CN109652479B - Method for improving antioxidant capacity of dendrobe polysaccharide - Google Patents

Method for improving antioxidant capacity of dendrobe polysaccharide Download PDF

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Publication number
CN109652479B
CN109652479B CN201910073553.2A CN201910073553A CN109652479B CN 109652479 B CN109652479 B CN 109652479B CN 201910073553 A CN201910073553 A CN 201910073553A CN 109652479 B CN109652479 B CN 109652479B
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polysaccharide
dendrobium
antioxidant capacity
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CN109652479A (en
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白文艳
陈洁梅
黄九九
刘伟
古志平
吴保林
乔晓颖
陈丽花
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Guangzhou Meihujian Biotechnology Co ltd
Guangzhou Opseve Cosmetics Co ltd
South China Agricultural University
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Guangzhou Meihujian Biotechnology Co ltd
Guangzhou Opseve Cosmetics Co ltd
South China Agricultural University
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/04Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Abstract

The invention discloses a microbial fermentation method for improving the oxidation resistance of dendrobium polysaccharide, belonging to the field of plant active ingredient function improvement. The method effectively improves the inoxidizability of the dendrobium polysaccharide by inoculating beneficial microorganisms, degrading polysaccharide with high molecular weight into polysaccharide with low molecular weight which is beneficial to absorption of organisms by utilizing microbial fermentation, and determining the total antioxidant capacity of fermentation liquor and stock solution. The method has the advantages of simple process, convenient operation, mild reaction conditions, environmental protection and easy implementation and popularization; the dendrobe polysaccharide obtained by the method has small molecular weight, strong oxidation resistance and wide application prospect in the fields of research and development of functional medicines and foods and the like, and is beneficial to organism absorption.

Description

Method for improving antioxidant capacity of dendrobe polysaccharide
Technical Field
The invention relates to the field of function improvement of plant active ingredients, in particular to a method for improving the antioxidant capacity of dendrobe polysaccharide.
Background
Dendrobe (Dendrobium) is one of the earliest recorded orchidaceae plants in ancient literature in China, has the effects of tonifying stomach, promoting fluid production, nourishing yin and clearing heat, and contains polysaccharide as a main active ingredient. In the last decade, related research work mainly focuses on the research on extraction methods, chemical components, pharmacological actions, fingerprint spectra and the like of dendrobium officinale (Yao morning, research progress of dendrobium officinale polysaccharides, Chinese national folk medicine 2015, 24 (15): 6-7).
Polysaccharide is a polymer formed by connecting more than 10 monosaccharides through glycosidic bonds, widely exists in animal, plant and microorganism tissues, and is one of basic substances for maintaining life and normal operation (high sensitivity and the like, Chinese medicine plant polysaccharide resists skin aging). Researches find that the polysaccharide and the glycoconjugate participate in and mediate the regulation of various life phenomena of cells (Liuyao and the like, the research on the bioactivity of plant polysaccharide progresses, Jiangsu agricultural science, 2013, 41 (1): 1-4).
The human body has various free radicals and extremely strong oxidizing capability, unsaturated fatty acids of various biological membranes can be subjected to peroxidation to form lipid peroxide, and serious consequences such as nucleic acid cross-linking error, protein decomposition, DNA mutation and the like are caused, so that the biological membrane has the functions of resisting aging and various diseases. Scientific studies have also found that increased levels of oxidative stress are associated with many diseases. At present, people generally consider that the plant polysaccharide has the effects of scavenging free radicals and resisting aging. The mechanism of action may be explained by (1) direct action on ROS itself. (2) The polysaccharide molecule acts on the antioxidant enzyme. (3) The polysaccharide molecule complexes the metal ions necessary to generate ROS. (4) Promoting the release of SOD from the cell surface. With the intensive research on polysaccharides, it has been found that polysaccharides have many bioactive effects. Has good effects in resisting tumor, enhancing immunity, resisting virus, resisting inflammation, lowering blood sugar, reducing blood lipid, resisting aging, and promoting fracture healing. The method is probably related to the fact that the polysaccharide shows stronger antioxidant activity in various experimental systems (Liuyao and the like, the research progress of the biological activity of plant polysaccharide, Jiangsu agricultural science, 2013, 41 (1): 1-4).
Generally, in the polysaccharide leaching liquor directly extracted, the polysaccharide has large molecular weight and poor water-soluble effect, and is not beneficial to organisms to absorb into the body and exert the biological activity of the organisms, so that the application of the polysaccharide is limited to a certain extent, the polysaccharide is degraded to a certain extent, and the biological activity of the polysaccharide can be maintained and even improved, thereby having important significance. In the polysaccharide degradation method, the microbial degradation method does not need a large amount of reaction reagents, has mild reaction conditions and is environment-friendly. The degradation product is safe, and can be used as raw material for producing cosmetics, food and medicine.
Disclosure of Invention
The invention aims to provide a method for improving the antioxidant capacity of polysaccharide, which improves the antioxidant capacity of polysaccharide in a microbial fermentation mode, reduces the molecular weight of polysaccharide without influencing the main components of polysaccharide, is beneficial to the absorption of organisms and the improvement of biological activity, does not need a large amount of reaction reagents, has mild reaction conditions and is environment-friendly.
The purpose of the invention is realized by the following technical scheme:
a method for improving the antioxidant capacity of dendrobe polysaccharide comprises the following steps:
(1) extracting herba Dendrobii polysaccharide with hot water;
(2) activating microbial strains;
(3) preparing a seed solution;
(4) and (5) inoculating and fermenting.
Preferably, the microorganism is bacillus subtilis. Is preserved by the research center for plant germplasm protection and utilization of the university of south China agriculture, and is purchased from Bacillus subtilis with the number of 1.821 in the China general microbiological culture Collection management center.
Preferably, the specific process of step (1) is as follows: taking fresh dendrobium, slicing, drying to constant weight, grinding and sieving, and mixing according to the weight ratio of 1: adding the dendrobium dry powder and distilled water into 80(g: mL) material-liquid ratio, heating in a water bath at 80-90 ℃ for 1-2 h, filtering, repeatedly extracting filter residues once, combining extracting solutions obtained in two times, adding absolute ethyl alcohol until the final concentration of alcohol is 80%, standing overnight, filtering to obtain crude polysaccharide precipitate, redissolving the crude polysaccharide precipitate by using distilled water, adjusting the concentration of the redissolved solution to 10mg/mL, taking 50mL of the redissolved solution into a reaction container, and sterilizing for later use.
Preferably, the specific process of step (2) is as follows: selecting the preserved bacterial colony with inoculating needle, streaking on plate, culturing at 37 deg.c in constant temperature incubator for 24 hr, selecting single bacterial strain for slant preservation or continuous streaking amplification.
Preferably, the specific process of step (3) is as follows: scraping 3 rings of strains from a plate cultured for 24h to 100mL of nutrient broth culture medium, and culturing for 24h at constant temperature of 37 ℃ and 180r/min by using a shaking table to obtain a seed solution.
Preferably, the specific process of step (4) is as follows: adding 1mL of seed solution into polysaccharide solution to be fermented, shaking and culturing at 37 deg.C for 180r/min in a constant temperature shaking incubator, and sterilizing at 121 deg.C for 20min after 16 hr.
Preferably, the dendrobium stem is dendrobium officinale, dendrobium nobile, dendrobium chrysotoxum, dendrobium fimbriatum, dendrobium rosa rugosa, dendrobium nobile and dendrobium sarcandicum.
The invention has the following beneficial effects:
the invention solves the problem that the consumption of the dendrobium is reduced and the dendrobium has the same oxidation resistance under a certain application cost condition, so that the consumption of the polysaccharide is greatly reduced, basic data is provided for the development and utilization of the dendrobium polysaccharide, and the economic benefit of dendrobium polysaccharide processing enterprises is increased. Meanwhile, after microbial fermentation, the macromolecular polysaccharide is degraded into small molecules which are beneficial to the absorption of organisms, the medicinal value of the dendrobium polysaccharide is improved, and the dendrobium polysaccharide has important significance for the research and development of functional medicines and foods. The method has the advantages of simple operation, low cost, no need of a large amount of reaction reagents, mild reaction conditions and simple operation.
Drawings
FIG. 1 is a comparison of the antioxidant capacity of the biofermented polysaccharides of example 1 with that of a polysaccharide stock.
FIG. 2 is a comparison of the antioxidant capacity of the biofermented polysaccharides of example 2 with that of the polysaccharide stock solutions.
Detailed Description
The following will clearly and completely describe the technical solutions in 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 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.
The present invention will be described in further detail with reference to specific embodiments in order to make the above objects, features and advantages more apparent and understandable.
Example 1
Agar plate medium: 20g/L of agar powder (dry powder), 3g/L of beef extract and 10g/L, NaCl 5g/L of peptone, wherein the pH value is 7.4 +/-0.2; liquid culture medium: 3g/L beef extract, 10g/L, NaCl 5g/L peptone and pH value of 7.4 +/-0.2. The microorganism used is bacillus subtilis which is purchased from bacillus subtilis with the number of 1.821 in China general microbiological culture collection center, and the fermentation is carried out according to the following steps:
(1) extracting the dendrobium officinale polysaccharide by a hot water extraction method: taking 100g of fresh dendrobium, slicing, placing in a dryer for drying treatment at 105 ℃, drying for 5min at 65 ℃ to constant weight (about 41.31g), grinding, sieving with a 40-mesh sieve, and mixing according to the weight ratio of 1: adding 20g of dendrobium dry powder and 1600mL of distilled water into 80(g: mL) of material-liquid ratio, heating in a water bath at 80 ℃ for 1h, filtering, repeatedly extracting filter residues once, combining extracting solutions obtained in two times, adding about 3.00L of absolute ethyl alcohol until the final concentration of alcohol is 80%, standing overnight at 4 ℃, filtering to obtain about 13.77g of crude polysaccharide precipitate, adding 5L of distilled water for redissolution, adjusting the concentration of the redissolved solution to 10mg/mL, taking 50mL of the redissolved solution into a 100mL conical flask, placing at 121 ℃, and sterilizing for 20min for later use.
(2) Activating microbial strains: selecting the preserved bacterial colony with inoculating needle, streaking on plate, culturing at 37 deg.c in constant temperature incubator for 24 hr, selecting single bacterial strain for slant preservation or continuous streaking amplification.
(3) Preparing a seed solution: scraping 3 rings of strains from a plate cultured for 24h to 100mL of nutrient broth culture medium, and culturing for 24h at constant temperature of 37 ℃ and 180r/min by using a shaking table to obtain a seed solution.
(4) Inoculating and fermenting: adding 1mL of seed solution into polysaccharide solution to be fermented, shaking and culturing at 37 deg.C for 180r/min in a constant temperature shaking incubator, and sterilizing at 121 deg.C for 20min after 16 hr.
Measuring the total antioxidant capacity of the fermentation liquor: the total antioxidant activity was measured by ABTS free radical scavenging ability, and the result is shown in FIG. 1, using polysaccharide stock solution without fermentation as control. ABTS oxidized to form a stable blue-green material with a maximum absorption peak at 734 nm. The oxidation resistance of the measured substance enables the blue-green substance to fade in different degrees, and the total oxidation resistance of the measured substance is calculated according to the change of the light absorption value.
As can be seen from FIG. 1, the total antioxidant capacity of the biofermented polysaccharides was enhanced compared to the stock solution. When the fermentation time is 16h and the polysaccharide concentration is 2.5mg/mL, the clearance rate of ABTS free radicals is 14.48%, and the antioxidant capacity is high. The stock solution had 6.21% clearance of ABTS free radicals. Compared with the original liquid, the total antioxidant capacity of the fermented product for 16 hours is improved by 2.3 times.
Example 2
Agar plate medium: 20g/L of agar powder (dry powder), 3g/L of beef extract and 10g/L, NaCl 5g/L of peptone, wherein the pH value is 7.4 +/-0.2; liquid culture medium: 3g/L beef extract, 10g/L, NaCl 5g/L peptone and pH value of 7.4 +/-0.2. The microorganism used is bacillus subtilis which is purchased from bacillus subtilis with the number of 1.821 in China general microbiological culture collection center, and the fermentation is carried out according to the following steps:
(1) extracting the dendrobium officinale polysaccharide by a hot water extraction method: taking 200g of fresh dendrobium, slicing, drying at 105 ℃ for 5min to constant weight (about 50.34g) at 65 ℃, grinding, sieving with a 40-mesh sieve, and mixing according to the weight ratio of 1: adding 30g of dendrobium dry powder and 2400mL of distilled water into 80(g: mL) feed liquid ratio, heating in a water bath at 90 ℃ for 1h, filtering, repeatedly extracting filter residues once, combining two extracting solutions, adding about 4.60L of absolute ethyl alcohol until the final concentration of alcohol is 80%, standing overnight at 4 ℃, filtering to obtain about 18.10g of crude polysaccharide precipitate, adding 5L of distilled water to redissolve the precipitate, adjusting the concentration to 10mg/mL, taking a conical flask of 50mL to 100mL, placing at 121 ℃, and sterilizing for 20min for later use.
The rest of the procedure was the same as in example 1.
As can be seen from FIG. 2, the total antioxidant capacity of the biofermented polysaccharides was enhanced compared to the stock solution. When the fermentation time is 16h and the polysaccharide concentration is 2.5mg/mL, the clearance rate of ABTS free radicals is 15.13%, and the antioxidant capacity is high. The stock solution has 6.81 percent of clearance rate of ABTS free radicals. Compared with the original liquid, the total antioxidant capacity of the fermented product for 16 hours is improved by 2.22 times.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (1)

1. A method for improving the antioxidant capacity of dendrobium officinale polysaccharide is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting Dendrobium officinale polysaccharide with hot water;
(2) activating microbial strains;
(3) preparing a seed solution;
(4) inoculating and fermenting;
the specific process of the step (1) is as follows: taking fresh dendrobium, slicing, drying to constant weight, grinding and sieving, and mixing according to the weight ratio of 1: adding 80(g: mL) of material-liquid ratio into the dendrobium dry powder and distilled water, heating in a water bath at 80-90 ℃ for 1-2 h, filtering, repeatedly extracting filter residues once, combining extracting solutions obtained in two times, adding absolute ethyl alcohol until the final concentration of alcohol is 80%, standing overnight, filtering to obtain crude polysaccharide precipitate, redissolving the crude polysaccharide precipitate by using distilled water, adjusting the concentration of the redissolved solution to 10mg/mL, taking 50mL of the redissolved solution into a reaction container, and sterilizing for later use;
the specific process of the step (2) is as follows: selecting the preserved bacterial colony with an inoculating needle, streaking on a plate, culturing for 24h at 37 ℃ in a constant-temperature incubator, and selecting a single strain for slant preservation or continuous streaking amplification;
the specific process in the step (3) is as follows; scraping the strain from a plate cultured for 24h to 100mL of nutrient broth culture medium, and culturing for 24h at the constant temperature of 37 ℃ in a shaking table and under the condition of 180r/min to obtain seed liquid;
the specific process in the step (4) is as follows: adding 2% seed solution into conical flask, shaking culture at 37 deg.C for 16 hr, and sterilizing at 121 deg.C for 20 min;
the microorganism is bacillus subtilis which is purchased from bacillus subtilis with the number of 1.821 in China general microbiological culture collection center.
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