A method for preparing fermented mare milk based on multi-omics technology optimization and application thereof

Abstract

This invention discloses a fermented mare's milk preparation method optimized based on multi-omics technology. First, transcriptomics technology is used to analyze differentially expressed genes of *Kluyveromyces martensii* and *Lactobacillus fermentum* in the mare's milk environment, screening out key functional genes related to lipid metabolism and flavor synthesis, such as FAD2, ADH2, and ATF1, and establishing a predictive model of strain metabolic capacity for precise screening. Then, an innovative temperature- and oxygen-controlled fermentation process is employed, dividing the fermentation process into three stages: pre-fermentation, mid-fermentation, and post-fermentation. Temperature (35℃, 37℃, 39℃) and dissolved oxygen content (5%, 2%, 0.5%) are optimized in each stage to guide metabolic flow in the desired direction. Combined with an intelligent pH-ORP dual-feedback control system, the fermentation status is monitored in real time and parameters are automatically adjusted to ensure batch-to-batch consistency of product quality. Finally, ultrasonic-assisted cell disruption technology (40kHz / 200W / 5min) is introduced to improve the physicochemical properties and flavor stability of the raw milk.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, specifically to a method for preparing fermented mare's milk based on multi-omics technology optimization and its application. Background Technology

[0002] Mare's milk, an animal dairy product with high nutritional value and unique functional properties, is rich in various vitamins, minerals, and immune-active factors. It can be used as an ideal alternative food for people with cow's milk protein allergies and has a long history of consumption and broad market prospects in Xinjiang, Inner Mongolia, and other regions of my country. However, the volatile flavor compound decanoic acid in mare's milk contributes as much as 19.61% to the overall flavor, resulting in an unpleasant sweaty odor. This characteristic severely restricts the market promotion and consumer acceptance of mare's milk and its fermented products.

[0003] Currently, research on the volatile flavor characteristics of mare's milk and the impact of fermentation on flavor compounds is relatively limited both domestically and internationally. Traditional fermentation processes mainly rely on experience-based operations, lacking systematic strain screening methods and precise process parameter control. Specifically, existing technologies have the following shortcomings: First, traditional strain screening methods are inefficient. They typically employ a trial-and-error screening approach, requiring numerous preliminary experiments and failing to predict the metabolic performance of strains in actual fermentation environments. This results in long screening cycles, high costs, and low success rates, making it difficult to quickly identify strains that can effectively regulate decanoic acid content and enhance flavor, a key bottleneck restricting the improvement of fermented mare's milk flavor. Second, fermentation process parameter control is rudimentary. Key parameters such as temperature, oxygen, and pH are often set constantly or manually adjusted, unable to be dynamically adjusted according to the fermentation process. This leads to poor batch-to-batch product consistency, large fluctuations in decanoic acid content, and unstable formation of superior flavor compounds, hindering industrial-scale production. Therefore, there is an urgent need to develop a fermented mare's milk preparation method that can effectively reduce decanoic acid content, increase superior flavor compounds, improve physicochemical properties and antioxidant capacity, while also being industrially feasible. Summary of the Invention

[0004] The purpose of this invention is to provide a fermented mare's milk preparation method optimized based on multi-omics technologies. Transcriptomics analysis reveals the metabolic mechanisms of the fermented strains, screening out key functional genes related to lipid metabolism and flavor compound synthesis. An innovative temperature- and oxygen-controlled fermentation process is employed, dividing the fermentation process into three stages: pre-fermentation, mid-fermentation, and post-fermentation, with optimized temperature and oxygen parameters for precise control of metabolic flow. An intelligent pH-ORP dual-feedback control system is introduced to monitor the fermentation status in real time and automatically adjust process parameters, ensuring batch-to-batch consistency of product quality. Finally, ultrasonic-assisted cell-wall disruption technology is used to process the raw milk, improving the stability and retention rate of flavor substances.

[0005] A method for preparing fermented mare's milk optimized based on multi-omics technology includes the following steps:

[0006] (1) Take fresh mare's milk and sterilize it;

[0007] (2) Ultrasonic-assisted cell wall disruption is performed on sterilized mare's milk;

[0008] (3) Use transcriptomics technology to analyze differentially expressed genes in Kluyveromyces marxoiris and Lactobacillus fermentum and screen key metabolic genes;

[0009] The Kluyveromyces martensii is BNCC187341 and Lactobacillus fermentum is BNCC194390. If their homologous strains can exhibit transcriptional characteristics that are completely consistent with these two strains, the key metabolic genes include FAD2 gene, ADH2 gene and ATF1 gene.

[0010] (4) Mix Kluyveromyces martensii and Lactobacillus fermentum at a ratio of 1:3 to 3:1 and inoculate them into the treated mare's milk. The total inoculation amount is 3% v / v, of which the inoculation amount of Kluyveromyces martensii is 1×10⁻⁶. 5 CFU / mL, Lactobacillus fermentum inoculum 1×10⁻⁶ 6 CFU / mL, with an inoculation ratio of 1:2 or 2:1;

[0011] (5) A controlled-temperature and controlled-oxygen segmented fermentation process is adopted for the fermentation of the compound bacteria. The fermentation process is divided into a pre-stage, a middle stage, and a post-stage. The specific parameters of the segmented fermentation are as follows:

[0012] The initial stage involves a temperature of 34-36℃, a dissolved oxygen content of 5-8%, and a duration of 10-14 hours.

[0013] The intermediate stage temperature is 36-38℃, dissolved oxygen content is 2-3%, and the time is 6-10 hours.

[0014] The later stage temperature is 38-40℃, dissolved oxygen content is below 0.5%, and the time is 3-6 hours;

[0015] (6) Fermentation parameters are adjusted in real time by a smart pH-ORP dual feedback control system. The pH control range of the system is 4.0-4.6, and the ORP control range is 100-180mV.

[0016] (7) Fermented mare's milk is obtained after fermentation is completed.

[0017] In step (2):

[0018] The parameters for the ultrasonic-assisted cell wall disruption treatment are: frequency 30-50kHz, power 150-250W, time 3-8 minutes, and pulse ratio 1:1.

[0019] The intelligent pH-ORP dual feedback control system also includes an automatic adjustment function. When the pH is lower than the set lower limit, it automatically reduces the stirring rate and increases the aeration rate. When the ORP is higher than the set upper limit, it automatically increases the nitrogen input.

[0020] After fermentation, the decanoic acid content of fermented mare's milk decreased by more than 95%, the content of volatile ester flavor compounds increased by more than 50% compared with unfermented mare's milk, the sensory score was greater than 85 points, and the DPPH free radical scavenging rate was greater than 50%.

[0021] The sterilization process described in step (1) is pasteurization or ultra-high temperature instantaneous sterilization.

[0022] Sugars are added as a carbon source before fermentation, and the sugars are selected from one or more of glucose, lactose, and sucrose.

[0023] Fermented mare's milk was prepared using the method described above.

[0024] The fermented mare's milk has a sensory score greater than 85 points, a DPPH free radical scavenging rate greater than 50%, and a batch-to-batch consistency deviation of less than 5%.

[0025] In the fermented mare's milk, decanoic acid contributes less than 5% to the flavor, while ester flavor compounds contribute more than 40% to the total flavor.

[0026] Specifically:

[0027] like Figure 1 As shown, this invention is the first to apply transcriptomics technology to the field of screening strains for fermented mare's milk. By extracting total RNA from Kluyveromyces marxianus and Lactobacillus fermentum in the mare's milk fermentation environment, differentially expressed gene analysis was performed using a high-throughput sequencing platform to screen out key functional genes related to lipid metabolism and flavor compound synthesis.

[0028] Specifically, this invention screened the following key genes:

[0029] FAD2 gene: Encodes ω-6 fatty acid desaturase, which is involved in the synthesis pathway of unsaturated fatty acids. Upregulation of its expression can significantly increase the content of polyunsaturated fatty acids (PUFAs) and improve the nutritional value of products.

[0030] ADH2 gene: encodes alcohol dehydrogenase, which is involved in the synthesis of alcohol flavor compounds. Upregulation of its expression can promote the conversion of ethanol to ethyl ester and increase the content of ester aroma compounds.

[0031] ATF1 gene: encodes acetyltransferase, a key enzyme in the synthesis of ester compounds. Upregulation of its expression can significantly increase the yield of ester flavor compounds.

[0032] Based on the expression levels of the aforementioned key genes, this invention establishes a model for predicting the metabolic capacity of bacterial strains. This model can predict the metabolite profiles of strains under different fermentation conditions, enabling precise screening and process optimization. The application of this model increases strain screening efficiency by more than three times and shortens the screening cycle by 60%.

[0033] like Figure 2 As shown, this invention innovatively proposes a "three-stage temperature-controlled oxygen" fermentation process, which dynamically adjusts temperature and dissolved oxygen parameters based on changes in the metabolic characteristics of the bacterial strain during fermentation, guiding the metabolic flow in the expected direction:

[0034] Pre-fermentation stage (12 hours): Temperature controlled at 35℃, dissolved oxygen content controlled at 7%. This stage focuses on cell proliferation; the higher temperature and oxygen conditions promote the rapid growth and reproduction of *Kluyveromyces martensii* and *Lactobacillus fermentum*, with cell density reaching 10-1 within 12 hours. 8 With a concentration of CFU / mL or higher, a sufficient biomass foundation is laid for the subsequent synthesis of flavor compounds.

[0035] During the fermentation stage (8 hours): the temperature is controlled at 37℃, and the dissolved oxygen content is controlled at 3%. This stage is crucial for the synthesis of flavor compounds. Appropriately reducing the temperature and oxygen content can induce the strain to enter a secondary metabolic state, promoting the synthesis and accumulation of volatile flavor compounds such as esters, aldehydes, and alcohols. At the end of this stage, the concentration of the main flavor compounds in the fermented milk reaches its peak.

[0036] Post-fermentation stage (4 hours): Temperature controlled at 39℃, dissolved oxygen content controlled below 0.5%. This stage focuses on esterification and flavor maturation. Low temperature and low oxygen conditions promote the esterification of organic acids and alcohols, generating more ester aroma components, while simultaneously achieving the final optimization of flavor compounds.

[0037] like Figure 3 As shown, this invention develops an intelligent fermentation process monitoring system that collects the pH value and oxidation-reduction potential (ORP) of the fermentation system in real time through online sensors, and automatically adjusts parameters such as stirring rate and aeration rate using a preset control algorithm to achieve precise control of the fermentation process.

[0038] The system's control logic is as follows:

[0039] pH monitoring: When the pH value is lower than the set lower limit (preferably pH 4.5), the control system automatically reduces the stirring rate and appropriately increases the aeration rate to reduce further accumulation of organic acids. When the pH value is higher than the set upper limit (preferably pH 5.0), the stirring rate is appropriately increased to promote the metabolism of the strain.

[0040] ORP monitoring: When the ORP value is higher than the set upper limit (preferably 150mV), it indicates that the oxidizing power is too strong. The control system automatically increases the nitrogen input to reduce the dissolved oxygen content. When the ORP value is lower than the set lower limit (preferably 100mV), the ventilation is appropriately increased to maintain aerobic metabolism.

[0041] The system has a response time of less than 30 seconds and a control accuracy of ±0.1 pH unit and ±10 mVORP, which improves batch-to-batch product consistency by more than 80%.

[0042] like Figure 4 As shown, this invention introduces low-frequency ultrasonic-assisted cell-wall breaking technology in the pre-fermentation treatment stage. A 40kHz low-frequency ultrasonic wave is selected, with a power setting of 200W, a treatment time of 5 minutes, and a pulse ratio of 1:1 (1 second on, 1 second off).

[0043] The high-frequency mechanical vibrations generated by ultrasonic treatment can achieve the following effects:

[0044] Particle size reduction: Significantly reduced the particle size of protein micelles and fat globules in mare's milk, decreasing the peak particle size distribution from 3.5 μm to 2.2 μm, a reduction of 32%.

[0045] Protein structure activation: Increases the content of free sulfhydryl groups in proteins by 28%, exposes more hydrophobic amino acid sites, and enhances the binding ability with flavor compounds.

[0046] Flavor retention: Fermented milk prepared after ultrasonic treatment has a 41% higher retention rate of flavor substances during storage, and the flavor stability of the product during shelf life is significantly improved.

[0047] Additional features and advantages of this invention will be set forth in the description which follows, or may be learned by practicing the invention. Attached Figure Description

[0048] The technical solution and beneficial effects of the present invention will become apparent and readily understood from the following description in conjunction with the accompanying drawings, wherein:

[0049] Figure 1 This is a flowchart illustrating the multi-omics technology-assisted strain screening process of the present invention.

[0050] Figure 2 This is a graph showing the process parameters of the temperature- and oxygen-controlled fermentation process of the present invention.

[0051] Figure 3This is a block diagram illustrating the principle of the intelligent pH-ORP dual feedback control system of the present invention.

[0052] Figure 4 This is a diagram illustrating the effect of ultrasound-assisted cell wall disruption in this invention.

[0053] Figure 5 This is a comparison chart of product performance under different fermentation conditions according to the present invention;

[0054] Figure 6 This is a schematic diagram of the fermented mare's milk metabolic pathway of the present invention;

[0055] Figure 7 This is a GC-MS chromatogram comparing the aroma components of fermented mare's milk according to the present invention;

[0056] Figure 8 This is a schematic diagram of the monitoring interface of the intelligent fermentation control system of the present invention. Detailed Implementation

[0057] 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.

[0058] like Figure 6 , Figure 7 and Figure 8 As shown, a method for preparing fermented mare's milk optimized based on multi-omics technology includes the following steps:

[0059] (1) Take fresh mare's milk and sterilize it;

[0060] (2) Ultrasonic-assisted cell wall disruption is performed on sterilized mare's milk;

[0061] (3) Use transcriptomics technology to analyze differentially expressed genes in Kluyveromyces marxoiris and Lactobacillus fermentum and screen key metabolic genes;

[0062] Kluyveromyces martensii BNCC187341 and Lactobacillus fermentum BNCC194390 are homologous strains that exhibit transcriptional characteristics completely identical to those of the two strains. If these homologous strains exhibit transcriptional characteristics completely identical to those of the two strains (i.e., FAD2, ADH2, and ATF1 genes are all significantly upregulated in the mare's milk fermentation environment, with an upregulation factor of 3.0-fold), they also fall within the scope of protection of this invention. The key metabolic genes include the FAD2 gene, ADH2 gene, and ATF1 gene.

[0063] (4) Mix Kluyveromyces martensii and Lactobacillus fermentum at a ratio of 1:3 to 3:1 and inoculate them into the treated mare's milk. The total inoculation amount is 3% v / v, of which the inoculation amount of Kluyveromyces martensii is 1×10⁻⁶. 5CFU / mL, Lactobacillus fermentum inoculum 1×10⁻⁶ 6 CFU / mL, with an inoculation ratio of 1:2 or 2:1;

[0064] (5) A controlled temperature and oxygen segmented fermentation process is adopted for the fermentation of the compound bacteria. The fermentation process is divided into a pre-stage, a middle stage and a post-stage. The specific parameters for the segmented fermentation are as follows:

[0065] The initial stage temperature is 34-36℃, dissolved oxygen content is 5-8% (expressed as a relative percentage of online DO probe readings), and the time is 10-14 hours;

[0066] The intermediate stage temperature is 36-38℃, the dissolved oxygen content is 2-3% (expressed as a relative percentage of the online DO probe reading), and the time is 6-10 hours;

[0067] The temperature in the later stage is 38-40℃, the dissolved oxygen content is below 0.5% (expressed as a relative percentage of the online DO probe reading), and the time is 3-6 hours;

[0068] The preferred parameters are: pre-fermentation stage temperature 35 ℃, dissolved oxygen 6%, time 12 hours; mid-fermentation stage temperature 37 ℃, dissolved oxygen 2.5%, time 8 hours; post-fermentation stage temperature 39 ℃, dissolved oxygen 0.5%, time 4 hours.

[0069] (6) Fermentation parameters are adjusted in real time through an intelligent pH-ORP dual feedback control system. The pH control range of the system is 4.0-4.6, and the ORP control range is 100-180mV.

[0070] (7) Fermented mare's milk is obtained after fermentation is completed.

[0071] In step (2):

[0072] The parameters for ultrasonic-assisted cell disruption are: frequency 30-50kHz, power 150-250W, time 3-8 minutes, and pulse ratio 1:1.

[0073] It should be noted that the preferred parameters for ultrasonic-assisted cell disruption are a frequency of 40 kHz, a power of 200 W, and a time of 5 minutes.

[0074] The intelligent pH-ORP dual feedback control system also includes an automatic adjustment function. When the pH is lower than the set lower limit, it automatically reduces the stirring rate and increases the aeration rate. When the ORP is higher than the set upper limit, it automatically increases the nitrogen input.

[0075] After fermentation, the decanoic acid content of fermented mare's milk decreased by more than 95%, the content of volatile ester flavor compounds increased by more than 50% compared with unfermented mare's milk, the sensory score was greater than 85 points, and the DPPH free radical scavenging rate was greater than 50%.

[0076] The sterilization process in step (1) is pasteurization or ultra-high temperature instantaneous sterilization.

[0077] Sugars are added as a carbon source before fermentation. The sugars are selected from one or more of glucose, lactose, and sucrose.

[0078] A fermented mare's milk, prepared by a specific method.

[0079] The fermented mare's milk has a sensory score of over 85 points, a DPPH free radical scavenging rate of over 50%, and a batch-to-batch consistency deviation of less than 5%.

[0080] Decanoic acid contributes less than 5% to the flavor of fermented mare's milk, while ester flavor compounds contribute more than 40% to the total flavor.

[0081] Example 1: Multi-omics-assisted strain screening and metabolic mechanism analysis

[0082] Step 1: Strain Activation: Kluyveromyces martensii (BNCC187341, purchased from the Beina Biotechnology Culture Collection Center) and Lactobacillus fermentum (BNCC194390, purchased from the Beina Biotechnology Culture Collection Center) were inoculated into MRS and YEPD media respectively, and cultured at 37°C for 24 hours for activation before use. The Kluyveromyces martensii and Lactobacillus fermentum used in this invention are limited to the specific strains mentioned above. Homologous strains exhibiting transcriptional characteristics completely identical to these two strains (i.e., significant upregulation of FAD2, ADH2, and ATF1 genes in the mare's milk fermentation environment, with upregulation folds of not less than 3.0-fold, 2.6-fold, and 2.3-fold, respectively) also fall within the scope of protection of this invention.

[0083] Step 2: Transcriptomics analysis: The activated strain was inoculated into sterilized mare's milk at a 2% inoculum and cultured at 37°C for 12 hours to reach the logarithmic growth phase. The bacterial cells were then collected, and total RNA was extracted using the TRIzol method and sent to a sequencing company for transcriptome sequencing (RNA-seq).

[0084] Step 3: Differentially expressed gene screening: Differential expression analysis was performed using DESeq2 software. Genes with significant differential expression were screened based on the criteria of FoldChange ≥ 2 and p-value < 0.05. KEGG pathway enrichment analysis revealed that differentially expressed genes were significantly enriched in the fatty acid metabolism pathway (map00071) and the alcohol metabolism pathway (map00040).

[0085] Step 4: Validation of key genes: Three key genes, FAD2, ADH2 and ATF1, were selected for qRT-PCR validation. The results showed that the expression of these three genes was significantly upregulated by 3-fold in the mare's milk fermentation environment, which was consistent with the transcriptome sequencing results.

[0086] Step 5: Establishment of Metabolic Prediction Model: Based on the correlation analysis between key gene expression data and metabolite profiles, the model construction method, validation data, and applicable conditions are clarified, as follows:

[0087] ① Model building method:

[0088] Using the fold changes (X1, X2, X3) of FAD2, ADH2, and ATF1 gene expression obtained from qRT-PCR validation in Step 4 as independent variables, and the corresponding ester content (Y) detected by gas chromatography-mass spectrometry (GC-MS) as the dependent variable, linear regression analysis was performed using SPSS software. The optimal fitting equation was screened by stepwise regression, and irrelevant variables were eliminated to determine the regression coefficients and constant terms. Finally, a regression prediction model was established: Y = 0.35X1 + 0.28X2 + 0.22X3 + 1.8 (where X1, X2, and X3 are the fold changes of FAD2, ADH2, and ATF1, respectively, and Y is the predicted ester content, in μg / mL).

[0089] ② Model validation data:

[0090] Fifteen independent fermentation samples (covering different inoculation ratios and fermentation temperature gradients) were selected for validation. The actual fold increase of three key genes in the samples was substituted into the model to calculate the predicted values ​​of esters. These predicted values ​​were then compared with the actual values ​​detected by GC-MS. The validation results showed that the average relative error between the predicted and actual values ​​was 11.2%, and the model's coefficient of determination R0 was [value missing]. 2 =0.83, with a prediction accuracy of 87%, indicating that the model has a good fit and high prediction reliability.

[0091] ③ Model Applicability Conditions: This model is only applicable to the co-fermentation system of Kluyveromyces martensii (BNCC187341) and Lactobacillus fermentum (BNCC194390) as defined in this invention. The applicable fermentation environment is as follows: fresh and unspoiled mare's milk raw materials, sterilization method is pasteurization or ultra-high temperature instantaneous sterilization, fermentation pH is controlled at 4.5, and fermentation temperature is 37℃. When the above applicable conditions are exceeded, the model prediction accuracy will decrease and the parameters need to be recalibrated.

[0092] Example 2: Temperature- and oxygen-controlled segmented fermentation process

[0093] Step 1: Raw material pretreatment. Take fresh mare's milk, pasteurize it (63℃, 30 minutes), and then cool it to room temperature for later use.

[0094] Step 2: Inoculation of the strain. The selected Kluyveromyces martensii and Lactobacillus fermentum were mixed and inoculated at a ratio of 1:2, with a total inoculation volume of 3% (v / v), of which the Kluyveromyces martensii inoculation volume was 1×10⁻⁶. 5CFU / mL, Lactobacillus fermentum inoculum 1×10⁻⁶ 6 CFU / mL.

[0095] Step 3: Segmented fermentation control (Note: In this invention, all dissolved oxygen contents are expressed as a relative percentage of online DO probe readings)

[0096] Initial stage: Open the ventilation valve to supply oxygen, maintain dissolved oxygen at 5-8%, temperature at 35℃, and incubate for 12 hours.

[0097] Intermediate stage: Reduce aeration rate, maintain 3% solubility, temperature 37℃, and incubate for 8 hours.

[0098] Later stage: Switch to nitrogen protection, maintain dissolved oxygen below 0.5%, temperature 39℃, and incubate for 4 hours.

[0099] Step 4: Fermentation endpoint detection After fermentation, the product indicators were tested: decanoic acid content decreased by 97.2%, ester content increased by 58%, sensory score was 85.6 points, and DPPH free radical scavenging rate was 52.3%.

[0100] Example 3: Intelligent pH-ORP Dual Feedback Control System

[0101] The intelligent monitoring system used in this invention includes the following components:

[0102] pH online sensor: Measurement range 3.0-7.0, accuracy ±0.1;

[0103] ORP online sensor: Measurement range -500 to +500mV, accuracy ±10mV;

[0104] Programmable Logic Controller (PLC): Responsible for data acquisition and control algorithm calculation;

[0105] Actuators include a variable frequency stirring motor, a solenoid valve, and a gas mass flow meter;

[0106] The control program uses a PID algorithm, with the following parameters:

[0107] The proportional gain (P) is 2.8, the integral gain (I) is 0.5, the derivative gain (D) is 0.3, the sampling period T is 10s, and the control output range is matched with the actuator (stirring speed corresponds to 50-500 rpm, gas flow rate corresponds to 0-2 L / min). The integral separation threshold is ±0.2 (when the absolute value of pH or ORP deviation is >0.2, the integral action is paused to avoid overshoot; when the deviation is ≤0.2, the integral action is resumed to eliminate steady-state error). Control logic thresholds: Based on the pH control range (4.0-4.6) and ORP control range (100-180mV) set in the claims, the control thresholds are set as follows: pH upper limit threshold 4.6, lower limit threshold 4.0, deviation trigger threshold ±0.1; ORP upper limit threshold 180mV, lower limit threshold 100mV, deviation trigger threshold ±10mV.

[0108] Specific control logic:

[0109] Based on the deviation between the real-time collected pH and ORP values ​​and the set values, the PID algorithm calculates and outputs control signals to automatically adjust the stirring speed (50-500 rpm) and gas flow rate (0-2 L / min) to achieve stable operation of the fermentation process;

[0110] When the pH is below the lower limit threshold (4.0) or the deviation is ≤-0.1, the stirring rate is automatically reduced (by 50 rpm each time, down to a minimum of 50 rpm) and the aeration rate is increased (by 0.2 L / min each time, up to a maximum of 2 L / min).

[0111] When the pH is higher than the upper limit threshold (4.6) or the deviation is ≥0.1, the stirring rate is automatically increased (by 50 rpm each time, up to a maximum of 500 rpm) and the aeration rate is reduced (by 0.2 L / min each time, down to a minimum of 0 L / min).

[0112] When the ORP is higher than the upper limit threshold (180mV) or the deviation is ≥10mV, the nitrogen input will be automatically increased (0.3L / min each time).

[0113] When the ORP is below the lower limit threshold (100mV) or the deviation is ≤-10mV, the nitrogen input will be automatically reduced (by 0.3L / min each time, down to 0L / min), while the air ventilation will be increased simultaneously.

[0114] Based on the deviation between the real-time collected pH and ORP values ​​and the set values, the stirring speed (50-500 rpm) and gas flow rate (0-2 L / min) are automatically adjusted to achieve stable operation of the fermentation process.

[0115] Example 4: Ultrasonic-assisted cell wall disruption treatment

[0116] Step 1: Use a 40kHz, 200W CNC ultrasonic cleaner with a 10L treatment tank.

[0117] Step 2: Parameter optimization. Place sterilized mare's milk (500mL) in the treatment tank and set the ultrasonic parameters as follows: power 200W, time 5min, pulse ratio 1:1, temperature 25℃.

[0118] Step 3: Sample testing after processing to assess the effectiveness of the treatment:

[0119] Particle size: The average particle size decreased from 3.5 μm to 2.2 μm (↓37%).

[0120] Free thiol groups: The content increased from 12.3 μmol / g to 15.7 μmol / g (↑28%).

[0121] Surface hydrophobicity: increased from 42.3 to 58.6 (↑39%);

[0122] Step 4: Fermentation verification After ultrasonic treatment, a fermentation experiment was conducted. Compared with the untreated group, the retention rate of flavor substances increased from 68% to 82% after 30 days of storage.

[0123] Table 1

[0124] Serial Number Technical indicators Existing technology (previous results from this laboratory) This invention Increase 1 Decanoic acid reduction rate 93.59% 97.2% +3.9% 2 Increase in esters - +58% Significant improvement 3 Sensory rating 78.39 points 85.6 points +9.2% 4 DPPH removal rate 33.07% 52.3% +58% 5 Product Consistency ±15% ±3% +80% 6 Fermentation cycle 24 hours 20 hours -17% 7 Flavor stability 68% 82% +21%

[0125] In summary, such as Figure 5 As shown, the fermented mare's milk preparation method optimized based on multi-omics technology disclosed in this invention and its application have at least the following beneficial effects:

[0126] 1. Significantly improved flavor and quality:

[0127] By using high-expression strains screened through multi-omics technology and combined with a three-stage temperature-controlled oxygen process, the decanoic acid content was reduced by 97.2%, while a large number of ester-based floral / fruity aroma substances were added, transforming the overall flavor of the product from "sweaty" to "ester-based".

[0128] 2. Comprehensive optimization of physicochemical properties:

[0129] Ultrasonic-assisted cell disruption technology reduces product particle size by 32%, makes the protein network structure more compact, improves water retention by 45%, and results in a finer product texture and better stability.

[0130] 3. Significantly enhanced antioxidant capacity:

[0131] Increased polyunsaturated fatty acid content and optimized metabolic pathways improved the DPPH free radical scavenging rate to 52.3%, significantly enhancing product functionality.

[0132] 4. Increased feasibility for industrialization:

[0133] The application of the intelligent pH-ORP dual feedback control system improves batch-to-batch product consistency by 80%, providing technical support for large-scale production.

[0134] 5. Increased production efficiency:

[0135] The optimized fermentation process shortens the fermentation cycle by 17%, increases the production capacity per unit time, and reduces production costs.

[0136] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing fermented mare's milk optimized based on multi-omics technology, characterized in that, Includes the following steps: (1) Take fresh mare's milk and sterilize it; (2) Ultrasonic-assisted cell wall disruption is performed on sterilized mare's milk; (3) Use transcriptomics technology to analyze differentially expressed genes in Kluyveromyces marxoiris and Lactobacillus fermentum and screen key metabolic genes; The Kluyveromyces martensii is BNCC187341 and Lactobacillus fermentum is BNCC194390. If their homologous strains can exhibit transcriptional characteristics that are completely consistent with these two strains, the key metabolic genes include FAD2 gene, ADH2 gene and ATF1 gene. (4) Mix Kluyveromyces martensii and Lactobacillus fermentum at a ratio of 1:3 to 3:1 and inoculate them into the treated mare's milk. The total inoculation amount is 3% v / v, of which the inoculation amount of Kluyveromyces martensii is 1×10⁻⁶. 5 CFU / mL, Lactobacillus fermentum inoculum 1×10⁻⁶ 6 CFU / mL, with an inoculation ratio of 1:2 or 2:1; (5) A controlled-temperature and controlled-oxygen segmented fermentation process is adopted for the fermentation of the compound bacteria. The fermentation process is divided into a pre-stage, a middle stage, and a post-stage. The specific parameters of the segmented fermentation are as follows: The initial stage involves a temperature of 34-36℃, a dissolved oxygen content of 5-8%, and a duration of 10-14 hours. The intermediate stage temperature is 36-38℃, dissolved oxygen content is 2-3%, and the time is 6-10 hours. The later stage temperature is 38-40℃, dissolved oxygen content is below 0.5%, and the time is 3-6 hours; (6) Fermentation parameters are adjusted in real time by a smart pH-ORP dual feedback control system. The pH control range of the system is 4.0-4.6, and the ORP control range is 100-180mV. (7) Fermented mare's milk is obtained after fermentation is completed.

2. The preparation method according to claim 1, characterized in that, In step (2): The parameters for the ultrasonic-assisted cell wall disruption treatment are: frequency 30-50kHz, power 150-250W, time 3-8 minutes, and pulse ratio 1:

1.

3. The preparation method according to claim 1, characterized in that, The intelligent pH-ORP dual feedback control system also includes an automatic adjustment function. When the pH is lower than the set lower limit, it automatically reduces the stirring rate and increases the aeration rate. When the ORP is higher than the set upper limit, it automatically increases the nitrogen input.

4. The preparation method according to any one of claims 1-2, characterized in that, After fermentation, the decanoic acid content of fermented mare's milk decreased by more than 95%, the content of volatile ester flavor compounds increased by more than 50% compared with unfermented mare's milk, the sensory score was greater than 85 points, and the DPPH free radical scavenging rate was greater than 50%.

5. The preparation method according to any one of claims 1-4, characterized in that, The sterilization process described in step (1) is pasteurization or ultra-high temperature instantaneous sterilization.

6. The preparation method according to any one of claims 1-10, characterized in that, Sugars are added as a carbon source before fermentation, and the sugars are selected from one or more of glucose, lactose, and sucrose.

7. A fermented mare's milk, characterized in that, It is prepared by the preparation method according to any one of claims 1-6.

8. The fermented mare's milk as described in claim 7, characterized in that, The fermented mare's milk has a sensory score greater than 85 points, a DPPH free radical scavenging rate greater than 50%, and a batch-to-batch consistency deviation of less than 5%.

9. The fermented mare's milk as described in claim 7 or 8, characterized in that, In the fermented mare's milk, decanoic acid contributes less than 5% to the flavor, while ester flavor compounds contribute more than 40% to the total flavor.

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