CN108192931A - A kind of method that combined-enzyme method extraction black garlic prepares black garlic polyphenol - Google Patents
A kind of method that combined-enzyme method extraction black garlic prepares black garlic polyphenol Download PDFInfo
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Abstract
The present invention relates to chemical technology fields, and in particular to a kind of method that combined-enzyme method extraction black garlic prepares black garlic polyphenol.The method that a kind of combined-enzyme method extraction black garlic of the present invention prepares black garlic polyphenol, including（1）The pretreatment of black garlic（2）The preparation of complex enzyme liquid（3）Complex enzyme is added to the water generation complex enzyme liquid（4）Enzyme-deactivating（5）The preparation of black garlic polyphenol.The method that a kind of combined-enzyme method extraction black garlic of the present invention prepares black garlic polyphenol has simple for process, mild condition, Extraction solvent is water, without using organic solvent, the advantages of black garlic polyphenol extraction efficiency is high, and black garlic polyphenol prepared by the present invention has Hydroxyl radical-scavenging ability, DPP free radical scavenging abilities and reducing power.
Description
Technical field
The present invention relates to chemical technology fields, and in particular to a kind of combined-enzyme method extraction black garlic prepares the side of black garlic polyphenol
Method.
Background technology
Garlic is time-honored medical and edible dual purpose plant, not only has the function of good broad-spectrum sterilization, and it is antitumor,
It is anti-oxidant, improve immune, prevention of cardiovascular disease, reducing blood lipid, liver protecting etc. and have the effect of certain.Black garlic also known as hair
Ferment black garlic is with fresh raw garlic, belt leather manufactured food after fermenting in fermenting case, anti-inflammatory, anti-oxidant, defaecation, adjusting blood glucose
Blood fat and other effects is definite, is emerging integration of drinking and medicinal herbs product, since the raw material of black garlic is increased income abundant, clear efficacy, industrialization
Research is respectively provided with impetus to agricultural, food service industry, and research and development have a extensive future.
Black garlic is fermented through garlic, the effect of hot fermentation, remains fractions of active ingredient in garlic, meanwhile, to big
The content of Multiple components also produces influence in garlic, and total phenol and total acid content significantly increase, and compared with garlic, black garlic taste changes
The problems such as being apt to, also eliminating the GI irritation of garlic.About the study of active components of black garlic, it is mostly focused on polyphenol, sugar, ammonia
The ingredients such as base acid.The polyphenol native compound various as a kind of activity, the content in black garlic are relatively high.Using polyphenol as
Index carries out active constituent extraction, the purifying process of black garlic, has feasibility.The extraction of Polyphenols in Chinese medicine, natural drug,
The methods of purifying process, can be used solvent extraction, supercritical extract, chromatographic isolation.But there are 1) need ethyl alcohol for the above method
Wait organic solvents, solvent-oil ratio is big, 2) prolonged heating extraction, solvent recovery program are needed, 3) or need by spy
The deficiencies of different equipment, extraction purification program is complicated.And polyphenol component extraction, purifying process in relation to black garlic, there is not yet system is ground
Study carefully report.In particular by Enzymatic Extraction black garlic, black garlic polyphenol is obtained, both at home and abroad without correlative study.
Enzyme is the substance being widely present in nature and organism, physiological reaction necessary to enzyme digestion reaction is also life entity,
Therefore, enzymolysis and extraction has simple for process, mild condition, black garlic polyphenol extraction efficiency height as a kind of common extracting method
The advantages that, extractive technique can be provided for intensive processing, the product development of black garlic.
Invention content
In order to solve the technical issues of above-mentioned, the object of the present invention is to provide a kind of combined-enzyme method extraction black garlics to prepare black garlic
The method of polyphenol, intensive processing, product development for black garlic provide extractive technique.
In order to realize above-mentioned purpose, present invention employs following technical solutions：
A kind of method that combined-enzyme method extraction black garlic prepares black garlic polyphenol, includes the following steps：
1) pretreatment of black garlic；Black garlic is processed into kibble；
2) preparation of complex enzyme liquid：Complex enzyme is added to the water generation complex enzyme liquid；
3) combined-enzyme method extracts：It is added in the complex enzyme liquid that step 2) obtains in the black garlic that step 1) processing is obtained, into
Row extraction；
4) enzyme-deactivating：High-temperature inactivation complex enzyme after extraction；
5) preparation of black garlic polyphenol：The extracting solution for inactivating complex enzyme that step 4) is obtained is concentrated or drying obtains black garlic
Polyphenol extracting solution or extract, the black garlic polyphenol extracting solution or extract detected using detection method.
The pretreatment of the step 1) black garlic.Including high-pressure homogenization, high speed shear method, cal rolling method, pulverizer
A variety of methods such as comminuting method by pretreatment, obtain the broken coarse granule of black garlic.
Complex enzyme in the step 2) is prepared by cellulase, mannase and pectase.
The mass fraction that the complex enzyme liquid cellulase accounts for complex enzyme liquid is 1~5%, and mannase accounts for compound
The mass fraction of enzyme solution is 1~10%, and the mass fraction ratio that pectase accounts for complex enzyme liquid is 1~10%.
The complex enzyme liquid and black garlic mass ratio in the step 3) are 5:1 to 25:1.
25-45 DEG C of the Extracting temperature extracted in the step 3).
The extraction time extracted in the step 3) is 40-80min.
Detection method in the step 5) is spectrophotometry.
The present invention has the characteristics that following as a result of above-mentioned experimental method and applying step：
1. this method has simple for process, mild condition.This method only relates to enzymolysis and extraction, and black garlic is through simple pre- place
Reason, except high-temperature inactivation and solvent concentration need hot conditions after multiplex-enzyme extraction, other are room temperature extraction conditions.
2. Extraction solvent is water, without using organic solvent.
3. compared with solvent extraction process, the present invention uses enzyme so that the present invention is high with black garlic polyphenol extraction efficiency
Advantage.
4. being tested through antioxidation in vitro, black garlic polyphenol prepared by the present invention has Hydroxyl radical-scavenging ability, DPP free radicals
Scavenging activity and reducing power.
Description of the drawings
Fig. 1 multiplex-enzyme extractions black garlic prepares the process flow chart of black garlic polyphenol；
The 3D response surface designs (black garlic Dove content and liquid ratio and Extracting temperature) of Fig. 2A combined-enzyme methods extraction black garlic polyphenol；
The 3D response surface designs (black garlic Dove content and liquid ratio and extraction time) of Fig. 2 B combined-enzyme methods extraction black garlic polyphenol；
The 3D response surface designs of Fig. 2 C combined-enzyme methods extraction black garlic polyphenol are (when black garlic Dove content is with Extracting temperature and extraction
Between)；
Fig. 3 black garlics polyphenol is to the clearance rate of hydroxyl radical free radical；
Fig. 4 black garlics polyphenol is to the clearance rate of DPPH free radicals；
The total reducing power of Fig. 5 black garlic polyphenol.
Specific embodiment
Embodiment 1
1. the pretreatment of black garlic.Using modes such as extruding, shearing or homogenate, black garlic is ground into kibble；Take black garlic
250g, scissors shreds into the coarse granule of soya bean size, spare.
2. selection and the ratio optimization of enzyme, select different enzymes, carried out according to the ratio of positive quadraturing design test optimization compound
The preparation of enzyme；
Optimum amount when (1) three kind of enzyme is used alone
Black garlic is taken (to shred) 60.0g, is divided into 12 parts (each 5 grams) of decile, 12 parts of black garlics are put into 12 100mL triangles burns
Bottle in, in 12 conical flasks add in mass fraction be 0.1%, 0.5%, 1.0%, 1.5%, 2.0% cellulase (according to
It is secondary marked as C1~C5), the mannase (label successively of mass percent 0.1%, 0.5%, 1.0%, 1.5%, 2.0%
For H1~H5) and mass percent be 0.1%, 0.5%, 1.0%, 1.5%, 2.0% pectase (successively marked as M1~M5)
75 grams each (being 15 to 1 according to complex enzyme liquid and black garlic mass ratio), natural pH value is placed in 40 DEG C of waters bath with thermostatic control, extraction
60min shook 1min every 15 minutes, is cooled to room temperature filtering, 100mL is settled to distilled water to obtain the final product.
In the accurate extracting solution each 1mL to 5mL volumetric flasks for drawing above-mentioned steps and obtaining, distilled water is added to be settled to 5mL, is protected from light
1min is stood, it is accurate respectively to draw in 0.5ml to 10ml volumetric flasks, it is then respectively adding 1mL forint phenol reagent and 2mL sodium carbonate
Solution (mass fraction 15%) is settled to 10mL with distilled water, 1h is protected from light under room temperature, and extinction is measured at 760nm wavelength
Angle value measures black garlic polyphenol content (n=3) according to regression equation, determines therefrom that cellulase, mannonase pectase list
Optimum amount when solely using.
Experimental result
In the cellulase (C of various concentration1~C5), mannase (H1~H5) and pectase (M1~M5) under effect,
Black garlic polyphenol content the results show that three kinds of enzymes in concentration C3、H3、M3, i.e., when mass percent is 1.0%, a concentration of 10.0mg/g
The polyphenol content highest of extraction.
(2) complex enzyme optimum proportioning orthogonal test
On the basis of optimised quantity, that is, concentration 1% when three kinds of cellulase, mannonase pectase enzyme independent roles,
The horizontal orthogonal test of 3 factor 3 is designed, selects orthogonal arrage L9(34) experimental design (table 1) is carried out, using black garlic polyphenol content as finger
Mark determines the optimal combination ratio (dosage) of cellulase, mannonase pectase these three enzymes.
1 factor level table of table
According to above-mentioned L9(34) orthogonal arrage prepare different proportion three kinds of complex enzyme liquids, it is spare.Black garlic (shredding) is taken respectively
5.0g (accurate weighed) is placed in 100mL conical flasks, adds in 60 grams of above-mentioned complex enzyme liquids (according to complex enzyme liquid and black garlic quality
Than being 15 to 1), natural pH value is placed in 40 DEG C of waters bath with thermostatic control, extracts 60min, was shaken 1min every 15 minutes, is cooled to room
Temperature filtering, 100mL is settled to distilled water to obtain the final product.Each sample is repeated 3 times (n=3).By above-mentioned black garlic polyphenol content measure side
Method measures absorbance value, calculates black garlic polyphenol content, the results are shown in Table 2.
2 orthogonal experiments of table
3 analysis of variance table of table
| Soruces of variation | Sum of square of deviations | Degree of freedom | F values | F critical values | Factor influences |
| A | 0.721 | 2 | 0.532 | 6.940 | Not significantly |
| B | 1.992 | 2 | 1.468 | 6.940 | Not significantly |
| C | 0.646 | 2 | 0.476 | 6.940 | Not significantly |
| Error | 2.71 | 4 |
It is shown by table 2-3 results：Factor influences B>C>A, B, C, A factor there are no significant difference, wherein factor A influence most
Small, finally determining optimum proportioning is A2B3C3, i.e. three kinds of enzyme optimal combination are cellulase 2%, mannase 3%, pectin
Enzyme 3%.
(3) response phase method optimization multiplex-enzyme extraction technique
Single factor experiment
Black garlic is taken (to shred) 5.0g (accurate weighed) respectively to be placed in 100mL conical flasks, adds in 75 grams (according to complex enzyme
Liquid and black garlic mass ratio are 15 to 1) complex enzyme liquid (mass fraction for 2% cellulase, 3% mannonase, 3% pectin
Enzyme), natural pH value is placed in 40 DEG C of waters bath with thermostatic control, is extracted 30min, 60min, 90min, 120min respectively, is shaken every 15min
It shakes 1min, is cooled to room temperature filtering, 100mL is settled to distilled water to obtain the final product.Each sample is repeated 3 times (n=3), by above-mentioned black
Garlic polyphenol content assay method measures absorbance value, calculates black garlic polyphenol content.As a result it shows：The extracted amount of black garlic polyphenol is at any time
Between extension accordingly increase, and tend to balance in 60min or so.
Black garlic is taken (to shred) 5.0g (accurate weighed) respectively to be placed in 200mL conical flasks, by complex enzyme liquid and black garlic matter
Amount is than being 5:1、10:1、15:1、20:1 and 25:1 it is separately added into that (mass fraction is 2% cellulase, 3% mannonase
3% pectase) complex enzyme liquid, natural pH value is placed in 40 DEG C of waters bath with thermostatic control, extracts 60min respectively, is shaken every 15min
1min is cooled to room temperature filtering, and 100mL is settled to distilled water to obtain the final product.Each sample is repeated 3 times (n=3), by above-mentioned black garlic
Polyphenol content assay method measures absorbance value, calculates black garlic polyphenol content.As a result it shows：Black garlic polyphenol extracted amount is with liquid material
The increase of ratio and increase, but when liquid ratio be more than 15:When 1, black garlic polyphenol extracted amount is on a declining curve instead.
Black garlic is taken (to shred) 5.0g (accurate weighed) respectively to be placed in 100mL conical flasks, by complex enzyme liquid and black garlic matter
Amount is than (15:1) complex enzyme liquid of (mass fraction is 2% cellulase, 3% mannonase, 3% pectase) is separately added into,
Natural pH value, be respectively placed in 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, in 45 DEG C of waters bath with thermostatic control, extract 60min respectively, be cooled to room temperature
Filtering, 100mL is settled to distilled water to obtain the final product.Each sample is repeated 3 times (n=3), by above-mentioned black garlic polyphenol content assay method
Absorbance value is measured, calculates black garlic polyphenol content.With the continuous raising of Extracting temperature, polyphenol extracted amount has apparent raising,
But after 40 DEG C, polyphenol extracted amount is on a declining curve instead as temperature increases.
Response phase method optimizes the extraction conditions of black garlic polyphenol：
On the basis of single factor experiment, according to Box-Behnken experimental design principles, setting black garlic dosage 5.0g is (accurate
It is really weighed), mass fraction is 2% cellulase, the complex enzyme liquid of 3% mannonase, 3% pectase, and natural pH value is chosen
Extraction time (A), 3 factors of liquid ratio (B) and Extracting temperature (C) are as variable is investigated, using the horizontal response surface of 3 factor 3
Analytic approach using black garlic polyphenol content as response, optimizes multiplex-enzyme extraction black garlic polyphenol technique.Each sample repeats 3
Secondary (n=3) measures absorbance value by above-mentioned black garlic polyphenol content assay method, calculates black garlic polyphenol content.Factor is shown in level
Table 4, testing program is with the results are shown in Table 5.
The response surface optimization experimental factor of 4 combined-enzyme method of table extraction black garlic polyphenol is horizontal
| It is horizontal | A/ extraction times (min) | B/ liquid ratios | C/ Extracting temperatures (DEG C) |
| -1 | 30 | 10 | 35 |
| 0 | 60 | 15 | 40 |
| 1 | 90 | 20 | 45 |
5 combined-enzyme method of table extracts the response surface experimental program and result of black garlic polyphenol
Quadratic polynomial regression fit is carried out to 5 test data of table using 8.0 softwares of Design-Expert, it is more to obtain black garlic
Phenol content is to the mathematical regression model of A, B, C：
Y=8.08+0.072A+0.094B+0.087C+0.11AB+0.048AC+0.032BC-0.97A 2-0.63B2-
0.59C2。
Y is black garlic polyphenol content (mg/g) in formula, and A is extraction time (min), and B is liquid ratio, and C is Extracting temperature (DEG C).
Variance analysis, as can be seen from Table 6, the F=192.44 of the regression model, P are carried out to the model of gained<
0.0001, secondary model is notable used by illustrating this experiment.In this model, lose and intend item P>0.05, mistake plan inspection is not notable, says
It is bright to exist without mistake pseudo-factor.The coefficient of multiple correlation R of equation2=0.9960, Adj-R2=0.9908, illustrate model regression equation
Degree of fitting is good.
6 combined-enzyme method of table extracts the results of analysis of variance of black garlic polyphenol
| Source | Sum of square of deviations | Degree of freedom | It is square | F values | P values |
| Model | 8.07 | 9 | 0.90 | 192.44 | <0.0001 |
| It loses and intends | 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 design of extracting method is shown in Fig. 2.It can be seen that increasing over time, extraction takes the lead in rising to be declined again.When
During temperature rise, extraction takes the lead in rising to be declined again；When liquid ratio increase, extraction takes the lead in rising to be declined again.Pass through response phase method
Multivariate regression models prediction is carried out, obtaining optimal Extraction Processes condition is：Extraction time 61.31min, liquid ratio 15.40:
1 (mL/g), 40.38 DEG C of Extracting temperature.Black garlic polyphenol extracted amount predicted value is 8.087mg/g under this condition.
Verification test
Accurate to weigh black garlic 5.0g, adjustment sets each parameter as extraction time 61min, complex enzyme liquid and black garlic mass ratio
15:1,40 DEG C of Extracting temperature, black garlic polyphenol extracted amount is 8.125mg/g (RSD=0.18%, n=3) on this condition, and pre-
Measured value relative error is 0.47%, it is good predictive to show that the model established has, preferred extraction conditions repeatability is good.
Combined-enzyme method extracts.Black garlic particle add in complex enzyme, according to response surface design experiment optimize optimum extraction process,
It extracts.
The black garlic 5.0g shredded is weighed, according to complex enzyme liquid and black garlic mass ratio 15:1,40 DEG C of extractions of Extracting temperature are black
Garlic, extraction time 61min.
Enzyme-deactivating, high-temperature inactivation complex enzyme.
After extraction time, extraction system is warming up to 80 DEG C rapidly, maintains 10min inactivators.(4) black garlic polyphenol
It prepares, extracting solution is concentrated or drying obtains black garlic polyphenol extracting solution or extract.
Extracting solution is concentrated into terms of black garlic raw material, and content is the concentrate of 1g/ml.
Multiplex-enzyme extraction method, with extraction (solid-liquid ratio 1：20,50% ethanol solution, 85 DEG C extraction 1.5h), alcohol reflux
Method (solid-liquid ratio 1：20,50% ethanol solution is heated to reflux 1.5h) and ultrasound assisted extraction method (solid-liquid ratio 1：20,50% ethyl alcohol
Solution, ultrasonic extraction 1.5h) compare, the content of black garlic polyphenol and extract are relatively shown in Table 7 in extracting solution.
7 Different Extraction Method black garlic Dove content of table and extract compare
| Number | Method | Determination of Polyphenols (mg/g) | Extract (g/g) |
| 1 | Enzyme process assisted extraction | 8.125 | 0.688 |
| 2 | Extraction | 7.219 | 0.552 |
| 3 | Ultrasonic wave added method | 3.235 | 0.344 |
| 4 | Circumfluence method | 2.185 | 0.291 |
Embodiment 2
(1) pretreatment of black garlic：Using modes such as extruding, shearing or homogenate, black garlic is ground into kibble；Take black garlic
100g, scissors shreds into the coarse granule of soya bean size, spare.
(2) selection of enzyme and ratio optimization select different enzymes, are carried out according to the ratio of positive quadraturing design test optimization compound
The preparation of enzyme：
The 2% of complex enzyme liquid quality, mannase, which is accounted for, according to cellulase accounts for the 3% of complex enzyme liquid quality, pectase
3% ratio of complex enzyme liquid quality is accounted for, cellulase 30g, mannase 45g and pectase 45g is weighed, water is added to be configured to
1500 grams of complex enzyme liquid.It is spare.
(3) multiplex-enzyme extraction：
The black garlic of step (1) is added in the complex enzyme liquid of step (2), 40min is extracted in 40 DEG C of waters bath with thermostatic control.
(4) enzyme-deactivating, high-temperature inactivation complex enzyme：
After extraction time, extraction system is warming up to 80 DEG C rapidly, maintains 10min inactivators.
(5) preparation of black garlic polyphenol：Extracting solution is concentrated or drying obtains black garlic polyphenol extracting solution or extract.
Extracting solution is concentrated into terms of black garlic raw material, and content is the concentrate of 1g/ml.
Embodiment 3
(1) pretreatment of black garlic：Using modes such as extruding, shearing or homogenate, black garlic is ground into kibble；Take black garlic
100g, scissors shreds into the coarse granule of soya bean size, spare.
(2) selection of enzyme and ratio optimization select different enzymes, carry out the preparation of complex enzyme in proportion：
The 1% of complex enzyme liquid quality, mannase, which is accounted for, according to cellulase accounts for the 1% of complex enzyme liquid quality, pectase
1% ratio of complex enzyme liquid quality is accounted for, cellulase, mannase and each 15g of pectase is weighed, water is added to be configured to 1500
Gram complex enzyme liquid.It is spare.
(3) multiplex-enzyme extraction：
The black garlic of step (1) is added in the complex enzyme liquid of step (2), 61min is extracted in 40 DEG C of waters bath with thermostatic control.
(4) enzyme-deactivating, high-temperature inactivation complex enzyme：
After extraction time, extraction system is warming up to 80 DEG C rapidly, maintains 10min inactivators.
(5) preparation of black garlic polyphenol：Extracting solution is concentrated or drying obtains black garlic polyphenol extracting solution or extract.
Extracting solution is concentrated into terms of black garlic raw material, and content is the concentrate of 1g/ml.
(6) nigrotic acid assay
Different proportion multiplex-enzyme extraction method black garlic polyphenol and extract compare
| Number | Method | Determination of Polyphenols (mg/g) | Extract (g/g) |
| 1 | The implementation case | 6.984 | 0.574 |
| 2 | The enzyme extraction method of optimization | 8.125 | 0.688 |
Embodiment 4
(1) pretreatment of black garlic：Using modes such as extruding, shearing or homogenate, black garlic is ground into kibble；Take black garlic
100g, scissors shreds into the coarse granule of soya bean size, spare.
(2) selection of enzyme and ratio optimization select different enzymes, carry out the preparation of complex enzyme in proportion：
The 5% of complex enzyme liquid quality, mannase, which is accounted for, according to cellulase accounts for the 10% of complex enzyme liquid quality, pectase
10% ratio of complex enzyme liquid quality is accounted for, cellulase 75g, mannase and each 150g of pectase is weighed, water is added to prepare
Into 1500 grams of complex enzyme liquid.It is spare.
(3) multiplex-enzyme extraction：
The black garlic of step (1) is added in the complex enzyme liquid of step (2), 80min is extracted in 40 DEG C of waters bath with thermostatic control.
(4) enzyme-deactivating, high-temperature inactivation complex enzyme：
After extraction time, extraction system is warming up to 80 DEG C rapidly, maintains 10min inactivators.
(5) preparation of black garlic polyphenol, extracting solution is concentrated or drying obtains black garlic polyphenol extracting solution or extract：
Extracting solution is concentrated into terms of black garlic raw material, and content is the concentrate of 1g/ml.
(7) nigrotic acid assay：
Different proportion multiplex-enzyme extraction method black garlic polyphenol and extract compare
| Number | Method | Determination of Polyphenols (mg/g) | Extract (g/g) |
| 1 | The implementation case | 6.315 | 0.724 |
| 2 | The enzyme extraction method of optimization | 8.125 | 0.688 |
Larger due to the use of compound enzyme amount, extract extract yield when containing the amount of enzyme is higher, but polyphenol content
Significantly lower than the polyphenol content obtained by optimization ratio multiplex-enzyme extraction.
It is attached：Black garlic polyphenol accident antioxidant activity research
1st, Hydroxyl radical-scavenging ability
In 10mL tool plug scale test tubes, it is separately added into the ferrous sulfate of the 9.0mmol/L of 1.0mL, 1.0mL successively
9.0mmol/L salicylic acid solutions, shake up, and accurate addition 0,0.2,0.4,0.6,0.8,1.0mL mass concentrations are 50 μ g/mL respectively
Black garlic polyphenol solution and 2.0,1.8,1.6,1.4,1.2,1.0mL water, be eventually adding the hydrogen peroxide of the 8.8mmol/L of 1mL
Solution keeps the temperature 60min at 25 DEG C, and absorbance value is measured at 510nm.Using ascorbic acid (vitamin C) as positive control,
Distilled water is used as blank control, and parallel determination 3 times (n=3) calculates clearance rate.
Hydroxyl radical free radical clearance rate=[1- (A1-A2)/A0] × 100%
Wherein A0 is by the absorbance (water replaces test sample solution) of control group, and A1 is the absorbance of test group, and A2 is sample
The absorbance of product (water replaces hydrogenperoxide steam generator).
It is calculated when black garlic polyphenol mass concentration is respectively 2.0,4.0,6.0,8.0,10.0 μ g/mL pair by above-mentioned formula
The clearance rate of hydroxyl radical free radical is 9.72%, 18.83%, 26.51%, 35.51%, 41.17%.Using SPSS16.0 softwares into
Row independent samples t test, P<0.05 represents that difference is statistically significant.Hydroxyl radical-scavenging capability result is shown in Fig. 3, in experiment matter
Measure in concentration range, black garlic polyphenol and ascorbic acid are respectively provided with the effect for removing hydroxyl radical free radical, and clearance rate with quality it is dense
The increase of degree and increase, but black garlic polyphenol is significantly lower than the clearance rate of hydroxyl radical free radical in the ascorbic acid of identical mass concentration,
Statistically significant (the P of the two comparing difference<0.05).
2nd, DPP free radical scavenging abilities
In 10mL tool plug scale test tubes, precision adds in the 0.04mg/mLDPPH ethanol solutions of 2.0mL, accurate respectively to move
The black garlic polyphenol solution that 20,40,60,80,100,120 μ L mass concentrations is taken to be 125 μ g/mL, adds water to 5mL, is sufficiently mixed, room
Temperature is protected from light 30min, and 517nm wavelength measures absorbance, is denoted as A1, DPPH ethanol solutions are replaced with isometric absolute ethyl alcohol,
The black garlic polyphenol extracting solution of different quality concentration is added in, with method time-and-motion study absorbance, is denoted as A2, it is right as the positive using ascorbic acid
According to parallel determination 3 times (n=3) calculates clearance rate.
DPPH free radical scavenging activities=1-A1/A2× 100%
Wherein A1 is sample absorbance, and A2 is the absorbance of negative control
When being respectively 0.5,1.0,2.0,2.5,3.0 μ g/mL by above-mentioned formula calculating black garlic polyphenol mass concentration, to DPPH
The clearance rate of free radical is respectively 13.29%, 24.59%, 36.65%, 48.28%, 61.52%, 73.19%.DPP free radicals
Scavenging activity is shown in Fig. 4, and in test mass concentration range, black garlic polyphenol and ascorbic acid are respectively provided with the work for removing DPPH free radicals
With, and clearance rate is increased with the increase of concentration, and under identical mass concentration, the clearance rate of black garlic polyphenol is apparently higher than
Ascorbic acid, the statistically significant (P of the two comparing difference<0.05).
3rd, total reducing power
It is accurate to pipette the black garlic polyphenol solution that 20,40,60,80,100,120 μ L mass concentrations are 125 μ g/mL, it adds water to
0.5mL, phosphate buffer solution, the 2.0mL1% potassium ferricyanides of 1.0mLPH6.6 are sequentially added, is kept the temperature in 50 DEG C of water-baths
20min postcoolings add in 1.0mL10% trichloroacetic acids, 0.5mL0.1% ferric trichlorides, shake up, stand 10min at room temperature, with
Ascorbic acid is positive control, and absorbance is measured at 700nm, and parallel determination 3 times (n=3) is averaged.As a result black garlic is more
When phenol mass concentration is respectively 0.5,1.0,2.0,2.5,3.0 μ g/mL, absorbance is respectively 0.141,0.281,0.459,
0.564、0.732、0.935.Total reducing power result is shown in Fig. 5, in test mass concentration range, with black garlic polyphenol and ascorbic acid
Mass concentration increase and absorbance significantly increases, prompt both be respectively provided with good reducing power, and the reduction of black garlic polyphenol
Ability is better than ascorbic acid, the statistically significant (P of the two comparing difference<0.05).
Claims (7)
1. a kind of method that combined-enzyme method extraction black garlic prepares black garlic polyphenol, which is characterized in that include the following steps：
1）The pretreatment of black garlic；Black garlic is processed into kibble；
2）The preparation of complex enzyme liquid：Complex enzyme is added to the water generation complex enzyme liquid；
3）Combined-enzyme method extracts：By step 1）It handles and step 2 is added in obtained black garlic）In obtained complex enzyme liquid, carried
It takes；
4）Enzyme-deactivating：High-temperature inactivation complex enzyme after extraction；
5）The preparation of black garlic polyphenol：By step 4）The obtained extracting solution of inactivation complex enzyme is concentrated or drying obtains black garlic polyphenol
Extracting solution or extract, the black garlic polyphenol extracting solution or extract detected using detection method.
2. the method that a kind of combined-enzyme method extraction black garlic according to claim 1 prepares black garlic polyphenol, it is characterised in that：Institute
State step 2）In complex enzyme be prepared by cellulase, mannase and pectase.
It is 3. special according to the method that a kind of combined-enzyme method extraction black garlic of claims 1 or 2 any one of them prepares black garlic polyphenol
Sign is：The mass fraction that the complex enzyme liquid cellulase accounts for complex enzyme liquid is 1 ~ 5%, and mannase accounts for complex enzyme
The mass fraction of liquid is 1 ~ 10%, and the mass fraction ratio that pectase accounts for complex enzyme liquid is 1 ~ 10%.
4. the method that a kind of combined-enzyme method extraction black garlic according to claim 1 prepares black garlic polyphenol, it is characterised in that：Institute
State step 3）In the complex enzyme liquid and black garlic mass ratio be 5:1 to 25:1.
5. the method that a kind of combined-enzyme method extraction black garlic according to claim 1 prepares black garlic polyphenol, it is characterised in that：Institute
State step 3）In 25 to 45 DEG C of the Extracting temperature extracted.
6. the method that a kind of combined-enzyme method extraction black garlic according to claim 1 prepares black garlic polyphenol, it is characterised in that：Institute
State step 3）In the extraction time extracted be 40-80min.
7. the method that a kind of combined-enzyme method extraction black garlic according to claim 1 prepares black garlic polyphenol, it is characterised in that：Institute
State step 5）In detection method be spectrophotometry.
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| CN113017046B (en) * | 2021-03-17 | 2023-11-28 | 湖北将军红健康产业股份有限公司 | Preparation method of black garlic juice, preparation method of black garlic vinegar and black garlic vinegar prepared by preparation method |
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