CN112812199A - Response surface method-based polymeric grass polysaccharide for optimizing enzyme-assisted extraction and preparation method and application thereof - Google Patents

Response surface method-based polymeric grass polysaccharide for optimizing enzyme-assisted extraction and preparation method and application thereof Download PDF

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CN112812199A
CN112812199A CN202110067351.4A CN202110067351A CN112812199A CN 112812199 A CN112812199 A CN 112812199A CN 202110067351 A CN202110067351 A CN 202110067351A CN 112812199 A CN112812199 A CN 112812199A
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polysaccharide
comfrey
response surface
assisted extraction
extraction
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尚红梅
张南翼
郭洋
张鹤翔
曹梓航
刘梦雪
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Jilin Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents

Abstract

The invention discloses a comfrey polysaccharide based on a response surface method and optimized enzyme-assisted extraction, and a preparation method and application thereof, and belongs to the technical field of raw material deep processing technology. The preparation method of the polymeric grass polysaccharide comprises the following steps: the method comprises the following steps: extracting; step two: precipitating with ethanol to remove impurities; step three: deproteinizing; step four: dialyzing to remove salt; step five: DEAE-52 cellulose column chromatography; step six: sepharose CL-6B agarose gel column chromatography; the invention also provides application of the polysaccharide in regulating and controlling the abundance of probiotics in chicken intestinal tracts, and establishes a complete and feasible technical route for researching the polysaccharide, such as extraction, separation and purification, physicochemical properties and biological activity.

Description

Response surface method-based polymeric grass polysaccharide for optimizing enzyme-assisted extraction and preparation method and application thereof
Technical Field
The invention relates to the technical field of deep processing technology of plant raw materials, in particular to a comfrey polysaccharide based on response surface method optimized enzyme-assisted extraction and a preparation method and application thereof.
Background
Intestinal health refers to the common action of various physiological activities, intestinal flora and physiological functions to maintain the intestinal homeostasis. Intestinal health is closely related to animal production performance, and in the animal production industry, intestinal health and animal health are synonymous terms. Among the factors that maintain intestinal health, the intestinal flora plays a key role in animal health and production as a regulator of animal metabolic reactions, immune system and health. Good gut flora is crucial for optimal growth performance of poultry, while unfavorable gut flora may promote gut infections, leading to decreased growth rate and increased mortality.
In recent years, the development of natural growth promoters as a substitute for antibiotics in animal husbandry has become a hotspot. In fact, most natural growth promoters work by modifying the intestinal flora. Polysaccharide is a biological macromolecule and is a substrate selectively utilized by intestinal flora. The polysaccharide can change intestinal flora, promote gastrointestinal balance, and improve host health.
Comfrey (Symphytum officinale L.) is a perennial herb of the family lithospermaceae and is widely used worldwide as a kind of pasture and medicinal plant. Polysaccharides are one of the bioactive components of polymeric grasses. The research on the polysaccharide of the polyrhachis has not been deep enough at present. Only individual scholars analyze the hot water extraction, in-vitro oxidation resistance and blood sugar reducing activity of the polysaccharide of the comfrey, and the optimization of the polysaccharide enzyme auxiliary extraction method of the comfrey and the research on the influence of the polysaccharide enzyme auxiliary extraction method on the cecal intestinal flora of the chickens are not carried out.
Disclosure of Invention
The invention aims to provide a preparation method of polyrhachis vicina Roger polysaccharide based on optimized enzyme-assisted extraction by a response surface method, which comprises the steps of firstly obtaining the optimal extraction condition by taking the yield of the polyrhachis vicina Roger polysaccharide as a response value through a single-factor test and a response surface optimization test, verifying, and then preparing the polyrhachis vicina Roger polysaccharide according to the optimal extraction condition, so that the average value of the yield of the final polyrhachis vicina Roger polysaccharide is 20.32%, and the theoretical value obtained by an approximate model is 20.81%; and a fermentation model is established by taking the content of the caecum of the chickens as a raw material, and the influence of the polysaccharide of the polyrhachis vicina on the composition of the flora of the culture system is researched.
In order to test the above objects, the present invention provides a method for preparing polyrhachis vicina Roger polysaccharide based on response surface optimization and enzyme-assisted extraction, comprising the following steps:
the method comprises the following steps: extraction of
Soaking the comfrey powder in water, adding 1% of complex enzyme, regulating the pH value to 6, soaking to obtain an extracting solution, filtering the extracting solution, and concentrating the filtrate to obtain a concentrated solution; wherein the soaking time is 60min, and the dosage ratio of the water to the comfrey powder is 20 mL/g;
step two: precipitating with ethanol to remove impurities
Precipitating the concentrated solution with ethanol to remove water-soluble impurities to obtain crude polysaccharide precipitate of herba Euphorbiae Humifusae;
step three: deproteinization
Removing protein from the obtained crude polysaccharide of the comfrey by adopting a Sevag method;
step four: desalting by dialysis
Removing inorganic salt and micromolecular substances from the crude polysaccharide of the polymerized grass after the protein is removed by a dialysis method to obtain desalted polymerized grass polysaccharide;
step five: DEAE-52 cellulose column chromatography
Subjecting the obtained desalted polymeric grass polysaccharide to DEAE-52 cellulose column chromatography, collecting and combining the collected liquid of main peak, concentrating, and lyophilizing;
step six: sepharose CL-6B agarose gel column chromatography
And (3) carrying out Sepharose CL-6B agarose gel column chromatography on the obtained freeze-dried polymeric grass polysaccharide, collecting and merging eluent of main peaks, concentrating, and freeze-drying to obtain the polymeric grass crude polysaccharide.
Preferably, the polymeric grass powder in the first step is obtained by the following method:
cleaning herba Potentillae Discoloris, oven drying, pulverizing, and sieving to obtain herba Potentillae Discoloris powder.
Preferably, the drying temperature is 50 ℃.
Preferably, the screen used for said screening has a size of 1 mm.
Preferably, the soaking temperature in the first step is 50 ℃.
Preferably, the complex enzyme in the step one comprises cellulase, pectinase and papain.
Preferably, the mass ratio of the cellulase to the pectinase to the papain is 1: 1: 1.
preferably, the concentration of the ethanol added in the alcohol precipitation and impurity removal in the second step is 75-80%.
In addition, the invention also provides the polymeric grass polysaccharide prepared by the preparation method.
In addition, the invention also provides application of the polyrhachis vicina in regulating and controlling the abundance of probiotics in chicken intestinal tracts.
The invention has the advantages of
Compared with the prior art, the method adopts the process of response surface optimization to extract the polymeric grass polysaccharide, so that the average value of the yield of the polymeric grass polysaccharide reaches 20.32 percent, which is similar to the theoretical value of 20.81 percent calculated by Design Expert Software 8.0.6, and the polymeric grass polysaccharide is subjected to alcohol precipitation, deproteinization and desalting, and then subjected to column chromatography twice to obtain better purification effect; the comfrey polysaccharide extracted by the preparation method provided by the invention has the activity of promoting the proliferation of lactic acid bacteria; the invention establishes a complete and feasible technical route for researching the extraction, separation and purification, physicochemical properties and biological activity of the polysaccharide of the comfrey.
Drawings
FIG. 1 is a chromatographic elution graph of a polymeric grass polysaccharide DEAE-52 cellulose column in example 1 provided by the present invention;
FIG. 2 is the Sepharose CL-6B agarose gel column chromatography elution graph of the polysaccharide of the grass of example 1;
FIG. 3 is a graph showing the effect of liquid-to-feed ratio on polysaccharide yield in response surface optimization test provided by the present invention;
FIG. 4 is a graph showing the effect of extraction time on the yield of comfrey polysaccharide in response surface optimization experiments provided by the present invention;
FIG. 5 is a graph showing the effect of the amount of complex enzyme on the yield of comfrey polysaccharide in response surface optimization experiments provided by the present invention;
FIG. 6 is a graph showing the effect of pH on the yield of comfrey polysaccharide in response surface optimization experiments provided by the present invention;
FIG. 7 is an infrared spectrum of the polymerized grass polysaccharide of example 1;
FIG. 8 is a chromatogram of the molecular weight of comfrey polysaccharide of example 1;
FIG. 9 is a chromatogram of a monosaccharide standard of example 1 provided in the present invention;
FIG. 10 is a chromatogram of monosaccharide composition of polysaccharide of Polyporus frondosa of example 1;
FIG. 11 is a graph of carbohydrate consumption rate of the polymeric grass polysaccharide fermentation group of example 1 provided by the present invention;
FIG. 12 is a graph of petals of the polymeric grass polysaccharide operating sorting units (OTUs) at different fermentation times according to example 1 of the present invention;
FIG. 13 is a graph of principal axis analysis (PCoA) based on weighted UniFrac distance in the fermentation test of comfrey polysaccharide according to example 1 of the present invention;
FIG. 14 is a chart showing the composition of microorganisms at genus level in the fermentation test of comfrey polysaccharide according to example 1 of the present invention.
Detailed Description
Firstly, the invention provides a response surface method-based preparation method for optimizing enzyme-assisted extraction of comfrey polysaccharide, which comprises the following steps:
the method comprises the following steps: extraction of
Washing, drying, crushing and sieving the comfrey, wherein the drying temperature of the comfrey is preferably 50 ℃, and the size of the sieve is preferably 1mm, so as to obtain comfrey powder; soaking the obtained polymeric grass powder in water, adding a complex enzyme and adjusting the pH value to obtain an extracting solution, wherein the soaking temperature of the polymeric grass powder is preferably 50 ℃, the soaking time of the polymeric grass powder is preferably 60min, the dosage ratio of water to the polymeric grass powder is preferably 20mL/g, the complex enzyme is used as a water extraction condition, the dosage is preferably 1%, the specific components and the mass ratio of the complex enzyme are cellulase, pectinase and papain are 1: 1, and the pH value is preferably adjusted to 6; filtering the extract after soaking, and concentrating the filtrate to obtain a concentrated solution;
step two: precipitating with ethanol to remove impurities
Slowly adding anhydrous ethanol into the concentrated solution to obtain a mixed solution, wherein the final concentration of the ethanol is preferably 75-80%, preferably standing at 0-4 ℃ for 12-24h, then centrifuging, the centrifuging speed is preferably 3000-3500r/min, the centrifuging time is preferably 10-15min, and removing the supernatant after centrifuging to obtain a polymeric grass polysaccharide precipitate;
step three: deproteinization
Dissolving the obtained polymeric grass polysaccharide precipitate in water, wherein the mass-to-water volume ratio of the polymeric grass polysaccharide is 1-1.5 g: 100-; the repeated deproteinization is preferably performed for 5-8 times, and the crude polysaccharide is obtained by rotary evaporation vacuum concentration, wherein the rotary evaporation vacuum concentration temperature condition is preferably 50-60 ℃.
Step four: desalting by dialysis
The proper amount of deproteinized crude polysaccharide solution of the comfrey is filled in a dialysis bag, a space of 1/3-1/2 is preferably reserved after two ends of the dialysis bag are tied, the specification of the dialysis bag is preferably 1400-2000Da molecular weight cutoff, after the dialysis is carried out by distilled water at room temperature, the distilled water is completely replaced for dialysis, and the dialysis time of the first distilled water at room temperature is preferably as follows: 16-18h, wherein the time of the clean distilled water for dialysis is preferably 10-12h, and the crude polysaccharide of the comfrey is obtained by concentration and freeze-drying after dialysis.
Step five: DEAE-52 cellulose column chromatography
Dissolving crude polysaccharide of comfrey in water, preferably adjusting the concentration to 80-100mg/mL, sampling, performing chromatography on DEAE-52 cellulose column (3.5cm × 20cm), preferably eluting with 1-1.5mL of distilled water, sequentially eluting with NaCl solutions (0.1, 0.3, 0.5, 1.5mol/L) with different concentrations, preferably at a flow rate of 2mL/min, and collecting the eluate (8 mL/tube);
the polysaccharide content of each tube is determined by adopting a phenol-sulfuric acid method: mixing each tube of eluate (60 μ L) with concentrated sulfuric acid (150 μ L) and phenol solution (30 μ L, 6%, w/v) for reaction, preferably for 30min, recording reaction absorbance at 490nm after reaction, drawing elution curve, combining the collected liquid of main elution peak, concentrating, and lyophilizing.
Step six: sepharose CL-6B agarose gel column chromatography
Dissolving the lyophilized polysaccharide in water, preferably adjusting the concentration to 50-100mg/mL, loading to Sepharose CL-6B Sepharose column (2.6cm × 100cm), preferably 1-2mL, eluting with distilled water, preferably 0.9mL/min, collecting at flow rate of 4.5min for 1 tube, preferably 4.05mL for each tube;
the polysaccharide content of each tube is determined by adopting a phenol-sulfuric acid method:
mixing each tube of eluate (60 μ L), concentrated sulfuric acid (150 μ L) and phenol solution (30 μ L, 6%, w/v) for reaction, preferably for 30min, recording reaction absorbance at 490nm, drawing elution curve, mixing the collected solutions of main elution peaks, concentrating, and lyophilizing.
In addition, the invention also provides the polymeric grass polysaccharide obtained by the preparation method.
In addition, the invention also provides application of the polyrhachis vicina polysaccharide extracted by optimizing the enzyme assistance based on the response surface method in regulating and controlling the abundance of probiotics in the intestinal tract of the chicken.
The present invention is described in further detail below with reference to specific examples, in which the starting materials are all commercially available.
Example 1
Cleaning herba comfreae, oven drying at 50 deg.C, pulverizing, and sieving with 1mm sieve to obtain herba comfreae powder; soaking the obtained polymeric grass powder in water at 50 ℃ for 60min, simultaneously adding 1% of complex enzyme and adjusting the pH value to 6 to obtain an extracting solution, wherein the dosage ratio of the water to the polymeric grass powder is 20mL/g, and the specific components and the mass ratio of the complex enzyme are cellulase, pectinase and papain which are 1: 1; filtering the extract after soaking, and concentrating the filtrate to obtain a concentrated solution;
step two: precipitating with ethanol to remove impurities
Slowly adding 75% anhydrous ethanol into the concentrated solution to obtain a mixed solution, standing at 4 deg.C for 12h, centrifuging at 3000r/min for 15min, and removing supernatant to obtain polysaccharide precipitate;
step three: deproteinization
Dissolving the obtained polysaccharide precipitate in water, adding Sevag reagent (n-butyl alcohol: chloroform: 1:4) with the same volume as the water at the mass-to-volume ratio of 1g to 100mL, shaking vigorously for 15min, centrifuging at 5000r/min for 10min, demixing the liquid after centrifugation, carefully absorbing the supernatant, repeatedly deproteinizing for 8 times, and performing rotary evaporation and vacuum concentration at 50 ℃ to obtain the deproteinized polysaccharide.
Step four: desalting by dialysis
And (3) filling a proper amount of deproteinized crude polysaccharide solution of the comfrey into a dialysis bag with the specification of 1400Da, binding two ends of the dialysis bag, leaving a space of 1/3, dialyzing with distilled water at room temperature for 18h, then completely exchanging the distilled water, dialyzing for 10h, concentrating after dialysis, and freeze-drying to obtain the crude polysaccharide of the comfrey.
Step five: DEAE-52 cellulose column chromatography
Dissolving crude polysaccharide of comfrey in water, regulating concentration to 100mg/mL, loading 1mL of the crude polysaccharide into DEAE-52 cellulose column (3.5cm × 20cm), eluting with distilled water, sequentially eluting with NaCl solutions (0.1, 0.3, 0.5 and 1.5mol/L) with different concentrations at a flow rate of 2mL/min, and collecting eluate (8 mL/tube) by tube;
the polysaccharide content of each tube is determined by adopting a phenol-sulfuric acid method: mixing each tube of eluate (60 μ L) with concentrated sulfuric acid (150 μ L) and phenol solution (30 μ L, 6%, w/v) for reaction for 30min, recording reaction absorbance at 490nm after reaction, drawing elution curve, combining the collected liquids of main elution peaks as shown in figure 1, concentrating, and lyophilizing.
Step six: sepharose CL-6B agarose gel column chromatography
Dissolving the lyophilized polysaccharide in water, adjusting the concentration to 50mg/mL, loading 1mL into Sepharose CL-6B Sepharose column (2.6cm × 100cm), performing chromatography with distilled water as eluent at flow rate of 0.9mL/min, collecting 1 tube at 4.5min, and collecting 4.05mL in each tube;
the polysaccharide content of each tube is determined by adopting a phenol-sulfuric acid method:
mixing each tube of eluate (60 μ L), concentrated sulfuric acid (150 μ L) and phenol solution (30 μ L, 6%, w/v) for reaction for 30min, recording reaction absorbance at 490nm after reaction, drawing elution curve, combining the eluates of main elution peaks as shown in figure 2, concentrating, and lyophilizing. Response surface method optimization enzyme-assisted extraction technology investigation of comfrey polysaccharide
Based on a single-factor test, a Box-Behnken method of Design Expert Software 8.0.6 Software is used for experimental Design, and the yield of the polymeric grass polysaccharide is used as a response value to obtain the optimal extraction condition.
1. Single factor test
Cleaning herba Centellae, oven drying at 50 deg.C, pulverizing, and sieving with 1mm sieve; weighing 1g of a comfrey powder sample, placing the comfrey powder sample in a 50mL centrifuge tube for a single-factor experiment, and observing the influence of each extraction factor on the extraction of the comfrey polysaccharide by taking the comfrey polysaccharide yield as an index; the extraction temperature was 50 ℃. Fixing other conditions, and respectively examining the liquid-material ratio (X)1) (10, 15, 20, 25, 30mL/g), extraction time (X)2) (50, 60, 70, 80, 90min), and the amount of complex enzyme (X)3) (each enzyme: comfrey powder ═ 0.5, 1.0, 1.5, 2.0, 2.5%, w/w), pH (X)4) (3, 4, 5, 6, 7) influence on yield of comfrey polysaccharide.
2. Response surface optimization test
On the basis of a single-factor test, the Box-Behnken method of Design Expert Software 8.0.6 Software is used for optimizing main factors influencing polysaccharide yield, 4-factor 3-level test Design is carried out, the polysaccharide yield of the comfrey is taken as a response value, the optimal extraction condition is obtained, and verification is carried out. The experimental design factors and levels are shown in table 1.
TABLE 1Box-Behnken design factors and horizon table
Figure BDA0002904629920000081
3. Test results
(1) Liquid to feed ratio
As shown in figure 3, the diffusion speed of the solvent into the polymeric grass cells is increased along with the increase of the liquid-to-material ratio, and the yield of the polymeric grass polysaccharide reaches a peak value when the yield is 20 mg/mL. However, when the liquid-to-feed ratio is further increased, the yield of the polymeric grass polysaccharide is not increased. Thus, the liquid-to-material ratio for extracting the polymeric grass polysaccharide is determined to be 20 mg/mL.
(2) Extraction time
As shown in FIG. 4, the yield of comfrey polysaccharide increases with the extraction time. When the extraction time is 60min, the yield of the comfrey polysaccharide is the highest. While extraction times of 70, 80 and 90min reduced the yield of comycosin. Thereby determining that the response surface of the comfrey polysaccharide has the optimized extraction time of 60 min.
(3) Amount of complex enzyme
As shown in figure 5, when the amount of the complex enzyme is 1%, the yield of the polysaccharide reaches the maximum, and when the amount of the complex enzyme is further increased to 1.5%, 2% or 2.5%, the yield of the polysaccharide is not further increased. Therefore, the optimal usage amount of the complex enzyme is 1%.
(4)pH
As shown in the attached figure 6, the yield of the comfrey polysaccharide shows a trend that the yield increases and then decreases with the increase of the pH, and the yield of the comfrey polysaccharide reaches a peak value when the leaching pH is 6, so that the response surface of the comfrey polysaccharide is determined to be the optimal extraction pH of 6.
4. 29 sets of tests were obtained using the Box-Behnken design and the results are shown in Table 2.
TABLE 2Box-Behnken test design and results
Figure BDA0002904629920000082
Figure BDA0002904629920000091
The yield (Y) of the polysaccharide of the polyrhachis vicina to the liquid-material ratio (X) is obtained by using the design of a Box-Behnken response surface method1) Extraction time (X)2) Dosage of complex enzyme (X)3)、pH(X4) Second order polynomial regression equation of (1):
Figure BDA0002904629920000092
the regression model analysis of variance is shown in table 3. The mismatch term was not significant (P-0.1584), indicating that the predictive model had better fit. Regression model significance (P)<0.0001). A lower coefficient of variation (c.v. ═ 4.93%) indicates that the fitted model is reproducible. Determining the coefficient (R)2) 0.9599, indicating that the regression model can predict the yield of the comfrey polysaccharide. All quadratic coefficients, linear coefficients and X2X3The P values of the two are all less than 0.05, which shows that the coefficients have obvious influence on the yield of the polymeric grass polysaccharide. The magnitude of the F value corresponding to the factor indicates the degree of influence of the factor on the test index. The F values corresponding to the liquid-material ratio, the extraction time, the compound enzyme dosage and the pH value are respectively 15.81, 8.06, 7.84 and 9.60. Therefore, the sequence of factors affecting the extraction of the comfrey polysaccharide is as follows: liquid to feed ratio (X)1)>pH(X4)>Extraction time (X)2)>Amount of Complex enzyme (X)3). According to a quadratic polynomial equation, the optimal extraction parameters of the polysaccharide of the comfrey are a liquid-material ratio of 21mL/g, extraction time of 62min, the using amount of complex enzyme of 1.1 percent and a pH value of 6.
TABLE 3 regression model analysis of variance
Figure BDA0002904629920000101
5. Verification test
The method is characterized in that a verification test is carried out according to the optimal scheme in the test design, and 3 times of repeated tests are carried out, so that the average value of the yield of the comfrey polysaccharide is 20.32%, and the theoretical value obtained by approximating a model is 20.81%. Therefore, the regression model obtained by the research can be used for predicting the yield of the comfrey polysaccharide.
Second, IR Spectroscopy of the polysaccharide prepared in example 1
The comfrey polysaccharide was mixed with dry KBr powder and tableted. Measuring range of infrared spectrometer is 4000-400 cm-1Infrared spectrograms were generated using Origin 8.5 software, as shown by the IR spectrogram of FIG. 7, 3407cm-1The absorption peak at (A) represents the stretching vibration of O-H; 1619cm-1The absorption peak at (a) represents the stretching of C ═ O; 2921cm-1The absorption peaks indicate the C-H stretching and bending vibration; 1077cm-1The absorption peak indicates that the polymeric grass polysaccharide exists in the pyranose form; 1416cm-1The peak at (a) is due to the curvature of C-H; at 840cm-1The bands indicate that beta-glycosidic linkages may exist between the saccharide units in the polymeric grass polysaccharide.
Thirdly, the polymeric grass polysaccharide prepared in example 1 is analyzed for molecular weight
The molecular weight of the comfrey polysaccharide was determined by gel permeation chromatography equipped with a multi-angle laser light scattering detector and a differential detector (HPSEC-MALLS-RI, Wyatt Technology, Santa Barbara, Calif., USA). Using a Shodex OH-pak SB-806 column (8.0 mm. times.300 mm, Showa Denko, Tokyo, Japan), equilibration at 35 ℃ and 200. mu.L of sample were introduced. The mobile phase contains 0.02 percent of NaN3And 0.2mol/L KH2PO4The flow rate of the deionized water (2) was 0.5 mL/min. The molecular weight of the sample was calculated using ASTRA V4.09.07(Wyatt Technology), curve-fitted using Berry extrapolation, and the dn/dc value was 0.147 mL/g; FIG. 8 is a molecular weight chromatogram of a comfrey polysaccharide, wherein LS is a multi-angle laser light scattering detector; dRI is a differential detector; the comfrey polysaccharide has a molecular weight of 6.25 kDa.
Fourthly, monosaccharide composition analysis is carried out on the polymeric grass polysaccharide prepared in the example 1
The monosaccharide composition of the comfrey polysaccharide is determined by adopting a high performance liquid chromatography. The detection wavelength of the ultraviolet detector is 245 nm; the chromatographic column is a C18 chromatographic column (Sepax Amerhyst, 4.6mm multiplied by 250mm,5 μm, Delaware, USA), the column temperature is 25 ℃, the mobile phase is a mixture of phosphate buffered saline (0.1mol/L, pH 7) and acetonitrile, the ratio is 80:20(v/v), the flow rate is 1ml/min, the sample injection amount is 10 μ L, and the attached figure 9 is a monosaccharide standard chromatogram, which is specific: 1. mannose; 2. galacturonic acid; 3. glucose; 4. galactose; 5. arabinose; FIG. 10 is a chromatogram of monosaccharide composition of polysaccharide from Polyporus frondosus, specifically: 2. galacturonic acid; 3. glucose; 4. galactose; 5. arabinose; referring to FIG. 9, it can be seen that comycotina polysaccharides consist of galacturonic acid (17.01 mol%), arabinose (16.15 mol%), galactose (20.83 mol%) and glucose (46.01 mol%).
Fifthly, the in vitro fermentation characteristics of the polymeric grass polysaccharide prepared in example 1 are analyzed, and the specific method is as follows:
(1) preparation of caecum content pulp
The fermentation model is established by taking the content of the cecum as a raw material. Selecting 12 healthy domestic chickens (Gallus domesticus) of 6 months old (6 male and female), mixing caecum contents, preparing caecum content slurry (20%, w/v) with the solution, and preparing modified normal saline containing NaCl (8.5g/L) and cysteine-HCl (0.5 g/L); the caecum content slurry was homogenized for 1min at 10000rpm/min using a homogenizer and then filtered through 4 layers of gauze.
(2) Preparation of fermentation Medium
Fermentation Medium (1L) containing NaCl (0.1g) and K2HPO4(0.04g), yeast extract (2g), CaCl2(0.01g)、MaSO4·7H2O (0.01g), peptone (2g), cysteine-HCl (0.5g), Tween 80(2mL), KH2PO4(0.04g), resazurin (1%, w/v, 1mL), vitamin K (10. mu.L), crimson (0.02g), bile salts (0.5g), NaHCO3(2g) The pH of the fermentation medium was adjusted to 6.8 with 0.1mol/L HCl solution. The fermentation medium is sterilized (30min,121 deg.C) and used.
(3) Fermentation of comfrey polysaccharide
A fermentation medium (16mL) containing comfrey polysaccharide (12.5mg/mL) was mixed with caecum content slurry (4 mL; 20%, w/v), and the resulting mixture was used as comfrey polysaccharide fermentation group. Fermentation medium (16mL) was mixed with caecal content slurry (4 mL; 20%, w/v) as a blank. The fermentation process was carried out in an anaerobic incubator at 37 ℃. Collecting fermentation samples at different fermentation time (0, 6, 12, 24, 48h), centrifuging at 8000rpm/min and 4 deg.C for 15 min. The supernatant was used to determine total sugar content. The precipitate was used for analysis of the microbial composition. The fermentation experiments for the blank group were repeated 3 times. The fermentation experiment of the polysaccharide group of comfrey was repeated 4 times.
(4) Carbohydrate digestibility determination
And (3) determining the total sugar content in the collected fermentation sample by adopting a phenol-sulfuric acid method. The in vitro fermentation carbohydrate consumption rate was calculated according to the following formula:
Figure BDA0002904629920000121
total sugar content in the formulathThe total sugar content of the samples is 6h, 12h, 24h and 48h, the consumption rate of the carbohydrate in the polysaccharide fermentation group is shown in figure 11, and the most effective utilization time of the carbohydrate is 6h to 12h in the fermentation period; when the fermentation time is 48 hours, the carbohydrate consumption rate of the polymeric grass polysaccharide fermentation group is 47.06 +/-1.79%, which indicates that the chicken cecal bacteria can utilize the polymeric grass polysaccharide as a carbon source.
(5) Determination of microbial composition of fermentation broth
Selecting 11 groups of fermentation samples to carry out microbial composition determination; raw cecal content pulp (OR), blank fermentation samples were collected at 0h, 6h, 12h, 24h and 48h (named T0B, T6B, T12B, T24B and T48B, respectively), polymerized grass polysaccharide fermentation samples were collected at 0h, 6h, 12h, 24h and 48h (named (T0P, T6P, T12P, T24P and T48P, respectively), PowerFecal was usedTMDNA isolation kits (MO BIO Laboratories Inc, Carlsbad, CA, US) extract total bacterial DNA from each sample. Each sample was sequenced by Nordheim Gene Co., Ltd (Beijing, China) and the V4 hypervariable region of the bacterial 16S rRNA gene was analyzed. Amplicons of each sample were sequenced on the Illumina HiSeq 2500 platform (iiiuma, San Diego, US), and different species screening, beta diversity and alpha diversity analyses were performed according to operational classification units (OTUs).
The results are shown in FIG. 12, the total number of 428 OTUs in 11 groups of caecal flora is higher than that in the blank group at 0, 6, 12, 24 and 48h, which indicates that the addition of the comycose in the fermentation system can cause the change of the microbial composition; comparing the microflora between the assay samples at the genus level using principal coordinate analysis (PCoA); converting a distance matrix of weighted or unweighted UniFrac values among the samples into a group of new orthogonal coordinate axes; the weighted UniFrac distance of PCoA is shown in FIG. 13, and the distances between the groups T6P, T12P, T24P and T48P and the blank groups (T6B, T12B, T24B and T48B) are far, which indicates that the polysaccharide has a regulating effect on the microbial structure.
The coverage represents the coverage of the example library; as can be seen from Table 4, the coverage of 10 fermentation samples was greater than 0.99, indicating that the sequencing results reflect the actual condition of the samples. The shannon index and the Simpson index are positively correlated with the microbial diversity, and the Chao1 and the ACE index are positively correlated with the microbial abundance; at 0, 6, 12, 24 and 48h, the shannon index and the simpson index of the polysaccharide fermentation group of the comfrey are lower than those of the blank group, which is probably related to the competitive action of the dominant flora; at 6h, the indexes of Chao1 and ACE of the polysaccharide fermentation group are higher than those of the blank group, and the indexes of Chao1 and ACE of the polysaccharide fermentation group at 6h are also higher than those of other fermentation times under different fermentation times; these results indicate that the addition of comfrey polysaccharide to the fermentation system can increase the microbial abundance by 6 h.
TABLE 3 Alpha diversity of fermentation samples
Figure BDA0002904629920000131
Figure BDA0002904629920000141
The values in table 3 are expressed as mean ± standard deviation; blank group n is 3; the polymeric grass polysaccharide fermentation group n is 4.
Mean differences were significant for different capital letters within the same column (P < 0.05).
The mean difference of different lower case letters within the same row was significant (P < 0.05).
To fully compare the relative abundance of microbial composition at the genus level, the relative abundances of the first 35 bacteria per fermentation time are shown in the heatmap, as shown in fig. 14, with an increasing trend in the relative abundance of Lactobacillus (Lactobacillus) in the polymeric grass polysaccharide fermentation group.

Claims (10)

1. A preparation method of polyrhachis vicina Roger polysaccharide based on optimized enzyme-assisted extraction by a response surface method is characterized by comprising the following steps:
the method comprises the following steps: extraction of
Soaking the comfrey powder in water, adding 1% of complex enzyme, regulating the pH value to 6, soaking to obtain an extracting solution, filtering the extracting solution, and concentrating the filtrate to obtain a concentrated solution; wherein the soaking time is 60min, and the dosage ratio of the water to the comfrey powder is 20 mL/g;
step two: precipitating with ethanol to remove impurities
Precipitating the concentrated solution with ethanol to remove water-soluble impurities to obtain crude polysaccharide precipitate of herba Euphorbiae Humifusae;
step three: deproteinization
Removing protein from the obtained crude polysaccharide of the comfrey by adopting a Sevag method;
step four: desalting by dialysis
Removing inorganic salt and micromolecular substances from the crude polysaccharide of the polymerized grass after the protein is removed by a dialysis method to obtain desalted polymerized grass polysaccharide;
step five: DEAE-52 cellulose column chromatography
Subjecting the obtained desalted polymeric grass polysaccharide to DEAE-52 cellulose column chromatography, collecting and combining the collected liquid of main peak, concentrating, and lyophilizing;
step six: sepharose CL-6B agarose gel column chromatography
And (3) carrying out Sepharose CL-6B agarose gel column chromatography on the obtained freeze-dried polymeric grass polysaccharide, collecting and merging eluent of main peaks, concentrating, and freeze-drying to obtain the polymeric grass crude polysaccharide.
2. The method for preparing comfrey polysaccharide based on response surface method optimized enzyme assisted extraction as claimed in claim 1, wherein the comfrey powder in step one is obtained by the following method:
cleaning herba Potentillae Discoloris, oven drying, pulverizing, and sieving to obtain herba Potentillae Discoloris powder.
3. The method for preparing polyrhachis vicina polysaccharide based on optimized enzyme-assisted extraction by the response surface method as claimed in claim 2, wherein the drying temperature is 50 ℃.
4. The method for preparing comfrey polysaccharide based on optimized enzyme-assisted extraction by the response surface method as claimed in claim 2, wherein the size of the screen used for sieving is 1 mm.
5. The method for preparing polyrhachis vicina polysaccharide based on optimized enzyme-assisted extraction by the response surface method as claimed in claim 1, wherein the soaking temperature in the first step is 50 ℃.
6. The method for preparing comfrey polysaccharide based on response surface method optimized enzyme assisted extraction as claimed in claim 1, wherein the complex enzyme in step one comprises cellulase, pectinase and papain.
7. The method for preparing comfrey polysaccharide based on response surface method optimized enzyme assisted extraction as claimed in claim 6, wherein the mass ratio of cellulase, pectinase and papain is 1: 1: 1.
8. the method for preparing comfrey polysaccharide based on response surface method optimized enzyme assisted extraction as claimed in claim 1, wherein the concentration of ethanol added in the alcohol precipitation impurity removal in step two is 75-80%.
9. The polymeric grass polysaccharide produced by the method of claim 1.
10. Use of the polymeric grass polysaccharide of claim 9 to modulate the abundance of probiotics in the chicken gut.
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