CN111000875A - Extraction method of kelp polyphenol - Google Patents
Extraction method of kelp polyphenol Download PDFInfo
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- A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
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- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The invention belongs to the technical field of plant extraction, and discloses a method for extracting kelp polyphenol. The method comprises the following steps: s1Freezing and thawing the kelp by a freezing and thawing method; s2Adding pectinase strain fermentation liquor into the unfrozen kelp, inoculating and fermenting, and fermenting to obtain a pigment liquid layer; s3And adding an ethanol solution into the pigment layer, carrying out ultrasonic treatment, filtering, and drying the filtrate to obtain the kelp polyphenol. The extraction method has the advantages of reduced 95% ethanol consumption, improved extraction rate and oxidation resistance, and promotes industrialization of herba Zosterae Marinae polyphenol.
Description
Technical Field
The invention relates to the technical field of plant extraction, and particularly relates to a method for extracting kelp polyphenol.
Background
Marine plant seaweeds contain polyphenols, and researchers refer to various phenolic compounds isolated and purified from seaweeds as seaweed phenols (algae polyphenols). Marine phenolic compounds have strong biological activity, which is the hot research in recent years, and seaweed phenolic compounds can be classified into simple phenolic compounds and polyphenol compounds according to the molecular structure characteristics. The kelp resources in China are very rich, the kelp cultivation becomes one of the mainstream industries of the seawater cultivation in China, the yield is at the top of the world, the kelp polyphenol has the effects of oxidation resistance, tumor resistance, bacteria resistance, chemical defense and the like, and great development and utilization values are achieved, but the problems of easy oxidation, instability, complex extraction components, low extraction rate and the like of the kelp phenolic substances restrict the industrial preparation and production of the kelp polyphenol.
The extraction of kelp polyphenol mainly uses dried, crushed and sieved kelp powder as raw material, and adopts a certain concentration ethanol solution extraction method, but the polyphenol usually forms stable compounds with protein and polysaccharide in the kelp body in the form of hydrogen bond and hydrophobic bond, so that the extraction rate is low, and at present, the ultrasonic extraction method, microwave extraction method, metal ion precipitation method and supercritical CO extraction method are mostly adopted2The extraction method comprises a fluid extraction method, a biological enzymolysis extraction method and the like, wherein the ultrasonic-assisted ethanol extraction method is the most common method, and the method can loosen the structure of the kelp by ultrasonic oscillation, release polyphenol substances and improve the extraction efficiency. However, all the existing extraction methods aim at the problems that the treatment of all kelp raw materials is low in effective concentration of polyphenol, more reagents are required to be added, the purity of the polyphenol in an extract is low and the like, so that the technical problem of low extraction rate of the polyphenol is still not solved, the antioxidant performance of the obtained kelp polyphenol is relatively low, the preparation and application of the kelp polyphenol as a functional ingredient are restricted, and therefore, the exploration of a new extraction method of the kelp polyphenol has important significance.
Disclosure of Invention
In order to solve the problem of low extraction rate and oxidation resistance of the kelp polyphenol caused by extracting the kelp polyphenol from the kelp raw material slurry in the prior art, the invention provides an extraction method of the kelp polyphenol.
In order to solve the technical problems, the invention adopts the following technical scheme:
the extraction method of the kelp polyphenol specifically comprises the following steps:
S1freezing and thawing the kelp by a freezing and thawing method; preferably, the kelp is frozen for 6 to 12 hours at the temperature of between 18 ℃ below zero and 20 ℃ below zero and is unfrozen at the temperature of between 20 and 25 ℃.
S2Adding pectinase strain fermentation liquor into the unfrozen kelp, inoculating and fermenting, adding water after fermentation, stirring, and filtering to obtain a filter residue pigment layer;
S3and adding an ethanol solution into the pigment layer, performing ultrasonic treatment, filtering, and drying the filtrate to obtain the kelp polyphenol.
The content of kelp polyphenol in a kelp pigment layer is about 95-98%, the inventor tries to directly extract kelp polyphenol from kelp pigment, but many methods are successful, and the inventor tests and surprisingly discovers that kelp is frozen and thawed first, the kelp polyphenol extraction method provided by the invention comprises the steps of firstly treating kelp by a freeze-thaw method, freezing and thawing the kelp, forming large ice crystals in the freezing process, destroying the structure of the kelp pigment layer, flowing out water after thawing, forming a gap between the pigment layer and kelp meat, better playing a role in subsequent pectinase fermentation, fully contacting and degrading pectin in the pigment layer by pectinase, loosening and dispersing the compact texture of the original kelp, loosening the pigment layer, facilitating separation, destroying the structure of the kelp pigment layer, degrading the bound kelp polyphenol by pectinase, the kelp polyphenol in the pigment layer is easy to extract when the pigment layer is extracted by combining ethanol and ultrasonic wave, the finally used extraction solvent is less in dosage, and the extraction rate can be obviously improved.
If the fresh kelp is adopted as the raw material, the fresh kelp can be directly cut into strips or blocks and then frozen; when the kelp is dry kelp, soaking the kelp in water, wherein the mass ratio of the dry kelp to the water is 1: 1-3, the soaking time is 1-3 hours, draining the water after cleaning, cutting the kelp into strips or blocks, and freezing the strips or blocks. When the herba Zosterae Marinae is fresh herba Zosterae Marinae, cleaning, cutting into strips or blocks, and freezing.
The pectinase according to the present invention is not particularly limited and may be selected from those commonly used in the art, and preferably, the pectinase is selected from Aspergillus niger (Aspergillus niger).
Preferably, the pectinase can be expanded and cultured in the invention, so that the concentration of the pectinase bacteria liquid reaches 106～108CFU/mL, spraying the bacterial solution with the mass of 1-5% of that of the fresh kelp or the rehydrated kelp on the unfrozen kelp, kneading to enable the bacterial solution to be better contacted with the pectin, and fermenting for 30-90 min at the temperature of 20-25 ℃; more preferably, the fermented kelp and water are mixed and stirred according to the mass ratio of 1: 0.5-1.
To step S3Adding 40-80g of 95% ethanol into the pigment layer obtained in the step (b), adding water to make the mass concentration of the ethanol solution reach 40-70%, and then carrying out ultrasonic auxiliary treatment with the ultrasonic treatment power of 500-2000W for 5-15min at the temperature of 20-40 ℃. According to the method, the kelp is treated by adopting a freeze-thaw method, the kelp is frozen and then thawed, large ice crystals are formed in the freezing process, the structure of a kelp pigment layer is damaged, and kelp polyphenol in the pigment layer is easily extracted when the pigment layer is extracted by adopting ethanol and ultrasonic waves in combination.
The kelp polyphenol mainly exists in the pigment layer, the content of the kelp polyphenol in the kelp pigment layer is about 95-98%, the invention adopts a freeze thawing method and a pectinase fermentation method to complete better separation of the pigment layer and the kelp meat, and finally adopts an alcohol extraction method combined with ultrasonic waves to extract the kelp polyphenol in the pigment layer. According to the technical scheme, the use amount of 95% ethanol in the extraction method of the kelp polyphenol provided by the invention is reduced to 1/3-1/2 of that in the traditional extraction method, the extraction rate is improved by 10% -25%, and through functional evaluation, the oxidation resistance is improved by about 10%, so that the industrial utilization of the kelp polyphenol is promoted.
Drawings
FIG. 1 is a graph showing the results of the measurement of antioxidant activity of sea tangle polyphenol according to the present invention;
FIG. 2 is a graph showing the comparison of antioxidant ability of polyphenol from Laminaria japonica according to example 4 of the present invention.
Detailed Description
The invention discloses a method for extracting kelp polyphenol, which can be realized by appropriately improving process parameters by taking the contents of the kelp polyphenol as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The following detailed description of the invention refers to specific embodiments thereof for better understanding by those skilled in the art.
Example 1 extraction of Laminaria Polyphenol
S1Cutting 100g of fresh kelp into strips by a freeze thawing method, freezing for 6h at-18 ℃, and then thawing in air at 20 ℃.
S2Culturing Aspergillus niger (Aspergillus niger) in an enlarged manner to reach Aspergillus niger bacterial liquid concentration of 106～108CFU/mL, spraying the bacterial solution on the thawed kelp according to the mass of 1% of the fresh kelp, fermenting at the temperature of 20 ℃ for 30min, mixing the fermented kelp with water according to the mass ratio of 1:0.5, stirring and kneading, and filtering to obtain a filter residue pigment layer (the shape can be shown in figure 1);
S3adding the pigment layer into 40g of 95% ethanol by mass concentration to make the concentration of the ethanol reach 40%, then carrying out ultrasonic treatment with the ultrasonic treatment power of 500W and the time of 5min at the temperature of 30 ℃, filtering the extracting solution after ethanol extraction, wherein the filtrate is crude kelp polyphenol extracting solution, precooling the crude kelp polyphenol extracting solution for 6h at the temperature of minus 20 ℃, and carrying out freeze drying to obtain 2.9g of crude kelp polyphenol and the extraction rate of the kelp polyphenol of 2.9%.
Example 2 extraction of Laminaria Polyphenol
S1Drying seaSoaking the kelp in water at a mass ratio of 1:2 for 2h, washing, draining off water, cutting 100g of rehydrated kelp into strips, freezing at-20 ℃ for 12h, and thawing in air at 25 ℃.
S2Culturing Aspergillus niger (Aspergillus niger) in an enlarged manner to reach Aspergillus niger bacterial liquid concentration of 106～108CFU/mL, spraying the bacterial liquid on thawed kelp according to the mass of 3% rehydrated kelp, fermenting for 90min at the temperature of 25 ℃, mixing the fermented kelp and water according to the mass ratio of 1:1, stirring and kneading, and filtering to obtain a filter residue pigment layer (the shape can be shown in figure 1);
S3adding the pigment layer into 80g of 95% ethanol with mass concentration to enable the concentration of the ethanol to reach 70%, then carrying out ultrasonic treatment with ultrasonic treatment power of 2000W, time of 15min and temperature of 40 ℃, filtering an extracting solution obtained after ethanol extraction, wherein a filtrate is a kelp polyphenol crude extracting solution, precooling the filtrate for 12h at-40 ℃, and carrying out freeze drying to obtain a kelp polyphenol crude product, wherein the extraction rate of the kelp polyphenol is 3.2g, and the extraction rate is 3.2%.
Example 3 extraction of Laminaria Polyphenol
S1Cutting fresh herba Zosterae Marinae into strips, freezing at-19 deg.C for 9 hr, and thawing in 22 deg.C air.
S2Culturing Aspergillus niger (Aspergillus niger) in an enlarged manner to reach Aspergillus niger bacterial liquid concentration of 106～108CFU/mL, spraying the bacterial liquid on the thawed kelp, fermenting at 23 ℃ for 60min, mixing the fermented kelp with water according to the mass ratio of 1:0.8, stirring and kneading to obtain a pigment layer (the shape can be shown in figure 1);
S3adding the pigment layer into 60g of 95% ethanol with mass concentration to enable the concentration of the ethanol to reach 55%, then carrying out ultrasonic treatment with the ultrasonic treatment power of 1000W and the time of 10min at the temperature of 20 ℃, filtering the extracting solution after ethanol extraction, wherein the filtrate is the crude kelp polyphenol extracting solution, precooling the crude kelp polyphenol extracting solution for 9h at the temperature of minus 30 ℃, and carrying out freeze drying to obtain a crude kelp polyphenol product with the extraction rate of 3.0g and the extraction rate of 3.0 g.
Comparative example 1 extraction of Laminaria japonica Polyphenol Using conventional method
Weighing 100g of fresh kelp, drying, crushing, sieving by a 40-60 mesh sieve, adding 100g of 95% ethanol, adding water until the concentration of the ethanol is 50-70%, carrying out ultrasonic treatment for 60-120min, raising the temperature to 50-60 ℃, precooling the extracted kelp polyphenol extracting solution for 9h at-30 ℃, carrying out freeze drying to obtain a kelp polyphenol crude product, and carrying out multiple extraction, wherein the extraction rate is 2.0-2.5%. The amount of 95% ethanol used and the extraction rate of polyphenol from kelp were compared with those of examples 1 to 3, and the results are shown in Table 1.
TABLE 1 extraction rate of sea tangle polyphenols under different extraction methods
The data in table 1 show that the ethanol used in examples 1-3 is significantly less than the ethanol used in the comparative examples, and the extraction rate of the polyphenol from kelp obtained by the preparation method provided in examples 1-3 is significantly higher than that of the traditional process. The invention only extracts the pigment layer, releases the kelp polyphenol in a combined state through degrading by pectinase, and combines the kelp polyphenol with ultrasonic treatment, so that the polyphenol can be efficiently extracted at a lower temperature in a shorter time.
Example 4 assay of antioxidant Properties of kelp polyphenols
The kelp polyphenol obtained in example 1 and the kelp polyphenol obtained in comparative example 1 were subjected to oxidation resistance detection by the following detection method:
(1) the diphenyl picryl hydrazine radical free radical (α -diphenyl- β -pentacylhydrazine, DPPH) and methanol solution are mixed to be purple, and the maximum absorption value is 517 nm.
Free radical scavenging rate ═ a0-(Ai-Ai0))/A0×100％ (2)
AiThe absorbance was measured after mixing 1mL of DPPH solution and 1mL of sample solutions of different concentrations. A. thei0The absorbance of the mixture is 1mL of sample solution and 1mL of methanol, and the background absorption of the sample to be detected is obtained. A. the01mL of DPPH solution and 1mL ofThe absorbance of the ionized water mixture was used as a blank control.
(2) Hydroxyl radical scavenging
Respectively placing 50 mu L of samples with different concentrations in a 96-well plate, and then adding 50 mu L of 0.75mmol/L phenanthroline solution; 100 μ L of 0.2mol/L PBS solution with pH value of 7.4; 50 μ L of 0.75mmol/L FeSO4A solution; 50 μ L volume fraction 0.01% H2O2The solution is evenly mixed and then placed in a water bath at 37 ℃ for constant temperature 30min, the absorbance of the sample is detected at the wavelength of 536nm, water is used as a blank control group, and Vc is used as a positive control group.
Clearance rate ═ 1- (A)1-A2)/A0)％ (3)
A0The absorbance of the sample was replaced with water; a. the1Is the absorbance of the sample; a. the2For substituting H for sample and distilled water2O2Absorbance of (b).
The oxidation resistance of the kelp polyphenol is measured by adopting the DPPH method and the hydroxyl radical scavenging method, the final detection result is shown in figure 2, and figure 2 shows that the oxidation resistance of the kelp polyphenol obtained by the traditional method is obviously inferior to that of the kelp polyphenol obtained by the embodiment method, and the oxidation resistance of the kelp polyphenol is improved by about 10%. Comparative example 1 the traditional extraction method is adopted to extract the whole kelp, the reagent is more, the ultrasonic auxiliary treatment is added, the time is long, the heat production is more, the temperature is high, the partial oxidation loss of polyphenol is caused, and the biological activity is low; the kelp is treated by adopting a freeze thawing method, the kelp is frozen and then thawed, large ice crystals are formed in the freezing process, the structure of a kelp pigment layer is damaged, and bound kelp polyphenol is released through pectinase degradation, so that the kelp polyphenol in the pigment layer is easily extracted when the pigment layer is extracted by adopting ethanol and ultrasonic waves in combination, at the moment, the ultrasonic time is short, the heat release is less, the overhigh temperature caused by long-time ultrasonic treatment is avoided, the ultrasonic temperature can be kept at 20-40 ℃, and the activity of the kelp polyphenol is furthest maintained while the extraction rate is increased.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The extraction method of the kelp polyphenol is characterized by comprising the following steps:
S1freezing and thawing the kelp by a freezing and thawing method;
S2adding pectinase strain fermentation liquor into the unfrozen kelp, inoculating and fermenting, adding water and stirring after fermentation, and filtering to obtain a filter residue pigment layer;
S3and adding an ethanol solution into the pigment layer, carrying out ultrasonic treatment, filtering, and drying the filtrate to obtain the kelp polyphenol.
2. The method for extracting polyphenol from kelp as claimed in claim 1, wherein the step S is a step1And freezing the kelp at-18 to-20 ℃ for 6 to 12 hours.
3. The method for extracting polyphenol from kelp as claimed in claim 1 or 2, wherein step S is a step of1And thawing at 20-25 deg.C.
4. The method for extracting polyphenol from kelp as claimed in claim 1, wherein when the kelp is fresh kelp, the kelp is cut into strips or pieces and then frozen; when the kelp is dry kelp, soaking the kelp in water to obtain rehydrated kelp, wherein the mass ratio of the dry kelp to the water is 1: 1-3, the soaking time is 1-3 hours, draining the water after cleaning, cutting the kelp into strips or blocks, and freezing the strips or blocks.
5. The method for extracting polyphenol from kelp as claimed in claim 1, wherein the pectinase is selected from aspergillus niger (aspergillus niger).
6. The method for extracting polyphenol from kelp as claimed in claim 1, 4 or 5, wherein the pectinase is enlarged and cultured so that the concentration of the pectinase bacteria liquid reaches 106～108CFU/mL, spraying the bacterial liquid with the mass of 1-5% of that of the fresh kelp or the rehydrated kelp on the thawed kelp, and fermenting at the temperature of 20-25 ℃ for 30-90 min.
7. The method for extracting polyphenol from kelp as claimed in claim 6, wherein the fermented kelp is mixed with water in a mass ratio of 1:0.5-1, kneaded under stirring, and filtered to obtain a pigment layer of residue.
8. The method for extracting polyphenol from kelp as claimed in claim 1, wherein the step S is a step3Adding 40-80g of 95% ethanol by mass, adding water to make the final ethanol concentration reach 40-70%, and performing ultrasonic auxiliary treatment at 500-2000W for 5-15min at 20-40 deg.C.
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| PCT/CN2020/089964 WO2021098164A1 (en) | 2019-11-18 | 2020-05-13 | Method for extracting kelp polyphenols |
| ZA2022/04640A ZA202204640B (en) | 2019-11-18 | 2022-04-25 | Method for extracting kelp polyphenols |
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| WO2021098164A1 (en) * | 2019-11-18 | 2021-05-27 | 青岛科技大学 | Method for extracting kelp polyphenols |
| CN114521647A (en) * | 2022-03-23 | 2022-05-24 | 福建农林大学 | Kelp probiotic fermentation product with anti-aging function and preparation method thereof |
| CN115380918A (en) * | 2022-08-29 | 2022-11-25 | 福州大学 | Response surface method optimized extraction method of kelp polyphenol |
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| CN113667030B (en) * | 2021-08-11 | 2022-10-25 | 华南理工大学 | Method for decoloring and purifying osmanthus fragrans crude polysaccharide |
| CN113491670B (en) * | 2021-08-18 | 2023-09-12 | 合肥中龙神力动物药业有限公司 | Detoxifying oral liquid for livestock and preparation method thereof |
| CN115228133B (en) * | 2022-07-28 | 2024-04-12 | 黑龙江八一农垦大学 | Method for extracting combined polyphenol in coarse cereals by continuous compound enzyme method |
| CN115645312B (en) * | 2022-10-11 | 2023-12-12 | 江苏海洋大学 | Application of sargassum pallidum polyphenol in preparing whitening cosmetics and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021098164A1 (en) * | 2019-11-18 | 2021-05-27 | 青岛科技大学 | Method for extracting kelp polyphenols |
| CN114521647A (en) * | 2022-03-23 | 2022-05-24 | 福建农林大学 | Kelp probiotic fermentation product with anti-aging function and preparation method thereof |
| CN114521647B (en) * | 2022-03-23 | 2024-04-26 | 福建农林大学 | Kelp probiotics fermented product with auxiliary antioxidant capacity improving function and preparation method thereof |
| CN115380918A (en) * | 2022-08-29 | 2022-11-25 | 福州大学 | Response surface method optimized extraction method of kelp polyphenol |
| CN115380918B (en) * | 2022-08-29 | 2024-01-26 | 福州大学 | Method for optimized extraction of kelp polyphenol by response surface method |
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