A method and kit for rapid detection of multiple key enzyme activities and microorganisms in Daqu

Abstract

The present application provides a kind of key enzyme activity and microorganism of multiple Daqu combined rapid detection method and kit, belong to Daqu fermentation process quality control technical field.The method includes multiple key enzyme activity of Daqu and Daqu microbial content detection, based on " multi-enzyme co-extraction-microscale color-detection of whole board", efficient detection technology, with pH4.6 acetic acid-sodium acetate as extraction medium, only one extraction treatment can be applied to saccharifying power, liquefying power, protease activity three enzyme activity detection single enzyme extract of Daqu sample, and on this basis, the detection condition is optimized, and the detection time is significantly shortened;In addition, the method also develops to realize the technology of " one operation, synchronous growth, synchronous detection " of multiple microorganisms, by introducing targeted inhibition strategy, for different core microorganisms of Daqu, optimization of selective liquid medium, add appropriate antibacterial peptide, realize the one-time synchronous culture of multiple strains, simultaneous detection, improve detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology for Daqu fermentation process, specifically relating to a rapid detection method and kit for the combined activity of multiple key enzymes and microorganisms in Daqu. Background Technology

[0002] As a key saccharification and fermentation agent in the brewing process of Baijiu (Chinese liquor), the quality of Daqu (a type of starter culture) directly determines the flavor, yield, and quality stability of the liquor. The content and activity of core enzymes (such as saccharification power, liquefaction power, and protease activity) and key microorganisms (lactic acid bacteria, yeast, and molds) are core indicators for evaluating Daqu quality and crucial for regulating its quality during fermentation and ensuring the quality of the finished product. Therefore, achieving rapid and accurate detection of core enzyme activity and key microorganisms in Daqu is of great significance for the production control and quality improvement of brewing enterprises.

[0003] Currently, the detection methods for enzyme activity and microorganisms in Daqu (a type of starter culture) have many shortcomings, making it difficult to meet the needs of rapid quality control in industrial production. Specific problems are as follows:

[0004] Firstly, the current standard methods QB / T 4257-2011 and GB / T 23527-2009 require differentiated enzyme extraction processes and enzyme activity detection procedures for the detection of the three core functional enzyme activities in Daqu (a type of starter culture): saccharification power, liquefaction power, and protease activity. For example, saccharifying enzyme and liquefying enzyme solutions require extraction at 35°C for 1 hour using an acetate-sodium acetate buffer solution at pH 4.6, while protease solutions require extraction at 40°C using a buffer solution at pH 3.0-10.5. Furthermore, saccharifying enzyme activity requires manual titration combined with visual endpoint assessment, while liquefying enzyme activity requires visual endpoint assessment with manual timing. Therefore, the detection of these three enzyme activities necessitates independent sample pretreatment and enzyme reaction detection. This not only involves cumbersome procedures and high reagent consumption, but also, due to the multiple extraction and detection processes, each operation is performed by a single person. The total time for determining the three enzyme activities typically exceeds 6-8 hours, resulting in low detection efficiency and failing to meet the rapid quality control requirements in Daqu production. Simultaneously, existing step-by-step extraction and detection methods are prone to errors due to batch variations and fluctuations in operating conditions, limiting the parallelism and comparability of different enzyme activity detection results and failing to efficiently and accurately reflect the true enzyme activity level of Daqu samples. In recent years, although the detection conditions of spectrophotometric methods for enzyme activity have been optimized and improved, different enzyme activities still need to be extracted and reacted step by step, which takes a long time and cannot meet the real-time monitoring requirements of hundreds of samples produced daily in the brothel.

[0005] Secondly, in the field of microbial detection, simultaneous screening and quantitative analysis of multiple strains of different species is one of the core requirements for improving detection efficiency and reducing detection costs. In existing detection methods, due to the inherent differences in the physiological and metabolic characteristics of different microorganisms, their growth cycles and reproduction rates vary significantly. This necessitates that existing detection processes involve batch-wise and time-segmented inoculation, culture, and detection for different strains. Multiple independent tests require repeated inoculation operations, occupying culture equipment, and adding reagents, significantly extending the overall detection cycle and failing to meet the needs of rapid detection scenarios. Furthermore, repeated operations increase the risk of human error, and multiple batches of testing consume more reagents, consumables, and equipment resources, significantly increasing detection costs. Regarding the optimization of selective culture media, existing technologies often involve adding antibiotics to inhibit non-target strains, and antimicrobial peptides have also been applied to culture media to inhibit other bacteria. However, existing antimicrobial peptides and antibiotics are mostly broad-spectrum inhibitors with poor specificity, unable to distinguish between harmful and beneficial bacteria, and easily damage the target strain.

[0006] Therefore, developing a rapid detection method for the activity of multiple enzymes and the content of microorganisms in Daqu (a type of starter culture) to make the detection of core enzyme activity and key microorganisms in Daqu faster, simpler to operate, and more accurate has become an urgent technical problem to be solved in the field of quality control of Daqu fermentation process. Summary of the Invention

[0007] This invention provides a rapid detection method for multiple key enzyme activities and microbial content during the fermentation process of Daqu (a type of starter culture). The method involves rapid detection methods for enzyme activities such as saccharification power, liquefaction power, and protease activity, as well as rapid detection methods for the viable content of lactic acid bacteria, yeast, and mold. This method overcomes the shortcomings of existing methods for detecting enzyme activity and microorganisms in Daqu, such as the need to extract different enzyme solutions through different treatments, long enzyme activity reaction times, long time consumption for traditional viable microbial counting, high operational intensity, and poor antibacterial specificity of traditional selective culture media relying on broad-spectrum antibiotics.

[0008] This invention proposes a rapid detection method for the quality of Daqu (a type of starter culture), including the detection of multiple key enzyme activities and microbial content, comprising the following steps: The detection of multiple key enzyme activities includes: extracting the Daqu sample once with an acetate-sodium acetate buffer or a sodium lactate buffer at pH 4.6 to obtain a Daqu enzyme extract suitable for detecting glycation power, liquefaction power, and protease activity. Then, the glycation power, liquefaction power, and protease activity are simultaneously detected using a three-channel partition of a 48-well plate. The glycation power, liquefaction power, and protease activity of the sample are calculated by combining the standard curve. Microbial content detection includes: inoculating Daqu samples into selective liquid culture media suitable for lactic acid bacteria, yeast, and mold, respectively, and culturing them; simultaneously detecting the absorbance values ​​after culturing using an ELISA reader; and calculating the content of each microorganism by combining the results with a standard curve; among which, Preparation of selective liquid culture medium for lactic acid bacteria: Based on MRS, add 3~6 mg / L AMP-338 antimicrobial peptide, 1000~2000 mg / L potassium sorbate, and 10~14 mg / L nystatin to make selective liquid culture medium for Daqu lactic acid bacteria. Preparation of selective liquid culture medium for yeast: Based on YPD, add 3~6 mg / L LAB-129 antimicrobial peptide, and add organic acid at a volume ratio of acetic acid to lactic acid of 1:3, wherein the volume fraction of acetic acid is 0.1% and the volume fraction of lactic acid is 0.3%, as the selective liquid culture medium for Daqu yeast. Preparation of selective liquid culture medium for molds: Add 3~6 mg / L LAB-129 antimicrobial peptide and 5~60 g / L sodium chloride to the Bengal Red medium and adjust the pH to 4.0~7.0 to prepare selective liquid culture medium for Aspergillus macrocephala.

[0009] Furthermore, the specific assays for several key enzyme activities include: A1 Daqu sample pretreatment: Preparation of enzyme extract for Daqu enzyme activity test: Add an acetate-sodium acetate buffer solution or a sodium lactate buffer solution with pH 4.6 to the Daqu sample, make up to volume, filter, and collect the filtrate, which is the Daqu enzyme extract; A2 Saccharification power colorimetric reaction: Take the Daqu enzyme extract obtained in A1, add starch solution to carry out the enzymatic reaction, add NaOH solution to terminate the enzymatic reaction, and obtain the reaction solution; take the obtained reaction solution, add 3,5-dinitrosalicylic acid colorimetric reagent, and carry out the colorimetric reaction in a boiling water bath to obtain the test solution; A3 Liquefaction Power Colorimetric Reaction: In the experimental group, the starch solution was preheated, and then the Daqu enzyme extract obtained in A1 was added to the preheated starch solution to carry out an enzymatic reaction, resulting in a reaction solution. The blank control group followed the same operating steps as above, except that the Daqu enzyme extract was replaced with an equal volume of acetate-sodium acetate buffer solution at pH 4.6. Iodine-potassium iodide solution was added to the reaction solutions of both the experimental group and the blank control group, and the mixture was stirred to carry out a colorimetric reaction, resulting in the test solution. A4 protease activity colorimetric reaction: In the experimental group, the Daqu enzyme extract obtained from A1 was preheated, and then casein solution was added for enzymatic reaction. The reaction was terminated by adding trichloroacetic acid solution. After filtration, sodium carbonate solution and Folin reagent were added to the filtrate in sequence, and a colorimetric reaction was carried out at an isothermal temperature to obtain the test solution. In the blank group, the Daqu enzyme extract obtained from A1 was preheated, and then trichloroacetic acid solution was added for enzymatic reaction. Then casein solution was added, mixed well, filtered, and then sodium carbonate solution and Folin reagent were added to the filtrate in sequence, and a colorimetric reaction was carried out at an isothermal temperature to obtain the test solution. A5 microplate reader absorbance values: A 48-well plate was used for three-channel partitioned reaction, with each channel corresponding to the detection of glycation power, liquefaction power and protease activity. The test solutions obtained from the A2 color reaction, A3 color reaction and A4 color reaction were respectively taken into the corresponding channels of the 48-well plate, and the three channels were detected simultaneously. The absorbance values ​​of each reaction well were recorded in sequence. A6 Standard Curve Plotting: Prepare a series of standard solutions of corresponding concentrations for three detection indicators: glycation power, liquefaction power, and protease activity. The standard for glycation power detection is glucose solution, the standard for liquefaction power detection is starch solution, and the standard for protease activity detection is tyrosine solution. Take each series of standard solutions of different concentrations and incubate and develop them according to the enzyme activity detection conditions corresponding to A2, A3, and A4, respectively. Plot the standard curves for glucose, starch, and tyrosine with the concentration of the standard solution on the x-axis and the absorbance value on the y-axis. A7 uses the absorbance value of the test solution measured in A5 to substitute into the glucose standard curve, starch standard curve, and tyrosine standard curve obtained in A6 to calculate the content of the corresponding product or substrate in the test solution; then, the saccharification power, liquefaction power, and protease activity of Daqu are calculated using the corresponding formulas.

[0010] Furthermore, the detection of microbial content specifically includes: B4. Plotting the standard curve: The *Aspergillus oryzae* suspension was serially diluted to obtain a 10⁻⁶ standard curve. -1 ~10 -6 The bacterial suspensions were diluted at various ratios and inoculated into selective liquid culture media for lactic acid bacteria, yeast, and mold, respectively. The absorbance values ​​were measured using an ELISA reader. After incubation, the bacterial suspensions were diluted and spread onto the corresponding basal culture media plates for plate counting. Standard curves were plotted with absorbance values ​​on the x-axis and the viable cell count on the y-axis to obtain standard curves for lactic acid bacteria, yeast, and mold, respectively. B5 Microbial Quantitative Detection: A 48-well plate was used for three-channel partitioned reactions, with each channel corresponding to the detection of lactic acid bacteria, yeast, and mold, respectively. Three different selective liquid culture bases were taken and added to the selective culture media of lactic acid bacteria, yeast, and mold, respectively, in the 48-well plate. After covering the plate, the plates were incubated in an incubator for 12-15 h. The absorbance values ​​of each reaction well were recorded simultaneously using an ELISA reader. Finally, the absorbance values ​​were substituted into the standard curves of lactic acid bacteria, yeast, and mold, respectively, to calculate the number of different microorganisms in the ELISA.

[0011] Furthermore, A1 satisfies at least one of the following conditions: (1) The ratio of Daqu sample to buffer solution is 2.5 g: 4~6 ml; then the volume is adjusted to 50 mL; (2) It also includes, after adjusting the volume, shaking, and then placing it in a constant temperature water bath at 37~43℃ for immersion for 15-20 minutes. (3) Preparation of Daqu samples includes crushing the Daqu blocks and sieving them for later use; and / or A2 satisfies at least one of the following conditions: (1) The volume ratio of Daqu enzyme extract to starch solution is 1:4~6, wherein the mass-volume concentration of starch solution is 1.8~2.2g / 100ml; (2) The conditions for the enzyme-catalyzed reaction are to react in a constant temperature water bath at 35~40℃ for 15~20 min; (3) The volume ratio of Daqu enzyme extract to NaOH solution is 1:0.5, wherein the mass-volume concentration of NaOH solution is 20 g / 100ml; (4) The volume ratio of the reaction solution to the 3,5-dinitrosalicylic acid colorimetric reagent is 2:4~6; (5) Boiling water bath color development reaction for 5-6 minutes; (6) also includes the following steps: after the colorimetric reaction in a boiling water bath, the mixture is cooled and then diluted to a fixed volume.

[0012] Furthermore, A3 satisfies at least one of the following conditions: (1) The volume ratio of starch solution to Daqu enzyme extract is 1:10, wherein the mass-volume concentration of starch solution is 1.8~2.2 g / 100ml; (2) The preheating treatment is specifically preheating at 35℃ for 8 to 10 minutes; (3) The enzyme-catalyzed reaction is specifically carried out at 35-40℃ for 15-18 minutes; (4) The volume ratio of the reaction solution to the iodine-potassium iodide solution is 1:1~2; and / or A4 meets at least one of the following conditions: (1) The volume ratio of Daqu enzyme extract, casein solution, and trichloroacetic acid solution is 1:1:2~3; (2) The preheating treatment is specifically heating in a 40℃ constant temperature water bath for 2~3 minutes; (3) The enzyme-catalyzed reaction is specifically carried out in a constant temperature water bath at 35~40℃ for 10 min; (4) The volume ratio of filtrate, sodium carbonate solution, and Folin reagent is 1~2:5:1; (5) The constant temperature color development reaction is specifically carried out in a constant temperature water bath at 40℃ for 10 min.

[0013] Furthermore, A5 satisfies at least one of the following conditions: (1) The detection wavelength for saccharification power is 550 nm, the detection wavelength for liquefaction power is 660 nm, and the detection wavelength for protease activity is 680 nm. (2) The three-channel partitioned reaction using a 48-well plate is as follows: a 48-well plate is used to carry out the three-channel partitioned reaction, and a single 48-well plate is divided into three independent channels along the longitudinal direction, each channel containing 16 reaction wells.

[0014] Furthermore, A7 satisfies at least one of the following conditions: (1) Formula 1, Saccharification power (mg / (g·h)) = reducing sugar concentration × N / (m / V1×T×V2 / V3); Wherein: reducing sugar concentration (mg / ml) is obtained from glucose standard curve; N is dilution factor; m is the mass of oven-dried Daqu (g); V1 is the final volume of Daqu treatment (ml); T is enzyme reaction time (h); V2 is the volume of filtrate used in enzyme reaction (ml); V3 is the total volume of enzyme reaction (including sodium hydroxide solution used to terminate the reaction, ml); (2) Formula 2, liquefaction force = (W0-W) / cvt; Where: W0 is the starch content of the test sample calculated from the starch standard curve (g / L); W is the starch content of the blank group calculated from the starch standard curve (g / L); c is the concentration of enzyme extract (g / mL); v is the volume of enzyme extract added to the reaction system (mL); t is the enzyme-catalyzed reaction time (min). (3) Formula 3, protease activity (U / g) = ((A0-A) × V×4 × n) / (10 × W); Where: A0 is the tyrosine concentration of the test sample obtained from the tyrosine standard curve (μg / mL); A is the tyrosine concentration of the blank group measured from the tyrosine standard curve (μg / mL); V is the sample pretreatment volume (mL); 4 is the total volume of the colorimetric reaction reagent (mL); n is the enzyme solution dilution factor; 10 is the enzyme-catalyzed reaction time (min); W is the mass of the oven-dried Daqu sample participating in the reaction (g).

[0015] Furthermore, B4 satisfies at least one of the following conditions: (1) The absorbance value was measured at a wavelength of 600 nm using an ELISA reader; (2) The culture was carried out at a constant temperature of 30℃ for 12-15 h; (3) Preparation of Daqu bacteria suspension: Take Daqu sample, dilute with physiological saline, mix, centrifuge and take the supernatant, which is Daqu bacteria suspension; B5 meets at least one of the following conditions: (1) Divide a single 48-well plate into three independent channels along the longitudinal direction, each channel containing 16 reaction wells; (2) Add the *Aspergillus oryzae* suspension to selective culture media for lactic acid bacteria, yeast, and mold, respectively, wherein the inoculum amount of *Aspergillus oryzae* suspension is 1-3%; (3) The specific culture is to place the container in an incubator at 25-40℃ after covering it with a lid.

[0016] The present invention also provides a kit for rapid detection of the activity of multiple key enzymes and microbial content in Daqu as described above.

[0017] Furthermore, the kit includes: 1) The core components of the enzyme activity assay kit adapted to the above detection methods include: ① Sample pretreatment components: Acetic acid-sodium acetate buffer solution or sodium lactate buffer solution at pH 4.6; ② Enzyme activity detection substrate system Substrate for saccharification power assay: starch solution with a mass-volume concentration of 1.8–2.2 g / 100 ml; Substrate for liquefaction force testing: starch solution with a mass-volume concentration of 1.8~2.2 g / 100ml; Protease activity assay substrate: 10 g / L casein solution; ③ Reaction color development and termination system Colorimetric reagents: 3,5-dinitrosalicylic acid colorimetric reagent, iodine-potassium iodide solution, sodium carbonate solution, and Folin reagent; Termination reagents: NaOH solution and trichloroacetic acid solution with a mass-volume concentration of 20 g / 100 ml; ④ Standard curve plotting components: glucose standard, starch standard, tyrosine standard; ⑤ Detection carrier and auxiliary components: 48-well plate, deionized water; 2) The core components of the microbial detection kit adapted to the above detection methods include: ① Sample pretreatment components: sterile physiological saline; ② Selective culture medium system Selective liquid culture medium for lactic acid bacteria: Based on MRS medium, add 3~6 mg / L AMP-338 antimicrobial peptide, 1000~2000 mg / L potassium sorbate, and 10~14 mg / L nystatin; Selective liquid culture medium for yeast: Based on YPD medium, add 3~6 mg / L LAB-129 antimicrobial peptide, and add acetic acid and lactic acid in a volume ratio of 1:3, wherein the volume fraction of acetic acid is 0.1% and the volume fraction of lactic acid is 0.3%. Selective liquid culture medium for molds: Based on Bengal Red liquid culture medium, add 3~6 mg / L LAB-129 antimicrobial peptide, 5~60 g / L sodium chloride and adjust the pH to 4.0~7.0; ③ Detection carrier and auxiliary components: 48-well plate, pH adjuster.

[0018] This invention has the following advantages: This invention proposes a rapid detection method for multiple key enzyme activities and microbial content in Daqu (a type of Chinese liquor). It comprehensively considers the impact of inconsistent buffer pH, temperature, and time required for different enzyme extraction solutions, as well as the differences in targeted inhibition culture media and growth cycles required by different microbial genera during the testing of enzyme activity indicators such as saccharification power, liquefaction power, and protease activity in Daqu. Based on existing detection methods, it optimizes the conditions and develops a highly efficient detection technology of "multi-enzyme co-extraction - micro-color development - full plate reading," a method for "one-time operation, simultaneous growth, and simultaneous detection" of multiple microorganisms, and a detection method of "dual 48-well plate three-channel partitioned reaction." This achieves integrated extraction and simultaneous detection of saccharification power, liquefaction power, and protease activity in Daqu, as well as simultaneous screening and quantitative analysis of multiple microorganisms. It achieves the goal of rapidly and accurately quantifying the key enzyme activities and viable microbial counts in Daqu, meeting the rapid quality control needs of industrial production sites in the brewing industry and providing efficient and reliable technical support for the precise control of Daqu production. This method is characterized by accurate, rapid, efficient, high accuracy, high precision, and high sensitivity, overcoming the shortcomings of traditional detection methods such as cumbersome operation, long cycle, large error, high cost, and lack of dedicated reagent kits. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This illustrates the effect of different extraction times on the saccharification power, liquefaction power, and protease activity of the samples in Experimental Example 2 of this invention. Figure 2 This is the curve showing the effect of the boiling water bath color development time on the saccharification power in Experiment Example 4 of the present invention; Figure 3 The curve showing the effect of the amount of iodine-potassium iodide solution added on the liquefaction force in Experiment Example 5; Figure 4 This illustrates the effect of different immersion temperatures on the saccharification power, liquefaction power, and protease activity of the samples in Experiment Example 6 of this invention. Figure 5This is a comparison of lactic acid bacteria colony counts under different treatment groups in Experiment Example 8 of the present invention; Figure 6 This is a comparison of the number of yeast colonies isolated from different samples using different optimized culture media in Experiment Example 8 of the present invention; Figure 7 This is a comparison of the number of mold colonies isolated from different samples using different optimized culture media in Experiment Example 8 of the present invention; Figure 8 This is the growth curve of the core microorganisms in the low-temperature Daqu (a type of starter culture) in Experiment Example 8 of this invention; Figure 9 The growth curves of yeast at different inoculum amounts in Experiment Example 8 of this invention are shown. Figure 10 This is a design diagram of a 48-well microplate for the determination of Daqu enzyme activity according to the present invention; Figure 11 This is a design diagram of a 48-well ELISA plate for the determination of microorganisms in Daqu (a type of starter culture) according to the present invention. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0022] The present invention will now be described in detail with reference to the embodiments.

[0023] This invention proposes a rapid detection method for the quality of Daqu (a type of starter culture), including the detection of multiple key enzyme activities and microbial content, comprising the following steps: The detection of multiple key enzyme activities includes: extracting the Daqu sample once with an acetate-sodium acetate buffer or a sodium lactate buffer at pH 4.6 to obtain a Daqu enzyme extract suitable for detecting glycation power, liquefaction power, and protease activity. Then, the glycation power, liquefaction power, and protease activity are simultaneously detected using a three-channel partition of a 48-well plate. The glycation power, liquefaction power, and protease activity of the sample are calculated by combining the standard curve. Microbial content detection includes: inoculating Daqu samples into selective liquid culture media suitable for lactic acid bacteria, yeast, and mold, respectively, and culturing them; simultaneously detecting the absorbance values ​​after culturing using an ELISA reader; and calculating the content of each microorganism by combining the results with a standard curve; among which, Preparation of selective liquid culture medium for lactic acid bacteria: Based on MRS, add 3~6 mg / L AMP-338 antimicrobial peptide, 1000~2000 mg / L potassium sorbate, and 10~14 mg / L nystatin to make selective liquid culture medium for Daqu lactic acid bacteria. Preparation of selective liquid culture medium for yeast: Based on YPD, add 3~6 mg / L LAB-129 antimicrobial peptide, and add organic acid at a volume ratio of acetic acid to lactic acid of 1:3, wherein the volume fraction of acetic acid is 0.1% and the volume fraction of lactic acid is 0.3%, as the selective liquid culture medium for Daqu yeast. Preparation of selective liquid culture medium for molds: Add 3~6 mg / L LAB-129 antimicrobial peptide and 5~60 g / L sodium chloride to the Bengal Red medium and adjust the pH to 4.0~7.0 to prepare selective liquid culture medium for Aspergillus macrocephala.

[0024] In one embodiment of the present invention, the detection of multiple key enzyme activities specifically includes: A1 Daqu sample pretreatment: Preparation of enzyme extract for Daqu enzyme activity test: Add an acetate-sodium acetate buffer solution or a sodium lactate buffer solution with pH 4.6 to the Daqu sample, make up to volume, filter, and collect the filtrate, which is the Daqu enzyme extract; A2 Saccharification power colorimetric reaction: Take the Daqu enzyme extract obtained in A1, add starch solution to carry out the enzymatic reaction, add NaOH solution to terminate the enzymatic reaction, and obtain the reaction solution; take the obtained reaction solution, add 3,5-dinitrosalicylic acid colorimetric reagent, and carry out the colorimetric reaction in a boiling water bath to obtain the test solution; A3 Liquefaction Power Colorimetric Reaction: In the experimental group, the starch solution was preheated, and then the Daqu enzyme extract obtained in A1 was added to the preheated starch solution to carry out an enzymatic reaction, resulting in a reaction solution. The blank control group followed the same operating steps as above, except that the Daqu enzyme extract was replaced with an equal volume of acetate-sodium acetate buffer solution at pH 4.6. Iodine-potassium iodide solution was added to the reaction solutions of both the experimental group and the blank control group, and the mixture was stirred to carry out a colorimetric reaction, resulting in the test solution. A4 protease activity colorimetric reaction: In the experimental group, the Daqu enzyme extract obtained from A1 was preheated, and then casein solution was added for enzymatic reaction. The reaction was terminated by adding trichloroacetic acid solution. After filtration, sodium carbonate solution and Folin reagent were added to the filtrate in sequence, and a colorimetric reaction was carried out at an isothermal temperature to obtain the test solution. In the blank group, the Daqu enzyme extract obtained from A1 was preheated, and then trichloroacetic acid solution was added for enzymatic reaction. Then casein solution was added, mixed well, filtered, and then sodium carbonate solution and Folin reagent were added to the filtrate in sequence, and a colorimetric reaction was carried out at an isothermal temperature to obtain the test solution. A5 microplate reader absorbance values: A 48-well plate was used for three-channel partitioned reaction, with each channel corresponding to the detection of glycation power, liquefaction power and protease activity. The test solutions obtained from the A2 color reaction, A3 color reaction and A4 color reaction were respectively taken into the corresponding channels of the 48-well plate, and the three channels were detected simultaneously. The absorbance values ​​of each reaction well were recorded in sequence. A6 Standard Curve Plotting: Prepare a series of standard solutions of corresponding concentrations for three detection indicators: glycation power, liquefaction power, and protease activity. The standard for glycation power detection is glucose solution, the standard for liquefaction power detection is starch solution, and the standard for protease activity detection is tyrosine solution. Take each series of standard solutions of different concentrations and incubate and develop them according to the enzyme activity detection conditions corresponding to A2, A3, and A4, respectively. Plot the standard curves for glucose, starch, and tyrosine with the concentration of the standard solution on the x-axis and the absorbance value on the y-axis. A7 uses the absorbance value of the test solution measured in A5 to substitute into the glucose standard curve, starch standard curve, and tyrosine standard curve obtained in A6 to calculate the content of the corresponding product or substrate in the test solution; then, the saccharification power, liquefaction power, and protease activity of Daqu are calculated using the corresponding formulas.

[0025] In step A1 of this invention, the inventors discovered that by using a pretreatment temperature of 40℃ and a time of 15min, simultaneous micro-quantitative detection of multiple indicators can be achieved. The innovative use of multi-enzyme co-extraction in the highly efficient detection technology of "multi-enzyme co-extraction-micro-color development-full plate reading" allows for the simultaneous enrichment of multiple target enzymes with only a single immersion, effectively reducing sample volume and operation steps. Furthermore, the measured results of saccharification power, liquefaction power, and protease activity are highly consistent with traditional national standard methods, providing efficient and stable technical support for the rapid determination of the quality of low-temperature Daqu (a type of starter culture).

[0026] In one embodiment of the present invention, in step A1, an acetate-sodium acetate buffer solution with pH 4.6 is used to extract the enzyme solution required for the enzyme activity tests of saccharification power, liquefaction power, and protease. Further, the preparation method of the pH 4.6 acetate-sodium acetate buffer solution includes: weighing 164g of anhydrous sodium acetate, dissolving it in water, adding 114mL of glacial acetic acid, and diluting with water to 1000mL.

[0027] In one embodiment of the present invention, in A1, the ratio of the amount of Daqu sample to the amount of buffer solution is 2.5 g: 4~6 ml; the volume is adjusted to 50 mL.

[0028] In one embodiment of the present invention, the preparation of the Daqu sample includes crushing the Daqu block and sieving it for later use.

[0029] In one embodiment of the present invention, in A1, the Daqu sample is weighed with an accuracy of 0.001 g.

[0030] In one embodiment of the present invention, in A1, the Daqu sample is diluted to 100 mL with physiological saline.

[0031] In one embodiment of the present invention, A1 further includes, after being brought to a constant volume, shaking, and then being placed in a constant temperature water bath at 37~43℃ for immersion for 15-20 minutes.

[0032] Preferably, in A1, after shaking, it is placed in a 40℃ constant temperature water bath for immersion for 15 minutes.

[0033] In one embodiment of the present invention, in A1, the mixing specifically involves oscillating in a shaker at 37°C for 30 minutes.

[0034] In step A2 of this invention, the saccharification power is tested using the 3,5-dinitrosalicylic acid (DNS) colorimetric method. Under alkaline heating conditions, the reducing sugar generated by the enzymatic reaction undergoes a redox reaction with the DNS reagent to produce an orange-red product. The absorbance of this product is positively correlated with the reducing sugar content, and the saccharification power is quantified accordingly.

[0035] In one embodiment of the present invention, in A2, the volume ratio of the Daqu enzyme extract to the starch solution is 1:4-6, wherein the mass-volume concentration of the starch solution is 1.8-2.2%. Preferably, the volume ratio of the Daqu enzyme extract to the starch solution is 1:5; and the mass-volume concentration of the starch solution is 2%. It should be noted that throughout this text, unless otherwise specified, the mass-volume concentration refers to the percentage of solute mass (g) to the total solution volume (mL). For example, a starch solution with a mass-volume concentration of 2% means that 100 mL of solution contains 2 g of starch. A NaOH solution with a mass-volume concentration of 20% means that 100 mL of solution contains 20 g of sodium hydroxide.

[0036] In one embodiment of the present invention, in A2, the conditions for the enzymatic reaction are: reaction in a constant temperature water bath at 35~40℃ for 15~20 min.

[0037] In one embodiment of the present invention, in A2, the volume ratio of Daqu enzyme extract to NaOH solution is 1:0.5, wherein the mass-volume concentration of NaOH solution is 20%.

[0038] In one embodiment of the present invention, in A2, the volume ratio of the reaction solution to the 3,5-dinitrosalicylic acid (DNS) colorimetric reagent is 2:4~6. Specifically, it can be 2:4.

[0039] In one embodiment of the present invention, in A2, the volume adjustment specifically means adjusting the volume to 50.0 mL.

[0040] In one embodiment of the present invention, in A2, the time for the color development reaction begins from the time the boiling water boils.

[0041] In one embodiment of the present invention, in A2, the color development reaction is carried out in a boiling water bath for 5-6 minutes.

[0042] In one embodiment of the present invention, A2 further includes a boiling water bath color development reaction followed by cooling and then volume dilution.

[0043] In this embodiment of the invention, a preferred substrate concentration (starch solution concentration of 2%) and reaction color development time (5 min) are used, resulting in a more complete enzymatic reaction and significantly improved stability of absorbance detection values. Furthermore, the national standard for saccharification uses a titration method, which requires approximately 90 min. However, this invention, by replacing the saccharification and titration steps in the national standard with a color development reaction, reduces the operation time to only about 20 min.

[0044] In step A3 of this invention, the liquefaction power is tested using the iodine colorimetric method. Starch forms a characteristic blue complex with iodine. After the liquefying enzyme hydrolyzes the starch, the blue complex decreases, the absorbance of the system decreases, and the change in absorbance is positively correlated with the amount of starch hydrolyzed. Based on this, the liquefaction power can be quantified.

[0045] In one embodiment of the present invention, in A3, the volume ratio of starch solution to Daqu enzyme extract is 1:10, wherein the mass-volume concentration of starch solution is 1.8~2.2%. Preferably, the mass-volume concentration of starch solution is 2.0%.

[0046] In one embodiment of the present invention, in A3, the preheating treatment specifically involves preheating at 35°C for 8 minutes.

[0047] In one embodiment of the present invention, in step A3, the enzymatic reaction is specifically carried out at 35-40°C for 15 min. Preferably, the enzymatic reaction is carried out at 35°C for 15 min. In this embodiment of the present invention, the enzymatic reaction at 35°C yields the best results.

[0048] In one embodiment of the present invention, in A3, the volume ratio of the reaction solution to the iodine-potassium iodide solution is 1:1 to 2. Preferably, the volume ratio of the reaction solution to the iodine-potassium iodide solution is 1:1. In this embodiment of the present invention, when the volume ratio of the reaction solution to the iodine-potassium iodide solution is 1:1, the colorimetric reaction between the iodine-potassium iodide solution and the starch reaches saturation, and the corresponding absorbance detection value reaches its peak, resulting in optimal detection accuracy. The concentration of the iodine-potassium iodide solution is 40 g / L.

[0049] In step A4 of this invention, the protease activity is detected using the Folin-Ciocalteu method: the protease catalyzes the hydrolysis of casein to generate tyrosine. Under alkaline conditions, tyrosine undergoes a redox reaction with Folin-Ciocalteu reagent to generate a stable blue compound. The absorbance of this compound at a specific wavelength is positively correlated with the tyrosine content, thereby enabling the quantitative detection of protease activity.

[0050] In one embodiment of the present invention, in A4, the casein solutions corresponding to different proteases have different pH values. Preferably, the casein solution corresponding to the acidic protease has a pH of 3.0, the casein solution corresponding to the neutral protease has a pH of 7.5, and the casein solution corresponding to the alkaline protease has a pH of 10.5.

[0051] In one embodiment of the present invention, in A4, the volume ratio of the Daqu enzyme extract, casein solution, and trichloroacetic acid solution is 1:1:2~3. The casein solution has a concentration of 10.0 g / L, and the trichloroacetic acid solution has a concentration of 65.4 g / L.

[0052] In one embodiment of the present invention, in A4, the preheating treatment specifically involves heating in a 40°C constant temperature water bath for 2-3 minutes.

[0053] In one embodiment of the present invention, in A4, the enzymatic reaction is specifically carried out in a constant temperature water bath at 35~40℃ for 10 min.

[0054] In one embodiment of the present invention, in A4, the volume ratio of filtrate, sodium carbonate solution, and Folin reagent is 1~2:5:1. The concentration of the sodium carbonate solution is 42.4 g / L.

[0055] In one embodiment of the present invention, in A4, the constant temperature colorimetric reaction is specifically carried out in a constant temperature water bath at 40°C for 10 min.

[0056] In step A5 of this invention, an enzyme-linked immunosorbent assay (ELISA) reader is used to simultaneously detect the glycation power test solution, the liquefaction power test solution, and the protease activity test solution to quickly obtain the absorbance values ​​of the three test solutions.

[0057] In one embodiment of the present invention, in step A5, 1 mL of the test solution obtained from the colorimetric reaction in step A2, 1 mL of the test solution obtained from the colorimetric reaction in step A3, and 1 mL of the test solution obtained from the colorimetric reaction in step A4 are respectively taken into the corresponding channels of the 48-well plate, and the three channels are simultaneously detected.

[0058] In one embodiment of the present invention, in A5, the detection wavelength for saccharification power is 550 nm, the detection wavelength for liquefaction power is 660 nm, and the detection wavelength for protease activity is 680 nm.

[0059] In one embodiment of the present invention, in A5, the three-channel partitioned reaction using a 48-well plate is specifically as follows: the three-channel partitioned reaction is performed using a 48-well plate, and a single 48-well plate is divided into three independent channels along the longitudinal direction, each channel containing 16 reaction wells.

[0060] In one embodiment of the present invention, the specific partition layout of the 48-hole plate in A5 is as follows: Figure 10Wells A1-8 and B1-8 are for glycation power detection, wells C1-8 and D1-8 are for liquefaction power detection, and wells E1-8 and F1-8 are for protease activity detection. Spacing is provided between each channel to prevent cross-contamination.

[0061] In step A7 of this invention, the absorbance value of the test solution is substituted into the glucose standard curve, starch standard curve, and tyrosine standard curve respectively to calculate the content of the corresponding product or substrate in the test solution; then, the saccharification power, liquefaction power, and protease activity of Daqu are calculated by the corresponding formulas.

[0062] In one embodiment of the present invention, Formula 1, saccharification power (mg / (g·h)) = reducing sugar concentration × N / (m / V1×T×V2 / V3); Wherein: reducing sugar concentration (mg / ml) is obtained from glucose standard curve; N is dilution factor; m is the mass of oven-dried Daqu (g); V1 is the final volume of Daqu treatment (ml); T is enzyme reaction time (h); V2 is the volume of filtrate used in enzyme reaction (ml); V3 is the total volume of enzyme reaction (including sodium hydroxide solution used to terminate the reaction, ml); Formula 2, liquefaction force = (W0 - W) / cvt; Where: W0 is the starch content of the test sample calculated from the starch standard curve (g / L); W is the starch content of the blank group calculated from the starch standard curve (g / L); c is the concentration of enzyme extract (g / mL); v is the volume of enzyme extract added to the reaction system (mL); t is the enzyme-catalyzed reaction time (min). Formula 3, Protease activity (U / g) = ((A0-A) × V×4 × n) / (10 × W); Where: A0 is the tyrosine concentration of the test sample obtained from the tyrosine standard curve (μg / mL); A is the tyrosine concentration of the blank group measured from the tyrosine standard curve (μg / mL); V is the sample pretreatment volume (mL); 4 is the total volume of the colorimetric reaction reagent (mL); n is the enzyme solution dilution factor; 10 is the enzyme-catalyzed reaction time (min); W is the mass of the oven-dried Daqu sample participating in the reaction (g). Compared with traditional enzyme activity detection methods, this invention improves the efficiency of enzyme activity detection by more than 5 times. The time required for the combined detection of the three core enzyme activity indicators is significantly reduced from 6-8 hours per person in traditional methods to ≤45 minutes per person (the detection method proposed in this application allows multiple steps to be performed simultaneously). Under the same experimental conditions, the daily throughput of a single microplate reader is 40 times that of the manual titration method in QB / T 4257-2011 and GB / T 23527-2009, which can fully meet the online real-time monitoring needs of koji production.

[0063] In one embodiment of the present invention, the detection of microbial content specifically includes: B4. Plotting the standard curve: The *Aspergillus oryzae* suspension was serially diluted to obtain a 10⁻⁶ standard curve. -1 ~10 -6 The bacterial suspensions were diluted at various ratios and inoculated into selective liquid culture media for lactic acid bacteria, yeast, and mold, respectively. The absorbance values ​​were measured using an ELISA reader. After incubation, the bacterial suspensions were diluted and spread onto the corresponding basal culture media plates for plate counting. Standard curves were plotted with absorbance values ​​on the x-axis and the viable cell count on the y-axis to obtain standard curves for lactic acid bacteria, yeast, and mold, respectively. B5 Microbial Quantitative Detection: A 48-well plate was used for three-channel partitioned reactions, with each channel corresponding to the detection of lactic acid bacteria, yeast, and mold, respectively. Three different selective liquid culture bases were taken and added to the selective culture media of lactic acid bacteria, yeast, and mold, respectively, in the 48-well plate. After covering the plate, the plates were incubated in an incubator for 12-15 h. The absorbance values ​​of each reaction well were recorded simultaneously using an ELISA reader. Finally, the absorbance values ​​were substituted into the standard curves of lactic acid bacteria, yeast, and mold, respectively, to calculate the number of different microorganisms in the ELISA.

[0064] In a preferred embodiment of the present invention, 5 mg / L AMP-338 antimicrobial peptide, 1000 mg / L potassium sorbate, and 10 mg / L nystatin were added to the MRS as a selective liquid culture medium for *Lactobacillus daquensis*. Under the aforementioned preferred conditions, the lactic acid bacteria count results were closest to the blank control group without the addition of antimicrobial agents, confirming that this addition regimen had the best inhibitory effect on non-target bacteria and had almost no adverse effect on the growth of lactic acid bacteria.

[0065] It should be noted that the MRS medium (De Man, Rogosa and Sharpe medium) contains the following per liter of water: 10.0 g peptone, 5.0–10.0 g beef extract, 4.0–5.0 g yeast extract, 20.0 g glucose, 1.0 mL Tween 80, 5.0 g sodium acetate trihydrate, 2.0 g diammonium citrate, 2.0 g dipotassium hydrogen phosphate heptahydrate, 0.58 g magnesium sulfate heptahydrate, and 0.25 g manganese sulfate tetrahydrate, with distilled water added to a final volume of 1 L. The pH of the medium is 6.2 ± 0.2.

[0066] In a preferred embodiment of the present invention, the yeast culture medium is prepared as follows: Based on YPD, 5 mg / L LAB-129 antimicrobial peptide is added, along with organic acids at a volume ratio of acetic acid to lactic acid of 1:3. The volume fraction of acetic acid is 0.1%, and the volume fraction of lactic acid is 0.3%, meaning 1 ml of liquid culture medium contains 1 ml of acetic acid and 3 ml of lactic acid. This serves as a selective liquid culture medium for *Saccharomyces cerevisiae*. Under the aforementioned preferred conditions, the yeast count results are closest to the blank control group without the added antimicrobial agent, confirming that this addition regimen has the best inhibitory effect on filamentous fungi without affecting yeast growth.

[0067] In this embodiment of the invention, the LAB-129 antimicrobial peptide is used to specifically inhibit Gram-positive and Gram-negative bacteria in the culture medium without affecting the growth of the target strain of yeast; non-target bacteria include lactic acid bacteria, Bacillus cereus, Bacillus subtilis, molds, etc.

[0068] It should be noted that each liter of the YPD medium contains 20.0 g of peptone, 10.0 g of yeast extract, and 20.0 g of glucose, with the remainder being distilled water as the solvent. The pH is the natural pH.

[0069] In a preferred embodiment of the present invention, the mold culture medium is prepared by adding 5 mg / L LAB-129 antimicrobial peptide and 50 g / L sodium chloride to Bengal red agar medium and adjusting the pH to 5.5, thus creating a selective liquid culture medium for Aspergillus flavus. Under the aforementioned preferred conditions, the inhibitory effect on non-target bacteria is optimal, and the impact on mold growth is minimal.

[0070] It should be noted that the AMP-338 antimicrobial peptide is disclosed in Chinese Patent CN120591174B (publication date: October 28, 2025), and its amino acid sequence is: DATSVIADGQLITVDSR. The LAB-129 antimicrobial peptide is disclosed in Chinese Patent CN120623287B (publication date: October 17, 2025), and its amino acid sequence is: NAVTALLKALRDLIEALKRKK.

[0071] It should be noted that each 1L of the Bengal Red medium contains: 5.0g peptone, 10.0g glucose, 1.0g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.033g Bengal Red, 20.0g agar, and distilled water to a final volume of 1L. The pH is 7.0±0.2.

[0072] In one embodiment of the present invention, in step B4, the preparation of Daqu bacteria suspension is carried out by taking Daqu sample, making up to a volume with physiological saline, mixing, centrifuging and taking the supernatant, which is Daqu bacteria suspension.

[0073] In one embodiment of the present invention, in B4, the absorbance value is measured by an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 600 nm.

[0074] In one embodiment of the present invention, in B4, the isothermal culture is performed at 35°C for 12-15 h.

[0075] In step B5 of this invention, a single 48-well plate is divided longitudinally into three independent channels, each containing 16 reaction wells. Spacing is provided between the channels to prevent cross-contamination.

[0076] In one embodiment of the present invention, the specific partition layout in B5 is as follows: Figure 11 Wells A1-8 and B1-8 are for lactic acid bacteria detection, wells C1-8 and D1-8 are for yeast detection, and wells E1-8 and F1-8 are for mold detection. Spacing is provided between each well to prevent cross-contamination. The 16 reaction wells in each well can be used simultaneously for parallel experiments and blank control setups, ensuring the repeatability and accuracy of the test results.

[0077] In one embodiment of the present invention, in B5, 1 ml of selective liquid culture medium is taken into a 48-well plate.

[0078] It should be noted that in step B5, the *Aspergillus oryzae* suspension is added to the selective culture media of lactic acid bacteria, yeast, and mold, respectively. The inoculum volume of the *Aspergillus oryzae* suspension is 1-3%. Preferably, the inoculum volume of lactic acid bacteria and mold is 1%, and the inoculum volume of yeast is 2%. It should be noted that 2% here means adding 2 volumes of yeast suspension to every 100 volumes of liquid culture medium, in volume percentage (v / v).

[0079] In one embodiment of the present invention, in B5, the cultivation specifically involves placing the container in an incubator at 25-40°C after covering it with a lid. A cultivation temperature of 30°C is preferred.

[0080] In one embodiment of the present invention, B5 is placed in an incubator at 25-40°C after being covered. Under the aforementioned preferred conditions, all three microorganisms can reach the initial stage of the stationary phase within 12 hours, ensuring consistent growth status during ELISA reader assays. This time is significantly shorter than the 24-48 hours in the prior art, greatly improving detection efficiency.

[0081] Compared with the traditional live bacteria counting method, the present invention significantly shortens the rapid quantitative detection cycle of microorganisms from 24-72 hours to 12 hours, improves the detection efficiency by 2-6 times, effectively reduces human operation error, and significantly improves the repeatability and accuracy of the detection results.

[0082] The results of this invention have both high accuracy and high precision. The spiked recovery rate is stable at 90%~110%. The linear correlation coefficient r of the test results with the national standard method is ≥0.995, and the relative standard deviation RSD is ≤3%. The detection accuracy is significantly better than the traditional manual titration method, effectively avoiding the human error introduced by visually judging the titration endpoint.

[0083] In summary, this invention addresses the industry pain points of low efficiency, cumbersome operation, and poor antibacterial specificity in detecting the core enzyme activity and microorganisms in Daqu (a type of Chinese liquor). It breaks through the technical constraints of traditional "multi-enzyme stepwise extraction, multi-bacterial batch detection, and broad-spectrum antibacterial agent action" technology. With "high-efficiency integration, precise targeting, and simultaneous detection" as the core inventive concept, it integrates three major technical dimensions: optimized enzyme extraction, targeted microbial inhibition, and high-throughput micro-quantity detection, to carry out systematic technical innovation and system reconstruction. An innovative enzyme activity detection system, comprising "multi-enzyme co-extraction - micro-color development - full plate reading," has been constructed, enabling the simultaneous high-throughput detection of multiple enzyme types and three enzyme activities through a single extraction and enrichment process. This reduces the detection time from 6-8 hours to ≤45 minutes, improving efficiency by over 8 times while maintaining high accuracy in line with national standards. A pioneering customized antimicrobial peptide targeted inhibition strategy has been developed, constructing antibiotic-free, highly specific and selective culture media. Combined with optimized culture conditions, simultaneous growth detection of three types of microorganisms within 12 hours has been achieved, significantly shortening the cycle compared to the traditional 24-72 hours. A dual 48-well plate with a three-channel partitioned detection mode has been designed, enabling integrated simultaneous detection of enzyme activity and multiple microbial indicators. This increases the detection throughput to 40 times that of traditional methods, effectively reducing reagent consumption and operational errors.

[0084] This invention organically integrates previously independent enzyme activity and microbial detection technologies to construct a highly efficient, accurate, and integrated rapid detection system for core quality indicators of Daqu (a type of starter culture). The resulting leap in efficiency, improved accuracy, and reduced costs far exceed what can be achieved by simple combinations of existing technologies. It provides a brand-new technical solution for real-time quality control of Daqu fermentation in the brewing industry and offers an innovative approach for integrated rapid detection of multiple indicators in the field of fermented foods. Its industrial application and technology promotion value are significant.

[0085] On the other hand, embodiments of the present invention also propose a kit for a rapid detection method of multiple key enzyme activities and microbial content in Daqu (a type of Chinese liquor), comprising: 1) The core components of the enzyme activity assay kit adapted to this detection method include: ① Sample pretreatment components: Acetic acid-sodium acetate buffer solution at pH 4.6; ② Enzyme activity detection substrate system Substrate for saccharification power assay: 2.0% starch solution (w / v); wherein the starch solution has a w / v concentration of 2%. The substrate for liquefaction force testing was a starch solution with a mass-to-volume concentration of 2.0%; wherein the mass-to-volume concentration of the starch solution was 2%. Substrate for protease activity assay: 10 g / L casein solution (adjust pH before volume adjustment: pH 3.0 for acidic protease, pH 7.5 for neutral protease, and pH 10.5 for alkaline protease).

[0086] ③ Reaction color development and termination system Colorimetric reagents: 3,5-dinitrosalicylic acid (DNS) colorimetric reagent (for glycation power detection), iodine-potassium iodide solution (for liquefaction power detection), sodium carbonate solution + Folin reagent (for protease activity detection); Termination reagents: 20% NaOH solution (for glycation power detection) and trichloroacetic acid solution (for protease activity detection).

[0087] ④ Standard curve plotting components: glucose standard (for saccharification power), starch standard (for liquefaction power), tyrosine standard (for protease activity).

[0088] ⑤ Detection carrier and auxiliary components: 48-well plate, deionized water.

[0089] 2) The core components of the microbial detection kit adapted to this detection method include: ① Sample pretreatment components: sterile physiological saline; ② Selective culture medium system (core component) Selective liquid culture medium for lactic acid bacteria: MRS medium, supplemented with 3~6 mg / L AMP-338 antimicrobial peptide, 1000~2000 mg / L potassium sorbate, and 10~14 mg / L nystatin; Yeast selective liquid culture medium: YPD medium, supplemented with 3-6 mg / L LAB-129 antimicrobial peptide, with an acetic acid to lactic acid mass ratio of 1:3; wherein the volume fraction of acetic acid is 0.1% and the volume fraction of lactic acid is 0.3%. Selective liquid culture medium for molds: Bengal Red liquid medium, with the addition of 3~6 mg / L LAB-129 antimicrobial peptide, 5~60 g / L sodium chloride and pH adjusted to 4.0~7.0.

[0090] ③ Detection carrier and auxiliary components: 48-well plate, pH adjuster, instruction manual (including dosage of each component, aseptic operation procedures, culture condition parameters, microplate reader detection parameters, standard curve plotting, and result calculation methods).

[0091] ④ Standard Curve Plotting Component It should be noted that all reagents used in this kit are consistent with those used in the detection method.

[0092] The kit described in this invention is based on a highly efficient detection technology of "multi-enzyme co-extraction-micro-color development-full plate reading," a multi-microorganism "one-time operation, simultaneous growth, simultaneous detection" technology, and an integrated detection layout technology for three-channel partitioned reactions within a 48-well plate. Coupled with corresponding standard curves, it enables rapid quantitative detection of enzyme activity indicators such as saccharification power, liquefaction power, and protease activity in Daqu (a type of starter culture), as well as rapid quantitative detection of lactic acid bacteria, yeast, and molds in Daqu. This kit is suitable for real-time quality control and finished product quality evaluation during the fermentation process of Daqu in the brewing industry, providing technical support for precise control of Daqu production.

[0093] The present invention will now be described in detail with reference to the embodiments.

[0094] Example 1 Rapid detection method for multiple key enzyme activities and microbial content in Daqu (a type of Chinese liquor). A1 Sample Pretreatment: Samples from each stage of low-temperature Daqu fermentation were taken and pretreated. Based on the measured moisture content of the Daqu sample, it was crushed and sieved. 2.5 g of Daqu sample was accurately weighed, accurate to 0.001 g, and 5 ml of pH 4.6 acetate-sodium acetate buffer solution was added. The volume was then adjusted to 50 mL, shaken evenly, and placed in a 40℃ constant temperature water bath for 15 min. After filtration through filter paper, the filtrate was collected as the enzyme extract for Daqu saccharification power, liquefaction power, and protease activity. Accurately weigh 10.0 g of Daqu sample, dilute to 100 mL with physiological saline from the microbial detection kit, place in a shaker at 37℃ and shake for 30 min, centrifuge and take the supernatant as Daqu suspension.

[0095] A2 Glycolysis Enzymatic Colorimetric Reaction: Take 1.0 mL of the enzyme extract obtained in step A1, add 5 mL of 2.0% starch solution, and react precisely in a 35℃ constant temperature water bath for 15 min. Immediately add 0.5 mL of 20% NaOH solution to terminate the enzymatic reaction. Take 2.0 mL of the above reaction solution, add 4.0 mL of DNS colorimetric reagent, mix well, heat precisely in a boiling water bath for 5 min (start timing from boiling), quickly cool to room temperature, and dilute to 50.0 mL with distilled water. After appropriate dilution, take 1 mL into a 48-well plate, measure the absorbance value at 550 nm using an ELISA reader, and record it. A3 Liquefaction Enzymatic Colorimetric Reaction: In the experimental group, 1 mL of 2.0% starch solution was placed in a test tube and preheated precisely in a 35℃ constant temperature water bath for 8 min. 10.0 mL of the enzyme extract obtained in step A1 was added, and after stirring evenly, the enzyme reaction was continued in a 35℃ constant temperature water bath for 15 min to complete the reaction of the experimental group. The same procedures were followed as in the experimental group, except that 10 mL of enzyme extract was replaced with an equal volume of pH 4.6 acetate-sodium acetate buffer solution as a blank control group. 1.0 mL of reaction solution from both the experimental group and the blank control group was taken, and 1 mL of iodine-potassium iodide solution (40 g / L) was added to each. The mixture was stirred and the color was developed. 1 mL of each solution was transferred to a 48-well plate, and the absorbance was measured at 660 nm using a microplate reader. The detection data were recorded. A4 protease activity enzymatic colorimetric reaction: Experimental group: Take 1.0 mL of the enzyme extract obtained in step A1, place it in a 40℃ constant temperature water bath for precise preheating for 2 min, add 1 mL of casein solution (concentration of 10.0 g / L) (the casein solution corresponding to acidic protease has a pH of 3.0), and continue to react in a 40℃ constant temperature water bath for 10 min; after the reaction, add 2 mL of trichloroacetic acid solution (concentration of 65.4 g / L), shake well and let stand for 10 min to terminate the reaction, filter and take 1 mL of filtrate, add 5 mL of sodium carbonate solution (concentration of 42.4 g / L) and 1 mL of Folin reagent in sequence, mix well and place in a 40℃ constant temperature water bath for precise color development for 10 min; Blank group: Take 1.0 mL of the enzyme extract obtained in step A1, preheat in a 40℃ water bath for 2 min, add 2 mL of trichloroacetic acid solution, react at 40℃ for 10 min, then add 1 mL of casein solution (the casein solution corresponding to acidic protease has a pH of 3.0), shake well and let stand for 10 min, filter, take 1 mL of filtrate, add 5 mL of sodium carbonate solution and 1 mL of Folin reagent in sequence, and react at 40℃ for 10 min. Take 1 mL of the colorimetric solution from the experimental group and the blank group respectively into a 48-well plate, measure the absorbance value at a wavelength of 680 nm using an ELISA reader, and record the detection data; A5 Glycosyl Power Standard Curve Construction: Prepare glucose standard solutions with concentrations of 0.02, 0.06, 0.10, 0.14, 0.18, and 0.22 mg / mL. Take 2 mL of each solution and add 4.0 mL of DNS colorimetric reagent. Mix thoroughly and heat precisely in a boiling water bath for 5 min (start timing from boiling point). After heating, immediately cool to room temperature and dilute to 50.0 mL with distilled water. Take 1 mL of each solution and transfer it to a 48-well plate. Measure the absorbance at 550 nm using a microplate reader. Plot the glucose standard curve with absorbance as the ordinate and glucose concentration as the abscissa. The standard curve for calculating the reducing sugar concentration (mg / ml) is y = 2.043x + 0.03248, R0 2 =0.9934, where x is the glucose concentration (mg / ml) and y is the absorbance. When the glucose content needs to be known after measuring the absorbance value, the measured absorbance value can be substituted into the aforementioned equation, and the obtained x is the glucose concentration.

[0096] Plotting the liquefaction power standard curve: Prepare starch standard solutions with concentrations of 0.00, 0.15, 0.30, 0.45, 0.60, and 0.90 g / L. Take 1.0 mL of each solution and add 1 mL of iodine-potassium iodide solution to each solution. Mix well and develop color. Take 1 mL of each solution and transfer it to a 48-well plate. Measure the absorbance at 660 nm using a microplate reader. Plot the starch standard curve with starch concentration on the x-axis and absorbance on the y-axis.

[0097] The standard curve for calculating starch content (g / L) is y = 1.148x + 0.04131, R0 2 =0.991, where x is the starch concentration (g / l) and y is the absorbance. When the starch concentration needs to be known after measuring the absorbance value, the measured absorbance value can be substituted into the aforementioned equation, and the obtained x is the starch concentration. Plotting the standard curve for protease activity: Prepare tyrosine standard solutions with concentrations of 0, 10, 20, 30, 40, 50, and 60 μg / mL. Take 1.0 mL of each solution and add 5.00 mL of sodium carbonate solution and 1.00 mL of Folin reagent, respectively. After shaking and mixing, incubate in a 40℃ water bath for 10 min. Transfer 1 mL of each solution to a 48-well plate. Use a test tube without tyrosine as a blank control. Measure the absorbance at 680 nm using an ELISA reader. Plot the tyrosine standard curve with tyrosine concentration on the x-axis and absorbance on the y-axis.

[0098] The standard curve for calculating the tyrosine concentration (μg / mL) is y = 0.00996x - 0.01896, R0 2 =0.9956, where x is the tyrosine concentration (μg / mL) and y is the absorbance. When the tyrosine concentration needs to be known after measuring the absorbance value, the measured absorbance value can be substituted into the aforementioned equation, and the obtained x is the tyrosine concentration. A6 Result Calculation: The saccharification power of low-temperature Daqu is calculated according to the following formula; Formula 1, Saccharification power (mg / (g·h)) = reducing sugar concentration × N / (m / V1×T×V2 / V3); In the formula: the reducing sugar concentration (mg / ml) is obtained from the standard curve; N is the dilution factor; m is the mass of the oven-dried Daqu (g); V1 is the final volume of Daqu treatment (ml); T is the enzymatic hydrolysis reaction time (h); V2 is the volume of filtrate used in the enzymatic hydrolysis reaction (ml); V3 is the total volume of the enzymatic hydrolysis reaction (including the sodium hydroxide solution used to terminate the reaction, ml); Calculation results: The liquefaction power of low-temperature Daqu (a type of Chinese liquor) is calculated according to the following formula; Formula 2, liquefaction force = (W0 - W) / cvt; In the formula: W0 - W is the starch consumption during the reaction process (g / L) obtained by conversion from the standard curve; c is the concentration of enzyme extract (g / mL); v is the volume of enzyme extract added to the reaction system (mL); t is the enzyme-catalyzed reaction time (min); Results calculation: The protease activity of low-temperature Daqu was calculated according to the following formula; Formula 3, Protease activity (U / g) = ((A0-A)×V×4×n) / (10×W); In the formula: A0 is the tyrosine concentration of the test sample obtained from the tyrosine standard curve (μg / mL); A is the tyrosine concentration of the blank group measured from the tyrosine standard curve (μg / mL); V is the sample pretreatment volume (mL); 4 is the total volume of the colorimetric reaction reagent (mL); n is the enzyme solution dilution factor; 10 is the enzyme reaction time (min); W is the mass of the oven-dried Daqu sample participating in the reaction (g). B) Microbiological testing: Preparation of B1 lactic acid bacteria culture medium: Based on MRS, add 5 mg / L AMP-338 antimicrobial peptide, 1000 mg / L potassium sorbate and 10 mg / L nystatin to make a selective liquid culture medium for Daqu lactic acid bacteria; Preparation of B2 yeast culture medium: Add 5 mg / L LAB-129 antimicrobial peptide to YPD, and add corresponding organic acids at an acetic acid to lactic acid ratio of 1:3. The volume fraction of acetic acid is 0.1% and the volume fraction of lactic acid is 0.3%, that is, 1L of liquid culture medium contains 1ml of acetic acid and 3ml of lactic acid, which serves as a selective liquid culture medium for Daqu yeast. Preparation of B3 mold culture medium: Add 5 mg / L LAB-129 antimicrobial peptide and 50 g / L sodium chloride to the Bengal Red medium and adjust the pH to 5.5 to make it a selective liquid culture medium for Aspergillus macrocephala. B4. Plotting the standard curve: Prepare a suspension of *Aspergillus oryzae* and perform serial dilutions to obtain a concentration of 10... -1 -10 - 6 Six CFU / g gradients were used. Each gradient of bacterial suspension was inoculated into selective liquid culture media for lactic acid bacteria, yeast, and mold, respectively, and incubated at 35°C for 12 hours. The absorbance was measured at 600 nm using an ELISA reader. The bacterial suspensions were then diluted appropriately and spread onto the corresponding basal culture media plates for plate counting. A standard curve was plotted with absorbance on the x-axis and viable cell count on the y-axis. The linear relationship between the OD value and the colony count is as follows: for lactic acid bacteria: y = 4.462x + 2.731(R) 2 =0.9938), Yeast: y=3.960x+1.428(R) 2 =0.9906), Mold: y=3.465x+0.5086(R) 2 =0.9874). Where x is the OD value of the culture solution, and y is the number of colonies in the culture solution; when the colony count is needed after measuring the OD value of the culture solution, the measured OD value of the culture solution can be substituted into the aforementioned equation, and the obtained y is the colony count.

[0099] B5 Microbial Quantitative Detection: 1 ml of each of the three different selective liquid culture media was added to a 48-well plate. The *Aspergillus oryzae* suspension was diluted to an appropriate ratio, and 10 μL (1% inoculum) was added to the selective medium for lactic acid bacteria and molds. 20 μL (2% inoculum) was added to the selective liquid medium for yeast. This process was performed aseptically. After capping, the plates were incubated at 30°C. OD values ​​were measured using a microplate reader after 12 hours. 600 OD 600 By substituting the data into the standard curve, the number of core microorganisms in the Daqu (a type of starter culture) can be obtained.

[0100] The final results of the detection of three enzyme activities and three microorganisms at different stages of Daqu fermentation are shown in Table 1.

[0101] Table 1

[0102] Experimental Example 1 Comparison of enzyme activity detection effects at different sample pretreatment temperatures This experiment investigated the effects of different pretreatment temperatures on the saccharification power, liquefaction power, and protease activity of Daqu (a type of starter culture), and verified the optimal pretreatment temperature parameters.

[0103] Five temperature gradient groups were set up for the experiment. All operations were the same as in Example 1, with the only variable being that the pretreatment temperature of the Daqu samples was replaced by 30℃, 35℃, 45℃, and 50℃ instead of 40℃. The results and precision data of saccharification power, liquefaction power, and protease activity are shown in Tables 2, 3, and 4, respectively.

[0104] Table 2

[0105] Table 3

[0106] Table 4

[0107] Note: In the table, "maximum precision" refers to the largest value obtained by dividing the absolute difference between any two parallel test results by the arithmetic mean of the two parallel tests. The meaning of maximum precision in the following embodiments is the same.

[0108] The above experimental data show that the pretreatment temperature of Daqu samples directly affects the detection results of saccharification power, liquefaction power, and protease activity. Among them, 40℃ is the optimal pretreatment temperature. Under this condition, the detection values ​​of the three enzyme activities all reach their peak values, the enzyme extraction efficiency is optimal, the enzyme activity is most complete, and the corresponding maximum precision values ​​are significantly lower than those of other temperature groups. The accuracy, repeatability, and stability of the detection results all reach the optimal level.

[0109] Experimental Example 2 Comparison of enzyme activity detection effects with different sample pretreatment times This experiment investigated the effects of different extraction times (15, 30, 45, and 60 min) on the extraction efficiency of Daqu saccharification power, liquefaction power, and protease activity.

[0110] The experiment used a temperature gradient group, and all operations were the same as in Example 1. The only variable was that in step A1, the heat preservation and impregnation time for the Daqu sample pretreatment was replaced with 30 min, 45 min, and 60 min, respectively, instead of 15 min. The results are shown below. Figure 1 .in, Figure 1 In this context, 'a' represents saccharification power. Figure 1 In this context, 'b' represents the liquefaction force. Figure 1 In this context, 'c' represents protease activity.

[0111] Figure 1 The results showed that when the extraction time fluctuated within the range of 15-60 min, neither the saccharification power nor the liquefaction power exhibited significant differences (P>0.05; ns indicates no significant difference). The protease activity showed a unique pattern of first decreasing and then increasing with time; compared to the conventional extraction time (60 min), the enzyme activity decreased significantly at 30 min (P<0.05). However, there was no significant change in protease activity at 15 min and 45 min (P>0.05). Therefore, to improve extraction efficiency and minimize operation time, an extraction time of 15 min was selected as the optimal pretreatment time to save processing time to the greatest extent.

[0112] Experimental Example 3 Comparison of detection effects of different starch concentrations in saccharification power testing This experiment investigated the effect of different starch concentrations on the extraction efficiency of saccharification power in Daqu (a type of starter culture).

[0113] The experiment was conducted using a concentration gradient group, with all operations identical to those in Example 1. The only variable was that in step A2, the mass-volume concentration of the starch solution in the enzymatic reaction system was replaced with 1.0%, 1.5%, 2.5%, and 3.0%, respectively, instead of 2.0%. The results are shown in Table 5.

[0114] Table 5

[0115] The experimental data above show that the starch substrate concentration has a significant impact on the saccharification power detection results. Among them, when the mass-volume concentration of the starch solution is 2.0%, the saccharification power detection value reaches its peak, and the corresponding maximum precision value is the lowest among all groups. This indicates that the enzymatic reaction is sufficient and complete at this substrate concentration, and the accuracy, repeatability and stability of the detection results are optimal. This is the optimal starch substrate concentration for saccharification power determination.

[0116] Test Example 4 Comparison of detection effects of different colorimetric reaction times in saccharification power assay This experiment investigated the effect of different colorimetric reaction times on the extraction efficiency of saccharification power in Daqu (a type of starter culture).

[0117] The experiment was conducted using gradient groups, with all operations identical to those in Example 1. The only variable was step A2, where the colorimetric reaction time was replaced with 2 min, 3 min, 4 min, 6 min, and 7 min instead of 5 min. The results are shown below. Figure 2 .

[0118] Depend on Figure 2 It is evident that boiling time directly affects the colorimetric intensity of the reducing sugar and DNS reagent. As the boiling time increases from 1 min to 5 min, the absorbance continuously rises, reaching a peak at 5 min, indicating that the colorimetric reaction is complete at this point. Further extending the boiling time results in a slight decrease in absorbance, which then tends to stabilize. This may be due to excessive heating causing partial degradation of the colorimetric product, reducing detection stability. Therefore, 5 min is selected as the optimal boiling time, ensuring sufficient colorimetric development while significantly shortening detection time and improving high-throughput detection efficiency.

[0119] Experimental Example 5 Comparison of detection effects of different amounts of iodine-potassium iodide solution in liquefaction force testing This experiment investigated the effect of different amounts of iodine-potassium iodide solution on the extraction efficiency of Daqu liquefaction.

[0120] The experiment was conducted using a gradient group, with all operations identical to those in Example 1. The only variable was the amount of iodine-potassium iodide solution used in step A3, which was 0.1, 0.2, 0.5, 0.6, 0.7, 0.8, 0.9, 1.5, 1.8, 2.0, 2.5, 3.0, 3.5, and 4.0 ml, respectively, replacing 1 ml. The results are shown below. Figure 3.

[0121] The experimental data above show that the amount of iodine-potassium iodide solution has a significant impact on the liquefaction power test results. Among them, the optimal amount of iodine-potassium iodide solution is 1 mL, which results in stable color development and the highest absorbance, and significantly reduces the amount of reagent used.

[0122] Experimental Example 6 Comparison of enzyme activity detection effects at different enzyme reaction temperatures This experiment investigated the effect of enzymatic reaction temperature on the extraction efficiency of Daqu saccharification power, liquefaction power, and protease activity.

[0123] The experiment was conducted using gradient groups, with all operations identical to those in Example 1. The only variable was that in step A3, the temperature of the enzymatic reaction was replaced with 30℃, 40℃, 45℃, and 50℃, respectively, instead of 35℃. The results are shown below. Figure 4 .

[0124] The results showed that the activities of all three enzyme classes exhibited a "first increase, then decrease" evolution pattern with increasing immersion temperature. The saccharification power remained high at 40℃ (no significant difference from the national standard of 35℃, ns), reaching as high as 850±92 U at 35℃ (P<0.05). Both increasing and decreasing the temperature led to a significant decrease in saccharifying enzyme activity (P<0.05). The liquefaction power was approximately 0.4±0.03 U at 30℃, increasing to 0.6-0.7±0.02 U at 40℃, with no significant difference from the national standard of 35℃. Subsequently, with increasing temperature, the liquefaction enzyme activity decreased significantly. The protease activity was approximately 15±2 U at 30℃, increasing to 30 U at 40℃, a significant difference (P<0.01). With increasing temperature (45℃, 50℃), the enzyme activity decreased to 20±3 U and 10±1 U, respectively, with the optimal value at 40℃ consistent with the national standard of 40℃. The "increase followed by decrease" pattern of activity among the three key enzyme types all point to the optimal temperature range of 35-40℃ for the low-temperature Daqu enzyme system. Ultimately, considering all factors, the optimal temperature for the enzymatic reaction was chosen to be 35℃.

[0125] Experimental Example 7 Comparison of detection effects of different colorimetric reaction times in protease activity assay This experiment investigated the effect of different colorimetric reaction times on the extraction efficiency of Daqu protease activity during protease activity assay.

[0126] The experiment was conducted using a gradient group, with all operations identical to those in Example 1. The only variable was step A4, where the 40℃ constant temperature water bath color development time was replaced with 5 min, 15 min, 20 min, and 25 min, respectively, instead of 10 min. The results are shown in Table 6.

[0127] Table 6

[0128] The experimental data above show that the colorimetric reaction time has a significant impact on the protease activity detection results. When the colorimetric reaction time is 10 min, not only are the protease activity values ​​stable, but there is also no significant difference in enzyme activity values ​​compared to the group with longer colorimetric reaction times. Furthermore, under this condition, the maximum precision is the lowest among all groups, and the accuracy, repeatability, and stability of the detection results are optimal. Selecting 10 min as the optimal colorimetric reaction time can effectively shorten the detection time while ensuring detection accuracy, significantly improving overall detection efficiency and better meeting the actual needs of rapid detection in production settings.

[0129] Experimental Example 8 Comparison of Optimization Conditions for Microbial Detection Methods (1) Optimization of lactic acid bacteria culture medium The procedure for detecting the microbial content in Daqu (a type of starter culture) is the same as in Example 1. The difference lies in that, based on the MRS basal medium, the concentration of AMP-338 antimicrobial peptide is fixed, and the ratio of the compound antimicrobial agent (potassium sorbate, nystatin, and polymyxin B) is varied to construct a highly efficient and selective separation system suitable for low-temperature Daqu lactic acid bacteria. See Table 7.

[0130] The results are as follows Figure 5 As shown, different antimicrobial agent formulations significantly affected the survival abundance of target lactic acid bacteria and the inhibitory efficacy against non-target bacteria. Using the MRS blank control group without antimicrobial agents as a safety benchmark, this experiment effectively verified the potential toxicity of high concentrations of antimicrobial agents to lactic acid bacteria. Data showed that the control group without antimicrobial agents (no antimicrobial peptides, potassium sorbate, nystatin, or polymyxin B added) achieved the highest colony abundance among all samples, but this represented the total mixed bacterial population and did not have selective separation significance. Treatment groups 1 and 3 had the second highest number of lactic acid bacteria colonies, while treatment groups 7 and 8, due to excessively high antimicrobial agent concentrations, had significantly lower colony counts than the control group, confirming a significant "over-inhibition" phenomenon and should be excluded.

[0131] Based on comprehensive colony count, morphology, and safety verification, treatment group 1 (5 mg / L AMP-338 antimicrobial peptide + 1000 mg / L potassium sorbate + 10 mg / L nystatin) was determined to be the optimal formulation. This culture medium can effectively reduce contamination by other microorganisms, achieve targeted enrichment and efficient separation of target bacteria, and maximize the preservation of the activity and abundance of lactic acid bacteria. This provides a reliable experimental basis for the subsequent isolation, purification, community analysis, and functional study of lactic acid bacteria in low-temperature Daqu (a type of starter culture).

[0132] In the figure, WQ to YQ are sample abbreviations for 7 consecutive stages in the low-temperature fermentation process of Daqu, namely: WQ: initial fermentation stage (or startering stage); SM: mold growth stage (or temperature rise stage); LM: mold drying stage (or temperature control stage); CH: Daqu formation stage (or high temperature stage); DH: high-heat stage (or early post-heat stage); HH: post-heat stage (or early storage stage); YQ: finished Daqu stage (or Daqu exit stage).

[0133] Table 7. Antibacterial agent dosage for different treatment groups

[0134] (2) Optimization of yeast culture medium The procedure was the same as that for detecting the microbial content in Daqu in Example 1, except that, based on YPD, different concentrations of LAB-129 antimicrobial peptide and the antibiotic ampicillin were set, while the concentration of organic acid was fixed, to screen the optimal culture medium formulation suitable for the directional isolation of Daqu yeast. See Table 8.

[0135] The results are as follows Figure 6 As shown. The control group (without added antimicrobial agents) had the highest colony count among all samples due to the absence of selective pressure from antimicrobial peptides, antibiotics, and organic acids, representing the total biomass of the mixed microbial community and thus lacking selective purification significance. Comparing the treatment groups, treatment group D (5 mg / L LAB-129 antimicrobial peptide + acetic acid: lactic acid volume ratio of 1:3) exhibited the best overall performance, with significantly higher yeast colony counts throughout the entire fermentation stage (WQ to YQ) compared to groups A, B, and C, while also having the lowest background contamination value. The advantage of this formulation lies in the effective inhibition of Gram-positive and Gram-negative bacteria by LAB-129 antimicrobial peptides. Simultaneously, the synergistic effect of acetic acid and lactic acid allows acetic acid to effectively kill bacteria and inhibit molds, solving the problem of contamination, while lactic acid maintains pH stability, reducing acid stress and thus protecting the target yeast. Treatment group D achieved a dynamic balance of "targeted contamination inhibition and precise yeast protection," perfectly adapting to the complex microbial environment of the entire fermentation process and effectively avoiding the technical pain points of incomplete or excessive yeast inhibition by a single acid.

[0136] Therefore, treatment group D (5 mg / L LAB-129 antimicrobial peptide + acetic acid: lactic acid volume ratio of 1:3) was determined to be the optimal selective culture medium for screening low-temperature Daqu yeast. This formulation can effectively eliminate interference from non-target bacteria, achieve accurate isolation and counting of yeast at all stages, and provide standardized technical support for the exploration of Daqu yeast resources and community succession research.

[0137] Table 8. Antibacterial agent dosage for different treatment groups

[0138] (3) Optimization of mold culture medium The procedure was the same as that for detecting the microbial content of Daqu in Example 1. The difference was that, based on Bengal Red medium, the concentration of LAB-129 antimicrobial peptide was fixed, and different sodium chloride concentrations and pH values ​​were set as dual-factor environmental parameters, as shown in Table 9.

[0139] The results are as follows Figure 7 As shown, different combinations of salt concentrations and pH gradients significantly affected the isolation efficiency of molds and their ability to inhibit other microorganisms. The number of mold colonies showed a trend of "first increasing and then decreasing" with changing salt concentration. A sodium chloride concentration of 5 g / L effectively eliminated salt-intolerant microorganisms through high osmotic pressure, reducing nutrient competition and achieving targeted enrichment of the target bacteria. However, when the concentration increased to 60 g / L, the high osmotic stress significantly inhibited mold hyphal extension and spore germination, leading to a decline in colony abundance. Regarding pH optimization, pH 5.5 showed global optimality throughout the entire fermentation stage (WQ to YQ), avoiding the damage to mold enzyme systems caused by the strongly acidic environment of pH 4.5 and overcoming the insufficient inhibitory effect of the slightly neutral environment of pH 6.0, precisely matching the slightly acidic growth preference of *Aspergillus davidii*. Figure 3-11 The results of mold colony counting during the fermentation of Daqu with different salt concentrations and pH gradients further verified the superiority and universality of the optimized formula "50 g / L sodium chloride + pH 5.5" in the selective separation of Daqu molds at low temperature and its applicability throughout the entire process.

[0140] Table 9. Antimicrobial agent dosage for different treatment groups

[0141] (4) Optimization of optimal inoculum size and culture time for lactic acid bacteria, yeast and mold To clarify the growth time sequence characteristics of the core microorganisms in low-temperature Daqu (a type of starter culture), this study used a fully automated growth curve analyzer to monitor the dynamic changes of OD600 of lactic acid bacteria, yeast, and mold within 0-48 h. The results showed that ( Figure 8 The three core microorganisms exhibited significantly different growth timelines and stage characteristics in the low-temperature Daqu system, and formed a clear synergistic metabolic pattern among them. Specifically, lactic acid bacteria had a relatively delayed growth cycle and a slow logarithmic growth rate, with OD600 stabilizing at 1.0-1.3 after 12 hours and fluctuating slightly in the later stages; yeast showed a short lag phase and explosive logarithmic growth characteristics, entering a rapid proliferation phase after 12 hours, and OD600 stabilizing at 2.0-2.5 after 24 hours, making it the group with the highest biomass and fastest growth rate among the three groups; molds exhibited a slow lag phase and a steady logarithmic growth pattern, with a rapid proliferation phase from 6 to 12 hours, and OD600 maintaining at 1.5-1.8 after 12 hours, showing a continuous and stable metabolic growth trend; Growth was regulated using an optimized inoculum size strategy. The optimized growth curve shows ( Figure 9 Different inoculum sizes significantly regulate the lag phase, logarithmic phase duration, and stationary phase initiation time of yeast. 1% inoculum (control group): The lag phase lasts up to 12 hours, entering the logarithmic growth phase only after 12 hours. The OD600 growth rate only slows down after 16 hours, with the initial stationary phase occurring at 16-18 hours. The growth rate is slow, and the temporal lag is significant. 2% inoculum: The lag phase is shortened to 8 hours, entering the logarithmic phase after 8 hours. OD600 growth slows down at 12 hours, with the initial stationary phase occurring at 12 hours, essentially consistent with the stationary phase initiation time of fast-growing strains. 5% inoculum: The lag phase is further shortened to 4 hours, rapidly entering the logarithmic phase after 4 hours. The OD600 growth rate slows significantly between 10-14 hours, with the initial stationary phase occurring at 14 hours. 10% and 15% inoculum: The lag phase was almost eliminated, and the logarithmic phase began after 2 hours. OD600 entered the stable phase after 8-10 hours. Although the stable phase was entered earlier, the excessively high inoculum resulted in an excessively high initial cell density, which limited the growth rate during the logarithmic phase. In the later stage, OD600 was slightly lower than that of the 5% inoculum group, and the phenomenon of premature depletion of nutrients was likely to occur.

[0142] Based on the characteristics of the overall growth curve, an inoculum size of 2% is the optimal control condition: this inoculum size allows the yeast to enter the initial stage of the stationary phase synchronously at 12 h, ensuring both temporal synchronization with lactic acid bacteria and molds and maintaining sufficient logarithmic growth amplitude, resulting in stable growth and meeting the needs of subsequent experiments.

[0143] Experimental Example 9 Simultaneous detection and accuracy verification of saccharification power, liquefaction power, and protease activity in Daqu (a type of starter culture). Experimental samples: Ten batches of Daqu samples at different fermentation stages (numbered D1-D10) were selected, crushed, and stored in a 4℃ refrigerator for later use; Experimental group (method of the present invention): Same as Example 1, the total detection time of the experimental group is ≤45min.

[0144] Control group (National Standard Methods - QB / T 4257-2011, GB / T 23527-2009): The three enzyme activities were independently extracted and detected using the corresponding national standard methods. Glycolytic enzyme activity was detected according to QB / T 4257-2011 "General Analytical Methods for Brewing Daqu," with a single enzyme detection time of 3-4 hours; liquefaction enzyme activity was detected according to QB / T 4257-2011 "General Analytical Methods for Brewing Daqu," with a single enzyme detection time of 2-3 hours; protease activity was detected according to GB / T 23527-2009 "Protein Preparations," with a single enzyme detection time of 2-3 hours. The total detection time for the control group was 7-10 hours.

[0145] The results of three enzyme activities in 10 batches of Daqu samples are shown in Table 10.

[0146] Table 10

[0147] As shown in Table 10, the average relative deviation between the glycation power detected by the experimental group using the method of this invention and the control group using the national standard method QB / T4257-2011 was only 3.9%, and the paired t-test result showed P=0.62>0.05, indicating that there was no significant difference in the glycation power detection results between the two groups, which meets the verification requirements for the accuracy of the detection method. At the same time, the relative deviations of the detection results of liquefaction power and protease activity from the corresponding national standard methods were all ≤5%, and the P values ​​were all greater than 0.05, proving that the method of this invention has high reliability for the detection of the three enzyme activities.

[0148] In terms of detection efficiency, the control group required 7-10 hours to independently detect the three enzyme activities in 10 batches of samples, while the experimental group, through "multi-enzyme co-extraction - micro-color development - full plate reading", only took 45 minutes in total, improving the detection efficiency by 89.28%, significantly shortening the detection cycle. At the same time, it eliminated the reagents, consumables and operation steps required for multiple extractions, reduced the detection cost, and solved the pain points of the cumbersome and time-consuming multi-enzyme activity detection process in the existing technology.

[0149] Experimental Example 10 Microbial detection method of the present invention and accuracy verification Preparation of Daqu (a type of starter culture) suspension: Accurately weigh 10.0 g of Daqu sample, dilute to 100 mL with physiological saline from the microbial detection kit, place in a shaker at 37℃ and shake for 30 min, centrifuge and take the supernatant as Daqu suspension for later use; The detection method provided by this invention; Selective liquid culture media: MRS medium + 5 mg / L AMP-338 antimicrobial peptide + 1000 mg / L potassium sorbate + 10 mg / L nystatin; YPD medium + 5 mg / L LAB-129 antimicrobial peptide + acetic acid and lactic acid in a 1:3 ratio (g / L); Bengal red medium + 5 mg / L LAB-129 antimicrobial peptide + 50 g / L sodium chloride + pH adjusted to 5.5; Add 1 mL of each of the three selective liquid culture media to the corresponding wells of a 48-well plate. Dilute the Aspergillus flavus suspension to a suitable concentration, and inoculate 10 μL of the 1% inoculum into the wells of the lactic acid bacteria and mold selective culture media, respectively. Inoculate 20 μL of the 2% inoculum into the wells of the yeast selective culture media. After capping, incubate at 35℃ for 12 h. Measure the OD of each well using a microplate reader. 600 By substituting the values ​​into the corresponding standard curve, the number of target core microorganisms in the Daqu (a type of starter culture) can be obtained. The results are shown in Table 8.

[0150] Compared with traditional flatbed counting: Common culture media: MRS medium; YPD medium; Bengal red medium; Take the bacterial culture from the above 48-well plates, dilute it appropriately, spread it on the above three ordinary culture medium plates, and count the plates after incubation.

[0151] The results are shown in Table 11.

[0152] Table 11

[0153] Experimental Example 11 Spiked quantitative detection validates method specificity Preparation of Daqu (a type of starter culture) suspension: Accurately weigh 10.0 g of Daqu sample, dilute to 100 mL with physiological saline from the microbial detection kit, place in a shaker at 37℃ and shake for 30 min, centrifuge and take the supernatant as Daqu suspension for later use; Preparation of spiked sample bacterial suspension: Take another equal amount of the above bacterial cells, that is, accurately weigh 20.0 g of Daqu sample, make up to 100 mL with physiological saline from the microbial detection kit, place in a shaker at 37℃ and shake for 30 min, centrifuge and take the supernatant as Daqu bacterial suspension for later use; Quantitative detection of microorganisms: See Example 1 for specific procedures; The results of the spiked quantitative test are shown in Table 12.

[0154] Table 12

[0155] Experimental Example 12 Specific inhibitory effect of adding LAB-129 antimicrobial peptide to selective yeast culture medium Preparation of Daqu (a type of starter culture) suspension: Accurately weigh 10.0 g of Daqu sample, dilute to 100 mL with physiological saline from the microbial detection kit, place in a shaker at 37℃ and shake for 30 min, centrifuge and take the supernatant as Daqu suspension for later use; Prepare the corresponding culture media according to the experimental groups, spread the above-mentioned *Aspergillus oryzae* suspension on each group in triplicate, and incubate at 30℃ for 48 hours. Observe the colony morphology, record the growth status using "+", "-", and "±", count the colonies, and calculate the growth rate of target bacteria and non-target bacteria. The experimental groups are as follows: 1. CK0 (blank control): YPD medium + acetic acid and lactic acid in a 1:3 ratio; 2. CK1 (antibiotic control): YPD medium + acetic acid and lactic acid in a 1:3 ratio + 200 mg / L ampicillin; 3. T1-T4 (LAB-129 experimental group): YPD medium + acetic acid and lactic acid in a 1:3 ratio + 1, 3, 5, and 7 mg / L LAB-129; The experimental results are shown in Table 13.

[0156] Table 13

[0157] Note: "±" indicates a growth rate of 10%-80%; "+" indicates a growth rate of ≥80%; "-" indicates a growth rate of ≤10%.

[0158] The results showed that LAB-129 antimicrobial peptides could precisely target and inhibit non-target bacteria in yeast selective culture media, while exhibiting good protective effects on the target yeast. Its specificity is mainly reflected in its ability to distinguish between target and non-target bacteria, inhibiting only non-target bacteria, and the inhibitory effect is concentration-dependent. Compared with traditional antibiotics (ampicillin), LAB-129 has advantages such as high specificity, good protective effect on target bacteria, and flexible concentration adaptability, effectively solving the problems of target bacteria damage and inaccurate inhibition of other bacteria caused by the broad-spectrum inhibition of traditional antimicrobial agents. The optimal application concentration of LAB-129 was 5 mg / L, at which concentration, complete inhibition of non-target bacteria (growth rate ≤10%) and normal growth of the target Saccharomyces cerevisiae (growth rate ≥80%) were achieved. This not only meets the requirement of "inhibiting contamination" in yeast selective culture but also ensures the growth activity of the target bacteria, providing new technical support for the optimization of yeast selective culture media and further demonstrating the application value and unique advantages of LAB-129 in yeast culture.

[0159] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid method for detecting the quality of Daqu (a type of starter culture), comprising the detection of multiple key enzyme activities and microbial content, characterized in that, Includes the following steps: The multiple key enzyme activity assays include: extracting the Daqu sample once with an acetate-sodium acetate buffer or a sodium lactate buffer at pH 4.6 to obtain a Daqu enzyme extract suitable for detecting glycation power, liquefaction power, and protease activity simultaneously; then using a 48-well plate with three channels to simultaneously detect glycation power, liquefaction power, and protease activity; and calculating the glycation power, liquefaction power, and protease activity of the sample using a standard curve. The microbial content detection includes: inoculating the Daqu sample into selective liquid culture media suitable for lactic acid bacteria, yeast, and mold, respectively, and culturing them; simultaneously detecting the absorbance values ​​after culturing using an enzyme-linked immunosorbent assay (ELISA) reader; and calculating the content of each microorganism by combining the results with a standard curve. Preparation of selective liquid culture medium for lactic acid bacteria: Based on MRS, add 3~6 mg / L AMP-338 antimicrobial peptide, 1000~2000 mg / L potassium sorbate, and 10~14 mg / L nystatin to make selective liquid culture medium for Daqu lactic acid bacteria. Preparation of selective liquid culture medium for yeast: Based on YPD, add 3~6 mg / L LAB-129 antimicrobial peptide, and add organic acid at a volume ratio of acetic acid to lactic acid of 1:3, wherein the volume fraction of acetic acid is 0.1% and the volume fraction of lactic acid is 0.3%, as the selective liquid culture medium for Daqu yeast. Preparation of selective liquid culture medium for molds: Add 3~6 mg / L LAB-129 antimicrobial peptide and 5~60 g / L sodium chloride to the Bengal Red medium and adjust the pH to 4.0~7.0 to prepare selective liquid culture medium for Aspergillus macrocephala.

2. The rapid detection method according to claim 1, characterized in that, The specific detection of the multiple key enzyme activities includes: A1 Daqu sample pretreatment: Preparation of enzyme extract for Daqu enzyme activity test: Add an acetate-sodium acetate buffer solution or a sodium lactate buffer solution with pH 4.6 to the Daqu sample, make up to volume, filter, and collect the filtrate, which is the Daqu enzyme extract; A2 Saccharification power colorimetric reaction: Take the Daqu enzyme extract obtained in A1, add starch solution to carry out the enzymatic reaction, add NaOH solution to terminate the enzymatic reaction, and obtain the reaction solution; take the obtained reaction solution, add 3,5-dinitrosalicylic acid colorimetric reagent, and carry out the colorimetric reaction in a boiling water bath to obtain the test solution; A3 Liquefaction Power Colorimetric Reaction: In the experimental group, the starch solution was preheated, and then the Daqu enzyme extract obtained in A1 was added to the preheated starch solution to carry out an enzymatic reaction, resulting in a reaction solution. The blank control group followed the same operating steps as above, except that the Daqu enzyme extract was replaced with an equal volume of acetate-sodium acetate buffer solution at pH 4.

6. Iodine-potassium iodide solution was added to the reaction solutions of both the experimental group and the blank control group, and the mixture was stirred to carry out a colorimetric reaction, resulting in the test solution. A4 protease activity colorimetric reaction: In the experimental group, the Daqu enzyme extract obtained from A1 was preheated, and then casein solution was added for enzymatic reaction. The reaction was terminated by adding trichloroacetic acid solution. After filtration, sodium carbonate solution and Folin reagent were added to the filtrate in sequence, and a colorimetric reaction was carried out at an isothermal temperature to obtain the test solution. In the blank group, the Daqu enzyme extract obtained from A1 was preheated, and then trichloroacetic acid solution was added for enzymatic reaction. Then casein solution was added, mixed well, filtered, and then sodium carbonate solution and Folin reagent were added to the filtrate in sequence, and a colorimetric reaction was carried out at an isothermal temperature to obtain the test solution. A5 microplate reader absorbance values: A 48-well plate was used for three-channel partitioned reaction, with each channel corresponding to the detection of glycation power, liquefaction power and protease activity. The test solutions obtained from the A2 color reaction, A3 color reaction and A4 color reaction were respectively taken into the corresponding channels of the 48-well plate, and the three channels were detected simultaneously. The absorbance values ​​of each reaction well were recorded in sequence. A6 Standard Curve Plotting: Prepare a series of standard solutions of corresponding concentrations for three detection indicators: glycation power, liquefaction power, and protease activity. The standard for glycation power detection is glucose solution, the standard for liquefaction power detection is starch solution, and the standard for protease activity detection is tyrosine solution. Take each series of standard solutions of different concentrations and incubate and develop them according to the enzyme activity detection conditions corresponding to A2, A3, and A4, respectively. Plot the standard curves for glucose, starch, and tyrosine with the concentration of the standard solution on the x-axis and the absorbance value on the y-axis. A7 uses the absorbance value of the test solution measured in A5 to substitute into the glucose standard curve, starch standard curve, and tyrosine standard curve obtained in A6 to calculate the content of the corresponding product or substrate in the test solution; then, the saccharification power, liquefaction power, and protease activity of Daqu are calculated using the corresponding formulas.

3. The rapid detection method according to claim 1, characterized in that, The microbial content detection specifically includes: B4. Plotting the standard curve: The *Aspergillus oryzae* suspension was serially diluted to obtain a 10⁻⁶ standard curve. -1 ~10 -6 The bacterial suspensions were diluted at various ratios and inoculated into selective liquid culture media for lactic acid bacteria, yeast, and mold, respectively. The absorbance values ​​were measured using an ELISA reader. After incubation, the bacterial suspensions were diluted and spread onto the corresponding basal culture media plates for plate counting. Standard curves were plotted with absorbance values ​​on the x-axis and the viable cell count on the y-axis to obtain standard curves for lactic acid bacteria, yeast, and mold, respectively. B5 Microbial Quantitative Detection: A 48-well plate was used for three-channel partitioned reactions, with each channel corresponding to the detection of lactic acid bacteria, yeast, and mold, respectively. Three different selective liquid culture bases were taken and added to the selective culture media of lactic acid bacteria, yeast, and mold, respectively, in the 48-well plate. After covering the plate, the plates were incubated in an incubator for 12-15 h. The absorbance values ​​of each reaction well were recorded simultaneously using an ELISA reader. Finally, the absorbance values ​​were substituted into the standard curves of lactic acid bacteria, yeast, and mold, respectively, to calculate the number of different microorganisms in the ELISA.

4. The rapid detection method according to claim 2, characterized in that, A1 satisfies at least one of the following conditions: (1) The ratio of Daqu sample to buffer solution is 2.5 g: 4~6 ml; then the volume is adjusted to 50 mL; (2) It also includes, after adjusting the volume, shaking, and then placing it in a constant temperature water bath at 37~43℃ for immersion for 15-20 minutes. (3) Preparation of Daqu samples includes crushing the Daqu blocks and sieving them for later use; and / or A2 satisfies at least one of the following conditions: (1) The volume ratio of Daqu enzyme extract to starch solution is 1:4~6, wherein the mass-volume concentration of starch solution is 1.8~2.2g / 100ml; (2) The conditions for the enzyme-catalyzed reaction are to react in a constant temperature water bath at 35~40℃ for 15~20 min; (3) The volume ratio of Daqu enzyme extract to NaOH solution is 1:0.5, wherein the mass-volume concentration of NaOH solution is 20g / 100ml; (4) The volume ratio of the reaction solution to the 3,5-dinitrosalicylic acid colorimetric reagent is 2:4~6; (5) Boiling water bath color development reaction for 5-6 minutes; (6) also includes the following steps: after the colorimetric reaction in a boiling water bath, the mixture is cooled and then diluted to a fixed volume.

5. The rapid detection method according to claim 2, characterized in that, A3 satisfies at least one of the following conditions: (1) The volume ratio of starch solution to Daqu enzyme extract is 1:10, wherein the mass-volume concentration of starch solution is 1.8~2.2 g / 100ml; (2) The preheating treatment is specifically preheating at 35℃ for 8 to 10 minutes; (3) The enzyme-catalyzed reaction is specifically carried out at 35-40℃ for 15-18 minutes; (4) The volume ratio of the reaction solution to the iodine-potassium iodide solution is 1:1~2; and / or A4 meets at least one of the following conditions: (1) The volume ratio of Daqu enzyme extract, casein solution, and trichloroacetic acid solution is 1:1:2~3; (2) The preheating treatment is specifically heating in a 40℃ constant temperature water bath for 2~3 minutes; (3) The enzyme-catalyzed reaction is specifically carried out in a constant temperature water bath at 35~40℃ for 10 min; (4) The volume ratio of filtrate, sodium carbonate solution, and Folin reagent is 1~2:5:1; (5) The constant temperature color development reaction is specifically carried out in a constant temperature water bath at 40℃ for 10 min.

6. The rapid detection method according to claim 2, characterized in that, A5 meets at least one of the following conditions: (1) The detection wavelength for saccharification power is 550 nm, the detection wavelength for liquefaction power is 660 nm, and the detection wavelength for protease activity is 680 nm. (2) The three-channel partitioned reaction using a 48-well plate is as follows: a 48-well plate is used to carry out the three-channel partitioned reaction, and a single 48-well plate is divided into three independent channels along the longitudinal direction, each channel containing 16 reaction wells.

7. The rapid detection method according to claim 2, characterized in that, A7 satisfies at least one of the following conditions: (1) Formula 1, Saccharification power (mg / (g·h)) = reducing sugar concentration × N / (m / V1×T×V2 / V3); Wherein: reducing sugar concentration (mg / ml) is obtained from glucose standard curve; N is dilution factor; m is the mass of oven-dried Daqu (g); V1 is the final volume of Daqu treatment (ml); T is enzyme reaction time (h); V2 is the volume of filtrate used in enzyme reaction (ml); V3 is the total volume of enzyme reaction (including sodium hydroxide solution used to terminate the reaction, ml); (2) Formula 2, liquefaction force = (W0-W) / cvt; Where: W0 is the starch content of the test sample calculated from the starch standard curve (g / L); W is the starch content of the blank group calculated from the starch standard curve (g / L); c is the concentration of enzyme extract (g / mL); v is the volume of enzyme extract added to the reaction system (mL); t is the enzyme-catalyzed reaction time (min). (3) Formula 3, protease activity (U / g) = ((A0-A) × V×4 × n) / (10 × W); Where: A0 is the tyrosine concentration of the test sample obtained from the tyrosine standard curve (μg / mL); A is the tyrosine concentration of the blank group measured from the tyrosine standard curve (μg / mL); V is the sample pretreatment volume (mL); 4 is the total volume of the colorimetric reaction reagent (mL); n is the enzyme solution dilution factor; 10 is the enzyme-catalyzed reaction time (min); W is the mass of the oven-dried Daqu sample participating in the reaction (g).

8. The rapid detection method according to claim 3, characterized in that, B4 meets at least one of the following conditions: (1) The absorbance value was measured at a wavelength of 600 nm using an ELISA reader; (2) The culture was carried out at a constant temperature of 30℃ for 12-15 h; (3) Preparation of Daqu (fermented starter culture) suspension: Take a Daqu sample, dilute it to volume with physiological saline, mix, centrifuge, and take the supernatant, which is the Daqu suspension; and / or B5 meets at least one of the following conditions: (1) Divide a single 48-well plate into three independent channels along the longitudinal direction, each channel containing 16 reaction wells; (2) Add the *Aspergillus oryzae* suspension to selective culture media for lactic acid bacteria, yeast, and mold, respectively, wherein the inoculum amount of *Aspergillus oryzae* suspension is 1-3%; (3) The specific culture is to place the container in an incubator at 25-40℃ after covering it with a lid.

9. A kit for the rapid detection method of multiple key enzyme activities and microbial content in Daqu (a type of Chinese liquor) according to any one of claims 1 to 8.

10. The reagent kit according to claim 9, characterized in that, include: 1) The core components of the enzyme activity assay kit adapted to the above detection methods include: ① Sample pretreatment components: Acetic acid-sodium acetate buffer solution or sodium lactate buffer solution at pH 4.6; ② Enzyme activity detection substrate system Substrate for saccharification power assay: starch solution with a mass-volume concentration of 1.8–2.2 g / 100 ml; Substrate for liquefaction force testing: starch solution with a mass-volume concentration of 1.8~2.2 g / 100ml; Protease activity assay substrate: 10 g / L casein solution; ③ Reaction color development and termination system Colorimetric reagents: 3,5-dinitrosalicylic acid colorimetric reagent, iodine-potassium iodide solution, sodium carbonate solution, and Folin reagent; Termination reagents: NaOH solution and trichloroacetic acid solution with a mass-volume concentration of 20 g / 100 ml; ④ Standard curve plotting components: glucose standard, starch standard, tyrosine standard; ⑤ Detection carrier and auxiliary components: 48-well plate, deionized water; 2) The core components of the microbial detection kit adapted to the above detection methods include: ① Sample pretreatment components: sterile physiological saline; ② Selective culture medium system Selective liquid culture medium for lactic acid bacteria: Based on MRS medium, add 3~6 mg / L AMP-338 antimicrobial peptide, 1000~2000 mg / L potassium sorbate, and 10~14 mg / L nystatin; Selective liquid culture medium for yeast: Based on YPD medium, add 3~6 mg / L LAB-129 antimicrobial peptide, and add acetic acid and lactic acid in a volume ratio of 1:3, wherein the volume fraction of acetic acid is 0.1% and the volume fraction of lactic acid is 0.3%. Selective liquid culture medium for molds: Based on Bengal Red liquid culture medium, add 3~6 mg / L LAB-129 antimicrobial peptide, 5~60 g / L sodium chloride and adjust the pH to 4.0~7.0; ③ Detection carrier and auxiliary components: 48-well plate, pH adjuster.

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