A complex starter with lipid-lowering activity and its application in the fermentation preparation of red sour soup

Abstract

The present application belongs to the field of microbial technology, and discloses a compound starter with lipid-lowering activity and application thereof in the fermentation preparation of red sour soup. The compound starter is composed of Pediococcus acidilactici MA8, Levilactobacillus zymae TA15 and a traditional starter. Cell experiments prove that the extracellular metabolites and bacterial bodies of Pediococcus acidilactici MA8 and Levilactobacillus zymae TA15 can regulate the expression of lipid metabolism related genes, reduce lipid accumulation, and have good lipid-lowering activity. The application of the compound starter to the fermentation of red sour soup can effectively improve the acid production efficiency, shorten the fermentation period, optimize the composition of organic acids and key flavor substances, improve the sensory quality of the product, and the comprehensive quality of the fermented product is better than that of the natural fermentation control group.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound fermentation agent with lipid-lowering activity and its application in the fermentation of red sour soup. Background Technology

[0002] With the increasing prevalence of high-fat diets among residents, the incidence of hyperlipidemia continues to rise, and various chronic complications caused by lipid metabolism disorders have become a significant public health issue. Compared to chemical drug interventions, lactic acid bacteria from natural food sources offer high safety and rich probiotic properties, making the regulation of lipid metabolism through dietary pathways a research hotspot in the field of functional foods.

[0003] Red sour soup is a traditional fermented food characteristic of southwestern my country. Existing research has confirmed that some lactic acid bacteria possess bioactivity that regulates lipid metabolism. Fermentation using functional lactic acid bacteria can enrich the functional properties of red sour soup. Current research has made preliminary explorations into the lipid-lowering function of lactic acid bacteria derived from red sour soup. For example, patent CN202310840682.6 discloses a strain of Bifidobacterium animalis BLH1 derived from red sour soup, confirming its potential to lower low-density lipoprotein cholesterol.

[0004] However, existing technologies still have certain limitations: traditional red sour soup relies heavily on natural fermentation, which results in long fermentation cycles and unstable product batch quality, making it difficult to meet the needs of standardized production; research on the lipid-lowering effects of lactic acid bacteria from red sour soup is still scarce, and there is little verification of its activity at the cellular level; at the same time, there are currently few compound fermenting agents that are suitable for the fermentation system of red sour soup and have both fermentation capabilities and lipid-lowering effects, and there is a lack of relevant research on the preparation of red sour soup using functional compound lactic acid bacteria fermentation, making it difficult to achieve the functional upgrade of traditional red sour soup. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention selects two lipid-lowering strains, Pediococcus lactis MA8 and Lactobacillus ta15, and combines them with Lactobacillus plantarum 9010 to prepare a compound fermentation agent. This agent is then applied to the fermentation of red sour soup, improving the problem of unstable quality in traditionally naturally fermented red sour soup. At the same time, it endows red sour soup with good lipid-lowering properties, thereby enhancing product quality and added value.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, this invention provides a lipid-lowering lactic acid bacteria compound, wherein the compound uses two strains of *Pediococcus acidilactici* MA8 and *Levilactobacillus zymae* TA15, both possessing excellent lipid-lowering activity, combined with *Lactobacillus plantarum* 9010. The three strains exhibit good compatibility and no antagonistic effects. The compound system combines excellent fermentation acid production performance with cholesterol and triglyceride scavenging activity, demonstrating significantly superior overall fermentation and functional characteristics compared to single strains and other compound combinations.

[0008] Among them, *Lactobacillus tamariscina* TA15 and *Pediococcus lactis* MA8 showed good effects in regulating lipid metabolism and improving lipid accumulation in high-fat diets. Both the bacterial cells and extracellular metabolites of these strains effectively inhibited the formation and accumulation of lipid droplets in HepG2 cells. Their mechanism of action involves differential regulation of lipid metabolism-related genes, downregulating the expression of key lipid synthesis genes such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), acetyl-CoA carboxylase (ACC), and sterol regulatory element-binding transcription factor 1 (SREBF1), while upregulating the expression of lipid breakdown and lipid clearance-related genes such as peroxisome proliferator-activated receptor α (PPARα) and low-density lipoprotein receptor (LDLR). The bacterial cells exerted a lipid-lowering effect by activating the adenosine monophosphate-activated protein kinase (AMPK) gene pathway, while the lipid-lowering effect of the extracellular metabolites was independent of AMPK gene regulation.

[0009] In a second aspect, this invention provides the application of a compound fermenting agent with both fermentation properties and lipid-lowering functions in red sour soup. The compound fermenting agent is composed of *Pediococcus lactis* MA8, *Lactobacillus faciesensis* TA15, and *Lactobacillus plantarum* 9010.

[0010] The preparation method for red sour soup using this compound fermentation agent is as follows:

[0011] The compound fermentation agent was inoculated into the red sour soup fermentation substrate at an inoculation amount of 10. 4 ~10 8 The concentration of CFU / mL is controlled at a fermentation temperature of 25–35℃. The mixture is allowed to ferment statically for 3–11 days. After fermentation, red sour soup is obtained.

[0012] Preferably, the red sour soup fermentation substrate includes tomatoes, peppers, and auxiliary ingredients.

[0013] Preferably, the auxiliary materials include one or more of the following: ginger, garlic, edible salt, glutinous rice flour, and white wine.

[0014] Preferably, the red sour soup fermentation substrate is prepared by sterilization of the following ingredients in a mass ratio of tomato: chili: ginger: garlic: salt: glutinous rice flour: white wine = 100: 20: 10: 5: (0.72~3.62): 2: 5.

[0015] The application of the compound fermentation agent in the preparation of products (including drugs and functional foods) for regulating blood lipids and preventing hyperlipidemia.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) Excellent in vitro lipid-lowering effect. The *Pediococcus lactis* MA8 and *Lactobacillus ta15* used in this invention can effectively improve lipid accumulation induced by high lipids at the hepatocyte level, reduce intracellular lipid droplet formation, and alleviate lipid metabolism abnormalities, thus exhibiting excellent in vitro lipid-lowering effects.

[0018] (2) Outstanding value in fermentation applications. Applying compound lactic acid bacteria starter to the fermentation of red sour soup can stabilize the fermentation process and shorten the fermentation time. The fermentation cycle is 3 to 11 days, which can effectively improve the defects of unstable quality and obvious batch differences in traditional natural fermentation. At the same time, it can optimize the organic acid composition and flavor substance structure of the product, improve the flavor characteristics and overall sensory quality, and is more suitable for standardized and large-scale production applications.

[0019] The Pediococcus acidilactici MA8 strain was deposited on May 19, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No. 66353. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0020] The Levilactobacillus zymae TA15 was deposited on May 19, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 66351, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0021] The plant lactobacillus ( Lactobacillus plantarum Strain 9010 was deposited on August 19, 2011, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 5172, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. This strain has been disclosed in Chinese Patent CN102978134A. Attached Figure Description

[0022] Figure 1 The results are from a pairwise streak compatibility test of the three lactic acid bacteria strains.

[0023] Figure 2 The effects of simvastatin and its extracellular metabolites on the survival rate of HepG2 cells were investigated, including (a) the effect of simvastatin on the survival rate of HepG2 cells, (b) the effect of extracellular metabolites of Pediococcus lactis MA8 on the survival rate of HepG2 cells, and (c) the effect of extracellular metabolites of Lactobacillus ta15 on the survival rate of HepG2 cells.

[0024] Figure 3 The effects of different sample interventions on Oil Red O staining of HepG2 cells induced by high lipid were: (a) control group, (b) high lipid model group, (c) positive drug group, (d) MA8 extracellular metabolite treatment group, (e) MA8 extracellular metabolite prevention group, (f) TA15 extracellular metabolite treatment group, (g) TA15 extracellular metabolite prevention group, (h) MA8 cell intervention group, and (i) TA15 cell intervention group.

[0025] Figure 4 The effects of different sample interventions on the total cholesterol (TC) and triglyceride (TG) content in high-lipid-induced HepG2 cells are shown in (a) and (b) TC content.

[0026] Figure 5 The effects of extracellular metabolites of MA8 and TA15 and bacterial cells on the relative gene expression levels induced by high lipids in HepG2 cells are shown in (a) HMGCR, (b) ACC, (c) SREBF1, (d) PPARα, (e) LDLR, and (f) AMPK.

[0027] Figure 6 The figure shows the results of single-factor process optimization for fermenting red sour soup with compound fermentation agent, including (a) the effect of inoculum amount on total fermentation acid; (b) the effect of salt addition amount on total fermentation acid; (c) the effect of fermentation time on total fermentation acid; and (d) the effect of compound inoculum ratio on total fermentation acid.

[0028] Figure 7 This is a comparison of the sensory quality of red sour soup fermented with compound fermenting agents and red sour soup fermented naturally. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0030] Example 1: Screening of the Optimal Combination

[0031] 1.1 Screening and Evaluation of Optimal Lactic Acid Bacteria Combinations

[0032] Based on previous strain screening results, *Pediococcus lactis* MA8 and *Lactobacillus ta15*, selected as the core experimental strains, were chosen as the control strain, and *Lactobacillus plantarum* 9010 was selected as the control strain. A compounding scheme was designed using the single-factor variable method, setting up a single-strain control group, a two-strain compounding group, and a three-strain compounding group, with a total inoculation volume of 3% (v / v). Blank MRS broth medium was used as a negative control. After fermentation, acid production capacity, cholesterol clearance capacity, and triglyceride clearance capacity were used as evaluation indicators. A comprehensive scoring method was used to quantitatively evaluate each strain compounding combination, ultimately selecting the optimal lactic acid bacteria compounding combination that combines excellent acid production characteristics with superior lipid-lowering activity.

[0033] Acid production characteristics determination: Acid production characteristics are an important indicator for strain screening. The acid production capacity of the strain was determined by acid-base titration: The activated strain was inoculated into MRS liquid medium at a 3% (v / v) inoculum and incubated at 37℃ for 16 h; 10.0 mL of bacterial solution was placed in a 100 mL volumetric flask, and water was added to the mark and mixed well. 20.0 mL of the diluted bacterial solution was placed in an Erlenmeyer flask, 2-4 drops of phenolphthalein indicator were added, and titrated with 0.1 mol / L NaOH standard solution until a faint red color appeared and did not fade within 60 s as the endpoint. The acid production was calculated according to formula (1-1).

[0034] (1-1)

[0035] In the formula: V1 represents the volume of NaOH standard solution consumed in the titration of the fermentation broth sample, mL; V2 represents the volume of NaOH standard solution consumed in the titration of the control group (blank culture medium), mL; C represents the concentration of NaOH in the titrant, 0.1 mol / L; k represents the lactic acid conversion factor, 0.090; F represents the dilution factor; M represents the mass of the sample, g.

[0036] A comparative analysis of the acid-producing capacity, cholesterol scavenging capacity, and triglyceride scavenging capacity of the various experimental bacterial strains revealed that the three-strain combination of *Pediococcus lactis* MA8, *Lactobacillus faciesiensis* TA15, and *Lactobacillus plantarum* 9010 exhibited the best overall performance, possessing both excellent fermentation acid-producing characteristics and lipid-lowering function. This optimal combination produced 25.37 g / kg of acid, achieved a cholesterol scavenging rate of 53.24%, and a triglyceride scavenging rate of 33.08%, significantly outperforming other single-strain and combined bacterial strain systems, making it the best lactic acid bacteria combined fermentation combination.

[0037] Table 1. Comparison of acid-producing capacity, cholesterol clearance capacity, and triglyceride clearance capacity of different lactic acid bacteria combinations.

[0038] Note: The overall score uses the range-standardized summation method; first, the acid production, cholesterol clearance rate, and triglyceride clearance rate are respectively standardized according to their ranges, using the following formula:

[0039] (1-2)

[0040] The three standardized results are then added together to obtain the comprehensive score.

[0041] 1.2 Antagonistic compatibility test between optimal compound strains

[0042] To verify whether there are mutual inhibitory effects among the strains in the optimal combination obtained through screening and to clarify the compatibility of the strain combinations, the streak cross method was used to detect the antagonistic effects between the strains. Each strain to be tested was activated and cultured separately to obtain fresh bacterial suspensions in the logarithmic growth phase. The activated bacterial suspensions were then streaked pairwise on MRS solid medium using an inoculation loop and incubated at 37°C for 24 h. The presence of colonies at the intersection of the streaks was observed.

[0043] The results of the antagonistic relationship between the strains are as follows: Figure 1 As shown, the colonies of the three lactic acid bacteria in the cross-stretched areas were continuous and uniform, with no inhibition zones or growth inhibition observed. The results indicate that there was no antagonistic interaction among the three lactic acid bacteria strains, and their compatibility was good. Therefore, they can be used as a combination of strains for further research and application in compound fermentation.

[0044] Example 2: Regulatory effects of Pediococcus lactis MA8 and Lactobacillus ta15 on lipid metabolism in HepG2 cells.

[0045] 2.1 Cell resuscitation and passage

[0046] Under aseptic conditions, frozen HepG2 cells were removed from the liquid nitrogen container and quickly thawed in a 37°C water bath for 1–2 min. After thawing, the cell suspension was transferred to a centrifuge tube, 2 mL of DMEM complete medium was added, and the supernatant was discarded after centrifugation. The cell pellet was resuspended in 3 mL of fresh complete medium, and after thorough mixing, it was transferred to a culture flask and incubated at 37°C in a 5% CO2 incubator. When the cell confluence reached 80%–90%, the old medium was discarded, the cells were washed twice with PBS, and 1 mL of 0.25% trypsin solution was added. The cells were digested at 37°C for 1 min. After observing under a microscope that the cells had shrunk and become rounded, 3 mL of complete medium was added to stop the digestion. A single-cell suspension was prepared by pipetting and passaged at a 1:3 ratio. The results showed that the cells in the logarithmic growth phase were stable and could meet the requirements of all subsequent experiments.

[0047] 2.2 Establishment and validation of a high-fat HepG2 cell model

[0048] 2.2.1 Preparation of composite induction solution

[0049] Palmitic acid (PA) and oleic acid (OA) were dissolved in 0.1 mol / L NaOH solution and distilled water respectively to prepare stock solutions. These stock solutions were then mixed with BSA solution to prepare OA-PA composite induction solutions of different concentrations at a ratio of OA:PA = 2:1. The solutions were then filtered through a 0.22 μm filter membrane for sterilization and stored at low temperature for later use.

[0050] 2.2.2 Preparation of Composite Induction Solution

[0051] HepG2 cells were administered at a rate of 1×10⁻⁶. 5 Cells were seeded per well in a 96-well plate, with a final volume of 100 μL per well. A blank control well was included. After culturing at 37℃ and 5% CO2 for 24 h, the cells adhered to the plate. They were then washed with PBS, and a series of concentrations of composite induction solutions were added for further 24 h of culturing. After incubation with CCK-8 reagent in each well, the absorbance was measured at 450 nm, and cell viability was calculated using formula (2-1).

[0052] (2-1)

[0053] In the formula: A S A represents the absorbance of the experimental group; c A represents the absorbance value of the control group; A0 represents the absorbance value of the blank group.

[0054] The results showed that within the concentration range of 200–800 μmol / L, cell viability was ≥ 90%, with no significant cytotoxicity. However, when the concentration increased to 1000 μmol / L, cell viability decreased significantly, failing to meet experimental requirements. It is generally considered that when cell viability is above 90%, the test substance has no significant toxicity to cells and will not interfere with normal cellular physiological processes; therefore, a composite induction solution of 200–800 μmol / L was selected for subsequent experiments.

[0055] 2.2.3 Oil Red O staining

[0056] HepG2 cells were administered at a rate of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates. After culture and adhesion, the culture medium was discarded, the cells were washed with PBS, and fixed with Oil Red O fixative. After washing with isopropanol, Oil Red O staining solution was added, followed by rinsing with isopropanol until the interstitial tissue was clear, and then washing with distilled water. After counterstaining the cell nuclei with hematoxylin and returning them to blue, the intracellular lipid deposition was observed under a microscope.

[0057] The results showed that, compared with the blank control group, the number of lipid droplets and staining intensity in the cells of each concentration treatment group first increased and then decreased with the increase of inducer concentration. The 800 μmol / L group had the most obvious intracellular lipid deposition, with dense lipid droplets merging into large clumps, and the high lipid model construction effect was the best.

[0058] 2.2.4 Determination of TC and TG content

[0059] After treating HepG2 cells with different concentrations of inducers for 24 h, the culture medium was discarded, and the cells were digested with trypsin and collected by centrifugation. The precipitate was resuspended in PBS and sonicated on ice (5 s / time, 5 s interval, total time 3 min) until the cells were completely lysed. The TC and TG contents in the cell homogenate were measured using the corresponding kits.

[0060] The results showed that the compound inducer had little effect on the intracellular TC content of HepG2 cells, with no significant overall fluctuation. However, the TG content increased with increasing inducer concentration, indicating a significant lipid accumulation effect. Excessively high concentrations of the compound inducer could adversely affect cells, interfering with normal cellular physiological states and lipid metabolism. Based on a comprehensive analysis of cell viability, Oil Red O staining, and lipid accumulation results, the 400 μmol / L induction condition demonstrated good cell growth, uniform and regular lipid droplet distribution, moderate lipid accumulation, and stable model establishment, making it suitable for constructing a HepG2 hyperlipidemic cell model.

[0061] 2.3 Effects of Lactic Acid Bacteria Extracellular Metabolites and Cells on High-Lipid HepG2 Cells

[0062] 2.3.1 Sample Preparation

[0063] Preparation of extracellular metabolites: Centrifuge the activated lactic acid bacteria culture, collect the supernatant, filter it through a 0.22 μm filter membrane to remove bacteria, and obtain sterile fermentation supernatant.

[0064] Cell preparation: Centrifuge the activated lactic acid bacteria solution and discard the supernatant; wash the cell pellet with PBS buffer and resuspend it, adjusting the concentration of the suspension to 7.00 ± 0.05 lg CFU / mL for later use.

[0065] 2.3.2 Experimental Grouping

[0066] The study included a normal control group, a high-lipidemia model group, a positive control group (10–50 μmol / L simvastatin), an extracellular metabolite treatment group, a prevention group (DMEM medium containing 5%, 10%, 20%, and 40% sterile supernatant), and a bacterial intervention group (7.00 ± 0.05 lg CFU / mL). The control group consisted of normally cultured cells. The model group was treated with 400 μmol / L OA-PA composite induction solution for 24 h to construct a high-lipidemia cell model. The positive control group, extracellular metabolite treatment group, and bacterial intervention group were induced with the composite induction solution for 24 h, followed by 24 h of sample culture. The extracellular metabolite prevention group was first treated with sample for 24 h, then cultured with OA-PA composite induction solution for another 24 h.

[0067] 2.3.3 Screening of Intervention Sample Concentration

[0068] The effects of different concentrations of simvastatin, MA8 and TA15 extracellular metabolites on the survival rate of HepG2 cells were detected by the CCK-8 assay to determine the optimal intervention concentration.

[0069] The results are as follows Figure 2 As shown, simvastatin at concentrations of 10–30 μg / mL had no significant toxicity to HepG2 cells, and 30 μg / mL was selected as the optimal concentration for positive control. 5% (v / v) extracellular metabolites of MA8 and TA15 had no significant toxicity to cells, and the extracellular metabolite of MA8 at this concentration could significantly improve cell survival rate, so 5% (v / v) was selected as the optimal concentration for action of the extracellular metabolite.

[0070] 2.3.4 Effects of Lactic Acid Bacteria Extracellular Metabolites and Cells on Lipid Accumulation in HepG2 Cells

[0071] After intervention according to the above grouping, Oil Red O staining was used to observe intracellular lipid deposition, and the intracellular TC and TG contents were measured at the same time.

[0072] The results are as follows Figure 3 As shown, compared with the model group (group b), intervention with extracellular metabolites of MA8 and TA15 and bacterial cells significantly reduced intracellular lipid droplet deposition, with no obvious fusion clusters; among them, the MA8 extracellular metabolite treatment group showed the best intervention effect, effectively reducing intracellular lipid accumulation. Results are as follows... Figure 4 As shown, the levels of TC and TG in each intervention group were significantly lower than those in the model group (P < 0.05); among them, the TA15 extracellular metabolite group had the best effect on downregulating TC, while the MA8 bacterial group had the best effect on downregulating TG. Both can effectively reduce intracellular lipid accumulation.

[0073] 2.3.5 Effects of extracellular metabolites and bacterial cells of lactic acid bacteria on the expression of lipid metabolism genes in high-lipid HepG2 cells

[0074] RNA was extracted from HepG2 cells using an RNA extraction kit. RNA concentration and purity were measured using a Nanodrop 2000c micro spectrophotometer. The qualified total RNA was used to prepare cDNA templates using a reverse transcription kit, and real-time quantitative PCR was performed according to the kit instructions. The reaction mixture was 20 μL, containing 2 μL of cDNA template, 0.40 μL each of forward and reverse primers, and the remaining components were added according to the kit's recommended amounts.

[0075] Using GAPDH as an internal reference gene, 2 -△△Ct The relative expression levels of HMGCR, ACC, SREBF1, PPARα, LDLR, and AMPK genes were calculated using a method shown in Table 2.

[0076] Table 2 Primer sequences for real-time quantitative PCR of lipid metabolism-related genes

[0077]

[0078] The results are as follows Figure 5 As shown, both bacterial cells and extracellular metabolites of the two strains downregulated the expression of key lipid synthesis genes such as HMGCR, ACC, and SREBF1, while upregulating the expression of lipid breakdown and clearance genes such as PPARα and LDLR. Only the bacterial cell intervention group of the two strains upregulated AMPK gene expression, while their extracellular metabolites had no significant effect on AMPK gene expression. Therefore, the lipid-lowering effects of the two strains may be related to the regulation of the expression of genes related to lipid synthesis, breakdown, and clearance.

[0079] Example 3: Application of compound fermentation agent in red sour soup

[0080] 3.1 Experimental Materials and Basic Conditions

[0081] Fresh tomatoes, red peppers, ginger, garlic, salt, glutinous rice flour, and white wine; compound freeze-dried bacterial powder (Lactococcus lactis MA8: Lactobacillus ta15: Lactobacillus plantarum 9010); process parameters not specifically specified shall be performed in accordance with conventional methods in this field.

[0082] 3.2 Fermentation process of red sour soup

[0083] 3.2.1 Raw material processing and ingredient mixing

[0084] Select fresh tomatoes and red peppers, removing the stems and any remaining leaves; peel, wash, drain, and then crush ginger and garlic. Mix them thoroughly according to the following mass ratio: tomatoes: peppers: ginger: garlic: salt: glutinous rice flour: white wine = 100: 20: 10: 5: 3: 2: 5. After mixing, package the mixture and sterilize it at 90℃ for 15 minutes, then cool it to room temperature for later use.

[0085] 3.2.2 Fermentation Grouping and Operation

[0086] Two fermentation treatments were set up: a natural fermentation group (NF group, without exogenous lactic acid bacteria inoculation) and a combined direct-inoculation fermentation group (DVS group, inoculated with combined freeze-dried bacterial powder under aseptic conditions); the final inoculation concentration of the DVS group was 10. 6 CFU / g, strain ratio (viable cell count ratio) was MA8∶TA15∶9010=1∶1∶1. All samples were sealed and placed in a 30℃ constant temperature incubator for static fermentation for 7 days.

[0087] 3.3 Optimization of Red Sour Soup Fermentation Process

[0088] 3.3.1 pH and total acid determination

[0089] pH was measured directly using a pH meter; total acid content was determined according to GB 12456-2021, with the following specific implementation method: Accurately weigh 5g of fermented red sour soup sample, add ultrapure water and dilute to 50.0g, shake well, and titrate with 0.100 mol / L NaOH standard titrant until the solution turns slightly red and remains so for 30s without fading, recording the volume consumed. Calculate the total acid content using formula (3-1):

[0090] (3-1)

[0091] In the formula: V1 represents the volume of NaOH standard solution consumed in the titration of the fermentation broth sample, mL; V2 represents the volume of NaOH standard solution consumed in the titration of the control group, mL; C represents the concentration of NaOH in the titrant, 0.1 mol / L; K represents the lactic acid conversion factor, 0.090; F represents the dilution factor; M represents the mass of the sample, m.

[0092] 3.3.2 Single-factor optimization of fermentation process

[0093] Single-factor experiments were conducted to investigate the inoculation amount (10) 4 ~10 8 The effects of CFU / mL, salt addition (0.5%~2.5%), fermentation time (3~11 d), and starter culture ratio on the fermentation quality of red sour soup were investigated, with pH and total acidity as evaluation indicators to determine the optimal range of process parameters.

[0094] The results are as follows Figure 6 As shown, the results of the single-factor experiment indicated that at an inoculation dose of 10... 6 With CFU / mL, salt addition of 1.5%, and fermentation time of 7 days, the total acid content of each inoculant ratio reached over 20 g / kg. Among them, under the inoculant ratio of 1:2:2, the red sour soup produced the highest acid content among all groups, showing better fermentation characteristics.

[0095] 3.3.3 Orthogonal optimization of fermentation process

[0096] Based on the results of the single-factor experiments, a three-factor, three-level orthogonal experiment was designed with salt addition (A), inoculum amount (B), and fermentation time (C) as optimization factors (see Table 3). The optimal fermentation process parameters were determined with total acid content as the evaluation index.

[0097] Table 3. Factors and levels in the orthogonal experiment of fermenting red sour soup with compound microbial agents

[0098]

[0099] The results are shown in Table 4. The order of factors affecting the total acid content of red sour soup is A (salt addition) > C (fermentation time) > B (inoculation amount). The theoretically optimal combination is A1B2C2, that is, salt addition of 1.0% and inoculation amount of 10%. 6 CFU / mL, fermentation time 7 days. Red sour soup fermentation was carried out under these optimal conditions. After fermentation, the titratable acid content was determined according to the acid production characteristic determination method in Example 1 of this invention. The results showed that under these conditions, the total acid content of the red sour soup produced by the three-strain compound inoculant reached 23.13 g / kg, with a fermentation endpoint pH of 3.19; the total acid content of the natural fermentation control group was 15.46 g / kg, with a fermentation endpoint pH of 3.74. The fermentation effect of the compound inoculant of this invention is significantly increased.

[0100] Table 4. Orthogonal optimization results of fermentation of red sour soup with compound microbial agents

[0101]

[0102] 3.4 Quality Analysis of Fermented Red Sour Soup Products

[0103] 3.4.1 Determination of organic acids

[0104] The organic acid content in red sour soup was determined by HPLC. The specific operation was as follows: 10 g of red sour soup fermentation broth was weighed, and ultrapure water was added to a final volume of 40 mL. The mixture was centrifuged at 5000 r / min for 15 min at 4℃. The supernatant was filtered through a 0.22 μm aqueous filter membrane before being added to the HPLC. A Rezex ROA-Organic Acid H⁺ ion exchange column was used with 5 mmol / L dilute sulfuric acid as the mobile phase, a detection wavelength of 210 nm, an injection volume of 10 μL, a flow rate of 0.5 mL / min, and a column temperature of 40℃. A mixed standard stock solution was prepared, serially diluted, and a standard curve was plotted for quantitative analysis.

[0105] The results are shown in Table 5. Nine organic acids were detected in the fermented red sour soup. The overall content of various organic acids in the DVS group was higher than that in the NF group. Lactic acid content reached as high as 47.21 g / kg, 2.09 times that of the NF group. Furthermore, the DVS group detected citric acid, acetic acid, and tartaric acid, which were not present in the NF group. Malic acid content was significantly increased, while oxalic acid content decreased slightly, resulting in a fuller and smoother sour taste in the red sour soup. In conclusion, the DVS group can promote the accumulation of characteristic organic acids in red sour soup, making the product's sour taste fuller and smoother, and its overall quality is superior to that of the NF group.

[0106] Table 5. Differences in organic acid content between fermented red sour soup in the DVS and NF groups.

[0107]

[0108] Note: ND indicates not detected.

[0109] 3.4.2 Determination of volatile flavor compounds

[0110] Volatile flavor components were determined using HS-SPME-GC-MS. 1 g of sample was weighed and placed in a 20 mL headspace vial. After sealing and heating to equilibrium, the sample was extracted with an extraction head, thermally desorbed at 250 °C, and then detected on a chromatographic column. Qualitative identification was completed by searching and matching in a mass spectrometry database, and the relative content was calculated using the peak area normalization method.

[0111] The results are shown in Table 6. 40 volatile substances were identified in the NF group and 43 in the DVS group. The DVS group had a richer variety of alcohols and esters, and no irritating ketones were detected in the NF group. It also contained unique heterocyclic compounds, which made the aroma of the red sour soup more mellow, full, and richer in layers.

[0112] Table 6-1 Relative contents of key volatile flavor compounds in fermented red sour soup of DVS and NF groups

[0113]

[0114] Table 6-2 Relative contents of key volatile flavor compounds in fermented red sour soup of DVS and NF groups (continued)

[0115]

[0116] Note: ND indicates not detected.

[0117] 3.4.3 Sensory evaluation

[0118] Referring to existing food sensory evaluation standards and combining the quality characteristics of red sour soup products, four sensory indicators—appearance, texture, taste, and aroma—were selected to conduct a comprehensive sensory evaluation of the fermented red sour soup. The specific scoring criteria are shown in Table 7.

[0119] Table 7 Sensory Evaluation Criteria for Red Sour Soup

[0120]

[0121] The results are as follows Figure 7 As shown, the DVS group of red sour soup scored higher than the NF group in all four dimensions of appearance, taste, aroma and aftertaste. Its color was uniform and bright red, its sour taste was pure and rich, its fermentation aroma was harmonious, and its aftertaste was sweet and comfortable. It had no obvious browning or off-flavors, and its overall sensory quality was better than the NF group, resulting in higher product acceptance.

[0122] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A complex starter having a lipid-lowering activity, characterized in that, The compound starter culture consists of Pediococcus acidilactici MA8, Levilactobacillus zymae TA15, and a traditional starter culture.

2. The compound fermentation agent according to claim 1, characterized in that, The traditional starter culture is Lactobacillus plantarum 9010.

3. The application according to claim 2, characterized in that, In the compound fermentation agent, the ratio of viable counts of Pediococcus lactis MA8, Lactobacillus ta15 and Lactobacillus plantarum 9010 is (1~10):(1~10):(1~10).

4. The application according to claim 3, characterized in that, In the compound fermentation agent, the ratio of viable counts of Pediococcus lactis MA8, Lactobacillus ta15 and Lactobacillus plantarum 9010 is (1~2):(1~2):(1~2).

5. The application of the compound fermentation agent according to claim 1, 2, 3 or 4 in the fermentation preparation of red sour soup.

6. The application according to claim 5, characterized in that, The preparation method of the red sour soup is as follows: The complex starter culture is inoculated into a red vinegar fermentation substrate at an inoculation amount of 10 4 ~ 10 8 CFU / mL, the fermentation temperature is controlled at 25~35℃, the static fermentation is performed for 3~11 days, and the red vinegar is obtained after the fermentation is completed.

7. The application according to claim 6, characterized in that, The fermentation substrate for the red sour soup includes tomatoes, peppers, and other ingredients.

8. The application according to claim 7, characterized in that, The auxiliary ingredients include one or more of the following: ginger, garlic, edible salt, glutinous rice flour, and white wine.

9. The application according to claim 8, characterized in that, The red sour soup fermentation substrate is prepared by mixing ingredients in the following mass ratio: tomato: chili: ginger: garlic: salt: glutinous rice flour: white wine = 100: 20: 10: 5: (0.72~3.62): 2: 5, and then sterilizing to obtain the red sour soup fermentation substrate.

10. The use of the compound fermentation agent according to claim 1, 2, 3 or 4 in the preparation of products for regulating blood lipids and preventing hyperlipidemia.

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