Lactobacillus brevis and application thereof

Abstract

This invention discloses a strain of *Lactobacillus brevis* and its applications, belonging to the field of food microbiology technology. The invention provides a strain of *Lactobacillus brevis* CHOL1, isolated from sauerkraut. After safety evaluation, this strain is suitable for use in food. It exhibits a strong ability to degrade nitrite and can also react with myoglobin to generate nitrosomyoglobin. Inoculation of this strain into fermented meat products revealed that it can reduce nitrite and nitrosamine residues while improving the color of the meat products. As a starter culture, this strain can improve the quality and safety of fermented meat products and can serve as a natural and effective substitute for nitrite in fermented meat products.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus brevis CHOL1 and its application in fermented meat products, belonging to the field of food microbiology technology. Background Technology

[0002] Nitrites, a common substance in fermented meat products, originate from three main sources: firstly, nitrite food additives; secondly, nitrate impurities in salt generated during curing through microbial reduction; and thirdly, nitrites formed from proteins in meat products through microbial metabolism and digestion by *Nitrostomia nitrosamines*. Although nitrites play a role in meat products by fixing color, improving texture, inhibiting the growth of foodborne pathogens, and acting as antioxidants, they can also react with amines as precursors to form highly carcinogenic nitrosamines. Furthermore, the accumulation of nitrosamines in the body poses a significant health hazard. Therefore, finding alternative methods to replace the functions of nitrites and reduce nitrite and nitrosamine residues in meat products is crucial for improving the safety of fermented meat products.

[0003] Studies have confirmed that lactic acid bacteria have the ability to promote myoglobin color development and reduce nitrite and nitrosamines. Their application in fermented sausages is expected to partially replace the function of nitrite, reducing the amount of nitrite used; their application in fermented fish products can reduce nitrite and nitrosamine residues. Currently, studies have screened lactic acid bacteria that can promote myoglobin color development or degrade nitrite. For example, Li et al. screened a strain of *Lactobacillus fermentum* that can convert myoglobin into nitrosomyoglobin, promoting the color development of meat products; Wang et al. found that inoculating *Lactobacillus sakei* can degrade nitrite in meat products. However, no lactic acid bacteria strain has yet been screened that not only has a strong ability to degrade nitrite but also promotes myoglobin color development and improves the color of meat products. Furthermore, as nitrosamines are a common and potent carcinogen in meat, reducing their residues in meat products is crucial for human health. However, no studies have reported that single-strain lactic acid bacteria fermentation can achieve the triple effect of degrading nitrite, promoting the color development of meat products, and reducing nitrosamine residues in meat products. For fermented fish, especially mandarin fish, no studies have reported that inoculating with lactic acid bacteria alone can simultaneously degrade nitrite and nitrosamines. Therefore, our aim is to screen for a lactic acid bacterium with outstanding color development and strong nitrite degradation capabilities. Applying this bacterium to fermented meat products such as fermented sausages and fermented fish can not only promote color development and degrade nitrite residues, but also reduce nitrosamines in meat products, thereby improving the quality and safety of fermented meat products. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a strain of Lactobacillus brevis and its application, aiming to solve the technical problem that lactic acid bacteria, as fermentation agents, cannot simultaneously degrade nitrite and nitrosamine residues and improve the color of meat products.

[0005] The first technical solution provided by this invention is a strain of Lactobacillus brevis CHOL1, which has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.64656, deposit date of May 20, 2024, and deposit address of Guangdong Provincial Center for Microbial Culture Collection, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0006] The Lactobacillus brevis CHOL1 grew well in MRS solid medium and produced round, smooth, milky-yellow colonies after 24 hours of incubation at 37°C.

[0007] The second technical solution provided by the present invention is a microbial preparation containing Lactobacillus brevis CHOL1 as described in the first technical solution.

[0008] In some embodiments, the microbial preparation is a solid or liquid preparation, such as a lyophilized powder or fermentation broth.

[0009] In some embodiments, the amount of *Lactobacillus brevis* CHOL1 added to the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

[0010] Furthermore, in the microbial preparation, the amount of *Lactobacillus brevis* CHOL1 added is not less than 1×10⁻⁶. 7 CFU / g or 1×10 7 CFU / mL.

[0011] The third technical solution provided by the present invention is a fermentation agent containing Lactobacillus brevis CHOL1 as described in the first technical solution or a microbial preparation as described in the second technical solution.

[0012] In some embodiments, other additives or microbial preparations may also be added to the fermentation agent.

[0013] In some embodiments, the amount of *Lactobacillus brevis* CHOL1 added to the starter culture is not less than 1 × 10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

[0014] Furthermore, the amount of *Lactobacillus brevis* CHOL1 added to the starter culture is not less than 1 × 10⁻⁶. 7CFU / g or 1×10 7 CFU / mL.

[0015] In some embodiments, the preparation of the fermenting agent is specifically as follows: a single colony of *Lactobacillus brevis* CHOL1 is picked and inoculated into 10 mL of MRS liquid medium, and cultured at 37°C and 200-250 rpm for 24 h to obtain bacterial solution A; then 3 mL of bacterial solution A is taken and inoculated into 150 mL of MRS liquid medium, and cultured at 37°C and 200-250 rpm for 24 h to obtain bacterial solution B; bacterial solution B is centrifuged at 8000 rpm / min for 5 min and the bacterial cells are collected; the *Lactobacillus brevis* CHOL1 bacterial cells are diluted with a 0.9% sodium chloride aqueous solution to prepare the bacterial solution.

[0016] The fourth technical solution provided by the present invention is the application of Lactobacillus brevis CHOL1 as described in the first technical solution, or the microbial preparation as described in the second technical solution, or the fermentation agent as described in the third technical solution in the preparation of fermented meat products.

[0017] In some embodiments, the fermented meat products include, but are not limited to, fermented sausages, fermented fish (especially mandarin fish), etc.

[0018] The fifth technical solution provided by the present invention is the role of Lactobacillus brevis CHOL1 as described in the first technical solution, or the microbial preparation as described in the second technical solution, or the fermentation agent as described in the third technical solution in the degradation of nitrite and / or nitrosamines.

[0019] The sixth technical solution provided by the present invention is a method for reducing nitrite and / or nitrosamines in fermented meat products. The method involves introducing the Lactobacillus brevis CHOL1 described in the first technical solution, or the preparation described in the second technical solution, or the starter culture described in the third technical solution into the fermentation system of the fermented meat products.

[0020] In some embodiments, the concentration of Lactobacillus brevis CHOL1 in the fermentation system is 1 × 10⁻⁶. 6 ~1×10 8 CFU / g.

[0021] In some embodiments, the fermented meat products include, but are not limited to, fermented sausages, fermented fish (especially mandarin fish), etc.

[0022] In some embodiments, the preparation of the fermented sausage includes the following steps:

[0023] S1. Freshly slaughtered lean pork and fat are minced in a meat grinder with a 1.5cm aperture sieve plate and mixed according to the ratio of lean meat to fat of 9:1-7:3.

[0024] S2. Based on the total weight of lean and fatty meat in step S1 as 100%, add 1.5-2.5 wt% salt, 0.2%-0.5% wt% MSG, 0.5%-1.5 wt% white wine, 1.0-4.0% wt% white sugar, 1.0-1.5 wt% soy sauce, 0.5-1.5% wt% chili powder, and 0.01 wt% sodium nitrite. Inoculate with the *Lactobacillus brevis* CHOL1 described in the first technical solution, the preparation described in the second technical solution, or the fermentation agent described in the third technical solution. After stirring evenly, marinate at room temperature for 30 minutes and then stuff into sausages.

[0025] S3. The sausages obtained in step S2 are fermented at 10-15℃ for 10-12 days to obtain air-dried sausages.

[0026] In some embodiments, the preparation of the fermented fish meat includes the following steps:

[0027] S1. Clean the fresh mandarin fish by removing its internal organs, scales, and gills;

[0028] S2. Weigh out drinking water equal to the weight of the mandarin fish obtained in step S1, pour it into a container, and add 3wt% salt, 1wt% scallion, 0.6wt% ginger, 0.1wt% star anise, 0.05wt% fennel, 0.05wt% cumin, 0.01wt% chili, and 0.01wt% Sichuan pepper, based on 100% of the weight of the drinking water. Then, inoculate with Lactobacillus brevis CHOL1 as described in the first technical solution, the microbial preparation as described in the second technical solution, or the fermentation agent as described in the third technical solution.

[0029] S3. Soak the mandarin fish obtained in step S1 in the fermentation liquid, press the fish body with stones, and ferment at 12℃ for 5 days.

[0030] In some embodiments, the preparation of the fermented fish meat includes the following steps:

[0031] S1. Remove the internal organs, scales, and gills from the fresh mandarin fish, drain it, and weigh it.

[0032] S2, preparation of auxiliary materials: weigh out 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% Sichuan pepper, 0.002% chili powder, 0.6% fresh ginger, and 1% scallion according to the fish body weight, and inoculate with Lactobacillus brevis CHOL1 as described in the first technical solution, the microbial preparation as described in the second technical solution, or the fermentation agent as described in the third technical solution;

[0033] S3. Apply the auxiliary materials evenly to the inner and outer surfaces of the mandarin fish, place it in a sealed container, and ferment at 12℃ for 7 days.

[0034] Compared with existing methods, the present invention has the following advantages:

[0035] This invention provides a strain of *Lactobacillus brevis* CHOL1, isolated from sauerkraut. After safety evaluation, this strain is suitable for food application. It exhibits strong nitrite degradation capabilities and can also react with myoglobin to generate nitrosomyoglobin. Inoculation of dried sausage with this strain reduced nitrite and nitrosamine residues while improving the color of the meat product. Inoculation of fermented mandarin fish also reduced nitrite and nitrosamine residues. As a starter culture, this strain can improve the quality and safety of fermented meat products and can serve as a natural and effective substitute for nitrites in fermented meat products.

[0036] Preservation of biological materials

[0037] A strain of the aforementioned Lactobacillus brevis, CHOL1, has been deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC NO.64656, deposited on May 20, 2024, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Attached Figure Description

[0038] Figure 1 Photograph of MRS solid medium culture of Lactobacillus brevis CHOL1;

[0039] Figure 2 Color results of inoculating Lactobacillus brevis with myoglobin in a simulated system. Detailed Implementation

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0041] All culture media used in this invention were prepared using conventional methods. Unless otherwise specified, the molecular biology operations involved in the examples refer to Sambrook J et al., eds., Science Press, 2002, Molecular Cloning: A Laboratory Manual (3rd Edition); or to the product instruction manual.

[0042] The culture media involved in the following examples are as follows:

[0043] MRS liquid culture medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7 ± 0.2.

[0044] MRS solid medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5 ± 0.2.

[0045] YPD solid medium: peptone 10.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, agar 14.0 g / L.

[0046] YPD liquid medium: peptone 20.0 g / L, glucose 20.0 g / L, yeast extract 10.0 g / L, pH 6.5 ± 0.2.

[0047] In the comparative examples below, *Lactobacillus plantarum* CICC 20242, CICC 22205, CICC 20264, CICC 20038, CICC 20659, CICC 22220, *Pediococcus pentosaceus* CICC 22227, CICC 21862, *Lactobacillus brevis* CICC 6139, CICC 24450, CICC 20297, CICC 20269, *Leuconostoc mesenteriae* CICC 21725, CICC 21861, *Lactobacillus acidophilus* CICC 6255, CICC 20248, CICC 20244, *Streptococcus thermophilus* CICC 20364, CICC 25032, CICC 20367, and *Staphylococcus equi* CICC 10435 were purchased from the China Industrial Microbial Culture Collection Center; *Lactobacillus plantarum* ATCC... 8014, Pediococcus pentosaceus ATCC 33316, Staphylococcus aureus ATCC 51136, ATCC 51137, Staphylococcus equi ATCC 43958, Staphylococcus xylose ATCC 29971, 21145 were purchased from Jitai Biotechnology; Saccharomyces cerevisiae CGMCC 2.1364, 2.148 were purchased from Guangdong Provincial Microbial Culture Collection Center.

[0048] Example 1: Isolation and identification of Lactobacillus brevis CHOL1.

[0049] (1) Collection of strains: isolated from homemade sauerkraut.

[0050] (2) Identification of strain CHOL1:

[0051] The genome of strain CHOL1 was extracted for 16S rDNA identification. The genome extraction method was performed using the glass bead method as described in "A Concise Guide to Molecular Biology Experiments".

[0052] The PCR conditions are as follows:

[0053] The amplification system consisted of: 25 μL of 2×Taq Master Mix, 2 μL of primer 27F, 2 μL of primer 1492R, 19 μL of sterile water, and 2 μL of template. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 55℃ annealing for 15 s; 72℃ extension for 1 min; 72℃ for 5 min; 30 cycles; and indefinite reaction at 4℃. After PCR, the PCR products were detected by agarose gel electrophoresis (1.0%). Bright bands indicated successful PCR amplification of the genome, which was then ready for sequencing.

[0054] The 16S rDNA sequence of strain CHOL1 is shown in SEQ ID NO:1.

[0055] BLAST n analysis of the sequencing results revealed that this bacterium shared the highest homology (99.93%) with *Levilactobacillus brevis*. Therefore, this strain was identified as *Levilactobacillus brevis* and named *Levilactobacillus brevis* CHOL1. *Levilactobacillus brevis* CHOL1 grew well in MRS solid medium, and after 24 hours of incubation at 37°C, [the following results were observed]. Figure 1 As shown, the colonies of Lactobacillus brevis are round, smooth, and milky yellow.

[0056] Based on morphological and 16S rDNA identification, strain CHOL1 has been tentatively identified as *Levilactobacillus brevis* and deposited on May 20, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), accession number GDMCC NO. 64656. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China.

[0057] Example 2: Safety evaluation of Lactobacillus brevis CHOL1.

[0058] (1) Pathogenicity test of Lactobacillus brevis CHOL1

[0059] The pathogenicity test of the strains was carried out in accordance with the provisions of the "Technical Guidelines for Safety Testing and Evaluation of Microbial Strains for Raw Materials of Health Food (2020 Edition)" and 10 mice were selected for each group of experiments.

[0060] Table 1. Results of pathogenicity test of Lactobacillus brevis CHOL1 (by gavage)

[0061]

[0062]

[0063] Note: Lowercase letters in the table indicate significant differences in male mice, and uppercase letters indicate significant differences in female mice.

[0064] The experimental results show that there was no significant difference between the experimental group and the control group, therefore it can be concluded that the strain is not pathogenic.

[0065] (2) Drug resistance evaluation experiment of Lactobacillus brevis CHOL1:

[0066] The methods for evaluating the antibiotic resistance of *Lactobacillus brevis* CHOL1 and the cutoff values ​​for each antibiotic were based on the European Food Safety Authority's 2012 guideline, "Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance." Activated *Lactobacillus brevis* CHOL1 was used as the indicator bacterium, and the minimum inhibitory concentration (MIC) of each antibiotic was determined using a serial two-fold dilution method. The antibiotic resistance of the strain was evaluated by comparing the MIC with the cutoff value. When the MIC was equal to or less than the predetermined cutoff value, the strain was considered sensitive to the antibiotic; when the MIC was greater than the predetermined cutoff value, the strain was considered resistant to the antibiotic.

[0067] Table 2 Results of antibiotic susceptibility testing for Lactobacillus brevis CHOL1

[0068]

[0069] The results showed that Lactobacillus brevis CHOL1 was sensitive to antibiotics such as ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, tetracycline, chloramphenicol, and clindamycin, indicating that the strain has good safety.

[0070] (3) Hemolysis test of Lactobacillus brevis CHOL1

[0071] Culture medium preparation: Columbia agar, 5% defibrinated sheep blood.

[0072] Take approximately 10 units of Lactobacillus brevis CHOL1. 8 CFU / mL was streaked onto Columbia agar plates containing 5% defibrinated sheep blood and incubated at 37°C for 24 h. Hemolysis around the colonies was then observed. Staphylococcus aureus ATCC 25923 was used as a positive control.

[0073] The results showed that Staphylococcus aureus ATCC 25923 exhibited a well-defined, completely transparent hemolytic zone of approximately 6-8 mm around its colonies, typical of β-hemolysis. In contrast, the Lactobacillus brevis CHOL1 colonies provided in this invention did not show a hemolytic zone, indicating that the strain was non-hemolytic and a safe strain.

[0074] Example 3: The method for determining the nitrite degradation ability of Lactobacillus brevis CHOL1 is as follows:

[0075] (1) Preparation of the sodium nitrite standard curve: Pipette 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, 1.00 mL, 1.50 mL, 2.00 mL, and 2.50 mL of sodium nitrite standard working solution (5 μg / mL) into 15 mL centrifuge tubes. Add 2 mL of 4 g / L p-aminobenzenesulfonic acid solution to the tubes, mix well, let stand for 3-5 min, then add 1 mL of 2 g / L naphthylethylenediamine hydrochloride solution, add water to 10 mL, mix well, and let stand for 15 min. Measure the absorbance at 538 nm and plot the standard curve. Prepare a reagent blank simultaneously.

[0076] (2) Determination of nitrite degradation capacity of Lactobacillus brevis CHOL1: Frozen Lactobacillus brevis CHOL1 was cultured on MRS solid medium at 37℃ for 1 day. Single colonies were picked from the plates and inoculated into MRS liquid medium for 1 day. Then, with an initial OD of 1.5, 1% was inoculated into MRS liquid medium containing 100 μg / mL sodium nitrite and cultured at 37℃ for 36 h. After culture, the bacterial solution was centrifuged at 10000 rpm / min for 5 min. 1 mL of the bacterial supernatant was placed in a 15 mL centrifuge tube, 2 mL of p-aminobenzenesulfonic acid was added, and after standing for 5 min, 1 mL of naphthylethylenediamine hydrochloride solution was added, followed by 6 mL of deionized water. After mixing, the solution was allowed to stand for 15 min, and the absorbance was measured at a wavelength of 538 nm. The nitrite content was calculated according to the standard curve. The blank control group was MRS liquid medium supplemented with 100 μg / mL sodium nitrite. The formula for calculating the nitrite degradation rate is as follows:

[0077]

[0078] In the formula, the units for the initial nitrite content and the nitrite content after cultivation are both μg / mL.

[0079] The determination showed that Lactobacillus brevis had a nitrite degradation rate of 99.03% in MRS liquid culture medium containing sodium nitrite, indicating that Lactobacillus brevis CHOL1 has a strong nitrite degradation ability.

[0080] Example 4: The method for determining the colorimetric ability of Lactobacillus brevis CHOL1 is as follows:

[0081] Myoglobin stock solution (20 mg / mL) was incubated at 50°C for 30 min. After incubation, the myoglobin stock solution was centrifuged (10000g, 5 min, 4°C). The supernatant was filtered through a 0.22 μm filter and added to MRS liquid medium to prepare a simulated system with a myoglobin concentration of 2.0 mg / mL. 40 μL of bacterial culture was inoculated into this system, and the mixture was anaerobic at 37°C for 18 h. After centrifugation (10000g, 5 min, 4°C), the color was measured. An MRS broth system containing myoglobin and supplemented with 0.015% sodium nitrite was used as a positive control, and an MRS liquid medium system containing myoglobin was used as a blank control.

[0082] The results of the colorimetric ability test of Lactobacillus brevis CHOL1 are attached. Figure 2 As shown, group 1 consisted of CHOL1 from *Lactobacillus brevis*, group Y was the positive control group, and group C was the blank group. The a* / b* value of *Lactobacillus brevis* after culturing in the MRS liquid medium system containing myoglobin was 1.42±0.08, the a* / b* value of the positive control group with added sodium nitrite was 1.20±0.20, and the a* / b* value of the blank group was 0.41±0.03. This indicates that *Lactobacillus brevis* can convert myoglobin into a red myoglobin derivative, even achieving a color-developing ability similar to that of sodium nitrite.

[0083] Example 5: Lactobacillus brevis CHOL1 at 7×10 6 CFU / g was inoculated into air-dried intestines.

[0084] S1: Freshly slaughtered lean pork and fat are minced in a meat grinder with a 1.5cm aperture screen plate and mixed according to the ratio of lean meat to fat = 9:1.

[0085] S2: Preparation of bacterial culture:

[0086] A single colony of *Lactobacillus brevis* CHOL1 was picked and inoculated into 10 mL of MRS liquid medium and cultured at 37°C and 200-250 rpm for 24 h to obtain bacterial suspension A. Then, 3 mL of bacterial suspension A was inoculated into 150 mL of MRS liquid medium and cultured at 37°C and 200-250 rpm for 24 h to obtain bacterial suspension B. Bacterial suspension B was centrifuged at 8000 rpm / min for 5 min to collect the bacterial cells. The *Lactobacillus brevis* CHOL1 cells were diluted with a 0.9% sodium chloride aqueous solution to a concentration on the order of 10-1. 6 CFU / mL bacterial culture

[0087] S3: Based on 100% total weight of lean and fatty meat, add 1.5 wt% salt, 0.5 wt% MSG, 1.5 wt% white wine, 1.0 wt% white sugar, 1.5 wt% soy sauce, 0.5 wt% chili powder, 0.01 wt% sodium nitrite, and Lactobacillus brevis CHOL1 bacterial solution; wherein the Lactobacillus brevis CHOL1 bacterial solution is prepared at a concentration of 7 × 10⁻⁶.6 Inoculate the meat with CFU / g, mix well, marinate at room temperature for 30 minutes, and then stuff into sausages.

[0088] S4: Fermentation: The sausages obtained in S3 are fermented at 15°C for 12 days to make air-dried sausages, which are named Example 5.

[0089] Example 6: Lactobacillus brevis CHOL1 at 5 × 10 7 CFU / g was inoculated into air-dried intestines.

[0090] In addition to S2, the specific implementation method involves diluting Lactobacillus brevis CHOL1 cells with a 0.9% sodium chloride aqueous solution to a concentration on the order of 10. 7 "CFU / mL bacterial suspension", "Lactobacillus brevis CHOL1 bacterial suspension in S3 at 5×10" 7 Except for the difference between "CFU / g inoculated into meat" and Example 5, the rest of the procedure was carried out in accordance with the method described in Example 5.

[0091] Example 7: Lactobacillus brevis CHOL1 at 3×10 8 CFU / g inoculated into dried sausage

[0092] In addition to S2, the specific implementation method involves diluting Lactobacillus brevis CHOL1 cells with a 0.9% sodium chloride aqueous solution to a concentration on the order of 10. 8 "CFU / mL bacterial suspension", "Lactobacillus brevis CHOL1 bacterial suspension in S3 at 3×10" 8 Except for the difference between "CFU / g inoculated into meat" and Example 5, the rest of the procedure was carried out in accordance with the method described in Example 5.

[0093] Example 8: Lactobacillus brevis CHOL1 at 7×10 6 CFU / mL was inoculated into wet-cured stinky mandarin fish.

[0094] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0095] S2: Preparation of bacterial culture

[0096] A single colony of *Lactobacillus brevis* CHOL1 was picked and inoculated into 10 mL of MRS liquid medium and cultured at 37°C and 200-250 rpm for 24 h to obtain bacterial culture A. Then, 3 mL of bacterial culture A was inoculated into 150 mL of MRS liquid medium and cultured at 37°C and 200-250 rpm for 24 h to obtain bacterial culture B. Bacterial culture B was centrifuged at 8000 rpm / min for 5 min to collect the bacterial cells. The *Lactobacillus brevis* CHOL1 cells were diluted with a 0.9% sodium chloride aqueous solution to a concentration of 10... 7 CFU / mL bacterial culture

[0097] S3: Fermentation broth preparation: Weigh drinking water equal to the weight of the mandarin fish obtained in step S1, pour it into a container, and add 3wt% salt, 1wt% scallion, 0.6wt% ginger, 0.1wt% star anise, 0.05wt% fennel, 0.05wt% cumin, 0.01wt% chili pepper, 0.01wt% Sichuan pepper, and Lactobacillus brevis CHOL1 bacterial culture, based on the weight of the drinking water as 100%. The concentration of Lactobacillus brevis CHOL1 in the fermentation broth is 7×10⁻⁶. 6 CFU / mL.

[0098] S4: Fermentation: The mandarin fish obtained in step S1 is immersed in the fermentation liquid, the fish is pressed down with stones, and fermented at 12°C for 5 days. The product is named Example 8.

[0099] Example 9: Lactobacillus brevis CHOL1 at 4 × 10 7 CFU / mL was inoculated into wet-cured stinky mandarin fish.

[0100] In addition to S2, the specific implementation method involves diluting Lactobacillus brevis CHOL1 cells with a 0.9% sodium chloride aqueous solution to a concentration of 10... 8 The concentration of Lactobacillus brevis CHOL1 in the fermentation broth in S3 is 4 × 10⁻⁶ CFU / mL bacterial solution. 7 Except for the difference in "CFU / mL" compared to Example 8, all other procedures were carried out in accordance with the implementation method of Example 8.

[0101] Example 10: Lactobacillus brevis CHOL1 at 7×10 6 CFU / mL was inoculated into dried and salted mandarin fish.

[0102] S1: Remove the internal organs, scales, and gills from the fresh mandarin fish, drain it, and weigh it.

[0103] S2: Same as Example 8S2.

[0104] S3: Preparation of auxiliary ingredients: Based on the fish weight, weigh out 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% Sichuan peppercorns, 0.002% chili powder, 0.6% fresh ginger, 1% scallions, and Lactobacillus brevis CHOL1 bacterial solution. The concentration of Lactobacillus brevis CHOL1 is 7 × 10⁻⁶. 6 CFU / g.

[0105] S4: Apply the auxiliary materials evenly to the inner and outer surfaces of the mandarin fish, place it in a sealed container, and ferment it at 12°C for 7 days. The product is named Example 10.

[0106] Example 11: Lactobacillus brevis CHOL1 at 4 × 10 7 CFU / mL was inoculated into dried and salted mandarin fish.

[0107] S1: Remove the internal organs, scales, and gills from the fresh mandarin fish, drain it, and weigh it.

[0108] S2: The preparation of bacterial culture is the same as in Example 8S1.

[0109] S3: Preparation of auxiliary ingredients: Based on the fish body weight, weigh out 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% Sichuan peppercorns, 0.002% chili powder, 0.6% fresh ginger, 1% scallions, and Lactobacillus brevis CHOL1 bacterial solution. The concentration of Lactobacillus brevis CHOL1 is 4 × 10⁻⁶. 7 CFU / g.

[0110] S4: Apply the auxiliary materials evenly to the inner and outer surfaces of the mandarin fish, place it in a sealed container, and ferment it at 12°C for 7 days. The product is named Example 11.

[0111] Comparative Example 1: Add 0.01% sodium nitrite and let it ferment and air dry.

[0112] S1: Freshly slaughtered lean pork and fat are minced in a meat grinder with a 1.5cm aperture screen plate and mixed according to the ratio of lean meat to fat = 9:1.

[0113] S2: Based on the total weight of lean and fatty meat as 100%, add 1.5wt% salt, 0.5wt% MSG, 1.5wt% white wine, 1.0wt% white sugar, 1.5wt% soy sauce, 0.5wt% chili powder, and 0.01wt% sodium nitrite. Mix well and marinate at room temperature for 30 minutes before stuffing into sausages.

[0114] S3: Fermentation: The sausages obtained in S2 are fermented at 15℃ for 12 days to make air-dried sausages, and the product is named Comparative Example 1.

[0115] Comparative Example 2: Add 0.015% sodium nitrite and let it ferment and air dry.

[0116] The specific implementation method is the same as Comparative Example 1, wherein the amount of sodium nitrite added is 0.015% of the total mass of lean and fatty meat.

[0117] Comparative Example 3: Wet-process naturally fermented stinky mandarin fish

[0118] S1. Clean the fresh mandarin fish by removing its internal organs, scales, and gills.

[0119] S2. Preparation of fermentation liquid: Weigh out drinking water of the same mass as the mandarin fish obtained in step S1, and add 3wt% salt, 1wt% scallion, 0.6wt% ginger, 0.1wt% star anise, 0.05wt% fennel, 0.05wt% cumin, 0.01wt% chili and 0.01wt% Sichuan pepper, based on the mass of drinking water as 100%.

[0120] S3: Fermentation: The mandarin fish obtained in S1 was immersed in the fermentation liquid obtained in S2, the fish was pressed down with stones, and fermented at 12℃ for 5 days. The resulting product was named Comparative Example 3.

[0121] Comparative Example 4: Dry-fermented stinky mandarin fish

[0122] S1: Remove the internal organs, scales, and gills from the fresh mandarin fish, drain it, and weigh it.

[0123] S2: Preparation of auxiliary ingredients: Based on the weight of the fish, weigh out 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% Sichuan pepper, 0.002% chili powder, 0.6% fresh ginger, and 1% scallion.

[0124] S3: Apply the auxiliary materials evenly to the inner and outer surfaces of the mandarin fish, place it in a sealed container, and ferment it at 12℃ for 7 days. The product is named Comparative Example 4.

[0125] Comparative Example 5: A microbial compound preparation containing Lactobacillus plantarum CICC 20242, Pediococcus pentosaceus CICC 22227, and Lactobacillus brevis CICC6139 was inoculated into air-dried intestines.

[0126] S1: Same as Example 5S1.

[0127] S2: Preparation of bacterial culture:

[0128] Single colonies of *Lactobacillus plantarum* CICC 20242, *Pediococcus pentosaceus* CICC 22227, and *Lactobacillus brevis* CICC 6139 were picked and inoculated into 10 mL of MRS liquid medium and cultured at 37°C and 200-250 rpm for 24 h to obtain bacterial suspensions A1, A2, and A3. Subsequently, 3 mL of each of suspensions A1, A2, and A3 were inoculated into 150 mL of MRS liquid medium and cultured at 37°C and 200-250 rpm for 24 h to obtain bacterial suspensions B1, B2, and B3. The bacterial cells of suspensions B1, B2, and B3 were collected by centrifugation at 8000 rpm / min for 5 min. The B1, B2, and B3 bacterial cells were diluted with 0.9% sodium chloride aqueous solution to a final concentration of 10 μL. 6 CFU / mL bacterial culture

[0129] S3: Fermentation broth preparation: Fermentation broth was prepared by mixing *Lactobacillus plantarum* CICC 20242, *Pediococcus pentosaceus* CICC 22227, and *Lactobacillus brevis* CICC6139 bacterial cultures in a 1:1:1 ratio. The final concentration of the fermentation broth was 10. 6 CFU / mL.

[0130] S4: Based on 100% total weight of lean and fatty meat, add 1.5 wt% salt, 0.5 wt% MSG, 1.5 wt% baijiu (Chinese white liquor), 1.0 wt% sugar, 1.5 wt% soy sauce, 0.5 wt% chili powder, and 0.01 wt% sodium nitrite to form a mixed fermentation liquid; wherein the mixed fermentation liquid is the fermentation liquid prepared in S3, which is fermented at 7×10 6 Inoculate the meat with CFU / g, mix well, marinate at room temperature for 30 minutes, and then stuff into sausages.

[0131] S5: Fermentation: The sausages obtained in S4 are fermented at 15℃ for 12 days to make air-dried sausages, and the product is named Comparative Example 5.

[0132] Comparative Example 6: A mixed starter culture was prepared by adding Lactobacillus plantarum CICC 22205, Pediococcus pentosaceus CICC 21862, Leuconostoc mesenteriae CICC 21725, Lactobacillus acidophilus CICC 6255, and Lactobacillus brevis CICC 24450 in a volume ratio of 4:4:2:3:4 and then inoculated into air-dried intestines.

[0133] S1: Same as Example 5S1.

[0134] S2: Preparation of bacterial culture:

[0135] Single colonies of *Lactobacillus plantarum* CICC 22205, *Pediococcus pentosaceus* CICC 21862, *Leuconostoc mesenteriae* CICC 21725, *Lactobacillus acidophilus* CICC 6255, and *Lactobacillus brevis* CICC 24450 were picked and prepared into bacterial suspensions B1, B2, B3, B4, and B5 according to the method described in S2 of Comparative Example 5.

[0136] S3: Fermentation broth preparation: A fermentation broth was prepared by mixing *Lactobacillus plantarum* CICC 22025, *Pediococcus pentosaceus* CICC 21862, *Leuconostoc mesenteriae* CICC 21725, *Lactobacillus acidophilus* CICC 6255, and *Lactobacillus brevis* CICC 24450 in a ratio of 4:4:2:3:4. The final concentration of the fermentation broth was 10. 6 CFU / mL.

[0137] The implementation methods of S4 and S5 are the same as those in Comparative Example 5. 、 S5.

[0138] Comparative Example 7: A mixed starter culture was prepared by adding Lactobacillus plantarum CICC 20264, Pediococcus pentosaceus CICC 21862, Leuconostoc mesenteriae CICC 21725, Lactobacillus acidophilus CICC 20248, and Lactobacillus brevis CICC 20297 in a volume ratio of 4:4:2:3:4 and then inoculating it into air-dried intestines.

[0139] S1: Same as Example 5S1.

[0140] S2: Preparation of bacterial culture:

[0141] Single colonies of *Lactobacillus plantarum* CICC 20264, *Pediococcus pentosaceus* CICC 21862, *Leuconostoc mesenteriae* CICC 21725, *Lactobacillus acidophilus* CICC 20248, and *Lactobacillus brevis* CICC 20297 were picked and prepared into bacterial suspensions B1, B2, B3, B4, and B5 according to the implementation method of S2 in Comparative Example 5.

[0142] S3: Fermentation broth preparation: Fermentation broth was prepared by mixing *Lactobacillus plantarum* CICC 20264, *Pediococcus pentosaceus* CICC 21862, *Leuconostoc mesenteriae* CICC 21725, *Lactobacillus acidophilus* CICC 20248, and *Lactobacillus brevis* CICC 20297 bacterial cultures in a ratio of 4:4:2:3:4. The final concentration of the fermentation broth was 10. 6 CFU / mL.

[0143] The implementation methods of S4 and S5 are the same as those of Comparative Examples 5S4 and S5.

[0144] Comparative Example 8: A mixed starter culture was prepared by adding Lactobacillus plantarum ATCC 8014, Pediococcus pentosaceus ATCC 33316, Leuconostoc mesenteroides CICC21861, Lactobacillus acidophilus CICC 20244, and Lactobacillus brevis CICC 20269 in a volume ratio of 4:4:2:3:4 and then inoculating it into air-dried intestines.

[0145] S1: Same as Example 5S1

[0146] S2: Preparation of bacterial culture:

[0147] Single colonies of *Lactobacillus plantarum* ATCC 8014, *Pediococcus pentosaceus* ATCC 33316, *Leuconostoc mesenteriae* CICC21861, *Lactobacillus acidophilus* CICC 20244, and *Lactobacillus brevis* CICC 20269 were selected and bacterial suspensions (B1, B2, B3, B4, and B5) were prepared according to the method described in S2 of Comparative Example 5.

[0148] S3: Fermentation broth preparation: Fermentation broth was prepared by mixing *Lactobacillus plantarum* ATCC 8014, *Pediococcus pentosaceus* ATCC 33316, *Leuconostoc mesenteriae* CICC 21861, *Lactobacillus acidophilus* CICC 20244, and *Lactobacillus brevis* CICC 20269 in a ratio of 4:4:2:3:4. The final concentration of the fermentation broth was 10. 6 CFU / mL.

[0149] The implementation methods of S4 and S5 are the same as those of Comparative Examples 5S4 and S5.

[0150] Comparative Example 9: Add Streptococcus thermophilus CICC 20364 to dried intestines.

[0151] Except for the inoculation strain being Streptococcus thermophilus CICC 20364, the rest of the implementation methods are the same as in Example 5.

[0152] Comparative Example 10: Add thermophilic streptococcus CICC 25032 to dried intestines.

[0153] Except for the inoculated strain being Streptococcus thermophilus CICC 25032, the rest of the implementation methods are the same as in Example 5.

[0154] Comparative Example 11: Add thermophilic streptococcus CICC 20367 to dried intestines.

[0155] Except for the inoculated strain being Streptococcus thermophilus CICC 20367, the rest of the implementation methods are the same as in Example 5.

[0156] Comparative Example 12: Fish fermentation Staphylococcus aureus ATCC 51136 was added and inoculated into air-dried intestines.

[0157] Except for the inoculation strain being fish-fermenting Staphylococcus aureus ATCC 51136, the rest of the implementation methods are the same as in Example 5.

[0158] Comparative Example 13: Fish fermentation Staphylococcus aureus ATCC 51137 was added and inoculated into air-dried intestines.

[0159] Except for the inoculation strain being fish-fermenting Staphylococcus aureus ATCC 51137, the rest of the implementation methods are the same as in Example 5.

[0160] Comparative Example 14: Staphylococcus equi ATCC 43958 was added and inoculated into air-dried intestines.

[0161] Except for the inoculation strain being Staphylococcus equi ATCC 43958, the rest of the implementation methods are the same as in Example 5.

[0162] Comparative Example 15: Staphylococcus aureus CICC 10435 was added and inoculated into air-dried intestines.

[0163] Except for the inoculation strain being Staphylococcus equi CICC 10435, the rest of the implementation methods are the same as in Example 5.

[0164] Comparative Example 16: Lactobacillus plantarum CICC 20038 was added and inoculated into air-dried intestines.

[0165] Except for the inoculated strain being Lactobacillus plantarum CICC 20038, the rest of the implementation methods are the same as in Example 5.

[0166] Comparative Example 17: Lactobacillus plantarum CICC 20242 was added and inoculated into air-dried intestines.

[0167] Except for the inoculation strain being Lactobacillus plantarum CICC 20242, the rest of the implementation methods are the same as in Example 5.

[0168] Comparative Example 18: Lactobacillus plantarum CICC 20659 was added and inoculated into air-dried intestines.

[0169] Except for the inoculated strain being Lactobacillus plantarum CICC 20659, the rest of the implementation methods are the same as in Example 5.

[0170] Comparative Example 19: Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylose ATCC29971 were inoculated into wet-cured stinky mandarin fish in equal proportions.

[0171] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0172] S2: Preparation of bacterial culture

[0173] First, bacterial cells were prepared as follows: Single colonies of *Lactobacillus plantarum* CICC 22220 and *Staphylococcus xylose* ATCC 29971 were picked and inoculated into 10 mL of MRS liquid medium and cultured at 37℃ and 200-250 rpm for 24 h to obtain bacterial solutions A1 and A2. Then, 3 mL of bacterial solutions A1 and A2 were inoculated into 150 mL of MRS liquid medium and cultured at 37℃ and 200-250 rpm for 24 h to obtain bacterial solutions B1 and B2. After centrifuging bacterial solutions B1 and B2 at 8000 rpm / min for 5 min, the bacterial cells were collected. The *Lactobacillus plantarum* CICC 22220 and *Staphylococcus xylose* ATCC 29971 bacterial cells were diluted with a 0.9% sodium chloride aqueous solution to a final concentration of 10. 7 CFU / mL bacterial culture

[0174] The preparation method of Saccharomyces cerevisiae CGMCC 2.1364 cells is as follows: A single colony of Saccharomyces cerevisiae CGMCC 2.1364 is picked and inoculated into 10 mL of YPD solid medium, and cultured at 30℃ and 200-250 rpm for 24 h to obtain bacterial solution A3; then, 3 mL of bacterial solution A3 is inoculated into 150 mL of YPD liquid medium, and cultured at 30℃ and 200-250 rpm for 24 h to obtain bacterial solution B3. After centrifuging bacterial solution B3 at 8000 rpm / min for 5 min, the cells are collected. The Saccharomyces cerevisiae CGMCC 2.1364 cells are diluted with a 0.9% sodium chloride aqueous solution to a concentration of 10... 7 CFU / mL bacterial culture

[0175] S3: Fermentation Broth Preparation: Weigh out drinking water equal to the weight of the mandarin fish obtained in step S1, pour it into a container, and add 3wt% salt, 1wt% scallion, 0.6wt% ginger, 0.1wt% star anise, 0.05wt% fennel, 0.05wt% cumin, 0.01wt% chili pepper, 0.01wt% Sichuan pepper, and Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylose ATCC 29971 bacterial cultures in equal volume ratios to obtain a solution of 7×10⁻⁶. 6 CFU / mL.

[0176] S4: Fermentation: The mandarin fish obtained in step S1 was immersed in the fermentation liquid, the fish was pressed down with stones, and fermented at 12℃ for 5 days. The product was named Comparative Example 19.

[0177] Comparative Example 20: Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylose CICC21145 were inoculated into wet-cured stinky mandarin fish in equal proportions.

[0178] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0179] S2: Preparation of bacterial culture

[0180] The preparation methods for Lactobacillus plantarum CICC 20242 and Staphylococcus xylose CICC 21145 bacterial suspensions were the same as those for Lactobacillus plantarum CICC 22220 and Staphylococcus xylose ATCC 29971 bacterial suspensions in Comparative Example 19S2.

[0181] The preparation method of Saccharomyces cerevisiae CGMCC 2.1364 bacterial culture was the same as that of Comparative Example 19S2.

[0182] S3: Fermentation broth preparation: The fermentation broth of Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylose CICC 21145 was prepared in accordance with the fermentation broth preparation method of Comparative Example 19S3.

[0183] S4 is the same implementation method as Comparative Example 19.

[0184] Comparative Example 21: Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylose ATCC29971 were inoculated into dried and salted stinky mandarin fish in equal proportions.

[0185] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0186] S2: Preparation of Compound Bacterial Solution

[0187] First, bacterial cells were prepared as follows: Single colonies of *Lactobacillus plantarum* CICC 22220 and *Staphylococcus xylose* ATCC 29971 were picked and inoculated into 10 mL of MRS liquid medium and cultured at 37℃ and 200-250 rpm for 24 h to obtain bacterial solutions A1 and A2. Then, 3 mL of bacterial solutions A1 and A2 were inoculated into 150 mL of MRS liquid medium and cultured at 37℃ and 200-250 rpm for 24 h to obtain bacterial solutions B1 and B2. After centrifuging bacterial solutions B1 and B2 at 8000 rpm / min for 5 min, the bacterial cells were collected. The *Lactobacillus plantarum* CICC 22220 and *Staphylococcus xylose* ATCC 29971 bacterial cells were diluted with a 0.9% sodium chloride aqueous solution to a final concentration of 10. 7 CFU / mL bacterial culture

[0188] The preparation method of Saccharomyces cerevisiae CGMCC 2.1364 cells is as follows: A single colony of Saccharomyces cerevisiae CGMCC 2.1364 is picked and inoculated into 10 mL of YPD solid medium, and cultured at 30℃ and 200-250 rpm for 24 h to obtain bacterial solution A3; then, 3 mL of bacterial solution A3 is inoculated into 150 mL of YPD liquid medium, and cultured at 30℃ and 200-250 rpm for 24 h to obtain bacterial solution B3. After centrifuging bacterial solution B3 at 8000 rpm / min for 5 min, the cells are collected. The Saccharomyces cerevisiae CGMCC 2.1364 cells are diluted with a 0.9% sodium chloride aqueous solution to a concentration of 10... 7 CFU / mL bacterial culture

[0189] Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylose ATCC 29971 bacterial cultures were mixed in equal volume ratios to prepare a 7×10⁻⁶ solution. 6 A compound bacterial solution with CFU / mL.

[0190] S3: Preparation of auxiliary ingredients: Based on the fish body weight, weigh out 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% Sichuan peppercorns, 0.002% chili powder, 0.6% fresh ginger, and 1% scallions, and prepare a compound bacterial solution; the amount of bacterial solution added is 7 × 10⁻⁶. 6 CFU / g.

[0191] S4: Apply the auxiliary materials evenly to the inner and outer surfaces of the mandarin fish, place it in a sealed container, and ferment it at 12℃ for 7 days. The product is named Comparative Example 21.

[0192] Comparative Example 22: Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylose CICC21145 were inoculated into dried and salted stinky mandarin fish in equal proportions.

[0193] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0194] S2: Preparation of bacterial culture

[0195] The preparation methods for Lactobacillus plantarum CICC 20242 and Staphylococcus xylose CICC 21145 bacterial suspensions were the same as those for Lactobacillus plantarum CICC 22220 and Staphylococcus xylose ATCC 29971 bacterial suspensions in Comparative Example 21S2.

[0196] The preparation method of Saccharomyces cerevisiae CGMCC 2.1364 bacterial culture was the same as that of Comparative Example 21S2.

[0197] Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylose ATCC 29971 bacterial cultures were mixed in equal volume ratios to prepare a 7×10⁻⁶ culture. 6 A compound bacterial solution with CFU / mL.

[0198] S3, S4: Same as comparative example 21S3, S4.

[0199] Test case

[0200] The embodiments and comparative examples of the present invention were tested:

[0201] I. Determination of Nitrite Residue

[0202] To determine the nitrite residue in the examples and comparative examples, the nitrite was determined spectrophotometrically according to GB 5009.33-2016 "National Food Safety Standard - Determination of Nitrite and Nitrate in Food". The specific method is as follows:

[0203] (1) Nitrite extraction: Weigh 5g (accurate to 0.001g) of homogenized sample (if water is added during preparation, the amount should be calculated accordingly), place it in a 250mL stoppered conical flask, add 12.5mL of 50g / L saturated borax solution, add about 150mL of water at approximately 70℃, mix well, heat in a boiling water bath for 15min, remove and cool in a cold water bath, and place to room temperature. Quantitatively transfer the above extract to a 200mL volumetric flask, add 5mL of 106g / L potassium ferrocyanide solution, shake well, then add 5mL of 220g / L zinc acetate solution to precipitate the protein. Add water to the mark, shake well, let stand for 30min, remove the upper layer of fat, filter the supernatant with filter paper, discard the initial 30mL of filtrate, and keep the filtrate for later use.

[0204] (2) Nitrite determination: Pipette 40.0 mL of the above filtrate into a 50 mL stoppered colorimetric tube. Separately, pipette 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, 1.00 mL, 1.50 mL, 2.00 mL, and 2.50 mL of sodium nitrite standard working solution (5 μg / mL) into separate 50 mL stoppered colorimetric tubes. Add 2 mL of 4 g / L p-aminobenzenesulfonic acid solution to each tube, mix well, and let stand for 3-5 min. Then add 1 mL of 2 g / L naphthylethylenediamine hydrochloride solution to each tube, dilute to the mark with water, mix well, and let stand for 15 min. Using a 1 cm cuvette, adjust the zero point with the zero tube, and measure the absorbance at a wavelength of 538 nm. Plot a standard curve for comparison. Perform a reagent blank simultaneously.

[0205] The residual amounts of nitrite in the examples and comparative examples are shown in Table 3. The results show that for dried sausages, the residual amounts of nitrite in Examples 5, 6, and 7 were even lower, reaching as low as 1.78 mg / kg. Furthermore, the nitrite content in the fermented mandarin fish inoculated with *Lactobacillus brevis* was reduced to undetectable levels. This is attributed to the high nitrite degradation capacity of *Lactobacillus brevis* CHOL1, which effectively reduces nitrite residue when inoculated into fermented meat products.

[0206] Table 3. Residual sodium nitrite in dried sausage (mg / kg)

[0207]

[0208]

[0209] II. Determination of Nitrosamine Residue

[0210] Based on the results of nitrite residue determination, experimental groups capable of degrading nitrite were screened for further determination of nitrosamine residue. The specific determination method is as follows:

[0211] Sample pretreatment: Weigh 10g of homogenized sample, add 10uL of NDPA-d14 internal standard (2μg / L), add 15mL of acetonitrile, vortex to mix, freeze at -20℃ for 30min, then add 4g of magnesium sulfate and 1g of sodium chloride, shake for 30s, centrifuge at 8000rpm / min at 4℃ for 10min; take 6mL of the supernatant and add it to a 15mL centrifuge tube containing 50mg PSA, 150mg C18E and 900mg anhydrous sodium sulfate, vortex thoroughly for 1min, centrifuge at 8000rpm / min at 4℃ for 10min, take 5mL of the supernatant and concentrate it to 1mL with nitrogen, filter through a 0.22um organic filter membrane, ready for analysis.

[0212] GC parameters: carrier gas: helium; constant flow rate: 1.0 mL / min; temperature program: hold at 40 °C for 0.5 min, increase to 115 °C at a rate of 6 °C / min, hold for 1 min, increase to 150 °C at a rate of 15 °C / min, hold for 0.5 min, increase to 180 °C at a rate of 2 °C / min, hold for 2 min; solvent delay: 8.5 min;

[0213] MS parameters: Ionization method: EI; Ion source temperature: 240℃; Inlet temperature: 240℃; Quadrupole temperature: 150℃; Interface temperature: 240℃; Detection mode: MRM; Gain factor: 1.

[0214] The results of nitrosamine residue determination in the examples and comparative examples are shown in Table 4. As can be seen from the table, the residue levels of each nitrosamine and the total amount of nitrosamines in the dried sausage after adding *Lactobacillus brevis* CHOL1 were lower than those in the comparative example, indicating that *Lactobacillus brevis* CHOL1 can effectively reduce nitrosamine residue in dried sausage. Furthermore, compared with the comparative example, inoculation with *Lactobacillus brevis* significantly reduced the residue level of nitrosamines in fermented mandarin fish. Therefore, inoculating fermented meat products with *Lactobacillus brevis* CHOL1 can better reduce nitrosamine residue in fermented meat.

[0215] Table 4. Nitrosamine residues in dried intestines (μg / kg)

[0216]

[0217]

[0218] III. Colorimetric Measurement

[0219] Based on the results of nitrosamine residue determination, groups that can reduce nitrosamine residue were screened and their color was further determined. The specific determination method is as follows:

[0220] The color of dried sausage and stinky mandarin fish was determined using an UltraScan Pro colorimeter. The instrument was set to reflective mode and calibrated using a white standard plate. The a* / b* value was used as the color index for the evaluation examples and comparative examples. Each sample was measured in parallel 6 times, and the average value was calculated.

[0221] Table 5 shows the colorimetric values ​​of the examples and comparative examples. In practical applications, the a* / b* value can more accurately reflect the results observed by the naked eye; the larger the a* / b* value, the more vibrant the product color. The colorimetric results show that *Lactobacillus brevis* significantly improved the a* / b* value of the dried sausage. This is attributed to the excellent color-developing ability of *Lactobacillus brevis*, which can serve as a natural bacterial agent to partially replace nitrite. Inoculation with *Lactobacillus brevis* had no significant effect on the color of the stinky mandarin fish, possibly because its myoglobin content is low, and the effect of *Lactobacillus brevis* on it is not obvious.

[0222] Table 5. Colorimetric values ​​(a* / b*)

[0223]

[0224]

[0225] Based on the above results, compared with existing studies, the *Lactobacillus brevis* CHOL1 provided by this invention, as a fermenting agent for fermented meat products such as dried sausages and fermented mandarin fish, can significantly reduce residual sodium nitrite and nitrosamines in fermented meat, thereby improving the safety of fermented meat products. Furthermore, the excellent color-developing ability of *Lactobacillus brevis* CHOL1 helps to improve the color of fermented sausages and enhance product quality.

[0226] 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 Lactobacillus brevis ( Levilactobacillus brevis CHOL1, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 20, 2024, with accession number GDMCC NO.64656.

2. A microbial preparation containing the Lactobacillus brevis CHOL1 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The amount of the Lactobacillus brevis CHOL1 added in the microbial preparation is not less than 1 x 10 6 CFU / g or 1 x 10 6 CFU / mL.

4. A fermenting agent, characterized in that, The fermentation agent contains Lactobacillus brevis CHOL1 as described in claim 1, or contains a microbial preparation as described in any one of claims 2 to 3.

5. The use of Lactobacillus brevis CHOL1 as described in claim 1, or the microbial preparation as described in any one of claims 2 to 3, or the starter culture as described in claim 4 in the preparation of fermented meat products.

6. The application according to claim 5, characterized in that, The fermented meat products include fermented sausages and fermented fish.

7. The use of Lactobacillus brevis CHOL1 as described in claim 1, or the microbial preparation as described in any one of claims 2 to 3, or the fermentation agent as described in claim 4 in the degradation of nitrite.

8. A method for reducing nitrite in fermented meat products, characterized in that, The method involves introducing the Lactobacillus brevis CHOL1 as described in claim 1, or the microbial preparation as described in any one of claims 2-3, or the fermenting agent as described in claim 4 into the fermentation system of fermented meat products.

9. The method according to claim 8, characterized in that, In the fermentation system, the concentration of Lactobacillus brevis CHOL1 is 1 x 10 6 ~ 1 x 10 8 CFU / g.

10. The method according to claim 8, characterized in that, The fermented meat products include fermented sausages and fermented fish.

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