Fermented lactobacillus mucus capable of degrading mycotoxin as well as composition and application of fermented lactobacillus mucus

Abstract

The invention discloses lactobacillus mucus capable of degrading mycotoxin and a composition and application of the lactobacillus mucus, and belongs to the technical field of microorganisms, the lactobacillus mucus FS95 is preserved in the China Center for Type Culture Collection on December 22, 2025, the preservation number is CCTCC NO: M20252983, and the lactobacillus mucus can degrade vomitoxin and / or aflatoxin. The invention also discloses a lactobacillus mucilaginosus FS95 fermentation composition and an application of the lactobacillus mucilaginosus FS95 fermentation composition in preparation of fermented food. The lactobacillus mucus FS95 is high in growth performance, good in heat resistance and acid production performance and fast in acid production; the strain has the advantages of high efficiency in degradation of aflatoxin, vomitoxin and nitrite, good water activity resistance, great application prospect in degradation of fungaltoxin, and great application prospect in preparation of fermented food.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a fermenting Lactobacillus that can degrade fungal toxins, its composition, and its application. Background Technology

[0002] Aflatoxins are a class of secondary metabolites mainly produced by fungi such as *Aspergillus flavus* and *Aspergillus parasiticus*. Structurally, these toxins are structural analogs composed of difuran rings and coumarin. Aflatoxins are highly teratogenic, carcinogenic, and mutagenic, and widely contaminate agricultural products and foods such as peanuts, corn, cottonseed, rice, dried fruits, and milk. More than twenty types of aflatoxins have been identified, among which aflatoxin B1 (AFB1) has the widest distribution and is the most toxic. In 1993, the International Agency for Research on Cancer (IARC) of the World Health Organization classified AFB1 as a Group 1 carcinogen.

[0003] Currently, the commonly used methods for removing AFB1 are mainly physical and chemical methods. Physical methods include sorting, rinsing, high-temperature heating, radiation, and solvent extraction. These methods are either labor-intensive and inefficient, or they may damage the nutritional components of agricultural products. Chemical methods utilize oxidants, sodium hydroxide, and other chemical reagents to react with the toxins, thereby reducing their toxicity. However, these methods have significant limitations. Many reagents can harm the skin, eyes, and respiratory tract of operators, and chemical residues are difficult to remove, affecting the quality and safety of agricultural products and food.

[0004] Vomitoxin, also known as deoxynivalenol, is a fungal toxin. It is primarily produced by fungi of the genus *Fusarium*. It is widely found in various grains and their products, such as wheat, corn, and rice. Vomitoxin can be produced when grains encounter suitable conditions for fungal growth and reproduction during growth, harvesting, storage, or processing. Ingesting food containing vomitoxin may cause gastrointestinal symptoms such as nausea, vomiting, abdominal pain, and diarrhea. In severe cases, it can even lead to immune system disorders, growth retardation, and other problems, posing various health risks.

[0005] Microbial detoxification has become a research hotspot in recent years. This method mainly uses microorganisms such as bacteria and fungi and their metabolites to remove toxins such as AFB1 from food. This detoxification method has the advantages of being non-polluting to raw materials, highly specific, avoiding the regeneration of toxins, having mild degradation conditions, strong specificity, and high detoxification efficiency. Therefore, it is an efficient and safe detoxification method.

[0006] Different microorganisms exhibit significantly different detoxification efficiencies. Many microorganisms can efficiently remove AFB1, such as the strain disclosed in CN110042072A that degrades aflatoxin B1 (CGMCC No. 17142, biological material: LH-F001; classification name: Rummeliibacillus stabekisii), with a flavourinum degradation rate exceeding 90%. However, this Rummeliibacillus is not a strain that can be added to food. Therefore, developing strains capable of degrading aflatoxin and vomitoxin for use in foods such as fermented bean paste is of great significance. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a strain of Lactobacillus fermentans FS95 that can degrade fungal toxins, as well as new applications of fermentation compositions of Lactobacillus fermentans FS95, specifically in the degradation of aflatoxin and / or vomitoxin, and especially in the application of Lactobacillus fermentans FS95 strain and its metabolites in the preparation of fermented foods.

[0008] One of the technical solutions adopted by this invention to solve its technical problem is:

[0009] A strain of *Limosilactobacillus fermentum* capable of degrading mycotoxins, wherein *Limosilactobacillus fermentum* FS95 was deposited at the China Center for Type Culture Collection on December 22, 2025, with accession number CCTCC NO: M20252983, and the mycotoxins that *Limosilactobacillus fermentum* can degrade include vomitoxin and / or aflatoxin.

[0010] Limosilactobacillus fermentum, also known as fermenting lactobacillus, has heterofermentation characteristics, strong acid production capacity, strong salt tolerance, strong growth capacity, and high acid resistance. It also has high nitrite degradation performance. In 2011, it was included in my country's "List of Microbial Strains that Can Be Used in Food". The Limosilactobacillus fermentum FS95 screened by the applicant of this invention can efficiently degrade aflatoxin and vomitoxin, with a degradation rate of up to 47.47% for aflatoxin and 89.39% for vomitoxin.

[0011] Limosilactobacillus fermentum FS95 was isolated from fermented broad beans from Pixian County, Sichuan Province. The purified strain was identified by 16S rRNA, and the 16S rRNA sequence is shown in SEQ ID NO:1. The 16S rRNA sequence was then compared using NCBI BLAST, and the similarity to Limosilactobacillus fermentum in Genebank was greater than 99%, confirming that the strain was Limosilactobacillus fermentum FS95. Based on the 16S rRNA gene sequence alignment results, a Neighbor-Joining phylogenetic tree was constructed with Carnobacterium maltaromaticum ATCC 27865 (JF749288) as an outbranch, and the strain was named Limosilactobacillus fermentum FS95.

[0012] The colonies formed by fermenting Lactobacillus mucinus FS95 of this invention on MRS medium are smooth, with complete edges, milky white color, shiny appearance and soft texture, and appear rod-shaped under a microscope; the suitable growth temperature is 37-39℃ and the growth pH is 3.0-7.0.

[0013] One of the technical solutions adopted by the present invention to further solve its technical problem is:

[0014] A fermentation composition is prepared by fermenting *Lactobacillus FS95*, a fungicide-degrading bacteria.

[0015] The fermentation composition includes live Lactobacillus FS95, inactivated bacterial cells, fermentation broth supernatant, fermentation precipitate, or spray-dried / lyophilized powder; the fermentation composition is prepared by fermenting Lactobacillus FS95 in MRS medium.

[0016] Preferably, the fermentation supernatant of *Lactobacillus myxoides* FS95 is prepared by fermenting *Lactobacillus myxoides* FS95 in MRS medium at 37°C for 24 hours, activating it twice, transferring it to fresh MRS medium at a 2% inoculum, culturing it under the same conditions for 12 hours, collecting the bacterial cells by centrifugation, and then inoculating it into MRS medium at a 2% inoculum again, culturing it at 37°C for 16-24 hours, collecting the fermentation supernatant by low-temperature centrifugation (10000 r / min, 10 min, 4°C), and then inactivating the supernatant by high-temperature inactivation (121°C, 15 min), concentration, and spray drying.

[0017] Another technical solution adopted by the present invention to further solve its technical problem is:

[0018] Probiotic preparations, including the aforementioned *Lactobacillus mucinus* FS95 or fermentation composition that can degrade mycotoxins, wherein the content of *Lactobacillus mucinus* FS95 in the probiotic preparation is ≥2×10⁻⁶. 7 CFU / g, or, the content of *Lactobacillus fermentum* FS95 in the probiotic preparation is ≥2×10⁻⁶. 7 CFU / ml.

[0019] Another technical solution adopted by the present invention to further solve its technical problem is:

[0020] Application of fermented Lactobacillus mucinus FS95 in the degradation of aflatoxin and / or vomitoxin.

[0021] Application of fermentation composition of Lactobacillus myxobolus FS95 in the preparation of fermented foods.

[0022] The fermented foods include broad bean paste or broad bean sauce, chili sauce, fermented milk, pickled vegetables, fermented black beans, bean sprouts, soybean paste, fish sauce, pickled fish, cured meat, and ham.

[0023] Another technical solution adopted by the present invention to further solve its technical problem is:

[0024] A type of fermented soybean paste, prepared by fermentation with the aforementioned Lactobacillus mucilaginosus.

[0025] The method for preparing the fermented soybean paste includes the following steps:

[0026] S1. Chili mash production: Fresh red chilies are selected, cleaned, crushed, mixed with edible salt, and placed in a chili mash fermentation tank for preservation and sealing to make chili mash. The edible salt content of the chili mash is (20±2)%, (W / W).

[0027] S2. Broad Bean Fermentation: Broad beans are shelled, impurities removed, soaked, and mixed with wheat flour and soybean meal. Following process requirements, Aspergillus oryzae (Hu Niang 3.042) is inoculated and cultured to produce fermented broad beans. Then, the above-mentioned fermented Lactobacillus FS95 bacterial powder (live count ≥1×10⁻⁶) is added. 9 Dissolve CFU / g in 15-17% saline solution at a specified mass ratio (e.g., 1-2:1000) (i.e., final concentration of Lactobacillus fermentum FS95 ≥ 1 × 10⁻⁶ CFU / g). 6 CFU / ml), the chopped petals are mixed with the inoculated brine (the water content of the mixture is 30-40%), and then placed in a tank for incubation at 35±5℃. The fermentation cycle is 35-50 days to produce mature petals.

[0028] S3. Post-fermentation: By weight ratio, add 60-80% chili mash and 20-40% cooked chili peppers to the fermentation tank and stir evenly. Stir for 30 minutes every 7 days. After more than 3 months of natural post-fermentation, Pixian Doubanjiang (Pixian chili bean paste) is produced, which is red and oily, mellow in taste and rich in soy sauce aroma.

[0029] Preferably, the viable count of the fermented Lactobacillus mucinus FS95 bacterial powder is ≥1×10⁻⁶. 9 CFU / g, wherein the mass ratio of the fermented Lactobacillus mucinus FS95 bacterial powder to brine is 1~2:1000.

[0030] The beneficial effects of the fermented Lactobacillus mucilaginosus FS95 of this invention, which can degrade mycotoxins, are as follows:

[0031] The present invention relates to a fermentation-promoting *Lactobacillus mucilaginosus* FS95 strain that can degrade mycotoxins, exhibiting strong growth performance and a viable cell count as high as 2.03 × 10⁻⁶. ^9 CFU / mL; it can grow well at 37-50℃, has good acid production performance and fast acid production; it has strong degradation of aflatoxin and nitrite, and good water activity resistance. It has great application prospects in aflatoxin degradation and great application prospects in the preparation of fermented foods.

[0032] After 16 hours of fermentation, the acid content reaches as high as 0.82g / 100ml. It has the advantages of rapid acid production, an acid production rate of 0.153 pH / h, and sustained acid production, making it an excellent fermentation agent.

[0033] The fermentation strain of Lactobacillus mucinus FS95 exhibits superior salt tolerance compared to other strains under culture conditions of 8% (MRS) and 10% (MRS) salt concentrations. After 16 hours of culture in 8% sodium chloride (MRS) at 37℃, the OD value is 0.603, demonstrating extremely strong salt tolerance.

[0034] Fermented Lactobacillus FS95 can efficiently degrade aflatoxin on MRS medium, with a degradation rate as high as 47.47%; the degradation rate of vomitoxin is as high as 89.39%; and the degradation rate of nitrite is as high as 93.75%.

[0035] Lactobacillus fermentans FS95 can tolerate a growth environment with a water activity of 0.940 and can be used in the production of fermented foods such as broad beans.

[0036] Experiments have verified that the fermented Lactobacillus FS95 or the composition of fermented Lactobacillus FS95 of the present invention can efficiently degrade vomitoxin, aflatoxin and nitrite. Therefore, fermented Lactobacillus FS95 has broad application prospects in the preparation of fermented foods such as broad bean paste.

[0037] Preservation of biological materials

[0038] Limosilactobacillus fermentum FS95 was deposited at the China Center for Type Culture Collection (CCTCC) on December 22, 2025, with accession number CCTCC NO:M20252983, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0039] Figure 1 —This is a colony diagram of the fermenting Lactobacillus mucinus FS95, which is capable of degrading mycotoxins according to the present invention;

[0040] Figure 2 —A microscopic morphological image of the fermenting Lactobacillus mucilaginosus FS95, which is capable of degrading fungal toxins according to the present invention;

[0041] Figure 3 —A phylogenetic tree of the fermenting Lactobacillus mucilaginosus FS95, which can degrade mycotoxins according to the present invention;

[0042] Figure 4 —This is a growth curve of the fermented Lactobacillus mucilaginosus FS95, which is capable of degrading mycotoxins according to the present invention;

[0043] Figure 5 —This is a diagram showing the acid production of the fermenting Lactobacillus mucilaginosus FS95, which is capable of degrading mycotoxins according to the present invention;

[0044] Figure 6 —A standard curve diagram for the determination of aflatoxin content using the aflatoxin kit of the present invention;

[0045] Figure 7 —This is a standard curve diagram for the determination of vomitoxin content in this invention;

[0046] Figure 8 —This is a standard curve diagram for the determination of nitrite content in this invention. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0048] The following are the culture media involved in the embodiments of the present invention:

[0049] MRS medium (g / L): peptone 10.0, beef meal 5.0, yeast powder 4.0, glucose 20.0, Tween 80 1.0, K2HPO4·7H2O 2.0, anhydrous sodium acetate 5.0, triammonium citrate 2.0, MgSO4·7H2O 0.2, MnSO4·H2O 0.05, agar pH 6.2±0.2 (with 15g / L agar powder for solid medium).

[0050] Physiological saline (g / L): 0.85 NaCl, dissolved in 1 L of water.

[0051] After preparing the above reagents, place them in an autoclave and sterilize at 121℃ for 20 minutes.

[0052] Example 1

[0053] Screening and identification of Lactobacillus fermentans FS95

[0054] Limosilactobacillus fermentum FS95 was deposited at the China Center for Type Culture Collection on December 22, 2025, with accession number CCTCC NO:M20252983.

[0055] 1) Screening of Lactobacillus fermentans FS95 strain

[0056] Samples were collected from fermented broad beans grown in Pixian County, Sichuan Province. 1g of the collected sample was weighed and placed in 9mL of sterile physiological saline. After thorough mixing by shaking, the sample was serially diluted 10-fold and spread onto MRS solid medium. It was then strictly anaerobic incubated at 37℃ for 48 hours. Single colonies of different shapes and sizes were visually observed and streaked for purification at least four times. Microscopic observation and the calcium dissolution method preliminarily identified the bacteria as lactic acid bacteria. The purified strain was stored in 45% glycerol at -80℃ for future use.

[0057] 2) Strain morphology and microscopic observation

[0058] See the colony morphology of Lactobacillus fermentans FS95 strain. Figure 1 The colonies are smooth with intact edges, milky white in color, shiny, and have a soft texture. See [microscopic morphology description] for details. Figure 2 The fermenting Lactobacillus mucinus FS95 is rod-shaped.

[0059] 3) Molecular biological identification of strains

[0060] The purified *Limosilactobacillus fermentum* FS95 strain was identified by 16S rRNA, and the 16S rRNA sequence of this strain is shown in SEQ ID NO: 1. The obtained 16S rRNA sequence was aligned using NCBIBLAST, and the similarity to *Limosilactobacillus fermentum* in Genebank was greater than 99%, confirming that this strain is *Limosilactobacillus fermentum* FS95. Based on the 16S rRNA gene sequence alignment results, a Neighbor-Joining phylogenetic tree was constructed with *Carnobacterium maltaromaticum* ATCC 27865 (JF749288) as an outbranch. The phylogenetic tree is shown below. Figure 3 As shown, it is named Limosilactobacillus fermentum FS95.

[0061] 4) Determination of growth performance and acid production performance

[0062] The *Lactobacillus fermentum* FS95 strain was inoculated into MRS liquid medium at a 2% inoculum and incubated at 37°C. OD values ​​were measured at 2, 4, 6, 8, 10, 12, and 16 hours. 600 and pH value.

[0063] Table 1. Growth performance test of Lactobacillus FS95 strain during fermentation.

[0064]

[0065] From Table 1 and Figure 4 It can be seen that the OD of fermented Lactobacillus FS95 after 16 hours of culture was [missing information]. 600 The pH reached 6.24, with a short fermentation time, strong environmental adaptability, and vigorous vitality, exhibiting strong growth performance; after fermentation of *Lactobacillus mucilaginosus* FS95 in MRS liquid medium at 37℃ for 16 hours, the pH reached 3.76 (e.g., ...). Figure 5 As shown in the figure, the acid production rate (ΔpH / Δt) is 0.153 pH / h, which means that the fermentation of Lactobacillus mucilaginosus FS95 has good acid production performance and a relatively fast acid production rate.

[0066] 5) Determination of the heat resistance of the strain

[0067] The *Lactobacillus fermentum* FS95 strain was inoculated into MRS liquid medium at a 2% inoculum size and cultured at 37℃, 40℃, 45℃, and 50℃ for 16 h to determine the OD. 600 and pH value.

[0068] Table 2. Heat resistance test of Lactobacillus fermentans FS95 strain

[0069]

[0070] When fermenting broad beans in tanks or jars, the local temperature inside the tank can reach up to 50℃ in summer due to solid-state heat conduction. Therefore, the fermentation inoculant needs to have good temperature tolerance. As shown in Table 2, *Lactobacillus mucilaginosus* FS95 grows and produces acid well at 37-50℃. The OD of *Lactobacillus mucilaginosus* FS95 cultured at 50℃ for 16 hours was... 600 A value of 4.75 indicates that Lactobacillus FS95 is suitable for fermenting fermented soybean paste.

[0071] 6) Determination of salt tolerance of strains

[0072] The *Lactobacillus fermentans* FS95 strain, which exhibits superior growth performance, was activated in MRS medium and then inoculated at a rate of 2% into MRS liquid medium containing 8% and 10% sodium chloride. After incubation at 37°C for 16 hours, the OD and pH values ​​of the fermentation broth were measured. Three replicates were prepared for each group, with MRS liquid medium without added sodium chloride serving as a control.

[0073] The salt tolerance of *Lactobacillus fermentans* FS95 is shown in Table 3. *Lactobacillus fermentans* FS95 exhibited good salt tolerance under culture conditions of 8% (MRS) and 10% (MRS). Based on the initial salt tolerance OD value (2% inoculum) of 0.125, after standing for 16 hours under 8% and 10% salt concentrations, the OD values ​​were 0.603 and 0.435, respectively, indicating that *Lactobacillus fermentans* FS95 can tolerate a 10% sodium chloride culture environment. According to the pH results, the acid production performance of *Lactobacillus fermentans* FS95 under 8% (MRS) and 10% (MRS) salt concentrations was comparable to that in conventional MRS medium.

[0074] Table 3 Salt tolerance test of Lactobacillus fermentans FS95

[0075]

[0076] 7) Determination of acid resistance of strains

[0077] After activating Lactobacillus fermentum FS95 in MRS medium, it was inoculated at a rate of 10% into sterile physiological saline with lactic acid concentrations of 0.8% and 1.0%. After being incubated at 37°C for 2 hours, the OD and pH values ​​of the fermentation broth were measured.

[0078] Table 4. Acid resistance test of Lactobacillus FS95 during fermentation.

[0079]

[0080] Based on the initial lactic acid tolerant OD value (at an inoculum of 10%) of 0.624, Table 4 shows that the OD values ​​of *Lactobacillus fermentatus* FS95 under culture conditions with lactic acid concentrations of 0.80% and 1.00% were 0.648 and 0.644, respectively. These values ​​showed almost no change compared to the initial lactic acid tolerant OD value. Therefore, *Lactobacillus fermentatus* FS95 exhibits good lactic acid tolerance under culture conditions with lactic acid concentrations of 0.80% (MRS) and 1.00% (MRS), meaning it can tolerate a culture environment with a lactic acid concentration of 1.00%. Based on the pH results, *Lactobacillus fermentatus* FS95 also showed good acid production performance under culture conditions with lactic acid concentrations of 0.80% (MRS) and 1.00% (MRS).

[0081] 8) Determination of the aflatoxin degradation performance of the strain

[0082] After activating Lactobacillus fermentum FS95 in MRS medium, based on previous viable cell count data, the culture was divided into 10... 8 The inoculum was inoculated into MRS liquid medium containing AFB1 standard (AFB1 added amount was 10 mg / L, the actual concentration was subject to the final determination), and cultured in a shaker at 37℃ for 72 h. The aflatoxin content in the fermentation broth was determined (AFB1 absorbance was detected at 450 nm using an enzyme-linked immunosorbent assay reader), with 3 parallel samples.

[0083] The standard curve obtained from the aflatoxin assay kit for aflatoxin content determination is shown below. Figure 6 As shown, the standard curve meets the experimental standard (R²). 2 >0.98), which can be used to detect aflatoxin (AFB1) antagonism in fermenting Lactobacillus mucilaginosus FS95.

[0084] Table 5. Aflatoxin degradation performance test of Lactobacillus mucilaginosus FS95

[0085]

[0086] According to the results of Lactobacillus fermentum FS95 antagonizing AFB1 shown in Table 5, Lactobacillus fermentum FS95 has good antagonistic performance against aflatoxin, and the degradation rate of aflatoxin (AFB1) by the strain is 47.47%.

[0087] According to the requirements of GB 2761-2017 National Food Safety Standard - Limits of Mycotoxins in Food, "The microbial limit of aflatoxin B1 in fermented soy products shall not exceed 5.0 μg / Kg." As shown in Table 5, in a culture system containing 10.6509 mg / AFB1, the degradation rate of aflatoxin AFB1 can reach up to 47.47% (reaction time of 72 h), which can degrade 5.0559 mg of AFB1.

[0088] 9) Determination of the antagonistic performance of the strain against vomitoxin

[0089] After activating Lactobacillus fermentum FS95 in MRS medium, based on previous viable cell count data, the culture was divided into 10... 8 The inoculum was inoculated into MRS liquid medium containing vomitoxin (DON) standard (vomitoxin added at 100 µg / L, the actual concentration is subject to final determination), and cultured in a shaker at 37℃ for 72 h. The vomitoxin content in the fermentation broth was then determined (the absorbance of AFB1 was measured at 450 nm using an ELISA reader).

[0090] The standard curve was constructed using the direct competition ELISA kit method. The absorbance at 0 µg / L was designated B0, and the absorbance at other concentrations was designated B. The logarithm of the reference concentration was plotted on the x-axis, and the absorbance ratio (B / B0) was plotted on the y-axis. Figure 7 As shown.

[0091] The test standard curve meets the experimental standard (R). 2 >0.99), which can be used to detect the antagonistic effect of fermenting Lactobacillus mucin FS95 on vomitoxin.

[0092] Table 6. Degradation performance test of vomitoxin by fermented Lactobacillus mucinus FS95

[0093]

[0094] According to the results of Lactobacillus fermentum FS95 antagonizing vomitoxin shown in Table 6, Lactobacillus fermentum FS95 has excellent anti-vomitoxin performance, and the degradation rate of vomitoxin by the strain is 89.39%.

[0095] 9) Determination of the strain's ability to degrade nitrite

[0096] After activating Lactobacillus fermentum FS95 in MRS medium, it was inoculated at a rate of 5% into MRS liquid medium with a nitrite content of 30 mg / L. After incubation at 37℃ for 12 h, the nitrite content in the fermentation broth was determined according to GB5009.33-2016.

[0097] The standard curve determined by the nitrite content detection kit is as follows: Figure 8 As shown.

[0098] Table 7. Performance test of fermented Lactobacillus mucilaginosus FS95 in degrading nitrite.

[0099]

[0100] As shown in Table 7, the fermentation of Lactobacillus mucilaginosus FS95 exhibits excellent performance in degrading nitrite, with a degradation rate as high as 93.75% (30 mg / L).

[0101] 9) Determination of the water activity of the strain

[0102] Water activity, the ratio of the vapor pressure of free water in food to the vapor pressure of pure water, is a key environmental factor for microbial growth. Water activity directly affects cell membrane integrity, enzyme activity, and the efficiency of metabolite production. In the fermentation environment of fermented soybeans, high salt content leads to decreased water activity, creating osmotic stress on lactic acid bacteria and affecting their survival and fermentation efficiency. Understanding the water activity tolerance of lactic acid bacteria is crucial for screening for suitable bacteria for fermentation. Since salt content affects the moisture content of the fermentation environment, using sodium chloride as a water activity regulator is simple to operate, inexpensive, and suitable for practical applications, making it a suitable choice for preparing culture media with water activity tolerance.

[0103] According to the draft of the "Guiding Principles for the Application of Water Activity in Microbial Control of Non-sterile Drugs (First Public Notice)," the water activity of saturated sodium chloride (26.5%) is 0.753 ± 0.001. The relationship between sodium chloride concentration and water content is shown in Table 8.

[0104] Table 8. Relationship between sodium chloride and water activity

[0105]

[0106] Table 8 shows that sodium chloride concentration and moisture content are negatively correlated. Fermented Lactobacillus FS95 can tolerate a culture environment with 10% sodium chloride, at which point the tolerable water activity is 0.940.

[0107] According to publicly available data in the literature, the water activity of finished fermented broad bean paste ranges from 0.749 to 0.803. During the production process, the water activity decreases with increasing salt content. In the koji-making stage, the water activity is 0.950–0.970, which is suitable for mold growth. In the early fermentation stage, the water activity is 0.900–0.930, a stage dominated by lactic acid bacteria. In the later fermentation stage, the water activity drops to 0.850–0.880, eventually reaching 0.749–0.803 in the finished product. This indicates a significant variation in moisture content between the koji-making stage and the early fermentation stage. This is the critical water activity range for fermented broad bean paste (0.930–0.950), within which lactic acid bacteria play a major role. Therefore, the most critical point for assessing the strain's tolerance to water activity is 0.940. As mentioned earlier, *Lactobacillus mucilaginosus* FS95 can tolerate a water activity of 0.940.

[0108] In summary, *Lactobacillus fermentum* FS95 exhibits strong environmental adaptability and vigorous vitality, along with excellent heat resistance. It also demonstrates good salt and acid resistance, enabling it to efficiently degrade aflatoxin, vomitoxin, and nitrite. Furthermore, its good water activity tolerance allows it to adapt to the water activity environment of fermented products such as fermented soybean paste. This demonstrates its growth characteristics during the production of fermented soybean paste and its ability to control and degrade aflatoxin, vomitoxin, and nitrite during the production process, thereby improving the quality and food safety of fermented soybean paste.

[0109] Therefore, the applicant proposes the application of Lactobacillus fermentans FS95 in the degradation of fungal toxins, especially in the preparation of fermented foods such as broad bean paste and chili sauce.

[0110] Example 2

[0111] A fermentation composition, obtained by fermenting *Lactobacillus fermentatus* FS95 as described in Example 1, comprises live *Lactobacillus fermentatus* FS95 bacterial powder; the live *Lactobacillus fermentatus* FS95 bacterial powder is obtained by centrifuging the fermentation broth obtained after centrifuging, precipitating, and freeze-drying the precipitate, wherein the live count of *Lactobacillus fermentatus* FS95 is 1.0 × 10⁻⁶. 9 CFU / g.

[0112] When a bottle of fermented soybean paste (250g) that has passed the AFB1 test is added with an ingredient that can antagonize AFB1 (the amount added is 2.5±1.5×10), ^8Theoretically, the highest AFB1 content in fermented soybean paste is 1.25 μg (the actual concentration should be lower than this). The initial AFB1 concentration in fermented soybean paste is 1.25 μg / 250g = 0.005 μg / g = 5 μg kg. If the bacterial strain (with a cell addition of 1 × 10⁻⁶) is used... 8 If the degradation rate of AFB1 (CFU / g) is 47.47% to evaluate the strain performance, then after a period of time (72h), the AFB1 content in the system should be 1.25-1.25×47.47%=0.6566μg. That is, the concentration of AFB1 in the fermented soybean paste after fermentation is 0.6566ug / 250g=2.6265mgKg≈2.6265μg / L. Trace amounts of AFB1 can be detected using an aflatoxin (AFB1) ELISA kit (sensitivity 1.0ng / mg). However, if the total degradation amount of fermented Lactobacillus FS95 is evaluated as 10.6509-5.5947=5.0562mg / Kg, then after a period of fermentation (72h), the amount of AFB1 that can be degraded by fermented Lactobacillus FS95 during fermentation is... The total amount is 5.0562 mg / Kg × 250g = 5.0562 μg / g × 250g = 1.2644g, which is greater than the initial content of AFBI in the fermented soybean paste (1.25 μg). That is, the content of AFB1 in the system should be degraded to close to 0 and cannot be detected by the aflatoxin (AFB1) ELISA kit.

[0113] Therefore, the applicant proposed the application of *Lactobacillus fermentatus* FS95 in fermented foods such as broad bean paste and chili sauce, and confirmed through actual product production that *Lactobacillus fermentatus* FS95 can efficiently degrade nitrite, vomitoxin, and aflatoxin (such as AFB1) in fermented foods such as broad bean paste and chili sauce, thereby improving the food safety performance of the products.

[0114] Example 3

[0115] The live *Lactobacillus fermentum* FS95 powder capable of degrading mycotoxins in this embodiment is obtained by centrifuging the fermentation broth of *Lactobacillus fermentum* FS95 on MRS medium and incubating it at 37°C for 16 hours, followed by freeze-drying or spray-drying to obtain the *Lactobacillus fermentum* FS95 powder. The live count of *Lactobacillus fermentum* FS95 is 1.5 × 10⁻⁶. 9 CFU / g.

[0116] The application of fermented Lactobacillus mucinus FS95 bacterial powder in the degradation of aflatoxin and vomitoxin in this embodiment.

[0117] In this embodiment, the live bacteria powder of *Lactobacillus fermentum* FS95 is used as a starter culture in fermented foods such as broad bean paste, chili sauce, pickles, and sauerkraut. The dosage is 2 × 10⁻⁶. 8 CFU / kg fermented food.

[0118] Example 4

[0119] The fermentation composition of *Lactobacillus fermentatus* FS95 in this embodiment is a mixture of live *Lactobacillus fermentatus* FS95 powder and freeze-dried fermentation supernatant powder in a weight ratio of 1:1. Specifically, *Lactobacillus fermentatus* FS95 is cultured on MRS medium at 37°C for 16 hours. The resulting fermentation broth is centrifuged, and the precipitate is then freeze-dried or spray-dried to obtain the *Lactobacillus fermentatus* FS95 powder. The live count of *Lactobacillus fermentatus* FS95 is 9 × 10⁻⁶. 8 CFU / g, its supernatant was freeze-dried to obtain the supernatant freeze-dried powder of fermented Lactobacillus mucilaginosus FS95.

[0120] The application of the fermentation composition of Lactobacillus mucinus FS95 in the preparation of fermented foods in this embodiment.

[0121] Example 5

[0122] The fermentation composition of this embodiment is a solid powder of fermented Lactobacillus myxoidus FS95 metabolites. Specifically, the fermented Lactobacillus myxoidus FS95 is cultured on MRS medium at 37°C for 16 hours, and the supernatant of the fermentation broth is obtained by centrifugation. The solid powder obtained by freeze-drying or spray-drying is the solid powder of fermented Lactobacillus myxoidus FS95 metabolites.

[0123] The fermentation composition in this embodiment is used in the preparation of fermented food, and the dosage is 500 mg / kg.

[0124] Example 6

[0125] The application of Lactobacillus fermentum FS95 in the preparation of fermented soybean paste: The Lactobacillus fermentum FS95 powder was prepared by the method described in Example 2.

[0126] The preparation method of fermented soybean paste includes the following steps:

[0127] S1. Chili mash production: Fresh red chilies are selected, cleaned, crushed, mixed with edible salt, and placed in a chili mash fermentation tank for preservation and sealing to make chili mash. The edible salt content of the chili mash is (20±2)%, W / W).

[0128] S2. Broad Bean Fermentation: Broad beans are shelled, impurities removed, soaked, and mixed with wheat flour and soybean meal. Following process requirements, Aspergillus oryzae (Hu Niang 3.042) is inoculated and cultured to produce fermented broad beans. Then, Lactobacillus FS95 fermentation powder (with a viable count of 1×10⁻⁶) is added. 9 Dissolve (CFU / g) in 15-17% saline at a specified mass ratio of 1:1000 (i.e., the final concentration of Lactobacillus fermentum FS95 is 1×10⁻⁶ CFU / g). 6 CFU / ml), the chopped petals are mixed with the inoculated brine (the water content of the mixture is 30-40%), and then placed in a tank for incubation at 35±5℃. The fermentation cycle is 35-50 days to produce mature petals.

[0129] S3. Post-fermentation: By weight ratio, add 70% chili mash and 30% cooked chili peppers to the fermentation tank and stir evenly. Stir for 30 minutes every 7 days. After more than 3 months of natural post-fermentation, Pixian Doubanjiang (Pixian chili bean paste) is produced, which is red and oily, mellow in taste and rich in soy sauce aroma.

[0130] To compare and analyze the degradation of aflatoxin and vomitoxin in fermented soybean paste by *Lactobacillus mucilaginosus* FS95, the applicant conducted the following experiments:

[0131] Control group: Traditional Pixian Doubanjiang (fermented broad bean paste) preparation method, including the following steps:

[0132] S1. Chili mash production: Fresh red chilies are selected, cleaned, crushed, mixed with edible salt, and placed in a chili mash fermentation tank for preservation and sealing to make chili mash. The edible salt content of the chili mash is (20±2)%, W / W).

[0133] S2. Broad bean mash production: Broad beans are shelled, impurities are removed, soaked, and mixed with wheat flour and soybean meal. Aspergillus oryzae (Hu Niang 3.042) is inoculated according to process requirements to produce fermented broad beans. Then, wheat flour (replacing the fermenting Lactobacillus FS95 powder in this example) is dissolved in 15-17% saline solution at a specified mass ratio of 1:1000. The fermented broad beans are mixed with the inoculated saline solution (the moisture content of the mixture is 30-40%) and placed in a fermentation tank at 35±5℃ for 35-50 days to mature, producing mature broad beans.

[0134] S3. Post-fermentation: By weight ratio, add 70% chili mash and 30% cooked chili peppers to the fermentation tank and stir evenly. Stir for 30 minutes every 7 days. After more than 3 months of natural post-fermentation, Pixian Doubanjiang (Pixian chili bean paste) is produced, which is red and oily, mellow in taste and rich in soy sauce aroma.

[0135] Experimental group: Doubanjiang (fermented broad bean paste) was prepared using the method described in this embodiment.

[0136] Three parallel samples of fermented soybean paste were prepared for both the control group and the experimental group for testing, and the average value of the test results was taken.

[0137] 1) Amino acid content

[0138] The amino acid content was determined by formaldehyde titration. Samples were taken from different locations in each group, and the average value was taken. The experimental results are shown in Table 9.

[0139] Table 9 Comparison of amino nitrogen content in fermented soybean paste prepared by different methods

[0140]

[0141] As shown in Table 9, the amino nitrogen content of Pixian Douban (fermented broad bean paste) prepared using fermented Lactobacillus mucilaginosus FS95 is 3.44 times that of the product without FS95.

[0142] 2) Aflatoxin determination

[0143] The aflatoxin content in the fermented soybean paste prepared by the two methods was determined according to the method provided in Example 1(8). The experimental results are shown in Table 10.

[0144] Table 10. Aflatoxin content in fermented soybean paste prepared by different methods

[0145]

[0146] Aflatoxin B1 (AFB1) is the most potent food contaminant discovered to date, making its content a crucial indicator of the quality of Pixian Doubanjiang (Pixian fermented broad bean paste). National regulations stipulate that the aflatoxin B1 content in Pixian Doubanjiang must be less than or equal to 5 mg / kg. Table 10 shows that the average aflatoxin B1 content in the control group was 4.74 mg / kg, while the average content in the experimental group was 0.32 mg / kg. The aflatoxin B1 content in the experimental group was only 6.75% of that in the control group, indicating that *Lactobacillus mucilaginosus* FS95 can efficiently degrade aflatoxin B1 during the preparation of Pixian Doubanjiang, thereby significantly reducing the aflatoxin AFB1 content in the bean paste.

[0147] 3) Vomitoxin assay

[0148] The vomitoxin content in the fermented soybean paste prepared by the two methods was determined according to the method provided in Example 1(9). The experimental results are shown in Table 11.

[0149] Table 11. Vomitoxin content in fermented soybean paste prepared by different methods

[0150]

[0151] Note: <10 indicates that the detection limit is less than 10 µg / kg.

[0152] Vomitoxin is one of the most potent toxic substances contaminating food. Flour is used in the production of Pixian Douban (fermented broad bean paste), and the vomitoxin content is a crucial indicator of flour quality. The national standard for vomitoxin in flour is ≤1000 µg / kg. Furthermore, traditional fermentation of Pixian Douban carries the risk of mycotoxin contamination. Table 11 shows that the average vomitoxin content in the control group was 18.35 µg / kg, while the average aflatoxin B1 content in the experimental group was less than 10 µg / kg. Although both the control and experimental groups had vomitoxin levels below 1000 µg / kg, the experimental group's levels were significantly lower than the control group. This indicates that *Lactobacillus mucilaginosus* FS95 can efficiently degrade vomitoxin during the preparation of Pixian Douban, reducing the risk of vomitoxin contamination.

[0153] 4) Evaluation of volatile aroma components

[0154] Sensory evaluation methods were used to analyze the aroma components. A sensory evaluation panel of 10 people evaluated the aroma and taste of Pixian Doubanjiang (Pixian chili bean paste) in both the control and experimental groups. Each person cast one vote for each indicator in either the control or experimental group.

[0155] Table 12 Evaluation of volatile aroma components in fermented soybean paste prepared by different methods

[0156]

[0157] Note: One vote per person; the more votes received, the better the aroma and taste.

[0158] As shown in Table 12, the experimental group received 127 votes, while the control group received 80 votes. The experimental group received 58.75% more votes than the control group. Therefore, the experimental group had more flavor-enhancing substances, especially ester aroma, soy sauce aroma, spicy aroma, fruit aroma, and umami flavor, which were much higher than those in the control group. This is because the Pixian Doubanjiang in the experimental group was inoculated with the dominant strain FS95. This strain grows vigorously, undergoes heterogeneous fermentation, has a long survival time, and secretes enzymes, which better decomposes the fats, starches, and proteins in the raw materials. At the same time, FS95 produces organic acids and other substances that inhibit the growth of harmful bacteria, thus producing more flavor-enhancing substances.

[0159] In summary, the fermented Lactobacillus mucinus FS95 agent of this invention produces soybeans with lower levels of aflatoxin and vomitoxin, both below the detection limit. In addition, the soybeans contain more amino nitrogen and flavor-enhancing substances, resulting in better product quality.

[0160] Example 7

[0161] The application of *Lactobacillus fumarate* FS95 in the preparation of chili sauce, wherein the amount of *Lactobacillus fumarate* FS95 added to the chili sauce is 1×10⁻⁶. 9 CFU / kg.

[0162] The fermentation composition of *Lactobacillus mucinus* FS95 of the present invention can be adjusted according to different applications, selecting appropriate components and dosages. For example, in Example 3, the dosage of *Lactobacillus mucinus* FS95 powder in the preparation of fermented food can be 5 × 10⁻⁶. 8 CFU / kg, 1×10 9 CFU / kg, the live bacteria powder of fermented Lactobacillus myxobolus FS95 and the freeze-dried powder of fermentation supernatant in Example 4 can also be used in the degradation of aflatoxin. The solid powder of metabolites of fermented Lactobacillus myxobolus FS95 in Example 5 can be used in the preparation of fermented food, and the dosage can also be 100mg / Kg, 400mg / Kg, 800mg / Kg or 1000mg / Kg. The changes in the above technical features can be understood and implemented by those skilled in the art through textual description, so no further drawings are required.

Claims

1. A fermenting *Lactobacillus mucilaginosus* capable of degrading mycotoxins, characterized in that, The fermenting *Lactobacillus* is *Limosilactobacillus fermentum* FS95, which was deposited at the China Center for Type Culture Collection on December 22, 2025, with accession number CCTCC NO: M20252983. The mycotoxins that *Lactobacillus fermentum* can degrade include vomitoxin and aflatoxin.

2. A fermentation composition, characterized in that, The fermentation composition is produced by fermenting *Lactobacillus mucinus* FS95, which is capable of degrading mycotoxins as described in claim 1, and includes live *Lactobacillus mucinus* FS95, inactivated bacterial cells, fermentation broth, fermentation precipitate, or spray-dried / lyophilized powder.

3. A probiotic preparation, characterized in that, The probiotic preparation comprises *Lactobacillus mucinus* FS95, which is capable of degrading mycotoxins as described in claim 1, and / or the fermentation composition as described in claim 2; the content of *Lactobacillus mucinus* FS95 in the probiotic preparation is ≥2 × 10⁻⁶. 7 CFU / g, or, the content of *Lactobacillus fermentum* FS95 in the probiotic preparation is ≥2×10⁻⁶. 7 CFU / ml.

4. The application of the fermentable Lactobacillus mucilaginosus FS95, as described in claim 1, in the degradation of aflatoxin and / or vomitoxin.

5. The use of the fermented Lactobacillus mucilaginosus FS95 as described in claim 1, which is capable of degrading mycotoxins, or the fermentation composition as described in claim 2, in the preparation of fermented foods.

6. The application as described in claim 5, characterized in that, The fermented foods include broad bean paste or broad bean sauce, chili sauce, fermented milk, pickled vegetables, fermented black beans, bean sprouts, soybean paste, fish sauce, pickled fish, cured meat, and ham.

7. A type of fermented soybean paste, characterized in that, The fermented soybean paste is prepared by fermentation with Lactobacillus mucilaginosus as described in claim 1.

8. The fermented soybean paste as described in claim 7, characterized in that, The method for preparing the fermented soybean paste includes the following steps: S1. Chili mash production: Fresh red chilies are selected, cleaned, crushed, mixed with edible salt, and placed in a chili mash fermentation tank for preservation and sealing to make chili mash. The edible salt content of the chili mash is (20±2)% (W / W). S2. Broad bean mash production: Broad beans are shelled, impurities are removed, soaked, and mixed with wheat flour and soybean meal. Aspergillus oryzae is inoculated according to process requirements to produce fermented broad beans. Then, the fermented Lactobacillus FS95 powder that can degrade mycotoxins as described in claim 1 is dissolved in 15-17% saline solution. The fermented broad beans are mixed with the inoculated saline solution and then placed in a tank for incubation at 35±5℃. The fermentation cycle is 35-50 days to produce mature broad beans. S3. Post-fermentation: By mass ratio, add 60-80% of the chili mash prepared in S1 and 20-40% of the cooked chili flakes prepared in S2 to the fermentation tank and stir evenly. Stir for 20-40 minutes every 5-8 days. After more than 3 months of natural post-fermentation, the fermented bean paste can be obtained.

9. The fermented soybean paste as described in claim 8, characterized in that, The viable count of the fermented Lactobacillus mucinus FS95 bacterial powder is ≥1×10⁻⁶. 9 CFU / g, wherein the mass ratio of the fermented Lactobacillus mucinus FS95 bacterial powder to brine is 1~2:1000.

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