CN111718428A - Method for preparing water-soluble polysaccharide by using dendrobium officinale fermentation liquor - Google Patents
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Abstract
The invention belongs to the technical field of food process preparation, and discloses a method for preparing water-soluble polysaccharide by using dendrobium officinale fermentation liquor, which comprises the steps of putting the dendrobium officinale fermentation liquor into a freeze dryer for freeze drying, adding deionized water and ethanol, and performing centrifugal freeze drying to obtain crude polysaccharide; dissolving the obtained crude polysaccharide in deionized water, adding Sevage reagent, shaking violently, standing, centrifuging, precipitating, and lyophilizing to obtain deproteinized polysaccharide; weighing deproteinized herba Dendrobii fermented polysaccharide, adding deionized water to dissolve, filtering, eluting, mixing all sugar-containing components, concentrating, and lyophilizing. The preparation method disclosed by the invention is simple and convenient to operate, low in energy consumption, environment-friendly, high in purity of the prepared polysaccharide, and beneficial to subsequent experimental research. The polysaccharide is obtained by alcohol precipitation and repeated freeze-drying, the structure of the polysaccharide is not damaged, and the activity of the polysaccharide is protected. The high-purity water-soluble polysaccharide prepared by the invention can promote the proliferation of macrophages and effectively relieve inflammation.
Description
Technical Field
The invention belongs to the technical field of food process preparation, and particularly relates to a method for preparing water-soluble polysaccharide by using dendrobium officinale fermentation liquor.
Background
Currently, the closest prior art: dendrobium officinale is a medicinal and edible herb in Dendrobium of Orchidaceae, is a traditional and rare Chinese medicinal material, and has the effects of tonifying stomach, promoting fluid production, nourishing yin, clearing heat, replenishing vital essence, tonifying kidney, strengthening essence, tonifying deficiency and the like. The polysaccharide is a main active ingredient in the dendrobium, and has the obvious effects of resisting inflammation, resisting oxidation in vitro, enhancing immunity, resisting tumors, reducing blood sugar and the like. The water-soluble polysaccharide is polymeric sugar with molecular weight larger than 10KD and capable of being dissolved in water, most of the polymeric sugar has obvious antitumor activity and immunoregulation effect, is widely existed in plants, fungi and algae, and can be used as an effect factor of a plurality of health-care foods. The dendrobium officinale in Wuzhishan Hainan is applied, the yield is the best, the mouth feel, the viscosity and the polysaccharide content are relatively high, particularly the glucan content is high, and in order to improve the application value of the dendrobium officinale, the content of water-soluble polysaccharide is improved through fermentation. The fermentation broth currently used is provided by a teacher at the institute of food institute of Hainan university, and is in industrial process production. At present, a plurality of methods for extracting and purifying dendrobium officinale polysaccharides and micromolecular active substances exist, but the extraction of high-purity water-soluble polysaccharides from dendrobium officinale fermentation liquor is not reported at present.
Before obtaining pure polysaccharide, crude polysaccharide is deproteinized, and Sevage reagent (V) is a commonly used method for deproteinizationChloroform：VN-butanol4: 1) because proteins are easily denatured, the denaturation is made to occur in a gel-like form by this property and then removed, which has the advantage of relatively gentle conditions and is not drastic. According to VChloroform：VN-butanol4: mixing at a ratio of 1, mixing with the sample, shaking, allowing protein to denature into gel in chloroform, standing at the junction of organic layer and water layer, and centrifuging. The enzymatic method can also remove proteins, such as pepsin, trypsin, etc., which can degrade the proteins in the sample partially or completely, and the Sevage method can be better used. Research shows that the freeze-thaw-Sevage combined method has high protein removal rate, but more polysaccharide is lost in the treatment process. Compared with the dendrobium officinale protostem, the impurity in the fermentation liquor is a little less, and the protease is easy to be changed, so the Sevage reagent is used for the effectBetter point of fruit
In summary, the problems of the prior art are as follows: the existing method for extracting high-purity water-soluble polysaccharide from dendrobium officinale fermentation liquor has not been reported. The polysaccharide is extracted from the dendrobium officinale directly, the polysaccharide is not reported to be extracted from the dendrobium officinale fermentation liquor, although the content of the polysaccharide extracted from the dendrobium officinale directly is high, the purification of more impurities is difficult, but the polysaccharide after fermentation has fewer impurities and is easy to purify, and the molecular weight is reduced, so the activity of the polysaccharide is increased.
The difficulty of solving the technical problems is as follows: the application of conventional methods for extracting polysaccharides in liquids has not been determined to be feasible at present.
The significance of solving the technical problems is as follows: the preparation method of the material provided by the invention is simple and convenient to operate, low in energy consumption, environment-friendly, high in purity of the prepared polysaccharide, high in purity of the polysaccharide measured by a phenol-sulfuric acid method up to about 95%, and beneficial to subsequent experimental research. The high-purity water-soluble polysaccharide prepared by the invention can promote the proliferation of macrophages and effectively relieve inflammation.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for preparing water-soluble polysaccharide by using dendrobium officinale fermentation liquor.
The invention is realized in such a way that a method for preparing water-soluble polysaccharide by using dendrobium officinale fermentation liquor comprises the following steps:
step one, extracting crude polysaccharide: and (3) freeze-drying the dendrobium officinale fermentation liquor in a freeze dryer, adding deionized water and ethanol, and carrying out centrifugal freeze-drying to obtain crude polysaccharide.
Step two, polysaccharide deproteinization: dissolving the obtained crude polysaccharide in deionized water, adding Sevage reagent, shaking vigorously, standing, centrifuging, precipitating, and lyophilizing to obtain deproteinized polysaccharide.
Step three, separation and purification: weighing deproteinized herba Dendrobii fermented polysaccharide, dissolving in deionized water, filtering with a needle filter, eluting, mixing all sugar-containing components, concentrating, and lyophilizing.
Further, the dendrobium officinale fermentation liquor is prepared by fermenting 5% of dendrobium officinale protostem (with right viscosity and easy filtration).
Further, in the first step, the method for extracting the crude polysaccharide specifically comprises the following steps:
(1) and (3) putting the dendrobium officinale fermentation liquor in a culture dish, and freeze-drying in a freeze dryer for 48 hours.
(2) Adding deionized water into the freeze-dried fermentation liquor, heating in a water bath at 90 ℃ to dissolve, and adding 3 times of 95% ethanol after complete dissolution to obtain flocculent precipitate.
(3) The flocculent precipitate was then centrifuged at 1500r/min for 20min in a centrifuge to remove excess supernatant.
(4) The flocculent precipitate was lyophilized in a lyophilizer for 48 hours to give the crude polysaccharide.
Further, in the second step, the method for deproteinizing polysaccharide specifically comprises:
adding a proper amount of deionized water into the crude polysaccharide obtained in the step one, performing ultrasonic treatment in an ultrasonic machine at the power of 60% at room temperature for 30 minutes, or heating in a water bath kettle at 90 ℃ to promote dissolution, and then adding Sevage reagent (V)Chloroform：VN-butanol4: 1) the ratio of polysaccharide solution and Sevage reagent was 3: volume 1 was mixed vigorously for 30 minutes.
(II) standing for 3 hours, and centrifuging at the rotating speed of 5000r/min for 10min to separate out the precipitate between the organic layer and the water layer. After separating, the organic layer was recovered, and 3 times of 95% ethanol was further added to the aqueous solution to obtain a precipitate.
And (III) continuously freeze-drying the precipitate obtained in the step (II) to obtain deproteinized polysaccharide. And (3) measuring the content of the polysaccharide by using a phenol-sulfuric acid method.
Further, in the third step, the method for separating and purifying specifically comprises:
1) weighing deproteinized herba Dendrobii fermented polysaccharide, adding appropriate amount of deionized water to dissolve completely, and filtering with needle filter with diameter of 0.22 μ L and diameter of 1.2 cm.
2) And (3) decolorizing and primarily separating the filtrate by using DEAE-52 anion exchange column chromatography, performing gradient elution by using deionized water and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8mol/L sodium chloride solution as an elution machine, and receiving in equal amount.
3) All sugar-containing fractions were combined, concentrated and lyophilized.
4) The decolorized polysaccharide was weighed, dissolved in an appropriate amount of deionized water, and filtered through a syringe filter with a pore size of 0.22. mu.L and a diameter of 1.2 cm.
5) The filtrate was further purified by SephadexG-100 gel chromatography using deionized water as the eluent at a flow rate of 0.6ml/min and received in equal amounts (3.0 ml/tube). The polysaccharide purity measured by phenol-sulfuric acid method is as high as about 95 percent by developing color by phenol-sulfuric acid method
6) Combining all sugar-containing components, concentrating, and lyophilizing.
7) Gel Permeation Chromatography (GPC) determines the molecular weight, and a single symmetrical peak is obtained to prove that the sample is pure polysaccharide. The polysaccharide separated by the method has high purity and less impurities, and maintains good biological activity.
In summary, the advantages and positive effects of the invention are: the invention can directly remove the protein from the polysaccharide obtained by soaking the fermentation liquor with alcohol to obtain the pure water-soluble polysaccharide, and the impurities such as starch, protein and the like in the polysaccharide can be reduced by repeated freeze thawing. The invention obtains the polysaccharide by alcohol precipitation and repeated freeze-drying, thus not damaging the structure of the polysaccharide and being beneficial to protecting the activity of the polysaccharide. The molecular weight of the polysaccharide after the dendrobium officinale is fermented can be reduced, so that the activity of the polysaccharide can be improved by reducing the molecular weight to a certain degree, and the immunocompetence of the polysaccharide can be improved to a certain degree.
The preparation method of the material provided by the invention is simple and convenient to operate, low in energy consumption, environment-friendly, high in purity of the prepared polysaccharide, high in purity of the polysaccharide measured by a phenol-sulfuric acid method up to about 95%, and beneficial to subsequent experimental research. The high-purity water-soluble polysaccharide prepared by the invention can promote the proliferation of macrophages and effectively relieve inflammation.
Drawings
FIG. 1 is a flow chart of a method for preparing water-soluble polysaccharide from a fermentation broth of Dendrobium officinale.
FIG. 2 is a flow chart of an implementation of the method for preparing water-soluble polysaccharide from fermentation broth of Dendrobium officinale according to the present invention.
FIG. 3 is an infrared spectrum of deproteinized polysaccharide provided by an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for preparing water-soluble polysaccharide by using dendrobium officinale fermentation liquor, and the invention is described in detail with reference to the attached drawings.
As shown in fig. 1, the method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation broth provided by the embodiment of the invention comprises the following steps:
s101: extracting crude polysaccharide: and (3) freeze-drying the dendrobium officinale fermentation liquor in a freeze dryer, adding deionized water and ethanol, and carrying out centrifugal freeze-drying to obtain crude polysaccharide.
S102: polysaccharide deproteinization: dissolving the obtained crude polysaccharide in deionized water, adding Sevage reagent, shaking vigorously, standing, centrifuging, precipitating, and lyophilizing to obtain deproteinized polysaccharide.
S103: separation and purification: weighing deproteinized herba Dendrobii fermented polysaccharide, dissolving in deionized water, filtering with a needle filter, eluting, mixing all sugar-containing components, concentrating, and lyophilizing.
The dendrobium officinale provided by the embodiment of the invention is produced in 5 months from Wuzhishan Hainan, and has the best yield and polysaccharide content, and the best mouthfeel, viscosity and glucan content.
The dendrobium officinale fermentation liquor provided by the embodiment of the invention is prepared by fermenting plant lactic acid bacteria stored in a screened Chinese academy of sciences.
The dendrobium officinale fermentation liquor provided by the embodiment of the invention is prepared by fermenting 5% of dendrobium officinale protostem (with right viscosity and easy filtration).
The technical solution of the present invention is further described with reference to the following examples.
Example 1: extraction of polysaccharides
In the method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation liquor provided by the embodiment 1 of the invention, the specific method for extracting polysaccharide comprises the following steps:
(1) weighing a proper amount of dendrobium officinale fermentation liquor, filling the dendrobium officinale fermentation liquor in a culture dish, freeze-drying the dendrobium officinale fermentation liquor for 48 hours, dissolving the dendrobium officinale fermentation liquor in deionized water, adding 95% ethanol in an amount which is 3 times the volume of the dendrobium officinale fermentation liquor, uniformly stirring, sealing and placing the mixture at room temperature for 12 hours.
(2) And (3) respectively filling the soaking solution of the ethanol into 50ml centrifuge tubes, and performing centrifugal separation to obtain solid precipitates, wherein the centrifugal rotation speed is 8000r/min, the centrifugation time is 20min, and the centrifugation temperature is 4 ℃.
(3) After centrifugation, the supernatant and the pellet were separated and the pellet was lyophilized in a lyophilizer for 48 h.
(4) The lyophilized sample was weighed to obtain crude polysaccharide.
Example 2: deproteinization
In the method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation liquor provided by embodiment 2 of the invention, the specific method for deproteinizing comprises the following steps:
(1) dissolving crude polysaccharide in deionized water, and heating in water bath or ultrasonic treating if the crude polysaccharide is not dissolved sufficiently.
(2) Sevage reagent (V) was then addedChloroform：VN-butanol4: 1) the ratio of polysaccharide solution and Sevage reagent was 3: volume 1 was mixed vigorously for 30 minutes.
(3) After standing for 3 hours, the mixture was centrifuged at 5000r/min for 10 minutes to form an organic layer and an aqueous layer.
(4) The organic layer and the aqueous layer were separated, and three times the amount of the absolute ethanol solution was further added to the aqueous layer, and the mixture was allowed to stand overnight until the precipitate was completely precipitated.
(5) And then, carrying out centrifugal separation on the water solution of the precipitate to obtain solid precipitate, wherein the centrifugal rotation speed is 8000r/min, the centrifugation time is 20min, and the centrifugation temperature is 4 ℃.
(6) And separating the supernatant from the precipitate after centrifugation, and freeze-drying the precipitate in a freeze dryer for 24 hours to obtain the deproteinized polysaccharide.
FIG. 3 is an infrared spectrum of polysaccharide after deproteinization in the example of the present invention, and the infrared spectrum shows that the stretching vibration of-OH at 3417cm-1 and the stretching vibration of C-H bond in-CH 2 or-CH 3 at 2927cm-1 are caused, and these two peaks are characteristic peaks of polysaccharide. The characteristic peak at 809cm-1 shows that the polysaccharide contains D-mannose.
Example 3: separating and purifying
In the method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation liquor provided by the embodiment 3 of the invention, the specific method for separating and purifying comprises the following steps:
(1) weighing deproteinized herba Dendrobii fermented polysaccharide, adding appropriate amount of deionized water to dissolve completely, and filtering with needle filter with diameter of 0.22 μ L and diameter of 1.2 cm.
(2) And (3) performing decoloration and primary separation on the filtrate by using DEAE-52 anion exchange column chromatography, performing gradient elution by using deionized water and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8mol/L sodium chloride solution as eluent respectively, and receiving in equal amount.
(3) All sugar-containing fractions were combined, concentrated and lyophilized.
(4) The separated polysaccharide was weighed, dissolved in an appropriate amount of deionized water, and filtered through a syringe filter having a pore size of 0.22. mu.L and a diameter of 1.2 cm.
(5) The filtrate was further purified by SephadexG-100 gel chromatography using deionized water as the eluent at a flow rate of 0.6ml/min and received in equal amounts (3.0 ml/tube). The polysaccharide is developed by a phenol-sulfuric acid method, and the purity of the polysaccharide measured by the phenol-sulfuric acid method is as high as about 95 percent.
(6) Combining all sugar-containing components, concentrating, and lyophilizing.
Example 4
The high-purity water-soluble polysaccharide prepared by the method for preparing the water-soluble polysaccharide by using the dendrobium officinale fermentation liquor can promote the proliferation of mouse macrophage RAW264.7 and down-regulate proinflammatory factor interleukin 6(IL-6) and tumor necrosis factor (TNF-alpha).
The technical effects of the present invention will be further described below with reference to specific experiments.
1. Detecting proliferation effect of polysaccharide on RAW264.7 cells by MTT method, collecting RAW264.7 cells in logarithmic growth phase, and performing quantitative analysis with 5 × 105Inoculating to 96-well cell culture plate, culturing in 100 μ L each well for 24 hr, and changing to polysaccharide concentration of Dendrobium officinale 0 (control), 25, 50, 100, 200, 400 μ g/mL-1The culture medium of (1) in 5% CO2And culturing for 48 hours in an incubator at 37 ℃, absorbing the culture solution, adding 20 mu l of MTT into each hole, continuously culturing for 4 hours under the conditions, abandoning the supernatant, adding 150 mu l of DMSO into each hole, oscillating for 10min on an oscillator, detecting the light absorption value by a microplate reader at the wavelength of 570nm, calculating the relative cell proliferation rate (the light absorption value of an experimental group-the light absorption value of a control group)/the light absorption value of the control group × 100%, and finding that the polysaccharide can promote the proliferation of macrophages.
2. Taking RAW264.7 cells 1 × 10 in logarithmic growth phase5The method comprises the steps of inoculating the cells in a 96-well cell culture plate, arranging a blank group, a model group and a treatment group, wherein the blank group is only added with a culture medium and cells, the model group is added with lipopolysaccharide dissolved in the culture medium, the treatment group is changed into polysaccharide solutions with different concentrations after the lipopolysaccharide is added for 6 hours, and after the lipopolysaccharide is cultured for 24 hours, supernatant is taken to measure the content of IL-6 and TNF- α, 5 repeated wells are carried out in each experimental group for 3 times, an ELISA kit is adopted, a specific operation method is carried out according to the kit specification, the absorbance is measured at 450nm, the content of IL-6 and TNF- α in the model group is increased, the content of IL-6 and TNF- α in the treatment group is decreased, and the polysaccharide in fermentation liquid can effectively relieve inflammation.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for preparing water-soluble polysaccharide by using dendrobium officinale fermentation liquor is characterized by comprising the following steps:
step one, extracting crude polysaccharide: freeze-drying the dendrobium officinale fermentation liquor in a freeze dryer, adding deionized water and ethanol, and performing centrifugal freeze-drying to obtain crude polysaccharide;
step two, polysaccharide deproteinization: dissolving the obtained crude polysaccharide in deionized water, adding Sevage reagent, shaking violently, standing, centrifuging, precipitating, and lyophilizing to obtain deproteinized polysaccharide;
step three, separation and purification: weighing deproteinized herba Dendrobii fermented polysaccharide, dissolving in deionized water, filtering with a needle filter, eluting, mixing all sugar-containing components, concentrating, and lyophilizing.
2. The method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation broth as claimed in claim 1, wherein in the first step, the method for extracting the crude polysaccharide specifically comprises:
(1) putting the dendrobium officinale fermentation liquor in a culture dish, and freeze-drying in a freeze dryer for 48 hours;
(2) adding deionized water into the freeze-dried fermentation liquor, heating in a water bath at 90 ℃ to dissolve the fermentation liquor, and adding 3 times of 95% ethanol after complete dissolution to obtain flocculent precipitate;
(3) centrifuging the flocculent precipitate obtained in the step (2) in a centrifuge to remove redundant supernatant;
(4) the flocculent precipitate was lyophilized in a lyophilizer for 48 hours to give the crude polysaccharide.
3. The method for preparing water-soluble polysaccharide by using the fermentation liquid of Dendrobium officinale Kimura et Migo as claimed in claim 2, wherein in the step (1), the fermentation liquid of Dendrobium officinale Kimura et Migo is prepared by fermenting 5% of Dendrobium officinale Kimura et Migo protostem.
4. The method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation liquor as claimed in claim 2, wherein in the step (3), the centrifugation speed is 1500r/min, and the centrifugation time is 20 min.
5. The method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation broth as claimed in claim 1, wherein in the second step, the method for deproteinizing polysaccharide specifically comprises:
(1) adding a proper amount of deionized water into the crude polysaccharide obtained in the step one, and then heating by utilizing ultrasound or water bath to promote dissolution; sevage reagent (V) was addedChloroform：VN-butanol4: 1) the ratio of polysaccharide solution and Sevage reagent was 3: 1 volume mixed vigorously for 30 minutes;
(2) standing for 3 hours, centrifuging in a centrifuge, and separating out precipitate between an organic layer and a water layer; after separating, recovering the organic layer, and continuously adding 3 times of 95% ethanol into the water solution to obtain a precipitate;
(3) continuously freeze-drying the precipitate obtained in the step (2) to obtain deproteinized polysaccharide; and (3) measuring the content of the polysaccharide by using a phenol-sulfuric acid method.
6. The method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation broth as claimed in claim 5, wherein in the step (1), the amount of the ionized water is 20-30 times of the volume of the polysaccharide; the method for promoting dissolution is ultrasonic treatment at room temperature of 60% for 30 min, or heating in water bath at 90 deg.C.
7. The method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation liquor as claimed in claim 5, wherein in the step (2), the centrifugal speed is 5000r/min, and the centrifugal time is 10 minutes.
8. The method for preparing water-soluble polysaccharide by using the dendrobium officinale fermentation broth as claimed in claim 1, wherein in the third step, the method for separating and purifying specifically comprises:
1) weighing deproteinized herba Dendrobii fermented polysaccharide, adding appropriate amount of deionized water to dissolve completely, and filtering with a needle filter with a pore diameter of 0.22 μ L and a diameter of 1.2 cm;
2) performing DEAE-52 anion exchange column chromatography for decolorization and primary separation;
3) mixing all sugar-containing components, concentrating, and lyophilizing;
4) weighing decolorized polysaccharide, dissolving with appropriate amount of deionized water, and filtering with a needle filter with pore diameter of 0.22 μ L and diameter of 1.2 cm;
5) further purifying the filtrate obtained in the step 4) by using a SephadexG-100 gel chromatographic column; developing by a phenol-sulfuric acid method;
6) combining all sugar-containing components, concentrating, and freeze-drying;
7) gel permeation chromatography GPC determines the molecular weight, obtains single symmetrical peak to prove the sample is pure polysaccharide.
9. The method for preparing water-soluble polysaccharide from the fermentation broth of Dendrobium officinale Kimura et Migo as claimed in claim 8, wherein in step 2), the gradient elution is performed with deionized water and 0.1-0.8mol/L NaCl solution as elution machine, and the elution is received in equal amount.
10. The method for preparing water-soluble polysaccharide from the fermentation broth of Dendrobium officinale Kimura et Migo of claim 8, wherein in step 4), deionized water is used as the eluent, the flow rate is 0.6ml/min, and the eluent is received in an equal amount of 3.0 ml/tube.
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| CN115819639A (en) * | 2023-01-10 | 2023-03-21 | 仲恺农业工程学院 | Preparation method and application of small-molecular-weight dendrobium officinale polysaccharide |
| CN115819639B (en) * | 2023-01-10 | 2024-03-22 | 仲恺农业工程学院 | Preparation method and application of dendrobium candidum polysaccharide with small molecular weight |
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