Lactobacillus parafarraginis with stable biogenic amine degradation ability and application thereof

Abstract

This invention discloses a strain of *Lactobacillus sarcodactylis* with stable biogenic amine degradation ability and its applications, belonging to the field of microbial fermentation technology. This invention screened a strain of *Lactobacillus sarcodactylis* L18 from fermented soybean paste, capable of degrading biogenic amines. This strain can simultaneously degrade eight common biogenic amines: tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. The *Lactobacillus sarcodactylis* L18 screened in this invention exhibits rapid growth, strong environmental adaptability, and stable amine-reducing performance. It can continuously exert its biogenic amine degradation effect under low to medium-high salt, acidic to weakly alkaline conditions, and common fermentation temperature conditions, making it particularly suitable for the effective control of biogenic amines such as tyramine in fermented foods.

Description

Technical Field

[0001] This invention relates to a strain of sausage-associated lactobacillus with stable biogenic amine degradation ability and its application, belonging to the field of microbial fermentation technology. Background Technology

[0002] Biogenic amines are a class of low-molecular-weight nitrogen-containing organic compounds widely found in food, typically generated from amino acids through the decarboxylation of microbial decarboxylases. Common biogenic amines include histamine, tyramine, putrescine, and cadaverine. While the human body can metabolize them in appropriate amounts, excessive accumulation in food and subsequent ingestion can pose serious health threats. For example, histamine can cause allergy-like symptoms such as headaches, rashes, and difficulty breathing; tyramine is associated with migraines and hypertensive crises; and putrescine and cadaverine not only enhance the toxicity of other biogenic amines but also act as precursors to nitrosamines, posing a potential carcinogenic risk. Therefore, controlling the content of biogenic amines in food is a crucial aspect of ensuring food safety.

[0003] Fermented foods are highly favored for their unique flavor, but the complex microbial communities and lengthy fermentation process also make them important carriers of biogenic amines. This is especially true in traditional fermented products made primarily from legumes. Because legumes are rich in protein, they are easily broken down into free amino acids by microorganisms during fermentation, which then further undergo decarboxylation to form biogenic amines. Typical fermented legume products include fermented soybean paste, fermented black beans, soy sauce, soybean paste, and various traditional bean pastes. These products typically employ open or semi-open natural fermentation processes, have long fermentation cycles, and involve complex microbial sources. Therefore, high levels of histamine, tyramine, and other biogenic amines are frequently detected in these products, posing certain food safety risks.

[0004] Currently, industry strategies for controlling biogenic amines in fermented foods mainly include screening high-quality raw materials, optimizing fermentation process parameters, inoculating with non-amine-producing fermenting agents, and employing post-treatment methods such as physical adsorption or chemical treatment. However, these methods all have their limitations. Raw material and process control cannot completely eliminate environmental microbial contamination; existing commercial fermenting agents primarily function to inhibit the growth of miscellaneous bacteria and generally lack the ability to actively degrade already formed biogenic amines; while adsorption or chemical post-treatment may damage product flavor and quality, and are also problematic in terms of efficiency and cost. Therefore, current technology still lacks an effective solution that can efficiently and specifically degrade already formed biogenic amines within the fermentation system or in the later stages of production without affecting product flavor and quality.

[0005] Therefore, isolating and obtaining a microbial strain capable of efficiently degrading various biogenic amines is of great application value for fundamentally improving the food safety of traditional fermented soybean products such as broad bean paste, fermented black beans, and soy sauce, and is of great significance for promoting the safe and high-quality development of the traditional fermented soybean industry. Summary of the Invention

[0006] To address the aforementioned problems, this invention has screened a strain of *Lactobacillus sarcodactylis* L18 from fermented soybean paste, capable of degrading biogenic amines. This strain can simultaneously degrade eight common biogenic amines: tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. The *Lactobacillus sarcodactylis* L18 screened in this invention exhibits rapid growth, strong environmental adaptability, and stable amine-reducing performance. It can continuously exert its biogenic amine degradation effect under low to medium-high salt, acidic to weakly alkaline conditions, and common fermentation temperature conditions, making it particularly suitable for the effective control of biogenic amines such as tyramine in fermented foods.

[0007] The first objective of this invention is to provide a strain of sausage-associated lactobacillus ( Companilactobacillus farciminis L18, the sausage-associated lactobacillus strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 27, 2026, with accession number GDMCC NO: 67690.

[0008] A second objective of this invention is to provide a microbial inoculant containing the aforementioned *Lactobacillus salina* L18.

[0009] A third objective of this invention is to provide the application of the aforementioned *Lactobacillus salina* L18 or the aforementioned microbial agent in the degradation of biogenic amines.

[0010] The fourth objective of this invention is to provide a method for preparing fermented soybean paste, comprising the following steps: The fermented broad bean paste was obtained by inoculating sausage-associated lactobacillus L18 into salt-containing broad bean koji. Among them, Lactobacillus sausage-associated with sausage L18 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 27, 2026, with the accession number GDMCC NO: 67690.

[0011] In one embodiment, the inoculum size of *Lactobacillus salviae* L18 is 10. 4 ~10 6 CFU / g broad bean koji; the fermentation is static fermentation at 28~35℃ for 30~60 days, with stirring once every 0~2 days for the first 0~7 days of fermentation, and then once every 4~7 days thereafter.

[0012] In one embodiment, the method for preparing salt-containing broad bean koji is to mix broad bean koji with brine at a ratio of 1~1.25:1~1.25 (w / w) to obtain salt-containing broad bean koji, wherein the salinity of the salt-containing broad bean koji is 6~12% (w / w).

[0013] A fifth objective of this invention is to provide a fermented soybean paste prepared by any of the methods described above.

[0014] The sixth objective of this invention is to provide a method for reducing the content of biogenic amines by using *Lactobacillus salviae* L18 to decompose biogenic amines. *Lactobacillus salviae* L18 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 27, 2026, with accession number GDMCC NO: 67690.

[0015] In one embodiment, the method for reducing the biogenic amine content in fermented soybean paste includes the following steps: Lactobacillus salina L18 was used at 10 4 ~10 6 The broad bean koji was inoculated into salt-containing broad bean koji at an inoculation rate of CFU / g, and statically fermented at 28~35℃ for 30~60 days to obtain broad bean paste. During fermentation, the mixture is stirred once every 0 to 2 days for the first 0 to 7 days, and then once every 4 to 7 days thereafter.

[0016] In one embodiment, the method for preparing salt-containing broad bean koji is to mix broad bean koji with brine at a ratio of 1~1.25:1~1.25 (w / w) to obtain salt-containing broad bean koji, wherein the salinity of the salt-containing broad bean koji is 6~12% (w / w).

[0017] Beneficial effects This invention screened a strain of *Lactobacillus sarcodactylis* L18 from fermented soybean paste, which has the ability to degrade biogenic amines. This strain can simultaneously degrade eight common biogenic amines, including tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine, with a total degradation rate of 72.53%. Specifically, the degradation rates of the above-mentioned biogenic amines by *Lactobacillus sarcodactylis* L18 are as follows: 52.46% (tryptamine), 61.38% (phenylethylamine), 78.94% (putrescine), 83.57% (cadaverine), 67.25% (histamine), 90.82% (tyramine), 70.63% (speridine), and 75.19% (sperine), respectively. After optimization, the degradation rates of the above-mentioned biogenic amines by lactic acid bacteria L18 can reach: 73.56% (tryptamine), 71.26% (phenylethylamine), 81.21% (putrescine), 88.31% (cadaverine), 70.32% (histamine), 95.36% (tyramine), 70.63% (spermine), and 73.36% (spermine).

[0018] The L18 strain of *Lactobacillus sarcodactylus* selected in this invention has the characteristics of rapid growth, strong environmental adaptability and stable amine reduction performance. It can continuously exert its bioamine degradation effect under low to medium-high salt, acidic to weakly alkaline and common fermentation temperature conditions, and is particularly suitable for the effective control of bioamines such as tyramine in fermented foods.

[0019] The L18 *Lactobacillus sarcodactylis* strain screened in this invention is particularly suitable for low-sodium fermentation systems. Specifically, in an inoculated fermentation system with a salinity of 12% (w / w), the degradation rate of total biogenic amines is 22.22%; in an inoculated fermentation system with a salinity of 9% (w / w), the degradation rate of total biogenic amines is 24.95%; and in an inoculated fermentation system with a salinity of 6% (w / w), the degradation rate of total biogenic amines is significantly improved, reaching 41.60%.

[0020] Preservation of biological materials sausage-associated lactobacillus ( Companilactobacillus farciminis L18, taxonomically named: Companilactobacillus farciminis It was deposited on January 27, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67690, and the deposit address is Guangdong Institute of Microbiology Culture Collection Center, China. Attached Figure Description

[0021] Figure 1 This is the standard curve for biogenic amines.

[0022] Figure 2 Morphological observation of Lactobacillus L18 associated with sausage.

[0023] Figure 3 Phylogenetic tree of the 16S rDNA sequence of Lactobacillus salina L18.

[0024] Figure 4 The growth and acid production curves of Lactobacillus L18 associated with sausage.

[0025] Figure 5 The effect of salinity on strain growth and tyramine degradation rate.

[0026] Figure 6 The effect of pH on strain growth and tyramine degradation rate.

[0027] Figure 7 The effect of temperature on strain growth and tyramine degradation rate.

[0028] Figure 8 The effect of inoculum size on strain growth and tyramine degradation rate.

[0029] Figure 9 The effect of substrate concentration on strain growth and tyramine degradation rate.

[0030] Figure 10 The degradation capacity of Lactobacillus salina L18 for eight biogenic amines under optimal culture conditions was measured.

[0031] Figure 11 The variation of biogenic amine content in sausage-associated lactobacillus L18 fermentation systems at different salinities. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0033] Raw material source: The fermented soybean paste is commercially available Pixian fermented soybean paste.

[0034] The 100 μg / mL biogenic amine mixed standard was purchased from the Tanmo Quality Inspection Standard Material Center. The standard contained tryptamine (Try), phenylethylamine (Phe), putrescine (Put), cadaverine (Cad), histamine (His), tyramine (Tyr), spermidine (Spd), and spermine (Spm).

[0035] The preparation process of mature broad bean koji is as follows: Selected broad beans are washed, soaked for 12 hours, and then steamed at 100 ℃ for 40 minutes. After cooling to room temperature, wheat flour is mixed in at a ratio of 17% (w / w), and the mixture is placed in a temperature-controlled koji-making room (28-30 ℃) for natural solid-state fermentation for 48 hours to produce mature broad bean koji.

[0036] The culture medium formulations involved in the examples are as follows: MRS medium: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, Tween 80 1.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, agar 15.0 g / L, final pH 6.2±0.2.

[0037] Nutrient agar (NA) medium: peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, final pH 7.2±0.2.

[0038] YPD medium: yeast extract 10.0 g / L, peptone 20.0 g / L, glucose 20.0 g / L, agar 15.0 g / L (solid medium), final pH approximately 6.5 ± 0.2.

[0039] The measurement methods involved in the examples are as follows: 1. Determination of biogenic amine content After the strain culture was completed, 1 mL of culture medium was taken, and 100 μL of internal standard solution (1,7-diaminoheptane, 100 μg / mL) was added. The volume was then adjusted to 5 mL with 0.4 mol / L perchloric acid solution. The mixture was incubated at 4 ℃ for 1 h, and then centrifuged at 10000 r / min at 4 ℃ for 10 min. 1 mL of the supernatant was taken to determine the biogenic amine content. 100 μL of internal standard solution (1,7-diaminoheptane, 100 μg / mL), 200 μL of 2 mol / L sodium hydroxide solution, and 300 μL of saturated sodium bicarbonate solution were added sequentially, and the mixture was thoroughly mixed. Then, 2.0 mL of derivatization reagent (10 mg / mL dansyl chloride-acetone solution) was added, and the mixture was vortexed for 30 s. The mixture was then incubated in a 40 ℃ water bath in the dark for 45 min. After the reaction was completed, 100 μL of 25% ammonia solution was added to terminate the reaction, and the mixture was vortexed for 30 s. The mixture was then allowed to stand at room temperature for 30 min in the dark. The volume was then adjusted to 5 mL with acetonitrile, and centrifuged at 8000 g for 5 min at 4 ℃. The supernatant was filtered through a 0.22 μm organic filter membrane. The filtrate was analyzed by high-performance liquid chromatography (HPLC, Agilent 1200 Infinity, USA) using an Agilent ZORBAX Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm). The chromatographic conditions were as follows: column temperature 30 ℃, detection wavelength 254 nm, injection volume 20 μL, and flow rate 1.0 mL / min. Mobile phase A was ultrapure water, and mobile phase B was acetonitrile, using a gradient elution program (see Table 1). Simultaneously, a standard curve was prepared using mixed standard solutions of biogenic amines at different concentrations to quantify the biogenic amines. Figure 1 ).

[0040] Table 1 Gradient elution program for high performance liquid chromatography

[0041] 2. Determination of the microstructure of the strain Fresh bacterial cells were prepared into smears and their morphology was observed under an optical microscope. Scanning electron microscopy (SEM) sample preparation was as follows: the bacterial strain was inoculated into MRS medium and cultured until OD2000. 600The bacterial cells were collected by centrifugation (10000 r / min, 5 min) at a depth of approximately 1.0 μL. After washing with physiological saline 2-3 times, they were fixed with pre-cooled 2.5% (v / v) glutaraldehyde and incubated at 4 ℃ for 24 h. Subsequently, they were washed with physiological saline and dehydrated sequentially with 30%, 50%, 70%, 90%, 100%, and 100% (v / v) ethanol, each step for 10 min. After dehydration, they were replaced with isoamyl acetate for 30 min, freeze-dried, and then sputter-coated with gold. The surface structure of the bacterial cells was observed using a scanning electron microscope in 10 kV high vacuum mode.

[0042] Example 1: Isolation, Screening and Identification of Strains 1. Isolation of strains Under aseptic conditions, 10 g of fermented soybean paste sample was weighed and added to 90 mL of sterile physiological saline. The mixture was then thoroughly homogenized in a sterile homogenizer bag to obtain an initial bacterial suspension. The obtained bacterial suspension was then serially diluted tenfold with sterile physiological saline to obtain 10... -1 ~10 -6 Diluted solutions were prepared. 100 μL of each serial dilution was spread onto different selective and non-selective solid culture media. Nutrient agar (NA) was used to isolate general bacteria, MRS agar to isolate lactic acid bacteria, and yeast extract peptone glucose agar to isolate yeast. The NA and YPD plates were incubated aerobically at 30°C for 24–48 h, while the MRS plates were incubated anaerobically at 30°C for 48–72 h. After incubation, representative single colonies were picked from well-distributed plates without obvious confluence, based on colony morphology, and streaked onto corresponding fresh solid culture media for purification. This purification process was repeated 2–3 times until morphologically consistent pure cultures were obtained. The purified strains were inoculated into the corresponding liquid culture media and cultured to the logarithmic growth phase. Sterile glycerol was added to a final concentration of 20% (v / v), mixed thoroughly, aliquoted into cryovials, numbered, and stored at -80°C for long-term preservation.

[0043] 2. Screening of strains The strains obtained from the above screening were inoculated into the corresponding culture medium at an inoculum rate of 2% (v / v) and cultured until OD500. 600 Seed culture was obtained at a concentration of 0.8. The seed culture was inoculated at a rate of 2% (v / v) into liquid culture medium containing 100 μg / mL of biogenic amine mixed standards and cultured at 30 °C for 72 h; the biogenic amine content was measured after the culture was completed.

[0044] Finally, one highly efficient biogenic amine-degrading lactic acid bacteria strain L18 and 17 other isolated strains were screened, and their biogenic amine degradation effects are shown in Table 2. The results showed that lactic acid bacteria L18 exhibited the best comprehensive degradation ability for multiple biogenic amines, simultaneously degrading eight common biogenic amines: tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine, with a total degradation rate as high as 72.53%. Specifically, the degradation rates of these biogenic amines by lactic acid bacteria L18 were: 52.46% (tryptamine), 61.38% (phenylethylamine), 78.94% (putrescine), 83.57% (cadaverine), 67.25% (histamine), 90.82% (tyramine), 70.63% (speridine), and 75.19% (sperine), respectively. Its overall degradation performance was significantly better than that of the other isolated lactic acid bacteria strains.

[0045] Further analysis of the 16S rRNA sequences of all lactic acid bacteria strains and comparison with the NCBI database showed that L12 and L13 were *Pediococcus lactis* (Lactococcus). Pediococcus acidilactici L14 is Pediococcus pentosaceus ( Pediococcus pentosaceus L15 is Lactobacillus plantarum ( Lactiplantibacillus plantarum L16 is Enterococcus faecalis ( Enterococcus faecium L17 and L18 are sausage-associated lactobacilli ( Companilactobacillus farciminis ).

[0046] Comparative analysis results showed that strains from different *Lactobacillus* genera exhibited significant differences in their biogenic amine degradation capabilities. Among them, strains from... Companilactobacillus Lactobacillus L18 of the genus *Lactobacillus* exhibits significantly superior overall degradation performance against various biogenic amines compared to other strains of the genus *Lactobacillus* (including...). Pediococcus genus, Lactiplantibacillus genus, Enterococcus genus and Tetragenococcus genus); at the same time, even within the same genus ( Companilactobacillus Compared with strains of the same genus, L18 still showed a better overall degradation effect than strain L17 of the same genus, demonstrating obvious strain-specific functional advantages.

[0047] The experimental results further demonstrate that L18 lactic acid bacteria has a broad-spectrum and efficient degradation ability for various structural types of biogenic amines, especially in the removal of high-risk biogenic amines such as tyramine, cadaverine, and spermidine. This shows that it has good application potential and industrialization value in the risk control and safety regulation of biogenic amine accumulation in fermented food systems.

[0048] Table 2. Degradation rates (%) of different biogenic amines by various fermented soybean paste isolates

[0049] 3. Identification of strains (1) Colony characteristics Lactic acid bacteria L18 were streaked onto MRS solid medium and incubated at 35 ℃ for 48 h. Colony morphology characteristics were recorded.

[0050] The results are as follows Figure 2 As shown, the results indicate that *Lactobacillus salina* L18 forms white, dot-like colonies on culture plates.

[0051] (2) Cell morphology Take fresh L18 lactic acid bacteria slices and observe the morphology of the bacteria under an optical microscope.

[0052] The results are as follows Figure 2 As shown, the results indicate that, as observed by optical microscopy and scanning electron microscopy, the bacterial cells have a rod-shaped structure, with obvious septa visible in the middle of the cells, and a uniform morphology.

[0053] (3) Biological identification Single L18 bacteria were picked and inoculated into 5 mL of MRS liquid medium and cultured with shaking at 35 °C until OD reached. 600 The value was 0.8. 1 mL of bacterial culture was centrifuged at 8000 g for 5 min to collect the bacterial cells. Genomic DNA was extracted using a rapid bacterial genomic DNA extraction kit, and DNA quality was assessed by 1% agarose gel electrophoresis.

[0054] Using extracted genomic DNA as a template, the 16S rRNA gene was amplified using universal primers 27-F and 1492-R.

[0055] The PCR reaction system was 25 μL, including: 12.5 μL of 2×PCR Mix, 1.0 μL (10 μM) each of primers 27-F and 1492-R, 1.0 μL of template DNA, and sterile deionized water to make up the volume.

[0056] The PCR amplification program was as follows: pre-denaturation at 95 °C for 5 min; followed by 30 cycles, each cycle consisting of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s; and finally extension at 72 °C for 10 min.

[0057] After confirmation by 1% agarose gel electrophoresis, the PCR products were sent to a sequencing company for bidirectional sequencing. The obtained 16S rRNA gene sequence was analyzed by BLAST alignment in the NCBI GenBank database. Sequences of the target strain and its closely related reference strains were selected, and multiple sequence alignment was performed using MEGA 11.0 software. A phylogenetic tree was constructed using neighbor-joining, and the results are shown below. Figure 3 As shown.

[0058] The lactic acid bacteria L18 was further identified as *Lactobacillus sarcodactylis* (Sausage-associated Lactobacillus). Companilactobacillus farciminis The strain was named *Lactobacillus sausageensis* L18. It was deposited on January 27, 2026, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO: 67690, located at the Guangdong Institute of Microbiology, China.

[0059] The 16S rDNA sequence of *Lactobacillus salina* L18 is shown below (SEQ ID NO: 1): 4. Preparation of microbial agents Single colonies of *Lactobacillus sarcodactylis* L18 were inoculated into MRS liquid medium and cultured at 35 °C and 150 rpm for 24 h to obtain a seed culture. The seed culture was then inoculated into MRS liquid medium at a 2% (v / v) inoculation rate and cultured at 35 °C and 150 rpm for 24 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 4 °C and 8000 rpm for 10 min to collect the bacterial cells. After washing twice with sterile physiological saline, the suspension was resuspended by adding 1 / 10 of the original volume of 10% skim milk powder as a preservative and incubated at 35 °C for 1 h. The resuspended suspension was pre-frozen at -80 °C for 4 h and then freeze-dried in a vacuum freeze dryer for 24 h to obtain the bacterial agent.

[0060] Example 2: Performance determination of Lactobacillus L18 associated with sausage 1. Optimization of bioamine degradation parameters Single colonies of *Lactobacillus sarcodactylis* L18 were picked and inoculated into MRS liquid medium. The culture was carried out at 35 °C and 150 rpm for 24 h. The bacterial cells were collected by centrifugation at 8000 × g for 5 min, and washed twice with sterile physiological saline. The OD of the bacterial suspension was adjusted. 600 A bacterial suspension was obtained by diluting the solution to 0.8, which was then used for subsequent assays.

[0061] (1) Determination of growth curve The bacterial suspension was inoculated into MRS liquid medium at an inoculum rate of 2% (v / v) and cultured at 35 ℃ and 120 rpm for 72 h. OD was measured periodically. 600 The value is used to plot the growth curve of the strain.

[0062] The results are as follows Figure 4 As shown, the results indicate that the strain grows rapidly and enters the stationary phase after about 12 hours of cultivation under suitable conditions, exhibiting a strong acid-producing capacity.

[0063] (2) Effects of salinity on strain growth and biogenic amine degradation rate The bacterial suspension was inoculated at a rate of 2% (v / v) into MRS medium containing 100 μg / mL tyramine standard (which exhibits the highest degradation rate for tyramine). The concentration of NaCl in the MRS medium was varied to 0%, 3%, 6%, 9%, 12%, 15%, and 18% (w / v), and the cultures were incubated at 35 ℃ and 120 rpm for 72 h. The OD of the strains was then measured. 600 And its ability to degrade tyramine.

[0064] The results are as follows Figure 5As shown, the results indicate that this strain can maintain good tyramine degradation ability in a salinity range of 0-12% (w / v), with a degradation rate of 50.81%-93.45%.

[0065] (3) Effects of pH on strain growth and biogenic amine degradation rate The bacterial suspension was inoculated at a rate of 2% (v / v) into MRS medium containing 100 μg / mL tyramine standard. The pH of the MRS medium was changed to 3, 4, 5, 6, and 7, respectively, and the cultures were incubated at 35 ℃ and 120 rpm for 72 h. The OD of the strains was then measured. 600 And its ability to degrade tyramine.

[0066] The results are as follows Figure 6 As shown, the results indicate that when the pH of the system is in the range of 4 to 9, the degradation ability of tyramine remains relatively stable, with a degradation rate of 71.24% to 92.16%.

[0067] (4) Effects of temperature on strain growth and biogenic amine degradation rate The bacterial suspension was inoculated at a rate of 2% (v / v) into MRS medium containing 100 μg / mL tyramine biogenic amine standard, and cultured at 25, 30, 35, 40, and 45°C at 150 rpm for 72 h. The OD of the strain was then measured. 600 And its ability to degrade tyramine.

[0068] The results are as follows Figure 7 As shown, the results indicate that the optimal temperature range for the growth and tyramine degradation of this strain is 30–40 °C.

[0069] (5) Effect of inoculum size on strain growth and biogenic amine degradation rate The bacterial suspensions were inoculated into MRS medium containing 100 μg / mL tyramine biogenic amine standard at inoculum values ​​of 1%, 3%, 5%, 7%, and 9% (v / v), respectively, and cultured at 35℃ and 150 rpm for 72 h. The OD of the strains was then measured. 600 And its ability to degrade tyramine.

[0070] The results are as follows Figure 8 As shown, the results indicate that the inoculum amount has little effect on the tyramine degradation ability of this strain, suggesting that it has good stability and applicability under different fermentation conditions.

[0071] (6) Effects of substrate concentration on strain growth and biogenic amine degradation rate The bacterial suspensions were inoculated at 2% (v / v) into MRS medium containing 25, 50, 75, 100, 150, and 300 μg / mL tyramine standard, respectively, and cultured at 35 ℃ and 150 rpm for 72 h. The OD of the strains was then measured.600 And its ability to degrade tyramine.

[0072] The results are as follows Figure 9 As shown, the results indicate that the degradation rate of tyramine increases significantly as the initial concentration of the substrate tyramine gradually increases in the range of 25–100 μg / mL, and the degradation rate tends to stabilize when the concentration of tyramine exceeds 100 μg / mL.

[0073] 2. Bioamine degradation performance test The bacterial suspension was inoculated at a rate of 3% (v / v) into MRS medium containing 100 μg / mL of eight biogenic amine standards (tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) (pH 7, NaCl concentration 0% (w / v)). The culture was incubated at 35 ℃ and 150 rpm for 72 h, and the OD of the strain was measured. 600 And the ability to degrade biogenic amines.

[0074] The results are as follows Figure 10 As shown, the results indicate that under optimal conditions, *Lactobacillus sarcodactylis* L18 can simultaneously degrade eight common biogenic amines: tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. Specifically, the degradation rates of these biogenic amines by *Lactobacillus sarcodactylis* L18 can reach 73.56% (tryptamine), 71.26% (phenylethylamine), 81.21% (putrescine), 88.31% (cadaverine), 70.32% (histamine), 95.36% (tyramine), 70.63% (speridine), and 73.36% (spermine), respectively.

[0075] In summary, the strain exhibits rapid growth, strong environmental adaptability, and stable amine-reducing performance. It can continuously exert its biogenic amine degradation effect under low to medium-high salt, acidic to weakly alkaline, and common fermentation temperature conditions, making it particularly suitable for the effective control of biogenic amines such as tyramine in fermented foods.

[0076] Example 3: Application of bacterial strains in degrading biogenic amines in fermented soybean paste 1. Raw material pretreatment Mature broad bean koji and sterile brine were thoroughly mixed at a ratio of 1:1 (w / w), and the total mass of a single fermentation system was controlled to be 4 kg. The final salinity of the system was adjusted to be 6%, 9% and 12% (w / w, of which 12% is the traditional fermentation salinity) by adjusting the brine concentration. After the materials were mixed evenly, they were dispensed into sterile sealed fermentation bottles to obtain salted broad bean koji.

[0077] Single colonies of *Lactobacillus sarcodactylis* L18 were picked and inoculated into MRS liquid medium. The culture was carried out at 30 °C and 150 rpm for 24 h. The bacterial cells were collected by centrifugation at 8000 × g for 5 min, and washed twice with sterile physiological saline. The concentration of the bacterial suspension was adjusted to 10. 4~10 6 CFU / mL.

[0078] (2) Fermentation by strain The suspension of Lactobacillus L18, a bacteria associated with sausage, was prepared at 1×10⁻⁶. 5 The inoculum of broad bean koji was inoculated into salt-containing broad bean koji at different salinities, and static fermentation was carried out at 30℃ for 50 days. For the first 7 days of fermentation, the mixture was stirred thoroughly once daily to promote uniform cell distribution, and then once weekly thereafter. After fermentation (50 days), each fermentation system was thoroughly mixed and sampled to determine the biogenic amine content.

[0079] The results are as follows Figure 11 As shown, the results indicate that this strain can effectively reduce the content of biogenic amines in the fermentation system under different salinity conditions. Compared with the naturally fermented sample without inoculation, in the inoculated fermentation system with a salinity of 12% (w / w), the degradation rate of tyramine was 50.69%, and the degradation rate of total biogenic amines was 22.22%; in the inoculated fermentation system with a salinity of 9% (w / w), the degradation rate of tyramine was 45.02%, and the degradation rate of total biogenic amines was 24.95%; while in the inoculated fermentation system with a salinity of 6% (w / w), the degradation rate of tyramine was 65.06%, and the degradation rate of total biogenic amines was significantly improved, reaching 41.60%.

[0080] Further analysis of the changes in different biogenic amine components revealed that, under 6% salinity conditions, Lactobacillus salina L18 exhibited a more significant degradation effect on biogenic amines with higher toxicity risks, with degradation rates of 60.43%, 65.06%, and 22.98% for histamine, tyramine, and phenylethylamine, respectively.

[0081] The above results indicate that Lactobacillus salivae L18, associated with sausages, is particularly suitable for low-salt or reduced-salt fermentation systems for fermented broad bean paste. It can significantly reduce the accumulation level of biogenic amines while ensuring smooth fermentation, thereby improving product safety.

[0082] Using the L18 sausage-associated lactobacillus described in this invention for inoculation and fermentation can effectively reduce the total content of biogenic amines in fermented soybean paste under different salinity conditions, and has a more significant degradation effect on highly toxic biogenic amines under low salinity conditions, making it particularly suitable for the safe production of reduced-salt fermented soybean paste and similar fermented foods.

[0083] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of sausage-associated lactobacillus ( Companilactobacillus farciminis L18, characterized in that, The *Lactobacillus* strain associated with sausage was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 27, 2026, with accession number GDMCC NO: 67690.

2. A microbial inoculant, characterized in that, The microbial agent contains the sausage-associated lactobacillus L18 as described in claim 1.

3. The application of the sausage-associated lactobacillus L18 as described in claim 1 or the microbial agent as described in claim 2 in the degradation of biogenic amines.

4. A method for preparing fermented soybean paste, characterized in that, Including the following steps: The fermented broad bean paste was obtained by inoculating sausage-associated lactobacillus L18 into salt-containing broad bean koji. Among them, Lactobacillus sausage-associated with sausage L18 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 27, 2026, with the accession number GDMCC NO: 67690.

5. The method according to claim 4, characterized in that, The inoculum size for *Lactobacillus salina* L18 in sausages was 10. 4 ~10 6 CFU / g broad bean koji; the fermentation is static fermentation at 28~35℃ for 30~60 days, with stirring once every 0~2 days for the first 0~7 days of fermentation, and then once every 4~7 days thereafter.

6. The method according to claim 4, characterized in that, The method for preparing salt-containing broad bean koji is to mix broad bean koji with brine at a ratio of 1~1.25:1~1.25 (w / w) to obtain salt-containing broad bean koji, wherein the salinity of the salt-containing broad bean koji is 6~12% (w / w).

7. The fermented soybean paste prepared by any one of claims 4 to 6.

8. A method for reducing the content of biogenic amines, characterized in that, The Lactobacillus salinae L18 was used to decompose biogenic amines. The Lactobacillus salinae L18 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 27, 2026, with accession number GDMCC NO: 67690.

9. The method according to claim 8, characterized in that, The method described above reduces the content of biogenic amines in fermented soybean paste, and includes the following steps: Lactobacillus salina L18 was used at 10 4 ~10 6 The broad bean koji was inoculated into salt-containing broad bean koji at an inoculation rate of CFU / g, and statically fermented at 28~35 ℃ for 30~60 days to obtain broad bean paste; During fermentation, the mixture is stirred once every 0 to 2 days for the first 0 to 7 days, and then once every 4 to 7 days thereafter.

10. The method according to claim 8, characterized in that, The method for preparing salt-containing broad bean koji is to mix broad bean koji with brine at a ratio of 1~1.25:1~1.25 (w / w) to obtain salt-containing broad bean koji, wherein the salinity of the salt-containing broad bean koji is 6~12% (w / w).

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