Rapid pickle fermentation method based on gradient temperature control and flora directional fermentation

Abstract

The invention discloses a rapid pickle fermentation method based on gradient temperature control and flora directional fermentation, and relates to the field of biological fermentation. Comprising the following steps: S100, chopping vegetables, performing cleaning, performing deastringency treatment, and performing draining for standby application; s200, lactococcus lactis, leuconostoc mesenteroides and lactobacillus plantarum are prepared into a three-layer embedding bacterial agent, and the three-layer embedding bacterial agent and the vegetables are put into saline water together; s300, nitrogen is introduced into a system in the step S200 to enable the dissolved oxygen content to be smaller than or equal to 0.5 mg / L, and gradient temperature control fermentation is performed in three stages; and S400, after fermentation is completed, adding mother water and then refrigerating. The three-layer embedding microbial agent is accurately matched with three-stage gradient temperature control fermentation, so that the fermentation period is shortened to be less than 78 hours, the residual quantity of nitrite is less than or equal to 0.5%, and the content of flavor substances is effectively increased.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a rapid fermentation method for kimchi based on gradient temperature control and directional fermentation of microbial communities. Background Technology

[0002] Traditional kimchi fermentation relies entirely on the natural succession of microbial communities in the environment. The fermentation process typically takes 15 to 30 days to complete, a lengthy process with high energy consumption, accounting for over 35% of the total cost. Because the microbial composition is significantly affected by factors such as season and region, the fermentation cycle can even extend to over 40 days in low-temperature winter conditions, resulting in a long-term utilization rate of less than 50%. Furthermore, harmful bacteria in the natural environment (such as nitrate-reducing bacteria) proliferate rapidly in the early stages of fermentation, causing nitrite levels to surge to a toxic peak of 50–80 mg / kg within 3 to 7 days, far exceeding the food safety limit (≤20 mg / kg), posing a clear carcinogenic risk. Simultaneously, the contamination rate of mold and other spoilage microorganisms is as high as 12%–18%, easily leading to quality problems such as bloating and softening of the product.

[0003] The randomness of the environmental microbial community leads to significant fluctuations in the proportion of lactic acid bacteria, ranging from 30% to 80%, resulting in severe dispersion of key indicators: pH values ​​deviate from the ideal range (3.2–3.6) by ±0.8 units, vegetable crispness loss rate reaches 15%–40%, and the content of core flavor substances (such as ethyl acetate and ethyl lactate) varies by more than 3 times. This batch-to-batch inconsistency seriously hinders the standardization and premiumization of products. Furthermore, traditional processes have failed to effectively tap the metabolic potential of functional strains, and the loss of high-value-added components has become a common problem in the industry.

[0004] To address these shortcomings, existing technologies have undertaken a series of explorations, but still suffer from systemic limitations. Regarding the application of microbial agents, the mechanical addition of fixed strain combinations leads to a mismatch between time and space: in the early stages of fermentation, the lack of rapidly producing acid-producing bacteria to suppress contaminating bacteria prolongs the start-up time; in the middle stages, the addition of flavor-producing bacteria is delayed, missing the optimal metabolic window; and in the later stages, the failure to introduce nitrite-degrading bacteria in a timely manner results in excessive residues.

[0005] Furthermore, safety assurance relies on end-product testing rather than process intervention, making it impossible to suppress nitrite formation in real time. Traditional processes and existing improved technologies both treat dynamic systems from a static perspective, making it difficult to synergistically optimize efficiency, safety, and quality. Therefore, developing an integrated method that can precisely coordinate the temporal expression of microbial community function with the dynamic adaptation of environmental parameters has become a key breakthrough in reconstructing the fermentation paradigm of kimchi. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid fermentation method for kimchi based on gradient temperature control and directional fermentation of microbial communities. By activating different functional microbial communities through three-stage gradient temperature control, combined with the sequential addition of compound microbial agents and multi-layer encapsulation technology, the fermentation cycle is shortened to less than 78 hours, while reducing the residual nitrite content to less than 0.5%, effectively improving the content of flavor substances, and solving the problems of long natural fermentation cycle, unstable flavor, and poor safety.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A rapid fermentation method for kimchi based on gradient temperature control and directional microbial fermentation includes the following steps:

[0009] S100. After chopping the vegetables, wash them, remove the bitterness, drain the water and set aside.

[0010] S200: Prepare a three-layer encapsulated bacterial agent by Lactococcus lactis, Leuconostoc mesenteroides and Lactobacillus plantarum, and add it to the brine along with vegetables;

[0011] S300: Nitrogen gas is introduced into the S200 system to make the dissolved oxygen content ≤0.5mg / L, and fermentation is carried out in three stages with gradient temperature control.

[0012] After fermentation of S400 is complete, add mother water and refrigerate.

[0013] The three-layer encapsulated microbial agent of this invention is precisely matched with the three-stage gradient temperature-controlled fermentation. By controlling the temperature of the three-stage gradient temperature-controlled fermentation, the three-layer encapsulated microbial agent is dissolved in layers and the microbial community is released and activated in stages. This ensures that the three functional fermentation strains grow into the dominant microbial community within a specific time sequence. The temperature gradient changes are matched with the growth and metabolic peaks of the three functional microbial communities, thereby shortening the fermentation cycle to less than 72 hours, reducing nitrite residue by more than 40%, and increasing the content of flavor substances.

[0014] Furthermore, in step S200, the preparation method of the three-layer embedded bacterial agent includes the following steps:

[0015] Step 1: Dissolve sodium alginate and xylooligosaccharide in deionized water, cool down, add Leuconostoc mesenteroides to make a mixture, add calcium chloride solution dropwise to the mixture, and solidify to form inner microspheres;

[0016] Step 2: Dissolve gelatin and lactitol in deionized water, cool down, add Lactobacillus plantarum to prepare an embedding solution, immerse the inner layer microspheres in the embedding solution, and then quickly transfer them to a low-temperature environment to solidify, thus preparing the middle layer embedded microspheres.

[0017] Step 3: Dissolve CMC and sucrose ester in deionized water, cool, add lactococcus lactis to prepare a coating solution, spray the coating solution evenly onto the surface of the middle layer embedded microspheres, and dry until the water content is ≤8% to obtain a three-layer embedded bacterial agent.

[0018] The inner layer microspheres have a particle size of 300–500 μm; the middle layer embedded microspheres have a particle size of 600–800 μm; and the outer layer coated microspheres have an overall particle size of 1000–1200 μm, with a coating layer thickness of 30–50 μm.

[0019] Step 3 is used to prepare the outer layer bacterial agent, whose encapsulation structure consists of a sodium carboxymethyl cellulose (CMC) and a sucrose ester oxygen barrier layer, and the encapsulated bacterial species is *Lactococcus lactis*. Step 2 is used to prepare the middle layer bacterial agent, whose encapsulation structure consists of a sodium alginate and xylooligosaccharides, and the encapsulated bacterial species is *Leuconostoc mesenteriae* PCY-3R18. Step 1 is used to prepare the inner layer bacterial agent, whose encapsulation structure consists of a gelatin-lactitol composite, and the encapsulated bacterial species is *Lactobacillus plantarum*.

[0020] Furthermore, in step 1, the mass ratio of sodium alginate, xylooligosaccharides, and deionized water is in the range of 2–3:1.0–1.5:100, and the dosage of Leuconostoc mesenteroides is 2 × 10⁻⁶. 9 ~3×10 9 The solution contains CFU / mL; the dissolution temperature is 50–60℃, and the temperature after cooling is 28–32℃; the mass fraction of calcium chloride in the system is 1.8–2.2%; the curing time is 25–35 min; the solution temperature is controlled at 23–27℃; and the pH value is 6.0–7.0.

[0021] Furthermore, in step 2, the mass ratio of gelatin, lactitol, and deionized water is in the range of 7–9:4–6:100, and the amount of *Lactobacillus plantarum* is 2.5 × 10⁻⁶. 9 ~3.5×10 9 The embedding solution was prepared at a concentration of CFU / mL, with a dissolution temperature of 37–42℃ and a subsequent cooling temperature of 23–26℃. The low-temperature curing environment was cold ethanol at 3–5℃, with a curing time of 15–20 min.

[0022] Furthermore, in step 3, the mass ratio of CMC, sucrose ester, and deionized water ranges from 1.6–2.0:0.4–0.6:100, and the amount of Lactococcus lactis used is 3.0 × 10⁻⁶. 9 ~4.0×10 9 The coating solution has a CFU / mL concentration, a dissolution temperature of 75–85℃, and a cooling temperature of 28–32℃; the spray rate is 1.5–2.5 mL / min, and the inlet air temperature is 30–40℃.

[0023] Further, in step S200, the solid-liquid ratio of vegetables to brine is 1:1.8-2.2, the salt content of brine is 2.5-3%, and the mass ratio of vegetables to three-layer encapsulated bacterial agent is 100:0.25-0.4.

[0024] Furthermore, in step S300, the three-stage gradient temperature-controlled fermentation method includes:

[0025] Stage 1: Ferment at 20-25℃ for 12-18 hours;

[0026] Stage 2: Fermentation at 10-15℃ for 24-36 hours;

[0027] Stage 3: Ferment at 15-18℃ for 18-24 hours.

[0028] Stage 1: After the outer layer of bacterial agent encapsulation structure comes into contact with the fermentation broth, the outer layer dissolves rapidly (<10 min), quickly occupies its ecological niche, and produces acid within 12 h, lowering the pH to below 4.5, inhibiting E. coli and other miscellaneous bacteria; at the same time, it consumes oxygen, creating an environment for anaerobic bacteria. Simultaneously, the low pH environment triggers the swelling process of the middle layer of gelatin.

[0029] Phase 2: When the pH of the fermentation broth drops below 4.5, the gelatin embedded in the middle layer of the bacterial agent will absorb water, swell, disintegrate, and release *Lactobacillus plantarum* cells under acidic conditions, with a release rate of 80% within 24 hours. Xylooligosaccharides act as prebiotics to promote cell proliferation. The synthesis of extracellular polysaccharides (dextran) increases the viscosity of the kimchi and produces flavor compounds such as diacetyl and acetoin.

[0030] At slightly lower temperatures (10–15°C), the growth rate of *Lactobacillus plantarum* slows down significantly. However, this actually shifts its metabolic pathway from rapid self-replication to more secondary metabolism. This low-temperature stress can effectively stimulate the activity of certain specific metabolic pathways in *Lactobacillus plantarum*, enabling it to synthesize flavor and texture substances such as diacetyl, acetoin, and extracellular polysaccharides more efficiently.

[0031] If stage 2 uses high temperatures (e.g., 30°C), *Lactobacillus plantarum* will grow excessively, rapidly consuming the sugars and other substrates in the culture medium. This leads to two problems: substrate waste, as the raw materials may be insufficient by the time stage 3 requires these substrates to degrade nitrite and synthesize GABA; and niche competition, where the rapidly growing *Lactobacillus plantarum* may inhibit the activity of *Leuconostoc mesenteroides*, which is released in later stages.

[0032] Stage 3: When the lactic acid accumulation is ≥5g / L and the temperature rises above 15℃, the sodium alginate-calcium alginate gel formed by sodium alginate and calcium chloride in the inner bacterial agent encapsulation structure, under the action of lactic acid and other organic acids, releases calcium ions (Ca... 2+ It will gradually be replaced by hydrogen ions (H) +The chelation and substitution of lactic acid molecules leads to the collapse of the gel network structure, slowly releasing Leuconostoc mesenteroides PCY-3R18 cells. Leuconostoc mesenteroides PCY-3R18 can continue to synthesize flavor compounds and various enzyme systems, enriching the layers of flavor.

[0033] At this point, the still-active *Lactobacillus plantarum* efficiently degrades nitrite at around 16°C (the peak temperature of its nitrite reductase activity) and synthesizes γ-aminobutyric acid (GABA).

[0034] Furthermore, in stage 2, calcium chloride of 0.1% to 0.3% of the initial weight of the vegetables was added and the vegetables were simultaneously treated with ultrasound at 35 to 40 kHz for 10 to 12 minutes.

[0035] Calcium chloride enhances cross-linking by forming "calcium bridges" with pectic acid in vegetable cell walls, matching the resonant frequency of plant cell walls at 35–40 kHz, thus improving calcium ion permeation efficiency; time 10–12 min: ensuring Ca 2+ The cross-linking reaction with pectin is completed at a rate greater than 90%.

[0036] Further, in step S400, after fermentation is complete, add 4-6% of the total mass of fermented kimchi as mother brine, vacuum pack, and refrigerate at 2-4℃ for 22-26 hours for post-fermentation; the mother brine contains ≥1×10⁻⁶ lactic acid bacteria. 8 The concentration of the sample is CFU / mL, and the pH is ≤3.8, with Lactobacillus plantarum accounting for 50%–70%.

[0037] Mother water, used as an inoculum for highly active lactic acid bacteria, constitutes 4-6% of the total mass of fermented kimchi, ensuring a lactic acid bacteria concentration ≥10. 8 The CFU / mL concentration is evenly distributed throughout the system, preventing localized over-acidity or uneven fermentation. The addition of mother water rapidly initiates post-fermentation, introducing active microbial communities to accelerate the synthesis of flavor compounds (such as ethyl lactate) while inhibiting unwanted microorganisms. Post-fermentation at 2–4℃ for 22–26 hours synergistically regulates microbial metabolism and enzymatic reactions, which is crucial for achieving complex flavors, textural stability, and enhanced safety in kimchi.

[0038] Further, in step S100, the specific method for de-astringency treatment includes: soaking in a citric acid solution with a mass fraction of 0.08-0.12% for 15-25 minutes to remove astringency, wherein the mass ratio of vegetables to the 0.08-0.12% citric acid solution is 1:2-3.

[0039] Citric acid removes the astringent taste of vegetables through two pathways: the carboxyl group of citric acid combines with the hydroxyl group of tannins to eliminate the astringent sensation; at the same time, it reduces the activity of polyphenol oxidase (PPO) by more than 50%, blocking the browning reaction.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] This invention utilizes a layered encapsulation process—core (Leuconostoc mesenteroides), shell (Lactobacillus plantarum), and coat (Lactococcus lactis)—to completely eliminate bacterial antagonism and resolve the acid stress damage issue present in the mixed encapsulation in Comparative Example 1. The outer layer utilizes CMC contact dissolution, the middle layer uses temperature-controlled phase transition of gelatin, and the inner layer uses sodium alginate for slow release, precisely matching the metabolic window of the bacterial community and avoiding the spatiotemporal misalignment and failure of free bacteria. This effectively ensures that the three functional fermentation strains grow into dominant communities within specific timeframes, allowing temperature gradient changes to match the growth and metabolic peaks of the three functional bacterial communities. This results in a fermentation cycle shortened to below 78 hours, nitrite residue reduced to below 0.5 mg / kg, and significantly increased flavor compound content: GABA content can reach 39.1 mg / 100g, and total flavor compounds can reach 1420 μg / g. In stage 2, 0.1–0.3% CaCl2 was added and sonicated at 35–40 kHz to promote the expansion of cell wall micropores, maintaining a fragility retention rate of >90%. Dynamic control of nitrogen filling and oxygen removal ensured dissolved oxygen levels ≤0.5 mg / L, inhibiting aerobic bacteria and reducing the contamination rate to <3%. Detailed Implementation

[0042] Example 1

[0043] The preparation method of the three-layer embedded bacterial agent includes the following steps:

[0044] Step 1: Leuconostoc mesenteroides PCY-3R18 was inoculated into MRS medium and cultured at 25°C for 24 hours (OD200). 600 =1.8); collect the bacterial sludge by centrifugation, and resuspend it in 0.1 mol / L PBS buffer (pH 7.0) to a final volume of 1×10⁻⁶. 10 CFU / mL.

[0045] At 55℃, 2.5g sodium alginate and 1.2g xylooligosaccharide were dissolved in 100mL deionized water. After cooling to 30℃, 33.3mL of Leuconostoc mesenteroides suspension was added to prepare a mixture. Calcium chloride solution was added dropwise to the mixture to make the mass fraction of calcium chloride in the system 2%. The solution temperature was controlled at 25℃ and the pH value was 6.5. The mixture was magnetically stirred at 200rpm for 30min to solidify and form inner layer microspheres. The mixture was rinsed 3 times with sterile water and temporarily stored at 4℃.

[0046] Step 2: Collect Lactobacillus plantarum by centrifugation (10,000 rpm, 10 min), and suspend the bacterial sludge in 10% skim milk as a preservative.

[0047] At 40℃, dissolve 8g gelatin and 5g lactitol in 100mL deionized water. After cooling to 25℃, add 3×10 11 CFU of Lactobacillus plantarum was used to prepare an embedding solution. The inner layer microspheres were immersed in the embedding solution for 10 seconds and then quickly transferred to cold ethanol at 4°C for 18 minutes to solidify, thus preparing the middle layer embedded microspheres.

[0048] Step 3: At 80℃, dissolve 1.8g CMC and 0.5g sucrose ester in 100mL deionized water. After cooling to 30℃, add 3.5×10 11 CFU (Cytobacter oxytococcus lactis) was used to prepare a coating solution. The middle layer of embedded microspheres was placed in a fluidized bed with an inlet air temperature of 35°C. The coating solution was sprayed at a rate of 2 mL / min to ensure uniform coverage of the outer layer. The microspheres were then dried to a moisture content of 6% to obtain a three-layer embedded bacterial agent.

[0049] Example 2

[0050] The preparation method of the three-layer embedded bacterial agent includes the following steps:

[0051] Step 1: Leuconostoc mesenteroides PCY-3R18 was inoculated into MRS medium and cultured at 25°C for 24 hours (OD200). 600 =1.8); collect the bacterial sludge by centrifugation, and resuspend it in 0.1 mol / L PBS buffer (pH 7.0) to a final volume of 1×10⁻⁶. 10 CFU / mL.

[0052] At 50℃, 2g of sodium alginate and 1g of xylooligosaccharide were dissolved in 100mL of deionized water. After cooling to 32℃, 26.7mL of Leuconostoc mesenteroides suspension was added to prepare a mixture. Calcium chloride solution was added dropwise to the mixture to make the mass fraction of calcium chloride in the system 1.8%. The solution temperature was controlled at 23℃ and the pH value was 6. The mixture was magnetically stirred at 200rpm for 25min to solidify and form inner layer microspheres. The mixture was rinsed 3 times with sterile water and temporarily stored at 4℃.

[0053] Step 2: Collect Lactobacillus plantarum by centrifugation (10,000 rpm, 10 min), and suspend the bacterial sludge in 10% skim milk as a preservative.

[0054] At 40℃, dissolve 7g gelatin and 7g lactitol in 100mL deionized water. After cooling to 25℃, add 2.5×10 11 CFU of Lactobacillus plantarum was used to prepare an embedding solution. The inner layer microspheres were immersed in the embedding solution for 8 seconds and then quickly transferred to cold ethanol at 3°C ​​for 15 minutes to solidify, thus preparing the middle layer embedded microspheres.

[0055] Step 3: At 75℃, dissolve 1.6g CMC and 0.4g sucrose ester in 100mL deionized water. After cooling to 28℃, add 4×10 11 CFU (Cytobacter oxytocin) was used to prepare a coating solution. The middle layer of embedded microspheres was placed in a fluidized bed with an inlet air temperature of 30°C. The coating solution was sprayed at a rate of 1.5 mL / min to ensure uniform coverage of the outer layer. The microspheres were dried to a moisture content of 8% to obtain a three-layer embedded bacterial agent.

[0056] Example 3

[0057] The preparation method of the three-layer embedded bacterial agent includes the following steps:

[0058] Step 1: Leuconostoc mesenteroides PCY-3R18 was inoculated into MRS medium and cultured at 25°C for 24 hours (OD200). 600 =1.8); collect the bacterial sludge by centrifugation, and resuspend it in 0.1 mol / L PBS buffer (pH 7.0) to a final volume of 1×10⁻⁶. 10 CFU / mL.

[0059] At 60℃, 2g of sodium alginate and 1.5g of xylooligosaccharide were dissolved in 100mL of deionized water. After cooling to 28℃, 40mL of Leuconostoc mesenteroides suspension was added to prepare a mixture. Calcium chloride solution was added dropwise to the mixture to make the mass fraction of calcium chloride in the system 2.2%. The solution temperature was controlled at 27℃ and the pH value was 7. The mixture was magnetically stirred at 200rpm for 35min to solidify and form inner layer microspheres. The mixture was rinsed 3 times with sterile water and temporarily stored at 4℃.

[0060] Step 2: Collect Lactobacillus plantarum by centrifugation (10,000 rpm, 10 min), and suspend the bacterial sludge in 10% skim milk as a preservative.

[0061] At 42℃, dissolve 9g gelatin and 6g lactitol in 100mL deionized water. After cooling to 26℃, add 3.5×10 11 CFU of Lactobacillus plantarum was used to prepare an embedding solution. The inner layer microspheres were immersed in the embedding solution for 12 seconds and then quickly transferred to cold ethanol at 5°C for 20 minutes to solidify, thus preparing the middle layer embedded microspheres.

[0062] Step 3: At 85℃, dissolve 1.8g CMC and 0.5g sucrose ester in 100mL deionized water. After cooling to 32℃, add 4×10 11 CFU (Cytobacter oxytococcus lactis) was used to prepare a coating solution. The middle layer of embedded microspheres was placed in a fluidized bed with an inlet air temperature of 40°C. The coating solution was sprayed at a rate of 2.5 mL / min to ensure uniform coverage of the outer layer. The microspheres were dried to a moisture content of 5% to obtain a three-layer embedded bacterial agent.

[0063] The performance parameters of the three-layer encapsulated bacterial agents prepared in Examples 1-3 are shown in Table 1.

[0064] Table 1 Performance parameters of the three-layer encapsulated bacterial agents prepared in Examples 1-3

[0065]

[0066] As shown in Table 1, the three-layer encapsulated bacterial agents prepared in Examples 1 to 3 all exhibited excellent performance in terms of structural integrity, bacterial survival rate, and release performance, with Example 1 being the optimal example.

[0067] Example 4

[0068] A rapid fermentation method for kimchi based on gradient temperature control and directional microbial fermentation includes the following steps:

[0069] S100, 100kg of fresh radishes are washed, cut into strips, treated to remove astringency, and drained for later use;

[0070] The specific method for removing astringency includes: soaking in a 0.1% citric acid solution for 20 minutes to remove astringency, with the mass ratio of vegetables to 0.1% citric acid solution being 1:2.5.

[0071] S200. Lactococcus lactis, Leuconostoc mesenteroides and Lactobacillus plantarum are prepared into a three-layer encapsulated bacterial agent. The preparation method is the same as in Example 1. The agent is added to the brine along with vegetables. The solid-liquid ratio of vegetables to brine is 1:2, the salt content of brine is 2.8%, and the mass ratio of vegetables to the three-layer encapsulated bacterial agent is 100:0.3.

[0072] S300: Nitrogen gas is introduced into the S200 system to make the dissolved oxygen content 0.4 mg / L, and fermentation is carried out in three stages with gradient temperature control.

[0073] The three-stage gradient temperature-controlled fermentation method includes: Stage 1: fermentation at 23℃ for 15 hours; Stage 2: fermentation at 12℃ for 30 hours;

[0074] Stage 3: Ferment at 16℃ for 21 hours.

[0075] In stage 2, calcium chloride was added at 0.2% of the initial weight of the vegetables and the vegetables were simultaneously treated with ultrasound at 38 kHz for 11 min.

[0076] S400. After fermentation, add 5% (by weight of the fermented kimchi) of mother brine, vacuum pack, and refrigerate at 3℃ for 24 hours to mature. The mother brine contains 2×10⁻⁶ lactic acid bacteria. 8 The concentration was CFU / mL, and the pH was 3.5. Of which, Lactobacillus plantarum accounted for 60%.

[0077] Example 5

[0078] A rapid fermentation method for kimchi based on gradient temperature control and directional microbial fermentation includes the following steps:

[0079] S100: Wash and cut 100kg of fresh chili peppers into strips, remove astringency, drain and set aside.

[0080] The specific method for removing astringency includes: soaking the vegetables in a 0.08% citric acid solution for 15 minutes to remove astringency, with the mass ratio of vegetables to the 0.08% citric acid solution being 1:2.

[0081] S200. Lactococcus lactis, Leuconostoc mesenteroides and Lactobacillus plantarum are prepared into a three-layer encapsulated bacterial agent. The preparation method is the same as in Example 1. The agent is added to brine along with vegetables. The solid-liquid ratio of vegetables to brine is 1:1.8, the salt content of brine is 2.5%, and the mass ratio of vegetables to the three-layer encapsulated bacterial agent is 100:0.25.

[0082] S300: Nitrogen gas is introduced into the S200 system to make the dissolved oxygen content 0.5 mg / L, and fermentation is carried out in three stages with gradient temperature control.

[0083] The three-stage gradient temperature-controlled fermentation method includes: Stage 1: fermentation at 20℃ for 12 hours; Stage 2: fermentation at 10℃ for 24 hours;

[0084] Stage 3: Ferment at 15℃ for 18 hours.

[0085] In stage 2, calcium chloride was added at 0.1% of the initial weight of the vegetables and the vegetables were simultaneously treated with ultrasound at 35 kHz for 10 min.

[0086] S400. After fermentation, add 4% (by weight of fermented kimchi) of mother brine, vacuum pack, and refrigerate at 2°C for 22 hours for post-fermentation. The mother brine contains 1×10⁻⁶ lactic acid bacteria. 8 The concentration was CFU / mL, and the pH was 3.8, with Lactobacillus plantarum comprising 50% of the composition.

[0087] Example 6

[0088] A rapid fermentation method for kimchi based on gradient temperature control and directional microbial fermentation includes the following steps:

[0089] S100, 100kg of fresh lotus root is washed, cut into strips, and drained after removing astringency.

[0090] The specific method for removing astringency includes: soaking the vegetables in a 0.12% citric acid solution for 25 minutes to remove astringency, with the mass ratio of vegetables to the 0.12% citric acid solution being 1:3.

[0091] S200. Lactococcus lactis, Leuconostoc mesenteroides and Lactobacillus plantarum are prepared into a three-layer encapsulated bacterial agent. The preparation method is the same as in Example 1. The agent is added to brine along with vegetables. The solid-liquid ratio of vegetables to brine is 1:2.2, the salt content of brine is 3%, and the mass ratio of vegetables to the three-layer encapsulated bacterial agent is 100:0.4.

[0092] S300: Nitrogen gas is introduced into the S200 system to make the dissolved oxygen content 0.3 mg / L, and fermentation is carried out in three stages with gradient temperature control.

[0093] The three-stage gradient temperature-controlled fermentation method includes: Stage 1: fermentation at 25℃ for 18 hours; Stage 2: fermentation at 15℃ for 36 hours;

[0094] Stage 3: Ferment at 18℃ for 24 hours.

[0095] In stage 2, calcium chloride was added at 0.3% of the initial weight of the vegetables and the vegetables were simultaneously treated with ultrasound at 40 kHz for 12 min.

[0096] S400. After fermentation, add 6% (by weight of the fermented kimchi) of mother brine, vacuum pack, and refrigerate at 4℃ for 26 hours to mature. The mother brine contains 2.5 × 10⁻⁶ lactic acid bacteria. 8 The concentration was CFU / mL, and the pH was 3.2. Of which, Lactobacillus plantarum accounted for 70%.

[0097] Comparative Example 1

[0098] Single-layer hybrid embedding:

[0099] Step 1: Preparation of mixed bacterial suspension

[0100] Lactococcus lactis, Leuconostoc mesenteroides PCY-3R18, and Lactobacillus plantarum were mixed at a live bacteria ratio of 2:1:2: Lactococcus lactis: 1.4 × 10⁻⁶ 11 CFU, Leuconostoc mesentery: 0.7 × 10⁻⁶ 11 CFU, Lactobacillus plantarum: 1.4 × 10⁻⁶ 11 CFU.

[0101] Collect the mixed bacterial sludge by centrifugation and resuspend it in 0.1 mol / L PBS buffer (pH 7.0) to a total concentration of 3.5 × 10⁻⁶. 11 CFU / mL.

[0102] Step 2: Sodium alginate encapsulation

[0103] Dissolve 2.5g of sodium alginate in 97.5g of deionized water (dissolve at 50℃), and cool to 30℃; add 33.3mL of mixed bacterial suspension (bacterial count ≈ 1.17 × 10⁻⁶). 10 CFU / mL), stir well; add 2% CaCl2 solution (under the same conditions as in Example 1), solidify for 30 min; rinse 3 times with sterile water, and store at 4°C.

[0104] Step 3: Fermentation Application

[0105] The process and parameters are the same as in Example 4.

[0106] Comparative Example 2

[0107] Double-layer embedding

[0108] Step 1: Preparation of inner layer microspheres, same as Step 1 in Example 1;

[0109] Leuconostoc mesenteroides was encapsulated in sodium alginate-xylooligosaccharide microspheres (particle size 300-500 μm).

[0110] Step 2: Middle layer coating (no outer layer)

[0111] Preparation of Lactobacillus plantarum embedding solution; same as step 2 in Example 1;

[0112] The inner microspheres were immersed in the embedding solution for 10 seconds and then cured with cold ethanol at 4°C for 18 minutes, forming only an "inner-middle" double-layer structure.

[0113] Omit the outer coating and dry directly to a moisture content of 6%.

[0114] Step 3: Fermentation Application

[0115] The process and parameters are the same as in Example 4.

[0116] Comparative Example 3

[0117] Unencapsulated, staged dosing (direct addition of free bacteria)

[0118] Experimental steps

[0119] Preparation of bacterial suspension (same strain and concentration as in Example 1):

[0120] Lactococcus lactis suspension: 3.5 × 10 11 CFU (0.1% skim milk protected); Leuconostoc mesentery suspension: 3.5 × 10⁻⁶ 10 CFU (PBS buffer); Lactobacillus plantarum suspension: 3.5 × 10⁻⁶ 11 CFU (0.1% skim milk protection)

[0121] Fermentation dosing process:

[0122] Phase 1: Add Lactococcus lactis suspension (0.15% of vegetable weight); Phase 2: Add Leuconostoc mesenteroides suspension (0.08%); Phase 3: Add Lactobacillus plantarum suspension (0.12%).

[0123] Other process conditions are the same as in Example 4.

[0124] Comparative Example 4

[0125] Constant temperature fermentation, fermentation parameters:

[0126] Temperature: 20℃ throughout; Time: 66 hours;

[0127] Other conditions, such as bacterial agent, saline, and nitrogen purging, are the same as in Example 4.

[0128] Comparative Example 5

[0129] Fermentation parameters:

[0130] Stage 1: Fermentation at 12℃ for 30 hours; Stage 2: Fermentation at 23℃ for 15 hours; Stage 3: Fermentation at 16℃ for 21 hours;

[0131] Other conditions are the same as in Example 4.

[0132] The performance parameters of the kimchi prepared by the fermentation methods of Examples 4-6 and Comparative Examples 1-5 are shown in Table 2.

[0133] Table 2. Performance parameters of kimchi prepared by the fermentation methods of Examples 4-6 and Comparative Examples 1-5.

[0134]

[0135] As shown in Table 2, the total fermentation cycle of Examples 4 to 6 is ≤78h, the residual nitrite content is ≤0.5mg / kg, the mold contamination rate is ≤2.3%, the GABA content can reach 39.1mg / 100g, the total flavor substances can reach 1420μg / g, the crispness retention rate is 94.7%, the release rate of Leuconostoc mesentery can reach 86.3%, and the survival rate of Lactobacillus plantarum reaches 91.5%.

[0136] Comparative Example 1 used a single-layer mixed encapsulation. During the early fermentation stage (0-12h), *Lactococcus lactis* excessively produced acid, causing the pH to rapidly drop to a strongly acidic environment <4.0. This resulted in acid stress damage to *Lactobacillus plantarum*, reducing its survival rate to 63.4%. Acid damage led to a loss of >80% of the nitrite reductase activity in *Lactobacillus plantarum*, with nitrite residue rising to 12.1 mg / kg. *Lactococcus lactis* consumed large amounts of glucose, resulting in a lack of substrate for GABA synthesis, with the final product being only 10.8 mg / 100g and the total flavor compounds only 563 μg / g. Microbial competition led to microecological imbalance, with a mold contamination rate of 13.7%. This demonstrates that the three-layer encapsulation structure of this invention can solve the problem of microbial antagonism.

[0137] In Comparative Example 2, without the CMC oxygen barrier layer, *Lactococcus lactis* was directly exposed to an environment with dissolved oxygen of 0.4 mg / L, resulting in a 45% decrease in survival rate under aerobic stress. The bacteria lost their oxygen-consuming capacity, leading to a buildup of dissolved oxygen up to 2.1 mg / L, providing favorable conditions for the proliferation of molds (strict aerobic bacteria), and increasing the contamination rate to 10.2%. *Leuconostoc mesenteroides* was inhibited by lactococcal lactin, resulting in a decrease in diacetyl synthesis and a reduction in total flavor compounds to 687 μg / g. Without the protection of the CMC outer layer, the middle gelatin layer was hydrolyzed by proteases, and the release rate of *Leuconostoc mesenteroides* was only 78.6%. The decreased survival rate of *Lactococcus lactis* and the inhibition of *Leuconostoc mesenteroides* led to a surge in nitrite residue to 9.8 mg / kg. This demonstrates the irreplaceable role of the CMC outer layer in oxygen barrier, enzymatic protection, and bacterial viability preservation, making it a core structure for controlling nitrite levels.

[0138] In Comparative Example 3, at pH 3.8 during stage 3, the inner membrane potential of free *Lactobacillus plantarum* collapsed after 30 minutes, and the survival rate decreased to 42.7%; 2% CaCl2 induced cell dehydration, and intracellular K+ decreased. + At a concentration of 80%, the release rate of Leuconostoc mesenteroides decreased to 34.2%, while the nitrite residue increased to 15.3 mg / kg; free bacteria adsorbed onto the vegetable surface to form a barrier, blocking Ca... 2+Infiltration led to a decrease in crispness retention to 65.8%. *Lactococcus lactis* excessively consumed glucose, while *Lactobacillus plantarum* lacked a carbon source for GABA synthesis, resulting in a GABA content of only 9.7 mg / 100g. *Lactococcus lactis* occupied an ecological niche but lacked flavor synthesis genes, while functional bacteria, due to damage, could not express key enzymes, resulting in a total flavor substance content of 498 μg / g. The lack of acid / salt buffering-targeted delivery-metabolic protection functions in the free-floating bacteria's encapsulation structure triggered a vicious cycle of bacterial damage, enzyme inactivation, and pathway collapse, indicating that the three-layer encapsulation structure is irreplaceable.

[0139] In Comparative Example 4, fermentation was carried out at a constant temperature of 20℃. Lactococcus lactis continuously dominated the fermentation process, inhibiting the growth of Leuconostoc mesenteroides, resulting in a total flavor compound content of only 702 μg / g. However, premature activation of Lactobacillus plantarum led to the missed window for nitrite degradation, resulting in a nitrite residue of 11.5 mg / kg and a GABA content of only 19.1 mg / 100g. This demonstrates that the three-stage temperature control process of this invention can effectively avoid the inhibitory effects between bacterial communities and effectively improve fermentation quality.

[0140] In Comparative Example 5, the low-temperature start-up in Stage 1 delayed fermentation; the sudden temperature increase to 23°C in Stage 2 triggered heat stress in the microbial community, resulting in a mortality rate of >40% for *Leuconostoc mesenteroides*, a release rate of only 51.3%, and a decrease in the survival rate of *Lactobacillus plantarum* to 74.6%. During the low-temperature stage (12°C), the enzyme activity of *Lactobacillus plantarum* was ≤30%, and GABA synthesis decreased to 18.7 mg / 100g. This demonstrates that the segmented temperature control of the present invention can perfectly match the three-layer encapsulation structure, promoting the orderly metabolism of the microbial strain.

Claims

1. A rapid fermentation method for kimchi based on gradient temperature control and directional microbial fermentation, characterized in that, Includes the following steps: S100. After chopping the vegetables, wash them, remove the bitterness, drain the water and set aside. S200: Prepare a three-layer encapsulated bacterial agent by Lactococcus lactis, Leuconostoc mesenteroides and Lactobacillus plantarum, and add it to the brine along with vegetables; S300: Nitrogen gas is introduced into the S200 system to make the dissolved oxygen content ≤0.5mg / L, and fermentation is carried out in three stages with gradient temperature control. After fermentation of S400 is complete, add mother water and refrigerate.

2. The fermentation method according to claim 1, characterized in that, In step S200, the preparation method of the three-layer embedded bacterial agent includes the following steps: Step 1: Dissolve sodium alginate and xylooligosaccharide in deionized water, cool down, add Leuconostoc mesenteroides to make a mixture, add calcium chloride solution dropwise to the mixture, and solidify to form inner microspheres; Step 2: Dissolve gelatin and lactitol in deionized water, cool down, add Lactobacillus plantarum to prepare an embedding solution, immerse the inner layer microspheres in the embedding solution, and then quickly transfer them to a low-temperature environment to solidify, thus preparing the middle layer embedded microspheres. Step 3: Dissolve CMC and sucrose ester in deionized water, cool, add lactococcus lactis to prepare a coating solution, spray the coating solution evenly onto the surface of the middle layer embedded microspheres, and dry until the water content is ≤8% to obtain a three-layer embedded bacterial agent.

3. The fermentation method according to claim 2, characterized in that, In step 1, the mass ratio of sodium alginate, xylooligosaccharides, and deionized water is 2–3:1.0–1.5:100, and the dosage of Leuconostoc mesenteroides is 2 × 10⁻⁶. 9 ~3×10 9 The solution contains CFU / mL; the dissolution temperature is 50–60℃, and the temperature after cooling is 28–32℃; the mass fraction of calcium chloride in the system is 1.8–2.2%; the curing time is 25–35 min; the solution temperature is controlled at 23–27℃; and the pH value is 6.0–7.

0.

4. The fermentation method according to claim 2, characterized in that, In step 2, the mass ratio of gelatin, lactitol, and deionized water is 7–9:4–6:100, and the amount of *Lactobacillus plantarum* used is 2.5 × 10⁻⁶. 9 ~3.5×10 9 The embedding solution was prepared at a concentration of CFU / mL, with a dissolution temperature of 37–42℃ and a subsequent cooling temperature of 23–26℃. The low-temperature curing environment was cold ethanol at 3–5℃, with a curing time of 15–20 min.

5. The fermentation method according to claim 2, characterized in that, In step 3, the mass ratio of CMC, sucrose ester, and deionized water ranges from 1.6–2.0:0.4–0.6:100, and the dosage of Lactococcus lactis is 3.0 × 10⁻⁶. 9 ~4.0×10 9 The coating solution has a CFU / mL concentration, a dissolution temperature of 75–85℃, and a cooling temperature of 28–32℃; the spray rate is 1.5–2.5 mL / min, and the inlet air temperature is 30–40℃.

6. The fermentation method according to claim 1, characterized in that, In step S200, the solid-liquid ratio of vegetables to brine is 1:1.8-2.2, the salt content of brine is 2.5-3%, and the mass ratio of vegetables to three-layer encapsulated bacterial agent is 100:0.25-0.

4.

7. The fermentation method according to claim 1, characterized in that, In step S300, the three-stage gradient temperature-controlled fermentation method includes: Stage 1: Ferment at 20-25℃ for 12-18 hours; Stage 2: Fermentation at 10-15℃ for 24-36 hours; Stage 3: Ferment at 15-18℃ for 18-24 hours.

8. The fermentation method according to claim 7, characterized in that, In stage 2, calcium chloride of 0.1%-0.3% of the initial weight of the vegetables was added and the vegetables were simultaneously treated with ultrasound at 35-40 kHz for 10-12 minutes.

9. The fermentation method according to claim 1, characterized in that, In step S400, after fermentation is complete, add 4-6% of the total mass of fermented kimchi as mother brine, vacuum pack, and refrigerate at 2-4℃ for 22-26 hours for post-fermentation; the mother brine contains ≥1×10⁻⁶ lactic acid bacteria. 8 The concentration of the sample is CFU / mL, and the pH is ≤3.8, with Lactobacillus plantarum accounting for 50%–70%.

10. The fermentation method according to claim 1, characterized in that, In step S100, the specific method for de-astringency treatment includes: soaking the vegetables in a citric acid solution with a mass fraction of 0.08-0.12% for 15-25 minutes to remove astringency, wherein the mass ratio of the vegetables to the 0.08-0.12% citric acid solution is 1:2-3.

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