Acetic fermentation process control method based on PQQ

Abstract

The invention provides a PQQ-based acetic fermentation process control method, which comprises the following steps: firstly, carrying out acetic fermentation to obtain a fermented beverage; measuring the PQQ content Vp and the acetic acid content V1 in the fermented beverage, taking the PQQ content Vp and the acetic acid content V1 as acetic acid fermentation markers, obtaining a fermentation index m according to the Vp and the V1, and obtaining an enzyme activity factor K according to the m; finally, the fermentation degree is judged according to the size of the enzyme activity factor K, when K is larger than 50%, it is judged that fermentation is over-fire, fermentation parameters are adjusted, and the step S1 is executed again for acetic fermentation; when K is smaller than 25%, fermentation is insufficient, fermentation parameters are adjusted, and acetic fermentation is conducted again; when K is larger than or equal to 25% and smaller than or equal to 50%, the fermentation degree is appropriate, and the acetic acid fermentation process control method based on PQQ is completed. The fermentation coenzyme and the acetic acid concentration are used as control parameters of the fermentation process, so that the control difficulty of the acetic acid fermentation process is greatly reduced, and the control efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of vinegar technology, and more specifically to a method for controlling the acetic acid fermentation process based on PQQ. Background Technology

[0002] Acetic acid fermentation is a type of oxidative fermentation, which refers to the process by which ethanol is oxidized into acetic acid by acetic acid bacteria.

[0003] Acetobacter is a Gram-negative bacterium with oval, rod-shaped cells that can be solitary, paired, or chain-like. Abnormal forms are easily observed in older cultures. It may or may not have peritrichous flagella. Acetobacter oxidizes ethanol to acetic acid and glucose to gluconic acid. Commonly used strains, such as Zhongke 1.41 and Huniang 1.01, are both genus Acetobacter.

[0004] The main mechanism of acetic acid fermentation is as follows: the oxidation of ethanol to acetic acid can be divided into the following two stages: (1) Ethanol is first oxidized to acetaldehyde under the catalysis of alcohol dehydrogenase. (2) Acetaldehyde absorbs water to form hydrated acetaldehyde, which is then oxidized to acetic acid by acetaldehyde dehydrogenase. Combining the above equations, the entire reaction equation is: CH3CH2OH + O2 → CH3COOH + H2O; In theory, 100g of alcohol can produce 130.4g of pure acetic acid, but in actual production, only 100g of acetic acid can be produced.

[0005] Acetic acid bacteria are aerobic bacteria, and fermentation is carried out under aeration conditions. The fermentation temperature is generally 25-30℃. Acetic acid bacteria require amino acids and vitamins, so in addition to ethanol, yeast juice, koji juice or other nitrogen-containing organic matter need to be added to the fermentation broth.

[0006] Detailed analysis of the acetic acid bacteria fermentation process 1. Ethanol fermentation period: TCA cycle is inhibited. - Main pathway: Ethanol → Acetaldehyde → Acetic acid (catalyzed by membrane-bound alcohol dehydrogenase and acetaldehyde dehydrogenase).

[0007] - Energy efficiency: Each molecule of ethanol oxidized produces 1 molecule of acetic acid, generating only 1 ATP (by simplifying the electron transport chain).

[0008] - Carbon flow direction: No acetyl-CoA is generated, so it does not enter the TCA cycle.

[0009] - Regulatory mechanism: High concentrations of ethanol and acidic environments (pH < 4) can inhibit key enzymes in the TCA cycle, such as α-ketoglutarate dehydrogenase.

[0010] - Prioritize the use of carbon sources for acetic acid synthesis (the target product for industry).

[0011] 2. The potential role of the TCA cycle - Stages of bacterial growth: - When utilizing carbon sources such as glucose and lactate, the TCA cycle is active, providing energy (ATP) and biosynthetic precursors (such as oxaloacetate).

[0012] - When ethanol is depleted: Some acetic acid bacteria initiate excessive acetic acid oxidation (through the acetyl-CoA → TCA cycle → CO2), but Zhongke 141 may weaken this pathway through selective breeding in order to maintain high acid production.

[0013] It is clear that acetic acid bacteria can only produce PQQ during the ethanol fermentation period, and the TCA cycle is inhibited.

[0014] Traditionally, acetic acid is considered a marker product of acetic acid bacteria. However, acetic acid can only measure the acidity of acetic acid drinks and cannot reflect the suitability of the acetic acid fermentation process. Therefore, a method is needed to measure the fermentation process and apply it to control the acetic acid fermentation process. Summary of the Invention

[0015] This invention aims to solve the control problem of acetic acid fermentation process and provides a PQQ-based method for controlling the acetic acid fermentation process. First, acetic acid fermentation is carried out to obtain a fermented beverage; then, the PQQ content (V) in the fermented beverage is measured. p And the acetic acid content V1, and the PQQ content V p Acetic acid content V1 is used as a marker of acetic acid fermentation, based on V... p The fermentation index m is obtained from V1, and then the enzyme activity factor K is obtained based on m. Finally, the fermentation degree is judged based on the magnitude of the enzyme activity factor K. When K > 50%, it is judged as over-fermentation, and the fermentation parameters are adjusted and acetic acid fermentation is repeated. When K < 25%, fermentation is insufficient, and the fermentation parameters are adjusted and acetic acid fermentation is repeated. When 25% ≤ K ≤ 50%, the fermentation degree is suitable. A method for controlling the acetic acid fermentation process based on PQQ is thus completed. This invention uses fermentation coenzyme and acetic acid concentration as control parameters for the fermentation process, which greatly reduces the difficulty of controlling the acetic acid fermentation process and improves the control efficiency.

[0016] This invention provides a method for controlling acetic acid fermentation process based on PQQ, comprising the following steps: S1. Perform acetic acid fermentation to obtain a fermented beverage; S2. Measure the PQQ content (V) in fermented beverages. p And the acetic acid content V1, and the PQQ content V p Acetic acid content V1 is used as a marker of acetic acid fermentation, based on V... pThe fermentation index m is obtained from V1, and then the enzyme activity factor K is obtained based on m; ; K= Where MOD is the remainder function, K is the remainder of the ratio of m to 20000, and K is expressed as a percentage; S3. Determine the degree of fermentation based on the value of enzyme activity factor K. When K > 50%, it is determined that the fermentation is over-fermented. Adjust the fermentation parameters and return to step S1 to repeat the acetic acid fermentation. When K < 25%, fermentation is insufficient. Adjust the fermentation parameters and return to step S1 to repeat the acetic acid fermentation. When 25%≤K≤50%, the fermentation degree is suitable, and a PQQ-based method for controlling the acetic acid fermentation process is completed.

[0017] The method for controlling the acetic acid fermentation process based on PQQ described in this invention, as a preferred embodiment, is that the fermented beverage in step S1 is a beverage obtained by introducing acetic acid bacteria ignition by mixing fermented liquor with fermentation base. In step S1, the acidity of the fermented beverage is above 0.8 degrees.

[0018] In the preferred embodiment of the acetic acid fermentation process control method based on PQQ described in this invention, in step S3, when K > 50%, the design value of the amount of fermented mash introduced is reduced, and the process returns to step S1. When K < 25%, increase the design value of the amount of fermented mash fed into the furnace and return to step S1.

[0019] The method for controlling the acetic acid fermentation process based on PQQ described in this invention, as a preferred approach, involves reducing the design value of the amount of fermented mash introduced and / or reducing the fermentation time when K > 50%.

[0020] The method for controlling the acetic acid fermentation process based on PQQ described in this invention, as a preferred embodiment, is that the fermented beverage in step S1 is any one of the following: buckwheat fermented beverage, sea buckthorn fermented beverage, and kudzu root fermented beverage.

[0021] The method for controlling the acetic acid fermentation process based on PQQ described in this invention, in a preferred embodiment, includes the following steps in step S1: S11. After enzymatic hydrolysis, enzyme inactivation, hot vortex sedimentation, and hot water top mash, yeast is introduced for fermentation to obtain cultured wine. S12. After grinding kudzu root into powder, kudzu root is obtained. Water, kudzu root, and malt are enzymatically hydrolyzed, liquefied, and cooled together, and then Daqu powder is added for fermentation to obtain kudzu root wine. S13. After diluting the alcohol content of the cultured wine, it is connected to the fruit vinegar generator. Then, acetic acid bacteria subsp. pasteurianum and acetic acid bacteria turbid variant are added. Kudzu root wine is continuously added until the acidity exceeds the specified degree. Then, it is divided to obtain fire mash wine. S14. Mix buckwheat hulls, kudzu root, kudzu root wine and fire mash wine together so that the acetic acid bacteria ignition can be directly introduced into the fermentation substrate. When mixing the mash, the moisture content, weight and oxygen content of the mash are controlled to adjust the acetic acid bacteria to the state with the fastest ignition time to obtain the mash. S15. Stir and mix to form a mash, allowing acetic acid bacteria to ferment and PQQ to accumulate until fermentation is complete, resulting in mature kudzu vinegar mash. S16. Extract the mature kudzu root vinegar mash at least twice at a temperature above 85 degrees Celsius, retain PQQ in the vinegar liquid until the acidity reaches the final specified acidity level to obtain the fermented beverage. In step S3, the enzyme activity factor K is adjusted by adjusting the mass percentage of the fermented liquor in step S14.

[0022] In the preferred embodiment of the acetic acid fermentation process control method based on PQQ described in this invention, in step S11, the alcohol content of the cultured wine is 5.0~6.0 degrees and the amino nitrogen content is greater than or equal to 0.1g / 100ml. In step S12, the alcohol content of the kudzu root wine is 4.0-4.5 degrees. In step S13, the alcohol content of the cultured wine is diluted to 4.0 degrees and then introduced into the fruit vinegar generator, and the acidity of the fermented wine is greater than 2 degrees. In step S14, buckwheat hulls, kudzu root, kudzu root wine, and fire mash wine are mixed together in a mass percentage ratio of 100:30:100:30. In step S16, the final acidity is specified as 0.8 degrees.

[0023] In the preferred embodiment of the acetic acid fermentation process control method based on PQQ described in this invention, the PQQ content is measured by spectrometry in step S2.

[0024] Alcohol dehydrogenase and aldehyde dehydrogenase are catalysts secreted by acetic acid bacteria during their activity. They are the most efficient biochemical agents for converting substances from ethanol to acetic acid, allowing microorganisms to absorb energy during this conversion process. Alcohol dehydrogenase and aldehyde dehydrogenase are the main enzymes involved in their activity. Their activity requires the participation of coenzymes to function properly, or the coenzyme determines the intensity of their activity. That is, without the main enzyme, the coenzyme alone cannot function, and without the coenzyme, the main enzyme is inactive and cannot convert substances. Coenzymes are small molecules that are acid-resistant, heat-resistant, and not easily inactivated. This coenzyme is PQQ, short for pyrroloquinoline quinone.

[0025] PQQ's unique molecular structure gives it the physicochemical advantages of vitamin C (reduction potential), riboflavin (redox reaction), and pyridoxal (carbonyl activity), and its redox potential is at least 100 times higher than that of vitamin C, menadione, and all isoflavones and polyphenols.

[0026] Redox reaction systems can repeatedly catalyze redox reactions, and the number of catalytic cycles depends in part on the chemical stability of the catalytic molecule. PQQ is a small quinone molecule with a relatively stable chemical structure, while many bioactive quinones or enediols (such as vitamin C) are less stable due to self-oxidation, polymerization, and changes in chemical structure. Its stable chemical structure and high redox potential make PQQ the most potent bioactive molecule for catalyzing redox reactions to date.

[0027] PQQ catalyzes up to 20,000 redox reactions, far exceeding the 4 reactions of vitamin C, and is 5,000 times that of vitamin C.

[0028] PQQ catalyzes up to 20,000 redox reactions. At its highest biological concentration, the ratio of acetic acid to PQQ is 20,000 times. When the acetic acid fermentation concentration reaches 1 g / 100 ml, the concentration of PQQ is only 0.5 ug / ml.

[0029] The fermentation index m was obtained by comparing VPQQ with Vacetic acid. The fermentation index m was then compared with 20000, and the remainder was taken to obtain the enzyme activity factor K, expressed as a percentage. The enzyme activity factor K reflects enzyme activity; a K value between 25% and 50% indicates stronger activity (when a certain fermentation concentration is reached, the bacterial fermentation concentration is 10...). 4 Rise to 10 7 (The concentration of acetic acid bacteria secreted enzymes reaches equilibrium). The fermentation concentration of acetic acid bacteria (acetic acid) can be estimated and compared with the concentration of PQQ used. During the growth phase of the culture, the number of acetic acid bacteria increases from 10... 4 Rise to 10 7 During this growth stage, acetic acid bacteria obtain energy through the tricarboxylic acid cycle and do not produce PQQ. The bacterial count rises to 10-1. 7 At this stage, acetic acid bacteria obtain energy by converting ethanol into acetic acid. The tricarboxylic acid cycle within the cell stops, and PQQ coenzyme combines with the main dehydrogenase to convert ethanol into acetic acid for energy. Only then can PQQ coenzyme migrate from the cell into the fermentation broth.

[0030] Therefore, in a deeper sense, acetic acid bacteria utilize enzymes to complete the material transformation, and coenzyme PQQ is the hallmark product of acetic acid bacteria fermentation.

[0031] The present invention has the following advantages: The dominant indicator of acetic acid bacteria fermentation is the production of acetic acid, while the latent indicator is the conversion of alcohol into acetic acid by dehydrogenases using a biochemical method. Dehydrogenases include a main enzyme and a coenzyme. Because the main enzyme is a large protein molecule, it is easily inactivated during its production, while the coenzyme is a small molecule, heat-resistant, acid-resistant, and structurally stable, and is retained during the conversion process. Therefore, the latent indicator of acetic acid bacteria fermentation is the production of fermentation coenzymes, and the concentrations of both are correlated with the degree of fermentation. Therefore, using the concentrations of fermentation coenzymes and acetic acid as control parameters for the fermentation process significantly reduces the difficulty of controlling acetic acid fermentation and improves control efficiency. Attached Figure Description

[0032] Figure 1 This is a flowchart of a PQQ-based method for controlling acetic acid fermentation. Figure 2 This is a diagram illustrating the PQQ content test in a PQQ-based acetic acid fermentation process control method. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Example 1

[0034] like Figure 1 As shown, a method for controlling acetic acid fermentation based on PQQ includes the following steps: S1. Acetic acid fermentation is carried out to obtain fermented beverages; in this embodiment, 1# is a buckwheat fermented beverage, 2# is a sea buckthorn fermented beverage, and 3# is a kudzu root fermented beverage. S2. Measure the PQQ content (V) of the fermented beverage. p And the acetic acid content V1, and the PQQ content V p Acetic acid content V1 is used as a marker of acetic acid fermentation, based on V... p The fermentation index m is obtained from V1, and then the enzyme activity factor K is obtained based on m; ; MOD is the modulo function, which expresses K as a percentage and takes only two decimal places.

[0035] Wherein, the unit of V1 and V p Convert to the same unit; The results are as follows: #1 Fermented Beverage: PQQ Content (V) p The concentration is 30 ng / ml; the acetic acid content V1 is 1.02 g / 100 ml; Substitute... In the middle, we get =340000; Substitute K= = In the middle, we get K=0; #2 Fermented Beverage: V p 45 ng / ml; V1 = 0.95 g / 100 ml; Substitute into In the middle, we get m=211111.11, substituting it into K= In the middle, we get K=56%; #3 Fermented Beverage: V p The value is 38 ng / ml; V1 = 0.90 g / 100 ml; substituting... In the middle, we get m=236842.11, substituting it into K= We obtained K=84%. S3. Determine the fermentation degree based on the enzyme activity factor K. For fermented beverage #1, K is 0, indicating a complete fermentation cycle with no active enzymes, suggesting over-fermentation. For fermented beverage #2, K is 56%, greater than 25% and slightly greater than 50%, indicating slightly over-fermentation. For fermented beverage #3, K is 84%, close to 80%, indicating over-fermentation; reduce the fermentation time appropriately. Example 2

[0036] like Figure 1 As shown, a method for controlling acetic acid fermentation based on PQQ includes the following steps: S1. Perform acetic acid fermentation to obtain a fermented beverage; Step S1 includes the following steps: S11. Preparation of cultured wine: Cultured wine is obtained by enzymatic hydrolysis, enzyme inactivation, hot vortex sedimentation, hot water top mash, and then inoculation with yeast for fermentation. The alcohol content of cultured wine is 5.0~6.0 degrees and the amino nitrogen content is greater than or equal to 0.1g / 100ml. S12. Preparation of kudzu root wine: After grinding kudzu root into powder, it is liquefied by enzymatic hydrolysis of water, kudzu root powder, and malt, cooled, and then fermented with daqu powder. The alcohol content of the kudzu root wine is 4.0-4.5 degrees. S13. Mix buckwheat hulls (100% by mass), kudzu root (blocks) (30% by mass), kudzu root wine (100% by mass), and simmered liquor (30% by mass) together. Directly introduce acetic acid bacteria into the fermentation substrate. During mixing, control the moisture content, weight, and oxygen content of the mash to adjust the acetic acid bacteria to the state with the fastest ignition time to obtain the mash. The weight of the fermented mash is 20-25 kg. In this embodiment, the weight of the fermented mash is 20 kg, and the weight of the mash that can be mixed is 260 kg. The automatic mixing time is half an hour, the fermentation start-up time is 8 hours, and the mash temperature increases from 18 degrees Celsius during mixing to 32 degrees Celsius. S14. Stir the mash to allow acetic acid bacteria to ferment. During the fermentation process, PQQ is enriched until the fermentation is completed to obtain mature kudzu vinegar mash. S15. The mature kudzu vinegar mash is extracted at least twice at a temperature above 85 degrees Celsius. During the extraction process, PQQ is retained in the vinegar liquid until the acidity reaches above 0.8 degrees Celsius, thus obtaining a fermented kudzu beverage.

[0037] S2. Measure the PQQ content (V) in kudzu root fermented beverage. p And the acetic acid content V1, and the PQQ content V p Acetic acid content V1 is used as a marker of acetic acid fermentation, based on V... p The fermentation index m is obtained from V1, and then the enzyme activity factor K is obtained based on m; ; K= ; where only the percentages after the decimal point are taken.

[0038] In this embodiment, K=84%.

[0039] S3. Determine the degree of fermentation based on the value of enzyme activity factor K. If K > 50%, the fermentation is over-fermented, so adjust and reduce the fermentation time. Adjustment Plan 1: Adjust the amount of fermented mash introduced in step S13 to 22 kg, and calculate the fermentation time as 8 hours before ignition. Subtract the 30 minutes of time spent igniting earlier from the total fermentation time, return to step S1, and re-ferment to obtain the PQQ content V. p The concentration is 35 ng / ml; the acetic acid content V1 is 0.95 g / 100 ml; substituting these values, we get: m = 271428.57, K = 57%, K is greater than 50%, so adjust for overheating.

[0040] Adjustment Plan 2: In step S13, adjust the amount of fermented mash added to 25 kg, while maintaining the total fermentation time at 24 hours. Return to step S1 and re-ferment to obtain the PQQ content V. p The concentration is 40 ng / ml; the acetic acid content V1 is 1.62 g / 100 ml; substituting these values, we get: m = 405000, K = 25%. The K value is greater than 25% and less than 50%, so the adjustment was successful.

[0041] In Examples 1 and 2, the detection method for pyrroloquinoline quinone (PQQ) using the ELISA detection kit is as follows: Detection principle The kit employs a competitive enzyme-linked immunosorbent assay (ELISA). Samples, standards, and HRP-labeled competitive antigen are added sequentially to microwells pre-coated with pyrroloquinoline quinone antigen, followed by incubation and thorough washing. The substrate TMB is used for color development; TMB is converted to blue by peroxidase, and then to yellow under acidic conditions. The color intensity is negatively correlated with the amount of pyrroloquinoline quinone (PQQ) in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader to calculate the sample concentration.

[0042] Sample collection, processing and preservation methods: 1. For each 25ml bottle of fermented buckwheat beverage, after opening the cap, draw up the liquid using a 25ml syringe, filter it using a 0.4um filter needle, and collect the filtrate into a beaker.

[0043] 2. Purchase standard fermented natto products from Tokyo Department Store. Take 10g of fermented natto and repeatedly soak it in 100ml of distilled water to dissolve it. Filter it using a 0.4um filter needle and collect the filtrate in a beaker.

[0044] 1# is the sample to be tested, and 2# is the standard sample.

[0045] Items to bring: ELISA reader (450nm) High-precision pipettes and tips: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL 37℃ constant temperature chamber Operating Precautions: Store the kit at 2-8℃ and allow it to equilibrate to room temperature for 20 minutes before use. Crystallization may occur in the concentrated washing solution taken from the refrigerator; this is normal. Heat in a water bath to completely dissolve the crystals before use.

[0046] Unused strips should be immediately returned to the resealable bag and stored in a sealed (low-temperature, dry) container.

[0047] When operating according to the instructions, the sample has been diluted 5 times. The final result is obtained by multiplying by 5 to get the actual concentration of the sample.

[0048] Strictly follow the incubation procedure as indicated in the instructions regarding the time, volume, and order of liquid addition.

[0049] Shake well before use for all liquid components.

[0050] reagent kit composition

[0051] Note: The concentrations of the standards (S0-S5) are, in order: 0, 0.5, 0.1, 2, 4, and 8 ppb. Preparation of reagents: Dilution of 20× Wash Buffer: Dilute with distilled water at a ratio of 1:20, that is, 1 part of 20× Wash Buffer to 19 parts of distilled water.

[0052] Board washing method: Manual plate washing: Shake off the liquid in the wells, fill each well with washing solution, let stand for 1 minute, shake off the liquid in the wells, and pat dry on absorbent paper. Repeat this washing process 5 times.

[0053] Automatic plate washer: Inject 350μL of washing solution into each well, soak for 1 minute, and wash the plate 5 times.

[0054] Operating steps: Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a self-sealing bag and return them to 4℃.

[0055] Set up standard wells and sample wells, and add 50 μL of standard at different concentrations to each standard well; Add 10 μL of the sample to be tested to the sample well first, then add 40 μL of sample diluent; do not add any to the blank well.

[0056] Except for the blank wells, add 50 μL of horseradish peroxidase (HRP)-labeled competitive antigen to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 min.

[0057] Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used).

[0058] Add 50 μL of substrate A and 50 μL of substrate B to each well and incubate at 37°C in the dark for 15 min.

[0059] Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0060] Result judgment: 1. Read the OD value of each well on a microplate reader with a wavelength of 450 nm within 15 minutes; 2. Percentage Binding Rate Calculation: Let the count of S0 tubes be B0, the count of each standard tube or sample tube be B, and the count of non-specific tubes be NSB. Then, the percentage binding rate is calculated as follows: B / B0 = (B - NSB) / (B0 - NSB) × 100% 3. Logit Calculation: The formula for calculating the logit value of each standard point or sample tube is as follows: logit = ln(B / B0) / (1-B / B0) 4. Divide the mean OD value of the standard by the mean OD value of the standard at point 0 to get the percentage binding rate of the standard point, and plot it on log-logit graph paper.

[0061] 5. Log-logit double logarithmic standard curve: On the graph paper, the first 1-9 from left to right represents the first decimal place, the second 1-9 represents the second decimal place, and the third 1-9 represents the third decimal place. The vertical axis of the graph paper represents percentages (1-99), that is, the percentage binding rate of each standard absorbance value. Draw a straight line passing through all points. The goal is to have as many points as possible on the line, with the remaining points evenly distributed on both sides of the line. Similarly, calculate the percentage binding rate of the sample from its absorbance value, and then find the point on the line corresponding to the binding rate on the vertical axis. The concentration on the horizontal axis corresponding to this point is the concentration of the sample; no conversion is needed.

[0062] 6. Manual Processing: Plot a standard curve (ideally a straight line) on logit-log graph paper with the standard concentration as the x-axis and the corresponding logit value as the y-axis. The concentration of the sample can be determined from the B / B0 value on the graph paper. If using ordinary graph paper, the inverse of the calculated value should be taken to obtain the final concentration value.

[0063] 7. Automatic processing: The computer automatically calculates the results using logit-log or four-parameter data processing modes.

[0064] 8. Sensitivity: 0.1 ppb 9. Legend: For example Figure 2 As shown.

[0065] Reagent kit performance: Accuracy: The correlation coefficient R between the linear regression of the standard and the expected concentration is greater than or equal to 0.9900.

[0066] Sensitivity: The minimum detectable concentration is less than 0.1 ppb.

[0067] Detection range: 0.1 - 8 ppb Specificity: It does not cross-react with other soluble structural analogs.

[0068] Repeatability: The coefficients of variation within and between plates are both less than 15%.

[0069] Storage: Store at 2-8℃, away from light and moisture.

[0070] Validity period: 6 months.

[0071] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for controlling the acetic acid fermentation process based on PQQ, characterized in that: Includes the following steps: S1. Perform acetic acid fermentation to obtain a fermented beverage; S2. Measure the PQQ content (V) of the fermented beverage. p And the acetic acid content V1, and the PQQ content V p Acetic acid content V1 is used as a marker of acetic acid fermentation, based on V... p The fermentation index m is obtained from V1, and then the enzyme activity factor K is obtained based on m; ； K= Where MOD is the modulo function, and K is expressed as a percentage; S3. Determine the degree of fermentation based on the value of enzyme activity factor K. When K > 50%, it is determined that the fermentation is over-fermented. Adjust the fermentation parameters and return to step S1 to repeat the acetic acid fermentation. When K < 25%, fermentation is insufficient. Adjust the fermentation parameters and return to step S1 to repeat the acetic acid fermentation. When 25%≤K≤50%, the fermentation degree is suitable, and a PQQ-based method for controlling the acetic acid fermentation process is completed.

2. The method for controlling the acetic acid fermentation process based on PQQ according to claim 1, characterized in that: The fermented beverage mentioned in step S1 is a beverage obtained by introducing acetic acid bacteria into the fermentation process after mixing the fermented liquor with the fermentation base. In step S1, the acidity of the fermented beverage is above 0.8 degrees.

3. The method for controlling the acetic acid fermentation process based on PQQ according to claim 2, characterized in that: In step S3, when K > 50%, reduce the design value of the amount of fermented mash fed into the machine and return to step S1; When K < 25%, increase the design value of the amount of fermented mash fed into the furnace and return to step S1.

4. The method for controlling the acetic acid fermentation process based on PQQ according to claim 3, characterized in that: When K > 50%, reduce the design value of the amount of fermented mash introduced and / or reduce the fermentation time.

5. The method for controlling the acetic acid fermentation process based on PQQ according to claim 2, characterized in that: The fermented beverage mentioned in step S1 is any one of the following: buckwheat fermented beverage, sea buckthorn fermented beverage, and kudzu root fermented beverage.

6. The method for controlling the acetic acid fermentation process based on PQQ according to claim 2, characterized in that: Step S1 includes the following steps: S11. After enzymatic hydrolysis, enzyme inactivation, hot vortex sedimentation, and hot water top mash, yeast is introduced for fermentation to obtain cultured wine. S12. After grinding kudzu root into powder, kudzu root is obtained. Water, kudzu root, and malt are enzymatically hydrolyzed, liquefied, and cooled together, and then Daqu powder is added for fermentation to obtain kudzu root wine. S13. After diluting the alcohol content of the cultured wine, it is connected to the fruit vinegar generator, and then acetic acid bacteria subsp. pasteuriana and acetic acid bacteria turbid variant are added. The kudzu root wine is continuously added until the acidity exceeds the specified degree, and then it is divided to obtain fire mash wine. S14. Buckwheat hulls, kudzu root, kudzu root wine and fire mash wine are mixed together so that the acetic acid bacteria ignition source is directly introduced into the fermentation substrate. During mixing, the moisture content, weight and oxygen content of the mash are controlled to adjust the acetic acid bacteria to the state with the fastest ignition time to obtain the mash. S15. Stir the mash to allow acetic acid bacteria to ferment and PQQ to accumulate until fermentation is complete, thus obtaining mature kudzu vinegar mash. S16. The mature kudzu vinegar mash is extracted at least twice at a temperature above 85 degrees Celsius, and PQQ is retained in the vinegar liquid until the acidity reaches the final specified acidity to obtain the fermented beverage. In step S3, the enzyme activity factor K is adjusted by adjusting the mass percentage of the fermented liquor mentioned in step S14.

7. The method for controlling the acetic acid fermentation process based on PQQ according to claim 6, characterized in that: In step S11, the alcohol content of the cultured wine is 5.0~6.0 degrees and the amino nitrogen content is greater than or equal to 0.1g / 100ml; In step S12, the alcohol content of the kudzu root wine is 4.0-4.5 degrees. In step S13, the alcohol content of the cultured wine is diluted to 4.0 degrees and then introduced into the fruit vinegar generator. The acidity of the fermented wine is greater than 2 degrees. In step S14, buckwheat hulls, kudzu root, kudzu root wine, and fire mash wine are mixed together in a mass percentage ratio of 100:30:100:

30. In step S16, the final specified acidity is 0.8 degrees.

8. The method for controlling the acetic acid fermentation process based on PQQ according to claim 1, characterized in that: In step S2, the PQQ content is measured by spectrophotometry.

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