CN115557830B - Method for extracting alkyl resorcinol from wheat bran based on ultrasonic-assisted enzymolysis - Google Patents
Method for extracting alkyl resorcinol from wheat bran based on ultrasonic-assisted enzymolysis Download PDFInfo
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- 229930188104 Alkylresorcinol Natural products 0.000 title claims abstract description 106
- 235000015099 wheat brans Nutrition 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 124
- 239000000843 powder Substances 0.000 claims abstract description 60
- 102000004190 Enzymes Human genes 0.000 claims abstract description 23
- 108090000790 Enzymes Proteins 0.000 claims abstract description 23
- 238000001556 precipitation Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 56
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 239000003480 eluent Substances 0.000 claims description 42
- 235000019441 ethanol Nutrition 0.000 claims description 40
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 39
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- 229910002027 silica gel Inorganic materials 0.000 claims description 34
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
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- PUNOCEUUYUXUGR-UHFFFAOYSA-N 5-Nonadecyl-1,3-benzenediol Chemical compound CCCCCCCCCCCCCCCCCCCC1=CC(O)=CC(O)=C1 PUNOCEUUYUXUGR-UHFFFAOYSA-N 0.000 description 2
- BBGNINPPDHJETF-UHFFFAOYSA-N 5-heptadecylresorcinol Chemical compound CCCCCCCCCCCCCCCCCC1=CC(O)=CC(O)=C1 BBGNINPPDHJETF-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
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- OHTBGMREZYLZQD-UHFFFAOYSA-N 5-tricosylresorcinol Chemical compound CCCCCCCCCCCCCCCCCCCCCCCC1=CC(O)=CC(O)=C1 OHTBGMREZYLZQD-UHFFFAOYSA-N 0.000 description 1
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
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Classifications
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for extracting alkyl resorcinol from wheat bran based on an ultrasonic-assisted enzymolysis method, and belongs to the field of natural product or raw material processing. The wheat bran powder is treated by enzyme, so that ARs are promoted to be released, phenolic acid compounds and other components are also promoted to be dissolved out, and the components are removed by subsequent alcohol precipitation, concentration and purification treatment, so that the interference of impurities in materials on alkyl resorcinol is reduced. The invention uses ultrasonic to assist ethanol extraction, the extract is clear, the viscosity is low, the impurity of the product is less, 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 biomarker in the grains is a special substance in the bran or embryo of the grains, can better reflect the content of the bran or embryo, and can be used for monitoring the production processes of dehulling, debranning and the like and identifying whether the whole grain product is adulterated or not. Wheat is one of the most consumed grains of global staple food, and is the most abundant grain source in the existing whole grain processed foods, so that the research on the whole grain markers is important, and relatively more grains are researched in the whole grain markers. Recent studies have found that a specific class of phenolic lipids, alkyl Resorcinol (ARs), exists in wheat grains and can be used as a marker for whole wheat foods. The alkyl side chains of ARs in cereals are typically saturated carbon chains containing from 17 to 25 odd numbered carbon atoms and can be expressed as 5-heptadecylresorcinol (ARC 17: 0), 5-nonadecylresorcinol (ARC 19: 0), 5-heneicosyl resorcinol (ARC 21: 0), 5-tricosyl resorcinol (ARC 23: 0), 5-pentacosyl resorcinol (ARC 25: 0), respectively. ARs have polar ends and hydrophobic alkyl side chains, are sparingly soluble in water, and are mainly soluble in organic solvents such as methanol (limited solubility when R > 19), ethanol, acetone, ethyl acetate, hexane, propanol, dichloromethane, and chloroform. ARs are themselves fluorescent.
ARs have amphipathy, and the amphiphilicity of the ARs endows the ARs with certain functionality, such as antioxidation, anti-tumor and the like, and have better improvement effect on various chronic diseases. Therefore, products with high amounts of ARs are increasingly gaining attention. ARs is specifically found in wheat, wheat-like bran, whereas embryo and endosperm are absent. After the bran-free dietary supplement is ingested by a human body, homologs or metabolites of the bran-free dietary supplement can be detected in biological samples such as blood plasma, and the homologs or metabolites of the bran-free dietary supplement cannot be detected in the biological samples such as blood plasma. And thus can be used as a biomarker for ingestion of whole wheat products.
Currently, the measurement methods of ARs mainly include spectrophotometry, liquid-phase fluorescence, gas chromatography, and the like. The detection method of ARs content in whole wheat flour in LS/T3244-2015 whole wheat flour adopts oscillation extraction method to extract ARs, spectrophotometry to detect. The oscillation extraction method in the line standard has the advantages of simple operation, low equipment requirement and the like, but has the defects of long extraction time (48 h), long pretreatment concentration time (4-6 h), low efficiency, high equipment loss caused by long-time operation and the like. At present, the grain industry standard generally selects a plurality of organic solvents such as n-propanol, ethyl acetate, methanol, acetone and the like for extracting ARs in grains, 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 easily extracted, more impurities are generated, and the subsequent experiments are not facilitated. Whereas the recovery of n-propanol to ARs was below 80%. ARs are used as amphiphilic molecules, the polarity of the ARs is similar to that of ethyl acetate, the total extract in the extract liquid is less, the recovery rate to be detected 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 sharp, and the ARs are harmful to human bodies. Therefore, it is very significant to develop a low-cost and high-yield method for extracting alkyl resorcinol from cereal bran.
Disclosure of Invention
[ technical problem ]
The invention aims to solve the technical problems of time consumption, low recovery rate and low purity of the existing method for extracting Alkyl Resorcinol (ARs).
Technical scheme
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: crushing wheat bran, and sieving with a 80-mesh sieve for standby;
(2) Enzymolysis: weighing 1g (accurate to 0.01 g) of the bran pretreated in the step (1) into a 50mL centrifuge tube, adding a phosphate buffer solution prepared in advance to ensure that the pH is stabilized at 5.0, respectively adding at least one of alpha-amylase, protease, cellulase and lignin enzyme, carrying out enzymolysis for 2 hours at 50 ℃, and boiling for 5 minutes to inactivate the enzyme;
(3) And (3) centrifuging: centrifuging at 4000rpm for 10min, leaching the bran residue collected after centrifugation with ethanol, precipitating the supernatant collected after centrifugation with ethanol, filtering after ethanol precipitation, collecting filtrate, and spin-drying the filtrate with a rotary evaporator at 45deg.C to obtain concentrated solution 1;
(4) Ethanol leaching: adding ethanol into the bran residue collected after the centrifugation in the step (3), carrying out ultrasonic leaching at room temperature for 1.5min with ultrasonic power of 300W, centrifuging the extract, and taking supernatant;
(5) Spin drying: spin-drying the supernatant obtained in the step (4) by a rotary evaporator to obtain a concentrated solution 2;
(6) Purifying by silica gel column chromatography: 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 into a 105 ℃ oven for high-temperature activation for 30min, then placing the silica gel powder into 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 mixture into a 50 ℃ oven for overnight drying, taking out, and cooling to the room temperature for standby;
selecting a 500 x 40mm normal pressure glass chromatographic column, sampling by adopting a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, gently shaking the chromatographic column until the surface of a filler is flat, loading the column to be 18cm in height, fixing the chromatographic column on a iron stand, lightly adding sample-mixed silica gel until the surface is flat, leaving the upper end of the chromatographic column at a certain height so as to accumulate eluent, performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10:1), performing rotary steaming every 100mL eluent, tracking by using TLC, analyzing by using HPLC and collecting the eluent;
(7) And (5) re-concentrating: collecting eluent, concentrating by rotary evaporation, blowing with nitrogen, and redissolving with 5mL chromatographic grade methanol;
(8) LC-MS detection analysis: after passing the reconstituted sample through 0.22. Mu.L of organic filter, 200. Mu.L was pipetted into a sample bottle for LC-MS analysis.
In the above method, preferably, in the step (2), the protease is 20. Mu.L/g, the α -amylase is 30. Mu.L/g, the cellulase is 60. Mu.L/g, the lignin enzyme is 5. Mu.L/g, the amount of protease added is 45mg, the amount of α -amylase added is 30mg, the amount of cellulase added is 15mg, and the amount of lignin enzyme added is 1.8g.
The method as described above, preferably, the specific operation of step (4) is: and (3) adding 20mL of ethanol into the bran residue collected after enzymolysis and centrifugation in the step (3), and carrying out ultrasonic leaching at room temperature for 1.5min, wherein the ultrasonic power is 300w.
The method as described above, preferably, the conditions of HPLC in step (6) are: (1) mobile phase: phase a 0.1% aqueous formic acid; phase B0.1% methanolic formate solution, flow rate: 1mL/min, column temperature: 30 ℃, sample injection amount: 10. Mu.L; (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.
The method as described above, preferably, the detection and analysis conditions of LC-MS in step (8) are:
LC conditions: ACQMITY MPLC BEN C 18 Chromatographic column (2.1 mm. Times.100 mm,1.7 μm); column temperature is 30 ℃; the sample injection amount is 5 mu L; mobile phase: phase A is ammonia water containing 0.1% (volume fraction), and phase B is acetonitrile; the flow rate is 0.5mL/min; elution gradient: 0-2 min, 100-15% of A and 0-85% of B; 2-4 min, 15-10% of A and 85-90% of B; 4-8 min, 10-5% of A and 90-95% of B; 8-9 min, 5-15% of A and 95-85% of B.
MS conditions: an electrospray ion source, a negative ion source; a multiple reaction detection mode; capillary voltage 1.2kV; taper hole airflow: nitrogen gas with a flow rate of 150L/h; the temperature of the ion source is 150 ℃; the desolvation gas temperature 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, small in harm to human bodies, and the extracted ARs products are safe and pollution-free.
2. The wheat bran powder is treated by enzyme, cellulose, starch, some proteins, lignin and other components in plant cells are degraded by the enzyme, so that the plant cell walls in the wheat bran raw material are damaged, the biological catalysis effect is exerted, the release of ARs is promoted, the dissolution of phenolic acid compounds, tannic acid, flavonoid compounds, saccharide compounds and other components is also promoted, water-soluble polysaccharide and other substances and a buffer solution form a mixed solution, and the mixed solution is removed by subsequent alcohol precipitation, concentration and purification treatment, so that the interference of impurities in the materials on alkyl resorcinol is reduced.
3. According to the invention, the residue collected after the bran enzymolysis liquid is centrifuged is extracted by utilizing ultrasonic assisted ethanol, the efficiency of ethanol extraction can be improved by the action of ultrasonic, more Ars are dissolved out, and the extraction cost is reduced while the toxicity caused by an organic solvent is avoided.
4. The invention uses ethanol to deposit the supernatant collected after the bran enzymolysis liquid is centrifugated, so that the impurities such as sugar substances and the like which are extracted and hydrolyzed can be removed by ethanol deposition, thereby achieving the aim of certain purification.
5. The invention uses ethanol for extraction, the extract is clear, the viscosity is low, the impurity content of the product is less, and the subsequent treatment is convenient.
Drawings
FIG. 1 is an LC-MS spectrum of ARC 17:0.
FIG. 2 effect of ultrasonic power on ARs leaching.
FIG. 3 effect of ultrasonic leaching time on ARs leaching amount.
FIG. 4 is a graph comparing ARs extraction in different bran (comparative examples 2-4).
FIG. 5 is a graph showing the comparison of ethyl acetate extraction and absolute ethanol extraction of wheat bran (comparative examples 1 to 2).
FIG. 6 is a graph showing the comparison of the extraction amount of wheat bran by ultrasonic enzymolysis with 100% ethanol (comparative example 1 and examples 1 to 4).
FIG. 7 is a graph showing comparison of the extraction amount of wheat bran subjected to ultrasonic enzymolysis with 75% ethanol (comparative examples 6 to 10).
FIG. 8 is a graph showing comparison of the extraction amount of wheat bran subjected to 50% ethanol ultrasonic enzymolysis (comparative examples 11 to 15).
FIG. 9 is a graph showing comparison of the extraction amount of wheat bran by ultrasonic enzymolysis with 25% ethanol (comparative examples 16 to 20).
Detailed Description
The wheat bran powder used in the following examples was milled to 80 mesh and then dried in a constant temperature oven for 6 hours to constant weight.
The proteases used in the examples below were 20 ten thousand U/g, alpha-amylase was 30 ten thousand U/g, cellulase was 60 ten thousand U/g and ligninase was 5 ten thousand U/g.
The detection and analysis conditions for the LC-MS of the following examples were:
LC conditions: ACQMITY MPLC BEN C 18 Chromatographic column (2.1 mm. Times.100 mm,1.7 μm); column temperature is 30 ℃; the sample injection amount is 5 mu L; mobile phase: phase A is ammonia water containing 0.1% (volume fraction), and phase B is acetonitrile; the flow rate is 0.5mL/min; elution gradient: 0-2 min, 100-15% of A and 0-85% of B; 2-4 min, 15-10% of A and 85-90% of B; 4-8 min, 10-5% of A and 90-95% of B; 8-9 min, 5-15% of A and 95-85% of B.
MS conditions: an electrospray ion source, a negative ion source; a multiple reaction detection mode; capillary voltage 1.2kV; taper hole airflow: nitrogen gas with a flow rate of 150L/h; the temperature of the ion source is 150 ℃; the desolvation gas temperature is 200 ℃ and the flow rate is 1000L/h.
The silica gel column chromatographic purification of the following examples is:
placing 100-200 mesh silica gel powder in a 105 ℃ 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 dish, uniformly stirring, placing in a 50 ℃ oven for overnight drying, taking out, and cooling to room temperature for standby;
selecting a 500 x 40mm normal pressure glass chromatographic column, sampling by adopting a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, gently shaking the chromatographic column until the surface of a filler is flat, fixing the chromatographic column on a frame table, lightly adding sample-stirring silica gel until the surface is flat, leaving a certain height at the upper end of the chromatographic column so as to accumulate eluent, and performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10:1).
Effect of ultrasonic power on ARs leaching amount:
(1) 1g of triticale bran powder (accurate to 0.01 g) was weighed separately into 5 50mL centrifuge tubes.
(2) 20mL of absolute ethyl alcohol is respectively added, then ultrasonic extraction is carried out for 1.5min at room temperature under the power of 100w, 200w, 300w, 400w and 500w respectively, and the extracting solution is centrifuged, and then the supernatant is taken.
(3) The supernatant was dried by spin-drying using a rotary evaporator at 45℃under a vacuum of 135Torr, and redissolved in methanol.
(4) After passing through 0.22 mu L of organic filter membrane, the re-dissolved sample liquid is subjected to LC-MS detection analysis to measure ARs concentration, and the result is shown in figure 2, wherein ARs obtained per gram of bran powder under different ultrasonic powers are 298.45+/-10.92 mug/g, 307.31 +/-8.37 mug/g, 314.22 +/-6.71 mug/g, 312.87 +/-5.64 mug/g and 308.73 +/-9.44 mug/g respectively.
Effect of ultrasonic leaching time on ARs leaching amount:
(1) 1g of triticale bran powder (accurate to 0.01 g) was weighed separately into 5 50mL centrifuge tubes.
(2) Respectively adding 20mL of absolute ethyl alcohol, respectively performing ultrasonic extraction for 0.5, 1.0, 1.5, 2.0 and 2.5min at room temperature and 300w of power, centrifuging the extract, and collecting supernatant.
(3) The supernatant was dried by spin-drying using a rotary evaporator at 45℃under a vacuum of 135Torr, and redissolved in methanol.
(4) The re-dissolved sample solution is subjected to an organic filter membrane of 0.22 mu L and then is subjected to LC-MS detection analysis to measure ARs concentration, and the result is shown in figure 3, wherein ARs obtained per gram of bran powder under different ultrasonic leaching time are 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 and 315.72 +/-9.49 mu g/g respectively.
Comparative example 1
(1) 1g of bran powder (accurate to 0.01 g) was weighed into a 50mL centrifuge tube.
(2) Adding 20mL of absolute ethanol, performing ultrasonic extraction (power is 200 w) at room temperature for 1.5min, centrifuging the extractive solution, and collecting supernatant.
(3) Spin-drying the supernatant at 45deg.C under 135Torr to obtain concentrated solution.
(4) The concentrate was purified by column chromatography on silica gel, spin distilled per 100mL of eluent, followed by TLC, analyzed by HPLC and the eluent was collected.
(5) The eluent is concentrated after rotary evaporation and redissolved by methanol.
(6) The re-dissolved sample solution is filtered by an organic filter membrane with the volume of 0.22 mu L, and then is subjected to LC-MS detection analysis to measure the ARs concentration, wherein the extraction quantity 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 of ARs in the concentrated solution in the step (3) compared with the mass of bran powder. The ARs content obtained after purification refers to the mass ratio of ARs in the eluent in step (4) to the mass of the concentrate in step (3).
Comparative examples 2, 3, 4, 5
(1) 1g of triticale (comparative example 2), wheat (comparative example 3), highland barley (comparative example 4), oat (comparative example 5) bran powder (accurate to 0.01 g) were weighed into a 50mL centrifuge tube.
(2) Ultrasonic extraction (power 300 w) was performed for 1.5min at room temperature with 20mL ethyl acetate, and the supernatant was obtained after centrifugation of the extract.
(3) Spin-drying the supernatant at 45deg.C under 175Torr to obtain concentrated solution.
(4) The concentrate was purified by column chromatography on silica gel, spin distilled per 100mL of eluent, followed by TLC, analyzed by HPLC and the eluent was collected.
(5) The eluent is concentrated after rotary evaporation and redissolved by methanol.
(6) The re-dissolved sample solution was passed through an organic filter membrane of 0.22. Mu.L, and then analyzed by LC-MS to measure ARs concentration, wherein the extraction amounts were 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), respectively, and the ARs contents obtained after purification by silica gel column chromatography were 137.3 to 143.6mg/g (comparative example 2), 101.3 to 103.8mg/g (comparative example 3) and 6.3 to 7.1mg/g (comparative example 4), respectively. The extraction amount refers to the mass of ARs in the concentrated solution in the step (3) compared with the mass of bran powder. The ARs content obtained after purification refers to the mass ratio of ARs in the eluent in step (4) to the mass of the concentrate in step (3).
Example 1
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed into 4 50mL centrifuge tubes.
(2) The pH of the mixture was adjusted to 5.0 by adding a pre-prepared phosphate buffer solution, 45mg of protease (20U/g) was added, and the mixture was subjected to enzymolysis at 50℃for 2 hours, followed by boiling for 5 minutes to inactivate the enzyme.
(3) Centrifuging at 4000rpm for 10min, leaching the bran residue collected after centrifugation with ethanol (see step 4), precipitating the supernatant collected after centrifugation with ethanol, filtering after ethanol precipitation, collecting filtrate, and spin-drying the filtrate with a rotary evaporator at 45deg.C to obtain concentrated solution 1;
(4) Adding 20mL of absolute ethyl alcohol into the bran residue collected after the centrifugation in the step (3), carrying out ultrasonic leaching (the power is 300 w) at room temperature for 1.5min, centrifuging the extracting solution, and taking the supernatant.
(5) And (3) spinning the supernatant obtained in the step (4) to near dryness by a rotary evaporator at 45 ℃ under the condition of a vacuum degree of 175Torr to obtain a concentrated solution 2.
(6) Combining the concentrated solutions 1 and 2;
placing 100-200 mesh silica gel powder in a 105 ℃ 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 dish, uniformly stirring, placing in a 50 ℃ oven for overnight drying, taking out, and cooling to room temperature for standby;
selecting a 500 x 40mm normal pressure glass chromatographic column, sampling by adopting a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, gently shaking the chromatographic column until the surface of a filler is flat, loading the column to be 18cm in height, fixing the chromatographic column on a iron stand, lightly adding sample-mixed silica gel until the surface is flat, leaving the upper end of the chromatographic column at a certain height so as to accumulate eluent, performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10:1), performing rotary steaming every 100mL eluent, tracking by using TLC, analyzing by using HPLC and collecting the eluent;
(7) Collecting eluent, concentrating by rotary evaporation, blowing with nitrogen, and redissolving with 5mL chromatographic grade methanol.
(8) LC-MS detection analysis:
mixing the re-dissolved sample solution with 0.22 mu L of organic filter membrane, and measuring ARs concentration by LC-MS detection analysis, wherein the ARs content obtained after extraction and purification is 362.68 mu g/g and 172.2-175.8 mg/g respectively. The extraction amount refers to the sum of the mass of ARs in concentrate 1 and concentrate 2 compared with the mass of the bran powder. The ARs content obtained after purification refers to the sum of the mass ratio of ARs in the eluent in the step (6) to the mass ratio of the concentrated solution 1 and the concentrated solution 2. The meaning of "extraction amount", "ARs content obtained after purification" in the subsequent examples is the same as in this example.
Example 2
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed into 4 50mL centrifuge tubes.
(2) Adding pre-configured phosphate buffer solution to adjust pH of the mixture to 5.0, adding 30mg of alpha-amylase, performing enzymolysis at 50deg.C for 2 hr, and boiling for 5min to inactivate enzyme.
(3) (8) the same as in example 1.
ARs extraction and ARs content obtained after purification were 383.42. Mu.g/g and 204.1-206.7 mg/g, respectively (5 replicates).
Example 3
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed into 4 50mL centrifuge tubes.
(2) Adding pre-configured phosphate buffer solution to adjust pH of the mixture to 5.0, adding 15mg of cellulase, performing enzymolysis at 50deg.C for 2 hr, and boiling for 5min to inactivate enzyme.
(3) (8) the same as in example 1.
ARs extraction and ARs content obtained after purification were 415.00. Mu.g/g and 234.1-236.7 mg/g, respectively.
Example 4
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed into 4 50mL centrifuge tubes.
(2) Adding pre-configured phosphate buffer solution to adjust pH of the mixture to 5.0, adding 1.8g lignin enzyme, performing enzymolysis at 50deg.C for 2 hr, and boiling for 5min to inactivate enzyme.
(3) (8) the same as in example 1.
ARs extraction and ARs content obtained after purification are 333.85 mug/g and 146.6-147.4 mg/g respectively.
TABLE 1 ARs content of enzymatic wheat bran with 100% ethanol as extraction solvent
Comparative example 6
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed into a 50mL centrifuge tube.
(2) 20mL of 75% ethanol was added and the ultrasonic extraction power was 300 w) at room temperature for 1.5min, and the extract was centrifuged to obtain the supernatant.
(3) Spin-drying the supernatant at 45deg.C by rotary evaporator to obtain concentrated solution.
(4) The concentrate was purified by column chromatography on silica gel, spin distilled per 100mL of eluent, followed by TLC, analyzed by HPLC and the eluent was collected.
(5) The eluent is concentrated after rotary evaporation and redissolved by methanol.
(6) The re-dissolved sample solution is filtered by an organic filter membrane with the volume of 0.22 mu L, and then is subjected to LC-MS detection analysis to measure the ARs concentration, wherein the extraction quantity is 240.48 mu g/g, and the ARs content obtained after purification is 123.4-129.3 mg/g. The extraction amount refers to the mass of ARs of the concentrated solution in the step (3) compared with the mass of the bran powder. The ARs content obtained after purification refers to the mass ratio of ARs in the eluent in step (4) to the mass of the concentrate.
Comparative examples 7, 8, 9, 10
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed accurately into 4 50mL centrifuge tubes.
(2) The pH of the mixture was adjusted to 5.0 by adding a pre-prepared phosphate buffer solution, and 45mg of protease (comparative example 4), 30mg of alpha-amylase (comparative example 5), 15mg of cellulase (comparative example 6) and 1.8g of lignin enzyme (comparative example 7) were added, respectively, and the mixture was subjected to enzymatic hydrolysis at 50℃for 2 hours, followed by boiling for 5 minutes to inactivate the enzymes.
(3) Centrifuging at 4000rpm for 10min, leaching the bran residue collected after centrifugation with ethanol (see step 4), precipitating the supernatant collected after centrifugation with ethanol, filtering after ethanol precipitation, and spin-drying the filtrate with a rotary evaporator at 45deg.C to obtain concentrated solution 1;
(4) Adding 20mL of 75% ethanol into the bran residue collected after the centrifugation in the step (3), performing ultrasonic leaching (power is 300 w) at room temperature for 1.5min, centrifuging the extracting solution, and taking the supernatant.
(5) And (3) spinning the supernatant obtained in the step (4) to near dryness by a rotary evaporator at 45 ℃ to obtain a concentrated solution 2. And combining the concentrated solutions, purifying by silica gel column chromatography, concentrating by rotary evaporation, and redissolving by methanol.
(6) Mixing the redissolved sample solution with 0.22 mu L of organic filter membrane, and measuring ARs concentration by LC-MS detection analysis, wherein the extraction amounts are 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); ARs content obtained after purification was 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), 122.1 to 126.3mg/g (comparative example 10), respectively. The extraction amount refers to the sum of the mass of ARs in concentrate 1 and concentrate 2 compared with the mass of the bran powder. The ARs content obtained after purification refers to the mass ratio of ARs in the silica gel column chromatography purification eluent to the mass of the concentrated solution.
TABLE 2 ARs content of enzymatic wheat bran with 75% ethanol as extraction solvent
Comparative example 11
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed accurately into a 50mL centrifuge tube.
(2) Adding 20mL of 50% ethanol, performing ultrasonic extraction (power 300 w) at room temperature for 1.5min, centrifuging the extractive solution, and collecting supernatant.
(3) Spin-drying the supernatant at 45deg.C by rotary evaporator to obtain concentrated solution.
(4) The concentrate was purified by column chromatography on silica gel, spin distilled per 100mL of eluent, followed by TLC, analyzed by HPLC and the eluent was collected.
(5) The eluent is concentrated after rotary evaporation and redissolved by methanol.
(6) The re-dissolved sample solution is filtered by an organic filter membrane with the volume of 0.22 mu L, and then is subjected to LC-MS detection analysis to measure the ARs concentration, wherein the extraction quantity is 140.75 mu g/g, and the ARs content obtained after purification is 64.7-67.3 mg/g. The extraction amount refers to the mass of ARs of the concentrated solution in the step (3) compared with the mass of the bran powder. The ARs content obtained after purification refers to the mass ratio of ARs in the eluent in step (4) to the mass of the concentrate.
Comparative examples 12, 13, 14, 15
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed into 4 50mL centrifuge tubes.
(2) The pH of the mixture was adjusted to 5.0 by adding a pre-prepared phosphate buffer solution, and 45mg of protease (comparative example 12), 30mg of alpha-amylase (comparative example 13), 15mg of cellulase (comparative example 14) and 1.8g of lignin enzyme (comparative example 15) were added, respectively, and the mixture was subjected to enzymatic hydrolysis at 50℃for 2 hours, followed by boiling for 5 minutes to inactivate the enzymes.
(3) Centrifuging at 4000rpm for 10min, leaching the bran residue collected after centrifugation with ethanol (see step 4), precipitating the supernatant collected after centrifugation with ethanol, filtering, and spin-drying the filtrate with a rotary evaporator at 45deg.C to obtain concentrated solution 1;
(4) Adding 20mL of 50% ethanol into the bran residue collected after the centrifugation in the step (3), performing ultrasonic leaching (power is 300 w) at room temperature for 1.5min, centrifuging the extracting solution, and taking the supernatant.
(5) And (3) spinning the supernatant obtained in the step (4) to near dryness by a rotary evaporator at 45 ℃ to obtain a concentrated solution 2.
(6) Combining the concentrated solutions 1 and 2;
placing 100-200 mesh silica gel powder in a 105 ℃ 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 dish, uniformly stirring, placing in a 50 ℃ oven for overnight drying, taking out, and cooling to room temperature for standby;
selecting a 500 x 40mm normal pressure glass chromatographic column, sampling by adopting a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, gently shaking the chromatographic column until the surface of a filler is flat, loading the column to be 18cm in height, fixing the chromatographic column on a iron stand, lightly adding sample-mixed silica gel until the surface is flat, leaving the upper end of the chromatographic column at a certain height so as to accumulate eluent, performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10:1), performing rotary steaming every 100mL eluent, tracking by using TLC, analyzing by using HPLC and collecting the eluent;
(7) Collecting eluent, concentrating by rotary evaporation, blowing with nitrogen, and redissolving with 5mL chromatographic grade methanol.
(8) Mixing the redissolved sample solution with 0.22 mu L of organic filter membrane, and measuring ARs concentration by LC-MS detection analysis, wherein the extraction amounts are 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; ARs content obtained after purification was 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), 72.4 to 75.3mg/g (comparative example 15), respectively. The extraction amount refers to the sum of the mass of ARs in concentrate 1 and concentrate 2 compared with the mass of the bran powder. The ARs content obtained after purification refers to the mass ratio of ARs in the silica gel column chromatography purification eluent to the mass of the concentrated solution.
TABLE 3 ARs content of enzymatic wheat bran at 50% ethanol extraction solvent
Comparative example 16
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed accurately into a 50mL centrifuge tube.
(2) Adding 20mL 25% ethanol, performing ultrasonic extraction (power 300 w) at room temperature for 1.5min, centrifuging the extractive solution, and collecting supernatant.
(3) The supernatant is dried by spin drying through a rotary evaporator at 45 ℃ to obtain concentrated solution. Purifying the concentrated solution by silica gel column chromatography, concentrating by rotary evaporation, and redissolving by methanol.
(4) The re-dissolved sample solution is filtered by an organic filter membrane with the volume of 0.22 mu L, and then is subjected to LC-MS detection analysis to measure the ARs concentration, 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 refers to the mass of ARs of the concentrated solution in the step (3) compared with the mass of the bran powder. The ARs content obtained after purification refers to the mass ratio of ARs in the silica gel column chromatography purification eluent to the mass of the concentrated solution.
Comparative examples 17, 18, 19, 20
(1) 1g of black wheat bran powder (accurate to 0.01 g) was weighed into 4 50mL centrifuge tubes.
(2) The pH of the mixture was adjusted to 5.0 by adding a pre-prepared phosphate buffer solution, and 45mg of protease (comparative example 17), 30mg of alpha-amylase (comparative example 18), 15mg of cellulase (comparative example 19) and 1.8g of lignin enzyme (comparative example 20) were added, respectively, and the mixture was subjected to enzymatic hydrolysis at 50℃for 2 hours, followed by boiling for 5 minutes to inactivate the enzymes.
(3) Centrifuging at 4000rpm for 10min, leaching the bran residue collected after centrifugation with ethanol (see step 4), precipitating the supernatant collected after centrifugation with ethanol, filtering, and spin-drying the filtrate with a rotary evaporator at 45deg.C to obtain concentrated solution 1;
(4) Adding 20mL of 25% ethanol into the bran residue collected after the centrifugation in the step (3), performing ultrasonic extraction (with the power of 300 w) at room temperature for 1.5min, centrifuging the extract, and taking the supernatant.
(5) And (3) spinning the supernatant obtained in the step (4) to near dryness by a rotary evaporator at 45 ℃ to obtain a concentrated solution 2.
(6) Combining the concentrated solutions 1 and 2;
placing 100-200 mesh silica gel powder in a 105 ℃ 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 dish, uniformly stirring, placing in a 50 ℃ oven for overnight drying, taking out, and cooling to room temperature for standby;
selecting a 500 x 40mm normal pressure glass chromatographic column, sampling by adopting a dry method, pouring activated silica gel powder into a clean and dry chromatographic column, gently shaking the chromatographic column until the surface of a filler is flat, loading the column to be 18cm in height, fixing the chromatographic column on a iron stand, lightly adding sample-mixed silica gel until the surface is flat, leaving the upper end of the chromatographic column at a certain height so as to accumulate eluent, performing gradient elution by using petroleum ether-ethyl acetate (v/v, 10:1), performing rotary steaming every 100mL eluent, tracking by using TLC, analyzing by using HPLC and collecting the eluent;
(7) Collecting eluent, concentrating by rotary evaporation, blowing with nitrogen, and redissolving with 5mL chromatographic grade methanol.
(8) After the reconstituted sample was mixed with 0.22. Mu.L of an organic filter membrane, the concentration of ARs was measured by LC-MS detection analysis, and the extraction amounts 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; ARs content obtained after purification was 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), 21.3 to 22.2mg/g (comparative example 20), respectively. The extraction amount refers to the sum of the mass of ARs in concentrate 1 and concentrate 2 compared with the mass of the bran powder. The ARs content obtained after purification refers to the mass ratio of ARs in the silica gel column chromatography purification eluent to the mass of the concentrated solution.
TABLE 4 ARs content of enzymatic wheat bran with 25% ethanol as extraction solvent
Conclusion: the results show that the wheat bran is subjected to enzymolysis by using protease, alpha-amylase, cellulase and lignin enzyme and is extracted by ultrasound, so that the ARs extraction amount can be remarkably improved to a certain extent, and from the concentration of an extraction solvent, absolute ethanol is more than 75 percent ethanol is more than 50 percent ethanol is more than 25 percent ethanol; from the enzyme effect, cellulase > alpha-amylase > protease > lignin enzyme. The invention reduces the extraction cost and obviously improves the yield while avoiding toxic components brought by the organic solvent.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 of:
(1) Pretreatment: crushing wheat bran;
(2) Enzymolysis: weighing crushed bran, adding phosphate buffer solution, and adding at least one of alpha-amylase, protease, cellulase and lignin enzyme for enzymolysis;
(3) And (3) centrifuging: centrifuging the enzymolysis liquid after enzymolysis is finished, leaching bran residues collected after centrifugation by absolute ethyl alcohol, precipitating supernatant collected after centrifugation by ethyl alcohol, filtering and collecting filtrate after alcohol precipitation, and spin-drying the filtrate by a rotary evaporator to obtain concentrated solution 1 for later use;
(4) Ethanol leaching: adding absolute ethyl alcohol into the bran residue collected after the centrifugation in the step (3), carrying out ultrasonic leaching at room temperature, centrifuging an extracting solution, and taking supernatant;
(5) Spin drying: spin-drying the supernatant obtained in the step (4) by a rotary evaporator to obtain a concentrated solution 2;
(6) Purifying by silica gel column chromatography: mixing the concentrated solution 1 and the concentrated solution 2, purifying by silica gel column chromatography, and collecting eluent;
(7) And (5) re-concentrating: concentrating the eluent collected in the step (6) by rotary evaporation, blowing with nitrogen to dry, and re-dissolving with 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 as claimed in claim 1, wherein the step (1) is to crush the wheat bran and pass through a 80 mesh sieve for standby.
3. The method for extracting and detecting alkylresorcinol from wheat bran according to claim 1, wherein the step (2) is enzymolysis: weighing 1g of the bran pretreated in the step (1) into a 50mL centrifuge tube, adding a phosphate buffer solution to stabilize the pH at 5.0, adding at least one of alpha-amylase, protease, cellulase and lignin enzyme, and carrying out enzymolysis for 2h at 50 ℃.
4. The method for extracting and detecting alkylresorcinol from wheat bran as claimed in claim 1, wherein the ethanol leaching step (4): adding ethanol into the bran residue collected after centrifugation in the step (3), carrying out ultrasonic leaching at room temperature for 1.5min with ultrasonic power of 300W, centrifuging the extract, and collecting supernatant.
5. The method for extracting and detecting alkyl resorcinol in wheat bran according to claim 1, wherein the step (6) is characterized in that firstly, 100-200 meshes of silica gel powder is placed in a 105 ℃ oven for activation for 30min, then is placed in a dryer for cooling to room temperature, a concentrated solution sample with the same quality as the silica gel powder is weighed and poured into an evaporation dish together with the silica gel powder, and is stirred uniformly, placed in a 50 ℃ oven for overnight drying, taken out and cooled to room temperature for standby;
selecting a 500 x 40mm normal pressure glass chromatographic column, adopting a dry method for sample loading, and selecting a volume ratio of 10:1 in petroleum ether-ethyl acetate, spin-evaporating every 100mL of eluent, tracking with TLC, analyzing with HPLC and collecting the eluent.
6. The method for extracting and detecting alkylresorcinol in wheat bran according to claim 1, wherein the adding amount of cellulase in the step (2) is 15mg/g wheat bran powder, and the cellulase activity is 60 ten thousand U/g.
7. The method for extracting and detecting alkylresorcinol from wheat bran according to claim 1, wherein the conditions of HPLC in step (6) are: (1) mobile phase: phase a 0.1% aqueous formic acid; phase B0.1% methanolic formate solution, flow rate: 1mL/min, column temperature: 30 ℃, sample injection amount: 10. Mu.L; (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. The method for extracting and detecting alkylresorcinol from wheat bran according to claim 1, wherein the detection and analysis conditions of LC-MS in step (8) are:
LC conditions: ACQMITY MPLC BEN C 18 Chromatographic column (2.1 mm. Times.100 mm,1.7 μm); column temperature is 30 ℃; the sample injection amount is 5 mu L; mobile phase: phase A is 0.1% ammonia water, and phase B is acetonitrile; the flow rate is 0.5mL/min; elution gradient: 0-2 min, 100-15% of A and 0-85% of B; 2-4 min, 15-10% of A and 85-90% of B; 4-8 min, 10-5% of A, 90-95% of B; 8-9 min, 5-15% of A and 95-85% of B;
MS conditions: an electrospray ion source, a negative ion source; a multiple reaction detection mode; capillary voltage 1.2kV; taper hole airflow: nitrogen gas with a flow rate of 150L/h; the temperature of the ion source is 150 ℃; the desolvation gas temperature is 200 ℃ and the flow rate is 1000L/h.
9. The method for extracting and detecting alkylresorcinol from wheat bran as claimed in claim 1, wherein the alpha-amylase is added in the amount of 30mg/g of wheat bran powder in the step (2), and the alpha-amylase activity is 30 ten thousand U/g.
10. The method for extracting and detecting alkylresorcinol in wheat bran as claimed in claim 1, wherein the protease added in the step (2) is 45mg/g wheat bran powder, and the protease activity is 20 ten thousand U/g.
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| 彭田园 ; 赵仁勇 ; 王香玉 ; .超声波辅助提取全麦粉中烷基间苯二酚的工艺优化.河南工业大学学报(自然科学版).2017,38(04),14-19. * |
| 超声波辅助提取全麦粉中烷基间苯二酚的工艺优化;彭田园;赵仁勇;王香玉;;河南工业大学学报(自然科学版);38(04);14-19 * |
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