Preparation method of bacteria-enzyme synergistic fermentation red sour soup

Abstract

This invention discloses a method for preparing red sour soup through synergistic fermentation of bacteria and enzymes, belonging to the field of food processing technology. The method includes the following steps: mixing and pulping raw materials, adding a compound enzyme preparation for enzymatic hydrolysis, and then inoculating with *Lactobacillus plantarum* NR1-7 for fermentation after hydrolysis to obtain red sour soup. This invention effectively overcomes the shortcomings of traditional natural fermentation and single fermentation methods by constructing a synergistic fermentation system of *Lactobacillus plantarum* and a compound enzyme preparation. Based on the genetic characteristics of bacteriocin synthesis, quorum sensing regulation, and carotenoid biosynthesis in the *Lactobacillus plantarum* NR1-7 genome, this invention ensures stable acidity, bright red color, and long-lasting antibacterial and preservative effects during the product's shelf life, achieving an industrial upgrade of traditional red sour soup fermentation technology towards standardization, functionalization, and high quality.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a method for preparing red sour soup through synergistic fermentation of bacteria and enzymes. Background Technology

[0002] Red sour soup is a traditional fermented food with strong local characteristics in Guizhou, my country. It is usually made from red chili peppers and tomatoes as the main ingredients, supplemented with glutinous rice, ginger, garlic, etc., and is made through natural fermentation. It has a rich, sour and mellow flavor and a bright red color. It is not only widely used as an everyday condiment, but also has attracted much attention due to its rich content of organic acids, phenols, flavonoids and various active substances, making it one of the hot topics in the industrial development of traditional fermented foods.

[0003] Currently, the production of red sour soup still relies primarily on natural fermentation. Natural fermentation depends on the raw materials and wild microbial communities in the environment, which presents several insurmountable drawbacks. First, natural fermentation is slow to start, has a long fermentation cycle, and results in significant batch-to-batch quality variations, making standardized and large-scale production difficult. Second, the complex microbial community in the natural fermentation system poses a high risk of contamination by other microorganisms, easily leading to problems such as excessive nitrite levels and flavor degradation, posing food safety hazards. Furthermore, during traditional natural fermentation, functional components in the raw materials, such as polysaccharides, polyphenols, flavonoids, capsaicin, and gingerol, are largely lost due to excessive microbial metabolism or oxidation, resulting in a significant reduction in the product's nutritional value and health benefits.

[0004] To improve the quality and safety of red sour soup, researchers have attempted to introduce artificially inoculated pure lactic acid bacteria for enhanced fermentation. While the introduction of lactic acid bacteria can accelerate acid production and inhibit some spoilage bacteria, the degradation efficiency of macromolecules (such as proteins, cellulose, and pectin) in the raw materials is limited under a single-strain fermentation system. This results in insufficient release of flavor precursors and functional active ingredients, leading to a thin product flavor and minimal improvement in functional activity indicators (such as antioxidant capacity). Other studies have used external enzyme preparations to assist fermentation, but simple enzymatic hydrolysis lacks the synergistic effect of subsequent microbial metabolic transformation, contributing little to the accumulation of flavor substances and secondary metabolites.

[0005] More importantly, current technologies lack systematic research and clear technical solutions on how to construct an efficient and controllable "microorganism-enzyme" synergistic fermentation system to simultaneously shorten the fermentation cycle of red sour soup, improve the retention rate of functional components, inhibit the formation of harmful substances (such as nitrite), and stabilize the product's shelf-life quality. Therefore, developing a novel fermentation process for red sour soup that can balance fermentation efficiency, flavor quality, functional activity, and safety is of great significance for promoting the industrial upgrading of traditional fermented foods. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing red sour soup through synergistic fermentation of bacteria and enzymes, so as to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a method for preparing red sour soup through synergistic fermentation of bacteria and enzymes, comprising the following steps: mixing and pulping the raw materials, adding a compound enzyme preparation for enzymatic hydrolysis, and inoculating with Lactobacillus plantarum NR1-7 for fermentation after enzymatic hydrolysis to obtain red sour soup.

[0008] The second technical solution of the present invention is a red sour soup prepared by the preparation method.

[0009] Based on the above technical solution, the present invention has the following technical effects: This invention effectively overcomes the shortcomings of traditional natural fermentation and single fermentation methods by constructing a synergistic fermentation system of *Lactobacillus plantarum* and a compound enzyme preparation. This synergistic process efficiently degrades macromolecules in raw materials through enzymatic pretreatment, providing abundant precursors for the rapid proliferation and metabolism of *Lactobacillus plantarum* NR1-7, significantly shortening the fermentation cycle and accelerating acid production. Simultaneously, it enhances the competitive advantage of lactic acid bacteria, significantly reduces nitrite content during fermentation, and improves product safety. Furthermore, the synergistic fermentation, through a dynamic balance between enzymatic release and microbial transformation, significantly increases the retention rate of key functional active ingredients in red sour soup, such as polysaccharides, total phenols, total flavonoids, capsaicin, and 6-gingerol, compared to natural fermentation and inoculation-only fermentation. It also effectively promotes the targeted accumulation of small-molecule bioactive peptides, aromatic amino acid derivatives, and phenylpropane secondary metabolites, thereby endowing the product with superior DPPH and ABTS free radical scavenging capabilities and a more harmonious and full-bodied flavor profile. Furthermore, based on the genetic characteristics of bacteriocin synthesis, quorum sensing regulation, and carotenoid biosynthesis in the Lactobacillus plantarum NR1-7 genome, this invention ensures that the product maintains stable acidity, bright red color, and long-lasting antibacterial and preservative effects during its shelf life, thus realizing the industrial upgrade of traditional red sour soup fermentation process towards standardization, functionalization, and high quality. Attached Figure Description

[0010] Figure 1 The effect of fermentation time on the quality of red sour soup co-fermented by bacteria and enzymes.

[0011] Figure 2 The effect of fermentation temperature on the quality of red sour soup co-fermented by bacteria and enzymes.

[0012] Figure 3 The effect of inoculum quantity on the quality of red sour soup co-fermented by bacteria and enzymes.

[0013] Figure 4 To investigate the effects of different treatment methods on protease activity during the fermentation of red sour soup.

[0014] Figure 5 To investigate the effects of different treatment methods on cellulase activity during the fermentation of red sour soup.

[0015] Figure 6 To investigate the effects of different treatment methods on β-glucosidase activity during the fermentation of red sour soup.

[0016] Figure 7 To investigate the effects of different treatment methods on the polysaccharide content during the fermentation of red sour soup.

[0017] Figure 8 The effects of different treatment methods on the content of catechin (A), chlorogenic acid (B), ferulic acid (C), rutin (D), kaempferol (E), and gallic acid (F) during the fermentation of red sour soup were investigated.

[0018] Figure 9 The effects of different treatment methods on the total phenol content during the fermentation of red sour soup.

[0019] Figure 10 To investigate the effects of different treatment methods on the total flavonoid content during the fermentation of red sour soup.

[0020] Figure 11 To investigate the effects of different treatment methods on capsaicin content during the fermentation of red sour soup.

[0021] Figure 12 To investigate the effects of different treatment methods on the 6-gingerol content during the fermentation of red sour soup.

[0022] Figure 13 To investigate the effects of different treatment methods on the GABA content during the fermentation of red sour soup.

[0023] Figure 14 To investigate the effects of different treatment methods on DPPH free radical scavenging activity during the fermentation of red sour soup.

[0024] Figure 15 To investigate the effects of different treatment methods on the ABTS free radical scavenging activity during the fermentation of red sour soup.

[0025] Figure 16 To investigate the effects of different treatment methods on the vitamin C content during the fermentation of red sour soup.

[0026] Figure 17 To investigate the effects of different treatment methods on pH during the fermentation of red sour soup.

[0027] Figure 18 To investigate the effects of different treatment methods on the total acid content during the fermentation of red sour soup.

[0028] Figure 19 To investigate the effects of different treatment methods on the reducing sugar content during the fermentation of red sour soup.

[0029] Figure 20 To investigate the effects of different treatment methods on the nitrite content during the fermentation of red sour soup.

[0030] Figure 21 To investigate the effects of different treatment methods on the amino acid nitrogen content during the fermentation of red sour soup.

[0031] Figure 22 To investigate the effects of different treatment methods on the soluble solids content during the fermentation of red sour soup.

[0032] Figure 23 To investigate the effects of different treatment methods on the soluble protein content during the fermentation of red sour soup.

[0033] Figure 24 To investigate the effects of different treatment methods on the dietary fiber content during the fermentation of red sour soup.

[0034] Figure 25 Principal component analysis (PCA) plots of red sour soup fermentation samples under different treatment methods. Note: EZ indicates enzymatic hydrolysis only; EZ-I indicates enzymatic hydrolysis + inoculation group; LEZ indicates enzymatic hydrolysis only (1 / 10) group; LEZ-I indicates enzymatic hydrolysis (1 / 10) + inoculation group; I indicates inoculation only group; NF indicates natural fermentation group; QC indicates quality control sample. Detailed Implementation

[0035] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0036] This invention provides a method for preparing red sour soup through synergistic fermentation of bacteria and enzymes, comprising the following steps: mixing and pulping the raw materials, adding a compound enzyme preparation for enzymatic hydrolysis, and inoculating with Lactobacillus plantarum NR1-7 for fermentation after enzymatic hydrolysis to obtain red sour soup.

[0037] In some specific implementation schemes, the raw materials, by weight, include: 40-60 parts red chili peppers, 20-30 parts tomatoes, 5-15 parts glutinous rice flour, 3-8 parts ginger, 3-8 parts garlic, 1-5 parts white sugar, and 1-5 parts white wine.

[0038] In some specific implementations, the compound enzyme preparation consists of pectinase, cellulase and protease; the enzymatic hydrolysis treatment is carried out at a temperature of 35-45°C for 8-16 hours.

[0039] In some specific implementations, the amount of pectinase added is 30-70 U / g of raw material, the amount of cellulase added is 30-70 U / g of raw material, and the amount of protease added is 50-150 U / g of raw material.

[0040] In some specific embodiments, the inoculum size of *Lactobacillus plantarum* is 2% (m:m), and the bacterial concentration is 3 × 10⁻⁶. 9 CFU / mL; the fermentation temperature was 31℃ and the fermentation time was 6 days.

[0041] In some specific implementations, a step of sterilizing the raw materials is included before the enzymatic hydrolysis treatment.

[0042] The present invention also provides a red sour soup prepared by the preparation method described above.

[0043] In this embodiment of the invention, the red chili peppers, tomatoes, glutinous rice flour, ginger, garlic, white sugar, and white wine were all purchased from Fuwangjia Supermarket in Huaxi District, Guiyang City, Guizhou Province.

[0044] Pectinase (food grade), cellulase (food grade), and protease (food grade) were purchased from Shandong Longket Enzyme Co., Ltd.

[0045] Lactobacillus plantarum ( Lactiplantibacillus plantarum NR1-7 has been disclosed in patent CN120436282A, with accession number CCTCC NO: M 20211541.

[0046] Example 1 1. Preparation of Red Sour Soup The preparation process of red sour soup is as follows: First, prepare the ingredients according to the following weight ratio: 50% red chili peppers, 25% tomatoes, 10% glutinous rice flour, 5% ginger, 5% garlic, 2% sugar, and 3% white wine. Wash the red chili peppers, tomatoes, ginger, and garlic thoroughly and remove any impurities. Remove the stems from the tomatoes and cut them into chunks. Peel the ginger and garlic. Then, mix all the ingredients together and blend them into a smooth paste using a food processor.

[0047] During the enzymatic hydrolysis stage, pectinase (50 U / g), cellulase (50 U / g), and protease (100 U / g) were added to the mixed slurry, and enzymatic hydrolysis was carried out for 12 hours in a 40℃ water bath. After enzymatic hydrolysis, activated NR1-7 inoculum was inoculated for fermentation. The inoculum needed to be activated in MAS liquid medium at 37℃ for 30 hours beforehand, and the inoculum concentration was controlled at 3×10⁻⁶. 9 CFU / g, and then fermented in a closed system.

[0048] Polysaccharides were determined using the phenol-sulfuric acid method, and total acidity in food was determined according to GB 12456-2021.

[0049] This experiment invited 20 experienced teachers and students majoring in food-related fields to form a sensory evaluation group. In a well-lit and well-ventilated food laboratory, they conducted sensory evaluations of the products, scoring them on four aspects: color, texture, flavor, and taste. The total score was 20 points, with higher scores indicating a more popular product.

[0050] Using total acid, polysaccharide content, and sensory evaluation scores as indicators, single-factor experiments were conducted to investigate fermentation time (2, 4, 6, 8, 10 days), fermentation temperature (24, 28, 32, 36, 40℃), and inoculum size (1, 2, 3, 4, 5%, m:m, bacterial concentration 3×10⁻⁶). 9 The effect of CFU / mL on red sour soup.

[0051] Based on the results of the single-factor experiments, using total acid, polysaccharide, and sensory score as response values, Design-Expert10 was used to optimize fermentation time, fermentation temperature, and inoculum size.

[0052] 2. Results of the single-factor experiment The effect of fermentation time on the quality of red sour soup co-fermented by bacteria and enzymes, such as Figure 1 As shown, with the extension of fermentation time, the total acid content and sensory score both showed a trend of first increasing and then decreasing, while the polysaccharide content decreased significantly. The quality of red sour soup is the result of a dynamic balance between substrate consumption, microbial metabolism, and product accumulation. Fermentation time that is too short or too long is not conducive to quality optimization. Controlling fermentation to terminate at the peak stage of total acidity and sensory score is the key to achieving a harmonious balance between flavor and acidity. In production, the fermentation endpoint must be strictly controlled to avoid quality deterioration.

[0053] The effect of fermentation temperature on the quality of red sour soup co-fermentation by bacteria and enzymes, such as Figure 2 As shown, with increasing fermentation temperature, both total acid content and sensory score exhibited a trend of first increasing and then decreasing, while polysaccharide content continued to decline. 28-36℃ is the optimal temperature range for co-fermentation of *Lonicera japonica* and enzymes, ensuring both high total acid content and good sensory quality while maintaining a moderate polysaccharide degradation rate. Temperatures that are too high or too low can lead to metabolic imbalance; therefore, fermentation temperature must be strictly controlled during production, and the matching of enzymatic hydrolysis and fermentation processes should be optimized to further balance acidity, flavor, and polysaccharide retention.

[0054] The effect of inoculum quantity on the quality of red sour soup co-fermentation by bacteria and enzymes is as follows: Figure 3 As shown, as the inoculum increased from 1% to 5%, the total acid content and sensory score both showed a trend of first increasing and then decreasing, while the polysaccharide content continued to decrease.

[0055] 3. Response Surface Optimization Experiment Based on the results of single-factor experiments, a three-factor, three-level response surface analysis was conducted using total acid, polysaccharide, and sensory score as response values, and fermentation time, fermentation temperature, and inoculum quantity as the factors.

[0056] Based on the combined response values ​​of total acid, polysaccharides, and sensory scores, the optimal response results were obtained as follows: fermentation time 5.55 days, fermentation temperature 31.02℃, and inoculum quantity 2%. Under these optimized conditions, the predicted total acid value after fermentation was 21.49 g / L, the predicted polysaccharide value was 9.99 mg / mL, and the predicted sensory score was 16.92.

[0057] To verify the accuracy of the optimized experimental results, the results needed to be validated and the optimized fermentation conditions revised: fermentation time 6 days, fermentation temperature 31℃, and inoculum size 2%. Under these conditions, the total acid content of the red sour soup was 20.1 g / L, the polysaccharide content was 11.84 mg / mL, and the sensory score was 17.

[0058] Example 2 1 Experimental Methods To explore the economic viability of co-fermentation by bacteria and enzymes, a low-enzyme-hydrolysis (1 / 10) treatment group (LEZ and LEZ-I) was added to the standard enzyme hydrolysis dosage to evaluate the impact of reducing enzyme dosage on fermentation efficiency and product quality, providing a reference for cost control in industrial production. This example sets up the following experimental groups: (1) Enzyme hydrolysis only (EZ): The sample is enzymatically hydrolyzed but not inoculated with bacteria for fermentation.

[0059] (2) Enzymatic hydrolysis + inoculation group (EZ-I): The sample was enzymatically hydrolyzed and inoculated with NR1-7 for fermentation.

[0060] (3) Enzymatic hydrolysis only (1 / 10) group (LEZ): The sample was enzymatically hydrolyzed, and the amount of enzyme added was 1 / 10 of the normal dose. No inoculation and fermentation were performed.

[0061] (4) Enzymatic hydrolysis (1 / 10) + inoculation group (LEZ-I): The sample is enzymatically hydrolyzed, and the amount of enzyme added is 1 / 10 of the normal dose. It is then inoculated with NR1-7 for fermentation.

[0062] (5) Group I: The sample was not enzymatically digested and was directly inoculated into NR1-7 for fermentation.

[0063] (6) Natural fermentation group (NF): The sample is not enzymatically digested or inoculated with microorganisms, and fermentation is carried out by microorganisms in the natural environment.

[0064] The preparation method of the red sour soup for each experimental group is the same as in Example 1.

[0065] The protease activity was determined according to the national standard GB / T 23527.1—2023, and the cellulase activity was determined according to the national standard GB / T 2583—2023. The determination of β-glucosidase activity was performed according to reference (Su et al., 2021). Polysaccharide determination was performed using the phenol-sulfuric acid method. The determination of phenolic substances was performed according to reference (Abdo et al., 2022). The determination of total phenols was performed according to reference (Vodnar et al., 2017). The determination of total flavonoids was performed according to reference (Vodnar et al., 2017). The determination of capsaicin was performed according to GB / T 21266-2007. The determination of 6-gingerol was performed according to reference (Ning Erjuan et al., 2018). The determination of GABA was performed according to reference (Pu et al., 2019). The determination of antioxidants was performed according to reference (Qiu et al., 2023). The determination of vitamin C was performed according to the high performance liquid chromatography method in GB5009.86-2016 "National Food Safety Standard - Determination of Ascorbic Acid in Food". LC-MS non-targeted metabolomics was also used for determination.

[0066] 2. Experimental Results 2.1 Changes in protease activity The effects of different treatment methods on protease activity during the fermentation of red sour soup, such as Figure 4 As shown, the protease activity of all treatment groups decreased with the extension of fermentation time, but the magnitude and pattern of the decrease varied significantly among the different treatment groups. The bacterial-enzyme synergistic treatment group exhibited the best and most stable protein degradation ability.

[0067] The synergistic fermentation of bacteria and enzymes demonstrated a significant advantage in maintaining protease activity. Exogenous enzymatic pretreatment provided the system with ample initial enzyme activity, while inoculation with lactic acid bacteria delayed the decline in enzyme activity through metabolic regulation. The synergistic effect of these two processes enabled the continuous and efficient operation of the protein hydrolysis process. This result provides enzymatic assurance for the deep hydrolysis of red sour soup protein substrates and lays the material foundation for the formation of flavor compounds.

[0068] 2.2 Changes in cellulase activity The effects of different treatment methods on cellulase activity during the fermentation of red sour soup, such as Figure 5 As shown, the cellulase activity in the enzymatic hydrolysis group and the bacterial-enzyme synergistic group decreased with the extension of fermentation time, while the low enzyme amount group showed trace activity in the early stage of fermentation and then quickly dropped to zero. The inoculated group and the natural fermentation group showed no activity throughout the entire process.

[0069] The addition of exogenous enzymes is the main source of cellulase activity in the red sour soup fermentation system. The synergistic treatment of bacteria and enzymes effectively delayed the decline in cellulase activity and maintained a more sustained degradation capacity through the metabolic regulation of lactic acid bacteria. Under low enzyme levels, enzyme activity was rapidly depleted, making it difficult to support effective degradation. This result highlights the advantages of synergistic bacteria-enzyme treatment in resource utilization efficiency and provides an enzymatic basis for the efficient deconstruction of plant cell walls during red sour soup fermentation.

[0070] 2.3 Changes in β-glucosidase activity The effects of different treatment methods on β-glucosidase activity during the fermentation of red sour soup, such as Figure 6 As shown, there were significant differences in the changes in β-glucosidase activity among the treatment groups, with the bacterial-enzyme synergistic treatment group exhibiting unique dynamic characteristics.

[0071] The addition of exogenous enzymes is the main source of β-glucosidase activity in the red sour soup fermentation system. Synergistic treatment of bacteria and enzymes significantly increased β-glucosidase activity in the mid-fermentation stage, forming a distinct activity peak, through the regulation of lactic acid bacteria metabolism. Although the activity level decreased somewhat in the later stages, it remained at a high level. This dynamic process demonstrates a synergistic mechanism where enzymatic hydrolysis provides the driving force for microbial growth, and microbial metabolism, in turn, enhances enzyme activity. This provides enzymatic support for the efficient degradation of plant cell wall polysaccharides and the generation of fermentable sugars during red sour soup fermentation.

[0072] 2.4 Changes in polysaccharide content The changes in polysaccharide content in red sour soup under different fermentation methods are as follows: Figure 7 As shown in the figure. The results indicate that the synergistic fermentation of bacteria and enzymes showed a significant polysaccharide recovery capacity in the later stage of fermentation, which was higher than that of the enzymatic hydrolysis alone, the inoculation alone, and the natural fermentation group. This suggests that the synergistic fermentation of bacteria and enzymes can not only slow down polysaccharide degradation, but also promote the synthesis of new polysaccharides by microorganisms.

[0073] Compared to the enzyme-only group, the higher final value in the synergistic group reflects the contribution of microorganisms; compared to the inoculation-only group, the higher initial value in the synergistic group reflects the key role of enzymatic hydrolysis. Therefore, the synergistic effect of bacteria and enzymes establishes a dynamic balance between enzyme release, cell utilization, and extracellular polysaccharide synthesis, optimizing polysaccharide retention. Compared to traditional processes, the polysaccharide retention rate of bacteria-enzyme synergistic fermentation (79.43%) is 41.71% higher than that of natural fermentation (56.05%), demonstrating that this technology can effectively solve the problem of polysaccharide loss in traditional fermentation and improve the functional quality of red sour soup.

[0074] 2.5 Changes in phenolic content The results showed that enzymatic hydrolysis and inoculation treatment exhibited a good synergistic effect, providing an important basis for regulating the functional quality of traditional fermented foods through targeted fermentation technology.

[0075] 2.6 Changes in total phenol content The results show that ( Figure 9 The synergistic fermentation of bacteria and enzymes demonstrates significant advantages in promoting the release of phenolic substances and maintaining stability. While the total phenolic content in the synergistic group was slightly lower than the highest value in the enzymatic hydrolysis-only group, the overall trend was more stable, and it was higher than that in the natural fermentation and inoculation-only groups. This indicates that the synergistic fermentation of bacteria and enzymes releases and transforms phenolic substances more efficiently through the complementary effect of microbial metabolism and enzymatic hydrolysis. The synergistic fermentation of bacteria and enzymes significantly increased the total phenolic content of red sour soup and enhanced fermentation stability, maintaining high efficiency while reducing enzyme dosage. This helps reduce production costs, improves process economy, and provides a scientific basis for the modernization and improvement of traditional fermented foods.

[0076] 2.7 Changes in total flavonoid content like Figure 10 As shown, this study investigated the promoting effect of bacterial-enzyme co-fermentation on the functional components of red sour soup by comparing the effects of different treatments on the total flavonoid content. Bacterial-enzyme co-fermentation, through enzymatic pretreatment to optimize substrate availability and combined with the metabolic activity of *Lactobacillus plantarum*, significantly promoted the accumulation of total flavonoids in red sour soup. This result provides a theoretical basis for the development of high-quality fermentation technology for red sour soup.

[0077] 2.8 Changes in capsaicin content like Figure 11 As shown, bacterial-enzyme co-fermentation significantly slows down the degradation of capsaicin and improves its retention rate, exhibiting a clear synergistic effect. Bacterial-enzyme co-fermentation not only optimizes fermentation efficiency but also significantly inhibits capsaicin degradation and improves the retention rate of bioactive components.

[0078] 2.9 Changes in 6-gingerol content like Figure 12 As shown, this study determined the effects of different treatments on the 6-gingerol content in red sour soup. The results showed that different pretreatments and fermentation methods produced significant differences. Simply relying on natural fermentation or mild enzymatic hydrolysis without controlling the microbial community leads to highly unstable 6-gingerol content; although it may accumulate in the short term, it is ultimately lost due to excessive degradation. Inoculation with *Lactobacillus plantarum* is a key strategy for achieving stable retention of 6-gingerol.

[0079] 2.10 Changes in GABA content like Figure 13 As shown, this study measured the changes in GABA content during the fermentation of red sour soup under different treatments, aiming to elucidate the independent and synergistic effects of enzymatic hydrolysis and *Lactobacillus plantarum* inoculation on GABA production. In this red sour soup fermentation system, the net change in GABA was consumption rather than enrichment. Inoculation with *Lactobacillus plantarum* is an effective strategy to stabilize GABA content and slow down its degradation; high-dose enzymatic hydrolysis may accelerate GABA loss; the synergistic effect of enzymatic hydrolysis and inoculation was not significant in this system.

[0080] 2.11 Changes in DPPH free radical scavenging activity like Figure 14 As shown, this study determined the effects of different treatments on DPPH scavenging rate during the fermentation of red sour soup and systematically evaluated the contributions of enzymatic hydrolysis and *Lactobacillus plantarum* inoculation to antioxidant activity. Fermentation is the core process for enhancing the antioxidant activity of red sour soup. Inoculation with *Lactobacillus plantarum* is the preferred strategy for rapidly enriching antioxidants. Low-dose enzymatic hydrolysis and its synergistic effect can significantly enhance antioxidant activity in the early stage of fermentation. Although natural fermentation starts more slowly, it can ultimately achieve a high level of antioxidant activity.

[0081] 2.12 Changes in ABTS free radical scavenging activity like Figure 15 As shown, this study systematically evaluated the effects of enzymatic hydrolysis and Lactobacillus plantarum inoculation on the formation of antioxidant activity by analyzing the changes in the ABTS free radical scavenging rate of red sour soup under different fermentation treatments.

[0082] Fermentation significantly enhances the ABTS free radical scavenging capacity of red sour soup, with the highest increase exceeding 3 times; inoculation with Lactobacillus plantarum accelerates the formation of antioxidant activity, with particularly significant effects in the early stages of fermentation; although the natural fermentation system starts up more slowly, it can eventually reach an antioxidant level comparable to that of pure culture fermentation; these findings provide important theoretical basis for optimizing the production process of red sour soup and scientifically explaining the mechanism of its antioxidant activity formation.

[0083] 2.13 Changes in Vitamin C Content like Figure 16 As shown, this study measured the dynamic changes in vitamin C content in red sour soup under different fermentation treatments. The fermentation process of red sour soup helps retain and accumulate vitamin C. Synergistic treatment with bacteria and enzymes, especially the combination of low-dose enzymatic hydrolysis and inoculation, significantly improved the nutritional value of the product through a synergistic mechanism of substrate optimization via appropriate enzymatic hydrolysis and inoculation-led fermentation.

[0084] 2.14 LC-MS Non-targeted Metabolomics Data Analysis During the fermentation of red sour soup, the metabolite profile underwent significant and orderly changes. The results of comprehensive heatmap cluster analysis clearly show that the sample fermented for 6 days was significantly better than the unfermented initial sample in terms of metabolite richness and flavor compound accumulation.

[0085] Firstly, the accumulation of amino acids and small peptides in the fermented samples showed a significant advantage. Dozens of dipeptides and tripeptides, such as Ile-Val-Leu, Ile Ile Leu, Leu Ser Phe, Asp Val Leu, Gly-Ile-Leu, and Asn Ile Phe, exhibited a continuous upward trend from low levels at day 0 of fermentation to high levels at days 3 and 6. These small-molecule peptides are products of deep protein degradation, and their enrichment directly demonstrates that enzymatic pretreatment combined with Lactobacillus plantarum fermentation can effectively convert large-molecule proteins in the raw materials into more easily absorbed, flavor-active small-molecule nitrogen-containing compounds. Small peptides and free amino acids are the core material basis for umami, richness, and complex flavor. Therefore, the substantial increase in these substances after 6 days of fermentation signifies a fundamental improvement in the flavor quality of the red sour soup.

[0086] Secondly, regarding organic acid metabolism, the fermentation process achieves a positive transformation from raw material organic acids to fermented organic acids. Citric acid content was highest in the sample at day 0 of fermentation, gradually decreasing as fermentation progressed to days 3 and 6. This reflects that the citric acid inherent in raw materials such as tomatoes and peppers was utilized as a carbon source or converted into other organic acids by lactic acid bacteria during fermentation. Simultaneously, the continuously increasing levels of organic acid metabolites such as 2-methylcitric acid and 2-hydroxy-4-methylpentanoic acid are direct products of lactic acid bacteria activity. This metabolic transformation—consuming raw material organic acids and generating fermented organic acids—ensures that the fermented red sour soup not only retains its sour characteristics but also constructs a more complex and mellow sourness system, avoiding the sharpness of simple raw material acids. This is the key to the flavor enhancement of traditional fermented foods.

[0087] In summary, fermentation treatment completely reshaped the metabolite profile of red sour soup. Samples fermented for 6 days showed significantly better results than samples fermented for 0 days in terms of the abundance of small peptides, the complexity of organic acids, and the activity of the overall metabolic network. This metabolic evolution ultimately translates into an improvement in the sensory quality of red sour soup, manifested as a richer umami flavor, a more mellow sour taste, and a more complex flavor profile. This fully validates the effectiveness and necessity of the enzymatic hydrolysis + Lactobacillus plantarum fermentation process used in this study in improving the quality of red sour soup.

[0088] KEGG pathway enrichment analysis was performed on three key time points (0, 3, and 6 days) of fermentation in the enzymatic hydrolysis (1 / 10) and inoculated groups to reveal the dynamic evolution of the metabolic network during Lactobacillus plantarum fermentation. The fermented samples (3 and 6 days) showed significantly better metabolic activity and functional integrity than the unfermented samples, and the 6-day fermentation group exhibited further maturation and optimization of the metabolic system compared to the 3-day group.

[0089] Compared to day 0 of fermentation, analysis on day 3 showed that the citric acid cycle (TCA) was the most significantly changed pathway with the highest enrichment ratio. Although the day 0 sample underwent enzymatic pretreatment, releasing large amounts of monosaccharides, amino acids, and organic acids, microbial metabolism had not yet started, and the TCA cycle was relatively quiescent. However, by day 3, *Lactobacillus plantarum* had entered a period of vigorous metabolism, efficiently generating energy and biosynthetic precursors through the TCA cycle using the abundant carbon sources released by enzymatic hydrolysis, providing power for cell growth, reproduction, and secondary metabolism. This signifies that the sample had transformed from a nutrient-rich but metabolically inert raw material system into a highly active bioreaction system. High enrichment was also observed in glyoxylic acid and dicarboxylic acid metabolism, as well as phenylalanine metabolism. The former corresponds to the degradation and transformation of raw material lipids, while the latter indicates the initiation of the synthesis of volatile flavor compounds such as phenylethanol and benzaldehyde, which can be metabolized and degraded into phenolic acids by gut microbiota. The significant enrichment of other carbon fixation pathways and the two-component system further confirms that the microorganisms in the day 3 fermentation sample were actively adapting to the environment and actively acquiring carbon sources. As an important signal transduction system, the two-component system regulates a variety of physiological and metabolic processes, such as bacteriocin synthesis, proteolysis, acid resistance, and antibiotic resistance. Its enrichment indicates that microorganisms have initiated adaptive regulatory mechanisms in the fermentation environment.

[0090] Compared to day 0 of fermentation, analysis on day 6 showed that the TCA cycle remained dominant, indicating that *Lactobacillus plantarum* maintained vigorous energy metabolism activity throughout the entire fermentation cycle. Nucleotide metabolism followed closely with the second highest enrichment ratio, reflecting the continued active nucleic acid metabolism of microorganisms in the later stages of fermentation, where raw material nucleic acids were degraded into small molecules such as nucleosides and bases, serving as both nitrogen sources and flavor precursors. Phenylalanine metabolism maintained a high enrichment level, confirming the continuous synthesis of volatile flavor compounds. The significant enrichment of other carbon fixation pathways and alanine, aspartic acid, and glutamate metabolism indicated that the utilization of carbon and nitrogen sources by microorganisms became more diversified and refined in the later stages of fermentation. The enrichment of ABC transporters and purine metabolism reflected the continued activity of transmembrane transport and genetic material metabolism, respectively, revealing that the metabolic network of the 6-day fermented red sour soup sample was fully mature, with energy metabolism, flavor compound synthesis, and genetic information processing all in a highly active state.

[0091] Compared to day 3 of fermentation, analysis on day 6 showed that nucleotide metabolism had the highest enrichment ratio, while purine and pyrimidine metabolism were also highly enriched, indicating that microbial nucleic acid metabolism activity reached its peak in the mid-to-late stages of fermentation, with cell proliferation accompanied by DNA / RNA synthesis and turnover. The significant enrichment of ABC transporters, coupled with the functional coupling of nucleotide metabolism, reflects the active transmembrane transport of substances maintained by microorganisms in the later stages of fermentation. Overexpression of ABC transporters increased intracellular ATP concentration, thus protecting cells from acid damage in the early stages of acid stress. The enrichment of cysteine ​​and methionine metabolism indicates that the day 6 fermentation sample has a unique advantage in sulfur-containing flavor synthesis. The presence of tryptophan metabolism and the sulfur transport system further confirms the refined regulation of secondary metabolism and flavor synthesis in the later stages of fermentation.

[0092] In summary, from day 0 to day 6 of fermentation, the red sour soup underwent a complete metabolic evolution process involving energy activation, network construction, and refined optimization. The fermented sample comprehensively surpassed the unfermented sample in terms of energy metabolism, amino acid conversion, nucleotide metabolism, and flavor synthesis potential. The sample fermented for 6 days further demonstrated functional differentiation of the metabolic system and refined refinement of the flavor network, validating the significant effectiveness of the microbial-enzyme co-fermentation process in improving the quality of red sour soup from a systems biology perspective.

[0093] Example 3 1 Experimental Methods Place the red sour soup sample on white paper and measure the color difference using a calibrated colorimeter: L * (Brightness), a* (red-green intensity), b* (yellow-blue intensity), and ΔE.

[0094] The reducing sugar content of red sour soup was determined using the 3,5-dinitrosalicylic acid method (DNS method). The nitrite content was determined according to GB 5009.33-2016. The determination of amino acid nitrogen in food was performed using a saccharimeter, in accordance with GB 5009.235-2016. The determination of soluble protein was performed according to the reference (Bradford, 1976). The dietary fiber extraction protocol followed the method described by Jia et al. (Jia et al., 2019). The determination of organic acids was performed according to GB 5009.157-2016, "National Food Safety Standard: Determination of Organic Acids in Food". Non-targeted metabolomics analysis was also conducted.

[0095] 2. Experimental Results 2.1 pH Changes like Figure 17As shown, this study elucidates the contribution of synergistic bacterial-enzyme treatment to fermentation start-up speed and acidification efficiency by measuring the dynamic changes in pH value during the fermentation of red sour soup under different treatments. Synergistic bacterial-enzyme treatment significantly accelerates the fermentation process of red sour soup and shortens the production cycle. Its mechanism of action involves enzymatic hydrolysis optimizing the substrate and inoculation dominating acidification; both are indispensable. Low-dose enzymatic hydrolysis achieves the best synergistic effect, demonstrating its economic efficiency and high efficiency.

[0096] 2.2 Changes in total acid content like Figure 18 As shown, inoculation with *Lactobacillus plantarum* is the dominant factor in acid production in red sour soup, while enzymatic pretreatment plays a powerful role by providing pre-digested nutrient substrates for the bacteria. Synergistic bacterial-enzyme treatment, especially the full-volume enzymatic hydrolysis synergistic group, showed the best performance in both fermentation rate and final acid production.

[0097] 2.3 Color Changes The effects of different treatment methods on color indicators (L, a, b*, ΔE) during the fermentation of red sour soup showed that the interaction between enzymatic hydrolysis and inoculation had a significant impact on the product color. Among them, the synergistic treatment of bacteria and enzymes (EZ-I) exhibited a better red-yellow color in the middle and late stages of fermentation. Enzymatic hydrolysis pretreatment disrupted the cell wall structure and promoted the release of pigment precursors such as capsanthin and lycopene; the metabolism of *Lactobacillus plantarum* affected the pigment state and color development by changing the pH and redox environment. The low enzyme synergistic group showed outstanding redness, while only the inoculated group and the natural fermentation group showed weak color formation, highlighting the key role of enzymatic hydrolysis pretreatment in improving the color quality of red sour soup.

[0098] 2.4 Changes in reducing sugar content like Figure 19 As shown, this study measured the changes in reducing sugar content in red sour soup after 0, 3, and 6 days of fermentation under different treatments. The synergistic treatment group with bacteria and enzymes showed unique advantages in the middle and late stages of fermentation, especially in maintaining and increasing the reducing sugar level, which was significantly better than the single treatment group and the natural fermentation group.

[0099] 2.5 Changes in nitrite content like Figure 20 As shown, this study systematically evaluated the regulatory effects of enzymatic hydrolysis, *Lactobacillus plantarum* inoculation, and their synergistic effects on the safety of red sour soup fermentation by measuring the dynamic changes in nitrite content during fermentation in different treatment groups. The results showed that the synergistic treatment of bacteria and enzymes could rapidly and significantly reduce nitrite content.

[0100] 2.6 Determination of amino acid nitrogen like Figure 21As shown, this study measured the dynamic changes of amino acid nitrogen in red sour soup under different treatment methods to elucidate the synergistic mechanism of bacterial-enzyme co-fermentation on protein transformation and flavor precursor accumulation. Bacterial-enzyme co-fermentation ensures a continuous nitrogen source supply through enzymatic hydrolysis, while efficiently assimilating precursors and directing them to the flavor metabolic network, avoiding ineffective amino acid accumulation. This is conducive to the formation of a harmonious and rich complex flavor, providing a core technological strategy for the production of high-quality red sour soup.

[0101] 2.7 Changes in soluble solids like Figure 22 As shown, the analysis of soluble solids content during the fermentation of red sour soup reveals that different treatment methods have a decisive influence on its changing trend, highlighting the unique advantages of microbial-enzyme synergistic treatment in promoting substance dissolution and transformation. Microbial-enzyme synergistic fermentation, through the powerful degradation by exogenous enzymes, continuously injects soluble components into the system. This not only offsets the decline caused by microbial consumption but also achieves additional growth through microbial metabolic contributions, ensuring that the product obtains higher substance concentrations and richer flavor precursors. This is the material basis for improving the sensory and flavor fullness of red sour soup.

[0102] 2.8 Changes in soluble proteins The effects of different treatment methods on the soluble protein content during the fermentation of red sour soup, such as Figure 23 As shown in the figure, the soluble protein content of each treatment group showed a continuous decreasing trend with the extension of fermentation time, but the rate of decrease and the final value differed significantly. Among them, the bacterial-enzyme synergistic treatment group showed the best protein retention capacity.

[0103] 2.9 Changes in dietary fiber content like Figure 24 As shown, the dietary fiber content of the red sour soup samples in each treatment group remained relatively stable during fermentation, without showing a consistent upward or downward trend. This indicates that dietary fiber, as a structurally stable polysaccharide component, was not significantly degraded or transformed in this fermentation system.

[0104] 2.10 Changes in organic acid content This embodiment measured the dynamic changes of organic acids during the fermentation of red sour soup under different treatments. The results showed that the fermentation process and treatment methods have a decisive influence on the formation of organic acids. Among them, the synergistic treatment of bacteria and enzymes showed the core advantages in driving the efficient accumulation of lactic acid and shaping a harmonious acidity spectrum. The core of the regulation of organic acid metabolism in red sour soup by synergistic fermentation of bacteria and enzymes is not simply to increase the total amount of lactic acid, but to optimize lactic acid accumulation (rapid start-up) and coordinate the overall balance of the organic acid spectrum. This achieves a rapid start and stable finish in the fermentation process, shaping an acidity spectrum dominated by mild lactic acid and with a variety of characteristic acid flavors in harmony. This provides a key chemical basis for improving the layering and acceptability of the flavor quality of red sour soup.

[0105] 2.11 Multivariate Statistical Analysis To comprehensively analyze the effects of microbial-enzyme co-fermentation on the metabolites of red sour soup, this study employed UPLC-QTOF-MS non-targeted metabolomics to analyze six groups of samples. The quality control samples showed tight clustering in the PCA plot, indicating good instrument stability and reliable data; a total of 3773 metabolites were identified. PCA analysis revealed significant separation in the metabolomics profiles of the six groups. The first principal component (PC1, 24.7%) mainly reflected differences in enzyme addition; the groups with conventional enzyme levels (EZ, EZ-I) and low enzyme levels (LEZ, LEZ-I) were clearly separated along the PC1 axis. The second principal component (PC2, 22.6%) mainly reflected whether inoculation was involved; the inoculated groups (EZ-I, LEZ-I, I) and the uninoculated groups (EZ, LEZ, NF) were clearly separated along the PC2 axis. The enzymatic hydrolysis + inoculation group (EZ-I) was located between the pure enzymatic hydrolysis group (EZ) and the pure inoculation group (I), indicating that its metabolic characteristics were influenced by both. Overall, all treatment groups were clearly separated from the naturally fermented group, indicating that artificial intervention significantly altered the metabolic profile of red sour soup.

[0106] 2.12 Screening of key differential metabolites To comprehensively analyze the effects of bacterial-enzyme synergistic fermentation on the metabolites of red sour soup, this study conducted a comprehensive analysis of the top 20 metabolites with the largest fold differences among the six groups. The results showed that bacterial-enzyme synergistic treatment exhibited a systematic and multi-level regulatory effect on the metabolic network of red sour soup.

[0107] The most significant and consistent effect of the synergistic treatment of bacteria and enzymes was the promotion of the accumulation of specific bioactive peptides. L-lysine-L-alanine-L-phenylalanine was significantly upregulated in multiple synergists, becoming a marker product. Furthermore, peptides such as tyrosine-arginine-lysine, phenylalanyl-aspartic-alanine, and prolyl-valine-methionine were also enriched in different synergists. This demonstrates that the free amino acids and small peptide fragments released by the enzyme were efficiently utilized and reassembled by *Lactobacillus plantarum* to synthesize functional peptides that are difficult to accumulate with a single treatment. The upregulation of nitrogen-containing compounds such as glutamine-arginine, DL-arginine, and D-ornithine further reflects the enhancement of nitrogen metabolism by the synergistic system.

[0108] In the synergistic group, nivermectin C, bitter substances, and various flavonoid glycosides and phenolic esters were upregulated (EZ-I vsI), indicating that enzymatic hydrolysis helps release cell wall-bound phenolic substances, which are then retained in the fermentation environment. Compared with natural fermentation, the synergistic group showed a significant reduction in simple phenols such as 4-hydroxystyrene and methoxybenzyl alcohol, as well as complex oxidized lipid phenolic products. This suggests that the synergistic system may inhibit phenolic degradation and byproduct formation caused by other microorganisms or oxidation, thus contributing to the maintenance of flavor purity and stability.

[0109] In the bacterial-enzyme synergistic group, oxidized fatty acids such as 9,10-dihydroxystearic acid and 11-hydroxy-eicosadienoic acid were downregulated compared to the enzymatic hydrolysis-only group, possibly because *Lactobacillus plantarum* consumed or transformed these oxidative intermediates. Lipid-derived aroma components such as cis-3-hexenyl acetate and cis-jasmone were upregulated compared to the inoculation-only group, indicating that lipid precursors provided by enzymatic hydrolysis promoted the biosynthesis of aroma substances. Lysophosphatidylcholine was often downregulated in the synergistic group, reflecting the dynamic balance between membrane lipid metabolism and cell growth.

[0110] In the naturally fermented group, carbohydrate derivatives such as maltotriose and D-sorbitol were generally enriched, while these substances showed a downregulation trend in the co-fermentation group. This indicates that in single-strain fermentation dominated by *Lactobacillus plantarum*, carbon flow is more efficiently directed to acid-producing pathways such as lactic acid fermentation, reducing the accumulation of intermediate sugar alcohols or oligosaccharides. Raffinose was upregulated in the low-enzyme co-fermentation group (LEZ-I vsI), suggesting that mild enzymatic hydrolysis may selectively release specific functional oligosaccharides.

[0111] Nucleic acid metabolites such as xanthine nucleotides and 2'-O-methyladenosine were downregulated compared to the enzymatic digestion-only group, which is related to their utilization during the growth and reproduction of *Lactobacillus plantarum*, reflecting active microbial nucleic acid metabolism. 3,3'-Diaminodipropylamine was upregulated in multiple synergistic groups, and as a polyamine, it is associated with microbial stress response and cell growth regulation. L-tyramine was significantly downregulated in the synergistic group (especially compared to natural fermentation), indicating that single-strain fermentation can effectively inhibit the activity of contaminating microorganisms that decarboxylate amino acids and produce amines, thus improving food safety. Reduced glutathione was upregulated under some synergistic conditions, and the presence of isoascorbic acid suggests that synergistic fermentation helps to form or retain antioxidant active ingredients.

[0112] 2.13 KEGG enrichment analysis of differential metabolites To systematically elucidate the regulatory mechanism of bacterial-enzyme co-fermentation on the metabolic network of red sour soup, this study conducted KEGG pathway enrichment analysis on the differential metabolites of six groups.

[0113] Phenylacetane biosynthesis is the most crucial and stable metabolic target in the synergistic process between bacteria and enzymes. The enrichment factor of this pathway is positively correlated with the intensity of enzymatic hydrolysis. This indicates that the phenolic acids and flavonoid precursors released by enzymatic hydrolysis are efficiently converted by *Lactobacillus plantarum*, which is the core metabolic basis for the characteristic color, flavor, and antioxidant activity of red sour soup.

[0114] The secondary metabolic potential of microorganisms was fully stimulated. The biosynthesis pathways of macrolide antibiotics and non-ribosomal peptide siderophores were continuously enriched in the synergistic group compared with the natural fermentation and enzymatic hydrolysis group. This confirmed that the abundant substrates provided by enzymatic hydrolysis pushed the metabolic state of Lactobacillus plantarum from survival mode to synthetic mode, synthesizing siderophores, polyketides and other microbial functional products that surpass the traditional flavor.

[0115] The sphingolipid signaling pathway and sphingolipid metabolism were significantly enriched in the synergistic group, the inoculated group alone, and the natural fermentation group, indicating that enzymatic hydrolysis treatment profoundly affects the cell membrane composition and signal sensing ability of Lactobacillus plantarum by changing the physical properties of the matrix, thus achieving an upgrade from nutrient supply to physiological regulation.

[0116] Nucleotide metabolism and purine / pyrimidine metabolism were highly enriched in the synergistic group compared with the inoculation-only group and the enzymatic digestion-only group, reflecting that the microbial nucleic acid synthesis and energy metabolism tend to be more active in the environment of abundant precursor release by enzymatic digestion, which provides a basis for accelerating biosynthesis and proliferation.

[0117] Aromatic amino acid metabolism (biosynthesis of phenylalanine, tyrosine, and tryptophan) was significantly enriched in the synergistic group compared to the naturally fermented group, providing a material basis for the synthesis of flavor compounds and bioactive precursors. Arginine and proline metabolism were enriched in the low-enzyme synergistic group compared to the inoculated group alone, suggesting that low-intensity enzymatic hydrolysis directionally regulates nitrogen metabolism. Lipid-related pathways such as arachidonic acid metabolism and α-linolenic acid metabolism were continuously enriched in multiple comparisons, indicating that bacteria and enzymes synergistically promote the conversion and functionalization of raw material lipids, affecting flavor formation and textural stability.

[0118] Microbial-enzyme co-fermentation achieves a systematic reshaping of the metabolic network of red sour soup through the synergistic effects of three core pathways: phenylpropane biosynthesis (flavor basis), macrolide biosynthesis (microbial function), and sphingolipid signaling pathway (physiological regulation). This technology transforms complex raw material components into fermented products with prominent functional components, harmonious flavor, and high safety through controllable and efficient biotransformation, providing a solid metabolomics basis for the modernization and upgrading of traditional fermented foods.

[0119] 2.14 Comprehensive Analysis of Key Metabolic Pathways This embodiment reveals that bacterial-enzyme co-fermentation, compared to single fermentation, reshapes the metabolic network of red sour soup in multiple dimensions. Among them, the phenylpropane biosynthesis, macrolide antibiotic biosynthesis, and sphingolipid signaling pathway were significantly enriched in comparisons between different groups and showed clear regulatory patterns with changes in process conditions, which best systematically demonstrate the metabolic advantages and core regulatory mechanisms of bacterial-enzyme co-fermentation.

[0120] Example 4 To systematically evaluate the effects of different pretreatment methods and sterilization processes on the shelf-life quality of red sour soup, this study added sterilized and non-sterilized control groups to the experimental groups in Example 2. The sample preparation methods for each group are as follows: Sterilization Group: The raw material slurry for red sour soup was first sterilized (112℃, 1 min) to eliminate interference from natural microorganisms in the raw material. After sterilization, each group underwent enzymatic hydrolysis and inoculation treatment, followed by fermentation at the optimal process parameters (fermentation time 6 days, fermentation temperature 31℃, inoculation rate 2%). After fermentation, the finished red sour soup was obtained and placed in a 35℃ constant temperature incubator for accelerated storage experiments. Samples were taken at 0, 15, 30, 45, 60, 75, and 90 days to test various indicators.

[0121] Unsterilized control group: To investigate the effect of sterilization process on the storage stability of red sour soup, an enzymatic hydrolysis (1 / 10) + inoculation group was added. The samples in this group were not sterilized, but were directly enzymatically hydrolyzed and inoculated for fermentation according to the above method. After the fermentation was completed, the finished product was obtained and accelerated storage experiment was carried out under the same conditions.

[0122] This embodiment systematically evaluated the dynamic effects of different pretreatment methods on the acidity, polysaccharides, antioxidant activity, and sensory quality of red sour soup during a 90-day storage period through an accelerated storage experiment at 35℃. Combined with whole-genome sequencing of *Lactobacillus plantarum* NR1-7, the genetic basis of its quality regulation was revealed at the molecular level. The main results are as follows: Shelf-life quality analysis showed that inoculation with *Lactobacillus plantarum* was the core factor determining the acidity stability of red sour soup. The sterilized inoculated group exhibited excellent acidity stability with pH fluctuations ≤0.06 and a total acidity decrease ≤15% during a 90-day storage period. While the unsterilized inoculated group showed more efficient acid production in the short term, its acidity stability was inferior to the sterilized group in the later stages. The synergistic treatment of bacteria and enzymes showed limited polysaccharide protection in the early storage period (15-45 days), while the low-enzyme synergistic group showed better polysaccharide retention in the later storage period. Regarding antioxidant activity, the unsterilized synergistic group showed the best activity maintenance capacity during long-term DPPH storage, while the antioxidant activity of ABTS was more temperature-sensitive and rapidly declined during accelerated storage. Sensory evaluation results showed that the synergistic treatment group maintained the highest sensory score throughout the entire storage period, with the unsterilized synergistic group performing best in the later storage period.

[0123] Genome-wide analysis revealed six gene clusters for the biosynthesis of secondary metabolites in the NR1-7 genome. Two RiPP-like bacteriocin biosynthesis systems were identified on chromosomes and plasmids, both containing lagD (ABC transporter) and lcnD (secretory protein) genes, providing direct genetic evidence for the sustained specific antibacterial effect of NR1-7 during the later stages of the red sour soup's shelf life. Regions 1.2-1.4 constitute the biosynthetic pathways for terpenes and polyketides. The crtM / crtN gene cluster in Region 1.4 is responsible for carotenoid synthesis, closely related to the formation of the red color and antioxidant activity of red sour soup. Region 1.5 encodes the Agr quorum sensing system, regulating the temporal expression of secondary metabolites such as bacteriocins, enabling the strain to optimize resource allocation based on population density.

[0124] COG functional classification analysis showed that the NR1-7 genome was highly enriched with core functional genes such as carbohydrate metabolism, amino acid metabolism, and nucleotide metabolism, which ensured the rapid acid production capacity of the strain in the early stage of fermentation. At the same time, the antimicrobial peptide synthesis system, multidrug efflux pump, antibiotic resistance protein and phage defense system encoded by the defense mechanism (category V) gene formed a spatiotemporal synergy with the core metabolism, together constituting a complete biological preservation system.

[0125] In summary, *Lactobacillus plantarum* NR1-7 possesses a complete genetic pathway for synthesizing bacteriocins, carotenoids, and polyketides. Its Agr quorum sensing system regulates the temporal expression of secondary metabolites, and the presence of two bacteriocinonin systems (chromosome + plasmid) significantly enhances its antibacterial potential. These genetic characteristics are highly consistent with its phenotype of rapid acid production, maintaining stable acidity, inhibiting spoilage bacteria, preserving red color, and exhibiting antioxidant activity during the shelf life of red sour soup. This provides a solid molecular theoretical basis for the application of bacterial-enzyme co-fermentation technology in the standardized and safe production of red sour soup.

[0126] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing red sour soup through synergistic fermentation of bacteria and enzymes, characterized in that, Includes the following steps: After mixing and pulping the raw materials, a compound enzyme preparation is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, Lactobacillus plantarum NR1-7 is inoculated for fermentation to obtain red sour soup.

2. The preparation method according to claim 1, characterized in that, The ingredients, by weight, include: 40-60 parts red chili peppers, 20-30 parts tomatoes, 5-15 parts glutinous rice flour, 3-8 parts ginger, 3-8 parts garlic, 1-5 parts white sugar, and 1-5 parts white wine.

3. The preparation method according to claim 1, characterized in that, The compound enzyme preparation is composed of pectinase, cellulase and protease; the enzymatic hydrolysis treatment is carried out at a temperature of 35-45℃ for 8-16 hours.

4. The preparation method according to claim 3, characterized in that, The amount of pectinase added is 30-70 U / g of raw material, the amount of cellulase added is 30-70 U / g of raw material, and the amount of protease added is 50-150 U / g of raw material.

5. The preparation method according to claim 1, characterized in that, The inoculum size of *Lactobacillus plantarum* was 2% (m:m), and the bacterial concentration was 3 × 10⁻⁶. 9 CFU / mL; the fermentation temperature was 31℃ and the fermentation time was 6 days.

6. The preparation method according to claim 1, characterized in that, Prior to the enzymatic hydrolysis treatment, the process also includes a step of sterilizing the raw materials.

7. The red sour soup prepared by the preparation method according to any one of claims 1-6.

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