A compound microbial agent, fermented glutinous rice ball chili sauce and preparation method and application thereof

Abstract

The application discloses a kind of compound microbial inoculant, fermented Xiba chili sauce and its preparation method and application, it is related to food fermentation technical field, preparation method includes four steps of rehydration, crushing, mixing, bacteria inoculation fermentation.No salt is added when mixing, 25% vol~35% vol alcohol of white spirit is added to make the initial ethanol volume fraction of mixture 4%~8%;The compound microbial inoculant composed of plant lactobacillus P158 and LYA31 is inoculated according to the ratio of viable bacteria 1:0.8~1.2, first main fermentation at 30~35 DEG C to pH value not higher than 4.2, then post-mature at 20~25 DEG C, total fermentation 90d~120d.The application realizes directional fermentation in the system of not adding salt completely, by compounding plant lactobacillus P158 and plant lactobacillus LYA31, with white spirit to build ethanol selective barrier, so that the content of capsaicin and nitrite is significantly reduced.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, specifically to a compound microbial agent, fermented glutinous rice chili sauce, its preparation method and application. Background Technology

[0002] Ciba chili is a unique chili product from the Sichuan, Chongqing, and Guizhou regions. It gets its name from its resemblance to soft, glutinous rice cakes after processing. It's an important ingredient in hot pot broth, spicy chicken, chili oil, and other specialty foods. The traditional method involves selecting moderately spicy and fragrant dried chilies, removing the stems, washing them, and soaking them in water or hot water to soften them. Then, pound them together with ginger and garlic in a mortar or pestle until they reach a sticky, glutinous consistency. Finally, salt is added for seasoning.

[0003] Existing technical solutions for glutinous rice cakes and chili sauce mainly have the following three problems: Firstly, high concentrations of salt are commonly used for preservation and flavoring. For example, the preparation method of Ciba chili sauce disclosed in existing technology CN101664164A involves crushing chili peppers into a paste, pouring it into an earthenware jar, adding salt, mixing evenly, covering and marinating for 12-16 hours, and then stir-frying it in hot oil at 180-190℃. The pickled chili sauce and its preparation method disclosed in CN105995917A involve placing 1000 parts by weight of water, 250 parts of Erjingtiao chili peppers, 250 parts of millet chili peppers, 70 parts of pickling salt, 20 parts of white wine, 20 parts of white sugar, spices, and lactic acid bacteria in a fermentation jar and sealing it at room temperature for 3 days. The salt content of the pickling system is approximately 4.3%. The above method uses a large amount of salt, resulting in a salty and heavy finished product. Furthermore, the nitrates carried in the salt and raw materials are easily reduced to nitrites during the pickling process, posing a food safety hazard. At the same time, the high-salt environment also inhibits the growth of lactic acid bacteria, slowing down the fermentation process.

[0004] Secondly, the fermentation process is uncontrolled, leading to unstable product quality. Traditional glutinous rice chili paste relies heavily on natural fermentation by microorganisms in the raw materials and environment, or is simply pounded and salted before use. The composition of the microbial community fluctuates with batches and seasons, resulting in a dark color, unstable hygiene, and short shelf life in the finished product. Existing technology CN106690228A discloses glutinous rice chili paste for hot pot base and a rich and mellow hot pot base and its preparation method. Although the aim is to retain a moderate spiciness and remove the pungent taste, the method involves selecting chili varieties and optimizing the stir-frying and braising processes, without introducing directional fermentation.

[0005] Third, the strong irritation of the gastrointestinal tract by capsaicin remains unresolved. Capsaicin is the main spiciness component of chili peppers; its ingestion can trigger physiological reactions such as sweating and diarrhea, and promote the secretion of inflammatory factors. While fermented chili paste can reduce the spiciness compared to dried chili peppers, the total amount of capsaicin remains the same, and consuming large quantities can still easily cause intestinal discomfort. Some studies have attempted to use Bacillus strains to enhance the fermentation of fermented chili paste to degrade capsaicin; however, Bacillus is not a dominant bacterium in traditional chili fermentation systems, and its application requires sterilization of the material to eliminate competition from background bacteria, making industrial implementation difficult. Furthermore, these strains lack the ability to rapidly produce acid, failing to simultaneously address the issues of nitrite control and shelf-life extension.

[0006] Therefore, how to enable lactic acid bacteria to proliferate normally and dominate fermentation in an ethanol-containing system without adding any salt, while simultaneously reducing capsaicin and nitrite levels, is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides a compound microbial agent, fermented glutinous rice chili sauce, and its preparation method and application. It provides a compound microbial agent that can synergistically and directionally ferment in a salt-free, ethanol-containing system, and uses this compound microbial agent to prepare fermented glutinous rice chili sauce with significantly reduced capsaicin and nitrite content.

[0008] The technical solution of the present invention is as follows: In one aspect, a compound microbial agent is provided, comprising *Lactobacillus plantarum* P158 and *Lactobacillus plantarum* LYA31 with a live bacteria ratio of 1:0.8~1.2; Lactobacillus plantarum P158, its taxonomic name is Lactobacillus plantarum ( Lactobacillus plantarum This strain was deposited on October 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 28616. The strain has been disclosed in existing technology CN117814456A and is available to the public from the aforementioned depository institution.

[0009] Lactobacillus plantarum LYA31, its taxonomic name is Lactobacillus plantarum ( Lactobacillus plantarum This strain was deposited on August 28, 2020, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 20578. The strain has been disclosed in existing technology CN112458003A and is available to the public from the aforementioned depository institution.

[0010] Secondly, a method for preparing fermented glutinous rice chili sauce is provided, comprising the following steps: S1. Rehydration: Take dried chili peppers, soak them in hot water at 90℃~100℃ for 3min~10min, remove them, drain and cool them to obtain rehydrated chili peppers; S2. Crushing: Crush the rehydrated chili peppers to a particle size of no more than 10mm, and crush the garlic, pickled ginger and pickled Erjingtiao peppers to a particle size of 2mm~4mm respectively. S3. Mixing: By weight, take 40-50 parts rehydrated chili peppers, 5-10 parts garlic, 5-10 parts pickled ginger, 4-10 parts pickled Erjingtiao peppers, 10-20 parts sweet citrus, 20-30 parts baijiu (Chinese liquor) with an alcohol content of 25% vol-35% vol, and 0.5-5 parts mixed spices. Do not add salt. Mix evenly to obtain a mixture with an initial ethanol volume fraction of 4%-8%. S4. Inoculation and Fermentation: Inoculate the above-mentioned compound microbial agent into the mixture to achieve a lactic acid bacteria count of 1×10⁻⁶. 6 CFU / g ~ 1×10 7 The product is CFU / g, sealed, and fermented at 30℃~35℃ for 7d~10d until the pH value is no higher than 4.2. Then it is fermented at 20℃~25℃ for a total fermentation time of 90d~120d to obtain fermented glutinous rice chili sauce.

[0011] Thirdly, a fermented glutinous rice cake chili sauce is provided, prepared using the method described above; this fermented glutinous rice cake chili sauce contains no added salt, has a pH value not higher than 4.2, a total acid content (calculated as lactic acid) not less than 1.5g / 100g, and a lactic acid bacteria count not less than 1×10⁻⁶. 5 The CFU / g content, nitrite content not exceeding 0.10 mg / kg, and capsaicin content not exceeding 0.15 g / kg are all acceptable.

[0012] Fourthly, the invention provides an application of the aforementioned compound microbial agent in the preparation of fermented glutinous rice chili sauce.

[0013] Fifthly, the application of the fermented glutinous rice chili sauce described above in the preparation of hot pot base or compound seasoning is provided.

[0014] The present invention has the following beneficial effects: 1. This invention achieves, for the first time, targeted lactic acid bacteria fermentation of chili paste without the addition of any salt. By combining *Lactobacillus plantarum* P158 and *Lactobacillus plantarum* LYA31, and by controlling the amount and alcohol content of the baijiu (Chinese liquor), the initial ethanol volume fraction of the mixture falls within the range of 4% to 8%. This range is sufficient to inhibit coliform bacteria, molds, and other miscellaneous bacteria, thus replacing salt as a preservative, while also remaining within the tolerance range of the compound microbial agent of this invention. Compared to the less than 1% ethanol level corresponding to the amount of baijiu used in existing chili fermentation technologies, this invention achieves a significantly higher ethanol content in the system. The volume fraction was increased by an order of magnitude, thus transforming ethanol from a flavoring agent into a preservative, allowing for the complete elimination of salt. At an ethanol volume fraction of 8%, the survival rate of the compound microbial agent of this invention reached 77.89%. Furthermore, when Lactobacillus plantarum P158 and Lactobacillus plantarum LYA31 were combined at a live count ratio of 1:0.8~1.2, especially 1:1, they exhibited a significant synergistic effect. Their degradation rate of capsaicin, acid production rate, lactic acid bacteria accumulation, and inhibitory effects on nitrite and coliforms were all superior to any single bacterium or an equivalent amount of commercial strains. 2. This invention significantly reduces the capsaicin content in Ciba chili. After fermentation with the compound microbial agent of this invention, the total amount of capsaicin decreased from 0.197 g / kg to 0.143 g / kg, dihydrocapsaicin decreased from 0.115 g / kg to 0.083 g / kg, and capsaicin decreased from 0.104 g / kg to 0.075 g / kg. The product's irritation to the gastrointestinal tract is significantly reduced. 3. This invention significantly reduces the nitrite content in the product. Since no salt is introduced into the system, the main external pathway for nitrate introduction is eliminated at the source. The nitrite content in the finished product is reduced from 0.16 mg / kg to 0.07 mg / kg, and the safety is significantly improved. 4. This invention employs a segmented temperature-controlled process for primary fermentation and secondary fermentation. During the primary fermentation stage, acid is rapidly produced at 30℃~35℃, causing the pH value of the system to drop below 4.2 within 7d~10d, forming an acid-alcohol dual barrier together with ethanol. During the secondary fermentation stage, the temperature is lowered to 20℃~25℃ for a long period of slow transformation, which not only ensures the continuous degradation of capsaicin-like substances, but also avoids color deterioration and over-ripening of flavor caused by high-temperature long-cycle fermentation. 5. Under the condition of not adding any salt, the total acid content of the product is increased from 0.74g / 100g to 1.76g / 100g, the number of lactic acid bacteria is increased from 3.291gCFU / g to 5.121gCFU / g, and the amino acid nitrogen content is increased from 0.18g / 100g to 0.20g / 100g. While extending the shelf life, it also enriches probiotics and flavor substances. 6. The manufacturing process of this invention is simple, does not require sterilization of materials, and retains the rich nutrients in the raw materials. It can be directly applied to the production of hot pot base and compound seasonings, and has good prospects for industrial application. Attached Figure Description

[0015] Figure 1 A comparison chart showing the survival rates of the compound bacterial agent of this invention and commercial bacterial strains under different ethanol concentrations; Figure 2 This is a graph showing the growth curves of the compound microbial agent of this invention and the commercial strains, as well as the pH changes of the fermentation broth. Figure 3 This is a graph showing the change in capsaicin content during the fermentation process of Example 1; Figure 4 The graphs show the rate of change in body weight and the Disease Activity Index (DAI) scores for each group of mice, where L represents the low-dose group, M represents the medium-dose group, H represents the high-dose group, U represents the unfermented group, and C represents the blank control group. Figure 5 The bar chart shows the relative abundance of gut microbiota at the phylum level in each group of mice. L represents the low-dose group, M the medium-dose group, H the high-dose group, U the unfermented group, and C the blank control group. Figure 6 The bar chart shows the relative abundance of gut microbiota genera in each group of mice. L represents the low-dose group, M the medium-dose group, H the high-dose group, U the unfermented group, and C the blank control group. Figure 7 The heatmap shows the species abundance clustering of the gut microbiota at the genus level in each group of mice, where L represents the low-dose group, M represents the medium-dose group, H represents the high-dose group, U represents the unfermented group, and C represents the blank control group. Figure 8 The evolutionary clade diagram of the differential species LEfSe in the gut microbiota of mice in each group is shown, where L is the low-dose group, M is the medium-dose group, H is the high-dose group, U is the unfermented group, and C is the blank control group. Figure 9 The graph shows the levels of colonic inflammatory factors in mice in each group, where L represents the low-dose group, M represents the medium-dose group, H represents the high-dose group, U represents the unfermented group, and C represents the blank control group. Figure 10 The graphs show the serum MPO and NF-κB P65 levels in mice in each group, where L represents the low-dose group, M represents the medium-dose group, H represents the high-dose group, U represents the unfermented group, and C represents the blank control group. Figure 11 The graph shows the short-chain fatty acid content in the colon contents of mice in each group. L represents the low-dose group, M represents the medium-dose group, H represents the high-dose group, U represents the unfermented group, and C represents the blank control group. Detailed Implementation

[0016] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0017] I. Instructions for Preservation of Biological Materials Lactobacillus plantarum P158, its taxonomic name is Lactobacillus plantarum ( Lactobacillus plantarum This strain was deposited on October 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 28616. The strain has been disclosed in existing technology CN117814456A and is available to the public from the aforementioned depository institution.

[0018] Lactobacillus plantarum LYA31, its taxonomic name is Lactobacillus plantarum ( Lactobacillus plantarum This strain was deposited on August 28, 2020, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 20578. The strain has been disclosed in existing technology CN112458003A and is available to the public from the aforementioned depository institution.

[0019] II. Biological characteristics of the strain The main biological characteristics of *Lactobacillus plantarum* P158 and *Lactobacillus plantarum* LYA31 are as follows: both are Gram-positive, non-spore-forming short rods, facultatively anaerobic, catalase-negative, with an optimal growth temperature of 30℃~37℃ and an optimal growth pH of 5.5~6.5. They can homofermentate with glucose, fructose, etc. as carbon sources to produce lactic acid.

[0020] Among them, *Lactobacillus plantarum* P158 is more tolerant to ethanol in the system and quickly initiates acid production, while *Lactobacillus plantarum* LYA31 is a diacetyl-producing strain (see CN112458003A). While synergistically producing acid, it imparts a mild fermented flavor to the product. As shown in Experiment 2, the degradation effect of capsaicin-like substances is relatively limited when used alone. However, when the two strains are combined at a live bacteria ratio of 1:0.8~1.2, especially 1:1, to form the compound bacterial agent of this invention, in a salt-free fermentation system of glutinous rice chili with an ethanol volume fraction of 4%~8%, its ethanol tolerance survival rate, acid production rate, capsaicin degradation rate, and inhibition effect on nitrite and coliform bacteria are significantly better than any single strain or an equal amount of commercial strains, showing a significant synergistic effect (see Experiment 1 and Experiment 2 for details).

[0021] III. Preparation of Microbial Agents Lactobacillus plantarum P158 and Lactobacillus plantarum LYA31 were inoculated into MRS liquid medium and activated by static incubation at 37°C for 18 h, with two generations of activation. The activated bacterial solution was then transferred to MRS liquid medium at a 2% inoculation rate and incubated at 37°C until the viable count reached 1×10⁻⁶. 9 Collect bacterial cells by centrifugation at 8000 r / min for 10 min with a concentration of CFU / mL or higher. Wash twice with sterile physiological saline and resuspend. Measure the viable cell count separately. Mix the bacterial suspensions of the two strains at a viable cell ratio of 1:1 to obtain the compound bacterial agent of this invention.

[0022] Example 1 This embodiment provides a method for preparing fermented glutinous rice cake chili sauce, including the following steps: S1. Rehydration: Take clean, new-generation dried chili peppers, soak them in 95℃ hot water for 6 minutes, remove them, drain them, and let them cool naturally to obtain rehydrated chili peppers. S2. Crushing: Crush the rehydrated chili peppers through a crusher equipped with a 10mm screen, and crush the garlic, pickled ginger, and pickled Erjingtiao peppers to a particle size of 3mm. S3. Mixing: By weight, take 45 parts rehydrated chili peppers, 8 parts garlic, 8 parts pickled ginger, 7 parts pickled Erjingtiao peppers, 15 parts sweet citrus, 25 parts baijiu (Chinese white liquor) with an alcohol content of 30% vol, and 2 parts mixed spices. The mixed spices include Sichuan peppercorns, star anise, fennel, and cinnamon in a mass ratio of 2:1:1:1. No salt is added. Mix evenly to obtain the mixture. The initial ethanol volume fraction of the mixture is determined to be 6.5%. S4. Inoculation and Fermentation: Inoculate the above-mentioned compound microbial agent into the mixture to achieve a lactic acid bacteria count of 5 × 10⁻⁶. 6 The product was in CFU / g, sealed in jars, and fermented at 33℃ for 8 days. The pH value dropped to 4.05. Then it was transferred to 23℃ for 112 days of post-fermentation, for a total fermentation time of 120 days, to obtain fermented glutinous rice chili sauce.

[0023] Example 2 The process is basically the same as in Example 1, except that: in step S1, the hot water temperature is 90℃ and the soaking time is 10 minutes; in step S3, 40 parts of rehydrated chili peppers, 5 parts of garlic, 5 parts of pickled ginger, 4 parts of pickled Erjingtiao peppers, 10 parts of sweet citrus, 20 parts of 25% vol baijiu (Chinese white liquor), and 0.5 parts of mixed spices are used, with an initial ethanol volume fraction of 4.2%; in step S4, the lactic acid bacteria count is 1×10⁻⁶. 6 The CFU / g was fermented at 30℃ for 10 days, followed by a post-fermentation at 20℃ for 80 days, for a total fermentation time of 90 days.

[0024] Example 3 The process is basically the same as in Example 1, except that: in step S1, the hot water temperature is 100℃ and the soaking time is 3 minutes; in step S3, 50 parts of rehydrated chili peppers, 10 parts of garlic, 10 parts of pickled ginger, 10 parts of pickled Erjingtiao peppers, 20 parts of sweet citrus, 30 parts of 35% vol liquor, and 5 parts of mixed spices are used, with an initial ethanol volume fraction of 7.9% in the mixture; and in step S4, the lactic acid bacteria count is 1×10⁻⁶. 7 CFU / g, primary fermentation at 35℃ for 7 days, followed by secondary fermentation at 25℃ for 103 days, for a total fermentation time of 110 days.

[0025] Comparative Example 1 It is basically the same as Example 1, except that no microbial agent is added and no fermentation is carried out. After the mixture is mixed, the traditional Ciba chili is obtained.

[0026] Comparative Example 2 The method is basically the same as in Example 1, except that the compound microbial agent is replaced with a commercial *Lactobacillus plantarum* strain with the same number of live bacteria, hereinafter referred to as the commercial strain; the commercial strain is a commercially available *Lactobacillus plantarum* (…). Lactobacillus plantarum Fermentation inoculant.

[0027] Comparative Example 3 It is basically the same as Example 1, except that the compound microbial agent is replaced with a single microbial agent of Lactobacillus plantarum P158 with the same number of live bacteria.

[0028] Comparative Example 4 It is basically the same as Example 1, except that the compound bacterial agent is replaced with a single bacterial agent of Bacillus plantarum LYA31 with the same number of live bacteria.

[0029] Comparative Example 5 The mixture is basically the same as in Example 1, except that instead of adding white wine, 8 parts of salt are added, and the initial ethanol volume fraction of the mixture is 0%.

[0030] Comparative Example 6 It is basically the same as Example 1, except that segmented temperature control is not used, and the entire process is carried out at a constant temperature of 33°C and sealed for 120 days.

[0031] Experimental Example 1: Determination of Ethanol Tolerance of Microbial Agents After activating the compound bacterial agent of the present invention and the commercial strain used in Comparative Example 2, they were inoculated at a 2% inoculum into MRS liquid medium containing 4%, 8%, 12%, 16%, and 20% ethanol by volume, respectively, and cultured at 30°C for 48 h. The relative growth of the culture medium (D80 value) at a specific wavelength was measured with the culture medium without added ethanol as a control. The results are shown in Table 1.

[0032] Table 1. Tolerance results of strains under different ethanol contents. As shown in Table 1, the growth of the strain decreased with increasing ethanol content. When the ethanol volume fraction was 4%, the relative growth D80 values ​​of the commercial strain and the compound bacterial agent of this invention were both above 96%, indicating that *Lactobacillus plantarum* grew well in a 4% ethanol environment. When the ethanol volume fraction was 8%, the growth activity weakened, but the compound bacterial agent still maintained above 80%, while the commercial strain decreased to 73.92%. When the ethanol volume fraction continued to increase, the growth environment of the strain changed significantly, and its growth was significantly inhibited, resulting in reduced activity.

[0033] Further preparation of bacterial suspensions was carried out, and each suspension was inoculated at a 2% inoculum into MRS liquid medium with ethanol volumes of 8%, 9%, 10%, 11%, and 12% (v / v), respectively. The suspensions were incubated at 30°C for 48 hours, and the viable cell count was determined. The results are shown in Table 2. The survival rate was calculated based on the viable cell count of the control group (K) without added ethanol, and the results are as follows: Figure 1 As shown.

[0034] Table 2. Viable cell counts of bacterial strains under different ethanol contents (×10⁻¹⁰) 8 CFU / mL From Table 2 and Figure 1 It can be seen that the survival rate of the bacterial strain gradually decreases with increasing ethanol content. When the ethanol volume fraction increases from 8% to 12%, the survival rate drops from over 70% to 20%-30%. At an ethanol volume fraction of 8%, the survival rate of the compound bacterial agent of this invention is 77.89%, while that of commercial strains drops to 66.75%. At an ethanol volume fraction of 9%, the survival rate of the compound bacterial agent is 68.53%, while that of commercial strains is 53.11%. At an ethanol volume fraction of 10%, both drop below 50%. The survival rate of the compound bacterial agent is consistently higher than that of commercial strains at all ethanol concentrations, indicating that the compound bacterial agent of this invention has stronger ethanol tolerance.

[0035] The above results indicate that the present invention limits the initial ethanol volume fraction of the mixture to 4%~8%. When it is below 4%, ethanol is insufficient to replace salt to play an antibacterial and preservative role. When it is above 8%, the survival rate of the compound microbial agent of the present invention drops to below 50%, making it difficult to start the main fermentation, slowing down acid production, and easily leading to contamination by miscellaneous bacteria and fermentation failure.

[0036] In addition, by Figure 2 It can be seen that the growth trend of the selected strains follows a certain pattern: in the initial stage, the strains have just adapted to the environment and have begun to grow, with no significant changes in pH value and strain growth; as time changes, the strains begin to grow rapidly between 4h and 12h, the pH value decreases significantly, and the bacterial growth also increases significantly. In comparison, the compound bacterial agent has a stronger acid-producing ability.

[0037] Experimental Example 2: Verification of the Synergistic Effect of Compound Microbial Agent Formulation Following the process described in Example 1, *Lactobacillus plantarum* P158 single strain (Comparative Example 3), *Lactobacillus plantarum* LYA31 single strain (Comparative Example 4), and compound bacterial agents composed of the two at viable cell ratios of 3:1, 2:1, 1:1, 1:2, and 1:3 were inoculated. A separate Comparative Example 1 (uninoculated and unfermented) and a commercial bacterial strain group (Comparative Example 2) were also included. After 120 days of fermentation in each inoculated group (samples were taken directly from Comparative Example 1 after mixing), the degradation rate of capsaicin, the number of days required for pH to drop to 4.2, total acid content, lactic acid bacteria count, peak nitrite level, and coliform count were measured. The effect of the compounding ratio on the fermentation effect was investigated. The results are shown in Table 3. The degradation rate of capsaicin was calculated based on the total amount of capsaicin in the unfermented sample of Comparative Example 1.

[0038] Table 3. Effects of different microbial agent ratios on the quality of fermented glutinous rice cake chili sauce. Note: The degradation rate of capsaicin and the time required for pH to drop to 4.2 in Comparative Example 1 (which was not inoculated or fermented) are not applicable and are indicated by "—".

[0039] The above results indicate that: (1) Compared with Comparative Example 1, which was not inoculated or fermented, the degradation rate of capsaicin, total acid content, and number of lactic acid bacteria in each inoculated fermentation group were significantly increased, while the peak nitrite and number of coliform bacteria were significantly reduced, indicating that directional lactic acid bacteria fermentation is the key to achieving capsaicin degradation, acid production and antibacterial effects, and improved safety; (2) Under the condition of equal viable count inoculation, the various indicators of the compound group of *Lactobacillus plantarum* P158 and *Lactobacillus plantarum* LYA31 were better than those of the single strain groups (Comparative Example 3, Comparative Example 4) and the commercial strain group (Comparative Example 2), and the degradation rate of capsaicin, total acid content, and number of lactic acid bacteria in the compound group exceeded the simple average level of the two single strain groups (e.g., viable count). When the ratio is 1:1, the degradation rate of capsaicin is 27.4%, which is significantly higher than the average of 19.8% for single strains of *Lactobacillus plantarum* P158 (21.3%) and single strains of *Lactobacillus plantarum* LYA31 (18.3%). This indicates that the two strains have a synergistic effect in the co-fermentation system. (3) As the ratio of viable bacteria of *Lactobacillus plantarum* P158 to viable bacteria of *Lactobacillus plantarum* LYA31 changes from 3:1 to 1:3, the various indicators show a trend of first being better and then worse. The optimal value is reached when the ratio of viable bacteria is 1:1 (i.e., Example 1). At this time, the time required for the pH value to drop to 4.2 is the shortest (7.0d), the degradation rate of capsaicin and the total acid content are the highest, and the peak value of nitrite and the number of coliform bacteria are the lowest. In summary, the synergistic effect of *Lactobacillus plantarum* P158 and *Lactobacillus plantarum* LYA31 in a 1:1 ratio of live bacteria is the most significant. This combination can rapidly establish an acid-alcohol dual barrier without the addition of salt, while simultaneously addressing the degradation of capsaicin, the enrichment of probiotics, and the inhibition of nitrite and other bacteria. Therefore, the preferred live bacteria ratio of *Lactobacillus plantarum* P158 to *Lactobacillus plantarum* LYA31 in this invention is 1:1.

[0040] Experimental Example 3: Determination of Capsaicin Content According to GB / T 21266-2007 "Determination of Capsaicin and Expression of Spiciness in Chili Peppers and Chili Pepper Products", the samples of Example 1 and Comparative Example 1 were tested and the test results of each group were compared. Statistical analysis showed that P<0.05, indicating that the difference in sample means was statistically significant. The specific results are shown in Table 4.

[0041] Table 4. Results of Capsaicin Content Determination The test results show that, after fermentation with *Lactobacillus plantarum* P158 and *Lactobacillus plantarum* LYA31 in Example 1, the capsaicin content of the glutinous rice chili was significantly reduced, indicating that the present invention can effectively improve the original irritation of glutinous rice chili by using customized process and special strains.

[0042] Experimental Example 4: Comparative Determination of Various Physicochemical Indicators The determination was made according to GB 2762-2022 "National Food Safety Standard - Limits of Contaminants in Food"; the total acid content was determined by the acid-base indicator titration method in GB 12456-2021 "National Food Safety Standard - Determination of Total Acidity in Food"; the total bacterial count and lactic acid bacteria count were determined with reference to GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count" and GB 4789.35-2023 "National Food Safety Standard - Microbiological Examination of Food - Examination of Lactic Acid Bacteria", respectively; the reducing sugar content was determined by the direct titration method in GB 5009.7-2016 "National Food Safety Standard - Determination of Reducing Sugar in Food"; the amino acid nitrogen content was determined by the pH meter method in GB 5009.235-2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food"; and the nitrite content was determined by the spectrophotometric method in GB 5009.33-2025 "National Food Safety Standard - Determination of Nitrite and Nitrate in Food". Comparing the test results of each group, statistical analysis showed that P<0.05, indicating that the difference in sample means was statistically significant. The specific results are shown in Table 5.

[0043] Table 5. Results of Physicochemical Indicators Measurement The test results show that, after fermentation with Lactobacillus plantarum P158 and Lactobacillus plantarum LYA31 in Example 1, the total acid content, number of lactic acid bacteria and amino acid nitrogen content of the glutinous rice chili sauce were significantly increased, while the nitrite content was greatly reduced, thus improving the safety of the glutinous rice chili sauce and extending its shelf life.

[0044] Example 5: Changes in capsaicin content during fermentation Following the method described in GB / T 21266-2007, samples were taken and tested at different time points during the fermentation process of Example 1. The results are as follows: Figure 3 As shown.

[0045] The test results show that the chili sauce of this invention utilizes *Lactobacillus plantarum* P158 and *Lactobacillus plantarum* LYA31 for fermentation. The total amount of capsaicin in the initial fermentation mixture was approximately 0.20 g / kg (comparable to the unfermented sample in Comparative Example 1). During the first 7 days of fermentation, the capsaicin content remained relatively stable. From days 7 to 15, the content increased due to the release of capsaicin from the chili matrix during the initial fermentation stage, reaching a peak of approximately 0.24 g / kg around day 15. After day 15, the content began to decrease continuously due to the degradation effect of the bacterial strains. By day 120, the capsaicin content had decreased to approximately 0.14 g / kg, a significant reduction compared to the initial fermentation level. This indicates that the present invention can effectively reduce the capsaicin content, thereby minimizing intestinal irritation.

[0046] Test Example 6 Gavage Test in Mice SPF-grade adult healthy male BALB / c mice, 6 weeks old, weighing 18g to 22g, 50 in total, were purchased from Shudaqiang (Chengdu) Biotechnology Co., Ltd. The experiment was carried out after one week of adaptive feeding. The animal experiment was approved by the Animal Ethics Committee of Sichuan Agricultural University (No.: 20250436), and all operations during animal handling complied with animal ethics standards.

[0047] The mice were conventionally reared and randomly divided into 5 groups, 10 mice in each group, namely: low-dose group, medium-dose group, high-dose group, unfermented group and blank control group. The test simulated continuous 5-day intake of Ciba chili. The gavage doses were 1.5g / kg body weight for the low-dose group, 3.0g / kg body weight for the medium-dose group, and 6.0g / kg body weight for the high-dose group. The unfermented group was gavaged with the same amount of the sample of Comparative Example 1 as the high-dose group, and the blank control group was gavaged with an equal volume of normal saline. The gavage volume of each group was 10mL / kg body weight, once a day. Samples were collected 24 hours after the end of continuous 5-day gavage, and the mice were dissected to obtain serum, colon tissue samples, colon contents and feces. The test results of each group were compared, and statistical analysis showed that P<0.05, indicating that the difference in sample means was statistically significant.

[0048] (1) Body weight change and DAI value of mice It can be seen from the test results ( Figure 4 ) that the mice in the blank control group were in good physical condition, with stable weight change, and the DAI fluctuated around 0 point. The mice in the low-dose group were in good physical condition, their body weight began to decrease slightly on the 4th day, the overall DAI change was not obvious, and the body weight decreased by about 1% compared with the blank control group, with no significant difference. The mice in the unfermented group showed phenomena such as reduced activity, dull coat color and weight loss from the first day, the symptoms gradually aggravated, loose stools and occult blood appeared, and the body weight decreased to 89% on the sixth day, which was statistically different from the high-dose group and the medium-dose group (P<0.05). Among the fermented groups, the weight loss trend of mice in the medium-dose group and the high-dose group was relatively slow, and the weight decreased to 98% and 91% respectively on the sixth day, with a statistical difference (P<0.05). It shows that compared with unfermented Ciba chili, the Ciba chili sauce of the present invention can reduce its influence on the body weight and DAI value of mice.

[0049] (2) Structural analysis of intestinal flora in mice It can be seen from the test results ( Figures 5 to 8 ) that feeding the Ciba chili sauce of the present invention can significantly increase the abundance of probiotics such as Lactobacillus , Akkermansia and other probiotics, as well as Lachnospiraceae , Ruminococcaceae and other short-chain fatty acid-producing bacteria, and effectively reduce the abundance of Helicobacter , Eubacterium fissicatena group and MucispirillumThe abundance of pathogens that cause intestinal inflammation.

[0050] (3) Mouse colon inflammatory factors Based on the test results ( Figure 9 The results showed that the level of inflammation in the intestines of mice in the fermented group was significantly lower than that in the unfermented group (P<0.05), and the levels of TNF-α, IL-6, and IL-1β in the colon of mice in the fermented group were all significantly lower than those in the unfermented group. IL-10 is a core anti-inflammatory cytokine that inhibits excessive inflammatory responses, regulates the balance of the immune system, and prevents inflammatory damage to normal tissues; its level is negatively correlated with the level of inflammation. Fermented chili paste significantly increased the IL-10 level in the gastrointestinal tract of mice.

[0051] (4) MPO and NF-κB P65 levels in mouse serum Based on the test results ( Figure 10 The results showed that the MPO level in the serum of mice in the unfermented group was significantly higher than that in the fermented group and the blank control group, increasing by 11.28% compared to the high-dose group, and by 18.22% and 16.48% compared to the medium-dose group and the low-dose group, respectively (P<0.05). This indicates that feeding mice with unfermented chili paste significantly increased MPO levels compared to fermented chili paste. The NF-κB P65 level in mice in the unfermented group was significantly higher than that in the blank control group, the low-dose group, and the medium-dose group, increasing by 66.25%, 49.18%, and 33.4%, respectively (P<0.05), indicating that the chili paste of this invention can effectively slow down the increase in NF-κB P65 levels in mice.

[0052] (5) Short-chain fatty acid content Based on the test results ( Figure 11 As can be seen, the content of the three short-chain fatty acids in the unfermented group mice was significantly lower than that in the low-dose group and the blank control group, and the content of acetic acid and butyric acid was significantly different from that in the medium-dose group. This indicates that after fermentation with the specific process and special strains of this invention, the glutinous rice chili can reduce the original spiciness and irritation of chili to a certain extent. Compared with the unfermented glutinous rice chili, the glutinous rice chili of this invention has a more alleviating effect on the gastrointestinal irritation of mice.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, all equivalent or simple variations made in accordance with the principles described in the present invention are included within the protection scope of the present invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the structure of the present invention or exceed the scope defined in the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A compound microbial agent, characterized in that, Including *Lactobacillus plantarum* P158 and *Lactobacillus plantarum* LYA31 with a viable count ratio of 1:0.8~1.2; Lactobacillus plantarum P158, its taxonomic name is Lactobacillus plantarum ( Lactobacillus plantarum It was deposited at the China General Microbiological Culture Collection Center on October 12, 2023, with accession number CGMCC No. 28616; Lactobacillus plantarum LYA31, its taxonomic name is Lactobacillus plantarum ( Lactobacillus plantarum It was deposited on August 28, 2020, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.20578.

2. The compound microbial agent according to claim 1, characterized in that, The viable count ratio of *Lactobacillus plantarum* P158 to *Lactobacillus plantarum* LYA31 was 1:

1.

3. A method for preparing fermented glutinous rice chili sauce, characterized in that, Includes the following steps: S1. Rehydration: Take dried chili peppers, soak them in hot water at 90℃~100℃ for 3min~10min, remove them, drain and cool them to obtain rehydrated chili peppers; S2. Crushing: Crush the rehydrated chili peppers to a particle size of no more than 10mm, and crush the garlic, pickled ginger and pickled Erjingtiao peppers to a particle size of 2mm~4mm respectively. S3. Mixing: By weight, take 40-50 parts rehydrated chili peppers, 5-10 parts garlic, 5-10 parts pickled ginger, 4-10 parts pickled Erjingtiao peppers, 10-20 parts sweet citrus, 20-30 parts baijiu (Chinese liquor) with an alcohol content of 25% vol-35% vol, and 0.5-5 parts mixed spices. Do not add salt. Mix evenly to obtain the mixture. The initial ethanol volume fraction of the mixture is 4%-8%. S4. Inoculation and fermentation: The compound microbial agent described in claim 1 or 2 is inoculated into the mixture to make the lactic acid bacteria count 1×10⁻⁶. 6 CFU / g ~ 1×10 7 The product is CFU / g, sealed, and fermented at 30℃~35℃ for 7d~10d until the pH value is no higher than 4.

2. Then it is fermented at 20℃~25℃ for a total fermentation time of 90d~120d to obtain fermented glutinous rice chili sauce.

4. The preparation method according to claim 3, characterized in that, In step S3, the mixed spices include Sichuan peppercorns, star anise, fennel seeds, and cinnamon in a mass ratio of 2:1:1:

1.

5. The preparation method according to claim 3, characterized in that, In step S4, the primary fermentation temperature is 32℃~34℃, the secondary fermentation temperature is 22℃~24℃, and the total fermentation time is 100d~120d.

6. A fermented glutinous rice cake chili sauce, characterized in that, The fermented glutinous rice chili sauce is prepared by any one of claims 3 to 5; it contains no added salt, has a pH value not higher than 4.2, a total acid content (calculated as lactic acid) not less than 1.5 g / 100 g, and a lactic acid bacteria count not less than 1 × 10⁻⁶. 5 The CFU / g content, nitrite content not exceeding 0.10 mg / kg, and capsaicin content not exceeding 0.15 g / kg are all acceptable.

7. The application of the compound microbial agent according to claim 1 or 2 in the preparation of fermented glutinous rice chili sauce.

8. The application of the fermented glutinous rice chili sauce according to claim 6 in the preparation of hot pot base or compound seasoning.

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