Method for detecting fermentation power of yeast for making hard liquor

Abstract

The invention provides a method for detecting the fermenting power of yeast for making hard liquor on one hand, which comprises the following steps of: mixing a glucose solution serving as a fermentation substrate with a yeast sample to be detected, fermenting to obtain an ethanol-containing solution to be detected, and calculating the fermenting power according to the ethanol concentration of the solution to be detected. The method provided by the invention is applied to quality evaluation of the yeast for making hard liquor, and the fermentation performance of the yeast for making hard liquor is represented by detecting the fermentation power of the yeast for making hard liquor. The detection method provided by the invention has the beneficial effects of high detection accuracy, low self error, convenient and simple detection process and low detection cost.

Description

Technical Field

[0001] This invention belongs to the field of brewing technology, specifically relating to a method for detecting the fermentation power of Daqu (a type of starter culture). Background Technology

[0002] The unique aroma and taste of Maotai-flavor liquor are closely related to the high-temperature koji (fermented starter culture) produced using a unique process. In the production of Maotai-flavor liquor, the high-temperature koji not only inoculates the mash with microbial communities but also promotes the development of a more complex aroma. Furthermore, the high-temperature koji acts as a secondary feeder.

[0003] High-temperature daqu (a type of starter culture) is one of the main microbial sources for fermentation of mash. The saccharification of mash and the production of ethanol are closely related to the microbial community of high-temperature daqu. To more intuitively demonstrate the alcohol-producing capacity of daqu, the China National Light Industry Council proposed the concept of fermentation power as a physicochemical indicator in 2011, defining it as the ability of microorganisms in daqu to convert sugar raw materials into carbon dioxide and ethanol. [1] .

[0004] Accurate detection of ethanol concentration has always been a challenge in the industry. During the fermentation of starch using traditional Chinese koji (a type of starter culture), various compounds are produced. Traditional hydrometer methods, which treat this as a mixture of ethanol and water, clearly ignore the influence of other byproducts. Furthermore, due to its high volatility and relatively stable chemical properties, many chemical reactions involving ethanol can also occur with other alcohols. Propanol and butanol are byproducts of microbial alcoholic fermentation, making the development of a high-precision method for ethanol concentration detection difficult. However, in recent years, the widespread use of enzyme-linked immunosorbent assay (ELISA) has improved this situation to some extent. Alcohol dehydrogenase (ADH) is a zinc-containing metalloenzyme with broad substrate specificity. Alcohol dehydrogenase, using nicotinamide adenine dinucleotide (NAD) as a coenzyme, catalyzes the reversible reaction between primary alcohols and aldehydes: CH3CH2OH + NAD. + →CH3CHO+NADH+H + It is widely present in humans and mammals and is responsible for the body's ethanol metabolism. Due to its high precision and rapid response, it has been widely used in rapid ethanol detection kits.

[0005] Biosensor analyzers are detection instruments that use the change in membrane voltage generated by the reaction between a specific enzyme membrane and its corresponding substrate to determine the concentration of the substrate. They have high detection accuracy, fast detection speed, and relatively simple operation, and are currently widely used in the detection of various chemical substances such as glucose, acetic acid, ethanol, and glutamic acid.

[0006] In existing technologies, the detection methods for the fermentation power of Daqu (a type of koji) mainly fall into two categories: the traditional carbon dioxide reduction method and the optimized ultraviolet spectrophotometry and gas chromatography methods developed in recent years. The carbon dioxide reduction method primarily determines the fermentation power by detecting the mass difference of the fermentation apparatus before and after fermentation. However, several articles have pointed out that this method ignores the carbon dioxide produced by the respiration of other microorganisms in Daqu. Since the carbon dioxide content produced by microbial respiration in different Daqu samples is difficult to estimate, it inevitably has a certain degree of error compared to methods that directly detect ethanol concentration. [2] The detection indicators are difficult to directly reflect the actual alcohol production capacity of Daqu (a type of starter culture). Ultraviolet spectrophotometry detects ethanol concentration through a specific colorimetric reaction between chromate ions in potassium dichromate and a mixture of ethanol and concentrated sulfuric acid. Gas chromatography, on the other hand, is a colorimetric separation method using gas as the mobile phase. However, both ultraviolet spectrophotometry and gas chromatography are colorimetric methods. During fermentation, Daqu will migrate some pigments from microorganisms and Daqu powder into the saccharification liquid. Therefore, both methods require distillation before detection, which inevitably increases the error caused by manual operation. [3] Furthermore, due to various reasons such as complex operation and high instrument requirements, the colorimetric comparison method has not been widely adopted.

[0007] However, the fermentation power and saccharification power of Daqu (a type of starter culture) are both important quantitative indicators of Daqu's alcohol production capacity. Accurate detection of these indicators is beneficial for determining production process parameters during Daqu production and for the precise construction of large-scale AI models for alcohol production. Furthermore, the quality of Daqu, as a "catalyst" and "inoculant" in baijiu production, significantly impacts the final product. Therefore, the industry urgently needs to develop a highly economical, accurate, and easy-to-operate method for detecting the fermentation power of Daqu. Summary of the Invention

[0008] In view of the above-mentioned problems in the existing technology, the present invention provides a method for detecting the fermentation power of Daqu (a type of starter culture), comprising the following steps:

[0009] (1) Using glucose solution as a fermentation substrate, it was mixed with the Daqu sample to be tested and then fermented.

[0010] (2) The test solution was obtained after fermentation and treatment;

[0011] (3) Detect the ethanol concentration of the test liquid and calculate the fermentation power according to the fermentation power calculation formula of Daqu.

[0012] In some implementations, in step (1), 10g of the Daqu sample to be tested is added to every 100mL of fermentation substrate for fermentation; in some implementations, the fermentation is a sealed constant temperature fermentation, and in some implementations, the temperature of the sealed constant temperature fermentation is 38-42℃ and the time is 24-120h; in some implementations, the temperature of the sealed constant temperature fermentation is 40℃ and the time is 72h.

[0013] In some embodiments, the concentration of the glucose solution in step (1) is 5.0-16.2°Bx; in some embodiments, the concentration of the glucose solution in step (1) is 12.6°Bx.

[0014] In some embodiments, the substrate includes a buffer salt system comprising a hydrogen phosphate and a chloride salt; in some embodiments, the hydrogen phosphate is one or more of Na₂HPO₄·12H₂O, NaH₂PO₄, KH₂PO₄, and K₂HPO₄; in some embodiments, the chloride salt is one or more of NaCl, KCl, and NH₄Cl; in some embodiments, the buffer salt system comprises Na₂HPO₄·12H₂O, NaCl, KCl, and KH₂PO₄; in some embodiments, the buffer salt system is obtained by mixing Na₂HPO₄·12H₂O: NaCl: KCl: KH₂PO₄ in a mass ratio of 5:10:0.3:3.

[0015] The addition of inorganic salts as a buffer solution in this invention can effectively reduce the increase in acidity of the fermentation broth caused by the inability of CO2 to escape from the confined space. By maintaining the solution in a weakly acidic environment suitable for yeast fermentation throughout the fermentation process, it can effectively reduce abnormal fermentation phenomena caused by the death of yeast cells due to excessive acidity.

[0016] In some embodiments, the test liquid treatment process in step (2) includes the following steps: centrifuging the fermented sample to separate the supernatant; in some embodiments, the supernatant is filtered; in some embodiments, the filtration process involves filtering the supernatant through a microporous membrane; in some embodiments, the microporous membrane is an aqueous microporous membrane with a pore size of 0.22 μm-0.8 μm; in some embodiments, the microporous membrane is an aqueous microporous membrane with a pore size of 0.22 μm.

[0017] In some embodiments, the centrifugation conditions are 4000-6000 rpm for 5-15 min; in some embodiments, the centrifugation conditions are 5000 rpm for 10 min.

[0018] In some implementations, the ethanol concentration in step (3) is directly detected by an ethanol biosensor; in some implementations, the detection process is as follows: after calibration with a special standard solution before detection, a certain amount of the test solution is taken into the ethanol biosensor, and the ethanol concentration in the test solution is tested.

[0019] In some implementations, step (3) of the detection process further includes adjusting the pH value of the test solution before detection; in some implementations, the pH value of the test solution is adjusted to be in the range of 5-8.

[0020] In some implementation schemes, the formula for calculating the fermentation power of Daqu in step (3) is: Fermentation power is calculated using the formula: U=C×V,

[0021] In the formula: U—fermentation power of the sample, in U; ​​C—ethanol concentration detected by the biosensor, in g / L; V—solution volume, in L.

[0022] Where U represents the unit g ethanol·(g Daqu·72h), that is, 1U=1g ethanol·(g Daqu·72h);

[0023] Fermentation power is defined as the amount of ethanol produced by 1 kg of Daqu (a type of starter culture) using 1 L of 12.6°Bx glucose solution as a substrate.

[0024] This invention provides an application of the method, which includes: Daqu quality evaluation, Daqu production process optimization, or Daqu quality comparison.

[0025] The fermentation power of Daqu (a type of starter culture) is its ability to convert sugar raw materials into ethanol, which directly reflects the alcohol production capacity of Daqu. By detecting the fermentation power through the method provided in this invention, the fermentation performance of Daqu can be directly reflected. In the production process of Daqu, the production process can be optimized by detecting the fermentation power of Daqu, and the production process in the starter culture process can be determined to obtain Daqu with high fermentation power. Furthermore, for different Daqu, their quality can be evaluated by detecting their fermentation power, thereby comparing different Daqu.

[0026] The detection method provided by this invention has the following beneficial effects:

[0027] (1) High detection accuracy and low self-error: The fermentation power of Daqu is calculated by directly detecting the ethanol concentration, and no distillation operation is required before detection, which avoids interference from other random errors such as human error, and the detection accuracy is high.

[0028] (2) The detection process is convenient and simple: Daqu and glucose saccharification liquid are directly mixed and fermented, and then the ethanol sensor can be used to easily measure the ethanol content. No complicated pretreatment is required. The reagents used in the detection process are safe, avoiding experimental risks and instrument damage. Attached Figure Description

[0029] Figure 1 A flowchart illustrating the operation of the method for detecting the fermentation power of Daqu (a type of Chinese koji) based on an ethanol biosensor method provided by the present invention.

[0030] Figure 2 This paper compares the fermentation power differences of the same Daqu sample detected by the ethanol biosensor method described in Example 1 of this invention with those detected by the CO2 reduction method, ultraviolet spectrophotometry, and gas chromatography. The figure uses a combination of box plots and violin plots to show the data dispersion and density distribution. The box plot shows the interquartile range (IQR) of the data, with the lower edge representing the 25th percentile (Q1) and the upper edge representing the 75th percentile (Q3). The horizontal line in the middle of the box plot represents the median. The violin plot shows the density of data under different fermentation powers through its width; a wider width indicates more data around that value. NS. indicates no statistically significant difference between the two groups.

[0031] Figure 3 The correlation between fermentation power of distilled and undistilled samples under different saccharification solution concentrations;

[0032] Figure 4 The CO2 reduction method was used to detect the difference in fermentation power of the same Daqu sample between sorghum saccharification liquid and glucose saccharification liquid. The figures show the data dispersion and density distribution using a combination of box plots and violin plots. The box plots represent the interquartile range (IQR), with the lower edge representing the 25th percentile (Q1) and the upper edge representing the 75th percentile (Q3). The horizontal line in the box plot represents the median, and the points in the box plot represent the average. The violin plots show the density of data under different fermentation powers through their width; a wider width indicates more data points near that value. NS. indicates no statistically significant difference between the two groups.

[0033] Figure 5 The difference in fermentation power of the same Daqu (a type of starter culture) sample was detected using an ethanol biosensor method for sorghum saccharification liquid and glucose saccharification liquid. The figures show the data dispersion and density distribution using a combination of box plots and violin plots. The box plots represent the interquartile range (IQR), with the lower edge representing the 25th percentile (Q1) and the upper edge representing the 75th percentile (Q3). The horizontal line in the box plot represents the median, and the points in the box plot represent the average. The violin plots show the density of data under different fermentation powers through their width; a wider width indicates more data points near that value. * indicates a statistically significant difference between the two groups.

[0034] Figure 6 This paper presents a response surface methodology for analyzing the effect of saccharification liquid concentration and fermentation time on fermentation power in the ethanol biosensor method described in Example 1 of this invention. Detailed Implementation

[0035] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0036] Experimental materials: Biosensor standard solution, buffer solution, and anhydrous glucose (C6H4O) provided by the Technical Center of Guizhou Xijiu Co., Ltd. (28°19′22.3″N, 106°12′35.7″E). 12 O6) and analytical grade (AR) chemical reagents such as Na2HPO4·12H2O and KH2PO4.

[0037] Experimental subjects: High-temperature Daqu mixed samples and white Daqu samples with a storage period of 0 months provided by the No. 2 Daqu Production Workshop of Guizhou Xijiu Co., Ltd. (28°19′22.3″N, 106°12′35.7″E).

[0038] Example 1

[0039] This embodiment provides a method for detecting the fermentation power of Daqu (a type of starter culture) based on an ethanol biosensor.

[0040] Preparation of 12.6°Bx saccharification solution: Accurately weigh 13.217g of anhydrous glucose into a 250mL beaker, add 50mL of water to dissolve it, and transfer the solution to a volumetric flask. Rinse the beaker wall with a wash bottle containing pure water and slowly pour the washing solution into the volumetric flask. Repeat the above cleaning operation more than 3 times. Use a dropper to draw a small amount of pure water to make up the volume to 100mL and shake well. Pour the solution into a 250mL Erlenmeyer flask, then add 1g Na2HPO4·12H2O, 2g NaCl, 0.06g KCl and 0.6g KH2PO4 and mix well. Then place the flask in a 90℃ water bath and heat for 10min to sterilize.

[0041] Sealed constant temperature fermentation: Accurately weigh 10.00g of the Daqu sample to be tested, pour the sample into the prepared saccharification solution, seal the mouth of the conical flask with a rubber stopper, and seal the gap of the flask mouth with PM sealing film for 3 turns. Place the prepared sample to be fermented in a 40℃ constant temperature incubator for 72h.

[0042] Sample pretreatment: Transfer all fermented samples to 50mL centrifuge tubes and balance the weight on a balance (do not wash out the remaining liquid from the bottle wall during centrifugation to avoid diluting the ethanol concentration). Centrifuge at 5000rpm for 10min. Use a needleless medical syringe to extract a portion of the supernatant from the centrifuge tube and filter it through an aqueous microporous membrane (0.22μm). Store the supernatant in a 1.5mL EP tube for later use. Because the ethanol concentration in the sample is low and ethanol is highly volatile, the sample processing should be accurate and rapid. The processed sample should be tested within 2 hours. Before testing, the EP tube cap should be tightly closed and stored in a -20℃ refrigerator to prevent ethanol evaporation.

[0043] Sample determination and calculation: Take the standard solution for the M-100 biosensor analyzer, use a pipette to draw up the standard solution (not less than 500 μL) and add it to the sample cup. Place the standard solution in the calibration position. Take the filtered sample in the sample cup (not less than 500 μL) and place it in the sample position. (If the sample pH is in the range of 5 to 8, the supernatant after filtration can be directly used for testing; if it is not in this pH range, the sample solution must be adjusted to this pH range before testing). After the standard and sample are placed, first click the "Test" icon in the main menu interface of the Silman Biosensor Analyzer (M-100), then click the cup number where the sample is located. The instrument will automatically perform calibration and complete the test.

[0044] Fermentation force is calculated using the formula: U = C × V.

[0045] In the formula: U—fermentation power of the sample, in U(g ethanol·(g Daqu·72h)); C—ethanol concentration detected by the biosensor, in g / L; V—solution volume, in L. The calculation results are rounded to three decimal places.

[0046] Fermentation power is defined as the amount of ethanol produced by 1 kg of Daqu (a type of starter culture) using 1 L of 12.6°Bx glucose solution as substrate, which is 1 unit, i.e., 1 U = 1 g ethanol·(g Daqu·72h).

[0047] Example 2

[0048] This embodiment compares and verifies the detection differences between the ethanol biosensor method described in Example 1 and the CO2 reduction method, ultraviolet spectrophotometry, and gas chromatography.

[0049] (1) Experimental methods

[0050] High-temperature Daqu mixed samples and white Daqu samples with a storage period of 0 months were selected and fermented in saccharification liquid at 5.0–16.2°Bx. The fermentation power was detected simultaneously using the ethanol biosensor method, CO2 reduction method, ultraviolet spectrophotometry, and gas chromatography described in Example 1.

[0051] 1. CO2 reduction method analysis procedure; the procedure for this method is described in the references. [1] :

[0052] 1.1 Preparation of saccharification solution: Take one part sorghum flour, add five parts tap water and cook for 1 to 2 hours. Add amylase according to the instructions for use to liquefy the solution. After liquefaction, add one part 60°C warm water and 5% of the raw material amount of saccharifying enzyme (50,000 activity units). Stir well and saccharify at 60°C for 3 to 4 hours. Test with dilute iodine solution until no blue color appears. Then heat to 90°C, filter with fine white cloth, measure the sugar content of the solution, and adjust the concentration to different ranges of 5.0 to 16.2°Bx using sterile water before use.

[0053] 1.2 Measure 50 mL of the saccharified liquid into a 100 mL Erlenmeyer flask, plug it with a cotton stopper, and wrap it with parchment paper. Separately wrap the fermentation plug with parchment paper. Place both flasks in a steam sterilizer and sterilize at 0.1 MPa for 20 min. After cooling to approximately 28°C, add 0.5 g of koji powder under aseptic conditions. Simultaneously perform a blank test (without adding koji powder). Fill the fermentation plug with the solution and inject approximately 5 mL of sulfuric acid. Seal the flask, sealing the stopper with paraffin wax. Wipe the outer wall of the flask dry and weigh it using an analytical balance with a sensitivity of 0.0001 g. Record the reading as M1 (for the blank test, the reading is M3). Place the fermentation plug in a 30°C incubator and ferment for 72 h. Remove the fermentation plug, gently shake it to release carbon dioxide, and weigh it. Record the reading as M2 (for the blank test, the reading is M4).

[0054] 1.3. Calculation Results

[0055] The fermentation power of the sample is calculated using the formula X1 = (M1 - M2) - (M3 - M4), where:

[0056] X1—Fermentation power of the sample, in U (gCO2·(g Daqu·72h)); M1—Total mass of fermentation plug and contents before fermentation, in grams (g); M2—Total mass of fermentation plug and contents after fermentation, in grams (g); M3—Total mass of fermentation plug and contents before fermentation in the blank test, in grams (g); M4—Total mass of fermentation plug and contents after fermentation in the blank test, in grams (g); Calculation results are rounded to two decimal places.

[0057] 2. Ultraviolet spectrophotometric analysis procedure; the procedure for this method is described in the references. [3] :

[0058] 2.1 Culture Medium Preparation: Weigh 100g of sorghum used in brewing and pour it into a 1000mL beaker. Add 500mL of distilled water to the sorghum. Preheat the water bath to 100℃ and steam the sorghum in the water bath for 1 hour. After steaming, cool the steamed sorghum to 60℃, then weigh 2.5g of amylase and add it to the sorghum to liquefy it. After liquefaction for 5 minutes, add 100mL of 60℃ warm water. Weigh 1.0g of saccharifying enzyme and add it to the liquefied sorghum, then place it in a 60℃ water bath. After about 3 hours in the water bath, test with dilute iodine solution until no blue color appears, then stop the water bath and raise the temperature to 90℃. Filter the solution through a fine white cloth, measure the sugar content, and adjust the sugar content of the filtrate to 5.0–16.2°Bx with glucose solution to obtain the saccharified solution.

[0059] 2.2 Fermentation: Accurately measure 100 mL of the saccharified liquid into a 150 mL Erlenmeyer flask using a 100 mL graduated cylinder. Stopper the flask with cotton, wrap the mouth with kraft paper, and wrap the fermentation plug with kraft paper. Place both flasks simultaneously in a high-temperature sterilizer. Sterilization conditions: pressure 0.1 MPa, time 20 min. After sterilization, cool to approximately 28℃, and add 1.0 g of Daqu (fermentation starter) powder in a clean bench. Separately, use the sterilized saccharified liquid and fermentation plug without adding Daqu powder as a blank. Fill the fermentation plug with 15 mL of 2.61 mol / L sulfuric acid and place it in a 30℃ incubator for 72 h of fermentation. After incubation, remove the fermentation apparatus.

[0060] 2.3 Drug Preparation:

[0061] Ethanol standard solution: Accurately pipette 1.00 mL of analytical grade anhydrous ethanol into a 50 mL volumetric flask, add 48 mL of distilled water, shake well, and then dilute to the mark with distilled water. Shake well and set aside.

[0062] Potassium dichromate solution: Weigh 10.00g of analytical grade potassium dichromate, dissolve it in a small amount of water, and make up to 250mL.

[0063] Concentrated sulfuric acid: analytical grade, purity 95%–98%;

[0064] NaOH solution: concentration 0.1 mol / L.

[0065] 2.4 Sample Preparation: After fermentation, transfer the fermentation broth to a 500mL distillation flask, add 25mL of distilled water, and shake well. Neutralize and titrate with 0.1mol / L sodium hydroxide until the solution is neutral. Add a few boiling chips to the flask, connect the distillation apparatus, and distill. Collect the distillate in a 100mL volumetric flask and cool it in an ice-water bath. Distill slowly until the distillate level is close to the mark, then remove the volumetric flask and place it in a 20℃ water bath for 30 minutes. Finally, dilute to the mark with distilled water and shake well.

[0066] 2.5 Standard Curve Plotting: Prepare seven 25 mL A-grade colorimetric tubes. Add 2.00 mL of potassium dichromate solution and 3.0 mL of concentrated sulfuric acid to each tube, mix well, and cool. Then, add 0.00, 0.20, 0.40, 0.60, 0.80, 1.00, and 1.25 mL of ethanol standard solution sequentially, mix well, react for 15 min, add water to the mark, mix again, zero the tube with tube 0, adjust the spectrophotometer wavelength to 585 nm, measure the absorbance of each tube, and plot the standard curve.

[0067] 2.6 Sample Detection: Take a clean 25 mL A-grade colorimetric tube, add 2.00 mL of potassium dichromate solution and 2.50 mL of concentrated sulfuric acid, mix well, and cool. After cooling, add 1.00 mL of the prepared sample and dilute to the mark. Simultaneously perform a blank sample experiment (i.e., the distillate sample without added koji). Zero the tube using tube 0 of the standard curve, and determine the ethanol content of the blank sample (C2) at a wavelength of 585 nm, and the ethanol content of the sample (C1).

[0068] 2.7 Calculation Results:

[0069] Fermentation power is calculated using the formula X2 = (C1 / 100 - C2 / 100) × 100, where:

[0070] X2—Fermentation power of the sample, in mL of ethanol·(g Daqu·72h); C1—Ethanol content of the sample, in %; C2—Ethanol content of the blank sample, in %; 100 in C1 / 100 and C2 / 100—Conversion factor; 100—Volume of culture medium, in mL; Calculation results are retained to two decimal places.

[0071] 3. Gas chromatography; the procedure for this method is described in the references. [3] :

[0072] 3.1 Culture medium preparation: Weigh 100g of sorghum used in brewing and pour it into a 1000mL beaker. Add 500mL of distilled water to the sorghum. Preheat the water bath to 100℃ and steam the sorghum in the water bath for 1 hour. After steaming, cool the steamed sorghum to 60℃, then add 2.5g of amylase to liquefy it. After liquefaction, add 100mL of 60℃ warm water. Add 1.0g of saccharifying enzyme and place it in a 60℃ water bath for 3-4 hours (test with dilute iodine solution until no blue color appears). Raise the temperature to 90℃. Filter with fine white cloth, measure the sugar content of the solution, and adjust the sugar content of the filtrate to 5.0-16.2°Bx with glucose solution to obtain the saccharified solution.

[0073] 3.2 Fermentation: Measure 100 mL of saccharified liquid into a 150 mL Erlenmeyer flask, plug it with cotton, and wrap it with kraft paper. Separately wrap the fermentation plug with kraft paper, and place both in a steam sterilizer. Sterilize at 0.1 MPa for 20 min. When cooled to about 28℃, add 1.0 g of koji powder under aseptic conditions. At the same time, perform a blank experiment (without adding koji powder). Fill the fermentation plug with 15 mL of 2.61 mol / L sulfuric acid, and place it in a 30℃ incubator for fermentation for 72 h.

[0074] 3.3 Sample Preparation: After fermentation, transfer the fermentation broth to a 500mL distillation flask, add 25mL of distilled water, and shake well. Neutralize and titrate with 0.1mol / L sodium hydroxide until the solution is neutral. Add a few boiling chips, connect the distillation apparatus, and distill. Collect the distillate in a 100mL volumetric flask, cool the flask in an ice bath, and slowly distill until close to the mark. Remove the flask and place it in a 20℃ water bath for 30 minutes. Dilute to the mark with distilled water and shake well. Set aside for later use.

[0075] 3.4 Preparation of Standard Solutions: Accurately measure 0.3945 g of anhydrous ethanol, kept at 20℃, and dilute to 10 mL to prepare a stock solution with a concentration of 39.45 g / L. Pipette 1.25 mL, 2.5 mL, and 5.0 mL of the stock solution and dilute to 10 mL volumetric flasks to form a standard curve (1) with gradient concentrations of 4.9313 g / L, 9.8625 g / L, 19.7250 g / L, and 39.4500 g / L, respectively. Accurately measure 0.3888 g of anhydrous ethanol, kept at 20℃, and dilute to 100 mL to prepare a stock solution with a concentration of 3.888 g / L. Pipette 0.5 mL, 1.0 mL, 2.0 mL, and 4.0 mL of the mother liquor into 10 mL volumetric flasks, respectively, to form standard curves with gradient concentrations of 0.1994 g / L, 0.3888 g / L, 0.7776 g / L, and 1.5552 g / L. The standard curves are determined based on the ethanol content in the samples.

[0076] 3.5 Preparation of internal standard solution: Accurately measure 0.3146 g of n-propanol at a constant temperature of 20℃ and dilute to 100 mL.

[0077] 3.6 Sample Detection: Add 1 mL of the prepared sample to a vial, then add 0.1 mL of internal standard solution to the vial, and detect on an Agilent 7890B instrument. Detection conditions: Column: CP-Wax57CB; 50m 0.25mm 0.2μm #CP97723; Detector: FID detector; Carrier gas: N2; Injection volume: 1 μL; Injection mode: constant pressure split; Carrier gas pressure: 15 psi; Split ratio: 40:1; Temperature program: initial temperature 35℃, hold for 4 min, then increase to 60℃ at a rate of 4℃ / min, then increase to 130℃ at a rate of 10℃ / min, and finally increase to 210℃ at a rate of 15℃ / min and hold for 20 min.

[0078] 3.7 Calculation Results: The spectrum was recorded by the recorder. A standard curve was plotted against the ethanol concentration using the ratio of the peak area of ​​the standard sample to the peak area of ​​the internal standard. The ethanol content in the sample was obtained by multiplying the value obtained from the standard curve by the dilution factor using the ratio of the peak area of ​​the sample group to the peak area of ​​the internal standard. The unit of fermentation power was finally obtained as: mL ethanol·(g Daqu·72h).

[0079] (2) Results and Analysis

[0080] The ethanol biosensor method, CO2 reduction method, ultraviolet spectrophotometry and gas chromatography were used to calculate the average fermentation power of each sample in 5 replicates at different sugar concentrations. The fermentation power of Daqu detected at 12.6°Bx is shown in Table 1.

[0081] Table 1. Average results of Daqu fermentation power test

[0082]

[0083] The principle by which fermentable sugars produce ethanol under the action of yeast is as follows: C6H 12 O6→2C2H5OH(ethanol)+2CO2+energy. Based on the fermentation principle and the density of ethanol at 20℃ being 0.789 g / cm3, the ethanol measured by the ethanol sensor method, UV spectrophotometer method, and gas chromatography can be converted to CO2 using the formula M(CO2)=ρ×V×0.956. The conversion of ethanol measured by the ethanol sensor method to CO2 is calculated using the formula M(CO2)=m(ethanol)×0.956. The average results of the converted fermentation power detection of Daqu are shown in Table 2, and the deviations in the detection results are shown in Table 3.

[0084] Table 2. Average results of Daqu fermentation power test after conversion.

[0085]

[0086] Table 3 Deviation of Daqu Fermentation Power Test Results

[0087] Repeated use of the same large-batch sample Ethanol sensor method <![CDATA[CO2 reduction method]]> Ultraviolet spectrophotometry Gas chromatography 1 0.0562 -0.3763 -0.0528 -0.0481 2 0.1040 0.1738 0.0075 0.0198 3 -0.0013 -1.0363 -0.1056 0.0575 4 -0.1159 -0.2363 0.1358 -0.1386 5 -0.0394 -0.7963 -0.0679 -0.0481 6 -0.0681 -0.9563 0 0.1254 7 0.0179 0.3438 0.0679 -0.0179 8 0.0466 2.8838 0.0151 0.0500 maximum deviation 0.1040 2.8838 0.1358 0.1254 minimum deviation -0.1159 -1.0363 -0.1056 -0.1386 average value 0.0562 0.8504 0.0566 0.0632

[0088] As can be seen from the average results of the Daqu fermentation power detection after conversion in Table 2 and the deviation of the detection results in Table 3, compared with the CO2 reduction method, the ethanol biosensor method described in Example 1 has a smaller fluctuation range and higher accuracy for the detection values ​​of the same Daqu sample.

[0089] There was no significant difference in the detection results between ultraviolet spectrophotometry, gas chromatography, and the ethanol biosensor method. This is because ultraviolet spectrophotometry and gas chromatography are already relatively accurate methods, but their procedures are cumbersome. According to actual laboratory testing time statistics, the ethanol biosensor method can complete the detection in 1 hour, while ultraviolet spectrophotometry and gas chromatography require 3 to 5 hours. Furthermore, ultraviolet spectrophotometry requires the addition of concentrated sulfuric acid to the cuvette, posing a safety hazard, while gas chromatography requires a large amount of standard solution, resulting in higher costs, and the gas chromatograph itself is also more expensive.

[0090] Statistical analysis was conducted using the ethanol biosensor method, CO2 reduction method, ultraviolet spectrophotometry, and gas chromatography in saccharified broth at 5.0–16.2°Bx, with five replicates for each sample. The average fermentation power was calculated, and statistical significance was determined. The results are as follows: Figure 2 The significance test used here is the Student's t-test. As can be seen from the figure, the data of the CO2 reduction method group is more dispersed, and some data deviate from the normal range. In the Student's t-test, this is considered to be random bias. Therefore, it is considered to be no different from the ethanol biosensor method in the significance test.

[0091] according to Figure 2 The results show that the CO2 reduction method has large fluctuations, while the ultraviolet spectrophotometry, gas chromatography, and ethanol biosensor method have smaller fluctuations.

[0092] Example 3

[0093] This embodiment verifies the impact of distillation on the detection results of the biosensor method.

[0094] (1) Experimental methods

[0095] Thirty high-temperature Daqu samples were selected, and the fermentation power of the Daqu was detected according to the ethanol biosensor method described in Example 1.

[0096] Non-distillation group: After sealed and constant-temperature fermentation in saccharification solutions of different concentrations, the samples were directly tested according to the steps in Example 1;

[0097] Distillation group: After sealed and constant-temperature fermentation in saccharification solutions of different concentrations, distillation was carried out, and the distillate was collected and tested according to the steps in Example 1;

[0098] The correlation between fermentation power of the two treatment methods was analyzed.

[0099] (2) Results and Analysis

[0100] The fermentation power of Daqu before and after distillation is highly positively correlated, as shown in the following results. Figure 3 This indicates that the distillation operation does not affect the detection accuracy of the biosensor method, and the distillation step can be omitted from this method.

[0101] In the detection process of ultraviolet spectrophotometry and gas chromatography, residual reducing sugars in the fermentation broth gelatinize during distillation, producing discoloration that can interfere with the detection results to some extent. However, the distillation step is a necessary step in both spectrophotometry and gas chromatography. The ethanol sensor method of this invention can simplify this step, which not only simplifies the operation process but also reduces experimental errors.

[0102] Example 4

[0103] This embodiment verifies the effect of sorghum saccharification liquid and glucose saccharification liquid as substrates at 12.6°Bx sugar content on the detection results of Daqu fermentation power.

[0104] (1) Experimental methods

[0105] 1.1 Preparation of two substrates:

[0106] Sorghum saccharification liquid: The brewing sorghum is ground into a fine powder of about 200 mesh using a grinder. Then, amylase and saccharifying enzyme are added in a ratio of 50:1 to hydrolyze the sorghum powder. After centrifugation at 5000 rpm for 5 min, the supernatant is taken.

[0107] Glucose saccharification solution (same as the 12.6°Bx saccharification solution in Example 1);

[0108] 1.2 Using white koji samples as the test objects, fermentation was carried out in two different substrates, and the fermentation power was detected by the CO2 reduction method and the ethanol biosensor method described in Example 1, respectively.

[0109] (2) Results and Analysis

[0110] At the same concentration, the fermentation power of sorghum saccharification liquid was higher than that of glucose solution; however, even when using sorghum saccharification liquid to test the fermentation power of the same Daqu sample, the results still showed significant differences, as shown in the following figures. Figure 4 and Figure 5 The significance test used here is also the Student's t-test. As can be seen from the figure, when using the CO2 reduction method, the data of the sorghum saccharification liquid group has a higher degree of dispersion. In the Student's t-test, it is considered that some data deviate from the normal range, which is a random bias. Therefore, it is considered to have no difference in the significance test.

[0111] In this case, when using the ethanol biosensor method, the data dispersion of the sorghum saccharification liquid group is also higher, and there are significant differences between the data of the sorghum saccharification liquid group and the glucose saccharification liquid group. It can be seen that the selection of different fermentation substrates has a significant impact on the detection of fermentation power, and glucose saccharification liquid is suitable for the ethanol biosensor method.

[0112] Example 5

[0113] After sorghum powder and soluble starch were hydrolyzed and saccharified using amylase and saccharifying enzyme, the residual starch was detected.

[0114] Method for detecting residual starch: After the sample was centrifuged at 5000 rpm for 10 min, the supernatant was separated into soluble sugar and starch using 80% ethanol. After acid hydrolysis of starch into glucose, the residual starch content in the supernatant of the sorghum saccharification liquid sample was detected by an enzyme-linked immunosorbent assay (ELISA) using the anthrone colorimetric method.

[0115] The test results showed that sorghum saccharification liquid had a higher residual starch content than soluble starch saccharification liquid, and the difference between different samples was greater. However, residual starch could be detected in both raw materials. The saccharification liquid prepared with glucose did not contain any starch and therefore did not have the problem of residual starch, making it more suitable as a substrate to reduce detection errors. The results are shown in Table 4-6.

[0116] Table 4. Residual starch content detected after complete enzymatic hydrolysis of sorghum powder.

[0117]

[0118] Table 5. Residual starch content detected after complete enzymatic hydrolysis of soluble starch.

[0119]

[0120] Table 6. Detection of starch content in glucose solution

[0121]

[0122]

[0123] Example 6

[0124] This embodiment optimizes the optimal fermentation conditions for the ethanol biosensor method through response surface methodology.

[0125] (1) Materials and Methods

[0126] Using white koji samples as the object, five saccharification liquid concentration gradients (5.0–16.2°Bx) and six fermentation time gradients (24–120 h) were set up. Fermentation power was tested according to the method in Example 1, and the optimal conditions were determined by response surface methodology.

[0127] (2) Results and Analysis

[0128] The test results are shown in Table 7 and Figure 6 As shown, the optimal fermentation time is 72 hours, and the optimal saccharification liquid concentration is 12.6°Bx; under these conditions, the fermentation power test results exhibit the best stability. The response surface curve is a smooth surface automatically generated based on the mean of the data using the response surface algorithm. The red dots in the figure represent the actual data portion that exceeds the predicted data of the response surface, without any data removal.

[0129] Table 7 Results of Daqu Fermentation Response Surface Analysis

[0130]

[0131]

[0132] Example 7

[0133] This embodiment verifies the effect of inorganic salt buffer solution in saccharification liquid on the fermentation process.

[0134] (1) Experimental procedure

[0135] Control group: No inorganic salts were added to the saccharified liquid, and fermentation was carried out according to the steps in Example 1;

[0136] Experimental group: Add the inorganic salts in the proportions described in Example 1 to the saccharification liquid and ferment according to the steps in Example 1.

[0137] The pH changes and final fermentation power during the fermentation process were detected in both groups.

[0138] (2) Results and Analysis

[0139] The pH and sugar concentration of the fermentation broth with and without buffer are shown in Table 8. The standard deviation of the fermentation power detection values ​​with and without buffer at different saccharification broth concentrations is shown in Table 9. It can be seen that the group with added inorganic salts can effectively alleviate the increase in acidity caused by CO2 accumulation in the closed environment, keep the fermentation broth in a suitable weakly acidic environment for yeast (pH 5-8), reduce yeast death caused by excessive acidity, significantly reduce abnormal fermentation phenomena, and improve detection stability.

[0140] Table 8. pH and sugar concentration of fermentation broth with and without buffer solution.

[0141]

[0142]

[0143]

[0144] Table 9 Standard deviation of fermentation power detection values ​​with and without buffer solution at different saccharification solution concentrations.

[0145]

[0146] As shown in Table 9, when testing fermentation power at the same saccharification liquid concentration, the standard deviation of fermentation power was much lower when buffer solution was added than when no buffer solution was added. In particular, at the optimal saccharification liquid concentration of 12.6°Bx, the standard deviation of fermentation power was as high as 0.739 without buffer solution, but decreased to 0.115 after adding buffer solution. It can be seen that adding buffer solution significantly reduces the detection error of fermentation power of Daqu.

[0147] References:

[0148] [1] China National Light Industry Council. General Analytical Methods for Brewing Daqu: QB / T 4257-2011 [S]. Beijing: Ministry of Industry and Information Technology of the People's Republic of China, 2011. [2011-12-20]. References:

[0149] [2] Xi Xianhui, Xu Yahui, Qu Yuqin, et al. Study on the method of detecting the fermentation power of Daqu by gas chromatography [J]. Brewing Technology, 2024, (06): 141-144. DOI: 10.13746 / j.njkj.2023260.

[0150] [3] Li Xiaojie, Lü Zhiyuan, Zhang Mengmeng, et al. Comparison of methods for detecting the fermentation power of Daqu by ultraviolet light, gas phase and carbon dioxide method [J]. Brewing, 2024, 51(02):122-126.

Claims

1. A method for detecting the fermentation power of Daqu (a type of starter culture), characterized in that, Includes the following steps: (1) Using glucose solution as a fermentation substrate, it was mixed with the Daqu sample to be tested and then fermented. (2) The test solution was obtained after fermentation and treatment; (3) Detect the ethanol concentration of the test liquid and calculate the fermentation power according to the fermentation power calculation formula of Daqu.

2. The method according to claim 1, characterized in that, In step (1), 10g of the Daqu sample to be tested is added to every 100mL of fermentation substrate for fermentation. Preferably, the fermentation is a sealed, constant-temperature fermentation; Preferably, the constant temperature sealed fermentation is carried out at a temperature of 38-42℃ for 24-120 hours. Preferably, the constant temperature sealed fermentation is carried out at a temperature of 40°C for 72 hours.

3. The method according to claim 1, characterized in that, The concentration of the glucose solution in step (1) is 5.0-16.2°Bx; Preferably, the concentration of the glucose solution in step (1) is 12.6°Bx.

4. The method according to claim 1, characterized in that, The substrate in step (1) also contains a buffer salt system, which includes one or both of hydrogen phosphate and chloride salts; Preferably, the hydrogen phosphate is one or more selected from Na2HPO4·12H2O, NaH2PO4, KH2PO4, and K2HPO4; Preferably, the chloride salt is one or more selected from NaCl, KCl, and NH4Cl; Preferably, the buffer salt system comprises Na2HPO4·12H2O, NaCl, KCl, and KH2PO4; Preferably, the buffer salt system is obtained by mixing Na2HPO4·12H2O:NaCl:KCl:KH2PO4 in a mass ratio of 5:10:0.3:

3.

5. The method according to claim 1, characterized in that, The test liquid processing in step (2) includes the following steps: centrifuging the fermented sample to separate the supernatant; Preferably, the supernatant is filtered; Preferably, the filtration process involves filtering the supernatant through a microporous membrane. Preferably, the microporous filter membrane is an aqueous microporous filter membrane with a pore size of 0.22 μm-0.8 μm; Preferably, the microporous filter membrane is an aqueous microporous filter membrane with a pore size of 0.22 μm.

6. The method according to claim 5, characterized in that, The centrifugation conditions are 4000-6000 rpm for 5-15 min; Preferably, the centrifugation conditions are 5000 rpm for 10 min.

7. The method according to claim 1, characterized in that, The ethanol concentration in step (3) is directly detected using an ethanol biosensor; Preferably, the detection process is as follows: before detection, the test solution is calibrated using a special standard solution, and then the test solution is aspirated into an ethanol biosensor to test and obtain the ethanol concentration contained in the test solution.

8. The method according to claim 7, characterized in that, The detection process in step (3) also includes adjusting the pH value of the test solution before detection; Preferably, the pH of the test solution is adjusted to be in the range of 5-8.

9. The method according to claim 1, characterized in that, The formula for calculating the fermentation power of Daqu in step (3) is: U=C×V, In the formula: U—fermentation power of the test liquid, in U; ​​C—ethanol concentration of the test liquid detected by the biosensor, in g / L; V—solution volume of the test liquid, in L.

10. The application of the method according to any one of claims 1-9, wherein the application includes: Evaluation of Daqu quality, optimization of Daqu production process, or comparison of Daqu quality.

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