CN108192931B - Method for preparing black garlic polyphenol by extracting black garlic through compound enzyme method - Google Patents
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Abstract
The invention relates to the technical field of chemical industry, in particular to a method for preparing black garlic polyphenol by extracting black garlic by a compound enzyme method. The invention relates to a method for preparing black garlic polyphenol by extracting black garlic with a compound enzyme method, which comprises (1) pretreatment of black garlic, (2) preparation of compound enzyme liquid, (3) adding compound enzyme into water to generate compound enzyme liquid, (4) enzyme inactivation and (5) preparation of black garlic polyphenol. The method for preparing the black garlic polyphenol by extracting the black garlic by the compound enzyme method has the advantages of simple process, mild conditions, water as an extraction solvent, no use of an organic solvent and high extraction efficiency of the black garlic polyphenol, and the black garlic polyphenol prepared by the method has the hydroxyl radical scavenging capacity, the DPP radical scavenging capacity and the reducing capacity.
Description
Technical Field
The invention relates to the technical field of chemical industry, in particular to a method for preparing black garlic polyphenol by extracting black garlic by a compound enzyme method.
Background
Garlic is a long-history medicinal and edible plant, not only has good broad-spectrum sterilization function, but also has certain efficacies in the aspects of resisting tumors, resisting oxidation, improving immunity, preventing cardiovascular diseases, reducing blood fat, protecting liver and the like. The black garlic is also called as fermented black garlic, is food prepared by fermenting fresh raw garlic with skin in a fermentation box, has exact effects of resisting inflammation, resisting oxidation, relaxing bowels, regulating blood sugar and blood fat and the like, is a new medicine-food homologous product, has a promoting effect on agriculture and food industry due to rich raw material sources and clear effects, and has wide research and development prospects.
The black garlic is prepared by fermenting the garlic, partial active ingredients in the garlic are reserved under the action of high-temperature fermentation, the content of various ingredients in the garlic is also influenced, the content of total phenols and total acids is obviously increased, the taste of the black garlic is improved compared with the garlic, and the problems of gastrointestinal irritation and the like of the garlic are solved. Active ingredient research on black garlic mostly focuses on ingredients such as polyphenol, sugar, amino acid, and the like. The polyphenol is used as a natural compound with various activities, and the content of the polyphenol in the black garlic is relatively high. The polyphenol is used as an index, and the process for extracting and purifying the active ingredients of the black garlic is feasible. The extraction and purification process of polyphenols in traditional Chinese medicine and natural medicine can adopt solvent extraction, supercritical extraction, chromatographic separation and other methods. However, the above methods have disadvantages such as 1) requiring an organic solvent such as ethanol and having a large solvent consumption, 2) requiring a long-term heating extraction and solvent recovery process, and 3) requiring a special facility and having a complicated extraction and purification process. The process for extracting and purifying the polyphenol components of the black garlic has not been reported in systematic research. Especially, the method for extracting the black garlic by adopting the enzyme method to obtain the black garlic polyphenol has no relevant research at home and abroad.
The enzyme is a substance widely existing in nature and organisms, and the enzymolysis reaction is also a necessary physiological reaction of the organisms, so the enzymolysis extraction is a common extraction method, has the advantages of simple process, mild conditions, high extraction efficiency of black garlic polyphenol and the like, and can provide an extraction technology for the fine and deep processing and product development of black garlic.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for preparing black garlic polyphenol by extracting black garlic by a compound enzyme method, and provides an extraction technology for deep processing and product development of black garlic.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing black garlic polyphenol by extracting black garlic by a compound enzyme method comprises the following steps:
1) pretreating black garlic; processing black garlic into coarse particles;
2) preparing a compound enzyme solution: adding the complex enzyme into water to generate a complex enzyme solution;
3) and (3) compound enzyme extraction: adding the black garlic obtained by the treatment in the step 1) into the compound enzyme liquid obtained in the step 2) for extraction;
4) enzyme inactivation: inactivating the complex enzyme at high temperature after extraction;
5) preparing black garlic polyphenol: concentrating or drying the extracting solution of the inactivated complex enzyme obtained in the step 4) to obtain a black garlic polyphenol extracting solution or extract, and detecting the obtained black garlic polyphenol extracting solution or extract by using a detection method.
The step 1) is the pretreatment of the black garlic. The method comprises a plurality of methods such as a high-pressure homogenizing method, a high-speed shearing method, a mechanical rolling method, a pulverizer crushing method and the like, and crushed coarse particles of the black garlic are obtained through pretreatment.
The complex enzyme in the step 2) is prepared from cellulase, mannase and pectinase.
In the compound enzyme solution, the mass fraction of cellulase in the compound enzyme solution is 1-5%, the mass fraction of mannase in the compound enzyme solution is 1-10%, and the mass fraction of pectinase in the compound enzyme solution is 1-10%.
The mass ratio of the compound enzyme liquid to the black garlic in the step 3) is 5:1 to 25: 1.
The extraction temperature for extraction in the step 3) is 25-45 ℃.
The extraction time in the step 3) is 40-80 min.
The detection method in the step 5) is a spectrophotometry.
The invention adopts the experimental method and the application steps, and has the following characteristics:
1. the method has the advantages of simple process and mild conditions. The method only relates to enzymolysis extraction, the black garlic is simply pretreated, except high-temperature conditions required by high-temperature inactivation and solvent concentration after complex enzyme extraction, and the other conditions are normal-temperature extraction conditions.
2. The extraction solvent is water, and organic solvent is not used.
3. Compared with a solvent extraction method, the method provided by the invention uses the enzyme, so that the method has the advantage of high extraction efficiency of the black garlic polyphenol.
4. Through in vitro antioxidant experiments, the black garlic polyphenol prepared by the invention has hydroxyl radical scavenging capacity, DPP (dipeptidyl peptidase) radical scavenging capacity and reducing capacity.
Drawings
FIG. 1 is a process flow diagram of a process for preparing black garlic polyphenol by extracting black garlic with complex enzyme;
FIG. 2A shows that the 3D response curve of black garlic polyphenol extracted by the compound enzyme method (content of black garlic polyphenol and liquid-material ratio and extraction temperature);
FIG. 2B shows that the 3D response curve of black garlic polyphenol extracted by the compound enzyme method (black garlic polyphenol content and liquid-to-material ratio and extraction time);
FIG. 2C shows the 3D response curve of black garlic polyphenol extracted by the complex enzyme method (content of black garlic polyphenol and extraction temperature and extraction time);
FIG. 3 shows the clearance of hydroxyl radical by polyphenol of black garlic;
FIG. 4 shows the DPPH radical scavenging rate of black garlic polyphenol;
FIG. 5 shows the total reducing power of polyphenol in black garlic.
Detailed Description
Example 1
1. And (4) pretreating the black garlic. Crushing the black garlic into coarse particles by adopting the modes of extrusion, shearing or homogenate and the like; 250g of black garlic is taken and cut into coarse particles with the size of soybean by scissors for standby.
2. Selecting different enzymes and preparing a complex enzyme according to the optimized proportion of an orthogonal design test;
(1) optimum dosage of three enzymes when used alone
Collecting black Bulbus Allii (minced), dividing into 12 parts (each 5 g), placing 12 parts into 12 100mL Erlenmeyer flasks, adding cellulase (sequentially labeled as C) with mass fraction of 0.1%, 0.5%, 1.0%, 1.5%, and 2.0% into 12 Erlenmeyer flasks1～C5) 0.1%, 0.5%, 1.0%, 1.5%, 2.0% by mass of mannanase (labeled as H in sequence)1～H5) And pectase (labeled as M in sequence) 0.1%, 0.5%, 1.0%, 1.5%, 2.0% by weight1～M5) 75g of each (according to the mass ratio of the compound enzyme solution to the black garlic of 15:1), placing the mixture in a constant-temperature water bath at 40 ℃ to extract for 60min, shaking for 1min every 15min, cooling to room temperature, filtering, and adding distilled water to a constant volume of 100mL to obtain the black garlic.
Accurately sucking the extracting solution obtained in the steps into volumetric flasks of 1mL to 5mL respectively, adding distilled water to fix the volume to 5mL, keeping the volumetric flasks in the dark for 1min, accurately sucking the extracting solution into volumetric flasks of 0.5mL to 10mL respectively, then adding 1mL of forskolin phenol reagent and 2mL of sodium carbonate solution (the mass fraction is 15%) respectively, fixing the volume to 10mL by using distilled water, reacting for 1h in the dark at normal temperature, measuring the absorbance value at the wavelength of 760nm, measuring the black garlic polyphenol content (n is 3) according to a regression equation, and determining the optimal dosage of the cellulase, the mannase and the pectinase when being used independently.
Results of the experiment
Cellulase at various concentrations (C)1～C5) Mannanase (H)1～H5) And pectinase (M)1～M5) Under the action, the content result of the black garlic polyphenol shows that the three enzymes are at the concentration C3、H3、M3That is, the content of polyphenol extracted is the highest when the mass percentage is 1.0% and the concentration is 10.0 mg/g.
(2) Composite enzyme optimal ratio orthogonal test
Designing an orthogonal test of 3-factor 3 level by taking the optimal amount, namely concentration 1%, of cellulase, mannase and pectinase when the three enzymes act independently as reference, and selecting an orthogonal table L9(34) Experiment design is carried out (table 1), and the optimal combination proportion (dosage) of the cellulase, the mannase and the pectinase is determined by taking the content of the black garlic polyphenol as an index.
TABLE 1 factor level table
According to the above-mentioned L9(34) Preparing three complex enzyme solutions with different proportions for later use in an orthogonal table. Respectively placing 5.0g (accurately weighed) of black garlic (cut into pieces) into a 100mL triangular flask, adding 60 g of the complex enzyme solution (according to the mass ratio of the complex enzyme solution to the black garlic being 15:1), placing the mixture in a constant-temperature water bath at 40 ℃ to extract for 60min, shaking for 1min every 15min, cooling to room temperature, filtering, and diluting to 100mL with distilled water to obtain the black garlic. Each sample was repeated 3 times (n-3). The absorbance value was determined according to the above method for measuring the polyphenol content of black garlic, and the polyphenol content of black garlic was calculated, and the results are shown in table 2.
TABLE 2 results of orthogonal experiments
TABLE 3 ANOVA TABLE
| Sources of variance | Sum of squares of deviation | Degree of freedom | F value | Critical value of F | Factor influence |
| A | 0.721 | 2 | 0.532 | 6.940 | Is not significant |
| B | 1.992 | 2 | 1.468 | 6.940 | Is not significant |
| C | 0.646 | 2 | 0.476 | 6.940 | Is not significant |
| Error of the measurement | 2.71 | 4 |
The results shown in tables 2-3 show that: the factors influencing B, C, A and B, C, A have no significant difference, wherein the factor A has the minimum influence, and the optimal proportion is finally determined to be A2B3C3, namely the optimal combination of the three enzymes is 2% of cellulase, 3% of mannase and 3% of pectinase.
(3) Response surface method optimized complex enzyme extraction process
Single factor test
Placing 5.0g (accurately called) of black garlic (cut into pieces) into a 100mL triangular flask, adding 75g (according to the mass ratio of the compound enzyme solution to the black garlic of 15:1) of compound enzyme solution (mass fractions of 2% cellulase, 3% mannase and 3% pectinase), naturally adjusting the pH value, placing in a constant-temperature water bath at 40 ℃, extracting for 30min, 60min, 90min and 120min respectively, shaking for 1min every 15min, cooling to room temperature, filtering, and diluting to 100mL with distilled water. Repeating each sample for 3 times (n is 3), measuring absorbance value according to the method for measuring the black garlic polyphenol content, and calculating the black garlic polyphenol content. The results show that: the extraction amount of black garlic polyphenol increases with time, and reaches equilibrium at about 60 min.
Putting 5.0g (accurately called) of black garlic (cut into pieces) into a 200mL triangular flask, adding the complex enzyme solution (2% of cellulase, 3% of mannase and 3% of pectinase in mass fraction) into the complex enzyme solution and the black garlic respectively according to the mass ratio of 5:1 to 10:1 to 15:1 to 20:1 and 25:1, putting the mixture into a constant-temperature water bath at 40 ℃ for extracting for 60min respectively, shaking for 1min every 15min, cooling to room temperature, filtering, and fixing the volume to 100mL by using distilled water. Repeating each sample for 3 times (n is 3), measuring absorbance value according to the method for measuring the black garlic polyphenol content, and calculating the black garlic polyphenol content. The results show that: the extraction amount of polyphenol of black garlic increases with the increase of the liquid-material ratio, but when the liquid-material ratio is more than 15:1, the extraction amount of polyphenol of black garlic is in a downward trend.
Respectively taking 5.0g (accurately called) of black garlic (cut into pieces) and placing the black garlic in a 100mL triangular flask, respectively adding the compound enzyme solution (mass fraction is 2% of cellulase, 3% of mannase and 3% of pectinase) according to the mass ratio of the compound enzyme solution to the black garlic (15:1), respectively placing the mixture in constant-temperature water baths of 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ for extracting for 60min, cooling to room temperature, filtering, and fixing the volume to 100mL by using distilled water to obtain the black garlic. Repeating each sample for 3 times (n is 3), measuring absorbance value according to the method for measuring the black garlic polyphenol content, and calculating the black garlic polyphenol content. The extraction amount of polyphenol is obviously increased along with the continuous increase of the extraction temperature, but after the temperature is over 40 ℃, the extraction amount of polyphenol is in a trend of decreasing along with the increase of the temperature.
The response surface method optimizes the extraction conditions of the black garlic polyphenol:
on the basis of a single-factor test, according to a Box-Behnken test design principle, setting the consumption of black garlic of 5.0g (accurately called as fixed), a compound enzyme solution with the mass fractions of 2% of cellulase, 3% of mannase and 3% of pectinase and a natural pH value, selecting 3 factors of extraction time (A), liquid-material ratio (B) and extraction temperature (C) as investigation variables, and optimizing the process for extracting the black garlic polyphenol by using the compound enzyme solution with the content of the black garlic polyphenol as a response value by adopting a 3-factor 3-level response surface analysis method. Repeating each sample for 3 times (n is 3), measuring absorbance value according to the method for measuring the black garlic polyphenol content, and calculating the black garlic polyphenol content. The factors and levels are shown in Table 4, and the protocol and results are shown in Table 5.
TABLE 4 level of response surface optimization test factors for extracting black garlic polyphenol by complex enzyme method
| Level of | A/extraction time (min) | B/liquid-to-material ratio | C/extraction temperature (. degree. C.) |
| -1 | 30 | 10 | 35 |
| 0 | 60 | 15 | 40 |
| 1 | 90 | 20 | 45 |
TABLE 5 Experimental scheme and results of response surface for extracting black garlic polyphenol by complex enzyme method
Performing quadratic polynomial regression fitting on the table 5 test data by using Design-Expert 8.0 software to obtain a mathematical regression model of the black garlic polyphenol content to A, B, C, wherein the mathematical regression model comprises the following steps:
Y＝8.08+0.072A+0.094B+0.087C+0.11AB+0.048AC+0.032BC-0.97A2-0.63B2-0.59C2。
wherein Y is the content of black garlic polyphenol (mg/g), A is the extraction time (min), B is the liquid-material ratio, and C is the extraction temperature (DEG C).
The resulting model was analyzed for variance, and as can be seen from table 6, the regression model had F192.44，P<0.0001, which shows that the secondary model adopted in the experiment is remarkable. In this model, the mismatching term P>0.05, no significant mis-simulation test, indicating no mis-simulation factor exists. Complex correlation coefficient R of equation2＝0.9960，Adj-R20.9908, the model regression equation is shown to have good fitness.
TABLE 6 analysis of variance of black garlic polyphenol extracted by complex enzyme method
| Source | Sum of squares of deviation | Degree of freedom | Mean square | F value | P value |
| Model (model) | 8.07 | 9 | 0.90 | 192.44 | <0.0001 |
| Mistaking | 0.023 | 3 | 7.1714×10‐3 | 3.25 | 0.1422 |
| Pure error | 9.487×10‐3 | 4 | 2.372×10‐3 | ||
| R2＝0.9960 | Adj‐R2＝0.9908 | Pred‐R2＝0.9525 |
The response surface of the extraction method is shown in fig. 2. It can be seen that the extraction rate rises first and then falls as time increases. When the temperature rises, the extraction rate rises firstly and then falls; when the liquid-material ratio is increased, the extraction rate is increased and then decreased. Performing multiple regression model prediction by a response surface method to obtain the optimal enzymatic extraction process conditions as follows: the extraction time is 61.31min, the liquid-material ratio is 15.40:1(mL/g), and the extraction temperature is 40.38 ℃. The predicted value of the extraction amount of the black garlic polyphenol under the condition is 8.087 mg/g.
Verification test
Accurately weighing 5.0g of black garlic, adjusting and setting parameters to be extraction time of 61min, setting the mass ratio of the compound enzyme liquid to the black garlic to be 15:1, and setting the extraction temperature to be 40 ℃, wherein the extraction amount of the black garlic polyphenol is 8.125mg/g (RSD is 0.18%, n is 3) under the condition, and the relative error with a predicted value is 0.47%, which indicates that the established model has good predictability and the repeatability of the preferred extraction conditions is good.
And (4) extracting by a compound enzyme method. And adding complex enzyme into the black garlic granules, and extracting according to an optimal extraction process optimized by a response surface design test.
Weighing 5.0g of cut black garlic, and extracting the black garlic at the extraction temperature of 40 ℃ for 61min according to the mass ratio of the compound enzyme solution to the black garlic of 15: 1.
Enzyme inactivation and high-temperature inactivation of the compound enzyme.
After the extraction time is over, the temperature of the extraction system is rapidly raised to 80 ℃, and the enzyme is inactivated for 10 min. (4) The preparation of the black garlic polyphenol comprises the step of concentrating or drying the extracting solution to obtain the black garlic polyphenol extracting solution or extract.
The extract is concentrated into a concentrated solution with the content of 1g/ml based on the raw material of the black garlic.
The content of polyphenol in black garlic in the extract and the extract thereof are shown in Table 7 in comparison with an extraction method (material-liquid ratio: 1: 20, 50% ethanol solution, extraction at 85 ℃ for 1.5h), an ethanol reflux method (material-liquid ratio: 1: 20, 50% ethanol solution, heating reflux for 1.5h) and an ultrasonic auxiliary extraction method (material-liquid ratio: 1: 20, 50% ethanol solution, ultrasonic extraction for 1.5 h).
TABLE 7 comparison of Polyfafene content in black garlic and extracts from different extraction methods
| Numbering | Method | Total polyphenol content (mg/g) | Extract (g/g) |
| 1 | Enzyme-assisted extraction | 8.125 | 0.688 |
| 2 | Leaching method | 7.219 | 0.552 |
| 3 | Ultrasound assisted method | 3.235 | 0.344 |
| 4 | Reflux method | 2.185 | 0.291 |
Example 2
(1) Pretreating black garlic: crushing the black garlic into coarse particles by adopting the modes of extrusion, shearing or homogenate and the like; 100g of black garlic is taken and cut into coarse particles with the size of soybean by scissors for standby.
(2) Selecting and proportioning optimizing enzymes, selecting different enzymes, and preparing the complex enzyme according to the optimized proportion of an orthogonal design test:
weighing 30g of cellulase, 45g of mannase and 45g of pectinase according to the proportion that the cellulase accounts for 2% of the mass of the complex enzyme liquid, the mannase accounts for 3% of the mass of the complex enzyme liquid and the pectinase accounts for 3% of the mass of the complex enzyme liquid, and adding water to prepare 1500 g of the complex enzyme liquid. And (5) standby.
(3) Complex enzyme extraction:
and (3) adding the black garlic obtained in the step (1) into the compound enzyme liquid obtained in the step (2), and extracting for 40min in a constant-temperature water bath at 40 ℃.
(4) Enzyme inactivation, high-temperature inactivation of complex enzyme:
after the extraction time is over, the temperature of the extraction system is rapidly raised to 80 ℃, and the enzyme is inactivated for 10 min.
(5) Preparing black garlic polyphenol: concentrating or drying the extracting solution to obtain black garlic polyphenol extracting solution or extract.
The extract is concentrated into a concentrated solution with the content of 1g/ml based on the raw material of the black garlic.
Example 3
(1) Pretreating black garlic: crushing the black garlic into coarse particles by adopting the modes of extrusion, shearing or homogenate and the like; 100g of black garlic is taken and cut into coarse particles with the size of soybean by scissors for standby.
(2) Selecting enzyme and optimizing the proportion, selecting different enzymes, and preparing the complex enzyme according to the proportion:
weighing 15g of each of cellulase, mannase and pectinase according to the proportion that the cellulase accounts for 1 percent of the mass of the complex enzyme liquid, the mannase accounts for 1 percent of the mass of the complex enzyme liquid and the pectinase accounts for 1 percent of the mass of the complex enzyme liquid, and adding water to prepare 1500 g of the complex enzyme liquid. And (5) standby.
(3) Complex enzyme extraction:
and (3) adding the black garlic obtained in the step (1) into the compound enzyme liquid obtained in the step (2), and extracting for 61min in a constant-temperature water bath at 40 ℃.
(4) Enzyme inactivation, high-temperature inactivation of complex enzyme:
after the extraction time is over, the temperature of the extraction system is rapidly raised to 80 ℃, and the enzyme is inactivated for 10 min.
(5) Preparing black garlic polyphenol: concentrating or drying the extracting solution to obtain black garlic polyphenol extracting solution or extract.
The extract is concentrated into a concentrated solution with the content of 1g/ml based on the raw material of the black garlic.
(6) Determination of Black acid content
Method for extracting black garlic polyphenol by using compound enzymes with different proportions and comparison of extracts
| Numbering | Method | Total polyphenol content (mg/g) | Extract (g/g) |
| 1 | This embodiment | 6.984 | 0.574 |
| 2 | Optimized enzyme extraction process | 8.125 | 0.688 |
Example 4
(1) Pretreating black garlic: crushing the black garlic into coarse particles by adopting the modes of extrusion, shearing or homogenate and the like; 100g of black garlic is taken and cut into coarse particles with the size of soybean by scissors for standby.
(2) Selecting enzyme and optimizing the proportion, selecting different enzymes, and preparing the complex enzyme according to the proportion:
according to the proportion that the cellulase accounts for 5 percent of the mass of the compound enzyme liquid, the mannase accounts for 10 percent of the mass of the compound enzyme liquid, and the pectinase accounts for 10 percent of the mass of the compound enzyme liquid, 75g of the cellulase, 150g of the mannase and 150g of the pectinase are weighed, and water is added to prepare 1500 g of the compound enzyme liquid. And (5) standby.
(3) Complex enzyme extraction:
and (3) adding the black garlic obtained in the step (1) into the compound enzyme liquid obtained in the step (2), and extracting for 80min in a constant-temperature water bath at 40 ℃.
(4) Enzyme inactivation, high-temperature inactivation of complex enzyme:
after the extraction time is over, the temperature of the extraction system is rapidly raised to 80 ℃, and the enzyme is inactivated for 10 min.
(5) Preparing black garlic polyphenol, concentrating or drying the extracting solution to obtain a black garlic polyphenol extracting solution or extract:
the extract is concentrated into a concentrated solution with the content of 1g/ml based on the raw material of the black garlic.
(7) And (3) black acid content determination:
method for extracting black garlic polyphenol by using compound enzymes with different proportions and comparison of extracts
| Numbering | Method | Total polyphenol content (mg/g) | Extract (g/g) |
| 1 | This embodiment | 6.315 | 0.724 |
| 2 | Optimized enzyme extraction process | 8.125 | 0.688 |
Because of using a large amount of compound enzyme, the extract contains enzyme, so that the yield is high, but the polyphenol content is obviously lower than that obtained by the compound enzyme extraction in an optimized proportion.
Attached: research on unexpected antioxidant activity of black garlic polyphenol
1. Hydroxyl radical scavenging ability
Adding 1.0mL of 9.0mmol/L ferrous sulfate and 1.0mL of 9.0mmol/L salicylic acid solution into a 10mL test tube with a plug scale in sequence, shaking up, accurately adding 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of black garlic polyphenol solution with the mass concentration of 50 mu g/mL and 2.0, 1.8, 1.6, 1.4, 1.2 and 1.0mL of water respectively, finally adding 1mL of 8.8mmol/L hydrogen peroxide solution, preserving the temperature for 60min at 25 ℃, and measuring the absorbance at 510 nm. Clearance was calculated by 3 replicates (n-3) with ascorbic acid (vitamin C) as a positive control and distilled water as a blank control.
Hydroxyl radical clearance ═ 1- (a1-a2)/a0 × 100%
Where A0 is the absorbance of the control (water instead of test sample solution), A1 is the absorbance of the test group, and A2 is the absorbance of the sample (water instead of hydrogen peroxide solution).
The clearance rates of hydroxyl free radicals are 9.72%, 18.83%, 26.51%, 35.51% and 41.17% when the mass concentration of the black garlic polyphenol is 2.0, 4.0, 6.0, 8.0 and 10.0 mu g/mL respectively according to the formula. Independent sample t-tests were performed using SPSS16.0 software, with P <0.05 indicating that the differences were statistically significant. The hydroxyl radical scavenging ability result is shown in figure 3, in the tested mass concentration range, the black garlic polyphenol and the ascorbic acid both have the effect of scavenging hydroxyl radicals, the scavenging rate is increased along with the increase of the mass concentration, but the scavenging rate of the black garlic polyphenol on the hydroxyl radicals is obviously lower than that of the ascorbic acid with the same mass concentration, and the comparison difference between the two has statistical significance (P < 0.05).
2. DPP radical scavenging ability
Precisely adding 2.0mL of 0.04mg/mLDPPH ethanol solution into 10mL test tube with plug scale, accurately transferring 20, 40, 60, 80, 100, 120 μ L of black Bulbus Allii polyphenol solution with mass concentration of 125 μ g/mL, adding water to 5mL, mixing, reacting at room temperature in dark for 30min, measuring absorbance at wavelength of 517nm, and recording as A1Replacing DPPH ethanol solution with equal volume of anhydrous ethanol, adding black Bulbus Allii polyphenol extractive solutions with different mass concentrations, and measuring absorbance by the same method, and recording as A2The clearance was calculated by 3-fold parallel assay using ascorbic acid as a positive control (n-3).
DPPH free radical clearance rate ═ 1-A1/A2×100％
Wherein A1 is the sample absorbance and A2 is the absorbance of the negative control
When the mass concentration of the black garlic polyphenol is respectively 0.5, 1.0, 2.0, 2.5 and 3.0 mu g/mL according to the formula, the clearance rates of DPPH free radicals are respectively 13.29%, 24.59%, 36.65%, 48.28%, 61.52% and 73.19%. The DPP free radical scavenging ability is shown in figure 4, in the tested mass concentration range, the black garlic polyphenol and the ascorbic acid both have the effect of scavenging DPPH free radicals, the scavenging rate is increased along with the increase of the concentration, and the scavenging rate of the black garlic polyphenol is obviously higher than that of the ascorbic acid under the same mass concentration, and the comparison difference of the two has statistical significance (P < 0.05).
3. Total reducing power
Accurately transferring 20, 40, 60, 80, 100 and 120 mu L of black garlic polyphenol solution with the mass concentration of 125 mu g/mL, adding water to 0.5mL, sequentially adding 1.0mL of phosphate buffer solution of H6.6 and 2.0mL of 1% potassium ferricyanide, preserving the temperature in a 50 ℃ water bath for 20min, cooling, adding 1.0mL of 10% trichloroacetic acid and 0.5mL of 0.1% ferric trichloride, shaking uniformly, standing at room temperature for 10min, taking ascorbic acid as a positive control, measuring the absorbance at 700nm, carrying out parallel measurement for 3 times (n is 3), and taking the average value. As a result, the absorbance values were 0.141, 0.281, 0.459, 0.564, 0.732, and 0.935 when the mass concentrations of the black garlic polyphenol were 0.5, 1.0, 2.0, 2.5, and 3.0. mu.g/mL, respectively. The total reducing power result is shown in figure 5, in the test mass concentration range, the absorbance is obviously increased along with the increase of the mass concentration of the black garlic polyphenol and the ascorbic acid, which indicates that the black garlic polyphenol and the ascorbic acid both have good reducing power, the reducing power of the black garlic polyphenol is superior to that of the ascorbic acid, and the comparison difference between the black garlic polyphenol and the ascorbic acid has statistical significance (P < 0.05).
Claims (2)
1. A method for preparing black garlic polyphenol by extracting black garlic by a compound enzyme method is characterized by comprising the following steps:
1) pretreating black garlic; processing black garlic into coarse particles;
2) preparing a compound enzyme solution: adding the complex enzyme into water to generate a complex enzyme solution;
3) and (3) compound enzyme extraction: adding the black garlic obtained by the treatment in the step 1) into the compound enzyme liquid obtained in the step 2) for extraction;
4) enzyme inactivation: inactivating the complex enzyme at high temperature after extraction;
5) preparing black garlic polyphenol: concentrating or drying the extracting solution of the inactivated complex enzyme obtained in the step 4) to obtain a black garlic polyphenol extracting solution or extract, and detecting the obtained black garlic polyphenol extracting solution or extract by using a detection method; the complex enzyme in the step 2) is prepared from cellulase, mannase and pectinase; in the compound enzyme solution, the mass fraction of cellulase in the compound enzyme solution is 1-5%, the mass fraction of mannase in the compound enzyme solution is 1-10%, and the mass fraction ratio of pectinase in the compound enzyme solution is 1-10%; the mass ratio of the compound enzyme liquid to the black garlic in the step 3) is 5:1 to 25: 1; the extraction temperature for extraction in the step 3) is 25 to 45 ℃; the extraction time in the step 3) is 40-80 min.
2. The method for preparing black garlic polyphenol by extracting black garlic with the compound enzyme method according to claim 1, which is characterized in that: the detection method in the step 5) is a spectrophotometry.
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| CN105838570A (en) * | 2016-05-20 | 2016-08-10 | 徐州工程学院 | Preparation method of black garlic, mulberry and purple sweet potato composite fruit vinegar |
| CN106474322A (en) * | 2016-12-07 | 2017-03-08 | 黑龙江省科学院大庆分院 | Black garlic polyphenol oral liquid containing Eurycoma longifolia extract product and preparation method thereof |
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| CN104664384A (en) * | 2015-03-10 | 2015-06-03 | 山东农业大学 | Preparation method and application of black garlic fermentation liquid |
| CN105838570A (en) * | 2016-05-20 | 2016-08-10 | 徐州工程学院 | Preparation method of black garlic, mulberry and purple sweet potato composite fruit vinegar |
| CN106474322A (en) * | 2016-12-07 | 2017-03-08 | 黑龙江省科学院大庆分院 | Black garlic polyphenol oral liquid containing Eurycoma longifolia extract product and preparation method thereof |
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