CN114316080B - Method for improving extraction rate and bioactivity of grifola frondosa crude polysaccharide - Google Patents
Method for improving extraction rate and bioactivity of grifola frondosa crude polysaccharide Download PDFInfo
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Abstract
The invention discloses a method for improving the extraction rate and the biological activity of grifola frondosa polysaccharide, which comprises the following steps: adding hydrogen peroxide and ascorbic acid into a solution consisting of grifola frondosa powder and deionized water to react; concentrating the obtained reaction solution, adding absolute ethyl alcohol, adding deionized water into the obtained precipitate for redissolving to obtain a crude polysaccharide solution; and (3) freeze-drying the obtained crude polysaccharide solution to obtain the grifola frondosa degradation crude polysaccharide (DGFP). The obtained grifola frondosa degradation crude polysaccharide (DGFP) has high in-vitro antioxidant activity.
Description
Technical Field
The invention belongs to the technical field of chemistry and chemical engineering, and relates to a method for degrading grifola frondosa polysaccharide by combining hydrogen peroxide and ascorbic acid so as to increase the extraction rate and enhance the biological activity of the grifola frondosa polysaccharide.
Background
Grifola frondosa, marketed in China, japan and other Asian countries as a medicinal and edible fungus. Grifola frondosa is an edible and medicinal fungus commonly known as dancing bamboo and black bone. Regular intake of Grifola frondosa can enhance immune system, lower blood sugar, and improve spleen, stomach and nerve functions. Researches show that the grifola frondosa has various biological activities such as antioxidation, immunoregulation, anticancer, anti-inflammatory and the like. Various bioactive compounds have been obtained from Maitake mushroom fruiting bodies and liquid culture mycelia, and many biological activities have been confirmed. Various active substances isolated from fungi have been described in the literature, polysaccharides being one of the important substances. Research has proved that the polysaccharide extracted from Grifola frondosa shows antioxidant property and has the ability of scavenging free radicals; therefore, grifola frondosa can be used as raw material of functional food with antioxidant effect.
The polysaccharide extracted from maitake mushroom has large molecular weight and relatively low bioactivity. And therefore further improvements are needed.
106478834A discloses a subcritical water extraction method of polysaccharide from Grifola frondosa, which comprises oven drying, pulverizing, subcritical extracting, coarse filtering, holding temperature, centrifuging, concentrating, purifying, decolorizing, precipitating, drying, washing, drying, secondary extracting, etc., by using subcritical water with good penetrating and dissolving capacity, cell wall polysaccharide and subcritical water are fully contacted and dissolved out, concentrating by water bath method, purifying Grifola frondosa polysaccharide by combining Sevag method, recovering once centrifuged precipitate, and extracting Grifola frondosa polysaccharide, thereby not only increasing extraction amount of Grifola frondosa polysaccharide, but also greatly reducing waste of raw materials, and significantly improving extraction efficiency and purity of Grifola frondosa polysaccharide. The extraction method needs a professional subcritical extraction kettle, needs to adjust the critical pressure, and has higher extraction condition requirement.
105732836A, discloses an extraction process of Grifola frondosa polysaccharide and its application in preparing medicine for repairing gastric mucosa wound, and adopts water extraction → ultrafiltration → alcohol precipitation method to obtain Grifola frondosa polysaccharide. According to the process, after supernatant liquid is obtained through centrifugation, the supernatant liquid needs to be collected by an ultrafiltration separation component, the steps are complex, and more loss is caused.
The traditional hot water extraction method of the grifola frondosa crude polysaccharide is to extract the grifola frondosa defatted powder under the condition of hot water after mixing water and the grifola frondosa defatted powder according to a certain proportion, and has the defects that the yield of the obtained grifola frondosa polysaccharide is low, and the molecular weight of the obtained polysaccharide is large.
Disclosure of Invention
The invention aims to provide a method for improving the extraction rate and the biological activity of the crude polysaccharide of grifola frondosa.
In order to solve the technical problems, the invention provides a method for improving the extraction rate and the biological activity of grifola frondosa polysaccharide, which comprises the following steps:
s1, adding hydrogen peroxide and ascorbic acid into a solution consisting of grifola frondosa powder (grifola frondosa defatted powder) and deionized water to form a reaction system, and reacting (heating extraction reaction) the reaction system for 3 +/-0.5 hours at 65-105 ℃ under the stirring condition to obtain a reaction solution;
the molar ratio of hydrogen peroxide to ascorbic acid =1 (1 ± 0.1);
grifola frondosa powder: the feed-liquid ratio of the deionized water is =1 (30 +/-5);
H 2 O 2 the concentration of (A) is 5-25 mmol/L; the concentration of Vc is 5-25 mmol/L;
s2, concentrating the reaction solution obtained in the step S1 to 30-35% of the original volume to obtain a concentrated solution;
adding absolute ethanol into the concentrated solution, standing at 0-5 deg.C for 24 + -2 h (all day), and centrifuging (8000 + -1000rpm, 20 + -5 min) to obtain precipitate; the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1.5-4.5;
adding deionized water into the obtained precipitate for redissolution to obtain a crude polysaccharide solution; concentrating the solution: deionized water =0.8 to 1.2 by volume;
and (3) freeze-drying the obtained crude polysaccharide solution at (-40-60 ℃ for 22-26 hours) to obtain the grifola frondosa degradation crude polysaccharide (DGFP).
As an improvement of the method for improving the extraction rate and the biological activity of the grifola frondosa polysaccharide, the reaction system comprises the following components:
H 2 O 2 the concentration of (A) is 20mmol/L, and the concentration of Vc is 20mmol/L;
the reaction system reacts for 3h at 95 ℃ under the stirring condition to obtain reaction liquid.
The method for improving the extraction rate and the biological activity of the grifola frondosa polysaccharide is further improved as follows:
in the step S2:
concentrating the reaction solution obtained in the step S1 to 1/3 of the original volume to obtain a concentrated solution;
adding anhydrous ethanol into the concentrated solution, standing at 4 deg.C for 24 hr, and centrifuging (8000rpm, 20min) to obtain precipitate;
the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1;
taking the precipitate to be redissolved by deionized water, wherein the volume ratio of the concentrated solution to the deionized water is 0.8;
and (3) freeze-drying the obtained crude polysaccharide solution at (-50-60 ℃ for 24 hours) to obtain the grifola frondosa degradation crude polysaccharide (DGFP).
The method for improving the extraction rate and the biological activity of the grifola frondosa polysaccharide is further improved as follows:
the biological activity of the grifola frondosa polysaccharide is antioxidant activity.
The invention utilizes hydrogen peroxide and ascorbic acid to generate hydroxyl free radicals which are very active and can react with hydrogen atoms of polysaccharide (hydrogen absorption reaction), thereby causing the breakage of glycosidic bonds and further reducing the molecular weight of the polysaccharide.
Compared with the prior art, the invention has the technical advantages that:
1. compared with the crude polysaccharide (GFP) of the grifola frondosa obtained by the traditional hot water extraction, the extraction rate (DGFP) of the grifola frondosa degraded crude polysaccharide obtained by the invention is obviously improved;
2. compared with the crude polysaccharide (GFP) of the grifola frondosa obtained by the traditional hot water extraction, the in-vitro antioxidant activity of the grifola frondosa degraded crude polysaccharide (DGFP) is obviously improved.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the results of DPPH radical scavenging ability of crude polysaccharide of Grifola Frondosa (GFP), polysaccharide of Grifola frondosa degradation (DGFP 1-1) obtained in example 1-1 of the present invention, and ascorbic acid (experiment 1).
FIG. 2 shows the results of ABTS radical scavenging ability of crude polysaccharide of Grifola Frondosa (GFP), polysaccharide of Grifola frondosa degradation (DGFP 1-1) obtained in example 1-1 of the present invention, and ascorbic acid (experiment 2).
FIG. 3 shows the results of the measurement of hydroxyl radical scavenging ability of Grifola frondosa crude polysaccharide (GFP), grifola frondosa degraded polysaccharide (DGFP 1-1) obtained in example 1-1 of the present invention, and ascorbic acid (experiment 3).
FIG. 4 shows the results of the total reducing power test of Grifola frondosa crude polysaccharide (GFP), grifola frondosa degraded polysaccharide (DGFP 1-1) obtained in example 1-1 of the present invention, and ascorbic acid (experiment 4).
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
In the present invention, the rotation speed of the stirring is generally 1000 to 1500rpm.
The preparation method of the defatted powder of the grifola frondosa adopts a conventional technology, and specifically comprises the following steps: mixing the grifola frondosa powder and absolute ethyl alcohol according to a material-liquid ratio of 1g: refluxing for 2h at 90 deg.C for 20ml, vacuum filtering to obtain powder, repeating for three times, and conventionally drying to obtain Maitake defatted powder.
Example 1-1:
s1, mixing the degreased powder of the grifola frondosa and deionized water according to a material-liquid ratio of 1:30 Mixing (by weight ratio) to obtain Grifola frondosa solution;
adding hydrogen peroxide and ascorbic acid into the grifola frondosa solution to form a reaction system; the amount ratio (molar ratio) =1 of hydrogen peroxide to ascorbic acid: 1, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system are both 20mmol/L;
heating and stirring the reaction system at 95 ℃ for reaction for 3h, thereby realizing the degradation of the grifola frondosa crude polysaccharide and obtaining a reaction solution;
s2, rotationally evaporating and concentrating the reaction liquid obtained in the step S1 at 60 ℃ to 1/3 of the original volume to obtain a concentrated liquid;
adding anhydrous ethanol into the concentrated solution, standing at 4 deg.C for a whole day (24 h), and centrifuging (8000rpm, 20min) to obtain precipitate; the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1;
taking the precipitate and redissolving the precipitate by using deionized water; the volume ratio of the concentrated solution to the deionized water is 0.8;
the obtained complex solution is frozen and dried for 24 hours at the temperature of minus 60 ℃ to obtain the crude polysaccharide (DGFP 1-1) degraded by the grifola frondosa, and the yield is 15.83 percent.
The yield is 100 percent (the mass of the degraded crude polysaccharide of the grifola frondosa obtained after freeze drying/the mass of the degreased powder of the grifola frondosa)
The composition and molecular weight of DGFP1-1 are shown in Table 1
TABLE 1 comparison of the composition and molecular weight of the degraded polysaccharide DGFP1-1 and the crude polysaccharide
Note that Glc, gal, man, fuc, rib, glcUA, glcN represent glucose, galactose, mannose, fucose, ribose, glucuronic acid and glucosamine, respectively.
Example 1-2, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system of step S1 of example 1-1 were adjusted to 15mmol/L; the reaction system is heated and stirred at 65 ℃ for reaction for 3h to obtain reaction liquid. The remaining amounts were the same as in example 1-1, and a Grifola frondosa degraded crude polysaccharide (DGFP 1-2) was obtained in a yield of 8.6%.
Examples 1 to 3, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system of step S1 of examples 1 to 1 were adjusted to 15mmol/L; the reaction system is heated and stirred at 75 ℃ for reaction for 3h to obtain reaction liquid. The remaining amounts were the same as in example 1-1, and a Grifola frondosa degraded crude polysaccharide (DGFP 1-3) was obtained in a yield of 11.50%.
Examples 1 to 4, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system of step S1 of examples 1 to 1 were adjusted to 15mmol/L; the reaction system is heated and stirred at 85 ℃ for reaction for 3h to obtain reaction liquid. The remaining phases were identical to example 1-1, giving crude polysaccharide from Grifola frondosa degradation (DGFP 1-4) in a yield of 11.82%.
Examples 1 to 5, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system of step S1 of examples 1 to 1 were adjusted to 15mmol/L; the reaction system is heated and stirred at 105 ℃ to react for 3h to obtain a reaction solution. The remaining amounts were the same as in example 1-1, and the Grifola frondosa degradation crude polysaccharide (DGFP 1-5) was obtained in a yield of 13.56%.
Examples 1 to 6, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system of step S1 of example 1 to 1 were adjusted to 15mmol/L; the obtained reaction system is heated and stirred at 95 ℃ to react for 3h, so as to obtain reaction liquid. The remaining amounts were the same as in example 1-1, and the Grifola frondosa degradation crude polysaccharide (DGFP 1-6) was obtained in a yield of 15.82%.
Examples 1 to 7, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system of step S1 of example 1 to 1 were adjusted to 5mmol/L; the obtained reaction system is heated and stirred at 95 ℃ to react for 3 hours to obtain reaction liquid. The remaining amounts were the same as in example 1-1, and the crude polysaccharide from Grifola frondosa degradation (DGFP 1-7) was obtained in a yield of 13.29%.
Examples 1 to 8, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system of step S1 of example 1 to 1 were adjusted to 10mmol/L; the obtained reaction system is heated and stirred at 95 ℃ to react for 3h, so as to obtain reaction liquid. The remaining amounts were the same as in example 1-1 to obtain a Grifola frondosa degraded crude polysaccharide (DGFP 1-8) with a yield of 15.30%.
Examples 1 to 9, the concentrations of hydrogen peroxide and ascorbic acid in the reaction system of step S1 of example 1 to 1 were adjusted to 25mmol/L; the obtained reaction system is heated and stirred at 95 ℃ to react for 3h, so as to obtain reaction liquid. The remaining amounts were the same as in example 1-1, and the Grifola frondosa degradation crude polysaccharide (DGFP 1-9) was obtained in a yield of 15.99%.
Comparative example 1, changing "hydrogen peroxide and ascorbic acid were added" to "hydrogen peroxide and ascorbic acid were not added" in example 1-1, i.e., the amounts of hydrogen peroxide and ascorbic acid used were both 0, and the remaining amounts were the same as in example 1-1, to obtain crude polysaccharide of Grifola Frondosa (GFP) in a yield of 11.51%.
Experiment 1,
The literature (Espin J C, soler-Rivas C, wichs H J, et. Anthocynin-based natural colors: a new source of anti-additive activity for foodstuff [ J ] is adopted]The grifola frondosa degradation polysaccharide (DGFP 1-1) obtained in example 1-1 was tested for DPPH radical scavenging ability by the method reported in Journal Agricultural Food Chemistry,2000,48 (5): 1588-1592) and compared with the grifola frondosa crude polysaccharide. As can be seen from FIG. 1, the DPPH radical scavenging ability of the Grifola frondosa degrading polysaccharide (DGFP 1-1) is significantly improved compared to the Grifola frondosa crude polysaccharide. IC of Grifola frondosa crude polysaccharide (GFP) and Grifola frondosa degradation polysaccharide (DGFP 1-1) 50 1.149mg/mL and 0.248mg/mL, respectively. However, the DPPH radical scavenging activity of DGFP1-1 is still less than that of vitamin C (IC 50 of 0.005 mg/mL).
Experiment 2,
The literature (Xiao H, fu X, cao C, et al. Sulfonated modification, characterization, antioxidant and polyglycolic activities of polysaccharides from Sargassum pallidum [ J ] is adopted].International Journal of Biological Macromolecules,2019,121:407-414.Jeddou K B,Chaari F,Maktouf S,et al.Structural,functional,and antioxidant properties of water-soluble polysaccharides from potatoes peels[J]Food Chemistry,2016,205, 97-105.) A method was reported to test the ability of Grifola frondosa degrading polysaccharide (DGFP 1-1) obtained in example 1-1 to scavenge free radicals in ABTS, and to compare it with the crude polysaccharide of Grifola frondosa. As can be seen from FIG. 2, the scavenging ability of ABTS free radicals is significantly improved after the degradation of the crude polysaccharide of Grifola frondosa. However, the ABTS free radical activity of DGFP1-1 is still less than that of vitamin C. cG for scavenging superoxide anion free radical by GFP and DGFP1-1 50 Are respectively 1.162mg/mL and 0.567mg/mL。
Experiment 3,
The literature (Chen H W, chen A H, shao Y, et. Studies on the antioxidant capacity of zinc rich exopolysaccharide of yeast deficiencies [ J ] is used]Food and Fermentation Industries,2009,35 (6): 54-57.Deng C, hu Z, fu H T, hu M H, et al, chemical analysis and antioxidant activity in the vision of a β -D-glucan isolated from a dictionary index. International Journal of Biological Macromolecules,2012,51, 70-75.) the Grifola frondosa degraded polysaccharide (DGFP 1-1) obtained in example 1-1 was tested for hydroxyl radical scavenging ability and compared with undegraded polysaccharide. As can be seen from FIG. 3, the hydroxyl radical scavenging ability of the degraded polysaccharide of Grifola frondosa is significantly improved. However, the superoxide anion radical activity of DGFP1-1 is still less than that of vitamin C. IC for eliminating GFP and DGFP1-1 hydroxyl free radical 50 3.532mg/mL and 2.627mg/mL, respectively.
Experiment 4,
The Grifola frondosa-degrading polysaccharide (DGFP 1-1) obtained in example 1-1 was tested for reducing ability by a method reported in the literature (Yen G, chemical H.antibiotic activity of varied amounts of extracts in relation to the anti-inflammatory Chemistry [ J ]. Journal of Agricultural and Food Chemistry,1995, 43. As can be seen from FIG. 4, the reducing power of the degraded polysaccharide of Grifola frondosa is significantly improved.
The grifola frondosa degradation polysaccharide (DGFP 1-1) obtained in example 1-1 and the grifola frondosa crude polysaccharide (GFP-D) obtained in comparative example 1 were examined by the methods described in the above experiments 1 to 4, and the results were compared with those of example 1-1, as shown in Table 2 below
TABLE 2 comparison of antioxidant Activity of degraded polysaccharide obtained in example 1-1 and crude polysaccharide obtained in comparative example 1
Comparative example 2-1, the concentration of hydrogen peroxide and the concentration of ascorbic acid in the reaction system of step S1 of example 1-1 were adjusted to 25mmol/L and 10mmol/L, that is, the usage ratio (molar ratio) =5 of hydrogen peroxide and ascorbic acid: 2; the rest is equivalent to embodiment 1. The yield was 13.44%.
Comparative example 2-2, the concentration of hydrogen peroxide and the concentration of ascorbic acid in the reaction system in step S1 of example 1-1 were adjusted to 10mmol/L and 25mmol/L, that is, the amount ratio (molar ratio) =2:5; the rest is equivalent to embodiment 1. The yield was 13.51%.
The grifola frondosa polysaccharides obtained in the above 2 comparative examples were subjected to the antioxidant activity test at a concentration of 1mg/mL, and the results are compared with DGFP1-1 in Table 3 below.
TABLE 3 comparison of antioxidant Activity of degraded polysaccharide obtained in example 1-1 and crude polysaccharide obtained in comparative example 1
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 (4)
1. The method for improving the extraction rate and the biological activity of the grifola frondosa polysaccharide is characterized by comprising the following steps of:
s1, adding hydrogen peroxide and ascorbic acid into a solution consisting of grifola frondosa powder and deionized water to form a reaction system, and reacting the reaction system for 3 +/-0.5 h at 95-105 ℃ under the stirring condition to obtain a reaction solution;
the molar ratio of hydrogen peroxide to ascorbic acid =1 (1 ± 0.1);
grifola frondosa powder: the feed-liquid ratio of the deionized water is =1 (30 +/-5);
H 2 O 2 the concentration of (b) is 20 to 25mmol/L; the concentration of Vc is 20 to 25mmol/L;
s2, concentrating the reaction liquid obtained in the S1 to 30-35% of the original volume to obtain a concentrated liquid;
adding absolute ethyl alcohol into the concentrated solution, standing for 24 +/-2 hours at 0-5 ℃, and then centrifuging to obtain a precipitate; the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1;
adding deionized water into the obtained precipitate for redissolution to obtain a crude polysaccharide solution; concentrating the solution: deionized water =0.8 to 1.2 by volume ratio;
and (3) freeze-drying the obtained crude polysaccharide solution to obtain the grifola frondosa degradation crude polysaccharide (DGFP).
2. The method of claim 1, wherein the extraction rate and biological activity of the polysaccharide are increased by:
in the reaction system:
H 2 O 2 the concentration of (A) is 20mmol/L, and the concentration of Vc is 20mmol/L;
the reaction system reacts for 3 hours at 95 ℃ under the condition of stirring to obtain reaction liquid.
3. The method of claim 2, wherein the extraction rate and bioactivity of the grifola frondosa polysaccharide are as follows:
in the S2:
concentrating the reaction solution obtained in the step S1 to 1/3 of the original volume to obtain a concentrated solution;
adding absolute ethyl alcohol into the concentrated solution, standing at 4 ℃ for 24h, and then centrifuging to obtain a precipitate;
the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1;
taking the precipitate, and redissolving the precipitate by using deionized water, wherein the volume ratio of the concentrated solution to the deionized water is 0.8;
and (3) freeze-drying the obtained crude polysaccharide solution to obtain the grifola frondosa degradation crude polysaccharide (DGFP).
4. The method for improving the extraction rate and the biological activity of the grifola frondosa polysaccharide according to any one of claims 1 to 3, wherein the method comprises the following steps:
the biological activity of the grifola frondosa polysaccharide is antioxidant activity.
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