CN109628524B

CN109628524B - Method for enhancing biological activity of sargassum fusiforme polysaccharide

Info

Publication number: CN109628524B
Application number: CN201910026070.7A
Authority: CN
Inventors: 周涛; 杨斯淇
Original assignee: Zhejiang Gongshang University
Current assignee: Zhejiang Gongshang University
Priority date: 2019-01-11
Filing date: 2019-01-11
Publication date: 2021-11-05
Anticipated expiration: 2039-01-11
Also published as: CN109628524A

Abstract

The invention discloses a method for enhancing biological activity of sargassum fusiforme polysaccharide, which comprises the following steps: carrying out degradation reaction on crude sargassum fusiforme polysaccharide in a buffer solution with the pH value of 4.5-7.0 under the action of a complex enzyme consisting of pectinase and glucoamylase; removing enzyme from the obtained reaction solution, filtering, concentrating the obtained filtrate, dialyzing with a dialysis bag with molecular weight cutoff of 3500Da, concentrating the obtained dialysate, and freeze-drying to obtain the sargassum fusiforme compound zymolytic polysaccharide ESFP. The method of the invention can not only obviously improve the antioxidant activity of the sargassum fusiforme polysaccharide and the binding capacity of cholic acid, but also enhance the immunoregulation activity of the sargassum fusiforme polysaccharide on macrophages.

Description

Method for enhancing biological activity of sargassum fusiforme polysaccharide

Technical Field

The invention belongs to the technical field of chemistry and chemical engineering, and relates to a method for enhancing biological activity of sargassum fusiforme polysaccharide by carrying out enzymolysis on the sargassum fusiforme polysaccharide.

Background

The Cyrtymenia Sparsa contains various bioactive components with health benefits, especially acidic polysaccharide, and has antiviral, antifungal, antibacterial, antioxidant, antiinflammatory, blood lipid reducing and antitumor effects. Since the sargassum fusiforme is low in price and widely distributed, the sargassum fusiforme has good research prospect when being processed into functional food or developed into a medicine with low side effect. The content of carbohydrate in the sargassum fusiforme is high, but the bioactivity of crude polysaccharide in the sargassum fusiforme is relatively low. The biological activity of the polysaccharide is closely related to the molecular structure of the polysaccharide, and comprises glycosidic bonds, monosaccharide composition, sulfate group content, molecular weight and the like. High molecular weight polysaccharides may prevent their penetration into cells to perform a given function due to lower water solubility, while lower molecular weight sulfated polysaccharides show higher biological activity. Because the functional characteristics of polysaccharides obtained by degrading active polysaccharides by different methods are different, it is very important to select proper extraction and degradation methods to improve various biological activities of the polysaccharides to a greater extent. At present, most of the existing methods for degrading sargassum fusiforme polysaccharide are oxidation degradation method and acid degradation method, and the method for degrading sargassum fusiforme polysaccharide by adopting enzyme method is rarely reported.

Disclosure of Invention

The invention aims to solve the technical problem of providing a novel method for improving the biological activity of sargassum fusiforme for degrading polysaccharide.

In order to solve the technical problems, the method for enhancing the biological activity of the sargassum fusiforme polysaccharide (the method for carrying out compound enzyme degradation on sargassum fusiforme crude polysaccharide) comprises the following steps:

1) and (3) degradation:

firstly, dissolving crude Sargassum Fusiforme Polysaccharide (SFP) in a buffer solution with the pH value of 4.5-7.0, then adding a complex enzyme consisting of pectinase and glucoamylase, and then adding the buffer solution for dilution, wherein the concentration of the crude Sargassum Fusiforme Polysaccharide (SFP) in the obtained system is 2-5 mg/mL, and the total concentration of the complex enzyme is 20-80U/mL; the pectase is: the activity ratio of the saccharifying enzyme is 6: 0-0: 6;

carrying out oscillation reaction on the system at the temperature of 40-65 ℃ for 1-5 h, thereby realizing the degradation of crude polysaccharide of the sargassum fusiforme;

2) removing enzyme (high-temperature enzyme removal) from the reaction solution obtained in the step 1), and then filtering, and concentrating the obtained filtrate to 20-30% of the original volume (namely, about 1/4); dialyzing for 24-48 h by using a dialysis bag with the molecular weight cutoff of 3500Da, concentrating the obtained dialysate (the solution remained in the dialysis bag), and freeze-drying at (-50-60 ℃ for concentration and drying to constant weight) to obtain the sargassum fusiforme compound enzymolysis polysaccharide (ESFP).

As an improvement of the method for enhancing the biological activity of sargassum fusiforme polysaccharide of the invention:

the concentration of crude polysaccharide (SFP) of the sargassum fusiforme in the obtained system is 4.0mg/mL, and the total concentration of complex enzyme is 68.4U/mL; the pectase is: the activity ratio of the saccharifying enzyme is 3.3:1, and the pH value of the buffer solution is 6.0;

oscillating and reacting for 3 hours at the temperature of 54.2 ℃; the sargassum fusiforme compound zymolytic polysaccharide ESFP1-1 is obtained.

The molecular weight of ESFP1-1 is 86.8 kDa. Compared with undegraded Sargassum Fusiforme Polysaccharide (SFP), the oxidative activity, cholic acid binding activity and immunoregulation effect of the polysaccharide are obviously improved.

As a further improvement of the method for enhancing the biological activity of sargassum fusiforme polysaccharide of the invention:

the buffer solution with the pH value of 4.5-7.0 is acetic acid-sodium acetate buffer solution with the pH value of 4.5-7.0, and citric acid-sodium citrate buffer solution.

As a further improvement of the method for enhancing the biological activity of sargassum fusiforme polysaccharide of the invention: separating and purifying the sargassum fusiforme compound zymolytic polysaccharide ESFP 1-1; the method comprises the following steps:

roughly dividing:

selecting Cellulose DEAE-52 chromatographic column (specification is 60cm x phi 2.6cm, effective length is 50 cm);

weighing 400 + -40 mg of sargassum fusiforme composite zymolytic polysaccharide ESFP1-1, dissolving in 7mL of deionized water, centrifuging to obtain supernatant, uniformly adding the supernatant to the upper surface of a chromatographic column filler, eluting with deionized water and NaCl solutions with the concentrations of 0.1mol/L, 0.3mol/L, 0.5mol/L and 0.8mol/L in sequence (when detecting that an eluent corresponding to the eluent does not contain polysaccharide, the eluent is replaced by another eluent for elution, and the ultraviolet absorption value at 490nm can be detected by adopting a sulfuric acid-phenol method separation tube), wherein the flow rate is 1mL/min,

respectively collecting 4 eluents corresponding to the 4 eluents of NaCl solution of 0.1mol/L, 0.3mol/L, 0.5mol/L and 0.8mol/L, concentrating under reduced pressure, dialyzing, desalting, and freeze-drying to obtain four primarily purified polysaccharide components of DEAE-52 of the sargassum fusiforme composite enzymolysis polysaccharide;

secondly, subdivision:

selecting Sephadex G-100 gel chromatographic column (the specification of the chromatographic column is 60cm multiplied by phi 1.6cm, and the effective volume of the column is 50cm multiplied by phi 1.6 cm);

the four primarily purified polysaccharide fractions, which were roughly separated by Cellulose DEAE-52, were subjected to the following procedures, respectively:

weighing 100 +/-10 mg of primarily purified polysaccharide components, dissolving in 3mL of deionized water, centrifuging to obtain a supernatant, adding the supernatant into a Sephadex G-100 gel chromatographic column, eluting with deionized water at the flow rate of 0.20mL/min, combining polysaccharide eluates (ultraviolet absorption value at 490nm can be detected tube by using a sulfuric acid-phenol method, drawing an elution curve of the Sephadex G-100 chromatographic column, generally 20min per tube), concentrating under reduced pressure, and freeze-drying to obtain four purified components (purified polysaccharides) of the sargassum fusiforme composite enzymatic hydrolysis polysaccharide, which are respectively named as ESFP1, ESFP2, ESFP3 and ESFP 4.

Their molecular weights were 75.5, 86.3, 76.2 and 86.5kDa, respectively.

The invention also provides the application of the degraded sargassum fusiforme polysaccharide and the purified polysaccharide obtained by the method in the preparation of functional food base materials.

The invention adopts Cellulose DEAE-52 column chromatography and Sephadex G-100 gel chromatography to separate and purify sargassum fusiforme zymolytic polysaccharide (ESFP) so as to obtain a purified polysaccharide component with better activity.

The composition and molecular weight data of the obtained sargassum fusiforme zymolytic polysaccharide and sargassum fusiforme crude polysaccharide (SFP) used as a raw material are shown in Table 1.

The invention has the following technical effects:

1. compared with undegraded crude Sargassum Fusiforme Polysaccharide (SFP), the in-vitro antioxidant activity, cholic acid binding activity and immunological activity of RAW264.7 macrophages (which have obvious promotion effect on proliferation, phagocytosis and NO molecule release of macrophages) of the sargassum fusiforme zymolytic polysaccharide (ESFP) are obviously improved.

The invention adopts a compound enzyme method of pectinase and glucoamylase to degrade the sargassum fusiforme polysaccharide for the first time; the enzymatic degradation has the advantages of mild action conditions, small influence on substituents such as sulfate groups on the polysaccharide and the like.

2. Four separated and purified components (ESFP1, ESFP2, ESFP3 and ESFP4) obtained by separating and purifying sargassum fusiforme degraded polysaccharide (ESPY) have better antioxidant activity and immunocompetence of RAW264.7 macrophage.

In conclusion, the method of the invention can not only obviously improve the antioxidant activity of the sargassum fusiforme polysaccharide and the binding capacity of cholic acid (combined with the bile acid, can accelerate the decomposition of cholesterol in vivo and effectively reduce the content of cholesterol in serum and liver of a human body so as to achieve the effect of reducing blood fat), but also enhance the immunoregulation activity of the sargassum fusiforme polysaccharide on macrophages.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a result of determination of DPPH radical scavenging ability of complex Enzymatic Sargassum Fusiforme Polysaccharide (ESFP) and sargassum fusiforme crude polysaccharide (SFP) obtained in example 1-1 of the present invention.

FIG. 2 is a result of measurement of superoxide anion radical scavenging ability of complex enzymatic degraded Hizikia fusiforme polysaccharide (ESFP) and crude Hizikia fusiforme polysaccharide (SFP) obtained in example 1-1 of the present invention.

FIG. 3 shows the measurement results of the hydroxyl radical scavenging ability of the complex enzymatic hydrolyzed Hizikia fusiforme polysaccharide (ESFP) and crude Hizikia fusiforme polysaccharide (SFP) obtained in example 1-1 of the present invention.

FIG. 4 shows the results of measurement of the reducing power of the complex enzymatic hydrolyzed Hizikia fusiforme polysaccharide (ESFP) and crude Hizikia fusiforme polysaccharide (SFP) obtained in example 1-1 of the present invention.

FIG. 5 shows the effect of each of the isolated and purified fractions ESFP1, ESFP2, ESFP3, ESFP4 and crude Hizikia fusiforme polysaccharide (SFP) on the proliferation of RAW264.7 macrophages, which are obtained from example 2-1 of the present invention.

FIG. 6 shows the effect of each of the isolated and purified fractions ESFP1, ESFP2, ESFP3, ESFP4 and crude Hizikia fusiforme polysaccharide (SFP) on the phagocytic activity of RAW264.7 cells of the complex enzymatically hydrolyzed Hizikia fusiforme polysaccharide (ESFP) obtained in example 2-1 of the present invention.

FIG. 7 is a graph showing the effect of each of the isolated and purified fractions ESFP1, ESFP2, ESFP3, ESFP4 and Hizikia fusiforme crude polysaccharide (SFP) of the complex enzymatic hydrolyzed Hizikia fusiforme polysaccharide (ESFP) obtained in example 2-1 of the present invention on NO releasing activity of RAW264.7 cells.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1-1:

1g of crude Hizikia fusiforme polysaccharide (SFP) was weighed out and dissolved in 100mL of a citric acid-sodium citrate buffer solution (pH 6.0), pectinase (3 ten thousand U/g, 437.4mg) and glucoamylase (10 ten thousand U/mL, 39.8. mu.L) (activity ratio 3.3:1) were added, and then the solution was diluted to 250mL with the same acetic acid-sodium acetate buffer solution so that the final polysaccharide concentration in the resulting system was 4mg/mL and the final enzyme concentration was 68.4U/mL, and the system was reacted for 3 hours with shaking (frequency 220r/min) in a water bath at 54.2 ℃. Heating in water bath at the temperature of more than 90 ℃ for 15 minutes to inactivate enzyme, filtering, concentrating the clear liquid (filtrate) to 1/4 with the original volume by rotary evaporation (60-70 ℃), dialyzing for 48 hours by using a dialysis bag with the molecular weight cutoff of 3500Da, concentrating the dialysate (the solution left in the dialysis bag) by rotary evaporation (60-70 ℃) to 25% of the volume of the dialysate), freezing (50-60 ℃) and drying for 24 hours to obtain 921mg of the sheet sargassum fusiforme enzymolysis polysaccharide (ESFP1-1), wherein the degradation rate of the polysaccharide obtained under the condition is 18.6%.

The degradation rate (reducing sugar content in degraded polysaccharide-reducing sugar content in undegraded crude polysaccharide)/(total sugar content-reducing sugar content in undegraded crude polysaccharide) × 100%.

The composition and molecular weight of ESFP1-1 are shown in Table 1.

TABLE 1 comparison of the composition and molecular weight of the degraded polysaccharide ESFP1-1 and the crude polysaccharide

Note that Fuc, Gal, Man, Glc, Xyl and stand for fucose, galactose, mannose, glucose and xylose, respectively.

Examples 1 to 2:

the shaking reaction time was changed to 4h, and the remainder was the same as in example 1-1, to obtain 882mg of Hizikia fusiforme zymolytic polysaccharide (ESFP1-2), the degradation rate of the polysaccharide obtained under this condition was 18.7%.

Examples 1 to 3:

changing the oscillation reaction time to 1h, wherein the activity ratio of pectinase to glucoamylase is 3:1, and the balance is equal to that in example 1-1, 913mg of sargassum fusiforme zymolytic polysaccharide (ESFP1-3) is obtained, and the degradation rate of the polysaccharide obtained under the condition is 13.8%.

Examples 1 to 4:

changing the oscillation reaction time to 1h, the reaction temperature to 50 ℃, the final concentration of the complex enzyme to 70U/mL, the activity ratio of the pectinase to the glucoamylase to be 2:1, and the balance being equal to the example 1-1, obtaining 904mg of sargassum fusiforme zymolytic polysaccharide (ESFP1-4), wherein the degradation rate of the obtained polysaccharide under the condition is 9.4%.

Examples 1 to 5:

changing the oscillation reaction time to 1h, the reaction temperature is 60 ℃, the final concentration of the complex enzyme is 80U/mL, the rest is the same as that of the example 1-1, 896mg of the sargassum fusiforme zymolytic polysaccharide (ESFP1-5) is obtained, and the degradation rate of the polysaccharide obtained under the condition is 8.9%.

Comparative example 1, the activity ratio of pectinase to saccharifying enzyme in example 1-1 is changed from 3:1 to 4: 1; the final enzyme concentration is unchanged; the rest was equivalent to example 1-1. 878mg of sargassum fusiforme enzymolysis polysaccharide (ESFP-1D) is obtained, and the degradation rate of the polysaccharide obtained under the condition is 17.5%.

Comparative example 2, the activity ratio of pectinase to saccharifying enzyme in example 1-1 is changed from 3:1 to 2: 1; the final enzyme concentration is unchanged; the rest was equivalent to example 1-1. 892mg of sargassum fusiforme enzymolysis polysaccharide (ESFP-2D) is obtained, and the degradation rate of the polysaccharide obtained under the condition is 15.9%.

Example 2-1,

400mg of the sargassum fusiforme compound zymolytic polysaccharide (ESFP1-1) obtained in the example 1-1 is dissolved in 7mL of deionized water and centrifuged (at the rotating speed of 6000r/min for 10 minutes) to obtain supernatant, the supernatant is uniformly added on the upper surface of a chromatographic column packing, and is eluted by 5 eluents of deionized water, 0.1mol/L, 0.3mol/L, 0.5mol/L and 0.8mol/L NaCl solution respectively through DEAE Cellulose-52 (the specification of the column is 60cm multiplied by phi 2.6cm, and the effective length is 50cm), and the flow rate is controlled to be 1 mL/min; collecting each tube for 11min with automatic collector; detecting the ultraviolet absorption value of the eluent at 490nm by a sulfuric acid-phenol method separation tube to determine whether the eluent contains polysaccharide, and when detecting that the eluent corresponding to the eluent does not contain polysaccharide, eluting with the next eluent; the eluent (containing sugar, the volume of which is 770, 440, 330 and 264mL) corresponding to the 4 eluents of NaCl solution of 0.1mol/L, 0.3mol/L, 0.5mol/L and 0.8mol/L is respectively collected. The above 4 eluents were treated as follows: concentrating to 25% of the original volume by using a rotary evaporator (60-70 ℃), dialyzing and desalting (dialyzing for 36h by using a dialysis bag with the molecular weight cutoff of 1000 Da), and freeze-drying the dialyzate (the solution left in the dialysis bag) (drying to constant weight at the temperature of-60 ℃), so as to correspondingly obtain four separated components (four primarily purified polysaccharide components) of the sargassum fusiforme zymolytic polysaccharide.

Remarking: since the eluate (330mL) obtained by elution with deionized water had a lower polysaccharide content, no further separation was performed.

And respectively carrying out the following operations on the four separated components: further purifying with SephadexG-100 column (column size 60 cm. times. phi.1.6 cm, effective length 50cm), eluting with deionized water, and purifying to obtain four components ESFP1, ESFP2, ESFP3 and ESFP 4.

The method comprises the following specific steps:

100mg of the separated component (polysaccharide component of the primary purification) is dissolved in 3mL of deionized water, centrifuged (at the rotating speed of 6000r/min for 12 minutes) to obtain supernatant, the supernatant is loaded into a Sephadex G-100 gel chromatographic column and eluted by 200mL of deionized water, the flow rate is 0.20mL/min, and one tube is collected every 20 min. The absorbance at 490nm was measured tube by tube using the sulfuric acid-phenol method and the Sephadex G-100 column elution curve was plotted (for confirmation of the number of tubes collected and the purity of the sample, indicating only one component if a single peak). And combining the polysaccharide eluates, performing rotary evaporation and concentration (concentrating to 25% of the original volume), and performing freeze drying (-60 ℃ for drying to constant weight), thereby respectively obtaining each purified component of the sargassum fusiforme compound zymolytic polysaccharide, which is named as ESFP1, ESFP2, ESFP3 and ESFP 4. Their molecular weights were 75.5, 86.3, 76.2 and 86.5kDa, respectively. The composition and molecular weight are shown in Table 2.

TABLE 2 composition and molecular weight of the four purified polysaccharide fractions

Experiment 1,

Adopts literature (separation and purification of Lujianghong, laminarin, structural characteristics and application research in cigarette [ D)]University of southern China, 2013.) reported a method of testing DPPH radical scavenging ability of the complex enzymolyzed polysaccharide of hizikia fusiforme obtained in example 1-1 and crude polysaccharide of hizikia fusiforme, and comparing them. As shown in FIG. 1, DPPH free radical is generated after crude polysaccharide of Cyrtymenia Sparsa is degraded by complex enzymeThe cleaning ability is significantly improved. IC of SFP and ESFP₅₀0.768mg/mL and 0.53mg/mL, respectively. The clearance of ESFP at a concentration of 5mg/mL is equivalent to the clearance of Vc at 0.1 mg/mL. However, the DPPH radical scavenging activity of ESFP is still less than that of vitamin C.

Experiment 2,

The literature (Marklund S, Marklund G. Involution of the superoxide reaction in the oxidation of pyromalol and a genetic analysis for superoxide dispersion [ J ] is used]European Journal of biochemistry, 1974,47: 469-474) reported a method of testing the superoxide anion radical scavenging ability of the complex enzymatically hydrolyzed polysaccharides of Hizikia fusiforme obtained in example 1-1 and crude Hizikia fusiforme polysaccharides and comparing them. As can be seen from FIG. 2, the scavenging ability of the sargassum fusiforme compound zymolytic polysaccharide to superoxide anion free radicals is obviously higher than that of sargassum fusiforme crude polysaccharide. When the concentration is 5mg/mL, the clearance rates of SFP and ESFP are 73.9 percent and 85.12 percent respectively, and the IC of SFP and ESFP₅₀Respectively 0.723mg/mL and 0.412 mg/mL, which shows that the enzymatic hydrolysis process improves the superoxide anion radical scavenging capacity of the sargassum fusiforme polysaccharide.

Experiment 3,

The complex enzyme-hydrolyzed polysaccharide of Hizikia fusiforme obtained in example 1-1 and crude polysaccharide of Hizikia fusiforme were tested for their ability to scavenge hydroxyl radicals and compared by the method reported in the literature (YANG W F, WANG Y, LI X P, et al. As can be seen from FIG. 3, the hydroxyl radical scavenging ability of the sargassum fusiforme complex zymolytic polysaccharide is higher than that of sargassum fusiforme crude polysaccharide. When the concentration of SFP and ESFP is 5mg/mL, the clearance rate of hydroxyl free radical is respectively as follows: 39.15% and 50.1%. Wherein the ESFP clearance corresponds to the clearance of Vc of 0.02 mg/mL.

Experiment 4,

The complex enzyme-hydrolyzed polysaccharides of Hizikia fusiforme obtained in example 1-1 were tested for reducing power and compared with crude Hizikia fusiforme polysaccharides by a literature method (Yen G, chemical H.antibiotic activity of vacuum tea extracts in relation to the anti-inflammatory Chemistry [ J ]. Journal of Agricultural and Food Chemistry,1995,43: 27-32.). As can be seen from FIG. 4, when the concentrations of the two polysaccharides are the same, the absorbance of ESFP is significantly higher than that of SFP, which indicates that compared with the polysaccharide before enzymolysis, the reduction capability of the polysaccharide ESFP after enzymolysis to ferric iron is significantly improved, and the polysaccharide has stronger reduction capability in the polysaccharides under the premise of taking Vc as positive control.

Experiment 5,

The complex enzyme-hydrolyzed polysaccharides of hizikia fusiforme obtained in example 1-1 and crude hizikia fusiforme polysaccharides were measured for their in vitro bile acid binding ability and compared by literature methods (Niu, y.g., Xie, z.h., Zhang, h., Sheng, y., & Yu, L.L. (2013). Effects of structural modifications on physical and biological acids-binding properties of psyllium.

The result shows that the sargassum fusiforme compound zymolytic polysaccharide (ESFP) has stronger binding capacity to free sodium cholate and sodium chenodeoxycholate, which is respectively equal to 53 percent and 47 percent of the binding capacity of isocholyamine; the bile acid binding capacity of crude polysaccharide of Cyrtymenia Sparsa (SFP) is inferior, and is 50% and 38% of the binding capacity of cholestyramine.

Examples 1-2 to 1-5 the enzymolyzed polysaccharides (ESFP1-2) to (ESFP1-5) obtained in comparative examples 1 to 2 were measured according to the methods described in the above experiments 1 to 5, and the results were compared with those of example 1-1 and are shown in Table 3 below.

TABLE 3 comparison of antioxidative Activity of degraded polysaccharides obtained in examples 1-1 to 1-5 and comparative examples 1 and 2

Experiment 6-1, measurement of macrophage proliferation regulating action

To each well of a 96-well plate, 95. mu.L of cell suspension (2X 10 macrophage concentration of RAW 264.7) was added⁵one/mL), 5 μ L each of the LPS solution and the ESFP1, ESFP2, ESFP3, ESFP4 solutions at different concentrations were added (final concentration of LPS of 1, 5, 10 μ g/mL; ESFP1, ESFP2, ESFP3,Final concentration of ESFP4 was 10, 50, 250, 500, 1000. mu.g/mL), the mixture was cultured at 37 ℃ in a cell culture incubator with 5% CO₂Under the culture conditions of (3), 24 hours and 48 hours, respectively. The experiment was performed in parallel with 6 groups. After 24h incubation, the 96-well plates were removed from the cell incubator, 10. mu.L of MTT solution (5mg/mL) was added to each well, carefully mixed and incubated in the cell incubator for 4 hours. The culture medium in each well was carefully aspirated to prevent cell layer rupture. The OD at 570nm of each well was determined by adding 100. mu.L of MTT solution (5mg/mL) to each well and gently shaking to dissolve all the formazan crystals. The same procedure was followed for 48 h.

The experiment was performed with different concentrations of Lipopolysaccharide (LPS) as a positive control and water as a blank control (i.e. at a concentration of 0).

The results obtained are shown in FIG. 5, from FIG. 5 it can be seen that: after 24h of culture, when the concentration is lower, each polysaccharide component has obvious proliferation activity on RAW264.7 macrophage, but after reaching a certain concentration, the cell proliferation activity is reduced along with the increase of the concentration. After 48h of culture, the cell proliferation activity trend is similar with concentration, but the cell proliferation activity of the four purified polysaccharides (ESFP1-4) is higher than that of the crude polysaccharide SFP and the degraded polysaccharide ESFP.

Experiment 6-2 measurement of macrophage phagocytosis

To each well of a 96-well plate, 95. mu.L of cell suspension (RAW264.7 macrophage concentration 1X 10)⁶one/mL), 5 μ L each of the LPS solution and the ESFP1, ESFP2, ESFP3, ESFP4 solutions at different concentrations were added (final concentration of LPS of 1, 5, 10 μ g/mL; ESFP1, ESFP2, ESFP3 and ESFP4 were added to the culture medium at final concentrations of 10, 50, 250, 500 and 1000. mu.g/mL), and the mixture was cultured at 37 ℃ in a cell culture incubator with 5% CO₂Under the culture conditions of (3), 24 hours and 48 hours, respectively. The experiment was performed in parallel with 6 groups. After 24h incubation, the 96-well plates were removed from the cell incubator, the cell culture fluid was aspirated, and 100. mu.L of neutral red solution (0.075%) was added to each well at 37 ℃ with 5% CO₂Culturing for 0.5 h. The neutral red solution was discarded and the residual unbound neutral red in the wells was washed off with pre-warmed PBS buffer, 100. mu.L each (which was added slowly to the wells to prevent adherence when washed with PBS)Cell blowing up). PBS was discarded from each well, 150. mu.L of cell lysate was added, and the reaction was carried out at 37 ℃ for 1 hour. After mixing by gentle shaking, the OD value of each well at a wavelength of 570nm was measured by a microplate reader. The same procedure was followed for 48 h.

In this experiment, LPS of different concentrations was used as a positive control, and water was used as a blank control.

The results obtained are shown in FIG. 6, and from FIG. 6, it can be seen that: after 24h of culture, the ESFP1, ESFP2 and ESFP in 6 samples have good effect of promoting RAW264.7 cell phagocytosis, wherein the ESFP2 has prominent effect when the concentration is 500 mug/mL, and the OD value reaches 0.165, which is not only far larger than the OD values of other concentrations, but also higher than the positive control of each concentration. The result after 48h of culture also shows that the phagocytosis of macrophages for degrading the polysaccharide ESFP is stronger than that of the crude polysaccharide SFP, and the change trend of the phagocytosis of macrophages along with the concentration of the polysaccharide is similar to that after 24h of culture.

Experiment 6-3, determination of Effect of regulating macrophage to release NO molecule

To each well of a 96-well plate, 95. mu.L (1X 10) of the cell suspension was added⁶Suspension of RAW264.7 macrophage cells in concentration), 5. mu.L each of ESFP1, ESFP2, ESFP3 and ESFP4 solutions in different concentrations (1, 5, 10, 50, 250, 500 and 1000. mu.g/mL, respectively) were added, and the culture temperature was 37 ℃ in a cell culture incubator with 5% CO₂Under the culture conditions of (3), 24 hours and 48 hours, respectively. The experiment was performed in parallel with 6 groups. After 24 hours of culture, the 96-well plate was taken out from the cell incubator, the cell culture solution was transferred to another new 96-well plate, 100. mu.L of Griess reagent (Griess A: Griess B ═ 1:1) was added to each well, the mixture was gently shaken and mixed, and then left to stand in the dark for 10min, and the OD at 570nm was measured. The same procedure was followed for 48 h.

The results obtained are shown in FIG. 7, and from FIG. 7, it can be seen that: after 24h of culture, the effect of ESFP on promoting the release of NO factors from RAW264.7 cells is far better than that of crude polysaccharide SFP. ESFP1, ESFP2 and ESFP in 6 samples have better effect of promoting NO factor release of RAW264.7 cells. The ESFP2 has a prominent effect when the concentration is 1000 mug/mL, and the concentration of NO in cell fluid reaches 75.77 mug M, which is far higher than the influence of positive control LPS. After 48h of culture, the influence on NO release of RAW264.7 cells is sequentially (according to peak values): ESFP2> ESFP1> ESFP3> ESFP 4. ESFP2 was found to have the strongest activity of promoting NO secretion from cells, and the concentration of NO reached 90.11. mu.M at an addition concentration of 1000. mu.g/mL. The concentration of NO in the cell fluid significantly increased after 48h of culture compared to 24h of culture.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims

1. The method for enhancing the biological activity of the sargassum fusiforme polysaccharide is characterized by comprising the following steps:

1) and (3) degradation:

firstly, dissolving crude sargassum fusiforme polysaccharide in a buffer solution with the pH value of 6.0, then adding a complex enzyme consisting of pectinase and glucoamylase, and then adding the buffer solution for dilution, wherein the concentration of the crude sargassum fusiforme polysaccharide in the obtained system is 4.0mg/mL, and the total concentration of the complex enzyme is 68.4U/mL; the pectase is: the saccharifying enzyme activity ratio =3.3: 1;

carrying out oscillation reaction on the system at the temperature of 54.2 ℃ for 3h, thereby realizing the degradation of crude polysaccharide of the sargassum fusiforme;

2) removing enzyme from the reaction solution obtained in the step 1), filtering, and concentrating the obtained filtrate to 20-30% of the original volume; dialyzing for 24-48 h by using a dialysis bag with the molecular weight cutoff of 3500Da, concentrating the obtained dialysate, and freeze-drying to obtain the sargassum fusiforme compound zymolytic polysaccharide ESFP.

2. The method for enhancing the biological activity of sargassum fusiforme polysaccharide as claimed in claim 1, wherein:

the buffer solution with the pH of 6.0 is acetic acid-sodium acetate buffer solution with the pH of 6.0 and citric acid-sodium citrate buffer solution.

3. The method for enhancing the biological activity of sargassum fusiforme polysaccharide according to claim 1 or 2, wherein: separating and purifying the sargassum fusiforme compound zymolytic polysaccharide ESFP; the method comprises the following steps:

roughly dividing:

selecting a Cellulose DEAE-52 chromatographic column;

weighing 400 +/-40 mg of sargassum fusiforme compound zymolytic polysaccharide ESFP, dissolving in 7mL of deionized water, centrifuging to obtain supernatant, uniformly adding the supernatant to the upper surface of a chromatographic column filler, sequentially eluting with deionized water and NaCl solutions with the concentrations of 0.1mol/L, 0.3mol/L, 0.5mol/L and 0.8mol/L at the flow rate of 1mL/min,

secondly, subdivision:

selecting a Sephadex G-100 gel chromatographic column;

weighing 100 +/-10 mg of primarily purified polysaccharide components, dissolving in 3mL of deionized water, centrifuging, taking supernatant, loading in a Sephadex G-100 gel chromatographic column, eluting with deionized water at the flow rate of 0.20mL/min, combining polysaccharide eluates, concentrating under reduced pressure, and freeze-drying to obtain four purified components of the sargassum fusiforme composite enzymolysis polysaccharide, which are named as ESFP1, ESFP2, ESFP3 and ESFP4 respectively.

4. Use of the sargassum fusiforme complex zymolytic polysaccharide ESFP prepared according to the method of claim 1 or 2 or the purified fraction prepared according to the method of claim 3 in the preparation of a functional food base.