CN112898262A - Method for synchronously extracting flavones and polysaccharides from Chinese prickly ash leaves - Google Patents
Method for synchronously extracting flavones and polysaccharides from Chinese prickly ash leaves Download PDFInfo
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- 235000011949 flavones Nutrition 0.000 title claims abstract description 82
- 150000004676 glycans Chemical class 0.000 title claims abstract description 82
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 82
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 82
- 238000000034 method Methods 0.000 title claims abstract description 27
- 150000002213 flavones Chemical class 0.000 title claims description 22
- 241001079064 Zanthoxylum schinifolium Species 0.000 title 1
- 238000000855 fermentation Methods 0.000 claims abstract description 62
- 230000004151 fermentation Effects 0.000 claims abstract description 62
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- 244000089698 Zanthoxylum simulans Species 0.000 claims abstract description 41
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- 239000006002 Pepper Substances 0.000 claims description 18
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- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 description 4
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- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 3
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- BNGXYYYYKUGPPF-UHFFFAOYSA-M (3-methylphenyl)methyl-triphenylphosphanium;chloride Chemical compound [Cl-].CC1=CC=CC(C[P+](C=2C=CC=CC=2)(C=2C=CC=CC=2)C=2C=CC=CC=2)=C1 BNGXYYYYKUGPPF-UHFFFAOYSA-M 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
Abstract
The invention discloses a process for synchronously extracting flavone and polysaccharide from Chinese prickly ash leaves, wherein the Chinese prickly ash leaves are fermented by mixing aspergillus niger and bacillus subtilis, and the cell walls of the Chinese prickly ash leaves are damaged by cellulase generated by the growth and metabolism of the aspergillus niger and the bacillus subtilis, so that the dissolution of the flavone and the polysaccharide is promoted, and the extraction rate is improved; by adopting a novel extraction method combining microbial fermentation technology and ultrasonic extraction, flavone and polysaccharide are synchronously obtained from the zanthoxylum bungeanum leaves, so that the consumption of time and energy is reduced, and the cost is saved.
Description
Technical Field
The invention relates to the technical field of extraction of effective components of plants, in particular to a method for synchronously extracting flavones and polysaccharides from Chinese prickly ash leaves.
Background
At present, flavone and polysaccharide in pepper leaves are mainly extracted by a hot water extraction method, an ultrasonic method, a supercritical extraction method, a microwave extraction method and the like, but the extraction rate is low, single components are mostly extracted, the resource utilization rate is low, the cost is high, the microbial fermentation technology mainly utilizes various biological enzymes secreted by microorganisms in the growth and metabolism process to destroy the structure of plant cells, so that active components can be dissolved out to a large extent, the extraction efficiency is improved, and Trichoderma reesei, Aspergillus niger, actinomycetes, Bacillus subtilis and the like can produce enzymes such as cellulase, protease and the like, so that the plant cell walls can be destroyed, and nutrient substances can be dissolved out in a large amount.
The method synchronously extracts flavone and polysaccharide from the zanthoxylum bungeanum leaves by combining an aspergillus niger and bacillus subtilis mixed fermentation technology with an ultrasonic extraction technology, and optimizes fermentation extraction conditions by adopting an orthogonal test so as to realize the extraction of the flavone and the polysaccharide in the zanthoxylum bungeanum leaves to the maximum extent.
Disclosure of Invention
In order to achieve the purpose, the invention provides the following technical scheme: a method for synchronously extracting flavones and polysaccharides from Chinese prickly ash leaves is characterized by comprising the following process steps:
preprocessing Chinese prickly ash leaves
Drying folium Zanthoxyli Bungeani in a constant temperature drying oven at 55-65 deg.C, pulverizing, and sieving with 60 mesh sieve to obtain folium Zanthoxyli Bungeani powder;
② preparation of fermentation medium
Weighing a proper amount of pepper leaf powder, adding ultrapure water according to a material-liquid ratio, uniformly mixing, sterilizing for 15-25min at the temperature of 110-130 ℃, cooling to room temperature, inoculating activated bacillus subtilis and aspergillus niger mixed seed liquid according to a corresponding inoculation amount and strain proportion, placing in a constant-temperature oscillation incubator, performing fermentation culture, and setting corresponding temperature and rotating speed to perform fermentation culture;
③ extraction of flavone
After fermentation, adding 60% ethanol, placing in an ultrasonic cleaning machine, extracting for 30min under the conditions of ultrasonic power of 100W and ultrasonic frequency of 40kHz, and filtering at 60 ℃;
extraction of polysaccharide
And (4) adding ultrapure water with 4 times volume into the filter residue obtained in the step (III), placing the filter residue in an ultrasonic cleaning machine, extracting for 30min under the conditions of ultrasonic power of 100W and ultrasonic frequency of 40kHz, and filtering and concentrating at the temperature of 60 ℃.
Further, preferably, the feed-to-liquid ratio is 1:20(g: mL).
Further, preferably, the inoculation amount is 5%.
Further, preferably, the ratio of the Aspergillus niger to the Bacillus subtilis mixed seed liquid strains is 1: 2.
Further, preferably, the temperature of the fermentation culture is 31 ℃, the rotation speed is 150r/min, and the fermentation time is 3 d.
Compared with the prior art, the invention has the beneficial effects that:
(1) aspergillus niger and bacillus subtilis are mixed and fermented for the first time to obtain pepper leaves, and cellulase generated by growth and metabolism of the Aspergillus niger and the bacillus subtilis is used for destroying cell walls of the pepper leaves, promoting dissolution of flavone and polysaccharide and improving extraction rate;
(2) by adopting a novel extraction method combining microbial fermentation technology and ultrasonic extraction, flavone and polysaccharide are synchronously obtained from the zanthoxylum bungeanum leaves, so that the consumption of time and energy is reduced, and the cost is saved.
Drawings
FIG. 1 shows the influence of feed liquid ratio on the contents of flavone and polysaccharide in Zanthoxylum bungeanum leaf;
FIG. 2 is the effect of inoculation amount on the contents of flavones and polysaccharides in zanthoxylum bungeanum leaves;
FIG. 3 shows the effect of strain ratio on the content of flavones and polysaccharides in Zanthoxylum bungeanum leaves;
FIG. 4 is the effect of fermentation temperature on the content of flavones and polysaccharides in Zanthoxylum bungeanum leaves;
FIG. 5 shows the effect of fermentation time on the content of flavones and polysaccharides in Zanthoxylum bungeanum leaf
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
1 materials and methods
1.1 Experimental methods
1.1.1 pretreatment of Zanthoxylum bungeanum leaves
Selecting fresh folium Zanthoxyli, removing rotten leaves and dead leaves, washing with running water to remove surface impurities, oven drying in a 60 deg.C constant temperature drying oven, pulverizing, and sieving with 60 mesh sieve to obtain folium Zanthoxyli powder. The folium Zanthoxyli powder is stored at room temperature in a dark and dry environment.
1.1.2 preparation of fermentation Medium
Weighing a proper amount of pepper leaf powder, adding ultrapure water according to a material-liquid ratio, uniformly mixing, sterilizing at 121 ℃ for 20min, cooling to room temperature, inoculating activated bacillus subtilis and aspergillus niger mixed seed liquid according to a corresponding inoculation amount and strain proportion, placing in a constant-temperature oscillation incubator, setting corresponding temperature and rotating speed, and performing fermentation culture.
1.1.3 extraction of flavones
After fermentation, adding 60% ethanol, placing in an ultrasonic cleaning machine, extracting for 30min under the conditions of ultrasonic power of 100W and ultrasonic frequency of 40kHz, and the temperature is 60 ℃. Filtering and measuring the content of the flavone in the solution.
1.1.4 extraction of polysaccharides
Adding ultrapure water 4 times the volume of the filter residue 1.3.3, placing in an ultrasonic cleaning machine, and extracting for 30min at 60 deg.C under the conditions of ultrasonic power of 100W and ultrasonic frequency of 40 kHz. Filtering and concentrating, taking 5.0mL of concentrated solution to fix the volume to 50mL, and determining the polysaccharide content in the solution.
1.1.5 measurement of polysaccharide content
The total sugar content is determined by adopting a phenol-sulfuric acid method: 0, 0.2, 0.4, 0.6, 0.8, 1.0mL of the standard solution and 1.0mL of the sample solution were removed respectively and placed in a 10mL test tube with a plug, and 1mL was supplemented with ultrapure water. Adding 1.0mL of 5% redistilled phenol, shaking, adding 5.0mL of concentrated sulfuric acid, mixing by vortex, heating in a water bath at 100 ℃ for 15min, and cooling with running water to room temperature to measure the absorbance value at 490 nm. And drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance value as an ordinate, and obtaining a standard equation as follows: 4.4461x +0.0323 (R)20.999) and calculating the total sugar content in the sample according to the formula.
In the formula: c, the total sugar concentration in the extracting solution is mg/mL; v, extracting solution volume; n, dilution multiple; m, leaf mass of Zanthoxylum bungeanum, g.
The content of reducing sugar is determined by a DNS method: 0.0 mL, 0.2 mL, 0.4mL, 0.6 mL, 0.8 mL, 1.0mL of the standard solution and 1.0mL of the sample solution were placed in 10mL stoppered tubes, and distilled water was added to 2 mL. Respectively adding 1.5mL DNS reagent, heating in water bath at 100 deg.C for 5min, cooling to room temperature with running water, supplementing 10mL with ultrapure water, and mixing. The absorbance values were measured at a wavelength of 540 nm. And drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance value as an ordinate, and obtaining a standard equation as follows: 5.2487x +0.0588 (R)20.9992) and calculated according to the formulaThe content of reducing sugar in the product.
In the formula: c, reducing sugar concentration in the extracting solution is mg/mL; v, extracting solution volume; n, dilution multiple; m, leaf mass of Zanthoxylum bungeanum, g.
Polysaccharide content-total sugar content-reducing sugar content
1.1.6 measurement of Total Flavonoids content
The content of the total flavone is measured by adopting an ultraviolet spectrophotometry: accurately weighing 10.0mg rutin standard, dissolving with 60% ethanol, and diluting to 100mL to obtain 0.1mg/mL rutin standard solution.
Respectively putting 0.0 mL, 1.0mL, 2.0mL, 3.0 mL, 4.0 mL, 5.0mL rutin standard solution and 2.0mL sample solution in a 10mL test tube with a plug, adding 60% ethanol solution to complement to 5mL, adding 0.3mL 5% sodium nitrite solution, shaking uniformly, standing for 6min, adding 0.3mL 10% aluminum nitrate solution, shaking uniformly, standing for 6min, adding 4mL 5% sodium hydroxide solution, shaking uniformly, and standing for 15 min. The absorbance values were determined at a wavelength of 510 nm. And drawing a standard curve by taking the concentration of the rutin as a horizontal coordinate and the absorbance value as a vertical coordinate, and obtaining a standard equation as follows: y is 12.518x-0.0048 (R)20.9998) and calculating the content of the total flavone in the sample according to a formula.
In the formula: c, the concentration of the flavone in the extracting solution is mg/mL; v, extracting solution volume; n, dilution multiple; m, leaf mass of Zanthoxylum bungeanum, g.
1.1.7 Single factor test for extracting flavone and polysaccharide from Chinese prickly ash leaves by microbial fermentation
1.1.7.1 selection of ratio of liquid to feed
Weighing a proper amount of pepper leaf powder, respectively adding water according to the material-liquid ratio of 1:5, 1:10, 1:15, 1:20 and 1:25(g: mL), sterilizing at 121 ℃ for 20min, cooling to room temperature, simultaneously inoculating a mixed seed liquid of aspergillus niger and bacillus subtilis with the strain ratio of 1:3 according to the total inoculation amount of 5%, and fermenting at 150r/min and 31 ℃ for 3 d.
1.1.7.2 selection of inoculum size
Weighing a proper amount of pepper leaf powder, adding water according to a material-liquid ratio of 1:15, sterilizing at 121 ℃ for 20min, cooling to room temperature, inoculating a mixed seed liquid of aspergillus niger and bacillus subtilis with a strain ratio of 1:3 simultaneously according to total inoculation amounts of 1%, 3%, 5%, 7% and 9%, and fermenting at 150r/min and 31 ℃ for 3 d.
1.1.7.3 selection of the proportion of species
Weighing a proper amount of pepper leaf powder, adding water according to a material-liquid ratio of 1:15, sterilizing at 121 ℃ for 20min, cooling to room temperature, then respectively inoculating mixed seed liquid of aspergillus niger and bacillus subtilis according to a total inoculation amount of 5% and a strain ratio of 1:1, 1:2, 1:3, 2:1 and 3:1, and fermenting at 150r/min and 31 ℃ for 3 d.
1.1.7.4 selection of fermentation temperature
Weighing a proper amount of pepper leaf powder, adding water according to a material-liquid ratio of 1:15, sterilizing at 121 ℃ for 20min, cooling to room temperature, simultaneously inoculating a mixed seed liquid of aspergillus niger and bacillus subtilis with a strain ratio of 1:3 according to a total inoculation amount of 5%, and fermenting at 25 ℃, 28 ℃, 31 ℃, 34 ℃ and 37 ℃ for 3d at 150 r/min.
1.3.7.5 selection of fermentation time
Weighing a proper amount of pepper leaf powder, adding water according to a material-liquid ratio of 1:15, sterilizing at 121 ℃ for 20min, cooling to room temperature, simultaneously inoculating a mixed seed liquid of aspergillus niger and bacillus subtilis with a strain ratio of 1:3 according to a total inoculation amount of 5%, and fermenting at 150r/min and 31 ℃ for 1d, 2d, 3d, 4d and 5d respectively.
1.1.8 orthogonal test for extracting flavone and polysaccharide from Chinese prickly ash leaves by microbial fermentation
On the basis of a single-factor test, 3 factors of the material-liquid ratio, the inoculation amount and the fermentation temperature are selected, and the content of the flavones and the polysaccharides in the pepper leaves is taken as a response value. The other test conditions are that the ratio of the strains is 1:2, the fermentation time is 3d, the rotating speed is 150r/min, the pH is natural, and the test is carried out according to an orthogonal design table.
TABLE 1 orthogonal experimental design
1.1.9 in vitro antioxidant Activity assay
Dpph.scavenging activity: according to Blois[20,21]The method is slightly modified, 2.0mL of DPPH solution and 2.0mL of flavone and polysaccharide sample solutions with different concentrations are put into a 10mL test tube with a plug, shaken evenly and placed for 60min, and the absorbance value A of the sample solution is measured at the wavelength of 517nm0The absorbance values A of 2.0mL of 80% ethanol solution and 2.0mL of sample solutions of flavone and polysaccharide of different concentrations were measured in the same manner1And absorbance values A of 2.0mL of 80% ethanol solution and 2.0mL of DPPH solution2VC is used as a reference substance. Three times of analysis, taking the average value, and calculating DPPH & clearance according to the following formula:
OH scavenging Activity: method according to Ma[22-24]Slightly modified by the same inventors, 1.5mL of 5mM phenanthroline solution was placed in a 10mL stoppered tube, 2.0mL of phosphate buffer solution was added, and 1.0mL of 7.5mM ferric sulfate (FeSO) was added4) The solution is subjected to constant volume, heated in water bath at 37 ℃ for 1h, and the absorbance value A is measured at the wavelength of 510mn0(ii) a 1.5mL of 5mM phenanthroline solution was placed in a 10mL stoppered tube, 2.0mL of phosphate buffer solution was added, and 1.0mL of 7.5mM ferric sulfate (FeSO) was added4) The solution was added 1.0mL of 1% (v/v) H2O2Fixing the volume, heating in water bath at 37 ℃ for 1h, and measuring the absorbance value A1(ii) a 1.5mL of 5mM phenanthroline solution was placed in a 10mL stoppered tube, 2.0mL of phosphate buffer solution was added, and 1.0mL of 7.5mM ferric sulfate (FeSO) was added4) Adding flavone and polysaccharide sample solutions with different concentrations, and adding 1.0mL 1% (v/v) H2O2Fixing the volume, heating in water bath at 37 ℃ for 1h, and measuring the absorbance value A2VC is used as a reference substance. Three times of analysis, taking the average value, calculating the OH clearance according to the following formula:
reduction capability: method according to Chen et al[23,25]The method of (1) was modified slightly by mixing 1.0mL of each concentration of the flavone sample solution, polysaccharide sample solution, negative control (blank), and positive control (VC), 1.0mL of 200mM phosphate buffer solution (pH 6.6) and 0.5mL of 1% (w/v) potassium ferricyanide, and heating in a 50 ℃ water bath for 20 min. Cooling, adding 10% (w/v) trichloroacetic acid, centrifuging at 3000r/min for 10min, collecting 2.0mL supernatant, adding 2.0mL water, mixing, adding 0.4mL 0.1% ferric chloride, standing for 10min, and measuring absorbance at 700 nm. The larger the absorbance value, the stronger the reducing power, the three times of analysis, and the average value was taken.
1.2 statistical analysis of data
Statistical analysis of the data was performed using SPSS 22.0 software. The experimental data are expressed as mean ± sd, with significant statistical significance as P < 0.05.
2 results and analysis
2.1 results of single factor experiments on microbial fermentation extraction
2.1.1 Effect of feed liquid ratio on flavone and polysaccharide content in Zanthoxylum bungeanum leaf
As can be seen from FIG. 1, the feed-liquid ratio has a significant effect (P <0.05) on the contents of flavone and polysaccharide within a certain range, and when the feed-liquid ratio is less than 1:15(g: mL), the contents of the flavone and polysaccharide increase with the increase of the feed-liquid ratio, probably because the concentration difference between the raw material and the solution increases with the increase of the feed-liquid ratio, the flavone and polysaccharide in the prickly ash leaves are promoted to be dissolved out; when the feed-liquid ratio is 1:15, the contents of flavone and polysaccharide reach maximum values, respectively 19.26mg/g and 16.10 mg/g; however, the feed-liquid ratio is continuously increased, and the content of both materials is in a trend of gradually decreasing, because the feed-liquid ratio is too large, the concentration of nutrient substances is reduced, the growth and metabolism of microorganisms are influenced, and the fermentation and extraction of flavone and polysaccharide are not facilitated.
2.1.2 Effect of inoculum size on Flavonoids and polysaccharides content of Zanthoxylum bungeanum leaf
As can be seen from FIG. 2, when the inoculation amount is less than 5%, the contents of flavone and polysaccharide increase with the increase of the inoculation amount, probably because the inoculation amount is increased, the fermentation effect of the microorganism effectively improves the dissolution of flavone and polysaccharide in the pricklyash leaves. When the fermentation inoculation amount is 5%, the contents of flavone and polysaccharide reach peak values, and are 19.39mg/g and 17.31 mg/g. However, as the inoculation amount is increased, the content of flavone and polysaccharide is reduced, probably because the microorganism number is in a supersaturated state, and the flavone and polysaccharide are decomposed and utilized, so that the content is reduced. 2.1.3 Effect of Strain ratio on Flavonoids and polysaccharides content of Zanthoxylum bungeanum leaf
As can be seen from FIG. 3, when the ratio of Aspergillus niger to Bacillus subtilis is 1:2, the maximum content of flavone and polysaccharide is 16.45mg/g and 15.38mg/g, which indicates that under this ratio of strains, Aspergillus niger and Bacillus subtilis reach a better symbiotic state, and can promote the dissolution of flavone and polysaccharide to the maximum extent. However, continuing to increase the ratio of B.subtilis, the content of flavones and polysaccharides tends to decrease, probably because the growth is slow when A.niger is low; in addition, excessive bacillus subtilis has an inhibition effect on the growth and the propagation of aspergillus niger, so that the release of flavone and polysaccharide in the pepper leaves is not facilitated.
2.1.4 Effect of fermentation temperature on Flavonoids and polysaccharides in Zanthoxylum bungeanum leaf
The fermentation temperature has obvious influence on the content of flavone and polysaccharide (P is less than 0.05), and the temperature can influence the fermentation and extraction of the flavone and polysaccharide of the pepper leaves by influencing the activity and growth metabolism of microorganisms. As can be seen from FIG. 4, the content of flavone and polysaccharide increases with the temperature increase in the range of 25 ℃ to 31 ℃, and the peak values of flavone and polysaccharide reached 19.03mg/g and 16.82mg/g when the fermentation temperature was 31 ℃. In the range of 31 ℃ to 37 ℃, the content of flavone and polysaccharide is reduced along with the increase of temperature, because the activity of the strain is reduced and the metabolic capability is reduced due to the over-low or over-high temperature, which has adverse effect on the fermentation action of microorganisms.
2.1.5 Effect of fermentation time on Flavonoids and polysaccharides in Zanthoxylum bungeanum leaf
As can be seen from FIG. 5, the contents of flavone and polysaccharide gradually increased with the increase of fermentation time, reached maximum values of 15.95mg/g and 13.55mg/g respectively at 3d of fermentation, and the contents of polysaccharide and flavone both gradually decreased with the increase of fermentation time. The reason is that at the beginning of fermentation, nutrient substances are sufficient, the growth and metabolism of microorganisms are fast, and flavone and polysaccharide in the pepper leaves are dissolved out in a large amount along with the fermentation of the microorganisms; however, as the fermentation continues, the nutrient substances in the fermentation system are consumed, and the partially dissolved flavone and polysaccharide are decomposed and utilized, so that the content of the flavone and polysaccharide is reduced.
2.2 results of orthogonal test for microbial fermentation extraction
According to the results of the single-factor test, the feed-liquid ratio, the inoculation amount and the fermentation temperature have obvious influence on the contents of flavone and polysaccharide in the zanthoxylum bungeanum leaves, so that the three factors are optimized by the orthogonal test, the flavone content and the polysaccharide content are respectively taken as response values to carry out the orthogonal test without interaction, and the orthogonal test design and the test results are shown in table 2.
TABLE 2 orthogonal optimization test results and analysis
Note: x, Y respectively taking flavone content and polysaccharide content as response values;
respectively the average value of the flavone content or the polysaccharide content of each factor under the same level; r is
The range of the three data in each column is extremely poor, namely the maximum value minus the minimum value, and the R value can reflect the influence degree of each factor on the flavone content or the polysaccharide content. The unit mg/g represents the amount of flavone and polysaccharide extracted from each gram of dry pricklyash leaves
As can be seen from Table 2, the main relationship among the factors affecting the flavone content of Zanthoxylum bungeanum leaf is: b (inoculum size)>A (feed liquid ratio)>C (fermentation temperature); the main factors influencing the flavone content of the zanthoxylum bungeanum leaves are as follows: b (inoculum size)>C (fermentation temperature)>A (ratio of material to liquid). Thus, the optimal fermentation conditions for flavones are A3B2C2The optimal fermentation conditions for the polysaccharide are A1B2C2. The analysis result of t-test shows that the polysaccharide content and the flavone content between the two groups have no significant difference, namely A3B2C2The content of the flavone is obviously higher than A1B2C2And (4) grouping. Therefore, select A3B2C2The optimal fermentation conditions for fermenting and extracting the flavones and the polysaccharides of the zanthoxylum bungeanum leaves are provided.
2.3 validation test of optimal Process
Combining the orthogonal test results of flavone and polysaccharide, and selecting the optimal fermentation condition A3B2C2Three verification experiments were carried out (feed-liquid ratio 1:20, fermentation temperature 31 ℃, fermentation time 3d), and control group (no inoculation of microorganism, other conditions being the same). The results show that the flavone content of the optimal condition set is 19.15 mg/g; the polysaccharide content was 17.62mg/g, all higher than the other test groups. And compared with the control group (the contents of flavone and polysaccharide are respectively 11.80mg/g and 11.59mg/g), the extraction amount is respectively improved by 62.29 percent and 52.03 percent. Therefore, according to the result of the single-factor test, the optimal conditions for fermenting and extracting the flavones and the polysaccharides from the Chinese prickly ash leaves are as follows: the ratio of feed to liquid is 1:20(g: mL), the inoculation amount is 5%, the ratio of strains is 1:2, the fermentation temperature is 31 ℃, and the fermentation time is 3 d.
2.4 in vitro antioxidant Activity
The in vitro antioxidant activity of the optimal group of flavones and polysaccharides was examined and the results are shown in table 3. Therefore, the flavones and the polysaccharides in the zanthoxylum bungeanum leaves have certain capacity of removing DPPH and OH, and the reducing power (0.79) is higher than that (0.71) of the positive control VC with the same concentration. In conclusion, the flavones and polysaccharides of the zanthoxylum bungeanum leaves have good in-vitro antioxidant activity.
3 conclusion
The method adopts a microbial fermentation extraction technology to synchronously extract flavone and polysaccharide in the zanthoxylum bungeanum leaves, and adopts an orthogonal test to optimize fermentation extraction conditions on the basis of a single-factor test to obtain the optimal fermentation conditions: the method comprises the steps of mixing pepper leaf powder with water according to a material-liquid ratio of 1:20(g: mL), inoculating 5% of a mixed seed liquid of Aspergillus niger and Bacillus subtilis according to a total inoculation ratio of 1:2, and performing fermentation extraction at 31 ℃ for 3 days under the condition of 150r/min, wherein the obtained content of flavone and polysaccharide in pepper leaves is maximum and is respectively 19.15mg/g and 17.62mg/g, and compared with a control group which does not use a microbial fermentation technology, the extraction amount is respectively improved by 62.29% and 52.03%. Provides valuable theoretical reference for the comprehensive development and utilization of the pepper leaves.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A method for synchronously extracting flavones and polysaccharides from Chinese prickly ash leaves is characterized by comprising the following process steps:
preprocessing Chinese prickly ash leaves
Drying folium Zanthoxyli Bungeani in a constant temperature drying oven at 55-65 deg.C, pulverizing, and sieving with 60 mesh sieve to obtain folium Zanthoxyli Bungeani powder;
② preparation of fermentation medium
Weighing a proper amount of pepper leaf powder, adding ultrapure water according to a material-liquid ratio, uniformly mixing, sterilizing for 15-25min at the temperature of 110-130 ℃, cooling to room temperature, inoculating activated bacillus subtilis and aspergillus niger mixed seed liquid according to a corresponding total inoculation amount and strain proportion, placing in a constant-temperature oscillation incubator for fermentation culture, and setting corresponding temperature and rotating speed for fermentation culture;
③ extraction of flavone
After fermentation, adding 60% ethanol, placing in an ultrasonic cleaning machine, extracting for 30min under the conditions of ultrasonic power of 100W and ultrasonic frequency of 40kHz, and filtering at 60 ℃;
extraction of polysaccharide
And (4) adding ultrapure water with 4 times volume into the filter residue obtained in the step (III), placing the filter residue in an ultrasonic cleaning machine, extracting for 30min under the conditions of ultrasonic power of 100W and ultrasonic frequency of 40kHz, and filtering and concentrating at the temperature of 60 ℃.
2. The method for synchronously extracting flavones and polysaccharides from zanthoxylum bungeanum leaves according to claim 1, wherein the feed-liquid ratio is 1:20(g: mL).
3. The method for synchronously extracting flavones and polysaccharides from zanthoxylum bungeanum leaves as claimed in claim 1, wherein the inoculation amount is 5%.
4. The method for synchronously extracting flavones and polysaccharides from zanthoxylum bungeanum leaves according to claim 1, wherein the ratio of the Aspergillus niger to the Bacillus subtilis mixed seed liquid strain is 1: 2.
5. The method for synchronously extracting flavones and polysaccharides from zanthoxylum bungeanum leaves as claimed in claim 1, wherein the fermentation culture temperature is 31 ℃, the rotation speed is 150r/min, and the fermentation time is 3 d.
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