阅读说明：本技术 一种葡萄糖发酵乳酸中发酵液残糖的检验方法 (Method for detecting residual sugar in fermentation liquor in glucose fermentation lactic acid ) 是由 何志华 贾惠丽 朱明伟 樊永乐 胡佳辉 樊希文 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种葡萄糖发酵乳酸中发酵液残糖的检验方法,涉及乳酸检测方法领域,包括步骤一：提取离心发酵液上清液A；步骤二：稀释上清液A,将步骤一制得的上清液A,进行5-50倍数的稀释,放置入生物传感仪检测葡萄糖浓度；步骤三：滴定实验,参照生物传感仪葡萄糖浓度做滴定；步骤四,浓度计算,当发酵液中还原糖含量≤2.5％时,S＝(V0-V)/0.2×0.1,可大幅度提高葡萄糖发酵乳酸中发酵液残糖含量的准确性和平行性。(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.)

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.

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.