Fermented grain reducing sugar detection method based on titrator

Abstract

The invention provides a method for detecting reducing sugar in fermented grains based on a titrator, which is used for quantitatively analyzing reducing sugar in a fermented grain sample by adopting a full-automatic potentiometric titration technology in combination with an accurate chemical analysis method, and specifically comprises the following steps: S1, preparing an instrument and a reagent; preparing instruments and reagents required by detection; s2, preparing a solution; preparing a sodium thiosulfate standard solution, a sulfuric acid solution, a potassium iodide solution, a solution A and a solution B for later use; s3, sample treatment; treating a to-be-detected sample for later use; s4, determining the content of reducing sugar in the fermented grains through a full-automatic potentiometric titration method; sequentially carrying out pre-titration, blank titration and sample titration, and calculating to obtain a final result. The method has obvious advantages in the aspects of data management, operation convenience and the like, and can replace a manual titration method to be applied to determination of the reducing sugar content in the fermented grains.

Description

Technical Field

[0001] The present invention relates to the technical field of wine packaging, and in particular to a titrator-based method for detecting reducing sugar in fermented grains. Background Art

[0002] In the brewing process, the reducing sugar content in the fermented grains of sauce-flavor liquor is an important factor affecting its fermentation quality. By measuring the reducing sugar content in the fermented grains, the saccharification degree of the fermented grains can be understood, thereby improving the fermentation conditions of the fermented grains. For a long time, the classical chemical method used to determine the reducing sugar in the fermented grains has mostly been the Fehling reagent method [1]. However, this method is affected by many factors such as heating temperature and boiling time, and requires a high level of operator proficiency, otherwise it is easy to cause large errors [2]. For this reason, we analyzed the entire process of reducing sugar determination in the fermented grains, explored the various factors affecting the determination of reducing sugar in the fermented grains, and reduced the error of reducing sugar determination in the fermented grains by improving the operating method. In recent years, due to the high accuracy and precision of the automatic titrator, which can minimize the influence of human factors, the potentiometric titration method has gradually become a new and reliable method for reducing sugar determination and has been used by more and more testing departments [3]. However, there is still a lack of systematic methodological research on the determination of reducing sugar in the fermented grains by this method. In this study, the content of reducing sugar in mash was determined by a fully automatic potentiometric titrator and the results were compared with those determined by manual titration.

[0003] [1] Ye Haihui, He Xiufen, Wang Xiulan. Improvement of Fehling's reagent titration method for determining reducing sugars[J]. Tropical Agricultural Sciences, 2001, (03): 9-11.

[0004] [2] Zhao Dong, Niu Guangjie, Zhao Xudong, et al. Discussion on the analysis method of reducing sugar in mash[J]. Brewing, 2013, 40(02): 88-90.

[0005] [3] Ye Hu, Chen Ying, Zhao Xiaofeng, et al. Automatic potentiometric titration method for determination of total sugar in solid foods[J]. Occupation and Health, 2012, 28(18): 2244-2246. DOI: 10.13329 / j.cnki.zyyjk.2012.18.001. Summary of the Invention

[0006] In order to solve the above problems, the present invention discloses a method for detecting reducing sugar in fermented grains based on a titrator.

[0007] The specific plan is as follows:

[0008] A titrator-based method for detecting reducing sugar in fermented grains uses fully automatic potentiometric titration technology combined with precise chemical analysis methods to quantitatively analyze reducing sugar in fermented grains samples. The method specifically includes the following steps:

[0009] S1. Preparation of instruments and reagents: Prepare the instruments and reagents required for testing;

[0010] S2. Solution preparation: prepare sodium thiosulfate standard solution, sulfuric acid solution, potassium iodide solution, solution A and solution B and set aside;

[0011] S3, sample processing: process the sample to be tested and set aside;

[0012] S4. Determine the reducing sugar content in the mash by fully automatic potentiometric titration; perform pre-titration, blank titration and sample titration in sequence, and calculate the final result.

[0013] Furthermore, the instruments and reagents in step S1 are as follows:

[0014] Instruments include: CT90 fully automatic potentiometric titrator, ORP redox electrode, PMK224ZH / E electronic balance, HH-6 digital display constant temperature water bath;

[0015] Reagents include: sodium thiosulfate (Na2S2O3·5H2O), sodium carbonate (Na2CO3), sulfuric acid (H2SO4), copper sulfate (CuSO4·5H2O), potassium iodide (KI), potassium sodium tartrate (C4H4O6KNa·4H2O), sodium hydroxide (NaOH), glucose (C6H 12 O6) and deionized water.

[0016] Furthermore, the solutions and preparation methods in step S2 are as follows:

[0017] 0.1 mol / L sodium thiosulfate standard solution: Weigh 26 g of sodium thiosulfate pentahydrate and 0.2 g of sodium carbonate, dissolve in 1 L of water, boil slowly for 10 minutes, cool, and filter after two weeks;

[0018] Sodium hydroxide solution: weigh 8 g of NaOH solid, dissolve it in water and dilute to 100 mL;

[0019] 16% (mass fraction) sulfuric acid solution: Measure 90 mL of concentrated sulfuric acid and dilute to 1 L;

[0020] 30% (mass fraction) potassium iodide solution: weigh 150g KI, dissolve it in water, add 25mL NaOH, and dilute to 500mL with water;

[0021] Fehling's solution (Solution A): Weigh 20.96 g of copper sulfate pentahydrate and dissolve it in water. Add 1 mL of sulfuric acid solution and dilute to 500 mL with water.

[0022] Fehling's solution (Solution B): Weigh 173 g of potassium sodium tartrate tetrahydrate and 40 g of NaOH and dissolve them in water. Mix the two solutions and dilute to 500 mL.

[0023] Furthermore, the samples and processing methods in step S3 are as follows:

[0024] Sample: Maotai-flavor liquor mash (Guizhou Guijiu Group Co., Ltd.);

[0025] Treatment method: After the sample is evenly mixed, it is divided into quarters and 10.0 (+0.01) g of the mash sample is placed in a beaker. 100 mL of deionized water is added and stirred. After standing for 30 minutes (stirring every 10 minutes), the sample is filtered and the filtrate is set aside.

[0026] Furthermore, the pre-titration, blank titration and sample titration in step S4 specifically include:

[0027] S41. Pre-titration: Accurately pipette Fehling's reagent (5 mL each of Solution A and Solution B / 10 mL of Solution A and 5 mL of Solution B) into the titration cup. Heat and boil in a water bath for 5 minutes. Rapidly cool with cold water and place the sample tray for titration. Using the software, add 10 mL of sulfuric acid solution, 10 mL of potassium iodide solution, and 20 mL of deionized water to the titration cup. Select dynamic potentiometric titration as the titration mode and an ORP combination electrode as the electrode. Set the pre-addition volume to 0 and titrate with 0.1 mol / L sodium thiosulfate standard solution to the maximum isoelectric point to obtain the pre-titration volume.

[0028] S42. Blank titration: except that the preset volume is 1 mL less than the pre-titration volume, the rest of the operation is the same as the pre-titration. When the maximum isoelectric point is reached, the final blank volume is obtained.

[0029] S43. Sample titration: Accurately pipette Fehling's reagent (5 mL each of Solution A and Solution B / 10 mL of Solution A and 5 mL of Solution B) into the titration cup, add 2 mL of sample filtrate, heat in a water bath and boil for 5 minutes, then quickly cool with cold water. Using the software, add 10 mL of sulfuric acid solution, 10 mL of potassium iodide solution, and 20 mL of deionized water. Set the appropriate pre-addition volume and titrate with 0.1 mol / L sodium thiosulfate standard solution to the maximum isoelectric point to obtain the sample titration volume.

[0030] Furthermore, the result calculation method in step S4 is:

[0031] When Fehling's reagent A and B are mixed, a sky-blue copper hydroxide precipitate is generated.

[0032] CuSO4+2NaOH→Cu(OH)2↓+Na2SO4

[0033] Reacts with potassium sodium tartrate to form a dark blue complex potassium sodium copper tartrate

[0034]

[0035] Potassium sodium copper tartrate is reduced by reducing sugar to form a red cuprous oxide precipitate

[0036]

[0037] Under acidic conditions, the remaining potassium sodium copper tartrate can precipitate Cu 2+

[0038]

[0039] Cu 2+ With I in potassium iodide -1 The reaction forms I2, which can be titrated with Na2S2O3 standard solution. The reducing sugar content in the mash can be calculated based on the Na2S2O3 consumption.

[0040] 2Cu 2+ +4I - →2CuI↓+I2

[0041] I2+2Na2S2O3→Na2S4O6+2NaI

[0042] The beneficial effects of the present invention are as follows: the linear relationship of the method of the present invention is verified by the measurement results of glucose standard solutions with different concentrations, R 2 =0.999; Four samples of fermented grains from different production teams were measured using both the fully automated potentiometric titration method and the manual titration method. The RSD of the fully automated potentiometric titration method was between 0.30% and 0.97%, demonstrating that the fully automated potentiometric titration method outperformed the manual titration method in terms of repeatability. Error and correlation analysis of the test results from the two methods revealed a relative error range of -1.38% to 0.78%, with no significant difference between the two methods. This method demonstrates significant advantages in data management and ease of operation, and can replace manual titration methods for the determination of reducing sugar content in fermented grains. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a linear test graph of the method in the embodiment. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0045] The present invention provides a titrator-based method for detecting reducing sugar in fermented grains. By adopting a fully automatic potentiometric titration technique in combination with an accurate chemical analysis method, the reducing sugar in the fermented grains sample is quantitatively analyzed. The method specifically comprises the following steps:

[0046] S1. Preparation of instruments and reagents: Prepare the instruments and reagents required for testing;

[0047] S2. Solution preparation: prepare sodium thiosulfate standard solution, sulfuric acid solution, potassium iodide solution, solution A and solution B and set aside;

[0048] S3, sample processing: process the sample to be tested and set aside;

[0049] S4. Determine the reducing sugar content in the fermented grains by fully automatic potentiometric titration; perform pre-titration, blank titration, and sample titration in sequence, and calculate the final result;

[0050] S5. Determine the reducing sugar content in the fermented grains by manual titration. Sample preparation, sample pre-titration, formal titration, and calculation shall comply with the provisions of DB 34 / T 2264-2014 "Analysis Methods for Fermented Grains from Solid-State Fermentation";

[0051] S6. Establishing a linear relationship for the titration reaction: Weigh 0.204 g, 0.502 g, 0.801 g, 1.102 g, 1.405 g, and 1.705 g of glucose, dissolve in water, and dilute to a 100 mL volumetric flask. Perform the sample determination according to 2.2.2, performing a linear regression analysis with the glucose mass as the abscissa and the titration volume as the ordinate, and draw a curve.

[0052] S7. Precision and accuracy experiment: Take 10.0(+0.01)g of fermented mash from 4 different teams as samples, accurately weigh them, perform sample pretreatment according to 2.1, and determine the reducing sugar content in the samples by automatic potentiometric titration and manual titration respectively. Calculate the relative standard deviation (RSD), and perform error and correlation analysis on the results of the two methods.

[0053] In this embodiment, the instruments and reagents in step S1 are as follows:

[0054] Instruments include: CT90 fully automatic potentiometric titrator, ORP redox electrode, PMK224ZH / E electronic balance, HH-6 digital display constant temperature water bath;

[0055] Reagents include: sodium thiosulfate (Na2S2O3·5H2O), sodium carbonate (Na2CO3), sulfuric acid (H2SO4), copper sulfate (CuSO4·5H2O), potassium iodide (KI), potassium sodium tartrate (C4H4O6KNa·4H2O), sodium hydroxide (NaOH), glucose (C6H 12 O6) and deionized water.

[0056] Furthermore, the solutions and preparation methods in step S2 are as follows:

[0057] 0.1 mol / L sodium thiosulfate standard solution: Weigh 26 g of sodium thiosulfate pentahydrate and 0.2 g of sodium carbonate, dissolve in 1 L of water, boil slowly for 10 minutes, cool, and filter after two weeks;

[0058] Sodium hydroxide solution: weigh 8 g of NaOH solid, dissolve it in water and dilute to 100 mL;

[0059] 16% (mass fraction) sulfuric acid solution: Measure 90 mL of concentrated sulfuric acid and dilute to 1 L;

[0060] 30% (mass fraction) potassium iodide solution: weigh 150g KI, dissolve it in water, add 25mL NaOH, and dilute to 500mL with water;

[0061] Fehling's solution (Solution A): Weigh 20.96 g of copper sulfate pentahydrate and dissolve it in water. Add 1 mL of sulfuric acid solution and dilute to 500 mL with water.

[0062] Fehling's solution (Solution B): Weigh 173 g of potassium sodium tartrate tetrahydrate and 40 g of NaOH and dissolve them in water. Mix the two solutions and dilute to 500 mL.

[0063] Furthermore, the samples and processing methods in step S3 are as follows:

[0064] Sample: Maotai-flavor liquor mash (Guizhou Guijiu Group Co., Ltd.);

[0065] Treatment method: After the sample is evenly mixed, it is divided into quarters and 10.0 (+0.01) g of the mash sample is placed in a beaker. 100 mL of deionized water is added and stirred. After standing for 30 minutes (stirring every 10 minutes), the sample is filtered and the filtrate is set aside.

[0066] Furthermore, the pre-titration, blank titration and sample titration in step S4 specifically include:

[0067] S41. Pre-titration: Accurately pipette Fehling's reagent (5 mL each of Solution A and Solution B / 10 mL of Solution A and 5 mL of Solution B) into the titration cup. Heat and boil in a water bath for 5 minutes. Rapidly cool with cold water and place the sample tray for titration. Using the software, add 10 mL of sulfuric acid solution, 10 mL of potassium iodide solution, and 20 mL of deionized water to the titration cup. Select dynamic potentiometric titration as the titration mode and an ORP combination electrode as the electrode. Set the pre-addition volume to 0 and titrate with 0.1 mol / L sodium thiosulfate standard solution to the maximum isoelectric point to obtain the pre-titration volume.

[0068] S42. Blank titration: except that the preset volume is 1 mL less than the pre-titration volume, the rest of the operation is the same as the pre-titration. When the maximum isoelectric point is reached, the final blank volume is obtained.

[0069] S43. Sample titration: Accurately pipette Fehling's reagent (5 mL each of Solution A and Solution B / 10 mL of Solution A and 5 mL of Solution B) into the titration cup, add 2 mL of sample filtrate, heat in a water bath and boil for 5 minutes, then quickly cool with cold water. Using the software, add 10 mL of sulfuric acid solution, 10 mL of potassium iodide solution, and 20 mL of deionized water. Set the appropriate pre-addition volume and titrate with 0.1 mol / L sodium thiosulfate standard solution to the maximum isoelectric point to obtain the sample titration volume.

[0070] Furthermore, the result calculation method in step S4 is:

[0071] When Fehling's reagent A and B are mixed, a sky-blue copper hydroxide precipitate is generated.

[0072] CuSO4+2NaOH→Cu(OH)2↓+Na2SO4

[0073] Reacts with potassium sodium tartrate to form a dark blue complex potassium sodium copper tartrate

[0074]

[0075] Potassium sodium copper tartrate is reduced by reducing sugar to form a red cuprous oxide precipitate

[0076]

[0077] Under acidic conditions, the remaining potassium sodium copper tartrate can precipitate Cu 2+

[0078]

[0079] Cu 2+ With I in potassium iodide -1 The reaction forms I2, which can be titrated with Na2S2O3 standard solution. The reducing sugar content in the mash can be calculated based on the Na2S2O3 consumption.

[0080] 2Cu 2+ +4I - →2CuI↓+I2

[0081] I2+2Na2S2O3→Na2S4O6+2NaI

[0082] In this embodiment, the results of the method of the present invention and the manual titration method are analyzed as follows:

[0083] 1. Linear relationship of titration reaction

[0084] The sample determination method of the present invention is used for determination, and the titration consumption volume is recorded as 19.046mL, 17.185mL, 15.032mL, 12.574mL, 10.488mL, and 8.373mL. A curve is drawn with glucose as the horizontal axis and the titration volume as the vertical axis, as shown in FIG. Figure 1 As shown in the figure, it can be seen that the method of the present invention has a good linear relationship, and the regression equation is y=-7.2199x+20.665, R 2 =0.999.

[0085] 2. Repeatability of fully automatic potentiometric titration

[0086] The determinations were performed using both fully automated potentiometric and manual titration methods. Each sample was measured three times in parallel. The results are shown in Table 1. The relative standard deviations for the fully automated potentiometric titration method ranged from 0.30% to 0.97%, while those for the manual titration method ranged from 0.76% to 1.25%. This indicates that the fully automated potentiometric titration method has superior repeatability to the manual titration method. This may be due to the fact that the manual titration method relies on manual endpoint determination, which is subject to numerous factors. The fully automated potentiometric titration method, on the other hand, uses the instrument to automatically identify the endpoint based on potential changes, and the titration speed is precisely controlled by the instrument, significantly reducing human error and resulting in better stability.

[0087] Table 1 Repeatability test of potentiometric titration

[0088]

[0089] 3. Consistency of results between automatic potentiometric titration and manual titration

[0090] The results of the automatic potentiometric titration method and the manual titration method are shown in Table 2. It can be seen from the table that the relative error of the automatic potentiometric titration method is -1.38%-0.78% compared with the manual titration results. The results of the two methods are subjected to correlation analysis, and R 2 =0.9983, indicating that the reducing sugar content determined by the automatic potentiometric titration method and the manual titration method have a good correlation and no significant difference. Therefore, the automatic potentiometric titration method can be used to determine the reducing sugar content in fermented grains.

[0091] Table 2 Results of automatic potentiometric and manual titration

[0092]

[0093] 4. Conclusion

[0094] The present invention applies the full-automatic potentiometric titrator to the determination of reducing sugar content in fermented grains. By studying glucose standard solutions of different concentrations, the full-automatic potentiometric titration method is used to determine the results and draw a curve. 2=0.999, the results show that the method has a good linear relationship; the results of the automatic potentiometric titration method and the manual titration method are compared. The relative standard deviation of the automatic potentiometric titration method is 0.30%-0.97%, and the relative standard deviation of the manual titration method is 0.76%-1.25%. The relative error range of the two methods is -1.38%-0.78%, R 2 =0.9983.

[0095] In summary, it can be judged that the fully automatic potentiometric titration method has good precision and accuracy, and has a high correlation with the results of the manual titration method. Combined with the characteristics of the instrument itself, it shows obvious advantages in data management and operational convenience. Therefore, the fully automatic potentiometric titration method can replace the manual titration method in the determination of reducing sugar content in mash.

[0096] The technical means disclosed in the solutions of the present invention are not limited to those disclosed in the above-mentioned embodiments, but also include technical solutions composed of any combination of the above-mentioned technical features. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for detecting reducing sugar in fermented grains based on a titrator, characterized in that: The quantitative analysis of reducing sugars in fermented grains samples was performed using fully automated potentiometric titration technology combined with precise chemical analysis methods. The specific steps include: S1. Preparation of instruments and reagents: Prepare the instruments and reagents required for testing; S2. Solution preparation: prepare sodium thiosulfate standard solution, sulfuric acid solution, potassium iodide solution, solution A and solution B and set aside; S3, sample processing: process the sample to be tested and set aside; S4. Determine the reducing sugar content in the mash by fully automatic potentiometric titration; perform pre-titration, blank titration and sample titration in sequence, and calculate the final result.

2. The method for detecting reducing sugar in fermented grains based on a titrator according to claim 1, wherein: The instruments and reagents in step S1 are as follows: Instruments include: CT90 fully automatic potentiometric titrator, ORP redox electrode, PMK224ZH / E electronic balance, HH-6 digital display constant temperature water bath; Reagents include: sodium thiosulfate, sodium carbonate, sulfuric acid, copper sulfate, potassium iodide, potassium sodium tartrate, sodium hydroxide, glucose, and deionized water.

3. The method for detecting reducing sugar in fermented grains based on a titrator according to claim 2, wherein: The solutions and their preparation methods in step S2 are as follows: 0.1 mol / L sodium thiosulfate standard solution: Weigh 26 g of sodium thiosulfate pentahydrate and 0.2 g of sodium carbonate, dissolve in 1 L of water, boil slowly for 10 minutes, cool, and filter after two weeks; Sodium hydroxide solution: weigh 8 g of NaOH solid, dissolve it in water and dilute to 100 mL; 16% mass fraction sulfuric acid solution: Measure 90mL of concentrated sulfuric acid and dilute to 1L; 30% potassium iodide solution: Weigh 150g KI, dissolve it in water, add 25mL NaOH, and dilute to 500mL with water; Solution A is Fehling's solution: Weigh 20.96 g of copper sulfate pentahydrate and dissolve it in water. Add 1 mL of sulfuric acid solution and dilute to 500 mL with water. Solution B is Fehling's solution: Weigh 173 g of potassium sodium tartrate tetrahydrate and 40 g of NaOH separately and dissolve them in water. Mix the two solutions and make up to 500 mL.

4. The method for detecting reducing sugar in fermented grains based on a titrator according to claim 3, wherein: The samples and processing methods in step S3 are as follows: Sample: fermented grains of Maotai-flavor liquor; Treatment method: After the sample is evenly mixed, it is divided into quarters and 10.0 (+0.01) g of the mash sample is taken into a beaker. 100 mL of deionized water is added and stirred. After standing for 30 minutes, it is filtered and the filtrate is set aside.

5. The method for detecting reducing sugar in fermented grains based on a titrator according to claim 4, wherein: The pre-titration, blank titration and sample titration in step S4 specifically include: S41. Pre-titration: Accurately pipette Fehling's reagent into the titration cup, wherein 5 mL of solution A and 5 mL of solution B are added; 10 mL of solution A and 5 mL of solution B are added; and the mixture is heated and boiled in a water bath for 5 min. Rapidly cool the mixture with cold water, and the sample tray is placed for titration. 10 mL of sulfuric acid solution, 10 mL of potassium iodide solution, and 20 mL of deionized water are controlled to be added to the titration cup. The titration mode is dynamic potentiometric titration, and the electrode is an ORP combination electrode. The pre-addition volume is set to 0. Titrate with 0.1 mol / L sodium thiosulfate standard solution to the maximum isoelectric point to obtain the pre-titration volume. S42, blank titration: except that the preset volume is 1 mL less than the pre-titration volume, the rest of the operation is the same as the pre-titration. When the maximum isoelectric point is reached, the final blank volume is obtained; S43. Sample titration: Accurately pipette Fehling's reagent into the titration cup, wherein 5 mL of Solution A and 5 mL of Solution B are added; 2 mL of sample filtrate is added, and the mixture is heated and boiled in a water bath for 5 min. Rapidly cool with cold water, and 10 mL of sulfuric acid solution, 10 mL of potassium iodide solution, and 20 mL of deionized water are added in a controlled manner. Set an appropriate pre-addition volume, and titrate with 0.1 mol / L sodium thiosulfate standard solution to the maximum isoelectric point to obtain the sample titration volume.

6. The method for detecting reducing sugar in fermented grains based on a titrator according to claim 5, wherein: The result calculation method in step S4 is: When Fehling's reagent A and B are mixed, a sky-blue copper hydroxide precipitate is generated. CuSO4+2NaOH→Cu(OH)2↓+Na2SO4 Reacts with potassium sodium tartrate to form a dark blue complex potassium sodium copper tartrate Potassium sodium copper tartrate is reduced by reducing sugar to form a red cuprous oxide precipitate Under acidic conditions, the remaining potassium sodium copper tartrate can precipitate Cu 2+ Cu 2+ With I in potassium iodide -1 The reaction forms I2, which can be titrated with Na2S2O3 standard solution. The reducing sugar content in the mash can be calculated based on the Na2S2O3 consumption. 2Cu 2+ +4I - →2CuI↓+I2 I2+2Na2S2O3→Na2S4O6+2NaI.

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