CN117187319A - Peach gum polysaccharide and fermentation extraction method and application thereof - Google Patents
Peach gum polysaccharide and fermentation extraction method and application thereof Download PDFInfo
- Publication number
- CN117187319A CN117187319A CN202311131647.3A CN202311131647A CN117187319A CN 117187319 A CN117187319 A CN 117187319A CN 202311131647 A CN202311131647 A CN 202311131647A CN 117187319 A CN117187319 A CN 117187319A
- Authority
- CN
- China
- Prior art keywords
- peach gum
- fermentation
- gum polysaccharide
- polysaccharide
- galactanase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 103
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- 238000000855 fermentation Methods 0.000 title claims abstract description 58
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- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P37/08—Antiallergic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
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- Molecular Biology (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Pulmonology (AREA)
- Physics & Mathematics (AREA)
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Abstract
The invention discloses a fermentation extraction method of peach gum polysaccharide, which comprises the following steps: step one, mixing soaked peach gum, ultrapure water and yeast dry powder of 1, 3-expressible galactosan enzyme according to the proportion of 1:5-100:0.01-0.1, stirring and fermenting; stopping stirring when the viscosity of the fermentation liquid is reduced to 200-1000 mPa.s, pouring out the fermentation liquid, adding absolute ethanol until the mass fraction of the ethanol is 60-90%, standing, and removing the supernatant; and step three, washing the lower precipitate by adopting ethanol, and performing vacuum filtration and freeze drying to obtain the peach gum polysaccharide. The invention also discloses peach gum polysaccharide and application of the peach gum polysaccharide in preparing anti-allergic and antipruritic skin care products. The fermentation and extraction method of peach gum polysaccharide has low energy consumption and is environment-friendly. The peach gum polysaccharide obtained by the method has the effects of resisting allergy and relieving itching.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to peach gum polysaccharide, and a fermentation extraction method and application thereof.
Background
The peach gum has various medicinal values, and has the main effects of reducing blood fat, reducing blood sugar, moisturizing skin and the like in the aspects of medicine and cosmetology. The peach gum has large molecular weight, high crosslinking structure and difficult dissolution. The preparation of the soluble peach gum polysaccharide can be generally carried out by adopting a water swelling hydrolysis extraction method, an improved alcohol extraction method, a high pressure method and the like, and although the solubility of the peach gum polysaccharide is improved, the molecular weight is greatly reduced, the uniformity of the product is poor, the molecular weight distribution of the product is wider, the skin barrier or intestinal mucosa system has certain barrier and barrier effect on the absorption of the peach gum polysaccharide, and the bioavailability is lower. Therefore, a method for effectively utilizing peach gum polysaccharide by organisms is needed to be researched so as to promote the application of peach gum in medicine or beauty and skin care. In view of the fact that the polysaccharide in the organism is mostly a polymer with sugar units, the polysaccharide is cracked into oligosaccharide through a specific method, so that the molecular weight of the product can be effectively reduced, the absorption of the organism is facilitated, and the biological activity of the product is also expected to be improved. In recent years, research on microbial enzyme-producing and splitting peach gum polysaccharide has been developed, foreign research has been carried out to separate crude enzymes secreted in and out of cells of aspergillus flavus and capable of hydrolyzing peach gum polysaccharide from aspergillus flavus cultures taking dissolved peach gum polysaccharide as a carbon source, and research has been carried out in China to screen strains with high expression of galactanase from strains splitting carrageenan for peach gum splitting and polysaccharide extraction.
However, screening and preservation of highly active, highly stable strains of galactanase enzymes is a challenging task. The expression level of the natural strain causes a decrease in activity and stability with an increase in the number of generations. The natural strain requires specific culture medium conditions, so that the introduction of new impurities into the components and the metabolites produced during fermentation is unavoidable, increasing the difficulty of purification of the polysaccharide at a later stage and bringing uncontrollable risks to the use of the product.
Disclosure of Invention
In order to solve the problems, the invention utilizes yeast engineering bacteria, introduces a galactanase gene sequence through a DNA recombination technology, takes crude extracted peach gum polysaccharide as a nutrient culture solution, takes saccharide components as a carbon source and trace plant proteins as a nitrogen source, maintains the growth of the yeast, and enables the yeast to produce the galactanase so as to form extracellular proteins secreted outside cell walls, but does not release into the culture solution. So as to directionally enzymolysis the crosslinked peach gum polysaccharide, release polysaccharide monomers with smaller molecular weight, and improve the bioavailability of the polysaccharide. Therefore, the first purpose of the invention is to provide a fermentation extraction method of peach gum polysaccharide, so as to solve the problems of poor uniformity and wider molecular weight distribution of products in the existing extraction method of peach gum polysaccharide. The second object of the invention is to provide peach gum polysaccharide obtained by the fermentation and extraction method. The third object of the invention is to provide an application of the peach gum polysaccharide.
In order to achieve the above purpose, the invention adopts the following technical scheme:
as a first aspect of the present invention, a method for fermentation extraction of peach gum polysaccharide, comprising the steps of:
step one, mixing soaked peach gum, ultrapure water and saccharomycete dry powder capable of expressing 1, 3-galactanase according to the proportion of 1:5-100:0.01-0.1, and then pouring the mixture into a fermentation tank for constant temperature and constant speed stirring fermentation;
stopping stirring when the viscosity of the fermentation liquid is reduced to 200-1000 mPa.s, pouring out the fermentation liquid, adding absolute ethanol until the mass fraction of the ethanol is 60-90%, standing, and removing the supernatant;
washing the lower precipitate with ethanol, vacuum filtering, freeze drying to obtain peach gum polysaccharide,
the NCBI GenBank of the amino acid sequence of 1, 3-galactanase is KAA8623185.1.
According to the invention, the ratio of peach gum, ultrapure water and yeast dry powder capable of expressing 1, 3-galactanase in the step one is 1:10:0.01.
Preferably, in the second step, absolute ethyl alcohol is added until the mass fraction of the ethyl alcohol is 70%, and the mixture is kept still.
According to the invention, the preparation method of the saccharomycete dry powder capable of expressing 1, 3-galactanase in the step one comprises the following steps:
A. preparation of yeast clone: extracting genome DNA of an expression vector 1, 3-galactanase, linearizing, and then electroconverting into competent pichia pastoris; obtaining a yeast strain capable of expressing 1, 3-galactanase through MD plate culture;
b: purifying the saccharomycetes capable of expressing the 1, 3-galactanase in the step A, and preparing the saccharomycetes into saccharomycetes dry powder.
According to the invention, the fermentation temperature in the first step is 30 ℃, the rotating speed is 50-500 rpm, and the fermentation time is 5-20h.
As a second aspect of the present invention, a peach gum polysaccharide is obtained by extraction using the fermentation extraction method of peach gum polysaccharide described above.
As a third aspect of the invention, the use of a peach gum polysaccharide as described above for the preparation of a medicament for the treatment of allergy and itching.
As a fourth aspect of the invention, the use of a peach gum polysaccharide as described above for the preparation of an anti-allergic and antipruritic skin care product.
As a fifth aspect of the invention, a pharmaceutical composition comprises a peach gum polysaccharide as described above.
As a sixth aspect of the invention, a cosmetic composition comprises a peach gum polysaccharide as described above.
The fermentation and extraction method of peach gum polysaccharide has the beneficial effects that:
1. according to the invention, the yeast engineering bacteria are utilized, and the 1, 3-galactosan enzyme gene sequence is introduced through a DNA recombination technology, so that the yeast expresses the galactosan enzyme to form extracellular proteins secreted outside the cell wall, but the extracellular proteins are not introduced into the culture solution, so that the introduction of new impurities can be avoided, and the purification of peach gum polysaccharide in subsequent procedures is facilitated; meanwhile, the invention has simple operation and formula operation by utilizing the strain of the yeast engineering bacteria, and the strain is stable, so that the yeast dry powder can be prepared in advance for long-term storage;
2. the fermentation method is adopted, the fermentation and purification processes are simple, the fermentation process is low in energy consumption and emission, and the environment is protected;
3. in the fermentation process, saccharomycetes use saccharide substances in peach gum as a carbon source, protein components in peach gum as a nitrogen source, and other culture medium components are not required to be introduced. The waste protein components are degraded, so that the purification of the peach gum polysaccharide in the later stage is facilitated;
4. the peach gum polysaccharide extracted by the method has higher hydrolysis degree, low viscosity and narrower molecular weight range; can improve the anti-allergic and antipruritic effects of peach gum polysaccharide;
5. and (3) adding the saccharomycete dry powder into a fermentation tank, and stirring, wherein the stirring can be used for rapidly carrying out enzymolysis on peach gum and extracting polysaccharide in the peach gum.
The peach gum polysaccharide extracted by the fermentation method has the beneficial effects that: has the effects of rapidly resisting sensitization, relieving itching, diminishing inflammation and relieving pain on the skin. Therefore, the method has wide application value in the fields of medicine and cosmetology.
Drawings
FIG. 1 is a chromatogram of peach gum polysaccharide obtained by the water extraction method of example 1.
FIG. 2 is a chromatogram of peach gum polysaccharide obtained by the fermentation method of example 2.
FIG. 3 is a graph showing the results of the proliferation of macrophages promoted by peach gum polysaccharide of example 4.
FIG. 4 is a graph showing the results of the peach gum polysaccharide anti-allergic and red-removing test of example 9.
Fig. 5 is a block diagram showing the itching relieving test of human skin by peach gum polysaccharide according to example 10.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of engineering Yeast Dry powder
1) Preparation of yeast clones, according to the amino acid sequence of 1, 3-galactanase (sequence from NCBI: genBank: KAA 8623185.1), and digested and ligated with nucleic acid to obtain linearized DNA with the signal peptide removed, and DNA synthesis and vector construction were purchased from Shanghai New Yopulorganism, and the linearized DNA was dissolved in 5-10. Mu.LTE. mu.L of commercial competent Pichia pastoris GS115 (purchased from Tian Gen) was mixed with 10. Mu.g of linearized DNA and transferred to a pre-chilled 0.2cm electric beaker. Place on ice for 5min. Machine parameters were set, 1ml of pre-chilled 1M sorbitol was immediately added to the cup, the contents transferred to a sterile centrifuge tube into 200 μl aliquots, plated on MD plates (basal glucose medium) and incubated plates at 30 ℃ until clonal generation.
2) Expression and purification of galactanase. Monoclonal was picked up, inoculated into 25ml BMGY Medium (Buffered Glycerol-complex Medium), shake flask 250ml, fermented at 30℃and 250rpm for 5 hours to a density value of bacteria OD 600=4, centrifuged at 3000g at room temperature for 5 minutes, cells were collected, the supernatant was removed, and cells were resuspended to OD 600=1.0 with BMMY Medium for induction expression. Adding the above culture into 1L shake flask, covering with two layers of sterilized gauze or cheesecloth, and placing into a shaking table for continuous growth. Every 24 hours, methanol was added to a final concentration of 0.5% to continue induction. After 3 days of continuous culture, the expression of galactanase was detected by SDS-PAGE electrophoresis. The cells were collected at a centrifugation speed of 4000 rpm. The cells were washed with PBS to prepare a medium composition. And finally, drying the thalli into bacterial powder by using a freeze dryer. The content of 1, 3-galactanase in each gram of dry bacterial powder is 0.2 gram. And (5) refrigerating and preserving for standby.
Example 2 high pressure Water extraction method for peach gum polysaccharide
After sundries are removed from peach gum, about 100g of peach gum is taken, and is soaked with proper amount of water overnight, ultrapure water is added according to the feed-liquid ratio of 1:20, and the peach gum is extracted by stirring with hot water at 80 ℃ for 4 hours for 2 times. Because the viscosity of the solution is high, filtering with 2 layers of gauze, centrifuging the filtrate for 15min at 8000r/min, pouring out the supernatant, performing reduced pressure rotary evaporation to about 1/3 of the original volume, pouring out, adding absolute ethanol until the mass fraction of the ethanol is 90%, standing overnight, and pouring out the supernatant. Washing the lower sediment with absolute ethyl alcohol, decompressing, pumping and filtering, and freeze-drying to obtain peach gum polysaccharide. The total saccharide content of peach gum polysaccharide was determined using the phenol sulfuric acid method, and the standard curve was determined using glucose.
The recovery rate of the polysaccharide is 98 percent by a phenol sulfuric acid method, the protein content of the polysaccharide is 0.12 percent by a coomassie brilliant blue method, and the polysaccharide consists of galactose, xylose, arabinose, mannose and glucose by HPLC-ELSD chromatographic analysis, wherein the molar ratio is 18.12:13.64:54.37:2.58:10.75. the solution was diluted to 50mg/ml with water and the viscosity of the solution was found to be 4000 Pa.s.
The HPLC-SEC chromatographic analysis results are shown in FIG. 1.
The results in FIG. 1 show that the aqueous extraction method extracts polysaccharide with a retention time of 7.3 minutes.
EXAMPLE 3 extraction of peach gum polysaccharide by fermentation
After sundries are removed from peach gum, about 100g of peach gum is soaked with proper amount of water overnight, and the feed liquid ratio of the peach gum to ultrapure water to the yeast dry powder expressing galactanase is 1:10:0.01 ultrapure water and yeast dry powder (expressed with the galactosan enzyme) are added, then poured into a 5L fermentation tank, 0.5 percent methanol is added, the temperature is regulated to 30 ℃, the rotating speed is 250rpm, and the fermentation time is 10 hours. The consistency of the feed solution was measured and the fermentation was stopped when the consistency was reduced below 500 mpa.s. The feed liquid was poured out and centrifuged at 6000rpm for 10 minutes with a high-speed centrifuge. The bacterial precipitate at the bottom is discarded, and microfiltration is performed by using a roll filter (pore size of 0.22 μm) to remove residual bacterial and granular components without causing bacterial contamination. Absolute ethanol is added until the mass fraction of the ethanol is 70%, and the supernatant is poured off after standing overnight. Washing the lower precipitate with 75% ethanol, vacuum filtering, and lyophilizing to obtain peach gum polysaccharide. The total saccharide content of peach gum polysaccharide was determined using the phenol sulfuric acid method, and the standard curve was determined using glucose. Protein content was determined using coomassie brilliant blue G250 method.
The recovery rate of polysaccharide is 98% by the phenol sulfuric acid method. The protein content was 0.02% as measured by coomassie brilliant blue method. The vegetable protein content is significantly reduced. The crude polysaccharide consisted of galactose, xylose, arabinose, mannose, glucose by HPLC-ELSD chromatography with a molar ratio of 20.35:17.86:50.84:1.57:9.57.
the HPLC-SEC chromatographic analysis results are shown in FIG. 2.
The results in FIG. 2 show that the fermentation process extracts polysaccharide with a retention time of 8.5 minutes.
Comparison of the fermentation process of example 3 with the conventional water extraction process of example 2:
(1) The recovery of polysaccharide obtained by both methods is close, but the protein content of the fermentation process is significantly reduced, meaning that the polysaccharide purity is higher.
(2) The composition of glucose and arabinose in the polysaccharide component in the fermentation method is reduced, and the galactose and xylose contents are increased.
(3) The peak time of polysaccharide molecules obtained by the fermentation method is longer than that of polysaccharide molecules obtained by the water extraction method, which means that the molecular weight is lower, the width of the peak is narrower, the molecular weight distribution is narrower, and the molecular size is more uniform.
EXAMPLE 4 extraction of peach gum polysaccharide by fermentation
After sundries are removed from peach gum, about 100g of peach gum is soaked with proper amount of water overnight, and the feed liquid ratio of the peach gum to ultrapure water to the yeast dry powder expressing galactanase is 1:5:0.001 adding ultrapure water and saccharomycete dry powder, pouring into a 5L fermentation tank, adding 5% methanol, regulating the temperature to 30 ℃, stirring at 50rpm, and slowly stirring for fermentation time of 20h. The consistency of the feed solution was determined and the fermentation was stopped after the consistency had fallen below 1000 mpa.s. The feed liquid was poured out and centrifuged at 6000rpm for 10 minutes with a high-speed centrifuge. The bacterial precipitate at the bottom is discarded, and microfiltration is performed by using a roll filter (pore size of 0.22 μm) to remove residual bacterial and granular components without causing bacterial contamination. Absolute ethanol is added until the mass fraction of the ethanol is 70%, and the supernatant is poured off after standing overnight. Washing the lower precipitate with 75% ethanol, vacuum filtering, and lyophilizing to obtain peach gum polysaccharide.
EXAMPLE 5 extraction of peach gum polysaccharide by fermentation
After sundries are removed from peach gum, about 100g of peach gum is taken, and is soaked with proper amount of water overnight, and the ratio of the peach gum to the water is 1:100:0.01 of ultrapure water and saccharomycete dry powder are added, then poured into a 5L fermentation tank, 5 percent of methanol is added, the temperature is regulated to 30 ℃, the rotating speed is 500rpm, and the fermentation time is 5 hours. The consistency of the feed solution was measured and the fermentation was stopped after the consistency had decreased below 200 mpa.s. The feed liquid was poured out and centrifuged at 6000rpm for 10 minutes with a high-speed centrifuge. The bacterial precipitate at the bottom is discarded, and microfiltration is performed by using a roll filter (pore size of 0.22 μm) to remove residual bacterial and granular components without causing bacterial contamination. Absolute ethanol is added until the mass fraction of the ethanol is 70%, and the supernatant is poured off after standing overnight. Washing the lower precipitate with 75% ethanol, vacuum filtering, and lyophilizing to obtain peach gum polysaccharide.
EXAMPLE 6 macrophage proliferation promotion
To test the biological activity of polysaccharide extracted by the fermentation method in vitro, the present example determines the biological effect of polysaccharide by comparing the methods of stimulating macrophage proliferation. 200 mu L of RAW 264.7 mouse macrophage cells in logarithmic growth phase are inoculated into a 96-well plate, and the cell concentration is 10 multiplied by 10 3 Individual wells/wells, CO at 37 DEG C 2 After overnight incubation in the incubator, the supernatant was aspirated after cell attachment, 200. Mu.L of the polysaccharide solution prepared in example 2 at a concentration of 10. Mu.g/ml, 40. Mu.g/ml and 80. Mu.g/ml, respectively, and 200. Mu.L of the polysaccharide solution prepared in example 3 at a concentration of 10. Mu.g/ml, 40. Mu.g/ml and 80. Mu.g/ml, respectively, were added, and incubation in the incubator was continued for another 24 hours. Then 20. Mu.L of CCK-8 solution was added, and after incubation for 2 hours, absorbance was measured at 490 nm. The positive control group was lipopolysaccharide of 10. Mu.m. Proliferation rate (%) = (a/a) 0 -1) x 100. The results are shown in FIG. 3.
The results show that: the peach gum polysaccharide extracted by both methods can show the effect of promoting macrophage proliferation. Among them, the effect of the peach gum polysaccharide extracted in example 3 is significantly better than that of the peach gum polysaccharide extracted in example 2, and the effect is most obvious at 40 mug/mL. The effect of water extraction is equivalent to that of lipopolysaccharide. There was a significant difference from the blank.
Conclusion: the peach gum polysaccharide extracted by the fermentation method has better effect of promoting macrophage proliferation.
EXAMPLE 7 action against dextran-induced skin itch in mice
To determine the effect of the polysaccharide extracted from the different examples, the bioavailability was assessed using an anti-itch method. 80 mice were divided into 10 groups at random, and administered by gavage at the doses listed in Table 1. For 5 consecutive days, 30min after the last administration, dextran was intravenously injected at 0.95mg/kg in each mouse tail. The itching is indicated by the scratching of the head, the scratching of the trunk and the mouth of the mice. In order to calculate the itching degree more accurately, the paw or mouth is designed to touch the body part for 1 time, the itching action is performed but the body part is not touched for 0.2 time, and the itching times and the itching duration of the mice within 30 minutes are recorded. The results are shown in Table 1.
Table 1 action of peach gum polysaccharide on resisting dextran-induced skin itch of mice (X+ -s)
Note that: p <0.05, P <0.01 compared to the blank group
The results show that the peach gum polysaccharide extracted by the water extraction method and the fermentation method has obvious antipruritic effect on the systemic skin itch model of mice, namely, the model has obvious antipruritic effect on the exogenous dextran-induced release endogenous histamine animal itch model, and the duration of itch is shorter. Among them, the peach gum polysaccharides extracted in example 3 and example 5 have more remarkable antipruritic effect, and have remarkable improvement effect on main skin allergy symptoms.
Example 8 Effect on histamine induced increases in capillary permeability in mice
80 mice were divided into 10 groups at random, and administered by gavage at the doses listed in Table 2. After the last administration for 30min, injecting 10 mug/mL histamine into abdomen to form a small skin hill, immediately injecting 0.5% Evans blue 0.01mL/g into tail vein, killing animals after 30min, peeling two blue dyed skin sheets by using a puncher with the diameter of 0.8mm, putting into 3mL physiological saline-acetone (3:7) mixed solution, putting into a constant temperature water bath pot at 37 ℃ for 24h, centrifuging the leaching solution, taking supernatant, and measuring absorbance by using a spectrophotometer (610 nm). The results are shown in Table 2.
TABLE 2 vascular permeability increasing effect of peach gum polysaccharide (X.+ -.s)
Note that: p <0.05, P <0.01 compared to the blank group
The results show that: the peach gum polysaccharide extracted in the example 3 and the example 5 has obvious inhibition effect on capillary permeability increase caused by histamine, and can reduce or eliminate allergic symptoms. The inhibition rate of example 3 is better.
Example 9 human skin anti-sensitization test
6 skin healthy volunteers were recruited. Cleaning arm skin, and air drying. 4 areas (total 8 areas) were selected on the front of each arm, the outline was drawn with a marker, and 3X 3cm filter paper was cut and attached to the arm. The test was performed once per day. The test was performed for a total of 50 days. 3ml of a 1.5% histamine solution was pipetted onto each piece of filter paper. The filter paper was allowed to absorb the solution uniformly. The histamine solution was allowed to rest on the arm for 30 minutes. The filter paper is then removed and the skin surface is rinsed of histamine solution. Seven areas with similar red and swollen and itching degrees were selected. 1mg of peach gum polysaccharide of examples 2-5 was applied respectively. Two positive controls were each coated with 1mg of oat kernel extract. 100 μl of 0.1% paeonol was used for the drug control group. The blank group was smeared with physiological saline. After 30 minutes, skin itching and surface redness were observed. The results are shown in FIG. 4, table 3 and Table 4.
TABLE 3 human skin anti-sensitization test
Remarks: each group was tested once a day for a total of 50 days. I.e. the total number of test points per group is 50.
TABLE 4 human skin anti-sensitization test
Remarks: drop test point duty cycle = drop test point number/total number of test points.
The results show that the peach gum polysaccharide extracted in the embodiment 3 and the embodiment 5 has obvious functions of resisting allergy and removing redness, and can obviously relieve the symptoms of skin itch, pimple and redness.
Example 10 itching time test of human skin
6 skin healthy volunteers were recruited. Cleaning arm skin, and air drying. 3 areas (total 6 areas) were selected on the front of each arm, the outline was drawn with a marker, and 3X 3cm filter paper was cut and attached to the arm. 3ml of a 1.5% histamine solution was pipetted onto each piece of filter paper. The filter paper was allowed to absorb the solution uniformly. The histamine solution was allowed to rest on the arm for 30 minutes. The filter paper is then removed and the skin surface is rinsed of histamine solution. Four areas with similar red and swollen and itching degrees were selected. Coated with 1mg/ml of the polysaccharide prepared in example 3 and the polysaccharide prepared in example 2, respectively. The positive control used 1mg/ml oat kernel extract and 0.1% paeonol solution. The blank group was smeared with physiological saline. Touching the skin at different time points, the skin itching is felt. The results are shown in FIG. 5.
The results in fig. 5 show that the polysaccharide prepared in example 3 can rapidly relieve skin itch, and can significantly improve skin itch within 5 minutes and completely inhibit skin itch within 20 minutes.
In conclusion, the peach gum polysaccharide extracted by the fermentation method removes protein components in plants in the fermentation process, improves the purity of the polysaccharide, and has obvious anti-allergy and antipruritic effects, so that the peach gum polysaccharide can be better applied to the fields of beauty and skin care and anti-allergy antipruritic medicines.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof. For example, the sequences of the examples of the present invention are merely illustrative of the invention, and one skilled in the art can redesign primers and probes for detecting other target gene sequences in accordance with the principles of the present invention.
Claims (9)
1. The fermentation extraction method of the peach gum polysaccharide is characterized by comprising the following steps of:
step one, mixing soaked peach gum, ultrapure water and saccharomycete dry powder capable of expressing 1, 3-galactanase according to the proportion of 1:5-100:0.01-0.1, and then pouring the mixture into a fermentation tank for constant temperature and constant speed stirring fermentation;
stopping stirring when the viscosity of the fermentation liquid is reduced to 200-1000 mPa.s, pouring out the fermentation liquid, adding absolute ethanol until the mass fraction of the ethanol is 60-90%, standing, and removing the supernatant;
washing the lower precipitate with ethanol, vacuum filtering, freeze drying to obtain peach gum polysaccharide,
the NCBI GenBank of the amino acid sequence of 1, 3-galactanase is KAA8623185.1.
2. The method for fermentation extraction of peach gum polysaccharide according to claim 1, wherein the ratio of peach gum, ultrapure water and yeast dry powder capable of expressing 1, 3-galactanase in the step one is 1:10:0.01.
3. The method for extracting and fermenting peach gum polysaccharide according to claim 1, wherein the method for preparing the saccharomycete dry powder capable of expressing 1, 3-galactanase in the first step comprises the following steps:
A. preparation of yeast clone: extracting genome DNA of an expression vector 1, 3-galactanase, linearizing, and then electroconverting into competent pichia pastoris; obtaining a yeast strain capable of expressing 1, 3-galactanase through MD plate culture;
b: purifying the saccharomycetes capable of expressing the 1, 3-galactanase in the step A, and preparing the saccharomycetes into saccharomycetes dry powder.
4. The method for fermentation and extraction of peach gum polysaccharide according to claim 1, wherein the fermentation temperature in the first step is 30 ℃, the rotation speed is 50-500 rpm, and the fermentation time is 5-20h.
5. A peach gum polysaccharide, characterized in that it is obtained by extraction by fermentation extraction of the peach gum polysaccharide according to any one of claims 1-4.
6. Use of the peach gum polysaccharide according to claim 5 for preparing a medicament for treating allergy and itching.
7. Use of the peach gum polysaccharide according to claim 5 for preparing an anti-allergic and antipruritic skin care product.
8. A pharmaceutical composition comprising the peach gum polysaccharide of claim 5.
9. A cosmetic composition comprising the peach gum polysaccharide of claim 5.
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| JP4267696B2 (en) * | 1996-03-01 | 2009-05-27 | ノボザイムス アクティーゼルスカブ | Enzyme having galactanase activity |
| CN101701198B (en) * | 2009-10-27 | 2012-06-06 | 暨南大学 | Peach gum hydrolase producing strain and application in preparation of peach gum polysaccharide thereof |
| CN102372789B (en) * | 2010-08-25 | 2013-06-05 | 上海辉文生物技术有限公司 | Peach gum polysaccharide, its extract, preparation method and application thereof |
| CN104651340A (en) * | 2015-03-04 | 2015-05-27 | 暨南大学 | Peach gum polysaceharide lyase with microbacterium source and separation and purification method and application of peach gum polysaceharide lyase |
| CN105061617B (en) * | 2015-07-31 | 2017-06-30 | 华中农业大学 | A kind of extraction process of peach gum polysaccharide and its application |
| CN105753998B (en) * | 2016-01-23 | 2018-04-10 | 暨南大学 | A kind of peach gum polysaccharide catabolite PGP 1 and its preparation method and application |
| CN105669874B (en) * | 2016-02-02 | 2017-11-28 | 暨南大学 | A kind of peach gum polysaccharide catabolite PGP 2 and its preparation method and application |
| CN107286268A (en) * | 2017-08-02 | 2017-10-24 | 杨俊� | A kind of peach gum polysaccharide extracting method |
| CN110511969A (en) * | 2019-09-06 | 2019-11-29 | 上海应用技术大学 | Natural peach gum fermentation prepares the method for peach gum extract and its applies in cosmetics |
| CN111235197A (en) * | 2020-03-04 | 2020-06-05 | 美尔健（深圳）生物科技有限公司 | High-efficiency anti-allergic itching-relieving peach gum polysaccharide and fermentation extraction method and application thereof |
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2020
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2021
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| CN112725392A (en) | 2021-04-30 |
| CN111235197A (en) | 2020-06-05 |
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