Method for synchronously detecting daucoside, free cyanide and total cyanide in baijiu brewing raw materials

Abstract

The invention discloses a method for synchronously detecting daucoside, free cyanide and total cyanide in baijiu brewing raw materials, and belongs to the field of baijiu. According to the method, the phosphate buffer solution is used for extraction, oscillation extraction, enzymolysis extraction and the like are combined, so that the detection of the danioside, the free cyanide and the total cyanide is synchronously realized, and the method is high in specificity, high in accuracy and high in operation stability.

Description

Technical Field

[0001] This invention relates to a method for simultaneously detecting stigmosiderin, free cyanide, and total cyanide in raw materials for baijiu (Chinese liquor) brewing, and belongs to the field of baijiu. Background Technology

[0003] The main cyanogenic glycoside in sorghum, a raw material for Baijiu brewing, is stigmosiderin, which is produced by the metabolism of L-tyrosine. Under suitable conditions, stigmosiderin can release free hydrogen cyanide (HCN) through hydrolysis, which then reacts with gaseous ethanol during distillation to form cyanide esters (EC). Besides stigmosiderin, urea, naturally present in the brewing raw materials, can also decompose into ammonia and cyanic acid at high temperatures, which can also react with ethanol to form EC. Therefore, the sources of cyanide precursors in Baijiu production mainly include two forms: bound (such as stigmosiderin) and free (such as HCN and cyanic acid). However, there is currently a lack of systematic research on the detection methods for these different types of cyanide in the Baijiu brewing system; most studies focus on the detection and limit of EC in the final product after distillation, while neglecting the dynamic changes and controllability of precursor substances in upstream materials.

[0004] Regarding research on total cyanide, the commonly used quantitative method for detecting cyanide in grain products, as specified in GB 5009.36-2023 "National Food Safety Standard: Determination of Cyanide in Food", is distillation spectrophotometry. However, this method generally suffers from problems such as complex procedures, long processing time, sample stability issues, and poor recovery rates, making it difficult to meet the needs of dynamic monitoring of multiple batches and stages of raw materials for solid-state brewing of baijiu.

[0005] Regarding free cyanide, the pretreatment procedure for gas chromatography in the national standard cannot effectively distinguish free cyanide in fermented grain samples. On the one hand, the current national standard method has not conducted specific adaptation studies for cyanide in the fermented grain system; on the other hand, if the ultrasonic pretreatment step for grain samples is still used, it can easily lead to the decomposition of stigmosiderin in the raw materials for baijiu, thereby introducing additional cyanide and affecting the accuracy of the test results.

[0006] Therefore, finding a method to simultaneously detect succinosides, free cyanide, and total cyanide in baijiu brewing raw materials is a key foundation for improving the safety and quality control of baijiu. Summary of the Invention

[0007] [Technical Issues] There is still a lack of systematic research on the detection methods for these different types of cyanide (stigmosiderin, free cyanide, and total cyanide) in the Baijiu brewing system; Conventional methods can only detect one of the following: succinoside, free cyanide, and total cyanide; simultaneous detection is not possible.

[0008] [Technical Solution] To address the aforementioned problems, this invention provides a method for the simultaneous detection of stigmosiderin, free cyanide, and total cyanide in baijiu (Chinese liquor) brewing raw materials. Specifically, this invention combines extraction with phosphate buffer, shaking extraction, and enzymatic extraction to simultaneously detect stigmosiderin, free cyanide, and total cyanide. Furthermore, this method exhibits high specificity, high accuracy, and strong operational stability.

[0009] The first objective of this invention is to provide a method for simultaneously detecting stigmosiderin, free cyanide, and total cyanide in raw materials for Baijiu (Chinese liquor) brewing, comprising the following steps: (1) Add phosphoric acid aqueous solution to the raw materials for brewing liquor to be tested, extract by vortexing, centrifuge, take part of the supernatant as analyte A, and use it to detect the free cyanide content; mix the remaining supernatant and precipitate evenly to form a mixture; (2) Extract the mixture from step (1) by ultrasonication, centrifuge to obtain supernatant; take a portion of the supernatant as test solution B to detect the content of stigmosiderin; (3) Add phosphate buffer, sodium hydroxide solution and β-glucosidase solution to the supernatant in step (2), incubate, add sodium hydroxide solution, let stand, and obtain test solution C, which is used to detect the total cyanide content.

[0010] In one embodiment of the present invention, the raw material for brewing the liquor to be tested in step (1) is one of sorghum, sorghum slurry, and mash.

[0011] In one embodiment of the present invention, the concentration of the phosphoric acid aqueous solution in step (1) is 0.05-0.15M and the pH is 2-4; more preferably, the concentration of the phosphoric acid aqueous solution is 0.1M and the pH is 3.

[0012] In one embodiment of the present invention, the ratio of the raw material to be tested for brewing liquor and the phosphoric acid aqueous solution in step (1) is 5g:15-20mL; more preferably 5g:16mL.

[0013] In one embodiment of the present invention, the vortex oscillation in step (1) is a vortex oscillation at room temperature (20-30℃) for 20-40 minutes, and more preferably a vortex oscillation for 30 minutes.

[0014] In one embodiment of the present invention, the centrifugation in step (1) is 4000-6000 rpm, centrifuged at 4°C for 4-6 min; more preferably, it is 5000 rpm, centrifuged at 4°C for 5 min.

[0015] In one embodiment of the present invention, the uniform mixing in step (1) is achieved by vortex oscillation at room temperature (20-30°C) for 4-6 minutes. More preferably, vortex oscillation is performed for 5 minutes.

[0016] In one embodiment of the present invention, in step (1), analyte A is determined by headspace gas chromatography-mass spectrometry, and the result is substituted into the standard curve (y=5.9481x+173.94(R)). 2 =0.9998 (x is the cyanide concentration; y is the peak area) to calculate the free cyanide content.

[0017] In one embodiment of the present invention, the ultrasonic extraction in step (2) is performed at 55-65 W and 35-45 kHz for 25-35 min; more preferably, it is performed at 60 W and 40 kHz for 30 min.

[0018] In one embodiment of the present invention, the centrifugation in step (2) is 10,000-15,000 rpm, centrifuged at 4°C for 8-12 min; more preferably, it is 12,000 rpm, centrifuged at 4°C for 10 min.

[0019] In one embodiment of the present invention, in step (2), the test solution B is determined by liquid chromatography-ion trap mass spectrometry, and the result is substituted into the standard curve (y=1003x-2112.4(R)). 2 =0.9999, x is the concentration of styracin; y is the peak area) to calculate the styracin content.

[0020] In one embodiment of the present invention, the ratio of the supernatant, phosphate buffer, first added sodium hydroxide solution, β-glucosidase solution, and second added sodium hydroxide solution in step (3) is 0.2 mL: 0.3-0.4 mL: 0.1-0.2 mL: 0.05-0.15 mL: 0.1-0.3 mL; more preferably 0.2 mL: 0.35 mL: 0.15 mL: 0.1 mL: 0.2 mL.

[0021] In one embodiment of the present invention, the pH of the phosphate buffer in step (3) is 6.5-7.5, and more preferably 7.

[0022] In one embodiment of the present invention, the sodium hydroxide solution added for the first time in step (3) is an aqueous sodium hydroxide solution with a concentration of 0.01-0.1 mol / L, more preferably 0.05 mol / L.

[0023] In one embodiment of the present invention, the concentration of the β-glucosidase solution in step (3) is 0-10 U / mL, more preferably 5 U / mL; the solvent is a phosphate buffer with pH = 6.0.

[0024] In one embodiment of the present invention, the incubation in step (3) is carried out at 25-35°C for 5-30 minutes; more preferably, it is carried out at 30°C for 15 minutes.

[0025] In one embodiment of the present invention, the sodium hydroxide solution added for the second time in step (3) is an aqueous sodium hydroxide solution with a concentration of 0.5-1.5 mol / L, more preferably 1 mol / L.

[0026] In one embodiment of the present invention, the standing time in step (3) is 5-15 minutes at room temperature (20-30°C), and more preferably 10 minutes.

[0027] In one embodiment of the present invention, the test solution C in step (3) is determined by headspace gas chromatography-mass spectrometry, and the result is substituted into the standard curve (y=5.9481x+173.94(R)). 2 =0.9998 (x is the cyanide concentration; y is the peak area) to calculate the total cyanide content.

[0028] The second objective of this invention is the application of the method described herein in the field of detecting harmful substances in liquor.

[0029] [Beneficial Effects] (1) The method of the present invention can realize the simultaneous detection of stigmosiderin, free cyanide and total cyanide in the raw materials for brewing liquor; that is, the content of stigmosiderin, free cyanide and total cyanide can be tested in one sample. The operation process is integrated, the consumables are unified and it is convenient to perform multi-index joint testing.

[0030] (2) In this invention, phosphate buffer is used as the extraction reagent. The sample A is obtained by shaking extraction and used to detect the free cyanide content. The sample B is obtained by ultrasonic extraction and used to detect the stigmosiderin content. Then, the bound cyanide is released to the maximum extent by a combination of enzymatic hydrolysis and alkaline hydrolysis to obtain the sample C, which is used to detect the total cyanide content. The detection effect is accurate.

[0031] (3) The method of the present invention is applicable to solid brewing substrates such as sorghum, sorghum slurry, and fermented mash, and solves the drawback of the national standard method being unable to distinguish between cyanogenic glycosides and free cyanide; the detection limit is low, the steps are simplified, and it is suitable for large-scale sample processing and monitoring.

[0032] (4) The method of the present invention has excellent accuracy and precision. The recovery rate of free cyanide spiked is greater than 85%, and the RSD is controlled within 10%, which shows good accuracy and precision.

[0033] (5) In the method of the present invention, the recovery rate of mossin was verified by spiked recovery, reaching 83.53-96.4%, with RSD<6%, indicating good precision; the recovery rate of free cyanide spiked sample reached greater than 85%, with RSD<6%; the recovery rate of total cyanide spiked sample reached greater than 85%, with RSD<8%, and the detection range covered most of the bound precursors in brewing raw materials in mash. Attached Figure Description

[0034] Figure 1 The effect of shaking time on the extraction of downstream cyanide from different samples.

[0035] Figure 2 The effect of ultrasonic treatment time on the extraction of mossin from different samples.

[0036] Figure 3 The effect of ultrasonic extraction time (10-40 min) on the total cyanide method parameters.

[0037] Figure 4 The effect of β-glucanase concentration (0-10 U / mL) on the total cyanide method parameters.

[0038] Figure 5 The effect of enzymatic reaction time (5-30 min) on the total cyanide method parameters.

[0039] Figure 6 The effect of alkaline hydrolysis duration (0-20 min) on the total cyanide method parameters. Detailed Implementation

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0041] Test method: 1. The free cyanide content was determined by headspace gas chromatography-mass spectrometry; Specifically as follows: Take 0.2 mL of analyte A into a headspace vial, add 9.8 mL of ultrapure water, add 0.2 mL of phosphoric acid aqueous solution (1:5, volume ratio), then add 0.2 mL of chloramine T solution (10 g / L), immediately cap and seal, shake for 5 min, and wait for GC-MS detection.

[0042] Instrument headspace conditions: The headspace equilibrium temperature was 60℃; the sampling needle temperature was 65℃; the headspace heating time was 10 min; the injection interval was 16 min; and the injection volume was 1 mL.

[0043] Instrumental chromatographic conditions: The chromatographic column was a DB-FFAP capillary column (60 m × 0.25 mm × 0.25 μm); the column flow rate was 1.4 mL / min; the column temperature was 40.0℃; the column oven temperature program was as follows: 40℃ held for 2 min, then increased to 60℃ at a rate of 20℃ / min and held for 2 min; then increased to 200℃ at a rate of 50℃ / min and held for 2 min.

[0044] The injection mode was split (10:1); the injection port temperature was 200℃; the carrier gas was He; and the purge flow rate was 3.0 mL / min.

[0045] Instrument mass spectrometry conditions: The ion source temperature was 230℃; the interface temperature was 220℃; the solvent delay time was 3 min; the scanning ion m / z was 61, 63, 35, and 26; and the quantitative ion m / z was 61.

[0046] 2. The content of stigmosiderin was determined by liquid chromatography-ion trap mass spectrometry; Specifically as follows: Take 1 mL of the test solution B into a liquid chromatography bottle and filter it through a 0.22 μm aqueous PTEE filter membrane. Analyze the stigmosiderin content of the sample using a triple quadrupole linear ion trap liquid chromatography-mass spectrometry system (SCIEX, USA).

[0047] Instrument LC conditions: Column temperature: 40℃; Mobile phase A: water (5mM ammonium acetate); Mobile phase B: acetonitrile; The flow rate was 0.3 mL / min, and the injection volume was 2 μL. The elution conditions were: 0-0.5 min, 5% B; 0.5-7.5 min, 5-40% B; 7.5-8 min, 40-95% B; 8-9 min, 95% B; 9-9.1 min, 95-5% B; and 9.1-12 min, 5% B. Instrument MS conditions: ESI ion source, positive ion mode; The capillary voltage was set to 2.7 kV, the cone voltage to 20 V, the collision energy to 12 V, the source temperature to 120℃, the desolventizing temperature to 300℃, the desolventizing gas flow rate to 700 L / h, the cone gas flow rate to 50 L / h, and the collision gas flow rate to 0.15 mL / min.

[0048] The MS detector was set to MRM mode, with m / z 329→180 and 329→132 selected as the qualitative ion pair for cyanogenic glycosides and m / z 329→132 as the quantitative ion pair.

[0049] 3. The total cyanide content was determined by headspace gas chromatography-mass spectrometry; Specifically as follows: Take 0.2 mL of the analyte C into a headspace vial, add 9.8 mL of ultrapure water, add 0.2 mL of phosphoric acid aqueous solution (1:5, volume ratio), then add 0.2 mL of chloramine T solution (10 g / L), immediately cap and seal, shake for 5 min, and wait for GC-MS detection.

[0050] Instrument headspace conditions: The headspace equilibrium temperature was 60℃; the sampling needle temperature was 65℃; the headspace heating time was 10 min; the injection interval was 16 min; and the injection volume was 1 mL.

[0051] Instrumental chromatographic conditions: The chromatographic column was a DB-FFAP capillary column (60 m × 0.25 mm × 0.25 μm). The column flow rate was 1.4 mL / min; the column temperature was 40.0℃. The column oven temperature program is as follows: 40℃ held for 2 min, then increased to 60℃ at a rate of 20℃ / min and held for 2 min; then increased to 200℃ at a rate of 50℃ / min and held for 2 min.

[0052] The injection mode was split (10:1); the injection port temperature was 200℃; the carrier gas was He; and the purge flow rate was 3.0 mL / min.

[0053] Instrument mass spectrometry conditions: The ion source temperature was 230℃; the interface temperature was 220℃; the solvent delay time was 3 min; the scanning ion m / z was 61, 63, 35, and 26; and the quantitative ion m / z was 61.

[0054] Raw materials used in the examples: 1. Sorghum soaked feed is obtained by soaking sorghum. Samples were collected from a soy sauce-flavored liquor producer in Jiangsu Province; all samples were sealed and stored at 4℃ to ensure their stability. The specific steps for soaking the grain are as follows: (1) First water addition: Add 95℃ hot water to the sorghum, the amount of water added is 34% of the total mass of the sorghum, immediately stir manually, then pile it in a tray lined with water-absorbing sponges to simulate the actual mountain-like structure, and place it in a 40℃ constant temperature box for continuous stacking culture. (2) Second water addition: 90 min after the first water addition, add 95℃ hot water to the sorghum, the amount of water added is 12% of the total mass of the sorghum, and immediately stir manually; then pile it in a tray lined with absorbent sponges to simulate the actual mountain-like structure, and place it in a 40℃ constant temperature box for continuous stacking culture. (3) Third water addition: 360 min after the second water addition, add 95℃ hot water to the sorghum, the amount of water added is 10% of the total mass of the sorghum, and immediately stir manually; then pile the sample in a tray lined with absorbent sponges to simulate the actual mountain-like structure, and place it in a 40℃ constant temperature box for continuous stacking culture until the grain is moistened, for a total of 54 h. The mixture was stirred again at 24 and 36 hours after the first water injection.

[0055] 2. The phosphate buffer solution was prepared by mixing 0.1 mol / L phosphate aqueous solution and 0.1 mol / L sodium phosphate aqueous solution.

[0056] 3. Before testing the sorghum feed, it needs to be homogenized for 55 seconds and washed for 20 seconds.

[0057] 4. In the examples and comparative examples, the solvent used in the solutions is water unless otherwise specified; the concentration of the solutions is a mass percentage unless otherwise specified; the temperature of the operations is room temperature (20-30°C) unless otherwise specified.

[0058] 5. Prepare fresh β-glucosidase solution every 3 days and store it frozen.

[0059] Example 1: Construction of Standard Curve 1. Construction of cyanide standard curve: Take an appropriate amount of the cyanide analysis standard material (KCN, 10 mg / L) in water and dilute it with pure water to prepare standard working solutions with concentrations of 0, 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, and 5.00 mg / L.

[0060] The cyanide content was determined according to the method (headspace gas chromatography-mass spectrometry), and a cyanide concentration curve was plotted.

[0061] The standard curve is as follows: y = 5.9481x + 173.94 (R) 2 =0.9998 (x is the cyanide concentration; y is the peak area).

[0062] The total cyanide content and free cyanide content are measured using the same substances, only at different times and in different forms. Therefore, both are calculated using the aforementioned standard curve.

[0063] Based on the calibration curves for free cyanide and total cyanide, and combined with the response data of blank samples, the limit of detection (LOD) was calculated using the "3σ / slope" method. In the test, the standard deviation of the blank sample response was 2.1, which, when substituted into the calculation, yielded a LOD of approximately 1.06 μg / L; the limit of quantitation (LOQ) calculated using the 10σ / slope method was 3.53 μg / L.

[0064] 2. Establishment of the standard curve for stigmosiderin: A 10 mg / L standard solution of stigmosiderin was prepared using pure water, and then diluted with pure water to prepare working standard solutions with concentrations of 0, 0.01, 0.02, 0.05, 0.10, 0.20, 0.50, and 1.00 mg / L. The stigmosiderin content was determined according to the method (using liquid chromatography-ion trap mass spectrometry), and cyanide concentration curves were plotted.

[0065] The standard curve is as follows: y = 1003x - 2112.4 (R) 2 =0.9999, where x is the concentration of styracin; y is the peak area.

[0066] The quantitative analysis of stigmosiderin was performed using liquid chromatography-mass spectrometry (LC-MS), and a calibration curve was established. Based on the instrument response characteristics and repeatability data from low-concentration samples, the method limit of quantitation (LOQ) was determined to be 7.8 μg / L, and the limit of detection (LOD) to be 2.4 μg / L.

[0067] Example 2 Test Method A method for simultaneous detection of stigmosiderin, free cyanide, and total cyanide in raw materials for Baijiu (Chinese liquor) brewing includes the following steps: (1) Take 5g of the raw material for Baijiu brewing (sorghum slurry), add 16mL of 0.1M phosphate aqueous solution with pH 3, vortex at room temperature for 30min for extraction, centrifuge at 5000rpm and 4℃ for 5min, take 0.2mL of supernatant as analyte A, and use it to detect the free cyanide content; vortex the remaining supernatant and precipitate at room temperature for 5min to form a mixture; (2) Extract the mixture from step (1) by ultrasonic extraction at 60 W and 40 kHz for 30 min, centrifuge at 12000 rpm and 4℃ for 10 min to obtain the supernatant; take 1 mL of the supernatant as test solution B to detect the content of stigmosiderin. (3) Take 0.2 mL of the supernatant from step (2), add 0.35 mL of phosphate buffer (pH = 7.0), 0.15 mL of 0.05 mol / L sodium hydroxide aqueous solution, and 0.1 mL of 5 U / mL β-glucosidase solution (solvent is phosphate buffer with pH 6), incubate at 30°C for 15 min, then add 0.2 mL of 1 mol / L sodium hydroxide aqueous solution, let stand at room temperature for 10 min to obtain test solution C, which is used to detect the total cyanide content; Among them, analyte A was determined by headspace gas chromatography-mass spectrometry, and the result was substituted into the standard curve (y=5.9481x+173.94 (R²)). 2 =0.9998 (where x is the cyanide concentration and y is the peak area) to calculate the free cyanide content; The test solution B was determined using liquid chromatography-ion trap mass spectrometry (LC-IMS), and the results were substituted into the standard curve (y = 1003x - 2112.4 (R²)). 2 =0.9999, x is the concentration of styracin; y is the peak area) to calculate the styracin content; The test solution C was determined using headspace gas chromatography-mass spectrometry, and the results were substituted into the standard curve (y=5.9481x+173.94 (R0). 2 =0.9998 (x is the cyanide concentration; y is the peak area) to calculate the total cyanide content.

[0068] Example 3 Precision Verification According to GB / T 32465 2015 Requirements for Validation and Internal Quality Control of Chemical Analysis Methods and GB / T27417 The 2017 Guidelines for Conformity Assessment and Validation of Chemical Analysis Methods specify the precision validation of the method in Example 2. Specifically as follows: Six portions of sorghum feed (with known contents of sorghum glycoside, free cyanide, and total cyanide of 64.85, 3.40, and 8.35 mg / kg, respectively) were taken as parallel samples and tested according to the method in Example 2 to evaluate the precision.

[0069] The test results are as follows: Table 1 Precision of Free Cyanide Content

[0070] Table 2 Precision of Total Cyanide Content

[0071] Table 3 Precision of stigmoside content

[0072] As can be seen from Tables 1-3, the RSD of the precision test is less than 10%, which meets the precision requirements.

[0073] Example 4 Accuracy Verification According to GB / T 32465 2015 Requirements for Validation and Internal Quality Control of Chemical Analysis Methods and GB / T27417 The 2017 Guidelines for Conformity Assessment and Validation of Chemical Analysis Methods verify the accuracy of the method in Example 2. Specifically as follows: Take three portions of sorghum feed (the contents of sorghum glycoside, free cyanide, and total cyanide are known to be 64.85, 3.40, and 8.35 mg / kg, respectively), and add standard solutions of the target analyte at three different spiking levels to the samples, with concentrations of 80% (low concentration), 100% (medium concentration), and 120% (high concentration) of the target content, respectively.

[0074] The tests were conducted according to the method in Example 2. Each group of samples was processed using the same extraction and detection procedures and then used for recovery rate and accuracy evaluation, in triplicate.

[0075] The test results are as follows: Table 4. Accuracy of Free Cyanide Content in Raw Materials for Maotai-Flavor Baijiu Brewing

[0076] Table 5. Accuracy of Total Cyanide Content in Raw Materials for Sauce-Flavored Baijiu Brewing

[0077] Table 6. Accuracy of strychnine content in raw materials for brewing sauce-flavored baijiu

[0078] As can be seen from Tables 4-6, the spiked recovery experiment results show that the recovery rate is between 80% and 120%, and the RSD is less than 10%, indicating that the method has high accuracy and good repeatability, meeting the requirements for the detection of raw materials for liquor.

[0079] Example 5 Parameter Optimization 1. Effect of shaking time on downstream cyanide extraction from different samples The raw material was sorghum slurry (with known contents of sorghum glycoside, free cyanide, and total cyanide of 64.85, 3.40, and 8.35 mg / kg, respectively).

[0080] The oscillation time in step (1) of Example 2 was adjusted to 5, 10, 20, 30 and 60 min, while the rest remained the same as in Example 2.

[0081] Test results are as follows Figure 1 .

[0082] from Figure 1 It can be seen that the extraction effect and efficiency are best when the oscillation time is 30 minutes.

[0083] 2. Effect of ultrasonic treatment time on the extraction of stigmosiderin from different samples The raw material was sorghum slurry (with known contents of sorghum glycoside, free cyanide, and total cyanide of 64.85, 3.40, and 8.35 mg / kg, respectively).

[0084] The extraction time in step (2) of Example 2 was adjusted to 0, 10, 20, 30, and 60 min, while other steps remained the same as in Example 2.

[0085] Test results are as follows Figure 2 .

[0086] from Figure 2 It can be seen that the extraction effect is best when the extraction time is 30 minutes.

[0087] 3. Effect of ultrasonic extraction time in step (2) of the total cyanide method parameters (10–40 min) The raw material was sorghum slurry (with known contents of sorghum glycoside, free cyanide, and total cyanide of 64.85, 3.40, and 8.35 mg / kg, respectively).

[0088] The ultrasonic extraction time in step (2) of Example 2 was adjusted to 10, 20, 30, and 40 min, while the other steps remained the same as in Example 2.

[0089] Test results are as follows Figure 3 .

[0090] from Figure 3 It can be seen that the ultrasonic extraction effect is best when the extraction time is 30 minutes.

[0091] 4. Effect of β-glucanase concentration (0–10 U / mL) on total cyanide method parameters The raw material was sorghum slurry (with known contents of sorghum glycoside, free cyanide, and total cyanide of 64.85, 3.40, and 8.35 mg / kg, respectively).

[0092] The concentration of the β-glucosidase solution in step (3) of Example 2 was adjusted to 0, 2, 5, and 10 U / mL, while other aspects remained the same as in Example 2.

[0093] Test results are as follows Figure 4 .

[0094] from Figure 4 It can be seen that the extraction effect is best when the concentration of β-glucosidase solution is 5 U / mL.

[0095] 5. Effect of enzymatic reaction (incubation) time (5–30 min) on total cyanide method parameters The raw material was sorghum slurry (with known contents of sorghum glycoside, free cyanide, and total cyanide of 64.85, 3.40, and 8.35 mg / kg, respectively).

[0096] The incubation time in step (3) of Example 2 was adjusted to 5, 10, 15 and 30 min, while the rest remained the same as in Example 2.

[0097] Test results are as follows Figure 5 .

[0098] from Figure 5 It can be seen that the extraction effect is best when the incubation time is 15 minutes.

[0099] 6. Effect of alkaline hydrolysis duration (standing time) (0–20 min) on total cyanide method parameters The raw material was sorghum slurry (with known contents of sorghum glycoside, free cyanide, and total cyanide of 64.85, 3.40, and 8.35 mg / kg, respectively).

[0100] The settling time in step (3) of Example 2 was adjusted to 0, 5, 10, and 20 minutes, while the rest remained the same as in Example 2.

[0101] Test results are as follows Figure 6 .

[0102] from Figure 6 It can be seen that the extraction effect is best when the settling time is 10 minutes.

[0103] Example 6: Consistency Verification of Three Cyanide Species Data Based on Molar Mass Conversion This embodiment aims to verify the accuracy and methodological consistency of the method of the present invention in the synergistic quantitative detection of multiple forms of cyanide by quantitatively determining the contents of stigmosiderin, free cyanide and total cyanide, and combining the molar mass of each component.

[0104] The sorghum samples after sorghum slurry were analyzed in parallel using the LC-MS and GC-MS detection methods established above, and the mass concentrations (mg / kg) of sorghum glycoside, free cyanide and total cyanide in each sample were obtained.

[0105] To achieve a unified conversion of cyanide content across the three forms, molar concentration is used as the benchmark, referencing the molar mass of each substance: stigmosiderin has a molar mass of 311.3 g / mol; free cyanide (as expressed in CN...) - The molar mass (calculated) is 26.02 g / mol. The conversion formula is as follows: C mol (μmol / g) = C mass (mg / kg) ÷ M (g / mol) Where C mass M is the mass concentration (mg / kg). Molar mass (g / mol) The calculation results are as follows: C_total cyanide = 0.3209 μmol / g; C_dhurrin = 0.2084 μmol / g; C_free cyanide = 0.1307 μmol / g The measured values ​​of stigmosiderin and free cyanide were converted to molar concentrations, summed, and compared with the molar concentration corresponding to the total cyanide detection result. The error was controlled within ±5%, verifying that: C_total cyanide ≈ C_dhurrin + C_free cyanide The results fully demonstrate that the three types of cyanide detection methods established in this invention can achieve synergistic quantification of cyanogenic glycosides and free cyanide in samples, exhibiting good complementarity and data consistency, and are suitable for simultaneous monitoring of different cyanide forms throughout the entire process of Baijiu brewing.

[0106] Comparative Example 1: National Standard Method - Distillation-Spectrophotometry Traditional distillation-spectrophotometry (using isonicotinic acid-pyrazolone as the colorimetric reagent) has the following drawbacks: (1) The recovery rate is unstable, especially in complex matrices (such as fermented mash samples), where the recovery rate is often below 80%, and in some samples it is even below 70%, resulting in poor accuracy.

[0107] (2) The method has low sensitivity, with detection limits generally in the range of 0.5–1 mg / kg and quantification limits relatively high, making it difficult to meet the requirements for accurate quantification of low-content cyanide.

[0108] (3) Sample processing is complicated, requiring long-term steam distillation, which is time-consuming and labor-intensive, and is not suitable for multi-sample detection.

[0109] In comparison, the total cyanide detection method established in this invention has the following significant advantages: (1) Based on the combination of β-glucosidase hydrolysis and alkaline hydrolysis, the bound cyanogenic glycosides and cyanohydrins in the sample can be fully converted into free cyanide; (2) GC-MS was used to detect derivatized cyanide, with a detection limit as low as 0.003 mg / kg and a quantitation limit as low as 0.01 mg / kg, significantly improving sensitivity; (3) The sample processing procedure is simplified, requiring no distillation, only ultrasonic extraction, enzymatic hydrolysis and static alkaline hydrolysis, which is suitable for large-scale detection of multiple samples; (4) The recovery rate is stable, with an average recovery rate of over 85% and an RSD of less than 8%, significantly improving reproducibility and accuracy.

[0110] The comparison shows that the traditional distillation-colorimetric method is not suitable for the accurate determination of total cyanide in complex matrices, while the method of the present invention can achieve comprehensive, rapid and accurate detection of multiple forms of cyanide in solid-state fermentation samples of baijiu.

[0111] Comparative Example 2: National Standard Gas Chromatography-Mass Spectrometry The core issues of the national standard gas chromatography method include: (1) Ultrasonic pretreatment of samples under pure water conditions will cause hydrolysis of cyanogenic glycosides, increase the content of free cyanide, and result in higher detection values. Therefore, the method cannot accurately detect free cyanide. (2) It has poor adaptability to complex solid fermentation materials (such as wine mash), low extraction rate and poor repeatability.

[0112] The free cyanide detection method of the present invention is based on the following optimized design: (1) Extraction was performed using a phosphate buffer solution with pH < 4, which effectively maintained the stability of cyanogenic glycosides, prevented decomposition, and truly reflected the actual level of free cyanide in the sample; (2) The spiked recovery rate of sorghum feed and mash samples was over 85%, and the RSD was less than 4%. The stability and reliability of the method were better than the national standard method.

[0113] By comparison, we can see that: The national standard gas chromatography method is unsuitable for determining free cyanide in baijiu raw material samples due to its inability to distinguish cyanide speciation under certain pretreatment conditions and its limited detection targets. However, the detection method of this invention achieves accurate identification and quantification of actual free cyanide in baijiu brewing raw materials, and is suitable for solid sample detection scenarios.

[0114] Comparative Example 3 The pH of the phosphoric acid aqueous solution in step (1) of Example 2 was adjusted to 6, while the rest remained the same as in Example 2.

[0115] turn out: Under these extraction conditions, the free cyanide content in the sample was significantly higher than in Example 2, while the stigmosiderin content was significantly lower. Further comparison of the total cyanide determination results showed similarities to Example 2. This phenomenon indicates that at pH 6, residual β-glucosidase in the raw material may be partially activated, causing premature decomposition of stigmosiderin during extraction to produce free cyanide. This blurs the boundaries between different forms of cyanide, affecting their accurate differentiation and independent quantification. Therefore, in the extraction system at pH = 6, stigmosiderin undergoes non-specific hydrolysis, severely interfering with the accurate determination of free cyanide and stigmosiderin, and reducing the method's distinguishing ability.

[0116] Table 7

[0117] Comparative Example 4 Adjust the order of steps (1) and (2) in Example 2; that is, swap the order of "oscillation" and "ultrasound" in the sample pretreatment steps, that is, perform ultrasonic extraction first, and then perform vortex oscillation. Everything else remains the same as in Example 2.

[0118] turn out: This change in order led to significant differences in the detection results for various types of cyanide. In Comparative Example 4, the detection result for stigmosiderin was 61.71 ± 1.99 mg / kg, slightly lower than the 64.85 ± 3.02 mg / kg in the Example; free cyanide decreased significantly from 3.40 ± 0.27 mg / kg in the Example to 2.57 ± 0.08 mg / kg, indicating insufficient extraction due to inadequate initial mixing. The total cyanide content also decreased from 8.35 ± 0.20 mg / kg in the Example to 7.76 ± 0.21 mg / kg, still within a reasonable range, but the detection stability and logical consistency declined.

[0119] Therefore, the processing sequence of oscillation followed by ultrasound is more conducive to sample homogenization, improving extraction efficiency and method accuracy.

[0120] Table 8

[0121] Comparative Example 5 The phosphoric acid aqueous solution in step (1) of Example 2 was adjusted to a methanol aqueous solution with a volume fraction of 80%, while the rest remained the same as in Example 2.

[0122] The results showed that although the use of 80% methanol extraction solution had a relatively small effect on stigmosiderin (60.31 ± 2.21 mg / kg vs 64.85 ± 3.02 mg / kg) and free cyanide (3.22 ± 0.18 mg / kg vs 3.40 ± 0.27 mg / kg), with a limited decrease, a more significant decrease was observed in the total cyanide content (3.21 ± 0.29 mg / kg vs 8.35 ± 0.20 mg / kg). This suggests that the methanol system mainly affects the effectiveness of the enzymatic hydrolysis stage. β-glucosidase's catalytic activity is most stable in aqueous buffer, while high concentrations of organic solvents (such as methanol) can lead to enzyme denaturation and loss of activity, thus failing to effectively hydrolyze potential precursors such as stigmosiderin to generate hydrocyanic acid, ultimately affecting the release of total cyanide. Therefore, although the initial extraction had little effect on some components, the failure of the enzymatic conversion step was the main reason for the significantly lower total cyanide content compared to the examples.

[0123] This result further demonstrates that the use of aqueous phosphoric acid solution in this invention has better adaptability and analytical accuracy, especially when enzymatic hydrolysis steps are involved.

[0124] Table 9

[0125] Comparative Example 6 The order of steps (2) and (3) in Example 2 was changed. That is, the mixture obtained in step (1) was first directly subjected to total cyanide detection treatment (enzyme addition and alkaline hydrolysis), and then subjected to ultrasonic treatment. The rest was the same as in Example 2.

[0126] turn out: The contents of stigmosiderin (63.12 ± 1.65 mg / kg vs. 64.85 ± 3.02 mg / kg in the example) and free cyanide (3.39 ± 0.06 mg / kg vs. 3.40 ± 0.27 mg / kg) were basically the same, indicating that changing the order had little impact on the detection of these two forms. However, the total cyanide content decreased significantly to 2.46 ± 0.22 mg / kg, which was significantly lower than 8.35 ± 0.20 mg / kg in the example. This phenomenon indicates that, without ultrasonic treatment, bound precursors such as stigmosiderin are difficult to release into the supernatant, resulting in a significant reduction in enzymatic hydrolysis efficiency, thereby affecting the accuracy of total cyanide determination.

[0127] These results highlight the crucial role of the sequence of sonication followed by enzyme addition in this method for the complete release and accurate quantification of total cyanide.

[0128] Table 10

[0129] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for simultaneous detection of stigmosiderin, free cyanide, and total cyanide in raw materials for Baijiu (Chinese liquor) brewing, characterized in that, Includes the following steps: (1) Add phosphoric acid aqueous solution to the raw materials for brewing liquor to be tested, extract by vortexing, centrifuge, take part of the supernatant as analyte A, and use it to detect the free cyanide content; mix the remaining supernatant and precipitate evenly to form a mixture; (2) Extract the mixture from step (1) by ultrasonication, centrifuge to obtain supernatant; take a portion of the supernatant as test solution B to detect the content of stigmosiderin; (3) Add phosphate buffer, sodium hydroxide solution and β-glucosidase solution to the supernatant in step (2), incubate, add sodium hydroxide solution, let stand, and obtain test solution C, which is used to detect the total cyanide content.

2. The method according to claim 1, characterized in that, In step (1), the raw materials for brewing the liquor to be tested are sorghum, sorghum slurry, and mash.

3. The method according to claim 1, characterized in that, In step (1), the ratio of the raw materials for the liquor brewing to be tested to the phosphoric acid aqueous solution is 5g:15-20mL.

4. The method according to claim 1, characterized in that, In step (1), the vortex oscillation is a vortex oscillation at room temperature (20-30℃) for 20-40 minutes.

5. The method according to claim 1, characterized in that, In step (1), the mixture is homogenized by vortexing at room temperature (20-30℃) for 4-6 minutes.

6. The method according to claim 1, characterized in that, In step (2), ultrasonic extraction is performed at 55-65 W and 35-45 kHz for 25-35 min.

7. The method according to claim 1, characterized in that, In step (3), the ratio of the amount of supernatant, phosphate buffer, sodium hydroxide solution added for the first time, β-glucosidase solution, and sodium hydroxide solution added for the second time is 0.2 mL: 0.3-0.4 mL: 0.1-0.2 mL: 0.05-0.15 mL: 0.1-0.3 mL.

8. The method according to claim 1, characterized in that, The sodium hydroxide solution added for the first time in step (3) is an aqueous sodium hydroxide solution with a concentration of 0.01-0.1 mol / L.

9. The method according to claim 1, characterized in that, In step (3), the incubation is carried out at 25-35℃ for 5-20 minutes; the standing is carried out at room temperature (20-30℃) for 5-15 minutes.

10. The application of the method according to any one of claims 1-9 in the field of detecting harmful substances in liquor.

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