim 3, which is characterized in that the sea grass polysaccharide degrading enzyme as follows into
It is prepared by row：
Step 1：The feed series bacillus that deposit number is CGMCC NO.12912 is activated, seed liquor is made；
Step 2：The seed liquor is seeded in culture medium, fermented acquisition fermentate；
Step 3：Supernatant is taken after the fermentate is centrifuged, after degerming, concentration, desalination, dry obtained sea grass polysaccharide degradation
Enzyme.
5. preparation method according to claim 3, which is characterized in that the sea grass polysaccharide of the purifying and the sea grass polysaccharide
The mass ratio of degrading enzyme is 50:1.
6. the preparation method of the low molecular weight sea grass polysaccharide of a kind of sulphation, which is characterized in that include the following steps：
A) low molecular weight sea grass polysaccharide is scattered in organic solvent, adds esterifying reagent and reacted, obtain reaction solution, institute
Low molecular weight sea grass polysaccharide is stated as the low molecular weight sea grass polysaccharide or claim 3~5 any one described in claims 1 or 2
The low molecular weight sea grass polysaccharide that the preparation method is prepared；
B) lye is added in into the reaction solution and neutralized, obtain neutralizer；
C) neutralizer is centrifuged successively, dialyses, concentrates and alcohol precipitation, obtain refined solution；
D) refined solution is centrifuged successively, weight is molten and freeze-drying obtains the low molecular weight sea grass polysaccharide of sulphation.
7. preparation method according to claim 6, which is characterized in that the esterifying reagent is made as follows
It is standby：
It is 2 by volume ratio:After 1 anhydrous pyridine is mixed with chlorosulfonic acid, heating stirring obtains esterifying reagent.
8. a kind of low molecular weight sea grass polysaccharide for the sulphation that preparation method as claimed in claims 6 or 7 is prepared, special
Sign is that the weight average molecular weight of the low molecular weight sea grass polysaccharide of the sulphation is 5.99 × 104Da；Sulfate radical degree of substitution is
0.81, the low molecular weight sea grass polysaccharide of the sulphation is rhamnose by molar ratio：Glucose：Galactolipin：Xylose：Arabinose
=1.09：1.00：0.06：0.10：0.11 monosaccharide composition.
9. the low molecular weight sea grass polysaccharide of sulphation according to claim 8, which is characterized in that low point of the sulphation
The polydispersity coefficient of son amount sea grass polysaccharide is 2.501, and the number-average molecular weight of the low molecular weight sea grass polysaccharide of the sulphation is
2.40×104Da；The Z-average molecular weight of the low molecular weight sea grass polysaccharide of the sulphation is 1.574 × 105Da。
10. a kind of antioxidant, which is characterized in that will including the low molecular weight sea grass polysaccharide described in claims 1 or 2 or right
It asks described in the low molecular weight sea grass polysaccharide or claim 6 or 7 that the preparation method described in 3~5 any one is prepared
The low molecule of sulphation described in the low molecular weight sea grass polysaccharide or claim 8 or 9 of the sulphation that preparation method is prepared
Measure sea grass polysaccharide.
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