CN112362769A - Method for detecting ergothioneine content in edible fungi - Google Patents
Method for detecting ergothioneine content in edible fungi Download PDFInfo
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Abstract
The invention provides a method for detecting the ergothioneine content in edible mushrooms, and belongs to the technical field of analytical chemistry. The edible fungus to be detected comprises a fresh sample or a dry sample, and an extraction solvent is selected according to the fresh sample and the dry sample, wherein the extraction solvent adopted by the fresh sample is a formic acid-methanol solution, a formic acid-water solution or a formic acid-methanol-water solution, the extraction solvent adopted by the dry sample is a methanol-water solution, an ultrasonic or oscillation extraction method is adopted, the operation is simple, the extraction effect is good, and then the content of the ergothioneine in the extraction solution is analyzed by utilizing a hydrophilic interaction chromatography-ultra high performance liquid chromatography (HILIC-UPLC), so that the accurate and rapid determination of the content of the ergothioneine in the edible fungus to be detected can be realized. In addition, the method provided by the invention has good repeatability and high separation degree.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for detecting the ergothioneine content in edible fungi.
Background
Ergothioneine (ERGOTHIONEINE, ERT) was discovered in 1909 in a fungus, ergot (Claviceps purpurea), is a rare natural chiral amino acid, is an important physiologically active substance in organisms, and has multiple functions of resisting oxidation, scavenging free radicals, preventing ultraviolet radiation damage, regulating intracellular redox reaction, participating in intracellular energy regulation, serving as a cell physiological protective agent and the like. Because of the specific biological functions and pharmacological activities of ergothioneine, such as: the copper-based compound has the advantages of resisting inflammation, preventing degeneration of nervous tissues, preventing DNA and protein from being oxidized and damaged due to copper, inhibiting developmental defects, protecting liver and the like, generating no toxic or side effect after being ingested by human and animals, belonging to products generally regarded as safe, showing good application prospects in the fields of cosmetics, functional foods, pharmacy, therapeutics, biomedicine and the like, and being an active compound with high added value.
At present, the detection of ergothioneine at home and abroad has no unified and effective standard. The ergothioneine is very soluble in water, so the extraction solvent is generally deionized water or an aqueous organic solvent. The reflux extraction method is a classical extraction method of ergothioneine, and is characterized in that Schoenbergen (Schoenbergen, Zhao Yanmin, Linjiwei, and the like) is orthogonally designed and optimized to extract the ergothioneine in oyster mushroom leftovers, food research and development, 2017,38(03):40-45.) is adopted to extract the ergothioneine in the oyster mushroom leftovers by reflux at the temperature of 100 ℃ by using distilled water as a solvent, and the extraction process is optimized by an orthogonal method, wherein the optimal extraction process condition is as follows: pH 8, feed-liquid ratio 1: 6(g/mL), the extraction time is 2h, the extraction times is 2 times, and the extraction rate reaches 87.45 percent under the condition. However, the reflux extraction method takes a long time and is complicated to operate.
Regarding the detection method of ergothioneine, High Performance Liquid Chromatography (HPLC) and high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) are reported to be used for detecting the content of ergothioneine in mycelium, fermentation liquor, blood and tissues. More methods for detecting ergothioneine are established to meet different requirements, and the method has very important practical significance for the research and application of the ergothioneine.
Disclosure of Invention
The invention aims to provide a method for detecting the ergothioneine content in edible fungi, which is simple to operate and can realize accurate and rapid determination of the ergothioneine content in the edible fungi.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting the ergothioneine content in edible fungi, which comprises the following steps:
(1) extracting the edible fungi to be detected by adopting an extraction solvent to obtain an extracting solution; the extraction is ultrasonic extraction or oscillation extraction; wherein the edible fungi to be detected comprise fresh samples or dry samples; when the fresh sample is extracted, the adopted extraction solvent is formic acid-methanol solution, formic acid-water solution or formic acid-methanol-water solution; when the dry sample is extracted, the adopted extraction solvent is methanol-water solution;
(2) performing hydrophilic interaction-ultra high performance liquid chromatography analysis on the extracting solution obtained in the step (1) to obtain an edible fungus liquid phase analysis result to be detected; obtaining the ergothioneine content of the edible fungi to be detected according to the standard curve of the ergothioneine content and the analysis result of the edible fungi liquid to be detected; wherein the chromatographic conditions of the ultra-high performance liquid chromatography comprise:
a chromatographic column: an XBridge BEH HILIC column or a Zic HILIC column;
mobile phase: the mobile phase comprises a phase A and a phase B, wherein the phase A is a formic acid-acetonitrile solution, and the volume fraction of formic acid in the formic acid-acetonitrile solution is 0.05-0.2%; the phase B is a formic acid-water solution, and the volume fraction of formic acid in the formic acid-water solution is 0.05-0.2%; the volume ratio of the phase A to the phase B is 80: 20-90: 10;
a detector: an ultraviolet detector or a diode array detector.
Preferably, the variety of the edible fungi to be detected comprises oyster mushroom, white jade mushroom, pleurotus eryngii, shiitake mushroom, needle mushroom, straw mushroom, chicken mushroom, hypsizygus marmoreus, agaricus bisporus, hypsizygus marmoreus, tremella, ganoderma lucidum, cordyceps militaris, edible fungus, hericium erinaceus, agaricus blazei, agaric, agrocybe cylindracea, shiitake mushroom or morchella esculenta.
Preferably, in the step (1), the volume fraction of formic acid in the formic acid-methanol solution is 0.1%, the volume fraction of formic acid in the formic acid-water solution is 0.1%, the volume fraction of formic acid in the formic acid-methanol-water solution is 0.1%, and the volume fraction of methanol is 30-70%; the volume fraction of methanol in the methanol-water solution was 70%.
Preferably, the ultrasonic extraction of the edible fungi to be detected comprises the following steps:
crushing the edible fungi to be detected, mixing the obtained crushed sample with an extraction solvent, and extracting under an ultrasonic condition to obtain an extraction slurry;
and carrying out solid-liquid separation on the extraction slurry to obtain an extracting solution.
Preferably, when the edible fungi to be detected is a fresh sample, the crushed sample is in a slurry state, and the using amount ratio of the crushed sample to the extraction solvent is (1.00-3.00) g: (30-90) mL; the extraction temperature is 25-40 ℃, the extraction time is 5-20 min, and the extraction power is 50-120W.
Preferably, when the edible fungi to be detected is a dry sample, the particle size of the crushed sample is less than 425 μm, and the using amount ratio of the crushed sample to the extraction solvent is (0.50-2.00) g: (30-90) mL; the extraction temperature is 25-40 ℃, the extraction time is 20-40 min, and the extraction power is 50-120W.
Preferably, the solid-liquid separation mode is centrifugation, the rotation speed of the centrifugation is 3000-5000 r/min, and the centrifugation time is 3-10 min.
Preferably, the flow rate of the mobile phase is 0.4 mL/min.
Preferably, the column temperature of the chromatography column is 40 ℃.
Preferably, the hydrophilic interaction-ultra high performance liquid chromatography analysis is performed at a detection wavelength of 262 nm.
The invention provides a method for detecting the ergothioneine content in edible fungi. The edible fungi to be detected comprise fresh samples or dry samples, and extraction solvents are selected according to the fresh samples and the dry samples, wherein the extraction solvents adopted by the fresh samples are formic acid-methanol solutions, formic acid-aqueous solutions or formic acid-methanol-aqueous solutions, the extraction solvents adopted by the dry samples are methanol-aqueous solutions, an ultrasonic or oscillation extraction method is adopted, the operation is simple, the extraction effect is good, then the content of the ergothioneine in the extracting solution is analyzed by using hydrophilic interaction-ultra high performance liquid chromatography (HILIC-UPLC), the accurate and rapid determination of the content of the ergothioneine in the edible fungi to be detected can be realized, the detection limit of the ergothioneine is 0.010mg/g for the fresh samples, and the dry samples are 0.020 mg/g; the quantitative fresh limiting sample is 0.020mg/g, and the dry sample is 0.040 mg/g; the peak emergence time of ergothioneine is 3-5 min. In addition, the method provided by the invention has good repeatability and high separation degree.
Drawings
FIG. 1 is a liquid chromatogram of ergothioneine in an embodiment of the invention, wherein a is a liquid chromatogram of a 10mg/L ergothioneine standard working solution, b is a liquid chromatogram of a dry tremella sample, c is a liquid chromatogram of a fresh shiitake mushroom sample, d is a liquid chromatogram of a fresh pleurotus eryngii sample, and e is a liquid chromatogram of a fresh flammulina velutipes sample.
FIG. 2 is a linear plot of ergothioneine standard solutions in an example of the invention.
Detailed Description
The invention provides a method for detecting the ergothioneine content in edible fungi, which comprises the following steps:
(1) extracting the edible fungi to be detected by adopting an extraction solvent to obtain an extracting solution; the extraction is ultrasonic extraction or oscillation extraction; wherein the edible fungi to be detected comprise fresh samples or dry samples; when the fresh sample is extracted, the adopted extraction solvent is formic acid-methanol solution, formic acid-water solution or formic acid-methanol-water solution; when the dry sample is extracted, the adopted extraction solvent is methanol-water solution;
(2) performing hydrophilic interaction-ultra high performance liquid chromatography analysis on the extracting solution obtained in the step (1) to obtain an edible fungus liquid phase analysis result to be detected; obtaining the ergothioneine content of the edible fungi to be detected according to the standard curve of the ergothioneine content and the analysis result of the edible fungi liquid to be detected; wherein the chromatographic conditions of the ultra-high performance liquid chromatography comprise:
a chromatographic column: an XBridge BEH HILIC column or a Zic HILIC column;
mobile phase: the mobile phase comprises a phase A and a phase B, wherein the phase A is a formic acid-acetonitrile solution, and the volume fraction of formic acid in the formic acid-acetonitrile solution is 0.05-0.2%; the phase B is a formic acid-water solution, and the volume fraction of formic acid in the formic acid-water solution is 0.05-0.2%; the volume ratio of the phase A to the phase B is 80: 20-90: 10;
a detector: an ultraviolet detector or a diode array detector.
The invention adopts an extraction solvent to extract the edible fungi to be detected, so as to obtain an extracting solution. The variety of the edible fungi to be detected is not particularly limited, and any edible fungi needing to detect the content of ergothioneine, which are well known to those skilled in the art, can be specifically selected from oyster mushroom, white jade mushroom, pleurotus eryngii, lentinus edodes, needle mushroom, straw mushroom, chicken mushroom, hypsizygus marmoreus, agaricus bisporus, hypsizygus marmoreus, tremella, lucid ganoderma, cordyceps militaris, edible fungus, hericium erinaceus, agaricus blazei murill, edible fungus, agrocybe cylindracea, lentinus edodes or morchella esculenta. In the invention, the edible fungus matrix is relatively complex, and the method provided by the invention can avoid the determination of the ergothioneine content by the complex edible fungus matrix, and the ergothioneine separation degree is high.
In the invention, the edible fungi to be detected comprise fresh samples or dry samples. The invention aims at the pertinence selection of an extraction solvent for a fresh sample and a dry sample, and particularly, the ergothioneine is a water-soluble compound, has higher polarity, contains water in the fresh sample, so that the water-containing extraction solvent can be adopted or not, and the water-containing extraction solvent is required for the dry sample. In the invention, when the fresh sample is extracted, the extraction solvent adopted is formic acid-methanol solution, formic acid-water solution or formic acid-methanol-water solution, and in consideration of hydrophilic interaction, a chromatograph is sensitive to a sample injection solution and may have strong solvent effect, so the invention preferably adopts the formic acid-methanol solution as the extraction solvent of the fresh sample, the volume fraction of formic acid in the formic acid-methanol solution is preferably 0.1%, the volume fraction of formic acid in the formic acid-water solution is preferably 0.1%, the volume fraction of formic acid in the formic acid-methanol-water solution is preferably 0.1%, the volume fraction of methanol is preferably 30-70%, and specifically 30%, 50% or 70%; when the dry sample is extracted, the adopted extraction solvent is methanol-water solution, and the volume fraction of methanol in the methanol-water solution is preferably 70%.
In the invention, the extraction is preferably ultrasonic extraction or oscillation extraction, wherein the temperature control is inconvenient in the oscillation extraction process, the temperature control is convenient in the ultrasonic extraction process, and the ultrasonic extraction is preferably adopted in the invention in order to ensure that the extraction efficiency is more stable. In the present invention, the method for performing ultrasonic extraction on the edible fungi to be detected preferably comprises the following steps:
crushing the edible fungi to be detected, mixing the obtained crushed sample with an extraction solvent, and extracting under an ultrasonic condition to obtain an extraction slurry;
and carrying out solid-liquid separation on the extraction slurry to obtain an extracting solution.
In the invention, when the edible fungi to be detected is a fresh sample, the crushed sample is preferably in a slurry state, and specifically, the fresh sample is crushed into a uniform slurry state by a multifunctional food processor; the preferable dosage ratio of the crushed sample to the extraction solvent is (1.00-3.00) g: (30-90) mL, more preferably 2.00 g: 40 mL; the extraction temperature is preferably 25-40 ℃, and more preferably 25-30 ℃; the extraction time is preferably 5-20 min, and more preferably 10 min; the power of the ultrasonic wave is preferably 50-120W, and more preferably 80-100W; the solid-liquid separation mode is preferably centrifugation, the rotation speed of the centrifugation is preferably 3000-5000 r/min, more preferably 4000r/min, the centrifugation time is preferably 3-10 min, more preferably 5min, and the supernatant obtained by the centrifugation is the extracting solution.
In the invention, when the edible fungi to be detected is a dry sample, the granularity of the crushed sample is preferably less than 425 μm, specifically, the crushed sample can pass through a 425 μm standard mesh screen, and the part under the mesh screen is taken as the crushed sample; the preferable dosage ratio of the crushed sample to the extraction solvent is (0.50-2.00) g: (30-90) mL, more preferably 1.00 g: 40 mL; the extraction temperature is preferably 25-40 ℃, and the extraction time is preferably 20-40 min, and more preferably 30 min; the power of the ultrasonic wave is preferably 50-120W.
In the invention, the solid-liquid separation mode is preferably centrifugation, the rotation speed of the centrifugation is preferably 3000-5000 r/min, more preferably 4000r/min, the time of the centrifugation is preferably 3-10 min, more preferably 5min, and the supernatant obtained by the centrifugation is the extracting solution.
In the present invention, the shaking extraction preferably comprises vortex shaking or shaking table shaking, the vortex shaking is preferably performed at a rotation speed of 2000r/min, and the shaking table shaking is preferably performed at a rotation speed of 200 r/min; in the present invention, the shaking extraction is performed under room temperature conditions, and the other operating conditions are preferably those referred to as ultrasonic extraction.
After the extracting solution is obtained, performing hydrophilic interaction ultra-high performance liquid chromatography analysis on the extracting solution to obtain an edible fungus liquid phase analysis result to be detected; obtaining the ergothioneine content of the edible fungi to be detected according to the standard curve of the ergothioneine content and the analysis result of the edible fungi liquid to be detected; wherein the chromatographic conditions of the ultra-high performance liquid chromatography comprise:
a chromatographic column: an XBridge BEH HILIC column or a Zic HILIC column;
mobile phase: the mobile phase comprises a phase A and a phase B, wherein the phase A is a formic acid-acetonitrile solution, and the volume fraction of formic acid in the formic acid-acetonitrile solution is 0.05-0.2%; the phase B is a formic acid-water solution, and the volume fraction of formic acid in the formic acid-water solution is 0.05-0.2%; the volume ratio of the phase A to the phase B is 80: 20-90: 10;
a detector: an ultraviolet detector or a diode array detector.
In the present invention, the extraction solution is preferably subjected to microfiltration membrane filtration before the detection of the sample application, and the microfiltration membrane is preferably an organic microfiltration membrane, more preferably a 0.22 μm organic microfiltration membrane.
In the present invention, when the edible fungus to be tested is a fresh sample, obtaining the extracting solution preferably further includes: and transferring the extracting solution into a volumetric flask, fixing the volume by using the extracting solvent, shaking up, taking a small amount of diluted extracting solution, and measuring by using an ultra-high performance liquid chromatograph after the extracting solution passes through a microfiltration membrane.
In the present invention, when the edible fungus to be tested is a dry sample, obtaining the extracting solution preferably further includes: transferring the extracting solution into a volumetric flask, fixing the volume by using the extracting solvent, shaking up, taking a small amount of diluted extracting solution, drying, redissolving by using a formic acid-methanol solution, and measuring by using an ultra-high performance liquid chromatograph after passing through a microfiltration membrane; in the present invention, the drying is preferably nitrogen blow drying; in the present invention, the volume fraction of formic acid in the formic acid-methanol solution used in the reconstitution is preferably 0.1%, and the amount of the formic acid-methanol solution used in the reconstitution is not particularly limited and may be selected according to actual needs.
In the invention, the chromatographic column is an Xbridge BEH HILIC column or a Zic HILIC column, wherein the grain size of the filler in the Zic HILIC column is relatively large, and the peak width of the ergothioneine is large when separation analysis is carried out, so the Xbridge BEH HILIC column is preferably adopted in the invention.
In the present invention, the model of the ultra high performance liquid chromatograph used for performing the hydrophilic interaction ultra high performance liquid chromatography is an ultra high performance liquid chromatography (Waters Acquity UPLC) apparatus equipped with an ultraviolet detector or a diode array detector, and the manufacturer prefers Waters (Waters).
In the present invention, when the hydrophilic interaction ultra high performance liquid chromatography is performed, the volume fraction of formic acid in the a phase is preferably 0.1%; the volume fraction of formic acid in phase B is preferably 0.1%; the volume ratio of the A phase to the B phase is preferably 88: 12.
In the present invention, when the hydrophilic interaction ultra high performance liquid chromatography is performed, the flow rate of the mobile phase is preferably 0.4 mL/min; the column temperature of the chromatographic column is preferably 40 ℃; the detection wavelength is preferably 262 nm; the amount of sample is preferably 3. mu.L.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The instrumentation, materials and reagents used in the following examples are as follows:
1. instrumentation and equipment
Ultra-high performance liquid chromatography (Waters Acquity UPLC) equipped with uv detector or diode array detector, pipette gun (eppendorf), ultrasonic cleaner, centrifuge, nitrogen blow dryer, vortex mixer, multifunctional food processor, sample grinder, 425 μm standard sieve, balance with sensing 0.01g and 0.0001 g.
2. Materials and reagents
Ergothioneine (C)9H15N3O2S, CAS number: 67747-09-5, purity not less than 98%) from TRC; acetonitrile (chromatographically pure), methanol (chromatographically pure) was purchased from Shanghai Tantake Technology, Inc.; formic acid (chromatographically pure) was purchased from Sigma-Aldrich.
Fresh samples of edible fungi: oyster mushroom, white beech mushroom, Pleurotus eryngii, Lentinus edodes, needle mushroom, straw mushroom, chicken mushroom, Hypsizygus marmoreus, Agaricus bisporus, Hypsizygus marmoreus, Stropharia rugoso-annulata;
edible fungus dry sample preparation: tremella, lucid ganoderma, cordyceps militaris, edible fungus, hericium erinaceus, agaricus blazei, agaric, agrocybe cylindracea, mushroom and morel.
EXAMPLE 1 formulation of standard solution of ergothioneine
Preparing an ergothioneine standard stock solution: weighing 10mg (accurate to 0.0001g) of ergothioneine standard, dissolving with water, and metering to 10mL to obtain ergothioneine standard stock solution (1000mg/L), and storing at-18 deg.C for use.
In this example, the retention time of a standard stock solution of ergothioneine was determined. Because the solubility of 1000mg/L ergothioneine standard stock solution is very high, the ergothioneine standard stock solution cannot be directly measured on a machine, a proper amount of ergothioneine standard stock solution is diluted to 50mg/L each time, and the peak area of the ergothioneine standard solution is measured on the machine. The samples were diluted and measured every 15 days, and the peak area was observed to change, and the results are shown in Table 1. As can be seen from Table 1, the standard stock solution of ergothioneine with the mass concentration of 1000mg/L is stored at-18 ℃, the peak area does not change obviously within 2 months, and the effective period of the standard stock solution of ergothioneine is set to be 2 months.
TABLE 1 ergothioneine Standard stock solutions preservation Life time data
Preparing an ergothioneine standard working solution: accurately transferring a certain amount of ergothioneine standard stock solution, and gradually diluting with formic acid-methanol solution with formic acid volume fraction of 0.1% to obtain a series of standard working solutions with mass concentrations of 1.00mg/L, 2.00mg/L, 5.00mg/L, 10.0mg/L and 50.0 mg/L; prepared immediately after use.
Example 2 optimization of the chromatographic separation conditions for ergothioneine
1. And determining the detection wavelength. Scanning the ergothioneine standard working solution with the concentration of 10.0mg/L within the wavelength range of 200-400 nm to obtain the ergothioneine with a significant characteristic absorption peak at 262nm, and setting the detection wavelength to be 262 nm.
2. And (4) determination of a chromatographic column. The chromatographic columns used in the prior art for the detection of ergothioneine include C18 columns or hydrophilic columns. The invention is verified by four types of chromatographic columns, namely waters BEH C18 (marked as a column No. 1), waters Atlantis T3 (marked as a column No. 2), waters Xbridge BEH HILIC (marked as a column No. 3) and Merck Zic HILIC (marked as a column No. 4), ergothioneine hardly remains on the column No. 1 and the column No. 2, but the retention effect and the separation effect on the column No. 3 and the column No. 4 are better, but the filler particle size in the column No. 4 is relatively larger, the peak width of the ergothioneine is larger when separation analysis is carried out, so that the column No. 3 is selected for carrying out subsequent condition optimization and experiments.
3. And (4) determination of a mobile phase. The detection of ergothioneine in the prior art generally uses acetonitrile and aqueous ammonium acetate as mobile phases. As ergothioneine is easy to generate stronger interaction with residual silanol groups on a chromatographic column to cause tailing, the invention adds formic acid into the mobile phase, and particularly selects a formic acid-acetonitrile solution (the volume fraction of the formic acid is 0.1%) and a formic acid-aqueous solution (the volume fraction of the formic acid is 0.1%) as the mobile phase, and the results show that the ergothioneine standard working solution with the concentration of 10mg/L is taken as a sample, and the response of the sample is stronger and the peak shape is symmetrical when the formic acid-acetonitrile solution and the formic acid-aqueous solution are taken as the mobile phase.
Then, the separation effect of the ergothioneine and impurities in the actual sample under the conditions of different flow rates (0.3, 0.4, 0.5 or 0.6mL/min) is considered, and the result shows that when the flow rate is 0.4mL/min, the ergothioneine has better separation degree, and other impurity peaks in the actual sample basically have no interference with the ergothioneine peak. The graph of the UV chromatogram separation of the ergothioneine standard working solution and the content determination of the actual sample is shown in figure 1.
In summary, the chromatographic conditions finally established are: a chromatographic column: xbridge BEH HILIC column (100X 2.1mm, 1.7 μm); column temperature: 40 ℃; mobile phase: the A phase is formic acid-acetonitrile solution (formic acid volume fraction is 0.1%), the B phase is formic acid-water solution (formic acid volume fraction is 0.1%), and the volume ratio of the A phase to the B phase is 88: 12; flow rate: 0.4 mL/min; detection wavelength: 262 nm; sample introduction amount: 3 μ L.
Example 3 HPLC Standard Curve of ergothioneine plotting and determination of detection and quantitation limits
Under the chromatographic conditions finally determined in example 2, ergothioneine standard working solutions were sequentially injected from low concentration to high concentration, and a standard curve was plotted according to the obtained peak areas and the corresponding concentrations (mg/L) of the standard working solutions, as shown in FIG. 2. The result shows that the ergothioneine is in the range of 1.00-50.0 mg/L, and the concentration and the peak area have a good linear relation (see table 2).
TABLE 2 regression equation and correlation coefficient for ergothioneine standard working solutions
In the chromatogram (a in fig. 1) obtained from an ergothioneine standard solution (concentration of S/N < 20), the detection Limit (LOD) was 3-fold signal-to-noise ratio (S/N ═ 3); the minimum concentration at which the precision and the accuracy both meet the requirements when an actual sample is measured is taken as a limit of quantitation (LOQ), the detection limit of the ergothioneine is 0.010mg/g for a fresh sample, and the detection limit of the ergothioneine is 0.020mg/g for a dry sample; the quantitative fresh-limiting sample is 0.020mg/g, and the dry sample is 0.040 mg/g.
Example 4 calculation of ergothioneine content in edible fungi
The ergothioneine content in the sample is calculated by mass fraction w, the value is expressed by milligram per gram (mg/g) and is calculated according to the formula (1):
in equation (1):
a is the peak area of ergothioneine in the sample solution;
as-peak area of ergothioneine in standard solution;
ρ -the mass concentration of ergothioneine in the standard solution in milligrams per liter (mg/L);
v-final volumetric volume of sample in milliliters (mL);
m-mass of the sample in grams (g);
f-sample dilution factor.
The calculation result retains three significant digits, and when the result is less than 0.100mg/g, two significant digits are retained.
Example 5 determination of solvent for extraction of ergothioneine in edible fungi
The ergothioneine is a water-soluble compound with larger polarity, and the influence of 5 extraction solvents on the extraction efficiency of the ergothioneine in fresh edible fungus samples is discussed in the embodiment, wherein the 5 extraction solvents are specifically:
the No. 1 extraction solvent is formic acid-water solution, and the volume fraction of formic acid is 0.1%;
the 2# extraction solvent is formic acid-methanol-water solution, the volume fraction of formic acid is 0.1%, and the volume fraction of methanol is 30%;
the 3# extraction solvent is formic acid-methanol-water solution, the volume fraction of formic acid is 0.1%, and the volume fraction of methanol is 50%;
the No. 4 extraction solvent is formic acid-methanol-water solution, wherein the volume fraction of formic acid is 0.1%, and the volume fraction of methanol is 70%;
the 5# extraction solvent was formic acid-methanol solution with a formic acid volume fraction of 0.1%.
Respectively adopting No. 1-5 # extraction solvents (40mL) to ultrasonically extract fresh samples (the mass is 2.00g) of the edible fungi into slurry for 30min under the conditions of 100W power and 25 ℃, centrifuging for 5min at 4000r/min, transferring supernatant into a 50mL volumetric flask, fixing the volume to 50mL by using corresponding extraction solvents, shaking uniformly, taking 1mL of extracting solution, passing through a 0.22 mu m filter membrane, determining by using an ultra-high performance liquid chromatograph, and inspecting the extraction effect of the extraction solvents according to the content of ergothioneine in the fresh samples of the edible fungi, wherein each extraction solvent is subjected to parallel test for 6 times, and the result is shown in Table 3.
The results show that the extraction efficiency of the fresh edible fungi represented by the flammulina velutipes is not very different by adopting the extraction solvents. However, considering that the chromatograph is sensitive to the sample solution due to the hydrophilic interaction and a strong solvent effect may occur, the 5# extraction solvent is used as the extraction solvent of the fresh edible fungus sample.
Influence of extraction solvent # 31-5 on ergothioneine extraction effect of fresh edible fungus sample
Unit: mg/g
Note: unless otherwise specified, all samples were fresh samples, as follows.
This example discusses the effect of 5 extraction solvents on the ergothioneine extraction efficiency of dried edible fungi samples, wherein the 5 extraction solvents are specifically:
the No. 6 extraction solvent is water;
the 7# extraction solvent is methanol-water solution, and the volume fraction of methanol is 30%;
the No. 8 extraction solvent is methanol-water solution, and the volume fraction of methanol is 50%;
the No. 9 extraction solvent is methanol-water solution, and the volume fraction of methanol is 70%;
the 10# extraction solvent was methanol.
Respectively adopting 6# to 10# extraction solvents (40mL) to ultrasonically extract a dried edible fungus sample (which is crushed and then passes through a 425-micrometer standard mesh screen, the part which is used after being dried is taken, the mass is 1.00g) for 30min under the conditions of 100W power and 25 ℃, centrifuging for 5min at 4000r/min, transferring supernatant into a 50mL volumetric flask, fixing the volume to 50mL by using the corresponding extraction solvents, shaking uniformly, taking 1mL of extracting solution, drying by using nitrogen, re-dissolving by using 1mL of formic acid-methanol solution (the volume fraction of formic acid is 0.1%), passing through a 0.22-micrometer filter membrane, determining by using an ultra-high performance liquid chromatograph, inspecting the extraction effect of the extraction solvents according to the content of ergothioneine in the dried edible fungus sample, and performing parallel tests 6 times on each extraction solvent, wherein the result is shown in Table 4.
The result shows that the extraction efficiency of the ergothioneine is highest when the 9# extraction solvent is adopted for the edible fungus dry sample represented by the tremella.
TABLE 46 # 10# Effect of the extraction solvent on the ergothioneine extraction Effect of the dried edible fungus samples
Unit: mg/g
Example 6 determination of the method of extraction of ergothioneine from edible fungi
In this example, the influence of the ultrasonic method, the vortex oscillation method and the table oscillation method on the ergothioneine extraction effect is discussed, specifically, dry tremella, fresh flammulina velutipes, fresh pleurotus eryngii and fresh mushrooms randomly purchased in the market are used as samples (all of which are used after being crushed), extraction solvents are respectively selected according to the results in example 5 to extract a fresh sample and a dry sample, that is, a formic acid-methanol solution (marked as a 0.1% formic acid-methanol solution) with a formic acid volume fraction of 0.1% is used as an extractant for the fresh sample, and a methanol-water solution (marked as a 70% methanol water solution) with a methanol volume fraction of 70% is used as an extractant for the dry sample, and the extraction method is as follows:
an ultrasonic method: accurately weighing 2.00g of sample (fresh sample) into a 50mL centrifuge tube, adding 40mL of the 0.1% formic acid methanol solution, carrying out ultrasonic extraction for 30min at the power of 100W and at the temperature of 25 ℃ in an ultrasonic cleaner, centrifuging for 5min at 4000r/min, transferring the supernatant into a 50mL volumetric flask, carrying out constant volume determination to 50mL by using the 0.1% formic acid methanol solution, shaking uniformly, taking 1mL of extract, and passing through a 0.22 mu m filter membrane for determination by an ultra-high performance liquid chromatograph.
Accurately weighing 1.00g of sample (dry sample) in a 50mL centrifuge tube, adding 40mL of 70% methanol aqueous solution, carrying out ultrasonic extraction for 30min at 25 ℃ under the power of 100W in an ultrasonic cleaner, centrifuging for 5min at 4000r/min, transferring supernatant into a 50mL volumetric flask, carrying out constant volume to 50mL by using the 70% methanol aqueous solution, shaking uniformly, taking 1mL of extract nitrogen for blow-drying, re-dissolving by using 1mL of formic acid-methanol solution (the volume fraction of formic acid is 0.1%), and carrying out determination by using an ultra-high performance liquid chromatograph after passing through a 0.22 mu m filter membrane.
Vortex oscillation method: accurately weighing 2.00g of sample (fresh sample) into a 50mL centrifuge tube, adding 40mL of the 0.1% formic acid methanol solution, performing vortex extraction for 30min by a vortex instrument (the rotation speed is 2000r/min, the temperature is room temperature), centrifuging for 5min at 4000r/min, transferring the supernatant into a 50mL volumetric flask, performing constant volume to 50mL by using the 0.1% formic acid methanol solution, shaking uniformly, taking 1mL of extract, and passing through a 0.22 mu m filter membrane for determination by an ultra-high performance liquid chromatograph.
Accurately weighing 1.00g of sample (dry sample) into a 50mL centrifuge tube, adding 40mL of 70% methanol aqueous solution, performing vortex extraction for 30min by a vortex instrument (the rotating speed is 2000r/min, the temperature is room temperature), centrifuging for 5min at 4000r/min, transferring supernatant into a 50mL volumetric flask, performing constant volume to 50mL by using the 70% methanol aqueous solution, shaking uniformly, drying 1mL of extract nitrogen by blowing, re-dissolving by using 1mL of formic acid-methanol solution (the volume fraction of formic acid is 0.1%), and passing through a 0.22 mu m filter membrane for determination by an ultra-high performance liquid chromatograph.
Shaking table oscillation method: accurately weighing 2.00g of sample (fresh sample) into a 50mL centrifuge tube, adding 40mL of the 0.1% formic acid methanol solution, shaking and extracting for 30min by a shaking table instrument (the rotating speed is 200r/min, the temperature is room temperature), centrifuging for 5min at 4000r/min, transferring the supernatant into a 50mL volumetric flask, fixing the volume to 50mL by using the 0.1% formic acid methanol solution, shaking uniformly, taking 1mL of extract, and passing through a 0.22 mu m filter membrane for determination by an ultra-high performance liquid chromatograph.
Accurately weighing 1.00g of sample (dry sample) into a 50mL centrifuge tube, adding 40mL of 70% methanol aqueous solution, shaking and extracting for 30min by a shaking table instrument (the rotating speed is 200r/min, the temperature is room temperature), centrifuging for 5min at 4000r/min, transferring supernatant into a 50mL volumetric flask, fixing the volume to 50mL by using the 70% methanol aqueous solution, shaking uniformly, taking 1mL of extract for drying, redissolving by using 1mL of formic acid-methanol solution (the volume fraction of formic acid is 0.1%), and passing through a 0.22 mu m filter membrane for determination by an ultra-high performance liquid chromatograph.
The results of the 3 methods are shown in Table 5. The results show that the 3 methods have no obvious difference in the extraction effects of ergothioneine in dried tremella, fresh flammulina velutipes, fresh pleurotus eryngii and fresh shiitake mushrooms. However, the shaking table oscillation method and the vortex oscillation method cannot control the temperature and can only be carried out at room temperature, while the ultrasonic method can control the temperature and has more stable extraction efficiency, so the ultrasonic method is adopted for extraction.
TABLE 5 Effect of different extraction methods on ergothioneine extraction
Unit: mg/g
Example 7 determination of the extraction time of ergothioneine in edible fungi
This example discusses the effect of ultrasonic extraction on ergothioneine extraction at 5min, 10min, 20min, 30min and 40min, and the results are shown in Table 6. As can be seen from the table, after 10min of ultrasonic treatment, the content of ergothioneine in the samples does not change obviously even if the extraction time is prolonged by taking needle mushrooms, pleurotus eryngii and mushrooms as representative fresh samples, so the extraction time is selected for 10 min; taking tremella as a representative dry sample, and after the ultrasonic treatment is carried out for 30min, even if the extraction time is prolonged, the content of ergothioneine in the sample has no obvious change, so that the extraction time is selected for 30 min.
TABLE 6 Effect of different extraction times on the ergothioneine extraction Effect
Unit: mg/g
According to the analysis results of the embodiments 5-7, the method for extracting ergothioneine from edible fungi is finally determined as follows:
fresh sample preparation: accurately weighing 2.00g of sample into a 50mL centrifuge tube, adding 40mL of the 0.1% formic acid methanol solution, carrying out ultrasonic extraction for 10min at 25 ℃ under the power of 100W in an ultrasonic cleaner, centrifuging for 5min at 4000r/min, transferring the supernatant into a 50mL volumetric flask, carrying out constant volume to 50mL by using the 0.1% formic acid methanol solution, shaking uniformly, taking 1mL of extract, and passing through a 0.22 mu m filter membrane for determination by an ultra-high performance liquid chromatograph.
Drying a sample: accurately weighing 1.00g of sample in a 50mL centrifuge tube, adding 40mL of 70% methanol aqueous solution, carrying out ultrasonic extraction for 30min at 25 ℃ under the power of 100W in an ultrasonic cleaner, centrifuging for 5min at 4000r/min, transferring supernatant into a 50mL volumetric flask, carrying out constant volume to 50mL by using the 70% methanol aqueous solution, shaking uniformly, taking 1mL of extract nitrogen for drying, re-dissolving by using 1mL of formic acid-methanol solution (the volume fraction of formic acid is 0.1%), and passing through a 0.22 mu m filter membrane for determination by an ultra-high performance liquid chromatograph.
Example 8 Called recovery of ergothioneine from edible fungi
And (3) performing an addition recovery test by adopting a matrix and standard addition method, and determining the recovery rate and the accuracy of the method. Selecting dry tremella, fresh mushroom, fresh needle mushroom and fresh pleurotus eryngii with known ergothioneine content, adding 3 mass concentrations into each sample, preparing 6 parallel samples for each mass concentration, simultaneously measuring the ergothioneine content in a control sample, determining the measured amount of the added ergothioneine by using a subtraction method, and comparing the measured amount with the added amount to calculate the addition recovery rate of the ergothioneine. The ergothioneine content of the four edible fungi is greatly different, so that the following rules are followed when the four edible fungi are added and recovered: and performing labeling recovery according to the content of the labeled sample by 0.1 time, 0.5 time and 1 time, and performing 6 parallels on each labeled sample. The detailed results are shown in Table 7. The results in the table show that the standard recovery rates of the ergothioneine in the edible fungus samples are all 80.0-120.0%, and the method is proved to be high in accuracy and capable of meeting the analysis requirements.
TABLE 7 ergothioneine spiking recovery test results
Example 9 repeated experiments on the determination of ergothioneine in edible fungi
The dry tremella, fresh mushroom, fresh needle mushroom and fresh pleurotus eryngii samples are respectively subjected to parallel determination for 5 times according to the method, and the arithmetic mean, the intra-day variation coefficient and the inter-day variation coefficient are obtained, so that the result shows that the method has better precision (see table 8). As can be seen from table 8, the absolute difference in test results between the replicates in the laboratory did not exceed 15% of the arithmetic mean.
TABLE 8 ergothioneine repeatability test results
Example 10 determination of ergothioneine content in edible fungi
(1) Sample preparation
Fresh sample: the fresh sample is purchased in a certain supermarket or a production base in Shanghai; taking a fresh sample of edible fungi with a weight of not less than 1000g, lightly wiping off surface attachments with clean gauze, cutting, placing in a multifunctional food processor for crushing, and storing in a sealed container at-18 ℃ for later use.
Drying the sample: purchasing dry samples from bagged or boxed commodities in a certain supermarket at Shanghai, and counting sample sources according to the production places marked on the commodity packages; taking not less than 200g of dried edible fungus sample, placing in a sample pulverizer to pulverize, passing through a 425 μm standard mesh screen to prepare a sample to be tested, and storing in a sealed container at normal temperature for later use.
(2) Extraction of
Fresh sample: accurately weighing 2.00g of sample into a 50mL centrifuge tube, adding 40mL of the 0.1% formic acid methanol solution, carrying out ultrasonic extraction for 10min at 25 ℃ under the power of 100W in an ultrasonic cleaner, centrifuging for 5min at 4000r/min, transferring the supernatant into a 50mL volumetric flask, carrying out constant volume to 50mL by using the 0.1% formic acid methanol solution, shaking uniformly, taking 1mL of extract, and passing through a 0.22 mu m filter membrane for determination by an ultra-high performance liquid chromatograph.
Drying the sample: accurately weighing 1.00g of sample in a 50mL centrifuge tube, adding 40mL of 70% methanol aqueous solution, carrying out ultrasonic extraction for 30min at 25 ℃ under the power of 100W in an ultrasonic cleaner, centrifuging for 5min at 4000r/min, transferring supernatant into a 50mL volumetric flask, carrying out constant volume to 50mL by using 70% methanol aqueous solution, shaking uniformly, taking 1mL of extracting solution for drying, re-dissolving by using 1mL of 0.1% formic acid methanol solution, and passing through a 0.22 mu m filter membrane for determination by using an ultra-high performance liquid chromatograph.
(3) Measurement of
Chromatographic conditions: a chromatographic column: an XBridge BEH HILIC column (100 × 2.1mm, 1.7 μm), or a comparable-specification chromatographic column; column temperature: 40 ℃; mobile phase: the A phase is formic acid-acetonitrile solution (formic acid volume fraction is 0.1%), the B phase is formic acid-water solution (formic acid volume fraction is 0.1%), and the volume ratio of the A phase to the B phase is 88: 12; flow rate: 0.4 mL/min; detection wavelength: 262 nm; sample introduction amount: 3 μ L.
Drawing a standard curve: and (3) measuring by using a ergothioneine series standard working solution by referring to the chromatographic conditions, and drawing a standard curve regression equation of the ergothioneine by taking the mass concentration of the standard working solution as a horizontal coordinate and taking a peak area as a vertical coordinate.
Quantitative determination: and (4) performing qualitative determination according to the retention time, wherein the relative deviation of the retention time of the sample and the standard substance is not more than 2%, and performing quantitative determination by an external standard method. And (3) the response value of ergothioneine in the sample liquid to be detected is within the range of the standard curve, and if the response value exceeds the range of 1.00-50.0 mg/L of the standard working curve, the sample liquid is diluted and then subjected to sample injection analysis.
Fourthly, blank test: the procedure was followed except for the non-weighed samples.
Calculating a result: the ergothioneine content in the test sample is calculated as mass fraction w, and the value is expressed in milligrams per gram (mg/g) according to the formula (1).
4. Determination of ergothioneine in edible fungi
The ergothioneine content of the edible fungi was determined according to the above procedures and procedures, and the results are shown in Table 9.
TABLE 9 analysis results of ergothioneine content in edible fungus samples
Note: in Table 9, "/" indicates no detection.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for detecting the ergothioneine content in edible fungi comprises the following steps:
(1) extracting the edible fungi to be detected by adopting an extraction solvent to obtain an extracting solution; the extraction is ultrasonic extraction or oscillation extraction; wherein the edible fungi to be detected comprise fresh samples or dry samples; when the fresh sample is extracted, the adopted extraction solvent is formic acid-methanol solution, formic acid-water solution or formic acid-methanol-water solution; when the dry sample is extracted, the adopted extraction solvent is methanol-water solution;
(2) performing hydrophilic interaction-ultra high performance liquid chromatography analysis on the extracting solution obtained in the step (1) to obtain an edible fungus liquid phase analysis result to be detected; obtaining the ergothioneine content of the edible fungi to be detected according to the standard curve of the ergothioneine content and the analysis result of the edible fungi liquid to be detected; wherein the chromatographic conditions of the ultra-high performance liquid chromatography comprise:
a chromatographic column: an XBridge BEH HILIC column or a Zic HILIC column;
mobile phase: the mobile phase comprises a phase A and a phase B, wherein the phase A is a formic acid-acetonitrile solution, and the volume fraction of formic acid in the formic acid-acetonitrile solution is 0.05-0.2%; the phase B is a formic acid-water solution, and the volume fraction of formic acid in the formic acid-water solution is 0.05-0.2%; the volume ratio of the phase A to the phase B is 80: 20-90: 10;
a detector: an ultraviolet detector or a diode array detector.
2. The method according to claim 1, wherein the edible fungi to be tested comprise oyster mushroom, white jade mushroom, pleurotus eryngii, shiitake mushroom, flammulina velutipes, straw mushroom, chicken mushroom, hypsizygus marmoreus, agaricus bisporus, hypsizygus marmoreus, stropharia rugoso-annulata, tremella, ganoderma lucidum, cordyceps militaris, dictyophora phalloidea, hericium erinaceus, agaricus blazei, agaricus, agrocybe cylindracea, shiitake mushroom or morchella esculenta.
3. The method according to claim 1, wherein in the step (1), the volume fraction of formic acid in the formic acid-methanol solution is 0.1%, the volume fraction of formic acid in the formic acid-water solution is 0.1%, the volume fraction of formic acid in the formic acid-methanol-water solution is 0.1%, and the volume fraction of methanol is 30-70%; the volume fraction of methanol in the methanol-water solution was 70%.
4. The method according to any one of claims 1 to 3, wherein the ultrasonic extraction of the edible fungi to be tested comprises the following steps:
crushing the edible fungi to be detected, mixing the obtained crushed sample with an extraction solvent, and extracting under an ultrasonic condition to obtain an extraction slurry;
and carrying out solid-liquid separation on the extraction slurry to obtain an extracting solution.
5. The method according to claim 4, wherein when the edible fungi to be tested is a fresh sample, the crushed sample is in a slurry state, and the dosage ratio of the crushed sample to the extraction solvent is (1.00-3.00) g: (30-90) mL; the extraction temperature is 25-40 ℃, the extraction time is 5-20 min, and the extraction power is 50-120W.
6. The method according to claim 4, wherein when the edible fungi to be tested is a dry sample, the particle size of the crushed sample is less than 425 μm, and the using amount ratio of the crushed sample to the extraction solvent is (0.50-2.00) g: (30-90) mL; the extraction temperature is 25-40 ℃, the extraction time is 20-40 min, and the extraction power is 50-120W.
7. The method according to claim 4, wherein the solid-liquid separation mode is centrifugation, the rotation speed of the centrifugation is 3000-5000 r/min, and the time of the centrifugation is 3-10 min.
8. The method of claim 1, wherein the flow rate of the mobile phase is 0.4 mL/min.
9. The method of claim 1, wherein the column temperature of the chromatography column is 40 ℃.
10. The method of claim 1, wherein the hydrophilic interaction-ultra high performance liquid chromatography is performed at a detection wavelength of 262 nm.
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