Method for detecting sugar content of molasses by combining microbial fermentation with refractometer

Abstract

The invention relates to the technical field of molasses sugar content detection, and discloses a method for detecting molasses sugar content by combining microbial fermentation with a refractometer, and the method comprises the following steps: S1, preparing reagent materials; s2, diluting the permeophilic yeast suspension and sterile normal saline according to a ratio of 1: 10; s3, treating a molasses sample to be detected; s4, setting a measuring pipe; s5, setting a control tube; and S6, determination: calculating the sugar content of the molasses according to a formula X sugar content = (X0-X1) * V / m. Compared with a traditional polysaccharide detection kit method, the method has the advantages that the detection time is greatly shortened, long enzymolysis time, standard curve making and OD value detection time are not needed, only simple instruments such as a refractometer are used, and the method for detecting the sugar content of the molasses is extremely low in cost and relatively high in efficiency, and has a wide application prospect. The method has excellent accuracy and stability, is low in detection threshold, and can be widely applied to molasses production, transaction and processing industries.

Description

Technical Field

[0001] The invention relates to the technical field of molasses sugar content detection, in particular to a method for detecting the sugar content of molasses by using microbial fermentation combined with a refractometer. Background Art

[0002] The main purpose of molasses sugar content testing is to determine the sugar content in molasses, which is of great significance for evaluating the quality of molasses, determining its use and formulating related production processes. The refractometer uses the principle of refraction of light when passing through solutions of different concentrations to measure the refractive index of the solution and then deduce the sugar content in the solution.

[0003] The conventional sugar content detection cycle is too long, and because the sugars in molasses are mostly mixed sugars with complex polymerization units, they are not suitable for conventional sugar content detection methods. Therefore, the simplest and most extensive refractometer detection is still used in the molasses trading process.

[0004] The essence of a refractometer is to detect the soluble solid content, and it is only suitable for detecting and judging the sugar content of a sugar solution with a relatively single ingredient. However, many other substances in molasses have an impact on the solid content, so the deviation in sugar content detection using a refractometer is very large.

[0005] Most companies that use molasses as raw material to produce high value-added products use a bio-enzyme total sugar detection kit, which uses imported enzyme preparations to hydrolyze sugars with different degrees of polymerization into glucose, and then uses ultraviolet spectrophotometry to detect glucose to obtain the sugar content of molasses. This method has a high detection cost, and production companies generally cannot afford to frequently test molasses from different sources and batches, so they can only conduct representative sampling, which brings a lot of uncertainty to the fermentation industry and process, and has a great impact on production and quality.

[0006] Therefore, we need to propose a method of detecting the sugar content of molasses by combining microbial fermentation with a refractometer, which can accurately detect the sugar content of molasses and reduce the detection cost. Summary of the invention

[0007] The object of the present invention is to provide a method for detecting the sugar content of molasses by combining microbial fermentation with a refractometer. Compared with the traditional polysaccharide detection kit method, the detection time is greatly shortened, and there is no need for long enzymatic hydrolysis time, standard curve preparation, and OD value detection time. Only simple instruments such as refractometers are used. The method is an extremely low-cost and high-efficiency method for detecting the sugar content of molasses, has excellent accuracy and stability, and has a low detection threshold. The method can be widely used in molasses production, trading, and processing industries to solve the problems raised in the above-mentioned background technology.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer, comprising the following steps:

[0009] S1. Preparation of reagent materials: 1 tube of 50 ml osmophilic yeast suspension, 1 bottle of 500 ml 0.9% sterile saline;

[0010] S2. Dilute the osmophilic yeast suspension with sterile saline at a ratio of 1:10 to make the solution concentration 3×10 5 ~3×10 7 CFU / mL;

[0011] S3. Treat the molasses sample to be tested: weigh 5 g of molasses stock solution, add pure water to make up to 50 mL, centrifuge at 5000 r / min for 2 min to remove mud, take the supernatant to be tested, and obtain the sample treatment test solution;

[0012] S4. Setting a measuring tube: adding a sample treatment liquid to be tested and an osmophilic yeast suspension into the measuring tube, and culturing at 36° C. for 2 h to allow the yeast to ferment and consume sugars, and taking the supernatant for testing after centrifugation, that is, obtaining the supernatant in the measuring tube;

[0013] S5. Setting a control tube: adding the sample treatment solution to be tested and the osmophilic yeast suspension to the control tube, and boiling to inactivate the yeast, and taking the supernatant to be tested after centrifugation, that is, obtaining the supernatant of the control tube;

[0014] S6. Determination: Take the supernatant of the test tube and the control tube, and place them on the refractometer for determination. According to the formula X 含糖量 =(X0-X1)*V / mCalculate the sugar content of molasses and record the value.

[0015] Preferably, in step S2, the dilution process of the osmophilic yeast suspension is carried out under sterile conditions, including the following process:

[0016] S21. Take a sterile test tube or conical flask and add sterile saline;

[0017] S22. Use a sterile pipette or pipette to draw up the osmophilic yeast suspension, add it into a sterile test tube or conical flask and mix it with sterile saline;

[0018] S23. Use a vortex shaker to shake the test tube or conical flask to thoroughly mix the osmophilic yeast suspension with sterile saline.

[0019] Preferably, in step S3, processing the molasses sample to be tested comprises the following process:

[0020] S31, accurately weigh 5 g of molasses stock solution using an electronic balance, and transfer the weighed molasses stock solution to a sterile container;

[0021] S32, add pure water to the sterile container to a total volume of 50 ml, and stir with a magnetic stirrer to fully dissolve and mix the molasses;

[0022] S33, transferring the dissolved molasses solution to a centrifuge tube, and centrifuging at 5000 r / min for 2 min to remove insoluble sand and impurities;

[0023] S34. After centrifugation, transfer the supernatant to another sterile container to avoid bringing in sediment in preparation for subsequent sugar content detection.

[0024] Preferably, in step S4, when setting the measuring tube, 5 ml of the sample is taken to treat the test liquid, and 5 ml of the osmophilic yeast suspension is added to the test liquid. After culturing, the liquid is centrifuged at a speed of 8000 r / min for 2 min using a centrifuge.

[0025] Preferably, in step S4, setting the assay tube includes the following process:

[0026] S41. Use a sterile pipette to accurately draw 5 ml of the treated test solution;

[0027] S42, adding 5 ml of the diluted osmophilic yeast suspension to the sampled test tube;

[0028] S43, placing the test tube in a constant temperature water bath or incubator and culturing at 36°C for 2 hours to allow the yeast to ferment and consume the sugar;

[0029] S44. After the culture is completed, centrifuge at 8000r / min for 2 minutes to separate the supernatant and the bacteria;

[0030] S45. Transfer the supernatant after centrifugation to another sterile test tube to avoid bringing in bacteria, and use it as the supernatant of the assay tube for testing.

[0031] Preferably, in step S5, when setting the control tube, 5 ml of the sample is taken to treat the test solution, and 5 ml of the osmophilic yeast suspension is added to the test solution. After inactivation, the solution is centrifuged at 8000 r / min for 2 min using a centrifuge.

[0032] Preferably, in step S5, setting the control tube includes the following process:

[0033] S51. Use a sterile pipette to accurately draw 5 ml of the treated test solution;

[0034] S52, adding 5 ml of the diluted osmophilic yeast suspension to the sampled test tube;

[0035] S53. Immediately place the test tube in boiling water and boil for 3-8 minutes to inactivate the yeast;

[0036] S54, after inactivation, centrifuge at 8000r / min for 2min to separate the supernatant and the inactivated bacteria;

[0037] S55. Transfer the supernatant after centrifugation to another sterile test tube to avoid bringing in bacteria, and use it as the supernatant of the control tube for testing.

[0038] Preferably, formula X 含糖量 =(X0-X1)*V / m, X0 is the blank sugar Brix value, Bx; X1 is the sugar Brix value after yeast fermentation, Bx; V is the dilution multiple; m is the sample volume, in g.

[0039] Preferably, in step S6, the sugar content determination includes the following process:

[0040] S61. Calibrate the refractometer and ensure that it is in good working condition;

[0041] S62. Use a sterile pipette or pipette to accurately draw 2-4 ml of supernatant from the assay tube and control tube respectively;

[0042] S63, dropping the supernatant of the measuring tube and the supernatant of the control tube onto the prism of the refractometer, so that the droplets are evenly distributed and cover the entire surface of the prism;

[0043] S64. Close the shading plate of the refractometer, adjust the light source to allow light to pass through the prism and the droplet, observe and record the sugar hammer value displayed on the refractometer, and to ensure the accuracy of the measurement, perform multiple measurements on each sample, take the average value, and record the value.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] Compared with the traditional polysaccharide detection kit method, the detection time of the present invention is greatly shortened, and there is no need for long enzymatic hydrolysis time, standard curve preparation, and OD value detection time. In addition, only simple instruments such as refractometers are used. The present invention is a molasses sugar content detection method with extremely low cost and high efficiency, and has excellent accuracy and stability, and a low detection threshold, and can be widely used in molasses production, trading, and processing industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a flowchart of the present invention;

[0047] Figure 2 It is a flowchart of the dilution of the osmophilic yeast suspension of the present invention;

[0048] Figure 3 A flowchart of the present invention for processing a molasses sample to be tested;

[0049] Figure 4 A flowchart of setting up a measuring tube for the present invention;

[0050] Figure 5 A flowchart of setting a control tube for the present invention;

[0051] Figure 6 The figure is a flowchart of the supernatant of the test tube and the supernatant of the control tube according to the present invention. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] See also Figure 1-6 The present invention provides a technical solution: a method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer, comprising the following steps:

[0054] S1. Preparation of reagent materials: 1 tube of 50 ml osmophilic yeast suspension, 1 bottle of 500 ml 0.9% sterile saline;

[0055] S2. Dilute the osmophilic yeast suspension with sterile saline at a ratio of 1:10 to make the solution concentration 3×105~3×107 CFU / mL;

[0056] The materials required for this step include: osmophilic yeast suspension (50 mL), sterile physiological saline (0.9%, 500 mL), sterile test tubes or conical flasks, sterile pipettes or pipettes, sterile culture dishes, and a vortex oscillator.

[0057] In step S2, the dilution process of the osmophilic yeast suspension is carried out under sterile conditions and using sterile tools to avoid cross infection, including the following process:

[0058] S21. Take a sterile test tube or conical flask and add sterile saline (e.g. 5 ml);

[0059] S22. Use a sterile pipette or pipette to draw up the osmophilic yeast suspension (e.g., 0.5 ml), add it to a sterile test tube or conical flask, and mix it with sterile saline;

[0060] At this point, the bacterial suspension has been diluted 10 times (5 mL of normal saline + 0.5 mL of bacterial suspension = 5.5 mL of diluent, but the volume of the added bacterial suspension is usually ignored during calculation, and it is approximately considered to be diluted 10 times).

[0061] S23. Use a vortex shaker to shake the test tube or conical flask to thoroughly mix the osmophilic yeast suspension with sterile saline.

[0062] Optionally, in order to verify whether the concentration of the diluted bacterial suspension meets expectations, a small amount of the diluted bacterial suspension may be taken for inoculation and culture, and the number of colonies may be counted.

[0063] The diluted bacterial suspension should be used as soon as possible to avoid prolonged storage that may lead to reduced microbial activity or contamination.

[0064] S3. Treat the molasses sample to be tested: weigh 5 g of molasses stock solution, add pure water to make up to 50 mL, centrifuge at 5000 r / min for 2 min to remove mud, take the supernatant to be tested, and obtain the sample treatment test solution;

[0065] The materials used in this step include: molasses stock solution, pure water, a centrifuge, a sterile container (such as a centrifuge tube), an electronic balance, a pipette or a straw, and a magnetic stirrer.

[0066] In step S3, processing the molasses sample to be tested includes the following process:

[0067] S31, accurately weigh 5 g of molasses stock solution using an electronic balance, and transfer the weighed molasses stock solution to a sterile container;

[0068] S32, add pure water to the sterile container to a total volume of 50 ml, and stir with a magnetic stirrer to fully dissolve and mix the molasses;

[0069] S33, transferring the dissolved molasses solution to a centrifuge tube, and centrifuging at 5000 r / min for 2 min to remove insoluble sand and impurities;

[0070] S34. After centrifugation, transfer the supernatant to another sterile container to avoid bringing in sediment in preparation for subsequent sugar content detection.

[0071] During the molasses sample processing, maintain sterile operation to avoid contamination. If the molasses stock solution contains a large amount of solid particles or sticky substances, a longer dissolution time and stronger stirring may be required to ensure sufficient dissolution. The treated test solution should be used as soon as possible to avoid long-term storage that may cause changes in composition or contamination.

[0072] The materials needed to set up the test tubes and control tubes include: the treated test solution (the supernatant after centrifugation to remove impurities), the diluted osmophilic yeast suspension, sterile test tubes or centrifuge tubes, a constant temperature water bath or incubator (for maintaining the culture temperature), a centrifuge, a refractometer or other instruments for measuring the sugar hammer.

[0073] S4. Setting a measuring tube: adding a sample treatment liquid to be tested and an osmophilic yeast suspension into the measuring tube, and culturing at 36° C. for 2 h to allow the yeast to ferment and consume sugars, and taking the supernatant for testing after centrifugation, that is, obtaining the supernatant in the measuring tube;

[0074] In step S4, when setting the measuring tube, take 5 ml of the sample to treat the test solution, add 5 ml of the osmophilic yeast suspension to the test solution, and after culturing, use a centrifuge to centrifuge at a speed of 8000 r / min for 2 minutes.

[0075] In step S4, setting the measuring tube includes the following process:

[0076] S41. Use a sterile pipette to accurately draw 5 ml of the treated test solution;

[0077] S42, adding 5 ml of the diluted osmophilic yeast suspension to the sampled test tube;

[0078] S43, placing the test tube in a constant temperature water bath or incubator and culturing at 36°C for 2 hours to allow the yeast to ferment and consume the sugar;

[0079] S44. After the culture is completed, centrifuge at 8000r / min for 2 minutes to separate the supernatant and the bacteria;

[0080] S45. Transfer the supernatant after centrifugation to another sterile test tube to avoid bringing in bacteria, and use it as the supernatant of the assay tube for testing.

[0081] S5. Setting a control tube: adding the sample treatment solution to be tested and the osmophilic yeast suspension to the control tube, and boiling to inactivate the yeast, and taking the supernatant to be tested after centrifugation, that is, obtaining the supernatant of the control tube;

[0082] In step S5, when setting the control tube, take 5 ml of the sample to treat the test solution, add 5 ml of the osmophilic yeast suspension to the test solution, inactivate it, and centrifuge it at a speed of 8000 r / min for 2 minutes.

[0083] In step S5, setting the control tube includes the following process:

[0084] S51. Use a sterile pipette to accurately draw 5 ml of the treated test solution;

[0085] S52, adding 5 ml of the diluted osmophilic yeast suspension to the sampled test tube;

[0086] S53. Immediately place the test tube in boiling water and boil for 3-8 minutes to inactivate the yeast;

[0087] S54, after inactivation, centrifuge at 8000r / min for 2min to separate the supernatant and the inactivated bacteria;

[0088] S55. Transfer the supernatant after centrifugation to another sterile test tube to avoid bringing in bacteria, and use it as the supernatant of the control tube for testing.

[0089] S6. Determination: Take the supernatant of the test tube and the control tube, and place them on the refractometer for determination. According to the formula X 含糖量 =(X0-X1)*V / mCalculate the sugar content of molasses and record the value.

[0090] The materials needed for the determination process of the supernatant of the test tube and the supernatant of the control tube include: the supernatant of the test tube, the supernatant of the control tube, a refractometer, a sterile pipette or pipette, a sterile test tube (for placing the test liquid), a notebook and a pen (for recording the determination results).

[0091] Formula X 含糖量 =(X0-X1)*V / m, X0 is the sugar brix value of the blank (the sugar brix value of the control tube, indicating the sugar content in the unfermented molasses solution), Bx; X1 is the sugar brix value after yeast fermentation (the sugar brix value of the measuring tube, indicating the sugar content after yeast fermentation), Bx; V is the dilution multiple; m is the sample sampling amount, in g.

[0092] The sugar hammer value is an indicator of the soluble solids content in the solution and has a certain correlation with the sugar content.

[0093] In step S6, the sugar content determination includes the following process:

[0094] S61. Calibrate the refractometer and ensure that it is in good working condition;

[0095] S62. Use a sterile pipette or pipette to accurately draw 2-4 ml of supernatant from the assay tube and control tube respectively;

[0096] S63, dropping the supernatant of the measuring tube and the supernatant of the control tube onto the prism of the refractometer, so that the droplets are evenly distributed and cover the entire surface of the prism;

[0097] S64. Close the shading plate of the refractometer, adjust the light source to allow light to pass through the prism and the droplet, observe and record the sugar hammer value displayed on the refractometer, and to ensure the accuracy of the measurement, perform multiple measurements on each sample, take the average value, and record the value.

[0098] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer, characterized in that: The following steps are involved: S1. Preparation of reagent materials: 1 tube of 50 ml osmophilic yeast suspension, 1 bottle of 500 ml 0.9% sterile saline; S2. Dilute the osmophilic yeast suspension with sterile saline at a ratio of 1:10 to make the solution concentration 3×10 5 ~3×10 7 CFU / mL; S3. Treat the molasses sample to be tested: weigh 5 g of molasses stock solution, add pure water to make up to 50 mL, centrifuge at 5000 r / min for 2 min to remove mud, take the supernatant to be tested, and obtain the sample treatment test solution; S4. Setting a measuring tube: adding a sample treatment liquid to be tested and an osmophilic yeast suspension into the measuring tube, and culturing at 36° C. for 2 h to allow the yeast to ferment and consume sugars, and taking the supernatant for testing after centrifugation, that is, obtaining the supernatant in the measuring tube; S5. Setting a control tube: adding the sample treatment solution to be tested and the osmophilic yeast suspension to the control tube, and boiling to inactivate the yeast, and taking the supernatant to be tested after centrifugation, that is, obtaining the supernatant of the control tube; S6. Determination: Take the supernatant of the test tube and the control tube, and place them on the refractometer for determination. According to the formula X 含糖量 =(X0-X1)*V / mCalculate the sugar content of molasses and record the value.

2. The method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer according to claim 1, characterized in that: In step S2, the dilution process of the osmophilic yeast suspension is carried out under sterile conditions, including the following steps: S21. Take a sterile test tube or conical flask and add sterile saline; S22. Use a sterile pipette or pipette to draw up the osmophilic yeast suspension, add it into a sterile test tube or conical flask and mix it with sterile saline; S23. Use a vortex shaker to shake the test tube or conical flask to thoroughly mix the osmophilic yeast suspension with sterile saline.

3. The method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer according to claim 1, characterized in that: In step S3, processing the molasses sample to be tested includes the following process: S31, accurately weigh 5 g of molasses stock solution using an electronic balance, and transfer the weighed molasses stock solution to a sterile container; S32, add pure water to the sterile container to a total volume of 50 ml, and stir with a magnetic stirrer to fully dissolve and mix the molasses; S33, transferring the dissolved molasses solution to a centrifuge tube, and centrifuging at 5000 r / min for 2 min to remove insoluble sand and impurities; S34. After centrifugation, transfer the supernatant to another sterile container to avoid bringing in sediment in preparation for subsequent sugar content detection.

4. The method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer according to claim 1, characterized in that: In step S4, when setting the measuring tube, take 5 ml of the sample to treat the test solution, add 5 ml of the osmophilic yeast suspension to the test solution, and after culturing, use a centrifuge to centrifuge at a speed of 8000 r / min for 2 minutes.

5. The method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer according to claim 1, characterized in that: In step S4, setting the measuring tube includes the following process: S41. Use a sterile pipette to accurately draw 5 ml of the treated test solution; S42, adding 5 ml of the diluted osmophilic yeast suspension to the sampled test tube; S43, placing the test tube in a constant temperature water bath or incubator and culturing at 36°C for 2 hours to allow the yeast to ferment and consume the sugar; S44. After the culture is completed, centrifuge at 8000r / min for 2 minutes to separate the supernatant and the bacteria; S45. Transfer the supernatant after centrifugation to another sterile test tube to avoid bringing in bacteria, and use it as the supernatant of the assay tube for testing.

6. The method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer according to claim 1, characterized in that: In step S5, when setting the control tube, take 5 ml of the sample to treat the test solution, add 5 ml of the osmophilic yeast suspension to the test solution, inactivate it, and centrifuge it at a speed of 8000 r / min for 2 minutes.

7. The method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer according to claim 1, characterized in that: In step S5, setting the control tube includes the following process: S51. Use a sterile pipette to accurately draw 5 ml of the treated test solution; S52, adding 5 ml of the diluted osmophilic yeast suspension to the sampled test tube; S53. Immediately place the test tube in boiling water and boil for 3-8 minutes to inactivate the yeast; S54, after inactivation, centrifuge at 8000r / min for 2min to separate the supernatant and the inactivated bacteria; S55. Transfer the supernatant after centrifugation to another sterile test tube to avoid bringing in bacteria, and use it as the supernatant of the control tube for testing.

8. The method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer according to claim 1, characterized in that: Formula X 含糖量 =(X0-X1)*V / m, X0 is the blank sugar Brix value, Bx; X1 is the sugar Brix value after yeast fermentation, Bx; V is the dilution multiple; m is the sample volume, in g.

9. The method for detecting the sugar content of molasses by microbial fermentation combined with a refractometer according to claim 1, characterized in that: In step S6, the sugar content determination includes the following process: S61. Calibrate the refractometer and ensure that it is in good working condition; S62. Use a sterile pipette or pipette to accurately draw 2-4 ml of supernatant from the assay tube and control tube respectively; S63, dropping the supernatant of the measuring tube and the supernatant of the control tube onto the prism of the refractometer, so that the droplets are evenly distributed and cover the entire surface of the prism; S64. Close the shading plate of the refractometer, adjust the light source to allow light to pass through the prism and the droplet, observe and record the sugar hammer value displayed on the refractometer, and to ensure the accuracy of the measurement, perform multiple measurements on each sample, take the average value, and record the value.

Images