CN115836730A - Method for enriching polyphenol with function of regulating intestinal flora from grifola frondosa polysaccharide extraction waste liquid - Google Patents
Method for enriching polyphenol with function of regulating intestinal flora from grifola frondosa polysaccharide extraction waste liquid Download PDFInfo
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Abstract
The invention provides a method for enriching polyphenol with the function of regulating intestinal flora from a grifola frondosa polysaccharide extraction waste liquid, which comprises the following steps: taking an alcohol precipitation solution obtained by extracting water and precipitating the grifola frondosa polysaccharide with alcohol as a raw material solution, recovering ethanol, filtering, concentrating in vacuum, adjusting pH, and adsorbing by using macroporous adsorption resin; washing the macroporous adsorbent resin column bed layer after adsorbing polyphenol with deionized water to remove impurities, eluting with ethanol solution, collecting eluate, recovering ethanol, vacuum concentrating, and drying to obtain Grifola frondosa polyphenol enrichment substance. The enrichment method has the advantages of simple and convenient operation, high repeatability, good selectivity of the used macroporous resin, low cost, easy regeneration treatment and the like; the Grifola frondosa polyphenol obtained by enrichment has high yield and purity, can effectively improve the diversity of intestinal flora and promote the growth of intestinal beneficial flora, and has good application prospect in preparing intestinal flora regulators.
Description
Technical Field
The invention belongs to the field of bioseparation engineering research, and particularly relates to a method for enriching polyphenol with a function of regulating intestinal flora from a grifola frondosa polysaccharide extraction waste liquid.
Background
The intestinal tract is an important digestive absorption site of the human body, particularly microorganisms in the intestinal tract, carry about 150 times of genes of the known human genome, and are considered as another 'important organ' of the human body. The intestinal microorganisms participate in basic biological processes of human bodies, and occurrence and development of various chronic diseases such as inflammatory bowel diseases, obesity, diabetes, alcoholic liver diseases, cancers and the like are closely related to intestinal microorganism disorders. The food-derived polyphenol can regulate intestinal microbial structure, change intestinal microenvironment (such as lower intestinal pH), stimulate macrophage or lymphocyte in intestinal tissue to resist cancer, and has positive regulation effect on human intestinal health.
Grifola frondosa (Grifola frondosa), also known as chestnut mushroom and Polyporus frondosus, is a valuable and rare edible and medicinal macrofungi in Polyporus, and researches show that the Grifola frondosa has various pharmacological and biological activities of resisting tumors, improving immunity, reducing blood sugar, resisting viruses and radiation, scavenging free radicals and the like. The polyphenol substance is one of the important active components of the grifola frondosa, and has strong in vitro antioxidant activity, antibacterial effect and lipid metabolism disorder improving effect.
At present, there are several methods for extracting Polyporus frondosus polyphenol reported in literature and patent publications. CN105732250A discloses a preparation method of a high-purity grifola frondosa polyphenol component, which is characterized in that a grifola frondosa polyphenol crude component is prepared by an ultrasonic-assisted extraction technology, biomacromolecules such as polysaccharide and protein in the grifola frondosa polyphenol crude component are removed by an ultrafiltration membrane interception technology, and finally, a series separation and purification technology of macroporous adsorption resin NKA-II column chromatography and preparative high performance liquid chromatography is adopted to prepare various high-purity grifola frondosa polyphenol compound monomers.
At present, the research on extraction conditions of grifola frondosa polysaccharides and polyphenols is limited to single extraction (Shajiayong, chen Zhichao, zhu Yoxian, and the like. Different grifola frondosa polysaccharides extraction and in-vitro antioxidant activity research [ J ]. Food industry, 2022, 43 (8): 59-63; lv Xucong, gu Ruibo, li Yan, and the like. Therefore, after the grifola frondosa polysaccharide is extracted, the grifola frondosa polyphenol in the extraction waste liquid is enriched, so that a new way is provided for the comprehensive utilization of grifola frondosa, better economic benefits and social benefits can be obtained, and the environmental protection pressure can be effectively relieved.
The polyphenol has the functions of resisting aging, resisting radiation, eliminating free radicals, and the like, and has good effects on preventing and treating cardiovascular and cerebrovascular diseases. Research shows that polyphenol is the main active component playing a role in antioxidation in edible fungi, chen Xiangdong and the like (Chen Xiangdong, liu Xiaowen, wu Wutong. Maitake polyphenol extraction and activity research [ J ]. Food and biotechnology reports, 2005 (4): 26-30) separate powdery substances from maitake mycelia, and the maitake polyphenol crude extract is proved to be mainly polyphenol substances by applying a spectrum technology and a chemical identification test, and has stronger antioxidation by adopting erythrocyte oxidative hemolysis and malonaldehyde (malondiadehyde) tests.
Patent publication CN114989319A discloses a grifola frondosa polysaccharide with function of regulating intestinal flora, and a preparation method and application thereof, wherein grifola frondosa sporophore polysaccharide is extracted by an alkaline method, and is combined with operations of microfiltration, ultrafiltration, drying and the like to obtain the grifola frondosa polysaccharide, and the obtained grifola frondosa polysaccharide has function of regulating intestinal flora and has good application in preparation of intestinal flora regulators. The alcohol precipitation solution obtained by extracting water and precipitating the grifola frondosa polysaccharide with alcohol is taken as a raw material solution, and a process for enriching and recovering the grifola frondosa polyphenol by using a macroporous resin adsorption method is developed, so that the function of expanding the grifola frondosa polyphenol for regulating intestinal flora has important research significance and application value.
Disclosure of Invention
The invention aims to provide a preparation method for enriching polyphenol from a grifola frondosa polysaccharide extraction waste liquid and application of the preparation method in regulating intestinal flora.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for enriching polyphenol with the function of regulating intestinal flora from a grifola frondosa polysaccharide extraction waste liquid comprises the following steps:
(1) Taking an alcohol precipitation solution obtained by extracting and precipitating grifola frondosa polysaccharide with water as a raw material solution, recovering ethanol, filtering to remove insoluble substances, and performing vacuum concentration to ensure that the total polyphenol content of the raw material solution is 1.0-1.2 mg/mL to be used as an upper column solution;
(2) Adjusting the pH value of the effluent to 4.0, introducing sample at the flow rate of 2-3 BV/h, passing through a chromatographic column which is pretreated and is filled with macroporous adsorption resin by a wet method, controlling the flow rate, collecting the effluent in parts, and stopping sample adsorption when the concentration of total polyphenol in the effluent reaches 50.0 mu g/mL;
(3) Washing the macroporous adsorption resin column bed layer after adsorbing polyphenol with deionized water to remove impurities, eluting with 50-70% volume fraction ethanol solution, collecting eluent, recovering ethanol, vacuum concentrating, and drying to obtain the Grifola frondosa polyphenol concentrate.
The types of the macroporous resin in the step (2) are AB-8, X-5 and NKA-9.
The macroporous adsorption resin column bed layer after adsorbing polyphenol in the step (3) of the invention is washed by deionized water to remove impurities at the flow rate of 2BV/h, and the volume consumption of deionized water for removing impurities is 3BV.
After the macroporous adsorption resin column bed layer after adsorbing polyphenol in the step (3) of the invention is washed by deionized water to remove impurities, the elution flow rate of the ethanol eluent is 2BV/h, and the volume consumption of the eluent is 2BV.
The grifola frondosa polyphenol prepared by the invention is used for carrying out an intragastric administration experiment on mice, and the treatment is as follows: 70 female Kunming mice of SPF grade 6 weeks old were normally bred for one week to acclimate to the mouse house and then randomly divided into seven groups of 10 mice each, namely a normal control group (KB), the Grifola frondosa polyphenol of example 1 (GFT-1), the Grifola frondosa polyphenol of example 2 (GFT-2), and the Grifola frondosa polyphenol of example 3 (GFT-3). During the experiment, mice were subjected to regular gavage for 14 consecutive days, and the gavage dose of each group was 400 mg/kg. D. The polyphenol solution of Grifola frondosa of different groups is prepared by normal saline, normal control group is perfused with normal saline with the same volume, mice freely ingest drinking water during feeding period, and the weight change of each group of mice is recorded during experiment period.
Collecting fresh mouse excrement on the last day of experiment week 2, cooling with liquid nitrogen, storing at-80 ℃ for later use, sending to Shanghai Senno gene science and technology limited company for high-throughput sequencing through an Illumina MiSeq sequencing platform, and evaluating the regulation capacity of the grifola frondosa polyphenol prepared by the invention on the intestinal flora of the mouse according to the change of the intestinal flora of the mouse.
The invention has the advantages and positive effects that:
1) The raw material adopted by the invention is grifola frondosa, which is a common edible fungus with homology of medicine and food, is convenient and easy to obtain, and the utilized raw material liquid is extracted from the extraction waste liquid of polysaccharide extracted from grifola frondosa, so that the method has the characteristics of saving and environmental protection.
2) The chemical regulators used in the process of enriching the polyphenol are harmless substances, and can be widely applied to industrial production of the edible fungi; the method has the advantages of simple operation, high repeatability, good selectivity of the used macroporous resin, low cost, easy regeneration treatment and the like.
3) The yield of the grifola frondosa polyphenol enriched by the method reaches more than 80 percent, the purity of the grifola frondosa polyphenol enriched by the method reaches more than 60 percent, and a mouse model test shows that the feeding of the total polyphenol substances extracted from the grifola frondosa polysaccharide extraction waste liquid prepared by the method can obviously stimulate the growth of probiotics such as Lactobacillus and Akkermansia which are original beneficial bacteria, inhibit the growth of harmful bacteria such as Corynebacterium and Fei Kelan Mum (Facklamia), effectively regulate the function of intestinal flora, and have better application in preparing an intestinal flora regulator.
Drawings
FIG. 1 is a process flow diagram of the process for extracting waste liquid enriched polyphenols from grifola frondosa polysaccharides;
FIG. 2 is an infrared spectrum of a Grifola frondosa polyphenol concentrate prepared according to the present invention (GFT-1 is Grifola frondosa polyphenol obtained in example 1, GFT-2 is Grifola frondosa polyphenol obtained in example 2, and GFT-3 is Grifola frondosa polyphenol obtained in example 3);
FIG. 3 is a Venn diagram showing the specific sequence of intestinal flora of mice fed with the enriched Grifola frondosa polyphenol obtained according to the present invention (KB is blank control, GFT-1 is Grifola frondosa polyphenol obtained according to example 1, GFT-2 is Grifola frondosa polyphenol obtained according to example 2, and GFT-3 is Grifola frondosa polyphenol obtained according to example 3);
FIG. 4 is a graph showing the relative abundance changes at the level of the phylum of the mouse intestinal flora when the mouse is fed with the enriched grifola frondosa polyphenol prepared according to the present invention (KB is blank control, GFT-1 is grifola frondosa polyphenol prepared according to example 1, GFT-2 is grifola frondosa polyphenol prepared according to example 2, and GFT-3 is grifola frondosa polyphenol prepared according to example 3, firmicutes, bacteroidetes, actinomycetes, proteobacteria);
FIG. 5 is a graph showing the relative abundance of beneficial (A) and harmful (B) bacteria in the intestinal flora of mice when the mice were fed with the enriched Grifola frondosa polyphenol prepared according to the present invention (KB is blank control, GFT-1 is Grifola frondosa polyphenol prepared according to example 1, GFT-2 is Grifola frondosa polyphenol prepared according to example 2, GFT-3 is Grifola frondosa polyphenol prepared according to example 3, lactobacillus (Lactobacillus), akkermansia (Akkermansia), corynebacterium (Corynebacterium), fei Kelan Mum (Facklamia));
FIG. 6 composition thermogram of the species at the level of the mouse gut flora after feeding the mouse with enriched grifola frondosa polyphenol according to the present invention (KB as blank control, GFT-1 as grifola frondosa polyphenol obtained in example 1, GFT-2 as grifola frondosa polyphenol obtained in example 2, GFT-3 as grifola frondosa polyphenol obtained in example 3, roseburia (Roche), jeotgalicaccus (temporary name of Mediterranean), bacteroides (Bacteroides), staphylocccus (Staphylococcus), psychrobacter (Bacillus), sporosarcina (sarcina), corynebacterium (Corynebacterium), fallamia (Fei Kelan M), alcaligenes (Alcaligenes), prevotella (Prevotella), acinetobacter), lactobacillus (Oscillus), lactobacillus (Arctirium), lactobacillus (Akebiae (Lactobacillus), and Acinetobacter), lactobacillus (Arctium), and Lactobacillus (Akebiae (Arctium).
Detailed Description
The following experimental procedures were applied throughout the examples:
grifola frondosa polyphenol content determination Folin phenol method is used for determining the content of total phenols in Grifola frondosa, namely gallic acid is used as a standard substance, the absorbance value of each sample is determined by determining the standard curve of the gallic acid, the concentration of the sample is calculated by a regression equation, the determination is repeated for 3 times, and the result is represented by the average value +/-standard deviation.
(1) Determination of gallic acid Standard Curve
Accurately weighing 0.01g of gallic acid to a constant volume of 100mL, respectively putting 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5mL into a 25mL volumetric flask, adding 6.0mL of distilled water, uniformly mixing, adding 0.5mL of Fulin phenol reagent, then adding 2.0mL of 10% sodium carbonate solution, fixing the volume of the distilled water, keeping out of the sun for 2h, measuring the light absorption value at 760nm, repeating the operation for 2 times, and drawing a standard curve. And drawing a gallic acid standard curve by taking the absorbance as an ordinate and the concentration of the gallic acid as an abscissa.
(2) Determination of polyphenol content
Weighing 0.01g of freeze-dried grifola frondosa polyphenol powder, placing the powder in a 25mL volumetric flask, adding 60% ethanol for constant volume, fully shaking for dissolution, absorbing 2mL of the solution, adding 0.5mL of Fulin phenol reagent, adding 2.0mL of 10% sodium carbonate solution, measuring an absorbance at 760nm after keeping out of the sun for 2 hours after constant volume, repeating the operation for 3 times to obtain an average value of the absorbance, substituting the average value of the absorbance of a sample to be measured into a gallic acid standard curve, and calculating polyphenol concentration and polyphenol content according to an equation.
(3) Infrared spectroscopic analysis of Polyporus frondosus polyphenol
Weighing 5mg of grifola frondosa polyphenol powder prepared according to the invention, mixing with KBr powder, tabletting, and performing by using a Fourier infrared spectrometer at 4000-400 cm -1 The scanning is performed over a range of wavelengths.
The invention is further described below with reference to examples:
example 1
Grifola frondosa polyphenol enrichmentPreparing a (GFT-1) extract, namely measuring 200mL of alcohol precipitation liquid obtained by water extraction and alcohol precipitation of grifola frondosa polysaccharide, recovering ethanol under reduced pressure until no alcohol smell exists, filtering to remove insoluble substances, concentrating the total polyphenol content of a raw material liquid to 1.0mg/mL in vacuum, and adjusting the pH value of the concentrated grifola frondosa polyphenol to 4.0 by using 4mol/L NaOH. Introducing sample at a flow rate of 2BV/h, passing through a chromatographic column which is pretreated and is filled with macroporous adsorption resin by a wet method, controlling the flow rate, collecting effluent liquid in parts, and stopping sample adsorption when the concentration of total polyphenol in the effluent liquid reaches 50.0 mug/mL; washing the macroporous adsorption resin column bed layer after adsorbing polyphenol with deionized water with the flow rate of 3BV being 2BV/h to remove impurities, eluting with ethanol solution with the volume fraction of 50% of 2BV at the flow rate of 2BV/h, collecting eluent, recovering ethanol, concentrating in vacuum, and drying to obtain the grifola frondosa polyphenol enrichment (GFT-1), wherein the yield of the grifola frondosa polyphenol is 81.4%, and the purity is 62.3%. The obtained GFT-1 was subjected to infrared measurement, and an infrared scanning spectrum (FIG. 2) was shown at 3400cm -1 The left and right strong broad peaks are the stretching vibration absorption of hydroxyl, 3000cm -1 About 1600-1400 cm of stretching vibration of unsaturated C-H bond of benzene ring -1 Has characteristic absorption of benzene ring, and is judged to be phenolic substance.
Example 2
Preparing a grifola frondosa polyphenol enrichment (GFT-2) by weighing 200mL of an alcohol precipitation solution obtained by extracting and precipitating grifola frondosa polysaccharide with water, recovering ethanol under reduced pressure until no alcohol smell exists, filtering to remove insoluble substances, concentrating the total polyphenol content of a raw material solution to 1.2mg/mL in vacuum, and adjusting the pH value of the concentrated solution of the grifola frondosa polyphenol to 4.0 by using 4mol/L NaOH. Introducing sample at a flow rate of 2BV/h, passing through a chromatographic column which is pretreated and is filled with macroporous adsorption resin by a wet method, controlling the flow rate, collecting effluent liquid in parts, and stopping sample adsorption when the concentration of total polyphenol in the effluent liquid reaches 50.0 mug/mL; washing the macroporous adsorption resin column bed layer after adsorbing polyphenol with deionized water with the flow rate of 2BV/h of 3BV for removing impurities, eluting with an ethanol solution with the volume fraction of 60% with the flow rate of 2BV/h, collecting eluent, recovering ethanol, concentrating in vacuum, and drying to obtain the grifola frondosa polyphenol enrichment (GFT-2), wherein the yield of the grifola frondosa polyphenol is 82.1%, and the purity is 61.9%. The obtained GFT-2 was subjected to infrared measurement, and an infrared scanning spectrum (FIG. 2) was shown at 3400cm -1 Strength of right and leftThe broad peak is the stretching vibration absorption of hydroxyl, 3000cm -1 About 1600-1400 cm of stretching vibration of unsaturated C-H bond of benzene ring -1 Has characteristic absorption of benzene ring, and is judged to be phenolic substance.
Example 3
Preparing a grifola frondosa polyphenol enrichment (GFT-3) by weighing 200mL of alcohol precipitation liquid obtained by carrying out water extraction and alcohol precipitation on grifola frondosa polysaccharide, recovering ethanol under reduced pressure until no alcohol smell exists, filtering to remove insoluble substances, concentrating the total polyphenol content of a raw material liquid to 1.1mg/mL in vacuum, and regulating the pH value of the concentrated grifola frondosa polyphenol to 4.0 by using 4mol/L NaOH. Introducing sample at a flow rate of 2BV/h, passing through a chromatographic column which is pretreated and is filled with macroporous adsorption resin by a wet method, controlling the flow rate, collecting effluent liquid in parts, and stopping sample adsorption when the concentration of total polyphenol in the effluent liquid reaches 50.0 mug/mL; washing the macroporous adsorption resin column bed layer after adsorbing polyphenol with deionized water with the flow rate of 2BV/h and the volume fraction of 3BV for removing impurities, eluting with ethanol solution with the volume fraction of 70% with the flow rate of 2BV/h, collecting eluent, recovering ethanol, concentrating in vacuum, and drying to obtain the grifola frondosa polyphenol concentrate (GFT-3), wherein the yield of the grifola frondosa polyphenol is 82.3%, and the purity is 61.7%. The obtained GFT-3 was subjected to infrared measurement, and an infrared scanning spectrum (FIG. 2) was shown at 3400cm -1 The left and right strong broad peaks are the stretching vibration absorption of hydroxyl, 3000cm -1 About 1600-1400 cm of stretching vibration of unsaturated C-H bond of benzene ring -1 Has characteristic absorption of benzene ring, and is judged to be phenolic substance.
Example 4
Influence of different adsorption resins on static adsorption-elution performance of grifola frondosa polyphenol, accurately weighing 1.5g of each of 3 types of pretreated resins, respectively placing the resins into 150mL triangular bottles with stoppers, respectively adding 30mL of grifola frondosa polyphenol column-loading liquid, oscillating (180 r/min) for 24h at room temperature until adsorption balance is reached, measuring the total concentration of grifola frondosa polyphenol in the solution at the moment, and calculating saturated adsorption capacity. Filtering the resin after adsorption, desorbing with 70mL of 95% ethanol with volume fraction, oscillating for 24h, measuring the total polyphenol concentration in the desorption solution, and calculating the elution amount and the desorption rate. The results of measuring the static adsorption-elution performance of each resin under the same experimental conditions are shown in Table 1.
TABLE 1 static saturated adsorption-desorption performance measurement results of macroporous adsorption resin on Polyphenol of Grifola frondosa
As can be seen from Table 1, the resins AB-8, X-5 and NKA-9 all have a static saturation adsorption capacity of more than 50mg/g for Grifola frondosa polyphenol, and can be used in the adsorption separation process of Grifola frondosa polyphenol.
Example 5
Influence of ethanol concentration on desorption effect 20mL of resin selected by static experiment was measured, pretreated and loaded into a 1.2X 30cm glass chromatographic column. Adsorbing according to the above optimal adsorption conditions, washing the macroporous adsorbent resin column bed layer with deionized water to remove impurities, and desorbing Polyporus frondosus with ethanol of different concentrations as shown in Table 2.
TABLE 2 influence of ethanol concentration on Grifola frondosa polyphenol desorption rate
As is clear from Table 2, the desorption rate was improved with the increase in the ethanol concentration within a certain range. When the concentration of the ethanol is 10%, the desorption rate is only 27.6%; when the ethanol concentration of the volume fraction is increased to 50-70%, the desorption rate reaches over 80%, and the effect is good. After that, the volume fraction concentration of the ethanol is increased, although the desorption rate is slightly increased, the change is not obvious, the cost is too high for production, the volatility is large, and the difficulty of controlling operation and conditions is increased. Therefore, 50% -70% ethanol is selected for elution.
Example 6
Evaluation of Grifola frondosa polyphenol intestinal flora regulatory function after gavage of the Grifola frondosa polyphenol-enriched samples (GFT-1, GFT-2, GFT-3) prepared in examples 1 to 3, respectively, for 14 days, the feces of mice in the blank control group (KB group) and Grifola frondosa polyphenol group of examples 1 to 3 were analyzed by small 16S rRNA high-throughput sequencing. The specific method comprises the following steps:
70 female Kunming mice of SPF grade 6 weeks old were normally bred for one week to acclimate to the mouse house and then randomly divided into seven groups of 10 mice each, namely a normal control group (KB), the Grifola frondosa polyphenol of example 1 (GFT-1), the Grifola frondosa polyphenol of example 2 (GFT-2), and the Grifola frondosa polyphenol of example 3 (GFT-3). During the experiment, mice were subjected to regular gavage for 14 consecutive days, and the gavage dose of each group was 400 mg/kg. D. The polyphenol solution of Grifola frondosa of different groups is prepared by normal saline, normal control group is perfused with normal saline with the same volume, mice freely ingest drinking water during feeding period, and the weight change of each group of mice is recorded during experiment period.
And (3) feeding the mice for the last day of the 2 nd week, collecting fresh mouse excrement, cooling the excrement by liquid nitrogen, preserving the excrement at the temperature of minus 80 ℃ for later use, and sending the excrement to Shanghai Senno gene technology company for high-throughput sequencing through an Illumina MiSeq sequencing platform.
As shown in Table 3, compared with a control group (KB), after the grifola frondosa polyphenol concentrates (GFT-1, GFT-2 and GFT-3) obtained under different gastric gavage conditions are used by each group of mice, the relevant indexes of Chao1, objective species, shannon and Simpson are significantly different (P is less than 0.05), and the grifola frondosa polyphenols of different examples improve the richness and diversity of intestinal flora of the mice, which indicates that the grifola frondosa polyphenol concentrate has an effect of improving the diversity of the intestinal flora of the mice.
TABLE 3 analysis of the Alpha index diversity of intestinal flora in mice after feeding Grifola frondosa polyphenol enrichment
Note: KB-blank control, GFT-1-Polyphenol from Grifola frondosa prepared in example 1, GFT-2-Polyphenol from Grifola frondosa prepared in example 2, GFT-3-Polyphenol from Grifola frondosa prepared in example 3
After sequencing of bacterial 16S rRNA in the gut of each group of mice, the sequences obtained were merged and divided into Amplicon Sequencing Variants (ASV). Based on the obtained ASV abundance matrix, R software was used to calculate the number of ASVs shared by each sample group, and using Venn (Venn) plots, the number of ASVs shared and unique among different sample groups was shown. As can be seen from fig. 3, 11887 ASVs were detected in the blank control group and the grifola frondosa polyphenol group of the different examples, wherein the total ASVs are only 511, accounting for 4.30%, and the grifola frondosa polyphenol group is significantly increased in number of ASVs specific to each group, which indicates that the intestinal flora of mice perfused with grifola frondosa polyphenol is significantly different from the blank control group in the category of intestinal flora, and the intestinal flora structure thereof is significantly changed.
Two dominant phyla in the mouse intestinal flora are respectively firmicutes and bacteroidetes, and account for more than 90% of the relative abundance of the composition of the intestinal bacteria. From FIG. 4, the predominant bacteria status of Bacteroides and firmicutes of Polyphenol of Grifola frondosa group of different examples was unchanged compared with the blank control group, but the relative abundance of firmicutes, actinomycetes and Proteobacteria was decreased, the number of Bacteroides was increased, and the ratio of firmicutes to Bacteroides was decreased significantly. Studies have shown that an increase in the firmicutes/bacteroidetes ratio is closely associated with the occurrence and development of various diseases such as inflammatory bowel disease, obesity and constipation. Experimental data show that the grifola frondosa polyphenol can regulate the intestinal flora structure, and the grifola frondosa polyphenol can have the potential of improving related disease symptoms when being used appropriately.
Significance analysis at the genus level for the blank control group and the example group is shown in FIGS. 5-A and 5-B. Compared with a blank control group KB, the relative abundance of bacteroides and akkermansia in the intestinal flora of the Grifola frondosa polyphenol enriched group of different examples is remarkably increased (P is less than 0.001); different examples the relative abundance of corynebacterium and Fei Kelan of embacter in the polyphenolic group of grifola frondosa was reduced. Therefore, the grifola frondosa polyphenol can stimulate the proliferation of the original beneficial bacteria in intestinal tracts, such as lactobacillus, akkermansia and the like, and inhibit the growth of harmful bacteria in corynebacterium, fei Kelan Mum and the like. Therefore, the grifola frondosa polyphenol has the potential of optimizing the host intestinal flora structure and exerting the probiotic function on mice.
The heat maps were used to further compare species composition differences between samples by species composition analysis using abundance data from the genus at the top 20 th of the mean abundance to achieve a display of species abundance distribution trends for each sample. The heat map represents the size of the data in shades of color. Different color variations may be used to represent the given information. The darker the color, the greater the abundance of the species. As can be seen from FIG. 6, at the genus level, the numbers of Lactobacillus, ackermansia and Bifidobacterium of the Grifola polyphenol enriched group were significantly increased (P < 0.05). The experiment proves that the mice can obviously stimulate the growth of probiotics such as lactobacillus and akkermansia which are original beneficial bacteria, inhibit the growth of harmful bacteria such as corynebacterium and Fei Kelan Mum and effectively regulate the function of intestinal flora through the intervention of grifola frondosa polyphenol.
As can be seen from Table 4, the difference between the Grifola frondosa polyphenol enriched group and the blank control group in different examples is very significant (P < 0.01); the GFT-1 group and the GFT-3 group also have very significant difference (P is less than 0.01); the GFT-2 grifola frondosa polyphenol group and the GFT-3 polyphenol group have significant difference (P is less than 0.05), so that the appropriate dose of grifola frondosa polyphenol can significantly regulate the intestinal flora of mice.
TABLE 4 differential analysis between intestinal flora groups of mice fed with Grifola frondosa polyphenol enrichment
Note: KB-blank control, GFT-1-Grifola frondosa obtained in example 1, GFT-2-Grifola frondosa obtained in example 2, and GFT-3-Grifola frondosa obtained in example 3.
Claims (5)
1. A method for enriching polyphenol with the function of regulating intestinal flora from a grifola frondosa polysaccharide extraction waste liquid is characterized by comprising the following steps:
(1) Taking an alcohol precipitation solution obtained by extracting and precipitating grifola frondosa polysaccharide with water as a raw material solution, recovering ethanol, filtering to remove insoluble substances, and performing vacuum concentration to ensure that the total polyphenol content of the raw material solution is 1.0-1.2 mg/mL to be used as an upper column solution;
(2) Adjusting the pH value of the column loading solution to 4.0, injecting sample at the flow rate of 2-3 BV/h, passing through a chromatographic column which is pretreated and is filled with macroporous adsorption resin by a wet method, controlling the flow rate, collecting the effluent liquid in parts, and stopping loading adsorption when the concentration of total polyphenol in the effluent liquid reaches 50.0 mug/mL;
(3) Washing the macroporous adsorption resin column bed layer after adsorbing the polyphenol with deionized water to remove impurities, eluting with an ethanol solution with the volume fraction of 50-70%, collecting the eluent, recovering the ethanol, concentrating in vacuum, and drying to obtain the ash tree flower polyphenol enrichment.
2. The method for enriching polyphenols having the function of regulating intestinal flora from the waste liquid obtained from the extraction of the polysaccharides from Grifola frondosa according to claim 1, wherein the types of the macroporous resins in the step (2) are AB-8, X-5 and NKA-9.
3. The method for enriching polyphenols having the function of regulating intestinal flora from the waste liquid from the extraction of the polysaccharides from grifola frondosa according to claim 1, wherein the flow rate of washing the macroporous adsorbent resin column bed layer after adsorbing the polyphenols with deionized water to remove impurities is 2BV/h, and the amount of deionized water to remove impurities is 3BV in step (3).
4. The method according to claim 1, wherein the macroporous adsorbent resin column bed layer after adsorbing the polyphenols in step (3) is washed with deionized water to remove impurities, the elution flow rate of the ethanol eluent is 2BV/h, and the amount of the eluent is 2BV.
5. The method for enriching polyphenols from the waste liquid of Grifola frondosa polysaccharide extract to adjust the function of intestinal flora according to claim 1, wherein the enriched polyphenols have the obvious function of adjusting intestinal flora, can increase the diversity and abundance of intestinal flora, remarkably stimulate the growth of probiotic bacteria such as Lactobacillus and Ackermansia, and inhibit the abundance of harmful bacteria such as Corynebacterium and Fei Kelan Mum.
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| CN114832022A (en) * | 2022-06-16 | 2022-08-02 | 湖北省农业科学院农产品加工与核农技术研究所 | Preparation of phellinus igniarius sporocarp phenolic active substance and application of phellinus igniarius sporocarp phenolic active substance in regulation of intestinal flora and uric acid metabolism |
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| CN114832022A (en) * | 2022-06-16 | 2022-08-02 | 湖北省农业科学院农产品加工与核农技术研究所 | Preparation of phellinus igniarius sporocarp phenolic active substance and application of phellinus igniarius sporocarp phenolic active substance in regulation of intestinal flora and uric acid metabolism |
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