CN112730734A - Method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid - Google Patents
Method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid Download PDFInfo
- Publication number
- CN112730734A CN112730734A CN202011528892.4A CN202011528892A CN112730734A CN 112730734 A CN112730734 A CN 112730734A CN 202011528892 A CN202011528892 A CN 202011528892A CN 112730734 A CN112730734 A CN 112730734A
- Authority
- CN
- China
- Prior art keywords
- solution
- glucose
- titration
- fermentation
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000008103 glucose Substances 0.000 title claims abstract description 118
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 112
- 238000000855 fermentation Methods 0.000 title claims abstract description 94
- 230000004151 fermentation Effects 0.000 title claims abstract description 94
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 50
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 27
- 239000004310 lactic acid Substances 0.000 title claims abstract description 27
- 238000004448 titration Methods 0.000 claims abstract description 104
- 239000006228 supernatant Substances 0.000 claims abstract description 35
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 238000007865 diluting Methods 0.000 claims abstract description 14
- 238000002474 experimental method Methods 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 60
- 239000012086 standard solution Substances 0.000 claims description 57
- 238000009835 boiling Methods 0.000 claims description 37
- 239000003153 chemical reaction reagent Substances 0.000 claims description 35
- RSJOBNMOMQFPKQ-UHFFFAOYSA-L copper;2,3-dihydroxybutanedioate Chemical compound [Cu+2].[O-]C(=O)C(O)C(O)C([O-])=O RSJOBNMOMQFPKQ-UHFFFAOYSA-L 0.000 claims description 35
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 claims description 22
- 229960002378 oftasceine Drugs 0.000 claims description 22
- 238000010438 heat treatment Methods 0.000 claims description 21
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000011324 bead Substances 0.000 claims description 18
- 239000011521 glass Substances 0.000 claims description 18
- 239000012085 test solution Substances 0.000 claims description 18
- 235000015073 liquid stocks Nutrition 0.000 claims description 6
- 239000011550 stock solution Substances 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 238000000227 grinding Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 9
- 229960001484 edetic acid Drugs 0.000 description 17
- 239000000203 mixture Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 239000002253 acid Substances 0.000 description 4
- 241000282324 Felis Species 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- KVZLHPXEUGJPAH-UHFFFAOYSA-N 2-oxidanylpropanoic acid Chemical compound CC(O)C(O)=O.CC(O)C(O)=O KVZLHPXEUGJPAH-UHFFFAOYSA-N 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229940100486 rice starch Drugs 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/16—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using titration
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid, which relates to the field of lactic acid detection methods and comprises the following steps: extracting supernatant A of the centrifugal fermentation liquid; step two: diluting the supernatant A, diluting the supernatant A prepared in the step one by 5-50 times, and placing the diluted supernatant A into a biosensor to detect the concentration of glucose; step three: in the titration experiment, the glucose concentration of the biosensor is titrated; and step four, calculating the concentration, wherein when the content of reducing sugar in the fermentation liquor is less than or equal to 2.5%, S is (V0-V)/0.2 multiplied by 0.1, and the accuracy and the parallelism of the content of residual sugar in the fermentation liquor in the glucose fermentation lactic acid can be greatly improved.
Description
Technical Field
The invention relates to the field of lactic acid detection methods, in particular to a method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid.
Background
Lactic acid (2-hydroxypropionic acid) is a compound, the production method is mostly a bacterial fermentation method, the raw materials of the fermentation method are corn starch, sweet potato starch, rice starch, cellulose and the like, the main way is that sugar is adjusted to about 6 pH under the action of lactic acid bacteria, the fermentation period is 35-40 hours, in the process of producing lactic acid by fermenting lactic acid bacteria, the optimal fermentation end time can be controlled by measuring the residual sugar in the fermentation liquor, the existing method for measuring the residual sugar in the fermentation liquor is a Felin sugar-fixing method, the reaction of Felin and reducing sugar in the Felin sugar-fixing method is influenced by factors such as the pH value of a solution, the heating temperature, the stirring strength, the evaporation capacity, the titration speed and the like, moreover, the judgment of the titration end point is greatly influenced by human factors, and meanwhile, the chromaticity and the pH value in the fermentation liquor influence the end point judgment of the Fehling glucose determination method and influence the accuracy of a measurement result.
Disclosure of Invention
The invention aims to solve the problem that the existing method for measuring the residual sugar content in the fermentation liquor is inaccurate, and provides a method for testing the residual sugar content in the fermentation liquor in the glucose fermentation lactic acid, which can greatly improve the accuracy and the parallelism of the residual sugar content in the fermentation liquor in the glucose fermentation lactic acid.
In order to achieve the above purpose, the invention adopts the technical scheme that:
a method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid comprises the following steps:
the method comprises the following steps: extracting supernatant A of the centrifugal fermentation liquid, when glucose is fermented by lactic acid for 35 hours, taking 20ml of the fermentation liquid, putting the fermentation liquid into a centrifuge, and centrifuging for 4-6 min under the centrifugal condition that the rotating speed is 3200-3800 r/min to obtain the supernatant A; the purpose of centrifugation is that the fermentation liquor is turbid, and the direct dilution with the fermentation liquor can influence the detection result of the biosensor analyzer and further can block the pipeline.
Step two: diluting the supernatant A, diluting the supernatant A prepared in the step one by 5-50 times, and placing the supernatant A into a biosensor analyzer to detect the concentration of glucose; because the concentration range of the detected glucose of the biosensor is 1g/L, the error of the detection result can be below 0.2% only when the biosensor is diluted in the detection range of the biosensor.
Step three: a titration experiment, wherein titration is carried out according to the glucose concentration detected by the biosensor;
1) blank titration, namely adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator into a dry 250mL conical flask, titrating by using 0.05mol/L EDTA (ethylene diamine tetraacetic acid) standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is converted into orange; accurately adding alkaline copper tartrate reagents B and A, each 5mL of which contains a plurality of glass beads, adding 10mL of 1g/L glucose standard solution in advance, shaking up, heating in an electric furnace to boil within 2 minutes, boiling for 30 seconds, and dropping the glucose standard solution at a constant speed until the color of the glucose standard solution is changed from blue to colorless, thus obtaining the titration end point. Recording the milliliter value V0 of consuming 1g/L glucose standard solution before and after boiling to ensure that the whole titration process is completed within 3 minutes;
2) performing preliminary titration, precisely sucking 0.20mL of fermentation liquid, adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator, titrating with 0.05mol/L EDTA (ethylene diamine tetraacetic acid) standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange. Accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, pre-adding a proper amount of glucose standard solution of which the volume is 8mL according to the glucose content of a biosensor, uniformly shaking the mixture, placing the mixture on an electric furnace, heating the mixture to boil the mixture within 2 minutes, after boiling the mixture for 30 seconds, and uniformly titrating the mixture by using 1g/L of the glucose standard solution until the mixture is colorless from blue, thus obtaining the titration end point. Recording the milliliter value Vm of 1g/L glucose standard solution consumed before and after boiling for formal titration reference to ensure that the whole titration process is completed within 3 minutes;
3) formally titrating, precisely absorbing 0.20mL of fermentation liquor, adding 10mL of water, 3mL of 20% KOH test solution and 20mg of calcein indicator, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange; and accurately adding 5mL of alkaline copper tartrate reagent B and 5mL of alkaline copper tartrate reagent A respectively, adding a plurality of glass beads, adding 1mL of glucose standard solution which is less than the formal titration consumption Vm, shaking up, placing on an electric furnace, heating to boil within 2 minutes, after boiling for 30 seconds, titrating at a constant speed by using the glucose standard solution until the color of the glucose standard solution is changed from blue to colorless, thus obtaining the titration end point. Recording the milliliter value V of 1g/L glucose standard solution consumed before and after boiling to ensure that the whole titration process is completed within 3 minutes;
and step four, calculating a formula. (V0-V)/0.2 × 0.1;
wherein, S- -glucose content,%; v0- -mL of glucose standard consumed by blank titration;
V-mL of consumed glucose standard solution for formal titration; 0.2-mL of pre-fermentation liquid stock solution.
In order to further optimize the present invention, the following technical solutions may be preferably selected:
preferably, the centrifugal speed in the first step is 3500r/min, and the centrifugal time is 5 min.
Preferably, in the second step, the sample solution is diluted by 50, that is, 2ml of the supernatant solution is accurately absorbed by a pipette, and the volume is determined to be 100ml of the volumetric flask by RO water and is mixed uniformly.
Preferably, in the third step, the calcein indicator is prepared by mixing and grinding 1g of calcein and 100g of sodium chloride in parts by weight.
The invention has the beneficial effects that:
1. because the glucose is fermented to produce acid, when the residual sugar content in the fermentation liquor is titrated along with the increase of the fermentation period, the sample liquid can splash when the sample liquid is detected to be heated because the acid exists in the fermentation liquor, and the method solves the problem that the sample liquid splashes outwards in the residual sugar titration heating process and prevents potential safety hazards brought by the splashing process.
2. As the fermentation of glucose produces acid, the end point color changes to purple when the fermentation liquor is detected due to the fact that the acid content in the fermentation liquor is high along with the increase of the fermentation period, the problem of color change is avoided through the method, and the end point color of residual glucose is directly changed from blue to colorless, so that the judgment is more direct.
3. The invention improves the detection error caused by the influence of color judgment and sample liquid splashing, and the data error control range is less than or equal to 0.1 percent.
Detailed Description
Example 1:
a method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid comprises the following steps:
the method comprises the following steps: extracting supernatant A of the centrifugal fermentation liquid, when glucose is fermented by lactic acid for 35 hours, taking 20ml of the fermentation liquid, putting the fermentation liquid into a centrifuge, and centrifuging for 5min under the centrifugal condition that the rotating speed is 3500r/min to obtain supernatant A;
step two: diluting the supernatant A, diluting the supernatant A prepared in the step one by 5 times, and placing the supernatant A into a biosensor to detect the concentration of glucose;
step three: in the titration experiment, the glucose concentration of the biosensor is titrated;
1) blank titration, namely adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator into a dry 250mL conical flask, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is converted into orange; accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, adding 10mL of 1g/L glucose standard solution in advance, shaking up, heating in an electric furnace to boil within 2 minutes, after boiling for 30 seconds, dropping the glucose standard solution at a constant speed until the purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value V0 of the total consumed glucose standard solution before and after boiling;
2) performing preliminary titration, precisely sucking 0.20mL of fermentation liquid, adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator, titrating with 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange. Accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, pre-adding a proper amount of glucose standard solution of which the volume is 8mL according to the glucose content of a biosensor, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed of 1g/L until purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value Vm of the consumed glucose standard solution before and after boiling to be used as formal titration reference;
3) formally titrating, precisely absorbing 0.20mL of fermentation liquor, adding 10mL of water, 3mL of 20% KOH test solution and 20mg of calcein indicator, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange; accurately adding 5mL of alkaline copper tartrate reagent B and alkaline copper tartrate reagent A, adding a plurality of glass beads, adding 1mL of glucose standard solution which is less than the formal titration consumption Vm, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed by using the glucose standard solution until purple disappears and ensuring that the titration is finished within 3 minutes in the whole process, and recording the milliliter number V of the consumed glucose standard solution before and after boiling;
step four, calculating the concentration, wherein when the content of reducing sugar in the fermentation liquor is less than or equal to 2.5 percent, S is (V0-V)/0.2 multiplied by 0.1;
wherein, S- -glucose content,%; v0- -mL of glucose standard consumed by blank titration;
V-mL of consumed glucose standard solution for formal titration; 0.2-mL of pre-fermentation liquid stock solution.
Example 2:
a method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid comprises the following steps:
the method comprises the following steps: extracting supernatant A of the centrifugal fermentation liquid, when glucose is fermented by lactic acid for 35 hours, taking 20ml of the fermentation liquid, putting the fermentation liquid into a centrifuge, and centrifuging for 5min under the centrifugal condition that the rotating speed is 3500r/min to obtain supernatant A;
step two: diluting the supernatant A, diluting the supernatant A prepared in the step one by 10 times, and placing the diluted supernatant A into a biosensor to detect the concentration of glucose;
step three: in the titration experiment, the glucose concentration of the biosensor is titrated;
1) blank titration, namely adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator into a dry 250mL conical flask, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is converted into orange; accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, adding 10mL of 1g/L glucose standard solution in advance, shaking up, heating in an electric furnace to boil within 2 minutes, after boiling for 30 seconds, dropping the glucose standard solution at a constant speed until the purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value V0 of the total consumed glucose standard solution before and after boiling;
2) performing preliminary titration, precisely sucking 0.20mL of fermentation liquid, adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator, titrating with 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange. Accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, pre-adding a proper amount of glucose standard solution of which the volume is 8mL according to the glucose content of a biosensor, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed of 1g/L until purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value Vm of the consumed glucose standard solution before and after boiling to be used as formal titration reference;
3) formally titrating, precisely absorbing 0.20mL of fermentation liquor, adding 10mL of water, 3mL of 20% KOH test solution and 20mg of calcein indicator, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange; accurately adding 5mL of alkaline copper tartrate reagent B and alkaline copper tartrate reagent A, adding a plurality of glass beads, adding 1mL of glucose standard solution which is less than the formal titration consumption Vm, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed by using the glucose standard solution until purple disappears and ensuring that the titration is finished within 3 minutes in the whole process, and recording the milliliter number V of the consumed glucose standard solution before and after boiling;
step four, calculating the concentration, wherein when the content of reducing sugar in the fermentation liquor is less than or equal to 2.5 percent, S is (V0-V)/0.2 multiplied by 0.1;
wherein, S- -glucose content,%; v0- -mL of glucose standard consumed by blank titration; V-mL of consumed glucose standard solution for formal titration; 0.2-mL of pre-fermentation liquid stock solution.
Example 3:
a method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid comprises the following steps:
the method comprises the following steps: extracting supernatant A of the centrifugal fermentation liquid, when glucose is fermented by lactic acid for 35 hours, taking 20ml of the fermentation liquid, putting the fermentation liquid into a centrifuge, and centrifuging for 5min under the centrifugal condition that the rotating speed is 3500r/min to obtain supernatant A;
step two: diluting the supernatant A, diluting the supernatant A prepared in the step one by 20 times, and placing the diluted supernatant A into a biosensor to detect the concentration of glucose;
step three: in the titration experiment, the glucose concentration of the biosensor is titrated;
1) blank titration, namely adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator into a dry 250mL conical flask, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is converted into orange; accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, adding 10mL of 1g/L glucose standard solution in advance, shaking up, heating in an electric furnace to boil within 2 minutes, after boiling for 30 seconds, dropping the glucose standard solution at a constant speed until the purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value V0 of the total consumed glucose standard solution before and after boiling;
2) performing preliminary titration, precisely sucking 0.20mL of fermentation liquid, adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator, titrating with 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange. Accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, pre-adding a proper amount of glucose standard solution of which the volume is 8mL according to the glucose content of a biosensor, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed of 1g/L until purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value Vm of the consumed glucose standard solution before and after boiling to be used as formal titration reference;
3) formally titrating, precisely absorbing 0.20mL of fermentation liquor, adding 10mL of water, 3mL of 20% KOH test solution and 20mg of calcein indicator, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange; accurately adding 5mL of alkaline copper tartrate reagent B and alkaline copper tartrate reagent A, adding a plurality of glass beads, adding 1mL of glucose standard solution which is less than the formal titration consumption Vm, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed by using the glucose standard solution until purple disappears and ensuring that the titration is finished within 3 minutes in the whole process, and recording the milliliter number V of the consumed glucose standard solution before and after boiling;
step four, calculating the concentration, wherein when the content of reducing sugar in the fermentation liquor is less than or equal to 2.5 percent, S is (V0-V)/0.2 multiplied by 0.1;
wherein, S- -glucose content,%; v0- -mL of glucose standard consumed by blank titration; V-mL of consumed glucose standard solution for formal titration; 0.2-mL of pre-fermentation liquid stock solution.
Example 4:
a method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid comprises the following steps:
the method comprises the following steps: extracting supernatant A of the centrifugal fermentation liquid, when glucose is fermented by lactic acid for 35 hours, taking 20ml of the fermentation liquid, putting the fermentation liquid into a centrifuge, and centrifuging for 5min under the centrifugal condition that the rotating speed is 3500r/min to obtain supernatant A;
step two: diluting the supernatant A, diluting the supernatant A prepared in the step one by 50 times, and placing the supernatant A into a biosensor to detect the concentration of glucose;
step three: in the titration experiment, the glucose concentration of the biosensor is titrated;
1) blank titration, namely adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator into a dry 250mL conical flask, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is converted into orange; accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, adding 10mL of 1g/L glucose standard solution in advance, shaking up, heating in an electric furnace to boil within 2 minutes, after boiling for 30 seconds, dropping the glucose standard solution at a constant speed until the purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value V0 of the total consumed glucose standard solution before and after boiling;
2) performing preliminary titration, precisely sucking 0.20mL of fermentation liquid, adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator, titrating with 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange. Accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, pre-adding a proper amount of glucose standard solution of which the volume is 8mL according to the glucose content of a biosensor, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed of 1g/L until purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value Vm of the consumed glucose standard solution before and after boiling to be used as formal titration reference;
3) formally titrating, precisely absorbing 0.20mL of fermentation liquor, adding 10mL of water, 3mL of 20% KOH test solution and 20mg of calcein indicator, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange; accurately adding 5mL of alkaline copper tartrate reagent B and alkaline copper tartrate reagent A, adding a plurality of glass beads, adding 1mL of glucose standard solution which is less than the formal titration consumption Vm, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed by using the glucose standard solution until purple disappears and ensuring that the titration is finished within 3 minutes in the whole process, and recording the milliliter number V of the consumed glucose standard solution before and after boiling;
step four, calculating the concentration, wherein when the content of reducing sugar in the fermentation liquor is less than or equal to 2.5 percent, S is (V0-V)/0.2 multiplied by 0.1;
wherein, S- -glucose content,%; v0- -mL of glucose standard consumed by blank titration; V-mL of consumed glucose standard solution for formal titration; 0.2-mL of pre-fermentation liquid stock solution.
Residual sugar detection experiment:
1. reduce the potential safety hazard of sample liquid splashing caused by heating and boiling the sample in the experiment
2. And the result is more direct after the color reaches the end point, so that the data error caused by the misjudgment of the end point is improved.
3. The formula of the recovery rate and the results are shown in the table
Normalized recovery% ((normalized results-sample results)/normalized rate 100%)
4. Analysis of results
According to the analysis of the experimental results: with the fermentation process, the glucose content is slowly reduced, the detection result of the detection method is stable, and the standard addition recovery rate is high, so that the method is feasible for detecting the glucose in the fermentation liquor.
Claims (4)
1. A method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid is characterized by comprising the following steps:
the method comprises the following steps: extracting supernatant A of the centrifugal fermentation liquid, when glucose is fermented by lactic acid for 35 hours, taking 20ml of the fermentation liquid, putting the fermentation liquid into a centrifuge, and centrifuging for 4-6 min under the centrifugal condition that the rotating speed is 3200-3800 r/min to obtain the supernatant A;
step two: diluting the supernatant A, diluting the supernatant A prepared in the step one by 5-50 times, and placing the diluted supernatant A into a biosensor to detect the concentration of glucose;
step three: in the titration experiment, the glucose concentration of the biosensor is titrated;
1) blank titration, namely adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator into a dry 250mL conical flask, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is converted into orange; accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, adding 10mL of 1g/L glucose standard solution in advance, shaking up, heating in an electric furnace to boil within 2 minutes, after boiling for 30 seconds, dropping the glucose standard solution at a constant speed until the purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value V0 of the total consumed glucose standard solution before and after boiling;
2) performing preliminary titration, precisely sucking 0.20mL of fermentation liquid, adding 10mL of water, 3mL of 20% KOH test solution and 22mg of calcein indicator, titrating with 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange. Accurately adding alkaline copper tartrate reagents B and A, each 5mL of the alkaline copper tartrate reagents B and A, adding a plurality of glass beads, pre-adding a proper amount of glucose standard solution of which the volume is 8mL according to the glucose content of a biosensor, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed of 1g/L until purple color disappears and ensuring that the titration is completed within 3 minutes in the whole process, and recording the milliliter value Vm of the consumed glucose standard solution before and after boiling to be used as formal titration reference;
3) formally titrating, precisely absorbing 0.20mL of fermentation liquor, adding 10mL of water, 3mL of 20% KOH test solution and 20mg of calcein indicator, titrating by using 0.05mol/L EDTA standard titration solution, observing in a backlight mode, and stopping titration when the yellow-green fluorescence is changed into orange; accurately adding 5mL of alkaline copper tartrate reagent B and alkaline copper tartrate reagent A, adding a plurality of glass beads, adding 1mL of glucose standard solution which is less than the formal titration consumption Vm, uniformly shaking the solution, placing the solution on an electric furnace, heating the solution to boil within 2 minutes, after boiling for 30 seconds, titrating the solution at a constant speed by using the glucose standard solution until purple disappears and ensuring that the titration is finished within 3 minutes in the whole process, and recording the milliliter number V of the consumed glucose standard solution before and after boiling;
step four, calculating the concentration, wherein when the content of reducing sugar in the fermentation liquor is less than or equal to 2.5 percent, S is (V0-V)/0.2 multiplied by 0.1;
wherein, S- -glucose content,%; v0- -mL of glucose standard consumed by blank titration;
V-mL of consumed glucose standard solution for formal titration; 0.2-mL of pre-fermentation liquid stock solution.
2. The method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid as claimed in claim 1, wherein: in the first step, the centrifugal speed is 3500r/min, and the centrifugal time is 5 min.
3. The method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid as claimed in claim 1, wherein: in the second step, after the supernatant is accurately aspirated by a pipette, 10-fold dilution is performed.
4. The method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid as claimed in claim 1, wherein: in the third step, the calcein indicator is prepared by mixing and grinding 1 part of calcein and 100 parts of sodium chloride in parts by weight.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011528892.4A CN112730734A (en) | 2020-12-22 | 2020-12-22 | Method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011528892.4A CN112730734A (en) | 2020-12-22 | 2020-12-22 | Method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112730734A true CN112730734A (en) | 2021-04-30 |
Family
ID=75605907
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011528892.4A Pending CN112730734A (en) | 2020-12-22 | 2020-12-22 | Method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112730734A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113325139A (en) * | 2021-06-04 | 2021-08-31 | 通辽梅花生物科技有限公司 | Method for detecting content of trehalose in amino acid fermentation liquor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080095703A1 (en) * | 2005-03-07 | 2008-04-24 | Johannes Coy | Method For Checking And Controlling The Mammalian Lactic Acid Fermentation Process / Aerobic Glucose Fermentation Metabolic Pathway In Mammalian Organism |
| CN103898016A (en) * | 2014-03-25 | 2014-07-02 | 华中农业大学 | High-yield lactic acid bacterium and method for preparing calcium lactate by fermenting eggshells with same |
| KR20150042309A (en) * | 2013-10-08 | 2015-04-21 | 한국화학연구원 | Method for the production of sugar form waste Curcuma longa as a biomass, and lactic acid and ethanol through fermentation |
| CN110079562A (en) * | 2019-04-11 | 2019-08-02 | 华中农业大学 | A kind of method that immobilization fermentation egg-shell meal prepares calcium lactate |
-
2020
- 2020-12-22 CN CN202011528892.4A patent/CN112730734A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080095703A1 (en) * | 2005-03-07 | 2008-04-24 | Johannes Coy | Method For Checking And Controlling The Mammalian Lactic Acid Fermentation Process / Aerobic Glucose Fermentation Metabolic Pathway In Mammalian Organism |
| KR20150042309A (en) * | 2013-10-08 | 2015-04-21 | 한국화학연구원 | Method for the production of sugar form waste Curcuma longa as a biomass, and lactic acid and ethanol through fermentation |
| CN103898016A (en) * | 2014-03-25 | 2014-07-02 | 华中农业大学 | High-yield lactic acid bacterium and method for preparing calcium lactate by fermenting eggshells with same |
| CN110079562A (en) * | 2019-04-11 | 2019-08-02 | 华中农业大学 | A kind of method that immobilization fermentation egg-shell meal prepares calcium lactate |
Non-Patent Citations (1)
| Title |
|---|
| 中华人民共和国卫生部 中国国家标准化管理委员会: "《GB/T 5009.7-2008 食品中还原糖的测定》", 中国轻工业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113325139A (en) * | 2021-06-04 | 2021-08-31 | 通辽梅花生物科技有限公司 | Method for detecting content of trehalose in amino acid fermentation liquor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106932532A (en) | A kind of analysis method for detecting COD in water | |
| CN112730734A (en) | Method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid | |
| CN101358230B (en) | Method for measuring carboxymethylcellulose enzyme activity | |
| CN105296595B (en) | A kind of bioenzyme activity detection method based on nanogold growth | |
| CN106353309B (en) | A method of yeast beta-dextran content in detection modulation cream | |
| CN102980856B (en) | Carboxymethyl cellulase activity determination method | |
| CN107831127A (en) | A kind of method of sugared content in detection feed | |
| CN110923291B (en) | Method for detecting activity of glucose oxidase for feed | |
| WO2024148766A1 (en) | Method for determining activity of glucose oxidase | |
| CN104020169A (en) | Chemical detection method for dissolved organic matters in organic fertilizer | |
| CN106979929B (en) | Method for rapidly determining reaction performance of dissolving pulp | |
| CN107991252A (en) | A kind of stabilizer for α-hydroxybutyrate dehydrogenase assay kit and preparation method thereof | |
| CN106404994B (en) | It makes wine into the detection method of cellar grain grain gelatinization degree | |
| CN114136960A (en) | Method for measuring resistant starch content of rice in batches | |
| CN104807764A (en) | Alkaline xylanase activity determination method | |
| CN113310782A (en) | Digestion method for determining selenium in agricultural products and determination method for selenium content in agricultural products | |
| CN110133179A (en) | A kind of reduced sugar Test paper and its application method | |
| CN114807297A (en) | Fermented grain detection method based on biosensor | |
| CN106338573B (en) | A method of measuring organic acid content in agricultural digester gas engineering anaerobic fermentation system | |
| CN113358528B (en) | Method for detecting acetylcholinesterase and inhibitor thereof based on pendant drop method | |
| CN103529021A (en) | Quick analyzing method of total sugar | |
| CN110609033B (en) | Method for detecting enzyme value of sucrose invertase in honey | |
| CN115326638B (en) | Detection method of amylase in oyster sauce and application thereof | |
| CN117607207A (en) | Daqu saccharification force measuring method based on glucose oxidase | |
| CN114486870B (en) | Nanometer enzyme functionalized paper-based sensor for rapid blood glucose detection and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210430 |