CN113215206B - Preparation and purification method of grifola frondosa polysaccharide with high antioxidant activity - Google Patents
Preparation and purification method of grifola frondosa polysaccharide with high antioxidant activity Download PDFInfo
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Abstract
The invention discloses a preparation and purification method of Grifola frondosa polysaccharide with high in vitro antioxidant activity, which is characterized in that farnesol is added during the initial fermentation or the fermentation process of Grifola frondosa liquid. The method of the invention promotes the change of the polysaccharide yield of the Grifola frondosa fermentation liquor by adding the farnesol in the fermentation process of the Grifola frondosa mycelium liquid, obviously improves the yield of the acidic polysaccharide component produced by the fermentation of the Grifola frondosa, obviously improves the polysaccharide yield of the Grifola frondosa fermentation liquor on the basis of not increasing the original fermentation period, has the maximum improvement amplitude of 149 percent, increases the content of the acidic polysaccharide after separation and purification from 7.4 percent to 58.2 percent, obviously improves the in vitro antioxidant activity of the acidic polysaccharide after separation and purification compared with a control group, and has high safety, no toxicity and high stability of the farnesol as an external additive, thereby laying the foundation for the industrialized production of the polysaccharide of the Grifola frondosa fermentation liquor.
Description
Technical Field
The invention belongs to the technical field of extraction of grifola frondosa polysaccharide, and particularly relates to a preparation and purification method of grifola frondosa polysaccharide with high antioxidant activity.
Background
The Grifola frondosa polysaccharide is the main bioactive component of Grifola frondosa fermentation liquor. A large number of pharmacological and pharmacodynamic studies prove that the grifola frondosa fermentation broth polysaccharide has good effects on resisting tumors and HIV viruses, and preventing and treating diabetes, hypertension, hyperlipidemia, obesity, senile dementia and the like, and some aspects even exceed the clinically applied lentinan, coriolus versicolor polysaccharide and schizophyllan polysaccharide. In addition, the grifolan has many active functions beneficial to the human body. It can improve anoxia tolerance, enhance immunity, scavenge free radicals, resist radiation, and enhance bone marrow and liver activity, thereby prolonging life. Grifola frondosa polysaccharide can be divided into mycelium structural polysaccharide and fermentation broth polysaccharide secreted to the outside of cells. The two have great difference in structural hierarchy, preparation method and physiological activity. At present, the research on the grifola frondosa is mainly focused on the extraction process of the polysaccharide of the fruit body of the grifola frondosa, and the research reports on liquid submerged fermentation are less. The medical value and the health value of the grifola frondosa polysaccharide are closely concerned in the medical field and the food industry, are deeply loved and accepted by people, and have wide application prospects. The efficacy of grifola frondosa has been widely accepted, but the yield of the acidic polysaccharide component of grifola frondosa limits the market application to meet the increasing demand of people.
Farnesol is a quorum sensing molecule of fungi, naturally exists in essential oil of acacia, jasmine and the like, is mainly used for preparing spices or preparing edible essence, and has the advantages of being natural, safe and reliable. Farnesol has physiological effects of regulating and controlling the formation of fungal hyphae, the formation of cell membranes, oxidative stress, the outflow of medicaments and the like. Some ROS can be induced to be generated by the farnesol in the liquid fermentation process of the coriolus versicolor, and the increase of the ROS content can change the environment of the coriolus versicolor fermentation liquid to a certain extent, so that the change of the mycelium shape of the coriolus versicolor, the formation of cell membranes and the generation of active substances are influenced. Determining the in vitro antioxidant activity of the polysaccharide, namely, adding farnesol in the fermentation process of the grifola frondosa liquid to observe the yield change of the polysaccharide in the grifola frondosa fermentation liquid, separating and purifying the polysaccharide to obtain the polysaccharides with different components of the grifola frondosa, and determining the in vitro antioxidant activity of the polysaccharides.
At present, the method for producing the polysaccharide from the Grifola frondosa fermentation liquor by using the liquid submerged fermentation technology has the characteristics of short production period, low production cost, high economic benefit and the like, and is an effective method for obtaining the polysaccharide from the Grifola frondosa fermentation liquor at present. Various methods for extracting grifola frondosa polysaccharide are reported in documents and patent publications, and patent publication CN105669876A discloses a process for extracting grifola frondosa polysaccharide, wherein grifola frondosa polysaccharide is obtained by using dry grifola frondosa sporocarp as a raw material for extracting polysaccharide and adopting methods such as water extraction, alkali extraction, ultrafiltration, alcohol precipitation and the like. Patent publication CN1752109A discloses a method for preparing grifolan by enzymolysis, which comprises the steps of carrying out enzymolysis on grifola frondosa mycelia in a fermentation broth by virtue of an enzyme system existing in the grifola frondosa mycelia, adding cellulase, beta-glucanase, pectinase and neutral protease into the fermentation broth for enzymolysis, carrying out enzyme deactivation, filtering, concentrating, alcohol precipitating, separating and the like to obtain the grifolan, carrying out enzymolysis on the grifola frondosa fungus fermentation broth by skillfully utilizing the enzyme system of the mycelia and an additional mixed enzyme system under proper conditions, and obviously improving the content of the grifolan in a grifolan crude polysaccharide product after the enzymolysis. Patent publication CN104403014A discloses a preparation method for increasing the yield of Grifola frondosa polysaccharide, which increases the extraction rate of Grifola frondosa polysaccharide through proper enzymolysis and alcohol precipitation technologies, macroporous adsorption resin decolorization, ultrafiltration membrane filtration and other steps. Patent publication CN101880700A discloses a liquid fermentation method for increasing the yield of ganoderma lucidum polysaccharide, which utilizes the liquid culture of ganoderma lucidum cells, and adds rare earth elements praseodymium, neodymium or lanthanum into the culture medium to increase the yield of ganoderma lucidum polysaccharide. Patent publication CN110128558A discloses a preparation method of a mulberry polysaccharide-iron chelate with antioxidant activity, the invention prepares an iron supplement with antioxidant activity, which is beneficial to human body absorption, so that the chelate can play an antioxidant role while treating iron-deficiency anemia, thereby achieving double health care functions. Patent publication CN109988251A discloses a method for preparing flammulina velutipes acidic polysaccharide with antioxidant activity, the method comprises adding ethanol and alkaline solution into flammulina velutipes mycelium or fruiting body to obtain flammulina velutipes acidic polysaccharide. The method has the advantages of high extraction yield, no need of heating for extraction, small damage to active ingredients, simple operation, no corrosion to equipment and the like.
By contrast, the present patent application is substantially different from the above patent publications.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation and purification method of grifola frondosa polysaccharide with high antioxidant activity.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for preparing and purifying Grifola frondosa (Grifola frondosa) polysaccharide with high antioxidant activity in vitro comprises adding farnesol at the beginning of fermentation or during fermentation of Grifola frondosa liquid.
Moreover, the Grifola frondosa is Grifola frondosa with the preservation number of CGMCC No.5.404, and the preservation unit is as follows: china general microbiological culture Collection center, national institute of sciences, 3, west road, 1, north Chen, the area facing the sun, Beijing.
Furthermore, the method is to add farnesol at 0-2 d.
And the final concentration of the farnesol is 0.2-1.0 mmol/L.
In addition, the method is to inoculate the grifola frondosa into the fermentation medium and add farnesol into the fermentation medium.
Further, the liquid fermentation medium contains per L: 22g of glucose, 3g of peptone, 1.2g of potassium dihydrogen phosphate, 0.8g of magnesium sulfate heptahydrate and vitamin B 1 0.12g, natural pH.
Moreover, the method comprises the following specific steps:
(1) inoculating the grifola frondosa seed solution into a fermentation culture medium, adding farnesol into the fermentation culture medium at the initial fermentation or during the fermentation of the grifola frondosa, and fermenting for 8d at the temperature of 25-28 ℃ at 160 r/min;
(2) taking the fermentation liquor after the grifola frondosa mycelia are separated according to the following fermentation liquor: the volume ratio of 95% ethanol is 1: 4, mixing with 95% ethanol, precipitating with ethanol at 4 deg.C overnight, centrifuging, removing supernatant, dissolving the precipitate with distilled water, and dissolving at below 60 deg.C for 8 hr to obtain fermentation broth polysaccharide solution;
(3) freeze-drying the obtained polysaccharide solution of the fermentation liquor, removing protein and decoloring, and freeze-drying; preparing 5mg/mL polysaccharide solution, purifying by DEAE-650M ion exchange column chromatography, eluting with distilled water, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L and 1.0mol/L NaCl solution in sequence, and collecting corresponding components; purifying the polysaccharide solution with dextran gel column to obtain corresponding components, and obtaining the Grifola frondosa polysaccharide with high in vitro antioxidant activity.
Further, the inoculation was carried out at an inoculation amount of 10% by volume.
An edible and medicinal fungus, wherein the bioactive component of the fungus is exopolysaccharide prepared by the method.
The invention has the following advantages and positive effects:
1. the method of the invention promotes the change of the polysaccharide yield of the Grifola frondosa fermentation liquor by adding the farnesol in the fermentation process of the Grifola frondosa mycelium liquid, obviously improves the yield of the acidic polysaccharide component produced by the fermentation of the Grifola frondosa, obviously improves the polysaccharide yield of the Grifola frondosa fermentation liquor on the basis of not increasing the original fermentation period, has the maximum improvement amplitude of 149 percent, increases the content of the acidic polysaccharide after separation and purification from 7.4 percent to 58.2 percent, obviously improves the in vitro antioxidant activity of the acidic polysaccharide after separation and purification compared with a control group, and has high safety, no toxicity and high stability of the farnesol as an external additive, thereby laying the foundation for the industrialized production of the polysaccharide of the Grifola frondosa fermentation liquor.
2. The crude polysaccharide of Grifola frondosa obtained by the method of the invention is dissolved and purified by anion exchange chromatography and gel chromatography to obtain an acidic polysaccharide component with molecular weight of 2.02 x 10 6 Da. The grifola frondosa strain used in the invention is China general microbiological culture Collection center (CGMCC) No. 5.404. The polysaccharide purified by the method has obvious dosage effect on the removal effect of DPPH free radicals, ABTS free radicals, hydroxyl (OH-) free radicals and superoxide anion (O2-) free radicals, and the reducing capability is also improved. The DPPH free radical clearance of the prepared grifola frondosa polysaccharide component is improved from 49.82% to 71.68%, and is improved by 44%; the ABTS free radical clearance is improved from 65.43 percent to 98.38 percent and is improved by 50 percent; the clearance rate of hydroxyl free radicals is improved from 78.5 percent to 99.46 percent and is improved by 27 percent; the scavenging rate of superoxide anion free radicals is improved from 94.65% to 98.1%, and is improved by 3.6%; the reducing power is improved from 0.23 to 0.38The height is 65 percent higher. The antioxidant activity of the grifola frondosa polysaccharide prepared and purified by the method is remarkably increased relative to that of a control group, so that a practical basis is provided for the application of extracellular polysaccharide in improving the biological antioxidant activity, a new direction is provided for the research of improving the in-vitro antioxidant activity of the grifola frondosa polysaccharide, and the method is more beneficial to industrial production and application.
3. The exogenous additive farnesol used in the method is a quorum sensing molecule of the fungus, has the physiological effects of regulating and controlling the mycelium formation of the grifola frondosa fungus, regulating and controlling the cell membrane formation, the oxidative stress, regulating the medicine outflow and the like, and is used for improving the in-vitro antioxidant activity of the grifola frondosa extracellular polysaccharide.
Drawings
FIG. 1 is a graph showing the variation of the fermentation broth of Grifola frondosa without farnesol; wherein, (a) is a fermentation broth polysaccharide yield graph; (b) is a dry weight graph of the bacteria; (c) is a graph of residual sugar content; (d) a graph of pH change of the fermentation liquor is shown;
FIG. 2 is a graph showing the variation of the parameters of the present invention in which 0-0.2mmol/L farnesol is added during fermentation for 0-2 d; wherein, farnesol is added when the Grifola frondosa mycelia enter the fermentation stage for 0-2d, the dry weight of the Grifola frondosa is reduced, but the yield of polysaccharide in fermentation liquor is increased; the pH value is slightly higher than that of the fermentation liquor of the deep fermentation of the grifola frondosa liquid without the farnesol; wherein, (a) is a polysaccharide yield graph of fermentation liquor; (b) is a dry weight graph of the bacteria; (c) is a graph of residual sugar content; (d) a graph of pH change of the fermentation liquor is shown;
FIG. 3 is a graph showing the variation of the parameters of the present invention in which 0.2-0.4mmol/L farnesol was added during fermentation 0-2 d; wherein, the fermentation is carried out for 0-2d, and then the exogenous additive farnesol is added to obviously improve the yield of the polysaccharide in the Grifola frondosa fermentation liquor; the pH value is slightly higher than that of the fermentation liquor of the deep fermentation of the grifola frondosa liquid without the farnesol; wherein, (a) is a fermentation broth polysaccharide yield graph; (b) a dry weight graph of the bacteria is shown; (c) is a graph of residual sugar content; (d) is a graph of pH change of the fermentation liquor;
FIG. 4 is a graph showing the variation of the parameters of the present invention in which 0.4-0.6mmol/L farnesol was added during fermentation for 0-2 days; wherein, farnesol is added into the mycelium for 0-2 days of fermentation, so that the yield of the polysaccharide in the Grifola frondosa fermentation liquor can be improved; the pH value is slightly higher than that of the fermentation liquor of the deep fermentation of the grifola frondosa liquid without the farnesol; wherein, (a) is a fermentation broth polysaccharide yield graph; (b) is a dry weight graph of the bacteria; (c) is a graph of residual sugar content; (d) is a graph of pH change of the fermentation liquor;
FIG. 5 is a graph showing the variation of the parameters of the present invention in which 0.6-0.8mmol/L farnesol was added during fermentation for 0-2 days; wherein, the farnesol is added when the mycelium is fermented to 0-2d, so that the yield of the polysaccharide in the grifola frondosa fermentation liquor can be obviously improved, and the glucose is almost exhausted when the fermentation is finished, so that the full utilization is realized. The pH value is slightly higher than that of the fermentation liquor of the deep fermentation of the grifola frondosa liquid without the farnesol; wherein, (a) is a fermentation broth polysaccharide yield graph; (b) is a dry weight graph of the bacteria; (c) is a graph of residual sugar content; (d) is a graph of pH change of the fermentation liquor;
FIG. 6 is a graph showing the variation of the parameters of the present invention in which 0.8-1.0mmol/L farnesol was added during fermentation for 0-2 days; the farnesol is added when the mycelium is fermented to 0-2d, so that the yield of the grifola frondosa fermentation liquid polysaccharide can be improved, but the influence effect is gradually weakened; the overall change of the pH value is larger, and the pH value is higher than that of fermentation liquor of deep fermentation of grifola frondosa liquid without farnesol; wherein, (a) is a fermentation broth polysaccharide yield graph; (b) is a dry weight graph of the bacteria; (c) is a graph of residual sugar content; (d) a graph of pH change of the fermentation liquor is shown;
FIG. 7 is a graph showing the variation of polysaccharide content, mycelium polysaccharide content and total sugar content in Grifola frondosa fermentation broth after fermentation of a control group and the variation of polysaccharide content, mycelium polysaccharide content and total sugar content in Grifola frondosa fermentation broth after fermentation of a farnesol group added at a concentration of 0.6-0.8mmol/L after fermentation for 0-2 days; the farnesol is added to promote the secretion of the grifola frondosa mycelium polysaccharide into the fermentation liquor, so that the content of the grifola frondosa mycelium polysaccharide is reduced, and the content of the grifola frondosa fermentation liquor polysaccharide is increased;
FIG. 8 is a graph showing the elution curve of ion exchange column chromatography of polysaccharide samples from Grifola frondosa fermentation broth under different fermentation conditions; the acidic polysaccharide component in the polysaccharide of the Grifola frondosa fermentation liquor can be obviously increased by adding farnesol in the fermentation process of the Grifola frondosa; wherein, (a) is the elution curve chart of ion exchange column chromatography of the polysaccharide sample of the Grifola frondosa fermentation liquor without adding farnesol; (b) is an ion exchange column chromatography elution curve diagram of a grifola frondosa fermentation broth polysaccharide sample added with 0.6-0.8mmol/L farnesol during fermentation for 0-2 days;
FIG. 9 is a graph of in vitro antioxidant activity of different components of grifola frondosa fermentation broth polysaccharide under different fermentation conditions in accordance with the present invention; the farnesol is added in the fermentation process of the grifola frondosa, so that the in-vitro antioxidant activity of the medium and acidic polysaccharide components in the grifola frondosa fermentation liquid polysaccharide can be obviously improved, and the antioxidant activity of the acidic polysaccharide is obviously improved particularly; wherein (a) is a diagram of the scavenging capacity of different components of the polysaccharide of the Grifola frondosa fermentation broth on DPPH free radicals; (b) the scavenging capacity of different components of the polysaccharide of the Grifola frondosa fermentation liquor on ABTS free radicals is shown; (c) the scavenging ability of different components of the grifola frondosa fermentation broth polysaccharide on hydroxyl free radicals; (d) the scavenging ability of different components of the polysaccharide of the Grifola frondosa fermentation liquor on superoxide anion free radicals; (e) the reducing power of different components of the polysaccharide of the Grifola frondosa fermentation liquor; the neutral polysaccharide and the acidic polysaccharide without the addition of the farnesol group and the neutral polysaccharide and the acidic polysaccharide with the addition of the farnesol group are respectively recorded as: EPS-C-0M, EPS-C-0.2M, EPS-F-0M, EPS-F-0.2M;
FIG. 10 is a photograph showing the proof of purchase of the strain of Fomitopsis mairei having a preservation number of CGMCC No.5.404, used in the present invention;
FIG. 11 is a photograph of a purchase order of biological materials with a accession number of CGMCC No.5.404 as used in the present invention.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products unless otherwise specified, the methods used in the invention are all conventional methods in the field if not specified, and the quality of each substance used in the invention is conventional quality.
A method for preparing and purifying Grifola frondosa polysaccharide with high antioxidant activity in vitro by adding farnesol at the initial fermentation stage or during fermentation of Grifola frondosa liquid.
Preferably, the Grifola frondosa is Grifola frondosa with the preservation number of CGMCC No.5.404, and the preservation unit is as follows: the general microbiological culture collection center of the China Committee for culture Collection of microorganisms, institute of biological research, China academy of sciences, No. 3, West Lu 1, Beijing, on the North Chen of the Chaoyang district, is shown in FIGS. 10 and 11.
Preferably, the method is to add farnesol at 0-2 d.
Preferably, the final concentration of the farnesol is 0.2-1.0 mmol/L.
Preferably, the method comprises the steps of inoculating grifola frondosa into a fermentation medium, and adding farnesol into the fermentation medium.
Preferably, the liquid fermentation medium contains per L: 22g of glucose, 3g of peptone, 1.2g of monopotassium phosphate, 0.8g of magnesium sulfate heptahydrate and vitamin B 1 0.12g, natural pH.
Preferably, the method comprises the following specific steps:
(1) inoculating the grifola frondosa seed solution into a fermentation culture medium, adding farnesol into the fermentation culture medium at the initial fermentation or during the fermentation of the grifola frondosa, and fermenting for 8d at the temperature of 25-28 ℃ at 160 r/min;
(2) taking the fermentation liquor after the grifola frondosa mycelium is separated according to the following fermentation liquor: the volume ratio of 95% ethanol is 1: 4, mixing with 95% ethanol, precipitating with ethanol at 4 deg.C overnight, centrifuging, removing supernatant, dissolving the precipitate with distilled water, and dissolving at below 60 deg.C for 8 hr to obtain fermentation broth polysaccharide solution;
(3) freeze-drying the obtained polysaccharide solution of the fermentation liquor, then removing protein and decoloring, and freeze-drying; preparing 5mg/mL polysaccharide solution, purifying by DEAE-650M ion exchange column chromatography, eluting with distilled water, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L and 1.0mol/L NaCl solution in sequence, and collecting corresponding components; purifying the polysaccharide solution with corresponding components by a sephadex column to obtain corresponding components, and obtaining the grifola frondosa polysaccharide with high in vitro antioxidant activity.
Preferably, the inoculation is carried out at an inoculum size of 10% by volume.
The edible and medicinal fungi have bioactive components of extracellular polysaccharide prepared by the method.
Specifically, the preparation and detection examples are as follows:
in the present invention, the activation of grifola frondosa, the seed culture, and the culture with fermentation broth, the culture medium and the culture conditions used in the culture with fermentation broth are all conventional culture medium and conventional culture conditions in grifola frondosa culture, which are well known to those skilled in the art.
A liquid fermentation method for preparing grifola frondosa polysaccharide with high in vitro antioxidant activity and improving the yield of grifola frondosa fermentation broth polysaccharide comprises the following steps:
(1) adding exogenous additive farnesol in the liquid fermentation process of the grifola frondosa mycelia;
(2) adding farnesol in the process of Grifola frondosa liquid fermentation broth, and performing fermentation culture for 8d to obtain fermentation broth and Grifola frondosa mycelia;
(3) after the grifola frondosa mycelia and the fermentation broth are separated, the polysaccharide of the fermentation broth is extracted.
Preferably, the farnesol is added for fermentation when the grifola frondosa enters the fermentation stage for 0-2 d.
Preferably, the concentration of the farnesol added in the step of the first step is 0.6-0.8 mmol/L.
Preferably, the first step is performed for 0-2 days of the liquid fermentation of the grifola frondosa mycelia by adding farnesol.
Preferably, the step three of extracting the polysaccharide in the fermentation liquor comprises the following steps:
taking 1 volume and 4 times volume of fermentation liquor, standing with 95% ethanol, precipitating at 4 deg.C overnight, centrifuging, removing supernatant, dissolving precipitate with distilled water, and dissolving at 60 deg.C for 8 hr to obtain fermentation liquor polysaccharide.
More specifically, the preparation and detection are as follows:
the method is suitable for any Grifola frondosa strain liquid submerged fermentation.
And (3) measuring polysaccharide in fermentation liquor: taking 1 and 4 times of 95% ethanol of the fermentation broth, standing, precipitating with ethanol at 4 deg.C overnight, centrifuging, removing supernatant, dissolving the precipitate with distilled water, dissolving at constant temperature below 60 deg.C for 8 hr, and measuring polysaccharide in the fermentation broth of Grifola frondosa by phenol-sulfuric acid method.
And (3) separation and purification of fermentation liquor polysaccharide: dissolving the polysaccharide in the fermentation liquid, mixing with Sevage reagent (chloroform and n-butanol with volume of 4:1) at volume ratio of 3:1, magnetically stirring for 20min, removing protein, dialyzing, and lyophilizing; dissolving the polysaccharide without protein, decolorizing with macroporous resin, and lyophilizing; and (3) separating and purifying the polysaccharide after protein removal and decoloration through DEAE-650M ion exchange column chromatography, eluting with 0-1.0 mol/L NaCl solution, and separating polysaccharide components with different properties.
Example 1 (control group)
Culturing the grifola frondosa seeds: cutting Grifola frondosa grown on PDA plate into 1cm 2 And 3-5 small blocks of the small blocks are taken and inoculated into a seed culture medium with a liquid loading volume of 100mL/250mL triangular flask, and cultured for 5 days at the temperature of 28 ℃ at 160 r/min.
Fermenting and culturing the grifola frondosa: 150mL of fermentation medium is added into a 500mL triangular flask, the inoculation amount is 15mL of seed solution, and the mixture is cultured for 8 days at the temperature of 28 ℃ at the speed of 160 r/min.
The seed culture medium contains 200g of potato, 20g of glucose, 5g of peptone, 2g of monopotassium phosphate, 1g of magnesium sulfate heptahydrate and vitamin B per L 1 0.02g, pH is natural. The fermentation medium contains 22g of glucose, 3g of peptone, 1.2g of monopotassium phosphate, 0.8g of magnesium sulfate heptahydrate and vitamin B per L 1 0.12g, pH natural.
The extraction method and the determination method of the grifola frondosa polysaccharide comprise the following steps:
mixing 1mL of the fermentation broth with 95% ethanol with 4 times of volume, precipitating with ethanol at 4 ℃ for 12h, centrifuging at 10000rpm for 10min, collecting precipitate, and redissolving the precipitate with distilled water to obtain fermentation broth polysaccharide solution. And (3) adopting a phenol-sulfuric acid method to measure the polysaccharide content of the polysaccharide solution of the Grifola frondosa fermentation liquor. The polysaccharide content of the Grifola frondosa fermentation liquor is determined to be (0.5036 + -0.0011) g/L, and various parameters are shown in figure 1.
Example 2
The culture medium and the culture method are the same as the culture method in the example 1, and the differences are that the fermentation culture of the grifola frondosa is carried out for the 0 th to the 2 th days, the exogenous additive farnesol is added for 0 to 0.2mmol/L, and the fermentation liquid of the grifola frondosa after the farnesol is added is continuously fermented for the 8 th days. And (4) obtaining the grifola frondosa mycelia after the fermentation is finished, and extracting fermentation liquor polysaccharide in the fermentation liquor. The extraction method and the determination method of the polysaccharide in the Grifola frondosa fermentation liquor are the same as those in example 1, the content of the polysaccharide in the Grifola frondosa fermentation liquor is determined to be (0.8371 +/-0.0532) g/L, the content is increased by 66 percent compared with a control group, and various parameters are shown in figure 2.
Example 3
The culture medium and the culture method are the same as the culture method in the embodiment 1, and the difference is that the fermentation culture of the grifola frondosa is carried out for the 0 th to the 2 th days, the exogenous additive farnesol is added for 0.2 to 0.4mmol/L, and the fermentation liquid of the grifola frondosa after the farnesol is added is continuously fermented for the 8 th days. And (4) obtaining the grifola frondosa mycelia after the fermentation is finished, and extracting fermentation liquor polysaccharide in the fermentation liquor. The extraction method and the determination method of the polysaccharide in the Grifola frondosa fermentation liquor are the same as those in example 1, the polysaccharide content in the Grifola frondosa fermentation liquor is determined to be (1.0058 +/-0.0128) g/L, the polysaccharide content is increased by 98 percent compared with that in a control group, and various parameters are shown in figure 3.
Example 4
The culture medium and culture method are the same as example 1, except that the fermentation culture of Grifola frondosa is performed at 0-2d, and exogenous additive farnesol is added at 0.4-0.6 mmol/L. And continuously fermenting the Grifola frondosa fermentation liquor added with the farnesol until 8 d. And (4) obtaining the grifola frondosa mycelia after the fermentation is finished, and extracting fermentation liquor polysaccharide in the fermentation liquor. The extraction method and the determination method of the polysaccharide in the Grifola frondosa fermentation liquor are the same as those in example 1, the content of the polysaccharide in the Grifola frondosa fermentation liquor is determined to be (1.1381 +/-0.062) g/L, the content is increased by 126 percent compared with that in a control group, and various parameters are shown in figure 4.
Example 5
The culture medium and the culture method are the same as the culture method in the embodiment 1, and the difference is that the fermentation culture of the grifola frondosa is carried out for the 0 th to the 2 th days, the exogenous additive farnesol is added for 0.6 to 0.8mmol/L, and the fermentation liquid of the grifola frondosa after the farnesol is added is continuously fermented for the 8 th days. After the fermentation is finished, obtaining the grifola frondosa mycelium and extracting fermentation broth polysaccharide in the fermentation broth. The extraction method and the determination method of the polysaccharide in the Grifola frondosa fermentation liquor are the same as those in example 1, the content of the polysaccharide in the Grifola frondosa fermentation liquor is determined to be (1.2528 +/-0.0079) g/L, the content is increased by 149 percent compared with a control group, and various parameters are shown in figure 5.
Example 6
The culture medium and the culture method are the same as the culture method in the example 1, and the differences are that the fermentation culture of the grifola frondosa is carried out for the 0 th to the 2 th days, the exogenous additive farnesol is added for 0.8 to 1.0mmol/L, and the grifola frondosa fermentation liquor added with the farnesol is continuously fermented for the 8 th days. After the fermentation is finished, obtaining the grifola frondosa mycelium and extracting fermentation broth polysaccharide in the fermentation broth. The method for extracting and measuring the polysaccharide in the Grifola frondosa fermentation liquor is the same as that in example 1, the content of the polysaccharide in the Grifola frondosa fermentation liquor is measured to be (1.0833 +/-0.0022) g/L, the content is increased by 115 percent compared with that in a control group, and various parameters are shown in figure 6.
Example 7
A liquid fermentation method for improving polysaccharide yield of Grifola frondosa fermentation liquor comprises the following steps:
the grifola frondosa mycelium is activated, cultured in seed liquid and cultured by liquid submerged fermentation.
And (3) performing fermentation culture on the grifola frondosa at the 0-2d, and adding 0.6-0.8mmol/L of exogenous additive farnesol.
And continuously fermenting the Grifola frondosa fermentation liquor added with the farnesol until 8 d.
After the fermentation is finished, obtaining the grifola frondosa mycelium and extracting fermentation broth polysaccharide in the fermentation broth.
Deproteinizing and decolorizing crude extracellular polysaccharide of Grifola frondosa, purifying by DEAE-650M ion exchange column chromatography, and collecting corresponding polysaccharide components.
The method for determining different polysaccharide components of the grifola frondosa polysaccharide sample purified and collected by DEAE-650M ion exchange column chromatography comprises the following steps:
collecting polysaccharide solutions eluted by NaCl solutions with different concentrations by using an automatic collector, collecting 8mL of polysaccharide solution in each tube, collecting 100 tubes of NaCl solution with each concentration, and measuring the polysaccharide content of the polysaccharide solution in each tube.
And (3) adopting a phenol-sulfuric acid method to measure the polysaccharide content of polysaccharide solutions in different tubes.
As shown in fig. 7 and 8: determining that the neutral polysaccharide content of the polysaccharide sample of the Grifola frondosa fermentation liquor without farnesol is 78.2 percent and the acidic polysaccharide content is 7.4 percent after the polysaccharide sample is purified and collected by DEAE-650M ion exchange column chromatography; the polysaccharide sample of the Grifola frondosa fermentation liquor added with farnesol is purified and collected by DEAE-650M ion exchange column chromatography, the content of neutral polysaccharide is 33.5%, and the content of acidic polysaccharide is 58.2%.
Example 8
In example 5, compared with example 1, the deep fermentation of the grifola frondosa liquid is 8 days, and the fermentation broth polysaccharide is produced by adding 0.6-0.8mmol/L of exogenous additive farnesol, and is remarkably improved compared with example 1 through determination. The in vitro antioxidant activities of the different polysaccharides obtained from Grifola frondosa by the two fermentation methods are different, as shown in FIG. 9: neutral polysaccharide (2.5g/L, marked as EPS-C-0M) which is purified and collected by DEAE-650M ion exchange column chromatography and sephadex column chromatography of a polysaccharide sample of the Grifola frondosa fermentation liquor without farnesol has 49.82% of clearance rate on DPPH free radicals, 65.43% of clearance rate on ABTS free radicals, 78.5% of clearance rate on hydroxyl free radicals and 0.23 of reducing power; the acidic polysaccharide (2.5g/L, recorded as EPS-C-0.2M) collected by purifying a polysaccharide sample of the Grifola frondosa fermentation liquor without the farnesol by DEAE-650M ion exchange column chromatography and sephadex column chromatography has the clearance rate of 64.96% to DPPH free radicals, 96.8% to ABTS free radicals, 83.96% to hydroxyl free radicals and 0.33% reducing force. Neutral polysaccharide (2.5g/L, recorded as EPS-F-0M) which is purified and collected by DEAE-650M ion exchange column chromatography and sephadex column chromatography of a polysaccharide sample of the Grifola frondosa fermentation liquor added with farnesol has the clearance rate of 58.47 percent to DPPH free radicals, 88.10 percent to ABTS free radicals, 98.67 percent to hydroxyl free radicals and 0.35 percent of reducing power; acidic polysaccharides (2.5g/L, recorded as EPS-F-0.2M) collected by purifying a polysaccharide sample of the Grifola frondosa fermentation liquor without adding farnesol by DEAE-650M ion exchange column chromatography and sephadex column chromatography have the clearance rate of 71.68 percent on DPPH free radicals, 98.38 percent on ABTS free radicals, 99.46 percent on hydroxyl free radicals, 98.1 percent on superoxide anion free radicals and 0.38 percent of reducing power.
The yield of the polysaccharide of the Grifola frondosa fermentation liquor is increased compared with that of the polysaccharide of the Grifola frondosa fermentation liquor obtained by liquid fermentation in example 1 under the conditions that fermentation culture is carried out with different addition amounts of farnesol in the same fermentation stage and the total duration of the liquid submerged fermentation of the Grifola frondosa is not changed.
Among examples 2 to 6, the most preferable example is example 5 in which 0.6 to 0.8mmol/L of farnesol as an exogenous additive was added at fermentation time 0 to 2 days.
At this time, the polysaccharide yield of the Grifola frondosa fermentation broth can be improved by at least 148.77% compared to that of example 1 without adding farnesol.
In example 7, the crude extracellular polysaccharide of Grifola frondosa was deproteinized and decolorized, and then purified by DEAE-650M ion exchange column chromatography to collect the corresponding polysaccharide fractions. The content of acidic polysaccharide in the polysaccharide sample of the fermentation liquor after the culture of the grifola frondosa fermentation liquor by adding farnesol is increased from 7.4 percent to 58.2 percent by the purification and collection of DEAE-650M ion exchange column chromatography.
In example 8, compared with the non-added farnesol group, the clearance rate of DPPH free radical is increased from 49.82% to 58.47%, the clearance rate of ABTS free radical is increased from 65.43% to 88.10%, the clearance rate of hydroxyl free radical is increased from 78.50% to 98.67%, the clearance rate of superoxide anion free radical is increased from 94.65% to 96.23%, and the reduction capacity is increased from 0.23 to 0.35 in the neutral polysaccharide after separation and purification of the polysaccharide sample of Grifola frondosa induced by farnesol; compared with the neutral polysaccharide component without the farnesol, the acidic polysaccharide after the separation and purification of the polysaccharide sample of the grifola frondosa induced by the farnesol has the advantages that the clearance rate of DPPH free radicals is increased from 64.96% to 71.68%, the clearance rate of ABTS free radicals is increased from 96.80% to 98.38%, the clearance rate of hydroxyl free radicals is increased from 83.96% to 99.46%, the clearance rate of superoxide anion free radicals is increased from 95.00% to 98.10%, the reduction capacity is increased from 0.33 to 0.38, and the antioxidant activity of the acidic polysaccharide component with the farnesol is increased from 49.82% to 71.68% and is increased by 44%; the clearance rate of ABTS free radicals is increased from 65.43 percent to 98.38 percent, and is improved by 50 percent; the clearance rate of hydroxyl free radicals is increased from 78.50% to 99.46%, and is improved by 27%; the scavenging rate of superoxide anion free radicals is increased from 94.65% to 98.10%, which is increased by 3.6%; the reduction capacity is increased from 0.23 to 0.38, which is increased by 65%.
The data in the above figures were measured and recorded starting from 2d with farnesol added at different times. The data includes two processes of Grifola frondosa mycelium liquid fermentation and farnesol addition fermentation. Meanwhile, the optimal range of the fermentation time length of the farnesol can be calculated. The polysaccharide obtained from the two fermentations is separated and purified to obtain different polysaccharide components, and the in vitro antioxidant activity of the acidic polysaccharide component obtained by farnesol induction is obviously improved.
In conclusion, when the grifola frondosa is fermented for 0-2 days, the farnesol is added for fermentation, so that the yield of polysaccharide in fermentation liquor generated by submerged fermentation of grifola frondosa liquid, particularly the yield of acidic polysaccharide, can be effectively increased, and the in-vitro antioxidant activity of each component of the grifola frondosa can be increased by farnesol induction.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, alterations and modifications are possible without departing from the spirit and scope of this disclosure and appended claims, and accordingly, the scope of this disclosure is not limited to the embodiments disclosed.
Claims (7)
1. A method for preparing and purifying Grifola frondosa (Grifola frondosa) polysaccharide with high in vitro antioxidant activity is characterized in that: the method comprises the steps of adding farnesol at the initial fermentation or in the fermentation process of the grifola frondosa liquid;
the final concentration of the farnesol is 0.2-1.0 mmol/L.
2. The method for preparing and purifying grifolan having high antioxidant activity in vitro as claimed in claim 1, wherein: the Grifola frondosa is Grifola frondosa with the preservation number of CGMCC No.5.404, and the preservation unit is as follows: china general microbiological culture Collection center, Beijing, national institute of sciences, No. 3, Xilu No. 1, Beicheng, Chaoyang, China general microbiological culture Collection center.
3. The method for preparing and purifying grifolan having high antioxidant activity in vitro as claimed in claim 1, wherein: the method is to add farnesol at 0-2 d.
4. The method for preparing and purifying grifola frondosa polysaccharide with high antioxidant activity in vitro as claimed in claim 1, wherein: the method comprises the steps of inoculating grifola frondosa into a fermentation medium, and adding farnesol into the fermentation medium.
5. The method for preparing and purifying grifola frondosa polysaccharide with high antioxidant activity in vitro as claimed in claim 4, wherein: the liquid fermentation medium contains per L: 22g of glucose, 3g of peptone, 1.2g of potassium dihydrogen phosphate, 0.8g of magnesium sulfate heptahydrate and vitamin B 1 0.12g, natural pH.
6. The method for preparing and purifying grifolan having high antioxidant activity in vitro as claimed in claim 1, wherein: the method comprises the following specific steps:
(1) inoculating the grifola frondosa seed liquid into a fermentation culture medium, adding farnesol into the fermentation culture medium at the initial fermentation or in the fermentation process of grifola frondosa, and fermenting for 8d at the temperature of 25-28 ℃ at 160 r/min;
(2) taking the fermentation liquor after the grifola frondosa mycelium is separated according to the following fermentation liquor: the volume ratio of 95% ethanol is 1: 4, mixing with 95% ethanol, precipitating at 4 ℃ overnight, centrifuging, removing supernatant, dissolving precipitate with distilled water, and dissolving at below 60 ℃ for 8h to obtain fermentation broth polysaccharide solution;
(3) freeze-drying the obtained polysaccharide solution of the fermentation liquor, then removing protein and decoloring, and freeze-drying; preparing 5mg/mL polysaccharide solution, purifying by DEAE-650M ion exchange column chromatography, eluting with distilled water, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L and 1.0mol/L NaCl solution in sequence, and collecting corresponding components; purifying the polysaccharide solution with dextran gel column to obtain corresponding components, and obtaining the Grifola frondosa polysaccharide with high in vitro antioxidant activity.
7. The method for preparing and purifying grifola frondosa polysaccharide with high antioxidant activity in vitro as claimed in claim 6, wherein: the inoculation is carried out at an inoculum size of 10% by volume.
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