Method for preparing conjugated linoleic acid yoghourt through double-system separation and fermentation and monitoring device

Abstract

The invention discloses a method for preparing conjugated linoleic acid yoghourt through double-system separation and fermentation and a monitoring device, and belongs to the technical field of functional dairy products. The method comprises the following steps: (1) after milk lactase is subjected to enzymolysis, inoculating exopolysaccharide-producing lactic acid bacteria for fermentation to prepare exopolysaccharide-rich fermented milk; (2) inoculating the vegetable oil enzymatic hydrolysate with probiotics with linoleic acid isomerization capacity under anaerobic conditions for fermentation to prepare CLA fermentation liquor, monitoring torque changes at different heights in real time by adopting a fermentation state monitoring device in the fermentation process, and starting a fine adjustment intervention device for graded remediation when the torque difference exceeds a threshold value; (3) mixing the CLA fermentation liquor with a multi-wall material, homogenizing and spray-drying to prepare CLA microcapsules; and (4) compounding the fermented milk with the CLA microcapsules to obtain the functional yoghourt. Mutual interference of two fermentation systems is avoided through a double-system separation fermentation technology, the demulsification problem in the anaerobic fermentation process is prevented in combination with an automatic remediation system, and efficient conversion of CLA is ensured.

Description

Technical Field

[0001] This invention relates to the field of functional dairy product technology, specifically to a method for preparing low-allergenic functional yogurt by directionally synthesizing conjugated linoleic acid through a dual-system separation fermentation, and a matching intelligent fermentation monitoring device. Background Technology

[0002] Conjugated linoleic acid (CLA) offers numerous health benefits, including anti-cancer properties, weight loss, and immune regulation, making it an ideal functional lipid. However, its natural content is extremely low. Currently, CLA production methods primarily include chemical synthesis and microbial transformation. Chemical synthesis easily produces isomer mixtures and solvent residues, posing food safety risks. While microbial transformation is relatively safer, it suffers from low conversion efficiency (typically <30%), long fermentation cycles, and difficulty in controlling isomer ratios. Particularly when CLA is converted in yogurt substrates, high concentrations of free fatty acids can easily cause rancidity, textural degradation, and whey separation, impacting product quality and industrial production.

[0003] Existing microbial transformation methods face multiple technical challenges: direct addition of high concentrations of linoleic acid has significant cytotoxicity to lactic acid bacteria, inhibiting cell growth; yogurt-producing strains with excellent fermentation characteristics lack efficient CLA transformation capabilities, while the fermentation characteristics of high-CLA-producing strains cannot meet the requirements of yogurt production, making it difficult to balance product quality and functionality; existing strains cannot synthesize specific CLA isomers in a targeted manner, resulting in high randomness in product composition and uncontrollable functional activity; and the fermentation process lacks effective monitoring methods, leading to poor batch-to-batch stability.

[0004] Traditional fermentation monitoring relies primarily on conventional parameters such as pH, dissolved oxygen, and temperature, lacking specialized monitoring for the stability of oil-water emulsion systems. While existing technologies, such as CN 108949517 A, have addressed the oxygen contamination problem associated with anaerobic bacterial inoculation, their monitoring systems are static and lack real-time response to dynamic changes in the fermentation process. In multiphase fermentation systems, relying solely on nitrogen purging and pressure control is insufficient to guarantee long-term stability, especially when the viscosity of the fermentation broth changes; traditional stirring monitoring cannot accurately reflect differences in mixing states at different heights. Existing technologies lack intelligent control algorithms based on multi-parameter fusion, relying excessively on human experience, resulting in poor process reproducibility.

[0005] The development of CLA functional yogurt also faces challenges in nutritional blending and stability: the lack of synergistic design between CLA and other functional components (extracellular polysaccharides, probiotics, amino acids, etc.) makes it impossible to maximize nutritional benefits; as an unsaturated fatty acid, CLA is easily oxidized and degraded during storage, and existing antioxidant protection measures have limited effectiveness; the application of microencapsulation technology is immature, and there is a lack of systematic research on the selection of encapsulation materials, process optimization, and evaluation of encapsulation efficiency. In particular, how to maintain the structural integrity of microcapsules and the controlled release effect in the acidic environment of yogurt remains a technical challenge.

[0006] Therefore, there is an urgent need to develop integrated CLA functional yogurt production technology, which can achieve efficient CLA conversion, stable product quality control, and synergistic functional effects through methods such as strain-directed screening, dual-system separation fermentation, intelligent process monitoring, microencapsulation stabilization, and precise nutrient blending. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing conjugated linoleic acid (CLA) yogurt through a dual-system separation fermentation. This method employs dual-system separation fermentation technology, using a fermentation state monitoring device to monitor the emulsion stability during the CLA fermentation process in real time, and using a fine-tuning intervention device for automatic remediation. This effectively solves the technical problem of easy emulsion breakdown in oil-water two-phase emulsion systems during anaerobic fermentation, ensuring efficient CLA conversion while maintaining the homogeneous and stable state of the fermentation system. Finally, a CLA-rich, viable bacteria-rich, and highly functional conjugated linoleic acid yogurt is prepared using microencapsulation technology.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing functional yogurt using directional fermentation to synthesize conjugated linoleic acid includes the following steps: Step 1: Prepare fermented milk rich in extracellular polysaccharides After lactose hydrolysis of milk, it is inoculated with lactic acid bacteria that produce extracellular polysaccharides for fermentation to obtain fermented milk rich in extracellular polysaccharides. Step 2: Preparation of CLA fermentation broth The vegetable oil hydrolysate was inoculated with probiotics with linoleic acid isomerization ability under anaerobic conditions for fermentation. During the fermentation process, a fermentation status monitoring device was used to monitor the torque changes of the fermentation liquid at different heights in real time. When the torque difference exceeded the preset threshold, a fine-tuning intervention device was activated to remedy the situation, and CLA fermentation liquid was obtained. The fermentation status monitoring device includes: Multiple sets of stirring components installed at different heights in the fermenter; Torque conversion and acquisition component installed on the stirring assembly; A torque amplification external lead assembly that amplifies the torque signal and converts it into vertical displacement, which is then transmitted to the outside of the fermenter. A monitoring component for monitoring changes in torque; Step 3: Preparation of CLA microcapsules CLA fermentation broth was mixed with wall material, and then homogenized and dried to obtain CLA microcapsules. Step 4: Product compounding The fermented milk rich in extracellular polysaccharides prepared in step one was mixed with the CLA microcapsules prepared in step three to obtain conjugated linoleic acid functional yogurt.

[0009] Preferably, in step one, the lactose hydrolysis process is controlled with the pH value within the range of 6.5-6.7 and the hydrolysis time is 1.5-2 hours, so that the lactose content is reduced to below 0.5%.

[0010] Preferably, in step one, the fermentation is controlled by a temperature gradient: the temperature is maintained at 35-39℃ in the early stage of fermentation, increased to 36-40℃ in the middle stage, and increased to 38-42℃ in the later stage.

[0011] Preferably, in step two, the vegetable oil is walnut oil, flaxseed oil, or a vegetable oil rich in linoleic acid.

[0012] Preferably, in step two, the vegetable oil hydrolysate is prepared by mixing vegetable oil, water and emulsifier and then homogenizing under high pressure to obtain an emulsion, followed by adding lipase for enzymatic hydrolysis.

[0013] Preferably, in step two, the torque conversion acquisition component of the fermentation state monitoring device includes: a sliding guide rail disposed on the outer periphery of the fixed connecting ring of the stirring blade; a sliding block installed on the sliding guide rail; an elastic member with one end connected to the sliding block and the other end fixed to the fixed connecting ring; and a pull rope with one end connected to the free end of the stirring blade and the other end connected to the sliding block.

[0014] Preferably, in step two, the torque amplification external lead assembly includes: a lever mounting base fixedly mounted on the fixed connecting ring; a lever whose one end engages with the inclined surface of the sliding block and whose other end is hinged to the lever mounting base; a transmission ring coaxial with the stirring main shaft and located above the fixed connecting ring; and a rigid transmission rod located above the transmission ring and driven by the transmission ring.

[0015] Preferably, in step two, the remedial procedure of the fine-tuning intervention device includes: a first-level remedial measure, which involves switching the stirring mode to pulse stirring, reducing the fermentation temperature by 0.5-1°C, and activating the ultrasonic auxiliary system on the tank wall; and a second-level remedial measure, which involves adding emulsifier to the fermentation liquid based on the first-level remedial measure.

[0016] Preferably, in step three, the wall material includes β-cyclodextrin, sodium alginate, sucrose ester, and maltodextrin.

[0017] Preferably, in step three, the preparation process of the CLA microcapsules includes: heating the CLA fermentation broth to 55-65°C under vacuum conditions, adding wall material, and then homogenizing it through high-speed shearing, ultrasonic treatment, and microfluidic jet to obtain a nanoemulsion, followed by spray drying to obtain microcapsule powder.

[0018] Compared with the prior art, the present invention has the following beneficial effects: Dual-system separation fermentation technology: The fermentation of extracellular polysaccharides and CLA are carried out separately, avoiding mutual interference between the two fermentation systems. This ensures both high yield of extracellular polysaccharides and efficient conversion of CLA.

[0019] Unique fermentation status monitoring device: It adopts the mechanical multi-layer torque sensing principle, and reflects the stability of the emulsion in real time by monitoring the torque difference of the stirring layer at different heights. It avoids the reliability problem of electronic sensors in corrosive fermentation environments, and has high monitoring accuracy and is intuitive and visual.

[0020] Fine-tuning intervention device: Automatically initiates a graded recovery program based on torque differences, effectively preventing demulsification of oil-water two-phase emulsions during anaerobic fermentation by adjusting stirring mode, temperature, and adding emulsifiers, thus ensuring stable CLA fermentation.

[0021] Microcapsule encapsulation technology: Using multi-wall composite encapsulation technology under vacuum conditions, combined with high-speed shearing, ultrasonic treatment and microfluidic homogenization, the CLA encapsulation rate reaches more than 87% and the retention rate reaches more than 92%, which significantly improves the stability and bioavailability of CLA.

[0022] Product Quality: The final product has a high CLA content (>500mg / 100g) and a sufficient number of live bacteria (>10). 8 It contains CFU / g and is rich in various functional components such as extracellular polysaccharides and galactooligosaccharides, and has good sensory quality and a long shelf life (6 months). Attached Figure Description

[0023] Figure 1 Flowchart of the overall preparation process for conjugated linoleic acid functional yogurt; Figure 2 Final product compounding and quality control flowchart; Figure 3 Schematic diagram of the multi-layer stirring assembly and torque conversion and acquisition assembly; Figure 4 Enlarged view of a portion of the torque amplification external lead assembly; Figure 5 Schematic diagram of the overall structure of the fermenter and monitoring device; Figure 6 Front view of the observation cover and scale display system.

[0024] Numbering on the map: 01-02 series: 01 stirring spindle; 02 stirring blades; 021 blade body; 022 fixed connecting ring.

[0025] 100 series: 100 fermentation tank.

[0026] 200 Series: 200 Fermentation Status Monitoring Device; 210 Stirring Component; 220 Torque Conversion and Acquisition Component; 221 Sliding Guide Rail; 222 Sliding Block; 223 Elastic Component; 224 Pull Rope; 230 Torque Amplification External Lead Component; 231 Lever Mounting Base; 232 Lever; 233 Transmission Ring; 234 Rigid Transmission Rod; 240 Monitoring Component; 241 Scale Display Sleeve; 242 Observation Cover.

[0027] Detail numbers: 2221 Sliding block inclined surface; 2331 Transmission ring protrusion; 2332 Transmission ring concave part.

[0028] 300 Series: 300 fine-tuning intervention device; 310 vertical tube. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0030] Example: A method for preparing conjugated linoleic acid (CLA) yogurt through a dual-system separation fermentation process is disclosed. This method employs a dual-system separation fermentation technology, using two independent systems: an extracellular polysaccharide fermented milk prepared from milk hydrolysate and a CLA fermentation broth prepared from walnut oil hydrolysate. The functional product is ultimately prepared through microencapsulation technology and a compounding process. In the crucial step of fermenting the walnut oil hydrolysate, a fermentation monitoring and fine-tuning intervention device is employed. This device integrates a multi-layer torque monitoring system, an electrical impedance spectroscopy analyzer, a NIR detector, and an intelligent control algorithm. This allows for real-time monitoring of the stability of the easily separable oil-water two-phase emulsion system during anaerobic fermentation. Through graded early warning and automatic remedial measures, the risk of emulsion demulsification is controlled, ensuring CLA conversion while maintaining the homogeneous and stable state of the fermentation system.

[0031] Step 1: Preparation of milk hydrolysate and directional fermentation of extracellular polysaccharides Take 1000L of fresh whole milk (protein content 3.2%, fat content 3.6%, lactose content 4.8%) and pump it into a 1500L stainless steel enzymatic hydrolysis tank equipped with a temperature control jacket, pH electrode, temperature probe, and variable frequency stirring system. Start the steam heating system to heat the milk to 85℃ and maintain it for 15 seconds for pasteurization. During the pasteurization process, maintain a low stirring speed of 50rpm to ensure uniform heating. Immediately after pasteurization, switch to cooling mode and circulate 10℃ cooling water through the jacket to reduce the milk temperature to 55℃ within 10 minutes, with a temperature control accuracy of ±2℃. Once the temperature stabilizes at 55℃, start the aseptic feeding system and add a pre-prepared and sterilized (0.22μm) lactase solution quantitatively via a peristaltic pump. The amount of lactase (enzyme activity 2000U / g) added is 0.15% of the milk weight, i.e., 1.5kg. Simultaneously with enzyme addition, the automatic pH adjustment system was activated, and the pH value was controlled within the range of 6.6±0.1 by adding 10% NaOH solution dropwise. The stirring speed was adjusted to 120 rpm, and timed enzymatic hydrolysis began. During enzymatic hydrolysis, a 5 mL sample was automatically taken every 30 minutes, and the lactose content was monitored online. When the enzymatic hydrolysis reached 1.5 hours, the lactose content dropped below 0.5%; enzymatic hydrolysis continued for 2 hours, and the lactose content dropped to 0.01%, while the galactooligosaccharide content reached 2.3 g / L. After enzymatic hydrolysis was completed, the temperature was quickly raised to 85℃ and maintained for 5 minutes for enzyme inactivation, and then rapidly cooled to 37℃ before proceeding to the fermentation process.

[0032] The prepared milk hydrolysate was pumped into a 2000L fermenter equipped with a spiral guide plate and a bottom flow booster. The fermenter was pre-sterilized by steam at 121℃ for 30 minutes and protected under sterile positive pressure. Glyceryl tubes of *Lactobacillus plantarum* CCFM1073 (deposited at the Microbial Culture Collection Center of Jiangnan University, China) were taken from an -80℃ ultra-low temperature freezer and activated in MRS medium at 37℃ for 18 hours until the logarithmic growth phase (OD600=2.5). The cells were collected by centrifugation, washed twice with sterile physiological saline, resuspended, and the cell concentration was adjusted to 5×10^9 CFU / mL. 50L of the bacterial suspension was added to the fermenter through a sterile inoculation port to achieve an initial inoculation volume of 5×10^7 CFU / mL. The temperature gradient control program was initiated: From 0-4 hours, the temperature was maintained at 37℃ to promote rapid cell growth; during this stage, the cells mainly utilize glucose and galactose for proliferation. From 4-8 hours, the temperature was increased to 38℃ to enter the extracellular polysaccharide synthesis phase; this temperature is favorable for the expression of polysaccharide synthase systems. From 8-12 hours, the temperature was increased to 40℃ to promote the release and accumulation of extracellular polysaccharides. The fermentation process employed an intermittent stirring mode, stirring at 100 rpm every 30 minutes, followed by a 30-minute pause. This mode ensured the uniformity of fermentation while avoiding excessive shearing that could damage the polysaccharide structure. At the 10th hour of fermentation, the apparent viscosity reached 950 mPa·s, and the pH dropped to 4.35. After 12 hours of fermentation, the pH change rate was less than 0.02 / h, and the CO2 production rate dropped to 15% of the baseline level. Based on the multi-parameter fusion algorithm, the fermentation was determined to have reached its endpoint. A rapid cooling program was immediately initiated, introducing 0℃ ice water through the jacket to lower the fermentation broth temperature to 4℃ within 20 minutes, while simultaneously reducing the stirring speed to 30 rpm to maintain suspension. Sampling and testing showed that the extracellular polysaccharide content reached 856 mg / L (determined by phenol-sulfuric acid method), the viable bacteria count was 2.3 × 10^9 CFU / mL (plate count method), the pH value was 4.32, the product was uniformly milky white, had good fluidity and stability, and no whey separation occurred.

[0033] Step 2: Preparation of walnut oil enzymatic hydrolysate and CLA-directed fermentation In an emulsification workshop equipped with a high-pressure homogenizer, temperature control system, and online particle size analyzer, 100 kg of refined walnut oil (62% linoleic acid content, purchased from Shandong Luhua Group), 200 kg of purified water, and 5 kg of sucrose ester (HLB value 12, food grade) were added to a 500 L high-speed dispersion tank. First, a coarse emulsion was formed by high-speed dispersion at 3000 rpm for 10 minutes at 60℃. Then, it was pumped into a high-pressure homogenizer for two-stage homogenization, with the first stage pressure set at 25 MPa and the second stage at 5 MPa, repeated three times. During homogenization, the emulsion particle size was monitored using an online laser particle size analyzer, ultimately yielding an emulsion with a D50 of 2.8 μm and a span of 1.6.

[0034] The emulsion was transferred to a 500L enzymatic hydrolysis tank equipped with a ribbon stirrer and pasteurized at 75°C for 15 minutes, with stirring maintained at 80 rpm to prevent stratification. After sterilization, it was rapidly cooled to 37°C, and 1.5 kg of pre-prepared Aspergillus oryzae lipase solution (enzyme activity 1000 U / g) was added through a 0.22 μm sterile filter. The pH was adjusted to 7.2 with 5M NaOH solution. Enzymatic hydrolysis was carried out under constant temperature of 37°C and stirring at 200 rpm. Samples were taken hourly and the free fatty acid content was analyzed by gas chromatography. After 1 hour of enzymatic hydrolysis, the free fatty acid content reached 8.5%, after 2 hours it reached 14.2%, and after 3 hours it reached 17.8%, of which free linoleic acid accounted for 61% of the total free fatty acids. After enzymatic hydrolysis, the temperature was rapidly raised to 85°C and maintained for 10 minutes to inactivate the enzyme, and then cooled to 37°C. An online turbidimeter showed that the emulsion had good stability and no stratification.

[0035] 300L of the prepared walnut oil hydrolysate was transferred into an anaerobic fermentation system, which includes a main fermenter 100, an anaerobic workstation, a fermentation status monitoring device 200, a fine-tuning intervention device 300, a dissolved oxygen monitoring system, and an online NIR detection device.

[0036] The fermentation status monitoring device 200 includes multiple sets of stirring components 210 at different heights, consisting of a stirring main shaft 01 and several stirring blades 02, installed in the fermentation tank 100; at least one torque conversion and acquisition component 220 installed on the stirring blades 02 in each set of stirring components 210; a torque amplification and external lead component 230 that receives the torque acquired by the torque conversion and acquisition component 220 and performs feedback conversion and amplification to display on the Z-axis; and a monitoring component 240 that monitors the changes in the amplified and displayed torque by comparison.

[0037] The stirring main shaft 01 in the stirring assembly 210 is connected to the output shaft of the power source (usually an externally mounted motor) at one end and is a free end extending from the outside to the inside (usually extending from the top to the bottom of the fermentation tank 100) at the other end. The stirring blades 02 are usually two horizontally symmetrical blades connected to the stirring main shaft 01. Each set is usually located at a different horizontal height. The stirring blades 02 include blade body 021 and a fixed connecting ring 022 that connects to the root of blade body 021 and is sleeved on the stirring main shaft 01.

[0038] The torque conversion and acquisition component 220 includes a sliding guide rail 221 disposed on the outer periphery of a fixed connecting ring 022, a sliding block 222 mounted on the sliding guide rail 221, an elastic member 223 whose one end is movably connected to the sliding block 222 (e.g., hinged) and the other end is connected to a mounting seat fixed on the fixed connecting ring 022, forming a movable (e.g., hinged) connection, and a pull rope 224 whose one end is connected to the free end of the stirring blade 02 and the other end is connected to the sliding block 222. The upward-facing surface of the sliding block 222 is an inclined surface 2221, used to transmit power to the torque amplification and external transmission component 230.

[0039] The torque amplification external assembly 230 includes a lever mounting base 231 located above the inclined plane 2221 and fixedly mounted on the fixed connecting ring 022; a lever 232 (the lever 232 is divided at the hinge point, with the length of the end away from the inclined plane being more than twice the length of the other end) that slides and / or rolls with the inclined plane 2221 at one end and extends upward and is hinged to the lever mounting base 231 near the inclined plane 2221 at the other end); a transmission ring 233 coaxial with the stirring main shaft 01 and located above the fixed connecting ring 022; and a rigid transmission rod 234 located above the transmission ring 233 and driven by the transmission ring. The upper end face of the transmission ring 233 has a protruding portion 2331 with an inclined plane or arc shape for contacting the rigid transmission rod 234 to move it upward; and a concave portion 2332 with an inclined plane or arc shape for contacting the end of the lever 232 away from the inclined plane to rotate it. It should be noted that the length of each rigid drive rod 234 will be matched according to the height position of the mating drive ring 233, and each rigid drive rod 234 extends upward to a uniform height outside the fermenter 100. Preferably, the rigid drive rod 234 is equipped with casters.

[0040] The monitoring component 240 includes a scale display sleeve 241 fitted onto the portion of each rigid transmission rod 234 located outside the fermenter 100, so that all rigid transmission rods 234 can be initially aligned to the same starting point, such as zero point; an observation cover 242 is provided in the center for the stirring spindle 01 to pass through, and is detachably fixed to the upper surface of the fermenter cover. The position in the observation cover 242 corresponding to the scale display sleeve 241 (generally horizontally) is partially or completely covered with transparent material; a follow-up mounting bracket is fixedly connected to the stirring spindle 01 at one end and extends outward at the other end to form a camera mounting part, and the imaging equipment, generally a camera, is positioned and mounted on the follow-up mounting bracket.

[0041] Working principle and monitoring process of fermentation status monitoring device 200: This monitoring device employs a mechanical multi-layer torque sensing principle, visually monitoring the resistance torque of the fermentation broth on the stirring blades by converting it into vertical displacement. The working process is as follows: (1) Torque acquisition stage: When the viscosity of the fermentation liquid changes, the resistance torque of the stirring blade 02 at different heights changes accordingly. This torque is transmitted to the sliding block 222 through the pull rope 224, causing the sliding block to generate radial displacement along the sliding guide rail 221. The displacement magnitude is proportional to the torque.

[0042] (2) Signal amplification and transmission stage: The inclined surface 2221 of the sliding block 222 pushes the lever 232 to rotate around the lever mounting base 231. Due to the lever ratio design (the length of the end away from the inclined surface is more than half that of the end near the inclined surface), the torque signal is mechanically amplified. The far end of the lever 232 pushes the transmission ring 233 to rotate, and the protrusion 2331 on the transmission ring converts the rotation into the vertical upward motion of the rigid transmission rod 234.

[0043] (3) Signal external display stage: The rigid transmission rods 234 of each layer transmit the torque signal to the outside of the fermenter in the form of vertical displacement, and the torque is quantitatively read through the scale display sleeve 241. The torque difference of the stirring layers at different heights is intuitively reflected in the height difference of each transmission rod.

[0044] (4) Automatic monitoring and recording stage: The camera fixed on the follow-up mounting bracket captures the changes in the readings of each scale display sleeve 241 in real time, and automatically reads the values ​​and records the torque change curve through the image recognition algorithm. When the torque difference between the upper, middle and lower layers exceeds 3%, the system automatically issues an early warning; when the difference reaches 10%, the oil phase separation alarm is triggered and the automatic remedial program is started.

[0045] The advantages of this device are: ① It avoids the reliability issues of electronic sensors in corrosive fermentation environments; ② It achieves high-precision monitoring through mechanical amplification; ③ Multi-layer synchronous monitoring can promptly detect emulsion stratification trends; ④ The visual display allows operators to intuitively judge the fermentation status. This monitoring device can control torque changes within a reasonable range, ensuring the stability of the emulsion system during CLA fermentation.

[0046] The dissolved oxygen monitoring system employs multi-point electrochemical sensors to monitor the dissolved oxygen concentration at different locations within the fermenter in real time, maintaining an anaerobic environment (<0.1ppm). The NIR detection device uses near-infrared spectroscopy to detect changes in the composition of the fermentation broth online and monitor the CLA formation process.

[0047] Anaerobic fermentation process and fine-tuning intervention device workflow: First, the fermenter was anaerobically treated: a vacuum was applied to -0.075 MPa for 30 minutes to remove air from the tank, then high-purity nitrogen (99.999%) was introduced to atmospheric pressure, and this process was repeated three times. Finally, a nitrogen positive pressure of 0.01 MPa was maintained, and nitrogen was continuously introduced at a flow rate of 0.1 vvm to maintain the anaerobic environment. In an anaerobic glove box, *Bifidobacterium breve* FBJCP2M1 (deposited at the Microbial Culture Collection Center of Jiangnan University, China) was removed from -80°C and inoculated into pre-reduced modified MRS medium (with 0.5 g / L cysteine ​​hydrochloride and 0.5 g / L ascorbic acid added), and anaerobically cultured at 37°C for 20 hours until the OD600 reached 3.0. The cells were collected, washed and resuspended with anaerobic PBS buffer, and the bacterial concentration was adjusted before being added to the fermenter through an anaerobic transfer chamber, resulting in an initial inoculum of 1 × 10^8 CFU / mL.

[0048] The fermentation temperature was controlled at 37±0.5℃, and the pH was maintained within the range of 7.0±0.2 by an automatic titration system (10% NaOH and 10% lactic acid).

[0049] When the fermentation status monitoring device 200 detects a torque difference exceeding a preset threshold, the fine-tuning intervention device 300 automatically initiates a graded remedial program: The first stage involves switching the constant-speed stirring to pulse stirring mode, operating in a cyclical pattern of 80 rpm (2 min) - 100 rpm (2 min) - 120 rpm (2 min) - 100 rpm (2 min) - 80 rpm (2 min); simultaneously, the fermentation temperature is slowly reduced from 37℃ to 36℃ at a rate of 0.5℃ / 10 min; and the tank wall ultrasonic auxiliary system is activated, operating at a low power of 50W in pulse mode (30 seconds on / 90 seconds off) for gentle treatment. The second stage, based on the first stage, involves supplementing the fermentation broth with 0.3% sucrose ester and 0.2% lecithin via the feeding system, evenly dispersing them into the fermentation liquid through the vertical pipe 310 atomizing nozzle.

[0050] After 4 hours of fermentation, the CLA concentration reached 1.2 g / L, with a free linoleic acid consumption rate of 15%; after 8 hours, the CLA concentration was 3.1 g / L, with a free linoleic acid consumption rate of 28%; after 12 hours, the CLA concentration was 4.5 g / L, with a free linoleic acid consumption rate of 42%; and after 16 hours, the CLA concentration reached 5.3 g / L, with a free linoleic acid consumption rate of 42%, achieving the target conversion rate. Throughout the fermentation process, the fermentation status monitoring device 200 showed that the torque difference between the upper, middle, and lower layers remained within 8%, indicating good emulsion stability. At the end of fermentation, samples were taken for GC-MS analysis, which showed a total CLA content of 5.28 g / L, of which c9,t11-CLA accounted for 76.3%, t10,c12-CLA accounted for 18.5%, and other isomers accounted for 5.2%, with product quality meeting expectations.

[0051] Step 3: CLA microcapsule encapsulation After fermentation, the CLA fermentation broth was not separated but directly encapsulated in the original fermenter. First, under vacuum (-0.08 MPa), the fermentation broth was slowly heated from 37°C to 60°C at a rate of 2°C / min. The vacuum effectively prevented CLA oxidation during the heating process. Once the temperature stabilized at 60±1°C, the wall material addition system was activated. This system consisted of four independent powder feeders and a Venturi mixer. β-cyclodextrin powder was added through feeder 1 at a rate of 100 g / min, achieving a final concentration of 13%. The wall material was drawn into the fermentation broth by the negative pressure of the Venturi tube, preventing powder agglomeration. After the β-cyclodextrin is completely dissolved (approximately 15 minutes), sodium alginate (feeder 2, final concentration 3.5%), sucrose ester (feeder 3, final concentration 6.5%), and maltodextrin (feeder 4, final concentration 2.5%) are added sequentially, with each wall material added 10 minutes apart, while maintaining stirring at 200 rpm to ensure uniform dissolution. After all wall materials have been added, the pH is adjusted to 7.0 ± 0.1 using 2M NaOH solution.

[0052] Subsequently, a high-speed shear dispersion system was activated. This system includes a rotor-stator type high-speed shear machine installed at the bottom of the tank, which shears at 10,000 rpm for 5 minutes, during which the temperature is controlled within the range of 60±2℃ by jacket cooling water. After shearing, three ultrasonic transducers (frequency 20kHz, power 150W / each) installed on the tank wall were activated and treated in pulse mode (30 seconds of operation, 30 seconds of interval) for 10 minutes. Ultrasonic treatment can further reduce the emulsion particle size and promote the embedding effect between the wall material and CLA. The treated emulsion was pumped into a microfluidic homogenizer through a circulation pipeline and treated through a Y-type interactive cavity at a pressure of 30MPa, circulated 3 times. After each treatment, the particle size distribution was detected by an online laser particle size analyzer, and finally a nanoemulsion with a D50 of 285nm and a PDI of 0.18 was obtained.

[0053] Microcapsule drying was performed using a spray drying tower equipped with two-fluid nozzles. The drying parameters were set as follows: inlet air temperature 170℃, outlet air temperature 80±2℃, feed flow rate 15mL / min, atomization pressure 0.3MPa, and nitrogen as the atomizing gas (to reduce oxidation). 0.02% vitamin E was added to the feed solution as an antioxidant before feeding. The spray drying process lasted approximately 4 hours, yielding a pale yellow microcapsule powder, which was immediately packaged and sealed under nitrogen purging. Sampling and analysis showed: microcapsule moisture content 3.8%, encapsulation efficiency 87.3% (calculated from surface oil and total oil content), CLA retention 92.5% (compared to before encapsulation), particle size distribution D50 312nm (measured after rehydration), and a Zeta potential of -28.5mV, indicating good system stability. Scanning electron microscopy revealed that the microcapsules were regularly spherical with smooth, crack-free surfaces, indicating ideal encapsulation. The microcapsule powder was dispersed in water to form a 5% suspension. After being stored at 37°C for 7 days, the CLA retention rate was still above 90%, while the retention rate of the unencapsulated CLA control group was only 45%. Microencapsulation increased the CLA retention rate by 45 percentage points.

[0054] Step 4: Product compounding and quality evaluation The final product is prepared in a constant-temperature compounding tank (2000L, equipped with a double-jacketed refrigeration system) in a cleanroom. First, the compounding tank and all piping are disinfected with 75% alcohol, then rinsed with sterile water, and finally dried with sterile air. The refrigeration system is activated to lower the tank temperature to 4°C and maintain it at a constant temperature, with fluctuations controlled within ±0.5°C. 1400L of the prepared fermented milk rich in extracellular polysaccharides is pumped into the compounding tank through sterile piping, and the anchor stirrer is turned on at a low speed of 30 rpm. On a sterile workbench, 260kg of CLA microcapsule powder is added in batches to 50L of 4°C sterile water, and pre-dispersed using a magnetic stirrer at 200 rpm for 10 minutes, taking care to avoid excessive foaming. The pre-dispersed microcapsule suspension is slowly added to the compounding tank using a peristaltic pump at a flow rate of 5L / min, with the addition point below the liquid surface to reduce air ingress. The entire addition process takes approximately 50 minutes.

[0055] After all the microcapsules were added, stirring continued for 30 minutes to ensure uniform dispersion. During this time, observation was performed through the sight glass on the tank wall to confirm that there was no obvious particle aggregation or stratification. Then, the following functional ingredients were added sequentially: 15 kg of L-histidine (dissolved in 100 L of water), 45 kg of fructooligosaccharides (prepared as a 50% solution), 5 kg of pectin (pre-dissolved and cooled in water at 80°C), and 2 kg of natural vanilla flavoring. After all ingredients were added, the stirring speed was increased to 50 rpm, and mixing was continued for 30 minutes. The pH of the product was monitored using an online pH meter at this point, and then adjusted to 4.35 ± 0.05 using a 10% citric acid solution. The prepared solution was then piped to a low-pressure homogenizer for gentle homogenization at 15 MPa. Note that this pressure will not damage the microcapsule structure; it only serves to further improve the product texture. After homogenization, the product is rapidly cooled to 4°C using a plate heat exchanger and then enters an aseptic filling machine. The filling specification is 200mL / bottle, the filling speed is 3000 bottles / hour, and the filling accuracy is ±2mL.

[0056] The final product underwent quality evaluation, and the test results are as follows: In terms of physicochemical indicators, the protein content was 3.05% (Kjeldahl method), fat content was 3.42% (Soxhlet extraction method), CLA content was 523 mg / 100g (GC-MS determination), extracellular polysaccharide content was 92 mg / 100g (phenol-sulfuric acid method), lactose content was 0.007% (HPLC method), and histidine content was 58 mg / 100g (amino acid analyzer). All indicators met or exceeded the design requirements. In terms of microbiological indicators, the viable count of *Lactobacillus plantarum* was 1.8 × 10^8 CFU / g, the viable count of *Bifidobacterium breve* was 8.5 × 10^7 CFU / g, the total viable count was 2.65 × 10^8 CFU / g, coliform bacteria were <3 CFU / g, molds and yeasts were <10 CFU / g, and pathogenic bacteria were not detected. The product meets national food safety standards. In terms of sensory evaluation, the product is uniformly milky white with a slight yellow tinge, has a delicate and smooth texture without any grainy feel, a moderately sweet and sour taste, a light fermented milk aroma, and no off-flavors or unpleasant tastes. The overall score is 92 points (out of 100).

[0057] Stability evaluation showed that, under 4°C storage conditions, the CLA retention rates on days 7, 14, and 21 were 98.2%, 95.6%, and 91.3%, respectively; microcapsule sedimentation rates were 0.8%, 1.5%, and 2.1%, respectively; pH ​​remained stable within the range of 4.30-4.38; no whey separation or stratification occurred; and the viable cell count remained above 10^7 CFU / g at day 21. Accelerated testing (7 days at 37°C is equivalent to 6 months at 4°C) showed that the product quality still met the standards, and the predicted shelf life is up to 6 months.

[0058] Although this disclosure has been described in detail with reference to only a limited number of embodiments, it should be readily understood that this disclosure is not limited to these disclosed embodiments. Rather, this disclosure may be modified to incorporate any number of variations, alterations, substitutions, combinations, subcombinations, or equivalent arrangements not described above but commensurate with the scope of this disclosure. Furthermore, while various embodiments of this disclosure have been described, it should be understood that aspects of this disclosure may include only some of the described embodiments.

Claims

1. A method for the preparation of a conjugated linoleic acid yoghurt by a dual system separation fermentation, characterized in that, It comprises the following steps: Step one: preparation of the exopolysaccharide-rich fermented milk After the lactose hydrolysis of milk, the exopolysaccharide-producing lactic acid bacteria are inoculated for fermentation to obtain the exopolysaccharide-rich fermented milk; Step two: preparation of the CLA fermentation broth The plant oil hydrolysate is inoculated with probiotics with linoleic acid isomerization capacity under anaerobic conditions for fermentation, and a fermentation state monitoring device is used to monitor the torque changes of the fermentation broth at different heights in real time. When the torque difference exceeds the preset threshold, a fine intervention device is started to remedy, and the CLA fermentation broth is obtained; The fermentation state monitoring device comprises: A plurality of stirring assemblies installed at different heights in the fermentation tank; A torque conversion acquisition assembly installed on the stirring assembly; The torque signal is amplified and converted into vertical displacement and transmitted to the torque amplification external lead assembly outside the fermentation tank; A monitoring assembly for monitoring torque changes; Step three: preparation of CLA microcapsules The CLA fermentation broth is mixed with the wall material, and then homogenized and dried to obtain the CLA microcapsules; Step four: product compounding The exopolysaccharide-rich fermented milk prepared in step one is mixed with the CLA microcapsules prepared in step three to obtain conjugated linoleic acid functional yogurt.

2. The production method according to claim 1, characterized by, In step one, the lactose hydrolysis process controls the pH value in the range of 6.5-6.7, and the enzyme hydrolysis time is 1.5-2 hours, so that the lactose content is reduced to below 0.5%.

3. The preparation method according to claim 1, characterized in that, In step one, the fermentation adopts temperature gradient control: the temperature is maintained at 35-39℃ in the early stage, increased to 36-40℃ in the middle stage, and increased to 38-42℃ in the later stage.

4. The preparation method according to claim 1, characterized in that, In step two, the plant oil is walnut oil, flaxseed oil or plant oil rich in linoleic acid.

5. The preparation method according to claim 1, characterized in that, In step two, the plant oil hydrolysate is prepared by the following method: plant oil, water and emulsifier are mixed and subjected to high-pressure homogenization to obtain an emulsion, and lipase is added for enzyme hydrolysis.

6. The method of claim 1, wherein, In step two, the torque conversion acquisition assembly of the fermentation state monitoring device comprises: A sliding guide rail arranged on the outer periphery of the fixed connection ring of the stirring paddle; A sliding block mounted on the sliding guide rail; A spring member connected to the sliding block at one end and fixed to the fixed connection ring at the other end; A pull rope connected to the free end of the stirring paddle at one end and connected to the sliding block at the other end.

7. The production method according to claim 6, wherein In step two, the torque amplification external lead assembly comprises: A lever mounting seat fixedly installed on the fixed connection ring; A lever hinged at one end to the inclined surface of the sliding block and at the other end to the lever mounting seat; A transmission ring coaxial with the stirring main shaft and located above the fixed connection ring; A rigid transmission rod driven by the transmission ring and located above the transmission ring.

8. The method of claim 1, wherein, In step two, the remedial program of the fine intervention device comprises: First-level remediation: switch the stirring mode to pulse stirring, reduce the fermentation temperature by 0.5-1℃, and start the tank wall ultrasonic auxiliary system; Second-level remediation: on the basis of the first-level remediation, supplement emulsifier to the fermentation broth.

9. The method of claim 1, wherein, In step three, the wall material comprises β-cyclodextrin, sodium alginate, sucrose ester and malt dextrin.

10. The method of any one of claims 1-9, wherein, In step three, the preparation process of the CLA microcapsules comprises: heating the CLA fermentation broth to 55-65℃ under vacuum conditions, adding the wall material, and then subjecting to high-speed shearing, ultrasonic treatment and microfluidic homogenization to obtain a nanoemulsion, and then subjecting to spray drying to obtain microcapsule powder.

11. A fermentation state monitoring device for a fermentation process, characterized in that It comprises: A plurality of stirring assemblies are installed at different height positions in the fermenter, each of the stirring assemblies comprises a stirring spindle and stirring blades fixed on the stirring spindle; A torque conversion acquisition assembly is installed on the stirring blades, which is used to convert the torque received by the stirring blades into a radial displacement signal; A torque amplification and external transmission assembly is used to amplify the radial displacement signal and convert it into an axial displacement signal transmitted to the outside of the fermenter; A monitoring assembly is used to display and record the axial displacement signal, thereby achieving monitoring of the torque changes of the fermentation liquid at different heights.

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