Lactobacillus collinoides and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2026-04-15
- Publication Date
- 2026-06-09
Smart Images
Figure CN122168478A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbiology, and specifically relates to a strain of *Lactobacillus pachyphylla* and its applications. Background Technology
[0002] Flavor is a core factor determining the sensory quality and consumer acceptance of traditional fermented soy products such as fermented bean curd. The final formation and stability of fermented bean curd's flavor quality spans the post-ripening and subsequent shelf-life stages. Aroma compounds are mainly composed of esters and alcohols, with esters playing a crucial role in imparting fruity, sweet, and ester-like flavors. However, traditional fermented bean curd production often employs an open-air natural fermentation process, resulting in a complex microbial community structure and significant batch-to-batch fluctuations. This leads to unstable product flavor quality, and the content of desirable flavor compounds such as esters is easily affected by the continuous action of microorganisms throughout the shelf life, making it difficult to guarantee consistent quality. Therefore, screening for superior fermentation strains that can specifically enhance key flavor compounds such as esters in fermented bean curd is of great significance for improving its flavor quality and product consistency.
[0003] During the shelf life of fermented bean curd, the growth of harmful microorganisms is common, with powdered Miller's yeast being one of the most frequent contaminants. Powdered Miller's yeast exhibits strong salt tolerance and can proliferate rapidly in the high-salt environment of fermented bean curd. Its metabolic activity produces undesirable flavor compounds such as 2-octanone, 2-nonanone, and 1,2,3-trimethoxybenzene, which impart a sour and astringent taste, severely damaging the sensory quality and product stability of fermented bean curd. Currently, the industry's main methods for controlling harmful yeasts still rely on adding chemical preservatives or increasing the amount of salt. However, with the increasing consumer demand for natural, healthy, and low-salt foods, reducing the use of chemical additives has become a key direction for the transformation and upgrading of the fermented bean curd industry.
[0004] Lactic acid bacteria are one of the dominant bacterial groups during the shelf life of fermented bean curd, making a core contribution to the formation of its characteristic flavor. *Ligilactobacillus pobuzihii*, initially isolated and identified by Chen et al. from the traditional fermented food *Pobuzi*, was published as a new species of the genus *Lactobacillus* in 2010 and reclassified into the genus *Ligilactobacillus* in 2020 after systematic taxonomic revision. Existing studies have shown that *Ligilactobacillus pobuzihii* has better salt tolerance than general lactic acid bacteria and can grow well in saline environments. In traditional fermented shrimp yogurt, *Ligilactobacillus pobuzihii* showed a significant positive correlation with several key volatile flavor compounds, playing a dominant role in the synthesis of esters and acids, demonstrating its potential as a flavor-enhancing starter culture. The related genus *Ligilactobacillus salivarius* also showed good flavor-improving ability in fermented soy milk. This indicates that strains of the genus *Ligilactobacillus* have broad application value in enhancing the flavor of plant-based fermented foods. However, there are currently no reports of using *Ligilactobacillus pobuzihii* to simultaneously inhibit powdered *Miller's yeast* and enhance the flavor of fermented bean curd.
[0005] Chinese invention patent application CN202111039168.X discloses a bioreactor for high-density cultivation of *Lactobacillus pyogenes* and its usage method. This patent application addresses the problems of large pH fluctuations, uneven nutrient distribution, and low cultivation density in existing *Lactobacillus pyogenes* cultivation equipment by providing a bioreactor that stabilizes the pH and nutrient composition of the culture medium through a multi-layered carrier frame, a fluid-infusing cotton core, and a circulating culture medium system, effectively increasing the cultivation density of *Lactobacillus pyogenes*. However, this patent application only solves the problem of high-density cultivation equipment for *Lactobacillus pyogenes* and does not address the specific functional applications of this strain in fermented food systems, nor does it reveal its inhibitory effect on powdered *Miller's yeast* or its potential application in improving the flavor of fermented bean curd. Summary of the Invention
[0006] To address the problems existing in the prior art, the primary objective of this invention is to provide a strain of *Lactobacillus pachymansi*. This *Lactobacillus pachymansi* can inhibit the growth of powdered *Miller's yeast* and has a high ability to generate volatile flavor compounds, giving fermented bean curd a rich ester aroma and a lower astringency.
[0007] Another object of the present invention is to provide the application of the aforementioned *Lactobacillus thuringiensis* combined with *Lactobacillus* in the production of fermented bean curd.
[0008] The objective of this invention is achieved through the following technical solution:
[0009] A strain of *Ligilactobacillus pobuzihii*, named *Ligilactobacillus pobuzihii* LXMMF5, with accession number GDMCC NO: 68066, was deposited on April 10, 2026, at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0010] A microbial preparation containing the aforementioned *Lactobacillus pachymansi* and *Lactobacillus*.
[0011] The preferred dosage form of the microbial preparation is liquid, powder, or granules.
[0012] The application of the above-mentioned *Polygonum multiflorum* combined with *Lactobacillus* or the above-mentioned microbial preparations in the production of fermented bean curd products.
[0013] A method for producing fermented bean curd products includes the following steps: inoculating fermented bean curd that has completed post-ripening with the above-mentioned Lactobacillus pachycarpa, and fermenting it to obtain fermented bean curd products.
[0014] The aforementioned *Lactobacillus thuringiensis* and *Lactobacillus* are preferably activated and cultured, and the resulting bacterial solution is then added to the fermented bean curd after ripening.
[0015] The preferred activation procedure is to inoculate the above-mentioned *Lactobacillus thuringiensis* and *Lactobacillus* into MRS broth medium and then incubate it anaerobically at 25–35°C.
[0016] The preferred temperature for anaerobic culture is 29–31°C.
[0017] The preferred anaerobic culture time is 24–48 hours.
[0018] The concentration of the bacterial solution is 6–10 LogCFU / mL; preferably 7–8 LogCFU / mL.
[0019] The inoculation amount of the bacterial solution is 1-10% v / v; preferably 1-5% (v / v).
[0020] The fermentation temperature is preferably 20–35°C; more preferably 24–26°C.
[0021] The fermentation time is 60 to 100 days; more preferably 80 days.
[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0023] (1) The culture conditions of the combined Lactobacillus LXMMF5 and the bacterial suspension for inoculation disclosed in this invention are simple, and the preparation method of the bacterial suspension for inoculation is simple, which has great application potential in the fermented food industry.
[0024] (2) The LXMMF5 of the combined Lactobacillus pumilum and the present invention are isolated and purified from fermented bean curd and are tolerant to the production environment of high-salt products such as fermented bean curd, soy sauce and soy sauce.
[0025] (3) The LXMMF5 of the combined Lactobacillus dysenteriae disclosed in this invention is sensitive to ampicillin, gentamicin, kanamycin, streptomycin, clindamycin, tetracycline, erythromycin and chloramphenicol, and has no drug resistance genes or virulence genes, and has broad application prospects.
[0026] (4) The supernatant of the combination of *Lactobacillus simulans* and *Lactobacillus LXMMF5* disclosed in this invention has a good inhibitory effect on powdered *Milleria mollissima* isolated from fermented bean curd in YPD medium, and has the potential to prepare related biological agents.
[0027] (5) The LXMMF5 of the combined Lactobacillus pumilum and the present invention can significantly increase the odor activity value (OAV) of key volatile flavor substances in fermented bean curd, which can significantly increase the content of volatile flavor substances with flavor effects, especially esters, thereby improving the flavor of fermented bean curd and giving it a rich ester aroma and floral and fruity aroma, which has good application prospects. Attached Figure Description
[0028] Figure 1This is a RAPD typing diagram of *Lactobacillus pachymansi* combined with *Lactobacillus
[0029] Figure 2 The growth curve of *Lactobacillus thuringiensis* combined with *Lactobacillus* in sterile MRS broth containing 8% NaCl (w / v).
[0030] Figure 3 This is the phylogenetic tree of *Lactobacillus pachycarpa* and *Lactobacillus LXMMF5* in this invention.
[0031] Figure 4 This is a colony morphology diagram of the combined Lactobacillus LXMMF5 and Pseudomonas aeruginosa of the present invention.
[0032] Figure 5 This is a microscopic image of the bacterial cell morphology of *Lactobacillus simulans* combined with *Lactobacillus LXMMF5* according to the present invention.
[0033] Figure 6 This is a genomic map of the *Lactobacillus* LXMMF5 co-integrated with *Polygonum multiflorum* in this invention.
[0034] Figure 7 This is a diagram showing the annotation results of secondary metabolites of the present invention, combining *Lactobacillus* LXMMF5 with *Lactobacillus* in the antiSMASH database.
[0035] Figure 8 The growth curves of powdered Miller's yeast in the supernatant after culturing with different proportions of Pseudomonas aeruginosa and Lactobacillus LXMMF5 for 24 h. Detailed Implementation
[0036] To better understand the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the embodiments do not constitute any limitation on the scope of protection of the present invention.
[0037] In the following examples and comparative examples:
[0038] MRS broth medium: per 1L of distilled water, 10.0 g casein digest, 2.0 g triammonium citrate, 10.0 g beef extract, 0.2 g magnesium sulfate (MgSO4·7H2O), 4.0 g yeast extract, 0.05 g manganese sulfate (MnSO4·4H2O), 5.0 g sodium acetate, 2.0 g dipotassium hydrogen phosphate, 20.0 g glucose, 1.08 g Tween-80, and 80 g sodium chloride (NaCl). The solvent is distilled water, pH is natural, and the medium is sterilized at 121℃ for 15 min. MRS solid medium is prepared by adding 2 g / 100mL agar powder to the broth medium.
[0039] YPD broth medium: 10.0 g yeast extract, 20.0 g peptone, and 20.0 g glucose per 1 L of distilled water, with distilled water as the solvent, natural pH, sterilized at 121°C for 15 min.
[0040] LSM medium preparation: Mix the prepared IST medium and MRS broth medium at a volume ratio of 9:1, adjust the pH to 6.8, and autoclave at 121℃ for 15 min.
[0041] Example 1: Screening and identification of *Polygonum multiflorum* combined with *Lactobacillus*
[0042] (1) Isolation and purification of strains
[0043] Take 5g of fermented bean curd sample, add 45mL of sterile physiological saline, and grind thoroughly in a mortar to obtain a grinding solution. Dilute 1mL of the grinding solution serially to 9mL of sterile physiological saline. Spread three suitable serial dilutions onto MRS solid medium and incubate at 30℃ for 48h. Select single colonies with different morphologies and further purify them using the streak plate method. Repeat this process twice until single colonies with uniform morphology are obtained from the same plate. Incubate the purified single colonies anaerobically at 30℃ for 48h in MRS liquid medium and store with glycerol at -80℃.
[0044] (2) 16S rDNA sequencing identification
[0045] Genomic DNA was extracted using a bacterial DNA extraction kit and amplified by PCR. Universal bacterial primers 27F and 1492R were used. The PCR reaction mixture consisted of: 2 μL template DNA, 2 μL primer 27F (10 μM), 2 μL primer 1492R (10 μM), 20 μL 2×PCR mix, and 24 μL ultrapure water. The PCR program was: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 30 s, 46℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles; 72℃ further extension for 10 min; and 4℃ indefinite cycling. After the PCR products showed clear and specific bands upon electrophoresis, the remaining samples were sent to Sangon Biotech (Guangzhou) Co., Ltd. for 16S rDNA sequencing. The obtained sequences were submitted to the NCBI database and homology analysis was performed using BLAST.
[0046] Analysis using NCBI's BLAST tool revealed a total of 186 bacterial strains, of which 42 strains were highly similar to Ligilactobacillus pobuzihii, and were identified as Ligilactobacillus pobuzihii.
[0047] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';
[0048] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3';
[0049] (GTG)'5:5'-GTGGGTGGTGGTGGTG-3'.
[0050] (3) RAPD typing of Plastrum lactis combined with Lactobacillus
[0051] DNA was extracted from 42 strains of *Lactobacillus* and *Bacillus* using a bacterial sample genomic DNA extraction kit. Primer (GTG)'5 was used. The reaction mixture consisted of 3 μL template DNA, 25 μL 2×PCR mix, 3 μL primer (GTG)'5, and 19 μL ultrapure water. Amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 40℃ annealing for 30 s, 72℃ extension for 8 min, for 30 cycles; followed by a final extension at 72℃ for 16 min. After amplification, the PCR products were subjected to agarose gel electrophoresis to separate the amplified fragments. Electrophoresis conditions were: 120 V for 40 min, and the presence of specific bands was detected using a UV gel imaging system.
[0052] The 42 strains of *Lactobacillus pachyphylla* were clustered into 6 types with 90% accuracy. Specifically, 37 strains clustered into type I, LXMMF2 into type II, LXMMF5 into type III, LXMMF17 into type IV, LXMMD5 into type V, and LXMMF25 into type VI (see [link to relevant documentation]). Figure 1 To reduce repetitive work in subsequent experiments, three representative strains will be selected from category I, and one representative strain will be selected from each of the remaining five categories, for a total of eight strains for further research.
[0053] (4) Test for bioamines produced by *Lactobacillus thuringiensis* combined with *Lactobacillus*
[0054] Based on amino acid decarboxylase culture medium, 0.5% (w / v) of L-tyrosine, L-lysine hydrochloride, L-ornithine hydrochloride, L-histidine, and L-arginine were added to respectively to detect the production of tyramine, cadaverine, putrescine, histamine, and spermine. The solution was dissolved thoroughly in distilled water and the pH was adjusted to 6.8. The activated bacterial suspension (8 LogCFU / mL) was inoculated at 3% (v / v) into the above five amino acid culture media and anaerobically cultured at 30℃ for 48 h. The color changes of the culture media were observed. Controls included: uninoculated culture medium (blank control) and culture medium inoculated with bacterial suspension but without added amino acids (negative control). A turbid and purple culture medium was considered positive; a yellow or unchanged culture medium was considered negative.
[0055] Table 1 shows that two strains produced both histamine and putrescine: LXMMF32 and LXMMD5; two strains produced only histamine: LXMMF25 and LXMMC18; and the remaining strains did not produce any of the five biogenic amines. Therefore, LXMMF2, LXMMF5, LXMMF16, and LXMMF17 were selected for further screening.
[0056] Table 1 Results of the colorimetric reaction of biogenic amines in the strains
[0057]
[0058] (5) Study on the growth characteristics of *Lactobacillus pachymansi* and *Lactobacillus rubrum* in a high-salt environment
[0059] The *Lactobacillus thuringiensis* and *Lactobacillus* were activated twice consecutively at 30℃ to obtain a seed culture. The seed culture was inoculated at a 5% (v / v) inoculation rate into sterile MRS broth containing 8% NaCl (w / v) and incubated statically at 30℃ for 48 h. Samples were taken at 0, 8, 12, 16, 20, 24, 32, 40, and 48 h, and the absorbance (OD) at 600 nm was measured using uninoculated MRS liquid medium as the zeroing point. 600nm ). (with OD) 600nm The values are plotted on the ordinate and the incubation time on the x-axis to create growth curves for the four bacterial strains. Results are shown below. Figure 2 LXMMF5 grows relatively quickly and can enter the logarithmic growth phase more rapidly. It has the highest OD value during the stationary phase, showing good high salt adaptability and growth stability. Therefore, LXMMF5 was selected for research.
[0060] (6) Construction of a phylogenetic tree of *Lactobacillus pachymansi* and *Lactobacillus*
[0061] Analysis using NCBI's BLAST tool revealed that strain LXMMF5 (its 16S rDNA sequence is shown below) is highly similar to *Ligilactobacillus pobuzihii*, thus identifying LXMMF5 as *Ligilactobacillus pobuzihii*, and constructing a phylogenetic tree (see [link]). Figure 3 ).
[0062]
[0063] The strain was named Lactobacillus dysporidis LXMMF5 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 10, 2026, with accession number GDMCC NO: 68066.
[0064] (7) Observation of colony and cell morphology
[0065] LXMMF5 strain was cultured on MRS solid medium using the streak plating method, and the morphology of single colonies was observed. Gram staining was used to observe the bacterial morphology under a microscope. LXMMF5 colonies on MRS solid medium appeared milky white, small, and round (see...). Figure 4 Gram staining was positive, and under microscopic observation, the cells of *Lactobacillus rubrum* and *Lactobacillus* appeared as long rods (see...). Figure 5 ).
[0066] Example 2: Antibiotic Resistance
[0067] According to the European Food Safety Authority (EFSA) regulations on drug susceptibility testing, the microbroth dilution method was used for drug resistance phenotype testing. The selected antibiotics were ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol, prepared with gradient concentrations of 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. *Lactobacillus pachymansi* and *Lactobacillus aspergillus* stored at -80℃ were activated twice with MRS broth medium, adjusting the bacterial suspension concentration to 8 LogCFU / mL. 100 μL of each gradient concentration of antibiotics and 100 μL of bacterial suspension were added to 96-well plates. A bacterial suspension containing the highest concentration of antibiotic and LSM medium was used as a positive control, while LSM medium without bacterial suspension served as a negative control. The plates were incubated at 30℃ for 48 h, and the absorbance was measured at 625 nm. The drug resistance phenotype of different strains was determined based on the minimum inhibitory concentration (MIC) inflection point specified by EFSA. The MIC inflection points of *Lactobacillus subtilis* combined with *Lactobacillus* were: ampicillin 4 μg / mL, gentamicin 16 μg / mL, kanamycin 64 μg / mL, streptomycin 64 μg / mL, erythromycin 1 μg / mL, clindamycin 1 μg / mL, tetracycline 8 μg / mL, and chloramphenicol 4 μg / mL.
[0068] The drug resistance phenotype detection results of *Lactobacillus thuringiensis* combined with *Lactobacillus* LXMMF5 were as follows: ampicillin 2 μg / mL, gentamicin 8 μg / mL, kanamycin 32 μg / mL, streptomycin 32 μg / mL, erythromycin 0.5 μg / mL, clindamycin 0.5 μg / mL, tetracycline 4 μg / mL, and chloramphenicol 2 μg / mL. It can be seen that all the detected values are less than or equal to the inflection point value specified by EFSA, therefore LXMMF5 is sensitive to all eight tested antibiotics.
[0069] Example 3: Whole Genome Information Analysis
[0070] 1. Detection Method
[0071] The *Lactobacillus* LXMMF5 was activated twice in MRS broth medium, centrifuged at 8000×g for 5 min, and the supernatant was discarded. High-quality genomic DNA was extracted and its purity, concentration, and integrity were checked using Nanodrop, Qubit, and 0.35% agarose gel electrophoresis. Large DNA fragments were recovered using the BluePippin automated nucleic acid recovery system. Library construction was performed using the SOK-LSK109 ligation kit, including DNA damage repair and end repair, magnetic bead purification, adapter ligation and magnetic bead purification, Qubit library quantification, and finally, the high-quality library DNA was sequenced. The raw data underwent quality control, filtering out low-quality and excessively short (<2000bp) reads. The filtered reads were assembled de novo using Canu v1.5, and the assembly results were corrected using Raconv 3.4.3 software. Circularization and start site adjustment were performed using Circler v1.5.5, and error correction was performed on the assembled draft genome using Pilon v1.22. The assembled, qualified data can be used for genomic component analysis, functional analysis, and genome mapping. Gene prediction was performed using Prodigal v2.6.3, repetitive sequence alignment was performed using RepeatMasker v4.0.5, IslandPath-DIMOB v0.2 was used to predict gene islands in the genome, and finally, Circos v0.66 was used to draw a genome circummap. Drug resistance genes were annotated using the CARD database, virulence genes were annotated using the VFDB database, and secondary metabolite information was annotated using the antiSMASH database.
[0072] 2. Results
[0073] (1) Basic Information
[0074] The genome size of *Lactobacillus pachycarpa* LXMMF5 is 2,350,840 bp, containing 2,145 coding genes with a total length of 1,937,538 bp and a GC content of 38.12%. Eighteen rRNAs and 62 tRNAs were predicted. Nine gene islands were predicted for this strain, with a total length of 99,830 bp and an average length of 11,092 bp. A genome loop map of LXMMF5 was constructed using the assembled and predicted genome information (see [link to webpage]). Figure 6 ).
[0075] Figure 6 In the diagram, the outermost ring represents positive-strand genes, followed by negative-strand genes. The function of each gene is obtained through COG annotation, which can be found in the corresponding information on the right side of the diagram. The middle layer represents ncRNA, black represents tRNA, and red represents rRNA. The two layers further inward represent GC content and GC offset, respectively.
[0076] (2) Analysis of drug resistance genes
[0077] The whole genome information was compared with the CARD database, and no related antibiotic resistance genes were found.
[0078] (3) Virulence gene analysis
[0079] The whole genome information was aligned to the VFDB database, and the screening criteria were set as sequence similarity ≥85%, sequence coverage ≥85%, and Evalue <0.05. No related virulence genes were found.
[0080] (4) Secondary metabolite analysis
[0081] The whole genome sequence was uploaded to the antiSMASH database, and the T3PKS gene cluster was annotated (see...). Figure 7 The product was identified as a chalcone-type polyketide after comparison with the NCBI database. Chalcones, as biosynthetic precursors of flavonoids, possess a variety of important biological activities. In terms of antibacterial activity, chalcone derivatives exhibit inhibitory effects against pathogens such as Staphylococcus aureus and Escherichia coli, with some derivatives showing minimum inhibitory concentrations (MICs) as low as 62.5 μg / mL, and can enhance the antibacterial effects of traditional antibiotics. In terms of anti-inflammatory activity, 4'-hydroxychalcone exerts its anti-inflammatory effect by inhibiting the NF-κB signaling pathway. In terms of antitumor activity, chalcones can induce tumor cell apoptosis and inhibit angiogenesis.
[0082] Example 4: Inhibition effect on representative strains of powdered Miller's yeast isolated from fermented bean curd.
[0083] (1) Powdered Miller's yeast LXMP18 (this strain has been disclosed in the literature "Wang Jingwen et al. Screening, identification and fermentation characteristics of functional yeast in high-salt dilute soy sauce [J]. Food Science, 2021, 42(22):91-97.", corresponding to strain SWJS W323-M. farinosa in the literature) was cultured with YPD and activated twice continuously at 30℃ and 200rpm, and the bacterial concentration was adjusted to 8 LogCFU / mL.
[0084] The *Lactobacillus pachymansi* and *Lactobacillus* LXMMF5 were activated twice, and the bacterial concentration was adjusted to 8 LogCFU / mL to obtain the *Lactobacillus pachymansi* seed culture. The seed culture was inoculated into MRS broth at a 5% (v / v) inoculum and cultured at 30℃ for 24 h. After centrifugation at 5000 rpm for 1 min, the supernatant was filtered through a 0.22 μm aqueous microporous membrane for sterilization, and the supernatant from the *Lactobacillus pachymansi* culture was collected. 2.5%, 5%, and 10% (v / v) of the supernatant were added to YPD medium, respectively, and powdered *Miller's yeast* LXMP18 was inoculated at a 3% (v / v) inoculum, with an equal volume of sterile water added as a control. The culture was incubated at 30℃ and 200 rpm, and the OD value at 600 nm was measured every 4 h.
[0085] This study investigated the inhibitory effects of *Lactobacillus pachymansi* LXMMF5 and *Lactobacillus pachymansi* LXMMC18 (a strain isolated separately in the laboratory) on powdered *Milleria pulveratum*.
[0086] (2) Results
[0087] The growth inhibition of powdered Müller's yeast by the culture supernatant of *Lactobacillus thuringiensis* combined with *Lactobacillus* LXMMF5 was observed. Figure 8 .Depend on Figure 8 It can be seen that the culture supernatant of *Lactobacillus thuringiensis* combined with *Lactobacillus* LXMMF5 has a significant antibacterial effect on powdered *Miller's yeast* isolated from fermented bean curd. After 24 hours of culture, the inhibition rates of 2.5%, 5%, and 10% (v / v) of the supernatant were 12.14%, 20.14%, and 29.49%, respectively, indicating that the inhibitory effect is better with the increase of the amount of supernatant added.
[0088] When the supernatant of *Lactobacillus thuringiensis* combined with *Lactobacillus* LXMMC18 was added at 2.5%, 5%, and 10% (v / v), the inhibition rates on the growth of *Miller's yeast* powder at 24 h were 1.35%, 2.83%, and 5.94%, respectively, and the antibacterial effect was not significant.
[0089] Example 5: Flavor of fermented bean curd inoculated with *Lactobacillus thuringiensis* and *Lactobacillus simulans* LXMMF5
[0090] (1) The Lactobacillus pachymansi LXMMF5 was activated twice consecutively, and the bacterial concentration was adjusted to 8 LogCFU / mL to obtain the Lactobacillus pachymansi seed culture. The Lactobacillus pachymansi seed culture was inoculated into freshly ripened fermented bean curd obtained from the factory at an inoculation rate of 5% (v / v), and the container was sealed. It was then allowed to stand at 25℃ for 80 days.
[0091] (2) The main volatile components in the sample were determined by SPME-GC-MS, with anisaldehyde as the internal standard (final concentration 0.5 ppm). The capillary column used for volatile component separation was HP-INNOWax (30 m × 250 μm, inner diameter and thickness 0.25 μm); the solid phase microextraction (SPME) needle was a divinylbenzene-carboxylic acid-polydimethylsiloxane fiber head (50 / 30 μm DVB / CAR / PDMS). An Agilent 7890B gas chromatograph (GC) and a 7000C triple quadrupole mass spectrometer (MS) were used. The SPME needle was pretreated at 60 °C for 30 min. 0.5 g of the sample to be tested, 0.2 g of NaCl, and 2 mL of distilled water were added to a glass headspace vial (20 mL), and incubated at 60 °C for 20 min. The aged extraction needle was inserted into the headspace of the headspace vial at 60°C for 45 min, and then immediately held at 250°C for 5 min in the GC inlet. The temperature programming was as follows: initial temperature 40°C, held for 3 min, then increased to 230°C at 5°C / min, and finally held at 230°C for 8 min. The split ratio was set to 5:1, the mass spectrometry recording range was 25–500 m / z, and the scan duration was 0.2 s. Helium (99.9995% purity) was used as the carrier gas at a flow rate of 1 mL / min. The interface temperature and ion source temperature were 250°C and 230°C, respectively. Volatile flavor compounds were identified by comparing mass spectra and retention indices from the NIST20.L database. The concentration of volatile flavor compounds was calculated based on the mass concentration of the internal standard with the closest retention index and the peak area ratio between each volatile flavor compound and the internal standard. The odor activity value (OAV) was calculated by dividing the concentration of volatile flavor compounds in water by the odor threshold. The OAV value is calculated by dividing the concentration of each volatile compound by the reported odor threshold.
[0092] The contribution of volatile compounds to the overall flavor profile was objectively assessed using their odor activity (OAV) values. OAV = volatile compound concentration / odor threshold. Generally, volatile flavor compounds with an OAV > 1 are considered to contribute more to the overall flavor and are defined as key volatile flavor compounds. Thirteen key volatile flavor compounds were identified in fermented soybean paste inoculated with *Lactobacillus thuringiensis* and *Lactobacillus* LXMMF5, as shown in Table 2. Table 2 shows that the main key volatile flavor compound is ethyl octanoate (OAV = 80927.09), followed by ethyl hexanoate (OAV = 6142.46), which imparts a rich fruity and floral aroma to the fermented bean curd. In addition, 2-pentylfuran (OAV = 835.28), 1-octen-3-ol (OAV = 476.9), and ethyl butyrate (OAV = 93.27) also had high OAV values.
[0093] Table 2 Key volatile flavor compounds in fermented bean curd inoculated with LXMMF5
[0094]
[0095] Note: Odor descriptions can be found at https: / / www.flavornet.org / flavornet.html, https: / / www.perflavory.com / , or https: / / www.femaflavor.org / flavor-library; thresholds for volatile flavor compounds can be found in the 2nd edition of "Compilation of Compound Odor Thresholds".
[0096] Comparative Example 1
[0097] (1) The fermented bean curd after the later fermentation was directly sealed and left to stand at 25°C for 80 days to obtain the sample of Comparative Example 1.
[0098] (2) SPME-GC-MS analysis was performed on the sample of Example 1. A total of 14 key volatile flavor compounds were identified, as shown in Table 3. As can be seen from Table 3, the main key volatile flavor compounds were ethyl octanoate (OAV=76917.19), followed by ethyl hexanoate (OAV=5308.85), ethyl 2-methylbutyrate (OAV=1572.48), 2-pentylfuran (OAV=759.11), and 1-octen-3-ol (OAV=566.34).
[0099] Table 3 Key volatile flavor compounds of Comparative Example 1
[0100]
[0101] Compared with control example 1, fermented bean curd inoculated with *Polygonum multiflorum* and *Lactobacillus LXMMF5* significantly increased the OAV value of esters, meaning it significantly increased the content of flavor-characteristic volatile esters, especially ethyl octanoate and ethyl hexanoate, imparting a rich brandy aroma and tropical fruit sweetness to the fermented bean curd. Simultaneously, the addition of phenylacetaldehyde imparted hyacinth and almond sweetness, and significantly reduced the OAV value of raw, green flavor compounds such as 1-octen-3-ol and ethyl 2-methylbutyrate, eliminating the raw, astringent taste of the fruit peel. This transformed the overall flavor profile into a harmonious and balanced complex of "fruity-wine-floral" aromas, significantly improving the flavor quality and market value of the fermented bean curd.
[0102] As can be seen from the above examples, the present invention has isolated a strain of Lactobacillus pachymansi LXMMF5 with inhibitory effect on powdered Miller's yeast from fermented bean curd. The culture conditions are simple and the safety is good (Examples 1 and 2). It has a good inhibitory effect on powdered Miller's yeast isolated from fermented bean curd (Example 4). Moreover, after inoculation into fermented bean curd, it can significantly improve the flavor of fermented bean curd (Example 5 and Comparative Example 1). This is mainly reflected in: significantly increasing the OAV value of esters that impart wine and fruit aroma to fermented bean curd and aldehydes that impart floral and almond aroma, and significantly reducing the OAV value of raw green taste substances, eliminating the astringent taste of fruit peel.
[0103] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A strain of *Lactobacillus pachyphylla*, characterized in that: The name of the Lactobacillus pobuzihii is LXMMF5, with accession number GDMCC NO: 68066. It was deposited on April 10, 2026 at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. A microbial preparation, characterized in that: Contains the *Lactobacillus pachymansi* as described in claim 1.
3. The microbial preparation according to claim 2, characterized in that: The dosage form of the microbial preparation is liquid, powder, or granules.
4. The use of the *Lactobacillus* compounded with *Lactobacillus* as described in claim 1 or the microbial preparation as described in any one of claims 2 to 3 in the production of fermented bean curd products.
5. A method for producing fermented bean curd products, characterized in that... The process includes the following steps: inoculating the fermented bean curd that has completed its post-ripening with Lactobacillus as described in claim 1, and then fermenting it to obtain the fermented bean curd product.
6. The method for producing fermented bean curd products according to claim 5, characterized in that: The bacterial solution obtained by activating and culturing *Lactobacillus thuringiensis* is added to the fermented bean curd after it has finished ripening.
7. The method for producing fermented bean curd products according to claim 6, characterized in that: The activation process involves inoculating the *Lactobacillus pachymansi* and *Lactobacillus* into MRS broth medium and then incubating it anaerobically at 25–35°C.
8. The method for producing fermented bean curd products according to claim 7, characterized in that: The anaerobic culture time is 24–48 hours.
9. The method for producing fermented bean curd products according to claim 7, characterized in that: The concentration of the bacterial solution is 6–10 LogCFU / mL; The inoculation amount of the bacterial solution is 1-10% v / v.
10. The method for producing fermented bean curd products according to claim 5, characterized in that: The fermentation temperature is 20–35°C; The fermentation time is 60 to 100 days.