Sample pretreatment method for detecting content of total trans-2-nonenal in malt

Abstract

The invention relates to the technical field of malt detection, and particularly discloses a sample pretreatment method for detecting the content of total trans-2-nonenal in malt. The method comprises the steps of malt crushing, enzyme deactivation treatment, low-temperature stirring extraction, centrifugal separation, pH adjustment, headspace bottle sample preparation, nitrogen blowing oxygen discharge, high-temperature potential trans-2-nonenal release, internal standard addition and the like. Through measures such as 70 DEG C constant-temperature water bath enzyme deactivation, low-temperature operation below 5 DEG C, nitrogen blowing protection and the like, the influence of enzymatic and non-enzymatic oxidation reactions on a detection result is remarkably reduced, and the detection authenticity and reliability are improved. Experiments show that the method can effectively protect the target substance, and the detection result is closer to the true value.

Description

Technical Field

[0001] The present invention belongs to the technical field of malt detection, and particularly relates to a sample pretreatment method for detecting the total content of trans-2-nonenal in malt. Background Art

[0002] Trans-2-nonenal is one of the key aging flavor substances in beer, and its content gradually increases with the extension of the storage time of finished beer. When the concentration of trans-2-nonenal in beer reaches more than 0.08 μg / L, it will endow the beer with an unpleasant smell similar to corrugated cardboard, significantly affecting the flavor and freshness of the beer. In addition, there is a synergistic effect between trans-2-nonenal and other aging flavor substances, and this synergistic effect will reduce the perception threshold of other aging flavor substances, thereby further destroying the overall flavor characteristics of the beer. It should be noted that trans-2-nonenal mainly exists in the form of binding with amines (i.e., trans-2-nonenal precursor) in the early stage of finished beer storage, and gradually transforms into the free state and releases the characteristic flavor with the extension of the storage time. Therefore, accurately detecting the total content of trans-2-nonenal in malt (including the total amount of free and bound forms) is of great significance for predicting the freshness of beer and optimizing the malt quality.

[0003] However, in the actual detection process, the measurement result of the total content of trans-2-nonenal in malt is easily significantly affected by the sample pretreatment process. Research shows that the following problems mainly exist in the sample pretreatment process: First, the enzymatic reaction of fatty acid oxidase during the malt extraction process will lead to the generation of additional trans-2-nonenal, thus interfering with the authenticity of the detection result; Second, the extraction temperature has a significant impact on the stability of trans-2-nonenal. Under high temperature conditions, trans-2-nonenal may undergo oxidative decay, resulting in an underestimation of its content; Third, a non-enzymatic oxidation reaction may occur when the sample is in contact with air, especially during the detection process of headspace-gas chromatography-mass spectrometry, and the sample needs to undergo high-temperature treatment, which further exacerbates the oxidation risk of trans-2-nonenal. Therefore, how to effectively control the influence of these factors on the detection result has become a key technical problem for accurately determining the total content of trans-2-nonenal in malt.

[0004] In view of the above problems, the prior art has not provided a systematic and efficient solution. Although some studies have tried to reduce the interference of external factors on the detection result by improving the sample pretreatment method, these methods often fail to comprehensively consider the combined effects of multiple factors such as enzymatic reaction, temperature conditions and oxygen contact, resulting in a large deviation in the detection result. Therefore, it is urgent to develop a sample pretreatment method that can effectively inhibit enzymatic oxidation and non-enzymatic oxidation reactions to improve the authenticity and reliability of the detection of the total content of trans-2-nonenal in malt. The present invention is an innovative solution proposed based on this need. Summary of the Invention

[0005] In view of the problems existing in the detection process of the total trans - 2 - nonenal content in malt, the present invention proposes an optimized sample pretreatment method. The said problems mainly include that the enzymatic reaction of fatty acid oxidase leads to a high detection result, the high - temperature conditions during extraction and detection trigger non - enzymatic oxidation reactions resulting in the loss of target substances, and the oxidation risk caused by the contact of the sample with air. Therefore, the present invention provides a systematic sample pretreatment technical solution, aiming to effectively reduce the influence of the above factors on the detection result, thereby improving the authenticity and reliability of the detection.

[0006] To solve the above problems, the present invention provides a sample pretreatment method for detecting the total trans - 2 - nonenal content in malt. The technical solution of the present invention includes the following steps: S1. Crushing the malt sample; S2. Weighing and mixing the fine malt powder with water; S3. Inactivating fatty acid oxidase; S4. Stirring extraction in an ice - water bath and centrifuging at low temperature; S5. Adjusting the pH of the malt extract to 4.0; S6. Preparing the headspace vial sample; S7. Blowing oxygen out with nitrogen; S8. Rapidly stirring and dissolving sodium chloride; S9. Releasing potential trans - 2 - nonenal at high temperature; S10. Adding the internal standard solution under the protection of nitrogen blowing; S11. Detecting with a headspace - gas chromatography - mass spectrometry instrument; S12. Data calculation. Among them, each step is carefully designed to effectively protect and extract the target substance.

[0007] In step S1, a Miag DLF U - type disk mill is used to crush the malt sample, and the disk spacing is set to 0.2 mm; the particle size range of the obtained fine malt powder is 80 - 100 mesh; to ensure that the malt sample is fully crushed into a fine powder state. The said crushing operation can significantly increase the contact area between the target substance and the solvent in the subsequent extraction process, thereby improving the extraction efficiency.

[0008] Furthermore, in step S2, 4 - 5 g of fine malt powder (accurate to 0.01 g) is weighed and placed in a stoppered iodine flask, and 40 mL of water at 72 °C is added. After sealing with a stopper and gently shaking, the fine malt powder and water are fully mixed. The use of the high - temperature water not only initially inhibits the activity of fatty acid oxidase but also ensures sufficient contact between the fine malt powder and water, laying a foundation for subsequent operations.

[0009] Particularly, in step S3, the iodine flask is placed in a 70 °C constant - temperature water bath and kept warm for 5 min, and then rapidly cooled to room temperature. The design of the high - temperature water bath achieves the complete inactivation of fatty acid oxidase through heat treatment, preventing it from continuing to catalyze the formation of trans - 2 - nonenal in subsequent operations, thereby reducing the detection error caused by enzymatic reactions.

[0010] Further, in step S4, add 1 magnetic rotor into the iodine flask, seal it with a stopper and place it in an ice-water bath. Stir at a speed of 750 rpm for 15 min, and during this period, control the water bath temperature below 5°C. After stirring, transfer the mixed mash to a centrifuge tube and centrifuge it at a low temperature of 7500 rpm for 10 min at 5°C. Separate the supernatant as the malt extract. The low-temperature operation inhibits the oxidation reaction of trans-2-nonenal and improves the extraction efficiency and stability at the same time.

[0011] In step S5, use 85% phosphoric acid to adjust the pH of the malt extract to 4.0. The design of the acidic environment helps to stabilize trans-2-nonenal, reduce its loss in subsequent operations, and thus improve the accuracy of the detection results.

[0012] Further, in step S6, add 1.5 g of sodium chloride, 1 magnetic rotor, and 5 mL of the malt extract with a pH of 4.0 into the headspace vial in sequence. The addition of sodium chloride provides a more comprehensive analysis basis for subsequent detection by improving the release efficiency of trans-2-nonenal.

[0013] In step S7, insert the needle of the nitrogen blower into the headspace vial at a position about 10 mm above the liquid level and blow nitrogen for 3 min, and try to keep the liquid level calm during this period. After nitrogen blowing is completed, lift the nitrogen blower needle to a position 10 - 20 mm above the headspace vial opening and tighten the bottle cap under nitrogen blowing protection. The nitrogen blowing operation reduces the oxidation risk of trans-2-nonenal in subsequent operations by removing oxygen in the headspace vial.

[0014] Further, in step S8, place the sealed headspace vial on a magnetic stirrer and stir it quickly to dissolve sodium chloride completely as soon as possible. Quick stirring not only improves the dissolution efficiency of sodium chloride but also further promotes the release of trans-2-nonenal.

[0015] In step S9, place the sealed headspace vial in a constant temperature water bath at 90°C for 2 h, and then place it in a refrigerator at 4°C for static cooling. The high-temperature condition promotes the conversion of potential trans-2-nonenal from the bound state to the free state, and cooling helps to stabilize the sample state and prepare for subsequent detection.

[0016] Further, in step S10, open the bottle cap under nitrogen blowing protection, add 100 μL of the internal standard solution of p-fluorobenzaldehyde with a concentration of 5 mg / L, and then tighten the bottle cap to seal. The addition of the internal standard solution is used to calibrate the detection results and improve the accuracy of quantitative analysis.

[0017] In step S11, the prepared sample is detected using a headspace-gas chromatography-mass spectrometry (HS-GC-MS). The specific detection conditions are as follows: The chromatographic column is HP-5MS (30m × 0.25mm i.d. × 0.25μm); the column temperature program is an initial temperature of 35°C held for 2 min, then increased to 100°C at a rate of 5°C / min, and then increased to 270°C at a rate of 10°C / min; the injection port mode is a split / splitless injection port, splitless mode, valve opening after 0.5 min, and the temperature is 240°C; the carrier gas is helium, and the column flow rate is 1.0 mL / min; the mass spectrometry conditions are a quadrupole mass analyzer, and the temperature is 150°C; the temperature of the electron impact ion source is 230°C, and the voltage is 70 eV; the scanning range is 33 - 280 amu, and selected ion monitoring mode is used for quantification; the headspace sampler conditions are an incubation temperature of 40°C, an extraction time of 40 min, a desorption time of 5 min, and a stirrer speed of 250 rpm.

[0018] In step S12, according to the detection results of the headspace-gas chromatography-mass spectrometry, the following formula is used for calculation:

[0019] Total trans-2-nonenal content in malt:

[0020]

[0021] Where X 2 is the concentration of trans-2-nonenal in the malt extract, in μg / L; A is the peak area of trans-2-nonenal; X s is the content of p-fluorobenzaldehyde, taking 0.1 μg / L; A s is the peak area of p-fluorobenzaldehyde; f is the relative correction factor.

[0022] Total trans-2-nonenal content in dry basis malt:

[0023]

[0024] Where X is the total trans-2-nonenal content in dry basis malt, in μg / 100g; m is the weight of the malt weighed for extraction, in g; X 1 is the moisture content of the malt, in %; X 2 is the content of trans-2-nonenal in the extract, in μg / L.

[0025] The beneficial effects of the present invention are as follows. By systematically optimizing the sample pretreatment steps, the enzymatic reaction of fatty acid oxidase and non-enzymatic oxidation reaction on the detection results are significantly reduced. The technical solution realizes the effective protection and extraction of the target substance through key steps such as enzyme inactivation treatment in a constant temperature water bath at 70 °C, low-temperature stirring extraction below 5 °C, pH 4.0 acidic environment adjustment, nitrogen blowing for oxygen removal, and high-temperature release of potential trans-2-nonenal. Experimental verification shows that the detection results of Group A1 using the method of the present invention are closer to the true value, and the higher the extraction temperature, the more obvious the attenuation of total trans-2-nonenal, further proving the scientificity and reliability of the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic flow chart of the sample pretreatment method for detecting the content of total trans-2-nonenal in malt of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The present invention provides a sample pretreatment method for detecting the content of total trans-2-nonenal in malt, in combination with Figure 1 The specific embodiments of the present invention will be described in detail. Figure 1 It is a schematic flow chart of the present invention. The Arabic numeral labels in the reference signs correspond to the equipment or operation components involved in each step. The following will be gradually described in combination with the drawings and embodiments.

[0028] Embodiment:

[0029] A sample pretreatment method for detecting the content of total trans-2-nonenal in malt is provided, and the steps are as follows:

[0030] First, use a Miag DLF U disk type crusher to crush the malt sample to be tested, set the disk spacing to 0.2 mm, and ensure that the malt sample is fully crushed into fine powder state. The purpose of this step is to increase the contact area between the target substance and the solvent in the subsequent extraction process, thereby improving the extraction efficiency. In actual operation, the crushed malt fine powder needs to be immediately transferred to a stoppered iodine flask to avoid oxidation reactions caused by long-term exposure to air.

[0031] Subsequently, weigh 4 - 5 g of fine malt powder, accurate to 0.01 g, and place it in a stoppered iodine flask. Add 40 mL of water at 72 °C to the iodine flask, stopper and seal it, then gently shake to fully mix the fine malt powder with water. The use of high-temperature water not only initially inhibits the activity of fatty acid oxidase but also ensures sufficient contact between the fine malt powder and water, laying the foundation for subsequent operations. After mixing, place the iodine flask in a 70 °C constant-temperature water bath and keep it warm for 5 min to achieve complete inactivation of fatty acid oxidase. This heat treatment process destroys the spatial structure of fatty acid oxidase, completely preventing its ability to catalyze the formation of trans-2-nonenal, thereby reducing the detection error caused by enzymatic reactions. After the insulation ends, quickly cool the iodine flask to room temperature, and the cooling process can be accelerated by using a normal-temperature water bath or rinsing the outer wall with normal-temperature water.

[0032] After completing the enzyme inactivation treatment, add 1 magnetic rotor to the iodine flask and stopper and seal it. Subsequently, place the iodine flask in an ice-water bath, control the water bath temperature not exceeding 5 °C, and at the same time use a magnetic stirrer to stir at a speed of 750 rpm for 15 min. The design of the low-temperature water bath aims to inhibit the oxidation reaction of trans-2-nonenal and at the same time promote the release of the target substance from the malt matrix into the aqueous phase. After stirring, transfer the mixed mash to a centrifuge tube and centrifuge at 7500 rpm for 10 min at 5 °C, and separate the supernatant as the malt extract. The low-temperature centrifugation operation further reduces the risk of loss of trans-2-nonenal during the separation process and at the same time improves the clarity of the extract.

[0033] After obtaining the malt extract, adjust its pH to 4.0 with 85% phosphoric acid. The introduction of an acidic environment helps to stabilize the chemical properties of trans-2-nonenal and prevent its degradation or transformation during subsequent operations. After adjustment, pipette 5 mL of the malt extract into a 20 mL headspace vial, and sequentially add 1.5 g of sodium chloride and 1 magnetic rotor to the headspace vial. The addition of sodium chloride increases the release efficiency of trans-2-nonenal by increasing the ionic strength of the solution, providing a more comprehensive analysis basis for subsequent detection.

[0034] Next, perform the nitrogen purging and oxygen removal operation. Insert the nitrogen purging needle into the headspace vial about 10 mm above the liquid level and purge with nitrogen for 3 min. Try to keep the liquid surface calm during this process to avoid splashing of the sample or mixing of gas caused by violent disturbance. After nitrogen purging, lift the nitrogen purging needle to a position 10 - 20 mm above the headspace vial opening and tighten the bottle cap under nitrogen purging protection. This step significantly reduces the oxidation risk of trans-2-nonenal in subsequent operations by removing oxygen from the headspace vial. Subsequently, place the sealed headspace vial on a magnetic stirrer and stir quickly to dissolve sodium chloride completely as soon as possible. Quick stirring not only improves the dissolution efficiency of sodium chloride but also further promotes the release of trans-2-nonenal.

[0035] After stirring is completed, place the sealed headspace vial in a constant temperature water bath at 90 °C for 2 h. The design of the high temperature condition is to promote the conversion of potential trans-2-nonenal from the bound state to the free state, thereby improving the accuracy of the detection results. After the heat preservation is completed, place the headspace vial in the refrigerator and let it stand and cool at 4 °C to stabilize the sample state and prepare for subsequent detection.

[0036] After cooling is completed, open the bottle cap under the protection of nitrogen blowing, and add 100 μL of p-fluorobenzaldehyde internal standard solution with a concentration of 5 mg / L, then tighten the bottle cap and seal it. The addition of the internal standard solution is used to calibrate the detection results and improve the accuracy of quantitative analysis. Finally, use a headspace-gas chromatography-mass spectrometry instrument to detect the prepared sample. The specific detection conditions are as follows: The chromatographic column is HP-5MS (30 m × 0.25 mm i.d. × 0.25 μm); the column temperature program is an initial temperature of 35 °C for 2 min, rising to 100 °C at a rate of 5 °C / min, and then rising to 270 °C at a rate of 10 °C / min; the injection port mode is a split / splitless injection port, splitless mode, the valve is opened after 0.5 min, and the temperature is 240 °C; the carrier gas is helium, and the column flow rate is 1.0 mL / min; the mass spectrometry conditions are a quadrupole mass analyzer, with a temperature of 150 °C; the temperature of the electron impact ion source is 230 °C, and the voltage is 70 eV; the scanning range is 33 - 280 amu, and the selected ion mode is used for quantification; the headspace sampler conditions are a heat preservation temperature of 40 °C, an extraction time of 40 min, a desorption time of 5 min, and a stirrer speed of 250 rpm.

[0037] According to the detection results of the headspace-gas chromatography-mass spectrometry instrument, calculate the total trans-2-nonenal content in malt according to the following formula:

[0038] Concentration of trans-2-nonenal in malt extract:

[0039]

[0040] Where X 2 is the concentration of trans-2-nonenal in the malt extract, in μg / L; A is the peak area of trans-2-nonenal; X s is the content of p-fluorobenzaldehyde, taking 0.1 μg / L; A s is the peak area of p-fluorobenzaldehyde; f is the relative correction factor.

[0041] Total trans-2-nonenal content in dry malt

[0042]

[0043] Where X is the total trans-2-nonenal content in dry malt, in μg / 100 g; m is the weight of the malt weighed for malt extraction, in g; X 1 is the moisture content of the malt, in %; X2 It is the content of trans-2-nonenal in the extract, with the unit of μg / L.

[0044] Through the above systematic sample pretreatment technical solution, the present invention realizes the effective control of the enzymatic reaction of fatty acid oxidase, non-enzymatic oxidation reaction, and the oxidation risk caused by the contact of the sample with air.

[0045] To verify the influence of different pretreatment conditions on the content of total trans-2-nonenal, the experiments in this example were divided into Group A and Group B, and different pretreatment conditions were used for comparative analysis. Through this grouping design, the specific influence of pretreatment conditions on the content of target compounds can be more intuitively revealed, providing a scientific basis for subsequent process optimization. In Group A, 4-5 g of fine malt powder (accurate to 0.01 g) was weighed and placed in a stoppered iodine flask. 40 mL of water at 72 °C was added, and after stoppered and sealed, it was gently shaken to mix the malt powder and water evenly. Then the iodine flask was placed in a 70 °C constant temperature water bath and kept warm for 5 min. Four groups of samples were prepared according to this operation and were respectively labeled as A1, A2, and A3. In Group B, 4-5 g of fine malt powder (accurate to 0.01 g) was weighed and placed in a stoppered iodine flask. 40 mL of water at 3 °C was added, and after stoppered and sealed, it was gently shaken to mix the malt powder and water evenly.

[0046] During the extraction process of total trans-2-nonenal, in Group A1, the iodine flask was placed in a normal temperature water bath or the outer wall was rinsed with normal temperature water to cool it to room temperature as soon as possible. 1 magnetic rotor was added to the iodine flask and stoppered and sealed, then it was placed in an ice water bath and magnetically stirred at a speed of 750 rpm for 15 min, and the water bath temperature should not be higher than 5 °C during this period. In Group A2, the iodine flask was placed in a normal temperature water bath or the outer wall was rinsed with normal temperature water to cool it to about 40 °C as soon as possible. 1 magnetic rotor was added to the iodine flask and stoppered and sealed, then it was placed in a 40 °C constant temperature water bath and magnetically stirred at a speed of 750 rpm for 15 min. In Group A3, 1 magnetic rotor was added to the iodine flask and stoppered and sealed, then it was placed in a 70 °C constant temperature water bath and magnetically stirred at a speed of 750 rpm for 15 min. In Group B, 1 magnetic rotor was added to the iodine flask and stoppered and sealed, then it was placed in an ice water bath and magnetically stirred at a speed of 750 rpm for 15 min, and the water bath temperature should not be higher than 5 °C during this period.

[0047] After extraction, use a low-temperature centrifuge to centrifuge at 7500 rpm for 10 min at 5 °C, and separate the supernatant, which is the malt extract. Then, adjust the pH of the malt extract to 4.0 with 85% phosphoric acid. Pipette 5 mL from it into a 20 mL headspace vial, and then sequentially add 1.5 g of sodium chloride and 1 magnetic stir bar to the headspace vial. Before the potential release reaction of trans-2-nonenal, insert the needle of the nitrogen blowing instrument about 10 mm above the liquid level in the headspace vial, blow nitrogen for 3 min (try to keep the liquid surface calm during nitrogen blowing), then lift the nitrogen blowing needle to a position 10 - 20 mm above the mouth of the headspace vial, and tighten the bottle cap under nitrogen blowing protection. Place the headspace vial on a magnetic stirrer and stir rapidly for 15 min to dissolve the sodium chloride.

[0048] Subsequently, place the sealed headspace vial in a constant temperature water bath at 90 °C for 2 h, and then place it in a refrigerator at 4 °C for static cooling. Under nitrogen blowing protection, open the bottle cap and add 100 μL of the internal standard solution of p-fluorobenzaldehyde with a concentration of 5 mg / L, then tighten the bottle cap to seal it, and obtain the final sample solution to be measured.

[0049] Use a headspace-gas chromatography-mass spectrometry instrument to detect the sample solution to be measured. The chromatographic column is HP-5MS (30 m × 0.25 mm i.d. × 0.25 μm). The initial column temperature is maintained at 35 °C for 2 min, then raised to 100 °C at a rate of 5 °C / min, and finally raised to 270 °C at a rate of 10 °C / min. Split / splitless injection port, splitless mode, valve opening after 0.5 min, and the temperature is 240 °C. The carrier gas is helium, and the column flow rate is 1.0 mL / min. The temperature of the quadrupole mass analyzer is 150 °C, the temperature of the electron impact ion source is 230 °C, and the voltage is 70 eV. The scanning range is 33 - 280 amu, and the selected ion mode is used for quantification. The conditions of the headspace sampler are a holding temperature of 40 °C, an extraction time of 40 min, a desorption time of 5 min, and a stirrer speed of 250 rpm. Quantitative analysis is carried out by the internal standard method. The experimental results are shown in Table 1 below:

[0050] Table 1

[0051]

[0052] According to the experimental results in Table 1, it can be known that during the extraction operation of total trans-2-nonenal, if appropriate enzyme inactivation measures are not taken, lipoxygenase will continue to function and produce more trans-2-nonenal. Therefore, the experimental data results of group A1 are slightly lower than those of group B. Comparing the results of groups A1 - A3, it can be seen that the higher the extraction temperature, the more obvious the attenuation of total trans-2-nonenal, indicating that trans-2-nonenal is very sensitive to temperature in the presence of oxygen. Experimental verification shows that the detection results of group A1 using the method of the present invention are closer to the true value, and the higher the extraction temperature, the more obvious the attenuation of total trans-2-nonenal, further proving the scientificity and reliability of the method of the present invention.

[0053] It should be noted that the above-described embodiments should be understood as illustrative and not as limiting the scope of protection of the present invention. The scope of protection of the present invention is subject to the claims. For those skilled in the art, without departing from the essence and scope of the present invention, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention.

Claims

1. A sample pretreatment method for detecting the total trans-2-nonenal content in malt, characterized in that: The following steps are involved: S1. Sample pre-treatment: Use Miag DLFU disc mill to grind malt sample, weigh 4-5g malt fine powder, add 40mL of 72℃ water to mix, inactivate enzyme in 70℃ constant temperature water bath for 5min, and then cool; S2, extraction and separation: adding a magnetic rotor to the above solution, sealing it, stirring it in an ice water bath, and then centrifuging it at 5°C and 7500 rpm for 10 min to obtain a malt extract; S3, solution adjustment and preparation: add phosphoric acid to the above malt extract to adjust the pH of the malt extract, take 5 mL of the extract and add it to a headspace bottle containing sodium chloride and a magnetic rotor; S4, oxygen removal and dissolution: insert the needle of the nitrogen blower into the headspace bottle 10mm away from the liquid surface and blow nitrogen for 3 minutes, then lift the needle to 10-20mm above the bottle mouth, tighten the bottle cap under the protection of nitrogen blow, and then stir quickly to dissolve the sodium chloride; S5. Target release and test preparation: Place the sealed headspace bottle in a constant temperature water bath to convert the potential trans-2-nonenal into a free state, then cool it to 4°C, add p-fluorobenzaldehyde internal standard solution under nitrogen protection and seal it; S6. Detection and analysis: Use headspace-gas chromatography-mass spectrometry to detect samples, and set specific chromatographic column, column temperature, injection port, carrier gas, mass spectrometer and headspace injector conditions for detection and analysis.

2. The sample pretreatment method according to claim 1, characterized in that: When the Miag DLFU disc mill is used in step S1, the disc spacing is set to 0.2 mm; the particle size range of the obtained malt fine powder is 80-100 mesh.

3. The sample pretreatment method according to claim 1, characterized in that: In step S2, the temperature of the ice water bath is controlled below 5°C and the stirring rate is 750 rpm for 15 min.

4. The sample pretreatment method according to claim 1, characterized in that: In step S3, 85% phosphoric acid is used to adjust the pH of the malt extract to 4.

0.

5. The sample pretreatment method according to claim 1, characterized in that: The mass of sodium chloride in step S3 is 1.5 g.

6. The sample pretreatment method according to claim 1, characterized in that: In step S5, the headspace bottle is placed in a constant temperature water bath at 90° C. for 2 hours.

7. The sample pretreatment method according to claim 1, characterized in that: The concentration of the p-fluorobenzaldehyde internal standard solution added in step S5 is 5 mg / L, and the amount added is 100 μL.

8. The sample pretreatment method according to claim 1, characterized in that: In step S1, the fatty acid oxidase of malt is inactivated by adding water at a temperature of 72°C to the iodine volumetric flask. The iodine volumetric flask is sealed with a stopper and placed in a 70°C constant temperature water bath for 5 minutes. The iodine volumetric flask is then placed in a room temperature water bath or the outer wall is rinsed with room temperature water to cool it to room temperature as quickly as possible.

9. The sample pretreatment method according to claim 1, characterized in that: In step S2, in view of the possible oxidative attenuation of trans-2-nonenal at high temperature, stirring extraction is performed under ice-water bath conditions (water bath temperature is not higher than 5° C.), and the supernatant is centrifuged at 5° C.

10. The sample pretreatment method according to claim 1, characterized in that: In step S6, in order to prevent the high-temperature non-enzymatic oxidation reaction of trans-2-nonenal that may occur during the detection of trans-2-nonenal on a high-performance gas chromatograph, a second nitrogen blow protection measure is taken during the process of adding the internal standard solution.

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