Detection method for the content of bioactive peptides in wine

Abstract

The present invention belongs to the field of detection technology, and particularly relates to a detection method for the content of bioactive peptides in wine. Aiming at the technical problems that there are few reports on the detection method for the content of bioactive peptides in wine and the detection sensitivity is low, the present invention provides a detection method for the content of bioactive peptides in wine, which is to purify and enrich the bioactive peptides in wine through an SPE column, and then perform qualitative and quantitative determination by using an ultra-high performance liquid chromatography tandem mass spectrometer. The method of the present invention can accurately determine the content of bioactive peptides in wine samples, provide reliable technical support for the analysis and research of bioactive substances in wine, and has important significance for studying the health promotion effect of wine.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a method for detecting the content of bioactive peptides in wine. Background Art

[0002] 98% of the components in Chinese liquor are water and ethanol, and the other approximately 2% of trace components determine the flavor type and style of Chinese liquor and play bioactive functions. The trace components mainly include volatile components such as alcohols, phenols, acids, esters, nitrogen-containing compounds and sulfur-containing compounds. Among them, the types, aroma thresholds, interactions, etc. of volatile components in different flavor type Chinese liquors have been systematically reported; non-volatile components such as free amino acids, minerals, vitamins, peptides, etc., as important trace components in Chinese liquor, have gradually become the research focus of scholars. Existing research has found that a tripeptide PHP was isolated and identified from the original liquor of Gujing strong flavor type Chinese liquor and the original liquor of Guojing sesame flavor type Chinese liquor, and 4 polypeptides AKRA, DRAR, PPDG and PHP were identified from the original liquor of Guojing sesame flavor type Chinese liquor and the original liquor of Jingzhi sesame flavor type Chinese liquor, among which AKRA and DRAR have very strong antioxidant capabilities. Moreover, undistilled wines such as yellow rice wine and rice wine contain more and richer components such as bioactive peptides.

[0003] Therefore, the separation, identification and bioactivity verification of peptides in wine are an important direction for the research of healthy Chinese liquor, providing scientific evidence for the health promotion effect of liquor. However, at present, there are few reported methods for determining the content of bioactive peptides in wine, and the detection precision and accuracy still need to be improved. Summary of the Invention

[0004] Aiming at the technical problems that there are few reported methods for detecting the content of bioactive peptides in wine and the detection sensitivity is low, the present invention provides a method for detecting the content of bioactive peptides in wine, which is to purify and enrich the bioactive peptides in wine through an SPE cartridge, and then perform qualitative and quantitative determination by an ultra-high performance liquid chromatography tandem mass spectrometer.

[0005] The specific technical solution is as follows:

[0006] The method for detecting the content of bioactive peptides in wine includes the following steps:

[0007] (1) Preparation of the sample to be tested: After the wine is pretreated with an SPE cartridge, the sample to be tested is obtained;

[0008] (2) Preparation of the mixed standard working solution of bioactive peptides: Gradiently prepare the mixed standard working solution of bioactive peptides;

[0009] (3) Detection of the sample to be tested: Use ultra-high performance liquid chromatography-mass spectrometry to determine the bioactive peptide standard working solution and the sample to be tested, and quantify by the external standard method.

[0010] Among them, in step (1), the pretreatment method is as follows: After the wine passes through an activated SPE small column, it is rinsed, eluted, dried, re-dissolved with water, mixed evenly and then filtered to obtain the sample to be tested.

[0011] Preferably, 10 - 100 mL of the wine sample, after being enriched by the SPE small column, has a volume of 2 mL.

[0012] Further, in the pretreatment, the SPE small column is activated successively with 3 - 10 mL of water and 3 - 10 mL of 100% methanol.

[0013] Further, in the pretreatment, it is rinsed with 3 - 10 mL of water, eluted with 3 - 10 mL of 100% methanol, and concentrated to dryness by nitrogen blowing.

[0014] Further, in the pretreatment, the amount of water for re-dissolution is 1 - 5 mL.

[0015] Further, in the pretreatment, it is filtered with a 0.22 μm filter membrane.

[0016] Among them, the source of the wine is at least one of distilled wine and fermented wine.

[0017] Preferably, the distilled wine is at least one of Chinese liquor, brandy, whisky, vodka, rum, gin, tequila or fruit distilled wine.

[0018] Preferably, the fermented wine is at least one of yellow rice wine, beer, wine, fruit wine, sake, rice wine, raw wine or milk wine.

[0019] Among them, the active peptides include at least one of YE, AAPK, TPF, DRAR, AYI and DREI.

[0020] The amino acid sequence of the active peptide segment YE is tyrosine - glutamic acid, the amino acid sequence of the peptide segment AAPK is alanine - alanine - proline - lysine, the amino acid sequence of the peptide segment TPF is threonine - proline - phenylalanine, the amino acid sequence of the peptide segment DRAR is aspartic acid - arginine - alanine - arginine, the amino acid sequence of the peptide segment AYI is alanine - tyrosine - isoleucine, and the amino acid sequence of the peptide segment DREI is aspartic acid - arginine - glutamic acid - isoleucine.

[0021] Among them, in step (2), the gradient concentrations of the active peptide mixed standard working solution are 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 50 μg / L.

[0022] Among them, in step (3), the ultra - performance liquid chromatography - mass spectrometry method uses an ultra - performance liquid chromatography tandem mass spectrometer, and its chromatographic conditions are as follows:

[0023] Chromatographic column: Waters C 18 or T3 chromatographic column;

[0024] Preferably, the chromatographic column specifications are: 50mm×2.1mm, 1.7μm; 100mm×2.1mm, 1.7μm or 150mm×2.1mm, 1.7μm;

[0025] Flow rate: 0.2 mL / min;

[0026] Column temperature: 35 °C;

[0027] Sample injection volume: 5 μL;

[0028] Mobile phase A is 0.05% - 0.2% formic acid solution, mobile phase B is acetonitrile, and the flow rate is 0.2 - 0.4 mL / min;

[0029] Preferably, mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, and the flow rate is 0.2 mL / min;

[0030] The elution conditions are that mobile phase B is 2% - 5% from 0 - 3 min, mobile phase B is increased to 20% from 3 - 5 min, mobile phase B is increased to 40% from 5 - 7 min, mobile phase B is increased to 100% from 7 - 8 min, and mobile phase B is restored to the initial state of 2% and maintained for 1 min from 8 - 9 min.

[0031] The mass spectrometry conditions are as follows:

[0032] Electrospray ionization source, positive ion mode ESI + ;

[0033] Capillary voltage 3.0 kV, cone voltage 50 kV;

[0034] Desolvation temperature 350 °C;

[0035] Desolvation gas flow rate 700 L / min; cone counter gas flow rate 150 L / hr;

[0036] Collision cell pressure 7.0 Bar.

[0037] Beneficial effects: After the bioactive peptides in the wine are purified and enriched by an SPE cartridge, ultra-high performance liquid chromatography tandem mass spectrometry is used for qualitative and quantitative determination. Under the conditions of the present invention, the correlation coefficient of the regression equation of the six bioactive peptide standard working solutions is >0.995, and the detection limit is 0.1 μg / L to 2.0 μg / L. The method of the present invention can accurately determine the content of bioactive peptides in wine samples, providing reliable technical support for the analysis and research of bioactive substances in wine. At the same time, the wine samples are purified and enriched by an SPE cartridge, avoiding the loss of bioactive peptides caused by conditions such as temperature and negative pressure. The present invention can be applied to the determination of peptide content in wine, which is of great significance for studying the health promotion effect of wine. Description of the Drawings

[0038] Figure 1 It is the spectrogram of the active peptide YE standard product;

[0039] Figure 2 It is the spectrogram of the active peptide AAPK standard product;

[0040] Figure 3 It is the spectrogram of the active peptide TPF standard product;

[0041] Figure 4 It is the spectrogram of the active peptide DRAR standard product;

[0042] Figure 5 It is the spectrogram of the active peptide AYI standard product;

[0043] Figure 6 It is the spectrogram of the active peptide DREI standard product. Detailed Embodiments

[0044] After the bioactive peptides in the wine are purified and enriched by an SPE cartridge, ultra-high performance liquid chromatography tandem mass spectrometry is used for qualitative and quantitative determination. At present, there is no technical solution for pretreating wine with an SPE cartridge and using ultra-high performance liquid chromatography tandem mass spectrometry for qualitative and quantitative detection of active peptides YE, AAPK, TPF, DRAR, AYI, and DREI.

[0045] The specific embodiments of the present invention for pretreating wine samples with an SPE cartridge include:

[0046] Take the wine sample and mix it evenly. The SPE cartridge is activated with 3 - 10 mL of water and 3 - 10 mL of 100% methanol. Transfer 10 - 100 mL of the wine sample to the SPE cartridge for purification under normal pressure. When no more sample flows out, wash the cartridge with 3 - 10 mL of water, and then elute the SPE cartridge with 3 - 10 mL of 100% methanol. Collect the eluate, dry it with nitrogen, add 1 - 5 mL of water for reconstitution, mix evenly, and filter with a 0.22 μm filter membrane to obtain the test solution.

[0047] The wine sample of the present invention is purified and enriched by SPE cartridges, which can avoid the loss of bioactive peptides caused by conditions such as temperature and negative pressure.

[0048] For the wine sample pretreated by SPE cartridges in the present invention, the sample and the standard working solution are determined according to the conditions of ultra-high performance liquid chromatography tandem mass spectrometry. The concentration of the sample solution is corrected with the standard curve, and the external standard method is used for quantification.

[0049] In one embodiment of the present invention, the chromatographic conditions adopted are: chromatographic column: waters C 18 or T3 chromatographic column; flow rate: 0.2 mL / min; column temperature: 35 °C; injection volume: 5 μL; mobile phase A is 0.05% - 0.2% formic acid solution, mobile phase B is acetonitrile, and the flow rate is 0.2 - 0.4 mL / min; the elution conditions are that mobile phase B is 2% - 5% from 0 to 3 min, mobile phase B is increased to 20% from 3 to 5 min, mobile phase B is increased to 40% from 5 to 7 min, mobile phase B is increased to 100% from 7 to 8 min, and mobile phase B is restored to the initial state of 2% and maintained for 1 min from 8 to 9 min.

[0050] In a preferred embodiment of the present invention, the specifications of the chromatographic column are: 50 mm × 2.1 mm, 1.7 μm; 100 mm × 2.1 mm, 1.7 μm or 150 mm × 2.1 mm, 1.7 μm;

[0051] In a preferred embodiment of the present invention, mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, and the flow rate is 0.2 mL / min.

[0052] In one embodiment of the present invention, the mass spectrometry conditions adopted are: electrospray ionization source, positive ion mode ESI + ; capillary voltage 3.0 kV, cone voltage 50 kV; desolvation temperature 350 °C; desolvation gas flow rate 700 L / min; cone counter gas flow rate 150 L / hr; collision cell pressure 7.0 Bar.

[0053] The solution of the present invention will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For those reagents or instruments not specified regarding the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0054] Example 1 Determination of the Contents of 6 Bioactive Peptides in Wine

[0055] 1. Treatment of Wine Sample

[0056] Take the wine sample and mix it thoroughly. Activate the SPE column with 3 mL of methanol and 3 mL of water. Transfer 50 mL of the wine sample to the SPE column for purification under normal pressure. When no more sample flows out, wash the column with 5 mL of water, then elute the SPE column with 5 mL of methanol. Collect the eluate, dry it with nitrogen, re-dissolve it in 2 mL of water, mix well, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0057] 2. Instrument determination

[0058] Determine the sample and the standard working solution according to the conditions of the ultra-high performance liquid chromatography tandem mass spectrometer. The standard working solution is prepared as shown in Table 3 below. Calibrate the concentration of the sample solution with the standard curve and quantify by the external standard method.

[0059] Chromatographic column: waters C 18 Chromatographic column (100 mm × 2.1 mm, 1.7 μm); Flow rate: 0.2 mL / min; Column temperature: 35 °C; Injection volume: 5 μL.

[0060] Mobile phase: Mobile phase A is 0.1% formic acid solution, and mobile phase B is acetonitrile. The flow rate is 0.2 mL / min;

[0061] The elution conditions are as follows: from 0 to 3 min, mobile phase B is 2% - 5%; from 3 to 5 min, mobile phase B is increased to 20%; from 5 to 7 min, mobile phase B is increased to 40%; from 7 to 8 min, mobile phase B is increased to 100%; from 8 to 9 min, mobile phase B returns to the initial state of 2% and is maintained for 1 min.

[0062] Mass spectrometry conditions: The ionization mode is electrospray ionization source, positive ion mode ESI + ; Capillary voltage 3.0 kV, cone voltage 50 kV, desolvation temperature 350 °C, desolvation gas flow rate 700 L / min, cone counter gas flow rate 150 L / hr, collision cell pressure 7.0 Bar.

[0063] Table 1 Mass spectrometry parameters of 6 active peptides

[0064] Serial number Polypeptide amino acid sequence Parent ion Daughter ion Orifice voltage Collision energy 1 YE 311.3 136.2，148.2* 40 15，15 2 AAPK 386.5 226.5，244.5* 40 20，20 3 TPF 364.5 199.5，263.5* 40 15，15 4 DRAR 259.5 202.0，247.0* 40 10，15 5 AYI 366.4 207.4，235.4* 40 12，12 6 DREI 532.5 255.5，304.5* 40 30，30

[0065] 3. Linear range

[0066] The 6 active peptides are prepared by biochemical synthesis, and the purity of each is greater than 95%.

[0067] Accurately weigh 1 mg of each active peptide standard, dissolve it in water and make up the volume to 10 mL to prepare a single-standard stock solution with a concentration of 100 mg / L, and store it at -18°C. Accurately pipette each single-standard stock solution, dilute it with water, and prepare a mixed standard working solution with concentrations of 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, and 50 μg / L. Filter it through a 0.22 μm filter membrane and determine it by liquid chromatography-mass spectrometry.

[0068] 4. Detection limit and quantification limit

[0069] Add the active peptide to be detected to the blank matrix. When the signal-to-noise ratio of the added concentration measurement is 3 (S / N = 3), calculate the quantification limit based on three times the signal-to-noise ratio. The detection limit (LOD) and quantification limit (LOQ) of each active peptide are shown in Table 2.

[0070] Table 2 Linear range, detection limit and quantification limit of active peptides

[0071]

[0072]

[0073] 5. Recovery rate and precision

[0074] The accuracy of the method was evaluated by two parameters: spiked recovery rate and determination precision. The recovery rate of the method was verified by spiking the blank matrix. The concentrations of the three spiking levels were 5 μg / L, 10 μg / L, and 25 μg / L respectively. Each spiking level was determined in triplicate, and the average recovery rate and the corresponding standard deviation were calculated. The verification of the determination precision of the method was carried out by performing six parallel determinations at the spiking level of 10 μg / L, and the determination standard deviation was calculated.

[0075] Table 3 Determination results of recovery rate and precision of active peptides

[0076]

[0077] 6. Sample determination

[0078] Collect 9 kinds of wine samples, including 3 kinds of white wine, 3 kinds of raw wine, and 3 kinds of yellow rice wine, and determine the content of active peptides in them. The results are shown in Table 4 below. The content of active peptides in yellow rice wine and raw wine is higher than that in finished white wine.

[0079] Table 4 Results of active peptide content in wine

[0080]

[0081] Note: nd indicates below the detection limit.

[0082] After the above method validation, the detection method of the present invention can provide a rapid and accurate determination method for the quantification of active peptides in wine.

[0083] It should be noted that the specific features, structures, materials, or characteristics described in this specification may be combined in a suitable manner in any one or more embodiments. In addition, without contradiction, those skilled in the art may combine and combine the different embodiments described in this specification and the features of different embodiments.

Claims

1. Detection method for the content of bioactive peptides in wine, characterized in that: It includes the following steps: (1) Preparation of the sample to be tested: After the wine is pretreated with an SPE cartridge, the sample to be tested is obtained; (2) Preparation of the mixed standard working solution of bioactive peptides: Gradient preparation of the mixed standard working solution of bioactive peptides; (3) Detection of the sample to be tested: The mixed standard working solution of bioactive peptides and the sample to be tested are determined by ultra-high performance liquid chromatography-tandem mass spectrometry, and quantitative analysis is carried out by the external standard method; Among them, the bioactive peptides include YE, AAPK, TPF, DRAR, AYI, and DREI; Among them, the ultra-high performance liquid chromatography-tandem mass spectrometry method uses an ultra-high performance liquid chromatography-tandem mass spectrometer, and its chromatographic conditions are as follows: Chromatographic column: waters C 18 or T3 chromatographic column; Mobile phase A is a 0.05% - 0.2% formic acid solution, and mobile phase B is acetonitrile; The elution conditions are as follows: From 0 to 3 minutes, mobile phase B is 2% - 5%; from 3 to 5 minutes, mobile phase B is increased to 20%; from 5 to 7 minutes, mobile phase B is increased to 40%; from 7 to 8 minutes, mobile phase B is increased to 100%; from 8 to 9 minutes, mobile phase B is restored to the initial state of 2% and maintained for 1 minute; The mass spectrometry conditions are as follows:

2. The detection method of the content of bioactive peptides in wine according to claim 1, characterized in that: In step (1), the pretreatment method is: After the wine passes through the activated SPE cartridge, it is rinsed, eluted, dried, re-dissolved with water, mixed well and filtered to obtain the sample to be tested.

3. The detection method of the content of bioactive peptides in wine according to claim 2, characterized in that: 10 - 100 mL of the wine sample, after being enriched by the SPE cartridge, has a volume of 2 mL.

4. The detection method of the content of bioactive peptides in wine according to claim 2 or 3, characterized in that: In the pretreatment, 3 - 10 mL of water and 3 - 10 mL of methanol are used to activate the SPE cartridge in sequence; 3 - 10 mL of water is used for rinsing, 3 - 10 mL of methanol is used for elution, and it is concentrated to dryness by nitrogen blowing; the amount of water for re-dissolving is 1 - 5 mL; filtration is carried out using a 0.22 μm filter membrane.

5. The detection method of the content of bioactive peptides in wine according to claim 1, characterized in that: The source of the wine is at least one of distilled wine or fermented wine.

6. The detection method of the content of bioactive peptides in wine according to claim 5, characterized in that: The distilled wine is at least one of Chinese liquor, brandy, whisky, vodka, rum, gin, tequila, or fruit distilled wine; the fermented wine is at least one of yellow rice wine, beer, wine, fruit wine, sake, rice wine, raw wine, or milk wine.

7. The detection method for the content of bioactive peptides in wine according to claim 1, wherein: In step (2), the gradient concentrations of the mixed standard working solution of bioactive peptides are 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, and 50 μg / L.

8. The method for detecting the content of bioactive peptides in wine according to claim 1, wherein: In step (3), the ultra-high performance liquid chromatography-tandem mass spectrometry method uses an ultra-high performance liquid chromatography-tandem mass spectrometer, and its chromatographic conditions are as follows: The specifications of the chromatographic column are: 50 mm × 2.1 mm, 1.7 μm; 100 mm × 2.1 mm, 1.7 μm or 150 mm × 2.1 mm, 1.7 μm; Column temperature: 35 °C; Sample injection volume: 5 μL; Flow rate: 0.2 - 0.4 mL / min.

9. The detection method of the content of bioactive peptides in wine according to claim 8, characterized in that: The flow rate is 0.2 mL / min.

10. The detection method for the content of bioactive peptides in wine according to claim 8, characterized in that: The mass spectrometry conditions are as follows: Electrospray ionization source, positive ion mode ESI + ; Capillary voltage 3.0 kV, cone voltage 50 kV; Desolvation temperature 350 °C; Desolvation gas flow rate 700 L / min; cone orifice backflush gas flow rate 150 L / hr; Collision cell pressure 7.0 Bar.

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