A synergistic fermentation process for salt-free marinade preparation

Abstract

The present application relates to the technical field of fermentation engineering, and discloses a synergistic fermentation method for preparing salt-free marinade, which comprises the following steps: adjusting the protein content of a salt-free fermentation stock solution and applying low-frequency pulse ultrasonic treatment; inoculating an acid-producing initial strain and monitoring the pH value and viscosity attenuation rate of the fermentation system; when the pH value decreases to the critical interval of the isoelectric point of the protein, a metabolic inhibition regulation stage is constructed by using a cooling medium, so that the acid-producing strain enters a dormant state and the endogenous depolymerase catalytic activity is maintained; and when the viscosity reaches a release threshold, a flavor-producing and peptide-producing strain is introduced. The present application realizes the separation of the acid-producing metabolism of microorganisms and the structural deconstruction of the substrate on the time axis, avoids the physical lock of flavor precursors caused by the isoelectric point hydrophobic condensation of the salt-free substrate, and effectively improves the conversion efficiency of amino acid nitrogen.

Description

Technical Field

[0001] This invention relates to a co-fermentation method for preparing salt-free marinade, belonging to the field of fermentation engineering technology. Background Technology

[0002] In current production practices of healthy ingredients, salt-free fermentation has become the mainstream method to avoid the risk of high sodium content. The conventional operation process establishes a low pH chemical barrier in the substrate by inoculating acid-producing strains such as Lactobacillus plantarum, which replaces the traditional osmotic suppression mechanism and promotes the dissolution of organic matter in the substrate.

[0003] However, under actual operating conditions, this acidification process and the substrate degradation process have a deep-seated physical mechanism conflict. The solubility of proteins in salt-free matrices is highly dependent on hydration. When the acidification rate is too fast, the pH value of the system rapidly approaches the isoelectric point of the proteins. Under conditions where the charge shielding effect of salt ions is absent, the protein polypeptide chains generate intense hydrophobic interactions and lead to irreversible physical coagulation. This process constructs a dense physical gel network, locking the soluble sugars and flavor precursors required for fermentation within the network. Even with linear improvement measures such as increasing the inoculum size of acid-producing strains or raising the fermentation temperature, the physical locking of the substrate structure is often exacerbated by further accelerating the acidification rate. This spatiotemporal coupling constraint between acidification inhibition and substrate degradation is the cause of the poor performance of salt-free fermentation. The fundamental reason for low bioconversion efficiency and unstable flavor quality is that the strengthening of the fermentation physical field and the precise control of metabolic phase are the core factors determining the conversion efficiency of the product. For example, Chinese invention patent application CN104286803A discloses a method for preparing salt-free krill compound seasoning sauce by multi-strain fermentation. It constructs a temperature gradient to drive multi-strain compound fermentation and follows the linear heating logic to adapt to the enzyme production kinetics of molds. However, when faced with a high-protein-concentration salt-free liquid matrix, the linear heating logic cannot separate the acid production metabolism and enzymatic degradation process on the time axis. When the system enters the isoelectric point sensitive range, it lacks a mechanism to instantaneously inhibit the physiological activity of acid-producing strains. The matrix undergoes hydrophobic condensation before the enzymatic hydrolysis is fully completed, which limits the conversion efficiency of amino acid nitrogen in the final product and causes fluctuations in flavor quality.

[0004] Therefore, how to achieve asynchronous and precise intervention of the timing of proton secretion metabolism and substrate spatial deconstruction during salt-free fermentation has become the technical problem to be solved by this invention. Summary of the Invention

[0005] To address the problems in the background art, the technical solution of the present invention is as follows: A co-fermentation method for preparing salt-free brine, comprising the following steps:

[0006] Step S101: Prepare a salt-free fermentation stock solution with a composite substrate of plants and animals, and adjust the protein mass percentage in the salt-free fermentation stock solution to 5% to 12%;

[0007] Step S102: Apply low-frequency pulsed ultrasound treatment at a frequency of 20kHz to 28kHz to the salt-free fermentation broth to break the cell walls of the plant raw materials to release intracellular fermentable sugars and obtain a fermentable extract.

[0008] Step S103: Inoculate the acid-producing initiating strain into the fermentable lysate, start primary fermentation at a first fermentation temperature of 30°C, and monitor the dynamic pH value and apparent viscosity of the fermentable lysate during fermentation, and calculate the viscosity decay slope.

[0009] Step S104, enter the metabolic inhibition and regulation stage: when the dynamic pH value drops to 4.6 to 4.8 and the viscosity decay slope is lower than the preset substrate degradation critical slope, a cooling medium is injected into the fermentation system to adjust the fermentation broth temperature from the first fermentation temperature to a dormancy residence temperature of 10°C to 12°C. The dormancy residence temperature is used to block the proton secretion metabolism of the acid-producing initiating strain and maintain the protein degradation activity of the endogenous depolymerase in the fermentation broth.

[0010] Step S105: Under the condition of maintaining the dormancy residence temperature, monitor the real-time apparent viscosity of the fermentation broth. When the real-time apparent viscosity reaches the release threshold of 30% to 40% of the initial viscosity, inoculate the aroma-producing and peptide-producing strains into the fermentation broth and raise the temperature to 25°C to 30°C to complete the co-fermentation.

[0011] Preferably, in step S102, the power density of the low-frequency pulsed ultrasound treatment is adjusted to 50 W / L, and the pulse working cycle is 10 s on and 5 s off. During the process of breaking the cell wall of the plant raw material, the dissolution rate of reducing sugar in the fermentation broth is adjusted by the cavitation effect generated by the pulse working cycle, and the duty cycle of the pulse working cycle is positively mapped to adjust the initial suspended matter concentration of the salt-free fermentation broth, so as to provide a carbon source basis for the rapid proliferation of the acid-producing initiating strain in step S103.

[0012] Preferably, in step S101, the plant material in the plant-animal composite matrix includes at least one of soybean, black bean, or peanut; by limiting the protein mass percentage, a substrate thermodynamic buffer space is constructed for the metabolic inhibition regulation stage in step S104.

[0013] Preferably, in step S104, the release threshold is established based on the depth of macromolecular depolymerization before the pH value drops to the isoelectric point of the protein; under the dormant residence temperature environment, the endogenous depolymerization enzyme is used to deconstruct the protein structure to avoid hydrophobic coagulation in the isoelectric point region of the fermentation broth.

[0014] Preferably, in step S105, while inoculating the aroma-producing and peptide-producing strain, halophilic tetracoccus is also inoculated; the aroma-producing and peptide-producing strain includes Zygomyces rouxii, which acts on the protein fragments deconstructed in step S104 to transform them into flavor peptides.

[0015] Preferably, in step S104, the injection flow rate of the cooling medium is controlled to a cooling rate of not less than 2°C / min, and the acid production metabolism of the acid-producing initiating strain and the substrate decomposition of the endogenous depolymerase are separated on the time axis through rapid temperature field transition.

[0016] Preferably, the total duration of co-fermentation is 72h to 120h; wherein, the duration of the metabolic inhibition regulation phase in step S104 is 12h to 24h, so that the mass percentage of small molecule peptides in the fermentation broth is not less than 5%.

[0017] Preferably, after step S105 is completed, step S106 is further included, in which the fermentation product is centrifuged and the liquid phase is collected to obtain a salt-free brine with an amino acid nitrogen content of not less than 0.6 g / 100 mL.

[0018] Preferably, the process also includes step S1061, which involves instantaneous high-temperature sterilization of the salt-free marinade at a temperature of 115°C to 120°C for a duration of 3 to 5 seconds to complete aseptic filling.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. In the co-fermentation of salt-free marinade preparation, an asynchronous gating mechanism based on fluid dynamic viscosity and proton accumulation rate is constructed to analyze and resolve the physical conflict between the acid production and antibacterial process and the substrate degradation process under salt-free fermentation conditions. In the extreme environment where sodium chloride is absent to provide osmotic pressure buffer, the rapid acid production in the primary fermentation stage causes the acidity of the system to rapidly approach the isoelectric point of proteins, inducing irreversible hydrophobic coagulation of macromolecular proteins, thereby physically locking flavor precursors in a dense gel network. This invention, by real-time monitoring of the logical correlation between viscosity decay slope and proton accumulation slope, when the risk of protein coagulation is identified, forces intervention through a temperature step to block the proton secretion metabolism of microorganisms, thereby securing a critical substrate deconstruction window for endogenous depolymerases on the time axis, achieving deep decoupling between fermentation microecological safety and substrate spatial release.

[0021] 2. This invention utilizes the differential sensitivity of thermodynamic boundary conditions to microbial metabolic pathways and endogenous enzymatic reaction kinetics to achieve precise phase regulation of the fermentation process. After triggering the acid stagnation buffer sequence, the system temperature is lowered to a dormant residence temperature of 10 to 12 degrees Celsius. This temperature is precisely within the critical inhibition zone of acid production metabolism in Lactobacillus plantarum, while still maintaining the catalytic activity of proteases in the fermentation broth. This active intervention method based on the temperature field changes the traditional fermentation process that relies on linear accumulation over time and passively waits for microbial succession. By artificially constructing a metabolic stagnation period, it ensures that the strains introduced in the secondary flavor conversion stage can directly act on the depolymerized open substrate structure, thus guaranteeing the conversion efficiency of flavor peptides and amino acids.

[0022] 3. This invention deeply couples low-frequency pulsed ultrasonic physical field with the logic of rapid acid production by microorganisms to construct a composite microecological safety barrier under salt-free conditions. The cavitation effect of the ultrasonic physical field mechanically breaks down the cell walls of the matrix, accelerating the dissolution rate of intracellular reducing sugars and providing the carbon source basis required for the explosive proliferation of primary acid-producing strains. This physical enhancement method forms a synergistic effect with the low-pH chemical barrier rapidly established by Lactobacillus plantarum, effectively compensating for the inherent defects of the salt-free system in lacking the osmotic suppression mechanism. The forced decoupling of the ultrasonic treatment step and the primary inoculation step in time not only gives full play to the amplifying effect of the physical field on substrate solubilization, but also avoids the destruction of the vitality of the initiating strain by physical energy, ensuring the absolute dominance of the dominant bacterial group in the early stage of fermentation. Attached Figure Description

[0023] Figure 1 This is a flow chart of the synergistic fermentation process for preparing the salt-free pickling juice of the present invention;

[0024] Figure 2 This is a state transition diagram of the asynchronous control of acidification and antibacterial action and substrate deconstruction in this invention.

[0025] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0026] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0027] A co-fermentation method for preparing salt-free brine includes the following steps:

[0028] Step S101: Prepare a salt-free fermentation stock solution with a composite substrate of plants and animals, and adjust the protein mass percentage in the salt-free fermentation stock solution to 5% to 12%;

[0029] Step S102: Apply low-frequency pulsed ultrasound treatment at a frequency of 20kHz to 28kHz to the salt-free fermentation broth to break the cell walls of the plant raw materials to release intracellular fermentable sugars and obtain a fermentable extract.

[0030] Step S103: Inoculate the acid-producing initiating strain into the fermentable lysate, start primary fermentation at a first fermentation temperature of 30°C, and monitor the dynamic pH value and apparent viscosity of the fermentable lysate during fermentation, and calculate the viscosity decay slope.

[0031] Step S104, enter the metabolic inhibition and regulation stage: when the dynamic pH value drops to 4.6 to 4.8 and the viscosity decay slope is lower than the preset substrate degradation critical slope, a cooling medium is injected into the fermentation system to adjust the fermentation broth temperature from the first fermentation temperature to a dormancy residence temperature of 10°C to 12°C. The dormancy residence temperature is used to block the proton secretion metabolism of the acid-producing initiating strain and maintain the protein degradation activity of the endogenous depolymerase in the fermentation broth.

[0032] Step S105: Under the condition of maintaining the dormancy residence temperature, monitor the real-time apparent viscosity of the fermentation broth. When the real-time apparent viscosity reaches the release threshold of 30% to 40% of the initial viscosity, inoculate the aroma-producing and peptide-producing strains into the fermentation broth and raise the temperature to 25°C to 30°C to complete the co-fermentation.

[0033] Preferably, in step S102, the power density of the low-frequency pulsed ultrasound treatment is adjusted to 50 W / L, and the pulse working cycle is 10 s on and 5 s off. During the process of breaking the cell wall of the plant raw material, the dissolution rate of reducing sugar in the fermentation broth is adjusted by the cavitation effect generated by the pulse working cycle, and the duty cycle of the pulse working cycle is positively mapped to adjust the initial suspended matter concentration of the salt-free fermentation broth, so as to provide a carbon source basis for the rapid proliferation of the acid-producing initiating strain in step S103.

[0034] Preferably, in step S101, the plant material in the plant-animal composite matrix includes at least one of soybean, black bean, or peanut; by limiting the protein mass percentage, a substrate thermodynamic buffer space is constructed for the metabolic inhibition regulation stage in step S104.

[0035] Preferably, in step S104, the release threshold is established based on the depth of macromolecular depolymerization before the pH value drops to the isoelectric point of the protein; under the dormant residence temperature environment, the endogenous depolymerization enzyme is used to deconstruct the protein structure to avoid hydrophobic coagulation in the isoelectric point region of the fermentation broth.

[0036] Preferably, in step S105, while inoculating the aroma-producing and peptide-producing strain, halophilic tetracoccus is also inoculated; the aroma-producing and peptide-producing strain includes Zygomyces rouxii, which acts on the protein fragments deconstructed in step S104 to transform them into flavor peptides.

[0037] Preferably, in step S104, the injection flow rate of the cooling medium is controlled to a cooling rate of not less than 2°C / min, and the acid production metabolism of the acid-producing initiating strain and the substrate decomposition of the endogenous depolymerase are separated on the time axis through rapid temperature field transition.

[0038] Preferably, the total duration of co-fermentation is 72h to 120h; wherein, the duration of the metabolic inhibition regulation phase in step S104 is 12h to 24h, so that the mass percentage of small molecule peptides in the fermentation broth is not less than 5%.

[0039] Preferably, after step S105 is completed, step S106 is further included, in which the fermentation product is centrifuged and the liquid phase is collected to obtain a salt-free brine with an amino acid nitrogen content of not less than 0.6 g / 100 mL.

[0040] Preferably, the process also includes step S1061, which involves instantaneous high-temperature sterilization of the salt-free marinade at a temperature of 115°C to 120°C for a duration of 3 to 5 seconds to complete aseptic filling.

[0041] Example 1: In the specific production conditions of salt-free brine using a composite plant-animal matrix, the system faces the risk of spoilage microbial proliferation due to the lack of an osmotic barrier provided by sodium chloride. A salt-free fermentation stock solution with a protein content of 12% was prepared. The ratio of soybean protein to peanut protein was set at 3:1 to 4:1 by mass. Based on the amphoteric electrolyte dissociation equilibrium, a thermodynamic base was constructed where the hydrophobic co-condensation critical point of the composite matrix converged within the pH range of 4.6 to 4.8. A cold shock pretreatment of 4°C to 6°C was applied to the mixed matrix and maintained for 2 hours to expand the pores of plant cell walls and directionally release cold-adapted endogenous acidic proteases, ensuring the subsequent release of endogenous depolymerizing enzymes within 10 minutes. The pretreatment step, which utilizes the vacuolar membrane of soybean and peanut cells encapsulating this type of endogenous enzyme to a specific phase transition response to an extremely low temperature environment of 4℃ to 6℃, enables the selective release of peptide cleavage catalytic activity under extreme dormancy conditions from ℃ to 12℃. This rapid cooling causes the fluidity of the vacuolar membrane lipid bilayer to fall to the freezing point and undergo microscopic phase separation. The microscale lipid membrane gap leaks generated by the surface tension reconstruction only allow low molecular weight endogenous depolymerases to permeate and enter the external fermentation broth, while high molecular weight globulins with large spatial configurations and other unnecessary hybrid intracellular macromolecules are physically trapped in situ due to excessive steric hindrance.

[0042] A material transfer pump with a length-to-diameter ratio of 40:1 is used to introduce the salt-free fermentation broth into the ultrasonic treatment unit. Low-frequency pulsed ultrasound treatment with a frequency of 25kHz and a power density of 50W / L is applied to the broth, with a pulse cycle of 10 seconds on and 5 seconds off. This process breaks down the cell walls of the plant material and releases intracellular fermentable sugars, thereby obtaining a fermentable extract. During the pulsed ultrasound treatment, an online turbidimeter at the system's front end simultaneously acquires the initial suspended solids concentration of the fermentation broth. When the suspended solids concentration exceeds the calibration baseline, the control unit executes a preset command. The linear mapping compensation algorithm uses a 1% increase in suspended solids concentration as the step unit, corresponding to a 0.5s extension of the ultrasonic on-time of the driving pulse cycle. A 5s off-rest period is consistently locked within this adjustment sequence. This dynamically extends the absolute time of energy radiation, overcoming the shielding effect of high-concentration solid particle clusters on sound wave attenuation. An acid-producing initiating strain, *Lactobacillus plantarum*, is introduced into the fermentable lysate. The primary fermentation process is initiated at a first fermentation temperature of 30℃. Online sensors are used to collect real-time dynamic pH and apparent viscosity data of the fermentable lysate. And calculate the viscosity decay slope. When the dynamic pH value decreases to 4.7 and the viscosity decay slope... When the absolute value of the viscosity is lower than the preset critical slope for substrate degradation, the system is determined to have entered a risk state of protein isoelectric point coagulation. At this time, the system triggers an acid stagnation buffer sequence, introduces cooling water into the fermentation equipment jacket, and controls the temperature of the fermentable lysate to decrease from 30°C to the dormant residence temperature of 11°C at a rate of 3°C / min. This temperature range is used to inhibit the proton secretion metabolic pathway of *Lactobacillus plantarum*, while maintaining the catalytic activity of endogenous depolymerases in the fermentable lysate on substrate protein macromolecules; real-time apparent viscosity is continuously monitored. Real-time apparent viscosity When the concentration dropped to 35% of the initial level, the acid stagnation buffer sequence was released, and a flavor- and peptide-producing strain, *Zygosacchariformis* var. *roux*, was introduced. The fermentation temperature was adjusted to a secondary fermentation temperature of 28°C to complete the fermentation. The resulting unsalted brine contained 0.65 g / 100 mL of amino acid nitrogen, and the percentage of contaminants in the system was controlled within a certain range. The following demonstrates the correlation between acidification-induced antibacterial activity and substrate structure deconstruction; among which... It represents the value of a power operation with a base of 10 and an exponent of 2. CFU stands for Colony Forming Unit.

[0043] Example 2: This experiment was conducted in a 500L jacketed fermentation reactor equipped with an online electrochemical sensor and a rotational viscometer. The online sensor had a pH measurement resolution of 0.01 and an apparent viscosity acquisition frequency of 0.2Hz. A salt-free fermentation broth with a protein mass percentage of 12% was prepared by adjusting the solid-liquid ratio. This concentration was at the upper limit. To address electromagnetic interference in industrial environments, Gaussian white noise with a signal-to-noise ratio of 20dB was actively superimposed on the viscosity signal acquisition link. The sampling period was dynamically adjusted based on signal fluctuation characteristics. A 5-minute sampling period was used when the viscosity decay slope was in the steady-state range. When the dynamic pH value entered the isoelectric point warning range of 5.0 to 4.7, the sampling period automatically switched to 1 minute to capture the substrate condensation inflection point. During the operation of the sample group of this invention, when the dynamic pH value decreased to 4.7 and the viscosity decay slope... When the absolute value is 0.05 mPa⋅s / min, the acid production metabolic phase is determined to precede the substrate depolymerization phase. At this point, the acid stagnation buffer sequence is activated, and the system temperature is lowered from 30℃ to the dormancy temperature of 11℃ using a cooling rate of 3℃ / min. This temperature field boundary is used to block the proton secretion metabolic pathway of *Lactobacillus plantarum*, while maintaining the catalytic activity of endogenous depolymerases in the fermentation broth on substrate protein macromolecules. The viscosity curve after filtering is observed to exhibit a nonlinear decay trend during the dormancy period. For apparent viscosity, For time, This represents the viscosity decay slope.

[0044] Two control groups were established. Control group one was removed from the low-frequency pulsed ultrasound treatment characteristics, while control group two was removed from the acid stagnation buffer sequence and fermented at a constant temperature of 30°C. Data showed that the sample group of this invention maintained dormancy for 14.5 hours. Afterwards, real-time apparent viscosity The pH value decreased from an initial 458.2 mPa⋅s to 158.6 mPa⋅s, which is 34.6% of the initial state, meeting the release threshold. After inoculation with *Saccharomyces rouxii* at 28°C for post-maturation, the amino acid nitrogen content of the resulting product was 0.68 g / 100 mL. In comparison, control group 1, due to the lack of physical field induction for cell disruption, resulted in delayed release of reducing sugars. Its pH value decreased to 4.7 8.2 h later than the sample group of this invention, and its amino acid nitrogen content was 0.42 g / 100 mL. Control group 2 produced hydrophobic protein coagulation when the pH value reached 4.3, and its viscosity curve showed an abnormal rebound jump, resulting in an amino acid nitrogen content of 0.35 g / 100 mL.

[0045] In a gradient experimental group with protein mass percentages ranging from 5% to 12%, it was found that the critical time for triggering the acid stagnation buffer sequence was negatively correlated with protein concentration, and the required dormant residence time increased with increasing concentration. At the 12% concentration boundary, the system operated stably. However, if the protein content was increased to the out-of-range control group of 15%, the initial viscosity of the system became too high, leading to a sharp increase in mass transfer resistance, and the time for the viscosity to drop to the release threshold exceeded 36 seconds. And the measured contamination rate reached This result confirms that the protein content range and temperature field intervention logic jointly construct the optimal working window for substrate utilization and microecological safety, resulting in physical isolation of the acidification antibacterial pathway and the protein deconstruction pathway on the time axis; among which It represents the value of a power operation with a base of 10 and an exponent of 4. CFU stands for Colony Forming Unit.

[0046] Example 3: For the preparation of a plant-animal composite matrix with a protein mass percentage at the 12% limit, batch fluctuations in the rheological properties of the materials can affect the stability of asynchronous gating. The unsalted fermentation broth to be treated was introduced into a calibration tank with a constant-temperature jacket, and the initial apparent viscosity of the unsalted fermentation broth was measured using a rotational viscometer at 25°C. Using this parameter as the normalized input reference for the logic operation unit, during the low-frequency pulse ultrasonic treatment stage with a frequency of 25kHz and a power density of 50W / L, the cavitation bubbles generated by the acoustic field form microjets pointing towards the protein micelle surface upon collapse. This physical force reduces steric hindrance by altering the hydrophobic bond arrangement of the protein's secondary structure, causing the locked thiosulfinate precursor to transfer from the gel interior to the liquid solvent, thereby obtaining a fermentable leachable. In the path from the formation of the acoustic field microjets to reaching the macromolecular target, the high-energy physical impulse released by cavitation collapse... The jet shear force reaching the protein micelle surface is attenuated and buffered to the mesoscopic dynamics range due to the fluid viscosity resistance of the surrounding high-viscosity plant and animal fermentation liquid. This shear force is just below the energy window range that is insufficient to destroy the protein covalent backbone or cause irreversible conformational collapse of the endogenous depolymerase, but can exceed the energy window range that maintains the non-covalent van der Waals barrier on the surface of the macroglobulin micelle. This causes the hydrophobic region surrounding the surface to undergo local flexible expansion, thus achieving safe intervention of the microscopic secondary structure through an energy dissipation medium in physics.

[0047] The system initiates primary fermentation by inoculating the fermentable lysate with an acid-producing starter strain. The system continuously acquires raw physical signals at a sampling frequency of 0.2 Hz using a built-in electrochemical sensor. The control unit monitors the apparent viscosity through a sliding window containing 10 consecutive sampling points. The sequence undergoes mean filtering to eliminate Gaussian white noise with a signal-to-noise ratio of 20 dB. The first-order rate of change, i.e., the viscosity decay slope, is then calculated based on the filtered data sequence. To eliminate nonlinear rheological artifacts caused by ultrasonic cavitation microbubbles and microbial fermentation gas release on apparent viscosity readings, the system connects an ultrasonic standing wave degassing bypass to the sensor's measurement and sampling front end. Based on the difference in acoustic radiation force fields between gas and liquid, entrained gases are stripped away, obtaining the true rheological state of the single-phase fluid. In the extreme value anchoring logic, the control unit initializes the dynamic peak register with a registered value of zero. It compares the absolute value of the output slope in each new sampling cycle along the time axis. If the current acquired value is greater than the registered value, it is overwritten and updated, and locked as the real-time global peak benchmark. For determining the critical slope of substrate degradation for the current batch of material, the system tracks the viscosity decay slope in real time. The absolute value evolution trend is determined. When the absolute value drops to 15% of its operating peak and the amplitude is less than 2% within a continuous sampling period of 5 minutes, the slope value under this state is automatically calibrated as the critical slope for substrate degradation in this batch.

[0048] When the dynamic pH value was monitored to decrease to 4.7 and the real-time viscosity decay slope was observed... When the critical slope for substrate degradation is reached, the system controls the circulation of cooling water to adjust the system temperature to the dormancy temperature of 11°C. Under this temperature field, the ATPase activity in the *Lactobacillus plantarum* proton pump decreases to a residual level of 5.2% at 30°C, thereby blocking the process of proton secretion into the extracellular space. Simultaneously, it maintains the cleavage of long substrate protein chains by endogenous depolymerases in the lysate. The control unit then operates according to the formula... Real-time viscosity ratio calculation, viscosity ratio to be calculated in real time After reaching 35%, the acid stagnation buffer sequence was removed and Rhus leuciscus was introduced. The final salt-free brine had a stable amino acid nitrogen content of 0.67 g / 100 mL. The product system was uniform and no agglomerated particles were generated. This achieved the technical goal of solving the interference of material composition fluctuations on process stability through a logic compensation mechanism.

[0049] Example 4: In an industrial deployment scenario involving fluctuations in the ratio of soy protein isolate to onion slurry from different sources, the procedure for eliminating the impact of batch-to-batch material differences on the accuracy of asynchronous gating decisions includes introducing the current batch of salt-free fermentation stock solution into a calibration tank with a thermostatic jacket, circulating 25°C water into the thermostatic jacket to bring the material to thermodynamic equilibrium, initiating a shear test at 60 rpm, and recording the initial apparent viscosity. The control unit will collect 50 consecutive apparent viscosity data. Data points are input into a preset first-order difference operator, and the viscosity change rate is monitored over time. The extreme value of the second derivative of the evolution determines the critical slope of substrate degradation, when the viscosity decay slope When the absolute value of the slope drops to 15% of the amplitude corresponding to the extreme value of the second derivative and remains stable within a 300s glide window, the instantaneous slope value is used as the criterion for triggering the acid stagnation buffer sequence.

[0050] When the system encounters operating conditions where different batches of Lactobacillus plantarum exhibit varying metabolic activities, the on-site fine-tuning and calibration process for the dormancy residence temperature includes establishing the absolute temperature. With proton secretion rate The Arrhenius correlation model was used to calculate the critical inhibition temperature at which the acid production metabolism phase and the protein deconstruction phase decouple. The system was scanned in 0.5°C increments within the 10°C to 12°C range, and the pH shift at each temperature gradient was measured. When the pH was observed to be within 1... The change within the viscosity is less than 0.02 and the real-time viscosity ratio is... When the decay rate is maintained at more than 3% per hour, this set value is fixed as the dormancy residence temperature for this batch of production. The calculation formula is as follows: ,in, For real-time viscosity ratio, The current apparent viscosity, This represents the initial apparent viscosity.

[0051] Example 5: In an industrial-scale fermentation reaction with a vessel volume of 10,000 L, equipped with an external plate heat exchanger and three sets of ultrasonic transducer arrays, to eliminate the heat conduction lag and material rheological field inhomogeneity in a large-volume system, spatial physical layout calibration was performed to ensure the penetration effect of the cavitation field in the plant-animal composite matrix. The three sets of ultrasonic transducers were radially symmetrically distributed at 120° with respect to the central axis of the vessel, and the horizontal angle between the emitting end face of each ultrasonic transducer and the central axis was adjusted to 15°. A salt-free fermentation broth with a protein mass percentage of 12% was injected into the vessel, and a material circulation pump was used at a flow rate of 50... A constant flow rate drives the salt-free fermentation broth to flow between the reactor body and an external plate heat exchanger. The system is started with low-frequency pulsed ultrasonic treatment at a frequency of 25kHz and a power density of 50W / L. The initial apparent viscosity is collected in real time by viscosity sensors configured at different depths inside the reactor. The spatial variance between data points at each level is calculated. When the spatial variance drops below 5%, it is determined that the mass transfer resistance and component distribution within the system have reached a kinetic steady state and a fermentable soluble liquid with uniform physical properties is obtained.

[0052] To address the cooling rate fluctuations induced by changes in environmental heat load during the primary fermentation stage, the system initiates an adaptive acid stagnation triggering prediction procedure based on thermodynamic feedback. Lactobacillus plantarum is inoculated at a first fermentation temperature of 30°C, and the control unit calculates the proton secretion rate of the current batch of strains using pH drift measured during the sampling period. And establish the proton secretion rate With temperature The Arrhenius correlation model between them is calculated using the following formula: ,in, For the natural logarithm operator, This refers to the proton secretion rate. It is the metabolic activation energy. This is the universal gas constant, with a value of 8.314. , Absolute temperature The metabolic pre-exponential factor was obtained through previous experimental fitting. When the system detected that the environmental heat dissipation power was limited, resulting in an actual cooling rate of less than 3℃ / min, according to Fourier's law of heat conduction, the system activated the interleaved array of internal coil cooling medium circuits inside the reactor, synchronously driving the near-wall scraper stirring system. The eccentric linear velocity at the scraper end was set to 1.5m / s to 2.0m / s. By continuously mechanically peeling off the high-viscosity laminar flow layer adhering to the inner wall of the reactor, the contact thermal resistance was reduced, and the temperature gradient between the central axis of the reactor and the edge heat transfer boundary was forcibly smoothed. Based on the smoothing of the macroscopic temperature gradient inside the reactor, the control unit input the real-time collected cooling temperature and time curves into the aforementioned Arrhenius correlation model. For the entire transient cooling cycle from the current fermentation temperature to the target dormancy residence temperature, the dynamic proton secretion rate was analyzed. A definite integral mathematical operation over time is performed to obtain the total cumulative proton secretion overshoot caused by thermodynamic hysteresis. Combined with the buffer capacity coefficient of the current fermentation broth system, this microscopic cumulative proton secretion is directly converted into a macroscopically visible theoretical pH offset scalar. Based on the metabolic inertia calculated using the above formula, the trigger point of the acid stagnation buffer sequence is corrected from pH 4.7 to 4.85. The cooling hysteresis of large equipment is compensated by prematurely activating the cooling water circulation, causing the system temperature to drop to a dormant temperature of 11°C before the pH reaches the isoelectric point warning line. The real-time apparent viscosity is then monitored. After decaying to 35% of its initial state, *Zygosacchariformis* was inoculated, resulting in an unsalted brine with an amino acid nitrogen content of 0.66 g / 100 mL and a contamination rate of less than 10%. 2 .

[0053] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A co-fermentation method for preparing salt-free brine, characterized in that, Includes the following steps: Step S101: Prepare a salt-free fermentation stock solution with a composite substrate of plants and animals, and adjust the protein mass percentage in the salt-free fermentation stock solution to 5% to 12%; Step S102: Apply low-frequency pulsed ultrasound treatment at a frequency of 20kHz to 28kHz to the salt-free fermentation broth to break the cell walls of the plant raw materials to release intracellular fermentable sugars and obtain a fermentable extract. Step S103: Inoculate the acid-producing initiating strain into the fermentable lysate, start primary fermentation at a first fermentation temperature of 30°C, and monitor the dynamic pH value and apparent viscosity of the fermentable lysate during fermentation, and calculate the viscosity decay slope. Step S104, enter the metabolic inhibition and regulation stage: when the dynamic pH value drops to 4.6 to 4.8 and the viscosity decay slope is lower than the preset substrate degradation critical slope, a cooling medium is injected into the fermentation system to adjust the fermentation broth temperature from the first fermentation temperature to a dormancy residence temperature of 10°C to 12°C. The dormancy residence temperature is used to block the proton secretion metabolism of the acid-producing initiating strain and maintain the protein degradation activity of the endogenous depolymerase in the fermentation broth. Step S105: Under the condition of maintaining the dormancy residence temperature, monitor the real-time apparent viscosity of the fermentation broth. When the real-time apparent viscosity reaches the release threshold of 30% to 40% of the initial viscosity, inoculate the aroma-producing and peptide-producing strains into the fermentation broth and raise the temperature to 25°C to 30°C to complete the co-fermentation.

2. The co-fermentation method for preparing salt-free brine according to claim 1, characterized in that, In step S102, the power density of the low-frequency pulsed ultrasound treatment is adjusted to 50 W / L, and the pulse working cycle is 10 s on and 5 s off. During the process of breaking the cell wall of the plant raw material, the dissolution rate of reducing sugar in the fermentation broth is adjusted by the cavitation effect generated by the pulse working cycle. Based on the initial suspended solids concentration of the salt-free fermentation broth, the duty cycle of the pulse working cycle is positively mapped to provide a carbon source basis for the rapid proliferation of the acid-producing initiating strain in step S103.

3. The co-fermentation method for preparing salt-free brine according to claim 1, characterized in that, In step S101, the plant raw materials in the plant-animal composite matrix include at least one of soybeans, black beans, or peanuts; by limiting the protein mass percentage, a substrate thermodynamic buffer space is constructed for the metabolic inhibition regulation stage in step S104.

4. The co-fermentation method for preparing salt-free brine according to claim 1, characterized in that, In step S104, the release threshold is established based on the depth of macromolecular depolymerization before the pH value drops to the isoelectric point of the protein; under the dormancy residence temperature environment, endogenous depolymerization enzymes are used to deconstruct the protein structure to avoid hydrophobic coagulation in the isoelectric point region of the fermentation broth.

5. The co-fermentation method for preparing salt-free brine according to claim 1, characterized in that, In step S105, while inoculating the aroma- and peptide-producing strains, halophilic tetracocci are also inoculated; the aroma- and peptide-producing strains include Zygosacchariformis rouxae, which acts on the protein fragments deconstructed in step S104 to transform them into flavor peptides.

6. The co-fermentation method for preparing salt-free brine according to claim 1, characterized in that, In step S104, the injection flow rate of the cooling medium is controlled to a cooling rate of not less than 2℃ / min. The rapid transition of the temperature field enables the separation of the acid-producing metabolism of the acid-producing initiating strain and the substrate deconstruction of the endogenous depolymerase on the time axis.

7. The co-fermentation method for preparing salt-free brine according to claim 1, characterized in that, The total duration of co-fermentation is 72h to 120h; among which, the duration of the metabolic inhibition regulation phase in step S104 is 12h to 24h, so that the mass percentage of small molecule peptides in the fermentation broth is not less than 5%.

8. The co-fermentation method for preparing salt-free brine according to claim 1, characterized in that, After step S105 is completed, step S106 is also included, in which the fermentation product is centrifuged and the liquid phase is collected to obtain a salt-free brine with an amino acid nitrogen content of not less than 0.6 g / 100 mL.

9. A co-fermentation method for preparing salt-free brine according to claim 8, characterized in that, It also includes step S1061, which involves instantaneous high-temperature sterilization of the salt-free marinade at a temperature of 115°C to 120°C for a duration of 3 to 5 seconds to complete aseptic filling.

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