CN115557830A - Method for extracting alkylresorcinol from wheat bran based on ultrasonic-assisted enzymolysis method - Google Patents
Method for extracting alkylresorcinol from wheat bran based on ultrasonic-assisted enzymolysis method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/685—Processes comprising at least two steps in series
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
- C07C37/72—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
- C07C37/82—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by solid-liquid treatment; by chemisorption
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention discloses a method for extracting alkylresorcinol from wheat bran based on an ultrasonic-assisted enzymolysis method, and belongs to the field of natural product or raw material processing. The invention uses enzyme to treat wheat bran powder to promote the release of ARs and the dissolution of phenolic acid compounds and other components, and then the components are removed by subsequent alcohol precipitation, concentration and purification treatment, so as to reduce the interference of impurities in the materials on the alkylresorcinol. The invention uses ultrasonic-assisted ethanol extraction, the extract is clear, the viscosity is low, the product has few impurities, and the subsequent treatment is convenient.
Description
Technical Field
The invention belongs to the field of natural product or raw material processing, and particularly relates to a method for extracting alkyl resorcinol from wheat bran based on an ultrasonic-assisted enzymolysis method.
Background
The biomarkers in the grain are specific substances in the bran or germ of the grain, and can better reflect the content of the bran or germ so as to monitor the production processes of hulling, debranning and the like and identify whether the whole grain product is adulterated. Wheat is one of grains which are consumed as the most staple food in the world, and is also the grain source with the most quantity in the existing whole grain processed food, so that the wheat is the key point for researching whole grain markers and is also the grain which is relatively researched more in the whole grain markers. Recent researches show that Alkylresorcinol (ARs) which is a special phenolic lipid exists in wheat grains and can be used as a marker of whole wheat food. The alkyl side chains of ARs in cereals are typically saturated carbon chains containing 17 to 25 odd numbered carbon atoms and can be represented as 5-heptadecylresorcinol (ARC 17: 0), 5-nonadecylresorcinol (ARC 19: 0), 5-heneicosylresorcinol (ARC 21: 0), 5-tricosylresorcinol (ARC 23: 0), 5-pentacosylresorcinol (ARC 25: 0), respectively. ARs have polar ends and hydrophobic alkyl side chains, are slightly soluble in water, are mainly soluble in methanol (solubility limited when R > 19), ethanol, acetone, ethyl acetate, hexane, propanol, dichloromethane and chloroform. ARs are fluorescent themselves.
The ARs have amphipathy, and the amphipathy endows the ARs with certain functionality, such as oxidation resistance, tumor resistance and the like, and has better improvement effect on various chronic diseases. Therefore, products with high amounts of ARs are increasingly gaining attention. ARs are specifically present in the bran of wheat, etc., but are not contained in the embryo and endosperm. After being taken by human body, the polypeptide can be detected in biological samples such as plasma and the like, and when people eat bran-free meals, the polypeptide cannot be detected in the biological samples such as the plasma and the like. And thus can be used as a biomarker for whole wheat product intake.
At present, the methods for measuring ARs mainly include spectrophotometry, liquid-phase fluorescence, gas chromatography, and the like. The method for detecting the content of ARs in whole wheat flour in LS/T3244-2015 'whole wheat flour' adopts an oscillation extraction method to extract ARs, and adopts a spectrophotometry method to detect. The oscillation extraction method in the row mark has the advantages of simple operation, low requirement on equipment and the like, but has the defects of long extraction time (48 h), long pretreatment concentration time (4-6 h), low efficiency, large equipment loss caused by long-time operation and the like. At present, the extraction solvent of ARs in grains is generally extracted by using various organic solvents such as n-propanol, ethyl acetate, methanol, acetone and the like according to the grain industry standard, and researches show that the mutual solubility of the methanol, the acetone and the water is strong, and the recovery rate is extremely low, because the water-soluble protein is extremely easy to extract, more impurities are generated, and the subsequent experiments are not facilitated. While the recovery rate of the ARs by the n-propanol is below 80 percent. The ARs are used as amphiphilic molecules, the polarity of the ARs is similar to that of ethyl acetate, few extracts in the extract are extracted, the recovery rate to be measured is high, the baseline interference is small, but the ethyl acetate is used as an organic reagent, the extraction cost is high, the smell is pungent, and the ARs are harmful to human bodies. Therefore, the development of a method for extracting the alkylresorcinol from the cereal bran with low cost and high yield is very significant.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problems of time consumption, low recovery rate and low purity of the existing method for extracting the Alkylresorcinol (ARs).
[ solution ]
The invention provides a method for extracting alkyl resorcinol from wheat bran based on an ultrasonic-assisted enzymolysis method, which mainly comprises the following steps:
(1) Pretreatment: firstly, crushing wheat bran, and sieving the crushed wheat bran with a 80-mesh sieve for later use;
(2) Enzymolysis: weighing 1g (accurate to 0.01 g) of bran pretreated in the step (1) in a 50mL centrifuge tube, adding a prepared phosphate buffer solution in advance to stabilize the pH to 5.0, respectively adding at least one of alpha-amylase, protease, cellulase and ligninase, carrying out enzymolysis for 2h at 50 ℃, and then boiling for 5min to inactivate the enzymes;
(3) Centrifuging: centrifuging at the rotating speed of 4000rpm for 10min, extracting bran residues collected after centrifuging by using ethanol, precipitating supernate collected after centrifuging by using ethanol, filtering and collecting filtrate after ethanol precipitation, and spin-drying the filtrate by using a rotary evaporator at the temperature of 45 ℃ to obtain concentrated solution 1 for later use;
(4) Ethanol leaching: adding ethanol into the bran residues collected after centrifugation in the step (3), performing ultrasonic extraction at room temperature for 1.5min at the ultrasonic power of 300W, centrifuging the extracting solution, and taking supernate;
(5) And (3) spin-drying: spin-drying the supernatant obtained in the step (4) by a rotary evaporator to obtain a concentrated solution 2;
(6) Silica gel column chromatography purification: mixing the concentrated solution 1 and the concentrated solution 2, purifying by silica gel column chromatography,
firstly, placing 100-200 meshes of silica gel powder in a drying oven at 105 ℃ for high-temperature activation for 30min, then placing the silica gel powder in a dryer for cooling to room temperature, weighing a concentrated solution sample with the same mass as the silica gel powder and the silica gel powder, pouring the concentrated solution sample and the silica gel powder into an evaporation dish, uniformly stirring, placing the evaporation dish in the drying oven at 50 ℃ for overnight drying, taking out, and cooling to room temperature for later use;
selecting a 500 × 40mm normal-pressure glass chromatographic column, loading the sample by a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, slightly shaking the chromatographic column until the surface of a filler is smooth, wherein the height of the packed column is 18cm, fixing the chromatographic column on an iron support, slightly adding sample-mixing silica gel until the surface is smooth, leaving a certain height above the chromatographic column for eluent to accumulate, performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10), performing rotary evaporation on each 100mL of eluent, tracking by using TLC, analyzing by using HPLC, and collecting the eluent;
(7) And (3) re-concentration: collecting eluent, performing rotary evaporation concentration, blowing to dry by nitrogen, and re-dissolving by using 5mL chromatographic grade methanol;
(8) LC-MS detection analysis: after the reconstituted sample was filtered through a 0.22. Mu.L organic filter, 200. Mu.L of the resulting solution was pipetted into a sample vial for LC-MS analysis.
In the method as described above, preferably, in the step (2), the protease is 20 ten thousand U/g, the α -amylase is 30 ten thousand U/g, the cellulase is 60 ten thousand U/g, and the ligninase is 5 ten thousand U/g, and the amount of the protease to be added is 45mg, the amount of the α -amylase to be added is 30mg, the amount of the cellulase to be added is 15mg, and the amount of the ligninase to be added is 1.8g.
The method as described above, preferably, the specific operation of step (4) is: and (4) adding 20mL of ethanol into the bran residues collected after enzymolysis and centrifugation in the step (3), and performing ultrasonic extraction at room temperature for 1.5min, wherein the ultrasonic power is 300w.
The method as described above, preferably, the conditions of HPLC in the step (6) are: (1) mobile phase: phase A0.1% aqueous formic acid; phase B0.1% methanoic acid in methanol, flow rate: 1mL/min, column temperature: 30 ℃, sample size: 10 mu L of the solution; (2) the mobile phase elution procedure was: 0min, 15-25% of phase A and 75-85% of phase B; 5min, 15-10% of phase A and 75-90% of phase B; 10min, 5-10% of phase A and 90-95% of phase B; 15min, 0-5% of phase A and 95-100% of phase B; 35min, 15-25% of phase A and 75-85% of phase B; (3) a detector: the detection wavelength was 280nm using a DAD detector.
In the method as described above, preferably, the detection analysis conditions of LC-MS in the step (8) are:
LC conditions: ACQMITY MPLC BEN C 18 Chromatography columns (2.1 mm. Times.100mm, 1.7. Mu.m); the column temperature is 30 ℃; the sample injection amount is 5 mu L; mobile phase: the phase A is ammonia water containing 0.1 percent (volume fraction), and the phase B is acetonitrile; the flow rate is 0.5mL/min; elution gradient: 0-2min, 100-15% of A and 0-85% of B; 2-4 min, 15-10% of A,85％～90％ B；4～8min，10％～5％ A，90％～95％ B；8～9min，5％～15％ A，95％～85％ B。
MS conditions: electrospray ion source, negative ion source; a multi-reaction detection mode; the capillary voltage is 1.2kV; taper hole airflow: nitrogen at the flow rate of 150L/h; the ion source temperature is 150 ℃; the temperature of the desolvation gas is 200 ℃, and the flow rate is 1000L/h.
[ advantageous effects ]
1. The method is simple and easy to implement, low in cost, high in yield and small in harm to human bodies, and the extracted ARs products are safe and pollution-free.
2. The invention uses enzyme to treat wheat bran powder, uses the enzyme to degrade cellulose, starch, some protein and lignin and other components in plant cells, thereby destroying plant cell walls in the bran raw material, playing a biological catalysis role, promoting the release of ARs, promoting the dissolution of phenolic acid compounds, tannic acid, flavonoid compounds, carbohydrate compounds and other components, leading water-soluble polysaccharide and other substances to form mixed solution with buffer solution, and then removing the mixed solution through subsequent alcohol precipitation, concentration and purification treatment, thereby reducing the interference of impurities in the materials on alkylresorcinol.
3. According to the method, the residues collected after the centrifugation of the bran enzymatic hydrolysate are extracted by using ultrasound to assist ethanol, the efficiency of ethanol extraction can be improved under the action of ultrasound, more Ars can be dissolved out, and the extraction cost is reduced while the toxicity caused by an organic solvent is avoided.
4. The supernatant fluid collected after the centrifugation of the enzymatic hydrolysate of the bran is subjected to alcohol precipitation by ethanol, so that impurities such as saccharides and the like extracted and hydrolyzed can be removed by alcohol precipitation to achieve the purpose of certain purification.
5. The invention uses ethanol for extraction, the extract is clear, the viscosity is low, the product has less impurities, and the subsequent treatment is convenient.
Drawings
Figure 1 is an LC-MS spectrum of ARC17: 0.
FIG. 2 Effect of ultrasound power on ARs leaching amount.
FIG. 3 Effect of ultrasonic leach time on ARs leach yield.
FIG. 4 is a graph comparing the amount of ARs extracted from different brans (comparative examples 2 to 4).
FIG. 5 is a graph comparing ethyl acetate extraction and absolute ethanol extraction of wheat bran (comparative examples 1 to 2).
Fig. 6 is a comparison graph of the extraction amount of wheat bran subjected to 100% ethanol ultrasonic enzymolysis (comparative example 1 and examples 1 to 4).
FIG. 7 is a comparison of the extraction yields of wheat bran by ultrasonic enzymolysis with 75% ethanol (comparative examples 6 to 10).
FIG. 8 is a graph showing the comparison of the extraction amount of wheat bran subjected to ultrasonic enzymolysis with 50% ethanol (comparative examples 11 to 15).
FIG. 9 is a comparison of the extraction yields of wheat bran by ultrasonic enzymolysis with 25% ethanol (comparative examples 16 to 20).
Detailed Description
The following examples used bran flour produced by milling wheat bran to 80 mesh and then dried in a constant temperature oven for 6 hours to constant weight.
The following examples used 20 ten thousand U/g protease, 30 ten thousand U/g alpha-amylase, 60 ten thousand U/g cellulase and 5 ten thousand U/g ligninase.
The following detection and analysis conditions for LC-MS of the examples were:
LC conditions: ACQMITY MPLC BEN C 18 Chromatography columns (2.1 mm. Times.100mm, 1.7. Mu.m); the column temperature is 30 ℃; the sample injection amount is 5 mu L; mobile phase: the phase A is ammonia water containing 0.1 percent (volume fraction), and the phase B is acetonitrile; the flow rate is 0.5mL/min; elution gradient: 0-2min, 100-15% of A and 0-85% of B; 2-4min, 15-10% of A, 85-90% of B; 4-8min, 10% -5% of A and 90% -95% of B; 8-9min, 5-15% of A and 95-85% of B.
MS conditions: electrospray ion source, negative ion source; a multi-reaction detection mode; capillary voltage 1.2kV; taper hole airflow: nitrogen at the flow rate of 150L/h; the ion source temperature is 150 ℃; the temperature of the desolvation gas is 200 ℃, and the flow rate is 1000L/h.
The silica gel column chromatography purification of the following examples was:
placing 100-200 meshes of silica gel powder in a 105 ℃ drying oven for high-temperature activation for 30min, then placing in a dryer for cooling to room temperature, weighing a sample with the same mass as the silica gel powder and the silica gel powder, pouring the sample and the silica gel powder into an evaporation vessel, uniformly stirring, placing in a 50 ℃ drying oven for overnight drying, taking out, and cooling to room temperature for later use;
selecting a 500 x 40mm normal-pressure glass chromatographic column, loading a sample by a dry method, pouring activated silica gel powder into the clean and dry chromatographic column, slightly shaking the chromatographic column until the surface of the filler is flat, wherein the height of the packed column is 18cm, fixing the chromatographic column on an iron support, slightly adding sample-mixing silica gel until the surface is flat, leaving a certain height of the upper end of the chromatographic column for eluent accumulation, and performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10).
Influence of ultrasonic power on ARs leaching amount:
(1) Separately, 1g of triticale bran powder (to the nearest 0.01 g) was weighed into 5 50mL centrifuge tubes.
(2) Respectively adding 20mL of absolute ethyl alcohol, then respectively carrying out ultrasonic extraction for 1.5min at room temperature with the power of 100w, 200w, 300w, 400w and 500w, centrifuging the extracting solution and taking the supernatant.
(3) The supernatant was rotary-dried by a rotary evaporator at 45 ℃ under a vacuum of 135Torr, and then redissolved with methanol.
(4) The ARs concentration of the re-dissolved sample solution was measured by LC-MS detection analysis after passing through 0.22. Mu.L of organic filter membrane, and the results are shown in FIG. 2, wherein the ARs obtained per gram of bran powder under different ultrasonic powers were 298.45. + -. 10.92. Mu.g/g, 307.31. + -. 8.37. Mu.g/g, 314.22. + -. 6.71. Mu.g/g, 312.87. + -. 5.64. Mu.g/g, 308.73. + -. 9.44. Mu.g/g, respectively.
Influence of ultrasonic leaching time on the amount of ARs leached:
(1) Separately, 1g of triticale bran powder (to the nearest 0.01 g) was weighed into 5 50mL centrifuge tubes.
(2) Respectively adding 20mL of absolute ethyl alcohol, then respectively carrying out ultrasonic extraction for 0.5, 1.0, 1.5, 2.0 and 2.5min at the power of 300w at room temperature, centrifuging the extracting solution and taking the supernatant.
(3) The supernatant was dried by spin drying with a rotary evaporator at 45 ℃ under a vacuum of 135Torr and then reconstituted with methanol.
(4) The ARs concentration of the re-dissolved sample solution was measured by LC-MS detection analysis after passing through 0.22. Mu.L of organic filter membrane, and the results are shown in FIG. 3, and the ARs obtained per gram of bran powder at different ultrasonic extraction times were 303.19. + -. 10.16. Mu.g/g, 312.73. + -. 8.64. Mu.g/g, 321.82. + -. 6.84. Mu.g/g, 316.47. + -. 5.77. Mu.g/g, 315.72. + -. 9.49. Mu.g/g, respectively.
Comparative example 1
(1) Weigh 1g of bran powder (accurate to 0.01 g) into a 50mL centrifuge tube.
(2) Adding 20mL of anhydrous ethanol, performing ultrasonic extraction (power of 200 w) at room temperature for 1.5min, centrifuging the extractive solution, and collecting supernatant.
(3) The supernatant was rotary-dried by a rotary evaporator at 45 ℃ under a vacuum of 135Torr to obtain a concentrated solution.
(4) The concentrate was purified by silica gel column chromatography, rotary evaporated per 100mL of eluent, followed by TLC, analyzed by HPLC and the eluent was collected.
(5) The eluent is re-dissolved in methanol after rotary evaporation and concentration.
(6) And (3) passing the redissolved sample solution through a 0.22 mu L organic filter membrane, detecting and analyzing by LC-MS to measure the ARs concentration, wherein the extraction amount is 320.41 mu g/g, and the ARs content obtained after purification is 132.3-135.8 mg/g. The extraction amount refers to the mass ratio of ARs in the concentrated solution obtained in the step (3) to the mass of the upper bran powder. The content of ARs obtained after purification refers to the mass ratio of ARs in the eluent of the step (4) to the mass of the concentrated solution of the step (3).
Comparative examples 2, 3, 4, 5
(1) Weigh 1g triticale (comparative example 2), wheat (comparative example 3), highland barley (comparative example 4), oat (comparative example 5) bran flour (to the nearest 0.01 g) into a 50mL centrifuge tube.
(2) Adding 20mL ethyl acetate, ultrasonic extracting at room temperature (power 300 w) for 1.5min, centrifuging the extractive solution, and collecting supernatant.
(3) The supernatant was subjected to spin-drying by a rotary evaporator at 45 ℃ under a vacuum of 175Torr to obtain a concentrated solution.
(4) The concentrate was purified by silica gel column chromatography, rotary evaporated per 100mL of eluent, followed by TLC, analyzed by HPLC and the eluent was collected.
(5) The eluent is re-dissolved in methanol after rotary evaporation and concentration.
(6) After passing the redissolved sample solution through 0.22 mu L of organic filter membrane, the ARs concentration is measured by LC-MS detection analysis, the extraction amount is 271.13 mu g/g (comparative example 2), 242.54 mu g/g (comparative example 3), 12.60 mu g/g (comparative example 4) and 8.39 mu g/g (comparative example 5), wherein the ARs content obtained after silica gel column chromatography purification is 137.3-143.6 mg/g (comparative example 2), 101.3-103.8 mg/g (comparative example 3) and 6.3-7.1 mg/g (comparative example 4). The extraction amount refers to the mass ratio of the ARs in the concentrated solution in the step (3) to the mass of the upper bran powder. The content of ARs obtained after purification refers to the mass ratio of ARs in the eluent of the step (4) to the mass of the concentrated solution of the step (3).
Example 1
(1) Weigh 1g triticale bran powder (accurate to 0.01 g) into 4 50mL centrifuge tubes.
(2) Adding a pre-prepared phosphate buffer solution to adjust the pH of the mixture to 5.0, adding 45mg protease (20 ten thousand U/g), performing enzymolysis at 50 deg.C for 2 hr, and boiling for 5min to inactivate the enzyme.
(3) Centrifuging at 4000rpm for 10min, extracting bran residue with ethanol (see step 4), precipitating supernatant with ethanol, filtering, collecting filtrate, and spin-drying the filtrate at 45 deg.C with rotary evaporator to obtain concentrated solution 1;
(4) Adding 20mL of absolute ethyl alcohol into the bran residue collected after centrifugation in the step (3), performing ultrasonic extraction (power is 300 w) at room temperature for 1.5min, centrifuging the extracting solution, and taking supernatant.
(5) And (4) rotating the supernatant obtained in the step (4) to be nearly dry through a rotary evaporator under the conditions of 45 ℃ and 175Torr of vacuum degree to obtain a concentrated solution 2.
(6) Mixing the concentrated solutions 1 and 2;
placing 100-200 meshes of silica gel powder in a 105 ℃ drying oven for high-temperature activation for 30min, then placing in a dryer for cooling to room temperature, weighing a concentrated solution sample with the same mass as the silica gel powder and the silica gel powder, pouring the concentrated solution sample and the silica gel powder into an evaporation vessel, uniformly stirring, placing in a 50 ℃ drying oven for overnight drying, taking out, and cooling to room temperature for later use;
selecting a 500 x 40mm normal-pressure glass chromatographic column, loading a sample by a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, slightly shaking the chromatographic column until the surface of a filler is flat, wherein the height of the packed column is 18cm, fixing the chromatographic column on an iron support, slightly adding sample-mixing silica gel until the surface is flat, leaving a certain height at the upper end of the chromatographic column for eluent accumulation, performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10), performing rotary evaporation on each 100mL of eluent, tracking by using TLC, analyzing by using HPLC, and collecting the eluent;
(7) Collecting eluent, carrying out rotary evaporation concentration, blowing and drying by blowing nitrogen, and re-dissolving by using 5mL chromatographic grade methanol.
(8) LC-MS detection analysis:
the ARs concentration is measured by LC-MS detection analysis after the redissolved sample liquid is mixed with 0.22 mu L of organic filter membrane, and the extraction amount and the ARs content obtained after purification are 362.68 mu g/g and 172.2-175.8 mg/g respectively. The extraction amount refers to the mass ratio of ARs in concentrated solution 1 and concentrated solution 2 to the mass of testa Tritici powder. The content of ARs obtained after purification is the sum of the mass of the concentrated solution 1 and the mass of the concentrated solution 2 in the mass ratio of the ARs in the eluent of the step (6). The meanings of "extraction amount" and "ARs content obtained after purification" in the subsequent examples are the same as in the present example.
Example 2
(1) Weigh 1g triticale bran powder (accurate to 0.01 g) into 4 50mL centrifuge tubes.
(2) Adding a pre-prepared phosphoric acid buffer solution to adjust the pH of the mixture to 5.0, adding 30mg of alpha-amylase, performing enzymolysis at 50 deg.C for 2h, and boiling for 5min to inactivate the enzyme.
(3) Examples (8) to (8) are the same as example 1.
The amount of extracted ARs and the amount of ARs obtained after purification were 383.42. Mu.g/g and 204.1-206.7 mg/g, respectively (5 replicates).
Example 3
(1) Weigh 1g of triticale bran powder (to the nearest 0.01 g) into 4 50mL centrifuge tubes.
(2) Adding pre-prepared phosphoric acid buffer solution to adjust pH of the mixture to 5.0, adding 15mg cellulase, performing enzymolysis at 50 deg.C for 2 hr, and boiling for 5min to inactivate enzyme.
(3) Examples (8) to (8) are the same as example 1.
The extraction amount of ARs and the content of ARs obtained after purification are 415.00 mu g/g and 234.1-236.7 mg/g respectively.
Example 4
(1) Weigh 1g of triticale bran powder (to the nearest 0.01 g) into 4 50mL centrifuge tubes.
(2) Adding pre-prepared phosphoric acid buffer solution to adjust pH of the mixture to 5.0, adding 1.8g ligninase, performing enzymolysis at 50 deg.C for 2 hr, and boiling for 5min to inactivate enzyme.
(3) Examples (8) to (8) are the same as example 1.
The extraction amount of ARs and the content of ARs obtained after purification are 333.85 mu g/g and 146.6-147.4 mg/g respectively.
TABLE 1 ARs content of enzymatically hydrolyzed wheat bran when the extraction solvent is 100% ethanol
Comparative example 6
(1) Weigh 1g triticale bran powder (accurate to 0.01 g) into a 50mL centrifuge tube.
(2) Adding 20mL of 75% ethanol, performing ultrasonic extraction at room temperature with the ultrasonic extraction power of 300 w), extracting for 1.5min, centrifuging the extracting solution, and taking supernatant.
(3) And (4) carrying out rotary drying on the supernatant through a rotary evaporator at the temperature of 45 ℃ to obtain a concentrated solution.
(4) The concentrate was purified by column chromatography on silica gel, rotary evaporated per 100mL of eluent, followed by TLC, analyzed by HPLC and the eluent was collected.
(5) The eluent is re-dissolved in methanol after rotary evaporation and concentration.
(6) And (3) passing the redissolved sample solution through a 0.22 mu L organic filter membrane, and then detecting and analyzing by LC-MS to measure the ARs concentration, wherein the extraction amount is 240.48 mu g/g, and the ARs content obtained after purification is 123.4-129.3 mg/g. The extraction amount is the mass ratio of the ARs in the concentrated solution obtained in the step (3) to the mass of the bran powder. The content of ARs obtained after purification refers to the mass ratio of ARs in the eluate obtained in step (4) to the mass of the concentrate.
Comparative examples 7, 8, 9, 10
(1) Accurately weigh 1g of triticale bran powder (to the nearest 0.01 g) into 4 50mL centrifuge tubes.
(2) The pH of the mixture was adjusted to 5.0 by adding a phosphoric acid buffer solution prepared in advance, and 45mg of protease (comparative example 4), 30mg of alpha-amylase (comparative example 5), 15mg of cellulase (comparative example 6), and 1.8g of ligninase (comparative example 7) were added, respectively, and enzymatic hydrolysis was performed at 50 ℃ for 2 hours, followed by boiling for 5min to inactivate the enzymes.
(3) Centrifuging at 4000rpm for 10min, extracting bran residue with ethanol (step 4), precipitating supernatant with ethanol, filtering, and spin-drying the filtrate at 45 deg.C with rotary evaporator to obtain concentrated solution 1;
(4) Adding 20mL of 75% ethanol into the bran residues collected after centrifugation in the step (3), performing ultrasonic extraction (with the power of 300 w) at room temperature for 1.5min, centrifuging the extracting solution, and taking supernate.
(5) And (5) rotating the supernatant obtained in the step (4) to be nearly dry through a rotary evaporator at the temperature of 45 ℃ to obtain a concentrated solution 2. Mixing the concentrated solutions, purifying by silica gel column chromatography, concentrating by rotary evaporation, and dissolving with methanol.
(6) The ARs concentration was measured by LC-MS detection analysis after mixing the reconstituted sample solution with 0.22. Mu.L of organic filter membrane, and the extraction amounts were 274.95. Mu.g/g (comparative example 7), 292.25. Mu.g/g (comparative example 8), 312.56. Mu.g/g (comparative example 9), and 251.25. Mu.g/g (comparative example 10), respectively; the ARs contents obtained after purification were 127.6 to 133.1mg/g (comparative example 7), 137.9 to 142.4mg/g (comparative example 8), 144.7 to 146.9mg/g (comparative example 9), and 122.1 to 126.3mg/g (comparative example 10), respectively. The extraction amount is the mass ratio of the sum of the ARs in the concentrated solution 1 and the concentrated solution 2 to the mass of the epithelial powder. The ARs content obtained after purification refers to the mass ratio of ARs in the eluent purified by silica gel column chromatography to the mass of the concentrated solution.
TABLE 2 ARs content of enzymatically hydrolyzed wheat bran with 75% ethanol as extraction solvent
Comparative example 11
(1) Accurately weigh 1g of triticale bran powder (to the nearest 0.01 g) into a 50mL centrifuge tube.
(2) Adding 20mL 50% ethanol, performing ultrasonic extraction at room temperature (power is 300 w) for 1.5min, centrifuging the extractive solution, and collecting supernatant.
(3) And (4) carrying out rotary drying on the supernatant through a rotary evaporator at the temperature of 45 ℃ to obtain a concentrated solution.
(4) The concentrate was purified by silica gel column chromatography, rotary evaporated per 100mL of eluent, followed by TLC, analyzed by HPLC and the eluent was collected.
(5) The eluent is re-dissolved in methanol after rotary evaporation and concentration.
(6) And (3) passing the redissolved sample solution through a 0.22 mu L organic filter membrane, and then detecting and analyzing by LC-MS to measure the ARs concentration, wherein the extraction amount is 140.75 mu g/g, and the ARs content obtained after purification is 64.7-67.3 mg/g. The extraction amount is the mass ratio of the ARs in the concentrated solution obtained in the step (3) to the mass of the bran powder. The content of ARs obtained after purification refers to the mass ratio of ARs in the eluate obtained in step (4) to the mass of the concentrate.
Comparative examples 12, 13, 14, 15
(1) Weigh 1g of triticale bran powder (to the nearest 0.01 g) into 4 50mL centrifuge tubes.
(2) The pH of the mixture was adjusted to 5.0 by adding a phosphoric acid buffer solution prepared in advance, and 45mg of protease (comparative example 12), 30mg of alpha-amylase (comparative example 13), 15mg of cellulase (comparative example 14), and 1.8g of ligninase (comparative example 15) were added, respectively, and enzymatic hydrolysis was performed at 50 ℃ for 2 hours, followed by boiling for 5min to inactivate the enzymes.
(3) Centrifuging at 4000rpm for 10min, extracting bran residue with ethanol (step 4), precipitating supernatant with ethanol, filtering, and spin-drying the filtrate at 45 deg.C with rotary evaporator to obtain concentrated solution 1;
(4) Adding 20mL of 50% ethanol into the bran residues collected after centrifugation in the step (3), performing ultrasonic extraction at room temperature (the power is 300 w), wherein the extraction time is 1.5min, centrifuging the extracting solution, and taking supernate.
(5) And (4) rotating the supernatant obtained in the step (4) to be nearly dry through a rotary evaporator at the temperature of 45 ℃ to obtain a concentrated solution 2.
(6) Mixing the concentrated solutions 1 and 2;
placing 100-200 meshes of silica gel powder in a drying oven at 105 ℃ for high-temperature activation for 30min, then placing the silica gel powder in a dryer for cooling to room temperature, weighing a concentrated solution sample with the same mass as the silica gel powder and the silica gel powder, pouring the concentrated solution sample and the silica gel powder into an evaporation dish, uniformly stirring, placing the evaporation dish in the drying oven at 50 ℃ for overnight drying, taking out, and cooling to room temperature for later use;
selecting a 500 x 40mm normal-pressure glass chromatographic column, loading a sample by a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, slightly shaking the chromatographic column until the surface of a filler is flat, wherein the height of the packed column is 18cm, fixing the chromatographic column on an iron support, slightly adding sample-mixing silica gel until the surface is flat, leaving a certain height at the upper end of the chromatographic column for eluent accumulation, performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10), performing rotary evaporation on each 100mL of eluent, tracking by using TLC, analyzing by using HPLC, and collecting the eluent;
(7) Collecting the eluent, carrying out rotary evaporation concentration, blowing the eluent to dry by nitrogen, and redissolving by 5mL of chromatographic grade methanol.
(8) The above-mentioned reconstituted samples were mixed with 0.22. Mu.L of an organic filter and then subjected to LC-MS detection analysis to measure ARs concentrations, which were 162.75. Mu.g/g (comparative example 12), 189.48. Mu.g/g (comparative example 13), 217.56. Mu.g/g (comparative example 14), and 155.77. Mu.g/g (comparative example 15), respectively; the ARs contents obtained after purification were 73.3 to 78.1mg/g (comparative example 12), 87.9 to 94.4mg/g (comparative example 13), 104.1 to 105.6mg/g (comparative example 14), and 72.4 to 75.3mg/g (comparative example 15), respectively. The extraction amount is the mass ratio of the sum of the ARs in the concentrated solution 1 and the concentrated solution 2 to the mass of the epithelial powder. The ARs content obtained after purification refers to the mass ratio of ARs in the eluent purified by silica gel column chromatography to the mass of the concentrated solution.
TABLE 3 ARs content of enzymatically hydrolyzed wheat bran with 50% ethanol as extraction solvent
Comparative example 16
(1) Accurately weigh 1g of triticale bran powder (to the nearest 0.01 g) into a 50mL centrifuge tube.
(2) Adding 20mL of 25% ethanol, performing ultrasonic extraction at room temperature (power is 300 w) for 1.5min, centrifuging the extracting solution, and taking supernatant.
(3) And (4) carrying out rotary drying on the supernatant by a rotary evaporator at the temperature of 45 ℃ to obtain a concentrated solution. Purifying the concentrated solution by silica gel column chromatography, concentrating by rotary evaporation, and re-dissolving with methanol.
(4) And (3) passing the redissolved sample solution through a 0.22 mu L organic filter membrane, and then detecting and analyzing by LC-MS to measure the ARs concentration, wherein the extraction amount is 61.11 mu g/g, and the ARs content obtained after purification is 27.3-28.2 mg/g. The extraction amount is the mass ratio of the ARs in the concentrated solution obtained in the step (3) to the mass of the bran powder. The content of ARs obtained after purification refers to the mass ratio of ARs in the eluent purified by silica gel column chromatography to the mass of the concentrated solution.
Comparative examples 17, 18, 19, 20
(1) Weigh 1g of triticale bran powder (to the nearest 0.01 g) into 4 50mL centrifuge tubes.
(2) The pH of the mixture was adjusted to 5.0 by adding a phosphoric acid buffer solution prepared in advance, and 45mg of protease (comparative example 17), 30mg of alpha-amylase (comparative example 18), 15mg of cellulase (comparative example 19), and 1.8g of ligninase (comparative example 20) were added, respectively, and enzymatic hydrolysis was performed at 50 ℃ for 2 hours, followed by boiling for 5min to inactivate the enzymes.
(3) Centrifuging at 4000rpm for 10min, extracting bran residue with ethanol (step 4), precipitating supernatant with ethanol, filtering, and spin-drying the filtrate at 45 deg.C with rotary evaporator to obtain concentrated solution 1;
(4) Adding 20mL of 25% ethanol into the bran residue collected after centrifugation in the step (3), performing ultrasonic leaching (with the power of 300 w) at room temperature for 1.5min, centrifuging the extracting solution, and taking a supernatant.
(5) And (4) rotating the supernatant obtained in the step (4) to be nearly dry through a rotary evaporator at the temperature of 45 ℃ to obtain a concentrated solution 2.
(6) Mixing the concentrated solutions 1 and 2;
placing 100-200 meshes of silica gel powder in a drying oven at 105 ℃ for high-temperature activation for 30min, then placing the silica gel powder in a dryer for cooling to room temperature, weighing a concentrated solution sample with the same mass as the silica gel powder and the silica gel powder, pouring the concentrated solution sample and the silica gel powder into an evaporation dish, uniformly stirring, placing the evaporation dish in the drying oven at 50 ℃ for overnight drying, taking out, and cooling to room temperature for later use;
selecting a 500 × 40mm normal-pressure glass chromatographic column, loading the sample by a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, slightly shaking the chromatographic column until the surface of a filler is smooth, wherein the height of the packed column is 18cm, fixing the chromatographic column on an iron support, slightly adding sample-mixing silica gel until the surface is smooth, leaving a certain height above the chromatographic column for eluent to accumulate, performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10), performing rotary evaporation on each 100mL of eluent, tracking by using TLC, analyzing by using HPLC, and collecting the eluent;
(7) Collecting the eluent, carrying out rotary evaporation concentration, blowing the eluent to dry by nitrogen, and redissolving by 5mL of chromatographic grade methanol.
(8) The above-mentioned redissolved sample solutions were mixed with 0.22. Mu.L of an organic filter and analyzed by LC-MS detection to measure ARs concentrations, and the amounts of extraction were 72.84. Mu.g/g (comparative example 17), 75.34. Mu.g/g (comparative example 18), 89.31. Mu.g/g (comparative example 19), and 57.22. Mu.g/g (comparative example 20), respectively; the ARs contents obtained after purification were 36.7 to 38.2mg/g (comparative example 17), 37.9 to 39.4mg/g (comparative example 18), 43.9 to 46.2mg/g (comparative example 19), and 21.3 to 22.2mg/g (comparative example 20), respectively. The extraction amount refers to the mass ratio of ARs in concentrated solution 1 and concentrated solution 2 to the mass of testa Tritici powder. The ARs content obtained after purification refers to the mass ratio of ARs in the eluent purified by silica gel column chromatography to the mass of the concentrated solution.
TABLE 4 ARs content of enzymatically hydrolyzed wheat bran with 25% ethanol as extraction solvent
And (4) conclusion: the results show that the enzymolysis of the wheat bran by using protease, alpha-amylase, cellulase and ligninase and the ultrasound-assisted two-step extraction can obviously improve the extraction amount of ARs to a certain extent, and from the concentration of an extraction solvent, the absolute ethyl alcohol is more than 75 percent and the 50 percent and the 25 percent are more than 25 percent; from the enzyme effect, cellulase > alpha-amylase > protease > ligninase. The invention reduces the extraction cost and obviously improves the yield while avoiding toxic components brought by organic solvent.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The method for extracting and detecting the alkyl resorcinol in the wheat bran is characterized by mainly comprising the following steps:
(1) Pretreatment: firstly, crushing wheat bran;
(2) Enzymolysis: weighing the crushed bran, adding a phosphoric acid buffer solution, and adding at least one of alpha-amylase, protease, cellulase and ligninase for enzymolysis;
(3) Centrifuging: after enzymolysis is finished, centrifuging the enzymolysis liquid, extracting bran residues collected after centrifugation by using ethanol, precipitating supernate collected after centrifugation by using ethanol, filtering and collecting filtrate after ethanol precipitation, and spin-drying the filtrate by using a rotary evaporator to obtain concentrated solution 1 for later use;
(4) Ethanol leaching: adding ethanol into the bran residues collected after centrifugation in the step (3), performing ultrasonic extraction at room temperature, centrifuging the extracting solution, and taking supernatant;
(5) And (3) spin-drying: spin-drying the supernatant obtained in the step (4) by a rotary evaporator to obtain a concentrated solution 2;
(6) Silica gel column chromatography purification: mixing the concentrated solution 1 and the concentrated solution 2, purifying by silica gel column chromatography, and collecting eluate;
(7) And (3) re-concentration: carrying out rotary evaporation and concentration on the eluent collected in the step (6), blowing and drying by blowing nitrogen, and then re-dissolving by using chromatographic grade methanol;
(8) LC-MS detection analysis: the reconstituted sample was filtered and analyzed by LC-MS.
2. The method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein in the step (1), the wheat bran is crushed and sieved by a 80-mesh sieve for standby.
3. The method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein the step (2) comprises enzymolysis: weighing 1g of the bran pretreated in the step (1) in a 50mL centrifuge tube, adding a phosphoric acid buffer solution to stabilize the pH value at 5.0, adding at least one of alpha-amylase, protease, cellulase and ligninase, and performing enzymolysis for 2 hours at 50 ℃.
4. The method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein the step (4) of ethanol extraction: and (4) adding ethanol into the bran residues collected after centrifugation in the step (3), performing ultrasonic extraction at room temperature, wherein the ultrasonic time is 1.5min, the ultrasonic power is 300W, centrifuging the extracting solution, and taking supernate.
5. The method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein in step (6), silica gel powder of 100-200 meshes is firstly placed in an oven at 105 ℃ for activation for 30min, then placed in a dryer for cooling to room temperature, a concentrated solution sample with the same mass as the silica gel powder is weighed, poured into an evaporation dish together with the silica gel powder and stirred uniformly, placed in an oven at 50 ℃ for overnight drying, taken out, and cooled to room temperature for later use;
selecting a 500 × 40mm normal-pressure glass chromatographic column, adopting a dry method for sample loading, and selecting a material with a volume ratio of 10:1 petroleum ether-ethyl acetate, rotary evaporation of each 100mL of eluate, follow-up using TLC, analysis using HPLC and collection of the eluate.
6. The method for extracting and detecting alkylresorcinol in wheat bran according to claim 1Characterized in that the addition amount of the cellulase in the step (2) is 15mg/g Wheat bran powder The activity of the cellulase is 60 ten thousand U/g.
7. A method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein the HPLC conditions in the step (6) are as follows: (1) mobile phase: phase A0.1% aqueous formic acid; phase B0.1% methanoic acid in methanol, flow rate: 1mL/min, column temperature: 30 ℃, sample introduction: 10 mu L of the solution; (2) the mobile phase elution procedure was: 0min, 15-25% of phase A and 75-85% of phase B; 5min, 15-10% of phase A and 75-90% of phase B; 10min, 5-10% of phase A and 90-95% of phase B; 15min, 0-5% of phase A and 95-100% of phase B; 35min, 15-25% of phase A and 75-85% of phase B; (3) a detector: the detection wavelength was 280nm using a DAD detector.
8. A method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein the detection and analysis conditions of LC-MS in the step (8) are as follows:
LC conditions: ACQMITY MPLC BEN C 18 Chromatography columns (2.1 mm. Times.100mm, 1.7. Mu.m); the column temperature is 30 ℃; the sample size is 5 mu L; mobile phase: the phase A is 0.1% ammonia water, and the phase B is acetonitrile; the flow rate is 0.5mL/min; elution gradient: 0 to 2min,100 to 15% by weight A,0 to 85% by weight B; 2-4 min, 15-10% by weight, A, 85-90% by weight, B; 4-8min, 10% -5%, A,90% -95%, B; 8-9min, 5-15% of A, 95-85% of B;
MS conditions: electrospray ion source, negative ion source; a multi-reaction detection mode; the capillary voltage is 1.2kV; taper hole airflow: nitrogen at the flow rate of 150L/h; the ion source temperature is 150 ℃; the temperature of the desolvation gas is 200 ℃, and the flow rate is 1000L/h.
9. The method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein the amount of the alpha-amylase added in the step (2) is 30mg/g Wheat bran powder And the activity of the alpha-amylase is 30 ten thousand U/g.
10. Root of herbaceous plantThe method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein the protease is added in an amount of 45mg/g in the step (2) Wheat bran powder The activity of the protease is 20 ten thousand U/g.
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