Method for measuring content of fatty acid in sorghum

Abstract

The invention provides a method for measuring the content of fatty acid in sorghum. The method is efficient, easy to operate and capable of measuring various fatty acids. The method comprises the steps that S10, sorghum is smashed and sieved, and smashed sorghum is obtained; s20, taking the crushed sorghum according to a first preset weight to obtain a crushed sorghum sample to be detected, adding a sulfuric acid-methanol solution into the crushed sorghum sample, carrying out water bath reflux, and after the reflux is finished, centrifuging and taking supernate; and S30, adding a potassium hydroxide-methanol solution to the supernatant, and carrying out a heat-preservation reaction for a first preset duration in a water bath to obtain a reaction product liquid containing fatty acid methyl ester. And S40, adding an extraction solvent into the reaction product liquid to extract fatty acid methyl ester, oscillating and ultrasonically extracting for a second preset time to obtain an extracting solution containing fatty acid methyl ester. And S50, carrying out qualitative and quantitative determination on fatty acid methyl ester in the extracting solution by adopting a gas chromatograph to obtain a fatty acid methyl ester determination result, and calculating the content of various fatty acids in the crushed sorghum sample according to the fatty acid methyl ester determination result and a corresponding fatty acid content conversion calculation relation.

Description

Technical Field

[0001] The present application relates to the technical field of fatty acid content detection in sorghum, and in particular to a method for determining the fatty acid content in sorghum. Background Art

[0002] The wine industry needs to detect the fatty acid content in sorghum efficiently and accurately to ensure the standardization of the entire winemaking process and the quality of the wine. The current fatty acid content detection method is relatively cumbersome and uses many types of reagents. The detection method can only detect a small number of fatty acids in sorghum, and the detection accuracy of the determination method is unknown.

[0003] Therefore, there is an urgent need for a method for determining the fatty acid content in sorghum. Summary of the invention

[0004] The present application provides a method for determining the fatty acid content in sorghum, comprising:

[0005] S10: crushing sorghum and sieving to obtain crushed sorghum;

[0006] S20: a step of hydrolyzing bound fatty acids and extracting fatty acids, taking crushed sorghum according to a first preset weight to obtain a crushed sorghum sample to be tested, adding a sulfuric acid-methanol solution to the crushed sorghum sample, refluxing in a water bath, and centrifuging to obtain a supernatant after the refluxing is completed;

[0007] S30: fatty acid methyl esterification step, adding potassium hydroxide-methanol solution to the supernatant, keeping the reaction in a water bath for a first preset time, and obtaining a reaction product liquid containing fatty acid methyl ester;

[0008] S40: a fatty acid methyl ester extraction step, adding an extraction solvent to the reaction product liquid to extract the fatty acid methyl ester, and oscillating and ultrasonically extracting for a second preset time to obtain an extract containing fatty acid methyl ester;

[0009] S50: using a gas chromatograph to perform qualitative and quantitative determination of fatty acid methyl esters in the extract to obtain the determination results of fatty acid methyl esters, and calculating the contents of various fatty acids in the broken sorghum sample according to the conversion calculation relationship between the determination results of fatty acid methyl esters and the corresponding fatty acid contents.

[0010] In some optional embodiments of the present application, in step S10:

[0011] The sorghum was crushed with a hammer cyclone mill and passed through a 200-mesh sieve to obtain ground sorghum.

[0012] In some optional embodiments of the present application, in step S20:

[0013] Reflux in a water bath at 80°C for 2h to 4h.

[0014] In some optional embodiments of the present application, reflux is carried out in a water bath at 80° C. for 2 h to 3 h.

[0015] In some optional embodiments of the present application, reflux in a water bath at 80° C. for 2 h, 2.5 h or 3 h;

[0016] In some optional embodiments of the present application, in step S20: the centrifugation condition is centrifugation at 8000 rpm for 5 minutes.

[0017] In some optional embodiments of the present application, 20 ml of sulfuric acid-methanol solution is added to every 1 g of the broken sorghum sample to soak the broken sorghum sample.

[0018] In some optional embodiments of the present application, the concentration of the sulfuric acid-methanol solution is 1.0 mol / L to 2 mol / L.

[0019] In some optional embodiments of the present application, the concentration of the sulfuric acid-methanol solution is 1.0 mol / L.

[0020] In some optional embodiments of the present application, in step S30:

[0021] Add 0.7 mol / L to 1.1 mol / L potassium hydroxide-methanol solution to the supernatant, and keep the reaction in a water bath at 30°C to 50°C for a first preset time to obtain a reaction product liquid containing fatty acid methyl esters. The first preset time is 30 min to 60 min.

[0022] In some optional embodiments of the present application, 0.7 mol / L potassium hydroxide-methanol solution is added to the supernatant, and the reaction is kept in a 30°C water bath for a first preset time to obtain a reaction product liquid containing fatty acid methyl esters, and the first preset time is 45 minutes.

[0023] In some optional embodiments of the present application, in step S40: the extraction solvent is n-hexane, oscillation is 1 minute and ultrasonic extraction is performed for a second preset time, and the second preset time is 5 minutes.

[0024] In some optional embodiments of the present application, 1 ml of the supernatant obtained by centrifugation in step S20 is taken, and 1 ml of potassium hydroxide-methanol solution is added to the 1 ml of supernatant. 3 mL of n-hexane is added in step S40.

[0025] In some optional embodiments of the present application, in step S50, the analysis conditions of the gas chromatography include:

[0026] A DB-Fast FAME column was used, and the parameters of the DB-Fast FAME column were 30m×0.25mm, 0.25um;

[0027] The carrier gas was nitrogen, 12 psi, constant pressure;

[0028] The injection port used split injection, the injection port temperature was 250 °C, and the split ratio was 100:1;

[0029] Column oven temperature program: initial temperature 80°C, maintained for 0.5 min, increased to 165°C at 40°C / min, maintained at 165°C for 1 min, increased to 230°C at 4°C / min, maintained at 230°C for 4 min;

[0030] The detection parameters of hydrogen flame ionization detector FID are as follows: operating temperature 260°C, hydrogen flow rate 40 mL / min, air flow rate 400 mL / min; nitrogen is used as the tail gas with a flow rate of 25 mL / min.

[0031] In some optional embodiments of the present application, the sorghum is wine sorghum.

[0032] In some optional embodiments of the present application, the method for determining the fatty acid content in sorghum can obtain the content of at least 37 fatty acids in the crushed sorghum sample, and the 37 fatty acids are butyric acid, caproic acid, caprylic acid, caprylic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, cis-9-tetradecanoic acid, pentadecanoic acid, cis-10-pentadecanoic acid, hexadecanoic acid, cis-9-hexadecanoic acid, heptadecanoic acid, cis-10-heptadecanoic acid, octadecanoic acid, trans-9-octadecanoic acid, cis-9-octadecanoic acid, trans, trans-9,12-octadecadienoic acid, cis, cis-9,12-octadecadienoic acid, eicosanoic acid, cis, cis, cis-6,9,12-decanoic acid, and cis-9,12-octadecadienoic acid. Octatrienoic acid, cis-11-eicosanonoic acid, cis,cis,cis-9,12,15-octadecatrienoic acid, heneicosanoic acid, cis,cis-11,14-eicosadienoic acid, docosanoic acid, cis,cis,cis-8,11,14-eicosatrienoic acid, cis-13-docosanonoic acid, cis-11,14,17-eicosatrienoic acid, cis-5,8,11,14-eicosatetraenoic acid, tricosanoic acid, cis-13,16-docosadienoic acid, tetracosanoic acid, cis-5,8,11,14-eicosapentaenoic acid, cis-15-tetracosnonoic acid and cis-4,7,10,13,16,19-docosahexaenoic acid.

[0033] In some optional embodiments of the present application, the conversion calculation relationship in step S50 is as shown in Formula 1:

[0034]

[0035] In formula 1:

[0036] X i - The content of each fatty acid in the broken sorghum sample, in grams per 100 grams (g / 100g);

[0037] msi —The concentration of each standard substance in the standard assay solution, in grams per liter (g / L);

[0038] A i - Peak area of ​​each fatty acid methyl ester in the extract;

[0039] F FAME-FA - Conversion coefficient of each fatty acid methyl ester into fatty acids;

[0040] V1—the volume of the added extraction solvent, in mL;

[0041] V2—the volume of sulfuric acid-methanol solution added, in mL;

[0042] V3-the volume of the supernatant used for fatty acid methyl esterification in step S30, in mL;

[0043] 1000—conversion factor;

[0044] A Si - Peak area of ​​each fatty acid in the standard test solution;

[0045] m—the weight of the broken sorghum sample, in grams (g);

[0046] 100—Coefficient for converting the content to the content per 100 g of sample.

[0047] The method for determining the fatty acid content in sorghum provided in the embodiment of the present application adopts sulfuric acid-methanol solution to hydrolyze and extract the bound fatty acids in sorghum, which can better hydrolyze triglycerides and extract fatty acids. The methylation of fatty acids with potassium hydroxide-methanol solution ensures that the fatty acids are completely methylated, which is conducive to the detection of the types and contents of fatty acids in sorghum. Subsequently, the fatty acid methyl esters are extracted with an extraction solvent and then subjected to gas chromatography detection to avoid the acidity or alkalinity in the test liquid affecting the subsequent gas chromatography detection, thereby increasing the total amount of fatty acids that can be measured and increasing the types of fatty acids that can be measured. Fewer types of detection reagents are used, which reduces the detection cost and simplifies the determination process. The method for determining the fatty acid content in sorghum provided in the embodiment of the present application can measure 37 kinds of fatty acids in sorghum, and there are many types of fatty acids that can be measured, and it can be used to specifically detect sorghum for wine. DETAILED DESCRIPTION

[0048] The present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0049] The present application provides a method for determining the fatty acid content in sorghum, comprising:

[0050] S10: crushing sorghum and sieving to obtain crushed sorghum;

[0051] S20: a step of hydrolyzing bound fatty acids and extracting fatty acids, taking crushed sorghum according to a first preset weight to obtain a crushed sorghum sample to be tested, adding a sulfuric acid-methanol solution to the crushed sorghum sample, refluxing in a water bath, and centrifuging to obtain a supernatant after the refluxing is completed;

[0052] S30: Fatty acid methyl esterification step, adding potassium hydroxide-methanol solution to the supernatant, keeping the reaction in a water bath for a first preset time, and obtaining a reaction product liquid containing fatty acid methyl ester.

[0053] S40: a fatty acid methyl ester extraction step, in which an extraction solvent is added to the reaction product liquid to extract the fatty acid methyl ester, and the fatty acid methyl ester is extracted by oscillation and ultrasonic extraction for a second preset time to obtain an extract containing fatty acid methyl ester.

[0054] S50: The fatty acid methyl ester in the extract is qualitatively and quantitatively determined by gas chromatography to obtain the fatty acid methyl ester determination result, and the content of various fatty acids in the broken sorghum sample is calculated according to the fatty acid methyl ester determination result and the corresponding fatty acid conversion coefficient.

[0055] In some optional embodiments of the present application, in step S10:

[0056] The sorghum was crushed with a hammer cyclone mill and passed through a 200-mesh sieve to obtain ground sorghum.

[0057] In some optional embodiments of the present application, in step S20:

[0058] Reflux in a water bath at 80°C for 2h to 4h.

[0059] In some optional embodiments of the present application, reflux is carried out in a water bath at 80° C. for 2 h to 3 h.

[0060] In some optional embodiments of the present application, reflux in a water bath at 80° C. for 2 h, 2.5 h or 3 h;

[0061] In some optional embodiments of the present application, in step S20: the centrifugation condition is centrifugation at 8000 rpm for 5 minutes.

[0062] In some optional embodiments of the present application, 20 ml of sulfuric acid-methanol solution is added to every 1 g of the broken sorghum sample to soak the broken sorghum sample.

[0063] In some optional embodiments of the present application, the concentration of the sulfuric acid-methanol solution is 1.0 mol / L to 2 mol / L.

[0064] In some optional embodiments of the present application, the concentration of the sulfuric acid-methanol solution is 1.0 mol / L.

[0065] In some optional embodiments of the present application, in step S30:

[0066] Add 0.7 mol / L to 1.1 mol / L potassium hydroxide-methanol solution to the supernatant, and keep the reaction in a water bath at 30°C to 50°C for a first preset time to obtain a reaction product liquid containing fatty acid methyl esters. The first preset time is 30 min to 60 min.

[0067] In some optional embodiments of the present application, 0.7 mol / L potassium hydroxide-methanol solution is added to the supernatant, and the reaction is kept in a 30°C water bath for a first preset time to obtain a reaction product liquid containing fatty acid methyl esters, and the first preset time is 45 minutes.

[0068] In some optional embodiments of the present application, in step S40: the extraction solvent is n-hexane, oscillation is 1 minute and ultrasonic extraction is performed for a second preset time, and the second preset time is 5 minutes.

[0069] In some optional embodiments of the present application, 1 ml of the supernatant obtained by centrifugation in step S20 is taken, and 1 ml of potassium hydroxide-methanol solution is added to the 1 ml of supernatant. 3 mL of n-hexane is added in step S40.

[0070] In some optional embodiments of the present application, in step S50, the analysis conditions of the gas chromatography include:

[0071] A DB-Fast FAME column was used, and the parameters of the DB-Fast FAME column were 30m×0.25mm, 0.25um;

[0072] The carrier gas was nitrogen, 12 psi, constant pressure;

[0073] The injection port used split injection, the injection port temperature was 250 °C, and the split ratio was 100:1;

[0074] Column oven temperature program: initial temperature 80°C, maintained for 0.5 min, increased to 165°C at 40°C / min, maintained at 165°C for 1 min, increased to 230°C at 4°C / min, maintained at 230°C for 4 min;

[0075] The detection parameters of hydrogen flame ionization detector FID are as follows: operating temperature 260°C, hydrogen flow rate 40 mL / min, air flow rate 400 mL / min; nitrogen is used as the tail gas with a flow rate of 25 mL / min.

[0076] In some optional embodiments of the present application, the sorghum is wine sorghum.

[0077] In some optional embodiments of the present application, the method for determining the fatty acid content in sorghum can obtain the content of at least 37 fatty acids in the crushed sorghum sample, and the 37 fatty acids are butyric acid, caproic acid, caprylic acid, caprylic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, cis-9-tetradecanoic acid, pentadecanoic acid, cis-10-pentadecanoic acid, hexadecanoic acid, cis-9-hexadecanoic acid, heptadecanoic acid, cis-10-heptadecanoic acid, octadecanoic acid, trans-9-octadecanoic acid, cis-9-octadecanoic acid, trans, trans-9,12-octadecadienoic acid, cis, cis-9,12-octadecadienoic acid, eicosanoic acid, cis, cis, cis-6,9,12-decanoic acid, and cis-9,12-octadecadienoic acid. Octatrienoic acid, cis-11-eicosanonoic acid, cis,cis,cis-9,12,15-octadecatrienoic acid, heneicosanoic acid, cis,cis-11,14-eicosadienoic acid, docosanoic acid, cis,cis,cis-8,11,14-eicosatrienoic acid, cis-13-docosanonoic acid, cis-11,14,17-eicosatrienoic acid, cis-5,8,11,14-eicosatetraenoic acid, tricosanoic acid, cis-13,16-docosadienoic acid, tetracosanoic acid, cis-5,8,11,14-eicosapentaenoic acid, cis-15-tetracosnonoic acid and cis-4,7,10,13,16,19-docosahexaenoic acid.

[0078] In some optional embodiments of the present application, the conversion calculation relationship in step S50 is as shown in Formula 1:

[0079]

[0080] In formula 1:

[0081] X i - The content of each fatty acid in the broken sorghum sample, in grams per 100 grams (g / 100g);

[0082] m si —The concentration of each standard substance in the standard assay solution, in grams per liter (g / L);

[0083] A i - Peak area of ​​each fatty acid methyl ester in the extract;

[0084] F FAME-FA - Conversion coefficient of each fatty acid methyl ester into fatty acids;

[0085] V1—the volume of the added extraction solvent, in mL;

[0086] V2—the volume of sulfuric acid-methanol solution added, in mL;

[0087] V3-the volume of the supernatant used for fatty acid methyl esterification in step S30, in mL;

[0088] 1000—conversion factor;

[0089] A Si - Peak area of ​​each fatty acid in the standard test solution;

[0090] m—the weight of the broken sorghum sample, in grams (g);

[0091] 100—Coefficient for converting the content to the content per 100 g of sample. [Specific embodiment]

[0093] 1. Reagents

[0094] Methanol: analytical grade, purchased from Tianjin Komiou Chemical Reagent Co., Ltd.

[0095] Potassium hydroxide: analytical grade, purchased from Tianjin Komiou Chemical Reagent Co., Ltd.

[0096] Sulfuric acid: analytical grade, purchased from Chengdu Jinshan Chemical Reagent Co., Ltd.

[0097] n-Hexane: chromatographic grade, Supelco (Chromatography Department).

[0098] 37 fatty acid methyl ester standard samples were purchased from Supelco (Chromatography Department). The 37 fatty acids are butyric acid, hexanoic acid, octanoic acid, caprylic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, cis-9-tetradecanoic acid, pentadecanoic acid, cis-10-pentadecanoic acid, hexadecanoic acid, cis-9-hexadecanoic acid, heptadecanoic acid, cis-10-heptadecanoic acid, octadecanoic acid, trans-9-octadecanoic acid, cis-9-octadecanoic acid, trans, trans-9,12-octadecadienoic acid, cis, cis-9,12-octadecadienoic acid, eicosanoic acid, cis, cis, cis-6,9,12-octadecatrienoic acid, cis-1 1-eicosamonoenoic acid, cis, cis, cis-9,12,15-octadecatrienoic acid, heneicosanoic acid, cis, cis-11,14-eicosadienoic acid, docosanoic acid, cis, cis, cis-8,11,14-eicosatrienoic acid, cis-13-docosamonoenoic acid, cis-11,14,17-eicosatrienoic acid, cis-5,8,11,14-eicosatetraenoic acid, tricosanoic acid, cis-13,16-docosadienoic acid, tetracosanoic acid, cis-5,8,11,14-eicosapentaenoic acid, cis-15-tetracosmonoenoic acid, cis-4,7,10,13,16,19-docosahexaenoic acid.

[0099] 2. Equipment

[0100] Crushing: BLH-5601 hammer cyclone mill (Zhejiang Bethlehem Instrument Equipment Co., Ltd.).

[0101] Balance: AR2140 electronic balance (Mettler Toledo (Shanghai) Co., Ltd.).

[0102] Water bath reflux: DH2880Pro intelligent water bath condensation reflux extractor (Dehechuangrui Scientific Instrument (Qingdao) Co., Ltd.).

[0103] Centrifugation: 5804 centrifuge (Eppendorf).

[0104] Gas chromatograph: GC7820 Agilent gas chromatograph.

[0105] Gas chromatography system: 7820GC / FID.

[0106] Inlet: The inlet adopts split injection, the inlet temperature is 250℃, and the split ratio is 100:1.

[0107] A DB-Fast FAME column was used, and the parameters of the DB-Fast FAME column were 30m×0.25mm, 0.25um;

[0108] Carrier gas: nitrogen, 12 psi, constant pressure.

[0109] Column oven temperature program: initial temperature 80°C, maintain for 0.5 min, increase to 165°C at 40°C / min, maintain at 165°C for 1 min, increase to 230°C at 4°C / min, maintain at 230°C for 4 min.

[0110] The detection parameters of hydrogen flame ionization detector FID are as follows: operating temperature 260°C, hydrogen flow rate 40 mL / min, air flow rate 400 mL / min; nitrogen is used as the tail gas with a flow rate of 25 mL / min.

[0111] Injection volume: 1uL.

[0112] Ultrasonic extraction: POWERSONIC 620 ultrasonic cleaning machine (Shanghai Chenhui Instrument Co., Ltd.).

[0113] 3. Determination steps of fatty acid content in sorghum

[0114] S10: crushing sorghum and sieving to obtain crushed sorghum;

[0115] S20: a step of hydrolyzing bound fatty acids and extracting fatty acids, taking crushed sorghum according to a first preset weight to obtain a crushed sorghum sample to be tested, adding a sulfuric acid-methanol solution to the crushed sorghum sample, refluxing in a water bath, and centrifuging to obtain a supernatant after the refluxing is completed;

[0116] S30: Fatty acid methyl esterification step, adding potassium hydroxide-methanol solution to the supernatant, keeping the reaction in a water bath for a first preset time, and obtaining a reaction product liquid containing fatty acid methyl ester.

[0117] S40: a fatty acid methyl ester extraction step, in which an extraction solvent is added to the reaction product liquid to extract the fatty acid methyl ester, and the fatty acid methyl ester is extracted by oscillation and ultrasonic extraction for a second preset time to obtain an extract containing fatty acid methyl ester.

[0118] S50: using a gas chromatograph to perform qualitative and quantitative determination of the fatty acid methyl ester in the extract to obtain a fatty acid methyl ester determination result, and calculating the content of various fatty acids in the broken sorghum sample according to a conversion calculation relationship between the fatty acid methyl ester determination result and the corresponding fatty acid content.

[0119] The method for determining the fatty acid content in sorghum provided in the embodiment of the present application adopts sulfuric acid-methanol solution to hydrolyze and extract the bound fatty acids in sorghum, which can better hydrolyze triglycerides and extract fatty acids. The methylation of fatty acids with potassium hydroxide-methanol solution ensures that the fatty acids are completely methylated, which is conducive to the detection of the types and contents of fatty acids in sorghum. Subsequently, the fatty acid methyl esters are extracted with an extraction solvent and then subjected to gas chromatography detection to avoid the acidity or alkalinity in the test liquid affecting the subsequent gas chromatography detection, thereby increasing the total amount of fatty acids that can be measured and increasing the types of fatty acids that can be measured. Fewer types of detection reagents are used, which reduces the detection cost and simplifies the determination process. The method for determining the fatty acid content in sorghum provided in the embodiment of the present application can measure 37 kinds of fatty acids in sorghum, and there are many types of fatty acids that can be measured, and it can be used to specifically detect sorghum for wine.

[0120] In some examples, the conversion coefficients of fatty acid methyl ester and corresponding fatty acid are as shown in Table 1 below: FAME-FA .

[0121] Table 1 Conversion coefficients between fatty acid methyl esters and fatty acids

[0122]

[0123]

[0124] F FAME-FA is the conversion factor of fatty acid methyl esters into fatty acids.

[0125] 4. Study on the operating conditions of each step in the determination method

[0126] 4.1 Concentration of sulfuric acid-methanol solution

[0127] This section uses the acidity of the sulfuric acid-methanol solution after the final hydrolysis to measure whether the hydrolysis is complete. Hydrolysis consumes sulfuric acid in the solution, and consumption stops after the hydrolysis is completed. The consumption of sulfuric acid at different concentrations is measured.

[0128] The sorghum was crushed and sieved to obtain broken sorghum. Specifically, the sorghum was crushed with a hammer cyclone mill and passed through a 200-mesh sieve to obtain a broken sorghum sample.

[0129] Prepare 0.5 mol / L, 1.0 mol / L, 1.5 mol / L and 2 mol / L sulfuric acid-methanol solutions respectively.

[0130] The study found that 20 mL of sulfuric acid-methanol solution had a better wetting effect on 1 g of crushed sorghum sample.

[0131] Four experimental groups were set up, each of which included a blank experiment and a sample experiment. For each sample experiment, 1 g of the broken sorghum sample was taken, and 20 mL of sulfuric acid-methanol solution was added (the sample experiments in each experimental group corresponded to sulfuric acid-methanol solutions with concentrations of 0.5 mol / L, 1.0 mol / L, 1.5 mol / L and 2 mol / L, respectively) and refluxed on an 80°C water bath for 4 h (4 h was a sufficiently long reflux time). The blank experiment in each experimental group was to take 20 mL of sulfuric acid-methanol solution of the same concentration as the sample experiment and reflux it on an 80°C water bath for 4 h. After the blank experiment and the sample experiment were refluxed in each experimental group, 3 mL of the solution that had been refluxed in the blank experiment and the sample experiment was taken and titrated with 0.1 mol / L sodium hydroxide solution to calculate the amount of sulfuric acid consumed by hydrolysis.

[0132] The titration results of the four experimental groups are shown in Table 2

[0133] Table 2

[0134]

[0135] As shown in Table 2, after the concentration reaches 1.0 mol / L, the amount of sulfuric acid consumed by hydrolysis is basically the same, indicating that 1 g of the crushed sorghum sample has been fully hydrolyzed, so the concentration of the sulfuric acid-methanol solution can be selected to be 1.0 mol / L to 2.0 mol / L. In some embodiments, the sulfuric acid-methanol solution is selected to be 1.0 mol / L.

[0136] 4.2 Potassium hydroxide-methanol concentration, water bath time and water bath temperature

[0137] An orthogonal experiment was designed to determine the potassium hydroxide-methanol concentration, water bath time and water bath temperature to obtain the most sufficient conditions for methylation of fatty acids, so as to optimize the fatty acid methylation step and increase the total amount of fatty acids measured by the determination method.

[0138] Table 3 Orthogonal experimental conditions

[0139]

[0140] As shown in Table 3, a three-factor three-level orthogonal experiment was set up.

[0141] Nine groups of orthogonal experiments were set up, namely, Examples 1 to 9. The determination method of each example was carried out according to the following steps, except that the potassium hydroxide-methanol concentration, water bath time and water bath temperature respectively corresponded to the parameters set in the orthogonal experiment:

[0142] S10: crushing the sorghum and sieving to obtain broken sorghum. Specifically, crushing with a hammer cyclone mill and passing through a 200-mesh sieve to obtain broken sorghum.

[0143] S20: hydrolyzing bound fatty acids and extracting fatty acids, taking 1 g of broken sorghum to obtain a broken sorghum sample to be tested, adding 20 ml of 1.0 mol / L sulfuric acid-methanol solution to the broken sorghum sample, refluxing in a water bath at 80° C. for a sufficient time (for example, 4 hours), and centrifuging to obtain a supernatant after the reflux is completed. Specifically, the supernatant is obtained after centrifugation at 8000 rpm for 5 minutes.

[0144] S30: Fatty acid methyl esterification step, adding 1 ml of a potassium hydroxide-methanol solution of a preset concentration to 1 ml of the supernatant that has been centrifuged, and reacting in a water bath under set conditions for a first preset time to obtain a reaction product liquid containing fatty acid methyl esters.

[0145] S40: a step of extracting fatty acid methyl esters, adding 3 ml of n-hexane as an extraction solvent to the reaction product solution to extract fatty acid methyl esters, shaking for 1 minute and ultrasonically extracting for 5 minutes to obtain an extract containing fatty acid methyl esters.

[0146] S50: using a gas chromatograph to perform qualitative and quantitative determination of fatty acid methyl esters in the extract to obtain the determination results of fatty acid methyl esters, and calculating the contents of various fatty acids in the broken sorghum sample according to the conversion calculation relationship between the determination results of fatty acid methyl esters and the corresponding fatty acid contents.

[0147] Table 4 Orthogonal experiment results

[0148]

[0149] Table 5 The fatty acid contents of each embodiment measured in the orthogonal experiment

[0150]

[0151]

[0152]

[0153] As shown in Table 4 and Table 5, the orthogonal test results show that the combination of A1, B2 and C1 is the optimal experimental condition, that is, the potassium hydroxide-methanol concentration is 0.7 mol / L, the water bath time is 45 min, and the water bath temperature is 30°C. As shown in Table 6 below, according to this parameter design, the total amount of fatty acids measured in Example 10 is 3.536 g / 100 g. In the determination method of Example 10, the potassium hydroxide-methanol concentration is 0.7 mol / L, the water bath time is 45 min, and the water bath temperature is 30°C, and the rest is the same as Examples 1 to 9.

[0154] Table 6 The fatty acid contents measured in Example 10

[0155]

[0156]

[0157] 4.3 Hydrolysis extraction reflux time

[0158] The hydrolysis extraction reflux time corresponding to Examples 11 to 15 is set to 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, respectively. The determination methods of Examples 11 to 15 are the same as those of Example 10 except for the hydrolysis extraction reflux time.

[0159] Table 7 The fatty acid contents measured in Examples 11 to 15

[0160]

[0161]

[0162] It can be seen that the total amount of fatty acids in the hydrolysis extraction reflux time rises to a relatively high 3.5g / 100g area after 2.0h (including 2.0h), and the fatty acid content corresponding to 2.0h, 2.5h and 3.0h fluctuates slightly around 3.5 in the area near 3.5g / 100g. It can be considered that the measured fatty acid content tends to be stable, indicating that the hydrolysis extraction reflux time can range from 2.0h to 3.0h. Considering saving the measurement time, 2.0h can be used as a better hydrolysis extraction reflux time. The hydrolysis extraction reflux time here is the reflux time in step S20.

[0163] 5. Study on the hydrolysis and extraction effect of sulfuric acid-methanol solution on broken sorghum samples

[0164] The determination method of Example 16:

[0165] S10: crushing the sorghum and sieving to obtain broken sorghum. Specifically, crushing with a hammer cyclone mill and passing through a 200-mesh sieve to obtain broken sorghum.

[0166] S20: hydrolyzing bound fatty acids and extracting fatty acids, taking 1 g of broken sorghum to obtain a broken sorghum sample to be tested, adding 20 ml of 1.0 mol / L sulfuric acid-methanol solution to the broken sorghum sample, refluxing in a water bath at 80° C. for 2 hours, and centrifuging to obtain a supernatant after the reflux is completed. Specifically, the supernatant is obtained after centrifugation at 8000 rpm for 5 minutes.

[0167] S30: Fatty acid methyl esterification step, add 1 mL of 0.7 mol / L potassium hydroxide-methanol solution to 1 mL of the supernatant that has been centrifuged, shake gently to prevent the liquid from adhering to the wall, and keep the reaction in a 30° C. water bath for 45 minutes to obtain a reaction product liquid containing fatty acid methyl ester.

[0168] S40: a step of extracting fatty acid methyl esters, wherein 3 mL of n-hexane is added to the reaction product solution as an extraction solvent to extract fatty acid methyl esters, and the mixture is shaken for 1 minute and ultrasonically extracted for 5 minutes to obtain an extract containing fatty acid methyl esters.

[0169] S40: using a gas chromatograph to perform qualitative and quantitative determination of fatty acid methyl esters in the extract to obtain the fatty acid methyl ester determination results, and calculating the contents of various fatty acids in the broken sorghum sample based on the fatty acid methyl ester determination results and the corresponding fatty acid content conversion calculation relationship.

[0170] The determination method of comparative example 1:

[0171] Except for adding pure methanol solution in step S30, the rest of the steps are the same as the determination method of Example 16.

[0172] Table 8 Fatty acid contents measured in Example 16 and Comparative Example 1

[0173]

[0174]

[0175] Table 8 shows that the total amount of fatty acids in the broken sorghum sample measured in Comparative Example 1 without using potassium hydroxide-methanol solution for methyl esterification is significantly lower than that in Example 16 using potassium hydroxide-methanol solution for fatty acid methyl esterification, indicating that only part of the fatty acids are methylated when the sorghum is hydrolyzed and extracted with sulfuric acid-methanol solution. If the fatty acids need to be completely methylated, potassium hydroxide-methanol solution needs to be used for further methyl esterification to increase the total amount of fatty acids that can be measured and increase the types of fatty acids measured.

[0176] 6. Test accuracy of the determination method

[0177] The broken sorghum samples were reselected and the fatty acid content in the samples was determined by the determination method in Example 16 of the present application. The test results were compared with the national standard method "GB 5009.168-2016 National Food Safety Standard Determination of Fatty Acids in Foods". Since the main fatty acids in sorghum are octadecanoic acid, cis-9-octadecenoic acid, and cis, cis-9,12-octadecadienoic acid, the content differences of these three substances were mainly compared. The comparison results are shown in Table 8 below.

[0178]

[0179] It can be seen that the maximum relative error of the main substance is 3.06%, which is far lower than the national standard requirement of less than 10% for test precision. The accuracy of the determination method provided in the embodiment of the present application is feasible.

[0180] In general, the embodiments of the present application provide a method for determining the fatty acid content in sorghum that is efficient, accurate, and uses a small amount of reagents. The crushed sorghum sample is first hydrolyzed with a sulfuric acid-methanol solution, which can better hydrolyze triglycerides and extract fatty acids. The fatty acids are then methylated with a potassium hydroxide-methanol solution to ensure that the hydrolyzed fatty acids are fully methylated to ensure that the fatty acids obtained in subsequent measurements are rich in variety and high in content. The use of an extraction solvent such as n-hexane to extract fatty acid methyl esters avoids damage to the chromatographic column when the test liquid is acidic or alkaline and is directly measured on the machine. In the embodiments of the present application, only four reagents (methanol, potassium hydroxide, sulfuric acid, and n-hexane) were used to complete the detection of sorghum fatty acids. This method can determine at least 37 fatty acids in sorghum, with a large number of types.

[0181] The above specific implementations do not constitute a limitation on the protection scope of this application. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of this application should be included in the protection scope of this application.

Claims

1. A method for determining the fatty acid content in sorghum, characterized in that: include: S10: crushing the sorghum and sieving to obtain crushed sorghum; S20: a step of hydrolyzing bound fatty acids and extracting fatty acids, taking the crushed sorghum according to a first preset weight to obtain a crushed sorghum sample to be tested, adding a sulfuric acid-methanol solution to the crushed sorghum sample, refluxing in a water bath, and centrifuging to obtain a supernatant after the refluxing is completed; S30: a fatty acid methyl esterification step, adding a potassium hydroxide-methanol solution to the supernatant, and keeping the reaction in a water bath for a first preset time to obtain a reaction product liquid containing fatty acid methyl ester; S40: a fatty acid methyl ester extraction step, adding an extraction solvent to the reaction product liquid to extract the fatty acid methyl ester, and oscillating and ultrasonically extracting for a second preset time to obtain an extract containing fatty acid methyl ester; S50: using a gas chromatograph to perform qualitative and quantitative determination of the fatty acid methyl ester in the extract to obtain a fatty acid methyl ester determination result, and calculating the content of various fatty acids in the broken sorghum sample according to a conversion calculation relationship between the fatty acid methyl ester determination result and the corresponding fatty acid content.

2. The method for determining the fatty acid content in sorghum according to claim 1, characterized in that: In the step S10: The sorghum was crushed with a hammer cyclone mill and passed through a 200-mesh sieve to obtain ground sorghum.

3. The method for determining the fatty acid content in sorghum according to claim 2, characterized in that: In the step S20: Reflux in a water bath at 80°C for 2h to 4h; Preferably, reflux in a water bath at 80°C for 2 to 3 hours; preferably, reflux in a water bath at 80°C for 2 hours, 2.5 hours or 3 hours; Preferably, in the step S20: The centrifugation condition was 8000 rpm for 5 minutes.

4. The method for determining the fatty acid content in sorghum according to claim 1, characterized in that: 20 ml of sulfuric acid-methanol solution was added per 1 g of the crushed sorghum sample to soak the crushed sorghum sample.

5. The method for determining the fatty acid content in sorghum according to claim 1, characterized in that: The concentration of the sulfuric acid-methanol solution is 1.0 mol / L to 2 mol / L; Preferably, the concentration of the sulfuric acid-methanol solution is 1.0 mol / L.

6. The method for determining the fatty acid content in sorghum according to claim 1, characterized in that: In step S30: Adding 0.7 mol / L to 1.1 mol / L potassium hydroxide-methanol solution to the supernatant, and keeping the reaction in a water bath at 30° C. to 50° C. for a first preset time to obtain a reaction product liquid containing fatty acid methyl esters, wherein the first preset time is 30 min to 60 min; Preferably, 0.7 mol / L potassium hydroxide-methanol solution is added to the supernatant, and the reaction is carried out in a 30° C. water bath for a first preset time to obtain a reaction product liquid containing fatty acid methyl esters, wherein the first preset time is 45 minutes.

7. The method for determining the fatty acid content in sorghum according to claim 1, characterized in that: In the step S40: the extraction solvent is n-hexane, the oscillation is 1 minute and the ultrasonic extraction is performed for a second preset time, and the second preset time is 5 minutes; Preferably, the step S30 comprises: taking 1 ml of the supernatant obtained by centrifugation in step S20, and adding 1 ml of potassium hydroxide-methanol solution to the 1 ml of the supernatant, and adding 3 mL of n-hexane in step S40.

8. The method for determining the fatty acid content in sorghum according to claim 1, characterized in that: In step S50, the analysis conditions of gas chromatography include: A DB-Fast FAME column was used, and the parameters of the DB-Fast FAME column were 30m×0.25mm, 0.25um; The carrier gas was nitrogen, 12 psi, constant pressure; The injection port used split injection, the injection port temperature was 250 °C, and the split ratio was 100:1; Column oven temperature program: initial temperature 80°C, maintained for 0.5 min, increased to 165°C at 40°C / min, maintained at 165°C for 1 min, increased to 230°C at 4°C / min, maintained at 230°C for 4 min; The detection parameters of hydrogen flame ionization detector FID are as follows: operating temperature 260°C, hydrogen flow rate 40 mL / min, air flow rate 400 mL / min; nitrogen is used as the tail gas with a flow rate of 25 mL / min.

9. The method for determining the fatty acid content in sorghum according to claim 1, characterized in that: The sorghum is sorghum for wine; Preferably, the method for determining the fatty acid content in sorghum can obtain the content of at least 37 fatty acids in the crushed sorghum sample, and the 37 fatty acids are butyric acid, caproic acid, octanoic acid, caprylic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, cis-9-tetradecanoic acid, pentadecanoic acid, cis-10-pentadecanoic acid, hexadecanoic acid, cis-9-hexadecanoic acid, heptadecanoic acid, cis-10-heptadecanoic acid, octadecanoic acid, trans-9-octadecanoic acid, cis-9-octadecanoic acid, trans, trans-9,12-octadecadienoic acid, cis, cis-9,12-octadecadienoic acid, eicosanoic acid, cis, cis, cis-6,9,12-octadecanoic acid, and cis-9,12-octadecadienoic acid. trienoic acid, cis-11-eicosamonoenoic acid, cis,cis,cis-9,12,15-octadecatrienoic acid, heneicosanoic acid, cis,cis-11,14-eicosadienoic acid, docosanoic acid, cis,cis,cis-8,11,14-eicosatrienoic acid, cis-13-docosamonoenoic acid, cis-11,14,17-eicosatrienoic acid, cis-5,8,11,14-eicosatetraenoic acid, tricosanoic acid, cis-13,16-docosadienoic acid, tetracosanoic acid, cis-5,8,11,14-eicosapentaenoic acid, cis-15-tetracosmonoenoic acid and cis-4,7,10,13,16,19-docosahexaenoic acid.

10. The method for determining the fatty acid content in sorghum according to claim 1, characterized in that: The conversion calculation relationship in step S50 is shown in Formula 1: In formula 1: X i - The content of each fatty acid in the broken sorghum sample, in grams per 100 grams (g / 100g); m si —The concentration of each standard substance in the standard assay solution, in grams per liter (g / L); A i - Peak area of ​​each fatty acid methyl ester in the extract; F FAME-FA - Conversion coefficient of each fatty acid methyl ester into fatty acids; V1—the volume of the added extraction solvent, in mL; V2—the volume of sulfuric acid-methanol solution added, in mL; V3-the volume of the supernatant used for fatty acid methyl esterification in step S30, in mL; 1000—conversion factor; A Si - Peak area of ​​each fatty acid in the standard test solution; m—the weight of the broken sorghum sample, in grams (g); 100—Coefficient for converting the content to the content per 100 g of sample.

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