CN108239176B - Low-molecular-weight enteromorpha polysaccharide and preparation method thereof, sulfated low-molecular-weight enteromorpha polysaccharide and preparation method and application thereof - Google Patents

Low-molecular-weight enteromorpha polysaccharide and preparation method thereof, sulfated low-molecular-weight enteromorpha polysaccharide and preparation method and application thereof Download PDF

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CN108239176B
CN108239176B CN201611226588.8A CN201611226588A CN108239176B CN 108239176 B CN108239176 B CN 108239176B CN 201611226588 A CN201611226588 A CN 201611226588A CN 108239176 B CN108239176 B CN 108239176B
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enteromorpha polysaccharide
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刘晨光
李嘉欣
丁松
于钰
孙茜
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Qingdao Youdo Bioengineering Co ltd
Ocean University of China
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Abstract

The invention provides enteromorpha polysaccharide with low molecular weight, wherein the weight average molecular weight of the enteromorpha polysaccharide with low molecular weight is 4.48 multiplied by 104Da; the low molecular weight enteromorpha polysaccharide is prepared from rhamnose: glucose: galactose: xylose: arabinose 1.65: 1.00: 0.09: 0.57: 0.17 monosaccharide composition. The invention adopts an enzymolysis method to obtain the low molecular weight enteromorpha polysaccharide with specific molecular weight and monosaccharide composition, and the low molecular weight enteromorpha polysaccharide has higher antioxidant activity. In addition, the low-molecular-weight enteromorpha polysaccharide is further subjected to sulfation modification on the basis of the low-molecular-weight enteromorpha polysaccharide, and the obtained sulfated low-molecular-weight enteromorpha polysaccharide also has good antioxidant activity.

Description

Low-molecular-weight enteromorpha polysaccharide and preparation method thereof, sulfated low-molecular-weight enteromorpha polysaccharide and preparation method and application thereof
Technical Field
The invention belongs to the technical field of enteromorpha extracts, and particularly relates to low-molecular-weight enteromorpha polysaccharide and a preparation method thereof, and sulfated low-molecular-weight enteromorpha polysaccharide and a preparation method thereof.
Background
The active molecule has one or several unpaired electron radicals or atoms, and has certain stability, high oxidizing property, active chemical property and other features. Under normal conditions, free radicals have the regulation effect on cell proliferation, differentiation, apoptosis and necrosis; rely on free radicals and oxidation processes to generate energy from numerous body tissues; the oxidation and antioxidation of the body are in dynamic balance. However, various exogenous and endogenous oxidative stress reactions affect the dynamic balance of free radicals in organisms, and particularly, excessive active oxygen is generated when the organisms are ill, so that the organisms are in an oxidative stress state. Excessive free radicals and metabolites thereof in the organism cause cell metabolic dysfunction and damage, and the excessive free radicals attack biomacromolecules such as DNA, protein, carbohydrate and the like to generate various different consequences, so that the free radicals are closely related to the generation of various diseases, such as cancer, cardiovascular diseases, rheumatoid arthritis and aging of the organism caused by the increase of the external age of atherosclerosis. Free radicals in the body are generally scavenged by antioxidants. The antioxidants commonly used today are t-butyl-p-cresol formulation (BHA) and Butylhydroxytoluene (BHT). Although BHA and BHT have excellent antioxidant effects, with the increasing awareness of environmental protection, it is becoming suspected that such chemically synthesized antioxidants may induce liver damage and cause cancer. Therefore, the search for novel antioxidants from natural products is an important direction.
In recent years, people have made intensive studies on the antioxidant activity of algal polysaccharides and oligosaccharides. However, as people have studied the composition, properties, structure and function of polysaccharides, they have paid attention to the following problems: polysaccharides are not all biologically active when found in nature; some polysaccharides are not favorable for exerting the biological activity due to the obstacles such as structure or physicochemical properties; some polysaccharides have good drug effect, but also can generate adverse reaction and even toxic and side effects; some polysaccharides isolated from natural organisms have high molecular weight but weak activity and are desired to be further improved. Therefore, it is an essential approach to solve the above problems to degrade or structurally modify polysaccharides in a certain way.
The enteromorpha prolifera is common green algae in Qingdao coastal areas, is rich in resources and easy to collect. The polysaccharide is used as the main effective component of enteromorpha, and the research on the biological activity of the enteromorpha is increasing day by day. According to literature reports and previous works of the applicant, the enteromorpha polysaccharide has antioxidant activity, but the antioxidant activity is obviously insufficient. Therefore, a method for improving the antioxidant activity of enteromorpha polysaccharide is found, and the utilization value of enteromorpha is favorably improved.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a low molecular weight enteromorpha polysaccharide and a preparation method thereof, and a sulfated low molecular weight enteromorpha polysaccharide and a preparation method thereof.
The invention provides enteromorpha polysaccharide with low molecular weight, wherein the weight average molecular weight of the enteromorpha polysaccharide with low molecular weight is 4.48 multiplied by 104Da; the low molecular weight enteromorpha polysaccharide is prepared from rhamnose: glucose: galactose: xylose: arabinose 1.65: 1.00: 0.09: 0.57: 0.17 monosaccharide composition.
Preferably, the polydispersity of the low molecular weight enteromorpha polysaccharide is 1.648, and the number average molecular weight of the low molecular weight enteromorpha polysaccharide is 2.72 multiplied by 104Da; the Z-average molecular weight of the low-molecular-weight enteromorpha polysaccharide is 2.326 multiplied by 105Da。
The invention also provides a preparation method of the enteromorpha polysaccharide with low molecular weight, which comprises the following steps:
A) mixing the purified enteromorpha polysaccharide with enteromorpha polysaccharide degrading enzyme prepared by fermenting feed bacillus with the preservation number of CGMCC NO.12912, performing enzymolysis reaction, and then sequentially performing enzyme deactivation and centrifugation to obtain supernatant;
B) and precipitating the supernatant with ethanol, centrifuging to obtain a low-molecular-weight enteromorpha polysaccharide solution, and concentrating, dialyzing and freeze-drying the low-molecular-weight enteromorpha polysaccharide solution to obtain the low-molecular-weight enteromorpha polysaccharide.
Preferably, the enteromorpha polysaccharide-degrading enzyme is prepared according to the following method:
step 1: activating feed paenibacillus with a preservation number of CGMCC NO.12912 to prepare seed liquid;
step 2: inoculating the seed liquid into a culture medium, and fermenting to obtain a fermentation product;
and step 3: centrifuging the fermented product, taking supernatant, sterilizing, concentrating, desalting, and drying to obtain the Enteromorpha polysaccharide degrading enzyme.
Preferably, the mass ratio of the purified enteromorpha polysaccharide to the enteromorpha polysaccharide degrading enzyme is 50: 1.
The invention also provides a preparation method of the sulfated enteromorpha polysaccharide with low molecular weight, which comprises the following steps:
a) dispersing low-molecular-weight enteromorpha polysaccharide into an organic solvent, and adding an esterification reagent for reaction to obtain a reaction solution, wherein the low-molecular-weight enteromorpha polysaccharide is the low-molecular-weight enteromorpha polysaccharide in the claim 1 or 2 or the low-molecular-weight enteromorpha polysaccharide prepared by the preparation method in any one of the claims 3 to 5;
b) adding alkali liquor into the reaction liquid for neutralization to obtain a neutralized liquid;
c) sequentially centrifuging, dialyzing, concentrating and precipitating the neutralized solution with ethanol to obtain a purified solution;
d) and sequentially carrying out centrifugation, redissolution and freeze drying on the purified solution to obtain the sulfated enteromorpha polysaccharide with low molecular weight.
Preferably, the esterification reagent is prepared according to the following method:
mixing anhydrous pyridine and chlorosulfonic acid in a volume ratio of 2:1, and heating and stirring to obtain the esterification reagent.
The invention also provides the sulfated enteromorpha polysaccharide with the low molecular weight prepared by the preparation method, wherein the weight average molecular weight of the sulfated enteromorpha polysaccharide with the low molecular weight is 5.99 multiplied by 104Da; the sulfate radical substitution degree is 0.81, and the sulfated enteromorpha polysaccharide with low molecular weight is prepared from the components of rhamnose: glucose: galactose: xylose: arabinose 1.09: 1.00: 0.06: 0.10: 0.11 monosaccharide composition.
Preferably, the sulfated enteromorpha polysaccharide with low molecular weight has a polydispersity of 2.501 and a number average molecular weight of 2.40 × 104Da; the Z-average molecular weight of the sulfated enteromorpha polysaccharide with low molecular weight is 1.574 multiplied by 105Da。
The invention also provides an antioxidant which comprises the low-molecular-weight enteromorpha polysaccharide or the low-molecular-weight enteromorpha polysaccharide prepared by the preparation method, or the sulfated low-molecular-weight enteromorpha polysaccharide prepared by the preparation method or the sulfated low-molecular-weight enteromorpha polysaccharide.
Compared with the prior art, the invention provides the enteromorpha polysaccharide with low molecular weight, and the weight average molecular weight of the enteromorpha polysaccharide with low molecular weight is 4.48 multiplied by 104Da; the low molecular weight enteromorpha polysaccharide is prepared from rhamnose: glucose: galactose: xylose: arabinose 1.65: 1.00: 0.09: 0.57: 0.17 monosaccharide composition. The invention adopts an enzymolysis method to obtain the low molecular weight enteromorpha polysaccharide with specific molecular weight and monosaccharide composition, and the low molecular weight enteromorpha polysaccharide has higher antioxidant activity. In addition, the low-molecular-weight enteromorpha polysaccharide is further subjected to sulfation modification on the basis of the low-molecular-weight enteromorpha polysaccharide, and the obtained sulfated low-molecular-weight enteromorpha polysaccharide also has good antioxidant activity.
Drawings
FIG. 1 is a gradient elution curve of Enteromorpha polysaccharide DEAE Sepharose Fast Flow stage;
FIG. 2 is a Sepharose 6B gel permeation chromatogram of Enteromorpha polysaccharide;
FIG. 3 is an infrared spectrum of a low molecular weight Enteromorpha polysaccharide and a sulfated low molecular weight Enteromorpha polysaccharide;
FIG. 4 is the superoxide radical scavenging activity of low molecular weight Enteromorpha polysaccharide and sulfated low molecular weight Enteromorpha polysaccharide;
FIG. 5 is hydroxyl radical scavenging activity of low molecular weight Enteromorpha polysaccharide and sulfated low molecular weight Enteromorpha polysaccharide;
FIG. 6 is DPPH free radical scavenging activity of low molecular weight Enteromorpha polysaccharide and sulfated low molecular weight Enteromorpha polysaccharide;
FIG. 7 is the reducing power of low molecular weight Enteromorpha polysaccharide and sulfated low molecular weight Enteromorpha polysaccharide;
figure 8 is a graph of the metal chelating capacity of low molecular weight enteromorpha polysaccharides and sulfated low molecular weight enteromorpha polysaccharides.
Detailed Description
The invention provides enteromorpha polysaccharide with low molecular weight, wherein the weight average molecular weight of the enteromorpha polysaccharide with low molecular weight is 4.48 multiplied by 104Da; the low molecular weight enteromorpha polysaccharide is prepared from rhamnose: glucose: galactose: xylose: arabinose 1.65: 1.00: 0.09: 0.57: 0.17 monosaccharide composition.
The polydispersity number of the low molecular weight enteromorpha polysaccharide is 1.648, and the number average molecular weight of the low molecular weight enteromorpha polysaccharide is 2.72 multiplied by 104Da; the Z-average molecular weight of the low-molecular-weight enteromorpha polysaccharide is 2.326 multiplied by 105Da。
The invention also provides a preparation method of the enteromorpha polysaccharide with low molecular weight, which comprises the following steps:
A) mixing the purified enteromorpha polysaccharide with enteromorpha polysaccharide degrading enzyme prepared by fermenting feed bacillus with the preservation number of CGMCC NO.12912, performing enzymolysis reaction, and then sequentially performing enzyme deactivation and centrifugation to obtain supernatant;
B) and precipitating the supernatant with ethanol, centrifuging to obtain a low-molecular-weight enteromorpha polysaccharide solution, and concentrating, dialyzing and freeze-drying the low-molecular-weight enteromorpha polysaccharide solution to obtain the low-molecular-weight enteromorpha polysaccharide.
In the invention, the enteromorpha polysaccharide degrading enzyme prepared by fermenting paenibacillus feed with the preservation number of CGMCC NO.12912 is prepared according to the following method:
step 1: activating feed paenibacillus with a preservation number of CGMCC NO.12912 to prepare seed liquid;
step 2: inoculating the seed liquid into a culture medium, and fermenting to obtain a fermentation product;
and step 3: centrifuging the fermented product, taking supernatant, sterilizing, concentrating, desalting, and drying to obtain the Enteromorpha polysaccharide degrading enzyme.
Step 1, before activation, a pretreatment step is also included; the pretreatment is to streak and culture the paenibacillus foeniculus with the preservation number of CGMCC NO.12912 on a solid culture medium containing enteromorpha polysaccharide for 5 to 7 days.
In the embodiment of the invention, the culture medium for activating the paenibacillus foraging with the preservation number of CGMCC NO.12912 in the step 1 comprises:
Figure BDF0000012742860000041
the pH value of the culture medium is 6.0-8.0, and preferably 7.0.
In a culture medium for activating the strain provided by the invention, the peptone is tryptone or casein peptone; the yeast extract is yeast extract or yeast extract powder; preferably yeast extract powder.
In some embodiments, the culture medium that activates the bacterial species provided herein comprises
Figure BDF0000012742860000042
Figure BDF0000012742860000052
The culture medium adopted for fermentation in the step 2 is provided by the invention and comprises the following components:
Figure BDF0000012742860000051
the carbon source is selected from alpha-cyclodextrin, beta-cyclodextrin, dextrin, starch, amygdalin, arbutin, cellobiose, D-fructose, D-galactose, gentiobiose, alpha-D-glucose, alpha-D-lactose, maltose, mannose, D-mannitol, D-mannose, melezitose, melibiose, alpha-methyl-D-galactoside, beta-methyl-D-galactoside, 3-methyl-glucose, alpha-methyl-D-glucoside, beta-methyl-D-glucoside, alpha-methyl-D-mannoside, isomaltulose, D-psicose, D-raffinose, salicin, D-sorbitol, D-glucoside, D-mannoside, D-, Stachyose, sucrose, D-tagatose, D-trehalose, turanose, acetic acid, beta-hydroxybutyric acid, methyl pyruvate, pyruvic acid, monomethyl succinate, glycerol, adenosine, 2 '-deoxyadenosine, inosine, thymidine, uridine, adenosine-5' -phosphate, thymidine-5 '-phosphate, uridine-5' -phosphate, 6-phospho-fructose or 6-phospho-glucose;
the carbon source in the culture medium is selected from glucose, xylose, rhamnose, fructose, maltose, sucrose, lactose, starch and enteromorpha polysaccharide. In some embodiments, the carbon source of the medium is enteromorpha polysaccharide.
The concentration of the carbon source is 0.4 g/L-4 g/L. Specifically 0.4g/L, 0.6g/L, 0.8g/L, 1g/L, 2g/L, 3g/L or 4 g/L. The concentration of the carbon source affects the growth of the bacteria and the activity of the produced enzyme, the growth of the bacteria becomes worse with the increase of the concentration of the carbon source, and the activity of the enzyme shows a tendency of increasing and then decreasing with the increase of the concentration of the carbon source.
The nitrogen source is selected from NaNO3、NH4Cl、NH4NO3Urea, yeast extract, peptone, gelatin. In the examples of the present invention, the nitrogen source is peptone and yeast extract. In some embodiments, the yeast extract is a yeast extract powder; the peptone is tryptone or casein peptone. The mass ratio of the peptone to the yeast extract is (4-16): (2-14).
In the embodiment of the invention, the concentration of the peptone is 4 g/L-16 g/L. Specifically 4g/L, 6g/L, 8g/L, 10g/L, 12g/L, 14g/L or 16 g/L. The concentration of the peptone influences the growth condition of thalli and the activity of enzyme production, the growth condition of the thalli shows a trend of increasing firstly and then decreasing with the increase of the concentration of the peptone, the highest value of the growth condition of the thalli is 10g/L, and the activity of the enzyme shows a trend of increasing firstly and then decreasing with the increase of the concentration of the peptone, and the highest value of the activity of the enzyme is 6 g/L.
Similarly, the concentration of the yeast extract powder is 3g/L to 15 g/L. Specifically 3g/L, 5g/L, 7g/L, 9g/L, 11g/L, 13g/L or 15 g/L. The concentration of the yeast extract powder affects the growth condition of the thallus and the activity of the produced enzyme, the growth condition of the thallus shows a trend of increasing firstly and then decreasing with the increase of the concentration of the yeast extract powder, the highest value of the growth condition of the thallus shows a trend of 9g/L, and the activity of the enzyme shows a trend of increasing firstly and then decreasing with the increase of the concentration of the yeast extract powder, and the highest value of the activity of the enzyme shows a trend of 7 g/L.
In the embodiment of the invention, the concentration of NaCl is 5 g/L-35 g/L; specifically 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L or 35 g/L. Experiments show that the concentration of NaCl influences the growth condition of the thallus and the activity of the produced enzyme, and the growth condition of the thallus is worsened along with the increase of the concentration of NaCl, and the activity of the enzyme is also reduced.
In the examples of the present invention, Na2HPO4The concentration of (A) is 1 mmol/L-9 mmol/L; specifically 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L or 9 mmol/L. Experiments show that Na2HPO4The concentration of (A) affects the growth of the cells and the activity of the enzyme produced, and is dependent on Na2HPO4The growth of the cells becomes worse with increasing concentration of Na2HPO4The activity of the enzyme is increased and then decreased at the increased concentration, and the highest value is 6 mmol/L.
Experiments show that the pH value of the culture medium influences the growth condition of thalli and the activity of produced enzyme, and in the embodiment of the invention, the pH value of the culture medium is 6.0-8.0. Specifically 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0. The highest value of the thallus growth and the enzyme activity is when the pH value is 7.0.
FePO4The hydrate of (a) is iron phosphate heptahydrate; MgSO (MgSO)4The hydrate of (a) is magnesium sulfate heptahydrate.
Examples of media for verifying the effect include:
Figure BDF0000012742860000061
proved by verification, the culture medium with better effect comprises:
Figure BDF0000012742860000062
Figure BDF0000012742860000071
more preferably, the culture medium comprises:
Figure BDF0000012742860000072
the medium used for the pretreatment is a solid medium prepared by adding agar to the medium used for the fermentation, and specifically, the solid medium comprises:
Figure BDF0000012742860000073
the activation condition in the step 1 is 24-36 ℃, and the shake culture is carried out for 12-24 h; the rotating speed of the shaking table is 150-210 rpm.
The fermentation condition in the step 2 is 24-36 ℃, and the shake culture is carried out for 24-48 h; the rotating speed of the shaking table is 150-210 rpm.
In the examples, the fermentation temperature, the rotation speed, the inoculum size and the fermentation volume were screened.
Wherein the fermentation temperature is 20 deg.C, 24 deg.C, 28 deg.C, 32 deg.C, 36 deg.C. The results show that 28 ℃ is the optimum fermentation temperature.
The rotation speed of the fermentation was 40rpm, 80rpm, 120rpm, 160rpm, 200rpm, and 240 rpm. The results show that 200rpm is the optimum rotation speed.
The inoculation amount is 1-8%, specifically 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%. The results revealed that the optimum inoculum size was 3%.
The fermentation volume is 50 mL-120 mL, specifically 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 110mL, 120 mL. The results show that the optimal fermentation volume is 70 mL.
The research of the invention shows that the activity and the amount of the enteromorpha polysaccharide degrading enzyme obtained by 24h fermentation can reach higher level under the appropriate condition.
The rotation speed of the centrifugation in the step 3 is 6000rpm, and the time is 20 min.
The centrifugation, sterilization, concentration and desalination in step 3 are all carried out at 4 ℃.
The sterilization adopts filtration sterilization, and the filter diameter is 0.22 mu m.
The concentration is carried out by ultrafiltration.
Desalting by dialysis.
The pore size for dialysis was 8000-14000 Da.
The components of the dialysate include: 5mMPBS buffer
The drying is vacuum freeze drying.
The enteromorpha polysaccharide degrading enzyme produced by the strain and fermentation provided by the invention has good enzyme activity, can degrade enteromorpha polysaccharide, and can prepare enteromorpha polysaccharide oligosaccharides with different polymerization degrees according to different parameters. The activity of the enteromorpha polysaccharide degrading enzyme provided by the invention can reach 1.03U/mL.
Mixing the obtained Enteromorpha polysaccharide degrading enzyme prepared by fermenting the Paenibacillus feed with the preservation number of CGMCC NO.12912 with the purified Enteromorpha polysaccharide to obtain a mixture.
The purified enteromorpha polysaccharide is prepared according to the following method:
1) extracting polysaccharide in enteromorpha by using a hot water extraction method to obtain crude enteromorpha polysaccharide;
2) mixing the enteromorpha crude polysaccharide with water to obtain an enteromorpha crude polysaccharide aqueous solution, and eluting the enteromorpha crude polysaccharide aqueous solution through a DEAE Sepharose Fast Flow weak anion exchange chromatographic column to obtain an eluent;
3) concentrating and dialyzing the eluent, and purifying by using a Sepharose 6B gel chromatographic column to obtain a purified enteromorpha polysaccharide aqueous solution;
4) and concentrating, dialyzing and freeze-drying the purified enteromorpha polysaccharide aqueous solution to obtain the purified enteromorpha polysaccharide.
The hot water extraction method is specifically operated according to the following method:
mixing the cleaned enteromorpha polysaccharide with water, and heating and extracting to obtain a reaction product;
filtering the reaction product to obtain a filtrate;
decoloring the filtrate, removing odor, and concentrating to obtain a concentrated solution;
filtering the concentrated solution, centrifuging and cooling to obtain gel;
freezing the gel, mixing the frozen gel with ethanol with the volume concentration of more than or equal to 95%, and dehydrating to obtain dehydrated polysaccharide;
and drying the dehydrated polysaccharide to obtain the enteromorpha polysaccharide.
The method comprises the following steps of firstly mixing cleaned enteromorpha polysaccharide with water, heating and extracting to obtain a reaction product.
The cleaning of the enteromorpha polysaccharide is carried out according to the following method:
soaking and stirring the enteromorpha polysaccharide in water, wherein the soaking time is preferably 10-40 min, filtering with gauze after soaking is finished, and removing filtrate.
The number of times of cleaning is preferably 3-4.
Mixing the obtained cleaned enteromorpha polysaccharide with water, and heating and extracting, wherein the extraction temperature is 80-100 ℃, and the material-liquid ratio is 1: 20-1: 40, the extraction pH is 6-7, and the extraction time is 2-3 hours.
According to the extraction method, the yield of the obtained enteromorpha polysaccharide is high, and the obtained enteromorpha polysaccharide solution is easy to shift to a gel state from a sol state.
Most preferably, the extraction conditions are that the temperature is 90 ℃, the pH value is 6, the extraction time is 3h, and the material-liquid ratio is 1: 40.
The reaction product was filtered to obtain a filtrate, and the obtained filtrate was further filtered with a 500-mesh silk screen to remove insoluble impurities.
The filtrate was then concentrated to one fifth of the original volume to give a concentrated solution. So that the filtrate forms gel, or the extracted filtrate is added with the cleaned enteromorpha again for secondary extraction, so as to improve the polysaccharide content in the filtrate and enable the filtrate to form gel as soon as possible.
And after the concentrated solution is obtained, heating the concentrated solution to 40-50 ℃, and then centrifuging the concentrated solution for 5-15 minutes at the rotating speed of 5000-10000 r/min, thereby further removing insoluble substances in the polysaccharide extracting solution.
And (3) placing the centrifuged polysaccharide solution at 4 ℃ to obtain gel.
And freezing the gel, mixing the frozen gel with ethanol with the volume concentration of more than or equal to 95%, and dehydrating to obtain dehydrated polysaccharide.
The freezing temperature is-20 ℃, and the freezing time is more than or equal to 8 hours. The number of times of dehydration is preferably 2-3.
The enteromorpha polysaccharide obtained by adopting the dehydration mode can protect the structure of the polysaccharide, thereby being more beneficial to the formation of microspheres.
And drying the dehydrated polysaccharide to obtain the crude enteromorpha polysaccharide.
The drying temperature is 50 ℃, and the drying time is 4-5 hours.
And finally, crushing the enteromorpha polysaccharide.
After obtaining the crude enteromorpha polysaccharide, mixing the crude enteromorpha polysaccharide with water to obtain an aqueous solution of the crude enteromorpha polysaccharide, and eluting the aqueous solution of the crude enteromorpha polysaccharide by using a DEAE Sepharose Fast Flow weak anion exchange chromatographic column to obtain an eluent;
specifically, crude enteromorpha polysaccharide is taken, dissolved by using a proper amount of water, and centrifuged to remove insoluble substances, and the supernatant passes through a DEAE Sepharose Fast Flow weak anion exchange chromatographic column to separate and purify the crude polysaccharide, wherein the specific separation and purification method comprises the following steps:
sequentially eluting 2 column volumes with distilled water, 2 column volumes with 0.5mol/L NaCl and 3 column volumes with 1.07mol/L NaCl, and drawing an elution curve by a sulfuric acid-phenol method. The 0mol/L NaCl eluate was pooled according to the elution profile.
After obtaining an eluent, concentrating and dialyzing the eluent, and purifying by using a Sepharose 6B gel chromatographic column to obtain a purified enteromorpha polysaccharide aqueous solution;
the method of concentration and dialysis is not particularly limited in the present invention, and may be a method known to those skilled in the art.
Concentrating and dialyzing the eluent, and further purifying by using a Sepharose 6B gel chromatographic column by the following specific method:
and (3) taking water as a mobile phase, and combining the eluates according to an elution curve at the flow rate of 1mL/min to obtain the purified enteromorpha polysaccharide aqueous solution.
And concentrating, dialyzing and freeze-drying the purified enteromorpha polysaccharide aqueous solution to obtain the purified enteromorpha polysaccharide.
Mixing the purified enteromorpha polysaccharide with enteromorpha polysaccharide degrading enzyme prepared by fermenting feed bacillus with the preservation number of CGMCC NO.12912, performing enzymolysis reaction, and then sequentially performing enzyme deactivation and centrifugation to obtain supernatant;
the mass ratio of the purified enteromorpha polysaccharide to the enteromorpha polysaccharide degrading enzyme is 25: 1-100: 1.
In the invention, the temperature of the enzymolysis reaction is 30-60 ℃, and the time of the enzymolysis reaction is 60-96 h.
And (4) inactivating enzyme and centrifuging the product after enzymolysis to obtain supernatant.
The enzyme deactivation is carried out by boiling with boiling water for 10min, which is protein inactivation.
The rotating speed of the centrifugation is 8000rpm, and the time of the centrifugation is 10 min.
And after obtaining supernatant liquid, carrying out alcohol precipitation on the supernatant liquid, centrifuging to obtain a low-molecular-weight enteromorpha polysaccharide solution, and concentrating, dialyzing and freeze-drying the low-molecular-weight enteromorpha polysaccharide solution to obtain the low-molecular-weight enteromorpha polysaccharide.
Specifically, 95% ethanol was added to the supernatant to precipitate and remove unreacted polysaccharide. And (3) performing rotary evaporation and concentration on the supernatant, dialyzing (less than or equal to 2000Da), and freeze-drying to obtain the low-molecular-weight enteromorpha polysaccharide (DUP).
The invention also provides a preparation method of the sulfated enteromorpha polysaccharide with low molecular weight, which comprises the following steps:
a) dispersing low-molecular-weight enteromorpha polysaccharide into an organic solvent, and adding an esterification reagent for reaction to obtain a reaction liquid, wherein the low-molecular-weight enteromorpha polysaccharide is the low-molecular-weight enteromorpha polysaccharide or the low-molecular-weight enteromorpha polysaccharide prepared according to the preparation method;
b) adding alkali liquor into the reaction liquid for neutralization to obtain a neutralized liquid;
c) sequentially centrifuging, concentrating and precipitating the neutralized solution with ethanol to obtain a purified solution;
d) and sequentially carrying out centrifugation, redissolution and freeze drying on the purified solution to obtain the sulfated enteromorpha polysaccharide with low molecular weight.
Firstly, dispersing low-molecular-weight enteromorpha polysaccharide in an organic solvent, and then adding an esterification reagent for reaction to obtain a reaction solution.
The esterification reagent is prepared according to the following method:
mixing anhydrous pyridine and chlorosulfonic acid in a volume ratio of 2:1, and heating and stirring to obtain the esterification reagent.
Specifically, the anhydrous pyridine is cooled to 0 ℃ in an ice-water bath, chlorosulfonic acid is slowly added under magnetic stirring, stirring is continuously carried out for 30min at 95 ℃ to fully dissolve the faint yellow solid, and the faint yellow solid is refrigerated in a refrigerator after sealing and is effective within 1 week.
The organic solvent is preferably formamide.
In the present invention, the reaction is specifically:
suspending low-molecular-weight enteromorpha polysaccharide powder in an organic solvent, dropwise adding the esterification reagent under magnetic stirring, and reacting at 95 ℃ for 3h to obtain a reaction solution.
After the reaction is finished, cooling the reaction solution to 0 ℃ in an ice-water bath, and adding a 4mol/L NaOH solution which is pre-cooled to 0 ℃ into the reaction solution for neutralization to obtain a neutralized solution;
then, centrifuging, dialyzing, vacuum rotary evaporation concentrating the neutralization solution, and adding 95% ethanol with 3 times volume for alcohol precipitation to obtain a purified solution;
and centrifuging, redissolving and freeze-drying the purified solution to obtain the sulfated enteromorpha polysaccharide (SDUP) with low molecular weight.
In the present invention, the Degree of Substitution (DS) for sulfate is calculated according to the formula of formula (I):
DS ═ 1.62 × S%/(32-1.02 × S%), formula (I);
in the formula (I), S% is the mass fraction of sulfur atoms.
The invention also provides the sulfated enteromorpha polysaccharide with the low molecular weight, which is prepared by the preparation method and has the weight average molecular weight of 5.99 multiplied by 104Da; the sulfate radical substitution degree is 0.81, and the sulfated enteromorpha polysaccharide with low molecular weight is prepared from the components of rhamnose: glucose: galactose: xylose: arabinose 1.09: 1.00: 0.06: 0.10: 0.11 monosaccharide composition.
The polydispersity of the sulfated enteromorpha polysaccharide with low molecular weight is 2.501, and the number average molecular weight of the sulfated enteromorpha polysaccharide with low molecular weight is 2.40 multiplied by 104Da; the Z-average molecular weight of the sulfated enteromorpha polysaccharide with low molecular weight is 1.574 multiplied by 105Da。
The invention also provides an antioxidant which is selected from the low molecular weight enteromorpha polysaccharide or the sulfated low molecular weight enteromorpha polysaccharide.
The invention adopts an enzymolysis method to obtain the low molecular weight enteromorpha polysaccharide with specific molecular weight and monosaccharide composition, and the low molecular weight enteromorpha polysaccharide has higher antioxidant activity. In addition, the low-molecular-weight enteromorpha polysaccharide is further subjected to sulfation modification on the basis of the low-molecular-weight enteromorpha polysaccharide, and the obtained sulfated low-molecular-weight enteromorpha polysaccharide also has good antioxidant activity.
For further understanding of the present invention, the low molecular weight enteromorpha polysaccharide and the preparation method thereof, the sulfated low molecular weight enteromorpha polysaccharide and the preparation method and application thereof provided by the present invention are illustrated below with reference to the following examples, and the protection scope of the present invention is not limited by the following examples.
Example 1 Enteromorpha polysaccharide extraction
Cleaning the dry Enteromorpha prolifera powder with tap water (removing salt and other impurities), and drying at 60 deg.C for use. Extracting polysaccharide by a hot water extraction method, according to the ratio of enteromorpha: adding distilled water into water (w/v): 1:30, heating and stirring at 100 ℃ for extracting for 2h, filtering by using 200-mesh bolting silk, repeatedly extracting filter residue once, combining two crude filtrates, and measuring the volume. Standing the coarse filtrate, cooling to gel, cutting into strips, and freezing in a refrigerator at-20 deg.C. And taking out the rubber strips after the rubber strips are completely frozen, breaking the rubber strips, adding 95% alcohol with the volume 1.5 times that of the crude filtrate, and standing the mixture at room temperature to freeze and dehydrate ice blocks. Filtering with 200 mesh silk, squeezing out water and alcohol by hand. And further dehydrating the filter residue by using a certain amount of 95% alcohol, and then drying at 60 ℃ to obtain the enteromorpha crude polysaccharide.
Example 2 separation and purification of Enteromorpha polysaccharide
Separating and purifying the crude polysaccharide by using a DEAE Sepharose Fast Flow weak anion exchange chromatographic column, collecting fractions by using a partial collector, and detecting the total sugar content by using a sulfuric acid-phenol method. According to the linear elution result, respectively eluting 2 column volumes with distilled water, 2 column volumes with 0.5mol/L NaCl, 3 column volumes with 1.07mol/L NaCl, 2 column volumes with 4mol/L NaCl, and washing 6 column volumes with deionized water (in order to remove redundant salt solution); column volume: 60 mL; sample loading amount: 30 mg; flow rate: 3.5 mL/min. Collecting part of the sample by a collector, detecting the total sugar content by a sulfuric acid-phenol method, and plotting the elution volume, the absorbance and the salt concentration. The results are shown in FIG. 1, and FIG. 1 is a gradient elution curve of the Enteromorpha polysaccharide DEAE Sepharose Fast Flow stage. Collecting distilled water eluate, dialyzing to remove salt, concentrating, lyophilizing, and storing in a drier.
The polysaccharide fraction obtained by anion exchange chromatography on DEAE Sepharose Fast Flow was further purified by Sepharose 6B gel column chromatography (2.6X 80 cm). The eluent was water at a flow rate of 1 mL/min. Fractions were collected using a fraction collector (10 mL/tube); the total sugar content was determined by the sulfuric acid-phenol method, and the absorbance was measured at 490nm and plotted as absorbance-elution time. The results are shown in FIG. 2, and FIG. 2 is a gel permeation chromatogram of Sepharose 6B of Enteromorpha polysaccharide. Mixing the components with high sugar content, dialyzing, desalting, lyophilizing, and storing in a drier.
Example 3 preparation of Enteromorpha polysaccharide degrading enzyme
1) And pretreatment: marking the feed Paenibacillus with the preservation number of CGMCC NO.12912 on an enteromorpha polysaccharide solid culture medium, and culturing for 5d at 28 ℃;
the solid culture medium comprises 2g/L of enteromorpha polysaccharide, 6g/L of peptone, 7g/L of yeast extract powder, 5g/L of NaCl and Na2HPO46mmol/L,MgSO4 1g/L，FePO4·7H2O0.05 g/L and agar 15 g/L.
2) And activating: inoculating 1-3 rings of the solid medium strain into a test tube of seed culture solution, and culturing at 28 ℃ and 200rpm for 12 h.
The culture solution comprises 2g/L of enteromorpha polysaccharide, 6g/L of peptone, 7g/L of yeast extract powder and 5g/L of NaCl.
3) And fermenting: inoculating the seed culture medium into a fermentation culture medium, and culturing for 24h at 28 ℃ of a shaking table to obtain a fermentation product.
The fermentation medium comprises 2g/L of enteromorpha polysaccharide, 6g/L of peptone, 7g/L of yeast extract powder, 5g/L of NaCl and Na2HPO46mmol/L,MgSO4 1g/L，FePO4·7H2O is 0.05 g/L. After the fermentation was completed, the OD of the bacterial liquid was measured600The value is 1.223, and the obtained bacterial liquid is centrifuged at 6000rpm for 20min to obtain fermentation supernatant; and (3) filtering the obtained fermented supernatant with a 0.22-micron filter membrane for sterilization, performing ultrafiltration concentration, dialyzing for desalting, and performing vacuum freeze drying at the temperature of 4 ℃ to obtain the enteromorpha polysaccharide degrading enzyme.
And (3) measuring enzyme activity: the detection method of the enzyme activity is a DNS method, and the enzyme activity is defined as follows: 1mL of enzyme solution produced 1. mu.g of reducing sugars as one activity unit for 1min, resulting in 1.03U/mL.
Example 4 preparation of Low molecular weight Enteromorpha polysaccharide
The preparation of the enteromorpha polysaccharide with low molecular weight is carried out by an enzyme hydrolysis method. Reacting the enteromorpha polysaccharide degrading enzyme prepared in the embodiment 3 with the enteromorpha polysaccharide prepared in the embodiment 2 at a mass ratio of 1:50 at 45 ℃ for 72h, boiling with boiling water for 10min to inactivate protein, centrifuging at 8000rpm for 10min, collecting supernatant, adding 95% ethanol for precipitation, and removing unreacted polysaccharide. And (3) concentrating the supernatant through rotary evaporation, dialyzing (less than or equal to 2000Da), and freeze-drying to obtain the enteromorpha polysaccharide (DUP) with low molecular weight. The infrared spectrum test of the low molecular weight enteromorpha polysaccharide is shown in figure 3, and figure 3 is an infrared spectrum chart of the low molecular weight enteromorpha polysaccharide and sulfated low molecular weight enteromorpha polysaccharide.
Example 5 sulfated Low molecular weight Enteromorpha polysaccharide
The sulfation modification adopts chlorosulfonic acid-pyridine method. Chlorosulfonic acid reacts violently with water and even explodes, so the operation process needs to avoid water contact.
1) The esterification reagent is prepared by adding 200mL anhydrous pyridine into 500mL round bottom flask, cooling to 0 deg.C in ice water bath, slowly adding 100mL chlorosulfonic acid under magnetic stirring, stirring at 95 deg.C for 30min to obtain yellowish solid, dissolving completely, sealing, and refrigerating in refrigerator for 1 week.
2) Synthesis of sulfated Enteromorpha polysaccharide with Low molecular weight 1g of Enteromorpha polysaccharide powder prepared in example 4 was suspended in 40ml of formamide, 7.5ml of an esterification reagent was added dropwise under magnetic stirring, and reacted at 95 ℃ for 3 h. And after the completion, cooling to 0 ℃ in an ice water bath, adding a 4mol/L NaOH solution which is pre-cooled to 0 ℃ to neutralize the pH value of the solution, centrifuging, dialyzing, performing vacuum rotary evaporation and concentration, adding 95% ethanol with the volume being 3 times that of the solution, centrifuging, re-dissolving, and freeze-drying to obtain the sulfated enteromorpha polysaccharide (SDUP) with low molecular weight. The infrared spectrum test of the sulfated enteromorpha polysaccharide with low molecular weight is carried out, the result is shown in figure 3, and figure 3 is an infrared spectrum chart of the enteromorpha polysaccharide with low molecular weight and the sulfated enteromorpha polysaccharide with low molecular weight.
Degree of sulfate substitution (DS) calculation: DS ═ 1.62 × S%/(32-1.02 × S%)
S%: mass fraction of sulfur atoms.
Example 6 chemical Studies
(1) The total sugar content is measured by phenol-sulfuric acid method. Drawing a standard curve: accurately preparing a 0.1mg/mL rhamnose standard solution. 0.0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4mL of standard solution was aspirated, and the final volume was adjusted to 0.5mL with water. 0.3mL of 6% phenol and 1.5mL of concentrated sulfuric acid were added, shaken, heated in a water bath at 100 ℃ for 15min, cooled and then the absorbance A was measured at 490 nm. A standard curve is plotted with rhamnose concentration (mg/mL) as abscissa and A as ordinate.
The low molecular weight Enteromorpha polysaccharide prepared in example 4 and the sulfated low molecular weight Enteromorpha polysaccharide sample prepared in example 5 were prepared into 5mg/mL solutions, respectively, and diluted to 0.1mg/mL when used. Taking 0.5mL, repeating the above standard operation, and calculating total sugar content with standard curve.
(2) The content of uronic acid is determined by sulfuric acid-carbazole method. Carbazole reagent: 0.1g of carbazole was dissolved in 90mL of 95% ethanol, and the volume was 100 mL. Sulfuric acid-borax solution: 0.9534g of borax is dissolved in 10mL of water, stirred and dissolved, 90mL of concentrated sulfuric acid is added into ice water bath, and the mixture is evenly mixed and then stands overnight for standby. Drawing a standard curve: the standard solution of the glucuronic acid with the concentration of 0.25mg/mL is accurately prepared. Respectively sucking 0, 10, 20, 30, 40, 50, 65, 70 and 80 mu L of standard solution, adding water to the final volume of 100 mu L, adding 0.6mL of sulfuric acid-borax solution and 20 mu L of carbazole, shaking up, heating at 100 ℃ for 10min, cooling, and measuring the absorbance A at 530 nm. A standard curve is drawn by taking the concentration of glucuronic acid (mg/mL) as the abscissa and the absorbance as the ordinate. And (3) determination of a sample: at a suitable concentration of sample, 100. mu.L of the sample was taken and processed as described above. Uronic acid content in the sample was calculated as a standard curve.
(3) The content of sulfate radical is measured by barium chloride-gelatin method. The sulfate radical standard solution is prepared by accurately weighing potassium sulfate which is dried to constant weight at 105 ℃, dissolving the potassium sulfate by using 1mol/L hydrochloric acid solution, and preparing the sulfate radical standard solution with the concentration of 0.6 mg/mL. 0.5% gelatin solution 2.5g gelatin is weighed, dissolved in 500mL feed water at about 70 deg.C, left to stand overnight, and stored at 4 deg.C for further use. Barium chloride-gelatin solution: 0.5g of barium chloride was weighed and dissolved in 100mL of a 0.5% gelatin solution which had been left to stand overnight, and stored at 4 ℃ for further use. Drawing a sulfate standard curve: accurately sucking 0.00 mL, 0.04 mL, 0.08 mL, 0.12 mL, 0.16 mL and 0.20mL of sulfate radical standard solution into a test tube, supplementing the test tube with 1mol/L HC1 solution to 0.20mL, respectively adding 3.8mL, 8% trichloroacetic acid and 1.0mL of barium chloride-gelatin solution into each tube, shaking uniformly, standing, reacting for 15min at room temperature, and measuring an absorbance A1 at a wavelength of 360 nm; the barium chloride-gelatin solution was replaced by a gelatin solution of the same volume of 0.5%, and the absorbance A was measured at a wavelength of 360nm2. With sulfate radical content as abscissa (A)1-A2) The absorbance of (A) is plotted on the ordinate as a standard curve. Adding 1mol/L HCl into the low molecular weight Enteromorpha prolifera polysaccharide prepared in example 4 and the sulfated low molecular weight Enteromorpha prolifera polysaccharide prepared in example 5, hydrolyzing at 100 ℃ for 6h, adding 3.8mL, 8% trichloroacetic acid and 1.0mL barium chloride-gelatin solution, shaking, standing, reacting at room temperature for 15min, and measuring the absorbance A at the wavelength of 360nm1(ii) a The barium chloride-gelatin solution was replaced with the same volume of 0.5% gelatin solution. Determination of the light absorption A at 360nm2And calculating the sulfate radical content by taking sodium sulfate as a standard substance as a standard curve.
(4) Protein content of samples of the low molecular weight enteromorpha polysaccharide prepared in example 4 and the sulfated low molecular weight enteromorpha polysaccharide prepared in example 5 were determined by coomassie brilliant blue method.
The following results are obtained in table 1:
TABLE 1 physicochemical Properties of Low molecular weight Enteromorpha polysaccharide and sulfated derivative polysaccharide thereof
Figure BDF0000012742860000151
In Table 1, Nd is not detected. The yield is the yield of the sulfated enteromorpha polysaccharide prepared from the enteromorpha polysaccharide with low molecular weight.
EXAMPLE 7 monosaccharide composition determination
The monosaccharide composition was determined by PMP pre-column derivatization high performance liquid chromatography (PMP-HPLC). Preparing PMP solution with the concentration of 0.5mol/L by using methanol; preparing a standard solution of mixed monosaccharide (L-rhamnose, D-xylose, D-glucose, D-galactose and D-arabinose) with the concentration of 10 mg/mL; the low molecular weight enteromorpha polysaccharide prepared in example 4 and the sulfated low molecular weight enteromorpha polysaccharide sample prepared in example 5 are respectively hydrolyzed completely by trifluoroacetic acid solution, and after the hydrolysate is dried by rotary evaporation at 40 ℃, CH is added repeatedly3The OH was evaporated to dryness until excess trifluoroacetic acid was removed. The sample after evaporation to dryness was dissolved in 100. mu.L of water to make a 10mg/mL sample solution. Taking 100 mu L of each of the sample and the standard monosaccharide solution, adding 120 mu L of 0.5mol/mL PMP solution and 100 mu L of 0.3mol/L NaOH solution, carrying out water bath reaction at 70 ℃ for 1h, cooling to room temperature, adding 100 mu L of hydrochloric acid solution with the concentration of 0.3mol/L for neutralization, extracting with dichloromethane for 3 times, taking supernatant, and filtering with a 0.22 mu m microporous filter membrane to obtain the derivative sample and the standard product. The chromatographic conditions are as follows: a chromatographic column: an Agilent TC-C18 chromatographic column; column temperature: 30 ℃; mobile phase: 0.02mol/ml phosphate buffer (pH6.8)/CH3CN is 83:17 (V/V). Gradient elution was (0min, 17% B;5min, 18% B; 10min, 19% B; 30min, 20% B). Elution loading: 20 mu L of the solution; flow rate: 1.0 mL/min; a detector: UV (250 nm). The monosaccharide composition and the molar ratio in the sample can be known according to the peak-out time and the peak area ratio. The following results of table 2 were obtained:
TABLE 2 monosaccharide components of low molecular weight Enteromorpha polysaccharides and sulfated derivative polysaccharides thereof
Figure BDF0000012742860000161
Example 8 molecular weight determination
The molecular weight is determined by high performance gel permeation liquid chromatography (HPGPC). The molecular weight of the polysaccharide was determined by High Performance Gel Permeation Chromatography (HPGPC). Under HPGPC conditions, the analytical column is SB-806HQ and SB-804HQ series column; the mobile phase is 0.1mol/L NaNO3(ii) a The flow rate is 1 mL/min; the column temperature was 25 ℃; the sample injection amount is 20 mu L; and detecting by a difference detector. Injecting 20 mu L of sample respectively, and drawing a standard curve by GPC software according to different peak emergence times of 6 standard samples with different molecular weights. And (3) measuring the sample, namely precisely weighing the low-molecular-weight enteromorpha polysaccharide prepared in the example 4 and the sulfated low-molecular-weight enteromorpha polysaccharide sample prepared in the example 5 respectively, adding a mobile phase to dissolve the samples to prepare a 5% solution, centrifuging to obtain a supernatant, injecting 20 mu L of the supernatant, and using GPC software to correspond to a standard curve according to the peak-out time. Calculating the peak molecular weight of the sample, and calculating the molecular weight distribution width coefficient (D) according to the peak width. The following results of table 3 were obtained:
TABLE 3 molecular weights and polydispersity indices of low molecular weight Enteromorpha polysaccharide and sulfated derivative polysaccharide thereof
Figure BDF0000012742860000162
Example 9 superoxide radical scavenging Activity assay
The activity experiment for scavenging superoxide radicals of the low molecular weight Enteromorpha polysaccharide prepared in example 4 and the sulfated low molecular weight Enteromorpha polysaccharide prepared in example 5 adopts NADH-PMS-NBT method.
In particular toThe operation is as follows: samples (0.01-0.5mg/mL) were prepared at various concentrations using Tris-HCl buffer (pH8.0). 0.1ml of each sample was taken and 1ml of 557. mu.M NADH (in 16mM Tris-HCl, pH8.0), 1ml of 45. mu.M PMS (in 16mM Tris-HCl, pH8.0), 1ml of 108. mu.M NBT (in 16mM Tris-HCl, pH8.0) was added. Reacting at room temperature for 5min, and measuring the absorbance at 560nm to obtain A1；A0All reagents but no sample; a. the2Samples and reagents but replacing NBT with Tris-HCl.
Superoxide radical clearance calculation formula:
superoxide radical clearance (%) - [1- (a)1-A2)/A0]×100％
The results are shown in fig. 4, and fig. 4 is a graph of superoxide radical scavenging activity of low molecular weight enteromorpha polysaccharides and sulfated low molecular weight enteromorpha polysaccharides. As can be seen from FIG. 4, when the sample concentration is 2mg/mL, the superoxide radical scavenging activities of the low molecular weight enteromorpha polysaccharide and the sulfated low molecular weight enteromorpha polysaccharide are 72.48% and 93.06%, respectively, and the clearance of Vc at the same concentration is 82.95%. The clearance rate of superoxide radical is obviously enhanced and is higher than Vc with the same concentration after the enteromorpha polysaccharide is sulfated and modified.
Example 10 hydroxyl radical scavenging Activity experiment
Experiment on hydroxyl radical scavenging activity of low molecular weight Enteromorpha polysaccharide prepared in example 4 and sulfated low molecular weight Enteromorpha polysaccharide prepared in example 5
Preparing polysaccharide samples (0.25-10.0mg/mL) with different concentrations, each sample 2mL, adding FeSO 5mmol/L4(2mL), 5mmol/L ethanolic salicylate solution (2 mL); then 2ml of 5mmol/LH is added2O2. Water bath at 37 deg.C for 30min, cooling to room temperature, and measuring absorbance at 510nm as A1Ascorbic acid was used as a positive control. A. the0Is a reagent (no sample); a. the2Is a free hydroxyl radical.
Hydroxyl radical clearance calculation formula:
hydroxyl radical clearance rate ═ 1- (A)1-A2)/A0]×100％
The results are shown in fig. 5, fig. 5 for hydroxyl radical scavenging activity of low molecular weight enteromorpha polysaccharide and sulfated low molecular weight enteromorpha polysaccharide. As can be seen from fig. 5, when the sample concentration is lower than 4mg/mL, the clearance rate of hydroxyl radicals of the low molecular weight enteromorpha polysaccharide and the sulfated low molecular weight enteromorpha polysaccharide is significantly higher than Vc; when the concentration of the sample is higher than 4mg/mL, the clearance rate of hydroxyl radicals of the low-molecular-weight enteromorpha polysaccharide and the sulfated low-molecular-weight enteromorpha polysaccharide is lower than Vc; the clearance rates of hydroxyl radicals of the low-molecular-weight enteromorpha polysaccharide, the sulfated low-molecular-weight enteromorpha polysaccharide and Vc are 54.29%, 73.21% and 100% respectively at the concentration of 10 mg/mL. The hydroxyl radical clearance rate of the low molecular weight enteromorpha polysaccharide and the sulfated low molecular weight enteromorpha polysaccharide is higher than Vc under low concentration; and at high concentration, the clearance rate of hydroxyl radicals of the enteromorpha polysaccharide after sulfation modification is lower than that of Vc with the same concentration, but is higher than that of the hydroxyl radicals of the unmodified enteromorpha polysaccharide.
EXAMPLE 11DPPH radical scavenging experiment
Prepare 0.2mmol/L DPPH ethanol solution for use. Polysaccharide concentrations (0.25-10mg/mL) were prepared at different concentrations. 1ml of sample was taken and 2ml of DPPH and 2ml of 95% ethanol were added. Mixing the reaction solution vigorously, placing in a dark room for reaction for 30min, and measuring the light absorption value at 517nm as A1。A0The reaction solution contained 2ml of DPPH and 3ml of ethanol. A. the21ml and 4ml of 95% ethanol. DPPH free radical clearance rate calculation formula of polysaccharide:
DPPH radical scavenging ratio (%) - [1- (A)1-A2)/A0]×100％
The results are shown in fig. 6, fig. 6 is a graph of DPPH free radical scavenging activity of low molecular weight enteromorpha polysaccharides and sulfated low molecular weight enteromorpha polysaccharides. As can be seen from fig. 6, DPPH radical clearance of low molecular weight enteromorpha polysaccharide and sulfated low molecular weight enteromorpha polysaccharide is lower than Vc; but when the concentration of the sample is 10mg/mL, the DPPH free radical clearance rate of the sulfated enteromorpha polysaccharide with low molecular weight is close to Vc; the DPPH free radical clearance rates of the enteromorpha polysaccharide with low molecular weight, the sulfated enteromorpha polysaccharide with low molecular weight and Vc are respectively 43.67%, 93.26% and 98.05% under the condition that the concentration is 10 mg/mL. The DPPH free radical clearance rate is obviously enhanced after the enteromorpha polysaccharide is modified by sulfation.
Example 12 reduction force measurement
Samples (0.25-10.0mg/mL) were prepared at different concentrations using 0.2mmol/mL phosphate buffer (pH 6.6). The specific method is that 1.0mL of sample is taken, 1mL (1%, w/v) of potassium ferricyanide is added, and the temperature bath is carried out for 20min at 50 ℃. The reaction was then stopped by the addition of 2.0ml of trichloroacetic acid (10%, w/v). Then, 1.2ml of ferric chloride (0.1%, w/v) was added to the reaction solution, and the absorbance at 700nm was measured. The larger the absorbance, the stronger the reducing power. The results are shown in fig. 7, fig. 7 is the reducing power of low molecular weight enteromorpha polysaccharide and sulfated low molecular weight enteromorpha polysaccharide. As can be seen from fig. 7, the reducing capacities of the low molecular weight enteromorpha polysaccharide, the sulfated low molecular weight enteromorpha polysaccharide and Vc at a concentration of 10mg/mL are 0.9713, 2.0449 and 3.8013, respectively; the reducing power of the sulfated enteromorpha polysaccharide with low molecular weight under the same concentration is not as high as Vc, but is obviously higher than that of the enteromorpha polysaccharide without sulfation modification and with low molecular weight. The sulfated and modified enteromorpha polysaccharide with low molecular weight can obviously enhance the reducing capability.
Example 13 determination of Metal-chelating ability
The metal chelating ability of polysaccharides was determined with slight modification of the literature references. Samples (0.2-10mg/mL) were prepared at different concentrations, and 2mM FeCl was added2(0.1mL) and 5mM phenanthroline (0.4mL), and the mixture is mixed uniformly and placed at room temperature for 10 min. EDTA as a positive control, absorbance was measured at 562 nm. The metal ion chelating capacity calculation formula is as follows:
metal chelating ability (%). 100%. times (A)0-A)/A0
A0The absorbance value of no sample is shown, and A is the absorbance value of the sample mixed liquid.
The results are shown in fig. 8, fig. 8 is a graph of the metal chelating capacity of low molecular weight enteromorpha polysaccharides and sulfated low molecular weight enteromorpha polysaccharides. As can be seen from FIG. 8, the metal ion chelating capacities of the low molecular weight Enteromorpha polysaccharide, the sulfated low molecular weight Enteromorpha polysaccharide and EDTA at a concentration of 10mg/mL are 97.69%, 66.78% and 99.70%, respectively; the metal ion chelating ability of the low molecular weight enteromorpha polysaccharide under the same concentration is obviously higher than that of the sulfation modified low molecular weight enteromorpha polysaccharide and is close to that of EDTA. The metal ion chelating capacity of the sulfated and modified low-molecular-weight enteromorpha polysaccharide is reduced.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (8)

1. The preparation method of the enteromorpha polysaccharide with low molecular weight is characterized by comprising the following steps:
A) mixing the purified enteromorpha polysaccharide with enteromorpha polysaccharide degrading enzyme prepared by fermenting feed bacillus with the preservation number of CGMCC NO.12912, performing enzymolysis reaction, and then sequentially performing enzyme deactivation and centrifugation to obtain supernatant;
B) precipitating the supernatant with ethanol, centrifuging to obtain a low molecular weight enteromorpha polysaccharide solution, and concentrating, dialyzing and freeze-drying the low molecular weight enteromorpha polysaccharide solution to obtain low molecular weight enteromorpha polysaccharide;
the weight average molecular weight of the low molecular weight enteromorpha polysaccharide is 4.48 multiplied by 104Da; the low molecular weight enteromorpha polysaccharide is prepared from rhamnose: glucose: galactose: xylose: arabinose 1.65: 1.00: 0.09: 0.57: 0.17 monosaccharide composition.
2. The preparation method according to claim 1, wherein the low molecular weight Enteromorpha polysaccharide has a polydispersity of 1.648 and a number average molecular weight of 2.72 x 104Da; the Z-average molecular weight of the low-molecular-weight enteromorpha polysaccharide is 2.326 multiplied by 105 Da。
3. The preparation method according to claim 1, wherein the enteromorpha polysaccharide-degrading enzyme is prepared according to the following method:
step 1: activating feed paenibacillus with a preservation number of CGMCC NO.12912 to prepare seed liquid;
step 2: inoculating the seed liquid into a culture medium, and fermenting to obtain a fermentation product;
and step 3: centrifuging the fermented product, taking supernatant, sterilizing, concentrating, desalting, and drying to obtain the Enteromorpha polysaccharide degrading enzyme.
4. The preparation method according to claim 1, wherein the mass ratio of the purified enteromorpha polysaccharide to the enteromorpha polysaccharide-degrading enzyme is 50: 1.
5. A preparation method of sulfated enteromorpha polysaccharide with low molecular weight is characterized by comprising the following steps:
a) dispersing low-molecular-weight enteromorpha polysaccharide into an organic solvent, and adding an esterification reagent for reaction to obtain a reaction solution, wherein the low-molecular-weight enteromorpha polysaccharide is prepared by the preparation method according to any one of claims 1 to 4; the esterification reagent is prepared according to the following method: mixing anhydrous pyridine and chlorosulfonic acid in a volume ratio of 2:1, and heating and stirring to obtain an esterification reagent;
b) adding alkali liquor into the reaction liquid for neutralization to obtain a neutralized liquid;
c) sequentially centrifuging, dialyzing, concentrating and precipitating the neutralized solution with ethanol to obtain a purified solution;
d) and sequentially carrying out centrifugation, redissolution and freeze drying on the purified solution to obtain the sulfated enteromorpha polysaccharide with low molecular weight.
6. A sulfated low molecular weight Enteromorpha polysaccharide prepared by the preparation method of claim 5, wherein the sulfated low molecular weight Enteromorpha polysaccharide has a weight average molecular weight of 5.99 x 104 Da; the sulfate radical substitution degree is 0.81, and the sulfated enteromorpha polysaccharide with low molecular weight is prepared from the components of rhamnose: glucose: galactose: xylose: arabinose 1.09: 1.00: 0.06: 0.10: 0.11 monosaccharide composition.
7. The sulfated low molecular weight Enteromorpha polysaccharide of claim 6, wherein the sulfated low molecular weight Enteromorpha polysaccharide is characterized in that the sulfated low molecular weight Enteromorpha polysaccharideThe polydispersity number of the Enteromorpha polysaccharide is 2.501, and the number average molecular weight of the sulfated Enteromorpha polysaccharide with low molecular weight is 2.40 multiplied by 104Da; the Z-average molecular weight of the sulfated enteromorpha polysaccharide with low molecular weight is 1.574 multiplied by 105 Da。
8. An antioxidant, characterized by comprising the low molecular weight Enteromorpha polysaccharide prepared by the preparation method of any of claims 1 to 4, or the sulfated low molecular weight Enteromorpha polysaccharide prepared by the preparation method of claim 5 or the sulfated low molecular weight Enteromorpha polysaccharide of claim 6 or 7.
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CN109666604B (en) * 2018-12-24 2022-05-06 光明乳业股份有限公司 Highly concentrated lactobacillus plantarum proliferation agent and preparation method and use method thereof
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103352016A (en) * 2013-06-27 2013-10-16 中国海洋大学 Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha
CN103451119A (en) * 2012-06-03 2013-12-18 中国海洋大学 Alteromonas and method thereby for producing gel-type enteromorpha polysaccharide degrading enzyme by using Alteromonas
CN103951761A (en) * 2014-05-13 2014-07-30 浙江工商大学 Method for degrading enteromorpha prolifera polysaccharides by enzymic method
CN104560774A (en) * 2014-06-30 2015-04-29 中国海洋大学 Method for preparing block oligosaccharide containing rich rhamnose sulfate from Enteromorpha polysaccharide
CN105399854A (en) * 2015-12-17 2016-03-16 浙江工商大学 Method used for improving antioxidant activity and antibacterial activity enteromorpha polysaccharide at same time

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8426185B2 (en) * 2008-01-31 2013-04-23 Battelle Energy Alliance, Llc Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103451119A (en) * 2012-06-03 2013-12-18 中国海洋大学 Alteromonas and method thereby for producing gel-type enteromorpha polysaccharide degrading enzyme by using Alteromonas
CN103352016A (en) * 2013-06-27 2013-10-16 中国海洋大学 Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha
CN103951761A (en) * 2014-05-13 2014-07-30 浙江工商大学 Method for degrading enteromorpha prolifera polysaccharides by enzymic method
CN104560774A (en) * 2014-06-30 2015-04-29 中国海洋大学 Method for preparing block oligosaccharide containing rich rhamnose sulfate from Enteromorpha polysaccharide
CN105399854A (en) * 2015-12-17 2016-03-16 浙江工商大学 Method used for improving antioxidant activity and antibacterial activity enteromorpha polysaccharide at same time

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Alteromonas sp. A321产浒苔多糖降解酶及酶学性质研究";李银平;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20150115(第01期);第B018-73页 *
"不同取代度的硫酸化肠浒苔多糖抗氧化活性研究";刘玉凤等;《食品工业科技》;20160624(第19期);第142-147、152页 *

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