CN115251383B - Method for continuously preparing two highland barley flavone extracts with different purposes from highland barley and application - Google Patents
Method for continuously preparing two highland barley flavone extracts with different purposes from highland barley and application Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/385—Concentrates of non-alcoholic beverages
- A23L2/39—Dry compositions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0292—Treatment of the solvent
- B01D11/0296—Condensation of solvent vapours
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/04—Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs
- C12G3/05—Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs with health-improving ingredients, e.g. flavonoids, flavones, polyphenols or polysaccharides
- C12G3/055—Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs with health-improving ingredients, e.g. flavonoids, flavones, polyphenols or polysaccharides extracted from plants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Botany (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Non-Alcoholic Beverages (AREA)
Abstract
The invention relates to a method for continuously preparing highland barley flavone extracts with two different purposes from highland barley rice and application thereof, which utilizes isoelectric precipitation technology and enzymolysis technology to treat highland barley flavone extract to obtain highland barley flavone extract A and highland barley flavone extract B, and the prepared highland barley flavone extract A is applied to solid beverage or solid health food, and the highland barley flavone extract B is applied to wine body, and both highland barley flavone extracts have better oxidation resistance.
Description
Technical Field
The invention relates to the technical field of natural product extraction and application, in particular to a method for continuously preparing two highland barley flavone extracts with different purposes from highland barley and an application way thereof.
Background
Highland barley, also known as naked barley, is an important highland cereal crop and is widely distributed in northwest mountain areas, qinghai areas, tibet areas and the like in China. Highland barley is a main grain crop for Tibetan people, and has higher highland barley yield and rich nutritive value due to high Tibetan altitude and sufficient illumination. Highland barley is generally peeled and processed into highland barley rice or highland barley feed for human and livestock. Modern researches have found that active substances such as beta-glucan, flavonoid compounds and the like in highland barley have good health care effects, wherein the flavonoid compounds have strong oxidation resistance and assist in improving human immunity. Therefore, the method has very important significance for developing and utilizing highland barley.
At present, the highland barley flavone is extracted from highland barley seedlings, highland barley bran, highland barley rice and the like. Highland barley seedlings and highland barley bran are taken as extraction raw materials to prepare highland barley flavone extracts, and highland barley rice is taken as raw materials to prepare highland barley flavone extracts more easily. The highland barley rice contains rich protein and starch, after ethanol is used for extraction, the extract is concentrated to separate out a large amount of alcohol-soluble protein, polysaccharide and starch along with the reduction of the ethanol content, the substances are tightly combined with flavonoid substances, the precipitate rich in flavonoid components is discarded after centrifugation, and the separation effect of centrifugation on suspended polymers in the extract is very little, so that the column is blocked when the extract is purified by macroporous resin, the purification is difficult, and the yield of the highland barley rice flavone extract is low.
The highland barley flavone extract is prepared from highland barley seedlings serving as raw materials through the modes of alcohol extraction, macroporous resin adsorption, concentration, alcohol precipitation and the like, highland barley rice is not used in the whole preparation process, protein in the highland barley seedlings is not removed, a small amount of macroscopic insoluble precipitation is caused when the highland barley flavone extract is applied to wine bodies at normal temperature, and a large amount of insoluble precipitation is caused at low temperature, so that the sales of wine products are influenced. In addition, highland barley seedlings are taken as raw materials, so that the yield of highland barley in the current year is reduced, and national grain safety is not facilitated. The highland barley seedlings are taken as raw materials, the highland barley flavone extract is prepared by clarifying the extracting solution after alcohol extraction through a ceramic membrane, separating through an ultrafiltration membrane and desalting and concentrating through a nanofiltration membrane, and the preparation process needs to use three membranes for operation, so that the cost is high, and the industrial production is not facilitated.
In recent years, few reports on the preparation of highland barley flavone extracts for wine by using highland barley rice are presented, and no report is presented on the preparation method of highland barley flavone extracts for wine which do not generate sediment under the condition of low temperature.
Disclosure of Invention
The invention aims to provide a method for continuously preparing highland barley flavone extracts with two different purposes from highland barley and application thereof, wherein the prepared highland barley flavone extract A is applied to solid beverage or solid health food, the highland barley flavone extract B is applied to wine body, and both highland barley flavone extracts have better oxidation resistance. The invention greatly improves the yield of the highland barley flavone extract, not only contributes to the deep processing of highland barley, but also improves the planting income.
A method for continuously preparing two highland barley flavone extracts with different purposes from highland barley comprises the following steps:
Coarsely crushing highland barley, then performing condensation reflux extraction for 1-3 times by using ethanol solution with the mass fraction of 30-80%, extracting at 70-95 ℃ for 0.5-2.5 hours, filtering the extracting solution and merging the extracting solution;
concentrating the extracting solution until the ethanol concentration of the recovery solution is less than 2%, centrifuging for 10-30min at the rotating speed of 5000-10000r/min, and collecting precipitate and centrifuging supernatant;
step three, mixing the precipitate in the step two with ethanol solution according to the mass volume ratio of 1:5-15g/mL, regulating the pH of the mixed solution to 4-6.5, heating, condensing and refluxing for 1-3h at 70-95 ℃, centrifuging, collecting supernatant, regulating the pH to be neutral, and concentrating the supernatant until the ethanol concentration of the recovery solution is less than 2%;
Step four, combining the centrifugal supernatant obtained in the step two and the step three, regulating the pH to 4.5, performing isoelectric precipitation for 10-20 hours, centrifuging, collecting supernatant, and regulating the pH to be neutral or slightly alkaline;
step five, adding alpha-amylase and saccharifying enzyme into the supernatant, wherein the mass ratio of the compound enzyme to the highland barley used is 1-10:1000, carrying out enzymolysis for 1-3 hours at 30-70 ℃ under the condition of enzymolysis pH=4-7, then passing through a macroporous resin column, flushing the column with pure water until effluent is not turbid, and eluting the column with 3-10 times of 20-60% ethanol to obtain eluent;
step six, concentrating the eluent into dry paste powder to obtain highland barley flavone extract A;
And seventhly, adding 5-10 times of 30-60% ethanol into the highland barley flavone extract A, heating to 70-95 ℃ for condensing and refluxing for 1-3h, standing for 12-24h at-20 ℃, centrifugally collecting supernatant, concentrating, and performing vacuum freeze drying or low-temperature vacuum drying to obtain the highland barley flavone extract B.
Preferably, in the first step, the mass fraction of ethanol used in the condensation reflux extraction is 60%, the extraction temperature is 85 ℃, the extraction time is 1.5h, and the extraction times are 3 times.
Preferably, in the second step, the centrifugal speed is 7000r/min and the centrifugal time is 15min.
Preferably, in the third step, the mass-volume ratio of the precipitate to the added ethanol solution is 1:10g/mL, the acidic pH value is 5.5, the condensing reflux temperature is 85 ℃, the mass fraction of the ethanol solution is 60%, and the condensing reflux time is 1.5h.
Preferably, in the fourth step, the isoelectric precipitation time is 15 hours.
Preferably, in the fifth step, the mass ratio of the complex enzyme to the highland barley is 5:1000, the enzymolysis time is 3 hours, the enzymolysis temperature is 55 ℃, the enzymolysis pH is 6, the mass fraction of the ethanol eluent is 60%, and the elution multiple is 8.
Preferably, in the seventh step, the mass fraction of the ethanol solution is 60%, the mass-volume ratio of the highland barley flavone extract A to the ethanol solution is 1:8, the condensation reflux temperature is 85 ℃, the condensation reflux time is 1.5 h, the mixture is kept stand for 24 hours at the temperature of minus 20 ℃, and the supernatant is dried by vacuum freeze drying.
The highland barley flavone extract A is added into solid beverage or solid health food at a ratio of 1-5mg/g.
The addition amount of highland barley flavone extract B in wine is 5mg/L.
The invention has the beneficial effects that:
1. the invention reuses the precipitation waste generated after the ethanol extraction and concentration of the highland barley, and obviously improves the yield of the highland barley flavone extract.
2. The invention can prepare two highland barley flavone extracts with different purposes at the same time, wherein highland barley flavone extract A can be dissolved in water and low-level ethanol solution, but a little precipitation occurs. The highland barley flavone extract B is easily dissolved in 30-60% wine, and no macroscopic precipitate is formed when the temperature is higher than-20deg.C after the wine is added. The color difference value of the highland barley flavone extract B is obviously lower than that of the highland barley flavone extract A in the step six.
3. The invention utilizes isoelectric precipitation technology and enzymolysis technology to treat highland barley flavone extract, avoids the blockage of insoluble components such as protein, polysaccharide and the like in the highland barley flavone extract on a resin column, and prolongs the service cycle of the resin.
4. The highland barley flavone extract obtained by the invention has stronger antioxidant activity.
5. The raw materials used in the invention are green and pollution-free, so that pollution of the chemical agent to highland barley flavone extract is avoided, and the method has wide application prospect.
Drawings
FIG. 1 is a graph showing the trend of the adsorption capacity of macroporous resin to highland barley flavone;
FIG. 2 is a graph showing the tendency of highland barley flavone extract to DPPH radical scavenging ability in various examples.
Detailed Description
Example 1
The embodiment provides a method for continuously preparing two highland barley flavone extracts with different purposes from highland barley, which comprises the following steps:
Firstly, coarsely crushing 2kg of highland barley, then performing condensation reflux extraction for 2 times by using an ethanol solution with the mass fraction of 50%, extracting at 80 ℃ for 2.5 hours, filtering the extracting solution and combining the extracting solutions. Concentrating the extractive solution until the ethanol concentration of the recovered solution is less than 2%, centrifuging at 10000r/min for 10min, collecting precipitate, and centrifuging supernatant. Mixing the precipitate with 60% ethanol solution according to the mass volume ratio of 1:15g/mL, regulating the pH of the mixed solution to 5.5, heating, condensing and refluxing for 1h at 85 ℃, centrifuging, collecting supernatant, regulating the pH to be neutral, and concentrating the supernatant until the ethanol concentration of the recovered solution is less than 2%. Combining the two obtained centrifugal supernatants, regulating pH to 4.5, isoelectric precipitation for 15h, centrifuging, collecting supernatant, and regulating pH to neutrality. Adding alpha-amylase and saccharifying enzyme into the supernatant, wherein the mass ratio of the compound enzyme to the highland barley rice is 3:1000, carrying out enzymolysis for 1h at 50 ℃ under the condition of enzymolysis pH=6, then passing through a macroporous resin column, washing the column with pure water until effluent liquid is not turbid, and eluting the column with 6 times of 50% ethanol to obtain eluent. Concentrating the eluent into dry paste powder to obtain highland barley flavone extract A. Adding 10 times of 30% ethanol into highland barley flavone extract A, heating to 85deg.C, condensing and refluxing for 3 hr, standing at-20deg.C for 24 hr, centrifuging to collect supernatant, concentrating, and vacuum freeze drying to obtain highland barley flavone extract B.
Example 2
The embodiment provides a method for continuously preparing two highland barley flavone extracts with different purposes from highland barley, which comprises the following steps:
Firstly, coarsely crushing 2kg of highland barley, then performing condensation reflux extraction for 3 times by using an ethanol solution with the mass fraction of 60%, extracting at the temperature of 85 ℃ for 1.5 hours, filtering the extracting solution and combining the extracting solutions. Concentrating the extractive solution until the ethanol concentration of the recovered solution is less than 2%, centrifuging at 7000r/min for 15min, collecting precipitate, and centrifuging to obtain supernatant. Mixing the precipitate with 60% ethanol solution according to the mass volume ratio of 1:10g/mL, regulating the pH of the mixed solution to 5.5, heating, condensing and refluxing for 1.5h at 85 ℃, centrifuging, collecting supernatant, regulating the pH to be neutral, and concentrating the supernatant until the ethanol concentration of the recovered solution is less than 2%. Combining the two obtained centrifugal supernatants, regulating pH to 4.5, isoelectric precipitation for 15h, centrifuging, collecting supernatant, and regulating pH to neutrality. Adding alpha-amylase and saccharifying enzyme into the supernatant, wherein the mass ratio of the compound enzyme to the highland barley rice is 5:1000, carrying out enzymolysis for 3 hours at 55 ℃ under the condition of enzymolysis pH=6, then passing through a macroporous resin column, washing the column with pure water until effluent liquid is not turbid, and eluting the column with 8 times of 60% ethanol to obtain eluent. Concentrating the eluent into dry paste powder to obtain highland barley flavone extract A. Adding 8 times of 60% ethanol into highland barley flavone extract A, heating to 85deg.C, condensing and refluxing for 1.5 hr, standing at-20deg.C for 24 hr, centrifuging to collect supernatant, concentrating, and vacuum freeze drying to obtain highland barley flavone extract B.
Comparative example 1
This comparative example is different from example 2 in that the following operations are not performed: mixing the precipitate with 60% ethanol solution according to the mass volume ratio of 1:10g/mL, regulating the pH of the mixed solution to 5.5, heating, condensing and refluxing for 1.5h at 85 ℃, centrifuging, collecting supernatant, regulating the pH to be neutral, and concentrating the supernatant until the ethanol concentration of the recovered solution is less than 2%. Concentrating the extracting solution until the ethanol concentration of the recovering solution is less than 2%, centrifuging for 15min at a rotating speed of 7000r/min, and performing subsequent operations such as isoelectric precipitation, enzymolysis and the like on the collected centrifugal supernatant.
Comparative example 2
This comparative example is different from example 2 in that the two obtained centrifugal supernatants were combined and then subjected to subsequent operations directly through a macroporous resin column without isoelectric precipitation and enzymatic hydrolysis.
Comparative example 3
Referring to the method in the invention patent CN101361932B, the highland barley flavone extract is prepared by the following steps: taking 2kg of highland barley, coarsely pulverizing, adding 40 times of ethanol solution with concentration of 50%, extracting at 80deg.C for 2 times, each time for 2 hr to obtain extractive solution; concentrating until the ethanol concentration in the recovered extract is less than 10%, standing for 1h, filtering with 400 mesh filter cloth to obtain filtrate, flowing the filtrate through a glass adsorption column filled with AB-8 and having a diameter of 10 cm and a height of 120 cm, and washing with pure water 10 times the volume of the filling material to colorless; eluting with ethanol with volume 20 times of the filling volume and concentration of 50%, and concentrating to obtain extract; finally, the highland barley flavone extract is obtained by using 95% ethanol to carry out hot melting and cooling to normal temperature, separating out precipitate, centrifugally filtering at a rotation speed of 5000 rpm, and then drying (the drying temperature is 80 ℃).
Comparative example 4
Compared with example 2, the comparative example is different in that 2kg of highland barley rice is replaced with 2kg of highland barley seedlings.
Test example 1
Determination of total flavone content in samples of each example and comparative example A and yield calculation.
Preparation of test solution: about 25mg of the sample is precisely weighed, placed in a 50mL volumetric flask, added with a proper amount of 60% ethanol, uniformly shaken, subjected to ultrasonic treatment for 10 minutes to completely dissolve the sample, cooled and then fixed in volume to be used as a sample solution.
Preparing a reference substance solution: taking rutin reference substance 20mg, precisely weighing, placing in 50mL volumetric flask, adding appropriate amount of methanol, dissolving with ultrasound, cooling, adding methanol to scale, and shaking. Precisely measuring 25mL, placing in a 50mL volumetric flask, adding water to dilute to a scale, and shaking uniformly.
Preparation of a standard curve: precisely measuring 1mL, 2mL, 3mL, 4mL, 5mL and 6mL of control solution, respectively placing into 25mL measuring flask, respectively adding water to 6.0mL, adding 1mL of 5% sodium nitrite solution, mixing well, and standing for 6 minutes. Then, 1mL of a 10% aluminum nitrate solution was added thereto, and the mixture was shaken and left for 6 minutes. 10mL of 4% sodium hydroxide solution is added, water is added to the scale, the mixture is shaken well, and the mixture is left to stand for 15 minutes. The corresponding reagent is used as a blank, absorbance is measured at a wavelength of 500nm according to an ultraviolet-visible spectrophotometry, absorbance is used as an ordinate, concentration is used as an abscissa, a standard curve is drawn, and the standard curve is abs= 0.02179C-0.00372, and r 2 = 0.9971.
And (3) measuring and calculating: taking a sample solution, filtering with filter paper, precisely measuring 2mL of a continuous filtrate, placing the filtrate into a 25mL measuring flask, and measuring absorbance according to the method under the preparation item of a standard curve from 'adding water to 6 mL'.
The total flavone content in the sample was calculated according to the following formula (1.1):
Wherein:
w-content (%) of total flavone in the sample;
C-determining the concentration of total flavonoids in the test solution (mg/L) from a standard curve:
V—sample dilution volume (mL);
m-sample mass (mg).
Sample yield calculation: the sample yield was calculated as follows (1.2):
Wherein:
H-sample yield (%);
m-mass of sample (g) obtained:
m-mass of raw material used (g).
Table 1 total flavone content and yield of samples in each of examples and comparative examples
From table 1, it can be seen that the flavone content of highland barley flavone extract a and highland barley flavone extract B in example 2 is not significantly different from that of comparative example 1, but the product yield is significantly higher than that of comparative example 1, which indicates that the recovery of the precipitated waste generated by ethanol extraction and concentration of highland barley can significantly improve the yield of highland barley flavone extract. The flavone content and the product yield of the highland barley flavone extract A and the highland barley flavone extract B in the example 2 are obviously higher than those of the comparative example 2, which shows that isoelectric precipitation and enzymolysis operations can improve the flavone content and the yield of the products. The flavone content and the product yield of the highland barley flavone extract A and the highland barley flavone extract B in the example 2 are obviously higher than those of the highland barley flavone extract in the comparative example 3, which shows that the preparation method of the highland barley flavone extract of the invention patent CN101361932B is better than that of the highland barley flavone extract of the invention patent CN101361932B, and the invention can obtain products with two purposes at the same time. In the example 2, the flavone content and the product yield of the highland barley flavone extract A and the highland barley flavone extract B are both obviously higher than those of the comparative example 4, which shows that the highland barley rice raw materials have higher effects of preparing the highland barley flavone extract than that of highland barley seedlings.
Test example 2
Low temperature stability test
In order to further illustrate that the highland barley flavone extract obtained in the invention has better low-temperature stability, samples in each example and comparative example are selected and added into wine (the addition amount is 2 mg/L) to carry out the following low-temperature stability experiment. And respectively standing for 30d, 180d and 365d at the temperature of 15 ℃ below zero to observe whether macroscopic precipitation appears in the wine body.
Table 2 low temperature stability of samples in examples and comparative examples
As can be seen from Table 2, examples 1, 2, 1, 2 and 4 did not generate macroscopic precipitate at low temperature, while comparative example 3 had precipitate, indicating that highland barley flavone extract B prepared by the present invention had higher low temperature stability, and isoelectric precipitation and enzymolysis operations were not responsible for the absence of precipitate. In addition, the highland barley flavone extract prepared by the method in the invention patent CN101361932B does not have low-temperature stability, which proves that the invention has the advantage of low-temperature stability compared with the invention patent CN 101361932B.
Test example 3
Service life test of macroporous resin adsorption sample solution
And (3) taking the highland barley total flavone specific adsorption quantity as an index, observing the specific adsorption quantity change of the macroporous resin after repeated adsorption elution, and setting the specific adsorption quantity to be reduced to below 65% and ending the service life of the macroporous resin.
The test method comprises the following steps: the macroporous resin D101 is soaked in 95% ethanol for 3 hours, then the macroporous resin is washed with 95% ethanol until effluent liquid of the macroporous resin is not turbid, and then a large amount of pure water is used for washing until no alcohol smell exists. 6 parts of the treated wet resin (dry weight about 100 g) were taken and packed into 6 glass resin columns, respectively. And (3) taking a proper amount of extracting solution to be put on a column according to the methods in each example and comparative example, carrying out dynamic adsorption on extracting solution flowing out from the lower part of the resin column at a flow rate of 10mL/min after static pre-adsorption for 1.5 hours, and collecting effluent liquid for measuring the total flavone content. Eluting the resin with 1L of purified water and 60% ethanol respectively after adsorption, and finally washing the resin with a proper amount of purified water until the effluent liquid does not contain ethanol, thereby completing 1-time use. The above operation was repeated to obtain the specific adsorption amount for each time.
The specific adsorption amount was calculated according to formula 1.3:
And (3) taking the adsorption times as an abscissa and the specific adsorption quantity as an ordinate, and making an adsorption capacity change trend chart, see fig. 1.
As can be seen from FIG. 1, the specific adsorption capacity of the resin produced for the first time is about 98%, and the adsorption capacity of the resin is degraded along with the accumulated increase of the adsorption capacity of the resin, the specific adsorption capacity of the resin produced in example 1, example 2, comparative example 1 and comparative example 4 is lower than 65% after 60 times of production, and the specific adsorption capacity of the resin produced in comparative example 2 and comparative example 2 is lower than 65% after about 30 times of production, which means that the isoelectric point precipitation and enzymolysis steps can remarkably improve the service life of the resin, and the highland barley flavone extract prepared by the preparation method of the invention can prolong the service life of the resin by 2 times compared with the use of the invention patent CN101361932B, thereby greatly reducing the regeneration cost of the resin.
Test example 4
Determination of the ability of highland barley flavone extract A and highland barley flavone extract B to clear DPPH free radical in different examples
The experimental steps are as follows:
(1) Preparation of DPPH solution (0.25 mM): accurately weighing 4.929mg DPPH, dissolving in a 50mL volumetric flask, adding absolute ethyl alcohol to fix the volume to 50mL, refrigerating and keeping in dark place;
(2) Preparation of positive control solution: preparing vitamin C into mother solution (0.24 mg/mL), and performing gradient dilution to obtain 0.12, 0.10, 0.08, 0.06, 0.04 and 0.02mg/mL;
(3) Preparation of sample solution: preparing highland barley flavone extracts prepared in each example into mother liquor (10 mg/mL), and performing gradient dilution to obtain 8, 6, 4 and 2mg/mL;
(4) Color reaction: uniformly mixing 1mL of DPPH ethanol solution (0.25 mM) with 1mL of absolute ethanol, carrying out light-shielding reaction at 25 ℃ for 30min, measuring the light absorption value at 517nm, taking absolute ethanol as a blank, and marking the light absorption value as A; uniformly mixing 1mL of DPPH ethanol solution (0.25 mM) with 1mL of sample solution, carrying out light-shielding reaction at 25 ℃ for 30min, measuring the light absorption value at 517nm, taking absolute ethanol as a blank, and marking the light absorption value as B; uniformly mixing 1mL of absolute ethyl alcohol with 1mL of sample solution, carrying out light-shielding reaction for 30min at 25 ℃, measuring the light absorption value of the sample solution at 517nm, taking absolute ethyl alcohol as a blank, and marking the light absorption value as C; and Vc is used as a control group.
(5) DPPH radical scavenging (%) was calculated as shown in equation 1.4:
DPPH radical scavenging Rate (%) = [ A- (B-C) ]/Ax100% (1.4)
The DPPH radical scavenging ability of the highland barley flavone extract in different examples is shown in figure 2.
From fig. 2, it can be seen that the highland barley flavone extract a and highland barley flavone extract B in example 1 and example 2 have strong oxidation resistance.
Test example 5
Color difference of highland barley flavone extract in wine in different examples and comparative examples
The highland barley flavone extracts in the different examples and comparative examples were added to the wine body with purified water as a control group, and color difference measurement was performed using a color difference meter according to an addition amount of 5 mg/L.
TABLE 3 color difference of samples in examples and comparative examples
L ﹡ represents the brightness of the wine body, the sign is + which indicates the color of the wine body is white, and the sign is-which indicates the color of the wine body is dark; a ﹡ represents the redness and the greenness of the wine body, the sign is + which indicates that the color of the wine body is redder, and the sign is-which indicates that the color of the wine body is greener; b ﹡ represents yellow Lan Du of the wine body, the symbol is + indicating that the color of the wine body is yellow, and the symbol is-indicating that the color of the wine body is blue. It can be seen from Table 3 that the color and brightness of the wine body after the highland barley flavone extract A in example 2 is added into the wine body is lower than that of the highland barley flavone extract B, and the color of the wine body added with the highland barley flavone extract B is yellowish red. The wine body added with the highland barley flavone extract A and the highland barley flavone extract B in the comparative example 4 presents bluish green color, which is probably because more chlorophyll is extracted in the highland barley flavone extract prepared by taking highland barley seedlings as raw materials by adopting the invention patent CN101361932B, so that the wine body presents bluish green color when the highland barley flavone extract is applied. Generally, flavonoids are in a reddish-yellow color, and wine bodies are in a reddish-yellow color which is easily accepted by consumers. Therefore, the highland barley isoflavone extract prepared by the invention is better than the invention CN101361932B in color.
In summary, the foregoing embodiments are merely illustrative of the preferred embodiments of the present invention and do not include all embodiments of the invention. Various modifications and changes may be made by one skilled in the art without departing from the spirit and scope of the invention, which is therefore intended to be covered by the appended claims.
Claims (4)
1. A method for continuously preparing two highland barley flavone extracts with different purposes from highland barley, which is characterized by comprising the following steps:
Coarsely crushing highland barley, then performing condensation reflux extraction by using an ethanol solution, filtering and collecting an extracting solution; the mass fraction of ethanol used for condensation reflux extraction is 30-80%, the extraction temperature is 70-95 ℃, the extraction time is 0.5-2.5h, and the extraction times are 1-3 times;
Concentrating the extracting solution until the ethanol concentration of the recovering solution is less than 2%, centrifuging, and collecting precipitate and centrifuging supernatant;
Adding an ethanol solution into the precipitate obtained in the step two, regulating the pH to be acidic, heating, condensing, refluxing, centrifuging, collecting supernatant, regulating the pH to be neutral, and concentrating the supernatant until the ethanol concentration of the recovery liquid is less than 2%; the mass volume ratio of the sediment to the added ethanol solution is 1:5-15g/mL, the acidic pH value is 4-6.5, the condensing reflux temperature is 70-95 ℃, the mass fraction of the ethanol solution is 30-80%, and the condensing reflux time is 1-3h;
Step four, combining the centrifugal supernatant obtained in the step two and the step three, regulating the pH to 4.5, performing isoelectric precipitation for 10-20 hours, centrifuging, collecting supernatant, and regulating the pH to be neutral or slightly alkaline;
Step five, adding complex enzyme into the supernatant for enzymolysis, then passing through a macroporous resin column, flushing the column with pure water until effluent is not turbid, and eluting the column with ethanol to obtain eluent; the composite enzyme is alpha-amylase and saccharifying enzyme, the mass ratio of the composite enzyme to highland barley is 1-10:1000, the enzymolysis time is 1-3h, the enzymolysis temperature is 30-70 ℃, the enzymolysis pH is 4-7, the mass fraction of ethanol eluent is 20-60%, and the elution multiple is 3-10;
step six, concentrating the eluent into dry paste powder to obtain highland barley flavone extract A;
Step seven, adding ethanol into the highland barley flavone extract A for condensation reflux, then standing for a period of time at the temperature of minus 20 ℃, centrifugally collecting supernatant, concentrating and drying to obtain highland barley flavone extract B; the mass fraction of the ethanol solution is 30-60%, the mass volume ratio of the highland barley flavone extract A to the ethanol solution is 1:5-10, the condensation reflux temperature is 70-95 ℃, the condensation reflux time is 1-3h, the mixture is stood for 12-24h at the temperature of minus 20 ℃, and the supernatant fluid is dried by vacuum freeze drying or low-temperature vacuum drying.
2. The method according to claim 1, wherein in the second step, the centrifugal speed is 5000-10000r/min and the centrifugal time is 10-30min.
3. The application of highland barley flavone extract A prepared by the method of claim 1 or 2, which is characterized in that the highland barley flavone extract A is applied to solid beverage or solid health food, and the adding proportion is 1-5mg/g.
4. The use of highland barley flavone extract B prepared by the method of claim 1 or 2, characterized in that it is applied to wine, the addition amount is 1-5mg/L, and no precipitation is generated at-20 ℃.
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