Rapid qualitative detection method for cellulase in white granulated sugar
The method of detecting cellulase in white sugar by measuring the viscosity of sodium carboxymethyl cellulose solution solves the problems of speed and accuracy, simplifies the detection steps, reduces costs, and is applicable to the detection of cellulase in single-batch and multi-batch white sugar samples.
Patent Information
- Application Number
- CN202511249138.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-03
- Publication Date
- 2025-10-31
AI Technical Summary
Current technology cannot quickly and accurately detect whether white sugar contains cellulase, leading to product quality problems and corporate losses during the production process.
The viscosity detection method using sodium carboxymethyl cellulose solution determines whether cellulase is present by comparing the viscosity change of the white sugar sample to be tested with that of the standard solution. The detection process is simple, requiring only a viscometer, and is suitable for single-batch and multi-batch samples.
It enables rapid and accurate qualitative detection of cellulase, simplifies the detection process, reduces costs, and improves detection efficiency. It is suitable for cellulase detection in white sugar during the production process.
Smart Images
Figure SMS_1 
Figure SMS_2
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cellulase detection technology, specifically a rapid qualitative detection method for cellulase in white sugar. Background Technology
[0002] Cellulase (β-1,4-glucan-4-glucan hydrolase) is a collective term for a group of enzymes that degrade cellulose to produce glucose. It is not a monomeric enzyme but a multi-component enzyme system that works synergistically; it is a complex enzyme. It mainly consists of exo-β-glucanase, endo-β-glucanase, and β-glucosidase, acting on cellulose and its derivatives to break down cellulose into oligosaccharides or monosaccharides and proteins.
[0003] Because the current national standard for white sugar, GB / T 317-2018, does not specify requirements for enzymes, cellulase can be introduced into downstream food production as residues from white sugar production. In the production of liquid products using sodium carboxymethyl cellulose as the viscosity base, if the white sugar contains cellulase, it will enzymatically degrade cellulose and cellulose derivatives, causing the product to lose its viscosity and become substandard, resulting in losses for the company.
[0004] The following are some commonly used methods for detecting cellulase: I. Phenol-sulfuric acid method The phenol-sulfuric acid method is one of the classic methods for determining cellulase activity. Its principle is that cellulase hydrolyzes cellulose to produce reducing sugars, which can react with reagents phenol and sulfuric acid to produce a measurable color.
[0005] II. Arginine Method The arginine method is another commonly used method for determining cellulase activity. Its principle is as follows: cellulase hydrolyzes cellulose to produce reducing sugars, which react with arginine to produce urinary glycosamines. These urinary glycosamines then react with phenolamines to form measurable pigments.
[0006] III. Rheological Method Rheology is a method for determining enzyme activity by utilizing the change in system viscosity caused by cellulase degradation of substrate.
[0007] IV. Fluorescence Method The fluorescence method is a novel approach for determining cellulase activity. Its principle is that cellulase can hydrolyze cellulose to produce fluorescent substances, and the fluorescence intensity of these substances is directly proportional to the activity of the cellulase.
[0008] The above-mentioned detection methods are all activity detection methods, and they have drawbacks such as requiring numerous testing instruments and reagents, high precision requirements, complex testing procedures, long processing times, and high testing costs. In the white sugar application industry, these methods cannot quickly identify whether white sugar contains cellulase during incoming inspection. Summary of the Invention
[0009] The purpose of this invention is to provide a rapid qualitative detection method for cellulase in granulated sugar, addressing the problems existing in current cellulase activity detection methods. This method overcomes the shortcomings of existing cellulase activity detection methods, enabling simultaneous acceptance testing of 2-5 batches of granulated sugar samples, rapidly screening out granulated sugar samples with cellulase residues, and achieving high accuracy.
[0010] To achieve the above-mentioned objectives, the specific technical solution of the present invention is as follows: A rapid qualitative detection method for cellulase in granulated sugar includes the following steps: 1) Preparation of standard solution: To test N (N≥1) portions of white sugar samples: Heat RO water to 75-85℃, turn on the stirrer, slowly add (1+N)×5.000g sodium carboxymethyl cellulose, adjust the stirrer to a suitable speed, and continue stirring for 10-20 minutes to ensure the material is fully dissolved. Cool down to 40-60℃, and bring the volume to (1+N)×900ml with RO water. Stir well, and slowly add the prepared 10% citric acid monohydrate solution to adjust the pH to 4.5-6.5 to obtain sodium carboxymethyl cellulose solution. Divide the obtained sodium carboxymethyl cellulose solution into 1+N portions. 2) Viscosity test of sodium carboxymethyl cellulose standard solution: Place one portion of sodium carboxymethyl cellulose solution under a stirrer, turn on the stirrer, add 100.0g of standard white sugar until completely dissolved, cover or seal with plastic wrap, and keep in a water bath at 40-60℃ for 30 minutes. Then test its viscosity value. After testing 5 times, take the average value as C0. 3) Viscosity detection of sodium carboxymethyl cellulose test solution: Place the other N portions of sodium carboxymethyl cellulose solution under a stirrer, turn on the stirrer, add 100.0g of each of the other white sugar samples to be tested until completely dissolved, cover or seal with plastic wrap, and keep in a water bath at 40-60℃ for 90 minutes. Then test its viscosity value. After testing 5 times, take the average value as C1. The constant temperature in steps 2) and 3) must be consistent, and the temperature difference between the materials when testing viscosity should be ≤0.5℃.
[0011] 4) Result determination: I: C1≥C0, the white sugar cellulase test is negative, meaning the sample does not contain cellulase; II: C1 < C0, the white sugar to be tested is positive for cellulase, that is, the white sugar to be tested contains cellulase.
[0012] Furthermore, the rapid qualitative detection method for cellulase in white sugar also includes a parallel sample preparation step added between step 3) and step 4): that is, parallel samples are set up according to steps 1) to 3).
[0013] Furthermore, the rapid qualitative detection method for cellulase in granulated sugar is also applicable to the rapid qualitative detection of cellulase in mixed samples of multiple batches of granulated sugar.
[0014] Furthermore, a rapid qualitative detection method for cellulase in mixed samples of multiple batches of granulated sugar includes the following steps: Steps 1)-2), the aforementioned steps 1)-2). 3'): After keeping the other sodium carboxymethyl cellulose test solutions at a constant temperature in a water bath for 90 minutes, the viscosity value was measured. The average value was taken from 5 measurements and recorded as C1'. 4') Result determination: Ⅰ: C1'≥C0, the white sugar cellulase test is negative, that is, the sample to be tested does not contain cellulase; II: C1' < C0, the white sugar to be tested is positive for cellulase, that is, the white sugar to be tested contains cellulase; After the mixed sample tests positive, a separate batch is then tested for identification, i.e., the identification test is performed according to steps 1) to 4) of the aforementioned rapid qualitative detection method for cellulase in white sugar.
[0015] Furthermore, a rapid qualitative detection method for cellulase in multiple batches of mixed white sugar samples includes a parallel sample preparation step added between step 3') and step 4'): that is, parallel samples are set up according to steps 1) to 3').
[0016] Furthermore, in both the rapid qualitative detection method for cellulase in a single batch of white sugar samples and the rapid qualitative detection method for cellulase in a mixed sample of multiple batches of white sugar, in step 1), the sodium carboxymethyl cellulose must meet the relevant requirements of GB1886.232-2016, and its viscosity value must be between 500 (mPa·s) and 1400 (mPa·s); the standard white sugar is white sugar that does not contain cellulase.
[0017] Furthermore, in both the rapid qualitative detection method for cellulase in a single batch of white sugar samples and the rapid qualitative detection method for cellulase in a mixed sample of multiple batches of white sugar, the citric acid monohydrate is of analytical grade, and the mass percentage of the citric acid monohydrate solution is 10%.
[0018] Compared with existing technologies, the beneficial effects of this invention are: (i) The detection method is simple and easy to implement, and the only instrument required is a viscometer; (ii) It has a fast detection speed and is suitable for detecting cellulase in white sugar during the production process, with high accuracy.
[0019] (iii) This method is applicable not only to the rapid qualitative detection of cellulase in a single batch of mixed white sugar samples, but also to the rapid qualitative detection of cellulase in multiple batches of mixed white sugar samples. Detailed Implementation
[0020] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.
[0021] Any feature disclosed in this specification (including the claims and abstract) may be replaced by other equivalent or similar features, unless specifically stated otherwise. That is, unless specifically stated otherwise, each feature is merely one example of a series of equivalent or similar features.
[0022] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0023] Example 1: Rapid qualitative detection method for cellulase in white sugar samples (2 white sugar samples to be tested) 1. Preparation of standard solution 1.1 Material Weighing: Sodium carboxymethyl cellulose (viscosity 978 mPa·s) 15.0008 g; Citric acid monohydrate 10.0007g; Standard sample: 100.03g of white granulated sugar (without cellulase).
[0024] 240312 Sample of white granulated sugar 100.05g (white granulated sugar produced by XX white granulated sugar manufacturer on March 12, 2024); 230809 White granulated sugar sample 100.02g (white granulated sugar produced by XX white granulated sugar manufacturer on August 9, 2023); 1.2 Citric acid monohydrate standard solution: 10.0007 g of analytical grade citric acid monohydrate was dissolved in RO water and diluted to 100 ml. The solution was shaken well and then set aside.
[0025] 1.3 Preparation of sodium carboxymethyl cellulose solution: Two samples of granulated sugar were tested: Approximately 2000 ml of RO water was heated to 77°C, and sodium carboxymethyl cellulose was slowly added while stirring. The mixture was then dissolved for 17 minutes until fully dissolved. Stirring was stopped, and RO water was added to bring the volume to 2700 ml. The solution was then cooled to 40.5°C. Stirring was restarted, and citric acid monohydrate standard solution was slowly added dropwise to adjust the pH of the solution to 4.97.
[0026] Divide the prepared sodium carboxymethyl cellulose solution into three equal portions, numbered 0#, 1#, and 2#.
[0027] 2. Detection of viscosity C0 value of sodium carboxymethyl cellulose standard solution 2.1 Treatment of sodium carboxymethyl cellulose standard solution Turn on the stirrer and add all the standard white sugar to the 0# sodium carboxymethyl cellulose solution until completely dissolved. Then, cover the mouth of the beaker with plastic wrap and place it in a water bath at a set temperature of 40.5℃ for 30 minutes.
[0028] 2.2 Viscosity value C0 detection Five sets of viscosity values were tested using a constant-temperature standard solution (testing conditions: material temperature 40.3℃; viscometer rotor #2, speed 60rpm). The five sets of C0 values were 86.50 mPa·s, 86.00 mPa·s, 86.50 mPa·s, 87.00 mPa·s, and 86.00 mPa·s, respectively. The average value was taken as 86.4 mPa·s.
[0029] C0 = 86.4 (mPa·s) 3. Detection of viscosity value (C1) of sodium carboxymethyl cellulose test solution 3.1 Treatment of sodium carboxymethyl cellulose test solution Start stirring. For the remaining two portions of sodium carboxymethyl cellulose solution, add 100.05g of white sugar (240312) to be tested in sample #1 and 100.02g of white sugar (230809) to be tested in sample #2. Stir each for 5 minutes until completely dissolved. Then, seal the mouth of the beaker with plastic wrap and place it in a constant temperature water bath at 40.5℃ for 90 minutes.
[0030] 3.2 Viscosity (C1) Detection Five sets of viscosity values were measured for the test liquid after being kept at a constant temperature. Test conditions: material temperature 40.3℃, viscometer rotor #2, rotation speed 60 rpm. Results are shown in the table below.
[0031] 4. Result Determination 240312 White Sugar: Cl=96.5C0=86.4 C1 > C0, this batch of white sugar is cellulase negative, meaning it does not contain cellulase; 230809 White granulated sugar: Cl=7.30, C0=86.4 C1 < C0, this batch of white sugar is positive for cellulase, meaning it contains cellulase.
[0032] 5. Result Comparison: The cellulase activity of two white sugar samples, 240312 and 230809, was determined using the filter paper method according to GB / T23881-2009. The results are as follows: Sample 240312, white granulated sugar: Not detected. The white sugar sample 230809: 0.4 U / g.
[0033] Example 2: Rapid qualitative detection method for cellulase in white sugar sample (1 mixed white sugar sample to be tested) 1. Preparation of standard solution 1.1 Material Weighing: Sodium carboxymethyl cellulose (viscosity 1056 mPa·s) 10.0001 g; Citric acid monohydrate 10.0004g; Standard sample: 100.06g of granulated sugar (without cellulase).
[0034] 240312 White granulated sugar sample 20.03g (white granulated sugar produced by XX white granulated sugar manufacturer on March 12, 2024); 230809 White granulated sugar sample 20.02g (white granulated sugar produced by XX white granulated sugar manufacturer on August 9, 2023); 240323 White granulated sugar sample 20.02g (white granulated sugar produced by XX white granulated sugar manufacturer on March 23, 2024); 240504 White granulated sugar sample 20.00g (white granulated sugar produced by XX white granulated sugar manufacturer on May 4, 2024); Sample 240519, 20.01g of white granulated sugar (white granulated sugar produced by XX white granulated sugar manufacturer on May 19, 2024). A total of 100.08g of white sugar from five batches other than the standard white sugar was mixed together and used as the white sugar sample to be tested.
[0035] 1.2 Citric acid monohydrate standard solution: 10.0004 g of analytical grade citric acid monohydrate was dissolved in RO water and diluted to 100 ml. The solution was shaken well and then set aside.
[0036] 1.3 Preparation of sodium carboxymethyl cellulose solution: To test one sample of granulated sugar: Heat approximately 1200 ml of RO water to 75°C, start stirring, and slowly add sodium carboxymethyl cellulose, continuing to dissolve for 18 minutes until fully dissolved. Turn off stirring, add RO water to bring the volume to 1800 ml, and cool the solution to 40.3°C. Start stirring again, and slowly add citric acid monohydrate standard solution dropwise to adjust the pH of the solution to 5.05.
[0037] Divide the prepared sodium carboxymethyl cellulose solution into two equal portions, numbered 0# and 1# respectively.
[0038] 2. Detection of viscosity C0 value of sodium carboxymethyl cellulose standard solution 2.1 Treatment of sodium carboxymethyl cellulose standard solution Turn on the stirrer and add 100.06g of standard white sugar to the 0# sodium carboxymethyl cellulose solution. Stir for 6 minutes until completely dissolved. Then, cover the mouth of the beaker with plastic wrap and place it in a water bath at a temperature of 40.5℃ for 30 minutes.
[0039] 2.2 Viscosity value C0 detection Five sets of viscosity values were tested using a standard solution at a constant temperature.
[0040] The testing conditions were: material temperature 40.3℃; viscometer rotor number 2, speed 60rpm.
[0041] The five C0 values were 92.30 mPa·s, 92.00 mPa·s, 90.80 mPa·s, 91.50 mPa·s, and 91.80 mPa·s, respectively, and the average value was 91.68 mPa·s.
[0042] C0 = 91.68 (mPa·s) 3. Detection of viscosity value (C1) of sodium carboxymethyl cellulose test solution 3.1 Treatment of sodium carboxymethyl cellulose test solution Turn on the stirrer, add the mixed white sugar sample to the No. 1 sodium carboxymethyl cellulose solution, stir for 6 minutes until completely dissolved, then cover the mouth of the beaker with plastic wrap and place it in a constant temperature water bath at 40.5℃ for 90 minutes.
[0043] 3.2 Viscosity (C1) Detection Five sets of viscosity values were measured using the test liquid after it was kept at a constant temperature.
[0044] Testing conditions: material temperature 40.3℃, viscometer rotor number 2, speed 60rpm.
[0045] The five sets of measured values were 38.80 mPa·s, 37.4 mPa·s, 38.9 mPa·s, 37.00 mPa·s, and 38.40 mPa·s, respectively. The average value was 38.1 mPa·s. C1 = 38.1 mPa·s 4. Result Determination C1=38.1 mPa·sC0=91.68(mPa·s) If C1 < C0, the mixed sample is positive for cellulase, meaning it contains cellulase.
[0046] Qualitative detection of cellulase in 5 batches of white sugar samples 5.1 Material Weighing Sodium carboxymethyl cellulose (viscosity 1056 mPa·s) 30.0008 g; Standard sample: 100.00g of granulated sugar (without cellulase).
[0047] 240312 Sample of white granulated sugar 100.01g (white granulated sugar produced by XX white granulated sugar manufacturer on March 12, 2024); 230809 White granulated sugar sample 100.00g (white granulated sugar produced by XX white granulated sugar manufacturer on August 9, 2023); 240323 White granulated sugar sample 100.02g (white granulated sugar produced by XX white granulated sugar manufacturer on March 23, 2024); 240504 Sample of white granulated sugar 100.00g (white granulated sugar produced by XX white granulated sugar manufacturer on May 4, 2024); Sample 240519, 100.01g of white granulated sugar (white granulated sugar produced by XX white granulated sugar manufacturer on May 19, 2024). 5.2 Preparation of sodium carboxymethyl cellulose solution: Five samples of white sugar were tested: Approximately 4000 ml of RO water was heated to 85°C, and with stirring, 30.0008 g of sodium carboxymethyl cellulose was slowly added, continuing to dissolve for 20 minutes until fully dissolved. Stirring was then stopped, and RO water was added to bring the volume to 5400 ml. The solution was then cooled to 40.4°C. Stirring was restarted, and citric acid monohydrate standard solution was slowly added dropwise to adjust the pH of the solution to 5.01.
[0048] The prepared sodium carboxymethyl cellulose solution was divided into 6 equal portions, numbered as follows: 0 # ① # 、② # ③ # ④ # ⑤ # .
[0049] 5.3 Viscosity value C0 detection 5.3.1 Treatment of sodium carboxymethyl cellulose standard solution Turn on the stirrer and add 100.00g of standard white sugar to the 0# sodium carboxymethyl cellulose solution. Stir for 6 minutes until completely dissolved. Then, cover the mouth of the beaker with plastic wrap and place it in a water bath at a temperature of 40.5℃ for 30 minutes.
[0050] 5.3.2 Viscosity value C0 detection Five sets of viscosity values were tested using a standard solution at a constant temperature.
[0051] The testing conditions were: material temperature 40.3℃; viscometer rotor number 2, speed 60rpm.
[0052] The C0 values for the five groups were 92.00 mPa·s, 92.10 mPa·s, 90.40 mPa·s, 91.70 mPa·s, and 91.00 mPa·s, respectively. The average value was 91.44 mPa·s.
[0053] C0 = 91.44 (mPa·s) 5.4 Detection of C1 viscosity value of sodium carboxymethyl cellulose test solution 5.4.1 Treatment of sodium carboxymethyl cellulose test solution Turn on the stirrer and add the white sugar samples from batches 240312, 230809, 240323, 240504, and 240519 to ① respectively. # -⑤ # Stir each of the sodium carboxymethyl cellulose solution for 6 minutes until completely dissolved, then seal the mouth of the beaker with plastic wrap and place it in a constant temperature water bath at 40.5℃ for 90 minutes.
[0054] 5.4.2 Viscosity (C1) Detection ① # -⑤ # Five sets of viscosity values were measured for each sodium carboxymethyl cellulose test solution.
[0055] Testing conditions: material temperature 40.3℃, viscometer rotor number 2, rotation speed 60rpm.
[0056] The test results are shown in the table below.
[0057] 6. Result Determination Sample 240312, white granulated sugar, C1=92.90, C0=91.44, C1>C0; cellulase was negative in the white granulated sugar. Sample 230809, white granulated sugar, showed C1=8.50 and C0=91.44, with C1<C0 indicating positive cellulase in the white granulated sugar. Sample 240323, white granulated sugar, C1=94.96, C0=91.44, C1>C0; cellulase was negative in the white granulated sugar. Sample 240504, white granulated sugar, C1 = 99.02, C0 = 91.44. C1 > C0. Cellulase was negative in the white granulated sugar. Sample 240519, white granulated sugar, C1=96.82, C0=91.44, C1>C0; cellulase was negative in the white granulated sugar. The mixed sample of white sugar from batch 230,809 contained cellulase.
[0058] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.
[0059] This background section is provided to generally present the context of the invention. The work of the currently named inventors, the work to the extent described in this background section, and aspects of this section that did not constitute prior art at the time of application are neither expressly nor impliedly acknowledged as prior art to the invention.
Claims
1. A rapid qualitative detection method for cellulase in granulated sugar, characterized in that... Includes the following steps: 1) Preparation of standard solution: To test N samples of white sugar: Heat RO water to 75-85℃, turn on the stirrer, slowly add (1+N)×5.000g sodium carboxymethyl cellulose, continue stirring for 10-20min to ensure complete dissolution, cool to 40-60℃, and bring the volume to (1+N)×900ml with RO water. Stir well, slowly add citric acid monohydrate to adjust the pH of the solution to 4.5-6.5, obtaining sodium carboxymethyl cellulose solution; divide the obtained sodium carboxymethyl cellulose solution into 1+N equal portions; 2) Viscosity test of sodium carboxymethyl cellulose standard solution: Place one portion of sodium carboxymethyl cellulose solution in a stirrer, turn on the stirrer, add 100.0g of standard white sugar until completely dissolved; after complete dissolution, cover or seal with plastic wrap and keep in a water bath at 40-60℃ for 30 minutes, then test its viscosity value, and take the average value after 5 tests as C0. 3) Viscosity detection of sodium carboxymethyl cellulose test solution: Place the other sodium carboxymethyl cellulose solution in a stirrer, turn on the stirrer, add 100.0g of each of the other white sugar samples to be tested until completely dissolved, cover or seal with plastic wrap, and keep in a water bath at 40-60℃ for 90 minutes. Then measure its viscosity value. After 5 tests, take the average value as C1. 4) Result determination: I: C1≥C0, the white sugar cellulase test is negative, meaning the sample does not contain cellulase; II: C1 < C0, the white sugar to be tested is positive for cellulase, that is, the white sugar to be tested contains cellulase.
2. The rapid qualitative detection method for cellulase in granulated sugar according to claim 1, characterized in that: The method also includes a parallel sample preparation step added between step 3) and step 4): that is, parallel samples are set up according to steps 1) to 3).
3. The rapid qualitative detection method for cellulase in granulated sugar according to claim 1, characterized in that: The method is also applicable to the rapid qualitative detection of cellulase in mixed samples of multiple batches of white sugar.
4. The rapid qualitative detection method for cellulase in granulated sugar according to claim 3, characterized in that: Steps 1)-2) are the same as in claim 1; 3'): The sodium carboxymethyl cellulose test solution was kept at a constant temperature of 40-60℃ for 90 minutes and the viscosity value was measured. The average value was taken after 5 measurements and recorded as C1'. 4') Result determination: Ⅰ: C1' ≥ C0, the white sugar cellulase test is negative, that is, the sample to be tested does not contain cellulase; II: C1' < C0, the white sugar to be tested is positive for cellulase, that is, the white sugar to be tested contains cellulase; If the white sugar sample is a mixture of multiple batches, after the mixed sample tests positive, a single batch should be tested for identification, i.e., the identification test should be performed according to the steps in claim 1.
5. The rapid qualitative detection method for cellulase in granulated sugar according to claim 4, characterized in that: The method further includes a parallel sample preparation step added between step 3') and step 4'): that is, parallel samples are set up according to steps 1) to 3').
6. The rapid qualitative detection method for cellulase in granulated sugar according to any one of claims 1-5, characterized in that: In step 1), the sodium carboxymethyl cellulose must meet the relevant requirements of GB1886.232-2016, and its viscosity value must be between 500 (mPa·s) and 1400 (mPa·s); the standard white sugar is white sugar without cellulase.
7. The rapid qualitative detection method for cellulase in granulated sugar according to claim 6, characterized in that: The citric acid monohydrate solution has a mass percentage of 10%; the citric acid monohydrate used is of analytical grade.
8. The rapid qualitative detection method for cellulase in granulated sugar according to claim 6, characterized in that: The constant temperature in steps 2) and 3) must be consistent, and the temperature difference of the material when testing viscosity should be ≤0.5℃.
9. The rapid qualitative detection method for cellulase in granulated sugar according to claim 6, characterized in that: N≥1。