Pickled vegetable rapid fermentation method based on pH response release active peptide microcapsules

Abstract

The invention discloses a rapid pickle fermentation method based on pH-responsive release bioactive peptide microcapsules, and belongs to the technical field of food fermentation. The method comprises the following steps: pretreating raw materials, putting into a jar, preparing fermentation liquor, and sealing and fermenting. The method is characterized in that pH response type active peptide microcapsules are added into the fermentation liquor; according to the microcapsule, active peptide is embedded by taking calcium alginate as a matrix, and a chitosan / calcium alginate polyelectrolyte compound film layer is formed on the surface through electrostatic self-assembly. At the initial stage of fermentation (pH gt: 5.5), the film layer structure is compact, active peptides are effectively locked, and natural succession of microbial communities is prevented from being interfered; when the fermentation is in the middle stage, the lactic acid bacteria produce acid to reduce the pH value to a critical point of 4.5-5.0, the membrane layer is protonized, swelled and collapsed, and active peptides are released to promote the proliferation of the lactic acid bacteria. According to the method, the fermentation period can be greatly shortened, rich flavors generated by metabolism of various microorganisms are reserved to the maximum extent, and the technical problem that fermentation acceleration and flavors are difficult to consider at the same time is solved.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, specifically a rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules. Background Technology

[0002] Kimchi fermentation is a complex biochemical process that relies on the natural microbial community attached to the surface of vegetables, primarily involving lactic acid fermentation. This process not only gives kimchi its unique tangy flavor and crisp texture but also effectively inhibits the growth of pathogens, improving food safety. However, traditional natural fermentation methods have the following problems: long fermentation cycles (usually requiring weeks or even months), large fluctuations in product quality, and susceptibility to contamination by other microorganisms leading to spoilage.

[0003] To address the aforementioned issues, the main methods currently employed are as follows: ① Inoculation with starter cultures: This involves artificially adding selected or compound lactic acid bacteria strains (such as *Lactobacillus plantarum* and *Pediococcus pentosaceus*) to dominate the fermentation process, aiming to accelerate acidification, inhibit unwanted microorganisms, and ensure the reproducibility of fermentation. However, this technology has limitations: exogenous strains must compete with complex indigenous microbial communities, and their colonization and dominance are not always guaranteed; furthermore, the addition of single or a few strains may lead to a homogenized product flavor, losing the richness of traditional fermented kimchi flavor. ② Controlling fermentation parameters: This involves influencing the microbial growth rate by controlling physical parameters such as fermentation temperature and oxygen content. For example, moderately increasing the initial fermentation temperature can accelerate lactic acid bacteria metabolism. While effective, this method has a narrow control range; excessively increasing the temperature may promote the growth of unwanted microorganisms, and it also has high energy costs and limited ability to finely control the final flavor. ③ Adding exogenous functional additives: This aims to promote the growth of beneficial bacteria or inhibit unwanted microorganisms by adding specific substances. This mainly includes: adding nutrient substrates, such as adding active peptides, glucose, vitamins, and soy protein to provide extra nutrition for lactic acid bacteria, shortening the fermentation cycle of kimchi, reducing nitrite content, and inhibiting the deterioration of kimchi quality; or adding antibacterial substances, such as adding nisin to target and inhibit spoilage bacteria and pathogens.

[0004] However, through in-depth research and practice, it has been found that the existing exogenous additive technology still has the following shortcomings: (1) Inflexible adjustment: The current addition method is to add all of them at once, which cannot intelligently supplement lactic acid bacteria according to the actual changes in the fermentation process (such as pH drop). (2) Affects the fermentation process: Once the initial microbial environment of kimchi fermentation environment is dominated by miscellaneous bacteria, adding them too early may promote the growth of miscellaneous bacteria, inhibit the growth of lactic acid bacteria, disrupt the normal fermentation rhythm, or even directly lead to fermentation failure. (3) Affects the flavor: It is easy to bring unpleasant bitterness or off-flavor to the product, and the premature start of lactic acid metabolism of lactic acid bacteria leads to an overly sharp and prominent sour taste, lacking the complex and mellow flavor formed by the metabolism of other microorganisms at the beginning of fermentation, making the kimchi taste monotonous.

[0005] In conclusion, developing an intelligent delivery system capable of sensing the fermentation process and precisely releasing functional factors at the optimal time is crucial for overcoming the shortcomings of existing technologies and upgrading the fermented food industry. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the aforementioned technologies, the primary objective of this invention is to provide a rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules. This invention encapsulates active peptides in pH-responsive microcapsules, allowing them to begin releasing during the mid-fermentation stage (when the pH drops to 4.5-5.0 and lactic acid bacteria have established dominance). This precisely targets the lactic acid bacteria for nutrition, reducing the risk of fermentation failure while optimizing both fermentation speed and flavor.

[0007] The objective of this invention is achieved through the following solution: A rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules, characterized in that pre-treated fresh vegetables are placed in a jar, fermentation liquid is added, and the jar is sealed and placed in a cool place for fermentation. The fermentation broth is prepared from salt, Lactobacillus plantarum, pH-responsive active peptide microcapsules, and water.

[0008] Preferably, in the fermentation broth, the amount of salt added is 2.0%~4.0% of the fermentation broth mass; the amount of pH-responsive active peptide microcapsules added is 0.1%~0.8% of the fermentation broth mass; the plant-based lactic acid bacteria are provided by freeze-dried Lactobacillus plantarum powder, the amount of freeze-dried Lactobacillus plantarum powder added is 0.01%~0.08% of the fermentation broth mass, and the viable count is (3~8)×10⁻⁶. 9 CFU / g.

[0009] Preferably, the pH-responsive active peptide microcapsules are prepared by the following method: S1. Dissolve the active peptide in a weakly alkaline buffer solution to prepare a peptide solution, and then mix it with sodium alginate solution to form a mixed dispersion. S2. The mixed dispersion is atomized into a calcium chloride solution to form calcium alginate gel microspheres and then solidified; S3. The cured gel microspheres are mixed with chitosan acetic acid solution and reacted. After washing and drying, the pH-responsive active peptide microcapsules are obtained.

[0010] Preferably, in S1, the weakly alkaline buffer solution is a phosphate buffer solution with a pH of 7.0 to 8.0; The active peptide is one or more of soybean peptides, casein peptides, and whey peptides; the peptide solution has a mass-volume concentration of 5% to 15%. The sodium alginate solution has a mass-volume concentration of 2.5% to 3.5%. The volume ratio of the peptide solution to the sodium alginate solution is 1:(2~4).

[0011] Preferably, in S2, the atomization is carried out using the spray gel method, the mass-volume concentration of the calcium chloride solution is 3.0%~5.0%, and the solution is allowed to stand and solidify for 20~40 minutes.

[0012] Preferably, in S3, the chitosan acetic acid solution is prepared by adding 0.5% to 1.5% by mass of chitosan to an acetic acid solution with a volume concentration of 0.5% to 1.0%, and the pH value is 5.5 to 6.0.

[0013] Preferably, the pretreatment refers to selecting fresh, rot-free, and pest-free vegetables, washing off the surface mud and sand with clean water, soaking them in a 2% to 3% salt solution, then taking them out, washing them, draining them, and setting them aside.

[0014] Preferably, the weight ratio of the fermentation liquid to the vegetables is (1~5):1, and the vegetables are at least one of mustard greens, cowpeas, and Chinese cabbage.

[0015] Preferably, the microcapsules rapidly release the active peptides when the pH is < 5.5.

[0016] The kimchi obtained by the fermentation method described in this invention.

[0017] The core mechanism of this invention lies in utilizing the inherent dynamic change in pH value from high to low during kimchi fermentation. By constructing pH-responsive microcapsules, intelligent controlled release of active peptides is achieved. In the early stage of fermentation (pH > 5.5), the chitosan molecules in the polyelectrolyte complex (PEC) membrane layer of the microcapsule shell have a low degree of protonation, and the molecular chains contract. The membrane structure formed by the combination with the calcium alginate matrix is ​​dense and stable, effectively locking in the internal active peptides, protecting them from loss, and avoiding interference with the natural succession of the microbial community. This ensures the production of rich flavor precursors synthesized by various microorganisms in the early stage. When fermentation reaches the middle stage, the acid produced by lactic acid bacteria metabolism causes the pH value to continuously decrease. When the pH value drops to the critical point (4.5~5.0), the degree of amino protonation of chitosan molecules increases sharply, and the interchain repulsion increases. This disrupts the electrostatic balance of the PEC membrane layer, causing the network structure to swell and disintegrate rapidly, achieving rapid release of active peptides. This provides nutrients to promote the proliferation and acid production of lactic acid bacteria, thereby shortening the fermentation cycle while obtaining a high-quality product with a mild sour taste and rich flavor layers.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Achieve precise and controllable release of nutrients: By constructing pH-responsive microcapsules, the static defects of the existing "direct addition" strategy are effectively overcome, enabling nutrients to be intelligently released when the environmental pH value drops to a specific threshold, achieving "on-demand supply", thereby significantly improving the utilization rate of active factors and promoting the growth of target lactic acid bacteria in a precise and efficient manner.

[0019] (2) Significantly shorten the fermentation cycle: By providing precise nutritional support in the above key stages, the metabolic activity and proliferation rate of lactic acid bacteria are accelerated, thereby effectively reducing the pH value of the fermentation system, significantly shortening the fermentation time, and improving production efficiency.

[0020] (3) To preserve the rich initial fermentation flavor to the greatest extent: By delaying the release of nutrient factors, the natural succession of the microbial community is avoided from being disturbed too early in the fermentation stage. This is intended to ensure the normal growth and metabolic activities of various microorganisms (such as yeast, acetic acid bacteria, etc.) in the early stage of fermentation, so that they can fully generate flavor substances such as esters and alcohols, thereby avoiding the problem of single flavor and overly sharp sourness in the final product, and obtaining high-quality pickled vegetables with mellow sourness, rich flavor layers and strong mellowness. Attached Figure Description

[0021] Figure 1 The release curves of the active peptide microcapsules in Examples Q1-Q3 are shown. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0023] Example Q1 A method for preparing pH-responsive release bioactive peptide microcapsules is as follows: S1. Preparation of Mixed Dispersion: Prepare 0.05 M, pH 8.0 phosphate buffer (PBS): Accurately weigh 0.27 g of potassium dihydrogen phosphate (KH2PO4) and 1.42 g of disodium hydrogen phosphate (Na2HPO4), dissolve in approximately 90 mL of ultrapure water, stir thoroughly to dissolve, adjust the pH to 8.0 with 1 M NaOH solution, and finally bring the volume to 100 mL with ultrapure water. Dissolve soybean peptides in the buffer to prepare a peptide solution with a mass concentration of 8.0%, and mix with 3.0% (w / v) high-G content sodium alginate solution (where high G content refers to sodium alginate with a guluronic acid (G) molar fraction ≥65%) at a volume ratio of 1:3, stir evenly to form a homogeneous and workable dispersion.

[0024] S2. Microcapsule gelation molding: The above dispersion was atomized into a 4.0% (w / v) calcium chloride solution using a spray gelation method to form calcium alginate gel microspheres, which were then allowed to stand for 30 minutes to solidify.

[0025] S3. pH-responsive membrane construction: The cured gel microspheres were contacted with a 1.0% (w / v) chitosan acetic acid solution (chitosan was dissolved in 0.8% (v / v) acetic acid solution at 1.0% (w / v) and the pH was adjusted to 5.7) to form a polyelectrolyte complex (PEC) membrane on the surface of the microspheres through electrostatic self-assembly.

[0026] S4. Post-processing: After the reaction is complete, collect the microcapsules, wash and dry them to obtain active peptide microcapsule powder.

[0027] The encapsulation efficiency of the active peptide microcapsules prepared in Example Q1 was determined to be 89.0±1.5%, and the loading was 40.5±1.2%.

[0028] Example Q2 A method for preparing pH-responsive release bioactive peptide microcapsules is as follows: S1. Preparation of the mixed dispersion: The preparation method of the weakly alkaline buffer solution is the same as in Example Q1. Soy peptides are dissolved in the buffer solution to prepare a peptide solution with a mass concentration of 6.0%, and mixed with a 3.4% (w / v) high-G sodium alginate solution at a volume ratio of 1:3.5. The mixture is stirred evenly to form a homogeneous and workable dispersion.

[0029] S2. Microcapsule gelation molding: The above dispersion was atomized into a 4.5% (w / v) calcium chloride solution using the spray gelation method to form calcium alginate gel microspheres, which were then allowed to stand for 38 minutes to solidify.

[0030] S3. pH-responsive membrane construction: The cured gel microspheres were contacted with a 1.3% (w / v) chitosan acetic acid solution (chitosan was dissolved in 0.9% (v / v) acetic acid solution at 1.3% (w / v) and the pH was adjusted to 5.6) to form a polyelectrolyte complex (PEC) membrane on the surface of the microspheres through electrostatic self-assembly.

[0031] S4. Post-processing: After the reaction is complete, collect the microcapsules, wash and dry them to obtain active peptide microcapsule powder.

[0032] The encapsulation efficiency of the active peptide microcapsules prepared in Example Q2 was determined to be 94.5±1.0%, and the loading capacity was 32.8±1.0%.

[0033] Example Q3 A method for preparing pH-responsive release bioactive peptide microcapsules is as follows: S1. Preparation of the mixed dispersion: The preparation method of the weakly alkaline buffer solution is the same as in Example Q1. Soy peptides are dissolved in the buffer solution to prepare a peptide solution with a mass concentration of 13.0%, and mixed with a 2.7% (w / v) high-G sodium alginate solution at a volume ratio of 1:2.2. The mixture is stirred evenly to form a homogeneous and workable dispersion.

[0034] S2. Microcapsule gelation molding: The above dispersion was atomized into a 3.2% (w / v) calcium chloride solution using the spray gelation method to form calcium alginate gel microspheres, which were then allowed to stand for 25 minutes to solidify.

[0035] S3. pH-responsive membrane construction: The cured gel microspheres were contacted with a 0.7% (w / v) chitosan acetic acid solution (chitosan was dissolved in 0.6% (v / v) acetic acid solution at 0.7% (w / v) and the pH was adjusted to 5.9) to form a polyelectrolyte complex (PEC) membrane on the surface of the microspheres through electrostatic self-assembly.

[0036] S4. Post-processing: After the reaction is complete, collect the microcapsules, wash and dry them to obtain active peptide microcapsule powder.

[0037] The encapsulation efficiency of the active peptide microcapsules prepared in Example Q3 was determined to be 82.0±2.0%, and the loading was 58.9±1.8%.

[0038] Three types of active peptide microcapsules were placed in a buffer solution with continuously changing pH to simulate the release process in a kimchi fermentation system, resulting in the following... Figure 1 The release curves shown are as follows: Example Q1 (Balanced Type) exhibits the best pH-responsive release characteristics. It shows minimal initial leakage (24h: ~9%) and rapid, complete release during the pH drop phase (15%~85% within 54~66h), making it the preferred choice for rapid fermentation. Example Q2 (High Encapsulation Type) offers better sealing, with an extremely low initial release rate (24h: ~5%); its mid-term release response is moderate and stable, providing optimal protection for high-value, easily depleted bioactive peptides. Example Q3 (High Loading Type) can hold more internal bioactive peptides; however, it experiences more initial leakage (24h: ~25%), which not only leads to waste but may also promote the growth of unwanted microorganisms. In summary, Example Q1 offers better encapsulation and pH response, making it suitable for subsequent kimchi production.

[0039] A rapid fermentation method for kimchi based on pH response and release of bioactive peptides, the specific process of which is as follows: Example 1 (1) Raw material pretreatment: Select 5 kg of fresh mustard greens that are free from rot and pests, wash the surface mud and sand with clean water, soak in 2.5% salt water for 30 seconds, take them out, wash them, drain them, and set them aside. (2) Putting into the fermentation container: The mustard greens obtained in step (1) are tightly packed into the fermentation container to remove as much air as possible, which is conducive to the formation of an anaerobic fermentation environment, and then put into use. (3) Fermentation broth preparation: Add salt (3.0% of the total mass of the fermentation broth) and freeze-dried Lactobacillus plantarum powder (0.03% of the total mass of the fermentation broth, with a viable count of 5.0 × 10⁻⁶) to 4.85 kg of water. 9 The active peptide microcapsule powder prepared in Example Q1 (at a rate of 0.3% of the total mass of the fermentation broth) and the active peptide microcapsule powder prepared in Example Q1 were thoroughly stirred and dispersed to prepare a total of 5 kg of fermentation broth.

[0040] (4) Fermentation treatment: Add the fermentation liquid to the jar containing mustard greens in step (2), seal it, and place it in a cool place to ferment.

[0041] Comparative Example 1 No freeze-dried Lactobacillus plantarum powder was added to the fermentation broth, and the remaining steps were the same as in Example 1.

[0042] Comparative Example 2 No microcapsules were added to the fermentation broth, and the remaining steps were the same as in Example 1.

[0043] Example 2 (1) Raw material pretreatment: Select 5kg of fresh mustard greens that are free from rot and pests, wash the surface mud and sand with clean water, soak in 3.0% salt water for 30 seconds, take them out, wash them, drain them, and set them aside. (2) Putting into the fermentation container: The mustard greens obtained in step (1) are tightly packed into the fermentation container to remove as much air as possible, which is conducive to the formation of an anaerobic fermentation environment, and then put into use. (3) Fermentation broth preparation: Add salt (4.0% of the total mass of the fermentation broth) and freeze-dried Lactobacillus plantarum powder (0.03% of the total mass of the fermentation broth, with a viable count of 5.0 × 10⁻⁶) to 4.85 kg of water. 9 The active peptide microcapsule powder prepared in Example Q1 (at an amount of 0.5% of the total mass of the fermentation broth) was thoroughly stirred and dispersed to prepare a total of 5 kg of fermentation broth.

[0044] (4) Fermentation treatment: Add the fermented liquid to the jar containing mustard greens in step (2), seal it, and place it in a cool place to ferment.

[0045] Comparative Example 3 The active peptide microcapsule powder was replaced with an equal mass of unencapsulated soybean peptide powder, and the remaining steps were exactly the same as in Example 2.

[0046] Example 3 (1) Raw material pretreatment: Select 5kg of fresh, rot-free, and pest-free cowpeas, wash the surface mud and sand with clean water, soak in 3.0% salt water for 30 seconds, take them out, wash them, drain them, and set them aside. (2) Put into the fermentation container: Pack the cowpeas obtained in step (1) tightly into the fermentation container to remove as much air as possible, so as to facilitate the formation of an anaerobic fermentation environment, and set aside for later use; (3) Fermentation broth preparation: Add salt (2.0% of the total mass of the fermentation broth) and freeze-dried Lactobacillus plantarum powder (0.03% of the total mass of the fermentation broth, with a viable count of 5.0 × 10⁻⁶) to 4.85 kg of water. 9 The active peptide microcapsule powder prepared in Example Q1 (0.1% of the total mass of the fermentation broth) and the active peptide microcapsule powder prepared in Example Q1 were thoroughly stirred and dispersed to prepare a total of 5 kg of fermentation broth.

[0047] (4) Fermentation treatment: Add the fermented liquid to the jar containing mustard greens in step (2), seal it, and place it in a cool place to ferment.

[0048] Comparative Example 4 The amount of active peptide microcapsule powder added was 0.05%, and the other steps were the same as in Example 3.

[0049] Comparative Example 5 The amount of active peptide microcapsule powder added was 0.8%, and the other steps were the same as in Example 3.

[0050] Test Results I. pH Changes Table 1. pH changes during the fermentation process of Examples 1-3 and Comparative Examples 1-5

[0051] As shown in Table 1, among all groups, Comparative Example 1 (without Lactobacillus plantarum inoculation) had the highest pH value and the slowest fermentation rate throughout the entire fermentation cycle, approaching natural fermentation. This indicates that exogenous dominant lactic acid bacteria are a prerequisite for the rapid start-up of kimchi and the promotion effect of microcapsules. Compared with Comparative Example 2, the pH value of Example 1 rapidly decreased from 5.20 to 3.32 from day 5 to day 15 of fermentation, a decrease of 1.88, which is much larger than the 1.16 decrease in Comparative Example 2. This indicates that when the pH of the kimchi system decreases to the response value, the soybean peptides contained in the microcapsules are rapidly released, promoting the growth of lactic acid bacteria and thus accelerating acid production, resulting in a significant drop in pH.

[0052] Comparing Example 2 and Comparative Example 3, the latter showed a rapid pH decrease (pH=4.90) in the early stage of fermentation (day 5), while Example 2 had a higher pH value (5.10) during the same period. However, in the middle stage of fermentation (day 15), the pH value of Example 2 decreased to 3.27, lower than that of Comparative Example 3 (3.45), and continued until the end of fermentation (day 30, 3.18 vs 3.22). This indicates that the direct addition of soybean peptide powder can promote the rapid growth of lactic acid bacteria and reduce pH in the early stage of fermentation. However, due to the continuous consumption of nutrients, the acid production efficiency of the kimchi in the direct addition group was lower in the later stage of fermentation compared with the precise supply of nutrients in the later stage of microcapsules. This indicates that the time-controlled release capability of pH-responsive microcapsules enables them to precisely provide nutrients in the key stage when lactic acid bacteria become the dominant bacteria, thereby achieving more efficient and thorough acidification.

[0053] Furthermore, a clear dose-response relationship was observed between the amount of microcapsules added. Comparative Example 2 (no microcapsules added) and Comparative Example 4 (0.05% added) showed little difference in pH value throughout the fermentation cycle. Moreover, the pH value of Example 3 (0.1% added) on day 20 of fermentation was 3.28, significantly lower than that of Comparative Example 4 (3.59), indicating that 0.1% is the minimum effective addition amount to accelerate kimchi fermentation. The final pH values ​​of Example 2 (0.5%) and Comparative Example 5 (0.8%) were not significantly different (pH values ​​on day 30 were 3.18 and 3.17 respectively), indicating that fermentation efficiency reached its maximum when the addition amount reached 0.5%, and adding more than 0.8% did not provide any benefit. In summary, this invention achieves precise and efficient control of the kimchi fermentation process by synergistically using *Lactobacillus plantarum* and 0.1%–0.5% pH-responsive microcapsules.

[0054] II. Changes in the number of lactic acid bacteria Table 2. Changes in the number of lactic acid bacteria during fermentation in Examples 1-3 and Comparative Examples 1-5 (unit: 1g CFU / mL)

[0055] As shown in Table 2, compared with all groups, Comparative Example 1 (without Lactobacillus plantarum) consistently had the lowest number of lactic acid bacteria, which directly corresponds to its slow pH decrease. Comparing Example 1 and Comparative Example 2, the number of lactic acid bacteria in Example 1 was significantly higher than that in Comparative Example 2 during the early to mid-stages of fermentation (5–15 days), indicating that after the pH decreased to 5.0, the soybean peptides released from the microcapsules were utilized by the lactic acid bacteria as nutrients for rapid growth and acid production.

[0056] Comparing Example 2 and Comparative Example 3, the number of lactic acid bacteria in Comparative Example 3 (directly added peptides) rapidly increased to 8.2 lg CFU / mL on day 5, higher than that in Example 2 (7.9 lg CFU / mL). However, after day 10, the number of lactic acid bacteria in Comparative Example 3 plateaued and began to decline. Example 2, on the other hand, showed a significant growth peak on day 10, reaching 9.2 lg CFU / mL, and maintained a high bacterial count thereafter. This indicates that the unencapsulated active peptides were rapidly consumed by the microbial community (including lactic acid bacteria and other microorganisms) in the early stages of fermentation. Later, when nutrients were depleted, the bacterial growth plateaued and began to decline due to acid stress. The microcapsules, effectively blocking the active peptides at pH > 5.0, provided a relatively natural competitive environment for various microorganisms. As *Lactobacillus plantarum* grew and metabolized naturally, the environmental pH gradually decreased to 4.5-5.0, causing the microcapsules to swell and release soybean peptides, which were then utilized by the lactic acid bacteria to the maximum extent, achieving a second growth peak.

[0057] The bacterial count change in Comparative Example 4 (0.05% microcapsules) was not significantly different from that in Comparative Example 2 (no addition), indicating that this addition amount had no significant effect. However, the bacterial count in Example 3 (0.1% microcapsules) was significantly higher than that in Comparative Example 4 at all time points, clearly defining the minimum effective addition amount. In the three groups of Examples 1 (0.3%), Example 2 (0.5%), and Comparative Example 5 (0.8%), the number of lactic acid bacteria remained at a very high level with no significant difference (approximately 9.0–9.3 lg CFU / mL) throughout the mid-to-late stages of fermentation (days 10–30). This indicates that when the microcapsule addition reaches 0.5%, the promoting effect on lactic acid bacteria approaches saturation, and excessive addition cannot further expand the bacterial community dominance.

[0058] III. Flavor Compound Analysis Table 3. Changes in flavor compound content during fermentation in Examples 1-3 and Comparative Examples 1-5.

[0059] As shown in Table 3, the lactic acid content (5.5 g / kg) of Comparative Example 1 (without added Lactobacillus plantarum) was significantly lower than that of the other groups. This directly corresponds to its low number of lactic acid bacteria and slow pH decrease, indicating that natural fermentation is slower than inoculation fermentation, and it takes longer to start fermentation in the early stage (establishing the dominance of lactic acid bacteria). Comparative Example 3 (with direct addition of peptides) had the highest lactic acid content (8.8 g / kg) but the lowest acetic acid content (0.8 g / kg). The data indicates that in the early stages of fermentation, rapid peptide supply leads to rapid and singular homolactic fermentation by lactic acid bacteria, potentially prematurely inhibiting other acid-producing metabolic pathways (such as heterolactic fermentation or acetic acid production). Furthermore, the rapid pH drop prematurely inhibits the synthesis of flavor precursors such as ethanol, benzaldehyde, and acetic acid by yeast and other microorganisms. In contrast, Examples 1 and 2, while maintaining high levels of lactic acid (8.5-8.6 g / kg, mild acidity), accumulated more acetic acid (1.2-1.3 g / kg), ethanol (0.5 g / kg), and benzaldehyde (12.8-13 μg / kg, nutty aroma). The mechanism lies in the slow release of soybean peptides, which avoids excessive intervention in the initial fermentation stage, ensuring the necessary metabolic activities of alcohol-producing microorganisms (such as yeast) in the early fermentation phase, and generating more aroma precursors. Subsequently, under the combined action of acetic acid produced by lactic acid bacteria metabolism and ethanol produced by yeast metabolism, a rich amount of esters (ethyl acetate content 12~15.5mg / kg, banana and pineapple aroma) are synthesized through esterification reaction. In the middle stage of fermentation, lactic acid bacteria synthesize a large amount of lactic acid (8~8.6g / kg) under the precise supply of soybean peptides, so the kimchi has a relatively mellow and rich flavor.

[0060] Furthermore, a high level of methanethiol (the pungent smell of garlic or onion) was detected in Comparative Example 3, while it was not detected or detected in very low amounts in other groups. This may be because peptides themselves contain a certain amount of methionine, which can serve as a precursor for the synthesis of methanethiol. Moreover, in the early stages of fermentation, peptides are indiscriminately utilized by other bacteria, leading to the accumulation of methanethiol and negatively impacting the flavor of kimchi. In contrast, the Example 1 uses intelligent release of precise nutrient lactic acid bacteria to avoid indiscriminate utilization.

[0061] The flavor profile data also showed a clear dose-response effect and saturation phenomenon. The flavor profiles of Example 3 (0.1% microcapsules) were significantly better than those of Comparative Example 4 (0.05% microcapsules), clearly defining the lower limit of the effective dose. The flavor profile contents of Example 1 (0.3%), Example 2 (0.5%), and Comparative Example 5 (0.8%), especially ethyl acetate, a marker of flavor complexity, did not increase linearly with increasing dosage. The ethyl acetate content of Example 1 (15.5 mg / kg) was even higher than that of Comparative Example 5 (13.5 mg / kg), indicating that a dosage of 0.3% was sufficient to optimize flavor metabolism pathways, and excessive addition was unnecessary.

[0062] IV. Sensory Evaluation Score Analysis Table 4 Sensory scores (points) of Examples 1-3 and Comparative Examples 1-5 after 30 days of fermentation.

[0063] As shown in Table 4, the sensory score (53 points) of Comparative Example 1 (without starter) was significantly lower than all other groups, with lower scores in the core indicators of "acidity", "aroma" and "taste". This indicates that the lack of exogenous dominant lactic acid bacteria prevents the fermentation system from starting quickly and from forming the basic flavor that qualified kimchi should have in a short period of time.

[0064] In the successfully fermented groups, Comparative Example 3 (direct addition) scored significantly lower (64 points) than Example 2 (79 points). The product from Comparative Example 3 exhibited a "single flavor" deficiency; Example 2, while achieving rapid fermentation, also achieved superior sensory characteristics of "mild sourness and rich flavor." This demonstrates that the pH-responsive microcapsules proposed in this invention can accelerate the fermentation process of kimchi while producing better kimchi flavor.

[0065] The trend of sensory scores was consistent with that of physicochemical indicators: the flavor was significantly improved from Comparative Example 4 (67 points) to Example 3 (74 points); Example 1 (81 points) and Example 2 (79 points) reached the best and most stable high score plateau; Comparative Example 5 scored 77 points and had a slightly bitter taste, indicating that the addition of too many microcapsules would bring unpleasant flavor. From the perspective of acceptability, the optimal addition range of microcapsules was finally determined to be 0.1% to 0.5%.

[0066] Test Standard Description: The encapsulation efficiency, loading capacity, and release curve of the active peptide microcapsules obtained in Examples Q1-Q3 above, and the determination methods for pH, total acid, lactic acid bacteria count, and volatile flavor compounds of the kimchi obtained in Examples 1-3 and Comparative Examples 1-5 are as follows: I. Performance Determination of Active Peptide Microcapsules Experiment 1 Encapsulation efficiency and loading capacity determination Accurately weigh an appropriate amount of active peptide microcapsule powder and record the weight as m. Dissolve the sample in 10 mL of distilled water and vortex for 2 minutes to elute the active peptides adhering to the surface of the microcapsules. Then centrifuge the mixture at 8000 rpm for 10 minutes and collect the supernatant 1. Redisperse the precipitate after centrifugation in 10 mL of phosphate-buffered saline (PBS) at pH 7.4 and vortex at 45°C (150 rpm) for 1 hour to allow the microcapsules to completely disintegrate and release the encapsulated active peptides. Centrifuge again at 8000 rpm for 10 minutes and collect the supernatant 2.

[0067] The absorbance values ​​of supernatant 1 and supernatant 2 were measured at a wavelength of 570 nm using the ninhydrin colorimetric method. A standard curve was prepared using soybean peptide standard solutions of the same concentration gradient, and the peptide concentration (mg / mL) was calculated based on the standard curve.

[0068] calculate:

[0069] Surface peptide content Ws: calculated from the absorbance of supernatant 1 and the standard curve.

[0070] Total peptide content Wt: calculated from the absorbance of supernatant 2 and the standard curve.

[0071] Experiment 2 Release Curve In vitro release curve determination of active peptide microcapsules Prepare phosphate-citrate buffer solutions with pH values ​​of 6.0, 5.0, and 4.0 using 0.1 M disodium hydrogen phosphate solution and 0.1 M citric acid solution, respectively, and add 0.9% (w / v) sodium chloride to each solution to maintain a certain ionic strength. Accurately weigh the microcapsule powder equivalent to 10.0 mg of active peptide (denoted as Mt), redisperse it in an appropriate amount of pH 6.0 buffer solution, and transfer it to the upper layer of an ultrafiltration tube.

[0072] Place the dialysis bag or ultrafiltration tube into an Erlenmeyer flask containing 50 mL of pH 6.0 buffer. Seal the flask and place it in a constant temperature shaking incubator at 30°C and 100 rpm for shaking release.

[0073] Phase 1 (simulating the initial fermentation stage, 0-24 hours): At the set time points (e.g., 1, 2, 4, 8, 12, 24 hours), take out 2 mL of the entire release medium (or the lower layer filtrate of the ultrafiltration tube) and immediately add an equal volume of fresh pH 6.0 buffer solution at the same temperature to maintain a constant total volume.

[0074] Phase 2 (simulating mid-fermentation, 24-48 hours): After sampling at 24 hours, quickly replace all remaining release medium in the conical flask with 50 mL of fresh pH 5.0 buffer solution and continue shaking. Take 2 mL samples at time points (e.g., 28, 32, 36, 48 hours) and replenish with an equal volume of pH 5.0 buffer solution.

[0075] The third stage (simulating the late stage of fermentation, 48-72 hours): After sampling at the 48th hour, the release medium was completely replaced with 50 mL of fresh buffer solution with pH 4.0, and shaking was continued until 72 hours. Samples were taken at time points (e.g., 52, 60, 72 hours).

[0076] Active peptide concentration determination: The concentration of active peptides in the samples taken at each time point was determined using the ninhydrin colorimetric method. The specific steps are as follows: a. Take 1.0 mL of sample solution, add 1.0 mL of 2% (w / v) ninhydrin solution, and mix well.

[0077] b. Heat in a boiling water bath for 15 minutes, then quickly cool to room temperature.

[0078] c. Dilute to 10 mL with distilled water and measure the absorbance at a wavelength of 570 nm.

[0079] Based on the pre-plotted standard curve of active peptides, the concentration of active peptides in each sample (Cn, μg / mL) was calculated.

[0080] Cumulative release rate calculation: Calculate the cumulative release rate (Q, %) of the active peptide at each time point using the following formula:

[0081] in: Q n : Cumulative release rate (%) at the nth time point.

[0082] V: Total volume of the released medium (mL).

[0083] C i : The peptide concentration (mg / L) measured at the i-th time point.

[0084] V s : The volume (mL) of each sample taken.

[0085] C n : The peptide concentration (mg / L) measured at the nth time point.

[0086] M t Total mass (mg) of active peptides in microcapsules.

[0087] Release curve drawing: Plot the in vitro release curves of the active peptide microcapsules of Examples Q1, Q2, and Q3 with time (hours) on the x-axis and cumulative release rate (%) on the y-axis.

[0088] II. Determination of fermentation indicators for kimchi Experiment 3 pH and Total Acidity Determination Samples were taken daily at regular intervals during fermentation. 10.0 g of kimchi sample was homogenized with 90 mL of sterile distilled water, allowed to stand for 30 minutes, and then the pH value of the homogenized solution was measured directly using a calibrated precision pH meter.

[0089] Accurately weigh 5 g of sample into a 100 mL Erlenmeyer flask, add ultrapure water to dilute to 50 g, mix well, and then titrate with 0.1 mol / L sodium hydroxide standard solution, shaking constantly while adding the solution. Observe the pH meter reading at the same time, and record the volume of standard sodium hydroxide solution consumed when the endpoint is reached (pH value is 8.30 ± 0.1). Measure the same sample twice. Calculate the total acid content in the sample using equation (4).

[0090]

[0091] In the formula, χ represents the total acid content in the sample, in g / kg; c: The accurate value of the concentration of the sodium hydroxide standard titration solution, in mol / L; V1: The volume (mL) of sodium hydroxide standard solution consumed during the titration of the sample solution; V2: Volume (mL) of sodium hydroxide standard solution consumed in the blank test; K: represents the conversion factor for lactic acid, 0.09; F represents the dilution factor of the sample solution; m is the mass of the sample (g).

[0092] Experiment 4 Determination of total lactic acid bacteria count Referencing the national standard GB 4789.35-2016 "National Food Safety Standard - Microbiological Examination of Food - Examination of Lactic Acid Bacteria", 25 g of pickled vegetable sample was aseptically taken and placed in 225 mL of sterile physiological saline to homogenize into a 1:10 sample homogenate. The homogenate was then serially diluted 10-fold. Two to three suitable dilutions were selected, and 1 mL of each dilution was pipetted into sterile Petri dishes. MRS agar medium cooled to 46°C was poured over the dilutions, mixed thoroughly, and allowed to solidify. The plates were then inverted and incubated in an anaerobic incubator at (36±1)°C for (48±2) hours. Plates with colony counts between 30 and 300 CFU were used for colony counting. The final result is expressed as colony forming units per gram (CFU / g).

[0093] Experiment 5: Determination of Volatile Flavor Compounds in Pickled Mustard Greens The content of volatile flavor compounds in the pickled mustard greens obtained in Examples 1-3 and Comparative Examples 1-5 was determined. Method: 1 g of pickled mustard green homogenate sample was accurately weighed and placed into a 20 mL headspace vial. 20 μL of 1.738 ppm 2-methyl-3-heptanone was added as an internal standard. The headspace vial was then sealed with a clamp cap fitted with silicone insulation.

[0094] HS-SPME conditions: 75 μm CAD / PDMS extraction head, extraction temperature 65℃, extraction time 45 min, desorption temperature 250 ℃, desorption time 3 min.

[0095] GC conditions: Column TR-5ms (60 m × 0.25 mm × 0.25 μm); carrier gas: high-purity He, purity 99.999%; flow rate: 1.00 mL / min; injection port temperature: 250 ℃; splitless injection mode. Temperature program: initial temperature 40 ℃, hold for 2 min; increase to 120 ℃ at 5 ℃ / min, hold for 2 min; then increase to 220 ℃ at 7 ℃ / min, hold for 5 min.

[0096] MS conditions: Electron impact (EI) source, electron energy 70 eV, ion source temperature 250℃, transfer line temperature 250℃, mass scan range 33~350 m / z, scan rate 3.00 scans / s.

[0097] Data processing: Based on the NIST 17 database, the volatile flavor compounds in the sample homogenate were qualitatively analyzed by mass spectrometry and quantified to the content of flavor compounds in μg / mL. The relative content of volatile flavor compounds was calculated using equation (5): Volatile flavor compound content = peak area of ​​volatile flavor compounds / peak area of ​​internal standard × content of internal standard (5) Experiment 6 Sensory Evaluation Scoring Sensory evaluation was conducted on the pickled mustard greens obtained in Examples 1-3 and Comparative Examples 1-5. Evaluation method: After the pickled greens were fermented and matured, 20 people were asked to conduct sensory evaluation and score the prepared pickled greens according to their personal taste and flavor preferences.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules, characterized in that, After pre-treating the fresh vegetables, put them into a jar, add the fermentation liquid, seal it, and place it in a cool place to ferment. The fermentation broth is prepared from salt, Lactobacillus plantarum, pH-responsive active peptide microcapsules, and water.

2. The rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules according to claim 1, characterized in that, In the fermentation broth, the amount of salt added is 2.0% to 4.0% of the broth's mass. The pH-responsive active peptide microcapsules were added at a concentration of 0.1%–0.8% of the fermentation broth mass; the plant-based lactic acid bacteria were provided by freeze-dried Lactobacillus plantarum powder, which was added at a concentration of 0.01%–0.08% of the fermentation broth mass, with a viable count of (3–8) × 10⁻⁶. 9 CFU / g.

3. The rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules according to any one of claims 1 or 2, characterized in that, The pH-responsive bioactive peptide microcapsules were prepared by the following method: S1. Dissolve the active peptide in a weakly alkaline buffer solution to prepare a peptide solution, and then mix it with sodium alginate solution to form a mixed dispersion. S2. The mixed dispersion is atomized into a calcium chloride solution to form calcium alginate gel microspheres and then solidified; S3. The cured gel microspheres are mixed with chitosan acetic acid solution and reacted. After washing and drying, the pH-responsive active peptide microcapsules are obtained.

4. The rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules according to claim 3, characterized in that, In S1, the weakly alkaline buffer solution is a phosphate buffer solution with a pH of 7.0~8.0; The active peptide is one or more of soybean peptides, casein peptides, and whey peptides; the peptide solution has a mass-volume concentration of 5% to 15%. The sodium alginate solution has a mass-volume concentration of 2.5% to 3.5%. The volume ratio of the peptide solution to the sodium alginate solution is 1:(2~4).

5. The rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules according to claim 4, characterized in that, In S2, the atomization is carried out using the spray gel method, the mass-volume concentration of the calcium chloride solution is 3.0%~5.0%, and it is allowed to stand and solidify for 20~40 minutes.

6. The rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules according to claim 5, characterized in that, In S3, the chitosan acetic acid solution is prepared by adding 0.5% to 1.5% by mass of chitosan to an acetic acid solution with a volume concentration of 0.5% to 1.0%, and the pH value is 5.5 to 6.

0.

7. The rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules according to claim 3, characterized in that, The pretreatment refers to selecting fresh, rot-free, and pest-free vegetables, washing off the surface mud and sand with clean water, soaking them in a 2% to 3% salt solution, then taking them out, washing them, draining them, and setting them aside.

8. The rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules according to claim 3, characterized in that, The weight ratio of the fermentation liquid to the vegetables is (1~5):1, and the vegetables are at least one of mustard greens, cowpeas, and Chinese cabbage.

9. The rapid fermentation method for kimchi based on pH-responsive release of active peptide microcapsules according to claim 3, characterized in that, When pH < 5.5, the microcapsules rapidly release active peptides.

10. Kimchi obtained by the fermentation method according to any one of claims 1 to 9.

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