CN115380918B - Method for optimized extraction of kelp polyphenol by response surface method - Google Patents
Method for optimized extraction of kelp polyphenol by response surface method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/03—Algae
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/70—Comminuted, e.g. emulsified, fish products; Processed products therefrom such as pastes, reformed or compressed products
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
The invention discloses a method for optimally extracting kelp polyphenol by a response surface method, which comprises the following steps: cleaning, drying and crushing kelp; adding deionized water into kelp powder, adding pectase, and performing enzymolysis to obtain a mixture 1; adding absolute ethyl alcohol into the mixture 1, and carrying out ultrasonic treatment to obtain a mixture 2; placing the mixture 2 into a microwave oven for low-fire heating to obtain a mixture 3; and centrifuging the mixture 3, and collecting supernatant to obtain the crude extract of kelp polyphenol. The kelp polyphenol extracted by the method has the functions of resisting bacteria and oxidization and inhibiting the activity of cathepsin L cysteine protease, can be applied to preparing daily chemical products, foods, health-care products or medicines with antibacterial effect, can be applied to improving the texture characteristics of minced fillet gel products, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of seaweed source active ingredient extraction and aquatic product processing, and particularly relates to a method for optimally extracting kelp polyphenol by a response surface method.
Background
Phaeophyta (Phaeophyta) is a group of algae with high economic value, about 1500 kinds of algae are counted, and the individuals are large; they are mainly distributed in the cold and warm ocean, with only a few brown algae growing in fresh water. Brown algae is an edible marine algae plant widely used in human production and life, and common edible brown algae include kelp, sargassum fusiforme, undaria pinnatifida and the like.
Kelp was first described in Jiayou Ben Cao (Jiayou Ben Cao): it can be used as a drainage medicine for promoting the growth of women and treating wind. "has rich edible and medicinal values, such as preventing and treating goiter, reducing blood pressure and blood fat, reducing weight, resisting cancer, detumescence and diuresis, etc.
Kelp generally grows on undersea rocks, and the growth is mainly affected by temperature and illumination, but does not cause any serious damage. It is therefore speculated that kelp may produce certain active ingredients such as kelp polyphenols, laminarin, mannitol, fatty acids, terpenes, indoles, etc. to protect against external uv radiation, oxidative damage, natural enemy predation, etc. Many researches show that the brown algae polyphenol has various biological activities such as antioxidation, antibiosis, anti-inflammatory, anticancer, antivirus and the like, and is a good source of natural oxidant.
In recent years, the research on the extraction process of brown algae polyphenols has been increased, and besides the classical solvent extraction method, there are various methods such as ultrasonic extraction, microwave extraction, enzymolysis and microbiological method. The research direction is mainly focused on the optimization of the extraction process. However, the problems of low extraction rate, low purity, resource waste and the like are found after the methods are applied to the extraction of kelp polyphenol. Currently, few studies on extraction of polyphenols from brown algae, especially brown algae polyphenols from kelp; in addition, enzyme-ultrasonic-microwave combined extraction of laminarin is not reported at present, and further research and development are needed to be conducted so as to facilitate comprehensive utilization of laminarin resources.
In order to realize deep development and comprehensive utilization of kelp, solve the problems of single product, low added value and the like in industry and industry, and research on the extraction process of kelp polyphenol, the technical problem to be solved is currently and urgently needed to be solved, effectively accords with the sustainable development concept, enriches the theoretical basis of the commercialization of brown algae, and further improves the economic added value of the seaweed.
Disclosure of Invention
In order to solve and optimize the problems in the existing kelp polyphenol extraction method, the invention aims to provide a method for optimizing and extracting kelp polyphenol by a response surface method.
The invention realizes the aim through the following technical scheme:
the invention firstly provides a method for optimally extracting kelp polyphenol by a response surface method, which comprises the following steps:
(1) Cleaning herba Zosterae Marinae, oven drying at 50-60deg.C, and pulverizing to obtain herba Zosterae Marinae powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35 (m: v) adding deionized water, adding pectase, wherein the amount of pectase is 0.5-1.0% of the weight of herba Zosterae Marinae powder, the enzyme activity of pectase is 500U/g, adjusting pH value of enzymolysis environment to 3-4, and performing enzymolysis at 50-60deg.C for 45-120 min to obtain mixture 1;
(3) Adding absolute ethyl alcohol with the final concentration of 60vol% into the mixture 1 in the step (2), and performing ultrasonic treatment for 30min under the conditions of ultrasonic power of 350w, ultrasonic frequency of 28KHz and ultrasonic temperature of 30-50 ℃ to obtain a mixture 2;
(4) Placing the mixture 2 in the step (3) into a microwave oven, and heating with low fire for 3min under the condition of power of 360W to obtain a mixture 3;
(5) Centrifuging the mixture 3 in the step (4) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
Further, in the step (2), the pectase is used in an amount of 0.8% by weight of the kelp powder, the pH value of the enzymolysis environment is 3.0, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 55 min.
Furthermore, the use amount of the pectase is 0.8% of the weight of the kelp powder, the pH value of the enzymolysis environment is 3.0, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 55min, which is optimized by a response surface method, and the specific steps are as follows:
(1) Single factor experiment: designing a plurality of groups of single-factor experiments according to four single factors of the dosage of pectase, the pH value of an enzymolysis environment, the enzymolysis temperature and the enzymolysis time;
(2) Response surface method optimization: taking the polyphenol content in the kelp polyphenol crude extract as a response value, carrying out a response surface experiment on the basis of the single factor experiment in the step (1) by adopting a Box-Behnken principle, and simulating to obtain a multiple regression equation as follows:
Y=3.352+0.002A-0.130B-0.020C-0.016D-0.011AB+0.035AC-0.034AD+0.062BC+0.021BD+0.035CD-0.238A 2 -0.239B 2 -0.140C 2 -0.133D 2 ；
wherein the response value Y is the polyphenol content in the crude extract of kelp polyphenol, and the variable parameter A is the pectase addition amount, B is the enzymolysis time, C is the enzymolysis temperature and D is the enzymolysis environment pH value;
(3) Solving the regression equation in the step (2) to obtain the optimal extraction process conditions of the polyphenols in the kelp: the dosage of pectase is 0.8% of the weight of the kelp powder, the pH value of the enzymolysis environment is 3.05, the enzymolysis temperature is 50.85 ℃, and the enzymolysis time is 54.56 min; and then the actual operation is taken as a constraint condition, and the optimal extraction process parameters are obtained by correction: the pectase dosage is 0.8% of the kelp powder weight, the pH value of the enzymolysis environment is 3.0, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 55 min.
The invention also provides kelp polyphenol optimally extracted by a response surface method, and the kelp polyphenol is prepared by the method.
The invention also provides application of the kelp polyphenol in preparing daily chemical products, foods, health-care products or medicines with an antibacterial effect.
The invention also provides application of the kelp polyphenol in preparing an antioxidant.
The invention also provides application of the kelp polyphenol in preparing cysteine protease inhibitor.
The invention also provides application of the kelp polyphenol in improving the texture and characteristics of the minced fillet gel.
The invention has the remarkable advantages that:
according to the invention, the regression relation between a plurality of variables and one or more dependent variables is adopted by a response surface analysis method, and the regression relation optimization of the extraction conditions and the extraction results of the kelp polyphenol is obtained by regression analysis, so that the optimal extraction process of the kelp polyphenol is determined, and the result shows that compared with the traditional leaching method, ultrasonic method, microwave method, ultra-micro method and biological enzyme method, the total phenol content in the crude kelp polyphenol extract obtained by an enzyme-ultrasonic-microwave combined method is obviously increased, so that the enzyme-microwave combined extraction method is a more efficient and green preparation method. In addition, the kelp polyphenol extracted by the invention has antibacterial activity, antioxidant activity and cysteine protease inhibition activity, and can also improve the texture characteristics of minced fillet gel products.
Drawings
Fig. 1: influence of different extraction methods on the polyphenol content of kelp.
Fig. 2: single factor experimental results.
Fig. 3a and 3b: a response surface diagram and a contour diagram (3 a) of the interaction of the pectase dosage and the enzymolysis time on the polyphenol content, and a response surface diagram and a contour diagram (3 b) of the interaction of the pectase dosage and the enzymolysis temperature on the polyphenol content.
Fig. 3c and 3d: a response surface diagram and a contour diagram (3 c) of the interaction of the pectase dosage and the pH of the enzymolysis environment on the polyphenol content, and a response surface diagram and a contour diagram (3 d) of the interaction of the enzymolysis time and the enzymolysis temperature on the polyphenol content.
Fig. 3e and 3f: and a response surface diagram and a contour diagram (3 e) of the interaction of the enzymolysis time and the enzymolysis environment pH to the polyphenol content, and a response surface diagram and a contour diagram (3 f) of the interaction of the enzymolysis environment pH and the enzymolysis temperature to the polyphenol content.
Fig. 4: antibacterial activity of crude extract of kelp polyphenol.
Fig. 5: bacteriostatic activity of various levels of laminarin components. a-crude extract; b-petroleum ether phase; c-ethyl acetate phase; d-n-butanol phase; 1-sample concentration was 500. Mu.g/mL, 2-sample concentration was 250. Mu.g/mL, 3-sample concentration was 125. Mu.g/mL, and 4-sample concentration was 75.5. Mu.g/mL.
Fig. 6: antioxidant activity of the various components of laminarin.
Fig. 7: the inhibition activity of cysteine proteinase by the components of each level of laminarin.
Fig. 8: influence of crude extract of kelp polyphenol on texture and characteristics of minced fillet gel.
Detailed Description
In order to facilitate understanding of the foregoing disclosure, the technical solutions of the present invention will be further described with reference to the specific embodiments, but the following examples are only limited to the present invention and do not represent the scope of the present invention defined by the claims.
Example 1
The enzyme-ultrasonic-microwave combined method for extracting kelp polyphenol comprises the following steps:
(1) Cleaning herba Zosterae Marinae, oven drying at 50-60deg.C, and pulverizing to obtain herba Zosterae Marinae powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35 Adding deionized water according to the ratio of (M: v), adding pectase (the pectase dosage is 0.75% of the kelp powder weight, and the pectase activity is 500U/g), adjusting pH to 3.5 with 1M HCl, and performing enzymolysis in a magnetic stirrer at 50deg.C and rotation speed of 300 r/min for 60 min to obtain mixture 1;
(3) Adding absolute ethyl alcohol with the final concentration of 60vol% into the mixture 1 in the step (2), and performing ultrasonic treatment for 30min under the conditions of ultrasonic power of 350w, ultrasonic frequency of 28KHz and ultrasonic temperature of 50-60 ℃ to obtain a mixture 2;
(4) Placing the mixture 2 in the step (3) into a microwave oven, and heating with low fire for 3min under the condition of power of 360W to obtain a mixture 3;
(5) Centrifuging the mixture 3 in the step (4) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
The normal temperature leaching process of extracting kelp polyphenol includes the following steps:
(1) Cleaning herba Zosterae Marinae, oven drying at 50-60deg.C, and pulverizing to obtain herba Zosterae Marinae powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35 Adding deionized water according to the ratio of (m: v), adding absolute ethyl alcohol with the final concentration of 60vol%, and leaching 6 h at room temperature to obtain a mixture 1;
(3) Centrifuging the mixture 1 in the step (2) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
The ultrasonic extraction of kelp polyphenol comprises the following steps:
(1) Cleaning herba Zosterae Marinae, oven drying at 50-60deg.C, and pulverizing to obtain herba Zosterae Marinae powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35 Adding deionized water in the ratio of (m: v), adding absolute ethyl alcohol with the final concentration of 60vol%, and performing ultrasonic treatment for 30min under the conditions of ultrasonic power of 350w, ultrasonic frequency of 28KHz and ultrasonic temperature of 50-60 ℃ to obtain a mixture 1;
(3) Centrifuging the mixture 1 in the step (2) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
The microwave process of extracting kelp polyphenol includes the following steps:
(1) Cleaning herba Zosterae Marinae, oven drying at 50-60deg.C, and pulverizing to obtain herba Zosterae Marinae powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35 Adding deionized water according to the ratio of (m: v), adding absolute ethyl alcohol with the final concentration of 60vol%, and heating for 3min with low fire in a microwave oven under the condition of power of 360W to obtain a mixture 1;
(3) Centrifuging the mixture 1 in the step (2) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
The ultra-micro method for extracting kelp polyphenol comprises the following steps:
(1) Cleaning herba Zosterae Marinae, oven drying at 50-60deg.C, and pulverizing to obtain herba Zosterae Marinae powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35 Adding deionized water in the ratio of (m: v), adding absolute ethyl alcohol with the final concentration of 60vol%, and performing ultrasonic treatment for 30min under the conditions of ultrasonic power of 350w, ultrasonic frequency of 28KHz and ultrasonic temperature of 50-60 ℃ to obtain a mixture 1;
(3) Placing the mixture 1 in the step (2) into a microwave oven, and heating with low fire for 3min under the condition of power of 360W to obtain a mixture 2;
(4) Centrifuging the mixture 2 in the step (3) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
The biological enzyme process of extracting kelp polyphenol includes the following steps:
(1) Cleaning herba Zosterae Marinae, oven drying at 50-60deg.C, and pulverizing to obtain herba Zosterae Marinae powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35 Adding deionized water according to the ratio of (M: v), adding pectase (the pectase dosage is 0.75% of the kelp powder weight, the pectase activity is 500U/g), adjusting pH to 3.5 with 1M HCl, and performing enzymolysis in a magnetic stirrer at 50deg.C and rotation speed of 300 r/min for 60 min to obtain mixture 1;
(3) Centrifuging the mixture 3 in the step (2) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
All the five extraction methods are carried out under the condition of light shielding.
The polyphenol content (calculated by total phenol content) in the kelp crude extract is determined by using a Fu Lin Fen colorimetric method. As shown in figure 1, compared with the traditional leaching method, ultrasonic method, microwave method, ultra-micro method and biological enzyme method, the total phenol content in the crude extract of laminarin obtained by the enzyme-ultrasonic-microwave combined method is obviously increased.
Example 2
(1) The extraction process for extracting laminarin by an enzyme-ultrasonic-microwave combined method involves 4 key conditions: pectase addition amount, enzymolysis time, enzymolysis temperature and enzymolysis environment pH value. The following one-factor experiments were performed in four ways:
(1) the method for extracting laminarin is basically the same as the enzyme-ultrasonic-microwave combined method provided in the example 1, except that in the step (2), the enzymolysis time is fixed at 60 min, the enzymolysis temperature is 50 ℃ and the enzymolysis environment pH is 3.5, so as to study single-factor experiments of different pectase addition amounts. The results are shown in FIG. 2 a: the addition amount of pectase is 0.75% of the weight of herba Zosterae Marinae powder.
(2) The procedure of the method for extracting laminarin is basically the same as that of the enzyme-ultrasonic-microwave combined method provided in the example 1, except that in the step (2), the addition amount of the immobilized pectase is 0.75% of the weight of laminarin powder, the enzymolysis temperature is 50 ℃, and the enzymolysis environment pH is 3.5, so that single-factor experiments of different enzymolysis time are studied. As shown in FIG. 2b, the enzymolysis time was 60 min.
(3) The method for extracting laminarin is basically the same as the enzyme-ultrasonic-microwave combined method provided in the embodiment 1, except that in the step (2), the addition amount of the immobilized pectase is 0.75% of the weight of laminarin powder, the enzymolysis time is 60 min, and the enzymolysis environment pH is 3.5, so that single-factor experiments of different enzymolysis temperatures are studied. As a result, the enzymatic hydrolysis temperature was found to be optimal at 60℃as shown in FIG. 2 c.
(4) The method for extracting laminarin is basically the same as the enzyme-ultrasonic-microwave combined method provided in the embodiment 1, except that in the step (2), the addition amount of the immobilized pectase is 0.75% of the weight of laminarin powder, the enzymolysis time is 60 min, and the enzymolysis temperature is 50 ℃, so as to study single-factor experiments of different enzymolysis environments pH. As a result, the pH of the enzymatic hydrolysis environment was 3.5 as shown in FIG. 2 d.
(2) Response surface test optimization of kelp polyphenol extraction process conditions:
the Design-Expert V8.0.6 is utilized to carry out a Box-Behnken Design response surface test, based on a single factor test result, the pectinase addition amount A (wt%), the enzymolysis time B (h), the enzymolysis temperature C (DEG C) and the enzymolysis environment pH value D are taken as independent variables, the polyphenol content Y (mg/g) is taken as a response value Design test, a four-factor three-level response surface test is designed, the set result is shown in a table 1, and a multiple regression equation is established:
Y=3.352+0.002A-0.130B-0.020C-0.016D-0.011AB+0.035AC-0.034AD+0.062BC+0.021BD+0.035CD-0.238A 2 -0.239B 2 -0.140C 2 -0.133D 2 。
other process parameters and operating procedures were the same as the enzyme-ultrasonic-microwave combined extraction method provided in example 1. The response surface analysis scheme and experimental results are shown in table 2. The analysis of variance results are shown in Table 3.
Table 1: test factor and level setting
Table 2: response surface analysis scheme and experimental result
Table 3: model analysis of variance
(3) Analysis and optimization of test results:
drawing analysis is carried out according to the regression equation, a response surface diagram and a contour diagram of the regression equation are obtained, the response surface diagram and the contour diagram are shown in fig. 3, and the following conclusion is obtained: the optimal process conditions for obtaining the polyphenol content under different extraction process conditions are as follows: pectase addition amount: 0.80 weight percent, enzymolysis time 54.56 min, enzymolysis temperature 50.85 ℃ and enzymolysis environment pH 3.05; with reference to actual operation, the modified pectase addition amount is 0.80 and wt%, the enzymolysis temperature is 50 ℃, the enzymolysis time is 55min, and the enzymolysis environment pH is 3.0. Under the correction condition, the theoretical polyphenol content of the crude extract of the kelp polyphenol is 3.37 mg/g and is close to the theoretical value of 3.36 mg/g.
Example 3
The kelp polyphenol crude extract is prepared by adopting the modified process conditions of the example 2, and the specific steps are as follows:
(1) Cleaning herba Zosterae Marinae, oven drying at 50-60deg.C, and pulverizing to obtain herba Zosterae Marinae powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35 Adding deionized water according to the ratio of (M: v), adding pectase (the pectase dosage is 0.8% of the kelp powder weight, the pectase activity is 500U/g), adjusting pH to 3.0 with 1M HCl, and performing enzymolysis in a magnetic stirrer at 50deg.C and rotation speed of 300 r/min for 55min to obtain mixture 1;
(3) Adding absolute ethyl alcohol with the final concentration of 60vol% into the mixture 1 in the step (2), and performing ultrasonic treatment for 30min under the conditions of ultrasonic power of 350w, ultrasonic frequency of 28KHz and ultrasonic temperature of 50-60 ℃ to obtain a mixture 2;
(4) Placing the mixture 2 in the step (3) into a microwave oven, and heating with low fire for 3min under the condition of power of 360W to obtain a mixture 3;
(5) Centrifuging the mixture 3 in the step (4) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
The kelp polyphenol crude extract is subjected to rotary evaporation and concentration to prepare an extract, ethanol is recovered, the extract is dried, and then the extract is prepared into a DMSO solution with the concentration of 100 mg/mL, and further the extract is continuously diluted into a corresponding required concentration by using PBS buffer solution (0.2M, ph=6.8).
Selecting staphylococcus aureus ATCC 12600 as indicator bacteria, inoculating the bacterial liquid into LB liquid culture medium, culturing at 37deg.C and 180 rpm to logarithmic phase, and regulating the absorbance of bacterial liquid to OD with fresh LB liquid culture medium 600 About 0.4 to about 0.5, and the cell number is about 10 8 cfu/mL is absorbed, 400 mu L of bacterial suspension is added into about 20mL of LB solid culture medium which is cooled to about 50 ℃ after melting, and then the dish is rapidly and lightly rocked on a plane, so that bacterial liquid and the LB solid culture medium are fully and uniformly mixed and then are horizontally placed, and the bacterial liquid and the LB solid culture medium are cooled. After cooling, smooth small holes with a diameter of 3 mm were uniformly punched in the agar plate, and 30. Mu.L of kelp polyphenol sample solution was added to each hole. And standing for 10min after the sample liquid is added to ensure that the liquid to be measured is uniformly dispersed. Placing the sample-added bacteria-containing flat plate into a constant temperature incubator at 37 ℃ for culturing 12-24And (6) observing a bacteriostasis result after h.
The result shows that the inhibition zone of the kelp polyphenol extract with the concentration of 5 mg/mL is 6.92 mm, and the inhibition zone of the kelp polyphenol extract with the concentration of 100 mg/mL is 10.93 mm, which shows that the kelp polyphenol extract has better inhibition activity on gram-positive bacteria and food-borne pathogenic microorganisms.
Example 4
The crude extract of laminarin in example 3 is concentrated by rotary evaporation, ethanol is recovered, and the rest water-containing part is subjected to solvent fractionation extraction, and the specific implementation scheme is as follows: adding petroleum ether with equal volume or excessive amount, shaking to fully and uniformly mix, standing, collecting lower water-containing component after obvious layering, continuously extracting with petroleum ether, repeating for three times until extraction is complete, and rotary evaporating the collected supernatant to remove petroleum ether to obtain petroleum ether phase; the aqueous fraction was further extracted with ethyl acetate, n-butanol, and the remainder was aqueous phase. There are 5 components up to this point, crude Extract (CE), petroleum ether Phase (PEE), ethyl acetate phase (EAE), n-butanol phase (n-BE) and aqueous phase (WE), respectively.
The minimum inhibitory concentration (Minimal inhibitory concentration, MIC) and minimum bactericidal concentration (Minimal bactericidal concentration, MBC) of each component against staphylococcus aureus ATCC 12600 were determined using a double isocratic dilution 96-well plate method. Briefly, the cell number was adjusted to 10 according to example 4 8 cfu/mL, further diluted to 10 5 cfu/mL for use. After each component is diluted by LB liquid culture medium in gradient, 50 mu L of bacterial liquid is added into the holes of a 96-well plate to be uniformly mixed, and then the mixture is cultured for 24 h at 37 ℃, and the OD of the bacterial liquid is measured by an enzyme-labeled instrument 600 The MIC of each component for the strain is obtained at a concentration corresponding to no bacterial growth. As shown in Table 4, each of the components showed different degrees of bacteriostatic activity against the bacterium; wherein the ethyl acetate phase and the n-butanol phase show good antibacterial activity, the MIC is 250 mug/mL, the MIC of the crude extract and the MIC of the petroleum ether phase are 500 mug/mL, and the MIC of the water phase is more than 1 mg/mL.
Table 4 minimum inhibitory concentration of each component
MBC was determined by inclined-plane drip method, 0.05. 0.05 mL drop was pipetted from all clarified tubes in MIC assay onto agar solid medium, and after incubation at 37℃for 24 h, the corresponding extract phase concentration in the sterile-drop grown medium area was MBC. As a result, as shown in FIG. 5, the MBC of the petroleum ether phase and the ethyl acetate phase was 500. Mu.g/mL, and the MBC of the other components was more than 1 mg/mL.
Example 5
The antioxidant free radical scavenging capacity of each of the components of example 4 was determined using a two-fold isocratic dilution method. Vitamin C (Vc) is used as a positive control, and the antioxidant capacity of each level of kelp polyphenol component is compared with Vc. As shown in FIG. 6, each of the various components of kelp polyphenol and Vc has a certain antioxidant capacity, and the antioxidant activity is enhanced with the increase of the concentration. The ethyl acetate phase component after fractional extraction has the best DPPH free radical scavenging ability and half inhibition concentration (IC 50 ) 1.74 mg/mL; the n-butanol phase component has good ABTS free radical scavenging ability, and half inhibition concentration (IC 50 ) 61.7 μg/mL, indicating that kelp polyphenol extract can be used as a natural antioxidant source.
Example 6
Measurement of cysteine protease inhibitory Activity of each fraction of measurement example 4. The results are shown in FIG. 7, which shows that as the concentration of the kelp polyphenol extract increases, the cysteine protease inhibitory activity thereof increases continuously; at the same concentration, the PEE and EAE components showed greater inhibitory activity in total. Calculated, half maximal Inhibitory Concentration (IC) of CE, PEE, EAE, n-BE, WE 50 ) The method comprises the following steps of: 0.63, 0.18, 0.44, 1.17, 3.79, mg/mL.
Fresh grass carp removing heads, tails, bones, red meat, fascia and the like are purchased in a supermarket, the endogenous protease in the fish meat is kept as much as possible after 3 times of water washing and 2 times of low-concentration (0.125%) brine rinsing, minced fillet is obtained, 8% (w/w) kelp polyphenol crude extract is added into the minced fillet, and the operation process is kept at a low temperature (within 10 ℃). Two-stage heating (heating at 37deg.C for 30 min; heating at 90deg.C for 30 min) to obtain minced fish gel, immediately cooling with crushed ice, storing in a refrigerator at room temperature, thawing at normal temperature, and measuring texture. As shown in fig. 8, the gel strength and hardness of the minced fillet gel after adding the crude extract of kelp polyphenol are significantly higher than those of the blank group, and the properties of the minced fillet gel can be significantly improved.
The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above examples. Modifications, variations, or substitutions by one of ordinary skill in the art are intended to be included within the scope of the present invention.
Claims (1)
1. A method for optimally extracting kelp polyphenol by a response surface method is characterized by comprising the following steps of: the method comprises the following steps:
(1) Cleaning kelp, drying at 50-60 ℃, and crushing to obtain kelp powder;
(2) Weighing kelp powder according to a feed liquid ratio of 1:35, adding deionized water, adding pectase, wherein the dosage of pectase is 0.8% of the weight of the kelp powder, the enzyme activity of pectase is 500U/g, adjusting the pH value of the enzymolysis environment to 3.0, and performing enzymolysis at 50deg.C for 55min to obtain a mixture 1;
(3) Adding absolute ethyl alcohol with the final concentration of 60% into the mixture 1 in the step (2), and performing ultrasonic treatment for 30min under the conditions of ultrasonic power of 350w, ultrasonic frequency of 28KHz and ultrasonic temperature of 30-50 ℃ to obtain a mixture 2;
(4) Placing the mixture 2 in the step (3) into a microwave oven, and heating with low fire for 3min under the condition of power of 360W to obtain a mixture 3;
(5) Centrifuging the mixture 3 in the step (4) at 10000 rpm and 4 ℃ for 15min, and collecting supernatant to obtain crude extract of kelp polyphenol.
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