Lactobacillus pentosus M34-1, complex microbial inoculant and application thereof in fermentation of red vinegar

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to a pentose lactis bacillus M34-1, a compound microbial inoculant and application of the compound microbial inoculant in fermentation of red sour soup. The pentose lactis bacillus M34-1 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 39925. The compound microbial inoculant is composed of lactis bacillus MX14, Pichia membranifaciens PY33 and pentose lactis bacillus M34-1. The titratable acid content reaches 22.70 g / kg after 7 days of fermentation of the red sour soup by using the compound microbial inoculant, which is 18.0% higher than that of the MX14 / PY33 double-bacterium control, and the viable cell count of PY33 at the end of fermentation is close to that of the double-bacterium control; the obtained red sour soup has high total organic acid content, the composition of free amino acids is improved, and different volatile substance composition characteristics are formed. The present application has the advantages of strong acidification capacity, good strain compatibility and relatively balanced quality control effect, and can be used for controllable fermentation production of the red sour soup.

Description

Technical Field

[0001] This invention belongs to the field of food microbial fermentation technology, specifically relating to a strain of Lactobacillus pentosus M34-1, a compound microbial agent containing the strain, and the application of the strain and the compound microbial agent in the fermentation of red sour soup. Background Technology

[0002] Red sour soup is a representative traditional fermented food in southwestern my country. It is typically made from tomatoes and chili peppers as the main ingredients, supplemented with glutinous rice flour, salt, white wine, ginger, and garlic, and fermented by microorganisms. It is characterized by a harmonious balance of sour, savory, and spicy flavors, along with a prominent fermented aroma. Traditional red sour soup relies heavily on natural fermentation, which is easily affected by factors such as ingredient composition, ambient temperature, and operating conditions, resulting in problems such as slow fermentation initiation, insufficient acidification, significant batch-to-batch variations, and unstable flavor.

[0003] Existing technologies have disclosed a compound microbial inoculant composed of *Lactobacillus plantarum* and *Pichia pastoris*, which can promote the acidification of red sour soup. However, to further improve fermentation efficiency and product quality, further improvements to the compound inoculant are needed. The addition of exogenous lactic acid bacteria is uncertain: it may enhance fermentation performance through synergistic metabolism, or it may inhibit the growth of yeast PY33 due to nutrient competition or acid stress, thereby affecting the stability of the fermentation system and the formation of flavor compounds. Therefore, screening lactic acid bacteria with both strong acid-producing capacity and good compatibility in compound fermentation is of great significance for constructing an efficient and stable compound fermentation system. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Lactobacillus pentosaccharide M34-1 with excellent acid-producing performance and good compatibility in compound fermentation.

[0005] Another objective of this invention is to provide a compound microbial agent comprising Lactobacillus pentosus M34-1, Lactobacillus plantarum MX14, and Pichia pastoris PY33.

[0006] Another object of the present invention is to provide a method for preparing the aforementioned composite microbial agent.

[0007] Another object of the present invention is to provide the application of the strain and the compound microbial agent in the fermentation of red sour soup, and a method for preparing red sour soup using the compound microbial agent.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Lactobacillus pentosaccharis*, wherein the strain is *Lactobacillus pentosaccharis* M34-1, and is classified as *Lactobacillus pentosaccharis* (… Lactiplantibacillus pentosusIt was deposited on July 29, 2026 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39925.

[0009] The Pentosacchariformis M34-1 was isolated from a traditional acidic food sample and identified as Pentosacchariformis through colony morphology observation, 16S rRNA gene sequence comparison and phylogenetic analysis.

[0010] Secondly, the present invention provides the application of the Pentosacchari lactobacillus M34-1 in the preparation of red sour soup, the application including at least one of increasing the titratable acid content of red sour soup and promoting lactic acid accumulation.

[0011] Thirdly, the present invention provides a compound microbial agent for fermenting red sour soup, the compound microbial agent comprising *Lactobacillus pentosus* M34-1, *Lactobacillus plantarum* MX14, and *Pichia pastoris* PY33. The compound microbial agent can increase the titratable acid, lactic acid, and total organic acid content of red sour soup, and maintain the survival of *Pichia pastoris* PY33 in the fermentation system while enhancing acidification capacity.

[0012] Further, the volume ratio of the bacterial culture of *Lactobacillus plantarum* MX14, *Pichia pastoris* PY33, and *Lactobacillus pentosus* M34-1 is (0.5~2):(0.5~2):(0.5~2), preferably (0.8~1.2):(0.8~1.2):(0.8~1.2), and more preferably 1:1:1. The viable cell count in the bacterial culture is adjusted or cultured to 1×10⁻⁶. 8 CFU / mL or higher concentration.

[0013] Furthermore, the viable cell counts of the three strains in the bacterial solution before mixing were each no less than 1×10⁻⁶. 8 CFU / mL. Preferably, the bacterial concentration is 10. 8 ~10 10 CFU / mL, preferably 10 8 ~10 9 CFU / mL.

[0014] Furthermore, the preparation method of the composite microbial agent includes: culturing *Lactobacillus plantarum* MX14, *Pichia pastoris* PY33, and *Lactobacillus pentosus* M34-1 respectively, collecting the bacterial cells and resuspending them in 0.9% sterile physiological saline, and adjusting the viable count of each bacterial solution to not less than 1×10⁻⁶. 8 After CFU / mL, the mixture is prepared by mixing at a volume ratio of 1:1:1 to obtain the composite microbial agent.

[0015] Fourthly, the present invention provides a method for preparing red sour soup using the aforementioned compound microbial agent, comprising one or more of the following steps: S1. Mix tomatoes, fresh red peppers, glutinous rice flour, salt, white wine, ginger and garlic and homogenize them to obtain the red sour soup raw material paste. S2, heat-treat the red sour soup raw material slurry obtained in step S1, and then cool it for later use; S3, inoculate the aforementioned compound microbial agent into the red sour soup raw material slurry obtained in step S2; S4 involves fermenting the inoculated red sour soup raw material slurry to obtain the red sour soup product.

[0016] Preferably, in step S2, the raw material slurry is heat-treated at 105°C for 15 min and then cooled to room temperature; Preferably, in S3, the above-mentioned compound microbial agent is added at a total inoculum amount of 5% (v / w), that is, 5 mL of compound microbial agent is added for every 100 g of raw material slurry; Preferably, S4 is fermented at 30 ℃ for 7 days to obtain the red sour soup product.

[0017] Furthermore, based on the quality of tomatoes, the red sour soup raw material paste includes: Glutinous rice flour 1%~5% (w / w), salt 1%~6% (w / w), white wine 3%~10% (v / w), fresh red chili peppers 10%~30% (w / w), ginger 5%~15% (w / w), and garlic 2%~10% (w / w).

[0018] Preferably, the red sour soup base includes: 1.5%~3% (w / w) glutinous rice flour, 2%~4% (w / w) salt, 4%~6% (v / w) white wine, 15%~25% (w / w) fresh red chili peppers, 8%~12% (w / w) ginger and 4%~6% (w / w) garlic.

[0019] More preferably, the red sour soup base includes: 2% (w / w) glutinous rice flour, 3% (w / w) salt, 5% (v / w) white wine, 20% (w / w) fresh red chili peppers, 10% (w / w) ginger, and 5% (w / w) garlic.

[0020] Beneficial effects The *Lactobacillus pentosus* M34-1 obtained by screening in this invention has a strong acid-producing ability and can form a well-compatible three-strain compound fermentation system with *Lactobacillus plantarum* MX14 and *Pichia pastoris* PY33.

[0021] The compound microbial agent was used to ferment red sour soup. On day 7 of fermentation, the titratable acid content reached 22.70 g / kg, which was 18.0% higher than that of the MX14 / PY33 dual-strain control group. At the end of fermentation, the number of viable PY33 cells in the three-strain group was similar to that in the dual-strain control group, indicating that the addition of M34-1 did not significantly inhibit the survival of PY33.

[0022] The total organic acid content of the red sour soup obtained by the three-strain compound fermentation reached 45.25 g / kg, of which the lactic acid content reached 24.98 g / kg; the total free amino acid content was at a high level, and the sweet amino acid content was high, which is conducive to enriching the flavor composition of the red sour soup.

[0023] Under the same MX14 / PY33 fermentation background, different candidate lactic acid bacteria exhibited different complex fermentation characteristics. The M20 group had a higher total sugar consumption rate, but the number of viable PY33 cells decreased at the end of fermentation; the M3 group had a higher reducing sugar consumption rate, but the final titratable acid content was lower than that of the M34-1 group. In contrast, the M34-1 tri-strain group, while increasing the titratable acid and organic acid content, was able to maintain PY33 survival well, demonstrating a more balanced overall fermentation performance in terms of acidification capacity, substrate utilization, and strain compatibility.

[0024] In summary, the compound microbial agent provided by this invention has strong acidification ability, good strain compatibility and relatively balanced quality control effect, and can be used for the controllable and standardized fermentation production of red sour soup. Attached Figure Description

[0025] Figure 1 Colony morphology of Lactobacillus pentosus M34-1 on MRS solid medium.

[0026] Figure 2 A phylogenetic tree of Lactobacillus pentosus M34-1 constructed based on the 16S rRNA gene sequence.

[0027] Figure 3 The acid-producing properties of red sour soup fermented by different single lactic acid bacteria and by a combination of three bacteria were compared. Figure 3 A~ Figure 3 F represents the change in titratable acid content during fermentation. Figure 3 G~ Figure 3 I represents the titratable acid content at the fermentation endpoint. Figure 3 J~ Figure 3 L represents the acid production rate during the early stage of fermentation.

[0028] Figure 4 Comparison of fermentation performance of red sour soup under different fermentation treatments, among which Figure 4 A represents the endpoint pH and titratable acid content of red sour soup under different treatments. Figure 4 B represents the number of viable lactic acid bacteria. Figure 4 C represents the number of viable PY33 cells. Figure 4 D represents the total sugar content. Figure 4 E represents the reducing sugar content. Figure 4 F represents the nitrite content.

[0029] Figure 5 The composition and content of flavor compounds in fermented red sour soup by different bacterial strains, among which Figure 5 A represents the absolute content of organic acids. Figure 5 B represents the relative content of organic acids. Figure 5 C represents the absolute content of free amino acids. Figure 5 D represents the relative content of free amino acids.

[0030] Figure 6 The composition and content of volatile compounds in fermented red sour soup by different strains, among which Figure 6 A represents the absolute content of different types of volatile compounds. Figure 6 B represents the relative content of different types of volatile compounds. Figure 6 C represents the clustering heatmap of monomeric volatile compounds.

[0031] Figure 7 Production process of red sour soup compound microbial agent. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention. Culture media, reagents, and instruments not specifically described are all commercially available, and experimental methods not specifically described are conventional methods in the art.

[0033] Example 1: Preliminary screening of candidate lactic acid bacteria for red sour soup fermentation Thirty strains of lactic acid bacteria were isolated from 17 traditional fermented food samples. The titratable acid content at the end of fermentation in red sour soup and the average acid production rate in the early stage of fermentation were used as the main screening indicators to evaluate the single-strain fermentation ability of the 30 lactic acid bacteria and their fermentation performance in a composite system composed of Lactobacillus plantarum MX14 and Pichia pastoris PY33.

[0034] The lactic acid bacteria strains include isolates from different traditional pickled food samples, among which strain M34-1 was derived from a pickled radish sample from Pingle County, Guilin City, Guangxi Zhuang Autonomous Region.

[0035] The fermentation substrate for red sour soup is prepared as follows: Weigh 1000 g of tomatoes, add 200 g of fresh red chili peppers, 100 g of ginger, 50 g of garlic, 20 g of glutinous rice flour, 30 g of salt and 50 mL of white wine, mix well and treat at 105 ℃ for 15 min, then cool to room temperature for later use.

[0036] Each type of lactic acid bacteria and *Lactobacillus plantarum* MX14 to be screened were inoculated into MRS liquid medium and cultured at 37 ℃ for 24 h; *Pichia pastoris* PY33 was inoculated into PDB liquid medium and cultured at 30 ℃ for 24 h.

[0037] After cultivation, the cultures of each strain were centrifuged at 4 ℃ and 10000 r / min for 10 min, the supernatant was discarded, and the cells were resuspended in 0.9% sterile physiological saline. The concentration of the bacterial suspension was initially adjusted based on the turbidity of the suspension, and the plate count method was used for verification to ensure that the viable cell count of each suspension reached approximately 1×10⁻⁶. 8 CFU / mL.

[0038] A single-strain fermentation group for the lactic acid bacteria to be screened and a three-strain complex fermentation group consisting of the lactic acid bacteria to be screened, MX14, and PY33 were set up. In the three-strain complex fermentation group, the volume ratio of the bacterial culture of MX14, PY33 and the corresponding lactic acid bacteria to be screened was 1:1:1.

[0039] The total inoculum amount for each treatment was 5% (v / w). After inoculation, fermentation was carried out at 30 °C for 7 days, and the titratable acid content was determined from 0 to 7 days of fermentation.

[0040] Titratable acids were determined by titration with a standard sodium hydroxide solution and expressed as lactic acid. The average acid production rate during the early stage of fermentation was calculated using the following formula: Average acid production rate = (titerable acid content on day 3 - titratable acid content on day 0) ÷ 3.

[0041] The acid-producing properties of red sour soup fermented by different single lactic acid bacteria and by three-strain compound fermentation are as follows: Figure 3 As shown. By Figure 3 A~ Figure 3 F indicates that the initial titratable acid content of each treatment group ranged from 4.51 to 5.68 g / kg. During fermentation, the titratable acid content increased rapidly mainly within 0 to 3 days, and the growth rate gradually decreased after 3 days, eventually stabilizing in the later stage of fermentation. This suggests that the acidification process of the red sour soup mainly occurred in the early stage of fermentation.

[0042] Depend on Figure 3 G~ Figure 3 As shown in L and Table 1, there are significant differences in the endpoint titratable acid content and the initial acid production rate of different lactic acid bacteria in single-strain fermentation and three-strain combined fermentation systems.

[0043] Among the 30 lactic acid bacteria strains, most strains did not show a significant acidification enhancement effect after being added to the MX14 / PY33 system, and some strains even reduced the acidification capacity of the complex system; while some strains were able to further promote acid accumulation.

[0044] Table 1. Evaluation of acidification performance of different candidate lactic acid bacteria in the MX14 / PY33 co-fermentation system Among them, the MX14 / PY33 / M34-1 tribacterial combination showed high acidification performance, with a titratable acid content of 21.85 g / kg on day 7 and an average acid production rate of 5.16 g / (kg·d) from day 0 to day 3.

[0045] Based on the comprehensive endpoint of titratable acid content, early acid production rate, and compatibility with the complex system, five dominant candidate strains were screened from 30 lactic acid bacteria strains and numbered M34-1, M34-2, M3, M20, and M8-2, respectively, for subsequent re-screening and evaluation.

[0046] Example 2: Secondary screening of candidate lactic acid bacteria and identification of Lactobacillus pentosaccharide M34-1 To further evaluate the compatibility of candidate lactic acid bacteria in the combined fermentation system of *Lactobacillus plantarum* MX14 and *Pichia pastoris* PY33, the five candidate lactic acid bacteria obtained in Example 1 were re-screened and evaluated.

[0047] M34-1, M34-2, M3, M20, and M8-2 were combined with MX14 and PY33 to form a three-strain co-fermentation system, with the MX14 / PY33 dual-strain fermentation group serving as a control. The raw material formulation, bacterial concentration, total inoculum size, and fermentation conditions were kept consistent across all treatments.

[0048] After fermentation, the titratable acid content, viable lactic acid bacteria count, viable PY33 count, sugar content, organic acid content, and free amino acid content of each treatment group were measured to evaluate the role of different candidate lactic acid bacteria in the compound fermentation system.

[0049] The results showed that different candidate lactic acid bacteria exhibited certain differences in the MX14 / PY33 basic system, with some strains showing good compatibility for co-fermentation. Considering both the endpoint acidification level and the stability of the co-fermentation system, M34-1 was selected as the target for further identification.

[0050] (1) Observation of colony morphology of strain M34-1 The selected dominant strain M34-1 was inoculated into MRS solid medium and cultured at 37 °C for 24 h.

[0051] like Figure 1 As shown, strain M34-1 forms milky white colonies on MRS plates. The colonies are round, with neat edges and smooth surfaces.

[0052] (2) Identification of 16S rRNA gene sequence Strain M34-1 was inoculated into MRS liquid medium and cultured at 37 °C for 24 h. 1 mL of the bacterial culture was placed in a 1.5 mL sterile centrifuge tube, and the bacterial cells were collected by centrifugation. Genomic DNA of strain M34-1 was extracted using Plant Zol (TransGen kit).

[0053] Extracted genomic DNA was amplified using bacterial universal primers 27F (SEQ ID NO: 1 5′-AGAGT TTGAT CCTGGCTCAG-3′) and 1492R (SEQ ID NO: 2 5′-TACGG CTACC CTTGT TACGA-3′). PCR conditions were as follows: pre-denaturation at 94℃ for 5 min, followed by 25 cycles of denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, and extension at 72℃ for 1.5 min. After each cycle, a final extension at 72℃ for 7 min was performed. Detection was performed using 3% agarose gel electrophoresis, and the amplified samples were subsequently sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0054] The 16S rRNA gene sequence of strain M34-1 was compared with relevant sequences in the NCBI database using BLAST homology, and a phylogenetic tree was constructed using sequences from relevant reference strains. For example... Figure 2 As shown, strain M34-1 clustered with the reference strain *Lactobacillus pentosaccharis* in the same branch. Based on colony morphology, sequence alignment, and phylogenetic analysis, strain M34-1 was identified as *Lactobacillus pentosaccharis* (…). L. pentosus ).

[0055] The 16S rRNA gene sequence of M34-1 is shown in SEQ ID NO:3: Example 3: Establishment of a co-fermentation system of MX14 / PY33 and candidate lactic acid bacteria The candidate lactic acid bacteria M34-1, M34-2, M3, M20 and M8-2 obtained from Example 1, as well as Lactobacillus plantarum MX14, were inoculated into MRS liquid medium and cultured at 37°C for 24 h; Pichia pastoris PY33 was inoculated into PDB liquid medium and cultured at 30°C for 24 h.

[0056] After the culture was completed, the bacterial cells of each strain were collected by centrifugation, resuspended in 0.9% sterile physiological saline, and the viable count of each bacterial suspension was adjusted to not less than 1×10^8 CFU / mL.

[0057] The preparation method of red sour soup raw material slurry is as follows: weigh 1000 g of tomatoes, add 200 g of fresh red chili peppers, 100 g of ginger, 50 g of garlic, 20 g of glutinous rice flour, 30 g of salt and 50 mL of white wine, mix and homogenize, treat at 105 ℃ for 15 min, cool to room temperature, and dispense into sterile fermentation containers to obtain red sour soup raw material slurry.

[0058] A dual-strain fermentation group consisting of MX14 / PY33 and a three-strain co-fermentation group consisting of candidate lactic acid bacteria and MX14 / PY33 were set up. In the three-strain co-fermentation group, the bacterial suspensions of MX14, PY33 and candidate lactic acid bacteria were mixed at a ratio of 1:1:1. The total inoculum amount of each treatment was 5% (v / w). After inoculation, fermentation was carried out at 30℃ for 7 days for subsequent evaluation of the co-fermentation performance.

[0059] Example 4: Performance evaluation of candidate lactic acid bacteria co-fermentation and determination of dominant strain M34-1 Based on the candidate lactic acid bacteria co-fermentation system constructed in Example 3, this embodiment conducts a secondary screening evaluation of five candidate lactic acid bacteria strains, namely M34-1, M34-2, M3, M20 and M8-2, according to the grouping method, raw material formula, bacterial concentration, total inoculum amount and fermentation conditions described in Example 3. Each treatment is set up in 3 parallels.

[0060] During fermentation, the titratable acid content, viable lactic acid bacteria count, viable PY33 count, total sugar content, and reducing sugar content of each treatment group were measured. After fermentation, the composition of organic acids, free amino acids, and volatile compounds was further measured to comprehensively evaluate the composite fermentation performance of different candidate lactic acid bacteria in the MX14 / PY33 basic system.

[0061] Table 2. Main performance evaluation of different candidate lactic acid bacteria co-fermentation systems The acidification properties, cell count, sugar content, and nitrite content of red sour soup under different fermentation treatments are as follows: Figure 4As shown. By Figure 4 As shown in Table A and Table 2, there were differences in the endpoint pH and titratable acid content among different fermentation treatments. On day 7 of fermentation, the titratable acid content of the MX14 / PY33 / M34-1 tri-strain group reached 22.70 g / kg, the highest among all tri-strain combined fermentation groups; the titratable acid content of the MX14 / PY33 dual-strain control group was 19.24 g / kg. Compared with the dual-strain control group, the titratable acid content of the MX14 / PY33 / M34-1 tri-strain group increased by 18.0%, indicating that the addition of M34-1 can further enhance the acidification capacity of the MX14 / PY33 basic fermentation system.

[0062] Depend on Figure 4 C indicates that the survival of PY33 varied in different co-fermentation systems. On day 7 of fermentation, the viable PY33 counts in the MX14 / PY33 / M34-1 tri-strain group and the MX14 / PY33 dual-strain control group were 4.67 ± 0.06 log CFU / g and 4.64 ± 0.03 log CFU / g, respectively, with no significant difference (p > 0.05), indicating that the addition of M34-1 did not significantly inhibit the survival of PY33 at the end of fermentation. In contrast, the viable PY33 count in the MX14 / PY33 / M20 group was 3.77 ± 0.02 log CFU / g, a decrease of 0.87 log CFU / g compared to the dual-strain control group. These results suggest that M34-1 has good compatibility with MX14 and PY33 in co-fermentation.

[0063] Depend on Figure 4 D and Figure 4 As shown in Figure E, the total sugar and reducing sugar contents of all treatment groups decreased during fermentation. The total sugar consumption rate and reducing sugar consumption rate of the MX14 / PY33 / M34-1 tri-strain group were 50.7% and 61.5%, respectively. The total sugar consumption rate of the MX14 / PY33 / M20 group was 59.8%, but its PY33 viable cell count was significantly reduced; the reducing sugar consumption rate of the MX14 / PY33 / M3 group was 67.1%, but its endpoint titratable acid content was lower than that of the M34-1 tri-strain group. This indicates that the MX14 / PY33 / M34-1 tri-strain group exhibits good overall performance in terms of sugar utilization, acid accumulation, and strain compatibility. Figure 4 As can be seen from F, the nitrite content at the fermentation endpoint of each treatment group was at a low level, indicating that the fermentation system did not cause significant accumulation of nitrite.

[0064] Organic acids in red sour soup from different fermentation treatments were determined by high-performance liquid chromatography (HPLC). The composition and content of organic acids and free amino acids in red sour soup from different fermentation treatments are shown below. Figure 5 As shown. By Figure 5 A and Figure 5As shown in section B, nine organic acids were detected in all treated samples: oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid, and propionic acid. The total organic acid content of the MX14 / PY33 / M34-1 tri-strain group reached 45.25 g / kg, with lactic acid content reaching 24.98 g / kg, the highest value among all treatment groups in this example. The lactic acid content of the M34-1 single-strain group was 24.75 g / kg, indicating that M34-1 has a strong lactic acid production capacity; after co-fermentation with MX14 and PY33, the total organic acid content further increased.

[0065] Free amino acids in red sour soup from different treatments were determined using liquid chromatography-mass spectrometry. Figure 5 C and Figure 5 As shown in Figure D, the total free amino acid content of each treatment group ranged from 10.49 to 27.25 g / kg, with the MX14 / PY33 / M34-1 tri-strain group reaching a relatively high level of 27.21 g / kg. All treatment groups were primarily composed of umami amino acids such as glutamic acid and aspartic acid, accounting for 59.13% to 71.37% of the total free amino acids. The sweet amino acid content of the MX14 / PY33 / M34-1 tri-strain group reached 4.85 g / kg, the highest among all treatment groups in this embodiment. These results indicate that the tri-strain fermentation system can increase the total free amino acid content and improve the composition of umami and sweet amino acids.

[0066] The composition and content of volatile compounds in red sour soup after different fermentation treatments are as follows: Figure 6 As shown. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the volatile compounds in the red sour soup from each treatment. Figure 6 A and Figure 6 As indicated by B, a total of 150 volatile compounds were detected in all samples, including alcohols, hydrocarbons, esters, ketones, aldehydes, sulfur-containing compounds, acids, ethers, and phenols. The total volatile compound content of the MX14 / PY33 / M34-1 tri-strain group reached 189.32 mg / kg, which was 3.0% higher than the MX14 / PY33 dual-strain control group and 21.2% higher than the M34-1 single-strain group. The contents of alcohols, hydrocarbons, esters, and sulfur-containing compounds in this tri-strain group were all at relatively high levels.

[0067] Depend on Figure 6 As can be seen from C, the composition of volatile compounds of red sour soup obtained by fermentation of different strain combinations is different. The MX14 / PY33 / M34-1 three-strain group has different volatile compound composition characteristics from the two-strain control group and the M34-1 single-strain group.

[0068] In summary, the MX14 / PY33 / M34-1 three-strain compound fermentation system can increase the titratable acidity and total organic acid content of red sour soup, effectively utilize sugars, and maintain the survival of PY33 while enhancing acidification capacity. Simultaneously, it can increase the total amount of free amino acids, improve the composition of umami and sweet amino acids, and regulate the composition of volatile compounds. Therefore, the compound microbial agent composed of M34-1 with MX14 and PY33 has strong acidification capacity, good strain compatibility, and a relatively balanced quality control effect, making it suitable for the controlled and standardized fermentation production of red sour soup.

[0069] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A strain of *Lactobacillus pentosaccharide*, characterized in that, The pentosaccharide lactobacillus is pentosaccharide lactobacillus ( Lactiplantibacillus pentosus M34-1 was deposited on July 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.39925.

2. The application of the Lactobacillus pentosus M34-1 as described in claim 1 in the preparation of red sour soup.

3. A compound microbial inoculant for fermenting red sour soup, characterized in that, include: The *Lactobacillus pentosus* M34-1, *Lactobacillus plantarum* MX14, as described in claim 1, with the accession number CGMCC No. 33391; and *Pichia pastoris* PY33, with the accession number CGMCC No. 33392.

4. The compound microbial agent according to claim 3, characterized in that, The volume ratio of the bacterial culture of *Lactobacillus plantarum* MX14, *Pichia pastoris* PY33, and *Lactobacillus pentosus* M34-1 was (0.5~2):(0.5~2):(0.5~2).

5. The compound microbial agent according to claim 3 or 4, characterized in that, The viable cell counts of *Lactobacillus plantarum* MX14, *Pichia pastoris* PY33, and *Lactobacillus pentosus* M34-1 in the bacterial culture before mixing were each no less than 1 × 10⁻⁶. 8 CFU / mL.

6. The compound microbial agent according to claim 4, characterized in that, The compound microbial agent is a bacterial suspension, culture medium, or fermentation culture containing bacterial strains.

7. The application of the compound microbial agent according to any one of claims 3 to 6 in the preparation of red sour soup.

8. The application according to claim 7, characterized in that, The compound microbial agent is used for at least one of the following: (1) Increase the titratable acid content of red sour soup; (2) Increase the content of lactic acid and total organic acids in red sour soup; (3) Maintain the survival of Pichia pastoris PY33 in the red sour soup fermentation system; (4) Increase the content of sweet amino acids in red sour soup.

9. A method for preparing red sour soup using a compound microbial agent, characterized in that, Includes the following steps: S1, mix tomatoes, fresh red peppers, glutinous rice flour, salt, white wine, ginger and garlic and homogenize them to obtain red sour soup raw material slurry; S2, heat-treat the red sour soup raw material slurry obtained in step S1 and cool it for later use; S3, inoculate the compound microbial agent according to any one of claims 3 to 6 into the red sour soup raw material slurry obtained in step S2; S4, ferment the inoculated red sour soup raw material slurry to obtain red sour soup product.

10. The method according to claim 9, characterized in that, Based on the quality of tomatoes, the red sour soup raw material slurry includes: 1.5%~3% (w / w) glutinous rice flour, 2%~4% (w / w) salt, 4%~6% (v / w) white wine, 15%~25% (w / w) fresh red chili peppers, 8%~12% (w / w) ginger, and 4%~6% (w / w) garlic; the heat treatment conditions in step S2 are 105 ℃ for 15 min; the total inoculation amount of the compound microbial agent in step S3 is 5% (v / w), and the *Lactobacillus plantarum* MX14, *Pichia pastoris* PY33, and *Lactobacillus pentosus* M34-1 are mixed in a 1:1:1 volume ratio before inoculation; the fermentation temperature in step S4 is 30 ℃, and the fermentation time is 7 days.

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