CN115251383A - Method for continuously preparing highland barley flavone extracts with two different purposes from highland barley and application - Google Patents
Method for continuously preparing highland barley flavone extracts with two different purposes from highland barley and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
- 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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- 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)
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- Genetics & Genomics (AREA)
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Abstract
The invention relates to a method for continuously preparing highland barley flavone extracts with two different purposes from highland barley and application thereof, wherein the highland barley flavone extract is treated by isoelectric point precipitation technology and enzymolysis technology to obtain a highland barley flavone extract A and a highland barley flavone extract B, the prepared highland barley flavone extract A is applied to solid beverage or solid health-care food, and the highland barley flavone extract B is applied to wine, and the two 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 highland barley flavone extracts with two different purposes from highland barley and an application approach thereof.
Background
The highland barley is also called naked barley, is an important highland cereal crop and is widely distributed in northwest mountainous regions, qinghai, tibet and other regions of China. The highland barley is a main grain crop of Tibet people, and is high in yield and rich in nutritional value due to high Tibet altitude and sufficient illumination. The highland barley is usually processed into highland barley rice or highland barley feed for people and livestock to eat by peeling treatment. Modern researches find that active substances such as beta-glucan, flavonoid compounds and the like in the highland barley have good health-care efficacy, wherein the flavonoid compounds have strong oxidation resistance and assist in improving the immunity of a human body. Therefore, the development and utilization of the highland barley are of great significance.
Currently, the extraction sources of the highland barley flavone comprise highland barley seedlings, highland barley bran, highland barley rice and the like. The highland barley flavone extract is prepared simply by taking highland barley seedlings and highland barley bran as extraction raw materials, and the highland barley flavone extract is prepared difficultly by taking highland barley rice as a raw material. The highland barley contains abundant protein and starch, after the highland barley is extracted by using ethanol, the extract is concentrated to separate out a large amount of alcohol-soluble protein, polysaccharide and starch along with the reduction of the content of the ethanol, the substances can be tightly combined with flavonoid substances, the precipitate rich in the flavonoid component after centrifugation can be discarded, and the effect of the centrifugation on the separation of a suspension polymer in the extract is very small, so that the extract can block a column when macroporous resin purification is carried out, the purification is difficult, and the yield of the highland barley flavone extract is low.
In the invention patent CN101361932B, highland barley seedlings are used as raw materials, highland barley flavone extract is prepared by alcohol extraction, macroporous resin adsorption, concentration, alcohol precipitation and other modes, highland barley rice is not used in the whole preparation process, and protein in the highland barley seedlings is not removed, so that a small amount of macroscopic insoluble precipitate can be generated when the product is applied to a wine body at normal temperature, and a large amount of insoluble precipitate is generated under low temperature conditions, thereby affecting the sale of wine products. In addition, the highland barley seedlings are used as raw materials, so that the yield of the highland barley in the current year is reduced, and the national grain safety is not facilitated. In the invention patent CN101696381A, highland barley seedlings are used as raw materials, the extractive solution after alcohol extraction is subjected to ceramic membrane clarification, ultrafiltration membrane separation and nanofiltration membrane desalination and concentration to prepare the highland barley flavone extract, three membranes are required to be used for operation in the preparation process, the cost is high, and the method is not beneficial to industrial production.
In recent years, few reports of highland barley flavone extracts for preparing wine by using highland barley rice appear, and no report is found about a preparation method of highland barley flavone extracts for wine, which does not generate precipitates 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, and the prepared highland barley flavone extract A is applied to solid beverage or solid health-care food, and the prepared highland barley flavone extract B is applied to wine, and the two highland barley flavone extracts have better oxidation resistance. The invention greatly improves the yield of the highland barley flavone extract, makes a contribution to the fine and deep processing of highland barley and improves the planting income.
A method for continuously preparing highland barley flavone extracts with two different purposes from highland barley comprises the following steps:
step one, coarsely crushing highland barley, then carrying out condensation reflux extraction for 1-3 times by using 30-80% ethanol solution in percentage by mass, extracting at the temperature of 70-95 ℃ for 0.5-2.5h, filtering the extracting solution, and combining the extracting solution;
step two, concentrating the extracting solution until the ethanol concentration of the recovered solution is less than 2%, centrifuging for 10-30min under the condition of the rotating speed of 5000-10000r/min, and collecting precipitate and centrifuging supernatant;
step three, mixing the precipitate obtained in the step two with an ethanol solution according to the mass-volume ratio of 1:5-15g/mL, adjusting the pH of the mixed solution to 4-6.5, then heating, condensing and refluxing for 1-3h at 70-95 ℃, centrifuging, collecting a supernatant, adjusting the pH to be neutral, and concentrating the supernatant until the ethanol concentration of a recovery solution is less than 2%;
step four, combining the centrifugal supernatant obtained in the step two and the step three, adjusting the pH value to 4.5, precipitating at an isoelectric point for 10-20h, centrifuging, collecting the supernatant, and adjusting the pH value to be neutral or alkalescent;
adding alpha-amylase and saccharifying enzyme into the supernatant, wherein the mass ratio of the complex enzyme to the used highland barley is 1-10, carrying out enzymolysis for 1-3h at 30-70 ℃ under the condition that the enzymolysis pH =4-7, then passing through a macroporous resin column, washing the column with pure water until the effluent liquid is not turbid, and eluting the column with 3-10 times of 20-60% ethanol to obtain an 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 xanthone extract A, heating to 70-95 ℃, condensing and refluxing for 1-3h, standing at-20 ℃ for 12-24h, centrifuging, collecting supernatant, concentrating, and performing vacuum freeze drying or low-temperature vacuum drying to obtain a highland barley xanthone 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.
Preferably, in the fourth step, the isoelectric point precipitation time is 15 hours.
Preferably, in the fifth step, the mass ratio of the complex enzyme to the highland barley is 5 to 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 1.5h, standing is carried out for 24h at the temperature of minus 20 ℃, and the drying mode of the supernatant is vacuum freeze drying.
The proportion of the highland barley xanthone extract A in the solid beverage or solid health food is 1-5mg/g.
The addition amount of the highland barley flavone extract B in the wine body is 5mg/L.
The invention has the following beneficial effects:
1. the invention recycles the sediment waste generated by extracting and concentrating the highland barley with ethanol, thereby obviously improving the yield of the highland barley flavone extract.
2. The invention can simultaneously prepare two highland barley xanthone extracts with different purposes, wherein the highland barley xanthone extract A can be dissolved in water and low-alcohol solution, but a little precipitate appears. The highland barley xanthone extract B is easily dissolved in 30-60% wine body, and no macroscopic precipitate is separated out when the temperature is higher than-20 deg.C after the highland barley xanthone extract B is added into the wine body. 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 sixth step.
3. The method treats the highland barley xanthone extracting solution by using an isoelectric point precipitation technology and an enzymolysis technology, avoids blockage of insoluble components such as protein, polysaccharide and the like in the highland barley xanthone extracting solution 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, the pollution of the chemical agent to the highland barley xanthone extract is avoided, and the method has wide application prospect.
Drawings
FIG. 1 is a graph showing the trend of adsorption capacity change of macroporous resin on highland barley flavone;
FIG. 2 is a graph showing the tendency of the highland barley flavone extract to remove DPPH free radicals in different examples.
Detailed Description
Example 1
The embodiment provides a method for continuously preparing highland barley flavone extracts with two different purposes from highland barley, which comprises the following steps:
firstly, coarsely crushing 2kg of highland barley, then carrying out condensation reflux extraction by using an ethanol solution with the mass fraction of 50%, extracting for 2 times at the extraction temperature of 80 ℃ for 2.5h, 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 rotation speed of 10000r/min for 10min, and collecting precipitate and centrifuging supernatant. And mixing the precipitate with a 60% ethanol solution according to a mass-volume ratio of 1. Combining the two obtained centrifugal supernatants, adjusting pH to 4.5, precipitating at isoelectric point for 15h, centrifuging, collecting supernatant, and adjusting pH to neutral. Adding alpha-amylase and saccharifying enzyme into the supernatant, wherein the mass ratio of the compound enzyme to the used highland barley is 3:1000, carrying out enzymolysis for 1h at 50 ℃ under the condition that the enzymolysis pH is =6, then passing through a macroporous resin column, washing the column with pure water until the effluent liquid is not turbid, and eluting the column with 6 times of 50% ethanol to obtain the eluent. Concentrating the eluate to obtain dry extract powder to obtain semen Avenae Nudae flavone extract A. Adding 10 times of 30% ethanol into the highland barley flavone extract A, heating to 85 deg.C, condensing and refluxing for 3h, standing at-20 deg.C for 24h, centrifuging, collecting supernatant, concentrating, and vacuum freeze drying to obtain highland barley flavone extract B.
Example 2
The embodiment provides a method for continuously preparing highland barley flavone extracts with two different purposes from highland barley, which comprises the following steps:
firstly, coarsely crushing 2kg of highland barley, then carrying out condensation reflux extraction by using an ethanol solution with the mass fraction of 60%, extracting for 3 times at the extraction temperature of 85 ℃ for 1.5h, filtering the extracting solution and combining the extracting solution. Concentrating the extractive solution until the ethanol concentration of the recovered solution is less than 2%, centrifuging at 7000r/min for 15min, and collecting precipitate and supernatant. Mixing the precipitate with 60% ethanol solution at a mass-volume ratio of 1. Combining the two obtained centrifugal supernatants, adjusting the pH to 4.5, precipitating at isoelectric point for 15h, centrifuging, collecting the supernatant, and adjusting the pH to neutral. Adding alpha-amylase and saccharifying enzyme into the supernatant, wherein the mass ratio of the complex enzyme to the used highland barley is 5:1000, performing enzymolysis for 3h at 55 ℃ under the condition that the enzymolysis pH =6, then passing through a macroporous resin column, washing the column with pure water until the effluent liquid is not turbid, and eluting the column with 8 times of 60% ethanol to obtain an eluent. Concentrating the eluate into dry extract powder to obtain semen Avenae Nudae flavone extract A. Adding 8 times of 60% ethanol into the highland barley flavone extract A, heating to 85 deg.C, condensing and refluxing for 1.5h, standing at-20 deg.C for 24h, centrifuging, collecting supernatant, concentrating, and vacuum freeze drying to obtain highland barley flavone extract B.
Comparative example 1
This comparative example is compared with example 2, with the difference that the following operations are not carried out: mixing the precipitate with 60% ethanol solution at a mass-volume ratio of 1. Only concentrating the extracting solution until the ethanol concentration of the recovered solution is less than 2%, centrifuging for 15min at the rotating speed of 7000r/min, and performing subsequent operations such as isoelectric point precipitation, enzymolysis and the like on the collected centrifugal supernatant.
Comparative example 2
Compared with the embodiment 2, the difference of the comparative example is that after the centrifugal supernatants obtained twice are combined, isoelectric point precipitation and enzymolysis are not carried out, and the subsequent operation is carried out by directly passing through a macroporous resin column.
Comparative example 3
The highland barley xanthone extract is prepared by the method in patent CN101361932B of the invention, and the steps are as follows: pulverizing semen Avenae Nudae 2kg, adding 40 times of 50% ethanol solution, extracting at 80 deg.C for 2 times (each time for 2 hr) to obtain extractive solution; then concentrating until the concentration of ethanol in the recovered extracting solution is less than 10%, standing for 1h, filtering with 400-mesh filter cloth to obtain filtrate, allowing the filtrate to flow 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 with a volume 10 times that of the filling material until colorless; eluting with 50% ethanol with 20 times of the filling volume, and concentrating to obtain extract; and finally, using 95% ethanol for hot dissolution, cooling to normal temperature, separating out precipitates, carrying out centrifugal filtration at the rotating speed of 5000 r/min, and then drying (the drying temperature is 80 ℃) to obtain the highland barley flavone extract.
Comparative example 4
Compared with the example 2, the difference of the comparative example is that 2kg of highland barley rice is replaced by 2kg of highland barley seedlings.
Test example 1
Determination of the content of Total Flavonoids A and calculation of the yield in the samples of the examples and comparative examples.
Preparation of a test solution: taking a sample of about 25mg, precisely weighing, placing in a 50mL volumetric flask, adding a proper amount of 60% ethanol, shaking up, carrying out ultrasonic treatment for 10 minutes to completely dissolve the sample, and cooling to a constant volume to obtain a test solution.
Preparation of a control solution: taking 20mg of rutin reference substance, precisely weighing, placing in a 50mL volumetric flask, adding an appropriate amount of methanol, ultrasonically dissolving, cooling, adding methanol to scale, and shaking up. Precisely measuring 25mL, placing in a 50mL volumetric flask, adding water to dilute to a scale, and shaking up.
Preparation of a standard curve: precisely measuring 1mL, 2mL, 3mL, 4mL, 5mL and 6mL of the reference substance solution, respectively placing in 25mL measuring bottles, adding water to 6.0mL and 5% sodium nitrite solution 1mL, mixing, and standing for 6 minutes. Then, 1mL of 10% aluminum nitrate solution was added thereto, and the mixture was shaken and allowed to stand for 6 minutes. Adding 10mL of 4% sodium hydroxide solution, adding water to the scale, and shakingMix and stand for 15 minutes. Measuring absorbance at 500nm wavelength by ultraviolet-visible spectrophotometry using corresponding reagent as blank, drawing standard curve with absorbance as ordinate and concentration as abscissa, wherein the standard curve is Abs =0.02179C-0.003722＝0.9971。
Measurement and calculation: taking the sample solution, filtering with filter paper, precisely measuring 2mL of the subsequent filtrate, placing in a 25mL measuring flask, and measuring the absorbance according to the method under the preparation item of the standard curve from 'adding water to 6 mL'.
The total flavone content in the sample was calculated as follows (1.1):
in the formula:
w-content of total flavonoids in the sample (%);
c, determining the concentration (mg/L) of the total flavone in the test solution according to the standard curve:
v-sample dilution volume (mL);
m-sample mass (mg).
And (3) calculating the yield of the sample: the sample yield was calculated according to the following formula (1.2):
in the formula:
h-sample yield (%);
m-mass of sample obtained (g):
m-mass of raw material used (g).
TABLE 1 Total Flavonoids content and yield of the samples in the examples and comparative examples
It can be seen from table 1 that the flavone contents of the highland barley xanthone extract a and the highland barley xanthone extract B in example 2 are not significantly different from those in comparative example 1, but the product yield is significantly higher than that in comparative example 1, which indicates that the yield of the highland barley xanthone extract can be significantly increased by reusing the precipitation waste generated by ethanol extraction and concentration of highland barley. 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 obviously higher than those of the comparative example 2, which shows that the flavone content and the yield in the product can be improved by isoelectric precipitation and enzymolysis operation. In 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 highland barley flavone extract in comparative example 3, which shows that the preparation method of the highland barley flavone extract of the patent is superior to that of the highland barley flavone extract in patent CN101361932B, and the invention can simultaneously obtain products with two purposes. In the embodiment 2, the flavone content and the product yield of the highland barley flavone extract A and the highland barley flavone extract B are obviously higher than those of the comparative example 4, which shows that the effect of preparing the highland barley flavone extract from the highland barley raw materials is higher 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, the samples in the examples and the comparative examples are selected and added into the wine body (the adding amount is 2 mg/L) to carry out the following low-temperature stability experiment. Standing at-15 deg.C for 30d, 180d and 365d respectively to observe whether macroscopic precipitate appears in the wine body.
TABLE 2 Low temperature stability of the samples of the examples and comparative examples
As can be seen from Table 2, no macroscopic precipitates were generated in examples 1 and 2, comparative examples 1 and 2, and comparative examples 4, while no macroscopic precipitates were generated in comparative example 3, which indicates that the highland barley flavone extract B prepared by the present invention has high low temperature stability, and isoelectric point precipitation and enzymatic hydrolysis operations are not the cause of no precipitates. In addition, the highland barley flavone extract prepared by the method in the invention patent CN101361932B does not have low-temperature stability, which shows that the highland barley flavone extract has the advantage of low-temperature stability compared with the invention patent CN101361932B.
Test example 3
Service life test of macroporous resin adsorption sample solution
And (3) observing the change of the specific adsorption capacity of the macroporous resin after multiple times of adsorption and elution by taking the specific adsorption capacity of the highland barley total flavonoids as an index, and ending the service life of the macroporous resin when the set specific adsorption capacity is reduced to below 65%.
The test method comprises the following steps: soaking D101 type macroporous resin with 95% ethanol for 3 hr, washing with 95% ethanol until the effluent liquid is not turbid, and washing with a large amount of pure water until no alcohol smell is detected. 6 parts of the treated wet resin (about 100g dry weight) were packed in 6 glass resin columns, respectively. According to the method in each example and comparative example, a proper amount of extract is taken to be loaded on a column, the extract flows out from the lower part of the resin column at the flow rate of 10mL/min after static pre-adsorption for 1.5h to carry out the dynamic adsorption process, and the effluent is collected to be used for measuring the content of the total flavone. And after adsorption, respectively eluting the resin with 1L of purified water and 60% ethanol, and finally washing the resin with a proper amount of purified water until an effluent liquid does not contain ethanol, so that the resin is used for 1 time. The above operations were repeated to obtain the specific adsorption amounts for each time.
The specific adsorption capacity was calculated according to equation 1.3:
the adsorption capacity change trend chart is drawn by taking the adsorption times as the abscissa and the specific adsorption amount as the ordinate, and is shown in figure 1.
As can be seen from figure 1, the specific adsorption capacity of the resin produced for the first time is about 98%, the resin adsorption capacity is degraded along with the cumulative increase of the resin adsorption capacity, after 60 times of production, the specific adsorption capacity of the resin is lower than 65% in examples 1, 2, 1 and 4, and after about 30 times of production, the specific adsorption capacity of the resin is lower than 65% in comparative examples 2 and 2, which shows that the service life of the resin can be remarkably prolonged by the steps of isoelectric point precipitation and enzymolysis, and the service life of the resin prepared by the preparation method of the invention can be prolonged by 2 times by using the highland barley xanthone extract compared with the invention patent CN101361932B, and the regeneration cost of the resin is greatly reduced.
Test example 4
Determination of DPPH free radical clearance capability of highland barley rice flavone extract A and highland barley rice flavone extract B in different embodiments
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 a constant volume of 50mL, and refrigerating and storing in a dark place;
(2) Preparation of positive control solution: preparing mother liquor (0.24 mg/mL) from vitamin C, and diluting to 0.12, 0.10, 0.08, 0.06, 0.04, 0.02mg/mL by gradient;
(3) Preparation of sample solution: respectively preparing the highland barley flavone extracts prepared in the embodiments into mother liquor (10 mg/mL), and diluting the highland barley flavone extracts into 8, 6, 4 and 2mg/mL through gradient dilution;
(4) And (3) color development reaction: uniformly mixing 1mL of DPPH ethanol solution (0.25 mM) and 1mL of absolute ethanol, carrying out dark reaction at 25 ℃ for 30min, measuring the light absorption value at 517nm, and marking the light absorption value as A by taking the absolute ethanol as a blank; uniformly mixing 1mL of DPPH ethanol solution (0.25 mM) and 1mL of sample solution, reacting at 25 ℃ in a dark place for 30min, measuring the light absorption value at 517nm, and marking the light absorption value as B by taking absolute ethyl alcohol as a blank; uniformly mixing 1mL of absolute ethyl alcohol with 1mL of sample solution, carrying out a light-shielding reaction at 25 ℃ for 30min, measuring the light absorption value at 517nm, and marking the light absorption value as C by taking the absolute ethyl alcohol as a blank; and Vc was used as a control group.
(5) DPPH free radical clearance (%) was calculated according to equation 1.4:
DPPH radical scavenging ratio (%) = [ A- (B-C) ]/A × 100% (1.4)
The DPPH free radical scavenging ability of the highland barley flavone extract in different examples is shown in FIG. 2.
It can be seen from fig. 2 that the highland barley flavone extract a and the highland barley flavone extract B in examples 1 and 2 both have strong oxidation resistance.
Test example 5
The color difference of the highland barley flavone extract in the wine body in different examples and comparative examples
Purified water is used as a control group, the highland barley flavone extracts in different embodiments and comparative examples are added into wine, and a colorimeter is used for performing colorimetry according to the addition amount of 5mg/L.
TABLE 3 color difference of samples in examples and comparative examples
L﹡The sign is + to indicate that the color of the wine body is white, and the sign is-to indicate that the color of the wine body is dark; a is﹡The red and green degree of the wine body is represented, the sign is + to represent that the color of the wine body is red, and the sign is-to represent that the color of the wine body is green; b﹡The wine body is represented by yellow-blue degree, the sign is + to indicate that the wine body is yellow, and the sign is-to indicate that the wine body is blue. It can be seen from Table 3 that the wine body color brightness of the highland barley flavone extract A in example 2 added into the wine body is lower than that of the highland barley flavone extract B, and the wine body color of the highland barley flavone extract B added into the wine body 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 is blue-green, which is probably because the wine body is blue-green when the highland barley flavone extract is applied because more chlorophyll is subjected to an extracting solution when the highland barley flavone extract is prepared by taking highland barley seedlings as a raw material in the invention patent CN101361932B. Generally, flavonoids appear yellowish red, and wine bodies appear yellowish red which is easily accepted by consumers. Thus, it is possible to provideThe highland barley flavone extract prepared by the invention patent is superior to the invention patent CN101361932B in color.
In summary, the above-described embodiments are merely illustrative of the preferred embodiments of the present invention and do not encompass all of the scope of the invention. Various changes and modifications may be effected therein by one of ordinary skill in the pertinent art without departing from the scope or spirit of the present invention, as defined by the appended claims.
Claims (9)
1. A method for continuously preparing highland barley flavone extracts with two different purposes from highland barley is characterized by comprising the following steps:
step one, coarsely crushing highland barley, then carrying out condensation reflux extraction by using an ethanol solution, filtering and collecting an extracting solution;
step two, concentrating the extracting solution until the ethanol concentration of the recovered solution is less than 2%, centrifuging, and collecting precipitate and centrifuging supernate;
step three, adding an ethanol solution into the precipitate obtained in the step two, adjusting the pH value to acidity, then heating, condensing, refluxing, centrifuging, collecting supernatant, adjusting the pH value to neutrality, and concentrating the supernatant until the ethanol concentration of the recovered solution is less than 2%;
step four, combining the centrifugal supernatant obtained in the step two and the step three, adjusting the pH to 4.5, carrying out isoelectric point precipitation for a period of time, centrifuging and collecting the supernatant, and adjusting the pH to be neutral or alkalescent;
adding complex enzyme into the supernatant for enzymolysis, then passing through a macroporous resin column, washing the column with pure water until the effluent liquid is not turbid, and then eluting the column with ethanol to obtain an eluent;
step six, concentrating the eluent into dry paste powder to obtain highland barley flavone extract A;
and seventhly, adding ethanol into the highland barley flavone extract A, condensing and refluxing, standing for a period of time at the temperature of minus 20 ℃, centrifugally collecting supernatant, concentrating and drying to obtain a highland barley flavone extract B.
2. The method as claimed in claim 1, wherein in the first step, the mass fraction of ethanol used in the 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.
3. 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.
4. The method according to claim 1, wherein in the third step, the mass volume ratio of the precipitate to the added ethanol solution is 1:5-15g/mL, the acidic pH value is 4-6.5, the condensation reflux temperature is 70-95 ℃, the mass fraction of the ethanol solution is 30-80%, and the condensation reflux time is 1-3h.
5. The method according to claim 1, wherein in the fourth step, the isoelectric precipitation time is 10-20h.
6. The method as claimed in claim 1, wherein in the fifth step, the type of the complex enzyme is alpha-amylase and saccharifying enzyme, the mass ratio of the complex enzyme to the highland barley is 1-10, the enzymolysis time is 1-3h, the enzymolysis temperature is 30-70 ℃, the enzymolysis pH is 4-7, the mass fraction of the ethanol eluent is 20-60%, and the elution multiple is 3-10.
7. The method as claimed in claim 1, wherein in the seventh step, 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 condensing reflux temperature is 70-95 ℃, the condensing reflux time is 1-3h, the mixture is allowed to stand at-20 ℃ for 12-24h, and the supernatant is dried by vacuum freeze drying or low temperature vacuum drying.
8. The application of the highland barley flavone extract A prepared by the method of any one of claims 1 to 7 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 to 5mg/g.
9. The application of the highland barley flavone extract B prepared by the method of any one of claims 1 to 7 is characterized in that the highland barley flavone extract B is applied to wine in an amount of 1 to 5mg/L and does not precipitate at a low temperature of-20 ℃.
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