CN113481242A - Method for improving biological activity of plant polyphenol through microbial transformation - Google Patents
Method for improving biological activity of plant polyphenol through microbial transformation Download PDFInfo
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Abstract
The invention discloses a method for improving the bioactivity of plant polyphenol through microbial transformation. The method comprises the steps of extracting plant polyphenol by an ultrasonic-assisted alcohol extraction method, using Aspergillus niger as a transformation strain, culturing the Aspergillus niger in a Sabouraud's liquid culture medium for 1-3 days, adding the extracted plant polyphenol into the culture medium, continuously culturing for 2-4 days, collecting bacterial liquid, and extracting by ethyl acetate to obtain a transformation product. The invention takes plant natural extracts rich in polyphenol as substrates, utilizes enzyme generated in the microbial metabolic process to carry out catalytic reaction on the substrates, modifies and modifies the chemical structure of the substrates, can obtain compounds with high bioavailability, high bioactivity and high stability, and provides more high-quality natural product sources for developing novel health-care foods and medicines.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for improving the bioactivity of plant polyphenol through microbial transformation.
Background
The polyphenol is a compound which takes phenol as a basic skeleton and has a polyhydroxy structure, widely exists in roots, stems, leaves, fruits and other parts of plants, and is a plant secondary metabolite which has a complex structure and multiple biological activities. At present, thousands of phenolic compounds are found in plants, including flavonoids, tannins, phenolic acids, anthocyanins, and the like. The polyphenol substance has important biological activities of oxidation resistance, blood sugar reduction, antibiosis and the like, in vitro oxidation resistance activity of phenolic substances extracted from some plants is even obviously higher than that of ascorbic acid, and in vitro blood sugar reduction activity is superior to acarbose which is a main component of oral hypoglycemic drugs, so that the plant polyphenol is developed into health food or drugs, and the polyphenol substance has the remarkable advantages of high activity, safety, no toxic or side effect and the like, and has great potential in the aspect of treating human chronic diseases.
At present, a great variety of plants rich in polyphenol substances can be excavated for extracting and separating polyphenol, and related extraction technologies are quite mature. However, natural phenols extracted from plants have limited practical applications due to problems such as poor solubility, high hydrophobicity, low bioavailability, and unstable properties. Therefore, modifying the structure of the phenolic substance by chemical reaction or microbial transformation and other methods to change the solubility and polarity of the phenolic substance, improve the bioavailability and improve the biological activity and stability of the phenolic substance has become a research hotspot of plant polyphenol application. The phenolic substance structure modification is carried out by utilizing chemical reaction, and the defects of easy introduction of chemical substances which are not beneficial to human health, environmental pollution, complex reaction steps, high cost and the like exist. In contrast, microorganisms metabolize to produce abundant enzymes, can efficiently catalyze plant polyphenols to carry out methylation, desugarization conversion, dehydroxylation and other reactions, thereby changing the structure of phenols, and has the advantages of mild reaction conditions, environmental protection, few byproducts, low cost and the like. Therefore, the microbial transformation method is an ideal method for improving the characteristics of plant polyphenol so as to be widely applied to products.
Disclosure of Invention
The invention aims to provide a method for improving the biological activity of plant polyphenol through microbial transformation, which is a microbial transformation method for efficiently improving the bioavailability, the biological activity and the stability of natural plant polyphenol and can improve the availability of plant polyphenol.
In order to achieve the purpose, the invention adopts the following technical scheme: extracting plant polyphenol by ultrasonic wave assisted alcohol extraction, adopting Aspergillus niger as a conversion strain, culturing Aspergillus niger in a Sabouraud's liquid culture medium at 28 deg.C and 180r/min for 1-3 days, adding the extracted plant polyphenol into the culture medium, continuously culturing for 2-4 days, collecting bacterial liquid, and extracting with ethyl acetate to obtain a conversion product.
The method for improving the biological activity of the plant polyphenol through microbial transformation specifically comprises the following steps:
(1) extracting natural plant polyphenol: drying the plant for extracting polyphenol at 50 ℃, crushing by a crusher, sieving by a 40-mesh sieve, adding 60-90% ethanol solution into the powder according to the material-liquid ratio of 1:20-1:160, carrying out ultrasonic treatment at the power of 100-.
(2) Culturing Aspergillus niger: aspergillus niger was inoculated on a Sabouraud's dextrose agar medium plate and cultured in an incubator at 28 ℃ for 5-7 days until black spores grew on the plate. Washing Aspergillus niger spores from the plate with sterile water, and diluting the spore solution to 10%6-107 CFU/mL, adding spore liquid into Sabouraud's dextrose liquid culture medium at a volume ratio of 1:90-110, and culturing at 28 deg.C and 180r/min for 1-3 days.
(3) Aspergillus niger transformation: preparing the polyphenol extract into 10-20 mg/mL solution with sterile water, adding the polyphenol solution into the bacterial solution of the step (2) at a volume ratio of 1:9-11, and culturing at 28 deg.C and 180r/min for 1-4 days.
(4) And (3) collecting a conversion product: and (4) removing the bacteria from the bacterial liquid obtained in the step (3) by using a vacuum filtration method to obtain a culture solution. Mixing the culture solution with ethyl acetate saturated water solution at a volume ratio of 1:1.8-2.2, sufficiently shaking for more than 10 min, collecting ethyl acetate layer, vacuum rotary evaporating at 45-50 deg.C to remove ethyl acetate, and vacuum freeze drying to obtain the converted product.
According to the technical scheme, the polyphenol-rich plant natural extract is used as a substrate, and enzyme generated in the microbial metabolism process is used for carrying out catalytic reaction on the substrate, so that the chemical structure of the substrate is modified, the bioavailability of the substrate is improved, and the bioactivity and stability of the substrate are improved.
The beneficial results of the invention are: 1. according to the invention, by using the advantages of Aspergillus niger as an enzyme-producing treasury and converting natural plant polyphenol by using a rich enzyme system generated by metabolism of Aspergillus niger, a compound with high bioavailability, high bioactivity and high stability can be obtained, and more high-quality natural product sources are provided for developing novel health-care foods and medicines; 2. the invention adopts a microbial conversion method to replace a chemical reaction method to modify the structure of a natural plant product, and has the advantages of simple process, mild conditions and more environmental protection; 3. the invention provides a new method for developing and utilizing a plurality of plants rich in polyphenol, which is beneficial to improving the additional value of the plants and improving the product quality and benefit.
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FIG. 1 is a graph of the effect of time on the conversion of the polyphenol extract from the leaves of Myrciaria dubia by Aspergillus niger.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
Example 1
Method for improving biological activity of plant polyphenol through microbial transformation
(1) Extracting natural plant polyphenol: picking fresh jaboticaba leaves, drying at 50 ℃, crushing by a crusher, sieving by a 40-mesh sieve, adding 70% ethanol solution into the powder according to a feed-liquid ratio of 1:80, carrying out ultrasonic treatment at a power of 150W for 20 min, then oscillating for 1 h, centrifuging for 10 min at 5000 g, collecting supernatant, carrying out vacuum rotary evaporation at 45 ℃ to remove ethanol to the utmost, and finally carrying out vacuum freeze drying to obtain the jaboticaba leaf polyphenol extract.
(2) Culturing Aspergillus niger: inoculating Aspergillus niger to a Sabouraud's dextrose agar culture medium plate, culturing at 28 deg.C in an incubator for 5 days until black spores grow on the plate, washing Aspergillus niger spores from the plate with sterile water, and diluting the spore solution to 106 CFU/mL, adding the spore liquid into a Sabouraud's dextrose liquid culture medium according to the volume ratio of 1:100, and culturing for 2 days under the conditions of 28 ℃ and 180 r/min.
(3) Aspergillus niger transformation: preparing the polyphenol extract into 10 mg/mL solution with sterile water, adding the polyphenol solution into the bacterial solution obtained in the step (2) at a volume ratio of 1:10, and culturing at 28 deg.C and 180r/min for 2 days.
(4) And (4) removing the bacteria from the bacterial liquid obtained in the step (3) by using a vacuum filtration method to obtain a culture solution. Mixing the culture solution and ethyl acetate saturated aqueous solution at a volume ratio of 1:2, fully oscillating for 20 min, collecting an ethyl acetate layer, carrying out vacuum rotary evaporation at 45 ℃ to remove ethyl acetate to the utmost, and finally carrying out vacuum freeze drying to obtain the jaboticaba leaf polyphenol conversion product.
As shown in FIG. 1, under the conditions tested in this example, Aspergillus niger transformed jaboticaba leaf polyphenol extract 2 d increased the total antioxidant capacity by 61.64%.
Example 2
(1) Extracting natural plant polyphenol: picking fresh guava leaves, drying at 50 ℃, crushing by a crusher, sieving by a 40-mesh sieve, adding an 80% ethanol solution into the powder according to a material-to-liquid ratio of 1:100, carrying out ultrasonic treatment at 100W for 10 min, then oscillating for 1 h, centrifuging for 10 min by a centrifugal force of 5000 g, collecting supernatant, carrying out vacuum rotary evaporation at 45 ℃ to remove ethanol to the utmost, and carrying out vacuum freeze drying to obtain the natural plant polyphenol extract.
(2) Culturing Aspergillus niger: aspergillus niger was inoculated on a Sabouraud's dextrose agar medium plate and cultured in an incubator at 28 ℃ for 7 days until black spores grew on the plate. Washing Aspergillus niger spores from the plate with sterile water, and diluting the spore solution to 10%7 CFU/mL, spore liquid was added to a Sabouraud's dextrose liquid medium at a volume ratio of 1:100, and cultured at 28 ℃ at 180r/min for 3 days.
(3) Aspergillus niger transformation: preparing 20 mg/mL solution of polyphenol extract with sterile water, adding polyphenol solution into the bacterial solution of step (2) at a volume ratio of 1:10, and culturing at 28 deg.C and 180r/min for 3 days.
(4) And (3) collecting a conversion product: and (4) removing the bacteria from the bacterial liquid obtained in the step (3) by using a vacuum filtration method to obtain a culture solution. Mixing the culture solution with ethyl acetate saturated water solution at a volume ratio of 1:2, fully shaking for more than 30 min, collecting ethyl acetate layer, vacuum rotary steaming at 45 deg.C to remove ethyl acetate, and vacuum freeze drying to obtain guava leaf polyphenol conversion product.
Claims (7)
1. A method for improving the bioactivity of plant polyphenol by microbial transformation is characterized in that: extracting plant polyphenol by ultrasonic wave assisted alcohol extraction, adopting Aspergillus niger as a transformation strain, culturing Aspergillus niger in a Sabouraud's liquid culture medium for 1-3 days, adding the extracted plant polyphenol into the culture medium, continuously culturing for 2-4 days, collecting bacterial liquid, and extracting with ethyl acetate to obtain a transformation product.
2. The method of claim 1, wherein the microbial transformation of plant polyphenols comprises: the method comprises the following steps:
(1) extracting plant polyphenol: drying the plant for extracting polyphenol, pulverizing with a pulverizer, sieving, adding ethanol solution into the powder according to the material-liquid ratio of 1:20-1:160, performing ultrasonic treatment, oscillating, centrifuging, collecting supernatant, performing vacuum rotary evaporation to remove ethanol to dryness, and finally performing vacuum freeze drying to obtain natural plant polyphenol extract;
(2) culturing Aspergillus niger: inoculating Aspergillus niger on a Sabouraud's dextrose agar culture medium plate, culturing for 5-7 days at 28 ℃ until Aspergillus niger spores grow on the plate, washing the Aspergillus niger spores from the plate by using sterile water, diluting the obtained spore liquid, then adding the diluted spore liquid into the Sabouraud's dextrose broth culture medium according to the volume ratio of 1:90-110, and culturing for 1-3 days at 28 ℃ and 180 r/min;
(3) aspergillus niger transformation: preparing the natural polyphenol extract into 10-20 mg/mL polyphenol solution with sterile water, adding the polyphenol solution into the bacterial liquid obtained in the step (2) according to the volume ratio of 1:9-11, and continuously culturing for 1-4 days at 28 ℃ and 180 r/min;
(4) and (3) collecting a conversion product: removing thalli from the bacterial liquid obtained in the step (3) to obtain a culture solution, mixing the culture solution with an ethyl acetate saturated aqueous solution in a volume ratio of 1:2, fully oscillating, collecting an ethyl acetate layer, carrying out vacuum rotary evaporation to remove ethyl acetate to the greatest extent, and finally carrying out vacuum freeze drying to obtain a conversion product.
3. The method of claim 2, wherein the microbial transformation of plant polyphenols comprises: in the step (1), the concentration of the ethanol solution is 60-90%.
4. The method of claim 2, wherein the microbial transformation of plant polyphenols comprises: in the step (1), the ultrasonic power is 100-.
5. The method of claim 2, wherein the microbial transformation of plant polyphenols comprises: in the step (2), the concentration of the diluted spore liquid is 106-107 CFU/mL。
6. The method of claim 2, wherein the microbial transformation of plant polyphenols comprises: in the step (4), the volume ratio of the culture solution to the ethyl acetate saturated aqueous solution is 1: 1.8-2.2.
7. The method of claim 2, wherein the microbial transformation of plant polyphenols comprises: in the step (1) and the step (4), the temperature of vacuum rotary evaporation is 45-50 ℃.
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Citations (6)
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- 2021-07-19 CN CN202110814705.7A patent/CN113481242A/en active Pending
Patent Citations (7)
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| US20070244189A1 (en) * | 2004-12-27 | 2007-10-18 | Pokka Corporation | Antioxidant Material, Anti-Deterioration Agent and Food or Beverage |
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Non-Patent Citations (1)
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